Cold-sensitive regulatory mutants of simian virus 40

J. Mol. Biol.

(1980) 140, 129-142

Cold-sensitive Regulatory Mutants of Simian Virus 40 DANIEL DIMAIO

AND DANIEL

NATHANS

Department of Microbiology Johns Hopkins University School of Medicine Baltimore, Md 21205, U.S.A. (Received 3 January

1980)

A series of conditionally or partially defective simian virus 40 muta.nts has been constructed with lesions within the regulatory segment, of the viral genome neai the BgZI clea.vage site which includes the origin of replication of viral DNA. A single-stranded gap was positioned in this region by controlled, approximately I, followed synchronous nick translation from the BgZI site with DNA polymerase by enzymatic extension of the nick into a small gap. The gapped DNA was either deaminated with sodium bisulfite or converted into linear form by digestion with nuclease S,. After infection of cell monolayers with the modified DNA, viable mutants containing deletions or base substitutions within the regulatory segment were isolated with high efficiency, and their mutational sites were mapped by restriction analysis and nucleotide sequencing. One set of mutants had overlapping deletions extending from the start of t,he T antigen coding sequence to within 32 base-pairs of the BgZI site. Most of these mutants were cold-sensitive, i.e. formed small plaques at 32°C relative to wild-type virus, as were several mutants with base substitutions in the same region. Other mutants

formed small plaques at all temperatures tested. A second series of deletion and base substitution mutations has been mapped t,o the late side of the BglI site. The late deletion closest to the replication origin diverged from t’he wild-type sequence 33 base-pairs from the RgZI site, and formed wild-type plaques; however, a mutant with base substitut,ions in the same region was cold-sensitive. Since all the detectable mutational alterations map outside known proteincoding sequences, we infer that these mutants are probably defective in DNA

replication, transcription, RNA processing, and/or translation. The frequency of cold-sensitive mutants suggests that this phenotypa may reflect changes in proteinPnucleic acid interactions involved in rrgulat,ory phenomena.

1. Introduction The general organization of the genorne of simian virus 40 (SV40) has been determined by a variety of genetic and biochemical techniques (for a review, see Kelly & Nathans, 1977; Fareed & Davoli, 1977). With the availability of the complete nucleotide sequence of SV40 DNA, it is now possible to correlate specific messenger RNAs and polypeptides with the DNA sequences encoding them (Reddy et al., 1978; Fiers et al., 1978). These assignments reveal that bet’ween the initiation codon for T antigen (transcribed counter-clockwise on the standard map) and the start of the late genes (transcribed clockwise) there is a stretch of 625 nucleotide pairs that codes for no known protein product. This DNA segment contains the origin of viral 129

0022~2836/SO/170129-14

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1980 Academic

Press

inc.

(London)

Ltd.

131

rm,4 replication

I).

111MAl0

AND

I). SATHANS

(Tk~nna It Sa.thans.

197-L: Farraccl it nl.. 197%), squ(~n(~c’~ WWS~OII~Pt crl., 1978). and probably signals inrolretl in initiation of t,ranscription (Lauh of rrl.. 1979). RNA processin,g. anti rihosomo bintling to mRX’A. T antigen-binding sit-es have also been idtntificd in this rrgulatory sqqntqt (Reed ef (II.. 1975; Tjian. 1978a.O) and have been implicat,ed in the initiation of’ I-ir;lI DPU‘A replication (Shortlc & Xat’hans. 1979). and possibly in the regulation of’ (tart!, and late transcript,ion (Tegtmt~yr r’t nl., 1975: Reed d al.. 1976: Khoury & May. ing to the 5’ c~titls of’ mRNXs

(Ghost1

1878).

In an at,tempt t)o identify the -Jarious regulat)ory elements in this segment, of’ t,hc viral genome, wvp are const’ructing mutants with alterations at pre-selected sitrs in t,his region and correlabing their nucleotide changes with the molecular and biological properties of the mut)ants. In an rarlier publicat’ion Shortlc & Kat,hans (1979) described the const’ruction by local mutagenesis of mutant)s with bast substit,utions at t,he replication origin. The mutations are in a &-dominant, element that rqu1att.s the rat,e of viral DNA replication. thus operationally identifying the ori signal. Herr we report, on the construct8ion and initial characterization of mut,ants \jith deletions or base subst,itutions on eit,her side of the replicaCon origin. The mutat,ional akerations have been defined by nucleot,idc sequence analysis and have been shown to tie within the regulatory segment. AMarry of the mutants are partially or corldit,ionallJ defective. the most, common phenotype bring cold sensitivity. It should therefore bc possible to correlat)e t’he scquencc~ changes with defects in specific rrgulatory events,

2. Materials and Methods (a) Cells and vivttses SV40 form 1 DNA was preparccl f’ronl BSCQO cells inf@cted with SV40 strain 776, as described earlier (Brockman K: Nathans, 1974), and purific~d by electrophoresis. Z’uql nntl AluI were the gifts from G. Ket,ner and D. Shortle, respectively. All other restriction rndonuclensts wprc purchased from commercial sources. Most restriction nndonucleasc reactions were carried out. at 37°C in 20 mM-Tris.HCl (pH 7.4), 7 mM-i&&l,, 7 mMmereaptoethanol, 0.0 1 y/, (w/v) gelatin. TaqI reactions were incubated at 5O”C, and HilzdIII reactions included 40 mM.XaCl. All prcparativc, rclnctions wctrc tjerminatod b\; the addition of EDTA to 20 m&r; the DNA was t~xtractcd with phenol and dialyzed int)o 15 mw-N&l, 1.5 mu-sodium cit,rotcx. Elcctrophorcsis of DPjA in polyacrylamidc or agarost’ gels was carritltl out, as describrd previously (Danna K- Nathans, 1971 : Sharp et al., 1973) and the DNA was eluted from agaros? prls using KI (Smith, 1980). (b) ~Vick translation Nick translation was carried 0111 at 14°C in 50 ITIM-KPO, (pH 7.4), 10 mM-MgCl,, 0.4 EM-dnoxyribonurleosidc triphosphntc,s (plus trnrrr amounts of [rx-32P]dTTP OI [r-32PJdCTP). All nick translation reactions contained SV40 DNA wit,h a single nick at thr BgZI site (Shortlc & Nathans, 1978) and sntlrrating amounts of rndonuclease-fret Escherichiu coli DNA polymcmsc I (Worthington). The amount of enzyme sufficient to saturate the DNA was determined by measuring perchloric acid-insoluble radioactivity 1972) after a 10.min incubatjion at different cnzymt in reaction portions (Englund, concent,rat,ions. In a t)ypical reaction cont,aining 5 pg of DNA in 200 ~1 total vol. 30 units of polymerase gave a maximal rate: of incorporation of nuclcotides. To analyze the nick translational products, portions of‘ th(L reaction wcw made 20 mu in EDTA and incubated for 5 min at 70°C. Then 5 pg of carrier transf(,r RNA was added, and the DNA was precipitated with ethanol after thr addition of ammonium acet,atr (Maxam & Gilbert,.

REGULATORY

MUTANTS

OF

SV40

121

1980). The precipitate was dried under vacuum and dissolved in 10 mM-Tris.HCl (pH 8.0), 1 mm-EDTA. After digestion with restriction enzyme(s), the DNA wa,s electrophoresed in polyacrylamide gels. In some experiments the duplex fragments were first denatured and electrophoresis was carried out in denaturing gels (McMaster & Carmichael, 1977). (c) Mutagenesis

of nick-tramlated

DNA

Nick-t,ranslatcd DNA labeled with 32P nucleotide was gapped using endonuclease-fret Micrococcus luteus DNA polymerase (Shortle & Nathans, 1978). No radioactive nucleotide was released, as assayed on PEI-cellulose (Kelly & Smith, 1970). Part of the gapped DNA was Imearized wit,h nuclease S, (Miles). The amount of S1 just, sufficient to convrrt essentially all of t)he gapped circular DNA into linear molecules was determined for each preparation of DNA. In a typical reaction, gel-purifird DNA (0.5 pg/ml) in 0.26 ml was digested with lo4 units of S1 for 30 min at 22°C in st.andard rcaaction buffer (Vogt, 1973). After extraction with phenol, full length linear DNA was purified by clectrophoresis in agarosr. For bisulfite mutagenesis, gapped circular DNA was rcnctrd with sodium bisulfite under conditions estimated to deaminate 300,, of exposed cytosinc residues (Shortle & Nathans, 1978). Deaminat,ed DNA was purified (as form II DNA) by cxlcctrophorcsis in a 1.49, (w/v) agarose gel and used direct)ly for transfection. (d) Transfection Subconfluent layers of BSC40 cells in 6-cm dishes were transfccted with in vitro-modified DNA by means of the DEAE-dextran method (McCutchan & Pagano, 1968) and replicate dishes incubated at 32”C, 37”C, and 40°C. Cells were stained with neutral red after the plaques had developed for 9 days at 37”C, or 4O”C, or for 15 days at 32°C. Individual plaques were picked into 150 ~1 of minimal Eagle’s medium with 2(,$$ (v/v) fetal calf serum and stored in microwell dishes. Virus from each plaque was then replated at 32”C, 37”C, or 40°C. Plaques were picked at the least restrictive temperature for a given isolate and retested by restriction analysis of progeny DNA (see below). Mutant stocks were prepared from those plaques yielding single mutants with no evidence of wild-type SV40 contamination (as judged by restriction analysis). (e) Screen

for

mutants

Viral DNA uniformly labeled with 32P was extracted from cells grown microwells and infected wit,h individual plaques (Shortle et al., 1979). The DNA digested with restriction endonucleases followed by electrophoresis in 4% or acrylamide gels and autoradiography. To assist in visualizat.ion of smaller DNA DNA samples were treated with RNAase A (200 pg/ml) and RNAase T, (50 1 h at 37°C following restriction. (f) DNA

in 12 mm was then So/b (w/v) fragments, pg/ml) for

sequencing

Hind111 or HinfI fragments of mutant DNA were labeled at their 5’ ends with [Y-~~P]ATP and polynucleotide kinase, or at their 3’ ends with [or-32P]dATP (plus cold dGTP for the H&f1 fragments) and M. Zuteus DNA polymerase. After digestion with HpaII the fragment to be sequenced (HindIII-H&I, map co-ordinates 0.65 to 0.73; or HinfI-HpaII, map co-ordinates 0.645 to 0.73) was iaolatcd by electrophoresis, degraded by the method of Maxam & Gilbert (1980), and the products subjected to electrophoresis in ult)rathin, denaturing polyacrylamide gels (Sanger & Coulson, 1978).

3. Results (a) Outline

of the method for generating

mutations

around the replication

origin

To generate mutations around the replication origin targets for local mutagenesis were created adjacent to the BglI cleavage site at map co-ordinate 0.661 as shown in Figure 1. Form I SV40 DNA was treated with BgZI in the presence of ethidium

13”

D. DrMAIO

AND

-,\, 4fz.il Infect cells 4

D. NATHANS

Endonucleose S, / ~’

Deletion mutants

FIG. 1. Generation of mutations around the origin of replication.

See text for details.

bromide to produce a single strand scission at’ the enzyme cleavage site, and the nick “translated” under controlled conditions to an adjacent position by the combined 5’ --f 3’ exonuclease and polymerase activities of E. coli DNA polymerase L (Kelly et al., 1970). The nick was then extended into a small gap by the exonucleolyt,ic activity of M. luteus DNA polymerase. To construct deletion mutants, the gapped DNA was converted into linear molecules by digestion of its single-stranded segment with nuclease S,. To construct base substitution mutants, cytosine residues in the single-stranded region were deaminated by treatment with sodium bisulfite (Shortle & Nathans, 1978). In each case monolayers of BSC40 cells were transfected with the modified DNA, and the resulting plaques were screened for the presence of plaque morphology mutants and for viral genomes with altered restriction patterns. was

(b) Controlled nick translation Since positioning of the target for local mutagenesis requires controlled, approximately synchronous nick translation, we describe this step of the procedure more fully. The reaction was carried out at low temperature both to control its rate and to minimize strand displacement and template switching (Kornberg, 1974). At saturating enzyme, a temperature of 14”C, and low concentrations of the four deoxynucleoside triphosphates (0.4 PM), measured incorporation of nucleotides into BglI-nicked SV40 DNA wa,s proportional to time of incubation, approximating five to ten nucleotides per minute per molecule of DNA. To analyze the products of this reaction, 32P-labeled nucleotide was digested nick-translated DNA containing incorporated with various restriction enzymes and the products were analyzed by electrophoresis in polyacrylamide gels before and after denaturation, as shown in Figure 2. After digestion with PwuII + HtiuLdIII, nearly all of the radioactivity was in the 342 basepair fragment F containing the Bgll site. After denaturation, the radioactivit’y was found in two size classes of fragments, each about equally labeled, the larger corresponding to strands elongated toward the early gene region, and the smaller corresponding to strands elongated toward the lat,e gene region (Figs 2 and 3). As seen in Figure 3, for any given time of nick translation, each size class consists of a set of fragments of similar length, indicating approximate synchrony of the elongation

REGULATORY

MUTANTS

Denature

OF

SV4O

133

f

I

Fro. 2. Characterization of nick-translated DNA. The circle represents the PwuII + Hind111 cleavage map of SV40 DNA, oriented with map co-ordinate O.O/l.O at the top. The early gene region extends approx. 180’ counter-clockwise from the BgZI site; and the late region, 180” clockwise from the BgZI site. The lines below the circle are an enlargement of the genome near the BgZI site. Molecules with a nick in either strand are shown, and the jagged lines represent nucleotides with 3sP incorporated during nick translation. As described in the text, nick-translated DNA was digested with the indicated restriction endonuoleases and electrophoresed with or without denaturation.

reaction. From the decrease in mobility of fragments with increasing time of incubation, we estimate an average rate of extension of about five nucleotides per minute in this particular experiment. This value was confirmed by digesting nick-translated DNA with BglI (see Fig. 2) and determining the length of labeled single-stranded fragments by gel electrophoresis under conditions used for nucleotide sequence analysis (Sanger & Coulson, 1978). For the subsequent generation of deletion mutants, to be described below, DNA was nick-translat)ed a distance of about 35 and 55 nucleotides from the BglI site; for the generation of base substitution mutants, only DNA nick-translated about 35 nucleotides was used. (c) Construction

and detection of mutants

To construct mutants from nick-translated DNA the nick was first converted to a small gap as noted in Figure 1 and described in detail elsewhere (Shortle & Nathans, 1978). The gapping reaction could be followed by noting the decrease in mobility of PvuII - Hind111 fragment F caused by the presence of a gap. After gap formation, the DNA was linearized with nuclease S, or deaminated with sodium bisulfite, as described in Materials and Methods. The products were used directly for transfection of BSC40 cell monolayers, i.e. the linear DNA was not, cyclized, nor were gaps filled in enzymatically, prior to transfection. Two procedures were used t,o detect SV40 mutants among the resulting viral plaques. First, plaques formed at 32”C, 37°C or 40°C were re-plated at each of the three temperatures to look for temperature-dependent mutants or plaque morphology mutants. Second, 32P-labeled viral DNA extracted from cells infected with virus from

134

Il.

DI~IAIC)

Origin

AND

1).

NATHANS

-

215

-

70

-

63 -

FIG. 3. Analysis of single strand lengths of nick-translated DNA. After nick translation for 1.5, 3, or 5 min in the presence of [a-3ZP]dCTP plus unlabeled deoxyribonucleoside triphosphates, the product was digested with PvuII + HindIII, deproteinized, and denatured in dimethyl sulfoxide/glyoxal prior to electrophoresis in a 4% polyacrylamide gel (McMaster & Carmichael, 32P-labeled SV40 DNA similarly treated after BgZI + PvuII + 1977). The first track is uniformly HindI digestion. The numbers are the length in nucleotides of marker fragments. wt, wild type. (Because of the opposed stagger at the ends of the 68 base-pair fragment produced by Hind111 + BgZI digestion, upon denaturation this fragment gives rise to 2 discrete polynucleotides of length 70 and 63 bases.)

individual plaques was analyzed by digestion wit,h various restriction enzymes followed by electrophoresis of t’he resulting fragments and autoradiography. Of 58 plaques generated with linear DNA that were tested for t,he presence of plaque morphology mutants, 16 yielded mutants that were cold-sensitive (cs class), i.e. produced small plaques relative to wild-type SV4O at 32”C, but wild.type size plaques at 37°C and 40°C. Of the 33 plaques generated with deaminated DNA that were

REGULATORY

MUTANTS

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SV40

135

tested, seven were cold-sensitive and three formed small plaques at 32”C, 37°C and 40°C (SJJclass). Two examples of cold-sensitive mutant, plaques are shown in Figure 4. For restriction analysis, labeled viral DNA was first digested with PvuII +HindIII +BgZI. This combination of enzymes produces from wild-type DNA seven major fragment.s, including a 269 base-pair fragment on t,he late side of the BgZI site and a 68 base-pair fragment on the early side (see Fig. 2). Such analysis would detect mutant,s with delet’ions of approximately 3 base-pairs or greater on the early side of t,he BgbI site or about 10 base-pairs or greater on the late side, or rnutant,s t,hat had

Wild-typo

c,

1096

c,

1066

FIG. 4. Plaque morphology mutants. Dilutions of wild-type SV40 and mutant stocks were used to infect monolayers of BSC40 cells. Plates were incubated at 33”C, 37°C or 40°C, and after being stained with neutral red, plaques were photographed on t’he indicated day post-infection.

lost or gained a restriction site. Examples of rest,riction patterns of representative mutants are shown in Figure 5. Mutant dl 1091 has a deletion of about 20 base-pairs on the late side of the BglI site: mutant cs 1085 has a deletion of about 20 base-pairs on the early side of the BglI sit,e; cs 1088 appears to have a delet)ion also, but digestion wit,h other enzymes indicated that instead of a deletion cs 1088 had acquired new HindIII sites. Most isolates were similarly analyzed with HaeIII, AZuI and Hid. In an attempt to identify base substitutions that would create new restriction sites as predictled from the wild-type nucleotide sequence, DNAs from putative bisulfiteinducted mutants were digested with Hind11 and TaqI in addition to t,he enzymes noted above. In several instances new restriction sites and/or loss of pre-existing sites were detected. The overall results of restriction analysis are given in Table 1. Of 111 plaques surveyed by the above methods, 43 contained detectable alterations only in the targeted area: 26 of the 58 plaques derived from linearized DNA yielded deletion

138

1). DIMAIO

496

-

447

-

269

-

AND

D. NATHANS

30 -

Pm. 5. Restriction endonuclease digest of mutant DNAs. Uniformly s2P-labeled form I DNA prepared from wild-type or mutant virus-infected cells was digested with PvuII + Hind111 + BgZI and electrophoresed in an 8% polyacrylamide gel. The numbers refer to the length in base-pairs of the wild-type (wt) fragments. Arrows indicate positions of wild-type fragments closest to the BgZI site on the early and on the late side.

and of a total of 53 plaques derived from l&sulfite-treated DNA, 12 yielded viruses with altered restriction sites without detectable deletions, and five yielded viruses with small deletions. About 694, of the plaques surveyed contained mutants with detectable genomic alterations outside the BglI region: we presume these arose from randomly gapped DNA. mutants;

REGULATORY

Results of restriction

analysis:

Approximate distance of nick translation and in &TO mutagenesis 35 bases, nuclease 65 bases, nuclease 35 bases, bisulfite 7 Mutations

mapping

MUTANTS

Number of plaques screened ~ _-

Early

25 33 53 the

SV40

TABLE 1 distribution of mutations

S1 S,

between

OF

Number side?

137

around the BglI of mutants Late sidef

7 6 10 II

BgZI site and the HiTIf

A/D

junction

site

detected: Others .___-

2 11 7’1

4 2

at nucleotide

position

1

5055. 1 Mutations mapping between the RgZ site $ Deletions or x-e-arrangements mapping 11All 10 mutants contained an HaeIII addition, 2 of the mutants had also gained side of the BgZI sitr. 71 Two mutants had gained a new Hind11 deletions on the late side.

(cl) Nucleotide

and the PvuII A/C junction at nucleotide position 190. outside PvuII + Hind111 fragment F. E/M fusion fragment and no detectable deletion. In one or more new HindIII-cleavage sites on the early site near

the

sequences of mutad

BglI site. The other

5 contained

small

DNds

In order t.o define the genomic lesions of mutants more precisely, the nucleotide sequence around the BgZI site was determined by the Maxam-Gilbert method (Maxam & Gilbert, 1980) for a number of mutants with changes det,ected by restriction analysis. Table 2 lists the mutants analyzed, how they were generated, and their plaque morphologies. Included also in the Table are six mutants with base-pair substitutions at the BgZT site, previously reported by Shortle & Nathans (1979). Figure 6 presents t,he result’s of nucleotide sequence analyses. On the basis of sequence changes, deletion mutants were of two types: simple deletions and delet’ions with nucleotide subst,itutions. These t’wo classes of deletion mutant,s generated by cell-mediated cyclization of linear SV40 DNA have been noted (Thimmappaya & Shenk, 1979; Volckaert’ et al., 1979: M. Gutai, personal communication), and presumably reflect different mechanisms of cyclization. As seen in %igurc 6, mutat)ions on the late side of the BgZI site extend in the aggregate from position 5191 t,o position 4, and none affect plaque morphology. However, cs 1096, whose DNA has t,wo base substitutions within the segment missing in two of the deletion mutants, is cold-sensitive. On t’he early side of the BglI site deletions extend from position 5083 to posit’ion 5125. Many of these mutants are cold-sensitive. Of the three sequenced mutants wit,h base substitmions on the early side, two have multiple G.G to T+A transitions associated with cold-sensitive morphology (cs 1968 and cs 1989): and one has a single t,ransit,ion without change in plaque size. Each of t,hese mutants has one or more altered restriction sites, as shown in Figure 6. The high frequency of multiple base substitutions in the mutants exa,mined so far may be due to the fact that we selected mutants with altered restriction patterns for nucleotide sequence analysis. In any event, sequence analysis confirms the presence of mutations within the targeted regulatory segment and defines precise mutational changes to be correlated with the biological properties of the various mutants.

Early

side

.

GGCCGAAGCGGCC ‘CGGCTTCGCCGG GGCCGAGTCGGCC CCGGCTCAGCCGG GGCCGAGGCAGCC CCGGCTCCGTCGG GGCCGAGGCGACC CCGGCTCCGCTGG

‘o33

‘* OS ‘o34 Or ‘OX sp

‘03’

GGCAGAGGCGGCC CCGTCTCCGCCGG

lo”

GGTCGAGGCGGCC CCAGCTCCGCCGG

shp

c* ‘03’

”

log6

H,“tdlI

GTCiACCATGGA CAGTTGGTKCT

Late

side

REGULATORY

MUTANTS

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SV40

139

TABLE 2 Viable mutants with nucleotide changes near the BglI Mutant designationx cs cs cs cs cs dl cs cs cs ar

1081 1082 1083 1084 1085 1086 1087 1088 1089 1090

ar 1026s shp 1027Q: sp 1030$ cs 103 19: cs 1033s C8 1034s dl dl dl dl dl cs

1091 1092 1093 1094 1095 1096

Mutagenesis procedure

Site selection 55 35 35 55 35 35 55 35 35 35

base base base base base base base base base base

-5’ + 5’ + 5’ -+ 5’ + 3’ + 3’ --f 55 35 55 55 55 35

3’ 3’ 3’ 3’ 5’ 5’

nick nick nick nick nick nick nick nick nick nick gap gap gap gap gap gap

base base base base base base

at at at at at at

nick nick nick nick nick nick

s, S s: s1

translation translation translation translation translation t,ranslation translation translation translation translation the the the the the the

BgZI BglI BgZI BgZI BgZI BgZI

''5 1 s,

S Bis&ite Bisulfite Bisulfite site site site site site site

translation translation translation t,ranslation translation translation

t The nucleotide sequences are shown in Fig. 6. $ cs, cold-sensitive; dl, deletion; sp, small plaque; $ From Shortle C Nathans (1979). iI s, small; wt, wild-type; shp, sharp-edged.

site?

Plaque 32°C

morphology 37°C

s s 8 S S wt S S S

wt

Bisulfite Hisulfite Bisulfitr Bisulfte

u-t shp

wt shp

S

S

s

M’t

Bisuifite

s

wt

Bisuliitr

s

wt

S1 s1 S1 s, S Bisuifite

wt wt wt wt wt

wt, wt wt wt wvt w-t

shp, sharp-edged;

:

S

ar, altered

restriction.

4. Discussion An extension of the method for local mutagenesis is described in this paper. namely, a procedure for positioning the target, for mutagenesis at a pre-selected region of DNA near a defined restriction site. By controlled, approximately synchronous nick translation with DNA polymerase 1 a single strand scission at, a restricbion site can be moved to a desired position 10 to 100 nucleotides or so on either side of the original nick. The nick can t.hen be extended into a small gap by appropriat,e exonucleolyt~ic digestion, and in the case of SV40 the circular gapped DNA can be linearized at bhe gap t,o generate deletion mutants: alternatively the gapped DNA can be treated with a single strand-specific mutagen such as bisulfite, t,o generate base substitution mutations. The synchrony of nick translation is imperfect so that targets for mutagenesis extend over tens of nucleotides in the population of DNA molecules. The distribution of mutations may reflect variations in rate of nick translation dependent on t,he local sequence of the DNA (Fig. 3). The generation of two sets of mutants, one set with mutations on one side of the initial restriction cut. and the second set with mutations on the other side is unavoidable. but since the mutants are easily cloned and mapped, the t,wo types can be sorted out,. Another possible limitation (unless t,he

11(l

I). DrMAIO

AKD

D. NATHANS

same region of DNA can be approached from t’wo dire&ions) is that only one DNA strand in a given region can be mut,agenized in the hisulfite procedurr. Shortle & Nathans (1978) found that) local bisulfite mutagenesis of SV40 DNA at a unique resbriction sitr followed by enrichment for molecules resistant t,o restrict,ion at tllat, site results in the generation of viable mutant viruses with a frequency of 70 t,o 8OOj, of plaques surveyed. In t’he procedure described in t.his paper, no such enrichment was possible. Nonetheless, based on the frequency of loss of the Hap111 site at. nucleotide positions 5109 to 5112. we estimate t,hat under the conditions employed. the efficiency with which bisulfitc-induced mutants were generated was approximately 400/, Our motivation for constructing mutations adjacent to the BgZI site of SV40 DNA is to identify and characterize regulat,ory elements in the DNA segment t,hat lies between early and late genes and contains the origin of DNA replication, binding sites for T antigen, possible promoters for early and late t,ranscription, and possible sites for rrgulat,ion of transcript,ion and RNA processing. Since thcrc is no specific selection for mutant,s with base changes nit,hin these regulatory element,s. lye have chosen tJo creat’e mutations throughout, t,his region biochemically and then assess the physiological effects of each defined mutation. The mutants we have isolated so far have small deletions and/or base sub&itutions (generally multiple) clustered in t,wt, discrete regions outside of protein-coding sequences and adjacent to the previously localized origin of viral DNA replication, as expected from the way t,hc mutant,s were generated. At present wc know the nucleotide sequence changes at the mutational sites. t,hat t’he mutants are viable, and that many are cold-sensitive. The fact that the mutants described in this paper are viable indicates that the deleted regions of DNA (from position 5083 to 5125 on the early side of the BgZI site and from position 5191 to 4 on the lat,e side) contain no uniquely occurring signals essential to lytic growth. This conclusion regarding dispensable sequences confirms and extends other reports on similar SV40 and polyoma mutants (Subramanian 6t Shenk, 1978; Wells el aZ.. 1979: Magnusson & Berg. 1979: Bendig & Folk, 1979). On the early side of the Rgll site SV40 DNA is rich in invert,ed and direct, repeats (Subramanian et al., 1977). some of which are conserved in the DNA of other papovaviruses (Dhar et al., 1978; Friedman et al., 1978). Some deletions regenerate one copy of such repeated sequences. It is therefore possible that redundant signals exist in this region and that deletions that remove all copies of a given sequence would not be viable. To illustrate this point we note that mutants cs 1082, cs 1083, cs 1084, and cs 1085 all delete much of the highest affinity T antigen-binding site described by Tjian (1978a), including nucleotides which appear to interact directly with T antigen (Tjian, 19783). One interpretation of these data is that this binding site is not required for viability. Another possibility is that the redundant sequence A-A-A-A-G-C-C-T, present once in all the mutants, is the essential part of the site. Thorough mutational analysis of the SV40 regulatory segment will require the generation of mutants throughout this region of the genome, many of which are likely to be non-viable. We have therefore begun to clone and propagate new SV40 mutants as pBR322/SV40 recombinants in E. coli. Such mutant genomes require no SV40 funct,ions for growth in E. coli, but can be tested for SV40 activities by transfection of cultured cells (Pipas et al., 1979).

REGULATORY

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SV40

141

It is striking that most mutants with alterations on the early side of the regulatory segment are partially defective or cold-sensitive. Assuming that plaque phenotype is related to the observed mutations (testable by marker rescue or in vitro recombinaDion) we interpret the partial or conditional defectiveness to mean that nucleotide sequence signals involved in regulating virus development have been modified and are therefore less active than the wild-type signals. The frequent occurrence of coldsensitive mutants, noted earlier for SV40 ori mutants (see Table 2) and for certain lat,e deletion mutants (C.-J. Lai 8: G. Khoury, personal communication) suggests that’ the signals (in the form of DNA or RNA) are involved in temperature-dependent int,eractions with viral or cellular proteins, or in the proper folding of RNA transcripts. In the former case we hypothesize that regulatory proteins are less able to accommodate an altered signal at low temperature. Physiological and biochemical experiments with the defective mutants may help define t’he nature of the underlying regulatory events. We thank David Shortle for valuable critique. This research was supported by a grant from the National Cancer Institute, United States Public Health Service (5 PO1 CA16519). One of us (D. D.) is a pre-doctoral student in the Medical Scientists Training Program of the United States National Institutes of Health (GM7309).

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