J M Bishop

Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.

Six monoclonal antibodies have been isolated from mice immunized with synthetic peptide immunogens whose sequences are derived from that of the human c-myc gene product. Five of these antibodies precipitate p62c-myc from human cells, and three of these five also recognize the mouse c-myc gene product. None of the antibodies sees the chicken p110gag-myc protein. All six antibodies recognize immunoblotted p62c-myc. These reagents also provide the basis for an immunoblotting assay by which to quantitate p62c-myc in cells.

Neoplastic transformation by truncated alleles of human NOTCH1/TAN1 and NOTCH2.

The Notch genes of Drosophila melanogaster and vertebrates encode transmembrane receptors that help determine cell fate during development. Although ligands for Notch proteins have been identified, the signaling cascade downstream of the receptors remains poorly understood. In human acute lymphoblastic T-cell leukemia, a chromosomal translocation damages the NOTCH1 gene. The damage apparently gives rise to a constitutively activated version of NOTCH protein. Here we show that a truncated version of NOTCH1 protein resembling that found in the leukemic cells can transform rat kidney cells in vitro. The transformation required cooperation with the E1A oncogene of adenovirus. The transforming version of NOTCH protein was located in the nucleus. In contrast, neither wild-type NOTCH protein nor a form of the truncated protein permanently anchored to the plasma membrane produced transformation in vitro. We conclude that constitutive activation of NOTCH similar to that found in human leukemia can contribute to neoplastic transformation. Transformation may require that the NOTCH protein be translocated to the nucleus. These results sustain a current view of how Notch transduces a signal from the surface of the cell to the nucleus.

Detection of Avian Tumor Virus-Specific Nucleotide Sequences in Avian Cell DNAs

The effect of unlabeled cellular DNA upon the reassociation kinetics of labeled double-stranded DNA made by DNA polymerase from avian tumor viruses has been used to measure virus-specific nucleotide sequences in cells. Multiple copies of these sequences were found equally in normal chick cells, in chick cells transformed in culture, and in Rous tumor cells. Copies were also present equally in cells harboring chick helper factor and group-specific antigen, and in cells lacking those characteristics. Quail DNA also contains multiple copies of these sequences, but no copies were detected in HeLa-cell DNA or salmon-sperm DNA.

Neoplastic transformation by the human gene N-myc

Amplification and abundant expression of a gene known as N-myc are found frequently in advanced stages of human neuroblastoma and may play a role in the genesis of several malignant human tumors. Previous studies have shown that N-myc can cooperate with a mutant allele of the proto-oncogene c-Ha-ras to transform embryonic rat cells in culture. Here we show that N-myc can also act alone to elicit neoplastic growth of an established line of rat fibroblasts (Rat-1). We used recombinant DNA vectors to express either N-myc or its kindred gene c-myc in transfected cells. Both genes caused morphological transformation, anchorage-independent growth, and tumorigenicity. We noticed two variables that appeared to influence the ability to isolate cells transformed by N-myc and c-myc: the abundance in which the genes were expressed and biological selection to eliminate untransformed cells from the cultures. Our findings sustain the belief that N-myc is an authentic proto-oncogene, lend further credibility to the role of N-myc in the genesis of human tumors, and establish a convenient assay that can be used to explore further the properties of both N-myc and c-myc.

The src protein contains multiple domains for specific attachment to membranes.

The proteins encoded by the oncogene v-src and its cellular counterpart c-src (designated generically here as pp60src) are tightly associated with both plasma membranes and intracellular membranes. This association is due in part to the amino-terminal myristylation of pp60src, but several lines of evidence suggest that amino-terminal portions of the protein itself are also involved. We now report that pp60src contains at least three domains which, in conjunction with myristylation, are capable of mediating attachment to membranes and determining subcellular localization. We identified these domains by fusing various portions of pp60src to pyruvate kinase, which is normally a cytoplasmic protein. Amino acids 1 to 14 of pp60src are sufficient to mediate both myristylation and the attachment of pyruvate kinase to cytoplasmic granules. In contrast, amino acids 38 to 111 mediate association with the plasma membrane and perinuclear membranes, whereas amino acids 204 to 259 mediate association primarily with perinuclear membranes. We conclude that pp60src contains independent domains that target the protein to distinctive subcellular locations and thus may facilitate diverse biological functions of the protein.

RNA-directed DNA synthesis by virions of Rous sarcoma virus: Further characterization of the templates and the extent of their transcription☆

Abstract Four species of RNA found in Rous sarcoma virus have been tested for their roles as templates for RNA-directed DNA synthesis by detergent-activated virions of RSV: the high molecular-weight subunits of the 70S RNA complex, 4S and 5S RNAs contained in the 70S complex, and 7S RNA extracted from purified virions. All three forms of low molecular-weight RNA are at least partially transcribed, but the principal templates for DNA synthesis are the high molecular-weight subunits of 70S RNA. DNA synthesized by virions of RSV contains nucleotide sequences representative of most or all of the viral genome, but the majority of these sequences are present in very small proportions. These results further substantiate the occurrence of preferential transcription from a limited region of the viral genome as defined previously for double-stranded enzymatic product by measurement of reassociation kinetics. By contrast, single-stranded DNA synthesized by virions of RSV in the presence of actinomycin D is a complete and relatively uniform copy of the entire viral genome. The latter observation facilitates both the study of nucleic acid homologies among RNA tumor virus genomes and the execution of competition hybridizations to determine the portion of a viral genome present in various populations of cellular RNA. An example of each of these procedures is given.

The PVT gene frequently amplifies with MYC in tumor cells.

The line of human colon carcinoma cells known as COLO320-DM contains an amplified and abnormal allele of the proto-oncogene MYC (DMMYC). Exon 1 and most of intron 1 of MYC have been displaced from DMMYC by a rearrangement of DNA. The RNA transcribed from DMMYC is a chimera that begins with an ectopic sequence of 176 nucleotides and then continues with exons 2 and 3 of MYC. The template for the ectopic sequence represents exon 1 of a gene known as PVT, which lies 50 kilobase pairs downstream of MYC. We encountered three abnormal configurations of MYC and PVT in the cell lines analyzed here: (i) amplification of the genes, accompanied by insertion of exon 1 and an undetermined additional portion of PVT within intron 1 of MYC to create DMMYC; (ii) selective deletion of exon 1 of PVT from amplified DNA that contains downstream portions of PVT and an intact allele of MYC; and (iii) coamplification of MYC and exon 1 of PVT, but not of downstream portions of PVT. We conclude that part or all of PVT is frequently amplified with MYC and that intron 1 of PVT represents a preferred boundary for amplification affecting MYC.

Effects of translocations on transcription from PVT

We have previously described a transcription unit on human chromosome 8, designated as PVT, that is consistently disrupted by the minority forms of translocations [t(2;8) and t(8;22)] in Burkitts lymphoma. PVT begins 57 kilobase pairs downstream of the proto-oncogene MYC and is more than 200 kilobase pairs in length. In order to explore the pathogenic impact of translocations affecting PVT, we have characterized further the structure and transcription of the locus. In normal cells, PVT is transcribed into a variety of RNAs, the diversity of which remains unexplained. Alleles of PVT affected by translocations give rise to additional RNAs. These RNAs arise from a fusion of the first exon of PVT on chromosome 8 to the constant region of an immunoglobulin light chain on either chromosome 2 or chromosome 22. We have found no evidence that any of the normal or abnormal transcripts of PVT give rise to a protein. Our results suggest that the pathogenic effects of the variant translocations in Burkitts lymphoma are not executed by a gene situated in a vicinity of the chromosomal breakpoints. Instead, our data leave open the possibility that the effects of the translocations may be mediated by activation of the relatively distant MYC gene.

Organization of the endogenous proviruses of chickens: Implications for origin and expression

Abstract Eleven of the endogenous proviruses of white leghorn chickens have been mapped with restriction endonucleases and specific nucleic acid hybridization reagents. The restriction maps of these endogenous proviruses have been compared with restriction maps of avian sarcoma virus (ASV) and Rous-associated virus O (RAV-O), an endogenous virus which is spontaneously released by cells from certain lines of chickens. Endogenous proviruses have the same basic structure as proviruses acquired by exogenous infection; the gene order is the same in the provirus as in viral RNA, and the ends of the provirus form a characteristic direct repeat which contains sequences derived from both ends of viral RNA. The endogenous proviruses can thus be described “cell DNA-3′5′- gag-pol-env -3′5′-cell DNA,” where 3′ and 5′ denote sequences homologous to the 3′ and 5′ ends of viral RNA. The endogenous proviruses of chickens are more closely related to RAV-O than to ASV, based on restriction maps and on hybridization with reagents specific for the 3′ ends of RAV-O and ASV. However, all but two of the endogenous proviruses lack at least one of the two Sst I sites in RAV-O DNA (see the preceding paper) and can thus be distinguished from RAV-O by digestion with Sst I. One of the two exceptions, ev -2, is found in the DNA of line 7 2 and line 100 chickens and is genetically linked to the production of RAV-O. The only other provirus (which we call B) having both the Sst I sites in RAV-O DNA was seen only once in a line 100 sample. Of the nine remaining elements, six had large deletions. Three proviruses ( ev -4, ev -6, and an element we call A) are missing the left 3′5′ repeat and have sustained deletions extending varying distances into gag or pol . One of these, ev -6, is associated with the gs − chf + phenotype; the phenotype can be explained from the structure of the provirus. ev -3 has both terminal repeats intact but has sustained a deletion near the gag-pol boundary. This provirus is associated with the gs + chf + phenotype and the structure of the provirus could account for the peculiar RNA and protein associated with this phenotype. We have also found two elements which apparently consist of sequences present only in the 3′5′ terminal repeat unit, with no other associated virus specific sequences. Such structures might arise by homologous recombination between the terminal repeats of a normal provirus.

Isolation of duplicated human c-src genes located on chromosomes 1 and 20.

The oncogene (v-src) of Rous sarcoma virus apparently arose by transduction of the chicken gene known as c-src(chicken). We isolated DNA fragments representative of two src-related loci from recombinant DNA bacteriophage libraries of the human genome. One of these loci, c-src1(human), appeared to direct the synthesis of a 5-kilobase polyadenylated RNA that presumably encodes pp60c-src(human). Probes specific for the other locus, c-src2(human), did not hybridize to polyadenylated RNA prepared from a variety of human cell lines. Partial nucleotide sequence determinations of the loci demonstrated that c-src1(human) is highly related to chicken c-src and that c-src2(human) is slightly more divergent. The sequences imply that the final two coding exons of each human locus are identical in length to those of chicken c-src and that the location of an amber stop codon is unchanged in all three loci. c-src1(human) has been mapped to chromosome 20, and the second locus is located on chromosome 1. We conclude that c-src1(human) is the analog of c-src(chicken) and that the duplicated locus, c-src2(human), may also be expressed.