Ribosomal RNA synthesis and processing in a particulate site in the HeLa cell nucleus

A particulate fraction has been isolated from detergent-prepared HeLa cell nuclei. The fraction consists largely of organelles that resemble the nucleoli of intact cells. The 45S RNA that is precursor to 28S and 18S ribosomal RNA is associated with the fraction. The 32S RNA that is labeled after the 45S RNA and is the apparent precursor to 28S RNA is also associated with the fraction. The nucleoplasm contains 28S RNA that behaves as an intermediate between the 32S nucleolar RNA and the 28S cytoplasmic RNA.     

Structure of RNA in ribosomes

The 50S and 30S ribosomes and 23S and 16S RNA were hydrolyzed with ribonuclease A. The rate constants and number of fragments produced were determined for each reaction. The conformation of 23S RNA changes when the RNA is extracted from the ribosome. Specific regions of the RNA in 50S and 30S ribosomes are protected from hydrolysis by the ribosomal proteins.     

RIBOSOMAL RNA ON THE SURFACE OF RIBOSOMES

Ribonuclease from pancreas releases nucleotide from Escherichiacoli ribosomes while not altering significantly the base composition of the total ribosomal RNA. Ribonuclease hydrolyzes ribsomal RNA without destroying the structure of 70S or 30S and 50S ribosomes. The RNA in 30S and 50S ribosomes appears more sensitive to the action of ribonuclease. The data suggest that RNA may be a surface component of the ribosome.     

Quantitation of RNA tumor viruses and viruslike particles in human milk by hybridization to polyadenylic acid sequences

RNA tumor viruses and viruslike particles from human milk are quantitated by hybridization of the polyadenylic acid regions in their 60S to 70S RNA to radioactive polyribouridylic acid of known specific activity. The length of the polyadenylic acid region in the 60S to 70S RNA of the human milk particle is identical to that of the known oncogenic RNA viruses.     

Detergent-solubilized RNA polymerase from cells infected with foot-and-mouth disease virus

The foot-and-mouth disease virus RNA polymerase complex was dissociated from cellular membranes with deoxycholate in the presence of dextran sulfate. The soluble polymerase complex was active in the cell-free synthesis of virus-specific RNA; solubilization of the complex permitted direct analysis of the cell-free reaction mixtures without recourse to RNA extraction. A major RNA-containing component found early during cell-free incubation ranged from approximately 140 to 300S. The final major products of the cell-free system were 37S virus RNA, 20S ribonuclease-resistant RNA, and a 50S component containing RNA.     

5S RNA synthesized by Escherichia coli in presence of chloramphenicol: different 5'-terminal sequences

Escherichia coli cells, grown in the presence of chloramphenicol, synthesize a low molecular weight RNA (CM-5S RNA) not bound to ribosomes which is similar to ribosomal 5S RNA. Oligonucleotide patterns derived from ribonuclease digests of 5S RNA and of CM-5S RNA are indistinguishable except that the 5'-terminal oligonucleotides differ. Whereas the nucleotide sequence of the 5'-terminus of normal 5S RNA is (p)U(p)G-, there are three alternate sequences of the 5'-terminus of CM-5S RNA: (p)U(p)U(p)G-, (p)U(p)U(p)U(p)G-, and (p)A(p)U(p)U(p)U(p)G-.     

Synthesis of 5S and 4S RNA in metaphase-arrested HeLa cells

The continued synthesis of both 5S and 4S RNA in metaphase-arrested HeLa cells is demonstrated; 5S RNA is apparently synthesized at approximately 74 percent of the interphase rate, while 4S RNA is synthesized at approximately one-third the rate. The ratio of uridine incorporation to RNA methylation is used to correct for the alteration in the specific activity of the pyrimidine pool during metaphase arrest.     

岩白菜幼叶总RNA的提取方法研究

为了筛选出提取岩白菜幼叶总RNA的最佳方法,为岩白菜的转录组测序提供技术支撑,本文采用6种试剂盒法提取岩白菜幼叶的总RNA,通过凝胶电泳和BIO-RAD核酸蛋白检测仪检测提取的RNA样品的质量和纯度,并对提取效果进行了分析.结果表明:用Trizol Kit、Trizol A+ Kit、Transzol Up Kit提取的幼叶总RNA没有28S rRNA和18S rRNA条带;用inunPREP Plant RNA Kit提取的RNA具有28S rRNA和18S rRNA条带,但条带模糊、含量低;用Transzol Plant Kit和EasyPure Plant RNA Kit提取的RNA具有28S rRNA和18S rRNA这2条清晰的条带,但后者提取的总RNA的得率和纯度更高.综合考虑后认为EasyPure Plant RNA Kit法是快速提取岩白菜幼叶总RNA的最佳方法.     

Structure of the S15,S6,S18-rRNA complex: assembly of the 30S ribosome central domain

The crystal structure of a 70-kilodalton ribonucleoprotein complex from the central domain of the Thermus thermophilus 30S ribosomal subunit was solved at 2.6 angstrom resolution. The complex consists of a 104-nucleotide RNA fragment composed of two three-helix junctions that lie at the end of a central helix, and the ribosomal proteins S15, S6, and S18. S15 binds the ribosomal RNA early in the assembly of the 30S ribosomal subunit, stabilizing a conformational reorganization of the two three-helix junctions that creates the RNA fold necessary for subsequent binding of S6 and S18. The structure of the complex demonstrates the central role of S15-induced reorganization of central domain RNA for the subsequent steps of ribosome assembly.     

不同方法提取狗脊蕨叶片总RNA的比较分析

[目的]为构建狗脊蕨叶cDNA文库,保存珍贵基因资源,克隆和研究与有效成分合成相关的基因等提供技术参考。[方法]以狗脊蕨叶片为材料,采用CTAB法、一步法、酚-SDS法和RNA试剂盒提取分离法提取总RNA,通过比较分析确定最优方法。[结果]这4种方法都能提取出182、8 S的RNA,但CTAB法和试剂盒提取分离法的总RNA质量较高,CTAB法还提取出清晰的5S RNA。一步法和SDS法带型模糊,有降解现象。CTAB法提取的狗脊蕨叶片总RNA质量较好,28S1、8S和5S条带清晰,且无明显降解。CTAB提取法OD260/OD280的值在1.93~2.06,试验中其OD260/OD280值为2.012,接近2.0,纯度较高。一步法和SDS法OD260/OD280>2.0,试剂盒法OD260/OD280<2.0。[结论]CTAB法适合提取的狗脊蕨叶片总RNA,质量较好,可用于cDNA合成、文库构建等后续分子生物学试验。     

Secondary structure of ribosomal RNA

Infrared spectra were obtained for 16S and for 23S ribosomal RNA's in D(2)O solutions. The percentage of each base in the paired and unpaired regions of the RNA was determined from the spectra. The secondary structures of 16S and 23S ribosomal RNA's (from Escherichia coli) are significantly different from each other and are also different from those of yeast ribosomal RNA, formylmethionyl-transfer RNA, and the anticodon fragment of this transfer RNA.     

罗汉果果实总RNA提取方法的比较研究

针对罗汉果果实富含多糖和多酚类物质的特点,以广西罗汉果代表种质青皮果种的果实为材料,比较了改良Trizol法、改良异硫氰酸胍法、改良CTAB法和常规Trizol法4种不同的总RNA提取方法的提取效果。结果表明,改良Trizol法所提取的RNA呈现28S rRNA、18S rRNA和5S rRNA 3条清晰的谱带,且28S条带宽度接近18S的2倍,纯度高（D260/D280=201;D260/D230=202）,完整性好（RIN=950）,得率为（26000±1947） μg·g-1。RT\|PCR结果进一步表明,改良Trizol法提取的RNA完全能够用于后续的分子生物学研究。     

半夏叶片总RNA的提取研究

[目的]为了获得高产高质的半夏RNA,为半夏分子生物学方面的研究打下基础。[方法]采用改进的CTAB法提取半夏叶片RNA。用紫外分光光度计测定所提RNA样品的OD260、OD280和OD230的值,用RNA浓度计算公式:RNA(μg/μl)=OD260×40×稀释倍数/1000,求出RNA浓度。[结果]紫外分光光度计的测定结果表明:OD260/OD280介于1.7～2.0之间。用改进的CTAB法提取的半夏叶片RNA质量较好,经变性胶电泳后,有较明显的3条带,分别是28S、18S和5S,28S、18S清晰可见,5S稍有弥散,但在亮度上28S与18S差别不大。[结论]此方法简便易行,成本较低,适合于富含多糖、多酚植物材料的RNA提取。     

鸡脂肪组织总RNA提取方法的优化和RT-PCR检测

提取完整的RNA是基因表达分析中的基础,但由于脂肪组织含有大量的油脂,所以从脂肪组织中提取高质量的RNA有一定的困难。在本研究中,报道了经过优化TRIzol试剂操作程序成功地从肉鸡脂肪中分离出了完整、高质量、纯度好的总RNA。用该方法提取的RNA显示清晰的28S、18S和5S三条带,OD260/OD280均值为1.893,浓度均值为263μg/mL。保温试验与RT-PCR扩增鸡LPL和GAPDH基因的分析结果也证明了所提取的总RNA质量较高。     

辣椒叶片RNA提取方法研究

以经过紫外线诱导处理的辣椒叶片为材料,分别用Trizol试剂快速提取法、尿素提取法和酚 SDS提取法提取其总RNA,比较各RNA产率、纯度及电泳图谱等,结果表明,尿素法提取的RNA28S和18S条带较为清晰,还可得到23S和16S带,较少有降解;而另2种方法所获得的RNA纯度较低,降解严重,琼脂糖电泳图谱通常只出现1条带.     

新型试剂-D．S．G在烟草RNA提取与纯化中的应用

核酸提取是分子生物学实验的基础,提取高质量的核糖核酸(RNA)是构建cDNA文库和进行基因表达研究工作的前提。笔者比较了TRIREAGEN法、D.S.G法、异硫氰酸胍氯化铯密度梯度分离法、D.S.G和TRIREAGENT结合法、TRIREAGENT和D.S.G结合法等5种烟草RNA提取方法,结果表明D.S.G和TRIREAGENT两种试剂的配合使用均可有效地去除烟草植物细胞壁中的多聚糖及其它成份,获取较高质量的RNA。这说明新型试剂-D.S.G与TRIREAGENT的配合使用有利于获得高质量的RNA,但D.S.G单独使用效果并不佳。     

草石蚕块茎总RNA提取及质量分析

采用3种方法提取草石蚕块茎的总RNA,以期筛选出适合草石蚕块茎总RNA提取的最佳方法。结果表明,RNAiso Reagent法提取草石蚕块茎总RNA的28S和18S条带清晰明亮,RNA无降解,无DNA污染,质量较好。王艳红法和TRIzolR Reagent一步提取法提取总RNA的28S和18S条带较暗,提取的总RNA有降解现象。根据紫外分光光度计检测结果分析,3种方法提取草石蚕块茎总RNA的纯度都较好。从提取RNA的产量来看,RNAiso Reagent法提取总RNA的浓度最高,为1112μg/mL,王艳红法次之,TRIzolR Reagent一步提取法最低。因此,RNAiso Reagent法是草石蚕块茎总RNA提取的最佳方法。     

白菜总RNA的高效提取方法及常见问题分析

[目的]建立从白菜叶片组织中快速提取高质量RNA的方法,为进行LD—PCR、AnP、SRAP及其他分子生物学试验奠定基础。[方法]通过增加高速离心时间,用3％H2O2处理试验器皿等措施对Trizol试剂法进行优化,建立了一种简单、快捷、经济、高效的RNA提取方法。[结果]在1．5h左右可得到高质量的总RNA,经琼脂糖凝胶电泳、蛋白核酸分析仪检测,提取的总RNA具有清晰的28S、18S、5S3条带,且28S的宽度是18S的2倍左右,0D260／DD280比值为1．90—2．16。进一步以提取的总RNA为模板成功进行了单链cDNA的合成和LD—PCR。[结论]改良的Trizol试剂法所提取的RNA纯度和完整度极高,可用于LD—PCR、SRAP、基因克隆及表达分析等后续分子生物学试验。     

阿魏侧耳菌丝体RNA提取方法的比较与研究

采用一步法、异硫氰酸胍—巯基乙醇联合变性法和STE法3种方法,分别对阿魏侧耳总RNA的提取效果进行比较。结果表明,一步法难以提取RNA,异硫氰酸胍—巯基乙醇联合变性法提取RNA效果不理想,存在蛋白污染,这2种方法不适于富含多糖的阿魏侧耳总RNA的提取;STE法提取阿魏侧耳总RNA质量高、完整性好、成功率高,经琼脂糖凝胶电泳、紫外分光光度计检测,提取的总RNA具有清晰的28S,18S,5S三条带,且28S的亮度是18S的2倍左右,OD260/OD280比值为18～20,可作为阿魏侧耳总RNA提取的首选方法。用此方法来提取阿魏侧耳液体发酵不同时期的RNA,可以满足进一步分子生物学研究的要求。