Cloning, expression and genetic mapping of the mouse SH3 domain protein, SH3D2B.

Abstract

The LP-BM5 mixture of murine leukemia viruses causes an immunodeficiency syndrome in mice termed MAIDS (reviewed in Morse et al. 1992). The etiological agent in the mixture is a replication-defective virus (BM5def) that encodes only a single protein. This protein is a variant of the normal viral Gag precursor that is cleaved into the internal virion capsid proteins MA (matrix, p15), p12, CA (capsid, p30), and NC (nucleocapsid, p10). The BM5def Pr60Gag differs from the Gag polyprotein of nonpathogenic ecotropic virus in the carboxyterminus of MA and throughout much of p12. In an attempt to understand how these altered Gag products contribute to the development of immunodeficiency, we used the yeast two-hybrid system to identify cellular proteins that interact with the Pr60Gag (Kim et al. submitted). This approach identified several genes, with one, designated Y1, represented in 10 independent captures. Of the 10 yeast two-hybrid clones, 8 were identical in length (1.45 kbp), and 2 were longer (1.6 and 1.8 kbp). One of the 8 clones (Y1) was sequenced in its entirety, and the 58 ends of the two longer clones were sequenced until the sequence overlapped with that of Y1. Searches of the databanks identified homology with a mouse cDNA, SH3D2B (accession #U58885; Sparks et al. 1996). The composite sequence corresponds to bp 258 to 1966 of SH3D2B, and therefore lacks the 58-most 0.2 kb of SH3D2B, but includes the C-terminal SH3 domain. Comparison of the two sequences identified only two bp differences over a stretch of 1709 bp: a C → T transition resulting in a change of SH3D2B AA 285 from Ala to Val, and, at SH3D2B nt 1963, a T → G change in the 38 untranslated region. Since Y1 was cloned from a C57BL/Ka T-cell lymphoma and SH3D2B was obtained from an NIH Swiss library, it is likely that these two amino acid differences represent polymorphisms between strains. Recently the human homology of SH3D2B, termed EEN (extra eleven nineteen) by the authors, was found to be part of a novel MLL fusion protein in a patient with acute myeloid leukemia (So et al. 1997). EEN is part of a family of genes that contain a C-terminal SH3 domain. This suggests that alterations in SH3D2B expression may contribute to cell transformation. In addition, the human homolog has been independently cloned by PCR with degenerate primers designed to amplify SH3 domain-containing cDNAs (Giachino et al. 1997). These authors termed the SH3D2B human homolog SH3GL1. A multitissue RNA blot (Promega, Madison, Wis.) was hybridized with a nick-translated 1.4-kb XhoI Y1 insert as probe. Transcripts of 2.4 kb were detected at high levels in testis and at low levels in all other tissues examined (Fig. 1). This is comparable to the 2.6-kb transcript reported to be the predominant transcript in human tissues with EEN as probe (So et al. 1997), and to the 2.7-kb single transcript found ubiquitously in human tissues with SH3GL1 as probe (Giachino et al. 1997). Sh3d2b was genetically mapped by analysis of two sets of genetic crosses: (NFS/N or C58/J × M. m. musculus) × M. m. musculus (Kozak et al. 1990), (NFS/N × M. spretus) × M. spretus or C58/J (Adamson et al. 1991). DNAs from the progeny of these crosses have been typed for over 1000 markers, which map to all 19 autosomes and the X Chromosome (Chr) including the Chr 17 markers Tik (T viral integration site), Stp2 (sulfotransferase, phenol preferring 2), C3 (complement component 3), Trp53-ps (transformation related protein 53 pseudogene), and Hprt-ps1 (hypoxanthine phosphoribosyltransferase—pseudogene 1). The probes and polymorphisms used to type these markers have been described previously (Miyazaki et al. 1995; Khan et al. 1995). The additional locus Vav (vav oncogene) was typed as a ScaI polymorphism in the M. spretus crosses and as a BglII polymorphism in the M. m. musculus crosses with a mouse Vav probe obtained from Dr. I.J. Lee (NIAAA, Bethesda, Md.). The Y1 insert was used as hybridization probe on Southern blots and identified EcoRI fragments of 17.5 kb in NFS/N and C58/J and 10.5 kb in M. m. musculus, and SstI fragments of 13.0