分离、定位单考口片段的方法与意义

分离、定位单考口片段的方法与意义唐清（华西医科大学医学遗传组）人与动物基因组上的ＤＮＡ顺序按其是否重复可分为２大类,若同一段顺序在基因组内重复出现多次,则这段顺序称为重复顺序;若某一顺序在基因组内只出现一次,则这段顺序称为单拷贝顺序或单考贝片段。单拷...     

山葡萄成熟果皮cDNA文库的构建及ESTs初步分析

以山葡萄成熟果皮为材料,采用Creator~（TM） SMART~（TM） cDNALibraryConstruction技术构建了cDNA文库。经鉴定,文库的库容量为1.001×10~6,重组率为94.1 1%,插入片段在0.25～1.9 kb。经随机测序,获得935个有效ESTs,聚类拼接后得到636条单基因簇（unigenes）,包括107个重叠群（contigs）和529个单拷贝（singlets）。这些数据为今后山葡萄功能基因分离克隆和功能基因组学研究奠定了基础。     

玉米中抗病基因myb1和NDR1同源序列的荧光原位杂交物理定位

以烟草和拟南芥中的单拷贝抗病基因ｍｙｂ１和ＮＤＲ１作探针,利用荧光原位杂交的方法分别对这两个基因在玉米（Ｚｅａ ｍａｙｓ Ｌ．）和烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）、玉米和拟南芥（Ａｒａｂｉｄｏｐｓｉｓ ｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）中的同源性做了研究。杂交结果表明ｍｙｂ１和ＮＤＲ１的同源序列分别位于玉米第８、５染色体,单个信号位置表明０这两个基因的同源序列在玉米基因组中只有     

编码人疱疹病毒－6型核糖核苷酸还原酶和P41蛋白基因的克隆和序列测定

本文报道人疱疹病豢－６型（ＨＨＶ－６）ｐＳＴＹ２８ＤＮＡ片段的序列测定。应用分子克隆、缺损突变体（Ｄｃｌｅｔｉｏｎｍｕｔａｎｔ）制备和序列测定等技术,完成了３．９ｋｂＨＨＶ－６ｐＳＴＹ２８ＤＮＡ片段的全序列测定。经ＤＮＡＳＩＳ核酸蛋白软件分析,该片段含有两个开读框架（ＯＲＦ）核糖核苷酸还原酶（ＲＩＲ）ＯＲＦ有２４１４个核苷酸,可编码８０５个氨基酸;Ｐ４１蛋白由１１００个核苷酸组成。与其他疱疹病毒作氨基酸同源性比较,ＨＨＶ－６ＲｉＲ与人巨细胞病毒（ＨＣＭＶ）有高度同源性,最适记分（Ｏｐｔｉｍｉｚｅｄｓｃｏｒｅ）达４５９。实验结果支持Ｅｓｆｔａｔｈｉｏｕ提出的论点,ＨＨＶ－６属于β－疱疹病毒。     

HSP70基因上游调控序列对GST基因在耻垢分枝杆菌中表达效率的 影响

将外源基因———日本血吸虫２６ｋＤ抗原（Ｓｊ２６ＧＳＴ）基因克隆到大肠杆菌分枝杆菌穿梭质粒中,构建成四个不同的表达截体,研究它们在耻垢后分枝杆菌（Ｍｙｃｏｂａｃｔｅｒｉｕｍｓｍｅｇｍａｔｉｓ）中的表达效率。首先将含有结核杆菌热休克蛋白７０（ＨｅａｔＳｈｏｃｋＰｒｏｔｅｉｎ,ＨＳＰ７０）的启动子的质粒ｐＭＴ７０用ＮｃｏＩ切,进行两种不同的修饰后,得到不同的ＳＤ序列;将Ｓｊ２６ＧＳＴ基因克隆进去。再将含ＨＳＰ７０启动子和Ｓｊ２６ＧＳＴ基因的片段切下,克隆到分枝杆菌大肠杆菌穿梭质粒ｐＢＣＧ２０００中,筛选出不同ＳＤ序列、不同方向和不同拷贝数的分枝杆菌表达载体四个。所表达的重组天然Ｓｊ２６ＧＳＴ（ｒＳｊ２６ＧＳＴ）为可溶性蛋白,在ＳＤＳＰＡＧＥ上分子量为２６ｋＤ处可见明显的表达蛋白带。通过薄层扫描分析,发现表达质粒中双拷贝启动子外源基因组合,表达效率最高,是单拷贝组合的１６倍,占分枝杆菌菌体总蛋白的２８％。而不同的克隆方向和不同的ＳＤ序列（两者相差３个碱基）对表达效率的影响不明显。     

SV40PolyA片段串联拷贝数目影响GFP报告基因表达

Alu元件（约280bp）是人类基因组中的重要重复序列,串联Alu呈长度依赖性下调GFP报告基因表达,在Alu串联序列上游正向或反向插入SV40PolyA（简称PolyA,240bp）,解除Alu串联序列对GFP基因的抑制作用.PCR法扩增PolyA反序（PolyAas）不同位置的60bp片段,插入pAlu14质粒（14个Alu正向串联插入pEGFP-C1）的GFP基因和Alu14之间,瞬时转染HeLa细胞,通过荧光显微镜观察和Northern检测,1F1R（PolyAas5′端第1个60bp片段）和4F4R（自PolyAas5′端计算第4个60bp片段）不能活化基因;2F2R和3F3R（PolyAas中间的2段）可以解除Alu14对GFP基因的抑制作用.将2F2R和3F3R各自反复首尾串联,分别插入pAlu14质粒GFP基因和Alu串联序列之间,瞬时转染HeLa细胞,用插入4个2F2R的pAlu28,插入4个3F3R的pAlu18,插入4个3F3R的pAlu28作长度对照,以排除由于插入片段长度增加可能对结果造成的干扰,发现2～4个拷贝的2F2R和3F3R活化基因作用高于一个拷贝的同样片段,但是多于8拷贝以后,活化GFP基因作用减弱.本实验证明,SV40 PolyAas至少含有两段活化基因序列,活化基因序列（2F2R,3F3R）的最适活化基因条件需要合适的拷贝数.     

杜氏盐藻DCA1启动子内GT重复序列在盐诱导调控中的作用

为了研究杜氏盐藻双拷贝碳酸酐酶（DCA1）启动子中高度重复的GT序列在盐诱导表达时的调控作用,设计不同的引物,通过PCR法获得6条不同长度的DCA1启动子片段,分别与gus报告基因融合后构建6个表达载体;电击法转化杜氏盐藻细胞。组织化学染色和荧光定量法检测GUS在不同盐浓度下的瞬时表达。结果显示,DCA1启动子内高度重复的GT序列无论与其上游、下游或上下游片段同时结合均能驱动gus基因的表达,并且其表达受氯化钠浓度调控,其中和上下游均结合时活性最强;无GT重复序列的融合片段及GT 重复的下游片段也能驱动gus基因的表达,但其表达不受氯化钠浓度调控;而GT重复的上游片段不能驱动gus基因的表达。结果提示:盐藻DCA1启动子中高度重复的GT序列在盐诱导调控中起重要作用,可能为一种新型的盐诱导元件。     

对微分离的大豆单染色体的克隆及其PCR产物的原位杂交分析

通过玻璃针分离法从大豆（Ｇｌｙｃｉｎｅ ｍａｘ Ｌ．）根尖细胞中期分裂相中显微分离出一条染色体,经Ｓａｕ３Ａ人工接头介导的两轮ＰＣＲ后,将其第二轮扩增产物克隆到质粒载体上,构建了单染色体质粒文库,经分析,该微克隆文库包含约２００００个重组子。随机挑选１７８个重组子进行鉴定,证明该文库的插入片室主要介绍于２００－１８００ｂｐ大小８３０ｂｐ;其中,中、高拷贝重复序列占４４％,单、低拷贝序列占５６％。微     

稻胚凝集素基因的克隆,序列分析及表达

以水稻基因组ＤＮＡ为模板,以特异引物经聚合酶链式反应方法扩增出稻胚凝集素基因并克隆到Ｅ．ｃｏｌｉ质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ（＋）的ＳｍａⅠ位点。序列分析表明,克隆到的基因片段大小为７８１ｂｐ,没有内含子,编码１条长２２７个氨基酸、分子量约２３ｋＤ的肽链,其中Ｎ－端２８个氨基酸是信号肽。与报道的稻胚凝集素ｃＤＮＡ序列进行顺序同源性比较,发现它们之间有很高的同源性（９９．７４％）,其编码区第１６７     

利用微分离黑麦1R染色体的体外扩增产物进行染色体着染研究

首先对显微分离出的黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体进行了两轮Ｓａｕ３Ａ连接接头介导的ＰＣＲ扩增（ＬＡ＿ＰＣＲ）。经Ｓｏｕｔｈｅｒｎ杂交证实这些染色体扩增片段来源于基因组ＤＮＡ之后,再利用１Ｒ染色体的第二轮扩增产物、黑麦基因组ＤＮＡ、ｒＤＮＡ基因为探针,与其根尖细胞中期分裂相进行染色体原位杂交,发现微分离的１Ｒ染色体体外扩增产物中包含大量的非该染色体特异性重复序列,而其信息量却较黑麦总基因组少;当以适量的黑麦基因组ＤＮＡ进行封阻时,微分离染色体的体外扩增产物成功地被重新定位在中期分裂相的一对１Ｒ染色体上,说明微分离１Ｒ染色体的ＰＣＲ扩增产物中的确包含了该染色体特异性的片段。此外,以从１Ｒ染色体微克隆文库中筛选出的一单、低拷贝序列和一高度重复序列分别为探针,染色体原位杂交检测发现,这一高度重复序列可能为端粒相关序列;而单、低拷贝序列却未检测到杂交信号。这些结果从不同侧面反映出染色体着染技术是证实微分离、微切割染色体的真实来源及筛选染色体特异性探针的有利工具。建立了可供参考的植物染色体着染实验体系,为染色体微克隆技术在植物中的进一步应用提供了便利。     

A method to recognize distant repeats in protein sequences

An automated algorithm is presented that delineates protein sequence fragments which display similarity. The method incorporates a selection of a number of local nonoverlapping sequence alignments with the highest similarity scores and a graphtheoretical approach to elucidate the consistent start and end points of the fragments comprising one or more ensembles of related subsequences. The procedure allows the simultaneous identification of different types of repeats within one sequence. A multiple alignment of the resulting fragments is performed and a consensus sequence derived from the ensemble(s). Finally, a profile is constructed form the multiple alignment to detect possible and more distant members within the sequence. The method tolerates mutations in the repeats as well as insertions and deletions. The sequence spans between the various repeats or repeat clusters may be of different lengths. The technique has been applied to a number of proteins where the repeating fragments have been derived from information additional to the protein sequences. © 1993 Wiley-Liss, Inc.     

The Primary Structure of an Entamoeba histolyticaβ-Hexosaminidase A Subunit

ABSTRACT. An Entamoeba histolytica gene ( hex-A1 ) that encodes subunit A of the lysosomal enzyme β-hexosaminidase has been cloned and sequenced. The inferred 59 kDa hex-A1 protein has the same molecular weight and 32% amino acid residue identity with the human and mouse proteins and 28% residue identity with the Dictyostelium protein. Northern blot analysis identified a mRNA of approximately 1.6 kb, which is in agreement with the expected size of a mRNA encoding the 522 amino acid hex-A1 protein. Southern blot analysis indicated the presence of at least two β-hexosaminidase A subunit genes.     

Availability of short amino acid sequences in proteins

Much attention is being paid to protein databases as an important information source for proteome research. Although used extensively for similarity searches, protein databases themselves have not fully been characterized. In a systematic attempt to reveal protein-database characters that could contribute to revealing how protein chains are constructed, frequency distributions of all possible combinatorial sets of three, four, and five amino acids ("triplets," "quartets," and "pentats"; collectively called constituent sequences) have been examined in the nonredundant (nr) protein database, demonstrating the existence of nonrandom bias in their "availability" at the population level. Nonexistent short sequences of pentats were found that showed low availability in biological proteins against their expected probabilities of occurrence. Among them, six representative ones were successfully synthesized as peptides with reasonably high yields in a conventional Fmoc method, excluding the possibility that a putative physicochemical energy barrier in forming them could be a direct cause for the low availability. They were also expressed as soluble fusion proteins in a conventional Escherichia coli BL21Star(DE3) system with reasonably high yield, again excluding a possible difficulty in their biological synthesis. Together, these results suggest that information on three-dimensional structures and functions of proteins exists in the context of connections of short constituent sequences, and that proteins are composed of evolutionarily selected constituent sequences, which are reflected in their availability differences in the database. These results may have biological implications for protein structural studies.     

Sequencing and analysis of a genomic fragment provide an insight into the Dunaliella viridis genomic sequence

Dunaliella is a genus of wall-less unicellular eukaryotic green alga.Its exceptional resistancesto salt and various other stresses have made it an ideal model for stress tolerance study.However,very littleis known about its genome and genomic sequences.In this study,we sequenced and analyzed a 29,268 bpgenomic fragment from DunalieIla viridis.The fragment showed low sequence homology to the GenBankdatabase.At the nucleotide level,only a segment with significant sequence homology to 18S rRNA wasfound.The fragment contained six putative genes,but only one gene showed significant homology at theprotein level to GenBank database.The average GC content of this sequence was 51.1%,which was muchlower than that of close related green algae Chlamydomonas (65.7%).Significant segmental duplicationswere found within this fragment.The duplicated sequences accounted for about 35.7% of the entireregion.Large amounts of simple sequence repeats (microsatellites) were found,with strong bias towards(AC)_n type (76%).Analysis of other Dunaliella genomic sequences in the GenBank database (total 25,749bp) was in agreement with these findings.These sequence features made it difficult to sequence Dunaliellagenomic sequences.Further investigation should be made to reveal the biological significance of these uniquesequence features.     

窄叶鲜卑花(Sibiraea angustata)nrDNA ITS和cpDNA trnL-F序列分子进化特点的分析

应用PCR产物直接测序法分析了窄叶鲜卑花居群间nrDNA(核糖体DNA)ITS序列和cpDNA(叶绿体DNA)trnL-F的碱基差异,并与cpDNAtrnS-G序列和rpl20-rps12序列进行比较,从而初步研究两套植物基因组的变异速率。采用改良的CTAB法从硅胶干燥的窄叶鲜卑花叶片中提取总DNA,并对nrDNA ITS和cpDNAtrnL-F区域进行扩增、纯化、测序。nrDNA ITS序列共有601 bp,有变异位点3处,变异位点百分率为0.05%,(G+C)含量为41.4%。cpDNAtrnL-F序列共有927 bp,有变异位点1处,变异位点百分率0.01%,(G+C)含量为32.6%,两种序列的核苷酸多样性非常低。比较发现,窄叶鲜卑花nrDNA ITS区域较cpDNAtrnS-G序列和rpl20-rps12序列保守,变异速率较慢,比cpDNAtrnL-F序列变异速率稍快。通过对ITS序列单倍型(haplotype)进行分析发现,窄叶鲜卑花现有分布范围经历了居群近期范围扩张,与叶绿体基因组(trnS-G和rpl20-rps12序列)得出的结论一致。因此,窄叶鲜卑花nrDNA ITS序列适合该种的谱系地理学研究。     

A systematic search for protein signature sequences.

Signature sequences are contiguous patterns of amino acids 10-50 residues long that are associated with a particular structure or function in proteins. These may be of three types (by our nomenclature): superfamily signatures, remnant homologies, and motifs. We have performed a systematic search through a database of protein sequences to automatically and preferentially find remnant homologies and motifs. This was accomplished in three steps: 1. We generated a nonredundant sequence database. 2. We used BLAST3 (Altschul and Lipman, Proc. Natl. Acad. Sci. U.S.A. 87:5509-5513, 1990) to generate local pairwise and triplet sequence alignments for every protein in the database vs. every other. 3. We selected "interesting" alignments and grouped them into clusters. We find that most of the clusters contain segments from proteins which share a common structure or function. Many of them correspond to signatures previously noted in the literature. We discuss three previously recognized motifs in detail (FAD/NAD-binding, ATP/GTP-binding, and cytochrome b5-like domains) to demonstrate how the alignments generated by our procedure are consistent with previous work and make structural and functional sense. We also discuss two signatures (for N-acetyltransferases and glycerol-phosphate binding) which to our knowledge have not been previously recognized.     

EMBOSS软件包序列分析程序应用实例

本文介绍欧洲分子生物学开放软件包EMBOSS序列分析程序应用实例.第1节简单介绍EMBOSS软件包的概况和基本用法.第2节介绍格式转换、序列提取、序列变换和序列显示等常用序列处理程序.第3节介绍序列比对程序,包括双序列比对、多序列比对和点阵图程序.第4节介绍常用核酸序列分析程序,可用于核苷酸组分统计、开放读码框分析、C...     

基于核糖体基因的鳞钙皮菌种内分子关系分析

为探讨不同地域的鳞钙皮菌Didymium squamulosum种内分子亲缘关系,通过PCR扩增鳞钙皮菌子实体及原质团DNA,得到SSU、ITS1-5.8S-ITS2 rRNA基因区域,并以SSU、5.8S rRNA基因片段构建NJ亲缘关系树。     

二倍体与二基体

DiploidandDihasoidShenShuxiang(NantongAgricultureSchoolofJiangsurovince226007)在作物遗传学的教科书上,二倍体的概念都是这样的：在相物体细胞的染色体数目中,包含着两套染色体,一套来自父本的花粉细胞,另一套来自母本的卵细胞。凡是体细胞中含有步套染色体的植物（2n）统称为二倍体。按照这个定义,普通小麦、陆地棉等与水稻、玉米一样,都是二倍体。而实际上,普通小麦是“六倍体”,陆地棉是“四倍体”、由此可见,上述二倍体的概念是不够确切的．为了弄清利么是二倍体,我们先来了解一厂一倍体的概念、在遗传学的染色体变…