The isolation of transcription factors from lambda gt11 cDNA expression libraries: human steroid 5 alpha-reductase 1 has sequence-specific DNA binding activity.
The Surf-1/Surf-2 bi-directional promoter contains binding sites for at least three transcription factors (Su1, Su2, and Su3). By screening a lambda gt11 HeLa cell cDNA expression library with a concatenated Su2 factor binding site, we isolated a cDNA which encodes a protein with sequence-specific DNA binding activity. Gel retardation assays showed that the cloned factor binds specifically to the Su2 factor binding site present in the human Surf-1/Surf-2 promoter but not to an Su2 site containing mutated base pairs. Co-transfection experiments demonstrated that the cloned cDNA had little or no effect on the expression of a reporter gene under the control of multiple Su2 factor binding sites. Similarly a fusion protein in which the acidic activation domain of HSV VP16 was linked to the cloned factor had no effect, implying that the factor does not function as a DNA binding protein in vivo. DNA sequence analysis revealed that the cloned cDNA is identical to that of human steroid 5 alpha-reductase 1, an enzyme which converts testosterone to dihydrotestosterone. These results are discussed with respect to other putative transcription factors which have been isolated from cDNA expression libraries on the basis of their sequence-specific DNA binding activity.
Gaston
K
K
Eukaryotic Gene Organisation and Expression Laboratory, Imperial Cancer Research Fund, London, UK.
Fried
M
M
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification*
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004247:DNA; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase