人类基因组上的假基因

假基因是基因组上与编码基因序列非常相似的非功能性基因组DNA拷贝,一般情况都不被转录,且没有明确生理意义。假基因根据其来源可分为复制假基因和已加工假基因。迄今为止,明确鉴定的人类假基因多为已加工假基因,有8000个之多。在Swiss-Prot/TrEMBL收录的编码蛋白质的将近25500个基因序列中,约10％在基因组中有一个或多个近全长已加工假基因。其余的功能基因都没有已加工假基因。核糖体蛋白基因具有最多数量的已加工假基因,约有l700个(占已加工假基因数的22％),少数基因,如cyclophilinA、肌动蛋白(actin)、角蛋白(keratin)、GAPDH、细胞色素C(cytochromec)和nucleophosmin等则有很多份已加工假基因。总体上讲,假基因在人类染色体上的分布与染色体长度成比例,但已加工假基因在GC含量为41％～46％的染色体区域密度最高。已加工假基因的拷贝数和功能基因在生殖器官中的表达高度一致,说明许多假基因发生在胚胎阶段,另外也和基因中GC含量和基因大小密切相关。假基因的准确鉴定对基因组进化、分子医学研究和医学应用具有重要意义。     

利用Red/ET同源重组构建新型CCR5Δ32打靶载体

目的:目前应用TALENs或CRISPR-Cas基因编辑技术修饰CCR5基因以获得抗HIV功能的研究策略基本是破坏CCR5基因而不是制造CCR5Δ32这样天然存在的抗HIV基因型。为此,我们尝试通过细菌内同源重组技术构建一个新颖的CCR5Δ32的打靶载体,以便能够用于制备CCR5Δ32基因突变细胞系。方法:首先利用Red/ET同源重组系统将rpsl-neo片段插入细菌人工染色体(BAC)的CCR5基因中,随后再次利用同源重组将一个不含32 bp区域的非选择性52 bp片段替换该rpsl-neo片段,从而将BAC中的CCR5基因改造成CCR5Δ32基因型,最后将loxp-neo-loxp抗性基因插入CCR5基因的第2内含子区域,制备出含有抗性基因的CCR5Δ32打靶载体,该载体可在Cre酶作用下删除neo基因但只留下一个loxp在基因的内含子区。结果:rpsl-neo片段成功插入CCR5之Δ32区并为非选择性52 bp片段替换,loxp-neo-loxp成功插入内含子区。结论:通过Red/ET同源重组技术获得CCR5Δ32-loxp-neo-loxp打靶载体。     

细菌染色体第1类整合子捕获耐药性基因盒反应模型的构建

【背景】整合子在细菌耐药性的获得及传播中占据重要地位,对于整合反应检测方法的改良及反应机制的研究,可以加深我们对细菌耐药性产生和播散的理解,为遏制耐药菌株的产生和播散提供新的途径。【目的】在细菌染色体上构建第1类整合子反应模型,用于评价整合酶介导的基因盒位点特异性重组。【方法】 PCR分别扩增含氯霉素耐药基因cat的CM片段、含基因盒aadA5的LacA5片段、含整合子重组位点attI1及强可变区启动子的PcS片段和插入位点两侧的同源臂,重叠延伸聚合酶链反应连接上述5个片段制备整合子模型插入片段,通过同源重组将构建好的整合子模型片段插入大肠埃希菌JM109染色体中。转入高表达第1类整合酶的质粒pHSint,在链霉素平板上筛选发生整合的菌株,并经聚合酶链反应和测序验证。【结果】构建的整合子模型片段经测序与预期一致,整合子模型片段成功插入大肠埃希菌JM109染色体中。转入高表达整合酶的质粒pHSint后,在链霉素平板上成功筛选出基因盒aadA5发生整合的菌株,经聚合酶链反应扩增并测序与预期一致。【结论】在大肠埃希菌染色体上成功构建第1类整合酶介导基因盒位点特异性重组反应模型,为进一步揭示整合子捕获耐药性基因盒的反应机制奠定基础。     

假基因的组成、分布及其分子进化

假基因(pseudogene)是指基因组中与正常基因序列相似, 但是缺乏功能的DNA 序列。通过序列同源性搜索, 可以收集基因组中假基因的群体特性、染色体分布和同源家族等特性。假基因很好地保留了数百万年前基因组中祖先基因的分子记录, 被视为“基因化石”, 因此假基因在进化和比较基因组学中是重要的资源。应用假基因和基因比较体系, 可以探究生物基因的进化史和基因组稳定性。如: 用Ka/Ks比值确定假基因的自然选择压、物种亲缘关系和进化距离, 分析假基因自身的进化趋势, 探讨DNA 突变的成因等。     

假基因的组成、分布及其分子进化

假基因（pseudogene）是指基因组中与正常基因序列相似,但是缺乏功能的DNA序列.通过序列同源性搜索,可以收集基因组中假基因的群体特性、染色体分布和同源家族等特性.假基因很好地保留了数百万年前基因组中祖先基因的分子记录,被视为＂基因化石＂,因此假基因在进化和比较基因组学中是重要的资源.应用假基因和基因比较体系,可以探究生物基因的进化史和基因组稳定性.如：用Ka/Ks比值确定假基因的自然选择压、物种亲缘关系和进化距离,分析假基因自身的进化趋势,探讨DNA突变的成因等.     

鲸类视觉退化分子机制的研究：基于GNAT2和CNGB3基因的进化分析

鲸类是一类次生性的水生哺乳动物,其陆生祖先大约53-56Ma从陆地返回海洋。为了适应水下的弱光环境,鲸类的光感受器以视杆细胞为主,视锥细胞功能大多退化,缺乏辨别颜色的能力,然而鲸类视觉退化的分子机制尚不清楚。本文选择在视锥细胞中表达且对光传导级联反应起重要作用的GNAT2和CNGB3基因,通过PCR扩增、测序以及在数据库中下载已有的基因序列,共获得8个鲸类代表性物种的同源序列。MEGA6.0软件比对发现侏儒抹香鲸和抹香鲸的GNAT2基因分别在148位和1012位插入了1个碱基 A,而抹香鲸的CNGB3分别在554位和1407位有1个碱基的缺失,导致提前终止密码子出现;另外,小须鲸CNGB3基因在1525-1527位出现了提前终止密码子,提示鲸类的这两个基因可能为假基因。通过I-TASSER在线预测GNAT2和CNGB3蛋白的三维结构,发现出现移码突变终止密码子的位置均位于这两个基因的重要功能域。另外,运用PAML软件的Branch model分析表明发生假基因的鲸类物种GNAT2和CNGB3基因出现选择压力放松,且假基因化可能是一个近期事件。侏儒抹香鲸、抹香鲸以及小须鲸的GNAT2和CNGB3基因出现假基因可能与其深潜习性以及完全的水生生境导致其色觉功能丢失相关。此外,Free-ratio model分析发现其他鲸类的ω值接近于1,说明GNAT2和CNGB3基因出现了选择压力放松,这可能是长期适应水生生境视觉退化的结果。     

鲸类视觉退化分子机制的研究：基于GNAT2和CNGB3基因的进化分析

鲸类是一类次生性的水生哺乳动物,其陆生祖先大约53-56Ma从陆地返回海洋。为了适应水下的弱光环境,鲸类的光感受器以视杆细胞为主,视锥细胞功能大多退化,缺乏辨别颜色的能力,然而鲸类视觉退化的分子机制尚不清楚。本文选择在视锥细胞中表达且对光传导级联反应起重要作用的GNAT2和CNGB3基因,通过PCR扩增、测序以及在数据库中下载已有的基因序列,共获得8个鲸类代表性物种的同源序列。MEGA6.0软件比对发现侏儒抹香鲸和抹香鲸的GNAT2基因分别在148位和1012位插入了1个碱基 A,而抹香鲸的CNGB3分别在554位和1407位有1个碱基的缺失,导致提前终止密码子出现;另外,小须鲸CNGB3基因在1525-1527位出现了提前终止密码子,提示鲸类的这两个基因可能为假基因。通过I-TASSER在线预测GNAT2和CNGB3蛋白的三维结构,发现出现移码突变终止密码子的位置均位于这两个基因的重要功能域。另外,运用PAML软件的Branch model分析表明发生假基因的鲸类物种GNAT2和CNGB3基因出现选择压力放松,且假基因化可能是一个近期事件。侏儒抹香鲸、抹香鲸以及小须鲸的GNAT2和CNGB3基因出现假基因可能与其深潜习性以及完全的水生生境导致其色觉功能丢失相关。此外,Free-ratio model分析发现其他鲸类的ω值接近于1,说明GNAT2和CNGB3基因出现了选择压力放松,这可能是长期适应水生生境视觉退化的结果。     

鲸类视觉退化分子机制的研究：基于GNAT2和CNGB3基因的进化分析

鲸类是一类次生性的水生哺乳动物,其陆生祖先大约53-56Ma从陆地返回海洋。为了适应水下的弱光环境,鲸类的光感受器以视杆细胞为主,视锥细胞功能大多退化,缺乏辨别颜色的能力,然而鲸类视觉退化的分子机制尚不清楚。本文选择在视锥细胞中表达且对光传导级联反应起重要作用的GNAT2和CNGB3基因,通过PCR扩增、测序以及在数据库中下载已有的基因序列,共获得8个鲸类代表性物种的同源序列。MEGA6.0软件比对发现侏儒抹香鲸和抹香鲸的GNAT2基因分别在148位和1012位插入了1个碱基 A,而抹香鲸的CNGB3分别在554位和1407位有1个碱基的缺失,导致提前终止密码子出现;另外,小须鲸CNGB3基因在1525-1527位出现了提前终止密码子,提示鲸类的这两个基因可能为假基因。通过I-TASSER在线预测GNAT2和CNGB3蛋白的三维结构,发现出现移码突变终止密码子的位置均位于这两个基因的重要功能域。另外,运用PAML软件的Branch model分析表明发生假基因的鲸类物种GNAT2和CNGB3基因出现选择压力放松,且假基因化可能是一个近期事件。侏儒抹香鲸、抹香鲸以及小须鲸的GNAT2和CNGB3基因出现假基因可能与其深潜习性以及完全的水生生境导致其色觉功能丢失相关。此外,Free-ratio model分析发现其他鲸类的ω值接近于1,说明GNAT2和CNGB3基因出现了选择压力放松,这可能是长期适应水生生境视觉退化的结果。     

鲸类视觉退化分子机制的研究：基于GNAT2和CNGB3基因的进化分析

鲸类是一类次生性的水生哺乳动物,其陆生祖先大约53-56Ma从陆地返回海洋。为了适应水下的弱光环境,鲸类的光感受器以视杆细胞为主,视锥细胞功能大多退化,缺乏辨别颜色的能力,然而鲸类视觉退化的分子机制尚不清楚。本文选择在视锥细胞中表达且对光传导级联反应起重要作用的GNAT2和CNGB3基因,通过PCR扩增、测序以及在数据库中下载已有的基因序列,共获得8个鲸类代表性物种的同源序列。MEGA6.0软件比对发现侏儒抹香鲸和抹香鲸的GNAT2基因分别在148位和1012位插入了1个碱基 A,而抹香鲸的CNGB3分别在554位和1407位有1个碱基的缺失,导致提前终止密码子出现;另外,小须鲸CNGB3基因在1525-1527位出现了提前终止密码子,提示鲸类的这两个基因可能为假基因。通过I-TASSER在线预测GNAT2和CNGB3蛋白的三维结构,发现出现移码突变终止密码子的位置均位于这两个基因的重要功能域。另外,运用PAML软件的Branch model分析表明发生假基因的鲸类物种GNAT2和CNGB3基因出现选择压力放松,且假基因化可能是一个近期事件。侏儒抹香鲸、抹香鲸以及小须鲸的GNAT2和CNGB3基因出现假基因可能与其深潜习性以及完全的水生生境导致其色觉功能丢失相关。此外,Free-ratio model分析发现其他鲸类的ω值接近于1,说明GNAT2和CNGB3基因出现了选择压力放松,这可能是长期适应水生生境视觉退化的结果。     

灵长类动物中TRIMCyp融合基因模式及对逆转录病毒复制的限制作用

TRIM5-CypA融合基因(TRIMCyp)是一种独特的TRIM5基因形式。迄今已发现新大陆猴中包括鹰猴在内的夜猴属所有代表种,以及在北平顶猴、巽他平顶猴、食蟹猴、印度恒河猴和熊猴等旧大陆猴中均存在这种基因融合现象,但在新大陆猴与旧大陆猴中的TRIMCyp融合基因的基因融合模式和表达剪接方式不同。新大陆猴TRIMCyp融合基因是由CypA假基因的cDNA序列通过LINE-1逆转座子介导的逆转座方式插入至TRIM5α基因的第7和第8外显子之间的内含子中形成,而旧大陆猴TRIMCyp融合基因则是由CypA假基因的cDNA序列以相似的逆转座方式插入至TRIM5基因的3’非翻译区(untranslatedregions,UTR)形成。TRIMCyp融合基因在不同灵长类动物中的存在比例、基因型、TRIMCyp融合蛋白的表达以及对逆转录病毒的限制活性均有所差异。鹰猴和平顶猴的TRIMCyp融合基因研究较多,鹰猴TRIMCyp融合蛋白可能以与TRIM5α相似机制限制HIV-1的感染,而平顶猴TRIMCyp融合蛋白则丧失了限制HIV-1的作用。这两个功能截然不同的融合基因为TRIM5α作用机制研究提供了难得的实验材料,也为建立HIV-1感染的新型灵长类动物艾滋病模型奠定了科学依据。该文综述了TRIMCyp融合基因在灵长类动物中的分布、存在形式及其限制逆转录病毒复制的作用机制等方面的研究情况。     

Processed pseudogenes are located preferentially in regions of low recombination rates in the human genome

The aim of this article is to demonstrate possible recombination‐associated evolutionary forces affecting the genomic distribution of processed pseudogenes. The relationship between recombination rate and the distribution of processed pseudogenes is analysed in the human genome. The results show that processed pseudogenes preferentially accumulate in regions of low recombination rates and this correlation cannot be explained by indirect relationships with GC content and gene density. Several explanatory models for the observation are discussed. A model of selection against ectopic recombination is tested based on the difference in distribution pattern between two classes of processed pseudogenes, which differ in the possibility of stimulating ectopic recombination. Our results indicate that the correlation between processed pseudogene density and recombination rate is probably results, in part, from the selection against ectopic recombination between closely located homologous processed pseudogenes. We also found a length effect in processed pseudogene distribution, namely long processed pseudogenes are located more preferentially in regions of low recombination rates than short ones.     

Transposons but not retrotransposons are located preferentially in regions of high recombination rate in Caenorhabditis elegans

We analyzed the distribution of transposable elements (TEs: transposons, LTR retrotransposons, and non-LTR retrotransposons) in the chromosomes of the nematode Caenorhabditis elegans. The density of transposons (DNA-based elements) along the chromosomes was found to be positively correlated with recombination rate, but this relationship was not observed for LTR or non-LTR retrotransposons (RNA-based elements). Gene (coding region) density is higher in regions of low recombination rate. However, the lower TE density in these regions is not due to the counterselection of TE insertions within exons since the same positive correlation between TE density and recombination rate was found in noncoding regions (both in introns and intergenic DNA). These data are not compatible with a global model of selection acting against TE insertions, for which an accumulation of elements in regions of reduced recombination is expected. We also found no evidence for a stronger selection against TE insertions on the X chromosome compared to the autosomes. The difference in distribution of the DNA and RNA-based elements along the chromosomes in relation to recombination rate can be explained by differences in the transposition processes.     

Natural selection affects multiple aspects of genetic variation at putatively neutral sites across the human genome

A major question in evolutionary biology is how natural selection has shaped patterns of genetic variation across the human genome. Previous work has documented a reduction in genetic diversity in regions of the genome with low recombination rates. However, it is unclear whether other summaries of genetic variation, like allele frequencies, are also correlated with recombination rate and whether these correlations can be explained solely by negative selection against deleterious mutations or whether positive selection acting on favorable alleles is also required. Here we attempt to address these questions by analyzing three different genome-wide resequencing datasets from European individuals. We document several significant correlations between different genomic features. In particular, we find that average minor allele frequency and diversity are reduced in regions of low recombination and that human diversity, human-chimp divergence, and average minor allele frequency are reduced near genes. Population genetic simulations show that either positive natural selection acting on favorable mutations or negative natural selection acting against deleterious mutations can explain these correlations. However, models with strong positive selection on nonsynonymous mutations and little negative selection predict a stronger negative correlation between neutral diversity and nonsynonymous divergence than observed in the actual data, supporting the importance of negative, rather than positive, selection throughout the genome. Further, we show that the widespread presence of weakly deleterious alleles, rather than a small number of strongly positively selected mutations, is responsible for the correlation between neutral genetic diversity and recombination rate. This work suggests that natural selection has affected multiple aspects of linked neutral variation throughout the human genome and that positive selection is not required to explain these observations.     

The Distribution of L1 and Alu Retroelements in Relation to GC Content on Human Sex Chromosomes Is Consistent with the Ectopic Recombination Model

The distribution of Alu and L1 retroelements in the human genome changes with their age. Active retroelements target AT-rich regions, but their frequency increases in GC- and gene-rich regions of the genome with increasing age of the insertions. Currently there is no consensus on the mechanism generating this pattern. In this paper we test the hypothesis that selection against deleterious deletions caused by ectopic recombination between repeats is the main cause of the inhomogeneous distribution of L1s and Alus, by means of a detailed analysis of the GC distribution of the repeats on the sex chromosomes. We show that (1) unlike on the autosomes and X chromosome, L1s do not accumulate on the Y chromosome in GC-rich regions, whereas Alus accumulate there to a minor extent; (2) on the Y chromosome Alu and L1 densities are positively correlated, unlike the negative correlation on other chromosomes; and (3) in gene-poor regions of chromosome 4 and X, the distribution of Alus and L1s does not shift toward GC-rich regions. In addition, we show that although local GC content of long L1 insertions is lower than average, their selective loss from recombining chromosomes is not the main cause of the enrichment of ancient L1s in GC-rich regions. The results support the hypothesis that ectopic recombination causes the shift of Alu and L1 distributions toward the gene-rich regions of the genome. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Reviewing Editor: Dr. Deborah Charlesworth     

Natural selection in gene-dense regions shapes the genomic pattern of polymorphism in wild and domesticated rice

Levels of nucleotide variability are frequently positively correlated with recombination rate and negatively associated with gene density due to the effects of selection on linked variation. These relationships are determined by properties that frequently differ among species, including the mating system, and aspects of genome organization such as how genes are distributed along chromosomes. In rice, genes are found at highest density in regions with frequent crossing-over. This association between gene density and recombination rate provides an opportunity to evaluate the effects of selection in a genomic context that differs from other model organisms. Using single-nucleotide polymorphism data from Asian domesticated rice Oryza sativa ssp. japonica and ssp. indica and their progenitor species O. rufipogon, we observe a significant negative association between levels of polymorphism and both gene and coding site density, but either no association, or a negative correlation, between nucleotide variability and recombination rate. We establish that these patterns are unlikely to be explained by neutral mutation rate biases and demonstrate that a model of background selection with variable rates of deleterious mutation is sufficient to account for the gene density effect in O. rufipogon. In O. sativa ssp. japonica, we report a strong negative correlation between polymorphism and recombination rate and greater losses of variation during domestication in the euchromatic chromosome arms than heterochromatin. This is consistent with Hill-Robertson interference in low-recombination regions, which may limit the efficacy of selection for domestication traits. Our results suggest that the physical distribution of selected mutations is a primary factor that determines the genomic pattern of polymorphism in wild and domesticated rice species.     

The effects of recombination rate on the distribution and abundance of transposable elements

Transposable elements (TEs) often accumulate in regions of the genome with suppressed recombination. But it is unclear whether this pattern reflects a reduction in the efficacy of selection against deleterious insertions or a relaxation of ectopic recombination. Discriminating between these two hypotheses has been difficult, because no formal model has investigated the effects of recombination under the deleterious insertion model. Here we take a simulation-based approach to analyze this scenario and determine the conditions under which element accumulation is expected in low recombination regions. We show that TEs become fixed as a result of Hill-Robertson effects in the form of Muller's ratchet, but only in regions of extremely low recombination when excision is effectively absent and synergism between elements is weak. These results have important implications for differentiating between the leading models of how selection acts on TEs and should help to interpret emerging population genetic and genomic data.     

Recombination drives vertebrate genome contraction

Selective and/or neutral processes may govern variation in DNA content and, ultimately, genome size. The observation in several organisms of a negative correlation between recombination rate and intron size could be compatible with a neutral model in which recombination is mutagenic for length changes. We used whole-genome data on small insertions and deletions within transposable elements from chicken and zebra finch to demonstrate clear links between recombination rate and a number of attributes of reduced DNA content. Recombination rate was negatively correlated with the length of introns, transposable elements, and intergenic spacer and with the rate of short insertions. Importantly, it was positively correlated with gene density, the rate of short deletions, the deletion bias, and the net change in sequence length. All these observations point at a pattern of more condensed genome structure in regions of high recombination. Based on the observed rates of small insertions and deletions and assuming that these rates are representative for the whole genome, we estimate that the genome of the most recent common ancestor of birds and lizards has lost nearly 20% of its DNA content up until the present. Expansion of transposable elements can counteract the effect of deletions in an equilibrium mutation model; however, since the activity of transposable elements has been low in the avian lineage, the deletion bias is likely to have had a significant effect on genome size evolution in dinosaurs and birds, contributing to the maintenance of a small genome. We also demonstrate that most of the observed correlations between recombination rate and genome contraction parameters are seen in the human genome, including for segregating indel polymorphisms. Our data are compatible with a neutral model in which recombination drives vertebrate genome size evolution and gives no direct support for a role of natural selection in this process.     

On the abundance and distribution of transposable elements in the genome of Drosophila melanogaster

The abundance and distribution of transposable elements (TEs) in a representative part of the euchromatic genome of Drosophila melanogaster were studied by analyzing the sizes and locations of TEs of all known families in the genomic sequences of chromosomes 2R, X, and 4. TEs contribute to up to 2% of the sequenced DNA, which corresponds roughly to the euchromatin of these chromosomes. This estimate is lower than that previously available from in situ data and suggests that TEs accumulate in the heterochromatin more intensively than was previously thought. We have also found that TEs are not distributed at random in the chromosomes and that their abundance is more strongly associated with local recombination rates, rather than with gene density. The results are compatible with the ectopic exchange model, which proposes that selection against deleterious effects of chromosomal rearrangements is a major force opposing element spread in the genome of this species. Selection against insertional mutations also influences the observed patterns, such as an absence of insertions in coding regions. The results of the analyses are discussed in the light of recent findings on the distribution of TEs in other species.