Meiotic and Mitotic Recombination in Aspergillus and Its Chromosomal Aberrations

MEIOTIC AND MITOTIC RECOMBINATION IN Aspergillus AND ITS CHROMOSOMAL ABERRATIONS Etta Kafer Department of Biology, McGill University, Montreal, Canada

I. Introduction . . . . . . . . . . . . . . . . . . 11. Meiotic Crossing-over and Nondisjunction: Effects of Chromosomal Aberrations, Especially Translocations . . . . . . . . . . . A. Meiotic Crossing-over in Standard Crosses, . . . . . . . . B. Reduction of Crossing-over by Chromosomal Aberrations, Especially Reciprocal Translocations . . . . . . . . . . . . . C. Aneuploids from Controls, and from Crosses Heteroeygous for Reciprocal Translocations . . . . . . . . . . . . . . . . . D. Meiotic Nondisjunction Frequencies in Crosses Heteroeygous for Reciprocal Translocations . . . . . . . . . . . . . E. Interchromosomal Effects of Translocations in Meiosis . . . . . 111. Mitotic Recombination . . . . . . . . . . . . . . . A. Mitotic Crossing-over and Nondisjunction in Standard Diploids . . B. Crossing-over in the Centromere Area of Group I and Genetic Differences between Strains . . . . . . . . . . . . . . . . C. Mitotic Recombination in Triploids . . . . . . . . . . I). Induced Mitotic Recombination in Standard Diploids . . . . . E. Mitotic Recombination in Translocation Heterozygotes . . . . . F. Mitotic Crossing-over in Disomics from Translocation Crosses, Especially in “Stable” Disomics . . . . . . . . . . . IV. Genetic Mapping and the Use of Translocations . . . . . . . . A. Genetic Mapping of Mutants in Aspergilhs nidulans and Effects of Translocations . . . . . . . , . . . . . . . . . B. Frequencies, Detection, and Mapping of Translocations . . . . . C. Mapping of Centromeres and the Use of Translocations for Sequencing of Meiotic Fragments. . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

33 35 35 40

43 50 56 58 58

67 74 81 85 97 100 101 107 116 124

I. Introduction

When mitotic recombination and the parasexual cycle were first demonstrated in Aspergillus nidulans, it was expected and hoped that these processes would permit mapping and breeding in commercially used asexual fungi, like Penicillium chrysogenum, Aspergillus oryzae, etc. 33

34

ETTA KAFER

(Pontecorvo et al., 1953 ; Pontecorvo, 1956). Indeed the feasibility of such work, and the existence of somatic fusion of cells and nuclei followed by mitotic segregation, have been demonstrated in a large number of species (Roper, 1966). These include not only important parasitic fungi, like Ascochyta imperfecta (Sanderson and Srb, 1965) or Ustilago hordei (Megginson and Person, 1974), but also mammalian cells and recently even those of higher plants (Gamborg et al., 1974). However, most of these investigations have not yet progressed to any practical stage, since various unexpected problems were encountered [for example, scarcity of spontaneous recombinants in Penicillium (Macdonald, 1971 ; Ball. 1973) 1. From the experience with Aspergillus nidulans (Kafer, 1965), it seems likely that chromosomal aberrations, especially translocations, which are induced simultaneously with desired mutations and recombinants, produce a major problem for genetic analysis by mitotic recombination, even though these do not explain all unexpected features of mitotic segregation encountered with asexual species. Such aberrations are frequently induced in fungi by practically all commonly used mutagens, especially the relatively high and multiple doses of irradiation which have been applied in many asexual species (even low doses of UV induce a considerable frequency of translocations in Neurospora, as reported by Perkins, 1974). Since heterozygous translocations prevent random recovery of chromosomes in mitotic haploids from diploids, it is obvious that mitotic analysis of asexual species becomes frustratingly complicated by induced translocations, especially if chromosome numbers are small [e.g., possibly only three in Penicillium (Ball, 1973) 1. Detailed genetic analysis can, therefore, be successful if mutants are obtained spontaneously (e.g., selected by resistance to analogs and inhibitors) or with mutagens that cause no or few aberrations (e.g., chemicals that induce mainly base-pair substitutions). So far few mutagens of this type are known that are effective in fungi [except possibly nitrous acid (Abbondandolo and Bonatti, 1970) ] ; however, low doses of highly mutagenic chemicals, like nitrosoguanidine (NG) , also appear to produce relatively fewer chromosomal aberrations than point mutations [ e.g., permitting fast progress of mitotic mapping in the slime mold Dictyostelium, where mainly NG-induced mutants are being used (Katz and Kao, 1974; Kessin et al., 1974) 1. Although unrecognized aberrations complicate genetic analysis, translocations, once identified by their effects on meiotic and mitotic nondisjunction, can be used to advantage in mitotic mapping of markers and centromeres. This certainly is the case for Aspergillus nidulans, where meiotic recombination also occurs, so that translocations can be combined with suitable markers. In this species, meiotic recombination frequencies are so high that markers of the same chromosome arm often are com-

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

35

pletely unlinked in crosses; such markers can be sequenced either by mitotic crossing-over, or by means of overlapping translocations (Pontecorvo and Kafer, 1958; Kafer, 1958, 1975). I n asexual species, such use of translocations is bound to be very difficult and will probably be restricted to a few favorable cases. One major problem is that in most fungi, mitotic, and even meiotic, chromosomes are too small for recognition of translocations cytologically. However, new techniques might solve this problem, as has recently been the case for mammalian chromosbmes, which has led to fast progress in the mapping of translocations and deletions and to their use for mitotic mapping of genes in human cells (Jacobs et al., 1974; Ruddle, 1973; Shows and Brown, 1975). One great advantage of techniques that use chromosomal aberrations for genetic mapping is the fact that the obtained results are qualitative, and often comlementary to evidence from meiotic mapping [as has been demonstrated in many species of higher plants, e.g., in maize by Kasha and Burnham (1965) and in Drosophila, as in phages and bacteria, even at the microlevel within genes, e.g., by Welshons and Keppy (1975) 1. In addition, chromosomal aberrations may give information on basic processes, e.g., those of pairing and disjunction [as demonstrated in Drosophila by Grell (1962, 1967) ; or in maize by Burnham et al. (1972) 1. This investigation, therefore, had three purposes : 1. T o analyze the effects of one, or two, overlapping, reciprocal translocations on meiotic crossing-over and nondisjunction ; 2. To identify the processes of mitotic recombination in diploids with and without translocations, in triploids, and also in disomics from single and double translocation crosses, as well as the effects of inducing agents on these ; 3. T o assess the various methods of genetic mapping, the uses of translocations for mapping, and the various problems arising from chromosomal aberrations for mapping by the parasexual cycle. All the techniques and information on mutants used in these investigations have been written up in detail and are available from the author on request (origins of most of the translocations are shown in Table 1 ; for other strains, see Barratt et al., 1975). II. Meiotic Crossing-over and Nondisjunction: Effects of Chromosomal Aberrations, Especially Translocations

A. MEIOTIC CROSSING-OVER IN STANDARD CROSSES 1. Homogeneity of Linkage Values

Meiotic recombination in standard strains of Aspergillus nidulans produces typical values between linked markers, which differ no more

TABLE 1 Mitotic and Meiotic Tests for Translocations (T) in Original or Progeny Strains of UV-Induced Mutants'

UVinduced mutant

Residual genotype

FGSC No.*

Linkage group of mutant andarm

ActAl

riboA yA; nicB

23 1

I11 L

adE2O~

biA pabaA biA

50 429

I

adG14 anAl

biA; p A biA

choAla

biA (AcrA; lysB; choA+)

lacAl luAl lysB6

YA; P w A biA biA; smA

lysD2O

biA; sB pabaA y A

35 31d 1 354 58d 5.5 66 395 418

"1

I L I L

R1 VI R I L V L

Mitotic test in diploidsc Recombination between groups Deduced translocation None

T1(ZZ;VZZ) None None

No. of Lowest hap% loids found -

-

-

67

3

51 81

Groups with


Meiotic test in crossesC Frequencies of disomics for T-groups No./Total

%

0/1126

<0.1

1I;VII

17/ 958 -

1.8 -

36 34

None None

0/2660

-

-


T1(Z;VZZ)

48 -

0

1;VII

-

-

29/1187

None None None

48 39 31

31 36 26

None None None

0/1603 -


27/1437 60/1499

1.9 4.0

TI (ZZZ;VIZ)

31 125

1II;VII

-

-

-

biA biA

219 34

IV L I11 L

~ h e n A 2 ~ riboA adG y A proA1 pabaA y A

304 332

III

phenAP proAl

biA; nirA biA

260 32

111 It I L

pr0A2~

biA riboA g A ; AclA

111 414

I")

pyroA4 riboAl riboB2

biA biA biA; ActA w A 3

33 158

sAl'

biA anA y A ; w A 2 ; pyroA adG; pyroA; chaA

370 40

biA; chaA2 pabaA; chaA2

372 4 13

biA b i A ; lysB

398

methGl methH2

sAaa sD6O

3d

"I

IV It I L VIII It

None None

80 41

T1(I;VZZZ) T1(Z;ZV)e

None

T1 (ZZ;ZZZj None None None

32 -

"1

1;IV -

-

-

22

0

1I;III

50

2.5 22

34 40 18

None None None

-

-

-

29

T2(I;VZZZ)

For translocation-free progeny, see Barratt et al. (197,5), Section I. FGSC, Fungal Genetics Stock Center. c A few of these results are included in Kafer (196.5) or in Upshall and Kafer (1974). d Poor strain, containing additional morphological mutants. c Independently identified earlier by Sinha (1967). a

3 -

-

86

TI (V;VZZZ)

None None

1;VIII

T1 (V;VZ)

II1

37 34

V ;\? Y;VIII 1;VIII

7/ 8.57

0.8

19/1648 0/1603

1.2 <1.0

34/ 867

3.9

71 310

-

14/ 398

2.6 2.4

18/1786 23/ 347

1.0 4.2

-

1.8

-

14/ 799

m

2

0

g

E z

5U g+I

5p 0 0

9

2

u2

2

k

x

-iz 3

3.

38

ETTA KAFER

from cross to cross than expected from random sampling. This is exemplified by Table 2, where values from different laboratories are compared and approximate averages are calculated [only one of our 13 values deviates enough to be significant a t the 57%level, while 1/20 are expected to do so by chance; standard errors are calculated as in Kafer (1958) using angular transformation and weighting with the total number of tested progeny (Snedecor and Cochran, 1967); since for important crosses usually 400 progeny are tested rather than the routine sample of 200, and in some of the most informative crosses even 600-1000, it did not seem suitable to use the computer program of Dorn (1972), which produces nonweighted averages]. This uniformity of recombination values in Aspergillus is almost unique, which in part must be due to the fact that in this homothallic species all strains could be started from a single haploid nucleus (Pontecorvo et al., 1953). Except for induced mutations, all strains are therefore naturally isogenic. Aspergillus also shows extremely long genetic maps-e.g., compared to Neurospora-with three to four meiotically unlinked regions in several chromosomes or even arms. This relatively high rate of meiotic recombination may also, a t least in part, be a consequence of the high level of isogenicity. But it probably is also species specific, just like the differences observed in various mammalian species [ e.g., man showing higher chiasma frequencies than mouse, even though mice used for such investigations are usually more inbred (Ford and Clegg, 1969) ; or the extremely high frequency of chiasmata in amphibians correlated with relatively low chromosome numbers (e.g., Wallace, 1974) 1. 2. Lack of Chiasrna Interference

Aspergillus also differs from many other genetically well analyzed organisms by its complete lack of (positive) chiasma interference (Strickland, 1958), or even production of slight negative interference in certain crosses (Kafer, 1958) or under certain conditions (Elliott, 1960b). These results agree with the computer-analyzed data of Dorn (1972), which show no interference for crosses between markers in three of the linkage groups, and slight negative interference (i.e,, values of > 1 for coincidence = observed double crossovers/expected) in the two other groups (contrary to a statement of Dorn, referred to by Clutterbuck, 1974). I n one of these latter cases, namely in the crosses involving group IV, apparent negative interference might possibly be due to a reciprocal translocation Tl (IV;VIII) associated with frA 1 (Kiifer, 1975) as pointed out by Clutterbuck (1974). However, such effects are more likely caused by inversions than by translocations, since the latter reduce double as well as single crossing-over.

TABLE 2 Combined Recombination Frequencies ( %) in Controls and Heterozygous Translocation Crosses for Intervals in the Right Half of Linkage Group Io Markers and intervals Crosses

riboA

Control crosses

-

anA

19.2 f 1 . 3 (960)

19.4 & 1 . 0 (1446) 17.4 & 1 . 5 (3121) Combined values (Total)

18.3 (5.527)

-

adG

6.4 f 0 . 8 (960)

6 . 7 f 0.5 (1022) 8.2 f 0 . 5 (1284)

-

proA

25.3 k 1.4 (960)

29.8 & 1.2 (2747) 33.9 f 1.05 (2591)

7.2

30.6 (6298)

(3266)

- .-

pabaA

-

yA

-

biA

Reference

6.9 k 1.4 (960) 8.8 (1119)

13.6 f 1 . 1 (960) 16.5 (623)

3 . 1 f 0.6 (960) 5.5 (623)

Elliott (1960b)

11.1 f 1 . 3 (593) 7.9 f 1.2 (3938) 8.7 k 0.3 (3854)

16.8 f 1.2 (938) 15.7 f 1.0 (4228) 16.7 k 0.5 (3126)

6.9 f 0 . 5 (2962) Tj.7 5 0.4 (4098) 5.8 k 0 . 3 (2549)

Dorn (1972)

8.4 (10,464)

16.0 (987.5)

6.8 (11,192)

5.5b (901)

11.4b (1611)

5.4 (812)

Elliott (1960b)

Kafer (1958) Kiifer, Unpublished

33.1 Crosses heterozygous

for TI(Z;VZZ) or TZ(1;VIZZ)

12. I* (921)

20. 4b (543)

Totals tested are given in parentheses. Difference from control average significant at 1 % level. c 5% level.

-

a

b

w

CD

40

ETTA KAFER

Additional evidence for lack of interference is also contained in the values of Table 3 (Section 11, B, 2), where control recombination frequencies for all pairwise combinations of five markers of group I are shown; these can be analyzed for coincidence in six groups of three markers each. The obtained coincidence values are very close to 1 in five of the six cases (two of them slightly less than 1, namely, 0.91 and 0.85, and three slightly larger than 1, namely 1.06, 1.17, 1.25), and only one indicates possible negative interference (coincidence value of 1.75, for the two adjacent intervals suA - 12.2% - gnlD - 18.7% - fpaB, compared t o 24.3% for suA - f p a B ) . The published meiotic data from different laboratories are therefore in excellent agreement and indicate that, in standard crosses under standard conditions, distribution of multiple crossing-over in Aspergillus nidulans is usually random. As a consequence, the values of meiotic linkage maps are approximately additive only for small intervals, ca. up to 15 centiMorgans (cMo), while for three adjacent but more distant markers, the frequency of recombinants between the outside markers is considerably less than the sum for the other two pairs (see Section IV, on mapping).

B. REDUCTION OF CROSSING-OVER BY CHROMOSOMAL ABERRATIONS, ESPECIALLY RECIPROCAL TRANSLOCATIONS The effects of chromosomal aberrations on meiotic recombination are well known and are of two general types: ( 1 ) if single (or equivalent) crossing-over leads to the formation of inviable products, then most recovered types are noncrossovers or double crossovers; or (2) if aberrations interfere with meiotic pairing and chiasma formation, all crossovers, single as well as multiple ones, are reduced in frequency. 1. Inversions

The best-known crossover suppressors are inversions, which are commonly used in Drosophila and also are now being developed for mutagenicity tests in mice (Evans and Phillips, 1975). They act mainly, but not exclusively, by the first mechanism (Roberts, 1967). In most other organisms inversions have been isolated only rarely, probably mainly because of their relatively cryptic cytological and genetic properties (as demonstrated, e.g., in mice by Roderick and Hawes, 1974). I n fungi so far only three pericentric inversions have been identified in Neurospora; these inversions all produce inviable crossover progeny in heterozygous crosses, as is evident from the occurrence of asci with two white abortive spores (Newmeyer and Taylor, 1967; Turner et al., 1969). I n Aspergillus, only a single case of a likely inversion has been encountered, which

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

41

seems to be associated with lysA1. This inversion appears to overlap with T1 (VZ;VIZ) (“Ab VI” in Pollard et nl., 1968) but probably not T1 (V;VZ),as indicated by the finding that it produces many stable and partially stable aneuploids in crosses only with the former. No other methods for detection of inversions are available in this species which produces neither ascospore patterns nor cytologically identifiable chromosomes, and where reduced recombination frequencies would be detectable only in relatively well-mapped segments. 2. Translocations

I n Drosophila many translocations with one break fairly distal on a chromosome arm have been shown to reduce meiotic recombination even more effectively. In such cases single as well as double crossovers are absent, probably owing to synaptic disturbances (Roberts, 1970, 1972). I n Aspergillus, reduction of meiotic recombination has become evident around the break points of all translocations that have been mapped so far. For markers that are fairly closely linked to the breaks, the map distances in heterozygous crosses are often reduced to about half (see Fig. 15). To check whether such reductions were also observed in more distant intervals, recombination was analyzed in detail in linkage group I, in which the breaks of two translocations were mapped, namely that of T2(Z;VZZZ), close but proximal to suA (Ma and Kafer, 1974), and that of T1 (Z;VZZ), very close and probably distal to fpnB (Kafer, 1975). A special set of crosses was analyzed in which the same multiply marked standard strain was crossed to a standard strain, and to both translocations [namely: fpaB galD suAadE SulA riboA anA pabaA y A adE biA; FGSC No. 483 (Barratt et al., 1975) 1. Homozygous translocation crosses heterozygous for these same markers were also carried out (except for fpaR in the Tl(Z;VZZ) cross, because no recombinant containing this marker in coupling with this translocation has been obtained so far). The results for markers distal on I L and also for the most proximal ones on VII and VIII, combined with similar data that were homogeneous, are presented in Table 3 (and for intervals to the right of riboA in Table 2; data in both tables are based on totals of 1000-2000 tested in most cases, and always on totals of over 500). Since all values from homozygous crosses were indistinguishable from controls, these have been omitted, except for the intervals between the markers riboA - SulA - fwA - facC, which are on the same chromosome in homozygous T2(Z;V1ZI) crosses but not in controls (sD50 cannot be used as a marker in T x T crosses because these are homozygous for sD) . From the data in Table 3 it can be seen that recombination in intervals

42

.

ETTA KAFER

TABLE 3 Interchromosomal Linkages, and Reduced Recombination Frequencies ( %) for Intrachromosomal Intervals, in Crosses Heterozygous for Tf (Z;VZZ)or Ti2(Z;VZZZ)a Crosses heterozygous for Tf (Z;VZZ) Linkage group I riboA

group

SulA

I

suA galD fpaB

1

fpaB1- ,*'"I

SulA

suA

galD

16.0 (19.2)'

27.1 (42.0)

28.9 (41.6)

29.3 (44.0)

22.1 (35.1)

25.3 (37.3)

26.4 (42.7)

(12.2)

(24.3)

(19.2) 15.4 (35.1)

28.1 (41.6)

21.8 (37.3)

(12.2)

SD

27.3 (44.0)

21.8 (42.7)

16.5 (24.3)'

(18.7)'

2.8

6.1

VIII

fwA

facC

2(6;

:;:1

(4

(#I

13.4 (74

33.7 20.2 [45.31* [48.31

16.8 (2)

16.2

33.0 21.8 [42.8]* [50.2]

13.8

17.6 (2)

22.4

(#)

suA

galD

fpaB

riboA

SulA

(2)

32.4 (#)

t

(2)

(18.7)

I

Group

34.8

19.2

25.8 (42.0)

L

Group VII

(#)

22.3

(#I

(Z)

\ 10.5

-$"

Linkage group I Crosses heterozygous for T2(Z;VZZZ)

13.7

6.6 (8.8)"

SD

fwA

Group VIII

Values for controls in parentheses (19.2)*; for homozygous T in brackets [45.3]. Symbols for degree of significance: a = difference not significant; * = significantly different a t 5% level; all other values significantly different at 1% level. # No linkage; on different chromosomes. -+ = positions of T-breaks. a

b

close to the breaks shows highly significant differences between controls and heterozygous translocation crosses ; for the more distant intervals recombination values are also somewhat smaller in translocation crosses, but not significantly so. I n Table 2, the values from crosses heterozygous for either translocation are compared to the average standard values for

RECOMBINATION

AND TRANSLOCATIONS IN

43

Aspergillus

intervals to the right of riboA, and in such tests significant differences show up for all except the most distant intervals. However, in longer chromosomes these effects do not spread beyond intervals that show free recombination; e.g., no effects of these translocations are found for markers distal on VII R, or VIII R, respectively (data not shown). 3. Double Translocation Crosses

Even in crosses between two overlapping translocations, recombination is normal in distant, long intervals of the involved groups. On the other hand, for shorter intervals, closer to the breaks, the obtained values are usually even more reduced than in single translocation crosses [as shown also for Drosophila by Robinson and Curtis (1972), who in a double translocation cross, found very much reduced recombination in one differential segment and none at all in the other]. For example, in a cross of T1 ( V ; V I ) X T l (V1;VIl)(cross 2141, genotypes in Table 6) meiotic recombination between the four markers of linkage group VI (lacA - b w A - s B - s b A ) appears completely normal, i.e., no significant linkage was found for any pair except s B - sbA, and the value for the latter did not differ from controls. On the other hand, in cross 2146, linkages of markers around the breaks, e.g., galD and phenB of T l ( I ; V I I ) or palF - lysD - malA of T1 (ZI1;Vll) became very tight. FROM CONTROLS, AND FROM CROSSES C. ANEUPLOIDS HETEROZYGOUS FOR RECIPROCAL TRANSLOCATIONS

1. Typical Disomics from Standard Crosses

I n Aspergillus, ascospores from crosses between haploid strains are usually haploid, but rare aneuploids or diploids are also found (Pritchard, 1954), and in Neurospora ascospores are the best source of diploids (Smith, 1974). Most aneuploids from standard crosses are n 1 types, but occasionally n 2 or 2n 1 colonies are encountered. The frequency of aneuploids of all these types from standard crosses or selfed cleistothecia is about 0.1-0.2% [Upshall and Kafer, 1974; the lower values of Pollard et al. (1968), are now known t o be due to storage of the ascospore suspension a t 4OC, which leads to fairly rapid loss of all disomics]. Eight different disomic types corresponding to eight mitotic linkage groups can be distinguished visually (Kafer, 1961 ; Kafer and Upshall, 1973). These different types show very different rates of abnormal growth, with n 1 for group I V (“n IV”) often so normal that it is hard to detect, especially when morphological mutants segregate, and n 1 for group V I I I ( [ [ n VIII”) growing so poorly that often i t can hardly be seen and is easily overgrown by normal colonies. These differences are expected to

+

+

+

+

+

+

+

44

ETTA KAFER

influence the frequencies of their recovery, especially in ascospore platings of high density. 2. Disomics from Crosses Heterozygous for One Translocation

In meiosis, heterozygous reciprocal translocations produce unbalanced products even with 2 : 2 disjunction of centromeres, but these all have deficiencies and none are likely to survive in haploid organisms (also in the mouse, but not in man, these are invariably lethal; Ford and Clegg, 1969). I n addition, 3:l and 4:O disjunctions occur with increased frequencies, so that additional specific aneuploids are produced. Recovery of such aneuploids depends on two main factors: (a) they have t o survive long enough to be classified; (b) for genetic analysis they also have to produce identifiable phenotypes. a. Viability. In haploid organisms hypoploids are usually not viable whereas hyperploids show varying degrees of viability. For example, in yeast, viability is excellent up to about four extra chromosomes (Parry and Cox, 1970; James and Inhaber, 1975) and in Neurospora disomics are very unstable, but these and large duplications show very good viability and cannot be detected visually except when incompatibility alleles become heterozygous (e.g., Perkins, 1975;Smith, 1975). I n higher organisms, hypoploidy for small elements may be tolerated in some cases [as in certain tertiary monosomics of the tomato of Khush and Rick (1967)1, especially in species with a few disproportionately small chromosomes [e.g., in Drosophila, where the large autosomes are not recovered in trisomics, but haplo and triplo types can be obtained for chromosomes X and IV from translocation crosses (e.g. Grell, 1959)1. b. Identification. Aneuploid products may be recognizable by a system that uses genetic markers plus phenotypic criteria [such as patroclinous males and matroclinous females after X-nondisjunction, produced for example, by elevated temperature in Drosophila (Grell, 1971a)1. Or identification may be possible from special features of the general appearance alone, presumably resulting from imbalance a t many loci; as for the famous trisomic series of Datura (Blakeslee and Belling, 1924) or similarly recognizable types in tomato [Rick and Khush (1968),but not in the related potato (Kessel and Rowe, 1974)l. I n fungi other than Aspergillus, such phenotypic specificity is either absent or hard to recognize (as in yeast ; James et al., 1974). c. Recovery. I n Aspergillus only four of the potential 34 types of aneuploids from any T X cross (Ford and Clegg, 1969) are expected to be fairly viable, namely the hyperploid products from 3:1 disjunctions. There are four n 1 types, disomic either for one of two normal chromosomes or for one of the two translocation chromosomes [as shown for TI (VZ;VII) and T l ( I I I ; V I I ) (Kafer, 1975)1. Such n 1 always result

+

+

+

RECOMBINATION AND TRANSLOCATIONS IN

45

Aspergillus

from primary meiotic nondisjunction so that the “typical looking” disomics with a normal chromosome extra contain both translocation chromosomes in the balanced haploid set, while the two types of translocation disomics contain both standard chromosomes (mitotic crossingover occasionally produces exceptions to this rule). The viability of the “typical looking” types does not differ from that of standard n 1, while the translocation disomics have specific phenotypes and different but specific viabilities for every translocation. They differ much more than the standard n 1, since many translocations produce chromosomes that are either much longer or much shorter than the standard ones. Some of these translocation disomics are therefore even less viable than n V I I I and only rarely recovered [e.g., one type from crosses with T1 (Z;VZZ) or T,?(Z;VlII),namely the n 1 which is disomic for the longer translocation chromosome, in which most of the long arms of V I I or V I I I are attached to almost the whole chromosome I (see Fig. 15)]. Generally, both types of translocation disomics are expected t o survive for translocations between linkage groups for which viable n 2 have repeatedly been obtained [like I11 or I V with each other, or with 11, V, VI, or V I I (Pollard et al., 1968; E. Kafer, unpublished)]. On the other hand, phenotypic recognition of highly viable types like disomics for very short translocation chromosomes may become very difficult or impossible, since these may not show differences distinct enough to be identified in single colonies. This is postulated to be the case for the other, short, translocation chromosomes of both T1 (Z;VZZ) and TZ(I;VZZZ) (above), as well as for the missing type of T1 ( V ; V I ) (Fig. 1 and Table 4 ; genotypes in Table 5 ) . The progressive reduction of phenotypic effects of all or part of a chromosome is best exemplified by comparison of disomics with duplications, as generated by nonreciprocal translocations (e.g., for group 111 ; Bainbridge, 1970; Clutterbuck, 1970). Even though some correlation between the severity of effects on viability, i.e., growth rate and conidiation, and the size of the duplicated segments is generally found, it is not expected to be consistent or accurate, since the genes responsible are not likely to be distributed at random in a chromosome. Indeed it is obvious that such correlation does not provide accurate information on chromosome sizes: for example, group VII is longer than many others, but has the least phenotypic effect as judged by the difficulties of identifying 2 n + V I I . More precise information, like that for Drosophila produced by Lindsley et al. (1972) is, however, not available, and will be difficult to obtain except for fairly large segments.

+

+

+

+

+

3. Disomics from Crosses between Overlapping Translocatwns

From crosses between two reciprocal translocations, new disomic types are obtained when one of the breaks of each translocation is located in

46

ETTA KAFER

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

47

the same chromosome (the extreme case where both breaks are in the same chromosome will be discussed in the next section). For example, one new disomic type is expected as a consequence of meiotic crossing-over between the breaks, which produces a new “double-translocation” chromosome as the complementary product t o a normal homolog for the linkage group involved in both translocations [called “tripartite” by Tuleen (1973)working with barley translocations]. I n addition, and much more striking, new types of almost stable disomics are formed. These arise frequently, and three types are expected. One type, the most frequent of these, is genetically and phenotypically a segregant that contains the equivalent of two normal homologs of the common linkage group, each being split up into two segments, so that the extra chromosome cannot be lost (Figs. 2a and 11). Such “stable” n 1, disomic for a standard chromosome, are produced, for example, in the absence of meiotic crossingover by nondisjunction in the “common” group when the four translocation chromosomes segregate to one pole and the two normal homologs to the other [very frequent trisomics, 2n 1 for the “common” chromosome, from double-T crosses have been demonstrated in the mouse (White et al., 1974)1. The other two types can be formed after meiotic crossing-over between the breaks, which produces the new “tripartite” chromosome plus a standard one (Fig. 3).If both these crossover products segregate to the same pole, which may occur frequently if they have centromeres of different groups, no third element exists that would produce a viable haploid type; however, if they segregate with the two residual chromosomes from either parent (namly, one normal and one translocation chromosome in either case) stable disomics with the equivalent of one extra translocation chromosome are produced (Figs. 2b and 3 show one of these, which has been recovered repeatedly). Such disomics have been analyzed in detail from the relatively wellmarked crosses shown in Table 6. For the three crosses of T1 (Z;VZZ) to either T1 (VZ;VIZ) or T1 (ZZZ;VZZ) detailed results are shown in Table 7. All the listed types could be identified visually, and their heterozygous markers confirmed the phenotypic classification. For TI (Z;VZZ), which has one break extremely distal in group I and the other proximal in V I I R (see Fig. 14), one type of translocation disomic is usually inviable and the other probably not identifiable (as mentioned above). Since T1 (Z;VZI)

+

+

FIG. 1. Platings of disomics from a cross heteroeygous for TI(V;VI).(a and b) Typical-looking disomics: (a) n V; (b) n VI. (c) Only type of recovered translocation disomic, n V-VI, disomic for centromere of V. (d) Haploid. FIG.2. Transfers of “stable” disomics from a cross of TI(Z;VZI)x TI(ZIZ;VZI) showing rare haploid sectors. (a) Stable n + VII, p a l F / + , producing dark paZF and lighter pal’ haploid sectors. (b) “Stable” translocation disomic, n VII-111.

+

+

+

+

TABLE 4 Frequencies of Disomics in Crosses Heterozygous for Reciprocal Translocations

Cross No. T1(V;V I ) 9 Crosses. 19.53 2130 2131 21.52 2132 All Tl(V;VZ)

Total number of colonies

Total disomics, all types

Relative frequencies of specific n T-disomics n V-VI

+

~

(%)

(No.)

n+V

4557 2750 1628 1872 3120 3695

2.5% 2.3% 2.7% 8.9%. 3.9% 4.1%

(112) (64) (44) (166) (121) (153)

48% 19% 26% 57%. 24% 27%

(49) (13) (11) (90) (29) (41)

17622

3.7%

(660)

37% (233)

26% 62% 62% 37%. 72% 56%

n

(3ga) (30) (26) (58) (87) (84)

+VI

14% 19% 12% 6% 4% 17%

51 % (324)

+ 1types

Typical disomics (absolute %)

T-disomics n +VI-V

(15) (13) (5) (10) (5) (26)

n+V

?a

-

-

-

12% (74)

1

n +\'I

1.1 0.5 0.7 4.W 0.9 1.1

0.33 0.47 0.31 0.53 0.23 0.70

1.3

0.42

Total specific disomics

T1(VI;VZZ)

(%)

(No.)

n

+ VI

n

+ VI-VII

~~

n ~

+ VII

n

+ VII-VI

12

+ VI

n

+ VII

~~~

4 Crossesa 5 Crosses* 5 recent crosses

3643 12740 6090

2.7% 5.0% 4.8%

(100) (643) (290)

35% (35) 42% (270) 42% (121)

13% (13) 16% (102) 11% (31)

30% (30) 23% (145) 27% (79)

22% (22) 20% (126) 16% (45)

1.0 2.1 2.0

0.8 1.1 1.3

All Tl(VZ;VZZ)

22473

4.6%

(1033)

41 % (426)

14% (146)

25% (254)

19% (193)

1.9

1.1

z* r3

w %-

cj

I

RECOMBINATION AND TRANSLOCATIONS IN

49

Aspergillus

TABLE 5 Genotypes of Crosses Heterozygous for T1 (V;VZ) (Markers on Groups I’ and 1’1 Only)

Cross of origin for T-strain

Linkage groups

Cross No.

T

(noT)

1953

T

2152

(noT)

T 2130 2131

VI

V

(noT)

x

T (noT)

T 2132

(noT)

2136

(noT)

T

+ + + + + ; + + + + facA + + ; lacA + + + facA + + lacA + lysB p A + + riboD ; + + + + facA + + lacA + + + + + + lacA bwA + + facA + + lacA + + + lysB p A + hxA riboD ;

; ;

+ + facA + + + + hxA riboD + + facA + + ZysB p A + hxA riboD

ZysB

Homozygous T1( V ; V I ) cross T p A facA 2153 T lysB

+

+

+ + +

; ;

; ;

: ;

ribOD ;

+

;

lacA bwA

+ +

sB sB

+ +

+ + sB + + sbA sB sbA + 4+ sbA

(990, not shown)

1953 1953 2133 2130

+ + 4lacA bwA + sbA + + + + + + 4- sbA lacA bwA + +

2130

2131 2132

is one of the parents in all these crosses, this considerably reduced the numbers of aneuploid types and facilitated analysis (even though the other two translocations each produced two recognizable types of translocation disomics). For example, no types, stable or sectoring, disomic for the tripartite crossover chromosome I-VII-VI or I-VII-111 (Le., with centromere of group I) are expected to be viable; and only two, rather than three, types of stable disomics were likely to be obtained (as was found, Table 7).One of these stable types is a typical-looking n VII in all crosses (Fig. 2a), and the other is “disomic” for the translocation chromosome with the centromere of VII [namely, in the two crosses with T1 (ZZ1;VZZ) disomic for the VII-I11 chromosome as shown in Figs. 2b and 31. As can be seen in Fig. 2, both types of “stable” disomics have extremely large disomic centers, and these show the phenotypes of the much smaller centers of the corresponding sectoring disomics (published recently, Kafer, 1975); but they only produce very rare haploid sectors, since haploids can be formed only after mitotic crossing-over between the breaks in VII has occurred (Fig. 3 and, below, Fig. 12 and Table 17).

+

50

ETTA KAFER

PARENTS

I-VII-III

---- + Act'

lllN 8-

VII tcrossover)

t IL

Mitotic Crossing Over

I

I-v11

111

VII-l

F I ~ .3. Meiotic crossing-over between the breaks in a cross of Tf(1;VII)x Tl(IIZ;VZI) (cross 2146) and formation of stable translocation disomic n + VII-I11

by meiotic nondisjunction ; mitotic crossing-over in one of the intervals between T-breaks (see Table 17) leading to formation of a normally unstable disomic, able to produce haploid sectors of a crossover type.

D. MEIOTICNONDISJUNCTION FREQUENCIES IN CROSSES HETEROZYGOUS FOR RECIPROCAL TRANSLOCATIONS 1. Single Translocation Crosses

Since reciprocal translocations cause disturbances in pairing a t meiosis, it is not surprising that nondisjunction for the two involved chromosomes is

++

+I

u

a

+% - '",+

H

Aspergillus

?+ +; ++

++

d

+.f 1

+ I ;+ $+

._.-

R

& + +?j + +

rq .c

X

._..

z+ ++

.- .-

++

+%

s + % E ++ ++ -

3.

+ $ g + +? +; + % + $1 +%

++ %+

+g

%+

+;

++ +; EL

rq % +

&+

4

L

1

$+

H

rq

jt

j + I1 + Y

RECOMBINATION AND TRANSLOCATIONS IN

Y

F

H

$L +

JY +

J+ Y

51

TABLE 7 Frequencies and Types of Disomics from Crosses between Partially Overlapping Translocations: TI (ZL;VZZR)Crossed to Either TI (VZR;VZZR)or TI (ZZZL;VZZR) Normally unstable disomics with frequent haploid sectors Cross (No.) and 2nd T involved

Total Total prog- abnoreny mals

Mainly n 1. n

+

+ VI

n+ VI-VII

+

n VII-VI

Cross- All unover' stable n V I I types n

+

"Stable" disomics

+ VII

n

+

VII-VI

Both types"

Other abnormals

Ef:

x TI (VZ;VZZ) (1944) Nos. Frequencies

331

154 46.5%

x TI (ZIZ;VZZ) (1946) (2146)

413 236 -

Total Nos. 649 Frequencies

n

+ I11

7

29

n+ 111-VII

n+ VII-111

21

67 20.2%

50

29

79 23.9%

8 2.4%

n+ VII-I11

145

5

8

131 -

5 -

1 -

20 10 -

35 28 -

35 35 -

103 79 -

16 34 -

19 18 -

276 42.5%

10

9

30

63

70

182 28.0%

50

37

35

7

52 -

-0

87 13.4%

7 1.1%

The very inviable n + I-VII type from TI (Z;VZZ), the corresponding stable disomic, and the crossover types reciprocal to + V I I with the resulting "tripartite" chromosome extra, have been obtained only rarely, and could not be identified with certainty. a

n

7

3

2 w

B

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

53

greatly increased. Aspergillus presents an especially suitable system for the genetic analysis of these effects, since all eight types of standard disomics can be recognized visually and, provided ascospores are plated a t low densities, such aneuploids can be recovered fairly reliably. For all known translocations it was found that the frequencies of disomy for the heteromorphic groups increased 50- to 100-fold in heterozygous crosses, the highest values being found in those cases where all of the four expected disomics are recovered (Upshall and Kafer, 1974). When one or both types of translocation disomics are not recoverable or identifiable (see above), these obtained values for aneuploid frequencies are minimal estimates of the nondisjunction frequencies. On the other hand, typical looking disomics that contain an extra standard chromosome have known viabilities, and their absolute and relative frequencies show characteristic values typical for each translocation (Table 4).Such frequencies seem to show a fairly good inverse correlation with the distance of the T-break from the centromere; that is, the closer the break to the centromere the higher the resulting nondisjunction in meiosis (Kafer, 1975). This relationship may make i t possible to predict the approximate position of the breaks of new translocations from their aneuploid frequencies in heterozygous crosses, especially if similar information is available from crosses heterozygous for mapped cases that have one break in the same chromosome arm [this has been attempted for T1 (V ;V I),especially the break in group V I , and its n+VI frequencies are compared to those of T1 (VZ;VII) in Table 4; T1 ( V ; V l ) has subsequently been mapped, and details of the relevant crosses are given in Table 5 and in Section TV on mapping]. 2. Aneuploid Frequencies in Crosses between Translocations

When two translocations are intercrossed, the types and frequencies of aneuploids depend very much on the relative positions of the four breaks. There are three possibilities (as outlined in detail by Curtis and Robinson, 1971): (a) they may all be located in different chromosomes; (b) in the opposite extreme, the same two chromosomes are involved in both translocations; or (c) the breaks map in three chromosomes. Results from these three types of crosses in Aspergillus are shown in Table 8. a. Breaks in Four Groups. As expected, when all four breaks map in different groups, no interaction is detectable and the observed frequencies of disomics are not significantly different from the sum of the values obtained for each translocation alone (in Table 8, part a ) : three of the four cases fall well within the expected range, while in one cross, 2159, aneuploids are slightly but not significantly higher than expected (namely 10.5%, compared to 4% plus 3.5%, giving a x2 of about 2.8, P = 0.10).

54

ETTA KAFER

TABLE 8 Aneuploid Frequencies ( %) in Crosses between Two Translocations and Variation with Plating Densities5 Type of cross and translocations involved (a) Breaks in four different chromosomes Tl(I1;III)X Tl(V;VI) T2(I;VIII) X Tl(V1;VII) T2(I;VIII) X Tl(II1;VII) Tl(II1;VII) X Tl(1V;VIII) (b) Two breaks each in two chromosomes Tl(1L;VIIR) X TB(1L;VIIR) Tl(1R;VIII) X TB(1L;VIIIR) (c) Breaks in three chromosome arms, two breaks in same one Mapped translocations

Tl(1;VII) X T1(V1;VII) Tl(1;VII) X Tl(II1;VII) Tl(V1;VII) X Tl(II1;VII) T2(I;VIII) X Tl(1;VII) TP(1;VIII) X Tl(1V;VIII)

Arm of overlap, distance

Cross

-

2159 1822 2156 2158

-

1790 18% (131/725) 1656c 2 % (23/1038)

No.

Plating densities High

-

VII It Fairly close

Low

10.5 % (20/ 189) 7 . 1 % (29/407) 8 . 5 % (18/2ll) 7 . 5 % (33/430) 16% (68/420)“ -

1788 29 % (258/902) 1944 Fairly distant 1946 35 % (145/413) 2146 29 % (306/1054) Fairly close 2145 I L, very close 1645” 11 % (171/1555) 2142 25% (165/711) VIII R, very 2143 13% (112/844) distant 2157 1943b 10% (35/35@)

47 % (154/331) 55 % (131/236) 46 % (165/362) 20% (76/390) 14 % (46/320) -

VIII VIII

14 % (30/220) -

Crosses of unmapped T1 (V;VZZZ) (distances unknown) and

TI ( V;VZ)

Tl(V;VIII) Tl(V;VIII) Tl(V;VIII) Tl(V1;VII)

X X X X

TB(1;VIII) Tl(1V;VIII) Tl(V;VI) Tl(V;VI)

V

VI R, close?

213Sb 22% 2137b 11% 213gb 13 % 2141b 27%

(160/75@) (57/500b) (41/300b) (145/54@)

Observed numbers are given in parentheses. Totals estimated from number of colonies with “recombinant” color, since partly selfed, twin cleistothecia. Results of A. Upshall (unpublished).

b. Breaks in Two Groups. The situation is quite different when both breaks map in the same two groups or even in the same arm, so that types and frequencies of the aneuploids depend on the relative positions of breaks within a chromosome or arm. Such crosses have been used in vari-

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

55

ous organisms, either to identify the chromosome arms in which breaks are located (as in barley; Kasha and Burnham, 1965) or to generate specific duplications [ e.g., in Neurospora using ascospore patterns (Perkins, 1974) ; or in Drosophila for gene dosage studies (O’Brien and Gethmann, 1973) 1. Only two pairs of translocations of this type are available in Aspergillus. One of these, T l ( I ; V I I I ) x T2(Z;VIIZ), showed no detectable interaction (cross 1656, Table 8,b). This agrees with the postulated positions of the breaks in group I, namely a t opposite ends of both arms, and indicates a similar relationship in VIII [one break of Tl(1;VIZI) maps distal in I R (Klfer, 1965), but the other break has not yet been mapped, because of extremely low aneuploid frequencies obtained in certain crosses to markers of group VIII (Upshall and Kafer, 1974) 1. I n the other cross (1790 in Table 8,b) an unexpectedly found 1;VII translocation which showed breaks in the same chromosome regions as T1 ( I ; V l l ), was crossed to the latter to check for identity. Since 16-18% aneuploids were observed, it is evident that a new TZ(I;VZI) was present [ T 2 ( I ; V I I )could have originated in an ancestral UV-treated multitranslocation strain, or in one of the parents which is a reverted duplication strain; the latter produced another derivative, which contained a T (1;V;VII;VIIZ) complex, after a few years of occasional subculturing]. c. Crosses between Translocations with Breaks in Three Groups. When one break of each of the two translocations that are crossed together is in the same chromosome, it is expected that all three chromosome pairs involved will form joint pairing figures in meiosis. These can be used to determine breaks cytologically in some species. Such pairing results in increased disturbance of normal disjunction for the various centromeres, especially those of the chromosome pair involved in both translocations. A high frequency of nondisjunction is therefore expected, leading to the formation of various aneuploid types (as explained in detail in Section 11, C, 3 ) . Three main variables must influence the overall frequencies: (i) the distance of the more proximal break from the centromere, since proximity seems t o increase nondisjunction ; (ii)the distance between the breaks, especially their relative position to the centromere, i.e., whether they are located on the same or opposite arms; (iii) the viability of the various aneuploid products. A large number of such crosses have been analyzed, because it was hoped that these relationships could be used to get approximate estimates of the positions of the breaks for unmapped cases, as is possible by cytological techniques in higher plants, like barley (Tuleen and Gardenhire, 1974). The crosses between translocations with mapped breaks used for this investigation are grouped according to the arm of overlap (which corresponds either to VII R, I L or VIII R, Table 8,c). Some of the pairs of

56

ETTA KAFER

translocations were crossed more than once, using various suitably marked strains (genotypes in Table 6, for crosses that were analyzed in detail). Since rather large differences were found in such pairs, repeated platings from the same crosses were also carried out. From the obtained results, it became evident that a major variable was the plating density. This high variability obscured all minor differences between pairs of similar crosses. I n addition, since three variables interact, similar values can result from quite different situations. This is demonstrated in Table 8,c, where no distinction is possible between the first two listed cases, both involving T1 (Z;VZZ),even though the distance between the breaks in VII R must be about twice as long in the second [cross to T1 (ZIZ;VZZ)]as in the first [to T1 (V1;VZZ)1, and no meiotic linkage is detectable in standard crosses for any of these intervals. And for the crosses with Ti?(Z;VIZI)no significant difference in aneuploid frequencies is detectable for crosses with overlap in I L compared to those in VIII R, even though the two situations are quite different: in the first case the two breaks are quite close, but both distal in I L, while in the second the breaks are exceedingly far apart, but one is quite proximal in VIII R (Fig. 14, and Table 8,c, crosses 1645 and 2142, compared to crosses 2143 and 2157). Such results, therefore, have no predictive value, unless they are quite extensive, and only if several similar cases are well analyzed and can be compared and used as standards. And even well established results may fit several situations [as do those of the unmapped translocations TI (V;VZ)ahd T1 (V;VIZZ),quite apart from the fact that the latter can hardly be used when they differ no more from each other and from the crosses between mapped translocations than repeats of the same cross in several cases]. I n conclusion, tentative mapping based on results from crosses with overlapping translocations is probably not worth while in most cases. The effort needed to get consistent values in several such pairs is hardly greater than that needed for mapping by other methods (especially since partly selfed cleistothecia are frequent, which unpredictably changes the survival rate of ascospore samples). On the other hand, very extreme cases can be recognized even in small samples: breaks are probably distal in opposite arms when barely additive values of disomic frequencies are found; or fairly close and in proximal areas, when 4 0 4 0 % abnormals are produced in double-?' crosses. E. INTERCHROMOSOMAL EFFECTS OF TRANSLOCATIONS IN MEIOSIS Since the heterozygous translocation crosses of Aspergillus show many of the features investigated in detail in Drosophila, it is of interest to

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

57

consider whether the general mechanism postulated in the latter organism also may apply to Aspergillus. Based on an extensive analysis of effects by recombination-reducing aberrations on the disjunction of other chromosomes, two phases of pairing have been postulated for the meiosis of Drosophila: one early one, leading to and reinforced by chiasma formation, and a second one, “distributive pairing,” which involves all noncrossover chromosomes and may result in pairing of heterologous types (Grell, 1962, 1967). According to this hypothesis, reciprocal translocations may not only produce increased nondisjunction of the involved chromosomes, but, indirectly, also of others, especially if these have affinities and pair with chromosomes of the rearranged type. This is expected to lead to increased recovery of n 1 for other linkage groups, as well as to increased frequencies of n+ 2 among the T-specific types [observations in support of a general occurrence of these kinds of effects have also been reported in man (Grell, 1971b) 1. I n Aspergillus, occasionally some translocation crosses seemed to give higher than expected aneuploid frequencies for noninvolved chromosomes. For example, the early crosses of T1 ( V ; V I ) gave an unusually high frequency of “other” n 1 types, mainly disomics for group 111, some of them originating from n V I11 types. Similarly, a well-analyzed cross heterozygous for Tl(1;VZZ) gave unusually many n 2 types (namely 11 out of 4400 progeny, or almost 10% of all 129 recovered aneuploids; and in this case also over half of the n 2 were disomic for group 111). However, such observations have remained entirely sporadic and their overall frequencies do not conform to the expectations based on Grell’s hypothesis. They may, on the other hand, be comparable to results by Rick and Gill (1973), who obtained various aneuploids, usually trisomic for shorter chromosomes, from crosses with primary trisomics of tomato. But if it is taken into account that in various situations n I11 types are the most frequent [e.g., among aneuploids isolated from diploids (Pollard et al., 1968), or among n 2 types from any source, see above], the inconsistent recovery of n+ 2 types may be attributable to the very poor viability of most types. Consequently, double disomics would be viable and expected only in certain favorable cases, and their low frequencies could not be used as an argument for or against any hypothesis. As a further check, crossing-over in n and n+ 1 progeny has been compared in a random sample of values from crosses heterozygous for T1 (ZZZ;VZZ) or T l (V;VZ). It was found that in the 27 randomly chosen comparisons from these crosses, recombination is as high in disomics as it is in haploids (namely, values were larger in n 1 than in haploids for 14 of the 27 analyzed pairs of markers, and smaller in 13). This agrees with the general similarities of values between n and n 1 obtained from

+

+ + +

+

+

+

+

+

+

58

ETTA KAFER

all other crosses and may be analogous to results with Y-autosome translocations obtained by Novitski (1975). From all the combined evidence, it seems possible that there is no regular occurrence of distributive pairing in Aspergillus; since noncrossover chromosomes hardly ever seem to exist, this is not really surprising. I n extreme situations, with very high or no crossing-over, distributive pairing may not be needed [as indicated by the lack of it in Drosophila males; or in the cases of “uncoupled” mutants which lack distributive pairing but do not affect exchange, or the disjunction of bivalents that have undergone exchanges (Carpenter, 1973) 1. Ill. Mitotic Recombination

A. MITOTICCROSSING-OVER AND NONDISJUNCTION IN STANDARD DIPLOIDS Several types of mitotic segregation which lead to spotting or variegation have been demonstrated in various organisms. I n Aspergillus, two main types occur spontaneously: (1) Mitotic crossing-over, which seems to occur in all fungi, and possibly all Diptera, generalizing from cases in Drosophila (Stern, 1936) and the house fly (Nothiger and Dubendorfer, 1971). So far it has been reported only in very few higher plants and animals (e.g., recently in Tradescantia by Christianson, 1975). (2) Mitotic nondisjunction, which seems to be a universal process, the reported frequencies of which vary mainly with the ease of identification. Absolute Frequencies. I n higher organisms it is extremely difficult t o estimate the frequencies of these processes, since the somatic cells involved show various rates and extents of division during development, and clone size may also be affected by the mutants used for detection. I n fungi, where vegetative growth is fairly uniform and mononucleate units can be sampled (e.g., in conidia) approximate estimates can be made, provided proper correction for clonal distribution is applied [ Luria and Delbriick (1943), as shown for crossing-over in the right arm of group I1 (Kafer, 1958) 1 . The incidence of mitotic crossing-over using color markers has been estimated in two similar sets of experiments. It varies from about 0.1 to 0.3% per chromosome arm, probably correlated with arm length (Kafer, 1961), giving a total estimate of over 3% [since a t least 6 of the established 13 arms are very long, and probably only 3 relatively short (Clutterbuck, 1974) ; see Fig. 151 . Nondisjunction, based mainly on aneuploid frequencies, probably is also a t least 2 3 % , considering the difficulties of identification and lower survival [ as evident

IN

RECOMBINATION AND TRANSLOCATIONS

59

Aspergillus

from the fact that none of these types were recovered in similar experiments of Pontecorvo et al. (1954) 1. Such information is valuable mainly for estimates of coincidence of the various types of segregation, e.g., double crossing-over vs. nondisjunction (see below). For all other purposes, mitotic recombination frequencies between genes in diploids are so low, and such determinations are so laborious, that more convenient measures of relative frequencies are used by most investigators, and also in all other experiments discussed here. 1. Mitotic Crossing-over

Mitotic crossing-over occurs a t the four-strand stage of mitosis, SO that there are two possibilities for the segregation of centromeres (Stern, 1936; Fig. 4). Since mitotic recombination is rare, products are usually selected. Namely, intergenic crossovers are obtained as segregants with increased resistance or striking phenotypes, when fully or partially recessive markers become homozygous (type I segregation) ; and interallelic crossovers are selected as wild-type recombinants between different alleles which cause a requirement or sensitivity to an inhibitor (type I1 segregation). The latter type of selection can lead to recovery of both strands

I

B ;'

+

su

+

+

SUI

Ps;

+

+ 4

Y

+ +

independent exchanges at 4-strand stage of mitosts

bx-" cha 4

Two possible types of segregation of centromeres

I

Twin-spots. homozygous distal to CO .1

30 pius

PIUS

2.

-

40

a Parental

phenotypes Heterozygous segregants. unchanged

1.

4o

Reciprocal 2.

cr0-m30

sy

:

+

+ 8YI

+BuI

:

u:

rib

i

+

: :

i

;,bO

pro 1

- .

IYS

-

I

4

+

ly.

7.t

I

i5 i

/

Cha

fw

i7 i Pro 2

\

I

+

+ +

cha C b

-v

FIG. 4. Reciprocal mitotic crossing-over and genotypes of various segregants resulting from coincidence of exchanges in I L and VIII R, considering the two possible types of segregation of centromeres in mitosis.

60

ETTA KAFER

involved in the exchange [in about half of the cases, Fig. 4 (Roper and Pritchard, 1955) 1 . However, such recombinants frequently result not from reciprocal crossing-over, but from gene conversion [as shown even in Drosophila (Kelly, 1974)l. Because of the ease of selection, allelic recombination is used extensively as a tool for measuring recombination frequencies [e.g., in various uvs mutants of Ustilago (Holliday, 1971) ] or for the mapping of noncomplementing alleles [induced in yeast, e.g., by sunlamp irradiation (Lawrence and Christensen, 1974) 1. Also, its frequencies are relatively high, often barely ten times rarer than meiotic ones. However, because mitotic conversion occurs with relatively high frequencies and does not seem to be coupled to outside marker exchange, sequencing of outside markers or centromeres is not easily possible from segregation of markers in selected allelic recombinants (Davies et al., 1975). This method has therefore been tried only once (for group IV; Kafer 1958), but not used further, and only intergenic crossing over is discussed here. In selected intergenic crossovers, only one of the two strands involved in an exchange is recovered. I n Aspergillus, these have been identified first by color (Pontecorvo et al., 1953, 1954) and later by resistance (Roper and Kiifer, 1957), and selection of the latter type is so far the only method available in Dictyostelium (Williams et al., 1974; Gingold and Ashworth, 1974; Katz and Kao, 1974). However, in systems that select by visual criteria it is possible, with suitable markers in repulsion, to recover all four strands: namely, one crossover and one parental each in two “reciprocal” diploid segregants, as “twin spots” (Stern, 1936 ; Wood and Kiifer, 1967) ; (Fig. 4 ) . Indeed, since no other process is known that would produce two fully viable segregants with this type of complementarity, such twin spots are the one conclusive criterion for somatic crossing-over in some species of higher organisms [e.g., in soybeans (Vig and Paddock, 1968) 1, Relative Frequencies, Distribution. As in Drosophila, mi totic crossingover in Aspergillus is concentrated in the centromere areas and shows characteristic distribution in each arm which differs from that of meiotic crossing-over when large enough samples are compared (e.g., Table 10). However, even small samples can be used in a qualitative way to determine the sequence of markers on a chromosome arm, provided a distal marker is available for selection. One basic problem is to identify crossover segregants and distinguish them from those produced by other processes. This is especially difficult in the first stages of analysis, as exemplified by the problem of interpretation encountered by Pontecorvo and Roper before haploids were distinguished (in Pontecorvo et al., 1953). And even a t later stages, nondisjunctional segregants are often

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

61

impossible to identify with certainty ; for example, after definite mapping of the centromere to the “left” of sD and coA ( M a and Klfer, 1974; Clutterbuck and Cove, 1974), it is now known that some of the co ribo cha “nondisjunctional” segregants of Kafer (1961) must have been crossover types. This latter distinction is not crucial for the sequencing of markers. But it is essential for the mapping of centromeres, as is good recovery of diploid vs. haploid segregants and uniform viability of all segregant types (discussed in detail in the section on genetic mapping). Specially marked diploids have now been prepared that facilitate visual distinction of the various types of segregants and therefore make it easier to observe and measure the relative frequencies of the various types of segregation (Table 9 ) . All of them contain two color mutants in repulsion on the right arm of linkage group VIII, fawn (fwA) and chartreuse (chaA) ; in addition they have two or three selective markers in coupling on the two arms of group I (including the suppressor suAadE on I L, and yA = yellow on I R in all cases). Therefore, if markers on I L are selected (e.g., suA), all haploids are either fawn (yA;fwA) or yellow chartreuse (yA;chaA) and can easily be distinguished from diploid segregants, which may be green crossovers (wild-type color) or yellow (yA/yA) nondisjunctionals. Very rarely, resulting from two events of recombination, diploids may be chartreuse (chaA/chaA) or fawn (fwA/ fwA) ; the latter can be visually distinguished from lighter fawn, yA;fwA, haploids only under optimal conditions. The distribution of crossing-over in both arms of group I is shown in Table 10 (results of diploids 2196 and 2200, genotypes in Table 9, and of diploid 859, genotype in Table 11). These results demonstrate the general agreement, but also the discrepancy in the prod-centromere area, compared to the similar published results of Pontecorvo and Kafer (1958; diploids Y and Z, Table 10; extensive analysis to explain these discrepancies is discussed below). Diploid 2196 is heterozygous for fpaB distal on I L, which permits selection of resistant segregants on complete medium (CM) supplemented with p-fluorophenylalanine (pfp). This extends the mitotic map of I L and was intended to provide a simple method for teaching of mitotic recombination, since, on media containing pfp, all sectors, even haploid ones, are usually large enough to be tested without prior purification. However, selection on CM + p f p produces such a predominance of haploids that in two large samples analyzed here, only the few diploid segregants shown in Table 10 were obtained. These results are, therefore, very approximate and, judging from larger samples published previously ( M a and Klfer, 1974), the value for interval I1 may be underestimated. Selection for f p a B is therefore very suitable when haploids

62

ETTA KAFER

TABLE Genotypes of Diploids for Teaching Diploid

No.

475 2196

2198

2201 2200

Linkage groups

Haploids FGSCNo.

473 475 474

fpu-sensitive

1 f p a B galD

suA

+

riboA

a n A pabaA

+ galD s u A + riboA a n A + + + SulA + + + galD s u A + riboA a n A (s+) + + SulA + + 479 -

yA

+ + + SulA + + + + f p a B galD s u A + riboA a n A pabaA y A + + + SulA + + + + diploids

513 466

yA

+

+

pro-94

biA

+

+

+

are wanted, but not economical for quantitative analysis of crossing-over (the reverse is true for selection using sulfite-requiring mutants, like sB or sC on selenate media ; see Tables 19 and 20). 2. Nondisjunction

Haploid and diploid segregants resulting from misdistribution of whole chromosomes may arise from several processes, especially when inducing agents are used (see below). I n Aspergillus, spontaneous nondisjunction usually involves missegregation of single or few chromosomes, producing trisomics as primary products (see Fig. 5 ) . Such trisomics have reduced growth rate, but produce diploid nondisjunctional segregants repeatedly by chromosome loss or %econdary nondisjunction.” The determination of absolute frequencies of nondisjunction is therefore very approximate (see above), and it is not surprising that even their relative frequencies (e.g., compared to diploid crossovers) may differ under different conditions. Haploid segregants are likely to be the result of several steps of chromosomal loss, presumably starting with the 2 n - 1 products which only survive in heterokaryotic condition (Kafer, 1961) (for viability and phenotypes of aneuploids see above, “meiotic” aneuploids) . Cytological evidence for these postulated steps has recently been produced by Brody and Williams ( 1974) in Dictyostelium, where diploids produced aneuploid nuclei of all classes, from 2 n 2 down to n 1. However, no recognizable aneuploid clones could be maintained even from spores with sizes falling between the usual diploid or hapoid ones. Observed Belative Frequencies of (Diploid Crossover a n d ) Nondisjunctional Segregants. For the left arm of group I , using suA as a selective

+

+

+ +

biA

adE biA adE lysF pabaA y A

+

pabaA

adE adE adE adE

+

RECOMBINATION AND TRANSLOCATIONS IN

63

Aspergillus

9 and Induction of Segregation Linkage groups

I

I1

I11

v

IV

+ + ++ + + + AcrA w A ActA pyroA facA

+

+

+ --

+

+

+

+ - -+ -

+

AcrA

ActA

+

+

+

+

Selection of

su

+

+

suI

+ lacA

+ sB

+

+

+ +

VIII

s D f OliA choA

+ +

+

choA

w

A

+

+

+ + riboB chaA s D f w A + + + + + chaA

+ + + + + + + + + + + choA + + + chaA lacA sB + + + + + + + + + +

AcrA ActA pyroA facA a d E+ --adE AcrA pyroA facA

+

VII

VI

lacA

sB

+

OliA

+

+

+

chaA

z: Nondisjunction of single chromosomes ( 2 steps)

0

I

Double Nondisjunct ion

I

Yad

+

+ ad

bi

su

+

su

+

-

Yad + Y d +

+ ad

bi

trisom

/i

loss of extra homolog

+

su 2n

-

-

Y

d

+

ad

+

nondislunctional ~y

su

+

+

SUI

parental 2n

y

A

v

+ a d bi

FIG.5. Mitotic nondisjunction in group I producing sulsu, y/y segregants: either double nondisjunction, or two steps of single nondisjunction with an “unstable” trisomic 2n I intermediate.

+

TABLE 10: Relative Frequencies of Mitotic Crossing-over in Various Intervals of Both Arms in Group 10 DIploid and soled

Selective marker

I

I I I U su

fpaB gal

+

m B gal

or

&

S

U

S u l +

+

+

S d +

+

+

+

+

+

+

+

gal

sUA

1pMB ad

sUA

+

suA

fPaB gal

+

gal

su

m B gal +

rib0 rib0

an

ribo

+

--

pro

an

+

+

+

fiaB gal

s

S su

t

+

+

-

u

l

+

+

+

d

+

-

-

+

rib0

+

+

an

pro

--

+

I

z

o

s

7%

5%

Y

II

1 6

+

+

m

38%

+

+ +

N

20%

5%

(12

+

+

v

18%

5%

(6

+

Y

+ V

+

+

+

+

+

+

Y

x

J y s Paba Y Paba Y

paaa Y

+

Y

+

+

Y

I Y Pnba ~ Y

Observed numbers are given in parentheses.

10% (24

+ 48)

17%

+ 44)

14%

I

21%

Y

1

+

%

+ +

vmIx

-

lntervab

X

1 marker 859 Y a n d Z SuA SUA

Paba Y

+

~ Y S

I

+

su +

+

---

po

ribo an

+

=

+

+

su

fpaB gal +

+

+

~YS

-

+ + s u l + +

JpaB gal su + + + & YA

+

+

SUA

an pro

+

su i

A

--

V V I V I I V I I I M

ribo

S d +

+ +

JpaB

YY

+

rn W

+

Meiotic

1 VI

+

29%

68%

(127)

(73)

(169

20)

6!%

(102)

51%

(30)

+ 244)

16

I11% (998)

yA (1) <0.5%

YA 3%

vm

“4%

(164)

49%

48%

Ix

3%

(62)

18%

10%

X

16%

(123)

33%

33%

y/y double Co

23%

7%

nondisjunctionals

(3)

Totals:

(374)

1%

2

p: 6s96

>28 (407)

YA 5%

68%

(1) <0.5%

M

22%

-

1%

6%

5%

(181)

(310)

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

65

marker, yellow ( y A / y A ) nondisjunctional diploids showed frequencies of 6 1 0 % (not shown in Table 10, these are “yellow suppressed” sectors, among all LLsuppressed’’ sectors from diploids of Table 9 ; totals inspected, over 2000). These values vary slightly according to the method used for the choosing of “random” samples of sectors: yellow nondisjunctional sectors are conspicuous and easier to identify than green crossover sectors, so that the former are isolated preferentially when “all” sectors are counted rather than the “first” large one from each colony. However, from seven other diploids, which did not show such color differences, a truly random and similar average of 6.5% nondisjunctionals was obtained among suA/suA diploid segregants (120/1883 sectors tested). For the right arm of group I , nondisjunctional types among yellow segregants, selected as single heads from colonies of various diploids on CM, seem to show a lower frequency, which generally ranges from 1 to 6% (see y A / y A segregants in Tables 10 and 12). The overall average was found to be 3.7% (239 nondisjunctionals among 6367 yellow segregants isolated from 30 diploids) ; that is, only slightly more than half that found among suA/suA segregants. This is contrary to expectation, since the distance from the centromere to suA on the left arm of I is much longer than that on the right arm to y A , so that nondisjunctional types should be a relatively higher fraction among the y A / y A than among suA/suA. The discrepancy may, however, be the result of different conditions of selection which are expected to affect growth of unbalanced 2n 1 types and, consequently frequencies of secondary nondisjunction. However, it is hard to imagine more favorable conditions than exist in the heteroknryotic situation on CM when yellow heads are picked ; but i t has been found that growth conditions of heterozygous suA/+ diploids on medium lacking adenine seem to be unexpectedly favorable for aneuploids [especially suA/suA/+ types, but others as well (Kafer, 1976) 1. Because of such variables which influence recovery, i t therefore does not seem possible to use the incidence of nondisjunctionals as a common base line for comparisons of crossing-over frequencies in both arms of a chromosome, as theoretically should be the case.

+

3. Double Crossing-over us. Nondisjunction, and Coincidence

Considering the relative frequencies of mitotic crossing-over and nondisjunction, i t was concluded (and assumed above) that segregants homozygous for all markers of a chromosome are much more likely to be of nondisjunctional origin. Crossovers, resulting from double or consecutive crossing-over in both arms, are expected to be much rarer, in spite of the relatively high concentration of crossing-over close to the centromere. Evidence for this assumption has been obtained recently, using

66

ETTA KAFER

diploids of Table 9, which contain markers facilitating rough estimates of double crossing-over or coincident segregation. I n these diploids, 2-strand double crossovers with exchanges in both arms of group I or nondisjunctionals will be yellow diploid segregants, if suAadE is used as a selective marker; but, in addition, four-strand doubles are also recognizable since y+/y+ are darker green and such diploids are easily identified (because in these diploids all haploids are lighter colored). Assuming no chromatid interference, these two types, y A / y A or should be equally frequent (and so far we have no evidence against this assumption). The values obtained for four-strand double crossing-over ranged from zero to about 1% with an approximate average of 0.245% (in over 20 samples of about 300 each). Double crossing-over is, therefore, no more frequent than expected from random coincidence of single exchanges (unless two-strand doubles across the centromere are disproportionately frequent). To obtain a further estimate of the relative frequencies of nondisjunction and two-strand double crossing-over (other than across the centromere) “suppressed” yellow segregants (suA/suA y A / y A ) from diploid 2200 were tested. Among these it was found that a minimum of 6.5% (8/127) were definitely double crossovers (not homozygous for pro) ; this value leads to a minimum estimate of 0.4% double crossovers among all suA/suA segregants, well within the range indicated above ; therefore, nondisjunction is about ten times more frequent than double crossingover in this chromosome. Coincidence of mitotic crossing over in different chromosomes is also found with frequencies no greater than expected from random occurrence of single exchanges (but in most cases nondisjunction was distinguishable from crossing-over only for group I ) . For example, diploids of Table 9 permit a rough estimate of coincidence in I L and VIII R, when suA/suA sectors are inspected: all “suppressed” chartreuse (suA/suA; chaA/chaA) sectors are the result of crossing over in I L plus crossing over in VIII R (or nondisjunction, but presumably no more than in ca. 10% of the cases). Their frequency was found to be roughly 0.5% (3/413 in one tested sample with presumably slightly preferential isolation). Coinfor AcrA) among cidence of segregation for AcrA (producing segregants selected for a marker of group I has been observed with similarly low frequencies (5/1476 segregants from three different diploids = 0.3%). These results therefore indicate that multiple events of mitotic recombination occur a t random in Aspergillus. This was found not only for spontaneous segregants, but also for induced ones (Shanfield and Kafer,

+/+,

+/+

RECOMBINATION AND TRANSLOCATIONS IN

67

Aspergillus

1971). In Drosophila, on the other hand, results by Garcia-Bellido (1972) suggest that after induction with X-rays double-crossover types are too frequent to be due to independent events in all cases.

B. CROSSING-OVER IN THE CENTROMERE AREAOF GROUPI GENETICDIFFERENCES BETWEEN STRAINS

AND

When mitotic analysis of T1 (ZL;VIIR) was started (Kafer, 1962), i t soon became obvious that the mitotic mapping of proA and of the centromere of group I presented special problems. From the control (diploid 600, which is p r o A l / + , Table 1 1 ) results similar t o those shown above for diploid 859 (Table 10) were obtained: namely, significantly more of the “suppressed” (suA/suA) crossovers were homozygous for proA1 than obtained previously, and considerably fewer proA/proA were found among the “yellow” crossovers than observed previously in diploids Y and Z (Pontecorvo and Kafer, 1958). To trace the possible mutation and to check whether genetic or technical differences caused these discrepancies, twelve proA1 strains from the two proA1 pedigrees were combined with suitable tester strains and checked for segregation of proA1, by selecting “suppressed” as well as “yellow” crossovers. As a first finding, i t could be shown that when high levels of proline were used in the selective medium lacking adenine (rather than normal levels of arginine, as used previously) all diploids heterozygous for proA1 in coupling with suAadE and y A , produced significant frequencies of “suppressed” pro/pro crossovers in the left arm, which always were considerably higher than those of yellow pro/pro crossovers on the right. It was concluded, that the centromere must, therefore be located t o the right of proA, rather than to the left as deduced from earlier results [and also concluded by Strickland (1958) on the basis of results from unordered tetrads]. It became clear that only poor viability under the conditions used previously, had failed to show up the relatively high amount of mitotic crossing-over on the left arm between proA and the centromere which occurred in all diploids. However, if this was the case, an explanation would have to be found for the “yellow” pro/pro “crossovers” (still heterozygous for all other markers on the left arm, e.g., suAadE) . These diploid pro-segregants could possibly have arisen by “double” events (like crossing-over between proA and suAadE, followed by nondisjunction ; as previously assumed for the corresponding “suppressed” pro segregants of diploid Z ) ; but, since they were consistently found with frequencies about half that of normal nondisjunctional yellow segregants, they are much more frequent than expected from chance

TABLE 11 Genotypes of proA/+ Diploids (Groups I and I1 Only) Diploid Nos.

Available testers: FGSC Nos.

1408" 600 348 854 1388" 863 348 85 1

+

+

anA

luA

suA riboA

+

+

+

850

+ proA1

+

anA

+

+

suA

+

+

+

proAl

anA

adG

+

+

+

proA11

+ +

+

+ suA

12760

proA1

+

suA 1276"

864

+

s u A riboA

859

I1

Linkage groups

+ suA

riboA fpaA91

+ + + + + +

anA

+

+

+

anA

luA

+

+

anA

luA

+

+

+ proA1

+ proA2

+ pro-94

-

0

0

-

m

rn

0

lysF P a b a A

+ + + +

+

yA

adE

+

+

adE

biA

pabaA

yA

adE

+

+

+ +

adE

biA

adE

biA

YA

adE

pabaA

AcrA

+

+

wA

AcrA

+

. + +

0

wA

wA

M

e e

P

m

+

pabaA

YA

+

+ +

+

+

+

adE

biA

+

wA

+

+

yA

adE

+

Acra

wA

+

+

0

AcrA

wA

+

+

-

AmA

wA

+

+

+

+

+

adE

biA

+

+

yA

adE

+

+

+

+

adE

biA

+

+

yA

adE

+

AcrA

+

+

+

P

4

M

s 0

0

867

866

((

suA 50

+ suA

871

suA

+ +

+

+

+

+

+ +

+ +

+

pro-94

+

proA2

+

+

868

suA

+

+

869

suA

+ +

+ + + +

55

+ 865

suA

896

suA

+ +

+

riboA

897

{{

509

suA

+

Tester strain lost, Montreal number.

+

+ +

ZuA

+ + adG

+

+ + +

proA2

+

+

-

1

-

pro-94 proA2

+ pro-94 proA2

+ pro-94

-

+

+

YA

+ +

+

+

adE

yA

adE

YA

adE

+

+

yA

adE

yA

adE

+ +

+

+

+

yA

adE

yA

adE

+

+ +

adE

+

yA

adE

+ + + + + + + + +

+ + + + + + + +

+

+

+ biA

+ + biA

+

AcrA

wA

+

+

Acra

wA

AcrA

wA

+

+

AcrA

wA

AcrA

wA

+

+

AcrA

wA

AcrA

wA

+

+

AcrA

wA

-

A

70

ETTA KAFER

coincidence of crossing-over and nondisjunction. I n addition, however many other markers were present (e.g., the close markers EysF in I R and luA in I L) in no case was there evidence for any type of segregation in these yellow or suppressed pro-types other than “single crossing-over” on either side of proA (the genotypes of all, or a t least ten, pro segregants from both selections from every test diploid were analyzed by haploidization) . Since such ambiguous results might possibly be produced by diploids heterozygous for an intrachromosomal aberration, it was provisionally assumed that a small pericentric inversion had been induced together with proA1 and closely linked to it. To explain the ambiguous results, all test diploids would have to be heterozygous and the aberration would not be present in any of the pro+ tester strains used. This actually might be expected if the inversion was small enough and meiotic recombination reduced enough, that it remained virtually associated with proA1. As a first check of this hypothesis two other available proA mutants, proA2 and proA11, were analyzed for mitotic segregation. The results were very similar to those obtained with proAl (Table 1 2 ) . Therefore, if a chromosomal aberration was the cause of the ambiguous results with proA1, this same aberration would also have to be present in proA2 (and proA11) and presumably originate in biAl in which both proA1 and 2 had been induced. The only strain definitely without i t would then be the original wild type, mutants induced in it like yA2, and possibly progeny from backcrosses to the former. To test this prediction, a new pro mutant was induced in the original wild-type strain. In several tests this mutant, pro-94, gave consistent results, namely a considerable frequency of ‘Lsuppressed’l pro/pro mitotic crossovers and practically no “yellow” ones (e.g., diploid 2200, Tables 9 and 10; pro-94 is closely linked to proA2, but probably an allele of p r o R ) . To demonstrate clearly that a pericentric inversion was present in the biAl strain it was necessary to construct a diploid homozygous for the aberration and heterozygous for pro. I n such a “biA” diploid proA should map consistently to the right of the centromere, just as it mapped to the left in homozygous “wild type”; only structurally heterozygous diploids should produce ambiguous results. Pairs of diploids were therefore constructed using the most suitable pro+ tester strains, each against both the original proA2 and the pro-94 strains (in which suAadE, y A adE, had been introduced by mitotic crossing-over in distal segments, Fig. 6). These pairs of diploids are shown in Table 11. The results, obtained when crossovers were selected on both arms of group I, are given in Table 12. They do not show the pattern expected from a small inversion in the centromere area of the original birl-strain. Only the diploid homozygous for “wild type” centromere area (diploid 865)

TABLE 12 Frequencies of pro Crossovers among Segregants Selected on I L (suA or fpaA) or I R ( y A ) in Diploids Containing Different pro Strains Combined with Various Tester Strains ~

Centromere type -+ type 1 Selection: Tester strain FGSC No. “Wild type” Mixed

17

Suppressed suA/suA Diploid No. 865

% -15%

864

“biA”

55

868 -13%

“biA”

50

866

Mixed (Fd

(Fraction)

897 2 3 . 5 % (71/302)

Centromere type +

1

863 “Mixed” 348 or (Fs) M 1276

(Nondisjunctionals %)

Diploid No.

(3 %)

869

1. I % (8/715)

(2%)

1 . 0 % (3/292)

(1.3%)

%

(Fraction)

(46 incl. N)4 221 1 . 5 % (3/196)

13% (190/1483)

Suppressed

Yellow ( y A / y A )

(67 incl. N). 283 1 . 0 % (2/207)

18% (187/992)

(M 1276 lost) 509

(Fs)

“biA-type” (proA2)

“Wild-type” (pro-94)

9 % (10/109)

%

(Fraction)

850

(4.2%)

896

12% (48/339)

4 . 3 % (16/368)

(3.5%)

867

”Mixed” (proAll) Selected fpaA/fpaA

(Fraction)

(Nondisjunctionals %)

(19 incl. N). 2 . 1 % (6/281) 136 11 % (63/597) 2 . 6 % (13/478)

871

1 . 8 % (12/653) (3.5%)

%

Yellow

-’%

-10%

(43 incl. N). 238 ~

.

10% (119/1228)

-

(4.6%)

_

5% (21/426)

(5.4%)

“biA-type” (proAl) Yellow

2 % (6/251)

(3.1%)

851

8 % (18/217)

5 % (7/141)

(7.5%)

a suA pro/++ diploids homozygous for yA and adE, were used t o select suA/suA types, among which pro crossovers could not be distinguished from nondisjunctionals (N).

72

ETTA KAFER

+ proA- strain -

-

Marker strain suA yA adE

-

-

3) selected white

C>

proA

+

bjA

AUA

WA

haplold:

FIG.6. Stepwise insertion of selective markers .yA (and simultaneously adE) distal on I R, and suAadE distal on I L, into pro strains by mitotic crossing-over, followed by haploidisation. gives unambiguous results: these again place pro consistently onto the left arm, provided 1% exceptional pro-yellow types are disregarded as possible coincidence of two events of segregation. All other diploids give significant frequencies of “yellow” pro/pro “crossovers,” even though considerably less than “suppressed” pro/pro. These results, therefore, rule out the existence of a pericentric inversion in the original biAl strain but, on the other hand, definitely implicate genetic differences between biA1 and the original wild type ~ b sthe basis of the different results obtained with proA1 (or proA2) compared to pro-94. Since only diploids with “wild type” centromeres give results in agreement with current models of mitotic crossing-over, it looks as if the original biA 1 strain contains a mutation that affects mitotic segregation close to the prod-centromere area. Because no testable hypothesis comes to mind, details of these results are given here as accurately as possible for future consideration: Twelve different derived proAl strains were checked for mitotic crossing over in 18 different diploids. These produced on the average 9.3 k 1.1% of %uppressed” pro/pro crossovers (among 4913 selected ; range 5-15%). From 13 of these diploids, “yellow” crossovers could also be selected (total tested 1880) ; these were homozygous for the proA allele in coupling with yA with an average frequency of 3.4 f 0.5%.However, there appeared to be differences between different proA1-strains ; e.g., the “early” proAy+strain from the published diploid Z gave the highest values of apparent

RECOMBINATION AND TRANSLOCATIONS IN

Aspergil lus

73

crossing over to the left of proA among yA/yA (when tested in three different diploids, including diploid 854, genotype in Table 11): i t showed a range of 5-7% among a total of 452 yA/yA (which all had t o be analyzed for heteroxygosis of proA1 by selection of suA-wA haploids, since in these diploids proA was in replusion to yA). On the other hand, the derived proAl-“control” strain for the translocation work, when tested in four different diploids (one of them being diploid 600, Table 11) showed significantly lower values, ranging from 1.1 to 3.6% (average 1.8%, 539 yA/yA tested; x2 = 10.5, P < 0.01). Most other strains of the pedigree between these two proAl-strains gave, however, intermediate values (average 4.0%, 689 yA/yA tested from 6 diploids). The following procedure was used to check the original proA11 (pabaAl yA2; pyroA4) strain for proA-segregation. T o avoid all crossing, a new fpaA-allele (fpaA91) was induced in this strain by nitrosoguanidine treatment, so that fpaA/fpaA crossover segregants could be isolated on the left arm of group I, as well as yellow ones on I R (diploid 863, Table 12; comparable to the selection in both arms after introduction of suAadE on the left arm, and yA ad23 on the right arm, into proA2 and pro-94 strains, Fig. 6, and Table 11). The general trends emerging from a closer inspection of the data of Table 12 are the following: All diploids which contain suA in coupling with pro-94 of “wild type” origin, give higher frequencies of “suppressed” pro mitotic crossovers on I L (ranging from 13 to 23%, average 15.8%), than the corresponding diploids with proA2 of “biAl” origin (which show a range of 9-1276, average 10.8%). In addition, all the former diploids show lower frequencies of “yellow” pro/pro crossovers on I R (1.0-1.8%) than the latter (range from 2 to 570, average 3.4%). While for any one pair of diploids these differences are barely significant a t the 5% level, the combined values show highly significant differences (x2 > 10, P < 0.01, as expected from the fact that there is no overlap of values). I n addition, the centromere type of the pro+ ‘V.ester” strain also seems to influence results, since less yellow pro exceptional types are produced when pro-94 is combined with a tester having a “wild type” centromere (diploid 865) than when the tester is a “biA”-type strain (diploids 866 and 868). When all data are considered, it seems clear that only diploids with “wild type” centromeres give unambiguous results as expected from reciprocal mitotic crossing-over, while none of the diploids with “biA”-type centromeres conform to these patterns, presumably due to some type of mutation (that may also cause the correlated higher nondisjunction). To speculate on the kind of mutation which would cause the observed results one could postulate that i t creates a weak alternate centromere position (“spindle fiber” attached to the left of proA) which would result

74

ETTA KAFER

in about 1.5-20/0 single crossovers on the “right” arm when the mutation is heteroaygous (diploids 969, 866, 868), and twice that much when it is homoaygous (diploids 854,Z and 867,896, etc.) . At this time it is difficult to visualize how this or any other alternate hypothesis could be tested, since the possibilities of mitotic analysis have been pretty well exhausted, and the cytology of mitosis is obviously too imprecise to be a suitable tool (Robinow and Caten, 1969). The only remaining possibility, namely investigation of the genetic differences between pro-94 and various proA mutants and the anomalous centromere behavior in the latter, by means of meiotic tetrad analysis might, however, be worthwhile. While previously no markers close to any other centromeres were known so that the centromere distances computed from unordered tetrads were, by necessity, very approximate (Strickland, 1958) markers very close to their centromeres have now been identified in both arms of groups 11, 111, and IV, and new potentially linked ones have become known also for all other groups (Fig. 15; Clutterbuck, 1974). Mutations causing changes, e.g., in heterochromatic content, that affect meiotic crossing over as well as nondisjunction, have been observed in several organisms (in maize, by Ward, 1973; or in Drosophila, by Carpenter and Sandler, 1974). I n the above case in Aspergillus, however, extensive crossing of the described strains with “wild type” or “biA” centromere areas to the same tester strains has not revealed any effect of the postulated mutation on meiotic recombination (results are included in Table 2 ) . Whether any other of the reported cases that show reduction of repetitive sequences in the centromere areas correlated with increases of nondisjunction, or diffuse centromeres, are relevant here is hard to guess, even though at least in one case differences between meiosis and mitosis were observed (Comings and Okada, 1972; Laird, 1973). C. MITOTICRECOMBINATION IN TRIPLOIDS Triploids of Aspergillus nidulans have been obtained previously. Provided they were heteroaygous for conidial color markers, it could be seen (Fig. 7) that they are considerably less stable than diploids, and that they produce a high frequency of color segregants, probably by chromosomal segregation (Elliott, 1956; E. Kafer, unpublished). However, i t turned out to be impossible to obtain quantitative information, as long as mainly recessive markers were available, even when almost every homolog contained a marker (as shown for one of these early triploids, triploid 407, Table 13). These triploids were constructed with the aim of recogniaing as many segregants as possible by having two mutant alleles of most

TABLE 13 Genotypes of Triploids Triploid HomoNo. logs

I

Linkage groups :

m

Il

a 407

b C

a

2300

b C

Groups :

Iv a

407

b C

a

2300

b C

+ +

V

GPYYrOA

QymA

---. ---. methG

+

+

methG

+ +

+

QymA

M

VI

==-L- + +

+

nicA

+

nicA

+

VIII

sB

+-

+

+

+

+

+

+

+ +

+

+ + +

+

m'cB

choA

+

-++

choA

+

+

0

+ + +

+

+

nicA

+

+

facA

+ + + + + sbA lacA sB

+

+ +

+ malA

+ +

OIiA

+

choA

+ +

+

-+

+ + i

riboB

+ +

chd ~~

s D f w A +

+ + +

+

nboB

~

chaA

+ +

nboB chaA

76

ETTA KAFER

recessive markers present in the triploid. Crossing-over or nondisjunction would then produce segregants differing in their requirements or color from the original triploid, so that such segregants could fairly easily be recognized. However, it turned out that segregation frequencies are so high, that the problem is not to obtain spontaneous segregants, but to distinguish the various types obtained. To assess the relative frequencies of crossing over vs. chromosomal loss or nondisjunction, crossovers should be distinguishable from nondisjunctionals, and triploids from diploids and haploids. Since it is impossible to prove whether a segregant is homoor hemizygous for all recessive markers of a homolog, such distinctions were difficult or impossible for most segregants from triploid 407. The situation is quite different for the recently constructed triploid 2300 (Table 13). It contains not only more recessive color mutants than triploid 407 (five rather than three), but also a relatively large number of mutants that can be recognized in the heterozygous condition ( f p a B , SulA, suAadE if adE is present, IodA, AcrA, ActA, sB, and OZiA). These can be used to distinguish diploid or triploid segregants from haploid ones, since in the former usually a t least one of them remains heterozygous; also loss of homologs containing these mutants can easily be followed. In addition, mutants are generally present only as a single allele which makes identification of all homologs in haploids possible (except for some white haploids, Table 15; the haploids selected on CM+pfp were isolated to check the genotype of the triploid). Only the mutant rib& of group VIII was present in two homologs, since it served as a balancing marker to obtain the heterokaryon between a diploid containing two of the homologs (b/c) and the haploid strain (a) from which the triploid was selected. I n addition, yA2 and yA91 (a recently obtained olive mutant) are noncomplementing alleles, which, however, can easily be distinguished in haploids (but not in the various diploid or triploid combinations) . Haploids and diploids were also distinguished visually from triploids (Fig. 8 ) . The results obtained with this triploid (2300) demonstrate very clearly that chromosome loss is occurring extremely frequently in triploids of A. nidulans. When conidia from the newly synthesized triploid colonies were plated onto complete medium, very high frequencies of unstable aneuploid colonies of many different types were obtained (30-3576 in two platings with totals of about 200 each). I n addition, most triploid colonies produced one or two color patches or segments large enough to be seen by the naked eye (see Fig, 7b) compared to 1-2 per 100 diploid colonies (Wood and Kafer, 1969) (Fig. 7a), and practically all of these resulted from chromosome loss. To obtain a random sample of segregants, all easily visible color segments from the triploid colonies of the first plating were purified by

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

77

FIG.7. Low-density platings of conidia on complete medium: (a) diploid; (b) triploid. FIG.8. Shape and arrangement of conidial heads permitting visual classification of (a) diploids, (b) triploids, as well as (c) haploid segregants.

78

ETTA KAFER

streaking, and the resulting stable types were tested for markers and for ploidy (285 segregants from 145 triploid-looking colonies, Table 14). Since few segregants remained triploid, chromosome loss is obviously the major process a t work, while nondisjunction is quite rare and mitotic crossing-over even more so. This is also evident from the large frequency of aneuploids found, not only among the plated conidia which resulted in sectoring usually hyperdiploid colonies, but also as precursors of many of the isolated color segregants. Triploids of A . nidulans therefore constitute an excellent source of aneuploids (comparable to triploid meiosis in yeast, Parry and Cox, 1970). a. Random Loss of Chromosomes. That the frequent loss of chromosomes was basically a random process was demonstrated in two ways: (i) All haploids, which constituted about one-third of all color segregants, were classified for loss of homologs for every linkage group. The resulting frequencies then were compared with that expected on the basis of random loss (Table 15). As can be seen from these results, very few cases are found where the observed frequencies differ significantly from the expected ones, and in three of these cases the known reduced viability of the corresponding mutants is likely to be the main cause (homologs Ia, Ic, and I I I b ; only for one, sbA on VIb, does the extremely low recovery remain unexplained). (ii) All diploid yellow segregants (mainly yA2/yA91) were checked for loss of any of the mutants conferring resistance as an indication of loss of one of the homologs of groups 11,111,VI, TABLE 14 Relative Frequencies of Various Types of Spontaneous Color Segregants from Triploid Colonies of 2300

Crossovers Color

Haploids

Yellow-olive

23

Darker or paler green White Fawn Chartreuse Yellow-cha

3 33 16 2 9

Total Nos. and frequencies

86 30 %

Chromosomaltype segregants

Total

2n

3n

2n

3n

No.

%

7

3

151

16

200

12%

25 33

9% 12%

i2

> 10 > 4%

(max. 9%)

> 167 > 60%

285

TABLE 15 Relative Frequencies of Recovery of the Three Homologs a, b, or c in Haploids from Triploid No. 2300

a

F!

Bis

m

Linkage groups and homologs

I a

b

I1

I11

IV

-c

a

b

c

a

b

c

a

b

V

VI

VII

VIII

--c

a

b

c

a

b

c

a

b

c

2el

(a, b a o r c ) b c

(b orc)

Totals (No.)

8 Z

.+ r3

No. from CM So 64 14b 33 19 34 30 22 34 41 lga 26 36 26 24 46 6" 34 33 26 27 29 (1) 23 llb (22) No. from 68c 1 2 24 24 23 48 Oc 23 27 15. 29 29 22 20 37 10 33 28 18 25 21 (8) 21 9' (12) C M pfp Total No. 76c 65 16 57 43 57 78 22c 57 68 34a 55 65 48 44 83 7a 67 61 44 52 50 (9) 44 20 (34)

+

Combined % x2

P

a

4gC 41 10 45.1' <.005

36 28 36 50 14c 36 43 22 35 41 31 28 53 4a 43 39 28 33 32 2.4 0.9 11.3e 4.7 61.P 2.8 >0.25 >.5 <0.01 -0.1 <0.005 >0.25

Homolog with reduced recovery.

63 <0.1 >0.99

* Small chartreuse or haploid green color segments are difficult to detect in the light green heterozygous triploid.

+

Ratios distorted in sample from CM pfp, which selects for fpaB on Ia, and against phenA on IIIb. d x2 refers to haploids from C M only. c Significant deviations from expected random recovery.

86 71 157

p r

0

P

z 3

0

2

P P '1

3. c1

80

ETTA KAFER

and VII. It was found that also in this case, the pattern of marker loss agrees well with that expected from random loss of one of the three homologs: The various combinations of loss of the same number of markers ( 1 or 2 or 3) occurred with similar frequencies, while th*eaverage number of cases for loss of any specific combination of markers decreased as the number of lost markers increased (from 0 to 4 ) . b. Mitotic Crossing-over. To check for mitotic crossing over vs. nondisjunction, all yellow nonhaploid segregants were analyzed for markers on both arms of group I. It was found that 10% of these (17/167) were still triploid, but only very few of them (3/17) were 3n crossovers (still heterozygous for fpaB as well as SulA). In addition, seven of the 151 diploid yellow segregants were crossovers (namely six fpaB+,SulA+ but not proA, and one f p a B SulA+ but not anA or p a b a i l ) . For these it is difficult to be sure whether crossing over occurred in the 3n, 2n or aneuploid stage. But there is no doubt that mitotic crossover segregants from triploids are exceedingly rare. c. Nondisjunction. The majority of the few obtained yellow triploid segregants (13/16) apparently are of a nondisjunctional type, in which loss of homolog Ic usually was compensated for by nondisjunction of I b (the most viable homolog, which was also the one most frequently recovered in haploids). As expected, few (1/144) of the yellow diploid segregants resulted from nondisjunction (i.e., were homozygous for all markers of I a or I b ) , since in triploid 2300 simple loss of homolog I a (carrying the only y+ allele) produced yellow segregants. This indicated that the observed process is not one of random distribution of chromosomes, but more likely one of preferential division of nuclei of lower ploidy. Therefore, all results agree, and they indicate that triploids of Aspergillus show relatively low frequencies of mitotic crossing over but randomly lose chromosomes a t a high rate. Triploids cannot be transferred since even in freshly synthesized 3n colonies up to a third of the conidia are already aneuploid. Such aneuploids are mainly hyperdiploid, and diploid segregants represent a relatively stable intermediate stage, similar to relatively stable clonal variants in mammalian cell lines (e.g., Terzi and Hawkins, 1975). I n addition, haploid segregants are also more frequent than in diploids. Such triploids appear, therefore, to be even less competitive than triploid cells in man (Mittwoch and Delhanty, 1972). The whole segregation pattern rather resembles that found in hybrid mammalian cells used for mapping of human genes, and is quite different from the situation in yeast, for example, where polyploids and even aneuploids are relatively stable [as analyzed in detail recently by Campbell et al. (1975) ].

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

81

D. INDUCED MITOTICRECOMBINATION IN STANDARD DIPLOIDS Since spontaneous somatic segregation is very rare, induction of such recombination is often resorted to, especially when it is used as a tool for analysis of various problems. The main question in these cases is whether the inducing agents actually increase the frequency of spontaneous recombination, or produce segregants hy entirely different mechanisms, or both. In either of the latter two cases the problem arises how to distinguish segregants that look identical but are the end products of different types of processes. This is all the more difficult because the end result depends on the relative viabilities of the intermediate products, like aneuploids or heterozygous deletions, and these differ greatly from species to species. Generalizations from one organism or system t o another are, therefore, difficult even if these are relatively closely related (as shown, for example, by the entirely different aneuploid tolerance in yeast, Aspergillus, and Ustilago). In addition, the results depend on the developmental stage of the treated nuclei and cells, especially in higher organisms like Drosophila, but also in fungi, (e.g., in Aspergillus) where quiescent or germinating conidia, or growing hyphae may be exposed t o inducing agents. The final results also depend on relative frequencies of induced and spontaneous recombination, and the abilities of various segregants to conipete with normal cells. For example, in Drosophila, X-rays are routinely used to increase mitotic crossing-over not only in developmental, hut also in behavioral studies (Hotta and Benzer, 1972) ; and evidence for reciprocal crossingover is based mainly on the production of “twin spots” when markers in repulsion on thc same chromosome arm are used (Stern, 1936). It is well known that X-rays also produce chromosomal aberrations and breakage which can be demonstrated by cytological techniques (e.g., Gatti et al., 1974), but survival of such nuclei is poor and no visible spots are expected from most of these (Stern and Tokunaga, 1971). Therefore, only secondary and very subtle differences between spontaneous and induced crossing-over show u p ; in this system these differences are quite impossible to trace directly to any primary effects of ionizing radiation [e.g.) differential effects of “Minutes” (Ferrus, 1975) ; or differences in relative distances in mitotic recombination maps (Garcia-Bellido, 1972) 1. Indirect deductions from relative frequencies of stable products, however, are possible. This is demonstrated by Vig (1973a,b) working with chlorophyll mutants in soybean, which so far is the best-analyzed system in higher plants. Several types of primary effects are deduced froin results in hornozygous controls compared to heterozygotes, which give spontaneous twin spots after treatment with various inhibitors of DNA

82

ETTA KAFER

or protein synthesis. Taking into account the known cytological effects of these chemicals on plant chromosomes, the following effects were distinguished: (1) induced mitotic crossing-over, indicated by proportional increases in twin and single spots (e.g., mitomycin C) ; (2) induced nondisjunction, recognized by lesser increases of twin spots than single spots (e.g., by sodium azide) ; and (3) segmental aneuploidy or terminal deletions by increases of certain single spots only [e.g., by fluorodeoxyuridine (FUdR) 1. a. Selective Methods. In fungi, induced segregation is being used in commercially important asexual species (e.g., in Penicillium, Ball 1973). I n such situations it is more important to obtain segregants with desired properties than to understand their mode of induction. Fungal systems, however, have the advantage that induced segregants can be investigated further, since subculturing [recently also achieved in higher plants (Dulieu, 1975) ] and analysis of their genotypes is possible by haploidization. Also, segregants can be enriched by selective techniques, which, on the other hand, have the disadvantage of restricting recovery to certain stable types. Much interesting information is lost in this way, not only about reciprocal types but also about unstable intermediate products, which could be recovered with appropriate methods. But when very rare events are being investigated, as in the case of recombination between alleles, selective methods may permit isolation of informative types not obtainable in any other way [ e.g., the long-stretch conversions obtained by Bandiera et al. (1973) 1. Also for fast tests (e.g., of possible environmental mutagens) , labor-saving selective techniques are very valuable [e.g., the efficient one of Bignami et al. (1974) 1. Or visual selection for color sectors can be used (Kappas et al., 1974) in a system comparable to that of Vig in soybean, provided well-marked diploids are used [like those in the top part of Table 9 (KBfer et al., 1976) 1. b. Nonselective Detailed Analysis. With nonselective techniques, analysis of the actual effects of inducing agents is possible in Aspergillus nidulans, since these permit to isolate unstable precursor products of induced segregation. In addition, their genotypes and their likely mode of origin can be deduced from the types of secondary segregants that any one primary type is able to produce. Using this information, and comparing the stable segregants obtained from different selective situations, it is then possible to deduce the interplay between induced primary effects and spontaneous secondary ones, as modified by competition between differentially viable types (induced poorly viable and spontaneously recovered segregants) . Such analysis is very time consuming and, therefore, can be carried out only on a small scale. To avoid competition between types with different growth rates, single

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

83

nuclei should ideally be treated and grown under optimal conditions. I n

A . nidulans conidia are uninucleate and suitable on that account, but

many agents act only on dividing nuclei. In most cases, therefore, quiescent and germinating conidia should be treated. The latter are only partly synchronized even in controls, and understandably less so when treatment delays germination (Scott and Alderson, 1974). The approximate time of the ,first mitosis can be recognized by the first signs of bulging in conidia which start producing germ tubes. For short treatments, effective chemicals can be added briefly before that time, during presumptive periods of DNA replication; less reactive ones may have to be present during the whole “preincubation” period, and either type can be removed by diluting and plating onto solid media at a time when many nuclei are in the first or second division. I n low-density platings normal diploid colonies can be distinguished from modified types, which represent aneuploid segregants or induced mutations, and the level of killing can be determined. If well-marked diploids are used, the following frequently encountered types can be distinguished: 1. Colonies of mutant color or with large mutant segments (often “twin” halves or quarters) that result from induced mitotic crossing-over. Testing of a few well-spaced spots around the periphery of normal colonies permits detection of segregation for biochemical markers and may reveal events at the half-chromatid level (Wood and Kafer, 1969). Such twin segments are induced, e.g., by UV, FUdR, or mitomycin C. But, as expected from work with other organisms (e.g., Ustilago; Esposito and Holliday, 1964), great differences are observed in relative rates compared to killing, or to induction of other segregant types (Shanfield and Kafer, 1971). 2. Visually identifiable trisomics as a result of mitotic nondisjunction. These show mainly a chromosomal type of segregation of markers, and give 1 : 2 ratios for recessive :dominant alleles, if the recessive one is present on two homologs [no specifically nondisjunction inducing agent has been identified; nitrosoguanidine (NG) produced a fair number of trisomics ; however, Na-azide successfully used by Vig (1973a) has not yet been checked]. 3. Abnormally growing types which, from single nuclei, produce a variety of haploid and multiply nondisjunctional diploid patches or sectors (but few and relatively small areas of hyperploid conidia). These seem to result from malfunction of spindles and show a general misdistribution of chromosomes. Such “breakdown” types are exceedingly rare among spontaneous segregants, but very frequent after treatment with agents that induce haploids [ e.g., p-fluorophenylalanine (pfp) (Morpurgo, 1963; Shanfield and Kafer, 1971)l. Evidence for effects on spindles has

84

ETTA KAFER

been obtained by Crackower (1972) using cytological methods for the fungicide griseofulvin, which also induces haploid segregants (Kappas and Georgopoulos, 1974). 4. Other abnormally growing types that frequently produce segregant sectors and often contain semidoininant lethals, e.g., terminal deletions. These are the most difficult to recognize since many resemble aneuploids, and produce stable segregants of the usual three euploid types [which often is interpreted as induced segregation (e.g., Kafer, 1960) 1. Only detailed analysis of a sufficient number of cases shows that they produce a wide variety of phenotypes, each one resulting from specific reduction in growth rate and conidiation (specific for the monosomic segment) and each with a characteristic pattern of subsequent spontaneous segregation [that is, ratios of crossovers and nondisjunctionals of a few types, and of haploids, are found to differ in each case (Kafer, 1963, 1969) 1. 5. I n normally growing survivors completely recessive lethals and chromosomal translocations are further detectable by haploidization, provided markers are present in all linkage groups (Kafer and Chen, 1964; Clutterbuck, 1974). That different techniques can produce different information in the same organism and different results in different species, is best illustrated by the effects of pfp in several Aspergillus species and Ustilago. While in A. nidulans pfp-treated conidia on CM produce abnormal colonies with a wealth of haploid patches in which aneuploids are relatively inconspicuous, diploid colonies grown on Chi containing pfp often form large bulges that are aneuploid and secondarily show conidiating haploid patches or sectors. A few of the most frequent and viable aneuploids, like 2n I1 or 2n V are even regularly obtained among purification streaks, as are also disomics for the same groups (Kafer and Upshall, 1973). Such specific and quite stable dieomics have also been observed in Ustilago violacea by Day and Jones (1971). They find that in this spccies resistant mutants are being selected for a t a fairly high rate, possibly in line with the observation of Talmud and Lewis (1974) that in some eukaryotes pfp is highly mutagenic. However, in A. nidulans, haploids induced by pfp do not show increased resistance (McCully and Forbes, 1965). If also in other Aspergillus species aneuploids are a relatively long-lasting stage in the production of haploids when segregants are selected from CM containing pfp, this could explain partly the observed, relatively high, rates of crossing-over in A. niger (Lhoas, 1967) or in A. flavus (Papa, 1973) : recently it has been found that disomics of A . nidulans show much higher rates than diploids for mitotic crossingover between markers of opposite arms (Section 111, F ; Upshall et al., 1977).

+

+

RECOMBINATION

AND TRANSLOCATIONS IN

Aspergillus

85

E. hlITOTIC RECOMBINATION I N TRANSLOCATION HETEROZYGOTES Diploids heterozygous for reciprocal translocations are expected to produce unbalanced products when crossovers are selected in one of the structurally heterozygous chromosome arms (see Fig. 9 ) . Such a crossover will be trisomic for one of the two translocated segments and monosomic for the other. Provided this unbalanced segregant is able to grow and produce further mitotic segregation, balanced diploid types, which form better growing sectors, will be produced. These result from an occasional event of mitotic crossing-over (or nondisjunction) in the other of the two involved chromosome arms. Theoretically such selected 2-step crossovers should therefore give information on the positions of the translocation breaks (Kafer, 1962). However, it has been found that results are usually more complex than expected and rarely useful for the mapping of T-breaks for the following reasons: Monosomy reduces viability drastically; standard 2n - 1 types, for example, are known to exist only as transient nuclear stages in heterokaryotic condition in the mycelium, but never to form viable conidia. The selected unbalanced crossovers, therefore, show reduced growth rates to various degrees, and recovery of these primary types becomes difficult or impossible except when the monosomic segments are small. In addition, the phenotypes of any viable crossovers are unknown, and their genotypes are usually indistinguishable from those of their second-order nondisjunctional segregants, so that the latter may be mistaken for primary types (Kafer, 1976). Therefore, it usually is impossible to identify the various segregant types before a translocation is well mapped, so that suitable markers can be placed onto the various chromosome segments. 1. Types of Recovered Crossovers and Steps of Segregation

a. Recovery and Viability of Primary Crossover Types. Since mitotic crossovers from translocation heterozygotes are monosomic for one translocated segment and trisomic for the other, it depends very much on the size and genetic content of these segments, which primary crossovers are detectable and/or grow enough to produce further stable segregants. Reciprocal crossing-over in either of the two heteromorphic chromosome arms is expected to produce a twin pair of unbalanced crossovers. Such twins are reciprocal for their imbalance, each one being trisomic for that translocation segment for which the other is monosomic. It was found, however, that, in both of the two cases chosen for detailed analysis, only some of the expected types could be selected. Half of them (one type of imbalance) was probably far too unbalanced to produce conidia on the selective media. This, most likely, is due to the fact that in both cases

86

ETTA KAFER

, * n v I

+

fpaA anA

A

VII

+

\ : - -

%A

+ +

,

Balanced TO VIO f

+

bA .

A

+

’

YA

+

phene

Selected croswer

+ -momK)somic W l t r i s o m r : segment malA

tho.

pink fluffy-aconial unbalanced type

+ SU*

biA

fpaA anA

7

slightly fluffy green. lrisomlc (tho*)

su’

malA

cho*

fpaA anA YA

+

darker green diploid

FIG.9. Genotypes of segregants resulting from three steps of segregation in diploids heterozygous for Tl(Z;VZZ) with jpaA in coupling (and suAadE = su in repulsion), selected on CM pfp: step 1. Mitotic crossing over in I L producing “fluffy’’ unbalanced types, fpaA/jpaA. Steps 2 and 3. Spontaneous steps of nondisjunction in group VII, resulting in well-growing trisomic 2n VII sectors that produce stable diploid segments, segregant for markers in VII R.

+

+

RECOMBINATION AND TRANSLOCATIONS

IN

Aspergillus

87

one break is fairly distal, and the second one is quite proximal in a long chromosome arm. Therefore, only crossovers monosomic for the smaller distal segment and trisomic for the very long one, have been recovered, but never the reciprocal types monosomic for the latter segments. Two translocations were chosen for analysis, both with one break in I L in which three convenient selective markers are located (suAadE, f p a A , and fpaB, see Fig. 15). I n the case of T2(Z;VIZI), the distal two of these markers are translocated (Ma and Kafer, 1974) so that selection becomes possible in both involved arms and all the viable crossovers could be selected (two from each diploid). But such primary crossovers, selected from T2(I;VIZZ)/+ diploids, were difficult to recover and to identify, since they grew very poorly and barely produced conidia; in addition, they always were outgrown by their spontaneous second- and third-order diploid segregants (Kafer, 1976). I n the case of Tl(1;VII)on the other hand, the break in I L is distal to all three selective markers (Kafer, 1975), and only one expected type of primary crossovers could be isolated: Selection was possible only in I L, and the selective marker had to be in coupling with the translocation (shown for fpaA in Fig. 9 ) . I n this case, the monosomic segment of I L is sufficiently smaller so that the primary crossovers grow quite well and show up as distinct sectors on selective media. Their conidiation is, however, reduced and they look “pink” and “fluffy,” so that any second-order diploid sectors show up clearly. On the other hand, when selective markers are used in repulsion to T1 (I;VII),prolonged incubation produces patches or small sectors of two unstable types that show unexpected genotypes and segregation patterns. Evidence has now been obtained (presented in the next section) that these unexpected types originate as translocation monosomics, which apparently are viable in this case, because the VII-I T-chromosome is so very small. b. Steps of Segregation in Primary Crossovers. Details of the sequence of steps of segregation, following selection of primary crossovers from T/+ diploids, are shown in Fig. 9 for Tl(I;VII).I n the first selected step, segregation usually results from mitotic crossing-over, nondisjunction being relatively rare (see Table 16). These primary crossovers are selected because they lose the dominant wild type allele, and, in the case of T1 (I;VII), they become homozygous for the selective mutant. They are unbalanced, because they are trisomic for the long segment of VII R attached distal on I L, and monosomic for the tip of I L. They grow quite well (as mentioned above), and their “pink-fluffy” appearance is very similar to that of the standard disomics, n 1 for group VII. Like the n VII types, they conidiate almost normally on thin medium especially if grown a t room temperature rather than a t 37OC. When

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transferred or replated, such unbalanced crossovers regularly produce green sectors (looking like the colonies in Fig. l l e ) . Most of these turn out to be unstable and are slightly abnormal 2 n + V I I types, arising presumably by nondisjunction in VII (step 2, Fig. 9 ) . Occasionally diploid dark green sectors are produced by compensating crossing-over in VII R (not shown in Fig. 9 ) . The 2n VII intermediates are mainly identified by their phenotypes [in comparison to standard 2n VII (Kafer and Upshall, 1973)l ; in addition, marker segregation in their stable diploid dark green sectors confirms the postulated genotypes (step 3, Fig. 9 ) . Segregants of this type have been analyzed in large numbers from diploids heterozygous for various markers and for T1 (1;T711)in coupling with the selective marker suAadE [a recessive suppressor used for selection from diploids homozygous for adE, on media lacking adenine; details are shown in Kafer (1976) 1. Recently, diploids with suAadE in repulsion, but fpaA in coupling to T1 ( I ; V I I ) , have also been successfully used to obtain unbalanced fpaA/fpaA crossovers. Such a diploid (as shown in Fig. 9, but heterozygous for several additional markers) was grown on media containing pfp, and conidia were “needle-plated” from 50 apparent or real sectors. Among these, 34 fpA-resistant, near-diploid segregants were obtained. They all showed one of the two expected phenotypes: (i) 20 of them were phenotypically indistinguishable from the pink, poorly conidiating but well growing, primary crossovers obtained previously when suAadE in coupling with T l ( 1 ; V I I ) was used for selection. These segregants produced the familiar two- or three-step patterns, with light green 2n VII intermediates and dark green diploid sectors; (ii) the other 14 represented such light-green secondary segregants, namely 2n VII trisomics, with the occasional darker diploid sectors. As expected, segregation for markers on I L was found in the first step, and primary crossovers were either anA/anA or ansi/+ depending on the position of the exchange (and nondisjunction was rare; see Fig. 9 and Table 16). Subsequently markers of VII R segregated in the last step, when, e.g., cho+ on the trisomic segment in VII was lost, so that thc two choA alleles on the segments attached to I L become expressed. In conclusion, it has been possible to select primary unbalanced crossovers from all diploids heterozygous for T l ( 1 ; V I I ) and Td(l;VIII),but in all cases only half of the possible types were viable enough to be recoverable. This is not unexpected, since in both translocations one break is located quite distal, the other close, to a centromere, creating translocation segments of very different lengths. I n general, it is expected that translocations with two distal breaks will produce viable crossovers of all expected types, while those with two proximal breaks may well pro-

+

+

+

+

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duce none. The former is probably the case for T1 (VI;VII), since four reciprocally segregating unbalanced types were recovered from a translocation heterozygote, among large samples of nonselected conidia (Kafer, 1961). I n addition, as expected, these four likely crossover types formed pairs of identical phenotypes (however, with markers available a t the time genetic evidence was very incomplete), In all cases, viable unbalanced crossovers show distinct phenotypes and reduced growth rate. Therefore, even very rare types of compensating crossing-over between the break and the centromere show up as large diploid sectors. I n addition, nondisjunction is unusually frequent in the primary unbalanced types, especially when breaks are proximal; and trisomics are regularly found. These grow sufficiently better to produce conspicuous unstable sectors that form stablediploid sectors by chromosome loss in a third step. Formation of trisomics can only be inferred from the changes in growth rate and phenotype, since nondisjunction rarely produces a genetically detectable change. However, it is confirmed by the exclusively nondisjunctional segregation in the next step. For each translocation the situation is expected to be different. The relative growth rates and conidiation of the four chromosome segments produced by the two breaks, when mono- or trisomic, determine the chances for recovery and recognition of the various types. In Aspergillus, inviability seems to be the real problem, while the various phenotypes are unusually well distinguishable, so that cytological identification can be dispensed with. 2. Translocation Monosornics

From both, diploids heterozygous for T l ( 1 ; V I I ) and for T1 (VI;VIZ), unexpected unbalanced segregants have been obtained which mainly produce stable haploid sectors. These are now believed to be translocation monosomics. Usually these types are rare, but in one case, when the selective marker suAadE is used in repulsion to T1 (I;V II),they can be selected-even if only indirectly-on media lacking adenine : they grow slightly better because they produce stable suAadE segregants and are detected because no selected suAadE crossovers are viable and competing in this case. For detailed analysis, therefore, a diploid of the type shown a t the top of Figs. 9 and 10 was used (but with many additional markers). Such diploids previously have been found to produce unexpected unstable segregants of two types, both of them with stable suAadE sectors: (i) very slow growing segregants with a new phenotype which produce mainly haploid sectors ; and (ii) segregants of the unbalanced “pink-fluffy” type which are still heterozygous for suAadE, but form diploid suAadE sectors either by crossing over in I L or nondisjunction of I (Kafer, 1976). During the most recent study of these types, it has

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finally been possible to demonstrate conclusively that the first of these types originates by loss of the small translocation chromosome, and the second is usually produced by nondisjunction of chromosome VII in the fikt one (steps 1 and 2, Fig. 10). While the first type produces mainly conspicuous stable haploid sectors, the second forms mainly diploid sectors, either by one step of mitotic crossing-over in I L, or by two Balanced

VII

-,

1 ) Loss of ViI-l T- chromosome

2n:

TI ( 1 . v /suAadE ~

I

\

indirect 8election

on media

lacking adenine

\

J. Ts a

Crosslng Over in vIIR

2 ) Nondisjunction of normal VII

I

VII

Irisam I C A

J*

manosom~e segments el

"pink-fluffy"

(relatively

SU

su

Phen

unbalanced types

3 ) Nondisjunction

frequent)

green

2n

yellow 2n

FIG.10. Genotypes of segregants produced in four steps of segregation from repulsion diploids TI(I;VZI)/suAadE on media lacking adenine: (1) Loss of the very small VII-I translocation chromosome producing slow-growing translocation monosomics, mainly with normal haploid sectors (not shown) ; (2) nondisjunction of the normal VII in T-monosomics resulting in much faster growing, but fluffy, unbalanced types (crossing-over proximal in VII R occasionally produces this type in one step) ; (3) crossing-over in I L of the unbalanced type produces normal suA/suA diploids. while nondisjunction of I results in a light green translocation trisomic (yA/yA/+) ; (4) loss of the long I-VII T-chromosome from the latter results in balanced yellow 272 sectors.

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steps of nondisjunction of I (steps 3 and 4 of Fig. 10). Identification of these four steps is possible by changes in phenotypes (Fig. l l ) ,and at the same time genetic markers segregate in some of them. The finding that one of the regularly obtained unexpected types can produce the other (step 2) explains the recurring problems of classification of the two types, since every so often, some cases seemed to change overnight from one to the other, especially on further incubation. a. Phenotypic Evidence. The relationship between the unexpected unstable types first became evident, when some of the postulated translocation monosomics were plated a t very low densities on unusually thick medium to improve photographic recording. Under these conditions “abnormal” centers of all types conidiate less and show up much better, and unstable sectors have a chance to produce second- and third-order sectors. Therefore, it suddenly was easy to see that T-monosomics not only produce stable haploid (and rare diploid) sectors, but quite frequently show sectors or large areas of the unstable “pink-fluffy” type (Figs. llb-d) . It also became evident, that any diploid sectors always arise from these unstable intermediates, in one or two further steps of segregation (Figs. l l c and l l d ) . On thin media, such “pink-fluffy” areas, which may surround the centers, often become indistinguishable from it (except on transillumination) so that only euploid segregants show up as sectors (Fig. l l a ) ; in such cases monosomics look quite different from those shown in Fig. l l b but are almost indistinguishable from stable disomics (Fig. 2a). To confirm that the unstable intermediates can always be found proximal to any diploid sectors, conidia from such areas were replated: the expected “pink-fluffy” unstable type was obtained in all cases (Fig. l l e ) . These colonies produced diploid sectors much more readily than the original T-monosomic (Fig. l l d ) , and such 2n sectors always were either green, or yellow originating from a light-green unstable intermediate. The latter must be a translocation trisomic type which produces only yellow 2n sectors, nondisjunctional for group I (replated in Fig. l l f ) . b. Genetic Evidence. The original Tl ( I ; V I I )/suAadE diploid was plated at low densities on media lacking adenine and incubated for over a week. When sectorlike areas of improved growth were formed by most colonies, abnormal conidia from their proximal parts were plated on complete media (as described recently in detail; Kafer, 1976). The result was that in the majority of cases (18/23 platings) slow-growing types with haploid sectors were recovered, in a few cases simultaneously with faster growing “pink-fluffy” types. When tested for genetic segregation all these segregants were found to be only mutant for phenB, as expected from loss of phenB+ due to loss of the small T-chromosome (step 1, Fig.

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10). The other platings (5/23) showed only colonies of the pink-fluffy type; of these, four had segregated for phenB, and one had not. The latter presumably remained heterozygous for phenB as a consequence of mitotic crossing-over between phenB and the break proximal in VII R (alternative to steps 1 and 2, Fig. 10; tests for the leaky mutant phenB are quite difficult but are clear-cut in the resulting stable sectors; with nicB used in coupling to nzaZA, these are liomozygous for nicB and therefore completely negative on media lacking nicotinic acid if they are phenB+, but partially growing if they are phenB) . The postulated translocation monosomics always produced stable haploid sectors, under crowded conditions exclusively so (450 colonies showed 390 haploid sectors, 140 of which were tested). Such haploids were mutant for all markers of I and V I I in coupling with suAadE and segregated randomly for markers on all other chromosomes, as expected from translocation monosomics. In addition, in low-density platings, sectors of the unstable pink-fluffy type were frequent (about half as many as haploid ones were identified). No marker segregation could be demonstrated for this step (step 2, Fig, 10). This agrees with the hypothesis that the pink-fluffy types result from nondisjunction of the normal VII-chromosome, hemizygosis for phenB not being distinguishable from homozygosis. From the pink-fluffy unstable sectors (total about 200 from the 450 colonies) over 100 green diploid sectors were obtained in a final step of segregation. When tested, these all showed segregation for suAadE (loss of suA+) and other markers in coupling on I L, dependent on the position of the exchange (results are combined with earlier ones from the same diploid in Table 16) ; in addition all recessive markers distal to the break on VII R in coupling to suA (e.g., maZA) simultaneously showed up, owing to the loss of the trisomic segment of VII R which contained their wildtype alleles (Fig. 10, alternative to steps 3 and 4 ) . These segregants therefore arose by mitotic crossing-over in I L. Alternatively, yellow diploid sectors usually grew from segments Gf lightish-green areas. ForFIG. 11. Phenotypes and patterns of sectoring in segregants from diploids

T1(I;VZl)/suAndE selected on media lacking adenine and replated on complete

medium (genotypes in Fig. 10). (a-c) Replated T-monosomics in different growth conditions: (a) on thin medium, 2n - 1 center surrounded by unbalanced type, both conidiating, with white n sectors; (b) on thick medium, incubated 6 days, with haploid and unbalanced fluffy sectors; (c) incubated 9 days, large “fluffy” areas, one with third-order green crossover sector. (d) Unbalanced, “fluffy” types, one with 2n - 1 center, the other with 2n green crossover sectors and two unstable Ttrisomic segments with yellow 2n sectors. (e) Replated “pink-fluffy” unbalanced type with green trisomic and yellow 2n sectors. ( f ) Replated translocation trisomic with yellow 2n sectors (step 4, Fig. 10).

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mation of the latter showed no marker segregation, but a pronounced change in phenotype, as expected from nondisjunction of the normal I-chromosome (step 3, Fig. 10). The light-green types are therefore translocation trisomics, and as expected, on replating they produced only yellow, mainly diploid suA yA, sectors (Fig. llf). Like the green crossovers, these stable yellow sectors show simultaneous segregation for markers on I (in this case all markers proximal to the break on I L as well as those on I R) and on VII R (distal to the break), as expected from loss of the long I-VII translocation chromosome (39 tested; step 4, Fig. 10). In conclusion, all evidence is consistent and indicates that from diploids T1 (Z;VZZ)/suAadE, translocation monosomics are obtained on media lacking adenine, even though these segregants are not homo- or hemizygous for suAadE. It appears likely that their growth is favored by the following three factors: (i) their stable sectors all are hemi- or homozygous for suAadE and nuclei of this type in the mycelium would confer a selective advantage and be responsible for the improved growth; (ii) loss of the VII-I translocation chromosome would be relatively frequent and the resulting monosomic relatively viable, because this chromosome is extremely small; (iii) inviability of the selected crossover types from this diploid prevents competition. No such translocation monosomics have ever been obtained from diploids heterozygous for Td(Z;VIZZ), since in this case suAadE is translocated and point iii would not apply. On the other hand, the diploid heterozygous for TI (VZ;VZZ), which was analyzed in detail previously (Kafer, 1961), indeed produced almost identical looking types, among random samples of conidia. These presumed translocation monosomics always were heterozygous for markers on six normal chromosomes and for choA of VII R which is translocated to VI R, but not for the mutants on the other VIV I I translocation chromosome. This indicates that in this case, it is also the rearranged chromosome with centromere area of VII, which is lacking in the monosomic. This explains the similarity of the phenotypes of the monosomics; but since in this translocation the break in VII R is further distal (see Fig. 15), it is somewhat surprising that the viability also appears to be quite similar. 3. Frequencies of Different Types of Mitotic Segregation in Translocation Heterox ygotes

So far, no accurate data bearing on effects of translocations on mitotic recombination are available. All measurements of absolute frequencies of the primary products of crossing-over vs. nondisjunction are unreliable, and in addition they are valid only for the specific translocations used,

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mainly for the following two reasons: (a) viabilities of primary segregants resulting from these two processes differ tremendously, crossovers being much more unbalanced than the primary trisomics from nondisjunction; (b) these absolute viabilities as well as their relative abilities to compete with normal colonies depend very much on the position of the breaks, i t . , the lengths and types of mono- or trisomic segments. Attempted measurements of such frequencies, by analysis of the frequencies of various disomics from translocation heterozygotes (Pollard e t al., 1968), or the frequencies of primary unbalanced segregants in random samples of plated conidia from T6(I;VZZZ)/+ diploids (unpublished data), are therefore, of very limited use. The only somewhat indicative data are relative frequencies of crossovers and nondisjunctional types, especially if translocation heterozygotes are used, in which both types of segregants show relatively good viabilities in comparison with controls. The disadvantage is, that such relatively viable segregants are only obtained when brcaks arc distal, and therefore when effects are minimal. This was the case for diploids heterozygous for Ti!(Z;VIIZ) (Kafer, 1976), and for diploids with T1 (Z;VlZ) in coupling to the selective marker (Table 16). The obtained results suggest that, as expected, nondisjunction frequencies as well as the distribution of crossing over, are influenced by the translocation, even though for I L the effects are quite small. That is, in heterozygous translocation diploids where pairing and crossing-over in the regions of the breaks is presumably reduced, crossingover tends to become relatively more frequent in the intervals closer to the centromere, and mitotic nondisjunction appears to be slightly increased (but samples are too small to demonstrate significant differences). The situation is much more abnormal, however, in the unbalanced firstorder segregants, where nondisjunction may be relatively frequent. However, in such cases the frequencies of diploid crossover sectors are compared with those of diploid nondisjunctional ones resulting from two steps of nondisjunction (see Table 16, SUA selection). The latter vary somewhat with plating densities and may represent a maximum value in cases where one event of primary nondisjunction (one segment of the T-trisomic type) produces more than one distinct yellow secondary nondisjunctional sector. The alternative is to compare the frequencies of dark green crossover sectors with those of translocation trisomic type (in replated unstable types by visual classification). I n this way an attempt was made to compare nondisjunction in the two different heteromorphic chromosomes of T l (Z;VIZ) a t two different temperatures ( 2 5 O and 37OC). While it was found that crossover sectors showed no significant differences at the two temperatures, nondisjunctional sectors were in-

TABLE 16 Nondisjunction and Crossing-over in TI (Z;VZZ) Diploids and Controls

Type of diploid

NondisSelective No. of junction tested in group I marker on I crossovers (%) su

Relative frequencies of crossing-over in various intervals of I L

33 % 34 % -

76

6% 5-9 % 8%

TIT

suA

580

8-12 %

21 %

T/

suA fPaA

57 98

10-15% 11 %

22 % -

15-25 %

22 % 23 %

Controls

+

TI +

2nd-step segregants

suA fpaA

{5:;

riboA

-57

.

-

anA

14 %

*

14 %

-

%

28 %

I

-50%

%

-

centromere

* 9%

@%

* 77 %

5%

proA 53 % *

64%

I

-61

-

c

15 % P

*

95 % +

16 %

*

M

3*

w $

B

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creased 3-5 times in numbers and 2-3 times in relative frequencies. This applied equally for both chromosomes I and VII. However, nondisjunction was relatively much higher in group VII, where the break is proximal ( 6 0 4 5 % ; total of 218 colonies inspected) than in group I where the break is distal ( 1 6 3 5 % ; total of 419 colonies). This agrees with results in higher plants where especially very small translocation chromosomes show relatively high nondisjunction frequencies. These results show that, while the effect of the translocation in the original balanced heterozygote may be quite small, exceedingly high frequencies of nondisjunctional segregants may be isolated, since primary segregants usually show much higher nondisjunction, especially if breaks are proximal. Unfortunately no suitable genetic markers were available in the above cases on the small translocation chromosomes VII-I of T1 (Z;VZZ), or VZZZ-I of T2(Z;VZZZ), to check on their nondisjunction frequencies in the original heterozygous diploids, and, translocation trisomics with these chromosomes extra are not likely to be phenotypically recognizable. On the other hand, the translocation monosomics found regularly as segregants from some of these diploids indicate that, also in Aspergillus, loss of the small translocation chromosome is relatively frequent.

F. MITOTICCROSSING-OVER IN DISOMICS FROM TRANSLOCATION CROSSES,ESPECIALLY IN “STABLE”DISOMICS 1. Normally Unstable Disomics

In Aspergillus nidulans mitotic crossing-over in disomics seems to be much more frequent than that observed in diploids. The highest values obtained in disomics from translocation crosses were those observed as sectors of unusual color in disomics for group I which were heterozygous for y A and T1 (Z;VZZ). These showed about 8% recombination between the translocation break near fpaB in I L and yA in I R (Kafer, 1975; see also Fig. 15). Even higher values were observed for standard n 1 for the same group (between suAadE and yA) and somewhat lower values between markers on both arms of other groups (Upshall et al., 1977). This would suggest an average level of 3-570 mitotic crossing-over per arm in disomics, compared to 0.1-0.3% in diploids. These differences are in agreement with the inverse correlation between stability and frequency of mitotic recombination (in diploids and disomics of Neurosporu, Aspergillus, and yeast) as postulated by Smith (1974), even though no logical reason for such a relationship has been proposed. Such differences, therefore, have to be taken into account when comparisons are made between different species, i.e., crossover frequencies in diploids should be

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compared separately from those in aneuploids or derived haploids [which probably would reduce the differences between A . nidulans and A . niger (Lhoas, 1967) ; as mentioned above]. Selected Crossovers, Distribution, and Coincidence of Recombination. Unstable disomics from translocation crosses usually produce only one type of haploid sector, even if many markers are heterozygous, because such disomics are structurally heteromorphic (see Section 11, C on translocation disomics). Heterozygosis, therefore, only becomes evident when the common balanced haploids contain the recessive allele, so that the growth responses of the disomic center differs from that of the sector. From such heterozygous n 1 types, crossovers can be selected by transferring them to a medium that selects against one recessive allele present in the haploid noncrossover sectors. The frequencies of such selected crossover sectors depend on the distance of the “selective” marker to the translocation break. They are quite rare, except when selective marker and break map on opposite arms. That is, as in diploids and standard disomics, crossing over occurs mainly close to the centromeres. Indeed, among the selected rare types, double crossovers with an additional exchange in the centromere area have been observed fairly frequently (the relatively small samples analyzed showed no significant difference from frequencies expected on the basis of random coincidence).

+

2. “Stable” Disomics

Three types of unusually stable disomics are expected from crosses between translocations that have one break each in the same chromosome (Section 11, C ) . The recovery of such types depends on their viabilities and phenotypic distinctness [and only two such types were obtained from crosses of Tl(I;VIZ) to other translocations with a break in VII R, Table 711. Very rarely the extra chromosome material of such “stable” disomics is lost and normal haploid sectors are produced (Fig. 2 ) . This is expected to be possible after the occurrence of a reciprocal mitotic crossing-over in the segment between the two breaks (as diagrammed in Figs. 3 and 12). Such a mitotic exchange in a “stable” n 1 which is disomic, e.g., for the two segments of a standard chromosome, produces as one of the two complementary products a new type of “tripartite” chromosome, and as the other a normal (or standard) chromosome; the latter can then be lost to form a balanced haploid containing both translocations (Fig. 12 and Table 17). These “stable disomics” are almost indistinguishable phenotypically from unstable duplication types in which the duplicated material involves a large segment of the same linkage group (Bainbridge and Roper, 1966). Such duplications also form rare haploid sectors, but apparently lose the extra chromosome segment by some un-

+

/-\ 1

RECOMBINATION AND TRANSLOCATIONS IN

99

Aspergillus

malA*

OitA’

I-VII

A,-~A

---+*--

- +.

wl-lll

Ill-VII

. eF’.

OioA

.

pIF

lY.0

,, ,,’

“stable“n+VII

Pairing and mitotic crossing over

I-VII

YA

+

normally unstable n + V I I lost

. 7 I-VII-Ill

._ly.0

\2y+

regularly

VII-l

III-vII

double T/ haploid

FIG.12. Stable n + V I I from cross of Tl(I;VII)x Tf(ZZI;VII)(2146) resulting irom meiotic nondisjunction, and mitotic crossing-over in the disomic segment between the two breaks in VII R, leading to formation of normally unstable n VII able t o form haploid “crossover” sectors.

+

orthodox mechanism [termed mitotic nonconformity in Aspergillus by Roper and Nga, (1969)l. This process appears to be either the consequence of nonrandom chromosome breaks in some cases [e.g., in Neurospora (Perkins, 1972, 1975) ] or of transposition of some segments causing loss of others (Azevedo and Roper, 1970). It was therefore of interest to analyze in detail the genetic changes associated with the loss of the disomic material in the “stable disomics” obtained here. For a detailed analysis the best-marked disomics with n VII phenotype (Fig. 2a) were chosen from a cross between 7’1 (1;VZI) and T l ( I I I ; V I I ) (cross 2146, Table 6 ) . It was found that six (of the 34, Table 7) cases with this phenotype were still heterozygous for all markers of VII R in the parental arrangement (since meiotic recombination was only partially reduced in the segment between the two breaks which must be over 100 cMo units long). These six disomics were used to inoculate the centers of a large number of CM plates, and all obvious haploid sectors were isolated (1-2 per plate). Tests of these haploids produced the results shown in Table 17. The segregation of markers in these haploids from stable disomics clearly supports the postulated mechanism of single mitotic crossing-over,

+

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TABLE 17 Types and Relative Frequencies of Mitotic Crossover Haploids from Stable n VII Disomics from Cross 2146 (Table 6): TI (ZL;VZZR) X TI (ZZZL;VZZR)

+

T-break niR mddk p w e Intervals 1 - 4 in T-overlap

MIA

+

palF T-break

Crossing-over in intervals: Genotypes of sectors:

+

Stable n VII isolate No. 273 222 244 211 252 256

1

2

3

Oli + pal

++ pal

+sF pal

3 0 10 6 6 4 29 23 %

16 0 6 3 2 0 27 21 %

5 5 7 2 3 -6 28 22 %

4 +sF+

Number of haploid sectors 30 7 37 18 14 21 127

Relative frequencies :

6 2 14 7 3 11 43 34 %

followed by loss of the normal VII crossover product. I n addition, the data show that mitotic exchanges occurred with similar frequencies in the four marked intervals between the breaks, which are all on the same arm and of roughly similar size (the slightly more frequent type is the most viable one, since it is the only one which is p a l F + ) . IV. Genetic Mapping and the Use of Translocations

A large variety of methods combining genetic, cytological, and biochemical techniques are used in various organisms for the mapping of genes to specific chromosome segments. Since the methods based on mitotic recombination have been new and especially useful in Aspergillus nidulans, emphasis will be mainly on these: in addition, techniques making use of translocations for genetic mapping, in conjunction with meiotic and mitotic recombination, are considered in detail. These latter techniques logically are the same as the ones employed for the mapping of translocations ; similarly, mapping of markers to chromosome arms leads to mapping of centromeres.

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A. GENETICMAPPINGOF MUTANTSIN Aspergillus nidulans A N D EFFECTS OF TRANSLOCATIONS The procedure for genetic mapping in A . nidulans takes into account that meiotic distances are relatively large, so that markers on the same chromosome or even on the Same arm frequently are unlinked in crosses (as discussed in Section 11, A ) . Mapping therefore generally proceeds in two or three stages: (1) Identification of the mitotic linkage group or syntenic group, i.e., mapping to one of the eight chromosomes; (2) meiotic mapping in crosses to a selection of widely spaced markers of that, chromosome; and (3) if this second step does not identify the precise location within the linkage group and exact location is of special interest, additional mapping to chromosome arms or segments by one or more further methods, which are less generally applicable: (a) mitotic crossingover in standard diploids heterozygous for suitable markers ; (b) mitotic recombination in homozygous translocations diploids ; or (c) mapping by means of disomics from heterozygous translocation crosses. 1. Mapping of Mutants to One of the Eight Chromosomes

(Which Detects Translocutions) The first step of mapping of any new marker consists of a check for mitotic linkage to markers on all eight chromosomes, in haploids which are selected from heterozygous test diploids. This mapping technique was a logical extension of the work of Pontecorvo et al. (1954), who demonstrated the usefulness of haploid segregants for identification of all markers which are located on the same chromosome. It became feasible after, and was developed simultaneously with, the mapping of the first pairs of markers on all eight chromosomes (Kafer, 1958). This method is relatively easy and much more efficient than meiotic mapping, so that in addition to the almost 200 markers actually placed on the current Aspergillus map, almost half as many are mapped just to one of the eight groups (Clutterbuck, 1974). Similar mapping of markers to whole chromosomes is also possible by means of disomics, especially in organisms where these are stable (such as yeast, Mortimer and Hawthorne, 1973). a. Mapping Procedure. The mitotic mapping technique involves the following three steps: (i) The mutant strain has to be combined into a heterokaryon with a suitably marked “mitotic mapping strain” (Barratt et al., 1965, 1975; or “master strain,” McCully and Forbes, 1965). From the heterokaryon, the test diploid can readily be isolated (Roper, 1952). Since simple standard strains are usually used to obtain new mutants (e.g. biA in various colors, to obtain nutritional mutants by the “bi-

102

ETTA KAFER

starvation” technique; Pontecorvo et al., 1953), we find it worthwhile to first cross all induced mutants to a “meiotic tester” strain. I n such crosses, we can not only introduce markers that facilitate heterokaryon formation, but, in addition, eliminate any of the frequently induced morphological mutants, and check for gross aberrations (which increase aneuploid frequencies, Upshall and Kafer, 1974). (ii) HapZoids are selected by one or two of the methods for which the test diploid is suitable: either by “double selection” using markers on two different chromosomes to obtain some of the rare spontaneous haploids resulting from mitotic nondisjunction (Kafer, 1958) ; or by induced haploidization on media containing chemicals that interfere with mitosis and favor the growth of haploid types (Lhoas, 1961; Hastie, 1970). (iii) The segregation of markers in the haploids is analyzed, and any cases of complete linkage rather than free recombination are identified. These procedures generally map new mutants to one of the eight mitotic linkage groups provided they are not cytoplasmic ones [like some of the oligomycinresistant mutants of Rowlands and Turner (1973)l. If any unusual linkages are found, selection of haploids by more than one method, or analysis of differently marked diploids distinguishes cases due to translocations from those caused by special conditions, like poor viabilities or interaction of mutants [e.g., suppression of f p a A or fpaB by Zys mutants, as found here, and similarly in Neurospora (Kinsey and Stadler, 1969)l. b. Identification of Mitotic Linkage Groups with Chromosomes. Many methods are known that permit assignment of linkage groups to chromosomes. For example, mono-, di-, or trisomics can be used, usually in somatic cells (since they are often sterile). This is possible only if the mitotic chromosomes are sufficiently distinct (such identification has recently been achieved with biochemical techniques in yeast, for chromosome and group I, which contain many rRNA genes; Finkelstein et al., 1972). Or meiotic pairing figures of zygotes heterozygous for translocations can permit identification, if two translocations are used that involve one common chromosome that is identifiable in meiotic prophase {as has become feasible in mice with the recently improved staining methods for mammalian chromosomes (Miller and Miller, 1972) ; or in cytologically more favorable material like the mosquito (Bhalla et al., 1974) 1. In Aspergillus nidulans cytological identification of chromosomes is obviously difficult, since problems were encountered even in counting the total number of meiotic chromosomes: only with the help and experience gained with the larger chromosomes of Neurospora (McClintock, 1945) was the haploid chromosome number recognized as eight (Elliott, 1960a) rather than four (Pontecorvo et al., 1953). Mitotic chromosomes are even harder to count, and in addition they are somatically paired, so that diploids can be recognized only by the increased amount of total

RECOMBINATION AND TRANSLOCATIONS IN

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DNA (Robinow and Caten, 1969). This rules out the use of aneuploids, which in Aspergillus are unstable and never go through meiosis. The meiotic method using translocations, which has turned out to be feasible, if difficult, in Neurospora (Barry and Perkins, 1969) would seem to be the most promising approach, especially if rings of translocations could be used that would tie up all but a few chromosomes. However, even though translocation pairing figures can in some cases be identified clearly, determination of the relative sizes of all eight chromosomes and identification of specific ones in translocation crosses, is probably not feasible with current cytological techniques (E. R. Boothroyd, personal communication). It seems, therefore, unprofitable to attempt numbering of chromosomes by size, and the unconventional practice of referring to chromosomes with the number of the corresponding linkage groups (which is done by some investigators using Aspergillus, and regularly for yeast) probably is the most sensible procedure under the circumstances. 2. Meiotic Mapping of Mutants

For linkage groups with many mapped markers and few stretches of 50% crossing-over, meiotic linkages can usually be found. This is especially true for relatively short ones, like IV or V, but also applies to the well-sequenced groups I, 11, and VIII (Clutterbuck, 1974; see also Fig. 15). On the other hand, meiotic linkages are often not found in the remaining groups, and in these cases the alternative techniques, described below for sequencing of markers and centromeres, are especially useful. Furthermore, meiotic mapping of new markers may be unsuccessful not only because of the meiotically long distances, but also because generally no positive, or cven slightly negative, interference is observed (Section 11, A ) . Three-point crosses of loosely linked markers often do not clearly establish their sequence, nor do crosses of two closely linked ones to more distant markers [e.g., the position of dilA closely linked to argB and proximal of methH (Fig. 14) is only tentatively established, even though 400 progeny were tested from a cross heterozygous for these mutants]. To map loosely linked markers, certain translocations can be very useful, because recombination is reduced in heterozygous crosses, and a t least markers linked to the breaks can often be sequenced more easily (Section 11, B ) ; in addition, the orientation of distal segments may become apparent. One such case is illustrated in Fig. 13, which shows the values for intra- and interchromosomal recombination in crosses heterozygous for T1 (ZV;VZZZ). These results clearly map sE between chaA and nirA and place nirA distal to all other markers, confirming mitotic crossing-over data of A. Niklewicz (Gravel et al., 1970; details shown in M a and Kafer, 1974). On the other hand, when translocations are present that are not recog-

104

ETTA KAFER

pabaB 29%,+

(680

12% 11063)

FIG.13. Meiotic recombination frequencies (‘36) between markers of groups IV and VIII in crosses heterozygous for !l’l(ZV;VZZZ) (values based on 1-5 samples of about 200 each; total tested number in parentheses).

nized or not sufficiently taken into account, sequencing based on results from heterozygous crosses may lead to erroneous maps. A case in point is the map of group 111, where reduction of recombination by unidirec+ VZZZ) by Clutterbuck, tional translocations (observed, e.g., for T,%?(ZII 1970) was more extreme than originally assumed (Bainbridge, 1970; Kafer, 1975). In this regard it is interesting to note that, even though it is stated that in the recent map “translocations have been taken into account where necessary” (Clutterbuck, 1974), all the quoted discrepancies are in areas where markers tightly linked to translocations are known to have been used for mapping [at least in some of the crosses: namely, suCpro and T1 (ZZZ + VZZZ) in 111; frAl and TI (ZV;VZZZ) in IV R ; sD50 and T,%?(Z;VIZI)proximal in VIII R, evident also in the data of Dorn (1972) ; and one additional case in VII R, namely markers around the break of T1 (ZZZ;VZZ) associated with ZysD20; see below, subsection 3 , ~ .

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

105

3. Mapping of Markers to Specific Chromosome Arms or Segments

Of the three methods that permit identification of the chromosome arm or segment in which a mutant is located, two make use of mitotic recombination, one ( a ) of mitotic crossing-over in standard diploids; the other (b) of mitotic haploidization and crossing-over in homozygous translocation diploids ; the third (c) uses disomics from heterozygous translocation crosses. This last type of analysis in addition identifies suitable strains containing markers in coupling with translocations, which then can be used for mitotic mapping in homozygous translocation diploids. Such diploids are useful also for centromere mapping when selective markers are translocated to new groups (as in diploids of Table 19). Of the three techniques, only the first has been in general use for some time. However, since it is efficient mainly in linkage groups in which selective markers are located distally on both arms, as in groups I and 11, it has only rarely been used for mapping in other groups (for example, for the mapping of centromeres in groups I11 and I V ) . The other two techniques are even more restricted and can be applied only when suitable, mapped translocations are available ; these, therefore, have only recently been used in special cases. a. Sequencing b y Mitotic Crossing-over in Standard Diploids. Mapping by mitotic crossing-over is most efficient, when recessive mutants are used in coupling (or dominant ones in repulsion) to the selective marker and to a marker on the opposite arm. A small sample of crossover segregants will then identify the relative position of any meiotically unlinked mutants, since results are basically qualitative: the new mutant is located either distal to the selective marker, if all crossovers are homozygous for i t ; or on the other arm, if the new mutant is homozygous only simultaneously with the marker on the opposite arm (i.e., in nondisjunctional segregants) ; or proximal, if it is homozygous in a fraction of crossover segregants. However, if centromeres have not yet been mapped with certainty, or when segregants are so rare that they approach mutation frequencies, results are much less easy to interpret (see below, Section IV, C on centromere mapping). b. Mapping to Chromosome Segments b y Mitotic Analysis of Homozygous Translocation Diploids. When mapped translocations are available with a break in the chromosome to which a marker has been assigned, these can be used for mapping to scgrnents in several ways; the simplest is mapping by mitotic analysis of homozygous translocation diploids (Ma and Kafer, 1974). This is relatively easy once strains with the new mutant and suitable markers in coupling with the translocation have been

106

ETTA KAFER

constructed, or when a translocation is tightly linked to a marker. From diploids heterozygous for markers and homozygous for the translocation, haploids can then be isolated that map any new mutant to one of the two segments defined by the translocation break. Selection of mitotic crossovers may, in addition, define the sequence of markers in the distal translocated segment or in the residual part of that arm, if a suitable selective system is available. Such mapping has recently been carried out in many cases, usually either preceding or confirming results obtained by other methods. For example, the sequence palF - lysD - malA, choA was firmly established in this way, when over 100 haploids from three diploids homozygous for Tl(III;VII)with the break a t lysD were analyzed: in all cases palF was completely linked to the proximal markers phenB and sF, but not to the translocated ones, malA, choA, or nicB. These results agree with those obtained from translocation disomics, not only from crosses with T1 (III;VII)but also with Tl (VI;VII).[In addition, in the latter palF is the only one of these markers that shows definite, if slight, meiotic linkage to sF or pantoB close to the break, and similar linkage was also found for O l d , on the other side of the break (Fig. 15) (Klfer, 1975).] I n Table 19 below, results from diploids homozygous for T 1 (VI;VII) confirm that, while palF in this translocation is on the same distal segment as maZA, choA and nicB, the mutant OliA maps proximal (since it segregates with phenB, the only nontranslocated marker in Tl(I;VII); for results based on mitotic crossovers from these diploids, see Section IV, C ) . c. Mapping by Means of Disomics from Heterozygous Translocation Crosses. This method also depends on the availability of a translocation that has one mapped break in the relevant linkage group and, in addition, produces a reasonable frequency of n 1, especially translocation disomics. From heterozygous translocation crosses, four types of disomics are expected and found for some translocations (see Section 11, C, 2, c) : two types of “typical looking” n+ 1 types, which contain one of the two involved standard chromosomes in addition to a haploid set including both translocation chromosomes ; and two types of translocation disomics, each disomic for one of the two rearranged chromosomes and containing in addition a balanced standard haploid set. Since haploid sectors from typical looking disomics usually contain the translocation, these are very useful for construction of homozygous diploids. However, because of the relatively high rate of mitotic crossing-over that may occur a t the fourstrand stage in disomics, cases sometimes are found that do not agree with this general rule, but seem to be homomorphic as well as homozygous [e.g., apparent double crossovers around the breaks are often of this type (Kafer, 1975) 1.

+

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

107

If one, or preferably two, recognizable types of translocation disomics are found, these can be checked for heterozygous markers (especially in cases with recessive alleles in haploid sectors). Since any unmapped mutant will be found to be heterozygous in only one of the two types, such results usually assign the marker to one of the two segments defined by the translocation break (exactly like those obtained with haploids from homozygous T-diploids) . This type of mapping corresponds to methods in various higher plants that show visually identifiable aneuploids, e.g., translocation trisomics in tomato (Khush and Rick, 1967) or secondary trisomics in Datura (Carlson, 1972). I n addition to this qualitative mapping, which depends on the recovery of translocation disomics, it is possible to check for meiotic linkages to the translocation breaks, using both types of disomics, namely, translocation disomics and/or typical looking disomics (as explained in detail in the next section). Also meiotic linkages of the new mutant to normally unlinked markers may show up in such crosses because of the resulting reduction of crossing-over (Section 11,B). Such mapping was, for example, first obtained for ad1 and s A on 111L, which both showed very close meiotic linkages to the break of Tl(II1;VII)in disomics (and to each other in haploids) ; since only adI was heterozygous simultaneously with the proximal marker A c t A in one type of disomic, and sA was heterozygous in the other, these results mapped sA close, but distal t o adI on I11 L (see Fig. 14; as confirmed subsequently by the haploids from the homozygous T-diploids mentioned above). Similarly, the first mapping of OliA to a proximal segment of VII R has been obtained with translocation disomics (40 isolated) from two crosses heterozygous for Tl (VI;VII). Of the 17 that. produced haploid OZi+sectors, five were heterozygous for OliA simultaneously with phenB, but not for pantoB or any of the other markers of VII R ; and all these five disomics showed the phenotypes typical for disomics heterozygous for the proximal part of VII R and the distal segment of VI R, thus mapping OZiA proximal to the break on VII [as shown also in T/T diploids, Table 19; OZiA has recently been renamed O K (A. J. Clutterbuck, personal communication) ].

B. FREQUENCIES, DETECTION, AND MAPPING OF TRANSLOCATIONS The various steps for mapping of translocation breaks are basically the same as those outlined in the preceding section for mapping of mutants: ( 1 ) Translocations are detected, and the linkage groups involved can be determined by isolation of haploids from heterozygous diploids, Table 19; OZiA has recently been renamed OliC (A. J. Clutterin the standard mapping methods, (Kafer, 1958; McCully and Forbes,

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ETTA KAFER

1965) , but not the previous modification used by Forbes (1959) 1. (2) I n favorable cases, meiotic mapping may be possible, especially when small linkage groups or well-mapped arms are involved. (3) Mitotic recombination in diploids and analysis of disomics from T-crosses can be used for further analysis, and combined results may permit to map the breaks.

1. Zdentification and Frequencies of Translocatwns a. Detection. The detection of translocations in well-marked diploids is easy (Kafer, 1962) except when unexpected interactions occur, either between certain types of mutants and selective media, or between markers under certain conditions [e.g., inviability of morphological mutants on CM with pfp (Kafer, 1965; Bainbridge and Roper, 1966) ; or inhibition of f p a mutants by 2ys mutants (Section IV, A, p. 102) ; or inviability of paba on CM containing sulfonilamide (Bignami et al., 1974) ; etc.]. As mentioned above, these can be recognized if more than one system of selection or more than one type of mapping strain are used: consistent linkage of all markers of two groups, provided both reciprocal types of haploids can be recovered, clearly identifies the chromosomes containing the breaks. Crosses to standard strains also reveal most translocations, since they cause increased nondisj unction, which produces disomics for the involved groups (demonstrated in Upshall and Kafer, 1974). Results from tests using both these methods, which identified nine UV-induced translocations, are shown in Table 1. It should, therefore, be relatively easy to keep all strains translocation-free, except those being used specifically for translocation work. However, while this has been achieved to a considerable extent for most of the retained Montreal strains, it also has become obvious that such a procedure is much too time-consuming when translocations are not of primary interest. This is the case because unexpected or undesired translocations remain in many strains for the following reasons: widespread distribution of early translocations in the strains of all laboratories ; high rate of induction of translocations with most mutagenic treatments ; spontaneous occurrence of new translocations even in tested strains, especially those with “reverted” duplications ; mix-up of strains of origin with indistinguishable phenotypes ; tight linkage of desired mutants to induced translocations; etc. It seems, therefore, that as a general rule aberrations have to be expected in many strains, and that the best procedure is probably to check strains immediately before they are needed, a t least for those genetic investigations where translocations could interfere. b. Association of Mutants with Breaks. I n 5 of the 16 analyzed cases it has not been possible to separate an induced mutant from a chromosomal aberration present in the same strain, even though analysis was quite extensive. Some of these may represent cases of position effects of

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

109

the breaks [as shown for brZA12 and T 2 ( N I + V I I I ) by Clutterbuck, (1970) 1, while others are known to be due to close linkage; the latter must be the case for pantorll, induced in a strain containing T I (ZII + V I I I ) and not yet separated from it. Three other cases are more likely of the former type, since mutant and translocation were induced simultaneously and remained inseparable when over 100 haploid or disomic progeny from crosses were tested (namely, lysD20 and sD50, Table 1 ; and frA1, Fig. 13) ; this, however, does not constitute conclusive proof (as, e.g., available in Drosophila; Lefevre, 1973). c. Frequencies of Induced Translocations. Induction of balanced reciprocal translocations without any phenotypic effects is much more frequent than was originally assumed, not only after irradiation with Xor 7-rays, but also after UV treatment (Kafer, 1965). It appears now that ultraviolet light at a level of killing used in Aspergillus work ( 2 4 % survival) induces translocations with frequencies of 15% to 25% (9/21 in Table 1, which are all well analyzed cases, plus 1/21 further UVinduced mutants for which the original strain was not analyzed). Analysis of random samples of UV-treated strains produced slightly lower values (A. Upshall, personal communication). These results are in line with similar observations in Neurospma [somewhat lower UV dose, much less killing because conidia are multinucleate and 8% aberrations (Perkins, 1974) 1. Lower frequencies of UV-induced translocations have been found after treatment of diploids (Kiifer and Chen, 1964), but this could be due to somatic pairing which, a t least in Drosophila, leads mainly to fusion of homologs rather than to interchromosomal translocations when breaks are induced (Gatti et al., 1974). Whether some of the powerful chemical mutagens currently in use also frequently induce chromosomal aberrations is not yet known. So far only a few mutants induced by nitrosoguanidine (NG) have been checked, and no translocations have been found, in line with results by Malling and DeSerres (1970) in Neurospora. Also the recent progress in mitotic mapping in slime molds using mainly NG-induced mutants, indicates that translocations may be relatively rare in this case (Rothman and Alexander, 1975). However, NG is known to cause breaks in higher organisms (in human cells found by Kelly and Legator, 1970) and possibly also in diploids of Aspergillus (Shanfield and Kafer, 1971). Certainly EMS has been shown to induce translocations in mice, where such aberrations are recognized as the main cause of sterility (Cacheiro et aZ., 1974).

2. Meiotic Mapping As expected, meiotic mapping of translocation breaks is usually difficult in Aspergillus, even though the reduction of recombination in heterozy-

110

ETTA KAFER

gous crosses is most helpful. Homozygous crosses, furthermore, have barely been usable, except in cases where a self-fertile marker is associated with the break. Such markers also greatly enhance meiotic mapping in heterozygous crosses (as shown for Tl(1V;VIII) associated with jrAl in Fig. 13).

3. Mapping of Translocation Breaks to Specific A m s or Segments a. Mapping of Breaks to Specific Chromosome Arms. This can be relatively easy in heterozygous diploids when a selective marker is available in the same arm as one break. In such diploids, mitotic crossovers are either abnormal because unbalanced, or entirely absent if inviable (for problems with this method, see Section 111, E). b. Haploids and Mitotic Crossovers from Homozygous T / T Diploids. Such segregants easily map the breaks, but accurate mapping is possible only if the closest markers can be used, which therefore has to be in coupling with the translocation. This is most readily obtained when a marker is either induced closely linked to or associated with one of the breaks; or, on the other hand, when even the closest markers are barely meiotically linked. In other cases, the closest markers will be recognized more likely by the difficulties encountered in getting them into coupling (Le., by recognizable linkage in disomics, see below). However, results in haploids from T/T diploids are always clear-cut and useful for confirmation; and they are especially useful when only one marker is found linked to the breaks, and/or when no translocation disomics are obtained. c. Mapping of T-Breaks in Translocation Disomics. Such mapping of translocation breaks is no different from mapping of new mutants (discussed above) ; all translocation disomics are checked for heterozygous markers, and if two types have been obtained, these show complementary patterns: one type is heterozygous (with characteristic frequencies) for all the markers of the distal translocated segment of one chromosome and also for the nontranslocated markers and the centromere area of the other; the second type is found heterozygous for all other markers of the two involved chromosomes. These results, therefore, qualitatively map the translocation breaks if the sequence of all mapped markers is known [as shown for T l ( V ; V l ) and markers of Group V, Table 18, discussed below]. d. Unidirectional Translocations. Mapping these translocations by identification of heterozygous markers in phenotypically recognizable duplication types is basically the same technique as the previous one (Bainbridge, 1970, Clutterbuck, 1970 ; Perkins, 1972). The details differ because duplication types are much more frequent (one-third of the viable progeny); their haploid sectors, on the other hand, are much rarer-i.e., their heterozygous centers are more stable and much easier

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

111

to test [like the stable disomics from crosses between overlapping translocations (Fig. 2) ]. I n duplication types all heterozygous markers belong to the same linkage group and they indicate the extent of the translocated segment. e. Meiotic Linkage of Markers to Breaks in All Types of Disomics. Since the majority of haploid sectors from T-disomics obtained in heterozygous T-crosses contain the standard chromosome set, and those from typical-looking n 1 contain the rearranged set, their sectors show up meiotic linkages of markers to the transolcation breaks in the form of complementary allele ratios. Testing one sector from each disomic and comparing marker segregation in the sectors of the two groups of disomics, therefore, identifies meiotic linkages of markers to the translocation breaks (Kafer, 1975).

+

4. Example, Mapping

of T1 (V ;V I)

Genetic mapping of translocations by a combination of all the described methods is illustrated here, using the case of the reciprocal translocation T1 (V;VZ). This translocation was mapped in detail to check on predictions of approximate break positions, based on disomic frequencies in crosses heterozygous for translocations (Kafer, 1975). Details are given here to demonstrate not only how a combination of methods is used to advantage, but also to show some of the problems encountered in this type of work. The translocation T1 (V ;V I) was discovered in an F, progeny of a cross with the UV-induced sAl mutant of group 111, and subsequently also demonstrated in the original W-treated strain (Table 1 ) . Eleven other sAl descendants were checked for the translocation, and seven were found to show increased frequencies of disomics, especially of n V and n VI (range 1-5% in relatively small platings), while four others showed less than 0.5% and therefore were translocation free (total of 9 heterozygous translocation crosses in Table 4 ) . Larger platings were made from five well marked crosses heterozygous for T1 (V ;V I) (genotypes in Table 5 ) . These definitely produced only one type of translocation disomic, which grows almost normally and produces large conidiating aneuploid centers (Fig. l c ) , rather than the two variants postulated earlier on the basis of the nine small samples (Upshall and Kafer, 1974). Tests for heterozygous markers confirmed that all translocation disomics were of the same type; they were found to be heterozygous for lysB, but not facA, hxA, riboD of group V (bottom of Table 18). They also were heterozygous for the distal markers of VI, namely sB and sbA, but not for the proximal marker lacA (Fig. 15). These results place the breaks of TI (V;VI) between lysB and facA on V (where the sequence has been

+

+

CL CI

XI

TABLE 18 Frequencies of Recombination between Breaks of TI (V;VI)and Relevant Markers in All Types of Disomics, and Heterozygous Markers in Translocation Disomics, from Crosses Heterozygous for T1(V;VZ)

Cross No.

+

Total TZ 1 (Typical disomics Translocation disomics)

1953 2130 2152 2132 2131

Total disomics:

+5630) (16 + 26) 42 (35 + 87) 122 (67 + 84) (26

151 (100 58) 158

+

+

VL ZysB

pA

-

-

- proxima1 - V R facA

+ 7) 29 % (7 + 6)

- distal hxA ribOD -

lacA

+

-

proximal - VI R - distal bwA sB sbA

(14 18) 57 % (4 7) 26% 31 % ( 5 25) (3 10) (9 22) (17 38) (15 44) 25% 11% 25% 45 % 48 % 13) (18 26) (22 24) (24 34) (32 33) (35 44) (15 29 % 31% 38% 43% 52 % 18 % (23 20) (4 11) (16 22) (23 24) (45 26) (45 35) 9% 24% 30% 45% 27 % 51 %

+ +

+

+

+

(9

+

+ +

+

+ +

+ +

+

+

+

+

+

+ +

(15 15) 54 % (9 14) (9 16) 55% 60% (15 +48) 52 % (33 38) 47 % (49 30) (37 34) 50% 45%

+

+

+

+

+

(244 285) 529 Crossovers/total : Combined percent:

117/431 27%

T-disomics: heterozygous/tested 140/127

28/280 10%

144/529 27%

01 /124a 0/73

105/309 34%

0/57

191/431 44% 0/96

1

250/487 51%

39/193

0/91

O?/llOa

90%

Conidial color of disomic centers is difficult to recognize in T-disomics, especially in the case of bwA.

195/378 52%

167/351 48%

47/67

36/56

M

3P

2

$

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

113

established by meiotic recombination) and between lacA and sB (sequenced by mitotic crossing over; see Section C, p. 120). Heterozygosis for the color mutants p A and bwA could not be determined visually in these brownish disomic types, and mapping with respect to these two markers was carried out subsequently by other methods. Only in one cross were contradictory results obtained, namely many similar looking unusual disomics often heterozygous for lysB and f a c A ; however, since in this cross a progeny strain from a double translocation cross had been used, it probably contained a differently modified chromosome V (and since all other crosses gave consistent results in large Samples, this cross was excluded from the final analysis and Table 18). To obtain meiotic mapping of markers to the breaks, all types of disomics were analyzed for marker frequencies in their haploid sectors. Table 18 gives the numbers of observed recombinants between the breaks and the markers of V and VI, listed separately for “typical looking” disomics (namely n + V and n + V I combined) and for translocation disomics. The pooled frequencies clearly demonstrate meiotic linkage of the translocation breaks to bwA, but not to any other marker on VI. On the other hand, all markers on V show meiotic linkage to the break on V. The obtained values map this break in the middle between 1ysB and f a c A , close to p A but probably proximal to it, since p A maps closer to f a c A (27%) than to 2ysB (40%; Fig. 15). These results agree with the above approximate mapping indicated by the heterozygous markers in the T-disomics. They are also confirmed by meiotic intergroup linkages obtained in these crosses : namely, bwA becomes meiotically linked to some of the markers on V, closest to p A and less close, but equidistant, to lysB and facA (31% recombination in each case). Such intergroup linkages between markers located close to the breaks usually show smaller values than expected from the distance estimated for each marker to its break, because disomics show a relatively high level of mitotic crossingover (as discussed in Section 111, F) . To map the breaks of T1 (V;VZ) with respect to the linked color markers, p A in V, and bwA on VI, hoinozygous translocation diploids were constructed (Table 19, diploids 2208 PA/+, 2182 and 2184 bwA/+). The haploids isolated from these diploids clearly mapped p A on the translocated piece, i.e., the T-break to “left” and proximal to p A in V, in agreement with the meiotic results. On the other hand, b w A was mapped on the proximal part of VI R , since it segregated in haploids together with lacA and was now unlinked to the translocated markers sB and sbA. This must mean, that bzcA and p A are located on the same translocation chromosome in T1 (V;VZ). To confirm this hypothesis, and to measure the meiotic distance between them, cross 2153, homozygous

TABLE 19 Haploid Segregants from Diploids Homozygous for TI (V;VZ)or TI (VZ;VZZ) v - VI VI - v lysB - sB sbA lacA bwA - p A facA hxA riboD VII - VI VI - VII phenB OliA - sB sbA IacA bwA - pantoB palF malA choA nicB

Markers

2184 2208 2211

sB

a

+

a

+

6 + 6 a

+

6 a 6 +

TI (VI;VZZ)

1

- -

+- +++ -+ -+ - ++

I" - +

a

1996 1997

a I; a - + - -

1998

a - + - -

a

6

+ ++ +- -+ - -

- + ++ -+ +- +- - + + - -

6 - -

45

6 + + + + + +

6 - + + + + + a - + +--+ 6 + + + + + +

a

a + + - - + -

+- +

++ + +

VI a

Parental

Recombinants

V-VI a VI-V a VI-V b

Total numbers of haploids of four types:

1992

6

a

V I I - VI

++

Total

VI - v

v - VI

TI ( V ;VI) 2182

Numbers and types of haploids

Arrangement of markers on homologs a/b

Diploid No.

7

10

44"

16

16

54

18

44" -

16 57

187

- VII

-+ + ----

Parental

V-VI b VI-V a VI-V b 12

16

5

7

12

14

10

8 46

11 42

42

VII-VI a VI-VII a VI-VII b

9

VII-VI b VI-VII a VI-VII b

6 + - + + + + +

42

7

22

3

lo

b

64

24

11

13

16

39

18

6

11

4

36

2 51

5 44

3 30

26

a t

b a i;

++ +- +- ++ +- +- -+ ++ ++ ++ ++ +- -+- + _ _ + - - - + ++ +++++

Totals:

Two rare crossover types from selenate selection not included.

181

%

H

2 E 1

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

115

for T1 (V;VZ), but heterozygous pA X bwA was carried out (genotypes in Table 5 ) . It was possible to distinguish all four color combinations visually, and slight but significant linkage was found between PA and bwA (314 recombinants, 4476, among a total of 703 classified). As expected, this value is considerably higher than the corresponding one in heteroxygous T-crosses [estimated a t less than 35% in cross 2136, in which double-mutant types pA;bwA were difficult to classify (Table 5 ) 1. 5. Approximate Mapping of T-Breaks, Based on Disomic Frequencies in Crosses By comparing the absolute frequencies of “typical looking” disomics in crosses heterozygous for all mapped translocations, an inverse correlation of the distances from breaks to centromeres was clearly apparent (Kafer, 1975) ; that is, meiotic nondisjunction was high when breaks were proximal and low when they were distal. As mentioned in Section 11, D, 1, it should be feasible to use this relationship and to predict the approximate position of the breaks for an unmapped translocation from aneuploid frequencies in heteroxygous crosses (especially if these could be compared t o other, mapped, cases with breaks in the same arm). Such predictions were attempted for T1 ( V ; V I ) ,which now has been mapped. The absolute frequencies of typical-looking disomics from all TI (V;VZ) crosses are shown in Table 4. On the average, these values are fairly high for n V, and about half as much for n VI (ca. 1% and 0.50/0, respectively, after correction for abnormal viabilities in cross 2131). These results place one of the breaks proximal in group V, which, considering the above mapping results, may well be the case; on the other hand, the break in VI R appears to be located quite distal in VI, and certainly much further distal than that of T1 (VI;VZZ),since the latter gives many more n VI in heterozygous crosses (Table 4). This contradicts the meiotic mapping results, which place both breaks close to each other in VI, distal but linked to bwA. The difference in the n VI frequencies is, therefore, not explained and the postulated correlation is not found; rather, breaks in close proximity on the same arm show quite different frequencies of typical disomics. This finding is rather disappointing, even though not entirely unexpected. It indicates that additional factors may influence nondisjunction frequencies of centromeres of heteromorphic pairs. One such factor could well be the length of the attached translocated segment, which is very much longer in the case of T1 (V1;VII) than T1 (V;V1).However, if the situation is indeed more complicated and based on a t least two unknown variables, predictions are likely to be very inaccurate, possibly even misleading.

+

+

+

+

116

ETTA KAFER

C. MAPPINGOF CENTROMERES AND THE USE OF TRANSLOCATIONS FOR SEQUENCING OF MEIOTIC FRAGMENTS While centromeres probably now could be mapped meiotically by analysis of unordered tetrads because quite a lot of markers close to their centromeres have become available, no such analysis has been attempted in recent years. In Aspergillus, early results from standard crosses between markers of groups I-IV indicated loose linkage of markers to three of the centromeres (Strickland, 1958). All of these have been confirmed by mitotic mapping, even though the actual values calculated for the meiotic distances do not agree exactly with current estimates (three of them being smaller, and one considerably larger). All recent centromere mapping is, therefore, based on mitotic crossing-over or on special techniques that make use of translocations. These latter are of two types: The centromere position can be postulated either on the basis of mapped translocation breaks if breaks are very close to the centromeres; or centromeres can be mapped by mitotic crossing-over in diploids homozygous for translocations ‘in cases where the latter transfer suitable selective markers to new linkage groups. 1. Mapping of Centromeres by Mitotic Crossing-over in Standard

Diploids For reliable mapping of centromeres by mitotic crossing-over, selection should ideally be practicable in both chromosome arms. When all types of selection are considered (including, for example, selection for auxotrophy), this probably is the case in Aspergillus for all groups in which markers on both arms have been isolated. However, in the case of acrocentric chromosomes, or when all available markers are located on the same arm, centromere mapping presents more of a problem (as is the case for markers in groups VI, VII, and VIII used here). Also, if selection is possible in only one arm, it is practically impossible to distinguish between crossover segregants and nondisjunctional ones, unless more than one marker is available on the other arm. Fortunately, crossingover is relatively frequent close to the centromere. Therefore, two markers, which only rarely, and always simultaneously, become homozygous (especially if they are not closely linked in meiosis) can usually be identified as markers on the opposite arm. Hence the rare homozygous segregants are probably of nondisjunctional origin. In other cases, where selected crossovers apparently show recombination between all “proximal” markers, the centromere position cannot definitely be identified. It can, however, be tentatively assigned if values for the most ‘Lproximal” interval are either extremely large (over 30%) or very small (less than

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

117

570). Such guesses are somewhat easier if the same selective system has been used in less ambiguous situations, and the relative viabilities of all types can be judged. No expected percent value for nondisjunctionals can be given because the relative frequencies of nondisjunctionals among selected diploid segregants vary not only with the relative distance of the selective marker from the centromere, but also with the different conditions of selection (values of less than 3% and over 25% have been obtained). It seems that under different conditions, the absolute frequencies of nondisjunction vary considerably and in unpredictable ways (as evident for chromosome I : Section 111, A, 2 ) . This is understandable if different conditions permit growth and further segregation of trisomics, which always are the primary product, to different degrees. Also it is possible that certain selective media actually induce “double” nondisjunction (e.g., p-fluorophenylalanine when used for selection of fpaA or fpaB, since it causes general missegregation of chromosomes). Additional problems also arise when selective or other markers are used that become homoaygous by recombination not much more frequently than by mutation [as, e.g., ActA, see below; or as found for two of the markers recently used in Dictyostelium (Mosses et al., 1975)]. Problems naturally also may be caused by intrachromosomal aberrations, e.g., inversions (or minor ones like the one postulated for the centromere area of group I in the biA strain: Section 111,B). Any other aspects of centromere mapping will be discussed by presenting as examples the details for the centromers of groups I to I V and VI, which all were mapped by mitotic crossing-over in standard diploids. a. Linkage Group I . In Aspergillus nidulans good selective markers are available distal in both arms only in two groups: groups I and 11. On I R, selection for yellow conidial heads (yA) has been used extensively even though it is rather laborious ; however, relatively efficient selection is possible on the other arm (using suAadE, and fpaA or fpaB). The centromere position between lysF - yA on the one hand and suAadE or fpaA - 1uA on the other, is therefore easy to demonstrate; unexpectedly, however, the mapping between proA and lysF in certain strains does not give completely unambiguous results (see Section 111,B). b. Group 11. This centromere was first mapped between wA and thiA by selecting for AcrA on I1 L, and acrB on I1 R (Kafer, 1958), but it has more recently been mapped between two closer markers by Clutterbuck (1974), namely palcA and riboE, the color mutant ygA being used as a selective marker on I1 R. c. Group IZI. The best selective marker in this group is sC, which has recently been mapped close and distal to ad1 on I11 L (Kafer, 1975). On selenate media, homozygous sC segregants can easily be obtained (Jan-

118

ETTA KAFER

sen, 1975; Arst, 1968). Jansen’s results show the sequence of markers on the SC arm (clearly the left arm, see Fig. 14) as cnzH sC - dilA - gaZA, and the centromere between galA and phenA was identified by obtaining a few phenA crossovers after selection for auxotrophy (among homozygous sC, 16% of nondisjunctionals were obtained). We have used selection for SC in several diploids heterozygous for a variety of markers. The two most informative ones are shown in Table 20 (diploids 1873 and 1877). The obtained results confirm and extend those of Jansen; they map the centromere between ActA and phenA (as postulated by Bainbridge, 1970) and place SuBpro on the same arm as phenA, that is to the right of p h e d (no recombination between phenA and SuBpro was obtained in sC/sC segregants, so that the types that are homozygous for all markers are recognized as nondisjunctionals ; their frequencies, 15-20y0, agree with those obtained by Jansen). In addition galE, a d l , and methH are confirmed as markers of the left arm (as placed by Dorn, 1967) ; however, galE is shown to be definitely located distal to sC, and methH, galA, and ActA proximal to it [as shown in Figs. 14 and 15 and in the most recent map of Clutterbuck (1974) ; confirming evidence for the position of suBpro has recently also been published by Klimczuk and Wegledski (1974) 1. Selection of crossovers using ActA (Bainbridge, 1970) on very high levels of actidione, was found to be very difficult, since most of the rare sectors turned out to be haploids, or heterokaryons between haploids and the parental diploid. From one diploid, with a d l , ActA, and phenA in coupling, no diploid actidione-resistant crossovers could be obtained. From a second one, with sC, galA, ActA, and phenA in coupling, a few

-

I

I

edE

meaB 28

14

I I

.-I

I 39'

-

1 m H

I I

27-1

I

i 3.8 ;d' sA

I 145

I galA

methH dilA 9

1 !I

28

I

I ActA

phenA

1

I--

I

6'

11.

1

-

suBpro 24.

I

1

11. -*

FIG. 14. Linkage map of group 111: Meiotic recombination (%) from standard crosses, except for distances adjacent to break of Tl(ZZZ;VZZ); * = well established values, standard errors
TABLE 20 Diploid Mitotic Recombinants from Diploids 1873 and 1877, Heteroaygous for sC and Other Markers of Group 111 Diploid 1873

Diploid 1877

cross-

Meiotic overs in units interval

Intervakl 2 galE SC No. of egregants

+

40

1

'0

5

2

0

50

3 s28}2,

10

SO

4

2

1'

5 - } g

10

8

25

6

(1 9

Nondisjunctionals

z 1'

15 0

0

+

+

3

+

d methU

5

4

dilA

+

+

8

7

8

+ - p .h - , -

gola ActA

9

d

+

i

-

I

+

SuBpro

1

2

3

4

5

SC f

+

+ -

- -

t

6

7 8 9

-

- - -+ " + + + +

:Gr:!

ApprOX.

combined eettmate

gpnts

Distal

+ -

sc+

+ + +

sc+

+

- sc+

+

-

-

-

-

-

s c + s c +

s c i

s c +

+

s c +

+

s c +

+

s c i

+

s c +

+

i

+ + + -

-

+

-

+

-

+ + +

+ - - + i-

= , +-

-

+

+ - $ ++ - +

-- +

-

I + s c + - -

35-45%

1-3%

-

+ I*sc+++--li-

+ s c + + + + +

-

i

+

d

+

+

+

I + s c + + + - -

-

+

10-15%

+ +

-

2-5%

+- +

+ - )

+ * c + + + - - + I

cated type is dentified in this way.

+ + + +

+I :- + l+sc+++--g+-

+

+

-

0%

+ a + + + - +- -

+

I

154

15-208

1 I I

Other arm 15-2096

120

ETTA KAFER

crossovers homozygous for ActA, as well as sC and gaZA but not phenA, were isolated. Prototrophic sectors were also isolated, but after extensive purification only two of these were genuine diploid segregants; one of the two was haploidized, and it turned out to be still heterozygous, containing the Act+ allele as well as a mutation to a much more resistant allele in the other homolog. d. Group IV. The centromere of group I V has originally been mapped between methG and pyroA in a diploid homozygous for biA, using “bistarvation” for selection (Kafer, 1958) ; and the data with T l (1V;VIII) (Fig. 13) showed that jrAl and all markers to the right (paZC - pabaB pyrod) are on the same arm. To map the centromere more accurately, diploids heterozygous for the new suppressor of adE20, w C l ladE20, were constructed with all these markers (except j r A l ) in coupling to suCadE. Since suCadE is very closely linked to jrAl in crosses heterozygous for TI (1V;VlII), it was expected t o map proximal on the right arm. However, the following results (of K. Schafer) map the centromere between suCadE and jrA 1 : all (ca. 300) diploid suCadE segregants were found to be also homozygous for methG, placing this marker distal to suCadE; on the other hand, only a fraction (27%) were homozygous for markers of the right arm (paZC - pabaB - pyroA) and, as expected, always simultaneously homozygous for all of them. These latter segregants must therefore be nondisjunctional diploids and their high frequency a result of the very close position of suCadE to the centromere. These results confirm the mapping of the markers paZC to pyroA on the right arm of IV, and map the centromere to the right of suCadE, hence between suCadE and jrA; but suCadE, like methG‘, must be located on the left arm. e. Group VI. The centromere of this group was mapped to the left of ZacA, using sB (on selenate media) for selection, in diploids heterozygous for sB in coupling with ZacA, b.wA, and sbA (Fig. 15). All the obtained diploid segregants were homozygous for sbA as well as sB, placing sbA distal to sB. In addition, many segregants (ca. 65%) were also homozygous for bwA and over half of these lacA/ZacA. This confirms the sequence lacA - bwA - sB,sbA deduced from the mapping of T l ( V I ; V I I ) (Kafer, 1975) and of TI ( V ; V I )(above) and places the centromere to the “left” of lacA and of all other markers used here (unless nondisjunction is as high as 30% in this case).

-

2. The Use of Mapped Translocations for Positioning of Centromeres and Sequencing of Meiotic Fragments

The mapping of reciprocal translocations can produce two types of information if incompletely sequenced linkage groups are involved: (a)

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

121

Markers that are translocated together map on the same chromosome tip or arm, while all other markers of that group are located on the residual part of that chromosome, which must contain the centromere. (b) Results from several translocations with breaks in the same arm identify overlaps and may sequence meiotically unlinked markers or meiotic fragments of that arm. a. The centromere of VIII was identified to the left of sD50 and all other markers used here (Fig. 15), when the break of T2(I;VIZZ) was mapped a t sD and all other markers of VIII were found to be translocated to linkage group I ( M a and Kafer, 1974) ; if diploids homozygous for this translocation are used, mapping of the centromere of VIII in relation to any markers proximal to sD will be easy, since in such diploids two selective markers, suAadE and fpaB, are translocated to VIII R ) . Similarly the first evidence for the position of the centromere of group V to the “left” of pA - f a c A - hxA -riboD was obtained from the mapping of the translocation T1 (V;VZ), since all these markers are translocated t o the centromere-ZacA segment of group VI (Tables 18 and 19 and Fig. 15). b. The mapping of the breaks of T1 (Z;VIZ), T1 (VI;VII), and T1 (ZII;VZZ) lead to the following sequencing of meiotic fragments of two groups: on VI, lacA - bwA - sB, sbA; and on VII, probably all on the same arm, phenB - OliA - sF,pantoB - palF - lysD - malA - choA - nicB (Kafer, 1975).

5. Mapping of the Centromeres by Mitotic Crossing-over in T / T Diploids To map the centromeres of V and VII, the two translocations that transfer the selective marker sB to these chromosomes have been used in T/T diploids, heterozygous for sB and a variety of markers. Segregants homo- or hemizygous for sB were selected on selenate media. Table 19 gives the genotypes of these diploids and of the mitotic haploids isolated from them, which confirmed the positions of the breaks with respect to markers on the two segments. Three diploids homozygous for T l (V;VZ) were used to map the centromere of group V (2182, 2184, and 2211) and these gave the following types of diploid segregants. All sB/sB scgregants (total ca. 400) were simultaneously homozygous for sbA, placing sbA distal to sB in the translocated segment from group VI (as expected from the corresponding results in standard diploids mentioned above). None of these sB/sB segregants was homozygous for any other marker but lysB [even though about 90 could have shown homozygosis for lacA or bwA, two markers proximal on VI R ; and 100-200, homozygosis for either facA, hxA, or

122

ETTA KAFER

111

14

hiA

*

;

-

so0 47

bwA

I

38

.8

-500

nbd)

38

17

k A

VI

frA

27

V

sbA

41'

20

35

VII

fscA

COA

Vlll

@ 21'

T2(1;VIII)

CnlB

5

fr0

0

4

r,ka

50

pal0 chaA

.E

I

I

FIG.15. Sequence of markers and mapped translocation breaks in all eight chromosomes and meiotic recombination frequencies between adjacent markers in percent. Values above the lines are from recent standard crosses; below, from heterozygous translocation crosses; * = well established values, based on totals >low, standard errors less than 10%; "(4", etc.) = tentative values based on totals <5W. For linkage group 11, no new mapping data were available, and those of Kafer (1958) are shown.

RECOMBINATION AND TRANSLOCATIONS IN

Aspergillus

123

riboD of group V which presumably are translocated to VI, judging from their mapping on the “other” translocation chromosome of T1 (V;VZ)1. The mutant, lysB was found to be homozygous only rarely ( 1 4 % in three samples of 50-100 each from diploid 2211, the only diploid with sB and lysB in coupling). These results indicate that the centromere of V is probably located between lysB and the break of T1 ( V ; V I ) ,proximal to P A : such a low frequency of lysB/lysB types is more likely to be due to nondisjunction than to crossing-over (provided the viability of ZysB types was normal on selenate media; diploids with sB in coupling to nicA, which maps very close to lysB, could be used to check this; or, even more conclusive, haploidization of sB/sB segregants from diploids 2184 and 2208, which carry sB and l y s B in repulsion; the latter might be rather time consuming, but would avoid construction of new strains with nicA, as well as sB and the translocation in coupling). The centrornere of group VIZ, on the other hand, was mapped by the break of TI (1;VIZ) as to the left of all markers used here, except possibly phenB which is located proximal, but fairly closely linked, t o the break (Fig. 15). To identify the position of the centromere either t o the left of phenB, or between phenB and the translocation break, two diploids homozygous for Tl(VZ;VZZ) and heterozygous for phenB and sB in coupling were constructed (diploids 1997 and 1998, Table 19). When homozygous sB segregants were selected on selenate, over 25% of these were found to be also homozygous for phenB. This most likely maps the ccntromere to the left of phenB (as in the case of lacA on VI above). I n addition the sB/sB mitotic crossovers (from diploid 1997) segregated for OZiA: namely, about 70% of these segregants were still heterozygous for OliA as well as phenB, a few of them were homozygous for OliA+but not for phenB, while all homozygous phenB also were homozygous for OliA’. This confirms the mapping of OZiA proximal to the break of T1 (VZ;VZZ) and definitely places it distal to phenB, that is, between phenB and sF or pantoB, on the standard map. All markers of VII used so far, therefore, map on one long “right” arm of this group, which may possibly represent an acrocentric chromosome. In conclusion, these recent results show that the use of translocations, and the various methods of mapping that they make possible, has led to a considerable change in the overall maps of Aspergillus (some of these results are already taken into account in the recent map of Clutterbuck, 1974). Not only have all centromeres now been placed, even if somewhat tentatively, but in addition most of the unlinked meiotic fragments have been sequenced. However, several of these fragments contain groups of meiotically linked markers that still need to be oriented. This applies specifically t o fragments on which only one marker was used in the translocation mapping; a second one a t the other end might possibly

124

ETTA KAFER

have shown up additional meiotic linkages in heterozygous translocation crosses. This, for example, is the case for group V I ; however in early attempts to use the markers lysA, nicC, and pacC, difficulties of various types were encountered (aberrations, excessive selfing, etc.). It seems quite likely that crosses of the latter two and e.g., molA (Clutterbuck, 1974) to the sequenced markers used here, and heterozygous for one of the translocations with breaks in VI, would produce some new meiotic linkages. Or, failing this, diploids heterozygous for sB and the above markers in coupling could sequence a t least some of them, even if-at the worstall of them were located on the unidentified “left” arm. The only other nonoriented fragment a t this time is that containing nicB (as well as palD and f l A ) distal on VII R. Since early crosses heterozygous for palD, choA, and T1 (V1;VIZ) did not produce any detectable linkage between these mutants, the only other useful experiment might be to use flA in a similar cross (such morphological mutants were not used here, because they make classification of disomics impossible). It is clear, however, that even complete sequencing of markers that would orient these known fragments would not in any sense finish or establish the total AspergiZlus map, since it is still expanding a t a considerable rate. New, loosely linked markers are being found not only a t the ends of mapped groups, but not infrequently have to be placed onto completely unlinked meiotic fragments. And it seems that the overall rate of increase in size has barely changed from the first map (Kafer, 1958) to the second one (Dorn, 1967; increase >600 units in 9 years) compared to the next one (Clutterbuck, 1974; increase >450 units in 7 years). It is, therefore, quite clear that the genetic map of Aspergillus nidulans is still far from being “saturated,” and considerable increases in the near future will presumably result just from the meiotic mapping of the over 75 genes, which a t this time are mapped only to one of the eight linkage groups (Clutterbuck, 1974). ACKNOWLEDGMENTS The excellent technical assistance of Mrs. P. Marshall and the expert photography of Mr. R. Lamarche are greatfully acknowledged. Thanks are due also to Drs. E. R. Boothroyd, A. J. Clutterbuck, and A. Upshall for help in the preparation of this publication. This work was supported by operating grant No. 2564 from the National Research Council of Canada.

REFERENCES Abbondandolo, A., and Bonatti, S. 1970. The production by nitrous acid of complete and mosaic mutations during defined nuclear stages in cells of Schizosaccharomyces pombe. Mutat. Res. 9, 59-69.

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125

Arst, H. N. 1968. Genetic analysis of the first steps of sulphate metabolism of Aspergillus nidulans. Nature (London) 219, 265-270. Azevedo, J. L., and Roper, J. A. 1970. Mitotic non-conformity in Aspergillus: Successive and transposable genetic changes. Genet. Res. 16, 79-93. Bainbridge, B. W. 1970. Genetic analysis of an unequal chromosomal translocation in Aspergillus nidulans. Genet. Res. 15, 317-326. Bainbridge, B. W., and Roper, J. A. 1966. Observations on the effects of a chromosome duplication in Aspergillus nidulans. J. Gen. Microbiol. 42, 417-424. Ball, C. 1973. Improvement of penicillin productivity in Penicillium chrysogenum by recombination. In “Genetics of Industrial Microorganisms” (Z. VanEk, Z. HOE k l e k , and J. Cudlin, eds.), Vol. 2, pp. 227-237. Elsevier, Amsterdam. Bandiera, M., Armaleo, D., and Morpurgo, G. 1973. Mitotic intragenic recombination as a consequence of heteroduplex formation in Aspergillus nidulans. Mol. Gen. Genet. 122, 137-148. Barratt, R. W., Johnson, G. B., and Ogata, W. N. 1965.Wild-type and mutant stocks of Aspergillus nidulans. Genetics 52, 233-246. Barratt, R. W., Ogata, W. N., and Kiifer, E. 1975. Fungal Genetics Stock Center: Aspergillus Stocklist (1st revision, July 1975). Aspergillus Newsl. 13, 23-58. Barry. E. G., and Perkins, D. D. 1969. Position of linkage group V markers in chromosome 2 of Neurospora crassa. J. Hered. 60, 12&125. Bhalla, S. C., Cajaiba, A. C. I., Carvalho, W. M. P., and Santos, J. M. 1971. Translocations, inversions and correlation of linkage groups to chromosomes in the mosquito Culex pipiens fatignns. Can. J . Genet. Cytol. 18, 837-350. Bignami, M., Morpurgo, G., Pagliano, R., Carere, A., Conti, G., and Di Giuseppe, G. 1974. Nondisjunction and crossing over induced by pharmaceutical drugs in Aspergillus nidulans. Mutat. Res. 26, 159-170. Blakeslee, A . F., and Belling, J. 1924. Chromosomal mutations in the Jimson weed (Datura stramonium).J . Hered. 15, 194-206. Brody, T., and Williams, K. L. 1974. Cytological analysis of the parasexual cycle in Dictyostelium discoideum. J. Gen. Microbiol. 82, 371-383. Burnham, C. R.,Stout, J. T., Weinheimer, W. H., Kowles, R. V., and Phillips, R. L. 1972.Chromosome pairing in maize. Genetics 71, 111-126. Cacheiro, N. L., Russell, L. B., and Swartout, M. S.1974. Translocations, the predominant cause of total sterility in sons of mice treated with mutagens. Genetics 76,7%91. Campbell, D. A., Fogel, S., and Lusnak, K. 1975. Mitotic chromosome loss in a disomic haploid of Saccharomyces cerevisiae. Genetics 79, 383-396. Carlson, P. S. 1972. Locating genetic loci with aneuploids. MoZ. Gen. Genet. 114, 273-280. Carpenter, A. T. C. 1973. A meiotic mutant defective in distributive disjunction in Drosophila melanognster. Genetics 73, 39W28. Carpenter, A. T. C., and Sandler, L. 1974. On recombination-defective meiotic mutants in Drosophila melanogaster. Genetics 76, 45g475. Christianson, M. L. 1975. Mitotic crossing-over as an important mechanism of floral sectoring in Tradescantia. Mutat. Res. 28, 389-395. Clutterbuck, A. J. 1970. A variegated position effect in Aspergillus nidulans. Genet. Res. 16, 303-316. Clutterbuck, A. J. 1974. Aspergillus nidulans. In “Handbook of Genetics” (R. C. King, ed.), Vol. 1, pp. 447-510. Plenum, New York. Clutterbuck, A . J., and Cove, D. J. 1974. The genetic loci of Aspergillus nidulans. In “Handbook of Microbiology” (H. Lechevalier, ed.), pp. 665476. Chem. Rubber Publ. Co., Cleveland, Ohio.

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