Expression of the chloroplast ribosomal RNA genes of Euglena gracilis during chloroplast development

Abstract

The cellular content and transcription program of the chloroplast rRNA genes of E. gracilis Z were determined during the light-induced development of chloroplasts by hybridization of total cell DNA or RNA to purified 3H-labeled chloroplast ribosomal DNA ([3H]ct rDNA). Pancreatic DNase activated, partially purified chloroplast rDNA was enzymatically labeled in vitro by E. coli DNA polymerase I with [3H]TTP as a substrate. The [3H] DNA was denatured and hybridized with a vast excess of purified chloroplast 16 and 23S rRNA. The rRNA-[3H]ct rDNA hybrid was isolated by chromatography on hydroxylapatite. The [3H]ct rDNA was purified and characterized by the kinetics of its renaturation with chloroplast DNA and rRNA, and by the thermal stability of [3H]DNA-DNA and [3H]DNA-RNA hybrids. [3H]ct rDNA was hybridized in trace amounts to cellular RNA or DNA isolated from Euglena cells 0, 4, 8, 12, 24, 48 and 72 h after the onset of chloroplast development. From a comparison of the kinetics of hybridization with hybridization of standards of known kinetic complexity quantitative estimates of the cellular rRNA and rDNA gene content were made. Chloroplast rRNA increases from 2-26% of the cellular RNA during development, while the percentage of cellular DNA represented by ct rDNA increases 2- to 3-fold. Correcting for the change in cellular RNA and DNA content during development, the number of copies of the rRNA gene increases less than 2-fold, while the number of copies of rRNA per cell increases 6-fold. The results are consistent with either a transcriptional activation of the ribosomal genes or an increased rRNA stability during development.