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Biomedical subjects

H W Chan

Publications and source records attributed to H W Chan.

At least 73 records · Page 4Linked to original sources
PubMed1981

Detection and cloning of murine leukemia virus-related sequences from African green monkey liver DNA.

By using low-stringency nucleic acid hybridization conditions and specific subgenomic segments of the AKR ecotropic provirus as probes, murine leukemia virus (MuLV)-related sequences were detected in African green monkey (AGM) liver DNA. The MuLV-reactive segments present in restricted AGM DNA ranged from 1.9 kilobases (kb) to greater than 10 kb in size. On the basis of this finding, a 17-kb segment was cloned from a partial EcoRI AGM library in lambda Charon 4A which shared nearly 5 kb of homology with AKR ecotropic MuLV DNA. The MuLV-related sequences detected in restricted preparations of AGM DNA or present in the cloned monkey DNA reacted with probes mapping 2.0 to 7.0 kb from the 5' terminus of the AKR ecotropic provirus. The AGM clone also contained repeated sequences that flanked the MuLV-related segment. Labeled, subgenomic, MuLV-reactive segments of the monkey clone hybridized to multiple restriction fragments of AGM liver DNA, indicating the presence of several copies of the MuLV-related sequences.

Animals↗
PubMed1980

Haemoprotein- and transition metal ion-catalysed oxidation of linoleic acid. Selectivity of the position of oxygenation.

Oxidation of linoleic acid in aqueous buffers favoured the formation of the 13 positional isomers of hydroperoxylinoleic acid. The reaction which was catalysed by haemoproteins and Fe(II) and Cu(II) ions showed positional selectivity that was similar to lipoxygenase-catalysed reactions but yielded equal proportions of both enantiomers of the hydroperoxides. There was little selectivity when methyl linoleate was used as the substrate or when linoleic acid was oxidised in organic solvents. The results indicated that positional selectivity was, at least in part, due to the conformation of the fatty acid molecule in aqueous media. Implication of the selectivity in a non-enzymic reaction is discussed especially in relation to its effect on the determination of lipoxygenase specificities.

Buffers↗
PubMed1980

Identification of ecotropic proviral sequences in inbred mouse strains with a cloned subgenomic DNA fragment.

A specific probe for detecting ecotropic murine leukemia virus sequences was constructed by cloning a 500-base-pair DNA segment, corresponding to a portion of the env region of the AKR ecotropic virus, in a pBR322/Escherichia coli K-12 host/vector system. This probe was used to screen the cellular DNAs of six inbred strains of mice for the presence of ecotropic retroviral DNA sequences by the Southern blot hybridization procedure. Three copies of ecotropic viral DNA were detected in AKR/N (a high-ecotropic virus strain) and two were found in BALB/c (a low-ecotropic virus strain) DNAs. As expected, no sequences reactive with this probe were found in NFS mouse DNA (a virus-negative strain). However, cellular DNA sequences that reacted strongly with the ecotropic-specific DNA probe were detected in certain NZB, C57L, and 129 mice (all virus-negative strains). In contrast to the reactive sequences in AKR and BALB/c, the reactions were chiefly associated with EcoRI segments that were subgenomic in size.

Animals↗
PubMed1980

Transfection of molecularly cloned Friend murine leukemia virus DNA yields a highly leukemogenic helper-independent type C virus.

Unintegrated viral DNA was isolated via the Hirt procedure from mouse fibroblasts newly infected with Friend murine leukemia virus (F-MuLV) clone 201, a biologically cloned helper virus isolated from stocks of F-MuLV complex. A physical map of the unintegrated in vivo linear viral DNA was generated for several restriction endonucleases. The supercoiled viral DNA was digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized viral DNA was then inserted into lambda gtWES.lambda B at the EcoRI site and cloned in an approved EK2 host. Eight independent lambda-mouse recombinants were identified as containing F-MuLV DNA inserts by hybridization with F-MuLV 32P-labeled complementary DNA. One of the F-MuLV DNA inserts was 9.1 kilobases (kb) and had the same restriction enzyme sites as the unintegrated linear F-MuLV DNA. Six inserts were 8.5 kb; each lacked a single copy of the terminally redundant sequences of the unintegrated linear viral DNA. One insert was 8.2 kb and contained a 0.9-kb deletion. After digestion with EcoRI, one recombinant DNA preparation containing an 8.5-kb insert was infectious for NIH 3T3 cells. Undigested recombinant DNA was not infectious. The infectivity of the EcoRI-digested DNA followed multihit kinetics, indicating that more than one molecule was required to register as an infectious unit. The virus isolated from this transfection (F-MuLV-57) was NB-ecotropic, helper-independent, and formed XC plaques. Inoculation of this virus into newborn NIH Swiss mice induced leukemia and splenomegaly in greater than 90% of animals within 3 to 4 weeks. The gross and microscopic abnormalities induced by F-MuLV clone 57 were identical to those seen with the original parent stocks of F-MuLV clone 201. These results indicate that this helper-independent F-MuLV can induce a rapid nonthymic leukemia in the absence of the spleen focus-forming virus.

Animals↗
PubMed1980

Identification of ecotropic proviral sequences in twelve inbred mouse strains.

A specific probe for detecting ecotropic murine leukemia virus sequences was constructed by cloning a 400 base-pair DNA, corresponding to a portion of the env region of the AKR ecotropic virus. We have employed this probe to detect the presence or absence of ecotropic retroviral DNA sequences in 12 inbred strains of mice using the Southern blotting hybridization procedure. Our results show that between 2 and 6, and 1-2 copies of the ecotropic viral genome are present in mice with high or low incidence of leukemia, respectively. Ecotropic viral sequences are either absent or present in partial copies in mouse strains which heretofore failed to yield ecotropic viruses.

Animals↗
PubMed1979

Molecular cloning of polyoma virus DNA in Escherichia coli: oncogenicity testing in hamsters.

Inoculation of suckling hamsters with 2 x 10(8) live cells of Escherichia coli K12 strain chi1776, carrying the complete genome of polyoma virus in a recombinant plasmid, failed to induce tumors in any of 32 recipients. Also, lambda phage DNA and particles with a monomeric insert of polyoma DNA did not induce tumors. Purified recombinant plasmid DNA, as well as phage particles and DNA containing a head-to-tail dimer of polyoma DNA, showed a low degree of oncogenicity, comparable to that of polyoma DNA prepared from mouse cells. These findings support the previous conclusions, based on infectivity assays in mice, that propagation of polyoma virus DNA as a component of recombinant DNA molecules in E. coli K12 reduces its biologic activity many orders of magnitude relative to the virus itself.

Animals↗
PubMed1979

Molecular cloning of polyoma virus DNA in Escherichia coli: plasmid vector system.

A series of recombinant plasmids containing polyoma virus (PY) DNA were constructed, and their biological activity was evaluated in mice and in cultured mouse cells. While all of the recombinants studied contain the complete, potentially infectious viral DNA, in no case was the intact recombinant PY-plasmid DNA, or live Escherichia coli containing the recombinant plasmids, capable of inducing PY infection of mice, either by feeding or by parenteral injection.

Animals↗
PubMed1979

Molecular cloning of polyoma virus DNA in Escherichia coli: lambda phage vector system.

The biological activity of recombinant phage and recombinant phage DNA containing monomeric or dimeric polyoma DNA inserts was examined in mice and cultured mouse cells. Recombinant preparations containing a single copy of viral DNA were invariably noninfectious; molecules containing a dimeric polyoma DNA insert were at least seven orders of magnitude less infectious than polyoma virions after parenteral inoculation. No infection was detected with any recombinant preparation after oral administration.

Animals↗
PubMed1979

Biological activity of polyoma viral DNA in mice and hamsters.

The biological activity of polyoma viral DNA was evaluated in mice and hamsters. Viral DNA administered parenterally is about 4 to 5 logs less efficient than polyoma virions in establishing infection in mice. Supercoiled viral DNA was infectious for mice after parenteral administration, giving mean infective doses of 10(-3) to 10(-4) microgram. However, animals fed microgram quantities of polyoma DNA I did not become infected. Linearization of viral DNA with R.EcoRI or R.BamHI, which are single-cut enzymes cleaving in the early and late regions of the genome, respectively, reduced the infectivity for mice approximately fivefold. Approximately 10% of newborn hamsters inoculated intraperitoneally with polyoma DNA I developed tumors. In contrast, the same amount of viral DNA which had been cleaved in the early region with R.EcoRI induced tumors in 50% of inoculated hamsters.

Animals↗
PubMed1979

Molecular cloning of the Harvey sarcoma virus closed circular DNA intermediates: initial structural and biological characterization.

Supercoiled Harvey sarcoma virus (Ha-SV) DNA was extracted from newly infected cells by the Hirt procedure, enriched by preparative agarose gel electrophoresis, and digested with EcoRI, which cleaved the viral DNA at a unique site. The linearized Ha-SV DNA was then inserted into lambda gtWESlambda B at the EcoRI site and cloned in an approved EK2 host. Ha-SV DNA inserts from six independently derived recombinant clones have been analyzed by restriction endonuclease digestion, molecular hybridization, electron microscopy, and infectivity. Four of the Ha-SV DNA inserts were identical, contained about 6.0 kilobase pairs (kbp), and comigrated in agarose gels with the infectious, unintegrated, linear Ha-SV DNA. One insert was approximately 0.65 kbp smaller (5.35 kbp) and one was approximately 0.65 kpb larger (6.65 kpb) than the 6.0 kpb inserts. R-looping with Ha-SV RNA revealed that the small (5.35 kbp) insert contained one copy of the Ha-SV RNA. Preliminary restriction endonuclease digestion of the recombinant DNAs suggested that the middle-size inserts contained a 0.65-kbp tandem duplication of sequences present only one in the small-size insert; this duplication corresponded to the 0.65-kpb terminal duplication of the unintegrated linear Ha-SV DNA. The large-size insert apparently contained a tandem triplication of these terminally located sequences. DNA of all three sized inserts induced foci in NIH 3T3 cells, and focus-forming activity could be rescued from the transformed cells by superinfection with helper virus. Infectivity followed single-hit kinetics, suggesting that the foci were induced by a single molecule.

Animals↗
PubMed1977

Autoxidation of methyl linoleate. Separation and analysis of isomeric mixtures of methyl linoleate hydroperoxides and methyl hydroxylinoleates.

The mixture of conjugated diene hyperperoxide isomers obtained from autoxidation of methyl linoleate was separated by high performance liquid chromatography (HPLC). Four major isomers were obtained from adsorption chromatography and identified as the 9 and 13 positional isomers having the trans-trans and cis-trans configurations. The latter geometrical isomers have the trans double bond adjacent to the hydroperoxide group. The hydroxy compounds (methyl hydroxylinoleates) obtained from the hydroperoxides by NaBH4 reduction were similarly separated but with improved resolution. This is the first instance of the complete separation of these compounds and provides a rapid method for their analysis. Unlike adsorption chromatography, reversed-phase chromatography separates the mixtures only according to the geometrical isomerism of the double bonds and not according to the position of the hydroxy or hydroperoxide function.

Chromatography, High Pressure Liquid↗
PubMed1977

A simple method for the preparation of pure 9-D-hydroperoxide of linoleic acid and methyl linoleate based on the positional specificity of lipoxygenase in tomato fruit.

Incubation of linoleic acid with crude homogenate of tomato fruit gave a high yield (69%) of linoleic acid hydroperoxides with a ratio of 9- to 13-hydroperoxide isomers of 96:4. After chromatography of the products, as free acids or methyl esters, hydroperoxides with 9- to 13-isomeric ratios of greater than 99:1 were obtained. The major product was characterized as 9-D-hydroperoxy-octadeca-trans-10,cis-12-dienoic acid. The results demonstrate the positional specificity of lipoxygenase from tomato fruit.

Linoleic Acids↗