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W J Brammar

Publications and source records attributed to W J Brammar.

At least 73 records · Page 4Linked to original sources

Functions antagonistic to plasmid formation by lambda N- chromosomes.

It was concluded in the preceding paper that lambda N- cI- genomes probably failed to form plasmids within infected Escherichia coli cells because they leakily express functions that act to destabilize the plasmid state. This prediction was investigated by examining the effect upon plasmid-forming ability of the loss of possible anti-plasmid functions. The loss of Ter function was found to allow long-term plasmid formation, although the efficiency of initial plasmid formation and the heritable stability without selection were low. The combined loss of the int, red and gam gene functions also promoted plasmid growth, although the absence of Ter lambda was necessary. In contrast, the presence of Ter80 (due to an h80 substitution) did not prevent plasmid formation when the int, red and gam genes were absent, indicating that Ter80 does not attack the closed-circular form of the lambda chromosome. The combined loss of the ter, int, red and gam gene functions facilitated fully efficient inheritance of the lambda N- cI- plasmids in the absence of selection, although the efficiency of initial plasmid formation remained low. However, cells harbouring such plasmids suffered a decline in viability, indicating that the plasmids expressed a function (or more than one) that acts to debilitate the host cells--presumably an effect that is increased with this genotype because the modified lambda N- cI- plasmids are inherently more stable. The possible involvement of the lambda S and kil functions in destabilization is discussed.

Autoradiography↗

A programme for the construction of a lambda phage.

Infection of a sensitive host by the lambdoid coliphages can cause death of the bacterial cell by lysis or can lead to a lysogenic cell, with the viral DNA stably integrated into the host chromosome. These alternative responses both require the coordination of several host and phage functions, and lambda infection follows a well controlled developmental plan. The lytic and lysogenic pathways of lambdoid infection are reviewed, with emphasis on the variety of control mechanisms involved in the commitment to a particular pathway.

Bacteriolysis↗

Molecular cloning of two distinct renin genes from the DBA/2 mouse.

We report the molecular cloning of cDNA copies of DBA/2 mouse submaxillary gland (SMG) renin mRNA, which were used to probe Southern transfers of mouse genomic DNA. The results suggested either that there is a single renin gene containing a large intron in that part of the gene corresponding to the probe, or that there are two distinct renin genes. We have shown that the latter is the case by cloning and isolating two similar but distinct renin genes from DBA/2 mouse DNA. Restriction maps of the regions containing the two renin genes are presented, together with nucleotide sequence data locating a complete exon coding for amino acids 268-315 of mouse SMG renin.

Animals↗

Transcriptional termination sites in the b2 region of bacteriophage lambda that are unresponsive to antitermination.

A bacteriophage lambda cloning vector carrying the trp/lacW205 substitution is described. The vector facilitates the fusion in vitro of genetic control signals to the lacZ structural gene of Escherichia coli. This system was used to define transcriptional termination sites in the lambda b2 region. This region contains termination sites that are unresponsive to the lambda antiterminating proteins pQ and pN.

Bacteriophage lambda↗

The cis-specificity of the Q-gene product of bacteriophage lambda.

A trp/lacW205 substitution, fused to the late region of bacteriophage lambda, provided a convenient assay for phage late gene expression in the presence or absence of lambda pQ. Comparison of lacZ expression from Q+ and Q- phages showed that late gene expression was markedly Q-dependent (263-fold difference). A cis/trans comparison of lambda pQ action showed a 180-fold difference in lacZ expression. The results suggest that pQ in only significantly active when supplied in cis to its site of action.

Bacteriophage lambda↗

Bacillus licheniformis penicillinase synthesized in Escherichia coli contains covalently linked fatty acid and glyceride.

DNA sequence analysis of the structural gene for Bacillus licheniformis penicillinase has revealed a tetrapeptide sequence of Leu-Ala-Gly-Cys within the NH2-terminal part of the precursor form of penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6). The same tetrapeptide occurs in the signal sequence of the prolipoprotein of Escherichia coli, and the cysteine residue in the tetrapeptide of prolipoprotein is modified to form glyceride-cysteine which becomes the NH2 terminus of Braun's lipoprotein. On the basis of labeling, with [2-3H]glycerol, [3H]palmitate, [35S]methionine, and [35S]sulfuric acid, of an E. coli strain lysogenic for a lambda vector containing the penicillinase gene from B. licheniformis and of immunoprecipitation with rabbit antisera against purified B. licheniformis penicillinase, we conclude that B. licheniformis penicillinase synthesized in E. coli contains covalently linked glyceride and fatty acid. These results strongly suggest the operation of a modification system in E. coli, and presumably in other Gram-negative bacteria, which results in the formation of a glyceride-cysteine residue if the proper peptide sequence is present in the signal sequence of membrane proteins.

Bacillus↗

Molecular cloning of the gene for the beta-lactamase of Bacillus licheniformis and its expression in Escherichia coli.

The structural gene, pen, for the beta-lactamase of B. licheniformis has been cloned into a lambda vector and shown to be expressed at a low rate in E. coli. The cloned pen gene appears to be expressed from a promoter within the fragment of B. licheniformia DNA, since its rate of expression is not affected by the presence of the phage repressor, the absence of the phage's positive-control functions, or the position or orientation of the gene within the phage genome.

Bacillus↗

The construction in vitro of derivatives of bacteriophage lambda carrying the amidase genes of Pseudomonas aeruginosa.

The amidase genes of Pseudomonas aeruginosa were inserted into a lambda replacement vector following cleavage with the restriction endonuclease HindIII. The recombinant lambdaami was detected by enhanced growth of Escherichia coli around plaques of the recombinant phage on minimal medium containing acetamide as the nitrogen source. Low levels of amidase activity were detected in E. coli cultures infected with lambdaami and these were sufficient to allow growth with acetamide as nitrogen source. Lysis-defective derivatives of lambdaami were made by introducing Q-, S-, mutations. Cultures of E. coli infected with lambdaamiQ-S- synthesised amidase as the major protein. The amidase produced by these cultures was identical to that produced by PAC strains of P. aeruginosa in substrate specificty, thermal stability and immunological cross-reaction.

Amidohydrolases↗

In vitro insertion of the lambda attachment site into the plasmid RP4.

The region of the phage lambda chromosome containing the attachment site (P.P') and the genes int and xis, excised by the action of endonuclease R.EcoRI, has been inserted into the unique site for that enzyme on the promiscuous conjugative plasmid, RP4, generating the recombinant plasmid RP4att lambda. Transformants containing the hybrid plasmid were recognised by their ability to allow efficient lysogenization by phage lambda b2 (Weil and Signer, 1968; Echols et al., 1968) containing the mutant attachment site delta. P'. The construction and properties of the hybrid plasmid RP4att lambda are described.

Attachment Sites, Microbiological↗

Aberrant immunity behaviour of hybrid lambda imm21 phages containing the DNA of ColE1-type plasmids.

Hybrid lambda and lambda imm21 bacteriophages carrying various ColE1-type plasmids have been constructed in vitro. The lambda imm21/plasmid recombinants display aberrant immunity behaviour, giving clear plaques under conditions where the parental phages give turbid ones and being able to grow on homoimmune lysogens. lambda imm lambda/plasmid recombinants show no such unusual behaviour. Studies with hybrids of a lambda imm21 cITS phage carrying pMB9 DNA showed the operation of the plasmid's replication system to be the basic cause of the aberrant immunity behaviour. The plasmid replication system could act as a complete alternative to the phage system during vegetative phage growth. The probable reason that lambda imm21 phages show such altered phenotypes when carrying a functional plasmid replication origin, whereas lambda imm lambda and lambda imm434 (Mukai et al., 1978) phages do not, is the relative ease of titration of the phage 21 repressor to allow transcription from pR21. Various uses are considered for the altered phenotypic behaviour of lambda imm21/ColE1-type plasmid hybrids.

Bacteriocin Plasmids↗

Lambdoid phages that simplify the recovery of in vitro recombinants.

Derivatives of phage lambda are described for use as vectors for fragments of DNA generated with the HindIII and EcoRI restriction enzymes. With some vectors, hybrid molecules are recognised by a change from a turbid to a clear plaque morphology resulting from the insertion of a fragment of DNA into the lambda gene coding for the phage regressor. Other vectors contain a central, replaceable fragment of DNA which imparts a readily recognisable phenotype. This central fragment may include either a gene for a mutant transfer RNA (suppressor) or a part of the lacZ gene of E. coli able to complement a lacZ host. The appropriate lacZ host and indicator plates permit the ready distinction between recombinant and vector phages by the colour of the plaques.

Coliphages↗

The use of specialised transducing phages in the amplification of enzyme production.

Two types of lambdatrp phages have been used as model systems to investigate ways of optimising the expression of bacterial genes from transducing phage genomes. Excellent yields of trp enzymes were achieved by infecting a trpR- host with Q- or Q-S- derivatives of lambdatrpAM1, which expresses its trp genese exclusively from the trp promoter. The five trp geneproducts constituted more than 50% of the total soluble protein of infected cells under these conditions, and an even higher proportion of the protein synthesized after infection. In a trpR+ host, phage DNA replication was easily able to override tryptophan-mediated repression by titration of the trp promoter were equally productive, while having the advantage of being much simpler to construct and propagate. lambdatrp phages lacking the trp promoter were used to investigate ways of optimising gene expression initiated at the phage promoter, PL. Though very powerful, the latter promoter is more difficult to harness then the trp promoter. Derepression of transcription from PL by the use of cro- mutations is accompanied by poor replication of transducing phage DNA. Attempts to circumvent this difficulty using virulent of cro,cII double mutants have not been successful. Nevertheless, cells infected with a lambdatrp phage expressing its trp genes exclusively from PL made up to 16 per cent of their protein as trp gene-products.

Anthranilate Synthase↗

Control of cI gene expression in bacteriophage lambda imm434, studied in an immunity/trp fusion made in vitro.

The trp genes of a lambda imm434 trp-transducing phage have been fused to the immunity region by deletion, in vitro, of the DNA between two targets for the restriction enzyme R.EcoRI. The resulting phage has been used to study the control of expression of the cI gene in vivo. The constitutive rate of expression of the cI gene is between 2 and 5% of the maximally stimulated rate. The products of the cII and cIII genes enhance expression of cI on infection of a sensitive host. The requirement for the cII product is more stringent than that for the cIII protein. The phage 434 repressor present in a 434-immune cell stimulates the rate of cI expression from a superinfecting homoimmune phage about fifteen-fold. This result strongly suggests that repressor stimulates its own synthesis by a direct effect on transcription of the cI gene.

Chromosome Mapping↗