Kazuhiko Konishi | PubMed Health

PubMed2003

A highly sensitive detection method for somatic mutations in p53 gene.

A simple and highly sensitive detection method for somatic mutations, which is applicable to diagnostic samples is developed. This method detects somatic mutations that are lower than several percent in abundance. The unique feature with this method is that single-nucleotide primer extension is carried out in the absence of dye terminators that carry respective bases in the wild type gene. Data of mutation sites and bases in the target genes are obtained from databases. Primers of different nucleotide length are designed so that electrophoreses of the single-nucleotide primer extension products allow identification of respective mutations. Based on somatic mutation data in IARC TP53 mutation database, this method was applied to p53 somatic mutations. Results showed that extension reactions in 5 separate tubes allowed detection of mutations in human p53 gene for the most frequent 12 mutations in a nucleotide sequence of 14,049-14,522, which cover hot spot regions in exons 7 and 8. Confirming these results, reported mutations of p53 in human culture cells, MiaPaCa2, TE-6, RPMI8226, DLD1, and PC-3 are detected at 5% relative abundance in the sample DNA.

DNA Mutational Analysis↗

PubMed2002

Substrate regulation of calcium binding in Ca2+-ATPase molecules of the sarcoplasmic reticulum. I. Effect of ATP.

The effect of ATP on calcium binding of the Ca2+-ATPase of the sarcoplasmic reticulum has not been clarified. By comparing the calcium dependence of the ATPase activity and of phosphorylation of the ATPase molecules with that of calcium binding in the absence of ATP, we show the existence of two types of regulatory site of the enzyme molecules at which ATP binding variously improves the calcium binding performance of the molecules depending on the aggregation state of the molecules and pH; the two regulatory sites bind ATP at submillimolar (0.25 mm) and millimolar (5 mm) ATP, respectively. The results are discussed based on a model of two conformational variants (A and B forms) of the chemically equivalent ATPase molecules (Nakamura, J., and Furukohri, T. (1994) J. Biol. Chem. 269, 30818-30821). For example, in the sarcoplasmic reticulum membrane at pH 7.40, submillimolar ATP converted the calcium binding manner of the A form from noncooperative (Hill number (n(H)) of approximately 1) to cooperative (n(H) approximately 2), concurrent with a decrease in the apparent calcium affinity (K(0.5)) from 2-6 to 0.1-0.3 microm. The binding of the A form became almost the same as that of the B form (n(H) approximately 2, K(0.5) approximately 0.2 microm), which was not affected by ATP. Millimolar ATP further decreased the K(0.5) of the cooperative binding of the two forms to approximately 0.05 microm. Regulation of the calcium binding performance by ATP is discussed in terms of monomeric and oligomeric pathway models.

Animals↗