Euglena gracilis Chloroplast DNA Codes for Polyadenylated RNA

Abstract

Polyadenylated RNA, isolated from total cellular RNA of pbotoautotrophicafly grown Euglkna graciis, comprised 2.1% of the total cellular RNA and contained 6.2% polyadenylic acid. Polyadenylated RNA, labeled in vitro with "I, hybridized at saturating levels to an average 7.7% of the chloroplast DNA. In the presence of excess chloroplast rRNA, hybridization of polyadenylated RNA was reduced, but was still observed at a level corresponding to 2.8% of the chloroplast DNA. Polyadenylic acid was not detected in mRNA prepared from chloroplast polyribosomes, indicating a level of less than 0.1% polyadenylic acid in mature chloroplast mRNA. Of the total RNA isolated from cytoplasmic polyribosomes, 2.0% contained polyadenylic acid. This latter polyadenylated RNA did not hybridize to chloroplast DNA. In eukaryotes, most proteins coded by the nuclear DNA are translated from mRNAs which have poly(A) tracts at their 3'-termini (6). In contrast, major chloroplast proteins (24) are translated from mRNAs which are isolated from the non-poly(A)-RNA5 fraction (26,32). Even so, chloroplasts may contain small quantities ofpoly(A)-RNA. For example, the amount of this RNA isolatable from maize chloroplasts was I ,ug/kg leaves (14). However, the question of contamination of chloroplast preparations from higher plants and algae with cytoplasmic poly(A)-RNAs has not been completely resolved (14,26,32). In sum, any role for poly(A)-RNA in the case ofchloroplasts remains to be determined.Previously, we isolated chloroplast polyribosomes (2) and ctDNA (21) (27). The lysate was deproteinized by two extractions with phenolcresol (17) saturated with buffer (27) and containing 0.4 to 1.0%Yo tri-isopropyl naphthalene sulfonate (17,27). Polyribosomes were extracted similarly with phenol-cresol to prepare samples for isolation of poly(A)-RNA. One volume of 3.0 M K-acetate (pH 4.5) and 2 volumes of ethanol were added to the aqueous phase and the RNA was precipitated ovemight at -18 C. The precipitate was collected by centrifugation, washed twice with absolute ethanol, dissolved in 0.1 x SSC at a concentration of 2 to 5 mg/ ml, and stored at -70,C until used.The RNA was separated into poly(A)-RNA and non-poly(A)-RNA fractions by chromatography at room temperature on columns (0.9 x 3.0 cm) of poly(U)-Sepharose (Pharmacia) according to Lindberg and Perrson (18).Preparation of Chloroplast rRNA, DNA and Polyribosomes. Chloroplast rRNA and DNA were prepared from chloroplasts isolated from photoheterotrophic cells as described (2, 21). Each preparation of DNA, analyzed by centrifugation in an analytical CsCl gradient (28, 29) was free of contaminating nuclear or mitochondrial DNA.Chloroplast polyribosomes free of contaminating cytoplasmic or mitochondrial ribosomes were prepared from photoautotrophic cells essentially as described (2). Polyribosomes so prepared were previously shown to be chloroplast polyribosomes by their specific dissociation in buffer containing a low concentration of Mg2+, by their degradation patterns with RNase and with E...

A small portion of plastid transcripts is polyadenylated in the flagellate Euglena gracilis

“…The polyadenylation of plastid transcripts in E. gracilis was previously studied by hybridization experiments of labeled plastid RNA to plastid DNA, and it was concluded that plastid rRNAs were polyadenylated to some extent, while plastid mRNAs were not [38]. Using more precise PCR and quantitative PCR methods, in this study we have demonstrated that a small portion of plastid mRNAs is polyadenylated as well.…”

Polyadenylated RNA in two filamentous cyanobacteria.

“…3 that rRNA appears to hybridize to [3H]poly(U). It has been shown that chloroplast polyA+ RNA contains sequences whose hybridization to chloroplast DNA is competed by chloroplast rRNA (12). Recent analyses of polyA+ RNA from Nostoc sp.…”

Molecular Biology of Chloroplasts

“…W h e n isolated from fully-green Euglena, polyadenylated R N A hybridizes 2.8% of the chloroplast D N A (Milner et al, 1979). W h e n isolated from fully-green Euglena, polyadenylated R N A hybridizes 2.8% of the chloroplast D N A (Milner et al, 1979).…”

“…W h e n isolated from fully-green Euglena, polyadenylated R N A hybridizes 2.8% of the chloroplast D N A (Milner et al, 1979). O v e r 6 0 % , however, of the chloroplast polyadenylated R N A fraction from fully g r e e n Euglena contains sequences whose hybridization to chloroplast D N A is c o m p e t e d by chloroplast r R N A (Milner et al, 1979). O v e r 6 0 % , however, of the chloroplast polyadenylated R N A fraction from fully g r e e n Euglena contains sequences whose hybridization to chloroplast D N A is c o m p e t e d by chloroplast r R N A (Milner et al, 1979).…”

“…O v e r 6 0 % , however, of the chloroplast polyadenylated R N A fraction from fully g r e e n Euglena contains sequences whose hybridization to chloroplast D N A is c o m p e t e d by chloroplast r R N A (Milner et al, 1979). In Euglena at least, the n a t u r e of the chloroplast-coded polyadenylated RNAs r e m a i n s to be identified, because m a t u r e chloroplast m R N A s found in chloroplast polyribosomes a r e not polyadenylated (Milner et al, 1979). At least some of t h e chloroplast-coded polyadenylated RNAs have been considered to be m R N A s (Haff a n d B o g o r a d , 1976b; Verdier, 1979a,b), as a r e the polyadenylated RNAs coded by the nucleus.…”