In contrast to the established systems of plasmid-coded homologous ribosomal DNA (rDNA) cistrons in Escherichia coli little is known about the fate of heterologous rRNA. In order to study expression of foreign rDNA, rRNA cistrons from Proteus vulgaris were cloned in phage vector Charon 35, subcloned in pBR322 and transformed in E. coli. The inserts of two clones (pPM2 and pPM 24) were characterized by restriction analysis and Southern hybridization. Each of them harboured a complete rrn cistron. The location of rRNA genes of clone pPM2 was also verified by R-loop analysis. The 5' flanking region of the 16s rRNA of pPM2 was sequenced and compared to the E. coli counterparts. High-level homologies exist in the functional parts of this region, e.g. promoters, box A and RNAase I11 recognition site. The copy number of pPM2 and pPM14 was estimated to be 8 and 10, respectively. Clones showed a markedly reduced growth rate (generation time about 57 to 70 min) as compared to the non-transformed cells (generation time 40 min). rDNA cistrons of P . vulgaris were properly expressed and the transcripts are processed as demonstrated by the presence of 16s rRNA from P. vulgaris in both ribosomes and 30s ribosomal subunits isolated from the transformed E. coli cells. The fraction of heterologous rRNA in ribosomes was about 25%.
To trace the fate of heterologous rRNA operons in vivo, a complete rrn operon from P. vulgaris was transferred into the chromosome of E, coli. This was done by phage ? gt 11 and by plasmid pOM40 which promotes the integration of the cloned insert into the maiP locus. As derived from oligonucleotide analysis of 16S rRNA isolated from ribosomal 30S subunits the amount of heterologous 16S rRNA in the ribosomes of corresponding clones was determined to be about 5 % of the homologous E. coil 16S rRNA.Recently we reported the construction of recombinant plasmids by cloning ribosomal RNA operons from Proteus vulgaris in plasmid pBR322. Transformation of these plasmids in Escherichia coli lead to the formation of hybrid ribosomes in the corresponding clones (10) to which P, vulgaris 16S rRNA contributed about 25%. As demonstrated by in vitro transcription experiments (9) heterologous promoters are as efficiently recognized as the homologous promoters. In addition, in vitro translation by hybrid E, coil ribosomes, using the MS 2 system, has been shown to be as efficient as a homogeneous ribosome population from E. coil (unpublished). The aim of this study was to investigate the possibility of integrating a heterologous rRNA operon into the genome of E. coil to answer questions in the long run about (i) the maintenance of the almost perfect uniformity of the primary structure of rDNA genes located on different operons, and (ii) the fate of a heterologous ribosomal gene. The latter question is of crucial interest for modern rRNA based phylogeny (14).
Currently, there are four BSL-4 facilities built or planned in Germany, all falling under the legal regulations for recombinant DNA. The authorization of these facilities is the responsibility of the local authorities of the Federal States.This article reports on the commissioning of a highcontainment facility in Hamburg expected to go into service in 2011. A matrix of about 100 critical containment components has been compiled by the local authority that led the inspectorate through the commissioning process. In cases where no acceptance criteria for certain tests were specified in neither the German nor the European regulations, the commissioning authority had to determine the international state-of-theart standards which are often set by North American guidelines and regulations.This article describes in detail some typical findings from the commissioning process: inter alia filter testing, room integrity tests, and validation of gaseous room decontamination.
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