小麦丛矮病毒（WRSV）的近全长cDNA基因文库的构建

用大肠杆菌Poly（A）聚合酶,在纯化的小麦丛矮病毒的单链基因组RNA3＇末端加多聚腺苷酸,以此RNA作模板,12－18寡聚脱氧胸核着酸作引物,合成cNDA后,分别用限制性内切酶PstI和EcoRI对该cDNA酶切,同时再分别用PstI单酶和SmaI与EcoRI双酶对pUC载体酶切,经连接、转化感受态细胞、筛选和酶切鉴定后,共得到10种不同大小的非定向克隆,其大小之和约14kb,同时还得到定向克隆47个。已知N蛋白基因3＇端含有SacI酶切位点,故在用限制性内切酶SacI对10种插入片段进行分析后发现,仅有约6kb的插入片段含有SacI位点,再用合成的与WRSVNN蛋白mRNA3＇端顺序相同的引物对该克隆进行顺序分析证明,它含有WRSVN蛋白、G蛋白、M蛋白和部分L蛋白基因。对47个定向克隆进行顺序测定后,用DNA顺序分析软件将它与其它几种弹状病毒基因组的3＇前导（leader）和5＇拖尾（trailer）RNA进行比较后发现,有两个定向克隆分别含有它们的3＇前导或5＇拖尾的高保守区顺序,构建了一个近全长的小麦丛矮病毒（WRSV）cDNA文库。     

Inhibition of protein synthesis by double-stranded RNA in reticulocyte lysates: evidence for activation of an endoribonuclease.

Double-stranded RNA is a potent inhibitor of protein synthesis in rabbit reticulocyte lysates. Three lines of evidence suggest that at least part of this inhibitory activity is due to activation of a nuclease which degrades mRNA: (1) In the presence of emetine reticulocyte polysomes are partially degraded to structures containing 1–3 ribosomes; (2) 34S Mengo-virus RNA is degraded to fragments sedimenting at less than 18S; (3) The template activity of globin mRNA extracted from the lysates is reduced by 90% when compared to appropriate controls. The ability of double-stranded RNA to activate a nuclease in the reticulocyte system is very similar to that observed in extracts from interferon treated cells and probably involves formation of the unusual oligonucleotide pppA^2′ p^5′ A^2′ p^5′ A.     

The use of spun columns in the purification of double-stranded cDNA appropriate for tailing

A reliable estimation of the efficiency of adding homopolymeric tails to double-stranded cDNA molecules by terminal deoxynucleotidyl transferase is extremely important in the construction of cDNA libraries. Appreciable differences in transformation efficiency result when the homopolymer tails to be annealed are of inadequate length. We report here that the use of a Sephadex G-50 spun column to remove oligo(dT)12-18 frequently coprecipitating in ethanol with the cDNA results in a more dependable estimation of tail lengths.     

Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus.

DNA polymerase alpha, delta and epsilon can be isolated simultaneously from calf thymus. DNA polymerase delta was purified to apparent homogeneity by a four-column procedure including DEAE-Sephacel, phenyl-Sepharose, phosphocellulose, and hydroxylapatite, yielding two polypeptides of 125 and 48 kDa, respectively. On hydroxylapatite DNA polymerase delta can completely be separated from DNA polymerase epsilon. By KCl DNA polymerase delta is eluted first, while addition of potassium phosphate elutes DNA polymerase epsilon. DNA polymerases delta and epsilon could be distinguished from DNA polymerase alpha by their (i) resistance to the monoclonal antibody SJK 132-20, (ii) relative resistance to N2-[p-(n-butyl)phenyl]-2-deoxyguanosine triphosphate and 2-[p-(n-butyl)anilino]-2-deoxyadenosine triphosphate, (iii) presence of a 3'----5' exonuclease, (iv) polypeptide composition, (v) template requirements, (vi) processivities on the homopolymer poly(dA)/oligo(dT12-18), and (vii) lack of primase. The following differences of DNA polymerase delta to DNA polymerase epsilon were evident: (i) the independence of DNA polymerase epsilon to proliferating cell nuclear antigen for processivity, (ii) utilization of deoxy- and ribonucleotide primers, (iii) template requirements in the absence of proliferating cell nuclear antigen, (iv) mode of elution from hydroxylapatite, and (v) sensitivity to d2TTP and to dimethyl sulfoxide. Both enzymes contain a 3'----5' exonuclease, but are devoid of endonuclease, RNase H, DNA helicase, DNA dependent ATPase, DNA primase, and poly(ADP-ribose) polymerase. DNA polymerase delta is 100-150 fold dependent on proliferating cell nuclear antigen for activity and processivity on poly(dA)/oligo(dT12-18) at base ratios between 1:1 to 100:1. The activity of DNA polymerase delta requires an acidic pH of 6.5 and is also found on poly(dT)/oligo(dA12-18) and on poly(dT)/oligo(A12-18) but not on 10 other templates tested. All three DNA polymerases can be classified according to the revised nomenclature for eukaryotic DNA polymerases (Burgers, P.M. J., Bambara, R. A., Campbell, J. L., Chang, L. M. S., Downey, K. M., Hübscher, U., Lee, M. Y. W. T., Linn, S. M., So, A. G., and Spadari, S. (1990) Eur. J. Biochem. 191, 617-618).     

DNA polymerase and thymidine kinase activities in MC-29 virus-induced transplantable hepatoma and the effect of cytostatic treatment on these activities

The activity of DNA polymerases and thymidine kinase was compared in the MC-29 leukosis virus-induced transplantable hepatoma and in the livers of rats treated with cyclophosphamide (CP), cytosine-arabinoside (ara-C) and 5-fluoro-uracil (5-FU). The specific activity of DNA polymerase was twenty times greater in the MC-29 leukosis virus-induced hepatoma, while thymidine kinase was only 3–5 times greater than in the liver.All three enzymes showed Michaelis-Menten kinetics in their substrate and template saturation curves. The template utilization of DNA polymerases from hepatoma and from liver was compared. Both had higher activities on a poly(dA) · poly(dT) template at pH 8.0, than on DNA at pH 7.5. After chromatography on a phosphocellulose column two polymerases were separated. The first peak eluted by 0.15 M KCl preferred DNA as template (polymerase α). The second eluted by 0.5 M KCl worked better on poly(dA) · poly(dT) (polymerase β). Thymidine kinase was eluted by 0.25 M KCl. Inhibition by N-ethylmaleimide (NEM) showed the polymerase α to be sensitive and the polymerase β to be resistant to the sulfhydryl blocking agent; similar to the respective enzymes of other eukaryotic cells. The specific acitivity of DNA polymerase decreased after CP treatment at 6 h and 72 h and after ara-C treatment at 72 h. The specific activities of thymidine kinase were not changed significantly in response to the drug administrations.     

Ribosomal RNA genes of Trypanosoma brucei: mapping the regions specifying the six small ribosomal RNAs

There are six small ribosomal RNAs in trypanosome ribosomes. sRNA3 and sRNA5 of Trypanosoma brucei brucei have been partially sequenced. Sequence homologies indicate that sRNA3 is 5.8S RNA and sRNA5 is 5S RNA of T. b. brucei. The regions specifying these two, and the remaining four small RNAs, have been identified within clones of rRNA genes and in the genome. Five of the small RNAs, 1, 2, 3, 4 and 6, hybridise exclusively within the major rRNA gene repeat. A map of the regions specifying these small RNAs is presented. sRNA3 (5.8S RNA) hybridises to a region corresponding to the transcribed spacer of other eukaryotes. sRNA1 hybridises to a region between sequences specifying the two large subunit RNA molecules of 2.3 kb and 1.8 kb. Sequences specifying sRNAs 2 and 4 are present near the sequence specifying sRNA1, while sRNA6 appears to be specified 3' to the sequence specifying the 1.8-kb RNA sequence. In addition regions of secondary hybridisation for small RNAs 2, 3, 4 and 6 have also been identified. Though sRNA5 (5S RNA) hybridises within the major rRNA repeat, a separate 5S RNA gene repeat with unit size of 760 bp is also present. It is 10 to 20 times more abundant than the major rRNA gene repeat.     

Human cytomegalovirus. III. Virus-induced DNA polymerase.

Infection of WI-38 human fibroblasts with human cytomegalovirus (CMV) led to the stimulation of host cell DNA polymerase synthesis and induction of a novel virus-specific DNA polymerase. This cytomegalovirus-induced DNA polymerase was purified and separated from host cell enzymes by DEAE-cellulose and phosphocellulose column chromatographies. It can be distinguished from host cell enzymes by chromatographic behavior, template primer specificity, sedimentation property, and the requirement of salt for maximal activity. This virus-induced enzyme has a sedimentation coefficient of 9.2S and is found in both the nuclei and cytoplasm of virus-infected cells, but not in uninfected cells. This enzyme could efficiently use activated calf-thymus DNA, oly(dA)-oligo(dT)12-18, and poly(dC)-oligo(dG)12-18 as template primers, especially poly(dA)-oligo(dT)12-18, but it could not use poly(rA)-oligo(dT)12-18, poly(rC)-oligo(dG)12-18, or oligo(dT)12-18. The enzyme requires Mg2+ for maximal activity, is sensitive to p-hydroxymercuribenzoate, and is not a zinc metalloenzyme. In addition, the cytomegalovirus-induced DNA polymerase activity can be enhanced by adding 0.06 to 0.12 M NaCl or 0.03 to 0.06 M (NH4)2SO4 to the reaction mixture.     

Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H.

A model RNA template-primer system is described for the study of RNA-directed double-stranded DNA synthesis by purified avian myeloblastosis virus DNA polymerase and its associated RNase H. In the presence of complementary RNA primer, oligo(rI), and the deoxyribonucleoside triphosphates dGTP, dTTP, and dATP, 3'-(rC)30-40-poly(rA) directs the sequential synthesis of poly(dT) and poly(dA) from a specific site at the 3' end of the RNA template. With this model RNA template-primer, optimal conditions for double-stranded DNA synthesis are described. Analysis of the kinetics of DNA synthesis shows that initially there is rapid synthesis of poly(dT). After a brief time lag, poly(dA) synthesis and the DNA polymerase-associated RNase H activity are initiated. While poly(rA) is directing the synthesis of poly(dT), the requirements for DNA synthesis indicate that the newly synthesized poly(dT) is acting as template for poly(dA) synthesis. Furthermore, selective inhibitor studies using NaF show that activation of RNase H is not just a time-related event, but is required for synthesis of the anti-complementary strand of DNA. To determine the specific role of RNase H in this synthetic sequence, the primer for poly(dA) synthesis was investigated. By use of formamide--poly-acrylamide slab gel electrophoresis, it is shown that poly(dT) is not acting as both template and primer for poly(dA) synthesis since no poly(dT)-poly(dA) covalent linkages are observed in radioactive poly(dA) product. Identification of 2',3'-[32P]AMP on paper chromatograms of alkali-treated poly(dA) product synthesized with [alpha-32P]dATP as substrate demonstrates the presence of rAMP-dAMP phosphodiester linkages in the poly(dA) product. Therefore, a new functional role of RNase H is demonstrated in the RNA-directed synthesis of double-stranded DNA. Not only is RNase H responsible for the degradation of poly(rA) following formation of a poly(rA)-poly(dT) hybrid but also the poly(rA)fragments generated are serving as primers for initiation of synthesis of the second strand of the double-stranded DNA.     

Hybridization of Oligo(dT) to RNA on nitrocellulose

Quantitation of mRNA immobilized on nitrocellulose filters is an essential aspect of some studies in molecular biology. Hybridization of oligo(dT)₁₈ to the poly(A) tails of mRNA can be used to measure filter-bound mRNA and thus provides a basis for comparing abundance of specific mRNAs. Hybridization rate of ³²P-labeled oligo(dT)₁₈ in 0.75 M NaCl, 75 mM sodium citrate, pH 7 (5 × SSC) to immobilized RNA was maximal at 25°C. Filters were fully hybridized under these conditions within 1 hr when the oligo(dT)₁₈ concentration was 10 pmol/ml or higher. Salt dependence of the dissociation temperature (T_d) of oligo(dT)₁₈:RNA duplex on filters was described by the equation T_d = 42 − 20log₁₀[molar Na⁺] (°C). With stringent washing of the duplex (four 5-min washes in 2 × SSC at room temperature), oligo(dT)₁₈ gave no signal with plasmid DNA, rRNA, or tRNA. We have found that olig(dT)₁₈ can be used to normalize signal strengths rapidly and conveniently from total or oligo(dT)-selected eukaryotic RNA.     

Altered fatty acid composition in regulatory mutants of Streptomyces cinnamonensis

Abstract cDNA-RNA liquid hybridization analysis was used to compare the RNA sequence homology between two members of the Nudaurelia β virus family, Trichoplusia ni virus ( T.ni V) and Dasychira pudibunda virus ( D.p V). Heterologous hybridization experiments demonstrated that these viruses shared little sequence homology. Using oligo(dT) chromatography and oligo(dT)_12–18 as a primer for cDNA synthesis it was shown that neither T.ni V nor D.p V RNA genomes possess a poly(A) tract at the 3' end.