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J C Gray

Publications and source records attributed to J C Gray.

At least 37 records · Page 2Linked to original sources
PubMed2000

Assembly of the Rieske iron-sulphur protein into the cytochrome bf complex in thylakoid membranes of isolated pea chloroplasts.

The assembly of the Rieske iron-sulphur protein into the cytochrome bf complex was examined following import of 35S-labeled precursor protein by isolated pea chloroplasts. Rieske protein assembled into the cytochrome bf complex was resolved from unassembled Rieske protein and from other membrane complexes by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. Four mutant forms of the Rieske protein were able to assemble into the cytochrome bf complex in isolated chloroplasts. These were a triple substitution mutant, C107S/H109R/C112S, replacing conserved residues involved in the ligation of the [2Fe-2S] centre; the mutant Delta45-52 which removed a glycine-rich region predicted to form a flexible hinge between the hydrophobic membrane-associated region and the hydrophilic lumenal domain; and mutants Delta168-173 and Delta177-179 which removed two C-terminal regions, which are highly conserved in chloroplast and cyanobacterial Rieske proteins. This indicates that the [2Fe-2S] cluster, the glycine-rich region and the C-terminal region are not essential for stable assembly of the Rieske protein into the cytochrome bf complex in isolated chloroplasts.

Biological Transport↗
PubMed2000

The role of amino-acid residues in the hydrophobic patch surrounding the haem group of cytochrome f in the interaction with plastocyanin.

Soluble turnip cytochrome f has been purified from the periplasmic fraction of Escherichia coli expressing a truncated petA gene encoding the precursor protein lacking the C-terminal 33 amino-acid residues. The protein is identical [as judged by 1H-NMR spectroscopy, midpoint redox potential (+ 365 mV) and electron transfer reactions with plastocyanin] to cytochrome f purified from turnip leaves. Several residues in the hydrophobic patch surrounding the haem group have been changed by site-directed mutagenesis, and the proteins purified from E. coli. The Y1F and Q7N mutants showed only minor changes in the plastocyanin-binding constant Ka and the second-order rate constant for electron transfer to plastocyanin, whereas the Y160S mutant showed a 30% decrease in the overall rate of electron transfer caused in part by a 60% decrease in binding constant and partially compensated by an increased driving force due to a 27-mV decrease in redox potential. In contrast, the F4Y mutant showed increased rates of electron transfer which may be ascribed to an increased binding constant and a 14-mV decrease in midpoint redox potential. This indicates that subtle changes in the hydrophobic patch can influence rates of electron transfer to plastocyanin by changing the binding constants and altering the midpoint redox potential of the cytochrome haem group.

Amino Acid Sequence↗
PubMed2000

The role of individual lysine residues in the basic patch on turnip cytochrome f for electrostatic interactions with plastocyanin in vitro.

The role of electrostatic interactions in determining the rate of electron transfer between cytochrome f and plastocyanin has been examined in vitro with mutants of turnip cytochrome f and mutants of pea and spinach plastocyanins. Mutation of lysine residues Lys58, Lys65 and Lys187 of cytochrome f to neutral or acidic residues resulted in decreased binding constants and decreased rates of electron transfer to wild-type pea plastocyanin. Interaction of the cytochrome f mutant K187E with the pea plastocyanin mutant D51K gave a further decrease in electron transfer rate, indicating that a complementary charge pair at these positions could not compensate for the decreased overall charge on the proteins. Similar results were obtained with the interaction of the cytochrome f mutant K187E with single, double and triple mutants of residues in the acidic patches of spinach plastocyanin. These results suggest that the lysine residues of the basic patch on cytochrome f are predominantly involved in long-range electrostatic interactions with plastocyanin. However, analysis of the data using thermodynamic cycles provided evidence for the interaction of Lys187 of cytochrome f with Asp51, Asp42 and Glu43 of plastocyanin in the complex, in agreement with a structural model of a cytochrome f-plastocyanin complex determined by NMR.

Aspartic Acid↗
PubMed2000

The ycf 9 (orf 62) gene in the plant chloroplast genome encodes a hydrophobic protein of stromal thylakoid membranes.

There are still some open reading frames, orfs, with unknown function in the higher plant chloroplast genome. Of these conserved orfs, designated as ycfs (hypothetical chloroplast open reading frames), one is ycf 9 (orf 62) in the transcription unit with the psbC and psbD genes. The aim of this work was to investigate the function of ycf 9 by insertional inactivation of the gene with a selectable marker cassette, consisting of the aadA coding region connected to the trc promoter and rrnB terminator. This cassette was inserted 19 bp downstream from the start of the coding region of the tobacco ycf 9 gene. Two DNA constructs with the aadA cassette in opposite orientations were precipitated on 1 micron gold particles and delivered into leaves of Nicotiana tabacum, cultivar Samsun, by the biolistic method. Spectinomycin-resistant plants regenerated following bombardment with only the construct containing the aadA gene in the opposite orientation as ycf 9. In spite of several subsequent regeneration cycles on spectinomycin, the transplastomic plants did not reach homoplasmicity. This suggests that the ycf 9 gene product is essential for chloroplast function. Using a polyclonal antibody raised against the inner part of the gene product, the polypeptide was localized in the stromal thylakoid membranes of chloroplasts.

Amino Acid Sequence↗
PubMed2000

The pea light-independent photomorphogenesis1 mutant results from partial duplication of COP1 generating an internal promoter and producing two distinct transcripts.

The pea lip1 (light-independent photomorphogenesis1) mutant shows many of the characteristics of light-grown development when grown in continuous darkness. To investigate the identity of LIP1, cDNAs encoding the pea homolog of COP1, a repressor of photomorphogenesis identified in Arabidopsis, were isolated from wild-type and lip1 pea seedlings. lip1 seedlings contained a wild-type COP1 transcript as well as a larger COP1' transcript that contained an internal in-frame duplication of 894 bp. The COP1' transcript segregated with the lip1 phenotype in F(2) seedlings and could be translated in vitro to produce a protein of approximately 100 kD. The COP1 gene in lip1 peas contained a 7.5-kb duplication, consisting of exons 1 to 7 of the wild-type sequence, located 2.5 kb upstream of a region of genomic DNA identical to the wild-type COP1 DNA sequence. Transcription and splicing of the mutant COP1 gene was predicted to produce the COP1' transcript, whereas transcription from an internal promoter in the 2.5-kb region of DNA located between the duplicated regions of COP1 would produce the wild-type COP1 transcript. The presence of small quantities of wild-type COP1 transcripts may reduce the severity of the phenotype produced by the mutated COP1' protein. The genomic DNA sequences of the COP1 gene from wild-type and lip1 peas and the cDNA sequences of COP1 and COP1' transcripts have been submitted to the EMBL database under the EMBL accession numbers AJ276591, AJ276592, AJ289773, and AJ289774, respectively.

Arabidopsis Proteins↗
PubMed2000

A study of interval breast cancer within the NHS breast screening programme.

AIM: To define the biological nature and malignant potential of interval cancers presenting to a breast unit within the NHS breast screening programme. METHODS: 112 interval cancers were compared with matched, screen detected and symptomatic cancers in terms of their radiographic, histopathological, and immunohistochemical features. RESULTS: Interval cancers, strictly defined, showed no characteristic radiographic pattern. In terms of size, vascular invasion, lymph node status, and prognosis they were intermediate between screen detected and symptomatic cancers. Within the interval cancers there was an excess of grade 1 and grade 3 tumours, and lesions with a high Ki67 index but immunohistochemistry otherwise failed to discriminate between the three groups. Inclusion of data from false negative "interval cancers" did not significantly alter the results. CONCLUSIONS: Interval cancers are more aggressive than screen detected cancers but in general less aggressive than symptomatic cancers. However, within a heterogeneous group, occasional interval cancers are exceptionally malignant.

Aged↗
PubMed1999

Characterization of a single-copy Arabidopsis gene encoding a protein showing limited similarity to the N-terminus of the mammalian clathrin-assembly protein AP180.

The Arabidopsis 194 gene encoding a protein containing sequence similarity to an N-terminal region of the clathrin-assembly protein AP180 has been identified in a 4.9-kb region of genomic DNA upstream of the gene encoding the high mobility group protein HMG-I/Y. The gene consists of 12 exons and 11 introns, identified by comparison with partial cDNAs and using the NetPlantGene programme, and encodes a protein of 584 amino acid residues. The C-terminal region of the protein contains 8 tandem repeats of a 17-amino-acid-residue sequence. Southern blot analysis of genomic DNA from Columbia and Landsberg ecotypes of Arabidopsis indicates the presence of a single copy of the 194 gene. The 194 gene is expressed in all organs of Arabidopsis including roots, stems, leaves, flowers, and developing siliques, as determined by northern blot analysis.

Adaptor Proteins, Vesicular Transport↗
PubMed1999

A galinstan expansion femtosyringe for microinjection of eukaryotic organelles and prokaryotes.

A galinstan expansion femtosyringe enables femtoliter to attoliter samples to be introduced into prokaryotes and subcellular compartments of eukaryotes. The method uses heat-induced expansion of galinstan (a liquid metal alloy of gallium, indium, and tin) within a glass syringe to expel samples through a tip diameter of about 0.1 microm. The narrow tip inflicts less damage than conventional capillaries, and the heat-induced expansion of the galinstan allows fine control over the rate of injection. We demonstrate injection of Lucifer Yellow and Lucifer Yellow-dextran conjugates into cyanobacteria, and into nuclei and chloroplasts of higher organisms. Injection of a plasmid containing the bla gene into the cyanobacterium Phormidium laminosum resulted in transformed ampicillin-resistant cultures. Green fluorescent protein was expressed in attached leaves of tobacco and Vicia faba following injection of DNA containing its gene into individual chloroplasts.

Alloys↗
PubMed1999

Two MAR DNA-binding proteins of the pea nuclear matrix identify a new class of DNA-binding proteins.

Four MAR-binding proteins of 60, 65, 70 and 72 kDa have been detected by South-Western blotting and isolated from pea nuclear matrices. Two cDNAs encoding the 60 and 65 kDa proteins (MARBP-1 and MARBP-2) were isolated from a pea leaf cDNA library by screening with a PCR product obtained using degenerate primers based on an amino acid sequence from the 60 kDa protein. The proteins of 560 and 550 amino acids are 86% identical and contain several KKD/E repeats near the C-terminus. Escherichia coli-expressed MARBP-1 specifically binds A/T-rich MAR DNA. The interaction of MARBP-1/MARBP-2 with MAR DNA involves novel DNA-binding motifs. The MARBP-1 and MARBP-2 genes are expressed in a range of pea tissues and are encoded by genes at different loci. MARBP-1 and MARBP-2 are homologous to yeast nucleolar proteins Nop56p and Nop58p, which are involved in ribosome biogenesis, and to similar highly conserved proteins in other eukaryotes and in archaebacteria. MARBP-1 and MARBP-2 may have multifunctional roles in chromatin organisation and ribosome biogenesis.

Amino Acid Sequence↗
PubMed1999

An Arabidopsis gene encoding a chloroplast-targeted beta-amylase.

beta-Amylase is one of the most abundant starch degrading activities found in leaves and other plant organs. Despite its abundance, most if not all of this activity has been reported to be extrachloroplastic and for this reason, it has been assumed that beta-amylases are not involved in the metabolism of chloroplast-localized transitory leaf starch. However, we have identified a novel beta-amylase gene, designated ct-Bmy, which is located on chromosome IV of Arabidopsis thaliana. Ct-Bmy encodes a precursor protein which contains a typical N-terminal chloroplast import signal and is highly similar at the amino acid level to extrachloroplastic beta-amylases of higher plants. Expression of the ct-Bmy cDNA in E. coli confirmed that the encoded protein possesses beta-amylase activity. CT-BMY protein, synthesized in vitro, was efficiently imported by isolated pea chloroplasts and shown to be located in the stroma. In addition, fusions between the predicted CT-BMY transit peptide and jellyfish green fluorescent protein (GFP) or the entire CT-BMY protein and GFP showed accumulation in vivo in chloroplasts of Arabidopsis. Expression of the GUS gene fused to ct-Bmy promoter sequences was investigated in transgenic tobacco plants. GUS activity was most strongly expressed in the palisade cell layer in the leaf blade and in chlorenchyma cells associated with the vascular strands in petioles and stems. Histochemical staining of whole seedlings showed that GUS activity was largely confined to the cotyledons during the first 2 weeks of growth and appeared in the first true leaves at approximately 4 weeks.

Amino Acid Sequence↗
PubMed1999

Allele-specific interactions between ttg and gl1 during trichome development in Arabidopsis thaliana.

Trichome development in Arabidopsis thaliana is a well-characterized model for the study of plant cell differentiation. Two genes that play an essential role in the initiation of trichome development are GL1 and TTG. Mutations in either gene prevent the initiation of most trichomes. The GL1 gene encodes a myb-related transcription factor. Mutations in TTG are pleiotropic, affecting anthocyanins, root hairs, and seed coat mucilage in addition to trichomes. Six ttg alleles were examined and shown to form a hypomorphic series. The severity of all aspects of the ttg phenotype varied in parallel in this allelic series. The weakest allele, ttg-10, causes frequent clusters of adjacent trichomes, suggesting a role for TTG in inhibiting neighboring cells from choosing the trichome fate. This allele results from a mutation in the 5'-untranslated region of ttg and creates an out-of-frame upstream AUG codon. The ttg-10 allele shows several unusual genetic interactions with the weak hypomorphic gl1-2 allele, including intergenic noncomplementation and a synthetic glabrous phenotype. These interactions are specific for the gl1-2 allele. The implication of these results for current models of trichome development is discussed.

Alleles↗
PubMed1999

The TRANSPARENT TESTA GLABRA1 locus, which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein.

The TRANSPARENT TESTA GLABRA1 (TTG1) locus regulates several developmental and biochemical pathways in Arabidopsis, including the formation of hairs on leaves, stems, and roots, and the production of seed mucilage and anthocyanin pigments. The TTG1 locus has been isolated by positional cloning, and its identity was confirmed by complementation of a ttg1 mutant. The locus encodes a protein of 341 amino acid residues with four WD40 repeats. The protein is similar to AN11, a regulator of anthocyanin biosynthesis in petunia, and more distantly related to those of the beta subunits of heterotrimeric G proteins, which suggests a role for TTG1 in signal transduction to downstream transcription factors. The 1.5-kb TTG1 transcript is present in all major organs of Arabidopsis. Sequence analysis of six mutant alleles has identified base changes producing truncations or single amino acid changes in the TTG1 protein.

Alleles↗
PubMed1999

Neural and vascular anatomy of the menisci of the human knee.

STUDY DESIGN: Review of literature. OBJECTIVES: To review the general anatomy, vascular anatomy, healing potential, neural anatomy, and sensory functions of the menisci of the human knee. BACKGROUND: Recent research has revealed important roles and functions of the menisci of the human knee. METHODS AND MEASURES: A Medline search was performed using the following title and key words: menisci, meniscus, meniscal, vascular, blood, neural, nerve, anatomy, healing, sensory, mechanoreceptors, proprioception, nociceptors, surgery, meniscectomy, repair, and rehabilitation. The references from each article obtained were then reviewed in order to find additional articles not already located through the Medline search. RESULTS: In adults, the blood supply to the menisci of the knee reaches the outer 10% to 33% of the body of the menisci. This portion of the menisci is capable of inflammation, repair, and remodeling. Neural innervation with nociceptors and type I, II, and III mechanoreceptors reaches the outer 66% of the body of the menisci. The anterior and posterior horns of the menisci have a rich supply of both blood vessels and nerves. CONCLUSIONS: The menisci of the human knee are an important source of proprioceptive information regarding the position, direction, velocity, and acceleration and deceleration of the knee. Rehabilitation following injury or surgery to the menisci of the knee should, therefore, incorporate a proprioceptive retraining program that respects both the abilities and inabilities of different portions of the menisci to follow through with repair and remodeling.

Adult↗
PubMed1999

Diagnosis of intermittent vascular claudication in a patient with a diagnosis of sciatica.

BACKGROUND AND PURPOSE: The purpose of this case report is to illustrate the importance of medical screening to rule out medical problems that may mimic musculoskeletal symptoms. CASE DESCRIPTION: This case report describes a woman who was referred with a diagnosis of sciatica but who had signs and symptoms consistent with vascular stenosis. The patient complained of bilateral lower-extremity weakness with her pain intensity at a minimal level in the region of the left sacroiliac joint and left buttock. She also reported numbness in her left leg after walking, sensations of cold and then heat during walking, and cramps in her right calf muscle. She did not report any leg pain. A medical screening questionnaire revealed an extensive family history of heart disease. Examination of the lumbar spine and nervous system was negative. A diminished dorsalis pedis pulse was noted on the left side. Stationary cycling in lumbar flexion reproduced the patient's complaints of lower-extremity weakness and temporarily abolished her dorsalis pedis pulse on the left side. OUTCOMES: She was referred back to her physician with a request to rule out vascular disease. The patient was subsequently diagnosed, by a vascular specialist, with a "high-grade circumferential stenosis of the distal-most aorta at its bifurcation." DISCUSSION: This case report points out the importance of a thorough history, a medical screening questionnaire, and a comprehensive examination during the evaluation process to rule out medical problems that might mimic musculoskeletal symptoms.

Adult↗
PubMed1998

Characterization of a cDNA encoding the thylakoidal processing peptidase from Arabidopsis thaliana. Implications for the origin and catalytic mechanism of the enzyme.

We have identified and sequenced a cDNA containing a complete open reading frame for a putative 340-amino acid precursor of the thylakoidal processing peptidase from Arabidopsis thaliana. The predicted amino acid sequence of the protein includes regions highly conserved among Type I leader peptidases and indicates that the enzyme uses a serine-lysine catalytic dyad mechanism. Phylogenetic analysis indicated a common ancestry of the enzyme with those from oxygenic photosynthetic prokaryotes, suggesting that the cDNA encoded the chloroplast enzyme. The catalytic domain was overexpressed in Escherichia coli, generating a product capable of cleaving the thylakoid-transfer domain from a chloroplast protein. Antibodies to the overexpressed polypeptide cross-reacted with a 30-kDa thylakoid membrane protein.

Amino Acid Sequence↗
PubMed1998

Characterisation and promoter analysis of the Arabidopsis gene encoding high-mobility-group protein HMG-I/Y.

The single-copy gene encoding the Arabidopsis HMG-I/Y protein was isolated and characterised. The gene encodes a protein of 204 amino acid residues and contains a single intron of 73 bp. Primer extension analysis indicates that transcription starts 115 bp upstream of the translation start and the leader sequence contains a short open reading frame of 13 amino acid residues. The 5'-upstream region of 2117 bp and several 5' deletions were fused to the beta-glucuronidase (GUS) reporter gene and transferred to tobacco by Agrobacterium-mediated transformation. Analysis of transgenic tobacco plants containing HMG-I/Y promoter regions of -2117, -1468 and -707 from the translation start detected GUS activity in all organs examined, including roots, stems, leaves and floral organs. Deletion from -707 to -185 resulted in a 20-30-fold reduction in GUS activity in roots and stems, indicating the presence of important quantitative regulatory elements in this region.

Amino Acid Sequence↗
PubMed1998

A/T-rich sequences act as quantitative enhancers of gene expression in transgenic tobacco and potato plants.

The role of an A/T-rich positive regulatory region (P268, -444 to -177 from the translation start site) of the pea plastocyanin gene (PetE) promoter has been investigated in transgenic plants containing chimeric promoters fused to the beta-glucuronidase (GUS) reporter gene. This region enhanced GUS expression in leaves of transgenic tobacco plants when fused in either orientation to a minimal pea PetE promoter (-176 to +4) and in roots when fused in either orientation upstream or downstream of a minimal cauliflower mosaic virus 35S promoter (-90 to +5). The region was also able to enhance GUS expression in microtubers of transgenic potato plants when placed in either orientation upstream of a minimal class I patatin promoter (-332 to +14). Dissection of P268 revealed that cis elements responsible for enhancing GUS expression from the minimal PetE promoter were distributed throughout P268. Multiple copies of a 31 bp A/T-rich sequence from within P268 and of a 26 bp random A/T sequence were able to enhance GUS expression from the minimal PetE promoter, indicating that A/T-rich sequences are able to act as quantitative, non-tissue-specific enhancer elements in higher plants.

Base Sequence↗