双翅目昆虫（黑腹果蝇和冈比亚按蚊）内含子丢失的比较分析

内含子插入和丢失的进化动力及机制尚存在许多疑问。通过对真核生物的105个同源基因的蛋白质高度保守区域内含子-外显子结构的研究,对人Homo sapiens、小鼠Mus musculus、大鼠Rattus norvegicus、黑腹果蝇Drosophila melanogaster、冈比亚按蚊Anopheles gambiae和秀丽隐杆线虫Caenorhabditis elegans的3 574个内含子、1 001个的内含子保守位点进行分析,推断出不同系统中内含子的变化途径。发现在进化早期,脊椎动物、双翅目昆虫和线虫的共同祖先中含有大量内含子,在进化过程中,双翅目昆虫和线虫发生了大量的内含子丢失,甚至在双翅目昆虫中内含子丢失较线虫更严重。线虫获得的内含子略多于丢失的内含子, 而在双翅目昆虫中则显示出内含子的丢失明显多于内含子的获得。该结果合理地解释了内含子在脊椎动物、线虫及昆虫中数量的分布呈下降趋势。     

中国汉族个体HLA-A、-B基因全长序列的测定及调控区多态性

文章利用20个中国汉族个体样本建立了稳定精确的HLA-A、-B基因全长序列的克隆测序方法, 获得HLA-A 10个等位基因4.2 kb序列, HLA-B 6个等位基因3.7 kb序列, 序列涵盖了两个基因的所有外显子、所有内含子、5′启动子区以及3′非翻译区(3′UTR)。A*1153是文章发现的一个新等位基因, B*151101的内含子序列、5个HLA-A以及2个HLA-B等位基因的5′启动子序列和3′UTR序列为国际上首次报道, 其他等位基因均延伸了IMGT/HLA数据库中释放的全长序列。文章首次在中国汉族个体中测定了IMGT/HLA数据库中没有覆盖的HLA-A、-B基因的上游5′启动子以及下游3′UTR区域的多态性模式。HLA-A基因5′启动子延伸区域共发现26个SNPs和一处3 bp(AAA/-)的插入/缺失, 3′UTR延伸区域共发现14个SNPs; HLA-B基因5′启动子延伸区域共发现5个SNPs和一处1 bp(T/-)的插入/缺失, 3′UTR延伸区域共发现8个SNPs。通过对两个基因的5′启动子、外显子以及3′UTR的系统发育树分析, 发现两个基因调控区与外显子的进化关系有所不同, HLA-A基因除A*24020101外, 其他等位基因两端调控区与外显子连锁比较紧密, HLA-B基因两端调控区与外显子之间则发生了较为频繁的重组事件。     

水稻minE基因的克隆及分析

 植物叶绿体与原核生物分裂机制相似,其中MinE蛋白在细菌分裂过程中具有重要作用. 为了研究植物MinE蛋白在叶绿体分裂过程中的功能及其进化,利用RT PCR技术克隆了水稻叶绿体分裂相关基因OsMinE,并在GenBank登录（No. AY496951）.OsMinE基因cDNA全长1 035 bp,其ORF为711 bp,编码236个氨基酸.与原核生物MinE蛋白相比,水稻OsMinE具有明显延伸的N端与C端.其N端102个氨基酸残基为预测的叶绿体导肽序列,C端延伸保守,推测赋予植物MinE蛋白新的功能.植物minE基因结构分析显示,水稻、拟南芥、杨树都仅含有1个内含子,且插入位置及相位相同.这表明,该内含子可能在单子叶、双子叶植物分化前产生.水稻OsMinE基因在大肠杆菌细胞中的表达严重影响了细胞的分裂,初步证明了水稻MinE蛋白与原核细胞MinE蛋白功能类似.水稻OsMinE基因的克隆为进一步研究叶绿体的分裂机制奠定了基础.     

茉莉酸信号途径中转录抑制因子JAZ蛋白家族的分子进化分析

茉莉酸在植物的生长发育、应激反应和次生代谢过程中起着重要的调控作用。转录抑制因子JAZ（Jasmonate ZIM-domain）蛋白则是茉莉酸信号从SCF^coi1受体复合物向下游茉莉酸应答基因转导的纽带。采用比较基因组学的方法。从多谱系的角度对植物JAZ蛋白家族进行分子进化分析并取得以下研究结果。（1）在藻类植物、苔藓植物、蕨类植物、裸子植物及单、双子叶植物6个不同谱系的15种代表植物基因组中,鉴定了82个JAZ同源基因,其中在低等藻类植物基因组中没有鉴定到JAZ同源基因,提示JAZ家族基因可能起源于陆生植物。（2）系统发育分析表明,在植物基因组中JAZ蛋白家族可分为10个保守的亚家族,而谱系特异扩增尤其是串联重复和区段重复可能是陆生植物JAZ家族基因扩增与进化的主要机制,并导致多个谱系特异的JAZ亚家族产生。（3）基因结构分析表明,JAZ家族基因含有0一7个数目不等、62—4222bp长度不等的内含子,提示在植物基因组进化过程中,JAZ家族基因可能发生内含-丢失或内含子插入缺失,进而导致基因外显子．内含子结构的多样性。该研究结果将为植物JAZ蛋白家族的深入研究提供参考。     

动物肌动蛋白基因中内含子的来源及存在意义的探讨

对动物界演化过程中肌动蛋白家族内含子插入位置分布的演化规律作了分析,并对相同插入位置的内含子序列按同亚型和不同亚型作了比较。结果得出:从整个肌动蛋白家族的外显子序列高度保守性推断整个肌动蛋白家族可能是从共同的祖先蛋白进化而来的;从同亚型肌动蛋白内含子序列的类似性随进化距离而变化,但在短进化距离的物种间,类似性都较高,不同亚型肌动蛋白内含子序列的类似性都较低,即使是同一物种(如人),类似性也远低于同亚型但进化距离较近的物种,由此可推断,同亚型肌动蛋白的内含子序列可能从共同祖先进化,不同亚型肌动蛋白的内含子序列从不同祖先进化,综上推断可导出内含子可能是在蛋白异化过程中获得的:还发现内含子在肌动蛋白家族编码基因中位置的分布随进化方向不同而逐步形成两种截然不同的模式,由此提出了内含子的位置分布与动物演化方向之间可能具有某种必然联系,为内含子的存在提出了某种依据。     

基因的克隆及表达分析

DNA重组激活基因（Recombination activating genes RAG）是脊椎动物特异性免疫反应的关键基因,也是脊椎动物进化分析的标记基因之一。鲫鱼具有很强的适应性和抗病能力,是我国广泛养殖的重要经济淡水鱼;由于具有不同的倍性和丰富的遗传多样性,又是研究鱼类基因组进化的独特材料。本研究用PCR方法扩增、克隆了鲫鱼的Rag基因。鲫鱼Rag1基因从起始密码到终止密码总长4188bp,由三个外显子和两个内含子组成,其中开放阅读框长3192bp,编码1063个氨基酸。Rag2基因从起始密码到终止密码总长1593bp,没有内含子,只有单一的编码区,编码530个氨基酸。Rag1和Rag2基因的ORF和氨基酸序列在不同鱼类中的对比结果表明其在进化过程中非常保守。不同鱼类Rag1基因的第二内含子也是高度保守的,转录因子结合位点分析表明在第二内含子的保守区域中有许多转录因子的可能结合位点。其中有一段在所有已知鱼类中都存在的保守区域是与性腺发育相关的转录因子SRY和SOX5的可能结合位点,提示Rag1基因的表达可能与性腺发育具有相关性。用RT-PCR方法进行的组织特异性表达分析表明Rag1基因在鲫鱼成体的头肾和精巢都能检测到表达,提示Rag基因不仅主导了免疫组织中的DNA重组,也可能参与了生殖细胞的DNA重组。RT-PCR检测证明Rag1基因在鲫鱼胚胎发育至第5天开始表达,在第7天鲫鱼胚胎的胸腺原基中可检测到Rag1基因mRNA的杂交信号,在第9天鲫鱼胚胎的胸腺原基中可以检测到很强的Rag1 mRNA原位杂交信号,说明该时期可能是鲫鱼免疫基因重组的活跃时期。     

科技消息

西德的Antoine等新发现摇蚊(Chironomus thummithummi)的血红蛋白基因没有内含子。脊椎动物的肌红蛋白基因及血红蛋白基因,在同一位置上都有二个内含子,这是自脊椎动物诞生以来,在几亿年进化期间,被稳定地保留下来的。豆科植物也存在与血红蛋白类似的豆血红蛋白,它的基因有3个内含子,其中二个与脊椎动物的非常相似。这可解释为,在远古时期珠蛋白基因是有3个内含子的,其中一个在进化中消失了。摇蚊有12种血红蛋白,其氨基酸顺序与脊椎动物血红蛋白的β链约有16％的氨基酸一致。这与人肌红蛋白和八目鳗珠蛋白的一致度(19％)几乎相同。toine等分析了摇蚊珠蛋白基因中的4个(A、B、C     

昆虫的珠蛋白基因无内含子

西德的Antoine等新发现摇蚊(Chironomus thummi thummi)的血红蛋白基因没有内含子。脊椎动物的红蛋白基因及血红蛋白基因,在同一位置上都有二个内含子,这是自脊椎动物诞生以来,在几亿年进化期间,被稳定地保留下来的。豆科植物也存在与血红蛋白类似的豆血红蛋白,它的基因有3个内含子,其中二个与脊椎动物的非常相似。这可解释为,在远古时期珠蛋白基因是有3个内含子的,其中一个在进化中消失了。摇蚊有12种血红蛋白,其氨基酸顺序与脊椎动物血红蛋白的β链约有16％的氨基酸一致。这与人肌红蛋白和八目鳗珠蛋白的一致度(19％)几乎相同。toine等分析了摇蚊珠蛋白基因中的4个(A、B、c     

鲫鱼Rag基因的克隆及表达分析

DNA重组激活基因(Recombination activating genes RAG)是脊椎动物特异性免疫反应的关键基因,也是脊椎动物进化分析的标记基因之一.鲫鱼具有很强的适应性和抗病能力,是我国广泛养殖的重要经济淡水鱼;由于具有不同的倍性和丰富的遗传多样性,又是研究鱼类基因组进化的独特材料.本研究用PCR方法扩增、克隆了鲫鱼的Rag基因.鲫鱼Rag1基因从起始密码到终止密码总长4188bp,由三个外显子和两个内含子组成,其中开放阅读框长3192bp,编码1063个氨基酸.Rag2基因从起始密码到终止密码总长1593bp,没有内含子,只有单一的编码区,编码530个氨基酸.Rag1和Rag2基因的ORF和氨基酸序列在不同鱼类中的对比结果表明其在进化过程中非常保守.不同鱼类Rag1基因的第二内含子也是高度保守的,转录因子结合位点分析表明在第二内含子的保守区域中有许多转录因子的可能结合位点.其中有一段在所有已知鱼类中都存在的保守区域是与性腺发育相关的转录因子SRY和SOX5的可能结合位点,提示Rag1基因的表达可能与性腺发育具有相关性.用RT-PCR方法进行的组织特异性表达分析表明Rag1基因在鲫鱼成体的头肾和精巢都能检测到表达,提示Rag基因不仅主导了免疫组织中的DNA重组,也可能参与了生殖细胞的DNA重组.RT-PCR检测证明Rag1基因在鲫鱼胚胎发育至第5天开始表达,在第7天鲫鱼胚胎的胸腺原基中可检测到Rag1基因mRNA的杂交信号,在第9天鲫鱼胚胎的胸腺原基中可以检测到很强的Rag1 mRNA原位杂交信号,说明该时期可能是鲫鱼免疫基因重组的活跃时期.     

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。     

Periodicities in coding and noncoding regions of the genes

Gene population statistical studies of protein coding genes and introns have identified two types of periodicities on the purine/pyrimidine alphabet: (i) the modulo 3 periodicity or coding periodicity (periodicity P3) in protein coding genes of eukaryotes, prokaryotes, viruses, chloroplasts, mitochondria, plasmids and in introns of viruses and mitochondria, and (ii) the modulo 2 periodicity (periodicity P2) in the eukaryotic introns. The periodicity study is herein extended to the 5' and 3' regions of eukaryotes, prokaryotes and viruses and shows: (i) the periodicity P3 in the 5' and 3' regions of eukaryotes. Therefore, these observations suggest a unitary and dynamic concept for the genes as for a given genome, the 5' and 3' regions have the genetic information for protein coding genes and for introns: (1) In the eukaryotic genome, the 5' (P2 and P3) and 3' (P2 and P3) regions have the information for protein coding genes (P3) and for introns (P2). The intensity of P3 is high in 5' regions and weak in 3' regions, while the intensity of P2 is weak in 5' regions and high in 3' regions. (2) In the prokaryotic genome, the 5' (P3) and 3' (P3) regions have the information for protein coding genes (P3). (3) In the viral genome, the 5' (P3) and 3' (P3) regions have the information for protein coding genes (P3) and for introns (P3). The absence of P2 in viral introns (in opposition to eukaryotic introns) may be related to the absence of P2 in 5' and 3' regions of viruses.     

小鼠基因内含子中CpG岛和几个转录调控元件分析

内含子在基因转录调控中的作用已多次被实验报道,然而对其参与调控的普遍性还缺乏足够的理论支持。本研究利用计算分析方法,对小鼠基因内含子中的CpG岛(CpGisland)、TATA框(TATAbox)、CAAT框(CAATbox)以及GC框(GCbox)等元件的出现频率进行分析。结果发现,分别有56.01%、57.16%、65.88%和41.86%的第一内含子具有CpG岛、TATA框、CAAT框以及GC框,而它们在其它内含子中的平均出现频率则分别为14.07%、45.24%、49.91%和13.19%。即使考虑到不同位置的内含子,这些元件在第一内含子中的出现频率也显著高于它们在其它任何位置内含子中的出现频率。由于CpG岛、TATA框、CAAT框以及GC框均与基因的转录调控有关,据此推测小鼠第一内含子在基因转录调控过程中具有潜在的重要性。本研究结果为内含子参与转录调控提供了更多的理论依据。     

人与小鼠核糖体蛋白基因内含子中的转录调控位点分析

前期对酵母和果蝇核糖体蛋白(Ribosomal protein, RP)基因内含子序列中的寡核苷酸分析表明, 内含子中含有潜在的转录因子结合位点。为进一步发掘核糖体蛋白基因内含子参与转录调控的证据, 文章首先基于频率分析方法抽提出人和小鼠核糖体蛋白基因第一内含子中高频(Over-represented)出现的寡核苷酸片段 (亦称模体, Motif), 这些寡核苷酸中超过85%与已知的转录因子结合位点吻合, 是潜在的转录调控元件。对抽提出的寡核苷酸进行碱基组成分析, 发现95%以上的寡核苷酸富含碱基C和G, 而较少富含A和T。从寡核苷酸在内含子中的分布情况看, 它们相对靠近第一内含子的5′端, 即距离基因转录起始位点和上游区域较近。推测这些特征可能与基因转录调控有关。     

Do imprinted genes have few and small introns?

A gene is described as imprinted if its pattern of expression depends on whether it passed the previous generation in a male or female germ line. A recent paper⁽¹⁾ reports that imprinted genes have fewer and smaller introns than a control set of genes. The differences are striking but their interpretation is unclear. The loss of introns after a gene becomes imprinted is not sufficient to explain why imprinted genes have fewer introns than average, because related unimprinted genes also have few introns. Similarly, small introns appear to be a property of chromosomal region rather than of imprinting status itself, because neighboring unimprinted genes also have small introns.     

Intron size and exon evolution in Drosophila

We have found a negative correlation between evolutionary rate at the protein level (as measured by d(N)) and intron size in Drosophila. Although such a relation is expected if introns reduce Hill-Robertson interference within genes, it seems more likely to be explained by the higher abundance of cis-regulatory elements in introns (especially first introns) in genes under strong selective constraints.     

Complete, precise, and innocuous loss of multiple introns in the currently intronless, active cathepsin L-like genes, and inference from this event

Retrotransposition typically generates pseudogenes. Here we demonstrate a different fate of the retro-processed genes through a novel mechanism in which the retro-processed genes still maintain their sequence intactness and the original functions. We show that the shrimp cathepsin L (CatL) gene MeCatL has lost all of its five introns. Also, ProEPB, the ancestor of the CatL-like barley EPBs and rice REP1, has lost all of its three introns. The multiple introns in a gene might have been eliminated simultaneously and precisely at the original locus for the CatL-like genes of shrimp, barley, rice, Drosophila, and Theileria. We reason that retrotransposition is not responsible for the generation of a processed active intronless (PAI) gene when the gene product retains its sequence intactness and its original function. We propose that double-strand-break repair (DSBR) machinery might play a role in cDNA-mediated homologous recombination (cDMHR) that causes the loss of introns. The cDMHR/DSBR pathway is probably a fundamental mechanism for intron loss in PAI genes and in some asymmetric-intron genes.     

Widespread occurrence of spliceosomal introns in the rDNA genes of ascomycetes

Spliceosomal (pre-mRNA) introns have previously been found in eukaryotic protein-coding genes, in the small nuclear RNAs of some fungi, and in the small- and large-subunit ribosomal DNA genes of a limited number of ascomycetes. How the majority of these introns originate remains an open question because few proven cases of recent and pervasive intron origin have been documented. We report here the widespread occurrence of spliceosomal introns (69 introns at 27 different sites) in the small- and large-subunit nuclear-encoded rDNA of lichen-forming and free-living members of the Ascomycota. Our analyses suggest that these spliceosomal introns are of relatively recent origin, i.e., within the Euascomycetes, and have arisen through aberrant reverse-splicing (in trans) of free pre-mRNA introns into rRNAs. The spliceosome itself, and not an external agent (e.g., transposable elements, group II introns), may have given rise to these introns. A nonrandom sequence pattern was found at sites flanking the rRNA spliceosomal introns. This pattern (AG-intron-G) closely resembles the proto-splice site (MAG-intron-R) postulated for intron insertions in pre-mRNA genes. The clustered positions of spliceosomal introns on secondary structures suggest that particular rRNA regions are preferred sites for insertion through reverse-splicing.     

Conservation of the positions of metazoan introns from sponges to humans

Sponges (phylum Porifera) are the phylogenetic oldest Metazoa still extant. They can be considered as reference animals (Urmetazoa) for the understanding of the evolutionary processes resulting in the creation of Metazoa in general and also for the metazoan gene organization in particular. In the marine sponge Suberites domuncula, genes encoding p38 and JNK kinases contain nine and twelve introns, respectively. Eight introns in both genes share the same positions and the identical phases. One p38 intron slipped for six bases and the JNK gene has three more introns. However, the sequences of the introns are not conserved and the introns in JNK gene are generally much longer. Introns interrupt most of the conserved kinase subdomains I-XI and are found in all three phases (0, 1 and 2). We analyzed in details p38 and JNK genes from human, Caenorhabditis elegans and Drosophila melanogaster and found in most genes introns at the positions identical to those in sponge genes. The exceptions are two p38 genes from D. melanogaster that have lost all introns in the coding sequence. The positions of 11 introns in each of four human p38 genes are fully conserved and ten introns occupy identical positions as the introns in sponge p38 or JNK genes. The same is true for nine, out of ten introns in the human JNK-1 gene. The introns in human p38 and JNK genes are on average more than ten times longer than corresponding introns in sponges. It was proposed that yeast HOG1-like kinases (from i.e. Saccharomyces cerevisiae and Emericella nidulans) and metazoan p38 and JNK kinases are orthologues. p38 and JNK genes were created after the split from fungi by the duplication and diversification of the HOG1-like progenitor gene. Our results further support the common origin of p38 and JNK genes and speak in favor of a very early time of duplication. The ancestral gene contained at least ten introns, which are still present at the very conserved positions in p38 and JNK genes of extant animals. Four of these introns are present at the same positions in the HOG-like gene in the fungus E. nidulans. The others probably entered the ancestral gene after the split of fungi, but before the duplication of the gene and before the creation of the common, urmetazoan progenitor of all multicellular animals. A second gene coding for an immune molecule is described, the allograft inflammatory factor, which likewise showed a highly conserved exon/intron structure in S. domuncula and in human. These data show that the intron/exon borders are highly conserved in genes from sponges to human.     

Association of intron phases with conservation at splice site sequences and evolution of spliceosomal introns.

How exon-intron structures of eukaryotic genes evolved under various evolutionary forces remains unknown. The phases of spliceosomal introns (the placement of introns with respect to reading frame) provide an opportunity to approach this question. When a large number of nuclear introns in protein-coding genes were analyzed, it was found that most introns were of phase 0, which keeps codons intact. We found that the phase distribution of spliceosomal introns is strongly correlated with the sequence conservation of splice signals in exons; the relatively underrepresented phase 2 introns are associated with the lowest conservation, the relatively overrepresented phase 0 introns display the highest conservation, and phase 1 introns are intermediate. Given the detrimental effect of mutations in exon sequences near splice sites as found in molecular experiments, the underrepresentation of phase 2 introns may be the result of deleterious-mutation-driven intron loss, suggesting a possible genetic mechanism for the evolution of intron-exon structures.