Human and Mouse Homologs of theSchizosaccharomyces pombe rad17+Cell Cycle Checkpoint Control Gene

TheSchizosaccharomyces pombe rad17⁺cell cycle checkpoint control gene is required for S-phase and G2/M arrest in response to both DNA damage and incomplete DNA replication. We isolated and characterized the putative human (RAD17Sp) and mouse (mRAD17Sp) homologs of theS. pombeRad17 (Rad17Sp) protein. The humanRAD17Spopen reading frame (ORF) encodes a protein of 681 amino acids; themRAD17SpORF codes for a protein of 688 amino acids. ThemRAD17Spmessenger is highly expressed in the testis as a single 3-kb mRNA species. The human RAD17Sp and mRAD17Sp proteins are 24% identical and 46% similar to theS.pombeRad17Sp protein. Sequence homology was also noted with theSaccharomyces cerevisiaeRad24Sc (which is the structural counterpart ofS.pombeRad17Sp) and structurally related polypeptides fromCaenorhabditis elegans, Arabidopsis thaliana, Pyrococcus horikoshii,andDrosophila melanogaster.The degree of conservation between the mammalian RAD17Sp proteins and those of the other species is consistent with the evolutionary distance between the species, indicating that these proteins are most likely true counterparts. In addition, homology was found between the Rad17Sp homologs and proteins identified as components of mammalian replication factor C (RF-C)/activator 1, especially in several highly conserved RF-C-like domains including a “Walker A” motif. Using FISH and analysis of a panel of rodent–human cell hybrids, the humanRAD17Spgene (HGMW-approved symbolRAD17could be localized on human chromosome 5q13–q14, a region implicated in the etiology of small cell lung carcinoma, non-small-cell lung carcinoma, duodenal adenocarcinoma, and head and neck squamous cell carcinoma. Our results suggest that the structure and function of the checkpoint “rad” genes in the G2/M checkpoint pathway are evolutionary conserved between yeast and higher eukaryotes.     

Orthologues of the Caenorhabditis elegans Longevity Gene clk-1 in Mouse and Human

The clk-1 gene was isolated from the long-lived mutant of Caenorhabditis elegans and was suggested to play a biological role in longevity (Ewbank et al., 1997, Science 275: 980-983). The primary structure of CLK-1 showed a significant homology to Saccharomyces cerevisiae Coq7p/Cat5p, which is required for the biosynthesis of ubiquinone and the derepression of gluconeogenic genes. In the present study, we isolated and characterized human and mouse orthologues of the COQ7/CLK-1 gene. Sequence analysis of both the human and the mouse COQ7 cDNAs showed an open reading frame composed of 217 amino acids with calculated molecular mass of 24,309 and 24,044 Da, respectively. Homology search revealed that human COQ7 showed 85% identity to mouse COQ7, 89% identity to rat COQ7, 53% identity to C. elegans CLK-1, and 37% identity to S. cerevisiae Coq7p/Cat5p. Zoo blot analysis implied that the COQ7 gene was well conserved among mammal, bird, and reptile genomes. Tissue blot analysis showed that human COQ7 is dominantly transcribed in heart and skeletal muscle. Genomic analyses revealed that the human COQ7 gene is composed of six exons spanning 11 kb of human genome as a single-copy gene. Radiation hybrid mapping assigned the COQ7 gene to human chromosome 16p12.3-p13.11.     

青杄中sPPa1的cDNA序列克隆及其生物信息学分析

以青杄(Picea wilsonii)均一化cDNA文库为模板,通过RACE方法克隆得到青杄PPa1基因cDNA全长,对该cDNA序列、核苷酸序列的相似性、理化性质、疏水性、二级结构、三级结构及是否跨膜进行了分析预测;进行了多序列比对并构建了系统树,同时对PPa1在青杄各组织中的表达量进行了检测。结果表明:青杄PPa1基因共由216个氨基酸组成,分子量为24.55 kD,理论PI为5.83,属可溶性蛋白;二级结构主要由α-螺旋、不规则卷曲和β-折叠构成;PPa1在青杄花粉中表达量最高。研究为进一步研究青杄PPa1的功能奠定了基础。     

人LCRG1基因启动子的鉴定与初步分析

Laryngeal carcinoma related gene 1(LCRC1)是一个喉癌候选抑瘤基因,为进一步深入研究其转录调控机制,应用5'RACE技术确定了该基因的转录起始位点,然后在对人LCRG1基因进行生物信息学分析的基础上,通过PCR定向克隆和酶切亚克隆策略,构建了11种含不同长度LCRG1启动子荧光素酶报告基因重组体.启动子活性分析表明,-169～ 127区域的启动子活性最高.研究提示,LCRG1基因转录所必需的基因启动子序列在-169～ 127范围内.     

Cloning and Gene Mapping of the Mouse Homologue of theCBFA2T1Gene Associated with Human Acute Myeloid Leukemia

The humanCBFA2T1(also known asMTG8) gene, on chromosome 8, has been identified through its involvement in the t(8;21) chromosomal translocation, frequently found in acute myeloid leukemia. We report here the isolation and characterization of the mouse homologue of theCBFA2T1gene,Cbfa2t1h.Nucleotide sequence analysis ofCbfa2t1hcDNA clones revealed an open reading frame encoding a protein of 577 amino acids with an extremely high degree of amino acid identity (99.3%) to the human protein. The nucleotide sequence is also highly conserved between mouse and human in the 5′- and 3′-untranslated regions (87.0, 92.0, and 93.7% identities for 5′-untranslated, coding, 3′-untranslated regions, respectively). The 3′-untranslated region ofCbfa2t1hcontains a (CA)_ndinucleotide repeat, and the polymerase chain reaction amplification of the (CA)_nrepeat region revealed fragment length polymorphism among mouse strains. Using this polymorphism, we have mappedCbfa2t1hto mouse chromosome 4 close to the centromere using SMXA recombinant inbred strains and 106 intersubspecific backcross progenies of the (DBA/2 × Mae) × Mae cross. The chromosomal location was also confirmed by fluorescencein situhybridization.     

Cloning, Analysis, and Chromosomal Localization of Myoxin (MYH12), the Human Homologue to the Mouse dilute Gene

The mouse dilute gene encodes a novel type of nonmuscle myosin that structurally combines elements from both nonmuscle myosin type I and nonmuscle myosin type II. Phenotypically, mutations in the mouse dilute gene result not only in the lightening of coat color, but also in the onset of severe neurological defects shortly after birth. This may indicate that the mouse dilute gene is important in maintaining the normal neuronal function in the mouse. We report the isolation and sequencing of "myoxin" (MYH12), the human homologue of the mouse dilute gene, and its assignment to human chromosome 15.     

Characterization of a Mouse Gene (Fbxw6) That Encodes a Homologue of Caenorhabditis elegans SEL-10

The SCF complex is a type of ubiquitin ligase that consists of the invariable components SKP1, CUL1, and RBX1 as well as a variable component, known as an F-box protein, that is the main determinant of substrate specificity. The Caenorhabditis elegans F-box- and WD40-repeat-containing protein SEL-10 functionally and physically associates with LIN-12 and SEL-12, orthologues of mammalian Notch and presenilin, respectively. We have now identified a gene (which we call Fbxw6) that encodes a mouse homologue (F-box–WD40 repeat protein 6, or FBW6) of SEL-10 and is expressed mainly in brain, heart, and testis. Co-immunoprecipitation analysis showed that FBW6 interacts with SKP1 and CUL1, indicating that these three proteins form an SCF complex. Comparison of the genomic organization of Fbxw6, which is located on mouse chromosome 3.3E3, with that of mouse Fbxw1, Fbxw2, and Fbxw4 showed only a low level of similarity, indicating that these genes diverged relatively early and thereafter evolved independently.     

Sequence comparison of the Caenorhabditis elegans dpy-13 and col-34 genes, and their deduced collagen products

A 2232-nucleotide sequence spanning the col-34 gene from the nematode, Caenorhabditis elegans, is presented. This gene, which encodes a collagen protein (Clg), is transcribed from right to left with respect to the genetic map, and convergently with the nearby dpy-13 gene which also encodes a Clg. Both col-34 and dpy-13 have 5'-flanking elements in common with each other and also with other nematode Clg-encoding genes (clg). One element, variants of which are shared by col-7, col-19 and dpy-13, is predicted to be a target for a number of regulatory molecules, possibly including the ceh-18 product, a nematode POU-domain protein. The deduced amino acid sequence of Col-34 has a high degree of homology with the Dpy-13 collagen, although there are significant differences. In particular, one region of Dpy-13, which is predicted to have secondary structure different from Col-34, is altered by the recessive dpy-13(e225) mutation.     

MappingTNNC1, the Gene That Encodes Cardiac Troponin I in the Human and the Mouse

We have mapped the TNNC1 gene, whose protein product is the cardiac TnI protein. TnI is one of the proteins that makes up the troponin complex, which mediates the response of muscle to calcium ions. The human TNNC1 locus had been assigned to a large region of chromosome 19, and we have refined the mapping position to the distal end of the chromosome by amplification of DNAs from a chromosome 19 mapping panel. We have also mapped the mouse Tnnc1 locus, by following the segregation of an intron sequence through DNAs from the European Interspecific Backcross. Tnnc1 maps close to the centromere on mouse chromosome 7.     

中国水仙水杨酸甲酯合成酶基因克隆与表达分析

为了解中国水仙(Narcissus tazetta var. chinensis)花香气形成的分子机理,以花发育形成相关基因的SSH文库获得的cDNA片段为基础,利用RACE技术从中国水仙花朵中克隆了水杨酸甲酯合成酶基因,命名为NtSAMT1 (GeneBank No. JX273470),其cDNA全长1323 bp,包含1个1131 bp完整阅读框架,编码376个氨基酸,具有Methyltransf_7 superfamily蛋白保守区,与仙女扇(Clarkia breweri) SAMT蛋白(1m6eX)的三维结构相似。系统进化树分析表明,中国水仙与粳稻的SAMT蛋白亲缘关系最近。原核表达结果表明NtSAMT1在大肠杆菌中能高效表达。半定量RT-PCR分析表明,NtSAMT1在花蕾期就有表达,第1天完全开放时各部位均有表达,以雄蕊与雌蕊的表达量最高,第8天已检测不到表达。     

巴西橡胶树HbWRKY1基因的克隆及转化烟草的初步研究

利用RACE结合RT-PCR技术,从巴西橡胶树(Hevea brasiliensis)总RNA中扩增得到长度为1234 bp的WRKY基因cDNA全长编码序列。通过氨基酸同源性比对,该序列推导的氨基酸序列与蓖麻、白杨的WRKY同源性分别为79%和73%,表明分离的cDNA序列为橡胶树WRKY基因,命名为HbWRKY1。通过构建pCAMBIA1304-HbWRKY1植物表达载体,经农杆菌GV3101介导,将HbWRKY1基因导入烟草(Nicotiana tabacum)中,对所获得的潮霉素抗性烟草株系进行PCR鉴定。结果表明,HbWRKY1基因已整合到65株转基因植株中。干旱胁迫试验表明,HbWRKY1的过量表达可以明显提高转基因烟草对干旱胁迫的耐受能力。这说明WRKY基因与橡胶树抗旱能力之间存在一定的关系。     

棉花GhZIP4基因的克隆及表达分析

根据EST拼接的序列设计引物,利用RT-PCR和PCR方法,从陆地棉‘苏棉18’-cDNA和基因组DNA中分别克隆获得了GhZIP4基因片段.结果表明:(1)GhZIP4基因cDNA序列全长1 487 bp,包含1 269 bp ORF,编码422个氨基酸残基,其氨基酸序列具有典型的ZIP蛋白特征,预测具有8个跨膜结构域,第Ⅲ和第Ⅳ跨膜结构域间存在可变区,在可变区有2个富含His的结构域“HRHSHPHG”和“HSHGHGHD”.(2)氨基酸进化树分析显示,GhZIP4同拟南芥ZIP家族AtZIP4的相似性较高.(3)GhZIP4 DNA序列编码区全长1 778 bp,包含4个外显子和3个内含子,所有外显子/内含子交接点都遵从gt/ag剪接规则.(4)半定量分析显示,GhZIP4基因在茎中表达量最高,表明该基因有可能在某些金属离子地上部和根部的动态平衡分布过程中具有重要作用.     

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.     

HumanGFRA1: Cloning, Mapping, Genomic Structure, and Evaluation as a Candidate Gene for Hirschsprung Disease Susceptibility

Congenital aganglionic megacolon, commonly known as Hirschsprung disease (HSCR), is the most frequent cause of congenital bowel obstruction. Germline mutations in theRETreceptor tyrosine kinase have been shown to cause HSCR. Knockout mice forRETand for its ligand, glial cell line-derived neurotrophic factor (GDNF), exhibit both complete intestinal aganglionosis and renal defects. Recently, GDNF and GFRA1 (GDNF family receptor, also known as GDNFR-α), its GPI-linked coreceptor, were demonstrated to be components of a functional ligand for RET. Moreover,GDNFhas been implicated in rare cases of HSCR. We have mappedGFRA1to human chromosome 10q25, isolated human and mouse genomic clones, determined the gene's intron–exon boundaries, isolated a highly polymorphic microsatellite marker adjacent to exon 7, and scanned forGFRA1mutations in a large panel of HSCR patients. No evidence of linkage was detected in HSCR kindreds, and no sequence variants were found to be in significant excess in patients. These data suggest thatGFRA1's role in enteric neurogenesis in humans remains to be elucidated and that RET signaling in the gut may take place via alternate pathways, such as the recently described GDNF-related molecule neurturin and its GFRA1-like coreceptor, GFRA2.