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Biomedical subjects

M Perucho

Publications and source records attributed to M Perucho.

At least 91 records · Page 5Linked to original sources
PubMed1985

Inhibitory effect of interferon on the genetic and oncogenic transformation by viral and cellular genes.

The rodent established cell lines LTk- and NIH 3T3 have been used as recipients in gene transfer experiments to study the effect of interferon treatment on the genetic and oncogenic transformation by several genes of viral and cellular origin. Our results show that interferon severely inhibits, to a similar extent, the stable transformation of Ltk- and NIH 3T3 cells by the chicken thymidine kinase (tk) gene, Ecogpt gene, simian virus 40, v-Ha-ras, and human c-Ha-ras and c-Ki-ras oncogenes. These results are consistent with an inhibition by interferon at the level of stabilization or integration, or both, of exogenous DNA sequences in the recipient cells, with an apparent effect on gene expression.

Animals↗
PubMed1984

Activation of a human c-K-ras oncogene.

The human lung carcinomas PR310 and PR371 contain activated c-K-ras oncogenes. The oncogene of PR371 was found to present a mutation at codon 12 of the first coding exon which substitutes cysteine for glycine in the encoded p21 protein. We report here that the transforming gene of PR310 tumor contains a mutation in the second coding exon. An A----T transversion at codon 61 results in the incorporation of histidine instead of glutamine in the c-K-ras gene product. By constructing c-K-ras/c-H-ras chimeric genes we show that this point mutation is sufficient to confer transforming potential to ras genes, and that a hybrid ras gene coding for a protein mutant at both codons 12 and 61 is also capable of transforming NIH3T3 cells. The relative transforming potency of p21 proteins encoded by ras genes mutant at codons 12, 61 or both has been analyzed. Our studies also show that the coding exons of ras genes, including the fourth, can be interchanged and the chimeric p21 ras proteins retain their oncogenic ability in normal rodent established cell lines.

Amino Acid Sequence↗
PubMed1984

Isolation of transforming sequences of two human lung carcinomas: structural and functional analysis of the activated c-K-ras oncogenes.

Human lung tumors PR310 and PR371 maintained in nude mice contain activated c-K-ras oncogenes detectable by the ability of their DNAs to induce the morphological transformation of NIH 3T3 mouse fibroblasts. Using phage libraries constructed with DNA from NIH 3T3 mouse fibroblast transformants, we have isolated human sequences that span greater than 40 kilobase pairs of the c-K-ras oncogene. Based on the conservation of these human sequences in mouse fibroblast transformants, we conclude that the transforming ability of the oncogene activated in these tumors resides within a 43- to 46-kilobase-pair DNA region. No clear differences were observed between the structures of the PR310 and PR371 cloned oncogene sequences. Nucleotide sequence analysis in concert with DNA transfection experiments suggests that the PR371 oncogene has been activated by a single base change in the first exon, which results in the substitution of cysteine for glycine in position 12 of the predicted amino acid sequence. The genetic alteration responsible for the transforming activity of the PR310 oncogene, however, does not reside in the first exon. These results indicate that the activation of the c-K-ras oncogene in human lung cancer can occur by different mutational events.

Amino Acid Sequence↗
PubMed1983

Isolation and preliminary characterization of the transforming gene of a human neuroblastoma cell line.

DNA from the human neuroblastoma cell line SK-N-SH is capable of inducing foci of transformed NIH 3T3 cells after DNA-mediated gene transfer. Using genetic selection with the Escherichia coli sup F gene, we have isolated human sequences from mouse cells responsible for the oncogenic transformation. These sequences are present in all human DNAs surveyed and no gross rearrangements of these sequences are found in SK-N-SH cells. Although clearly distinct from two other human transforming genes present in bladder, lung, and colon carcinoma cell lines, all three transforming gene sequences may be related members of the ras gene family.

Animals↗
PubMed1983

Three human transforming genes are related to the viral ras oncogenes.

Three distinct transforming genes present in human tumor cell lines are all related to the viral oncogenes of Harvey and Kirsten murine sarcoma viruses, designated v-H-ras and v-K-ras, respectively. The transforming gene of a bladder carcinoma cell line has been shown to be a human homolog to v-H-ras [Parada, L. F., Tabin, C. J., Shih, C. & Weinberg, R. A. (1982) Nature (London) 297, 474-478; Santos, E., Tronick, S. R., Aaronson, S. A., Pulciani, S. & Barbacid, M. (1982) Nature (London) 298, 343-347]. The transforming gene common to one colon (SK-CO-1) and two lung carcinoma (SK-LU-1 and Calu-1) cell lines is the same human homolog of v-K-ras as is the transforming gene previously identified in a lung carcinoma cell line Lx-1 [Der, C. J., Krontiris, T. G. & Cooper, G. M. (1982) Proc. Natl. Acad. Sci. USA 79, 3637-3640]. The transforming gene of SK-N-SH neuroblastoma cells is weakly homologous to both v-H-ras and v-K-ras. NIH 3T3 cells transformed with the SK-N-SH transforming gene contain increased levels of a protein serologically and structurally related to the protein products of the v-H-ras and v-K-ras genes. Therefore, it represents a third member of the ras gene family, which we have called N-ras. Based on the homology with the v-ras genes, we have established the orientation of transcription and approximate coding regions of the cloned human K-ras and N-ras genes.

Animals↗
PubMed1982

Isolation and preliminary characterization of a human transforming gene from T24 bladder carcinoma cells.

DNA from T24, a cell line derived from a human bladder carcinoma, can induce the morphological transformation of NIH 3T3 cells. Using techniques of gene rescue to clone the gene responsible for this transformation, we have found that it is human in origin, less than 5 kilobase pairs in size and is homologous to a 1,100-base polyadenylated RNA species found in T24 and HeLa cells. Blot analysis indicates extensive restriction endonuclease polymorphism near this gene, in human DNAs.

Animals↗
PubMed1982

Enrichment of histone H5-synthesizing polysomes by indirect immunoadsorption. Purification of H5 mRNA.

A method of purifying H5 mRNA from immature hen erythrocytes is described. Polysomes from red blood cells of anemic animals were allowed to react with affinity-chromatography-purified rabbit anti-H5 antibodies and immunoadsorbed to cellulose-insolubilized sheep and anti-rabbit antibodies. This method produced a 10- to 15-fold enrichment of the H5 mRNA, which was further purified by sucrose gradient centrifugation, poly(U)-Sepharose chromatography, and size fractionation in denaturing polyacrylamide gels. The H5 mRNA activity was essentially pure, as shown by translation in a reticulocyte cell-free system and immunoprecipitation with H5-antibodies. The proportion of H5 mRNA has been estimated to lie in the range 0.4-0.6% of all cellular mRNAs. These values are in agreement with the estimated relative synthesis of H5 in the immature erythrocyte.

Animals↗
PubMed1981

Human-tumor-derived cell lines contain common and different transforming genes.

We have screened different cultured cell lines established from human tumors for the ability of their DNAs to induce transformed foci in NIH/3T3 cells. Based on restriction endonuclease digestions and the presence of human sequences in mouse transformants, we conclude that five of these human tumor cell lines contain a gene or genes capable of transforming mouse cells and that at least three different transforming genes are present in these five lines. Three cell lines, two derived from lung carcinomas and one derived from a colon carcinoma, transfer the same or closely related human genes. If these transforming genes are mediating the tumorigenic state of the human cells, then our results indicate that overlapping pathways leading to tumorigenesis may arise independently.

3T3 Cells↗
PubMed1981

The somatic replication of DNA methylation.

We have tested the hypothesis that DNA methylation patterns are replicated in the somatic cells of vertebrates. Using M-Hpa II, the modification enzyme from Haemophilus parainfluenzae which methylates the internal cytosine residues in the sequence 5'CCGG 3' GGCC, we methylated bacteriophage phi X174 RF DNA and the cloned chicken thymidine kinase (tk) gene in vitro and then introduced these DNAs and unmethylated controls into tk- cultured mouse cells by DNA-mediated transformation. Twenty-five cell generations later, the state of methylation of transferred DNA was examined by restriction endonuclease analysis and blot hybridization. We conclude that methylation at Hpa II sites is replicated by these cultured cells but not with 100% fidelity. We have also noted that methylation of the cloned chicken tk gene decreases its apparent transformation efficiency relative to unmethylated molecules.

Animals↗
PubMed1980

Isolation of the chicken thymidine kinase gene by plasmid rescue.

We have used the bacterial plasmid pBR322 as a vehicle to isolate genes coding for selectable markers from higher eukaryotes. In this way, we have obtained the chicken thymidine kinase (tk) gene as a 2.2-kilobase EcoRI/HindIII insert in BR322. The cloned gene transforms tk- animal cells with an efficiency equal to that of the cloned herpes simplex virus-1 tk gene.

Animals↗
PubMed1980

Study of the interaction of glyceraldehyde-3-phosphate dehydrogenase with DNA.

Glyceraldehyde-3-phosphate dehydrogenase binds to homologous and heterologous single-stranded but not double-stranded DNA. Binding to RNA, poly(A) and poly(dA-dT) has also been observed. Enzyme binding to these nucleic acids leads to the formation of an insoluble complex which can be sedimented at low speed. The interaction of glyceraldehyde-3-phosphate dehydrogenase with DNA is strongly inhibited by NAD and NADH but not by NADP. Adenine nucleotides, which inhibit the dehydrogenase activity by competing with NAD for its binding site (Yang, S.T. and Deal, W.C., Jr. (1969) Biochemistry 8, 2806--2813), also inhibit enzyme binding to DNA, whereas glyceraldehyde-3-phosphate and inorganic phosphate are non-inhibitory. These results suggest that DNA interacts through the NAD binding sites of glyceraldehyde-3-phosphate dehydrogenase. In accordance with this idea, it was found that DNA also binds to lactate dehydrogenase, an enzyme containing a similar dinucleotide binding domain, and that this binding is inhibited by NADH. A study of the base specificity of the DNA-glyceraldehyde-3-phosphate dehydrogenase interaction using dinucleoside monophosphates shows that inhibition of DNA binding by the dinucleotides requires the presence of a 3'-terminal adenosine and is greater when the 5'-terminus contains a pyrimidine instead of a purine. These results suggest that the dinucleotides bind at the NAD site of the dehydrogenase and that the enzyme would interact preferentially with PypA dinucleotides present in the nucleic acid.

Animals↗
PubMed1980

Histone H5 messenger RNA is polyadenylated.

In most known systems, histone mRNA lacks the poly(A) sequence at the 3' end of the molecule typical of most mRNAs. Furthermore, the synthesis of histones, unlike that of most proteins, is tightly coupled to DNA synthesis. Nevertheless, histone synthesis occurs in amphibian oocytes in the absence of DNA synthesis. Moreover, it has recently been found that in amphibian oocytes most of the histone mRNA is polyadenylated, and the polyadenylate is probably removed during maturation of the oocyte. Histone H5, an H1-like tissue-specific histone occurring only in nucleated erythrocytes, is also atypical in that it is synthesised in the absence of DNA synthesis during maturation of the red blood cells. We report here that H5 mRNA is polyadenylated.

Animals↗
PubMed1980

Genetic and physical linkage of exogenous sequences in transformed cells.

The calcium phosphate precipitation method of Graham and van der Eb (1973) is an efficent means of introducing DNA into culturaed animal cells. Cells which incorporate one selectable marker are also likely to incorporate one selectable marker are also likely to incorporate sequences from the carrier DNA. Both selected and unselected markers are found integrated in the high molecular weight nuclear DNA of the host. In the present study, we demonstrate that exogenously acquired sequences are gentically linked, segregating and amplifying coordinately, and that their flanking sequences derive primarily from the carrier species rather than the host species. Based on these results, we propose that, upon transformation, the host cell ligates incorporated DNA into a large concatameric structure which may at times be as large as 2000 kilobases. From blotting data alone we cannot determine whether this structure is chromosomal or extrachromosomal in location.

Animals↗
PubMed1980

Transformation of mammalian cells with an amplifiable dominant-acting gene.

We have transferred a mutant hamster gene coding for an altered dihydrofolate reductase to wild-type cultured mouse cells by using total genomic DNA from methotrexate-resistant Chinese hamster ovary A29 cells as donor. By demonstrating the presence of hamster gene sequences in transformants we have provided direct evidence for gene transfer. Transformants selected for increased resistance to methotrexate contain increased amounts of the newly transferred gene. We have used this mutant dhfr gene to introduce the Escherichia coli antibiotic resistance plasmid pBR322 into animal cells. Amplification of the dhfr sequences results in amplification of the pBR322 sequences as well. The use of this gene may allow the introduction and amplification of virtually any genetic element in various new cellular environments.

Animals↗
PubMed1977

Identification of the mammalian DNA-binding protein P8 as glyceraldehyde-3-phosphate dehydrogenase.

The DNA-binding protein P8 from transformed hamster fibroblasts (line NIL-1-hamster sarcoma virus) has been purified to homogeneity by DNA-cellulose and phosphocellulose chromatography. The molecular weight of dissociated P8 is 36000, the same as that reported for the subunits of glyceraldehyde-3-phosphate dehydrogenase, and the mobility of these proteins in polyacrylamide gels is identical. The amino acid composition of P8 is very similar to that of glyceraldehyde-3-phosphate dehydrogenase. When assayed for glyceraldehyde-3-phosphate dehydrogenase activity the P8 preparation had a specific activity of 54.6 units/mg, a value comparable to that of the crystalline enzyme from several sources. Furthermore, serum prepared against P8 crossreacts with glyceraldehyde-3-phosphate dehydrogenase from hamster muscle. These results show that P8 is glyceraldehyde-3-phosphate dehydrogenase. The interaction of P8 from transformed fibroblasts and glyceraldehyde-3-phosphate dehydrogenase from hamster and rabbit muscle with DNA has been studied using a Millipore filtration technique. These proteins have affinity for single-stranded DNA but not for double-stranded DNA.

Amino Acids↗