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Biomedical subjects

A E Simon

Publications and source records attributed to A E Simon.

At least 19 recordsLinked to original sources
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

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

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↗
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↗
PubMed1983

High-frequency mutation at the adenine phosphoribosyltransferase locus in Chinese hamster ovary cells due to deletion of the gene.

Evidence for a two-step model to explain the high-frequency expression of the recessive phenotype at the autosomal adenine phosphoribosyltransferase (APRT; EC 2.4.2.7) locus in Chinese hamster ovary (CHO) cells was given by Simon et al. [Simon, A. E., Taylor, M. W., Bradley, W. E. C. & Thompson, L. (1982) Mol. Cell. Biol. 2, 1126-1133]. This model proposed a high-frequency event, leading to allelic inactivation or a loss of gene function, and a low-frequency event, causing a structural alteration of the APRT protein. Either event could occur first, resulting in two classes of heterozygotes. We have analyzed the low-frequency event that gave rise to the class 2 aprt heterozygote D416 and the high-frequency event that led to APRT- cells derived from D416. Genomic Southern blots of Msp I- or Hpa II-digested DNA from wild-type CHO, aprt heterozygote D416, and two APRT- cell lines derived from D416 indicate a loss of a specific Msp I/Hpa II recognition sequence at one aprt locus in the heterozygote that correlates with the production of the electrophoretically altered APRT protein found in D416. The APRT- mutants are homozygous for the loss of this Msp I/Hpa II site. By using an additional CHO gene as an internal control, it was determined that the APRT- mutants contain only a single copy of the altered aprt gene. Thus, the high-frequency event that produces APRT- mutants derived from D416 is not an inactivation event but rather a deletion of the wild-type aprt gene.

Adenine Phosphoribosyltransferase↗