cDNA Cloning and Characterization of a Human Sperm Antigen (SPAG6) with Homology to the Product of the Chlamydomonas PF16 Locus

Serum from an infertile male with high-titer anti-sperm antibodies was used to identify a novel human sperm antigen by screening of a testis expression library. The clone, initially designated Repro-SA-1 (HUGO-approved symbol SPAG6), was found to encode a sequence highly enriched in testis. The deduced amino acid sequence of the full-length cDNA revealed striking homology to the product of the Chlamydomonas reinhardtii PF16 locus, which encodes a protein localized to the central pair of the flagellar axoneme. The human gene encodes 1.8- and 2.8-kb mRNAs highly expressed in testis but not in prostate, ovary, spleen, thymus, small intestine, colon, peripheral blood leukocytes, heart, brain, placenta, liver, muscle, kidney, and pancreas. The gene was mapped to chromosome 10p11.2-p12. Antibodies raised against SPAG6 sequences localized the protein to the tails of permeabilized human sperm. Both the Chlamydomonas protein and SPAG6 contain eight contiguous armadillo repeats, which place them in a family of proteins known to mediate protein-protein interactions. The cloning of the human homologue of the Chlamydomonas PF16 locus provides a new avenue to explore the role of the axoneme central pair in human sperm function.     

Molecular characterization of two isoforms of ZFAND3 cDNA from the Japanese quail and the leopard gecko, and different expression patterns between testis and ovary

Zing finger AN1-type domain 3 (ZFAND3), also known as testis expressed sequence 27 (Tex27), is a gene found in the mouse testis, but its physiological function is unknown. We identified the full-length sequences of two isoforms (short and long) of ZFAND3 cDNA from Japanese quail and leopard gecko. This is the first cloning of avian and reptilian ZFAND3 cDNA. The two isoforms are generated by alternative polyadenylation in the 3'UTR and have the same ORF sequences encoding identical proteins. There were highly conserved regions in the 3'UTR of the long form near the polyadenylation sites from mammals to amphibians, suggesting that the features for determining the stability of mRNA or translation efficiency differ between isoforms. The deduced amino acid sequence of ZFAND3 has two putative zinc finger domains, an A20-like zinc finger domain at the N-terminal and an AN1-like zinc finger domain at the C-terminal. Sequence analysis revealed an additional exon in the genomic structures of the avian and reptilian ZFAND3 genes which is not present in mammals, amphibians, or fish, and this exon produces additional amino acid residues in the A20-like zinc finger domain. Expression analysis in Japanese quail revealed that the expression level of ZFAND3 mRNA was high in not only the testis but also the ovary, and ZFAND3 mRNA was expressed in both spermatides of the testis and oocytes of the ovary. While the short form mRNA was mainly expressed in the testis, the expression level of the long form mRNA was high in the ovary. These results suggest that ZFAND3 has physiological functions related to germ cell maturation and regulatory mechanisms that differ between the testis and ovary.     

Molecular cloning, expression, and chromosomal mapping of human chondroitin 4-sulfotransferase, whose expression pattern in human tissues is different from that of chondroitin 6-sulfotransferase

Chondroitin 4-sulfotransferase (C4ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of the N-acetylgalactosamine residues of chondroitin. We previously reported the cloning of C4ST cDNA from mouse brain. We here report the cloning and expression of human C4ST cDNA. The cDNA was isolated from a human fetal brain cDNA library by hybridization with a DNA probe prepared from rat poly(A)(+) RNA used for the cloning of mouse C4ST cDNA. The cDNA comprises a single open reading frame that predicts a Type II transmembrane protein composed of 352 amino acids. The protein has an amino acid sequence homology of 96% with mouse C4ST. When the cDNA was introduced into a eukaryotic expression vector and transfected in COS-7 cells, the sulfotransferase activity that transfers sulfate to both chondroitin and desulfated dermatan sulfate was overexpressed. Northern blot analysis indicated that human C4ST mRNAs (6.0 and 1.9 kb) are expressed ubiquitously in various adult human tissues. Dot blot analysis has shown that human C4ST is strongly expressed in colorectal adenocarcinoma and peripheral blood leukocytes, whereas strong expression of human chondroitin 6-sulfotransferase (C6ST) is observed in aorta and testis. These observations suggest that the expression of C4ST and C6ST may be controlled differently in human tissues. The C4ST gene was localized to chromosome 12q23.2-q23.3 by fluorescence in situ hybridization.     

Isolation, localization, and cloning of a kainic acid binding protein from frog brain

Excitatory amino acids (EAA) are major neurotransmitters in the vertebrate central nervous system. EAA receptors have been divided into three major subtypes on the basis of electrophysiological and ligand binding studies: N-methyl-D-aspartate, kainate, and quisqualate receptors. To understand their molecular properties, we undertook a project aimed at isolation and cloning of these receptor subtypes. We purified a kainate binding protein (KBP) from frog brain, in which kainate binding sites are about fortyfold more abundant than in rat brain, using domoic acid affinity chromatography, and made monoclonal and polyclonal antibodies to the purified protein. These antibodies immunoprecipitate the frog KBP but not KBPs from other species. Immunocytochemical analyses show that KBP has a synaptic and extrasynaptic localization in frog optic tectum, with most labeling being extrasynaptic. The cDNA encoding frog brain KBP was isolated by screening a frog brain cDNA library with oligonucleotide probes that were based on the amino acid sequence of the purified protein. The deduced amino acid sequence of the KBP has a hydrophobic profile similar to those of other ligand-gated ion channel subunits, such as the nicotinic acetylcholine receptor, the GABAA receptor, and the glycine receptor. Frog brain KBP is very similar (36% amino acid identity to the carboxyl half) to rat brain kainate receptor, suggesting that these two proteins evolved from a common ancestor. The function of KBP in frog brain remains a major question. Preliminary results showed that Xenopus laevis oocytes injected with KBP RNA did not produce a detectable electrophysiological response when perfused with kainate. These results suggest that additional subunits may be required to form a functional receptor or that KBP is not functionally related to a neurotransmitter receptor.     

利用电子差异展示方法克隆人类睾丸高表达新基因SPATA11

利用NCBI中的电子差异展示(digital differential display,DDD)软件,比较来自睾丸(包括睾丸癌)与来自其它组织的EST文库,从筛查人类睾丸中高表达而在其他组织中不表达或低表达的差异ESTs入手,成功克隆了一个在人类睾丸中高表达的新基因SPATA11．RT-PCR实验证实其在成人睾丸高表达．序列分析表明该基因含4个外显子,基因组跨越2．6kb,定位于19pl3．3．cDNA编码一个含221个氨基酸,相对分子质量为24．5kD的新蛋白．Northern杂交结果显示：该基因含有1．1kb大小的唯一转录本,主要在睾丸中强表达．肝脏、肺、卵巢和肾脏中有微弱表达．而其他组织中该基因无表达．     

Cloning and expression pattern of the human NDRG3 gene

We report the cloning and expression pattern of a novel N-myc downstream-regulated gene 3 (NDRG3), located on human chromosome 20q11.21-11.23. The NDRG3 cDNA is 2588 base pair in length, encoding a 363 amino acid polypeptide highly related to mouse Ndr3 protein. Northern blot reveals that NDRG3 is highly expressed in testis, prostate and ovary. By in situ hybridization, the NDRG3 mRNA was localized to the outer layers of seminiferous epithelium, indicating that it may play a role in spermatogenesis.     

A new approach for the detection of multiple protein kinases using monoclonal antibodies directed to the highly conserved region of protein kinases

To explore the protein kinase family enzymes expressed in cells, we attempted to generate antibodies that could detect a wide variety of protein kinases. For the production of such antibodies, synthetic peptides corresponding to amino acid sequences of a highly conserved subdomain (subdomain VIB) of the protein kinase family were used for immunization. Among the various peptide antigens, a peptide with 16 amino acids, CVVHRDLKPENLLLAS, effectively produced polyclonal antibodies with broad cross-reactivities to protein kinases. Two monoclonal antibodies, designated M8C and M1C, detected a variety of protein kinases such as calmodulin-dependent protein kinase II, calmodulin-dependent protein kinase IV, cAMP-dependent protein kinase, and mitogen-activated protein kinases, on Western blotting. The antibodies also immunoprecipitated various protein kinases in cell extracts. Furthermore, these antibodies could be used for detection of positive clones in the expression cloning of various protein kinases. Among 39 positive clones obtained from mouse brain cDNA library, 36 clones were identified as cDNA clones for various known and novel protein serine/threonine kinases, suggesting that the antibodies reacted highly specifically with various protein kinases. These results indicate that the present monoclonal antibodies directed to multiple protein kinases will be a powerful tool for the detection of a variety of known and novel protein kinases in cells.     

Molecular cloning of rat liver 3α-hydroxysteroid dehydrogenase and identification of structurally related proteins from rat lung and kidney

3α-Hydroxysteroid dehydrogenase and related enzymes play important roles in the metabolism of endogenous compounds including androgens, corticosteroid, prostaglandins and bile acids, as well as drugs and xenobiotics such as benzo(a)pyrene. Complementary DNA clones encoding 3α-hydroxysteroid dehydrogenase were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. A full-length cDNA clone of 1286 base pairs contained an open reading frame encoding a protein of 322 amino acids with an estimated M(w) of 37 kD. When expressed in E. coli, the encoded protein migrated to the same position on SDS-polycrylamide gels as the enzyme in rat liver cytosols. The protein expressed in bacteria was highly active in androsterone oxidation in the presence of NAD as cofactor and this activity was inhibited by indomethacin, a potent inhibitor of 3α-hydroxysteroid dehydrogenase. The predicted amino acid sequence of 3α-hydroxysteroid d dehydrogenase was related to sequences of several other aldo-keto reductases such as bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase and frog lens epsilon-crystallin, suggesting that these proteins belong to the same gene family. Recently, we have found that monoclonal antibodies against 3α-hydroxysteroid dehydrogenase also recognized multiple antigenically related proteins in rat lung, kidney and testis. Further screening of liver, lung and kidney cDNA libraries using these monoclonal antibodies as probes resulted in the isolation of additional five different cDNAs encoding proteins with high degree of structural homology to rat liver 3α-hydroxysteroid dehydrogenase.     

Cloning and characterization of the mouse Interleukin Enhancer Binding Factor 3 (Ilf3) homolog in a screen for RNA binding proteins

In a screen for RNA-binding proteins expressed during murine spermatogenesis, we have identified a cDNA that encodes a protein of 911 amino acids that contains two copies of the double-stranded RNA-binding motif and has 80% identity with human Interleukin Enhancer Binding Factor 3 (ILF3). Linkage and cytogenetic analyses localized the Ilf3 cDNA to a portion of mouse Chr 9, which shows conserved synteny with a region of human Chr 19 where the human ILF3 gene had been previously localized, supporting that we had cloned the murine homolog of ILF3. Northern analysis indicated the Ilf3 gene is ubiquitously expressed in mouse adult tissues with high levels of expression in the brain, thymus, testis, and ovary. Polyclonal antibodies detected multiple protein species in a subset of the tissues expressing Ilf3 RNA. Immunoreactive species are present at high levels in the thymus, testis, ovary, and the spleen to a lesser extent. The high degree of sequence similarity between the mouse ILF3 protein and other dsRNA binding motif-containing proteins suggests a role in RNA metabolism, while the differential expression indicates the mouse ILF3 protein predominantly functions in tissues containing developing lymphocyte and germ cells. Received: 21 October 1998 / Accepted: 15 January 1999     

Molecular cloning, mapping and characterization of the human neurocalcin delta gene (NCALD)

We identified a new human gene that encodes a cognate of the bovine neurocalcin delta from a human fetal brain cDNA library; hence we named it human neurocalcin delta (NCALD) gene. The deduced polypeptide product of the cDNA is 22 kDa in size, and its amino acid sequence is 100% and 99% identical to that of the bovine and chicken neurocalcin, respectively. Northern blots showed that the NCALD gene is more abundantly expressed in brain, testis, ovary and small intestine. Tissue in situ hybridization confirmed the existence of the NCALD mRNA in the adult human testis. Radiation hybrid panel mapping localized the gene to chromosome 8 between molecular markers D8S270 and D8S257.     

Type XXVI collagen,a new member of the collagen family,is specifically expressed in the testis and ovary

HSP47 is a collagen-specific molecular chaperone that specifically recognizes and binds to the triple helical domain of various types of collagens. Here we report the cloning of the entire coding region of a novel collagen-like protein by yeast two-hybrid screening of a 17.5-day whole mouse embryo cDNA library using HSP47 as a bait. The cDNA of this protein and its deduced amino acid sequence are 2,690 bp and 438 amino acids long, respectively. The protein contains two clusters of Gly-X-Y collagenous repeats and three noncollagenous domains. Northern blot analysis showed that its mRNA is specifically expressed in the testis and ovary in adult tissues and that expression in these tissues is highest in the neonate. Biochemical characterization of this protein revealed that its proline residues are hydroxylated, it undergoes N-linked glycosylation, it forms trimers, and it is secreted in vitro. Immunohistochemical studies showed that the myoid cells and the pre-theca cells synthesized it in the testis and ovary, respectively, resulting in the accumulation of this protein in the extracellular spaces of these organs. These observations suggest that this protein is a new member of the collagen protein family. We thus designated this protein as type XXVI collagen.     

The inducible blood--brain barrier specific molecule HT7 is a novel immunoglobulin-like cell surface glycoprotein.

The unique properties of brain endothelial cells, which form the blood-brain barrier, are reflected by the expression of specific cell surface molecules. We report here the purification, cloning and expression of one such molecule which is recognized by HT7 monoclonal antibodies. The HT7 antigen is a highly glycosylated 45-52 kd protein localized in brain endothelial cells, kidney epithelial cells and erythroblasts. The protein was purified to homogeneity from plasma membrane proteins isolated from all three sources using immunoaffinity chromatography and reverse phase HPLC. The amino-terminal amino acid sequences of the proteins were found to be identical. Based on amino acid sequence information, specific primers were designed and the polymerase chain reaction was used to obtain a full length cDNA clone. The nucleotide sequence encoded a novel glycoprotein with two C2-like immunoglobulin related domains, one transmembrane domain and a cytoplasmic tail. Expression of the transfected cDNA in COS cells resulted in the appearance of the HT7 antigen on the surface of these cells. On the basis of our results we propose that the protein may be a receptor involved in cell surface recognition at the blood-brain barrier.     

Cloning and characterization of a novel human SPRYD4 gene encoding a putative SPRY domain-containing protein.

We report here the cloning and characterization of a novel human SPRYD4 gene which encodes a SPRY domain containing protein. The SPRYD4 gene is isolated from the human brain cDNA library, and mapped to 12q13.2 by searching the UCSC genomic database. The SPRYD4 cDNA is 1201 base pairs in length and contains an open reading frame encoding 207 amino acids. The SPRYD4 gene consists of two exons and encodes a putative protein with a SPRY domain ranging from 86 to 203 amino acids. The RT-PCR analysis reveals that SPRYD4 is ubiquitously expressed in 18 human tissues. However, it is strongly expressed in kidney, bladder, brain, thymus and stomach, while weakly expressed liver, testis, uterus, spleen and lung. Subcellular localization demonstrates that SPRYD4 protein is localized in the nuclear when overexpressed in COS-7 cell.     

Identification of a brain-specific 27/26-kDa extracellular protein with the monoclonal antibody to differentiated PC12h pheochromocytoma cells

We have searched for brain-specific extracellular molecules using a library of monoclonal antibodies against surface antigens of differentiated PC12h cells. One of the monoclonal antibodies, PCH42-14, recognized a 27/26-kDa protein of 10-week-old rat brain on immunoblotting. PCH42-14 antigen was detected only in brain, especially in cerebrum, olfactory bulb, mesencephalon, hippocampus, medulla oblongata, and spinal cord. On hippocampal neuron culture, PCH42-14 antigen existed extracellularly along with the neuronal extensions.