Characterization of a murine high-affinity thiamine transporter, Slc19a2

Thiamine-responsive megaloblastic anemia with deafness and diabetes (TRMA) is a rare autosomal recessive disorder of thiamine transport. Previous studies have demonstrated that the disease is caused by mutations in the SLC19A2 gene encoding a high-affinity thiamine transporter. We hypothesize that thiamine transport, mediated by SLC19A2, plays a role in the development and or maintenance of several organ systems, in particular the erythropoietic, auditory, and glucose homeostasis systems. To investigate the transporter further, we cloned the murine Slc19a2 locus and characterized the resulting protein. Murine Slc19a2 is a 498 amino acid protein, with 12 predicted transmembrane domains. The gene spans approximately 13kb with 6 exons, structurally identical to that of the human homolog. We localized the Slc19a2 gene to mouse chromosome 1, a region syntenic to human chromosome 1q23 that contains the TRMA locus. Transient expression of Slc19a2 in HEK293T cells resulted in specific uptake of [3H] thiamine, confirming a thiamine transporter function. Western blot analysis of mouse tissues reveals a wide distribution of Slc19a2 protein. Immunohistochemistry studies indicate that Slc19a2 is expressed on the cell surface and intracellularly, and is specifically localized to a subpopulation of cells in cochlea, small intestine, and pancreas.     

Targeted disruption of Slc19a2, the gene encoding the high-affinity thiamin transporter Thtr-1, causes diabetes mellitus,sensorineural deafness and megaloblastosis in mice

Thiamin-responsive megaloblastic anemia syndrome (TRMA) is characterized by diabetes mellitus, megaloblastic anemia and sensorineural deafness. Mutations in the thiamin transporter gene SLC19A2 cause TRMA. To generate a mouse model of TRMA, we developed an Slc19a2 targeting construct using transposon-mediated mutagenesis and disrupted the gene through homologous recombination in embryonic stem cells. Erythrocytes from Slc19a2(-/-) mice lacked the high-affinity component of thiamin transport. On a thiamin-free diet, Slc19a2(-/-) mice developed diabetes mellitus with reduced insulin secretion and an enhanced response to insulin. The diabetes mellitus resolved after 6 weeks of thiamin repletion. Auditory-evoked brainstem response thresholds were markedly elevated in Slc19a2(-/-) mice on a thiamin-free diet, but were normal in wild-type mice treated on that diet as well as thiamin-fed Slc19a2(-/-) mice. Bone marrows from thiamin-deficient Slc19a2(-/-) mice were abnormal, with a megaloblastosis affecting the erythroid, myeloid and megakaryocyte lines. Thus, Slc19a2(-/-) mice have provided new insights into the TRMA disease pathogenesis and will provide a tool for studying the role of thiamin homeostasis in diabetes mellitus more broadly.     

Chromosomal localization to 19q13.3, partial genomic structure and 5′ cDNA sequence of the human symplekin gene

Exon trapping from cosmids mapping to chromosome 19q13.3 yielded 6 exonic sequences that matched the human symplekin gene, which encodes a tight junction-related protein. One exonic sequence identified a 4.0 kb brain cDNA clone, R6E1, which contained 302 bp 5′ to the originally reported 3.7 kb symplekin cDNA. A portion of this novel 5′ sequence matched an additional trapped exonic sequence which was obtained from the most telomeric cosmid analyzed. The symplekin gene thus lies in a telomeric-to-centromeric direction on 19q13.3. Only three cosmids from a large 19q13.3 contig hybridized with R6E1, thereby assigning the symplekin gene to a 40 kb region immediately telomeric to gene 59 and the DM protein kinase gene. The 5′ end of the R6E1 clone has a potential initiation codon with a strong Kozak sequence and Northern blot analysis detected a 4.2 kb signal in most human tissues, indicating that R6E1 may be a complete cDNA sequence. Based on the trapped exonic sequences, twelve exon-intron boundaries were predicted. The sequence data in this paper have been deposited in GenBank, accession number U88726.     

Molecular cloning and characterization of the human orthologue of the oppo 1 gene encoding a sperm tail protein

We report here the molecular cloning and characterization of a human orthologue of oppo 1, a mouse gene encoding a male germ-cell-specific sperm tail protein, and the organization of its genomic structure. The mRNA of the human oppo 1 gene (h-oppo 1) was expressed exclusively in the testis, and the 30 kDa protein encoded by the mRNA was detected in human testis and sperm. Immunohistochemical analyses showed that human OPPO 1 protein was localized in the flagellae of ejaculated sperm. A human genomic DNA database search indicated that the h-oppo 1 gene mapped to chromosome 17. The genomic structure of h-oppo 1 showed differences in exon/intron usage, the sequence of the 5'-flanking region, and the first intron was rich in Alu repeats as compared with the mouse oppo 1 gene. Comparison of the two genomic sequences indicated that human oppo 1 has evolved independently, resulting in substantial differences in the genomic structure after the human-mouse split, whereas the sequence of the basic functional unit of the oppo 1 gene seems to have been relatively well conserved.     

Molecular cloning and nucleotide sequence of cDNA encoding a functional murine low-density-lipoprotein receptor

The low-density-lipoprotein (LDL) receptor is an important mediator of mammalian cholesterol metabolism; its congenital absence in humans is characterized by hypercholesterolemia, atherosclerosis, and coronary artery disease. We report here the identification and cloning of a cDNA encoding the murine LDL receptor. The cDNA insert is 4467 base pairs in length and the deduced amino acid sequence bears 78.2% homology with the reported human sequence. This murine cDNA was subcloned into a retroviral-based expression vector, LmLSN1, and transfected into 3T3 cells. The production of functional LDL receptor was confirmed by ligand binding of DiI-LDL cholesterol.Sequence data from this article has been deposited with the EMBL/GenBank Libraries under Accession No. X64414.     

Molecular cloning and characterization of a novel ABC transporter gene in the human pathogen Trichophyton rubrum.

A gene encoding an ABC transporter in the dermatophyte Trichophyton rubrum, TruMDR1, was cloned by PCR using degenerate primers. The open reading frame of TruMDR1 is 4838 bp long and the deduced amino acid sequence shows high homology with ABC transporters involved in drug efflux of other fungi. The effect of chemicals on the expression level of mRNAs of this gene was analysed by Northern blot. An increase in expression level was observed when the fungus was exposed to ethidium bromide, ketoconazole, cycloheximide, fluconazole, griseofulvin, imazalil and itraconazole, suggesting the participation of this gene in drug efflux in this dermatophyte. The identification of a gene potentially involved in cellular detoxification in a pathogenic fungus is the first step towards knowing molecular events related to antifungal resistance.     

cDNA cloning, genomic structure and mRNA expression pattern of porcine type I interferons receptor 2 gene

Type I interferons (IFN) are important mediators of the host defence against viruses through binding to the cell surface receptors, among which the binding to type I IFN receptor 2 (IFNAR2) is the very first step initiating a complex signal transduction cascade. By using RT–PCR and 5′ RACE approaches, we obtained porcine IFNAR2 cDNA, the nucleotide identity of its coding region is 57.53%, 67.45%, 74.07% and 74.63% to those of mouse, human, sheep and cattle, respectively; and the deduced protein of which shares 38.18%, 55.29%, 62.01% and 63.39% identity to those of mouse, human, sheep and cattle, respectively. The genomic structure of porcine IFNAR2 gene consists of nine exons and eight introns. Porcine IFNAR2 mRNA expression was detected in all tissues examined, being strong in the spleen, small intestine, cerebrum and uterus tissues and relatively weak in the stomach tissues. As compared with piglets, the expression of IFNAR2 mRNA was significantly higher in both liver and spleen of Laiwu adult pigs (P < 0.01); in Duroc pigs, however, significantly higher IFNAR2 mRNA expression was only found in adult liver (P < 0.05). In Duroc × Landrace × Yorkshire commercial pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV), the expression of IFNAR2 mRNA in lung tissue was significantly down‐regulated as compared to uninfected ones (P < 0.05).     

Male infertility and thiamine-dependent erythroid hypoplasia in mice lacking thiamine transporter Slc19a2

Thiamine-responsive megaloblastic anemia with diabetes and deafness (TRMA) is an autosomal recessive disease caused by mutations in the high-affinity thiamine transporter gene SLC19A2. To study the role of thiamine transport in the pathophysiology of TRMA syndrome and of each of the component disorders, we created a targeted disruption of the Slc19a2 gene in mice. Slc19a2 -/- mice are viable and females are fertile. Male -/- mice on a pure 129/Sv background are infertile with small testes (testis/body weight=0.13 +/- 0.04 knockout vs. 0.35 +/- 0.05 wild type, P<0.000005). The lack of developing germ cells beyond primary spermatocytes suggests an arrest in spermatogenesis prior to meiosis II. Nuclear chromatin changes indicative of apoptosis are present. No mature sperm are found in the tubules or epididymis. This phenotype suggests a previously unknown role for thiamine transport in spermatogenesis and male fertility. Slc19a2 -/- mice on a pure 129/Sv background develop reticulocytopenia after two weeks on thiamine-depleted chow with a virtual absence of reticulocytes in the peripheral blood (0.12% knockout vs. 2.58% wild type, P=0.0079). Few erythroid precursors are found in the bone marrow. Contrary to human TRMA syndrome, we see no evidence of megaloblastosis or ringed sideroblasts in the bone marrow of Slc19a2 -/- mice in thiamine-replete or thiamine-deficient dietary states. Phenotypic differences between TRMA patients and Slc19a2 -/- mice might be explained by dissimilar tissue expression patterns of the transporter, as well as by differing metabolic needs and possible different species-specific contributions of the related thiamine transporter Slc19a3.     

Complete cDNA sequence and genomic organization of a human pancreas-specific gene homologous to Caenorhabditis elegans sel-1

We have isolated the complete cDNA of a human SEL-1L gene, termed TSA305, that is abundantly expressed only in the pancreas. The cDNA contained an open reading frame of 2382 nucleotides, encoding a deduced protein of 794 amino acids whose predicted sequence showed 46% identity and 64% similarity with SEL-1 of Caenorhabditis elegans. SEL-1 is thought to be a negative regulator of the NOTCH, LIN-12, and GLP-1 receptors, which are required for differentiation and maturation of cells as well as cell–cell interactions during development in C. elegans. The degree of homology among these proteins suggests that the TSA305 gene product may be a member of the SEL-1 family and therefore involved in downregulation of mammalian Notch signaling. Direct sequencing revealed at least 20 coding exons in TSA305. We localized the gene to chromosome bands 14q24.3–q31 by radiation hybrid (RH) mapping and fluorescence in situ hybridization (FISH). The IDDM11 locus has been mapped in this region, and TSA305 may represent a candidate gene for predisposition in some families whose insulin-dependent diabetes is not linked to the HLA locus. Received: March 29, 1999 / Accepted: May 11, 1999     

Characterization and genomic sequence of the murine 60 kD Ro gene

Autoantibodies binding 60 kD Ro (or SS-A) are commonly found in patients with systemic lupus erythematosus and Sj?gren's syndrome. While many studies have examined the autoimmune response directed against this RNA-protein, its function is still uncertain. As part of a broad effort to better understand animal models of anti-Ro autoimmunity we have characterized the murine 60 kD Ro gene. Southern blot analysis of mouse genomic DNA suggests that the 60 kD Ro gene is a single copy gene. The complete sequence of the gene was determined from three overlapping genomic lambda phage clones (GenBank accession number AF065398). The murine 60 kD Ro gene spans approximately 23 kb and consists of 8 or 9 exons. DNA sequence analysis revealed the presence of multiple B1 repetitive units. It maps in synteny with the human 60 kD Ro gene. Therefore, the isolation and characterization of the 60 kD Ro gene will be instrumental for future studies on protein function and the role this protein plays in the development of autoimmune responses.     

Molecular cloning and characterization of a novel human gene homologous to the murine ecotropic retroviral receptor.

A novel human cDNA homologous to the murine ecotropic retroviral receptor was cloned from a cDNA library derived from a human T-cell line. The human cDNA is highly homologous to the murine counterpart (87.6% amino acid identity), and its sequence predicts a protein with 629 amino acids (approximately 68 kDa), which is 7 amino acids more than the murine counterpart (622 amino acids). The predicted protein is highly hydrophobic and contains 14 potential transmembrane-spanning domains. No other gene and protein with significant homology to the cloned human gene and the predicted protein were identified by a computer-based search of sequence data banks other than the murine T-cell early activation gene (52.5% amino acid identity) and the murine ecotropic retroviral receptor gene. The human gene is ubiquitously expressed in human tissues and conserved among mammalian species. The genomic gene was also isolated from a cosmid library derived from human lymphocytes, and its organization was elucidated. The gene mapped to human chromosome 13.     

cDNA cloning of murine interleukin-HP1: homology with human interleukin 6

Interleukin-HP1 (HP1) is a murine T cell-derived lymphokine, originally described as a growth factor for B cell hybridomas and plasmacytomas, that was recently shown to stimulate growth and differentiation of normal B and T lymphocytes. Here, we describe a cDNA for HP1 that was isolated from a library prepared using mRNA of a murine helper T cell clone activated with a clonotypic antibody. The cDNA, which hybridizes with a mRNA of approximately 1300 bp, encodes a polypeptide consisting of 211 amino acids with a typical signal sequence of 24 residues followed by 187 amino acids, which form the mature protein (Mr = 21,710). No N-glycosylation site but several potential O-glycosylation sites were identified in the predicted sequence. Comparison of the cDNA sequence of HP1 with that of human interleukin 6 disclosed a homology of 65% at the DNA level and of 42% at the protein level with a maximum of 57% for the segment spanning residues 42-102 of mature HP1. Considering the functional homology that was previously established between these two proteins, we therefore propose that HP1 be renamed murine interleukin 6.     

Identification and characterization of the cynomolgus monkey chromodomain gene cynCDY,an orthologue of the human CDY gene family

Microdeletions within the AZF (azoospermia factor) a, b and c regions of the Y chromosome can be detected worldwide in 1-10% of infertile men. AZFc, containing genes such as DAZ, CDY, RBMY and others, is most frequently deleted and associated with oligo- or azoospermia. The function of the different genes within AZFc is not yet understood. Here we report the identification and first characterization of the cynomolgus monkey (Macaca fascicularis) homologue of the human CDY gene. cynCDY encodes a 541 aa protein, which like human CDY possesses two putative functional domains: an N-terminal chromodomain, possibly involved in heterochromatin interactions, and a C-terminal domain showing similarity to enoyl-CoA-isomerase, which is involved in fatty acid oxidation. Northern analysis and in-situ hybridization experiments revealed testis- and stage-specific expression of cynCDY mRNA, mainly confined to round and elongating spermatids. Fluorescence in-situ hybridization (FISH) performed on monkey metaphase chromosomes displayed exclusively Y-specific signals in Yq12.1. Using fibre FISH, short signal stretches that indicate the presence of three CDY copies could be visualized, although their integrity or function remains unknown. cynCDY is similar to human CDY with features of a retrotransposon, but different in the 3'UTR. It seems to represent a more ancestral form of CDY and its characterization yields insights into the evolution of candidate genes for AZF.     

Cloning of the human and murine ROM1 genes: genomic organization and sequence conservation

Rom-1 and peripherin are related membrane proteins of the photoreceptorouter segments. Both proteins are located at the rims of thephotoreceptor disks, where they may act jointly in disk biogenesis.Mutations in the gene (RDS) encoding peripherin cause autosomaldominant retinitis pigmentosa, autosomal dominant punctata albescensand butterfly macular degeneration in man, and retinal degenerationslow in mice. To facilitate ROM1 mutation and linkage analysisin inherited retinal diseases, we cloned and characterized thehuman and murine ROM1 genes. In both species, the ROM1 codingregion is contained within     

Molecular cloning,complete sequence,and biological characterization of a xenotropic murine leukemia virus constitutively released from the human B-lymphoblastoid cell line DG-75

A previously undetected retrovirus has been isolated from the human Epstein-Barr virus (EBV)-negative, B-lymphoblastoid DG-75 cell line, widely used for EBV gene transfection studies. The complete 8207-base genome of the DG-75 retrovirus was molecularly cloned from viral mRNA and sequenced (Accession No. AF221065). Northern blot analysis with probes specific for the putative RU-5, gag, pol, and env regions identified a full-length viral RNA and spliced env mRNA. DG-75 viral RNA was isolated from the DG-75 cell sublines UW and KAR, but not from the HAD subline. The DG-75 retrovirus was isolated with primer-binding sites that match tRNA(Thr) and tRNA(Gln2). Homology searches revealed homology to (i) xenotropic NZB-9-1 env mRNA, (ii) Moloney-MLV pol region, and (iii) a truncated Evi-2 endogenous proviral sequence gag and pol region. Viral interference and infectivity assays confirmed the xenotropic nature of the DG-75 retrovirus. The DG-75 retrovirus is the first isolate of an exogenous xenotropic MLV in which the full-length genomic sequence has been characterized.     

Mouse histamine N-methyltransferase: cDNA cloning, expression, gene cloning and chromosomal localization

OBJECTIVE: Histamine N-methyltransferase (HNMT) catalyzes the Ntau-methylation of histamine. We set out to clone a mouse liver HNMT cDNA and the mouse HNMT gene as steps toward characterizing molecular genetic mechanisms involved in the regulation of this important histamine-metabolizing enzyme. DESIGN: A PCR-based strategy was used to clone both the mouse HNMT cDNA and the gene encoding that cDNA, Hnmt. The cDNA was used both to express recombinant mouse HNMT and to determine the chromosomal localization of Hnmt. RESULTS: The mouse liver HNMT cDNA was 1657 bp in length with an 888 bp open reading frame (ORF) that encoded a 296 amino acid protein with a predicted Mr value of approximately 32.5 kDa. The amino acid sequence of the encoded protein was 84% identical to that of human kidney HNMT. Mouse HNMT was expressed in COS-1 cells, and its apparent Km values for histamine and S-adenosyl-L-methionine (Ado-Met), the two cosubstrates for the reaction, were 5.3 and 5.8 microM, respectively. The mouse HNMT gene, Hnmt, spanned approximately 25 kb and had 7 exons. Its structure differed from that of the human gene primarily by the presence of an additional exon at the 5'-terminus. Hnmt mapped to mouse chromosome 2 in an area of conserved synteny to human chromosome 2q, the location of the human gene (2q22) on the basis of fluorescence in situ hybridization. CONCLUSIONS: Cloning and functional characterization of the mouse HNMT cDNA and gene will now make it possible to study in the mouse molecular genetic mechanisms involved the regulation of this important histamine-metabolizing enzyme.     

人类肝脏cDNA文库的构建及hHGF基因的筛选、克隆与表达

目的构建人类肝脏的cDNA文库，从中筛选hHGF基因并进行克隆与表达。方法从人类胎儿肝脏中快速分离提取出mRNA；将已提取出的mRNA构建成cDNA文库；从中筛选出hHGF（hepatocyte growth factor）基因后，进行克隆并测序确定；构建表达质粒pBV221-hHGF后转化大肠杆菌BL21（DE3α），以异丙基硫代-B-D-半乳糖苷（IPTG）诱导，收集菌体后提取蛋白样品并对其进行SDS-PAGE电泳及蛋白免疫印迹检测。结果成功地构建了人类肝脏的cDNA文库；经筛选得到约2200bp的hHGF基因；对其进行测序确认；成功地构建该基因的表达型质粒，并对表达蛋白进行蛋白免疫印迹检测，检测出其表达产生的蛋白质相对分子量在100000左右。结论人类肝脏cDNA文库成功地构建后可用于筛选获取完整的cDNA基因，从中筛选、克隆hHGF基因，并测序确认，进行表达蛋白质检测，结果正确，以上工作为进一步深入地进行与人类肝脏cDNA文库及hHGF相关的实验研究奠定了一定的基础。     

Porcine Fas-ligand gene: genomic sequence analysis and comparison with human gene.

Thymic Fas-ligand (FasL) cDNA and hepatic FasL genomic sequences were obtained from a 2-month-old LW pig. From these nucleotide sequences, amino acid sequence was deduced and compared with FasL sequences obtained from various animals. This comparison reveals that porcine FasL is closer to that of human, macaca and cat, and differs more from mouse and rat. The extracelluar domains of porcine and human FasL proteins appear to be functionally compatible. The complete genomic DNA sequence of porcine FasL was also compared with its human counterpart. Exons showed 80-89% nucleotide homology between pig and human, while introns showed 64-69% nucleotide homology. Sequence comparison by Harr plot analysis revealed many stretches within introns having identical sequences, suggesting that the sites may have unidentified common functions. One potential extra exon between exons 2 and 3 was located within porcine intron 2. This potential exon has no counterpart in human FasL intron 2. Whether or not this extra exon can be expressed and could cause additional immunological responses remains to be investigated. For future xenotransplantation, it is important to compare porcine and human genomic sequences, and to investigate their system compatibilities.