Cloning and characterization of porcine insulin gene

The complete porcine preproinsulin cDNA and 1022 bp of its 5'-flanking region have been cloned by PCR-based technology and characterized. The porcine insulin gene has the same structure of three exons and two introns as that found in all insulin genes sequenced to date. Northern blot analysis of isolated adult porcine islets demonstrated an increase in steady-state insulin mRNA levels in response to high concentrations of glucose. Highly conserved cis-acting elements were found in the 5'-flanking region of the porcine insulin gene including multiple E and A elements as well as a cAMP responsive element (CRE). Tissue-specific activity of the proximal promoter was confirmed by transient transfection of the promoter/reporter gene constructs. This information now makes it possible for regulation and expression of the porcine insulin gene to be analyzed.     

The rat peptidylarginine deiminase-encoding gene: structural analysis and the 5'-flanking sequence.

Genomic clones of the rat peptidylarginine deiminase (PAD)-encoding gene (PAD) were isolated, and the gene organization was analyzed by restriction mapping and nucleotide sequencing. The PAD spans more than 50 kb and contains 16 exons and 15 introns. The lengths of the introns from 0.5 kb to more than 16.5 kb. A 1.7-kb sequence in the 5'-flanking region was determined. S1 nuclease mapping revealed two putative cap sites 79 and 81 bp upstream from the N-terminal ATG codon of PAD, which had been determined by amino acid sequence analysis. This ATG was confirmed to be the translation start site, since no other ATG codon was found in the open reading frame downstream from the cap sites. The 5'-flanking sequence contains four potential SP1-binding sites, a putative Pit-1/GHF-1-binding site, four short sequences either identical or homologous to the sequences in the promoter regions of rat or human growth hormone encoding genes, as well as a sequence similar to an estrogen-responsive element. However, neither a typical TATAA box, nor CCAAT box is present. These results provide important clues for elucidating the mechanism of female-specific and/or sex cycle-dependent gene expression.     

中国荷斯坦牛POU1F1基因与PRL基因的多态性及其聚合效应对产奶性状的影响

文章采用DNA测序、PCR-RFLP和CRS-PCR技术对979头中国荷斯坦牛POU1F1基因与PRL基因进行研究,发现了3个新SNPs,分别是POU1F1基因第二外显子G1178C、PRL基因5侧翼区A906G和A1134G。采用SAS统计软件GLM程序,利用最小二乘法拟合线性模型,分析基因多态性与产奶性状的关系。结果表明:POU1F1基因1178位点GC基因型在产奶量、乳蛋白量、乳脂量方面均为优良基因型。PRL基因5侧翼区906位点AG基因型在产奶量方面为优良基因型,1134位点不同基因型产奶性状差异不显著。对PRL基因5侧翼区的906位点和POU1F1基因的1178位点进行基因互作分析,结果在乳脂率、乳蛋白率、产奶量、乳蛋白量和乳脂量方面各基因型组合之间均未观察到显著差异,说明基因聚合效应并不是单基因效应的简单相加,基因聚合效应在分子育种中具有更重要的意义。     

Human plasminogen activator inhibitor-1 gene. Promoter and structural gene nucleotide sequences

We have determined the nucleotide sequence of the human plasminogen activator inhibitor-1 (PAI-1) gene and significant stretches of DNA which extend into its 5'-and 3'-flanking DNA regions; a total sequence of 15,867 base pairs (bp) is presented. The sequenced 5'-flanking DNA (1,520 bp) contains the essential eukaryotic cis-type proximal regulatory elements CCAAT and TATAA; the more distal 5'-flanking DNA region, as well as some introns, contain sequence elements which share identities with known eukaryotic enhancer elements. A major finding is the identification of a large region of shared nucleotides (comprising of about 520 bp) between the 5'-flanking DNAs of PAI-1 and tissue-type plasminogen activator genes. The length of the PAI-1 5'-untranslated region was found to be 145 bp as determined by nuclease analysis. The remaining PAI-1 structural gene consists of amino acid coding regions (containing a total of 1,206 bp, coding for the 23 amino acids of the signal peptide and 379 amino acids of the mature PAI-1 protein), 8 intron regions (a total of 8,978 bp), and a long 3'-untranslated region of about 1,800 bp which contains several polyadenylation sites. Two types of repetitive DNA elements are located within the PAI-1 structural gene and flanking DNAs: we have found 12 Alu elements and 5 repeats of a long poly (Pur) element. These Alu-Pur elements may represent a subset of the more abundant Alu family of repetitive sequence elements.     

Molecular cloning and nucleotide sequence of a gene encoding a cotton palmitoyl-acyl carrier protein thioesterase.

A cotton genomic clone containing a 17.4-kb DNA segment was found to encompass a palmitoyl-acyl carrier protein (ACP) thioesterase (Fat B1) gene. The gene spans 3.6 kb with six exons and five introns, and is apparently the first plant FatB acyl-ACP thioesterase gene to be completely sequenced. The six exons are identical in nucleotide sequence to the open reading frame of the corresponding cDNA, and would encode a preprotein of 413 amino acids. The preprotein can clearly be identified as a FatB acyl-ACP thioesterase from its similarity to the deduced amino acid sequences of other FatB thioesterase preproteins. A 5'-flanking region of 914 bp was sequenced, with the potential TATA basal promoter 324 bp upstream from the ATG initiation codon. The 5'-flanking sequence also has a putative CAAT box and two presumptive basic region helixloop-helix (bHLH) elements with the consensus motif CANNTG (termed an E box), implicated as being a positive regulatory element in seed-specific gene expression.     

苹果一个锌指蛋白基因的cDNA克隆及其表达特性分析(英文)

A cDNA library was created from stem apex tissue from Jonathan apples (Malus domestica Borkh.), harvested in June to August, during which the plant transitions from vegetative growth to reproductive growth. From this library, we isolated an expressed sequence tag (EST) sequence containing a zinc finger motif, using this sequence, a 779 bp cDNA fragment was obtained by using 5‘ RACE, and a final full-length cDNA encoding an apple zinc finger protein (named MdZF1; GenBank accession number AB116545) was obtained by further PCR. This zinc finger motif of MdZF1 has high homology with INOETERMINATE1 (ID1) gene from maize which seemed to be involved in the transition to flowering. Northern blot and RT-PCR analyses showed that the MdZF1 expressed in the root, stem, leaves, shoot apex and floral organs of the apple, with expression levels higher in root, stem, leaves and floral shoot apex than that in floral organs (sepals, petals, stamens and pistils). Genomic Southern analysis showed that there was a single copy gene in apple genome.     

苹果一个锌指蛋白基因的cDNA克隆及其表达特性分析

采集了处于营养生长向生殖行长转化期(6~8月)的红玉苹果(Malus domestica Borkh.)茎顶,构建了其cDNA文库,并从中分离得到了一个具有锌指结构的EST序列,又通过5`RACE的方法,从cDNA文库中找到了其上游779bp的cDNA片段.最后用PCR的方法获得了苹果锌指蛋白的全长cDNA,并命为MdZF1.该cDNA序列已在GenBank登录,登录号为AB116545.MdZF1的锌指结构域与玉米的开花转换基因ID1有高度同源性.通过对苹果不同组织、器官的Northem和RT-PCR分析表明MdZF1在根、茎、叶、顶芽以及花器官(萼片、花瓣、雄蕊、雌蕊)中都有表达.Southern分析表明MdZF1的基因组中是以单拷贝存在的.     

Identification of a fat cell enhancer: analysis of requirements for adipose tissue-specific gene expression.

The molecular basis for adipose-specific gene expression is not known. To approach the problem of adipocyte gene expression, we have analyzed in detail the capacity of the 5'-flanking region of the adipocyte P2 (aP2) gene to direct cell-type specific gene expression. Although the proximal promoter containing AP-1 and C/EBP binding sites is capable of directing differentiation-dependent gene expression in cultured adipocytes, these constructs are essentially inactive in the tissues of transgenic mice. We found that -5.4 kb of the 5'-flanking region were required to direct heterologous gene (chloramphenicol acetyl transferase; CAT) expression to the adipose tissue of transgenic mice. By deletion analysis, we identified a 520 bp enhancer at -5.4 kb of the aP2 gene. We show that this enhancer can direct high levels of gene expression specifically to the adipose tissue of transgenic mice. This enhancer also functions in a differentiation-dependent manner in cultured adipocytes and cannot be transactivated in preadipocytes by C/EBP. Molecular analysis indicates that several cis- and trans- acting acting elements, though not C/EBP, contribute to the specificity and potency of this enhancer.     

巴西橡胶树(Hevea brasiliensis)微管相关蛋白全长cDNA克隆及表达分析

从巴西橡胶树Hevea brasiliensis差减cDNA文库中分离到微管相关蛋白（Microtubule-associated protein,MAPs）基因片段,根据该基因片段序列信息,设计特异引物,采用cDNA末端快速扩增技术RACE（Rapid Amplification ofcDNA Ends）进行差异片段的5＇和3＇端的扩增,获得了长度为788bp的全长cDNA,该基因在GenBank中的登录号为AY461412.序列分析表明该基因包含完整的开放阅读框,编码144个氨基酸,与微管相关蛋白基因家族具有很高的同源性,推测该基因是微管相关蛋白基因.半定量RT-PCR检测证实它在胶乳中的表达强于叶中,胁迫处理（伤害及乙烯处理）使其表达上调.