Transcription of the chicken α 2 (Type I) collagen gene by homologous cell-free extracts

Abstract

We have used two methods to detect specific transcription of the chicken α2 (type I) collagen gene in cell-free extracts derived from Rous sarcoma virus-transformed chicken embryo fibroblasts. The first method is a modification of the S1 nuclease mapping procedure which utilizes a DNA probe labeled with ³²P at the 5' end of the HindIII linker originally used to clone the collagen promoter region into pBR322. The probe distinguishes newly made, specific RNA from endogenous RNA and nonspecific transcripts. Using this procedure we have found that chicken whole cell extracts support accurate initiation of transcription of the chicken α 2 (type I) collagen DNA template. Addition of either creatine phosphate, GTP, or UTP to concentrations of approximately 3 to 5 mM was found to stimulate RNA polymerase II transcription by 5-to 10-fold. The second method employs an avian myeloblastosis virus reverse transcriptase-catalyzed primary extension procedure, rendered in vitro-specific by use of a pBR322 fragment as primer. These two techniques should be useful for analyzing specific transcription in other types of cell-free extracts.