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W Mandecki

Publications and source records attributed to W Mandecki.

42 records · Page 3Linked to original sources
PubMed1982

A lac promoter with a changed distance between -10 and -35 regions.

A lac promotor mutant was constructed by filling in the protruding ends of the HpaII site located within the lac promotor. The mutation, named M42, is a two base pair insertion that changes the distance between the -10 and -35 regions from 18 to 20 residues. The activity of the mutant promotor measured in vivo is 15% of the wild-type promotor. The M42 promotor is sensitive to the catabolite repression in the manner similar to that of the wild type. Sequences of several deletions within the lac promotor are also given.

Base Sequence↗
PubMed1981

Position of the lacZX90 mutation and hybridization between complete and incomplete beta-galactosidase.

The position of the termination codon in lacZX90 was determined by isolation of a lac+ revertant. Lysine was found to replace tyrosine at position 1,012 of beta-galactosidase, indicating that X90 protein lacked the carboxyl-terminal 10 residues. A heat- and urea-sensitive hybrid enzyme was formed in vivo when supC, which supplies tyrosine to the position in the polypeptide corresponding to the nonsense codon, was used to suppress lacZX90. This result shows that suppression that adds back the original amino acid may not lead to the production of the wild-type enzyme if the latter is multimeric, because incomplete chains can be incorporated into the oligomer.

Amino Acid Sequence↗
PubMed1979

Expression of the lac operon in RNA polymerase mutants of Escherichia coli K12.

Three temperature-sensitive mutant strains for RNA polymerase beta or beta' subunits (carrying mutations tsx, A2R7 and R120) were used in order to investigate the dependence of the induced lac expression on stimulation by cyclic AMP after the shift to non-permissive temperature. High temperature lowered the rate of beta-galactosidase synthesis. However, the low rate of synthesis could be strongly increased by cyclic AMP (30, 2.4 and 5.7-fold increases for tsX, A2R7 and R120 mutants, respectively). At the permissive temperature stimulation by cyclic AMP was less than 1.4-fold (minimal medium supplemented with glycerol). The results suggest that the maximal expression of the lac operon is saturated, that is, a hypothetical increase in RNA polymerase or cAMP-CRP concentration in the cell with not enhance the expression. The concept of saturation explains why it was possible to increase the beta-galactosidase synthesis in conditions of limited promoter binding activity of RNA polymerase through increase in concentration of cyclic AMP-CRP complex in the cell (addition of cyclic AMP) to the values higher than that observed on glycerol.

Cyclic AMP↗
PubMedDate not supplied

High-resolution polyacrylamide gel electrophoresis of oligonucleotides using L-histidine buffer.

We have found that 50 mM L-histidine pH 7.6 as a buffer for gel electrophoresis greatly improves the resolution of oligonucleotides less than 70 residues long on denaturing polyacrylamide gels. The histidine buffer increases spacing between DNA bands on the gel about twofold in comparison with a standard buffer (89 mM Tris-borate, 2 mM EDTA pH 8.3). In addition, low conductivity of the histidine buffer results in a threefold reduction of the electrophoresis time. Conditions for electrophoresis were optimized by varying both histidine and acrylamide concentrations. Other polycationic compounds, such as spermidine and ethylenediamine, were also tested for improved resolution of oligonucleotides. Several hypotheses as to the factors influencing the separation of DNA on gels are presented.

Buffers↗