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G M Cooper

Publications and source records attributed to G M Cooper.

At least 109 records · Page 6Linked to original sources
PubMed1988

Identification of the protein product of the c-mos proto-oncogene in mouse testes.

The mouse c-mos proto-oncogene RNA is expressed primarily in mouse gonadal tissues and embryos. Until now, the c-mos protein has not been identified. Utilizing two different site-directed affinity purified anti-peptide antibodies, we have identified a 43 kDa c-mos protein in mouse testes and in germ cell preparations derived from testes. This 43 kDa testicular protein was found to be structurally related to a bacterially expressed c-mos protein by peptide mapping. Immunoblots of whole mouse sections were employed to establish that the c-mos protein is expressed primarily in the testes.

Animals↗
PubMed1987

Expression of c-mos RNA in germ cells of male and female mice.

We have investigated the cell types in mouse testis and ovary in which the c-mos protooncogene is normally transcribed. Blot hybridization analysis of electrophoretically fractionated RNAs from testes of mice with defects in germ-cell development and from prepubertal and adult mice indicated that c-mos was transcribed during male germ-cell development. Analysis of purified populations of spermatogenic cell types detected c-mos RNA in the earliest haploid postmeiotic germ cell, the round spermatid, indicating that c-mos was expressed transiently during spermatogenesis. c-mos RNA was detected by blot hybridization in the ovaries of prepubertal mice and decreased in relative concentration following gonadotropin-stimulated proliferation of granulosa cells. These results suggested that c-mos was transcribed in oocytes and were confirmed by detection of high levels of c-mos RNA in isolated grown oocytes. Thus, c-mos is expressed in both male and female germ cells, suggesting possible roles for this protooncogene in meiosis, germ-cell development, fertilization, and early embryogenesis.

Age Factors↗
PubMed1987

Activation of human raf transforming genes by deletion of normal amino-terminal coding sequences.

Three activated cellular raf genes have been detected by transfection of NIH 3T3 cells with human tumor DNAs. Blot hybridization analysis indicated that all three transforming raf genes had recombined with non-raf sequences in the vicinity of raf exon 7-intron 7, resulting in the deletion of about 40% of the normal coding sequence from the raf amino terminus. By cloning sequences upstream of the truncated raf loci we have shown that the rearrangements involve the fusion of three different 5' non-raf human sequences to the human raf gene. No rearrangements could be detected in the raf loci of the three original human tumor DNAs, suggesting that the raf genes were activated by DNA rearrangements occurring during transfection. Significant overexpression of raf mRNA was not evident in two of the three transformant lines, indicating that raf overexpression is not necessary and 5' truncation alone may be sufficient to activate the transforming potential of cellular raf genes.

Animals↗
PubMed1987

ret transforming gene encodes a fusion protein homologous to tyrosine kinases.

The ret transforming gene was activated by recombination between two unlinked segments of human DNA, most likely during transfection of NIH 3T3 cells. To further define this transforming gene, we isolated and sequenced ret cDNA clones. The nucleotide sequence indicates that the active ret transforming gene encodes a fusion protein with a carboxy-terminal domain which is 40 to 50% homologous to members of the tyrosine kinase gene family. This tyrosine kinase domain is preceded by a hydrophobic sequence characteristic of a transmembrane domain. Transcription of the ret tyrosine kinase sequence was detected in the SK-N-SH neuroblastoma, HL-60 promyelocytic leukemia, and THP-1 monocytic leukemia cell lines, but not in 25 other human tumor cell lines surveyed. The ret tyrosine kinase may thus represent a cell surface receptor which is expressed in a restricted range of human cells.

Amino Acid Sequence↗
PubMed1987

Structure/function analysis of ras using random mutagenesis coupled with functional screening assays.

We review the use of functional assays for the ras protein, p21, that have allowed us to screen for mutant ras genes encoding proteins defective in either interactions with guanine nucleotides or transforming activity. GTP binding and GTP-dependent autokinase activities were assayed directly on lysed bacterial colonies expressing p21. Mutants encoding ras proteins deficient in these activities were isolated after randomly mutagenizing a v-rasH expression vector. Transformation defective mutants were isolated by randomly mutagenizing a v-rasH retroviral shuttle vector. NIH cells were then infected with a stock of nonreplicating mutagenized retroviruses and nontransformed infected colonies were isolated. The mutant ras genes were then rescued from these cells for analysis. Characterization of these mutants defines domains of p21 involved in both biochemical and biological activities and addresses the role of guanine nucleotide binding in p21 function.

Animals↗
PubMed1987

Independently activated dbl oncogenes exhibit similar yet distinct structural alterations.

The dbl oncogene was initially isolated following transfection of NIH3T3 cells with DNA of a human diffuse B cell lymphoma. Its transcribed sequences were shown to be distributed over a 30-kb span within a molecularly cloned 45-kb segment of human DNA which contained the transforming gene. By restriction mapping, its transcribed region corresponded to that of its normal allele, except at the 5' end where a rearrangement involved transcribed dbl oncogene sequences from another locus. An independent isolate of a dbl-related transforming gene was obtained following transfection of NIH3T3 cells with DNA of a human nodular poorly differentiated lymphoma (NPDL). Physical mapping indicated that this transforming gene, designated NPDL-dbl, shared considerable homology with the dbl oncogene, but differed at both 5' and 3' termini. Its point of divergence from the normal allele at the 5' end was at least 10 kb upstream from that of the dbl oncogene. The oncogenes each expressed truncated transcripts compared to the 5.3-kb normal transcript. The dbl and NPDL-dbl oncogene translational products of 66 and 76 kDa, respectively, were consistent with their corresponding major 2.8- and 3.5-kb transcripts. It was not possible to detect evidence of the 5' structural rearrangements associated with these oncogenes in either of the original tumors. Thus, if these rearrangements were critical to their activation, they occurred in the process of gene transfer or in vivo in only a minority of tumor cells.

Animals↗
PubMed1987

Cellular oncogenes and cancer.

Human oncogenes have been identified either by the ability of normal or tumor DNAs to induce transformation of cells in culture or as the targets of chromosome translocations or DNA amplification in neoplasms. By the combination of these approaches, approximately 40 different genes have been implicated as potential contributors to the development of human neoplasms. The proteins which are encoded by these potential human oncogenes include plasma membrane proteins with tyrosine kinase activity, plasma membrane guanine nucleotide binding proteins, cytoplasmic proteins with serine/threonine kinase activity and nuclear proteins. In many tumors, more than 1 potential oncogene has been activated, suggesting that multiple genes may contribute to neoplasm pathogenesis. I will discuss the identification of these genes, their modes of activation, the diversity of their protein products and their potential roles in both neoplastic and normal cells.

Animals↗
PubMed1986

Biological and biochemical properties of human rasH genes mutated at codon 61.

Using site-directed mutagenesis, we have introduced mutations encoding 17 different amino acids at codon 61 of the human rasH gene. Fifteen of these substitutions increased rasH transforming activity. The remaining two mutants, encoding proline and glutamic acid, displayed transforming activities similar to the normal gene. Overall, these mutants vary over 1000-fold in transforming potency. Increased levels of p21 expression were required for transformation by weakly transforming mutants. The mutant proteins were unaltered in guanine nucleotide binding properties. However, all 17 different mutant proteins displayed equivalently reduced rates of GTP hydrolysis, 8- to 10-fold lower than the normal protein. There was no quantitative correlation between reduction in GTPase activity and transformation, indicating that reduced GTP hydrolysis is not sufficient to activate ras transforming potential.

Amino Acid Sequence↗
PubMed1986

Isolation of ras GTP-binding mutants using an in situ colony-binding assay.

We have developed a strategy to isolate mutant ras genes encoding proteins defective in GTP binding. Random in vitro mutagenesis of a v-Harvey (Ha)-ras expression vector was followed by an in situ GTP-binding assay on lysed bacterial colonies. Single amino acid substitutions at ras codon 83, 119, or 144 decreased the affinity of p21 for GTP by a factor of 25-100 primarily as a consequence of increased rates of dissociation of GTP from p21. Nevertheless, these mutant genes induced transformation of NIH 3T3 cells with efficiencies comparable to wild-type v-Ha-ras. In transformed cells, mutant p21s were phosphorylated to a degree similar to that of wild-type v-Ha-ras p21, suggesting that a decrease in affinity by a factor of 100 did not prevent the mutant ras protein from binding GTP in vivo. These results are discussed with respect to the role of GTP in the regulation of p21 function.

Amino Acid Sequence↗
PubMed1986

Monoclonal antibody specific for an activated RAS protein.

Activated RAS transforming genes that encode proteins (p21s) with amino acid substitutions at positions 12, 13, or 61 have been detected in 10-20% of human neoplasms. This report describes a monoclonal antibody (DWP) raised against a synthetic peptide corresponding to amino acids 5-16 of a mutated RAS gene encoding Val instead of Gly at position 12. DWP reacted in competition assays with peptides containing Val or Cys at position 12, but did not react with peptides containing Gly, Arg, Ser, Ala, Asp, or Glu at position 12. Immunoblot analysis of transformed NIH cells and human carcinoma cell lines showed that DWP reacts specifically with activated RAS proteins containing Val at position 12 and not with normal p21s or p21s activated by other amino acid substitutions at positions 12 and 61. Immunohistochemical studies showed that DWP-labeled transformed NIH cells and human carcinoma cells contained p21s with either Val or Cys at position 12 but not normal or other activated p21s. In contrast to the specificity seen with human carcinoma cell lines, analysis of formalin-fixed, primary carcinoma specimens indicated that positive immunoperoxidase staining with DWP did not necessarily correlate with immunoblot and transfection assays for the presence of activated RAS proteins. Immunohistochemical studies did show, however, that DWP preferentially binds human carcinoma cells.

Amino Acid Sequence↗
PubMed1986

Haemodynamic disturbances during anaesthesia in a patient receiving calcium channel blockers.

Haemodynamic changes (supraventricular tachycardia, decreases in arterial pressure) were observed during laryngoscopy and intubation of the trachea in a patient receiving nifedipine and verapamil. Before the induced stresses of laryngoscopy and tracheal intubation, these drugs had controlled the patient's arterial pressure and heart rate satisfactorily, and possible reasons why this was not so at the commencement of anaesthesia are discussed.

Anesthesia, General↗
PubMed1986

Comparison of isoflurane and halothane in outpatient paediatric dental anaesthesia.

Isoflurane was compared with halothane as the sole supplement to anaesthesia with nitrous oxide and oxygen for outpatient dental extractions in 80 children. Induction and maintenance of anaesthesia were satisfactory with both agents, although there was a higher incidence of coughing, salivation and laryngospasm in the group receiving isoflurane. However, in contrast to predictions from the physical properties of isoflurane and halothane, immediate recovery was significantly slower in patients who had received isoflurane. Recovery was complicated by coughing in a significant number of patients in the isoflurane group. The incidence of reported complications during later recovery was similar with both agents, apart from the complaint of non-specific postoperative aches in a significant number of patients to whom isoflurane was administered.

Anesthesia Recovery Period↗
PubMed1986

rasH mutants deficient in GTP binding.

Single amino acid substitutions were introduced into a region of the rasH protein (residues 116, 117, and 119) homologous to a variety of diverse GTP-binding proteins. Each of the mutant p21 proteins displayed a significant reduction (10- to 5,000-fold) in GTP binding affinity. Activated rasH proteins deficient in GTP binding were unaltered in their ability to morphologically transform NIH 3T3 cells.

Amino Acid Sequence↗
PubMed1985

The isolation and characterization of the Blym-1 transforming gene.

There are a number of similarities between chicken bursal lymphomas and human Burkitt's lymphomas. Both lymphomas are associated with viral infection, by LLV in bursal lymphomas and Epstein-Barr virus in Burkitt's lymphoma. Avian lymphoid leukosis virus integration is associated with enhanced c-myc expression, while the role EBV plays in tumorigenesis remains unclear. In Burkitt's lymphoma, however, c-myc activation does occur as a result of specific chromosomal translocations involving the human c-myc locus. Furthermore, the activated transforming genes detected by transfection of both bursal lymphoma and Burkitt's lymphoma DNAs are homologous members of the Blym family of genes. These similarities between chicken and human lymphomas provide evidence that viral involvement and oncogene activation are significant in tumor development and suggest they are involved in the multi-step progression to the neoplastic phenotype. The function of the Blym genes remains to be determined. Although the chicken and human Blym genes are only distantly related, they have maintained their homology to the amino-terminal regions of transferrins. This fact may reflect some functional constraint on the evolution of these genes. It is therefore possible that transforming genes such as Blym may function via a transferrin-related mechanism.

Animals↗
PubMed1985

Activation of a novel human transforming gene, ret, by DNA rearrangement.

A novel transforming gene was detected by transfection of NIH 3T3 cells with human lymphoma DNA. The tumor DNA induced a single focus in primary transfections, whereas DNAs of transformed NIH cells induced transformation with high efficiencies in secondary and tertiary assays. Molecular clones spanning about 37 kb of human sequence were isolated from tertiary transformant DNA. Blot hybridization indicated that the transforming gene consisted of two segments that were unlinked in both normal human and primary lymphoma DNAs. The two segments of human DNA were cotranscribed in transformed NIH cells but not in any human cells examined. The transforming gene thus appeared to be activated by recombination between two unlinked human DNA segments, possibly by cointegration during transfection.

Animals↗