The retroviral integration siteThe hematopoietic system is composed of a hierarchy of cells, ranging from the pluripotent stem cell, committed myeloid and lymphoid progenitor cells, to terminally differentiated blood cells (Metcalf 1988;Sachs 1987). The molecular mechanisms that control the developmental and proliferative decisions of the pluripotent hematopoietic stem cell are largely unknown. The extreme cellular heterogeneity of hematopoietic populations, combined with the low frequency of stem cells, has hampered experimental approaches to this problem. The generation of mutations that affect stem cell function and the molecular analysis of existing developmental mutations that control hematopoiesis (Pawson and Bernstein 1990) provide two powerful genetic strategies for identifying and characterizing the genes that control normal blood cell formation. In addition, because cellular genes involved in leukemic transformation may also have a role in regulating normal stem cell function, the identification of such genes may provide insights into both normal and leukemic hematopoiesis. Transformation by certain retroviruses can involve the integration of proviral DNA near or within specific cellular genes. Such integration events most frequently lead to the elevated expression of these genes (for review, see in Peters 1990), although the tumor suppressor gene p53 can also be inactivated by retroviral integration of the Friend leukemia virus (Ben-David et al. 1988, 1990b. The analysis of common retroviral integration events can, therefore, be used to identify and clone novel transforming genes involved in tumorigenesis. Using this approach, a large number of genes important in the leukemic transformation of hematopoietic cells, including have been isolated (Peters 1990).The Fli-1 and Spi-1 genes have been shown by us and others (Moreau-Gachelin et al. 1988; Ben-David et al. 1990) to be involved in erythroleukemia induction by various strains of Friend leukemia virus. Fli-1 (Friend leukemia integration-site 1) is rearranged in 75% of independently isolated erythroleukemic clones from mice inoculated at birth with the replication-competent Cold Spring Harbor Laboratory Press on May 9, 2018 -Published by genesdev.cshlp.org Downloaded from

Mutations at the mouse W/c-kit locus lead to intrinsic defects in stem cells of the melanocytic, hematopoietic, and germ cell lineages. W alleles vary in the overall severity of phenotype that they confer, and some alleles exhibit an independence of pleiotropic effects. To elucidate the molecular basis for these biological differences, we analyzed the c-k/t locus and the c-kit.associated autophosphorylation activities in five different W mutants representative of a range of W phenotypes. Mast cell cultures derived from mice or embryos homozygous for each W allele were deficient in c-kit autophosphorylation activity, the extent of which paralleled the severity of phenotype conferred by a given W allele both in vivo and in an in vitro mast cell coculture assay. The mildly dominant, homozygous viable alleles W 44 and W s7 were found to express reduced levels of an apparently normal c-k/t protein. In contrast, c-kit kinase defects conferred by the moderately dominant, homozygous viable alleles W 4~ or W ss or the homozygous lethal allele, W 37, were attributed to single-point mutations within the kinase domain of the c-kit polypeptide, which result in point substitutions of amino acid residues highly conserved in the family of protein tyrosine kinases. The nature and location of these amino acid substitutions account for the relative severity of phenotypes conferred by these W alleles and demonstrate that the pleiotropic developmental defects associated with the W/c-kit locus arise as the result of dominant loss-of-function mutations in a transmembrane receptor tyrosine kinase.

Friend murine leukemia virus (F-MuLV) induces erythroleukemia when inoculated into newborn BALB/c or NIH/Swiss mice. We have molecularly cloned F-MuLV host cell DNA junction fragments from an erythroleukemia cell line induced by F-MuLV to identify cellular genes involved in the leukemogenic process. One particular proviral integration site, Fli-1, is rearranged in 75% (9/12) of independently isolated erythroleukemia cell lines derived from either BALB/c or NIH/Swiss mice inoculated at birth with F-MuLV. Other hematopoietic neoplasms induced by F-MuLV, including myeloid (granulocytic) and lymphoid tumors, did not show rearrangements of the Fli-) locus. Similarly, none of 35 erythroleukemia cell lines induced by the Friend virus complexes (FV-A and FV-P) was rearranged at the Fli-1 locus. In contrast, no rearrangements were detected at the Sfpi-1 locus, a preferred site of integration in either FV-P-or FV-A-induced leukemias. Using recombinant inbred mice, the Fli-) locus was situated on mouse chromosome 9 close to the cellular protooncogene c-ets-1 . DNA and RNA analysis suggests, however, that Fli-1 is different from ets-). Thus, Fli-1 appears to define a distinct locus specifically involved in the induction of erythroid leukemias by F-MuLV.

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