Showing papers by "Geoffrey M. Cooper published in 1987"

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

Abstract: 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.

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

Abstract: 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.

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

Abstract: 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.

Independently activated dbl oncogenes exhibit similar yet distinct structural alterations.

Abstract: 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 53-kb normal transcript The dbl and NPDL-dbl oncogene translational products of 66 and 76 kDa, respectively, were consistent with their corresponding major 28- and 35-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

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

Abstract: 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.