Biomedical subjects

K. Wu

Publications and source records attributed to K. Wu.

3 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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PubMed1994

Folate Metabolism in Datura innoxia (In Vivo and in Vitro Folylpolyglutamate Synthesis in Wild-Type and Methotrexate-Resistant Cells).

In vivo folylpolyglutamate pools of the wild-type (Px4) and methotrexate-resistant (MTX161) Datura innoxia cell lines were detected by incorporation of [14C]p-aminobenzoate into folates. The folylpolyglutamate derivatives were cleaved to p-aminobenzoylpolyglutamates and separated according to glutamyl chain length by high-performance liquid chromatography. Hexaglutamates were the predominant form in both Datura cell lines. The proportions of individual folylpolyglutamates were unaffected by culturing the cells in medium containing products of one-carbon metabolism such as glycine, adenine, thymidine, or methionine. Radiolabeling of the hexaglutamates was greatly reduced in the presence of 10-8 M methotrexate (MTX) in the Px4 cells but not in the MTX161 cells. Tetrahydrofolate, 5, 10-methylenetetrahydrofolate, and folinic acid were effective substrates for the folylpolyglutamate synthetase from Datura cells in vitro, whereas MTX and folate were poor substrates. In vivo, MTX can be slowly converted into its polyglutamate derivatives up to MTXGlu4 or MTXGlu5 in Datura cells in the longer term. Significantly lower levels of MTX polyglutamates in MTX161 cells were found compared with those of Px4 cells during prolonged (10 d) exposure to MTX. Although in vivo and in vitro folylpolyglutamate synthesis was found to be similar in both cell lines, about a 4-fold increase in specific activity of [gamma]-glutamyl hydrolase (GGH) was detected in the MTX161 cell line. The increase in GGH in the resistant cells suggested that breakdown of polyglutamylated forms of MTX may play a role in acquired MTX resistance.

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PubMed1993

Methotrexate Resistance in Datura innoxia (Uptake and Metabolism of Methotrexate in Wild-Type and Resistant Cell Lines).

A wild-type Datura innoxia cell line (Px4) was used to select methotrexate-resistant cells through a stepwise procedure. Two independently selected cell lines, MTX161 and MTX132, were stable and shown to be 5 to 15 times more resistant to methotrexate than wild type. These methotrexate-resistant cells were similar to the wild-type cells in levels and kinetic properties of dihydrofolate reductase, the sensitivity of dihydrofolate reductase to methotrexate, the binding of [3H]methotrexate to soluble proteins, and the formation of methotrexate polyglutamate derivatives. High performance liquid chromatographic analyses indicated that methotrexate polyglutamylation is only slight and may not be significant in the toxicity of methotrexate to Datura cells. The uptake of methotrexate was also investigated in the wild-type and resistant cells. The Px4 cells exhibited a linear uptake that lasted for 1 to 7 h. The uptake was saturable, pH and energy dependent, and had a Km of 65.6 nM and a Vmax of 12.5 nmol h-1g-1 fresh weight. Neither MTX161 nor MTX132 exhibited the sustained uptake of methotrexate shown by the Px4 cells. As a result, there were greatly reduced concentrations of intracellular methotrexate in resistant cells. Resistant cell lines had 2- to 3-fold higher Km values for methotrexate uptake compared with Px4 cells. It is proposed that these cells became resistant as a result of a stable change in the membrane transport system for methotrexate.

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