人类1号染色体可变剪接与普通剪接基因同义密码子的使用分析I.同义密码子偏爱使用分析

人类1号染色体可变剪接(选择性剪接)基因344非冗余蛋白质编码序列(188183密码子)和普通剪接(非可变剪接)基因的386蛋白质编码序列(223116密码子)被用于研究人类密码子使用偏爱模式.全部密码子使用数据分析表明,人类可变剪接基因密码子的偏爱水平显著高于普通剪接基因.在人类1号染色体基因中,密码子第三位置的G C含量有很大的异质性(0.24～0.95),并且可变剪接基因密码子第三位置平均G C含量(64.66%)大于普通剪接基因(59.97%).Nc值对GC3s图显示密码子偏爱使用除了受核苷酸组成制约外,其它的因子可能也影响密码子的使用变化.此外,可变剪接基因中以G 或C结尾的密码子比普通剪接基因出现的频率高.密码子使用的差异可能是由可变剪接基因pre-mRNA特有的结构特征和多种剪接模式决定的.     

植物功能基因选择性剪接研究进展

选择性剪接是基因表达调控的重要机制,在植物发育、抗病和应对环境胁迫等方面起着重要的作用。近年来,新一代测序技术在植物基因组和转录组测序领域得到广泛应用,植物选择性剪接研究取得了一些新进展。本文就此进行了综述,包括选择性剪接占植物功能基因的比例、选择性剪接的类型及其调控机制等内容,并提出了今后植物上的研究重点应放在选择性剪接的功能方面。     

人类可变剪接基因上游非编码区结构特征分析

选择性剪接作为一种真核生物基因重要的表达调控机制备受关注,无论是剪接复合物的构成、剪接位点的选择以及本身的调控机制等都是当今研究的热点。然而对选择性剪接基因本身的转录水平调控却关注较少,这类基因是否存在特异的转录调控仍属未知。通过对3797个实验验证的人类选择性剪接基因的上游非编码区（主要是启动子区）识别提取,进而对其结构特征进行了分析。所得结果为进一步诠释选择性剪接基因非编码区功能提供基础,对揭示此类基因本身的转录调控机制具有重要的意义。     

应用软件Fgenesh预测水稻基因

 【目的】为生物基因组序列注释提供一定科学依据,加快序列注释进度和精确性。【方法】以粳稻日本晴（Oryza sativa L. ssp. japonica cv. Nipponbare）较长的6号染色体的序列为例,采用生物信息学手段,详细探索了Fgenesh（v2.0）对单子叶模式植物水稻基因的预测。【结果】预测的基因数涵盖了注释的基因数且两者相差不大;预测的基因以多外显子基因为主,占总基因的77.52%;预测基因的长度变化幅度很大;从显著匹配数上来看,Fgenesh对多外显子基因预测准确性较高,其中TIGR注释支持度达到100%,cDNA的支持度也在78%以上;从Fgenesh对多外显子基因不同位置的外显子预测来看,居间外显子和末端外显子的cDNA支持度较高;高支持度的多外显子基因中居间外显子的长度较短,而起始外显子和末端外显子较长;高支持度的单外显子基因长度多数较短;从外显子数目上看,高支持度多外显子基因的外显子数目主要集中在5以下。【结论】Fgenesh对水稻基因的预测有较高的准确性,但需要将预测结果与cDNA数据库进行序列比对,根据cDNA的支持情况对预测结果做必要的修订。     

肌肉生长发育可变剪接研究进展

为了解动物肌肉生长发育可变剪接调控过程最新研究,本文对近年来人、小鼠和其他多种动物肌肉生长发育的可变剪接调控的相关研究进行了整理与分析,总结了肌肉生长发育可变剪接的发生、调控蛋白多样性的分子机制、人和动物的肌肉发育过程中可变剪接的调控研究进展及可变剪接的数据量化方法.结果 表明:肌肉是表现出最高水平的组织特异性和保守可...     

The Prediction of Rice Gene by Fgenesh

This study has been carried out to give some scientific reasons for genome annotation, shorten the annotating time, and improve the results of gene prediction. Taking the sequence of the 6th chromosome, which has more length sequences than others, of Oryza sativa L. ssp. japonica cv. Nipponbare as analysis data in this research, the gene prediction of monocots module, rice, has been done by using Fgenesh ver. 2.0, and the predicting results have been explored particularly by bioinformatics methods. Results showed that the number of predicted genes for this chromosome was very close to the number of TIGR annotated genes. The majority of the predicted genes were multi-exon genes which had a percentage of 77.52. Length range was very big in the predicted genes. According to the significant match number, multi-exon genes can be predicted more veracity than single exon genes and the support can be reached up to 100% by TIGR annotation and up to 78% by cDNA. From the angle of predicted exons location of multi-exon genes, the internal exons and last exons had a high support of cDNA. The length of internal exons was relatively short in high （〉95% length, 〉78% similarity） cDNA and/or TIGR annotation support multi-exon genes, but the first exons and last exons were on the reverse. The majority of single exon genes which had more than 95% in length, and 78% in similarity support by cDNA and/or TIGR annotation was relatively short in length. From the angle of exon number, the majority of the multi-exon genes of high （〉 95% length, 〉 78% similarity） cDNA and/or TIGR annotation support had no more than 5 exon number. It was concluded that the rice gene prediction by Fgenesh was very good but needed modification manually to some extent according to cDNA support after aligning the predicting sequence of genes with cDNA database of rice.     

Predictive identification of exonic splicing enhancers in human genes

Specific short oligonucleotide sequences that enhance pre-mRNA splicing when present in exons, termed exonic splicing enhancers (ESEs), play important roles in constitutive and alternative splicing. A computational method, RESCUE-ESE, was developed that predicts which sequences have ESE activity by statistical analysis of exon-intron and splice site composition. When large data sets of human gene sequences were used, this method identified 10 predicted ESE motifs. Representatives of all 10 motifs were found to display enhancer activity in vivo, whereas point mutants of these sequences exhibited sharply reduced activity. The motifs identified enable prediction of the splicing phenotypes of exonic mutations in human genes.     

The snoRNA HBII-52 regulates alternative splicing of the serotonin receptor 2C

The Prader-Willi syndrome is a congenital disease that is caused by the loss of paternal gene expression from a maternally imprinted region on chromosome 15. This region contains a small nucleolar RNA (snoRNA), HBII-52, that exhibits sequence complementarity to the alternatively spliced exon Vb of the serotonin receptor 5-HT(2C)R. We found that HBII-52 regulates alternative splicing of 5-HT(2C)R by binding to a silencing element in exon Vb. Prader-Willi syndrome patients do not express HBII-52. They have different 5-HT(2C)R messenger RNA (mRNA) isoforms than healthy individuals. Our results show that a snoRNA regulates the processing of an mRNA expressed from a gene located on a different chromosome, and the results indicate that a defect in pre-mRNA processing contributes to the Prader-Willi syndrome.     

Regulated splicing of the Drosophila P transposable element third intron in vitro: somatic repression

In eukaryotic cells alternative splicing of messenger RNA precursors (pre-mRNA's) is a means of regulating gene expression. Although a number of the components that participate in regulating some alternative splicing events have been identified by molecular genetic procedures, the elucidation of the biochemical mechanisms governing alternative splicing requires in vitro reaction systems. The tissue specificity of P element transposition in Drosophila depends on the germline restriction of pre-mRNA splicing of the P element third intron (IVS3). Drosophila P element IVS3 pre-mRNA substrates were spliced accurately in vitro in heterologous human cell extracts but not in Drosophila somatic cell splicing extracts. Components in Drosophila somatic cell extracts that specifically inhibited IVS3 splicing in vitro were detected by a complementation assay. Biochemical assays for Drosophila RNA binding proteins were then used to detect a 97-kilodalton protein that interacts specifically with 5' exon sequences previously implicated in the control of IVS3 splicing in vivo. Inhibition of IVS3 splicing in vitro could be correlated with binding of the 97-kD protein to 5' exon sequences, suggesting that one aspect of IVS3 tissue-specific splicing involves somatic repression by specific RNA-protein interactions.     

苹果蠹蛾几丁质脱乙酰基酶2的基因克隆、表达模式和分子特性

【目的】研究苹果蠹蛾（Cydia pomonella）几丁质脱乙酰基酶2（chitin deacetylase 2,CDA2）的分子特性和表达模式,为新型杀虫剂靶标筛选提供理论依据。【方法】基于苹果蠹蛾转录组和基因组数据,通过同源比对获得苹果蠹蛾CDA2的相关信息;新鉴定的CpCDA2a和CpCDA2b与其他昆虫中已鉴定的CDA2氨基酸序列进行多序列比对,并采用MEGA7软件邻接法（neighbor-joining,NJ）构建系统发育树;通过生物信息软件预测CpCDA2a和CpCDA2b两个剪切体的蛋白功能域和三维结构并分析其差异,并通过在线软件对其分子量、等电点和亲/疏水性等蛋白理化性质及糖基化修饰位点等方面进行分析比较;采用荧光定量PCR检测CpCDA2a和CpCDA2b两个剪切体在苹果蠹蛾不同发育时期及其幼虫不同组织的表达情况,以及注射蜕皮激素后其表达量的变化趋势。【结果】获得了苹果蠹蛾CDA2的两个可变剪切体CpCDA2a和CpCDA2b的全长CDS序列及其内含子和外显子位置信息;分析发现两个剪切体的蛋白序列均具有信号肽、几丁质结合域（ChBD）、低密度脂蛋白受体域（LDLa）和几丁质脱乙酰基催化域（CDA）等功能域。多序列比对和聚类分析结果表明,不同昆虫间CDA2差异剪切类型非常类似,相同目昆虫的序列以较高的置信度聚为一支。三维结构模拟与比较发现两个剪切体的ChBD功能域在结构上存在较大差异;理化性质分析显示两个剪切体的亲/疏水性和糖基化位点也存在差异。不同发育时期表达量分析表明,CpCDA2a主要在幼虫期高表达,CpCDA2b主要在蛹的前期和中期高表达,而且在幼虫蜕皮前和蜕皮后CpCDA2a和CpCDA2b表达量均会显著上调;不同组织的表达量分析表明,CpCDA2a和CpCDA2b均在表皮中表达量最高,而CpCDA2b在头部的表达量也较高。对注射蜕皮激素后幼虫中CpCDA2a和CpCDA2b表达量分析显示,与对照相比在注射蜕皮激素后CpCDA2a的表达量在24 h和48 h均有所上调,但差异不显著,而CpCDA2b在24 h和48 h均显著上调。【结论】苹果蠹蛾CDA2的转录本存在两个可变剪切体CpCDA2a和CpCDA2b,根据对其分子特性、发育阶段和组织表达谱、以及注射蜕皮激素后表达动态等方面的综合分析,推测两者均参与苹果蠹蛾蜕皮和新表皮形成过程,但由于可变剪切的序列差异导致的蛋白结构和理化性质上的差异造成了两者在功能上的分化。     

内含子的识别和选择性剪切

真核生物内含子的一个显著特征是在很多生物中其5'和3'剪切位点的基本序列都具有相对很高的保守性,内含子从mRNA前体转录产物中的去除和伴随的外显子的连接称作mRNA前体的剪切,它是构成真核基因表达和基因调控水平的一个重要方面。这个过程由许多具有有限序列和特殊空间结构的顺式作用元件控制,由被称为剪切体的核糖核蛋白复合体来执行。以内含子的识别和由于识别造成的选择性剪切进行了综述,试图去理解造成选择性剪切的分子机理。     

烟草PR3b转录后剪切元件NRSE1与GUS融合表达后的可变剪切

【目的】烟草（Nicotiana tabacum L.）碱性几丁质酶基因PR3b在低烟碱突变体（nic1和nic2）中存在转录后mRNA可变剪切现象,但其可变剪切的发生机制仍不清楚。将PR3b的可变剪切元件NRSE1（nicotine- synthesis related splicing element 1）与GUS融合表达,分析NRSE1元件的独立可变剪切特性,以揭示其作用机制。【方法】利用PCR扩增方法获得PR3b cDNA序列中的NRSE1元件片段,并利用基因重组技术构建了烟草PR3b可变剪切元件NRSE1与GUS的融合表达载体。将融合表达载体导入农杆菌LBA4404后,通过农杆菌介导的叶盘转化法培育了表达NRSE1与GUS融合子的低烟碱突变体nic1和nic2及野生型烟草转基因植株;通过RT-PCR检测及GUS染色鉴定出阳性植株后,利用RT-PCR分析NRSE1与GUS融合表达后在低烟碱突变体和野生型烟草中的可变剪切特性;对转基因植株的幼苗进行乙烯（ET）和茉莉酸（JA）处理,通过GUS染色方法分析ET和JA处理对转基因植株中GUS活性的影响,并通过RT-PCR方法分析ET和JA处理对转基因植株中NRSE1与GUS融合子的可变剪切特性影响,以及对转基因植株中NRSE1与GUS融合子表达水平的影响。【结果】通过RT-PCR检测及GUS染色鉴定出表达NRSE1元件与GUS融合子的低烟碱突变体和野生型烟草转基因植株;RT-PCR检测及测序分析证明,NRSE1元件与GUS融合表达后仍能在低烟碱突变体发生高水平的可变剪切,剪切修饰区段的序列变化与烟草中PR3b的mRNA可变剪切修饰一致;利用ET和JA处理转基因植株进行的GUS染色表明,ET和JA处理对转基因植株的GUS活性有不同程度的影响;但利用ET和JA处理转基因植株进行的RT-PCR分析表明,ET和JA处理不改变NRSE1元件原有的诱导剪切特性,也不影响转基因植株中NRSE1元件与GUS融合子的表达水平。【结论】PR3b的可变剪切元件NRSE1与GUS在烟草中融合表达后,仍能在低烟碱突变体nic1和nic2中发生高水平的可变剪切;NRSE1在烟草中的可变剪切不依赖PR3b的其他mRNA区段,是烟草PR3b发生可变剪切的独立元件;ET和JA处理对NRSE1元件与GUS融合表达植株的GUS活性具有一定影响,可能存在翻译水平的调控作用。     

Mutations in U4atac snRNA, a component of the minor spliceosome, in the developmental disorder MOPD I

Small nuclear RNAs (snRNAs) are essential factors in messenger RNA splicing. By means of homozygosity mapping and deep sequencing, we show that a gene encoding U4atac snRNA, a component of the minor U12-dependent spliceosome, is mutated in individuals with microcephalic osteodysplastic primordial dwarfism type I (MOPD I), a severe developmental disorder characterized by extreme intrauterine growth retardation and multiple organ abnormalities. Functional assays showed that mutations (30G>A, 51G>A, 55G>A, and 111G>A) associated with MOPD I cause defective U12-dependent splicing. Endogenous U12-dependent but not U2-dependent introns were found to be poorly spliced in MOPD I patient fibroblast cells. The introduction of wild-type U4atac snRNA into MOPD I cells enhanced U12-dependent splicing. These results illustrate the critical role of minor intron splicing in human development.     

HTLV x-gene product: requirement for the env methionine initiation codon

The human T-cell leukemia viruses (HTLV) are replication-competent retroviruses whose genomes contain gag, pol, and env genes as well as a fourth gene, termed x, which is believed to be the transforming gene of HTLV. The product of the x gene is now shown to be encoded by a 2.1-kilobase messenger RNA derived by splicing of at least two introns. By means of S1 nuclease mapping of this RNA and nucleic acid sequence analysis of a complementary DNA clone, the complete primary structure of the x-gene product has been determined. It is encoded by sequences containing the env initiation codon and one nucleotide of the next codon spliced to the major open reading frame of the HTLV-I and HTLV-II x gene.