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Summary The steroid 21-hydroxylase enzyme (P450c21) is a member of the cytochrome P450 gene superfamily and is essential in the synthesis of cortisol and aldosterone. Defects in the P450c21B gene cause congenital adrenal hyperplasia (CAH), a common genetic disorder leading to virilization of newborn females. To avoid the standard cloning of mutant P450c21 genes from genomic libraries, we amplified the full-length genomic P450c21 genes by polymerase chain reaction (PCR). The amplification was followed by cloning and sequencing of a defective P450c21B gene. The strategy described here is generally applicable, thus making a simple characterization of the complete P450c21B gene possible. The method was tested in one patient suffering from the simple virilizing form of CAH. The sequence of three independent clones originating from the defective P450c21B showed that Ile at position 172 in exon 4 was substituted by Asn. The identical mutation also has been found in other patients with CAH. 相似文献
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C. M. Higgins R. M. Hall P. R. Campbell R. G. Dietzgen 《Plant Molecular Biology Reporter》2000,18(3):285-285
We report the application of a PCR-based method in conjunction with automated sequencing for the reliable detection and verification
of transgenes in crude extracts of leaf and callus tissue from different plant species. Transformed tissue can be identified
easily at any stage of the regeneration process, whether it is via embryogenesis or organogenesis. This allows researchers
to focus their attention and resources on truly transformed tissues and avoid unwittingly culturing untransformed tissues.
This protocol can also be used to rescue relatively large PCR products as well as duplexing the detection of transgenes. Direct
sequencing of the PCR products allows confirmation of the integrity of the transgenein planta. 相似文献
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Jerry A. Stitzel Stephen M. Campbell Allan C. Collins Michael J. Marks 《Journal of neurochemistry》1988,50(3):920-928
Two distinct binding sites with properties corresponding to those expected for nicotinic cholinergic receptors can be identified in brain by the specific binding of nicotine (or acetylcholine) and alpha-bungarotoxin. The effects of modification of these binding sites by treatment with the disulfide-reducing agent dithiothreitol were examined in tissue prepared from DBA mouse brains. Treatment with dithiothreitol reduced the binding measured with either ligand, and reoxidization of the disulfides fully restored binding. The effects of dithiothreitol treatment appeared to be due to a reduction in the maximal binding of nicotine and to a decrease in the binding affinity for alpha-bungarotoxin. Agonist affinity for the alpha-bungarotoxin binding site was reduced by treatment with low concentrations of dithiothreitol. The nicotine binding sites remaining after disulfide treatment displayed rates of ligand association and dissociation similar to those of unmodified tissue, but treatment of previously unmodified tissue with dithiothreitol accelerated the rate of nicotine dissociation. After reduction, both binding sites could be selectively alkylated with bromoacetylcholine. The results suggest that both putative nicotinic receptors in brain respond similarly to disulfide reduction and that their responses resemble those known for the nicotinic receptor of electric tissue. 相似文献
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A method is described which allows the user to track the influence which changes in nutritional state have on specific areas of intermediary metabolism. The approach relies on combined gas chromatography-gas flow proportional counting; data processing programs are used. To illustrate the operation and potential of the method, the effect of serum supplementation of the growth medium on overall fatty acid metabolism in HeLa cells is reported. 相似文献
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