Sequence and expression of human estrogen receptor complementary DNA

The mechanism by which the estrogen receptor and other steroid hormone receptors regulate gene expression in eukaryotic cells is not well understood. In this study, a complementary DNA clone containing the entire translated portion of the messenger RNA for the estrogen receptor from MCF-7 human breast cancer cells was sequenced and then expressed in Chinese hamster ovary (CHO-K1) cells to give a functional protein. An open reading frame of 1785 nucleotides in the complementary DNA corresponded to a polypeptide of 595 amino acids and a molecular weight of 66,200, which is in good agreement with published molecular weight values of 65,000 to 70,000 for the estrogen receptor. Homogenates of transformed Chinese hamster ovary cells containing a protein that bound [3H]estradiol and sedimented as a 4S complex in salt-containing sucrose gradients and as an 8 to 9S complex in the absence of salt. Interaction of this receptor-[3H]estradiol complex with a monoclonal antibody that is specific for primate ER confirms the identity of the expressed complementary DNA as human estrogen receptor. Amino acid sequence comparisons revealed significant regional homology among the human estrogen receptor, the human glucocorticoid receptor, and the putative v-erbA oncogene product. This suggests that steroid receptor genes and the avian erythroblastosis viral oncogene are derived from a common primordial gene. The homologous region, which is rich in cysteine, lysine, and arginine, may represent the DNA-binding domain of these proteins.     

Hormone-sensitive lipase: sequence, expression, and chromosomal localization to 19 cent-q13.3

Hormone-sensitive lipase, a key enzyme in fatty acid mobilization, overall energy homeostasis, and possibly steroidogenesis, is acutely controlled through reversible phosphorylation by catecholamines and insulin. The 757-amino acid sequence predicted from a cloned rat adipocyte complementary DNA showed no homology with any other known lipase or protein. The activity-controlling phosphorylation site was localized to Ser563 in a markedly hydrophilic domain, and a lipid-binding consensus site was tentatively identified. One or several messenger RNA species (3.3, 3.5, or 3.9 kilobases) were expressed in adipose and steroidogenic tissues and heart and skeletal muscle. The human hormone-sensitive lipase gene mapped to chromosome 19 cent-q13.3.     

The fragile X site in somatic cell hybrids: an approach for molecular cloning of fragile sites

Fragile X syndrome is a common form of mental retardation associated with a fragile site on the human X chromosome. Although fragility at this site is usually evident as a nonstaining chromatid gap, it remains unclear whether or not actual chromosomal breakage occurs. By means of somatic cell hybrids containing either a normal human X or a fragile X chromosome and utilizing two genes that flank the fragile site as markers of chromosome integrity, segregation of these markers was shown to be more frequent if they encompass the fragile site under appropriate culture conditions. Hybrid cells that reveal marker segregation were found to contain rearranged X chromosomes involving the region at or near the fragile site, thus demonstrating true chromosomal breakage within this area. Two independent translocation chromosomes were identified involving a rodent chromosome joined to the human X at the location of the fragile site. DNA analysis of closely linked, flanking loci was consistent with the position of the breakpoint being at or very near the fragile X site. Fragility at the translocation junctions was observed in both hybrids, but at significantly lower frequencies than that seen in the intact X of the parental hybrid. This observation suggests that the human portion of the junctional DNA may contain part of a repeated fragility sequence. Since the translocation junctions join heterologous DNA, the molecular cloning of the fragile X sequence should now be possible.     

Chromosomal location of human T-cell receptor gene Ti beta

A complementary DNA probe corresponding to the beta-chain gene of Ti, the human T lymphocyte receptor, has been molecularly cloned. The chromosomal origin of the Ti beta gene was determined with the complementary DNA by screening a series of 12 cell hybrid (mouse X human) DNA's containing overlapping subsets of human chromosomes. DNA hybridization (Southern) experiments showed that the human Ti beta gene resides on chromosome 7 and is thus not linked to the immunoglobulin loci or to the major histocompatibility locus in humans.     

狮头鹅AR基因克隆和组织表达

本研究旨在克隆鹅雄激素受体基因（Androgen receptor，AR），并了解其在不同组织中的表达情况.以狮头鹅为试验材料，采集下丘脑和睾丸组织样品，提取RNA逆转录后进行AR基因克隆，并用RACE扩增其cDNA全长序列，其它同采用实时荧光定量PCR的方法检测AR基因在各个组织中的表达情况.结果克隆获得AR基因全长cDNA序列，共得到4个转录本.通过氨基酸序列同源进行分析，发现狮头鹅AR基因在禽类和哺乳类动物中同源性较高，说明该基因在禽类和哺乳类进化保守.荧光定量PCR结果显示AR基因在狮头鹅12个组织中均有表达量，其中在腿肌、胸肌和睾丸表达量较高，表明AR基因可能参与调控狮头鹅公鹅的肌肉生长与繁殖.     

Genomic sequencing and methylation analysis by ligation mediated PCR

Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited because of the complexity of the mammalian genome. A newly developed genomic sequencing procedure in which a ligation mediated polymerase chain reaction (PCR) is used generates high quality, reproducible sequence ladders starting with only 1 microgram of uncloned mammalian DNA per reaction. Different sequence ladders can be created simultaneously by inclusion of multiple primers and visualized separately by rehybridization. Relatively little radioactivity is needed for hybridization and exposure times are short. Methylation patterns in genomic DNA are readily detectable; for example, 17 CpG dinucleotides in the 5' region of human X-linked PGK-1 (phosphoglycerate kinase 1) were found to be methylated on an inactive human X chromosome, but unmethylated on an active X chromosome.     

X染色体60149273位点在脂尾（臀）和瘦尾绵羊品种中的多态性及其基因定位

【目的】研究绵羊X染色体60149273位点在不同沉脂尾型绵羊品种中的多态性,为解析绵羊尾脂沉积性状的遗传机制提供基础数据。【方法】首先,采用PCR-SSCP分型方法检测X染色体60149273位点在不同脂尾类型绵羊品种的多态性,并借助PCR产物直接测序的方法确认各基因型相应序列,利用卡方检验的方法检测各基因型分布在不同品种中的平衡性。其次,利用生物信息方法将该SNP定位与绵羊androgen receptor（AR）,并使用电子克隆的方法克隆出绵羊AR全长编码序列。同时以阿勒泰羊臀部脂肪RNA为材料,采用RT-PCR的方克隆绵羊AR第3—8外显子序列,利用生物信息学方法分析序列同源性以及序列的物种进化保守性。【结果】①PCR-SSCP结果显示,在脂尾（臀）型绵羊品种阿勒泰羊和湖羊群体中X染色体60149273位点未检出多态性,GG基因型占100%;而瘦尾（臀）型绵羊品种中国美利奴和萨福克羊群体中则出现了多态性,GG基因型分别下降至10%和21%。②利用比较基因组学首次电子克隆出该SNP所属基因AR的全长编码区,并从阿勒泰羊臀脂mRNA中成功扩增羊源AR第3—8外显子片段,测序结果与电子克隆序列完全一致。③物种同源性比对进一步显示,AR在哺乳类动物中高度保守,哺乳动物各物种间同源性高达90%;进化树分析结果显示,牛、绵羊、海豚和猪等哺乳动物亲缘较近。【结论】首次发现绵羊AR第3内含子一处SNP在脂尾（臀）与瘦尾绵羊品种中存在较大差异,该SNP将可作为一个分子标记运用于低脂绵羊新品种的培育,同时该SNP所属AR也可作为一个重要候选功能基因应用于绵羊尾（臀）脂沉积分子机制的相关研究。     

Gene for human insulin receptor: localization to site on chromosome 19 involved in pre-B-cell leukemia

Consistent chromosomal translocations in neoplastic cells may alter the expression of proto-oncogenes that are located near the breakpoints. The complementary DNA sequence of the human insulin receptor is similar to those of the EGF receptor (erbB oncogene) and products of the src family of oncogenes. With in situ hybridization and Southern blot analysis of somatic cell hybrid DNA, the human insulin receptor gene was mapped to the distal short arm of chromosome 19 (bands p13.2----p13.3), a site involved in a nonrandom translocation in pre-B-cell acute leukemia.     

Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization

Several of the endothelial cell polypeptide mitogens that have been described probably play a role in blood vessel homeostasis. Two overlapping complementary DNA clones encoding human endothelial cell growth factor (ECGF) were isolated from a human brain stem complementary DNA library. Southern blot analysis suggested that there is a single copy of the ECGF gene and that it maps to human chromosome 5 at bands 5q31.3 to 33.2 A 4.8-kilobase messenger RNA was present in human brain stem messenger RNA. The complete amino acid sequence of human ECGF was deduced from the nucleic acid sequence of these clones; it encompasses all the well-characterized acidic endothelial cell polypeptide mitogens described by several laboratories. The ECGF-encoding open reading frame is flanked by translation stop codons and provides no signal peptide or internal hydrophobic domain for the secretion of ECGF. This property is shared by human interleukin-1, which is approximately 30 percent homologous to ECGF.     

斑马鱼尾加压素Ⅱ受体基因组织表达的研究

尾加压素Ⅱ受体(UrotensinⅡreceptor,UT)是一种7次跨膜G蛋白偶联受体,是目前发现的具有最强收缩血管作用的尾加压素Ⅱ的特异性受体。斑马鱼中存在4种不同的UT-like基因(uts2r1,uts2r2,uts2r3和uts2r4),分别位于斑马鱼4条不同染色体上。这4种uts2r基因核苷酸序列各不相同,uts2r1位于3号染色体上,其与12号染色体上的uts2r2基因蛋白序列相似度最高为47%,与6号染色体上的uts2r3和16号染色体上的uts2r4蛋白相似度分别是41.6%和33%。通过设计特异引物检测每种uts2r基因在斑马鱼成鱼各组织中的表达情况,结果显示3号染色体上的uts2r1基因在肾脏中表达量最高,6号染色体上的uts2r2基因在脊髓中表达量最高,12号染色体上的uts2r3基因在心脏中表达量最高,而16号染色体上的uts2r4基因在背肌中表达量最高。     

A human Y-linked DNA polymorphism and its potential for estimating genetic and evolutionary distance

A human DNA sequence (p12f2), derived from a partial Y-chromosome genomic library and showing homology with the X and Y chromosomes and with an undetermined number of autosomes, detected two Y-specific restriction fragment length variants on male DNA that had been digested with Taq I and Eco RI. These variants may have been generated through a deletion-insertion mechanism and their pattern of holoandric transmission indicates that they represent a two-allele Y-linked polymorphism (RFLP). By means of DNA from patients with inborn deletions in chromosome Y, this polymorphic DNA site was mapped to the interval Yq11.1-Yq11.22. The frequency of the rarest allele was about 35 percent in Algerian and Sardinian human males, whereas it was only 4 percent among Northern Europeans. The p12f2 probe also detected Y-specific DNA fragments in the gorilla and chimpanzee. In view of the monosomy of the Y chromosome in mammalian species, Y-linked RFLP's may prove to be more useful than autosomal or X-linked markers in estimating genetic distances within and between species.     

Chromosome mapping and expression of a putative testis-determining gene in mouse

Isolation and mapping of a mouse complementary DNA sequence (mouse Y-finger) encoding a multiple, potential zinc-binding, finger protein homologous to the candidate human testis-determining factor gene is reported. Four similar sequences were identified in Hind III-digested mouse genomic DNA. Two (7.2 and 2.0 kb) were mapped to the Y chromosome. Only the 2.0-kb fragment, however, was correlated with testis determination. Polymerase chain reaction analysis suggests both Y loci are transcribed in adult testes. A 3.6-kb fragment was mapped to the X chromosome between the T16H and T6R1 translocation breakpoints, and a fourth (6.0 kb) was mapped to chromosome 10. Hence, mYfin sequences have been duplicated several times in the mouse, although they are not duplicated in humans.     

Human apolipoprotein B: structure of carboxyl-terminal domains, sites of gene expression, and chromosomal localization

Apolipoprotein (apo-) B is the ligand responsible for the receptor-mediated catabolism of low density lipoproteins, the principal cholesterol-transporting lipoproteins in plasma. The primary structure of the carboxyl-terminal 30 percent (1455 amino acids) of human apo-B (apo-B100) has been deduced from the nucleotide sequence of complementary DNA. Portions of the protein structure that may relate to its receptor binding function and lipid binding properties have been identified. The apo-B100 messenger RNA is about 19 kilobases in length. The apo-B100 gene is expressed primarily in liver and, to a lesser extent, in small intestine, but in no other tissues. The gene for apo-B100 is located in the p24 region (near the tip of the short arm) of chromosome 2.     

Molecular cloning of complementary DNA encoding the avian receptor for vitamin D

Vitamin D3 receptors are intracellular proteins that mediate the nuclear action of the active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Two receptor-specific monoclonal antibodies were used to recover the complementary DNA (cDNA) of this regulatory protein from a chicken intestinal lambda gt11 cDNA expression library. The amino acid sequences that were deduced from this cDNA revealed a highly conserved cysteine-rich region that displayed homology with a domain characteristic of other steroid receptors and with the gag-erbA oncogene product of avian erythroblastosis virus. RNA selected via hybridization with this DNA sequence directed the cell-free synthesis of immunoprecipitable vitamin D3 receptor. Northern blot analysis of polyadenylated RNA with these cDNA probes revealed two vitamin D receptor messenger RNAs (mRNAs) of 2.6 and 3.2 kilobases in receptor-containing chicken tissues and a major cross-hybridizing receptor mRNA species of 4.2 kilobases in mouse 3T6 fibroblasts. The 4.2-kilobase species was substantially increased by prior exposure of 3T6 cells to 1,25(OH)2D3. This cDNA represents perhaps the rarest mRNA cloned to date in eukaryotes, as well as the first receptor sequence described for an authentic vitamin.     

核桃早实性相关SCAR标记(1-1500)基因末端序列的克隆

[目的]克隆SCAR标记的核桃早实性相关基因片段[1](AFLP早实分子标记转化成的SCAR标记)的末端序列,为验证其功能和早实核桃分子育种奠定基础.[方法]采用RACE技术对SCARE标记的核桃早实性相关基因片段设计特异性PCR引物,并扩增其末端序列.[结果]分别获得了长度为453 bp和463 bp的片段,通过与NCBI核酸数据库中已经发表的序列进行比对分析,发现该基因3'末端含有358个核苷酸非编码序列.其核酸序列与葡萄假定蛋白相应部位同源性为55.26;,与葡萄重叠群相应部位同源性为38.38;,与线虫枯粒Y38F2AR基因全序列相应部位相似性为40.86;,和野猪免疫球蛋白超家族成员相应部位的相似性为39.75;,具有poly(A)尾.5'末端含有248个核苷酸非编码序列,其核酸序列与葡萄重叠群基因组鸟枪全序列相应部位同源性为39.07;,与拟南芥基因组DNA 3号染色体相应部位的同源性为42.22;,与嗜热四膜虫假定蛋白基因序列相应部位相似性为44.53;,和斑马鱼DNA序列DKEY-120E17克隆2号连锁群全序列相应部位相似性为42.30;.[结论]试验采用的3'和5'末端快速扩增技术(3'RACE和5'RACE技术)能很好地扩增核桃早实性相关基因的末端序列.     

Reactivation of an inactive human X chromosome: evidence for X inactivation by DNA methylation

A mouse-human somatic cell hybrid clone, deficient in hypoxanthine-guanine phosphoribosyltransferase (HPRT) and containing a structurally normal inactive human X chromosome, was isolated. The hybrid cells were treated with 5-azacytidine and tested for the reactivation and expression of human X-linked genes. The frequency of HPRT-positives clones after 5-azacytidine treatment was 1000-fold greater than that observed in untreated hybrid cells. Fourteen independent HPRT-positive clones were isolated and analyzed for the expression of human X markers. Isoelectric focusing showed that the HPRT expressed in these clones is human. One of the 14 clones expressed human glucose-6-phosphate dehydrogenase and another expressed human phosphoglycerate kinase. Since 5-azacytidine treatment results in hypomethylation of DNA, DNA methylation may be a mechanism of human X chromosome inactivation.     

猪COPG2和MEST克隆、印记状况和组织表达分析

【目的】为了在猪中找到更多新的候选印记基因并分析它们在哺乳动物之间的保守性,以期为猪分子遗传育种提供基础分子生物学信息和分子标记。【方法】以长白与荣昌猪杂交F1 代一月龄个体为研究对象,通过比较生物学的方法,克隆COPG2和MEST全长cDNA,并分析基因的序列特点,然后利用 IMpRH(法国农业科学院的辐射杂种克隆板)分析COPG2和MEST在猪染色体上定位信息。通过RT-PCR产物直接测序方法分析了这些基因在一月龄F1代个体11个不同组织(心、胃、肌肉、肾脏、肺、肝、小肠、膀胱、舌头、脾和脂肪)的印记状况,并进一步利用Real-time PCR方法,分析其在一月龄F1代个体11个不同组织的表达情况。【结果】克隆得到2 817 bp COPG2和2 219 bp MEST序列。其中COPG2 包括2 616 bp 完整CDS(coding sequenc,编码序列)区域,分析表明其编码含871个氨基酸的蛋白质,MEST包括981 bp完整CDS区域,编码326个氨基酸的蛋白质。IMpRH分析结果表明,猪COPG2和MEST都位于猪18号染色体上并且与标记CL365941紧密连锁,LOD值分别为14.32和8.5。印记分析表明COPG2在11个组织中呈双等位表达,MEST在心脏、胃、肌肉、肾、肺、膀胱、舌头和脂肪中表达父方等位基因,但在肝脏、小肠和脾脏中呈双等位表达。荧光定量结果显示COPG2 和MEST总的表达量在一月龄个体各组织间存在着显著性差异(P＜0.01),其中COPG2和MEST在肾脏中表达量均高于其它各组织(P＜0.01)。【结论】在哺乳动物之间,COPG2序列较为保守但是印记状况却缺乏保守性;MEST序列和印记状况均较为保守,但MEST在猪中印记状况具有组织特异性。此外,染色体定位信息和印记状况证实了在猪的18号染色体上有由COPG2和MEST构成的新的印记域。