Analysis of a primer-independent GTF-I from Streptococcus salivarius

Abstract A glucosyltransferase (GTF) gene, designated gtfL , from Streptococcus salivarius was cloned and expressed in Escherichia coli and its nucleotide sequence determined. The GTF-L enzyme catalysed the synthesis of water-insoluble glucan in a primer-independent manner. The nucleotide sequence and derived amino acid sequence of GTF-L were similar in size and domain structure to previously sequenced glucosyltransferases. However, a 464-bp region of high variability was identified which could be selectively amplified from strains of S. salivarius by the polymerase chain reaction and could therefore form the basis for species identification. No sequence-specific motifs related to the solubility and linkage of the glucan product or its need for a dextran primer could be ascertained.     

The diversification of South American murid rodents: evidence from mitochondrial DNA sequence data for the akodontine tribe

Phylogenetic relationships based on 801 base pairs (bp) of the mitochondrial cytochrome b gene are examined for eight genera and 28 species of the akodontine tribe of South American murid rodents. The akodontine tribe comprises some 35% of the total diversity of the subfamily Sigmodontinae, but the current taxonomy at virtually all levels is uncertain because of inadequate generic diagnoses and assessments of variation and trends in traditional morphological characters. Monophyly of the tribe cannot be resolved by the sequence data, based on comparisons to outgroup taxa in three other tribes (Oryzomyini, Phyllotini, and Thomasomyini). However, highly corroborated monophyletic units within the group are obtained in a variety of both parsimony and distance analyses. These include a redefined and numerically dominant genus Akodon (with Microxus and Hypsimys as synonyms), Bolomys, Lenoxus, Oxymycterus, and a strongly supported assemblage that includes the central Andean Chroeomys and 'Akodon' andinus and the southern Abrothrix, 'Akodon' olivaceus, and the long-clawed mice of the genera Notiomys, Geoxus, and Chelemys. Sequence divergence within species is typically less than 5%, although levels can reach 10% for some highly polytypic forms. Divergence among genera within the tribe reaches 35% in corrected estimates, a level that is as great as that among representatives of different tribes. Changes in the current classification of akodontines are suggested based on these data, and the timing and place of origin of the tribe and its radiation is discussed.     

Polymerase chain reaction analysis of mitochondrial DNA polymorphism in N'Dama and Zebu cattle

A study of polymorphisms of mitochondrial DNA (mtDNA) of West African N'Dama (Bos taurus) and East African Zebu (B. indicus) cattle was carried out to obtain information on maternal phylogenetic relationships between these breeds. A relatively large sample size was made possible by using polymerase chain reaction (PCR) amplification of DNA prepared from small blood samples to generate fragments of two known polymorphic mtDNA regions, one within the gene encoding subunit 5 of NADH dehydrogenase and one encompassing the entire D-loop. This approach allowed us to achieve a higher resolution restriction analysis on mtDNA from more animals than would have been possible by conventional methods. PCR-amplified mtDNA of 58 animals from five populations was examined at 26 restriction sites by 16 enzymes. In this way 154 nucleotides of mtDNA were scanned for polymorphism. Six polymorphic sites were located by this means, five of which were within the D-loop and one of which was within the NADH dehydrogenase 5 gene. None of the polymorphisms observed could be con sidered typical of breed or type.     

基于DNA分子标记的潘氏闭壳龟遗传多样性分析

潘氏闭壳龟（Cuora pani）是中国特有种,国家二级重点保护野生动物。由于非法贸易加之栖息地破坏等,其野外种群数量十分稀少。本研究旨在利用线粒体基因与核基因标记揭示潘氏闭壳龟的遗传多样性和遗传结构,以期为中国潘氏闭壳龟的种群保护和管理提供科学依据。本次共研究14只潘氏闭壳龟个体,其中,6只原产地为四川广元,8只产地未知。结果表明,在14只潘氏闭壳龟样本中鉴定出Cyt b单倍型、ND4单倍型和R35单倍型各2个,且均有1个新单倍型;Cyt b、ND4和R35基因单倍型多样性分别为0.440、0.143和0.154,核苷酸多样性分别为0.000 41、0.000 19和0.000 63;各单倍型的平均遗传距离（p）均小于0.01。基于三个基因联合数据集的系统发育分析表明,潘氏闭壳龟与金头闭壳龟（C.aurocapitata）互为单系进化支,且中性检测和核苷酸错配分析显示,潘氏闭壳龟群体近期历史上可能未经历群体扩张事件。潘氏闭壳龟群体遗传多样性低,种内变异小,群体应对环境变化的能力低,建议应加强对中国潘氏闭壳龟野外种群的基础研究和保护力度,同时规范人工繁殖,以避免近亲繁殖和种群衰退。     

Relationships among didelphid marsupials based on sequence variation in the mitochondrial cytochrome B gene

Variation in the mitochondrial cytochrome b gene (nucleotide and amino acid sequences) is evaluated for 9 genera and 15 species of American opossums in the family Didelphidae, using the American caenolestid rat opossumLestoros and the New Guinean peroryctid bandicootEchimypera as outgroups. Phylogenetic analyses (parsimony and distance) strongly support the monophyly of the Didelphidae and delineate two major clades; (1)Didelphis andPhilander are strongly aligned sister taxa, withMetachirus weakly but consistently associated with them, and (2)Marmosa plusMicoureus, withMonodelphis falling outside that pair. The generaMarmosops, Caluromys, andGlironia exhibit varied relationships, depending upon the method of analysis and data (DNA or amino acid sequences) used, but generally are placed individually or in combinations near or at the base of the didelphid radiation. Some aspects of these relationships are consistent with current taxonomic views, but others are in marked contrast. Specifically, a clade comprised of the mouse opossumsMarmosa, Micoureus, andMarmosops is strongly rejected by log-likelihood analysis, contrary to expectations from some current classifications. Also, the woolly opossumsCaluromys andGlironia also do not form a sister-taxon relationship, as suggested by their placement in a subfamily separate from the remaining didelphids examined. However, such a relationship cannot be rejected from log-likelihood analyses. The relationships suggested fromcyt-b sequences are strongly concordant with those based on DNA-DNA hybridization analyses. In addition to systematic and phylogenetic properties, molecular evolution of the didelphid cytochrome b gene sequence is characterized according to nucleotide bias and rate differentials at each codon position and across the entire sequence.To whom correspondence should be addressed.     

Cloning single-stranded DNA

DNA sequence and expression analyses have greatly benefited from using M13 and pUC derived cloning vectors and their polycloning sites. A chronology of the original concepts and experiments is reviewed.     

Molecular cloning and complete nucleotide sequence of the repeated unit and flanking gene of the scallop Pecten maximus mitochondrial DNA: Putative replication origin features

In the bivalve mollusc Pecten maximus, the size of the mitochondrial DNA molecules ranges from 20 to 25.8 kbp. This variability is mainly correlated with the occurrence of a variable domain composed with two to five 1.6-kbp repeated units tandemly arrayed in the genome. DNA fragments spanning the 1,586-base-pair-long repeated element and the nearest flanking gene have been cloned and sequenced. This sequence was analyzed regarding its base composition and potential secondary structures. The repeated unit domain was positioned and oriented with regard to the known flanking gene. It ends 2 base pairs upstream relative to the beginning of the tRNA^gly gene. The peculiar properties of the repeated unit were compared with those of the 1,442-bp repeated element found in the mitochondrial genome of the deep sea scallop Placopecten magellanicus. This comparison provided evidence for the absence of nucleotide conservation, except for a small sequence engaged in a secondary structure, but argued for a strong pressure maintaining domains with specific nucleotide content. A possible role for the conserved sequence is discussed.Correspondence to: A. Rigaa     

中国和韩国黑线姬鼠两亚种的线粒体DNA限制性片段分析

本文利用印迹杂交技术对中国华北地区黑线姬鼠Ａｐｏｄｅｍｕｓａｇｒａｒｉｕｓｐａｌｉｄｉｏｒ和韩国黑线姬鼠Ａ．ａｇｒａｒｉｕｓｃｏｒｅａｅ共１０７号标本的线粒体ＤＮＡ（ｍｉｔｏｃｈｏｎｄｒｉａｌＤＮＡ,ｍｔＤ－ＮＡ）,通过８种限制性内切酶的消化,进行了限制性片段的分析。共检出３５种限制性片段和１２种单倍体类型。１２种单倍型在平均离散度为１．０１％时聚合为两个亚群：一个亚群为黑线姬鼠华北亚种,由采自中国４个不同地区的５１号标本的４种单倍型所组成;另一个亚群为黑线姬鼠朝鲜亚种,由采自韩国４个不同地区的５６号标本的８种单倍型所组成。黑线姬鼠华北亚种和朝鲜亚种在ｍｔＤＮＡ表型上表现出一定差异,这在分子水平上确立了两亚种的分类地位。为了进一步澄清黑线姬鼠种下分类的混乱,很有必要对中国其他地区的标本进行该项研究     

Evolution of the Mitochondrial Cytochrome Oxidase II Gene in Collembola

The sequence of the mitochondrial COII gene has been widely used to estimate phylogenetic relationships at different taxomonic levels across insects. We investigated the molecular evolution of the COII gene and its usefulness for reconstructing phylogenetic relationships within and among four collembolan families. The collembolan COII gene showed the lowest A + T content of all insects so far examined, confirming that the well-known A + T bias in insect mitochondrial genes tends to increase from the basal to apical orders. Fifty-seven percent of all nucleotide positions were variable and most of the third codon positions appeared free to vary. Values of genetic distance between congeneric species and between families were remarkably high; in some cases the latter were higher than divergence values between other orders of insects. The remarkably high divergence levels observed here provide evidence that collembolan taxa are quite old; divergence levels among collembolan families equaled or exceeded divergences among pterygote insect orders. Once the saturated third-codon positions (which violated stationarity of base frequencies) were removed, the COII sequences contained phylogenetic information, but the extent of that information was overestimated by parsimony methods relative to likelihood methods. In the phylogenetic analysis, consistent statistical support was obtained for the monophyly of all four genera examined, but relationships among genera/families were not well supported. Within the genus Orchesella, relationships were well resolved and agreed with allozyme data. Within the genus Isotomurus, although three pairs of populations were consistently identified, these appeared to have arisen in a burst of evolution from an earlier ancestor. Isotomurus italicus always appeared as basal and I. palustris appeared to harbor a cryptic species, corroborating allozyme data. Received: 12 January 1996 / Accepted: 10 August 1996