人类基因组盒式外显子和内含子保留的可变剪接位点预测

信使RNA的可变剪接是真核生物有别于原核生物的基本特征之一,信使RNA前体的可变剪接极大地丰富了高等真核生物蛋白质的多样性,并与生物体的组织特异性密切相关。文章对人类盒式外显子和内含子保留的一些基本特征进行了统计;根据剪接位点附近的单碱基、碱基二联体和三联体的保守性等特征,利用基于多样性指标的二次判别法,对盒式外显子和内含子保留的供体端和受体端可变剪接位点进行了预测。交叉检验结果表明,盒式外显子供体端和受体端的识别精度分别达到93％、84％以上的水平;内含子保留供体端和受体端的识别精度分别达到89％、81％以上的水平。     

细菌Ⅱ组内含子（GroupⅡintron）的转移机制

摘要: 约14年前在真核生物中发现的Ⅱ组内含子不仅具有催化功能,而且是可移动的逆转录元件。近年来研究发现细菌中也有可移动的Ⅱ组内含子,它们能够逆转录归巢进入同源无内含子的等位基因位点或者逆转录转座进入非等位基因位点。本文试图从细菌Ⅱ组内含子编码蛋白的活性功能域的组成与Ⅱ组内含子转移发生途径之间的联系,综述已知细菌Ⅱ组内含子主要的移动机制。同时,依据作者多年来研究海洋蓝细菌Ⅱ组内含子编码蛋白对Ⅱ组内含子剪接机理的结果,探讨了海洋蓝细菌Ⅱ组内含子是否会发生转移以及可能的转移方式,探讨了Ⅱ组内含子在生物基因组中发生转移的生物学意义。     

核酸-蛋白质结合能在剪切位点识别中的应用

把最大信息原理应用到核酸序列的保守位点分析中。利用最大信息原理,推导出了核酸和蛋白质特异性结合时的结合能表达式,并且估计了和蛋白质发生相互作用的核酸序列上的位点范围。为了检验此理论是否较为成功地反映了核酸和蛋白质结合时的实际情况,把它应用到基因内含子剪切位点的识别中,识别结果达到了较高的敏感性和特异性,这说明利用最大信息原理推导结合能表达式及估计核酸序列上参与反应的位点范围的理论是较为成功的。此研究结果一方面有助于核酸和蛋白质相互作用的理解,另一方面,也有助于和蛋白质发生相互作用的各种核酸序列的计算机识别研究。     

西藏小型猪H-FABP基因的PCR-RFLP研究

目的研究西藏小型猪心脏脂肪酸结合蛋白（H-FABP）基因5’-上游区和第二内含子内的遗传变异。方法应用PCR-RFLP技术测定30头西藏小型猪H-FABP的基因型。结果（1）在5’-上游区的Hinf I-RFLP位点上,西藏小型猪表现出多态性,等位基因h的频率为0．80;（2）在在第二内含子内的Hae Ⅲ-RFLP位点上,西藏小型猪均为DD纯合子;（3）在第二内含子内的Hinf I＊-RFLP位点上,除一头猪表现为bb基因型外,其余猪都表现为BB基因型,等位基因B的频率为0．97;（4）除Hae Ⅲ-RFLP位点外,在其余位点上西藏小型猪均处于Hardy-weinberg不平衡状态。（5）在Hinf I-RFLP中,西藏小型猪表现为中度多态性（0．25〈PIC〈0．5）,而在其他位点的RFLP中表现为低度多态（PIC〈0．25）。结论可以利用西藏小型猪H-FABP基因5’-上游区的多态遗传标记来分析其与肌内脂肪的关系。     

蛹虫草线粒体基因型快速检测体系的构建及线粒体遗传稳定性的初步研究

本研究的目的是建立一种快速确定蛹虫草菌株线粒体基因型的技术体系,并探讨蛹虫草连续传代培养后线粒体的遗传稳定性。从已知线粒体基因组的蛹虫草菌株中扩增线粒体内含子位点,将扩增产物混合并制作出两套DNA分子量标准,即在8个内含子位点分别具有内含子的8条扩增条带组成的M-I和在6个内含子位点分别缺失内含子的6条扩增条带组成的M-II。从待检测的蛹虫草菌株（包括3个已知和2个未知线粒体基因组的菌株）中扩增同样的（假定）内含子位点,然后通过琼脂糖凝胶电泳分别与制备好的两个DNA分子量标准进行比较,能够准确判断蛹虫草菌株的线粒体内含子分布模式,从而验证了所构建的线粒体基因型快速检测体系的有效性。选择10个蛹虫草组织分离菌株和8个单分生孢子菌株连续转接培养15代,没有发现线粒体内含子分布模式发生改变。本研究成功构建了快速检测蛹虫草线粒体基因型的技术体系,并发现蛹虫草线粒体具有很高的遗传稳定性,为开展蛹虫草线粒体遗传规律的研究奠定了基础。     

真核生物基因组长内含子递归剪接事件的分子机制

在高等真核生物基因组转录过程中,一次剪接可完成短内含子的去除,而较长内含子(10 kb)则需通过多次剪接方可去除。多次剪接去除长内含子的过程通常被称为递归性剪接。已有研究表明,递归性剪接事件与诸多生物学过程及疾病的发生发展有着密切的联系。近年来,关于递归性剪接的研究越来越多,研究者已经在果蝇(Drosophila)和多种脊椎动物基因组转录过程中发现了递归剪接事件,通过不同的生物信息学方法找到了多个递归剪接位点并进行了实验验证。目前国际上对递归性剪接的研究主要集中在递归剪接过程、剪接位点识别及其对生物学过程的影响等方面。本文针对真核生物基因组转录过程中递归剪接事件的分子机制和国内外研究现状进行了综述,旨在为深入理解RNA剪接过程分子机制提供参考。     

基于RNA-Seq数据识别果蝇剪接位点和可变剪接事件

完整基因结构的预测是当前生命科学研究的一个重要基础课题,其中一个关键环节是剪接位点和各种可变剪接事件的精确识别.基于转录组测序（RNA-seq）数据,识别剪接位点和可变剪接事件是近几年随着新一代测序技术发展起来的新技术策略和方法.本工作基于黑腹果蝇睾丸RNA-seq数据,使用TopHat软件成功识别出39718个果蝇剪接位点,其中有10584个新剪接位点.同时,基于剪接位点的不同组合,针对各类型可变剪接特征开发出计算识别算法,成功识别了8477个可变剪接事件（其中新识别的可变剪接事件3922个）,包括可变供体位点、可变受体位点、内含子保留和外显子缺失4种类型.RT-PCR实验验证了2个果蝇基因上新识别的可变剪接事件,发现了全新的剪接异构体.进一步表明,RNA-seq数据可有效应用于识别剪接位点和可变剪接事件,为深入揭示剪接机制及可变剪接生物学功能提供新思路和新手段.     

AT—AC内含子及其剪接机理的研究进展

ＡＴ┐ＡＣ内含子及其剪接机理的研究进展滕胜明镇寰（杭州大学生命科学学院,杭州３１００１２）关键词ＡＴ－ＡＣ内含子剪接机理最近发现了一种新类型的内含子,它在ｍＲＮＡ前体中存在的比例不到０．１％,其剪接位点高度保守的双核苷酸为ＡＴ和ＡＣ（ＡＴ－ＡＣ内含子...     

多样性指标用于基因中剪切位点的识别

根据基因剪切位点处的碱基保守性特征,和附近位点的碱基组成和关联特征,应用多样性指标和二次判别分析,对几类模式生物的基因结构进行统一的分析和预测,能够较好地识别外显子和内含子及其边界.计算结果表明,对于4类物种,线虫(C.elegans),拟南芥(A.thaliana), 果蝇(D.melanogaster)和人类(human),核苷酸水平的识别精度为92.5%～97.1%,外显子水平的识别敏感性为83.7%～94.5%,特异性为87.8%～97.1%.预测能力优于GeneSplicer等剪切位点检测软件.     

Prediction of human mRNA donor and acceptor sites from the DNA sequence

Artificial neural networks have been applied to the prediction of splice site location in human pre-mRNA. A joint prediction scheme where prediction of transition regions between introns and exons regulates a cutoff level for splice site assignment was able to predict splice site locations with confidence levels far better than previously reported in the literature. The problem of predicting donor and acceptor sites in human genes is hampered by the presence of numerous amounts of false positives: here, the distribution of these false splice sites is examined and linked to a possible scenario for the splicing mechanism in vivo. When the presented method detects 95% of the true donor and acceptor sites, it makes less than 0.1% false donor site assignments and less than 0.4% false acceptor site assignments. For the large data set used in this study, this means that on average there are one and a half false donor sites per true donor site and six false acceptor sites per true acceptor site. With the joint assignment method, more than a fifth of the true donor sites and around one fourth of the true acceptor sites could be detected without accompaniment of any false positive predictions. Highly confident splice sites could not be isolated with a widely used weight matrix method or by separate splice site networks. A complementary relation between the confidence levels of the coding/non-coding and the separate splice site networks was observed, with many weak splice sites having sharp transitions in the coding/non-coding signal and many stronger splice sites having more ill-defined transitions between coding and non-coding.     

人类基因组中可变和组成性剪接位点的预测

根据剪接位点的核酸序列保守特征,以及邻近位点的碱基组成和关联特性,结合一对可变剪接位点之间的距离参数和受体端剪接位点前30位碱基的GC和TC含量,利用结合多样性指标的二次判别方法（IDQD）,预测了人类基因组中可变和组成性内含子的供体端和受体端的剪接位点,对可变的供体端和受体端剪接位点,阈值ξ选择－2时,总的预测精度分别为87．9％和89．9％,对组成性的供体端和受体端剪接位点,阈值ξ选择－1,总的预测精度分别为92．8％和94.3％．     

A clean data set of EST-confirmed splice sites from Homo sapiens and standards for clean-up procedures.

A clean data set of verified splice sites from Homo sapiens are reported as well as the standards used for the clean-up procedure. The sites were validated by: (i) standard cleaning procedures such as requiring consistency in the annotation of the gene structural elements, completeness of the coding regions and elimination of redundant sequences; (ii) clustering by decision trees coupled with analysis of ClustalW alignments of the translated protein sequence with homologous proteins from SWISS-PROT; (iii) matching against human EST sequences. The sites are categorised as: (i) donor sites, a set of 619 EST-confirmed donor sites, for which 138 are either the sites or the regions around the sites involved in alternative splice events; (ii) acceptor sites, a set of 623 EST-confirmed acceptor sites, for which 144 are either the sites or the regions around the sites are involved in alternative splice events; (iii) genuine splice sites, a set of 392 splice sites wherein both the donor and acceptor sites had EST confirmation and were not involved in any alternative splicing; (iv) alternative splice sites, a set of 209 splice sites wherein both the donor and acceptor sites had EST confirmation and the sites or the regions around them were involved in alternative splicing. A set of nucleotide regions that can be used to generate a control set of false splice sites that have a high confidence of being non-functional are also reported.     

Distribution of exonic splicing enhancer elements in human genes

Ab initio prediction of functional exon splicing enhancer (ESE) elements based on RNA sequences present a challenge in the evaluation of the functional impacts of human genetic polymorphisms on splicing. To better understand the behavior of ESEs, we studied their distribution in human exons and introns for four known SR protein-binding motifs: SF2/SAF, SC35, SRp40, and SRp55. ESEs are enriched in regions in exons that are close to the splice sites, especially in the region 80 to 120 bases away from the ends of splice acceptor sites. Significant enrichment of ESEs is associated with weak splice acceptor sites but not weak donor sites. ESE density decreases at the 3 ends of long exons. ESEs are also enriched in introns with weak donor or acceptor sites. These characteristics of ESEs may help to predict functional ESE sites in RNA sequences.     

分支位点在真核基因受体位点识别中的作用

真核基因受体位点识别是剪接位点识别的一部分,也是基因识别中的重要环节,一直受到研究人员的关注。已有的研究结果显示受体位点的识别与分支位点有关,然而关于分支位点和受体位点识别的关系问题,目前还无人将其作为专门的问题予以深入研究。从受体位点识别出发,选取不同的受体位点序列长度,以神经网络为识别工具,对分支位点在受体位点识别中的作用做了深入研究和分析。实验结果表明,受体位点序列的特征信息集中在分支位点一例,因此分支位点在受体位点识别中具有重要作用。研究结果为受体位点识别问题中序列特征提取提供了依据。     

Activation-induced changes in alternate splice acceptor site usage

An AGCAG motif at 3' splice acceptor sites in a diverse set of genes creates alternate in-frame splice acceptor sites that produce alternate protein isoforms differing by a single amino acid. Among a group of over 12,000 EST-verified splice acceptor sites, only 74 genes were identified that contained the AGCAG motif, comprising about 0.7% of the total. In some cases the location of the single amino acid insertion occurs in a region with the potential to affect protein function. Analysis of cDNA from five different human genes of immunologic interest that contain the AGCAG motif, CD3zeta, CD79B, PLCgamma1, CD19, and CD32B (FcgammaRIIB), confirms that each gene encodes the two predicted splice variants. Variations occur in the splice variant ratio in all five of the genes tested during T and B cell activation, suggesting that the ratio is regulated by the cellular activation state. These results suggest that activation-induced variation in mRNA splicing may represent a mechanism for functional modulation of these proteins.     

Alternative splicing regulation at tandem 3' splice sites

Alternative splicing (AS) constitutes a major mechanism creating protein diversity in humans. Previous bioinformatics studies based on expressed sequence tag and mRNA data have identified many AS events that are conserved between humans and mice. Of these events, ~25% are related to alternative choices of 3′ and 5′ splice sites. Surprisingly, half of all these events involve 3′ splice sites that are exactly 3 nt apart. These tandem 3′ splice sites result from the presence of the NAGNAG motif at the acceptor splice site, recently reported to be widely spread in the human genome. Although the NAGNAG motif is common in human genes, only a small subset of sites with this motif is confirmed to be involved in AS. We examined the NAGNAG motifs and observed specific features such as high sequence conservation of the motif, high conservation of ~30 bp at the intronic regions flanking the 3′ splice site and overabundance of cis-regulatory elements, which are characteristic of alternatively spliced tandem acceptor sites and can distinguish them from the constitutive sites in which the proximal NAG splice site is selected. Our findings imply that AS at tandem splice sites and constitutive splicing of the distal NAG are highly regulated.     

One parameter to describe the mechanism of splice sites competition

The choice of a splice site is not only related to its own intrinsic strength, but also is influenced by its flanking competitors. Splice site competition is an important mechanism for splice site prediction, especially, it is a new insight for alternative splice site prediction. In this paper, the position weight matrix scoring function is used to represent splice site strength, and the mechanism of splice site competition is described by only one parameter: scoring function subtraction. While applying on the alternative splice site prediction, based on the only one parameter, 68.22% of donor sites and 70.86% of acceptor sites are correctly classified into alternative and constitutive. The prediction abilities are approximately equal to the recent method which is based on the mechanism of splice site competition. The results reveal that the scoring function subtraction is the best parameter to describe the mechanism of splice sites competition.