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J N Reeve

Publications and source records attributed to J N Reeve.

At least 109 records · Page 6Linked to original sources
PubMed1984

Mistranslation of the mRNA encoding bacteriophage T7 0.3 protein.

We have devised an experimental system using the T7 phage 0.3 protein to accurately quantitate in vivo errors in protein synthesis. The 0.3 protein is well suited for mistranslation studies because it is easy to purify, its precise amino acid and RNA sequences are known, and it contains no cysteine. Utilizing [35S]cysteine as a precursor we found an average of 1 cysteine residue misincorporated for every 43.5 molecules of 0.3 protein synthesized. Since there are 116 amino acids in 0.3 protein, 1 cysteine residue was misincorporated /5000 codons translated. If all 20 amino acids were misincorporated at the same frequency, the overall frequency of misincorporation of amino acids into 0.3 protein would be 4 X 10(-3)/codon translated. Parallel experiments measuring [35S]methionine incorporation into 0.3 protein supported the accuracy of our findings for cysteine misincorporation. We found an average of 5.7 methionine residues incorporated/molecule of 0.3 protein synthesized; the actual number from sequence data is known to be 6. Antibiotics which stimulate mistranslation (gentamicin and streptomycin) caused a modest increase in the number of cysteine residues misincorporated into 0.3 protein. The use of Escherichia coli strains, identical except for mutations in ribosomal protein genes known to affect the fidelity of translation, supported the contention that the errors being quantitated were mainly due to mistranslation rather than mistranscription .

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Bacteriophage Mu DNA circularizes following infection of Escherichia coli.

Mu DNA, isolated from infected cells or minicells, has been shown to be held by proteins in twisted and open circular forms. Circularization does not require protein synthesis in the infected cells. A 64,000-dalton polypeptide is injected into the infected cell with Mu DNA and co-sediments with Mu DNA through sucrose gradients. Circularization of the infecting Mu DNA does not require removal of the Escherichia coli DNA sequences which are attached to both ends of the Mu genome in the viral particle.

Bacteriophage mu↗
PubMed1982

Construction and use of SPP1v, a viral cloning vector for Bacillus subtilis.

A unique BamHI restriction site has been inserted into a nonessential region of the genome of a deletion mutant of phage SPP1. Construction of this phage, designated SPP1 v, required the in vitro conversion of a BclI site to a BamHI site. SPP1 v has been used as a vector phage to clone BamHI, BglII and BclI-generated restriction fragments of DNA. A direct selection for recombinants has been developed. Transfection with SPP1 v requires intact, genomic-length molecules, and cleavage with BamHI destroys the transfecting ability of this DNA. Recombinants in which the BamHI site has been destroyed by ligation to Bg/II or BclI-generated fragments of DNA become resistant to BamHI digestion after ligation and are active in transfection. Cloning of DNA containing BamHI sites has been accomplished by using the enzyme Bst1503I to methylate BamHI sites before insertion, and so to protect them during the BamHI digestion used to select against vector molecules. The in vitro construction of SPP1 v generated XmaIII sites directly adjacent to, and on both sides of the inserted BamHI site. This permits precise excision of cloned DNA even when cloning destroys the BamHI insertion site. Restriction-enzyme generated fragments of DNA in the size range of 0 to 4 Md have been cloned, including a full-length copy of plasmid pUB110, almost the complete sequence of plasmid pBR322, and a sequence of DNA that carried the lambda cos site.

Bacillus subtilis↗
PubMed1981

Cloning and expression of the Bacillus subtilis phage SPP1 in E. coli. I. Construction and characterization of lambda/SPP1 hybrids.

We have constructed lambda/SPP1 hybrid phages by in vitro ligation of EcoRI fragments of the Bacillus subtilis phage SPP1 DNA to a lambdoid bacteriophage vector. EcoRI digestion of SPP1 generated 15 DNA fragments of which 13 could be cloned. The SPP1 DNA of such hybrids was stably maintained and replicated in Escherichia coli, as indicated by marker rescue experiments in B. subtilis. EcoRI fragment 1 of SPP1 could not be cloned although subfragments of fragment 1 resulting from spontaneous deletions which occurred during the cloning regime were consistently obtained. A region within EcoRI fragment 1 responsible for its incompatibility with replication in E. coli was defined by these experiments.

Bacillus subtilis↗
PubMed1981

Cloning and expression of Bacillus subtilis phage SPP1 in E. coli. II. Expression of lambda/SPP1 hybrid phages in E. coli minicells.

In the preceding paper (Amann et al. 1981) we described the in vitro construction of hybrids between Escherichia coli phage lambda NM607 imm434 and B. subtilis phage SPP1. These lambda/SPP1 hybrids have been used to infect minicells produced by E. coli strain DS410. Analysis on polyacrylamide gels of 35S-methionine labeled proteins synthesized in infected minicells revealed the expression of both lambda and SPP1 genes. Infection of E. coli minicells carrying plasmid pGY101, which encodes and expresses the repressor gene of phage 434, results in the selective expression of the cloned SPP1 DNA. This has resulted in the assignment of 26 out of a total of 46 known SPP1 polypeptides (Mertens et al. 1979) to individual SPP1 DNA fragments. In addition, several lambda/SPP1 fusion peptides whose transcription either originates from lambda promoters or from promoters located on the inserted SPP1 fragment, were identified.

Bacillus subtilis↗
PubMed1981

phi X174-directed DNA and protein syntheses in infected minicells.

Phi X174-infected minicells, produced by Escherichia coli PC2251, synthesized 11 phi X174-encoded polypeptides. The infecting single-stranded viral genome was converted to a double-stranded, closed circular, replicative form (replicative form I). Little, if any, replicative form I replication took place, and synthesis of progeny single-stranded molecules could not be detected.

Bacteriophage phi X 174↗
PubMed1980

Identification of the repressor and repressor bypass (antirepressor) polypeptides of bacteriophage P1 synthesized in infected minicells.

P1 infected minicells synthesize approximately 50 phage-encoded polypeptides. Phage expression is temporally controlled, demonstrating phage polypeptides synthesized both early and late after infection. The P1 repressor, gpcl1 (Mr = 33,000), repressor bypass polypeptide, gpreb A (Mr = 27,500) and cistron 10 product, (gp10) (Mr = 64,000), have been identified by infection of minicells with P1 amber mutants. The beta-lactamase gene product (gpbla) carried by the closely related P7 and the chloramphenicol acetyl-transferase gene product (gpcat) carried by P1 Cm (in Tn9) have been demonstrated. Infection of minicells by P1virs or P1c4 mutants results in increased synthesis of gpreb A and a second polypeptide designated gpreb B (Mr = 40,000). The P1vir11 mutation leads to increased synthesis of a small polypeptide (Mr = 3,500) but does not affect the amount of gpc1 synthesized.

Coliphages↗
PubMed1980

Unusual behaviour of SPO1 DNA with respect to restriction and modification enzymes recognizing the sequence 5'-G-G-C-C.

SPO1 DNA contains only 5 cleavage sites for restriction enzymes which recognize and cleave the sequence 5'-G-G-C-C (HaeIII or BsuR). Fragments of SPO1 DNA cloned in E. coli to substitute 5'-hydroxymethyluracil (HMU) by thymine (T) remain resistant to HaeIII indicating that this unexpectedly small number of cleavages by HaeIII is not correlated with the presence of HMU in the normal phage DNA. It was previously shown that SPO1 is neither subject to B. subtilis R restriction (Trautner et al., 1974) nor modification in vivo (Günthert et al., 1975). We now show that SPO1 DNA can however be restricted and modified in vitro.

Bacteriophage lambda↗
PubMed1980

Synthesis of P1 ban protein in minicells infected by P1 mutants.

Phage P1 encodes a dnaB analog (ban) protein. Synthesis of ban protein has been studied in minicells infected by P1 mutants and has been identified as a polypeptide of 56,000 molecular weight by immunoprecipitation using antibody directed against E. coli dnaB protein. The amount of ban protein synthesized by P1 mutants increases in the order: P1 wild type, P1bac, P1crr, and P1bac crr. The relative amount of ban protein identified in P1bac- and P1bac crr-infected minicells is approximately the same as that previously found in dnaB.ban heteromultimers isolated from the corresponding P1 lysogens.

Coliphages↗
PubMed1979

Lambda encodes an outer membrane protein: the lom gene.

lambda infected minicells synthesize a polypeptide (M(r) = 20,500) which is incorporated almost exclusively into the outer membrane of the minicell envelope. The gene (lom = lambda outer membrane) encoding this polypeptide has been mapped in the non-essential region of the lambda genome between coordinates 39.4% and 40.7% of lambda.

Bacterial Outer Membrane Proteins↗
PubMed1979

Bacteriophage SPP1 polypeptides synthesized in infected minicells and in vitro.

Minicells produced by B. subtilis CU403divIVB1 and infected by SPP1 synthesize at least 46 polypeptides which can be separated by polyacrylamide gel electrophoresis. These polypeptides represent the expression of 86% of the SPP1 genome's coding capacity. Infection of minicells by sus mutants and deletion mutants of SPP1 has permitted a correlation of genetic location with gene product and has shown that SPP1 normally synthesizes at least 8 non-essential polypeptides. Restriction fragments of SPP1 produced by EcoRI digestion of SPP1 DNA have been purified and used as template DNA in a coupled transcription/translation system derived from E. coli to determine the polypeptides encoded by the individual fragments. SPP1 expression in minicells differs from SPP1 expression in nucleated cells (Esche, 1975) in that late syntheses are not dependent on phage DNA replication in infected minicells.

Bacillus subtilis↗
PubMed1978

DNA synthesis in toluene-treated bacteriophage-infected minicells or Bacillus subtilis.

Bateriophage (phi29, SPP1, or SPO1)-infected, toluene-treated minicells of Bacillus subtilis are capable of limited amounts of non-replicative DNA synthesis as measured by incorporation of [3H]dTTP into a trichloroacetic acid-precipitable form. The [3H]dTTP is covalently incorporated into small DNA fragments which result from the degradation of a small percentage of the infecting phage genomes (molecular weights in the range of 2 . 10(5)). Short exposure of the DNA molecules containing the incorporated [3H]dTMP to Escherichia coli exonuclease III results in over 90% of the E13H]dTMP being converted to a trichloroacetic acid-soluble form. The synthesis is totally dependent on host-cell enzymes and is not inhibited by the addition of chloramphenicol, rifampicin, nalidixic acid and mitomycin C and only slightly (approx. 20%) inhibited by the addition of 6-(p-hydroxyphenylazo)-uracil.

Bacillus subtilis↗