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PubMed1995

Chloroplast rRNA transcription from structurally different tandem promoters: an additional novel-type promoter.

Identification of transcription initiation sites in the promoter region of the tobacco chloroplast rRNA operon has been carried out by ribonuclease protection of in vitro capped RNAs and primer extension experiments. A promoter with typical chloroplast -10 and -35 motifs (P1) drives initiation of transcription from position -116 relative to the mature 16s rRNA sequence. In addition, we have found that a second primary transcript starts at position -64. This proximal promoter (P2) lacks any elements similar to those reported so far in chloroplast promoter regions, and hence P2 represents a novel-type promoter. Both transcripts are present in chloroplasts from green leaves and in non-photosynthetic proplastids from heterotrophically cultured cells (BY2), but their relative amounts appear to differ. The steady state level of the P2 transcript, with respect to P1, is higher in BY2 proplastids than in leaf chloroplasts.

Base Sequence↗

PubMed1997

Organization and nucleotide sequence analysis of the ribosomal gene set (rrnB) from Streptomyces lividans.

Streptomyces lividans (Sl) contains six ribosomal RNA (rRNA) gene sets, rrnA-F (Suzuki, Y., Ono, Y., Nagata, A. and Yamada, T. (1988) Molecular cloning and characterization of an rRNA operon in Streptomyces lividans TK21. J. Bacteriol. 170, 1631-1636). We have cloned the rrnB gene cluster. Physical mapping revealed that rrnB gene set is located on a 290 kb Asel fragment in the 11 to 12 o'clock region of the S. coelicolor A3(2) chromosome. The complete nucleotide (nt) sequence of Sl 23S rRNA has been determined. The structural gene of the Sl 23S rRNA codes for the 3108 nt RNA chain. The G+C content of the 23S rRNA is 57.3 mol%. The length of the spacer region between the 23S and 5S genes is 99 bp. Analysis of the sequences between the 16S and 23S genes and downstream of the 5S rRNA gene failed to identify any tRNA-like sequences. A secondary structure model of Sl 23 rRNA is proposed, based on the earlier published model of Gutell and Fox (Nucleic Acids Res. 16 (1988) 175-269).

Base Sequence↗

PubMed1995

Mycoplasma gallisepticum 16S rRNA genes.

The genome of Mycoplasma gallisepticum A5969 contains a truncated pseudogene for 16S rRNA in addition to a single unsplit rRNA-operon and a second discontinuous set of rRNA genes. Other M. gallisepticum strains tested do not possess the truncated gene. This gene is almost identical to full-size isolated 16S rRNA gene starting from at least 500 nucleotides upstream of the coding sequence and ending at the 977th nucleotide within the structural part of 16S rRNA.

Base Sequence↗

PubMed2001

Heterogeneity and compartmentalization of Pneumocystis carinii f. sp. hominis genotypes in autopsy lungs.

The extent and importance of genotype heterogeneity of Pneumocystis carinii f. sp. hominis within lungs have not previously been investigated. Two hundred forty PCR clones obtained from respiratory specimens and lung segments from three patients with fatal P. carinii pneumonia were investigated to detect genetic diversity in the internal transcribed spacer (ITS) region of the nuclear rRNA operon, the mitochondrial large-subunit (mtLSU) rRNA gene, and the dihydropteroate synthase-encoding gene. For two of the three examined patients, a mixture of different mtLSU rRNA and ITS genotypes was observed. Not all genotypes present in the lungs at autopsy were detected in the diagnostic respiratory samples. Compartmentalization of specific ITS and mtLSU rRNA sequence types was observed in different lung segments. In conclusion, the interpretation of genotype data and in particular ITS sequence types in the assessment of epidemiological questions should be cautious since genotyping done on respiratory samples cannot a priori be assumed to represent all genotypes present within the lung.

AIDS-Related Opportunistic Infections↗

PubMed1989

The structure and organization of the 16S ribosomal RNA gene from the archaebacterium Thermoplasma acidophilum.

The complete nucleotide sequence of the 16S rRNA gene from Thermoplasma acidophilum, as well as its 5' and 3' flanking regions, were determined by the dideoxynucleotide chain termination method. The 16S rRNA gene encodes 1471 nucleotides. The primary and secondary structures of T. acidophilum 16S rRNA both exhibit typical archaebacterial features. The sequence appears to be more closely related to 16S rRNAs of the methanogen--halophile group than to those of the thermoacidophile group. Secondary-structure comparisons generally support this relationship, although there are several examples in which the single-stranded loops in particular helices of T. acidophilum 16S rRNA more strongly resemble their counterparts in the 16S rRNA of Sulfolobus solfataricus, a member of the thermoacidophile group. In contrast to the polycistronic rRNA operons found in most organisms, the three rRNA genes from T. acidophilum occur in only a single copy per genome and appear to be physically unlinked. Consistent with this, the 16S rRNA gene is flanked by putative promoter and terminator sequences that are comparable to the transcription control signals from other archaebacterial genes. The sequence TATATATA, which is very similar to the archaebacterial promoter consensus TTTAT/AATA, is located 18 bases before the probable site of transcription initiation, TGCACAT. There is a potential transcription termination site immediately downstream from the gene that consists of a relatively stable stem and loop structure followed by stretches of Tresidues.

Amino Acid Sequence↗

PubMed2003

Regulation of the Escherichia coli rrnB P2 promoter.

The seven rRNA operons in Escherichia coli each contain two promoters, rrn P1 and rrn P2. Most previous studies have focused on the rrn P1 promoters. Here we report a systematic analysis of the activity and regulation of the rrnB P2 promoter in order to define the intrinsic properties of rrn P2 promoters and to understand better their contributions to rRNA synthesis when they are in their natural setting downstream of rrn P1 promoters. In contrast to the conclusions reached in some previous studies, we find that rrnB P2 is regulated: it displays clear responses to amino acid availability (stringent control), rRNA gene dose (feedback control), and changes in growth rate (growth rate-dependent control). Stringent control of rrnB P2 requires the alarmone ppGpp, but growth rate-dependent control of rrnB P2 does not require ppGpp. The rrnB P2 core promoter sequence (-37 to +7) is sufficient to serve as the target for growth rate-dependent regulation.

Amino Acids↗

PubMed2002

Physical and genetic map of Enterococcus faecium ATCC19434 and demonstration of intra- and interspecific genomic diversity in enterococci.

A physical map of the Enterococcus faecium ATCC19434 chromosome was constructed by NotI, I-CeuI and Sse8387I. The chromosome was a circular DNA of 2600 kb in size, and contained six rRNA operons (rrn). The locations and orientations of the six rrn operons and 24 different determinants were mapped. Genomes of three additional E. faecium strains were also analyzed by I-CeuI digestion, and the genome sizes were found to vary from 2550 to 2995 kb. We further investigated the genome sizes and number of rrn operons in four E. faecalis, one E. avium, and one E. durans strains. The genome sizes were larger than E. faecium: 3000-3250 kb in E. faecalis, 3445 kb in E. avium, and 3070 kb in E. durans. E. avium and E. durans contained six rrn operons as in E. faecium, but all the E. faecalis strains possessed four rrn operons.

Chromosomes, Bacterial↗

PubMed1994

Isolated P2 rRNA promoters of Escherichia coli are strong promoters that are subject to stringent control.

Ribosomal RNA synthesis in Escherichia coli is under stringent control. The seven rRNA operons are highly conserved and are each transcribed from two tandem promoters, P1 and P2, which are located about 120 base-pairs apart. In exponentially growing cells the majority of the transcripts are initiated at the P1 promoters. The P1 promoters are highly regulated, and are under stringent as well as growth rate controls. Here we demonstrate that transcription from the rrnA P1 promoter diminishes P2 expression. In the absence of P1, the P2 promoter acts as a rather strong promoter. Insertion of a transcription terminator between P1 and P2 eliminates the inhibition of P2 by P1, suggesting that the physical movement of RNA polymerase originating at P1 and progressing along the P2 promoter is necessary for the interference process to take place. Similarly to P1, the solitary P2 promoter is subject to stringent control.

Base Sequence↗

PubMedDate not supplied

[Adaptive reactions of mycoplasmas in vitro: "viable but unculturable forms" and nanocells of Acholeplasma laidlawii].

The adaptation of Acholeplasma laidlawii to conditions unfavorable for growth has been found to be accompanied by cell transformation into special morphological structures known as ultramicroforms (nanocells). The ratio of the cells of the two morphological types in the population depended on the growth conditions. Nanocells retained viability for a long time under conditions unfavorable for growth and showed resistance to stressors. Reduction in the cell size occurred due to unequal division, which involved the loss of cytoplasmic material. A. laidlawii ultramicroforms (nanocells) were able to restore proliferative activity and to revert to their initial vegetative form; they measured less than 0.2 microm and are the smallest cells known at present. Nanocells formed in vitro under exposure to abiogenic stressors may correspond to the A. laidlawii minibodies observed in infected plants upon exposure to biogenic stressors. The transformation of A. laidlawii cells into ultramicroforms was accompanied by condensation of the nucleoid, a change in the polypeptide spectrum, and a change in the availability of rRNA operons for in vitro amplification. All these changes are indicative of reorganization of the genetic and metabolic systems of mycoplasmas.

Acholeplasma laidlawii↗

PubMed2000

Organization of the ribosomal operon 165-235 gene spacer region in representatives of Neisseria gonorrhoeae.

Ribosomal rRNA gene fragments (rDNA) encompassing part of the 16S rDNA, the 16S-23S rDNA spacer region and part of the 23S rDNA of 229 Neisseria gonorrhoeae strains were enzymatically amplified using conserved primers. The fragments of approximately 1200 bp were subjected to restriction analysis with HinfI. This revealed 13 patterns (patterns I-XIII) of which patterns I (78 strains), II (32 strains), III (38 strains) and IV (56 strains) were the most abundant, comprising 89.1% of the strains. The obtained restriction patterns consisted of 3 to 8 bands, ranging in size from 32 to 854 bp. The sum of the obtained bands was about 1200 bp for patterns I, II, III, IV, V, IX, and XIII. However, for patterns VI, VII, VIII, X, XI and XII, the sum of the bands well exceeded the estimated size of approximately 1200 bp. We demonstrated that this results from sequence divergence in the 4 rRNA operons, present in the genome of N. gonorrhoeae, giving rise to patterns that are a combination of several other patterns.

Base Sequence↗

PubMed1998

Roles of multiple promoters in transcription of ribosomal DNA: effects of growth conditions on precursor rRNA synthesis in mycobacteria.

The roles of multiple promoters in the synthesis of rRNA under different conditions of growth were investigated, using two mycobacterial species as model organisms. When Mycobacterium smegmatis was grown under optimal conditions, its two rRNA operons contributed equally, with two promoters, one from each operon, being responsible for most transcripts. In stationary-phase growth or balanced growth under carbon starvation conditions, one operon (rrnAf) dominated and its three promoters contributed more equally to the generation of transcripts. Mycobacterium tuberculosis has a single operon with two promoters, one of which generated 80% of transcripts, at all stages of growth. We infer that each promoter functions independently according to its intrinsic strength when cells are growing slowly so that one operon with three promoters is roughly equivalent to three operons with one promoter; at high growth rates, occlusion effects reduce the efficiency of multiple promoters to that of a single promoter.

Culture Media↗

PubMed1996

Sequence and characterisation of a ribosomal RNA operon from Agrobacterium vitis.

One of the four ribosomal RNA operons (rrnA) from the Agrobacterium vitis vitopine strain S4 was sequenced, rrnA is most closely related to the rrn operons of Bradyrhizobium japonicum and Rhodobacter sphaeroides and carries an fMet-tRNA gene downstream of its 5S gene, as in the case of R. sphaeroides. The 16S rRNA sequence of S4 differs from the A. vitis K309 type strain sequence by only one nucleotide, in spite of the fact that S4 and K309 have very different Ti plasmids. The predicted secondary structure of the S4 23S rRNA shows several features that are specific for the alpha proteobacteria, and an unusual branched structure in the universal B8 stem. The 3' ends of the three other rrn copies of S4 were also cloned and sequenced. Sequence comparison delimits the 3' ends of the four repeats and defines two groups: rrnA/rrnB and rrnC/rrnD.

Base Sequence↗

PubMed2003

Molecular characterisation of Armillaria species from Zimbabwe.

Armillaria species are amongst the most important pathogens of trees and have a world-wide distribution. In recent years, the taxonomy of Northern Hemisphere Armillaria spp. has been extensively treated, but those occurring in Africa are poorly known. Previously, isolates of Armillaria from Zimbabwe have been grouped based on morphology and biochemical tests. In this study, six isolates representing the three previously characterized groups of Armillaria spp. occurring in Zimbabwe were analysed using DNA-based techniques. Three distinct clusters emerged from both PCR-RFLP and analysis of sequence data for the IGS-1 rRNA operon. The three groups corresponded to those previously identified based on morphology and biochemical tests. Differences in IGS-1 sequences strongly suggest that the Zimbabwean groups represent three distinct taxa. Isolates belonging to Group I, previously assumed to be to A. heimii, were similar to those identified as A. fuscipes from South Africa and La Reunion. Group II isolates resided in a clade apart from all other isolates and appear to represent A. heimii. The remaining isolates residing in Group III clustered with isolates from Zambia and Cameroon. These are different from A. heimii and A. fuscipes and apparently represent an undescribed taxon.

Agaricales↗

PubMed2002

Enumeration of total bacteria and bacteria with genes for proteolytic activity in pure cultures and in environmental samples by quantitative PCR mediated amplification.

Real-time quantitative PCR assays were developed for the absolute quantification of different groups of bacteria in pure cultures and in environmental samples. 16S rRNA genes were used as markers for eubacteria, and genes for extracellular peptidases were used as markers for potentially proteolytic bacteria. For the designed 16S rDNA TaqMan assay, specificity of the designed primer-probe combination for eubacteria, a high amplification efficiency over a wide range of starting copy numbers and a high reproducibility is demonstrated. Cell concentrations of Bacillus cereus, B. subtilis and Pseudomonas fluorescens in liquid culture were monitored by TaqMan-PCR using the 16S rDNA target sequence of Escherichia coli as external standard for quantification. Results agree with plate counts and microscopic counts of DAPI stained cells. The significance of 16S rRNA operon multiplicity to the quantification of bacteria is discussed.Furthermore, three sets of primer pair together with probe previously designed for targeting different classes of bacterial extracellular peptidases were tested for their suitability for TaqMan-PCR based quantification of proteolytic bacteria. Since high degeneracy of the probes did not allow accurate quantification, SybrGreen was used instead of molecular probes to visualize and quantify PCR products during PCR. The correlation between fluorescence and starting copy number was of the same high quality as for the 16S rDNA TaqMan assay for all the three peptidase gene classes. The detected amount of genes for neutral metallopeptidase of B. cereus, for subtilisin of B. subtilis and for alkaline metallopeptidase of P. fluorescens corresponded exactly to the numbers of bacteria investigated by the 16S rDNA targeting assay. The developed assays were applied for the quantification of bacteria in soil samples.

Bacillus cereus↗

PubMed2001

Clinical correlation of variations in the internal transcribed spacer regions of rRNA genes in Pneumocystis carinii f.sp. hominis.

OBJECTIVES: To analyse the importance of sequence variations in the internal transcribed spacer (ITS) regions 1 and 2 of the nuclear rRNA operon in AIDS patients with Pneumocystis carinii pneumonia (PCP). DESIGN AND METHODS: ITS 1 and 2 genotypes were determined in 162 bronchoalveolar lavage samples from 130 patients participating in a prospective cohort study of PCP. RESULTS: A total of 49 different ITS genotypes were detected. ITS genotype was not associated with the clinical severity or outcome of PCP. In 37 of 162 (23%) samples infection with two or more genotypes was observed. A genotype switch was detected in six of 10 patients (60%) with recurrent episodes of PCP. However, genotype changes were also seen in 10 of 19 patients (53%) who had repeated bronchoscopies within the same episode of PCP. The same ITS type was observed twice in 13 (46%) of the 28 patients with repeat bronchoscopies during single or recurrent episodes of pneumonia, but in only 14 of 81 (17%) randomly selected pairs (P < 0.01). CONCLUSION: Although the detection of ITS genotypes is not a random event, changes in genotype can be detected in a single episode of disease, with 23% of PCP patients being infected with more than one P. carinii genotype, thus complicating the use of this locus as a genetic marker to separate new infection from the reactivation of latent infection. ITS genotypes are not associated with the clinical severity of PCP.

AIDS-Related Opportunistic Infections↗

PubMed2004

Gene expression in Chromobacterium violaceum.

The repertoire of 4,431 open reading frames (ORFs), eight rRNA operons and 98 tRNA genes of Chromobacterium violaceum must be expressed in a regulated manner for successful adaptation to a wide variety of environmental conditions. To accomplish this feat, the organism relies on protein machineries involved in transcription, RNA processing and translation. Analysis of the C. violaceum genome showed that transcription initiation, elongation and termination are performed by the five well-known RNA polymerase subunits, five categories of sigma 70 factors, one sigma 54 factor, as well as six auxiliary elongation and termination factors. RNA processing is performed by a variety of endonucleases and exonucleases, such as ribonuclease H, ribonuclease E, ribonuclease P, and ribonuclease III, in addition to poly(A) polymerase and specific methyltransferases and pseudouridine synthases. ORFs for all ribosomal proteins, except S22, were found. Only 19 aminoacyl-tRNA synthetases were found, in addition to three aminoacyl-tRNA synthetase-related proteins. Asparaginyl-tRNA (Asn) is probably obtained by enzymatic modification of a mischarged aminoacyl-tRNA. The translation factors IF-1, IF-2, IF-3, EF-Ts, EF-Tu, EF-G, RF-1, RF-2 and RF-3 are all present in the C. violaceum genome, although the absence of selB suggests that C. violaceum does not synthesize selenoproteins. The components of trans-translation, tmRNA and associated proteins, are present in the C. violaceum genome. Finally, a large number of ORFs related to regulation of gene expression were also found, which was expected, considering the apparent adaptability of this bacterium.

Adaptation, Physiological↗

PubMed1997

Source of variation detected in ribotyping patterns of Haemophilus influenzae: comparison of traditional ribotyping, PCR-ribotyping and rDNA restriction analysis.

The pattern of EcoRI restriction fragments of chromosomal DNA that hybridize with a probe for genes encoding 16S and 23S rRNA is highly discriminatory for non-capsulate Haemophilus influenzae (NCHI). The source of variation detected by these probe-based ribotyping patterns was investigated by restriction analysis of rRNA operon (rrn) amplification products from nine representative strains. Digestion of rrn amplification products with EcoRI indicated one conserved EcoRI site within 16S rDNA and no EcoRI sites within the 16S-23S intergenic spacer region of the nine strains, and an EcoRI site at the 5' end of 23S rDNA from seven of the nine strains. Comparison of the EcoRI ribotyping patterns obtained with separate probes for 16S and 23S rDNA showed more variation with the 23S probe indicating variation in EcoRI sites downstream from the operon. Restriction analyses of 16S and 23S rDNA amplification products with AluI, HhaI, HaeIII and TaqI divided the nine 'traditional' ribotypes into a maximum of three and eight groups, respectively. Similar analyses of the 16S-23S intergenic regions (PCR-ribotyping) failed to distinguish any of the nine representative strains. Therefore, there is probably insufficient variation within the operon for it to form a good target for PCR-based typing methods. In contrast, 'traditional' ribotyping with cDNA from 16S plus 23S rRNA detects restriction site differences in the sequences flanking the operon, which show considerably more variation between strains. 'Traditional' ribotyping should therefore remain the standard for characterising NCHI in epidemiological investigations.

Bacterial Typing Techniques↗

PubMed1989

Termination of transcription by yeast RNA polymerase I.

Analysis of the termination of transcription by yeast RNA polymerase I (Pol I) using in vitro run-on experiments in both isolated nuclei and permeabilized cells demonstrated that Pol I does not traverse the whole intergenic spacer separating consecutive 37S operons, but terminates transcription before reaching the 5S rRNA gene, that is within NTS 1. In order to discriminate between processing and termination at the 3'-end generating sites previously identified in vivo in NTS 1 (T1, T2 and T3), fragments containing these sites were inserted into the middle of the reporter DNA of an artificial rRNA minigene. RNA isolated from yeast cells transformed with these minigenes was analyzed for the presence of transcripts derived from sequences both up- and downstream of the insert by Northern blot hybridization, reverse transcription analysis and S1 nuclease mapping. In accordance with previously obtained results T1 (+15 to +50) was found to behave as a processing site. T2 (+210) however was concluded to be an efficient, genuine Pol I terminator. In addition to T2, two other terminators were identified in NTS 1: T3A (at +690) and T3B (at +950). Surprisingly, when the 3' terminal part of NTS 2 was tested for its capacity to generate 3'-ends, another terminator (Tp) was found to be present at a position 300 bp upstream of the transcription initiation site of the 37S-rRNA operon.

Base Sequence↗