Chaoqun Wu

Molecular cloning and characterization of three novel lysozyme-like genes, predominantly expressed in the male reproductive system of humans, belonging to the c-type lysozyme/alpha-lactalbumin family.

Abstract Lysozymes, especially c-type lysozymes, are well-recognized bacteriolytic factors widely distributed in the animal kingdom and play a mainly protective role in host defense. The relatives of c-type lysozymes, alpha-lactalbumins, however, are only found in mammalian milk and possess a distinct biological function. These two proteins, having similar amino acid sequences, gene structure, and dimensional conformation, belong to the c-type lysozyme/alpha-lactalbumin family. Using human lysozyme as an information probe, we cloned four human cDNAs encoding homologues of human lysozyme; these were named LYZL2, LYZL4, LYZL6, and SPACA3 by the HUGO Gene Nomenclature Committee. Of these four, SPACA3 has been reported to code an intra-acrosomal sperm protein SLLP1. To our knowledge, the other three are reported here for the first time. Using Northern blot hybridization, including 16 different human tissues, we found that these four lysozyme-like genes were all highly expressed in the testis/epididymis. Further analysis of one, LYZL4, by in situ hybridization revealed that its mRNA was only detected in the epithelium of human epididymis, most abundantly in the caput, suggesting that LYZL4 plays a physiological role in male reproduction. By sequence analysis, we found that two essential catalytic residues of the human lysozyme were conserved in LYZL2 and LYZL6, whereas one site in LYZL4 and two sites in SPACA3 were replaced. The LYZL2, LYZL4, LYZL6, and SPACA3 genes were mapped to human chromosome 10p11.23, 3p21.33, 17q11.2, and 17q12, respectively, and displayed a similar genomic structure. Our data suggest that these four lysozyme-like genes, which have arisen from a common progenitor gene, play a major role in human reproduction.

GADD45γ, down-regulated in 65% Hepatocellular Carcinoma (HCC) from 23 Chinese patients, inhibits cell growth and induces cell cycle G2/M arrest for Hepatoma Hep-G2 cell lines

Growth-arrest and DNA-damage inducible (GADD) genes and Myeloid differentiation primary response (MyD) genes represent a family of genes that play a key role in negative control of cell growth. In the present study, following clone and location of human GADD45 γ (MyDL) gene, we have found that its mRNA expression level was down-regulated in 15/23 cases of clinic hepatocellular carcinoma (HCC) by comparing the northern hybridization results between the tumor tissues and adjacent normal tissues. Transient transfection of GADD45 γ cDNA with intact open reading frame sequence into the human hepatoma cells Hep-G2 resulted in dramatic growth suppression in colony formation assays. Furthermore, flow cytometry analysis indicated that GADD45 γ caused cell cycle arrest at G2/M transition when transfected into Hep-G2 cells. Therefore, the possible role of GADD45 γ in cell growth control was further confirmed in this paper.

Cloning and characterization of ARHGAP12, a novel human rhoGAP gene ☆

Rho GTPase activating proteins (GAPs) stimulate the intrinsic GTP hydrolysis activity of Rho family proteins. Here we report the cloning of two splice variants of a novel gene named ARHGAP12 (access number), which has an ORF of 2541bp. Profilescan search result showed that its putative protein contains five domains: rhoGAP, SH3, PH and two WW domains. ARHGAP12 is located in chromosome 10pter-cen and consists of 20 exons according to the Blastn result against high throughput genomic sequences (htgs). Reverse transcription PCR and Northern blot indicates that it ubiquitously expresses in human tissues as well as tumor cell lines, suggesting its basic roles in cells.

Transcriptomic analyses support the similarity of gene expression between brain and testis in human as well as mouse

We previously revealed similarity in gene expression patterns between human brain and testis, based on digital differential display analyses of 760 human Unigenes. In the present work, we reanalyzed the gene expression data in many tissues of human and mouse for a large number of genes almost covering the respective whole genomes. The results indicated that both in human and in mouse, the gene expression profiles exhibited by brain, cerebellum and testis are most similar to each other compared with other tissues.

Human Aurora-B binds to a proteasome α-subunit HC8 and undergoes degradation in a proteasome-dependent manner

Human Aurora/Ipl1-related kinase 2 (Aurora-B) is a key regulator of mitosis. Here human proteasome α-subunit C8 (HC8) was identified to interact with the Aurora-B by yeast two-hybrid screen. This finding was confirmed by GST pull-down assays and immunoprecipitation experiments. The Aurora-B protein level increased in HeLa cells cultured with proteasome inhibitor ALLN. Our data suggest that Aurora-B might undergo degradation by binding to HC8 in a proteasome-dependent manner during mitosis.

A novel human gene whose product shares homology with bovine brain-specific protein p25 is expressed in fetal brain but not in adult brain.

AbstractWe have cloned a novel human gene (C14orf5) from a fetal brain cDNA library that is located on chromosome 14 and consists of 4 exons. It encodes a protein of 170 amino acids that shares homology with human p25 alpha and bovine p25. Reverse transcription-polymerase chain reaction analysis indicated that it is highly expressed in liver and pancreas. Its transcripts could not be detected in adult brain but could be found in fetal brain.

Isolation and characterization of the human d-glyceric acidemia related glycerate kinase gene GLYCTK1 and its alternatively splicing variant GLYCTK2

Deficiency of human glycerate kinase leads to d-glycerate acidemia/d-glyceric aciduria. Through PCR cloning assisted by in silico approach, we isolated the human glycerate kinase genes—Glycerate Kinase 1 (GLYCTK1) and its alternatively splicing variant—Glycerate Kinase 2 (GLYCTK2), which might be associated with d-glycerate acidemia/d-glyceric aciduria. The locus of GLYCTK gene is mapped to 3p21. PCR amplification in seventeen human tissue cDNAs revealed that both GLYCTK1 and GLYCTK2 are expressed widely almost in all these tissues. The expression of mouse Glyctk in various tissues was demonstrated by in situ hybridization. Both GLYCTK1 and GLYCTK2 proteins are localized in cytosol, and GLYCTK2 proteins are specifically localized in mitochondria. Present results revealed the characteristic expression pattern of murine Glyctk in neural system, skeleton muscle, supporting that glycerate kinase is implicated in d-glycerate acidemia/d-glyceric aciduria.

Cloning and characterization of KLHL5, a novel human gene encoding a kelch-related protein with a BTB domain.

Most kelch family proteins contain two conserved domains, the BTB domain and the kelch repeat domain. Here we describe the cloning and characterization of a novel human KLHL5 gene. The 3488 bp cDNA encodes a kelch family protein homologous to the Drosophila kelch protein. It also contains the two conserved domains. Northern blot analysis reveals a single transcript. It is abundantly expressed in ovary, adrenal gland, and thyroid and less abundantly expressed in trachea, prostate, testis, lymph node, and spinal cord. KLHL5 was mapped to 4p13–4p15.1.

Expression of CDV-1R Gene in Mouse Epididymis as Revealed by in Situ Hybridization

We have previously studied mouse Cdv (carnitine deficiency-associated gene expressed in ventricle)-1 related gene Cdv-1R and its human counterpart CDV-1R, and revealed that mouse Cdv-1R was predominantly expressed in testis by multiple tissue northern analysis. To further localize the Cdv-1R mRNA in mouse testis and epididymis tissue, in situ hybridization study was reported in this article. In the adult mice, the Cdv-1R expression was intensively found in the epithelial cells of the caput and corpus epididymis, whereas it was moderately detected in the initial segment, and weakly in the cauda epididymis. In the seminiferous tubles of the testis, no obvious hybridization signals were observed above the background level. This Cdv-1R region-specific expression pattern in the epididimis suggests Cdv-1R may play an important role in sperm maturation. Moreover, considering the Cdv-1R has a similar expression distribution in epididymis to the OCTN2, it would appear that Cdv-1R might be involved in the carnitine pathway in the epididimis.

Identification and Characterization of LIW, a Novel Domain Involved in Animal NCKIPSDs and Some Uncharacterized Fungal Proteins

PSI-BLAST and HMM are two important tools for identification of novel protein domains, which can help to infer biological function and evolutionary relationships, to investigate new proteins, and to suggest future experiments. Additionally, the availability of public sequence databases and freely distributed tools for sequence analysis has meant that researchers from all over the world can use this approach (McEntyre and Gibson, 2004). The NCK-interacting protein with an SH3 domain (NCKIPSD), a Nckbinding protein expressed preferentially in neural tissues and testis, is involved in actin polymerization (Fukuoka et al., 2001), sarcomere assembly during cardiac myocyte differentiation (Lim et al., 2001), stress fiber formation induced by active mDia and regulation of actin polymerization (Satoh and Tominaga, 2001), interactions between intermediate filaments and late endosomal compartments (de Bernard et al., 2001), as well as in cell adhesion turnover in the downstream portion of the Rho-mDia pathway by interacting with Grb2 and Src (Satoh and Tominaga, 2001). NCKIPSD serves as an adaptor molecule to recruit other proteins to Nck at focal adhesions, which might be crucial for stable cell adhesion and can be dynamically modulated by SPIN90 phosphorylation that is dependent on cell adhesion and ERK activation (Lim et al., 2003). NCKIPSD is involved