The structure of the human c-fes/fps proto-oncogene.

We have determined the complete nucleotide sequence of a human DNA fragment of approximately 13 kbp, which was shown by Southern blot analysis to contain the entire v-fes/fps cellular homolog. The v-fes/fps homologous sequences were dispersed over 11 kbp in 18 interspersed segments which were flanked by splice junctions. Fusion of these segments created a DNA fragment in which coding regions similar to those observed in the viral oncogenes v-fes of the Gardner-Arnstein (GA) and Snyder-Theilen (ST) strains of feline sarcoma virus and v-fps found in Fujinami sarcoma virus could be identified. A potential initiation site in the first exon was found. About 200 nucleotides downstream of a translational stop codon in the v-fes/fps homologous region, a poly(A) addition signal was identified. The deduced amino acid sequence has a molecular weight of 93 390 dalton resembling NCP92, the recently described human c-fes/fps product. The topography of human c-fes/fps appeared to resemble that of chicken c-fps.     

Structure of the feline c-fes/fps proto-oncogene: genesis of a retroviral oncogene.

The nucleotide sequence of the feline c-fes/fps proto-oncogene was analyzed. Comparison with v-fes and v-fps revealed that all v-fes/fps homologous sequences were dispersed over 11 kilobase pairs in 19 interspersed segments. All segments, numbered exon 1 to exon 19 as in the chicken and human loci, were flanked by consensus splice junctions. The putative promoter region contained a CATT sequence and three CCGCCC motifs which were also found in the human locus at similar positions. About 200 nucleotides downstream of a translational stop codon in exon 19, a putative poly(A) addition signal was identified. Using the putative translation initiation codon in exon 2, a 93,000-molecular-weight protein could be deduced. This protein resembled very well the putative protein of the human c-fes/fps proto-oncogene (94% overall homology) and, although less well, the putative protein of the chicken c-fes/fps proto-oncogene (70% overall homology). As far as the feline c-fes/fps proto-oncogene sequences transduced to the Gardner-Arnstein (GA) and Snyder-Theilen (ST) strains of feline sarcoma virus (FeSV) are concerned, homology in deduced amino acid sequences between the GA- and ST-v-fes viral oncogenes and the proto-oncogene was 99%. Analysis of the recombination junctions between feline leukemia virus and v-fes sequences in GA- and ST-FeSV proviral DNA revealed for the left-hand junction the involvement of homologous recombination, presumably at the DNA level. The right-hand junction, which appeared identical in the GA-FeSV and ST-FeSV genomes, could have been the result of a site-specific recombination at the RNA level.     

Evolutionary placement of Xanthomonadales based on conserved protein signature sequences

Xanthomonadales comprises one of the largest phytopathogenic bacterial groups, and is currently classified within the gamma-proteobacteria. However, the phylogenetic placement of this group is not clearly resolved, and the results of different studies contradict one another. In this work, the evolutionary position of Xanthomonadales was determined by analyzing the presence of shared insertions and deletions (INDELs) in highly conserved proteins. Several distinctive insertions found in most of the members of the gamma-proteobacteria are absent in Xanthomonadales and groups such as Legionelalles, Chromatiales, Methylococcales, Thiotrichales and Cardiobacteriales. These INDELs were most likely introduced after the branching of Xanthomonadales from most of the gamma-proteobacteria and provide evidence for the phylogenetic placement of the early gamma-proteobacteria. Moreover, other proteins contain insertions exclusive to the Xanthomonadales order, confirming that this is a monophyletic group and provide important specific genetic markers. Thus, the data presented clearly support the Xanthomonadales group as an independent subdivision, and constitute one of the deepest branching lineage within the gamma-proteobacteria clade.     

Molecular cloning of the feline c-fes proto-oncogene and construction of a chimeric transforming gene

The feline c-fes proto-oncogene, different parts of which were captured in feline leukemia virus (FeLV) to generate the transforming genes (v-fes) of the Gardner-Arnstein (GA) strain of feline sarcoma virus (FeSV) and the Snyder-Theilen strain (ST) of FeSV, was cloned and its genetic organization determined. Southern blot analysis revealed that the c-fes genetic sequences were distributed discontinuously and colinearly with the v-fes transforming gene over a DNA region of around 12.0 kb. Using cloned c-fes sequences, complementation of GA-FeSV transforming activity was studied. Upon replacement of the 3' half of v-fesGA with homologous feline c-fes sequences and transfection of the chimeric gene, morphological transformation was observed. Immunoprecipitation analysis of these transformed cells revealed expression of high Mr fusion proteins. Phosphorylation of these proteins was observed in an in vitro protein kinase assay, and tyrosine residues appeared to be involved as acceptor amino acid.     

Characterization of a gene encoding a basic protein of the spermatid nucleus, TNP2, and its close linkage to the protamine genes in the bull.

During elongation and condensation of the spermatid nucleus, histones are replaced by spermatid-specific transition proteins (TNP). TNP1 is well characterized at the cDNA and at the genomic level and was found to be highly conserved during mammalian evolution (similarity between 83 to 98%). We here describe for the first time the nucleotide sequence and organization of the gene for TNP2. The gene was isolated from a bull cosmid library and was found to contain a single intron of 910 bp. The coding sequence consists of 390 bp and has a similarity of about 70% to that of the TNP2 cDNAs of mouse and rat. At the basis of amino-acid sequences, the bull TNP2 is 14 and 15 amino acids longer than that of mouse and rat, respectively, and the similarity is only 45% between bull and mouse and 42% between bull and rat. However, the evolutionary divergence has not occurred at the cost of basic amino acids which are of functional importance in DNA-protein interaction in the condensing spermatid nucleus. The TNP2 gene is closely linked to the protamine genes in the bull genome.     

Identification and genetic mapping of the murine gene and 20 related sequences encoding chromosomal protein HMG-17

HMG-17 is an abundant, nonhistone chromosomal protein that binds preferentially to nucleosomal core particles of mammalian chromatin. The human gene for HMG-17 has been localized to Chromosome (Chr) 1p, but the murine gene has not been previously mapped. Here we identify the murine functional gene, Hmg17, from among more than 25 related sequences (probably processed pseudogenes) and show that it is located on mouse Chr 4, in a region known to have conserved linkage relationships with human Chr 1p. We also report the map locations of 20 additional Hmg17-related sequences on mouse Chrs 1, 2, 3, 5, 7, 8, 9, 13, 15, 16, 17, 18, and X. The multiple, dispersed members of the Hmg17 multigene family can be detected efficiently with a single cDNA probe and provide useful markers for genetic mapping studies in mice.     

Expression of the mammalian c-fes protein in hematopoietic cells and identification of a distinct fes-related protein.

The avian c-fps and mammalian c-fes proto-oncogenes are cognate cellular sequences. Antiserum raised against the P140gag-fps transforming protein of Fujinami avian sarcoma virus specifically recognized a 92,000-Mr protein in human and mouse hematopoietic cells which was closely related in structure to Snyder-Theilen feline sarcoma virus P87gag-fes. This polypeptide was apparently the product of the human c-fes gene and was therefore designated p92c-fes. Human p92c-fes was associated with a tyrosine-specific protein kinase activity in vitro and was capable of both autophosphorylation and phosphorylation of enolase as an exogenous protein substrate. The synthesis of human and mouse p92c-fes was largely, though not entirely, confined to myeloid cells. p92c-fes was expressed to relatively high levels in a multipotential murine myeloid cell line, in more mature human and mouse granulocyte-macrophage progenitors, and in differentiated macrophage like cells as well as in the mononuclear fraction of normal and leukemic human peripheral blood. p92c-fes was not found in erythroid cells, with the exception of a human erythroleukemia line which retains the capacity to differentiate into macrophage like cells. These results suggest a normal role for the p92c-fes tyrosine kinase in hematopoiesis, particularly in granulocyte-macrophage differentiation. In addition, a distinct 94,000-Mr polypeptide, antigenically related to p92c-fes, was identified in a number of hematopoietic and nonhematopoietic human and mouse cells and was also found to be associated with a tyrosine-specific protein kinase activity.     

The gene encoding the splicing factor SF2/ASF is a proto-oncogene

Alternative splicing modulates the expression of many oncogene and tumor-suppressor isoforms. We have tested whether some alternative splicing factors are involved in cancer. We found that the splicing factor SF2/ASF is upregulated in various human tumors, in part due to amplification of its gene, SFRS1. Moreover, slight overexpression of SF2/ASF is sufficient to transform immortal rodent fibroblasts, which form sarcomas in nude mice. We further show that SF2/ASF controls alternative splicing of the tumor suppressor BIN1 and the kinases MNK2 and S6K1. The resulting BIN1 isoforms lack tumor-suppressor activity; an isoform of MNK2 promotes MAP kinase-independent eIF4E phosphorylation; and an unusual oncogenic isoform of S6K1 recapitulates the transforming activity of SF2/ASF. Knockdown of either SF2/ASF or isoform-2 of S6K1 is sufficient to reverse transformation caused by the overexpression of SF2/ASF in vitro and in vivo. Thus, SF2/ASF can act as an oncoprotein and is a potential target for cancer therapy.     

Physical and genetic mapping of a novel chromosome 19 ERCC1 marker showing close linkage with myotonic dystrophy.

Recent genetic linkage analyses have mapped the myotonic dystrophy locus to the region of 19q13.2-13.3 lying distal to the gene for creatine kinase subunit M (CKM). The human excision repair gene ERCC1 has also been mapped to this region of chromosome 19. A novel polymorphic DNA marker, pEO.8, has been isolated from a chromosome 19 ERCC1-containing cosmid that maps to a 300-kb NotI fragment encompassing both CKM and ERCC1. Genetic linkage analysis reveals close linkage between pEO.8 and myotonic dystrophy (DM) (zmax = 19.3, theta max = 0.01). Analysis of two key recombinant events suggests a mapping of DM distal to pEO.8 and CKM.     

Recognition of distantly related protein sequences using conserved motifs and neural networks.

A sensitive technique for protein sequence motif recognition based on neural networks has been developed. It involves three major steps. (1) At each appropriate alignment position of a set of N matched sequences, a set of N aligned oligopeptides is specified with preselected window length. N neural nets are subsequently and successively trained on N-1 amino acid spans after eliminating each ith oligopeptide. A test for recognition of each of the ith spans is performed. The average neural net recognition over N such trials is used as a measure of conservation for the particular windowed region of the multiple alignment. This process is repeated for all possible spans of given length in the multiple alignment. (2) The M most conserved regions are regarded as motifs and the oligopeptides within each are used to train intensively M individual neural networks. (3) The M networks are then applied in a search for related primary structures in a databank of known protein sequences. The oligopeptide spans in the database sequence with strongest neural net output for each of the M networks are saved and then scored according to the output signals and the proper combination that follows the expected N- to C-terminal sequence order. The motifs from the database with highest similarity scores can then be used to retrain the M neural nets, which can be subsequently utilized for further searches in the databank, thus providing even greater sensitivity to recognize distant familial proteins. This technique was successfully applied to the integrase, DNA-polymerase and immunoglobulin families.