CHARACTERIZATION OF THE MEVALONATE KINASE 5'-UNTRANSLATED REGION PROVIDES EVIDENCE FOR COORDINATE REGULATION OF CHOLESTEROL-BIOSYNTHESIS

Using a probe derived from the 5'-untranslated region of the human mev alonate kinase (MK) cDNA, we screened a lambda gt 11 genomic library a nd obtained a single clone containing the 5' untranslated region of th e gene, Nucleotide sequencing identified several putative regulatory e lements, including two Sp1 (GC box) elements and a CCAAT box, A canoni cal TATA box was not detected, Directly adjacent to one Spl element wa s a sterol regulatory element (SRE), 5'-CACCCCAG-3', which was a 7/8 b ase pair match to the consensus sequences identified in the genes enco ding 3-hydroxy-3-methylglutaryl-coenzyme A synthase and reductase, and the LDL receptor, There was no Spl element upstream of the SRE. North ern blot analysis in human CRL1508T cells revealed that quantities of MK poly A(+) RNA increased for cells grown in the presence of lipid-de ficient calf serum, and further increased upon addition of 1 mu M lova statin, Primer extension analysis with human poly A(+) RNA suggested a t least 4 transcription initiation sites downstream from the CCAAT box , To assess sterol responsiveness of transcription initiation, a 1.4 k b genomic fragment upstream of the translational start site was fused to the pSV2(cat) vector for transient expression in COS-7 cells, with chloramphenicol acetyltransferase (CAT) as the reporter gene, This con struct demonstrated modest levels of CAT expression which was induced > 2-fold when cells were grown in Lipoprotein-deficient calf serum, Ou r data provide further evidence for coordinate regulation of cholester ol biosynthesis in response to sterol, (C) 1998 Academic Press.