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Jan 6, 1981 - of yeast. Verification that the leuB gene present on pEH25 was responsible for ..... This work was supported by the National Research Council.
MOLECULAR AND CELLULAR BIOLOGY, Sept. 1981, p. 836-842 0270-7306/81/090836-07$02.00/0

Vol. 1, No. 9

Genetic Complementation of the Saccharomyces cerevisiae leu2 Gene by the Escherichia coli leuB Gene REGINALD K. STORMS,t * EUGENE W. HOLOWACHUCK,: and JAMES D. FRIESEN§ Department of Biology, York University, Downsview, Ontario, M3J 1P3, Canada Received 6 January 1981/Accepted 1 June 1981

The leucine operon of Escherichia coli was cloned on a plasmid possessing both E. coli and Saccharomyces cerevisiae replication origins. This plasmid, pEH25, transformed leuA, leuB, and leuD auxotrophs of E. coli to prototrophy; it also transformed leu2 auxotrophs of S. cerevisiae to prototrophy. f8-Isopropylmalate dehydrogenase was encoded by the leuB gene of E. coli and the leu2 gene of yeast. Verification that the leuB gene present on pEH25 was responsible for complementing yeast leu2 was obtained by isolating in E. coli several leuB mutations that resided on the plasmid. These mutant leuB- plasmids were no longer capable of complementing leu2 in S. cerevisiae. We conclude that S. cerevisiae is capable of transcribing at least a portion of the polycistronic leu operon of E. coli and can translate a functional protein from at least the second gene of this operon. The yeast Leu+ transformants obtained with pEH25, when cultured in miniimal medium lacking leucine, grew with a doubling time three to four times longer than when cultured in medium supplemented with leucine. The expression of yeast genes in Escherichia coli (11, 16), the observation that the same region of the Saccharomyces cerevisiae his3 gene apparently functions as a promoter in both S. cerevisiae and E. coli (17), and the expression of the E. coli ft-galactosidase (10) and chloramphenicol acetyltransferase (2) genes in yeast cells, suggest that some yeast auxotrophs might be complemented by the analogous E. coli gene. Should a gene included in a well-characterized E. coli operon complement the analogous S. cerevisiae auxotroph, it would provide an excellent opportunity to compare gene expression in the two organisms. In particular, one could determine whether S. cerevisiae and E. coli recognize the same or similar transcriptional and translational control regions, whether S. cerevisiae is capable of expressing genes that are organized in an operon, and whether specific E. coli proteins can function in S. cerevisiae. Because the leu operon has been well characterized in bacteria (5) and the leu2 gene of S. cerevisiae complements the leuB gene of E. coli (11), we chose to study the expression of the E. coli leuB gene in S. cerevisiae. ,B-Isopropylmalate dehydrogenase is encoded by both leuB and leu2 (11, 15). In this paper, we describe the cloning of the

E. coli leu operon and demonstrate the ability of the E. coli leuB gene to complement leu2 mutations of S. cerevisiae. This is the first published report of genetic complementation of a mutant eucaryotic gene with a procaryotic gene. MATERIALS AND METHODS Yeast and bacterial strains, plasmids, and phage. The plasmids used were pYF36, pYF85, and pYF91 (15). pYF36 is a plasmid containing all of pBR313 and the yeast leu2 gene. pYF85 is a composite plasmid capable of replication in both E. coli (because it contains pBR322) and S. cerevisiae (because it contains the 3.6-kilobase EcoRI fragment from the 2-

iLm yeast plasmid) (9). pYF91 is a composite plasmid

derived from pYF85 by insertion of the yeast leu2 gene. These plasmids (pYF36, pYF85, and pYF91) have been described in detail earlier (15). The S. cerevisiae strains used were the wild-type S288C and LL20 (leu2-3 leu2-112 his3-11 his3-15), the latter from G. R. Fink. The E. coli strains used were CSH73 (Alac Aleu), JF1161 (hsdr lac gal metB), JF 1754 (hsdr lac gal metB leuB hisB436), JF2320 (leuA371 AlacU169 relA), JF2321 (leuB401 AlacU169 relA), and JF2323 (leuD211 AlacU169 reLA). The source of E. coli leu deoxyribonucleic acid (DNA) was a transducing phage, A dleulO, isolated as described by Schrenk and Weisberg (13). Media. The complex and minimal media used for the growth of E. coli and S. cerevisiae were described earlier (15).

t Present address: Department of Biology, Concordia University, Montreal, Quebec, H3G 1M8, Canada. Hybridization conditions. Restriction endonuclet Present address: Department of Research, Queen's Medase-digested DNAs of S. cerevisiae, E. coli, phage, and ical Center, Honolulu, HI 96808. § Present address: Department of Medical Genetics, Uni- plasmids were transferred from agarose gels of nitrocellulose membranes as described by Southern (14). versity of Ontario, Toronto, Ontario, M5S 1A8, Canada. 836

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cerevisiae his3 gene (15) yielded plasmid pRS28 (Fig. 1). Transformation of E. coli with pEH25, pEH26, pRS27, and pRS28. E. coli strains (8). Restriction endonucleases and T4 ligase. Re- JF2320 (leuA), JF2323 (leuD), JF1754 (leuB), striction endonucleases and T4 ligase were purchased and CSH73 (Aleu) were transformed with from Bethesda Research Laboratories (Bethesda, pYF85, pEH25, and pRS27 (Table 1). The reMd.) or Boehringer-Mannheim (Montreal, Canada) and were used as recommended by the supplier. The H E B conditions for ligations were the same as previously Hybridization was as described by Dawid (4). Radioactive phage A dleulO DNA was used as a probe for E. coli leu sequences and was prepared by nick-translation, in accordance with the method of Maniatis et al.

described (15). Transformation. The procedure for the transformation of E. coli was essentially that described by Mandel and Higa (7). The procedure for the transformation of S. cerevisiae was that described by McNeil et al. (9). DNA preparation. Large batches of covalently closed circular plasmid DNA were isolated from E. coli essentially as described by Clewell and Helinski (1). S. cerevisiae DNA was isolated by the method of Cryer et al. (3). Plasmid mutagenesis. Plasmid pRS28 was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine (NG). Mutagenesis of an E. coli strain carrying pRS28 was carried out by adding 10 ,g of NG per ml 5 min after the amplification of plasmid DNA was initiated with chloramphenicol. After incubation at 37°C for 12 h, plasmid DNA was isolated as described above. Rapid isolation of plasmid DNA from E. coli and S. cerevisiae Plasmid DNA was isolated from S. cereviseae the method of Livingston and Hahne (6), except that glusulase was used instead of zymolyase. Plasmid DNA was isolated from E. coli as described previously (13).

RESULTTS Cloning the E. coli leucine operon. Several A dleu-transducing phage were isolated as described by Schrenk and Weisberg (13). These phage were screened for their ability to transduce araD, leuA, leuB, and Aleu mutants to prototrophy. One of these transducing phage, A dkeul0, was found to carry all of these markers and was used as a source of leu operon DNA. A dleulO DNA was digested with several restriction endonucleases known to cleave the plasmid pYF85 (Fig. 1) only once (i.e., BamHI, HindIII, and SaII) and ligated with pYF85 DNA digested similarly. These ligated DNA preparations were then used to transform E. coli strain JF1754, and Apr IeuB+ transfornants were selected. Only BamHI-cleaved A dleulO DNA yielded LeuB+ transformants. Two plasmids, pEH25 and pEH26 (Fig. 1), containing a 7.4-kilobase BamHI fragment in either orientation, were isolated. Removal of a HindIII fragment from pEH26 yielded the plasmid designated as pRS27 (Fig. 1). Plasmid pRS27 contained an E. coli DNA fragment flanked by HindIII and BamHI restriction endonuclease sites. Insertion in pRS27 of the BamHI fragment carrying the S.

El

H E B EB

pYF85

pEH26

E

A

Y~~~~ 15Kb.

\ Ap

lou

7.6 Kb.

B

B

E

E

H A'

IOU

a

pRS27

11.4 E \A

K b.

0D

FIG. 1. Structures of the plasmids used in this study. A single thin line on the circumference represents pBR322 DNA; a single thick line outside the main circumference represents E. coli leu DNA or yeast leu2 DNA; regions marked by a second thin line inside the main circumference represent yeast plasmid 2-p.m DNA; regions marked by a second thin line outside the main circumference represent yeast his3 DNA. The arrows indicate an intact Tcpromoter and the direction of transcription from this promoter. The position of EcoRI, BamHI, and HindIII restriction endonuclease sites are indicated by E, B, and H, respectively. pEH25 and pEH26 are identical, except that the E. coli BamHI fragment containing the leucine operon is in opposite orientations in the two plasmids. pRS27 was derived by digestion ofpEH26 with HindIII followed by ligation. pRS28 was constructed by inserting the yeast BamHI genomic fragment containing the his3 gene into the unique BamHI site of pRS27. Kb, Kilobase. A, B (within main circumference), and C, genes of leucine operon; A' and D', incomplete A and D genes.

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sults indicate that both pEH25 and pRS27 were

MOL. CELL. BIOL.

pRS28. Transformation of S. cerevisiae strain LL20 (leu2) with plasmids pYF85, pEH25, pRS27, pRS28, and pYF91 was carried out (Table 2). All plasmids except pYF85 and pEH26 were capable of transforming LL20 to leucine prototrophy at a high frequency (Table 2); therefore, the E. coli DNA cloned into pEH25, pRS27, and pRS28 was responsible for the ability of these plasmids to complement leu2. pRS28LL20 transformants selected for His' were all found to be Leu+ when replicated to a medium lacking leucine. Visible colonies from the pEH25, pRS27, and pRS28 Leu+ transformants took an average of 10 to 12 days to appear in the regeneration agar, whereas pYF91 Leu+ transformants or pRS28 His' transformants appeared in only 1.5 days (Table 2). These results suggest either that leuB was not efficiently expressed in S. cerevisiae or that the leuB-encoded enzyme did not function efficiently in yeast. pEH26 was unable to transform strain LL20 to Leu+, an unexpected result because pEH26 was identical to pEH25, except that the BamHI fragment containing the leucine operon was in the opposite orientation in the two plasmids (Fig. 1). This suggests that in S. cerevisiae, the leuB gene was not expressed from a promoter on the cloned E. coli DNA but was expressed from somewhere on the original plasmid pYF85. Alternatively, leuB could have been expressed from a region on the cloned E. coli DNA, but expression was prevented by pYF85 sequences upstream from the leuB gene in pEH26. Analysis of DNA isolated from the pEH25-yeast transformants. Several independent pEH25 transformants were isolated and grown to mid-log phase in medium lacking leucine; then total DNA was isolated from these

capable of transforming leuA, LeuB, and leuD mutants of E. coli to prototrophy. Therefore, all or a portion of all four genes of the leu operon were present on pEH25 and the smaller plasmid, pRS27. Transformation with pRS27 of the E. coli leuA strain JF2320 (Fig. 1), followed by selection on medium lacking leucine produced very few colonies relative to the numbers obtained when the same transformation was carried out by using pEH26 or when Apr transformants were selected by using pRS27 (Table 1). This suggests that removal of the HindIlI fragment from pEH26 in the construction of pRS27 deleted the promoter-proximal portion of the leuA gene and that acquisition of prototrophy by the leuA mutant occurred by recombination. This observation is supported by the DNA sequence analysis of leuA carried out by S. Wessler and J. Calvo (personal communication), who found that the HindIII site is within the leuA gene, approximately 100 base pairs from its 5' end. Apparently, pEH25 also lacks a portion of the leu operon because it did not complement CSH73 (Aleu), an E. coli strain carrying a deletion of the entire leu operon. However, pEH25 was capable of complementing the E. coli leuD point mutant, JF2323 (Table 1). These observations are consistent with the presence of a BamHI cleavage site in the leuD gene (the leuD protein fragment encoded by pEH25, pEH26, and pRS27 formed a functional complex with the leuD protein produced by the point mutant JF2323). A dleulO carried the intact leu operon because it complemented CSH73 (Aleu) and all the leucine point mutants. Transformation of S. cerevisiae with pYF85, pYF91, pEH25, pEH26, pRS27, and

TABLE 1. Transformnation and transduction of E. coli leu mutantsa JF1754 (leuB) JF2320 (leuA) JF2323 (leuD) Transformant Leu+ Ampr Amp' Leu+ Leu+ prb

pYF85 ............................... .................... pYF91 pEH25 .. . .. pEH26 ........... pRS27 ...... ... pRS29, pRS30, pRS31, pRS32d X dleulOf ..

312 590 433 211 414 664

0 850 278 183 380 0 +

210 682 30 21 263 332

0 0 18 27 2 7 +

148 260 29 65 194 82

0 0 37 187 215 156 +

CSH73 (Aleu)

Amp'

Leu+

208 732 1610 827 856

0 0 0 0 0

ND" +

a All transformations were carried out with 0.5 ,ug of plasmid DNA. The transformation frequencies ranged from 103 to 105 transformants per .tg of DNA. b Number of colonies that grew on LB agar (15) containing 50 ug of ampicillin per ml. C Number of colonies that grew when the same number of transformed cells as was plated on the ampicillincontaining LB agar was plated on agar lacking leucine. d Each value listed for this group indicates the mean value obtained with these plasmids. 'ND, Not deternined. f Lysogens of A dleulO and A cI857 S7 were constructed and 3creened for Leu+.

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TABLE 2. Frequency and efficiency of transformation of yeast strain LL20a No. of days for Transformants/yg

colonies to appear in regeneration

Doubling (h) in medium time containing:

agar

Plasmid

Leu'

His+

Leu+

His+

NAb

NA NA NA NA NA 1.5 1.5 NA

Histidine

Histi-

Leucine dine +

leucine

NA NA NA NA 2.4 2.2 6.7 NA 2.2 ND ND NDC pEH26 ............................... .0 10.2 NA 2.3 pRS27 .14,100 12 2.5 2.3 pRS28 .14,600 17,000 0 16,600 NA 2.5 2.2 pRS29, pRS30, pRS31, pRS32d ......... 2.2 NA 0 2.2 26 pYF36 .............................. a Yeast strain LL20 was transformed with the plasmids indicated. The cells from each transfornation were suspended in regenerating agar lacking leucine or histidine. These plates were then incubated at 300C for at least 60 days, when the total number of Leu+ and His+ colonies were determined. The number of colonies present within the regenerating agar was also scored each day. When Leu+ transformants were obtained, several of these were picked and grown for another 30 generations under the selective condition. Then four of these Leu+ isolates were selected, and their doubling times were determined in minimal medium containing histidine, leucine, or histidine plus leucine. b NA, Not applicable. ND, Not determined. d Each value listed for this group indicates the mean value obtained with these plasmids. 0 pYF85 .............................. pYF91 ...............................35,000 pEH25 ...............................11,500

0 0 0 0 0

1.5 9 NA 10 10 NA 2

c

transformants, from the yeast strain S288C and from the E. coli strain JF1161. The DNA was

digested with restriction endonuclease BamHI, and the DNA fragments were resolved by agarose gel electrophoresis (Fig. 2A). The DNA was then transferred to nitrocellulose filters (14) and hybridized with 32P-labeled X dleulO DNA. The results of these hybridizations (Fig. 2B) show that the 7.4-kilobase fragment present on pEH25 (Fig. 2B, lane 4) and pEH26 (Fig. 2B, lane 5) was also present in E. coli (Fig. 2B, lane 1) but was not present in S. cerevisiae (Fig. 2B, lane 2). On the other hand, the leucine-independent transformants of strain LL20 that were obtained by transformation with pEH25 all had the 7.4-kilobase E. coli DNA fragment (Fig. 2B, lane 3). The hybridization in lane 3 of Fig. 2B is typical of all four Leu+ transformants analyzed. Strain JF1754 (leuB hisB) was transformed with total DNA isolated from four pEH25 yeast transformants. All the Apr colonies obtained were also Leu+. Plasmids were isolated from 40 of these Apr transformants. The restriction endonuclease maps of these retrieved plasmids were indistinguishable from pEH25, except for a few that had acquired one copy of 2-tm DNA (data not shown). This phenomenon, the acquisition of an additional copy of the 2-am plasmid,

has been studied in detail (9, 15). Isolation of leuB- derivatives of pRS28. Mutagenized preparations of plasmid pRS28 were prepared as described above and were used

to transform JF1754. Apr colonies were selected and screened for the Leu- His' phenotype. Plasmids were isolated from four independent colonies and designated as pRS29, pRS30, pRS31, and pRS32. These plasmids were used to transform S. cerevisiae strain LL20, as well as the five E. coli leucine mutants. All four plasmids were capable of transforming E. coli leuA and leuD mutants to prototrophy but did not complement leuB (Table 1). In addition, these four leuB- mutant plasmids did not complement the leucine auxotrophy of S. cerevisiae strain LL20, although they did complement the his3 locus in that strain (Table 2). Restriction endonuclease analysis of the four leuB- mutant plasmids indicated that they were indistinguishable in structure from the original plasmid, pRS28 (data not shown). These results show that the leuB gene on plasmid pRS28 was responsible for complementing the yeast leu2 gene. Characteristics of strain LL20 transformed with pEH25. The ability to retrieve

the original plasmid (see above), the fact that plasmids pEH25, pRS27, and pRS28 transformed S. cerevisiae at a high frequency (Table 2), and the instability of the transformants (data not shown) indicate that the plasmids replicate autonomously in the yeast host. The growth rates in minimal medium lacking leucine of several pEH25, pRS27, and pYF91 transformants were compared with the growth rate of the parent strain, LL20, supplemented

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tive conditions.

A

B$

FIG. 2. (A) Restriction endonuclease BamHI digestion patterns of DNA samples. Lane 1, 2 pg of E. coli; lane 2, 5 pg of yeast strain LL20; lane 3, 5 pg of pEH25-transformed LL20; lane 4, 0.05 pg of pEH25; lane 5, 0.05 pg ofpEH26. (B) Autoradiography of 32P-labeled X dleulO DNA hybridized to a Southern blot (14) of the BamHI restriction digests shown in (A). The arrows in (A) and (B) mark the position of the 7.4-kilobase fragment.

with leucine. The pEH25, pRS27, and pYF91 transformants were grown for 30 generations in medium lacking leucine before the measurement of growth rates. In medium lacking leucine, pEH25 and pRS27 transformants grew with doubling times 3- and 4.3-fold greater, respectively, than those of pYF91 transformants or pEH25 transformants grown in medium supplemented with leucine (Table 2). Although pEH25 transformants grew with a doubling time of about 6.7 h, this occurred only after the cells had been growing for several generations under selective conditions. For example, the original pEH25 transformants did not appear as colonies in the regeneration agar for 10 days, whereas pYF91 transformants appeared after only 1.5 days. Upon restreaking, pEH25 transformants took only 2.5 days to appear as colonies. These observations indicate that pEH25 transformants initially grew very poorly but adapted or mutated during growth in the selec-

To determine whether these pEH25-transformed cultures had adapted to growth without leucine by a mutational event on the plasmid, plasmid DNA was retrieved from several pEH25-transformed LL20 cultures as described above. The retrieved plasmids were then used to transform LL20. These LL20 transformants appeared after 10 days, the same as for the original plasmids. It is possible that the cultures had adapted by a mutational event that occurred on the yeast genome. That such a mutation had not occurred was shown by curing several pEH25-LL20 transformants of the transforming plasmid by growth in yeast extract-peptone-dextrose medium for about 20 generations and screening for Leu- cells by replica plating. Three of these Leu- isolates were transformed with pEH25. These transformants also required 10 days before the first colonies appeared in the regeneration agar. It therefore appears that the pEH25 transformants examined did not adapt by a mutational event on the plasmid or on the yeast genome. DISCUSSION The data presented here demonstrate that the second gene of the E. coli leu operon, leuB, was functionally expressed in S. cerevisiae. Yeast leu2 mutant cells harboring plasmid pEH25 or pRS27 are capable of growth in the absence of leucine. Analysis of plasmids retrieved from several independent isolates of strain LL20 that harbor pEH25 indicates that the retrieved plasmids were indistinguishable from the original plasmid. Both the restriction endonuclease map and the ability of the retrieved plasmids to complement E. coli leuA, leuB, and leuD mutants were unaltered. These observations, plus the facts that pEH25 transformed LL20 to Leu+ at a high frequency and that all LL20 clones tranforned to His' by pRS28 are also Leu+ (data not shown), demonstrate that the transforming plasmid was capable of expressing the E. coli leuB gene in S. cerevisiae without alteration. However, it is not necessarily true that the leuB gene was transcribed from the same promoter region in S. cerevisiae as in E. coli. In fact, plasmid pRS27 had a deletion of the Leu promoter region, as was shown by the inability of pRS27 to complement leuA mutants with high effiency (Table 1). Moreover, the sequence data of Wessler and Calvo (personal communication) indicate that the HindIII restriction endonuclease site in the E. coli fragment carried by pEH25 was approximately 100 base pairs from the 5' end of the leuA structural gene. This indicates that in plasmid pRS27, transcription

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of the leuB gene, either in E. coli or S. cerevisae, between the parent and its daughter; successive did not originate from the leucine promoter. division with unequal segregation in the presRather, transcription must have started at a ence of selection for those cells with a higher sequence that was upstream from the HindIII plasmid copy number would result in an overall site, possibly a 2-,um promoter or a region within increase in plasmid copy number in the culture. the leu operon adjacent to leuB. Because neither plasmids retrieved from the Plasmid pEH26 did not transform LL20 to original LL2OpEH25 culture nor Leu- segregants leu+, although it did transforn E. coli leucine obtained from the adapted strains carried mumutants to prototrophy. These results suggest tations that enable new transformants to grow that in yeast, the leucine operon was not tran- quickly, the third mechanism appears to have scribed from its natural promoter and that the been responsible for the adaptation. leuB gene was translated from a message whose ACKNOWLEDGMENTS transcription was initiated in sequences outside We thank Isobel Matthews for technical assistance and the E. coli BamHI fragment. Removal of the HindIII fragment from pEH26 could create or Gordon Temple for photographic expertise. We are also grateto G. B. Kiss, R. E. Pearlnan, and Kathleen Cornish for make available a promoter from which the leuB ful valuable discussion and careful reading of the manuscript and gene of pRS27 could be expressed in S. cerevi- to Tina Sutherland and Krystyna Tarkowski for editorial siae.

Alternatively, the expression of the leuB gene from both pEH25 and pRS27 in S. cerevisiae may be from promoter sequences within the E. coli fragment, but in pEH26, expression of the leuB gene was inhibited by sequences adjacent to and upstream from the leuB gene. Because plasmid pRS27 was capable of transforming LL20 to Leu+, the HindIII fragment removed from pEH26 in the construction of pRS27 could contain the putative upstream sequences that prevented expression of leuB from pEH26 in yeast. The only sequences upstream from leuB and present on this HinduI fragment but not present upstream in pEH25 were those within the small HindIII-to-BamHI fragment. It is worth noting that this fragment contains a portion of the Tc promoter (Fig. 1) (12). An interesting possibility is that the bacterial Tc promoter prevents expression of leuB in pEH26. Preliminary results with a pRS27 derivative containing the his3 promoter cloned into the unique HindIII site of pRS27 show that the presence of a yeast promoter upstream and in the same reading direction as the leuB gene prevented the expression of leuB in S. cerevisiae. Experiments are in progress to determine the size, amount, and point of origin of the leu transcription products in cells carrying these various plasmids. pEH25 transformants adapted to a faster growth rate in the absence of leucine (Table 2). This adaptation could have resulted from one or more ofthree different mechanisms. One, a chro-

mosomal mutation occurred that increased the growth rate. This could have given rise to a steady decrease in the doubling time as the mutants accumulate in the population. Two, a mutation occurred on the plasmid, and this was responsible for the increased growth rate. Three, the increase was the result not of a mutational event but of unequal segregation of plasmids

assistance. This work was supported by the National Research Council (grant 3-640-299-10) and the National Cancer Institute of Canada. R.K.S. was supported in part by a National Research Council Postdoctoral Fellowship. E.W.H. was supported in part by a Province of Ontario Graduate Scholarship.

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13. Schrenk, W. J., and R. A. Weisberg. 1975. A simple method for making new transducing lines of coliphage A. Mol. Gen. Genet. 137:101-107. 14. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by electrophoresis. J. Mol. Biol. 98:503-517. 15. Storms, R. K., J. B. McNeil, P. S. Khandekar, G. An, J. Parker, and J. D. Friesen. 1979. Chimeric plasmids

MOL. CELL. BIOL. for the cloning of deoxyribonucleic acid sequences in Saccharomyces cerivisiae. J. Bacteriol. 140:73-82. 16. Struhl, K., J. R. Cameron, and R. W. Davis. 1976. Functional genetic expression of eukariotic DNA in E. coli. Proc. Natl. Acad. Sci. U.S.A. 73:1471-1475. 17. Struhl, K., and R. W. Davis. 1980. A physical, genetic and transcriptional map of the cloned his3 gene region of Saccharomyces cerivisiae. J. Mol. Biol. 136:309-332.