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Biomedical subjects

G Brady

Publications and source records attributed to G Brady.

46 records · Page 3Linked to original sources
PubMed1990

The chicken c-erbA alpha-product induces expression of thyroid hormone-responsive genes in 3,5,3'-triiodothyronine receptor-deficient rat hepatoma cells.

To determine the capacity of the chicken c-erbA (cTR-alpha) gene product in regulating expression of known thyroid hormone-responsive genes, both the cTR-alpha and the viral v-erbA genes were expressed in FAO cells, a rat hepatoma cell line defective for functional thyroid hormone receptors. Upon nuclear expression of the cTR-alpha protein the cells become responsive to thyroid hormone, as detected by expression of a number of genes (malic enzyme, phosphoenolpyruvate carboxykinase, and Na+/K(+)-ATPase) reported to be indirectly induced by the hormone in vivo. In addition, our data show that the c-erbA product directly activates the Moloney murine leukemia virus promoter in a ligand-dependent manner. The data show that the chicken c-erbA-alpha protein can modulate the expression of rat genes under either direct or indirect control by thyroid hormone.

Animals↗
PubMed1988

A selectable temperature-sensitive v-src Moloney retrovirus.

We have cloned the v-src gene of ts339B77 RSV into a new Moloney-based retroviral expression vector (YN). In the resulting construct (ts339YNsrc), the ts339 gene is transcribed from the viral LTR, while a selectable resitance marker, the Tn5 neomycin phosphotransferase (neor) gene, is transcribed from an internal thymidine kinase (Tk) promoter. G418 resistance and focus formation were induced in NIH3T3 cells at comparable efficiencies within the permissive temperature range (33-37 degrees C). At 39 degrees, on the other hand, focus induction was reduced 15-fold with no corresponding decrease in expression of G418 resistance. In cells infected with ts339YNsrc, phosphoproteins were elevated and similar in pattern on SDS PAGE regardless of whether the cells were grown at the permissive or restrictive temperature. The ts339YNsrc virus will be useful for the study of effects of v-src expression, and may also be of help in identifying relevant substrates of the v-src product.

3T3 Cells↗
PubMed1988

A point mutation in the DNA binding domain of the v-myb oncogene of E26 virus confers temperature sensitivity for transformation of myelomonocytic cells.

We have molecularly cloned a mutant of the v-myb, ets-containing E26 avian leukemia virus which is temperature sensitive for the transformation of myeloid cells. Cells infected with this mutant, ts21E26, are immature at 37 degrees C and can be induced to differentiate into resting, macrophage-like cells when shifted to 42 degrees C. The sequence of ts21E26 reveals a single relevant nucleotide alteration resulting in a threonine to arginine change in the highly conserved, putative DNA binding v-myb portion of the p135gag-myb-ets protein. Surprisingly, a ts21E26 viral construct in which the v-ets gene domain was deleted was only weakly temperature sensitive, although temperature sensitivity was largely restored in another v-ets deletion mutant whose 3' terminal sequences were replaced with those from the AMV v-myb gene. These results suggest that the temperature sensitive lesion in v-myb of ts21E26 alters the DNA binding capacity of p135 at 42 degrees C and that the primary structure of the C-terminus of this protein has an influence on the activity of sequences that are further upstream.

Animals↗
PubMed1985

Use of gene transfer and a novel cosmid rescue strategy to isolate transforming sequences.

Mouse Lewis Lung tumor DNA was ligated to a cosmid containing a geneticin (G418)/kanamycin resistance gene and transferred into NIH3T3 cells. Recipient cells were first selected for geneticin resistance and subsequently for their ability to grow as a tumour when injected into nude mice. By repeating this transfection procedure with DNA from resultant tumours, geneticin-resistant NIH3T3 cells were obtained which were tumorigenic and contained approximately 1-5 copies of the transferred cosmid. The functional oncogene was cloned by preparing cosmid libraries of third round tumour DNAs, using a cosmid which does not contain a kanamycin resistance gene. Due to the original linkage of the oncogene with the cosmid containing the kanamycin resistance gene, a series of kanamycin-resistant cosmids were isolated, five of which contained an active oncogene. Subsequent analysis showed that the oncogene present was highly related to the human N-ras gene. Using a DNA probe from the MLL N-ras gene, a non-transforming counterpart was isolated from mouse liver DNA. A comparison between the two N-ras genes showed that a mutation at the amino acid position corresponding to 61 in the human gene is responsible for transforming activity of the rescued gene.

Animals↗
PubMed1984

New cosmid vectors developed for eukaryotic DNA cloning.

A series of ColE1 and pSC101 cosmid vectors have been constructed suitable for cloning large stretches of DNA. All contain a single BamHI site allowing cloning of Sau3A, MboI, BglII, BclI , and BamHI-generated fragments. These vectors have the following characteristics: (i) they are relatively small (1.7-3.4 kb); (ii) the BamHI cloning site is flanked by restriction enzyme sites enabling direct cloning of unfractionated insert DNA without generating multiple insert or vector ligation products [ Ish - Horowitz and Burke, Nucl . Acids Res. 9 (1981) 2989-2998]; (iii) two vectors ( pHSG272 and pHSG274 ) contain a hybrid Tn5 KmR/ G418R gene which is selectable in both prokaryotic and eukaryotic cells, making them suitable for transferring DNA into eukaryotic cells, and (iv) the different prokaryotic selectable markers available in the other vectors described facilitate cosmid rescue of the transferred DNA sequences from the eukaryotic cell: CmR, ApR, KmR, ( pHSG429 ), CmR, ( pHSG439 ), colicin E1 immunity ( pHSG250 ), (v) the cosmid pHSG272 was used successfully to construct a shuttle vector based on the BPVI replicon [ Matthias et al., EMBO J. 2 (1983) 1487-1492].

Animals↗
PubMed1984

An alternative assay system for the detection of transforming genes.

We have developed an alternative assay system based on DNA mediated gene transfer (DMGT) for the detection of functional transforming genes which we find more sensitive and reliable than other assay systems. This approach involves selecting in tissue culture for cells which are expressing a drug-resistance marker co-transferred with the tumour DNA thus isolating the small number of cells which have taken up and are expressing exogenously added DNA. The ability of transformed cells present in this drug-resistant cell population to form tumours in athymic (nu/nu) nude mice was then used to assay for transforming genes. Using NIH3T3 and Rat 2 cells as recipients, tumours produced after DMGT with DNA from non-tumorgenic cells were only very rarely observed. Tumour induction was observed using DNA from a biopsy of a human ovary carcinoma and a spontaneous murine lung carcinoma. Two human retinoblastoma cell lines were negative for tumour induction. The transforming gene from the ovarian carcinoma was identified as c-K-ras2.

Animals↗
PubMed1983

A bovine papilloma virus vector with a dominant resistance marker replicates extrachromosomally in mouse and E. coli cells.

We describe the construction of a bovine papilloma virus-based vector (pCGBPV9) which contains a dominant selectable marker and replicates autonomously in both mouse and Escherichia coli cells. This vector contains the complete bovine papilloma virus genome, a ColE1 replication origin and a dominant selectable marker conferring resistance to kanamycin in bacteria and G418 in eukaryotic cells. A high number of G418R colonies are obtained after transfer of pCGBPV9 into mouse C127 cells. These G418R colonies contain vector DNA which replicates autonomously at approximately 10-30 copies per cell. The molecules are in most cases unrearranged and can be rescued into E. coli cells by bacterial transformation.

Animals↗
PubMed1983

Replication control mutations of plasmid R6-5 and their effects on interactions of the RNA-I control element with its target.

Nine high copy number mutations of plasmid R6-5, representing five phenotypically distinct groups, have been identified by DNA sequencing. In each mutant plasmid examined, a single nucleotide change was found. The effects of the mutations on possible gene products, and DNA-RNA secondary structure, were analyzed and compared with the observed phenotypes. The results of this study exclude the possibility that the primary plasmid replication control element, the product of the copA gene, is a polypeptide, and they are consistent with a model of plasmid replication control by the copA product which has the following features: (i) RNA-I, a short untranslated RNA molecule, is the product of the copA gene and regulates the frequency of initiation of plasmid replication, (ii) the hexanucleotide single-strand loop of the major hairpin of RNA-I is its active site, (iii) this active site functions by base pair interactions with its "target," its DNA template strand, or its complementary sequence on RNA-II, a transcript of opposite polarity that is the message of the repA gene, and (iv) the sequence and size of the loop, and the stability of the stem of the hairpin, are all critical factors that govern the functioning of RNA-I.

Base Sequence↗
PubMed1981

Regulation of DNA replication: "target" determinant of the replication control elements of plasmid R6-5 lies within a control element gene.

The replication control system of plasmid R6-5 has been investigated by characterization of high-copy-number mutant miniplasmids, development of an in vivo assay for the site of action or "target" of the replication control elements, and sequence analysis of the replication control regions of the wild-type plasmid and two copy-number mutant derivatives. These and other experiments have shown that three plasmid determinants--copA/incA, copB, and copT--are involved in DNA replication control. The products of the copB and copA/incA genes, a 9500-dalton basic polypeptide and either a 7200-dalton basic polypeptide or a short untranslated RNA molecule, respectively, are negative-acting elements that interact with the third element, their target, the copT DNA sequence, or its product to regulate the frequency of initiation of plasmid replication. The location of copT within the copA/incA gene and 1600 base pairs upstream from the origin of replication indicates that regulation is effected at a preinitiation stage of replication, such as the production of a primer or other initiation factor.

Base Sequence↗