Biomedical subjects

D L Court

Publications and source records attributed to D L Court.

60 records · Page 4Linked to original sources

PubMed1986

Transposition studies of mini-Mu plasmids constructed from the chemically synthesized ends of bacteriophage Mu.

We describe below the chemical synthesis of the right and left ends of bacteriophage Mu and characterize the activity of these synthetic ends in mini-Mu transposition. Mini-Mu plasmids were constructed which carry the synthetic Mu ends together with the Mu A and B genes under control of the bacteriophage lambda pL promoter. Derepression of pL leads to a high frequency of mini-Mu transposition (5.6 X 10(-2) which is dependent on the presence of the Mu ends and the Mu A and B proteins. Five deletion mutants in the Mu ends were tested in the mini-Mu transposition system and their effects on transposition are described.

Bacteriophage mu↗

PubMed1986

Murine leukemia virus pol gene products: analysis with antisera generated against reverse transcriptase and endonuclease fusion proteins expressed in Escherichia coli.

The organization of the murine leukemia virus (MuLV) pol gene was investigated by expressing molecular clones containing AKR MuLV reverse transcriptase or endonuclease or both gene segments in Escherichia coli and generating specific antisera against the expressed bacterial proteins. Reaction of these antisera with detergent-disrupted virus precipitated an 80-kilodalton (kDa) protein, the MuLV reverse transcriptase, and a 46-kDa protein which we believe is the viral endonuclease. A third (50-kDa) protein, related to reverse transcriptase, was also precipitated. Bacterial extracts of clones expressing reverse transcriptase and endonuclease sequences competed with the viral 80- and 46-kDa proteins, respectively. These results demonstrate that the antisera are specific for viral reverse transcriptase and endonuclease. Immunoprecipitation of AKR MuLV with antisera prepared against a bacterial protein containing only endonuclease sequences led to the observation that reverse transcriptase and endonuclease can be associated as a complex involving a disulfide bond(s).

AKR murine leukemia virus↗

PubMed1985

E. coli NusA protein binds in vitro to an RNA sequence immediately upstream of the boxA signal of bacteriophage lambda.

The NusA protein of Escherichia coli is a factor which mediates termination of transcription. In this paper, we demonstrate that the NusA protein can bind in vitro to a specific site on the mRNA of bacteriophage lambda. Several RNAs were synthesized by in vitro transcription of truncated lambda DNA templates, and the activity of NusA binding to these RNAs was examined by a Millipore filter-binding assay. RNAs containing the sequence immediately upstream of the boxA site were trapped on the filter by association with the NusA protein, but those lacking the site were not. Anti-NusA antibody inhibits this binding. To determine the binding site precisely, we developed a new method which we have named 'reverse-transcriptase mapping'. The RNA transcribed from the pL promoter was incubated with 32P-labelled DNA primer and NusA, and the primer-extension reaction was started by adding the reverse transcriptase. In this way, the primer extension was blocked at the position G of the boxA RNA sequence (5'CGCUCUUA 3'), indicating that the NusA-protection site is immediately upstream of boxA and includes the 5'-end C. The NusA protein purified from a temperature-sensitive nusA mutant defective in transcription termination showed reduced and thermolabile RNA-binding activity, suggesting that the RNA-binding activity is related to the physiological function of NusA.

Bacterial Proteins↗

PubMed1984

An accessory role for Escherichia coli integration host factor: characterization of a lambda mutant dependent upon integration host factor for DNA packaging.

Bacteriophage lambda grows lytically on Escherichia coli defective for integration host factor, a protein involved in lambda site-specific recombination and the regulation of gene expression. We report the characterization of a mutant, lambda cos154, that, unlike wild-type lambda, is defective for growth in integration host factor-defective E. coli. The cis-dominant mutation in lambda cos154 is a single base pair change in a region of hyphenated dyad symmetry close to the lambda left cohesive end; this mutation prevents DNA packaging. We propose the following two alternative roles for this site in lambda DNA packaging: (i) to bind an E. coli accessory protein required in the absence of integration host factor or (ii) to bind the phage-encoded terminase protein that is essential for DNA packaging.

Bacterial Proteins↗

PubMed1984

Analysis of two potential shuttle vectors containing herpes simplex virus defective DNA.

Two potential shuttle vectors which contained the identical herpes simplex virus type 1 (HSV-1) defective particle DNA (dDNA), but prokaryotic DNA of different origins, were examined for their stability when propagated in eukaryotic cells, and for their efficiency as shuttle vectors. Each chimeric molecule contained a 9.5 kilobase-pair (kb) EcoRI fragment (HSV12-7) representing a single unit of a class I HSV-1 dDNA. This dDNA was cloned into the bacteriophage lambda (lambda) vector lambda gtWES X lambda B' to create a 45.3 kb chimeric molecule (lambda gtWES::12-7), and into the plasmid vector pBR325, resulting in a 15.5 kb recombinant DNA molecule (pBR325::12-7). Each of these DNA molecules was transfected independently into African green monkey kidney cells which were then infected with wild-type HSV-1 helper virus. Both chimeric molecules were replicated and packaged into HSV-1 virions. However, regions of the lambda gtWES::12-7 chimeric DNA were rapidly deleted and rearranged, whereas the plasmid/HSV-1 DNA molecules were less rearranged. No intact lambda gtWES::12-7 DNA was recovered from HSV-1 virions as detected by infectivity of in vitro packaged DNA. However, pBR325::12-7 DNA isolated from HSV-1 virions was able to transform E. coli to ampicillin resistance. These results suggest additional considerations when designing single units of HSV-1 dDNA for use as vectors to accommodate large fragments of DNA.

Animals↗

PubMed1972

Isolation of plaque-forming, galactose-transducing strains of phage lambda.

Plaque-forming, galactose-transducing lambda strains have been isolated from lysogens in which bacterial genes have been removed from between the galactose operon and the prophage by deletion mutation.-A second class has been isolated starting with a lysogenic strain which carries a deletion of the genes to the right of the galactose operon and part of the prophage. This strain was lysogenized with a second lambda phage to yield a lysogen from which galactose-transducing, plaque-forming phages were obtained. These plaque-forming phages were found to be genetically unstable, due to a duplication of part of the lambda chromosome. The genetic instability of these partial diploid strains is due to homologous genetic recombindation between the two identical copies of the phage DNA comprising the duplication. The galactose operon and the duplication of phage DNA carried by these strains is located between the phage lambda P and Q genes.

Centrifugation, Density Gradient↗