Analysis of relative reversion frequencies for IS2 insertion mutations in the regulatory region of the galOPETK operon of Escherichia coli

Two previously characterized mutations in the galOPETK operon of Escherichia coli, galOP-3 and galOPE-490, contain IS2 insertions only 1 bp apart in the gal regulatory region; yet only the former yields Gal⁺ phenotypic revertants at a detectable frequency. We have shown that the galOPE-490 allele comprises two mutations—an IS2(I) insertion at bp+(2–6) (relative to the gal mRNA start site) plus a C/G to A/T transversion at bp+59. The latter creates an ochre stop codon and lies within the internal site of the bipartite gal operator; it acts as an operator mutation in an in vivo repressor titration assay. Analysis of a newly isolated allele (galOP-490^*) which retains the IS2 of galOPE-490 but is galE⁺ reveals a reversion frequency approximately 30-fold higher than that of galOP-3. Reversion of galOPE-490 is at least 10,000-fold lower and has not been detectable even under conditions conducive to enhanced double mutations in other systems.     

Stimulation in trans of synthesis of E. coli gal operon enzymes by lambdoid phages during low catabolite repression

Summary The infection of E. coli cells with different lambdoïd prophages triggers a stimulation of galactokinase synthesis when cells are grown in a medium giving rise to a mild catabolite repression (tryptone broth) with an inducer of the gal operon (fucose). These results show that during phage infection (or induction) some factor acting in trans is produced which is able to overcome efficiently catabolite repression of the kinase cistron. Using different strains of pbio252 (P^am, Q^am, H1), pbio256H1 and NNS7 we have concluded that the factor is the N gene product which is known for its anti- (rho) action.Studies of the whole gal operon in the same conditions show that epimerase unlike transferase and galactokinase is practically insensitive to catabolite repression by tryptone broth and that viral development has a low effect on it. This indicates that there is an internal modulation of gal operon expression. A mRNA termination site sensitive to the factor is known in the gal operon between galE and galT. Another site weaker than this one might exist between galE and operator-promoter region.     

Close vicinity of IS1 integration sites in the leader sequence of the gal operon of E. coli.

Summary Four insertions of IS1 in the leader sequence of the gal operon of E. coli have been analysed.Two of them occur at the same position, but in opposite orientations. The other two are inserted one nucleotide to one side and four nucleotides to the other side, respectively.In each case, nine base pairs of the leader sequence of the gal operon are duplicated directly, and are found flanking the termini of IS1 at its junction with the gal operon. These repeated sequences differ from each other as expected from the different insertion sites.     

High expression vectors for the production of recombinant single-chain urinary plasminogen activator from Escherichia coli

Summary An expression cassette containing a synonymous gene for human single-chain urokinase-type plasminogen activator (Rscu-PA) 5'-flanked by a trp promoter and the Shine-Dalgarno sequence of the xyl A operon of Bacillus subtilis and terminated by the terminators trp A and Tn10 was constructed and inserted into a pBR322 derivative to yield pBF160. When compared to pUK54 trp 207-1 containing the natural scu-PA gene without the Shine-Dalgarno sequence and terminator, the expression efficiency of pBF160 in Escherichia coli strains was improved by one order of magnitude. Replacement of the trp by the tac promoter (pBF171) did not affect expression. Inserting the Shine-Dalgarno sequence and Tn10 terminator into pUK54 trp 207-1 (pWH1320) slightly increased the expression level, whereas elimination of the Shine-Dalgarno sequence and the terminators from pBF160 with almost complete conservation of the synonymous structural gene (pBF191) significantly reduced the expression. Variation of the distance between the Shine-Dalgarno sequence and the start codon between 8 and 10 bp (pBF163) proved irrelevant. In conclusion, poor expression of mammalian genes in E. coli may result from both improperly designed regulatory elements and structural features of the coding region and therefore de-novo synthesis of the gene may be required to obtain satisfactory expression.     

Isolation of the mini insertions IS6 and IS7 of E. coli

Summary The mini IS elements IS6 and IS7 have been detected in constitutive gal ⁺ revertants of galOP-308::IS2 (I), in which the expression of the gal operon is turned off by IS2 in orientation I. Both, IS6 and IS7, are integrated into IS2 proximal to the gal structural genes. IS6 is 115 base pairs long and causes 50% constitutive expression of the gal genes. IS7 is only 65 base pairs long and the gal operon is expressed 20% constitutively compared to the gal ⁺ wild type operon. Both IS6 and IS7 are excised frequently, in the absence of selective pressure. These findings are discussed with respect to the evolution of gene expression.