Cloning and analysis of the pseudogene for human epinephrine synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT)

1. This gene completely lacks the intervening sequences. 2. This gene is truncated at the 5' end peptide encoding region by 433 base pairs (bp). 3. The 502 bp of this gene containing poly(A) signal are completely identical to the 3' half of mRNA encoding region of functional gene. 4. This gene has a poly(A) tail and is flanked by direct repeat of 6 bp. 5. Here we report for the first time the complete sequence of a human pseudogene for phenylethanolamine N-methyltransferase and this is the first report of cloning of pseudogene for catecholamine biosynthetic enzymes.     

Structure and evolution of the human prosaposin chromosomal gene.

The gene for prosaposin was characterized by sequence analysis of chromosomal DNA to gain insight into the evolution of this locus that encodes four highly conserved sphingolipid activator proteins or saposins. The 13 exons ranged in size from 57 to 1200 bp, while the introns were from 91 to 3812 bp in length. The regions encoding saposins A, B, and D each had three exons, while that for saposin C had only two. This sequence included the regions that encode the carboxy terminus of the signal peptide, the four mature prosaposin proteins, and the 3' untranslated region. Primer extension studies indicated that over 99% of the coding sequence was contained in these 19,985 bp. Use of PCR and reverse PCR techniques indicated that the most 5' coding approximately 140 bp contained large introns and at least two small exons. Analyses of the intronic positions in the saposin regions indicated that this gene evolved from an ancestral gene by two duplication events and at least one gene rearrangement involving a double crossover after introns had been inserted into the gene.     

The nucleotide sequence of the yeast MEL1 gene.

The complete nucleotide sequence of the MEL1 gene of the yeast, Saccharomyces cerevisiae, encoding alpha-galactosidase was determined. The nucleotide sequence contains an open reading frame of 1413 bp encoding a protein of 471 amino acids. Comparison with the known N-terminal amino acid sequence of the mature secreted protein indicated that alpha-galactosidase is synthesized as a precursor with an N-terminal signal sequence of 18 amino acids. The general features of this signal peptide resemble those of other yeast signal peptides. Molecular weight of the mature alpha-galactosidase polypeptide deduced from the nucleotide sequence is 50.049 kd. The 5' regulatory region has sequences in common with other yeast genes regulated by the GAL4-protein.     

牛RHOQ基因的电子克隆与生物信息学分析

利用电子克隆技术获得牛RHOQ基因cDNA序列,采用生物信息学方法对该基因及其编码蛋白的基本理化性质、疏水性、信号肽、二级结构和亚细胞定位等方面进行预测和分析。结果表明,牛RHOQ基因的cDNA序列全长1 517 bp,包含1个618 bp开放阅读框,编码205个氨基酸;其编码蛋白属疏水性蛋白,不存在信号肽及跨膜结构,定位于分泌系统囊泡;二级结构主要以无规则卷曲和α-螺旋为主。牛RHOQ基因编码蛋白可能具有生长因子功能,可能在神经再生和突触延伸过程中起重要作用。     

弗氏链霉菌丝氨酸蛋白酶基因的克隆及表达

从一株具有极强的降解羽毛能力的弗氏链霉菌菌株（Streptomyces fradiae var.k11）中纯化得到了一种丝氨酸蛋白酶SFP2。经蛋白测序,得到部分氨基酸序列,设计简并引物,PCR扩增得到部分基因序列,通过构建基因文库,获得了包括信号肽序列在内的完整的基因sfp2(EMBL收录号AJ784940),开放阅读框全长924bp,包括114bp的信号肽编码序列和810bp的酶原编码序列, 其中成熟蛋白编码基因长576bp,编码191个氨基酸,理论分子量为19.112kD。酶原编码基因和成熟蛋白编码基因均在大肠杆菌和枯草芽孢杆菌中得到了表达,酶原编码基因表达产物具有正常的生物学活性,证明了克隆基因的生物学功能。     

甘蔗细胞色素P450还原酶基因的电子克隆与分析

旨在为甘蔗细胞色素P450还原酶基因的实验克隆、功能鉴定及其应用提供参考。本研究以甘蔗类似细胞色素P450还原酶基因的EST序列CF576130.1为探针,在甘蔗EST数据库中进行检索,并基于电子克隆技术获得了甘蔗细胞色素P450还原酶基因（Cytochrome P450 reductase）的一条cDNA全长序列,命名为ScCYP450。采用生物信息学方法,对该基因编码蛋白从氨基酸组成、理化性质、亚细胞定位、跨膜结构域、疏水性/亲水性、高级结构及功能域等方面进行预测和分析。结果表明该基因全长1 821 bp,包含一个744 bp的开放阅读框,编码247个氨基酸,该基因编码蛋白定位于细胞内质网（膜）,为可溶性碱性蛋白,不存在信号肽,二级结构原件多为无规卷曲,含有多个保守功能域,主要功能为辅酶因子生物合成和翻译功能。     

Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis

The nucleotide sequence of the protective antigen (PA) gene from Bacillus anthracis and the 5' and 3' flanking sequences were determined. PA is one of three proteins comprising anthrax toxin; and its nucleotide sequence is the first to be reported from B. anthracis. The open reading frame (ORF) is 2319 bp long, of which 2205 bp encode the 735 amino acids of the secreted protein. This region is preceded by 29 codons, which appear to encode a signal peptide having characteristics in common with those of other secreted proteins. A consensus TATAAT sequence was located at the putative -10 promoter site. A Shine-Dalgarno site similar to that found in genes of other Bacillus sp. was located 7 bp upstream from the ATG start codon. The codon usage for the PA gene reflected its high A + T (69%) base composition and differed from those of genes for bacterial proteins from most other sequences examined. The TAA translation stop codon was followed by an inverted repeat forming a potential termination signal. In addition, a 192-codon ORF of unknown significance, theoretically encoding a 21.6-kDa protein, preceded the 5' end of the PA gene.     

Nucleotide sequence of the gene encoding the F72 fimbrial subunit of a uropathogenic Escherichia coli strain

The cloned DNA fragment encoding the F72 fimbrial subunit from the uropathogenic Escherichia coli strain AD110 has been identified. The nucleotide sequence of the structural gene and of 196 bp of the noncoding region preceding the gene was determined. The structural gene codes for a polypeptide of 188 amino acid residues, including a 21-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the F72 gene were compared with the reported sequences of the papA gene (B?ga et al., 1984). Both genes code for subunits of fimbriae that are involved in mannose-resistant hemagglutination (MRHA) of human erythrocytes. The available data show that there is absolute homology between the noncoding regions preceding both genes over 129 bp. The two proteins are homologous at the N terminus and C terminus; there is less, but significant, homology in the region between the N and C termini.     

Nucleotide sequence of the Clostridium thermocellum laminarinase gene.

The sequence presented (1022 bp) shows the Clostridium thermocellum laminarinase gene (lam1) and its flanking regions. The gene lam1 comprises an open reading frame of 726 nt, encoding a 242-aa protein with predicted Mr 27661. The ORF startswith the translation initiation codon ATG. This ATG codon is preceded at a spacing of 7 bp by a potential ribosome binding site (GGAGGT). A putative signal peptide was identified (the potential cleavage site is between position 27-28 aa). The comparison of the primary protein sequence with other beta-1, 3-1, 4-glucanases showed extensive homology for Bacillus amyloliqefaciens and Bacillus subtilis glucanases (identity-46.7%; similarity-57.0%).     

Amino acid sequence of the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis

The complete nucleotide sequence of the gene encoding the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis, and the derived amino acid sequence are presented. The nucleotide sequence of the gene reveals the existence of a typical bacterial signal peptide of 20 amino acid residues which is not found in the mature cytochrome subunit. The gene encoding the cytochrome subunit is preceded by the gene encoding the M subunit. Both genes overlap by 1 bp. The mature cytochrome subunit consists of 336 amino acid residues; 73% of its amino acid sequence was confirmed by protein sequencing work. The mol. wt of the cytochrome subunit including the covalently bound fatty acids and the bound heme groups is 40 500. The internal sequence homology is low, despite the symmetric structure of the cytochrome subunit previously shown by X-ray crystallographic analysis of the intact photosynthetic reaction centre. Sequence homologies to other cytochromes were not found.     

Pheromone 4 gene of Euplotes octocarinatus.

We have cloned and sequenced a 1.7 kb macronuclear chromosome encoding the pheromone 4 gene of Euplotes octocarinatus. The sequence of the secreted pheromone is preceded by a 42 amino acid leader peptide, which ends with a lysine residue. The sequence coding for the leader peptide contains information for a putative signal peptide and is interrupted by a 772 bp intron as shown by comparison with a cDNA clone. A 64 bp intron and a 145 bp intron interrupt the sequence coding for the secreted pheromone. The three introns contain typical 5' and 3' splice junctions and a putative branch point site. The small introns have a low GC content. The large intron has a GC content similar to that of the pheromone 4 gene exons. The amino acid sequence of pheromone 4, deduced from both the genomic DNA and the cDNA of pheromone 4, shows that the secreted pheromone consists of 85 amino acids. One of its amino acids is encoded by a UGA codon. Since it has been shown for pheromone 3 of E. octocarinatus that UGA is translated as cysteine, it is assumed that the UGA codon encodes cysteine in pheromone 4 as well. The 164 bp noncoding region upstream of the leader peptide is AT-rich and contains an inverted repeat capable of forming a stem-loop structure with a stem of 11 bp. The 151 bp noncoding region at the 3' end of the chromosome contains a putative polyadenylation sequence and an inverted repeat. The macronuclear molecule is flanked by telomeres and carries the pentanucleotide motif TTGAA, located at a distance of 17 nucleotides from the telomeres. This motif has been suggested to be involved in the formation of macronuclear chromosomes.     

来源于Bacillus subtilis的中性植酸酶基因的克隆及在大肠杆菌中的表达

通过ＰＣＲ的方法从Bacillus subtilis基因组中克隆了中性植酸酶基因nphy,ＤＮＡ全序列分析表明其结构基因全长１１５２个核苷酸（编码３８３个氨基酸）,５′端有一编码２６个氨基酸的信号肽序列。去除信号肽编码序列的nphy克隆到大肠杆菌ＩＰＴＧ诱导表达载体pTYB40上,在大肠杆菌中得到了高效表达,表达量达到大肠杆菌可溶性蛋白的４０％以上,表达产物具有生物学活性,证实了克隆到的中性植酸酶的基因有正常的生物学功能。     

Cloning and selective overexpression of an alkaline protease-encoding gene from Aspergillus oryzae.

The gene alpA encoding Aspergillus oryzae alkaline protease (ALP) was isolated from a genomic library of an industrial strain used in Thailand by using oligodeoxyribonucleotide probes based on the published cDNA sequence [Tatsumi et al., Agric. Biol. Chem. 52 (1988) 1887-1888]. The entire nucleotide sequence of the genomic clone obtained was determined. By comparison with the published cDNA sequence, it was found that ALP is encoded by four exons of 314, 445, 89 and 351 bp. Three introns, which interrupt the coding sequence, are 50, 59 and 56 bp in length. The gene contains a typical TATA box 103 bp upstream from the start codon, and a consensus polyadenylation signal, AATAAA, 189 bp from the stop codon. The alpA gene, introduced into a protease deficient strain (A. oryzae U1638) by cotransformation, directed the secretion of enzymatically active ALP into the culture medium. Cotransformants of the high-level ALP-producing strain U212 containing multiple copies of the alpA gene were able to secrete up to five times more ALP than the parental strain.     

Structure of the murine mb-1 gene encoding a putative sIgM-associated molecule

Genomic DNA clones containing the B cell-specific murine mb-1 gene were isolated and a 5.6-kb BamH I fragment was characterized. It is 5629 bp long and contains five exons: an exon containing the 5' untranslated and the coding sequence of the signal peptide, an exon of 294 bp, which contains most of the extracellular sequence of the MB-1 protein, a 119-bp long exon coding mainly for the transmembrane portion, and two exons of 69 bp and 427 bp encoding the cytoplasmic domain and the 3'-untranslated region, respectively. The mb-1 gene does not contain a "TATA box" found in many eukaryotic promoters. The 5'-flanking region has sequence stretches homologous to IgVH 5'-promoter regions and a bcl 2 intron sequence. It contains the decanucleotide sequence (ATGGCAAATA) almost identical to the octamer motif of IgVH promoters. A B cell-specific DNase I-hypersensitive site was found in the 3'-flanking region indicating that this region might be involved in B cell-specific expression of mb-1. Southern blot analysis of genomic liver DNA with the cloned mb-1 cDNA suggests the existence of another mb-1-related gene segment.     

大豆11S球蛋白Gy5(A3B4)的基因克隆和序列分析

大豆11Ｓ球蛋白(Ｇｌｙｃｉｎｉｎ)是大豆种子的主要贮藏蛋白,分子量为360ｋＤ,由6对相同的蛋白亚基(每对亚基的分子量约60ｋＤ)构成。每对亚基又是由一个酸性Ａ肽(35～45ｋＤ)和一个碱性Ｂ肽(22ｋＤ)通过二硫键连接而成。Ａ肽和Ｂ肽源自同一个基因,即首先由一个大的ｍＲ?..     

管氏肿腿蜂毒液过敏原3基因的克隆及表达分析

为研究管氏肿腿蜂Scleroderma guani毒液过敏原3基因SgA3的功能,通过RT-PCR克隆SgA3基因,采用荧光定量PCR分析其在不同发育阶段和组织中的表达特征,并利用载体pSUMO-Mut对其进行原核表达。克隆获得SgA3基因开放阅读框长699 bp,编码233个氨基酸,其中信号肽位于N端第1~23位氨基酸。多序列比对分析发现,SgA3与丽蝇蛹集金小蜂Nasonia vitripennis、粉蝶盘绒茧蜂Cotesia glomerata、红火蚁Solenopsis invicta和常见黄胡蜂Vespula vulgaris过敏原3的氨基酸序列一致性分别为50.44%、50%、48.89%和47.83%。荧光定量PCR结果表明,SgA3基因在雌成虫中高表达,且在毒液器官中的表达量显著高于雌成虫其他组织和雄成虫中的表达量。利用pSUMO-Mut表达载体,成功表达并纯化得到高纯度的SgA3重组蛋白。该研究结果为进一步研究SgA3基因的功能奠定了基础。     

Cloning of a mucin-desulfating sulfatase gene from Prevotella strain RS2 and its expression using a Bacteroides recombinant system

A gene encoding the mucin-desulfating sulfatase in Prevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into a Bacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.     

一个东亚钳蝎蝎毒素BmTXKβ基因的克隆及其基因表达调控

从东亚钳蝎 (ButhusmartensiiKarsch ,BmK)毒腺组织cDNA文库中分离的长链钾通道毒素BmTXKβcDNA序列 ,克隆了BmTXKβ基因组序列 .BmTXKβ基因含有一个长度为 886bp的内含子 ,定位于BmTXKβ成熟肽中 ,与其它蝎毒素基因内含子定位于信号肽的基因结构不同 .并且 ,BmTXKβ基因的内含子特征也与其它蝎毒素基因不同 .研究结果从基因水平上证实了BmTXKβ是一个新的蝎毒素样肽 .以BmTXKβcDNA序列为探针与蝎基因组DNASouthern杂交出现 2条特异性杂交带 .杂交结果为蝎毒素基因可能通过DNA重排、多拷贝或多基因家族来调控基因表达提供了证据 .