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91.
Expression of heteropolymeric ferritin improves iron storage in Saccharomyces cerevisiae 总被引:2,自引:0,他引:2
Kim HJ Kim HM Kim JH Ryu KS Park SM Jahng KY Yang MS Kim DH 《Applied and environmental microbiology》2003,69(4):1999-2005
Saccharomyces cerevisiae was engineered to express different amount of heavy (H)- and light (L)-chain subunits of human ferritin by using a low-copy integrative vector (YIp) and a high-copy episomal vector (YEp). In addition to pep4::HIS3 allele, the expression host strain was bred to have the selection markers leu2(-) and ura3(-) for YIplac128 and YEp352, respectively. The heterologous expression of phytase was used to determine the expression capability of the host strain. Expression in the new host strain (2805-a7) was as high as that in the parental strain (2805), which expresses high levels of several foreign genes. Following transformation, Northern and Western blot analyses demonstrated the expression of H- and L-chain genes. The recombinant yeast was more iron tolerant, in that transformed cells formed colonies on plates containing more than 25 mM ferric citrate, whereas none of the recipient strain cells did. Prussian blue staining indicated that the expressed isoferritins were assembled in vivo into a complex that bound iron. The expressed subunits showed a clear preference for the formation of heteropolymers over homopolymers. The molar ratio of H to L chains was estimated to be 1:6.8. The gel-purified heteropolymer took up iron faster than the L homopolymer, and it took up more iron than the H homopolymer did. The iron concentrations in transformants expressing the heteropolymer, L homopolymer, and H homopolymer were 1,004, 760, and 500 micro g per g (dry weight) of recombinant yeast cells, respectively. The results indicate that heterologously expressed H and L subunits coassemble into a heteropolymer in vivo and that the iron-carrying capacity of yeast is further enhanced by the expression of heteropolymeric isoferritin. 相似文献
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Molecular phylogeny of the major arthropod groups indicates polyphyly of crustaceans and a new hypothesis for the origin of hexapods 总被引:18,自引:6,他引:12
A phylogeny of the arthropods was inferred from analyses of amino acid
sequences derived from the nuclear genes encoding elongation factor-1 alpha
and the largest subunit of RNA polymerase II using maximum- parsimony,
neighbor-joining, and maximum-likelihood methods. Analyses of elongation
factor-1 alpha from 17 arthropods and 4 outgroup taxa recovered many
arthropod clades supported by previous morphological studies, including
Diplopoda, Myriapoda, Insecta, Hexapoda, Branchiopoda (Crustacea), Araneae,
Tetrapulmonata, Arachnida, Chelicerata, and Malacostraca (Crustacea).
However, counter to previous studies, elongation factor-1 alpha placed
Malacostraca as sister group to the other arthropods. Branchiopod
crustaceans were found to be more closely related to hexapods and myriapods
than to malacostracan crustaceans. Sequences for RNA polymerase II were
obtained from 11 arthropod taxa and were analyzed separately and in
combination with elongation factor-1 alpha. Results from these analyses
were concordant with those derived from elongation factor-1 alpha alone and
provided support for a Hexapoda/Branchiopoda clade, thus arguing against
the monophyly of the traditionally defined Atelocerata (Hexapoda +
Myriapoda).
相似文献
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Exposure of the outside of the isolated frog skin to a Ringer's solution, made hypertonic by the addition of mannitol, causes a rapid and sustained increase in transepithelial permeability through a structural distortion-a focal blistering-of the "tight" junctions of the outermost living cell layer. [(3)H]ouabain, used as an autoradiographic marker for the Na+-pump (Na+-K+-adenosine triphosphatase), is usually unable to penetrate the frog skin from the outside solution, but when added to a hypertonic mannitol- Ringer's solution in the outside bath it readily penetrates the epithelium, presumably through the opened shunt pathway. Radioautographic analysis of [(3)H]ouabain binding sites revealed that most of ouabain enters from the outside solution binds to the sites on the cell membranes of the stratum spinosum, as was the case when it was applied from the inside bath in an earlier study. The outer living cell layer, the first to be exposed to ouabain, does not appear to be the major site for the Na+-pump, and therefore, is not likely to be responsible for most of the active pumping of Na+. This result demonstrates that previous failure to show a high density of Na+-pump sites on the cells of the outermost layer, when [(3)H]ouabain was applied from the inside solution, was not due to the inability of the marker to reach these cells at a sufficient concentration to reveal all pump sites. These results provide further support for a model of Na+-transport across the frog skin which distributes the active pump step on the inward facing membranes of all living cells. 相似文献
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Trichloroethylene and chloroform degradation by a recombinant pseudomonad expressing soluble methane monooxygenase from Methylosinus trichosporium OB3b. 总被引:4,自引:2,他引:2
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Soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b can degrade many halogenated aliphatic compounds that are found in contaminated soil and groundwater. This enzyme oxidizes the most frequently detected pollutant, trichloroethylene (TCE), at least 50 times faster than other enzymes. However, slow growth of the strain, strong competition between TCE and methane for sMMO, and repression of the smmo locus by low concentrations of copper ions limit the use of this bacterium. To overcome these obstacles, the 5.5-kb smmo locus of M. trichosporium OB3b was cloned into a wide-host-range vector (to form pSMMO20), and this plasmid was electroporated into five Pseudomonas strains. The best TCE degradation results were obtained with Pseudomonas putida F1/pSMMO20. The plasmid was maintained stably, and all five of the sMMO proteins (alpha, beta, and gamma hydroxylase proteins, reductase, and component B) were observed clearly by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. TCE degradation rates were quantified for P. putida F1/pSMMO20 with a gas chromatograph (Vmax = 5 nmol per min per mg of protein), and the recombinant strain mineralized 55% of the TCE (10 microM) as indicated by measuring chloride ion concentrations with a chloride ion-specific electrode. The maximum TCE degradation rate obtained with the recombinant strain was lower than that of M. trichosporium OB3b but greater than other TCE-degrading recombinants and most well-studied pseudomonads. In addition, this recombinant strain mineralizes chloroform (a specific substrate for sMMO), grows much faster than M. trichosporium OB3b, and degrades TCE without competitive inhibition from the growth substrate. 相似文献