title: Identification of vitamin D target genes in human keratinocytes by subtractive screening.
abstract: 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2) D(3)) imposes cell cycle block in late G1 phase in cultured human keratinocytes. We wanted to identify early vitamin D target genes using a subtractive screening approach. Human foreskin keratinocytes were grown to about 70% confluence, treated with 2 x 10(-7) M 1alpha,25(OH)(2) D(3) or left untreated and RNA from both populations were isolated after 22h of incubation. cDNA was synthesised and cloned into plasmid vectors. For screening of the libraries, cDNA was amplified in vitro using T7 RNA polymerase and then the amplified RNA (driver, control population) and single stranded cDNA (tester) were used for subtractive hybridisation. Heterohybrids were then separated from single stranded nucleotides using a hydroxyapatite column. The radiolabeled single stranded cDNA was used for screening a colony blot. Positive clones were rescreened, plasmid DNA was isolated and used for verifying the results by Northern blot analysis, using RNA isolated from untreated keratinocytes, as well as RNA isolated after 6h, 12h and 24h of vitamin D treatment.
authors.affiliation: Department of Genetics and General Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria. pamela.moll@sbg.ac.at
authors.forename: Pamela Renate
authors.initials: PR
authors.lastname: Moll
authors.forename: Alfred
authors.initials: A
authors.lastname: Klausegger
authors.forename: Helmut
authors.initials: H
authors.lastname: Hintner
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Reischl
authors.forename: Michael
authors.initials: M
authors.lastname: Breitenbach
authors.forename: Klaus
authors.initials: K
authors.lastname: Richter
mesh_terms: D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D013382:Subtraction Technique
chemical_list: D018076:DNA, Complementary