Construction and characteristics of mini-Mu phages

The paper reports on the principles of construction, physical characterization and results of preliminary genetic investigation of hybrid plasmids containing Mu DNA sequences or deletion derivatives of phage Mu, the so-called mini-Mu phages. The mini-Mu were obtained by joining both phage ends within one plasmid in a regular orientation. A collection obtained by in vitro manipulations included 14 recombinant plasmids containing different DNA fragments of the Mu genome. Seven plasmids have both ends of phage Mu, three plasmids containing regularly oriented ends, i.e. mini-phages of different size: the mini-Mu5 (11 kb) within pRM8 plasmid, the mini-Mu4 Ap (18 kb) within pRM6 and the mini-mini-Mu (4.4 kb) within pRM5. The collection comprises mini-Mu phages with the gene kil inactivated after treatment with hydroxylamine. Biological properties of the hybrid plasmids have been preliminary studied.     

Fungal origin of PBV-group viruses isolated from Penicillium brevi-compactum]

Treatment of extracts from the disintegrated mycelium of Penicillium brevi-compactum with antisera against viruses PBV-1 and PBV-3 grown on bacteria results in a considerable decrease in the infectiveness of micellar preparations toward E. coli C. This is a specific reaction since treatment with a heterologic antiserum against phage T2 has no effect on the infectiveness of the micellar extracts. The kinetics of reassociation of DNA from PBV-3 and DNA from Penicillium brevi-compactum has shown that the value of Cot at half-reassociation of these DNAs is 13.3 times higher than the value of Cot at half-reassociation of DNA from PBV-3 and heterologic DNA from chicken embryos. As was found by calculations, DNA isolated from the fungal mycelium contains virus sequences at an amount of 3.7 virus genomes per cell. These data confirm the micellar origin of the viruses PBV-1 and PBV-3. The virus PBV-3 is supposed to be present in the cells in the form of prophage.     

Thermotoga neopolitina gene cluster, participating in degradation of starch and maltodextrins: expression of aglB and aglA gene in Escherichia coli, properties of recombinant enzymes

The aglB and aglA genes from the starch/maltodextrin utilization gene cluster of Thermotoga neapolitana were subcloned into pQE vectors for expression in Escherichia coli. The recombinant proteins AglB and AglA were purified to homogeneity and characterized. Both enzymes are hyperthermostable, the highest activity was observed at 85 degrees C. AglB is an oligomer of identical 55-kDa subunits capable of aggregation. This protein hydrolyses cyclodextrins and linear maltodextrins to glucose and maltose by liberating glucose from the reducing end of the molecules, and it is a cyclodextrinase with alpha-glucosidase activity. The pseudo-tetrasaccharide acarbose, a potent alpha-amylase and alpha-glucosidase inhibitor, does not inhibit AglB but, on the contrary, acarbose is degraded quantitatively by AglB. Recombinant AglB is activated in the presence of CaCl2, KCl, and EDTA, as well as after heating of the enzyme. AglA is a dimer of two identical 54-kDa subunits, and it hydrolyses the alpha-glycoside bonds of disaccharides and short maltooligosaccharides, acting on the substrate from the non-reducing end of the chain. It is a cofactor-dependent alpha-glucosidase with a wide action range, hydrolysing both oligoglucosides and galactosides with alpha-link. Thereby, the enzyme is not specific with respect to the configuration at the C4 position of its substrate. For the enzyme to be active, the presence of NAD+, DTT, and Mn2+ is required. Enzymes AglB and AglA supplement one another in substrate specificity and ensure complete hydrolysis to glucose for the intermediate products of starch degradation.     

Evolution of the response to heat shock in genus Drosophila

Thermotolerance was studied in a wide spectrum of Drosophila species and strains originating from different climatic zones and considerably differing from one another in the ambient temperature of their habitats. The species that lived in hot climate have a higher thermotolerance. Most species of the virilis group exhibited positive correlation between the HSP70 accumulation after heat exposure and thermotolerance; however, this correlation was absent in some species and strains. For example, the D. melanogaster Oregon R strain, which had the highest sensitivity to heat shock (HS) among all strains and species studied, displayed the maximum level of HSP70 proteins after HS. The patterns of induction of various heat shock protein (HSP) families after heat exposure in a wide spectrum of Drosophila species were compared. The results obtained suggest that the HSP40 and low-molecular-weight HSPs (lmwHSPs) play a significant role in thermotolerance and adaptation to hot climate. Polymorphism in hsp70 gene clusters of Drosophila and variation in the numbers of gene copies and hsp70 isoforms in group virilis were found. The evolutionary role of the variation in the number of hsp70 gene copies observed in the strains and species of genus Drosophila is discussed.     

Prediction of the three-dimensional structure of aflatoxin of Aspergillus flavus by homology modelling

Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus spp. The toxic effects of aflatoxins have adverse consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a protein containing a zinc-finger DNA-binding motif. AFLR-Protein three-dimensional model was generated using Robetta server. The modeled AFLR-Protein was further optimization and validation using Rampage. In the simulations, we monitored the backbone atoms and the C-α-helix of the modeled protein. The low RMSD and the simulation time indicate that, as expected, the 3D structural model of AFLR-protein represents a stable folding conformation. This study paves the way for generating computer molecular models for proteins whose crystal structures are not available and which would aid in detailed molecular mechanism of inhibition of aflatoxin.