J. Mol. Biol. (1977) 116, l-30

The Sequence of a Region of Bacteriophage +X174 DNA Coding for Parts of Genes A and B NIGEL

L. BROWN? AND MIICHAEL SMITHS

Medical Research Council Laboratory of Molecular Biology Hills Road, Cicmbridge CB2 2&H, England (Received 24 January 1977) Part of bacteriophage 4X174 DNA in the regions of genes A and B has been sequenced using the rapid method of Sanger & Coulson (1975) with confirmation b,y depurination analysis. The resulting sequence of 870 nucleotides codes for about half of the A protein, whose gene extends beyond either end of the sequence. In addition, over 80% of the B protein is coded by the same region. This, therefore, is the second region of the +X174 genome where two genes are translated from the same DNA using different reading frames. Possible locations of the A/B promoter and the A* protein translation “start” are discussed.

1. Introduction The nucleotide

sequences

of several

regions

of the bacteriophage

4X174

genome

have

been determined using the new, rapid “plus and minus” method of DNA sequencing (Sanger & Coulson, 1975). Most of these sequences have been determined with the aid of partial sequence data of the proteins from genes D, J, F, G and H of +X174 (Air et al., 1975,1976; Barrel1 et al., 1976; Sanger et al., 1977). Sequences in intercistronic regions have been obtained with the aid of more laborious DNA sequencing techniques (Fiddes, 1976; Air et al., 1976). Together these sequences account for about 3500 of the 5400 nucleotides in +X174 DNA. The remaining 1850 nucleotides code for genes A, B and C (Fig. 1). The corresponding proteins have not been available for peptide sequence determination, although the A protein has been extensively purified (Ikeda et al., 1976; A. Kornberg, unpublished data). Here we describe the determination of the sequence of a region of 870 nucleotides beginning near the left-hand end of restriction endonuclease HaeIII fragment 6a and ext’ending rightwards to just beyond the right-hand end of TaqI fragment 2 (Fig. 1). This region contains part of gene A and probably contains the translation start for the smaller gene A product (A* protein, Linney et al., 1972; Linney & Hayashi, 1973, 1974; Henry & Knippers, 1974; Denhardt, 1975), a promoter and a transcription start (Smith & Sinsheimer, 197h,b). A translation start, possibly for gene B, is also present (Ravetch et al., 1977; Brown & Smith, 1977). It was our objective to determine accurately the DNA sequence without the help of parallel peptide sequences or DNA sequence obtained by more laborious methods. t Present address: Department of Biochemistry, versit,y Walk, Bristol BS8 lTD, England. $ Present address: Department of Biochemistry, Columbia, Vancouver, B.C., Canada V6N lW5. 1

1

University

of Bristol,

Il’acu1t.v of Medicine,

Medical University

School, Uniof British

n.pc hp, amE

I

5c 5 2 Gene H om86tA)

1

4 8

I

1

I

I

I

3

500

3

1

I

600

om33tA)

6b

400 70 1

I 1

1

2

7b I161

I

300

6 30 I

4 60 I

I

700

I

III1

800

1000

1100 I

I203 II,

1300

1

8

I

1 I

5 3 6

3

I

I I

1

II

I700

9

7b

1

6c

I

I

I

[Gene0

17’ 16o’l6b

17

I

200 II

1900

I13 1‘1‘1‘1

2

4

6 4

1800

ochG(C)

1

k-18 ~8~9~+-10

I

l6m

8b

1500

5 17

151

1400

I omlE(A) om RE-I i A) om3!5(A) oml6(8) m3(A)

50

I

1 II 1 IO !12bl,Ll I201 15b’ ‘17 2 I I9 1101

900

Fig. 1. The restriction endonuclease map of the (appros.) 1850 nucleotides between the C-terminus of gene H of 4X174 and the N-terminus of gene D. This region codes for genes -4, B and C. Some representative mutants have been indicated in the positions to which they have been assigned by genetic of urn18 and a&5). The DNA sequence reported in this paper (870 nucleomapping (Weisbeek et al., 1976; see Baas et al., 19766 for more precise mapping tides) starts near the left-hand end of Hoe111 fragment 6a and extends rightward to the left end of TaqI fragment 7. n.p., nucleotidc pairs.

HoeM /-/ho1 Hoell HmdII HpaI HinfI MMI HphI WI

Hoi!.l

Ah1 TOOI

A-Gk-T T’C-G-A C’C-G-G G-G-C-C G-C-G% R-G-C-G& G-T-Y%-A-C G-T-TZA-A-C G’A-N-T-C G-A-A-G-A---E G-G-T-G-A---E C-T-G-CA?G

I

I

I

200

loo 0

SEQUENCE

OF PART

OF

4x174

GENES

il

BND

B

3

To this end, the plus and minus method (Sanger & Coulson, 1975) was refined to overcome the problem of estimating the number of nucleotides in a run of identical nucleotides and to minimise the effects of secondary structure in the DNA (Fiddes. 1976). In addition, the determination of the composition of the pyrimidine nucleotide tracts and the characterisation of the purine nucleotide adjacent to the 3’ end of each tract in each strand of defined short regions of $X174 replicative form I DNA has been used to confirm the sequence. The results allow the prediction of the sequence of the A protein in this region and suggest a possible translation start for the A* protein. Two possible locations for the promoter and transcription start have been identified. The coding of a ribosome binding site (Ravetch et al., 1977) within this region (Brown & Smith, 1977) allows the prediction that gene B is translated from the same DNA sequence as gene A, but using a different reading frame. This is the second set of overlapping genes in the $X174 genome, the fist being genes n and E (Barrel1 et al., 1976). These two sets of overlapping genes completely resolve the paradox of the genome size predicted from protein molecular weights being about 10% larger than the actual size (Barrel1 et al., 1976). They also resolve anomalies in the genetic data obtained from mutants mapping in these regions (e.g. Borrias et al., 1976)

2. Materials and Methods 4X174 am3 viral (plus) strand DNA, 4x174 am3 replicative form I DNA, +X174 am3 complementary strand DNA and 4X174 am16 viral strand DNA were gifts from C. A. Hutchison III. Restriction endonucleases AZ&, HapII, HaeII, HaeIII, HindII, HinfI, MBoII and P&I were prepared by methods similar to those described by Roberts et al. (1975). HhaI, HphI and TaqI were gifts from R. J. Roberts, J. R. Arrand and S. Sato, respectively. Restriction enzymes were stored at -20°C in 50% glycerol, 10 mM-pmercaptoethanol, 0.1 mM-EDTA, 10 mM-potassium phosphate (pH 7.4), at a concentration such that approximately 1 pl was required to completely digest 1 rg of X DNA in 1 h at 37°C (1 unit/pi). [a-32P]deoxynucleoside-5’-triphosphates were obtained from New England Nuclear and non-radioactive nucleoside-5’-triphosphates from P.-L. Laboratories. Escherichia coli polymerase I were obt,ained from BoehDNA polymerase I and the derived “Klenow” ringer. Bacteriophage T4 DNA polymerase preparations were gifts from A. Coulson and R. Kamen. The polymerases were stored in 50% glycerol at - 20°C at a concentrat,ion of about 2 units/pi. (a) Purification of deoxyribonucleoside-5’4riphosphates These were purified by ion-exchange chromatography on DEAE-cellulose (Whatman DE-23). Each nucleotide (approx. 10 pmol) was dissolved in water (1 to 2 ml) and applied to a column of DEAE-cellulose (500 mm x 10 mm) at room temperature. The nucleotides were eluted using a linear concentration gradient of NH,HCO, (total vol. 2 1) at a flow-rate of 2 ml/min (IO-ml fractions). For dATP the gradient was 0.1 M to 0.125 M (pH 8.2); for dGTP, 0.1 M to 0.15 M (pH 8.2) ; for dCTP and dTTP, 0.01 M to 0.10 M (pH 9.5) ; adjusted with concentrated NH,OH immediately prior to chromatography. Use of the pH 9.5 buffer is critical for the purification of dCTP and dTTP, since these nucleotides are not separated at lower pH. The purity of each triphosphate was examined by u.v. spectroscopy of individual fractions. The pure fractions were bulked and NH,HC03 and water removed by The nucleotide was dissolved in water repeated evaporation under reduced pressure. (5 pmol/ml) and treated with a small amount of freshly prepared Dowex 50 (NH:) to remove other cations. The resin was removed after A min and the nucleotide solution stored at - 20°C. (b) Primed synthesis of DNA for the plus-~ minus experiment The restriction endonuclease fragment primer (from 10 pg +X174 replicative form (purified electrophoretically, Galibert et al., 1974)) in water (10 ~1) and template

DNA

DNA

N. L.

4

BROWN

AND

M. SMITH

(1 pg, either viral or complementary strand $X174 DNA (purified as described by Brown et al., 1977)) in water (2 ~1) were mixed in a siliconised tube (50 mm x 10 mm) and then heated, in a sealed capillary, at 100°C for 3 min. 1 M-NaCl (1.25 ~1) and buffer A (1.25 ~1 100 M-i&$&, 10 mM-fi-mercaptoethanol, 200 mM-Tris.HCl, pH 7.4) were added and, after mixing, the solution was heat,rd at 68’C in a sealed capillary for 1 h to anneal the pritner aud template. ‘The solution was cooled to 5°C and to it was added a mixture of [cr-32P]dATP (200 pmol, 100 Ci/mmol, dry sample), dCTP, dGTP, dTTP (0.5 IuM, 2.5 ~1 of each), watar (1 ~1) and buffer A (1.5 ~1). DNA polymerasc I from E. coli (1 ~1, 2 to 3 units) was added and the mixture incubated at 5°C. Half the sample was quenched in 50 m>lEDTA (pH 7.5) (50 ~1) at 0°C after 1 mirl and the rest of the sample was quenched iu the same solution after 3 min. Water-saturated phenol (25 ~1) was added and the mixture shaken for 10 s on a Vortex mixer. After extraction with ether (2 x 250 ~1) and removal of residual ether by blowing air over the solution, it was applied to a column of Sephadex GlOO (3 mm x 200 mm). The column was eluted with 2 mM-Tris.HCl, 0.05 mM-EDTA (pH 7.5) (flow-rate approx. 50 pl/min). Elation was monitored with a Geiger counter and the faster moving polynucleotide fraction ( 100 to 150 ~1) collected in a small silicon&d tube and freeze-dried. About 10% of the radioactive nucleotide is incorporated into polynucleotide under these conditions and the extension of the primer ranges from zero t>o 150 to 200 nucleotides. For the plus-minus experiment, tho radioactive polytrncleotide is dissolved in water (20 to 30 ~1) immediately prior to use (see below). (c) The plus-minus

experiment

Eight samples (2 ~1 each) of the radioactive polynucleotide (above) were placed in capillary tubes together with the appropriate plus or minus mixtures (Sanger & Coulson, 1975) (2 ~1) and T4 DNA polymerase (1 ~1) at 37°C. Each plus mixture contained one of the deoxynucleoside-5’-t,riphosphates (0.5 mM) in 0.4 X buffer B (buffer B is 500 mM-N&l, 66 mivr-MgCl,, 66 mM-/?-mercaptoethanol, 66 mM-Tris.HCl, pH 7.4). Each minus mixture contained 3 of the deoxyribonucleotide triphosphates (0.025 mM) in 0.4 x buffer B. The amount of T4 DNA polymerase to be used was established by assay in trial plus and minus reactions, incubated at 37°C for 30 min. The amount of enzyme which gave a limit product under these conditions was used. After 30 min at 37”C, the datum restriction endonuclease (1 ~1, 0.5 to 1.0 unit) was added to each sample. (The datum restriction endonuclease is that which defines the 5’ end of the sequence under investigation.) In addition, to provide a reference pattern of oligonucleotides (graticule), a sample of the “radioactive polynucleotide” (2 to 4 ~1) and 0.4 x buffer B (2 ~1) was incubated at 37°C with the datum endonuclease (1 ~1). After 30 min the reactions were quenched by addition of a mixture (20 ~1) containing bromophenol blue (O.Ol%), xylene cyan01 FF (0.01%) and EDTA (0.025 M) in 90% formamide. The samples were heated at 90°C for 3 min to denature the DNA, and then cooled in ice-water. Each of the 9 samples was divided into 2 portions and each set of samples was applied in slots s-mm wide to a 12% acrylamide, 7 M-urea gel (1.5 mm x 200 mm x 400 mm; Sanger & Coulson, 1975) with TBE buffer (90 mM-Tris, 90 mM-sodium borat,e, 2.5 mM-EDTA, pH 8.3) (Peacock & Dingman, 1967). Electrophoresis was carried out in the type of apparatus described by Studier (1973) with TBE buffer in the electrode chambers. Electrophoretic conditions (600 V, 30 to 35 mA) were chosen with the specific objective of heating the gels to 50 to 60°C. For a given sample, power was applied until the bromophenol blue (equivalent to oligonucleotides ~15 in length) reached the bottom of the gel (approx. 4 11); power was applied to the second gel until the xylene cyan01 FF (equivalent to oligonucleotides ~55 in length) reached the bottom (about 8 h). Each gel was fixed in 10% acetic acid (10 min at 25OC), rinsed with distilled water, blotted dry on both sides, and covered, on a glass plate, with Saran-wrap. Aut,oradiography at room temperature for 24 h was uwlally sufficient to visualise the bands. (d) Location

of restriction

endonucleme

cleavage

sites

in the sequence

under

investigation

Two samples of radioactive polynucleotide (2 ~1) and a mixture of the 4 deoxyribonucleoside-5’-triphosphates (0.5 mM) in 0.4 x buffer B (2 ~1) together with T4 DNA polymeraae (1 ~1) were incubated at 37°C. To each sample was added the endonuclease

SEQUENCE

OF PART

OF

4x174

GENES

A AND

L3

,i

(1 ~1) whose site was under investigation and to one sample the datum restriction endonuclease (1 ~1). The samples were incubated at 37°C for 30 min. Tho reactions were then quenched and the samples denaturtxd and electropllor~t,ioall? fractionated in parallel with the plus and minus reactions. ((3) Ilepurin,ation

analysis

of defined

fragments

gerterated

hy primed

synthesis

A rcbstriction cndonuclease fragment primer (frown 4 rg +X174 replicative form) irl watc:r (4 ~1) and either viral or complementary strand t,cmplate DNA (0.5 pg in 1 ~1 water) were mixed and heated at lOOY1 for 3 min in a sealed capillary. Buffer A (0.5 ~1) and 1 ;11-NaCl (0.5 ~1) were added and the mixture was incubated at 67°C for 1 11to anneal the primer and template. The annealed mixture was divided into two, and half was added to an extension mixture (10 ~1) conta.ining [c(-32P]dATP (50 pmol, 100 Ci/mmol), dCTP. was equilibrated at 5°C and dTTP, dGTP (0.07 IIIM each) in 0.1 x buffer A. Tllc mixture the reaction was started by the addition of 1 to 1.5 units DNA polymerase I. Incubation was continued at 5°C for 30 min and stopped by the addition of 10 ~1 water-saturated phenol and 10 ~1 water with mixing. The aqueous layer uas removed and extracted wit11 water-saturated ether (4 x 250 ~1). The ether was removed by blowing air over the solutiou. Tho remaining half of the annealed mixture was treated in exactly the same way-, cxccpt in tllat [or-32P]dGTP and norl-radioactive dATP rt~placrd that [a-32P]dATP and tlGTl’, respoct,ively. Buffer B (0.1 vol.) and 1 or 2 restriction errzymtls (about twice the activity normall) required for this amount of DNA) were added to each reaction mixture, and the mixtlu-css incubated at 37°C until digestion was complete. Eacll react,ion was stopped wit11 5 ~1 of sol&on S (0.1 M-EDTA, 40% sucrose, 0.01 o/o bromopllcnol blue, O~Olo/;, xylene cyanol FF in tvater) and tile products werrt separated by elect,roplloresis in acrylamide slab gels (Soi;, 8yL or lOy~/,, acrylamide, depending on the size of tllcl product) in TBE b&&r. The products were visualised by autoradiography. Usually the primers. templates and restrictiorr e,lzymes were selected sucll that both strands (viral and complementary) of tlrr rcquircxd fragment were obtained in one set of experiments. Oftcan the required fragment was tjlrtl only band on the gel wit11 the same mobilit>- ill all 4 samples (dATP and d(:Tl’ label, \riral and complementary strands). The pieces of gel containing the required fragments were excised and lightly homogenised. The DNA was eluted by incubating the gel at 37°C in 0.5 u-NaCl, 0.1 M-Tris.HCl, 5 InnrEDTA, pH 7.5 (0.5 ml) for 12 11. The pieces of gel were removed by filtration tllrough glass wool. Transfer RNA (20 pg) was added to the filtrate as carrier, and the nucleic acids wer~~ precipitated by adding ethanol (2 vol.) and freezing. The precipitate was collected b> crutrifugation, and as much as possible of the supernatant was removed. The precipitate was washed quickly by resuspension in 80% ethanol, and then taken up in 50 ~1 wat)rr. A solut,ion of 3% diphenylamine in 98% formic acid (100 ~1) was added and the samplr MXS incubated at 37°C for 16 h. Water (100 ~1) was added and the sample extracted wit11 ether (4 x 2 vol.). The sample was dried down in a new siliconised tube, and then taken up in watr,r (approx. 7 ~1) and the depurination products separated by electrophoresis ant1 homocln-omatography as described by Ling (1972). The separated products were visualised by alltjoradiography. The molar ratios of the products were usually estimated from tilt. autoradiograph, or occasionally were determined by scraping off thr DEAF, laya containing each spot into fluor solution (0.4% BBOT (Koch-Light, Lt)d.) in tolnenc) and det,ermining the radioactivity in a Nuclear-Chicago Isocap 700 scintillat’ion countrr.

3. Results (a) The DNA

sequenw

The sequence of 870 nucleotides of 95X174 am3 viral DNA is summarised in Figure 2. As an illustration of the methods used for obtaining the sequence, the experimental data for the region from the Z6a/Z9 site to the R3/R8 site (nucleotides 254 to 313, inclusive) are presented in Figures 3,4 and 5. The plus and minus sequence determinations required to define this region were R8 priming on the viral strand and A4

M6(A4)/C(L)

M6(A4)/C(S)

WV(S)

RVW

Z9jV(S)

ZS/V(L)

ZS/V(L)

F5a(All)/V(S)

Fh(All)/V(L)

ZQ/W)

F5a(411)/V(L)

ZGa/C(L)

ZBb/C(L)

Z6b/C(S)

HinfI

3 T 5a 200

210

220

T.C.C.d.T.G.C.G.G.T.G.C.~4.C.T.T.T.A.T.G.C.G.G

T-G-G-T-A-C-A-G-C-T-B-8-T-G-G-C-C-G-T-C-T.T-C-A-T-T-T-C.C.A-T.G.C-G-G.T-G-C.A.C-T.T-T-A-T.G-C-G.G.A-C.A

190

G-G-G-T-C-G-C-d-A-G-G-C-T-A-d-T-G-A-T-T-C-A-C-A-C-G-C-C-G-A-C-T-G-C-T-A-T-C-A-G-T-.4-T-T-T-T-T-G-T-G-T.G.C-C-T-G-A-G-T-A-

T.G.~~.T.T.C.~4.C.~.C.G.C.~.G.~.C.T.G.C.’~.~~.T.(~.~4.G.T-~~.T.T.T-T.T-G-T-Q-T-G-C’-C

G-G-G-T-C-G-C-A-A-G-G-C-T-A-A-T-G-A-T-T-C-~4-C-A-C-G

T-A-A-T-C-C-C-A-A-T-G-C-T-T-T-G-C-G-T-G-A-C-T-A-T-T-T-T-C-G-T-G-A-T-.~-T-T-G-G-T-C-G-T-A-T-G-G-T-T.C-T-T-G-~‘-T-G-C-C-G-~~160 130 140 150

T..~.A.T.~.C.C.A~.~.T.G~~.~.~.~.~.C.G.T.G.~.C.T.~~.~.T.T.T.C.G.*.G.A.T.~.T.T.G.G.T.C.G.T.A.T.~.G.T.T.C’.T.T.(:.a_ W/V

&G-T-A.&".*

OF

c,-c,.i-&C,

PART

OF

4

-c-a

C-G-T-G-,-A-A-i-A-

"2

\--3 (3:

-7

?

+X174

GENES

A

B

13

" A 4 r.~~~4-T-G-T-C~C-A-T-i~~~C~A~A~C-T

-,-L-:.-T-i,?* ;,T

AND

-i---i--, C,T(G;

Gj

\ CT(Al

Fro. 6. Assignnrcnt of depurination products for the region from site Z6a/Z9 to R3/R8 (Table 2) to their positions in the DNA sequence of the region. All the depurination products could be quantitated, and are shown, except, T(G) (i.e. pTp labellcd with [a-32P]dGTP) from R8 priming on t,hc viral strand. Only those products larger than mononucleoside diphosphates are lebelled in the diagram. The terminal pyrimidine tract in the viral strand of this fragment (T,) is not labellt~d due to rlcavage by HindII, which leaves a terminal 3’.OH.

(MbolI-cleaved) priming on the complementary strand. The overlap of the two determinations consisted of nucleotides 267 to 292 inclusive. The specific fragment, used for depurination analysis Z6a/Z9-R3/R8 was generated by priming with R8 on the viral strand and Z6a on the complementary strand, followed by cleavage with HaeIII and HindII. The depurination analysis for t,his region (Fig. 5) is summarised in Table 3 and it completely confirms the sequence obtained by the plus and minus method (Fig. 6). All of the sequence presented here was determined and confirmed by these methods (Fig. 2 and Tables 1 to 10). The DNA sequence between nucleotides 1 and 197 (Fig. 2) may cont,ain one or two errors, due to the difficulties in quantitating depurination products from a large DNA fragment (fragment Z6b/6a-F3/5a, 213 TABLE 1 Depurination

Primer/template Label Product T

C ‘1 2

analysis of fragment Observed ZGb/complementary dATP dGTP

>4 (5) 4 1

('T i (‘2

4

T, CT, ,I?; c T

1 1 1

CT, C,Tz (‘,3T ‘1 G, (J,T, C’‘4 T

>4 (11) >4 (7) 3

>4 (7) 2

ZGblZSa- F3/F5u molar

yieldt F5a/viral$ tl ,\‘I’P

>4 (7) i4 (8) 2

>,4 (8) >4 (5)

dGTP

4 >4 2 4

(10)

1 1 2 1

1 1 2 1 1

7 For Tables 1 to 10, only the depurination observed, and in their predicted molar yields. accurately the products present in higher molar in parentheses. $ H&f1 fragment 58 was not used a.3 a primer HaeIII fragment 9 was used for priming on the was generated by cutting with HinfI and HaeIII.

2 1 products predicted from the gel sequence were Occasionally it was not possible to quantitate yield, and the predicted yields for these are given due to contamination with HinfI fragment 5b. viral st,rand. and the fragment for depurination