Edwin J. Crouse

Fractionation and identification of spinach chloroplast transfer RNAs and mapping of their genes on the restriction map of chloroplast DNA

Spinach chloroplast 4S RNAs has been separated by two-dimensional polyacrylamide gel electrophoresis into about 35 species. After extraction from the gel, 27 of these RNA species were identified by aminoacylation as tRNAs specific for 16 amino acids. Individual tRNAs were labeled in vitro with 125I and hybridized to DNA fragments obtained by digestion of spinach chloroplast DNA with KpnI, PstI, SalI and XmaI restriction endonucleases. A minimum of 21 genes corresponding to tRNAs for 14 different amino acids have been localized on the restriction endonuclease cleavage site map of the DNA molecule. Of these, 15 genes corresponding to tRNAs for 12 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. Each copy of this repeat region contains a set of genes for the ribosomal RNAs and a gene for tRNA2Ile in the spacer sequence between the 16S and 23S ribosomal RNAs. The genes for tRNA1Ile, tRNA2Leu and tRNA3Leu also map in the repeat region, but outside the ribosomal DNA unit. At present, two more chloroplast tRNAs (for Pro and Lys) have been identified, but not mapped, while 4 unidentified 4S RNAs have been mapped in the large single-copy region of the DNA molecule. Evidence is presented that isoaccepting tRNA species can be transcripts from different loci.

Homologies among ribosomal RNA and messenger RNA genes in chloroplasts, mitochondria and E. coli.

SummaryLabelled chloroplast rRNAs from Spinacia oleracea were hybridized to restriction endonuclease digests of chloroplast DNA from Oenothera hookeri and Euglena gracilis, to mitochondrial DNA of Acanthamoeba castellanii, and to DNA of the E. coli rrn B operon in the transducing phage lambda rifd18. The degree of homology is greatest for the 16S rRNA gene. Greater than 90% occurs between the two higher plant genes, 80% homology to the lower plant gene, 60%–70% homology to the bacterial gene, and 20% homology to the mitochondrial gene. The degree of hybridization varied considerably for the 23S and the 5S rRNA genes. Very high homology exists between the two higher plant genes, only about 50% homology for both the Euglena and bacterial genes, and no significant homology for the mitochondrial genes. These results show that any chloroplast (or E. coli) rRNA may be used as a probe to identify rRNA genes in other ctDNAs.Two RNA populations, each enriched for a different ctDNA-encoded mRNA, proved useful in the location of these genes on both higher plant ctDNAs. No significant hybridization was obtained using these probes to the Euglena ctDNA which seems to be too distantly related.

Chloroplast and Cyanobacterial Genomes, Genes and RNAs: a Compilation

The organization and expression of plastid genomes are among the most extensively studied rid& in plant molecular biology. Restriction endonuclease cleavage site maps have been constructed for the plastid genomes of many plant species. Genes for the rRNAs, for most of the tRNAs and for several proteins have been located on these physical maps. Furthermore, the nucleotide sequences of many of these genes, and/or the corresponding RNAs, have been determined. This article, which is an up-date of the information found in Bohnert et al., 1982 and Crouse et al., 1984, compiles the information on plastid genomes, genes and RNAs which has appeared in the literature. Some highlights of the data, along with the literature citations, are presented in tableform. In addition, related information on cyanobacteria is included. Table 1 lists the plastid genomes for which physical maps have been constructed, and indicates the genes which have been mapped. Sequenced genes for rRNAs, tRNAs and proteins are compiled in Tables 2, 3 and 4, respectively. Gene

Construction of the physical map of the chloroplast DNA of Phaseolus vulgaris and localization of ribosomal and transfer RNA genes

Construction of a physical map of the chloroplast DNA from Phaseolus vulgaris showed that this circular molecule is segmentally organized into four regions. Unlike other chloroplast DNAs which have analogous organization, two single-copy regions that separate two inverted repeats have been demonstrated to exist in both relative orientations, giving rise to two populations of DNA molecules. Hybridization studies using individual rRNA and tRNA species revealed the location of a set of rRNA genes and at least seven tRNA genes in each inverted repeat region, a minimum of 17 tRNA genes in the large single-copy region and one tRNA gene in the small single-copy region. The tRNA genes code for 24 tRNA species corresponding to 16 amino acids. Comparison of this gene map with those of other chloroplast DNAs suggests that DNA sequence rearrangements, involving some tRNA genes, have occurred.

Transfer RNA gene mapping studies on cyanelle DNA from Cyanophora paradoxa

SummaryThe 4S RNA of cyanelles from Cyanophora paradoxa strain LB 555 UTEX was fractionated by two-dimensional gel electrophoresis. Individual tRNA species were identified by aminoacylation, labeled in vitro and hybridized to restriction endonuclease fragments of cyanelle DNA. Hybridization experiments, using individual tRNA species, have revealed the location of two tRNA genes, coding for tRNAAla and tRNAIle, in each of the two spacer segments separating the 16S and 23S rRNA genes on the two inverted repeats (10 kbp each) and three tRNA genes in the small single-copy region (17 kbp) separating the two inverted repeats. A minimum of 14 tRNA genes in the large single-copy region (88.5 kbp) has also been found.Heterologous hybridization studies, using cyanelle tRNAs and chloroplast DNA from spinach, broad bean, or maize, indicate a high degree of homology between some tRNAs from cyanelles and chloroplasts.Although cyanelles are often condisered as having evolved from endosymbiotic cyanobacteria, the organization of tRNA genes on cyanelle DNA and the results of heterologous hybridization studies show that cyanelles are related to higher plant chloroplasts.

Transfer RNA genes ofZea mays chloroplast DNA

A minimum of 37 genes corresponding to tRNAs for 17 different amino acids have been localized on the restriction endonuclease cleavage site map of theZea mays chloroplast DNA molecule. Of these, 14 genes corresponding to tRNAs for 11 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. One tRNA gene is in the smaller single-copy region. Each copy of the large repeated sequence contains, in addition to the ribosomal RNA genes, 11 tRNA genes corresponding to tRNAs for 8 amino acids. The genes for tRNA2Ile and tRNAAla map in the ribosomal spacer sequence separating the 16S and 23S ribosomal RNA genes. The three isoaccepting species for the tRNAsLeu and the three for tRNAsSer, as well as the two isoaccepting species for tRNAAsn, tRNAGly, tRNAsIle, tRNAsMet, tRNAsThr, are shown to be encoded at different loci.Two independent methods have been used for the localization of tRNA genes on the physical map of the maize chloroplast DNA molecule: (a) cloned chloroplast DNA fragments were hybridized with radioactively-labelled total 4S RNAs, the hybridized RNAs were then eluted, and identified by two-dimensional polyacrylamide gel electrophoresis, and (b) individual tRNAs were32P-labelledin vitro and hybridized to DNA fragments generated by digestion of maize chloroplast DNA with various restriction endonucleases.

Transfer RNAs and tRNA genes of Vicia faba chloroplasts.

SummaryIsolated chloroplasts from broad bean and common bean were found to contain a minimum of 31 and 32 tRNA species, respectively. These individual chloroplast tRNAs were 32P-labeled in vitro and hybridized to DNA fragments obtained upon digestion of broad bean and common bean chloroplast DNAs with various restriction endonucleases. At least 30 tRNA genes were localized on the physical maps of the two chloroplast genomes. Comparison of the broad bean tRNA gene map to that of common bean revealed DNA sequence rearrangements, such as inversions, insertions/ deletions and duplications, within these two members of the Legu minosae family.

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

SummaryThe cytoplasmic and chloroplast tRNAs of Euglena gracilis Z strain were fractionated by two-dimensional gel electrophoresis and identified by aminoacylation. Purified chloroplast tRNAs, labeled in vitro with |32P|, 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.

Comparison of the cyanelle DNA from two different strains of Cyanophora paradoxa.

SummaryThe cyanelle DNA from two different strains of Cyanophora paradoxa (strain LB555UTEX and strain 1555) was investigated.The cyanelle DNA from both strains showed a buoyant density in neutral CsCI gradients of 1.692 g/cm3. The total molecular weight, as judged by restriction endonuclease analysis, of the two cyanelle DNAs differed. In strain LB555UTEX the size of the cyanelle DNA was equivalent to 127 ± 1 kb whereas in strain 1555 a size of 138 ± 1 kb was consistently found. The sizes of individual DNA fragments and the number of recognition sites for a particular restriction endonuclease appeared largely unrelated.A high amount of cross hybridization, as judged by reciprocal heterologous DNA hybridizations, however indicated a high degree of sequence homology between the two cyanelle DNAs. Under comparable conditions, cyanelle DNA hybridized nearly exclusively with the dG+dC-rich rRNA transcription units from plastid DNAs. Up to now conserved restriction endonuclease recognition sites between the two cyanelle DNAs were only observed within the cyanelle rRNA genes which are present twice on both cyanelle DNAs.

Mapping of tRNA Genes on the Circular DNA Molecule of Spinacia oleracea Chloroplasts

Chloroplasts contain their own complement of tRNAs, which are different from those of the cytoplasm and those of the mitochondria (Weil et al. 1977). The chloroplast tRNA structure is similar to that of prokaryotic tRNAs, at least for the two chloroplast tRNAs whose nucleotide sequences have been determined (Chang et al. 1976; Guillemaut and Keith 1977), and they are coded for by chloroplast DNA (Tewari and Wildman 1970; Gruol and Haselkorn 1976; Haff and Bogorad 1976; McCrea and Hershberger 1976; Schwartzbach et al. 1976). The genes coding for chloroplast rRNAs have been mapped on higher plant and algal chloroplast DNAs. In maize and spinach, the chloroplast rRNA genes are located on two inverted repeats (Bedbrook et al. 1977; Whitfeld et al. 1978); in Euglena, the chloroplast rRNA genes exist as three clustered tandem repeats (Gray and Hallick 1978; Rawson et al. 1978). But until now, the chloroplast tRNA genes have not been localized. In this paper we describe the fractionation and identification of spinach ( Spinacia oleracea ) chloroplast tRNAs and the first results of our efforts to map the tRNA genes on spinach chloroplast DNA (a circular molecule of ~ 90 × 10 6 daltons). Unbroken Chloroplasts (Herrmann et al. 1975) were used in the preparation of tRNAs, aminoacyl-tRNA synthetases, and high-molecular-weight DNA. The tRNAs were prepared by phenol treatment of detergent-lysed chloroplasts, precipitated by ethanol, dissolved in 1 M NaCl, diluted to 0.2 M NaCl, incubated with DNase, and purified on DEAE-cellulose columns (Burkard et al. 1970). Aminoacyl-tRNA synthetases were obtained...