NORMAL GENOMIC

• Neziri D et al.
• Cloning and molecular characterization of Dashurin encoded by C20orf116, aPCI-domain containing protein.
• Biochim Biophys Acta. 2010; 1800: 430-8
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• BACKGROUND: Characterization of gene products originating from undefinedopen reading frames and delineation of biological functions has become thetask after the human genome has been decoded. METHODS: We cloned the humanC20orf 116 and defined its transcript in liver, kidney and various brainregions by Northern analysis. Antibodies against recombinant protein usedfor immunofluorescence and immunoblots confirmed its expression in thesetissues. With the focus on kidney, its tubular expression and presence inglomerula were shown. RESULTS: A 28 aa long signal peptide predicted by insilico analysis is reflected by visualization of size variants ofapproximately 3kDa difference suggesting a signal peptidase cleavage ofthe proform. Cell compartment separation confirmed the presence ofDashurin in peroxisomes/mitochondria, microsomes, cytosol and nucleus.This is in line with green fluorescent protein (GFP)-Dashurin fusionprotein shuttling between cytosol and nucleus. Luciferase reporter studiesrevealed a 2-3 fold increase of promoter activities upon over-expression.Bioinformatic analysis identified a PCI-domain at the C-terminus providingprotein-protein interaction capabilities. CONCLUSION: Our present findingssuggest the involvement of Dashurin in gene transcription or mRNAtranslation. GENERAL SIGNIFICANCE: Dashurin shares the PCI-domain withthree multisubunit protein complexes (26S proteasome, COP9 signalosome andeIF3 translation initiation factor).

• Shimizu H, Taniguchi H, Hippo Y, Hayashizaki Y, Aburatani H, Ishikawa T
• Characterization of the mouse Abcc12 gene and its transcript encoding anATP-binding cassette transporter, an orthologue of human ABCC12.
• Gene. 2003; 310: 17-28
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• We have recently reported on two novel human ABC transporters, ABCC11 andABCC12, the genes of which are tandemly located on human chromosome16q12.1 [Biochem. Biophys. Res. Commun. 288 (2001) 933]. The present studyaddresses the cloning and characterization of Abcc12, a mouse orthologueof human ABCC12. The cloned Abcc12 cDNA was 4511 bp long, comprising a4101 bp open reading frame. The deduced peptide consists of 1367 aminoacids and exhibits high sequence identity (84.5%) with human ABCC12. Themouse Abcc12 gene consists of at least 29 exons and is located on themouse chromosome 8D3 locus where conserved linkage homologies havehitherto been identified with human chromosome 16q12.1. The mouse Abcc12gene was expressed at high levels exclusively in the seminiferous tubulesin the testis. In addition to the Abcc12 transcript, two splicing variantsencoding short peptides (775 and 687 amino acid residues) were detected.In spite of the genes coding for both ABCC11 and ABCC12 being tandemlylocated on human chromosome 16q12.1, no putative mouse orthologous genecorresponding to the human ABCC11 was detected at the mouse chromosome 8D3locus.

• Ma YX, Zhang SZ, Hou YP, Huang XL, Wu QQ, Sun Y
• Identification of a novel human zinc finger protein gene ZNF313.
• Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2003; 35: 230-7
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• A novel human zinc finger protein gene that contains both ring finger andC(2)H(2) domain was first isolated by mRNA differential display betweenthe testes of fertile adults and azoospermic patients followed by rapidamplification of cDNA ends (RACE). Total 6 exons of the human gene span a17,484 bp genomic DNA sequence that was mapped to chromosome 20q13 byfluorescence in situ hybridization. The mature processed mRNA encodes a228-amino acid protein with a C(3)HC(4) ring finger and three C(2)H(2)domains. Genomic analysis of the human gene identified two polyadenylationsignals in exon 6 resulting in alternative 3'-untranslated regions.Results of Northern blot and RT-PCR of RNAs extracted from multipletissues revealed that the gene has two transcripts of which the shortertranscript was expressed abundantly in fertile adult testes, but much lessin testes of azoospermic patient, fetus as well as other human tissues.These data suggest that the gene may play a role in human spermatogenesisand male fertility.

• Peng J et al.
• Identification of human CDV-1R and mouse Cdv-1R, two novel proteins withputative signal peptides, especially highly expressed in testis andincreased with the male sex maturation.
• Mol Biol Rep. 2002; 29: 353-62
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• Human systemic carnitine deficiency (SCD) is a hereditary disease causedby the mutation of OCTN2 and has the characteristics of cardiachypertrophy. Previous studies based on JVS mouse, an animal model of thisdisease, showed that Cdv-1 was highly expressed in ventricles of normalmouse, but was remarkably down-regulated in JVS mouse and can beup-regulated to normal level by breeding carnitine, which suggested Cdv-1was possibly involved in cardiac hypertrophy caused by carnitinedeficiency. In this study, the expression of human CDV-1, a homolog ofmouse Cdv-1, was undetectable in heart by northern hybridization. Theinconsistent expression levels of human CDV-1 and mouse Cdv-1 in heartimplied that cardiac hypertrophy in human SCD might not be associated withthe abnormal expression of CDV-1. Interestingly, another long transcriptsof the gene, Cdv-1R/CDV-1R, were cloned in the present study, in mouse andhuman, respectively. This long transcript predominantly expressed in bothhuman and mouse testis and its expression level was increased with testisdevelopment. Furthermore, we proved that the open reading frame ofCdv-1R/CDV-1R spans the exons 2 approximately 19 instead of exons 9approximately 19; and the peptide encoded by CDV-1R was composed of 676amino acids containing a putative signal peptide instead of 414 aminoacids described previously. In addition, it was proved that the expressionlevel of Cdv-1R in JVS mouse testis was as high as that in normal mousetestis, and both were not regulated by carnitine.

• Scarman AL et al.
• Organization and chromosomal localization of the murine Testisin geneencoding a serine protease temporally expressed during spermatogenesis.
• Eur J Biochem. 2001; 268: 1250-8
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• The recently characterized human serine protease, Testisin, is expressedon premeiotic testicular germ cells and is a candidate type II tumorsuppressor for testicular cancer. Here we report the cloning,characterization and expression of the gene encoding mouse Testisin,Prss21. The murine Testisin gene comprises six exons and five introns andspans approximately 5 kb of genomic DNA with an almost identical structureto the human Testisin gene, PRSS21. The gene was localized to murinechromosome 17 A3.3-B; a region syntenic with the location of PRSS21 onhuman chromosome 16p13.3. Northern blot analyses of RNA from a range ofadult murine tissues demonstrated a 1.3 kb mRNA transcript present only intestis. The murine Testisin cDNA shares 65% identity with human TestisincDNA and encodes a putative pre-pro-protein of 324 amino acids with 80%similarity to human Testisin. The predicted amino-acid sequence includesan N-terminal signal sequence of 27 amino acids, a 27 amino-acidpro-region, a 251 amino-acid catalytic domain typical of a serine proteasewith trypsin-like specificity, and a C-terminal hydrophobic extensionwhich is predicted to function as a membrane anchor. Immunostaining formurine Testisin in mouse testis demonstrated specific staining in thecytoplasm and on the plasma membrane of round and elongating spermatids.Examination of murine Testisin mRNA expression in developing spermconfirmed that the onset of murine Testisin mRNA expression occurred atapproximately day 18 after birth, corresponding to the appearance ofspermatids in the testis, in contrast to the expression of human Testisinin spermatocytes. These data identify the murine ortholog to humanTestisin and demonstrate that the murine Testisin gene is temporallyregulated during murine spermatogenesis.

• Naik UP, Naik MU, Eckfeld K, Martin-DeLeon P, Spychala J
• Characterization and chromosomal localization of JAM-1, a plateletreceptor for a stimulatory monoclonal antibody.
• J Cell Sci. 2001; 114: 539-47
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• We have previously reported the purification and characterization of a 32kDa platelet surface glycoprotein that is recognized by the stimulatorymonoclonal antibody, F11. The cDNA has been cloned and found to encode thehuman homolog of the murine junctional adhesion molecule, JAM; wetherefore named this human homolog JAM-1. Northern blot analysis indicatedthat JAM-1 mRNA is expressed as multiple species, the predominanttranscript being approximately 4.0 kb in size. Genetic mapping analysisusing fluorescence in situ hybridization (FISH) showed that it islocalized to chromosome 1q21.1-21.3. Recombinant JAM-1, when expressed inChinese hamster ovary (CHO) cells, localized to the cell membrane withintense staining where two adjacent cells actually made contact with eachother, suggesting that, similar to murine JAM, human JAM-1 may alsolocalize at the cell-cell junction. In well-spread cells, JAM-1co-localized with F-actin at the cell-cell contacts and at the membraneruffles, but not at the stress fibers. Interestingly, JAM-1 localizes onlyto the cell-cell junctions formed by two transfected cells and not to thecell-cell junctions formed by a transfected cell with an untransfectedcell, suggesting that JAM-1 may facilitate cell adhesion throughhomophilic binding. In addition, human platelets specifically bind to amonolayer of CHO cells expressing human JAM-1, further supportinghomophilic interactions. The results presented here indicate that JAM-1, areceptor for a platelet-activating antibody, is the human homolog of thejunctional adhesion molecule. JAM-1 is a single copy gene, which isconstitutively expressed on various tissues and cells, and may be involvedin cell to cell adhesion through homophilic interaction.

• Communi D, Suarez-Huerta N, Dussossoy D, Savi P, Boeynaems JM
• Cotranscription and intergenic splicing of human P2Y11 and SSF1 genes.
• J Biol Chem. 2001; 276: 16561-6
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• The P2Y(11) receptor is an ATP receptor positively coupled to the cAMP andphosphoinositide pathways. Ssf1 is a Saccharomyces cerevisiae nuclearprotein, which plays an important role in mating. The gene encoding thehuman orthologue of SSF1 is adjacent to the P2Y(11) gene on chromosome 19.During the screening of placenta cDNA libraries, we isolated a chimericclone resulting from the intergenic splicing between the P2Y(11) and SSF1genes. The fusion protein was stably expressed in CHO-K1 cells where itgenerated a cAMP response to ATP qualitatively indistinguishable from thatof the P2Y(11) receptor. According to both Western blotting and cAMPresponse, the expression of the fusion protein in the transfected cellswas clearly lower than that of the P2Y(11) receptor. Both P2Y(11) and SSF1probes detected a 5.6-kb messenger RNA with a similar pattern of intensityin each of 11 human tissues. The ubiquitous presence of chimerictranscripts and their up-regulation during granulocytic differentiationindicate that the transgenic splicing between the P2Y(11) and the SSF1genes is a common and regulated phenomenon. There are very few examples ofintergenic splicing in mammalian cells, and this is the first caseinvolving a G-protein-coupled receptor.

• Hirahara Y, Tsuda M, Wada Y, Honke K
• cDNA cloning, genomic cloning, and tissue-specific regulation of mousecerebroside sulfotransferase.
• Eur J Biochem. 2000; 267: 1909-17
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• We have isolated a mouse cDNA clone encoding3'-phosphoadenylylsulfate-galactosylceramide 3'-sulfotransferase(cerebroside sulfotransferase; CST; EC 2.8.2.11) from a kidney cDNAlibrary, using a human CST cDNA clone [Honke, K., Tsuda, M., Hirahara, Y.,Ishii, A., Makita, A. & Wada, Y. (1997) J. Biol. Chem. 272, 4864-4868] asa probe. A recombinant protein of the cloned cDNA showed CST activity. Thededuced protein is composed of the same 423 amino acids as human CST andits sequence exhibits 84% identity with that of the human counterpart.Northern-blot analysis and subquantitative reverse transcription-PCR(RT-PCR) analysis showed that the CST gene is preferentially transcribedin stomach, small intestine, brain, kidney, lung, and testis, in thatorder. To examine differences in transcripts in various tissues, weisolated CST cDNA clones from stomach, small intestine, brain, kidney, andtestis by 5'-RACE analysis. We found seven different nucleotide sequencesin the 5'-UTR, while the DNA sequences of all the isolated cDNA cloneswere identical in the coding region. In addition, we isolated CST genomicDNA clones from a mouse genomic library. The clones covered all the 5'-UTRsequences and coding exons including 3'-UTR. RT-PCR analyses of CST mRNAsfrom various tissues confirmed that CST transcripts aretissue-specifically spliced by alternative use of multiple exons 1. Theseobservations suggest that the tissue-specific expression of the CST geneis explained by alternative usage of multiple 5'-UTR exons flanked withtissue-specific promoters.

• Liu Q et al.
• Molecular cloning and tissue expression analysis of the beta subunit ofelongation factor 1 in the mouse.
• Biochem Genet. 2000; 38: 111-7

• Lansdell SJ, Millar NS
• Cloning and heterologous expression of Dalpha4, a Drosophila neuronalnicotinic acetylcholine receptor subunit: identification of an alternativeexon influencing the efficiency of subunit assembly.
• Neuropharmacology. 2000; 39: 2604-14
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• A neuronal nicotinic acetylcholine receptor (nAChR) subunit, Dalpha4, hasbeen identified and cloned from the fruit fly Drosophila melanogaster,together with several alternatively spliced transcripts. Intron-exonboundaries within the gene encoding Dalpha4 (nAcRalpha-80B) have beenidentified by comparison of cDNA and genomic sequence data. The influenceof amino acids encoded by alternatively spliced exons upon nicotinicradioligand binding and subunit-subunit co-assembly has been examined byheterologous expression in Drosophila S2 cells. The efficiency of subunitassembly has been shown to be influenced by amino acids surrounding thehighly conserved 15 amino acid cysteine-loop motif within the N-terminalextracellular domain of the nAChR Dalpha4 subunit. Extensive use has beenmade of publicly available data determined by the Berkeley DrosophilaGenome Project (BDGP). This includes expressed sequence tag (EST) data aswell as whole-embryo in situ hybridisation and polytene chromosome in situhybridisation data. BDGP in situ hybridisation data suggests that theDalpha4 mRNA is expressed within Drosophila brain and ventral nerve cordand demonstrates that the gene encoding this nAChR subunit is located atposition 80B on chromosome 3. The relationship between Dalpha4 and otherpreviously cloned nAChR subunits has been examined and the implicationsfor the nomenclature of insect nAChRs is discussed.

• Masat L et al.
• Mapping of the SWAP70 gene to mouse chromosome 7 and human chromosome11p15.
• Immunogenetics. 2000; 51: 16-9
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• The protein SWAP-70 was isolated as part of a DNA recombination complex inB lymphocytes, where it is predominantly expressed. In resting B cells,SWAP-70 is found in the cytoplasm; upon B-cell activation, it istransported both into the nucleus and to the cell membrane, where it isassociated with the B-cell receptor complex and may play a role in signaltransduction. In the nucleus, its involvement in heavy-chain class switchrecombination has been suggested. In this report, using restrictionfragment length polymorphism, simple sequence length polymorphism, andfluorescence in situ hybridization, we map the chromosomal localization ofthe mouse and the human genes to syntenic regions of mouse mid Chromosome(Chr) 7 and human Chr 11p15.

• Yanaka N et al.
• Insertional mutation of the murine kisimo locus caused a defect inspermatogenesis.
• J Biol Chem. 2000; 275: 14791-4
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• Spermatogenesis is a developmental process that occurs in several phasesand is regulated by a large number of gene products. An insertionaltransgenic mouse mutant (termed kisimo mouse) has been isolated thatresults in abnormal germ-cell development, showing abnormal elongatedspermatids in the lumina of seminiferous tubules. We cloned the disruptedlocus of kisimo and identified a novel testis-specific gene, THEG, whichis specifically expressed in spermatids and was disrupted in thetransgenic mouse. The yeast two-hybrid screening method revealed that THEGprotein strongly interacts with chaperonin containing t-complexpolypeptide-1epsilon, suggesting that THEG protein functions as aregulatory factor in protein assembly. Our findings indicate that thekisimo locus is essential for the maintenance of spermiogenesis and that agene expression disorder may be involved in male infertility.

• Yoshikawa T et al.
• Isolation of a cDNA for a novel human RING finger protein gene, RNF18, bythe virtual transcribed sequence (VTS) approach(1).
• Biochim Biophys Acta. 2000; 1493: 349-55
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• We have recently developed a novel database system, designated as thevirtual transcribed sequence (VTS) which efficiently extracts many genesfrom public human genome databases, and tested the feasibility of thisnovel computational approach (N. Miyajima, C. Burge, T. Saito, Biochem.Biophys. Res. Commun. 272 (2000) 801; http://host45.maze.co.jp/vts/). Inthis study, using the VTS approach, we isolated a cDNA for a novel humangene with RING finger motif (C(3)HC(4)), which is not deposited in publicEST databases. The isolated cDNA clone is 2163 bp in length, and containsan open reading frame of 452 amino acids. We designated the novel gene asRNF18. A database search showed that the RNF18 gene had the moderatesimilarity to SS-A/Ro52 protein, which is a ribonucleoprotein reactivewith autoantibodies in patients with Sjogren's syndrome and systemic lupuserythematosus. Tissue distribution analyses by Northern blot and RT-PCRmethods demonstrated that the RNF18 messenger RNA was preferentiallyexpressed in testis. The exon-intron boundaries of RNF18 gene weredetermined by aligning the cDNA sequence with the corresponding genomesequence. The isolated cDNA consists of eight exons that span about 11 kbof the genome DNA. The precise chromosomal location of the RNF18 gene wasdetermined by PCR-based radiation hybrid mapping, and the gene was locatedto centromere region of chromosome 11 between markers NIB1900 andD11S1350. Taken together, the VTS approach should provide a novel cDNAcloning strategy for isolating unidentified genes, which are not foundeven in EST databases but are detectable computationally.

• Bromme NC, Wex T, Wex H, Levy B, Lipyansky A, Bromme D
• Cloning, characterization, and expression of the human TIN-ag-RP geneencoding a novel putative extracellular matrix protein.
• Biochem Biophys Res Commun. 2000; 271: 474-80
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• The human gene encoding a novel tubulointerstitial nephritis antigen(TIN-ag)-related protein (TIN-ag-RP) was isolated, and its genomicorganization was determined. BLAST searches revealed the highest degree ofhomology to several mammalian TIN-ag orthologues, and a weak homology tocathepsin B-like proteases. The 12 kb gene was mapped by fluorescence insitu hybridization to chromosome 1p34.2-3, a locus neither related to thatof the human TIN-ag (6p11.2-12) nor to that of cathepsin B (8p22-23.1).The TIN-ag-RP is encoded in ten exons with introns ranging from 83 bp to 4kb. In addition, the gene contained one exon in the 5'UTR, but none in the3'UTR. Five of the 10 splice sites of the TIN-ag-RP gene were fullyconserved when compared to a related gene of C. elegans, whereas only onesplice site was identical to those found in cathepsin B genes.Furthermore, human TIN-ag-RP tagged with the T7-epitope, was expressed inHeLa cells, and was found to be localized in vesicular compartments aswell as secreted into the medium suggesting the involvement of theendosomal trafficking pathway. Based on the high degree of homology of theamino acid sequences and genomic organization between TIN-ag-RP andTIN-ag, we suggest that both molecules may form a distinct group or familyof TIN-ag-like proteins.

• Ardley HC, Rose SA, Tan N, Leek JP, Markham AF, Robinson PA
• Genomic organization of the human ubiquitin-conjugating enzyme gene,UBE2L6 on chromosome 11q12.
• Cytogenet Cell Genet. 2000; 89: 137-40
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• The human UBE2L6 gene encodes UbcH8(Kumar), a ubiquitin-conjugating enzyme(E2) highly simliar in primary structure to UbcH7 which is encoded byUBE2L3. Like UBC4 and UBC5 in yeast, these proteins demonstrate functionalredundancy. Herein we report the intron/exon structure of UBE2L6.Comparison of the genomic organization of UBE2L6 with UBE2L3 demonstratesthat these genes remain highly conserved at the genomic as well as at theprotein level. We also describe the chromosomal localization of UBE2L6,which maps to chromosome 11q12.

• Foussias G, Yousef GM, Diamandis EP
• Molecular characterization of a Siglec8 variant containing cytoplasmictyrosine-based motifs, and mapping of the Siglec8 gene.
• Biochem Biophys Res Commun. 2000; 278: 775-81
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• Through efforts to investigate the CD33-like subgroup of sialic acidbinding immunoglobulin-like lectins (Siglecs), which are believed to belocated on chromosome 19q13.4, we have identified the precise genomicregion containing the Siglec8 gene. It is located on chromosome 19q13.4,approximately 330 kb downstream of the Siglec9 gene. Further, we haveidentified a novel Siglec8 variant, named Siglec8-Long (Siglec8-L), whichdiffers in its last two exons from the previously published mRNA sequenceof Siglec8 (GenBank Accession No. AF195092). Both Siglec8 and Siglec8-Lare comprised of seven exons, of which the first five are identical,followed by marked differences in exon usage and mRNA splicing. The 499amino acid protein encoded by the Siglec8-L open reading frame has amolecular weight of 54 kDa. Like the other members of the CD33-likesubgroup of Siglecs, except for the previously published Siglec8,Siglec8-L also contains the two tyrosine-based motifs that have been foundto recruit both SH2 domain-containing tyrosine and inositol phosphatases.

• Larsson M et al.
• High-throughput protein expression of cDNA products as a tool infunctional genomics.
• J Biotechnol. 2000; 80: 143-57
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• A proteomics approach has been developed aimed to allow high throughputanalysis of protein products expressed from cDNA fragments (expressedsequence tags, ESTs). The concept relies on expression of gene products togenerate specific antibodies for protein analysis, such asimmunolocalization of the proteins on cellular and subcellular level. Toevaluate the system, 55 cDNA clones with predominantly unknown functionwere selected from a mouse testis cDNA-library. A bacterial expressionsystem was designed that allowed robust expression and easy purification.Protein levels between 15 and 80 mg l(-1) were obtained for 49 of theclones. Five clones were selected for immunization and all yieldedfunctional antibodies that gave specific staining in Western blotscreening of samples from various cell types. Furthermore, extensiveimmunolocalization information on subcellular level was obtained for threeof the five clones. All generated data were stored in a relationaldatabase, and are made available through a web-interface(http://www.biochem.kth.se/multiscale/), which also provides relevantlinks and allows homology searches from the original sequences. Thepossibility to allow analysis of gene products from whole genomes usingthis 'localization proteomics' approach is discussed.

• Van Eynde A, Perez-Callejon E, Schoenmakers E, Jacquemin M, Stalmans W, Bollen M
• Organization and alternate splice products of the gene encoding nuclearinhibitor of protein phosphatase-1 (NIPP-1).
• Eur J Biochem. 1999; 261: 291-300
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• Nuclear inhibitor of protein phosphatase-1 (NIPP-1) is one of two majorregulatory subunits of protein phosphatase-1 in mammalian nuclei. Wereport here the cloning and structural characterization of the humanNIPP-1 genes, designated PPP1R8P and PPP1R8 in human gene nomenclature.PPP1R8P (1.2 kb) is a processed pseudogene and was localized by in situhybridization to chromosome 1p33-32. PPP1R8 is an authentic NIPP-1 geneand was localized to chromosome 1p35. PPP1R8 (25.2 kb) is composed ofseven exons and encodes four different transcripts, as determined fromcDNA library screening, reverse transcriptase-PCR (RT-PCR) and/or EST(expressed sequence tag) database search analysis. NIPP-1alpha mRNArepresents the major transcript in human tissues and various cell lines,and encodes a polypeptide of 351 residues that only differs from thepreviously cloned calf thymus NIPP-1 by a single residue. The othertranscripts, termed NIPP-1beta, gamma and delta, are generated byalternative 5'-splice site usage, by exon skipping and/or by alternativepolyadenylation. The NIPP-1beta/delta and NIPP-1gamma mRNAs are expectedto encode fragments of NIPP-1alpha that differ from the latter by theabsence of the first 142 and 224 residues, respectively. NIPP-1gammacorresponds to 'activator of RNA decay-1' (Ard-1) which, unlikeNIPP-1alpha, displays in vitro and endoribonuclease activity and lacks anRVXF consensus motif for interaction with protein phosphatase-1. While theNIPP-1alpha/beta/delta-transcripts were found to be present in varioushuman tissues, the NIPP-1gamma transcript could only be detected in humantransformed B-lymphocytes.

• Thomas JW, Lee-Lin SQ, Green ED
• Human-mouse comparative mapping of the genomic region containing CDK6:localization of an evolutionary breakpoint.
• Mamm Genome. 1999; 10: 764-7

• Nonoguchi K et al.
• Cloning of human cDNAs for Apg-1 and Apg-2, members of the Hsp110 family,and chromosomal assignment of their genes.
• Gene. 1999; 237: 21-8
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• In mice, the Hsp110/SSE family is composed of the heat shock protein(Hsp)110/105, Apg-1 and Apg-2. In humans, however, only the Hsp110/105homolog has been identified as a member, and two cDNAs, Hsp70RY andHS24/p52, potentially encoding proteins structurally similar to, butsmaller than, mouse Apg-2 have been reported. To clarify the membership ofHsp110 family in humans, we isolated Apg-1 and Apg-2 cDNAs from a humantestis cDNA library. The human Apg-1 was 100% and 91.8% identical inlength and amino acid (aa) sequence, respectively, to mouse Apg-1. HumanApg-2 was one aa shorter than and 95.5% identical in sequence to mouseApg-2. In ECV304, human endothelial cells Apg-1 but not Apg-2 transcriptswere induced in 2 h by a temperature shift from 32 degrees C to 39 degreesC. As found in mice, the response was stronger than that to a 37-42degrees C shift. The human Apg-1 and Apg-2 genes were mapped to thechromosomal loci 4q28 and 5q23.3-q31.1, respectively, by fluorescencein-situ hybridization. We isolated cDNA and genomic clones encompassingthe region critical for the difference between Apg-2 and HS24/p52.Although the primer sets used were derived from the sequences common toboth cDNAs, all cDNA and genomic clones corresponded to Apg-2. Using asimilar approach, the relationship between Apg-2 and Hsp70RY was assessed,and no clone corresponding to Hsp70RY was obtained. These resultsdemonstrated that the Hsp110 family consists of at least three members,Apg-1, Apg-2 and Hsp110 in humans as well as in mice. The significance ofHS24/p52 and Hsp70RY cDNAs previously reported remains to be determined.

• Tang PZ, Tsai-Morris CH, Dufau ML
• A novel gonadotropin-regulated testicular RNA helicase. A new member ofthe dead-box family.
• J Biol Chem. 1999; 274: 37932-40
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• A gonadotropin-regulated testicular RNA helicase (GRTH) was identified andcharacterized. GRTH cloned from rat Leydig cell, mouse testis, and humantestis cDNA libraries is a novel member of the DEAD-box protein family.GRTH is transcriptionally up-regulated by chorionic gonadotropin viacyclic AMP-induced androgen formation in the Leydig cell. It has ATPaseand RNA helicase activities and increases translation in vitro. Thishelicase is highly expressed in rat, mouse, and human testes and weaklyexpressed in the pituitary and hypothalamus. GRTH is produced in bothsomatic (Leydig cells) and germinal (meiotic spermatocytes and roundhaploid spermatids) cells and is developmentally regulated. GRTHpredominantly localized in the cytoplasm may function as a translationalactivator. This novel helicase could be relevant to the control ofsteroidogenesis and the paracrine regulation of androgen-dependentspermatogenesis in the testis.

• Hsu SY
• Cloning of two novel mammalian paralogs of relaxin/insulin family proteinsand their expression in testis and kidney.
• Mol Endocrinol. 1999; 13: 2163-74
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• Based on sequence homology to insulin and relaxin, we have isolated twonovel genes of the insulin superfamily from mouse tissues. Because theseproteins show a high similarity to relaxin and relaxin-like factor (RLF orLey I-L), they were named as RIF1 (relaxin/insulin-like factor 1) and RIF2(relaxin/insulin-like factor 2). After RT-PCR, full-length cDNAs of RIF1and RIF2 were obtained from mouse testis and ovary, respectively. Inaddition, a putative human ortholog of RIF1 was isolated from humantestis. The deduced coding regions of mRIF1, mRIF2, and hRIF1 were 191,145, and 213 amino acids, respectively, and all three proteins contain atypical signal sequence for secretion at their amino terminus. Sequencecomparison indicated that RIFs encode proteins consisting of B and Asubunits connected by a long C domain peptide, and the deduced matureproteins of these putative ligands are most closely related to relaxin,RLF, and insulin from different species. Northern blot analysis showedthat RIF1 transcripts are approximately 1.2 kb in size and are expressedmainly in testis of mouse and human. In contrast, RIF2 message of 2.0 and1.2 kb are preferentially expressed in mouse kidney and are lower intestis, heart, and brain. In addition, immunohistochemical analysis showedthat testis expression of RIF1 is restricted to interstitial cellssurrounding seminiferous tubules. In kidney, the RIF2 message is localizedto selected epithelial cells of loop of Henle. The exclusive expressionpattern of RIF1 and related RLF in testis interstitial cells suggestedpotential physiological roles of these two distinct insulin/relaxin familyligands in testis function. Additionally, the spatial expression patternof RIF2 suggests a novel role of RIF2 in nephrophysiology. Identificationof RIF polypeptides expands the family of relaxin- and insulin-likehormones and allows future elucidation of the physiological role andhormonal mechanisms for these tissue-specific factors.

• Yu W et al.
• Characterization of three splice variants and genomic organization of themouse BMAL1 gene.
• Biochem Biophys Res Commun. 1999; 260: 760-7
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• The BMAL1 gene encodes a member of the basic helix-loop-helix/PER-ARNT-SIM(bHLH/PAS) family of transcription factors. It is a key regulator ofcircadian rhythms. Using sequence information from human BMAL1 (hBMAL1)cDNAs previously reported by our laboratory, we have isolated andcharacterized cDNAs encoding three splice variants of the mouse BMAL1(mBMAL1) gene. Of the three splice variants, mBMAL1b extends for 1878 bpin the coding sequence, which is 91% identical to that of hBMAL1b; itsdeduced amino acid sequence is 626 residues long and is 98% identical tothat of hBMAL1b, and sequence identities in the bHLH, PAS-A, and PAS-Bregions are 98, 100, and 100%, respectively. mBMAL1b' arises fromalternative usage of exon 2, which results in a 7-amino-acid insertion andalternative splice acceptor usage at the intron 9/exon 10 splice junction,which causes an alanine residue deletion. mBMAL1b' encodes 632 amino acidsand contains the bHLH/PAS domains. mBMAL1g' is generated by alternativesplice acceptor usage at the intron 6/exon 7 splice junction, whichresults in a 28-bp deletion adjacent to the 5' end of the PAS domain.Since the 28-bp deletion shifts the reading frame, mBMAL1g' is predictedto encode a product of only 222 amino acids that lacks the PAS domain. Thetissue distributions of the three splice variants showed some variation.The variations in the tissue distributions and predicted amino acidsequences suggest that the three splice variants may have differentfunctions. Direct sequencing of the genomic mBMAL1 clones indicated thatthe coding sequence of mBMAL1 spans 32 kb and includes 17 exons. Anunusual exon/intron donor sequence was found in intron 14, which beginswith GC at the 5' end. Comparison with the bHLH/PAS family genes revealedthat the intron/exon splice pattern of mBMAL1 most closely matches that ofthe mAhr, which suggests that BMAL1 and Ahr belong to the same subclassand may be derived from a common primordial gene.

• Bodeau-Pean S, Ravassard P, Neuner-Jehle M, Faucheux B, Mallet J, Dumas S
• A human tyrosine hydroxylase isoform associated with progressivesupranuclear palsy shows altered enzymatic activity.
• J Biol Chem. 1999; 274: 3469-75
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• A novel human tyrosine hydroxylase (HTH) messenger RNA subgroup generatedby alternative splicing and characterized by the absence of the third exonwas recently identified. The corresponding putative protein lacks 74 aminoacids including Ser31 and Ser40, two major phosphorylation sitesimplicated in the regulation of HTH activity. These mRNA species aredetected in adrenal medulla and are overexpressed in patients sufferingfrom progressive supranuclear palsy, a neurodegenerative disease mostlyaffecting catecholaminergic neurons of the basal ganglia. In the presentwork, an HTH protein isoform lacking exon 3 was identified in humanadrenal medulla. For this purpose, an antibody was raised against the HTHexon 3. The effect of the removal of exon 3 on the enzymatic activity ofHTH was studied in vitro by comparing a purified recombinant fusionprotein without exon 3 (glutathione S-transferase (GST)-HTHDelta3) to theequivalent protein containing exon 3 (GST-HTH3). In initial velocityconditions, GST-HTHDelta3 has 30% of the maximal velocity of GST-HTH3.Moreover, the skipping of exon 3 results in the absence of activation ofGST-HTH by heparin and increases by 10-fold the retroinhibition constantfor dopamine, demonstrating the involvement of exon 3 in the regulation ofHTH enzymatic activity. The identification of a variably expressed HTHisoform that lacks an exon implicated in activity regulation supports theview that HTH alternative splicing contributes to the functional diversitywithin the catecholaminergic system and may be implicated in someneurological diseases.

• Kohno N, Yamagata K, Yamada S, Kashiwabara S, Sakai Y, Baba T
• Two novel testicular serine proteases, TESP1 and TESP2, are present in themouse sperm acrosome.
• Biochem Biophys Res Commun. 1998; 245: 658-65
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• To identify a novel candidate(s) for acrosomal proteins that act on thesperm/egg interaction, a DNA fragment was PCR-amplified from a cDNAlibrary of acrosin-deficient mouse testis and then used as a probe toscreen a mouse testis cDNA library. Complementary DNA clones encoding eachof two similar but different serine proteases, TESP1 and TESP2, have beenidentified. The nucleotide sequences of these clones indicate that mouseTESP1 and TESP2 are initially synthesized as preproproteins of 367 and 366amino acids, respectively. Comparison of the two TESP sequences with thoseof typical serine proteases suggests that each TESP zymogen is probablyconverted into a two-chain mature enzyme consisting of light and heavychains covalently linked by a single pre-existing disulfide bond. Theconversion may be accomplished by another protease(s) with a trypsin-likecleavage specificity, since it is unlikely that the mature TESP1 and TESP2are capable of splitting the Lys-Ile bond between the light and heavychains. Northern blot analysis of total cellular RNA demonstrates that theTESP1 and TESP2 genes are expressed only in the testis, and thetranscripts are abundantly present in the haploid round spermatids.Moreover, immunocytochemical analysis of mouse cauda epididymal spermusing affinity-purified antibodies reveals that these two TESPs are bothlocalized in the sperm acrosome and are released during the acrosomereaction induced by calcium ionophore A23187. These findings provideadditional clues for elucidating the mechanisms involved in the sperm/egginteractions, including penetration of the zona pellucida by sperm.

• Pouresmaeili F, Morales CR, Oko R
• Molecular cloning and structural analysis of the gene encoding PERF 15protein present in the perinuclear theca of the rat spermatozoa.
• Biol Reprod. 1997; 57: 655-9
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• We have cloned the PERF 15 gene, which encodes the most abundant proteinof the perinuclear theca of rat spermatozoa. PERF 15 is related to thesuperfamily of lipophilic transport proteins. It has a molecular weight of15,060 and is present exclusively in the subacrosomal region of the spermhead. Northern blot analysis demonstrated that this gene transcribed PERF15 mRNA in meiotic and postmeiotic cells. The PERF 15 gene contains fourexons and three introns. Exon 1 codes for amino acids 1-24, exon 2 foramino acids 25-82, exon 3 for amino acids 83-116, and exon 4 for aminoacids 117-132. The three introns are composed of 2241, 547, and 164 basepairs (bp), respectively. The exon/intron boundaries are identical tothose found in the mouse myelin P2 gene, but there is no resemblance insize and sequence between the corresponding introns of the PERF 15 andmyelin P2 genes. Localization of the initiation transcription site byprimer extension showed that the 5'-untranslated region of this gene is 67bp upstream of the translation initiation site. Primer extension analysisalso suggests that there is one transcription start site for this gene.Inverse polymerase chain reaction generated a 204-bp fragment, locatedupstream of the translation initiation codon, that has some homology withregions of other mammalian genes.

• Ishino M, Ohba T, Inazawa J, Sasaki H, Ariyama Y, Sasaki T
• Identification of an Efs isoform that lacks the SH3 domain and chromosomalmapping of human Efs.
• Oncogene. 1997; 15: 1741-5
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• Efs was originally found by expression cloning of a mouse embryo cDNAlibrary through its Fyn-SH3 binding capacity (Ishino et al., Oncogene 11,2331-2338, 1995). Efs has characteristic regions important inintracellular signal transduction; these are an SH3 domain, a cluster ofputative ligands for SH2 domains and proline-rich sequences withSH3-binding consensus. In this paper, we report cDNA cloning of human Efsand a variant of it from a hippocampal cDNA library. The human Efs genewas mapped to chromosome 14q11.2-q12 by fluorescence in situhybridization. We identified two forms of human Efs, designated hEfs1 andhEfs2. hEfs1 represents the human counterpart of original mouse embryo Efs(mEfs1). hEfs2, the newly identified form, is identical to hEfs1, exceptfor its lack of the SH3 domain. hEfs1 and mEfs1 are 80% identical in theiramino acid sequences and 100% identical within the SH3 domain. Reversetranscription polymerase chain reaction analysis of adult mouse tissue RNAindicated expression of Efs2 and of Efs1 in various tissues. Evidencesuggesting the presence of the Efs2 protein in human tissue was obtainedby immunoprecipitation followed by immunoblotting with two differentanti-Efs antibodies. Possible functions of Efs2 are discussed.

• Yamagata T et al.
• Genome organization of human 48-kDa oligosaccharyltransferase (DDOST).
• Genomics. 1997; 45: 535-40
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• The enzyme oligosaccharyltransferase(dolichyl-diphosphooligosaccharide-protein glycosyltransferase; EC 2.4.1.119) (DDOST) catalyzes the transfer of a high-mannose oligosaccharide(GlcNac2Man9Glc3) from a dolichol-linked oligosaccharide donor(dolichol-P-GlcNac2Man9Glc3) onto the asparagine acceptor site within anAsn-X-Ser/Thr consensus motif in nascent polypeptide chains across themembrane of the endoplasmic reticulum. We isolated mouse and human DDOSTcDNAs from retinoic acid-treated mouse P19 EC cells and human NT-2 cells,respectively. DDOST mRNA is expressed intensely in heart and pancreas, butat lower levels in brain. Here we show that the human DDOST 48-kDa subunitgene (HGMW-approved symbol DDOST) is organized into 11 exons expandingabout 9 kb. This DDOST subunit gene is localized on chromosome 1p36.1 byfluorescence in situ hybridization analysis.

• Gaedigk R, Karges W, Hui MF, Scherer SW, Dosch HM
• Genomic organization and transcript analysis of ICAp69, a target antigenin diabetic autoimmunity.
• Genomics. 1996; 38: 382-91
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• Islet cell antigen p69 (ICAp69) is a target self-antigen in autoimmune(insulin-dependent) diabetes mellitus. Distributed over more than 100 kbon chromosome 6 (6{A1-A2}), the single murine genomic locus contains 14coding exons, 39-271 bp in length. The identified human and mouseintron-exon junctions are identical, with intron sizes ranging from 94 bpto 24 kb and with conserved flanking region intron sequences. cDNA cloningidentified alternatively spliced ICAp69 mRNA transcripts. Thepredominating alpha-transcripts lack exon 4, while beta-transcriptsinclude this exon, which codes translation termination in all readingframes and a truncated molecule following in vitro expression.gamma-Transcripts show splice removal of exons 8-12, whiledelta-transcripts exclude exon 11. Transcripts use alternativepolyadenylation signals including a less frequent ATTAAA sequence.5'-Untranslated cDNA and genomic sequencing and long PCR analysis suggestthe presence of more noncoding exons. All splice variants encode theconserved T-cell epitope (in exon 2) recognized by autoreactive T cells indiabetic children and diabetes-prone NOD mice.

• Choudhary SK et al.
• A haploid expressed gene cluster exists as a single chromatin domain inhuman sperm.
• J Biol Chem. 1995; 270: 8755-62
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• Mammalian spermiogenesis is marked by the initial disruption of thenuclear-histone-DNA complex by the transition proteins for ultimatereplacement with protamines. The genes for three of these low molecularweight basic nuclear proteins exist as a single linear array of PRM1,PRM2, and TNP2 on human chromosome 16p13.2. To begin to address themechanism governing their transcriptional potentiation, a region ofapproximately 40 kilo-bases of the human genome encompassing these geneswas introduced into the germ line of mice. Fluorescence in situhybridization and Southern analysis showed that this segment of the humangenome integrated into independent chromosomal sites while maintaining itsfidelity. Transcript analysis demonstrated that the expression of theendogenous mouse protamine Prm1 and Prm2 genes as well as the mousetransition protein Tnp2 gene were expressed along with their humantransgene counterparts. The pattern of expression of these transgenichuman genes within this multigenic cluster faithfully represented thatobserved in vivo. In addition, all members of this transgenic gene clusterwere expressed in proportions similar to those in human testis. Copynumber-dependent and position-independent expression of the transgenicconstruct demonstrated that the corresponding biological locus wascontained within this segment of the human genome. Furthermore, DNase Isensitivity established that in sperm the human PRM1-->PRM2-->TNP2 genicdomain was contained as an approximately 28.5-kilobase contiguous segmentbounded by an array of nuclear matrix associated topoisomerase IIconsensus sites. This is the first description of a multigenic malegamete-specific domain as a fundamental gene regulatory unit. A model ofhaploid-specific gene determination is presented.

• Cameron HS, Szczepaniak D, Weston BW
• Expression of human chromosome 19p alpha(1,3)-fucosyltransferase genes innormal tissues. Alternative splicing, polyadenylation, and isoforms.
• J Biol Chem. 1995; 270: 20112-22
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• The human alpha(1,3)-fucosyltransferase genes FUT3, FUT5, and FUT6 form acluster on chromosome 19p13.3. Expression was studied using reversetranscriptase-polymerase chain reaction, rapid amplification of cDNA ends,and Northern analyses. FUT3 and FUT6 were expressed at high levels, whileFUT5 expression was lower and restricted to fewer cell types.Alternatively spliced transcripts were identified for FUT3 and FUT6 inkidney, liver, and colon. A 2.37-kilobase pair (kb) FUT3 transcript,detected at high levels in kidney and colon, was absent in liver. FUT6expression was characterized by a 3.5-kb transcript present in kidney andliver, and a 2.5-kb transcript in colon and liver. Two polyadenylationsites were shown for FUT5, but absence of consensus sequences suggestsreduced efficiency for cleavage and polyadenylation. Two polyadenylationsites were also shown for FUT6, with the alternatively spliced downstreamsignal in tissues expressing high levels of FUT6. In these tissues,additional splicing results in isoforms with catalytic domain deletions.No detectable alpha(1,3)- or alpha(1,4)-fucosyltransferase activity wasfound in assays of cells transfected with FUT6 isoform cDNAs. Thus,tissue-specific post-transcriptional modifications are associated withexpression patterns of FUT3, FUT5, and FUT6.

• Xu L, He GP, Li A, Ro HS
• Molecular characterization of the mouse ribosomal protein S24 multigenefamily: a uniquely expressed intron-containing gene with cell-specificexpression of three alternatively spliced mRNAs.
• Nucleic Acids Res. 1994; 22: 646-55
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• A family of 16 genes encoding the mouse ribosomal protein S24 wasidentified, and four members from this family were cloned. A singleexpressed intron-containing S24 gene (termed mrpS24) and one pseudogene(mrpS24p) were completely sequenced and characterized. The mrpS24 gene hasseven exons and six introns spanning over 5.1 x 10(3) nucleotides (nt).The cap site of S24 was mapped to a G residue four nt upstream of apolypyrimidine tract and 15 nt downstream of a TATA-like (TATGA) element.The 5' region (-325 to +33) of the mrpS24 gene has a functional promoterthat was able to express the fused chloramphenicol acetyltransferase (CAT)reporter gene. Two different forms of mouse S24 cDNA clones werepreviously isolated. Sequence analysis showed that one of these cDNAclones (termed S24a) lacks the entire exon V sequence (18 nt), and thededuced amino acid sequence is missing a C-terminal lysine residue encodedby the other cDNA (S24b). The pseudogene mrpS24p is flanked by an 11-bpdirect repeat, and its sequence is almost identical to the S24 cDNAsequence, but it lacks two mini-exons, V and VI (20 nt), as in the casesof the human and rat S24 cDNAs. RT-PCR experiments demonstrated theexistence of a third form (S24c) that similarly lacks both of themini-exons, and suggested that different species of S24 mRNA might arisefrom alternative splicing of the mini-exons V and VI. Northern blotanalysis showed that S24 expression is down- and up-regulated duringadipocyte differentiation and in cellular transformation, respectively.RNase protection assays and RT-PCR experiments suggested that thesecell-specific changes of S24 mRNA levels are mainly due to fluctuations inS24c mRNA level. Our results provide the first indication that a ribosomalprotein gene is regulated by alternative usage of two mini-exons in acell-specific manner.

• Troxler RF et al.
• Localization of the gene for human heart fatty acid binding protein tochromosome 1p32-1p33.
• Hum Genet. 1993; 92: 563-6
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• Heart fatty acid binding protein (hFABP) is an abundant 14-kDa cytosolicprotein thought to be involved in trafficking of fatty acids from theplasma membrane to sites of beta-oxidation in mitochondria and peroxisomesand to the endoplasmic reticulum for lipid synthesis. A human hFABP cDNAisolated by polymerase chain reaction was used as a probe for in situhybridization to metaphase chromosomes. A fragment of the gene for humanhFABP was used as a probe for fluorescence in situ hybridization tometaphase chromosomes. The cDNA and genomic probes both localized the genefor human hFABP to chromosome 1p32-1p33.

• Levanon D, Danciger E, Dafni N, Groner Y
• Genomic clones of the human liver-type phosphofructokinase.
• Biochem Biophys Res Commun. 1986; 141: 374-80
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• Genomic clones of human liver phosphofructokinase (PFK) were isolated byscreening a gene bank enriched for chromosome 21 sequences with twosynthetic oligonucleotide probes designed from peptide sequences ofpurified human liver PFK. A 3.3 Kb fragment derived from the genomicclones was sub-cloned and designated to pG-PFKL 3.3. It hybridized with a3.5 Kb mRNA on Northern blots and was able to enrich selectively for liverPFK mRNA by hybrid-selection. These results demonstrated that the isolatedclones contain sequences homologous to human PFKL mRNA. When hybridized togenomic DNA blots pG-PFKL 3.3 reacted with the same 3.3 Kb BamHI fragmentin both human DNA and DNA of the mouse/human hybrid line WA17 whichcontains human chromosome 21 as the only human chromosome. These dataconfirm the assignment of the PFKL gene to chromosome 21.