Obtaining mice that carry human mitochondrial DNA transmitted to the progeny

To study human diseases associated with mutations in mitochondrial DNA one needs an animal model in which the distribution of abnormal mtDNA and its impact on the phenotype might be followed. We isolated human mitochondria from HepG2 cell culture and microinjected them into murine zygotes, upon which those were transplanted to the pseudopregnant mice. PCR with species-specific primers allowed detecting human mtDNA in the tissues of 7-13-day embryos. No serious alterations in the development of transmitochondrial embryos were noticed. Among various organs/tissues of the 13-day embryos, human mtDNA was detected only in the heart, skeletal muscles, and stomach, which is in line with its uneven distribution among the blastomeres of an early mouse embryo that we described previously. In four recipient females, the microinjected zygotes were allowed to develop to term, the four neonate males of their joint litter were sacrificed, and in three of them human mtDNA was detected in the heart, skeletal muscles, stomach, brain, testes, and bladder. Six females of that joint litter were grown and mated to intact males. In the progeny (F1) of one of the females two mice were carrying human mtDNA in the heart, skeletal muscles, stomach, brain, lungs, uterus, ovaries, and kidneys. The study confirms the possibility to obtain transmitochondrial mice carrying human mtDNA that is transmitted to the animals of the next generation. Our results also indicate that among the organs to which human mtDNA is distributed some are more likely to receive it than others.     

Binding of divalent copper and calcium ions to poly I

The effect ot Cu²⁺ and Ca²⁺ ions, on the ultraviolet differential (UVD) spectra of single-stranded poly I was studied and the coordination (Δε_b) and conformation (Δε_c) conponents of the spectra calculated The comparison of Δε_b and the UVD spectrum of protonated IMP leads to the conclusion that N(7) ot inosine-5'-monophosphate (IMP) is a coordinating site tor Ca²⁺ and Cu²⁺ ions on the polymer bases. The binding ot Ca²⁺ and Cu²⁺ ions causes differently directed displacements of the four absorption bands of poly I, which are observed in the wavenumber range (50-34) × 10³ cm⁻¹ The calculation of concentration dependencies tor the association constants (K“) ot Ca²⁺ and Cu²⁺ ions binding to poly I bases shows that the binding is cooperative The K“ values for the poly I + Ca²⁺ complex are two orders of magnitude lower than those for the poly 1 + Cu²⁺ complex At low ion concentrations, binding to the poly I phosphates predominates and increases the degree of the polynucleotide helicity. At higher concentrations the spectra are mainly affected by the ion binding to bases, which results in melting of the helical parts of poly I At Ca²⁺ concentrations exceeding 10⁻³ M light-scattering aggregates are formed. The degree of monomer order in them is close to that observed in multistranded helices of poly I     

Electrostatic potentials of E.coli genome DNA

Distribution of electrostatic potential of the complete sequence of E. coli genome was calculated. Comparative analysis of electrostatic patterns for 359 promoter and nonpromoter nucleotide sequences was carried out. It is found that nonpromoter regions are characterized by more homogeneous distribution of electrostatic potential with no common specific elements. Electrostatic patterns of promoter DNAs can be specified due to the presence of some distinctive motifs which may be involved as promoter signal elements in RNA-polymerase-promoter recognition.     

Phylogeny of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Genes in Haloalkaliphilic Obligately Autotrophic Sulfur-Oxidizing Bacteria of the Genus <Emphasis Type="Italic">Thioalkalivibrio</Emphasis>

Fragments of genes of the “green-like” form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of eight species of haloalkaliphilic obligately autotrophic sulfur-oxidizing bacteria of the genus Thioalkalivibrio have been revealed and sequenced using previously developed oligonucleotide primers. The data obtained are used for the construction of phylogenetic trees on the basis of nucleotide sequences of RuBisCO genes and their conceptual translations into amino acid sequences. Comparative analysis of the 16S rRNA and RuBisCO gene trees reveals discrepancies between their topologies. According to a RuBisCO gene analysis, the genus Thioalkalivibrio is not monophyletic, and its inner divergence conforms to the significant morphological differences observed between the species. Presumably, horizontal (interspecies) gene transfer was involved in the evolution of the genus Thioalkalivibrio.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 378–386.Original Russian Text Copyright © 2005 by Tourova, Spiridonova, Berg, Kuznetsov, Sorokin.     

Characterization of Molybdenum-Free Nitrate Reductase from Haloalkalophilic Bacterium Halomonas sp. Strain AGJ 1-3

Nitrate reductase from the haloalkalophilic denitrifying bacterium Halomonas sp. strain AGJ 1-3 was isolated and purified to homogeneity. The isolated enzyme belongs to a novel family of molybdenum-free nitrate reductases. It presents as a 130-140 kD monomeric protein with specific activity of 250 micromol/min per mg protein. The enzyme reduces not only nitrate, but also other anions, thus showing polyoxoanion reductase activity. Enzyme activity was maximal at pH 7.0 and 70-80 degrees C.     

The enlarged population of marginal zone/CD1d(high) B lymphocytes in nonobese diabetic mice maps to diabetes susceptibility region Idd11

The NOD mouse is an important experimental model for human type 1 diabetes. T cells are central to NOD pathogenesis, and their function in the autoimmune process of diabetes has been well studied. In contrast, although recognized as important players in disease induction, the role of B cells is not clearly understood. In this study we characterize different subpopulations of B cells and demonstrate that marginal zone (MZ) B cells are expanded 2- to 3-fold in NOD mice compared with nondiabetic C57BL/6 (B6) mice. The NOD MZ B cells displayed a normal surface marker profile and localized to the MZ region in the NOD spleen. Moreover, the MZ B cell population developed early during the ontogeny of NOD mice. By 3 wk of age, around the time when autoreactive T cells are first activated, a significant MZ B cell population of adult phenotype was found in NOD, but not B6, mice. Using an F2(B6 x NOD) cross in a genome-wide scan, we map the control of this trait to a region on chromosome 4 (logarithm of odds score, 4.4) which includes the Idd11 and Idd9 diabetes susceptibility loci, supporting the hypothesis that this B cell trait is related to the development of diabetes in the NOD mouse.     

Functional and Morphological Adaptation to Peptidoglycan Precursor Alteration in Lactococcus lactis

Cell wall peptidoglycan assembly is a tightly regulated process requiring the combined action of multienzyme complexes. In this study we provide direct evidence showing that substrate transformations occurring at the different stages of this process play a crucial role in the spatial and temporal coordination of the cell wall synthesis machinery. Peptidoglycan substrate alteration was investigated in the Gram-positive bacterium Lactococcus lactis by substituting the peptidoglycan precursor biosynthesis genes of this bacterium for those of the vancomycin-resistant bacterium Lactobacillus plantarum. A set of L. lactis mutant strains in which the normal d-Ala-ended precursors were partially or totally replaced by d-Lac-ended precursors was generated. Incorporation of the altered precursor into the cell wall induced morphological changes arising from a defect in cell elongation and cell separation. Structural analysis of the muropeptides confirmed that the activity of multiple enzymes involved in peptidoglycan synthesis was altered. Optimization of this altered pathway was necessary to increase the level of vancomycin resistance conferred by the utilization of d-Lac-ended peptidoglycan precursors in the mutant strains. The implications of these findings on the control of bacterial cell morphogenesis and the mechanisms of vancomycin resistance are discussed.     

Electrostatic properties of promoter recognized by E. coli RNA polymerase Esigma70

A comparative analysis of electrostatic patterns for 359 sigma70-specific promoters and 359 nonpromoter regions on electrostatic map of Escherichia coli genome was carried out. It was found that DNA is not a uniformly charged molecule. There are some local inhomogeneities in its electrostatic profile which correlate with promoter sequences. Electrostatic patterns of promoter DNAs can be specified due to the presence of some distinctive motifs which differ for different promoter groups and may be involved as signal elements in differential recognition of various promoters by the enzyme. Some specific electrostatic elements which are responsible for modulating promoter activities due to ADP-ribosylation of RNA polymerase alpha-subunit were found in far upstream regions of T4 phage early promoters and E. coli ribosomal promoters.     

Multiple-Locus Sequence Typing Analysis of Bacillus cereus and Bacillus thuringiensis Reveals Separate Clustering and a Distinct Population Structure of Psychrotrophic Strains

We used multilocus sequence typing (MLST) to characterize phylogenetic relationships for a collection of Bacillus cereus group strains isolated from forest soil in the Paris area during a mild winter. This collection contains multiple strains isolated from the same soil sample and strains isolated from samples from different sites. We characterized 115 strains of this collection and 19 other strains based on the sequences of the clpC, dinB, gdpD, panC, purF, and yhfL loci. The number of alleles ranged from 36 to 53, and a total of 93 allelic profiles or sequence types were distinguished. We identified three major strain clusters—C, T, and W—based on the comparison of individual gene sequences or concatenated sequences. Some less representative clusters and subclusters were also distinguished. Analysis of the MLST data using the concept of clonal complexes led to the identification of two, five, and three such groups in clusters C, T, and W, respectively. Some of the forest isolates were closely related to independently isolated psychrotrophic strains. Systematic testing of the strains of this collection showed that almost all the strains that were able to grow at a low temperature (6°C) belonged to cluster W. Most of these strains, including three independently isolated strains, belong to two clonal complexes and are therefore very closely related genetically. These clonal complexes represent strains corresponding to the previously identified species Bacillus weihenstephanensis. Most of the other strains of our collection, including some from the W cluster, are not psychrotrophic. B. weihenstephanensis (cluster W) strains appear to comprise an effectively sexual population, whereas Bacillus thuringiensis (cluster T) and B. cereus (cluster C) have clonal population structures.