Chloroplast ribosomal RNA genes in the chloroplast DNA of Euglena gracilis

Euglena chloroplast DNA has a buoyant density in CsCI of 1.686. Shearing this DNA produces a satellite band at density 1.700. The satellite, easily lost during preparative CsCI gradient centrifugation of chloroplast DNA, contains the genes for chloroplast ribosomal RNA. Pure Euglena chloroplast DNA is shown to contain one set of ribosomal RNA genes for each 90 × 10⁶ daltons of DNA.     

Mapping of the ribosomal RNA genes on spinach chloroplast DNA.

Spinach chloroplast ribosomal RNAs have been hybridized to restriction endonuclease fragments of spinach chloroplast DNA. All three RNA species (23S, 16S and 5S) hybridized to a single large fragment when the DNA was digested with either Sall or Pstl. Hybridization of 23S RNA to fragments produced by Smal yielded two radioactive bands which corresponded to the bi-molar 2.5 X 10(6) and 1.15 X 10(6) Mr fragments. 16S RNA also hybridized to two, bi-molar Smal fragments (3.4 X 10(6) and 2.5 X 10(6) Mr) and 5S RNA hybridized to the 1.15 X 10(6) Mr bi-molar Smal fragment. The 23S RNA and 16S RNA cistrons were each also shown to contain a single EcoRI site. From the data it was possible to conclude that the ribosomal RNA genes are located on the inverted repeat region of the spinach chloroplast DNA restriction map [1,2], that the sequence of the cistrons is 16S - 23S - 5S and that the size of the spacer between the 16S and 23S RNA cistrons is approximately 0.90 X 10(6) Mr.     

Site of synthesis of spinach chloroplast ribosomal proteins and formation of incomplete ribosomal particles in isolated chloroplasts

Chloroplast ribosomal proteins from spinach have been prepared in the presence of a protease inhibitor and some modifications have been introduced to the previous characterization of the 50S subunits (Mache et al., MGG, 177, 333, 1980): 33 ribosomal proteins are detected instead of 34. No change has been observed for the 30S subunits.Using a light-driven system of protein synthesis it is shown that up to ten ribosomal proteins of the 30S and eight proteins of the 50S subunits are made in the chloroplast.Newly synthesized ribosomal subunits have been analysed on CsCl gradients after sedimentation at equilibrium, allowing the separation of fully assembled subunits from incomplete ribosomal particles. Most of the newly made 50S subunits are fully assembled (=1.634). A small amount of incomplete 50S particles (=1.686) is detectable. Newly made 30S subunits (=1.598) and incomplete 30S particles (=1.691) are also observed. The ribosomal proteins of the incomplete 30S have been determined. They contain eight or nine of the 30S-proteins, seven of which are synthesized within the chloroplast. It is suggested that incomplete ribosomal particles resulted from a step in the assembly of ribosomal subunits.     

Nucleotide sequence of the single ribosomal RNA operon of pea chloroplast DNA

The nucleotide sequence of an 8 kbp region of pea ( Pisum sativum L.) chloroplast DNA containing the rRNA operon and putative promoter sites has been determined and compared to the corresponding sequences from maize, tobacco and the liverwort Marchantia polymorpha . The chloroplast DNA species of all vascular plants investigated, with the exception of a few legumes including pea, and of Marchantia contain an inverted repeat with an rRNA operon. The pea rRNA operon is the first sequenced rRNA operon from a plant with only one copy of the rRNA genes per molecule of chloroplast DNA. The organization of the operon is the same as for maize, tobacco and Marchantia . i.e. tRNA-Val gene/16S rRNA gene/spacer with intron-containing genes for tRNA-Ile and tRNA-Ala/23S rRNA gene/4.5S rRNA gene/5S rRNA gene. Current evidence suggests that the tRNA-Val gene may not be contranscribed with the other genes. For pea 16S, 23S, 4.5S and 5S rRNA have 1488, 2813, 105 and 121 nucleotides, respectively. The homologies of the entire operon (the tRNA-Val gene - 5S rRNA region) to those from tobacco, maize and Marchantia are 88, 82 and 79%, respectively. The corresponding homologies for tobacco/maize, tobacco/ Marchantia and maize/ Marchantia have similar values. The 16S and 23S rRNA genes from pea are more than 90% homologous to those from the 3 other species. We conclude that the fact that pea only has one set of rRNA genes per molecule of chloroplast DNA is apparently not correlated with any significant difference between the pea operon and the rRNA operons from tobacco, maize and Marchantia .     

Evidence for ribosomal RNA synthesis in pollen tubes in culture

The evidence is presented that pollen tubes ofNicotiana tabacum L. cultivated in shaken suspension do synthesize 5S, 18S and 28S RNA. Following incubation of pollen tubes in the presence of radioactive uracil or uridine, RNA was isolated from total pollen tube material after the removal of 4S RNA, from polysomes and from 80S ribosomal particles, and fractionated by density gradient centrifugation and MAK column chromatography. The results obtained further suggest a higher rate of 5S RNA synthesis with respect to 18S+28S RNA.     

Cellular content of chloroplast DNA and chloroplast ribosomal RNA genes in Euglena gracilis during chloroplast development.

The cellular content of chloroplast DNA in Euglena gracilis has been quantitatively determined. DNA was extracted from Euglena cells at various stages of chloroplast development and renatured in the presence of trace amounts of 3H-labeled chloroplast DNA. From the kinetics of renaturation of the 3H-labeled chloroplast DNA, compared with the kinetics of renaturation of excess nonradioactive chloroplast DNA, the fraction of cellular DNA represented by chloroplast DNA was calculated. The content of chloroplast DNA was found to increase from 4.9 to 14.6% of cellular DNA during light-induced chloroplast development. Correcting for the change in DNA mass per cell, the number of copies of chloroplast DNA is found to vary from 1400 to 2900 per cell. During this developmental transition, the cellular content of the chloroplast ribosomal RNA genes varies from 1900 to 5200 copies per cell. The ratio of the number of copies of rRNA genes to chloroplast genomes per cell remains in the range of 1-2 throughout chloroplast development, ruling out selective amplification of chloroplast rRNA genes as a means of regulation of rRNA gene expression. Direct measurement of the number of rRNA cistrons per 9.2 X 10(7) dalton genome yields a value of 1 or 2.     

Arrangement of the ribosomal RNA genes in chloroplast DNA of Leguminosae

Summary The circular chloroplast DNA from three species of plants in the taxonomic family Leguminosae were examined using electron microscopic techniques and restriction endonuclease digestion. Chloroplast DNAs from chickpea (Cicer arietinum), mung bean (Vigna radiata), and soy bean (Glycine max) were found to range in size from 119–151 kilobase pairs by contour length measurements. Sizes of the chloroplast DNAs have been further confirmed using different restriction endonucleases. Two of the chloroplast DNAs examined, soy bean and mung bean, contain a region approximately 15.9–18% of their monomer length that is repeated in reverse polarity. This repeated region separates a small unique region that ranges in size from 18.75–20.4 kilobase pairs and a large unique region that ranges in size from 73.4–85 kbp. This feature was not found in the chloroplast DNA of chickpea. R-loop hybridizations performed using chloroplast ribosomal RNAs demonstrate that the two ribosomal gene sets of the mung been and soy bean are arranged in inverted orientation within this repeated region. In contrast, the chickpea chloroplast DNA posesses a single ribosomal RNA gene set in the circular molecule. In all three chloroplast DNAs examined, the genes encoding the chloroplast 23S and 16S ribosomal RNA genes are separated by a spacer region which ranges in size from 2.2 to 2.48 kbp.     

Physical mapping of the pea chloroplast DNA and localization of the ribosomal RNA genes

The closed circular DNA of pea chloroplast has been digested with restriction endonucleases SalI, SmaI, BamHI, XbaI, XhoI, HindIII, and EcoRI. A physical restriction map of pea ctDNA has been constructed by mapping the SalI and SmaI sites. The pea ctDNA has been found to contain one set of ribosomal RNA genes by Southern hybridization of restriction endonuclease digest, R-loop studies, and DNA-DNA heteroduplex mapping. The 23 S and 16 S RNA genes are confined to a DNA region of 3.0 and 1.5 kbp, respectively. The two rRNA chains are separated by a spacer region of 2.2 kbp.     

Evaluation of nuclear ribosomal RNA and chloroplast gene markers for the DNA taxonomy of centric diatoms

Diatoms are highly diversified microeukaryotes with a taxonomy that has been under perpetual revision, particularly by means of molecular analyses. In this study, we evaluated typical genes to find the best molecular marker for the DNA taxonomy of centric diatoms, including Chaetoceros Ehrenberg, Cyclotella Kützing ex de Brébisson, Discostella Houk & Klee, Stephanodiscus Ehrenberg, and Thalassiosira Cleve. Our test genes included nuclear ribosomal RNA (e.g. small subunit, 5.8S, and large subunit [LSU]), and chloroplast genes (e.g. ribulose-1, 5-biphoshate carboxylase oxygenase and D1 protein of the photosystem II reaction centre core complex [psbA]). Calculated genetic divergence was highest for LSU ribosomal RNA D1-D5 (p-distance of 12.3), followed by 5.8S (7.7), ribulose-1, 5-biphoshate carboxylase oxygenase (7.4), small subunit (6.6), and psbA (3.7). The phylogenetic trees for individual genes effectively separated taxonomically tested centric diatoms with different phylogenetic resolutions; however, psbA was incongruent with others. These taxonomic resolving powers were in agreement with genetic divergences. Parsimony analysis showed that LSU evolved 1.97 times more rapidly than psbA and 1.07 times more rapidly than 5.8S. These results suggest that all of the tested genes except psbA can be used as taxonomic markers for centric diatoms and that LSU is the best molecular marker.     

Turnover-synthesis of chloroplast DNA in developing chloroplasts.

The mechanism for the turnover-synthesis of chloroplast DNA in the absence of net synthesis during the chloroplast maturation in Euglena gracilis was determined. DNA synthesis was measured by incorporation of32Pi into chloroplast DNA. The density label, 15N, was incorporated to examine the mechanism of turnover-synthesis. The newly synthesized segments represent a replacement of segments in the DNA containing 1.5 X 10(3) to 6.1 X 10(3) nucleotides. Twenty-three fragments of chloroplast DNA, generated by digestion with the restriction endonuclease EcoRI, became labeled with 32Pi. Turnover-synthesis, therefore, replaces segments throughout the molecule of chloroplast DNA.     

Molecular cloning of tobacco chloroplast ribosomal RNA genes

Summary Tobacco chloroplast ribosomal RNAs were shown to be hybridized with two EcoRI fragments of tobacco chloroplast DNA. These DNA fragments having molecular weights of 1.9x10⁶ and 2.8x10⁶ daltons were cloned using the bacterial plasmid pMB9 as a vector and E. coli HB101 as host bacteria. The recombinant plasmids containing either or both of these fragments were constructed and characterized.Abbreviations rRNA ribosomal RNA - EDTA ethylenediamine tetraacetic acid - SSC 0.15 M NaCl-0.015 M sodium citrate - EcoRI and HindIII restriction endonucleases isolated from E. coli RY13 and Haemophilus influenzae Rd, respectively