Identification, Characterization And Functional Studies Of Several Novel Human Genes

The fetal liver at 22 wk of gestation is a major site of fetal hematopoiesis in man, and is at the critical turning point between immigration and emigration of the hematopoietic system. The unique characteristics of the fetal liver at this stage are worthy of investigation. Based on large-scale sequencing of human fetal liver cDNA libraries, following routine bioinformatic strategy (e.g. Contig) and RACE, we have obtained 22 full-length cDNAs, which belong to 13 novel human genes. All of them have been deposited in GenBank database. Among these novel human genes, some were chosen for detailed investigation. The main results are obtained as follows:Human semaphorin 6C and semaphorin 6D: Two novel human transmembrane semaphorins, (HSA)SEMA6C (human orthologue of rat Sema6C) and (HSA)SEMA6D, that belong to the class VI subgroup of the semaphorin family have been cloned. The gene for SEMA6C is divided into 20 exons and mapped on chromosome Iq12-21.1. The SEMA6D gene contains 19 exons and assigned to chromosome 15q21.1. Northern blot and dot blot analysis have revealed that SEMA6C was primarily expressed only in skeletal muscle, whereas SEMA6D is expressed abundantly in kidney, brain and placenta, moderately in heart and skeletal muscle. Three isoforms of SEMA6C and five isoforms of SEMA6D derived from alternative splicing were identified and their expression were found to be regulated in a tissue- and development-dependent manner. Deletion analysis indicated that a sema domain and a PSI domain in their integrity are necessary for the appropriate posttranslation-modification and subcellular localization of those semaphorin proteins. The extracelluar domain of SEMA6C inhibited axonal extension of nerve growth factor (NGF)-differentiated PC12 cells, and induced growth cone collapse of chicken dorsal root ganglion (DRG), rat hippocampal neurons and rat cortical neurons in a dose-responsive manner. SEMA6D was also demonstrated to inhibit axonal extension of NGF-differentiated PC12 cells, and to induce the growth cone collapse of DRG and rat hippocampal neurons, but have no significant effect on thegrowth cones of rat cortical neurons. These data suggest that SEMA6C and SEMA6D might play distinct roles in neuronal development.Human NDRG2, NDRG3 and NDRG4: Three human cDNAs encoding NDRG2 (371aa), NDRG3 (375aa) and NDRG4 (339aa) we have cloned, are homologous to NDRG1. These three genes, together with NDRG1 (N-Myc downstream regulated), constitute the NDRG gene family. At amino acid level, the four members share 53-65% identity. Each of the four proteins contains an ct/p hydrolase fold as in human lysosomal acid lipase. Expression of the fusion proteins NDRG2/GFP, NDRG3/GFP and NDRG4/GFP in COS-7 cells showed that all of them are cytosolic proteins. Based on UniGene cluster analysis, the genes NDRG2, NDRG3 and NDRG4 are located at chromosome 14ql 1.1-11.2, 20ql2-11.23 and 16q21-22.1, respectively. Northern and dot blot analysis shows that all of the three genes are highly expressed in adult brain and almost not detected in the eight human cancer lines. In addition, in contrast to the relatively ubiquitous expression of NDRG1, NDRG2 is highly expressed in adult skeletal muscle and brain, NDRG3 highly expressed in brain and testis, and NDRG4 specifically expressed in brain and heart, suggesting that they might display different specific functions in distinct tissues.Human neural proliferation, differentiation and control factor l(hnpdcl): The hnpdcl gene (a novel human homologue of mouse npdcl) we isolated, which contains nine exons, was mapped to human chromosome 15. It encodes a polypeptide of 325 amino acids. Sequence analysis has shown that hNPDCl protein contains a putative signal peptide, a transmembrane segment, and a typical bipartite nuclear localization signal. Northern blot and dot blot hybridization indicates that hnpdcl mRNA is strongly expressed in adult brain, prostate, pituitary gland, and mammary glands. These results support that hNPDCl plays a role in the control of neural cell proliferation and differentiation, and suggest that it may be involved in the development of certain secretion glands.Human protein tyrosine phosphatase PTP-TD14: A human cDNA of 5248bp encoding a novel protein tyrosine phosphatase PTP-TD14(1636aa) has been isolated from fetal liver. The gene is located at chromosome 3p21.3, an area frequently deleted in many types of cancer, and composed of at least 26 exons and 25 intons. The phosphatase has unique features in its domain structure: a tyrosine phosphatasedomain, two SH3-binding motifs, two proline-rich region and an N-terminal domain similar to yeast BROl(a yeast protein that is involved in the mitogen-activated protein kinase signaling pathway). Northern blot and dot blot hybridizations indicate that it is expressed ubiquitously in human tissues and seven cancer cell lines. Thus, the phosphatase gene may be a candidate for one of the tumor suppressor genes located on 3 p21.3.Other six novel human genes with important biological functions: The full-length cDNAs of six novel human genes have been obtained. They are human basic kruppel-like factor (BKLF) gene, human c-Jun binding protein (JBP) gene, human hematopoietic zinc finger protein gene (Hzf), human liver membrane-bound protein (LMP) gene, human liver nuclear protein (LNP) gene, and human clathrin coat assembly protein (AP47) gene, which are mapped on Chr4pl5.2-pl6.1, Chrlql2-21.2, Chrl2, Chrlp32.3-33, Chrl5ql3.3-21.1 and Chrl9pl.28, respectively. The functional studies of these genes are in progress.