Investigation of physicochemical properties of arabinogalactan of different larch species

Samples of arabinogalactan (AG) were isolated by aqueous extraction from wood of different larch species (Larix sibirica Ledeb., Larix gmelinii (Rupr.) Rupr., Larix cajanderi Mayr., and Larix olgensis var. Koreana) and studied by HPLC, IR spectroscopy, and quantitative ¹³C NMR spectroscopy. The wood of the studied larch species was shown to contain significant amount of arabinogalactan (10–17% of the a.d.w. mass) and could be a source of its industrial production. The studied AG samples had similar structural and molecular-mass characteristics. This fact could facilitate the product standardization during its industrial production both from the wood of a single larch species and from mixed raw materials.     

Detection of Borrelia burgdorferi Sensu Stricto ospC Alleles Associated with Human Lyme Borreliosis Worldwide in Non-Human-Biting Tick Ixodes affinis and Rodent Hosts in Southeastern United States

Comparative analysis of ospC genes from 127 Borrelia burgdorferi sensu stricto strains collected in European and North American regions where Lyme disease is endemic and where it is not endemic revealed a close relatedness of geographically distinct populations. ospC alleles A, B, and L were detected on both continents in vectors and hosts, including humans. Six ospC alleles, A, B, L, Q, R, and V, were prevalent in Europe; 4 of them were detected in samples of human origin. Ten ospC alleles, A, B, D, E3, F, G, H, H3, I3, and M, were identified in the far-western United States. Four ospC alleles, B, G, H, and L, were abundant in the southeastern United States. Here we present the first expanded analysis of ospC alleles of B. burgdorferi strains from the southeastern United States with respect to their relatedness to strains from other North American and European localities. We demonstrate that ospC genotypes commonly associated with human Lyme disease in European and North American regions where the disease is endemic were detected in B. burgdorferi strains isolated from the non-human-biting tick Ixodes affinis and rodent hosts in the southeastern United States. We discovered that some ospC alleles previously known only from Europe are widely distributed in the southeastern United States, a finding that confirms the hypothesis of transoceanic migration of Borrelia species.     

Vanillin Resistance Induced by BssS Overexpression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Overexpression of the BssS gene, a biofilm formation regulator, in planktonic Escherichia coli cells has been shown to confer the vanillin-resistant phenotype Van^r to the bacteria. The MG1655P_L-tac-bssS strain started growing in liquid aerated LB medium with 2 g/L vanillin after a lag phase of 17 ± 2 h, whereas the original MG1655 strain did not grow under these conditions. The role of aldehyde reductase YqhD, a vanillin- degrading enzyme, in Van^r phenotype formation has been assessed. However, the Van^r trait in the MG1655P_L-tac-bssS strain primarily depended on autoinducer-2 (AI-2), which formed in E. coli cells with an intact luxS gene. We supposed that BssS acts together with autoinducer-2 (which presumably accumulated during the prolonged lag phase) to induce vanillin resistance determined by changes in the expression of a range of genes.     

Basic aspects of ovarian development in Drosophila melanogaster

Modern views of the development and structural organization of the female reproductive system in Drosophila melanogaster are reviewed. Special emphasis is placed on the generation and development of follicles in the germarium and the interactions of germline and somatic cells in the egg chamber. Detailed consideration is given to the main events that ensure and regulate the transport of mRNA, proteins, and organelles from nurse cells to the oocyte in the germarium and at later stages of egg chamber development.     

Genes Controlling Development: Morphoses, Phenocopies, Dimorphs, and Other Visible Expressions of Mutant Genes

We studied facultative dominant lethal mutations obtained earlier inDrosophila melanogaster. In some genotypes, these mutations were expressed as lethals, but in other genotypes they lacked this expression. The mutations were maintained in the following cultures: (1) females Muller-5 heterozygous for the mutation; (2) males crossed to attached-Xfemales; and (3) females and males homozygous for the mutation. During culturing, many mutations were found to give rise to phenotypically abnormal progeny. Generally, these abnormalities were morphoses involving various body parts; they were mostly asymmetric and nonheritable. Maternal and paternal effects in the formation of morphoses were observed. In four cases, dimorphic mutations were recorded: a female homozygous for the mutation had mutant phenotype whereas its male counterpart was phenotypically normal. The mutations were recessive with regard to the norm. New phenotypes behaving as mutations with incomplete penetrance arose during culturing. In cultures of mutant homozygotes phenocopies would appear en masse; they would persist for one or two generations and disappear. One wave of phenocopies succeeded another. Visible phenotypes appeared, which further behaved as ordinary recessive mutations. We concluded that these visible manifestations are characteristic for regulatory mutations controlling ontogeny. Their appearance is explained by the activation of new regulatory scenarios caused by blocking standard regulatory pathways.     

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.     

Genetic and Biochemical Characterization of Human AP Endonuclease 1 Mutants Deficient in Nucleotide Incision Repair Activity

Background

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3′-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5′ to a number of oxidatively damaged bases. At present, physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells.

Methodology/Principal Finding

We identified amino acid residues in the APE1 protein that affect its function in either the BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A amino acid substitutions revealed that all mutants exhibited greatly reduced NIR and 3′→5′ exonuclease activities, but were capable of performing BER functions to some extent. Expression of the APE1 mutants deficient in the NIR and exonuclease activities reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggesting that our APE1 mutants are able to repair AP sites. Finally, the human NIR pathway was fully reconstituted in vitro using the purified APE1, human flap endonuclease 1, DNA polymerase β and DNA ligase I proteins, thus establishing the minimal set of proteins required for a functional NIR pathway in human cells.

Conclusion/Significance

Taken together, these data further substantiate the role of NIR as a distinct and separable function of APE1 that is essential for processing of potentially lethal oxidative DNA lesions.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Preparative isolation of basic structural proteins of the influenza virus]

The schemes for preparative electrophoretic isolation and purification of major proteins from influenza virus are described. The viral envelope protein, hemagglutinin, two of its subunits, internal M and NP proteins of influenza viruses A/FPV/Rostock (H7N1), A/PR/8/34 (H1N1) and X-31 (H3N2) were obtained in preparative amounts and characterized by amino acid and N-terminus analyses.     

Repair of plasmid DNA treated with 8-methoxypsoralen and long-wave UV light (lambda=365 nm) in wild type and mutant rad2 cells of Saccharomyces cerevisiae

The method of repeated irradiation has been used to study excision of 8-MOP monoadducts from plasmid and chromosomal DNA in cells of wild type and rad2 mutant of Saccharomyces cerevisiae. The measurement of kinetics of monoadduct removal from chromosomal DNA in intact and competent yeast cells showed that monoadducts were excised in both types of cells with normal repair, but this process was blocked in intact and competent cells of the rad2 mutant. The survival of pYF91 plasmid treated in vitro with 8-MOP plus near UV-light has been studied in the cells of the wild type and in incision-defective rad2 mutant by the measurement of cell transformation frequency. Episomic pYF91 plasmid used in these experiments contained the yeast nuclear LEU2 gene, a portion of 2 mkm DNA and DNA of bacterial plasmid pBR322 with resistance to ampicillin. The pYF91 plasmid was treated with 8-MOP plus near UV-light in vitro, then unbound 8-MOP was removed by dialysis. This DNA was used for transformation. The transformed yeast cells were irradiated repeatedly. The quantitative alteration of the yield of transformants, depending on the time of keeping these yeast cells in complete liquid medium at 30 degrees C, prior to repeated irradiation, allowed to measure the kinetics of monoadduct excision from plasmid DNA. It was shown that monoadducts were removed equally effectively from plasmid DNA introduced into cells of the wild type and rad2 mutant. Possibly, the repair system of both these strains provides excision of monoadducts from plasmid DNA, but this process is blocked in the rad2 mutant, relatively to monoadduct excision from chromosomal DNA.