鼠细胞角蛋白Endo B的核苷酸序列分析

本文报导了由编码细胞角蛋白的基因组DNA文库中筛选的克隆的特点,介绍了其亚克隆和核苷酸序列分析.序列分析发现,Endo B基因由七个外显子和六个内含子组成.Endo B 基因家族至少由一个结构基因和一个假基因组成.     

热带爪蟾Hoxa11基因克隆及序列的进化分析

 H oxa1 1基因是控制脊椎动物肢发育的重要基因 .根据人和鼠 H oxa1 1基因外显子 区和 区的保守序列设计了简并引物 ,采用 PCR法从热带爪蟾基因组 DNA中扩增和克隆到了H oxa1 1基因 ,并测定了核苷酸序列 .克隆的热带爪蟾 H oxa1 1基因片段长 1 598bp,由外显子 、内含子和外显子 三部分组成 ,其中外显子 60 4 bp,外显子 49bp.将该片段的核苷酸序列与人、鼠、斑马鱼 H oxa1 1基因的相应区域进行比较 ,发现该基因的内含子长度存在明显差异 .斑马鱼、热带爪蟾、鼠和人的内含子长度分别为 632 bp,945bp,1 42 1 bp和 1 41 2 bp,随动物进化阶梯的提高而变长 .外显子 区则高度保守 ,都是 49bp,外显子 区在长度上呈现约 1 0 %的变异 .将热带爪蟾 H oxa1 1基因编码的氨基酸序列与人、鼠、斑马鱼进行比较 ,它们之间分别有 67.0 %、66.5%和 46.0 %的同源性 .热带爪蟾与哺乳动物的同源性高于鱼类 ,可能反映了脊椎动物从鳍到肢的进化过程中 ,H oxa1 1基因经历了较多的变异 .     

长春花钙调素基因克隆及其假基因的识别

以长春花[Catharanthus roseus(L.)G.Don]叶片cDNA和基因组DNA为模板,利用PCR技术扩增得到了长春花钙调素基因447 bp的全长编码cDNA序列和2个大小不同的DNA片段.序列分析表明,DNA长片段全长1 551 bp,由2个外显子和1个内含子构成,为长春花钙调素基因编码区DNA片段;DNA小片段全长447 bp,与447 bp的长春花钙调素基因cDNA核苷酸一致性高达87%,有56个碱基的差异,其中位于226 bp处的碱基A突变为T,即由AAG突变为终止密码子TAG使翻译提前终止.推测此447 bp的DNA小片段可能为长春花钙调素基因的假基因,命名为CCaMP1.     

银杏叶绿体petD基因的克隆与表达

根据黑松、云杉、菠菜与玉米叶绿体petD基因序列设计引物,以银杏叶绿体基因组DNA为模板,PCR扩增克隆了银杏叶绿体petD基因（GenBank登录号为DQ923066,命名为GbpetD）的序列。序列分析显示,GbpetD基因组DNA序列编码区长1243bp,含1个内含子和2个外显子,其外显子序列编码177个氨基酸。相似性比对显示,该基因编码区序列与云杉、台东苏铁、黑松、莴苣、木薯、北美落叶松的petD基因核苷酸同源性为84％-99％,氨基酸序列同源性为85％-93％。系统进化树分析结果表明GbpetD蛋白质与黑松、北美落叶松、云杉、苏铁等裸子植物的petD蛋白质聚类关系最近。半定量RT—PCR分析表明,GbpetD基因在银杏叶和茎中表达,在叶中表达量最大。     

扩展青霉PF898碱性脂肪酶基因组DNA的克隆及序列分析

扩展青霉 (Penicilliumexpansum)PF898可产生一种具有重要工业生产价值的碱性脂肪酶(PEL) .在通过 3′RACE和 5′RACE获得PEL完整的cDNA序列的基础上 ,通过PCR方法首次克隆了该脂肪酶的完整的基因组DNA序列 (GenBank登录号为AF330 6 35 ) .该脂肪酶DNA全长 14 0 4bp ,包括PEL编码区、3′非翻译区和部分 5′非翻译区基因的序列 .编码区DNA由 1135个碱基组成 ,含有 5个内含子 ,大小分别为 5 8bp、4 7bp、5 0bp、5 6bp和 6 9bp .在已报道的丝状真菌脂肪酶中 ,PEL基因的内含子数量最多 ,而其大小与其它丝状真菌脂肪酶基因的内含子一样 ,均为只有几十个碱基的小内含子 .PCR扩增获得的PLEDNA序列还包括由 195个碱基组成的 3′端非编码区序列 ,74个碱基的部分 5′端非编码区序列 .PELDNA全长序列中的 - 2 4至 - 2 7nt为TATAbox ,终止码TGA下游15 6nt出现AATAAA序列 ,TGA下游 182位出现poly(A)尾 ,为典型的真核基因结构 .同源性序列分析表明 ,PEL与其它真菌来源脂肪酶的基因组DNA序列同源性约为 39%～ 4 9% ,PEL内含子之间或PEL内含子与其它丝状真菌脂肪酶基因的内含子之间的序列同源性约 4 2 %～ 5 7% .     

荠菜LEAFY同源基因的克隆与进化分析

LEAFY同源基因是高等植物花的分生组织分化的重要调节基因。根据已发表的LEAFY同源基因序列保守区设计引物,以荠菜(Capsellabursa-pastoris(L.)Medic.)基因组DNA序列为模板,克隆了一条长2866bp的LEAFY同源基因。序列分析表明,该基因含有3个外显子和2个内含子,外显子编码424个氨基酸组成的多肽。其单个外显子核苷酸序列与拟南芥(Arabidopsisthaliana)LEAFY基因同源性在90%以上,氨基酸序列同源性为86%,而与琴叶拟南芥(Ara-bidopsislyrata)的氨基酸序列同源性高达90%。不同植物物种的LEAFY同源氨基酸序列在C端高度保守,而N端则有较大程度的变异。3个外显子进化速率不同可能是由于所受选择压力不同所致。存在于荠菜CapLFY基因346位上的精氨酸突变可能是造成荠菜两种生态型花期不同的原因。     

家蝇卵黄蛋白基因的克隆与结构序列分析

家蝇卵黄蛋白基因编码的卵黄蛋白是家蝇胚胎发育的重要营养来源 .根据 3种家蝇卵黄蛋白cDNA保守序列设计引物 ,用PCR技术从家蝇基因组DNA中扩增到大小为 76 8bp的mdYP1基因的部分DNA片段 .经地高辛标记成特异性探针 ,从构建的家蝇基因组文库中筛选出一个阳性克隆 ,并从该克隆中分离到大小为 3991bp的mdYP1基因组基因 .序列分析显示 ,该基因组序列含有约1 6kb的 5′ 上游区和 1 0kb的 3′ 下游区 ,编码区由一个 6 1bp的内含子和大小分别为 2 2 2bp和10 2 8bp的 2个外显子组成 .5′ 上游区含有典型的CAAT TATA盒 .     

酵母基因中转录正调控内含子序列特征的统计分析

大量实验研究显示,真核基因的许多内含子具有调控转录的功能,但是对此问题尚缺乏全面的研究.观察一些酵母基因的转录频率与基因的内含子序列后,发现一个值得注意的现象：转录频率高的基因内含子序列一般都比较长,而转录频率低的基因内含子一般都比较短.这提示高效转录基因的较长内含子中可能含有某些增强基因转录的特征性结构.于是选取两组酵母基因的内含子进行详细研究,第一组的基因具有较高的转录频率（>30）,第二组的基因转录频率较低（≤10）.对寡核苷酸（主要是四核苷酸、五核苷酸）的出现频率进行统计比较分析,探测到一批寡核苷酸,它们在第一组内含子中出现的频率显著高于在第二组内含子中出现的频率,同时也显著高于与第一组内含子相邻的外显子中的出现频率.其中一些寡核苷酸与实验研究得到的转录调控元件相同.从这批寡核苷酸在内含子和外显子序列中的分布看,高效转录基因内含子的序列结构确实有利于基因的转录.     

盾叶薯蓣环阿屯醇合酶全长基因的克隆与分析

环阿屯醇合酶(cycloartenol synthase,CAS)是薯蓣皂甙元生物合成途径中的第一个关键酶.以基因组DNA为模板,利用染色体步行和长距离PCR方法首次克隆了盾叶薯蓣CAS全长基因.序列分析比较结果表明,盾叶薯蓣CAS全长基因为7 192 bp,由18个外显子和17个内含子组成.外显子总长为2 280 bp,编码759个氨基酸,最长的外显子为198 bp,最短的为47 bp;内含子总长4 912 bp,最长的内含子为1 551 bp,最短的为68 bp.Southern blot杂交分析表明,CAS基因在盾叶薯蓣基因组中为单拷贝.     

甘蔗SoNIN1基因结构及其内含子信息分析

根据前期克隆得到的So NIN1基因的全长c DNA和部分启动子序列设计引物,应用PCR技术从甘蔗叶片g DNA中克隆So NIN1基因,并利用生物信息学方法分析基因的结构。结果表明,So NIN1基因的DNA序列长4 938 bp,包含6个外显子和5个内含子,Gen Bank登录号KF563902。在该基因5'非翻译区存在一个268 bp内含子序列,同时5个内含子序列中含有与低温、干旱和激素等胁迫响应相关的作用元件,这些可能对So NIN1基因转录表达起调控作用。依据So NIN1蛋白聚类和外显子-内含子基因结构可以将植物NINs分为两类。     

Noncoding DNA, isochores and gene expression: nucleosome formation potential

The nucleosome formation potential of introns, intergenic spacers and exons of human genes is shown here to negatively correlate with among-tissues breadth of gene expression. The nucleosome formation potential is also found to negatively correlate with the GC content of genomic sequences; the slope of regression line is steeper in exons compared with noncoding DNA (introns and intergenic spacers). The correlation with GC content is independent of sequence length; in turn, the nucleosome formation potential of introns and intergenic spacers positively (albeit weakly) correlates with sequence length independently of GC content. These findings help explain the functional significance of the isochores (regions differing in GC content) in the human genome as a result of optimization of genomic structure for epigenetic complexity and support the notion that noncoding DNA is important for orderly chromatin condensation and chromatin-mediated suppression of tissue-specific genes.     

The compositional properties of human genes

Summary The present work represents the first attempt to study in greater detail previously proposed compositional correlations in genomes, based on a body of additional data relating to gene localizations as well as to extended flanking sequences extracted from gene banks. We have investigated the correlations that exist between (1) the GC levels of exons of human genes, and (2) the GC levels of either intergenic sequences or introns associated with the genes under consideration. In both cases, linear relationships with slopes close to unity were found. The similarity of the linear relationships indicates similar GC levels in intergenic sequences and introns located in the same isochores. Moreover, both intergenic sequences and introns showed GC levels 5–10% lower than the corresponding exons. The above findings considerably strengthen the previously drawn conclusion that coding and noncoding sequences (both inter- and intragenic) from the same isochores of the human genome are compositionally correlated. In addition, we find linear correlations between the GC levels of codon positions and of the intergenic sequences or introns associated with the corresponding genes, as well as among the GC levels of codon positions of genes.     

Intergenics: A tool for extraction of intergenicregions

For the past one decade, there has been considerable explosion of interest in searching novel regulatory elements in the intergenic region between the protein coding regions. The microbial genomes are the most exploited in terms of intergenic (noncoding) regions due to its less complexity. We think, the increasing pace of genome sequencing calls for a tool which will be useful for the extraction of intergenic regions. IntergenicS (Intergenic Sequence) is a tool which can extract the intergenic regions of microbial genomes at NCBI. All the unannotated regions between annotated protein coding genes and noncoding RNA genes can be extracted. It also deals with the calculation of GC base composition of the intergenic regions. This will be a useful tool for the analysis of noncoding regions of both bacterial and archael genomes.     

Selection on Accessible Chromatin Regions in Capsella grandiflora

Accurate estimates of genome-wide rates and fitness effects of new mutations are essential for an improved understanding of molecular evolutionary processes. Although eukaryotic genomes generally contain a large noncoding fraction, functional noncoding regions and fitness effects of mutations in such regions are still incompletely characterized. A promising approach to characterize functional noncoding regions relies on identifying accessible chromatin regions (ACRs) tightly associated with regulatory DNA. Here, we applied this approach to identify and estimate selection on ACRs in Capsella grandiflora, a crucifer species ideal for population genomic quantification of selection due to its favorable population demography. We describe a population-wide ACR distribution based on ATAC-seq data for leaf samples of 16 individuals from a natural population. We use population genomic methods to estimate fitness effects and proportions of positively selected fixations (α) in ACRs and find that intergenic ACRs harbor a considerable fraction of weakly deleterious new mutations, as well as a significantly higher proportion of strongly deleterious mutations than comparable inaccessible intergenic regions. ACRs are enriched for expression quantitative trait loci (eQTL) and depleted of transposable element insertions, as expected if intergenic ACRs are under selection because they harbor regulatory regions. By integrating empirical identification of intergenic ACRs with analyses of eQTL and population genomic analyses of selection, we demonstrate that intergenic regulatory regions are an important source of nearly neutral mutations. These results improve our understanding of selection on noncoding regions and the role of nearly neutral mutations for evolutionary processes in outcrossing Brassicaceae species.     

Pattern of selective constraint in C. elegans and C. briggsae genomes.

Similarity between related genomes may carry information on selective constraint in each of them. We analysed patterns of similarity between several homologous regions of Caenorhabditis elegans and C. briggsae genomes. All homologous exons are quite similar. Alignments of introns and of intergenic sequences contain long gaps, segments where similarity is low and close to that between random sequences aligned using the same parameters, and segments of high similarity. Conservative estimates of the fractions of selectively constrained nucleotides are 72%, 17% and 18% for exons, introns and intergenic sequences, respectively. This implies that the total number of constrained nucleotides within non-coding sequences is comparable to that within coding sequences, so that at least one-third of nucleotides in C. elegans and C. briggsae genomes are under strong stabilizing selection.     

Genome-wide analysis of recombination machinery for spliceosomal introns gain

What caused spliceosomal introns gain remains an unsolved problem. To this, defining what spliceosomal introns arise from is critical. Here, the introns density of the genomes is calculated for four species, indicating:(1) sex chromosomes in mammals have lower intron densities, (2) despite that, the proportion of UTRs (untranslated regions) with introns in sex chromosomes is higher than other ones, and (3) AT content of introns is more similar to that of intergenic regions when these regions comprise the majority of a chromosome, and more similar to that of exons, when exons are the majority of the chromosome. On the other hand, introns have been clearly demonstrated to invade genetic sequences in recent times while sex chromosomes evolved from a pair of autosomes within the last 300 millions years. One main difference between sex chromosomes and autosomes in mammalian is that sex chromosomes recombination stopped. Thus, recombination might be the main determinant for eukaryotes gaining spliceosomal introns. To further prove that and avoid giving weak signal, the whole genomes from eight eukaryotic species are analyzed and present strong signal for above the trend (3) in three species (t-test, P = 0.55 for C. elegans, P = 0.72 for D. melanogaster and P = 0.83 for A. thaliana). These results suggest that the genome-wide coincidence as above (3) can only be caused by the large-scale random unequal crossover in eukaryote meiosis, which might have fueled spliceosomal introns but hardly occurred in prokaryotes.