Sequencing of the cholesteryl ester transfer protein 5' regulatory region using artificial transposons.
We have isolated and sequenced genomic clones encompassing more than 5 kb of the 5' flanking region of the cholesteryl ester transfer protein gene. This region contains multiple Alu repeats, a Mermaid repeat, and an extensive GA repeat, which made sequencing exceedingly difficult. To circumvent the problems that these repeats posed to traditional sequencing methodologies, we employed a novel transposon-facilitated technique, which greatly simplified sequencing of regions that had been difficult to accomplish otherwise. We utilized the artificial transposon, AT-2, a Bluescript derivative containing the dhfr gene and unique primer sites at both ends of the insertion DNA. Integration of the transposon occurred efficiently and covered the entire region of interest. Analysis of the sequence indicates a number of potential regulatory factor binding sites upstream of the previously characterized minimal promoter. The 5.7-kb regulatory region confers significant transcriptional activation in a conditionally transformed mouse hepatocyte line as compared to a minimal 137-bp promoter fragment. In addition, a tetranucleotide repeat of variable length that may provide a useful genetic marker has been identified 2 kb upstream of the CETP transcriptional start site.
Williams
S
S
Molecular Sciences Department, Pfizer Inc., Groton, CT 06340, USA.
Hayes
L
L
Elsenboss
L
L
Williams
A
A
Andre
C
C
Abramson
R
R
Thompson
J F
JF
Milos
P M
PM
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D053480:Cholesterol Ester Transfer Proteins; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics; D006023:Glycoproteins; D006801:Humans; D008099:Liver / Q000166:cytology; D051379:Mice; D018598:Minisatellite Repeats; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D017422:Sequence Analysis, DNA; D015533:Transcriptional Activation / Q000235:genetics
C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D004251:DNA Transposable Elements; D005819:Genetic Markers; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins