Molecular characterization and structural organization of D-elg, an ets proto-oncogene-related gene of Drosophila.

We have continued the molecular analysis of D-elg, a member of the Drosophila ets gene family. Based on the characterization of cDNA and genomic sequences, the D-elg gene contains five exons and four introns and produces a mRNA with an open reading frame of 464 amino acids. Consistent with this analysis, in vitro translation of a near full-length D-elg cRNA yields a protein with a molecular weight of approximately 56 kDa. D-elg shows significant homology to other ets proteins in the amino-terminal A domain and strong homology in the carboxy-terminal ETS domain. The D-elg protein is most similar to the alpha-subunit of the mouse GA-binding protein.     

Sequence and functional properties of Ets genes in the model organism Drosophila

Detailed molecular and genetic studies, coupled with the recent sequencing of the fly genome, have identified eight Ets-related genes in the model organism Drosophila. All show homology to genes in vertebrate species. Functional analyses of some of the Drosophila ets genes have revealed their essential roles in developmental processes such as metamorphosis, oogenesis, neurogenesis, myogenesis, and eye development. Such studies have yielded important insights into our understanding of the genetic control of hormonally-regulated gene expression, programmed cell death, and signal transduction during cell fate determination and differentiation. The developmental roles of E74 (ELF1), pointed (Ets 1), yan (TEL), and D-elg (GABPalpha) will be reviewed in this article. The context of their participation in signal transduction and gene regulation will also be discussed. The information should be of significant value to the study of related processes in higher organisms due to the growing evidence for the cross species conservation of developmental mechanisms.     

Genomic dispersal of the ets gene family during metazoan evolution.

Evolutionary homologs of the ets proto-oncogene have been discovered in the genomes of widely divergent eucaryote species from Drosophila to sea urchin to vertebrates. The prototype mammalian ets-1 and ets-2 genes are divided into three coding domains that differ in their rate of accumulation of sequence divergence. An analysis of sequence divergence of ets gene homologs in various species has produced a phylogenetic history of the ets gene family in the context of metazoan evolutionary radiation. A minimum of five duplication events of ets primordial genes were evident, namely (1) a duplication that separates primitive ets genes (Drosophila precursor of 74E, mouse PU.1 and human ELK1) from the ets-1, ets-2, erg ancestor; (2) and (3) two duplications that established separate ets, erg and elg/GABP-alpha lineages which occurred prior to invertebrate-vertebrate divergence; (4) divergence of ets-1 and ets-2 gene family also associated with vertebrate-invertebrate divergence; (5) duplication of ets-1 and ets-2 in Xenopus laevis to produce two ets-1 genes and two ets-2 genes during genomic tetraploidation in the recent ancestry of this species.     

The rgl-1 is a legitimate homologue of lethal giant larvae recessive oncogene in rat

We have cloned a rat homologue of the Drosophila recessive oncogene lethal (2) giant larvae from rat brain by RT-PCR using primers prepared from sequences conserved amongst lgl family genes. Sequence analysis predicts that the rat rgl-1 gene encodes a 1,036 amino acid polypeptide with a molecular weight of approximately 112 kDa, which contains a domain characteristic of WD-40 proteins. Northern blot analysis revealed that the highest expression of rgl-1 is detected in the testis, with moderate expression in ovary, brain, spleen, and kidney. Since there is a high degree of amino acid similarity among lgl proteins in various species, it is likely that there is an evolutionary relationship among these proteins. The amino acid identity of rgl-1 to Drosophila l(2)gl and mouse mgl-1 proteins showed 30.6 and 96.8%, respectively. The rat tomosyn, previously known as a homologue of Drosophila l(2)gl, showed much lower amino acid identity to Drosophila l(2)gl and mouse mgl-1 proteins (17.8 and 20%, respectively). Functional analysis showed that the expression of a rat rgl-1 cDNA in Saccharomyces cerevisiae missing sop genes, the yeast homologues of the Drosophila l(2)gl, restored partially the Na+ tolerance of the cell. Taken together, these results indicate that rgl-1, not tomosyn, is the legitimate homologue of lgl gene.     

Molecular characterization of hiwi,a human member of the piwi gene family whose overexpression is correlated to seminomas

The piwi family genes are highly conserved during evolution and play essential roles in stem cell self-renewal, gametogenesis, and RNA interference in diverse organisms ranging from Drosophila melanogaster and C. elegans to Arabidopsis. Here we report the molecular characterization of hiwi, a human member of the piwi gene family. hiwi maps to the long arm of chromosome 12, band 12q24.33, a genomic region that displays genetic linkage to the development of testicular germ cell tumors of adolescents and adults (TGCTs), i.e., seminomas and nonseminomas. In addition, gain of this chromosomal region has been found in some TGCTs. hiwi encodes a 3.6 kb mRNA that is expressed abundantly in the adult testis. It encodes a highly basic 861-amino-acid protein that shares significant homology throughout its entire length with other members of the PIWI family proteins in Drosophila, C. elegans and mammals. In normal human testes, hiwi is specifically expressed in germline cells, with its expression detectable in spermatocytes and round spermatids during spermatogenesis. No expresssion was observed in testicular tumors of somatic origin, such as Sertoli cell and Leydig cell tumors. Enhanced expression was found in 12 out of 19 sampled testicular seminomas-tumors originating from embryonic germ cells with retention of germ cell phenotype. In contrast, no enhanced expression was detected in 10 nonseminomas-testicular tumors that originate from the same precursor cells as seminomas yet have lost their germ cell characteristics. Finally, no enhanced expression was detected in four spermatocytic seminomas-testicular tumors that most likely originate from germ cells capable of partial meiosis. Thus, hiwi is specifically expressed in both normal and malignant spermatogenic cells in a maturation stage-dependent pattern, in which it might function in germ cell proliferation.     

Identification and characterization of human GUKH2 gene in silico

Drosophila Guanylate-kinase holder (Gukh) is an adaptor molecule bridging Discs large (Dlg) and Scribble (Scrib), which are implicated in the establishment and maintenance of epithelial polarity. Here, we searched for human homologs of Drosophila gukh by using bioinformatics, and identified GUKH1 and GUKH2 genes. GUKH1 was identical to Nance-Horan syndrome (NHS) gene, while GUKH2 was a novel gene. FLJ35425 (AK092744.1), DKFZp686P1949 (BX647246.1) and KIAA1357 (AB037778.1) cDNAs were derived from human GUKH2 gene. Nucleotide sequence of GUKH2 cDNA was determined by assembling 5'-part of FLJ35425 cDNA and entire region of DKFZp686P1949 cDNA. Human GUKH2 gene consists of 8 exons. Exon 5 (132 bp) of GUKH2 gene was spliced out in GUKH2 cDNA due to alternative splicing. GUKH2-REPS1 locus at human chromosome 6q24.1 and GUKH1-REPS2 locus at human chromosome Xp22.22-p22.13 are paralogous regions within the human genome. Mouse Gukh2 and zebrafish gukh2 genes were also identified. N-terminal part of human GUKH2, mouse Gukh2 and zebrafish gukh2 proteins were completely divergent from human GUKH1 protein. Human GUKH2 and GUKH1, consisting of eight GUKH homology (GKH1-GKH8) domains and Proline-rich domain, showed 28.5% total-amino-acid identity. GKH1, GKH4, GKH5, GKH7 and GKH8 domains were conserved among human GUKH1, human GUKH2 and Drosophila Gukh. Because human homologs of Drosophila dlg (DLG1-DLG7) as well as human homologs of Drosophila scrib (SCRIB, ERBB2IP and Densin-180) are cancer-associated genes, human homologs of Drosophila gukh (GUKH1 and GUKH2) are predicted cancer-associated genes.