蜜蜂5SrRNA核苷酸序列及与其他5SrRNA的比较

蜜蜂5SrRNA由119个核苷酸组成。与其他几种昆虫的5SrRNA比较,其序列的同源性在80％以上,具有较高的保守性。在二级结构的模式基础上,证明了单链突环区比双链螺旋区保守的现象;通过计算5SrRNA每年每个核苷酸位点上的碱基平均替换率,揭示了昆虫5SrRNA比脊椎动物的5SrRNA有较高的替换速率,其主要原因是双链螺旋区碱基高替换所致;并提出了用比较较保守的单链压的方法,修正计算碱基替换率的公式,以便由小分子rRNA推导出的遗传变异速率的结论更具有广泛性。     

呼吸道合胞病毒北京地区分离株G蛋白的基因分析

从经单克隆抗体证实为Ａ亚型的北京地区呼吸道合胞病毒（ＲＳＶ）分离株Ｂ７９中,用ＲＴ－ＰＣＲ扩增出编码Ｇ蛋白的基因片段,克隆至载体ｐＴＺ１８Ｒ中。经核苷酸序列测定证明,我国北京地区分离的Ａ亚型株Ｂ７９与ＲＳＶＡ亚型原型株（Ａ２株）Ｇ蛋白基因的核苷酸同源性为９３．８％,核苷酸的有义突变率达６５％。由核苷酸推导出氨基酸序列的同源性为８９．６％。氨基酸的变异主要集中在胞外区一个高度保守区的两端,而胞内区和跨膜区相对保守。本文探讨了我国北京地区ＲＳＶ分离株的Ｇ蛋白基因同原型株之间的变异,在疫苗研制中的意义。     

猛禽类15种鸟类线粒体tRNA基因序列及二级结构的比较研究

利用PCR方法扩增13个猛禽类物种线粒体基因组中3个主要的tRNA基因簇：IQM(tRNA^lle-tRNA^gln-tRNA^Met)、WANCY(tRNA^Trp-tRNA^Ala-tRNA^Asn-tRNA^Cys-tRNA^Tyr。)和HSL(tRNA^His-tRNA^Ser(AGY)-tRNA^Leu(CUN)),测序后结合GenBank已登陆的游隼和普通鵟相应序列探讨猛禽类共15种鸟类的分子系统进化。3个目的片段长度分别为212～214bp、353～362bp、205～208bp,通过比较这些tRNA基因序列和二级结构差异,发现可变核苷酸位点占47％,这些变异中67％出现在环区,且存在插入和(或)缺失。茎区相对保守,其中一些变异如双链的互补性碱基突变、G-U配对等对于维系tRNA二级结构的稳定性非常重要。以夜鹰为外群分别构建了15个猛禽类物种共11个线粒体tRNA基因全序列和茎区序列的NJ树和MP树．其中基于全序列的系统发育树分支具有较高的自引导值,因此该数据集所反映的猛禽类系统发育关系可能更接近真实水平。系统发育分析显示,隼形目鹰科更接近于鸱鹗科而不是隼科,而草鹗科的分类地位也与传统的形态学和DNA-DNA杂交数据的结论存在分歧。比较物种问tRNA基因二级结构发现,部分tRNA基因中的核苷酸插入和缺失特征在科水平具有共同衍征,提示这些特征对于猛禽类科间系统发育关系具有较高的参考价值。     

广东省水库3株水华微囊藻16s rRNA序列分析

微囊藻是有害淡水水华的主要藻种,但其形态分类困难,制约了相关研究的深入;16SrRNA序列分析广泛应用于微生物种类鉴定。测定广东3个水库的3株水华微囊藻16SrRNA基因部分序列,运用Mega3软件分析其遗传特征,结果表明,3株藻同源序列长度为1305bp,没有插入与缺失,序列中有3个变异位点,A T含量最大差异仅为0.2%,核苷酸相似性在99.85%以上,不能区分广东省水库同一藻种的不同藻株,表明水华微囊藻种内16SrRNA基因序列相当保守。要准确鉴定广东省水库微囊藻种类,丰富广东省水库微囊藻分子层面的分类资料,需要比较更多形态和地理来源的藻株,建立16SrRNA和其它基因序列核苷酸数据库,分析种间和种内差异,设计种专一性的分子探针。     

中国首例输入性中东呼吸综合征冠状病毒结构基因与附属基因的序列分析

针对中国首例实验室确诊输入性MERS-CoV感染病例,采用鼻咽拭子样品进行核酸提取、基因扩增与测序,获得MERS-CoV_ChinaGD01结构蛋白与附属蛋白编码基因,包括S基因、E基因、M基因、N基因、ORF3、ORF4a、ORF4b、ORF5和ORF8b基因序列。根据S基因进化分析发现中国输入性病例中MERS-CoV虽有少数位点变异,但仍与近年沙特流行株相近,属于MERS-CoV亚型5,N、E、M等结构基因核苷酸变异较少。附属蛋白进化分析表明:ORF3、ORF4a、ORF4b、ORF5均有一定的核苷酸替换,而ORF8b相对保守。总之,中国首例输入性MERSCoV与已发表序列相比总体表现为保守,但在部分基因序列上有一定的变异。这是中国首例输入性MERS-CoV的第一次基因分析结果报道,可为该MERS-CoV感染特点的进一步研究及相关疾病的防控提供参考。     

呼吸道合胞病毒在北京地区分离株G蛋白的基因分析

从经单克隆抗体证实为Ａ亚型的北京地区呼吸道合胞病毒（ＲＳＶ）分离株Ｂ７９中,用ＲＴ－ＰＣＲＴ扩增出编码Ｇ蛋白的基因片段,克隆至载体ｐＴＺ１８Ｒ中,经核苷酸序列测定证明,我国北京地区分离的Ａ亚型株Ｂ７９与ＲＳＶＡ亚型原型株（Ａ２株）Ｇ蛋白基因的核苷酸同源性为９３．８％,核苷酸的有义突变率为６５％,由核苷酸推导出氨基酸序列的同源性为８９．６％,氨基酸的变异主要集中在胞外区一个高度保守区的两端,而胞内区     

人类T细胞受体Vβ12基因编码区的等位多态性

T细胞抗原受体(TCR)β基因可变区核苷酸序列的变异,将影响到T细胞识别外来抗原的特异性。本文使用单链构象多态性(SSCP)技术,研究了TCRVβ12基因编码区的等位变异作用。在227例个体中,检出4个等位基因。它们按孟德尔定律分离和传递。在群体中的频率分别为0.4722、0.3166、0.2056和0.0056。核苷酸顺序分析表明存在1—3个核苷酸取代。但均为无声取代(Silent substitution),即氨基酸的顺序未改变。结果提示TCR Vβ12的蛋白质结构是高度保守的。     

猪apoC3基因的遗传变异分析

目的:探究猪apoC3基因多态性,为进一步探讨其对脂肪沉积的影响和表达调控等研究提供依据。方法:选取具有不同脂肪沉积特点的3个猪种(可乐猪、贵州白香猪和大约克猪)构建品种DNA池并进行测序,结合各SNP位点测序峰高比值估算等位基因频率,并利用在线软件对不同基因型的转录结合位点及mRNA二级结构进行预测。结果:在3个猪种的apoC3基因中共发现17个SNPs(2个位于5’侧翼区;1个位于外显子3中,为同义突变;1个位于3’非翻译区,其它13个分别位于3个不同内含子中),其中C813G变异增加了一个转录因子GATA-2结合位点,G2280A变异导致mRNA二级结构最小自由能增加0.4kkal/mol。结论:不同猪种间apoC3基因SNPs位点等位基因频率差异较大,而apoC3基因编码区相对保守。     

呼吸道合胞病毒B亚型分离株的G蛋白基因分析

对一株长春地区B亚型分离株(CC169)的G蛋白基因进行了 序列分析,结果表明：我国呼吸道合胞病毒(RSV)分离株CC169同RSV B亚型原型株CH18537的 核苷酸同源性为94%,核苷酸的有义突变率达65%。由核苷酸推导出氨基酸序列的同源性为894%,氨基酸的变异全部发生在胞外区,并主要集中在一个高度保守区的两端,胞内区和跨 膜区保守不变。氨基酸的变异导致了分离株既有糖基化位点的改变,又有蛋白长度的变异。 此外还初步探讨了我国RSV B 亚型分离株CC169的G蛋白基因同原型株之间的变异与疫苗研制 中的意义。     

阎氏菌科的16S rRNA可变区二级结构分析*

采用16SrRNA可变区二级结构图形分析,比较了阎氏菌科阎氏菌属典型种与微球菌亚目中几个相关科属典型种可变区二级结构的变化。结果表明,V3、V4存在明显的不同。将16SrRNA二级结构划分成不同的结构单元,提出在9个可变区中,至少要有2个不同的结构单元才可以定为新科,存在1个不同的结构单元可以定位新属;并认为16SrRNA可变区二级结构分析,可以作为一种辅助手段,应用于原核生物属以上水平的分类。     

Intragenomic and interspecific 5S rDNA sequence variation in five Asian pines

Patterns of intragenomic and interspecific variation of 5S rDNA in Pinus (Pinaceae) were studied by cloning and sequencing multiple 5S rDNA repeats from individual trees. Five pines, from both subgenera, Pinus and Strobus, were selected. The 5S rDNA repeat in pines has a conserved 120-base pair (bp) transcribed region and an intergenic spacer region of variable length (382-608 bp). The evolutionary rate in the spacer region is three- to sevenfold higher than in the genic region. We found substantial sequence divergence between the two subgenera. Intragenomic sequence heterogeneity was high for all species, and more than 86% of the clones within each individual were unique. The 5S gene tree revealed that different 5S repeats within individuals are polyphyletic, indicating that their ancestral divergence preceded the speciation events. The degrees of interspecific and intragenomic divergence among diploxylon pines are similar. The observed sequence patterns suggest that concerted evolution has been acting after the diversification of the two subgenera but very weak after the speciation of the four diploxylon pines. Sequence patterns in P. densata are consistent with hybrid origin. It had higher intragenomic diversity and maintained polymorphic copies of the parental types in addition to new and recombinant types unique to the hybrid.     

Chromosomal localization of 5S and 18S–5.8S–25S ribosomal DNA sites in five Asian pines using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was employed on mitotic metaphase chromosome preparations of five Asian Pinus species: Pinus tabuliformis, Pinus yunnanensis, Pinus densata, Pinus massoniana and Pinus merkusii, using simultaneously DNA probes of the 18S rRNA gene and the 5S rRNA gene including the non-transcribed spacer sequences. The number and location of 18S rDNA sites varied markedly (5-10 pairs of strong signals) among the five pines. A maximum of 20 major 18S rDNA sites was observed in the diploid genome (2n = 24) of P. massoniana. The 5S rDNA FISH pattern was less variable, with one major site and one minor site commonly observed in each species. The differentiation of rDNA sites on chromosomes among the five pines correlates well with their phylogenic positions in Pinus as reconstructed from other molecular data. P. densata, a species of hybrid origin, resembles its parents ( P. tabuliformis and P. yunnanensis), including some components characteristic of each parent in its pattern. However, the species is unique, showing new features resulting possibly from recombination and genome reorganization.     

Chromosomal localization of 5S and 18S rDNA in five species of subgenus Strobus and their implications for genome evolution of Pinus

BACKGROUND AND AIMS: Studying the genome structure of pines has been hindered by their large genomes and uniform karyotypes. Consequently our understanding of the genome organization and evolutionary changes in different groups of pines is extremely limited. However, techniques are now available that can surmount these difficulties. The purpose of this study was to exploit some of these techniques to characterize the genome differentiation between the two subgenera of Pinus: Pinus and Strobus. METHODS: Double-probe fluorescence in-situ hybridization (FISH) was used to localize the 5S and 18S rDNA loci on chromosomes of five species from the subgenus Strobus: P. bungeana, P. koraiensis, P. armandii, P. wallichiana and P. strobus. * KEY RESULTS: The rDNA FISH pattern varied considerably among the five species, with P. bungeana being the most distinct. By comparing the results obtained with those of previous rDNA FISH studies of members of the subgenus Pinus, several general features of rDNA loci distribution in the genus Pinus can be discerned: (a) species of subgenus Strobus generally have more rDNA loci than species of subgenus Pinus, correlating with their larger genomes in the subgenus Strobus; (b) there is a clear differentiation in 5S and 18S rDNA loci linkage patterns between the two subgenera; (c) variations in the rDNA FISH pattern correlate with phylogenetic relationships among species within the subgenus; (d) P. bungeana has fewer 18S rDNA sites than other pines investigated to date, but they give intense signals, and may reflect the primary distribution of the 18S-25S rDNA loci in the genus. CONCLUSIONS: The stable differentiation in rDNA FISH pattern between the subgenera suggests that chromosomal rearrangements played a role in the splitting of the two subgenera, and transpositional events rather than major structural changes are likely responsible for the variable rDNA distribution patterns among species of the same subgenus with conserved karyotypes.     

The Pines and Pine Forests of Xizang

Six species of pines are distributed in Xizang. They are: Pinus griffithii McClell., P. armandi Franch. and P. gerardiana Wall. of haploxylon pines and P. densata Mast., P. yttnnanensis French. and P. roxburghii Sarg. of diploxylon pines. According to the relation of these pines with water-temperature conditions, 4 ecological types may be divided: the warm-temp erate and wet type (P. griffithii), the warm-temperate and dry type (P. yunnanensis, P. roxburghii), the temperate-cold and moist type (P. armandi) and the temperate-cold and dry type (P. densata). The composition and structure of every pine community reflect the ecological environments of the given pine in the region. The main pine comnmnity in Xizang are P. griffithii forest and P. densata forest. The P. griffithii forest is distributed on the southern side of Himalayas, while the P. densata forest on the northern side of Himalayas and the southern part of Hengtuan mountains. This indicates that the Himalaya range is a clear boundary and there is difference in water-temperature condition between southern and northern parts. They belong different vegetation regions. The different distribution of other several pine forests reflects the difference of environmental conditions within these two regions. These facts have significance in the investigation of the regularity of vegetation distribution and vegetation division in Xizang. Besides, the vertical distribution of pines cannot be used as a marker to divide the altitudinal belts due to the wide range of adaptation of pines, though there must be regularity of vertical distribution too.     

Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.     

The structure of a single unit of ribosomal RNA gene (rDNA) including intergenic subrepeats in the Australian bulldog ant Myrmecia croslandi (Hymenoptera: Formicidae)

A complete single unit of a ribosomal RNA gene (rDNA) of M. croslandi was sequenced. The ends of the 18S, 5.8S and 28S rRNA genes were determined by using the sequences of D. melanogaster rDNAs as references. Each of the tandemly repeated rDNA units consists of coding and non-coding regions whose arrangement is the same as that of D. melanogaster rDNA. The intergenic spacer (IGS) contains, as in other species, a region with subrepeats, of which the sequences are different from those previously reported in other insect species. The length of IGSs was estimated to be 7-12 kb by genomic Southern hybridization, showing that an rDNA repeating unit of M. croslandi is 14-19 kb-long. The sequences of the coding regions are highly conserved, whereas IGS and ITS (internal transcribed spacer) sequences are not. We obtained clones with insertions of various sizes of R2 elements, the target sequence of which was found in the 28S rRNA coding region. A short segment in the IGS that follows the 3' end of the 28S rRNA gene was predicted to form a secondary structure with long stems.     

Systematic analysis and evolution of 5S ribosomal DNA in metazoans

Several studies on 5S ribosomal DNA (5S rDNA) have been focused on a subset of the following features in mostly one organism: number of copies, pseudogenes, secondary structure, promoter and terminator characteristics, genomic arrangements, types of non-transcribed spacers and evolution. In this work, we systematically analyzed 5S rDNA sequence diversity in available metazoan genomes, and showed organism-specific and evolutionary-conserved features. Putatively functional sequences (12 766) from 97 organisms allowed us to identify general features of this multigene family in animals. Interestingly, we show that each mammal species has a highly conserved (housekeeping) 5S rRNA type and many variable ones. The genomic organization of 5S rDNA is still under debate. Here, we report the occurrence of several paralog 5S rRNA sequences in 58 of the examined species, and a flexible genome organization of 5S rDNA in animals. We found heterogeneous 5S rDNA clusters in several species, supporting the hypothesis of an exchange of 5S rDNA from one locus to another. A rather high degree of variation of upstream, internal and downstream putative regulatory regions appears to characterize metazoan 5S rDNA. We systematically studied the internal promoters and described three different types of termination signals, as well as variable distances between the coding region and the typical termination signal. Finally, we present a statistical method for detection of linkage among noncoding RNA (ncRNA) gene families. This method showed no evolutionary-conserved linkage among 5S rDNAs and any other ncRNA genes within Metazoa, even though we found 5S rDNA to be linked to various ncRNAs in several clades.     

Variation in the nrDNA ITS of Pinus subsection Cembroides: implications for molecular systematic studies of pine species complexes.

The pinyon pines (Pinus subsection Cembroides), distributed in semiarid regions of the western United States and Mexico, include a mixture of relictual and more recently evolved taxa. To investigate relationships among the pinyons, we screened and partially sequenced 3000-bp clones of the nuclear ribosomal DNA internal transcribed spacer (ITS) region for 16 taxa from subsect. Cembroides and nine representatives from four other subsections of subgenus Strobus. Restriction digests of clones reveal within-individual heterogeneity, suggesting that concerted evolution is operating slowly on the ITS in pine species. Two ITS clones were identified as pseudogenes. Tandem subrepeats in the ITS1 form stem loops comparable to those in other genera of Pinaceae and may be promoting recombination between rDNA repeats, resulting in ITS1 chimeras. Within the pinyon clade, phylogenetic structure is present, but different clones from the same (or different) individuals of a species are polyphyletic, indicating that coalescence of ITS copies within individual genomes predates evolutionary divergence in the group. At the level of subsection and above, the ITS region corresponds well with morphological and cpDNA evidence. Except for P. nelsonii, the pinyons are monophyletic, with both subsect. Cembroides and P. nelsonii forming a clade with the foxtail and bristlecone pines (subsect. Balfourianae) of western North America.     

高山松及其亲本种油松和云南松DHAR基因的功能分化

高山松(Pinus densata)是油松(P. tabulaeformis)与云南松(P. yunnanensis)自然杂交产生的同倍性杂种, 分布于青藏高原东南缘, 占据了油松和云南松两个亲本种都不能正常生长的高海拔地带。为了揭示高山松、油松和云南松脱氢抗坏血酸还原酶(DHAR)基因的组成和功能分化, 分别从高山松、油松和云南松中克隆到2类DHAR基因(DHAR1与DHAR2)。组织表达模式分析表明, 这6个基因在根、韧皮部、叶和芽中均有表达; 通过系统发育分析发现, 高山松在物种形成过程中保留了油松的DHAR1拷贝以及云南松的DHAR2拷贝; 酶学性质分析则表明, 高山松与油松DHAR1蛋白对底物具有相似的催化活性、催化效率、最适pH和热力学稳定性, 但其催化活性比云南松DHAR1蛋白高约300倍。高山松DHAR2蛋白对底物的催化活性和热力学稳定性均高于油松DHAR2蛋白。高山松DHAR基因在生化功能上显示出优于或类似亲本DHAR, 这种优势功能的选择与杂种独特的生态适应性可能有重要的相关性。     

A minor class of 5S rRNA genes in Saccharomyces cerevisiae X2180-1B, one member of which lies adjacent to a Ty transposable element

In Saccharomyces cerevisiae the majority of the genes for 5S rRNA lie within a 9kb rDNA sequence that is present as 100-200 tandemly-repeated copies on Chromosome XII. Following our observations that about 10% of yeast 5S rRNA exists as minor variant sequences, we screened a collection of yeast DNA fragments cloned in lambda gt for 5S rRNA genes whose flanking sequences differed from those adjacent to 5S rRNA genes of the rDNA repeat. Three variant 5S rRNA genes were isolated on the basis of such dissimilarity to rDNA repeat sequences. They display a remarkable conservation of their DNA in the vicinity of the 5S coding region, and are examples of a minor form of 5S rRNA coding sequence present in a small number of copies in the yeast genome. These variant sequences appear to be transcribed as efficiently as 5S rRNA genes of the rDNA repeat. In one of our isolates of the variant sequence a Ty transposable element is inserted 145bp upstream of the initiation point for 5S rRNA synthesis.