External electric field control of electroosmotic flow in non-coated and coated fused-silica capillaries and its application for capillary electrophoretic separations of peptides.

The influence of an external electric field on the electroosmotic flow in the noncoated (bare) fused-silica capillaries and in the fused-silica capillaries with covalent coating of the inner surface by the polymer of a new acrylamido derivative, N-(acryloylaminoethoxy)ethyl-beta-D-glucopyranose, has been tested in the capillary electrophoretic separations of peptide analytes. The effect of magnitude and polarity of the external electric field on the flow-rate of the electroosmotic flow, the migration times of charged analytes and the separation efficiency and resolution of separations of synthetic oligopeptides, diglycine, triglycine, glycyl-proline and prolyl-glycine, by capillary zone electrophoresis has been evaluated. The effect of the external electric field on the velocity of the electroosmotic flow was much higher in the bare fused-silica capillaries than in the coated capillaries. Better separation of the analyzed peptides was achieved in the coated fused-silica capillaries. An external electric field proved to be an effective tool for control of the electroosmotic flow and for optimization of the speed and resolution of capillary electrophoretic separations of synthetic peptides.

Theory of the correlation between capillary and free-flow zone electrophoresis and its use for the conversion of analytical capillary separations to continuous free-flow preparative processes. Application to analysis and preparation of fragments of insulin.

A basic theoretical description of the correlation between capillary zone electrophoresis (CZE) and free-flow zone electrophoresis (FFZE) is presented. The theory of the correlation between CZE and FFZE results from the fact that both methods are based on the same separation principle, zone electrophoresis, and both are performed in the carrierless separation medium with the same composition of the background electrolyte. The equations describing the movement of the charged and noncharged particles in the d.c. electric field applied in the capillary and in the flow-through electrophoretic chamber are presented and used for the quantitative description of the correlation between CZE and FFZE. Based on the theory of the correlation between CZE and FFZE a procedure has been developed for conversion of analytical, microscale CZE separations into continuous preparative separation processes realized by FFZE. Practical application of the developed procedure is demonstrated by CZE analysis and FFZE preparation of an octapeptide fragment of human insulin.

In vitro and in vivo transcription from a computer predicted promoter.

A fragment (172 bp) of B. subtilis phage phi29 DNA, which does not contain a functional promoter for phage transcription, has been shown to direct transcription in the promoter-probe plasmid pPV33. The promoter candidate found in this fragment by the computer method of acceptability is compared with cryptic promoters selected by this computer method. It is characterized in vitro by electron microscopic visualization of RNA polymerase binding and 'run off' transcription, and in vivo by high resolution S1 mapping.

A novel method for promoter search enhanced by function-specific subgrouping of promoters--developed and tested on E.coli system.

A new method for evaluating some complex characteristics of the primary structure of E.coli promoters is proposed. The method, of nonparametric statistical significance, selects important conserved single-base positions in combination with 2-base coupling relations of identity and complementarity. The extended consensus of promoter characteristics thus obtained was used to scan unknown sequences for similarity with E.coli promoters. In terms of this method, a complete set of 244 E.coli promoters was shown to be structurally inconsistent. The set was then broken down into functionally homogeneous subsets of promoters to enhance the selectivity of the search for E.coli-specific promoter sequences, with a high significance level being attained.

Consensus symmetry pattern in E. coli promoter sequences.

A computer method was used to select two subgroups in 172 Escherichia coli promoter nucleotide sequences characterized by "standard" 17 bp and "non-standard" 17 +/- 2 bp spacing between the Pribnow box and -35 region. The conservation of the two-fold rotational (2f) and true palindrome (tp) symmetry relations was determined between nucleotide doublets in both promoter subgroups which represent consensus symmetry patterns. Statistically significant symmetries were primarily distinguished from those established by the nucleotide sequence conservation. The consensus symmetry pattern of standard promoters involves 45 of the 60 promoter nucleotide positions at which less strongly conserved and non-conserved sequences were mostly occupied (per se). They also show a high level of symmetry centre conservation. The conservation of the statistically significant 2f symmetry centres at positions -4.5 and -31.5 suggests partial conservation of the Pribnow box and -35 pentamer in the codogenic strand in an opposite orientation, respectively. The non-standard promoters differ from the standard ones by the consensus symmetry pattern and by the 2f and tp symmetry centre distribution with an overall lower degree of symmetry conservation in the -35 region. It has been suggested that the conserved symmetry relations provide an additional condition necessary for the specific interaction of RNA polymerase with the promoter sequence to initiate transcription.