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The number of rRNA genes in Escherichia coli

The three different ribosomal RNA species of Escherichia colt’ (5 S, 16 S and 23 S) originate from a single 30 S precursor RNA. The DNA coding for this 30 S rRNA is called an rRNA gene set (rm). It has long been known that these genes are redundant in bacteria, their number has been estimated on the basis of saturation hybridization experiments as being 510 (for a review see [ 1 ] ). The most reliable measurements suggested the existence of six rm genes [2]. Plasmids and transducing phages helped to map unambiguously five of these, which are now designated rrnA (85 min), rrnB (88 min), rrnC (83 min), rmD (71 min) and rmE (89 min) [3]. In this paper we present evidence strongly suggesting that the actual number of rRNA gene sets is seven in E. coli K12.

Cloning the modification methylase gene of Bacillus sphaericus R in Escherichia coli

The gene coding for the sequence-specific modification methylase methM . BspI of Bacillus sphaericus R has been cloned in Escherichia coli by means of plasmid pBR322. The selection was based on the expression of the cloned gene which rendered the recombinant plasmid resistant to BspI restriction endonuclease cleavage. The gene is carried by a 9 kb BamHI fragment and by a smaller 2.5 kb EcoRI fragment derived from the BamHI fragment. The Bsp-specific methylase level was found to be higher in the recombinant clones than in the parental strain. The methylase gene is probably located on the Bacillus sphaericus chromosome, and not on a plasmid known to be carried by this strain. The recombinant clones do not exhibit an BspI restriction endonuclease activity.

Structure of the Bacillus sphaericus R modification methylase gene

A 2.5 X 10(3) base-pair segment of Bacillus sphaericus R DNA cloned in Escherichia coli has previously been shown to carry the functional BspRI modification methylase gene. The approximate location of the gene on this DNA segment and its direction of transcription were established by subcloning experiments. The nucleotide sequence of the relevant region was determined by the Maxam-Gilbert procedure. An open reading frame that can code for a 424 amino acid protein was found. The calculated molecular weight (48,264) of this protein is in fair agreement with previous estimates (50,000 to 52,000). The synthesis of this protein was demonstrated in E. coli minicells. The initiation point of transcription by E. coli RNA polymerase was localized by in vitro transcription experiments. The open reading frame starts 29 base-pairs downstream from the transcription initiation site and it is preceded by a sequence showing extensive Shine-Dalgarno complementarity. Subcloning experiments and translation in minicells suggest that after removal of this translational initiation site, a secondary start site 29 amino acids downstream can also start translation in E. coli, and this shorter protein retains the methylase activity. The overall base composition of the gene and the codon usage indicate a strong preference for A.T base-pairs.

No mitochondrial haplotype was found to increase risk for alzheimer’s disease

BACKGROUND Seventy Alzheimers disease (AD) patients and 80 age- and sex-matched controls were analyzed for mitochondrial mutations T4336C and A3397G, reported to be associated with AD, and for mutations T4216C/G13708A characteristic for a normal human haplotype associated with increased frequency of occurrence of some hereditary diseases. The distribution of apolipoprotein E (apoE) alleles was also analyzed. METHODS Mitochondrial DNA was amplified by polymerase chain reaction, and the presence of mutations was detected by digestion with approximately chosen restriction endonucleases (restriction fragment length polymorphism). RESULTS One patient and 2 controls were found to belong to the T4336C/T1630C haplotype. No A3397G mutant was detected. The T4216C/G13708A haplotype occurred at 5/70 and 5/80 frequency in the two groups. Prevalence of the apoE4 allele was significantly higher in AD patients (25%) than in the control group (8.1%). CONCLUSIONS The T4336C/T16304C mutations were not found to associated with AD, and no predisposing mitochondrial haplotypes were found.

Identification of two new promoters probably involved in the transcription of a ribosomal RNA gene of Escherichia coli

The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to the 1821st nucleotide upstream from the beginning of the sequence coding for mature 16 S rRNA. In vitro transcription experiments indicated the presence of two new promoters in this region, located more than 1 kb upstream from the known P1 and P2 promoters of rrnB. Previous electron microscopic studies demonstrated that these sites bind RNA-polymerase very strongly. In vitro transcription, starting at these sites reads through the entire region into the rrnB gene without termination. A similar uninterrupted transcription into rrnB in vivo can be demonstrated by S1-mapping, and by fusing the DNA containing the new promoters (but not P1 and P2) to the lacZ gene. Thus it seems likely that these promoters (P3 and P4) belong functionally to the rrnB gene and play some role in its regulation of expression.

Mitochondrial mutation as a probable causative factor in familial progressive tubulointerstitial nephritis

Abstract Renal biopsy of two children and a maternal relative, diagnosed with severe progressive tubulointerstitial nephritis, has shown the presence of distorted mitochondria. Mitochondrial DNA from the blood of these patients was analysed. No major deletions were found, but an A to G mutation was detected in position 5656. It is proposed that this mutation might play a causative role in the renal disease of the patients.

Level of messenger RNA transcribed from the histidine operon in repressed, derepressed and histidine-starved Salmonella typhimurium

Abstract The level of the messenger RNA transcribed under various conditions from the histidine operon of Salmonella typhimurium has been estimated by means of RNA-DNA hybridization. The hybridization of labelled RNA to the DNA of the wild-type strain and to the DNA of a mutant strain carrying a large deletion in the histidine operon, have been compared. The level of histidine messenger was found to be low when the operon was repressed, and at least tenfold higher when it was derepressed. Under conditions of histidine starvation in a histidine auxotroph stringent strain, the synthesis of histidine messenger accounted for 27% of the total RNA synthesis. These results suggest that the regulation of the histidine operon is primarily transcriptional, and that in this case the coupling of transcription and translation is not obligatory.

Expression vectors based on the rac fusion promoter.

The -35 region of the rrnB P2 promoter and the -10 region of the lacZpo promoter-operator were fused to form the strong and regulatable rac promoter. Vectors were constructed that allow the attachment of protein-coding sequences to the beta-galactosidase alpha-peptide (LacZ alpha) in any reading frame. By introducing a high-copy-number mutation, the synthesis of a LacZ alpha-chloramphenicol acetyltransferase fusion protein reached more than 60% of total cell protein in Escherichia coli.

In vitro transcription of the ribosomal RNA genes of E. coli DNA.

SummaryBacterial ribosomal RNA synthesis was studied in an in vitro system in which the presence of heparin prevented reinitiation of transcription. The number of heparin-resistant binary complexes of RNA-polymerase and E. coli DNA depended strongly on the quality of the template. High-molecular weight DNA was a much superior template than DNA prepared by conventional techniques. Using this highmolecular weight DNA as template the amount of ribosomal RNA synthetized in one round of transcription was found to be 4–5 fold higher than the amount of rDNA present. Controls have shown that the transcription probably started at the proper initiation sites and no significant read-through form distant promoters contributed to this effect. If the binary polymerase-DNA complexes were dissociated in the presence of 0.5 M KC1 prior to transcription all RNA synthesis was strongly reduced but the proportion of rRNA increased in the transcript. However, in this case the amount of rRNA did not exceed the amount of rDNA. We propose that the promoters of the rRNA genes are complex structures, able to store 4–5 molecules of RNA polymerase and of these several polymerase only one is bound in an extremely salt-resistent form.

The structural basis of the high in vivo strength of the rRNA P2 promoter of Escherichia coli

Ribosomal RNA promoters of Escherichia coli are probably the strongest promoters in vivo and they can be used on plasmid vectors to express protein-coding sequences at a high rate. In fact, the P2 promoter of the rrnB gene is stronger (in vivo) than the tac promoter, which has a perfect consensus sequence. Conversion of the rrnB P2 promoter sequence to consensus significantly increases in vivo promoter strength. The removal of four nucleotides downstream of the -10 region also increases the strength of this promoter. On the other hand, shifting of the A + T-rich region upstream of this promoter by an 11-bp insertion drastically decreases in vivo activity. It is concluded that the two functionally important hexanucleotide sequences, -35 and -10, are necessary but not sufficient factors for the optimalization of in vivo promoter strength.