Conserved portion in bacterial ribosomal RNA

The conserved portion in bacterial ribosomal RNA was studied by the DNA-RNA hybridization method. The hybridization percentages were as follows: Bacillus subtilis DNA and B. subtilis 23S rRNA, 0.16; Escherichia coli DNA and E. coli 23S rRNA, 0.15; B. subtilis DNA and E. coli 23S rRNA, 0.03; E. coli DNA and B. subtilis 23S rRNA, 0.04. The RNA's extracted from the heterologous hybrids could be rehybridized with DNA's of B. subtilis and E. coli. The average chain lengths of the RNA's were estimated by sucrose density gradient centrifugation and Sephadex gel filtration. The results suggested that the size might be larger than 30 nucleotides. Nucleotide compositions of the RNA's in the hybrids were also studied. Both RNA's contained higher molar percentages of guanylic acid and cytidylic acid than the whole rRNA's.     

Large differences in substitutional pattern and evolutionary rate of 12S ribosomal RNA genes

We demonstrate using Drosophila, periodical cicadas, and hominid primates, that the molecular clock based on animal mitochondrial small- subunit (12S) rRNA genes ticks at significantly different relative rates depending on which taxa and which region of the gene are examined. Drosophila, which are commonly used as model taxa, are evolving in a highly peculiar manner with the majority of sites in the 3' half of the 12S gene apparently invariant. The analogous 3' half of the mitochondrial large-subunit rRNA gene (16S) appears to be similarly constrained. It is surprising that these regions that are already highly constrained in all animals should be even more constrained in Drosophila, especially when the Drosophila mitochondrial genome as a whole does not display a similar rate slowdown. This extreme 12S rate slowdown is not apparent in periodical cicadas or hominid primates and appears to be related to strong structural and functional constraints rather than a depressed mutation rate. Finally, the slow average rate of evolution in the third domain of Drosophila does not imply that the few variable sites lack multiple hits.      

Linkage of ribosomal RNA genes in Leptospira

We determined the linkage of 16S, 23S, and 5S rRNA genes in several strains of Leptospira and Leptonema by DNA-DNA hybridization. Almost all the hybridizations in all leptospires used in these experiments gave two radioactive bands and the results strongly suggest that the number of the 16S and the 23S rRNA genes in those strains is two, respectively. In contrast with the larger rRNAs, the number of 5S rRNA gene was different. In the strains of leptospires, L. biflexa, which were non-parasitic, there are two genes for 5S rRNA, whereas only one gene for 5S rRNA is carried in L. interrogans, which were originally isolated as parasitic. Southern hybridization experiments suggest that those rRNA genes are interspersed on the leptospiral chromosome.     

On the biosynthesis of bacterial ribosomal RNA

By comparison of the fingerprints of 5S and 23S ribosomal RNAs from Bacillus licheniformis with that of the precursor of 23S ribosomal RNA, it can be shown that 5S RNA is not a part of the precursor of 23S ribosomal RNA.     

Sequence polymorphisms in ribosomal RNA genes and variations in chromosomal loci of Oenothera odorata and O. laciniata

Numerous studies on Oenothera species have been investigated for the physiological and ecological characteristics. However, no such an information based on molecular cytogenetic has yet been introduced, in turn, is very essential for identifying sequence polymorphisms of rRNA genes with their loci on mitotic phases for further biological researches. In this study, sequence variations of rRNA genes in Oenothera odorata and O. laciniata were examined to identify informative factors as unique or repeat sequences in intra- and inter-specific variations. Intra-specific variation revealed that the sequences of the rRNA genes including the spacer regions were highly conserved revealing only a few variations. From the inter-specific variation, spacer regions of species were completely different as (1) non-homologous sequences in NTS and (2) different type repeat sequences in ITS 1, 2 and 5.8S rRNA, whereas the remaining coding regions were highly conserved. FISH was carried out on mitotic phases using the 5S rDNA of the analyzed sequences. From the interphase and metaphase chromosomes of the examined species, two loci of 5S rDNA in O. odorata and four loci in O. laciniata were confirmed on the telomeric region of the short arm. Due to the small size and unclear centromere of the chromosomes, karyotype could not be completed. However, we confirmed that the chromosomes are organized by meta- and acrocentric chromosomes and the chromosomes with identified loci were assumed to be paired by the location of loci at the telomeric region on the short arm with relative lengths.     

Comparative studies on bacterial 5S ribosomal RNA

The 5S rRNAs of Escherichia coli, Bacillus stearothermophilus, and B. subtilis were isolated and their molecular conformation examined. All three 5S rRNAs were similar with regard to nucleotide chain length, base composition and general configuration. Several major differences were apparent between the secondary and tertiary conformations of the 5S rRNA of E. coli and the genus Bacillus. Only minor differences were noted between those from the two Bacillus species. Each 5S rRNA species had a different 5′-terminal nucleotide: E. coli-U; B. stearothermophilus-C; B. subtilis-G.     

Amplification and sequencing of variable regions in bacterial 23S ribosomal RNA genes with conserved primer sequences

Published bacterial 23S ribosomal RNA sequences were aligned, and universally conserved regions flanking highly variable regions were looked for. In strategically positioned conserved regions, six oligonucleotides suitable for polymerase chain reaction (PCR) and sequencing were designed, allowing fast sequencing of four of the most variable 23S rRNA regions. Two other primers were designed for PCR amplification of nearly complete 23S rRNA genes. All these primers successfully amplified fragments of 23S rRNA genes from seven unrelated bacteria. Four primers were used to determine 938 bp of sequence forCampylobacter jejuni subsp.jejuni. These results indicate that the oligonucleotide sequences presented here are useful for PCR amplification and sequence determination of variable 23S rRNA regions for a broad variety of eubacterial species.     

Structure and variation in ribosomal RNA genes of pea

Summary A complete ribosomal DNA (rDNA) repeat unit has been cloned from the genome of Pisum sativum (garden pea) and used to construct a map containing a total of 58 cleavage sites for 23 different restriction enzymes. Regions encoding 18s and 25s ribosomal RNA (rRNA) were identified by R-loop analysis. A 180 bp sequence element is repeated eight times in the intergenic nontranscribed spacer (NTS) region, as defined by eight evenly spaced RsaI cleavage sites. Sequence heterogeneity among these elements (subrepeats) is indicated by the presence of an NcoI site within the five RsaI subrepeats distal to the 25s rRNA gene but not in the three subrepeats proximal to this gene, and also by the presence of an additional RsaI cleavage site in one subrepeat.The approximately 4000 copies of the rDNA repeat in the pea nuclear genome show considerable heterogeneity with respect to the length of the NTS region, and differences are also frequently observed between different genotypes. In both cases the length variation appears to be due primarily to differences in the number of subrepeat elements.Comparison of rDNA restriction maps for two pea genotypes separated for hundreds or perhaps thousands of generations reveals that they contain many rDNA identical repeat units. This data is consistent with the view that new rDNA variants are fixed only infrequently in the evolution of a species.Differences also exist between the rDNA repeats of a single genotype with respect to the degree of base modification at certain restriction sites. A large number of sites known to exist in the pea rDNA clone are not cleaved at all in genomic rDNA, or are cleaved in only some copies of the rDNA repeat. We believe these examples of incomplete cleavage results mostly from methylation, although it is difficult to rule out the possibility of sequence variation in all cases. Most putative modifications are best interpreted in terms of cytosine methylation in CG and CXG sequences, but at least one example is more consistent with adenine methylation.We also have constructed a more detailed restriction map of the wheat rDNA clone pTA71 and present a comparison of this map to our map of pea, pumpkin, and wheat in order to assess the amount of useful evolutionary information that can be obtained by comparison of such maps.     

Localization of the ribosomal RNA genes in Drosophila simulans

In situ hybridization of cloned rRNA genes from Drosophila melanogaster to D. simulans metaphase chromosomes shows that in the tested wild type strains both sex chromosomes contain a nucleolus organizer region. Silver grain counts support the published data that the X chromosomal rRNA gene number is significantly higher than the Y chromosomal.     

Systematic chromosomal deletion of bacterial ribosomal protein genes

Detailed studies of ribosomal proteins (RPs), essential components of the protein biosynthetic machinery, have been hampered by the lack of readily accessible chromosomal deletions of the corresponding genes. Here, we report the systematic genomic deletion of 41 individual RP genes in Escherichia coli, which are not included in the Keio collection. Chromosomal copies of these genes were replaced by an antibiotic resistance gene in the presence of an inducible, easy-to-exchange plasmid-born allele. Using this knockout collection, we found nine RPs (L15, L21, L24, L27, L29, L30, L34, S9, and S17) nonessential for survival under induction conditions at various temperatures. Taken together with previous results, this analysis revealed that 22 of the 54 E. coli RP genes can be individually deleted from the genome. These strains also allow expression of truncated protein variants to probe the importance of RNA-protein interactions in functional sites of the ribosome. This set of strains should enhance in vivo studies of ribosome assembly/function and may ultimately allow systematic substitution of RPs with RNA.