植物抗病毒病育种策略

为了得到抗病毒的寄主植物,植物育种学家进行了许多有益研究,形成了许多行之有效的抗病毒病育种策略。利用植物本身对病毒侵染所具有的一些免疫功能及其本身的一些抗性基因来获得抗性;利用来源于病毒自身基因的一些抗病性策略(PDR),如利用病毒外壳蛋白基因,病毒复制酶基因,病毒移动蛋白基因,病毒卫星RNA和反义RNA等,植物也可以获得抗性。近年来对由转录后RNA沉默引起的由RNA介导的病毒抗性策略(RMVR)也进行了深入地研究。除了PDR和RMVR以外,还有一些导致植物抗病毒的策略,包括利用美国商陆的病毒抗性蛋白(PAP),2',5’-寡腺苷酸合成酶,“植物抗体”以及病毒蛋白多肽来获得病毒抗性等。     

The effect of storage at different temperatures on the stability of Hepatitis C virus RNA in plasma samples.

One important issue related to Hepatitis C virus (HCV) RNA nucleic acid amplification testing (NAT) is the storage conditions of plasma samples in order to obtain reliable results. Many authors have reported that the storage conditions could affect the RNA stability and, hence, HCV RNA detection. We have studied HCV RNA stability in plasma samples after storage at different temperatures (-70, -20, 5 and 25 degrees C). Samples containing different HCV titres were stored and analysed by qualitative or quantitative NAT techniques at defined time points. At -20 degrees C, samples containing high HCV RNA titres were followed-up during approximately 2.6-2.7 years, samples with intermediate concentrations during approximately 1 year and samples with 100 International Units/millilitre (IU/ml) during 2.5 years. Independently of the HCV RNA concentration, the results show absence of decay in HCV RNA detectability. Samples stored at 25 degrees C maintain their HCV RNA titre during 14 days and samples at 5 degrees C were stable for at least 3 months.     

Mapping unintegrated avian sarcoma virus DNA: termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA.

Three major species of viral DNA have been observed in cells infected by retroviruses: a linear, double-stranded copy of a subunit of viral RNA; closed circular DNA; and proviral DNA inserted covalently into the genome of the host cell. We have studied the structures of the unintegrated forms of avian sarcoma virus (ASA) DNA using agarose gel electrophoresis in conjunction with restriction endonucleases and molecular hybridization techniques. The linear duplex DNA is approximately the same length as a subunit of viral RNA (approximately 10 kb) and it bears natural repeats of approximately 300 nucleotides at its termini. The repeats are composed of sequences derived from both the 3' and 5' termini of viral RNA in a manner suggesting that the viral DNA polymerase is transferred twice between templates. Thus the first end begins with a sequence from the 5' terminus of viral RNA and is permuted by about 100 nucleotides with respect to the 3' terminus of viral RNA; the linear DNA terminates with a sequence of about 200 nucleotides derived from the 3' end of viral RNA. We represent this structure, synthesized from right to left, as 3'5'-----3'5'. Two closed circular species of approximately monomeric size have been identified. The less abundant species contain all the sequences identified in linear DNA, including two copies in tandem of the 300 nucleotide 3'5' repeat. The major species lacks about 300 base pairs (bp) mapped to the region of the repeated sequence; thus it presumably contains only a single copy of that sequence. The strategies used to determine these structures involved the assignment of over 20 cleavage sites for restriction endonucleases on the physical maps of ASV DNA. Several strains of ASV were compared with respect to these sites, and the sites have been located in relation to deletions frequently observed in the env and src genes of ASV.     

Secondary structure of the r(CUUCGG) tetraloop.

The oligonucleotide r(GGACUUCGGUCC) has been observed to adopt a hairpin conformation by solution NMR and a double helical conformation by X-ray diffraction. In order to understand this apparent conflict, we used time-resolved fluorescence depolarization and 19fluorine NMR to follow the secondary structure of this dodecamer as the solution composition was changed stepwise from the NMR experimental conditions to those used for crystallization. Calculation of the dodecamer concentration in the crystal (180 mM strands) and the cation concentration needed for neutrality (>2 M) prompted investigation of a tethered species, in which two dodecamers are connected by a string of 4 nt, geometrically equivalent to approximately 100 mM strands, in 2.5 M NaCl. The RNA tetraloop and its DNA analog maintain a single-strand hairpin conformation in solution, even under the conditions used to grow the crystal. Under high salt conditions, the tethered RNA and DNA analogs of this sequence yield secondary components which could be the double helical conformation. Crystal contacts in addition to solvent changes and high RNA concentrations are needed to obtain the double helix as the predominant species.     

Synthesis of ribonucleotides and their participation in ribonucleic acid synthesis by Coxiella burnetii.

Synthesis of ribonucleic acid (RNA) by the deoxyribonucleic acid-dependent RNA polymerase of Coxiella burnetii required adenosine, uridine, guanosine, and cytidine 5'-triphosphates. Cell-free preparations of this obligate intracellular procaryotic parasite had competence to phosphorylate ribonucleoside mono- and diphosphates in the presence of exogenous adenosine and guanosine 5'-triphosphates to the corresponding di- and triphosphates. C. burnetii contained about 2 nmol of adenosine 5'-triphosphate per mg of protein, which could serve as a approximately P donor for in vivo synthesis of nucleoside triphosphates. The latter were then used as substrates in the synthesis of RNA in a coordinated metabolic system with C. burnetii RNA polymerase. It is suggested that during infection the rickettsiae might obtain the nucleotides necessary for RNA synthesis from the vacuoles in which C. burnetii proliferates.     

NMR evidence for the existence of two native conformations of 5S RNA

NMR spectra of the non-exchangeable protons in 5S RNA from E. coli show the existence of two distinct conformers of the molecule which meet the operational definition of "A form" or native 5S RNA. Both are easily distinguished spectroscopically from denatured, "B form" 5S RNA. The conditions which interconvert the two A form conformers strongly suggest that the transition between them gives rise to the low temperature optical melting transition first reported in 5S RNA by Kao and Crothers (1).     

Force unfolding kinetics of RNA using optical tweezers. II. Modeling experiments

By exerting mechanical force, it is possible to unfold/refold RNA molecules one at a time. In a small range of forces, an RNA molecule can hop between the folded and the unfolded state with force-dependent kinetic rates. Here, we introduce a mesoscopic model to analyze the hopping kinetics of RNA hairpins in an optical tweezers setup. The model includes different elements of the experimental setup (beads, handles, and RNA sequence) and limitations of the instrument (time lag of the force-feedback mechanism and finite bandwidth of data acquisition). We investigated the influence of the instrument on the measured hopping rates. Results from the model are in good agreement with the experiments reported in the companion article. The comparison between theory and experiments allowed us to infer the values of the intrinsic molecular rates of the RNA hairpin alone and to search for the optimal experimental conditions to do the measurements. We conclude that the longest handles and softest traps that allow detection of the folding/unfolding signal (handles approximately 5-10 Kbp and traps approximately 0.03 pN/nm) represent the best conditions to obtain the intrinsic molecular rates. The methodology and rationale presented here can be applied to other experimental setups and other molecules.     

Terminal uridylyl transferase of Vigna unguiculata: purification and characterization of an enzyme catalyzing the addition of a single UMP residue to the 3''-end of an RNA primer.

An enzyme which catalyzes the addition of a single UMP residue from UTP to the 3'-end of an RNA primer and which is referred to as terminal uridylyl transferase (TUT) has been extensively purified from the membrane fraction of vigna unguiculata leaves. The purification procedure involved (i) solubilization by cation depletion (ii) DEAE-Sepharose CL-6B column chromatography (iii) affinity chromatography of poly(U)-Sepharose 4B and (iv) glycerol gradient centrifugation. The molecular weight of the native enzyme was approximately 50,000 as determined by velocity sedimentation. Under conditions that were optimal for UMP-incorporation (5 mM Mg2+, low salt, 30 degrees C) TUT displayed a marked specificity for UTP as substrate, was unable to incorporate deoxyribonucleoside triphosphates and required a single-stranded oligo- or polyribonucleotide as primer. When oligoA20, tRNAasp of E. coli or alfalfa mosaic virus RNA 4 were used as primers at various substrate to primer ratio's, the vast majority of the product appeared to consist of primer molecules elongated with a single UMP residue as shown by polyacrylamide gelelectrophoresis and nearest neighbour analysis. We believe TUT to be a novel enzyme which has not been reported before and which may be a feasible tool in RNA sequencing as it enables the specific 3'-terminal labeling of RNA molecules.     

Mechanism of ribonucleic acid chain initiation. 1. A non-steady-state study of ribonucleic acid synthesis without enzyme turnover

A non-steady-state kinetic method has been developed to observe the initiation of long RNA chains by Escherichia coli RNA polymerase without the enzyme turnover. This method was used to determine the order of binding of the first two nucleotides to the enzyme in RNA synthesis with the first two nucleotides to the enzyme in RNA synthesis with poly(dA-dT) as the template. It was shown that initiator [ATP, uridyly(3'-5')adenosine, or adenyly(3'-5')uridylyl-(3'-5')adenosine] binds first to the enzyme-template complex, followed by UTP binding. The concentration dependence of UTP incorporation into the initiation complex suggests that more than one UTP molecule may bind to the enzyme-DNA complex during the initiation process. Comparison of the kinetic parameters derived from these studies with those obtained under steady-state conditions indicates that the steps involving binding of initiator or UTP during initiation cannot be rate limiting in the poly(dA-dT)-directed RNA synthesis. The non-steady-state technique also provides a method for active-site titration of RNA polymerase. The results show that only 36 +/- 9% of the enzyme molecules are active in a RNA polymerase preparation of high purity and specific activity. In addition, the minimal length of poly(dA-dT) involved in RNA synthesis by one RNA polymerase molecule was estimated to be approximately 500 base pairs.     

In vitro synthesis of a possible precursor RNA by purified vesicular stomatitis virus.

The RNA products synthesized $\text{in vitro}$ by the virion-associated RNA polymerase of purified vesicular stomatitis virus have previously been shown to contain two distinct 5′-terminal sequences. The mRNA species contain the blocked 5′-terminal G(5′)ppp(5′)A-A-C-A-G sequence and the initiated lead-in RNA segment (approximately 50 bases) contains the unblocked 5′ ppA-C-G sequence. In the present studies, using inosine 5′-triphosphate in place of GTP it is shown that RNA species as large as 14.5S contain an unblocked 5′-ppA-C-(I) sequence indicating that the GTP analogue permits synthesis of a possible precursor of viral mRNA $\text{in vitro}$.