Curr Genet (1988) 14:419-423 © Springer-Verlag 1988

Mapping of the RIB5 gene in Saccharomyces cerevisiae using UV light as an enhancer of rad52-mediated chromosome loss Maria de los Angeles Santos, Enrique A. Iturriaga, and Arturo P. Eslava Departamento de Microbiologla,Gen6tica, MedicinaPreventivay Salud Ptlblica, Universidadde Salamanca, E-37008 Salamanca, Spain

Summary. Rib5 mutants of S. cerevisiae are blocked at the end of the riboflavin biosynthetic pathway. Using UV light to increase rad52-mediated chromosome loss, we have assigned the rib5 mutation to chromosome II. Tetrad analysis of crosses between rib5 and other markers on chromosome II shows that the RIB5 gene is located on the right arm of this chromosome, closely linked to HIS7. Key words: S. cerevisiae - r a d 5 2 - c h r o m o s o m e UV light - Riboflavin

loss -

Introduction Riboflavin auxotrophic mutants have been isolated in only a few microorganisms. E. coli (Bacher et al. 1982), B. subtilis (Bacher et al. 1980), and yeast (Oltmanns and Bacher 1972) have been shown to have essentially similar riboflavin biosynthetic pathways. Riboflavindeficient mutants of Aspergillus nidulans have been genetically analyzed (Pontecorvo and co-workers, see K~ifer 1965) and four complementation groups (ribAribD) have been established among riboflavin-requiring mutants of Phycomyces blakesIeeanus (Orejas et al. 1987; Eslava 1987). Recently, the riboB locus of Aspergillus nidulans has been cloned (Oakley et al. 1987). Riboflavin auxotrophic mutants of S. cerevisiae were first isolated by Oltmanns and Lingens in 1967, and subsequent complementation analysis showed

Offprint requests to: A. P. Eslava

that six genes (ribl to rib5, and rib7) were involved in riboflavin biosynthesis (Oltmanns et al. 1969). The location of these genes on the genetic map is still unknown. Using growth and accumulation tests on all six complementation groups, it was possible to establish the pathway of riboflavin biosynthesis (Oltmanns et al. 1969; Oltmanns and Bacher 1972). The RIB5 gene of S. cerevisiae encodes riboflavin synthase: this enzyme consists of two or more subunits and is responsible for the last step in riboflavin biosynthesis (Plaut et al. 1974). Gene mapping is a more complex process in S. cerevisiae than in the majority of organisms that are subjected to genetic analysis owing to the high level of meiotic recombination and the large number of chromosomes found in this organism (O'Brien 1984). Classical linkage analysis is thus inadequate for assigning mutations to specific chromosomes, and different methods based on chromosome loss have consequently been developed to overcome this problem (Liras et al. 1978; Kawasaki 1979; Wood 1982; Klapholz and Esposito 1982). A method developed recently (Schild and Mortimer 1985; Hanic-Joyce 1985) makes use of the finding that chromosome loss in diploids homozygous for the rad52-1 mutation is spontaneous ,and that the frequency of loss can be increased by exposure to ionizing radiation (Mortimer et al. 1981; HanicJoyce 1985) or chemicals such as methyl methane sulfonate (MMS) (Schild and Mortimer 1985). In the present work, we demonstrate that UV light may also be used to increase chromosome loss and hence to map unknown mutations. Using rad52/rad52 diploids heterozygous for the unknown marker and known recessive chromosome mutations, we have increased the frequency of chromosome loss by exposure to UV light and have accordingly assigned the rib5 mutation to chromosome II.

420

Table 1. Yeast strains Strain

Genotype

Source

HK750 F575 F576 F577 F578 F579

Mate, rib5 Mata, tad52-1, adel (I), ura3 (V), his2 (VI), leul (VII), arg4 (VIII), aro7 (XVI) Mate, red52.1, leu2 (III) Mata, red52-1, ura3 (V) Mate, red52-1, leu2 (III), trpl (IV), metlO (VI), ura4 (XIII), his3 (XV), ade4 (XIII) Mata, red52-1, his7 (II), leu2 (III), lysl (IX), met5 (X) d, ade2 (XV), argl (XV), ade4 (XIII), ilv3 (X) Mate, rad52-1, leu2 (III), trp5 (VII), arg4 (VIII), his6 (IX), ilv3 (X), ural (XI), lys9 (XIV), met2 (XIV), ade2 (XV) Mate, gall (II), lys2 (II), tyrl (II), his7 (II), adel (I), ade2 (XV), ura (XI) Mate, tad52-1, rib5 Mate, red52-1, rib5 Mate, red52-2, leu2, trp5, arg4, his6, ilv3, ural, lys9, met2, ade2 Mate, tad52-1, rib5 Mata, red52-2, his7, leu2, lysl, met5, ade2, argl, ade4, ilv3

A. Bacher a F. del Rey b F. del Rey b F. del Rey b F. del Rey b F. del Rey b

F580 A364A AI2-4A AH O1 AI102

F. del Rey b F. del Rey b HK750 x F577 c

AI2-4A x F580c AI2-4A x F579

a Lehrstuhl ftir Organische Chemie und Biochemie, Technische Universit~it, Miinchen; FRG b Departamento de Microbiologia, Gen6tica, Medicina Preventiva y Salud Ptiblica, Facultad de Biologla, Universidad de Salamanca, E-37008 Salamanca, Spain c This work d Previously designated met6 (V). Chromosome loss method showed that the met6 mutation maps on chromosome X (see Results)

Materials and m e t h o d s

Strains. The genotypes and sources of yeast strains are shown in Table 1. Media. Yeast extract, peptone, and dextrose (YEPD) medium (Sherman et al. 1979) was supplemented when necessary with 10-20 mg/l of riboflavin. The minimal medium (A) consisted of: dextrose, 10 g; (NH4)2SO4, 1 g; KH2PO 4, 0.875 g;K2HPO4, 0.125 g; MgSO4+7H20, 0.5 g; NaC1, 0.1 g; CaCI2, 0.1 g; H3BO2, 0.5 mg; CuSO4 + 5H20, 0.04 mg; KI, 0.1 mg; FeC13 + 6H20 , 0.2 mg; MnSO4 + 4H20, 0.4 nag; ZnSO4 + 7H20, 0.4 mg; (NH4)2MoO 4, 0.2 mg; biotin, 0.l mg; deionized water, 11; agar (Dife0), 20 g if necessary (Oltmanns et al. 1969). The synthetic complete medium (AC) consisted of minimal medium plus the following nutritional requirements: S-adenine, 20 mg/ 1; uracil, 20 mg/1; L-tryptophan, 20 rag/l; L-histidine, 20 rag/l; L-arginine, 20 mg/1; L-methionine, 20 mg/1; L-tyrosine, 30 mg/ 1; L-leucine, 30 rag/l; L-lysine, 30 mg/1; L-isoleucine, 30 rag/l; L-phenylalanine, 50 mg/1; L-valine, 150 rag/l; riboflavin, 30 mg/1. Synthetic complete medium withotR a specific requirement (omission media, e.g., AC-leu) was used to determine the genotypes. Genetic methods. General yeast genetic methods such as mating, isolation of diploids, sporulation, asci dissection, genetic complementation, and the testing of genetic markers were according to Sherman et al. (1979). Mapping protocol. In this work, the rad52-induced chromosome loss procedure (Schild and Mortimer 1985; Hanic-Joyce 1985) was slightly modified: use was made of UV light instead of Xrays, -/-rays, or methyl methanesulfonate (MMS). Chromosome loss in diploid strains homozygous for the red52-1 mutation and heterozygous for rib5 and known chromosomal markers

was induced by exposing the cells to UV light. Following irradiation, the cells were incubated in YEPD medium at 30 °C and left in darkness for 7 days. Colonies were then patched onto YEPD master plates. Once the patches had grown, the master plates were replica-plated to A and AC media. Clones growing on AC medium and not on A medium were used to screen the expression of recessive markers in the omission media. A Chi-squared test revealed that 20%-30% of the initially patched colonies (see Results, Table 2) were sufficient for assessing the independent expression of rib5 mutation and the chromosomal markers.

Ultraviolet irradiation. A General Electric 15 W germicidal lamp was used as the UV source; doses were measured with a 254 nm UV Light Dosimeter (Spectronics, Westbury, New York). Exponentially growing rad52/rad52 diploid ceils were plated onto YEPD medium and immediately irradiated with 100 J/ m 2. A I.IV dose of 120 J/m 2 was used to distinguish between red52 and RAD52 meiotic segregation products.

Results UV light-increased chromosome loss in rad52/rad52 diploids The diploid strain AHO1, which is h o m o z y g o u s for red52-1 and h e t e r o z y g o u s for c h r o m o s o m a l markers, was irradiated with different doses o f U V light. Table 2 shows the increase in the expression o f recessive m u t a t i o n s w i t h increasing doses o f UV-irradiation: a m a x i m u m increase was f o u n d at 100 J / m 2 (0.281 events per colony). Doses o f 120 J / m 2 p r o d u c e d low

421 Table 2. Expression of heterozygous recessive markers in diploid AI101 Dose (J/m 2)

0

50 100

Survival a Total no. (%) of colonies examined

100 45 5

444 370 370

Number of colonies expressing recessive chromosomal markersb III

VII

VIII

IX

X

XI

XIV

XV

9.

leu2

trp5

arg4

his6

ilv3

ural

lys9met2

ade2

rib5

Total events

Events per colony

0.033 0.116 0.281

3

0

3

3

4

0

1

0

1

15

8 15

1 3

7 22

13 13

9 13

3 4

1 10

1 13

0 11

43 104

a Survivalbased on non-irradiated controls b Two colonies expressed only the met2 mutation. All leu2 colonies were also Mata, although three colonies were leu2-Mata. Lysine auxotrophs were always methionine-requiring. Two met2, but no lys9 colonies were found

Table 3. Coexpression of rib5 and chromosome markers in repulsion Chromosome

RIB5

rib5

Expected no. of colonies with coordinate expression of recessive markersa

HIS7 his7

178 69

73 0

14.7

< 0.01

III

LEU2 leu2

208 39

68 5

9.1

< 0.70

IX

LYS1 lysl

200 47

65 8

11.9

< 0.75

X

MET5-IL V3 met5C-ilv3

205 42

65 8

10.5

< 0.95

XIII

ADE4 ade4

193 54

67 7

12.6

< 0.30

XV

ADE2-ARG1 ade2-argl

210 37

57 16

11.2

< 0.70

II

Second marker

No. of colonies expressing pairs of markers

pb

a Expected number of colonies expressing rib5 and the second recessive marker if both markers were on different chromosomes b p values are calculated as Chi-squared test c Sehild and Mortimer (1985) found that the previously designated met6 (V) was in fact met5, which maps on chromosome X. Our results are in agreement with this findings since met5 always co-expresses with ilv3 (X)

viability (<1%) and were not tested. Table 2 also shows that exposure to UV light increased the expression of markers according to chromosome loss. The coordinate expression of met2 and lys9 in chromosome XIV, which is similar to that of leu2 and MATa in chromosome III, indicates ongoing chromosome loss events. There were only very few colonies in which one of the two markers on a chromosome was expressed while the other was not, and these can be clarified by the loss of chromosome fragments (Schild and Mortimer 1985; HanicJoyce 1985). The alternative, mitotic recombination, is unlikely since in rad52/rad52 diploids, the recombination mechanisms are disturbed (Game et al. 1980).

Assigning RIB5 to chromosome II

Analysis of 320 clones resulting from UV irradiation of diploid strain A I 1 0 2 (Table 1) showed that approximately the expected number of colonies coordinately expressed rib5-leu2, rib5-lysl, rib5-met5-ilv3, rib5ade4, and rib5-ade2-argl. No colonies were found which expressed ribS-his7, although 14 had been expected. Chi-squared analysis showed that the number of ribS-his7 coexpressing colonies differs significantly from what was expected (P < 1%), indicating that rib5 is located on chromosome IL

422 Table 4. Tetrad analysis data Chromosome arm

rib5-lys2 rib5-tyrl rib5-his7 his7-tyrl

II R

a b

Interval

Ascus type a

Map distance (cM)

PD

NPD

T

10 17 72 18

20 1 0 0

48 60 6 60

Unlinked 44.72 3.73 40.08

Published distance (cM)

42.5 b

PD, parental ditype segregation; NPD, nonparental ditype; T, tetratype Mortimer and Hawthorne (1966)

~-cyhl0

-lys2

-tyrl

-his7

~rib5

-MGL2

Fig. 1. Location of the RIB5 gene on the right arm of chromosome II

According to the chromosome-loss method, met6 in F579 was seen to map on chromosome X (see Table 3) and proven to be met5, a result in agreement with the findings of Schild and Mortimer (1985).

Tetrad analysis In order to elucidate the location of the rib5 marker on chromosome II, strains HK750 and A364 were crossed. Only the rib5, lys2, tyrl, and his7 markers were analyzed. Data were obtained from tetrads with four viable spores showing a 2:2 segregation for the four

markers. Analysis of 78 tetrads (Table 4) showed that rib5 is linked with his7 and tyrl, but is not linked with lys2. From the map distances, calculated according to Ma and Mortimer (1983), we conclude that rib5 is located 3.7 cM to the right of his7 (Fig. 1).

Discussion Diploids homozygous for the rad52-1 mutation have previously been shown to lose chromosomes mitotically. This spontaneous event can be increased by exposure to X-rays (Mortimer et al. 1981), methyl methanesulfonate treatment (Schild and Mortimer 1985), or 7-ray irradiation (Hanic-Joyce 1985). Data reported here show that UV irradiation also increases loss events (see Table 2) and can be successfully used to assign a mutation to one specific chromosome. The induction of chromosomal loss by UV irradiation has the advantages over the above-mentioned procedures in that UV light is available in most laboratories, does not require any special installation, and is easy to handle. The rad52-1 mutation was originally identified as being responsible for causing extreme sensitivity to Xrays while only slightly increasing the sensitivity to UV light (Resnick 1969). The RAD52 gene in S. cerevisiae has been shown to be involved in both recombination and DNA repair. In fact, physical linkage relationships are not altered by recombinational mechanisms in rad52/rad52 diploids (Ho 1975; Resnick and Martin 1976; Strike 1978; Game et al. 1980; Prakash et al. 1980; Malone and Esposito 1980; Saeki et al. 1981). The major source of UV-induced lethality in yeast are pyrimidine dimers (Cox and Game 1974). Among the three types of dark repair mechanisms of UVinduced pyrimidine dimers described in yeast, one occurs via recombinational events (Game 1983). If one assumes that pyrimidine dimers are partially repaired by a recombination mechanism and that the rad52-1 mutation blocks recombination (Game et al. 1980; Prakash et al. 1980; Saeki et al. 1980), an increase in

423 chromosome loss events following UV irradiation is quite plausible. The riboflavin biosynthetic pathway in S. eerevisiae has been elucidated by investigation of the accumulation products detected in single- and double riboflavindeficient mutants (Oltmanns et al. 1969; Oltmanns and Bacher 1972): six genes (rib1 to rib5, and rib7) are involved in this pathway. Tetrad analysis shows that genes RIB1, RIB7, and RIB2 are n o t linked to each other, although the position of these genes on the genetic map remains to be .elucidated. In the present work, we have located RIB5, which codes for the last enzyme in the pathway - the riboflavin synthetase (Baur et al. 1972) on chromosome II, 3.7 cM from the marker HIS7 (see Fig. 1). Experiments are currently in progress to map the rest of the genes involved in the biosynthesis of riboflavin in yeast. Preliminary results indicate that rib1 maps on chromosome II, rib3 on chromosome IV, and rib4 on chromosome XV. The isolation of the genes involved in the biosynthesis of riboflavin and the study of their regulation would presumably throw more light on the molecular framework of the pathway and would thus be of use in advancing the industrial production of this vitamin.

Acknowledgements. We are indebted to Prof. A. Bacher (Technische Universit~it MUnchen) for providing the riboflavin mutant strains and Prof. J. L. Revuelta and F. del Rey (Universidad de Salamanca) for their helpful suggestions. We also thank Fernando D~ez for technical assistance and Nick Skinner for helping in the writing. This work was supported by grants from the Comisi6n Asesora de Investigaci6n Cientlflca y T6cnica, grant No. PB86-0209, and Fondo de Investigaciones Sanitarias, grant No. 87/1268. E. A. I. held a graduate student fellowship from the Plan de Formaci6n de Personal Investigador of the Ministerio de Educaci6n y Ciencia.

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Communicated by B. S. Cox Received June 15, 1988