Cloning of rat TARC cDNA and analysis of tissue-specific mRNA expression

Thymus-and activation-regulated chemokine (TARC) is one that selectively controls the migration of type 2-helper T lymphocytes into inflammatory lesions. TARC is a CC chemokine and plays an essential role in recruiting CC chemokine receptor 4-positive Th2 cells to allergic lesions. We cloned TARC cDNA from rat thymus using RT-PCR. The rat TARC clone contained a full-length open reading frame encoding 93 amino acids that showed 83 and 66% homology with mouse and human homologs, respectively. The expression of TARC mRNA was mainly in the lymphoid organs, for example, the thymus, spleen, and lymph node. The recombinant TARC was expressed in Escherichia coli and purified in an active form. In addition, the purified rat TARC with S-tagged specifically binds to human CCR4 in CD4/CCR4-transfected HOS cells by cell-binding assay using flow cytometry. The TARC cDNA clones obtained in this study will be valuable for future studies on allergic diseases in rats.     

Cloning of rat TARC cDNA and analysis of tissue-specific mRNA expression

Thymus and activation-regulated chemokine (TARC) is one that selectively controls the migration of type 2-helper T lymphocytes into inflammatory lesions. TARC is a CC chemokine, and plays an essential role in recruiting CC chemokine receptor 4-positive Th2 cells to allergic lesions. We cloned TARC cDNA from rat thymus using RT-PCR. The rat TARC clone contained a full-length open reading frame encoding 93 amino acids that showed 83% and 66% homology with mouse and human homologs, respectively. The expression of TARC mRNA was mainly in the lymphoid organs, for example, the thymus, spleen, and lymph node. The recombinant TARC was expressed in Escherichia coli and purified in an active form. In addition, the purified rat TARC with S-tagged specifically binds to human CCR4 in CD4.CCR4-transfected HOS cells by Cell-binding assay using flow-cytometry. The TARC cDNA clones obtained in this study will be valuable for future studies on allergic diseases in rats.     

cDNA cloning and analysis of tissue-specific expression of mouse peroxisomal straight-chain acyl-CoA oxidase.

Straight-chain acyl-CoA oxidase is the first and rate limiting enzyme in the peroxisomal beta-oxidation pathway catalysing the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs, thereby producing H2O2. To study peroxisomal beta-oxidation we cloned and characterized the cDNA of mouse peroxisomal acyl-CoA oxidase. It consists of 3778 bp, including a 1983-bp ORF encoding a polypeptide of 661 amino-acid residues. Like the rat and human homologue the C-terminus contains an SKL motif, an import signal present in several peroxisomal matrix proteins. Sequence analysis revealed high amino-acid homology with rat (96%) and human (87%) acyl-CoA oxidase in addition to minor homology ( approximately 40%) with other related proteins, such as rabbit trihydroxy-cholestanoyl-CoA oxidase, human branched chain acyl-CoA oxidase and rat trihydroxycoprostanoyl-CoA oxidase. Acyl-CoA oxidase mRNA and protein expression were most abundant in liver followed by kidney, brain and adipose tissue. During mouse brain development acyl-CoA oxidase mRNA expression was highest during the suckling period indicating that peroxisomal beta-oxidation is most critical during this developmental stage. Comparing tissue mRNA levels of peroxisome proliferator-activated receptor alpha and acyl-CoA oxidase, we noticed a constant relationship in all tissues investigated, except heart and adipose tissue in which much more, and respectively, much less, peroxisome proliferator-activated receptor alpha mRNA in proportion to acyl-CoA oxidase mRNA was found. Our data show that acyl-CoA oxidase is an evolutionary highly conserved enzyme with a distinct pattern of expression and indicate an important role in lipid metabolism.     

黄河裸裂尻鱼肌肉生长抑制素基因克隆及表达分析

肌肉生长抑制素(myostatin,MSTN)属于转化生长因子β(TGF-β)超家族中的一个成员,是骨骼肌生长发育的负调控因子。该文以黄河裸裂尻鱼肌肉总RNA为模板,采用RT-PCT、5’-RACE和3’-RACE法获得MSTN基因全长cDNA序列为2180bp,包含长为1128bp的开放阅读框,编码375个氨基酸。以肌肉总DNA为模板,通过PCR法进一步获得了MSTN基因的2个内含子序列,分析表明,黄河裸裂尻鱼MSTN基因与其他脊椎动物具有相似的基因结构(包括3个外显子和2个内含子)。黄河裸裂尻鱼MSTN具有脊椎动物MSTN的共同序列特征,含有1个蛋白酶水解位点RXXR和8个位于TGF-β功能区域保守的半胱氨酸残基。氨基酸序列同源性分析表明,黄河裸裂尻鱼MSTN序列与其他鲤科鱼类MSTN具有较高的同源性;而与哺乳动物和禽类的MSTN同源性较低。系统发育分析表明,黄河裸裂尻鱼MSTN与其他鲤科鱼类聚于同一进化支。RT-PCR分析表明,该基因在黄河裸裂尻鱼9个被检组织中均有表达,但在心、肾、肠、精巢中表达量较高。Real-TimePCR分析显示,MSTN基因在胚胎中的相对表达量,随胚胎发育阶段的不同而有所差异,暗示MSTN的功能可能并不局限在对肌肉生长发育的负调控作用,可能还有其他功能。     

Molecular cloning and expression of rat betacellulin cDNA

The cDNA encoding an entire open reading frame of rat betacellulin has been cloned from rat kidney. Expression of this cDNA in COS7 cells showed a significant amount of mitogenic activity in the culture media. Western blotting of the cell lysates suggested that the membrane-anchored precursor was cleaved to release its ectodomain very efficiently.     

cDNA cloning and gene expression analysis of human myo-inositol 1-phosphate synthase

myo-Inositol 1-phosphate synthase (EC 5.5.1.4) (IPS) is a key enzyme in myo-inositol biosynthesis pathway. This study describes the molecular cloning of the full length human myo-inositol 1-phosphate synthase (hIPS) cDNA, tissue distribution of its mRNA and characterizes its gene expression in cultured HepG2 cells. Human testis, ovary, heart, placenta, and pancreas express relatively high level of hIPS mRNA, while blood leukocyte, thymus, skeletal muscle, and colon express low or marginal amount of the mRNA. In the presence of glucose, hIPS mRNA level increases 2- to 4-fold in HepG2 cells. hIPS mRNA is also up-regulated 2- to 3-fold by 2.5 microM lovastain. This up-regulation is prevented by mevalonic acid, farnesol, and geranylgeraniol, suggesting a G-protein mediated signal transduction mechanism in the regulation of hIPS gene expression. hIPS mRNA expression is 50% suppressed by 10mM lithium ion in these cells. Neither 5mM myo-inositol nor the three hormones: estrogen, thyroid hormone, and insulin altered hIPS mRNA expression in these cells.     

Effects of urocortin II on neonatal rat cardiac myocytes and non-myocytes

Urocortin (Ucn) II and III, homologous peptides of Ucn that are specific ligands for corticotropin-releasing hormone (CRH) type 2 receptor (CRH-R2), have recently been identified. The present study was designed to elucidate the effects of Ucn II, which is predominantly expressed in rodent heart, on neonatal rat cardiac myocytes (MCs) and cardiac non-myocytes (NMCs). Ucn II increased the incorporation of [³H]-leucine into MCs, as well as the accumulation of cAMP and the secretion of atrial natriuretic peptide. However, no significant changes were demonstrated in NMCs or an MC/NMC co-culture system. The effects of Ucn II were attenuated by astressin2-B, a specific antagonist of CRH-R2, and/or H89, an inhibitor of protein kinase A (PKA). These results indicate that Ucn II may be another endogenous cardiovascular substance that acts via CRH-R2 and the cAMP-dependent PKA pathway.     

猪Opn4基因cDNA克隆与表达分析

为揭示猪Opn4基因的结构并研究其在猪视网膜中昼夜表达差异与生长各阶段表达规律, 文章克隆了猪Opn4基因全长CDS区序列, 定量检测了Opn4基因在猪视网膜中昼夜的表达量以及其在不同生长阶段的表达量。结果表明, Opn4基因编码区序列为1 437 bp, 编码478个氨基酸, 分子式为C2398H3705N623O651S23; Opn4基因在白昼的表达量极显著高于夜晚(P<0.01); Opn4基因在出生前34 d胎儿视网膜中表达量最低(P<0.01); 1日龄时表达量最高(P<0.01); 1月龄、4月龄、84月龄猪视网膜中Opn4表达量差异不显著(P>0.05)。以上结果提示, Opn4基因可参与调控猪的生物节律, 并且Opn4主要参与出生后猪对外界光照的应答。     

苜蓿夜蛾保幼激素酯酶cDNA的克隆、序列分析及表达研究

【目的】研究苜蓿夜蛾Heliothis viriplaca保幼激素酯酶(Juvenile hormone esterase,JHE)的功能和作用机理。【方法】本试验提取苜蓿夜蛾的总RNA,并利用RT-PCR和RACE技术,扩增得到苜蓿夜蛾保幼激素酯酶基因的全长c DNA序列,命名为Hvjhe(Gen Bank登录号:JQ901384)。【结果】该基因含有3 106 bp,包括一个1 746 bp的开放阅读框,编码一个含581个氨基酸的多肽,分子量约为63.9 ku,多肽的等电点为5.11,且氨基酸序列具含有5个保幼激素酯酶特有的氨基酸保守模块。推导的氨基酸序列与烟芽夜蛾Heliothis virescens和棉铃虫Helicoverpa armigera中获得的保幼激素酯酶相似性较高,分别达到83%和82%。荧光定量PCR结果表明,该基因在苜蓿夜蛾不同发育时期和不同组织中都有m RNA水平的特异性表达,且在预蛹期表达量相对较高,取食期、蛹期期和成虫期表达量相对较低;在中肠和脂肪体内的表达量相对于其它组织较高。该基因在大肠杆菌Escherich coli表达系统中进行了诱导表达,经SDS-PAGE和Western blot检测结果表明,表达出与预测的蛋白分子量相符的融合蛋白。【结论】研究结果表明本试验获得了苜蓿夜蛾中一个新的保幼激素酯酶基因的c DNA序列。     

甘蓝夜蛾两种不同类型几丁质脱乙酰基酶基因的克隆与组织特异性表达

几丁质脱乙酰基酶(chitin deacetylase,CDA)是昆虫几丁质降解酶中的一种酶,可以将几丁质转化为壳聚糖,在昆虫几丁质代谢中具有重要作用.本研究以甘蓝夜蛾Mamestra brassicae5龄幼虫虫体为材料提取总RNA,利用RT-PCR和RACE技术,分别扩增得到甘蓝夜蛾的2类不同几丁质脱乙酰基酶基因的...     

重组腺病毒介导hMYH cDNA在原代培养大鼠心肌细胞中的表达及活性测定

构建携带hMYHcDNA的重组腺病毒表达载体,制备重组腺病毒,并在原代培养的大鼠心肌细胞中得到了表达,表达的hMYH蛋白具有结合并切割A∶8-OXO-G碱基错配的糖苷酶活性,对A∶G和A∶C错配具有较弱的结合活性和切割活性。该研究为进一步进行hMYH的基因治疗奠定了基础。     

cDNA cloning, gene organization and expression analysis of human peptidylarginine deiminase type VI

Peptidylarginine deiminase (PAD) catalyzes the post-translational modification of protein through the conversion of arginine to citrulline in the presence of calcium ions. Human, similar to rodents, has four isoforms of PAD (type I, II, III and IV/V), each of which is distinct in substrate specificity and tissue specific expression. In our large-scale sequencing project, we identified a new human PAD cDNA from a human fetal brain cDNA library. The putative protein encoded by this cDNA is designated hPADVI. Expression analysis of hPADVI showed that it is mainly expressed in adult human ovary and peripheral blood leukocytes. We conclude that hPADVI may be orthologous to mouse ePAD, basing on sequence comparison, chromosome localization and exon-intron structure analysis. PAD-mediated deimination of epithelial cell keratin resulting in cytoskeletal remodeling suggests a possible role for hPADVI in cytoskeletal reorganization in the egg and in early embryo development. This study describes a new important member of the human PAD family.     

Molecular cloning and expression of rat interleukin-1 alpha cDNA

A cDNA sequence coding for rat interleukin-1 alpha (IL-1 alpha) has been isolated from a cDNA library that was prepared with mRNA derived from LPS-stimulated rat peritoneal macrophages by using human IL-1 alpha cDNA as a probe. The rat cDNA encodes a 270 amino acid residue protein which is homologous (65%) to human IL-1 alpha. The rat cDNA sequence under SV40 early promoter directed the synthesis of biologically active IL-1 in monkey COS-1 cells. Rat IL-1 alpha mRNA is not expressed in spleen, lung, liver or brain, and is also not expressed in these organs of LPS-treated rat except spleen. This suggests that IL-1 alpha is not produced constitutively in various tissues and LPS is not sufficient to induce IL-1 alpha in most tissues. Our data indicate that the IL-1 activities which have been reported to be produced in the brain are not of alpha type. We have constructed a plasmid expressing the carboxy terminal 156 amino acids in Escherichia coli. Recombinant rat IL-1 alpha produced in COS cells or E. coli has cytotoxic activity against the human melanoma cell line A375S1 (GIF activity), which has been reported to be sensitive to human IL-1 alpha and IL-1 beta. This suggests that GIF activity is common to IL-1s derived from various sources.     

cDNA cloning and gene expression of ascorbate oxidase in tobacco

A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Northern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant.     

Molecular cloning and tissue-specific expression analysis of mouse spinesin, a type II transmembrane serine protease 5

We have previously reported novel serine proteases isolated from cDNA libraries of the human and mouse central nervous system (CNS) by PCR using degenerate oligodeoxyribonucleotide primers designed on the basis of the serine protease motifs, AAHC and DSGGP. Here we report a newly isolated serine protease from the mouse CNS. This protease is homologous (77.9% identical) to human spinesin type II transmembrane serine protease 5. Mouse spinesin (m-spinesin) is also composed of (from the N-terminus) a short cytoplasmic domain, a transmembrane domain, a stem region containing a scavenger-receptor-like domain, and a serine protease domain, as is h-spinesin. We also isolated type 1, type 2, and type 3 variant cDNAs of m-spinesin. Full-length spinesin (type 4) and type 3 contain all the domains, whereas type 1 and type 2 variants lack the cytoplasmic, transmembrane, and scavenger-receptor-like domains. Subcellular localization of the variant forms was analyzed using enhanced green fluorescent protein (EGFP) fusion proteins. EGFP-type 4 fusion protein was predominantly localized to the ER, Golgi apparatus, and plasma membrane, whereas EGFP-type 1 was localized to the cytoplasm, reflecting differential classification of m-spinesin variants into transmembrane and cytoplasmic types. We analyzed the distribution of m-spinesin variants in mouse tissues, using RT-PCR with variant-specific primer sets. Interestingly, transmembrane-type spinesin, types 3 and 4, was specifically expressed in the spinal cord, whereas cytoplasmic type, type 1, was expressed in multiple tissues, including the cerebrum and cerebellum. Therefore, m-spinesin variants may have distinct biological functions arising from organ-specific variant expression.     

构建定向T载体用于基因克隆和表达

传统的T载体克隆方法需要烦琐的后续步骤来筛选和鉴定重组子,并且无法实现目的基因的定向克隆。为了克服这些问题,本研究在pET-23a(+)的基础上构建了定向T载体pETG,首先通过定点诱变消除pET-23a(+)上的两个BfuⅠ位点得到PET-23aM;设计一对引物在5端各引入一个BfuⅠ位点,下游引物紧邻BfuⅠ位点引入13 bp的部分LacO序列,用该引物从pHBM2002上扩增Prrn-gfp表达盒,插入PET-23aM的NdeⅠ和XhoⅠ位点,得到定向T载体pETG。PCR扩增的目的基因通过下游引物引入7 bp剩余的LacO序列,该基因片段与BfuⅠ酶切制备的定向T载体连接、转化大肠杆菌DH10β感受态细胞,通过补加了X-gal的平板筛选蓝色重组子。质粒酶切和PCR鉴定表明蓝色菌落全部为定向插入的重组子,重组效率100%,利用本方法成功地定向克隆了103个人类肝蛋白编码基因cDNA,克隆过程无需复杂的步骤筛选鉴定重组子。随机选择了其中的8个基因的克隆进行表达,结果显示8个克隆均在大肠杆菌中获得成功表达。该结果表明定向T载体构建成功,并且该载体非常适合基因的克隆和表达。     

cDNA cloning and expression of an apoptosis-related gene, humanTFAR15 gene

By means of cDNA-RDA method, some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these fragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1 218 nucleotides and encodes 212 amino acids. The putative protein product of TFAR15 is partially homologous toC. elegans protein C14A4.11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung, and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-1 cells after GM-CSF withdrawal.In vitro analysis showed that the recombinant TFAR15 protein could inhibit the natural cell death of 293 cells, an embryonic kidney cell line.