Slipped Misalignment Mechanisms of Deletion Formation: In Vivo Susceptibility to Nucleases

Misalignment of repeated sequences during DNA replication can lead to deletions or duplications in genomic DNA. In Escherichia coli, such genetic rearrangements can occur at high frequencies, independent of the RecA-homologous recombination protein, and are sometimes associated with sister chromosome exchange (SCE). Two mechanisms for RecA-independent genetic rearrangements have been proposed: simple replication misalignment of the nascent strand and its template and SCE-associated misalignment involving both nascent strands. We examined the influence of the 3′ exonuclease of DNA polymerase III and exonuclease I on deletion via these mechanisms in vivo. Because mutations in these exonucleases stimulate tandem repeat deletion, we conclude that displaced 3′ ends are a common intermediate in both mechanisms of slipped misalignments. Our results also confirm the notion that two distinct mechanisms contribute to slipped misalignments: simple replication misalignment events are sensitive to DNA polymerase III exonuclease, whereas SCE-associated events are sensitive to exonuclease I. If heterologies are present between repeated sequences, the mismatch repair system dependent on MutS and MutH aborts potential deletion events via both mechanisms. Our results suggest that simple slipped misalignment and SCE-associated misalignment intermediates are similarly susceptible to destruction by the mismatch repair system.     

Electron and nuclear dynamics in many-electron atoms, molecules and chlorophyll-protein complexes: A review

It has been shown [V.A. Shuvalov, Quantum dynamics of electrons in many-electron atoms of biologically important compounds, Biochemistry (Mosc.) 68 (2003) 1333-1354; V.A. Shuvalov, Quantum dynamics of electrons in atoms of biologically important molecules, Uspekhi biologicheskoi khimii, (Pushchino) 44 (2004) 79-108] that the orbit angular momentum L of each electron in many-electron atoms is L = mVr = n? and similar to L for one-electron atom suggested by N. Bohr. It has been found that for an atom with N electrons the total electron energy equation E = −(Z^eff)²e⁴m/(2n²?²N) is more appropriate for energy calculation than standard quantum mechanical expressions. It means that the value of L of each electron is independent of the presence of other electrons in an atom and correlates well to the properties of virtual photons emitted by the nucleus and creating a trap for electrons. The energies for elements of the 1st up to the 5th rows and their ions (total amount 240) of Mendeleev' Periodical table were calculated consistent with the experimental data (deviations in average were 5 × 10^− 3). The obtained equations can be used for electron dynamics calculations in molecules. For H₂ and H₂⁺ the interference of electron-photon orbits between the atoms determines the distances between the nuclei which are in agreement with the experimental values. The formation of resonance electron-photon orbit in molecules with the conjugated bonds, including chlorophyll-like molecules, appears to form a resonance trap for an electron with E values close to experimental data. Two mechanisms were suggested for non-barrier primary charge separation in reaction centers (RCs) of photosynthetic bacteria and green plants by using the idea of electron-photon orbit interference between the two molecules. Both mechanisms are connected to formation of the exciplexes of chlorophyll-like molecules. The first one includes some nuclear motion before exciplex formation, the second one is related to the optical transition to a charge transfer state.     

Phylogeography of northern Dolly Varden Salvelinus malma malma based on analysis of mitochondrial DNA

The northern Dolly Varden, Salvelinus malma malma, is a typical representative of arctic fauna distributed in northeastern Asia and northwestern North America. Because its spawning habitats were affected by Pleistocene glacial advances over most of its natural range, S. m. malma is among the most interesting objects of phylogeographic and microevolutionary studies. We reconstructed the genealogy of mtDNA haplotypes from 27 Alaskan and Asian populations to study the influence of glacial and geological vicariance events on the contemporary population genetic structure, phylogeographic subdivision and distribution of the northern Dolly Varden. Analysis of restriction site states in three PCR‐amplified mtDNA regions (ND1/ND2, ND5/ND6, Cytb/D‐loop; 47% of the mitochondrial genome) resolved 75 haplotypes in 436 fish. Similar patterns of subspecific variation apparent from hierarchical diversity and nested clade analyses of mtDNA haplotypes identify weak spatial differentiation and low levels of divergence. Our results suggest that (1) demographic history has been influenced by historical range expansions and recent isolation by distance, (2) present populations from Asia and North America were colonized from one main Beringian Refugium, and (3) that this taxon's ancestral population probably experienced a bottleneck in the Beringian Refugium during the late Pleistocene (Wisconsin) glacial period.     

The Balkan chamois,an archipelago or a peninsula? Insights from nuclear and mitochondrial DNA

The Balkan chamois (Rupicapra rupicapra balcanica) is widespread on the Balkan Peninsula, along mountain massifs from Croatia in the north to Greece in the south and Bulgaria in the east. Knowledge on the genetic structure of Balkan chamois populations is limited and restricted to local studies. Therefore, the main objective of this study was to use nuclear (16 microsatellites) and mitochondrial (partial 376 base pairs control region) markers to investigate the genetic structure of this chamois subspecies throughout its distribution range and to obtain information on the degree of connectivity of the different (sub)populations. We extracted DNA from bone, dried skin and muscle tissue and successfully genotyped 92 individuals of Balkan chamois and sequenced the partial control region in 44 individuals. The Bayesian analysis suggested 3 genetic clusters and assigned individuals from Serbia and Bulgaria to two separate clusters, while individuals from the other countries belonged to the same cluster. Thirty new haplotypes were obtained from partial mitochondrial DNA sequences, with private haplotypes in all analyzed populations and only two haplotypes shared among populations, indicating the possibility of past translocations. The subspecies genetic composition presented here provides the necessary starting point to assess the conservation status of the Balkan chamois and allows the development of conservation strategies necessary for its sustainable management and conservation.

     

Barnase-barstar high affinity interaction phenomenon as the base for the heterogenous bioluminescence pseudorabies virus' immunoassay

The effective new variant of "sandwich" bioluminescent enzyme immunoassay (BEIA) for the sensitive detection of glycoprotein B (gB) of pseudorabies virus (PrV) was presently developed. The high affinity interaction of barnase-barstar protein pair and photoprotein obelin as bioluminescent marker were for the first time successfully applied to BEIA development. Preliminary the two monoclonal antibodies, 11/5 and 34/2, were raised against gB for ELISA PrV detection. Presently we used the same immuno-"sandwich" principle for BEIA. To do this the two different bioconjugates were elaborated. Recombinant barnase was chemically conjugated with monoclonal anti-PrV's gB IgG, and also barstar was fused in frame to obelin. The characteristics of BEIA method have been compared to ELISA PrV detection. We have shown the proposed here gB-BEIA was 40-fold more sensitive as opposed to gB-ELISA test. The construction might have a broad promise in multiple potential immunological applications.     

Evolutionary history of bacteriophages with double-stranded DNA genomes

Background

Reconstruction of evolutionary history of bacteriophages is a difficult problem because of fast sequence drift and lack of omnipresent genes in phage genomes. Moreover, losses and recombinational exchanges of genes are so pervasive in phages that the plausibility of phylogenetic inference in phage kingdom has been questioned.

Results

We compiled the profiles of presence and absence of 803 orthologous genes in 158 completely sequenced phages with double-stranded DNA genomes and used these gene content vectors to infer the evolutionary history of phages. There were 18 well-supported clades, mostly corresponding to accepted genera, but in some cases appearing to define new taxonomic groups. Conflicts between this phylogeny and trees constructed from sequence alignments of phage proteins were exploited to infer 294 specific acts of intergenome gene transfer.

Conclusion

A notoriously reticulate evolutionary history of fast-evolving phages can be reconstructed in considerable detail by quantitative comparative genomics.

Open peer review

This article was reviewed by Eugene Koonin, Nicholas Galtier and Martijn Huynen.     

Aggregation-Prone Motifs in Human Immunoglobulin G

Therapeutic antibodies of many different IgG subclasses (IgG1, IgG2 and IgG4) are used in the treatment of various cancers, rheumatoid arthritis and other inflammatory and infectious diseases. These antibodies are stored for long durations under high concentrations as required in the disease treatment. Unfortunately, these antibodies aggregate under these storage conditions, leading to a decrease in antibody activity and raising concerns about causing an immunological response. Thus, there is a tremendous need to identify the aggregation-prone regions in different classes of antibodies. We use the SAP (spatial-aggregation-propensity) technology based on molecular simulations to determine the aggregation-prone motifs in the constant regions of IgG1 classes of antibodies. Mutations engineered on these aggregation-prone motif regions led to antibodies of enhanced stability. Fourteen aggregation-prone motifs are identified, with each motif containing one to seven residues. While some of these motifs contain residues that are neighbors in primary sequence, others contain residues that are far apart in primary sequence but are close together in the tertiary structure. Comparison of the IgG1 sequence with those of other subclasses (IgG2, IgG3 and IgG4) showed that these aggregation-prone motifs are largely preserved among all IgG subclasses. Other broader classes of antibodies (IgA1, IgD, IgE and IgM), however, differed in these motif regions. The aggregation-prone motifs identified were therefore common to all IgG subclasses, but differ from those of non-IgG classes. Moreover, since the motifs identified are in the constant regions, they are applicable for all antibodies within the IgG class irrespective of the variable region. Thus, the motif regions identified could be modified on all IgGs to yield antibodies of enhanced stability.