Cloning and sequencing of the gene for the DNA-binding 17 K protein of Escherichia coli

117

Gene, 67 (1988) 117-124 Elsevier GEN 02441

Cloning and sequencing of the gene for the DNA-binding 17K protein of Escherichia coli (Recombinant phobicity;

DNA;

genomic fragment;

phage ,I library; plasmid vector; oligodeoxynucleotide

probe; hydro-

over-production)

A. Holck and K. Kleppe Department of Biochemistry and Laboratory of Biotechnology, University of Bergen, Bergen (Norway) Tel. + 47-(OS)200100 Received Revised

27 December 10 February

1987 1988

Accepted

18 February

1988

Received

by publisher

28 March

1988

SUMMARY

The skp gene encoding the 17 K protein, a basic DNA-binding nucleoid-associated protein of Escherichia coli, was cloned as part of a 2.3-kb genomic fragment. The gene was sequenced and a polypeptide of 161 amino acids (aa) was deduced from the nucleotide sequence. The primary translation product was processed by cutting off the N-terminal 20 aa residues, yielding a mature polypeptide of 141 aa. The M, of the mature polypeptide was 15 674. An E. coli transformant containing the skp gene on the plasmid pGAH317 was shown to overproduce the gene product some 20-fold.

INTRODUCTION

The DNA E. coli exists in the cell as an irregularly shaped body called the nucleoid. Several lines of evidence indicate that proteins may play important roles in the in vivo structure of this nucleoid. We have developed a method for isolating bacterial chromatin from the nucleoid (Sjastad et al., 1982). Correspondence to: Dr. Norwegian AS-NLH

Food (Norway)

Abbreviations:

17K protein,

protein

of approx.

phate;

acid(s);

17 kDa

ssb, single-strand Computer

at his

Institute,

present

P.O.

address:

Box 50, N-1432

bp, base pair; d, deletion;

a basic DNA-binding

ORF, open reading

Genetics

Holck

Tel. + 47-(02)940860.

aa, amino

inosine; otide(s);

A.

Research

of E. coli; kb, 1000 bp; nt, nucleframe;

binding;

Group;

I,

nucleoid-associated

SDS, sodium

UWGCG,

[ 1,designates

dodecyl

sul-

Univ. of Wisconsin plasmid-carrier

state.

The protein components of the bacterial chromatin have been thoroughly investigated (Lossius et al., 1984; Holck et al., 1987a). Purified DNA-protein complexes from bacterial chromatin have been shown to contain several different proteins, among these a group of basic proteins in the molecular size range of 16-18 kDa. Together, they comprise approx. 20% of the total DNA-binding proteins. One of these, the 17K protein, was also among the major DNA-cellulose-binding proteins of E. coli (Holck et al., 1987b). This protein was shown to be different from the ssb protein (Sigal et al., 1972), protein Hl (Cukier-Kahn et al., 1972), and HLPl (Lathe et al., 1980). The 17 K protein was purified to homogeneity, and the N-terminal sequence was determined by edman degradation. We describe here the cloning and the nucleotide sequence of the gene encoding the 17 K protein, here-

0378-l 119/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

118

after termed the skp gene. This represents a first step towards determining the three-dimensional structure of the 17 K protein and a better ~derst~ding of the nature of the DNA-protein interaction to elucidate the structure of the bacterial nucleoid.

(c) Constrnction coli library

MATERIALS AND METHODS

(a) Bacterial strains, enzymes and chemicals E. co& B, E. coIi K802 (hsdR _, hsdM+ , gal-, met-, supE) and E. co& HB 101 (F -, hsdS20 (rg , rn, ), recA13, ara-14, proA2, lacY1, galK2, rpsL20, (SmR), ~~1-5,mtl-1, supE44, a-) were from the laboratory stock of the Laboratory of Biotechnology, University of Bergen. E. coli JM105 (Alac-pro, thi, strA,

endA,

sbcB15,

hsdR4~F’~a~36,

preparation of plasmid DNA was according to Frei et al. (1985), except that the plasmids were purified by phenol-chloroform extractions instead of CsCl density-gradient centrifugations. Small-scale preparations of plasmids were prepared by the boiling method (Holmes and Quigley, 1981).

proAl?+,

lacl~ZAM15]) was a gift from Marine Genetics AS, Bergen. E. coli JA200 (F+, thr-1, leu36, trpE63, recA56, thi-1, ara-14, lacY1, galK2, gaET22, ~~1-5, m&l, 1-, supE44, glnV44) harbouring the plasmid pLC26-43, from the Clarke-Carbon E. coli hybrid

ColEl plasmid collection, was supplied by Dr. Barbara Bachmann at the E. coli Genetic Stock Center, New Haven, CT. Enzymes used in the cloning procedures were from New England Biolabs, Beverly, MA and Amersham International (U.K.). Phage IEMBL3 cloning vector and Packagene I DNA packaging system were from Promega Biotec, Madison, WI [ Y-~~P]ATP (3000 Ci/mmol) and [ a-35S]ATP (400 Ci/mmol) were supplied by Amersham International (U.K.). The Gemseq sequencing system was obtained from Promega Biotech. Colicin El was from Sigma Chemical Company, St. Louis, MO. (b) Isolation of DNA E. cob’ B DNA was deprotei~zed, RNase A-treated and purified by isopycnic density-gradient centrifugation in CsCl. Large-scale isolation of specific phages was carried out by precipitation of the lysates from 400 ml liquid cultures with polyethylene glycol. Phage partictes were disrupted by trea~~t with proteinase K in the presence of SDS (Vande Woude et al., 1979) and the released DNA was extracted with phenol-chloroform. Large-scale

and screening

of the ~~~~e~ie~iu

E. coli B DNA was partially digested with Sau3AI

to yield an average fragment length of 13-20 kb. AEMBL3 vector DNA was digested with BamHI foflowed by treatment with calf intestine alkaline phosphatase. The E. coli DNA fragments were ligated to the IEMBL3 arms and packed by a modification of the method of Sternberg et al. (1977), employing the Packagene 1 DNA packaging system. Recombin~t phages (5 x lo5 phages/pg DNA) were plated on E. coli K802 and screened by the method of Benton and Davis (197’7). Imprints on nitrocellulose filters were hybridized as described in section d, below. (d) Southern b~otti~

Genomic, plasmid and 1 DNA preparations were exhaustively digested with a number of restriction enzymes, and the fragments were electrophoresed on 0.7-1.5x agarose gels and blotted onto nitrocellulose according to the method of Southern (1975). The filters were hybridized with a 32P-labelled synthesized oligodeoxynucleotide (the 17 KI probe, a 29-mer) prepared on the basis of knowledge of the N-terminal amino acid sequence of the 17 K protein as shown in Fig. 1 (Holck et al., 1987b) or with a synthetic probe (the 17 KII probe, a 29”mer), hybridizing from nt position 436 to nt position 464 (see Fig. 3). The sequence of the 17KII probe was thus 5’-GAT-GCA-AAC-GCC-GTT-GCT-TACAAC-AGC-AG-3’. (e) Nucleotide

sequencing and computer analysis

DNA fragments were inserted into plasmid vectors pGEM3 or pGEM4 and sequenced according to the chain-termination procedure (Sanger et al., 1977) with the Gemseq sequencing system. The inserts were sequenced using oligodeoxynucleotides

119

from the two flanking and the 17KI Alternatively, M13mp18 essentially performed

SP6 and T7 promoter

and the 17KII DNA

probes

fragments

regions into

and M13mp19 vectors and sequenced as above. The computer analyses were on an IBM personal computer, using the

using

the

UWGCG

AND DISCUSSION

as primers.

were inserted

Staden-Plus sequence analysis programme (Amersham International, U.K.), and on a VAX/780 computer

RESULTS

programme

package

(a) Characterization

of the gene for the 17K protein

To identify and characterize the gene encoding the 17 K protein, and to find restriction sites suitable for cloning and subcloning

of the gene, purified chromo-

somal E. coli DNA was digested with a series of restriction enzymes either alone or in combinations.

(Devereux et al., 1984). The hydropathic index in Fig. 4 is plotted as described by Kyte and Doolittle

The resulting DNA fragments were subjected to electrophoresis and blotted onto nitrocellulose filters.

(1982), using their hydrophobicity

The filters were then hybridized

window

is slid along the sequence

values. In short, a and each amino

acid is assigned a value depending on the residue itself and on the adjacent residues. The diagram is scaled according to the maximum and minimum values in the chain. The charge index curve is obtained using a similar approach. The Robson prediction of the secondary structure is based on the method of Garnier et al. (1978). Protein structures are divided into four classes: turn, coil, sheet, helix. Each residue is assigned a probability of being found in each of these classes. The most likely structure (the highest probability) at each position is indicated by a dot in the Robson prediction plot. (f) Gel electrophoresis

and immunoblotting

One-dimensional SDS-polyacrylamide-gel electrophoresis was carried out using the buffer system of Laemmli and Favre (1973), with 15 y0 polyacrylamide gels. The gels were either stained using the silver staining method or electroblotted as described by Towbin et al. (1979). The antigen was detected employing monospecilic antibodies against the 17K protein and the Bio-Rad Immunoblot (GAR-HRP) Assay kit (Bio-Rad Laboratories, Munich, F.R.G.). The immunoblots were scanned with a Zeineh softlaser scanning densitometer (Biomed Instruments, Fullerton, CA) and the data were recorded on a Hewlett Packard Integrator 3390 A. Various amounts of E. coli overnight cultures were sonicated and the protein content was measured with the Bio-Rad protein assay prior to gel electrophoresis.

oligodeoxynucleotide

prepared

to a mixed synthetic on

the

basis

of

knowledge of the N-terminal amino acid sequence of the 17 K protein (the 17 KI probe). The amino acid sequence and the corresponding synthetic oligodeoxynucleotide are shown in Fig. 1. The probe gave one strong signal and did not hybridize to control DNA (human placenta DNA) confirming the specificity of the hybridization and indicating the presence of a unique single-copy gene. Taken together with the findings that the 17 K protein subunits migrate as one polypeptide upon two-dimensional gel electrophoresis and that the N-terminal sequence of the protein showed no ambiguities, the 17 K protein seemed to consist of four identical subunits in contrast to other known low-M, basic proteins suspected to interact with DNA such as HU which exists as heterotypic dimer (Rouviere-Yaniv and Kjeldgaard, 1979) and Hl, which exist in several forms: Hla, Hlb and Hlc (Spassky et al., 1984). The restriction map of the skp gene showed some similarities with that reported for the JivA gene (Bend& and Friesen, 1981). No signals were obtained when the 17 KI probe was hybridized to the plasmid pLC 26-43 reported to contain the JirA gene (Neidhardt et al., 1983). However, one cannot exclude the possibility that these proteins may show partial homology in other parts of the peptide chain.

NH*-Ala-Asp-Lys-Ile-Ala-Ile-Val-Asn-Met-Gly5'-GCI.GA+.;*ATI.GCI-ATI-GTPAAZ-ATG.GG-3' Fig. 1. The N-terminal

the corresponding nucleotide, inosine.

sequence

sequence

the 17KI

probe,

of the mature

17 K protein

of the mixed synthetic used to identify

and

oligodeoxy-

the &II gene. I,

120 All81

Haelll

Pstl

Sau3A Pstl

Pstl

Alul

Sau3A

Pstl

Alul

Sau3A Rsa I

Sau3A

-

-

17Kll

17KI

100

bp

Fig. 2. Restriction map of the skp gene and the strategy used for sequencing. The bold and the open lines indicate the structural gene and the signal sequence, respectively; transcription is from left to right. The long arrows indicate the direction and extent ofeach sequence determination. Short arrows labelled 17KI and 17KII indicate sequences homologous to the synthetic primers.

(b) Cloning and sequencing of the skp gene Partially Sau3AI-digested E. coli B DNA and IEMBL3 vector DNA were ligated and an E. coli library was constructed as described in MATERIALS AND METHODS, section c. Upon screening the library with the 17KI probe, 12 positive recombinants were found. A 2.3-kb HindIII-Sal1 fragment from one of the positive A clones was subcloned into pGEM3 plasmid vector, resulting in a recombinant plasmid, pGAH317, more suitable for further characterization and for direct sequencing of the gene (the Sal1 site originates from the AEMBL3 polylinker). The pGAH317 plasmid was then digested with restriction enzymes recognizing four bp to find fragments of suitable lengths for direct sequencing after subcloning into pGEM4 or M 13mp 18 and M13mp19 plasmid vectors. The results are summarized in Fig. 2, showing both the position of restriction sites and the sequencing strategy used for determining the skp gene. The positions of the restriction sites were confirmed by the sequence determination. The nucleotide sequence of the skp gene is shown in Fig. 3. A 483-bp ORF is coded from the alternative start codon GTG at nt position 25 to the stop codon at nt position 508. This sequence would encode a polypeptide of 161 aa. A subsequent cleaving at aa position 84 would yield a mature polypeptide consisting of 141 aa with an N-terminal sequence identical to that determined by edman degradation,

starting with the Ala encoded at aa position 85. The M,. of this polypeptide is 15 674, which is approximately the size of the 17 K subunit as determined by SDS-polyacrylamide-gel electrophoresis. The gene contains a nucleotide sequence of 60 nt at the 5’ end encoding a hydrophobic oligopeptide. It is thought that the signal peptide functions as a signal for the initial steps in the protein translocation. Such sequences are thus often, but not always, found on proteins destined for export or integration into membranes. However, when crude membranes and outer and cytoplasmic membranes were isolated (Bakken and Jensen, 1986) and subjected to immunoblotting, only trace amounts of the 17K protein were detected (results not shown). The significance of the hydrophobic N-terminal sequence of the nascent polypeptide thus remains unknown. One might speculate that the hydrophobic sequence of the nascent 17 K protein directs the protein to some kind of hydrophobic scaffold structure. Preceding the alternative start codon is a proposed ribosome-binding site almost identical to the Shine-Dalgarno consensus sequence (AGGAGGT), which would presumably act as a strong translation-stimulating sequence. This might be reflected in the abundance of the 17 K protein (1200 tetramers/cell). The end of the 17 K structural gene is followed by two regions of hyphenated dyad symmetry (nt 523-553 and nt 565-590) which may be involved in the termination of the 17K transcript. However, a conventional terminator structure where

121

50 30 10 AAATGGGATGGTAAGGAGTTTATTGTGAAAAAGTGGTTATTAGCTGCAGGTCTCGGTTTA MetlLysLysTrpLeuLeuAlaAlaGlyLeuGlyLeu 110 v 90 70 GCACTGGCAACTTCTGCTCAGGCGGCTGACAAAATTGCAATCGTCAACATGGGCAGCCTG AlaLeuAlaThrSerAlaGlnAlaAlaAspLy~IleAlaIleValAsnMetGlySerLeu 130 150 170 TTCCAGCAGGTAGCGCAGAAAACCGGTGTTTCTAACACGCTGGAAAATGAGTTCAAAGGC PheGlnGlnValAlaGlnLy~ThrGlyValSerAsnThrLeuGluAsnGluPheLysGly 190 210 230 CGTGCCAGCGAACTGCAGCGTATGGAAACCGATCTGCAGGCTAAAATGAAAAAGCTGCAG ArgAlaSerGluLeuGlnArgMetGluThrAspLeuGlnAlaLysMetLysLy5LeuGln 290 270 250 TCCATGAAAGCGGGCAGCGATCGCACTAAGCTGGAAAAAGACGTGATGGCTCAGCGCCAG SerMetLysAlaGlySerAspArgThrLy~LeuGluLysAspValMetAlaGlnArgGln 350 330 310 ACTTTTGCTCAGAAAGCGCAGGCTTTTGAGCAGGATCGCGCACGTCGTTCCAACGAAGAA ThrPheAlaGlnLysAlaGlnAlaPheGluGlnAspArgAlaArgArgSerAsnGluGlu 410 390 370 CGCGGCAAACTGGTTACTCGTATCCAGACTGCTGTGAAATCCGTTGCCAACAGCCAGGAT ArgGlyLysLeuValThrArgIleGlnThrAlaValLy~SerValAlaAsnSerGlnA~p 470 450 430 ATCGATCTGGTTGTTGATGCAAACGCCGTTGCTTACAACAGCAGCGATGTAAAAGACATC IleAspLeuValValAspAlaAsnAlaValAlaTyrAsnSerSerA~pValLysAspIle 530 510 490 ACTGCCGACGTACTGAAACAGGTTAAATAAGTAATGCCTTCAATTCGACTGGCTGATTTA * ThrAlaAspValLeuLysGlnValLysEnd 590 570 550 GCGCCGGTTGGATGCAGAACTACACGGTGATGGCGATATCGTCATCACCGGCGTTGCGTC Y_ w 650 630 610 CATGCAATCTGCACAAACAGGTCACATTACGTTCATGGTTAACCCAAAATACCGTGAGCA 6’70 TTTAGGCTTGTGCAGGCGT Fig. 3. Nucleotide The numbering indicated horizontal

sequence

of the skp gene and the deduced

amino acid sequence.

is from the 5’ end (with the last digits corresponding

by the downward divergent

arrows

arrowhead.

A possible

with the centers

Shine-Dalgarno

of symmetry

Only the antisense

to the nt). The cleavage sequence

is underlined,

strand

(mRNA-like)

site to yield the mature and dyad symmetries

is shown.

polypeptide

is

are indicated

by

shown by dots.

the dyad symmetries are followed by a G/C stretch and a T stretch as described by Rosenberg and Court (1980), was not found. A second ORF starting at nt position 552 was found out of phase 3’ of the proposed coding region of the skp gene. This ORF would encode a polypeptide of 37 aa. It is thus possible that the skp gene is

transcribed as part of a polycistronic mRNA. These questions will be settled only after the size of the RNA and the actual point of transcription termination have been determined in vitro. The sequence of the skp gene was unique when compared to 65 1 E. coli sequences (798 259 nt) from the EMBL data library.

122

TABLE

I

The search for codon

Amino

acid composition

Amino

acid

of Escherichia coli 17K protein

Subunit Amino

composition

Nucleotide

sequence b

table of codon frequency Ala

18

17

Arg

12

9

Asx Asp

18

(18) 11

Asn 0

0

Glx Glu

25

(22) 8

Gln 7

5

His

1

0

Ile

4

5

Leu

10

9 15

Lys

10

Met

3

5

Phe

4

4

Pro

ND

0 10

Ser

11

Thr

9

8

Trp

ND

0

Tyr

1

1

Val

12

13

145

141

Total

Fig. 3. Values

in parentheses

4o

are

(asp + asn)

I

A

in Fig. 3 turned

Only in this reading frame could the N-ter-

of the deduced

out to be the only one

protein

be identical

to that

The assignment of gene for the 17K

protein is thus convincingly confirmed. The evidence for this is further strengthened when comparing the deduced amino acid composition with that determined for the 17K protein. These results are shown in Table I. The predicted and determined values correspond well. The hydrophobicity and the charge distribution as well as the suggested secondary structure of the proposed nascent polypeptide are displayed in Fig. 4. The polypeptide is apparently divided into three domains. In the hydropathic plot both ends of the polypeptide chain show a hydrophobic character, whereas the middle part of the chain is clearly more hydrophilic. This is reflected also in the charge plot, which indicates that the charged areas are concentrated in the middle of the polypeptide chain. Likewise, a high degree of secondary structure is

or

(glu + gin).

0

suggested

found by Edman degradation. this sequence as the structural

a Values from Holck et al. (1987b). ND, not determined. b From

representative

probable. minal

14

Gly

supposedly

of the skp gene. When the found nucleotide sequence was translated in all three reading frames, the frame

7

Cys

in a nucleotide

sequence is a method for detecting coding sequences (Staden and McLachlan, 1982). The codon frequencies of the ompA, which codes for one of the most abundant E. coli proteins, was used to establish a

from:

acid analysis a

preference

I

I

I

1

I

I

i

r

2:

o”-.‘=

L -0

2

-40 4OrB

_I

-40 E* dF on IY

..

t -c

.. .

C-

h -

. .

..

s-

the dots, representing

I

I

I

I

I

I

20

40

60

80

100

120

140

one aa residue each, are separated section e.)

_ -

I

index (A), charge index (B) and suggested

chain. The second level indicates AND METHODS,

. . ..

...

.

-..........-........

Amino Fig. 4. Hydropathic

.

acid

secondary

number structure

of the proposed

nascent

into four levels. The top level suggests the positions

coils (c), the third sheets (s), and the bottom

level helix structures

polypeptide ofturns

(C). In panel C,

(t) in the polypeptide

(h). (For details, see MATERIALS

123

proposed towards the ends of the polypeptide, predominately random coils and sheet structures, whereas the middle part of the chain is dominated by a long a-helix structure. Pabo and Sauer (1984) have pointed out that DNA-binding proteins often use a-helical regions for DNA

recognition.

A possible

DNA

binding

would thus be in the area from about aa residues

site 65

to 80. This area also has a high number of basic amino acid residues. However, one should keep in mind that the method for prediction, although regarded among the better ones, was correct for only about 56% of the tested amino acid residues (Kabsch and Sander, 1983). (c) Expression of the 17K protein E. coli HB 10 1 transformed with plasmid pGAH3 17, containing the skp gene, was grown under conditions giving a high yield of plasmid. Total sonicated E. coli lysates were then subjected to SDSpolyacrylamide-gel electrophoresis, blotted onto nitrocellulose and the 17K protein detected with monospecific antibodies as described in MATERIALS AND METHODS, section f. The results are presented in Fig. 5. An approx. 20-fold increase in the amount of the 17 K protein was observed in the transformed bacteria compared to the non-transformed bacteria. High levels of expression are obviously not deleterious to the cells. A band corresponding to an &I,. of about 19000 was visible on the immunoblots. This band contained 20 times less protein than that corresponding to the 17 K protein and might well be the precursor of the mature polypeptide. These cells might be used to study the influence of the 17K protein on growth rate and on the nucleoid structure and appearance. Moreover, they will be a good source of 17 K protein for a thorough study of protein structure and protein-DNA interaction. (d) Conclusions Cloning and sequencing of DNA-binding proteins are initial steps in determining the tertiary and quaternary structure of a protein and obtaining a sound understanding of the nature of the proteinDNA interaction. Ultimately these studies may be of importance when elucidating the structure and organization of the bacterial nucleoid.

Fig. 5. Synthesis of 17 K protein. E. coli HB 101 was transformed with plasmid pGAH3 17 containing the gene for the 17 K protein. The bacteria were lysed and subjected to 0.1% SDS-15 % polyacrylamide gel electrophoresis and the 17K protein was identified by immunoblotting. Lanes A and B, silver-stained total E. coli protein. Lanes C and D, immunoblots of identical preparations as A and B, respectively. Lanes A and C, E. coli HB 101; lanes B and D, HBlOl[pGAH317].

The skp gene encoding the 17K protein, a basic DNA-binding protein of E. coli was identified and cloned using a synthetic oligodeoxynucleotide. This structural gene consisted of an ORF of 483 bp. The nascent translation product is processed yielding a polypeptide of 141 aa residues. A possible binding domain for DNA is in the hydrophilic area from about aa residues 65 to 80.

ACKNOWLEDGEMENTS

We want to thank Dr. A. Fjose and his group for preparing 1 and plasmid vectors and A. Nerland and R. Aasland for their help in sequencing and com-

124

puting. This study was supported

by grants from the

Norwegian Humanities.

for

Research

Council

Science

and

Lathe, R., But, H., Lecocq, J.-P. and Bautz, E.K.F.: Procaryotic histone-like

protein interacting

Natl. Acad.

Sci. USA 77 (1980) 3548-3552.

Lossius,

I., Sjastad,

dimensional present

with RNA polymerase.

K., Haarr,

L. and

gel electrophoretic

Kleppe,

separation

Proc.

K.: Two-

of the proteins

of Escherichia coli. J. Gen. Microbial.

in chromatin

130 (1984) 3153-3157. Neidhardt, REFERENCES

F.C., Vaughn,

Gene-protein

T.A. and Bloch,

P.L.:

index of Escherichia coli K-12. Microbial.

V., Phillips,

Rev.

47 (1983) 231-284. Bakken,

V. and Jensen,

H.B.:

Outer

membrane

proteins

Fusobacterium nucleatum Fevl. J. Gen. Microbial.

of

132 (1986)

1069-1078. Bendiak,

J.D.: Organization

of genes in the

four minute region of the Escherichia coli chromosome: dence that rpsB and tsf are co-transcribed.

evi-

Mol. Gen. Genet.

D.W.

and

Davis,

clones by hybridization

R.W.:

Screening

Igt

to single plaques

recombinant

in situ. Science 196

Cukier-Kahn,

R., Jacquet,

low molecular stimulate

from Escherichia coli that

proteins

DNA-directed

RNA synthesis:

Proc. Natl. Acad.

J., Haeberli,

P. and Smithies, 0.: A comprehensive

analysis programs

set

for the VAX. Nucl. Acids Res.

12 (1984) 387-395. Frei, E., Baumgartner,

S., Edstrbm,

J.-E. and Noll, M.: Cloning

of the extra sex combs gene of Drosophila and its identification by P-element-mediated

gene transfer.

EMBO

J. 4 (1985)

J., Osguthorpe,

accuracy

D.J. and Robson,

and implications

the secondary

structure

B.: Analysis

of simple methods

of globular proteins.

of the

of predicting

J. Mol. Biol. 120

DNA-

and

I., Aasland,

RNA-binding

R., Haarr, proteins

L. and Kleppe, of

chromatin

K.: from

Escherichia coli. Biochim. Biophys. Acta 908 (1987a) 188-199. Holck, A., Lossius,

I., Aasland,

and characterization

R. and Kleppe,

of the 17 K protein,

from Escherichia coli. Biochim.

protein

K.: Purification a DNA-binding

Biophys.

Acta

914

D.S. and Quigley,

preparation

of bacterial

M.: A rapid boiling method plasmids.

Anal. Biochem.

for the

114 (1981)

W. and Sander,

secondary

structure?

Kyte, J. and Doolittle, hydropathic

C.: How good are predictions

of protein

character

of a protein.

for displaying

the

J. Mol. Biol. 157 (1982)

Sanger, F., Nicklen,

U.K. and Favre,

teriophage

dimer. FEBS

M.: Maturation

inhibitors.

Proc. Natl. Acad.

Sigal, N., Delius, H., Kornberg, A DNA unwinding

with

Sci. USA 74

with DNA

Natl. Acad. Sjastad,

T., Gefter, M.L. and Alberts, B.:

protein

from Escherichia coli: its

isolated

and with DNA

polymerases.

Proc.

Sci. USA 69 (1972) 3537-3541.

K., Fadnes,

P., Kruger,

P.G., Lossius, I. and Kleppe, K.:

Isolation,

properties

and nucleolytic

degradation

of chroma-

tin from

Escherichia cob’. J. Gen.

Microbial.

128 (1982)

3037-3050. Southern,

E.M.: Detection separated

of specific

sequences

by gel electrophoresis.

among

DNA

J. Mol. Biol. 98

(1975) 503-517. Spassky,

A., Rimsky,

S., Garreau,

E. coli DNA-binding

H. and But,

H.: Hla,

protein which accumulates

compacts

an

in stationary

DNA in vitro. Nucl. Acids Res. 12

(1984) 5321-5340. Staden,

R. and McLachlan,

in identifying

A.D.: Codon preference

and its use

protein coding regions in long DNA sequences.

Nucl. Acids Res. 10 (1982) 141-156. Sternberg,

N., Tiemeier,

of a Dam

vector

D. and Enquist, containing

Towbin, H., Staehelin, fer of proteins

L.: In vitro packaging

EcoRI

T. and Gordon,

DNA

fragments

of

1 (1977) 255-280. J.: Electrophoretic

from polyacrylamide

trans-

gels to nitrocellulose

and some applications.

Proc. Natl. Acad.

Sci. USA 76 (1979) 4350-4354. M., Enquist,

Sullivan,

M. and

Fischinger,

Moloney

sarcoma

proviral

P.J.: DNA

Communicated

L.W., Nomura,

by J.K.C. Knowles.

S.,

Cloning

of integrated

sequences

in bacterio-

Sci. USA 76 (1979) 4464-4468.

of the head of bac-

T4. J. Mol. Biol. 80 (1973) 575-599.

Lett. 106 (1979)

S. and Coulson, A.R.: DNA sequencing

phage 1. Proc. Natl. Acad.

105-132. Laemmli,

Annu.

N.O.: Native Escherichia coli

J. and Kjeldgaard, is a heterotypic

Vande Woude, G.F., Oskarsson,

FEBS Lett. 155 (1983) 179-182. R.F.: A simple method

involved in

13 (1980) 319-353.

sheets: procedure

193-197. Kabsch,

sequences

of RNA transcription.

Escherichia coli and phage Pl. Gene

(198713) 49-54. Holmes,

and termination

phase, strongly

(1978) 97-120. A., Lossius,

Annu.

297-300.

fragments

979-987.

Holck,

Rouviere-Yaniv,

interaction

Sci. USA 69 (1972) 3643-3647. of sequence

M. and Court, D.: Regulatory

the promotion Rev. Genet.

recognition.

(1977) 5463-5467.

M. and Gros, F.: Two heat-resistant,

weight

R.T.: Protein-DNA

53 (1984) 291-321.

chain-terminating

(1977) 180-182.

Garnier,

and Sauer,

HU protein

181 (1981) 356-362.

Devereux,

CO.

Rev. Biochem. Rosenberg,

D.S. and Friesen,

Benton,

Pabo,