PubMed Health⌕ Search

Biomedical subjects

R Breathnach

Publications and source records attributed to R Breathnach.

At least 55 records · Page 3Linked to original sources
PubMed1988

The collagenase gene family in humans consists of at least four members.

Stromelysin is a collagenase-related connective-tissue-degrading metalloproteinase. We have detected RNAs capable of hybridizing to a rat stromelysin cDNA in 11 of 69 human tumours tested. Molecular cloning of cDNAs to these RNAs has identified them as a mixture of stromelysin RNA and a transcript of a hitherto-undescribed related human gene, the stromelysin-2 gene. We have also isolated cDNAs corresponding to a more distantly related new human gene, the pump-1 gene. A comparison of the cDNA-derived amino acid sequences of stromelysin-2 and pump-1 with the known sequences of stromelysin and collagenase reveals significant similarities, with conservation of sequence motifs believed to have functional importance in metalloproteinase action. We conclude that the collagenase gene family in humans consists of at least four members, and speculate that expression of these genes plays a role in cancer.

Amino Acid Sequence↗
PubMed1988

Evidence for expression of two distinct T cell receptor beta-chain transcripts in a human diphtheria toxoid-specific T cell clone.

We have cloned and sequenced the human T cell receptor alpha- and beta-chains cDNA present in a lambda gt10 library derived from a human CD4+ diphtheria toxoid-specific T cell clone, PH28. Two 1.3-kb beta-transcripts within frame V, D, J joining were found. These two functional beta-transcripts use two members of different V beta-families, V beta 3 and V beta 4, one joined to D beta 1-1, J beta 1-1, C beta 1, the other joined to D beta 1-1, J beta 2-1, C beta 2. Moreover, we have cloned the two different V beta germ-line counterparts whose sequences confirm that these two V beta are not pseudogenes. Analysis of T cell receptor beta and alpha rearrangements in PH28 showed that a single rearrangement on each chromosome could be assigned either at the J beta or the J gamma loci. These results strongly suggest an absence of allelic exclusion for 1.3-kb mRNA beta-transcripts in a human antigen-specific T cell clone.

Amino Acid Sequence↗
PubMed1988

Expression pattern of a gene for a secreted metalloproteinase during late stages of tumor progression.

We have reported that transin RNA, a 1.9-kb RNA coding for a novel, secreted proteinase, was overexpressed during the progression of benign mouse skin papillomas to malignant squamous cell carcinomas (SCCs) induced by a two-stage protocol (Proc Natl Acad Sci USA 83:9413, 1986). Recently a high degree of similarity has been demonstrated between rabbit stromelysin, a secreted metalloproteinase that degrades proteoglycans found in the basement membrane and the amino acid sequence predicted in rat transin cDNA. DNA sequencing of a mouse cDNA isolated from an SCC (initiated by 7,12-dimethylbenz[a]anthracene [DMBA] and promoted by 12-O-tetradecanoylphorbol-13-acetate [TPA]) showed greater than 85% nucleotide similarity and 90% amino acid similarity to the rat transin-1 cDNA nucleotide and predicted amino acid sequences. Using this mouse transin cDNA clone as a probe (labeled with 32P) we found enhanced levels of transin mRNA transcripts in SCCs induced by a protocol giving rise to metastatic tumors (repeated N-methyl-N-nitroso-N'-nitroguanidine [MNNG] treatments) compared with the level found in SCCs induced by a protocol that had a lower probability of giving rise to metastatic tumors (MNNG initiation followed by TPA promotion). A study of primary SCCs and metastatic lesions induced by repeated benzo[a]pyrene treatment showed that the levels of transin mRNA transcripts were reduced in the metastatic lesions in comparison to the primary tumors. Southern analysis of the DNA isolated from epidermis, papillomas, and SCCs indicated that neither transin gene amplification nor rearrangement accounted for increased levels of the transin mRNA transcripts. These data suggest a role for enhanced levels of transin production in the invasion and metastasis of chemically induced SCCs.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

Differential expression of preproenkephalin and transin mRNAs following oncogenic transformation: evidence for two classes of oncogene induced genes.

FR3T3 rat embryo fibroblast cells express preproenkephalin mRNA after transformation by polyoma virus middle T or Ha-ras oncogenes. This effect was not seen in another rat embryo fibroblast cell line (Rat-1) or in FR3T3 cells transformed by Rous sarcoma virus, bovine papilloma virus type I or SV40. The elevation in preproenkephalin mRNA levels is thus cell specific and oncogene specific. These results contrast with those obtained for transin mRNA, which was observed in both Rat-1 and FR3T3 cells transformed by a number of different oncogenes. The expression of transin RNA correlated with the expression of the transformed phenotype. We suggest that genes induced by oncogenes in a given cell will fall into two classes: those linked to the expression of the transformed phenotype (expression induced by all oncogenes conferring this phenotype) and those induced as a consequence of activation of a specific cellular signaling system (expression induced by a subset of oncogenes linked to the signaling system in question). This system may prove useful in distinguishing oncogene-induced events that are related to cellular transformation from those that are secondary to eliciting the transformed phenotype.

Animals↗
PubMed1987

Sequences coding for part of oncogene-induced transin are highly conserved in a related rat gene.

The transin gene is induced by oncogenes and epidermal growth factor (EGF). We report here the isolation of a related gene (transin-2 gene). The structures of these genes are very similar. Indeed, a stretch of 428 nucleotides of the transin gene containing both exon and intron sequences is 98% conserved in the transin-2 gene. However, the putative promoter regions of the genes show little sequence homology, apart from a short element related to a sequence involved in control of transcription by cyclic AMP or a tumour promoter. Expression of the transin-2 gene, unlike that of the transin gene, is not induced by EGF, dibutyryl cyclic AMP or cytochalasin D. Nevertheless, transin-2 RNA is expressed in several transformed rat embryo fibroblast cell lines, and can be induced by a tumour promoter. The proteins transin and transin-2 are approximately 71% homologous in sequence. Both proteins show significant sequence homology with two connective tissue degrading metalloproteases. These homologies raise the possibility that expression of transin and transin-2 in transformed cells might play a role in tumour invasion.

Amino Acid Sequence↗
PubMed1986

The mRNA coding for the secreted protease transin is expressed more abundantly in malignant than in benign tumors.

Transin RNA is a 1.9-kilobase RNA transcript induced by oncogenes in rat embryo fibroblast cell lines. We show that RNA species complementary to a cloned transin cDNA are present in mouse skin squamous cell carcinomas induced by a classical initiation-promotion protocol but not in premalignant, benign papillomas or in normal epidermis. A single application of a tumor promoting phorbol ester to normal epidermis elicits a transient increase in these RNA levels. Transin RNA encodes a secreted protease, an activity consistent with a functional role for enhanced expression of transin RNA in the progression of benign, encapsulated tumors to malignant, invasive carcinomas.

Animals↗
PubMed1986

Isolation of the oncogene and epidermal growth factor-induced transin gene: complex control in rat fibroblasts.

Various oncogenes or epidermal growth factor (EGF) induce transcription of a 1.9-kilobase RNA (transin RNA) in rat fibroblasts. The induction by EGF can be blocked by cycloheximide. Thus the response of the transin gene to EGF appears to require de novo protein synthesis. Transin RNA induction is specific to EGF, as neither insulin, platelet-derived growth factor, fibroblast growth factor, nor transforming growth factor beta could elicit the same response. However, transforming growth factor beta could block the EGF induction of transin RNA. Whereas the calcium ionophore A23187 and the tumor promoter TPA, either alone or administered together, did not increase transin RNA levels, TPA could synergise with a serum factor to effect such an increase. Dibutyryl cyclic AMP also induced transin RNA. Treatment of cells with the microfilament-disrupting agent cytochalasin B, but not the microtubule-disrupting agent colcemid, resulted in an increase in transin RNA levels, suggesting a role for the cytoskeleton in control of transin gene expression. The transin RNA does not contain repeated sequences and appears to be encoded by a single-copy gene. The protein sequence encoded by the last four exons of the transin gene shows some homology to two regions of the heme-binding protein hemopexin.

Animals↗
PubMed1985

Epidermal growth factor or serum stimulation of rat fibroblasts induces an elevation in mRNA levels for lactate dehydrogenase and other glycolytic enzymes.

We have isolated cloned cDNAs corresponding to five mRNAs whose level is increased following stimulation of quiescent rat fibroblasts by either epidermal growth factor or serum. Partial sequencing followed by a computer search of data banks has shown that the cloned cDNAs correspond to mRNAs encoding proteins with extensive homology to lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, enolase, triose phosphate isomerase, and actin. The complete nucleotide sequence of a rat fibroblast lactate dehydrogenase is presented.

Animals↗
PubMed1985

Epidermal growth factor and oncogenes induce transcription of the same cellular mRNA in rat fibroblasts.

We have isolated and sequenced a cloned cDNA corresponding to an mRNA present in significantly higher levels in rat cells transformed by polyoma virus, Rous sarcoma virus, and the cellular oncogene H-ras than in the normal parental cell lines. The mRNA transcript is also rapidly induced by the polypeptide growth factor epidermal growth factor, providing a new link between oncogenes and growth factors. Both the growth factor and the oncogenes control expression of the corresponding gene at the transcriptional level. Our results point to the existence of intracellular mechanisms that are common to the action of both growth factors and oncogenes.

Animals↗
PubMed1984

Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell line MCF-7.

We present the complete sequence of an mRNA which is induced by estrogen in the human breast cancer cell line MCF-7 [pS2 mRNA, Masiakowski et al., Nucleic Acids Res. 10, 7895-7903 (1982)]. Primer extension and cloning of double-stranded cDNA (ds-cDNA) into a vector designed to make full-length cDNA were used to determine the sequence of the fifteen 5'-terminal nucleotides which were not present in the original pS2 ds-cDNA clone. The mRNA sequence has a major open reading frame encoding 84 amino-acids, flanked by a 40 nucleotide 5'-untranslated region and a 198 nucleotide 3'-untranslated region preceding the polyA tail. The 3'-untranslated region contains a polyadenylation signal, AUUAAA, 14 nucleotides upstream from the polyA tail. The derived protein sequence contains a putative signal peptide region suggesting that the protein may be secreted. The nucleotide and derived amino-acid sequences were compared to previously determined sequences, particularly to those of hormone-regulated proteins and growth factors, and no obvious similarities were observed.

Amino Acid Sequence↗
PubMed1984

Selective amplification in methotrexate-resistant mouse cells of an artificial dihydrofolate reductase transcription unit making use of cryptic splicing and polyadenylation sites.

We have constructed a recombinant ( pMOP ) which is based on the bacterial plasmid pML2 and contains bovine papilloma virus type 1 (BPV 1) sequences linked to an artificial mouse dihydrofolate reductase (DHFR) transcription unit. This unit consists of the SV40 early gene promoter, a complete DHFR coding sequence and splice and polyadenylation sites from a rabbit beta-globin gene. Intact pMOP or a fragment thereof devoid of pML2 sequences will transform mouse cells to methotrexate resistance. The lines obtained contain approximately 200 copies of the DHFR transcription unit. In no case, however, did we find evidence of extrachromosomal maintenance of BPV 1 or DHFR sequences. One line, when selected for resistance to elevated levels of methotrexate, contained amplified copies of a 'novel' DHFR transcription unit resulting from fusion of two normal units. This line contained the DHFR RNA species present in the parent line plus some additional species. The structure of these various RNA species was determined and evidence found for the use of cryptic splice and polyadenylation sites.

Animals↗
PubMed1984

Synthetic donor and acceptor splice sites function in an RNA polymerase B (II) transcription unit.

We have synthesised a 32-bp oligonucleotide containing sequences conforming to the consensus sequences for donor and acceptor splice sites. The oligonucleotide has been inserted into an RNA polymerase B (II) transcription unit and the resulting recombinant used to study the splicing mechanism. Our findings are as follows: (i) the synthetic sites function when separated by several different prokaryotic or eukaryotic DNA fragments providing bulk intron sequence, (ii) intron size need not be greater than 29 bp, (iii) an AG dinucleotide 11 bp upstream from the invariant AG of an acceptor splice site renders the latter non-functional, and (iv) sequence changes distant from splice sites can affect the efficiency of their utilisation.

Animals↗
PubMed1983

Plasmids for the cloning and expression of full-length double-stranded cDNAs under control of the SV40 early or late gene promoter.

Okayama and Berg (1) have recently described a technique for the high efficiency cloning of full-length dscDNAs. We have constructed eukaryotic expression vectors compatible both with this technique (and with classical techniques) for dscDNA cloning. The vectors are such that recombinants obtained contain dscDNAs in the correct orientation downstream from a block of sequence comprising either the SV40 early or late gene promoter linked to a pair of splice sites from a rabbit beta-globin gene. A sequence encoding an SV40 polyadenylation site follows the dscDNA. We have used our vectors to make a library from chicken oviduct polyA(+) RNA using the Okayama and Berg technique. Ovalbumin recombinants occur in the library at the expected frequency and a high proportion contain full length copies of the ovalbumin mRNA. However, a similar result was not obtained for conalbumin recombinants. When recombinants are introduced into eukaryotic cells by either calcium phosphate coprecipitation or protoplast fusion, expression of chicken ovalbumin or conalbumin may be detected by indirect immunofluorescence. Under optimal conditions (use of SV40 late promoter and cos 7 cells) ovalbumin protein could be detected when the ovalbumin recombinant was present in only 2% of the protoplasts used for fusion. This suggests that colony banks obtained using our vectors could be screened in batches of 50 by protoplast fusion followed by a search for expression of a given protein using indirect immunofluorescence.

Animals↗
PubMed1982

Cloning of cDNA sequences of hormone-regulated genes from the MCF-7 human breast cancer cell line.

cDNA clones corresponding to a mRNA whose level is rapidly increased by addition of oestradiol to the culture medium have been isolated by differential screening of a cDNA library from the breast cancer cell line MCF-7, which contains oestrogen receptors. Such clones will be useful in studies of the DNA sequences required for hormonal induction and to determine whether expression of the corresponding gene is in any way related to the cancerous state. We have also obtained a cDNA clone for a messenger whose level is apparently decreased by steroid hormones.

Breast Neoplasms↗
PubMed1982

Recombinant DNA molecules comprising bovine papilloma virus type 1 DNA linked to plasmid DNA are maintained in a plasmidial state both in rodent fibroblasts and in bacterial cells.

Transformed cells obtained after transfecting FR3T3 rat fibroblasts with DNA of bovine papilloma virus type 1 ( BPV1 ) maintained only free copies of the viral genome. Transfection with BPV1 DNA inserted in a bacterial plasmid (pBR322 or pML2 ) did not produce transformants at a detectable rate, unless the viral sequences had been first excised from the plasmid. In contrast, transfer of the same plasmids by polyethylene glycol-induced fusion of bacterial protoplasts with FR3T3 rat or C127 mouse cells led to significant transformation frequencies. A total of eight cell lines were studied, three rat and five mouse transformants, obtained with various BPV1 - pML2 recombinants. In all cell lines, both BPV1 and plasmid sequences were maintained as non-integrated molecules, predominantly as oligomeric forms of the transforming DNA. In the three rat transformants and in two of the mouse lines, parts of the non-transforming viral region and some bacterial sequences were deleted. In the remaining three mouse lines, the monomeric repeat was a non-rearranged plasmid molecule which could be re-established as a plasmid in Escherichia coli after cleavage with "one-cut" restriction endonucleases and circularization of the molecule.

Animals↗
PubMed1981

Transformation of mouse fibroblasts to methotrexate resistance by a recombinant plasmid expressing a prokaryotic dihydrofolate reductase.

A recombinant plasmid has been constructed for the expression of inserted DNA sequences coding for polypeptide chains using the simian virus 40 early promoter and splicing and polyadenylylation signals from the rabbit beta-globin gene. The coding regions for two prokaryotic methotrexate-resistant dihydrofolate reductases were introduced into the expression vector. When mouse fibroblasts were exposed to these recombinant plasmids, it was possible to select methotrexate-resistant clones that had integrated the plasmids and produced a chimeric RNA coding for the prokaryotic enzyme.

Animals↗