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Biomedical subjects

R N Beachy

Publications and source records attributed to R N Beachy.

At least 91 records · Page 5Linked to original sources
PubMed1994

In vitro characterization of a cassette to accumulate multiple proteins through synthesis of a self-processing polypeptide.

The strategy for processing the polyprotein encoded by plant potyviruses has been mimicked by constructing an expression cassette based on the nuclear inclusion (Nla) proteinase from tobacco etch virus (TEV). This cassette (pPR01), includes the TEV Nla coding region flanked on each side by its heptapeptide cleavage sequence and cloning sites for the in frame insertion of two different open reading frames. pPR01 allows the synthesis, under the control of a single transcriptional promoter, of two proteins in equimolar amounts as part of a polyprotein which is cleaved into individual mature products by the TEV protease. In in vitro reactions the cassette functioned as expected when several different protein-coding sequences were used. The potential uses of pPR01 are discussed.

Amino Acid Sequence↗
PubMed1994

Nucleotide sequence and genome characterization of rice yellow mottle virus RNA.

The genome of rice yellow mottle virus (RYMV) is a single-stranded positive-sense RNA that is not polyadenylated, and has an M(r) of 1.4 x 10(6). We present here the 4550 nucleotide (nt) sequence of RYMV RNA, and its predicted genomic organization. The RYMV genomic RNA contains four open reading frames (ORFs). The first (nt 80 to 553) encodes a protein containing 157 amino acids with a predicted M(r) of 17.8K. No function has yet been attributed to this product. ORF2 (nt 608 to 3607) encodes a polyprotein of 999 amino acids, with a predicted M(r) of 110.7K. The first 134 amino acids of ORF2 are predicted to be the genome-linked protein, VPg, followed by the viral protease, the helicase and the RNA-dependent RNA polymerase. ORF3 is within the boundaries of ORF2 and is predicted to encode a polypeptide with 126 amino acids and an M(r) of 13.7K. No function has yet been attributed to this protein. ORF4 (nt 3447 to 4166), which overlaps the 3' terminus of ORF2, encodes a 26K protein. This polypeptide has been identified as the RYMV coat protein. The data presented here confirm that RYMV belongs to the sobemovirus group and thus is a member of the picorna-like family of plant viruses.

Amino Acid Sequence↗
PubMed1994

Complete nucleotide sequence of the geminivirus tomato yellow leaf curl virus, Thailand isolate.

The complete genome of a Thailand isolate of the geminivirus tomato yellow leaf curl virus (TYLCV-Th) has been cloned and the nucleotide sequence determined. The genome consists of two DNAs each slightly greater than 2700 nucleotides in length and designated A-DNA and B-DNA. The A-DNA contains six open reading frames (ORFs) capable of encoding proteins with M(r)s greater than 10K; two ORFs were located on the B-DNA. The predicted ORFs were found at positions on the genome similar to those identified in other geminiviruses. Based upon computer-assisted sequence comparisons with other geminiviruses, TYLCV-Th maintained the greatest degree of similarity with a tomato pathogen isolated in Australia, tomato leaf curl virus. Furthermore, sequence comparisons with a large number of geminiviruses confirm further divisions of the geminiviruses based upon geographical proximity of the viruses. Agroinoculation of Nicotiana benthamiana with a TYLCV-Th A-DNA clone containing a mutation in the capsid protein ORF demonstrated that the coat protein is not essential for TYLCV-Th infection but enhances the progression of the disease.

Base Sequence↗
PubMed1993

Nucleotide sequence and genomic organization of rice tungro spherical virus.

Rice tungro disease is caused by a combination of two viruses: rice tungro spherical virus (RTSV) and rice tungro bacilliform virus (Jones et al. (1991) J. Gen. Virol. 72, 757-761.). The genome of RTSV is a single-stranded polyadenylated RNA. We present here the 12,433-nucleotide complete sequence of RTSV genomic RNA and its deduced coding regions. This sequence contains a large open reading frame (ORF) which initiates following a 514-nucleotide 5' leader sequence and is capable of encoding a viral polyprotein of 390.3 kDa. Two viral subgenomic RNAs of ca. 1.2 and 1.4 kb, respectively, were detected in RTSV-infected leaf tissues and mapped by S1 nuclease protection assay. These RNAs were determined to be congruent with the genomic RNA sequence proximal to the 3' terminus and could contain up to two small ORFs in their 5' to 3' orientation. There are at least three capsid protein subunit cistrons near the N-terminus of the large ORF. A computer-aided search of the C-terminal half of the large ORF revealed conserved protein sequence motifs for a viral RNA polymerase, proteinase, and a putative NTP-binding protein. These sequence motifs are arranged in a manner that resembles those of picorna-like viruses. Taken together, these data indicate that RTSV is a distinct type of positive-strand RNA virus. The evolutionary relationships between RTSV and other picorna-like plant viruses are discussed.

Amino Acid Sequence↗
PubMed1993

Upstream regulatory sequences from two beta-conglycinin genes.

Genes encoding the beta-conglycinin seed storage proteins of soybean are expressed only in seeds during specific stages of development. The different subunits of beta-conglycinin, alpha', alpha and beta, are encoded by distinct members of a gene family. Yet there are marked differences in the regulation of the genes encoding the alpha'/alpha and beta subunits. Previous work (Chen et al., EMBO J 7: 297-302, 1988) identified a seed specific transcriptional enhancer upstream of a gene encoding the alpha' subunit. Mutations were made within this region to discern its functional components. Among those identified is a 62 bp region (between -77 and -140) that contains a vicilin box consensus sequence as well as a sequence that binds the soybean nuclear factor SEF4 in vitro. A second region, which contains a sequence homologous to the core of the legumin box consensus (i.e., CATGCAT-like or RY repeat element) at -246, was also shown to affect the activity of this enhancer in transgenic plants. A series of 5' terminal deletions were used to identify regulatory elements upstream of the beta subunit gene. Two regions were identified (from -553 to -442 and from -308 to -72) that, when deleted, led to a marked reduction in gene expression. Both of these elements contain sequences that bind SEF4 in vitro. The distal element also contains an AT-rich segment that recognizes a second nuclear factor, SEF1, in vitro. Neither of these elements contains any homology to the vicilin box consensus.

Antigens, Plant↗
PubMed1993

Geminivirus nomenclature: the need to set taxonomic standards.

The geminiviruses are plant viruses of significant economic impact. Different nomenclature has been used in various studies in descriptions of their genomes. We propose in this communication a uniform nomenclature to be used in future studies.

Plant Viruses↗
PubMed1993

Specificity of a promoter from the rice tungro bacilliform virus for expression in phloem tissues.

The major promoter region for the transcription of the genome of rice tungro bacilliform virus (RTBV), a newly described badnavirus, has been identified. Fragments of the RTBV genome upstream of the site of transcription initiation were isolated and tested for promoter activity using a beta-glucuronidase receptor gene (gusA). Assays of transient gusA expression were performed following introduction of the chimeric gene into protoplasts via electroporation. The chimeric RTBV-promoter: gusA gene was more active in rice protoplasts than in maize or tobacco protoplasts, but was weaker than gusA controlled by an enhanced 35S promoter from cauliflower mosaic virus. Analysis of gusA gene expression following introduction of chimeric reporter genes into intact leaves via micro-projectile bombardment indicated that the GUS activity is present primarily in vascular tissues. Transgenic rice plants carrying the chimeric gusA gene had GUS activity only in the phloem of the vascular bundles in the leaf. Tissue printing studies demonstrated that RTBV accumulates in the vascular bundles of infected rice leaves. The results of our study indicate that phloem-specific expression from the RTBV promoter is an intrinsic property of the viral promoter.

Base Sequence↗
PubMed1993

Analysis of a tobacco mosaic virus strain capable of overcoming N gene-mediated resistance.

The genome of Ob, a tobamovirus that overcomes the N gene-mediated hypersensitive response (HR), was cloned as a cDNA, and its nucleotide sequence was determined. The genomic organization of Ob is similar to that of other tobamoviruses, consisting of 6506 nucleotides and containing at least four open reading frames. These open reading frames encode a 126-kD polypeptide with a 183-kD readthrough product, a 30.6-kD movement protein, and an 18-kD coat protein. A bacteriophage T7 promoter sequence was fused to the full-length cDNA clone to obtain infectious RNA transcripts. These transcripts, when inoculated onto tobacco plants, induced disease symptoms indistinguishable from plants inoculated with Ob viral RNA. To determine which viral factor is responsible for the resistance-breaking character of Ob, a recombinant virus was constructed in which the movement protein gene of tobacco mosaic virus was replaced with that of Ob. Cultivar Xanthi NN tobacco plants infected with this virus responded with an HR, indicating that the Ob movement protein alone does not act to overcome the N gene-mediated response. Following mutagenesis of the infectious Ob cDNA clone with hydroxylamine, populations of transcripts from the mutagenized DNA were inoculated onto Xanthi NN tobacco, and a variant that induced the HR was identified. The mutant was analyzed and found to contain a single nucleotide change in the 126-kD gene. Recreating the mutation in the Ob cDNA clone by site-directed mutagenesis resulted in a virus that caused symptoms identical to the chemically induced mutant.

Amino Acid Sequence↗
PubMed1993

Genetically engineered protection against viruses in transgenic plants.

Transgenic plants carrying nucleotide sequences derived from plant viruses can exhibit increased resistance to viral disease. Many viral sequences confer some level of either resistance to infection or suppression of disease symptoms (tolerance). These include segments of viral genomes encoding capsid or coat proteins, sequences encoding proteins that are or may be subunits of the viral replicase, sequences incapable of encoding proteins, entire genomes of defective interfering viruses and satellite viruses, and complete genomes of mild strains of virus. The transgene may act on initiation of infection, replication of virus, spread of the infection throughout the plant, and symptom development. More than one of these processes can be impaired by a single transgene derived from a single viral gene. The level of protection ranges from very low to high, while the breadth of protection ranges from very narrow, where protection is only observed against closely related strains of the virus from which the transgene was derived, to moderately broad, extending to other viruses. Data are insufficient to establish a molecular mechanism of resistance for most of the described examples. In addition, although the use of a particular segment of the viral genome confers resistance in one virus-host system, analogous sequences from a different virus in another host may be ineffective.

Genetic Engineering↗
PubMed1992

Seed-specific repression of GUS activity in tobacco plants by antisense RNA.

beta-Conglycinin, the 7S storage protein of soybean, is expressed only in seeds and is regulated predominantly by gene transcription [5]. We applied an antisense strategy to modify expression of a beta-glucuronidase (uidA or gusA) gene in seeds using a promoter from a beta-conglycinin gene. Transgenic tobacco plants harboring the gusA gene under the control of the CaMV 35S promoter were retransformed with a gene construct comprising the beta-conglycinin promoter fused to the gusA gene in the antisense orientation. Double transformants were regenerated and transformation was confirmed by Southern blot hybridization. Seed-specific repression of GUS activity was observed in lines containing high copy numbers of the antisense gusA transgene. Suppression of GUS activity was correlated with the amounts of (-) sense gusA transcript detected and concomitantly with a decrease in gusA transcript levels. Furthermore, the amount of suppression of GUS activity was greatest during mid to late stages of seed development, when expression of the alpha' promoter is high. These results indicate that suppression of GUS activity is due to expression of the antisense gene.

Antigens, Plant↗
PubMed1992

Effects of terminal deletion mutations on function of the movement protein of tobacco mosaic virus.

A series of carboxy- and amino-terminal deletion mutations in the movement protein (MP) gene of tobacco mosaic virus (TMV) were ligated into a cloned TMV cDNA deleted for the endogenous MP gene. RNA transcripts were produced in vitro from clones carrying the various mutated MP genes. The effect of the deletion mutations on local and systemic movements of the infection was evaluated. Deletion of 9 or 33 amino acids from the carboxy terminus of the movement protein did not effect cell-to-cell movement as reflected by local lesion formation on Nicotiana tabacum cv. Xanthi NN plants. Deletion of 55 amino acids resulted in impaired MP that supported the formation of local lesions of 1 mm in diameter compared to lesions of 3-5 mm caused by the wild-type MP. Deletion of 74 amino acids (or more) from the carboxy terminus resulted in a protein that could not support virus movement. Modified viruses that contained repeated sequences in the 3' region of the MP gene lost the repeated sequences during replication and reverted to the wild type. This was evidenced by the size of the MP produced and by sequence analysis of reverse-transcribed PCR-amplified products, following infection by the modified virus. MP deleted for as few as 3 amino acids at the amino terminus could not support virus movement thus indicating that the amino-terminal domain is critical for MP activity.

Cell Wall↗
PubMed1992

Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants.

Expression of the tobacco mosaic virus 30-kD movement protein (TMV MP) gene in tobacco plants increases the plasmodesmatal size exclusion limit (SEL) 10-fold between mesophyll cells in mature leaves. In the present study, we examined the structure of plasmodesmata as a function of leaf development. In young leaves of 30-kD TMV MP transgenic (line 274) and vector control (line 306) plants, almost all plasmodesmata were primary in nature. In both plant lines, secondary plasmodesmata were formed, in a basipetal pattern, as the leaves underwent expansion growth. Ultrastructural and immunolabeling studies demonstrated that in line 274 the TMV MP accumulated predominantly in secondary plasmodesmata of nonvascular tissues and was associated with a filamentous material. A developmental progression was detected in terms of the presence of TMV MP; all secondary plasmodesmata in the tip of the fourth leaf contained TMV MP in association with the filamentous material. Dye-coupling experiments demonstrated that the TMV MP-induced increase in plasmodesmatal SEL could be routinely detected in the tip of the fourth leaf, but was restricted to mesophyll and bundle sheath cells. These findings are discussed with respect to the structure and function of plasmodesmata, particularly those aspects related to virus movement.

Cytoplasm↗
PubMed1991

Conserved amino acid sequences among plant proteins sorted to protein bodies and plant vacuoles. Can they play a role in protein sorting?

Amino acid sequence comparisons were made between the soybean alpha subunit of beta-conglycinin and 34 members of different plant protein families targeted to seed protein bodies or vacuoles. A number of short conserved amino acid sequences were identified in seed storage proteins, plant protease inhibitors and lectins, and the probable functions of these sequences are discussed. For proteins of known tertiary structure, these sequences map to the surface of the respective molecules. It is postulated that these regions produce a common secondary structure which could interact with other molecules involved in the sorting process. One of these regions, region A, is similar to the yeast carboxypeptidase Y (CPY) vacuolar targeting signal, and is present in both storage proteins and lectins. Computer modeling based upon the tertiary structure of concanavalin A (ConA) was used to generate models representing the structure of two highly related lectins from Dolichos biflorus, one of which is targeted to protein bodies and the other secreted. A different glycosylation pattern together with amino acid sequences upstream of the identified conserved amino acid sequences are predicted to modulate the presentation of the sorting domains in the lectins and be the determinant in the sorting of these lectins.

Amino Acid Sequence↗
PubMed1991

Multiple nuclear factors interact with upstream sequences of differentially regulated beta-conglycinin genes.

The expression of the alpha' and beta subunit genes of beta-conglycinin is differentially regulated during soybean embryo development. Although both are expressed solely in developing seeds during mid to late stages of embryo development, the alpha' subunit is expressed more highly on a per gene basis, and alpha' subunit mRNA begins to accumulate three to five days earlier than beta subunit mRNA. In cultured cotyledons, beta subunit gene(s) respond to changes in methionine or abscisic acid levels, whereas expression of the alpha' subunit gene(s) is unaffected by these changes. To investigate the mechanisms by which these genes are transcriptionally regulated, we examined the interactions of nuclear proteins with upstream sequences from the alpha' and beta subunit genes. Four distinct DNA binding factors were identified in nuclear extracts from developing soybean seeds. These factors are termed Soybean Embryo Factors (SEF) 1 through 4. SEF binding sites are distributed non-uniformly between the alpha' and beta subunit genes, and the amount of protein binding is modulated over the course of embryo development. DNA footprinting revealed the sequences recognized by three of these factors. Factors which behave in a manner similar to that of SEF3 were also identified in nuclear extracts from developing tobacco and sunflower seeds.

Antigens, Plant↗
PubMed1991

Transient gene expression in cassava using high-velocity microprojectiles.

The bacterial gene encoding beta-glucuronidase (GUS) was transiently expressed in cassava leaves following the introduction of the gene by microparticle bombardment. The DNA expression vector used to introduce the reporter gene is a pUC 19 derivative and consisted of a CaMV 35S promoter (P35S), the GUS coding region and 7S polyadenylation region. Several other promoters and regulating sequences were tested for efficiency in cassava leaves. Two derivatives of the P35S, one including a partial duplication of the upstream region of the P35S and the other containing a tetramer of the octopine synthase enhancer, were found to be expressed at three times the level of the P35S in cassava leaves. The ubiquitin 1 promoter from Arabidopsis thaliana was expressed at the same level as the P35S. No influence on the level of expression was observed when different 3' ends were used. The biolistic transient gene expression system in cassava leaves allows rapid analysis of gene constructs and can serve as a preliminary screen for chimeric gene function in the construction of transgenic cassava plants.

Cloning, Molecular↗
PubMed1991

Altered function of the tobacco mosaic virus movement protein in a hypersensitive host.

The N gene in Nicotiana sp. confers hypersensitive resistance to all strains of tobacco mosaic virus (TMV) and limits the rate of virus spread in infected leaves. To examine the role of the movement protein (MP) of TMV in the hypersensitive reaction (HR), transgenic Nicotiana tabacum cv. Xanthi-nc (genotype NN) plants that express the MP gene were produced and the molecular size exclusion limit of plasmodesmata in leaf mesophyll cells was monitored. At the HR-permissive temperature (24 degrees) movement from cell to cell of fluorescein isothiocyanate-labeled dextran of molecular mass 3.9 kDa was detected while 9.4-kDa molecules failed to move. At the HR-nonpermissive temperature (33 degrees) the 9.4-kDa probe moved readily from cell to cell. In contrast, in transgenic Xanthi (genotype nn) which express the MP gene the 9.4-kDa probe moved from cell to cell at 24 and 33 degrees. These results suggest that the N gene may modify the ability of the MP to alter plasmodesmata molecular exclusion limits, although expression of the TMV-MP gene alone did not induce the HR. Furthermore, when MP(+) Xanthi-nc tobacco lines were inoculated with a TMV that lacked a MP gene the HR was induced, and the concentration of MP in the transgenic lines was correlated with the degree of the HR.

Capsid Proteins↗