Multiple sequence elements are involved in the transcriptional regulation of the human squalene synthase gene.

The expression of human squalene synthase (HSS) gene is transcriptionally regulated in HepG-2 cells, up to 10-fold, by variations in cellular cholesterol homeostasis. An earlier deletion analysis of the 5'-flanking region of the HSS gene demonstrated that most of the HSS promoter activity is detected within a 69-base pair sequence located between nucleotides -131 and -200. ADD1/SREBP-1c, a rat homologue of sterol regulatory element-binding protein (SREBP)-1c binds to sterol regulatory element (SRE)-1-like sequence (HSS-SRE-1) present in this region (Guan, G., Jiang, G., Koch, R. L. and Shechter, I. (1995) J. Biol. Chem. 270, 21958-21965). In our present study, we demonstrate that mutation of this HSS-SRE-1 element significantly reduced, but did not abolish, the response of HSS promoter to change in sterol concentration. Mutation scanning indicates that two additional DNA promoter sequences are involved in sterol-mediated regulation. The first sequence contains an inverted SRE-3 element (Inv-SRE-3) and the second contains an inverted Y-box (Inv-Y-box) sequence. A single mutation in any of these sequences reduced, but did not completely remove, the response to sterols. Combination mutation studies showed that the HSS promoter activity was abolished only when all three elements were mutated simultaneously. Co-expression of SRE-1- or SRE-2-binding proteins (SREBP-1 or SREBP-2) with HSS promoter-luciferase reporter resulted in a dramatic increase of HSS promoter activity. Gel mobility shift studies indicate differential binding of the SREBPs to regulatory sequences in the HSS promoter. These results indicate that the transcription of the HSS gene is regulated by multiple regulatory elements in the promoter.

Binding of nuclear proteins to the enhancer elements of the rat apolipoprotein A-I gene.

To determine the cis- and trans-regulatory elements which control the expression of the apolipoprotein (apo) A-I gene, several DNA-protein binding assays, namely, gel mobility shift, exonuclease III protection, and exonuclease III footprinting assays, were employed to identify these elements. It is demonstrated that nuclear proteins of Hep G2 cells bind to five regions of DNA sequences between 252 and 149 base pairs upstream from the transcription initiation site of the rat apo A-I gene. Using South-Western blot analysis, it is determined that DNA-binding protein has a molecular mass of approximately 90 kDa. It is also shown that the DNA-binding protein was present in Hep G2 cells and rat livers but absent in rabbit livers. The results suggest that the lack of expression of the apo A-I gene in rabbit livers is due to the absence of this DNA-binding protein.

Identification of an enhancer-like element in the 5' flanking region of the rat apolipoprotein A-I gene.

In order to study the expression of the apolipoprotein (apo) A-I gene, we have isolated and characterized the structural gene encoding rat apo A-I. The 5' flanking sequence of the apo A-I gene was placed upstream of the coding sequence of the bacterial chloramphenicol acetyl transferase (CAT) gene, such that the expression of CAT activity in cultured cells is under the control of the promoter and regulatory sequences of the rat apo A-I gene. By transient transfection, nucleotide deletion and substitution methods, it was demonstrated that the nucleotide sequences between -464 and -148 upstream from the start of transcription of the rat apo A-I gene are required for the expression of this gene in Hep G2 cells and that these sequences function with an enhancer-like activity.

Rabbit apolipoprotein A-I mRNA and gene. Evidence that rabbit apolipoprotein A-I is synthesized in the intestine but not in the liver.

In order to study the tissue-specific expression of rabbit apolipoprotein (apo) A-I, a 923-base-pair clone, pRBA-502, complementary to rabbit apo A-I mRNA was identified from a rabbit intestinal cDNA library by hybrid-select translation and immunoprecipitation methods. Northern blot and dot-blot hybridization, utilizing 32P-labeled pRBA-502, revealed that the rabbit apo A-I gene is expressed in the intestine, not in the liver and that rabbit apo A-I mRNA is about 950 nucleotides in length. The entire nucleotide sequence of pRBA-502 has been determined and the complete amino acid sequence of the corresponding apo A-I has been deduced. The mRNA codes for a protein comprising 265 amino acids. Amino acids 1-18 and 19-24 of the primary translation product represent the presegment and prosegment, respectively, of apo A-I. Matured rabbit apo A-I contains 241 amino acids and has a molecular mass of 27612 Da. Using pRBA-502 as a probe, a 15.5-kb genomic fragment, which contains the entire apo A-I gene, was isolated from a rabbit liver genomic library. Sequence analysis of the gene shows that the 200 base pairs of the 5' upstream flanking region of the rabbit and human apo A-I genes showed 78% sequence homology. Like the human apo A-I gene, the rabbit apo A-I gene is interrupted by three intervening sequences. Except for two nucleotides in the fourth exon, the coding sequence of the rabbit liver apo A-I gene is identical to that of pRBA-502. Our data showed that the lack of expression of apo A-I gene in rabbit liver is not due to the alternation of rabbit liver apo A-I gene sequence and suggest that the expression of apo A-I gene in rabbit liver is regulated by a trans-acting regulating element(s).

The expression of HBsAg gene in yeast under GAL-10 promoter control.

The surface antigen gene of HBV (adr subtype) is inserted into the YEP 1 which is replicated and selected in E. coli and baker yeast Saccharomyces cerevisiae. The HBsAg gene can be expressed in yeast under GAL-10 promoter control. The protein synthesized in yeast is assembled into particles that have the same size and shape as particles isolated from plasma. One microgram surface antigen can be yielded in 100 ml culture.