Abstract
We have developed a spinach plastid run-on transcription system to determine the extent of transcriptional regulation of chloroplast genes during morphogenetic changes of the organelle. In contrast to transcription in a spinach chloroplast extract, which requires initiation of exogenously added genes (Gruissem, W., Greenberg, B. M., Zurawski, G., and Hallick, R. B. (1983) Cell 35, 815-828), RNA synthesis in the run-on system is not affected by heparin or different salt concentrations. Transcription is asymmetric, and the size of the run-on transcripts varies between 75 nucleotides and 8 kilobases. Quantitative filter hybridization studies included gene-specific probes for the ribosomal RNA genes and nine protein-coding genes. Based on the amounts of hybridizable run-on transcripts, these genes can be ordered according to their respective transcriptional activities. The relative transcriptional activities of psbA, rbcL, and atpB in the run-on assay correlate closely with their reported promoter strengths in vitro. The plastid run-on transcription assay has been applied to determine the transcriptional regulation of plastid genes. Hybridization of run-on transcripts to regions of the spinach chloroplast genome containing at least nine tRNA genes indicates that most or all loci are highly transcribed. No significant qualitative and quantitative differences are detected when run-on transcripts from plastids of etiolated and greening cotyledons are hybridized to total, restriction enzyme-digested chloroplast DNA, demonstrating limited transcriptional regulation during chloroplast development.
Highlights
We have developed a spinach plastid run-on tran- products in the dark [2]
An run-on system is not affected by heparin or different additional increase of psbA mRNA levels occurs during leaf salt concentrations
We have developed a new transcription system from isolated intact spinach plastids thaetnables us to quantify runon transcripts for individual chloroplast genes, providing
Summary
When intact chloroplasts were the characterization of run-on transcripts in hybridization assays, the added to thereaction mixture, a low level of RNA synthesis run-on transcription was carried out for 8 min, after which it was was consistently observed (Fig. 1).This is in agreement with terminated by addition of p1 of5% sodium sarcosinate, 50 mM Tris-HC1, pH 8.0,25 mM EDTA and extracted with phenol/chloroform/isoamyl alcohol. Filter Hybridizations-Chloroplast and plasmid DNAs were digested with restriction enzymes, fractionated in 0.9% agarose gels velope membrane limits the amount of nucleotide triphosphates available for efficient RNA synthesis in the organelle. + lated from standard reactions with disrupted young leaf chloroplasts and 100 pCi of [a-32P]UTP.The blot containing the and - DNA strands of rbcL, atpB, and M13mp DNA was hybridized with runon transcripts (total activity 7.2 X lo cpm) for 24 h.
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