Structure and Chromosomal Localization of the Human Homeobox Gene Prox 1

The genomic organization and nucleotide sequence of the human homeobox gene Prox 1 as well as its chromosomal localization have been determined. This gene spans more than 40 kb, consists of at least 5 exons, and encodes an 83-kDa protein. It shows 89% identity with the chicken sequence at the nucleotide level in the coding region, while the human and chicken proteins are 94% identical. Among the embryonic tissues analyzed (lens, brain, lung, liver, and kidney), the human Prox 1 gene is most actively expressed in the developing lens, similar to the expression pattern of the chicken Prox 1 gene. The Prox 1 gene was mapped to human chromosome 1q32.2–q32.3.     

Structure and Chromosomal Localization of the Human Salivary Mucin Gene, MUC7

We have isolated and characterized several MUC7 genomic clones encoding the human low-molecular-weight salivary mucin, MG2. The MUC7 gene spans ∼10.0 kb and comprises of three exons and two introns. Intron 1 is ∼1.7 kb long and is located in the 5′-untranslated region of the corresponding MUC7 cDNA. Intron 2 spans ∼6.0 kb and is located close to the boundary of the putative leader peptide and secreted protein. The entire region encoding the secreted peptide is located on exon 3, spanning ∼2.2 kb. The nucleotide sequence of sections of the MUC7 gene, including 1500 bp of the 5′-flanking region, was determined and analyzed for motifs identical or homologous to other known response elements. A modified RACE procedure was used to determine the 5′-end of the MUC7 mRNA. PCR, the human–hamster somatic cell hybrid panel PCRable DNAs kit, and anin situhybridization analysis on the complete metaphase chromosome spreads were used for the chromosomal localization of the MUC7 gene. It was mapped to chromosome 4q13–q21.     

Structure and Chromosomal Localization of the Human Stromal Cell-Derived Factor 1 (SDF1) Gene

Stromal cell-derived factors 1α and 1β are small cytokines belonging to the intercrine CXC subfamily and originally isolated from a murine bone-marrow stroma cell line by the signal sequence trap method. cDNA and genomic clones of human SDF1α and SDF1β (SDF1A and SDF1B) were isolated and characterized. cDNAs of SDF1α and SDF1β encode proteins of 89 and 93 amino acids, respectively. SDF1α and SDF1β sequences are more than 92% identical to those of the human counterparts. The genomic structure of the SDF1 gene revealed that human SDF1α and SDF1β are encoded by a single gene and arise by alternative splicing. SDF1α and SDF1β are encoded by 3 and 4 exons, respectively. Ubiquitous expression of the SDF1 gene, except in blood cells, was consistent with the presence of the GC-rich sequence in the 5′-flanking region of the SDF1 gene, as is often the case in the "housekeeping" genes. Although genes encoding other members of the intercrine family are localized on chromosome 4q or 17q, the human SDF1 gene was mapped to chromosome 10q by fluorescence in situ hybridization. Strong evolutionary conservation and unique chromosomal localization of the SDF1 gene suggest that SDF1α and SDF1β may have important functions distinct from those of other members of the intercrine family.     

Human Indolethylamine N-Methyltransferase: cDNA Cloning and Expression, Gene Cloning, and Chromosomal Localization

Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. We recently cloned and characterized the rabbit INMT cDNA and gene as a step toward cloning the cDNA and gene for this enzyme in humans. We have now used a PCR-based approach to clone a human INMT cDNA that had a 792-bp open reading frame that encoded a 263-amino-acid protein 88% identical in sequence to rabbit INMT. Northern blot analysis of 35 tissues showed that a 2.7-kb INMT mRNA species was expressed in most tissues. When the cDNA was expressed in COS-1 cells, the recombinant enzyme catalyzed the methylation of tryptamine with an apparent K_m value of 2.9 mM. The human cDNA was then used to clone the human INMT gene from a human genomic BAC library. The gene was 5471 bp in length, consisted of three exons, and was structurally similar to the rabbit INMT gene as well as genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. All INMT exon–intron splice junctions conformed to the “GT-AG” rule, and no canonical TATA or CAAT sequences were present within the 5′-flanking region of the gene. Human INMT mapped to chromosome 7p15.2–p15.3 on the basis of both PCR analysis and fluorescence in situ hybridization. Finally, two possible single nucleotide polymorphisms were identified within exon 3, both of which altered the encoded amino acid. The cloning and expression of a human INMT cDNA, as well as the cloning, structural characterization, and mapping of its gene represent steps toward future studies of the function and regulation of this methyltransferase enzyme in humans.     

小麦生长素结合基因TaABP1-D的表达与染色体定位

生长素影响了植物生长发育的诸多过程。生长素结合蛋白ABP1(auxin binding protein)作为一种生长素受体,在质膜上生长素诱导的快速反应中起重要作用。小麦中已经克隆获得了TaABP1-D,但其在细胞中的作用位置以及在染色体的定位情况仍不明确。本研究利用洋葱表皮细胞瞬时表达系统对小麦生长素结合基因TaABP1-D进行亚细胞定位,结果表明TaABP1-D蛋白为膜蛋白,存在于细胞质和细胞膜中;同时利用中国春缺体-四体材料和信息学方法,将TaABP1-D定位在小麦5D染色体长臂的近着丝粒位置上,距两侧EST标记BE490079和BE405060的遗传距离分别为0.51 c M和0.28 c M。     

家鸡G蛋白偶联受体85基因的结构、序列与组织表达分析

G蛋白偶联受体(GPCR)超家族是细胞膜上广泛存在的一类受体,是细胞跨膜信号转导的一类重要受体分子,参与许多生理过程调节。它们中仍有很多至今尚未找到内源性配体,这类受体被称为孤儿型受体。G蛋白偶联受体85(GPR85)是GPCR超家族中孤儿型受体的一员。目前,在非哺乳类脊椎动物中,针对GPR85的研究极少。本研究以家鸡Gallus gallus domesticus为模型,通过反转录PCR和RACE-PCR等方法从脑中克隆到GPR85基因的cDNA全长序列,揭示其基因结构,并用实时荧光定量PCR(qPCR)方法探究了该基因在家鸡各组织中的表达情况。结果显示:家鸡GPR85基因位于1号染色体上,由2个外显子组成,其编码区位于第2个外显子上,长为1 113 bp,可编码1个370个氨基酸的7次跨膜受体蛋白。家鸡GPR85与其他脊椎动物(人Homo sapiens、小鼠Mus musculus、大鼠Rattus norvegicus、热带爪蟾Xenopus tropicalis和斑马鱼Danio rerio)的GPR85具有高度的氨基酸序列一致性(>93%)。qPCR分析发现,GPR85基因mRNA在家鸡全脑、垂体、肾上腺、精巢中有较高表达,而在所检测的其他外周组织中表达极低。本研究首次揭示了家鸡GPR85基因的结构与表达特征,为后续探究GPR85基因在家鸡等非哺乳类中的生理功能奠定基础。     

Genomic Structure and Chromosomal Localization of Mouse Cyclin G1 Gene

Mouse genomic DNA harboring the full coding sequence of cyclin G1 was cloned and analyzed. The locations of five coding exons and the intron–exon boundary sequences were found to be conserved between the mouse and the human genes. Two putative binding sites for thep53tumor suppressor gene product were found around the first exon: one was located in the 5′ regulatory region, and the other was in the first intron. The mouse cyclin G1 gene was mapped to bands A5 to B1 of chromosomes 11 (11A5–B1) by FISH using genomic DNA clone as a biotinylated probe. The location of mouse cyclin G1 is syntenic to that of its human homologue, which we previously mapped to 5q32–q34 of chromosome 5. An additional faint signal was detected on chromosome 4 (4B1–C2), probably indicating the presence of a cyclin G1-related gene or pseudogene in the mouse genome.     

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 fluorescencein situhybridization, 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.     

Primary Structure of Human 11-cisRetinol Dehydrogenase and Organization and Chromosomal Localization of the Corresponding Gene

The universal chromophore of visual pigments in higher animals is 11-cisretinaldehyde. The final step in the biosynthetic pathway generating this compound is catalyzed by 11-cisretinol dehydrogenase, a membrane-bound enzyme abundantly expressed in the retinal pigment epithelium of the eye. In this work we demonstrate that the primary structure of human 11-cisretinol dehydrogenase is highly conserved with 91% identity to the bovine enzyme. The gene encoding 11-cisretinol dehydrogenase spans over ≈4.1 kb of DNA and is divided into four translated exons. Analysis of a panel of somatic cells hybrids and fluorescencein situhybridization on metaphase chromosomes revealed that the gene is located on chromosome 12q13–q14. Due to the unique role of 11-cisretinol dehydrogenase in the generation of visual pigments, it is a candidate gene for involvement in hereditary eye disease.     

Structural Organization of the Mouse and Human GALR1 Galanin Receptor Genes (GalnrandGALNR) and Chromosomal Localization of the Mouse Gene

The neuropeptide galanin elicits a range of biological effects by interaction with specific G-protein-coupled receptors. Human and rat GALR1 galanin receptor cDNA clones have previously been isolated using expression cloning. We have used the human GALR1 cDNA in hybridization screening to isolate the gene encoding GALR1 in both human (GALNR) and mouse (Galnr). The gene spans approximately 15–20 kb in both species; its structural organization is conserved and is unique among G-protein-coupled receptors. The coding sequence is contained on three exons, with exon 1 encoding the N-terminal end of the receptor and the first five transmembrane domains. Exon 2 encodes the third intracellular loop, while exon 3 encodes the remainder of the receptor, from transmembrane domain 6 to the C-terminus of the receptor protein. The mouse and human GALR1 receptor proteins are 348 and 349 amino acids long, respectively, and display 93% identity at the amino acid level. The mouseGalnrgene has been localized to Chromosome 18E4, homoeologous with the previously reported localization of the humanGALNRgene to 18q23 in the same syntenic group as the genes encoding nuclear factor of activated T-cells, cytoplasmic 1, and myelin basic protein.     

Complete cDNA Sequence, Genomic Structure, and Chromosomal Localization of the LPA Receptor Gene,lpA1/vzg-1/Gpcr26

Thelp_A1/Gpcr26locus encodes the first cloned and identified G-protein-coupled receptor that specifically interacts with lysophosphatidic acid. A murine full-length cDNA of size consistent with that seen on Northern blots (3.7 kb) was determined using 3′ rapid amplification of cDNA ends. Analysis of genomic clones revealed that the gene is divided into five exons, with one intron inserted in the coding region for transmembrane domain VI and one exon encoding the divergent 5′ sequence in another published cDNA clone variant (orphan receptor mrec1.3). This structure differs from the intronless coding region for a homologous receptor,Edg1,but is identical to another more similar orphan receptor (lp_A2) that has been deposited with GenBank. Using backcross analysis, both exons 1 and 4 mapped to a proximal region of murine Chromosome 4 indistinguishable from the vacillans gene. Exon 4 also mapped to a second locus on proximal Chromosome 6 inMus spretus,and this partial duplication was confirmed by Southern blot. The genomic structure indicates a distinct, divergent evolutionary lineage for thevzg-1/lp_A1subfamily of receptors compared to those of homologous orphan receptor genes.