基于线粒体COⅡ和Cytb基因序列的8种漠甲系统发育关系

对8种漠甲昆虫线粒体Cytb基因579bp和COⅡ基因585bp的序列片段进行联合分析。结果表明,8种甲虫在长度为1164bp的序列中碱基T,C,A,G的平均含量分别为33.7%,21.2%,33.5和11.6%,A+T平均含量明显高于G+C含量。密码子第三位点A+T含量高达77.2%,而该位点G的平均含量仅为4.0%。序列中碱基替换多发生于第三位点,转换略多于颠换,转换主要以C←T为主,颠换以A←T为主。以土甲族的Eumylada potanini为外群构建的系统发育树表明:漠王和漠甲以很高的置信值聚为一支,二者的关系与Bouchard最新的分类观点相吻合,即漠王族并入漠甲族成为一族;鳖甲族在支序图上另成一支,可视为一个单系群。     

基于Cyt b基因序列分析的松毛虫种群遗传结构研究

为了揭示松毛虫种群的遗传结构,采用DNA序列测定的方法测定了松毛虫不同种群的线粒体细胞色素b (Cyt b)基因的部分序列,并利用分子生物学软件分析其核苷酸组成、转换和颠换、氨基酸组成、遗传距离及亲缘关系。结果显示:在获得的Cyt b 基因387bp的序列中碱基A,T,C,G平均含量分别为40.1%、33.5%、9.5%、16.9%,A+T含量明显高于G+C含量表现出强烈的A、T偏向性,密码子第3位点的A+T含量高达86.5%,这种偏向性在种群间无明显差异。碱基替换主要发生在密码子第三位,转换大于颠换,且种群内替换高于种群间。该序列片段中共有39个核甘酸位点发生变异,遗传距离为0.000-0.100,显示出较小的遗传变异。蛋白质氨基酸由除谷氨酸以外的19种氨基酸组成。聚类分析结果表明马尾松毛虫和油松毛虫亚种遗传距离较近,种群间的遗传分化与生态环境有关。     

五种色蟌Cyt b基因序列及其系统发育关系

以昆虫mtDNA的Cytb基因作为分子遗传标记,用其特异性引物进行PCR扩增及DNA测序,共获得蜻蜓目束翅亚目色蟌科4属5种及1外群的Cytb基因部分序列(576bp),该片段中碱基T,C,A,G的平均含量分别为31.6,21.6,31.4和15.4%,A+T平均含量为63%,明显高于G+C含量(37%)。密码子第3位点的A+T平均含量较高,为66.4%。碱基替换多发生在密码子的第3位点。以巨齿尾溪蟌Bayadera melanopteryx作为外群构建系统发育树,结果显示,绿色蟌属Mnais和单脉色蟌属Matrona是单系群,绿色蟌属是单脉色属、细色蟌属Vestalis及艳色蟌属Neurobasis的姐妹群,是较早分化出来的一个类群。     

天然三倍体鲫鱼突变体萍乡肉红鲫线粒体细胞色素b基因序列分析

用细胞色素b(Cyt b)基因特异性引物,对萍乡肉红鲫(Carassius auratus var.pingxiangnensis)的线粒体Cyl b基因进行PCR扩增和双向测序.在12个个体中均得到序列一致的Cyt b基因全序列,长度为1 140 bp.其A、T、G、C含量分别为28.2%(321)、28.8%(328)、14.8%(170)和28.2%(321),A+T含量(57%)明显高于G+C含量(43%).与其他水生动物相同基因片段碱基含量相似.该基因中密码子第1位核苷酸中4种碱基组成较为均衡;第2位核苷酸中T的使用率较高,为41.8%,G的使用率较低,为13.2%;密码子第3位A的使用率较高,为39.7%,而G的使用率较低,仅为6.3%.BLAST结果显示,萍乡肉红鲫与其他鲫鱼类的Cyt b基因具有较高的同源性,其中与银鲫(C.a.gibelio)的相似性为93%,与普通鲫鱼(C.carassius)的相似性为98%.邻接法构建的分子系统进化树表明,萍乡肉红鲫与普通鲫鱼亲缘关系最近.     

基于psaA和psbA基因的红索藻目系统发育研究

对红索藻目植物棘刺红索藻(Thorea hispida)的psaA和psbA基因进行扩增和测序,并与其它类群比对分析,分别用最大简约法、邻接法和贝叶斯法构建系统发育树。结果显示,psaA和psbA基因测得的序列片段分别为825和920 bp,psaA基因A、T、C、G碱基含量分别为29.9%、35.8%、16.5%和17.8%,psbA基因A、T、C、G碱基含量分别为27.5%、35.3%、16.8%和20.4%,两个基因A+T含量均高于C+G含量,说明两个基因在进化上均有碱基的偏好性。用3种方法所构建的系统树拓扑结构基本一致,红索藻目植物均聚合于一个分支,独立于其它类群,支持红索藻目为一独立的目。     

叶甲亚科部分种类COⅡ基因序列分析及系统发育初探

以线粒体COⅡ基因作为分子标记,对叶甲亚科4种昆虫进行序列测定,获得该基因585 bp的序列片段,并结合GenBank中的8种同源序列进行分析,结果表明:12种叶甲序列片段变异率为45.6%,碱基T、C、A、G的平均含量分别为37.5%、16.5%、35.3%和10.7%, A+T平均含量为72.8%,明显高于G+C含量(27.2%).密码子第3位点A+T含量高达86.7%.碱基替换主要发生在C←→T和A←→T之间,第3位点的替换频率显著高于前两个位点.以中华萝摩叶甲Chrysocus chinensis为外群构建的系统发育树显示,金叶甲属和角胫叶甲属关系较近,弗叶甲属和叶甲属关系较近,圆肩属形成一个单系,位于系统树的基部,它们之间的关系为(圆肩属+(弗叶甲属+叶甲属)+(金叶甲属+角胫叶甲属)).属内种间的关系反映出叶甲的食性专化现象与其在分类系统上的地位是密切相关的.     

中国缘蝽科部分种类细胞色素b基因序列及系统进化研究

以线粒体细胞色素b(Cyt b)基因作为分子标记,首次对缘蝽科4亚科14种昆虫进行序列测定,获得Cyt b基因412 bp的序列片段,该片段中碱基T、C、A、G的平均含量分别为34.2%、11.4%、35.7 %和18.7 %,A T平均含量为69.9 %,明显高于G C含量(30.1 %).密码子第3位点的A T含量高达82.8%,亚科间序列变异大,有193个核苷酸位点发生变异,碱基替换多发生于第3位点.以筛豆龟蝽为外群构建系统发育树,表明在亚科级关系上,姬缘蝽亚科最原始,蛛缘蝽亚科次之,巨缘蝽亚科和缘蝽亚科亲缘关系较近,为较进化种类.     

基于线粒体Cyt b基因的全长序列探讨闭壳龟类的系统进化

采用PCR技术对淡水龟科具闭壳结构的黄缘盒龟、黄额盒龟、金头闭壳龟、潘氏闭壳龟、锯缘龟和白腹摄龟的线粒体Cytb基因的全长序列进行了PCR扩增和序列测定,并结合GenBank中16种淡水龟科物种的同源序列,进行了序列变异和系统发生分析。经C lustalX1.8软件对位排列后共有1154个位点,其中可变位点413个,简约信息位点301个;A＋T的平均含量（56.5%）高于G＋C（43.5%）。在氨基酸密码子中,第一位富含A,第二位富含T,第三位富含C;碱基转换/颠换率为5.97,碱基替换多发生在密码子第三位。以中华鳖和马来鳖为外群,通过最大简约法,最大似然法和贝叶斯法重建了分子系统树,均具有一致的拓扑结构,结果表明：金头闭壳龟和潘氏闭壳龟最先聚成一支,再和三线闭壳龟聚成一组,说明形态上相似的三种闭壳龟亲缘关系最近;闭壳龟属、盒龟属和单种锯缘龟属聚成一个单系的闭壳龟群,建议合并为闭壳龟属;齿缘龟属和果龟属聚为一支,它们与新的闭壳龟属关系较远,揭示闭壳结构的形成不是由一个共同祖先分化而来;乌龟属、花龟属和拟水龟属三属为并系起源,建议三属可以合并为一属。     

基于线粒体细胞色素b基因全序列的海南长臀鮠南渡江种群遗传变异分析

测定了海南南渡江15尾海南长臀鮠(Cranoglanis bouderius multiradiatus)的cytb基因全序列1138bp,发现3个可变位点和1个简约信息位点,共检测到4种单倍型。海南长臀鮠Cytb基因具有典型的起始密码子(ATG)和不完全终止密码子(T^**),共编码379个氨基酸,密码子第三位点G的含量仅2.9%,而C和A的含量分别为39.1%和38.5%,在第二位点上T的使用频繁高达41.3%,表明cytb基因在密码子碱基的使用具有偏倚性。海南长臀鮠基于Kimura-2-parameter模型的单倍型间遗传距离为0.0009～0.0017,在NJ系统树中没有明显的谱系结构,单倍型多样性(0.600)和核苷酸多样性(0.0006)都较低,表明海南长臀鮠遗传多样性非常贫乏,应及时采取有效措施保护海南南渡江种群。     

HLA-B*2736等位基因的序列分析

使用FLOW-SSO、PCR-SSP以及测序等分型技术, 发现一个与HLA-B*270401基因相关的未知基因。设计基因特异性引物单独扩增B*27基因的外显子2-5, 包括内含子2-4, 并进行双向测序, 分析与B*270401基因序列的差异。该基因的扩增产物为1 815 bp。与B*270401相比在外显子3和4共有10个碱基的改变, 从而使相应氨基酸发生错义或同义突变。碱基634 A→C (密码子130丝氨酸→精氨酸); 670 A→T (密码子142苏氨酸→丝氨酸); 683 G→T (密码子146色氨酸→亮氨酸); 698 A→T (密码子151谷氨酸→缬氨酸); 774 G→C (密码子176谷氨酸→天冬氨酸); 776 C→A (密码子177苏氨酸→赖氨酸); 781 C→G (密码子179谷氨酰胺→谷氨酸); 789 G→T (密码子181丙氨酸同义突变); 1 438 C→T (密码子206甘氨酸同义突变); 1 449 G→C (密码子210甘氨酸→丙氨酸)。在IMGT/HLA数据库中B*27组只有3个基因（B*270502 / 2706 / 2732）提交了内含子序列。该未知基因的内含子2序列与B*2706相同, 显示了与B*27组基因的同源性, 但其同源性在内含子3、4均未得到支持, 与B*27组基因相比, 内含子3的第106个碱基C→G, 碱基168缺失, 碱基179 G→A, 碱基536 G→A; 内含子4中碱基82 T→C。但其内含子3、4序列却与B*070201完全相同。该基因序列已提交GenBank, 编号为被DQ915176, 被WHO确认为HLA-B*2736等位基因。     

The rate and pattern of nucleotide substitution in Drosophila mitochondrial DNA.

The nucleotide sequences of a segment of mitochondrial DNA (mtDNA) have been determined for nine species or subspecies of the subgenus Drosophila of the genus Drosophila. This segment contains two complete protein-coding genes (i.e., NADH dehydrogenase subunit 1 and cytochrome b) and a transfer RNA gene (tRNA(ser)). The G+C content at third-codon positions for the two protein-coding genes was 1.5 times higher than that in the D. melanogaster species group, which belongs to the subgenus Sophophora. However, there was a substantial difference between the nucleotide frequencies of G and C. The number of nucleotide substitutions per silent site was more than three times higher than that for nuclear DNA, although it was only 60% of that for mammalian mtDNA. Both parametric and nonparametric analyses revealed a strong transition-transversion bias in nucleotide substitution, as was observed in mammalian mtDNA. Moreover, the rate of substitution of A and T for G and C is higher than that for the opposite direction. This bias seems to be responsible for the extremely A+T-rich base composition of Drosophila mtDNA. It is also noted that the rate of transitional change between A and G is higher than that between T and C.     

Microsatellite–Centromere Mapping in Large Yellow Croaker (<Emphasis Type="Italic">Pseudosciaena crocea</Emphasis>) Using Gynogenetic Diploid Families

The large yellow croaker (Pseudosciaena crocea) is an economically important marine fish in China. Inheritance of 22 heterozygous microsatellite loci was examined in normal crossed diploid families and meio-gynogenetic families in P. crocea. Two gynogenetic families were produced via inhibition of the second polar body in eggs fertilized with UV-irradiated sperm. The ratio of gynogenesis was proven to be 100% and 96.9% in the two families, respectively. Of the 22 examined loci, 4 showed a segregation distortion in both control and gynogenetic families. Microsatellite–centromere (M–C) map distances were examined using 18 loci with normal Mendelian segregation. Estimated recombination rates ranged between 0 and 1.0 under the assumption of complete interference. High recombinant frequencies between heterozygous markers and the centromere were found in large yellow croaker, as in other teleosts. The average recombination frequency was 0.586. Ten loci showed high M–C recombination with frequency greater than 0.67. M–C distances provide useful information for gene mapping in large yellow croaker.     

Simultaneous screening for three mutations in the ABCB1 gene

A noncoding C3435T mutation in exon 26 of the ABCB1 gene was found to be often associated with a G2677T(A) mutation in exon 21 encoding an Ala893Ser P-glycoprotein and with a noncoding C1236T mutation in exon 12. We developed a Pyrosequencing screening method that simultaneously detects all three mutations. After separate PCRs for each exon, the sequences around the potentially mutated nucleotide positions were simultaneously analyzed in a multiplex assay. The method was tested with DNA from 100 volunteers. Allele frequencies of the T1236, T2677, and T3435 alleles were 44, 42, and 50%, respectively. A mutation at position 3435 occurred together with a mutation at position 2677 or 1236 in 64 and 65% of the subjects, respectively. The most frequent haplotype, with 44.4%, was not mutated at all three positions, i.e., C1236, G2677, C3435. The second most frequent haplotype, with 37.1%, was mutated at all three positions, i.e., T1236, T2677, T3435. The most frequent genotype, with 36%, was heterozygously mutated at all three positions, i.e., C/T1236, G/T2677, C/T3435. The next most frequent genotypes were a homozygous nonmutated genotype, with 20%, and a homozygous mutated genotype, with 13%.     

Genetic analysis of structure and function in phage T4 tRNASer

We have determined the nucleotide sequences of 55 spontaneous mutations that inactivate a suppressor gene of phage T4 tRNASer. Most of the mutations caused substitutions or deletions of single nucleotides at 18 different positions in the tRNA. Two of three mutations that allowed the synthesis of mature tRNA had nucleotide substitutions at the junction of the dihydrouridine and anticodon stems, suggesting that this region of tRNASer is important for aminoacylation. The third mutation that synthesized tRNA had a nucleotide deletion in the anticodon loop, which presumably affected the translational capacity of the tRNA. We also sequenced 58 spontaneous reversion mutations derived from strains with the inactive suppressor genes. Some of these regenerated the initial tRNA sequence, while other generated a second-site mutation in the tRNA. These second-site mutations restored helical base-pairings to the tRNA that had been eliminated by the initial mutations. The new base-pairings involved G.C and A.U, and the A.C wobble pair at certain positions in the tRNA. This finding establishes the existence of A.C wobble pair in tRNA helices.     

G and T Nucleotide Contents Show Specie-Invariant Negative Correlation for All Three Codon Positions

Abstract

The nucleotide contents of the three codon positions show a number of statistical pairwise correlations, some of which are universal for all analysed genomes. Among the most prominent of these correlations are negative correlations between G and T contents found in genes of all species analysed. The pair A/C, which is complementary to G/T shows similar negative correlation in genes of most species. In the genes of several species including all mammalian genes studied, positive correlations between A and T contents, and G and C contents are found. Since these regularities are observed in all three codon positions they are connected with amino-acid content of proteins. Such correlations may origin from features of the mutation process or/and translation reading frame check. The well-known bias of the preference for G in the first codon position and its deficiency in the second is accompanied by opposite bias in T content. In the third codon position there is no general nucleotide preference, but its content is often biased with regard to GC content of the gene. G and T contents in this case are always shifted in the opposite directions Several ideas are drawn to explain this preference.     

Nucleotide sequence and characteristics of the gene for L-lactate dehydrogenase of Thermus caldophilus GK24 and the deduced amino-acid sequence of the enzyme

The gene for L-lactate dehydrogenase (LDH) (EC 1.1.1.27) of Thermus caldophilus GK24 was cloned in Escherichia coli using synthetic oligonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of the LDH was deduced from the nucleotide sequence. The deduced amino acid sequence agreed with the NH2-terminal and COOH-terminal sequences previously reported and the determined amino acid sequences of the peptides obtained from trypsin-digested T. caldophilus LDH. The LDH comprised 310 amino acid residues and its molecular mass was determined to be 32,808. On alignment of the whole amino acid sequences, the T. caldophilus LDH showed about 40% identity with the Bacillus stearothermophilus, Lactobacillus casei and dogfish muscle LDHs. The T. caldophilus LDH gene was expressed with the E. coli lac promoter in E. coli, which resulted in the production of the thermophilic LDH. The gene for the T. caldophilus LDH showed more than 40% identity with those for the human and mouse muscle LDHs on alignment of the whole nucleotide sequences. The G + C content of the coding region for the T. caldophilus LDH was 74.1%, which was higher than that of the chromosomal DNA (67.2%). The G + C contents in the first, second and third positions of the codons used were 77.7%, 48.1% and 95.5% respectively. The high G + C content in the third base caused extremely non-random codon usage in the LDH gene. About half (48.7%) the codons in the LDH gene started with G, and hence there were relatively high contents of Val, Ala, Glu and Gly in the LDH. The contents of Pro, Arg, Ala and Gly, which have high G + C contents in their codons, were also high. Rare codons with U or A as the third base were sometimes used to avoid the TCGA sequence, the recognition site for the restriction endonuclease, TaqI. Two TCGA sequences were found only in the sequence of CTCGAG (XhoI site) in the sequenced region of the T. caldophilus DNA. There were three segments with similar sequences in the two 5' non-coding regions, probably the promoter and ribosome-binding regions, of the genes for the T. caldophilus LDH and the Thermus thermophilus 3-isopropylmalate dehydrogenase.     

DNA序列进化过程中核苷酸替代的非独立性研究

本文评述了DNA序列间核苷酸替代数的估计方法,并通过对七个物种中组蛋白基因的比较对DNA进化的模型进行了考察。发现H2A基因第三位点上的碱基组成在物种间变异很大,并且跟H2A基因第一位点、H4基因第一、三位点及H2A上游,下游序列中的碱基组成有强正相关,提示DNA序列进化过程中存在着物种特异的区域性约束力。可能的原因是高等真核生物中GC含量升高,或者是染色体重组使这些同源序列位于不同的等质区段,从而受到不同的选择突变压。密码内各位点上核苷酸替代的相关性分析表明不同位点的替代是非独立的,其原因可能是一次替代事件引起多个位点的变化。文中讨论了这些结果对进化树推断的意义。     

SAIVGeM: spreadsheet analysis of immunoglobulin VH gene mutations

The analysis of mutations in immunoglobulin heavy chain variable (IGHV) region genes is a tedious process when performed by hand on multiple sequences. This report describes a set of linked Microsoft Excel files that perform several common analyses on large numbers of IGHV sequences. The spreadsheet analysis of immunoglobulin VH gene mutations (SAIVGeM) package determines the distribution of mutations among each nucleotide, the nature of the mutation at both the nucleotide and amino acid level, the frequency of mutation in the A/G G C/T A/T (RGYW) hotspot motifs of both strand polarity, and the distribution of replacement and silent mutations among the complementarity determining regions (CDRs) and the framework regions (FRs) of the immunoglobulin gene as defined by either the Kabat or IMGT conventions. These parameters are summarized and graphically presented where appropriate. In addition, the SAIVGeM package analyzes those mutations that occur in third positions of redundant codons. Because any nucleotide change in these positions is inherently silent, these positions can be used to study the mutational spectra without biases from the selection of protein structure.     

Variability of milk fat globule membrane protein gene between cattle and riverine buffalo.

A study on butyrophilin (BTN) gene was conducted to detect variability at nucleotide level between cattle and buffalo. Hae III PCR-RFLP was carried out in crossbred cattle and it revealed polymorphism at this locus. Three genotypes namely, AA, BB and AB and two alleles were observed with frequencies 0.78, 0.17, 0.04 and 0.87, 0.13, respectively. The sequences of different cattle, buffalo and sheep breeds have been reported in the EMBL gene bank with accession numbers: AY491468 to AY491475. The nucleotides, which have been substituted from allele A to B, were found to be C to G (71st nucleotide), C to T (86th nucleotide), A to T (217th nucleotide), G to A (258th nucleotide), A to C (371st nucleotide) and C to T (377th nucleotide). The nucleotide substitution at 71st, 86th and 377th position of the fragment were expected to be a silent mutation where as nucleotide changes at 217th, 258th and 371st positions were expected to be substituted by lysine with arginine, valine with isoleucine and leucine with proline in allele B. The differences of nucleotides and amino acids between cattle, buffalo and sheep breeds have been revealed and on the basis of nucleotide as well as protein variability the phylogenetic diagram have been developed indicating closeness between cattle and buffalo.