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Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1
Published on: May 27, 2016
[The sequence of the human PrP gene Exon I and its upstream segment has promoter-like activity]
1Institute of Virology, Chinese Academy of Preventive Medicine, Beijing 100052, China.
Objective:
The human PrP gene locates at the short-arm of the 20th chromosome. This article is was to map the promoter that transcribes the human PrP gene.
Methods:
The sequence of the human PrP gene Exon I and its upstream segment were amplified using PCR, and inserted into a CAT reporter plasmid pBL-CAT6 after sequence analysis. The values of the relative CAT expression under the control of this fragment were evaluated after transiently transfected into HeLa, COS7 and Sh-sy5y cell lines. The amounts of transcription activator SP1 in these three cell lines were calculated with band-shift assays.
Results:
Analysis of the human PrP gene Exon I and its upstream segment showed a GC rich sequence, with several potential SP1 binding sites, but without any TATA-box. Under the control of this fragment, the CAT expressions were 2-3 folds increased in transient transfection. Quantity band-shift assays revealed that SP1 was enriched in HeLa cells, but undetectable in COS7 and Sh-sy5y cells.
Conclusions:
The sequence of the human PrP gene Exon I and its upstream segment functions as a promoter-like sequence, probably being as a weak TATA-less promoter. Cells derived from different tissues contain different amount of SP1. The activity of this promoter-like sequence seems to be independent of SP1 presence.
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