IS2-mediated re-arrangement of the promoter sequence suppresses metabolic burden of the recombinant plasmid.

Recombinant plasmid pKA18 of the high expression bacterial system for penicillin amidase ('penicillin G acylase') bears the 3' end region of IS2 element. The IS2 sequence replaces the -35 region of promoter of pga and extends up to TAGTAT box at position -10 of the promoter region. It therefore forms a hybrid promoter of pga ppgaHT. A natural promoter ppgaWT was not detected on any recombinant plasmid isolated from recombinant strains of Escherichia coli constitutively producing penicillin amidase. PCR fragments carrying both types of promoters were cloned into the promoter-probe vector pET2 to compare their transcriptional activity: the activity of ppgaWT was 5x higher than that of ppgaHT. The same nucleotide "G" localized 28 nucleotides upstream of the translation start point was identified as the respective transcription start point of both mRNAs. An attempt was made to place the pga gene cloned on a plasmid under the control of the natural promoter: not a single clone expressing penicillin amidase was found among 150 transformants. High transcriptional activity of the natural promoter together with high pga gene dosage could result in a deleterious metabolic burden of the periplasmic enzyme.

Molecular cloning and expression of the pyranose 2-oxidase cDNA from Trametes ochracea MB49 in Escherichia coli.

A cDNA of a structural gene encoding pyranose 2-oxidase (P2O) from Trametes ochracea strain MB49 was cloned into Escherichia coli strain BL21(DE3) on a multicopy plasmid under the control of the trc promoter. Synthesis of P2O was studied in batch cultures in LB or M9-based mineral medium at 28 degrees C. While there was a low specific activity of P2O in LB medium, the enzyme was synthesised constitutively in mineral medium and represented 3% of the cell soluble protein (0.3 U mg(-1)). The effect of isopropyl beta-D-thiogalactoside on the expression of P2O was studied in mineral medium at 25 and 28 degrees C. The synthesis of P2O at 28 degrees C corresponded to 39% of the cell soluble protein but the major portion of P2O (93%) was in the form of non-active inclusion bodies (activity of P2O equalled 0.19 U mg(-1)). At 25 degrees C, the amount of P2O represented 14% of the cell soluble protein and the activity of P2O was 1.1 U mg(-1). The soluble enzyme represented 70% of the total amount of P2O.

Expression of the neutral protease gene from a thermophilic Bacillus sp. BT1 strain in Bacillus subtilis and its natural host: identification of a functional promoter.

The expression of the neutral protease gene (npr) from the thermophilic Bacillus sp. BT1 strain was studied in its natural host and in mesophilic Bacillus subtilis. In the thermophilic BT1 strain, the transcription of the protease gene is initiated from its own promoter, just 5' to the gene. In contrast, in heterologous B. subtilis this thermophilic npr promoter does not function, and expression of the npr gene results from transcription originating upstream of an adjacent gene, open reading frame X (ORF X). A functional promoter was identified 5' to ORF X that is required for efficient expression of the npr gene in Bacillus subtilis as verified by primer extension, reverse transcription-PCR, and 5' rapid amplification of cDNA ends experiments. These data suggest that transcriptional signals used in thermophilic Bacillus sp. BT1 strain are different from those used in B. subtilis.

Cloning and sequencing of the neutral protease-encoding gene from a thermophilic strain of Bacillus sp.

The neutral protease-encoding gene (npr) from the thermophilic strain Bacillus sp. BT1 was cloned and sequenced. A possible means of regulation of npr expression is suggested.

[Contribution to the kinetics of ethanol absorption and elimination in the presence of hydrocarbons (n-hexane) in the rat]. / Prispevek ke kinetice vstrebávání a eliminace ethanolu za prítomnosti uhlovodíku (n-héxanu) v krysím organismu.

Increased ethanol blood level follows the administering of ethanol mixed with single hydrocarbons in a similar way as after cannulation of ethanol with benzol Ethanol blood level changes are stipulated by hydrocarbons, reducing benzol compounds are not decisive for increasing influence. Kinetic relations indicate that resorption speed is not influenced substantially. Increased ethanol blood level in rat seems to be caused by restricted elimination.

[Affecting the levels of ethanol by administering gasoline with ethanol]. / Ovlivnení hladiny etanolu podáním benzínu vedle etanolu.

Administering of benzene alone into rat stomach through a tube does not increase the amount of reducing compounds in blood above the physiological level and does not influence the Widmark test in spite of the fact that benzene in blood in vitro produces reducing compounds. The administration of benzene alone to rats is not sufficient for any conspicuous reducing compounds in blood above the level corresponding to the administration of ethanol alone. This elevation is due to the increased ethanol serum level according to the result of gas chromatography. Benzene components may influence ethanol resorption, metabolism and elimination. The administration of benzene alone to rats is not sufficient for any conspicuous blood level of reducing compounds. Findings in rats represent a first stage information which may be important for forensic medical expertise.

Biochemical properties of alkaline phosphatase isoenzymes and their practical significance.

Certain physiochemical and biochemical properties of serum alkaline phosphatase isoenzymes are described concisely. Differences in their properties can be utilized in practice for the qualitative and quantitative determination of isoenzyme activities in sera in pathological states. Some of the methodological requirements of this differentiation by electrophoretic separation and inactivation-inhibition methods are evaluated.