红原鸡全基因组中微卫星分布规律研究

本文对红原鸡Gallus gallus全基因组中微卫星数量及分布规律进行了分析,查找到l～6个碱基重复类型的微卫星序列共282728个,约占全基因组序列(1.1Gb)的0.49%,分布频率为1/3.89kb,微卫星序列的长度主要在12～70个碱基长度范围内。第1、2、3条染色体上微卫星分布频率较高,而32号染色体上无微卫星分布。不同类型微卫星中,单碱基重复类型数目最多,为184192个,占总数的65.1%;其次是四、二、三、五、六碱基重复单元序列,分别占到总数的12.8%、9.7%、7.2%、4.6%、0.8%。T、A、AT、GTTT、AAAC、G、C、ATTT、AC、GT、AAAT、ATT、AAC、AAT、GTT、AG、CT、CTTT、AAAG、GTTTT、AAACA、AAGG、CCTT是红原鸡基因组中最主要的微卫星重复类型。本研究为红原鸡微卫星标记的分离筛选、遗传多样性的研究以及不同物种微卫星的比较分析奠定了基础。     

基于Roche 454 GS FLX高通量测序的叶城沙蜥基因组微卫星特征分析

叶城沙蜥Phrynocephalus axillaris是我国特有的一种小型爬行动物,广泛分布于新疆塔里木盆地、吐鲁番-哈密盆地和甘肃敦煌盆地。本研究利用Roche 454 GS FLX高通量测序技术进行叶城沙蜥微卫星位点筛选,获得了91 190条高质量序列。用Krait搜索微卫星位点,共得到1～6个碱基重复类型的完美型微卫星序列29 890个。不同类型微卫星中,单碱基重复类型数目最多,有14 630个,占总数的48. 95%,其次是二碱基,约占28. 60%,四碱基、三碱基、五碱基和六碱基分别占10. 73%、10. 48%、0. 92%和0. 32%。二碱基微卫星中AC重复类型数量最多,三碱基、四碱基、五碱基和六碱基中分别是ATC、AAAT、AAAAT和AATCCC。叶城沙蜥完美型微卫星中数量最多的11种重复拷贝类型分别为C、A、AC、AG、AAAT、ATC、AT、AAT、ATAG、AGG和AAC。本研究深化了对叶城沙蜥基因组的了解,并为以后开发和筛选大量高质量微卫星标记提供了数据支持,也为利用微卫星标记研究叶城沙蜥种群遗传结构和谱系地理模式奠定了基础。     

微卫星DNA标记及其在鱼类遗传多样性研究中的应用

微卫星DNA作为第二代分子遗传标记是高等真核生物基因组中种类多、分布广、具有高度的多态性和杂合度的分子标记,由于其具有多态性检出率高、信息含量大、共显性标记、实验操作简单、结果稳定可靠等优点,已经成为种群遗传学研究中被广泛应用的分子遗传标记。微卫星DNA标记技术在鱼类的群体遗传结构的分析、物种遗传多样性的鉴定以及遗传基因连锁图谱的构建等方面已初步得到应用。该文就微卫星技术的原理方法,在鱼类遗传多样性研究中的应用概况以及应用范围和注意事项等方面进行综述。为微卫星技术在鱼类遗传多样性研究中应用提供了理论参考。     

Interpretation, Stratification and Evidence for Sequence Variants Affecting mRNA Splicing in Complete Human Genome Sequences

Information theory-based methods have been shown to be sensitive and specific for predicting and quantifying the effects of non-coding mutations in Mendelian diseases. We present the Shannon pipeline software for genome-scale mutation analysis and provide evidence that the software predicts variants affecting mRNA splicing. Individual information contents (in bits) of reference and variant splice sites are compared and significant differences are annotated and prioritized. The software has been implemented for CLC-Bio Genomics platform. Annotation indicates the context of novel mutations as well as common and rare SNPs with splicing effects. Potential natural and cryptic mRNA splicing variants are identified, and null mutations are distinguished from leaky mutations. Mutations and rare SNPs were predicted in genomes of three cancer cell lines (U2OS, U251 and A431), which were supported by expression analyses. After filtering, tractable numbers of potentially deleterious variants are predicted by the software, suitable for further laboratory investigation. In these cell lines, novel functional variants comprised 6-17 inactivating mutations, 1-5 leaky mutations and 6-13 cryptic splicing mutations. Predicted effects were validated by RNA-seq analysis of the three aforementioned cancer cell lines, and expression microarray analysis of SNPs in HapMap cell lines.     

Genetic Variation and Discriminating Power of Four DNA Microsatellites in the Russian Population

The allele and genotype frequency distributions of four STRs (the LPL, vWA, FES/FPS, and F13B loci) commonly used in forensic medicine were studied with a sample of 200 ethnic Russians from Siberia. Genetic and molecular diversity of the four STRs was characterized in comparison with the American Caucasoid population. The set of the four STRs showed a high power of discrimination (PD = 0.99975). Comparison of the genetic variation at the four loci revealed a considerable difference between the Russian and American Caucasoid populations, precluding the use of data on allele frequencies in American Caucasoids for forensic testing in Russia. The results can be used as a reference in Siberia.     

后生动物线粒体基因组:起源、大小和基因排列进化

由于受到强烈的进化约束,后生动物线粒体基因组在大小和基因含量上一直保持稳定,相比之下核基因组则发生了巨大的改变。后生动物线粒体基因组结构的可塑性在一定程度上归功于可能由tRNA基因介导的基因重排事件,虽然亲缘关系密切的物种间也可能出现基因重排,但同门内的线粒体基因组仍趋向于具有类似的结构特征。我们对后生动物线粒体基因组的起源、大小和基因排列进化方面的特点进行了介绍。     

Evolution of MIR Elements Located in the Coding Regions of Human Genome

A search for new members of the mammalian interspersed repeat (MIR) family has been done over the coding regions of human genome from GenBank-116. Only 254 nucleotide sequences contained MIRs in coding regions, of which 45 MIR copies were unknown before, including 17 that occurred in translated gene regions. The program developed by the authors has been demonstrated to surpass the CENSOR program in the search power. The evolution of the MIR copies located in translated regions of human genome is discussed.     

Convergent Evolution of Human and Bovine Haptoglobin: Partial Duplication of the Genes

Haptoglobin (Hp) is a hemoglobin-binding plasma protein consisting of two types of chains, called α and β, which originate from a common polypeptide. In humans, but not in other mammals, Hp has been shown to occur in two allelic forms, Hp1 and Hp2, which differ in the length of the α-chain. The longer α-chain (in Hp2) seems to have arisen by an internal duplication of a gene segment coding for almost the entire α-chain of Hp1. In this article we show that Hp of cow (Bos taurus) contains an α-chain, the structure of which is similar to that of the human Hp2 α-chain. Furthermore, comparison of the structure of bovine Hp and human Hp2 suggests that the bovine gene arose by a duplication of the gene segment homologous to that duplicated in human Hp2. However, a phylogenetic analysis indicates that the two genes were formed independently. The evolutionary pressure that has led to the fixation of the Hps with a longer α-chain is not known. Reviewing Editor: Dr. Manyuan Long     

Molecular Analysis Using Mitochondrial DNA and Microsatellites to Infer the Formation Process of Japanese Native Horse Populations

To assess the genetic diversity of Japanese native horse populations, we examined seven such populations using mitochondrial DNA (mtDNA) and microsatellite analyses. Four reference populations of Mongolian horses and European breeds were employed as other equids. In the mtDNA analysis, the control region (D-loop) of 411 bp was sequenced, and 12 haplotypes with 33 variable sites were identified in the Japanese native horses. The phylogenetic tree constructed by haplogrouping and using worldwide geographic references indicated that the haplotypes of the Japanese native horses were derived from six equid clusters. Compared with the foreign populations, the Japanese native populations showed lower within-population diversity and higher between-population differentiation. Microsatellite analysis, using 27 markers, found an average number of alleles per locus of 9.6 in 318 native and foreign horses. In most native populations, the within-population diversity was lower than that observed in foreign populations. The genetic distance matrix based on allelic frequency indicated that several native populations had notably high between-population differentiation. The molecular coancestry-based genetic distance matrix revealed that the European populations were differentiated from the Japanese and Mongolian populations, and no clear groups could be identified among the Japanese native horse populations. The genetic distance matrices had few correlations with the geographic distribution of the Japanese native populations. Based on the results of both mtDNA and microsatellite analyses, it could be speculated that each native population was formed by the founder populations derived from Mongolian horses. The genetic construction of each population appears to have been derived from independent breeding in each local area since the time of population fission, and this was accompanied by drastic genetic drift in recent times. This information will help to elucidate the ancestry of Japanese native horses. An erratum to this article can be found at     

Evolution of human mitochondrial DNA: Evidence for departure from a pure neutral model of populations at equilibrium

Summary Human mitochondrial DNA (mtDNA) data from 18 populations have been carefully reexamined. A phylogeny of 77 mtDNA types found among the 1389 individuals analyzed for restriction fragment length polymorphisms (RFLPs) was established using the parsimony principle and compared to a UPGMA tree of the 18 populations. Both analyses agreed in separating African samples from the other populations, though the mtDNA type phylogeny suggested close relations between Africans and other continental groups. Conformity of observed mtDNA type frequency distributions with the infinite allele model was studied for 31 human populations. Several Oriental and Caucasoid populations were found to be overly homogeneous, generally due to an elevated frequency of one particular type. Contrastingly, all African samples conformed to the neutral model of populations at equilibrium and presented more diversified distributions. This suggested that part of the apparent African divergence was due to heterogeneous evolutionary processes and confirmed that some diversity reducing factors were at work in Caucasoids and Orientals. Several nonexclusive hypotheses accounting for the rejection of the neutrality tests were discussed. Alternative hypotheses concerning modern human emergence were also reviewed in the light of present results.     

蛋白质体外进化建库技术研究

蛋白质体外进化技术是蛋白质工程发展的一个里程碑,也是改造蛋白质的一种有效工具。它不仅具有重要的应用价值,而且有助于蛋白质结构与功能的研究。通过蛋白质体外进化技术已成功地改造了许多蛋白质,有些已应用于工农业生产。体外进化技术分为两步:建库和筛选。本文主要对蛋白质体外进化策略及对体外随机突变技术、DNA重组技术、利用活细胞自身修复系统构建突变文库等几种定向进化突变文库建立技术进行了介绍与论述,同时还对蛋白质体外进化技术的应用及与其它学科结合的研究前景进行了分析,为获得具有改进功能或全新功能的蛋白质提供理论基础。     

山羊草属异源多倍体植物基因组进化的ＲＡＰＤ分析

和２４个随机引物对山羊草属（Ａegilops L.)异源多倍体物种对其祖先二倍体物进行ＲＡＰＤ分析,对扩增出的３１３条带进行聚类分析发现,含Ｄ基因组的多倍体与二倍体祖先Ａe.squarrosa(DD)在聚类图上聚为一支;除Ａe.juvenalis(DDMMUU)聚到上一支外,含Ｕ基因组的多倍 与二倍体祖先Ae.umbellulata(ＵＵ）在聚类图上聚为另一支;多倍体与其他二倍体均不聚在一起,表明多倍体分别与Ａe.squarrosa(DD)、Ae.umbellulata(UU)具有较近的亲缘关系,这说明多倍体开之后,Ｄ和Ｕ基因组变化较小,而其他基因组则发生了较大的变化。     

Evidence for habitat fragmentation altering within-population processes in wombats

Habitat fragmentation and associated reduced dispersal of wildlife can lead to an accumulation of related individuals in fragments. The altered kin interactions and amplified chance of inbred matings has profound implications for mating and social systems, and ultimately population persistence. Nonetheless, within-fragment population processes are rarely studied. With this aim, we examined relatedness structure in two candidate isolated populations (Kulpara and Scrubby Peak) of southern hairy-nosed wombats ( Lasiorhinus latifrons ). Wombats were sampled by remote hair-trapping for genotyping at 14 microsatellite loci, enabling individual identification and estimation of space-use and associative behaviour with respect to relatedness. Genetic data indicated that Scrubby Peak was not strongly isolated, against predictions from landscape structure and history. In isolated Kulpara, inhibited female dispersal (normally the dispersing sex) was associated with high population density and altered kin relationships. First, female relatives preferentially coexisted, a radical departure from the previously reported active avoidance of female relatives in continuous habitat. This is consistent with females in altered habitat interacting with more- rather than less-related females to minimize the cost:benefit ratio of proximity to other wombats. Second, inbreeding avoidance appeared to be stronger at Kulpara than in conspecific populations with natural population structures. Although these adaptive behaviours may have contributed to persistence of the Kulpara population in the short term, they are unlikely to ensure its long-term viability in the face of ongoing isolation because they can act only to slow the rate of inbreeding and mitigate some of its negative impacts.     

Heterogeneous Nature and Distribution of Interruptions in Dinucleotides May Indicate the Existence of Biased Substitutions Underlying Microsatellite Evolution

Some aspects of microsatellite evolution, such as the role of base substitutions, are far from being fully understood. To examine the significance of base substitutions underlying the evolution of microsatellites we explored the nature and the distribution of interruptions in dinucleotide repeats from the human genome. The frequencies that we inferred in the repetitive sequences were statistically different from the frequencies observed in other noncoding sequences. Additionally, we detected that the interruptions tended to be towards the ends of the microsatellites and 5'-3' asymmetry. In all the estimates nucleotides forming the same repetitive motif seem to be affected by different base substitution rates in AC and AG. This tendency itself could generate patterning and similarity in flanking sequences and reconcile these phenomena with the high mutation rate found in flanking sequences without invoking convergent evolution. Nevertheless, our data suggest that there is a regional bias in the substitution pattern of microsatellites. The accumulation of random substitutions alone cannot explain the heterogeneity and the asymmetry of interruptions found in this study or the relative frequency of different compound microsatellites in the human genome. Therefore, we cannot rule out the possibility of a mutational bias leading to convergent or parallel evolution in flanking sequences.     

Alu Repeats in the Human Genome

Highly repetitive DNA sequences account for more than 50% of the human genome. The L1 and Alu families harbor the most common mammalian long and short interspersed elements. An Alu element is a dimer of similar, but not identical, fragments of total size about 300 bp, and originates from the 7SL RNA gene. Each element contains a bipartite promoter for RNA polymerase III, a poly(A) tract located between the monomers, a 3"-terminal poly(A) tract, and numerous CpG islands, and is flanked by short direct repeats. Alu repeats constitute more than 10% of the human genome and are capable of retroposition. Possibly, these elements played an important part in genome evolution. Insertion of an Alu element into a functionally important genome region or other Alu-dependent alterations of gene functions cause various hereditary disorders and are probably associated with carcinogenesis. In total, 14 Alu families differing in diagnostic mutations are known. Some of these, which are present in the human genome, are polymorphic and relatively recently have been inserted into new loci. Alu copies transposed during ethnic divergence of the human population are useful markers for evolutionary genetic studies.     

Experimental Evolution Yields Hundreds of Mutations in a Functional Viral Genome

Two lines of the bacteriophage T7 were grown to fix mutations indiscriminately, using a combination of population bottlenecks and mutagenesis. Complete genome sequences revealed 404 and 299 base substitutions in the two lines, the largest number characterized in functional microbial genomes so far. Missense substitutions outnumbered silent substitutions. Silent substitutions occurred at similar rates between essential and nonessential genes, but missense substitutions occurred at a higher rate in nonessential genes than in essential genes, as expected if they were less deleterious in the nonessential genes. Viral fitness declined during this protocol, and subsequent passaging of each mutated line in large population sizes restored some of the lost fitness. Substitution levels during these recoveries were less than 6% of those during the bottleneck phase, and only two changes during recovery were reversions of the original mutations. Exchanges of genomic fragments between the two recovered lines revealed that fitness effects of some substitutions were not additive—that interactions were accumulating which could lead to incompatibility between the diverged genomes. Based on these results, unprecedented high rates of nucleotide and functional divergence in viral genomes should be attainable experimentally by using repeated population bottlenecks at a high mutation rate interspersed with recovery. Present address (M. R. Badgett): Ambion, Inc., 2130 Woodward, Austin, TX 78744, USA Present address (D. Rokyta): Department of Biological Sciences, University of Idaho, Moscow, ID 83844, USA     

Covariation of Mitochondrial Genome Size with Gene Lengths: Evidence for Gene Length Reduction During Mitochondrial Evolution

Reduction of genome size and gene shortening have been observed in a number of parasitic and mutualistic intracellular symbionts. Reduction of coding capacity is also a unifying principle in the evolutionary history of mitochondria, but little is known about the evolution of gene length in mitochondria. The genes for cytochrome c oxidase subunits I–III, cytochrome b, and the large and small subunit rRNAs are, with very few exceptions, always found on the mitochondrial genome. These resident mitochondrial genes can therefore be used to test whether the reduction in gene lengths observed in a number of intracellular symbionts is also seen in mitochondria. Here we show that resident mitochondrial gene products are shorter than their corresponding counterparts in -proteobacteria and, furthermore, that the reduction of mitochondrial genome size is correlated with a reduction in the length of the corresponding resident gene products. We show that relative genomic AT content, which has been identified as a factor influencing gene lengths in other systems, cannot explain gene length/genome size covariance observed in mitochondria. Our data are therefore in agreement with the idea that gene length evolves as a consequence of selection for smaller genomes, either to avoid accumulation of deleterious mutations or triggered by selection for a replication advantage.     

Protein and Genome Evolution in Mammalian Cells for Biotechnology Applications

Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology.     

改进酶稳定性的定向进化技术

酶作为一种生物催化剂,以其独特的优良特性,在绿色化学和清洁生产中得到了广泛的应用。随着酶定向进化技术的建立和发展,通过定向进化改进酶稳定性的研究越来越多。详细综述了各种定向进化方法的特点及在提高酶稳定性方面的应用,并从结构和功能的角度进一步解释了相关机理。     

Genome screen for twinning rate QTL in four North American Holstein families

The objective of this study was to identify twinning rate quantitative trait loci (QTL) by typing pooled samples in a preliminary screening followed by interval mapping to test QTL effects. Four elite North American Holstein half-sib sire families with high twinning rate predicted transmitting abilities (PTA) were used in this study. Chromosomes 5, 7, 19 and 23 were not genotyped as these chromosomes were scanned for QTL in these families in a previous study. DNA was extracted from phenotypically extreme sons in each sire family. Two pools were prepared from sons of sires in each phenotypic tail, two each for high and low PTA levels for twinning rates. Each pool contained DNA from 4 to 15% of all sons of the sire depending on family. A total of 268 fluorescently labelled microsatellite markers were tested for heterozygosity in sires. About 135--170 informative markers per family were genotyped using pooled DNA samples. Based on the preliminary evidence for potential twinning rate QTL from pooled typing, interval mapping was performed subsequently on 12 chromosomal regions by family combinations. Evidence of QTL for twinning rate was found in one family on BTA 21 and 29 at a chromosome-wide P<0.05 and on BTA 8, 10 and 14 with a chromosome-wide P<0.01.