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N E Murray

Publications and source records attributed to N E Murray.

At least 55 records · Page 3Linked to original sources
PubMed1988

Enhanced recovery and restriction mapping of DNA fragments cloned in a new lambda vector.

In this paper we describe a modification to the lambda vector EMBL3 which greatly expedites the construction of restriction maps of cloned DNA sequences. In the modified vector, EMBL3cos, all the phage coding sequences are placed to the right of the cloning sites so that the left cohesive end is separated by only 200bp, rather than 20kb (as in conventional lambda vectors), from the inserted DNA fragment. We show that reliable restriction maps can be rapidly constructed from partial digests of clones made in this vector by labelling the left cohesive end with a complementary 32P-labelled oligonucleotide. In addition, we quantify the restriction of clones containing human DNA by the McrA and McrB systems of E. coli and show that the use of Mcr- plating strains can increase the yield of recombinant phage up to tenfold, to give cloning efficiencies of greater than or equal to 10(7) pfu/microgram of human DNA.

Bacteriophage lambda↗
PubMed1988

McrA and McrB restriction phenotypes of some E. coli strains and implications for gene cloning.

The McrA and McrB (modified cytosine restriction) systems of E. coli interfere with incoming DNA containing methylcytosine. DNA from many organisms, including all mammalian and plant DNA, is expected to be sensitive, and this could interfere with cloning experiments. The McrA and B phenotypes of a few strains have been reported previously (1-4). The Mcr phenotypes of 94 strains, primarily derived from E. coli K12, are tabulated here. We briefly review some evidence suggesting that McrB restriction of mouse-modified DNA does occur in vivo and does in fact interfere with cloning of specific mouse sequences.

5-Methylcytosine↗
PubMed1988

Distribution and diversity of hsd genes in Escherichia coli and other enteric bacteria.

We screened Salmonella typhimurium, Citrobacter freundii, Klebsiella pneumoniae, Shigella boydii, and many isolates of Escherichia coli for DNA sequences homologous to those encoding each of two unrelated type I restriction and modification systems (EcoK and EcoA). Both K- and A-related hsd genes were identified, but never both in the same strain. S. typhimurium encodes three restriction and modification systems, but its DNA hybridized only to the K-specific probe which we know to identify the StySB system. No homology to either probe was detected in the majority of E. coli strains, but in C. freundii, we identified homology to the A-specific probe. We cloned this region of the C. freundii genome and showed that it encoded a functional, A-related restriction system whose specificity differs from those of known type I enzymes. Sequences immediately flanking the hsd K genes of E. coli K-12 and the hsd A genes of E. coli 15T- were shown to be homologous, indicating similar or even identical positions in their respective chromosomes. E. coli C has no known restriction system, and the organization of its chromosome is consistent with deletion of the three hsd genes and their neighbor, mcrB.

Citrobacter↗
PubMed1987

Organization and sequence of the hsd genes of Escherichia coli K-12.

The nucleotide sequence of the hsdR and M genes, together with that for hsdS comprises an 8400 base segment spanning the entire hsd region of Escherichia coli K-12. The three hsd genes are transcribed in the same direction, but from two promoters. hsdR and hsdM are separated by 492 base-pairs, whereas the termination codon of hsdM overlaps the initiation codon of hsdS. pres precedes hsdR, and our data indicate a transcription termination signal in the interval between hsdR and pmod, as expected if transcription of hsdM and S is dependent on pmod. Transcription from pres is not influenced by the products of the hsdM and S genes, and the mechanism whereby restriction is prevented when the hsd region is transferred to a modification-deficient cell remains to be elucidated. A segment of the predicted amino acid sequence of the M polypeptide shares homology with a variety of adenine methylases and may identify part of the active site for methylation of specific adenine residues. The R polypeptide shows homology with a variety of ATPases, and pronounced regions of alpha-helical structure are predicted, one of which is amphipathic.

Amino Acid Sequence↗
PubMed1987

Reassortment of DNA recognition domains and the evolution of new specificities.

Type I restriction enzymes comprise three subunits only one of which, the S polypeptide, dictates the specificity of the DNA sequence recognized. Recombination between two different hsdS genes, SP and SB, led to the isolation of a system, SQ, which had a different specificity from that of either parent. The finding that the nucleotide sequence recognized by SQ is a hybrid containing components from both the SP and SB target sequences suggested that DNA recognition is carried out by two separable domains within each specificity polypeptide. To test this we have made the recombinant gene of reciprocal structure and demonstrate that it encodes a polypeptide whose recognition sequence, deduced in vivo, is as predicted by this model. We also report the sequence of the SB specificity gene, so that information is now available for the five known members of this family of enzymes. All show a similar organization of conserved and variable regions. Comparisons of the predicted amino acid sequences reveal large non-conserved areas which may not even be structurally similar. This is remarkable since these different S subunits are functionally identical, except for the specificity with respect to the DNA sequence with which they interact. We discuss the correlation of the variation in polypeptide sequence with recognition specificities.

Bacteriophage lambda↗
PubMed1986

Modification enhancement by the restriction alleviation protein (Ral) of bacteriophage lambda.

The product of the lambda ral gene alleviates restriction and enhances modification by the Escherichia coli K-12 restriction and modification system. An open reading frame (orf) located between genes N and Ea10 has been assigned to the ral gene. We have cloned this orf in a plasmid where its transcription is controlled by a thermolabile lambda repressor. Inactivation of the lambda repressor caused a 1000-fold reduction in K-specific restriction of unmodified lambda phage and a 100-fold increase in modification. In minicells transformed with ral+ plasmids, derepression resulted in the appearance of a polypeptide with a lower mobility than that predicted for a protein encoded by the orf attributed to ral; in a transcription and translation system in vitro DNA from a ral+ plasmid encoded a polypeptide with the same mobility. This polypeptide was absent when the plasmid DNA carried a mutant ral gene. The nucleotide sequence of this mutant gene defined two base changes, one of which inactivates the initiation codon of the orf. The K restriction endonuclease, which is also a K-specific methylase, is encoded by three genes designated hsdR, hsdM and hsdS, although the hsdR polypeptide is not essential for the methylase activity. We show that Ral enhances modification in a host strain lacking the entire hsdR gene, and lambda phages carrying the hsdM and S genes modify their own DNA inefficiently in the absence of Ral, despite the fact that derivatives of these phages provide efficient amplification of the K-specific methylase. Our data support a model in which, as a consequence of the interaction of Ral with either the hsdM or the hsdS polypeptide, the conformation of the enzyme is changed and the efficiency of methylation of unmodified target sites is enhanced. It has been postulated that Ral counteracts Rho, but in our experiments Ral did not relieve transcriptional polarity.

Bacteriophage lambda↗
PubMed1986

Two DNA recognition domains of the specificity polypeptides of a family of type I restriction enzymes.

The hsd genes of Salmonella typhimurium and Salmonella potsdam encode related type I restriction and modification systems designated SB and SP, respectively; the polypeptide encoded by the hsdS gene dictates the DNA sequence recognized. The hsdS genes of the SB and SP systems have a conserved sequence of around 100 base pairs flanked by two nonhomologous (variable) regions of around 500 base pairs. Recombination between the hsdS genes of SB and SP generated a system (SQ) with a different recognition specificity. We have localized the position of the crossover in the central conserved region by analysis of nucleotide sequences. Concomitant with the generation of a new combination of flanking variable regions is the recombination of minor differences in the central conserved region. A polypeptide domain encoded on the 5' side of the crossover dictates recognition of the trinucleotide component of the target sequence, and a second domain, encoded on the 3' side of the crossover, similarly governs recognition of the tetra- or penta-nucleotide component. Our analysis implicates at least parts of the variable regions in the determination of the specificity of interaction between protein and DNA. Furthermore, the trinucleotide components of the recognition sequences of S. typhimurium and Escherichia coli K-12 are identical, and the 5' segments of their hsdS genes are strikingly homologous rather than variable.

Amino Acid Sequence↗
PubMed1985

EcoA and EcoE: alternatives to the EcoK family of type I restriction and modification systems of Escherichia coli.

The genes (hsd A) encoding EcoA, a restriction and modification system first identified in Escherichia coli 15T-, behave in genetic crosses as alleles of the genes (hsd K) encoding the archetypal type I restriction and modification system of E. coli K12. Nevertheless, molecular experiments have failed to detect relatedness between the A and K systems. We have cloned the hsd A genes and have identified, on the basis of DNA homology, related genes (hsd E) conferring a new specificity to a natural isolate of E. coli. We show that the overall organization of the genes encoding EcoA and EcoE closely parallels that for EcoK. Each enzyme is encoded by three genes, of which only one, hsdS, confers the specificity of DNA interaction. The three genes are in the same order as those encoding EcoK, i.e. hsdR, hsdM and hsdS and, similarly, they include a promoter between hsdR and hsdM from which the M and S genes can be transcribed. The evidence indicates that EcoA and EcoE are type I restriction and modification enzymes, but they appear to identify an alternative family to EcoK. For both families, the hsdR polypeptide is by far the largest, but the sizes of the other two polypeptides are reversed, with the smallest polypeptide of EcoK being the product of hsd S, and the smallest for the EcoA family being the product of hsdM. Physiologically, the A restriction and modification system differs from that of K and its relatives, in that A-specific methylation of unmodified DNA is particularly effective.

Autoradiography↗
PubMed1985

T4 polynucleotide kinase; cloning of the gene (pseT) and amplification of its product.

The T4 gene (pseT) for polynucleotide kinase (pnk) has been cloned in lambda. Induction of a lambda E-W-S-cI857 prophage in which the pseT gene can be transcribed from the late lambda promoter, PR1, leads to greater than 100-fold amplification of pnk activity; pnk comprises approximately 7% of the total soluble cell protein. The purified enzyme, as expected, is both a 5'-kinase and a 3'-phosphatase. The amino acid sequence deduced from an open reading frame identified as the pseT gene contains a sequence which corresponds particularly well with that part of the adenine nucleotide binding site of adenylate kinase shown to form a flexible loop. A deletion mutant that lacks 5'-kinase activity, and possibly also 3'-phosphatase activity, has lost two amino acids from within the proposed loop structure. A second region of the pnk sequence shares homology with phosphoglycerate kinase, yeast inorganic pyrophosphatase and histone 2b from various organisms.

Amino Acid Sequence↗
PubMed1984

Genetic recombination can generate altered restriction specificity.

A recombinant strain, isolated following the transduction of an Escherichia coli recipient carrying the Salmonella typhimurium (SB) specificity genes with DNA from a donor having the Salmonella potsdam (SP) specificity, was shown [Bullas, L.R., Colson, C. & Van Pel, A. (1976) J. Gen. Microbiol. 95, 166-172] to have neither SB nor SP specificity but to encode a novel restriction specificity, SQ. The heteroduplex analysis of the hsdS (specificity) genes of the SB and SP restriction and modification systems described here identifies a conserved sequence of around 100 base pairs flanked by two nonhomologous regions each of approximately 500 base pairs. This organization parallels that previously deduced from the DNA sequences of the hsdS genes of the related E. coli K-12, B, and D restriction systems. The present heteroduplex analyses further show that the hsdS gene conferring the SQ specificity derives one nonhomologous region from the SB gene and the other from the SP gene, as predicted from genetic exchange within the conserved sequence. This finding supports the idea that two domains of an hsdS polypeptide, which are different for each specificity, may correlate with two regions of the DNA sequence recognized. It has been shown that the recognition sequences for E. coli K-12 and B each consist of two short oligonucleotide sequences interrupted by a nonspecific sequence. A similar organization is suggested for the Salmonella specificity systems, providing the potential for evolutionary diversification of restriction specificities as a result of recombination within the conserved sequence of the hsdS gene.

Bacteriophage lambda↗
PubMed1984

Gamma-T4 hybrid bacteriophage carrying the thymidine kinase gene of bacteriophage T4.

Among 32 lambda-T4 recombinant phages selected for growth on a thymidylate synthetase-deficient (thyA) host, 2 were shown to carry the T4 thymidine kinase (tk) gene. The lambda-T4tk phages contain two T4 HindIII DNA fragments (2.0 and 1.5 kilobases) that hybridize to restriction fragments of T4 DNA, encompassing the tk locus at 60 kilobases on the T4 map. The T4tk insert compensates for the simultaneous host deficiencies of thymidine kinase and thymidylate synthetase in a thymidine kinase-deficient (tdk) host growing in the presence of fluorodeoxyuridine when provided with thymidine and uridine. The lambda-T4tk hybrid phages specified five polypeptides with Mrs of 22,000 (22K), 21K, 14K, 11K, and 9K.

Bacteriophage lambda↗
PubMed1983

Sequence diversity among related genes for recognition of specific targets in DNA molecules.

Escherichia coli strains K12 and B, and a new strain designated D, each encode a characteristic restriction and modification enzyme. These enzymes (EcoK, EcoB and presumably EcoD) comprise three subunits of which one, that encoded by the so-called specificity gene (hsdS), is responsible for recognition of the DNA sequence specific to that system. The other two subunits, encoded by hsdR and hsdM, are interchangeable between systems, and the available molecular evidence suggests that the hsdR and hsdM genes are highly conserved. The DNA sequence of a segment of the hsd region that includes the hsdS gene has been determined for each of the three strains. The hsdS gene varies in length from 1335 to 1425 base-pairs and the only regions showing obvious homology, one of about 100 base-pairs and a second of about 250 base-pairs, are highly conserved. The remainder of each hsd S gene shares little, or no, homology with either of the other related specificity genes. Thus, the specificity subunits, though components of a family of closely related enzymes with very similar functions, have remarkably dissimilar primary structure.

Amino Acid Sequence↗
PubMed1982

Structural homologies among type I restriction-modification systems.

Structural homologies among different restriction systems of Escherichia coli and several Salmonella species have been investigated by immunological methods using antibodies prepared against two subunits of the E. coli K12 restriction enzyme, and by DNA hybridization experiments using different fragments of the E. coli K12 hsd genes as probes. The results with both techniques show a strong homology between the E. coli K12 and B restriction-modification systems, weaker but nevertheless marked homology between E. coli K12 and the Salmonella systems SB, SP, and SQ and, surprisingly, no homology between the E. coli K12 and A systems.

Base Sequence↗
PubMed1980

The hsd (host specificity) genes of E. coli K 12.

The hsd genes of E. coli K 12 have been cloned in phage lambda by a combination of in vitro and in vivo techniques. Three genes, whose products are required for K-specific restriction and modification, have been identified by complementation tests as hsdR, M and S. The order of these closely linked genes was established as R, M, S by analysis of the DNA of genetically characterised deletion derivatives of lambda hsd phages. The three genes are transcribed in the same direction but not necessarily as a single operon. Genetic evidence identifies two promoters, one from which transcription of hsdM and S is initiated and a second for the hsdR gene. The hsdR gene codes for a polypeptide of molecular weight approximately 130 000; hsdM for one of 62--65 000 and the hsdS gene was associated with two polypeptides of approximately 50 000. Circumstantial evidence suggest that one of these two polypeptides may be a degradation, or processed, derivative of the other. The hsdS polypeptide of E. coli B has a slightly higher mobility in an SDS-polyacrylamide gel than does that of E. coli K 12. A probe comprising most of the hsdR gene and all of the hsdM and S genes of E. coli K 12 shares extensive homology with the DNA of E. coli B but none with that of E. coli C.

Bacterial Proteins↗
PubMed1980

On the nature of sbcA mutations in E. coli K 12.

We have recently shown (Kaiser and Murray 1979) that many E. coli K 12 strains carry a defective prophage (Rac) located a few minutes clockwise of the trp operon on the genetic map. The Rac genome contains recE, the determinant for the ATP-independent exonuclease, ExoVIII. E. coli K 12 strains which carry sbcA mutations express recE constitutively. This paper describes an investigation of several such strains. We show that the SbcA phenotype may arise from more than one type of mutational change. The most readily explained SbcA phenotype is that of sbcA8 strains in which a large section of the Rac genome (including one hybrid attachment site and probably the prophage repressor gene) is deleted. Three sbcA- strains carry multiple (and probably tandemly repeated) copies of the Rac genome while two others carry a single Rac prophage that is indistinguishable in its hybridisation behaviour from that carried by sbcA+ strains.

Bacteriophage lambda↗
PubMed1980

Construction and characterization of Escherichia coli polA-lacZ gene fusions.

The promoter of the polA gene of Escherichia coli K-12 was fused to the lacZ gene by selecting deletions within a lambda lacZ polA transducing phage. Four fusions, deleting varying amounts of the polA gene, were characterized. The polA promoter was found to be approximately 3% as active as the fully induced lac promoter. This figure is compatible with the normal intracellular level of deoxyribonucleic acid polymerase I. No evidence was found for outogenous regulation of transcription from the polA promoter. Expression from this promoter was influenced by neither recA nor mitomycin C, but uvrD and uvrE mutations reduced expression slightly.

Bacteriophage lambda↗