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Genetic manipulation of a naturally competent bacterium, Helicobacter pylori.
Noto JM, Peek RM
(2012) Methods Mol Biol 921: 51-9
MeSH Terms: Chromosomes, Bacterial, DNA, Bacterial, Genetic Engineering, Helicobacter pylori, Mutagenesis, Site-Directed, Mutation, Transformation, Genetic
Show Abstract · Added September 3, 2013
Genetic manipulation of Helicobacter pylori facilitates characterization and functional analysis of individual H. pylori genes. This chapter discusses the methods involved in H. pylori chromosomal DNA isolation, mutagenesis of individual genes, and natural transformation.
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3 Members
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7 MeSH Terms
EPR spectroscopy and electrospray ionization mass spectrometry reveal distinctive features of the iron site in leukocyte 12-lipoxygenase.
Rapp J, Xu S, Sharp AM, Griffith WP, Kim YW, Funk MO
(2009) Arch Biochem Biophys 490: 50-6
MeSH Terms: Amino Acid Sequence, Animals, Arachidonate 12-Lipoxygenase, Electron Spin Resonance Spectroscopy, Escherichia coli, Hydrogen-Ion Concentration, Iron, Leukocytes, Lipid Peroxides, Molecular Weight, Oxidation-Reduction, Recombinant Proteins, Spectrometry, Mass, Electrospray Ionization, Sus scrofa, Temperature, Transformation, Genetic
Show Abstract · Added April 14, 2017
The procedure for the expression and purification of recombinant porcine leukocyte 12-lipoxygenase using Escherichia coli [K.M. Richards, L.J. Marnett, Biochemistry 36 (1997) 6692-6699] was updated to make it possible to produce enough protein for physical measurements. Electrospray ionization tandem mass spectrometry confirmed the amino acid sequence. The redox properties of the cofactor iron site were examined by EPR spectroscopy at 25K following treatment with a variety of fatty acid hydroperoxides. Combination of the enzyme in a stoichiometric ratio with the hydroperoxides led to a g4.3 signal in EPR spectra instead of the g6 signal characteristic of similarly treated soybean lipoxygenase-1. Native 12-lipoxygenase was also subjected to electrospray ionization mass spectrometry. There was evidence for loss of the mass of an iron atom from the protein as the pH was lowered from 5 to 4. Native ions in these samples indicated that iron was lost without the protein completely unfolding.
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1 Members
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16 MeSH Terms
A method for plasmid purification directly from yeast.
Singh MV, Weil PA
(2002) Anal Biochem 307: 13-7
MeSH Terms: DNA, Bacterial, DNA, Fungal, Escherichia coli, Plasmids, Saccharomyces cerevisiae, Sequence Analysis, DNA, Transfection, Transformation, Genetic
Show Abstract · Added March 5, 2014
A rapid technique for purifying plasmids from yeast Saccharomyces cerevisiae is described that yields high-quality DNA suitable for bacterial transformation, yeast transformation, and direct DNA sequencing. The method requires only small culture volumes and proprietary bacterial plasmid miniprep kits that allow one to simultaneously prepare a large number of samples in a very short period of time while avoiding the use of toxic organic chemicals. Both yeast single-copy CEN/ARS and high-copy 2micro shuttle plasmids can be isolated using this method. This technique is useful for plasmid purification from yeast two-hybrid experiments as well as yeast genetics and molecular biology experiments.
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8 MeSH Terms
Physiological consequences associated with overproduction of Mycobacterium tuberculosis FtsZ in mycobacterial hosts.
Dziadek J, Madiraju MV, Rutherford SA, Atkinson MA, Rajagopalan M
(2002) Microbiology 148: 961-71
MeSH Terms: Bacterial Proteins, Cell Division, Cloning, Molecular, Cytoskeletal Proteins, DNA Primers, Diploidy, GTP Phosphohydrolases, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Enzymologic, Mycobacterium smegmatis, Mycobacterium tuberculosis, Polymerase Chain Reaction, Recombinant Proteins, Species Specificity, Transformation, Genetic
Show Abstract · Added August 13, 2015
The ftsZ gene of Mycobacterium tuberculosis H37Rv has been characterized as the first step in determining the molecular events involved in the cell division process in mycobacteria. Western analysis revealed that intracellular levels of FtsZ are growth phase dependent in both M. tuberculosis and Mycobacterium smegmatis. Unregulated expression of M. tuberculosis ftsZ from constitutive hsp60 and dnaA promoters in M. tuberculosis hosts resulted in lethality whereas expression from only the hsp60 promoter was toxic in M. smegmatis hosts. Expression of ftsZ from the dnaA promoter in M. smegmatis resulted in approximately sixfold overproduction and the merodiploids exhibited slow growth, an increased tendency to clump and filament, and in some cases produced buds and branches. Many of the cells also contained abnormal and multiple septa. Expression of ftsZ from the chemically inducible acetamidase promoter in M. smegmatis hosts resulted in approximately 22-fold overproduction of FtsZ and produced filamentous cells, many of which lacked any visible septa. Visualization of the M. tuberculosis FtsZ tagged with green fluorescent protein in M. smegmatis by fluorescence microscopy revealed multiple fluorescent FtsZ foci, suggesting that steps subsequent to the formation of organized FtsZ structures but prior to septum formation are blocked in FtsZ-overproducing cells. Together these results suggest that the intracellular concentration of FtsZ protein is critical for productive septum formation in mycobacteria.
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15 MeSH Terms
Apoptosis triggered by Myc-induced suppression of Bcl-X(L) or Bcl-2 is bypassed during lymphomagenesis.
Eischen CM, Woo D, Roussel MF, Cleveland JL
(2001) Mol Cell Biol 21: 5063-70
MeSH Terms: ADP-Ribosylation Factor 1, Animals, Apoptosis, Blotting, Northern, Blotting, Western, Cell Survival, Cells, Cultured, Lymph Nodes, Mice, Mice, Knockout, Mice, Transgenic, Myeloid Cells, Proto-Oncogene Proteins c-bcl-2, Proto-Oncogene Proteins c-myc, RNA, Stem Cells, Time Factors, Transformation, Genetic, Tumor Suppressor Protein p53, bcl-X Protein
Show Abstract · Added March 5, 2014
Enforced Bcl-2 expression inhibits Myc-induced apoptosis and cooperates with Myc in transformation. Here we report that the synergy between Bcl-2 and Myc in transforming hematopoietic cells in fact reflects a Myc-induced pathway that selectively suppresses the expression of the Bcl-X(L) or Bcl-2 antiapoptotic protein. Myc activation suppresses Bcl-X(L) RNA and protein levels in cultures of primary myeloid and lymphoid progenitors, and Bcl-X(L) and Bcl-2 expression is inhibited by Myc in precancerous B cells from Emu-myc transgenic mice. The suppression of bcl-X RNA levels by Myc requires de novo protein synthesis, indicating that repression is indirect. Importantly, the suppression of Bcl-2 or Bcl-X(L) by Myc is corrupted during Myc-induced tumorigenesis, as Bcl-2 and/or Bcl-X(L) levels are markedly elevated in over one-half of all lymphomas arising in Emicro-myc transgenic mice. Bcl-2 and/or Bcl-X(L) overexpression did not correlate with loss of ARF or p53 function in tumor cells, indicating that these two apoptotic pathways are inactivated independently. Therefore, the suppression of Bcl-X(L) or Bcl-2 expression represents a physiological Myc-induced apoptotic pathway that is frequently bypassed during lymphomagenesis.
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20 MeSH Terms
The auxilin-like phosphoprotein Swa2p is required for clathrin function in yeast.
Gall WE, Higginbotham MA, Chen C, Ingram MF, Cyr DM, Graham TR
(2000) Curr Biol 10: 1349-58
MeSH Terms: Amino Acid Motifs, Amino Acid Sequence, Animals, Carrier Proteins, Cell Fractionation, Cell Membrane, Clathrin, Endocytosis, Golgi Apparatus, HSP70 Heat-Shock Proteins, Molecular Sequence Data, Phosphoproteins, Protein Binding, Protein Structure, Tertiary, Protein Transport, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sequence Alignment, Transformation, Genetic, Vacuoles, Vesicular Transport Proteins
Show Abstract · Added April 6, 2017
BACKGROUND - In eukaryotic cells, clathrin-coated vesicles transport specific cargo from the plasma membrane and trans-Golgi network to the endosomal system. Removal of the clathrin coat in vitro requires the uncoating ATPase Hsc70 and its DnaJ cofactor auxilin. To date, a requirement for auxilin and Hsc70 in clathrin function in vivo has not been demonstrated.
RESULTS - The Saccharomyces cerevisiae SWA2 gene, previously identified in a synthetic lethal screen with arf1, was cloned and found to encode a protein with a carboxy-terminal DnaJ domain which is homologous to that of auxilin. Like auxilin, Swa2p has a clathrin-binding domain and is able to stimulate the ATPase activity of Hsc70. The swa2-1 allele recovered from the original screen carries a point mutation in its tetratricopeptide repeat (TPR) domain, a motif not found in auxilin but known in other proteins to mediate interaction with heat-shock proteins. Swa2p fractionates in the cytosol and appears to be heavily phosphorylated. Disruption of SWA2 causes slow growth and several phenotypes that are very similar to those exhibited by clathrin mutants. Furthermore, the swa2Delta mutant exhibits a significant increase in membrane- associated or -assembled clathrin relative to a wild-type strain.
CONCLUSIONS - These results indicate that Swa2p is a clathrin-binding protein required for normal clathrin function in vivo. They suggest that Swa2p is the yeast ortholog of auxilin and has a role in disassembling clathrin, not only in uncoating clathrin-coated vesicles but perhaps in preventing unproductive clathrin assembly in vivo.
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22 MeSH Terms
The evolutionarily conserved Dim1 protein defines a novel branch of the thioredoxin fold superfamily.
Zhang YZ, Gould KL, Dunbrack RL JR, Cheng H, Roder H, Golemis EA
(1999) Physiol Genomics 1: 109-18
MeSH Terms: Amino Acid Sequence, Cell Cycle, Cell Cycle Proteins, Conserved Sequence, DNA, Recombinant, Evolution, Molecular, Fungal Proteins, G2 Phase, Green Fluorescent Proteins, HeLa Cells, Humans, Luminescent Proteins, Magnetic Resonance Spectroscopy, Microscopy, Fluorescence, Molecular Sequence Data, Mutation, Plasmids, Protein Folding, Protein Structure, Secondary, Recombinant Fusion Proteins, Schizosaccharomyces, Schizosaccharomyces pombe Proteins, Sequence Alignment, Sequence Homology, Amino Acid, Thioredoxins, Transfection, Transformation, Genetic
Show Abstract · Added March 5, 2014
Dim1 is a small evolutionarily conserved protein essential for G2/M transition that has recently been implicated as a component of the mRNA splicing machinery. To date, the mechanism of Dim1 function remains poorly defined, in part because of the absence of informative sequence homologies between Dim1 and other functionally defined proteins or protein domains. We have used a combination of molecular modeling and NMR structural analysis to demonstrate that approximately 125 of the 142 amino acids of human Dim1 (hDim1) define a novel branch of the thioredoxin fold superfamily. Mutational analysis of Dim1 based on the predicted fold indicates that alterations in the region corresponding to the thioredoxin active site do not affect Dim1 activity. However, removal of a very short carboxy-terminal extension generates a dominant negative form of the protein [hDim1-(1-128)] that when overproduced induces cell cycle arrest in G2, via a mechanism likely to involve alteration of Dim1 association with partner molecules. In sum, this study identifies the Dim1 proteins as a novel sixth branch of the thioredoxin superfamily involved in cell cycle.
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27 MeSH Terms
A two-dimensional support for selective binding of polyhistidine-tagged proteins: identification of a proliferating cell nuclear antigen point mutant with altered function in vitro.
Zaika A, Mozzherin DJ, Tan CK, Downey KM, Fisher PA
(1999) Anal Biochem 268: 193-200
MeSH Terms: Animals, Base Sequence, Binding Sites, Cattle, DNA, DNA Polymerase III, DNA Primers, Drosophila, Escherichia coli, Histidine, In Vitro Techniques, Paper, Point Mutation, Proliferating Cell Nuclear Antigen, Recombinant Proteins, Thymus Gland, Transformation, Genetic
Show Abstract · Added March 5, 2014
Whatman 3MM paper was chemically modified to generate nickel-charged iminodiacetic acid paper (Ni2+-IDA paper). Bacteria were transformed with Escherichia coli expression plasmids coding for either unmodified proliferating cell nuclear antigen (PCNA) or PCNA containing a genetically engineered polyhistidine tract (his-tag) located at its NH2 terminus. They were then grown, induced, and lysed, and macromolecules were transferred to Ni2+-IDA paper. After exhaustive washing, his-tagged PCNA but not unmodified PCNA remained bound to the paper. Moreover, bound his-tagged PCNA was biochemically active in an in situ DNA synthesis assay with exogenous template-primer and purified calf thymus DNA polymerase delta (pol delta). Ni2+-IDA paper was used to identify a PCNA- point mutant that, relative to wild-type PCNA, promotes increased DNA synthesis by pol delta beyond a model abasic template site. In addition, metal-charged IDA paper promises to be generally useful for functional screening of cells expressing cloned proteins.
Copyright 1999 Academic Press.
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17 MeSH Terms
Negative regulation of Wee1 expression and Cdc2 phosphorylation during p53-mediated growth arrest and apoptosis.
Leach SD, Scatena CD, Keefer CJ, Goodman HA, Song SY, Yang L, Pietenpol JA
(1998) Cancer Res 58: 3231-6
MeSH Terms: Animals, Antineoplastic Agents, Apoptosis, Blotting, Western, CDC2 Protein Kinase, Cell Cycle Proteins, Cell Division, Cyclin B, Cyclin B1, Down-Regulation, Embryo, Mammalian, Enzyme Activation, Fibroblasts, G2 Phase, Lymphoma, T-Cell, Mice, Nocodazole, Nuclear Proteins, Phosphorylation, Protein Conformation, Protein-Tyrosine Kinases, Rats, Transformation, Genetic, Tumor Cells, Cultured, Tumor Suppressor Protein p53
Show Abstract · Added February 15, 2016
The G2 cell cycle checkpoint protects cells from potentially lethal mitotic entry after DNA damage. This checkpoint involves inhibitory phosphorylation of Cdc2 at the tyrosine-15 (Y15) position, mediated in part by the Wee1 protein kinase. Recent evidence suggests that p53 may accelerate mitotic entry after DNA damage and that the override of the G2 checkpoint may play a role in the induction of apoptosis by p53. To determine the biochemical mechanism by which p53 inactivates the G2 checkpoint, the effects of p53 activation on Wee1 expression, Cdc2-Y15 phosphorylation, and cyclin B1-associated Cdc2 kinase activity were examined. Under conditions of either growth arrest or apoptosis, p53 activation resulted in the down-regulation of Wee1 expression and dephosphorylation of Cdc2. A parallel increase in cyclin B1/Cdc2 kinase activity was observed during p53-mediated apoptosis. Negative regulation of the Wee1 expression and Cdc2 phosphorylation by p53 was also evident in thymus tissue from p53+/+ mice but not from p53-/- mice. Inactivation of the G2 checkpoint may contribute to the tumor suppressor activity of p53.
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25 MeSH Terms
A requirement for NF-kappaB activation in Bcr-Abl-mediated transformation.
Reuther JY, Reuther GW, Cortez D, Pendergast AM, Baldwin AS
(1998) Genes Dev 12: 968-81
MeSH Terms: Animals, Apoptosis, Fusion Proteins, bcr-abl, Gene Expression Regulation, Gene Transfer Techniques, Humans, Leukemia, Myelogenous, Chronic, BCR-ABL Positive, Mice, Mice, Nude, NF-kappa B, Precursor Cell Lymphoblastic Leukemia-Lymphoma, Signal Transduction, Transcriptional Activation, Transfection, Transformation, Genetic, Tumor Cells, Cultured
Show Abstract · Added March 11, 2014
Bcr-Abl is a chimeric oncoprotein that is strongly implicated in acute lymphoblastic (ALL) and chronic myelogenous leukemias (CML). This deregulated tyrosine kinase selectively causes hematopoietic disorders resembling human leukemias in animal models and transforms fibroblasts and hematopoietic cells in culture. Bcr-Abl also protects cells from death induced on cytokine deprivation or exposure to DNA damaging agents. In addition, the antiapoptotic function of Bcr-Abl is thought to play a necessary role in hematopoietic transformation and potentially in leukemogenesis. The transcription factor NF-kappaB has been identified recently as an inhibitor of apoptosis and as a potential regulator of cellular transformation. This study shows that expression of Bcr-Abl leads to activation of NF-kappaB-dependent transcription by causing nuclear translocation of NF-kappaB as well as by increasing the transactivation function of the RelA/p65 subunit of NF-kappaB. Importantly, this activation is dependent on the tyrosine kinase activity of Bcr-Abl and partially requires Ras. The ability of Bcr-Abl to protect cytokine-dependent 32D myeloid cells from death induced by cytokine deprivation or DNA damage does not, however, require functional NF-kappaB. However, using a super-repressor form of IkappaBalpha, we show that NF-kappaB is required for Bcr-Abl-mediated tumorigenicity in nude mice and for transformation of primary bone marrow cells. This study implicates NF-kappaB as an important component of Bcr-Abl signaling. NF-kappaB-regulated genes, therefore, likely play a role in transformation by Bcr-Abl and thus in Bcr-Abl-associated human leukemias.
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16 MeSH Terms