Direct cloning of yeast genes from an ordered set of lambda clones in Saccharomyces cerevisiae by recombination in vivo.

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Copyright 0 1999 by the Genetics Society of America

Direct Cloning of Yeast Genes from an Ordered Set

of Lambda Clones in

Saccharomyces cerevisiae by Recombination

in

Vivo

James

R. Erickson and Mark Johnston

Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 6 3 1 1 0 Manuscript received March 9, 1992

Accepted for publication July 29, 1992

ABSTRACT

We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Esherichia coli-yeast shuttle plasmid carries URA3, ARS4

and C E N 6 , and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura+ transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement

the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E . coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3)

they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSNZ and M A K 7 , each of which presents one of these problems for cloning.

I

N

yeast, cloning a gene by complementation of a recessive mutation is usually a relatively simple matter. However, a number of factors can limit t h e ability to clone a particular gene. First, libraries am-

plified in Esherichia coli can be depleted of some genes. For example, RAD4 is lethal to bacteria (SIEDE a n d ECKARDT-SCHUPP 1986); K A R 2 confers a significant growth disadvantage upon the host (ROSE, MISRA a n d VOGEL 1989). Such problems are exacerbated with successive amplifications of a library. In addition, since many circulating libraries were constructed using partially digested, size selected yeast genomic

D N A ,

t h e availability of restriction sites greatly influences the probability of cloning any stretch of

DNA.

A

second factor that can contribute to difficulty in cloning a gene is the phenotype caused by the mutation used in screening of the library.

As

yeast genetics becomes increasingly sophisticated, the phenotypes of

mutations can be increasingly subtle. In some cases the phenotype of the mutant is too subtle to allow rare complementing clones to be identified. In other cases, the phenotypes of the mutant can only be scored after laborious manipulations, which severely reduces the number of transformants that can be screened for

the complementing gene.

All

these factors have made some yeast genes difficult t o isolate by conventional methods.

In this paper, we describe how we have solved these

Genetics 1 3 4 151-157 (May, 1993)

problems by adapting the method of MA et al. (1987)

for the purpose of cloning yeast genes. The method is based on the observation that a double-strand break in a plasmid can be repaired by cotransforming with a linear fragment containing

D N A

sequences that flank the double strand break (ORR-WEAVER, SZOS-

TAK a n d ROTHSTEIN 1 9 8 1 ; KUNES, BOTSTEIN a n d

Fox

1985). Yeast

D N A

sequences are rescued from

lambda clones by cotransforming yeast with the lambda clone and linearized plasmid that contains

phage lambda sequences that flank the cloned yeast

DNA.

T h e cloned yeast sequences are thus trans- ferred to a yeast centromere plasmid; the resulting colonies can then be screened for complementation.

This method takes advantage of the restriction map of the yeast genome compiled by RILES et al. (1 993), which provides lambda clones that cover 98% of the yeast genome. If the genetic map position of a particular gene is known, the lambda phage carrying it can be identified by testing for complementation of a mutation of only those lambda clones that span t h e region or, in the worst case, the entire chromosome.

We have successfully applied this method to clone three genes: GAL83 (MATSUMOTO, TOH-E a n d OSH-

IMA 1 9 8 1 ; MATSUMOTO, YOSHIMATSU a n d OSHIMA 1983), a gene apparently not present in any of four genomic plasmid libraries screened; SSN2 (CARLSON

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152 J. R. Erickson and M. Johnston

be complemented easily in a plate assay; and M A K 7

(WICKNER and LIEBOWITZ 1976), a gene whose phe- notypic effects are laboriously scored.

MATERIALS AND METHODS

Strains and media: E. coli strain DH5a (Bethesda Re- search Laboratories) was the host for all plasmids. Yeast strains used in complementation tests for identifying the various genes were: YM3033 (MATa, ura3-52, hid-200, ade2-101, lys2-801, ga180A538 gal83-2000, LEU2::GALl- lacZ); MCY447 (MATa, ura3-52, ade2-101, his4-539, snfl-

28, ssn2-4) obtained from M. CARLSON; 2778 (MATcu, mak7-1, ura3, met5 L-A-HN, M-a, rho") obtained from R.

WICKNER. Yeast strain 1074 (MATa, leul, karl-1, L-A-HN, M , ) was used to cytoduce killer RNA, and 5x47 (MATa/ MATa, hisl/+, trpl/+, u r d / + M-a) is the killer-sensitive

tester strain.

LB medium containing 100 pg/ml ampicillin was used to grow all bacterial strains harboring plasmids. Standard SD medium and supplements for yeast are described in ROSE, WINSTON and HIETER (1990). Amino acids and nucleotide base supplements were omitted where appropriate. 2-deoxy- D-galactose (2dGal) was added to 0.25%; 4.7 MB plates used to score the killer phenotype were described by WICKNER and LIEBOWITZ (1 976). Plates containing 5-flouro-orotic acid were described by BOEKE, LACROUTE and FINK (1984).

Plasmid construction: T h e CEN-ARS plasmid pBM2240

used to recover lambda clone inserts is based on pRS316 (SIKORSKI and HIETER 1989).?'he lambda DNA fragment flanking the yeast DNA insert on the right was an 858-bp BamHI-Xbal fragment generated by PCR using as template the lambda cloning vector MG3 (derived from the lambda cloning vector 147.1 (LOERAN and BRAMMER 1980)). The PCR was primed with the following oligonucleotides: 5'-

GCTCTAGAGATCTGCCAGATTACGCTCCTG-3' and

T h e PCR product was digested with BamHl and XbaI, gel purified and subcloned into the BamHI-XbaI site of pRS3 16. T h e lambda DNA fragment flanking the yeast DNA insert on the left was a 1.5-kb KpnI fragment from MG3 extending from nucleotide 17053-18556 (DANIELS et al. 1983). This fragment was subcloned in both orientations into the plasmid containing the BamHI-XbaI fragment. T h e orientations were verified by DNA sequencing. T h e plasmid containing the two lambda fragments i n the same relative orientation as they occur in lambda was designated pBM2240. T h e plasmid with the KpnI fragment in the opposite orientation was designated pBM2241. Plasmid pBM2384, a high-copy plasmid used to recover lambda clone inserts, was constructed by subcloning the Kpnl fragment and the BamHI- XbaI fragment of lambda from pBM2240 into the 2-pm- based vector pRS426.

DNA manipulations: Lambda clone DNA was prepared

from a 5-ml liquid lysate using Lambdasorb (Promega), according to the manufacturers suggested protocol for liquid lysates (Promega technical bulletin 32). Lysates were prepared by pulling an agar plug with the large bore end of

a Pasteur pipet and eluting in 20 mM Tris.CI, pH 8

+

20

mM MgCI, at 4" overnight. 100 pl of the phage eluate were combined with 100 pl 10 mM CaClp

+

10 mM MgC1.L and 100 pI of a saturated culture of E . coli strain C600 and incubated for 30 min at 37". Five ml of prewarmed NZC broth (per liter: 10 g NZ amine, 5 g NaCI, 5 g yeast extract, 1 g casamino acids, 2 g M g S 0 4 . 7 H 2 0 ) was added and incubated with vigorous aeration until the culture lysed (8- 10 hr). Cell debris was spun down and the supernatant was

5'-CGGGATCCCTGGAAGAAATACTCATAAGCC-3'.

treated with 0.5 PI 10 mg/ml DNase

+

2.5 PI 10 mg/ml RNase for 30 min at 37" prior to adding adsorbent. Recom- binant plasmids were recovered in E. coli from yeast lysed with glass beads (HOFFMAN and WINSTON 1987).

Yeast transformation and phenotype scoring: Yeast

transformations were performed by the spheroplast method (BURGERS and PERCIVAL 1987). In a typical experiment to rescue lambda clones, 100-200 ng of pBM2240 digested with EcoRI and XhoI and 1-2 pg of lambda clone DNA were used to transform yeast to Ura+. After digestion with EcoRI and XhoI, the vector DNA was phenol extracted, ethanol precipitated and resuspended in T E pH 8 at 100 ng/pl prior to transformation. Transformants were scored for the presence of the HIS3 or GAL83 genes by replica plating to SD- Ura-His or SD-Ura+ 0.25% SdGal, respectively. T h e clumpy growth of ssn2 mutants was scored after growth of transfornlants in liquid SD-Ura media. T h e phenotype of mak7 mutants was scored as described by WICKNER and

LEIBOWITZ (1976). To score for mak7 complementing activity, Ura+ transformants from each transformation plate were picked to SD-Ura. These isolates were grown overnight at 30" and mated with the killer and mitochondrial donor, 1074, to cytoduce killer plasmid. After 6 hr at 30" the mating mixture was streaked for single colonies onto SD- Ura-Leu to select for the inheritanced of the plasmid (Ura') and against the nucleus from the killer+ strain (Leu+). Suc- cessful cytoduction was scored by the ability to grow on YP

+

glycerol. Rho+ haploids were then printed to a lawn of the killer sensitive strain 5 x 4 7 . Halos indicating mainte- nance of the killer plasmid on 4.7 MB plates were scored after 2 days at 20". Five independent transformants were used to score the phenotype.

Enzyme assays: Beta-galactosidase (lacz) expression from the GALl-lad fusion, PRY 18 1 integrated at the LEU2 locus was assayed as described previously (YOCUM et al. 1984; FLICK and JOHNSTON 1990). Yeast were grown in SD-Ura medium containing either 2% glucose or 2% galactose. Enzyme activity for the recombinant p5083 is the average of four independent recombinants. Invertase was assayed as described previously (GOLDSTEIN and LAMPEN 1975; NEI- GEBORN and CARLSON 1984). Repressed cultures were grown to mid-log phase in 2% glucose, and cells were derepressed for 3 hr in SD-Ura+ 0.05% glucose. T h e results reported are the average of two assays.

RESULTS

Recovery of the HIS3 gene using pBM2240: To

establish that the yeast DNA in lambda clones can be recovered in a yeast plasmid, a lambda phage that was previously shown to contain the HZS3+ gene was used. A his3 mutant (YM3033) was cotransformed with DNA from the HZS3+-containing lambda clone (5380) and either with either of t w o different plasmid vectors containing homology to the arms of the lambda cloning vector flanking the inserted yeast DNA sequences. Plasmid pBM2240 (Figure 1) contains the lambda arm DNA fragments in the same relative orientation as they exist in the lambda clone; pBM2241 contains the lambda left arm fragment in the opposite orientation. Only pBM2240 is predicted to yield a closed circular plasmid through a double recombination event between the vector and the lambda clone.

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Cloning by Recombination in Yeast 153

FIGURE I.-Diagram of the recombination be-

a u ~

tween pBM2240 and a lambda clone, and the recombinant product. pBM2240 is based on the E. coli- yeast shuttle vector pRS3 16 (SIKORSKI and HIETER 1989). The shaded regions represent fragments of the lambda-cloning vector that were subcloned into pRS316 using the indicated restriction sites. The filled regions represent sequences required to select and maintain the plasmid in yeast. pBM2240 was digested with EcoRl and XhoI prior to cotransformation with lambda clone DNA. High-efficiency transformation is the result of recombination between the plasmid and the lambda clone. An analogous 2-pm-based high copy plasmid was also constructed (pBM2384, see MATERIALS AND METHODS).

[

Recombinant

)

TABLE 1

Recovery of HZS3+ from a lambda clone known to contain the

HZS3+ gene

gapped pBM2241 did not increase the frequency of transformation. These are the expected results if the double strand break in the vector is being repaired by recombination with the lambda clone, as diagramed in Figure 1. As expected, most of the Ura+ transformants are His+. Loss of the recombinant plasmid by selection on 5-FOA (BOEKE, LACROUTE and FINK 1984) resulted in the concomitant loss of the Ura+ and His+ phenotypes. This is in contrast to the two

His+ isolates obtained with pBM2241, in which the two phenotypes did not cosegregate. T h e identity of four recombinant plasmids was confirmed by restriction enzyme digestion (data not shown).

Recovery of GAL83: GAL83 is a gene required for glucose repression of GAL gene expression (MATSU-

MOTO, YOSHIMATSU and OSHIMA 1983; FLICK and JOHNSTON 1990). We believe that this gene is underrepresented or absent in yeast plasmid libraries amplified in E. coli because we have been unsuccessful in our repeated attempts to isolate it from several different libraries by genetic complementation. Because the map position of GAL83 is known (MATSUMOTO, YOSH-

Transforming DNA

Plasmid Lambda transformants Ura' rested

No. Ura' No. His' clones/no.

pBM2240 uncut None 2000 0154

pBM2240 cut None 45 0134

pBM2240 cut 5380 (100 ng) 200 20122 pBM2240 cut 5380 (2 pg) 1000 44/49

pBM2241 uncut None 1300 0152

pBM2241 cut None 85 0150

pBM2241 cut 5380 (2 p g ) 115 2/55

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154 J. R. Erickson and M . Johnston

Genetic Map

h

Y

z

I I

IF

6977 5083

H+tHtHi56OU -7150 ki4Hi 5313 k"Wi U678

M 1581

65UO 6224

5961

6793 P P W I k l 6 5 9 2

6697

Lambda

Clones PI-w" 4019

FIGURE 2.-Map of chromosome V sur- rounding GAL83. T h e location of CEN5 and H I S 1 were determined by hybridization (RILES et al. 1993). T h e location of GAL83

was deduced by determining which lambda clone complemented the ga183-2000 mutation (see MATERIALS AND METHODS). T h e lambda clones used spanned the interval from

6540-6793. T h e physical scale reflects the position of this interval in relation to the left end of Chromosome V. T h e physical scale ( i n kb) begins at the left telomere.

Scale

In

kb

TABLE 2

Cotransformation of YM3033 with pBM2240 and GAL83 regional lambda clones

Tral>sforming DNA

No. Ura' No. 2dGal' cIones/no. Plasrnid Lambda transforniants Urn+ tested

pBM2240 uncut None 2000 0/54

pBM2240 cut None 45 Of34

pBM2240 cut 4049 400 Of54

pBM2240 cut 5083 550 49 /56

pBM2240 cut 531 3 400 0 /54

pBM2240 cut 6224 400 0/52

pBM2240 cut 6540 400 O f 5 1

pBM2240 cut 6793 250 Of54

pBM2240 cut 6977 500 O f 5 1

pBM2240 cut 7 150 150 0 /24

TABLE 3

Recombinant plasmid containing sequences from the lambda clone 5083 restores wild-type regulation to a CALI-lac2 fusion

Beta-galactosidase activity in cells grown

in:

straln

Yeast

genotype Plasmid Glucose Galactose RdeVdnt

YM3033 _gal83 _pRS316 420 886

YM3033 _gal83 _p5083 18.5 825

YM2168 GAL83 _pRS316 30.2 603

IMATSU and OSHIMA 1983), it is amenable to cloning in pBM2240 by cotransforming with lambda clones carrying the relevant region from the yeast genome.

We employed the galactose analog 2-deoxy-D-galactose (2dGal) to test transformants for the presence of

GAL83. This compound is converted to a form that is toxic to yeast (2-deoxygalactose-1 -phosphate) by the action of galactokinase, the product of the GALl gene (PLATT 1984). Wild-type yeast grown in the presence of glucose are resistant to 2dCal due to glucose repression of GALl expression; gal83 mutants express GALl

under these conditions and are therefore sensitive to the analog.

To clone GAL83, eight lambda clones that encom-

pass the region to which GAL83 maps (between C E N 5

and H I S l , see Figure 2) were cotransformed with pBM2240 cut with EcoRI and XhoI. As expected, the inclusion of lambda DNA resulted in an increase in transformation efficiency (Table 2). T o screen for ga/8?-complementing activity, Ura+ transformants

were tested for sensitivity to 2dGal, which revealed that a single lambda clone, 5083, carries GAL83. This was verified by assays of GALl expression in the transformants that contain GAL83 (Table 3). These results delimit the location of GAL83 to the approximately 6 kb of DNA unique to this clone.

Cloning of SSN2: As a second demonstration of the utility of the lambda clone rescue technique, we employed it to isolate a gene whose mutant phenotype is too marginal to allow it to be isolated readily from a complex library. Such a gene is S S N 2 , whose mutations suppress the inability of s n f l mutants to grow on raffinose as a sole carbon source. Mutants of s n f l are unable to grow on raffinose because they are unable to derepress expression of invertase (encoded by

S U C 2 ) , the enzyme responsible for cleaving raffinose;

ssn2 mutants partially restore SUC2 expression in snfl mutants. Because mutations in ssn2 only modestly suppress the s n f l defect, they do not confer a readily selectable phenotype. T h e most easily scored phenotype of ssn2 mutants is their "clumpy" growth in liquid culture. DNA was prepared from individual lambda clones containing the region of chromosome IV to which S S N 2 maps and cotransformed with pBM2240 cut with EcoRI and XhoI (data not shown). Ura+ transformants were tested for clumpy growth and the ability to grow on YP Raffinose. A single lambda clone, 55 13, complemented the ssn2 mutant phenotypes (see Figure 3). This was confirmed by assaying

SUC2 expression in the transformants (Table 4). Cloning of MAK7: A third example of a gene that would be difficult to clone by screening libraries is

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Cloning by Recombination i n Yeast 1.55

Genetic Map

h

Ir

5 oc

2 x

Lambda

Clones

FIGURE 3.-Map of chromosome IV near S N F 1 . S S N 2 was shown to be tightly linked to S N F l (CARLSON et al. 1984). ‘The location of S N F l and

ADEB were determined by hybridization (RILES et al. 1993). The seven lanlbda clones between S N F l and

ADER were used with pBM2240 (clones numbered 8000 and higher are costnid clones). A single lambda clone, 551 3 , complemented the ssn2

phenotype. T h e physical scale ( i n kb) begins a t the left telomere.

Scale in kb

TABLE 4

Recombinant plasmid containing SSN2 restores the snfl

phenotype to SUC2

Units of invertase activity

Clun1py Strain Genotype Plasmid Repressed Ikrepressed growth

MCY447 snfl, ssn2 _p3984 0.40 1.2 Yes MCY447 s n f l , ssn2 _p5230 0.62 1.4 Yes MCY447 snfl, ssn2 _p5513 0.49 0.67 No YM2168 Wild-type None 1.3 80.3 N o

TABLE 5

Summary of cotransforming yeast strain 2778 with pBM2240 and M A K 7 regional lambda clones

Transforming D N A

N o . Ura’ Ability t u

Plasnlld Lanlbda tr-msforl11ants complement mak7

pBM2240 Uncut None 2000 ND

pBM2240 Cut None 20

pBM2240 Cut 1623 153 pBM2240 Cut 2590 91

pBM2240 Cut 3138 98 pBM2240 Cut 3289 89

pBM2240 Cut 4904 92

pBM2240 Cut 4992 8

pBM2240 Cut 5018 8

pBM2240 Cut 6357 110

pBM2240 Cut 6506 45

pBM2240 Cut 6525 49

- - - - - -

-

+

for replication of the double-stranded kilfer RNA plasmid (WICKNER and LIEBOWITZ 1976). Since the killer plasmid is not maintained in a mak mutant, potential transformant clones must have the killer plasmid reintroduced by cytoduction to score for complementing activity. This severely limits the number of transformants that can be tested for the presence of the gene.

M A K 7 had been previously mapped near the end of the left arm of chromosome VZZZ (WICKNER 1979). T h e 1 0 left-most lambda clones from chromosome

VZZZ were cotransforrned with pBM2240 into a mak7

mutant (see M A ~ r E R I A L s A N D METHODS). Ura+ trans-

formants produced from each lambda clone were

mated with a Karl nlutant to cytoduce the MI RNA plasmid into the transformants. T h e resultant cyto- ductants were printed to a lawn of a killer-sensitive strain to score their ability to maintain the killer plasmid (Makt phenotype). As seen in Table 5, lambda clone 6525 complemented the mak7 defect. Since the lambda clones 2590 and 6505 did not complement

mak7, the gene has been localized to an approximately 7-kb interval (see Figure 4).

I t is noteworthy that cotransforming with two

lambda clones (4992 and 50 18) actually decreased the frequency of transformation. Based on the genetic map, it is likely that the overlapping region between the t w o clones contains CEN8.

DISCUSSION

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156 J. R. Erickson and M. Johnston

Genetic Map

B

9

h

h rn

s

g

cr,

Lambda Clones

Scale

In

k b

FIGURE 4.-Map of chromosome VIIInear the left telomere. T h e location of S P O I l was determined by hybridization; the subtelomeric repeats X and Y hybridize to lambda clone 4904 (RILES et al. 1993). To clone M A K 7 , the 10 left- most lambda clones were used with pBM2240 to cotransform a mak7 mutant. A single lambda clone, 6525. complemented the mak7 defect. T h e position of CENR is based on transformation frequencies (see RESULTS). T h e physical scale (in kb) begins at the left telomere.

screened. This allows one to reduce greatly the com- plexity of the library being screened because each lambda clone is tested individually for complementing activity. I t would not be unreasonable to attempt to clone a gene that is only mapped to a chromosome by screening all of the lambda clones that span that chromosome for complementation by cotransformation. Such an approach is attractive because of the ease of mapping genes to chromosomes (WAKAM and SHERMAN 1990). For genes that lie on larger chromosomes, such as chromosome IV, which is represented by 114 lambda clones, it would be desirable to gather more genetic map information so that fewer lambda clones are needed. Once a gene has been localized to a lambda clone or overlapping clones, the actual location of the gene is often delimited to a fairly small interval, which simplifies subcloning the gene of interest.

This approach can also be used to convert lambda clones into high-copy yeast plasmids. We have constructed a 2-pm-based high copy plasmid similar to pBM2240. This plasmid (pBM2384, see MATERIALS

AND METHODS), when cut with EcoRI and Xhol and cotransformed with the GAL83-containing lambda clone 5083, yielded GAL83+ transformants at high frequency, similar to the results reported in Table 2.

There are three limitations that may prevent a particular gene from being cloned by this method. First, a few regions of the yeast genome are currently inaccessible to the technique because of gaps in the map for which no clones exist. Second, not all of the restriction map is based on lambda clones; in many cases, small gaps in the lambda clone map were spanned by cosmid clones. Such intervals cannot be recovered in pBM2240. We are in the process of developing a similar vector designed to rescue yeast sequences cloned in cosmid vectors. Third, it is im- portant to note that two different lambda vectors, MG3 and MG14, were used in constructing the library

used to generate the yeast restriction map. Most of the lambda clones are based on the MG3 vector, whose inserts pBM2240 was designed to recover. However, if one needed to recover a lambda clone from the MG14 vector, a new plasmid analogous to pBM2240 would have to be constructed. Neverthe- less, since most regions of the yeast genome can be recovered by this technique, it provides a useful alter- native to the use of plasmid libraries amplified in E. coli for cloning yeast genes. Finally, it should be noted that pBM2240 is not suited for recovering genes that are tightly CEN-linked because the recombinant plasmid would be dicentric. T h e 2-pm-based plasmid, pBM2384, would permit the recovery of lambda clones which contain centromeres.

We thank L. RILES for assistance with the lambda clones and map information and M. CARLSON and R. WICKNER for providing yeast strains. This work was funded by a grant from the NIH GM32540. J.R.E. was supported by by NIH institutional training grant T 3 2 GM 07076 to the Division of Biology and Biomedical Sciences.

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