Physical Mapping of Recombinant Plasmids Containing Broad Bean Chloroplast Ribosomal RNA Genes

Two BamHl fragments containing broad bean chloroplast rRNA genes were cloned using the bacterial plasmid pBR322 as a vector and Escherichia coli HB101 as host bacterial. Physical maps of the two cloned ct DNA BamHI fragments containing rRNA genes were constructed by cleavage with several restriction endonucleases and Southern blot hybridization with E. coli 16S-23S rRNAs. Recombinant plasmids pVFBI6 and pVFB32 contain a 16S rRNA sequence on the 4.70 kb BamHl fragment, a 23S rRNA sequence and 4.5S/5S rRNA sequences on the 5.65 kb BamHl fragment, respectively.     

Isolation of Euglena gracilis chloroplast 5S ribosomal RNA and mapping the 5S rRNA gene on chloroplast DNA.

Ribosomal RNA (5S) from Euglena gracilis chloroplasts was isolated by preparative electrophoresis, labeled in vitro with 125I, and hybridized to restriction nuclease fragments from chloroplast DNA or cloned chloroplast DNA segments. Euglena chloroplast 5S rRNA is encoded in the chloroplast genome. The coding region of 5S rRNA has been positioned within the 5.6 kilobase pair (kbp) repeat which also codes for 16S and 23S rRNA. There are three 5S rRNA genes on the 130-kbp genome. The order of RNAs within a single repeat is 16S-23S-5S. The organization and size of the Euglena chloroplast ribosomal repeat is very similar to the ribosomal RNA operons of Escherichia coli.     

Characterization of Paenibacillus popilliae rRNA operons

The terminal 39 nucleotides on the 3' end of the 16S rRNA gene, along with the complete DNA sequences of the 5S rRNA, 23S rRNA, tRNA(Ile), and tRNA(Ala) genes were determined for Paenibacillus popilliae using strains NRRL B-2309 and Dutky 1. Southern hybridization analysis with a 16S rDNA hybridization probe and restriction-digested genomic DNA demonstrated 8 copies of the 16S rRNA gene in P. popilliae strains KLN 3 and Dutky 1. Additionally, the 23S rRNA gene in P. popilliae strains NRRL B-2309, KLN 3, and Dutky 1 was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to occur as 8 copies. It was concluded that these 3 P. popilliae strains contained 8 rrn operons. The 8 operon copies were preferentially located on approximately one-half of the chromosome and were organized into 3 different patterns of genes, as follows: 16S-23S-5S, 16S-ala-23S-5S, and 16S-5S-ile-ala-23S-5S. This is the first report to identify a 5S rRNA gene between the 16S and 23S rRNA genes of a bacterial rrn operon. Comparative analysis of the nucleotides on the 3' end of the 16S rRNA gene suggests that translation of P. popilliae mRNA may occur in Bacillus subtilis and Escherichia coli.     

A restriction map of the ribosomal RNA genes and the short single-copy DNA sequence of the pearl millet chloroplast genome

The chloroplast rDNA genes of pearl millet (Pennisetum americanum) have been cloned and physically mapped. The chloroplast genome of the pearl millet contains two identical rRNA genes located on DNA sequences that are inverted with respect to one another and separated by 12 kb of single-copy DNA. The rRNA genes were positioned on a restriction endonuclease map by using as hybridization probes specific cloned rDNA sequences from the chloroplast DNA of the alga Euglena gracilis. The 16S and 23S rRNA genes were shown to be approx. 2 kb from one another, and the 5S RNA gene is immediately adjacent to the 23S tRNA gene.     

A pseudogene cluster in the leader region of the Euglena chloroplast 16S-23S rRNA genes.

The nucleotide sequence of a region (leader region) preceding the 5'-end of 16S-23S rRNA gene region of Euglena gracilis chloroplast DNA was compared with the homologous sequences that code for the 16S-23S rRNA operons of Euglena and E. coli. The leader region shows close homology in sequence to the 16S-23S rRNA gene region of Euglena (Orozco et al. (1980) J. Biol.Chem. 255, 10997-11003) as well as to the rrnD operon of E. coli, suggesting that it was derived from the 16S-23S rRNA gene region by gene duplication. It was shown that the leader region had accumulated nucleotide substitutions at an extremely rapid rate in its entirety, similar to the rate of tRNAIle pseudogene identified in the leader region. In addition, the leader region shows an unique base content which is quite distinct from those of 16S-23S rRNA gene regions of Euglena and E. coli, but again is similar to that of the tRNAIle pseudogene. The above two results strongly suggest that the leader region contains a pseudogene cluster which was derived from a gene cluster coding for the functional 16S-23S rRNA operon possibly by imperfect duplication during evolution of Euglena chloroplast DNA.     

Ribotyping of 16S and 23S rRNA genes and organization of rrn operons in members of the bacterial genera Gemmata,Planctomyces, Thermotoga,Thermus, and Verrucomicrobium

The number of organization of rrn genes of two members of the order Planctomycetales, Planctomyces limnophilus and Gemmata obscuriglobus, as well as three species from other bacterial phyla, namely Thermotoga maritima, Thermus aquaticus and Verrucomicrobium spinosum were examined by Southern blot hybridization analysis of restricted DNA with labeled 16S- and 23S rRNAs. Ribotyping analysis revealed that two species contain unlinked 16S- and 23S rRNA genes. Planctomyces limnophilus possessed two unlinked rrn genes which were separated from each other by at least 4.3 kb, and Thermus aquaticus had to unlinked 16S and 23S rRNA genes, separated from each other by at least 2.5 kb. Gemmata obscuriglobus exhibited five genes for which the organization could as yet not be determined because of the complex hybridization patterns. In the other two species, rrn genes clustered in operons. Thermotoga maritima had a single gene for each rRNA species which were separated by not more than 1.5 kb, while Verrucomicrobium spinosum had four copies of probably linked 16S and 23S rRNA genes with a maximal distance between 16S and 23S rRNA genes of 1.3 kb.     

Physical mapping of the ribosomal RNA genes of Mycoplasma capricolum.

Physical mapping of the rRNA genes of Mycoplasma capricolum was done by digestion of the mycoplasmal DNA with EcoRI, PstI and BglII and hybridization with nick-translated probes consisting of defined portions of the rrnB ribosomal RNA operon of Escherichia coli. The results indicate that the rRNA genes in the chromosome of M. capricolum are arranged in two clusters, each organized in the order 5'-16S-23S-5S-3', resembling the order of the genes in the rrnB operon, with no large spacer regions separating the genes in each cluster.     

Molecular cloning and characterization of an rRNA operon in Streptomyces lividans TK21.

The number of rRNA genes in Streptomyces lividans was examined by Southern hybridization. Randomly labeled 23 and 16S rRNAs were hybridized with BamHI, BglII, PstI, SalI, or XhoI digests of S. lividans TK21 DNA. BamHi, BglII, SalI and XhoI digests yielded six radioactive bands each for the 23 and 16S rRNAs, whereas PstI digests gave one band for the 23S rRNA and one high-intensity band and six low-density bands for the 16S rRNA. The 7.4-kilobase-pair BamHI fragment containing one of the rRNA gene clusters was cloned into plasmid pBR322. The hybrid plasmid, pSLTK1, was characterized by physical mapping, Southern hybridization, and electron microscopic analysis of the R loops formed between pSLTK1 and the 23 and 16S rRNAs. There were at least six rRNA genes in S. lividans TK21. The 16 and 23S rRNA genes were estimated to be about 1.40 and 3.17 kilobase pairs, respectively. The genes for the rRNAs were aligned in the sequence 16S-23S-5S. tRNA genes were not found in the spacer region or in the context of the rRNA genes. The G + C content of the spacer region was calculated to be approximately 58%, in contrast to 73% for the chromosome as a whole.     

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 .     

The nucleotide sequences of two tRNAAsn genes from tobacco chloroplasts

Recombinant plasmids which contain EcoRI fragments of tobacco chloroplast DNA carrying tRNA genes were constructed. Plasmids pTC211 and pTC293 contain the base sequences for tRNAAsn in their 1.4 and 1.1 Md EcoRI fragments, respectively. These two tRNA sequences are identical and are; 5'-TCCTCAGTAGCTCAGTGGTAGAGCGGTCGGCTGTTAACCGATTGGTCGTAGGTTCGAATCCTACTTGGGGAG-3'. Each tRNAAsn gene is located at about 0.9 kb apart from the distal end of each 5S rRNA gene and is coded for by the DNA strand opposite from that of the rRNA genes.     

Cotranscription and processing of 23S, 4.5S and 5S rRNA in chloroplasts from Zea mays

The termini of rRNA processing intermediates and of mature rRNA species encoded by the 3' terminal region of 23S rDNA, by 4.5S rDNA, by the 5' terminal region of 5S rDNA and by the 23S/4.5S/5S intergenic regions from Zea mays chloroplast DNA were determined by using total RNA isolated from maize chloroplasts and 32P-labelled rDNA restriction fragments of these regions for nuclease S1 and primer extension mapping. Several processing sites detectable by both 3' and 5' terminally labelled probes could be identified and correlated to the secondary structure for the 23S/4.5S intergenic region. The complete 4.5S/5S intergenic region can be reverse transcribed and a common processing site for maturation of 4.5S and 5S rRNA close to the 3' end of 4.5S rRNA was detected. It is therefore concluded that 23S, 4.5S and 5S rRNA are cotranscribed.     

Gene Dosage for 5S Ribosomal Ribonucleic Acid in Escherichia coli and Bacillus megaterium

The number of gene copies for 5S ribosomal ribonucleic acid (rRNA), relative to that for 16 and 23S rRNA, has been determined by deoxyribonucleic acid (DNA)-RNA hybridization for Escherichia coli and Bacillus megaterium. In both cases, the number of 5S rRNA genes equals the number of 16 or 23S rRNA genes. Rapid procedures for preparing extremely highly purified DNA suitable for DNA-RNA hybridization experiments and chemically pure 5S rRNA are described.     

Organization of the ribosomal RNA genes in Treponema phagedenis and Treponema pallidum.

The genomic DNA fragment which contains ribosomal RNA (rRNA) genes for Treponema phagedenis was cloned into bacteriophage vector lambda EMBL3. A restriction map of the fragment was constructed and the organization of the rRNA genes was determined. The fragment contained at least one copy of the 16S, 23S and 5S sequences and the genes are arranged in the order 16S-23S-5S. Southern hybridization using radiolabeled rRNA gene probes to genomic DNA from T. phagedenis strain Reiter and T. pallidum strain Nichols showed that these organisms have two radioactive fragments which hybridize to the probes in their genome. These results suggest that both pathogenic and non-pathogenic strains of Treponema may carry at least two sets of rRNA genes on their chromosomes.     

Euglena gracilis chloroplast ribosomal RNA transcription units. Nucleotide sequence polymorphism in 5 S rRNA genes and 5 S rRNAs

The three tandemly repeated ribosomal RNA operons from the chloroplast genome of Euglena gracilis Klebs, Pringsheim Strain Z each contain a 5 S rRNA gene distal to the 23 S rRNA gene (Gray, P.W., and Hallick, R.B. (1979) Biochemistry 18, 1820-1825). We have cloned two distinct 5 S rRNA genes, and determined the DNA sequence of the genes, their 5'- and 3'-flanking sequences, and the 3'-end of the adjacent 23 S rRNA genes. The two genes exhibit sequence polymorphism at five bases within the "procaryotic loop" coding region, as well as internal restriction endonuclease site heterogeneity. These restriction endonuclease site polymorphisms are evident in chloroplast DNA, and not just the cloned examples of 5 S genes. Chloroplast 5 S rRNA was isolated, end labeled, and sequenced by partial enzymatic degradation. The same polymorphisms found in 5 S rDNA are present in 5 S rRNA. Therefore, both types of 5 S rRNA genes are transcribed and are present in chloroplast ribosomes.