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Biomedical subjects

R N Beachy

Publications and source records attributed to R N Beachy.

At least 73 records · Page 4Linked to original sources
PubMed1996

The complete nucleotide sequence of yam mosaic virus (Ivory Coast isolate) genomic RNA.

The complete nucleotidic sequence of the yam mosaic virus (YMV) RNA was determined following the cloning of partial segments of the genome by reverse transcription and polymerase chain reactions (RT-PCR) using degenerate and/or specific oligonucleotide primers. YMV genomic RNA is 9,608 nucleotides in length and contains one open reading frame (ORF) encoding a polyprotein of 3,103 amino acids (aa) with a calculated Mr of 350,915. The 5' leader sequence of YMV RNA preceding the ORF is 134 nucleotides (nt) long while the 3' untranslated region (UTR) is 165 nt excluding the poly(A) tail. A computer algorithm predicted that the 3'UTR forms four stem loop structures which form a cloverleaf-like secondary structure. These structures apparently share some homologies with those observed in the 3'UTR of the potato virus Y-NL1 strain. Seven potential recognition sites for the NIa protease were found: one putative cleavage site for the P1 proteinase and one for the HC proteinase. The organization of the YMV genome is therefore similar to the other members of the genus Potyvirus based upon conserved sequence motifs common amongst members of this group. Despite its similarity with the other potyviruses in these conserved regions, YMV appears to be a distinct potyvirus species based upon a comparison of its sequence with those of other potyviruses.

Amino Acid Sequence↗
PubMed1996

Plant virus movement protein dynamics probed with a GFP-protein fusion.

A genetic fusion between the gene encoding green fluorescent protein (GFP) from the jellyfish Aequorea victoria, with that of the Ob-tobamovirus movement protein (MP) resulted in the expression of a fluorescent fusion protein (MP::GFP) that was fully biologically active in mediating the cell-to-cell spread of the Ob-virus. The MP::GFP fusion was used to follow in planta the subcellular trafficking of MP. GFP-tagged MP was transiently expressed and found to be associated with several subcellular compartments and structures including trans-wall structures, presumably plasmodesmata, and filament structures. The MP::GFP fusion can be used to monitor MP association with host proteins and structures, and for the isolation of interacting host components.

Animals↗
PubMed1996

Regeneration of transgenic cassava plants (Manihot esculenta Crantz) from microbombarded embryogenic suspension cultures.

A protocol was established for the introduction of DNA into embryogenic suspension-derived tissues of cassava via microparticle bombardment, for the selection of genetically transformed cells, and for the regeneration of fully transgenic plants from these cells. The plasmid DNA used for bombardment contained a gene encoding neomycin phosphotransferase (nptII) and a gene encoding beta-glucuronidase (uidA). Selection of bombarded tissue with paromomycin resulted in the establishment of putative transgenic embryogenic calli. In most of these calli, beta-glucuronidase was detected histochemically. Molecular analysis of paromomycin-resistant embryogenic calli and of plants regenerated from these calli, confirmed the stable integration of bombarded DNA into the cassava genome.

Biolistics↗
PubMed1996

Distribution of tobamovirus movement protein in infected cells and implications for cell-to-cell spread of infection.

The intercellular and intracellular distribution of the movement protein (MP) of the Ob tobamovirus was examined in infected leaf tissues using an infectious clone of Ob in which the MP gene was translationally fused to the gene encoding the green fluorescent protein (GFP) of Aequorea victoria. In leaves of Nicotiana tabacum and N. benthamiana, the modified virus caused fluorescent infection sites that were visible as expanding rings. Microscopy of epidermal cells revealed subcellular patterns of accumulation of the MP:GFP fusion protein which differed depending upon the radial position of the cells within the fluorescent ring. Punctate, highly localized fluorescence was associated with cell walls of all of the epidermal cells within the infection site, and apparently represents association of the fusion protein with plasmodesmata; furthermore, fluorescence was retained in cell walls purified from infected leaves. Within the brightest region of the fluorescent ring, the MP:GFP was observed in irregularly shaped inclusions in the cortical regions of infected cells. Fluorescent filamentous structures presumed to represent association of MP:GFP with microtubules were observed, but were distributed differently within the infection sites on the two hosts. Within cells containing filaments, a number of fluorescent bodies, some apparently streaming in cytoplasmic strands, were also observed. The significance of these observations is discussed in relation to MP accumulation, targeting to plasmodesmata, and degradation.

Biological Transport↗
PubMed1995

Interaction of tobamovirus movement proteins with the plant cytoskeleton.

The movement protein of tobacco mosaic tobamovirus and related viruses is essential for the cell-to-cell spread of infection and, in part, determines the host range of the virus. Movement protein (MP) was fused with the jellyfish green fluorescent protein (GFP), and a modified virus that contained this MP:GFP fusion protein retained infectivity. In protoplasts and leaf tissues, the MP:GFP fusion protein was detected as long filaments shortly after infection. Double-labeling fluorescence microscopy suggests that the MP interacts and coaligns with microtubules. The distribution of the MP is disrupted by treatments that disrupt microtubules, but not by cytochalasin B, which disrupts filamentous F-actin. Microtubules may target the MP to plasmodesmata, the intercellular channels that connect adjacent cells.

Biological Transport↗
PubMed1995

Inhibition of a plant virus infection by analogs of melittin.

An approach that enables identification of specific synthetic peptide inhibitors of plant viral infection is reported. Synthetic analogs of melittin that have sequence and structural similarities to an essential domain of tobacco mosaic virus coat protein were found to possess highly specific antiviral activity. This approach involves modification of residues located at positions analogous to those that are critical for virus assembly. The degree of inhibition found correlates well with sequence similarities between the viral capsid protein and the melittin analogs studied as well as with the induced conformational changes that result upon interaction of the peptides and ribonucleic acid.

Amino Acid Sequence↗
PubMed1995

Studies of coat protein-mediated resistance to TMV. I. The PM2 assembly defective mutant confers resistance to TMV.

Tobacco mosaic virus mutant PM2 contains two amino acid changes in coat protein sequence relative to the sequence of the coat protein of TMV U1. This results in unstable infectivity, inability to cause normal systemic infection, and accumulation of elongated open helixes of coat protein. Using site-directed mutagenesis we demonstrated that the characteristics of PM2 are due to the change of Thr28-->Ile, while the second change, Glu95-->Asp, had no apparent effect on virion structure or infectivity. Transgenic Nicotiana tabacum cv Xanthi NN and Xanthi nn plants that accumulate coat protein that contains one or both of the amino acid changes are as resistant to TMV infection as transgenic plants that contain wild-type TMV coat protein. The implication of these results on a model for coat protein-mediated resistance that involves the interaction of transgenic coat protein with the challenge virus is discussed.

Amino Acids↗
PubMed1995

Analysis of the proteolytic processing and activation of the rice tungro bacilliform virus reverse transcriptase.

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and member of the badnavirus subgroup. Open reading frame (ORF) 3 encodes the viral capsid protein, protease (PR), and reverse transcriptase (RT). A DNA fragment of ORF 3 that contains PR and RT sequences was previously expressed in insect cells to produce the PR/RT polyprotein that was processed to yield p62 and p55. p62 and p55 share common N-terminal amino acid sequences and exhibit reverse transcriptase activity. Mass spectrometry was employed to determine the precise molecular weight of the p62 and p55 proteins and enabled determination of the C-termini for both proteins. ORFs encoding either p62 or p55 were constructed and expressed in insect cells using the baculoviruses 62R-BBac and 55R-BBac, respectively. The recombinant p62R and p55R proteins were purified separately and shown to have the same enzymatic activities as previously reported for the processed p62 and p55. The putative active site of the PR was mutated (mpr), and the resulting mpr/RT ORF was expressed in insect cells using the baculovirus mpr/RT-BBac. The mpr/RT polyprotein was not processed in insect cells, resulting in the accumulation of the approximately 87-kDa mpr/RT polyprotein. This study further extends the understanding of p62 and p55 and clarifies the role of the RTBV PR in processing of the RT.

Animals↗
PubMed1995

A defective movement protein of TMV in transgenic plants confers resistance to multiple viruses whereas the functional analog increases susceptibility.

Transgenic tobacco plants that express a gene encoding a defective mutant of the tobacco mosaic virus (TMV) movement protein which are known to be resistant to several tobamoviruses were inoculated with viruses from different taxonomic groups to determine the breadth of resistance. There were significant delays in the time of appearance of disease symptoms and/or there was reduced systemic accumulation of virus in upper leaves of plants inoculated with tobacco rattle tobravirus, tobacco ringspot nepovirus, alfalfa mosaic alfamovirus, peanut chlorotic streak caulimovirus, and cucumber mosaic cucumovirus. Conversely, tobacco plants that express a gene encoding the functional tobacco mosaic virus wild-type movement protein accelerated symptom development, enhanced the severity of symptom formation, and/or increased the accumulation of these viruses and, additionally, TMV. Our results indicate that there are similar functions among the movement proteins of a number of plant viruses despite the apparent lack of sequence similarity between them.

Mosaic Viruses↗
PubMed1995

Plant virus expressing hybrid coat protein with added murine epitope elicits autoantibody response.

A modified tobacco mosaic virus (TMV) was used to direct the synthesis of TMV coat protein hybrids containing a 13 amino acid sequence of the murine zona pellucida ZP3 protein. This hybrid protein was found to be robust and accumulate to high concentrations in inoculated plants. Virus-like particles containing the hybrid coat protein could be readily purified from infected plant tissue. Parenteral immunization with these virus-like particles as antigen resulted in serum antibody recognizing ZP3 epitope in C57BL/6J and BALB/cBy mice. The antibodies recognized synthetic ZP3 peptide and the authentic murine ZP3 glycoprotein. Antibodies were recruited in vivo to the zone pellucida in female mice.

Animals↗
PubMed1995

The regulatory regions of the rice tungro bacilliform virus promoter and interacting nuclear factors in rice (Oryza sativa L.).

The Rice Tungro Bacilliform Virus (RTBV) promoter confers phloem-specific gene expression in transgenic rice plants. A series of promoter deletion mutants were fused with the Escherichia coli beta-glucuronidase A (uidA) reporter gene and introduced into transgenic rice plants. The RTBV promoter confers substantially stronger expression in shoots than in roots. A fragment of the promoter comprising nucleotides -164 to +45 relative to the transcriptional start site contains sufficient information for phloem-specific gene expression. Within this region, nucleotides -164 to -43 were essential for promoter function since deletion of this fragment dramatically reduced promoter activity. Gel-retardation assays identified two groups of rice nuclear factors (RNFG1 and RNFG2) that bind to the -164 to +45 promoter fragment. Competition and DNasel footprinting experiments indicated that RNFG1 bound to nucleotides -3 to +8 (Box I) while RNFG2 bound to nucleotides -53 to -39 (Box II). Interactions between the two groups of factors were observed. In addition, we found differences in the binding of nuclear factors from shoots versus from roots, in agreement with the different activities of the promoter in these two organs. It is proposed that binding of RNFG1 and RNFG2 between nucleotides -164 to +45 is essential for the tissue-specific expression of this promoter.

Base Sequence↗
PubMed1995

Tomato leaf curl geminivirus from India has a bipartite genome and coat protein is not essential for infectivity.

Genomes of two isolates of tomato leaf curl geminivirus from India (ToLCV-India) have been sequenced. ToLCV-India contains A and B components, both of which are required for systemic movement and symptom development. The two isolates have 94% sequence identify but one isolate gave mild symptoms in Nicotiana benthamiana and tomato. The genome organization of ToLCV-India is similar to other whitefly-transmitted geminiviruses (WTGs) with bipartite genomes. However, it contains an additional ORF, AV3, that has not been reported for other WTGs. Its coat protein (CP) sequence is highly homologous to that of Indian cassava mosaic virus (90%). Two mutations that truncated the CP after amino acids 65 or 172 did not affect systemic movement and symptom development in either N. benthamiana or tomato. However, the symptoms caused by mutant viruses were different from those in plants infected with unmodified viruses, and plants infected with the mutants had markedly reduced amounts of single-stranded viral DNA. Comparison of sequences and other biological features of ToLCV-India with other geminiviruses showed that ToLCV-India is a distinct virus and is related to the WTGs from the Old World. It is similar to African cassava mosaic virus in its requirement for B component and dispensability of coat protein for symptom development, unlike other geminiviruses that infect tomato in the Old World. It is proposed that ToLCV-India evolved more recently as compared to other geminiviruses that infect tomato in the Old World.

Base Sequence↗
PubMed1995

Studies of coat protein-mediated resistance to tobacco mosaic virus (TMV). II. Challenge by a mutant with altered virion surface does not overcome resistance conferred by TMV coat protein.

Transgenic tobacco plants expressing the coat protein (CP) gene of the U1 strain of tobacco mosaic virus (TMV) exhibit CP-mediated resistance (CP-MR) against some, but not all, tobamoviruses. To investigate the role of the amino acid sequences on the surface of the challenge virus in CP-MR, mutant strains of U1 TMV were constructed to contain the amino or carboxy termini of the CP of Sunn hemp mosaic tobamovirus (SHMV). The modified virus was unable to overcome CP-MR in transgenic plants that contained the TMV CP. In contrast, TMV in which the CP was replaced by the SHMV CP overcame CP-MR to the same extent as did SHMV. We conclude that CP-MR conferred by TMV CP involves interactions between amino acid sequences of the challenge viruses and the transgene protein other than those on the surface of the challenge virus.

Amino Acid Sequence↗
PubMed1995

Classification and identification of geminiviruses using sequence comparisons.

The genomes and ORFs of 36 geminiviruses were compared to obtain phylogenetic trees and frequency distributions of all possible pairwise comparisons with an objective to classify geminiviruses. Such comparisons show that geminiviruses form two distinct clusters of leafhopper-transmitted viruses that infect monocots (subgroup I) and whitefly-transmitted viruses that infect dicots (subgroup III), irrespective of the part of the genome considered. Of the two leafhopper-transmitted viruses that infect dicots, tobacco yellow dwarf virus has a sequence most similar to subgroup I viruses, and that of beet curly top virus differed depending upon the ORF considered. The distributions of identities within subgroups are significantly different suggesting that the taxonomic status of a particular isolate within a subgroup can be quantified. All the recognized strains of any one virus have greater than 90% sequence identity. It was observed that the 200 nucleotide intercistronic regions of geminiviruses are more variable than the remainder of the genome. The amino acid sequences of the coat protein (CP) of subgroup III viruses are more conserved than the remainder of the genome. However, a short N-terminal region (60-70 amino acids) of the CP is more variable than the rest of the CP sequence and is a close representation of the genome. PCR primers based on conserved sequences can be used to clone and sequence the N-terminal sequences of the CP of the geminiviruses; this sequence is sufficient to classify a virus isolate. A possible taxonomic structure for geminiviruses is proposed after considering the sequence comparisons and biological properties.

Amino Acid Sequence↗
PubMed1994

Influence of heterologous tobamovirus movement protein and chimeric-movement protein genes on cell-to-cell and long-distance movement.

Sunn-hemp mosaic tobamovirus (SHMV) moves slowly from cell to cell in Nicotiana tabacum cv. Xanthi, but fails to move long distance. To determine the role of the SHMV movement protein (MP) in cell-to-cell and long-distance movement in tobacco, the SHMV MP gene was inserted into a TMV-cDNA clone that had approximately the 5'-half of the endogenous MP gene deleted. RNA transcripts inoculated onto tobacco induced systemic infections by 8 days postinoculation. Sequence analysis of the MP genes from purified virus isolated from systemically infected leaf tissue indicated that chimeric SHMV/TMV MP genes had been generated through RNA-RNA recombination within the 3'-termini of the MP gene sequences. When exchanged for the MP gene of TMV, three of four chimeric MP genes analyzed provided long-distance movement function for the hybrid viruses in tobacco. Two of the three hybrid viruses that moved long distance showed enhanced cell-to-cell movement relative to a recombinant TMV that expressed the intact SHMV MP gene. These observations suggest that the C-terminus of the TMV MP contains a determinant that can influence cell-to-cell movement in tobacco. A recombinant virus, TLSM, that expressed the intact SHMV MP gene exhibited cell-to-cell movement that was intermediate to SHMV and TMV, but failed to produce coat protein and was defective in long-distance movement. To further examine the role of the SHMV MP gene in long-distance movement, transgenic N. tabacum cv. Xanthi that expressed the wild-type SHMV MP gene were generated and found to facilitate rapid and efficient long-distance movement of a TMV mutant that contained a dysfunctional MP gene. Therefore, the inability of SHMV to systemically infect tobacco is a function of virus components and sequences other than those encoded by the SHMV MP gene.

Base Sequence↗
PubMed1994

Rice tungro bacilliform virus encodes reverse transcriptase, DNA polymerase, and ribonuclease H activities.

Rice tungro bacilliform virus (RTBV) is a newly described badnavirus and proposed member of the plant pararetrovirus group. RTBV open reading frame 3 is predicted to encode a capsid protein, protease (PR), and reverse transcriptase (RT) and has the capacity to encode other proteins of as yet unknown function. To study the possible enzymatic activities encoded by open reading frame 3, a DNA fragment containing the putative PR and RT domains was used to construct the recombinant baculovirus PR/RT-BBac. Trichoplusia ni insect cells infected with PR/RT-BBac were used in pulse-labeling experiments and demonstrated synthesis of an 87-kDa polyprotein that corresponds in molecular mass to that predicted from the PR/RT DNA coding sequence. The 87-kDa polyprotein was processed with concomitant accumulation of 62-kDa (p62) and 55-kDa (p55) proteins. Amino-terminal sequencing of p62 and p55 determined that they mapped to the PR/RT domain and shared common amino termini. p62 and p55 were purified and exhibited both RT and DNA polymerase activities using synthetic primer/template substrates. Only p55 had detectable ribonuclease H activity, an activity intrinsic to all reverse transcriptases studied to date. Characterization of the RTBV RT provides a biochemical basis for classifying RTBV as a pararetrovirus and will lead to further studies of these proteins and their role in virus replication.

Amino Acid Sequence↗
PubMed1994

Tissue-specific and temporal regulation of a beta-conglycinin gene: roles of the RY repeat and other cis-acting elements.

Upstream regulatory sequences (URS) of the gene that encodes the alpha' subunit of beta-conglycinin, the 7S soybean seed storage protein, includes two RY repeat elements. The role of RY elements and sequences that bind soybean embryo factors 3 and 4 (SEF3 and SEF4; Allen et al., Plant Cell 1 (1989) 623-631) in regulating expression of the promoter was studied following site directed mutagenesis. Specific mutations introduced into these sequences abolished the in vitro binding activities of SEF3 and SEF4. The biological activities resulting from the mutations were determined in transgenic plants using two chimeric promoters comprising sequences from the CaMV 35S promoter and the alpha' subunit promoter. The uidA reporter gene was used to assess the levels of gene expression in transgenic plants. The mutations in the RY element and SEF3 and SEF4 binding sites had little effect on expression of the alpha' promoter. By contrast, mutations in the RY element had significant effect on gene expression when the URS from the alpha' promoter was ligated upstream of the core 35S promoter. Mutations in the RY element abolished the seed specific enhancing activity of the alpha' URS and caused expression of the chimeric promoter in leaves. These results indicate that the RY element plays a key role in seed-specific gene regulation in coordination with other cis-acting elements.

Antigens, Plant↗