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C H Shaw

Publications and source records attributed to C H Shaw.

At least 19 recordsLinked to original sources
PubMed1998

A chemotaxis cluster from Agrobacterium tumefaciens.

We report the DNA sequence of a 9.6-kb region of the Agrobacterium tumefaciens chromosome containing a putative 8-kb chemotaxis operon. The putative operon begins with orf1, whose predicted protein product shows strong sequence identity to methyl-accepting chemotaxis proteins (MCPs), followed by orf2, cheY1, cheA, cheR, cheB, cheY2, orf9, orf10. All of the identified homologues show a high degree of sequence conservation with their counterparts in the che operons from Sinorhizobium meliloti and Rhodobacter sphaeroides, and are arranged in a similar order. Mutations in orf1 and cheA result in impaired chemotaxis, whereas deletion of orf10, appears to have no effect on chemotaxis or motility. Although the putative operon does not contain a cheW homologue, heterologous probing and PCR using consensus primers indicates that cheW maps elsewhere in the Agrobacterium genome.

Agrobacterium tumefaciens↗
PubMed1997

Isolation and characterisation of a linked cluster of genes from Agrobacterium tumefaciens encoding proteins involved in flagellar basal-body structure.

We report the DNA sequence of 7205 bp of the Agrobacterium tumefaciens chromosome. This contains a putative operon encoding homologues of the flagellar rod and associated proteins FlgBCG and FliE, the L and P ring proteins (FlgHI) a possible flagellum-specific export protein FliP, and two proteins of unknown function, FlgA and FliL. Several of these genes have overlapping stop and start codons. Three non-flagellate Tn5-induced mutations map to this operon: fla-11 to the first gene, encoding the rod protein FlgB; fla-15 to flgA; and fla-12 to fliL. A site-specific mutation introduced into the final gene in this cluster, fliP, also resulted in a non-flagellate phenotype. This indicates that the operon is expressed, and that at least FlgB, FlgA, FliL and FliP are required for flagellar assembly in A. tumefaciens. The bulk of this operon is conserved in the same order in Rhizobium meliloti.

Agrobacterium tumefaciens↗
PubMed1997

The Agrobacterium tumefaciens motor gene, motA, is in a linked cluster with the flagellar switch protein genes, fliG, fliM and fliN.

We report the sequence of 3978 bp of the Agrobacterium tumefaciens chromosome which contains a putative operon encoding the homologues of the transmembrane proton channel protein MotA, and the flagellar switch proteins FliM, FliN and FliG. Two transposon insertions in fliG result in a non-flagellate phenotype, indicating that this gene at least is required for flagellar assembly.

Agrobacterium tumefaciens↗
PubMed1993

Timely culture for mycobacteria which utilizes a microcolony method.

For the isolation of mycobacteria from clinical specimens, we evaluated a method that used a thinly poured Middlebrook 7H11 agar plate (10 by 90 mm) that was examined microscopically. Inoculated plates were sealed, incubated, and examined at regular intervals for the appearance of microcolonies. Plates were examined microscopically, while still sealed, by focusing on the agar surface through the bottom of the plate and the agar. Plates were scanned at low power (x40 total magnification), and colony morphology was confirmed at intermediate power (x100 to x180 magnification). This method was compared with a traditional method that used macroscopic examination of standard mycobacterial media. By using all specimens submitted for mycobacterial culture over the duration of the study, the method was evaluated until 270 isolates of mycobacteria (Mycobacterium tuberculosis, n = 103; M. avium-M. intracellulare, n = 115; miscellaneous, n = 52) were detected. While the conventional method required an average of 23 days to the time of first detection of mycobacteria, the experimental method required an average of only 11 days. When limited to acid-fast stain-positive specimens that were culture positive for M. tuberculosis, the average interval to positivity was 7 days for the microcolony method compared with 17 days for the conventional method. With the experimental method, the microscopic colonial morphology allowed for the presumptive identification of M. tuberculosis colonies, which were distinguished by cording, and M. avium-M. intracellulare colonies, which were smooth and entire. Presumptive identification was complete for 83.5% of the M. tuberculosis isolates within 10 days and for 85% of the M. avium-M. intracellulare isolates within 11 days after inoculation. If the microcolony method was combined with a conventional tube medium, the composite would optimize for speed of recovery while providing the full sensitivity of the conventional method. In addition to reducing the interval to positivity, the microcolony method allows for the easy detection of mixed mycobacterial infections and yields a presumptive identification that facilitates the selection of a confirmatory gene probe test.

Bacteriological Techniques↗
PubMed1992

Cloning and characterisation of an oleosin gene from Brassica napus.

The sequence of an oleosin gene from Brassica napus has been determined. This gene contains a single intron of 437 bp and encodes a polypeptide of 195 amino acids. The oleosin gene product has an estimated molecular mass of 21.5 kDa and consists of a highly hydrophobic central domain flanked by relatively polar N- and C-terminal domains. The central domain is highly conserved between all oleosins sequenced to date and contains a run of periodically spaced leucine residues similar to that of a leucine-zipper motif. The gene has been shown to be expressed specifically in the embryo, maximally between 9 and 11 weeks after flowering, i.e. during the seed desiccation stage. Two transcriptional start sites have been mapped to -70 and -21 of the ATG and a putative ABA-responsive element and three repeated motifs have been identified in the promoter. These short promoter sequences could correspond to regulatory elements responsible for embryo-specific gene expression. Up to six genes exist in the oleosin gene family.

Amino Acid Sequence↗
PubMed1992

Sequence of an oleosin cDNA from Brassica napus.

Antibodies raised against purified rapeseed 19 kDa oleosin protein were used to screen an embryo-derived lambda gt11 expression library from Brassica napus. A near full-length cDNA clone, BnV, was isolated. The 781 bp cDNA contained an open reading frame of 549 bp followed by an untranslated region of 222 bp and a poly(A) region of 10 bp. Comparisons between this cDNA and a different oleosin cDNA previously isolated from the same library showed high degrees of sequence similarity in the central domain region and in the 3' untranslated region. Sequence similarities between the derived protein sequence of this cDNA and all other known oleosin protein sequences are discussed.

Amino Acid Sequence↗
PubMed1991

A class of amphipathic proteins associated with lipid storage bodies in plants. Possible similarities with animal serum apolipoproteins.

The lipid-storing tissues of plants contain many small (0.2-1 microns) lipid (normally triacylglycerol) droplets which are surrounded and stabilized by a mixed phospholipid and protein annulus. The proteinaceous components of the lipid storage bodies are termed oleosins and are not associated with any other cellular structures. The major oleosins of rapeseed and radish have been isolated by preparative SDS-PAGE and are respectively classes of 19 kDa and 20 kDa proteins. Both protein classes were N-terminally blocked for direct sequencing, but were partially sequenced following limited proteolytic digestion. The major rapeseed oleosin was made up of at least two 19 kDa polypeptides, termed nap-I and nap-II, which have closely related but different amino acid sequences. A single 20 kDa oleosin, termed rad-I, was found in radish. A near full length cDNA clone for a major rapeseed oleosin was sequenced and found to correspond almost exactly to the sequence of nap-II. The sequences of nap-I and rad-I show very close similarity to one another, as do the sequences of nap-II and the previously determined sequence for the major oleosin from maize. All four oleosins have a large central hydrophobic domain flanked by polar N- and C-terminal domains. Secondary structure predictions for the four oleosins are similar and a novel model is proposed based on a central hydrophobic beta-strand region flanked by an N-terminal polar alpha-helix and a C-terminal amphipathic alpha-helix. The possibility that oleosins exhibit structural and functional similarities with some animal apolipoproteins is discussed.

Amino Acid Sequence↗
PubMed1991

Swimming against the tide: chemotaxis in Agrobacterium.

Chemotaxis in bacteria is an excellent model for signal transduction processes. In Agrobacterium tumefaciens, the causative agent of crown gall tumour on wounded plants, it is a vital part of the organism's biology. A chromosomally-determined chemotaxis system causes the bacterium to be attracted into the rhizosphere by chemoattractants in plant exudates. By interfacing with this system, the multifunctional products of two Ti-plasmid encoded genes, virA and virG, allow the sensing of specific wound phenolics such as acetosyringone. This attracts Ti-plasmid harbouring A. tumefaciens to wound sites, where the higher acetosyringone concentrations lead to virA and virG-mediated induction of the vir-genes. The products of the induced genes, act in concert to effect transfer of the T-DNA to the plant cell.

Chemotaxis↗
PubMed1988

virA and virG are the Ti-plasmid functions required for chemotaxis of Agrobacterium tumefaciens towards acetosyringone.

Octopine and nopaline Ti-plasmids confer upon Agrobacterium tumefaciens C58C1 the ability to respond chemotactically to the vir-inducing phenolic wound exudate, acetosyringone. A. tumefaciens C58C1 containing Ti-plasmids with Tn5 insertions in virB, C, D or E exhibited marked chemotaxis towards acetosyringone. However, Ti-plasmids with mutations in virA or virG were unable to confer the responsive phenotype. Of the cosmid clones pVK219 (virAB) pVK221 (virBGC) pVK225 (virGCDE) and pVK257 (virABGC) mobilized to cured A. tumefaciens C58C1, only pVK257 bestowed acetosyringone chemotaxis. virA and virG are thus required for chemotaxis of A. tumefaciens towards acetosyringone. This suggests a multifunctional role for virA and virG: at low concentrations of acetosyringone they mediate chemotaxis and at higher concentrations they effect vir-induction.

Acetophenones↗
PubMed1988

Ti plasmid-specified chemotaxis of Agrobacterium tumefaciens C58C1 toward vir-inducing phenolic compounds and soluble factors from monocotyledonous and dicotyledonous plants.

Twelve phenolic compounds with related structures were analyzed for their ability to act as chemoattractants for Agrobacterium tumefaciens C58C1 and as inducers of the Ti plasmid virulence operons. The results divided the phenolic compounds into three groups: compounds that act as strong vir inducers and are chemoattractants for A. tumefaciens C58C1 harboring the nopaline Ti plasmid pDUB1003 delta 31, but not the isogenic cured strain; compounds that are at best weak vir inducers and are weak chemoattractants for Ti plasmid-harboring and cured A. tumefaciens C58C1; and compounds that are vir noninducers and are also nonattractants. A strong correlation between vir-inducing ability and Ti plasmid requirement for chemotaxis is thus established. In addition, chemical structure rules for vir induction and chemotaxis are outlined. Positive chemotaxis toward root and shoot homogenates from monocotyledonous and dicotyledonous plants was observed. At low extract concentrations, chemotaxis was enhanced by the presence of Ti plasmid. The chemoattractants do not derive from intact cell walls. Lack of attraction is not responsible for the apparent block to monocot transformation by A. tumefaciens.

Chemical Phenomena↗
PubMed1986

Light regulation of a SSRubisco-nos chimaeric gene: photoregulatory control sequences from a C3 plant function in cells of a CAM plant.

A SSRubisco-nos chimaeric gene has been constructed, in an oncogenic Ti-plasmid vector. A 900bp soybean SSRubisco upstream fragment, carrying CAAT and TATA boxes and transcription initiation point, was fused to the nos coding region, the fusion site being within the 5'-untranslated region. When this chimaeric construct was transferred to Kalanchoe cells, nos expression was shown to be light-regulated. Thus DNA sequences responsible for light-dark control of gene expression are wholly or partly contained within the 900bp soybean SSRubisco upstream region. Moreover, this is the first demonstration that photoregulatory elements in a gene derived from a C3 plant, function in cells of a plant exhibiting the CAM trait.

Base Sequence↗
PubMed1984

A functional map of the nopaline synthase promoter.

This paper describes the first functional map of a promoter expressed from the plant chromosome. We have constructed a series of overlapping deletion mutants within the region upstream of the Ti-plasmid encoded nopaline synthase (nos) gene. By monitoring nos expression in tumour tissue we have inferred a functional map of the nos promoter. The maximum length of sequence upstream of the transcription initiation point required to express wild type levels of nopaline synthase is 88 bp. Within this region, the "CAAT" box is essential for maximal activity; deletion of this sequence reduced apparent nos expression by over 80%. Presence of an intact or partial "TATA" box in the absence of the "CAAT" box supports a barely detectable level of nopaline synthase. Removal of all sequences upstream of the nos coding sequence results in no detectable activity.

Amino Acid Oxidoreductases↗
PubMed1984

The right hand copy of the nopaline Ti-plasmid 25 bp repeat is required for tumour formation.

At either end of the nopaline Ti-plasmid T-region resides a copy of a 25 bp repeated element. The normal T-DNA endpoint is 1 bp internal of the right copy, with the transcription initiation site of the nopaline synthase (nos) gene being approximately 300 bp away in the same direction. Here we describe results which demonstrate that deletion of any combination of sequences between the nos initiation site and the right copy of the 25 bp repeat does not affect oncogenicity. Thus a mutant retaining the right copy and only 3 bp internal of it is indistinguishable from the wild type parent in its oncogenic properties. However deletion of a further 39 bp, including complete removal of the right copy abolishes crown gall tumour formation on Kalanchöe and tobacco. From these results we infer that unlike the left border, the right copy of the 25 bp repeat is required for T-DNA transfer and/or integration. This is the first conclusive demonstration of the involvement of a copy of the repeats in this process.

Chromosome Deletion↗
PubMed1983

A general method for the transfer of cloned genes to plant cells.

This paper describes a method for the transfer to plant cells of any cloned gene, regardless of its termini or internal restriction enzyme cleavage sites. A broad host-range intermediate vector, pGV1117, was constructed containing HindIII-23, a right-end T-region fragment of the nopaline plasmid pTiC58. Using in vivo protection by EcoRI methylase and EcoRI linker ligation, a fragment of rabbit chromosomal DNA, carrying the beta-globin gene, was inserted into plasmid pGV1117. Following transmission to Agrobacterium tumefaciens, insertion of the gene into the T-region of pTiC58 occurred via in vivo recombination. Infection of axenic tobacco seedlings resulted in the transfer to the plant genome of an intact beta-globin gene, as part of the T-DNA. Although the gene was stably maintained during tissue culture, beta-globin-specific transcripts were not detected in the transformed plant cells.

Cloning, Molecular↗