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Analysis of a CHO Cell Line that Expresses a Peroxisomal 3-Hydroxy-3-methylglutaryl-CoA Reductase

William Engfelt

出版	University of California, San Diego, 1998
URL	http://books.google.com.hk/books?id=z4nWNT0AaHQC&hl=&source=gbs_api

註釋

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is present not only in the endoplasmic reticulum (ER) but also in the peroxisomes. To date no information is available regarding the function of the peroxisomal HMG-CoA reductase in cholesterol/isoprenoid metabolism and the structure of the peroxisomal HMG-CoA reductase has yet to be determined. We have identified a mammalian cell line that expresses only one HMG-CoA reductase protein and which is localized exclusively to peroxisomes to facilitate our studies on the function, regulation, and structure of the peroxisomal HMG-CoA reductase. This cell line was obtained by growing UT2 cells (which lack the ER HMG-CoA reductase), in the absence of mevalonate. The surviving cells exhibited a marked increase in a 90 kDa HMG-CoA reductase which was localized exclusively to peroxisomes. The UT2 cells grown in the absence of mevalonate containing the upregulated peroxisomal HMG-CoA reductase are designated UT2*. A detailed characterization and analysis of this cell line is presented in this study. The examination of UT2/UT2* cell cDNA and genomic DNA has led to the identification of two novel point mutations in intronic sequences of the ER HMG-CoA reductase gene. One mutation identified at the +1 position (G → A) of the 5 ′ splice site of exon 11-12 junction was shown to cause exon 11 skipping which resulted in the insertion of premature stop codons. A second mutation was also identified at the +5 position (G → A) of the 5′ splice site in the intron spanning exons 13 and 14. Furthermore, the data indicate that the two mutations in the reductase gene are present on the same allele. As demonstrated by RT-PCR of UT2 cell mRNA, the mutations produce aberrant spliced messages. If the aberrant messages were translated, truncated proteins of 44 kDa or 66 kDa would be predicted. More importantly, these truncated proteins would be expected not to have catalytic activity. Thus, the mutations identified in the ER reductase gene in UT2/UT2* cells indicate that neither a 97 kDa nor a 90 kDa reductase protein can be produced from this gene.