Showing papers by "Galina E. Pozmogova published in 1994"

An approach to construction of hybrid polypeptide molecules--homologue recombination method.

Abstract: Mapping of functionally important sites (FIS) is a problem of great importance in the study of protein molecules. Success in gene cloning and enlargement of the number of cloned genes has allowed new insights into this problem. Proteins belonging to a single family may exhibit consistent functional peculiarities. These make it feasible to map the FIS by recombining fragments of homologous genes. This process entails substitution of desired segments in gene under study for homologous ones from another representative of the family and subsequently comparing the wildtype and hybrid protein (1,2). Because structurally important residues are usually conserved it is likely that hybrid molecules formed will have maintained a correct and stable conformation. In choosing the segment to be substituted one should consider both the currently available data on the protein and/or any theoretical predictions (2,3). Eventually one may choose to substitute a great many segments that may overlap in aggregate the whole gene. In general two techniques are used for this purposesite-directed mutagenesis and PCR (1,4). These methods are time-consuming, elaborative and expensive, requiring the extensive chemical synthesis of oligonucleotides. In this article we present a new approach, the homologue recombination (HR) method, which allows a set of hybrid molecules to be produced in one experiment. The method is based upon the observation that the thermostable Vent-polymerase (New England Biolabs) which has 3'—5' proofreading exonuclease activity shortens the single-stranded DNA fragments synthesized in unidirectional PCR from their 3'-termini. An assortment of such fragments transcribed from a donor gene may be used as primers in the site-directed mutagenesis of a related acceptor gene. This will lead to the production of a number of hybrid genes. Segments of various lengths in the acceptor gene are substituted for homologous ones in the donor gene.

[Expression of a partially modified delta-endotoxin gene from Bacillus thuringiensis var. tenebrionis in transgenic potato plants].

Abstract: A modified gene (Bt77) of delta-endotoxin from Bacillus thuringiensis var. tenebrionis was constructed and cloned into pMON505. This binary transformation vector was introduced into Agrobacterium tumefaciens strains containing different helper disarmed Ti-plasmids, LBA4404, A281, and CBE21. These Agrobacterium strains were used to transform potato stem segments (S. tuberosum, cv Desiree, Resy, Temp, Granat). Regenerants were selected on kanamycin-containing media. The presence of the Bt77 sequence in plant genomic DNA was confirmed by PCR analysis. Bt gene expression was studied in regenerated plants. Western blot analysis revealed that transgenic plants produced the Bt protein in the range of 0.005-0.02% of total protein. Total protection against insect damage of leaf tissue from these plants was observed in laboratory bioassays with of Colorado beetle larvae. Transgenic plants showed incomplete protection from CB larvae.