Microbial sulfate reduction in a brackish meromictic steppe lake

Patterns of sulfate reduction were studied in water and sediments of Lake Shira, South Siberia, Russia. The lake was characterized by a high level of sulfate (91-116 mM). The concentration of hydrogen sulfide in the anoxic waters of the lake reached 0.6 mM. In summer the sulfate reduction rate in the water column, measured by radiometric technique, varied from 0.25 to 9.81 mol sulfate l^-1 d^-1. There were two peaks of sulfate reduction activity: just below the chemocline and near the sediment surface. Sulfate reduction rate in the profundal silts ranged from 4.1 to 90.6 mol l^-1 d^-1. The zone of the most active sulfate reduction was restricted to the surface sediment layers. The acceleration of sulfate reduction rate (up to 236 mol l^-1 d^-1) and the increase of density of viable sulfate reducers (up to 2 x 10⁵ cells ml^-1) were recorded in the littoral sediments adjacent to the mouth of the Son River and sewage discharge. It was apparently caused by the input of allochthonous organic substrates and also by a high environmental temperature. On an areal basis, sulfate reduction rate in the water was approximately 8 times higher than that in the profundal sediments. Sulfate reduction was the most important process of anaerobic oxidation of organic carbon in Lake Shira. In summer in the profundal zone of the lake, sulfate reducers were able to mineralize about 67% of the daily integrated primary production of phototrophic and chemotrophic organisms.     

Characterization of pig-mink cell hybrids: assignment of the TK1 and UMPH2 genes to pig chromosome 12

By fusion of thymidine kinase-deficient mink cells with pig leukocytes, a new type of cell hybrid was produced. It was demonstrated that pig chromosomes segregate in pig-mink hybrids and that hybrid cells contain no cytologically visible rearrangements between the chromosomes of parental species, or chromosome fragmentation. With a set of subclones of two primary hybrid clones, the genes for thymidine kinase-1 (TK1) and uridine 5-monophosphate hydrolase-2 (UMPH2) were assigned to pig Chromosome (Chr) 12. A cell line with a single pig Chr 8 on the background of mink chromosomes was established. This clone could serve as a source of DNA for building a chromosome-specific library of pig Chr 8. The data obtained suggest that pig-mink cell hybrids can be used for mapping of pig chromosomes.     

Genetics of esterases in Drosophila. IV. Slow-migrating S-esterase in Drosophila of the virilis group

A slow-migrating -esterase (S-esterase) is described which has been detected in Drosophila montana, Drosophila imeretensis, and some stocks of Drosophila virilis when mixtures of - and -naphthyl acetate are used as substrates in histochemical reactions after electrophoresis. Sexual dimorphism for S-esterase has been demonstrated. This esterase is contained in male genitalia only, predominantly in the ejaculatory bulb (waxy plug). It appears 3–4 days after emergence of flies. In hybrids between S⁺ and S⁰ species, the activity of the slow esterase is either decreased or inhibited. An autonomous synthesis of the S-esterase in the ejaculatory bulb was established by transplantation of imaginal genital discs into larvae of different Drosophila stocks. Based on analysis of physicochemical and immunochemical properties, S-esterase is suggested to be an independent fraction of esterase, possibly dimeric, which does not cross-react with -esterase antiserum.     

Restriction endonucleases: the detection of new producer strains and the isolation of enzyme preparations]

The method for analysis of microorganisms for the presence of the modification-restriction systems has been developed. The method has permitted to detect more than 10 new producing strains of restrictases including microorganisms of Rhizobium genus. Some of them are promising for practical use. It has been shown that using selection of clones the strain productivity can be increased. The purification process for the majority of restrictases has been proposed. Some physical and catalytic properties of new enzymes have been studied.     

In vitro and in vivo study of pluripotency in intraspecific hybrid cells obtained by fusion of murine embryonic stem cells with splenocytes

Hypoxanthine phosphoribosyltransferase–deficient (HPRT^-) mouse embryonic stem (ES) cells, HM-1 cells (genotype XY), were fused with adult female DD/c mouse spleen cells. As a result, a set of HAT-resistant clones was isolated. Four hybrid clones most similar in morphology and growth characteristics to the HM-1 cells were studied in detail with respect to their pluripotency. Of these, three clones contained 41–43 chromosomes, and one clone was nearly tetraploid. All the clones had the XXY set of sex chromosomes and expressed the HPRT of the somatic partner only. The hybrid clones shared features with the HM-1 cells, indicating that they retained their pluripotent properties: (1) embryonic ECMA-7 antigen, not TROMA-1 antigen, was present in most cells; (2) the hybrid cells showed high activity of endogenous alkaline phosphatase (AP); (3) all the hybrid clones were able to form complex embryoid bodies containing derivatives of all the embryonic germinal layers; (4) the hybrid cells contained synchronously replicating X chromosomes, indicating that they were in an active state; and (5) a set of chimeric animals was generated by injecting hybrid cells into BALB/c and C57BL/6J mouse blastocysts. Evidence for chimerism was provided by the spotted coat derived from 129/Ola mice and identification of 129/Ola glucose phosphate isomerase (GPI) in many organs. Thus the results obtained demonstrated that the hybrid cells retain their high pluripotency level despite the close contact of the “pluripotent” HM-1 genome with the “somatic” spleen cell genome during hybrid cell formation and the presence of the “somatic” X chromosome during many cell generations. The presence of HPRT of the somatic partner in many organs and tissues, including the testes in chimeric animals, shows that the “somatic” X chromosome segregates weakly, if at all, during development of the chimeras. There were no individuals with the 129/Ola genotype among the more than 50 offspring from chimeric mice. The lack of the 129/Ola genotype is explained by the imbalance of the sex chromosomes in the hybrid cells rendering the passage of hybrid cell descendants through meiosis in chimeras impossible. As a result, chimeras become unable to produce gametes of the hybrid cell genotype. Mol. Reprod. Dev. 50:128–138, 1998. © 1998 Wiley-Liss, Inc.     

High light field confinement for fluorescent correlation spectroscopy using a solid immersion lens

In this paper we present recent single molecule detection experiment using a solid immersion lens (SIL) for fluorescent correlation spectroscopy measurements. We compared the performance of the SIL in combination with an air objective (40x, numerical aperture (NA)=1.15) with a water immersion objective (40x, NA=0.6) in a confocal microscope system (ConfoCorr 1). Important parameters for single molecule experiments such as collection efficiency and excitation field confinement were investigated. Although the two set-ups have similar numerical aperture the measurements demonstrated higher field confinement and better collection efficiency for the SIL system in comparison to the conventional confocal set-up. Adding spherical aberrations shifts the sample volume up to 4 microm away from the plane surface of the SIL and conserves a diffraction limited focal volume. In this case the FCS autocorrelation demonstrates a free 3D diffusion of dye molecules in a highly confined light field.     

Fluorescent proteins as a toolkit for in vivo imaging

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, and its mutant variants, are the only fully genetically encoded fluorescent probes available and they have proved to be excellent tools for labeling living specimens. Since 1999, numerous GFP homologues have been discovered in Anthozoa, Hydrozoa and Copepoda species, demonstrating the broad evolutionary and spectral diversity of this protein family. Mutagenic studies gave rise to diversified and optimized variants of fluorescent proteins, which have never been encountered in nature. This article gives an overview of the GFP-like proteins developed to date and their most common applications to study living specimens using fluorescence microscopy.     

High-throughput RNAi screening in vitro: from cell lines to primary cells

Small interfering RNAs (siRNAs) are being used to induce sequence-specific gene silencing in cultured cells to study mammalian gene function. Libraries of siRNAs targeting entire human gene classes can be used to identify genes with specific cellular functions. Here we describe high-throughput siRNA delivery methods to facilitate siRNA library screening experiments with both immortalized and primary cells. We adapted chemical reverse transfection for immortalized adherent cell lines in a 96-well format. The method is fast, robust, and exceptionally effective for many cell types. For primary cells and immortalized cells that are recalcitrant to lipofection-based methods, we developed electropermeabilization (electroporation) conditions that facilitate siRNA delivery to a broad range of cell types, including primary human T-cells, hMSC, NHA, NDHF-Neo, HUVEC, DI TNC1, RPTEC, PC12, and K562 cells. To enable high-throughput electropermeabilization of primary cells, we developed a novel 96-well electroporation device that provides highly efficient and reproducible delivery of siRNAs. The combination of high-throughput chemical reverse transfection and electroporation makes it possible to deliver libraries of siRNAs to virtually any cell type, enabling gene function analysis and discovery on a genome scale.     

Visible and “cryptic” segregation of parental chromosomes in embryonic stem hybrid cells

Chromosome segregation of the parental chromosomes was studied in 20 interspecific hybrid clones obtained by fusion of Mus musculus embryonic stem cells with Mus caroli splenocytes. FISH analysis with labeled species specific probes and microsatellite markers was used for identification of the parental chromosomes. Cytogenetic analysis has shown significant intra- and interclonal variability in chromosome numbers and ratios of the parental chromosomes in the hybrid cells: six clones contained all M. caroli chromosomes, nine clones showed moderate segregation of M. caroli chromosomes (from 1 to 7), and five clones showed extensive loss of M. caroli chromosomes (from 12 to complete loss of all M. caroli autosomes). Both methods demonstrated cryptic segregation of the somatic partner chromosomes. For instance, five clones with near-tetraploid chromosome sets contained only few M. caroli chromosomes (from 1 to 8). The data obtained suggest that the tetraploid chromosome set per se is not a sufficient criterion for conclusion on the absence of chromosome loss in the hybrid cells. Note that cryptic chromosome segregation occurred at a high frequency in the examined hybrid clones. Thus, cryptic segregation should be borne in mind for assessing pluripotency and genome reprogramming of embryonic stem hybrid cells.__________Translated from Ontogenez, Vol. 36, No. 2, 2005, pp. 151–158.Original Russian Text Copyright © 2005 by Pristyazhnyuk, Temirova, Menzorov, Kruglova, Matveeva, Serov.     

Visible and "cryptic" segregation of parental chromosomes in embryonic stem hybrid cells

Chromosome segregation of the parental chromosomes was studied in 20 interspecific hybrid clones obtained by fusion of Mus musculus embryonic stem cells with Mus caroli splenocytes. FISH analysis with labeled species specific probes and microsatellite markers was used for identification of the parental chromosomes. Cytogenetic analysis has shown significant intra- and interclonal variability in chromosome numbers and ratios of the parental chromosomes in the hybrid cells: six clones contained all M. caroli chromosomes, nine clones showed moderate segregation of M. caroli chromosomes (from 1 to 7), and five clones showed extensive loss of M. caroli chromosomes (from 12 to complete loss of all M. caroli autosomes). Both methods demonstrated "cryptic" segregation of the somatic partner chromosomes. For instance, five clones with near-tetraploid chromosome sets contained only few M. caroli chromosomes (from 1 to 8). The data obtained suggest that the tetraploid chromosome set per se is not a sufficient criterion for conclusion on the absence of chromosome loss in the hybrid cells. Note that "cryptic" chromosome segregation occurred at a high frequency in the examined hybrid clones. Thus, "cryptic" segregation should be borne in mind for assessing pluripotency and genome reprogramming of embryonic stem hybrid cells.