Current Genetics (1982) 6:63-69

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© Springer-Verlag 1982

Fractionation and Identification of Euglena gracilis Cytoplasmic and Chloroplastic tRNAs and Mapping of tRNA Genes on Chloroplast DNA M. Kuntz, M. Keller, E. J. Crouse, G. Burkard, and J. H. Well Institut de BiologieMol6culaire et Cellulaire du CNRS, Universit6 Louis Pasteur, 15 rue Descartes, 67084 Strasbourg~ France

Summary. The cytoplasmic and chloroplast tRNAs of Euglena gracilis Z strain were fractionated by twodimensional gel electrophoresis and identified by aminoacylation. Purified chloroplast tRNAs, labeled in vitro with 13zPl, were hybridized to endonuclease restriction fragments of chloroplast DNA, allowing the corresponding tRNA genes to be localized on the physical map of Euglena chloroplast DNA. Key words: Euglena gracilis Z strain - tRNAs - Chloroplast tRNA genes - Hybridization

Introduction

Chloroplasts contain multiple copies of a circular DNA molecule. Several laboratories working on different strains of Euglena gracilis have constructed restriction endonuclease cleavage site maps of the chloroplast chromosome (reviewed in Hallick 1982). These physical maps can be used for the localization of chloroplast genes. Three tandem sets of rRNA genes have been mapped on the chloroplast DNA molecule of Euglena gracilis as well in the Z strain (Rawson et al. 1978; Gray and Hallick 1978; Jenni and Stutz 1978) as in the bacillaris strain (Helling et al. 1979). In addition to the three rRNA units, a supplementary 16S rRNA gene was mapped on the chloroplast DNA of Euglenagracilis strain Z (Jenni and Stutz 1979). From electron microscopy, this extra 16 rRNA gene was found to have the same orientation and length as the 16S rRNA genes in the three complete units (Koller and Delius 1982). But in another Euglena gracilis strain, only one rRNA unit was found (Wurtz and Buetow 1981).

Offprint requests to: J. H. Weil

Euglena chloroplast DNA also codes for a set of approximately 2 5 - 3 0 tRNAs (Schwartzbach et al. 1976; McCrea and Hershberger 1976; Gruol and Haselkorn 1976). Two tRNA genes, one for tRNA ne and one for tRNA Ma, were mapped and sequenced in the ribo, somal spacer located between the 16S and 23 S rRNA genes (Keller et al. 1980; Orozco et al. 1980;Graf et al. 1980). A mixture of tRNA Trp and tRNA Glu hybridized to the region proximal to the 5'-end of the 16S rRNA gene (Keller et al. 1980). From sequencing data (Orozco et al. 1980), this was shown to be due to a partial tRNA Trp gene. All three rRNA operons are believed to contain the same tRNA genes (Keller et al. 1980; Orozco et al. 1982a). In this study, the fractionation and identification of tRNAs from light-grown cells, dark-grown cells and isolated chloroplasts of Euglena gracilis Z strain are reported. The chloroplast tRNAs are shown to be distinct species from their cytoplasmic counterparts. Hybridization of the purified chloroplast tRNAs to restriction fragments of the chloroplast DNA was then used to localize tRNA genes on the physical map of the DNA molecule.

Materials and Methods

Isolation, Fractionation and Identification of tRNAs. Euglena graeilis Z strain (G6ttingen Collection n° 1224) was grown, either in continuous light or in continuous darkness, in a medium described by Vasconcelos et al. (1971). The total cellular tRNAs were isolated as previously described (Burkard et al. 1970). Chloroplasts were isolated according to the method of Ortiz et al. (1980) as modified by Ortiz and Stutz (1980). The chloroplast total tRNAs were extracted from purified chloroplasts as previously described (Burkard et al. 1970). Fractionation of the total tRNA populations into individual tRNA species was accomplished by two-dimensional potyaerylamide gel electrophoresis (Fradin et al. 1975; Burkaxdet al. 1982). 0172-8083/82/0006/0063/$ 01.40

M. Kuntz et al.: Mapping tRNA Genes on Euglenagraeilis Chloroplast DNA

64

Table 1. Identification of the cytoplasmic and chloroplast tRNA species ofEuglena graciIis tRNA specific for

Ala Arg Ash Asp Cys Glu

Cytoplasmic tRNA

Chloroplastic tRNA

Number ofisoaccepting species

Numbering in Fig. la Fig. lb

2 2 2 1 -

36, 44 _

33, 39 28, 31 24, 48 32

Number ofisoacepting species

Numbering in Fig. la

1 2 1 _

45 33, 46 _ _

-

-

1

-

--

--

1

--

Gin

Gly His Ile Leu Lys Met Phe Pro Set Thr Trp Tyr Val

2 1 2 3 3 2 1

63, 67 42 (43) 21, 23 14, 15, 28 22

44, 46 27 19, 21 14, 15, 18 28, 49, 50 23, 26 20

1 1 2 2 1 2 1

--

--

1

3 1 1 1 2

8, 11, 13 25 71 37 59, 60

After visualization of the RNA species by methylene blue staining, the spots were cut from the gel and the tRNAs extracted. The individual tRNA species were identified by aminoacylation (Burkard et al. 1970) using yeast or Euglena cytoplasmic aminoacyl-tRNA synthetases for cytoplasmic tRNAs and E. eoli or Euglena chloroplast aminoacyl-tRNA synthetases for chloroplast tRNAs (Well 1979).

Preparation of 132pi Labeled Chloroplast tRNAs. Total chloroplast tRNA or individual tRNA species were labeled in vitro after removal of the terminal adenosine from the 3'-end of the molecule using either periodate oxidation (in the case of total chloroplast tRNA) or snake venom phosphodiesterase (in the case of individual tRNA species). Labeling of the tRNA was performed enzymatically (Silberklang et al. 1977) using a 132pi ATP and yeast nucleotidyl transferase (Rether et al. 1974). These total or individual chloroplast 132pI tRNAs were used in hybridization experiments as previously described (Driesel et al. 1979).

Preparation of ChloroplastDNA. Euglena chloroplast DNA was isolated as described by Kopecka et al. (1977) or Driesel et al. (1979). Cleavage of the DNA by the restriction endonucleases Bali, BamHI, EcoRI, HaelI, KpnI or Pvull was carried out as recommended by the suppliers (Boehringer, Mannheim, and New-England Biolabs, Beverly). The cleavage products were separated on 0.7%-1.0% Seakem agarose slab gels (Marine Colloids Inc., Rockland), visualized with ethidium bromide under ultraviolet light and photographed using a Polaroid 665 film. The DNA fragments were transferred from the gel to nitrocellulose strips (Southern 1975) and hybridized to labeled tRNAs (Driesel et al. 1979).

10, 11, 13 23 53 22 40, 45

1 2 1 1 1

28 29, 30 8, 19 50 27 --

52 18 5 24

Results

Fractionation and Identification of Euglena tRNAs The total cellular t R N A p o p u l a t i o n from either lightgrown or dark-grown Euglena cells was fractionated b y two-dimensional polyacrylamide gel electrophoresis. The total t R N A p o p u l a t i o n from green cells was resolved into a b o u t 80 species and the t R N A p o p u l a t i o n from etiolated cells into a b o u t 60 species. Comparison o f the two patterns (Fig. l a and l b ) revealed that 29 spots c o n t a i n cytoplasmic tRNAs, specific for 16 a m i n o acids, as they can be aminoacylated using yeast or Euglena cytoplasmic a m i n o a c y l - t R N A synthetases (Table 1). Most o f the unidentified RNAs are either chloroplast or m i t o c h o n d f i a l t R N A species which were n o t aminoacylated b y yeast or Euglena cytoplasmic aminoacylt R N A synthetases. Of the identified cytoplasmic t R N A species, there are single t R N A species for 6 amino acids (aspartic acid, histidine, phenylalanine, t h r e o n i n e , t r y p t o p h a n and tyrosine). Two isoaccepting species were identified for 7 amino acids (alanine, arginine, asparagine, glycine, isoleucine, m e t h i o n i n e and valine) and three isoaccepting species were f o u n d for 3 amino acids (leucine, lysine and serine). In two cases, two different t R N A species co-migrated in the polyacrylamide gel.

M. Kuntz et al.: Mapping tRNA Genes on Euglena gracilis Chloroplast DNA

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Fig. la. Fractionation of total tRNAs from green cells of Euglena gracilis by two-dimensional polyacrylamide gel electrophoresis. The numbers refer to the tRNAs listed in Table 1. Circles with dotted lines correspond to weaker spots

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Fractionation and identification of Euglena gracilis cytoplasmic and chloroplastic tRNAs and mapping of tRNA genes on chloroplast DNA.

The cytoplasmic and chloroplast tRNAs of Euglena gracilis Z strain were fractionated by two-dimensional gel electrophoresis and identified by aminoacy...
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