Organization of three rRNA (rrn) operons from Sphingobium chungbukense DJ77

The nucleotide sequences of all three rRNA operons (rrnA, rrnB, and rrnC) of Sphingobium chungbukense DJ77 were determined. The three rrn operons have the same gene order (16S rRNA-tRNA^Ile-tRNA^Ala-23S rRNA-5S rRNA-tRNA^fMet). The nucleotide sequences were identical over a 5,468 bp region spanning the 16S rRNA gene to the 5S rRNA gene. Variability was observed in the 5S rRNA-tRNA^fMet spacer sequence of rrnB. The tRNA^fMet gene sequences were identical except for two bases (T₅₇₉₄ and A₅₈₇₁ in rrnB, T₅₉₄₂ and A₅₉₅₆ in rrnA, but C₅₉₄₂ and G₅₉₅₆ in rrnC). Comparative sequence analyses of ribosomal RNA operons from DJ77 with those of the class Alphaproteobacteria, to which the genus Sphingobium belongs, reveal close evolutionary relationships with other members of the order Sphingomonadales.     

Lack of polymorphism within the rRNA operons of group A streptococci

Polymerase chain reaction (PCR) ribotyping of many bacterial species has shown that polymorphism of the ribosomal RNA (rRNA) operons, within and between strains, is common. Restriction fragment length polymorphism (RFLP) analysis of the rRNA operons of thirty-two genetically and geographically distinct strains of group A streptococci (GAS) revealed that there are only two major HaeIII PCR-ribotypes. This variation is due to a single nucleotide change within the 16S–23S intergenic spacer regions of these operons. As in many other bacterial species, this spacer region in streptococci also contains the gene for tRNA^ala. Within each GAS isolate, hybridization results are consistent with the presence of six rRNA operons. Interestingly, for a given strain, irrespective of its origin, all six rRNA operons have the same RFLP pattern. This contrasts with the findings in many other bacterial species, where heterogeneity of the rRNA operons within a genome is a common feature. This lack of heterogeneity of rRNA operons in an organism that is known to acquire genetic sequences through horizontal transfer is intriguing. Received: 22 November 1996 / Accepted: 30 January 1997     

Isolation of Messenger Ribonucleic Acid for Myosin Heavy Chain

A chicken embryonic polysome fraction that contains 50–60 monoribosomes and synthesizes the heavy chains of myosin is separated from other polysomes of smaller sizes by centrifugation through two cycles of discontinuous and continuous sucrose gradients. The unique properties of the polyadenylic acid segment present at the 3′-end of eukaryotic messenger RNA (mRNA) were used to purify the mRNA for myosin heavy chain from the phenol-extracted total RNA obtained from this polysome fraction. The total RNA was filtered thro ugh millipore filters resulting in partition of the riboscmal RNA (rRNA) and mRNA species. This millipore-bound RNA fraction, which consists of the mRNA and some ribosomal RNAs, was eluted from the filters with sodium dodecyl sulfate (SDS). Subsequent chromatography of this fraction on a cellulose column gave two well-separated peaks: an unadsorbed peak of ribosomal RNAs which was eluted with buffers of high ionic strength and an adsorbed peak of mRNA which was eluted only with a buffer of low ionic strength. Polyacrylamide gel electrophoresis of the mRNA peak fraction showed a single band with no detectable amounts of other RNAs, the mRNA migrating slower than 28S rRNA. The product of in vitro translation of the purified mRNA using a homologous cell-free system was identified as the myosin heavy chain by the following criteria: coprecipitation with carrier myosin at low ionic strength; elution properties on DEAE-cellulose column; and comigration with the heavy chain in polyacrylamide gel electrophoresis. In order to demonstrate the fidelity of translation of the mRNA, ¹⁴C-labeled products of the in vitro translation were copurified with unlabeled myosin heavy chains added as a carrier. The mixture of polypeptides was then cleaved with CNBr and the resulting peptides were separated by molecular sieving. The correlation between the radioactivity and the UV absorbance in the separated peptides indicates that total synthesis of the myosin heavy chain was achieved.     

Sequence Heterogeneity of the Ten rRNA Operons in Clostridium perfringens

We have cloned and sequenced rRNA operons of Clostridium perfringens strain 13 and analyzed the sequence structure in view of the phylogenesis. The organism had ten copies of rRNA operons all of that comprised of 16S, 23S and 5S rDNAs except for one operon. The operons clustered around the origin of replication, ranging within one-third of the whole genome sequence as it is arranged in a circle. Seven operons were transcribed in clockwise direction, and the remaining three were transcribed in counter clockwise direction assuming that the gyrA was transcribed in clockwise direction. Two of the counter clockwise operons contained tRNA^Ile genes between the 16S and 23S rDNAs, and the other had a tRNA^Ile genes between the 16S and 23S rDNAs and a tRNA^Asn gene in the place of the 5S rDNA. Microheterogeneity was found within the rRNA structural genes and spacer regions. The length of each 16S, 23S and 5S rDNA were almost identical among the ten operons, however, the intergenic spacer region of 16S-23S and 23S-5S were variable in the length depending on loci of the rRNA operons on the chromosome. Nucleotide sequences of the helix 19, helix 19a, helix 20 and helix 21 of 23S rDNA were divergent and the diversity appeared to be correlated with the loci of the rRNA operons on the chromosome.     

Intragenomic 16S rDNA Divergence in <Emphasis Type="Italic">Haloarcula marismortui</Emphasis> Is an Adaptation to Different Temperatures

The halophilic archaeon Haloarcula marismortui contains three ribosomal RNA operons, designated rrnA, rrnB, and rrnC. Operons A and C are virtually identical, whereas operon B presents a high divergence in nucleotide sequence, having up to 135 nucleotide polymorphisms among the three 16S, 23S, and 5S ribosomal RNA genes. Quantitative PCR and structural analyses have been performed to elucidate whether the presence of this intragenomic heterogeneity could be an adaptation to the variable environmental conditions in the natural habitat of H. marismortui. Variation in salt concentration did not affect expression but variation in incubation temperature did produce significant changes, with operon B displaying an expression level four times higher than the other two together at 50°C and three times lower at 15°C. We show that the putative promoter region of operon B is also different. In addition, the predicted secondary structure of these genes indicated that they have distinct stabilities at different temperatures and a mutant strain lacking operon B grew slower at high temperatures. This study supports the idea that divergent rRNA genes can be adaptive, with different variants being functional under different environmental conditions (e.g., temperature). The same phenomenon could take place in other halophiles or thermophiles with intragenomic rDNA heterogeneity, where the use of 16S rDNA as a phylogenetic marker and indicator of biodiversity should be used with caution.     

Total Breakdown of Ribosomal and Transfer RNA in a Mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis>

STABLE RNA, including rRNA and tRNA, turns over slowly, if at all, during steady state growth of Escherichia coli^1–4. It is evident, however, that E. coli cells can degrade stable RNA, which is slowly broken down to various extents under such conditions as starvation phosphate^5,6, magnesium^7,8, potassium⁹ and nitrogen^10,11 The mutant described here very rapidly degrades what is essentially all its stable RNA when RNA synthesis is stopped in cultures at 42° C. The degradation can be prevented if antibiotics are added that inhibit breakdown of polysomes.     

Relative amounts of newly synthesized poly(A)+ and poly(A)- messenger RNA during development of Xenopus laevis

The relative amounts of newly synthesized poly(A)⁺ and poly(A)^? mRNA have been determined in developing embryos of the frog Xenopus laevis. Polysomal RNA was isolated and fractionated into poly(A)⁺ and poly(A)^? RNA fractions with oligo(dT)-cellulose. In normal embryos the newly synthesized polysomal poly(A)⁺ RNA has a heterodisperse size distribution as expected of mRNA. The labeled poly(A)^? RNA of polysomes is composed mainly of rRNA and 4S RNA. The amount of poly(A)^? mRNA in this fraction cannot be quantitated because it represents a very small proportion of the labeled poly(A)^? RNA. By using the anucleolate mutants of Xenopus which do not synthesize rRNA, it is possible to estimate the percentage of mRNA which contains poly(A) and lacks poly(A). All labeled polysomal RNA larger than 4S RNA which does not bind to oligo(dT)-cellulose in the anucleolate mutants is considered presumptive poly(A)^? mRNA. The results indicate that about 80% of the mRNA lacks a poly(A) segment long enough to bind to oligo(dT). The poly(A)⁺ and poly(A)^? mRNA populations have a similar size distribution with a modal molecular weight of about 7 × 10⁵. The poly(A) segment of poly(A)⁺ mRNA is about 125 nucleotides long. Analysis of the poly(A)^? mRNA fraction has shown that it lacks poly(A)₁₂₅.     

Immunospecific binding of capped mRNA of reovirus by an antibody to a cap structure; m7G(5′)pppGm

Antibody specific for m⁷G(5′)pppGm was elicited in rabbits. Binding of capped RNA by the antibody and specificity of the binding were assessed by radioimmunoassay using capped and uncapped mRNA of reovirus. More than 50% of capped mRNA, whereas less than 2% of uncapped mRNA were bound by the antibody. The ratios of the amount of competitor to that of m⁷G(5′)pppGm required for 50% inhibition of binding of capped mRNA was 1; 180; 430; 28,600; and 114,000 for m⁷G(5′)pppG, G(5′)pppGm, G(5′)pppG, yeast tRNA and rabbit rRNA respectively. Poly(A) showed no competition. By immunoaffinity chromatography, a 15-fold enrichment of capped mRNA was attained from the mixture of the RNA in a vast excess of rRNA.     

Identification of Mouse Haemoglobin Messenger RNA

RETICULOCYTE polyribosomes contain 9S RNA with many of the properties expected for the haemoglobin messenger RNA (mRNA)^1–12. Proof that this RNA is the haemoglobin (Hb) mRNA, however, can be obtained only by showing that it directs the synthesis of globin chains. Laycock and Hunt¹³ added an RNA isolated from rabbit reticulocytes to an E. coli cell-free preparation and observed the synthesis of material, with the properties of globin in the presence of N-acetylvalyl tRNA. We added the mouse reticulocyte 9S RNA to a rabbit reticulocyte cell-free system and have shown that material is synthesized which co-chromatographs with mouse globin β-chains¹⁴. We now present evidence that the material synthesized under the direction of the mouse 9S RNA is indeed mouse haemoglobin β-chains.     

Molecular cloning of the ribosomal RNA genes of the photosynthetic bacterium Rhodopseudomonas capsulata

Summary Chromosomal segments of Rhodopseudomonas capsulata carrying the ribosomal operons and cloned with the cosmid vector pHC79 have been identified by cross hybridization with ³²P-ATP labeled rRNAs. At least seven rRNA operons are present in the R. capsulata chromosome. By R-loop analyses of DNA-RNA hybrids, two distinct loop structures of sizes 1.50 kb and 2.52 kb corresponding to the 16S and 23S RNA molecules, respectively, were detected. Intact 23S RNA molecules can be isolated from R. capsulata ribosomes by sucrose density centrifugation. However, fragmentation of the 23S RNA molecule into a 16S-like molecule was observed during gel electrophoresis. Restriction mapping and hybridization of a 9 kb PstI fragment that contained one copy of the rRNA operon showed the following sequence of the RNA genes in R. capsulata 16S, 23S, and 5S. A spacer region of 0.91 kb was found between the 16S and the 23S RNA genes.     

Loss of Dispensable Endonuclease Activity in Relief of Polarity by <Emphasis Type="Italic">suA</Emphasis>

NONSENSE mutations in several microorganisms exert a pleio-tropic “polar” effect, reducing the level of expression of those genes in the same Operon which lie on the operator-distal side of the mutant gene'. Although ribosomes terminate translation and are quickly discharged from the messenger when they encounter a nonsense codon², they can efficiently re-attach to the messenger at the “start” codons of downstream genes, both in RNA phage^2–5 and in E. coli⁶. Thus, polarity must be caused by unavailability of the messenger template beyond the nonsense codon. In RNA phage, this unavailability has been attributed to a conformational masking of the messenger^2–5     

Separation of α- and β-Globin Messenger RNAs

THE 10S RNA fraction of reticulocytes from various species contains the haemoglobin messenger RNA^1–4. When this 10S RNA fraction is added to a cell-free system derived from reticulocytes or Krebs II ascites cells, it directs the synthesis of α and β chains of haemoglobin^5–8. The α and β messenger RNA molecules contained in this fraction, however, have not yet been separated and identified. When reticulocyte. RNA of mouse is subjected to electrophoresis on 6% polyacrylamide gels, the 10S fraction contains two major bands and three minor bands⁹, suggesting that the major lOS RNA bands contain the messenger RNAs for the α- and β-globin chains.