[Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. I. Construction of vector plasmids for transfer into plants and expression of a modified gene for beta-1,3-glucanase from Clostridium thermocellum in tobacco protoplasts]. / Konstruirovanie i analiz transgennykh rastenii Nicotiana tabacum L. s ékspressiruiushchimsia bakterial'nym genom beta-1,3-gliukanazy. I. Konstruirovanie vektorynykh plazmid dlia perenosa v rasteniia i ékspressiia modifitsirovannogo gena beta-1,3-gliukanazy iz Clostridium thermocellum v protoplastakh tabaka.

We constructed two vectors, pC27-glc and pC29-glc, that allow expression of the beta-1,3-glucanase gene (glc) in plant cells. The glc gene was previously cloned from anaerobic thermophilous bacterium Clostridium thermocellum. To increase the efficiency of expression, the N-terminal fragment of the glc gene encoding bacterial transient peptide was deleted, and hybrid variants of lacZ-glc were obtained. Analysis of expression of the hybrid genes in Escherichia coli showed that deletion of the fragment corresponding to 31 amino acids (a.a.) of beta-glucanase affected neither activity nor thermostability of the enzyme. The modified gene was subcloned into two vectors, pC27 and pC29, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter from Arabidopsis, respectively. Each of the resulting plasmids, pC27-glc and pC29-glc, was transfected into protoplasts of Nicotiana plumbaginifolia. Both the plasmids were shown to allow a high level of activity of the thermostable beta-1,3-glucanase. We plan to use the vectors obtained for transformation of agrobacteria and construction of transgenic plants.

[Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. II. Transgenic tobacco plants expressing the bacterial beta-glucanase gene from Clostridium thermocellum,--a model for studying the differential expression of stress response-related genes]. / Konstruirovanie i analiz transgennykh rastenii Nicotiana tabacum L. s ékspressiruiushchimsia bakterial'nym genom beta-1,3-gliukanazy. II. Transgennye rasteniia tabaka, ékspressiruiushchie bakterial'nye gen beta-gliukanazy iz Clostridium thermocellum,--model' dlia izucheniia differentsial'noi ékspressii genov stressovogo otveta.

The modified hybrid beta-1,3-glucanase gene (glc) of Clostridium thermocellum was expressed in tobacco Nicotiana tabacum. The glc gene was cloned into two plasmids, pC27-glc and pC29-glc, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter of Arabidopsis, respectively. These constructions were used for transformation of agrobacteria followed by transfer into plants. In transformed plants, each plasmid caused a high level of activity of thermostable bacterial glucanase not observed in reference plants. The plants obtained were used to study activation of some defense-related genes induced by their interaction with either tobacco mosaic virus (TMV) or a pathogenic fungus.

[Use of uracil-repair selection for extensive DNA sequence deletions]. / Primenenie uratsil-reparatsionnoi selektsii pri deletirovanii protiazhennoi posledovatel'nosti DNK.

A procedure for extensive deletion mutagenesis of DNA using the uracil repair system is exemplified by reconstruction of the pBR322 replication regulatory region cloned into M13tg131. By means of an oligonucleotide primer the 116-nucleotide fragment was excised and four nucleotides were introduced to form a BglII restriction site. Use of the uracil repair selection provided a 30-fold increase in the deletion mutagenesis efficiency.

Expression of the hAng gene in Escherichia coli; isolation and characterization of human recombinant Ser-(-1) angiogenin.

Recombinant human angiogenin has been synthesized in Escherichia coli with the aid of a human angiogenin gene (hAng) cloned by Neznanov et al (1990) from a human complementary DNA (cDNA) library. The gene has been expressed by use of a new type of expression vector called a 'TGATG vector' (plasmid pPR-TGATG-1; Mashko et al 1990a). The highest level of accumulation of the recombinant angiogenin (6%-8% of the total cell protein) was observed in E. coli strain BL21 carrying a temperature-amplifiable version of the plasmid. The synthesized polypeptide carries an additional serine residue at its N terminus in comparison with natural angiogenin. Furthermore, the initiator methionine residue of the recombinant protein is removed with high efficiency by E. coli terminal aminopeptidase. Simple procedures for purification of the recombinant angiogenin from the insoluble fraction of cell protein, and for refolding the protein allowed the isolation of almost 5 mg recombinant angiogenin g-1 wet bacterial biomass. The recombinant Ser-(-1) angiogenin displayed the same biological properties (specific RNAase activity and the ability to induce blood vessel growth on the sclera of experimental animals) as its natural counterpart isolated from human blood.