NOBOX基因的cDNA克隆及其特性分析

NOBOX(新生儿卵巢同源基因)是一个卵母细胞特异性表达的同源基因,在早期滤泡发生中起重要的作用。本研究结合电子克隆的方法,从猪卵母细胞中成功地克隆了NOBOX基因的全长cDNA序列(GenBank Accession No.FJ587509)。猪NOBOX基因的cDNA全长为1768 bp,包含1419 bp的开放阅读框。生物信息学分析表明NOBOX基因编码了472个氨基酸,分子量为51.08 kD,等电点为5.73。该蛋白定位于细胞核中,含有一个保守的结构域——cd00086。借助Clustalw软件,采用N-J算法构建了NOBOX蛋白的系统进化树,分析了不同物种间的进化关系。应用实时荧光定量PCR技术分析该基因在母猪不同组织、细胞及4种孤雌激活胚胎的表达模式,结果表明该基因在母猪各组织中均有不同程度的表达,其中在心脏、肾脏和卵母细胞中表达水平较高,推测其可能在心脏、肾脏和卵母细胞中发挥着重要的作用;NOBOX基因在胚胎发育阶段的表达水平高于G-V期的卵母细胞,表明在胚胎发育阶段pNOBOX的表达增强。     

Cloning and characterization of 414 polymorphic porcine microsatellites

We report the sequences, sizes, and number of alleles of 414 new porcine microsatellites that were cloned in our laboratory and 21 micro-satellites derived from GenBank DNA sequences. We also confirm the usefulness of porcine microsatellite primer pairs derived from short interdispersed elements.     

Cloning and expression of the porcine myogenin gene

Abstract

Myogenin is a member of the myoD family of myogenic regulatory factors which direct the initiation of myogenesis during skeletal muscle development. Myogenin is responsible for the differentiation of proliferating myoblasts into myotubes and the absence of this factor results in severe muscular deficiency. Using PCR primers designed from mammalian myogenin sequences, a 215 bp PCR product was amplified from porcine genomic DNA and found to be 89.7% homologous to mouse myogenin. A porcine cosmid library was screened with the myogenin PCR probe and a 6.5 kb portion of a cosmid containing the pig myogenin gene was sequenced. The sequence includes 5´ flanking region, the entire protein coding region composed of three exons and two introns and 3´ flanking region. The protein coding region was highly conserved (≥92.5%) with mouse and human myogenin coding regions. Transfection of pig myogenin into C3H10T1/2 cells, a multipotential fibroblast cell line, resulted in 28% myogenic conversion assayed by cell fusion to form multinucleated cells and synthesis of sarcomeric myosin. These results indicate that the cloned myogenin gene was capable of initiating myogenesis in a fibroblast cell line.     

Cloning and characterization of highly polymorphic porcine microsatellites.

A size-fractionated (200-400 bp) porcine genomic library was screened with the dinucleotide motifs (TG)n and (TC)n. The number of TG- and TC-positive clones was 83 and four, respectively, implying that the former motif is more frequent in the porcine genome, as previously reported in other species. Twenty-six TG-clones were sequenced, and the number of repeats varied between 16 and 42 with different compositions of the repetitive sequences; 17 clones had a perfect stretch of TG-repeats, four had imperfect stretches, and five had a compound structure with TG-repeats followed by TC-repeats. Primers for DNA amplification using the polymerase chain reaction (PCR) were synthesized for six loci. Ten unrelated individuals (two wild boars and eight domestic pigs of the Swedish Yorkshire breed) were screened for microsatellite polymorphism. All six microsatellite loci were polymorphic with two to seven alleles and observed heterozygosities in the range of 0.42-0.84; the inheritance of the observed polymorphism was confirmed by family studies. The characteristics of microsatellites make them highly suitable as genetic markers, and these microsatellites were isolated as a part of a pig gene mapping project.     

人端粒酶RNA基因的克隆与鉴定

以人血基因组ＤＮＡ为模板,合成两段２０个寡聚核苷酸为引物,经过ＰＣＲ扩增,得到４８０ｂｐ的片段,克隆到ｐＭＤ１８－Ｔ载体中,经电泳、酶切、ＰＣＲ鉴定后测定序列。序列分析表明氙克隆的人端粒酶ＲＮＡ（ｈｕｍａｎ ｔｅｌｏｍｅａｓｅ ＲＮＡ,ｈＴＲ）基因含有４８０ｂｐ,包括约４５０ｂｐ的编码模板区主序列和约３０ｂｐ的上游调控区序列,其中模板区的１１个核苷酸（５’－ＣＵＡＡＣＣＣＵＡＡＣ－３’）合成端粒亚     

Cloning, comparative characterization of porcine SCAP gene, and identification of its two splice variants

Sterol responsive element binding protein (SREBP) cleavage-activating protein (SCAP) is the key regulator of activation of SREBPs, which stimulate most enzymes in cholesterol and lipid synthesis. In order to investigate the molecular basis of lipid metabolism in the pig, a unique model for fat deposition, we isolated and characterized the porcine SCAP. The 4,096-bp full-length porcine SCAP cDNA contains an open reading frame of 3,840 bp. The predicted SCAP protein consists of 1,280 amino acids of 55–92% identity with its vertebrate counterparts. The porcine SCAP gene consists of at least 19 exons and 18 introns, which span over 13 kb of the genome. The porcine SCAP gene was mapped to chromosome 13q21–22 using a porcine-rodent somatic cell hybrid panel. Comparison of SCAP genomic structures from various species revealed intron losses in porcine, Tetraodon and fugu SCAP, and intron gains in cow and chicken SCAP. Moreover, we isolated two novel splicing SCAP variants with 193-bp (variant 2) in-frame deletion from testis and a variant with 291-bp (variant 3) in-frame deletion from liver and muscle, which may affect the function of the porcine SCAP. In conclusion, the intron gains and losses appear to have contributed to the shape of the modern SCAP family. The splice variants detected, first to be reported in any species, may be involved in the particulars of the fat metabolism in the pig. Our data lay foundation for further study of SCAP function in this species.     

人RHD基因的克隆和鉴定

目的克隆人RHD基因,并对其进行鉴定。方法以RhD阳性志愿者骨髓为材料,用TRIzol试剂提取总RNA;设计、合成人RHD基因扩增引物,RT-PCR方法扩增RHD基因片段;T／A克隆后将其亚克隆人pET28a（＋）载体中,经酶切、PCR和测序对重组质粒进行鉴定。结果骨髓总RNA被成功提取;RT-PCR成功扩增出RHD基因片段,其大小与预期约509bp基本一致;T／A克隆后再将其亚克隆,通过酶切和PCR证明RHD基因成功亚克隆入pET28a（＋）载体中;基因测序结果比对显示,与已公布的RHD基因（GenBank登录号为NM016124）序列基本一致,同源性为98％。结论成功克隆了RHD基因,这将为进一步研究奠定基础。     

Cloning and characterization of the human beta-glucuronidase gene

We have isolated a cosmid clone that contains GUSB, the human gene encoding beta-glucuronidase. The 21-kb gene contains 12 exons ranging from 85 to 376 bp in length. Exon 6 corresponds to the 153-bp deletion in the shorter of two types of cDNAs reported earlier, supporting the hypothesis that this cDNA arose by alternate splicing leading to exon skipping. The insert contains 4.2 kb of sequence upstream from the first exon and 6 kb 3' of the last exon. The clone expresses human beta-glucuronidase in stably transformed rat XCtk- cells. Comparison of the human gene organization with that recently reported for the murine beta-glucuronidase gene revealed that the intron/exon boundaries are identical. In the splice junctions, the most highly conserved regions are those identified as consensus sequences, and these are at least as highly conserved as bases encoding the translated portion of the gene.     

Cloning and characterization of the carp prolactin gene

A carp genomic DNA clone containing the carp prolactin (Prl) gene was isolated with carp Prl cDNA as a probe. The organization of the carp Prl gene was determined by restriction nuclease mapping and nucleotide sequencing. The Prl gene comprises approx. 2.8 kilobasepairs (kb) of DNA including the 5'-flanking region, five exons, four introns and the 3'-flanking region. Analysis of the 5'-flanking region reveals (1) the sequence TATATAAT at positions -38 to -31 upstream from the cap site which was found to be a guanine residue, and (2) the palindrome, CTCATTGCATATACAAATGAG at positions -79 to -59. The carp Prl gene matches with the reported cDNA except for one difference in coding region and five in the 3'-flanking region, while the encoded amino acid sequences are identical. The arrangement of exons and introns is very similar to that seen in carp GH as well as mammalian Prl, which, however, have much longer introns.     

水牛SRY基因的克隆及序列分析

根据荷斯坦牛SRY基因设计一对引物,采用聚合酶链式反应（PCR）技术,以中国沼泽型水牛（Swamp Buffalo）基因组DNA为模板,扩增得到SRY（Sex Deterimation region of Y chromosome）全序列约2005bp,其中1-504bp为5’启动子区,1196-2005bp为3’侧翼序列,在505-1195bp为SRY的外显子,编码229个氨基酸。在SRY HMG box区域设计探针,用地高辛标记后分别与雄性、雌性水牛基因组DNA进行Southern 杂交,结果显示该段序列只在雄性DNA样本中有杂交信号,证明SRY基因为雄性特异。BLAST比对结果显示与牛属动物SRY基因的同源性为96％,其中SRY基因HMG box区域同源性高达99%,说明SRY基因具有高度的进化保守性     

Cloning and characterization of microRNAs from porcine skeletal muscle and adipose tissue

MicroRNAs (miRNAs) are an abundant class of small regulatory RNAs that regulate the stability and translation of cognate mRNAs. Although an increasing number of porcine miRNAs has recently been identified, the full repertoire of miRNAs in pig remains to be elucidated. To identify porcine miRNAs potentially involved in myogenesis and adipogenesis, we constructed small RNA cDNA libraries from skeletal muscle and adipose tissue and identified 89 distinct miRNAs that are conserved in pig, of which 15 were new. Expression analysis of all newly identified and selected known porcine miRNAs revealed that some miRNAs were enriched in a tissue-specific manner, whereas others were expressed ubiquitously in the porcine tissues examined. Our results expand the number of known porcine miRNAs and provide useful information for further investigating the biological functions of miRNAs associated with growth and development of skeletal muscle or adipose tissue in pig.     

猪PILRA基因克隆及变异剪接体鉴定

Ⅱ型成对免疫球蛋白样受体(Paired immunoglobulin-like type 2 receptors, PILRs)是免疫球蛋白超家族成员之一,包括α和β两个亚型。PILRα在机体抵抗病原体入侵的免疫反应中发挥着重要作用,但目前尚未见关于猪PILRα的报道。为了分析其在猪抗病育种中的作用,本文克隆了猪PILRα编码基因(PILRA)并鉴定变异剪接体,利用实时荧光定量PCR方法构建其组织表达谱和诱导表达谱。结果表明,成功克隆了猪PILRA基因的3个变异剪接体V1~V3(GenBank登录号：KJ143679~81),预测的蛋白质多肽链分别长271 aa、254 aa和283 aa,且都具有免疫球蛋白结构域。各变异剪接体均在脾脏中表达量最高,肝脏、肺脏次之,在心脏、肾脏、胃、肌肉、淋巴、大肠、小肠和膀胱等组织中表达量较低或检测不到。Poly(I:C)能显著诱导变异剪接体V1的表达,但几乎不影响V2、V3的表达。研究结果为进一步揭示PILRA在猪抗病育种中的作用提供了基础。     

Isolation and characterization of the porcine growth hormone gene

A cosmid clone containing the entire porcine growth hormone (PGH) gene has been isolated using a full-length PGH cDNA as the hybridization probe. The gene within the cosmid was subcloned into plasmids and completely sequenced. The coding, promoter, and both 5'- and 3'-noncoding sequences of the PGH gene were found to be highly conserved when compared to the previously sequenced genes coding for rat, human and bovine growth hormones, and also to the human placental lactogen gene. The high degree of conservation between the 5'- and 3'-noncoding regions of the genes from these different species indicates that growth hormone genes may be evolving by some unusual mechanism. The PGH gene was found to contain the unusual variant GC donor splice site.     

Cloning,characterization, and embryonic expression analysis of the Drosophila melanogaster gene encoding insulin/relaxin-like peptide

Insulin is one of the key peptide hormones that regulates growth and metabolism in vertebrates. Evolutionary conservation of many elements of the insulin/IGF signaling network makes it possible to study the basic genetic function of this pathway in lower metazoan models such as Drosophila. Here we report the cloning and characterization of the gene for Drosophila insulin/relaxin-like peptide (DIRLP). The predicted protein structure of DIRLP greatly resembles typical insulin structure and contains features that differentiate it from the Drosophila juvenile hormone, another member of the insulin family. The Dirlp gene is represented as a single copy in the Drosophila melanogaster genome (compared to multiple copies for Drosophila juvenile hormone) and shows evolutionary conservation of genetic structure. The gene was mapped to the Drosophila chromosome 3, region 67D2. In situ hybridization of whole-mount Drosophila embryos with Dirlp antisense RNA probe reveals early embryonic mesodermal/ventral furrow expression pattern, consistent with earlier observation of the insulin protein immunoreactivity in Drosophila embryos. The in situ hybridization pattern was found to be identical to that obtained during immunohistochemistry analysis of the Drosophila embryos using various insulin monoclonal and polyclonal antibodies that do not recognize Drosophila juvenile hormone, supporting the idea that Dirlp is a possible Drosophila insulin ortholog. Identification of the gene for DIRLP provides a new approach for study of the regulatory pathway of the insulin family of peptides.     

Cloning and characterization of the gene for phosphatidylcholine synthase

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesized by either of two pathways, the CDP-choline pathway or the methylation pathway. In prokaryotes only the methylation pathway was thought to occur. Recently, however, we could demonstrate (de Rudder, K. E. E., Sohlenkamp, C., and Geiger, O. (1999) J. Biol. Chem. 274, 20011-20016) that a second pathway for phosphatidylcholine biosynthesis exists in Sinorhizobium (Rhizobium) meliloti involving a novel enzymatic activity, phosphatidylcholine synthase, that condenses choline and CDP-diacylglyceride in one step to form PC and CMP. Using a colony autoradiography method we have isolated mutants of S. meliloti deficient in phosphatidylcholine synthase and which are no longer able to incorporate radiolabeled choline into PC. Complementation of such mutants with a sinorhizobial cosmid gene bank, subcloning of the complementing fragment, and sequencing of the subclone led to the identification of a gene coding for a presumptive CDP-alcohol phosphatidyltransferase. Amplification of this gene and its expression in Escherichia coli demonstrates that it codes for phosphatidylcholine synthase. Genomes of some pathogens (Pseudomonas aeruginosa and Borrelia burgdorferi) contain genes similar to the sinorhizobial gene (pcs) for phosphatidylcholine synthase. Although pcs-deficient S. meliloti knock-out mutants show wild type-like growth and lipid composition, they are unable to perform rapid PC biosynthesis that normally is achieved via the phosphatidylcholine synthase pathway in S. meliloti wild type.     

Cloning and characterization of dehydrin gene from Ammopiptanthus mongolicus

Based on the sequence of an expressed sequence tag, the full-length cDNA of 1,008 nucleotides was cloned from Ammopiptanthus mongolicus by rapid amplification of cDNA ends. It was designated as AmDHN, encoding a protein of 183 amino acids. The calculated molecular weight of the AmDHN protein is 18.4 k Da, and theoretical isoelectric point is 5.78. The AmDHN localized in nucleus. Under normal growth conditions, differential expression of AmDHN exhibited that the expression was the highest in seeds and the lowest in flowers. AmDHN could be induced by NaCl, PEG6000, ABA and drought treatments. Salt and drought resistances of transgenic plants with overexpression of AmDHN are improved. Taken together, these results demonstrated that AmDHN could regulate the expression of abiotic-responsive genes and plays important roles in modulating the tolerance of plants to abiotic stresses.     

Cloning and functional characterization of the HRASLS2 gene

The HRAS-like suppressor 2 (HRASLS2) gene belongs to the H-REV107 gene family involved in the regulation of cell growth and differentiation. HRASLS2 is expressed at high levels in normal tissues of the small intestine, kidney, and trachea. We cloned HRASLS2 cDNA from human SW480 colon cancer cells. Most wild-type, and some N- and C-terminal truncated HRASLS2 (HRASLS2DeltaNDeltaC) were expressed as a granular pattern located at perinuclear region in HtTA cervical cancer cells, while truncation at the C-terminus only (HRASLS2DeltaC) resulted in a diffuse pattern. Wild-type HRASLS2 significantly suppressed colony formation of HeLa and HCT116 cells. HRASLS2DeltaNDeltaC significantly inhibited colony formation of HCT116 cells, but HRASLS2DeltaC did not affect cell growth. HRASLS2 suppressed the RAS-GTP levels and total RAS protein by 44% and 25%, respectively in HtTA cells; however, the suppression was not observed in truncated HRASLS2 variants. In conclusion, the HRASLS2 protein suppressed growth and RAS activities of cancer cells, and the C-terminal hydrophobic domain appeared to be indispensable for both activities.