Biomedical subjects

K. Ward

Publications and source records attributed to K. Ward.

4 recordsLinked to original sources

PubMed2002

A constitutive gene expression system derived from the tCUP cryptic promoter elements.

A limited number of constitutive promoters have been used to direct transgene expression in plants and they are often derived from non-plant sources. Here, we describe novel gene-regulatory elements which are associated with a cryptic constitutive promoter from tobacco, tCUP, and modifications that were made to create a strong gene-expression system that is effective across all living cell types from a wide range of plant species, including several important crops ( Arabidopsis, canola, flax, alfalfa, tobacco). The tCUP 5' untranslated region was mutated to eliminate translational interference by upstream ATGs, and the influence of the Kozak consensus sequence on the levels of a beta-glucuronidase (GUS) reporter gene activity was demonstrated. These modifications resulted in expression that was greatly enhanced in all organs. A TATA consensus sequence was added to the core promoter to complement an existing Initiator (Inr) sequence. Although this addition was known to elevate core promoter activity by 3-fold the additive effect on the overall gene-expression system was marginal in all of the transgenic plants tested. Two transcriptional enhancers were identified and the region containing them were oligomerized, yielding a significant increase in marker gene-expression in some but not all plant species. In general, the enhanced tCUP gene-expression system generated levels of GUS activity which exceeded that of the 35S promoter in most plant species and the elevation in activity occurred uniformly among the various plant organs. The potential benefit of cryptic elements for the construction of gene-expression systems for crop species is discussed

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PubMed1997

Importance of the Chiral Centers of Jasmonic Acid in the Responses of Plants (Activities and Antagonism between Natural and Synthetic Analogs).

The importance of the two chiral centers at C-3 and C-7 in the molecular structure of jasmonic acid in plant responses was investigated. We separated methyl jasmonate (MeJA) into (3R)- and (3S)-isomers with a fixed stereochemistry at C-3, but epimerization at C-7 is possible. The four isomers of the nonepimerizable analog 7-methyl MeJA were synthesized. These six esters and their corresponding acids were tested in three bioassays: (a) senescence in sunflower (Helianthus annuus) cotyledons; (b) proteinase inhibitor II gene expression in transgenic tobacco (Nicotiana tabacum) with [beta]-glucuronidase as a biochemical reporter; and (c) seed germination in Brassica napus and wheat (Triticum aestivum). The esters and acids had similar activities in the three assays, with the ester being more effective than its acid. The (3R)-stereochemistry was critical for jasmonate activity. Although activity was reduced after substituting the C-7 proton with a methyl group, the analogs with (3R,7R)- or (3R,7S)-stereochemistry were active in some of the assays. Although the four isomers of 7-methyl MeJA were inactive or only weakly active in the senescence assay, they could overcome the senescence-promoting effect of (3R)-MeJA. The strongest antagonistic effect was observed with the (3R,7S)-isomer.

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PubMed1995

In vivo evidence of increased malignant cell proliferation following surgery in ovarian cancer.

Flow cytometric evaluation of cellular DNA-ploidy and S-phase fraction was undertaken in serial post-operative peritoneal washings in 43 patients with histologically proven ovarian carcinoma, and 20 control patients. Five patients had FIGO stage I, two stage II and the remainder stage III/IV disease. Daily sampling of peritoneal fluid was performed commencing at day 0 (operation day) until the seventh post-operative day. Samples were analyzed fresh using a flow cytometer. Aneuploidy was detected in 168 (62.5%) of samples. Three patients had persistent diploidy throughout the study period, and were excluded from analysis. The mean aneuploid count rose from 24.7% [SEM (standard error of mean) = 4.6] on day 0 to 43.6% (SEM = 5.7) on day 4 (P < 0.008). The S-phase fraction for the aneuploid populations fell significantly (P < 0.02) from 4.9% (SEM = 1.2) on day 0 to 2.2% (SEM = 0.8) on day 1, but subsequently continued to rise. Cytologic evaluation confirmed malignant cells in 79% of aneuploid samples. Three patients in the control population had similar patterns to malignant conditions, two of these patients having benign ovarian cysts and ascites. These findings show an increased proliferative activity of aneuploid malignant cells following surgical intervention, as detected in peritoneal lavage samples of patients with ovarian cancer.

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PubMed1989

The Pea rbcS-3A Promoter Mediates Light Responsiveness but not Organ Specificity.

The expression of the pea rbcS-3A gene is responsive to light and restricted to chloroplast-containing cells. Previously, we identified transcriptional elements between -410 and -50 relative to the transcription start site as being sufficient for this regulated expression. Here we investigate the role of the promoter region (-50 to +15) in rbcS-3A expression. We demonstrate an interaction between the promoter and upstream sequences located between -189 and -166, which is important for high level expression. In addition we show that, when the rbcS promoter is driven by a weak test enhancer, it can confer light-responsive but not organ-specific expression upon a bacterial reporter gene.

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