Current Genetics (1982)6:93 98

Current Genetics © Springer-Verlag 1982

Genetic Mapping of arg, cpa, car and tsm Genes in Saccharomyces cerevisiae by Trisomic Analysis F. Hilger 1 , M. Pr6vot I , and R. Mortimer 2 1 Chairede Microbiologie, Facultg des Sciences Agronomiques de l'l~tat, 5800 Gembloux, Belgium 2 Department of Biophysics and Medical Physics, Donner Laboratory, University of California, Berkeley, California 94720, USA

Summary. By use of a set of 8 aneuploid strains of the yeast Saccharomyces cerevisiae, carrying from 1 to 5 identified disomic chromosomes, in crosses to a set of haploid strains collectively beating 11 unmapped genes, the following chromosome assignments were obtained for these unmapped genes: arg80 on XIII;arg3 on X;car2 on XII; cpal and tsm8740 on XV; tsm7269 (= rna6) on II; cpa2 on X or XV; arg82 and tsm4572 on III, IV or XVI; carl and arg81 on II, IV, VI, VII or XVI. Linkage tests between the unmapped genes and markers located on the chromosomes that had been designated as possible carriers by the previous analysis allowed 8 genes to be localized. The remaining three genes, cpa2, carl and arg81 (located on fragment F8), could not be positioned on any of the chromosomes indicated by the trisomic analysis, in spite of testing for linkage to markers covering most of the known regions of these chromosomes. Key words: Yeast - Genetic mapping - Trisomic analysis - Arginine

Introduction Many genetic and molecular studies on an organism are dependent upon the availability of detailed genetic maps. The current widespread interest in the yeast Saccharomyces cerevisiae as a simple eucaryotic cell is due, at least in part, to the availability of genetic maps and to the advanced genetic and molecular procedures established for this organism. For example, the recent cloning of centromeres from yeast was dependent on the availability of known markers very close to specific centro-

Offprint requests to: R. Mortimer

meres (Clarke and Carbon 1980; Fitzgerald-Hayes et al. 1981) and numerous studies on regulation, recombination, and gene cloning all are based on knowledge of the location of specific genes relative to each other and to their respective centromeres. While over 300 genes are now mapped in yeast, the current maps are far from complete or detailed. This is because yeast has alarge number of chromosomes and because frequencies of recombination in yeast are very high resulting in relatively large intergenic distances. We have decided that continued efforts at mapping in this organism are worthwhile and have employed a variety of techniques in these mapping studies (for review, see Mortimer and Schild 1981). In this report we describe the use of trisomic analysis to locate several previously unmapped genes. Trisomic analysis as a mapping method for yeast is based on the fact that genes that are present in a duplex condition (+/+/-) on a trisomic chromosome, will segregate 4+ : 0 - , 3+ : 1 - and 2+ : 2 - , whereas genes present in heterozygous condition ( + / - ) on a disomic chromosome segregate mainly 2+: 2 - . A very efficient approach to map a gene by trisomic analysis is to isolate a strain that is disomic for the chromosome that carries the unmapped gene. By crossing this strain to a set ofhaploids that carry markers on all the chromosomes, the chromosomal location of the unmapped gene is revealed by the chromosomal marker that shows trisomic segregation ratios. A general method to isolate a disome of the chromosome carrying an unmapped gene is to construct a diploid heterozygous for complementary or non-complementary alleles at this locus and then to select among the random spores from this diploid a strain that shows either a complementation response or heteroallelic reversion. We attempted to construct disomes of the argl and arg8 chromosomes by such procedures, but were unsuccessful (Hilger and Mortimer 1980). We then tried to isolate these disomes among the meiotic products of a trip0172-8083/82/0006/0093/$1.20

94

F. Hilger et al.: Mapping of Yeast Genes by Trisomic Analysis

Table 1. List of the unmapped mutations and their main characteristics Gene

Phenotype

Gene product or function

Reference

arg3

arginine requirement

ornithine carbamoyl transferase

Lacroute et al. 1965

cpal, cpa2

arginine requirement in the presence of ura2C or excess of uracil

carbamoyl phosphate synthetase

Lacroute et al. 1965

carl

arginine not catabolized

arginase

Wiame 1972

car2

ornithine not catabolized

ornithine transaminase

Wiame 1972

arg80 (argR1), arg81 (argR2), arg82 (argR3)

ornithine not catabolized (non-inducibilityof the car genes; non-repressibility of certain arg genes)

repressor?

B~chet et al. 1970

tsm45 72

thermosensitive growth; cdea

RNA synthesis

Thonart et al. 1976

tsm 7269 (rna6)

thermosensitive growth

tsm8 740

thermosensitive growth; edca

ribosome formation 9

Hartwell et al. 1970b b

a The edc phenotype of tsm4572 and tsm8740 was observed by F. Boutelet (unpubl.) b t8m7269 and tsm8740 were isolated and partially characterized by P. Thonart (unpubl.). tsm7269 was found to be allelic to rna6 (Hartwell et al. 1970)

loid that carried 2 complementary alleles (e.g.: argl-1/ argl-1/argl-2). We obtained several strains that were indeed disomic for the chromosome bearing argl or arg8, b u t all these strains had at least one other disomic chromosome. These results suggested that the apparent deleterious effects of disomy for one chromosome can be partially overcome by disomy for other chromosomes. Another more arduous way to map genes through use of disomes is to construct a set of strains that carry one or several identified disomic chromosomes (Mortimer and Hawthorne 1973). These disomic strains are then crossed to a haploid bearing the unmapped mutation. The segregation ratios observed for the mutation in these crosses (2+ : 2 - , or 4+ : 0 - , 3+: 1 - , and 2+: 2 - ) indicate one or several chromosomes as candidates for the gene to be mapped. Localization is then achieved by linkage tests to markers on these chromosomes. We followed this procedure in an attempt to map 8 genes involved in arginine metabolism plus 3 genes for which temperature-sensitive mutants had been isolated. We used the collection of disomes that had been isolated and characterized during the mapping study, mentioned above, on argl and arg8 (Hilger and Mortimer 1980). This work lead to the localization of 8 genes and to the exclusion of 3 other genes from most of the genomic regions described by the current genetic map (Mortimer and Schild 1980).

Materials and Methods The media, genetic procedures and most of the strains providing genetic markers for the various chromosomes have been described

(Hawthorne and Mortimer 1960, 1968; Mortimer and Hawthorne 1966, 1969, 1973; Hilger and Mortimer 1980). Table 1 presents the list of mutants to be mapped in this study; it also provides information about the isolation and characteristics of these mutants. The construction and analysis of the disomes ~T1, An20 and An24 have been described by Hilger and Mortimer (1980); An26, An30 and An31 were obtained and analyzed by the same procedures (unpubl.). 86EIC was obtained from J. Deschamps; Z14051C is described in Hilger and Mortimer (1980).

Results and Discussion

Trisomic Analysis Haploids carrying the unmapped genes were first crossed to an aneuploid such as An20 which has a high number of disomic chromosomes. When a regular 2+ : 2 - segregation was observed for an unmapped marker, it was concluded that this marker was n o t on one of the duplicated chromosomes in the aneuploid. The m u t a n t was then crossed to another aneuploid bearing another distribution of disomic chromosomes to. eliminate additional chromosomes. This was repeated until the gene had been excluded from all but one or a few chromosomes. When the unmapped marker showed trisomic segregation, i.e. 4 + : 0 - , 3+: 1 - and 2 + : 2 - tetrads, in the first cross, it was concluded that the corresponding gene was on one of the disomic chromosomes, present in the aneuploid. Then, the m u t a n t was crossed to other aneuploids differing from the first by one or two extra-chromosomes in order to narrow the location of the unmapped gene down to the lowest n u m b e r of specific chromosomes.

95

F. Hilger et al.: Mapping of Yeast Genes by Trisomic Analysis Table 2. Trisomic analysis results The genes to be mapped

arg80 arg81 arg82 arg3 carl cax2 cpal cpa2 tsm8740 tsm4572 tsm~269 (= rna6)

The aneuploid mapping strains and their extra-chromosomes ZT1

An20

An24

An26

An30

An31

III, IV* VIII, IX, IX, XV VIII, XI, X, XI, XV XV, XVI*

VIII*, IX, I, V, VIII* II, V, XI, XIII, XII, XIV, XIII* XV* XVII

Di

Tri Di

Tri

Tri Tri Tri

Di Di Di Tri Di Tri Tri Tri Di Di

Di Tri Tri

Di Di Di Tri Di Tri Di

Di

Di

Di Tri

Di

Di

Di

Di

Tri

86EIC XII

Chromosomes assumed to Z140-51C carry the unmapped genes VIII

Tri Di

XlI III, IV, VI, VII, XVI III, IV, XVI X III, IV, VI, VII, XVI XII XV X, XV XV III, IV, XVI II

Di = disomic segregation: + / - = (2+:2-) Tri = trisomic segregation: +/+/ = (4+:0-) (3+:1-) * = unstable disomic chromosome

The results in Table 2 show that in the most favorable situation, just two crosses were necessary to assign an unmapped gene to one chromosome (arg3) whereas in the least favorable case, five crosses were necessary to designate five chromosomes as possible carriers of an unmapped gene (arg81 and carl).

Linkage Studies The next mapping step was to cross the mutants to hap1old strains bearing markers on the chromosomes to which the unmapped gene had been assigned b y the previous analyses, in order to detect linkage. The positive results o f this study are shown in Table 3. Each o f the 6 genes -arg80, arg3, car2, cpal, tsm8740, tsm7269 -- which had been assigned to unique chromosomes have been mapped to specific loci on those chromosomes, arg82 and tsm4572 which each had been assigned to chromosomes III, IV or XVI, are located on IV and XVI, respectively. Three genes - cpa2, carl, arg81 - could not be positioned on one o f the chromosomes indicated as possible carriers o f these genes, cpa2 is excluded from all the estabfished chromosomes, except X and XV (Table 2). Linkage tests covering most of the known regions o f X and XV were negative (markers tested for linkage were: X - cdc6, ura2, arg3, ilv3, cdc8, cdc11; XV -argS, argl, pet1 7, ade2, his3, met7, cpal, prtl), arg81 and carl are excluded from all the chromosomes, except III, IV, VI,

VII, and XVI (Table 2). They are also excluded from most o f the established regions o f these 5 chromosomes b y linkage tests (markers tested for linkage were: III his4, leu2, MAT, thr4; IV - cdc9, trpl, arol, arg82, pet14, ade8 ; VI - his2, cdc14 ; VII - arieS, ly s5, met13, cyh2, trp5, leul, ade6, ade3; XVI - g a l 4 , aro 7, tsm4572. Furthermore, the exclusion o f arg81 from the estabfished chromosomes is substantiated by the fact that SUP5, which is shown to be linked to arg81 in this study (Table 3), has been excluded from 28 chromosome arms b y a mitotic segregation analysis (Mortimer and Hawthorne 1973). cpa2 and carl, as well as the fragment carrying arg81, m a y be considered to be candidates for positions on new chromosomes and, as may be inferred from the trisomic analysis data in Table 2, they would have to be placed on at least two different chromosomes.

Comments on Tetrad Analysis Data arg80 (XIII-R): The analysis o f the crossover patterns in individual tetrads confirms the order: een13-lys7-arg80. arg82 (IV-R): This gene is located b e t w e e n arol and petl4; its position with respect to horn2 andsup35 which are also in this region has not been determined. Based on the relative distances o f arg82, horn2 and sup35 from arol and pet14, arg82 is placed distal to horn2 andsup35.

96

F. Hilger et al.: Mapping of Yeast Genes by Trisomic Analysis

Table 3. Tetrad analysis data for arg80, arg81, arg82, arg3, car2, cpal, tsm8740, t s m 4 5 72, tsm 7269 Interval

Chromosome or Fragment

Segregation

Ascustype

FDS

SDS

13 15

18 16

PD

NPD

T

Map Distance cM

arg80 lys7 arg80

-

cenl3 (trpl) cenl3 (trpl) lys7

XIII

27

0

4

35.8 30.7 6.5

arg81

-

SUP5

F8

24

0

27

26.5

arg82 arg82 arg82 arol pet14

-

arol pet14 SUP2 pet14 SUP2

IV

43 47 18 19 24

0 0 7 0 2

16 42 68 42 37

13.6 23.6 71.2 34.4 38.9

arg3 arg3 arg3 ura2 ura2 cdc6

-

SUP7 ura2 cdc6 SUP7 cdc6 SUP7

X

24 49 29 7 27 6

0 0 8 2 0 4

37 48 82 44 38 33

30.3 24.7 62.2 59.2 29.2

car2 car2 ura4

-

ura4 gal2 gal2

XII

73 28 8

4 23 10

51 77 30

29.3

cpal cpal cpal

-

his3 met7 prtl

XV

10 19 4

2 0 7

34 21 16

55.0 26.2

tsrn8740 tsm8740ade2 -

acle2 cdc21 cdc21

XV

38 31 35

0 1 2

13 19 24

12.7 24.5 29.5

tsm4572 tsm4572aro7 -

aro7 rnak3 mak3

XVI

35 26 35

0 1 0

36 26 4

25.3 30.2 5.1

tsm7269 tsm7269gall -

gall lys2 lys2

II

8 32 18

0 0 4

23 42 43

37.1 28.4 57.2

PD = parental ditypes; NPD = non-parental ditypes; T = tetratypes; cM = centimorgan, map distance unit. For the interpretation of the data, see: Materials and Methods. Map distances determined by using the plots established by Mortimer and Schild (1980; Fig. 2 and 3), taking k = 0.3 for the x' v.s. SDS relation

a r g 3 (X-L): u r a 2 was localized on the left arm of chro-

c a r 2 (XII-R): This gene shows linkage to u r a 4 , which had

mosome X, distal to S U P 7 , on the basis of mitotic segregation data (Mortimer and Hawthorne 1973). Using super-triploid and mitotic mapping procedures, Wickner (1979) confirmed this localization of u r a 2 and showed that m a k l 7, although unlinked to u r a 2 , is distal to this marker. Kawasaki (1979) found c d c 6 to be linked to u r a 2 , but the orientation of the c d c 6 - u r a 2 segment with respect to c e n l O remained undetermined. The meiotic segregations observed in this study for a r g 3 , u r a 2 , c d c 6 a n d S U P 7 with respect to each other show that a r g 3 prorides the link between u r a 2 - c d c 6 a n d c e n l O v i a S U P 7 ; they also allow an unambiguous positioning of these genes. T h e order is c e n l O - S U P 7 - a r g 3 - u r a 2 - c d c 6 .

been placed on XII distal t o g a l 2 by trisomic and mitotic crossing-over analyses (Mortimer and Hawthorne 1973). Petes (1979) showed t h a t R D N 1 (the structural gene for rRNAs) is linked to g a l 2 , but not to u r a 4 , and also that R D N 1 is localized distal to g a l 2 . As c a r 2 does n o t show linkage to g a l 2 and as its relation to R D N 1 has n o t been investigated, the orientation of the u r a 4 - c a r 2 segment remains undetermined. This also applies to S U P 2 6 , which is tightly linked to u r a 4 (Ono et al. 1979b), b u t whose position with respect to c a r 2 has n o t been determined. (XV-R): The tetrad data clearly indicate that this gene lies distal to m e t 7 . c p a l does n o t show linkage to

cpal

97

F. Hilger et al.: Mapping of Yeast Genes by Trisomic Analysis IV

X

XII

V

V

XIII

XVI

F8

/

aro gal

1

L cdc

/

6

gal

2

()

cdc

21

his

3

1 arg

mak

3

aro

7

82

t._ssm s~74o lys pet

SUP 5

14

i

arg

7 80

a rg 81 met

7

prt

1

t sm

4572

ade 2

t s m 7269

arg

RDN

1

ura

4

3

lys2 SUP 2 SUP 7

I

car

2

11° cM I

Fig. 1. Map location of arg80, arg81, arg82, arg3, car2, cpal, tsm8740, tsm4572, and tsm7269 (= rna6). ( ) sequence not established with genes outside parentheses. - - - mitotic linkage

prtl, which thus remains at its position at the fight end of chromosome XV (Mortimer and Hawthorne 1973).

the Office of Health and EnvironmentalReseaxch of the U.S. Department of Energy under contract No. DE-ACO3-76SF00098.

tsm8740 (XV-R): This gene may be placedunambiguously between cdc21 and ade2. tsm4572 (XVI-R): This gene does not show centromerelinkage and the analysis of the crossing-over patterns in individual tetrads indicates the sequence mak3-aro7tsm4572. This assigns tsm4572 to the tight arm of XVI, distal to aro 7. We did n o t determine the relationship of tsm4572 to SUP16 or to tad56 which are also on XVI-R; it should be placed between these 2 genes on the basis of the distances determined in this study and those found for aro7-SUP16 (Ono et al. 1979a) and for aro7-rad56 (Game and Mortimer 1974).

tsm7269 (II-R): The segregation data allow this gene to be placed between gall and lys2, close to tsm134 and pho3,5; its position with respect to these latter markers has n o t been determined. The locations of these markers on the genetic map of Saccharomyces cerevisiae are summarized in Fig. 1.

Acknowledgment. This work was supported by a grant from the "Fonds National de la Recherche Scientifique", Belgium, and by

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98 Mortimer RK, Schild D (1981) Genetic mapping in Saccharomyces cerevisiae. In: Molecular Biology of the Yeast Saccharomyces. Cold Spring Harbor Laboratory Publications, pp 11-26 Ono B, Stewart JW, Sherman F (1979a) J Mol Biol 128:81-100 Ono B, Stewart JW, Sherman F (1979b)J Mol Biol 132:507-520 Petes JD (1979) J Bacteriol 138:185-192 Thonart P, B6chet J, Hilger F, Burny A (1976) J Bacteriol 125: 25-32

F. Hilger et al.: Mapping of Yeast Genes by Trisomic Analysis Wiame JM (1972) In: Horecker BL, Stadtman ER (eds) Current Topics in Cellular Regulation, vol IV. Academic Press, New York, pp 1-38 Wickner RB (1979) Genetics 92:803-821

Communicated by M. S. Esposito Received June 24, 1982