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G Bruening

Publications and source records attributed to G Bruening.

At least 19 recordsLinked to original sources
PubMed2002

An improved genetic linkage map for cowpea (Vigna unguiculata L.) combining AFLP, RFLP, RAPD, biochemical markers, and biological resistance traits.

An improved genetic linkage map has been constructed for cowpea (Vigna unguiculata L. Walp.) based on the segregation of various molecular markers and biological resistance traits in a population of 94 recombinant inbred lines (RILs) derived from the cross between 'IT84S-2049' and '524B'. A set of 242 molecular markers, mostly amplified fragment length polymorphism (AFLP), linked to 17 biological resistance traits, resistance genes, and resistance gene analogs (RGAs) were scored for segregation within the parental and recombinant inbred lines. These data were used in conjunction with the 181 random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), AFLP, and biochemical markers previously mapped to construct an integrated linkage map for cowpea. The new genetic map of cowpea consists of 11 linkage groups (LGs) spanning a total of 2670 cM, with an average distance of 6.43 cM between markers. Astonishingly, a large, contiguous portion of LG1 that had been undetected in previous mapping work was discovered. This region, spanning about 580 cM, is composed entirely of AFLP markers (54 in total). In addition to the construction of a new map, molecular markers associated with various biological resistance and (or) tolerance traits, resistance genes, and RGAs were also placed on the map, including markers for resistance to Striga gesnerioides races 1 and 3, CPMV, CPSMV, B1CMV, SBMV, Fusarium wilt, and root-knot nematodes. These markers will be useful for the development of tools for marker-assisted selection in cowpea breeding, as well as for subsequent map-based cloning of the various resistance genes.

Biomarkers↗
PubMed2000

Evidence for participation of RNA 1-encoded elicitor in Cowpea mosaic virus-mediated concurrent protection.

The cowpea (Vigna unguiculata) line Arlington, inoculated with Cowpea mosaic virus (CPMV), showed no symptoms, and no infectivity or accumulation of capsid antigen was detected at several days after inoculation. Coinoculation, but not sequential inoculation, of CPMV with similar concentrations of another Comovirus; Cowpea severe mosaic virus (CPSMV), resulted in reduced numbers of CPSMV-induced lesions. This apparent, CPMV-mediated reduction in number of CPSMV-induced infection centers was termed concurrent protection. We report results obtained by inoculating two nearly isogenic cowpea lines derived from a CPMV-susceptible cowpea crossed to Arlington, one line CPMV-susceptible and the other resistant. The CPMV virions B and M, encapsidating genomic RNAs 1 and 2, respectively, were extensively purified by gradient centrifugation. In the CPMV-resistant cowpea, either CPMV or CPMV B affected concurrent protection against CPSMV and against two distinct non-Comoviruses: Cherry leafroll virus and Southern bean mosaic virus. Adding CPMV M to the inoculum did not enhance CPMV-B-mediated protection. CPMV B was ineffective in protecting CPMV-susceptible cowpea. We postulate that CPMV-mediated concurrent protection is elicited in CPMV-resistant cowpea by a CPMV RNA-1-encoded factor and acts to reduce accumulation or spread of CPMV and certain coinoculated challenging viruses in or from the inoculated cell. Coinoculated CPMV did not protect CPMV-resistant cowpea against Tomato bushy stunt virus or Cucumber mosaic virus.

Comovirus↗
PubMed1997

Formation of circular satellite tobacco ringspot virus RNA in protoplasts transiently expressing the linear RNA.

The most abundant form of the satellite RNA of tobacco ringspot virus (sTRSV RNA) is a linear, unit length molecule of 359 nucleotide residues, designated L-(+)M. A postulated replication scheme for the satellite RNA has as its first, and apparently virus-independent, step the ligation of L-(+)M into the corresponding circular form C-(+)M. We transiently expressed L-(+)M wild type and L-(+)M mutants in tobacco protoplasts using an African cassava mosaic geminivirus vector. Measured extents of C-(+)M accumulation were correlated with computer-predicted folding to suggest wild-type secondary structure elements that might be deleted without reducing ligation. A 127-nucleotide residue mutant L-(+)M was created by replacing, with 7 and 3 residues, respectively, nucleotide residues 53-211 and 268-350, each of which was predicted to form a set of three adjacent imperfect stem-loops in wild-type L-(+)M. The mutant L-(+)M was found to be extensively ligated to C-(+)M in protoplasts and to retain a calculated helix of the wild-type molecule that incorporates the 3' terminal sequence. A trinucleotide in the 3' region was mutated so as to disrupt and restore, respectively, the calculated helix, reducing and restoring, respectively, C-(+)M formation. These results suggest that the 3' stem contributes to the suitability of the small L-(+)M molecules as a substrate for a protoplast RNA ligase and that computed folding of sTRSV RNA may be predictive of sTRSV RNA structure in vivo.

Base Sequence↗
PubMed1996

Chemical cleavage of 5'-linked protein from tobacco ringspot virus genomic RNAs and characterization of the protein-RNA linkage.

Each of the two genomic RNAs of tobacco ringspot nepovirus is known to have a 5'-linked protein, the VPg. We report a simplified analysis of the covalent VPg-RNA connection that allowed us to identify the 5' nucleotide residue of each genomic RNA and its phosphodiester link to a specific serine residue of the VPg, without resorting to in vivo labeling with 32P, in vitro radioiodination, or separation of the two genomic RNAs. Unfractionated genomic RNA was incubated with an oligodeoxyribonucleotide specific for the 5' region of either RNA 1 or RNA 2 and ribonuclease H. Reaction products were 3'-end-labeled and were fractionated by gel electrophoresis. The most highly labeled product derived from each genomic RNA was identified as a VPg-oligoribonucleotide (VPg-5'-oligo) by its sensitivity to proteinase. In a presumed beta-elimination reaction that apparently was more rapid than phosphodiester cleavage, incubation in alkaline sodium bicarbonate released a rapidly migrating product, 5'-oligo. Phosphatase-treated 5'-oligo accepted 5'-label in a polynucleotide kinase-catalyzed reaction, and uridylate was identified as the 5' terminal residue for both RNA 1 and RNA 2. Results from Edman degradation of the VPg suggest that the VPg is linked at serine 5 to the 5' uridylate of each genomic RNA.

Amino Acid Sequence↗
PubMed1995

A subgenomic RNA associated with cherry leafroll virus infections.

Cherry leafroll nepovirus (CLRV) genomic RNA 1 (8 kb) and genomic RNA 2 (7 kb) have 3' polyadenylate tracts and, extending 5' from the polyadenylate, nearly identical sequences of 1.6 kb termed the 3' common region. We observed RNAs 1 and 2 and a third RNA of 1.5 kb in nucleic acid extracts of CLRV-infected Nicotiana tabacum suspension cell protoplasts and Chenopodium quinoa plants, using a hybridization probe complementary to 1 kb of the 3' common region. The third RNA was partially purified by preparative gel electrophoresis and chromatography on an oligodeoxythymidylate column. Analyses of transcripts primed by a complementary oligodeoxyribonucleotide and of cDNA clones revealed that the third RNA corresponds to the 3' 1500 nucleotide residues of RNA 1. Hence we designate the newly characterized RNA as RNA 1A. RNA 1A was not detected as encapsidated RNA in extracts of either protoplasts or C. quinoa plants. The amount of accumulated RNA 1A declined between 24 and 48 hr after inoculation of protoplasts with CLRV virions, although CLRV RNAs 1 and 2 continued to accumulate. Other results were not consistent with cleaved RNA 1 being the origin of RNA 1A. RNA 1A has the properties of a subgenomic RNA, presumably synthesized from negative-sense RNA 1 as template.

Base Sequence↗
PubMed1995

Effective ribozyme delivery in plant cells.

Hammerhead ribozyme sequences were incorporated into a tyrosine tRNA (tRNA(Tyr)) and compared with nonembedded molecules. To increase the levels of ribozyme and control antisense in vivo, sequences were expressed from an autonomously replicating vector derived from African cassava mosaic geminivirus. In vitro, the nonembedded ribozyme cleaved more target RNA, encoding chloramphenicol acetyltransferase (CAT), than the tRNA(Tyr) ribozyme. In contrast, the tRNA(Tyr) ribozyme was considerably more effective in vivo than either the nonembedded ribozyme or antisense sequences, reducing CAT activity to < 20% of the control level. A target sequence (CM2), mutated to be noncleavable, showed no reduction in CAT activity in the presence of the tRNA(Tyr) ribozyme beyond that for the antisense construct. The reduction in full-length CAT mRNA and the presence of specific cleavage products demonstrated in vivo cleavage of the target mRNA by the tRNA(Tyr) ribozyme. The high titer of tRNA(Tyr) ribozyme was a result of transcription from the RNA polymerase III promoter and led to the high ribozyme/substrate ratio essential for ribozyme efficiency.

Base Sequence↗
PubMed1995

Trace amount of satellite RNA associated with tobacco ringspot virus: increase stimulated by nonaccumulating satellite RNA mutants.

The small satellite RNA of tobacco ringspot virus (sTRSV RNA) is dependent on tobacco ringspot virus (TRSV) for replication and encapsidation. sTRSV RNA has appeared during serial passage of certain TRSV strains in some hosts. Co-inoculation of bean with TRSV and either of two related, nonaccumulating mutants of sTRSV RNA induced the appearance of sTRSV RNA in a single passage (van Tol et al., 1991, Virology 180, 23-30). The sTRSV RNA obtained after serial passage and after co-inoculation have the same nucleotide sequence, designated the endogenous sequence. The endogenous sTRSV RNA nucleotide sequence differs from that of each of the nonaccumulating sTRSV RNA at three positions. In order to detect possible trace amounts of endogenous satellite RNA in virion RNA preparations, RNA from two TRSV isolates was subjected to reverse transcription and polymerase chain reaction of the transcript (RT-PCR), using primers with sTRSV RNA terminal sequences. The yield of RT-PCR product suggests that the virion RNA preparations contained approximately 0.1 fg of sTRSV RNA per microgram of virion RNA. The nucleotide sequence of the RT-PCR product corresponded to that of the endogenous sTRSV RNA. The endogenous sTRSV RNA of TRSV inocula appears to be latent, being maintained in very small amounts during serial passage of TRSV in some hosts but capable of dramatic increase during serial passage in other hosts or when TRSV was co-inoculated with either of two specific sTRSV RNA mutants. Ten other nonaccumulating sTRSV RNA mutants did not induce a detected increase in sTRSV RNA.

Base Sequence↗
PubMed1995

Increase of satellite tobacco ringspot virus RNA initiated by inoculating circular RNA.

A small satellite RNA of tobacco ringspot virus (sTRSV RNA) generates circular and linear molecules of unit length and repetitive sequence, linear multimers during replication. The phosphodiester junction joining the unit satellite RNA sequences in multimeric and circular RNA resisted base-catalyzed cleavage in circles but not in linear dimers. We postulate that junctions of multimeric satellite RNA form during synthesis of the polyribonucleotide chain, whereas those of circular RNA result from a ligation reaction that introduces a group blocking the junction 2'-hydroxyl. To test the relative effectiveness of linear and circular satellite RNAs in initiating replication, we inoculated onto bean (Phaseolus vulgaris cv Black Valentine) the four possible pairs of satellite RNA molecules, one member of each pair having the wild-type sTRSV RNA sequence and the other that of the replicating mutant 51AG/212CU, with each sequence provided as the unit circular or linear form. The relative amounts of wild-type and mutant satellite RNA sequence recovered from progeny virions reflected their relative abundances in the inoculum without regard to whether the sequence was supplied as a linear or a circular molecule. These results are consistent with models for the replication of the satellite RNA in which a circular form of the satellite RNA is a template for rolling circle transcription or is otherwise a replication intermediate or is readily converted to an intermediate. We also show that a circular form of a nonaccumulating satellite RNA mutant induced an increase in a satellite RNA that is endogenous to some tobacco ringspot virus virion preparations, as demonstrated previously for the linear form.

Base Sequence↗
PubMed1995

Transient gene expression of antisense RNA and coat protein-encoding sequences reduced accumulation of cherry leafroll virus in tobacco protoplasts.

Cherry leafroll nepovirus (CLRV) is the causative agent of blackline disease, which results in a fatal necrosis of the graft union of English walnut scion on certain rootstocks. Tobacco suspension cell protoplasts were electroporated with plasmid constructions, bearing or not bearing RNA-derived sequences, and, subsequently, were electroporated with CLRV virions or virion RNA Replication of CLRV in protoplasts was demonstrated by accumulation of both positive- and negative-sense CLRV genomic RNAs 1 and 2, capsid antigen, and virions. Three plasmids were tested for antiviral action. These have inserts that were derived from the coat protein gene, inserted in both orientations, and from the 3' terminal sequence that is nearly identical in RNA 1 and RNA 2, oriented for expression of antisense RNA. Plasmids were introduced into protoplasts 12 hr prior to introducing the virions or virion RNA. CLRV accumulation was reduced significantly by prior electroporation of plasmids intended to express coat protein or 3' antisense RNA, but not by electroporation of plasmid without insert or plasmid with coat protein encoding sequences in the antisense orientation. These results demonstrate the utility of transient expression in a protoplast system for comparing the efficacy of a variety of virus-derived and other sequences for their potential application in virus control strategies.

Capsid↗
PubMed1995

Long, nearly identical untranslated sequences at the 3' terminal regions of the genomic RNAs of cherry leafroll virus (walnut strain).

Hybridization analyses of cDNA clones derived from the two genomic RNAs, RNA1 and RNA2, of the walnut strain of the nepovirus cherry leafroll nepovirus (wCLRV) demonstrated a long region of high homology between the two viral RNAs. Subsequent mapping and nucleotide sequencing revealed a long, noncoding, presumably untranslated, region (3' UTR) immediately 5' of the terminal polyadenylate, a region that is almost identical in the two RNAs. This 3' UTR is 1567 nucleotide residues long in RNA1. Homologies of about 80% were found with corresponding regions of genomic RNAs from other strains of CLRV, but not with the corresponding regions of other nepovirus genomic RNAs.

Base Sequence↗
PubMed1994

Symptom severity of beet western yellows virus strain ST9 is conferred by the ST9-associated RNA and is not associated with virus release from the phloem.

The ST9 strain of beet western yellows virus (BWYV ST9) is unique among BWYV strains because it encapsidates not only its 5.6-kb genomic RNA but also a 2.8-kb RNA of distinct nucleotide sequence, designated as the ST9-associated RNA. We obtained isolates of BWYV ST9 that are free of the associated RNA by transfecting Nicotiana tabacum protoplasts with transcripts of an ST9 genomic cDNA clone. Aphids were fed on extracts of infected protoplasts and were transferred to young Shepherd's Purse (Capsella bursa-pastoris) plants. When the protoplast inoculum was ST9 genomic transcript or virion RNA of the L-1 strain of BWYV (free of the associated RNA), symptoms were mild and characteristic of BWYV L-1. When ST9-associated RNA was included in the inoculum with genomic RNA of either source, subsequently infected Shepherd's Purse plants showed the severe symptoms that are characteristic of BWYV ST9. Inclusion of ST9-associated RNA in the inoculum with ST9 genomic RNA increased the accumulation of capsid antigen and ST9 genomic RNA, relative to infections initiated with ST9 genomic RNA alone. Using gold-labeled antibody and electron microscopy, we assessed the distribution of virions in Shepherd's Purse plants. Regardless of whether the associated RNA was present, sites showing immunoreactivity above background levels were restricted to the phloem, suggesting that the increased BWYV ST9 titer and symptom severity that are correlated with the presence of the ST9-associated RNA are not due to escape of the infection from phloem limitation.

Animals↗
PubMed1993

Beet western yellows virus-associated RNA: an independently replicating RNA that stimulates virus accumulation.

Infections of plants by subviral RNA agents, alone or in association with virus genomic RNA molecules, are well known. The ST9 strain of beet western yellows virus encapsidates not only the 5.6-kilobase genomic RNA that is typical of luteoviruses, but also a 2.8-kilobase-associated RNA that has a distinct nucleotide sequence. The ST9-associated RNA has been postulated to be a satellite RNA, which by definition would be capable of replicating only in coinfections with beet western yellows virus or closely related viruses. To characterize the associated RNA, we inoculated protoplasts and leaves with in vitro transcripts of the virus genomic RNA and the ST9-associated RNA separately and in combination. Surprisingly, the ST9-associated RNA alone replicated efficiently in both protoplasts and leaves, and it stimulated accumulation of the virus genomic RNA in protoplasts. Thus, the ST9-associated RNA is a newly discovered type of plant infectious agent, which depends on its associated virus, beet western yellows virus, for encapsidation but not for replication.

Base Sequence↗
PubMed1993

Catalytically active geometry in the reversible circularization of 'mini-monomer' RNAs derived from the complementary strand of tobacco ringspot virus satellite RNA.

The less abundant polarity of the satellite RNA of tobacco ringspot virus, designated sTobRV(-)RNA, contains a ribozyme and its substrate. We demonstrate that the ribozyme can catalyze the ligation of substrate cleavage products and that oligoribonucleotides, termed 'mini-monomers' and containing little more than covalently attached ribozyme and substrate cleavage products, circularized spontaneously, efficiently and reversibly. The kinetics of ligation and cleavage of one such mini-monomer was consistent with a simple unimolecular reaction at some temperatures. Evidence suggests that the circular ligation product includes a 5 bp stem that is connected to a 4 bp stem by a bulge loop. Reduction of the bulge loop to one nt is expected to place the 4 and 5 bp helices in a nearly coaxial, rather than an angled or parallel, orientation. Such molecules did not circularize in a unimolecular reaction but did when incubated with second, trans-acting oligoribonucleotides that had either the original or a substituted 4 bp helix. These results suggest that a bulge loop that is too small prevents formation of geometry essential for unimolecular ligation. We suggest the term 'paperclip' to represent the arrangement of RNA strands in the region of sTobRV(-)RNA that participates in the cleavage and ligation reactions.

Base Sequence↗
PubMed1993

Evolution and replication of tobacco ringspot virus satellite RNA mutants.

The replication properties of linker insertion-deletion mutants of tobacco ringspot virus satellite RNA have been studied by amplification in plants infected with the helper virus. Sequence analysis of the cDNAs corresponding to the replicated forms shows that only one of the original mutated molecules replicates unaltered, and in general new variants accumulate. Depending on the location of the original mutation three types of sequence modifications were observed: (i) deletion of the mutated region followed by sequence duplication, (ii) sequence duplication and deletion outside of the mutated region and (iii) limited rearrangements at the site of mutation. The mutant that replicates without sequence changes accumulates linear multimeric forms suggesting that self-cleavage is affected although the sequence alteration does not involve the hammerhead catalytic domain. Alternative RNA conformations are likely to play a role in the origin of this phenotype and in the formation of sequence duplications. These results demonstrate the great structural flexibility of this satellite RNA.

Base Sequence↗
PubMed1993

Similar structure and reactivity of satellite tobacco ringspot virus RNA obtained from infected tissue and by in vitro transcription.

The 359 nucleotide residue (nt) satellite RNA of tobacco ringspot virus increases detectably only in association with replicating tobacco ringspot virus and becomes encapsidated in the virus coat protein. Results from previous reports are consistent with participation of rolling circle transcription in replication of the satellite RNA: (i) both the more abundant plus polarity satellite RNA, s(+)RNA and the complementary s(-)RNA occur in multimeric forms that self-cleave to release the unit length, 359 nt satellite RNA, (ii) circles of both s(+)RNA and s(-)RNA are present in extracts of infected tissue, and (iii) s(-)RNA, but not s(+)RNA, spontaneously and efficiently circularizes in vitro. Our analyses of RNA in tissue extracts suggest that s(+)RNA of all forms is about 100-fold more abundant than s(-)RNA. Nucleic acids were purified rapidly to minimize interconversion of linear and circular forms. For s(+)RNA and for s(-)RNA, the circular and the linear forms were detected in about equal amounts in tissue extracts. The linear s(-)RNA from tissue extracts was found to have the same 5'-terminal sequence as previously was found for s(-)RNA self-cleaved from in vitro transcripts. Like s(-)RNA synthesized in vitro, the circular and linear s(-)RNA from tissue extracts spontaneously and readily interconverted during incubation in vitro. In contrast, the bulk of the circular and linear forms of s(+)RNA were stable. The very limited interconversion of s(+)RNA forms in vitro suggests that circulation in vivo is enzymically catalyzed. Encapsidated satellite RNA was found to be composed of linear, unit length and multimeric forms, including previously undocumented s(-)RNA present in approximately the same relative abundance compared to s(+)RNA as was observed for RNA from tissue extracts. Circles were not detected in encapsidated RNA. We interpret our results in the context of a rolling circle model for satellite RNA replication.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

Cloned DNA copies of cowpea severe mosaic virus genomic RNAs: infectious transcripts and complete nucleotide sequence of RNA 1.

Cowpea severe mosaic virus (CPSMV) is a member of the comovirus group of messenger-sense RNA viruses with bipartite genomes, of which cowpea mosaic virus (CPMV) is the type member. Full-length copies of CPSMV RNA 1 were cloned in plasmids bearing a bacteriophage T7 promoter. Previously, similar clones of CPSMV RNA 2 had been obtained. A 5'-rUAUUAAAAUUUU sequence is common to RNA 1 and RNA 2. From two RNA 1 clones and four RNA 2 clones we excised non-CPSMV sequences so as to provide templates for in vitro transcripts that have only a single guanylate preceding CPSMV RNA sequences. Transcripts from the most active RNA 1 and RNA 2 clones, when mixed, showed about 5% of the infectivity of unfractionated CPSMV RNAs from virions. The longest, 1858 codon open reading frame of the 5957 nt CPSMV RNA 1 extends from an AUG at nt 257 to a UGA termination codon at nt 5831. The calculated molecular weight of the polyprotein is 208,000. Comparisons with the available amino acid residue (aa) sequence information from the complete CPMV RNA 1 sequence and the partial sequence of red clover mottle virus RNA 1 suggest that CPSMV RNA 1 specifies the expected set of five mature proteins: 32K proteinase cofactor, 58K presumed helicase, VPg 5'-linked protein of the genomic RNAs, 24K proteinase, and 87K presumed polymerase, separated by four cleavage sites. Of the determined and deduced cleavage sites of the three RNA 1 polyproteins, only that at the 24K/87K junction has a distinct aa pair in the CPSMV polyprotein. Of the five proteins, VPg and 87K show the greatest similarity between CPSMV and CPMV, with identities of 68 and 55%, respectively. Published mutational analysis of the CPMV 24K proteinase and alignment of aa sequences from three comoviruses suggest that cysteine-168, histidine-40 and glutamic acid-77 form the catalytic triad of the CPSMV 24K proteinase. Results are discussed in the context of the resistance that some cowpea (Vigna unguiculata) lines exhibit against CPMV but not against CPSMV.

Amino Acid Sequence↗