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1.
RNA as an RNA polymerase: net elongation of an RNA primer catalyzed by the Tetrahymena ribozyme 总被引：7，自引：0，他引：7
A catalytic RNA (ribozyme) derived from an intervening sequence (IVS) RNA of Tetrahymena thermophila will catalyze an RNA polymerization reaction in which pentacytidylic acid (C5) is extended by the successive addition of mononucleotides derived from a guanylyl-(3',5')-nucleotide (GpN). Cytidines or uridines are added to C5 to generate chain lengths of 10 to 11 nucleotides, with longer products being generated at greatly reduced efficiency. The reaction is analogous to that catalyzed by a replicase with C5 acting as the primer, GpNs as the nucleoside triphosphates, and a sequence in the ribozyme providing a template. The demonstration that an RNA enzyme can catalyze net elongation of an RNA primer supports theories of prebiotic RNA self-replication. 相似文献
2.
When DNA replication is inhibited during the synthesis (S) phase of the cell cycle, a signaling pathway (checkpoint) is activated that serves to prevent mitosis from initiating before completion of replication. This replication checkpoint acts by down-regulating the activity of the mitotic inducer cdc2-cyclin B. Here, we report the relation between chromatin structure and induction of the replication checkpoint. Chromatin was competent to initiate a checkpoint response only after the DNA was unwound and DNA polymerase alpha had been loaded. Checkpoint induction did not require new DNA synthesis on the unwound template strand but did require RNA primer synthesis by primase. These findings identify the RNA portion of the primer as an important component of the signal that activates the replication checkpoint. 相似文献
3.
甘蓝型油菜FAD2基因cDNA片段的克隆和序列分析 总被引：6，自引：3，他引：6
为了提高油菜的油酸含量，继而培育高油酸油菜品种，从甘蓝型油菜成熟叶片中分离总RNA，经反转录合成cDAN第一条链，以此为模板进行多聚酶链式反应，产物为一长654bp的DNA片段，经克隆和序列测定表明，其核苷酸序列与GenBank中甘蓝型油菜FAD2基因在比较区的同源性达100％。 相似文献
4.
昆虫细胞色素P 450与昆虫抗药性关系密切,本研究以烟实夜蛾5龄幼虫中肠的总RNA为模板,设计并合成简并引物,利用反转录-多聚酶链式反应(RT-PCR)扩增出了烟实夜蛾细胞色素P 450基因的cDNA片段,将其克隆到pMD 18-T载体进行序列测定,测序得到的491 bp的片断编码163个氨基酸残基,且该片断在阅读框内.与已公布的谷实夜蛾、棉铃虫和烟芽夜蛾细胞色素P 450氨基酸序列进行比较,其一致性分别为98.7%,98.7%和92.1%. 相似文献
5.
Multienzyme systems of DNA replication 总被引：48，自引：0，他引：48
Replication is accomplished by multienzyme systems whose operations are usefully considered in respect to three stages of the process: initiation, elongation, anid termination. 1) Initiation entails synthesis of a short RNA fragment that serves as primer for the elongation step of DNA synthesis. This stage, probed by SS phage DNA templates, reveals three distinctive and highly specific systems in E. coli. The Ml3 DNA utilizes RNA polymerase in a manner that may reflect how plasmid elements are replicated in the cell. The ?X174 DNA does not rely on RNA-polymerase, but requires instead five distinctive proteins which may belong to an apparatus for initiating a host chromosome replication cycle at the origin. The G4 DNA, also independent of RNA polymerase, needs simply the dnaG protein for its distinctive initiation and may thus resemble the system that initiates the replication fragments at the nascent growing fork. In each case it is essential that in vitro the DNA-unwinding protein coat the viral DNA and influence its structure. 2) Elongation is achieved in every case by the multisubunit, holoenzyme form of DNA polymerase III. Copolymerase III, which is an enzyme subunit, and adenosine triphosphate are required to form a proper complex with the primer template but appear dispensable for the ensuing chain growth by DNA polymerase (33). 3) Termination requires excision of the RNA priming fragment, filling of gaps and sealing of interruptions to produce a covalently intact phosphodiester backbone. DNA polymerase I has the capacity for excision and gapfilling and DNA ligase is required for sealing. What once appeared to be a simple DNA polymerase-mediated conversion of a single-strand to a duplex circle (34) is now seen as a complex series of events in which diverse multienzyme systems function. Annoyance with the difficulties in resolving and reconstituting these systems is tempered by the conviction that these are the very systems used ,by the cell in replicating its chromosome and extrachromosomal elements. Thus, understanding of the regulation of replication events in the cell, their localization at membrane surfaces and integration with cell division, and their coordination with phage DNA maturation and particle assembly will all be advanced by knowledge of the components of the replicative machinery. 相似文献
6.
湖北白猪IGF-1基因的克隆及序列分析 总被引：1，自引：0，他引：1
[目的]以湖北白猪肝脏作为试验材料,克隆出湖北白猪胰岛素生长因子（IGF-1）基因,并对其序列进行分析。[方法]采用Trizol法从湖北白猪的肝脏中提取总RNA,将其作为RT反应的模板,用P2（5＇-CAGGTAACTCGTGCAGAGCAAAGGA-3＇）引物合成IGF-1基因cDNA第一链,以其产物为模板,再以P1（5＇-CCCATCTCCCTGGATTTCTTTTTG-3＇）和P2为上下游引物扩增到大小约为607bp的产物,并将其克隆至pCRII载体上,经蓝白斑筛选、酶切、测序。[结果]经筛选、酶切、序列分析,表明该片段为IGF-1基因的cDNA克隆,它由607个核苷酸组成,其中1～145位是5＇端非翻译区,从第146位ATG起始密码子开始至第538位TAG终止密码子,539～607位是3＇端非翻译区,包含一完整的ORF,该ORF共有393核苷酸,编码一个推断由130个氨基酸组成的多肽。与GenBank中的序列比较,该序列与Muller等报道的猪IGF-1基因编码序列完全同源。[结论]成功克隆了湖北白猪IGF-1基因,并对其进行序列分析,证实了IGF-1基因的高度保守性,为采用转基因技术对湖北白猪进行育种奠定了基础和提供了技术依据。 相似文献
7.
[目的]以湖北白猪肝脏作为试验材料,克隆出湖北白猪胰岛素生长因子（IGF-I）基因,并对其序列进行分析。[方法]采用Trizol法从湖北白猪的肝脏中提取总RNA,将其作为RT反应的模板,用P2（5＇-CAGGTAACTCGTGCAGAGCAAAGGA-3＇）引物合成IGF-I基因cDNA第一链,以其产物为模板,再以P1（5＇-CCCATCTCCCTGGATTTCTTTTTG-3＇）和P2为上下游引物扩增到大小约为607 bp的产物,并将其克隆至pCRII载体上,经蓝白斑筛选、酶切、测序。[结果]经筛选、酶切、序列分析,表明该片段为IGF-I基因的cDNA克隆,它由607个核苷酸组成,其中1～145位是5＇端非翻译区,从第146位ATG起始密码子开始至第538位TAG终止密码子5,39～607位是3＇端非翻译区,包含一完整的ORF,该ORF共有393核苷酸,编码一个推断由130个氨基酸组成的多肽。与GenBank中的序列比较,该序列与Muller等报道的猪IGF-I基因编码序列完全同源。[结论]成功克隆了湖北白猪IGF-I基因,并对其进行序列分析,证实了IGF-I基因的高度保守性,为采用转基因技术对湖北白猪进行育种奠定了基础和提供了技术依据。 相似文献
8.
Bard J Zhelkovsky AM Helmling S Earnest TN Moore CL Bohm A 《Science (New York, N.Y.)》2000,289(5483):1346-1349
Polyadenylate [poly(A)] polymerase (PAP) catalyzes the addition of a polyadenosine tail to almost all eukaryotic messenger RNAs (mRNAs). The crystal structure of the PAP from Saccharomyces cerevisiae (Pap1) has been solved to 2.6 angstroms, both alone and in complex with 3'-deoxyadenosine triphosphate (3'-dATP). Like other nucleic acid polymerases, Pap1 is composed of three domains that encircle the active site. The arrangement of these domains, however, is quite different from that seen in polymerases that use a template to select and position their incoming nucleotides. The first two domains are functionally analogous to polymerase palm and fingers domains. The third domain is attached to the fingers domain and is known to interact with the single-stranded RNA primer. In the nucleotide complex, two molecules of 3'-dATP are bound to Pap1. One occupies the position of the incoming base, prior to its addition to the mRNA chain. The other is believed to occupy the position of the 3' end of the mRNA primer. 相似文献
9.
【目的】研究稻蝗属特异性DNA分子标记,为稻蝗属物种的分类鉴定提供快速有效的分子检测方法。【方法】基于稻蝗属物种及其近缘属种的大量RAPD-PCR结果,筛选出稻蝗属物种特异性的RAPD条带,对该特异RAPD条带进行克隆、测序。基于所测序列设计特异引物,以稻蝗属不同物种和蝗总科其它物种基因组DNA为模板进行PCR扩增。【结果】随机引物S823可在稻蝗属不同物种扩增出约650bp的RAPD条带,经对该条带的克隆、测序,发现在3个受试的稻蝗属物种中序列同源度达92.3%—96.6%,序列G+C含量大于15%,并富含大量的A、T重复区;基于已知序列设计的特异引物对稻蝗属7个物种可以扩增出目的条带(550bp),而对赤胫伪稻蝗及蝗总科其它物种均无扩增条带。【结论】鉴定了稻蝗属特异的RAPD条带,基于该条带序列设计的特异引物可用于稻蝗属物种的快速分子鉴定。 相似文献
10.
Rgh BNG基因是一种地黄(Rehmannia glutinosa)响应非生物胁迫和植物生长调节剂的基因。类Rgh BNG基因是一个与Rgh BNG基因核酸序列同源的地黄基因。克隆了地黄类Rgh BNG基因,预测其编码蛋白质的性质、结构和功能。用RNA提取试剂盒提取地黄总RNA,反转录成c DNA,根据已知地黄的Rgh BNG基因碱基序列设计上、下游引物,以c DNA为模板,利用PCR扩增产生包括Rgh BNG基因和类Rgh BNG基因在内的5个c DNA片段,其中类Rgh BNG基因全长1 974 bp,包含一个486 bp的ORF,编码161个氨基酸残基组成的蛋白质;蛋白质等电点为9.45,分子量为18.6 k Da,二级结构中延伸链占34.78%,无规则卷曲占33.54%,α-螺旋占21.74%,β-螺旋占9.94%,有两个可能的跨膜区,参与翻译和脂肪酸代谢。 相似文献
11.
根据GenBank已公布的禽流感病毒H9亚型血凝素蛋白编码基因,设计了一对引物(H1和H2),RT-PCR扩增禽流感病毒A/Duck/Shanghai/02/99(H9)的HA基因,扩增片段与载体pUCm-T的连接产物转化大肠杆菌感受态细胞,重组质粒pUCm-T-HA经限制性内切酶酶切和PCR鉴定后进行测序。BlastN分析结果显示该分离株血凝素编码基因与已发表的鸭源禽流感病毒A/Duck/Shantou/1605/01(H9N2)和A/Duck/Hong Kong/Y280/97(H9N2)血凝素编码基因的核甘酸序列同源性为98%,从而从分子水平确定了该分离株为H9亚型禽流感病毒。 相似文献
12.
A critical event in the origin of life is thought to have been the emergence of an RNA molecule capable of replicating a primordial RNA "genome." Here we describe the evolution and engineering of an RNA polymerase ribozyme capable of synthesizing RNAs of up to 95 nucleotides in length. To overcome its sequence dependence, we recombined traits evolved separately in different ribozyme lineages. This yielded a more general polymerase ribozyme that was able to synthesize a wider spectrum of RNA sequences, as we demonstrate by the accurate synthesis of an enzymatically active RNA, a hammerhead endonuclease ribozyme. This recapitulates a central aspect of an RNA-based genetic system: the RNA-catalyzed synthesis of an active ribozyme from an RNA template. 相似文献
13.
根据GenBank上发表的牛IL-2(M13204)基因序列,应用Primer Premier5引物设计软件自行设计一对引物,以ConA刺激培养24 h的奶牛外周血淋巴细胞为材料,从总RNA中扩增出奶牛IL-2基因,然后测序.结果表明克隆的奶牛IL-2基因大小477 bp,编码158个氨基酸,与GenBank上发表的牛IL-2基因序列比较,其核苷酸的同源性99.4%,与马、猪、猫、狗、山羊、绵羊的核苷酸的同源性分别为31.8%,32.0%,31.6%,30.3%,30.8%,31.0%. 相似文献
14.
Ribonuclease mitochondrial RNA processing, a site-specific endoribonuclease involved in primer RNA metabolism in mammalian mitochondria, requires an RNA component for its activity. On the basis of copurification and selective inactivation with complementary oligonucleotides, a 135-nucleotide RNA species, not encoded in the mitochondrial genome, is identified as the RNA moiety of the endoribonuclease. This finding implies transport of a nucleus-encoded RNA, essential for organelle DNA replication, to the mitochondrial matrix. 相似文献
15.
以递交到GenBank上的TMEM130蛋白基因序列(nm-152913)为模板设计引物,以人大脑cDNA文库为模板进行巢氏PCR扩增,并将PCR产物克隆到pMD18-T载体上,成功获得TMEM130蛋白基因编码序列,测序结果显示,获得的序列为TMEM130蛋白基因转录变异体2,其编码序列长1272bp。该基因定位在人类第7条染色体7q22.1区域,有3个转录变异体。生物信息学分析显示该序列可编码1个含423个氨基酸的蛋白质;Smartmode程序显示该蛋白质是1个跨膜蛋白,在100～250号氨基酸序列之间有1个与多囊肾病(PKD)相关的结构域。因此,鉴定TMEM130蛋白是一个与人类多囊肾病相关的蛋白。 相似文献
16.
Nair DT Johnson RE Prakash L Prakash S Aggarwal AK 《Science (New York, N.Y.)》2005,309(5744):2219-2222
The Rev1 DNA polymerase is highly specialized for the incorporation of C opposite template G. We present here the crystal structure of yeast Rev1 bound to template G and incoming 2'-deoxycytidine 5'-triphosphate (dCTP), which reveals that the polymerase itself dictates the identity of the incoming nucleotide, as well as the identity of the templating base. Template G and incoming dCTP do not pair with each other. Instead, the template G is evicted from the DNA helix, and it makes optimal hydrogen bonds with a segment of Rev1. Also, unlike other DNA polymerases, incoming dCTP pairs with an arginine rather than the templating base, which ensures the incorporation of dCTP over other incoming nucleotides. This mechanism provides an elegant means for promoting proficient and error-free synthesis through N2-adducted guanines that obstruct replication. 相似文献
17.
[目的]探讨SARS-COV结构蛋白的PCR扩增条件。[方法]采用正交试验对SARS全基因组进行PCR反应筛选目标片段,确定退火温度、模板浓度、聚合酶量、引物浓度P、CR延长时间对于PCR反应的影响。[结果]结果表明,在退火温度55~60°C、模板浓度5 mg/ml加入1μl、DNA聚合酶加入0.25μl、引物浓度25 pmol/LP、CR反应时间为60 s时,DNA回收量最高,为4.37μg。[结论]该研究为SARS冠状病毒核酸的转录和复制提供科学依据。 相似文献
18.
19.
调查表明云南省昆明市团结乡香石竹普遍发生病毒病。电镜负染检测采自团结乡香石竹病毒病病样,病叶汁液中观察到弯曲长线型的病毒粒体,长度约1000~1600 nm,直径约12 nm。对这些病样的叶片进行间接ELISA检测,所有样品与香石竹坏死斑点病毒抗血清都呈阳性反应。按照报道的长线形病毒属简并引物合成引物,采用RT-PCR法对血清反应呈阳性的香石竹总RNA扩增了1059ntsHSP70基因部分编码序列,将其克隆到pMD18-T载体上,并进行序列分析。根据测序结果设计引物建立RT-PCR扩增检测方法并测定其灵敏度。结果表明,使用简并引物和新设计的引物均能从CNFV感病香石竹组织中扩增出与预期大小一致的目标片段,而健康组织无此扩增产物。灵敏性测定结果表明该特异性引物RT-PCR可从稀释106植物总RNA中检测出病毒。 相似文献
20.
牛病毒性腹泻病毒为黄病毒科瘟病毒属代表种,其基因组为单股正链RNA,无Poly(A)尾。传统的3'-末端快速扩增方法不适用于牛病毒性腹泻病毒的3'-末端克隆测序。本研究根据牛病毒性腹泻病毒基因组3'-末端共有特征碱基,设计回文锚定引物CGend。以BVDVJZ05-1毒株为研究对象,进行了反转录及3'-末端克隆测序。结果表明,CGend的反转录效果优于随机引物和一般特异性下游引物,并且扩增得到了JZ05-1的3'-末端完整序列。本研究为瘟病毒属病毒反转录和3'-末端克隆测序提供了新思路。 相似文献