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Biomedical subjects

A E Simon

Publications and source records attributed to A E Simon.

At least 37 records · Page 2Linked to original sources
PubMed1998

Dissecting RNA recombination in vitro: role of RNA sequences and the viral replicase.

Molecular mechanisms of RNA recombination were studied in turnip crinkle carmovirus (TCV), which has a uniquely high recombination frequency and non-random crossover site distribution among the recombining TCV-associated satellite RNAs. To test the previously proposed replicase-driven template-switching mechanism for recombination, a partially purified TCV replicase preparation (RdRp) was programed with RNAs resembling the putative in vivo recombination intermediates. Analysis of the in vitro RdRp products revealed efficient generation of 3'-terminal extension products. Initiation of 3'-terminal extension occurred at or close to the base of a hairpin that was a recombination hotspot in vivo. Efficient generation of the 3'-terminal extension products depended on two factors: (i) a hairpin structure in the acceptor RNA region and (ii) a short base-paired region formed between the acceptor RNA and the nascent RNA synthesized from the donor RNA template. The hairpin structure bound to the RdRp, and thus is probably involved in its recruitment. The probable role of the base-paired region is to hold the 3' terminus near the RdRp bound to the hairpin structure to facilitate 3'-terminal extension. These regions were also required for in vivo RNA recombination between TCV-associated sat-RNA C and sat-RNA D, giving crucial and direct support for a replicase-driven template-switching mechanism of RNA recombination.

Binding Sites↗
PubMed1998

The effect of a drug and supply cost feedback system on the use of intraoperative resources by anesthesiologists.

UNLABELLED: We performed a randomized, prospective study to evaluate the use of a written feedback system in reducing the intraoperative costs of drugs and supplies used by anesthesiologists. Over 6 mo, 27 anesthesiology residents were randomized to feedback and control groups for their rotations in neurosurgical anesthesia. We recorded the cost of drugs and supplies for three procedures: carotid endarterectomy, lumbar decompression, and cervical decompression. For each study case, members of the feedback group received a written cost analysis showing their performance relative to the departmental average. Members of the feedback group had significantly lower costs for carotid endarterectomies ($79.98 +/- $15.20 vs $97.59 +/- $21.53) and for lumbar decompressions ($56.72 +/- $16.49 vs $76.05 +/- $20.11). The source of savings included lower use rates for propofol and etomidate and for patient warming devices. Analysis of data from recovery areas revealed a trend toward lower patient temperature in lumbar procedures performed by the feedback group. Three months after the feedback period, we collected a follow-up data set in the absence of feedback. This revealed a significant rebound in overall cost by the feedback group for both carotid endarterectomies and lumbar surgery. IMPLICATIONS: This is the first randomized, prospective evaluation of a cost management system in anesthesia. Using resident anesthesiologists, we showed that the written feedback of individualized performance data can be used to lower the overall cost of intraoperative drugs and supplies used for an anesthetic in the absence of mandated clinical guidelines.

Anesthesiology↗
PubMed1997

Analysis in vivo of turnip crinkle virus satellite RNA C variants with mutations in the 3'-terminal minus-strand promoter.

Turnip crinkle virus and its associated RNA, sat-RNA C, share similar, but not identical hairpins near their 3' ends and terminate with CCUGCCC-OH, which forms a single-stranded tail. With an in vitro transcription system containing partially purified TCV RdRp, the 3'-terminal 29 bases making up the hairpin and single-stranded tail were previously demonstrated to be required for transcription, and alterations in the stem, but not the loop, could affect template activity (C. Song and A. E. Simon, 1995, J. Mol. Biol. 254, 6-14). We have now analyzed sat-RNA C mutants in the 3' hairpin for ability to accumulate in vivo. While active templates in vitro were able to accumulate in vivo, some very weak templates in vitro were also able to accumulate in vivo without reversion or second-site alterations. Computer models of hairpin structure indicated that biologically active promoters could have hairpins less stable than wild type, with loops of variable length and sequence, and without a need for a 6-base single-stranded tail. In addition, transcripts containing compensatory exchanges in the upper stem region that had limited activity in vitro were biologically active in vivo, indicating that positioning of specific bases in the stem is not required to produce an active minus-strand promoter.

Carmovirus↗
PubMed1997

The coat protein of turnip crinkle virus is involved in subviral RNA-mediated symptom modulation and accumulation.

Some satellite (sat-) and defective interfering (DI) RNAs associated with plant viruses intensify or ameliorate the symptoms of the virus. We recently demonstrated that the TCV coat protein (CP) is involved in symptom modulation by sat-RNA C. Two additional subviral RNAs have now been tested for effect of the CP on symptom modulation. DI RNA G, which normally intensifies the symptoms of TCV, is able to attenuate symptoms if the TCV CP is replaced with the CP of cardamine chlorotic fleck virus. DI RNA G had no effect on the symptoms of TCV with a single base alteration in the CP open reading frame, unlike sat-RNA C, which was able to ameliorate the symptoms of the mutant TCV. Using a hybrid sat-RNA constructed from sat-RNA C and TCV (which shares a similar 3'-end region with DI RNA G), the 3'-terminal 53 bases of sat-RNA C were found to be involved in symptom attenuation, which was directly correlated with the lack of detectable viral genomic RNA in whole plants. Sat-RNA D had no effect on the symptoms of mutant or wild-type TCV. The accumulation of TCV subviral RNAs in plants and protoplasts was also found to be strongly influenced by the presence or absence of the wild-type TCV CP.

Base Sequence↗
PubMed1997

Analysis of the two subgenomic RNA promoters for turnip crinkle virus in vivo and in vitro.

Infection of plants or protoplasts with turnip crinkle virus (TCV), a monopartite RNA virus, results in the synthesis of the genomic RNA and two subgenomic (sg) RNAs. The transcription start site for the 1.45-kb sgRNA was previously mapped to position 2606 (J. C. Carrington, T. J. Morris, P. G. Stockley, and S. C. Harrison, (1987). J. Mol. Biol. 194, 265-276) corresponding to position 2607 in the TCVms isolate and the start site for the 1.7-kb sgRNA has now been mapped to position 2333 in TCVms. A 96-base sequence (90 bases upstream and 6 bases downstream) encompassing the transcription start site for the 1.45-kb sgRNA was sufficient for full promoter activity. Similarly, a 94-base sequence (90 bases upstream and 4 bases downstream) encompassing the start site was required for full activity of the 1.7-kb sgRNA promoter. The 1.45-kb sgRNA promoter, but not the 1.7-kb sgRNA promoter, was able to direct synthesis of a nontemplate RNA in vitro using partially purified TCV RNA-dependent RNA polymerase. Computer generated secondary structures for the two sgRNA promoters revealed an extensive hairpin just upstream from the transcription start site. Comparisons of corresponding sequences from related viruses indicates higher sequence conservation for the 1.45-kb sgRNA promoter compared with the 1.7-kb sgRNA promoter, despite the latter's location within the RNA-dependent RNA polymerase open reading frame.

Arabidopsis↗
PubMed1997

A novel 3'-end repair mechanism in an RNA virus.

Many positive-stranded RNA viruses contain short, single-stranded 3' ends that are vulnerable to degradation by host cellular RNases. Therefore, the existence of a 3'-end repair mechanism (analogous to cellular telomerases) must be required and/or advantageous for RNA viruses. Accordingly, we provide evidence suggesting that deletions of up to 6 nt from the 3' end of satellite (sat-) RNA C (a small parasitic RNA associated with turnip crinkle carmovirus) are repaired to the wild-type sequence in vivo and in vitro. The novel 3'-end repair mechanism involves the production of 4-8 nt oligoribonucleotides by abortive synthesis by the viral replicase using the 3' end of the viral genomic RNA as template. Based on our in vitro results, we postulate that the oligoribonucleotides are able to prime synthesis of wild-type negative-strand sat-RNA C in a reaction that does not require base pairing of the oligoribonucleotides to the mutant, positive-strand RNA template. The discovery of a 3'-end repair mechanism opens up new strategies for interfering with viral infections.

Carmovirus↗
PubMed1997

Satellite RNA-mediated resistance to turnip crinkle virus in Arabidopsis involves a reduction in virus movement.

Satellite RNAs (sat-RNAs) are parasites of viruses that can mediate resistance to the helper virus. We previously showed that a sat-RNA (sat-RNA C) of turnip crinkle virus (TCV), which normally intensifies symptoms of TCV, is able to attenuate symptoms when TCV contains the coat protein (CP) of cardamine chlorotic fleck virus (TCV-CPCCFV). We have now determined that sat-RNA C also attenuates symptoms of TCV containing an alteration in the initiating AUG of the CP open reading frame (TCV-CPm). TCV-CPm, which is able to move systemically in both the TCV-susceptible ecotype Columbia (Col-0) and the TCV-resistant ecotype Dijon (Di-0), produced a reduced level of CP and no detectable virions in infected plants. Sat-RNA C reduced the accumulation of TCV-CPm by < 25% in protoplasts while reducing the level of TCV-CPm by 90 to 100% in uninoculated leaves of Col-0 and Di-0. Our results suggest that in the presence of a reduced level of a possibly altered CP, sat-RNA C reduces virus long-distance movement in a manner that is independent of the salicylic acid-dependent defense pathway.

Arabidopsis↗
PubMed1997

Environmental and genetic effects on circadian clock-regulated gene expression in Arabidopsis.

Expression patterns of the cold-circadian rhythm-RNA binding (CCR) and chlorophyll a/b binding (CAB) protein genes have circadian rhythms with phases that are different from each other and are affected differently by cold (4 degrees C) treatment. Cycling of CCR and CAB RNA levels was observed in Arabidopsis seedlings grown for 5 days at 4 degrees C under a light/ dark photoperiod, although the cycling had reduced amplitude compared with normal growth conditions (20 degrees C). CCR RNA levels were elevated in the cold, whereas CAB RNA levels were reduced in the cold relative to levels in control seedlings. Cold pulses (4 degrees C for 12 or 20 hr) under continuous light affected the rhythms of CCR and CAB RNA levels in similar ways. The 12-hr cold pulse caused a 4-hr phase delay in both rhythms, whereas the 20-hr cold pulse resulted in a 12-hr phase delay in both rhythms. The timing of CAB expression 1 (toc1) mutation shortened the period of the CCR rhythm, matching previous results for the regulation of the CAB-luciferase (CAB-luc) transgene in this mutant. The results suggest that CCR and CAB share clock machinery but are regulated by downstream components that are affected differently by the cold. Also, the circadian clock regulating these genes in Arabidopsis has a cold-sensitive phase under continuous light conditions.

Arabidopsis↗
PubMed1997

RNA promoters located on (-)-strands of a subviral RNA associated with turnip crinkle virus.

Satellite (sat-) RNA C, one of the nonessential subviral RNAs of turnip crinkle virus (TCV), is dependent on the TCV-encoded RNA-dependent RNA polymerase (RdRp) for its replication. Earlier work showed that a stem-loop structure at the 3' end of (+)-strand sat-RNA C is required for synthesis of (-)-strands in vitro using a partially purified, template-specific TCV RdRp (Song C, Simon AE, 1995, J Mol Biol 254:6-14). Cis-sequences on (-)-strands of sat-RNA C that can serve as separate promoters in vitro have now been defined. Two promoter sequences are located on (-)-strand sat-RNA C, one comprising 11 bases located near the 3' end, and the other consisting of 14 bases located 41 bases from the 5' end. Both promoter sequences contain multiple consecutive C residues followed by multiple consecutive purines and have no obvious secondary structure, suggesting that, along with hairpin structures, specific primary sequences can be recognized by the TCV RdRp. The 3'-proximal promoter sequence directed synthesis from the 3' terminus using (-)-strand templates with the natural sat-RNA 3' end (AUCCC-3'). When plasmid-derived bases were present at the 3' ends of the templates, both promoter sequences could direct the RdRp to initiate transcription internally at the multiple consecutive C residues within the promoters. This result suggests that multiple consecutive C residues are important for transcription initiation and that natural 3'-end sequences, when located at 3' termini, help the RdRp to initiate at the 3' end of the molecule.

Carmovirus↗
PubMed1996

In vivo repair of 3'-end deletions in a TCV satellite RNA may involve two abortive synthesis and priming events.

RNA viruses that do not have the stabilizing features of poly(A) tails or amino acids covalently linked to their 3' ends must develop other means for protecting or repairing their genomes from damage caused by cellular RNases. We previously found that deletions in the single-stranded tails of a satellite RNA (sat-RNA D) associated with turnip crinkle virus are repaired in vivo (C. D. Carpenter and A. E. Simon, 1996, J. Virol. 70, 478-486). We now extend this analysis to show that sat-RNA D transcripts with 3'-end deletions of 5 bases give rise to wild-type sat-RNA, while deletions of 6 to 11 bases result in sat-RNA with additional deletions to the -14 position joined to internal TCV genomic RNA (or other) sequence followed by replacement of the terminal C1-2UGC1-3 motif. In addition, we have determined that the selection of internal TCV sequence used in the repair of sat-RNA D 3' ends is not random and generation of these short TCV segments likely involves primer-mediated synthesis of abortive products facilitated by base-pairing between internal regions of TCV genomic RNA and oligoribonucleotides generated by abortive cycling from the 3' end of the TCV genome.

Carmovirus↗
PubMed1996

Changes in locations of crossover sites over time in de novo generated RNA recombinants.

Recombinant RNAs generated in plants 3 weeks postinoculation with turnip crinkle virus (TCV) genomic RNA and an associated satellite RNA, sat-RNA D, have a majority of TCV crossover sites in a 24-nucleotide repeat (motif IIIA/IIIB) that forms part of a stable hairpin (Carpenter et al., 1995, J. Mol.Biol. 245, 608-622). To determine if parameters other than nucleotide sequence in the crossover region affect junction site selection, recombinants were assayed at various times postinoculation of plants and protoplasts. Populations of recombinants became progressively shorter in plants and larger in protoplasts. Levels of inoculated transcript and age of the plant were not substantial factors in the shifts in crossover site locations. The two most commonly cloned recombinant species were not amplified to detectable levels in protoplasts, suggesting that these molecules are not viable templates for replication. These results suggest that recombination between sat-RNA D and TCV is a very frequent event, and populations of recombinants are likely generated de novo in each infected cell and represent the original recombinant molecules rather than progeny of such molecules. Therefore, factors other than simple selection for recombinants that are more fit to replicate are probably responsible for the differences in junction sites in populations of sat-RNA D/TCV recombinants.

Binding Sites↗
PubMed1996

In vivo restoration of biologically active 3' ends of virus-associated RNAs by nonhomologous RNA recombination and replacement of a terminal motif.

Sequences at the 3' ends of plus-strand RNA viruses and their associated subviral RNAs are important cis elements for the synthesis of minus strands in vivo and in vitro. All RNAs associated with turnip crinkle virus (TCV), including the genomic RNA (4,054 bases) and satellite RNAs (sat-RNAs) such as sat-RNA D (194 bases), terminate with the motif CCUGCCC. While investigating the ability of in vivo-generated recombinants between sat-RNA D and TCV to be amplified in plants, we discovered that sat-RNA D, although truncated by as many as 15 bases in the chimeric molecules, was released from the chimeric transcripts and amplified to high levels. The "new" sat-RNA D molecules nearly all terminated with the motif (C1-2)UG(C1-3) (which may begin with 1 or 2 cytosines and end with 1, 2, or 3 cytosines), which was similar or identical to the natural sat-RNA D 3' end. The new sat-RNA D also contained between 1 and 22 bases of heterogeneous sequence upstream from the terminal motif, which, in some cases, was apparently derived from internal regions of either the plus or minus strand of the TCV genomic RNA. Since most of these internal genomic RNA sequences within TCV were not adjacent to (C1-2)UG(C1-3), at least two steps were required to produce new sat-RNA D 3' ends: nonhomologous recombination with the TCV genomic RNA followed by the addition or modification of the terminus to generate the (C1-2)UG(C1-3) motif.

Base Sequence↗
PubMed1995

Requirement of a 3'-terminal stem-loop in in vitro transcription by an RNA-dependent RNA polymerase.

Partially purified RNA-dependent RNA polymerase (RdRp) isolated from plants infected with turnip crinkle virus (TCV) is capable of template-dependent synthesis of TCV-associated RNAs. To determine the cis-sequences required for the synthesis of TCV satellite (sat-) RNA C (-) strands in vitro, templates containing interior deletions were subjected to transcription using RdRp-active fractions. Results indicated that the promoter for (-)-strand synthesis was contained within the 3'-terminal 29 bases of the (+)-strand. Structural probing by enzymatic digestion and chemical modification revealed the presence of a hairpin structure within this terminal region. Compensatory exchanges of four bases in the lower stem or alterations in the sequence and size of the loop region did not affect in vitro transcription, implying that the primary sequence in the loop and lower part of the stem is not important for interaction with the viral RdRp. However, single mutations in the base of the stem or double mutations in the upper stem strongly reduced template activity in vitro, suggesting that the stability of the hairpin is an important functional consideration. Relocation of the 3'-terminal 37 bases containing this stem-loop to inactive template RNA rendered the resultant hybrid RNA competent for in vitro transcription by RdRp activity, suggesting that the promoter for (-)-strand synthesis in vitro is completely contained within the 3'-terminal region.

Base Sequence↗
PubMed1995

Involvement of a stem-loop structure in the location of junction sites in viral RNA recombination.

Recombination between RNAs associated with turnip crinkle virus is thought to occur during plus-strand synthesis at motifs resembling the 5'-ends of genomic, subgenomic and satellite RNAs. Common structural regions encompassing the motifs have been found for major crossover sites on two different minus-strand templates, with junctions preferentially located in a single-stranded region at the 3' base of a hairpin. Base changes, deletions and compensatory alteration constructed in and around the hairpin in the region of the turnip crinkle virus genomic RNA involved in recombination support the importance of the hairpin for normal crossover site selection. This region of the genomic RNA is also important for replication of the viral genomic RNA in plants and protoplasts, suggesting a common link between sequences required for recombination and viral replication.

Base Sequence↗
PubMed1995

Symptom attenuation by a normally virulent satellite RNA of turnip crinkle virus is associated with the coat protein open reading frame.

Many satellite RNAs (sat-RNAs) can attenuate or intensify the symptoms produced by their helper virus. Sat-RNA C, associated with turnip crinkle virus (TCV), was previously found to intensify the symptoms of TCV on all plants in which TCV produced visible symptoms. However, when the coat protein open reading frame (ORF) of TCV was precisely exchanged with that of cardamine chlorotic fleck virus, sat-RNA C attenuated the moderate symptoms of the chimeric virus when Arabidopsis plants were coinoculated with the chimeric virus. Symptom attenuation was correlated with a reduction in viral RNA levels in inoculated and uninoculated leaves. In protoplasts, the presence of sat-RNA C resulted in a reduction of approximately 70% in the chimeric viral genomic RNA at 44 hr postinoculation, whereas the sat-RNA wa consistently amplified to higher levels by the chimeric virus than by wild-type TCV. TCV with a deletion of the coat protein ORF also resulted in a similar increase in sat-RNA C levels in protoplasts, indicating that the TVC coat protein, or its ORF, downregulates the synthesis of sat-RNA C. These results suggest that the coat protein or its ORF is a viral determinant for symptom modulation by sat-RNA C, and symptom attenuation is at least partly due to inhibition of virus accumulation.

Arabidopsis↗