Harnessing the intragenomic variability of rRNA operons to improve differentiation of Vibrio species.

Although the 16S rRNA gene is frequently used as a phylogenetic marker in analysis of environmental DNA, this marker often fails to distinguish closely related species, including those in the genus Vibrio. Here, we investigate whether inclusion and analysis of 23S rRNA sequence can help overcome the intrinsic weaknesses of 16S rRNA analyses for the differentiation of Vibrio species. We construct a maximum likelihood 16S rRNA gene tree to assess the use of this gene to identify clades of Vibrio species. Within the 16S rRNA tree, we identify the putative informative bases responsible for polyphyly, and demonstrate the association of these positions with tree topology. We demonstrate that concatenation of 16S and 23S rRNA genes increases the number of informative nucleotide positions, thereby overcoming ambiguities in 16S rRNA-based phylogenetic reconstructions. Finally, we experimentally demonstrate that this approach considerably improves the differentiation and identification of Vibrio species in environmental samples.

rRNA Operon Copy Number Can Explain the Distinct Epidemiology of Hospital-Associated Methicillin-Resistant Staphylococcus aureus.

The distinct epidemiology of original hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) and early community-associated MRSA (CA-MRSA) is largely unexplained. S. aureus carries either five or six rRNA operon copies. Evidence is provided for a scenario in which MRSA has adapted to the hospital environment by rRNA operon loss (six to five copies) due to antibiotic pressure. Early CA-MRSA, in contrast, results from wild-type methicillin-susceptible S. aureus (MSSA) that acquired mecA without loss of an rRNA operon. Of the HA-MRSA isolates (n = 77), 67.5% had five rRNA operon copies, compared to 23.2% of the CA-MRSA isolates (n = 69) and 7.7% of MSSA isolates (n = 195) (P < 0.001). In addition, 105 MSSA isolates from cystic fibrosis patients were tested, because these patients are repeatedly treated with antibiotics; 32.4% of these isolates had five rRNA operon copies. For all subsets, a correlation between resistance profile and rRNA copy number was found. Furthermore, we showed that in vitro antibiotic pressure may result in rRNA operon copy loss. We also showed that without antibiotic pressure, S. aureus isolates containing six rRNA copies are more fit than isolates with five copies. We conclude that HA-MRSA and cystic fibrosis isolates most likely have adapted to an environment with high antibiotic pressure by the loss of an rRNA operon copy. This loss has facilitated resistance development, which promoted survival in these niches. However, strain fitness decreased, which explains their lack of success in the community. In contrast, CA-MRSA isolates retained six rRNA operon copies, rendering them fitter and thereby able to survive and spread in the community.

Ribosomal multi-operon diversity: an original perspective on the genus Aeromonas.

16S rRNA gene (rrs) is considered of low taxonomic interest in the genus Aeromonas. Here, 195 Aeromonas strains belonging to populations structured by multilocus phylogeny were studied using an original approach that considered Ribosomal Multi-Operon Diversity. This approach associated pulsed-field gel electrophoresis (PFGE) to assess rrn operon number and distribution across the chromosome and PCR-temporal temperature gel electrophoresis (TTGE) to assess rrs V3 region heterogeneity. Aeromonads harbored 8 to 11 rrn operons, 10 operons being observed in more than 92% of the strains. Intraspecific variability was low or nul except for A. salmonicida and A. aquariorum suggesting that large chromosomic rearrangements might occur in these two species while being extremely rarely encountered in the evolution of other taxa. rrn operon number at 8 as well as PFGE patterns were shown valuable for taxonomic purpose allowing resolution of species complexes. PCR-TTGE revealed a high rate of strains (41.5%) displaying intragenomic rrs heterogeneity. Strains isolated from human samples more frequently displayed intragenomic heterogeneity than strains recovered from non-human and environmental specimens. Intraspecific variability ranged from 0 to 76.5% of the strains. The observation of species-specific TTGE bands, the recovery of identical V3 regions in different species and the variability of intragenomic heterogeneity (1-13 divergent nucleotides) supported the occurrence of mutations and horizontal transfer in aeromonad rrs evolution. Altogether, the presence of a high number of rrn operon, the high proportion of strains harboring divergent rrs V3 region and the previously demonstrated high level of genetic diversity argued in favor of highly adaptative capabilities of aeromonads. Outstanding features observed for A. caviae supported the ongoing process of adaptation to a specialized niche represented by the gut, previously hypothesized. 16S rRNA gene is an informative marker in the genus Aeromonas for both evolutionary and polyphasic taxonomic studies provided that multi-operon fingerprinting approaches are used.

[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.

Quantitative relationships for specific growth rates and macromolecular compositions of Mycobacterium tuberculosis, Streptomyces coelicolor A3(2) and Escherichia coli B/r: an integrative theoretical approach.

Further understanding of the physiological states of Mycobacterium tuberculosis and other mycobacteria was sought through comparisons with the genomic properties and macromolecular compositions of Streptomyces coelicolor A3(2), grown at 30 degrees C, and Escherichia coli B/r, grown at 37 degrees C. A frame of reference was established based on quantitative relationships observed between specific growth rates ( micro ) of cells and their macromolecular compositions. The concept of a schematic cell based on transcription/translation coupling, average genes and average proteins was developed to provide an instantaneous view of macromolecular synthesis carried out by cells growing at their maximum rate. It was inferred that the ultra-fast growth of E. coli results from its ability to increase the average number of rRNA (rrn) operons per cell through polyploidy, thereby increasing its capacity for ribosome synthesis. The maximum growth rate of E. coli was deduced to be limited by the rate of uptake and consumption of nutrients providing energy. Three characteristic properties of S. coelicolor A3(2) growing optimally ( micro =0.30 h(-1)) were identified. First, the rate of DNA replication was found to approach the rate reported for E. coli ( micro =1.73 h(-1)); secondly, all rrn operons were calculated to be fully engaged in precursor-rRNA synthesis; thirdly, compared with E. coli, protein synthesis was found to depend on higher concentrations of ribosomes and lower concentrations of aminoacyl-tRNA and EF-Tu. An equation was derived for E. coli B/r relating micro to the number of rrn operons per genome. Values of micro =0.69 h(-1) and micro =1.00 h(-1) were obtained respectively for cells with one or two rrn operons per genome. Using the author's equation relating the number of rrn operons per genome to maximum growth rate, it is expected that M. tuberculosis with one rrn operon should be capable of growing much faster than it actually does. Therefore, it is suggested that the high number of insertion sequences in this species attenuates growth rate to still lower values.

Analysis of conserved non-rRNA genes of Tropheryma whipplei.

The causative agent of Whipple's disease, Tropheryma whipplei, is a slow-growing bacterium that remains poorly-understood. Genetic characterization of this organism has relied heavily upon rRNA sequence analysis. Pending completion of a complete genome sequencing effort, we have characterized several conserved non-rRNA genes from T. whipplei directly from infected tissue using broad-range PCR and a genome-walking strategy. Our goals were to evaluate its phylogenetic relationships, and to find ways to expand the strain typing scheme, based on rDNA sequence comparisons. The genes coding for the ATP synthase beta subunit (atpD), elongation factor Tu (tuf), heat shock protein GroEL (groEL), beta subunit of DNA-dependent RNA polymerase (rpoB), and RNase P RNA (rnpB) were analyzed, as well as the regions upstream and downstream of the rRNA operon. Phylogenetic analyses with all non-rRNA marker molecules consistently placed T. whipplei within the class, Actinobacteria. The arrangement of genes in the atpD and rpoB chromosomal regions was also consistent with other actinomycete genomes. Tandem sequence repeats were found upstream and downstream of the rRNA operon, and downstream of the groEL gene. These chromosomal sites and the 16S-23S rRNA intergenic spacer regions were examined in the specimens of 11 patients, and a unique combination of tandem repeat numbers and spacer polymorphisms was found in each patient. These data provide the basis for a more discriminatory typing method for T. whipplei.

Unique architecture of the plastid ribosomal RNA operon promoter recognized by the multisubunit RNA polymerase in tobacco and other higher plants.

Expression of the plastid rRNA operon (rrn) during development is highly regulated at the level of transcription. The plastid rrn operon in most higher plants is transcribed by the plastid-encoded RNA polymerase (PEP), the multisubunit plastid RNA polymerase from PrrnP1, a sigma(70)-type promoter with conserved -10 and -35 core promoter elements. To identify functionally important sequences, the tobacco PrrnP1 was dissected in vivo and in vitro. Based on in vivo deletion analysis, sequences upstream of nucleotide -83 do not significantly contribute to promoter function. The in vitro analyses identified an essential hexameric sequence upstream of the -35 element (GTGGGA; the rRNA operon upstream activator [RUA]) that is conserved in monocot and dicot species and suggested that the -10 element plays only a limited role in PrrnP1 recognition. Mutations in the initial transcribed sequence (+9 to +14) enhanced transcription, the characteristic of strong promoters in prokaryotes. We propose that sigma interaction with the -10 element in PrrnP1 is replaced in part by direct PEP-RUA (protein-DNA) interaction or by protein-protein interaction between the PEP and an RUA binding transcription factor.

Chloroplast transformation with modified accD operon increases acetyl-CoA carboxylase and causes extension of leaf longevity and increase in seed yield in tobacco.

Acetyl-CoA carboxylase (ACCase) in plastids is a key enzyme regulating the rate of de novo fatty acid biosynthesis in plants. Plastidic ACCase is composed of three nuclear-encoded subunits and one plastid-encoded accD subunit. To boost ACCase levels, we examined whether overexpression of accD elevates ACCase production. Using homologous recombination, we replaced the promoter of the accD operon in the tobacco plastid genome with a plastid rRNA-operon (rrn) promoter that directs enhanced expression in photosynthetic and non-photosynthetic organs, and successfully raised the total ACCase levels in plastids. This result suggests that the level of the accD subunit is a determinant of ACCase levels, and that enzyme levels are in part controlled post-transcriptionally at the level of subunit assembly. The resultant transformants grew normally and the fatty acid content was significantly increased in leaves, but not significantly in seeds. However, the transformants displayed extended leaf longevity and a twofold increase of seed yield over the control value, which eventually almost doubled the fatty acid production per plant of the transformants relative to control and wild-type plants. These findings offer a potential method for raising plant productivity and oil production.

Sequence variability in the first internal transcribed spacer region within and among Cyclospora species is consistent with polyparasitism.

Cyclospora cayetanensis is a coccidian parasite which causes severe gastroenteritis in humans. Molecular information on this newly emerging pathogen is scarce. Our objectives were to assess genetic variation within and between human-associated C. cayetanensis and baboon-associated Cyclospora papionis by examining the internal transcribed spacer (ITS) region of the ribosomal RNA operon, and to develop an efficient polymerase chain reaction- (PCR)-based method to distinguish C. cayetanensis from other closely related organisms. For these purposes, we studied C. cayetanensis ITS-1 nucleotide variability in 24 human faecal samples from five geographic locations and C. papionis ITS-1 variability in four baboon faecal samples from Tanzania. In addition, a continuous sequence encompassing ITS-1, 5.8S rDNA and ITS-2 was determined from two C. cayetanensis samples. The results indicate that C. cayetanensis and C. papionis have distinct ITS-1 sequences, but identical 5.8S rDNA sequences. ITS-1 is highly variable within and between samples, but variability does not correlate with geographic origin of the samples. Despite this variability, conserved species-specific ITS-1 sequences were identified and a single-round, C. cayetanensis-specific PCR-based assay with a sensitivity of one to ten oocysts was developed. This consistent and remarkable diversity among Cyclospora spp. ITS-1 sequences argues for polyparasitism and simultaneous transmission of multiple strains.

Sequence microdiversity at the ribosomal RNA operons of Escherichia coli pyelonephritogenic strains.

OBJECTIVE: To determine whether Escherichia coli strains isolated from patients with uncomplicated acute pyelonephritis can be distinguished from those isolated from patients with complicated acute pyelonephritis on the basis of the genetic background. METHODS: In total, 103 E. coli strains isolated from patients with acute pyelonephritis (59 uncomplicated pyelonephritis (UAP) and 44 complicated pyelonephritis (CAP)) were characterized by RFLP of the intergenic spacer region 16S-23S rRNA, the presence of three alternative sequences found in the polymorphic V6 loop of the 16S rRNA gene, the presence of the pap gene, and antibiotic susceptibility. RESULTS: At similarity levels of 70%, four RFLP groups (alpha1, alpha2, beta1 and beta2) were discerned. Strains from UAP were statistically significant for alpha RFLP, with a strong association with the presence of the pap gene, V6-I sequence and antibiotic multisensitivity. Strains from CAP randomly belonged to the alpha or beta RFLP groups, with a very low presence of the pap gene, and random presence of V6 sequences, and were multiresistant to antibiotics. When the CAP strains were distributed according to underlying pathology, non-obstructive cases had RFLP and V6 polymorphisms similar to those of UAP cases, while obstructive cases were clearly distinct. CONCLUSIONS: UAP and non-obstructive CAP E. coli strains are sensitive to antimicrobials, show a high level of the pap gene and belong to the selective, homogeneous and highly protected molecular alpha2 group, where no recombinations, deletions or insertions are present. On the contrary, obstructive and vesicorenal reflux E. coli strains show significant antimicrobial resistance, high intercistronic heterogenicity (wide presence of block nucleotidic substitutions, deletions or insertions) and significantly lower virulence.

Degenerative minimalism in the genome of a psyllid endosymbiont.

Psyllids, like aphids, feed on plant phloem sap and are obligately associated with prokaryotic endosymbionts acquired through vertical transmission from an ancestral infection. We have sequenced 37 kb of DNA of the genome of Carsonella ruddii, the endosymbiont of psyllids, and found that it has a number of unusual properties revealing a more extreme case of degeneration than was previously reported from studies of eubacterial genomes, including that of the aphid endosymbiont Buchnera aphidicola. Among the unusual properties are an exceptionally low guanine-plus-cytosine content (19.9%), almost complete absence of intergenic spaces, operon fusion, and lack of the usual promoter sequences upstream of 16S rDNA. These features suggest the synthesis of long mRNAs and translational coupling. The most extreme instances of base compositional bias occur in the genes encoding proteins that have less highly conserved amino acid sequences; the guanine-plus-cytosine content of some protein-coding sequences is as low as 10%. The shift in base composition has a large effect on proteins: in polypeptides of C. ruddii, half of the residues consist of five amino acids with codons low in guanine plus cytosine. Furthermore, the proteins of C. ruddii are reduced in size, with an average of about 9% fewer amino acids than in homologous proteins of related bacteria. These observations suggest that the C. ruddii genome is not subject to constraints that limit the evolution of other known eubacteria.

Bacillus thermodenitrificans sp. nov., nom. rev.

A polyphasic study was performed on 10 soil isolates of thermophilic denitrifying Bacillus strains from different geographical areas. The presence of two main characteristic bands following amplification of the internal transcribed spacer (ITS) region of rrn operons suggests a close relatedness to 'Bacillus thermodenitrificans'. The isolates cluster around two strains of 'B. thermodenitrificans' in riboprint and fatty acid analyses, though differences occur at the strain level. Subsequent DNA-DNA reassociation studies including the 10 isolates, 'B. thermodenitrificans' DSM 465T and DSM 466, and Bacillus stearothermophilus ATCC 12980T and Bacillus thermoleovorans ATCC 43513T revealed such a high level of genomic relatedness between the isolates and the DSM strains (> 73% similarity) that they must be considered strains of the same taxon. The degree of DNA-DNA similarity between the 12 strains of 'B. thermodenitrificans' and the type strains of the other two phylogenetically neighbouring Bacillus species was significantly lower (21-43% similarity). Based upon phylogenetic, chemotaxonomic and phenotypic evidence, the designation of B. thermodenitrificans sp. nov., nom. rev. is proposed. The type strain of B. thermodenitrificans is DSM 465T.

The phenotype of mutations of the base-pair C2658.G2663 that closes the tetraloop in the sarcin/ricin domain of Escherichia coli 23 S ribosomal RNA.

The sarcin/ricin domain (SRD) in Escherichia coli 23 S rRNA is a part of the site for the association of elongation factors with ribosomes and for that reason is critical for the binding of aminoacyl-tRNA and for translocation during the reiterative elongation reactions of protein synthesis. The SRD has a GAGA tetraloop that is shut off by a Watson-Crick C2658 x G2663 pair. The contribution of this pair to the function of the ribosome has been evaluated by constructing mutations in the nucleotides and determining their phenotype. Constitutive expression of a plasmid-encoded rrnB operon with a G2663C transversion mutation that disrupts the Watson-Crick pair was lethal. Double transversion mutations, C2658G x G2663C and C2658A x G2663U, that reverse the polarity of the pyrimidine and the purine but restore the potential to form a canonical pair, were also lethal. Induction of transcription of 23 S rRNA with the same mutations, but encoded in a plasmid with a lambdaP(L) promoter and expressed at a lower level, retarded growth. The sedimentation profiles of ribosomes with transversion mutations in C2658 and/or G2663 are altered; the ratio of 50 S subunits to 30 S particles is changed and polysomes are reduced. Ribosomes with a G2663C, a C2658G x G2663C, or a C2658A x G2663U mutation in 23 S rRNA were not active in protein synthesis, indeed, they appeared to inhibit the activity of ribosomes with wild-type 23 S rRNA. Transversion mutations in the analogs of C2658 and G2663 decreased binding of EF-G to SRD oligoribonucleotides; the same mutations in 23 S rRNA decreased binding of the factor to intact ribosomes. The most severe phenotype, in growth, in protein synthesis, and in the binding of EF-G, was associated with a C2658G x G2663C mutation; it is surprising that this was more severe than an analogous C2658A x G2663U mutation. A double transition mutation, C2658U x G2663A, which is not known to have occurred in nature, had no effect on the growth of cells or on the function of ribosomes. The lethal phenotype of transversion mutations in C2658 and G2663 appears to derive from a loss of the capacity of ribosomes to bind EF-G and by indirection the EF-Tu ternary complex.

The phenotype of mutations of G2655 in the sarcin/ricin domain of 23 S ribosomal RNA.

The sarcin/ricin domain (SRD) in Escherichia coli 23 S rRNA forms a part of the site for the association of the elongation factors with the ribosome and hence is critical for the binding of aminoacyl-tRNA and for translocation. The domain is also the site of action of the eponymous toxins which catalyze covalent modification of single nucleotides that inactivate the ribosome. The conformation of the conserved guanosine at position 2655 is an especially prominent feature of the structure of the SRD: the nucleotide is bulged out of a helix and forms a base-triple with A2665 and U2656. G2655 in 23 S rRNA is protected from chemical modification when the elongation factors, EF-Tu and EF-G, are bound to ribosomes and the analog of G2655 in oligoribonucleotides is critical for recognition by the toxin sarcin and by EF-G. The contribution of G2655 to the function of the ribosome has been evaluated by constructing mutations in the nucleotide and determining the phenotype. Constitutive expression of a plasmid-encoded rrnB operon with a deletion of, or transversions in, G2655 is lethal to E. coli cells, whereas a defect in the growth of cells with a G2655A transition is observed only in competition with wild-type cells. The sedimentation profiles of ribosomes with mutations in G2655 are altered; most markedly by deletion or transversion of the nucleotide, less severely by transition to adenosine. Mutations of G2655 confer resistance to sarcin on ribosomes. Ribosomes with G2655Delta, G2655C, or G2655U mutations in 23 S rRNA are not active in protein synthesis, whereas those with the G2655A transition mutation suffer decreased activity.

The mediator for stringent control, ppGpp, binds to the beta-subunit of Escherichia coli RNA polymerase.

BACKGROUND: Inhibition of transcription of rRNA in Escherichia coli upon amino acid starvation is thought to be due to the binding of ppGpp to RNA polymerase. However, the nature of this interaction still remains obscure. RESULTS: Here, the azido-derivative of ppGpp was synthesized from azido-GDP and [gamma-32P]ATP by way of the phosphate transfer reaction of the RelA enzyme. The product was subsequently characterized by one and two-dimensional chromatography. The resulting compound [32P]azido-ppGpp, where the azido group is attached to the base moiety, was purified to homogeneity and was photo-crosslinked to Escherichia coli RNA polymerase. SDS-PAGE analysis of the azido-ppGpp-bound enzyme, tryptic digestion and Western blot analysis suggested that azido-ppGpp binds to the beta-subunit of RNA polymerase. CONCLUSION: It was observed that both the N-terminal and C-terminal domains of the beta-subunit were labelled with azido-ppGpp in the native enzyme. However, under denaturing conditions only the C-terminal part from amino acid residue 802 to residue 1211/1216/1223 was predominantly crosslinked to azido-ppGpp. The excess of unlabelled ppGpp competes with azido-ppGpp for binding to the enzyme. azido-ppGpp inhibits single-round transcription at the stringent promoter like rrnBP1. In addition, ribosomal protein genes were also found to be inhibited by N3ppGpp. On the other hand, transcription at the lac UV5 promoter remained unaffected upon the addition of azido-ppGpp.

Partial sequence of the rRNA operon of Pneumocystis carinii isolates from Puerto Rico

Several reports indicate geographic variation of isolates of Pneumocystis carinii hominis. We have sequenced the internal transcribed spacer (ITS) region and large subunit Group I intron of rRNA genes from P. carinii DNA obtained from two patients from Puerto Rico. Both can be subclassified as Type II, according to the sequence of the ITS region. A system capable of identifying individual isolates will be an essential tool for epidemiological studies of the organism. The amplification of DNA from fixed tissues may facilitate the processing of a large number of samples.

Organ-specific transcription of the rrn operon in spinach plastids.

The spinach rrn operon is used as a model system to study transcriptional regulation in higher plant photosynthetic and non-photosynthetic plastids. We performed capping experiments to determine whether P1, PC, or P2 promoters are employed for rrn transcription start sites in cotyledon and root tissues. By using a new method of analysis of capped RNA we demonstrate for the first time that 1) in both organs the rrn operon is expressed in a constitutive manner by cotranscription with the preceding tRNA(GAC)Val gene, and 2) the PC transcription start site is used only in cotyledons and leaves, i.e. we demonstrate the organ-specific usage of a plastid promoter. Both start sites, PC and that of the tRNA(GAC)Val cotranscript, lack Escherichia coli-like consensus sequences. The cotranscript is initiated 457 base pairs upstream of the tRNA(GAC)Val gene. The PC-specific DNA-binding factor, CDF2, is not detectable in root tissues confirming its regulatory role in PC-initiated rrn expression and the organ specificity of PC expression. Furthermore, our results show that rrn operon expression patterns differ in spinach and tobacco indicating species-specific transcriptional regulation of plant plastid gene expression.

Regulation of ribosomal RNA gene transcription during retinoic acid-induced differentiation of mouse teratocarcinoma cells.

We have examined the mechanism of regulation of rRNA synthesis in mouse F9 teratocarcinoma cells that were induced to differentiate by retinoic acid and dibutyryl cAMP. Ribosomal RNA (rRNA) synthesis was significantly reduced during differentiation of F9 cells into parietal endoderm cells. Nuclear run-on assay revealed that the rRNA gene transcription rates were reduced in differentiated cells, and this phenomenon could be mimicked by in vitro transcription assay using nuclear extracts prepared from F9 stem and F9 parietal endoderm cells. Analysis of the DNA-binding activities of two RNA polymerase I (pol I) transcription factors E1BF/Ku and UBF revealed decreased affinity for their cognate recognition sequences. Immunoblot analysis showed a marked reduction in the amounts of E1BF/Ku and UBF in the differentiated cells. Analysis of the steady-state RNA levels for the smaller subunit of E1BF/Ku and for UBF in differentiating F9 cells revealed decreased mRNA synthesis and increase in message level for the differentiation-specific marker laminin B1 with progression of the differentiated status of the cells. This study has demonstrated that differentiation of mouse F9 teratocarcinoma cells into parietal endoderm cells leads to diminished rRNA synthesis, which may be mediated by reduced DNA-binding activities and amounts of at least two pol I transcription factors.

Gradient of genomic diversity in the Pseudomonas aeruginosa chromosome.

In 545 Pseudomonas aeruginosa strains, mainly collected from patients with cystic fibrosis, SpeI-DraI macrorestriction fragment length diversity was scanned for using probes of known map position on the P. aeruginosa PAO chromosome. Southern analysis of the 60 unrelated clones uncovered a gradient of macrorestriction fragment length polymorphisms (RFLPs) from the origin of replication towards the auxotroph-poor region of the P. aeruginosa genome. Linkage disequilibrium between macrorestriction sites was conserved in the P. aeruginosa population in the region encompassed by the rrn operons. The oriC-reactive SpeI fragment was conserved in nearly all isolates examined. Few fragment length classes were seen for the alg60-, algR- and toxA-encoding SpeI fragments. Fragment size varied within one class by up to 20 kb. Two probes from the auxotroph-poor region detected a broad size range for the SpeI fragment, suggesting extensive genomic diversity in these regions. Subclonal variation of fragment size was detected at all investigated loci in at least one of the analysed clones, but within one particular clone, SpeI-RFLPs were found at only few loci.

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.