Isolation of progesterone-dependent complementary deoxyribonucleic acid fragments from rhesus monkey endometrium by sequential subtractive hybridization and polymerase chain reaction amplification.

摘要: The steroid sex hormone progesterone (P) induces the expression of a variety genes through signal transduction pathway mediated by P receptor, DNA-binding regulator transcription. To identify and gene networks that are dependent in rhesus endometrium, we used powerful technique employing subtractive hybridization coupled with polymerase chain reaction (PCR). Poly(A)+ RNA was isolated from both P-dominant (days 21-23 artificial menstrual cycles) estrogen (E)-dominant 9-13) endometrium. two classes were converted to cDNA, ligated EcoRI adaptors, amplified PCR using an adaptor-complimentary primer. E-dominant cDNA labeled biotin, hybridized excess (PcDNA), complexed streptavidin. Labeled cross-hybrid sequences common populations subtracted phenol-chloroform extraction. remaining fragments PCR. After four rounds hybridization/amplification, PcDNA analyzed for P-dependent semiquantitive Initial analysis revealed housekeeping undetectable but previously characterized retained. Three five clones sequenced at random library exhibited P-inducibility/dependency E PcDNA. One these, 835-basepair fragment designated H5, may represent novel gene, as no comparable homology could be found existing GenBank Swissprot databases. We estimate procedure described here resulted highly significant enrichment up-regulated tissue.