As shown by MORTIMER and HAWTHORNE (1966), the distribution of these markers on the genetic map is ad, on chroinosome I, hi,, le,, mating type and thr, ...
A NOVEL CHARACTER INDUCED I N YEAST BY P32DECAY: THE ABILITY TO MANIFEST HIGH FREQUENCIES OF ABNORMAL MEIOTIC SEGREGATIONS E. MOUSTACCIII, H. HOTTINGUER-DE-MARGERIE, A N D F. FABRE
Insiiiut du Radium, Biologie, Orsay (91) France Received August, 7, 1967
a haploid strain of Saccharomyces cereuisiae it has been observed that u??ih proportion of the cells surviving intense labelling with P3*were radioresistant mutants (Xr) ( MOUSTACCHI and MARCOVICH 1962; MOUSTACCHI 1964). Genetic analysis of these mutants has now shown that many of these possess a further character which results in abnormal segregations of various genetic markers after meiosis. This new character, which will be referred to as “AM”, abnormal meiosis, is not related to their property of radioresistance. For the convenience of the presentation, we shall assume here that “AM” behaves like a genetic determinant. The chromosomal or cytoplasmic localization of this factor is examined in the thesis of one of us (H. H. DE M.) . In a forthcoming publication, we will describe the genetic control of this property (“abnormal meiosis”) and the mitotic behaviour of‘the segregants from an A M / a m hybrid (H. HOTTINGUERDE-MARGERIE and E. M OUSTACCHI, in preparation). This communication reports the unusual patterns of meiotic segregations of various markers observed in strains bearing the AM character. It is demonstrated that an extra duplication of the genetic material during meiosis is probably responsible for these abnormal segregations. MATERIALS A N D METHODS
Yeast strains and genetic markers: The genetic markers used were recessive mutations controlling nutritional requirements for adenine (ad,, a d , , , ad,), uracil ( u r , ) , histidine (hil, h i 4 ) , tryptophan (tr,, t r 5 ) ,leucine (les,k,), threonine (thr,) and methionine ( m e t , ) . The cyI locus seems to be the structural gene for iso-I-cytochrome c (SHERMAN et al. 1966). The lack of reversions for the intracistronic mutant cy,. is indicative among other observations of a deletion (P. SLONIMSKI, personal communication). The segregation of the mating type alleles ( a and a) was also studied. As shown by MORTIMER and HAWTHORNE (1966), the distribution of these markers on the genetic map is ad, o n chroinosome I, hi,, le,, mating type and thr, on chromosome 111, tr, on chromosome IV, hi, on chromosome V, tr,, ad,,, and le, on chromosome VI1 and met, on fragment 3. Two of these markers, tr, and le, are closely linked to the centromere.
,
Strain
10018-1 10018-2 10191 10192 Grnetii F 57 : 9O%ql X December 1 9 b i
Received from
G. MAGNI G. MAGNI G. MACNI G. MAGNI
910
E. MOUSTACCHI 1069-1A 1069-2D NI23 DPI-IC DPI-1 CA S 416 B S 2073 B
et al.
a hi,le,thr4met,tr,ur,ad, (Y hi41e,thr4metltr,urlad, a hi, a w1-,hiltrl vl~,hi,trl a met,ur,tr,ad,ad,ad, a gal,le,ad,thr4
R. K. MORTIMER R. K. MORTIMER M. OCUR P. SLONIMSKI P. SLONIMSKI R. K. MORTIMER R. K. MORTIMER
Media: Yeast extract peptone (YEP), and synthetic (MGS) media were as described previously (MOUSTACCHI and MARCOVICH 1962). In order to achieve high specific activities of added P32, the amount of phosphate in the synthetic medium was reduced to 20 pg/ml. The buffering capacity of the medium was maintained by adding citrate buffer (0.1 M, p H 5.8). Media and procedures for presporulation and sporulation were these of MCCI-ARY et al. (1959). Isolation of the “AM” mutants: The procedure initially devised to induce radioresistant mutants was used (MOUSTACCHI 1965). Stationary phase cultures of haploid strains grown with aeration in the low phosphate synthetic medium were used to inoculate to a concentration of 5 x IO4 cells/ml, 3 ml of the same fresh medium to which had been added 50 to 100 pc of carrier free P32 phosphoric acid (specific activity 15 to 30 mc/mg). This culture was incubated at 28°C with aeration until the stationary phase was reached. Then the cells were harvested thoroughly washed and resuspended in 1 ml of ice cold saline (0.9% w/v NaC1). The initial number of cells was determined by plating on YEP. Measurements of radioactivity contained i n the cells were made on dry samples by means of an end-window Geiger Muller tube. The suspension was then stored at 0°C. The AM mutation is detected by crossing some survivors to P32 decay (10-2 survival, for instance) with a tester strain. When the tetrad analysis of these diploids shows an unusual pattern of segregation compared with the haploid parent it is kept for further study. Genetic analysis: Crosses of presumed mutants and tester strains were made from stationary phase cultures grown at 28°C with shaking in YEP. The diploids were selected by the prototroph and BURKHOLDER (199.9). After sporulation, tetrad analysis by recovery procedure of POMPER dissection were made with the aid of snail enzyme (JOHNSTON and MORTIMER1959). The nutritional requirements of clones derived from single spores were tested for by replica plating on synthetic media. The incubation temperature was 28°C. Estimation of DNA: The different cultures were taken in the same conditions, i.e. in stationary phase (#-hour culture). The proportion of budding cells was 2 to 5%. As it is known that budding cells have double the amount of DNA, these were counted as two cells. Cell counts were made using a Burker counting chamber. 10 ml of washed cell suspension containing 5 x 106 to 8 x 106 cells/ml were subjected to the preliminary extraction procedure of OGUR and ROSEN (1950). Extraction of DNA was carried out in 1.5 ml of 1.0 M perchloric acid at 70°C for 20 minutes. The DNA content of 1.0 ml of the supernatant was determined by the diphenylamine method of BURTON(1956) using a preparation of deoxyadenxine as standard. The amount of DNA per cell was calculated assuming an adeninethymine content of yeast DNA equal to 66% (estimated from melting temperature and buoyant density at 20°C). All extractions and DNA determinations were run in duplicate and were made at least three times for each strain. RES U LTS
Genetic analysis of AM/am hybrids: When an AM cell is crossed with a normal strain (am), both strains bearing a number of nutritional deficiencies, a high proportion of the asci obtained by sporulating the resultant hybrids deviate from the normal 2+:2- ratio seen in the controls (am x am). The wild type predominates among the segregants regardless of whether the nutritional marker was introduced #withthe AM or the normal am parent (Table 1 ) . Only 4+:0, 3+:1- and 2+:2- ratios are found. The 1+:3- and 0+:4- classes are lacking.
TABLE 1 Segregation of the genetic markers in am/am (controls) and AM/am hybrids ~
Types of asci Genotype of the hybnd
Genetic markers
4+ 0 34- 1-
2+ 2-
Spore germination (percent)
1 + 3-
0 4-
0 1 1
0 0 0
100
0 0 0
0 0 0
95
N123 x 10192 (control) ahi,AD T R a m _ _ ~ _ _ a HI ad,-, t r , a m
AD TR HI
NI23 x 10192-P2 a hi, A D T R a m _ _ _ _ _ ~ 01 HI ad,-7 ti-, AM
AD TR HI
1069-1A x 10192 (contrtd) a thr, hi, le, tr, A D megu r I T R a m ___-____ a T H R HI LE T R ad,,, ME U R tr, a m
AD HI LE ME THR TR UR
0 0 0 0 0 0 0
0 0 0 0 0 0 0
1 5 5 5 5 2 4
2 0 0 0 0 0 1
2 0 0 0 0 3 0
36
10691-lA x 10292P36 a thr, hi, le, tr, A D mel u r , T R a m a T H R HI LE T R ad,,, ME U R tr, AM
AD HI LE ME THR TR UR
2 9 9 4 6 3 1
4 4 2 7 6 5
8 3 4 5 4 5
2 0 1 0 0 2 1
0 0 0 0 0 0 0
68
S 2073-B x 10192 (control) a gal, le, ad,-, ad, th, a.m _________ a G A L LE A D A D T H om
AD LE THR TR
0 0 0 0
0 0 0 0
S 2073-B x 10192-P36 a gal, le, ad,_, ad, th, a.m a G A L LE A D A D T H AM
AD LE THR TR
0 3 6 1
'
NI23 x 10018-2 a hi, AD a m ~ a HI ad, a m
HI AD
NI23 x 10018-2P10 a hi, A D a m -a HI ad, AM
1 15 0 1 5 1 1 4
0 0 0
15 13 8 2 4 4 8 20 10 6
0
5
0 1 9 9 9
9 0 0 0
2
0 0 0 0
4 0 0 0
45
3 0 5
0 1
1 0
9 9
0 0
0 0
75
HI AD
19 21
25 16
5 12
0 0
0 0
86
DP1-IC x 10018-2 (control) a A D hi, t r , cy,_, a m ________ a ad, HI T R CY a m
AD TR HI
0
0 1 1 0 1 1 0 9
1 1 3
0 0 0
98
0 0
DP1-1C x 10018-2P10 a A D hi, t r , cy,-, a m __________ mad, HI T R C Y AM
CY, AD TR HI
7 3 4 5
2 3 1 3
0 0 0 0
0 0 0 0
43
_
2
2 0 0 0
0 1 1
(control)
1
6
0 2 3 0
912
E. MOUSTACCHI
et al.
The excess of 4+:0- and 3+:1- ratios is observed for all genes irrespective of the chromosomes involved (at least five are represented) and of the degree of centromere linkage. However the genes close to centromere show a relatively low proportion of tetrads in the 3+:1- classes. A known deletion mutant cyl-l (DR. P. SLONIMSKI, personal communication) gave the same abnormal segregation patterns as the point mutants. A large proportion of the spores derived from the DP1-1C X 10018-PI0 zygotes are able to synthesize the is0 1-cytochrome c (the cytochrome content of the cells has been estimated in DR. SLONIMSKI’S laboratory). The percentage of germination of the spores from the A M / a m hybrids presented in Table 1 is generally similar to that of the am x am controls. When the A M / a m diploid is homozygous for several recessive alleles, only 0+:4- asci are found after sporulation. In other words, A M does not act as a mutator gene inducing high rates of reversions to prototrophy during meiosis. Mating properties of the segregants from an AM/am hybrid: The monosporic clones derived from the different asci described in Table 1 were crossed with a and tester strains in order to determine the segregation of the mating type. Some segregants were able to mate with both a and testers while others failed to mate with either. By seeding these clones with unexpected mating properties in a sporulation medium, many segregants were observed to be capable of sporulation (Table 2). Therefore, they are presumably diploid o r nearly so. Tetrad analysis of test crosses using the segregants having a mating reaction and unable to sporulate, showed that these segregants were also diploid (aa or aa constitution) , aneuploid or haploid. The details of mating reactions and sporulation ability of the segregants from an A M / a m hybrid are given in Table 3. D N A determinations of the parents and hybrids from A M and control lines: As seen in Table 1, the segregation ratios in tetrads obtained from crosses of AM by normal are apparently consistent lwith those obtained from tetraploid hybrids, (Y
(Y
TABLE 2 Segregation of the ability to sporulate for the segregants derived f r o m a n AM/am hybrid Ascus type (sporulating : nonsporulating)
Hybnd
40
31
22
13
04
3
1
2
0
0
6
4
4
3
2
1
4
1
1
3
S2073 B x 1019%P36 ( a gal,Ze,ad,thL a m ) x ( a aZ,tr, A M )
0
0
2
0
5
DPI-1C x 10018-2P10 ( a c y l _ l h i j t r ja m ) x ( a ad, A M )
0
4
3
1
3
NI23 x 10018-2P10 ( a hi, a m ) x (aad, A M ) 1069-IA x 10192.P36 ( a ad,hi,le,th,me,tr,ur, S416B x 10192-P2 ( a met,ur,tr,ad,ad,ad,
am) am)
x
x
( a ad,tr,
AM)
( a ad,tr, A M )
913
A B N O R M A L M E I O T I C SEGREGATION I N YEAST
TABLE 3 Tests of direct sporulution or muting reaction of the segregants from DPI-IC ( am cylhi,tr,) x 10018 ( a AM ad,) hybrid Crossed by tester .\SCllS
1
2
Segregants
A B C D A B C D
19
A B C D
21
A
Sporulated direclly
+ ++
-
a -
..
-
(Y
-
+ ..
-
t
++
++
B C D 22
A B C D
23
A B C D
25
A B C D
26
A B C D
.. . .
-
34
while their counterparts from the control crosses show the features of diploid cells. In other words, it may be asked if we accidentally picked tetraploid clones when we used the prototrophic selection to obtain the hybrids or if tetraploidization occurs systematically before the meiotic process under the control of A M . T o
914
E. MOUSTACCHI
et al.
TABLE 4
D N A determination in parental lines ( a m and A M ) , hybrids (am/am and A M / a m ) and in four segregants derived from one A M / a m hybrid (DPf -14:x 10018-2P10) Strain
a cy, hi, tr, a m (DPI-1C) ( 1001 8-2) a hi, am (N123) aad, A M (10018-2P10) a/a cy,_,/CY hi,/HI tr,/TR &,/AD am/am (DPI-IC X 10018-2) z / a cy,-,/CY hi,/HI ir,/TR &,/AD am/AM (DP1-IC X 10018-2P10) a/a hi,/HI ad,/AD am/am (NI23 X 10018-2) a/a hi,/HI ad,/AD am/AM (N123 x 10018-2P10) 2A ADHITR 2B ADHITR 2C ADHITR 2 D ad-HITR
a ad, a m
DNA pg per cell ( 4 X 108) (mean value -C SE) Sporulation
9.2 f 1.00 10.1 & 1.08 10.5 f 0.93 9.9 f 0.97 23.8 f 2.10 21.2 f 2.07 22.6
* 2.05
20.7 f 2.00 19.1 f 1.95 19.5 k 2.05 24 & 2.10 22.2 2.10
*
++ ++
examine this possibility, D N A determinations were made of the parents and hybrids from A M and control lines grown vegetatively. As seen in Table 4,the D N A content of the A M parents is equal to that of the normal am cells. It is also clear that the A M / a m hybrids are diploid according to their D N A content, which is similar to that of the normal am/am clones. This result renders unlikely the previous hypothesis. The diploid or aneuploid constitution of the segregants derived from an A M / a m hybrid is confirmed by the D N A determination. The amount of D N A per cell of these segregants is close to that of a diploid cell; in some cases, it is even higher. Some spore clones that are unable to sporulate also have twice as much D N A as the haploid parent. Their ability to mate with an or a tester strain and their amount of D N A per cell makes it very probable that their composition is aa or U . Frequency of A M cells among Pse suruiuors: The precise determination of these frequencies is laborious because of the actual lack of a selective method. Eighteen surviving clones (strain 10192) from a P32decay leaving survivors were crossed to a tester am strain (N123)and the hybrids were selected by the prototroph recovery procedure. Ten of these hybrids yield asci with aberrant segregations for the three nutritional markers tr,, hi,). Control crosses were made from untreated cells (12zygotes tested) ; none of them have shown ratios deviating from 2+:2-. It may be asked if the disintegrations of P32incorporated in nucleic acids are responsible f o r the induction of a mutation from am to A M or for a selection of A M cells spontaneously present in a population. As the A M and am cells have the same sensitivity to the lethal effect of P3' decay, the hy(Y
ABNORMAL MEIOTIC SEGREGATION I N YEAST
915
pothesis of a selection of the AM cells is unlikely. We have detected these mutants by scoring a relatively law number of cells. Therefore we may assume that the structure which controls the AM character is particularly sensitive to P32decay or that its size is large. Stability of the A M ,character through mitosis: Once induced by P32decay, the AM character is stable, although not manifested during the vegetative growth of the mutant clone. Even after 500 mitotic generations, the AM cells, when crossed to normal haploids, will still give hybrids that yield asci with aberrant segregations. The transmission of this property through meiosis is described elsewhere (HOTTINGUER-DE-MARIGERIE, 1967; HOTTINGUER-DE-MARGERIE and E. MousTACCHI, in preparation). DISCUSSION
A high proportion of haploid yeast cells surviving heavy labelling with P3' have a novel character AM which is expressed after meiosis of an A M / a m hybrid by unusually high frequencies of abnormal segregations. Most of the segregants are capable of sporulation and have a DNA content higher than either parent. Several genetic causes may be, in yeast, at the origin of segregations deviating from the 2+:2- ratio normally observed: gene conversion (for review see ROMAN 1963), super-suppressors (HAWTHORNE and MORTIMER 1963), supernumerary mitosis followed by the inclusion of more than one nucleus in the spores ( WINGE and ROBERTS 1954), polyploidy (ROMAN et al. 1955). Owing to the high proportion of abnormal asci and the lack of 1+:3- and 0:4- ratios, gene conversion cannot account for our segregation data. Similarly the presence of super-suppressor genes cannot explain the diploid constitution of most of the F, segregants. In order to account for the large excess of prototrophs among the segregants of an A M / a m hybrid and the diploidization (or near-diploidization) of these F, clones, we have to presume that at some step of the cell cycle an excess of genetic material is accumulated. It may be asked if this synthesis of DNA occurs before (vegetative stage) , during, or after meiosis of an A M / a m hybrid. The possibility of its being before or after will be considered first. The almost normal spore viability, the large excess of the 4+:0 class of asci and the DNA content of the AM cells (equal to that of the normal am parental line) (Tables 1, 4) rule out a diploidization or aneuploidization of the AM parent disintegrations. under the influence of P2 The possibility of an accidental selection of a tetraploid hybrid or a systematic tetraploidization of an A M / a m cell during vegetative growth under the control of A M must be seriously considered, because the segregation data presented in Table 1 seem consistent with those obtained from tetraploid hybrids. For instance, the cumulative ratios for tr, and le,, which are close to the centromere, give 31 et ai. (4+:0), 5 (3+:1-) arid 14 ( 2 + : 2 - ) asci. From the estimation of ROMAN (1955) for 10% secon'd division segregation and assuming bivalent pairing, the expected ratios are 30.2 (4+:0), 6.1 (3+:1-) and 13.6 (2+:2-). For the distal genes hi,, ad, 7 , met,, thr, and ur,, the cumulative ratios are 87 (4+:0), 75
916
E. MOUSTACCHI et al.
(3+:1-) and 36 (2+:2-). The expectation on the assumption that the pairing prophase chromosomes exchange partners at random and that all genes are in duplex (AAaa) state is 88 (4+:0), 88 (3+:1-) and 22 (2+:2-) (i.e. 4:4:1). There is good agreement between the experimental data and the theoretical values. However, if we consider only one gene such as instead of several genes, the experimental values are 17 (4+:0), 17 (3f:l-) and 16 (2+:2-), against 22.2 (4+:0), 22.2 (3+:1-) and 5.5 (2+:2-) expected in the case of a tetraploid. In this case the chi-square test gives P .lo. On the other hand, the amount of DNA in the AM/am cells grown vegetatively is close to that of the normal am/am diploid cells. Unless we suppose a decreased extractibility of DNA in the AM/am clones (although two extractions 1~ perchloric acid gave the same results) this fact argues strongly against the hypothesis of a tetraploidization prior to meiosis. The frequency with which AM was found and the good repeatability of the results with different strains makes the hypothesis of an accidental selection of spontaneous tetraploid hybrids unlikely. Cytological and genetic evidence for supernumerary mitosis in some asci followed by a fusion at random of the extra nuclei has been reported (WINGE and ROBERTS 1954) ; asci with more than four spores have also been noticed. This process leads to an excess of prototrophs among the segregants. In the case of the AM property the proportion of abnormal asci is considerably higher than in the example studied by WINGEand ROBERTS (6 abnormal asci out of 37 analysed). Furthermore, the cytological examination of the cells during sporulation using GANESAN’S staining technique (1956) failed to show more than four nuclei, and in several hundreds of asci examined we have never seen more than four spores per ascus. Therefore, it is unlikely that an extra mitosis following the normal second division is responsible for the abnormal segregations of an AM/am hybrid. Diploidization of the segregants might be the consequence of a later event; for instance, if mating type were unstable (high mutation rate) after isolation of the monosporic clones, copulation could occur in the mixed mitotic progeny of an a or a cell. This process would give rise to diploid cells capable of sporulation. HOWever, this would explain neither the presence of 4+:0 and 3+:1- classes of asci for nutritional markers nor the presence of aa o r aa cells among the segregants. As none of the considered hypothesis are entirely satisfactory, we are led to consider the possibility of an extraduplication occurring during meiosis. It may be assumed that the first normal doubling of the DNA is followed by an extra duplication affecting the genome partially or entirely; a region heterozygous for a deletion such as cyI-I may as well be duplicated. We suppose that this event occurs in an early stage of meiosis because of the consistency of the experimental cumulative segregation ratios with those obtained from tetraploid lines. During pairing of the chromosomes, crossing over involving two or more chromatids takes place. At the next stage, each centromere, to which four chromatids are associated instead of two, may spilt into four centromeres and meiosis proceeds then as usual. If the entire genome is affected by this extra duplication, the four spores are viable and diploid. Irregular duplications and/or nondisjunctions may give rise to aneuploid spores.
ABR ORMAL M E I O T I C SEGREGATION I N YEAST
917
More complex models may be considered. but we fully realize that more information is needed. Particularly the precise determination of the timing of DNA synthesis during sporulation of AM/am hybrids compared to am/am diploids might indicate if the proposed mechanism is responsible for the abnormal segregations observed. Whatever may be the mechanism by which the excess of genetic material found in the ITl segregants of an AM/am hybrid originates, it is genetically controlled and it f;eemsof interest to follow its transmission through further meioses. We wish to thank DR. R. LARARJET and DR. H . MARCOWCH for their interest i n our work. We are grateful to DR. P. SLoiwMsKI who suggested to us the experiments on the effect of the AM M. 0. JOUANNEAU and of character on a deletion. The excellent technical assistance of MADAME is gratefully acknowledged. This research was sponsored in part by the U.S. MLLES. ENTERIC Atomic Energy Commission under contract AT (30-1) 2803, and by Euratom under contract 005-61-1. SUMMARY
A high proportion of haploid yeast cells surviving heavy labelling with P3’ have a novel character referred to as “AM’ which results in abnormal meiotic segregations. When an AM cell is crossed with a normal strain (am), and the resultant hybrid is sporulated, 4+:0,3$:1- and 2+:2- ratios are found f o r the nutritional markers iniroduced in the cross. Most of the segregants have a DNA content higher than either haploid parent and are capable of sporulation. A tetraploidization of the hybrid during vegetative growth seems unlikely. More probably an extra duplication of the genetic material during meiosis, controlled by AM, may be responsiblle for the abnormal segregations. LITERATURE CITED
BURTON,K., 1956 A study of the conditions and mechanism of diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62 : 315-321. GANESAN, A. T., 1959 The cytology of Saccharomyces. Compt. Rend. Trav. Lab. Carlsberg 31: 149-1 74.
HAWTHORNE, D. C.: and R. K. MORTIMER, 1963 Super-suppressx-s in yeast. Genetics 48: 617620. HOTTINGUER-DE-MARGERIE. H., 1967 Dkterminisme gknetique d’un type danomalies mkiotiques et mitotiques induites par les ddsintkgrations du 32P chez la Levure. Thkse, Facultk des Sciences, Paris. J. R., and R. K MORTIMER, 1959 Use of snail digestive juice in isolation of yeast JOHNSTON, spore tetrads. J. Bacteriol. 78:272.
MCCLARY, D. 0.. W. L. NULTY,and G. R. MILLER,1959 Effects of potassium versus sodium in the sporulation of Sacch.aromyces.J. Bacteriol. 7’8: 362-368. MORTIMER. R. K., and D. C . HAWTHORNE, 1966 Genetic mapping in Saccharomyces. Genetics 53: 165-173. MOUSTACCHI, E., 1965 Induction by physical and chemical agents of mutations for radioresistance in Saccharomyces cereuisicre. Mutation Res. 2 : 403-412. __ 1964 Facteurs de la radiosensibilitk des levures. Etude d’un mutant radiordsistant. Thkse, FacultC des Sciences. Paris.
918
E. MOUSTACCHI
et al.
MOUSTACCHI, E., and H. MARCOVICH, 1962 Effets des dksintbgrations du phosphore 32 incorpok dans la levure Saccharomyces cereuisiae. I. Induction d‘une mutation vers la rbsistance B l’effet lbtal des rayon X. Ann. Inst.Pasteur. 103: 841-865. OGUR,M., and G. ROSEN,1950 The nucleic acids of plant tissues. I. The extraction and estimation of DNA and RNA. Arch. Biochem. 25: 262-276. POMPER,S., and P. R. BURKHOLDER, 1949 Studies on the biochemical genetics of yeast. Proc. Natl. Acad. Sci. US. 35: 45-6.F. ROMAN, H., 1963 Gene conversion in fungi. pp. 209-221. Methodology in Basic Genetics. Edited by W. J. BURDETTE. Holden-Day, San Francisco. and S. M. SANDS,1955 Studies of polyploid Saccharomyces. I. ROMAN,H., M. M. PHILLIPS, Tetraploid segregation. Genetics 40: 546-561, F., J. W. STEWART, E. MARGOLIASH, J. PARKER, and W. CAMPBELL, 1966 The strucSHERMAN, tural gene for yeast cytochrome C. Proc. Natl. Acad. Sci. U.S. 5 5 : 1448-1504. WINGE,0., and C. ROBERTS, 1954 Causes of deviations from 2:2 segregations in the tetrads of monhybrid yeasts. Compt. Rend. Trav. Lab. Carlsberg, Ser. Physiol. 25: 285-329.
