YEAST VOL. 12: 411413 (1996)

-IV Yeast Mapping Reports

Physical Mapping of a Centromere-Proximal Region of Chromosome IV-L Defines the Placement of Genes USOl, MBPl, PSAl and SLCl D. C. J. GARDNER, G. C. TOMLIN, T. CELE, G. A. HAMILTON, C. M. JAMES, L. I. STATEVA AND S. G. OLIVER*

Department of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology (UMIST),” P. 0. Box 88, Manchester M60 1 QD. U. K

Received 16 July 1995; accepted 19 September 1995

A physical map of a 14.5 kb region close to the centromere on the left arm of chromosome IV of Saccharomyces cerevisiae is presented. This map has been constructed by restriction analysis of a clone from a YCpSO genomic library and by use of pre-existing and new sequence data from this region. The map reveals the following gene order (reading from the most centromere-distal to the most centromere-proximal locus): USOlIINTl-MBPl-PSAl- SLCl-YLAl and defines the size of the open reading frames and intergenic regions.

KEY WORDS ~ Saccharomyces cerevisiae; chromosome IV; USOl; INTI; M B P l ; PSA1; SLCl; YLAl

In the course of characterizing the SRBl gene, which is involved in the maintenance of osmotic stability in Saccharomyces cerevisiae (Venkov et al., 1974; Kozhina et al., 1979), we have isolated a clone from the Rose et al. (1987) YCp50 genomic library which contains a 14.5 kb fragment of yeast chromosome IV. Physical mapping and sequence analysis of this fragment demonstrated that it contains a number of known genes (USOI, MBPl and PSAI) and allowed the refinement of the current version of the S. cerevisiae physical map (edition 12; Mortimer et al., 1995). In this commu- nication, we report our restriction mapping and sequence analysis data for this clone and propose a revised version of this region of the chromosome IV map based on these results.

S. cerevisiae strain 7SLU (MATu ura3 leu2 srbl-I; Kozhina et al., 1979) was transformed with DNA from a genomic bank of S288C chromo- somal DNA cloned into the single-copy vector YCp50 (ATCC37415; Rose et al., 1987). Trans- formants capable of growth in the absence of an osmotic stabilizer (i.e. Srbl +) were selected and

*Corresponding author.

five clones were obtained, all of which contained the same 14.5 kb insert (SRB1-2; Figure 1). In a preliminary attempt to characterize this insert, five EcoRI fragments, of approximate sizes 0.4, 0.5, 1.1, 1.2 and 2.0 kb, were sub-cloned (see Figure 1) into pUC19 (Yanisch-Perron et al., 1985) and single-pass sequencing was performed on each end of the sub-fragments using the universal forward and reverse primers (Table 1). All clones and sub-clones were propagated in E. coli DH5u, an endA host strain. (See James et al. (1995) for a recent account of the sequencing methodology employed in our laboratory.) This identified por- tions of the MBPl (Koch et al., 1993) and SLCl (Nagiec et al., 1993) genes, allowing us to assign our clone to a centromere-proximal region of the left arm of chromosome IV (Mortimer et al., 1995).

To define precisely the extent of the cloned region, we sequenced the two ends of the original 14.5 kb insert, using custom primers designed to anneal to the YCp50 sequences on either side of the BamHI cloning-site (DG1 and DG3; Figure 1 and Table 1). These terminal sequences identi- fied genes USOIIINTI (Nakajima et al., 1991;

CCC 0749-503X/96/040411-03 0 1996 by John Wiley & Sons Ltd

412 D. C. J. GARDNER ET AL.

u19608

X54378/Lo3188 X74158 1-

L13282 * Centromere +

EE EE HH E E E H H H f E H S E II

i I psA7 ’ ’ ~ + L C l ~ H A

* * DG 1 DG2

+ * + f M13-F DG4 M13-R DG3

m1-2

- 0 1 2 3 4 5

kb

Figure 1. A physical map of the US01 - YLAl region on the left arm of chromosome IV. A restriction map showing the relative positions of EcoRI (E), Hind111 (H) and SalI (S) sites is presented, in which the open boxes represent the open reading frames of the indicated genes. Above this map, the extent of sequence data already entered into the public data libraries is shown, with the accession numbers of the relevant entries. Below the restriction map, the regions covered by the primary YCp50 clone (SRBI-2) and two of the sub-clones (SRBI -6a and SRB1-5) are indicated.

Table 1. Sequencing primers used in this study.

Name Oligonucleotide sequence Length ~~

DG1 5’-CTGCTCGCTTCGCTACTTGGAGC- 3’ 23-mer DG2 S’-ACTACACGGAAGTCAATCTCAC-3’ 22-mer DG3 S’-CGGTGATGTCGGCGATATAGG-3’ 21-mer DG4 S‘-AAGAATGCTGACTGTACGCG-3’ 20-mer MI3 forward S’-CGCCAGGGTTTTCCCAGTCACGAC-3’ 24-mer M13 reverse S’-AGCGGATAACAATTTCACACAGGA-3‘ 24-mer

Hostetter et al., 1993) and, again, SLCl (Nagiec et al., 1993). A primer (DG2; Table l), comp- lementary to the published INTI sequence allowed us to extend our sequence to the right, into a region which corresponded to the published se- quence for the MBPl gene (Koch et al., 1993). A similar approach was employed to extend the SLCl sequence leftwards, but this sequence re- vealed no homologies in the data libraries. Subse- quent searches of the data libraries revealed an overlap between the published MBPl sequence and a new entry (accession number U19608), cor-

responding to the gene PSAl (Benton et al., 1995). This PSAl sequence was used to design a primer (DG4; Table 1) which permitted extension of this sequence further to the right, although no further homology to data library entries was revealed. The PSAl sequence, plus our own restriction mapping, allowed us to predict the existence of a 2.25 kb Sun fragment containing the 3’ end of PSAl and its, so-far uncharacterized, centromere-proximal flanking region. This was sub-cloned into pUC19 (SRBl-6a; Figure 1) and the ends were sequenced using universal forward and reverse primers

MAPPING CHROMOSOME IV REGION 413

(Table 1; M13-F and M13-R in Figure l), con- firming the overlap with PSAl to the left and further extending the chromosome sequence to the right.

By combining our restriction maps of the original YCp50 clone (SRB1-2; Figure 1) and those of the sub-clones (SRB1-6a and SRB1-5; Figure l), together with published sequences and our own sequence data, we were able to assemble the map of this region of chromosome IV shown in Figure 1. We compared the results with the current genetic and physical maps of this region as pre- sented in the Stanford Saccharomyces Genome Information Resource (SGD; http:llgenome-www. stanford.edu/). None of the genes which we have assigned to this region has yet been mapped genetically (Mortimer et al., 1995). The Mortimer et al. (1995) physical map does not include assign- ments for USOIIINTI or PSAl and does not provide the relative order of SLCl and YLAl within the chromosome arm. Moreover, the ‘Mortimer’ map indicates a distance of some 35 kb for the MBPl-YLAl region, whereas our data indicate one of only 10 kb. Thus our revised map (Figure 1) permits the resolution of all of these issues and allows the refinement of the physical map of this region of the left arm of chromosome IV in terms of both gene order and intergenic distances.

ACKNOWLEDGEMENTS

We would like to thank Mark Nagiec and Ben Benton for their generous gifts of strains and clones and for much helpful advice. This work was supported by the BBSRC Biotechnology Directorate who provided a research project grant to S.G.O. and studentships to G.C.T. and G.A.H. (who is a student of the Advanced Centre for Biochemical Engineering, University College, London). T.C. was supported by the Canon Collins Educational Trust. We also thank Pencho Venkov and Eli Keshavarz-Moore for their support and encouragement.

REFERENCES Benton, B. K., Driscoll Plump, S., Roos, J., Lennarz,

W. J. and Cross, F. R. (1995). Overexpression of S. cerevisiae G1 cyclins restores viability of algl N-glycosylation mutants. Curr. Genet., in press.

Hostetter, M. K., Herman, D. J., Bendel, C. M., Tao, N. and Kendrick, K. E. (1993). An integrin analogue in Saccharomyces cerevisiae. Accession number LO3 188.

James, C. M., Indge, K. J. and Oliver, S . G. (1995). DNA sequence analysis of a 35 kb segment from Saccharomyces cerevisiae chromosome VII reveals 19 ORFs including RAD54, ACEIICUPZ, PMRI , RCKI, AMSI and CALIICDC43. Yeast, 11, 1413- 1419.

Koch, C., Moll, T., Neuberg, M., Ahorn, H. and Nasmyth, K. (1993). A role for the transcription factors Mbpl and Swi4 in progression from G1 to S phase. Science 261, 1551-1557.

Kozhina, T., Stateva, L. I. and Venkov, P. V. (1979). Genetic analysis of an osmotic sensitive Saccharomy- ces cerevisiae mutant W1160. Molec. Gen. Genet.

Mortimer, R. K., Cherry, J. M., Dietrich, F. S., Riles, L., Olson, M. V. and Botstein, D. (1995). Genetic and physical maps of S. cerevisiae, edition 12. http:ll genome-www.stanford.edu/.

Nagiec, M. M., Wells, G. B., Lester, R. L. and Dickson, R. C. (1993). A suppressor gene that enables Saccharomyces cerevisiae to grow without making sphingolipids encodes a protein that resembles an Escherichia coli fatty acyltransferase. J. Biol. Chem. 268, 221 56-22163.

Nakajima, H., Hirata, A., Ogawa, Y., Yonehara, T., Yoda, K. and Yamasaki, M. (1991). A cytoskeleton- related gene, USOI, is required for intracellular pro- tein transport in Saccharomyces cerevisiae. J. Cell Biol. 113, 245-260.

Rose, M. D., Novick, P., Thomas, J. H., Botstein, D. and Fink, G. R. (1987). A Saccharomyces cerevisiae genomic plasmid bank based on a centromere- containing shuttle vector. Gene 60, 237-243.

Venkov, P. V., Hadjiolov, A. A., Battaner, E. and Schlessinger, D. (1974). Saccharomyces cerevisiae sorbitol dependent fragile mutants. Biochem. Biophys. Res. Commun. 56, 559-604.

Yanisch-Perron, C., Vieira, J. and Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33, 103-119.

170, 351-354.