Genomic organisation and chromosomal assignment of ODF2 (outer dense fiber 2), encoding the main component of sperm tail outer dense fibers and a centrosomal scaffold protein

ODF2 (outer dense fiber 2) was first described as the main protein component of the sperm tail cytoskeleton, the outer dense fibers, but was shown recently to be a component of the centrosomal scaffold in chicken. In mouse two related ODF2 cDNA clones were isolated which have been suggested to be most likely the result of alternative splicing. We show here the exon/intron organisation of mouse ODF2 and demonstrate that alternative splicing results in related cDNA sequences and most likely explains, at least partially, the highly complex protein pattern detected on Western blots. ODF2 was mapped to rat chromosome 3 and more specifically by FISH analysis at bands 3q11-->3q12. In addition, we demonstrate that ODF2 is indeed a component of the centrosome and the mitotic spindle poles in mammals.     

A gene trap knockout of the abundant sperm tail protein, outer dense fiber 2, results in preimplantation lethality

Outer dense fiber 2 (Odf2) is highly expressed in the testis where it encodes a major component of the outer dense fibers of the sperm flagellum. Furthermore, ODF2 protein has recently been identified as a widespread centrosomal protein. While the expression of Odf2 highlighted a potential role for this gene in male germ cell development and centrosome function, the in vivo function of Odf2 was not known. We have generated Odf2 knockout mice using an Odf2 gene trapped embryonic stem cell (ESC) line. Insertion of a gene trap vector into exon 9 resulted in a gene that encodes a severely truncated protein lacking a large portion of its predicted coil forming domains as well as both leucine zipper motifs that are required for protein-protein interactions with ODF1, another major component of the outer dense fibers. Although wild-type and heterozygous mice were recovered, no mice homozygous for the Odf2 gene trap insertion were recovered in an extended breeding program. Furthermore, no homozygous embryos were found at the blastocyst stage of embryonic development, implying a critical pre-implantation role for Odf2. We show that Odf2 is expressed widely in adults and is also expressed in the blastocyst stage of preimplantation development. These findings are in contrast with early studies reporting Odf2 expression as testis specific and suggest that embryonic Odf2 expression plays a critical role during preimplantation development in mice.     

Characterization, chromosomal assignment, and tissue expression of a novel human gene belonging to the ARF GAP family

We have identified and characterized a novel human ADP-ribosylation factor GTPase-activating protein (ARFGAP1) gene that is related to other members of the ARF GAP family. The full-length cDNA for human ARFGAP1 was cloned following the identification of an EST obtained by large-scale cDNA library sequencing through a Blast search of public databases. Structurally, ARFGAP1 encodes a polypeptide of 516 amino acids, which contained a typical GATA-1-type zinc finger motif (CXXCX(16)CXXC) with the four cysteine residues that are highly conserved among other members of the ARF GAP family. The conserved ARF GAP domain may emphasize the biological importance of this gene. The ARFGAP1 gene, which contained 16 exons ranging from 0.5 to 9.3 kb, was mapped to human chromosome 22q13.2-q13.3 using radiation hybridization and in silico analyses. ARFGAP1 is strongly expressed in endocrine glands and testis. Interestingly, the expression of ARFGAP1 in testis is about sixfold higher than that in ovary, indicating a possible role of ARFGAP1 in the physiological function of sperm. Expression of ARFGAP1 in four human fetal tissues and seven cancer cell lines was also detected.     

Genomic cloning and chromosomal assignment of rat regucalcin gene

The gene for a Ca²⁺-binding protein regucalcin was cloned from a rat genomic library which was constructed in FIX II by screening with radiolabeled probe (complementary DNA of rat liver regucalcin). Positive clone had 19.9 kb insert of size and contained four exons of the gene coding for a rat regucalcin. These exons included the partial coding sequence (61.2% of open reading frame) and the entire 3-untranslated region of the gene. The nucleotide sequence of exons completely agreed with that of a rat regucalcin cDNA clone. The sequence analysis of the clone showed that the identifier sequence and two simple repeated sequences exist in the intron of the gene. Moreover, chromosomal location of the rat regucalcin gene was determined by direct R-banding fluorescencein situ hybridization (FISH) method with the 19.9 kb clone containing four exons. The regucalcin gene was localized on rat chromosome Xq11.1–12 proximal end.The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases with the following accession number D31662     

人类Connexin基因家族新成员-Cx44基因的克隆和特性分析

用绵羊和牛的Cx44基因(connexin 44 protein gene)序列对NCBI数据库进行Blast检索,得到一个相似性很高的人DNA序列(Human Genome Bank:AL138688),用GENSCAN程序分析AL138688,推测AL138688中包含一个编码区由1个外显子构成的基因——Cx44基因。人Cx44基因的开放阅读框为1320bp,推测编码435个氨基酸。用PROMOTORSCAN程序分析了其启动子。人Cx44基因与绵羊Cx44基因在1320bp有84.75％的一致性,其表达的蛋白有83％的一致性。用Map View将人的Cx44基因定位于13号染色体。     

Sequence and chromosomal assignment of a human novel cDNA: similarity to gamma-aminobutyric acid transporter

Gamma-aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the mammalian brain. Although initially thought to be confined to the central nervous system, GABAergic activity has also been described in other tissues throughout the body. In the present study, we report the cloning and localization of human GABA transporter cDNA and document its expression in various human tissues. A human liver cDNA library was initially screened by a 32P-labeled murine brain GABA transporter 3 (GAT-3) cDNA probe, and full-length cDNA was cloned by employing Marathon-Ready human kidney cDNA. The human GABA transporter cDNA encoded a 569 amino acid hydrophobic protein with 12 transmembrane domains (TMs). Search of published sequences revealed high homology with rat GAT-2, murine GAT-3 cDNA, human solute carrier family 6 member 13 (SLC6A13), and a human peripheral betaine/GABA transporter. Northern blot analyses demonstrated that the human GABA transporter is expressed strongly in the kidney and to a lesser extent in the liver and brain. The sequence was well matched with human chromosome 12p13.3, suggesting the human GABA transporter contains 14 exons. The above findings confirm the existence of and further characterize a specific GABA transporter in human tissues.     

Molecular cloning and chromosomal assignment of a human perforin (PFP) gene

Human perforin cDNA was isolated and the complete nucleotide sequence of the gene determined. The deduced amino acid sequence of human perforin showed 68.4% similarity to that of mouse perforin. RNA blot analysis of the human perforin gene revealed that the gene product is expressed preferentially in killer-type cells among cell lines tested, and in large granular lymphocytes among the peripheral blood mononuclear cells. In situ hybridization analysis with a human perforin cDNA probe revealed that the human perforin (PFP) gene is located on chromosome17q11-21. The nucleotide sequence data reported in this paper have been submitted to the GeBank nucleotide sequence database and have been assigned the accession number M28393.     

Differential expression of porcine sperm microRNAs and their association with sperm morphology and motility

MicroRNAs (miRNAs) are involved in nearly every biological process examined to date, but little is known of the identity or function of miRNA in sperm cells or their potential involvement in spermatogenesis. The objective was to identify differences in miRNA expression between normal porcine sperm samples and those exhibiting high percentages of morphological abnormalities or low motility. Quantitative RT-PCR was performed on sperm RNA to compare expression levels of 10 specific miRNAs that are predicted to target genes that code for proteins involved in spermatogenesis, sperm structure, motility, or metabolism. There were increases in the expression of four miRNAs, let-7a, -7d, -7e, and miR-22, in the abnormal group (P < 0.05), whereas miR-15b was decreased compared to controls (P < 0.05). Two miRNAs, let-7d and let-7e, were increased in the low motility group when compared to controls (P < 0.05). Bioinformatic analyses revealed that messenger RNA targets of the differentially expressed miRNAs encode proteins previously described to play roles in sperm function. Although the precise role of miRNA in sperm remains to be determined, their changes as associated with morphology and motility signify a critical biological function. Perhaps they are remnants of spermatogenesis, stored for a later role in fertilization, or are delivered to the oocyte to influence early embryonic development. Although there is no single cause of male infertility, the identification of miRNAs associated with sperm motility, structural integrity, or metabolism could lead to the development of a microarray or real time-based diagnostic assay to provide an assessment of male fertility status.     

The position of integration affects expression of the γ-globin-encoding gene linked to HS3 in transgenic mice

Proper expression of the human β-globin (βGlb) locus is dependent on the presence of a major regulatory element located upstream from the βGlb gene cluster, the locus control region (LCR). The LCR, as well as the individual DNase-I-hypersensitive sites from which it is composed, have been shown to provide position-of-integration-independent expression in transgenic mice. Here, we report that a transgenic founder carrying multiple integrations of a hypersensitive site 3::^Aγ globin gene (HS3::^Aγ) construct produced three types of progeny, one with zero ^Aγ expression in the adult stage, one with minimal ^Aγ expression (1% of ^Aγ-expressing cells) and one with abundant ^Aγ expression (100% ^Aγ-expressing cells). The possibility that these phenotypes were due to parental imprinting or to DNA rearrangements of the transgene or to point mutations of the HS3 core or the ^Aγ promoter were excluded. The pattern of inheritance of the three HS3::^Aγ transgene phenotypes indicate that the transgene has integrated into three different chromosomes. These results provide direct evidence that the HS3 of the LCR is not sufficient to protect the ^Aγ gene from position effects excerted by the surrounding chromatin.     

Isolation and chromosomal assignment of a novel human gene, CORO1C, homologous to coronin-like actin-binding proteins

We have isolated a gene, termed CORO1C (human coronin-like actin-binding protein 1C), that encodes a new member of the coronin-like family of proteins. The cDNA consists of 3,857 nucleotides, with an open reading frame of 1,422 bp encoding a 474 amino acid protein. The deduced amino acid sequence shared 65% identity with p57 (human coronin-like actin-binding protein), as well as 46% identity with coronin, a protein first isolated from the slime mold Dictyostelium discoideum. Computer analysis predicted that the product of the CORO1C gene would contain five WD repeats in its N-terminal region and a coiled-coil motif in its C-terminal region, both of which are conserved among coronin-like proteins. CORO1C was ubiquitously expressed in all human tissues examined, in contrast to other known coronin-like molecules, each of which is expressed in a tissue-specific manner. Immunocytochemical staining demonstrated that CORO1C was co-localized with F-actin; therefore, the gene product is likely to be important in cytokinesis, motility, and signal transduction, as are the other members of this molecular family. We assigned this novel gene to chromosome 12q24.1 by fluorescence in situ hybridization.