Mutations in the Chediak-Higashi syndrome gene (CHS1) indicate requirement for the complete 3801 amino acid CHS protein

Chediak-Higashi syndrome (CHS) is a rare, usually fatal, autosomal recessive disorder characterized by severe immunologic defects, reduced pigmentation, progressive neurologic dysfunction and a bleeding diathesis. The hallmark of CHS is giant organelles and giant granules in many different cell types, most likely the result of defective trafficking of specific organellar and granular proteins necessary for the normal genesis, structure or function of these cytoplasmic components. The CHS1 gene has recently been identified and shown to be homologous to the beige locus of the mouse; however, there has been disagreement as to the length of the functional CHS1 mRNA and protein. Here we report homozygous CHS1 gene mutations in two of the original probands we used to map the gene to 1q42-q44. One of these, a frameshift at codon 3197, supports our assertion that the functional CHS protein is a predicted 3801 amino acid polypeptide encoded by a 13.5 kb mRNA.     

Cyclosporine trough concentrations in predicting allograft rejection and renal toxicity up to 12 months after renal transplantation

We recently identified a novel gene (PB39) (HGMW-approved symbol POV1) whose expression is up-regulated in human prostate cancer using tissue microdissection-based differential display analysis. In the present study we report the full-length sequencing of PB39 cDNA, genomic localization of the PB39 gene, and genomic sequence of the mouse homologue. The full-length human cDNA is 2317 nucleotides in length and contains an open reading frame of 559 amino acids which does not show homology with any reported human genes. The N-terminus contains charged amino acids and a helical loop pattern suggestive of an srp leader sequence for a secreted protein. Fluorescence in situ hybridization using PB39 cDNA as probe mapped the gene to chromosome 11p11.1-p11.2. Comparison of PB39 cDNA sequence with murine sequence available in the public database identified a region of previously sequenced mouse genomic DNA showing 67% amino acid sequence homology with human PB39. Based on alignment and comparison to the human cDNA the mouse genomic sequence suggests there are at least 14 exons in the mouse gene spread over approximately 100 kb of genomic sequence. Further analysis of PB39 expression in human tissues shows the presence of a unique splice variant mRNA that appears to be primarily associated with fetal tissues and tumors. Interestingly, the unique splice variant appears in prostatic intraepithelial neoplasia, a microscopic precursor lesion of prostate cancer. The current data support the hypothesis that PB39 plays a role in the development of human prostate cancer and will be useful in the analysis of the gene product in further human and murine studies.     

Relation between coronary artery disease, aortic stiffness, and left ventricular structure in a population sample

With the use of the degenerated nucleotides that contain the conserved sequence of G protein-coupled receptor, we have identified a 648-bp clone (HDGRC02) from human genomic DNA with significant sequence homology to human neurotransmitter receptors. HDGRC02 was then used as a probe for the screening of full length gene. From human Lambda DASH II genomic library, a 1.6 Kb clone encoded a full length gene was isolated and named putative neurotransmitter receptor (PNR). PNR has a single open reading frame which predicts a 38.3 KD protein of 338 amino acids with seven transmembrane domain topography. The amino acid sequence of PNR exhibits considerable homology to the rat 5-HR1D receptor with 35% amino acid identity and 56% amino acid similarity. PNR also shows significant sequence homology to the 5-HT1D receptor from Japanese puffer fish fugu, to the 5-HT4L receptor from mouse, to the alpha-2 adrenergic receptor and to the D2 dopamine receptor. Northern blot analysis indicates that PNR is expressed in skeletal muscle and selected areas of the brain. A chromosome mapping study located the PNR gene with human chromosome band of 6q23. The findings in the present study demonstrate that PNR is a putative neurotransmitter receptor.     

Cloning, expression and mapping of a novel RING-finger gene (RNF5), a human homologue of a putative zinc-finger gene from Caenorhabditis elegans

The RING-finger is a unique zinc-chelating domain involved in mediating protein-protein interactions. The extensive sequence homology within the RING-finger domain allowed us to clone a novel member of the RING-finger family of genes. This cDNA clone, designated RNF5 (Ring-finger protein 5), contained an open reading frame of 540 nucleotides. Its predicted amino acid sequence revealed significant homology to a hypothetical protein encoded by Caenorhabditis elegans cosmid C16C10.7. The expression of RNF5 was detected in a variety of human tissues. The RNF5 gene was mapped by fluorescence in situ hybridization to chromosome 6p21.31. Radiation hybrid mapping further assigned RNF5 to a region proximal to the major histocompatibility complex (MHC) on chromosome 6. RNF5 is the third RING-finger gene identified in the region proximal to MHC raising the possibility that the RING-finger family of genes may exist as a cluster in this region.     

Cloning and comparative analysis of the human pre-T-cell receptor alpha-chain gene

In immature T cells the T-cell receptor (TCR) beta-chain gene is rearranged and expressed before the TCR alpha-chain gene. At this stage TCR beta chain can form disulfide-linked heterodimers with the pre-T-cell receptor alpha chain (pTalpha). Using the recently isolated murine pTalpha cDNA as a probe, we have isolated the human pTalpha cDNA. The complete nucleotide sequence predicts a mature protein of 282 aa consisting of an extracellular immunoglobulin-like domain, a connecting peptide, a transmembrane region, and a long cytoplasmic tail. Amino acid sequence comparison of human pTalpha with the mouse pTalpha molecule reveals high sequence homology in the extracellular as well as the transmembrane region. In contrast, the cytoplasmic region differs in amino acid composition and in length from the murine homologue. The human pTalpha gene is expressed in immature but not mature T cells and is located at the p21.2-p12 region of the short arm of chromosome 6.     

Point mutations causing Bloom's syndrome abolish ATPase and DNA helicase activities of the BLM protein

Bloom's syndrome (BS) is a rare human genetic disorder characterized by mutations within the BLM gene whose primary effects are excessive chromosome breakage and increased rates of sister chromatid interchange in somatic cells. We report the characterization of a murine protein (mBLM), highly related to the product of the human BLM gene. This protein exhibits an ATP-dependent DNA-helicase activity that unwinds DNA in a 3'-5' direction. Single amino acid substitutions found in BS cells, abolish both ATPase and helicase activities of this protein, indicating that defects in these BLM functions may be primarily responsible for BS establishment. These results provide the first evidence suggesting that the enzymatic activities of the BLM product are implicated in the upholding of genomic integrity.     

Isolation of an ovine genomic sequence containing the full-length angiotensin II type-1 receptor

We have isolated from a genomic library using PCR amplification an 1171 base sequence containing a putative ovine AT1-R protein coding sequence of 1080 bases. As expected the protein coding sequence is of greater than 99% homology to the partial protein coding sequence reported by Robillard et al, with only one base difference. Relative to other species, highest homology at the level of the cDNA protein coding sequence is to bovine (97.6%) and lowest homology to rat Type 1a (83.3%). The predicted protein amino acid sequence in turn encodes a protein with the properties of a seven alpha-helix transmembrane receptor (by TMPred) sharing closest homology (98.6%) to the bovine receptor and lowest to the rat Type 1a (90.2%). As expected from such a high degree of interspecies homology, amino acids identified by site-directed mutagenesis of the human or rat AT1A-R as involved in binding and action of AII are very highly conserved in the ovine sequence. In addition, both bovine and ovine AT1-R are known to exhibit lower affinity for DuP753 than human AT1-R, and in bovine AT1-R this has been suggested to coincide with the amino acid substitutions Ala->Thr (163) and Leu->Met (262) relative to the human sequence. Our ovine AT1-R cDNA sequence shares these same bovine substitutions.     

The murine Ext1 gene shows a high level of sequence similarity with its human homologue and is part of a conserved linkage group on chromosome 15

We have cloned and sequenced the murine homologue of the human EXT1 gene. At the protein level, these genes show almost complete identity as divergence is limited to only 5 amino acid positions that are scattered about the whole sequence. In addition, similarity searches identified a protein from chromosome III of C. elegans that shows significant similarity to the human and murine EXT/Ext genes. Using high resolution backcross mapping, the murine Ext1 was mapped at 26.55 cM between D15Mit143 and D15Mit153 on mouse chromosome 15. Therefore, Ext1 is part of an evolutionarily conserved linkage group including SDC2/Hspg1, TRHR/Trhr, EXT1/Ext1, MYC/Myc, and TG/Tgn.     

Isolation and chromosomal localization of GPR31, a human gene encoding a putative G protein-coupled receptor

The screening of a human genomic library with a chemokine receptor-like probe allowed us to obtain a putative member of the G protein-coupled receptor gene (GPCR) family, designated GPR31. Its deduced amino acid sequence encodes a polypeptide of 319 amino acids that shares 25-33% homology with members of the chemokine, purino, and somatostatin receptor gene families. Amino acid sequence comparison reveals that the best match in the protein databases is with the human orphan GPCR called HM74 (33% identity). Southern genomic analysis of the GPR31 gene shows a hybridization pattern consistent with that of a single-copy gene. Using fluorescence in situ hybridization, we have determined the chromosomal and regional localization of the GPR31 gene at 6q27. The GPR31 mRNA is expressed at low levels by several human cell lines of different cellular origins. The phylogenetic analysis suggests that the GPR31 receptor may represent a member of a new GPCR subfamily.     

Isolation and characterization of a rheumatoid arthritis-specific antigen (RA-A47) from a human chondrocytic cell line (HCS-2/8)

Two types of 47 kDa antigen specifically recognized by sera from rheumatoid arthritis (RA) patients were isolated from the membrane fraction of a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) by a 2-step procedure: preparative SDS-PAGE and reverse-phase HPLC. An N-terminal amino acid sequence in one of the 47 kDa antigens, named RA-A47, had 81% homology to that deduced from the DNA sequence of the colligin gene which is reported as human hsp47 gene, and 100% homology to that deduced from the DNA sequence of colligin-2 gene, a homologue of colligin. The RA-A47 cross-reacted with a monoclonal antibody raised against chick heat shock protein (Hsp) 47 and bound to gelatin. The expression of the ra-a47 gene was enhanced by heat shock treatment and TGF-beta stimulation. These findings suggest that RA-A47 is a Hsp47-like protein, presumably the product of the colligin-2 gene, and that a collagen-specific molecular chaperone(s) such as Hsp47 and/or RA-A47 is involved in cartilage destruction in RA.     

ICAAR, a novel member of a new family of transmembrane, tyrosine phosphatase-like proteins

We have isolated a cDNA from human foetal brain cDNA library which encodes a putative transmembrane protein bearing an intracellular protein tyrosine phosphatase (PTPase) like domain. The PTPase like domain contains an alanine to aspartate amino acid change relative to other PTPases in the catalytic core domain. This amino acid change is found in only three other known proteins, islet cell autoantigens; human, murine and rat IA-2, murine IA-2b and its rat orthologue phogrin, which have a similar overall structure to ICAAR, and the recently identified X-linked myotubular myopathy (MTM1) gene. ICAAR, IA-2 and IA-2b clearly represent a new family of PTP-like proteins for which catalytic activity has yet to be demonstrated. An abundant ICAAR mRNA is detectable in the brain and pancreas but not in the other normal human tissues surveyed. We have localised ICAAR to human chromosome 7q36.     

cDNA cloning and characterization of a rat spermatogenesis-associated protein RSP29

RSP29, a protein secreted by rat round spermatids, stimulates the secretory function of Sertoli cells in the testis. By making use of the N-terminal sequence homology of RSP29 and a human protein hDP1 that we had previously isolated, we cloned the full length cDNA sequence that encodes RSP29. The entire amino acid sequence of RSP29 showed significant homology with that of hDP1, which was later identified as glyoxalase II. Southern analysis showed that the RSP29 protein sequence is highly conserved in eukaryotes and possibly in prokaryotes. The RSP29 mRNA is expressed in many tissues but has an extremely high abundance in testis. These data suggest that RSP29 may have an important function in most tissues of enkaryotic organisms. The high expression of RSP29 in testis and its stimulatory effects on Sertoli cells suggest that RSP29 could be especially important in the regulation of spermatogenesis.     

Gene structure prediction using information on homologous protein sequence

In this paper a new approach for the prediction of protein coding gene structures is described. The principal scheme of prediction is as follows: first, the exons with the best potential are predicted in a sequence with unknown functions and a list of potential amino acid fragments coded by these exons is formed. Second, testing the homology between each amino acid fragment from the list and proteins from the SWISS-PROT database of amino acid sequences. One protein with the best homology is chosen out of all the homologous sequences. Third, reconstruction of the exon-intron structure, basing it on its homology with the chosen protein sequences. The method was tested on an independent control set (20 genes). The results were as follows: 21% of real exons were lost and 3% of non-real exons were found. This system can be used to refine the results of gene prediction systems, especially if highly homologous proteins are found in the amino acid sequence database.     

N-Acetyltransferase 2 (NAT2) and Glutathione S-Transferase μ (GSTM1) in Bladder-cancer Patients in a Highly Industrialized Area

The gene encoding the 33 kDa piroplasm surface protein of Theileria sergenti isolated in Korea was cloned and the nucleotide sequence was determined by dideoxy chain termination method. The cloned gene corresponds to 869 bp encoding an open reading frame 283 amino acids. Comparison of the sequence between Korean and Japanese isolates showed 99.4% homology rate in the nucleotide sequence and 98.9% homology rate in the amino acid sequence.     

Synthesis of GDP-L-fucose by the human FX protein

FX is a homodimeric NADP(H)-binding protein of 68 kDa, first identified in human erythrocytes, from which it was purified to homogeneity. Its function has been unrecognized despite partial structural and genetic characterization. Recently, on the basis of partial amino acid sequence, it proved to be the human homolog of the murine protein P35B, a tumor rejection antigen. In order to address the biochemical role of FX, its primary structure was completed by cDNA sequencing. This sequence revealed a significant homology with many proteins from different organisms. Specifically, FX showed a remarkable similarity with a putative Escherichia coli protein, named Yefb, whose gene maps in a region of E. coli chromosome coding for enzymes involved in synthesis and utilization of GDP-D-mannose. Accordingly, a possible role of FX in this metabolism was investigated. The data obtained indicate FX as the enzyme responsible for the last step of the major metabolic pathway resulting in GDP-L-fucose synthesis from GDP-D-mannose in procaryotic and eucaryotic cells. Specifically, purified FX apparently catalyzes a combined epimerase and NADPH-dependent reductase reaction, converting GDP-4-keto-6-D-deoxymannose to GDP-L-fucose. This is the substrate of several fucosyltranferases involved in the correct expression of many glyconjugates, including blood groups and developmental antigens.     

Novel allergen structures with tandem amino acid repeats derived from German and American cockroach

Cockroaches produce potent allergens that are an important cause of asthma. The two principal domiciliary cockroach species, Blattella germanica and Periplaneta americana, secrete major allergens, Bla g 1 and Per a 1. Here, we report the molecular cloning of three Bla g 1 cDNA clones, which showed 70% amino acid sequence identity with Per a 1. Plaque immunoassays with human IgE antibodies or murine monoclonal antibodies showed that these allergens were antigenically cross-reactive. The Bla g 1 sequences also showed homology to five previously undefined cockroach allergen sequences. An unusual feature of all these sequences was that they contained multiple tandem amino acid repeats of approximately 100 amino acid residues. Between one and seven repeat units were identified by dot-plot matrix analysis. The sequences also showed homology to a mosquito protein involved in digestion (ANG12 precursor) and to mitochondrial energy transfer proteins. High levels of Bla g 1 were found in cockroach hindgut and proventriculus. Amino acid sequencing of natural Bla g 1 and Per a 1 suggested that these allergens are cleaved by trypsin-like enzymes following secretion into the digestive tract. The repeat sequences appear to have evolved by duplication of an ancestral amino acid domain, which may have arisen from the mitochondrial energy transfer proteins.     

Nucleotide sequence of the VP4-encoding gene of an unusual human rotavirus (HCR3)

Human rotavirus strain HCR3 was isolated from the stool of a clinically normal infant and identified as a serotype G3 rotavirus; however, it could not be grouped into any known human VP4 genetic groups by a polymerase chain reaction assay. The fourth gene of strain HCR3, which encodes the outer capsid protein VP4, was sequenced. This gene is 2362 nucleotides in length and contains one open reading frame capable of encoding a protein of 776 amino acids. The VP4 protein of strain HCR3 shared 67.5-73.5% amino acid identity with those of strains KU, RV-5, 1076, and K8, representing four human genetic groups, and relatively high homology (84.7%) with a fifth genetic group represented by strain 69M, whose VP4 shows more similarity to animal than to human strains. Strain HCR3 shared higher VP4 amino acid homology with various animal rotaviruses, ranging from 74.5 to 89.4%. These observations suggest that the VP4 outer capsid protein of strain HCR3 represents a new VP4 genetic group that is more closely related to animal rotaviruses than to human rotaviruses.