Abstract Disclosed are discreet functionally translocatable DNA segments, termed Sterol Regulatory Elements (SRE's), which are fused to heterologous structural genes to provide sterol regulatory capability to the thus formed hybrid gene. The hybrid genes respond to sterols by decreasing the production of messenger RNA. The SRE segments contain as their primary functional nucleotide sequence, a 16 bp sequence referred to as direct repeat 2, isolated from the 5' regions of the human LDL receptor gene. DNA segments which include this 16 nucleotide long sequence similarly confer sterol regulatory capability to previously known promoters such as the HSV TK promoter. Also disclosed are discreet sequences which confer positive transcription promotion to heterologous structural genes and promoters without conferring sterol responsivity. Methods are disclosed for employing these genetic control elements in a myriad of embodiments which provide for a fine-tune control of heterologous genes. Methods are also disclosed for employing the SRE in a screening assay for drugs capable of stimulating the cell to synthesize LDL receptors.
A recombinant host cell transformed with a DNA containing a sterol regulatory element, wherein said sterol regulatory element comprises a nucleic acid sequence of: (a) 5'-A-A-A-A-T-C-A-C-C-C-C-A-C-T-G-C-3' or (b) 5'-G-C-A-G-T-G-G-G-G-T-G-A-T-T-T-T-3'.
Claim 2
Dependent Claim
29 words
A recombinant host cell transformed with a DNA containing a sterol regulatory element, wherein said sterol regulatory element comprises a nucleic acid sequence of: (a) 5'-A-A-A-A-T-C-A-C-C-C-C-A-C-T-G-C-3' or (b) 5'-G-C-A-G-T-G-G-G-G-T-G-A-T-T-T-T-3'.
Claim 3
Dependent Claim
21 words
The host cell of claim 1, wherein the sterol regulatory element is located on a recombinant DNA vector within the cell.
Claim 4
Dependent Claim
15 words
The host cell of claim 1, wherein the host cell is a eukaryotic host cell.
Claim 5
Dependent Claim
16 words
The host cell of claim 3, wherein the eukaryotic host cell comprises a mammalian host cell.
Claim 6
Dependent Claim
25 words
The host cell of claim 4, wherein the mammalian host cell comprises a Hep G2, VERO, HeLa, CHO, WI38, BHK, COS-7 or MDCK host cell.
Claim 7
Dependent Claim
87 words
The host cell of claim 1, further comprising a positive LDL receptor gene promoter element in operable linkage with said sterol regulatory element, wherein said positive LDL receptor gene promoter element comprises a nucleic acid sequence of: X n , wherein n=2-5, each X being independently selected from the DNA having the nucleotide sequence of: a) 5'-A-A-A-C-T-C-C-T-C-C-T-C-T-T-G-C-3'; b) 5'-A-A-A-C-T-C-C-T-C-C-C-C-C-T-G-C-3'; c) 5'-G-C-A-A-G-A-G-G-A-G-G-A-G-T-T-T-3'; or d) 5'-G-C-A-G-G-G-G-G-A-G-G-A-G-T-T-T-3'; with each X unit being separated by from 0-20 nucleotides selected from the group of nucleotides consisting of A, G, C, and T.
Claim 8
Dependent Claim
33 words
The host cell of claim 1, wherein the sterol regulatory element is positioned adjacent to a selected structural gene such that expression of the gene can be controlled by the sterol regulatory element.
Claim 9
Dependent Claim
14 words
The host cell of claim 7, wherein the structural gene comprises a reporter gene.