Biomedical subjects

A O Jackson

Publications and source records attributed to A O Jackson.

At least 37 records · Page 2Linked to original sources

PubMed1996

RNA-binding activities of barley stripe mosaic virus gamma b fusion proteins.

The barley stripe mosaic virus (BSMV) gamma-b gene encodes a 17 kDa cysteine-rich protein known to affect virulence and to have a role in regulating viral gene expression. We have constructed recombinant gamma-b-glutathione S-transferase fusion proteins in Escherichia coli and have determined the ability of the purified fusion proteins and various mutant derivatives to bind nucleic acids in vitro. Gel-shift analyses revealed that the wild-type gamma-b-fusion protein is able to bind RNA cooperatively. The binding affinity is highly selective for single-stranded RNA because double-stranded RNA, single-stranded and double-stranded DNA, and transfer RNA were unable to compete for binding with the labelled RNA probes. However, BSMV-specific sequence binding was not observed since a chloroplast RNA competed for binding with 32P-labelled transcripts derived from the BSMV genome. The first 44 amino acids of the 152 amino acid gamma-b fusion protein encompassing one of two cysteine-rich 'zinc finger-like' motifs, and a basic region separating the finger-like motifs are required for RNA binding. Site- specific amino acid substitutions within two groups of lysine and arginine residues located in the basic motif reduced the binding affinity of the fusion protein greatly, but cysteine and histidine substitutions designed to disrupt the finger-like motifs failed to have appreciable effects on binding. These findings indicate that the regulatory properties of gamma-b may be mediated in part by RNA binding activities.

Amino Acid Sequence↗

PubMed1996

Extraction of nuclei from sonchus yellow net rhabdovirus-infected plants yields a polymerase that synthesizes viral mRNAs and polyadenylated plus-strand leader RNA.

Although the primary sequence of the genome of the plant rhabdovirus sonchus yellow net virus (SYNV) has been determined, little is known about the composition of the viral polymerase or the mechanics of viral transcription and replication. In this paper, we report the partial isolation and characterization of an active SYNV polymerase from nuclei of SYNV-infected leaf tissue. A salt extraction procedure is shown to be an effective purification step for recovery of the polymerase from the nuclei. Full-length, polyadenylated SYNV N and M2 mRNAs and plus-strand leader RNA are among the products of the in vitro polymerase reactions. Polyadenylation of the plus-strand leader RNA in vitro is shown with RNase H and specific oligonucleotides. This is the first report of a polyadenylated plus-strand leader RNA for a minus-strand RNA virus, a feature that may reflect adaptation of SYNV to replication in the nucleus. Analysis of the SYNV proteins present in the polymerase extract suggests that the N, M2, and L proteins are components of the transcription complex. Overall, the system we developed promises to be useful for an in-depth characterization of the mechanics of SYNV RNA synthesis.

Base Sequence↗

PubMed1996

Plant virus gene vectors for transient expression of foreign proteins in plants.

The development of plant virus gene vectors for expression of foreign genes in plants provides attractive biotechnological tools to complement conventional breeding and transgenic methodology. The benefits of virus-based transient RNA and DNA replicons versus transgenic gene expression include rapid and convenient engineering coupled with flexibility for expeditious application in various plant species. These characteristics are especially advantageous when very high levels of gene expression are desired within a short time, although instability of the foreign gene in the viral genome can present some problems. The strategies that have been tested for foreign gene expression in various virus-based vectors include gene replacement, gene insertion, epitope presentation, use of virus controlled gene expression cassettes, and complementation. Recent reports of the utilization of virus vectors for foreign gene expression in fundamental research and biotechnology applications are discussed.

Journal Article↗

PubMed1996

Geographic distribution and serologic and genomic characterization of Morro Bay virus, a newly recognized bunyavirus.

More than 75,000 immature mosquitoes in three genera were collected from coastal California, reared to the adult stage, and tested for virus by plaque assay in Vero cell cultures. Twenty-six strains of Morro Bay (MB) virus, a newly recognized member of the California (CAL) serogroup, were isolated from Aedes squamiger, a pestiferous salt marsh mosquito species restricted to intertidal salt marshes in coastal California and Baja California. The geographic distribution of the isolates was 10 from San Luis Obispo County, one each from Santa Barbara and Orange Counties, and 14 from San Diego County. No virus isolations were made from 23,157 Ae. squamiger collected north of San Luis Obispo County (midpoint in the geographic range of this species in California). Thus, MB virus infection in Ae. squamiger appears to be restricted to the southern range of this species in California. Serum dilution neutralization tests indicated that MB virus represents a novel subtype of the California encephalitis (CE) serotype within the CAL serogroup. Comparative analyses of genomic sequence data from four geographically distinct MB virus isolates indicated that the isolates are genetically similar to each other and distinct from other CE serotype bunyaviruses. Phylogenetic analysis of nucleocapsid protein gene sequence data indicated that MB virus represents a distinct lineage within the CE serotype and thus supports the serologic classification of MB virus as a distinct CAL serogroup virus.

Aedes↗

PubMed1995

Tomato bushy stunt virus spread is regulated by two nested genes that function in cell-to-cell movement and host-dependent systemic invasion.

We have investigated the importance of two small nested genes (p19 and p22) located near the 3' end of the genome of tomato bushy stunt virus (TBSV) for infectivity in several hosts. Our results show that both genes are dispensable for replication and transcription and that the p19 gene encodes a soluble protein, whereas the p22 gene specifies a membrane-associated protein. Assays using TBSV derivatives that have the beta-glucuronidase gene substituted for the capsid protein gene demonstrate that p22 is required for cell-to-cell movement in all plants tested. Mutations inactivating p19 ameliorate the severe necrotic systemic symptoms elicited by wild-type TBSV in Nicotiana benthamiana and Nicotiana clevelandii, but p19 does not obviously affect movement in these hosts. However, in some local lesion hosts p19 influences the lesion diameter, which suggests that it has an auxiliary host-dependent role in movement. This notion is supported by the observation that p19 is required for long-distance spread of TBSV in spinach and for systemic infection of pepper plants. Thus, movement of TBSV is regulated by two nested genes; p22 governs cell-to-cell movement and p19 has a host-specific role in systemic invasion.

Base Sequence↗

PubMed1995

The effect of defective interfering RNAs on the accumulation of tomato bushy stunt virus proteins and implications for disease attenuation.

Tombusviruses, of which tomato bushy stunt virus (TBSV) is the type member, spontaneously generate defective interfering RNAs (DIs) that are known to interfere with viral accumulation and symptom development. We show that co-infection with TBSV and DIs causes a dramatic reduction in accumulation of TBSV subgenomic RNAs and corresponding TBSV proteins with a less dramatic reduced accumulation of the genomic RNA and the replicase proteins. Associated with this differentially regulated suppression was a greatly reduced expression of both the p19 protein, which is responsible for severe symptoms, and the p22 protein, which is associated with cell-to-cell movement of the virus. Therefore, the results suggest that the protective effect of DIs may be due to selective inhibition of p19 and p22 expression in addition to reduced replication of genomic RNA.

Genome, Viral↗

PubMed1995

Host effects and sequences essential for accumulation of defective interfering RNAs of cucumber necrosis and tomato bushy stunt tombusviruses.

Passage of cucumber necrosis virus (CNV) containing defective interfering (DI) RNAs through cucumber plants decreased the accumulation of DI RNAs to undetectable levels. Subsequent passages in two Nicotiana species (Nicotiana benthamiana or N. clevelandii) resulted in the appearance of DI RNA species that were larger than the DI RNAs observed during exclusive serial passages of CNV through the Nicotiana species. Sequence analysis of cloned cDNAs corresponding to the two DI RNA populations indicated that the smaller CNV-DI RNAs contained the four conserved regions (I through IV) of the genome typical of tombusvirus DI RNAs, whereas the larger DI RNAs were of similar organization but had a direct repeat of the middle portion of the molecule. This result suggests that the host has an influence on the type of DI RNA that accumulates during consecutive high multiplicity of infection passages. A comparative analysis of deletions targeting the individual conserved regions in both CNV and tomato bushy stunt virus (TBSV) DI RNAs revealed that only region III was completely dispensable for accumulation of either DI RNA species. More refined deletion analyses in regions I and II indicated that smaller segments of 75 and 35 nucleotides (nt), respectively, could be deleted without abolishing infectivity. The dispensable sequences in region II of both TBSV and CNV DI RNAs mapped to the top portion of a putative stem-loop structure. These studies indicate that both essential and nonessential sequences are conserved in DI RNAs. The essential sequences in regions I, II, and IV likely contain important cis-acting elements, whereas nonessential regions such as region III may play secondary roles such as optimally spacing cis-acting elements or maintaining the DI RNA at an overall size that is stable.

Base Sequence↗

PubMed1995

The tomato bushy stunt virus replicase proteins are coordinately expressed and membrane associated.

Two open reading frames at the 5'-end of the tomato bushy stunt virus genomic RNA are predicted to encode a 33-kDa (p33) protein and its 92-kDa (p92) readthrough product. From amino acid sequence comparisons with other small single-stranded RNA viruses, these proteins resemble viral components of the replicase-transcriptase complex. To investigate the accumulation of these proteins in the infected cell, two chimeric proteins were produced that expressed either a portion of p33 or the carboxy-terminal "half" of p92 fused with glutathione S-transferase, and polyclonal ascites fluids specific to p33 or p92 were elicited in mice. As expected, the anti-p33 antibody recognized p33 and the p92 readthrough protein, but the anti-p92 antibody was specific for p92. Immunoblot analyses revealed that at an early stage of infection both proteins were associated with the membrane fractions isolated from virus-infected plants, but later in the infection, prior to collapse of the tissues, these proteins were also associated with the cytoplasmic fraction. At all time points in plants and protoplasts p33 was about 20-fold more abundant than p92. A series of mutations derived from an infectious cDNA clone demonstrated that both the p33 and the p92 proteins were required for replication in protoplasts and the ratio of the two proteins was maintained in the replication-competent mutants. The wild-type amber (UAG) and in vitro-generated ochre (UAA) readthrough codon derivatives replicated in protoplasts. However, the tyrosine mutants (UAC or UAU) that were predicted to express only p92 were not viable in protoplasts.

Animals↗

PubMed1995

Determination and comparative analysis of the small RNA genomic sequences of California encephalitis, Jamestown Canyon, Jerry Slough, Melao, Keystone and Trivittatus viruses (Bunyaviridae, genus Bunyavirus, California serogroup).

The nucleotide sequences of the small (S) genomic RNAs of six California (CAL) serogroup bunyaviruses (Bunyaviridae: genus Bunyavirus) were determined. The S RNAs of two California encephalitis virus strains, two Jamestown Canyon virus strains, Jerry Slough virus, Melao virus, Keystone virus and Trivittatus virus contained the overlapping nucleocapsid (N) and non-structural (NSs) protein open reading frames (ORFs) as described previously for the S RNAs of other CAL serogroup viruses. All N protein ORFs were 708 nucleotides in length and encoded a putative 235 amino acid gene product. The NSs ORFs were found to be of two lengths, 279 and 294 nucleotides, which potentially encode 92 and 97 amino acid proteins, respectively. The complementary termini and a purine-rich sequence in the 3' non-coding region (genome-complementary sense) were highly conserved amongst CAL serogroup bunyavirus S RNAs. Phylogenetic analyses of N ORF sequences indicate that the CAL serogroup bunyaviruses can be divided into three monophyletic lineages corresponding to three of the complexes previously derived by serological classification. The truncated version of the NSs protein, which is found in five CAL serogroup bunyaviruses, appears to have arisen twice during virus evolution.

Amino Acid Sequence↗

PubMed1995

Identification of tomato bushy stunt virus host-specific symptom determinants by expression of individual genes from a potato virus X vector.

In this study, we analyzed the influence of two nested genes (p19 and p22) of tomato bushy stunt virus (TBSV) on disease symptoms in systemically infected plants and in local lesion hosts. The contribution of individual genes was determined by bioassays with an infectious clone of wild-type TBSV, with p19/p22 mutant derivatives, and by expression of individual TBSV genes from a heterologous potato virus X (PVX) vector. Our results showed that TBSV genes could be expressed at high levels from the PVX vector. The subcellular localization of these proteins as well as the ability of PVX-expressed p22 to trans complement TBSV cell-to-cell movement defective mutants indicate that the exogenously expressed proteins are functionally active. Inoculation studies with TBSV mutants and the PVX derivatives demonstrated that p19 induced a generalized necrosis upon systemic infection of Nicotiana benthamiana and N. clevelandii. In addition, p19 elicited the formation of local necrotic lesions in N. tabacum; however, in N. glutinosa and N. edwardsonii, the local lesion response was activated by p22. These results show that the p19 and p22 proteins of TBSV are important symptom determinants and that closely related plant species may contain different resistance genes that selectively respond to individual TBSV proteins.

Genes, Viral↗

PubMed1994

Multiple genetic determinants of barley stripe mosaic virus influence lesion phenotype on Chenopodium amaranticolor.

The ND18 and Type strains of barley stripe mosaic hordeivirus (BSMV) differ in the local lesion phenotypes they elicit on Chenopodium amaranticolor. The ND18 strain produces large necrotic lesions on this host by 3 to 4 days postinoculation, whereas the Type strain is less virulent and elicits chlorotic local lesions which appear about 2 weeks after inoculation. We have used infectious in vitro transcripts derived from full-length cDNA clones of these two BSMV strains to investigate the genetic basis for their differential virulence on C. amaranticolor. Pseudorecombination of the wild-type alpha, beta, and gamma genomic RNAs of each strain revealed that the lesion forming phenotype segregated with RNA gamma. Fine mapping of the phenotypic determinants on RNA gamma was carried out by constructing deletion mutants, chimeric recombinants, and point mutants. These experiments showed that three different genetic elements in the Type strain RNA gamma contribute significantly to its attenuated virulence on C. amaranticolor. In addition, pseudorecombination experiments using mutant Type strain gamma RNAs that were more virulent than native Type RNA gamma indicated that the clean segregation of the lesion forming phenotype observed with wild-type RNA gamma is fortuitous. This lesion phenotype is dependent on both the multiple attenuating determinants in the wild-type Type strain RNA gamma and the source of genomic RNAs alpha and beta in the inoculum. The complexity of these virulence determinants clearly illustrates the limitations of classical pseudorecombination as a tool for the genetic analysis of plant viruses.

Amino Acid Sequence↗

PubMed1994

Sequence analysis of the trailer region of sonchus yellow net virus genomic RNA.

The sequence of the 5' terminus (or the "trailer" region) of the minus-sense RNA genome of sonchus yellow net virus (SYNV) was determined by dideoxynucleotide termination sequencing of purified viral RNA and cloned cDNAs. The 5'-terminal nucleotide was identified by nuclease P1 digestion of 32P-end-labeled genomic RNA followed by polyethyleneimine cellulose chromatography. The trailer sequence occupies positions 13,561 to 13,720 relative to the 3' end of the genomic RNA and is composed of 160 nucleotides (nt) adjacent to a dinucleotide forming a portion of the "gene junction" sequence at the terminus of the L protein gene. The trailer sequence is longer than the 144-nt plus-strand leader RNA transcribed from the 3' end of the genomic RNA and is the longest trailer sequence yet reported among the nonsegmented negative-strand viruses. As is characteristic of other rhabdovirus genomes, the 3' and 5' termini of the SYNV genome are complementary and are capable of forming a panhandle structure involving 16 of the 18 terminal nucleotides. However, there is no obvious direct nucleotide sequence relatedness between the SYNV trailer sequence and those of animal rhabdoviruses and paramyxoviruses. The existence of a minus-strand leader RNA of the same polarity as the trailer sequence could not be detected in nucleic acid extracted from infected plants under hybridization conditions suitable for detection of the plus-strand leader RNA. In this regard, SYNV differs from vesicular stomatitis virus and is similar to other rhabdoviruses which also fail to accumulate detectable minus-strand leader RNAs.

Base Sequence↗

PubMed1994

Characterization and detection of sc4: a sixth gene encoded by sonchus yellow net virus.

The nucleotide sequence of a sixth gene (sc4) of the plant rhabdovirus sonchus yellow net virus (SYNV) was determined from viral genomic and poly(A)+ cDNA clones. The sc4 gene is 1196 nucleotides (nt) and has an open reading frame of 972 nt that is capable of encoding a protein of 324 amino acids. Primer extension analyses of poly(A)+ RNA isolated from infected plants indicate that the 5' end of the sc4 mRNA corresponds to nucleotide 2840, relative to the 3' end of the minus-sense genomic RNA and extends to nucleotide 4035. A 43-nt untranslated leader sequence precedes the predicted first AUG codon and a 181-nt untranslated sequence follows the translational stop codon. This gene is similar to the other SYNV genes in that it is flanked on each side by a conserved gene junction sequence. Polyclonal antibodies raised to an sc4 fusion protein react with a 37-kDa protein in virus-infected plants that is close to the predicted size of the sc4 protein. Western blot analyses of cellular fractionations from infected plants show that sc4 is membrane associated and sucrose density gradient analyses demonstrate that sc4 sediments in the same fractions as SYNV virions. Analysis of the sc4 open reading frame reveals that 16% of the amino acids are serine or threonine residues and that the protein has four potential consensus casein kinase II phosphorylation sites. The deduced amino acid sequence of sc4 also contains a motif related to alpha amylases and aspartic proteases. This completes the sequence determination of the 13,720-nt SYNV genome.

Amino Acid Sequence↗

PubMed1994

The barley stripe mosaic virus gamma b gene encodes a multifunctional cysteine-rich protein that affects pathogenesis.

Barley stripe mosaic virus contains seven genes, one of which specifies a 17-kD cysteine-rich protein, gamma b, that is known to affect virulence. To further characterize the role of gamma b in pathogenesis, we mutagenized sequences encoding amino acids within two clusters of cysteine and histidine residues in the cysteine-rich domain and a group of basic amino acids located between the clusters and determined the effects of these mutations on the symptom phenotype in barley. Three single amino acid substitutions in cluster 1 and two amino acid exchanges in the basic region caused bleached symptoms associated with pronounced elevations in accumulation of gamma b protein. In contrast, three single amino acid substitutions in cluster 2 and a mutation in the basic motif resulted in attenuated ("null") symptoms typical of those produced when the gamma b gene is deleted. Tissue infected with these "null" mutants accumulated slightly elevated amounts of the gamma b protein but significantly lower levels of coat protein and the putative movement protein beta b. Genetic complementation tests revealed that cluster 1 mutations are dominant over the wild-type gamma b gene, whereas those in cluster 2 are recessive. These results highlight the pivotal role of gamma b in pathogenesis and suggest that the two cysteine-rich clusters are functionally distinct and that they affect different aspects of disease development.

Amino Acid Sequence↗

PubMed1993

Serological analysis of barley stripe mosaic virus-encoded proteins in infected barley.

Polyclonal antisera raised against proteins or peptides corresponding to barley stripe mosaic virus (BSMV) open reading frames were used in immunoblot experiments to investigate the in vivo expression of BSMV-encoded proteins during infection of barley. Six of the seven putative gene products, whose functional roles have been defined in previous genetic studies, were detected at one or more stages of infection in barley tissue sampled at 4, 7, 10, and 14 days postinoculation (DPI). The alpha a, beta a, beta b, beta d, gamma a, and gamma b gene products were observed during the course of infection, but a protein corresponding to the beta c gene could not be identified. The 130-kDa alpha a and the 74-kDa gamma a proteins, which comprise the essential BSMV-encoded replicase components, differed in the time course of their expression and in their subcellular distribution. The highest concentration of the alpha a protein coincided with the appearance of leaf symptoms at 4 DPI and declined gradually as infection progressed. The alpha a protein was found primarily in the soluble protein fraction but some of the protein was also found in the membrane and cell wall fractions. In contrast, the gamma a protein peaked in concentration later at 7 to 10 DPI and was more abundant in membranous fractions. The cysteine-rich 17-kDa gamma b protein was located predominantly in the soluble fraction and its concentration remained relatively constant during the course of infection. The 25-kDa capsid protein (beta a) was present in the highest amounts in the soluble fraction at 4 DPI and it continued to increase in abundance throughout the sampling period. The transiently expressed 58-kDa beta b protein, which like beta c and beta d is required for systemic infection, was most abundant at the onset of symptoms, but declined precipitously in concentration as the infection progressed. Although larger amounts of beta b were present in the soluble fraction, the cell wall fraction contained a substantial portion estimated to be 30 to 40% of the protein. Affinity-purified antisera raised against a peptide corresponding to the hydrophobic 14-kDa beta d ORF revealed the presence of a BSMV-specific protein of the predicted size in membrane and cell-wall fractions. Like the beta b protein, the level of the beta d protein decreased dramatically as infection progressed, suggesting that the synthesis of these two proteins is coordinately expressed from the RNA beta subgenomic RNA.

Amino Acid Sequence↗

PubMed1993

Myoclonic spasms following intrathecal diamorphine.

The use of intrathecal diamorphine via an implanted portal system is described for pain control in a patient suffering from vertebral metastatic disease. The complication of myoclonic spasms affecting the lower half of the body occurred after 14 days, when increasing the bolus dose to 40 mg. The spasms lasted for 3 hr and then gradually subsided. Diamorphine was subsequently restarted at a lower dose of 15 mg twice daily. On increasing the dose to 20 mg diamorphine 10 days later, severe distressing myoclonic spasms recurred 20 min postinjection. Myoclonus could only be controlled by instituting a local anesthetic intrathecal block. The patient was finally managed with 20 mg diamorphine per day by intrathecal infusion, and the pain was reasonably well controlled for the following 10 weeks without any recurrence of myoclonic spasms.

Anesthesia, Local↗

PubMed1992

Structure of the L (polymerase) protein gene of sonchus yellow net virus.

The complete nucleotide sequence of the L protein gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, was determined by dideoxynucleotide sequencing of cloned cDNAs derived from the negative-strand genomic RNA. The L protein gene is composed of 6401 nucleotides (nt) located between positions 7158 and 13558 relative to the 3' end of the genomic RNA. Sequence analysis suggests that the complementary mRNA contains a 44 nt untranslated 5' leader sequence preceding an open reading frame of 6348 nucleotides that is capable of encoding a polypeptide of 2116 amino acids with a deduced molecular weight of 241,569 Da. The L protein is positively charged, has a high proportion of the amino acids Leu and Ile, and contains putative polymerase and RNA binding domains. Extended alignment of the SYNV L protein amino acid sequence with those of other nonsegmented negative-strand RNA virus polymerases reveals conservation of sequences within 12 blocks that appear sequentially along the protein. A cluster dendrogram derived from the L protein alignments indicates that SYNV is more closely related to animal rhabdoviruses than to the paramyxoviruses and that the animal rhabdoviruses have diverged less from each other than from SYNV.

Amino Acid Sequence↗

PubMed1992

RNA recombination in the genome of barley stripe mosaic virus.

Barley stripe mosaic Hordeivirus (BSMV) is a positive-strand RNA virus requiring three single-stranded RNAs (alpha, beta, and gamma) for infectivity. A terminal-sequence-dependent cloning strategy was used to clone the entire genome of the CV17 strain. Full-length gamma cDNA clones were obtained when oligonucleotides specific for the 5'-terminal sequence of RNA alpha were used in the cloning procedure, but not when RNA gamma-specific oligonucleotides were used. Sequence analysis of six putative gamma cDNA clones revealed that nucleotides 1-70 possess 89% homology with the first 70 nucleotides of RNA alpha. This leader region is separated from the gamma-specific coding region by an eight-base intervening sequence common to both CV17 RNAs alpha and gamma. Northern and Southern hybridization with oligonucleotide probes specific for either alpha or gamma leader sequences indicated that CV17 gamma cDNA clones are representative of native CV17 gamma RNAs. Furthermore, bioassays indicated that in vitro transcripts derived from these gamma cDNA clones were infectious when coinoculated with in vitro transcripts of full-length alpha and beta cDNA clones. Thus, the evidence suggests that RNA gamma of BSMV strain CV17 is a recombinant molecule which may have arisen as a result of natural recombination between RNAs alpha and gamma.

Base Sequence↗