Using PAC nested deletions to order contigs and microsatellite markers at the high repetitive sequence containing Npr3 gene locus.

Highly polymorphic di- and tetranucleotide repeats in and around Npr3, a potential candidate gene for hypertension, have been identified using a novel approach. Because this chromosomal site is rich in repetitive DNA and difficult to sequence, P1 artificial chromosomes were retrofitted with a loxP transposon to map the gene sequence within a clone using a series of nested deletions. Sequences from ends of deletions 1-3 kb apart identified a (CA)(20) and a (TA)(18)-(CA)(8) repeat 8 kb upstream and within an intron of Npr3, respectively. DNA from 17 individuals was analyzed for length polymorphisms in these and eight additional repeats identified in 200 kb of working draft sequence from this region in GenBank. The sequence contigs and microsatellite repeats from GenBank were ordered using the P1-derived artificial chromosome deletion series. Several of these repeats were found to vary considerably in length in the set of genomic DNA tested. Since this site in chromosome 5p has recently been implicated in disease in studies with genetically hypertensive rats, the microsatellite markers reported here will be useful for genetic analysis and may even be implicated in the disease process in humans. We discuss how these types of data are useful for interpreting draft DNA sequence coming out of the genome projects, and the utility of deletion clones as a resource for ordering contigs and gap filling.

Regulated expression of the human mutant ras gene after transfection of BALB/c mouse embryo fibroblast cells.

Four continuous cell lines constructed by transfecting BALB/c mouse fibroblast cells with an expression system that has the mutant c-Ha-ras gene under control of a truncated version of the mouse metallothionein-1 (mt-1) promoter were characterized for zinc-induced phenotype switching. These cells were selected for transformation in the presence of zinc, a known inducer of the mt-1 promoter. When the transfected cells were grown in medium depleted of zinc, there was a dramatic reduction in their soft agar cloning efficiency. Adding zinc back to the medium restored the transformed phenotype in a dose-dependent manner. Analysis of the intracellular p21 levels of induced and uninduced cells showed that zinc was modulating the expression of the transfected ras gene. In vivo studies done with syngeneic mice showed that zinc-induced cells were tumorigenic and formed metastatic lesions in the lungs of the inoculated animals.

Expression of Mason-Pfizer and simian type C viruses in the presence of 5-iododeoxyuridine and dexamethasone.

Production of infectious Mason-Pfizer monkey virus (M-PMV) was enhanced after treatment of the CMMT cell line with 2.5 x 10(-5) M dexamethasone phosphate (DXM). The reverse transcriptase (RT) activity and infectivity titers of treated culture fluids were enhanced by five- and tenfold, respectively. Along with stimulation of M-PMV synthesis, a simian type C virus (SCV) was also detected by electron microscopic and RT analyses. The SCV was serologically related to the endogenous baboon type C virus. 5-iododeoxyuridine (IUDR) also activated the SCV in the CMMT cell line while significantly inhibiting the production of infectious M-PMV. The activation of endogenous SCV by IUDR or DXM was transitory, since removal of these compounds from the growth medium resulted in the disappearance of SCV buds and the related RT activity; however, low levels of specific viral structural proteins continued to be synthesized intracellularly. Similarly, the enhancement of M-PMV production seen with DXM was lost when the treated cells were subcultured for 2 weeks in the absence of the hormone.

Interactions between endogenous baboon type-C virus and oncogenic viruses. I. Syncytium induction and development of infectivity assay.

Cells releasing the endogenous baboon virus (BV) can interact with human KC cells containing the Rous sarcoma virus (RSV) genome, resulting in cell fusion and syncytium formation. This interaction has been utilized in the development of a sensitive infectivity assay for BV. The titration pattern is of a one-hit type, demonstrating a linear relationship between virus concentration and number of syncytial plaques obtained in the KC co-cultivation assay. Endpoint titration comparisons indicate that the KC test is as sensitive as the immunofluorescence or the RNA-directed DNA-polymerase assays. Attempts to develop an XC test for BV failed, indicating that while BV can interact with the RSV genome it will do so in the human KC cells and not in the rat XC cells. Syncytia are also induced when KC cells are directly exposed to cell-free BV; however, a linear dose relationship is not obtained. When syncytium-positive KC cultures are passaged, the syncytia disappear and a chronic BV infection is established. These KC-BV cells then lose the ability to interact with either the endogenous cat RD-114 virus or the Mason-Pfizer virus which are known to form syncytia with KC cells.

The endogenous reverse transcriptase activity of Gibbon ape lymphoma virus: characterization of the DNA product.

The DNA product of the endogenous reverse transcriptase reaction of Gibbon ape lymphoma virus has been analyzed and characterized. Data show that in simultaneous detection assays in which the type and/or concentration of divalent cation is varied the best yield of rapidly-sedimenting DNA was obtained from reactions containing 1.5 mM Mn2+. This yield is ten-fold better than the yield observed at the optimal Mg2+ concentration (5.0mM). Evidence is presented to show that DNA synthesized at the optimal concentration of either of these cations consists of large pieces varying in size from 4 to 12S. This DNA hybridizes efficiently to homologous viral RNA (greater than 60 percent annealing) and protects at least two-thirds of GALV 70S [32P]RNA from ribonuclease digestion. The hybrids formed with homologous viral RNA are stable as evidenced by their thermal elution patterns from hydroxylapatite columns. In contrast, DNA synthesized in reactions in which the concentration of Mn2+ or Mg2+ was greater than optimal was predominantly 4S or smaller in size and displayed a low level of hybridization (less than 10 percent) to homologous viral RNA.