ABSTRACT
Decreased levels of the endogenous neuroprotectant kynurenic acid (KYNA) have been observed in the brain of Huntington's Disease (HD) patients and may be related to neuronal loss in this disorder. This reduction may be caused by a dysfunction of kynurenine aminotransferase II (KAT II), the major enzyme responsible for the synthesis of KYNA in the brain. Towards understanding the role of KAT II in HD, we isolated and characterized the cDNA sequence and determined the genomic organization of mouse KAT II (mKat-2). The full length mKat-2 cDNA is 1812 bp, encoding 425 amino acids, and shares 89.9% amino acid similarity with the rat Kat-2 sequence. The gene for mKat-2 is composed of 13 exons divided by 12 intronic sequences. Northern blot analysis demonstrated that mKat-2 mRNA is mainly expressed in kidney and liver. RT-PCR showed mKat-2 expression in the brain starting from at least d11 of embryonic development. An alternative isoform mKat-2beta, derived from the usage of novel exons, shows a different expression pattern from mKat-2. Western blot analysis of various mouse tissues shows a 40-kDa protein in brain, heart, kidney, and liver. In the kidney and liver an additional 45-kDa isoform was detected. Use of the BSS chromosomal mapping panel from The Jackson Laboratory indicates that the mKat-2 gene co-segregates with polymorphic markers D8Mit129 and D8Mit128 on mouse Chr 8. Knowledge of the genomic organization, the isoform tissue-specific expression patterns, the chromosomal localization of mKat-2, and the reagents generated here, will provide the tools for further studies and allow generation and characterization of mice that are nullizygous for mKat-2.
Subject(s)
Huntington Disease/enzymology , Huntington Disease/genetics , Isoenzymes/genetics , Lyases , Transaminases/genetics , Amino Acid Sequence , Animals , Base Sequence , Brain/enzymology , Chromosome Mapping , DNA Primers/genetics , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Exons , Gene Expression , Genome , Humans , Introns , Kidney/enzymology , Liver/enzymology , Mice , Molecular Sequence Data , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Sequence Homology, Amino Acid , Tissue DistributionABSTRACT
The premature chromosome condensation (PCC) assay has been proposed as a useful and rapid end point for biological dosimetry following accidental high-dose radiation overexposures. A major benefit of the PCC assay is that it does not require cells to divide for evaluation of cytogenetic damage. The PCC assay was performed on isolated human peripheral lymphocytes exposed in vitro to doses from 1 to 9 Gy of 250 kVp x-rays. The dose-response relationships of the frequency distribution and the yield of PCC fragments in cells were determined after one day of repair at 37 degrees C. A Qpcc approach, which involves the analysis of the yield of excess PCC fragments in damaged cells, was used to establish a dose-response calibration curve. This method is identical in concept to the Qdr technique introduced by Sasaki for partial-body exposure dose-estimates using asymmetrical chromosome aberrations (i.e., dicentrics and rings) in metaphase spreads of human lymphocytes. A simulated in vitro test of a partial-body exposure to a 6-Gy dose was performed. The results from this test provided dose estimates of 5.3 +/- 0.6, 4.7 +/- 0.6, 5.0 +/- 0.6 and 4.7 +/- 0.8 Gy for the 20, 30, 50 and 75 percent component of 6-Gy irradiated cells, respectively. An automated metaphase-finding system was evaluated for use with the PCC assay. This system helped to locate PCC spreads among the mitotic inducer Chinese hamster ovary (CHO) metaphase spreads, thereby facilitating rapid scoring of samples.(ABSTRACT TRUNCATED AT 250 WORDS)
