PubMed Health⌕ Search

Biomedical subjects

B Groner

Publications and source records attributed to B Groner.

At least 163 records · Page 9Linked to original sources
PubMed1986

The endogenous mouse mammary tumour virus locus Mtv-8 contains a defective envelope gene.

The Mtv-8 associated provirus, GR40, is not expressed in vivo. However, upon transfection into rat XC cells, the two MMTV specific mRNAs of 35S and 24S are transcribed. Further, the level of these transcripts is augmented when the transfected cells are grown in the presence of dexamethasone (Ponta et al., 1983). No virus can be detected in the medium of the transfected cells. Intracellular protein analysis of these transfected cells shows that although apparently authentic gag proteins are synthesized, a novel protein of 68 kDa is the only env related protein detectable. The sequence of the env gene of GR40 was determined and the predicted amino acid sequence of the env protein obtained. A premature termination codon is present 68 amino acids before the COOH-terminus of the known MMTV env precursor Pr73env. This would result in an env protein of about 68 kDa, in agreement with the size of the env protein found in GR40 transfected cells. The lack of the carboxy terminus may be responsible for the non-processing of the aberrant env precursor protein, p68.

Amino Acid Sequence↗
PubMed1986

Expression of anti-sense mRNA in H-ras transfected NIH/3T3 cells does not suppress the transformed phenotype.

We have attempted to reverse the transformed phenotype of cells expressing the H-ras oncogene. A plasmid in which the first exon of the H-ras oncogene was coupled to the SV40 early promoter in an anti-sense orientation was constructed. This construct was introduced into a clone of H-ras-transformed NIH/3T3 cells. Simultaneous expression of both the SV40 anti-sense construct and H-ras was observed. Anti-sense RNA was present in a 10-20-fold excess over sense H-ras RNA. Only a small fraction of the cytoplasmic RNA was present in a sense: anti-sense duplexed form. The expression of anti-sense H-ras RNA was not accompanied by a phenotypic reversion of transformed cells. The only phenotypic reversion we observed was accompanied by a loss of transfected H-ras sequences. The loss of transfected H-ras sequences occurs with a high frequency in cells supertransfected with the SV40 anti-sense construct.

Animals↗
PubMed1985

Hormonal regulation of cell surface expression of the major histocompatibility antigen H-2Ld in transfected cells.

The murine major histocompatibility antigens are cell surface glycoproteins which play an important role in the recognition of foreign antigens by cytotoxic T lymphocytes. Modulation of the level of expression of histocompatibility antigens could therefore be useful for the study of the interaction between the antigen presenting cells and T lymphocytes. The glucocorticoid hormone-inducible promoter, located in the long terminal repeat of mouse mammary tumor virus, was used to replace the promoter region of a cloned H-2Ld class I gene. The chimeric gene was introduced into cultured cells. Glucocorticoid induction of MMTV LTR H-2Ld mRNA could be shown by blot analysis. An S1 nuclease protection assay indicated that the transfected cells accurately initiate the chimeric mRNA. Immunoprecipitation of H-2Ld protein with a specific monoclonal antibody showed inducibility also at the cellular protein level. Fluorescence-activated cell sorter analysis monitored a 3-fold increase of H-2Ld on the cell surface when the transfected cells were grown in the presence of dexamethasone. This increase of H-2Ld expression was accompanied by a corresponding decrease on the cell surface of the endogenous H-2Kk.

Animals↗
PubMed1985

Production of mouse mammary tumor virus upon transfection of a recombinant proviral DNA into cultured cells.

We have investigated the intracellular proteins synthesized in rat XC and feline kidney cells transfected with endogenous mouse mammary tumor virus (MMTV) proviral DNA. The endogenous provirus GR40, associated with the Mtv-8 locus, directs the synthesis of gag proteins indistinguishable from those found in MMTV-infected cells. The env precursor Pr73env and the mature gp52 proteins could not be detected in these cells. Instead an env-related protein of 68K is synthesized. In contrast to this endogenous provirus, a cloned exogenous proviral variant directs the synthesis of apparently normal env proteins upon transfection into the same cell lines. These results suggest that the env gene of the endogenous MMTV provirus GR40 is defective. The exogenous proviral variant is not expected to synthesize virus particles since it carries a rearrangement in the gag gene. In order to obtain an MMTV provirus capable of correctly expressing both gag and env functions, we have constructed a hybrid endogenous-exogenous provirus containing the 5' long terminal repeat (LTR)-gag of GR40 and the pol-env-3' LTR of the exogenous provirus. Upon transfection into feline kidney cells, this hybrid provirus directed the synthesis of apparently authentic gag and env proteins. Further, virus particles can be detected in the culture medium of the transfected cells by electron microscopy. Viral proteins obtained from viral particles banded in a sucrose gradient were detected by immunoprecipitation.

Animals↗
PubMed1985

Hormonal response region in the mouse mammary tumor virus long terminal repeat can be dissociated from the proviral promoter and has enhancer properties.

The proviral DNA of mouse mammary tumor virus (MMTV) contains a regulatory region closely associated with its promoter, which subjects transcription to the control of glucocorticoid hormones. Delimitation analysis of a chimeric MMTV long terminal repeat-thymidine kinase gene (LTR-tk) has shown that the hormonal regulation sequence is confined to 202 nucleotides preceding the LTR-specific RNA initiation site. A second RNA initiation site (tk-specific mRNA) placed close to the regulatory MMTV sequence by in vitro recombination is also subjected to hormonal stimulation in transfected cells. A series of plasmids with deletions around the LTR cap site progressing from 3' to 5' was made and functionally tested. In vitro deletion of MMTV LTR sequences comprising the RNA initiation sequence and the "TATA" box do not effect hormonal regulation at the tk-specific mRNA start site. Nucleotides up to position -59 from the LTR initiation site could be deleted without influence on the glucocorticoid regulation, whereas deletions to position -65 abolished the hormonal effect on the tk gene transcription. A short MMTV LTR segment containing nucleotides -236 to -52 from the LTR initiation site was recombined with the tk gene or the alpha-globin gene. This fragment confers hormonal inducibility onto the heterologous genes over distances of 0.4 or 1.1 kilobases. The hormonal response region functions when it is placed either 5' or 3' of the regulated gene in both of the possible orientations and is reminiscent of an enhancer sequence.

Animals↗
PubMed1985

New acceptor cell for transfected genomic DNA: oncogene transfer into a mouse mammary epithelial cell line.

A line of mouse mammary epithelial cells (NMuMG) has been characterized for its ability to be stably transfected with exogenous DNA. A transfection frequency of at least 1 cell per 1,000 was obtained with the pSV2neo plasmid. Several thousand G418-resistant NMuMG cell clones can easily be generated in cotransfection of genomic DNA and pSV2neo. The NMuMG cells were isolated from normal mammary glands and do not form malignant lesions when injected into nude mice. We have cotransfected NMuMG cells with pSV2neo and genomic DNA from the human EJ bladder carcinoma line, a cell line which contains an activated c-rasH oncogene. When a pool of 4,700 G418-resistant colonies was injected into nude mice, tumors were obtained. These tumors contain a transfected human rasH gene. Genomic DNA transfection into a line of mouse epithelial cells, in combination with the selection of stable transfectants and tumor induction in nude mice, can be used to screen human tumor DNA for the presence of activated oncogenes.

Animals↗
PubMed1984

The methylation pattern of endogenous mouse mammary tumor virus proviral genes is tissue specific and stably inherited.

The methylation pattern of mouse mammary tumor virus (MMTV) proviral genes endogenous to the mouse strains C3H, 020, FM/JmsA, C57BL6, and BALB/c were investigated in various organs and mammary tumor tissue. Digestion of DNA with EcoRI or with EcoRI and HpaII followed by Southern blotting analysis and hybridization to a nick-translated MMTV DNA, allowed the distinction between the fully methylated and hypomethylated gene copies. MMTV proviral gene methylation was found to be organ specific, and the methylation pattern is stably inherited. The same proviral units present in different strains of mice exhibit the same organ-specific methylation patterns. Although proviral genes are normally heavily methylated in all tissues, hypomethylation of endogenous proviral genes was found in organs not known to express MMTV.

Animals↗
PubMed1984

The chromosomal integration site determines the tissue-specific methylation of mouse mammary tumour virus proviral genes.

Multiple endogenous mouse mammary tumour virus (MMTV) proviral genes are present at different chromosomal locations in inbred mouse strains. Proviral DNA methylation is location and tissue specific. The methylation patterns are stably inherited and appear to be conferred upon the viral DNA by the flanking mouse genomic DNA. In transformed cells, either mammary carcinoma cells, or cells immortalized by SV40 in vitro, the stable pattern of methylation is lost. Although hypomethylation of proviral genes, both in normal and in transformed tissue, accompanies MMTV-specific RNA expression, it is also observed in non-expressing tissues.

Animals↗
PubMed1984

Glucocorticoid hormone interactions with cloned proviral DNA of mouse mammary tumor virus.

The molecular details of glucocorticoid hormone regulation of expression of the mouse mammary tumor virus (MMTV) proviral gene have been investigated. Cloned proviral DNA was introduced into cultured cells by a gene transfer procedure. DNA acquired by transfection was shown to be expressed in a hormone regulated fashion. The proviral DNA was fragmented and recombined in vitro with an indicator gene to delimit the hormone response sequence. Inducibility of the indicator gene (thymidine kinase gene from Herpes Simplex Virus, tk) was observed upon recombination with the long terminal repeat (LTR) sequence of MMTV. Further delimitation of the LTR DNA demonstrated that 202 nucleotides located 5' of the RNA initiation site are sufficient to confer glucocorticoid regulation. In vitro interaction of LTR DNA with glucocorticoid hormone receptor complex, showed a preferential affinity to the same sequence which mediated hormonal regulation in transfected cells. Evidence for a direct receptor gene interaction in the process of gene induction was gained by the measurement of the kinetics of induction and the use of a glucocorticoid antagonist (RU 486). The induction of the transfected gene is very rapid, independent of simultaneous protein synthesis and requires a functional glucocorticoid receptor hormone complex.

Animals↗
PubMed1983

Correlation of glucocorticoid receptor binding sites on MMTV proviral DNA with hormone inducible transcription.

Steroid hormones, when complexed to their receptors, recognize and bind specific DNA sequences and subsequently induce increased levels of transcription. The mechanisms of steroid hormone action were analyzed by constructing chimeric DNA molecules from portions of mouse mammary tumor virus envelope and long terminal repeat (LTR) regions ligated to the thymidine kinase (tk) gene of herpes simplex virus. This construction allowed the tk gene to be expressed in a hormone-responsive fashion upon transfection into Ltk- cells. Comparison of transcription data with in vitro binding data showed that hormone-responsive transcription can be directly correlated to the presence of steroid hormone receptor binding sites on the DNA. There are at least two such receptor binding sites in the LTR region, one between -202 and -137 and another between -137 and -50 base pairs from the RNA cap site, as well as a site near the 5' end of the envelope region. These results strengthen the hypothesis that steroid-receptor complexes regulate genes primarily by binding to DNA sites near the promoter region and thereby modulate transcription.

Animals↗
PubMed1983

Transcription initiation of transfected mouse mammary tumor virus LTR DNA is regulated by glucocorticoid hormones.

A chimeric gene, recombined in vitro, containing a long terminal repeat (LTR) sequence from the proviral DNA of mouse mammary tumor virus and the thymidine kinase (tk) gene of Herpes Simplex Virus was introduced into L tk- cells. No transcription of LTR RNA was observed in transfected cells when glucocorticoid hormones were absent from the growth medium. Accumulation of LTR initiated RNA was measured upon hormone addition by the single strand specific nuclease RNA mapping procedure. The accumulation was rapid (detectable after 7.5 minutes), independent of simultaneous protein synthesis and mediated by a functional glucocorticoid receptor complex. Glucocorticoid hormones affect LTR transcription at the level of initiation. The rate of initiation (1.8 X 10(-2) molecules/cell/sec) and a half life of about 30 minutes could be calculated for LTR RNA. The half life of LTR RNA is independent of the presence of hormone.

Animals↗
PubMed1983

Subfragments of the large terminal repeat cause glucocorticoid-responsive expression of mouse mammary tumor virus and of an adjacent gene.

After transfection of mouse mammary tumor virus (MMTV) proviral DNA into cultured cells, the DNA is transcribed in a glucocorticoid-sensitive fashion. The large terminal repeat (LTR) region of MMTV is 1,328 nucleotides long and contains the regulatory information necessary for the hormonal response. We have constructed a MMTV LTR-thymidine kinase (tk) chimeric gene and have tested the biological activity of molecules containing various deletions in the LTR after transformation of LTK- APRT- mouse cells. In the TK+ transformants, both a LTR- tk chimeric RNA and an authentic tk RNA are correctly initiated and transcribed. The synthesis of the chimeric RNA as well as that of the tk RNA is hormonally regulated. A plasmid containing 202 nucleotides of LTR DNA 5' to the RNA initiation site is fully sensitive to glucocorticoids; 50 nucleotides still cause a residual inducibility.

Cell Transformation, Neoplastic↗
PubMed1983

A deletion mutant of mouse mammary tumour virus, lacking 516 nucleotides of the 5' long terminal repeat sequence, can be expressed in a hormone-responsive fashion.

In vitro manipulation of proviral DNA of mouse mammary tumour virus (MMTV) was used to construct mutants with defined deletions at the 5' end of the proviral gene. In the mutants 516, 1400 and 2000 nucleotides were removed from the 5' end. The deleted proviral DNA was tested for transcription and glucorticoid hormone regulation of viral RNA expression upon cotransfection into rat XC tk- cells with a thymidine kinase gene. Intact proviral DNA contained in the plasmid vector pBR322 and the deletion mutant pGR16 delta 516, missing 516 nucleotides of the 5' long terminal repeat (LTR) sequence, were transcribed in a hormone responsive fashion and produced RNA species of 35S and 24S. Deletion of the entire LTR sequence abolished MMTV transcription and the hormonal effect.

DNA, Viral↗