壳斗科植物叶绿体基因组结构及变异分析

利用生物信息学方法比较壳斗科6个属14个物种的叶绿体基因组间差异,以近缘物种榛为外类群构建系统进化树,揭示壳斗科叶绿体基因组的结构特征及变异规律。结果显示,14种壳斗科植物的叶绿体基因组均为双链环状分子结构,大小在160 kB左右,差异较小,最大仅差1 366 bp;基因顺序基本一致,而基因数量有所差异,infA、petG、rpl22、ycf1、ycf15等多个基因在部分物种中发生丢失;主要有32个蛋白编码基因长度发生变异,其原因是内含子的丢失、内含子或者编码区的长度改变,华南锥基因长度变异较大;4个IR边界相对保守,但锥栗、Castanea pumila、华南锥3个物种由于边界扩张导致rps19基因部分序列进入到IR区;以榛为外类群构建的系统发育树,各进化支支持率较高,分辨率较好。研究结果表明,叶绿体基因组可以用于分析关系较近与进化较快物种的系统发生问题,为系统发育和进化研究提供依据。     

基于CHD基因序列的18种猛禽鸟类系统发育关系

对18个猛禽CHD基因的一段内含子序列进行比较和分析.CHD-W和CHD-Z基因的多态性存在差异,CHD-W基因不适合种间系统发生学的研究.通过对CHD-Z基因扩增序列构建的NJ和ML树显示:隼科与其他猛禽物种关系较远;鹰科鸟类与鸮形目鸟类亲缘关系较近;在白腹鹞的分类地位上与传统形态学分类不一致;长耳鸮、领角鸮、花彩角鸮、西部鸣角鸮的分类地位存在分歧.鸮形目和隼形目鸟类的CHD-W基因大小有明显区别,支持形态学分类结果,与CHD-Z序列分析结果明显不同.     

异源四倍体鲫鲤及其原始父母本细胞周期蛋白B基因克隆与分子遗传分析

细胞周期蛋白(cyclin)B是真核细胞周期运转中调控G2期至M期转化的关键因子.本实验根据斑马鱼细胞周期蛋白 B1基因的剪切方式,设计3对特异于金鱼和银鲫细胞周期蛋白B基因外显子区兼并引物,首次扩增出异源四倍体鲫鲤及其原始亲本红鲫和鲤鱼细胞周期蛋白B基因2条大小分别约为2.4 kb和2.1 kb的片段.测序及比对分析表明：这3种鱼的细胞周期蛋白B基因2个片段均包含8个外显子和7个内含子.内含子剪切位点符合GT/AG规则,推测细胞周期蛋白B基因2个片段可能是细胞周期蛋白B基因的2种存在形式.异源四倍体鲫鲤与其原始父母本细胞周期蛋白B基因片段序列的比较结果表明：无论是外显子区还是内含子区,异源四倍体鲫鲤与其原始亲本都具有较高的遗传相似性,在1 025 bp的外显子序列中相同的碱基位点数达963个,为异源四倍体鲫鲤来源于红鲫和鲤鱼提供了分子证据;同时,异源四倍体鲫鲤与其原始亲本差异碱基位点的存在又表明这一独特的多倍体物种与其原始亲本存在着进化上的变异.此外,还分别以细胞周期蛋白B基因外显子和内含子序列构建了包括异源四倍体鲫鲤及其原始父母本在内的系统进化树.结果初步表明：对于亲缘关系较近的物种,用外显子和内含子序列构建的系统进化树与传统的物种进化树一致;而对于亲缘关系较远的物种,用内含子序列构建的进化树与传统的物种进化顺序存在较大差异.     

动物肌动蛋白基因中内含子的来源及存在意义的探讨

对动物界演化过程中肌动蛋白家族内含子插入位置分布的演化规律作了分析,并对相同插入位置的内含子序列按同亚型和不同亚型作了比较。结果得出:从整个肌动蛋白家族的外显子序列高度保守性推断整个肌动蛋白家族可能是从共同的祖先蛋白进化而来的;从同亚型肌动蛋白内含子序列的类似性随进化距离而变化,但在短进化距离的物种间,类似性都较高,不同亚型肌动蛋白内含子序列的类似性都较低,即使是同一物种(如人),类似性也远低于同亚型但进化距离较近的物种,由此可推断,同亚型肌动蛋白的内含子序列可能从共同祖先进化,不同亚型肌动蛋白的内含子序列从不同祖先进化,综上推断可导出内含子可能是在蛋白异化过程中获得的:还发现内含子在肌动蛋白家族编码基因中位置的分布随进化方向不同而逐步形成两种截然不同的模式,由此提出了内含子的位置分布与动物演化方向之间可能具有某种必然联系,为内含子的存在提出了某种依据。     

基于线粒体COⅠ和Cytb基因序列研究粉蝶科七属间的系统发生关系

【目的】为了探讨粉蝶科Pieridae 7属的系统进化关系。【方法】基于线粒体COⅠ(609 bp)和Cytb(393 bp)基因部分序列,以眼蝶科的2个物种为外类群,运用UPGMA和ME法重建分子系统树。【结果】联合基因构建的分子系统树显示:外群牧女珍眼蝶Coenonympha amaryllis(Cramer)和阿芬眼蝶Aphantopus hyperantu(Linnaeus)构成一独立支系,可以作为外群;云粉蝶属和粉蝶属姊妹关系构成一分支,亲缘关系较近;襟粉蝶属与钩粉蝶属形成姐妹关系,亲缘关系较近。【结论】成功重建了粉蝶科7属的系统进化关系。     

麝属分子系统进化地位的再分析

对麝Moschus spp.的分子系统进化地位进行了再研究.结果 表明,不同的基因及序列长度、分析中不同的物种数目、不同的分析方法对研究结果产生明显影响.在用线粒体Cyt b、16S rRNA基因及二者的连接序列分别构建的NJ、MP树中,都支持麝与鹿有更近的亲缘关系.在使用γ干扰素核基因的编码序列构建的NJ树中,显示麝与鹿也有较近的亲缘关系;而MP树则暗示麝与牛科有更近的亲缘关系.当选用包括林麝在内的18个物种,使用线粒体基因组重链上12个蛋白编码基因的核苷酸串连序列构建系统发生树时,在NJ、MP、ML和BI树中都支持麝与鹿有更近的亲缘关系,与选用23个物种得到的支持麝为鹿科/牛科二者共同姐妹群的结果存在明显差异.这可能是因为麝科与鹿科和牛科的亲缘关系较近,分歧时间较短,其分子片段所累积的进化信息较少,而且不同的分子片段进化速率不一致等造成的.因此,要彻底解决麝在偶蹄目中的进化地位必须要找到更适合的分子标记.     

中国特有果蝇 Drosophila curviceps 种亚组在Drosophila immigrans种组中的种系发生地位

果蝇immigrans种组中的curviceps种亚组是1992年新建立的中国特有果蝇类群。该种亚组中的物种主要分布在中国大陆和台湾。目前除了形态学水平的研究外,还没有其他证据支持建立该种亚组的合理性及其起源和种系发生地位。为了在DNA分子水平上探讨果蝇curviceps种亚组在果蝇immigrans种组中的种系发生地位,从而为今后更深入地研究中国特有果蝇,甚至为果蝇亚属的进化遗传学提供理论依据,测定了immigrans种组5个种亚组（nasuta、immigrans、hypocausta、quadrilineata、curviceps)中12个代表物种的rDNA的ITS1和部分Adh基因的序列。其中ITS1序列的长度为513－587bp,共有191个信息位点;Adh基因片段的长度在714－747bp之间,共99个信息位点。考虑到单个分子提供的信息较少,将两个分子的序列综合起来,组成一个较长的复合序列。分别根据ITS1,Adh和两个分子的复合序列排比（Alignment)结果,和最大简约法和邻接法构建分子系统树,其中根据复合序列构建的系统树与形态学研究结果最为一致。分子树显示curviceps种亚组的特种确定单独形成一个分枝,为种亚组级的分类阶元,支持了形态学将其建立为一个新种亚组。根据Kimura距离,估算了复合分子的替换速率约为每百万年1．48％,进而计算出5个种亚组的分 歧年代。结合各物种的地理分布,推测了immigrans种组的进化历史：curviceps种亚组与quadrilineata种亚组的亲缘关系最近,主要分布在中国南部的温带地区。它们之间的分歧时间大约为3．4百万年,是最年轻的两个种亚组。主要分布在苏门答腊及附近的热带地区的hypocausta种亚组的物种是最早分化出来的,与其他种亚组的分歧时间约为9．2百万年。该结果与形态学和生物地理学研究相吻合。值得一提是的,目前归属仍存在争议的物种D.neohypocausta,在分子系统树中与hypocausta种亚组的物种相距较远,而与immiagrasn种亚组的关系较近,但分枝置信度较低（＜50％）。由于还缺乏其他方面的证据,因此D.neohypocausta的归属有待今后的研究来作定论。     

基于CHD基因序列的隼形目12种鸟系统发育关系

对12个隼形目物种CHD基因的一段内含子序列进行比较和分析。CHD-W和CHD-Z基因的多态性存在差异,CHD-W基因不适合系统发生学的研究。通过对CHD-Z基因扩增序列构建的NJ和MP树,结果显示隼科鸟类与鹰科和鹳科鸟类关系较远,与传统形态分类和和鸟类新的分类系统不一致。在白腹鹞的分类地位上与传统形态学分类不一致,将白腹鹞归入鹰属一支。NJ树显示鸢属(黑鸢)与鵟属(毛脚鵟)亲缘关系较近,MP树显示鵟鹰属(灰脸鵟鹰)与鵟属(毛脚鵟)亲缘关系较近。NJ和MP树都显示雕属(金雕)和鹰雕属(鹰雕)与其他鹰科各属亲缘关系较远。     

中国地鼠线粒体Cyt b基因测序及其分子进化

目的测定中国地鼠线粒体DNA细胞色素b基因部分序列,分析其分子系统进化关系。方法提取中国地鼠肝脏的总基因组DNA。设计合成特异引物进行PCR扩增,经检测进行测序。用Blast与GenBank中啮齿类其他常用实验动物的物种细胞色素b基因进行同源序列比较,分析其碱基组成及变异情况,并用邻接法、最大简约法、最小进化法构建了分子系统树,在分子水平上探讨中国地鼠和常用啮齿类实验动物的进化关系。结果获得了中国地鼠线粒体Cytb基因的部分序列,共936bp。结论中国地鼠和金黄地鼠的亲缘关系最近,与小鼠、大鼠存在的差异相对大,与豚鼠的亲缘关系最远,与传统的分类地位基本吻合。     

美国红鱼生长激素基因的克隆及其遗传进化分析

采用PCR方法从美国红鱼肝脏中扩增出美国红鱼生长激素基因。该基因序列长为1 497 bp,与5个已报道的鲈亚目鱼类生长激素基因结构比较发现,他们的外显子和内含子之比均为6∶5,而且相对应的6个外显子大小一致。用Clustal X软件对美国红鱼和其他13种鲈形目鱼类生长激素基因序列进行比对,选取同源性较高的551 bp的编码区序列构建NJ和MP系统进化树,结果发现由其构建的MP和NJ树与根据形态特征构建的系统发育树基本一致,特别是在鲈形目鱼类科间及科以上分类阶元的系统发育研究方面比较一致。研究结果表明鱼类生长激素基因序列能够较真实反映近缘鱼类之间的关系,是进行鲈形目鱼类系统进化分析的较好遗传标志。     

RNomics: an experimental approach that identifies 201 candidates for novel, small, non-messenger RNAs in mouse

In mouse brain cDNA libraries generated from small RNA molecules we have identified a total of 201 different expressed RNA sequences potentially encoding novel small non-messenger RNA species (snmRNAs). Based on sequence and structural motifs, 113 of these RNAs can be assigned to the C/D box or H/ACA box subclass of small nucleolar RNAs (snoRNAs), known as guide RNAs for rRNA. While 30 RNAs represent mouse homologues of previously identified human C/D or H/ACA snoRNAs, 83 correspond to entirely novel snoRNAS: Among these, for the first time, we identified four C/D box snoRNAs and four H/ACA box snoRNAs predicted to direct modifications within U2, U4 or U6 small nuclear RNAs (snRNAs). Furthermore, 25 snoRNAs from either class lacked antisense elements for rRNAs or snRNAS: Therefore, additional snoRNA targets have to be considered. Surprisingly, six C/D box snoRNAs and one H/ACA box snoRNA were expressed exclusively in brain. Of the 88 RNAs not belonging to either snoRNA subclass, at least 26 are probably derived from truncated heterogeneous nuclear RNAs (hnRNAs) or mRNAS: Short interspersed repetitive elements (SINEs) are located on five RNA sequences and may represent rare examples of transcribed SINES: The remaining RNA species could not as yet be assigned either to any snmRNA class or to a part of a larger hnRNA/mRNA. It is likely that at least some of the latter will represent novel, unclassified snmRNAS:     

Processing of intron-encoded box C/D small nucleolar RNAs lacking a 5',3'-terminal stem structure

The C and D box-containing (box C/D) small nucleolar RNAs (snoRNAs) function in the nucleolytic processing and 2'-O-methylation of precursor rRNA. In vertebrates, most box C/D snoRNAs are processed from debranched pre-mRNA introns by exonucleolytic activities. Elements directing accurate snoRNA excision are located within the snoRNA itself; they comprise the conserved C and D boxes and an adjoining 5',3'-terminal stem. Although the terminal stem has been demonstrated to be essential for snoRNA accumulation, many snoRNAs lack a terminal helix. To identify the cis-acting elements supporting the accumulation of intron-encoded box C/D snoRNAs devoid of a terminal stem, we have investigated the in vivo processing of the human U46 snoRNA and an artificial snoRNA from the human beta-globin pre-mRNA. We demonstrate that internal and/or external stem structures located within the snoRNA or in the intronic flanking sequences support the accumulation of mammalian box C/D snoRNAs lacking a canonical terminal stem. In the intronic precursor RNA, transiently formed external and/or stable internal base-pairing interactions fold the C and D boxes together and therefore facilitate the binding of snoRNP proteins. Since the external intronic stems are degraded during snoRNA processing, we propose that the C and D boxes alone can provide metabolic stability for the mature snoRNA.     

In vitro assembly of the mouse U14 snoRNP core complex and identification of a 65-kDa box C/D-binding protein.

The eukaryotic nucleolus contains a diverse population of small nucleolar RNAs (snoRNAs) that have been categorized into two major families based on evolutionarily conserved sequence elements. U14 snoRNA is a member of the larger, box C/D snoRNA family and possesses nucleotide box C and D consensus sequences. In previous studies, we have defined a U14 box C/D core motif that is essential for intronic U14 snoRNA processing. These studies also revealed that nuclear proteins that recognize boxes C/D are required. We have now established an in vitro U14 snoRNP assembly system to characterize protein binding. Electrophoretic mobility-shift analysis demonstrated that all the sequences and structures of the box C/D core motif required for U14 processing are also necessary for protein binding and snoRNP assembly. These required elements include a base paired 5',3' terminal stem and the phylogenetically conserved nucleotides of boxes C and D. The ability of other box C/D snoRNAs to compete for protein binding demonstrated that the box C/D core motif-binding proteins are common to this family of snoRNAs. UV crosslinking of nuclear proteins bound to the U14 core motif identified a 65-kDa mouse snoRNP protein that requires boxes C and D for binding. Two additional core motif proteins of 55 and 50 kDa were also identified by biochemical fractionation of the in vitro-assembled U14 snoRNP complex. Thus, the U14 snoRNP core complex is a multiprotein particle whose assembly requires nucleotide boxes C and D.     

Identification of specific nucleotide sequences and structural elements required for intronic U14 snoRNA processing.

Vertebrate U14 snoRNAs are encoded within hsc70 pre-mRNA introns and U14 biosynthesis occurs via an intron-processing pathway. We have shown previously that essential processing signals are located in the termini of the mature U14 molecule and replacement of included boxes C or D with oligo C disrupts snoRNA synthesis. The experiments detailed here now define the specific nucleotide sequences and structures of the U14 termini that are essential for intronic snoRNA processing. Mutagenesis studies demonstrated that a 5', 3'-terminal stem of at least three contiguous base pairs is required. A specific helix sequence is not necessary and this stem may be extended to as many as 15 base pairs without affecting U14 processing. The spatial positioning of boxes C and D with respect to the terminal stem is also important. Detailed analysis of boxes C and D revealed that both consensus sequences possess essential nucleotides. Some, but not all, of these critical nucleotides correspond to those required for the stable accumulation of nonintronic yeast U14 snoRNA. The presence of box C and D consensus sequences flanking a terminal stem in many snoRNA species indicates the importance of this "terminal core motif" for snoRNA processing.     

A structural,phylogenetic, and functional study of 15.5-kD/Snu13 protein binding on U3 small nucleolar RNA

The 15.5-kD protein and its yeast homolog Snu13p bind U4 snRNA, U3 snoRNA, and the C/D box snoRNAs. In U4 snRNA, they associate with a helix-bulge-helix (K-turn) structure. U3 snoRNA contains two conserved pairs of boxes, C'/D and B/C, which were both expected to bind the 15.5-kD/Snu13 protein. Only binding to the B/C motif was experimentally demonstrated. Here, by chemical probing of in vitro reconstituted RNA/protein complexes, we demonstrate the independent binding of the 15.5-kD/Snu13 protein to each of the two motifs. Due to a highly reduced stem I (1 bp), the K-turn structure is not formed in the naked B/C motif. However, gel-shift experiments revealed a higher affinity of Snu13p for the B/C motif, compared to the C'/D motif. A phylogenetic analysis of U3 snoRNA, coupled with an analysis of Snu13p affinity for variant yeast C'/D and B/C motifs, and a study of the functionality of a truncated yeast U3 snoRNA carrying base substitutions in the C'/D and B/C motifs, revealed that conservation of the identities of residues 2 and 3 in the B/C K-turn is more important for Snu13p binding and U3 snoRNA function, than conservation of the identities of corresponding residues in the C'/D K-turn. This suggests that binding of Snu13p to K-turns with a very short helix I imposes sequence constraints in the bulge. Altogether, the data demonstrate the strong importance of the binding of the 15.5-kD/Snu13 protein to the C'/D and B/C motifs for both U3 snoRNP assembly and activity.     

Elements essential for processing intronic U14 snoRNA are located at the termini of the mature snoRNA sequence and include conserved nucleotide boxes C and D.

Essential elements for intronic U14 processing have been analyzed by microinjecting various mutant hsc70/Ul4 pre-mRNA precursors into Xenopus oocyte nuclei. Initial truncation experiments revealed that elements sufficient for U14 processing are located within the mature snoRNA sequence itself. Subsequent deletions within the U14 coding region demonstrated that only the terminal regions of the folded U14 molecule containing con- served nucleotide boxes C and D are required for processing. Mutagenesis of either box C or box D completely blocked U14 processing. The importance of boxes C and D was confirmed with the excision of appropriately sized U3 and U8 fragments containing boxes C and D from an hsc7O pre-mRNA intron. Competition studies indicate that a trans-acting factor (protein?) is binding this terminal motif and is essential for U14 processing. Competition studies also revealed that this factor is common to both intronic and non-intronic snoRNAs possessing nucleotide boxes C and D. Immunoprecipitation of full-length and internally deleted U14 snoRNA molecules demonstrated that the terminal region containing boxes C and D does not bind fibrillarin. Collectively, our results indicate that a trans-acting factor (different from fibrillarin) binds to the box C- and D-containing terminal motif of U14 snoRNA, thereby stabilizing the intronic snoRNA sequence in an RNP complex during processing.     

Different expression strategy: multiple intronic gene clusters of box H/ACA snoRNA in Drosophila melanogaster

The high degree of rRNA pseudouridylation in Drosophila melanogaster provides a good model for studying the genomic organization, structural and functional diversity of box H/ACA small nucleolar RNAs (snoRNAs). Accounting for both conserved sequence motifs and secondary structures, we have developed a computer-assisted method for box H/ACA snoRNA searching. Ten snoRNA clusters containing 42 box H/ACA snoRNAs were identified from D.melanogaster. Strikingly, they are located in the introns of eight protein-coding genes. In contrast to the mode of one snoRNA per intron so far observed in all animals, our results demonstrate for the first time a novel polycistronic organization that implies a different expression strategy for a box H/ACA snoRNA gene when compared to box C/D snoRNAs in D.melanogaster. Mutiple isoforms of the box H/ACA snoRNAs, from which most clusters are made up, were observed in D.melanogaster. The degree of sequence similarity between the isoforms varies from 99% to 70%, implying duplication events in different periods and a trend of enlarging the intronic snoRNA clusters. The variation in the functional elements of the isoforms could lead to partial alternation of snoRNA's function in loss or gain of rRNA complementary sequences and probably contributes to the great diversity of rRNA pseudouridylation in D.melanogaster.