Curr Genet (1991) 20:269-276 Current Genetics �9 Springer-Verlag 1991

Polymeric genes MEL8, MEL9 and MELIO- new members of -galactosidase gene family in Saccharomyces cerevisiae Gennadi Naumov i, Elena Naumova 1, Hilkka Turakainen 2., Pirkko Suominen 3, and Matti Korhola 3

1 All-Union Scientific Research Institute for Genetics and Selection of Industrial Microorganisms, 1 Dorozhnyi 1, Moscow 113545, USSR 2 Department of Genetics, University of Helsinki, Arkadiankatu 7, SF-00100 Helsinki, Finland 3 Research Laboratories of the Finnish State Alcohol Company (Alko Ltd), P.O. Box 350, SF-00101 Helsinki, Finland

Received May 22, 1991

Summary. We used a combinat ion of genetic hybridiza- tion analysis and electrokaryotyping with radioactively labelled M E L t gene probe hybridization to isolate and identify seven polymeric genes for the fermentat ion of melibiose in strain CBS 5378 of Saccharomyces cerevisiae (syn. norbensis). Four of the M E L genes, i.e. MEL3, MEL4, M E L 6 and MEL7, were allelic to those found in S. cerevisiae strain CBS 4411 (syn. S. oleaginosus) whereas three genes, i.e. MEL8, M E L 9 and MELIO oc- cupied new loci. Electrokaryotyping showed that all seven M E L genes in CBS 5378 were located on different chromosomes. The new MEL8, M E L 9 and MELIO genes were found on chromosomes XV, X/XIV and XII , respec- tively.

Key words: Saccharomyces cerevisiae - Melibiose fermen- tation - M E L - Polymeric genes

Introduction

Genetic studies using Saccharomyces cerevisiae currently deal only with a par t of the natural gene pool of this organism, as lines of the same origin are used in most laboratories throughout the world (Mort imer and John- ston 1986). Thus the natural genetic polymorphism of the yeast Saccharomyces is beyond the scope of most geneti- cists. Limited comparat ive genetic studies of yeast strains of different origins have revealed many gene families: polymeric SUC genes (Winge and Roberts 1958; N a u m o v 1972; Carlson et al. 1985), STA genes (Tamaki 1978; Pre- torius and Marmur 1988), complex polymeric M A L loci (Winge and Roberts 1958; N a u m o v 1977; Charron et al. 1989; Chow etal . 1989), complex systems of M G L genes (Hawthorne 1958; N a u m o v and Bashkirova 1985) and

* Present address." Research Laboratories of the Finnish State Al- cohol Company (Alko Ltd), P.O. Box 350, SF-00101 Helsinki, Fin- land

Offprint requests to." M. Korhola

transposable M A T (HML, H M R ) genes (Naumov and Tolstorukov 1974; Harash ima etal . 1974; Herskowitz 1988).

The present work is a continuation of our previous studies on the natural genetic polymorphism of melibiose fermentation (Liljestr6m 1985; Ruohola etal . 1986; Tubb etal . 1986a, b; Suominen 1988; N a u m o v 1989; Turakainen et al. 1988, 1991). We have previously identi- fied seven polymeric M E L genes encoding ~-galactosi- dase and responsible for melibiose fermentation in S. cerevisiae, and found five M E L genes in one strain, CBS 4411 (Naumov etal . 1990).

In this paper we report on three new M E L genes in the S. cerevisiae strain CBS 5378.

Materials and methods

Strains and culture conditions. The strains of S. cerevisiae used in the study are listed in Table 1. In addition to the Me1 + strains studied earlier (Naumov et al. 1990), we also used another strain, 5378-F2- 1D, originating from several successive monosporic breedings of the strain CBS 5378. Using this highly fertile strain, we have previously identified the homo-heterothallism genes HO, HMR and HML (Naumov and Tolstorukov 1974). Standard genetic techniques were used for the crosses, the isolation of spores and the testing for fermentation of melibiose and other sugars (Naumov et al. 1986, 1990; Naumov and Gudkova 1979). Mel + strains usually fermented melibiose (Serva Feinbiochemica, Heidelberg, FRG) after 1 day, but some strains took 2-7 days. The latter strains will be described in "Results". Mel- strains did not ferment melibiose during a test period of 10 days.

Isolation ofgenomic DNA. Total DNA from the yeast strains was isolated by the method described by Sherman et al. (1981), digested with EcoRI endonuclease (Boehringer, Mannheim, FRG) and sep- arated by electrophoresis in agarose gels.

Preparation of chromosomal DNA. Yeast cells were grown overnight, harvested, and washed with 50 mM EDTA, pH 7.5. The final pellet was suspended in 0.6 ml of an aqueous solution contain- ing citric acid (40raM), Na~PO 4 (120raM), pH6.0, EDTA (0.02 M), sorbitol (1.2 M) and DTT (5 raM). To this suspension was added 1 mI of 1% low melting point agarose with 4 mg of Novozym 234 (Novo Industri A/S, Copenhagen, Denmark) prepared in an

270

Table 1. Strains of Saccharomyces cerevisiae used

Strain Genotype Source

$288C MATe SUC2 mal mel gal2 CUP1 R.K. Mortimer (Berkeley, USA) X2180-1A MATa SUC2 mal reel gal2 CUP1 R.K. Mortimer YNN 295 MATe ura3 lys2 adel ade2 his trpI-A1 R.K. Mortimer N2 MATe SUC M A L MEL1 GAL E.A. Bevan (London, UK) VKM Y-1830 MATe(a) SUC M A L MEL2 GAL V.I. Kudryavtsev (Moscow, USSR) VKM Y-503 HO SUC real mel GAL V.I. Kudryavtsev (Moscow, USSR) CBS 4411 (VKPM Y-61) HO suc real MEL3 MEL4 MEL5 MEL6 MEL7 GAL N. van Uden (Portugal) CBS 5378 HO H M L e H M R e suc real M E L gal4 J. Santa Maria (Madrid, Spain)

(VKM Y-1232 = SBY 2314) OL0-11C MATe SUC2 MEL4 GAL Naumov et al. (1990) OL/-9D MATa SUC2 MEL5 GAL2 Naumov et al. (1990) OL2-4B MATa SUC2 MEL3 gal2 Naumov et al. (1990) OL2-5B MATe SUC2 MEL3 gal2 Naumov et al. (1990) OL2-7C MATa suc MEL3 gal2 Naumov et al. (1990) OL3-9B MATe SUC2 MEL4 gal2 Naumov et al. (1990) OL3-10D MATa SUC2 MEL4 gal2 Naumov et al. (1990) OL4-3A MATa suc MEL6 gal2 Naumov et al. (1990) OL8-9C MATe SUC2 MEL3 gal2 Naumov et al. (1990) OL8=9D MATe SUC2 MEL7 GAL Naumov et al. (1990) OLlI-11A MATa SUC2 MEL4 gal2 Naumov et al. (1990) OLl l - l lB MATe SUC2 MEL6 gal2 Naumov et al. (1990) OL12-1B MATe suc MEL6 gal2 Naumov et al. (1990) OLI3-7A MATa SUC2 MEL6 gal2 Naumov et al. (1990) OL13-7D MATe suc M E L 7 gal2 Naumov et al. (1990) OL14-9D MATe SUC2 MEL5 gal2 Naumov et al. (1990) ML2-3D MATe mal MEL2 gal2 Present study ML2-8C MATe M A L MEL2 GAL2 Present study

CBS, Centraalbureau voor Schimmelcultures Delft, Holland; VKM, All-Union Collection of Microorganisms, Moscow, USSR; VKPM, All-Union Collection of Industrial Microorganisms, Moscow, USSR; SBY, Seccion de Bioquimica, Instituto Nacional de Investigaciones Agrarias, Madrid, Spain

aqueous solution (CPE) containing citric acid (40 raM), Na2PO 4 (120 raM), pH 6.0 and EDTA (0.02 M) and cooled to 42~ The mixture was allowed to gel at 4 ~ overlaid with 1 ml of CPE and incubated at 30 ~ for 1 h. The overlay was then replaced with 1 ml of an aqueous solution containing EDTA (0.45 M), TRIS-HC1 (0.01 M, pHS.5), N-lauroyl sarcosine (1%) and proteinase K (Sigma Chemical Company, St. Louis, Mo., USA) (1 mg/ml), and the tubes were incubated at 37~ for 3 h. The overlay was then replaced with 0.5 M EDTA, pH 9, and the tubes stored at 4~ Agarose slices were washed in 0.05 M EDTA, pH 8.0 prior to frac- tionation of chromosomes by contour-clamped homogeneous elec- tric field (CHEF) gel electrophoresis.

CHEF gel electrophoresis. A CHEF-DRtmII apparatus (Bio-Rad Laboratories, Richmond, Calif., USA) was used to separate the chromosomal DNAs. Electrophoresis was carried out at 200 V and 14~ for 15 h with a switching time of 60 s and then for 8 h with a switching time of 90 s. A standard set of S. eerevisiae YNN 295 chromosomes was obtained commercially (Bio-Rad).

Southern blot analysis. Southern blot analysis of yeast DNA restric- tion fragments was carried out mainly according to Maniatis et al. (1982). After soaking the gels in 0.25 M HC1 for 30 min chromoso- mal DNA separated by CHEF was denatured, neutralized and transferred to nitrocellulose filters which were then baked at 80 ~ for 2 h. The MEL1 probe (2.8 kb BamHI-SalI fragment inserted in pGEM3; Ruohola et al. 1986) used for hybridization was prepared by in vitro transcription (Melton etal. 1984) and labelled with (e-32P)UTP (Amersham International plc, Amersham, UK). Hy- bridization was carried out in 50% formamide, 5 x SSC, 0.05 M sodium phosphate, pH 7.0, 0.05% BSA, 0.05% Ficoll, 0.05% PVP and 200 gg/ml denatured salmon sperm DNA (SSC is 0.15 M NaC1, 0.015 M trisodium citrate, pH 7.0) at 42~ overnight, after which the filters were washed twice for 30 min at 42~ with 2 x SSC con-

taining 0.1% SDS, and for 15 rain with 0.1 x SSC containing 0.1% SDS. Autoradiography X-ray film was exposed for 1-3 days. The TRP1 gene probe was a 0.6 kb XbaI-BglII fragment isolated from the plasmid pEMBLY r25 (Baldani and Cesareni 1985), containing most of the coding region plus 75 nucleotides of downstream se- quence. The ADC1 gene probe was a 1.5 kb HindIII-BamHI frag- ment isolated from pAAH5 (Ammeter 1983), containing 750 bp of promoter sequence plus 760 bp of coding sequence. The probe for the identification of chromosome XII was a 50-mer oligonucleotide complementary to the nontranscribed spacer of ribosomal DNA (Skryabin et al. 1984):

5'-TTTTTATTTCTTTCTAAGTGGGTACTGGCAGGAGC- CGGGGCCTAGTTTAG-3 r. The TRP1 and ADC1 probes and the oligonucleotide probe were labelled with digoxigenin-ll-dUTP us- ing the Nonradioactive DNA Labeling Kit (Boehringer, Mannheim, FRG). Hybridization was performed in 5 x SSC con- taining 0.1% N-lauroylsarcosine, 0.02% SDS and 1% blocking reagent at 68 ~ overnight, after which the filters were washed twice with 2 x SSC containing 0.1% SDS at room temperature for 5 min and with 0.1 x SSC containing 0.1% SDS at 68~ for 15 min. De- tection of hybridization was done with the Nonradioactive Kit. The filters were incubated in colour solution in the dark overnight.

Results

Analys i s o f F1

Tetrad analysis of hybr id (NR0) of the monospor i c highly fertile strain 5378-F2-1D (Mel § and a M e l - tester strain, X2180-1A, showed an a lmost absolute pre- dominance of Mel § segregants. A m o n g 69 tetrads only

271

Table 2. Identification of the genes MEL3, MEL4, MEL6 MELIO in Saccharomyces cerevisiae CBS 5378

Table 2 (continued)

Hy- Origin of Mel § No. of tetrads Genotypes brid hybrids segregating as

Mel § : Mel

Hy- Origin of Mel § No. of tetrads Genotypes brid hybrids segregating as

Mel § : Mel

4:0 2:0 3:1 4:0 2:0 3:1

NR0 5378 x X2180-1A NR1 NR0-4A x X2180-1A NR2 NR0-4B x $288C NR3 NR0-4C x $288C NR4 NR0-4D x X2180-lA NR5* NR2-3A x X2180-1A NR6 NR2-3B x X2t80-1A NR7 NR2-3C x X2180-1A NRS* NR2-3D x $288C NR9 NR4-4A x X2180-1A NR10 NR4-4B x $228C NR11 NR3-2A x X2180-1A NR12 NR3-2B x $288C NR13 NR3-2C x X2180-1A NR14 NR3-2D x $288C NR15 NR4-4C x $288C NR16 * NR4-4D x X2180-1A NR17 NR2-3B x OLI3-7A NR18 NR2-3B xOL2-4B NR19 NR3-2D xOL2-5B NR20 NR3-2D x OL12-1B NR21 NR4-4D x OL3-10D NR22 * NR4-4C x OL13-7D NR23 * NR4-4C x NR4-4D NR24 * NR2-3D x N2 NR25 * NR2-3D x ML2-8C NR26 * NR2-3D x OL2-5B NR27 * NR2-3D x OL3-9B NR28 * NR2-3D • OLI4-9D NR29 * NR2-3D x OLI1-11B NR30 * NR2-3B x NR2-3D NR31 NR2-3D x OL8-9D NR32 * NR5-10A x X2180-1A NR33 * NR5-10B x X2180-1A NR34 NR5-10C x $288C NR35 NR5-10D xS288C NR36 NR7-6B xX2180-1A NR37 NR7-6C x $288C NR38 NR7-6D x $288C NR39 NRl l -8A x $288C NR40 NRll-8BxX2180-1A NR41 NRl l -8Cx $288C NR42 NRI1-8DxX2180-1A NR43 NR12-7A• NR44 NR12-7C x $288C NR45 NR12-7D x $288C NR46 NR10-1Ax $288C NR47 NRI0-tB xX2180-1A NR48 NR10qC x X2180-tA NR49 NRI0-1D • $288C NR50 NR7-6C x N2 NR51 NR7-6C x ML2-3D NR52 NR7-6C x OL8-9C NR53 NR7-6Cx OL0-llC NR54 NR7-6C x OLI4-9D NR55 NR7-6C xOLI2-1B NR56 NR7-6C x OL13-7D NR57 NR7-6B xNR2-3D NR58 NR12-7CxN2 NR59 NR12-7C x ML2-8C NR60 NRI2-7C x OL2-5B NR61 NR12-7C x OL0-11C

66 0 3 (MEL)v/mel 24 0 8 (MEL)4/mel 15 1 6 (MEL)jmel 18 0 6 (MEL)4/meI 16 0 14 (MEL)a/mel 1 7 16 (MEL)2/mel

67Mel + :71 Mel MEL6/mel 2 6 7 (MEL)z/mel 0 24 0 MEL8/mel

63 Mel + : 18 Mel (MEL)2/mel 93 Mel + :41 Mel- (MEL)e/mel 2 5 18 (MEL)z/mel 8 4 15 (MEL)2/mel

11 0 11 (MEL) jmel 0 10 0 MEL3/mel

42 Mel + :47 Mel- MEL7/mel 39 Mel+ :38 Mel - MEL4/mel 26 0 0 MEL6/MEL6

3 9 13 MEL6/MEL3 27 0 0 MEL3/MEL3

5 5 13 MEL3/MEL6 25 0 0 MEL4/MEL4 24 0 0 MEL7/MEL7

7 2 10 MEL7/MEL4 3 4 10 MEL8/MEL1 5 5 /2 MEL8/MEL2 3 6 11 MEL8/MEL3 1 4 14 MEL8/MEL4 7 7 11 MEL8/MEL5 3 3 19 MEL8/MEL6

59 Me1 + : 15 Me1- MEL6/MEL8 4 4 16 MEL8/MEL7 0 21 0 MEL8/mel 0 22 0 MEL9/mel 0 24 0 MEL8/mel 0 21 0 MEL9/mel 0 17 0 MEL9/mel 0 23 0 MEL9/mel 0 23 0 MEL6/mel 0 22 0 MEL4/mel 0 24 0 MEL4/mel 0 24 0 MEL3/mel 0 25 0 MEL3/mel 0 26 0 MEL7/mel 0 26 0 MEL10/mel 0 23 0 MEL7/mel 0 24 0 MEL3/mel 0 23 0 MEL3/mel 0 24 0 MEL7/mel 0 24 0 MEL7/mel 4 6 15 MELg/MEL1 1 4 26 MEL9/MEL2 4 5 10 MEL9/MEL3 4 5 16 MEL9/MEL4 4 3 16 MEL9/MEL5 7 4 11 MEL9/MEL6 2 8 15 MEL9/MEL7 3 2 21 MEL9/MEL8 9 5 13 MEL10/MEL1 1 5 t4 MEL10/MEL2 2 4 13 MEL10/MEL3 2 3 15 MEL10/MEL4

NR62 NR12-TC x OL14-gD 7 4 17 MELI0/MEL5 NR63 NR12-7C x OLI2-tB 4 1 13 MELI0/MEL6 NR64 NR12-7C x OL8-9D 6 3 17 MELI0/MEL7 NR65 NR12-7C x NR5-10A 2 8 10 MELI0/MEL8 NR66 NR12-7C x NR7-6B 8 4 7 MEL10/MEL9 NR67 NR10-1B x OL2-7C 28 0 0 MEL3/MEL3 NR68 NRt0-1B x OL4-3A 5 3 17 MEL3/MEL6 NR69 NRI0-1D x OL13-7D 22 0 0 MEL7/MEL7 NR70 NRI1-8BxOL3q0D 21 0 0 MEL4/MEL4 NR71 NR5-10Cx ML2-8C 9 4 14 MEL8/MEL2

Additional genotypes: Segregants NR0-4A, NR0-4D, NR1-7A, NR1-7D, NR2-3A, NR2-3B, NR3-2A, NR3-2C, NR4-4A, NR4- 4D, NR5q0A, NR5-10B, NRT-6A, NR7-6B, NR9-4A, NR10-1B, NR10-1C, NRll-8B, NRtl-SD and NR12-7A had MATe, but NR0-4B, NR0-4C, NR1-7B, NR1-7C, NR2-3C, NR2-3D, NR3- 2B, NR3-2D, NR4-4B, NR4-4C, NR5-10C, NR5-10D, NR7-6C, NR7-6D, NRg-4B, NR9-4C, NR10-1A, NRI0-1D, NRtl-8A, NRll-SC, NR12-7C, NRI2-TD had MATa. All segregants are Suc § (SUC2) Gal- Mal- except NR0-4B, NR0-4D, NR2-3B, NR2-3D, NR4-4A, NR4-4D, NRg-4B (Suc) and NR0-4A, NR0- 4C, NR1-TA, NRI-7B (Gal+). Asterisks indicate delayed melibiose fermentation by hybrids and their segregants

three segregants were M e l - (Table 2), indicat ing that the strain CBS 5378 con ta ined at least five po lymer ic M E L genes.

In order to isolate and identify all M E L genes, we chose one comple te te t rad o f the hybr id N R 0 : 4A, 4B, 4C, 4D conta in ing Mel § segregants only. Analysis o f backcrosses (Table 2, N R I - N R 4 ) showed that each segre- gant o f the tetrad N R 0 - 4 con ta ined at least three M E L genes.

We have shown in a previous study ( N a u m o v et al. 1990) that the separa t ion o f yeast c h r o m o s o m e s by pulsed field gel e lect rophores is and their hybr id iza t ion to the rad ioac t ive MEL1 probe can be used to study the segregat ion o f M E L genes. In the present study, we used C H E F separa t ion and MEL1 hybr id iza t ion analysis in order to reduce the n u m b e r o f genetic crosses needed to isolate and identify all M E L genes present in a yeast strain.

F igure 1 shows the c h r o m o s o m a l pa t t e rn o f strain 5378-F2-1D and its segregants f rom the first cross with a M e l - strain, and the pa t te rn o f hybr id iza t ion with M E L 1 probe in com pa r i son with the strain CBS 4411 (MEL3 M E L 4 M E L 5 M E L 6 MEL7) . Fol lowing separa t ion on C H E F , the c h r o m o s o m e s were s tained with e th id ium b romide for pho tog raphy , t ransferred to ni t rocel lulose filters and hybr idized with the M E L 1 probe. The p robe showed hybr id iza t ion with four c h r o m o s o m a l bands f rom a m o n o s p o r i c isolate 4411-5A of strain CBS 4411. This strain carries five M E L genes, but its chr. X I I I con- ta ining M E L 6 comigra tes wi th chr. XVI conta in ing MEL3 ( N a u m o v et al. 1990). All four ch romosomal bands and two addi t iona l bands (chr. X / X I V and chr. VI I /XV)

272

showed hybridization with the MEL1 probe in strain 5378-F2-1D. One hybridizing chromosomal band con- taining chr. XIII and chr. XVI was found in all four seg- regants of the first cross with the Mel- strain, NR0-4, suggesting that (at least) two MEL genes were present in this doublet. Thus, like the strain CBS 4411, CBS 5378 may possess both the MEL3 and the MEL6 gene. The other five hybridizing bands showed regular 2 : 2 segrega- tion in the tetrad NR0-4. The hybridization pattern of this tetrad suggested that the segregants NR0-4A, NR0- 4B, NR0-4C and NR0-4D contained 4, 3, 4 and 3 MEL genes, respectively. These data allowed us to conclude that the parental strain 5378-F2-1D had at least seven polymeric MEL genes.

Fig. 1. Analysis of chromosomal DNA from strain CBS 5378 and its segregants in the first generation by contour-clamped homoge- neous electric field (CHEF) gel electrophoresis. Chromosomal DNA was prepared as described in Materials and methods. After etectrophoresis, the gel was stained with ethidium bromide for visu- alization of the chromosomes (left). The autoradiograph (right) shows the hybridization pattern with the MEL1 probe. For designa- tions of the segregants see Fig. 2

Analysis of F2

All polymeric MEL genes of the strain 5378-F2-1D were isolated in backcrosses with Mel- testers (Fig. 2). In the second generation we studied one complete tetrad from the four differernt hybrids: NR1-7A-D, NR2-3A-D, NR3-2A-D and NR4-4A-D. The backcrosses of the seg- regants with the Mel- parent showed that the segregants NR2-3B, NR2-3D, NR3-2D, NR4-4C and NR4-4D con- tained one MEL gene each as indicated by the monogenic

CBS 5378(Mel +) MEL3 MEL 4MEL6MEL 7MEL8MEL9MEL10

@ MEL6 l MELS I MEL9 | MELIO J

FMe~ l FM~ I MEL9 [Me~S l I MEL8 I L MEL9 J L MELIO J LMELIO~

X2180-1A $288C

| MEL6

~ N R 5

NR0

X2180-1A

7 61 MEL8 MEL9

| 1 7 4 1 7 4 [~E~ l r ~ MEL9 j LMEL9 ] MEL8

I I

MEL6 MEL9 MEL9 MEL6

X2180-1A(Mel-) mel3mel4mel6mel7mel8mel9mell O

[ME~ l MEIM | MEL7 | MELIO J

I Mpel F ~ l MEL4J IMEL71

N R l l ~ LMELI~

X2180-1A $288C

? MELz 1 M ~

MEilO ] NR12

*4 | 1 7 4 | MEL7 MELIO MELIO MEL7

.| MEL7

MEL4 MEL7

N R d e , -

[ ~ l M ~ ~ MEL4 ] LMEL7 ]

NR9

MEL3 MEL4 MEL4

NR13 NR10 /

MEL8 MEL9 M E L 8 MEL9 MEL4 MEL4 MEL3 MEL3 MEL4 MELIO - IMEL7 - ]FMEL4I MEL3 MEL3 MEL7 MEL7 IMELIO J LMEL7J

Fig. 2. Pedigree of the segregants studied from the cross CBS 5378 x X2180-1A and genetic isolation of MEL3, MEL4, MEL6, MEL7, MEL8, MEL9 and MELIO genes. Asterisks indicate strains in which genotype was determined by molecular methods only

273

Fig. 3A-C. Analysis of chromosomal DNA by CHEF from A the second and B, C the third generations of segregants descended from the strain CBS 5378

274

segregations patterns for melibiose fermentation (2 Mel + :2Mel-) (Table 2, NR6, NR8, NR14, NR15, NR16). Figure3A shows the chromosomal and hy- bridization patterns of this second generation of segre- gants. The segregant NR1-7C, which was not analysed genetically, only had one MEL gene, which was on chr. X or chr. XIV (comigrating). The segregant NR2-3B had its MEL gene on chr. XIII or XVI (upper band of the dou- blet migrating separately in this segregant). The segre- gant NR2-3D showed hybridization to MEL1 probe on chr. XV or VII (comigrating) and the segregant NR3-2D on chr. XIII or XVI (comigrating). The segregant NR4- 4D had its MEL gene on chr. XI.

The crosses with selected tester strains showed that the MEL genes in segregants NR2-3B and NR3-2D were allelic to MEL6 and MEL3, respectively, as no segrega- tion of ability to ferment melibiose (4 Mel + :0 Mel-) was found (Table 2, NRI7, NR19). The MEL gene in segre- gant NR4-4D was allelic to MEL4 (Table 2, NR21) and the MEL gene in segregant NR4-4C was allelic to MEL7 (Table 2, NR22) which was to be expected on the basis of their chromosomal location. Control crosses NR18, NR20, NR23 showed digenic segregation (Table2). MEL2 has been previously shown to be located on chr. VII in a strain of S. cerevisiae (Naumov et al. 1990). However, the cross of segregant NR2-3D (hybridization with MELI probe on chr. XV/VII) with MEL2 tester strain (ML2-8C) showed that their MEL genes were not allelic (Table 2, NR25). The segregant NR2-3D had a new gene, MEL8, evidenced by the fact that in hybrids with MEL1-MEL7 testers digenic segregation was found (Table 2, NR24-NR31). Thus, in two generations of seg- regants we identified five MEL genes: MEL3, MEL4, MEL6, MEL7 and MEL8.

Ana@s~ofF3

There were still at least two unidentified MEL genes in strain 5378-F2-1D. In order to identify all MEL genes of this strain it was necessary to determine the genotypes of at least three segregants of one complete tetrad in each of the hybrids NR0, NR2, NR3 and NR4. On the basis of backcrosses NR5, NR7 and NR9-NR12 (Table 2) and the molecular analysis (Fig. 3) segregants NR2-3A, NR2- 3C, NR4-4A, NR4-4B, NR3-2A and NR3-2B were likely to contain two MEL genes each whereas the segregant NR3-2C had three MEL genes (Table 2, NR13). With the exception of segregant NR3-2C, the MEL genes were isolated in the third generation. Analysis of backcrosses NR32-NR49 in a new generation confirmed that these segregants contained one MEL gene each (Table 2). Fig- ure 3 B, C shows the chromosomal pattern and the MEL! hybridization with the chromosomal DNA of the third generation of segregants containing isolated MEL genes. The genotypes of segregants NR9-4A-C were only deter- mined by the molecular method (Fig. 3 B). The same new chromosomal localization of the MEL gene was found in segregants NR7-6B, NR7-6C, NR5-10B and NR5-10D (Fig. 3 B). This gene was identified as MEL9 in crosses with the MEL1-MEL8 testers (Table 2, NR50-

NR57). The MEL gene of segregants NR12-7B and NR12-7C was found in the largest band, containing chro- mosomes IV and XII. Electrokaryotyping showed that this gene was similar in location to the MEL5 gene. But hybrid NR62 between segregant NR12-7C and MEL5 tester yielded digenic segregation (Table 2). In crosses with other MEL tester strains the new gene was identified as MELIO (Table 2, NR58-61, NR63-NR66).

Thus, in the third generation of segregants we identi- fied two new MEL genes: MEL9 and MELIO. For final identification of all MEL genes we had to determine genotypes of some segregants. The equal size of chromo- somes XIII and XVI made it difficult by CHEF analysis to identify genotypes of segregants NR10-1A, NRI0-1B and NR9-4A. It was not clear whether they carried MEL3 or MEL6. On the basis of recombination tests, the MEL gene of segregant NR10-1B was identified as MEL3 (Table 2, NR67, NR68). In accordance with molecular data (Figs. 1, 3) and the rules of meiosis, we were able to determine that the MEL gene in segregants NR10-1A, NR9-4A, NR4-4A, NR4-4B was MEL3 and that the MEL gene in segregants NR0-4A, NR1-7A, NR1-7B was MEL6. By genetic hybridization analysis we confirmed that segregants NR10-1D and NRll-8B car- ried the MEL7 and MEL4 genes, respectively (Table 2, NR69, NR70). Additional cross NR71 (Table 2) verified that the segregant NR5-10C did not contain MEL2 gene. Figure 2 shows the genotypes of all segregants studied by genetic and molecular methods.

Chromosomal assignment of new MEL genes

By electrokaryotyping we found that all seven MEL genes of the strain CBS 5378 were located on different chromosomes. A standard set of S. cerevisiae YNN 295 chromosomes was used for the numbering of chromo- somes (Mortimer et al. 1989). Three new MEL genes were found in double bands: MEL8 on chr. VII or XV, MEL9 on chr. X or XIV and MELIO on chr. IV or XII. In order to localize MEL8 gene, we had to analyse an additional hybrid (NR72). Strain NR2-3D (MEL8) was crossed with a monosporic culture of the Mel- strain VKM Y-503 having separate chr. VII and XV. Then, by electrokaryotyping we chose Mel + segregants where these two chromosomes were separate. Hybridization with the MEL1 probe and then with the ADC1 promoter probe (chr. XV) indicated the presence of MEL8 on chr. XV (not shown). To separate chr. IV and XII and to map MELIO, electrophoresis was carried out at a special protocol (200 V for 15 h with a switching time of 70 s followed by 8 h with a switching time of 120 s). The MELIO gene was found in the upper band of the separat- ed doublet, and hybridization with the rDNA oligonucle- otide probe confirmed the presence of chr. XII in the up- per band (not shown). The TRP1 gene probe (chr. IV) hybridized with the lower band of this doublet (not shown). To separate chromosomes X and XIV and to locate MEL9 gene, we crossed NR7-6C (MEL9) with two Mel- strains ($288C and YNN 295) having these two chromosomes separated. We analysed 21 Mel + segregants from these crosses, but none of them showed separation of

275

Fig. 4. Analysis of chromosomal DNA from the segregants con- taining isolated MEL genes. Ethidium bromide-stained chromo- somes are shown on the left and hybridization with the MEL1 probe on the right. Lane 1, YNN295 (Bio-Rad); lane 2, CBll (MEL1); lane 3, 1830-2-2 (MEL2); lane 4, OL0-11B (MEL3); lane 5, OL0-

chromosoms X and XIV. Figure 4 summarizes the results of chromosomal analysis of the segregants containing only one M E L gene each.

Restriction analysis

Total D N A from the strains C B l l , 1830-2-2 and segre- gants of CBS 4411 each carrying one of the genes MEL3- MEL7, the monosporic derivative of CBS 5378 and its segregants f rom the crosses with the Me l - strain was digested with restriction endonuclease Eco RI. The D N A fragments were then separated in an agarose gel and transferred to a nitrocellulose filter. The MEL1 probe was used for hybridization. The D N A from CBS 5378 gave a similar pat tern of EcoRI fragments hybridizing with MEL1 as did CBS 4411: three fragments of 6.9, 6.7 and 5.2 kb, respectively. Only the relative intensities of hybridization varied between the three fragments (not shown). In CBS 4411, the 6.9 kb fragment contains one M E L gene, and the 6.7 and 5.2 kb fragments contain two M E L genes each (Naumov et al. 1990). The 6.7 kb frag- ment of CBS 5378 hybridized more intensely with M E L 1 than did that of CBS 4411, suggesting the presence of more than two genes in the former fragment. Figure 5 shows that in CBS 5378, the 6.9 kb fragment contained one M E L gene (MEL8) and the 6.7 kb fragment four M E L genes (MEL3, MEL4 , M E L 6 and MELIO) . Two M E L genes, M E L 7 and MEL9, migrated with the 5.2 kb Eeo RI fragment. The sizes of Eco RI fragments contain- ing MEL3, M E L 6 and M E L 7 were identical in both strains. However, M E L 4 was in a 6.9 kb EcoRI fragment in CBS 4411 and in a 6.7 kb fragment in CBS 5378. EcoRI restriction sites are not found in the coding region of the genes but in the flanking regions. In MEL1, one site is 0.5 kb upstream from the translation start site and the other is 3 kb downstream from the stop codon (Nau- mov et al. 1990). Assuming that in both strains the M E L 4

11C (MEL4) ; lane 6, OL14-9D (MEL5); lane 7, OL13-7C (MEL6); lane 8, OL8-9D (MEL7); lane 9, 5378-F2-1D (S. norbensis); lane 10, NRll-8C (MEL3); lane 11, NRtI-8B (MEL4); lane 12, NR2-3B (MEL6); lane 13, NRI0-1D (MELT); lane i4, (503 x NR2-3D)-3B (MELS); lane 15, NR7-6C (MEL9); lane i6, NR12-7C (MELIO)

Fig. 5. Southern hybridization analysis of EcoRI-digested DNA from strains containing MEL1-MEL7: CBI1 (MELI, lane 1), 1830- 2-2 (MEL2, lane 2), OL8-9C (MEL3, lane 3), OLll-IIA (MEL4, lane 4), OLI-9D (MEL5, lane 5), OLII-tIB (MEL6, lane 6), OL8- 9D (MEL7, lane 7), and from CBS 5378 segregants containing only one MEL gene each: NR10-1B (MEL3, lane 8), NRll-8B (MEL4, lane9), NR7-6D (MEL6, lane lO), NRI0-1D (MEL7, lane 11), NR5-10C (MEL8, lane i2), NR5-10B (MEL9, lane 13), NRI2-7C (MELIO, lane 14). Genomic DNA was digested with EcoRI, sub- jected to electrophoresis in an agarose gel, transferred to nitrocellu- lose and hybridized with the MEL1 probe. The sizes of the restric- tion fragments are in kilobases

genes have an upstream EcoRI site identical to that of MEL1, the sizes of EcoRI fragments place the down- stream sites at 4 .7-4 .9 kb fi'om the stop codon of the M E L 4 gene, a distance less likely to be conserved with the gene.

The D N A from segregants containing one M E L gene (MEL3-MELIO) , originating f rom either CBS 4411 or CBS 5378, yielded only one hybridizing fragment after digestion with BamHI (8.5kb), HindIII (5.9kb), E c o R I + B a m H I (3.2 kb) or E c o R l + H i n d I I I (4.2 kb) (not shown). The fragment was identical in all segregants, indicating that the genes and their flanking regions are homologous sequences in which restriction sites have been conserved.

276

Discussion

The present study, using combined genetic and molecular analyses, identified for the first time a strain with seven polymeric MEL genes, three of which were new (MEL8- MELIO). We have previously studied the strain CBS 4411 containing five MEL genes (Naumov et al. 1990). Thus, 10 out of 16 chromosomes in Saccharomyces cerevisiae are now marked by MEL genes. In behaviour, the MEL genes of Mel + strains resemble the X and Y' sequences known to be located on almost all chromosomes and, obviously, migrate from one chromosome to another (Zakian and Blanton 1988; Jfiger and Philippsen 1989; Louis and Haber 1990a, b).

In the past few years a lot of attention has been focused on the question of microevolution of the polymeric genes SUC (Hohmann and Gozalbo 1988, 1989; Hohmann and Zimmermann 1986; Carlson et al. 1985), MAL (Charron and Michels 1988; Charron et al. 1989) and STA ~a- mashita et al. 1985, 1987; Pretorius et al. 1986). It now appears that the MEL gene family may be better suited for such studies. The MEL genes are unique, as they may result from concomitant evolution in one yeast cell, while other polymeric sugar fermentation genes studied are of unknown origin as a rule (Naumov 1988). There is usually one, very seldom two or three, SUC, MAL or STA poly- meric genes in one strain. The presence of many MEL genes in the same natural strain suggests their coevolution in one organism, whereas most other polymeric genes of sugar fermentation have probably evolved independently of each other. Thus, it is more expedient to investigate gene microevolution by studying polymeric MEL genes. Re- striction analysis and hybridization with the radioactive MELI probe showed that the MELI-MELIO genes are homologous sequences. Cloning and sequencing of the genes would help us to understand the evolution of the genes for melibiose fermentation. In this context it is inter- esting to study not only Mel +, but also Me l - strains. Strains that do not ferment sucrose, maltose or starch are known to contain silent sequences of fermentation genes (Carlson and Botstein 1983; Carlson etal. 1980, 1985; Charron and Michels 1988; Yamashita et al. 1985, 1987; Pretorius et al. 1986; del Castillo Agudo and Zimmermann 1986; Gozalbo and Hohmann 1989). Natural Mel - strains have not yet been studied by molecular method. However, five Mel - genetic lines are known to lack silent sequences of ~-galactosidase genes (Post-Beittenmiller et al. 1984; Casey and Rank 1988; Naumov et al. 1990). Three baker 's yeast strains have also been shown to be reel ~ (Liljestr6m- Suominen et al. 1988; Casey and Rank 1988).

The phenomenon of accumulation of MEL genes in some strains and absence of active MEL alleles in the majority of natural strains of S. cerevisiae is worthy of note of molecular evolutionists.

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Communicated by J. K. Z immermann