用mtDNA 12S rRNA序列变异检验鲤形目鱼类系统发育关系

通过对鲤形目鱼类5个科的代表类群的完整线粒体12S rRNA进行测序和分析,以检验目前的形态学假说。经序列比对后,有1000个位点,其中467个位点在茎区,533个在环区;有395个位点为变化位点,其中267个为系统发育信息位点。采用邻接法和最大简约法进行了系统发育分析,其结果支持鲤科鱼类成为一个单系群,非鲤科的鲤形目鱼类成为另一个单系群的观点,这与Siebert提出的假说相一致。鲤科鱼类包含3个主要的分支,即鱼丹系、鲤系和雅罗鱼系;但在非鲤科鲤形目鱼类中,其关系不能得到很好的解决,其中鳅科是复系,平鳍鳅科、条鳅亚科和花鳅亚科可能有较近的亲缘关系。     

基于线粒体16S rDNA序列探讨蛱蝶科(鳞翅目, 蝶亚目)主要分类群的系统发生关系

本文测定了蛱蝶科7亚科27种蛱蝶和斑蝶科2种蝴蝶的线粒体16S rRNA基因部分序列,并从GenBank中下载了6种蛱蝶的同源序列。以斑蝶科的幻紫斑蝶和绢斑蝶作外群,通过遗传分析软件对这些序列进行了比较分析,用邻接法和贝叶斯法重建了蛱蝶科的系统发育树,探讨了蛱蝶科主要类群间的系统发育关系。序列分析的结果显示:经比对处理后获得494bp长度序列,其中有可变位点206个,简约信息位点145个;A T平均含量78.4%,C G平均含量为21.6%,具A、T偏倚性。分子系统树显示:蛱蝶亚科并非单系群;蛱蝶族中眼蛱蝶属应移入斑蛱蝶族;闪蛱蝶和蛱蝶亚科与蛱蝶亚科具有较近的系统关系;结果支持豹蛱蝶和釉蛱蝶合为一亚科即釉蛱蝶亚科;支持将秀蛱蝶和蛱蝶亚科从线蛱蝶亚科中分离出来。     

从18S rRNA基因序列探讨盾腹吸虫的系统发育关系

盾腹亚纲吸虫被认为是寄生扁形动物中古老的类群,包括盾腹科、多萼科、裂杯科和皱腹科,科间系统发育关系尚存争议。本研究收集GenBank数据库中所有的盾腹吸虫18S rRNA基因序列,测定了三种盾腹吸虫的相应序列,分别采用最大简约法和最大似然法构建分子系统发育树。结果显示,多萼科的分类地位不成立,多萼属应还原到盾腹科;盾腹科的盾腹亚科和杯盾亚科均非单系,吸槽列数可能是平行进化特征,不能反映盾腹科各亚科间的系统发育关系。建议将具有边缘器的吸槽型腹吸盘,以及不具边缘器的吸杯型和皱褶型腹吸盘分别鉴定为盾腹科、裂杯科和皱腹科种类的共裔性状。     

基于18S rRNA基因序列的直翅目主要类群系统发育关系研究

基于78种直翅目昆虫的18S rRNA基因全序列构建了直翅目各主要类群间的系统发育关系。本研究的结果支持直翅目的单系性,但不支持蝗亚目和螽亚目各自的单系性;直翅目下除蜢总科和蝗总科外各总科的划分多数与Otte系统相一致;蜢总科的单系性得不到支持;蝗总科的剑角蝗科、斑腿蝗科、斑翅蝗科、网翅蝗科和槌角蝗科5科均不是单系群,各物种间的遗传距离差异不大,应合并为一科,即蝗科;本研究支持将Otte系统中蚱总科和螽蟖总科下各亚科级阶元提升为科级阶元;18S rRNA基因全序列可以作为划分科级阶元的工具,当位于同一分支上互成姐妹群的类群间的遗传距离超过1%时,这几个类群属于不同的科;但由于其在进化上的保守性,18S rRNA基因只能用于纲目等高级阶元间关系的研究,而由其获得的总科以下阶元间的关系并不可靠。     

基于线粒体ND1和16S rRNA基因序列探讨中国蝴蝶12科的系统发育关系(鳞翅目: 双孔次亚目: 蝶类)

对中国12科共32种代表蝶类的ND1基因和16S rRNA 基因进行了序列测定（包括新测30种ND1基因和9种16S rRNA基因）和比较分析, 同时采用邻接法、最大似然法和贝叶斯法构建了12科蝶类的系统发育树, 探讨了其高级分类群的系统发育关系。序列分析的结果显示: 经比对处理后的两个基因总长度为869 bp, 其中保守位点373个, 可变位点496个, 简约信息位点375个; A+T的平均含量为80.2％, 明显高于C+G的平均含量19.8％。分子系统树表明: 蛱蝶科不是单系群; 珍蝶类、斑蝶类和喙蝶类位于蛱蝶科内; 粉蝶科和凤蝶科具有共同祖先。据此建议: 绢蝶科应归入凤蝶科; 蚬蝶科归入灰蝶科; 珍蝶类、斑蝶类和喙蝶类作为蛱蝶科中的亚科, 眼蝶类从蛱蝶科中分离出来独立成科。另外, 环蝶类的系统分类地位还有待于进一步研究。     

基于28S rRNA D2序列的内茧蜂亚科的分子系统发育

首次利用同源28S rRNA D2基因序列对内茧蜂亚科Rogadinae （昆虫纲Insecta：膜翅目Hymenoptera：茧蜂科Braconidae）进行了分子系统学研究。本研究从95%～100%乙醇浸渍保存的标本中提取基因组DNA并扩增了10种内群种类和5种外群种类的28S rDNA D2片段并测序（GenBank序列号AY167645-AY167659）,利用BLAST搜索相关的同源序列, 采用了GenBank中13个种类的28S rRNA D2同源序列,然后据此进行分子分析。利用3个外群（共8个种类）和3种建树方法 (距离邻近法distance based neighbor joining, NJ; 最大俭约法maximum parsimony, MP; 和最大似然法maximum likelihood, ML)分析了内茧蜂亚科内的分子系统发育关系。结果表明,由分子数据产生的不同的分子系统树均显示内茧蜂亚科是一个单系群。内茧蜂亚科内依据形态和生物学特征的分群（族和亚族）及其系统发育关系得到部分支持。NJ、MP和ML分析结果均表明内茧蜂族Rogadini不是一个单系,而是一个并系,其余3族则得到不同程度的支持。内茧蜂族可分成2个分支：“脊茧蜂属Aleiodes+弓脉茧蜂属Arcaleiodes”和“沟内茧蜂属Canalirogas+锥齿茧蜂属Conspinaria+刺茧蜂属Spinaria+内茧蜂属Rogas”,二者不是姐妹群。脊茧蜂属Aleiodes和弓脉茧蜂属Arcaleiodes始终是姐妹群。脊茧蜂属Aleiodes是一个单系,并可分成2个姐妹分支,这与依据形态和生物学特征的亚属分群相一致。弓脉茧蜂属Arcaleiodes Chen et He,1991是一个独立的属。分支“沟内茧蜂属Canalirogas+锥齿茧蜂属Conspinaria+刺茧蜂属Spinaria+内茧蜂属Rogas”的单系性仅得到部分分子数据的支持;因形态特异（腹部成甲壳状）而列为亚族级的刺茧蜂属Spinaria,分子分析没有证实这一点。横纹茧蜂族Clinocentrini是个单系,并在内茧蜂亚科的系统发育中处于基部（原始）的位置。我们研究结果还表明,阔跗茧蜂属Yelicones和潜蛾茧蜂属Stiropius相对应的阔跗茧蜂族Yeliconini和潜蛾茧蜂族Stiropiini为2个独立的分支, 与形态和生物学的结果一致,但它们在内茧蜂亚科的系统发育的位置不明,有待今后进一步研究。     

基于叶绿体16S rRNA的绿色裸藻类系统发育及性状进化研究

该研究基于叶绿体16S rRNA基因序列,构建绿色裸藻类的系统发育树,并对绿色裸藻类植物8个形态性状进行祖先重建分析,以明确绿色裸藻类植物的系统演化关系,为研究该类植物的起源提供理论依据。结果表明：(1)贝叶斯法构建的绿色裸藻类系统发育树显示,双鞭藻属与拟双鞭藻属互为姐妹群,扁裸藻属、鳞孔藻属和盘裸藻属亲缘关系较近,而囊裸藻属和陀螺藻属亲缘关系较近,裸藻属、隐裸藻属、柄裸藻属和旋形藻属亲缘关系较近,表明裸藻属不是一个单系类群。(2)基于形态性状的祖先重建结果显示,绿色裸藻类相对原始的7个性状包括：表质柔软易变形,出现螺旋形线纹,细胞后端渐尖或尖尾刺状,无囊壳,叶绿体为片状、盾状或大盘状,具无鞘蛋白核,副淀粉粒为小颗粒状且数量不定,而鞭毛长度不能推断可能的祖先状态。(3)综合8种性状祖先重建结果发现,裸藻属和眼裸藻属植物具有所有原始性状,可能是最先出现的绿色裸藻类的祖先。     

基于16S rRNA基因序列探讨中国粉蛉科的系统发育关系

本研究测定了粉蛉科Coniopterygidae9属15种昆虫16SrRNA基因部分序列,对序列的碱基组成、转换/颠换比率、遗传距离、变异位点等进行分析。基于16SrRNA基因序列数据,分别采用邻接法（NJ）、最大简约法（MP）和最大相似法（ML）建立粉蛉科分子系统发育关系。研究结果表明：粉蛉亚科的粉蛉属Coniopteryx与重粉蛉属Semilalis是姊妹群,（虫齿）粉蛉属Conwentzia较前二者原始;囊粉蛉亚科的卷粉蛉属Helicoconis和隐粉蛉属Cryptoscenea的亲缘关系较近。曲粉蛉属Coniocompsa和异粉蛉属Heteroconis聚类在一起,但自展值较低。瑕粉蛉属Spiloconis和囊粉蛉属Aleuropteryx的位置在囊粉蛉亚科中不够稳定。     

九种绢丝昆虫线粒体12S rRNA基因的序列特征和系统发育分析

为探讨鳞翅目中绢丝昆虫之间的系统发育关系和分子进化特征，本研究测定了中国柞蚕Antheraea pernyi野生型和放养型的线粒体12S rRNA基因的部分序列，结合来自GenBank数据库的17条序列，对总共9种绢丝昆虫（2科3属）的12S rRNA基因序列进行了分析。利用软件MEGA 3.1进行碱基组成、变异位点的统计和分子进化分析，分别用类平均聚类法（UPGMA）、邻接法（NJ）、最小进化法（ME）、最大简约法（MP）重建系统发生树。测定的中国柞蚕野生型的12S rRNA基因序列（427 bp）与放养型“豫早1号”的序列完全一致。序列对齐后共鉴定80个变异位点，50个简约信息位点。碱基组成分析显示在科属间具有明显差异，AT含量蚕蛾科高于大蚕蛾科；在A和T碱基的使用上，大蚕蛾科偏好使用T，而蚕蛾科则偏好使用A。与动物中常见的以转换为主的碱基替换模式不同，所分析的9种昆虫中除桑蚕属内部为转换与颠换基本一致外，其余物种间均是颠换多于转换。进化分析支持柞蚕属、樗蚕属和桑蚕属的单系。基于UPGMA法的进化树支持琥珀蚕是柞蚕属的较原始类型，而NJ、ME和MP法则支持印度柞蚕是较原始的类型，因此，柞蚕属种间的进化关系尚需进一步研究。     

Interaction of ribosomal proteins S5, S6, S11, S12, S18 and S21 with 16 S rRNA

We have examined the effects of assembly of ribosomal proteins S5, S6, S11, S12, S18 and S21 on the reactivities of residues in 16 S rRNA towards chemical probes. The results show that S6, S18 and S11 interact with the 690-720 and 790 loop regions of 16 S rRNA in a highly co-operative manner, that is consistent with the previously defined assembly map relationships among these proteins. The results also indicate that these proteins, one of which (S18) has previously been implicated as a component of the ribosomal P-site, interact with residues near some of the recently defined P-site (class II tRNA protection) nucleotides in 16 S rRNA. In addition, assembly of protein S12 has been found to result in the protection of residues in both the 530 stem/loop and the 900 stem regions; the latter group is closely juxtaposed to a segment of 16 S rRNA recently shown to be protected from chemical probes by streptomycin. Interestingly, both S5 and S12 appear to protect, to differing degrees, a well-defined set of residues in the 900 stem/loop and 5'-terminal regions. These observations are discussed in terms of the effects of S5 and S12 on streptomycin binding, and in terms of the class III tRNA protection found in the 900 stem of 16 S rRNA. Altogether these results show that many of the small subunit proteins, which have previously been shown to be functionally important, appear to be associated with functionally implicated segments of 16 S rRNA.     

Further evidence that the ribosomal 30S proteins S3, S5, S9, S11, S12, and S18 possess specific 16S RNA binding sites

Summary E. coli ribosomal 16S RNA preparted by an acetic acid-urea extraction technique individually binds, in addition to the seven established proteins, 6 new 30S ribosomal proteins (S3, S5, S9, S12, S18 and S11) (Hochkeppel et al., 1976). In this communication we demonstrate the site specificity of these proteins. Binding curves of the individual proteins with acetic acid-urea 16S RNA show that the binding of all six proteins to the RNA reaches a plateau at 0.3–0.97 copies per 16S RNA molecule. No significant binding of these proteins to classical phenol extracted 16S RNA is observed, with the exception of S13 which binds 0.2 copies of protein per molecule of 16S RNA. Specificity of binding of these proteins is also demonstrated in chase experiments. The site specificity of individual [³H]-labeled 30S proteins bound to 16S RNA is tested by the addition of non-radioactive 30S total protein to the reaction mixture.     

Positions of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit of Escherichia coli

Neutron scattering distance data are presented for 33 protein pairs in the 30 S ribosomal subunit from Escherichia coli, along with the methods used for measuring distances between its exchangeable components. When combined with prior data, these new results permit the positioning of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit, completing the mapping of its proteins by neutron scattering. Comparisons with other data suggest that the neutron map is a reliable guide to the quaternary structure of the 30 S subunit.     

RNA-protein cross-linking in Escherichia coli 30S ribosomal subunits; determination of sites on 16S RNA that are cross-linked to proteins S3, S4, S5, S7, S8, S9, S11, S13, S19 and S21 by treatment with methyl p-azidophenyl acetimidate.

RNA-protein cross-links were introduced into E. coli 30S ribosomal subunits by treatment with methyl p-azidophenyl acetimidate. After partial nuclease digestion of the RNA moiety, a number of cross-linked RNA-protein complexes were isolated by a new three-step procedure. Protein and RNA analysis of the individual complexes gave the following results: Proteins S3, S4, S5 and S8 are cross-linked to the 5'-terminal tetranucleotide of 16S RNA. S5 is also cross-linked to the 16S RNA within an oligonucleotide encompassing positions 559-561. Proteins S11, S9, S19 and S7 are cross-linked to 16S RNA within oligonucleotides encompassing positions 702-705, 1130-1131, 1223-1231 and 1238-1240, respectively. Protein S13 is cross-linked to an oligonucleotide encompassing positions 1337-1338, and is also involved in an anomalous cross-link within positions 189-191. Protein S21 is cross-linked to the 3'-terminal dodecanucleotide of the 16S RNA.     

Interaction of proteins S16, S17 and S20 with 16 S ribosomal RNA

We have used rapid chemical probing methods to examine the effect of assembly of ribosomal proteins S16, S17 and S20 on the reactivity of individual residues of 16 S rRNA. Protein S17 strongly protects a compact region of the RNA between positions 245 and 281, a site previously assigned to binding of S20. Protein S20 also protects many of these same positions, albeit more weakly than S17. Strong S20-dependent protections are seen elsewhere in the 5' domain, most notably at positions 108, and in the 160-200 and 330 loop regions. Enenpectedly, S20 also causes protection of several bases in the 1430-1450 region, in the 3' minor domain. In the presence of the primary binding proteins S4, S8 and S20, we observe a variety of effects that result from assembly of the secondary binding protein S16. Most strongly protected are nucleotides around positions 50, 120, 300 to 330 and 360 in the 5' domain, and positions 606 to 630 in the central domain. In addition, numerous nucleotides in the 5' and central domains exhibit enhanced reactivity in response to S16. Interestingly, the strength of the S20-dependent effects in the 1430-1450 region is attenuated in the presence of S4 + S8 + S20, and restored in the presence of S4 + S8 + S20 + S16. Finally, the previously observed rearrangement of the 300 region stem-loop that occurs during assembly is shown to be an S16-dependent event. We discuss these findings with respect to assignment of RNA binding sites for these proteins, and in regard to the co-operativity of ribosome assembly.     

Apparent association constants for E. coli ribosomal proteins S4, S7, S8, S15, S17 and S20 binding to 16S RNA.

We have previously reported the development of a technique utilizing nitrocellulose filters, which rapidly separates ribosomal protein-ribosomal RNA complexes from unbound protein. We have used this technique to obtain binding data for the association of proteins S4, S7, S8, S15, S17, and S20 with 16S RNA. With the exception of protein S17, the association behavior for each of these proteins exhibits a single binding site with a unique binding constant. The apparent association constants have been calculated and have been found to have a range from 1.6 x 10(6) M-1 for protein S7 to 7.1 x 10(7) M-1 for protein S17. The Scatchard plot for the protein S17 binding data is biphasic, suggesting that within the RNA population two different binding sites exist, each with a different apparent association constant.     

S

在45只切断双侧缓冲神经的Sprague-Dawley 大鼠,应用细胞外记录方法, 观察了颈动脉内注射腺苷对76 个最后区 (AP) 神经元自发放电活动的影响。所得结果如下(1) 在记录到的42个自发放电单位中, 颈动脉内注射腺苷(25 μg/kg) 引起其中29个单位的放电频率由6.26±0.75 下降至4.74±0.76 spikes/s (P<0.01), 6 个单位放电频率由4.13±0.77增加至4.72±0.83 spikes/s (P<0.05),另外7个单位放电频率无明显变化, 而血压和心率在实验中无变化; (2)在应用非选择性腺苷受体拮抗剂8-苯茶碱(8-phenyltheophylline, 15 μg/kg) 的10个单位, 腺苷对放电的抑制效应可被完全阻断; (3) 应用选择性腺苷A     

Expression and purification of human ribosomal proteins S3, S5, S10, S19, and S26

The cDNAs for the human ribosomal proteins S3, S5, S10, S19, and S26 were introduced into a pET-15b vector and recombinant proteins containing an N-(His)(6)-fusion tag were expressed in high yields. To resolve the problem of frameshift during expression of S26 caused by the presence of tandem arginine codons in its mRNA that are rare in Escherichia coli, we substituted the rare AGA codon with the more frequent arginine codon (CGC) using a primer with this mutation for PCR amplification of S26 cDNA. All proteins were expressed mainly in the form of inclusion bodies and purified to homogeneity by metal affinity chromatography in one step (except for S3). Expression of the full-length S3 was accompanied by the formation of a low molecular weight polypeptide that was co-purified with S3 by metal affinity chromatography. Complete purification of S3 required an additional gel-filtration step. The proteins were refolded by stepwise dialysis. Both identity and purity of the proteins were confirmed by 2D PAGE. The proteins obtained could be used in a wide range of applications in biophysics, biochemistry, and molecular biology.