圆斑星鲽及相关种类线粒体DNA控制区结构分析

采用PCR产物直接测序法测定了圆斑星鲽(Verasper variegatus)的24个个体的线粒体控制区(Control region)核苷酸全序列, 并进行了结构分析。结果表明, 圆斑星鲽线粒体控制区核苷酸全序列具有长度多态性, 得到4种长度单元型, 主要表现为控制区中的串联重复序列的长度不同。对鲽形目鱼类如鲽科的条斑星鲽(Verasper moseri)、黄盖鲽(Limanda feruginea)、马舌鲽 (Reinhardtius hippoglossoides), 美洲拟庸鲽(Heppoglossoides platessoides )和鲆科的牙鲆(Paralichthys olivaceus)以及鳎科的欧洲鳎(Solea solea)、塞内加尔鳎(S. senegalensis)和沙鳎(S. lascari)的控制区的比较研究发现, 鲽形目鱼类的线粒体控制区均存在相似的结构, 即线粒体控制区可分为终止相关序列区(ETAS)、中央保守区(包括CSB-A、CSB-B、CSB-C、CSB-D、CSB-E、CSB-F)以及保守序列区(CSB1、CSB2、CSB3)和重复序列区(Repeat region)4个区域。通过与脊椎动物各个纲线粒体控制区序列的比较分析, 发现只有鲽形目(包括鲆、鲽类和鳎类)鱼类和两栖纲的无尾类在CSB-3之后存在相似的串联重复序列。     

鲤TYK2基因的克隆鉴定及组织表达分析

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E2F1 regulation of the human myo-inositol 1-phosphate synthase (ISYNA1) gene promoter

Human myo-inositol 1-phosphate synthase (IP synthase; E.C. 5.5.1.4), encoded by ISYNA1, catalyzes the de novo synthesis of inositol 1-phosphate from glucose 6-phosphate. It is a potential target for mood-stabilizing drugs such as lithium and valproate. But, very little is known about the regulation of human IP synthase. Here, we have characterized the minimal promoter of ISYNA1 and show that it is upregulated by E2F1. Upregulation occurs in a dose-dependent fashion and can be suppressed by ectopic expression of Rb. EMSA and antibody supershift analysis identified a functional E2F binding motif at -117. Complex formation at this site was competed by an excess of unlabeled Sp1 oligo consistent with the -117 E2F site overlapping an Sp1 motif. Because the -117 E2F motif is not a high-affinity binding site, we propose that the upregulation of ISYNA1 occurs through the cooperative interaction of several low-affinity E2F binding motifs present in the minimal promoter.     

黄颡鱼MSTN基因多态性及其与生长性状的相关性分析

肌肉生长抑制素基因(Myostatin,MSTN)属于转化生长因子β家族,其主要功能是负向调控肌肉的生长发育。采用PCR-SSCP技术对黄颡鱼MSTN基因进行单核苷酸多态性检测和分型,并与其生长性状进行关联分析。结果表明,在第一内含子部分检测到1个缺失位点和2个突变位点(T1003del、G1022A和T1063G),基因型分别为:AA、AB、CC、CD和DD;在第三外显子部分检测到1个突变位点(T132C),基因型分别为:EE和EF。关联性分析表明,AA基因型个体的全长、体长、体高、体厚、头长和体重显著大于CD和DD基因型(P<0.05),AA基因型雌性个体的全长、体长、体高、体厚、头长、尾柄高、尾柄厚和体重也显著大于DD基因型(P<0.05)。由此推断,AA基因型是影响雌性黄颡鱼生长性状的有利基因型,DD基因型是影响雌性黄颡鱼生长性状的不利基因型,可以尝试利用这两个位点对雌性黄颡鱼进行标记辅助选育。     

Cloning, characterization and promoter analysis of S-RNase gene promoter from Chinese pear (Pyrus pyrifolia)

The 5'-flanking region of the S(12)-, S(13)-, S(21)-RNase with a length of 854 bp, 1448 bp and 1137 bp were successfully isolated by TAIL-PCR from genomic DNA from 'Jinhua', 'Maogong' (Pyrus pyrifolia) and 'Yali' (Pyrus bretschneideri) genomic DNA. Sequence alignment and analysis of S(13)-, S(12)-, S(21)-RNase gene promoter sequences with S(2)-, S(3)-, S(4)-, S(5)-RNase 5'-flanking sequences indicated that a homology region of about 240 bp exists in the regions just upstream of the putative TATA boxes of the seven Chinese/Japanese pear S-RNase genes. Phylogenetic tree suggests that the homology region between the Chinese/Japanese pear and apple S-RNase gene promoter regions reflects the divergence of S-RNase gene was formed before the differentiation of subfamilies. Full length and a series of 5'-deletion fragments-GUS fusions were constructed and introduced into Arabidopsis thaliana plants. GUS activity were detected in S(12)-pro-(1 to 5)-GUS-pBll01.2 transgenic pistils and progressively decreased from S(12)-pro-1-GUS-pBI l01.2 to S(12)-pro-5-GUS-pBll01.2. No GUS activity was detected in S(12)-pro-6-GUS-pBll01.2 transgenic pistil and other tissues of non-transformants and all transgenic plants. The result suggested S(12)-RNase promoter is pistil specific expression promoter.     

Plasmodium falciparum: polymorphism in the MSP-1 gene in Indian isolates and predominance of certain alleles in cerebral malaria

Polymorphism in the block-2 region of merozoite surface protein-1 gene in 69 North Indian Plasmodium falciparum isolates was studied by PCR and RFLP using Dra-1 endonuclease. On the basis of molecular weight of the PCR products, considerable size polymorphism in target gene was seen and 69 isolates were classified into five allelic types. On RFLP, the isolates in three allelic types were further divided into two sub-allelic types each and thus eight genetic types could be identified. Interestingly, all five allelic types were identified in 47 isolates from uncomplicated (non-cerebral) malaria patients while only two allelic types (Type 2 and 3) were seen amongst 22 isolates from cerebral malaria patients. Furthermore, on RFLP, one subtype (2A) was predominantly seen in cerebral malaria patients and one subtype (3A) was exclusively found in cerebral malaria patients. These observations suggest that a few, comparatively more virulent isolates prevalent in an area may cause severe disease (cerebral malaria) which can be identified by molecular techniques like PCR-RFLP.     

Isolation and Characterization of OsGSTL1 Promoter from Rice

OsGSTL1 gene was isolated from the rice genomic library. Semi-quantitative RT-PCR analysis demonstrated that the expression of the OsGSTL1 in rice was not induced by chlorsulfuron, ethylene, abscisic acid, salicylic acid, and methyl jasmonate. In order to investigate the cis-elements of OsGSTL1 promoter, the promoter regions with different lengths were fused to the β-glucuronidase (GUS) reporter gene. All constructs were transformed into onion epidermal cells or A. thaliana plants to detect the expression patterns. In onion epidermal cells, the 160 bp fragment and longer ones were functional for directing GUS expression. In transgenic A. thaliana, the 2?155 bp upstream region of OsGSTL1 gene directed the GUS expression only in cotyledon after germination, but not in the root of young seedlings. In the later seedling, the 2?155 bp upstream region of OsGSTL1 gene directed GUS expression in roots, stems, and leaves. However, the GUS gene directed by a 1?224 bp upstream fragment is expressed in all the checked tissues. These results suggest that the spatiotemporal expression response elements of OsGSTL1 existed in the 5′-upstream region between −2?155 and −1?224 bp.     

A modified Plasmodium falciparum growth inhibition assay (GIA) to assess activity of plasma from malaria endemic areas

Plasma samples from patients undergoing treatment in malaria endemic countries often contain anti-malaria drugs, that may overstate effects of specific antibodies in growth inhibition assays (GIA). We describe a modified assay that uses drug resistant P. falciparum parasites (W2) that circumvents the requirement for dialyzing samples that may likely contain drugs such as chloroquine and sulfadoxine/pyrimethamine (SP).     

Identification and cloning of a gene encoding tannase (tannin acylhydrolase) from Lactobacillus plantarum ATCC 14917(T)

The gene tanLpl, encoding a novel tannase enzyme (TanLpl), has been cloned from Lactobacillus plantarum ATCC 14917(T). This is the first report of a tannase gene cloned from a bacterial source other than from Staphylococcus lugdunensis, which has been reported elsewhere. The open reading frame of tanLpl, spanning 1410 bp, encoded a 469-amino-acid protein that showed 28.8% identity to the tannase of S. lugdunensis with several commonly conserved sequences. These sequences could not be found in putative tannases reported for other bacteria and fungi. TanLpl was expressed in Escherichia coli DH5alpha from a pGEM-T expression system and purified. SDS-PAGE analysis indicated that purified TanLpl was a monomer polypeptide of approximately 50 kDa in size. Subsequent enzymatic characterization revealed that TanLpl was most active in an alkaline pH range at 40 degrees C, which was quite different from that observed for a fungal tannase of Aspergillus oryzae. In addition, the Michaelis-Menten constant of TanLpl was markedly lower than that of A. oryzae tannase. The evidence suggests that TanLpl should be classified into a novel family of tannases.     

Cloning and heterologous expression of a novel insecticidal gene (tccC1) from Xenorhabdus nematophilus strain

We have identified and cloned a novel toxin gene (tccC1/xptB1) from Xenorhabdus nematophilus strain isolated from Korea-specific entomophagous nematode Steinernema glaseri MK. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111058 Da. The toxin sequence shares 50-96% identical amino acid residues with the previously reported tccC1 cloned from X. nematophilus, Photorhabdus luminescens W14 P. luminescens TTO1, and Yersinia pestis CO92. The toxin gene was successfully expressed in Escherichia coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae (80% death of larvae within 2 days). Conclusively, the heterologous expression of the novel gene tccC1 cloned into E. coli plasmid vector produced recombinant toxin with high insecticidal activity.     

Molecular characterization of a truncated antigen (Wb14) of SXP-1 of Wuchereria bancrofti from four endemic regions in India

Wb14 of Wuchereria bancrofti, an orthologue of Brugia malayiSXP-1 and W. bancrofti SXP-1, was amplified from genomic DNA of W. bancrofti microfilaria collected from four distant geographical locations in India viz., Vellore, Bhubaneshwar, Pondicherry and Sevagram. The gene was sub-cloned in a prokaryotic vector pRSET and expressed in Escherichia coli as a truncated protein (∼23 kDa). The nucleotide sequence of the gene is 98% similar to that of WbSXP-1 and is found to be intron-less. However, the analysis and comparison of the derived amino acid sequence with WbSXP-1 showed that Wb14 is truncated at amino acid position 153. The distribution of the two genes in the studied four geographical locations indicated that WbSXP-1 is prevalent only in parasite samples from Sevagram while Wb14 is present in parasites from all the other locations. Only a limited polymorphism was observed in both the genes among the parasites from different geographical locations.