Professor Rosalind Smith

Unintegrated MAV-2(O) DNA was isolated from infected chicken embryo fibroblasts and inserted into the lambda bacteriophage vector lambda gtWES lambda B. Three x 10(6) bacteriophage plaques were screened, yielding a total of seven clones, six of which contained DNA representing the complete MAV-2(O) genome. Viral DNA was isolated from four of the clones and was used to transfect chicken embryo fibroblasts. All four clones produced virus as monitored by reverse transcriptase assay. When the four cloned viruses were inoculated into 10-day-old embryos, all hatched chickens developed osteopetrosis. One clone, lambda 9, induced osteopetrosis at a rate of onset and severity identical to that induced by the MAV-2(O) parental stock. This clone was selected for further study. To facilitate restriction mapping, the viral DNA from lambda 9 was subcloned into plasmid vector pUC 12 to construct a plasmid called p9. Cleavage of p9 DNA with single and multiple restriction endonucleases and hybridization with gene-specific probes identified the restriction fragments obtained. A comprehensive restriction map of cloned MAV-2(O) was generated and is compared with published maps and sequences of other avian retroviruses.

To determine whether c-fos is involved in avian leukosis virus-induced nephroblastoma, 28 tumors from chickens were analyzed for novel fos fragments. DNA from 1 of 16 clonal outgrowths (in chicken 6561) contained novel fos-related EcoRI and KpnI fragments which hybridized to both v-fos and viral probes. Oncogenicity tests using filtered 6561 tumor cell homogenates did not reveal a tumor-inducing transduction of c-fos. We conclude that c-fos is only an occasional target for proviral insertions or new transductions in avian leukosis virus-induced nephroblastoma. The results also identify a polymorphism in c-fos in K28 chickens and demonstrate that unintegrated viral DNA is not a general characteristic of avian leukosis virus-induced nephroblastoma.

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.

Nucleolar organizer regions (NORs) have been identified by means of an argyrophilic technique (Ag-NOR) in routinely processed, formalin-fixed paraffin sections of breast lesions. This method, which is novel in histopathology, reveals NORs as black dots in the nuclei of cells, by virtue of the argyrophilia of NOR-associated proteins. The number of Ag-NORs has been thought to be related to cellular activation and has recently been applied to non-Hodgkin's lymphomas and melanocytic skin lesions. It was found in the present study that the total number of Ag-NORs in malignant breast lesions significantly exceeded those of normal breast and benign lesions. The number of clumps of Ag-NORs, however, were not useful discriminators. Neither numbers of total Ag-NORs nor of clumps of Ag-NORs correlate with mitotic counts and it may be that their numbers relate to ploidy. It is suggested that the Ag-NOR technique will find increasing application as an adjunct to diagnostic histopathology. Copyright © 1988, Wiley Blackwell. All rights reserved

We sequenced two recombinant DNA clones constituting a single provirus of the milk-transmitted mouse mammary tumor virus characteristic of BR6 mice. The complete provirus is 9,901 base pairs long, flanked by 6 base-pair duplications of cellular DNA at the site of integration. Five extensive blocks of open reading frame corresponding to the gag gene, the presumed protease, the pol and env genes, and the open reading frame orf within the long terminal repeat of the provirus were readily discernible. Translation of gag, protease, and pol involved three different translational reading frames to produce the three overlapping polyprotein precursors Pr77, Pr110, and Pr160 found in virus-infected cells. Synthesis of the reverse transcriptase and endonuclease therefore required two separate frameshifts to suppress the termination codons at the ends of the Pr77 and Pr110 domains. Direct evidence is presented for translational readthrough of both stop codons in an in vitro protein synthesis system.

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 protooncogene 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.

The third (4-cell) and fourth (8-cell) cell cycles of early mouse development have been analysed in populations of blastomeres synchronized to the preceding cleavage division. DNA content was measured microdensitometrically. The entry of blastomeres into these cell cycles showed considerable heterogeneity both within and between individual embryos. This heterogeneity was greater in the fourth than in the third cell cycle. The component phases of the third cell cycle were estimated as G1 = 1h, S = 7h, and G2 + M = 2-5h, and those of the fourth cell cycle as G1 = 2h, S = 7h, and G2 + M = 1-3h.

In the course of analyzing sites of proviral integration in tumors induced by mouse mammary tumor virus (MMTV), we have isolated recombinant DNA clones corresponding to the 5' and 3' ends of four endogenous MMTV proviruses present in BALB/c and BR6 mice. This has permitted the structural characterization of each locus by detailed restriction mapping and the preparation of DNA probes specific for the cellular sequences flanking each provirus. These probes have been used to trace the segregation patterns of the proviruses, designated Mtv-8, Mtv-9, Mtv-17, and Mtv-21, in a panel of inbred strains of laboratory mice and to map Mtv-17 and Mtv-21 to mouse chromosomes 4 and 8, respectively. The unambiguous resolution of these four proviruses on Southern blots has greatly facilitated the analysis of other endogenous MMTV proviruses in these inbred mice.

The effects of aphidicolin, a reversible inhibitor of DNA polymerase alpha, both on replication and on development of the mouse embryo from the 2- and 4-cell stages to the compacted late 8-cell stage have been assessed. The continuous presence of aphidicolin from G1 of the 4-cell stage resulted in inhibition of DNA replication and prevention of division from 4 to 8 cells, but was without effect on the timing or incidence of cell flattening, surface polarization and cytoplasmic polarization. The continuous presence of aphidicolin from G1 of the 2-cell stage resulted in inhibition of DNA replication, division, and polarization. Some slight intercellular flattening in a few embryos did occur. If addition of aphidicolin was delayed by 10 h to early in G2 of the 2-cell stage, further rounds of replication were blocked and some embryos failed to cleave to 4-cells. Nevertheless, almost all embryos showed evidence of flattening and polarization regardless of cell number. In contrast, if aphidicolin was added in G1 of th 2-cell stage and removed after 10h, the cells showed delayed DNA replication, little evidence of division, and no cell flattening or polarization. We conclude that DNA replication at the 2-cell stage may be essential for the components of compaction studied, but that DNA replication at the 4- and 8-cell stages is not.

Approximately 50% of tumors induced by mouse mammary tumor virus (MMTV) contain an acquired provirus within a limited region of chromosomal DNA, termed int-2. We have extended our previous characterization of this locus and have mapped provirus integration sites in 21 independent tumors. Although integration occurs at multiple sites, proviruses within int-2 are distributed into two oppositely oriented groups whose transcription is directed away from a central domain. Provirus insertion in int-2 is accompanied by expression of RNA derived, at least in part, from this central domain. Since the RNA is not detected in normal mammary tissue, we conclude that MMTV integration activates the expression of a cellular gene within int-2 and that this event may contribute to tumorigenesis. © 1984.

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 cells. 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.

We have prepared specific probes for unique-sequence cellular DNA adjacent to each of the newly integrated proviruses in tumors induced by mouse mammary tumor virus (MMTV). The use of such probes to screen a large number of independent mammary tumors in the BR6 strain of mouse has indicated that in at least 17 out of the 40 tumors examined so far, an MMTV provirus has integrated into a common chromosomal domain. A 10 kb Eco RI fragment of single copy DNA from this region has been isolated and partially characterized by restriction enzyme mapping. Of the proviruses located within this fragment in different tumors, all but one are complete, in the same orientation, and clustered within about 3 kb of cellular DNA. These findings are consistent with an insertional mutagenesis model for tumorigenesis by MMTV, in which the integration of a provirus in a particular region of cellular DNA may activate a neighboring oncogene. The region we describe here appears to be different from that reported for mammary tumors in the C3H strain of mouse. © 1983.

In vitro protein synthesis and DNA sequence analysis indicate that mouse mammary tumor virus differs from other well-characterized retroviruses in that the long terminal repeat region of the provirus has the capacity to encode proteins. Different exogenously transmitted mouse mammary tumor virus strains and endogenous proviral units conserved this open reading frame feature in the long terminal repeat despite a variation in nucleotide sequence. The proteins encoded by the different long terminal repeats were clearly related, but showed minor variations in size and tryptic peptide maps. In each case, the largest in vitro product had a molecular weight of about 36,000 to 37,000, suggesting that the open reading frame sequences must extend for approximately 1,000 nucleotides beginning at the extreme 5' end of the long terminal repeat. The fact that the reading frame was conserved among these viruses argues in favor of an in vivo function for the open reading frame protein.

Chicken embryo cells infected with partial transformation mutants of Rous sarcoma virus were tested for tumor-forming ability in chickens and in nude mice. Cells transformed by each of these partial transformation mutants display different combination of transformation parameters. They therefore present a potentially favorable system for analyzing which properties of transformed cells are necessary for tumor formation. We found that the relative tumorigenicity of the virus mutants was generally similar in chickens and in nude mice, except that certain temperature-conditional mutants appeared to be sensitive to the differences in body temperature of the two experimental animals. (The body temperature of nude mice is 4 to 5°C lower than that of chickens). Thus, the nude mouse appears to be a suitable system for testing the tumorigenicity of transformed chicken cells. Because mice are nonpermissive for Rous sarcoma virus infection and replication, it was possible to recover the transformed chicken cells from the tumors in this host and to determine what phenotypic changes they had undergone during tumor development. We also examined the relationship between various cellular properties of the virus-infected chicken cells in vitro and their tumorigenicity in nude mice. The combined results of these two studies indicated that anchorage independence and plasminogen activator production were highly correlated with the tumor-forming ability of these cells, whereas loss of fibronectin did not correlate with tumorigenicity. Furthermore, the inability of the least tumorigenic virus mutant to stimulate the phosphorylation of a 36,000-M(r) target of pp60(src) raises the possibility that the 36,000-M(r) protein plays a role in tumor formation.

The integrated proviral DNA of RNA tumour viruses is bounded by long terminal repeat (LTR) segments of several hundred base pairs which result from duplication of sequences from both the 5' and 3' ends of the viral genome RNA1-3. Recently, DNA sequence analysis of the LTRs of several retroviruses has indicated that this region of the provirus may contain regulatory functions for viral RNA transcription 4-8. In this connection, mouse mammary tumour virus (MuMTV) is of particular interest in that transcription of the viral RNA can be specifically modulated by glucocorticoid hormones, suggesting that the site of steroid action may be contained within the LTR9. Moreover, the LTR of MuMTV is unusually long, extending for about 1,300 base pairs. We now present data which suggest that, in contrast with other retroviruses, the MuMTV LTR includes approximately 1,000 nucleotides in an open reading frame, capable of encoding a series of polypeptides which are quite distinct from any of the known structural components of MuMTV virions. These proteins can be expressed in vitro from cloned, recombinant DNA in which the MuMTV LTR has been inserted into the ampicillin-resistance gene of a bacterial plasmid. Tryptic peptide mapping has confirmed that these products are identical to the analogous series of proteins identified by in vitro translation of polyadenylated fragments of MuMTV genome RNA10. © 1981 Nature Publishing Group.

int-2 was discovered as a proto-oncogene transcriptionally activated by MMTV proviral insertion during mammary tumorigenesis in the mouse. Sequence analysis showed int-2 to be a member of the fibroblast growth factor family of genes. In normal breast and most other adult mouse tissues, int-2 expression was not detected except for low levels in brain and testis. However, using in situ hybridization, expression was found at a number of sites during embryonic development, from day 7 until birth. An analysis of the int-2 transcripts found in embryonal carcinoma cells revealed six major classes of RNA initiating at three promoters and terminating at either of two polyadenylation sites. Despite the transcriptional complexities, all size classes of RNA encompass the same open reading frame. Using an SV40 early promoter to drive transcription of an int-2 cDNA in COS-1 cells, several proteins were observed. These were shown to be generated by initiation from either of two codons: One, a CUG, leads to a product which localizes extensively to the cell nucleus and partially to the secretory pathway. In contrast, initiation at a downstream AUG codon results in quantitative translocation across the endoplasmic reticulum and the accumulation of products ranging in size from 27.5 x 103 M(r) to 31.5 x 103 M(r) in organelles of the secretory pathway. These proteins represented glycosylated and non-glycosylated forms of the same primary product with or without the signal peptide removed. These findings suggest the potential for a dual role of int-2; an autocrine function acting at the cell nucleus, and a possible paracrine action through a secreted product.