人免疫球蛋白重链可变区基因引物设计方法的改良

针对抗体胚系基因数据库的数据不断更新和完善,为获得人全部免疫球蛋白(Ig)重链可变区基因,改进引物设计方法,自主设计针对可变区基因高度保守的框架区1(FR1)和框架区4(FR4)的引物,提取未经免疫的健康人外周血单个核细胞,通过RT-PCR扩增重链可变区基因.其DNA序列与GenBank数据库和IMGT/V-QUEST软件比对,序列分析符合人免疫球蛋白重链基本框架结构,为胚系基因重排产生的序列.多个克隆的测序结果对比分析显示了良好的多样性.获得的重链序列为研制基因工程抗体及构建噬菌体抗体库奠定了物质基础,也为扩增其他物种Ig可变区基因的引物提供新的设计思路.     

Cloning of the mitochondrial genome of Anopheles quadrimaculatus

The entire 15 kilobase (kb) Anopheles quadrimaculatus mitochondrial DNA (mtDNA) was cloned as three EcoRI fragments in a bacteriophage vector and then subcloned into plasmid vectors. The cloned DNA was physically mapped with restriction endonucleases, and the maps were compared to the restriction patterns of native A. quadrimaculatus mtDNA. Several genes were mapped by sequencing the ends of A. quadrimaculatus mtDNA subclones and by hybridization with the previously characterized Aedes albopictus mtDNA clones. These portions of the genetic map were identical in gene order to those of Drosophila yakuba. The predicted amino acid sequence of the protein coding regions that were sequenced were between 72% and 98% homologous to D. yakuba. The cloned mtDNA will be useful as a probe for population genetic analysis of mosquitoes.     

Time of the deepest root for polymorphism in human mitochondrial DNA

Summary A molecular clock analysis was carried out on the nucleotide sequences of parts of the major noncoding region of mitochondrial DNA (mtDNA) from the major geographic populations of humans. Dates of branchings in the mtDNA tree among humans were estimated with an improved maximum likelihood method. Two species of chimpanzees were used as an outgroup, and the mtDNA clock was calibrated by assuming that the chimpanzee/human split occurred 4 million years ago, following our earlier works. A model of homogeneous evolution among sites does not fit well with the data even within hypervariable segments, and hence an additional parameter that represents a proportion of variable sites was introduced. Taking account of this heterogeneity among sites, the date for the deepest root of the mtDNA tree among humans was estimated to be 280,000±50,000 years old (±1 SE), although there remains uncertainty about the constancy of the evolutionary rate among lineages. The evolutionary rate of the most rapidly evolving sites in mtDNA was estimated to be more than 100 times greater than that of a nuclear pseudogene.     

Mapping Bombina mitochondrial genomes: the conundrum of Carpathian Bombina variegata (Anura: Discoglossidae)

Complete mitochondrial genomes (mtDNA) of five individuals representing two haplotypes of Bombina bombina and three of Bombina variegata were compared using restriction site maps. Phylogenetic analyses reveal three ancient mitochondrial lineages: (1) two very similar haplotypes A and B of B. bombina ; (2) almost identical haplotypes D and E of B. variegata ; and (3) haplotype C of B. variegata . Haplotype C is as different from haplotypes D/E as from A/B. These data are strikingly discordant with relationships based on morphology and allozymes. Haplotypes C and D/E represent a pre-Pleistocene mitochondrial divergence within B. variegata , nearly coincident with speciation between B. variegata and B. bombina . Geographical partitioning of the two divergent B. variegata mitochondrial lineages indicates repeated localization of the lineages in separate glacial refugia during the Pleistocene. That nuclear genes do not show a similar divergence, but rather indicate relatively free genetic exchange between populations with divergent mtDNAs, suggests that males dispersed much more widely than females during expansions from glacial refugia. Comparison of Bombina mtDNA maps with a restriction site map of Xenopus laevis mtDNA revealed 16 homologous sites; 12 of these may be nearly invariant across primitive anuran mtDNAs. Two distinct regions of heteroplasmy, representing two regions with variable numbers of sequence repeats [length variable (LV) regions], were characterized. Comparison with the Xenopus map places LV1, present in all five haplotypes, near the 5'-end of the control region, and LV2 present only in B. variegata , near the 3'-end. Although phylogenetic analyses did not group the two major B. variegata lineages together, presence of LV2 in both lineages supports placement of both within B. variegata .     

单引物预掺入PCR与抗体可变区基因的体外放大

用针对引导序列的5′-引物和针对恒定区的3′-引物,常规PCR程序只使所选5株单抗10个可变区cDNA中的4个得到放大.新设计的程序增设了一个反应时相:94℃ 1 min,37℃ 6-8min,循环1-3次,只加入5′-引物,补充3′-引物后,转入常规PCR循环,10个可变区cDNA均获放大.此程序被命名为“单引物预掺入PCR”.     

Searching for new morphological characters in the systematics of scleractinian reef corals: comparison of septal teeth and granules between Atlantic and Pacific Mussidae

Recent molecular analyses have challenged the traditional classification of scleractinian corals at all taxonomic levels suggesting that new morphological characters are needed. Here we tackle this problem for the family Mussidae, which is polyphyletic. Most of its members belong to two molecular clades composed of: (1) Atlantic Mussidae and Faviidae (except Montastraea) and (2) Pacific Mussidae (Cynarina, Lobophyllia, Scolymia, Symphyllia) and Pectiniidae. Other Pacific mussids (e.g. Acanthastrea) belong to additional clades. To discover new characters that would better serve as phylogenetic markers, we compare the skeletal morphology of mussid genera in different molecular‐based clades. Three sets of characters are considered: (1) macromorphology (budding; colony form; size and shape of corallites; numbers of septal cycles), (2) micromorphology (shapes and distributions of septal teeth and granules), and (3) microstructure (arrangement of calcification centres and thickening deposits within costosepta). Although most traditional macromorphological characters exhibit homoplasy, several new micromorphological characters are effective at distinguishing clades, including the shapes and distribution of septal teeth and granules, the area between teeth, and the development of thickening deposits. Arrangements of calcification centres and fibres differ among clades, but the fine‐scale structure of thickening deposits does not.     

MICROFAMILY (version 1): a computer program for detecting flanking‐region similarities among different microsatellite loci

Microsatellite flanking regions are not necessarily unique sequences, but they may group into sequence families. Microsatellites occurring within such families are likely to give multiple banding patterns during polymerase chain reaction amplifications. microfamily (version 1) is a program that detects flanking‐region similarities between different microsatellite‐containing sequences, thus allowing for potentially problematic sequences to be eliminated prior to primer design. The program also accomplishes some otherwise tedious sequence editing, such as checking for nonpermitted characters, and eliminates poorly readable extremities or potential vector/adapter contamination. microfamily is written in Perl and available for Linux and Windows systems at http://www.up.univ‐mrs.fr/local/egee/dir/meglecz/microfamily.html .     

白腊DREB基因克隆中简并引物的设计

根据拟南芥DREB2A基因序列进行GenBank内Blastx序列同源性查询,对其中同源性较高的25个氨基酸序列进行了分析,设计简并引物,应用RT-PCR技术克隆白腊DREB2A基因,得测长2473bp,与拟南芥DREB2A同源性为96.94%的基因片段,并在GenBank登记,注册号为AY536056。     

Polymorphism of the Noncoding Region of the Mitochondrial Genome in Three Kazakh Populations Inhabiting Different Areas of Kazakhstan and in DNA Samples from Ancient Populations of the Kazakhstani Altai

The polymorphism of the major noncoding region of mitochondrial DNA (mtDNA D loop, 528 bp) has been studied in samples from three modern Kazakh populations (from Almaty, the Semipalatinsk Region, and the Altai Mountains) and in DNA samples of ancient human populations of the Kazakhstani Altai. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis for 13 restriction sites, including BamHI, EcoRV, Sau3AI (one site each), KpnI (two sites), HaeIII (three sites), and RsaI (five sites) were used. The frequency distributions of all sites have been determined. The gene diversity (h) and the genetic distances between different Kazakh populations and other populations of the world have been calculated. The RFLP analysis of the mtDNA control region of fossil samples has been performed similarly to the analysis of modern mtDNA samples. Two fossil mtDNA samples from burial mound 11 are monomorphic with respect to all restriction sites analyzed.     

Species‐specific PCR primers for the mitochondrial genome control region hypervariable region 1 of the reef fish Lutjanus sebae

Lutjanus sebae is an important commercial and recreational fish found throughout the Indo‐West Pacific region. Using universal primers for the mitochondrial genome control region hypervariable region 1 (HVR1), we isolated a 385‐bp fragment of HVR1 then designed specific primers near each end of this sequence in the conserved regions flanking the hypervariable region. These primers were shown to yield good quantity and quality of product and to be species specific. Of 20 L. sebae studied, there were eight segregating sites and eight haplotypes. These primers will provide a useful tool for population genetics studies, identification of larvae and eggs and for wildlife forensics.     

基于cpDNA atpB-rbcL非编码序列分析桫椤种群遗传结构和遗传多样性,以贵州赤水桫椤国家级自然保护区为例

以PCR产物克隆后测序的方法测定了贵州赤水桫椤国家级自然保护区3个种群59个个体的叶绿体DNA (cpDNA) atpB-rbcL非编码区序列.序列长度介于727~732 bp之间,具长度多态性,A+T百分比含量较高,介于63.27%~63.89%之间.遗传多样性水平较低,表现出较高的单倍型多样性(h=0.639)和低的核苷酸多样性(Dij=0.00028)并存的特征,提示现有种群可能源自一个有效群体规模较小的种群的快速扩张,扩张时间尚短,不足以形成更为复杂的遗传结构.种群间的高基因流Nm、低遗传分化度FST、AMOVA分析以及DNA歧异度结果一致显示各种群间无明显的遗传分化.     

Population genetic structure of North Atlantic, Mediterranean Sea and Sea of Cortez fin whales, Balaenoptera physalus (Linnaeus 1758): analysis of mitochondrial and nuclear loci

Samples were collected from 407 fin whales, Balaenoptera physalus , at four North Atlantic and one Mediterranean Sea summer feeding area as well as the Sea of Cortez in the Pacific Ocean. For each sample, the sex, the sequence of the first 288 nucleotides of the mitochondrial (mt) control region and the genotype at six microsatellite loci were determined. A significant degree of divergence was detected at all nuclear and mt loci between North Atlantic/Mediterranean Sea and the Sea of Cortez. However, the divergence time estimated from the mt sequences was substantially lower than the time elapsed since the rise of the Panama Isthmus, suggesting occasional gene flow between the North Pacific and North Atlantic ocean after the separation of the two oceans. Within the North Atlantic and Mediterranean Sea, significant levels of heterogeneity were observed in the mtDNA between the Mediterranean Sea, the eastern (Spain) and the western (the Gulf of Maine and the Gulf of St Lawrence) North Atlantic. Samples collected off West Greenland and Iceland could not be unequivocally assigned to either of the two areas. The homogeneity tests performed using the nuclear data revealed significant levels of divergence only between the Mediterranean Sea and the Gulf of St Lawrence or West Greenland. In conclusion, our results suggest the existence of several recently diverged populations in the North Atlantic and Mediterranean Sea, possibly with some limited gene flow between adjacent populations, a population structure which is consistent with earlier population models proposed by Kellogg, Ingebrigtsen, and Sergeant.     

Generation of VNTRs and heteroplasmy by sequence turnover in the mitochondrial control region of two elephant seal species

We describe an unusual repetitive DNA region located in the 3′ end of the light (L)-strand in the mitochondrial control region of two elephant seal species. The array of tandem repeats shows both VNTR (variable-number tandem repeat) and sequence variation and is absent from 12 compared mammalian species, except for the occurrence in the same location of a distinct repetitive region in rabbit mtDNA and a similar repeat in the harbor seal. The sequence composition and arrangement of the repeats differ considerably between the northern elephant seal (Mirounga angustirostris) and the southern species (M. leonina) despite an estimated divergence time of 1 MY (based on an mtDNA-RNA gene and the nonrepetitive control region). Analysis of repeat sequence relationships within and between species indicate that divergence in sequence and structure of repeats has involved both slippagelike and unequal crossingover processes of turnover, generating very high levels of divergence and heteroplasmy. Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992     

云南猕猴mtDNA控制区全序列测定

目的测定云南猕猴线粒体DNA控制区全序列,对其进行鉴定及进化分析。方法利用PCR技术扩增猕猴线粒体DNA控制区全序列,结合GenBank中下载的猕猴参考序列（AY612638）,采用多个生物学软件对序列碱基组成、同源性、转换／颠换比等遗传信息进行分析,并基于邻接法（NJ）和最小进化法（ME）构建系统进化树。结果云南猕猴线粒体DNA控制区全长为（1084-1089）bp,A、T、G和c四种碱基平均含量分别为29．9％、26．9％、12．3％和30．9％,A＋T含量（56．8％）高于G＋C含量（43．2％）。所分析序列间的同源性为91．5％-99．5％,平均核苷酸变异率为4．5％,变异类型包括转换、颠换、插入和缺失4种形式,转换／颠换比值平均为26．1。进化树显示云南猕猴存在两个平行进化的姐妹分支。结论本研究获得了云南猕猴mtDNA控制区全序列,为猕猴进化关系研究及mtDNA控制区功能研究奠定基础。     

齐口裂腹鱼线粒体DNA控制区结构分析

利用直接测序法对齐口裂腹鱼(Schizothorax prenanti)线粒体DNA控制区进行了测序,并对其序列结构进行了分析.结果表明,齐口裂腹鱼线粒体控制区碱基组成中碱基A和T的含A明显高于G和C的含量,所有类型碱基组成中碱基G的含量最低,这与其他硬骨鱼类控制区碱基组成一致.通过与哺乳类和鲤形目鱼类控制区序列进行对...