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Biomedical subjects

L W Enquist

Publications and source records attributed to L W Enquist.

123 records · Page 7Linked to original sources
PubMed1980

Frequent site-specific deletion of coliphage lambda murine sarcoma virus recombinants and its use in the identification of a retrovirus integration site.

Stocks of hybrid lambda phages carrying the complete integrated provirus of either m1 or HT1 Moloney murine sarcoma virus, as well as flanking host sequences, frequently contain significant numbers of phages carrying a specific deletion. This deletion arises from a recombination event between the terminally repeated sequences in the provirus that deletes the unique Moloney murine sarcoma virus sequences bracketed by the terminally repeated sequences. Physical mapping has shown that the deletion phage retains one complete copy of the terminally repeated sequence and the flanking mink host sequences. One such deletion, lambdaHT1r+, was used to characterize a mink genomic DNA sequence that contains an HT1 Moloney murine sarcoma virus integration site. This integration site sequence from normal mink cells was also cloned into phage lambda. An analysis of the heteroduplexes between the integration site and the lambdaHT1r+ deletion indicated that no major rearrangement of host sequences occurred upon integration of the Moloney murine sarcoma provirus.

Animals↗
PubMed1979

Cloning of herpes simplex type 1 DNA fragments in a bacteriophage lambda vector.

DNA isolated from defective and nondefective virions of herpes simplex type 1 (HSV-1) (strain Patton) was digested with restriction endonucleases, and the resulting DNA fragments were inserted in the EK2 coliphage vector lambdagtWES . lambdaB. The recombinant DNA was encapsidated in vitro under P4 maximum containment conditions. These lambda-HSV1 hybrids were purified and amplified, and the DNA was isolated in the P4 facility. DNA, free of viable phage and bacteria, was removed from P4 conditions and analyzed. Represented among the hybrids studied to date are DNA fragments from about 50 percent of the normal HSV-1 genome. The hybrids derived from defective HSV-1 DNA fragments demonstrate the existence of many similar but not identical classes of defective genomes.

Chromosome Mapping↗
PubMed1979

Construction and characterization of a recombinant plasmid encoding the gene for the thymidine kinase of Herpes simplex type 1 virus.

We have constructed a hybrid plasmid by insertion of the thymidine kinase (TK) gene of Herpes simplex virus (HSV) type I at the BamHI site on Escherichia coli plasmid pBR322. The restriction endonuclease cleavage site map for the viral DNA fragment was determined for ten nucleases, and the insert in the recombinant plasmid has the same restriction nuclease digestion pattern as bona fide viral DNA. This result indicates that the plasmid contains an accurate copy of the viral DNA. The viral TK gene carried on the plasmid can be introduced into mammalian cells where it is expressed. This source of DNA with a selectable marker should be of considerable practical use in gene-transfer experiments in mammalian cells.

Chromosome Mapping↗
PubMed1979

Strand exchange in site-specific recombination.

The site-specific recombination system of phage lambda promotes crossovers at its attachment site (att). In this report we show that when phage are crossed in conditions where only the site-specific recombination system is active, a low frequency of crossovers can also be detected in a region that is close to but does not contain att. These crossovers require the phage int gene, the host hip gene, and the integrity of att. They are not detected if one of the parents carries a substitution of a heterologous attachment site (attB instead of attP). To explain these findings we suggest that site-specific recombination can proceed by exchange of single strands between the participating chromosomes at att and migration of the resulting junction outside of att.

Alleles↗
PubMed1979

Cloning of integrated Moloney sarcoma proviral DNA sequences in bacteriophage lambda.

We have identified integrated proviral DNA sequences of m1 and HT-1 isolates of Moloney sarcoma virus (MuSV) in EcoRI digests of transformed mink cell genomic DNA and have cloned these fragments in bacteriophage lambda. Both the lambda-HT1 phage recombinant, containing a 12.3-kilobase MuSV pair (kb) fragment, and the lambda-m1 phage recombinant, containing a 7.0-kb fragment, possess full copies of the sarcoma viruses along with 5' and 3' host flanking sequences. The MuSV proviral DNA sequences, 6.7 kb for HT-1 and 5.2 kb for m1, are colinear by heteroduplex microscopy with the 1.5-kb difference in size accounted for by two approximately equal to 0.8-kb deleted regions in m1. Both integrated viral genomes are terminally redundant and have integrated at the same site in the provirus but at different sites on the host chromosome. The host sequence flanking integrated HT-1 MuSV have been identified as a single EcoRI restriction fragment of 5.6 kb in normal mink cells.

Animals↗
PubMed1977

Cloning specific segments of the mammalian genome: bacteriophage lambda containing mouse globin and surrounding gene sequences.

We have developed a general approach to the cloning of specific segments of the mammalian genome that involves a two-step purification of EcoRI fragments of mammalian DNA and their in vitro insertion into a suitably constructed EK2 derivative of bacteriophage lambda. The combination of fragment purification, exclusion of parental-type recombinants, and simple phage screening techniques permits the isolation of virtually any gene segment for which there is an identifying hybridization probe. We illustrate the approach by describing the cloning of an approximately 7000-base-long segment of mouse DNA containing globin and surrounding gene sequences.

Animals↗
PubMed1975

Transfection of Escherichia coli spheroplasts. V. Activity of recBC nuclease in rec+ and rec minus spheroplasts measured with different forms of bacteriophage DNA.

The in vivo activity of the recBC nuclease was assayed by transfection of isogenic rec+ and rec minus spheroplasts with bacteriophage DNA of various origin and structure. The results indicate that the recBC nuclease can limit transfection at several stages during the production of an infective center; such limitations depend primarily on whether the DNA is in, or assumes, a nuclease-sensitive structure. The first stage of limitation can occur when a nuclease-sensitive transfecting molecule enters the spheroplast. Other potential limitation points occur during replication and maturation of the bacteriophage DNA. The initial stage can be bypassed by using recBC nuclease-resistant molecules such as circular forms. Through analysis of results with other DNA structures, we found that in vivo the effects of the double-strand exonucleolytic activity of the recBC nuclease predominated. The effects of the single-strand nuclease activities seem to be modified from those observed for the purified enzyme in vitro (Karu et al., 1974). Inside the cell, the single-strand exonuclease activity is very weak and the single-strand endonuclease activity is abolished almost completely.

Adenosine Triphosphatases↗
PubMed1969

Genetic homologies among Streptomyces violaceoruber strains.

Most of the genetic studies on streptomycetes have been done with cultures erroneously designated as Streptomyces coelicolor. To determine whether these cultures are genetically homologous with the S. violaceoruber nominifer, their deoxyribonucleic acids (DNA) were analyzed, and selected pairs of mutants were crossed. The four cultures used in genetic studies, and called S. coelicolor in the literature, were found to constitute a genospecies, based upon DNA hybridization and recombination tests. In addition, DNA from Actinopycnidium caeruleum formed extensive duplexes with S. violaceoruber DNA. S. violaceoruber cultures and A. caeruleum were distinctly different from the S. coelicolor nominifer.

DNA, Bacterial↗
PubMedDate not supplied

Biological interactions between herpesviruses and cyclooxygenase enzymes.

Decades ago, medical researchers noted that non-steroidal anti-inflammatory drugs (NSAIDs), for example aspirin and indomethacin, modulate primary herpesvirus infections and diminish reactivation of latent herpesvirus infections. NSAIDs inhibit cyclooxygenase (COX) enzymes, molecules necessary for generation of prostaglandins. Numerous studies indicate that herpesvirus infections elicit elevated levels of cyclooxygenase 2 (COX-2) with a resultant increase in prostaglandin E(2) levels (PGE(2)). Thus, the biochemical pathway underlying the anti-herpetic mechanism of NSAIDs is linked to the inhibition of COX. The precise roles of COX-2 and PGE(2) in the viral life cycle are unknown. However, among the alphaherpesvirus, betaherpesvirus and gammaherpesvirus subfamilies, evolutionarily conserved mechanisms ensure modulated expression of COX molecules, underscoring their importance in viral replication and virus-host interactions.

Animals↗