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At least 739 records · Page 41Linked to original sources
PubMed1990

Nuclease-resistant polyadenylated RNAs of significant size are detected by PCR in highly purified Creutzfeldt-Jakob disease preparations.

The molecular nature of the 'unconventional viruses' that cause slow, progressive brain deterioration is still poorly understood. As part of a reinvestigation of potential agent-specific nucleic acids, we developed a protocol for enriching agent-specific sequences. This protocol uses extensive micrococcal nuclease digestion followed by rate zonal sucrose sedimentation. Most of the infectivity in the gradient (84%) had a characteristic mean size of approximately 120S, and was resolved from 70% of a host glycoprotein (PrP) that can cosediment with infectivity. In infectious size fractions, nucleic acids were reduced approximately one million-fold with respect to starting brain homogenate, and specific purification of infectivity was approximately 100,000-fold with respect to nucleic acid. Using a novel polymerase chain reaction strategy, we were able to amplify RNA species in these fractions. Remarkably, host polyadenylated sequences of 1 to over 4 kb were detected in the nuclease-protected infectious fractions. These strategies set the stage for the identification of similar nucleic acids that may be specific for the CJD agent.

Base Sequence↗
PubMed1997

Turnip yellow mosaic virus RNA-dependent RNA polymerase: initiation of minus strand synthesis in vitro.

An RNA-dependent RNA polymerase (RdRp) activity was detergent-solubilized from the chloroplast membranes of Chinese cabbage leaves infected with turnip yellow mosaic virus (TYMV). The template-dependent, micrococcal nuclease-treated activity synthesized full-length minus strands from TYMV RNA and 3'-fragments as short as a 28-nucleotide-long RNA comprising the amino acid acceptor stem of the 3'-tRNA-like structure (TLS). Minus strands were shown to arise by de novo initiation with the insertion of GTP opposite the penultimate (C) residue of the 3'-terminal -CCA. The TYMV RdRp activity was template specific in that poly(A) RNA was not copied, and alfalfa mosaic virus (AIMV) RNA, which does not contain a 3'-TLS, was a very poor template. However, other viral RNAs with a 3'-TLS and in vitro transcripts of tRNAs were copied to varying degrees. Fully modified tRNAs were either inactive or poorly active templates, and AIMV 3'-RNA, even when provided with a 3'-terminal -ACCA, was not copied detectably. A potential role of the acceptor stem pseudoknot as a promoter element was assessed with mutations that drastically altered the structure and sequence of the pseudoknot, revealing only a twofold effect in decreasing template activity. The data show that RNAs with both a tRNA-like conformation and a -CCA 3'-terminus are potential templates for TYMV RdRp and suggest that promoter elements are not limited to the acceptor stem pseudoknot.

Base Sequence↗
PubMed1997

Evidence for heterologous encapsidation of potato spindle tuber viroid in particles of potato leafroll virus.

The aphid Myzus persicae (Sulz.) was shown to transmit potato spindle tuber viroid (PSTVd) to potato clone DTO-33 from source plants doubly infected with potato leafroll virus (PLRV) and PSTVd. Transmission was of the persistent type and did not occur when the insects were allowed to feed on singly infected plants. Only low levels of PSTVd were associated with purified PLRV virions, but its resistance to digestion with micrococcal nuclease indicates that the viroid RNA is encapsidated within the PLRV particles. Epidemiological surveys carried out at three locations in China revealed a strong correlation between PSTVd infection and the presence of PLRV, suggesting that PLRV can facilitate PSTVd spread under field conditions.

Capsid↗
PubMed1993

Detection of Staphylococcus aureus with biotinylated monoclonal antibodies directed against staphylococcal TNase complexed to avidin-peroxidase in a rapid sandwich enzyme-linked immunofiltration assay (sELIFA).

For rapid identification of Staphylococcus aureus, a monoclonal antibody (MAb)-biotin-avidin-peroxidase complex, directed against the S. aureus thermostable nuclease (TNase), was formed and used in a rapid three-step sandwich enzyme-linked immunofiltration assay (sELIFA) and a three-step sandwich enzyme-linked immunosorbent assay (sELISA). The MAb-peroxidase complex was formed by incubating the biotinylated MAbs with a streptavidin-peroxidase conjugate and the complex was purified by gel permeation chromatography. When compared with a four-step MAb-based sELISA described previously, this complex permitted one reagent step to be omitted in a three-step sELISA, and the test time was significantly reduced. The test sensitivity was slightly reduced in the three-step ELISA (detection limit 1.0-2.0 ng of TNase/ml) when compared to the four-step sELISA (detection limit 0.5-1.0 ng of TNase/ml). The sELIFA method was based on the filtration of bacterial culture supernates through nitrocellulose membrane disks pre-spotted with a MAb directed against the S. aureus TNase, followed by detection with the MAb-peroxidase complex (three-step sELIFA). A detection limit of 0.5-2.0 ng of TNase/ml was achieved with the three-step sELIFA, depending on the filtrate volume of culture supernates. The total test time was 10-15 min when pre-spotted and blocked membranes were used. A total of 85 bacterial strains was tested in the sELIFA. All the 28 S. aureus strains showed positive results, but none of the 57 non-S. aureus strains did so, although some of these produced thermostable nuclease activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗