扬子鳄种群的微卫星DNA多态及其遗传多样性保护对策分析

扬子鳄(Alligator sinensis)是中国特有的珍稀保护动物,为保护这一濒危物种,我国于80年代初在安徽宣州先后建立了扬子鳄繁殖研究中心和国家级扬子鳄自然保护区,现饲养种群存鳄数量已达10000余头。为了揭示扬子鳄种群的遗传结构,共采集了39个个体的样品,其中包括6件剥制标本,按代系不同,分为野生群、F1代饲养群及F1代饲养群,用微卫星DNA分子标记对其进行研究。分析结果显示：扬子鳄种群在微卫星水平表现出很低的遗传多样性,平均等位基因数A=2．38,平均有效等位基因数Ne=1．60,平均观察杂合度He=0．374,平均期望杂合度He=0．350,平均多态信息含量PIG=0．327,3个群体间A、Ne、Ho、He、PIC及各微卫星位点等位基因频率分布无显著差异,但F1代饲养群在Ami-μ-6和Ami-μ-222两个位点表现出极显著的遗传不平衡。扬子鳄种群遗传多样性水平低下的主要原因是近几十年来种群数量大幅减少造成,现阶段应将全部现存的扬子鳄作为一个整体加以保护。     

使用SSR和mtVNTR分子标记识别扬子鳄个体

扬子鳄是中国特有的濒危物种,为有效避免种群衰退,最大限度地保持该物种现有的遗传多样性,有必要对种群的个体进行个体识别研究,以便重建遗传谱系,指导现有繁育工作。应用SSR(Simple sequence repeats)与mtVNTR(Variable number tandem repeats on mitochondrial DNA)两种分子标记对扬子鳄39个个体进行了个体识别分析,结果显示：8个SSR座位的累计个体识别率与累计父权排除率分别达0．9968、0．7697,mtVNTR的个体识别率为0．9146,联合SSR与mtVNTR两种分子标记的累计个体识别率理论值达0．9997,并在实际分析中将39个扬子鳄个体完全区分开,其区分能力较RAPD(Random amplified polymorphic DNA)、AFLP(Amplified fragment length polymorphism)及mtDNA控制区5’端序列分析等分子标记要高。此外,SSR和mtvNTR还可对某些低频等位基因及其携带个体做有效的筛查,这对今后进行大量扬子鳄个体的分子标记识别和群体的遗传谱系建立等工作将具有一定实际意义[动物学报51(3)：501—506,2005]。     

三江源和祁连山国家公园雪豹种群的遗传结构分析

掌握遗传信息对濒危物种的保护和管理具有重要意义。本研究在我国雪豹重要分布区祁连山和三江源国家公园分别采集粪便样品,利用mtDNA的cyt b基因、微卫星多态性位点进行了雪豹的物种鉴定、个体识别和种群遗传结构评估。在采集286份疑似雪豹粪便样品中,成功的对86份雪豹样品进行了扩增鉴定,利用微卫星位点进行个体识别获得41只雪豹个体,其中祁连山国家公园26只,三江源国家公园15只。通过等位基因数、有效等位基因数、观测杂合度、期望杂合度、多态信息含量等指标进行种群遗传多样性评估,认为雪豹种群遗传多样性相对较低,但祁连山国家公园雪豹种群遗传多样性相对较高。STRUCTURE进行群体遗传结构分析表明,4个种群可以划分为3个遗传类群,祁连山国家公园的种群（YCW和QLS）与三江源国家种群(DC和SJ)的遗传差异,可能与种群间的地理隔离存在明显的相似性。     

都柳江鲤、鲫和草鱼种群mtDNA控制区及遗传多样性分析

采用PCR和DNA测序技术对贵州都柳江鲤(Cyprinus carpio)、鲫(Carassius auratus)和草鱼(Ctenopharyngodon idellus)种群的mtDNA控制区序列及遗传多样性进行了研究。获得了都柳江鲤、鲫和草鱼mtDNA控制区长度分别为899~901 bp、787 bp和901~905 bp的序列。该3种鱼类控制区碱基A、T含量较高,G含量最低。识别了该3种鱼类mtDNA控制区终止序列区、中央保守区和保守序列区等保守序列。其中,除CSB-2和CSB-3碱基组成相同外,其余核心序列碱基组成存在着差异。都柳江鲤、鲫和草鱼种群mtDNA控制区分别有24、24和11个多态位点,分属12、17和8个单倍型。都柳江鲤、鲫种群遗传多样性较高,草鱼种群遗传多样性较低。因此,有必要开展都柳江草鱼种群遗传多样性的保护。     

四川裂腹鱼乌江种群mtDNA控制区序列的遗传多样性分析

采用PCR扩增和直接测序法分析了四川裂腹鱼乌江种群32个个体mtDNA控制区的序列差异和遗传多样性.在该种群mtDNA控制区长度为464 bp的同源序列中,共计有9个变异位点,占总位点数的1.94%.32个个体共定义了9种单倍型,单倍型间平均遗传距离(P)为0.0060,单倍型多样度(Hd)为0.768.核苷酸多样性(π)为0.00324,平均核苷酸差异数(K)为1.500.用单倍型间遗传距离构建的NJ分子系统树聚为两个分支.结果提示四川裂腹鱼乌江种群遗传多样性匮乏,保护四川裂腹鱼乌江种群刻不容缓.     

黑麂皖-浙分布中心种群的遗传多样性

本文基于黑麂线粒体控制区序列对皖浙分布中心的4个黑麂种群的遗传多样性和基因流进行了研究。结果显示:4个种群的33个个体中有14个变异位点,占分析序列长度的2·91 %,并由此定义了12个单倍型;遗传多样性检测结果显示4个种群中开化种群具有最高的遗传多样性,应予以优先保护;4个种群间尚存在着一定的基因流,但存在可能由于遗传漂变而产生分化的危险。从Tajima’s D和Fu and Li’s D值的估算结果来看,这4个黑麂种群相对于中性进化的歧异度并没有明显的偏离,具有较为稳定的种群结构(P>0·1),没有明显的证据显示这4个黑麂种群间存在很强的平衡选择。     

黑龙江省不同山系狍种群遗传多样性分析

狍（Capreolus pygargus）为我国重要的经济动物,并且是国家一级保护动物——东北虎(Panthera tigris altaica)的主要食物之一。因此,深入了解狍各个地理单元内种群的遗传变异,可以为我们制定保护管理策略提供依据,进而恢复珍稀濒危物种的野外种群数量。对32个不同狍个体（来自3个不同山系）的线粒体DNA控制区的部分序列进行了测定和群体分析,发现了50个变异位点,定义了27种单倍型,其单倍型多样性(h)平均值为0.978、核苷酸多样性(π)平均值为0.022 60,种群总体遗传多样性较高;在3个地理单元的狍种群中,大兴安岭地区的狍种群具有较高的遗传多样性,应予以优先保护。从Tajima’s D和Fu & Li’s D值的估算结果表明,这3个狍种群与中性进化的歧异度相比,并没有明显的偏离(P> 0.1),无明显的证据显示这3个狍种群间存在很强的平衡选择。     

扬子鳄的保护遗传学研究进展

保护遗传学是主要研究与灭绝风险相关的遗传因素以及如何利用遗传学管理方法降低物种灭绝风险的科学,是保护生物学和分子遗传学的交叉学科.近几十年来,遗传学研究在生物多样性保护的理论和实践中发挥着越来越重要的作用.本文回顾了AFLP、mtDNA D-loop、RAPD、微卫星DNA、MHC等DNA分子标记技术在扬子鳄的样品采集、生物多样性、个体鉴定、繁殖管理、野外放归等保护遗传学方面研究所取得的一些进展.对扬子鳄保护的工作提出了建议:重建扬子鳄的谱系;加大对扬子鳄的放归力度;加强饲养种群之间的基因交流;借鉴密河鳄的管理经验.     

不同保存方法对川金丝猴粪便DNA提取效果的影响

目的:川金丝猴(Rhinopithecus roxellana)是我国特有珍稀物种,其粪便作为一种非损伤性样品,为珍稀濒危动物的种群数量调查、遗传多样性评价、亲缘关系、系统进化等研究带来了很大便利,本研究试图建立高效、简便的粪便样品保存方法。方法:在现有珍稀濒危动物粪便样品保存方法的基础上,分别使用干燥法、冷冻法和干燥-冷冻法保存川金丝猴的粪便样品,比较了不同保存方法的DNA提取效果,以及对mtDNA控制区片段的PCR扩增成功率和微卫星基因的PCR扩增效率。结果:干燥法、冷冻法和干燥-冷冻法三种不同保存方法保存粪便1周时间后,提取的粪便DNA样本扩增mtDNA片段的成功率均为92%,微卫星基因的扩增成功率分别为79%、78%、80%;保存2个月后,mtDNA片段扩增成功率分别为80%、76%和80%,微卫星基因扩增成功率分别为65%、61%、67%;保存6个月后,mtDNA片段扩增成功率分别为56%、52%和64%,微卫星基因扩增成功率分别40%、34%、46%。因此,随着保存时间的增长,三种方法的保存效率都将明显降低,但干燥-冷冻法得到的DNA样本扩增成功率相对较高。结论:粪便样品能够为川金丝猴的遗传多样性评价等相关研究提供有效信息,干燥-冷冻法保存能够更为有效的保证DNA的提取和基因扩增效率。     

基于粪便DNA的东北马鹿遗传多样性

东北马鹿(Cervus canadensis xanthopygus)为国家二级重点保护野生动物,近些年其种群数量急剧下降、分布区不断退缩、种群基因交流受阻,很多地区更是难觅其踪迹。亟需对其种群的遗传变化,特别是遗传多样性和近交衰退等种群遗传信息开展进一步评价,增强保护与管理的针对性。本研究在大、小兴安岭和长白山脉的6个重点研究区域,共收集409份疑似马鹿粪便样本。首先基于mtDNA Cyt b基因测序技术进行物种鉴定,并对鉴定为马鹿的阳性样本利用微卫星技术进行个体识别。结果共识别出172只东北马鹿个体;Cyt b基因序列共检测出14个变异位点和11个单倍型,单倍型多样性为0.849 (0.105～0.732),核苷酸多样性为0.678%(0.099%～0.775%)。10个微卫星位点检测出种群平均等位基因数为5.7 (5.2～7.2),有效等位基因数为3.3 (2.5～4.1),观测杂合度为0.687 (0.644～0.725),期望杂合度为0.619 (0.564～0.689),近交系数为-0.113 (-0.160～-0.037)。结果表明,东北马鹿种群遗传多样性处于中等水平,其...     

FBH1 Helicase Disrupts RAD51 Filaments in Vitro and Modulates Homologous Recombination in Mammalian Cells

Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.     

Human HEL308 localizes to damaged replication forks and unwinds lagging strand structures

HEL308 is a superfamily II DNA helicase, conserved from archaea through to humans. HEL308 family members were originally isolated by their similarity to the Drosophila melanogaster Mus308 protein, which contributes to the repair of replication-blocking lesions such as DNA interstrand cross-links. Biochemical studies have established that human HEL308 is an ATP-dependent enzyme that unwinds DNA with a 3' to 5' polarity, but little else is know about its mechanism. Here, we show that GFP-tagged HEL308 localizes to replication forks following camptothecin treatment. Moreover, HEL308 colocalizes with two factors involved in the repair of damaged forks by homologous recombination, Rad51 and FANCD2. Purified HEL308 requires a 3' single-stranded DNA region to load and unwind duplex DNA structures. When incubated with substrates that model stalled replication forks, HEL308 preferentially unwinds the parental strands of a structure that models a fork with a nascent lagging strand, and the unwinding action of HEL308 is specifically stimulated by human replication protein A. Finally, we show that HEL308 appears to target and unwind from the junction between single-stranded to double-stranded DNA on model fork structures. Together, our results suggest that one role for HEL308 at sites of blocked replication might be to open up the parental strands to facilitate the loading of subsequent factors required for replication restart.     

Studies on human DNA polymerase epsilon and GINS complex and their role in DNA replication

In eukaryotic cells, DNA replication is carried out by the coordinated action of three DNA polymerases (Pols), Pol α, δ, and ε. In this report, we describe the reconstitution of the human four-subunit Pol ε and characterization of its catalytic properties in comparison with Pol α and Pol δ. Human Pol ε holoenzyme is a monomeric complex containing stoichiometric subunit levels of p261/Pol 2, p59, p17, and p12. We show that the Pol ε p261 N-terminal catalytic domain is solely responsible for its ability to catalyze DNA synthesis. Importantly, human Pol (hPol) ε was found more processive than hPol δ in supporting proliferating cell nuclear antigen-dependent elongation of DNA chains, which is in keeping with proposed roles for hPol ε and hPol δ in the replication of leading and lagging strands, respectively. Furthermore, GINS, a component of the replicative helicase complex that is composed of Sld5, Psf1, Psf2, and Psf3, was shown to interact weakly with all three replicative DNA Pols (α, δ, and ε) and to markedly stimulate the activities of Pol α and Pol ε. In vivo studies indicated that siRNA-targeted depletion of hPol δ and/or hPol ε reduced cell cycle progression and the rate of fork progression. Under the conditions used, we noted that depletion of Pol ε had a more pronounced inhibitory effect on cellular DNA replication than depletion of Pol δ. We suggest that reduction in the level of Pol δ may be less deleterious because of its collision-and-release role in lagging strand synthesis.     

Distributions of Topological States in Circular DNA

A distinctive feature of closed circular DNA molecules is their particular topological state, which cannot be altered by any conformational rearrangement short of breaking at least one strand. This topological constraint opens unique possibilities for experimental studies of the distributions of topological states created in different ways. Primarily, the equilibrium distributions of topological properties are considered in the review. It is described how such distributions can be obtained and measured experimentally, and how they can be computed. Comparison of the calculated and measured equilibrium distributions over the linking number of complementary strands, equilibrium fractions of knots and links formed by circular molecules has provided much valuable information about the properties of the double helix. Study of the steady-state fraction of knots and links created by type II DNA topoisomerases has revealed a surprising property of the enzymes: their ability to reduce these fractions considerably below the equilibrium level.     

Topoisomerase II Regulates the Maintenance of DNA Methylation

The maintenance of DNA methylation in nascent DNA is a critical event for numerous biological processes. Following DNA replication, DNMT1 is the key enzyme that strictly copies the methylation pattern from the parental strand to the nascent DNA. However, the mechanism underlying this highly specific event is not thoroughly understood. In this study, we identified topoisomerase IIα (TopoIIα) as a novel regulator of the maintenance DNA methylation. UHRF1, a protein important for global DNA methylation, interacts with TopoIIα and regulates its localization to hemimethylated DNA. TopoIIα decatenates the hemimethylated DNA following replication, which might facilitate the methylation of the nascent strand by DNMT1. Inhibiting this activity impairs DNA methylation at multiple genomic loci. We have uncovered a novel mechanism during the maintenance of DNA methylation.     

The DDN Catalytic Motif Is Required for Metnase Functions in Non-homologous End Joining (NHEJ) Repair and Replication Restart

Metnase (or SETMAR) arose from a chimeric fusion of the Hsmar1 transposase downstream of a protein methylase in anthropoid primates. Although the Metnase transposase domain has been largely conserved, its catalytic motif (DDN) differs from the DDD motif of related transposases, which may be important for its role as a DNA repair factor and its enzymatic activities. Here, we show that substitution of DDN⁶¹⁰ with either DDD⁶¹⁰ or DDE⁶¹⁰ significantly reduced in vivo functions of Metnase in NHEJ repair and accelerated restart of replication forks. We next tested whether the DDD or DDE mutants cleave single-strand extensions and flaps in partial duplex DNA and pseudo-Tyr structures that mimic stalled replication forks. Neither substrate is cleaved by the DDD or DDE mutant, under the conditions where wild-type Metnase effectively cleaves ssDNA overhangs. We then characterized the ssDNA-binding activity of the Metnase transposase domain and found that the catalytic domain binds ssDNA but not dsDNA, whereas dsDNA binding activity resides in the helix-turn-helix DNA binding domain. Substitution of Asn-610 with either Asp or Glu within the transposase domain significantly reduces ssDNA binding activity. Collectively, our results suggest that a single mutation DDN⁶¹⁰ → DDD⁶¹⁰, which restores the ancestral catalytic site, results in loss of function in Metnase.     

Formation of a stable RuvA protein double tetramer is required for efficient branch migration in vitro and for replication fork reversal in vivo

In bacteria, RuvABC is required for the resolution of Holliday junctions (HJ) made during homologous recombination. The RuvAB complex catalyzes HJ branch migration and replication fork reversal (RFR). During RFR, a stalled fork is reversed to form a HJ adjacent to a DNA double strand end, a reaction that requires RuvAB in certain Escherichia coli replication mutants. The exact structure of active RuvAB complexes remains elusive as it is still unknown whether one or two tetramers of RuvA support RuvB during branch migration and during RFR. We designed an E. coli RuvA mutant, RuvA2(KaP), specifically impaired for RuvA tetramer-tetramer interactions. As expected, the mutant protein is impaired for complex II (two tetramers) formation on HJs, although the binding efficiency of complex I (a single tetramer) is as wild type. We show that although RuvA complex II formation is required for efficient HJ branch migration in vitro, RuvA2(KaP) is fully active for homologous recombination in vivo. RuvA2(KaP) is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2(KaP) for forks is decreased compared with wild type. Accordingly, RuvA2(KaP) is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2(KaP) is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2(KaP) is defective for stimulation of RuvB activity and stability of HJ·RuvA·RuvB tripartite complexes. This work demonstrates that the need for RuvA tetramer-tetramer interactions for full RuvAB activity in vitro causes specifically an RFR defect in vivo.     

A procedure for large-scale isolation of RNA-free plasmid and phage DNA without the use of RNase

A preparative procedure for the large-scale isolation of plasmid DNA without the use of RNAse is described. Crude plasmid DNA is prepared using a standard boiling method. High-molecular-weight RNA is removed by precipitation with LiCl, and low-molecular-weight RNA is removed by sedimentation through high-salt solution. The procedure is inexpensive, rapid, simple, and particularly suitable for processing several large-scale preparations simultaneously. A similar procedure has been developed for preparation of lambda-phage DNA.     

Contribution of the intrinsic curvature to measured DNA persistence length

The persistence length of DNA, a, depends both on the intrinsic curvature of the double helix and on the thermal fluctuations of the angles between adjacent base-pairs. We have evaluated two contributions to the value of a by comparing measured values of a for DNA containing a generic sequence and for an "intrinsically straight" DNA. In each 10 bp segment of the intrinsically straight DNA an initial sequence of five bases is repeated in the sequence of the second five bases, so any bends in the first half of the segment are compensated by bends in the opposite direction in the second half. The value of a for the latter DNA depends, to a good approximation, on thermal fluctuations only; there is no intrinsic curvature. The values of a were obtained from measurements of the cyclization efficiency for short DNA fragments, about 200 bp in length. This method determines the persistence length of DNA with exceptional accuracy, due to the very strong dependence of the cyclization efficiency of short fragments on the value of a. We find that the values of a for the two types of DNA fragment are very close and conclude that the contribution of the intrinsic curvature to a is at least 20 times smaller than the contribution of thermal fluctuations. The relationship between this result and the angles between adjacent base-pairs, which specify the intrinsic curvature, is analyzed.