Hybridization studies with RNA and DNA isolated from Euglena gracilis chloroplasts and mitochondria

Abstract

Volume 42 number 3 FEBS LETTERS June 1974 HYBRIDIZATION STUDIES WITH RNA AND DNA ISOLATED FROM EUGLENA GRACILIS CHLOROPLASTS AND MITOCHONDRIA Edwin J. CROUSE*, Jobst P. VANDREY** and Erhard STUTZ* Department of Biological Sciences, Northwestern University, Evanston, H 1. 60201, USA Received 26 March 1974 1. Introduction Euglena gracilis when grown in light contains chlor- oplasts and mitochondria. Both types of organelles contain their own set of duplex DNA [1-3] which re- plicate at a different pace than the nuclear DNA [4,5] does. These organelles also are equipped with functio- nal 70 S ribosomes containing 23 S, 16 S rRNA [6]. Organellar rRNA is considered to be a transcript of the respective organellar DNA. Euglena chloroplast DNA (chDNA) was shown to code for 23 S, 16 S rRNA [7-9]. The number of cistrons per chromosome, how- ever, is still in doubt and could be between one and three [9-11]. We have reported that the hybridization capacity of chDNA for 23 S, 16 S chloroplast RNA va- ries with the amount of a 'heavy' chDNA component, isolated from highly purified chloroplasts. This 'heavy' component, depending on the average fragment size, has a buoyant density of 1.692 to 1.701 g/cc (peak densi- ties) [12]. Since Euglena mitochondrial DNA (mtDNA) was reported to have densities in the range of 1.690 to 1.692 g/cc [1-3], the 'heavy' chDNA might be, at least partly, of mitochondrial origin. In order to clari- fy this point, we found it necessary to isolate and cha racterize mtDNA from Euglena gracilis and in particu- lar to measure the affinity of chloroplast rRNA for this DNA. Further, it seemed necessary to measure as well the hybridization capacity of mtDNA towards its own major RNA components since such an experi- ment was still lacking. Mitochondrial rRNA hybridizes with mtDNA to 3.7%, while only to 0.16% with chDNA under identi- cal incubation conditions. Ctdoroplast rRNA hybridi- zes with total chDNA, containing 30% of 1.692 g/cc DNA to 2.9%, while only 0,12% with mtDNA. We conclude that the 1.692 g/cc DNA found in chloro- plast DNA preparations is not of mitochondrial origin. 2. Materials and methods 2.1. Buffers Buffer I: 0.05 M Tris-HC1 (pH 7.9), 0.I KC1, 0.01 M MgC12, 5 mM 2-mercaptoethanol. Buffer II: 0.01 M Tris-HC1 (pH 7.9), 0.1 M KC1, 0.01 M MgC12. Buffer II1:0.1 M Tris-HC1 (pH 7.9), 2.5% sodium do decyl sulfate, 0.01 MEDTA. Buffer IV: 0.1 M NaC1, 0.05 M Na2 HPO4,0.05 M NaH2PO4,0.1 mM EDTA (pH 6.8). Buffer V: 0.04 M Tris-acetate (pH 7.9), 0.1 M sodium acetate, 1 mM magnesium acetate. New addresses. * Laboratoire de physiologie v~g6table et biochimie, Istitut de Botanique, Universite de Neuchfitel: rue de Chantemerle 18, CH-2000 NEUCHATEL, Suisse. ** Department of Pathology St. Louis University School of Medicine, St. Louis, Mo. 63104, USA. 2.2. Isolation and purification of mitochondrial RNA For the isolation of mitochondria, we used the aplastidic strain W3BUL (a gift from J. A. Schiff, Brandeis University). Cells were grown in a hetero- trophic medium [13] modified by using the trace ele- ment combination of Cramer and Meyers [ 14]. W3 BUI_ 262 North-Holland Publishing Company - Amsterdam