Transcriptional origin of Euglena chloroplast tRNAs

Abstract

tRNA*DNA hybridization studies indicate that Euglena chloroplast tRNAs are transcriptional products of the chloroplast genome, which contains approximately 28 tRNA cistrons. Hybridization with purified chioroplast tRNAPhe and tRNAAP shows that the chloroplast genome contains one cistron for each of these two species. No hybridization of chloroplast tRNA with nuclear DNA was observed. tRNAs from Euglena cytoplasm, Escherichia coli, and Agmenellum quadraduplicatum do not compete with chloroplast tRNA for hybridization with chloroplast DNA. Evidence is presented that photoinduction of chloroplast tRNAs is at the level of transcription rather than maturation of tRNA precursor molecules. The chloroplasts of Euglena gracilis contain tRNAs and aminoacyl-tRNA synthetases that are (i) induced by light and (ii) exclusively compartmentalized within these organelles (1-3). The synthetases are encoded by nuclear genes and are translated on cytoplasmic ribosomes (2). In the present report we have used tRNA-DNA hybridization to ascertain the intracellular localization of the structural genes for the chloroplast tRNAs.MATERIALS AND METHODS Euglena gracilis var. bacillaris and W3BUL, an ultravioletinduced mutant lacking detectable chloroplast structure and DNA, were used (4). The blue-green alga, Agmenellum quadraduplicatum (a gift from Dr. Lonnie 0. Ingram), was grown in ASP2 medium (5) and bubbled with 1% CO2 in air.Preparation of Nucleic Acids. Chloroplasts were isolated as described (2) except that the time of zonal centrifugation was 3 hr. They were stored at -80°. For the isolation of chloroplast DNA, the frozen chloroplasts were thawed in 0.15 M NaCl, 0.1 M Na2EDTA, and 0.1 M Tris-HCl (pH 8.0), and the DNA was extracted essentially as described (6) by Marmur using boiled pancreatic ribonuclease (100 Ag/ml, Sigma, Type IIIA) followed by treatment with predigested Pronase (500 gg/ml, Calbiochem). Nuclear DNA was isolated from W3BUL by the same procedure. After precipitation with isopropanol, the DNA was further purified by preparative CsCl gradient centrifugation (7). The gradients were fractionated so as to retain all DNA species. The amount of nuclear DNA in the chloroplast DNA fraction was determined from the density-gradient profiles.Chloroplast ribosomes and rRNA were extracted as described (8). rRNA was further purified by Sephadex G-100 chromatography in order to remove any residual tRNA. tRNA was isolated as described, using both DEAE-cellulose and Sephadex G-100 column chromatography (1, 3). Cytoplasmic tRNA was isolated from W3BUL, whereas chloroplast tRNA was extracted from purified chloroplasts (2). The chloroplast tRNA was free of contaminating cytoplasmic tRNA as determined by benzoylated DEAE-cellulose column chromaAbbreviations: SSC, 0.15 M NaCI-0.015 M sodium citrate (pH 7); x X SSC, concentration of the solution is x times that of SSC; tin, thermal dissociation ("melting") temperature.* A brief report of this work appeared in Plant Physiol. (1975) 56, S71. tography of the purified chloroplast...

Distribution of transfer RNA genes in thePisum sativum chloroplast DNA

“…A total of 30 tRNA genes were identified using these individual recombinant clones of ctDNA. This number is very close to that reported for Pisum sativum ctDNA (14) and to those found in other organisms (5,9,19,20).…”

“…The inability to localize the genes for three distinct aminoacyl tRNAs in ctDNA of Zea mays and Pisum sativum probably result from inadequate charging of the tRNAs rather than the lack of their genes in ctDNA (14,20). It was shown that there are about 25 tRNA genes in Euglena gracilis (19). Spinach tRNAs were fractionated into about 35 spots by two dimensional gel electrophoresis, and 21 tRNA genes were located (5).…”

Photo and Nutritional Regulation of Euglena Organelle Development

“…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).…”

“…Chloroplast encoded proteins are synthesized within the chloroplast using chloroplast ribosomes (Schwartzbach et al 1974) tRNAs, (Goins et al 1973;Hallick et al 1984;Reger et al 1970;Schwartzbach et al 1976a) and tRNA synthetases (Barnett et al 1976;Hecker et al 1974;Parthier et al 1972;Reger et al 1970). 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).…”

PHOTOCONTROL OF ORGANELLE BIOGENESIS IN Euglena

“…The chloroplast protein synthetic machinery consists of a set of tRNAs (Schwartzbach et al, 1976b;Hallick et al, 1984), tRNA synthetases (Parthier et al, 1972;Hecker et al, 1974;Reger et al, 1970), and ribosomes (Schwartzbach et al, 1974) which are distinct from their cytoplasmic counterparts. Low levels of chloroplast rRNA (Cohen and Schiff, 1976;Chelm et a/., 1977aChelm et a/., , 1978Chelm et a/., , 1979Rawson et a/., 1981) and tRNA (Reger et al, 1970;Goins et al, 1973;Schwartzbach e t a / .…”