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Infection of mouse embryos at 8 days of gestation with a replication-defective retrovirus carrying the human c-Ha-ras-1 oncogene led to efficient and rapid induction of hyperplastic lesions. Twenty-four percent of viable off-spring developed abnormal growths after infection with purified virus. The lesions contained a single integrated provirus and produced viral RNA and the Ha-ras oncogene product (p21). The latency period between the time of infection and appearance of the lesions suggested that secondary alterations in addition to activated ras were necessary for neoplasms to develop. The earliest and most abundant growths were cutaneous and appeared from 4 to 36 weeks of age, with a median of 4 weeks of age. A number of subcutaneous lesions also developed over the same time span but at a median of 18 weeks of age. The rapid development of cutaneous lesions in response to transduction of the ras oncogene contrasts with other studies in which adult skin required secondary treatment with promoters prior to ras induction of epithelial hyperplasia. These results demonstrate that infection of midgestation mouse embryos allows rapid analysis of oncogene potency in skin.
Mol Cell Biol 1989 Jan
PMID:Retroviral transduction of the human c-Ha-ras-1 oncogene into midgestation mouse embryos promotes rapid epithelial hyperplasia. 264 34

Infection of rat embryo cells with herpes simplex virus type 2 caused undermethylation of host cell DNA synthesized during infection. DNA made prior to infection was not demethylated, but some of its degradation products, including methyl dCMP, were incorporated into viral DNA. The use of mutant virus showed that some viral DNA synthesis appears to be required for the inhibition of methylation. Inhibition of methylation cannot be explained by an absence of DNA methyltransferase as the activity of this enzyme did not change during the early period of infection. Inhibition of host cell DNA methylation may be an important step in the transformation of cells by herpesviruses, and various transformed cell lines tested showed reduced levels of DNA methylation.
Mol Cell Biol 1988 Apr
PMID:Hypomethylation of host cell DNA synthesized after infection or transformation of cells by herpes simplex virus. 283 42

Adeno-associated virus (AAV) is a single-stranded DNA parvovirus that is dependent on adenovirus or herpesvirus for reproductive functions. We describe the construction of recombinant AAV vectors containing the chloramphenicol acetyltransferase gene or the neomycin phosphotransferase gene. These vectors carried their respective genes into a wide variety of cell types, including primary skin fibroblasts and hematopoietic cells. Infection efficiencies varied with cell type and ranged up to 3.0%. Coinfection of two different recombinant viruses was also used to introduce two different sequences simultaneously into a given cell. Finally, methods for obtaining recombinant AAV vectors with minimal contamination of wild-type virus are described. These various attributes of AAV vectors make them a viable DNA transduction system.
Mol Cell Biol 1988 Oct
PMID:Adeno-associated virus: a vector system for efficient introduction and integration of DNA into a variety of mammalian cell types. 284 25

A retrovirus shuttle vector is described that contains the dominant selectable neo gene which confers resistance to kanamycin in bacteria and to the drug G418 in animal cells. The bacterial supF gene and the origins of DNA replication from polyomavirus and the ColE1 replicon also have been included in this vector. Infection of normal rodent cells results in single-copy proviral integration, whereas infection of mouse (MOP) cells expressing polyoma large T antigen results in extrachromosomal replication of the DNA form of the virus. The copy number of the extrachromosomal circles in MOP cells varies from 0 to 100 copies per cell. G418-resistant MOP cells lose their drug-resistant phenotype after passage under nonselective conditions, suggesting that maintenance of the extrachromosomal circles is unstable. The extrachromosomal form of the virus can be recovered as plasmids in Escherichia coli. Two-thirds of the circles analyzed were found to be structurally intact. The others have undergone rearrangements including deletions and insertions. The bacterial supF gene was found to be intact in the majority of recovered plasmids. The data presented here suggest that these retroviruses should be useful as gene transfer vectors for animal cells in culture or in vivo.
Mol Cell Biol 1985 Feb
PMID:Characterization of a retrovirus shuttle vector capable of either proviral integration or extrachromosomal replication in mouse cells. 298 88

Bacteriophage lambda chromosomes are packaged in a polarized, sequential fashion from a multimeric DNA substrate. Mature chromosomes are generated when terminase introduces staggered nicks in the cohesive end sites (cos sites) bounding a chromosome. Packaging is polarized, to the initial and terminal cos sites for packaging a chromosome can be defined. To initiate packaging, terminase binds to cos at cosB, and subsequently cuts at cosN. To terminate packaging of a chromosome, a functional cosB is not required at the terminal cos. To explain this finding, it was proposed earlier that terminase scans for the terminal cosN, rather than any subsequent cosB, during packaging. In the work described here we performed helper packaging experiments to see whether processive action of terminase occurs during sequential packaging of lambda chromosomes. The helper packaging experiments involve trilysogens; strains carrying three prophages in tandem. Infection by a hetero-immune helper phage results in packaging of the repressed prophage chromosomes, since the prophage structure is analogous to the normal DNA substrate. Two chromosomes can be packaged from between the three cos sites of the prophages of a trilysogen. Both chromosomes are packaged even when the central cos is cosB-. Our interpretation of these data is that terminase is brought to the central cos by packaging; following cleavage of the central cos, the terminase remains bound to the distal chromosome; and terminase acts to begin packaging of the distal chromosome. The frequency at which terminase reads across the central cos to initiate packaging of the distal chromosome is in the range from 0.3 to 0.5 in our experiments. Reading across cos was found not to be greatly dependent on the state of cosB, indicating that cosB binding is only needed for packaging the first chromosome in a packaging series. A multilysogen was constructed in which the initial cos was cos+ and the distal cos sites were all cosB-. The initial and downstream chromosomes were found to be packaged. This result indicates that terminase that is brought to the central cos by packaging is not only able to initiate packaging of a downstream chromosome, but can also scan and terminate packaging of the downstream chromosome. A model is presented in which processive action of terminase is the basis for sequential packaging of lambda chromosomes.
J Mol Biol 1985 Dec 20
PMID:Processive action of terminase during sequential packaging of bacteriophage lambda chromosomes. 300 94

A derivative of the myeloproliferative sarcoma virus (Neor-MPSV) carrying the mos oncogene and dominant selection marker for neomycin resistance (Neor) was introduced into embryonal carcinoma and embryo-derived cell lines by transfection and infection using pseudotypes with Friend helper virus (Friend murine leukemia virus [F-MuLV]). Cells resistant to G418 (a neomycin analog) were cloned and expanded. Transductants retained an undifferentiated phenotype as judged by morphology, tumorigenicity, and cell-surface antigen analyses. Nucleic acid analysis of infectants revealed both Neor-MPSV and F-MuLV proviruses, although no virus was released. G418-resistant transductants remained nonpermissive for the expression of other proviruses and for subsequent superinfection. Northern analysis showed expression of full-length Neor-MPSV, as well as mos-specific subgenomic RNA. mos sequences were deleted from Neor-MPSV (Neor mos-1), and pseudotypes were used to infect embryonal carcinoma cells. No morphological differences were observed in either mos+ or mos- transductants as compared with parental cell lines. However, mos+ transductants showed an enhanced anchorage-independent growth compared with that of mos- transductants in agar cloning. PCC4 transductants were induced to differentiate with retinoic acid and superinfected with F-MuLV. Infection with viral supernatant in fibroblasts and in mice confirmed the rescue of biologically active Neor-MPSV.
Mol Cell Biol 1986 Jan
PMID:Viral transfer, transcription, and rescue of a selectable myeloproliferative sarcoma virus in embryonal cell lines: expression of the mos oncogene. 302 29

Trypanosoma cruzi infection in cultured human umbilical vein endothelial cells increased basal cellular calcium levels from 55 to 110 nM, as monitored with the fluorescent probe, fura-2. It also influenced intracellular calcium such that consistently higher total levels were observed in response to bradykinin, angiotensin II and norepinephrine, as compared to similarly treated uninfected cells. However, bradykinin and angiotensin II-dependent increases in calcium, when considered as the absolute increment or fold elevation over basal, were significantly lower in infected endothelial cells. Infection also influenced changes in calcium levels due to agents that operate independently of plasma membrane receptors. In the presence of ionomycin, the magnitude and rate of rise of intracellular calcium were decreased; additionally the calcium peak was delayed and the subsequent decline slowed. Similar to the results with bradykinin and angiotensin II, infection decreased both the increment in and fold stimulation of intracellular calcium in response to ionomycin. In contrast, infection altered only the total calcium stimulated in response to oligomycin; neither the fold stimulation of, nor increment in intracellular calcium was affected. These results indicate that (1) infection by T. cruzi alters calcium homeostasis in endothelial cells under basal and stimulated conditions; (2) both receptor-dependent and receptor-independent mechanisms are affected by infection. The possible contribution of altered calcium homeostasis induced by T. cruzi in the pathogenesis of chagasic cardiomyopathy is considered.
Mol Biochem Parasitol 1988 Jun
PMID:Alterations in intracellular calcium following infection of human endothelial cells with Trypanosoma cruzi. 304 42

The gene expression of nine phages of the T7 group was compared after infection of Escherichia coli B(P1). With the exception of phage 13a which grew normally, all of them infected E. coli B(P1) abortively. Differences were found in the efficiency of host killing which ranged from 100% for phage 13a to 37% for phage A1122. Infection by T7 prevented colony formation by about 70% of the cells but they showed filamentous growth until about 2 h after infection. It was shown by SDS-polyacrylamide gel electrophoresis and autoradiography of [35S]methionine-labelled phage-coded proteins that all phages except for 13a showed measurable expression only of the early genes. No correlation was observed between killing capacity and the pattern of gene expression, and the ability to hydrolyse S-adenosyl-methionine (SAM, a cofactor for the P1 restriction endonuclease) by means of a phage-coded SAMase. Mixed infection of E. coli B(P1) with 13a and T7 yielded mixed progeny indistinguishable from that observed after mixed infection of the normal host E. coli B. Genetic crosses with amber mutants of 13a and T7 showed that the 13a marker opo+ (overcomes P one), required for growth on B(P1), is located in the early region, to the left of gene 1 (RNA polymerase gene).
Mol Gen Genet 1988 Jun
PMID:Inhibition of gene expression of T7-related phages by prophage P1. 304 52

The diagnosis of virus infection by nucleic acid hybridization represents an alternative to classical virological diagnostic methods. One special technique termed 'filter in situ hybridization' consists of fixation of intact cells to nitrocellulose filters followed by hybridization with a labelled DNA probe. We demonstrate that filter in situ hybridization can be a simple and sensitive method for the detection of virus infection in cells. In an in vitro model system using a human B-lymphoma cell line infected by the lymphotropic papovavirus (LPV), it is shown that individual virus replicating cells can be detected by this method. Infection can be diagnosed even if only one out of 20,000 cells in a culture contains replicating virus. This assay may be of value as a diagnostic tool in other viral systems.
Mol Cell Probes 1988 Sep
PMID:Detection of individual virus-infected cells by filter in situ hybridization. 306 33

The BCR/ABL gene, formed by the Philadelphia chromosome translocation (Ph1) of human chronic myelogenous leukemia, encodes an altered ABL gene product, P210. P210 is strongly implicated in the malignant process of chronic myelogenous leukemia, but it precise role is unknown. Infection of long-term bone marrow cultures enriched for B-lymphoid cell types with a Moloney murine leukemia virus retroviral vector containing the BCR/ABL cDNA resulted in clonal outgrowths of immature B-lymphoid cells which expressed abundant P210 kinase activity. Surprisingly, infection of long-term myeloid lineage-enriched cultures also resulted in clonal outgrowths of immature B-lymphoid cells. The P210-expressing lymphoid cell lines resulting from either type of culture were resistant to the lethal effects of corticosteroids. These findings indicate that high levels of P210 expressed from a Moloney murine leukemia virus long terminal repeat preferentially stimulate the growth of immature B-lineage cells, and this effect is apparent even in myeloid lineage-enriched cultures, in which few if any lymphoid cells can be detected prior to infection.
Mol Cell Biol 1988 Oct
PMID:Selective transformation of primitive lymphoid cells by the BCR/ABL oncogene expressed in long-term lymphoid or myeloid cultures. 326 66


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