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Physical, Transcriptional and Genetical Mapping of a 24 kb DNA Fragment Located Between the PMAl and ATEl Loci on Chromosome VII from Saccharomyces cerevisiae ETIENNE CAPIEAUX, STANISLAW ULASZEWSKI*, ELISABETTA BALZI AND ANDRE GOFFEAU C'nirP dc Riochimie Physiologique, UniversitP Catholique de Louvain, 1348 Lou\.ain-la-Neuve. BeI,qiuni xlnstitute of Microbiology, Wroclaw IJniiwsiry, Przybvszewskiep 63, 51-148 Wroclaw, Poland
Received 28 July 1990; revised h November 1990
A physical map of a contiguous DNA fragment of 60 kb, extending from the centromerc to TRPS on the left arm of the chromosome VII of Sacr~huromyces cerevisioe. strain IL125-28, was established. Within a 3 1 kb region from PMAl towards 7RP.5, a total of 12 transcription products ranging from 0.6 to 3.6 kb were identified in cells grown exponentially o n rich medium. Near 87% of the DNA investigated was transcribed and on average one transcript, of 2.3 kb average Icngth. was detected every 2.7 kb of DNA. The physical and genetical distances between the markers CEN7, pmol. leul. pdrl and trpS were compared. A recombination frequency of 1 cM corresponds to an average distance of 3.3 kb between alleles in this region of chromosome V11. KEY WORDS-Saccharomvces
cereiisiae; chromosome VII: transcripts; genetic distances.
INTRODUCTION Exhaustive genetical mapping has been carried out on the region of chromosome VII from Sacchuromyces c*ere\isiae surrounding the PMAI and PDRl loci encoding the plasma membrane ATPase and pleiotropic drug resistance phenotype respectively (Mortimer et 01.. 1989).
The lack of discriminating power of the genetic approach does not allow to distinguish the numerous genetic loci for drug resistance reported in this region. We have therefore undertaken a physical mapping of this region (Balzi et ul., 1987, 1989, 1990) as well as systematic DNA sequencing (Chen et al., 1991a). We report here on the transcriptional mapping of the 24 kb rcgion spanning the PMAI and ATEl genetic loci on chromosome VI1 in order to answer the following questions: Are all open reading frames expressed? Is the gene density of this region different from that reported for other parts of the yeast genome? What is the size as well as the average length of the mRNAs? *Corrcspnnding author.
0749%503X/O1/03027546$05.00 010()1by John Wiley &Sons 1,td
Is the relation between genetical and physical distances in this region similar to the values reported for other genomic regions? MATERIALS AND METHODS Strains and media Escherichia coli JM 109 (recA 1, endA 1, xjrA96, [hi, hsdR17, relA I , k-,6, (lac-proAB) [F',traD36,proAB, ladq,lacZ6MIS] was used for transformation by the CaCI, procedure (Maniatis et al., 1982). Conventional LB medium was supplemented, when appropriate, with ampicillin ( 1 00 pg/ml). The S. cerevisiae strains used are described in Ulaszewski et al. (1987). The strains were grown at 30 "C in rich medium (YEPD) containing 1 % yeast extract (Difco), 1 o/c Bacto-peptone (Difco) and 2% glucose. For plating, the media were solidified with 2% Bacto-agar (Difco). Conventional genetical procedures for yeast-crossing, sporulation, and tetrad analysis were used. The genetical mapping
E. CAPIEAI!X E T A L
276 was performed according to Mortimer and Hawthorne (1975). From the results of the tetrad analysis, the distance between two markers was calculated in centimorgans as described by Snow (1979). Yeast transformation was carried out according to Dieckmann and Tzagoloff (1983).
DNA and RNA DNA constructions were carried out by standard techniques (Maniatis et al., 1982). DNA probes were labeled by nick-translation according to Rigby et aI. (1977). RNA was extracted from cells grown to a density of 3 x 107/ml by the procedure of Maccechini et al. (1979), and its concentration was determined by absorbance at 260 nm. Agarose gel electrophoresis, transfer of RNA from agarose gels to nitrocellulose paper. were carried out as described by Maniatis et ul. ( 1982). To determine the sizes of the transcripts identified by Northern hybridization, a lane containing total RNA was cut from each gel prior to blotting and stained with ethidium bromide to visualize the 18s and 2 5 s rRNA bands, whose sizes were taken to be 1.7 and 3.4 kb, respectively. RESULTS Physical analjsis We have subcloned 60 kb of DNA, extending from the centromere region to the TRPS gene on the left
1 6 -
subclones
-
15,
Transcriptional analjsis Within the 31 kb from the Hind111 site located from the PMAI promoter up to the first BamHI site located distal to ATE1 a total of 12 transcription products ranging from 0.6 to 3.6 kb were identified (Figure 2). Nine transcripts were unambiguously mapped in the 24 kb Hind111 fragment spanning PMAI and A T E ] . The mapping and the size of these transcripts allowed identification of those corresponding to the genes
I
- 5
l?181920M 21a1-+-21b 22
-
1Lb
arm of the chromosome VII. This tract comprises the inserts of two overlapping cosmidial plasmids, CosA and CosS (Figure 1). of 35 kb and 30 kb respectively. Both cosmids were issued from a genomic DNA library (kindly provided by Dr M.Guerineau, Universite de Paris Sud, Orsay. France) consisting of partial RamHI digests of S . cerevisiae strain IL1252B, cloned into the vector pHCG3 (Gerbaud et al., I98 I ) . CosA and CosS overlap in the LEUl region. CosA was initially used for cloning the pleiotropic drug resistance gene PDRI (Balzi et al., 1987) distal to L E U l . whereas CosS was isolated during the cloning of the plasma membrane ATPase gene P M A l , proximal to L E U l (Ulaszewski et al., 1987). The restriction maps of both cosmids were established and the subclones depicted in Figure 1, were used to locate the genes PMAl (Ulaszewski et 01.. 1087), LEUl (Skala et al., 1991), .sdI (Balzi ct (11.. 1989), PDRI (Balzi ct oi.,19871, PDR6 ( Chen et al., 1991a). ATEl (Balzi e f al., 1990) and TRPS (Balzi et al.. 1987) by complementation of mutant strains.
- 6
-9
?-
8-
10111
I
12a,-+
+ 4 1 2 b 13-
1 4 -
Figure I . The 60 kb region of chromosome VII. The inserts of the two overlapping cosmids. CosA (Balzi er a/..l987) and CosS (Ulas7ewski e r a / . . 1987) are shown. Physical and genetic maps are as previously descrihed (Balzi ef al.,1987, 1989. 19%; Ulaszewski er a/.,1987). Restriction sites presented for CosA are B u ~ I H I(B). llind111 (H). . M I (S). XhoI (X), SmaI (Sm). and S.sfI1 (St): for CosS only BamHI and Hind11 sites are presented. Under the cosmids, the following subclones are depicted and numbered as shown below within brackets: PA-X28 (la+lb), pA-X30 (2a+2b), PA-B6 ( 3 ) . pA-R6.5 (4). PA-B8 ( 5 ) . PA-B9 (6).pA-B3.5 (7). pA-B3 (8). pA-B8H (9).PAB8S(10),pA-X14(1l)(Balziera/.. 1987); pA-XS(12a+b).pA-H4(13)(Balziera/.. I~YO);pA-H6(14)(Bal7ieru/.. 1989):pSB12(15). CosS-Sub5.4 (16). CosS-Sub5 (17) (Ulaszewski cf a/.. 1987); pS1.6 (18). pE0.8 (19).pS4S(20), pS4K (2la+b). pS4B ( 2 2 ) .
Figure 2 RNA map of the PMAI-ATE1 region. (A) Restriction map with the approximate sizes (in kilobases), locations and identities of the transcribed regions (heavy lines). Restriction sites presented are: Hind111 (H), BarnHI (B), SalI ( S ) , SsrII (St), Xhol (X). Under the restriction map are depicted the DNA fragments used as radioactive probes for the Northern blot analysis shown in (B). (B) Autoradiograms of Northern blot hybridizations. Poly(A) RNA (lanes 1,3,8) and total RNA (lanes 2, 2’, 5.6, 7, 9, 10, 1I , 12) extracted from Succharomyces cerevisiae 1278b or GR350 (lane 6 ) were submitted to electrophoresis in an 1.5% agarose-formaldehyde gel and hybridized with the indicated DNA fragments. The transcript sizes indicated in kilobases were estimated as indicated in Materials and Methods. The intensities of the radioactive bands do not provide an estimation of the abundance of the RNA since probes with different specific activities were used.
1Kb
-
278
E. CAPIEAUX ET AL
PMAI (Ulaszewski et ul., 1987). LEUl (Skala et al., 1991). s d l (Balzi et al., 1989), PDRI (Balzi et 01.. 1987). PDR6 (Chen et al.. 1991a) and ATE1 (Balzi et al., 1990). The three unknown open reading frames YGL021, YGLO22. YGL023 described by Chen et 01. (1991b,c,d) could also be mapped precisely. However we have not mapped the three transcripts of 0.6, 0.9 and 2.7 kb identified in the BumHl fragment located distal to ATEI.
Genetical unu1ysi.s Classical tetrad analysis was carried out for genetic mapping of the mutations p m a l . leul. trp.5, pdrl and ade6. The strain S.cwe\isiae 20-584A (pmul-I) was crossed with a set of tester strains carrying centromere and auxotrophic markers as reported in Table 1. The results of 15 tetratypes for the crosses involving pmul and the centromere marker t r p l , indicated that the pmul gene is 4.0 cM away from a centromere. No recombination events were observed between leul and pmul after analysis of meiotic recombination in 400 asci. This is not surprising considering that the pmul-l mutation is located in the last third of the PMAI gene (Van Dyck et ul., 1990) and the leul mutation is located on the same strand in the first 153 bp from the 5' end of the LEU1 gene (Ulaszewski et ul., 1987). The two mutations are thus only 2.2 kb apart. The pdrl and leul loci are located 5.4 and 5.2 cM away from centromere VII. respectively. The positioning of p d r l , leul and pmal was estimated by a four point cross. The pmal mutation maps 2.3 and 9.3 cM from the pdr-1 and the trpS mutations respectively. The genetic distance between pdrl and trpS is 9.1 cM. The following genetic distances between the tested markers are thus estimated to be: 4.0 cM for centromere V11 topmal; less than 0.12 cM DIPLOID
GENE PAIR
US26, US53, US54 US6, US26 US6, US37 US62, US63 US62.US63 US^, US13, US62, US63, us202 US62, US63 US62, US63 US6. US7. US13, US62. US63 US7. US13 US7, US13. US202 US6, US13. US202
CEN7-pdrl CEN7-pmal CEN74eul trp5pdrl trohmal tipsieul leul-pdrl pmal-pdrl leul-pmal ade6-pmal ade6-leu1 ade6-trp5
Table I
for pmul to IC141; 2.3 cM for leu1 to pdrl and 9.1 cM for pdrl to trpS. DISCUSSION The 24 kb Hind111 DNA fragment spanning the PMAI and ATE1 genes has been totally sequenced. The sequence of LEUl (Skala et ul., 1991), PMAl (Serrano ct (11.. 1986; Capieaux et al., 1989; Van Dyck et al., 1990). s d l (Balzi et ol., 1989), PDKI (Balzi et ul., 1987) and ATEI (Balzi et ul., 1990) genes are published as well as those of PDR6 and of the 17 kb Hindlll fragment spanning the LEUl-ATE1 region (Chen et al., 1991a). Comparison of the transcript map and the DNA sequencing allows to conclude that all large open reading frames are expressed since a mRNA was detected for all open reading frames longer than 600 bp. The sensitivity of our method does not allow to detect smaller mRNA. Therefore additional smaller open reading frames, such as those described by Chen et al. (1991a). could well be also transcribed. On average. the difference between the size of a mRNA and that of the open reading frame predicted by DNA sequencing is about 300 nucleotides. The genes in the PMAI-TRP.5 region of chromosome V11 are closely packed. Taking into account only the detectable transcripts, there is one transcript of 2.3 kb average length every 2.7 kb of DNA within the 24 kb fragment spanning PMAI and ATEI. About 87% of the DNA region investigated is thus transcribed. The average intergenic sequence is 400 bp but varies from 218 (between scll and YGLO28) to 1232 bp (between YGL023 and PDR6). Four previous reports of transcript density, ranging from 67% to 85%. have been made for regions located on chromosome I, XV, and X of S.cerevisiuc (Struhl and Davis, 1981; Coleman et al., 1986; Steensma
TETRAD ANALYSIS TOTAL GENETIC MOLECULAR PD NPD T DISTANCE DISTANCE cM k SE (Kb) 100 83 89 86 32 37 139 0 0 231 246 0 162 0 162 0 400 0 46 2 54 3 49 6
22 15 8 31 53 56 8 8 0 66 75 77
205 190 77 170 284 302 170 170 400 114 132 132
5.4t1.4 4.021.0 5.2f1.8 9.1fl.l 9.3k1.1 93?1 1 23t1 1 2 3f1 1
>O 1tO
23.0 33.6 31 3 81 10 3 21
RECOMBINATION RATE (cM/Kb)
0.40 0.28
0 30 0 28 0 22 0 06
34.7t0.4 35.0+0.4 42.0C0.4
Comparison of genetic and molecular distances. The genetic distances were calculated from tetrad analysis according to Snow
(1989). Molecular distances were from the middle of the genes, except for pmal and leu1 mutations previously molecularly located. The strains used are described in Ulasrewski
( ' f
ul. (1987).
MAPPIKG O F A 24 K B DNA FRAGMENT FROM SACCHAHOMYCES CEfiEv'lSIAE
rt al., 1987; Barry et al., 1987). These studies, added
to ours, encompass a total of 83 kb of yeast DNA, among which a total of 35 long transcripts (tRNA excluded, Ty included), covering 80% of the DNA length were identified. An average transcript of 1.9 kb was detected every 2.4 kb. If we assume that this gene density is representative for the entire yeast DNA, which approximates 12,537 kb (rDNA excluded) ( Mortimer et al., 1989), a haploid S. cerevisiae genome should express about 5300 genes. This is a minimal estimate since, on one hand, small transcripts may not be detected and, on the other hand, some genes may be expressed in other growth conditions or genetic backgrounds than the ones investigated. Our results correlate well with early work carried out by genomic R-loop mapping which estimate the existence of 5000 transcribed regions in the entire yeast genome (Kaback et al., 1979). Comparison of the genetic distances with the physical distances between the markers CEN7, P M A I , L E U ] , PDRI and TRPS spread over 60 kb of chromosome VII, leads to the conclusion that in this centromeric region a recombination frequency of 1 cM unit corresponds to an average distance of 3.3 kb between alleles. This is slightly larger than the estimate of 2 4 4 kb/cM obtained by a much more global approach considering all genetic markers on all regions from all chromosomes (Mortimer et al., 1989) and it is more than twice the 1.59 kb/cM estimated for the entire chromosome I by Kaback et ul. (1989).
ACKNOWLEDGEMENTS This work was supported in part by grants to A. Gofteau from the Services de la Politique Scientifique: Action Science de la Vie and from the Fonds National pour la Recherche Scientifique, Belgium. NOTE During submission of this paper, we received the information that a transcriptional map of all the yeast chromosome 111 has been done by Yoshikawa and lsono (1990). By performing various Northern hybridization experiments, these authors identified 156 poly(A)' transcripts larger than 350 bp on the whole chromosome estimated to be 334.6 kb long. Also Melnick and Sherman (1990) reported the existence of six genes in a 7.5 kb segment located on chromosome X . These data fully justify the generalization of our data to the whole yeast genome.
279
REFERENCES Balzi, E., Chen, W., Capieaux, E., McCusker, J.H.. Haber. J.E. and Goffeau. A. (1989). The suppressor gene scll' of Saccharomyces cerevisiae is essential for growth. Gene 83,27 1-279. Balzi, E., Chen, W., Ulaszewski, S., Capieaux, E. and Goffeau, A. (1987). The multidrug resistance gene PDRl from Sacchuromyces cerevisiae. J. Biol. Chem. 262. 16871-1 6879. Balzi, E., Choder, M., Chen, W., Varshavsky. A. and Goffeau, A. (1990). Cloning and functional analysis of the arginyl-tRNA-protein transferasc gene ATE/ of Saccharomyces cerevisiae. J. Biol. Chem. 265. 7464-747 1 . Barry, K., Stiles, J.I., Pietras, D.F., Melnick. L. and Sherman, F. (1987). Physical analysis of the COR region: a cluster of six genes in Socrhoromj~ces cerevisiae. Mol. Cell. Biol. 7,632-638. Capieaux, E., Vignais, M.L., Sentenac, A. and Goffeau, A. (1989). The yeast Hi-TPase gene is controlled by the promoter binding factor TUF. J. Hiol. Chem. 264, 7437-7446. Chen, W., Balzi, E., Capieaux, E., Choder, M. and Goffeau, A. (1991a). The DNA sequencing of the 17 kb IiindIII fragment spanning the LEU/ and ATE1 loci on chromosome VII from Saccharomyes cc,rr\*i.siae reveals the PDR6 gene, a new member of the genetic network controlling pleiotropic drug resistance. Yeast 7, 287-299. Chen, W., Capieaux, E., Balzi, E. and Goffeau, A. ( 1991b). The YGUlzl gene encodes a putative membrane protein with a putative leucine zipper motif. Yeast 7, 301-303. Chen, W., Capieaux, E., Balzi, E. and Goffeau, A. (1091~). The YGL022 gene encodes a putative transport protein. Yeast 7, 305-308. Chen, W., Balzi, E.. Capieaux, E. and Goffeau, A. ( 1991 d). The YGL023 gene encodes a putative regulatory protein. Yeost 7,309-3 12. Colenan, K.G., Steensma, H.Y., Kaback. D.B. and Pringle, J Q. (1986). Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome. Mol. Cell. B i d . 6,45 16-4525. Dieckmann, C.D. and Tzagoloff, A. (1083). Transformation of nuclear respiratory deficient mutants of yeast. Methorls Enzymol. 97,355-360. Gerbaud, C., Elmerich, C., Tandeau de Masak, Pi., Chocat, P., Charpin, N., GuCrineau, M. and Aubert, J .P. ( I98 I ). Construction of new yeast vectors and cloning of the Nif (nitrogen fixation) gene cluster of Klehsiella pneumoniae in yeast. Current Genet. 3, 173-180. Kaback, D.B., Angerer, L.M. and Davidson, N. ( 1079). Improved methods for the formation and stabilization of R-loops. Nucleic Acids Res. 6, 2499-25 17. Kaback, D.B., Steensma, H.Y. and De Jonge, P. (1989). Enhanced meiotic recombination on the smallest chromosome of Saccharomyces cerevisiae. Proc. Null. Acad. Sci.USA 86,3694-3698.
280 Ivlaccechini, M.L.. Rudin, Y., Blobel, G. and Schatz, G. ( I 979). Impon of protcins into mitochondria: prccursor forms of the extramitochondrially made FI -ATPase subunits in yeast. Proc. Nu!/. Acad. Sci. USA 76, 343-347. Maniatis. T.. Fritsch. E.F. and Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, Laboratory. Ncw York. Melnick. L. and Sherman. F. (1990) Nucleotide sequence of the COR region-A cluster o f six genes in the yeast Socc&m)mycry r.ern3isioe.Gene 87,157-1 66. Mortimer, R.K. and Hawthorne. D.C. (1975). Genetic mapping in yeast. Methods Cell. Riol. 11, 221-233. Mortimer, R.K., Schild, D., Contopoulo, C.R. and Kans. J.A. ( 1989) Genetic map of Sacchromjc~c,.scerevisiae. Edition 10. Yeust 5, 3 2 1 4 0 4 . Rigby, P.W.J., Dieckmann, M.,Rhodes, C. and Berg, P. (1977). Labelling deoxyribonucleic acid to high specific activity in virro by nick-translation with DNA polymerase I. .I. Mol. B i d . 113, 237-25 I . Serrano. R., Kielland-Brandt, M.C. and Fink, G.R. (1986). Yeast plasma membrane ATPasc is essential for growth and has homology with (Na’ + K+). K’- and Ca”ATPases. Natirrr 319,680-693. Skala. J., Capieaux, E.. Balzi, E., Chcn, W. and Goffeau. A. (1991 ). Complete sequence of thc Suwharomyes
E. CAPIEAUX ETAL
cerevisiue LEU1 genc encoding isopropylmalate isomerase. Yeast 7,281-285. Snow, R. (1979) Maximum likelihood cstimation of linkage and inheritance from tetrad data. Genetics 92, 23 1-245. Steensma, H.Y., Crowley. J.C. and Kahack, D.B. (1987). Molecular cloning of chromosome 1 DNA from Soccharomyces cere\isiae: isolation and analysis of the CEN 1-ADE 1 -CDC I 5 region. M o l . Cell. Riol. 7. 4 1 0 419. Struhl, K. and Davis, W., (1981). Transcription of the his3 gene region in Saccharomyces cerevisiae. J . Mol. Riol. 152.535-552. Ulaszewski, S., Balzi, E. and Goffeau, A. (1987). Genetic and molecular mapping of the pmal mutation conferring vanadate resistance to the plasma membrane ATPase from Saccharornyce.s c.erevisioe. Mol. Gen. Gene!. 207, 3 8 4 6 . Van Dyck. L.. Petretski, J.H., Wolosker, H., Rodrigues. G. Jr., Schlesser, A,. Ghislain, M. and Goffeau, A. (1990). Molecular and biochemical characterization of thc Dio9 resistant pmal-1 mutation from S. cerevisiae. EM. J . 8ioc.h..submitted. Yashikawa, A. and Isono. K. (1990). Chromosome 111 of Sacchuromycw ccvwisioe: An ordered clone bank, a detailed restriction map and analysis of transcripts suggest the presencc of 160 genes. Yeast 6, 3 8 3 4 0 1 .
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Physical, Transcriptional and Genetical Mapping of a 24 kb DNA Fragment Located Between the PMAl and ATEl Loci on Chromosome VII from Saccharomyces cerevisiae ETIENNE CAPIEAUX, STANISLAW ULASZEWSKI*, ELISABETTA BALZI AND ANDRE GOFFEAU C'nirP dc Riochimie Physiologique, UniversitP Catholique de Louvain, 1348 Lou\.ain-la-Neuve. BeI,qiuni xlnstitute of Microbiology, Wroclaw IJniiwsiry, Przybvszewskiep 63, 51-148 Wroclaw, Poland
Received 28 July 1990; revised h November 1990
A physical map of a contiguous DNA fragment of 60 kb, extending from the centromerc to TRPS on the left arm of the chromosome VII of Sacr~huromyces cerevisioe. strain IL125-28, was established. Within a 3 1 kb region from PMAl towards 7RP.5, a total of 12 transcription products ranging from 0.6 to 3.6 kb were identified in cells grown exponentially o n rich medium. Near 87% of the DNA investigated was transcribed and on average one transcript, of 2.3 kb average Icngth. was detected every 2.7 kb of DNA. The physical and genetical distances between the markers CEN7, pmol. leul. pdrl and trpS were compared. A recombination frequency of 1 cM corresponds to an average distance of 3.3 kb between alleles in this region of chromosome V11. KEY WORDS-Saccharomvces
cereiisiae; chromosome VII: transcripts; genetic distances.
INTRODUCTION Exhaustive genetical mapping has been carried out on the region of chromosome VII from Sacchuromyces c*ere\isiae surrounding the PMAI and PDRl loci encoding the plasma membrane ATPase and pleiotropic drug resistance phenotype respectively (Mortimer et 01.. 1989).
The lack of discriminating power of the genetic approach does not allow to distinguish the numerous genetic loci for drug resistance reported in this region. We have therefore undertaken a physical mapping of this region (Balzi et ul., 1987, 1989, 1990) as well as systematic DNA sequencing (Chen et al., 1991a). We report here on the transcriptional mapping of the 24 kb rcgion spanning the PMAI and ATEl genetic loci on chromosome VI1 in order to answer the following questions: Are all open reading frames expressed? Is the gene density of this region different from that reported for other parts of the yeast genome? What is the size as well as the average length of the mRNAs? *Corrcspnnding author.
0749%503X/O1/03027546$05.00 010()1by John Wiley &Sons 1,td
Is the relation between genetical and physical distances in this region similar to the values reported for other genomic regions? MATERIALS AND METHODS Strains and media Escherichia coli JM 109 (recA 1, endA 1, xjrA96, [hi, hsdR17, relA I , k-,6, (lac-proAB) [F',traD36,proAB, ladq,lacZ6MIS] was used for transformation by the CaCI, procedure (Maniatis et al., 1982). Conventional LB medium was supplemented, when appropriate, with ampicillin ( 1 00 pg/ml). The S. cerevisiae strains used are described in Ulaszewski et al. (1987). The strains were grown at 30 "C in rich medium (YEPD) containing 1 % yeast extract (Difco), 1 o/c Bacto-peptone (Difco) and 2% glucose. For plating, the media were solidified with 2% Bacto-agar (Difco). Conventional genetical procedures for yeast-crossing, sporulation, and tetrad analysis were used. The genetical mapping
E. CAPIEAI!X E T A L
276 was performed according to Mortimer and Hawthorne (1975). From the results of the tetrad analysis, the distance between two markers was calculated in centimorgans as described by Snow (1979). Yeast transformation was carried out according to Dieckmann and Tzagoloff (1983).
DNA and RNA DNA constructions were carried out by standard techniques (Maniatis et al., 1982). DNA probes were labeled by nick-translation according to Rigby et aI. (1977). RNA was extracted from cells grown to a density of 3 x 107/ml by the procedure of Maccechini et al. (1979), and its concentration was determined by absorbance at 260 nm. Agarose gel electrophoresis, transfer of RNA from agarose gels to nitrocellulose paper. were carried out as described by Maniatis et ul. ( 1982). To determine the sizes of the transcripts identified by Northern hybridization, a lane containing total RNA was cut from each gel prior to blotting and stained with ethidium bromide to visualize the 18s and 2 5 s rRNA bands, whose sizes were taken to be 1.7 and 3.4 kb, respectively. RESULTS Physical analjsis We have subcloned 60 kb of DNA, extending from the centromere region to the TRPS gene on the left
1 6 -
subclones
-
15,
Transcriptional analjsis Within the 31 kb from the Hind111 site located from the PMAI promoter up to the first BamHI site located distal to ATE1 a total of 12 transcription products ranging from 0.6 to 3.6 kb were identified (Figure 2). Nine transcripts were unambiguously mapped in the 24 kb Hind111 fragment spanning PMAI and A T E ] . The mapping and the size of these transcripts allowed identification of those corresponding to the genes
I
- 5
l?181920M 21a1-+-21b 22
-
1Lb
arm of the chromosome VII. This tract comprises the inserts of two overlapping cosmidial plasmids, CosA and CosS (Figure 1). of 35 kb and 30 kb respectively. Both cosmids were issued from a genomic DNA library (kindly provided by Dr M.Guerineau, Universite de Paris Sud, Orsay. France) consisting of partial RamHI digests of S . cerevisiae strain IL1252B, cloned into the vector pHCG3 (Gerbaud et al., I98 I ) . CosA and CosS overlap in the LEUl region. CosA was initially used for cloning the pleiotropic drug resistance gene PDRI (Balzi et al., 1987) distal to L E U l . whereas CosS was isolated during the cloning of the plasma membrane ATPase gene P M A l , proximal to L E U l (Ulaszewski et al., 1987). The restriction maps of both cosmids were established and the subclones depicted in Figure 1, were used to locate the genes PMAl (Ulaszewski et 01.. 1087), LEUl (Skala et al., 1991), .sdI (Balzi ct (11.. 1989), PDRI (Balzi ct oi.,19871, PDR6 ( Chen et al., 1991a). ATEl (Balzi e f al., 1990) and TRPS (Balzi et al.. 1987) by complementation of mutant strains.
- 6
-9
?-
8-
10111
I
12a,-+
+ 4 1 2 b 13-
1 4 -
Figure I . The 60 kb region of chromosome VII. The inserts of the two overlapping cosmids. CosA (Balzi er a/..l987) and CosS (Ulas7ewski e r a / . . 1987) are shown. Physical and genetic maps are as previously descrihed (Balzi ef al.,1987, 1989. 19%; Ulaszewski er a/.,1987). Restriction sites presented for CosA are B u ~ I H I(B). llind111 (H). . M I (S). XhoI (X), SmaI (Sm). and S.sfI1 (St): for CosS only BamHI and Hind11 sites are presented. Under the cosmids, the following subclones are depicted and numbered as shown below within brackets: PA-X28 (la+lb), pA-X30 (2a+2b), PA-B6 ( 3 ) . pA-R6.5 (4). PA-B8 ( 5 ) . PA-B9 (6).pA-B3.5 (7). pA-B3 (8). pA-B8H (9).PAB8S(10),pA-X14(1l)(Balziera/.. 1987); pA-XS(12a+b).pA-H4(13)(Balziera/.. I~YO);pA-H6(14)(Bal7ieru/.. 1989):pSB12(15). CosS-Sub5.4 (16). CosS-Sub5 (17) (Ulaszewski cf a/.. 1987); pS1.6 (18). pE0.8 (19).pS4S(20), pS4K (2la+b). pS4B ( 2 2 ) .
Figure 2 RNA map of the PMAI-ATE1 region. (A) Restriction map with the approximate sizes (in kilobases), locations and identities of the transcribed regions (heavy lines). Restriction sites presented are: Hind111 (H), BarnHI (B), SalI ( S ) , SsrII (St), Xhol (X). Under the restriction map are depicted the DNA fragments used as radioactive probes for the Northern blot analysis shown in (B). (B) Autoradiograms of Northern blot hybridizations. Poly(A) RNA (lanes 1,3,8) and total RNA (lanes 2, 2’, 5.6, 7, 9, 10, 1I , 12) extracted from Succharomyces cerevisiae 1278b or GR350 (lane 6 ) were submitted to electrophoresis in an 1.5% agarose-formaldehyde gel and hybridized with the indicated DNA fragments. The transcript sizes indicated in kilobases were estimated as indicated in Materials and Methods. The intensities of the radioactive bands do not provide an estimation of the abundance of the RNA since probes with different specific activities were used.
1Kb
-
278
E. CAPIEAUX ET AL
PMAI (Ulaszewski et ul., 1987). LEUl (Skala et al., 1991). s d l (Balzi et al., 1989), PDRI (Balzi et 01.. 1987). PDR6 (Chen et al.. 1991a) and ATE1 (Balzi et al., 1990). The three unknown open reading frames YGL021, YGLO22. YGL023 described by Chen et 01. (1991b,c,d) could also be mapped precisely. However we have not mapped the three transcripts of 0.6, 0.9 and 2.7 kb identified in the BumHl fragment located distal to ATEI.
Genetical unu1ysi.s Classical tetrad analysis was carried out for genetic mapping of the mutations p m a l . leul. trp.5, pdrl and ade6. The strain S.cwe\isiae 20-584A (pmul-I) was crossed with a set of tester strains carrying centromere and auxotrophic markers as reported in Table 1. The results of 15 tetratypes for the crosses involving pmul and the centromere marker t r p l , indicated that the pmul gene is 4.0 cM away from a centromere. No recombination events were observed between leul and pmul after analysis of meiotic recombination in 400 asci. This is not surprising considering that the pmul-l mutation is located in the last third of the PMAI gene (Van Dyck et ul., 1990) and the leul mutation is located on the same strand in the first 153 bp from the 5' end of the LEU1 gene (Ulaszewski et ul., 1987). The two mutations are thus only 2.2 kb apart. The pdrl and leul loci are located 5.4 and 5.2 cM away from centromere VII. respectively. The positioning of p d r l , leul and pmal was estimated by a four point cross. The pmal mutation maps 2.3 and 9.3 cM from the pdr-1 and the trpS mutations respectively. The genetic distance between pdrl and trpS is 9.1 cM. The following genetic distances between the tested markers are thus estimated to be: 4.0 cM for centromere V11 topmal; less than 0.12 cM DIPLOID
GENE PAIR
US26, US53, US54 US6, US26 US6, US37 US62, US63 US62.US63 US^, US13, US62, US63, us202 US62, US63 US62, US63 US6. US7. US13, US62. US63 US7. US13 US7, US13. US202 US6, US13. US202
CEN7-pdrl CEN7-pmal CEN74eul trp5pdrl trohmal tipsieul leul-pdrl pmal-pdrl leul-pmal ade6-pmal ade6-leu1 ade6-trp5
Table I
for pmul to IC141; 2.3 cM for leu1 to pdrl and 9.1 cM for pdrl to trpS. DISCUSSION The 24 kb Hind111 DNA fragment spanning the PMAI and ATE1 genes has been totally sequenced. The sequence of LEUl (Skala et ul., 1991), PMAl (Serrano ct (11.. 1986; Capieaux et al., 1989; Van Dyck et al., 1990). s d l (Balzi et ol., 1989), PDKI (Balzi et ul., 1987) and ATEI (Balzi et ul., 1990) genes are published as well as those of PDR6 and of the 17 kb Hindlll fragment spanning the LEUl-ATE1 region (Chen et al., 1991a). Comparison of the transcript map and the DNA sequencing allows to conclude that all large open reading frames are expressed since a mRNA was detected for all open reading frames longer than 600 bp. The sensitivity of our method does not allow to detect smaller mRNA. Therefore additional smaller open reading frames, such as those described by Chen et al. (1991a). could well be also transcribed. On average. the difference between the size of a mRNA and that of the open reading frame predicted by DNA sequencing is about 300 nucleotides. The genes in the PMAI-TRP.5 region of chromosome V11 are closely packed. Taking into account only the detectable transcripts, there is one transcript of 2.3 kb average length every 2.7 kb of DNA within the 24 kb fragment spanning PMAI and ATEI. About 87% of the DNA region investigated is thus transcribed. The average intergenic sequence is 400 bp but varies from 218 (between scll and YGLO28) to 1232 bp (between YGL023 and PDR6). Four previous reports of transcript density, ranging from 67% to 85%. have been made for regions located on chromosome I, XV, and X of S.cerevisiuc (Struhl and Davis, 1981; Coleman et al., 1986; Steensma
TETRAD ANALYSIS TOTAL GENETIC MOLECULAR PD NPD T DISTANCE DISTANCE cM k SE (Kb) 100 83 89 86 32 37 139 0 0 231 246 0 162 0 162 0 400 0 46 2 54 3 49 6
22 15 8 31 53 56 8 8 0 66 75 77
205 190 77 170 284 302 170 170 400 114 132 132
5.4t1.4 4.021.0 5.2f1.8 9.1fl.l 9.3k1.1 93?1 1 23t1 1 2 3f1 1
>O 1tO
23.0 33.6 31 3 81 10 3 21
RECOMBINATION RATE (cM/Kb)
0.40 0.28
0 30 0 28 0 22 0 06
34.7t0.4 35.0+0.4 42.0C0.4
Comparison of genetic and molecular distances. The genetic distances were calculated from tetrad analysis according to Snow
(1989). Molecular distances were from the middle of the genes, except for pmal and leu1 mutations previously molecularly located. The strains used are described in Ulasrewski
( ' f
ul. (1987).
MAPPIKG O F A 24 K B DNA FRAGMENT FROM SACCHAHOMYCES CEfiEv'lSIAE
rt al., 1987; Barry et al., 1987). These studies, added
to ours, encompass a total of 83 kb of yeast DNA, among which a total of 35 long transcripts (tRNA excluded, Ty included), covering 80% of the DNA length were identified. An average transcript of 1.9 kb was detected every 2.4 kb. If we assume that this gene density is representative for the entire yeast DNA, which approximates 12,537 kb (rDNA excluded) ( Mortimer et al., 1989), a haploid S. cerevisiae genome should express about 5300 genes. This is a minimal estimate since, on one hand, small transcripts may not be detected and, on the other hand, some genes may be expressed in other growth conditions or genetic backgrounds than the ones investigated. Our results correlate well with early work carried out by genomic R-loop mapping which estimate the existence of 5000 transcribed regions in the entire yeast genome (Kaback et al., 1979). Comparison of the genetic distances with the physical distances between the markers CEN7, P M A I , L E U ] , PDRI and TRPS spread over 60 kb of chromosome VII, leads to the conclusion that in this centromeric region a recombination frequency of 1 cM unit corresponds to an average distance of 3.3 kb between alleles. This is slightly larger than the estimate of 2 4 4 kb/cM obtained by a much more global approach considering all genetic markers on all regions from all chromosomes (Mortimer et al., 1989) and it is more than twice the 1.59 kb/cM estimated for the entire chromosome I by Kaback et ul. (1989).
ACKNOWLEDGEMENTS This work was supported in part by grants to A. Gofteau from the Services de la Politique Scientifique: Action Science de la Vie and from the Fonds National pour la Recherche Scientifique, Belgium. NOTE During submission of this paper, we received the information that a transcriptional map of all the yeast chromosome 111 has been done by Yoshikawa and lsono (1990). By performing various Northern hybridization experiments, these authors identified 156 poly(A)' transcripts larger than 350 bp on the whole chromosome estimated to be 334.6 kb long. Also Melnick and Sherman (1990) reported the existence of six genes in a 7.5 kb segment located on chromosome X . These data fully justify the generalization of our data to the whole yeast genome.
279
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