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PubMed1986

Molecular cloning of the Mason-Pfizer monkey virus genome: biological characterization of genome length clones and molecular comparisons to other retroviruses.

The molecular cloning of the DNA provirus of Mason-Pfizer monkey virus (M-PMV) is described. Fourteen independent clones of integrated M-PMV proviruses were isolated from a human embryo kidney cell line that had been previously derived from a single cell clone infected with M-PMV. Characterization of these clones for size of insert, restriction pattern of flanking DNA, and presence of repetitive DNA in the flanking sequences revealed that 10 of the isolates were identical while the four remaining clones were unique. Three independent clones of unintegrated M-PMV proviruses containing a single copy of the long terminal repeat (LTR) were cloned from acutely infected human embryo kidney cells, Transfection assays revealed that 13 of 14 integrated proviruses and 2 of 3 unintegrated proviruses were capable of producing infectious virus. One of the integrated provirus clones (clone 6A) produced consistently higher titers of virus than all of the other clones in all assays used and in two different cell lines, indicating that it contained a mutation that enhances virus replication. The virus recovered after transfection was shown to be capable of inducing cell fusion in nontransformed cell lines, confirming that this property is associated with M-PMV. One of the clones was hybridized under conditions of varying stringency, to molecular clones of type B, C, and D retroviruses. These studies revealed M-PMV to be most closely related to squirrel monkey retrovirus (D-type virus) and more distantly related to mouse mammary tumor virus (B-type virus). Hybridization was also detected with clones from the pol gene region of a family of human endogenous sequences. No homology was detected with Rous sarcoma virus or most mammalian C-type viruses tested. The exceptions were baboon endogenous virus and RD114 in which previously identified homology in the env gene was confirmed. These results suggest that the type D and type B viruses can be linked together in a group of viruses of similar ancestral origin analogous to that recently proposed for the human T-cell leukemia viruses and bovine leukemia virus.

Animals↗
PubMed2002

Rapid non-genomic and genomic responses to progestogens, estrogens, and glucocorticoids in the endocrine pancreatic B cell, the adipocyte and other cell types.

Rapid biologic responses to injected steroids were described as early as 60 years ago. More recently, evidence has been presented that 17beta-estradiol given i.v. will double the uterine cAMP activity within 15 s (Proc Natl Acad Sci USA 1967;58:1711-8), and also that estrogens will bind to the outer surfaces of endometrial cells (Nature 1977;265:69-72), suggesting that these steroids can both engage and direct intracellular events. Unfortunately, studies of such rapid membrane effects of steroids have languished due to the accumulation of compelling data for the more slowly manifest actions of these compounds at the level of nuclear DNA. We report a number of observations in women, in experimental animals, and in isolated organ or cell systems using 17beta-estradiol, progesterone or glucocorticoids which provide ample evidence for rapid intracellular metabolic responses to these steroids, mediated by their actions at the cellular plasma membrane. Such rapid responses have been shown in various classic targets or not, such as the B cell of the endocrine pancreas and the fat cell. They involve plasma membrane binding, changes in membrane electrical activity, Ca2+ handling, G and Ras proteins, cAMP, cGMP, IP(3), DAG, phosphodiesterases, protein kinases, tyrosine kinases, ER kinases, and mitogen activated protein kinases (MAPks) and nitric oxide synthase. These recent findings are discussed in detail and should lead to a fuller understanding of the cellular effects of the steroid hormones.

Adipocytes↗
PubMed1988

Restriction endonuclease analysis of the genome of Campylobacter pylori with a rapid extraction method: evidence for considerable genomic variation.

We developed a rapid extraction method to analyze the chromosomal DNA of 84 isolates of Campylobacter pylori (formerly Campylobacter pyloridis) from 70 individuals. Only three of the nine endonucleases tested gave satisfactory digestions: HindIII, EcoRI, and SacI. The latter two produced mostly larger bands, whereas HindIII produced smaller bands, which allowed clearer comparisons between isolates. The isolates from 69 Australian subjects and one from England each had a unique profile. Isolates from a husband and wife were different, as were those from a brother and sister. In pairs of isolates from 11 individuals, the second isolate was markedly different in six subjects. The profile changes were not associated with changes in antibiotic susceptibility or with loss of catalase or urease activity, which can occur during storage of C. pylori. These restriction endonuclease profiles suggest considerable subspecies variation in C. pylori. Plasmid bands were found in undigested DNA from 40 of the 84 C. pylori isolates.

Campylobacter↗