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A E Simon

Publications and source records attributed to A E Simon.

At least 55 records · Page 3Linked to original sources
PubMed1995

Synthesis of novel products in vitro by an RNA-dependent RNA polymerase.

RNA-dependent RNA polymerase from turnip crinkle virus-infected turnip transcribes both strands of a virus-associated satellite RNA, sat-RNA C (356 bases), in vitro. While both plus- and minus-strand sat-RNA C can direct the synthesis of full-length complementary-strand products, transcription of minus-strand RNA also generates two non-template-sized products, L-RNA and S-RNA (C. Song and A. E. Simon, Proc. Natl. Acad. Sci. USA 91:8792-8796, 1994). Here we report that synthesis of L-RNA and S-RNA results from terminal elongation of the 3' end of the template. L-RNA has a panhandle structure and is composed of minus-strand template covalently linked to newly synthesized RNA complementary to its 5' 190 bases. S-RNA is composed of template covalently linked to its full-length complementary strand. All minus-strand templates tested yielded S-RNA. However, synthesis of L-RNA was affected by deletion of the 3' end of the minus-strand template or several internal regions and base alterations near the 5' end or in an internal sequence immediately upstream from the template-product junction that could potentially form a heteroduplex with the 3' end. Furthermore, mutations that disrupted or restored a stem-loop involved in RNA recombination in vivo affected the level of L-RNA produced in vitro, suggesting that the mechanisms for intramolecular formation of panhandle RNAs and intermolecular RNA recombination involve similar features.

Base Sequence↗
PubMed1994

RNA-dependent RNA polymerase from plants infected with turnip crinkle virus can transcribe (+)- and (-)-strands of virus-associated RNAs.

RNA-dependent RNA polymerase (RdRp) was solubilized from membranes of turnip infected with turnip crinkle virus (TCV), a single-stranded, monopartite RNA virus. The RdRp activity could be separated into three peaks by Sephacryl S500HR chromatography. RdRp from peak I, which contained substantial amounts of endogenous TCV genomic RNA, and peak II were template-specific, synthesizing full-length complementary strands of exogenous TCV subviral RNAs but not control RNA templates. Peak III RdRp was nonspecific, synthesizing full-sized products for all added RNA templates. Peak II RdRp transcribed several different TCV satellite (sat) and defective interfering RNA templates in both (+)- and (-)-sense orientations but did not transcribe (+)-strands of satellite RNAs associated with unrelated viruses. Monomeric-length sat-RNA C was synthesized from a template containing as many as 220 nonsatellite bases at the 3' ends of either (+)- or (-)-strands, indicating that the RdRp was able to recognize 3'-end sequences in an internal location. Deletion of 95-242 bases from the 3' end of (+)-strand sat-RNA C abolished the synthesis of template-length product. However, transcription of template-length products was not affected by the deletion of at least 257 bases from the 3' end of (-)-strand sat-RNA C template (leaving only the 100 5'-terminal residues), implying that different mechanisms exist for synthesis of (+)-and (-)-strand satellite RNA in vitro.

Plant Viruses↗
PubMed1994

Recombination between plus and minus strands of turnip crinkle virus.

Turnip crinkle virus (TCV), a small monopartite RNA virus, is associated with several subviral RNAs, some of which are the products of recombination between the genomic RNA and an unrelated satellite RNA (sat-RNA D). As part of a study on the nonrandom sequences found at the junctions of nearly all TCV recombinant molecules, we have characterized a novel recombinant (D/TCV950) that was formed from sat-RNA D and both the plus and minus strands of the genomic RNA. D/TCV950 (517 nt) is composed of sat-RNA D at the 5'-end, joined to four nontemplate derived uracil residues, and then three segments of TCV genomic RNA, one of which is from the complementary strand. Sequences at the right sides of the junctions were similar to sequences previously found at the right sides of junctions in other TCV-associated recombinant molecules. To our knowledge, this is the first report of a recombinant molecule formed from both coding and complementary strands of a single-stranded, positive-sense RNA virus.

Base Sequence↗
PubMed1994

Genes encoding glycine-rich Arabidopsis thaliana proteins with RNA-binding motifs are influenced by cold treatment and an endogenous circadian rhythm.

We have characterized the expression of two members of a class of Arabidopsis thaliana glycine-rich, putative RNA-binding proteins that we denote Ccr1 and Ccr2. Southern blot analysis indicates that Ccr1 and Ccr2 are members of a small gene family. Both Ccr1 and Ccr2 mRNA levels were influenced by a circadian rhythm that has an unusual phase for plants, with maximal accumulation at 6:00 PM and minimal accumulation at 10:00 AM. The level of CCR1 protein, however, remained relatively constant throughout the cycle. The transcript accumulation patterns of the Ccr1 and Ccr2 genes differed considerably from conditions that affect the expression of similar genes from maize, sorghum, and carrot. Levels of Ccr1 and Ccr2 mRNAs were unchanged in wounded plants, increased at least 4-fold in cold-stressed plants, and decreased 2- to 3-fold in abscisic acid-treated plants. Ccr1 transcript levels decreased in response to drought, whereas Ccr2 transcript levels increased under the same conditions. Based on the presence of additional Ccr transcripts in dark-grown plants, we propose that Ccr transcripts may be subjected to a light- or dark-mediated regulation.

Amino Acid Sequence↗
PubMed1994

Effect of template size on accumulation of defective interfering RNAs in protoplasts.

A turnip protoplast system has been used to study the effects of template size and sequence on the replication and/or stability of a small defective interfering (DI) RNA associated with turnip crinkle virus. Our results indicated that as little as a single base difference in the size of the molecule in some regions, rather than the specific sequence, affected the level of DI RNA accumulating in protoplasts.

Cloning, Molecular↗
PubMed1993

Sequences and structures required for recombination between virus-associated RNAs.

RNA recombination has been described for a number of viruses in the plant and animal kingdoms, but the mechanisms of selection of recombination sites are poorly understood. The nonrandom recombination between two subviral RNAs associated with turnip crinkle virus was used to study the requirement for specific sequences and structures in the generation of recombinant molecules. Single-base mutations that disrupted either the stem or the loop of one of the two computer-predicted stem-loop structures eliminated detectable recombinant molecules. However, recombinants were detected if compensatory mutations were generated that re-formed a stable hairpin structure. These results provide evidence for the necessity of specific structures in the formation of recombinant molecules in this system.

Base Sequence↗
PubMed1991

Recombination between satellite and genomic RNAs of turnip crinkle virus.

New recombinant molecules formed from satellite and genomic RNAs of turnip crinkle virus (TCV) have been characterized. Known collectively as sat-RNA CX, these molecules are composed of a nearly full-length segment of a previously characterized TCV satellite RNA (sat-RNA D) at the 5' end joined to variable lengths of TCV genomic RNA 3' terminal sequence. Sat-RNA CX molecules fall into two classes: molecules of 420 to 435 bases and larger species of 501 to 506 bases. The TCV sequence at the junction of the larger molecules is purine-rich and is similar to a motif found at the 5' ends of the TCV satellite RNAs and at the junctions of some TCV defective interfering RNAs. The TCV sequence at the junction of the smaller sat-RNA CX molecules is pyrimidine-rich and is similar to the sequence at the right side of a junction of one TCV defective interfering RNA as well as sequence immediately downstream of the internal initiation site of the 1.45-kb TCV subgenomic RNA. We propose that the latter motif is another putative signal recognized by the viral replicase during the generation of defective interfering and recombinant RNAs in the TCV system.

Base Sequence↗
PubMed1991

Formation of multimers of linear satellite RNAs.

A 22-base region of turnip crinkle virus satellite-RNA C (sat-RNA C) is involved in the accumulation of monomeric and dimeric forms. Deletions within the region inhibited the accumulation of sat-RNA C monomers. However, normal ratios of dimers to monomers occurred if the 22 bases were replaced by 22 unrelated bases or if the location of this region was altered. Therefore, these specific 22 bases are not involved in the accumulation of sat-RNA C monomers. Examination of the sequences at the junctions of multimers of all three turnip crinkle virus sat-RNAs revealed the deletion of bases corresponding to the 3' and 5' ends of monomeric units as well as the addition of nucleotides not present in monomers. Based on these results, we present a model to explain the formation of multimers of linear subviral RNAs associated with turnip crinkle virus. Our model suggests that multimers are formed by the reinitiation of replication by the replicase before release of the nascent strand. We have previously proposed the same mechanism for the formation of defective interfering RNAs, chimeric sat-RNAs, and sat-RNA recombinants in the turnip crinkle virus system (Cascone, Carpenter, Li, and Simon. (1990). EMBO J. 9, 1709-1715).

Base Sequence↗
PubMed1991

Mutations in a satellite RNA of turnip crinkle virus result in addition of poly(U) in vivo.

Turnip crinkle virus (TCV) is associated with many subviral RNAs including satellite (sat-) RNAs which require a helper virus for infectivity. When plants were inoculated with TCV and transcripts of TCV sat-RNA C containing deletions of 3 to 8 nucleotides beginning at position 100 and extending toward the 5' end, some of the sat-RNA isolated from plants migrated more slowly than expected on denaturing polyacrylamide gels. Cleavage of the sat-RNA into two segments by digestion with RNase H following hybridization to an oligonucleotide complementary to internal sat-RNA sequence indicated that the 5' one-third of the molecule was involved in the abnormal gel migration. Sat-RNAs derived from transcripts with a deletion of bases in position 96-100 were cloned. Sequencing of the cDNAs revealed that the aberrant migration of the sat-RNAs was due to the presence of variable lengths of poly(U) 10 nucleotides downstream from the deletion at a position which already contained five U residues. Deletions extending toward the 3' end in the same region did not result in poly(U) additions. Mutations in the original five U residues along with the 5' deletions also did not lead to poly(U) additions. The insertion of poly(U) in TCV sat-RNA C may be a new example of replicase stuttering with the distinction that it only occurs following specific upstream mutations.

Base Sequence↗
PubMed1991

In vivo accumulation of a turnip crinkle virus defective interfering RNA is affected by alterations in size and sequence.

Turnip crinkle virus is one of several single-stranded RNA plant viruses associated with defective interfering (DI) RNAs. A complete cDNA copy of a 344-base DI RNA (DI RNA G) was cloned downstream from a T7 RNA polymerase promoter. Transcripts synthesized in vitro were infectious when inoculated with helper virus on turnip plants. Studies of the infectivity of DI transcripts containing deletions, insertions, and single-base changes suggest that (i) in general, only the 5' two-thirds of the molecule can tolerate mutations; (ii) between 52 and 67 bases of terminal 5' sequence are required for infectivity; (iii) nucleotides in positions 68 to 138 are not specifically involved in RNA infectivity; (iv) DI RNA G molecules smaller than 327 bases are not amplified efficiently in plants.

Base Sequence↗
PubMed1990

Recombination between satellite RNAs of turnip crinkle virus.

Turnip crinkle virus (TCV) is associated with satellite (sat) RNAs (sat-RNA D, sat-RNA F), defective interfering (DI) RNAs (DI RNA G, DI1 RNA), and one RNA with properties of both sat-RNAs and DI RNAs (sat-RNA C). When plants were inoculated with TCV, sat-RNA D and in vitro sat-RNA C transcripts containing non-viable mutations in the 5' domain, recombinant sat-RNAs were recovered. These recombinants were composed of sat-RNA D at the 5' end and sat-RNA C sequences at the 3' end. Analysis of 20 independent recombination junctions revealed that unequal crossing-over had occurred in planta in a region of sequence similarity between the two sat-RNAs which resulted in the duplication of 3-16 nucleotides. Thirty percent of the sat-RNA recombinants also had one to three additional nucleotides inserted at the crossover junctions which did not correspond to either sat-RNA C or sat-RNA D sequence. The right side of the recombination junctions always began with one of three consecutive nucleotides of sat-RNA C. Based on the similarity between this sequence of sat-RNA C, the right side junction of DI RNA G and the 5' end of TCV, as well as the sequence similarity between right side junctions of DI1 RNA and sat-RNA C and the 5' end of the sat-RNAs, a replicase-driven copy choice mechanism is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1989

Turnip crinkle virus defective interfering RNAs intensify viral symptoms and are generated de novo.

Defective interfering (DI) RNAs have been isolated from a broad spectrum of animal viruses and have recently been identified in plant virus infections. Because of their ubiquitous nature, DIs are thought to play an important role in virus replication and yields. DI RNAs have now been found in association with a natural isolate of turnip crinkle virus (TCV-B) and are generated de novo after inoculation of turnip with virus derived from cloned transcripts. DI RNA G, naturally found in the TCV-B isolate, is a mosaic molecule with 5' and 3' viral segments and a repeat of 36 nucleotides at the beginning of the 3' segment. The 5'-terminal 21 nucleotides of DI RNA G were not similar to genomic TCV sequences but did resemble sequences found at the 5' end of other small RNAs associated with TCV (satellite RNAs). DI RNA G interferes with the accumulation of TCV genomic RNA and, unlike other DI RNAs, intensifies the symptoms of its helper virus. Infection of turnip with virus derived from cloned transcripts of TCV-B resulted in de novo generation of a DI RNA, DI1 RNA. DI1 RNA differed from DI RNA G by containing exact 5' and 3' ends of TCV as well as an internal virus segment.

Base Sequence↗
PubMed1988

Identification of regions affecting virulence, RNA processing and infectivity in the virulent satellite of turnip crinkle virus.

Turnip crinkle virus (TCV) supports a small family of satellite RNAs (RNAs C, D and F). RNA C is a virulent satellite, producing severe symptoms in host plants, while RNAs D and F are avirulent satellites. The virulent satellite (RNA C) has two major domains--a 5'-domain similar to the avirulent satellites and a 3'-domain similar to the 3'-end of the TCV genome. To demonstrate that the 3'-domain of RNA C determines virulence, a chimeric satellite was constructed composed mostly of the 5'-domain of the avirulent satellite (RNA F) and the 3'-domain of the virulent satellite (RNA C). To locate other functional regions, small DNA fragments were inserted or deleted at various sites in the cDNA of virulent satellite (RNA C). Most small internal deletions and insertions in the midsection of the molecule had no detectable effects while those near the 3'-end of RNA C destroyed infectivity. Modifications in a small region centering on an AGCAGC repeat in the domain of satellite homology blocked the accumulation of monomers and presumably the processing of RNA C. Other modifications in this region produced more intense symptoms. Hence, these experiments reveal regions of the satellite which determine virulence, are essential for infectivity, affect monomer accumulation (RNA processing) and modulate symptom expression.

Base Sequence↗
PubMed1987

Synthesis in vitro of infectious RNA copies of the virulent satellite of turnip crinkle virus.

RNA copies, synthesized in vitro, of the virulent satellite (RNA C) of turnip crinkle virus (TCV) infect plants when coinoculated with helper virus RNA. RNA C is a small linear RNA of about 355 bases which intensifies TCV symptoms in infected plants. Full-length cDNA copies of the satellite were inserted in an expression vector (for RNA synthesis in vitro) in such a way that RNA synthesized in vitro had the same 5'-end as the native satellite. Plus-strand RNA copies of the satellite in near-monomer and multimer form infected plants, while minus-strand RNA copies and DNA copies of the satellite RNA did not do so under the conditions tested. When plants were inoculated with RNAs synthesized in vitro from two independently cloned satellite cDNAs with base sequence and length differences, the products of infection corresponded in sequence to the different cRNAs used in the inocula. Satellite RNAs synthesized in vitro from either cDNA produced the same symptoms as the native satellite RNA.

Base Sequence↗
PubMed1986

The virulent satellite RNA of turnip crinkle virus has a major domain homologous to the 3' end of the helper virus genome.

RNA C (355 bases), RNA D (194 bases) and RNA F (230 bases) are small, linear satellite RNAs of turnip crinkle virus (TCV) which have been cloned as cDNAs and sequenced in this study. These RNAs produce dramatically different disease symptoms in infected plants. RNA C is a virulent satellite that intensifies virus symptoms when co-inoculated with its helper virus in turnip plants, while RNA D and RNA F are avirulent. RNA D and RNA F, the avirulent satellites, are closely related to each other except that RNA F has a 36-base insert near its 3' end, not found in RNA D. The 189 bases at the 5' end of RNA C, the virulent satellite, are homologous to the entire sequence of RNA D. However, the 3' half of RNA C, is composed of 166 bases which are nearly identical to two regions at the 3' end of the TCV helper virus genome. Hence, the virulent satellite is a composite molecule with one domain at its 5' end homologous to the other avirulent satellites and another domain at its 3' end homologous to the helper virus genome. All four TCV RNAs, RNAs C, D and F and the helper virus genome have identical 7 bases at their 3' ends. The secondary structure of RNA C deduced from the sequence can be folded into two separate domains - the domain of helper virus genome homology and the domain homologous to other TCV satellite RNAs. Comparative sequences of several different RNA C clones reveal that this satellite is a population of molecules with sequence and length heterogeneity.

Journal Article↗