HIV-1融膜肽的编码DNA序列比较

通过分析GenBank中的全部95个HIV-1完整基因组序列,设计融膜肽"探针序列",对所获得的95段融膜肽的编码DNA序列进行了翻译、对准和分析.得到融膜肽及其编码序列的"优势序列"及突变分布。 Abstract:Fusion peptide coding DNA sequences were retrieved from 95 HIV-1 complete genome entries of GenBank.Results of translation,alignment and analysis led to "the dominant sequence" and the mutation distribution of the fusion eptide coding DNA sequences.     

4种胡蜂前溶血肽原基因的克隆与序列比较

从雌性亚非马蜂、额斑黄胡蜂、墨胸胡蜂、大胡蜂毒腺中抽提总RNA,通过RT-PCR方法扩增得到4种胡蜂蜂毒前溶血肽原的cDNA,再将扩增产物克隆到pGEM⑥-Teasy vector上。测序结果表明：扩增得到的片段长度均为213bp,系蜂毒前溶血肽原编码区的cDNA。经序列比较,4种胡蜂蜂毒前溶血肽原之间的氨基酸序列同源性都超过95％。亚非马蜂、额斑黄胡蜂、墨胸胡蜂、大胡蜂各自与意大利蜜蜂蜂毒前溶血肽原氨基酸序列的同源性分别为95．8％、100％、97．2％、97．2％。结果表明：蜂毒前溶血肽原一级结构序列具有很高的保守性,尽管胡蜂和蜜蜂属于膜翅目不同的总科,但它们的前溶血肽原基因却非常相似。     

原核生物基因组肽编码sORFs分布及功能特征

近期,从非编码RNA中发现具有肽编码能力的小开放阅读框(sORFs),激发了人们对这种长期被忽略的基因组元件的研究兴趣,sORFs迅速成为当前重点研究领域.由于表达水平及丰度低、序列短等因素,对肽编码sORFs的有效研究方法及数据资源还很缺乏,现有研究仅集中在少数真核模式生物,对自然界中广泛存在的原核生物研究非常少,肽编码sORFs的发现为目前精准背景下的基因组注释提出严峻挑战.在此背景下,本文首先系统研究了80余种不同类型原核生物中长度小于100个氨基酸的肽编码sORFs分布及功能特征,并对不同长度区间sORFs的序列组成、分布及进化特征进行了对比分析.结果表明,肽编码sORFs在原核生物基因组普遍存在,随着序列长度的降低,其序列复杂度降低,行使的生物功能也相对集中.在此基础上,进一步结合当前肽编码sORFs研究现状,深入总结了肽编码sORFs研究存在的问题及挑战,为今后肽编码sORFs研究奠定了坚实理论基础.     

穿膜肽-LacI HPM载体的构建及其DNA结合活性测定

目的：借助穿膜肽TAT高效跨膜的特性和LacI前头肽突变体（LacI HPM）高亲和力结合DNA的特性,构建新型基因转导载体。方法：PCR扩增LacI、Lacl基因前头肽序列、前头肽序列突变体、TAT序列的编码基因,构建前头肽序列突变体和TAT的原核表达载体,可溶性表达TAT-LacI HPM融合蛋白并纯化,在缇冲液中氧化获得TAT-LacI HPM二聚体并浓缩,PCR检测二聚体融合蛋白与质粒的体外结合能力。结果：获得了pET-28a（-4-）-LacI HPM及pET-28a（＋）-TAT-LacI HPM表达质粒,表达纯化并获得二聚化融合蛋白,体外结合实验确定TAT-Lac／HPM二聚体融合蛋白与检测质粒DNA具有特异的高亲和力结合活性。结论：构建了穿膜呔TAT-LacI HPM,为进一步研究其作为新型DNA转运载体的可行性奠定了基础。     

大熊猫生长激素受体(GHR) cDNA 的克隆与序列分析

根据已报道的若干物种GHR 基因cDNA 序列设计引物, 利用RT- PCR 技术首次从大熊猫肝脏组织总RNA中扩增出GHR 基因编码区全长cDNA 序列, 克隆于pGEM®-T 载体后进行测序和序列分析。结果表明,大熊猫GHR 的ORF为1 917 bp , 编码638 个氨基酸的前体蛋白, 由18 个氨基酸的信号肽和620 个氨基酸的成熟肽组成,与人、狗、猪GHR 结构相似, 大熊猫GHR 成熟肽由246 个氨基酸的胞外区、24 个氨基酸的跨膜区和350 个氨基酸的胞内区组成, 并具GHR 的特征性结构。序列相似性比较显示, 大熊猫GHR 与哺乳类GHR 具有69 %～93 %的高序列相似性, 与爬行类和鸟类的序列相似性也达到60 % , 而与鱼类的序列相似性较低, 仅为30 %左右。与其它哺乳动物GHR 相比, 大熊猫GHR 在氨基酸序列上也存在明显的特异性。     

鳜小肽转运载体PepT1基因分子特征及其表达研究

小肽转运载体（PepT1）是低亲和力、高容量的肽转运载体,在小肽的吸收过程中发挥着重要的作用。研究采用同源克隆和RACE技术克隆了鳜鱼（Siniperca chuatsi） PepT1基因全长cDNA序列,其cDNA序列全长为2480 bp,包含43 bp的5'UTR序列,232 bp的3'UTR序列,以及2205 bp开放阅读框,编码735个氨基酸。 氨基酸序列同源性分析结果显示,鳜鱼与石斑鱼（Epinephelus aeneus）、鲈鱼（Dicentrarchus labrax）PepT1间同源性均为89%,与其他非鱼类物种的同源性则在46%56%。经预测,鳜鱼PepT1编码蛋白的分子量为64.8 kD,等电点为8.97,该蛋白具有与哺乳动物同源蛋白相似的12 个螺旋跨膜结构,并且在跨膜区9和10之间有一个大的外环;跨膜区氨基酸高度保守,并存在有5个膜外N-糖基化位点和3个膜内含蛋白激酶C基序的相同区域。实时荧光定量表达分析表明,鳜鱼PepT1基因在前肠和中肠中表达量显著高于后肠（P0.05）,这说明前、中肠是鳜鱼肠道吸收小肽的主要部位;在胚后不同发育阶段鳜鱼前肠均能检测到PepT1基因的表达,并且在10 g个体中表达量最高,之后随着体重的增加其表达量维持在一个稳定水平。本研究结果首次报道了鳜鱼PepT1基因全序列及其分子表达特征,为鱼类营养及生理学的研究提供有价值的参考资料。       

利用噬菌体展示文库筛选纤维素结合结构基元

从编码随机１５肽序列的噬菌体表面展示文库中,筛选得到了与纤维素有特异亲和能力的噬菌体克隆。序列分析结果表明,这些噬菌体克隆编码的氨基酸序列中芒香族氨基酸残基高度保守,与天然纤维素结合结构域的结构有一定的类似性。     

牦牛MT-I/-Ⅱ cDNA分子克隆及其蛋白质结构分析

利用基因特异引物YMTSP1和YMTSP2,通过RT-PCR从牦牛肝脏组织RNA中克隆出了牦牛MT-Ⅰ(Genbank Accession NoAY513744)和MT-Ⅱ(Genbank Accession NoAY513745)基因编码区全长.将牦牛MT-Ⅰ和MT-Ⅱ cDNA序列在CBI上进行同源性搜索发现,牦牛MT-Ⅰ/-Ⅱ编码区序列在不同哺乳动物中相当保守.牦牛MT-Ⅰ和MT-Ⅱ编码的MT-Ⅰ和MT-Ⅱ蛋白分别由61个氨基酸组成,其具有保守的短肽结构如C-X-C,C-C-X-C-C,C-X-X-C等,其决定MT蛋白分子的整个三维结构,在分子进化上十分保守.同时对牦牛MT的疏水性和跨膜区分析表明,牦牛MT蛋白可能不存在跨膜区,也不存在信号肽,是1种非分泌蛋白.并通过同源比较模建,预测和构建了牦牛MT-Ⅰ和MT-Ⅱ蛋白的分子空间结构,表明牦牛MT-Ⅰ和MT-Ⅱ由α-和β-两个结构域组成,在α-结构域含有5个Cys短肽结构,β-结构域有4个Cys短肽结构,且2个结构域由保守的三肽序列KKS相连.     

中华蜜蜂和意大利蜜蜂蜂毒前溶血肽原蛋白cDNA的克隆与序列分析

分别从中华蜜蜂Apis cerana cerana和意大利蜜蜂Apis mellifera工蜂毒腺中抽提总RNA,通过RT-PCR方法扩增,各得到了蜂毒前溶血肽原蛋白的cDNA,再将扩增产物克隆到pGEM-Teasy载体上,进行测序和序列分析。结果表明,所扩增到的这两个片段长度均为213 bp,均为编码蜂毒前溶血肽原的cDNA,并分别推导出两者所编码的氨基酸序列。经序列比较,中华蜜蜂前溶血肽原与意大利蜜蜂、印度蜜蜂Apis cerana indica前溶血肽原的同源性都为97%。所报道的中华蜜蜂蜂毒前溶血肽原的核苷酸序列的GenBank登录号为AF487907。     

北京地区人冠状病毒NL63 N和E蛋白基因序列及生物信息学分析

为了解北京地区新近发现的新型冠状病毒-人冠状病毒NL63(Human coronavirus NL63,HCoV-NL63)的N和E蛋白编码基因的特征,从经RT-PCR检测阳性的临床标本中扩增得到的HCoV-NL63 N蛋白和E蛋白编码基因序列,分别克隆至pCF-T和pUCm-T载体中并进行测序,同时运用生物信息学的方法,对北京HCoV-NL63阳性标本BJ8081 N和E蛋白编码基因的核苷酸和氨基酸序列与HCoV-NL63原型株及其他几种冠状病毒的N和E蛋白编码基因的核苷酸和氨基酸序列进行比较分析和种系进化分析;用SOPMA方法对BJ8081 N和E蛋白的二级结构进行了预测分析,并对N和E蛋白的其他生物学特性进行了预测分析.经序列比对分析发现,BJ8081 N蛋白氨基酸序列在78～85肽段(FYYLGTGP)内与所比较的其他冠状病毒N蛋白相应位置的氨基酸序列完全相同,提示此区段可能为包括HCoV-NL63在内的所有冠状病毒N蛋白的保守区域.在BJ8081 N蛋白氨基酸序列的100～121肽段可能是和基因组RNA相结合的位置;在BJ8081 E蛋白的15～37位氨基酸可能是E蛋白的跨膜区域.研究对BJ8081 N蛋白和E蛋白的编码基因序列进行了测定和生物信息学分析,为今后对HCoV-NL63的进一步深入研究奠定了基础.     

Isolation of two genomic sequences encoding the Mr = 14,000 subunit of rat prostatein

Complementary DNAs to rat ventral prostate poly(A) RNA were cloned into pBR322 by the "dG-dC tailing" procedure. Clones containing cDNAs to the mRNAs coding for each of the three subunits of a major secretory protein (prostatein) were identified by hybrid-arrested translation. A 457-nucleotide base pair cDNA (E45) and a portion of a 365-base pair cDNA (E85) were analyzed to determine the composite complete DNA coding sequence for the Mr = 14,000 (C3) subunit of prostatein. A sequence of 12-nucleotide bases (TTTGCTGCTATG) in the signal peptide of C3 was noted to be homologous to signal peptide nucleotide sequences reported in cDNAs coding for the other two prostatein subunits, Mr = 6,000 (C1) and 10,000 (C2). Complementary DNA coding for the C3 subunit was used as a hybridization probe to screen an EcoRI rat genomic DNA library. Two unique 12-kilobase genomic clones, each containing mRNA coding sequences within 2.5-3-kilobase fragments, were identified by restriction enzyme mapping and Southern blot analysis. Restriction enzyme sites within the coding regions of both genes were analogous to the cDNA. Differences in restriction enzyme sites in regions of intervening sequences and flanking DNA established the uniqueness of the two genes. It is suggested that both genes may be transcribed in vivo.     

Potentially functional regions of nucleic acids recognized by a Kohonen's self-organizing map

Computer recognition of short frnctional sites on DNA, suchas promoter regions or intron—exon boundaries, has recentlyattracted much interest. In this paper we have focused our attentionon the automatic recognition of relevant features of human nucleicacid sequences by means of an unsupervised artificial neuralnetwork model. Sixty messenger RNA and 31 genomic DNA sequenceswere analysed. The results showed that in mRNA, the minimalsimilarity 60 base pattern was guanine-and cytosine-rich andlocated in most sequences in a range of 250 bases from eitherthe middle point of the signal peptide coding region or fromthe start of the coding region. On DNA sequences a region definedby a cluster of minimal similarity patterns was present in manyof the analysed genes. This zone may be related to alternativesplicing and DNA methylation.     

Generating DNA sequences encoding tandem peptide repeats suitable for expression and immunological application

Tandem repeats of single short peptide sequences are useful for many purposes. Here we describe a method called ligation-PCR to construct DNA sequences encoding numerous tandem peptide repeats that can stably produce such repeats in both prokaryotic and eukaryotic cells. The method employs double-strand target monomers consisting of a short peptide coding sequences. These sequences contain 3-bp cohesive overhangs to ensure correct repeat orientation and reading frame during ligation. The ligation products are PCR amplified and directly cloned into a new TA-cloning vector, pZeroT. Constructs containing tandem 10-amino-acid myc-tag peptide coding sequence repeats that ranged from approximately 0.45–1.2 kb, representing 15–40 copies of the corresponding peptide, were successfully obtained by this method. When one of the constructs was subcloned into prokaryotic vector pET-28 c (+) and eukaryotic vector rGHpcDNA3.0, and introduced into E. Coli and COS-7 cells, respectively, proteins containing tandem myc-tag peptide repeats were expressed with expected molecular weights. Purified proteins from E. Coli could successfully stimulate a peptide specific immune response. This method provides a means to manipulate peptides at the nucleic acid level, and can serve as the basis for biological peptide synthesis, epitope-specific antibody production, and epitope-based DNA vaccine construction.     

Sequences encoding identical peptides for the analysis and manipulation of coding DNA

The use of sequences encoding identical peptides (SEIP) for the in silico analysis of coding DNA from different species has not been reported; the study of such sequences could directly reveal properties of coding DNA that are independent of peptide sequences. For practical purposes SEIP might also be manipulated for e.g. heterologous protein expression. We extracted 1,551 SEIP from human and E. coli and 2,631 SEIP from human and D. melanogaster. We then analyzed codon usage and intercodon dinucleotide tendencies and found differences in both, with more conspicuous disparities between human and E. coli than between human and D. melanogaster. We also briefly manipulated SEIP to find out if they could be used to create new coding sequences. We hence attempted replacement of human by E. coli codons via dicodon exchange but found that full replacement was not possible, this indicated robust species-specific dicodon tendencies. To test another form of codon replacement we isolated SEIP from human and the jellyfish green fluorescent protein (GFP) and we then re-constructed the GFP coding DNA with human tetra-peptide-coding sequences. Results provide proof-of-principle that SEIP may be used to reveal differences in the properties of coding DNA and to reconstruct in pieces a protein coding DNA with sequences from a different organism, the latter might be exploited in heterologous protein expression.     

The ompA signal peptide directed secretion of Staphylococcal nuclease A by Escherichia coli

The hybrid pre-enzyme formed by fusion of the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, to Staphylococcal nuclease A, a protein secreted by Staphylococcus aureus, is translocated across the cytoplasmic membrane of E. coli with concomitant cleavage of the signal peptide. A DNA fragment containing the coding sequence for the ompA signal peptide was initially ligated to a DNA fragment containing the coding sequence for nuclease A, with a linker sequence of 33 nucleotides separating the coding sequences. When this fused gene was induced, an enzymatically active nuclease was secreted into the periplasmic space; sequential Edman degradation of this protein revealed that the ompA signal peptide was removed at its normal cleavage site resulting in a modified version of the nuclease having 11 extra amino acid residues attached to the amino terminus of nuclease A. The 33 nucleotides between the coding sequences for the ompA signal peptide and the structural gene for nuclease A were subsequently deleted by synthetic oligonucleotide-directed site-specific mutagenesis. The nuclease produced by this hybrid gene was secreted into the periplasmic space and by sequential Edman degradation was identical to nuclease A. Thus, the ompA signal peptide is able to direct the secretion of fused staphylococcal nuclease A, and signal peptide processing occurs at the normal cleavage site. When the hybrid gene is expressed under the control of the lpp promoter, nuclease A is produced to the extent of 10% of the total cellular protein.     

Structure of the human neutrophil elastase gene

The gene for human neutrophil elastase (NE), a powerful serine protease carried by blood neutrophils and capable of destroying most connective tissue proteins, was cloned from a genomic DNA library of a normal individual. The NE gene consists of 5 exons and 4 introns included in a single copy 4-kilobase segment of chromosome 11 at q14. The coding exons of the NE gene predict a primary translation product of 267 residues including a 29-residue N-terminal precursor peptide and a 20-residue C-terminal precursor peptide. Analysis of the N-terminal peptide sequence suggests it contains a 27-residue "pre" signal peptide followed by a "proN" dipeptide, similar to that of other blood cell lysosomal proteases. The sequences for the mature 218-residue NE protein are included in exons II-V. The 5'-flanking region of the gene includes typical TATA, CAAT, and GC sequences within 61 base pairs (bp) of the cap site. The sequence 1.5 kilobases 5' to exon I contains several interesting repetitive sequences including six tandem repeats of unique 52- or 53-bp sequences. The 5'-flanking region also contains a 19-bp segment with 90% homology to a segment of the 5'-flanking region of the human myeloperoxidase (MPO) gene, a gene also expressed in bone marrow precursor cells and a protein stored in the same neutrophil granules as NE. In addition, like the MPO gene, the NE 5'-flanking region has several regions with greater than or equal to 75% homology to sequences 5' to c-myc, but there is no overlap between the NE-c-myc and MPO-c-myc homologous sequences.     

Clustering of identical oligomers in coding and noncoding DNA sequences.

We develop a quantitative method for analyzing repetitions of identical short oligomers in coding and noncoding DNA sequences. We analyze sequences presently available in the GenBank separately for primate, mammal, vertebrate, rodent, invertebrate and plant taxonomic partitions. We find that some oligomers "cluster" more than they would if randomly distributed, while other oligomers "repel" each other. To quantify this degree of clustering, we define clustering measures. We find that (i) clustering significantly differs in coding and noncoding DNA; (ii) in most cases, monomers, dimers and tetramers cluster in noncoding DNA but appear to repel each other in coding DNA. (iii) The degree of clustering for different sources (primates, invertebrates, and plants) is more conserved among these sources in the case of coding DNA than in the case of noncoding DNA. (iv) In contrast to other oligomers, we find that trimers always prefer to cluster. (v) Clustering of each particular oligomer is conserved within the same organism.     

Coding capacity of complementary DNA strands.

A Fortran computer algorithm has been used to analyze the nucleotide sequence of several structural genes. The analysis performed on both coding and complementary DNA strands shows that whereas open reading frames shorter than 100 codons are randomly distributed on both DNA strands, open reading frames longer than 100 codons ("virtual genes") are significantly more frequent on the complementary DNA strand than on the coding one. These "virtual genes" were further investigated by looking at intron sequences, splicing points, signal sequences and by analyzing gene mutations. On the basis of this analysis coding and complementary DNA strands of several eukaryotic structural genes cannot be distinguished. In particular we suggest that the complementary DNA strand of the human epsilon-globin gene might indeed code for a protein.     

Computer program recognition of a cDNA sequence specifying signal peptides]

An application of a computational analysis of cDNA sequences is presented in this paper. The goal is the identification of functional domains on sequence data. The results show the capability of this technique to identify a zone of DNA associated with the signal peptide coding region, whose biological function at DNA or RNA level is still unknown.     

Toxic and non-toxic strains of the cyanobacterium Microcystis aeruginosa contain sequences homologous to peptide synthetase genes

Abstract Toxic strains of Microcystis aeruginosa produce cyclic heptatoxins (microcystins) that are believed to be synthesized non-ribosomally by peptide synthetases. We analysed toxin-producing and non-toxic strains of M. aeruginosa with respect to the presence of DNA sequences potentially encoding peptide synthetases. Hybridizations of genomic DNA of various M. aeruginosa strains with PCR-amplificated fragments possessing homologies to adenylate-forming domains of peptide synthetase genes provided first evidence for the existence of corresponding genes in cyanobacteria. Furthermore we isolated and sequenced from genomic libraries overlapping fragments of M. aeruginosa DNA with a total length of 2982 bp showing significant homology to genes encoding peptide synthetases and hybridizing exclusively with DNA from toxic strains. Our results indicate that both toxic and non-toxic strains of M. aeruginosa possess genes coding for peptide synthetases and that hepatotoxin-producing and non-toxic strains differ in their content of genes for specific peptide synthetases.