Host cell properties and external pH affect proinsulin production by Saccharomyces yeast.

The expression of a hybrid gene encoding an alpha-factor prepro leader peptide-miniproinsulin (MPI) fusion [MPI is the same as the LysArg human insulin precursor described by Thim et al. (1986)] was tested in a series of isogenic yeast strains to investigate the influence of some genetic and physiological factors on heterologous production in yeast. We found that: (i) an MF alpha 1 gene disruption in haploid cells, as well as MF alpha 1 gene product expression in diploid cells, do not affect the MPI secretion level; (ii) under conditions of exogenous leucine availability, MPI production is hindered by leucine auxotrophy (a leu2 mutation); (iii) rho- mutations increase the per-cell MPI yield approximately three-fold; (iv) the MPI yield is apparently dependent on the pH of the culture medium: the higher the external pH, the larger the per-cell MPI yield.

[Isolation, purification, and characteristics of the new restriction enzymes BciBI and BciBII, produced by Bacillus circulans]. / Vydelenie, ochistka i kharakteristika novykh restriktaz BciBI i BciBII, produtsiruemykh Bacillus circulans.

New site-specific endonucleases BciBI and BciBII have been detected in Bacillus circulans. The enzymes were purified by fractionation of cell-free extract with polyethylene imine and ammonium sulphate (40-80% of saturation) followed by chromatography on DEAE-sepharose, blue-sepharose and phosphocellulose. The endonucleases BciBI and BciBII were separated only at the final step of the purification--by chromatography on the phosphocellulose column. The yields of BciBI and BciBII were 600 and 10,000 U/g of cells. It was found that restriction endonucleases BciBI and BciBII are isoschizomers of ClaI and BstNI, respectively.

[Specific endonuclease BbvBI from Bacillus brevis]. / Spetsificheskaia éndonukleza BbvBI iz Bacillus brevis.

A new restriction endonuclease BbvBI free from contaminating nonspecific nucleases and phosphatases was isolated from the Bacillus brevis cells. The enzyme was purified by fractionating the sonicated cell-free extract in a two-phase PEG/dextran system and subsequent chromatographies on DEAE-sepharose, blue sepharose and heparin sepharose. The endonuclease BbvBI displayed the maximal activity at 45 degrees C, pH between 8.0 and 8.5, MgCl2 concentration in the range of 5-10 mM and at the low ionic strength. It is shown that the enzyme cleaves the sequence G'GYPC'C, with the preferential cleavage of GGTACC and GGCACC sites as compared with GGTGCC and GGCGCC. Thus, the restriction endonuclease BbvBI is a true isoschizomer of nuclease BanI.

[The existence of the ribonucleoprotein branching enzyme in rabbit skeletal muscle and ribozyme corresponding to it]. / K voprosu o sushchestvovanii v skeletnykh myshtsakh krolika vetviashchego fermenta ribonukleoproteidnoi prirody i sootvetstvuiushchego ribozima.

Amylose isomerase (AI) preparations were isolated from rabbit muscles after Petrova et al., as well as by the additional fractionation steps. Their homogeneity, enzymatic activity and RNA, isolated from those preparations, were characterized. AI preparations, as described by Petrova et al., proved to be heterogeneous in respect to the protein and RNA; by using additional fractionation methods RNA and protein have been separated from each other, which proves that a homogeneous stable ribonucleoprotein complex, exerting AI activity, does not exist. It was shown by three independent methods that AI preparations isolated after Petrova do not display branching, but have amylolytic activity. RNA, isolated along with the AI preparations, proved to be mainly total tRNA degraded to different degrees. No RNA corresponding to the previously sequenced 2.5S RNA could be detected in these preparations. RNA preparations do not manifest neither branching, nor amylolytic activity. Our data prove that there is no ribozyme, whose existence has been suggested previously.

[BsoAI--a new site specific endonuclease from Bacillus coagulans]. / BcoAI--novaia sait-spetsificheskaia éndonukleaza iz Bacillus coagulans.

A new site-specific endonuclease was detected in toluene lysates of Bacillus coagulans AUCM B-732 and designated as BcoAI. The enzyme was purified by fractionation of the cell-free extract in the two-phase PEG/dextran system followed by chromatography on DEAE-sepharose and phosphocellulose and shown to be free of nonspecific nucleases and phosphatases. BcoAI has three cleavage sites on lambda DNA, but does not cleave SV40, pBR322 and pUC19 DNA. BcoAI recognizes the sequence 5' CAC decreases GTG 3' on double-stranded DNA and cleaves it as indicated by the arrow to yield blunt-ended DNA fragments. Thus, BcoAI is a true isoschizomer of PmaCI from Pseudomonas maltophila C.

[Localization of acid phosphatase in Saccharomyces cerevisiae and its export into culture media depends on the type of the N-terminal signal peptide]. / Lokalizatsiia kisloi fosfatazy Saccharomyces cerevisiae i ee éksport v kul'tural'nuiu sredu zavisiat ot tipa N-kontsevogo signal'nogo peptida.

The aim of this work was to study the character of intracellular distribution and efficiency of yeast acid phosphatase export depending on the type of the N-terminal signal peptide used. A number of plasmids carrying the acid phosphatase genes with different signal peptides sequences was constructed. The main site of the enzyme accumulation for the variant containing its own acid phosphatase signal peptide was the periplasm. Approximately the same pattern was observed when the hybrid signal peptide consisting of acid phosphatase signal peptide and alpha-factor preprosegment tandem was used. Unlike the above-mentioned systems the strain carrying acid phosphatase under the control of alpha factor preprosegment was able to export the enzyme into the culture medium. The experiments have shown the possibility of changing the final localization of secretory proteins by replacing the N-terminal signal peptide.

[The nature of N-terminal signal sequence determines the type of intracellular distribution and effectiveness of export of human growth hormone in Saccharomyces cerevisiae]. / Priroda N-kontsevoi signal'noi posledovatel'nosti opredeliaet kharakter vnutrikletochnogo raspredeleniia i éffektivnost' éksporta gormona rosta cheloveka u drozhzhei Saccharomyces cerevisiae.

Various N-terminal signal peptides (SP) were tested to investigate a human growth hormone (hGH) synthesis, processing and intracellular sorting in yeast. Maximal level of hGH was observed in the case when the mature hGH gene was placed under the control of PHO5 promoter. In this case about 90% of hGH was localized in the cytosol, but some portion was trustworthly detected in microsomes and periplasma in spite of the absence of SP. Addition of own or PHO5 SP resulted in lowering of the synthesis and a difficulty in the prehGH processing. In this case the immunoreactive products were localized mainly in periplasma and vacuoles and to a lesser degree in the cytosol. When hGH gene was placed under the control of the yeast MF alpha 1 promoter and alpha-factor preprosegment was used as SP more then a half (67%) of hGH processed correctly was exported in a medium, the rest was detected in vacuole (17%) and periplasma (8%).

[Behavior in a cell of artificial mini-chromosomes during induced transcription of centromeric DNA]. / Povedenie v kletke iskusstvennykh mini-khromosom pri indutsirovannoi transkriptsii tsentromernoi DNK.

Hybrid yeast plasmids were constructed, containing the centromere loci CEN3 under the control of two inducible yeast promoters--GAL10 and PHO5. It was shown, that during the induction of transcription from the GAL10 promoter the decrease in mitotic stability of minichromosome is affected both by partial disruption of centromere function by transcription and by influence of galactose on the number of residual cell divisions. In two strains the activity of GAL10 promoter was considerably higher than that of the PHO5 promoter. It is proposed to use the effect of minichromosome destabilization for evaluation of the relative promoter strength.

[Expression of a gene for hybrid protein containing the [Val8] amino acid sequence of calcitonin in yeast cells]. / Ekspressiia gena gibridnogo belka, soderzhashchego aminokislotnuiu posledovatel'nost' [Val8] kal'tsitonina, v kletkakh drozhzhei.

A recombinant plasmid has been constructed, which directs the synthesis of a hybrid protein, yeast repressible acid phosphatase [Val8]calcitonin, in yeast. The plasmid contains a truncated gene (pho5) acid phosphatase lacking 96 C-terminal amino acids replaced by the synthetic gene for human calcitonin and sequences required for the plasmid propagation in transformed yeast cells. A modified RIA method using immobilisation of protein extracts on solid supports was developed to monitor the expression of the hybrid protein. By use of this method, as well as by standard RIA of CNBr-cleaved protein extracts, synthesis of a calcitonin-related protein was detected in extracts of transformed strains grown under conditions inducing pho5 promoter.