粟酒裂殖酵母snR41 snoRNA的鉴定及其结构进化的意义

采用计算机分析和分子生物学实验的方法,在粟酒裂殖酵母Schizosaccharomyces pombe中发现和鉴定了一种新的反义snoRNA,命名为snR41,与酿酒酵母Sacharomyces cerevisiae snR41同源分子比较,粟酒裂殖酵母snR41只具有一个指导rRNA核糖甲基化的反义序列,这一发现进一步揭示了反义snoRNA同源分子在一级结构上的多样性,为研究反义SnoRNA结构及进化提供了新的线索。     

酵母snR72～78 snoRNA基因簇的研究

通过PCR分析发现Kluyveromyces delphensis中存在与酿酒酵母相似的snR72～78 snoRNA基因簇.对该基因簇进行序列测定并与酿酒酵母比较分析,发现基因簇中各个snoRNA编码区具有一定的保守性,但是基因间隔区却有完全不同的序列,这表明RNaseⅢ对snoRNA前体加工的识别信号存在于各snoRNA基因间隔区的高级结构中.进一步对另外4种酵母进行PCR分析,结果表明snR72～78 snoRNA基因簇是一种保守的基因组织,普遍存在于各种酵母中.     

拟南芥U59 snoRNA基因簇的结构与功能研究

在拟南芥（Ａｒａｂｉｄｏｐｓｉｓ ｔｈａｌｉａｎａ）中发现和鉴定了具有以义序列的ｓｎｏＲＮＡ组成的一个基因簇，该基因族由两个相同的ｓｎｏＲＮＡ基因组成。位于ｂｏｘＤ′上游的反义序列与人Ｕ５９ｓｎｏＲＮＡ的反义序列相同。因此命名为拟南芥Ｕ％９ａ和Ｕ５９ｂ。位于ｂｏｘＤ上游的反义序列在所有哺乳动物和酵母ｓｎｏＲＮＡ基因的反义序列中未找到同源拷贝，经低浓度的ｄＮＴＰ逆转录检验证实此反义序列并不指导拟南芥     

粗糙脉孢菌U3 snoRNA基因的鉴定及其表达和功能分析

通过对粗糙脉孢菌（Neurospora crassa）小RNA的cDNA文库筛选及基因组分析,鉴定了3个U3snoRNA基因,命名为NcU3A、NcU3B和NcU3C,这3个基因表达的U3snoRNA的大小分别为262,270和275nt．同时,Northern杂交还揭示出不同大小的U3snoRNA剪切变体,表明U3 snoRNA的表达存在复杂的转录后加工．粗糙脉孢菌U3snoRNA都具有物种间保守的与18SrRNA配对的功能区;NcU3A及其剪切变体还具有一段过去没有报道的与26SrRNA互补的反义序列,这段反义序列并不指导对应的rRNA位点的甲基化修饰,可能具有rRNA分子伴侣的作用．粗糙脉孢菌U3 snoRNA都是独立转录的基因,每个基因中均包含一个小内含子序列,其插入位点与酵母U3相似,表明它们古老的起源．研究结果对阐明U3 snoRNA的结构进化及功能多样性具有重要意义．     

粟酒裂殖酵母SnoRNA snR90基因缺失株的筛选鉴定

snR90是在粟酒裂殖酵母中发现的一种单基因编码的box H/ACA类snoRNA.在利用遗传手段在粟酒裂殖酵母基因组中敲除了snR90基因后,通过选择性培养基、PCR、Northern杂交这一系列方法筛选鉴定出snR90基因缺失株.该基因缺失株ΔsnR90的成功构建对snR90功能的分析很有的意义.     

结构语义学理论及其指导意义

结构语义学研究含蓄的语义关系。含蓄的语义关系表现在以下三个方面：上下义关系、反义关系和相对关系。研究上下义关系可以帮助学生措词准确、生动，同时扩大其词汇量；研究反义关系可以用于解释一些生僻用词；研究相对关系可以加深学生对词语的理解并加以灵活运用。     

反义多聚TAR对人免疫缺陷病毒I型TAT蛋白…

以ＨＩＶ－１ＴＡＲ的ＰＣＲ引物为自身模板，用ＰＣＲ法直接扩增得到多拷贝的ＴＡＲＤＮＡ同向串连体；构建了以ＨＩＶ－１ＬＴＲ片段为启动子，含有４．８和１５个拷贝的ＴＡＲＤＮＡ反主表达质粒；以荧光素酶基因为报告基因，检测了瞬时共转染体系中不同拷贝数的反义ＴＡＲＤＮＡ转录产和对Ｔａｔ反式激活作用的抑制作用，结果表明，反义多聚ＴＡＲＲＮＡ对Ｔａｔ自古以来生具有很强的抑制作用，其抑制程度与反义ＴＡＲ中联体的     

基于表达序列标签的萝卜ACA36基因簇鉴定

利用国际公用的表达序列标签（EST）数据库资源,运用生物信息学分析方法,在萝卜（Raphanus sativus）一条表达序列标签中发现一个新的snoRNA基因簇。此基因簇含有一个box H/ACA snoRNA基因和两个box C/D snoRNA基因,分别命名为RsACA36、RssnoR66和RsSNORD88。ACA36可形成典型的box H/ACA snoRNA双茎环二级结构;snoR66和SNORD88都具有典型的C盒与D盒保守元件,末端存在反向互补序列;三个snoRNA都含有能和核糖体RNA互补配对的反向互补序列。RsACA36和RsSNORD88均为在植物中首次发现。基于EST数据库、运用生物信息学方法鉴定snoRNA基因既保留了计算机RNA组学快捷、高效的优点,又使结果因为有了实验支撑而更直接、可信。     

血管内皮生长因子反义核酸增强HL60和K562细胞对As2O3的敏感性

目的：探讨血管内皮生长因子(VEGF)反义核酸能否提高HL60和K562细胞对三氧化二砷(As2O3)的敏感性。方法：采用经筛选所得的最优反义核酸(A7)，20个碱基经过全硫代修饰；以脂质体介导转染细胞，反义核酸和AsO3联合作用72h以后，用MTT法检测细胞生长情况，求IC50值；用ELISA法检测培养液中VEGF蛋白的浓度，用流式细胞仪检测细胞凋亡百分数。结果：VEGF反义核酸可显降低HL60、K562细胞对As2O3的IC50值，下调VEGF蛋白的表达，增加As2O3诱导的HL60、K562细胞凋亡作用。结论：VEGF反义核酸具有增强HL60和K562细胞对As2O3的敏感性，增强As2O3诱导的HL60和K562细胞凋亡作用；提示内源性VEGF蛋白具有使细胞产生耐药性的作用。     

粟酒裂殖酵母Z15 snoRNA的鉴定及结构与功能分析

运用计算机及实验的方法,在粟酒裂酵母（Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ ｐｏｍｂｅ）中发现和鉴定了一个新的,结构特殊的ｓｎｏＲＮＡ－Ｚ１５。该ｓｎｏＲＮＡ由一个独立转录的基因编码,位于酵母２号染色体上,Ｚ１５长达９２个核苷酸,属于反义ｓｎｏＲＮＡ家族,它除了具有典型的ｂｏｘＣ／Ｄ结构元素和末端配对区外,还含有分别与酿酒酵母Ｚ１５和Ｚ２相同的两个反义序列,反映了粟酒裂残酵母Ｚ１５ｓｎｏＲＮＡ基     

Identification and functional analysis of a novel box C／D snoRNA from Schizosaccharomyces pombe

By constructing and screening the Schizosaccharomyces pombe nuclear cDNA library, a novel small nucleolar RNAs (snoRNA) was identified. The novel snoRNA displays structural features typical of C/D box snoRNA family and possesses a 10-nt-long rRNA antisense element which is predicted to guide the 2‘-O-methylation of the fission yeast 25S rRNA at G940. As expected, the rRNA ribose-methyla- tion site predicted by the novel snoRNA was precisely mapped by a deoxynucleoside triphosphate concentration-dependent primer extension assay. The comparison of functional element of guide snoRNAs among eukaryotes reveals that the novel snoRNA is a partial counterpart of the budding yeast snR60 and was termed snR60-11, snR60-Ⅱ gene nested in the intron of a non-coding RNA gene with an unknown function, which is the first example of a yeast snoRNA encoded in an intron of a non-coding RNA gene. Furthermore, a number of yeast snR60 homologues were also identified from other fungi and fly. Our results reveal that snR60 exhibits diverse genomic organization in eukaryotes, implying the high mobility of snR60 gene in the course of evolution.     

Two closely linked rice U14 boxC/D snoRNAs

By analysis of the conserved elements in yeast U14 boxC/D snoRNA. the conserved elements in rice U14 boxC/D snoRNA have been speculated. Through computer search of the international rice genome database, two rice U14 snoRNA gene candidates are obtained. These two putative U14 snoRNA genes are closely linked on rice chromosome 2. The coding sequences of these two snoR-NAs exhibit the hallmark structure of boxC/D antisense snoRNA. They both have conserved boxC and boxD sequences and a 14nt-long complement to the sequence between 414nt and 427nt of rice 18S rRNA (according to GenBank accession no. X00755). The experimental evidence shows that these two snoRNAs are involved in the methylation of the complementary sequence of rice 18S rRNA. The existence and localization of these two snoRNAs are proved by RT-PCR and Northern blot. Further analysis shows that both of the newly found rice snoRNAs have high homology with maize U14 snoRNA. and they are named rice U14.1 snoRNA and U14.2 snoRNA respectively. The gene sequence encoding these two snoRNAs has been deposited in the GenBank database under accession number of AF332622.     

Identification and functional analysis of 23 novel box C／D snoRNAs from Oryza sativa

In a cDNA library generated from rice small nuclear RNAs,30box C/D small nucleolar RNAs (snoRNAs) were identiffied through preliminary screen.Except 7 known snoRNAs such as U14,all snoRNAs were identified in rice for the first time experimentally.Among the 23 novel snoRNAs,11 snoRNAs appear rice-specific,6 snoRNAs are unique to plants,the remaining 6 snoRNAs have their counterparts in both Arabidopsis and yeast or mammals according to the conserved antisense sequencs that guide 2‘-O-ribose methylation of rRNA,17 of the 23 novel snoRNAs were predicted to guide 24 2‘‘-O-ribose methylations at the specificsites of rice 5.8S,18S,25S rRNAs,among which 19 methylated sites were determined by primer extension at low dNTP concentrations.The remaining 6 snoRNAs devoid of rRNA antisense elements may represent novel snoRNA species in rice.The results show that constructing a cDNA library from small nuclear RNAs is an effective experimental approach for novel snoRNA is identification.The novel snoRNAs are important in elucidating the genomic organization and expression of plant snoRNA genes and the mechanism through which 2‘‘-O-ribose methylations took place in rRNAs.     

Identification and characterization of two novel box H/ACA snoRNAs from Schizosaccharomyces pombe

Ineukaryotes,alargenumberofsmallnucleolar RNAs(snoRNAs)accumulatedwithinthenucleolus playimportantrolesinprecursorribosomalRNA(pre RNA)processingandmaturation[1].AllsnoR NAs,withtheexceptionofRNaseMRP,canbe broadlydividedintotwoexpendinggroups,boxC/D andH/ACAsnoRNAs,basedonconservedsequence elements[2].BoxC/DsnoRNAscontaintwocon servedshortsequencemotifs,boxC(UGAUGA)and boxD(CUGA),locatedonlyafewnucleotidesaway fromthe5′and3′ends,respectively,generallyas partofatypical5′3…     

Z25, a novel intronic snoRNA encoded in mammalian nucleolin gene

A novel intronic small nucleolar RNA ( snoRNA) , termed Z25, was identified from mammals by-computer analysis and experimental sequence methods. Z25 is a 69 nucleotides long RNA containing typical boxC/D motifs, terminal stem and an 11-nucleotide sequence complementary to 18S rRNA. In theory, Z25 functions as an RNA guide for the 2'-0-ribose methylation of adenine at position 1678 (human 18S rRNA coordinate) in 18S rRNA. Z25 snoRNA gene was found to be located in the fifth intron of nucleolin gene of human, mouse and rat, demonstrating that the mammalian nucleoline gene is a host gene encoding multiple snoRNAs.     

A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis

Although the U3 small nucleolar RNA (snoRNA), a member of the box C/D class of snoRNAs, was identified with the spliceosomal small nuclear RNAs (snRNAs) over 30 years ago, its function and its associated protein components have remained more elusive. The U3 snoRNA is ubiquitous in eukaryotes and is required for nucleolar processing of pre-18S ribosomal RNA in all organisms where it has been tested. Biochemical and genetic analyses suggest that U3 pre-rRNA base-pairing interactions mediate endonucleolytic pre-rRNA cleavages. Here we have purified a large ribonucleoprotein (RNP) complex from Saccharomyces cerevisiae that contains the U3 snoRNA and 28 proteins. Seventeen new proteins (Utp1 17) and Rrp5 were present, as were ten known components. The Utp proteins are nucleolar and specifically associated with the U3 snoRNA. Depletion of the Utp proteins impedes production of the 18S rRNA, indicating that they are part of the active pre-rRNA processing complex. On the basis of its large size (80S; calculated relative molecular mass of at least 2,200,000) and function, this complex may correspond to the terminal knobs present at the 5' ends of nascent pre-rRNAs. We have termed this large RNP the small subunit (SSU) processome.     

酵母snoRNA基因簇启动子的比较分析

报道对酿酒酵母ｓｎｏＲＮＡ基因簇启动子序列的研究结果．通过计算机分析,发现酿酒酵母中Ｚ２Ｚ８和ＳｎＲ１９０Ｕ１４ｓｎｏＲＮＡ基因簇有相似的启动子结构．这两个ｓｎｏＲＮＡ基因簇都是由一个上游的启动子负责整个基因簇的转录,产生多个顺反子ｓｎｏＲＮＡ的前体,然后再加工成熟为各个独立的ｓｎｏＲＮＡ．在启动子保守序列ＴＡＴＡｂｏｘ的上游还发现２个ＲＡＰ１序列,表明ｓｎｏＲＮＡ基因簇的表达可以通过ＲＡＰ１元素与核糖体蛋白基因的表达协同调控．这是在ｓｎｏＲＮＡ基因的启动子中首次发现ＲＡＰ１调控元素．对ｓｎｏＲＮＡ基因簇的进化特点也进行了讨论．