A fine-scale chimpanzee genetic map from population sequencing

To study the evolution of recombination rates in apes, we developed methodology to construct a fine-scale genetic map from high-throughput sequence data from 10 Western chimpanzees, Pan troglodytes verus. Compared to the human genetic map, broad-scale recombination rates tend to be conserved, but with exceptions, particularly in regions of chromosomal rearrangements and around the site of ancestral fusion in human chromosome 2. At fine scales, chimpanzee recombination is dominated by hotspots, which show no overlap with those of humans even though rates are similarly elevated around CpG islands and decreased within genes. The hotspot-specifying protein PRDM9 shows extensive variation among Western chimpanzees, and there is little evidence that any sequence motifs are enriched in hotspots. The contrasting locations of hotspots provide a natural experiment, which demonstrates the impact of recombination on base composition.     

High-resolution mapping of crossovers reveals extensive variation in fine-scale recombination patterns among humans

Recombination plays a crucial role in meiosis, ensuring the proper segregation of chromosomes. Recent linkage disequilibrium (LD) and sperm-typing studies suggest that recombination rates vary tremendously across the human genome, with most events occurring in narrow "hotspots." To examine variation in fine-scale recombination patterns among individuals, we used dense, genome-wide single-nucleotide polymorphism data collected in nuclear families to localize crossovers with high spatial resolution. This analysis revealed that overall recombination hotspot usage is similar in males and females, with individual hotspots often active in both sexes. Across the genome, roughly 60% of crossovers occurred in hotspots inferred from LD studies. Notably, however, we found extensive and heritable variation among both males and females in the proportion of crossovers occurring in these hotspots.     

Genetics. Motivating hotspots

Only recently have we begun to characterize fine-scale recombination rates in mammals. In her Perspective, Przeworski discusses the work by Myers et al. in which linkage disequilibrium data have been used to produce a high-resolution recombination map for most of the human genome. More than 25,000 putative hotspots have been identified, as well as the first motifs that appear to influence their intensity.     

The fine-scale structure of recombination rate variation in the human genome

The nature and scale of recombination rate variation are largely unknown for most species. In humans, pedigree analysis has documented variation at the chromosomal level, and sperm studies have identified specific hotspots in which crossing-over events cluster. To address whether this picture is representative of the genome as a whole, we have developed and validated a method for estimating recombination rates from patterns of genetic variation. From extensive single-nucleotide polymorphism surveys in European and African populations, we find evidence for extreme local rate variation spanning four orders in magnitude, in which 50% of all recombination events take place in less than 10% of the sequence. We demonstrate that recombination hotspots are a ubiquitous feature of the human genome, occurring on average every 200 kilobases or less, but recombination occurs preferentially outside genes.     

A fine-scale map of recombination rates and hotspots across the human genome

Genetic maps, which document the way in which recombination rates vary over a genome, are an essential tool for many genetic analyses. We present a high-resolution genetic map of the human genome, based on statistical analyses of genetic variation data, and identify more than 25,000 recombination hotspots, together with motifs and sequence contexts that play a role in hotspot activity. Differences between the behavior of recombination rates over large (megabase) and small (kilobase) scales lead us to suggest a two-stage model for recombination in which hotspots are stochastic features, within a framework in which large-scale rates are constrained.     

Recombination and the nature of bacterial speciation

Genetic surveys reveal the diversity of bacteria and lead to the questioning of species concepts used to categorize bacteria. One difficulty in defining bacterial species arises from the high rates of recombination that results in the transfer of DNA between relatively distantly related bacteria. Barriers to this process, which could be used to define species naturally, are not apparent. Here, we review conceptual models of bacterial speciation and describe our computer simulations of speciation. Our findings suggest that the rate of recombination and its relation to genetic divergence have a strong influence on outcomes. We propose that a distinction be made between clonal divergence and sexual speciation. Hence, to make sense of bacterial diversity, we need data not only from genetic surveys but also from experimental determination of selection pressures and recombination rates and from theoretical models.     

The Hin invertasome: protein-mediated joining of distant recombination sites at the enhancer

The Hin protein binds to two cis-acting recombination sites and catalyzes a site-specific DNA inversion reaction that regulates the expression of flagellin genes in Salmonella. In addition to the Hin protein and the two recombination sites that flank the invertible segment, a third cis-acting recombinational enhancer sequence and the Fis protein, which binds to two sites within the enhancer, are required for efficient recombination. Intermediates of this reaction were trapped during DNA strand cleavage and analyzed by gel electrophoresis and electron microscopy in order to determine their structure and composition. The analyses demonstrate that the recombination sites are assembled at the enhancer into a complex nucleo-protein structure (termed the invertasome) with the looping of the three segments of intervening DNA. Antibody studies indicated that Fis physically interacts with Hin and that both proteins are intimately associated with the invertasome. In order to achieve this protein-protein interaction and assemble the invertasome, the substrate DNA must be supercoiled.     

网纹蟒非入侵式采样和线粒体基因组分析

采用非入侵式采样,提取网纹蟒Python reticulatus粪便和新鲜蛇蜕的基因组,探讨非入侵式采样在蛇类研究中的可能性。利用聚合酶链式反应（PCR）测定和拼接网纹蟒线粒体基因组全序列,结合GenBank中蟒科Pythonidae线粒体基因组全序列,对蟒科物种进行比较分析。结果发现:蛇蜕中提取的DNA优于粪便,液氮处理能提高DNA质量浓度。网纹蟒线粒体基因组全长17 641 bp,基因排布和结构同蟒科物种一致,但部分基因起始密码子和终止密码子存在差异。根据系统发育树分析推测,与蚺科Boidae相比,蟒科和闪鳞蛇科Xenopeltidae进化关系更接近。对蛇类物种进化过程的分析发现:热点区域存在2个相似度非常高的控制区,系统树上的拓扑结构呈簇状排列,推测2个控制区的结构来源于一个祖先。这种种内进化的模式为协同进化。     

RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination

The vast repertoire of immunoglobulins and T cell receptors is generated, in part, by V(D)J recombination, a series of genomic rearrangements that occur specifically in developing lymphocytes. The recombination activating gene, RAG-1, which is a gene expressed exclusively in maturing lymphoid cells, was previously isolated. RAG-1 inefficiently induced V(D)J recombinase activity when transfected into fibroblasts, but cotransfection with an adjacent gene, RAG-2, has resulted in at least a 1000-fold increase in the frequency of recombination. The 2.1-kilobase RAG-2 complementary DNA encodes a putative protein of 527 amino acids whose sequence is unrelated to that of RAG-1. Like RAG-1, RAG-2 is conserved between species that carry out V(D)J recombination, and its expression pattern correlates precisely with that of V(D)J recombinase activity. In addition to being located just 8 kilobases apart, these convergently transcribed genes are unusual in that most, if not all, of their coding and 3' untranslated sequences are contained in single exons. RAG-1 and RAG-2 might activate the expression of the V(D)J recombinase but, more likely, they directly participate in the recombination reaction.     

Hot spots for growth hormone gene deletions in homologous regions outside of Alu repeats

Familial growth hormone deficiency type 1A is an autosomal recessive disease caused by deletion of both growth hormone-1 (GH1) alleles. Ten patients from heterogeneous geographic origins showed differences in restriction fragment length polymorphism haplotypes in nondeleted regions that flanked GH1, suggesting that these deletions arose from independent unequal recombination events. Deoxyribonucleic acid (DNA) samples from nine of ten patients showed that crossovers occurred within 99% homologous, 594-base pair (bp) segments that flanked GH1. A DNA sample from one patient indicated that the crossover occurred within 454-bp segments that flanked GH1 and contained 274-bp repeats that are 98% homologous. Although Alu repeats, which are frequent sites of recombination, are adjacent to GH1, they were not involved in any of the recombination events studied. These results suggest that length and degree of DNA sequence homology are important in defining recombination sites that resulted in GH1 deletions.     

Synthesis of a site-specific DNA-binding peptide

The Hin recombinase binds to specific sites on DNA and mediates a recombination event that results in DNA inversion. In order to define the DNA-binding domain of the Hin protein two peptides 31 and 52 amino acids long were synthesized. Even though the 31mer encompassed the sequence encoding the putative helix-coil-helix-binding domain, it was not sufficient for binding to the 26-base pair DNA crossover site. However, the 52mer specifically interacted with the site and also effectively inhibited the Hin-mediated recombination reaction. The 52mer bound effectively to both the 26-base pair complete site and to a 14-base pair "half site." Nuclease and chemical protection studies with the 52mer helped to define the DNA base pairs that contributed to the specificity of binding. The synthetic peptide provides opportunities for new approaches to the study of the nature of protein-DNA interaction.     

转座子在害虫生物防治中的应用

转座子是一类散布在基因组中序列重复的DNA片段,它们可以不依靠同源重组而能在基因组中移动。综述了转座子的特征、分类、种类和分布,介绍了利用转座子获得的转基因昆虫和改良杀虫剂在害虫生物防治中的应用,并对转座子在害虫生物防治中的研究和应用前景进行展望。     

Recombinase-mediated gene activation and site-specific integration in mammalian cells

A binary system for gene activation and site-specific integration, based on the conditional recombination of transfected sequences mediated by the FLP recombinase from yeast, was implemented in mammalian cells. In several cell lines, FLP rapidly and precisely recombined copies of its specific target sequence to activate an otherwise silent beta-galactosidase reporter gene. Clones of marked cells were generated by excisional recombination within a chromosomally integrated copy of the silent reporter. By the reverse reaction, integration of transfected DNA was targeted to a specific chromosomal site. The results suggest that FLP could be used to mosaically activate or inactivate transgenes for analysis of vertebrate development, and to efficiently integrate transfected DNA at predetermined chromosomal locations.     

CRISPR/Cas9系统在植物基因组定点编辑中的研究进展

当外源DNA通过转基因技术导入植物细胞后,会以同源重组或非同源重组两种不同的方式整合到基因组中,进而获得相应的目标性状。外源DNA与受体细胞序列相同或相近的位点发生重新组合,从而整合到受体细胞的染色体上称之为同源重组;当发生了DNA双链断裂的细胞为了避免DNA或染色体断裂而造成DNA降解或对生命力的影响,而强行将2个DNA断端彼此连接在一起时则为非同源重组。发生非同源重组的细胞其基因组常出现核苷酸片段的插入和/或缺失以及其他突变等多种情况,使得研究者无法得到精确控制的突变结果;而发生同源重组的细胞基因组序列通常不变,通过加入同源重组的供体DNA,可以实现对基因组的精确修饰和改造。由于在植物中产生自发同源重组的概率很低,对植物基因组进行精确修饰和改造非常困难,位点特异性核酸酶的出现和应用,大大提升了同源重组的效率,使基因组编辑变得更加高效和精确,从而使得对包括植物在内的任何物种进行基因组编辑都将成为可能。锌指核酸酶（ZFN）和TALE核酸酶（TALENs）是能够使DNA的靶位点产生DNA双链断裂进而实现基因组定点编辑的常用系统,但在具体应用中发现这两种系统存在着许多缺陷和不足,如脱靶效应、与基因组进行特异结合与染色体位置及邻近序列有关等,另外技术难度大、构建组装时间长也限制了其应用。CRISPR/Cas系统广泛存在于细菌及古生菌中, 是机体长期进化形成的RNA指导的降解入侵病毒或噬菌体DNA的适应性免疫系统。Ⅱ型CRISPR/Cas系统经过密码子优化等改造后已成为继锌指核酸酶ZFNs和TALENs后的新型高效定点编辑的新技术,具有突变效率高、制作简单、易操作及成本低的特点。目前,该技术成功应用于人类细胞、斑马鱼、小鼠以及细菌的基因组精确编辑,编辑的类型包括基因的定点插入、小片段的缺失、多个位点同时突变、基因定点的indel突变等。目前,CRISPR/Cas系统在植物中的应用还比较有限,但该技术为植物基因工程的发展呈现了美好的前景。文中首先简要介绍了CRISPR/Cas系统的组成和基本原理,进而详细综述了该技术在植物内源基因和外源基因定点编辑中的应用,主要列举了自CRISPR/Cas系统改造成功以来利用该系统对单子叶和双子叶植物进行基因组定点编辑的案例,最后对基因组编辑技术在农业和植物基因工程上的应用进行了展望,希望能够为开展该领域研究的科研工作者提供参考。     

微卫星标记的基因组学研究进展

微卫星标记是真核生物基因组中分布均匀、含量高的一类短重复序列。本文从微卫星标记在基因组中的分布、微卫星标记的多态性、微卫星标记的进化、微卫星标记的功能及应用等几个方面对微卫星标记的基因组学研究进展进行综述。     

Frequent recombination in a saltern population of Halorubrum

Sex and recombination are driving forces in the evolution of eukaryotes. Homologous recombination is known to be the dominant process in the divergence of many bacterial species. For Archaea, the only direct evidence bearing on the importance or natural occurrence of homologous recombination is anecdotal reports of mosaicism from comparative genomic studies. Genetic studies, however, reveal that recombination may play a significant role in generating diversity among members of at least one archaeal group, the haloarchaea. We used multi-locus sequence typing to demonstrate that haloarchaea exchange genetic information promiscuously, exhibiting a degree of linkage equilibrium approaching that of a sexual population.     

Temporal fragmentation of speciation in bacteria

Because bacterial recombination involves the occasional transfer of small DNA fragments between strains, different sets of niche-specific genes may be maintained in populations that freely recombine at other loci. Therefore, genetic isolation may be established at different times for different chromosomal regions during speciation as recombination at niche-specific genes is curtailed. To test this model, we separated sequence divergence into rate and time components, revealing that different regions of the Escherichia coli and Salmonella enterica chromosomes diverged over a approximately 70-million-year period. Genetic isolation first occurred at regions carrying species-specific genes, indicating that physiological distinctiveness between the nascent Escherichia and Salmonella lineages was maintained for tens of millions of years before the complete genetic isolation of their chromosomes.     

大熊猫分布区珍稀濒危物种丰富度空间格局与热点区分析

分析物种丰富度格局和热点区是保护生物多样性的有效手段和途径。本文基于珍稀濒危物种名录信息,结合遥感影像、文献信息和专家经验,研究了大熊猫分布区内珍稀濒危物种及中国特有珍稀濒危物种的丰富度空间格局,确定了物种丰富度热点区,分析了大熊猫保护区对热点区保护的有效性。结果表明:大熊猫分布区内,与大熊猫同域分布的珍稀濒危物种有293种,包括哺乳动物109种、鸟类58种、爬行动物18种、两栖动物35种、高等植物73种;其中,IUCN极危物种11种、濒危物种48种、易危物种111种、近危物种74种,列入CITES附录Ⅱ2种,国家一级保护植物7种,国家二级保护植物36种;293种珍稀濒危物种中,133种为中国特有,19种为大熊猫分布区特有。大熊猫分布区中,岷山的文县和平武,邛崃山的汶川、宝兴和康定珍稀濒危物种丰富度最高,达到15~19种/km2,物种密集区呈块状或线状破碎化分布;秦岭山系、岷山山系和邛崃山系为中国特有珍稀濒危物种丰富度最高的集中分布区,丰富度为5~6种/km2;海拔1400~1600m和1800~2000m,分别为珍稀濒危物种和中国特有珍稀濒危物种丰富度最高的海拔区段,物种丰富度达186种和77种;800~2800m是珍稀濒危物种和中国特有珍稀濒危物种分布最密集的海拔范围,分别覆盖了88.4%的珍稀濒危物种和87.2%的中国特有珍稀濒危物种。大熊猫分布区有6个珍稀濒危物种丰富度热点区,占分布区总面积的10.5%,覆盖了分布区85%的珍稀濒危物种;中国特有珍稀濒危物种热点区,占分布区总面积的17.2%,覆盖了85%的中国特有珍稀濒危物种。大熊猫保护区,覆盖了27.6%的珍稀濒危物种热点区和21.4%的中国特有珍稀濒危物种热点区,保护了71.7%的珍稀濒危物种和70.7%的中国特有珍稀濒危物种。本研究结果可为提高大熊猫保护区的有效性和维护大熊猫种群的持续生存提供科学依据。      

DNA looping generated by DNA bending protein IHF and the two domains of lambda integrase

The multiprotein-DNA complexes that participate in bacteriophage lambda site-specific recombination were used to study the combined effect of protein-induced bending and protein-mediated looping of DNA. The protein integrase (Int) is a monomer with two autonomous DNA binding domains of different sequence specificity. Stimulation of Int binding and cleavage at the low affinity core-type DNA sites required interactions with the high affinity arm-type sites and depended on simultaneous binding of the sequence-specific DNA bending protein IHF (integration host factor). The bivalent DNA binding protein is positioned at high affinity sites and directed, by a DNA bending protein, to interactions with distant lower affinity sites. Assembly of this complex is independent of protein-protein interactions.     

Sequence-dependent pausing of single lambda exonuclease molecules

Lambda exonuclease processively degrades one strand of duplex DNA, moving 5'-to-3' in an ATP-independent fashion. When examined at the single-molecule level, the speeds of digestion were nearly constant at 4 nanometers per second (12 nucleotides per second), interspersed with pauses of variable duration. Long pauses, occurring at stereotypical locations, were strand-specific and sequence-dependent. Pause duration and probability varied widely. The strongest pause, GGCGAT TCT, was identified by gel electrophoresis. Correlating single-molecule dwell positions with sequence independently identified the motif GGCGA. This sequence is found in the left lambda cohesive end, where exonuclease inhibition may contribute to the reduced recombination efficiency at that end.