喉癌差异表达cDNA序列的分离与初步鉴定

分离和克隆人喉癌中新的相关基因将有助于提示喉癌的易感性与癌变机制。运用ｍＲＮＡ差异显示法对２例成人喉癌组织及配对癌旁正常组织的基因表达进行研究,分离到３５个差异显示片段;用反向Ｎｏｒｔｈｅｒｎ点杂交筛选到６个差异片段,经克隆、测序和匹配分析,得１２条不同ｃＤＮＡ序列,其中４条为新基因序列,另外８条与已知基因高度同源。将１２条ｃＤＮＡ序列固定在膜上,用来自喉癌和配对正常组织的总ｃＤＮＡ探针与其杂交和     

mRNA差异显示

分离差异表达的基因是研究生命调节过程的重要手段,mRNA差异显示技术是一种能成功分离差异表达基因的方法,文章对该方法的基本原理、方法步骤及其应用作了较为详细的介绍。     

差异表达基因分离技术的研究进展

分离并克隆差异表达基因是生命科学的研究热点.近年来,以差示筛选、扣除杂交等基本方法为基础,先后出现了抑制差减杂交,微阵列技术等多种分析差异表达基因的技术, 使差异表达基因分离方法不断完善.对这些方法的优缺点、发展趋势及应用前景进行了简要综述.     

茶树花粉特异蛋白基因CsPSP1的分离及序列分析

利用cDNA-AFLP技术比较了茶树[Camellia sinensis(L.)O.Kuntze cv.Wulong]花蕾发育早期和晚期的基因表达,结果表明存在明显差异。以E12和M20为引物对在晚期发育花蕾中筛选出一条281 bp特异表达的差异条带TDF53(transcipt-derived-fragment,TDF)。RT-PCR分析表明该片段只在晚期发育花蕾中特异表达。用RACE方法延伸其末端序列,克隆并测序获得全长cDNA序列(GenBank登录号:DQ887753)。该基因全长2079 bp,开放阅读框1701 bp,编码567个氨基酸,其分子量为63 kDa。序列和结构的同源性分析表明:该基因编码的氨基酸序列与烟草、油菜的花粉特异蛋白等同源性较高,由此推定,该基因为编码茶树花粉特异蛋白的基因,并将分离到的花粉特异蛋白基因命名为CsPSP1。     

草甘膦诱导表达的大豆与棉花EST序列的分离

利用差异显示PCR方法分离了63个草甘膦诱导后在大豆和棉花中差异表达的片段,测序分析结果表明属于33个草甘膦诱导的大豆和棉花EST序列。通过在GenBank中进一步比时研究发现:约85％的EST序列与水杨酸、冷、创伤、氧化等非生物胁迫诱导后表达库中的EST序列有高达95％以上的同源性,由此可推测这些基因参与了植物对非生物胁迫的反应过程。草甘膦诱导后高表达EST、序列的获得将有利于进一步分离相关非生物胁迫诱导表达基因及启动子,研究其转录调控的机理,有望建立草甘膦诱导系统。从而解决组成型表达造成外源基因在植物体所有发育阶段和所有组织部位表达,造成植物体能量浪费。     

高粱幼苗水分胁迫诱导表达差异cDNA的研究

以高粱为试验材料,用-0．7MPa的PEG-6000高渗溶液对其幼苗进行水分胁迫处理,利用mRNA差异显示技术分离得到53条高粱水分胁迫诱导表达的cDNA片段,其中包括5个完全诱导表达片段,43个上调片段和5个下调表达片段。经过Reverse Northern验证,筛选出13个差异表达的cDNA片段,并进行克隆测序。经GenBank查询,10个片段序列与已知序列有较高的同源性,3个片段同源性非常低,可能为新基因。     

宏基因组技术在开发未培养环境微生物基因资源中的应用

环境微生物宏基因组是一个巨大的基因资源库,但是仅有0.1%～1%的微生物在现有技术条件下是可培养的,因此致使未培养微生物基因资源的开发利用受到限制.宏基因组技术直接提取环境样品总DNA,避开了微生物分离培养的问题,极大扩展了微生物资源的利用空间,增加了获得新生物活性物质的机会.简要介绍了宏基因组的概念及宏基因组克隆技术的基本操作流程和技术要点,重点阐述了目的基因富集、核酸提取、载体和宿主系统选择、宏基因组文库筛选等"瓶颈"技术的研究进展.目的基因富集技术主要包括稳定同位素探针(SIP)、抑制消减杂交(SSH)和差异显示(DD)等.基因文库筛选分为序列依赖性筛选和非序列依赖性筛选,其中序列依赖性筛选包括特定基因PCR、反转录PCR (RT-PCR)、DNA微阵、亲和捕获等技术;非序列依赖性筛选主要指基于基因表达活性筛选和基因"陷阱"技术等.此外,介绍了一些近年来通过构建宏基因组文库筛选目的基因的应用实例.     

降低mRNA差异显示技术假阳性率的一种方法

为了探讨降低mRNA差异显示技术假阳性率的方法 ,进一步提高此技术的可靠性 ,提取了手术切除肝癌及非癌肝组织成对标本的总RNA ,逆转录获得cDNA片段 ,以mRNA差异显示方法筛选差异表达基因 ,选取较明显的一条差异表达条带 ,行进一步PCR扩增 .分别对PCR产物及其经TA克隆后随机挑选的 6个单克隆质粒DNA进行序列分析 ,并通过GenBank BLAST数据库进行序列的同源性比较 ,以Northern杂交予以来源确认 .自 72 0余条扩增条带中共选出 2 8条差异条带 .序列分析及同源性比较表明 ,所选择条带的PCR产物为一可能的新基因片段 ;而随机选择的 6个TA克隆质粒DNA中 ,有 4个为同一已知基因片段 ,一个为另一已知基因片段 ,一个为一可能的新基因片段 .同源性比较表明 ,PCR产物直接测序所得序列与TA克隆质粒DNA的 6个片段不具同源性 .结果表明 ,mRNA差异显示条带可能由 1条以上分子量相似的片段构成 ,直接对PCR产物行序列分析并以其为探针进行Northern杂交 ,是导致出现假阳性片段的原因之一 .将PCR产物进行TA克隆 ,对单克隆质粒DNA进行序列分析并以其为探针进行Northern杂交 ,可能是解决此问题的一种较好方法 .     

稻瘟菌侵染诱导水稻凝集素基因的表达

利用mRNA差异显示技术(DDRT-PCR),从非亲和性稻瘟菌生理小种131侵染的水稻品种爱知旭(Oryza sati-va L. cv.Aichi-asahi)叶片中分离了8个诱导差异表达的cDNA片段.对这8个差示片段进行了回收、重扩增和克隆,以其中一个长度为321碱基并与甘露糖结合水稻凝集素和水稻盐诱导蛋白基因高度同源的差示片段为探针,筛选水稻非亲和性cDNA文库,获得12个阳性克隆.序列测定和数据库查询表明该基因的cDNA与水稻凝集素基因的cDNA及盐诱导蛋白基因的cDNA核苷酸同源性高达96%,推定的氨基酸序列与甘露糖结合水稻凝集素的氨基酸序列一致,与水稻盐诱导蛋白仅相差2个氨基酸.Southern杂交显示该基因在水稻基因组中有两个同源拷贝数,Northern杂交表明非亲和性稻瘟菌侵染可强烈诱导该基因表达.因此推测该基因参与了水稻对稻瘟菌侵染的防御反应.     

水杨酸诱导的烟草抗性相关序列的分离

通过已分离鉴定的水杨酸诱导烟草‘云烟85’中与抗性相关的差异表达基因,采用差示筛选和反式Northern检测以及序列分析得到94个烟草差异表达的EST序列。经测序及同源性比较,其中87个有同源序列,7个为新序列;有51个与抗性相关,占总序列的54.3%,其中有系统获得抗性蛋白基因和病程相关蛋白基因等。     

玉米核雄性不育的分子机制研究与应用分析

玉米细胞核雄性不育突变体是研究花粉发育和减数分裂的理想材料,也是杂种优势利用的重要种质资源。随着分子生物技术的快速发展,部分玉米细胞核雄性不育基因陆续被成功克隆,为其在工程不育化杂交种生产中的应用奠定了基础。综述了近年来对玉米细胞核雄性不育的细胞学鉴定、基因克隆和分子机制的研究进展,并对其应用途径和前景进行了分析。     

Chromosomal and Genic Barriers to Introgression in Helianthus

The sexual transfer of genes between taxa possessing different structural karyotypes must involve the passage of genes through a chromosomal sterility barrier. Yet little is known about the effects of structural differences on gene introgression within or adjacent to the rearranged chromosomal fragments or about the patterns of introgression in collinear regions. Here, we employ 197 mapped molecular markers to study the effects of chromosomal structural differences on introgression in backcrossed progeny of the domesticated sunflower, Helianthus annuus, and its karyotypically divergent wild relative, H. petiolaris. Forty percent of the genome from the seven collinear linkages introgressed, whereas only 2.4% of the genome from the 10 rearranged linkages was transferred. Thus, chromosomal rearrangements appear to provide an effective mechanism for reducing or eliminating introgression in rearranged chromosomal segments. On the other hand, observations that 60% of the markers from within the collinear portion of the genome did not introgress suggests that genic factors also resist introgression in Helianthus. That is, selection against H. petiolaris genes in concert with linkage may have reduced or eliminated parts of the genome not protected by structural changes. Thus, barriers to introgression in Helianthus appear to include both chromosomal structural and genic factors.     

Assessing the patterns of linkage disequilibrium in genic regions of the human genome

We used the genotyping data generated by the International HapMap Project to study the patterns of linkage disequilibrium (LD) in human genic regions. LD patterns for 11,998 genes from 11 HapMap populations were identified by analyzing the distribution of haplotype blocks. The genes were prioritized using LD levels. The results showed that there were significant differences in the degree of LD between genes. Genes with high or low LD (the upper and lower quartiles of the LD levels) fell into different Gene Ontology functional categories. The high LD genes clustered preferentially in the metabolic process, macromolecule localization and cell-cycle categories, whereas the low LD genes clustered in the developmental process, ion transport, and immune and regulation system categories. Furthermore, we subdivided the genic region into 3'-UTR, 5'-UTR and CDS (coding region), and compared the different LD patterns in these subregions. We found that the LD patterns in low LD genes had a more interspersed block structure compared with the high LD genes. This was especially true in the CDS and 5'-UTR. The extent of LD was somewhat higher in 5'-UTRs compared with 3'-UTRs for both high and low LD genes. In addition, we assessed the overlap for the intragenic LD regions and found that the LD regions in high LD genes were more consistent among populations. Comprehensive information about the distribution of LD patterns in gene regions in populations may provide insights into the evolutionary history of humans and help in the selection of biomarkers for disease association studies.     

Gene-centric characteristics of genome-wide association studies

Background

The high-throughput genotyping chips have contributed greatly to genome-wide association (GWA) studies to identify novel disease susceptibility single nucleotide polymorphisms (SNPs). The high-density chips are designed using two different SNP selection approaches, the direct gene-centric approach, and the indirect quasi-random SNPs or linkage disequilibrium (LD)-based tagSNPs approaches. Although all these approaches can provide high genome coverage and ascertain variants in genes, it is not clear to which extent these approaches could capture the common genic variants. It is also important to characterize and compare the differences between these approaches.

Methodology/Principal Findings

In our study, by using both the Phase II HapMap data and the disease variants extracted from OMIM, a gene-centric evaluation was first performed to evaluate the ability of the approaches in capturing the disease variants in Caucasian population. Then the distribution patterns of SNPs were also characterized in genic regions, evolutionarily conserved introns and nongenic regions, ontologies and pathways. The results show that, no mater which SNP selection approach is used, the current high-density SNP chips provide very high coverage in genic regions and can capture most of known common disease variants under HapMap frame. The results also show that the differences between the direct and the indirect approaches are relatively small. Both have similar SNP distribution patterns in these gene-centric characteristics.

Conclusions/Significance

This study suggests that the indirect approaches not only have the advantage of high coverage but also are useful for studies focusing on various functional SNPs either in genes or in the conserved regions that the direct approach supports. The study and the annotation of characteristics will be helpful for designing and analyzing GWA studies that aim to identify genetic risk factors involved in common diseases, especially variants in genes and conserved regions.     

DNA rearrangement in orthologous orp regions of the maize, rice and sorghum genomes

The homeologous Orp1 and Orp2 regions of maize and the orthologous regions in sorghum and rice were compared by generating sequence data for >486 kb of genomic DNA. At least three genic rearrangements differentiate the maize Orp1 and Orp2 segments, including an insertion of a single gene and two deletions that removed one gene each, while no genic rearrangements were detected in the maize Orp2 region relative to sorghum. Extended comparison of the orthologous Orp regions of sorghum and japonica rice uncovered numerous genic rearrangements and the presence of a transposon-rich region in rice. Only 11 of 27 genes (40%) are arranged in the same order and orientation between sorghum and rice. Of the 8 genes that are uniquely present in the sorghum region, 4 were found to have single-copy homologs in both rice and Arabidopsis, but none of these genes are located near each other, indicating frequent gene movement. Further comparison of the Orp segments from two rice subspecies, japonica and indica, revealed that the transposon-rich region is both an ancient and current hotspot for retrotransposon accumulation and genic rearrangement. We also identify unequal gene conversion as a mechanism for maize retrotransposon rearrangement.     

Immobilization of Escherichia coli cells with penicillin-amidohydrolase activity on solid polymeric carriers

Whole cells of Escherichia coli containing the enzyme penicillinamidohydrolase EC 3.5.1.11 were immobilized on the surface of modified macroporous copolymers of glycidylmethacrylate with ethylenedimethacrylate and of copolymers of methacrylaldehyde (MA) with divinylbenzene (DVB) by means of glutaraldehyde. These polymeric carriers were modified before cell binding by using ammonia or polyamines, especially ethylenediamine and hexamethylenediamine (HMDA). The highest specific activity and the largest yield in cell immobilization were achieved with the macroporous copolymer of MA and DVB modified with HMDA. The material thus obtained was used in repeated conversions of benzylpenicillin to 6-aminopenicillanic acid in a stirred batch reactor.     

Epistasis and hybrid sterility in Saccharomyces

Hybrid sterility is thought to be due to deleterious epistatic interactions between genes from different species. Here we demonstrate that dominant genic incompatibility does not contribute to sterility in hybrids between Saccharomyces cerevisiae and five closely related species. Sterile diploids were made fertile by genome doubling to produce hybrid tetraploids. Based on these and previous results, we conclude that neither genic incompatibility nor classical chromosomal speciation models apply.     

Hybridization monitor: a method for identifying differences between complex genomes

We have developed a method to identify and amplify differential fragments between two complex genomes. This technique, named hybridization-monitored genome differential analysis (HMDA), incorporates a monitor system into a PCR-based solid subtraction hybridization that tracks the entire hybridization process. This is achieved by monitoring the subtraction progress using PCR analysis of the conserved sequence of 18S rDNA in the tester sample after each round of subtraction. Homologous fragments can then be eliminated when bound to the driver DNA immobilized on a solid membrane. The hybridization continues until the conserved DNA sequence of 18S rDNA can no longer be detected, and most of the unbound DNA fragments left in the liquid were mainly the tester-specific fragments, thus greatly decreasing the complexity of DNA template of PCR amplification, increasing the amplification efficiency of differences accordingly, and ensuring high positive efficiency and coverage across the tester genome. We have applied the technique in a comparison between the genomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe, which are two completely sequenced organisms. Results indicated that 95% of the subtracted clones have been confirmed to be different to the driver analyzed using the BLASTN homology alignment. With this technique, 240-fold enrichment of differences is obtained, and the coverage of the difference is up to 79%. These results indicate that HMDA can efficiently identify sequences that differ between two complex genomes.     

HETEROGENEOUS GENOMIC DIFFERENTIATION IN MARINE THREESPINE STICKLEBACKS: ADAPTATION ALONG AN ENVIRONMENTAL GRADIENT

Evolutionary divergence among populations occupying ecologically distinct environments can occur even in the face of on‐going gene flow. However, the genetic underpinnings, as well as the scale and magnitude at which this differentiation occurs in marine habitats are not well understood. We investigated the patterns and degree of genomic heterogeneity in threespine sticklebacks (Gasterosteus aculeatus) by assessing genetic variability in 20 nongenic and 20 genic (associated with genes important for freshwater adaptation) microsatellite loci in samples collected from 38 locations spanning the entire Baltic Sea coast to the North Sea boundary. Population divergence (F_ST ≈ 0.026) and structuring (five genetic clusters) was significantly more pronounced in the genic as compared to nongenic markers (F_ST ≈ 0.008; no genetic clusters). Patterns of divergence in the genic markers—45% of which were identified as outliers—correlated with local differences in salinity. Yet, a strong positive correlation between divergence in genic and nongenic markers, and their association with environmental factors suggests that adaptive divergence is reducing gene flow across the genome. Apart from providing a clear demonstration of heterogeneous genomic patterns of differentiation in a marine species, the results are indicative of adaptive population structuring across the relatively young Baltic Sea in spite of ample opportunities for gene flow.     

Gene Conversion Drives Within Genic Sequences: Concerted Evolution of Ribosomal RNA Genes in Bacteria and Archaea

Multiple copies of a given ribosomal RNA gene family undergo concerted evolution such that sequences of all gene copies are virtually identical within a species although they diverge normally between species. In eukaryotes, gene conversion and unequal crossing over are the proposed mechanisms for concerted evolution of tandemly repeated sequences, whereas dispersed genes are homogenized by gene conversion. However, the homogenization mechanisms for multiple-copy, normally dispersed, prokaryotic rRNA genes are not well understood. Here we compared the sequences of multiple paralogous rRNA genes within a genome in 12 prokaryotic organisms that have multiple copies of the rRNA genes. Within a genome, putative sequence conversion tracts were found throughout the entire length of each individual rRNA genes and their immediate flanks. Individual conversion events convert only a short sequence tract, and the conversion partners can be any paralogous genes within the genome. Interestingly, the genic sequences undergo much slower divergence than their flanking sequences. Moreover, genomic context and operon organization do not affect rRNA gene homogenization. Thus, gene conversion underlies concerted evolution of bacterial rRNA genes, which normally occurs within genic sequences, and homogenization of flanking regions may result from co-conversion with the genic sequence. Received: 31 March 2000 / Accepted: 15 June 2000