Retroviral insertional mutagenesis has been proposed as an efficient mechanism to turn on or to increase the expression of oncogenes in several avian or mammal models. Integration site studies of avian leukosis virus, murine leukaemia and murine mammary tumour viruses led to the coleutification of highly conserved genes whose expression is induced or increased during leukaemogenesis, probably through enhancer elements present in the retroviral long terminal repeats. This is reminiscent of the activation of cellular proto-oncogenes or putative oncogenes in numerous human tumours and leukaemias as a result of chromosomal translocations or DNA rearrangements. Here we report the characterization of a new putative oncogene isolated from a murine erythroleukaemia induced by the acute leukaemogenic retrovirus spleen focus forming virus (SFFV). An important and unusual feature of this genomic locus Spi-1 (for SFFV proviral integration) is that rearrangements due to SFFV integration were found in 95% of the erythroid tumours studied. A 4.0-kilobase messenger RNA was detected in rearranged tumours. No Spi-1 rearrangement was detected in other virally induced myeloid, lymphoid or erythroid tumours tested.

Eukaryotic cells seem to use GTP hydrolysis to regulate vesicular traffic in exocytosis and endocytosis. The best evidence for this comes from studies on the yeast Saccharomyces cerevisiae that have identified two small Ras-related GTP-binding proteins, Sec4p and Ypt1p, which control distinct stages of the secretory pathway. In mammalian cells the effects of a non-hydrolysable GTP analogue, GTP-gamma S, on different transport events have suggested that they also have proteins functionally related to yeast Sec4p and Ypt1p. The rab genes have recently been cloned and sequenced for rat and human and their proteins have highly conserved domains in common with Sec4p and Ypt1p (including a putative effector binding site). They are therefore good candidates for GTP-binding proteins involved in intracellular transport in mammalian cells. One of the Rab proteins (Rab1p) is the mammalian counterpart of Ypt1p (ref. 13). Here we report the localization of the protein Rab6p to the Golgi apparatus in several cell types. By immunolabelling and electron microscopy, Rab6p appears to be concentrated predominantly on the medial and trans cisternae and distributed over their entire surface.

Insertional mutagenesis of the spi-1 gene is associated with the emergence of malignant proerythroblasts during Friend virus-induced acute erythroleukemia. To determine the role of spi-1/PU.1 in the genesis of leukemia, we generated spi-1 transgenic mice. In one founder line the transgene was overexpressed as an unexpected-size transcript in various mouse tissues. Homozygous transgenic animals gave rise to live-born offspring, but 50% of the animals developed a multistep erythroleukemia within 1.5 to 6 months of birth whereas the remainder survived without evidence of disease. At the onset of the disease, mice became severely anemic. Their hematopoietic tissues were massively invaded with nontumorigenic proerythroblasts that express a high level of Spi-1 protein. These transgenic proerythroblasts are partially blocked in differentiation and strictly dependent on erythropoietin for their proliferation both in vivo and in vitro. A complete but transient regression of the disease was observed after erythrocyte transfusion, suggesting that the constitutive expression of spi-1 is related to the block of the differentiation of erythroid precursors. At relapse, erythropoietin-independent malignant proerythroblasts arose. Growth factor autonomy could be partially explained by the autocrine secretion of erythropoietin; however, other genetic events appear to be necessary to confer the full malignant phenotype. These results reveal that overexpression of spi-1 is essential for malignant erythropoiesis and does not alter other hematopoietic lineages.

Several oligonucleotide mixtures corresponding to a 6-amino acid sequence that is strictly conserved in all the ras and ras-related proteins (from various organisms) were tested for their ability to hybridize to 11 cloned members of the ras gene superfamily. Among these mixtures, a combination of two sets of partially complementary oligomers were able to hybridize to all the tested sequences. To identify members of the ras superfamily, we screened a rat brain cDNA library with these probes and isolated four genes, denoted rabl, -2, -3, and 4, encoding proteins homologous to the yeast YPT protein.Amino acid homology scores with YPT range from 75% for rabl to 37% for rab4, whereas the homologies with p21 ras and other ras-related proteins are =30%, and homologous residues were clustered in the regions involved in GTP/GDP binding. Another striking similarity shared by the rab and the other ras-related proteins is the conservation of at least one cysteine residue near the carboxyl-terminal end involved in the membrane binding of the ras proteins. rabi is a mammalian homolog of the yeast YPT gene, and the four rab genes constitute an additional branch of the ras gene superfamily that to our knowledge has not been described in higher eukaryotes.The c-Ha-ras (1) and c-Ki-ras (2) genes were first characterized as the cellular genes transduced in the Harvey and Kirsten sarcoma viruses. Later, these two genes as well as a third closely related one, N-ras (3), were found to be frequently activated to a transforming potential in mammalian tumors (for a review see ref. 4). Highly homologous genes have been isolated from a wide variety of organisms including Drosophila, Dictyostelium,. Two other genes, ral and R-ras, have been identified in mammals (9, 10), and the corresponding proteins share 50%o homology with the transforming ras proteins. The ras gene family has also been extended by fortuitous discoveries such as the YPT gene, found as an open reading frame between the tubulin and actin genes of yeast (11), and the rho genes, first identified in an Aplysia cDNA isolated for other purposes and later characterized in human, rat, and yeast genomes (12, 13). Rho and YPT proteins share '30% homology with the ras proteins.In mammals, all the known ras or ras-related genes code for 21-to 24-kDa proteins that share structural and biochemical homologies with the guanine nucleotide-binding regulatory (G) proteins (14) involved in mediation of signal transduction in a variety of receptor/effector systems. ras proteins bind GTP and GDP and exhibit a low GTPase activity, as do the G proteins (15, 16). Four homology boxes corresponding to the GTP binding site are highly conserved in all the ras-related proteins. Among them, a stretch of six residues: Asp-Thr-Ala-Gly-Gln-Glu (in positions 57-62 of Ki-ras) is strictly conserved. A computer search in a protein databank* did not detect any other protein possessing this sequence even among the G proteins or other nucleotide binding proteins; thus, this sequence could be specific fo...