119 Gene, 26 (1983) 119-126

Elsevier

GENE 902

Molecular cloning and characterization of the RADI gene of Succharomyces cerevisiae

(DNA repair; Escherichiu coli; yeasts; incision; excision; pyrimidine dimers; deletion mutants; UV sensitivity)

David R. Higgins, Satya Prakash, Paul Reynolds * and Louise Prakash *

Department of Biology, University of Rochester, Rochester, NY 14627 (U.S.A.) Tel. (716) 275-3847, and *Department of

Radiation Biology and Biophysics, University of Rochester, School of Medicine, Rochester, NY 14642 (U.S.A.) Tel. (716) 275-2656

(Received July 8th, 1983)

(Revision received and accepted September lst, 1983)

SUMMARY

We have cloned the RADI gene of Saccharomyces cerevisiae and physically mapped it to a 4.0-kb DNA

fragment from chromosome XVI. The RADI gene determines a transcript of 3.1 kb, and the direction of

transcription was found to be leftwards, from EcoRI towards BglII (Fig. 1). Deletions of the RADI gene were

made and were found to have no effect on viability of vegetative cells or spores, or on sporulation.

INTRODUCTION

In Escherichia coli, the uvrA, uvrB and uvrC genes

are required for the removal of ultraviolet light(UV)-

induced pyrimidine dimers from DNA (Boyce and

Howard-Flanders, 1964; Setlow and Carrier, 1964).

In addition to being extremely UV-sensitive, uvrA,

uvrB and uvrC mutants show enhanced sensitivity to

other DNA damaging agents which introduce distor-

tions in the DNA, such as nitroquinoline oxide

(Ikenaga et al., 1975), mitomycin C (Kondo et al.,

1970), and psoralen plus near UV light treatment

(Cole, 1971). The uvrA, uvrB and uvrC genes encode

proteins of J4, 114000 (Sancar et al., 1981a), 84000

(Sancar et al., 1981b) and 70000 (Sancar et al.,

198 Ic), respectively. The uvrABC endonuclease

Abbreviations: kb, kilobases or kilobase pairs; poly(A)+ ,

polyadenylated; tet, tetracycline; UV, ultraviolet light; [ 1, indi-

cates plasmid carrier state.

0378-l 119/83/$03.00 0 1983 Elsevier Science Publishers

activity has been reconstituted in vitro and shown to

incise UV-irradiated DNA, producing two cuts in

the damaged DNA strand, one on each side of the

pyrimidine dimer (Sancar and Rupp, 1983).

In eukaryotic organisms, excision repair seems to

require a large number of genes and is probably a

more complex process than in E. coli. In the yeast

S. cerevisiae, radl, rad2, rad3, rad4, rad7, radl0,

radl4, rad16, rad23 and mms19 mutants are defective

in excision of UV-induced pyrimidine dimers (Unrau

et al., 1971; Resnick and Setlow, 1972; Waters

and Moustacchi, 1974; Prakash, 1975; 1977a,b;

Reynolds, 1978; Prakash and P&ash, 1979; Miller

et al., 1982a), as well as of psoralen plus 360 nm

light-induced interstrand DNA crosslinks (Miller

et al., 1982a,b). Of these mutants, rudl, rud2, rud3,

rad4 and radl0 have been shown to be defective in

the incision step of excision repair (Wilcox and

Prakash, 198 1; Reynolds and Friedberg, 1981).

Attempts to characterize pyrimidine dimer-incising

120

activity in vitro from S. cerevisiae have not been

successful (Reynolds and Friedberg, 1980; Prakash,

L., unpublished results), perhaps because the activity

exists as part of a labile complex.

To identify and characterize the protein products

of the RAD genes involved in the incision process

and to examine the regulation of these genes following

treatment with DNA-damaging agents, we have

cloned several of these RAD genes. In this paper, we

report the isolation of the RADl gene by complemen-

tation of the radl mutants of S. cerevisiae and show

that the RADI gene determines a transcript of

3.1 kb. We also demonstrate that radl deletions are

viable, in contrast to rad3 deletions, which are reces-

sive lethals (Higgins et al., 1983).

MATERIALS AND METHODS

(a) Yeast and bacterial strains

The following yeast strains were used: 7799-K,

MATa his4-17 ura3-52 RAD’ (obtained from G.

Fink); DH25-lB, MATa his3-Al Zeu2-3 leu2-112

1~~1-1 trpl-289 ura3-52 radl-2; DH50-2B, HO his-

ura3-52 RAD’ ; DH50-9B, HO lys- ura3-52 radl-2;

LP2693-6C, MATa leu2-3 leu2-112 trpl-289 ura3-52

RAD’ ; B-635, MATa ~~~1-115 hisl-1 lys2 trp2

RAD’.

E. coli strain JM103 was used to propagate Ml3

derivative phages. Yeast strains containing plasmids

(indicated by square brackets around plasmid) were

maintained and grown on selective media.

(b) Plasmids

The yeast recombinant library used, obtained from

D. Botstein, consisted of partial Sau3A digests of

yeast genomic DNA ligated into the BamHI site of

the 7.6-kb hybrid plasmid YEp24 (Botstein et al.,

1979; Carlson and Botstein, 1982). YEp24 consists

of pBR322, a 2.2-kb EcoRI fragment of yeast 2 p

circle DNA, form B, conferring the ability to replicate

autonomously in yeast, and a l.l-kb Hind111 frag-

ment containing the yeast URA3 gene, for selection

in yeast.

YIPS, the 5.5kb plasmid used for integrative

transformation, is also derived from pBR322 and

contains the yeast URA3 gene. However, it does not

replicate autonomously in yeast since it lacks a yeast

replication origin sequence and can transform only

by homologous integration into the yeast genome

(Scherer and Davis, 1979; Botstein et al., 1979).

YIp5 contains unique EcoRI, HindIII, BamHI and

Sal1 sites.

YEpl3 is a 10.7-kb plasmid, which is also derived

from pBR322 and which replicates autonomously in

yeast because of the 2 p origin of replication; it

contains the yeast LEU2 gene for selection in yeast

(Broach et al., 1979). All plasmids were maintained

in E. coli strain HBlOl.

(c) Transformation procedures and UV irradiation

The spheroplast method of Hinnen et al. (1978) or

the treatment of intact yeast cells with lithium acetate

to promote DNA uptake (Ito et al., 1983) were used

for transformation of yeast. Response to UV irradia-

tion of various yeast strains was determined by the

spot test or from survival curves following UV irradia-

tion as described by Prakash and Prakash (1977).

(d) Other methods

Nucleic acids were purified and electrophoresed

as described previously (Higgins et al., 1983).

Restriction fragments were transferred to nitrocellu-

lose paper by the method of Southern (1975) and

hybridizations were carried out according to the

method of Maniatis et al. (1982), except that a single-

stranded probe with the 1.6-kb BglII-EcoRI frag-

ment of pDH2 (Fig. l), prepared as described below,

was used in the hybridization mixture. The specific

activity ofthe probe was 4.3 x 10’ cpm/pg DNA and

a total of 4.3 x 10h cpm was used in the hybridiza-

tion mixture.

Poly(A)+ RNA was prepared by binding total

RNA to oligo-deoxythymidylate cellulose (type 3,

Collaborative Research) in 10 mM Tris . HCl,

pH 7.5-0.5 M NaCl and eluting the bound

poly(A)+RNA in 10 mM Tris. HCl, pH 7.5 (Aviv

and Leder, 1972). RNA sa.mples were fractionated

by electrophoresis in formaldehyde agarose gels,

transferred to “gene screen paper” (New England

Nuclear Corp.) as described by the manufacturer

and hybridized with “P-labeled DNA (Maniatis

et al., 1982). The 1.6-kb BglII-EcoRI restriction

fragment of pDH2 (Fig. 1) was cloned into BamHI-

EcoRI restricted M13mp8 and M13mp9 (Messing

and Vieira, 1982), and the method of Hu and

Messing (1982) was used to prepare the labeled

single-stranded probes. About 6 x lo6 cpm were

used per RNA-DNA hybridization. All autoradio-

graphs were exposed on Kodak XL-l (X-omat) film

at - 70°C with DuPont Lightning Plus intensifying

screens.

RESULTS

(a) Isolation of the RADZ gene

The pool of yeast genomic DNA in YEp24 was

used to transform the uracil requiring (Ursa),

UV-sensitive (Rad - ) yeast strain DH25-1B (~~3-52

r&l-2) to uracil prototrophy (Ura’ ). Approx.

20000 transformants were screened for UV resist-

ance by spot testing and six UV-resistant (Rad’)

colonies were obtained. Subcloning and retesting of

these six transformants revealed that the genetic

markers present in the original untransformed strain,

as well as UV resistance, were retained. Plasmid

DNA from each of the six UV-resistant transfor-

mants was prepared by a modification of the Hirt

procedure (1967) and used to transform E. coli strain

HB 101 to ampicillin resistance. Plasmid DNA from

these transformed E. coli strains was then used to

121

transform the uru3-52 rudl-2 yeast strain DH25- 1B

to Ura+ . 60 Ura+ transformants were tested from

each of the six transformations and in all six cases,

the Ura+ transformants were also UV-resistant,

indicating that UV resistance was carried on the

plasmid. The six plasmids were designated pDH 1 to

pDH6. Plasmids pDH 1, pDH2, pDH3 and pDH5,

each containing an 8.5-kb insert of yeast DNA, gave

identical patterns of digestion with Hind111 and other

restriction enzymes, whereas plasmids pDH4 and

pDH6 contained an additional 3 kb of yeast DNA.

A restriction map of the yeast DNA insert containing

the rudl complementing function in plasmid pDH2

is given in Fig. 1. UV survival curves of various radl mutants, radl-2, radl-4, radl- 19 and radl-20, carry-

ing the plasmid pDH2 show normal levels of UV

resistance (Fig. 2).

(h) Genetic mapping of the cloned DNA segment

To verify that the yeast DNA insert in the pDH2

plasmid contained the yeast RADl gene and not a

suppressor of radl-2, or some other gene which

could complement radl-2 when present on a multi-

copy plasmid, we genetically mapped the 4.7-kb

HindIII-Sal1 fragment of pDH2 (Fig. 1). The 4.7-kb

HindIII-Sal1 fragment of plasmid pDH2 was ligated

into Hind111 + WI-restricted YIPS, a plasmid

containing the yeast URA3 gene but not the se-

quences required for autonomous replication in

yeast. Transformation with YIPS, therefore, occurs

Lb 6 i i j k j k ; i 1 Plarmtd Complementotion

E SII E SI H E SPSIB SII of rod!

__ __ pDH2 +

I pDH7

I I pDHII

__ 1 [+-- pDH2l +

__ I II-- pDH22 -

I I pDH23

Fig. 1. Restriction map ofthe yeast DNA insert in plasmid pDH2 and complementation pattern of pDH2 derivatives. Insert ofthe yeast

DNA segment in pDH2, given by the open bar, is in the BarnHI site of the ret gene of YEp24; the thin line represents YEp24 DNA.

* denotes the Sau3Al-BamHI junction of the fusion of insert with plasmid. Symbols for restriction enzymes are as follows: B, BarnHI;

Bg, BglII; E, EcoRI; H, HindIII; K, KpnI; SI, SacI; SII, SacII; Sa, SalI; Sp, SphI. The open bars below plasmid pDH2 represent DNA

segments of pDH2 cloned in various vectors, as indicated in the text. The 1.6-kb BglII-EcoRI fragment used to generate plasmid pDH23

was also inserted into M13mp8 and M13mp9 phages and used to generate single stranded probes for hybridization to yeast RNA to

determine the size and direction of the RADI transcript. The wavy line with the arrowhead below the DNA insert of plasmid pDH23

denotes the direction of the RADI transcript.

ooolM oooo’ UV FLUENCE (J/m’)

-0kFX-

UV FLUENCE (J/m*)

50

Fig. 2. Survival after UV irradiation of yeast strains harboring

various RADl insert containing plasmids. Cells were grown in

minimal medium supplemented with the necessary nutrients

except that plasmid bearing strains were grown in selective

medium. Irradiation was as described by Prakash and Prakash

(1977). (A) RAD’ and radl-2 strains with and without various

RADl plasmids. 0, Strain DH25-3A, leu2-3, leu2-112 ~I-289

ura3-52 RAD + ; 0, DH25-lB, MATa hb3-dl IeuZ-3 leu2-112

lysl-1 trpl-289 ~~3-52 radl-2; 0, DH25-lB[pDH2]; n , DH25-

lB[pDHZI]; A, DH25-lB[YEp24]; A, DH25_1B[pDH22]. (B)

Various rudl mutants with and without plasmid pDH2. 0,

Strain LP2790-7B, MATa rysZ RAD+; n , LP2809-3B, MATa his3-Al trpl-289 ura3-52 radl-4; A, LP2810-3B, MATa hW-Al

leu- ura3-52 radl-19; 0, LP2790-7A,Mata his- IeuZ-3 leu2-112

trp- ura3-52 radl-20; 0, LP2809-3B[pDH2]; A, LP2810-

3B[pDH2]; V, LP2790-7A[pDH2].

by homologous integration into the yeast genome

(Scherer and Davis, 1979). The resulting plasmid,

pDH7 (Fig. l), was used to transform strain DH25-

1B (ura3-52 r&l-2) to Ura+. The Ura+ transfor-

mants were UV-sensitive, indicating that the 4.7-kb

KndIII-Sal1 fragment did not contain the radl- complementing function. However, this DNA frag-

ment should show close linkage to the radl gene on

chromosome XVI, if the fragment originated from

that region of the chromosome. Three Ura+ radl-2 transformants in strain DH25-1B were crossed to

strain 7799-K (uru3-52 RAD + ) and diploids sporu-

lated. Genetic analysis of 34,28 and 24 tetrads from

these three crosses revealed 32, 25 and 22 parental

ditype tetrads (2 Ura+ Rad : 2 Ura- Rad +),

respectively, and 2,3 and 2 tetratype tetrads, respec-

tively. The close linkage ofthe URA3 gene present on

the plasmid pDH7 with the rudl gene indicated that

the plasmid pDH7 had integrated close to the radl site in the yeast genome. We also transformed

another strain, DH50-2B (ura3-52 RAD + ) to Ura +

with plasmid pDH7. Three Ura+ RAD’ transfor-

mants were crossed to the strain DH50-9B (ura3-52 radl-2) and diploids sporulated. Genetic analysis of

27 tetrads from each cross gave only parental ditype

tetrads: 2 Ura+ Rad’ and 2 Ura- Rad- spores.

These results again indicated that plasmid pDH7

had integrated near the radl locus and that the RADl gene was present on plasmid pDH2.

(c) Location of the RADl gene in the cloned DNA

insert

Plasmid pDH7, ‘containing the 4.7-kb HindIII-

Sal1 fragment of pDH2, did not complement radl-2, and neither did plasmid pDH 11, which contains the

5.3-kb BglII-BumHI fragment of pDH2 (Fig. 1)

ligated into BamHI restricted YEp13. We then con-

structed a deletion of the 2.8-kb Sac1 fragment

(Fig. 1) by subjecting pDH2 DNA to limit digest

with SacI, fractionating the products by electro-

phoresis in an agarose gel, and purifying the 13.3-kb

DNA fragment, which is deleted for the Sac1 frag-

ment, by electroelution. This DNA fragment was

then incubated with T4 DNA ligase and the mixture

used to transform E. coli strain HB 101 to ampicillin

resistance. The plasmid pDH21 (Fig. l), containing

a deletion of the 2.8-kb Sac1 fragment of pDH2, was

found to restore UV resistance to the radl-2 strain

DH25-1B (Fig. 2A). Another plasmid, pDH22,

deleted for the 4.1-kb Sac11 fragment (Fig. l), did not

restore UV resistance to the radl-2 strain (Fig. 2A).

All of these results taken together indicate that the

RADl gene extends from the left of the Bg111 site at

1.6 kb to somewhere between the Sac11 site at 3.3 kb

and Sac1 site at 4.0 kb (Fig. 1).

(d) The size of the RADZ transcript and transcription

direction

To determine the size of the RADl transcript and

the direction of transcription, the 1.6-kb BglII-EcoRI

fragment internal to the RADl gene was cloned into

BamHI-EcoRI restricted M 13mp8 and M 13mp9

phages (see Fig. 1 legend). The recombinant phage

containing the 1.6-kb BglII-EcoRI insert were used

123

as templates for DNA synthesis to generate radio-

actively labeled single-stranded probes, as described

by Hu and Messing (1982). A hybridization probe

primer (New England Biolabs), which anneals 45

nucleotides 5’ of the cloning site and therefore does

not prime DNA synthesis through the cloned insert,

was used to synthesize the complementary DNA

strand of either M 13mp8 or M13mp9. The

reaction, carried out in the presence of a radioactively

labeled deoxynucleoside triphosphate did not

proceed to completion under the conditions employ-

ed (Hu and Messing, 1982). Thus, the radioactively

labeled reaction product was single-stranded in the

region of the yeast insert, and the 5’ to 3’ orientation

of the RNA strand hybridizing to the single-stranded

DNA insert is opposite to the polarity of the DNA

strand with which it hybridizes. The labeled probes

were used in hybridizations to total yeast RNA from

the RAD’ strain B-635 and the rudl-2 strain

DH25- 1B with and without the pDH2 plasmid, and

to poly(A) + RNA from the RAD + strain B-635. The

RADl transcript is more abundant in the radl-2

strain containing the RADl gene on a multicopy

plasmid, pDH2 (Fig. 3A, lane 4) than in either the

A B kb

123 4

Fig. 3. Transcript ofthe RADl gene. Total RNA (20 fig per lane)

and 10 pg poly(A)‘RNA from yeast cells were fractionated on

formaldehyde-1.5%-agarose gels, transferred to gene screen

paper, and hybridized to the 1.6-kb BglII-EcoRI fragment of the

RADZ gene cloned into Ml3mp9. (A) Lane 1, total RNA from the

RAD+ strain B-635; lane 2, total RNA from the mdl-2 strain

DH25-1B; lane 3, total RNA from the radl-2 strain DH25-1B

containing the plasmid YEp24 without any additional yeast

DNA insert; lane 4, total RNA from the radl-2 strain DH25-IB

containing the radl complementing plasmid pDH2, the lower

narrow band being ribosomal RNA; (B) poly(A) + RNA from the

RAD + strain B-635.

RAD’ (Fig. 3A, lane 1) or radl-2 strain lacking the

pDH2 plasmid (Fig. 3A, lanes 2 and 3); the RADI

transcript is also more abundant in poly(A) + RNA

from the RAD+ strain (Fig. 3B) than in total RNA

from the RAD’ strain (Fig. 3, lane 1). The 3.1-kb

RADI transcript hybridizes to the 1.6-kb BglII-

EcoRI fragment of the RADI gene cloned in

M13mp9 (Fig. 3A, lane 4 and Fig. 3B), but not when

this fragment is cloned in M13mp8 (not shown).

Since the cloning sites of M13mp9 are arranged as

5’-BamHI-EcoRI-3’, and those of M13mp8 are

5’-EcoRI-BarnHI-3’, these results indicate that the

direction of transcription of the RADl gene is from

EcoRI to BglII (Fig. 1).

(e) Deletions of the RADl gene

Deletions of the RAD3 gene, which like the RADl

gene, is required for incising UV-damaged DNA, are

recessive lethals (Higgins et al., 1983). Deletions of

the RADl gene were generated by the method of

Shortle et al. (1982). The 1.6-kb BglII-EcoRI frag-

ment (see Fig. l), which is an internal fragment of the

RADl gene, was cloned into BumHI and EcoRI

restricted plasmid YIPS. The 6.7-kb plasmid thus

generated, pDH23 (Fig. l), was used to transform

the yeast strain 7799-5C (ura3-52 RAD + ) to Ura+ . Nine UV-sensitive Ura+ transformants were ob-

tained, which presumably resulted from integration

of the plasmid pDH23 at the RADI site in the yeast

genome, thereby creating deletions of the RADI

gene, as indicated in Fig. 4. Physical evidence for

integration of pDH23 at the RADl locus was

obtained by Southern analysis of yeast genomic

DNA from the recipient strain 7799-5C and the

Ura + transformants derived from it. Yeast genomic

DNA was restricted with BglII, fractionated by

electrophoresis in agarose gels, transferred to nitro-

cellulose by the method of Southern (1975), and then

hybridized to the nick translated 1.6-kb BgZII-EcoRI

fragment from pDH2. The size of the BglII fragment

from the recipient strain (Fig. 5, lane 1) hybridizing

to the RADI probe is 9.0 kb, and, as expected for

homologous integration at the RADI locus, the size

of the BglII fragment hybridizing to the RADI probe

from the 7799-5C[pDH23] transformants has in-

creased to 15.7 kb (Fig. 5, lanes 2 and 3).

In order to further ascertain that UV sensitivity in

these Ura + transformants was due to deletion at the

124

I- 16kb-

X

H HBg K E SII E SJ

CHROMOSOMAL RADl LOCUS . ..’ -f ’ a..

HOMOLOGOUS INTEGRATION AT RADl LOCUS

i H HBg K E E SII E SI

. . . . . . . .

Fig. 4. Generation of duplicated deletion of the RADI gene by homologous integration at the R.4DI locus of an internal segment of the

R&31 gene. The I&kb BglII-EcoRI fragment of pDH2 was ligated into BumHI-EcoRI restricted YIp5 to generate the plasmid pDH23.

As indicated diagramatically, integration by homologous recombination at the RAADI locus will generate a dupficated deletion at the

RADl locus flanking the YIp5 sequence. Symbols for restriction enzymes, as given in Fig. 1; thin line, pBR322 sequences; open segment,

RADl region DNA; hatched segment, yeast UK43 gene; heavy line, yeast chromosomal DNA.

1 2 3

kb

15.7

9.0

ML)1 locus, the Ura+ UV-sensitive tr~sform~ts were mated to the r&I-2 strain DH25-1B. The diploids were UV sensitive, as expected ifthe deletion mutation is allelic to the radl-2 mutation. Therefore, vegetatively growing haploid cells with a radl dele- tion are viable, unlike the rad3 deletion mutants. The radl deletion also had no effect on plating efficiency or growth rate. The rudl deletion mutants showed somewhat greater sensitivity to X-rays and methyl methanesulfonate than the radl-2 mutant, but similar levels of sensitivity as the other radl alleles examined, radl-4. radl-19 and radl-20. We then examined the viabiiity of radl deletion-bearing spores. Four Ura + UV-sensitive transformants in strain 7799-W were mated to the strain LP2693-6C (ura3-52 RAD + ), the diploids sporulated and 25-29 tetrads analyzed from each cross. In all four crosses, with the exception of

Fig. 5. Physical evidence for integration of plasmid pDH23

containing the I .6-kb BglII-EcoRI internal fragment ofthe RADl

gene at the RADl locus in the Yeast genome. . _ Plasmid pDH23 was used to transform the RAD’ ~~3-52

strain 7799-K to Ura +. Genomic DNA from the untransformed

strain (lane 1) and two W-sensitive transformallts (lanes 2 and

3) was purified and restricted with BglII. DNA fragments

separated by electrophoresis in agarose gels were transferred to

nitrocellulose and hybridized to 32P-labeled 1.6-kb BglII-EcoRI

fragment of pDH2.

one tetratype tetrad, only parental ditype tetrads showing 2 Ura+ Rad - and 2 Ural Rad + spores were obtained, demonstrating that Ura+ Rad - spores bearing the radl deletion are viable. The pre- dominance of parental ditype tetrads provides further genetic con~rmation for inte~ation of the plasmid pDH23 at the RAD1 site in strain 7799-5C. The radl Alradl A homozygotes were proficient in sporu- lation and produced viable spores.

DISCUSSION

We have cloned the RADl gene and used it to

determine the size of its transcript and the direction of transcription. The RADl gene encodes a 3.1-kb transcript and the direction of transcription is left- ward as shown in Fig. 1. Our subcloning results show that the RADI gene extends from the left of the BglII site at 1.6 kb to somewhere between the Sac11 site at 3.3 kb and Sac1 site at 4.0 kb (Fig. 1). The 3.1-kb RADl transcript could begin between the Sac11 and Sac1 sites on the right and end between the two Hind111 sites on the left (Fig. 1). The 3.1-kb transcript could code for a polypeptide of about 110 kDa1, similar in size to the uvrA protein ofE. coli.

We have also shown that deletions of the RADI

gene, created by integration of the internal 1.6-kb BglII-EcoRI fragment in the chromosomal RADI

gene, have no effect on viability or growth rate of vegetatively growing cells, or on sporulation or spore viability. Deletions of the RAD3 gene, which like the RADI gene is required for incising DNA containing pyrimidine dimers, are recessive lethals (Higgins et al., 1983). The RADl gene appears to be more specific to excision repair of damaged DNA, whereas the RAD3 gene is involved, in addition, in other vital cellular processes. Yeast cells containing the RADI

gene on a multicopy plasmid have much higher levels of RADI mRNA than cells with only the chromo- somal RADZ gene but show no alteration in growth rate or viability.

ACKNOWLEDGEMENTS

We thank Ravi Deshmukh for help with initial screening of transformants and Renata Polakowska for the Southern gel shown in Fig. 5. This investi- gation was supported by grants CA32514 and CA35035 to S.P. and GM19261 to L.P. from NIH.

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Communicated by S.R. Kushner