Cloning and identification of a novel human glioma differentiation related gene GDR1

In order to identify the genes associated with glioblastoma differentiation, some ESTs, expressed differentially in the control cell and the differentiated human glioblastoma cell line BT-325 induced by the all-trans retinoid acid, have been isolated by the method of DDRT-PCR. Of the 46 ESTs sequenced, 19 are from new genes. A full-length 1 535-bp cDNA, termed gene GDR1, has been isolated from the human cDNA library using the probe designed according to one of the novel ESTs, HGBB098. The open reading frame of GDR1 gene encodes a putative protein containing 334 amino acid residues. Blast against the current GenBank DNA and protein sequence database did not reveal significant homology with any known proteins. RT-PCR shows that GDR1 mRNA level increased in the differentiated BT-325 cells after being treated with RA. The different expression patterns of GDR1 mRNA in human tissues have been detected through the multiple tissue Northern blot hybridization.     

A novel method for evaluating and improving the ~1H-MRSI glioma data quality

Protonmagneticresonancespectroscopicimaging(1H MRSI)hasbeenintensivelystudiedtoquantita tivelyanalyzegliomaforclinician[1—3],including definingthetumorboundary,tumorpropertyandde terminingthetumorlevel.However,rawgliomadata of1H MRSIusuallyinvolvevariousartifacts,suchas thethermalnoise,eddycurrents,susceptibilityarti facts,rigidbodymotion,physiologicalpulsationflow andhardwareissues,whichsignificantlyaffectthe accuracyofthemeasuredresults.1H MRSIisdiffer entfrommagneticresonanceimaging(…     

Cloning, sequencing and function ofsanB, a gene related to nikkomycin biosynthesis ofStreptomyces ansochromogenes

A 6.0 kb DNA fragment related to nikkomycin biosynthesis was cloned from nikkomycin-producingStreptomyces ansochromogenes 7100. Sequence analysis showed that the 1.9 kbTth111 I fragment, a part of the 6.0 kb DNA fragment, contains one complete ORF designatedsanB (GenBank accession No. AF224501), which is composed of 1740 bp encoding a protein consisting of 580 amino acid residues. Its start codon is GTG at 100 bp position and stop codon is TGA at 1840-bp position. Database searching indicated that the deduced protein ofsanB is homologous to the histidinol-phosphate aminotransferase inStreptomyces coelicolor with 31% identities and 47% positives. Gene disruption was performed to study the function ofsanB. It was found that disruptants ofsanB lost the ability to synthesize nikkomycin, which reveals thatsanB is a novel gene essential for nikkomycin biosynthesis.     

睾丸组织高表达新基因THE的克隆及表达特征分析

以Homo.sapiens brain为库,选取编号为CA423810的EST序列,联网到NCBI调用Blast服务器分析,该EST序列是一个代表新基因的未知序列.以该序列作为电子探针,通过Internet采用Blast软件进行GenBank的EST数据库检索,获得了该序列的电子延伸产物EST重叠群.经与人类基因组草图进行序列校正,获得了全长为2 232bp的cDNA序列.利用NCBI的ORFfinder服务器,分析发现该序列具有完整的阅读框架,从而确定了基因的全长cDNA序列.该基因定位于染色体上的5q22,编码由388个氨基酸组成,分子量为44859的蛋白质.运用RTP-CR技术,以新基因电子克隆全长cDNA序列设计基因特异性引物,以11例癌组织及相应的正常组织的cDNA、人胎脑cDNA文库和人睾丸cDNA文库为模板扩增目的片段,并以看家基因GAPDH为内对照,检测目的基因的mRNA表达水平.研究结果表明,新基因只在睾丸组织中高表达,在直肠癌、结肠癌、宫颈癌、胃癌等的癌组织及相应的正常组织中都无明显的表达.因此将该新基因命名为睾丸组织高表达基因(testis high expression,THE).以上结果提示,THE基因可能是在人脑中表达的同一基因的不同剪接本.关键词检索UniGene数据.     

Cloning of human cytokine signal transduction inhibitor genehumSOCS-2

An EST (gb/AA115239) with high identity to the mouse cytokine signal transduction inhibitor genemmSOCS-2 was selected in GenBank EST database by the homologous screening method. The cDNA with the same sequence of the EST was got in human placenta cDNA library by PCR and a 1011 bp cDNA fragment was selected using above cDNA as probes to perform walking hybridization in placenta cDNA library. The cDNA fragment contains one 594 bp open reading frame (ORF) which encodes 198 amino acid residues. It was proved to be novel after NCBl database screening. Homology comparison showed that this gene has 93% identity tommSOCS-2 at the amino acid level and it has high identities to other related genes in SH₂ domain and SOCS box, so it was namedhumSOCS-2 and the accession number in GenBank is gb/AF020590. The expression analysis showed that the gene is expressed obviously higher in prostate than in other 15 human tissues.     

Cloning and characterization of a gene encoding phagerelated tail protein (PrTP) of endosymbiont Wolbachia

Wolbachia is an obligatory, maternally inherited intracellular bacterium, known to infect a wide range of arthropods. It has been implicated in causing cytoplasmic incompatibility (CI), parthenogenesis, the feminization of genetic males and male-killing in different hosts. However, the molecular mechanisms by which this fastidious bacterium causes these reproductive abnormalities have not yet been determined. In this study, we report on the cloning and characterization of the gene encoding phage-related tail protein (PrTP) from Wolbachia in Drosophila melanogaster CantonS (wMelCS) and from Wolbachia in Drosophila melanogaster yw67c23 (wMel) by representational difference analysis (RDA) and ligation-mediated PCR (LM-PCR). The functionality of a bipartite nuclear localization signal sequence (NLS) of the gene was also successfully tested in Drosophila S2 cells. PrTP expression in various strains of Wolbachia was investigated. Our results suggest that PrTP may not induce CI directly. However, the existence of prtp provided direct evidence of phage-mediated horizontal gene transfer (HGT) that might play an important role in a variety of reproductive abnormalities of Wolbachia.     

水稻中一个与COI1同源的基因克隆及其表达分析

利用同源序列分析,在水稻基因数据库中检索到了几条与拟南芥中的COI1基因具有高度同源性的EST(expression sequence tag)片段和相应的基因组序列．根据EST拼接和基因组比较结果设计引物,利用RT—PCR在水稻中得到与COI1高度同源的cDNA,并在基因组中推断出相应的基因,命名为RCOI1．RCOI1与COI1在氨基酸水平有74％的同源性．与COI1相似,RCOI1蛋白也具有典型的F—box和LRRs结构域．半定量RT—PCR显示该基因的表达受茉莉酸甲酯(MeJA)的诱导,说明这个基因可能参与水稻茉莉酸应答反应．该基因的发现对于探索单子叶植物的茉莉酸信号转导途径,以及研究该途径在农作物中的抗虫抗病和抵抗恶劣环境等方面的作用都有重要意义．     

Molecular cloning, identification and characteristics of NYD-SP9: Gene coding protein kinase presumably involved in spermatogenesis

Using cDNA microarray hybridization from a human testicular cDNA library, one gene exhibiting ten-fold difference at expression level between adult and embryo human testes was cloned and named NYD-SP9, which was believed to be involved in spermatogenesis. Southern blot hybridization results showed that NYD-SP9 expressed highly in testis but low in ovary. Protein motif analysis of this cDNA sequence revealed a cluster of phosphorylation sites, indicating its potential involvement in signal pathways during spermatogenesis. Furthermore, one transmembrane helix was predicted in N-terminal region, indicating that putative NYD-SP6 may be served as a transmembrane protein. The proximity of these potential phosphorylation sites to each other indicates that there may be interaction among these sites to regulate spermatogenesis. These findings suggested that protein kinase NYD-SP9 might play a role in male germ cell differentiation.     

Cloning and sequence analysis of Sox genes in a tetraploid cyprinid fish, Tor douronensis

A PCR survey for Sox genes in a young tetraploid fish Tor douronensis （Teleostei： Cyprinidae） was performed to access the evolutionary fates of important functional genes after genome duplication caused by polyploldizatlon event. Totally 13 Soxgenes were obtained in Tordouronensis, which represent SoxB, SoxC and SoxE groups. PhylogeneUc analysis of Sox genes in Tor douronensis provided evidence for fish-specific genome duplication, and suggested that Sox19 might be a teleost specific Sox gene member. Sequence analysis revealed most of the nucleoUde substitutions between duplicated copies of Soxgenes caused by tetreploldlzatlon event or their orthologues in other species are silent substitutions. It would appear that the sequences are under purifying selective pressure, strongly suggesting that they represent functional genes and supporting selection against all null allele at either of two duplicated loci of Sox4a, Sox9a and Sox9b. Surprising variations of the intron length and similarities of two duplicated copies of Soxga and Sox9b, suggest that Tor douronensis might be an allotetreploidy.     

Identification of a novel DNA molecule associated with Tobacco leaf curl virus

A circular DNA molecule, designated as DNAβ, was identified in tobacco plants infected with Tobacco leaf curl virus (TLCV) isolates Y5 and Y8 by PCR using primers based on the conserved region of the two reported DNAβ sequences of whitefly-transmitted geminiviruses (WTGs). The complete nucleotide sequences of DNAβ of Y5 and Y8 (TLCV DNAβ) were determined. Y5 DNAβ comprises 1333 nucleotides encoding 8 predicted ORFs with 4 ORFs in virion-sense DNA and 4 ORFs in complementary-sense DNA; Y8 DNAβ consists of 1338 nucleotides encoding 7 predicted ORFs with 4 ORFs in virion-sense DNA and 3 ORFs in complementary-sense DNA. TLCV DNAβ has little sequence homology to DNA-A of TLCV., except that it shares conserved TAATATTAC loop sequence with TLCV DNA-A. Sequence comparison showed that Y5 DNAβ shared 85% sequence homology with Y8 DNAβ, and both Y5 DNAβ and Y8 DNAβ had relatively low sequence identity (51%–65%) with the reported DNAβ molecules associated with Ageratum yellow vein virus and Cotton leaf curl virus. The immunotrapping PCR and whitefly transmission tests showed that DNAβ molecule could be encapsidated in virus particle and transmitted by Bemisia tabaci. This is the first report of DNAβ associated with WTGs in China.     

人类新基因CHID1的克隆与功能初步研究

使用生物信息学手段,从人公共蛋白质数据库的蛋白质序列中筛选到新的分泌蛋白基因CHID1(Chitinase domain containing 1),该基因定位于人11号染色体短臂15区5带(11p15.5),cDNA长1 182 bp,其编码蛋白共有393个氨基酸。转染COS-7细胞后Western blot实验显示:在细胞培养液中没有检测到CHID1蛋白。对CHID1基因在mRNA水平上进行组织分布考察发现:它在肺、肾、肝、心、胸腺中都有表达,肝中最高。细胞生长曲线揭示,Lovo-CHID1稳定细胞株有生长抑制的现象,而细胞周期分析发现细胞株在G1期发生阻滞。这表明CHID1有可能是个潜在的抑癌基因。     

尖镰胞菌细胞色素P450 55a1基因超表达载体的构建和水稻日本晴遗传转化

将细胞色素P450 55a1基因克隆到镰刀菌素植物超表达载体pCAMBIA1302中,构建了pCAMBIA1302-cyp55a1-gfp植物超表达载体,以水稻日本晴为遗传转化的受体对象,通过农杆菌介导侵染方法进行了遗传转化.结果表明：成功构建了细胞色素P450 55a1基因超表达载体,获得了多个细胞色素P450 55a1基因超表达的阳性植株,并以RT-PCR技术分析了阳性植株中细胞色素P450 55a1基因的表达水平.     

Transgenic tobacco plants expressing synthetic Cry1Ac and Cry1Ie genes are more toxic to cotton bollworm than those containing one gene

Transgenic tobacco plants carrying CrylAc, Crylle or both genes were obtained. In the leaves of transgenic plants carrying both genes, the contents of CrylAc and Crylle proteins were 0.173% and 0.131% of the total proteins, respectively. CrylAc protein content was 0.182 % and Cry1 le protein content was 0.124% of the total proteins in the leaves of transgenic plants containing only one Bt gene. Fresh leaves of transgenic tobacco and wild-type plants were used for the insect bioassay against wild-type and Cry1Ac-resistant cotton bollworm （Helicoverpa armigera）. The bioassay results showed that transgenic plants carrying both genes were significantly more toxic to wild-type and CrylAc-resistant cotton bollworm than those carrying CrylAc or Crylle alone. This study indicates that the higher toxicity of transgenic tobacco plants carrying both genes is caused by the cooperative function of both Bt proteins, thus providing a potential way to delay the development of insect resistance to transgenic crops.