Professor Rosalind Smith

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.

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 G 1 = 1h, S = 7h, and G 2 + M = 2-5h, and those of the fourth cell cycle as G 1 = 2h, S = 7h, and G 2 + 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.

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.

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 103M(r) to 31.5 x 103M(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.