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Biomedical subjects

W S Hayward

Publications and source records attributed to W S Hayward.

At least 19 recordsLinked to original sources
PubMed1997

Minimal truncation of the c-myb gene product in rapid-onset B-cell lymphoma.

Oncogenic activation of c-myb by insertional mutagenesis has been implicated in rapid-onset B-cell lymphomas induced by the nonacute avian leukosis virus EU-8. In these tumors, proviruses are integrated either upstream of the c-myb coding region or within the first intron of c-myb. Tumors with either type of integration contained identical chimeric mRNAs in which the viral 5' splice site was juxtaposed to the 3' splice site of c-myb exon 2 and myb exon 1 was eliminated. Both classes of integrations generated truncated Myb proteins that were indistinguishable by Western analysis. In contrast to most other examples of c-myb activation, the truncation consisted of only 20 N-terminal amino acids and did not disrupt either the DNA binding domain near the N terminus or the negative regulatory domain near the C terminus of Myb. The significance of the 20-amino-acid Myb truncation to tumorigenesis was tested by infection of chicken embryos with retroviral vectors expressing different myb gene products. While virus expressing either wild-type c-myb or c-myb mutated at the N-terminal casein kinase II sites was only weakly oncogenic at 10 weeks, the minimally truncated myb virus induced a high incidence of rapid-onset tumors, including B-cell lymphomas, sarcomas, and adenocarcinomas.

Alternative Splicing↗
PubMed1997

Genetic determinant of rapid-onset B-cell lymphoma by avian leukosis virus.

Infection of 10 day-old chicken embryos with the recombinant avian leukosis virus (ALV) EU-8 induces a high incidence of rapid-onset B-cell lymphoma by insertional activation of the c-myb gene. LR-9, a related ALV with differences from EU-8 in the gag and pol genes, induces rapid-onset lymphoma at only a low incidence. To localize the viral determinant(s) responsible for this biologic difference, we constructed and tested a series of reciprocal chimeras between EU-8 and LR-9 ALVs. The ability to induce rapid-onset lymphoma efficiently was localized to a 925-nucleotide (nt) region of the EU-8 gag gene. Sequence analysis of the region revealed a 42-nt deletion in EU-8 relative to LR-9, as well as some single-nucleotide changes. A mutant virus, delta LR-9, constructed by deleting these 42 nt from LR-9, also induced rapid-onset lymphoma at a high frequency, confirming the biologic significance of this deletion. This deletion removed nt 735 to 776, which lies within a cis-acting RNA element that negatively regulates splicing (NRS). The deletion was shown to cause an increase in splicing efficiency, which may lead to increased production of a truncated myb gene product from an ALV-myb readthrough RNA.

Animals↗
PubMed1997

bic, a novel gene activated by proviral insertions in avian leukosis virus-induced lymphomas, is likely to function through its noncoding RNA.

The bic locus is a common retroviral integration site in avian leukosis virus (ALV)-induced B-cell lymphomas originally identified by infection of chickens with ALVs of two different subgroups (Clurman and Hayward, Mol. Cell. Biol. 9:2657-2664, 1989). Based on its frequent association with c-myc activation and its preferential activation in metastatic tumors, the bic locus is thought to harbor a gene that can collaborate with c-myc in lymphomagenesis and presumably plays a role in late stages of tumor progression. In the present study, we have cloned and characterized two novel genes, bdw and bic, at the bic locus. bdw encoded a putative novel protein of 345 amino acids. However, its expression did not appear to be altered in tumor tissues, suggesting that it is not involved in oncogenesis. The bic gene consisted of two exons and was expressed as two spliced and alternatively polyadenylated transcripts at low levels in lymphoid/hematopoietic tissues. In tumors harboring bic integrations, proviruses drove bic gene expression by promoter insertion, resulting in high levels of expression of a chimeric RNA containing bic exon 2. Interestingly, bic lacked an extensive open reading frame, implying that it may function through its RNA. Computer analysis of RNA from small exon 2 of bic predicted extensive double-stranded structures, including a highly ordered RNA duplex between nucleotides 316 and 461. The possible role of bic in cell growth and differentiation is discussed in view of the emerging evidence that untranslated RNAs play a role in growth control.

Amino Acid Sequence↗
PubMed1997

Rapid induction of B-cell lymphomas by avian leukosis virus.

Avian leukosis viruses (ALVs) that induce rapid B-cell lymphomas integrate into the c-myb gene and produce an ALV-myb read-through RNA, which is spliced to produce a truncated Myb protein. The genetic determinants of such recombinant ALVs have been mapped to a 42-nt deletion within the gag gene. This deletion increases splicing efficiency since it is located within a negative regulator of splicing. We propose that the deletion leads to increased production of Myb protein by increasing splicing of an ALV-myb pre-mRNA.

Animals↗
PubMed1990

The block to transcription elongation is promoter dependent in normal and Burkitt's lymphoma c-myc alleles.

Aberrant c-myc expression patterns occur in human Burkitt's lymphoma cells, which consistently exhibit c-myc chromosomal translocations, mutations within and flanking the translocated allele, a loss of the block to transcription elongation in exon 1, and a promoter shift to use of the upstream P1 promoter. To define the mechanism responsible for the loss of transcription elongation blockage and resulting c-myc deregulation in Burkitt's lymphoma, we analyzed transcription patterns after transfer of normal and Burkitt's lymphoma c-myc alleles into murine cells and Xenopus oocyte germinal vesicles. We have determined that although the mutations within and surrounding several Burkitt's lymphoma c-myc alleles are not sufficient, in themselves, to abrogate the transcription elongation block, transcription initiation from the P2 promoter may be necessary to obtain the block to transcription elongation. To test directly the role of c-myc promoters in programming transcription elongation blockage, we analyzed transcription patterns from in vitro mutagenized c-myc genes containing deletions of either the P1 or P2 promoter. These data confirm that P1-initiated c-myc transcripts do not terminate at discrete sites near the 3' end of exon 1, whereas P2-initiated transcripts either terminate or read through the transcription block signals. Therefore, overexpression and/or constitutive expression from the c-myc P1 promoter may contribute to increased readthrough transcription in Burkitt's lymphoma cells and, hence, to aberrant expression patterns or levels of c-myc steady-state transcripts. In addition, the ability of normal cells to modulate c-myc P2-initiated transcription to either read through or to block elongation provides a fine control mechanism over c-myc steady-state RNA levels.

Alleles↗
PubMed1989

Structural organization of upstream exons and distribution of transcription start sites in the chicken c-myb gene.

We mapped and sequenced three upstream exons of the chicken c-myb gene and the regions flanking the first coding exon. We found multiple potential binding sites for transcription factors in the 5'-noncoding region, a T-rich stretch of 78 base pairs (bp) (68% T) in the first intron, and four fairly long open reading frames in the antisense direction of the first coding exon and its flanking regions. Three major transcription start sites, contained within a single 11-bp region, were identified by S1 nuclease analysis and primer extension. A sequence comparison of the avian and murine c-myb genes revealed a highly conserved sequence of 124 bp in the 5'-noncoding region. Its location between the putative transcription factor binding sites and the major transcription start sites suggests that it may play an important regulatory role in c-myb expression.

Animals↗
PubMed1989

Multiple proto-oncogene activations in avian leukosis virus-induced lymphomas: evidence for stage-specific events.

We have examined avian leukosis virus-induced B-cell lymphomas for multiple, stage-specific oncogene activations. Three targets for viral integration were identified: c-myb, c-myc, and a newly identified locus termed c-bic. The c-myb and c-myc genes were associated with different lymphoma phenotypes. The c-bic locus was a target for integration in one class of lymphomas, usually in conjunction with c-myc activation. The data indicate that c-myc and c-bic may act synergistically during lymphomagenesis and that c-bic is involved in late stages of tumor progression.

Animals↗
PubMed1988

Rapid induction of B-cell lymphomas: insertional activation of c-myb by avian leukosis virus.

EU-8 is a recombinant avian leukosis virus (ALV) constructed in vitro, which carries long terminal repeats and gag and pol genes from ring-necked pheasant virus and the env gene from UR2AV. Unlike either parent virus, when injected into 10-day-old chicken embryos, EU-8 induces a high incidence of clonally arising B-cell lymphomas within an unusually short latent period, often causing death within 5 to 7 weeks after infection. These tumors differ from the classic lymphoid leukosis induced by ALV in several respects, both biologically and at the molecular level. Most notably, in all of the EU-8-induced tumors examined, the provirus was integrated in the c-myb locus, and in no tumors were c-myc integrations found. Most of the proviral integrations were downstream of the initiation codon of c-myb and thus presumably resulted in some truncation of the c-myb gene product, although not to the same extent as has been found in other cases of c-myb activation. In addition, several of the proviruses were integrated well upstream of the c-myb coding region. This is the first report of ALV interaction with the c-myb proto-oncogene and the first report of c-myb activation resulting in tumors of lymphoid rather than myeloid origin, suggesting that the target cell specificity of transformation by the myb gene is not as restricted as previously believed.

Animals↗
PubMed1988

Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas.

We have determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myc genes contained missense mutations. This strongly supports the notion that the c-myc proto-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.

Animals↗
PubMed1987

Genetic determinants of neoplastic diseases induced by a subgroup F avian leukosis virus.

Two subgroup F avian leukosis viruses, ring-necked pheasant virus (RPV) and RAV-61, were previously shown to induce a high incidence of a fatal proliferative disorder in the lungs of infected chickens. These lung lesions, termed angiosarcomas, appear rapidly (4 to 5 weeks after infection), show no evidence of proto-oncogene activation by proviral integration, and are not induced by avian leukosis viruses belonging to other subgroups. To identify the viral sequences responsible for induction of these tumors, we constructed recombinant viruses by exchanging genomic segments of molecularly cloned RPV with those of a subgroup A leukosis virus, UR2AV. The ability to induce rapid lung tumors segregated only with the env sequences of RPV; the long terminal repeat of RPV was not required. However, recombinants carrying both env and long terminal repeat sequences of RPV induced lung tumors with a shorter latency. In several cases, recombinant viruses exhibited pathogenic properties differing from those of either parental virus. Recombinants carrying the gag-pol region of RPV and the env gene of UR2AV induced a high incidence of a muscle lesion termed infiltrative intramuscular fibromatosis. One recombinant, EU-8, which carries the gag-pol and LTR sequences of RPV, and the env gene of UR2AV, induced lymphoid leukosis after an unusually short latent period. The median time of death from lymphoid leukosis was 6 to 7 weeks after infection with EU-8 compared with approximately 5 months for UR2AV.

Animals↗
PubMed1987

5' long terminal repeats of myc-associated proviruses appear structurally intact but are functionally impaired in tumors induced by avian leukosis viruses.

B-cell lymphomas induced in chickens infected with avian leukosis viruses are characterized by integration of the virus within the cellular myc locus and alteration of c-myc expression. Although avian leukosis viruses are intact, replication-competent retroviruses, the structures of many myc-associated proviruses are altered by deletions, raising the possibility that proviral defectiveness plays an essential role in oncogenesis. We found that all myc-associated proviruses in 21 independent tumors had deletions, which were confined to the viral genome and did not extend into adjacent cellular sequences. Deletions were not random but, in at least 85% of the myc-associated proviruses, involved a region near the 5' end of the proviral genome where elements implicated in control of viral gene expression have been localized. A second class of deletions involved sequences in the 3' half of the viral genome and included the splice acceptor site used in generating viral env mRNA. Both the 5' and 3' long terminal repeats of myc-associated proviruses appeared to be structurally intact in most tumors, although the 5' long terminal repeats were not involved in expression of either U5-myc transcripts or detectable steady-state viral RNAs. A complex array of repeated sequence elements surrounded the junctions of the internal deletions in two myc-associated proviruses. The organization of the deleted proviruses was similar to that of deleted unintegrated viral molecules, consistent with a model in which deletions occurred prior to integration.

Avian Leukosis Virus↗
PubMed1985

Germ-line chromosomal localization of genes in chromosome 11p linkage: parathyroid hormone, beta-globin, c-Ha-ras-1, and insulin.

The chromosomal localization of genes for parathyroid hormone (PTH), beta-globin cluster, c-Ha-ras-1, and insulin, all of which have previously been assigned to the short arm of chromosome 11, generated considerable interest because of their association with development of disease states. Furthermore, the availability of recombination data from family studies made the determination of their physical location on the chromosome necessary. Several investigators have attempted this; however, controversy has arisen concerning the location of beta-globin, insulin, and c-Ha-ras-1 genes. Thus, while the results of some investigators suggested that all three genes are situated in the 11p15 region, data of other investigators placed the beta-globin and insulin genes close to the centromere and c-Ha-ras-1 in a more proximal region than 11p15. The subchromosomal position of the PTH gene remains to be determined. We have performed in situ hybridization of meiotic pachytene bivalents with 3H-labeled cloned genomic probes of PTH, beta-globin, and insulin genes and find their germ-line positions to be the following: PTH at 11p11.21, beta-globin at 11p11.22, and insulin at 11p14.1. These data, when considered with our recent germ-line assignment of the c-Ha-ras-1 gene to 11p14.1, indicate the following relative order on 11p: cen-PTH-beta-globin-c-Ha-ras-insulin or cen-PTH-beta-globin-insulin-c-Ha-ras. The former order is consistent with genetic evidence from linkage analysis of DNA polymorphisms adjacent to these genes segregating in families.

Chromosome Mapping↗
PubMed1984

Nucleotide sequence 5' of the chicken c-myc coding region: localization of a noncoding exon that is absent from myc transcripts in most avian leukosis virus-induced lymphomas.

We have determined the nucleotide sequence of the 2.2-kilobase-pair region upstream of the chicken c-myc coding exons. Using RNA blot analysis, we have localized a noncoding exon to a region that is separated from the c-myc coding sequences by an intron of 700-800 base pairs. In most avian leukosis virus-induced lymphomas proviral integration has occurred within, or downstream of, the first exon, thus presumably displacing the regulatory sequences that normally control c-myc expression. More than 70% of the integration sites were clustered in a 250-base-pair region in the first intron, immediately preceding the coding sequences. Sequences from the upstream noncoding exon were absent from the myc transcripts in these lymphomas; RNA transcripts from the normal c-myc allele were not expressed at detectable levels.

Animals↗
PubMed1984

Activation of a translocated c-myc gene: role of structural alterations in the upstream region.

The translocated c-myc gene in AW-Ramos, a Burkitt lymphoma cell line carrying the 8;14 translocation, is expressed at 2- to 5-fold higher levels than c-myc in lymphoblastoid cell lines. The translocation event has joined c-myc to the IgM switch region. As a consequence, a recently identified immunoglobulin transcriptional enhancer element is not linked to the translocated c-myc gene. Chromosomal recombination occurs approximately equal to 340 nucleotides upstream of the c-myc 5' cap site, leaving all three c-myc exons intact. The nucleotide sequences of the two coding exons in the translocated c-myc gene are identical to those of the normal c-myc gene. Nucleotide sequence analyses of the first, noncoding c-myc exon and of the region between this exon and the chromosomal recombination point reveal two single-base differences from normal c-myc. Our data indicate that altered expression rather than an altered gene product is responsible for c-myc activation in AW-Ramos cells and that this is a result of either loss of regulatory sequences located greater than 340 nucleotides upstream of c-myc or disruption of normal c-myc regulation by one or both base substitutions. Alternatively, unidentified enhancer-like sequences in the Ig locus may alter the expression of c-myc.

Base Sequence↗
PubMed1984

Mechanisms of oncogenesis by subgroup F avian leukosis viruses.

Subgroup F avian leukosis viruses, such as RAV-61 and ring-necked pheasant virus, are recombinants between exogenous chicken retroviruses and endogenous pheasant viruses and contain new envelope (env) genes. Chickens infected as 10-day-old embryos with subgroup F viruses develop fibrosarcomas, nephroblastomas, osteopetrosis, B-cell lymphomas, and a high incidence of a proliferative disorder involving the lung. Fibrosarcomas, nephroblastomas, and lymphomas appear after long latent periods (3 to 12 months). They contain discrete virus-cell junction fragments and are therefore clonal outgrowths of a single infected cell. Two ring-necked pheasant virus-induced B-cell lymphomas and an adenocarcinoma of the abdomen contained proviruses integrated at the c-myc locus and elevated levels of myc mRNA. At least four of the fibrosarcomas appeared to contain proviruses integrated at a common site, suggesting that a specific cellular gene may be involved in these tumors. The host gene has not been identified, however; 16 different oncogene probes failed to hybridize to fibrosarcoma junction fragments. In contrast to these neoplasms, lung lesions appeared rapidly (4 to 5 weeks), showed no evidence of clonality, and lacked long terminal repeat-initiated transcripts other than viral 35S and 21S mRNA. We conclude, therefore, that subgroup F retroviruses induce the proliferative disorder of the lung by a different mechanism.

Animals↗
PubMed1984

Localization of the cellular oncogenes ABL, SIS, and FES on human germ-line chromosomes.

The human germ-line positions of the oncogenes ABL, SIS, and FES, the cellular counterparts of the v-onc genes of Abelson murine leukemia virus, simian sarcoma virus, and feline sarcoma virus, respectively, have been determined by in situ molecular hybridization of 3H-labeled v-onc gene probes to meiotic pachytene chromosomes. The position of ABL at 9q34.1 corresponds to the breakpoint in chromosome 9 in the translocation that gives rise to the Philadelphia chromosome, t(9;22) (q34; q11); the position of SIS at 22q13.1 is distal to the breakpoint in this chromosome. FES at 15q26.1 is also distal to the breakpoint in chromosome 15 in the translocation commonly seen in acute promyelocytic leukemia, t(15;17) (q24;q22).

Abelson murine leukemia virus↗
PubMed1983

Localization of c-ras oncogene family on human germ-line chromosomes.

The c-ras family is a set of c-onc genes that are highly conserved in vertebrates. The genes in this family are homologous to the transforming genes of Harvey and Kirsten murine sarcoma viruses (v-Ha-ras and v-Ki-ras, respectively). Using an in situ molecular hybridization method, we detected three sites on the human pachytene chromosomes that exhibited significant hybridization to v-Ki-ras and v-Ha-ras probes. These were chromomere positions that corresponded to bands 11p14.1, 12p12.1, and 12q24.2 of somatic chromosomes. The relationship between these chromosomal sites and previously defined members of the human c-ras gene family is discussed. These chromosomal sites are known to be involved in specific chromosome changes in a variety of tumors and in several congenital disorders that predispose to neoplastic disease.

Autoradiography↗