Life time of some RNA products of rDNA intergenic spacer in HeLa cells.

In human cells, the intergenic spacers (IGS), which separate ribosomal genes, are complex approximately 30 kb-long loci. Recent studies indicate that all, or almost all, parts of IGS may be transcribed, and that at least some of them are involved in the regulation of the ribosomal DNA (rDNA) transcription, maintenance of the nucleolar architecture, and response of the cell nucleus to stress. However, since each cell contains hundreds not quite identical copies of IGS, the structure and functions of this locus remain poorly understood, and the dynamics of its products has not been specially studied. In this work, we used quantitative PCR to measure the expression levels of various rDNA regions at different times after inhibition of the transcription by Actinomycin D applied in high doses. This approach allowed us to measure real or extrapolated half-life times of some IGS loci. Our study reveals characteristic dynamic patterns suggestive of various pathways of RNA utilization and decay.

Cloning and characterization of the str operon and elongation factor Tu expression in Bacillus stearothermophilus.

The complete primary structure of the str operon of Bacillus stearothermophilus was determined. It was established that the operon is a five-gene transcriptional unit: 5'-ybxF (unknown function; homology to eukaryotic ribosomal protein L30)-rpsL (S12)-rpsG (S7)-fus (elongation factor G [EF-G])-tuf (elongation factor Tu [EF-Tu])-3'. The main operon promoter (strp) was mapped upstream of ybxF, and its strength was compared with the strength of the tuf-specific promoter (tufp) located in the fus-tuf intergenic region. The strength of the tufp region to initiate transcription is about 20-fold higher than that of the strp region, as determined in chloramphenicol acetyltransferase assays. Deletion mapping experiments revealed that the different strengths of the promoters are the consequence of a combined effect of oppositely acting cis elements, identified upstream of strp (an inhibitory region) and tufp (a stimulatory A/T-rich block). Our results suggest that the oppositely adjusted core promoters significantly contribute to the differential expression of the str operon genes, as monitored by the expression of EF-Tu and EF-G.

Indigenous plasmids in a production line of strains for penicillin G acylase derived from Escherichia coli W.

Three indigenous plasmids designated pRK1, pRK2 and pRK3 were identified among producers of penicillin G acylase (PGA) derived from the strain Escherichia coli W ATCC 9637. Their size and copy number (CN) in E. coli W were determined (kb; CN): pRK1 (80; 3.4), pRK2 (5.1; 71), and pRK3 (4.8; 13.7). Strain E. coli RE2 harboring these plasmids was used for selection of strains with reduced number of plasmids: the strain RE3 without plasmid pRK1 and the plasmid-less strain cERE3 were isolated. Indigenous plasmids did not code for the resistance determinants against 23 antibiotics and 10 heavy metals.