Gene. 114 (1992) 85-90 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

GENE 06411

85

Gene r i l l is the nearest downstream neighbour of bacteriophage T4 gene 31

(Recombinaat DNA; PCR amplification; nucleotide sequence; r mutations; sequence discrepancies; transitions; transver- sions)

Aulra Raudonikiene and Rimas Nivinskas

bastitute of Biochemisto,, Lithuanian Acadeno' of Sciences, Vilnius 232021 (Lithuania)

Received by A.J. Podhajska: 17 June 1991 Revised/Accepted: 30 December ! 991/3 ! J anuary 1992 Received at publishers: 4 February 1992

SUMMARY

The nucleotide sequence of the 2218-bp T4 DNA fragment encompassing gene 31 and five complete open reading frames (ORFs) is presented. We show here that one of these ORFs, ORF31.-1, located downstream from gene 31, is the rill gene. The position of the gene was established by comparison with the sequences of the rill gene mutants, r67, rES40 and rBB9. The ORF corresponding to the rill gene encodes a basic protein of 82 amino acids with an Mr of 9323 and a pl of 9.28. According to the Chou and Fasman [Adv. Enzymol. 47 (1978) 45-148] secondary structure prediction, the rill protein has a relatively high helical content. In addition, discrepancies with the overlapping sequences determined by other authors in this region are indicated.

INTRODUCTION

Rapid-lysis mutant r67 of gene rill, together with some other standard plaque morphology mutants have been widely used in early studies on the T4 genetic map (Edgar et al., 1962; Epstein et al., 1963; Streisinger et al., 1964; Mosig, 1968). Although ril l has been often used as a ref- ereace point for map distances from amber mutations in T4 genes, its own position on the genetic map has chdnged in the course of time. Streisinger et al. (1964) have shown r67 to be closely linked to gene 31 mutation, amN54 (1.5% recombinants), and mainly on the basis of these data, rlll

Correspondence to: Dr. R. Nivinskas, Institute of Biochemistry, Lithua- nian Academy of Sciences, Mokslininku 12, Vilnius 232021 (Lithuania) Tel. (0070122)35-91-46; Fax (0070122)227474.

Abbreviations: aa, amino acid(s): bp, bate pair(s); gpX, product of gene X; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonu- cleotide; ORF, open reading frame; P, promoter; PCR. polymerase chain reaction; pl, isoelectric point; RBS, ribosome-binding site(s); SD, Shine- Dalgamo (sequence); t, terminator of transcription; tsp, transcription start point(s); wt, wild type . . . .

originally was placed just upstream (clockwise) from gene 31. At this position, rlll can be found on i963-1976 maps of phage T4 (Epstein et ai., 1963; Streisinger et al., 1964; Mosig, 1968; Wood and Revel, 1976). However, discrep- ancies in the order of amber mutations, amNG71 and amN54, within gene 31 with respect to genes rill and 30 (Georgopoulos et al., 1972; Simon et al., 1974) suggested that the position of rlll on the T4 map may be incorrect. Revised location of the r67 mutation (based on two- and three-factor crosses) positioned rll l just downstream from gene 31 (Revel and Lielausis, 1978), with the order being cd-31-rlll-30 (Mathews et al., 1983).

Recently, the primary structure of gene 31 has been de- termined and the precise locations of its am mutations were shown (Nivinskas and Black, 1988; Keppel et al., 1990; Raudonikiene and Nivinskas, 1990). In this report, we present the sequence of the 2218-bp T4 DNA fragment containing gene 31 together with five complete ORFs, one of which, namely ORF31.-I, we show to be rlll. The location of r67 and two other ril l mutations is presented. Moreover, we indicate the discrepancies with the overlap- ping sequences determined by other authors in this region.

86

EXPERIMENTAL AND DISCUSSION

(a) Cloning and sequencing of the gene 31 region Gene 31 is located on a 3.1-kb EcoRl restriction frag-

ment (Fig. 1) lying within 130.3-127.2 kb on the genomic map of bacteriophage T4 (Kutt¢r and RQger, 1983). At- tempts to clone the entire 3.1-kb EcoRI DNA fragment (Mileham et al., 1980) as well as the overlapping 4.2-kb Bglll DNA fragment (Nivinskas, 1988) have failed, indi- cating that a gene product or a strong phage promoter (or both) lying in this region might be lethal for the host bac- teria. Furthermore, the overlapping region of these two DNA fragments has been shown to contain two T4 early promoter sites (128.6 and 128.2 kb) recognized by unmod- ified RNA polymerase in vitro (Gram et al., 1984). The 3.1-kb fragment has only been recovered as a 2.7-kb de- rivative in a phage 2 clone (607-10) (Mileham et al., 1980) containing a 1.9-kb EcoRI-Pstl subfragment with gene 31 (Nivinskas and Black, 1988b). Vectors for sequence anal- ysis were prepared by transferring a 1.9-kb EcoRI-PstI T4 DNA fragment from it clone (607-10) into M13mpl0 and M13mpll.

Our failure to clone the 1.2-kb PstI.EcoRI subfragment (obtained after PCR amplification) may be caused by the existence of the T4 strong early promoter, PE128.2, recently isolated and sequenced by Liebig and RQger (1989). The

establishment of this promoter sequence has permitted us to amplify, clone and sequence the region between the Pstl site and the PE128.2 (Fig. 1). According to the sequence upstream from the Pstl site and to that of PJ28.2, the two following oligo primers were synthesized: a 20-mer, 5'-AGCACGTGCGGTTCTTCGAG, identical to 1718- 1737 nt of the sequence shown in Fig. 2, and a 20-mer, 5'-CAATTATTTTACTACTTTCC, complementary to 2199-2218 nt of the same sequence (Fig. 2). The PCR am- plification was performed according to the protocol of S aiki et ai. (1988) on the entire T4 dC-containing DNA which was prepared using the T4 strain [56(amE5 l,dCTPase- )- denA(nd28,endoll - )-denB(delrllH23B,endolV- )-alcS ], as has been described previously (Nivinskas, 1988). To determine the sequence of the amplified fragment, we cloned it into plasmid vectors pTZ 18R and pTZI9R. The restric- tion map and a summary of the sequencing strategy for a 2218-bp DNA fragment containing gene 31 are presented in Fig. 1.

(b) Analysis of the sequence The nt sequence determined for the 2218-bp T4 DNA

fragment is shown in Fig. 2. In addition to the gene 31, five ORFs could be identified in the direction of early transcrip- tion. A summary of the identified ORFs and their charac- teristics is given in Table I. The deduced aa sequences

% P~12e.6 n 12e.2

I cd lal.21 3 1 . 1 i f - ' ~ ~ ~ I 31-31 I I 1 , i.o I

EcoR! Bglll Psti t 3.1-kb

I '

I I I ,

3O

EcoR! Bglll I

4.2-kb t

" . . . . 1~0 . . . . ' 'kb 0 2.0

Fig. I. The gene 31 region and the sequencing strategy. The schematic outline represents the genomic region between 131 and 125 kb on the physical map of bacteriophage T4 (Kutter and Rtlger, 1983). Shown are the positions of the restriction sites lor EcoRl, Bglil and Pstl, as well as the positions of T4 promoters and genes; size and position of the cd gene are as indicated in Maley et ai. (1990). The cloned 2218-bp T4 DNA fragment is shown below. The 1.9-kb EcoRI-Pstl T4 DNA fragment was obtained from a phage ). clone (607-10) (see section a, for details) and cloned into Ml3mpl0 and M 13mp 11 vectors. The Pstl-site downstream region of the 2218-bp fragment was PCR-amplified as desc~'ibed by S aiki et al. (1988) using two oligo primers (5'-AGCACGTGCGGTTCTTCGAG and 5'-CAATTATTTTACTACTITCC) and the entire T4 dC-containing DNA, and cloned into the pTZ18R and pTZ 19R plasmid vectors. Preparation of DNA restriction fragments and their ligation into cloning vectors were essentially as described in Maniatis et al. (1982). Sequences of T4 fragments cloned and subcloned into M13 phage vectors or into pTZ plasmid vectors were determined by the chain- termination method (Sanger et al., 1977), using the Klenow fragment of E. coil DNA polymerase I or the modified T7 DNA polymerase (Sequenase). The arrows indicate direction and length of the sequence determined. The scale at the bottom represents kb of the sequence shown in Fig. 2.

EcoRl

SD

ORP

31

.2

GAATTCTGTGGTGAATA ATG AAA TTT CGT TTG GTA AAA CTC ACA GOA ATT

Met Lys Phe Ar H Leu Val Lys Leu Thr Ala Ile

AGT TCT TAT TCT AAC GAG AAC ATC TCA TTT GCT GTA GAG TAT AAG AAA TAT

Ser Ser Tyr Ser Asn Glu Asn Ile Ser Phe Ala Val Glu Tyr Lys Lys Tyr

TTT TTC TCT AAA TGG AAA CAG TAT TAT AAG ACA AAT TGG GTT TGT ATT GAT

Phe Phe Her Lys Trp Lys Gln Tyr Tyr Lys Thr Asn Trp Val Cys lle Asp

AGA CCA TAT AGT TGG AAA TCT GAT TTA GAA AAA TGC CAA AAA TTA CTT TCC

Arg Pro Tyr Ser Trp Lys Ser Asp Leu Glu Lys Cys Gln Lys Leu Leu Ser

SD

ACC CTT AAA GAA CGT GGA ACA ACT CAT ATT AAA ACT GTA ATA GGT AAA TAA

Thr Leu Lys Glu Arg Gly Thr Thr His Ile Lys Thr Val Ile Gly Lys ***

O~

;1.1

ATG AAA CTG ACA ACT GAG CAG AAA GTA GCA ATT CGT GAA ATT TTG AAA ACT

Met Lys Leu Thr Thr Glu Gln Lys Val Ala Ile Arg Glu Ile Leu Lys Thr

NcoI

AAA TTG TCC ATG GGT GTT TCA AAC GTA GTT TTT GAA AAG TCT GAT GGT ACT

Lys Leu Ser Met Gly Val Set Ash Val Val Phe Glu Lys Ser Asp Gly Thr

ATT CGT ACT ATG AAA GGT ACT CGT GAT GCA GAC TTT ATG CCA ACC ATG CAA

Ile Arg Thr Met Lys Gly Thr Arg Asp Ala Asp Phe Met Pro Thr Met Gln

ACT GGC AAA TTG ACT GAA TCT ACT CGG AAA GAA TCT ACT GAC ATG ATT CCA

Thr Gly Lys Leu Thr Glu Ser Thr Arg Lys Glu Set Thr Asp Met Ile Pro

GTA TTT GAT GTT GAG CTT GGT GCG TGG CGA GGT TTT TCT ATT ~C AAA TTG

Val Phe Asp Val Glu Leu Gly Ala Trp Arg Gly Phe Ser Ile Asp Lys Leu

ATT TCC GTT AAT GGT ATG AAA GTT GAG CAT TTG CTT CAA TTT ATT GGT AAA

lie Ser Val Asn Gly Met Lys Val Glu His Leu Leu Gin Phe Ile Gly Lys

-~0

PM

-10

~

HpaI

SD

GENE 31

TA~ATGCTTTA~GAACTATTTG~TATTA~TAATTCATCTGTTAACAAAAAGGAAAAACG

ATG TCT

***

Met

S

er

amN

G71

GAA GTA CAA CA.__GG CTA CCA ATT CGT GCT GTC GGT GAA TAT GTT ATT TTA GTT

Glu Val Gln Gln Leu Pro Ile ArH Ala Val Gly Glu Tyr Val Ile Leu Val

TCT GAA CCT GCA CAA GCC GGT GAT GAA GAA GTT ACA GAA TCA GGA CTT ATT

Set Glu Pro Ala Gln Ala Gly Asp Glu Glu Val Thr Glu Ser Gly Leu Ile

ATC GGT AAA CGT GTT CAA GGT GAA GTT CCT GAA CTG TGT GTA GTT CAC TCT

Ile Gly Lys Arg Val Gln Gly Glu Val Pro Glu Leu Cys Val Val His Ser

GTO GGT OCT GAT GTT CCT GAA GGT TTC TGC GAA GTT GGT GAT TTG ACT TCT

Val Gly Pro Asp Val Pro Glu Gly Phe Cys Glu Val Gly Asp Leu Thr Ser

AsulI

CTT COA GTT GGT CAA ATT CGA AAT GTT CCG CAT CCT TTT GTA GCT CTG GGT

Leu Pro Val Gly Gln Ile ArH Ash Val Pro His Pro Phe Val Ala Leu Gly

amN54

CTT AAG CAG CCA AAA GAA ATT AAA CAA AAA TTC GTT ACC TGT CAC TAT AAA

Leu Lys Gln Pro Lys Glu Ile Lys Gln Lys Phe Val Thr Cys His Tyr Lys

PL

GCT ATT CCG TGT CTT TAT AAG TGAJTATAAAT~ATAATATGAATTGGGTGTCGGAATAA

Ala Ile Pro Cys Leu Tyr Lys ***

HpaI

BHIII

TAAGTTAACCGAACAATTCTATGTGGTAGTCTACAACTGAGAGATCTGTCGAAAGAAGATGAAATT

50

101

152

203

254

305

356

407

458

560

625

676

727

778

829

880

931

989

1055

SD

OR

P 31.-1 (rIII)

CA

GA

AG

AA

CG

TGA

CTA

CC

GA

GTT

TTA

ATC

TCTA

AC

GA

GA

ATT

TTTA

A ATG

AT

T A

AA

CA

A T

TA 1

11

7

Met Ile Lys Gln Leu

rBBg-T

A-rBB9

CAA C

AC GCT CTT GAA CTG CAA CGA AAC C~A TGG AAT AAT GGT CAC GAA AAC 1168

Gln His Ala Leu Glu Leu Gln Arg Ash A~ ~

Ash Ash Gly His Glu Ash

TAT GGC GCA TCT ATT GAT GTT GAA GCC GAA GCT CTT GAA ATC CTG CGT TAT 1219

Tyr Gly Ala Ser Ile Asp Val Glu Ala Glu Ala Leu Glu Ile Leu Arg Tyr

G-r6?

TTC AAA CAT CTG AAT CCT C~T CAA ACT GCA TTA GCT GOT GAG CTT CAG GAA 1270

Phe Lys His Leu Ash Pro Ala Gln Thr Ala Leu Ala Ala Glu Leu Gln Glu

G-rES40

AAA GAT GAA CTT AAA TAT GCT AAG CCT CTG GCT TCT GCT GCA CGA AKA GCA 1321

Lys Asp Glu Leu Lys Tyr Ala I~Ts Pro Leu Ala Ser Ala Ala Arg Lys Ala

G=rES40

GTT CGT CAC TTT GTG GTA ACA CTG AAG TAATTTATTGGAGATTCACTGCCTTAGTGTG

1379

Val

A

rg

His

P

ile

Val

V

al

Th

r L

eu ~

**

*

AGC TAAATCGAG@AGCCGTCGAACTGTCTGATTAATGATTTGCGAATCATTATAGTTTTAAGACCCC

1446

BstNI

GACAGTTTTACGGTGTACCTCTTGAATGTGAATGATGACGGGTTTATGGTTATCCTGGTC

GTTAAAT 1513

SD

ORF 31 .-2

ATCCAAAAACCTATGTTCCCCTTGAGGGCTTGCGCAGGCAATG

CCA ATA AGT CCT GCA TTT 1574

Met Pro Ile Ser Pro Ala Phe

TCA TTT AAA AGA GAA TTT ATA ATG GCA AAA CAA GCT AAA GCA AAG AAA GCA 1625

Ser Phe Lys ArE Glu Pile Ile Met Ala Lys Gln Ala Lys Ala Lys Lys AIa

GTT GAA AAG AAA GTT GGT GAT TCT AAA CGC GCT GGC TAC AAG CGT GGG TOG 1676

Val Glu Lys Lys Val Gly Asp Set Lys Arg Ala Gly Tyr Lys Arg Gly Ser

Eco72I

AAC TCT CGT ATC AAT CAA ACT GTT CA(} AAG ATC ATG CGC CGA GCA CGT GCG 1727

Ash Ser Arg Ile Ash Gln Thr Val Glu Lys Ile Met ArH ArH Ala Arg Ala

GTT CTT CGA GAT GAT GCT TCT CGT TTT GGT AAG CAG AAA GCA TAAGTTGAGGA 1780

Val Leu Arg Asp Asp Ala Ser ArH Phe Gly Lys Gln Lys Ala ***

-35

PE128.6

CTCCTTCGGGAGTCCTTTTTTATTTTCCAAAGATTGCACAAAGTTpTTTAC~GTATAGTTCCTT~

1847

-10

SD

ORF 31.-3 .~

GATAGTAT~ATCTTACACAAACAAAGGAGAATAAA

ATG AAA ACG ATT AAT CTG AAC GCT 1906

Met Lye Thr Ile Ash Leu Ash Ala

PstI

GOA GTT AAA ACT AAA TGC TTC AAT GGT AAA TAT GAT GAA ACT ATG TGG TTC

19

57

Ala Val Lys Thr Lys Cys Phe Asn Gly Lys Tyr Asp Glu Thr Met Trp Phe

TTA ATG GCA GTT GAA GGT GAT ATT ATT GAA GTA GAA ACA AOA GAA GGT ATG 2008

Leu Met Ala Val Glu Gly Asp Ile Ile Glu Val Glu Thr Thr Glu Gly Met

GGA ACA GAT TTC ACC TTT ACA ATT CAA GTT CAT AAT TTC TTT ACT GGT TGG 2059

Gly Thr Asp Phe Thr Phe Thr Ile Gln Val His Ash Phe Phe Thr Gly Trp

ATT TAT GAA TTG AAT ACA GTA ATO GTT GGA AAA ATT GAA CAA AAT GAA TTA 2110

Ile Tyr Glu Leu Ash Thr Val Ile Val Gly Lys Ile Glu Gln Ash Glu Leu

GGC GAA TGG CAT TAT GTT AOA GOT OGO OAA OGT GOA GAA OGO TTA ATT GAG 2161

Gly Glu Trp His Tyr Val Thr Ala ArH Gin ArH Ala Glu ArH Leu Ile Glu

AAG ATG AAA AAA GTT GGT AAA CTT GAC ATG CAG CAT TGG AAA GTA GTA AAA 2212

Lys Met Lys Lys Val Gly Lys Leu Asp Met Gln His Trp Lys Val Val Lys

TAATTG

Fig.

2.

Nuc

leot

ide

sequ

ence

of

the

2218

-bp

T4

DN

A f

ragm

ent

and

loca

tion

of

OR

Fs.

The

pos

itio

ns f

or s

ix c

ompl

ete

OR

Fs

are

show

n by

the

ded

uced

aa

sequ

ence

s. T

he n

umbe

r of

aa,

cal

cula

ted

M r,

pl

, an

d po

ssib

le R

BS

(S

D)

for

each

OR

F a

re l

iste

d in

Tab

le I

. C

onse

nsus

seq

uenc

es f

or t

he p

utat

ive

mid

dle

(PM

), l

ate

(Pt)

, an

d ea

rly

(PE

) pr

omot

ers

(see

als

o Fi

g. !

) ar

e bo

xed;

the

SD

seq

uenc

es

me

unde

rlin

ed.

Tw

o t.V

~ for

mid

dle

mod

e m

otA

-dep

ende

nt tr

ansc

ript

s ar

e m

arke

d w

ith

dow

nwar

d ar

row

s. F

acin

g ar

row

s in

dica

te i

nver

ted

repe

ats.

The

fit

chan

ges

corr

espo

ndin

g to

the

rB

B9,

r67

and

rE

S40

mut

atio

ns a

re i

ndic

ated

abo

ve t

he w

t se

quen

ce i

n bo

ld-f

ace

lett

erin

g. E

MB

L a

cces

sion

Nos

. ar

e M

2372

2, X

5453

6 an

d X

5854

4.

OO

-.J

88

TABLE I

Some characteristics of the ORFs found in the gene 31 region

ORF" First-last RBS region h nt

Stop codon

Number of aa encoded ~

Predicted Mr

Theoretical pl

31.2 18-251 31.1 255-560 31 620-952 31.-1 1103-1348 31.-2 1554-1769 31.-3 1883-2212

GAAUUCUGUGGUGAAUAAUG UAA 78 CUGUAAUAGGUAAAUAAAUG UAA 102 AACAAAAAGGAAAAACGAUG UGA 111 CUAACGAGAAUUI 'UUAAAUG UAA 82 GAGGGCUUGCGCAGGCAAUG UAA 72 AAACAAAGGAGAAUAAAAUG UAA !10

9396 11468 12078 9323 8170

12892

10.88 9.96 5.92 9.28

! !.98 6.83

" Only those ORFs found to be oriented in the direction of early transcription are shown. h Putative SD sequences and start AUG codons are underlined.

Number of aa encoded within the respective ORF; this included the start codon, AUG, but not the stop codon.

of the gene products are shown in Fig. 2. Also, we have cloned and overexpressed all these genes in the T7-RNA- polymerase-based system (Nivinskas et al., 1989; Raudon- il<iene and Nivinskas, 1990; A.R., unpublished results).

An inspection of the sequence reveals the presence of transcriptional control elements. The ORF31.-2 down- stream sequence contains the inverted repeat (nt 1776- 1797 in Fig. 2)immediately followed by a T-run. Sequences of this type are known to be efficient Rho factor- independent terminators of transcription (Rosenberg and Court, 1979; Brendel et al., 1986). The estimated ziG value ofthis structure is -60.7 kJ/mol. Moreover, 5 ' -CUUCGG hairpin structures placing the UUCG in the single-stranded loop recently had been shown to be extraordinarily stable (Tuerk et al., 1988). Interestingly, the hairpin found in our sequence differs from the hairpin downstream from the T4 gene, soc (Macdonald et al., 1984), only in the first nt. It should be also pointed out that our sequencing data seem to confirm the estimate of Gram et al. (1984) that tran- scription of this region in vitro (initiated at PEI31.7) ter- minates at t128.6.

In the intergene sequence between ORF31.-2 and ORF31.-3, we found a T4 early promoter (Fig. 2). The sequence of this promoter agrees perfectly with the se- quence of PE128.6, one of the 29 early promoters in T4, isolated and sequenced by Liebig and R0ger (1989). Most likely, the observed early promoter directs transcription of the downstream gene, ORF31.-3, which is followed by another early promoter, PE128.2, and a potential termina- tor, possibly representing a factor-dependent class of tran- scription terminators (Liebig and Rtlger, 1989). The middle mode promoter consensus sequence (Guild et al., 1988) is observed just upstream from gene 31 (Fig. 2). The tsp for a middle mode motA-dependent transcript starting at nt 595-596, as well as for an early transcript for gp31, were determined by Dr. N. Guild in primer extension studies (Nivinskas et al., 1989). Interestingly, the late promoter consensus sequence, 5'-TATAAATA (Kassavetis et al.,

1986), is also present in the sequenced region, 139 nt up- stream from the start codon of the ORF31.-I. Further investigation will be required to determine whether this sequence indeed is a functional late promoter.

Discrepancies with the overlapping sequences published by other authors are shown in Table II. In most cases differences are not so significant, although as a consequence of some of them, the reading frame of a certain gene is shifted. A minor difference, at 1294 nt, with the sequence of Prilipov et al. (1990), however, leads to the frame shift in ORF31.-1 and alters the C terminus of the deduced protein. In the following, we show that ORF31.-1 is, in fact, the coding sequence for rllL

TABLE II

Discrepancies with the overlapping sequences published by Keppel et al. (1990), Maley et al. (1990), and Prilipov et al. (1990)

Position" Difference b Reference ¢

2 ! A deletion Keppel ¢t al. (1990) 266 C instead of A Keppel et al. (1990) 268 A instead of C Keppel et al. (1990) 289 G insertion Keppel et al. (1990) 299 T inst,.ad of G Maley et al. (1990) 320 C instead of T Maley et al. (1990) 496 G instead of C Maley et al. (1990)

1294 G deletion Prilipov et al. (1990) 1541 G deletion PrUipov et al. (1990) 1779 G deletion Prilipov et al. (1990) 1784 C deletion Prilipov et al. (1990) 1877 C instead of A Prilipov et al. (1990)

Positions are according to the sequence shown in Fig. 2. h Differences found in the sequences of other authors as compared to our sequence. c The sequence determined by Keppel et al. (1990) overlaps with nt 18- 1036 of our sequence; the Maley et al. (1990)-determined sequence over- laps with nt 1-1036 ofour sequence; the sequence of Prilipov et al. (1990) overlaps w;.~h nt 1-1910 of our sequence.

(c) Identification of ri l l On the basis of mapping data (Streisinger et al., 1964,

Revel and Lielausis, 1978), rill was expected to be found within the DNA region studied here. In addition, already in 1962 (Edgar et al., 1962) the rill region has been pro- posed to be a very short genetic segment. However, the gene 31 downstream sequence includes three ORFs (Table I and Fig. 2) which all are small genetically unidentified genes. To confirm which of them might correspond to rill, we resequenced all three ORFs using the DNA from the rill mutant, r67. The rIII mutants were generously pro- vided by Dr. E.M. Kutter and Dr. W.B. Wood.

T4r67 DNA containing glueosylated hydroxymeth- yldeoxycytosine was isolated from concentrated r67 phage stock essentially as described by Kricker and Tindall (1989). This DNA was digested with restriction enzyme Ndel, and the total Ndel digest was used as a template for PCR amplification. The 1.2-kb DNA fragment including all three gene 31 downstream ORFs was amplified using two oligo primers: a 20-mer, 5 ' -TGGTAGTCTACAACT- GAGAG, identical to 1013-1032nt of the BglII-site upstream sequence shown in Fig. 2, and a 20-met, 5 ' - CAATTATTTTACTACTTTCC, complementary to the sequence in PE128.2 region, mentioned in section a. Fi- nally, the amplified fragment was cleaved with certain restriction endonucleases and ORF31.-I, ORF31.-2 and ORF31.-3 were separately cloned into plasmid vectors, pTZl8R and pTZ19R, and sequenced by the chain- termination method (Sanger et al., 1977).

The only difference between the nt sequence determined earlier and the sequence of the r67 mutant is a single A ~ G transition at nt position 1227 (Fig. 2). This would corre- spond to a His-~Arg mutational change at aa 42 in the protein encoded by ORF31.-I. Based upon the fact that only ORF31.-1 is altered by the r67 mutation (CAT~CGT) , we tentatively assign ORF31.-1 to be the rlll gene.

Since the starting material for sequencing the gene 31 region was a T4 DNA fragment from 2 clone (607-10) which has been constructed using T4 dC-containing DNA (Mileham et al., 1980), we now determined the nt sequence of rlll from the wt T4D. A comparison of the initially determined nt sequence (Fig. 2) with the sequence of wt T4D DNA revealed the rllI sequence to be absolutely identical.

In addition, we decided to find out whether this aa sub- stitution, namely ~is42--*Arg 42 in the protein encoded by ORF31.-1, results in r-type plaque morphology. Cells con- taining plasmids with the T4r67 DNA insert (ORF31.-1) were infected with the wt T4D and the progeny of the infection were plated on E. coil strain S/6/5 su- for r plaque selection. Phage stock prepared from a single r-type plaque was used to isolate DNA for the subsequent PCR

89

amplification, cloning and sequencing of a DNA fragment containing ORF31.-1. We had found the codon 42 of ORF3 l.-l to be CGT (Arg), as in the case ofthe r67 DNA. These results clearly demonstrate that ORF31 .-1 is the rlIl gene.

We also resequenced the corresponding DNA fragment from two other rlIl mutants, rES40 and rBB9. As a result, the rES40 mutation is due to an A-~G transition in the codon for aa 82, A A G ~ G A G , resulting in Glu replacing Lys at this point (Fig. 2). Moreover, the rES40 mutant carries another mutation, i.e., an A--,G transition in the codon for Lys 57, AAA-~AAG, resulting in no aa change. In the case of the rBB9 mutant, we found an A-~T trans- version in the codon for Ala Is, G C A ~ G C T , with no aa change, and also a G-~A transition in the codon for aa ~t', TGG-~TGA, resulting in an opal (nonsense) codon at this point (Fig. 2).

It is interesting to note that all three rill gene mutants differ in their plaque morphology (data not shown). Even though the rBB9 mutation terminating the rill gene protein at aa 16 permits the production of viable phages, the rBB9 mutant produces the smallest plaques among all the rIII mutants tested, whereas the rES40 mutant produces r-type morphology plaques which are even larger than r67 plaques.

(d) Conclusions We have determined the exact location of rill on the T4

phage genomic map. The nt sequence of rill predicts an 82-aa basic peptide with an Mr of 9323 and a pl of 9.28. Based on the rules described by Chou and Fasman (1978) for secondary structure prediction, the T4 rill protein con- tains 66°~, ~t-helix, 16~, fl-sheet and 18~/o fl-turn confor- mations (Fig. 3). The r67 and rES40 mutations in rill are not predicted to change the secondary structure of the rIII protein.

o o o o o o o o o o o o o o o i . 39 . I ~-o o o-~To o o-~.~-o . . . .

_,,9 89.... "o o o o o o o o o o o o o o o o o o o .:, o o o r.,~.~'xNvvvvv u ,~

= a-hellx

AAAA = p-sheet

.... = ~-turn

Fig. 3. Schematic diagram of the predicted sccondar) structure of rill protein. Residues are presented in x-helical, fl-sheet, and fl-turn confor- mations, as determined by the algorithm of Chou and Fasman (1978).

90

ACKNOWLEDGEMENTS

We wish to thank Dr. Elizabeth M. Kutter and Dr. William B. Wood for providing us with the r l l l gene mutants. We also thank Dr. Noreen E. Murray for the phage ~. clone (607-10).

REFERENCES

Brendel, V., Hamm, G.H. and Tcifonov, E.N.: Terminators of transcrip- tion with RNA polymerase from Esclwrichicl coil: what they look like and how to find them, J. Biomol. Struct. Dynam. 3 (1986) 705-723.

Chou, P.Y. and Fasman, G.D.: Prediction of the secondary structure el" proteins from their amino acid sequence. Adv. Enzymol. 47 (1978) 45-148.

Edgar, R,S., Feynman, R,P., Klein, S., Lielausis, I. and Steinberg, C.M.: Mapping experiments with r mutants of bacteriophage T4D. Genet- ics 47 (1962) 179-186.

Epstein, R.H., Belle, A., Steinberg, C.M., Kellenberger, E., Boy de la Tour, E., ChevaUey, R., Edgar, R.S., Susman, M., Denhardt, G.H. and Lielausis, A.: Physiological studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28 (1963) 375-394.

Georgopoulos, C.P., Hendrix, R.W., Kaiser, A.D. and Wood, W.B.: Role of the host cell in bacteriophage morphogenesis: effects of a bacterial mutation on T4 head assembly. Nature New Biol. 239 (1972) 38-41.

Gram, H., Liebig, H.-D., Hack, A,, Niggemann, E. and RUger, W.: A physical map of bacteriophage T4 including the positions of strong promoters and terminators recognized in vitro. Mol. Gen. Genet. 194 (1984) 232-240.

Guild, N., Gayle, M., Sweeney, R., Hollingsworth, T., Modeer, T. and Gold, L.: Transcriptional activation of bacteriophage T4 middle pro- meters by the mot/I protein. J. Mol. Biol. 199 (1988) 241-258.

Kassavetis, G.A., Zentner, P.G. and Geiduschek, E.P.: Transcription at bacteriophage T4 variant late promoters. An application of a newly devised promoter-mapping method involving RNA chain retraction. J. Biol. Chem. 261 (1986) 14256-14265.

Keppei, F., Lipinska, B., Ang, D. and Georgopoulos, C.: Mutational analysis of the phage T4 morphogenetic 31 gene, whose product inter- acts with the E,wherirl#cs eoli GroEL protein. Gene 86 (1990) 19-25.

Kricker, M.C, and Tindall, K.R,: Direct sequencing of bacteriophage T4 DNA with a thermostable DNA polymerase. Gene 85 (1989) 199- 204,

Kutter, E. and RIlgcr, W.: Map of the T4 genome and its transcription control sites, in: Mathews, C.K., Kutter, E.M., Mosig, G. and Berget, P.B. (Eds.), Bacteriophag~ 1"4. American Society for Microbiology, Washington, DC, 1983, pp. 277-290.

Liebig, H.-D, and ROger, W.: Bacteriophage 1"4 early promoter regions. Consensus sequences of promoters and ribosome-binding sites. J. Mol. Biol, 208 (1989) 517-536,

Macdonald, P.M., Kutter, E. and Mosig, G.: Regulation of a bacterio- phage T4 hie gene, soc, which maps in an early region. Genetics 106 (1984) 17-27.

Maley, G.F., Duceman, B.W., Wang, A.-M., Martinez, J. and Maley, F.: Cloning, sequence analysis, and expression of the bacteriophage T4 cd gene. J. Biol. Chem. 265 (1990)47-51.

Maniatis, T., Fritsch, E F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982.

Mathews, C.K., Kutter, E.M., Mosig, G. and Berget, P.B. (Eds.): Bac- teriophage T4. American Society for Microbiology, Washington, DC, 1983.

Mileham, A.J., Revel, H.R. and Murray, N.E.: Molecular cloning of the T4 genome, organization and expression of the frd-DNA ligase re- gion. Mol. Gen. Genet. 179 (1980) 227--239.

Mosig, G.: A map of distances along the DNA molecule of phage T4. Genetics 59 (1968) 137-151.

Nivinskas, R.H.: Molecular cloning of some Bglll fragments of bacte- riophage T4 DNA in the vector plasmid pSCC31. Genetika 24 (1988) 34-41.

Nivinskas, R. and Black, L.W.: Cloning, sequence, and expression of the temperature-dependent phage 1"4 capsid assembly gene 31. Gene 73 (1988a) 251-257.

Nivinskas, R.H. and Black, L.W.: Nucleotide sequence of bacteriophage T4 gene 31. Mol. Biol. (Mosc.) 22 (1988b) 1507-1516.

Nivinskas, R.H., Raudonikiene, A.A. and Guild, N.: New early gene upstream from the middle gene 31 of the bacteriophage T4: cloning and expression. Mol. Biol. (Moscow) 23 (1989) 739-749.

Prilipov, A.G., Mesyan~hinov, V., Aebi, U. and Kellenberger, E.: Clon- ing and sequencing of bacteriophage T4 genes between map positions 128.3-130.3. Nucleic Acids Res. 18 (1990) 3635.

Raudonikiene, A. and Nivinskas, R.: Nucleotide sequence of bacterio- phage 1"4 gene 31 region. Nucleic Acids Res. 18 (1990) 4280.

Revel, H,R. and Lielausis, I.: Revised location of the rill gene on the genetic map of bacteriophage T4. J. Virol. 25 (1978)439-441.

Rosenberg, M. and Court, D.: Regulatory sequences involved ir,-~'-~e pro- motion and termination of RNA transcription. Annu. Rev. Genet. 13 (1979) 319-353,

Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K,B. and Erlich, H.A.: Primer-directed enzymatic am- plification of DNA with a thermostable DNA polymerase. Science 239 ( ! 988) 487-491.

Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain- terminating inhibitors. Prec. Natl. Aead. Sei. USA 74 (1977)5463- 5467.

Simon, L.D., Snover, D. and Doermann, A.H.: Bacterial mutation af- fecting T4 phage DNA synthesis and tail production. Nature 252 (1974) 451-455.

Streisinger, G., Edgar, R.S. and Denhardt, G.H.: Chromosome structure in phage T4, I. Circularity of the linkage map. Prec. Natl. Acad. Sei. USA 51 (1964) 775-779.

Tuerk, C., Gauss, P., Thermes, C., Groebe, D.R., Gayle, M., Guild, N., Stormo, G., d'Aubenton-Carafa, Y., Uhlenbeck, O.C., Tinoco Jr., I., Brody, E.N. and Gold, L.: CUUCGG hairpins: extraordinarily sta- ble RNA secondary structures associated with various biochemical proces,,:es. Prec, Natl. Acad. Sei. USA 85 (1988) 1364-1368.

Wood, W.B, and Revel, H.R.: The genome of bacteriophage T4. Baete- riol. Rev. 40 (1976) 847-868.