Vladimir Glišin

Expression and characterization of human interferon-β1 in the methylotrophic yeast Pichia pastoris

We describe the heterologous expression of a human interferon‐β1 in the methylotrophic yeast Pichia pastoris. Biologically active recombinant human interferon‐β1 (rHuIFN‐β1) was secreted from shake‐flask‐grown P. pastoris cells into the medium using the Saccharomyces cerevisiae α‐mating factor prepro‐leader sequence at the level of (1–3)×105 i.u. (international units)/ml (6–12 mg/litre). An rHuIFN‐β1 with an N‐terminal sequence identical with that of native HuIFN‐β1 was purified and the specific activity was determined (2–3×107 i.u./mg). It was found that the secreted recombinant protein was partially N‐glycosylated.

The penicillin amidase of Arthrobacter viscosus (ATCC 15294)

The nucleotide (nt) sequence of the gene encoding penicillin G amidase (PA) of Arthrobacter viscosus strain ATCC 15,294 was determined. The sequence contained an open reading frame of 2406 nt with a G+C content of 37%. The deduced amino-acid sequence shows significant homology with other so far identified beta-lactam amidases of Gram- bacteria.

The primary structure of Providencia rettgeri penicillin G amidase gene and its relationship to other gram negative amidases

The nucleotide sequence of Penicillin G amidase (PA,E.C.3.5.1.11) of Providencia rettgeri was determined. We aligned our P. rettgeri PA with other known Gram negative periplasmically located beta-lactam amidases. The analysis revealed a high homology with other Enterobacteric amidases (60%-65%), while with similar Pseudomonas sp. amidases the homology exceeded 25%. These homologies indicate their common ancestry.

Synthesis and secretion of Providencia rettgeri and Escherichia coli heterodimeric penicillin amidases in Saccharomyces cerevisiae.

The Providencia rettgeri and Escherichia coli pac genes encoding heterodimeric penicillin G amidases (PAC) were successfully expressed in Saccharomyces cerevisiae. Furthermore, these recombinant enzymes are secreted from the yeast cell into the medium which is in contrast to bacterial hosts, where the enzymes are retained in the periplasm. Contrary to the P. rettgeri PAC-encoding gene, the E. coli pac is poorly expressed in yeast. The highest yield of P. rettgeri PAC was obtained with a multi-copy plasmid, resulting in of 1500units per liter. This yield is higher by an order of magnitude than that obtained in the best recombinant bacterial expression system. The recombinant P. rettgeri enzyme is only partially and selectively O-glycosylated. Only every sixth or seventh alpha-subunit is glycosylated, while the beta-subunit is not glycosylated at all. N-Glycosylation has not been detected.

A pool of nonpolysomal globin mRNAs in globin deficient reticulocytes of the anemic Belgrade rat

Abstract In anemia of the Belgrade rat ( b b ) reticulocytes contain less than half of the normal amount of mRNA for seven adult rat globin chains. cDNA hybridization measurements of the relative sizes of polysomal and nonpolysomal pools of globin mRNAs in these cells show that 45% of all globin mRNA molecules are not used at any given time in protein synthesis. This implies a translational control which ensures a production of globin chains in a correct ratio despite a severe mRNA unbalance.

DNA region responsible for transcriptional regulation of the Escherichia coli penicillin amidase (pac) gene by CRP and PAA

Transcriptional regulation of Escherichia coli ATCC11105 penicillin amidase gene (pac) by cAMP receptor protein (CRP) and phenylacetic acid (PAA) was studied by using operon fusions with divergent reporter gene (lacZ, and phoA) constructs. A 150 bp DNA segment essential for the regulation of pac gene transcription by CRP and PAA was defined.

Host dependenet inactivation by IS2 of induced E.coli penicillin amidase gene cloned on multicopy plasmids

Structural instability of the cloned penicillin acylase gene (pac) from E.coli ATCC11105 was studied under various physiological conditions. Structural changes which adversely affect the expression of penicillin acylase gene were selected for only under conditions in which the gene was active and fully induced. In E.coli strain YMC the predominant mutations were the insertions of the IS2 element at various sites within the 700 bp proximal portion of the pac gene. The results indicated that the induction of the plasmid cloned gene was the main factor which rendered it a target for inactivation by insertions of the host encoded IS2 elements. The mutational events were host specific and they were not influenced by mutual positions and orientations of key marker genes on the plasmid.

Towards a total analysis of polyribosome-associated ribonucleoprotein particles of sea urchin embryos

Abstract Sea urchin embryos were analysed for polyribosome-associated mRNA-protein particles. Simultaneously, experimental conditions were established under which these mRNA-protein complexes could be unambigously identified. The purification of polyribosomes through a 2-M sucrose layer and isolation of polyribosomes in media not containing detergents was essential. The analysis of polyribosome-released mRNA in CsCl isopycnic gradients revealed that the mRNA of sea urchin embryos is bound in polyribosomes in the form of ribonucleoprotein particles (densities about 1.45 g/cm 3 ) which contain a rather large (60 %) amount of proteins.

Molecular Biology in Embryology

Modern work in embryology puts the old controversy between preformation and epigenesis in a new light. The rapidly dividing cells of embryos contain many maternally preformed, high-molecular-weight components, including both enzymes and molecular building blocks to be used long after fertilization. All presently investigated cellular macromolecules, with the exception of DNA, are found in excess in fertilized egg cells, compared to levels in mature somatic cells. Many of these macromolecules, including an interesting population of stable RNA’s, remain constant in total amount through many successive cell divisions. Identical sets of genes in the cells of the early embryo later give rise to different cell types and lineages. Although some intermediate steps in this differentiation process have been identified, the ultimate cause of differential gene expression remains obscure. It is clear, however, that most differentiation is notthe result of loss of nuclear genetic information. Artificially induced changes in the pattern of cell division in some cases create a disorganized embryo, but in others development proceeds normally. The polarity of molecular organization in an egg’s cytoplasm appears to be critical to normal development. Cytoplasmic polarity may in turn result from the orientation of material in the cell nucleus. There seems to be an intrinsic geometry of chromosomes that permits a spatial display of information there to create significant, stable, and essential inhomogeneities and regionalizations of the cytoplasm. These, in turn, determine subsequent cleavage planes as cells divide, and the partitioning of information between daughter cells. The glossary in Chapter 5 will be helpful here also. In addition, for the nonbiological specialist, we offer the following, brief account of the main processes that relate DNA to proteins through RNA and that permit copying of genes during cell division.

Some properties of the 24S particle isolated from the cytoplasm of sea urchin eggs.

Abstract Discrete independent protein-RNA particles with a sedimentation constant of about 24S have been isolated from the cytoplasm of unfertilized sea urchin eggs or developing embryos. They contain about 8% of the total protein of the egg/embryo. The particles are 3–4% RNA by weight. Therefore, these particles bind the amount of RNA equal to about 4% of the total RNA of the egg/embryo. On the basis of labeling kinetics and sedimentation properties we tentatively conclude that this RNA represents the nonpolyribosome-bound RNA of the cytoplasm.