Transcription program of the chloroplast genome of Euglena gracilis during chloroplast development

Abstract

RNA transcription in Euglena gracifis chloroplasts has been characterized by hybridization of RNA from cells at different stages of chloroplast development to 3H-labeled chloroplast DNA restriction endonuclease fragments. Chloroplast DNA was digested into five fragments of 53,000, 35,000, 25,000, 10,000, and 6900 base pairs with Pst I. The 53,000-base-pair DNA When Euglena is grown in the dark on an oxidizable carbon source the cells lose the photosynthetic pigments and lamellae of the chloroplasts. The chloroplasts also lose the components of photosynthetic electron transport and the ability to fix CO2. In the dark, chloroplasts are replaced by proplastids, an organelle containing chloroplast DNA surrounded by a double membrane (1, 2). When dark-adapted cells are shifted to the light, chloroplast development begins whether the cells are dividing or not. Development is a temporal process, marked by the acquisition of properties of mature chloroplasts at specific intervals after the onset of light-induced development. The process is complete after 72 hr. with each cell having a full complement of mature chloroplasts (1, 2).We have been studying the changes in RNA transcription in the chloroplasts during light-induced development. Euglena chloroplast DNA, which has a genome size of 130,000-140,000 base pairs (130-140 kbp) (3, 4), is extensively transcribed at all developmental stages (5, 6). However, the extent of transcription varies significantly with both the developmental stage and the culture conditions. In exponentially growing cells on a phototrophic medium, there is an initial decrease in the fraction of the genome transcribed during the first 4 hr of growth in the light. Subsequently an induction of RNA synthesis occurs, reaching a maximum value 72 hr after the onset of development. At this time 23-26% of the DNA is transcribed, yielding RNA of 60-65 kilobase (kb) complexity (5, 7).Since these results were reported, a detailed restriction endonuclease map of the Euglena chloroplast genome has been published (4,8). With the map as reference, it has been possible to locate regions of the genome containing developmentally regulated transcription units. Specific restriction fragments from chloroplast DNA were purified, radioactively labeled to high specific activity in vitro, and utilized as hybridization probes for specific regions of the chloroplast genome. The restriction fragments used in this study were of known position on the chloroplast genome, and in aggregate represent the entire DNA sequence content. By using these hybridization probes, transcription was quantitatively studied at three stages: before induction of chloroplast development in dark-adapted cells, early during chloroplast development, and at a late stage of chloroplast development. From the results it can be concluded that the chloroplast genome contains several different types of transcription units, which can be characterized by their pattern of regulation. METHODSPreparation of RNA. The preparation of dark-adapted cultures ...

Selective in vitro transcription of chloroplast genes

“…The selective transcription of the sense strand of rDNA transcription units by the transcriptionally active chromosome (TAC) in vitro is consistent with the known in vivo transcription program of the chloroplast genome [9]. Also, with the more purified RNA polymerase in this complex, the in vitro transcripts consist predominantly of ribosomal RNAs.…”

“…The chloroplast DNA is well characterized, with the genes for 16s and 23s rRNAs [lo-121, 5s rRNA IS], and tRNAs [13-1.51 all mapped and localized. Also, the DNA has been the subject of in vivo transcription studies [8,9,16,17]. In addition, a tightly bound chloroplast DNA-dependent RNA polymerase can be isolated as a transcriptionally active chromosome (TAC) from Euglena chIoroplasts [ 181.…”

“…In Euglena gracilis, the major transcripts of the 145 kbp chloroplast genome are the 16s and 23s ribosomal RNAs [3,4], 5s rRNA 151, and at least 25 tRNAs [6,7]. These stable, abundant RNAs account for one-fourth of the total mass of RNA in light grown Euglena [8,9]. Chloroplast DNA therefore encodes the stable RNAs involved in the chloroplast translation apparatus.…”

Photo and Nutritional Regulation of Euglena Organelle Development

“…A large fraction of the Euglena chloroplast genome is transcribed in the dark and continues to be expressed in the light with the abundance of some transcripts increasing five-to tenfold upon light exposure (Chelm et al 1978(Chelm et al , 1979Chelm and Hallick 1976;Rawson and Boerma 1976a, b;Rawson et al 1981;Verdier 1979a, b). Some transcripts present in dark grown cells cannot be detected 4 h after light exposure but they are detectable 72 h after light exposure (Chelm et al 1979;Rawson and Boerma 1979).…”

“…Proplastids in dark grown cells have low levels of chloroplast rRNA (Chelm et al 1977b(Chelm et al , 1978(Chelm et al , 1979Cohen and Schiff 1976;Geimer et al 2009;Rawson et al 1981) and tRNA (Goins et al 1973;Reger et al 1970;Schwartzbach et al 1976a). The cellular content of chloroplast rRNA and chloroplast tRNA increases from approximately 5-25% of the cellular rRNA and tRNA content after 72 h of light exposure (Chelm et al 1977b(Chelm et al , 1978(Chelm et al , 1979Cohen and Schiff 1976;Rawson et al 1981;Schwartzbach et al 1976a). Transcriptome analysis suggests that the Euglena chloroplast genome is constitutively and not differentially expressed in the dark with light exposure increasing the transcription rate of all chloroplast genes proportionately (Geimer et al 2009).…”