Isolation of poly(A)+ RNA by paper affinity chromatography

Poly(A)+ RNA was isolated from in vitro short-term-labeled total cytoplasmic RNA of Ehrlich ascites tumor cells by oligo(dT) cellulose chromatography. This poly(A)+ RNA fraction was compared with a poly(A)+ RNA fraction isolated by a new procedure which involves specific binding of poly(A)+ RNA to messenger affinity paper (mAP) and its release in hot (70 degrees C) water. In typical experiments 10-11 micrograms (2.3%) of poly(A)+ RNA can be retained from 500 micrograms of total cytoplasmic RNA per cm2 of mAP in a quick one-step procedure. The poly(A)+ RNA preparations isolated by the two methods proved to be almost identical with respect to their fraction in total cytoplasmic RNA, specific radioactivities, sucrose gradient profiles, and translation assays. Since the isolation of poly(A)+ RNA by mAP is much less time consuming than that by oligo(dT) column chromatography and since the poly(A)+ RNA can be recovered from mAP in small volumes, which avoids further loss during precipitations, it can be advantageously used for preparative isolation of poly(A)+ RNA.     

Isolation and sequence determination of the 3'-terminal regions of isotopically labelled RNA molecules

The method which was developed for the selective isolation of 3′-terminal polynucleotides from large RNA molecules on columns of cellulose derivatives containing covalently bound dihydroxyboryl groups has been modified and adapted for use on radioactively labelled RNAs. The 3′-terminal polynucleotide fragments which result from specific ribonuclease digestion of isotopically detectable quantities of RNA can be selectively obtained in both high yield and purity by the modified procedure and can be subsequently analyzed by standard electrophoretic and chromatographic techniques. In addition, when the extent of enzymatic fragmentation of the RNA is controlled, the procedure permits the selective isolation of discrete “sets” of fragments of variable chain length, all of which derive from the 3′-terminus of the RNA molecule. These overlapping polynucleotides can be used directly to obtain extensive sequence information regarding the primary structure in the 3′-region of the RNA.     

Ordered aggregation of ribonucleic acids by the human immunodeficiency virus type 1 nucleocapsid protein

The nucleocapsid protein NCp7, which is the major genomic RNA binding protein of human immunodeficiency virus type 1, plays an important role in several key steps of the viral life cycle. Many of the NCp7 activities, notably the nucleic acid annealing and the genomic RNA wrapping ones, are thought to be linked to a nonspecific binding of NCp7 to its nucleic acid targets. The mechanism of these activities is still debated but several clues are in favor of an intermediate aggregation of nucleic acids by NCp7. To check and characterize the nucleic acid aggregating properties of NCp7, we investigated the interaction of NCp7 with the model RNA homopolymer, polyA, by quasielastic light scattering and optical density measurements. The ordered growth of monodisperse large particles independently of the nucleic acid size and the almost complete covering of polyA by NCp7 strongly suggested an ordered aggregation mechanism. The aggregate kinetics of growth in the optimum protein concentration range (≥2 μM) were governed by a so-called Ostwald ripening mechanism limited by transfer of NCp7-covered polyA complexes from small to large aggregates. The aggregation process was strongly dependent on both Na⁺ and Mg²⁺ concentrations, the optimum concentrations being in the physiological range. Similar conclusions held true when polyA was replaced by 16S + 23S ribosomal RNA, suggesting that the NCp7 aggregating properties were only poorly dependent on the nucleic acid sequence and structure. Finally, as in the NCp7 annealing activities, the basic regions of NCp7, but not the zinc fingers, were found critical in nucleic acid aggregation. Taken together, our data indicate that NCp7 is a highly efficient nucleic acid aggregating agent and strengthen the hypothesis that aggregation may constitute a transient step in various NCp7 functions. © 1997 John Wiley & Sons, Inc.     

Isolation and characterization of chromatin from Caulobacter crescentus

The subunit structure of Caulobacter crescentus chromatin has been proven by electron microscope studies. The use of EDTA-Na2 during the purification of the chromatin complex enhanced the removal of contaminating ribosomes and non-chromatin proteins. The preparation obtained by modified procedure contained RNA polymerase as one of the major proteins and three histone-like proteins (10 K, 17 K and a hitherto not described protein with mol. wt 14 K).     

迟钝爱德华氏菌RNA提取及痕量基因组DNA去除方法探究

迟钝爱德华氏菌EIB202是一类细胞壁结构特殊的革兰氏阴性菌,高质量RNA提取相对较难。为了从转录组水平研究这类致病菌的致病机理,需要摸索有效的RNA提取及RNA样品中痕量基因组DNA去除方法。对常规RNA提取步骤进行改进,增加PBS清洗、反复冻融及较高浓度溶菌酶处理等步骤;另外,利用小体系基因组DNA去除系统,Mg2+与Mn2+协同激活DNase I去除RNA样品中基因组DNA污染。利用优化方法提取的RNA在质量及浓度(1 740 ng/μL)方面均有了显著改善,并建立了一套完全去除RNA样品中痕量基因组DNA污染的程序。     

A new blotting medium for the simple isolation and identification of highly resolved messenger RNA.

A simple method has been developed that allows the rapid isolation and identification of highly resolved mRNA molecules. RNA species are separated by gel electrophoresis and then blotted on to a paper sheet to which polyuridylic acid has been covalently bound. This mRNA affinity paper ("mAP") specifically binds, in a reversible manner, polyA+ containing molecules. A replica picture of the agarose gel is thus obtained on the mAP, from which bound mRNA molecules can be eluted by heating in water. In addition to their simple isolation individual mRNA species, whilst still bound to mAP, can be identified by both "in-situ" hybridization and translation.     

An enzyme hydrolyzing the covalent bond between RNA and VPg of picornaviruses. Substrates and methods of analysis of activity

Some new substrates--RNA-VPg, RNA-peptide and a small fragment of RNA peptide--labelled at the peptide component with [125I]Bolton-Hunter reagent have been proposed for use in the isolation and characterization of the enzyme hydrolyzing the phosphodiester bond between the VPg protein and encephalomyocarditis virus RNA. A novel procedure for the analysis of the specific enzyme activity is based on thin-layer chromatography of hydrolytic products on silicagel or polyethylenimine cellulose. The molecular mass of the enzyme isolated by a modified procedure involving FPLC is 90-95 kD.     

Characterization and purification of messenger RNAs containing poly A in insect imaginal disks.

The presence of a fragment of polyA resistant to both T1 and p ribonucleases in mRNAs extracted from wing imaginal disks of an insect, Pieris brassicae, is reported. Its length was approximatively 150 nucleotides. PolyU sepharose affinity chromatography was subsequently used for purification of these polyA(+)mRNA molecules. Analyses on sucrose gradients showed a good recovery of poly(+)molecules characterized by their size (20-100 S) and a polydisperse pattern. These mRNAlike species represent 2-3 per cent of the total radioactivity incorporated into RNA in 3 hours of labeling. Sequential extractions were carried out to provide cytoplasmic RNA rich fractions (4 degrees C) and nuclear rich fractions (45 degrees C). When assayed for the presence of polyA(+)RNA, molecules extracted by these two sequential methods were found to be very similar in their polyA content.     

Structural basis for Pan3 binding to Pan2 and its function in mRNA recruitment and deadenylation

The conserved eukaryotic Pan2–Pan3 deadenylation complex shortens cytoplasmic mRNA 3′ polyA tails to regulate mRNA stability. Although the exonuclease activity resides in Pan2, efficient deadenylation requires Pan3. The mechanistic role of Pan3 is unclear. Here, we show that Pan3 binds RNA directly both through its pseudokinase/C‐terminal domain and via an N‐terminal zinc finger that binds polyA RNA specifically. In contrast, isolated Pan2 is unable to bind RNA. Pan3 binds to the region of Pan2 that links its N‐terminal WD40 domain to the C‐terminal part that contains the exonuclease, with a 2:1 stoichiometry. The crystal structure of the Pan2 linker region bound to a Pan3 homodimer shows how the unusual structural asymmetry of the Pan3 dimer is used to form an extensive high‐affinity interaction. This binding allows Pan3 to supply Pan2 with substrate polyA RNA, facilitating efficient mRNA deadenylation by the intact Pan2–Pan3 complex.     

巴西橡胶树顺式异戊烯基转移酶基因cDNA克隆及其序列特征分析

以巴西橡胶树(Hevea brasiliensis)胶乳的RNA为Tester;叶片RNA为Driver,利用抑制消减杂交法(suppressive subtractive hybridization,SSH)构建了一个胶乳特异表达基因差减文库.通过反式Northern点杂交(reverse Northern dot blots)筛选到一个与顺式异戊烯基转移酶基因(橡胶生物合成的关键酶基因)高度同源的阳性克隆R363.采用RACE方法获得该克隆的全长cDNA(GenBank登陆号:AY461414).序列分析表明,该基因长1156 bp,含有873 bp的阅读框,编码290个氨基酸,分子量约为32.9 kD,等电点为7.2,含有N-端跨膜螺旋区.同源性分析表明R363编码的蛋白质具有异戊烯基转移酶家族的特征,含有cis-异戊烯基链转移酶的5个高度保守区,推测R363可能是一种新的顺式-异戊烯基转移酶基因.Northern blot分析显示,R363在胶乳中高度表达,在叶中不表达.乙烯处理前后表达强度一致,表明该基因表达不为乙烯所诱导.     

Ribonuclease of Bacillus intermedius 7 P. Purification by chromatography on phosphocellulose and several characteristics of the homogeneous enzyme]

A new procedure for isolation of homogenous ribonuclease of Bac. intermedius from a commercial source is described. The yields of 140 mg of RNAse from 200 g of the enzymic powder were attained. The amino acid composition of the enzyme was determined. The RNAse contains neither the sulfhydryl groups nor the disulfide bonds and has only one histidine residue. At the same time the amount of aromatic amino acid residues is relatively high. The enzyme is highly resistant to heat and acid treatment but is less stable in an alkaline solution. The pH optimum of the RNAse for the RNA digestion is 8,5; the temperature optimum for this reaction is 37 degrees. A spectrophotometric method for the RNAse activity assay using polyA as a specific substrate was developed. The purified product provides a suitable starting material for structural studies.     

Effect of polynucleotide adsorption on characteristics of a planar phosphatidylcholine bilayer membrane

Interaction of DNA with planar bilayer phosphatidylcholine membrane in the presence of CaCl2 increases electric conductance of the membrane several times as a result of the formation of DNA-membrane complex. The same effect was observed in the cases of ribosomal RNA and synthetic homopolymers polyA, polyU and polyA X polyU double helix.     

An improved method for the isolation of polysomes from synchronous macroplasmodia of Physarum polycephalum

An improved method for the isolation of polysomes from synchronous macroplasmodia of Physarum polycephalum is described. The procedure involves preincubation and homogenization of the macroplasmodia in high ionic strength media supplemented with high concentrations of Mg²⁺ and ethylene glycol bis (β-aminoethyl ether) N,N′-tetraacetic acid. The polysomes recovered from plasmodial postmitochondrial lysates or heavy particle fractions prepared in this way are sensitive to RNase and EDTA and more than 90% of the total single ribosome population is present as polysomes. The polysomes obtained could also be utilized as a beginning point for the isolation of poly(A)-containing RNA which showed a mass average sedimentation value of 18 S. The development of a satisfactory procedure for the isolation of intact polysomes and poly(A)-containing RNA from macroplasmodia should facilitate studies on mRNA metabolism and translational activity during a naturally synchronous mitotic division cycle.     

The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems

The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications).     

Construction of cDNA coding for human von Willebrand factor using antibody probes for colony-screening and mapping of the chromosomal gene.

Von Willebrand Factor (vWF) mRNA was identified in fractionated polyA+ RNA preparations isolated from cultured human endothelial cells. Micro-injection of specific polyA+ RNA fractions in Xenopus laevis oocytes provoked the synthesis of a vWF-like product which could be detected with an immunoradiometric assay relying on Sepharose-linked monoclonal anti-vWF IgG and different radiolabeled monoclonal anti-vWF IgGs. A vWF-mRNA-containing polyA+ RNA preparation served as substrate for a size-selected cDNA-expression library of 60 000 colonies which was screened for the synthesis of antigens related to vWF, using polyclonal anti-vWF IgG and a second antibody conjugated with peroxidase. Eight positive colonies were detected of which two reacted strongly in the enzyme-linked assay. Immunoblotting of bacterial extracts of "expression clones" with a monoclonal anti-vWF IgG revealed polypeptides which size fits within the length of the cDNA insertions. Northern blotting of human endothelial RNA, employing fragments of vWF cDNA as probes, showed specific hybridization with a mRNA of about 9000 nucleotides. DNA-sequence analysis of a vWF-cDNA insertion revealed an open reading frame followed by a translation stopcodon. It is argued that the cDNA insertions encode the carboxy-terminal part of the vWF protein. vWF-cDNA probes were employed to map the von Willebrand factor gene on chromosome 12 using a panel of 35 human-rodent somatic cell hybrids.