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The sequence of the transforming region of the Moloney murine sarcoma virus genome has been determined by using molecularly cloned viral DNA. This region, 3.6 to 5.8 kilobase pairs from the left end of the molecule, contains the entire cellular insertion (src) sequence as well as helper viral sequences including the large terminal repeat (LTR). On the viral RNA strand, a long (1224 bases) open reading frame commenced to the left of the src-helper virus junction and terminated at a point 58 nucleotides into helper viral sequences to the right of src. Possible promoter and acceptor splice signals were detected in helper viral sequences upstream from this open reading frame. On the antiviral RNA strand, several promoter-like sequences, including one within the src region itself, were identified. However, no open reading frame downstream from these promoters was detected in the antiviral RNA strand. The LTR was found to contain promoter-like sequences as well as LTR was found to contain promoter-like sequences as well as mRNA capping and polyadenylylation signals. In addition, it possessed an 11-base inverted terminal repeat at each end. Thus, the structure of the Moloney murine sarcoma virus genome with an LTR at each end resembles that of prokaryotic transposable elements.

A probe derived from the 3' region of the BCR gene (breakpoint cluster region gene) detects four distinct loci in the human genome. One of the loci corresponds to the complete BCR gene, whereas the others contain a 3' segment of the gene. After HindIII cleavage of human DNA, these four loci are detected as 23-, 19-, 13-, and 9-kilobase-pair fragments, designated BCR4, BCR3, BCR2, and BCR1, respectively, with BCR1 deriving from the original complete BCR gene. All four BCR loci segregate 100% concordantly with human chromosome 22 in a rodent-human somatic cell hybrid panel and are located at chromosome region 22q11.2 by chromosomal in situ hybridization. The BCR2 and BCR4 loci are amplified in leukemia cell line K562 cells, indicating that they fall within the amplification unit that includes immunoglobulin lambda light chain locus (IGL) and ABL locus on the K562 Philadelphia chromosome (Ph1); additionally, in chronic myelogenous leukemia-derived mouse-human hybrids retaining a Ph1 chromosome in the absence of the 9q+ and normal chromosome 22, BCR2 and BCR4 loci are retained, whereas the 3' region of BCR1 and the BCR3 locus are lost, indicating that BCR3 is distal to BCR1 on chromosome 22. Similarly, in mouse-human hybrids retaining a Ph1 chromosome derived from an acute lymphoblastic leukemia-in the absence of the 9q+ and 22, only BCR2 and BCR4 loci are retained, indicating that the breakpoint in this acute lymphoblastic leukemia, as in chronic myelogenous leukemia, is proximal to the BCR1 3' region, but distal to the IGLC locus and the BCR2 and BCR4 3' loci. Thus, the order of loci on chromosome 22 is centromere----BCR2, BCR4, and IGL----BCR1----BCR3----SIS, possibly eliminating BCR2 and BCR4 loci as candidate targets for juxtaposition to the ABL gene in the acute lymphoblastic leukemia Ph1 chromosome.

Chronic myelogenous leukemia (CML) is a clonal hematologic malignancy characterized by a reciprocal translocation between chromosomes 9 and 22 [t(9;22)] in greater than 90% of cases. This translocation results in a short chromosome 22, termed the Philadelphia (Ph1 or 22q-) chromosome. Recently, the cellular oncogenes abl and sis were mapped to human chromosomes 9 and 22, respectively. Moreover, abl was shown to be translocated from chromosome 9 to 22 and sis from chromosome 22 to 9 in CML patients with t(9;22). These findings raised the possibility that one or both of these oncogenes is activated and directly involved in the development of the disease. We analyzed expression of the abl and sis oncogenes in leukemic cells from CML patients with t(9;22). We found that sis is not expressed but that abl is transcribed into an 8-kilobase RNA. This abl RNA is also present in two leukemic cell lines (EM2 and K562), which were derived from CML patients and contain the t(9;22). This 8-kilobase RNA is not detected in normal cells, in other human leukemias without t(9;22), or in human cell lines that lack t(9;22). The consistent presence of this abl RNA transcript in CML with t(9;22) suggests that it is a consequence of abl translocation and that it plays a role in the development of this leukemia.

Ras (Ha-Ras, Ki-Ras, N-Ras) is implicated in the regulation of various cell functions such as gene expression and cell proliferation downstream from specific extracellular signals. Here, we partially purified a Ras-interacting protein with molecular mass of about 180 kDa (p180) from bovine brain membrane extract by glutathione S-transferase (GST)-Ha-Ras affinity column chromatography. This protein bound to the GTP gamma S (guanosine 5'-(3-O-thio)triphosphate, a nonhydrolyzable GTP analog).GST-Ha-Ras affinity column but not to those containing GDP.GST-Ha-Ras or GTP gamma S.GST-Ha-Ras with a mutation in the effector domain (Ha-RasA38). The amino acid sequences of the peptides derived from p180 were almost identical to those of human AF-6 that is identified as the fusion partner of the ALL-1 protein. The ALL-1/AF-6 chimeric protein is the critical product of the t (6:11) abnormality associated with some human leukemia. AF-6 has a GLGF/Dlg homology repeat (DHR) motif and shows a high degree of sequence similarity with Drosophila Canoe, which is assumed to function downstream from Notch in a common developmental pathway. The recombinant N-terminal domain of AF-6 and Canoe specifically interacted with GTP gamma S.GST-Ha-Ras. The known Ras target c-Raf-1 inhibited the interaction of AF-6 with GTP gamma S.GST-Ha-Ras. These results indicate that AF-6 and Canoe are putative targets for Ras.

The unintegrated circular DNA form of Moloney murine sarcoma virus (MSV) has been cloned in bacteriophage lambda. Discrete deletions in the viral genome were shown to occur during propagation of recombinant phage in Escherichia coli. Heteroduplex and restriction enzyme analyses indicated the deletion of tandemly repeated sequences within certain of the cloned MSV DNA inserts. Cloned MSV DNA was used to prepare a probe composed of its acquired cellular (src) sequences, shown previously to be necessary for MSV transformation. Analysis of EcoRI digests of normal mouse cellular DNA revealed the presence of a single 14-kilobase-pair fragment containing these sequences which lacked contiguity with endogenous type C helper viral information of the same cells. Thus, the sarcoma virus-specific sequences of MSV are represented within the normal mouse genome in a manner analogous to that of a cellular gene.

The topography of endogenous type C viral sequences in mouse cellular DNA was investigated by EcoRI nuclease restriction and application of the Southern blotting technique. The DNAs from one outbred and five inbred strains were resolved into 20-35 fragments containing viral sequences, distributed in unique, though related, patterns for each mouse strain. Different normal tissues from the same animal were indistinguishable in their DNA patterns, suggesting that tissue differentiation is not associated with gross alteration in the topography of endogenous type C virus sequences. Tumor tissues from spontaneous lymphomas of AKR/J mice were similarly analyzed. In four out of seven individual tumors we detected the emergence of one or two new virus-containing DNA fragments. The mass of these fragment varied, indicating different insertion sites of the new viral sequences. The detection of these new viral sequences suggests that each tumor was composed of descendents of only one or a few cells.

Proviral DNA transcribed from the RNA of Moloney murine sarcoma virus was isolated from newly infected cells. Three forms of viral DNA were observed: (i) a linear double-stranded form of 3.4 X 10(6) daltons which constituted the major viral DNA species in the cell, and is thought to be a complete transcript (monomer) of viral RNA; (ii) a fast-sedimenting viral DNA bigger than the monomeric unit which can be either integrated provirls or concatamers; and (iii) covalently closed circles of monomer size representing 5% or less of the total viral DNA in the cell. The linear viral DNA was tested for its susceptibility to restriction endonucleases by electrophoretic analysis of the digestion products and their identification by hybridization with viral RNA or cDNA probes. The linear DNA is not cleaved by endonucleases EcoRI and BamHI. It is cleaved into two fragments by endonucleases HindIII and Hae II, and into three fragments by restriction endonuclease HincII. The fragments of the viral DNA added up to approximately 3.4 X 10(6) daltons; this and the uniform size of the linear DNA indicated that the viral DNA has unique ends and a complexity of 3.4 X 10(6) daltons. The different cleavage fragments were ordered with respect to each other and the 3' end of the viral RNA. It was observed that fragments from both ends of the linear DNA can be hybridized to sequence(s) at the 3' end of murine sarcoma virus RNA; this result suggested the possibility that a short redundant sequence exists at both termini of the genome.