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Biomedical subjects

H Ponta

Publications and source records attributed to H Ponta.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1984

Genetic changes in mammalian cells reminiscent of an SOS response.

Prior to the isolation of mammalian DNA repair genes and identification of their gene products, the comparison between the bacterial SOS response and various similar reactions in mammalian cells remains rather speculative. The increasing number of observed phenomena including enhanced DNA repair, virus induction, induced cellular differentiation, and neoplastic transformation, all following DNA damage or arrest of replication, are, however, suggestive of an SOS-like system of growth control and may form an entry into this fascinating area.

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

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↗
PubMed1982

Delimitation of a DNA sequence which confers inducibility by glucocorticoid hormones.

A chimeric long terminal repeat-thymidine kinase (LTR-tk) gene has been used to define the sequence requirements for glucocorticoid induction of gene expression. The original LTR-tk gene contains an entire mouse mammary tumor virus (MMTV) LTR preceding the tk gene. This gene can be expressed in a hormone-responsive fashion upon transfection into L tk--cells to produce a chimeric LTR-tk mRNA. Stepwise deletion of nucleotide sequences 5' of the viral RNA initiation site revealed that 202 nucleotides upstream of the viral cap site are sufficient for the hormonal regulation. Deletion of 5' sequences up to 59 nucleotides upstream of the viral cap site abolished RNA initiation in the LTR and hormonal induction.

Animals↗
PubMed1982

A B-lymphocyte-specific high-turnover protein: constitutive expression in resting B cells and induction of synthesis in proliferating cells.

A basic B-cell-specific protein of 35,000 daltons (XM1) is expressed at a high rate--comparable to actin--in resting splenic B lymphocytes of various species. Splenic or thymic T cells or fibroblasts do not express this protein. Upon polyclonal stimulation of B lymphocytes or in proliferating B-lymphocyte-derived cell lines, the synthesis of XM1 is switched off, but it is reinduced by agents that arrest replication, such as mitomycin C, hydroxyurea, ultraviolet light or gamma irradiation. Induction of XM1 after mutagen treatment is fast; the synthesis is regulated on the level of transcription or RNA processing. XM1 is turned over with half-life of less than 2 hr. XM1 is glycosylated. From compartment analysis and Triton solubilization it appears that XM1 is bound to the nuclear membrane.

Animals↗
PubMed1982

12-O-tetradecanoylphorbol 13-acetate-inducible proteins are synthesized at an increased rate in Bloom syndrome fibroblasts.

A set of proteins, which in normal fibroblasts were barely, if at all, detectable, were synthesized at an increased rate in fibroblasts from patients with Bloom syndrome (BS). The same set of proteins was induced in normal human fibroblasts by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). In BS cells, TPA caused a further 2-fold increase in the rate of synthesis. Production of these proteins was inhibited by the addition of fluocinolone acetonide to the culture medium. One of the proteins (XHF1) present at high levels in BS fibroblasts and in TPA-treated cells was also induced by irradiation with ultraviolet light. This protein was secreted into the culture medium. Most other TPA-inducible proteins were cytoplasmic. Among other human mutants prone to chromosome aberrations we found one of three tested cases of Fanconi anemia and one case of ataxia-telangiectasia that showed increased spontaneous rates of synthesis of the TPA-inducible proteins. In these cases, however, the induction by TPA was like that seen in healthy fibroblasts.

Bloom Syndrome↗
PubMed1981

The endogenous proviral mouse mammary tumor virus genes of the GR mouse are not identical and only one corresponds to the exogenous virus.

The endogenous proviral copies of mouse mammary tumor virus (MMTV) were selected from a gene library of GR mouse DNA. We obtained five different lambda. MMTV recombinant clones. Four of them correspond to the 3' Eco RI fragments of the endogenous proviruses an one comprises an intact MMTV provirus with 2 to 3 kb of flanking mouse genomic DNA. Heteroduplex formation followed by S1 digestion under stringent conditions shows that there is nucleotide sequence heterology among the cloned endogenous proviral copies. Only one endogenous proviral copy, associated with the mtv-2 locus, was found to be totally homologous to the exogenous proviral DNA.

Animals↗
PubMed1980

Development of Escherichia coli virus T1. The role of the proton-motive force.

In the interaction between Escherichia coli virus T1 and its host cell, which leads to reorientation of macromolecule synthesis, the alteration of the host cell membrane is an important step: The proton-motive force is rapidly reduced. This became apparent from selective changes in energy-coupled transports: proton-motive force- and ATP-dependent transports are inhibited in wild type cells. However, in ATPase-deficient (unc-) cells the ATP-driven transports are not affected by T1. The membrane potential is reduced by T1 as was measured by triphenylmethylphosphonium ion distribution. The potassium gradient is dissipated and calcium ions are accumulated by the cells. However, the residual (but reduced) membrane energy is essential for T1 development since the addition of uncouplers prevents any viral production. Consistently, the cell membrane remains intact after T1 infection: proteins, like beta-galactoside, amino acids, and alpha-methylglucoside cannot passively penetrate the membrane of T1-infected cells.

Adenosine Triphosphate↗