Abstract
The androgen-regulated 20-kDa protein gene consists of four exons that code for a major secretory protein of rat ventral prostate. Analysis of its potential cis-acting transcriptional regulatory elements revealed that a large intron 1 region (In-1) had stronger androgen response element (ARE) activity than did the 5'-flanking DNA. In cotransfected CV1 cells, In-1 and its most active subfragment In-1c functioned as AREs but not glucocorticoid response elements (GRE). Nevertheless several ARE/GRE-like partial palindromic sequences are present in In-1c, and it bound both androgen receptors and glucocorticoid receptors in mobility shift assays. A cluster of three ARE/GRE-like sequences contained within a 39-base pair sequence of In-1c had both ARE and GRE activities when analyzed as an isolated oligonucleotide, suggesting that other elements within In-1c determined its ARE specificity. In addition to ARE/GRE-like sequences, In-1c contains putative response elements for the transcription factors AP1, CREB, AP2, OCT-1, C/EBP, and a number of inverted and direct repeats. The ARE specificity of In-1c observed in CV1 cells was diminished in PC3 and HeLa cells transiently cotransfected with an androgen receptor or glucocorticoid receptor expression vector together with an In-1c reporter vector; however, the ARE activity of In-1c was greater than its GRE activity in these cell lines. Interestingly, a 131-base pair subfragment of In-1c retained ARE specificity in all three cell lines.
Highlights
A Complex Response Element in Intron1 of the Androgen-regulated ZO-kDa Protein Gene Displays Cell Type-dependent Androgen Receptor Specificity*
Analysis of its potential alocorticoid receptors, containsa high degree of sequence cis-acting transcriptionarlegulatory elements revealed that a large intron1region (In-1) had stronger androgen response element (ARE)activity thandid the 5’-flanking DNA
Dromic sequences are present in In-lc, and it bound AGTACGn,TGTTCT [9,10,13] within the first intron which both androgen receptors and glucocorticoid receptors functions as a glucocorticoid response element (GRE) in in mobility shift assays
Summary
In addition to non-GR residues present in T7X556, the resulting construct, pET16b-rGR-DBD, contained 19 non-GR amino and clone H8 from the HaeIII partiallydigested library ( cloned in Charon 4A at EcoRI site with EcoRI linker) When these genomic fragments were restricted with EcoRI,clone R6 yielded 7 bands and clone H8 11 bands on agarose gel electrophoresis. The 3' portion of the 20-kDa protein cDNA (nucleotides 353-809) hybridized with two fragments of clone H8 (1,255 bp and approximately 4 kilobases, respectively) (data not shown)C. lone H 8 contained a 258-bp EcoRI fragment into the expression vector pET16b. No increase in CAT activity was detected with the proteins rGR-DBD or rAR-DBD were eluted with 5ml of lysis buffer 5' fragment, nucleotides -901 to +8 Consensus sequences of transcription factorsare from Refs. 54 and 55
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