PubMed Health⌕ Search

Biomedical subjects

S A MacFarlane

Publications and source records attributed to S A MacFarlane.

At least 19 recordsLinked to original sources
PubMed2001

Tobravirus 2b protein acts in trans to facilitate transmission by nematodes.

Analysis of RNA2 of TRV PaY4 showed it to be recombinant, carrying 3'-terminal sequences derived from RNA1. Virus produced using an infectious cDNA clone of PaY4 RNA2 was nematode transmissible, demonstrating that natural TRV recombinant isolates are not necessarily defective. Mutations introduced into PaY4 RNA2 showed that the 2b gene, but not the 2c gene, is required for transmission by both Paratrichodorus pachydermus and P. anemones nematodes. Experiments examined whether infection of plants with two different virus clones would impact upon nematode transmission of either virus. Simultaneous inoculation with TRV clones expressing green or red fluorescent proteins revealed that mixing of the two virus populations did not occur, although, in roots, adjacent cells were found containing green- or red-tagged viruses. Subsequently, in similar experiments it was found that a TRV PaY4 2b mutant was transmitted when combined with wild-type TRV PaY4. Also, transmission of a 2b mutant of an in vitro TRV/PEBV recombinant virus (TRV-C1) occurred after coinfection with wild-type virus. Thus, the tobravirus 2b transmission protein is trans-acting. Although TRV PaY4 and TRV PpK20 are both transmitted by P. pachydermus, a 2b mutant of TRV PaY4 was not transmitted when coinoculated to plants with TRV PpK20.

Amino Acid Sequence↗
PubMed2001

'Worms' that transmit viruses.

Plant-parasitic nematodes transmit viruses that hit hard on global agriculture. Both the nematodes and the viruses they carry persist in the soil for a long time with the only remedy being highly toxic chemicals. Fundamental new research may offer the hope of developing safer control strategies.

Agriculture↗
PubMed2000

Efficient expression of foreign proteins in roots from tobravirus vectors.

Viral vectors were constructed from infectious cDNA clones of each of the three tobraviruses, tobacco rattle virus (TRV), pea early-browning virus (PEBV), and pepper ringspot virus (PepRSV). RNA2 of each of the three viruses was modified to carry an additional coat protein subgenomic promoter and was used to express green fluorescent protein (GFP) when inoculated to plants. The tobravirus expression vectors have a wide host range and were able to express GFP in, for example, Nicotiana species, tomato, pea, arabidopsis, and sugar beet. The TRV vector was able to invade and express GFP very efficiently in roots, whereas the widely used PVX vector was not.

Biotechnology↗
PubMed1999

The E116 isolate of Dutch pea early-browning virus is a recombinant virus.

The complete nucleotide sequence of RNA2 of the E116 isolate of Dutch pea early-browning virus (PEBV-D) was obtained from overlapping cDNA clones. The RNA was found to encode three open reading frames corresponding to, in 5' to 3' order, the coat protein, the 2b nematode transmission protein and the C-terminal part of the cysteine-rich 1b protein derived from RNA1. The 3' non-coding region of PEBV-D RNA2 was also shown to be derived from RNA1. This is the first demonstration that recombination of PEBV occurs in nature. Comparison of the amino acid sequences of the PEBV-D RNA2 proteins with those of British PEBV and several isolates of tobacco rattle virus reveals complex patterns of mixing of the genomes of these two viruses.

Amino Acid Sequence↗
PubMed1999

Similarities in the genome organization of tobacco rattle virus and pea early-browning virus isolates that are transmitted by the same vector nematode.

Although sequence data have been obtained for several tobravirus isolates, only two of these isolates are nematode-transmissible. Tobacco rattle virus (TRV) PpK20 is transmitted by Paratrichodorus pachydermus, whereas pea early-browning virus (PEBV) TpA56 is transmitted by Trichodorus primitivus. To clarify whether differences in the genome structure of these isolates are relevant to the specificity of interactions with particular vector nematodes, or merely reflect a taxonomic difference between TRV and PEBV, we have sequenced RNA2 of a new isolate of TRV (TpO1) that is transmitted by the same vector nematode as PEBV TpA56 but is not transmitted by the nematode vector of TRV PpK20. TRV TpO1 RNA2 encodes, in 5' to 3' order, a coat protein (CP), a 9K protein, a 2b (29K) protein and a 2c (18K) protein. Amino acid sequence comparison shows that both the CP and 2b proteins of TRV TpO1 resemble more closely the analogous proteins from PEBV TpA56 than those from TRV PpK20. Also, the TRV TpO1 9K protein has similarities with the PEBV 9K protein whereas this protein is lacking in TRV PpK20.

Amino Acid Sequence↗
PubMed1997

Viral determinants of pea early browning virus seed transmission in pea.

Pea early browning virus (PEBV) is a member of the genus, Tobravirus. It is transmitted by soil-inhabiting trichodorid nematodes and through seeds from diseased plants. By introducing mutations into the PEBV genome, we have studied the viral determinants of seed transmission in pea. Neither deleting a portion of the genome containing the three nonstructural genes in RNA2 nor the interuption of any of the three genes individually prevented PEBV seed transmission. However, a comparison of two PEBV isolates indicated a minor role for RNA2 or its products. In contrast, the removal of the coding sequence of the 12K gene in RNA1 almost completely abolished viral seed transmission. The virus lacking the 12K gene caused more severe symptoms on leaves and pods, and accumulated to a higher level than the wild-type virus in both types of tissues. However, the 12K deletion mutant accumulated poorly in anthers and carpels, and could not be detected in pollen grains and ovules. These results suggest that the 12K gene is involved in the infection of the gametic cells and hence the seed transmission of PEBV in pea.

Pisum sativum↗
PubMed1997

Replication of in vitro tobravirus recombinants shows that the specificity of template recognition is determined by 5' non-coding but not 3' non-coding sequences.

Natural recombinant tobacco rattle tobravirus (TRV) isolates contain sequences from a different tobravirus, pea early browning virus (PEBV). To characterize the sequence requirements for viable recombinant formation hybrid cDNA clones of RNA2 of PEBV and TRV were assembled. Inclusion of 320 nt from the 5' terminus of PEBV or 335 nt from the 5' terminus of TRV in the hybrid RNAs was sufficient to permit their replication by, respectively, PEBV RNA1 or TRV RNA1 regardless of the origin of the 3' terminal region. However, PEBV RNA1 but not TRV RNA1 was sometimes able to support low level replication of RNA2 containing the heterologous 5' terminal region. In vitro translation of PEBV transcripts containing 5' noncoding region deletions supported the hypothesis that in vivo the PEBV coat protein (CP) is expressed from a subgenomic RNA and that, therefore, in the recombinants the CP subgenomic promoter probably is recognized by the replicase of the heterologous virus.

Cloning, Molecular↗
PubMed1996

Multiple virus genes involved in the nematode transmission of pea early browning virus.

Mutations were introduced into four genes encoded by RNA2 of pea early browning virus (PEBV) to determine their possible involvement in the transmission of this virus by nematodes. Deletion of 28 amino acids from the C-terminus of the coat protein abolished the formation of virus particles. Deletion of 15 amino acids at the C-terminus did not affect particle formation but did abolish nematode transmission. In contrast, deletion of 13 amino acids immediately preceding the 16 C-terminal residues did not affect particle formation and decreased rather than abolished nematode transmission. A deletion in the gene encoding a 29-kDa protein and a frameshift mutation in the gene encoding a 23-kDa protein both abolished transmission without affecting virus particle formation. Mutations in an ORF encoding a 9-kDa protein, which is located on the genome between the coat protein gene and 29K gene, gave conflicting results. Removal of the AUG initiation codon from the 9K ORF had no effect on transmission, whereas the introduction of a frameshift mutation, which would prematurely terminate expression of the putative 9-kDa protein, decreased the frequency of transmission. The results show that the coat protein and probably all three of the other RNA2-encoded proteins play a role in the transmission of PEBV by vector nematodes.

Animals↗
PubMed1996

Rapid cloning of uncharacterised tobacco rattle virus isolates using long template (LT) PCR.

A rapid method was developed using long template (LT)-PCR to amplify the complete RNA2 of isolates of TRV for which no sequence data are available. The amplification makes use of a 5' terminal oligonucleotide which contains degeneracies corresponding to the sequences of several different TRV isolates, and a 3' oligonucleotide which is complementary to a sequence present in all known isolates. This method was used to show the high degree of sequence homology existing in the terminal regions of two uncharacterised TRV isolates (TPO3 and PAY4), and revealed the deletion of an 80-nucleotide sequence in the 5' terminal region of TPO3 RNA2.

Base Sequence↗
PubMed1996

Sequence analysis of wheat and oat furovirus capsid protein genes suggests that oat golden stripe virus is a strain of soil-borne wheat mosaic virus.

In northern blots, cDNA probes prepared to soil-borne wheat mosaic virus (SBWMV) RNA-1 and RNA-2 hybridized to RNA-1 and RNA-2, respectively, from a UK isolate of oat golden stripe virus (OGSV), as well as to their homologous RNAs. RT-PCR was used to amplify, clone and sequence a region of about 750 nucleotides spanning the capsid protein gene and part of the readthrough protein on RNA-2 from OGSV, a French isolate of SBWMV and two stable deletion mutants (Lab1 and Okl-7) of SBWMV isolates from Nebraska and Oklahoma respectively. There was very high (96.7-99.1%) nucleotide homology between all these sequences and the wild-type SBWMV sequences from Nebraska and Oklahoma. OGSV was more similar to SBWMV from France and Nebraska than were any of the isolates to SBWMV from Oklahoma. Of the few differences in the deduced amino acid sequences of the capsid proteins from the different isolates, OGSV differed from all SBWMV isolates only in one amino acid (isoleucine for valine at position 88). The high degree of similarity suggests that OGSV may best be classified as an oat strain of SBWMV.

Avena↗
PubMed1995

Molecular characterisation of UK isolates of barley yellow mosaic bymovirus.

Several isolates of barley yellow mosaic virus (BaYMV) from different sites in the UK, including some that were virulent on European resistant winter barley cultivars (resistance-breaking strain: BaYMV-2) and some that were not, were examined by RT-PCR, restriction mapping and sequencing of selected parts of the virus genome. Nucleotide and predicted amino acid sequences were determined for the 5'-terminal region, part of the NIa coding region and the coat protein coding region on RNA 1 and an area at the N-terminus of the 70-kDa protein coding region on RNA 2. The sequences differed from those previously reported for a BaYMV isolate from Japan and for two German isolates, one of which was of the BaYMV-2 strain. There were no strain-specific amino acid differences and the few, non-consecutive, nucleotide differences detected were probably not significant and were insufficient to develop a rapid diagnostic test to distinguish BaYMV-2 from other isolates. Restriction mapping of RNA 2 cDNA again showed no consistent strain-related differences. The differences previously reported between the two German isolates are probably not strain-related.

Base Sequence↗
PubMed1995

Sequence comparison of RNA2 of nematode-transmissible and nematode-non-transmissible isolates of pea early-browning virus suggests that the gene encoding the 29 kDa protein may be involved in nematode transmission.

A cDNA clone containing the complete coding region of RNA2 of the newly described TPA56 isolate of pea early-browning virus (English serotype, PEBV-E) has been obtained. A plasmid (pFLA56) containing this clone together with the 5' and 3' non-coding regions of PEBV isolate SP5 (the type isolate of PEBV-E) and flanked by the CaMV 35S promoter and NOS terminator is infectious when co-inoculated onto plants with pCaN1, an infectious clone of PEBV SP5 RNA1. Virus containing RNA2 derived from the cDNA clone of the TPA56 isolate is transmitted by Trichodorus primitivus nematodes, whereas virus containing RNA2 from a clone of the SP5 isolate is not transmitted. Sequencing revealed 11 differences out of 3374 nucleotides between the transmissible TPA56 and non-transmissible SP5 clones. However, only three of the base changes affected the amino acid sequences of virus gene products. A single, conservative amino acid change was present in the virus coat protein. Two non-conservative changes occurred in the protein of molecular mass 29.6 kDa expressed from an open reading frame located down-stream of the coat protein gene, suggesting that it has a function in nematode transmission and that changes in this protein prevent vector transmission of the SP5 isolate of PEBV.

Animals↗
PubMed1994

Detection and sequence analysis of a spontaneous deletion mutant of soil-borne wheat mosaic virus RNA2 associated with increased symptom severity.

Repeated passage of soil-borne wheat mosaic virus (SBWMV; wild-type Oklahoma field isolate; designated Okl-0) by manual inoculation resulted in deletion of part of SBWMV RNA2. Deletion was apparent in the population of RNA2 molecules after only 11 weeks in primary inoculated wheat plants (called Okl-1) and after 5 passages (Okl-5; 20 weeks) no full-length RNA2 remained. The extent of deletion in the Oklahoma isolate was compared with that in the previously studied Lab 1 (Nebraska) isolate. RNA2 from Okl-1, Okl-7, and Lab 1 were analyzed by RT-PCR amplification using sets of primer pairs which spanned all portions of the intact molecule. Lab 1 and the new stable isolate, Okl-7, were found to be deleted for 1058 and 759 nt, respectively, within the region encoding the coat protein-readthrough domain. However, the Okl-7 deletion was in a different position from the Lab 1 deletion. This suggests that deletions of SBWMV RNA2 which occur during serial manual inoculation are not directed toward production of a conserved, truncated form of the 84-kDa extended coat protein, but might reflect an RNA sequence-dependent event.

Amino Acid Sequence↗
PubMed1994

Heterologous encapsidation of recombinant pea early browning virus.

The coat protein gene of pea early browning virus (PEBV) was replaced with that of another tobravirus, tobacco rattle virus (TRV strain PPK20). The recombinant virus multiplied efficiently in the systemic host Nicotiana benthamiana and, on the local lesion host Phaseolus vulgaris, produced symptoms typical of PEBV rather than TRV showing that viral coat protein is not a determinant for lesion morphology. Both viral RNAs were encapsidated by TRV coat protein although the shorter particles (encapsidated RNA-2) did not form a discrete population. Evidence is presented to suggest involvement of nucleotide sequences upstream of the coat protein gene in virus particle assembly.

Antigens, Viral↗
PubMed1992

Plants transformed with a region of the 201-kilodalton replicase gene from pea early browning virus RNA1 are resistant to virus infection.

The 3' proximal portion of the gene encoding the 201-kDa putative replicase protein from the Tobravirus pea early browning virus (PEBV) can potentially be expressed separately as a 54-kDa protein. Nicotiana benthamiana plants transformed with the open reading frame (ORF) encoding the 54-kDa protein, designated 54K ORF, were resistant to infection by purified PEBV at inoculum doses of up to 1 mg/ml, the highest concentration tested. However, resistance was abolished by the introduction into the 54K ORF of mutations that would cause premature termination of translation. This suggests that the resistance mechanism requires the involvement of an intact 54-kDa protein. The 54K ORF-transformed plants were also resistant to infection by broad bean yellow band virus and an uncharacterized isolate of British PEBV (PGRO R) but were not resistant to infection by two other tobraviruses, pepper ringspot virus and the I6 isolate of tobacco rattle virus. Additionally, two variants of PEBV which overcame 54K ORF-mediated resistance have been isolated, the analysis of which might provide important information about both the resistance mechanism itself and the process of normal virus replication.

Amino Acid Sequence↗
PubMed1992

Modulation of proteoglycan metabolism by human fibroblasts maintained in an endogenous three-dimensional matrix.

This report describes synthesis and degradation of proteoglycans by human gingival fibroblasts growing in an endogenous three-dimensional matrix. Cells grown in the matrix cultures demonstrated a high rate of proteoglycan synthesis, varying between 2 and 4 times that of cells maintained in monolayer cultures. In addition, the relative amount deposited into the cell layer was increased in the matrix cultures, constituting 70% to 90% of the synthesized material during the first 24 h. Comparable levels for the monolayer cultures were 30% to 60%. The majority of the 35S-sulfate-labeled material in both matrix (80%) and monolayer (62%) cultures was susceptible to chondroitin ABC-lyase digestion. The major product was a low Mr (120,000) proteoglycan which could be immunoprecipitated by an antibody against PGII (decorin). In addition, the cells synthesized two chondroitin ABC-lyase-sensitive proteoglycans, one with Mr greater than 400,000, one with an apparent Mr of 250,000, as well as two heparan sulfate proteoglycans with Mr greater than 250,000. The low Mr dermatan sulfate, decorin, was also the major component deposited in the three-dimensional matrix, constituting about 60% of the total sulfate incorporation. In contrast, fibroblasts in monolayer cultures deposited only a small amount (13%) of decorin (PGII) in the cell layer, and the major proteoglycan in this compartment was heparin sulfate. The rate of release of the newly deposited proteoglycans was the same in the two culture conditions, although material released from the three-dimensional matrix cultures contained small Mr components indicating a higher degree of degradation. These studies show differences in proteoglycan metabolism by gingival fibroblasts grown in an endogenous matrix and in monolayer cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1991

Interleukin 1 beta induces rapid phosphorylation and redistribution of talin: a possible mechanism for modulation of fibroblast focal adhesion.

The majority of interleukin 1 (IL-1) receptors in human fibroblasts has been shown to be localized at focal adhesions. This study describes rapid alterations caused by IL-1 beta/IL-1-receptor interaction at these sites. Fibroblast monolayers, incubated with IL-1 beta and prepared for electron microscopy, showed successive loss of cell-substratum contact and fewer and less-pronounced processes. Immunocytochemistry revealed loss and redistribution of the talin staining initially observed after 5-15 min of IL-1 beta incubation. Similarly, the cytoskeleton showed a decrease in staining and a disorganization starting from 15 to 30 min after IL-1 addition, whereas extracellular fibronectin appeared largely unaffected. Prelabeling with [32P]phosphate showed a 2- to 3-fold increase in the level of talin phosphorylation, peaking at 15 min. Phospho amino acid analyses revealed a higher level of serine and threonine phosphorylation. The data suggest that the action of IL-1 beta on fibroblasts may be partially mediated by direct phosphorylation of talin via activation of a protein serine/threonine kinase, leading to changes in transmembrane linkage proteins and the cytoskeleton. Such alterations at focal adhesions may provide a mechanism by which IL-1 can rapidly modulate cell-matrix interactions during inflammation and wound healing.

Cell Adhesion↗