Linkage of conidial longevity determinant genes in Neurospora crassa

Mechanisms of Ageing and Development, 25 (1984) 63-77 Elsevier Scientific Publishers Ireland Ltd.

63

LINKAGE OF CONIDIAL LONGEVITY DETERMINANT GENES IN NEUROSPORA CRASSA

KENNETH D. MUNKRES and CHERYL A. FURTEK

Laboratory of Molecular Biology and Department of Genetics, The University of Wisconsin,Madison, Wisconsin 53706 (U.S.A.) (Received April 8th, 1983) (Revision received November 12th, 1983) SUMMARY The longevity of conidia of Neurospora crassa was previously defined as their ability to grow after aging in a constant environment. The heritable median lifespan of the wild type is 22 days. Heritable mutants with lifespans of 5 - 7 days were previously selected. The pleiotropic colony phenotype of the mutants greatly facilitates genetical analysis of their inheritance. Twenty-eight mutants were mapped by recombinational analysis. All but one were located at genes on one arm of the seven chromosomes, linkage group IR; the exception was at the terminus of linkage group VIR. Sixteen of the 17 genes on IR are collectively called the age-1 complex. The average and standard deviation of the distance of 13 intervals between adjacent genes in the complex was 4.9 + 1.1 map units; hence they appear to be spatially reiterated. Other observations indicate that the genes are also functionally redundant.

Key words: Neurospora; Longevity; Genes; Linkage

INTRODUCTION Survival of the conidia of Neurospora is defined as ability to grow after incubation of a mature culture at 30°C, 85-100% relative humidity, in cool white fluorescent light [1]. The median lifespan (mls) of a conidial population is defined as the age at which 50% survive. The heritable mls of wild type is 22 days. Heritable short-lived mutants (age-) with mls about 7 days were selected as either spontaneous occurrences or after near ultraviolet mutagenesis. Four lines of evidence indicate that defective formation of aerial cortidiophores of colonies of the mutants is a pleiotropic expression of the agegenes. This paper describes the linkage of 28 mutants with respect to one another and marker 0047-6374/84/$03.00 Printed and Published in Ireland

© 1984 Elsevier Scientific Publishers Ireland Ltd.

64 genes, employing classical methods of recombinational analysis. Preliminary accour~ts of this investigation have been reported [2-4]. The primary objectives of this investigation were to estimate: (1) the number of genes controlling conidial longevity and; (2) how the genes are distributed among the seven chromosomes. The principal conclusions are that there are many genes, essentially all localized on one arm of the seven chromosomes, and probably organized in a spatially repetitive sequence. The significance of these conclusions is discussed in light of biochemical, complementation, and dominance properties of the mutants, indicating functional redundance. MATERIALS AND METHODS Culture media

Mycelia are grown on Vogel's minimal medium (VM) [5] supplemented with 1.5% Difco Bacto agar. Agar medium in petri dishes contains 0.8% sorbose and 0.1% sucrose (VSS) to induce colonial growth [5]. Crosses

Slants of 5 ml of corn meal agar [5] in 18 X 150 mm test tubes are inoculated with conidia from a newly established culture and incubated in the dark at 25°C for 7 days. The protoperithecia are fertilized with fresh conidia and again incubated in the dark at 25°C for 14 days. During the 21-day period, the tubes are wrapped with aluminum foil to prevent desiccation of agar and cells. Then the foil is removed and the slants are incubated at room temperature in fluorescent light for 7 days. The perithecia and ascospores are suspended with the aid of a spatula in 5 ml of sterile water. The suspension is stirred in a mortar and pestle to release ascospores from undischarged perithecia and asci. Then 10 ml of water are added and the suspension is Fdtered through two layers of cheesecloth. The fdtrate, in a 15-ml conical centrifuge tube, is centrifuged at full speed for 5 min in a clinical centrifuge. The ascospore pellet is suspended in 5 ml of water containing 0.1% agar. The ascospore concentration is determined with a hemocytometer. The volume of the suspension is adjusted with the dilute agar solution such that a 0.1-ml aliquot contains about 50-100 ascospores. The ascospores are activated at 60°C for 60 min in a water bath and spread on a VSS agar dish with the aid of a glass rod and turntable. The plates are incubated at 35°C for 3 days in the dark in closed containers. Scoring the aer p h e n o ty p e

After wild-type colonies are incubated 4 days at 35°C in the dark, they begin to form aerial hyphae (conidiophores) bearing cortidia. Inversion of the plates, reduction of temperature to 20-25°C, and constant illumination with fluorescent light stimulate the developmental process, which we call aer ÷ (aerialogenesis)[1]. Other factors that influence the intensity of expression of the aer phenotype are colony density and depth of

65 the agar. A 9-cm petri dish should have at least 20 ml of VSS agar and no more than 100 colonies for maximum expression of aer +. The aer- phenotype is recognized by the failure to form conidiophores; conidia are borne on the colony surface. That phenotype is a pleiotropic character of the agemutants [ 1 ]. Since conidia of mature colonies are readily blown about, leading to crosscontamination, it is essential to employ a pedigree method for the selection of aermutants or for scoring that phenotype in crosses where other markers must also be scored. After 3 days incubation of the plates in the dark at 35°C, a small sterile, sharp spatula is used to excise a small wedge of the mycelium from a colony to a 1-ml VM slant. The colony isolation number is marked on both the bottom of the plate and the tube. The tube is cultured in the usual manner. The plates are incubated 4 days in an inverted position under fluorescent light at room temperature, at which time the aer phenotype is scored. Stocks Master stocks were stored on culture slants at --20°C. Linkage tester stocks were obtained from the Fungal Genetics Stock Center, Humboldt State University, Arcata, Califomia. The age mutants used here are listed in the tables. RESULTS The origin of the age mutants used, the number mapped, and the inferred number of genes are summarized in Table I. Table II summarizes the age phenotype of most of the mutants that were mapped. In all cases, the survival, S~o/So, which is an index of the rate of aging [ 1], is significantly less than that of wild type. Each mutant also exhibits the pleiotropic defect in colony morphology, aer- [ 1 ].

TABLE I ORIGIN AND GENE DISTRIBUTIONOF CONIDIALLONGEVITYMUTANTS Mutant isolation no.

Mutagen

No. mutants mapped

No. genes

1-36 (except 12, 13) 12, 13, 50, 53, 54

None Near UV

23 5

16a 4

Total 28

18 unique

aIncludes mutant no. 1 at age.2 locus on linkage group VI R and mutant no. 32 at age-3 locus on

linkage group IR.

66 TABLE II SURVIVAL OF 19 MUTANTS REPRESENTING 15 GENES IN THE age-1 COMPLEX OF N.

CRASSA Each mutant has been mapped and assigned a locus number in linkage group IR (Fig. 1). The first number is the original mutant isolation member; the second is the isolation number after backcross to wild tyl~e. Ss and Sis are the percentage viable conidla in a population of about 100 from cultures at maturity and after aging 10 days in the test environment, respectively, and are the average of triplicate measurements. The ratio S~/S o is an index of the longevity of the conidia. (If S s is observed to be greater than 100, it is assumed to be 100 for calculation of the ratio). The median lifespan of wild type is 22 days and the differential S~s/S o equals 90 ± 30. The mutants are characterized by Sa,/S, of 58 or less corresponding to a median life span of 11 days or less [ 1 ].

Locus no.

1.2 1.3 1.3 1.3 1.31 1.4 1.4 1.5 1.5 1.51 1.6 1.61 1.7 1.71 1.72 1.8 1.81 1.9 2.0 Wild type

Mutant no.

8-5 A 2-8 A 10a (so) a 31a (so) 15-1 A 5-1 A 54-6 A 9-11 A 4-9 A 50-16 A 6-18 A 17-2 A 3-10 a 19a-16a 18-16 A 16-2 A 21 A 14-4 A 26-18 A -

Survival Ss

S~,

S~o/S,

66 90 100 100 78 83 69 80 100 72 100 100 73 123 127 55 88 81 65 100 ± 30

19 23 45 40 23 10 34 40 52 40 28 36 38 28 48 9 38 47 20 90 • 30

29 26 45 40 29 12 49 50 52 55 28 36 52 28 48 16 43 58 31 90 ± 30

aBackcross progeny of two soft mutants (see text). The apparent s p o n t a n e o u s reversion frequencies o f the aer p h e n o t y p e o f 11 o f the m u t a n t s in Table II were previously r e p o r t e d [ 1 ]. Depending u p o n the size o f the conidial p o p u l a t i o n tested, the reversion frequencies were m i n i m a l l y less than 2 X 10 -4 and maximally 3 X 10 -3 per c o n i d i u m ; h e n c e it appears that the stability o f the p h e n o t y p e is n o t sufficiently l o w to c o n f o u n d r e c o m b i n a t i o n a l analysis. Initially, eight m u t a n t s were chosen for linkage analysis; isolates nos. i - 9

(no. 7 was

lost) (Tables I, II). Each was backcrossed once or twice to the original wild t y p e f r o m which it was selected. Previous studies indicated that b o t h the age- and aer- p h e n o t y p e s o f those m u t a n t s are inherited in a m e n d e l i a n , one-gene m a n n e r [ 1 ]. Backcross progeny o f the eight m u t a n t s were crossed to Perkins' [5] alcoy linkage

67 tester and the progeny were scored for all o f the five phenotypes. The results, in conjunction with those o f other subsequent crosses, indicated that seven o f the eight mutations were located on linkage group IR distal to the albino-1 marker. The exception, isolate no. 1, subsequently located on linkage group VIR, will be discussed later. Each o f the seven mutants located on IR and two UVA-induced mutants, nos. 12 and 13, were intercrossed. Random ascospores were plated on VSS agar. Recombination values were c o m p u t e d as twice the frequency of wild-type colonies, i.e. aer ÷, based on the total number of ascospores plated and assuming that the recombinational events were reciprocal. In a few crosses in which ascospore viability was high, minute, slowly emerging colonies, more abnormal than either m u t a n t parent, were observed at a frequency about that o f the wild-type recombinant colonies. We assume that they represent the double mutant recombinants and, therefore, at least in those instances, the recombination process was reciprocal. In other crosses, however, particularly those with low ascospore viability, the putative double mutants were infrequent or absent; hence, no systematic attempt was made to employ double mutant frequency in the analysis. Computation o f recombination frequency on the basis o f total viable ascospores was generally unsatisfactory because o f low viability, leading in many instances to nonsense frequencies o f 100% or greater. The results o f two-point recombinational analyses o f the nine mutants are in Table III. They were located at seven genes on linkage group IR (Fig. 1). The results o f other

TABLE III RECOMBINATION IN TWO-POINT CROSSES OF MUTANTS OF THE Age-I COMPLEX Cross a (locus assignment)

Mutant no.

1.1 x 1.2 1.1 " x 1.3 1.1 x 1.3 1.2 x 1.3 1.2 x 1.2 1.2 x 1.31 1.2 x 1.31 1.3 x 1.31 1.3 x 1.31 1.3 x 1.31 .1.2 x 1.4 1.3 x 1.4 1.31 x 1.4 1.4 x 1.4 1.4 x 1.4 1.1 x 1.5 1.1 x 1.5

12 X8 12 x 2 12 x 10 25 x2 25 x 8 25 x 15 8 x 15 2 x 15 10 × 15 31 x 15 8x5 2 x5 15 x 5 5x5 54 x 5 12 x4 12 X4

Total no. ascospores

Germination (%)

No. wild-type recombinants

Recombination b %

4824 483 315 1463 2670 704 3284 3284 5450 4048 890 550 600 2670 2700 600 1262

11 20

18

34 27

17

157 29 17 29 5 19 61 15 60 30 36 13 5 0 0 68 150

6.5 12 l 11 4.0 0.3 5.4 [ 3.'] 0.9 2.2 1.5 8.1 4.7 1.6 <0.07 <0.07 23[ 24

Average

11.5

4.6 1.5

23.5

68 TABLE III (continued) Cross a (lOCUS assignment]

Mutant no.

1.2 Xl.5 1.3 X 1.5 1.3 Xl.5 1.3 Xl.5 1.3 Xl.5 t.31 X 1.5 1.4 Xl.5 1.4 X 1.5 1.5 Xl.5 1.5 Xl.5 1.5 ×1.51 1.5 Xl.51 1.4 X l . 6 1.5 X l . 6 1.5 Xl.6 1.5 X l . 6 1.5 Xl.6 1.51X 1.6 1.6 X l . 6 1.6 X l . 6 1.1 x i . 6 1.1 X l . 6 1.5 Xl.61 1.5 ×1.61 1.5 Xl.61 1.5 X 1.61 1.6 X 1.61 1.2 X l . 7 1.2 ×1.7 1.4 X l . 7 1.5 Xl.7 1.5 Xl.7 1.6 Xl.7 1.61 X 1.7 1.2 X 1.71 1.5 X 1.71 1.5 Xl.71 1.61 X 1.71 1.7 Xl.71 1.7 Xl.71 1.5 X1.72 1.71 X 1.72 1.5 ×1.8 1.71 X 1.8 1.72 X 1.8 1.71 X 1.81 1.72 X 1.81

8X9 2×4 lOX4 2×9 10X9 15 X4 5X4 5 X9 4X4 4X9 4X50 4X50 5X6 4X6 9X6 4X13 4×27 50X6 27X6 13X6 12X13 12X6 4X17 4X17 4X53 4 X53 6 X53 8X3 25X3 5X3 4X3 9X3 6X3 53 X 3 8 X24 4 X24 4X19 17 X 19 3X19 3X24 4X18 19 X 18 4X16 19 X 16 18 X 16 19 X 36 18 × 36

Totalno. ascospores

Germination

(%)

No. wild.type recombinants

Recombination b %

970 1318 866 1140 500 294 2120 1622 2500 46000 815 2332 500 257 550 1196 2700 1780 2860 3560 717 450 660 640 1150 775 2888 1080 570 1430 6460 940 2850 2888 1770 805 500 1050 2128 900 828 2920 286 1600 330 1250 2000

41 100 47 60 56 57 58 55

2 100 7 45 9 58 4

41 20

8 2 50

50 28 69 0.7 15 51 78 4.3

19 5.9 40 16

!07 119 48 75 27 16 83 58 0 0 16 50 44 6 22 33 101 35 6 3 118 52 43 42 96 56 65 184 71 153 543 56 50 14 276 76 56 16 35 15 90 67 37 75 9 94 104

Average

22 11 13 11 11

1

13

7.5 0 0 3.9 t 4.3 18 4"7 8.0 I 5.5 7.5 3.9 0.4 0.08

33 t 23 13 t 17 14 4.5

34}

25 21 16.8 12 7.1 1.0 31 22 5.0 3.3 t 3.3 22 4.6 26 9.4 5.4 15 10.4

4.1

6.4

28

14

30

14

20

3.3

69 TABLE III (continued) O.oss a ~locus

M u t a n t no.

Total no.

Germination

ascospores

No. wild.type

%

assignment) 1.8 Xl.81 1.8 × 1.81 1.81 × 1.81 1.8 × 1.9 1.81 × 1.9 1.81 × 2.0 1.9 × 2.0

Recombination b

recombinants

16×36 16 × 21 36 X 21 16 × 14 21 × 14 21 × 26 14 X 26

2600 444 444 1065 720 660 490

93 95

62 10 0 116 37 64 29

4.81 4.5 <0.4 22 10.3 19.4 11.8

Average 4.6

aThe crosses are arranged in the table such that the locus to the left is also to the left (proximal) in the genetic map (Fig. 1). bThe percentage recombination is twice the frequency of wild-type recombinants based on total number of ascospores tested. In instances in which the cross was repeated or where the mutants appear to be sister alleles at a locus, the average of the recombination values was used for map construction. crosses, discussed below, permit orientation o f the map relative to the chromosome. By convention, the map is depicted with the left end proximal to the centromere. The genes are assigned a decimal number increasing from left to right. This unconventional notation is used because: (1) it provides an index to the relative map position of the genes; and (2) since the genes appear to be redundant by a number of criteria, it appears fallacious to assign each a unique number. The mutants are qualitatively indistinguishable in their age or aer phenotypes, exhibit the same enzymatic defects [6], fail to complement in heterokaryons [7], and are dominant to wild type [7]. The small recombination values between the seven genes were additive, permitting the construction of an internally consistent linkage map with unique gene sequence. Recombination values greater than 20% tended to be less than the sum o f the small intervals, as may be expected from chromosome interference; they are not used for map construction, but are included in the map as a semiquantitative confirmation of the gene sequence. Twenty-two additional age mutants were crossed to the double mutant, aur age-l.5 (aur is an allele o f albino-I). Random ascospores were plated, colonies were scored for the aur and aer phenotypes, and recombination frequencies were c o m p u t e d on the basis o f the total number o f ascospores plated. Examples o f the results o f those crosses are in Table IV. All except three o f the 22 new mutants appeared to be linked to the markers. In some crosses, the inequality of the frequencies o f the t w o recombinant classes, aur aer and + + , and the recombination frequencies permitted a tentative localization o f a mutation at an approximate distance to the right or left o f age-l.5. On the basis of such a prediction, a mutant was crossed to at least two o f the previously defined age loci o f appropriate distance to the left or right o f age-l.5 or, when order was ambiguous, to age loci b o t h right and left o f a g e - l . 5 (Table III). As a conservative approach, mutants were tentatively considered to be putative alleles

70

o~.~ ~o~ 0

do

~.~

~

~

~--~

1 I

0

~

~

°

i-~ I

1 I

~~.~

:l o o ~, . ~ •~ "~ ,-~

,

.-~ 1

~oo~ii oI _

~.~ ~

®

~-~

71 TABLE IV RECOMBINATION AND PROBABLE ORDER IN THREE-POINT CROSSES OF MUTANTS OF T H E age.1 COMPLEX TO THE aur MARKER Zygote genotype and recombination (%)a

aur + + age-l.l

18 aur + + age-l.3l

36

age-l.5 +

47 aur age-l.5 + +

42 aur age-l.5 + +

27 aur + + age-l.5

43 aur + + age-l.5

38 aur age-l.8 + +

22.6

No. ascospores tested

age-l.5 +

aer --+

Total

12

46

68

600 ,

4 12

10

18

28

510

4 15

26

24

50

2332

4 50

26

29

55

2704

4 27

25

18

43

660

4 17

70

26

96

1150

4 53

75

57

132

2300

19 4

23

14

37

286

16 4

48

14

62

2600

16 36

11

+ age-l.6

4.1 + age-l.61

13 + age-l.61

16.7 age-l. 71 +

11.4 age-l.8 +

26 + age-].8l

4.8

Mutant isolation no.

aur ++

23

aur age-l.5 + + age-l.51 48 4.3 aur age-l.5 + +

No. recombinants

aThe sequence and locus auignment was confirmed by the results of two-point crosses (Table III, Fig. 1).

if their recombination frequencies with near neighbor loci were similar; a conclusion that appears to be justified by the results o f direct recombinational tests o f allelism. Table III illustrates the results o f six crosses o f putative alleles at five l o c i ; t h e frequency o f wild-type colonies was either nil or less than 0.2% o f the total ascospores tested. The mechanism o f origin o f these wild progeny is not clear;however, we prefer the conservative view that they arose by either reversion or conversion o f heteroalleles, rather than by recombination o f closely linked genes. Not all cases o f putative allelism were analyzed by direct recombinational test. In the case o f putative alleles o f a g e - l . 3 , the crosses were sterile.

72 Crosses of a mutant by itself were generally sterile or barren; however, in fertile self-crosses of mutants 4 and 5, the frequency of wild-type colonies was less than 3 X 10 -4 (Table III). The low reversion frequency of the aer phenotype in conidial populations and the low frequency of wild-type progeny in either putative allele, or self-crosses indicates that the recombination data are not confounded by high reversion rates. Furthermore, the additivity of the map distances is not compatible with reversional origin of the wild-type progeny. Two crosses each of mutant no. 4 by mutants 12, 17, 50 and 53 were analyzed on separate occasions (Table Itl). The recombination values were reproducible, with variances ranging from 0% to 10% of the averages. The averages of recombination data from both duplicate crosses and those of"sister alleles with near neighbor loci (Table III) were used for the final construction of the genetic map, a procedure that improved the degree of additivity of the map intervals. Altogether, 16 genes were discovered in the age-1 complex, represented by 26 mutants. Eight of the 16 loci were represented by more than one allele, a fact that lends statistical confidence for map construction. There was a marked tendency of the map distances between adjacent loci to be about 5 units. There were only two instances in which another locus occurred within the 5-unit interval. Ignoring those exceptions, the average and standard deviation of the map distances of 13 intervals was 4.9 -. 1.1 (Table V). The map distance between next nearest neighbors was about twice that between nearest neighbors; that, of course, may be ex-

TABLE V MAP DISTANCES BETWEEN NEAREST NEIGHBOR GENES IN THE age-1 COMPLEX Gene interval (loci)

Map distance (map units)

1.1 - 1 . 2

1.2 -1.3 1.2 -1.31 1.3 -1.4 1.4 -1.5 1.5 -1.51 1.51-1.6 1.6 -1.61 1.61-1.71 1.7 -1.71 1.71-1.72 1.72-1.8 1.8 -1.81

6.5 4.0 5.4 4.7 7.5 4.1 3.9 4.5 5.0 3.3 4.6 5.4 4.8

Average Standard deviation

4.9 1.1

73 pected when the map distances are additive, but is also lends support to the hypothesis that the genes tend to be separated by a constant distance. The data in Table III are systematically arranged to assist map construction. In the first column of the table, loci to the left are at the left on the map. In addition, the position of the genes moves from left to right in the map as one proceeds down the column. The only exception to this rule are crosses between either the same mutant or alleles. In practice, each of the smallest map intervals is drawn to scale, using the averages of recombination values of duplicate or sister allele by I~eighbor crosses, if available. Then the larger intervals are drawn without considering the scale factor. Linkage o f genes in the age-1 complex with other genes in linkage group 1R Initial linkage analysis with Perkin's alcoy tester indicated that seven of the genes of the ageol complex were distal to the al-1 locus in linkage group IR, a conclusion that was confirmed by the results of three-point crosses involving two age loci and the aur marker, an allele of al-1 (Table IV). The results indicate that these age genes are far distal to aur. The following gene sequence is known to be far distal to al-l: so, aro-8, R, un-18 [8]. The observed linkage ofage-l.6 and age-1.71 with aro-8, R, and un-18 provides additional evidence for the location of the age-1 complex in IR (Table VI). On the basis of the foregoing and previously reported linkage data [8], it appeared that the soft(so) locus might be located at the left of the map. Two so mutants were studied by Perkins, et al. [8]: B-230 and P-1490. They concluded that the mutants were probably aUelic because they recombined with other markers at similar frequency and were non-complementary in heterokaryons. Female sterility prohibited a direct recombinational test of allelism.

TABLE VI RECOMBINATION IN TWO-POINT CROSSES OF age-I MUTANTS TO OTHER MARKERS IN LINKAGE GROUP IR Crossa

Mutant no.

Total no. ascospores tested

No. wild.type recombinants

Recombination b (%)

age.l.8 Xylo.4 age-l.9 Xylo-4 age-2.0 Xylo-4 age-l.6 Xaro-$ age-1.71 X R age.l.6 XR age-l. 71 X un.18

16, 32 14, 32 26, 32 13, DH8 16, 34508 3, 34508 16, ALS4

600 1464 509 1196 2200 2228 2600

90 189 31 2 125 36 5

30 26 12.1 0.3 11.4 3.3 0.4

aMarker symbols: ylo-4, yellow conidia; are, aromatic amino acid biosynthesis; R, round ascospore; un-I 8, unknown requirement temperature-sensitive. bRecombination is expressed as twice the wild-type recombinant frequency'based on total number of ascospores tested.

74 The two soft mutants were backcrossed to our wild-type stock, and five mutant progeny of each were selected. The original mutants and their progeny exhibited abnormal colony morphology and short-lived conidia like that of the age mutants [ 1] (Table II). Mutant no. 2 at the age-l.3 locus exhibits the so phenotype, dense compact conidiophores on a slant, unlike most of the age mutants; it was also female sterile anti would not complement with the so mutants. Backcross progeny of the so mutants, B-230 and P-1490, were assigned the code numbers 10 and 31, respectively, and crossed to age-l.1, age-1.31, and age-l.5. The recombination values were similar to those of mutant no. 2 in crosses to those loci (Table III); hence, it appears to be an allele of the so locus. The age-3 locus In a backcross of an UVA-induced age mutant to wild type, segregation of a mutation with yellow conidia was observed. The mutation segregated in a mendelian manner in a second backcross to wild type. The original mutant and its backcross progeny exhibited abnormally low conidial longevity; but, unlike the age mutants, its aer phenotype was normal [ 1 ]. The results of a three-point cross of the ylo mutant to aur are-8 indicated that it might be 30 map units distal to are-8; however, because of skewed aUelic ratios, the results were inconclusive. Table VI summarizes the results of three two-point crosses of the ylo mutant with the age loci 1.8, t .9, and 2.0 at the far right of the genetic map. The results are consistent with the far distal location o f y l o in linkage group IR, but additional three-point crosses with other markers must be analyzed to support that conclusion. The ylo-1 and ylo-2 loci in Neurospora are on other linkage groups [8]. Another locus, ylo-3, complements with ylo-1 or ylo-2, but its linkage relationship is unknown.

TABLE VII LINKAGE OF age-2 IN GROUP VIR Zygote genotype and recombination {%)

No. genotypes

Marker isolation

Recombinants

no.

Parental

chol-2 ylo-I ws-I + + + + age-2 35 45 8.2

26 I0 .

Singlesregion

Doublesregion

1

Triples Total

2

3

1.2

2-3

1-3

I

I0

I

8

I

0

1

48

11

14

I

9

0

3

I

49

47904 y30539y RP99 I

75 A cross of ylo-3 to our ylo mutant yielded 50% recombination; hence, it is tentatively called ylo-4 or the synonym, age-3, because of its distinct difference in morphological phenotype from that of the other age mutants.

Linkage of the age-2 locus in group VIR Mutant no. 1 could not be assigned to a linkage group by the use of the alcoy tester; however, in crosses tO the multicent [5 ] tester, it appeared to be linked to the ylo marker in group VI. (The presence of a segregating gratuitous morphological marker between the ylo and age loci facilitated the analysis). The results of a subsequent four-point cross clearly indicate that the age-2 locus is far right in group VI, 8.2 map units distal to the previous penultimate marker, ws-1 (Table VII). DISCUSSION Functionally related genes in Neurospora are generally distributed more or less randomly over the nuclear genome. Notable exceptions are the aro and qu~clusters [8]; hence, the most remarkable result of this study is the observation of 17 of 18 age loci on one arm of the seven chromosomes. Unlike the aro and qa clusters, however, the age loci are not contiguous. The map distances between adjacent genes are much larger than expected for contiguous genes and at least three genes with other functions are interspersed among the age loci. We have called the group of linked genes the age-1 complex [2,4,6]. With the present knowledge that other genes are interspersed, perhaps a more precise term is "gene family". There was a remarkable tendency for the map distance between adjacent genes to be about 5 map units; the average and standard deviation of 13 intervals was 4.9 + 1.1. We think that the genes of the age-1 complex probably are spatially and functionally redundant. Possibly they arose in evolution by gene duplication arising from unequal crossing over. Such a model provides a plausible framework for the interpretation of observations about unusual functional and mutational properties of the mutants. All age mutants representing the seven loci 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, and 1.7, were observed to be deficient in five antioxygenic enzymes: superoxide dismutase, catalase, glutathione peroxidase, cytochrome c peroxidase, and ascorbate free radical reductase [6]. It was proposed that the function of the genes is to regulate the synthesis of the enzymes and speculated that the large number of genes provides a mechanism for the amplification of regulatory signals in response to an increase in flux of free radicals and peroxides. These results illustrate the importance of these enzymes for conidial longevity and provide a molecular basis for the observed photosensitivity of the mutants [1]. Deficiency of these enzymes appears to render the conidia more susceptible to lethal macromolecular damage by photochemically generated free radicals and peroxides [6]. Complementation analysis indicated that all paired combinations of mutants represent-

76 ing the loci 1.3, 1.4, 1.5, 1.6, and 1.7 failed to complement for the aer phenotype [7]. Furthermore, five pairs of forced heterokaryons representing the loci 1.1, 1.2, 1.3, 1.5, and 1.8 also failed to complement. Mutants, 3, 4, 6, and 31 representing, respectively, the loci 1.7, 1.5, 1.6 and 1.3 were trans dominant to wild type for both the age and aer phenotypes in heterozygous duplications [7]. The mutant phenotypes were also dominant in forced heterokaryons of age-l.2 or -1.5 with wild type. Neurospora conidia are haploid. A priori, one may expect that any mutation of one of a family of redundant genes must be cis dominant to the others to be expressed. Cis dominance is inferred from the trans dominance to the wild-type genes in heterozygous chromosome duplications or heterokaryons. In summary, the enzymatic, complementation, and dominance properties all indicate that genes of the age-1 complex are functionally redundant. About 10% of the cells in a conidial or ascospore population of wild type are observed to be age- mutants [ 1 ]. On the basis of the present linkage studies, the average mutation frequency per gene per cell is about 0.006, a value that is many orders of magnitude greater than the forward mutation frequency of other genes. Conceivably, the mutant nuclei could have a selective replicative advantage during growth, leading to an inflated estimate of mutation frequency. Alternatively, one may speculate that in a situation involving a large number of closely linked redundant genes, DNA replication may frequently proceed out of register, leading to frequent replication errors. On the basis of the present studies, it appears likely that additional analysis of a larger collection o f age mutants would reveal additional loci on linkage group IR. Linkage analysis of another class of conidial longevity mutants would also be desirable. Such mutants, designated age +, have a lifespan and levels of antioxygenic enzymes greater than those of wild type [ 1,6 ]. ACKNOWLEDGEMENTS This research was supported by the College of Agriculture and Life Sciences, the Graduate School, and the National Institutes of Health (AG-O0930). This work con. stitutes a portion of a thesis by C.F. presented to the Graduate School of the University of Wisconsin in partial full'aliment of the master of science degree requirements. We thank W. Ogata for providing markers and linkage testers. We thank Drs. D.D. Perkins and R.W. Barratt for constructive criticism of this. ihvestigation. Contribution No. 2645 from the Laboratory of Genetics. REFERENCES 1 K.D. Munkres and C.A. Furtek, Selection of conidial longevity mutants of Neurospora crassa. Mech. Ageing Dev., in press. 2 K.D. Munkres, C.A. Furtek and E. Goldstein, Genetics of cellular longevityinNeurospora crassa. Age, 3 (1980) 108.

77 3 K.D. Mun~es, C.A. Furtek and R.S. Rana, Genetics of cellular longevity in Neurospora. II. A supergene cluster. Age, 4 (1981) 130. 4 C.A. Furtek, K.D. Munkres and R.S. Rana, Genetics of cellular longevity in Neurospora. III. Age, 5 (1982) 133. 5 R.H. Davis and F.J. de Serres, Genetic and microbiological techniques for Neurospora crassa. Methods Enzymol., 17A (1970) 79-143. 6 K.D. Munkres, R.S. Rana and E. Goldstein, Genetically determined conidial longevity is positively correlated with superoxide dismutase, catalase, gintathione peroxidase, cytochrome c peroxidase and ascorbate free radical reductase activities in Neurospora crassa. Mech. Ageing Dev., 24 (1984) 83-100. 7 K.D. Munkres, Dominance and complementation relationships of conidial longevity mutants of Neurospora crassa. Mech. Ageing Dev., in press. 8 D.D. Perkins, A. Radford, D. Newmeyer and M. Bj'brkman, Chromosomal loci of Neurospora crassa. Microbiol. Rev., 46 (1982) 426-570.