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A strain of Aspergillus nidulans producing a high frequency of disomics
[ 15 ] Trans. Br. mycol. Soc. 7S (1) 15-20 (1980)
Printed in Great Britain
A STRAIN OF ASPERGILLUS NIDULANS PRODUCING A HIGH FREQUENCY OF DISOMICS By A. C. GABRIELLI Institute of Biology, University of Campinas, Campinas, Brazil AND
J. L.
AZEVEDO
Institute of Genetics, University of Sao Paulo, Piracicaba, Brazil
A study was carried out of an Aspergillus nidulans strain which produces an average of 4'16 % disomies by conidial plating. This percentage is 20-35 times that of controls. Genetic analysis of the aneuploids revealed that they are disomics for linkage group III. Cytological analysis showed that disomic conidia have larger nuclei and are frequently binucleate. Since the only differences between the strain producing a high frequency of disomics and the strain from which it was derived is the presence of a mendelian gene designated v8, it was concluded that this gene is responsible for the high frequency of nondisjunction in linkage group III during mitosis. Variations in temperature and pH did not alter the frequency of disomics, but conidial age seemed to increase it. Haploid homokaryotic strains of Aspergillus nidulans are relatively stable when grown from mycelial inocula. However, when grown from conidia, they produce 0'1 to 0'3 % colonies with abnormal morphology which are phenotypically distinguishable from the parent strain (Upshall, 1966), and are usually disomic. Disomics for each of the eight chromosomes of A. nidulans have been obtained, and each has a characteristic phenotype which permits the identification of the linkage group involved (Kafer & Upshall, 1973). Chromosome aberrations in A. nidulans increase the frequency of nondisjunction during meiosis and there is an increase in disomics in the progeny of crosses involving one or more chromosomal aberrations. This does not happen in diploids heterozygotic for translocations (pollard, Kafer & Johnston, 1968). The first example of high frequency mitotic nondisjunction was described by Azevedo & Roper (1970) for an isolate designated as V8 which arose as a spontaneous sector from a haploid A. nidulans strain having a chromosomal duplication. This strain, when vegetatively propagated from conidia, produces a frequency of disomics which is much higher than those found in other haploid strains of A. nidulans. The phenotype of the disomics produced, together with preliminary genetic tests (Azevedo, 1971), showed disomy of linkage group III. Due to the high frequency of disomics, this strain is particularly suitable for a study of environmental factors which may influence the frequency of nondisjunction. The object of the research pre-
sented here was to determine the frequency of disomies obtained from the V8 strain under different conditions and to study the strain both genetically and cytologically. MATERIALS AND METHODS
Media The minimal medium (MM) was Czapek-Dox medium with 1 % (wIv) glucose. Complete medium (CM) was a complex medium containing yeast extract, hydrolysed casein, hydrolysed nucleic acid, vitamins, etc. (Pontecorvo et al., 1953). The solid media contained 1'5 % agar. Vitamins, amino acids and adenine were added to the minimal medium in order to classify meiotic segregants in the crossings performed. MM with galactose replacing glucose as a carbon source was also utilized. A medium containing ammonium acetate (Apirion, 1962) as the only source of carbon was also used. Strains The following A. nidulans strains were used: strain A, with a chromosome duplication (Nga & Roper, 1968); strain V8, derived from strain A and having a gene v8 located in linkage group IV and which has poor growth (Azevedo & Roper, 1970). Strain MSE of McCully & Forbes (1965), with genetic markers in all linkage groups, was also utilized. The genotypes of these strains are shown in Fig. 1 and all were stored at 5 °C on CM slopes.
0007-1536/80/2828-6230 $01.00 © 1980 The British Mycological Society
Disomics in Aspergillus nidulans
16 yA2
proAl pabaAti
n
(a)
•
•
+
•
•
+
+
•
Table
1.
adE20 biAl
Percentages of disomics produced by three A. nidulans strains
~-_~_~-
•
•
•
Strain
v8
•
A
MSE V8
••
•
V8 colonies, incubated for 10 days at 37° and from the edges were collected separately and the disomic frequency was determined as described above.
wA3
•
galAl III
•
pyroA4
Methods of genetic and cytological analysis
•
IV
(b)
facA303
•
V VI VII
sB3
•
lI/cB8
•
riboB2 VIII
% of disomics 0'21 0'12 4'16
yA2 adE20
suAl adE20 II
Total no. of Total of colonies disomics 11831 25 14 11323 1885 45 257
•
Fig. 1. Genotypesof used strains. (a) V8 strain with the genetic markers pro.A: and pabaA6 (proline and p-amino-benzoic acid requirements respectively) in linkage group I and the markers yA2 (yellow), adE20 (adenine requirement) and bi.At (biotin requirement) in the duplicate segment of the same linkage group. The determinant of deteriorated morphology (v8) is in linkagegroup IV. Linkage group II is represented by broken lines. Strain A alsoused in the present research is similar to V8 strain with exception of v8 which is absent in strain A. (b) Master Strain E (MSE) has genetic markers in all 8 linkage groups. adlleo, nicB8, pyroAs, riboli« and sB3 cause requirements for adenine,nicotinicacid, pyridoxine, riboflavin and thiosulphate respectively; SUAI adE20 is suppressor of adlizo; yA2 and wA3 (epistatic for yA2) cause yellow and white conidia respectively; galA: and facA]o] cause inability to use galactose and acetate as sole sources of carbon respectively.
The general genetie techniques were those of Pontecorvo et al. (1953). Heterokaryons were formed in liquid MM with 2 % CM. After 2 days incubation the mycelial mat was transferred to dishes of solid MM. Cleistothecia were obtained after 10 days incubation and cleaned of hyphae, Hiille cells and conidia. As there is a correlation between the size of cleistothecia and the proportion of hybrids, the largest were used (Baracho, Vencovsky & Azevedo, 1970). Cytological analysis was carried out by the HCI-Giemsa technique with modifications of strain concentration and staining time. The conidia were fixed in Helly's fixative for 15 min, hydrolysed in t N HCI at 60° for 15 min, washed twice for 5 min in water, and once in phosphate buffer, pH 7'0. They were then stained for 15min in diluted Giemsa solution (0' 1 ml stain/z ml phosphate buffer) and mounted in phosphate buffer, pH 7'0. Conidia and nuclei diameters were measured at the microscope with a light camera previously calibrated with a micrometrie scale. Two perpendicular diameters were traced and respective mean diameters were calculated. RESULTS AND DISCUSSION
Determination of disomic frequency
Conidia from each strain, grown on CM for four days at 37°, were suspended in Tween solution (0'1 % vjv) and plated on CM so that there were less than 50 colonies per plate. After three days incubation at 37°, the visually detectable haploid and disomic colonies were counted (Kafer & Upshall, 1973). The same method was followed to verify the effect of temperature and pH on disomie production by strain V8. However, two temperatures (28 and 37°) and four different pH values of CM were used (5'0, 6'0, 7'0 and 8'0) in addition to the usual 6'5 pH. The influence of conidial age on disomic production was also determined. In this case, the conidia from the central region of the
Frequency and analysis of disomics produced The percentages of disomics obtained from conidia of the V8 strain, the haploid strain (MSE) and of another strain, also haploid but having a chromosome duplication (strain A) indicated that strain V8 produced a large number of disomies, 20 to 35 times that found in strains used as controls (Table 1). Furthermore, most of the disomics, obtained from the V8 strain had a morphology (Fig. 2) characteristic of n + III disomics (Kafer & Upshall, 1973), while disomies for different linkage groups were obtained from the other two strains. Although the mean percentage of disornics was 4'16 for the V8 strain, the highest value obtained in 10 replicates was 4'8 %, and the lowest
A. C. Gabrielli and J. L. Azevedo
Fig. 2. Colonies from a conidial plating of V8 strain. V8 colonies and V8.1 unstable disomic colonies (arrowed) being produced. (x 2'5.)
Table
2.
Analysis of ascospores from the cross V8.1XMSE
Phenotype (morphology) Disomies (n+III) 57) X' f, Haploids 64 . = o·~o or a 1: 1 Total 121 J segregation. Galactose utilization Disomics (n+ III) galt
=
gal- =
Haploids
Total number gal! Total number gal-
55
+ 35)
Strain MSE V8 V8.1
Conidial diam*
Nuclear diam*
2'95 3'51 3,88
0'94 1'18 1'35
* Differences for conidia diameter and nuclei diameter among strains were significant (test F) at the 1 % level. LoS.D. (0'05) for nuclei = 0'0565 and L.S.D. (0'05) for conidia = 0'109.
2
gal" = 35 gal- = 29 = (55
Table 3. Diameters of conidia and nuclei (pm) of the disomic (V8.1), duplicated (V8) and stable haploid (MSE) strains
= 90}
= ( 2 + 29) = 31
X' = 0'02 for a 3: 1 segregation of galt t gal:
3'3 %. These were much higher than those obtained for the controls. As the only difference between strains A and V8 is the presence of a gene causing a change in colony of altered morphology (v8) which is located in linkage group IV (Azevedo & Roper, 1970), this gene appears to be responsible for the high frequency of disomics, possibly due to increased mitotic nondisjunction in linkage group III.
The frequency of disomics obtained from the conidia of several other strains does not exceed 0'3 % (Upshall, 1966) and the yield from ascospores is about 0'1-0'2 % (Upshall & Kafer, 1974; Kafer, 1977). In the presence of translocations, however, this percentage increases 10-20 fold, and 50-150 fold when the chromosome involved in the duplication is considered alone (Upshall & Kafer, 1974). Several disomies obtained from strain V8, all exhibiting a characteristic n + III morphology and designated as V8.1 according to the nomenclature proposed by Azevedo & Roper (1970), were crossed with the MSE strain. These disomics, like
Disomics in Aspergillus nidulans
18
all other A . nidulans disomics, are unstabl e, so that disomic segregants were obtained in onl y about 10 % of the h ybrid cleistothecia analysed. All ascospore progeny studied had a segregation ratio of 1 disomic: 1 haplo id . An alysis of the nutritional requirements of the disomic segregant s of these cros ses showed that all the genetic markers
had a m endelian segregation except the galactose m ark er , indicating th at linkage group III was invo lved. A typical anal ysis for one of the cro sses is found in Table 2 . M ost d isomic segregants of the V8 x l\1.SE cross grow on a medium containing galacto se as the only source of carbon . Some of th e colonies produce only gal " sectors while others
70
0 ---- MSE 60
50
I I I
,I
';; U
'"
t::
'0
_ _ -- vs
"
/ , / /
/ I I I / I I I
40
....
"
.0
E
'"
Z 30
D--
,
Disorni c ( V 8.])
\ \
,
\
\
\ I I I I I I
II ~ I I
'
I \ \
\
~O
10
0·54
0·63
0·84 0·99 ] , 14 1·29 Nuclei diamet er (I'm)
1-44
1·55
],77
Fig. 3. Variability in nuclear diameter of conidia of thr ee strains of A spergillus nidulans, (Results for 100 conidia of each strain.) Table 4. Influenc e of temperature on disomic production by strain V8 conidia plat ed at 37° (experiment A) and colonies grown at 37° and conidia plated at 28° and 37° (experlment B)
Experiment A Colonies grown at
Total no. of colonies
28° 37°
Total of disomics
% of disomies
175 3°1
3'87 3'5 6
869 1016
3'99
X'Test Mean effect 0'76 (1 d.f.) n.s. Heterogeneity 40.61 * (3 d.f.) Experiment B Colonies grown at 28° 37°
21754 235°3
4'3 2
X'Test Mean effect 3'05 (1 d.f.) n.s , Heterogen eity 53'32* (9 d.f.)
* Significant at the 1 % level; n.s,
= not significant.
A. C. Gabrielli and J. L. Azevedo Table 5. Percentages of disomics from V8 conidia when plated on media of different pH pH
Total number Total of of colonies disomies 183 4886 186 4 149 196 4864 145 3390 4382 159
5'0 6'0 6'5 7'0 8'0 X 2 test Mean effect 5'59 (4 d.f.) n.s. Heterogeneity 87'23* (16 d.f.)
% of disomics 3'75 4'4 8 4'03 4'28 3'64
* Significant at the 1 % level; n.s, = not significant.
Table 6. Percentages of disomics produced by conidia from V8 strain collected from the centre and edge of colonies Conidia collected Total no. of Total of % of disomics from colonies disomies 122 5 26 347 Centre Edge 17576 575 X2 test Mean effect 58-76* (1 d.f.) Heterogeneity 56'72* (9 d.f.) * Significant at the 1 % level.
produce sectors of two types (gal't and gal ' ), These results confirm the morphological observations which indicated that linkage group III was represented twice. The disomic strains were also studied cytologically. For this purpose, the diameters of 100 conidia and their nuclei were measured for the disomic strain V8.1. The controls were conidia of the V8 strain from which the disomic had originated, and of the haploid MSE strain. The results (Table 3) indicate that the three strains differ significantly with respect to conidial and nuclear diameter. As expected, higher values were found for strain V8.1, followed by strain V8 (having a chromosome duplication), and finally by MSE which is haploid and has no additional genetic material. Variability in nuclear diameterin the three strains (Fig. 3) is greatest, as expected for strains V8 and V8.1 which are unstable (Azevedo & Roper, 1970). The disomic strain also has binucleate or even trinucleate conidia. This may explain why conidia from the central region of disomic colonies show a variation in size ranging from that of haploid to diploid (pontecorvo & Kafer, 1958).
19
Effe ct of temperature, pH and incubation time on frequency of disomics produced by strain V8 The frequency of disomics was also determined by incubating strain V8 conidia at different temperatures, pH values and incubation times. Variations in temperature and pH had no effect on the frequency of disomies (T ables 4, 5). Conidia collected from the centre of a colony incubated for 10 days had a significantly higher frequency of disomics compared with conidia collected from the edges, where they were younger (T able 6). Cox & Bevan (1962) have previously suggested that colony age may influence the appearance of aneuploids in yeast. It is also interesting to point out that, at least at the observational level, the same effect is also found in man, where age has an effect on the frequency of trisomies in Down's syndrome, and in lymphocyte cultures from women more than 55 years old (Harnerton, 1971). These authors suggested the possibility that age produces a generalized reduction in mitotic efficiency, thus affecting chromosome distribution. In this study, it is also possible that the effect of the gene v8 in older conidia may be due to a lack or alteration of a substance which is necessary for the normal distribution of linkage group III in mitosis. It is also possible that, since the V8 strain has weak growth the conidia with a duplication of linkage group III may have a selective advantage. This fact may explain the high frequency of disomics produced by this strain and which increases with age.
We thank Professors Ivanhoe R. Baracho and Isaias O. Gera1di for their suggestions and help with statistical analysis . We are indebted to CNPq for financial assistance provided with grants SIP 04/052 and SIP 04/053. REFERENCES
D. (1962). A general system for the automatic selection of auxotrophs from prototrophs and vice-versa in miero-organisms. Nature, London 195, 959-961. AZEVEDO, J. L. (1971). Mitotic non-conformity in Aspergillus nidulans. Ph.D. Thesis, University of Sheffield. AZEVEDO, J. L. & ROPER, J. A. (1970). Mitotic nonconformity in Aspergillus: successive and transposable genetie changes. Genetical Research 16, 79-93· BARACHO, 1. R., VENCOVSKY, R. & AZEVEDO, J. L. (1970). Correlation between size and hybrid or selfed state of the c1eistothecia in Aspergillus nidulans. APIRION,
Transactions of the British Mycological Society 54,
1°9- 116.
20
Disomics in Aspergillus nidulans
Cox, B. S. & BEVA."I, E. A. (1962). Aneuploidy in yeast. N ew Phytologist 61, 342-355. HAMERToN, J. L. (1971). Human Cytogenetics. Ne w York: Academic Press. KAFER, E. (1977). Meiotic and mitotic recombination in Aspergillus nidulans and its chromosomal aberrations. Advances in Genetics 19, 33-131. KAFER, E. & UPSHALL, A. (1973). The phenotypes of the eight disomics and trisomies of Aspergillus nidulans. Journal of Heredity 64, 35-38. McCuLLY, K. S. & FORBES, E. (1965). The use of p-f1uorophenylalanine with ' master str ains' of Aspergillus nidulans for assigning genes to linkage groups. Genetical R esearch 6, 352-359. NGA, B. H . & ROPER, J. A. (1968). Vegetative nuclear division in Neurospora. American Journal of Botany 54, 735-748. POLLARD, D. R., KAFER, E. & JOHNSTON, M. T.
(1968). Influence of chromosomal aberrations on meiotic and mitotic nondisjunction in A spergillus nidulans. Genetics 60, 743-757. PONTECORVO, G. & KAFER, E. (1958). Genetic analysis based on mitotic recombination. Advances in Genetics 9, 71-104. PONTECORVO, G., ROPER, J. A., HEMMONS, L. M., McDoNALD, K. D . & BUFTON, A. W . J. (1953). The genetics of Aspergillus nidulans. Advances in Genetics 5,14 1 - 2 38. UPSHALL, A. (1966). Somatically unstable mutants of A spergillus nidulans, Nature, London 209, 11131115· UPSHALL, A. & KAFER, E. (1974). Detection and identification of translocations by increased specific nondisjunction in A spergillus nidulans. Genetics 76, 19-31.
(Received for publication 19 June 1979)
Printed in Great Britain
A STRAIN OF ASPERGILLUS NIDULANS PRODUCING A HIGH FREQUENCY OF DISOMICS By A. C. GABRIELLI Institute of Biology, University of Campinas, Campinas, Brazil AND
J. L.
AZEVEDO
Institute of Genetics, University of Sao Paulo, Piracicaba, Brazil
A study was carried out of an Aspergillus nidulans strain which produces an average of 4'16 % disomies by conidial plating. This percentage is 20-35 times that of controls. Genetic analysis of the aneuploids revealed that they are disomics for linkage group III. Cytological analysis showed that disomic conidia have larger nuclei and are frequently binucleate. Since the only differences between the strain producing a high frequency of disomics and the strain from which it was derived is the presence of a mendelian gene designated v8, it was concluded that this gene is responsible for the high frequency of nondisjunction in linkage group III during mitosis. Variations in temperature and pH did not alter the frequency of disomics, but conidial age seemed to increase it. Haploid homokaryotic strains of Aspergillus nidulans are relatively stable when grown from mycelial inocula. However, when grown from conidia, they produce 0'1 to 0'3 % colonies with abnormal morphology which are phenotypically distinguishable from the parent strain (Upshall, 1966), and are usually disomic. Disomics for each of the eight chromosomes of A. nidulans have been obtained, and each has a characteristic phenotype which permits the identification of the linkage group involved (Kafer & Upshall, 1973). Chromosome aberrations in A. nidulans increase the frequency of nondisjunction during meiosis and there is an increase in disomics in the progeny of crosses involving one or more chromosomal aberrations. This does not happen in diploids heterozygotic for translocations (pollard, Kafer & Johnston, 1968). The first example of high frequency mitotic nondisjunction was described by Azevedo & Roper (1970) for an isolate designated as V8 which arose as a spontaneous sector from a haploid A. nidulans strain having a chromosomal duplication. This strain, when vegetatively propagated from conidia, produces a frequency of disomics which is much higher than those found in other haploid strains of A. nidulans. The phenotype of the disomics produced, together with preliminary genetic tests (Azevedo, 1971), showed disomy of linkage group III. Due to the high frequency of disomics, this strain is particularly suitable for a study of environmental factors which may influence the frequency of nondisjunction. The object of the research pre-
sented here was to determine the frequency of disomies obtained from the V8 strain under different conditions and to study the strain both genetically and cytologically. MATERIALS AND METHODS
Media The minimal medium (MM) was Czapek-Dox medium with 1 % (wIv) glucose. Complete medium (CM) was a complex medium containing yeast extract, hydrolysed casein, hydrolysed nucleic acid, vitamins, etc. (Pontecorvo et al., 1953). The solid media contained 1'5 % agar. Vitamins, amino acids and adenine were added to the minimal medium in order to classify meiotic segregants in the crossings performed. MM with galactose replacing glucose as a carbon source was also utilized. A medium containing ammonium acetate (Apirion, 1962) as the only source of carbon was also used. Strains The following A. nidulans strains were used: strain A, with a chromosome duplication (Nga & Roper, 1968); strain V8, derived from strain A and having a gene v8 located in linkage group IV and which has poor growth (Azevedo & Roper, 1970). Strain MSE of McCully & Forbes (1965), with genetic markers in all linkage groups, was also utilized. The genotypes of these strains are shown in Fig. 1 and all were stored at 5 °C on CM slopes.
0007-1536/80/2828-6230 $01.00 © 1980 The British Mycological Society
Disomics in Aspergillus nidulans
16 yA2
proAl pabaAti
n
(a)
•
•
+
•
•
+
+
•
Table
1.
adE20 biAl
Percentages of disomics produced by three A. nidulans strains
~-_~_~-
•
•
•
Strain
v8
•
A
MSE V8
••
•
V8 colonies, incubated for 10 days at 37° and from the edges were collected separately and the disomic frequency was determined as described above.
wA3
•
galAl III
•
pyroA4
Methods of genetic and cytological analysis
•
IV
(b)
facA303
•
V VI VII
sB3
•
lI/cB8
•
riboB2 VIII
% of disomics 0'21 0'12 4'16
yA2 adE20
suAl adE20 II
Total no. of Total of colonies disomics 11831 25 14 11323 1885 45 257
•
Fig. 1. Genotypesof used strains. (a) V8 strain with the genetic markers pro.A: and pabaA6 (proline and p-amino-benzoic acid requirements respectively) in linkage group I and the markers yA2 (yellow), adE20 (adenine requirement) and bi.At (biotin requirement) in the duplicate segment of the same linkage group. The determinant of deteriorated morphology (v8) is in linkagegroup IV. Linkage group II is represented by broken lines. Strain A alsoused in the present research is similar to V8 strain with exception of v8 which is absent in strain A. (b) Master Strain E (MSE) has genetic markers in all 8 linkage groups. adlleo, nicB8, pyroAs, riboli« and sB3 cause requirements for adenine,nicotinicacid, pyridoxine, riboflavin and thiosulphate respectively; SUAI adE20 is suppressor of adlizo; yA2 and wA3 (epistatic for yA2) cause yellow and white conidia respectively; galA: and facA]o] cause inability to use galactose and acetate as sole sources of carbon respectively.
The general genetie techniques were those of Pontecorvo et al. (1953). Heterokaryons were formed in liquid MM with 2 % CM. After 2 days incubation the mycelial mat was transferred to dishes of solid MM. Cleistothecia were obtained after 10 days incubation and cleaned of hyphae, Hiille cells and conidia. As there is a correlation between the size of cleistothecia and the proportion of hybrids, the largest were used (Baracho, Vencovsky & Azevedo, 1970). Cytological analysis was carried out by the HCI-Giemsa technique with modifications of strain concentration and staining time. The conidia were fixed in Helly's fixative for 15 min, hydrolysed in t N HCI at 60° for 15 min, washed twice for 5 min in water, and once in phosphate buffer, pH 7'0. They were then stained for 15min in diluted Giemsa solution (0' 1 ml stain/z ml phosphate buffer) and mounted in phosphate buffer, pH 7'0. Conidia and nuclei diameters were measured at the microscope with a light camera previously calibrated with a micrometrie scale. Two perpendicular diameters were traced and respective mean diameters were calculated. RESULTS AND DISCUSSION
Determination of disomic frequency
Conidia from each strain, grown on CM for four days at 37°, were suspended in Tween solution (0'1 % vjv) and plated on CM so that there were less than 50 colonies per plate. After three days incubation at 37°, the visually detectable haploid and disomic colonies were counted (Kafer & Upshall, 1973). The same method was followed to verify the effect of temperature and pH on disomie production by strain V8. However, two temperatures (28 and 37°) and four different pH values of CM were used (5'0, 6'0, 7'0 and 8'0) in addition to the usual 6'5 pH. The influence of conidial age on disomic production was also determined. In this case, the conidia from the central region of the
Frequency and analysis of disomics produced The percentages of disomics obtained from conidia of the V8 strain, the haploid strain (MSE) and of another strain, also haploid but having a chromosome duplication (strain A) indicated that strain V8 produced a large number of disomies, 20 to 35 times that found in strains used as controls (Table 1). Furthermore, most of the disomics, obtained from the V8 strain had a morphology (Fig. 2) characteristic of n + III disomics (Kafer & Upshall, 1973), while disomies for different linkage groups were obtained from the other two strains. Although the mean percentage of disornics was 4'16 for the V8 strain, the highest value obtained in 10 replicates was 4'8 %, and the lowest
A. C. Gabrielli and J. L. Azevedo
Fig. 2. Colonies from a conidial plating of V8 strain. V8 colonies and V8.1 unstable disomic colonies (arrowed) being produced. (x 2'5.)
Table
2.
Analysis of ascospores from the cross V8.1XMSE
Phenotype (morphology) Disomies (n+III) 57) X' f, Haploids 64 . = o·~o or a 1: 1 Total 121 J segregation. Galactose utilization Disomics (n+ III) galt
=
gal- =
Haploids
Total number gal! Total number gal-
55
+ 35)
Strain MSE V8 V8.1
Conidial diam*
Nuclear diam*
2'95 3'51 3,88
0'94 1'18 1'35
* Differences for conidia diameter and nuclei diameter among strains were significant (test F) at the 1 % level. LoS.D. (0'05) for nuclei = 0'0565 and L.S.D. (0'05) for conidia = 0'109.
2
gal" = 35 gal- = 29 = (55
Table 3. Diameters of conidia and nuclei (pm) of the disomic (V8.1), duplicated (V8) and stable haploid (MSE) strains
= 90}
= ( 2 + 29) = 31
X' = 0'02 for a 3: 1 segregation of galt t gal:
3'3 %. These were much higher than those obtained for the controls. As the only difference between strains A and V8 is the presence of a gene causing a change in colony of altered morphology (v8) which is located in linkage group IV (Azevedo & Roper, 1970), this gene appears to be responsible for the high frequency of disomics, possibly due to increased mitotic nondisjunction in linkage group III.
The frequency of disomics obtained from the conidia of several other strains does not exceed 0'3 % (Upshall, 1966) and the yield from ascospores is about 0'1-0'2 % (Upshall & Kafer, 1974; Kafer, 1977). In the presence of translocations, however, this percentage increases 10-20 fold, and 50-150 fold when the chromosome involved in the duplication is considered alone (Upshall & Kafer, 1974). Several disomies obtained from strain V8, all exhibiting a characteristic n + III morphology and designated as V8.1 according to the nomenclature proposed by Azevedo & Roper (1970), were crossed with the MSE strain. These disomics, like
Disomics in Aspergillus nidulans
18
all other A . nidulans disomics, are unstabl e, so that disomic segregants were obtained in onl y about 10 % of the h ybrid cleistothecia analysed. All ascospore progeny studied had a segregation ratio of 1 disomic: 1 haplo id . An alysis of the nutritional requirements of the disomic segregant s of these cros ses showed that all the genetic markers
had a m endelian segregation except the galactose m ark er , indicating th at linkage group III was invo lved. A typical anal ysis for one of the cro sses is found in Table 2 . M ost d isomic segregants of the V8 x l\1.SE cross grow on a medium containing galacto se as the only source of carbon . Some of th e colonies produce only gal " sectors while others
70
0 ---- MSE 60
50
I I I
,I
';; U
'"
t::
'0
_ _ -- vs
"
/ , / /
/ I I I / I I I
40
....
"
.0
E
'"
Z 30
D--
,
Disorni c ( V 8.])
\ \
,
\
\
\ I I I I I I
II ~ I I
'
I \ \
\
~O
10
0·54
0·63
0·84 0·99 ] , 14 1·29 Nuclei diamet er (I'm)
1-44
1·55
],77
Fig. 3. Variability in nuclear diameter of conidia of thr ee strains of A spergillus nidulans, (Results for 100 conidia of each strain.) Table 4. Influenc e of temperature on disomic production by strain V8 conidia plat ed at 37° (experiment A) and colonies grown at 37° and conidia plated at 28° and 37° (experlment B)
Experiment A Colonies grown at
Total no. of colonies
28° 37°
Total of disomics
% of disomies
175 3°1
3'87 3'5 6
869 1016
3'99
X'Test Mean effect 0'76 (1 d.f.) n.s. Heterogeneity 40.61 * (3 d.f.) Experiment B Colonies grown at 28° 37°
21754 235°3
4'3 2
X'Test Mean effect 3'05 (1 d.f.) n.s , Heterogen eity 53'32* (9 d.f.)
* Significant at the 1 % level; n.s,
= not significant.
A. C. Gabrielli and J. L. Azevedo Table 5. Percentages of disomics from V8 conidia when plated on media of different pH pH
Total number Total of of colonies disomies 183 4886 186 4 149 196 4864 145 3390 4382 159
5'0 6'0 6'5 7'0 8'0 X 2 test Mean effect 5'59 (4 d.f.) n.s. Heterogeneity 87'23* (16 d.f.)
% of disomics 3'75 4'4 8 4'03 4'28 3'64
* Significant at the 1 % level; n.s, = not significant.
Table 6. Percentages of disomics produced by conidia from V8 strain collected from the centre and edge of colonies Conidia collected Total no. of Total of % of disomics from colonies disomies 122 5 26 347 Centre Edge 17576 575 X2 test Mean effect 58-76* (1 d.f.) Heterogeneity 56'72* (9 d.f.) * Significant at the 1 % level.
produce sectors of two types (gal't and gal ' ), These results confirm the morphological observations which indicated that linkage group III was represented twice. The disomic strains were also studied cytologically. For this purpose, the diameters of 100 conidia and their nuclei were measured for the disomic strain V8.1. The controls were conidia of the V8 strain from which the disomic had originated, and of the haploid MSE strain. The results (Table 3) indicate that the three strains differ significantly with respect to conidial and nuclear diameter. As expected, higher values were found for strain V8.1, followed by strain V8 (having a chromosome duplication), and finally by MSE which is haploid and has no additional genetic material. Variability in nuclear diameterin the three strains (Fig. 3) is greatest, as expected for strains V8 and V8.1 which are unstable (Azevedo & Roper, 1970). The disomic strain also has binucleate or even trinucleate conidia. This may explain why conidia from the central region of disomic colonies show a variation in size ranging from that of haploid to diploid (pontecorvo & Kafer, 1958).
19
Effe ct of temperature, pH and incubation time on frequency of disomics produced by strain V8 The frequency of disomics was also determined by incubating strain V8 conidia at different temperatures, pH values and incubation times. Variations in temperature and pH had no effect on the frequency of disomies (T ables 4, 5). Conidia collected from the centre of a colony incubated for 10 days had a significantly higher frequency of disomics compared with conidia collected from the edges, where they were younger (T able 6). Cox & Bevan (1962) have previously suggested that colony age may influence the appearance of aneuploids in yeast. It is also interesting to point out that, at least at the observational level, the same effect is also found in man, where age has an effect on the frequency of trisomies in Down's syndrome, and in lymphocyte cultures from women more than 55 years old (Harnerton, 1971). These authors suggested the possibility that age produces a generalized reduction in mitotic efficiency, thus affecting chromosome distribution. In this study, it is also possible that the effect of the gene v8 in older conidia may be due to a lack or alteration of a substance which is necessary for the normal distribution of linkage group III in mitosis. It is also possible that, since the V8 strain has weak growth the conidia with a duplication of linkage group III may have a selective advantage. This fact may explain the high frequency of disomics produced by this strain and which increases with age.
We thank Professors Ivanhoe R. Baracho and Isaias O. Gera1di for their suggestions and help with statistical analysis . We are indebted to CNPq for financial assistance provided with grants SIP 04/052 and SIP 04/053. REFERENCES
D. (1962). A general system for the automatic selection of auxotrophs from prototrophs and vice-versa in miero-organisms. Nature, London 195, 959-961. AZEVEDO, J. L. (1971). Mitotic non-conformity in Aspergillus nidulans. Ph.D. Thesis, University of Sheffield. AZEVEDO, J. L. & ROPER, J. A. (1970). Mitotic nonconformity in Aspergillus: successive and transposable genetie changes. Genetical Research 16, 79-93· BARACHO, 1. R., VENCOVSKY, R. & AZEVEDO, J. L. (1970). Correlation between size and hybrid or selfed state of the c1eistothecia in Aspergillus nidulans. APIRION,
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20
Disomics in Aspergillus nidulans
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(1968). Influence of chromosomal aberrations on meiotic and mitotic nondisjunction in A spergillus nidulans. Genetics 60, 743-757. PONTECORVO, G. & KAFER, E. (1958). Genetic analysis based on mitotic recombination. Advances in Genetics 9, 71-104. PONTECORVO, G., ROPER, J. A., HEMMONS, L. M., McDoNALD, K. D . & BUFTON, A. W . J. (1953). The genetics of Aspergillus nidulans. Advances in Genetics 5,14 1 - 2 38. UPSHALL, A. (1966). Somatically unstable mutants of A spergillus nidulans, Nature, London 209, 11131115· UPSHALL, A. & KAFER, E. (1974). Detection and identification of translocations by increased specific nondisjunction in A spergillus nidulans. Genetics 76, 19-31.
(Received for publication 19 June 1979)