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Sample records for acetyltransferase reporter gene

  1. Erythromycin induces expression of the chloramphenicol acetyltransferase gene cat-86.

    PubMed Central

    Rogers, E J; Lovett, P S

    1990-01-01

    The plasmid gene cat-86 specifies chloramphenicol-inducible chloramphenicol acetyltransferase in Bacillus subtilis. This gene, like the erythromycin-inducible erm genes, is regulated by translational attenuation. Here we show that cat-86 is also inducibly regulated by erythromycin. cat-86 does not confer resistance to erythromycin. PMID:2115875

  2. The chloramphenicol acetyltransferase gene of Tn2424: a new breed of cat.

    PubMed

    Parent, R; Roy, P H

    1992-05-01

    We have sequenced the gene coding for the chloramphenicol acetyltransferase of Tn2424 of plasmid NR79. This gene codes for a protein of 23,500 Da, and the derived protein sequence is similar to those of the chromosomal chloramphenicol acetyltransferases of Agrobacterium tumefaciens and Pseudomonas aeruginosa and of unidentified open reading frames, which may encode chloramphenicol acetyltransferases, adjacent to the ermG macrolide-lincosamide-streptogramin resistance gene of Bacillus sphaericus and the vgb virginiamycin resistance gene of Staphylococcus aureus. Weaker similarity to the LacA (thiogalactoside acetyltransferase) and CysE (serine acetyltransferase) proteins of Escherichia coli and the NodL protein of Rhizobium leguminosarum is also observed. There is no significant similarity to any other chloramphenicol acetyltransferase genes, such as that of Tn9. The Tn2424 cat gene is part of a 4.5-kb region which also contains the aacA1a aminoglycoside-6'-N-acetyltransferase gene; Tn2424 is similar to Tn21 except for the presence of this region. Sequences flanking the cat gene are typical of those flanking other genes inserted into pVS1-derived "integrons" by a site-specific recombinational mechanism.

  3. Regulatory region in choline acetyltransferase gene directs developmental and tissue-specific expression in transgenic mice.

    PubMed Central

    Lönnerberg, P; Lendahl, U; Funakoshi, H; Arhlund-Richter, L; Persson, H; Ibáñez, C F

    1995-01-01

    Acetylcholine, one of the main neurotransmitters in the nervous system, is synthesized by the enzyme choline acetyltransferase (ChAT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). The molecular mechanisms controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo are largely unknown. A previous report showed that a 3800-bp, but not a 1450-bp, 5' flanking segment from the rat ChAT gene promoter directed cell type-specific expression of a reporter gene in cholinergic cells in vitro. Now we have characterized a distal regulatory region of the ChAT gene that confers cholinergic specificity on a heterologous downstream promoter in a cholinergic cell line and in transgenic mice. A 2342-bp segment from the 5' flanking region of the ChAT gene behaved as an enhancer in cholinergic cells but as a repressor in noncholinergic cells in an orientation-independent manner. Combined with a heterologous basal promoter, this fragment targeted transgene expression to several cholinergic regions of the central nervous system of transgenic mice, including basal forebrain, cortex, pons, and spinal cord. In eight independent transgenic lines, the pattern of transgene expression paralleled qualitatively and quantitatively that displayed by endogenous ChAT mRNA in various regions of the rat central nervous system. In the lumbar enlargement of the spinal cord, 85-90% of the transgene expression was targeted to the ventral part of the cord, where cholinergic alpha-motor neurons are located. Transgene expression in the spinal cord was developmentally regulated and responded to nerve injury in a similar way as the endogenous ChAT gene, indicating that the 2342-bp regulatory sequence contains elements controlling the plasticity of the cholinergic phenotype in developing and injured neurons. Images Fig. 1 Fig. 2 PMID:7732028

  4. Genetic Variation at the N-acetyltransferase (NAT) Genes in Global Populations

    EPA Science Inventory

    Functional variability at the N-acetyltransferase (NAT) genes is associated with adverse drug reactions and cancer susceptibility in humans. Previous studies of small sets of ethnic groups have indicated that the NAT genes have high levels of amino acid variation that differ in f...

  5. Chloroplast-encoded serotonin N-acetyltransferase in the red alga Pyropia yezoensis: gene transition to the nucleus from chloroplasts.

    PubMed

    Byeon, Yeong; Yool Lee, Hyoung; Choi, Dong-Woog; Back, Kyoungwhan

    2015-02-01

    Melatonin biosynthesis involves the N-acetylation of arylalkylamines such as serotonin, which is catalysed by serotonin N-acetyltransferase (SNAT), the penultimate enzyme of melatonin biosynthesis in both animals and plants. Here, we report the functional characterization of a putative N-acetyltransferase gene in the chloroplast genome of the alga laver (Pyropia yezoensis, formerly known as Porphyra yezoensis) with homology to the rice SNAT gene. To confirm that the putative Pyropia yezoensis SNAT (PySNAT) gene encodes an SNAT, we cloned the full-length chloroplastidic PySNAT gene by PCR and purified the recombinant PySNAT protein from Escherichia coli. PySNAT was 174 aa and had 50% amino acid identity with cyanobacteria SNAT. Purified recombinant PySNAT showed a peak activity at 55 °C with a K m of 467 µM and V max of 28 nmol min-1 mg(-1) of protein. Unlike other plant SNATs, PySNAT localized to the cytoplasm due to a lack of N-terminal chloroplast transit peptides. Melatonin was present at 0.16ng g(-1) of fresh mass but increased during heat stress. Phylogenetic analysis of the sequence suggested that PySNAT has evolved from the cyanobacteria SNAT gene via endosymbiotic gene transfer. Additionally, the chloroplast transit peptides of plant SNATs were acquired 1500 million years ago, concurrent with the appearance of green algae.

  6. Chloroplast-encoded serotonin N-acetyltransferase in the red alga Pyropia yezoensis: gene transition to the nucleus from chloroplasts

    PubMed Central

    Byeon, Yeong; Yool Lee, Hyoung; Choi, Dong-Woog; Back, Kyoungwhan

    2015-01-01

    Melatonin biosynthesis involves the N-acetylation of arylalkylamines such as serotonin, which is catalysed by serotonin N-acetyltransferase (SNAT), the penultimate enzyme of melatonin biosynthesis in both animals and plants. Here, we report the functional characterization of a putative N-acetyltransferase gene in the chloroplast genome of the alga laver (Pyropia yezoensis, formerly known as Porphyra yezoensis) with homology to the rice SNAT gene. To confirm that the putative Pyropia yezoensis SNAT (PySNAT) gene encodes an SNAT, we cloned the full-length chloroplastidic PySNAT gene by PCR and purified the recombinant PySNAT protein from Escherichia coli. PySNAT was 174 aa and had 50% amino acid identity with cyanobacteria SNAT. Purified recombinant PySNAT showed a peak activity at 55 °C with a K m of 467 µM and V max of 28 nmol min–1 mg–1 of protein. Unlike other plant SNATs, PySNAT localized to the cytoplasm due to a lack of N-terminal chloroplast transit peptides. Melatonin was present at 0.16ng g–1 of fresh mass but increased during heat stress. Phylogenetic analysis of the sequence suggested that PySNAT has evolved from the cyanobacteria SNAT gene via endosymbiotic gene transfer. Additionally, the chloroplast transit peptides of plant SNATs were acquired 1500 million years ago, concurrent with the appearance of green algae. PMID:25183745

  7. Expression profiling of S. pombe acetyltransferase mutants identifies redundant pathways of gene regulation

    PubMed Central

    2010-01-01

    Background Histone acetyltransferase enzymes (HATs) are implicated in regulation of transcription. HATs from different families may overlap in target and substrate specificity. Results We isolated the elp3+ gene encoding the histone acetyltransferase subunit of the Elongator complex in fission yeast and characterized the phenotype of an Δelp3 mutant. We examined genetic interactions between Δelp3 and two other HAT mutants, Δmst2 and Δgcn5 and used whole genome microarray analysis to analyze their effects on gene expression. Conclusions Comparison of phenotypes and expression profiles in single, double and triple mutants indicate that these HAT enzymes have overlapping functions. Consistent with this, overlapping specificity in histone H3 acetylation is observed. However, there is no evidence for overlap with another HAT enzyme, encoded by the essential mst1+ gene. PMID:20096118

  8. Characterization of two acetyltransferase genes in the pyripyropene biosynthetic gene cluster from Penicillium coprobium

    PubMed Central

    Hu, Jie; Furutani, Ayako; Yamamoto, Kentaro; Oyama, Kazuhiko; Mitomi, Masaaki; Anzai, Hiroyuki

    2014-01-01

    Pyripyropenes potently and selectively inhibit acyl-CoA:cholesterol acyltransferase 2 (ACAT-2). Among multiple isomers of pyripyropene (A to R), pyripyropene A (PyA) has insecticidal properties in addition to its growth inhibition properties against human umbilical vein endothelial cells. Based on the predicted biosynthetic gene cluster of pyripyropene A, two genes (ppb8 and ppb9) encoding two acetyltransferases (ATs) were separately isolated and introduced into the model fungus Aspergillus oryzae, using the protoplast–polyethylene glycol method. The bioconversion of certain predicted intermediates in the transformants revealed the manner by which acetylation occurred in the biosynthetic pathway by the products expressed by these two genes (AT-1 and AT-2). The acetylated products detected by high-performance liquid chromatography (HPLC) in the extracts from AT-1 and AT-2 transformant clones were not present in the extract from the transformant clone with an empty vector. The HLPC charts of each bioconversion study exhibited high peaks at 12, 10.5 and 9 min, respectively. Further ultraviolet absorption and mass spectrometry analyses identified the products as PyE, PyO and PyA, respectively. AT-1 acetylated the C-1 of deacetyl-pyripyropene E (deAc-PyE), while AT-2 played an active role in acetylating the C-11 of 11-deAc-PyO and C-7 of deAc-PyA at two different steps of the biosynthetic pathway. PMID:26019565

  9. Molecular cloning of rice serotonin N-acetyltransferase, the penultimate gene in plant melatonin biosynthesis.

    PubMed

    Kang, Kiyoon; Lee, Kyungjin; Park, Sangkyu; Byeon, Yeong; Back, Kyoungwhan

    2013-08-01

    Because of the absence of an arylalkylamine N-acetyltransferase (AANAT) homolog in the plant genome, the proposal was made that a GCN5-related N-acetyltransferase superfamily gene (GNAT) could be substituted for AANAT. To clone rice serotonin N-acetyltransferase (SNAT), we expressed 31 rice GNAT cDNAs in Escherichia coli and screened SNAT activity by measuring N-acetyltryptamine after application with 1 mm tryptamine. GNAT5 was shown to produce high levels of N-acetyltryptamine in E. coli, suggesting a possible rice SNAT. To confirm SNAT activity, the GNAT5 protein was purified through affinity purification from E. coli culture. The purified recombinant GNAT5 showed high SNAT enzyme activity catalyzing serotonin into N-acetylserotonin. The values for Km and Vmax were 385 μm and 282 pmol/min/mg protein, respectively. An in vitro enzyme assay of purified SNAT showed N-acetylserotonin formation to be proportional to enzyme concentration and time, with peak activity at pH 8.8. High substrate concentrations above 1 mm serotonin inhibited SNAT activity. Finally, the mRNA level of SNAT was higher in shoots than in roots, but it was expressed constitutively, unlike N-acetylserotonin methyltransferase (ASMT), the terminal enzyme in melatonin synthesis. These results suggest that ASMT rather than SNAT is the rate-limiting enzyme of melatonin biosynthesis in plants.

  10. Histone acetyltransferase p300 promotes MKL1-mediated transactivation of catechol-O-methyltransferase gene.

    PubMed

    Liu, Zhipeng; Luo, Xuegang; Liu, Lei; Zhao, Wenwen; Guo, Shu; Guo, Yu; Wang, Nan; He, Hongpeng; Liao, Xinghua; Ma, Wenjian; Zhou, Hao; Zhang, Tongcun

    2013-12-01

    Previous studies have revealed that histone acetyltransferase p300 is recruited to the promoters of certain cardiac and smooth muscle specific genes to enhance the transactivation activity of myocardin, which is a master regulator in cardiovascular differentiation and development. Here, we found that the gene encoding catechol-O-methyltransferase (COMT), an important metabolic enzyme catalyzing the conversion of estrogen, is also a target gene of myocardin-related transcription factors (MRTFs). Megakaryoblastic leukemia 1 (MKL1, also named MRTF-A) and p300 could synergistically augment the expression of COMT gene, increase the metabolic rate of estrogen, and thus reduce the proliferation of MCF-7 breast cancer cells stimulated by estrogen. PMID:24096006

  11. No association between apolipoprotein E or N‐Acetyltransferase 2 gene polymorphisms and age‐related hearing loss

    PubMed Central

    Dawes, Piers; Platt, Hazel; Horan, Michael; Ollier, William; Munro, Kevin; Pendleton, Neil

    2014-01-01

    Objectives/Hypothesis Age‐related hearing loss has a genetic component, but there have been limited genetic studies in this field. Both N‐acetyltransferase 2 and apolipoprotein E genes have previously been associated. However, these studies have either used small sample sizes, examined a limited number of polymorphisms, or have produced conflicting results. Here we use a haplotype tagging approach to determine association with age‐related hearing loss and investigate epistasis between these two genes. Study Design Candidate gene association study of a continuous phenotype. Methods We investigated haplotype tagging single nucleotide polymorphisms in the N‐acetyltransferase 2 gene and the presence/absence of the apolipoprotein E ε4 allele for association with age‐related hearing loss in a cohort of 265 Caucasian elderly volunteers from Greater Manchester, United Kingdom. Hearing phenotypes were generated using principal component analysis of the hearing threshold levels for the better ear (severity, slope, and concavity). Genotype data for the N‐acetyltransferase 2 gene was obtained from existing genome‐wide association study data from the Illumina 610‐Quadv1 chip. Apolipoprotein E genotyping was performed using Sequenom technology. Linear regression analysis was performed using Plink and Stata software. Results No significant associations (P value, > 0.05) were observed between the N‐acetyltransferase 2 or apolipoprotein E gene polymorphisms and any hearing factor. No significant association was observed for epistasis analysis of apolipoprotein E ε4 and the N‐acetyltransferase 2 single nucleotide polymorphism rs1799930 (NAT2*6A). Conclusion We found no evidence to support that either N‐acetyltransferase 2 or apolipoprotein E gene polymorphisms are associated with age‐related hearing loss in a cohort of 265 elderly volunteers. Level of Evidence N/A. Laryngoscope, 125:E33–E38, 2015 PMID:25155015

  12. DNA hybridization and phosphinothricin acetyltransferase gene sequence detection based on zirconia/nanogold film modified electrode

    NASA Astrophysics Data System (ADS)

    Zhang, Wei; Yang, Tao; Jiang, Chen; Jiao, Kui

    2008-05-01

    This study reports a novel electrochemical DNA biosensor based on zirconia (ZrO 2) and gold nanoparticles (NG) film modified glassy carbon electrode (GCE). NG was electrodeposited onto the glassy carbon electrode at 1.5 V, and then zirconia thin film on the NG/GCE was fabricated by cyclic voltammetric method (CV) in an aqueous electrolyte of ZrOCl 2 and KCl at a scan rate of 20 mV/s. DNA probes were attached onto the ZrO 2/NG/GCE due to the strong binding of the phosphate group of DNA with the zirconia film and the excellent biocompatibility of nanogold with DNA. CV and electrochemical impedance spectroscopy (EIS) were used to characterize the modification of the electrode and the probe DNA immobilization. The electrochemical response of the DNA hybridization was measured by differential pulse voltammetry (DPV) using methylene blue (MB) as the electroactive indicator. After the hybridization of DNA probe (ssDNA) with the complementary DNA (cDNA), the cathodic peak current of MB decreased obviously. The difference of the cathodic peak currents of MB between before and after the hybridization of the probe DNA was used as the signal for the detection of the target DNA. The sequence-specific DNA of phosphinothricin acetyltransferase (PAT) gene in the transgenic plants was detected with a detection range from 1.0 × 10 -10 to 1.0 × 10 -6 mol/L, and a detection limit of 3.1 × 10 -11 mol/L.

  13. New O-acetyltransferase-deficient Ames Salmonella strains generated by specific gene disruption.

    PubMed

    Espinosa-Aguirre, J J; Yamada, M; Matsui, K; Watanabe, M; Sofuni, T; Nohmi, T

    1999-02-19

    CoASAc-dependent N-hydroxyarylamine O-acetyltransferase (OAT) is an enzyme involved in the intracellular metabolic activation of N-hydroxyarylamines derived from mutagenic nitroarenes and aromatic amines. The oat gene encoding the enzyme of S. typhimurium TA98 and TA100 was specifically disrupted and the sensitivities of the resulting strains, i.e., YG7130 and YG7126, to mutagens were compared with those of the conventional oat-deficient strains, i.e., TA98/1,8DNP6 and TA100/1,8DNP, respectively. The new oat-deficient strains and the conventional strains exhibited similar sensitivity against most of the chemicals tested: both strains YG7130 and strain TA98/1,8-DNP6 were resistant to mutagenicity by 1,8-dinitropyrene (1, 8-DNP), 1-nitropyrene, 2-amino-6-methyldipyrido[1,2-alpha:3', 2'-d]imidazole (Glu-P-1) and 2-amino-3-methyl-3H-imidazo[4, 5-f]quinoline (IQ); neither strain YG7130 nor strain TA98/1,8-DNP6 was resistant to the mutagenicity of 3-amino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2); strain YG7126 and strain TA100/1,8-DNP were refractory to the mutagenicity of 1,8-DNP. However, the order of the sensitivity against 2-nitrofluorene (2-NF) was TA98>YG7130>TA98/1, 8-DNP6 and TA100>YG7126>TA100/1,8-DNP. Since the strains YG7130 and YG7126 have chloramphenicol resistance (Cmr) gene in place of the chromosomal oat gene for gene disruption, the possible involvement of chloramphenicol acetyltransferase (CAT) encoded by the Cmr gene in the activation of 2-NF was examined. Strikingly, introduction of plasmid pACYC184 carrying the Cmr gene alone substantially enhanced the sensitivity of the conventional oat-deficient strains to 2-NF. These results suggest that the new strains as well as the conventional strains are useful to assess the roles of OAT in the metabolic activation of nitroaromatics and aromatic amines in S. typhimurium, and also that CAT has the ability to activate N-hydroxy aromatic amines to mutagens. PMID:10023048

  14. X-ray crystal structure of ornithine acetyltransferase from the clavulanic acid biosynthesis gene cluster.

    PubMed

    Elkins, Jonathan M; Kershaw, Nadia J; Schofield, Christopher J

    2005-01-15

    The orf6 gene from the clavulanic acid biosynthesis gene cluster encodes an OAT (ornithine acetyltransferase). Similar to other OATs the enzyme has been shown to catalyse the reversible transfer of an acetyl group from N-acetylornithine to glutamate. OATs are Ntn (N-terminal nucleophile) enzymes, but are distinct from the better-characterized Ntn hydrolase enzymes as they catalyse acetyl transfer rather than a hydrolysis reaction. In the present study, we describe the X-ray crystal structure of the OAT, corresponding to the orf6 gene product, to 2.8 A (1 A=0.1 nm) resolution. The larger domain of the structure consists of an alphabetabetaalpha sandwich as in the structures of Ntn hydrolase enzymes. However, differences in the connectivity reveal that OATs belong to a structural family different from that of other structurally characterized Ntn enzymes, with one exception: unexpectedly, the alphabetabetaalpha sandwich of ORF6 (where ORF stands for open reading frame) displays the same fold as an DmpA (L-aminopeptidase D-ala-esterase/amidase from Ochrobactrum anthropi), and so the OATs and DmpA form a new structural subfamily of Ntn enzymes. The structure reveals an alpha2beta2-heterotetrameric oligomerization state in which the intermolecular interface partly defines the active site. Models of the enzyme-substrate complexes suggest a probable oxyanion stabilization mechanism as well as providing insight into how the enzyme binds its two differently charged substrates. PMID:15352873

  15. The histone acetyltransferase p300 inhibitor C646 reduces pro-inflammatory gene expression and inhibits histone deacetylases

    PubMed Central

    van den Bosch, Thea; Boichenko, Alexander; Leus, Niek G. J.; Eleni Ourailidou, Maria; Wapenaar, Hannah; Rotili, Dante; Mai, Antonello; Imhof, Axel; Bischoff, Rainer; Haisma, Hidde J.; Dekker, Frank J.

    2016-01-01

    Lysine acetylations are reversible posttranslational modifications of histone and non-histone proteins that play important regulatory roles in signal transduction cascades and gene expression. Lysine acetylations are regulated by histone acetyltransferases as writers and histone deacetylases as erasers. Because of their role in signal transduction cascades, these enzymes are important players in inflammation. Therefore, applications of histone acetyltransferase inhibitors to reduce inflammatory responses are interesting. Among the few histone acetyltransferase inhibitors described, C646 is one of the most potent (Ki of 0.4 μM for histone acetyltransferase p300). C646 was described to regulate the NF-κB pathway; an important pathway in inflammatory responses, which is regulated by acetylation. Interestingly, this pathway has been implicated in asthma and COPD. Therefore we hypothesized that via regulation of the NF-κB signaling pathway, C646 can inhibit pro-inflammatory gene expression, and have potential for the treatment of inflammatory lung diseases. In line with this, here we demonstrate that C646 reduces pro-inflammatory gene expression in RAW264.7 murine macrophages and murine precision-cut lung slices. To unravel its effects on cellular substrates we applied mass spectrometry and found, counterintuitively, a slight increase in acetylation of histone H3. Based on this finding, and structural features of C646, we presumed inhibitory activity of C646 on histone deacetylases, and indeed found inhibition of histone deacetylases from 7 μM and higher concentrations. This indicates that C646 has potential for further development towards applications in the treatment of inflammation, however, its newly discovered lack of selectivity at higher concentrations needs to be taken into account. PMID:26718586

  16. X-ray crystal structure of ornithine acetyltransferase from the clavulanic acid biosynthesis gene cluster

    PubMed Central

    2004-01-01

    The orf6 gene from the clavulanic acid biosynthesis gene cluster encodes an OAT (ornithine acetyltransferase). Similar to other OATs the enzyme has been shown to catalyse the reversible transfer of an acetyl group from N-acetylornithine to glutamate. OATs are Ntn (N-terminal nucleophile) enzymes, but are distinct from the better-characterized Ntn hydrolase enzymes as they catalyse acetyl transfer rather than a hydrolysis reaction. In the present study, we describe the X-ray crystal structure of the OAT, corresponding to the orf6 gene product, to 2.8 Å (1 Å=0.1 nm) resolution. The larger domain of the structure consists of an αββα sandwich as in the structures of Ntn hydrolase enzymes. However, differences in the connectivity reveal that OATs belong to a structural family different from that of other structurally characterized Ntn enzymes, with one exception: unexpectedly, the αββα sandwich of ORF6 (where ORF stands for open reading frame) displays the same fold as an DmpA (L-aminopeptidase D-ala-esterase/amidase from Ochrobactrum anthropi), and so the OATs and DmpA form a new structural subfamily of Ntn enzymes. The structure reveals an α2β2-heterotetrameric oligomerization state in which the intermolecular interface partly defines the active site. Models of the enzyme–substrate complexes suggest a probable oxyanion stabilization mechanism as well as providing insight into how the enzyme binds its two differently charged substrates. PMID:15352873

  17. Implication of an Aldehyde Dehydrogenase Gene and a Phosphinothricin N-Acetyltransferase Gene in the Diversity of Pseudomonas cichorii Virulence

    PubMed Central

    Tanaka, Masayuki; Wali, Ullah Md; Nakayashiki, Hitoshi; Fukuda, Tatsuya; Mizumoto, Hiroyuki; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

    2011-01-01

    Pseudomonas cichorii harbors the hrp genes. hrp-mutants lose their virulence on eggplant but not on lettuce. A phosphinothricin N-acetyltransferase gene (pat) is located between hrpL and an aldehyde dehydrogenase gene (aldH) in the genome of P. cichorii. Comparison of nucleotide sequences and composition of the genes among pseudomonads suggests a common ancestor of hrp and pat between P. cichorii strains and P. viridiflava strains harboring the single hrp pathogenicity island. In contrast, phylogenetic diversification of aldH corresponded to species diversification amongst pseudomonads. In this study, the involvement of aldH and pat in P. cichorii virulence was analyzed. An aldH-deleted mutant (ΔaldH) and a pat-deleted mutant (Δpat) lost their virulence on eggplant but not on lettuce. P. cichorii expressed both genes in eggplant leaves, independent of HrpL, the transcriptional activator for the hrp. Inoculation into Asteraceae species susceptible to P. cichorii showed that the involvement of hrp, pat and aldH in P. cichorii virulence is independent of each other and has no relationship with the phylogeny of Asteraceae species based on the nucleotide sequences of ndhF and rbcL. It is thus thought that not only the hrp genes but also pat and aldH are implicated in the diversity of P. cichorii virulence on susceptible host plant species. PMID:24704843

  18. Effect of arylamine acetyltransferase Nat3 gene knockout on N-acetylation in the mouse.

    PubMed

    Sugamori, K S; Brenneman, D; Wong, S; Gaedigk, A; Yu, V; Abramovici, H; Rozmahel, R; Grant, D M

    2007-07-01

    Arylamine N-acetyltransferases (NAT) catalyze the biotransformation of many important arylamine drugs and procarcinogens. NAT can either detoxify or activate procarcinogens, complicating the manner in which these enzymes may participate in enhancing or preventing toxic responses to particular agents. Mice possess three NAT isoenzymes: Nat1, Nat2, and Nat3. Whereas Nat1 and Nat2 can efficiently acetylate many arylamines, few substrates appear to be appreciably metabolized by Nat3. We generated a Nat3 knockout mouse strain and used it along with our double Nat1/2(-/-) knockout strain to further investigate the functional role of Nat3. Nat3(-/-) mice showed normal viability and reproductive capacity. Nat3 expression was very low in wild-type animals and completely undetectable in Nat3(-/-) mice. In contrast, greatly elevated expression of Nat3 transcript was observed in Nat1/2(-/-) mice. We used a transcribed marker polymorphism approach to establish that the increased expression of Nat3 in Nat1/2(-/-) mice is a positional artifact of insertion of the phosphoglycerate kinase-neomycin resistance cassette in place of the Nat1/Nat2 gene region and upstream of the intact Nat3 gene, rather than a biological compensatory mechanism. Despite the increase in Nat3 transcript, the N-acetylation of p-aminosalicylate, sulfamethazine, 2-aminofluorene, and 4-aminobiphenyl was undetectable either in vivo or in vitro in Nat1/2(-/-) animals. In parallel, no difference was observed in the in vivo clearance or in vitro metabolism of any of these substrates between wild-type and Nat3(-/-) mice. Thus, Nat3 is unlikely to play a significant role in the N-acetylation of arylamines either in wild-type mice or in mice lacking Nat1 and Nat2 activities. PMID:17403913

  19. The UmGcn5 gene encoding histone acetyltransferase from Ustilago maydis is involved in dimorphism and virulence.

    PubMed

    González-Prieto, Juan Manuel; Rosas-Quijano, Raymundo; Domínguez, Angel; Ruiz-Herrera, José

    2014-10-01

    We isolated a gene encoding a histone acetyltransferase from Ustilago maydis (DC.) Cda., which is orthologous to the Saccharomyces cerevisiae GCN5 gene. The gene was isolated from genomic clones identified by their specific hybridization to a gene fragment obtained by the polymerase chain reaction (PCR). This gene (Umgcn5; um05168) contains an open reading frame (ORF) of 1421bp that encodes a putative protein of 473 amino acids with a Mr. of 52.6kDa. The protein exhibits a high degree of homology with histone acetyltransferases from different organisms. Null a2b2 ΔUmgcn5 mutants were constructed by substitution of the region encoding the catalytic site with a hygromycin B resistance cassette. Null a1b1 ΔUmgcn5 mutants were isolated from genetic crosses of a2b2 ΔUmgcn5 and a1b1 wild-type strains in maize. Mutants displayed a slight reduction in growth rate under different conditions, and were more sensitive than the wild type to stress conditions, but more important, they grew as long mycelial cells, and formed fuzz-like colonies under all conditions where wild-type strains grew in the yeast-like morphology and formed smooth colonies. This phenotype was not reverted by cAMP addition. Mutants were not virulent to maize plants, and were unable to form teliospores. These phenotypic alterations of the mutants were reverted by their transformation with the wild-type gene.

  20. The wheat transcription factor TaGAMyb recruits histone acetyltransferase and activates the expression of a high-molecular-weight glutenin subunit gene.

    PubMed

    Guo, Weiwei; Yang, Hua; Liu, Yongqiang; Gao, Yujiao; Ni, Zhongfu; Peng, Huiru; Xin, Mingming; Hu, Zhaorong; Sun, Qixin; Yao, Yingyin

    2015-10-01

    Glutenin proteins in wheat (Triticum aestivum L.) flour confer unique viscoelastic properties to dough products and, therefore, the concentration and composition of the glutenin proteins determine its end-use value. However, the mechanisms governing the glutenin gene expression remain elusive. In this study, we report that wheat TaGAMyb activates the high-molecular-weight glutenin subunit genes (TaGLU) through recruiting the histone acetyltransferase GCN5. By sequencing the promoters of TaGLU-1 genes from 40 modern wheat cultivars, we identified eight types of TaGAMyb binding motifs and verified these by electrophoretic mobility shift assays. The number of TaGAMyb binding motifs in TaGLU-1 genes is correlated with the abundance of glutenin in different cultivars. Chromatin immunoprecipitation plus polymerase chain reaction (ChIP-PCR) analysis reveals that TaGCN5 directly targets the promoters of TaGLU-1 genes in wheat endosperm. We find that TaGAMyb physically interacts with the wheat histone acetyltransferase TaGCN5 and also interacts with Arabidopsis thaliana AtGCN5. TaGAMyb ectopically expressed in Arabidopsis binds to the TaGLU-1Dy promoter on a TaGLU-1Dy transgene and activates its expression. AtGCN5 also targets the TaGLU-1Dy transgene and is involved in the establishment of acetylation at H3K9 and H3K14. These results demonstrate that TaGAMyb plays a dual role in activating expression of glutenin gene by directly binding to the TaGLU promoter and by recruiting GCN5 to modulate histone acetylation during wheat endosperm development.

  1. The human serotonin N-acetyltransferase (EC 2.3.1.87) gene (AANAT): Structure, chromosomal localization, and tissue expression

    SciTech Connect

    Coon, S.L.; Bernard, M.; Roseboom, P.H.

    1996-05-15

    Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, HGMW-approved symbol AANAT;EC 2.3.1.87) is the penultimate enzyme in melatonin synthesis and controls the night/day rhythm in melatonin production in the vertebrate pineal gland. We have found that the human AA-NAT gene spans {approx}2.5 kb, contains four exons, and is located at chromosome 17q25. The open reading frame encodes a 23.2-kDa protein that is {approx}80% identical to sheep and rat AA-NAT. The AA-NAT transcript ({approx}1 kb) is highly abundant in the pineal gland and is expressed at lower levels in the retina and in the Y79 retinoblastoma cell line. AA-NAT mRNA is also detectable at low levels in several brain regions and the pituitary gland, but not in several peripheral tissues examined. Brain and pituitary AA-NAT could modulate serotonin-dependent aspects of human behavior and pituitary function. 31 refs., 5 figs.

  2. Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean

    PubMed Central

    Guo, Bingfu; Guo, Yong; Hong, Huilong; Jin, Longguo; Zhang, Lijuan; Chang, Ru-Zhen; Lu, Wei; Lin, Min; Qiu, Li-Juan

    2015-01-01

    Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-PCR, and Western blot revealed that target genes have been integrated into genome and expressed effectively at both mRNA and protein levels. Furthermore, the glyphosate tolerance analysis showed that no typical symptom was observed when compared with a glyphosate tolerant line HJ06-698 derived from GR1 transgenic soybean even at fourfold labeled rate of Roundup. Chlorophyll and shikimic acid content analysis of transgenic plant also revealed that these two indexes were not significantly altered after glyphosate application. These results indicated that co-expression of G2-EPSPS and GAT conferred high tolerance to the herbicide glyphosate in soybean. Therefore, combination of tolerant and degraded genes provides a new strategy for developing glyphosate tolerant transgenic crops. PMID:26528311

  3. Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean.

    PubMed

    Guo, Bingfu; Guo, Yong; Hong, Huilong; Jin, Longguo; Zhang, Lijuan; Chang, Ru-Zhen; Lu, Wei; Lin, Min; Qiu, Li-Juan

    2015-01-01

    Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-PCR, and Western blot revealed that target genes have been integrated into genome and expressed effectively at both mRNA and protein levels. Furthermore, the glyphosate tolerance analysis showed that no typical symptom was observed when compared with a glyphosate tolerant line HJ06-698 derived from GR1 transgenic soybean even at fourfold labeled rate of Roundup. Chlorophyll and shikimic acid content analysis of transgenic plant also revealed that these two indexes were not significantly altered after glyphosate application. These results indicated that co-expression of G2-EPSPS and GAT conferred high tolerance to the herbicide glyphosate in soybean. Therefore, combination of tolerant and degraded genes provides a new strategy for developing glyphosate tolerant transgenic crops.

  4. LHX3 Interacts with Inhibitor of Histone Acetyltransferase Complex Subunits LANP and TAF-1β to Modulate Pituitary Gene Regulation

    PubMed Central

    Witzmann, Frank A.; Rhodes, Simon J.

    2013-01-01

    LIM-homeodomain 3 (LHX3) is a transcription factor required for mammalian pituitary gland and nervous system development. Human patients and animal models with LHX3 gene mutations present with severe pediatric syndromes that feature hormone deficiencies and symptoms associated with nervous system dysfunction. The carboxyl terminus of the LHX3 protein is required for pituitary gene regulation, but the mechanism by which this domain operates is unknown. In order to better understand LHX3-dependent pituitary hormone gene transcription, we used biochemical and mass spectrometry approaches to identify and characterize proteins that interact with the LHX3 carboxyl terminus. This approach identified the LANP/pp32 and TAF-1β/SET proteins, which are components of the inhibitor of histone acetyltransferase (INHAT) multi-subunit complex that serves as a multifunctional repressor to inhibit histone acetylation and modulate chromatin structure. The protein domains of LANP and TAF-1β that interact with LHX3 were mapped using biochemical techniques. Chromatin immunoprecipitation experiments demonstrated that LANP and TAF-1β are associated with LHX3 target genes in pituitary cells, and experimental alterations of LANP and TAF-1β levels affected LHX3-mediated pituitary gene regulation. Together, these data suggest that transcriptional regulation of pituitary genes by LHX3 involves regulated interactions with the INHAT complex. PMID:23861948

  5. Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean.

    PubMed

    Guo, Bingfu; Guo, Yong; Hong, Huilong; Jin, Longguo; Zhang, Lijuan; Chang, Ru-Zhen; Lu, Wei; Lin, Min; Qiu, Li-Juan

    2015-01-01

    Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-PCR, and Western blot revealed that target genes have been integrated into genome and expressed effectively at both mRNA and protein levels. Furthermore, the glyphosate tolerance analysis showed that no typical symptom was observed when compared with a glyphosate tolerant line HJ06-698 derived from GR1 transgenic soybean even at fourfold labeled rate of Roundup. Chlorophyll and shikimic acid content analysis of transgenic plant also revealed that these two indexes were not significantly altered after glyphosate application. These results indicated that co-expression of G2-EPSPS and GAT conferred high tolerance to the herbicide glyphosate in soybean. Therefore, combination of tolerant and degraded genes provides a new strategy for developing glyphosate tolerant transgenic crops. PMID:26528311

  6. Identification of a putative acetyltransferase gene, MMP0350, which affects proper assembly of both flagella and pili in the archaeon Methanococcus maripaludis.

    PubMed

    VanDyke, David J; Wu, John; Ng, Sandy Y M; Kanbe, Masaomi; Chaban, Bonnie; Aizawa, Shin-Ichi; Jarrell, Ken F

    2008-08-01

    Glycosylation is a posttranslational modification utilized in all three domains of life. Compared to eukaryotic and bacterial systems, knowledge of the archaeal processes involved in glycosylation is limited. Recently, Methanococcus voltae flagellin proteins were found to have an N-linked trisaccharide necessary for proper flagellum assembly. Current analysis by mass spectrometry of Methanococcus maripaludis flagellin proteins also indicated the attachment of an N-glycan containing acetylated sugars. To identify genes involved in sugar biosynthesis in M. maripaludis, a putative acetyltransferase was targeted for in-frame deletion. Deletion of this gene (MMP0350) resulted in a flagellin molecular mass shift to a size comparable to that expected for underglycosylated or completely nonglycoslyated flagellins, as determined by immunoblotting. Assembled flagellar filaments were not observed by electron microscopy. Interestingly, the deletion also resulted in defective pilus anchoring. Mutant cells with a deletion of MMP0350 had very few, if any, pili attached to the cell surface compared to a nonflagellated but piliated strain. However, pili were obtained from culture supernatants of this strain, indicating that the defect was not in pilus assembly but in stable attachment to the cell surface. Complementation of MMP0350 on a plasmid restored pilus attachment, but it was unable to restore flagellation, likely because the mutant ceased to make detectable flagellin. These findings represent the first report of a biosynthetic gene involved in flagellin glycosylation in archaea. Also, it is the first gene to be associated with pili, linking flagellum and pilus structure and assembly through posttranslational modifications. PMID:18539748

  7. Method to produce acetyldiacylglycerols (ac-TAGs) by expression of an acetyltransferase gene isolated from Euonymus alatus (burning bush)

    DOEpatents

    Durrett, Timothy; Ohlrogge, John; Pollard, Michael

    2016-05-03

    The present invention relates to novel diacylglycerol acyltransferase genes and proteins, and methods of their use. In particular, the invention describes genes encoding proteins having diacylglycerol acetyltransferase activity, specifically for transferring an acetyl group to a diacylglycerol substrate to form acetyl-Triacylglycerols (ac-TAGS), for example, a 3-acetyl-1,2-diacyl-sn-glycerol. The present invention encompasses both native and recombinant wild-type forms of the transferase, as well as mutants and variant forms. The present invention also relates to methods of using novel diacylglycerol acyltransferase genes and proteins, including their expression in transgenic organisms at commercially viable levels, for increasing production of 3-acetyl-1,2-diacyl-sn-glycerols in plant oils and altering the composition of oils produced by microorganisms, such as yeast, by increasing ac-TAG production. Additionally, oils produced by methods of the present inventions comprising genes and proteins are contemplated for use as biodiesel fuel, in polymer production and as naturally produced food oils with reduced calories.

  8. Acetate ester production by Chinese yellow rice wine yeast overexpressing the alcohol acetyltransferase-encoding gene ATF2.

    PubMed

    Zhang, J; Zhang, C; Qi, Y; Dai, L; Ma, H; Guo, X; Xiao, D

    2014-01-01

    Acetate ester, which are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction, are responsible for the fruity character of fermented alcoholic beverages such as Chinese yellow rice wine. Alcohol acetyltransferase (AATase) is currently believed to be the key enzyme responsible for the production of acetate ester. In order to determine the precise role of the ATF2 gene in acetate ester production, an ATF2 gene encoding a type of AATase was overexpressed and the ability of the mutant to form acetate esters (including ethyl acetate, isoamyl acetate, and isobutyl acetate) was investigated. The results showed that after 5 days of fermentation, the concentrations of ethyl acetate, isoamyl acetate, and isobutyl acetate in yellow rice wines fermented with EY2 (pUC-PIA2K) increased to 137.79 mg/L (an approximate 4.9-fold increase relative to the parent cell RY1), 26.68 mg/L, and 7.60 mg/L, respectively. This study confirms that the ATF2 gene plays an important role in the production of acetate ester production during Chinese yellow rice wine fermentation, thereby offering prospects for the development of yellow rice wine yeast starter strains with optimized ester-producing capabilities. PMID:25501183

  9. G9a-mediated methylation of ERα links the PHF20/MOF histone acetyltransferase complex to hormonal gene expression

    PubMed Central

    Zhang, Xi; Peng, Danni; Xi, Yuanxin; Yuan, Chao; Sagum, Cari A.; Klein, Brianna J.; Tanaka, Kaori; Wen, Hong; Kutateladze, Tatiana G.; Li, Wei; Bedford, Mark T.; Shi, Xiaobing

    2016-01-01

    The euchromatin histone methyltransferase 2 (also known as G9a) methylates histone H3K9 to repress gene expression, but it also acts as a coactivator for some nuclear receptors. The molecular mechanisms underlying this activation remain elusive. Here we show that G9a functions as a coactivator of the endogenous oestrogen receptor α (ERα) in breast cancer cells in a histone methylation-independent manner. G9a dimethylates ERα at K235 both in vitro and in cells. Dimethylation of ERαK235 is recognized by the Tudor domain of PHF20, which recruits the MOF histone acetyltransferase (HAT) complex to ERα target gene promoters to deposit histone H4K16 acetylation promoting active transcription. Together, our data suggest the molecular mechanism by which G9a functions as an ERα coactivator. Along with the PHF20/MOF complex, G9a links the crosstalk between ERα methylation and histone acetylation that governs the epigenetic regulation of hormonal gene expression. PMID:26960573

  10. Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene.

    PubMed

    Knowles, Joshua W; Xie, Weijia; Zhang, Zhongyang; Chennamsetty, Indumathi; Chennemsetty, Indumathi; Assimes, Themistocles L; Paananen, Jussi; Hansson, Ola; Pankow, James; Goodarzi, Mark O; Carcamo-Orive, Ivan; Morris, Andrew P; Chen, Yii-Der I; Mäkinen, Ville-Petteri; Ganna, Andrea; Mahajan, Anubha; Guo, Xiuqing; Abbasi, Fahim; Greenawalt, Danielle M; Lum, Pek; Molony, Cliona; Lind, Lars; Lindgren, Cecilia; Raffel, Leslie J; Tsao, Philip S; Schadt, Eric E; Rotter, Jerome I; Sinaiko, Alan; Reaven, Gerald; Yang, Xia; Hsiung, Chao A; Groop, Leif; Cordell, Heather J; Laakso, Markku; Hao, Ke; Ingelsson, Erik; Frayling, Timothy M; Weedon, Michael N; Walker, Mark; Quertermous, Thomas

    2015-04-01

    Decreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 "A" allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol-stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity.

  11. Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene

    PubMed Central

    Knowles, Joshua W.; Xie, Weijia; Zhang, Zhongyang; Chennemsetty, Indumathi; Assimes, Themistocles L.; Paananen, Jussi; Hansson, Ola; Pankow, James; Goodarzi, Mark O.; Carcamo-Orive, Ivan; Morris, Andrew P.; Chen, Yii-Der I.; Mäkinen, Ville-Petteri; Ganna, Andrea; Mahajan, Anubha; Guo, Xiuqing; Abbasi, Fahim; Greenawalt, Danielle M.; Lum, Pek; Molony, Cliona; Lind, Lars; Lindgren, Cecilia; Raffel, Leslie J.; Tsao, Philip S.; Schadt, Eric E.; Rotter, Jerome I.; Sinaiko, Alan; Reaven, Gerald; Yang, Xia; Hsiung, Chao A.; Groop, Leif; Cordell, Heather J.; Laakso, Markku; Hao, Ke; Ingelsson, Erik; Frayling, Timothy M.; Weedon, Michael N.; Walker, Mark; Quertermous, Thomas

    2015-01-01

    Decreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 “A” allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol-stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity. PMID:25798622

  12. Differential expression of histone deacetylase and acetyltransferase genes in gastric cancer and their modulation by trichostatin A.

    PubMed

    Wisnieski, Fernanda; Calcagno, Danielle Queiroz; Leal, Mariana Ferreira; Chen, Elizabeth Suchi; Gigek, Carolina Oliveira; Santos, Leonardo Caires; Pontes, Thaís Brilhante; Rasmussen, Lucas Trevizani; Payão, Spencer Luiz Marques; Assumpção, Paulo Pimentel; Lourenço, Laércio Gomes; Demachki, Sâmia; Artigiani, Ricardo; Burbano, Rommel Rodríguez; Smith, Marília Cardoso

    2014-07-01

    Gastric cancer is still the second leading cause of cancer-related death worldwide, even though its incidence and mortality have declined over the recent few decades. Epigenetic control using histone deacetylase inhibitors, such as trichostatin A (TSA), is a promising cancer therapy. This study aimed to assess the messenger RNA (mRNA) levels of three histone deacetylases (HDAC1, HDAC2, and HDAC3), two histone acetyltransferases (GCN5 and PCAF), and two possible targets of these histone modifiers (MYC and CDKN1A) in 50 matched pairs of gastric tumors and corresponding adjacent nontumors samples from patients with gastric adenocarcinoma, as well as their correlations and their possible associations with clinicopathological features. Additionally, we evaluated whether these genes are sensitive to TSA in gastric cancer cell lines. Our results demonstrated downregulation of HDAC1, PCAF, and CDKN1A in gastric tumors compared with adjacent nontumors (P < 0.05). On the other hand, upregulation of HDAC2, GCN5, and MYC was observed in gastric tumors compared with adjacent nontumors (P < 0.05). The mRNA level of MYC was correlated to HDAC3 and GCN5 (P < 0.05), whereas CDKN1A was correlated to HDAC1 and GCN5 (P < 0.05 and P < 0.01, respectively). In addition, the reduced expression of PCAF was associated with intestinal-type gastric cancer (P = 0.03) and TNM stages I/II (P = 0.01). The increased expression of GCN5 was associated with advanced stage gastric cancer (P = 0.02) and tumor invasion (P = 0.03). The gastric cell lines treated with TSA showed different patterns of histone deacetylase and acetyltransferase mRNA expression, downregulation of MYC, and upregulation of CDKN1A. Our findings suggest that alteration of histone modifier genes play an important role in gastric carcinogenesis, contributing to MYC and CDKN1A deregulation. In addition, all genes studied here are modulated by TSA, although this modulation appears to be dependent of the genetic background of the cell

  13. Synergism between the N-acetyltransferase 2 gene and oxidant exposure increases the risk of idiopathic male infertility.

    PubMed

    Yarosh, Sergey L; Kokhtenko, Elena V; Churnosov, Mikhail I; Ataman, Alexander V; Solodilova, Maria A; Polonikov, Alexey V

    2014-09-01

    N-acetyltransferase (NAT2) is a phase-II xenobiotic-metabolizing enzyme participating in the detoxification of toxic arylamines, aromatic amines and hydrazines. The present study was designed to investigate whether two common single-nucleotide polymorphisms (SNP) of the NAT2 gene (481C>T, rs1799929; 590G>A, rs1799930) are associated with susceptibility to idiopathic male infertility and to assess if the risk is modified by oxidant and antioxidant exposures. A total 430 DNA samples (203 infertile patients and 227 fertile men) were genotyped for the polymorphisms by PCR and restriction fragment length polymorphism. No association was found between the NAT2 polymorphisms and idiopathic male infertility. However, gene-environment interaction analysis revealed that a low-acetylation genotype, 590GA, was significantly associated with increased disease risk in men who had environmental risk factors such as cigarette smoking (OR 1.71, 95% CI 1.02-2.87, P = 0.042), alcohol abuse (OR 2.14, 95% CI 1.08-4.27, P = 0.029) and low fruit/vegetable intake (OR 1.68, 95% CI 1.01-2.79, P = 0.04). This pilot study found, as far as is known for the first time, that the polymorphism 590G>A of NAT2 is a novel genetic marker for susceptibility to idiopathic male infertility, but the risk is potentiated by exposure to various environmental oxidants.

  14. Pineal arylalkylamine N-acetyltransferase (Aanat) gene expression as a target of inflammatory mediators in the chicken.

    PubMed

    Piesiewicz, Aneta; Kedzierska, Urszula; Adamska, Iwona; Usarek, Michal; Zeman, Michal; Skwarlo-Sonta, Krystyna; Majewski, Pawel Marek

    2012-11-01

    Previously, we demonstrated that experimental peritonitis in chickens was attenuated by treatment with exogenous melatonin, while the developing inflammation decreased pineal AANAT activity. This suggested the existence of a bidirectional relationship between the activated immune system and pineal gland function. The aim of the present study was to identify the step(s) in the chicken pineal melatonin biosynthetic pathway that are affected by inflammation. Peritonitis was evoked by i.p. injection of thioglycollate solution, either 2h after the start, or 2h before the end of the light period, and the animals were sacrificed 4h later. The effect of inflammation on the expression of genes encoding enzymes participating in melatonin biosynthesis in the pineal gland, i.e. tryptophan hydroxylase 1 (Tph1), dopa decarboxylase (Ddc), arylalkylamine N-acetyltransferase (Aanat) and acetylserotonin O-methyltransferase (Asmt), was evaluated by qPCR. The pineal and serum melatonin concentration as well as the content of its precursors in the pineal gland were measured, along with the activity of the relevant biosynthetic enzymes. Developing peritonitis caused an increase in the pineal levels of the Tph1 mRNA during the night and the Asmt mRNA during the day, while nocturnal Aanat transcription was reduced. Both the pineal and serum melatonin level and the pineal content of N-acetylserotonin (NAS) were decreased during the night in birds with peritonitis. The amount and activity of pineal AANAT were significantly reduced, while the activity of HIOMT was increased under these experimental conditions. These results indicate that the observed decrease in MEL biosynthesis in chickens with developing inflammation is a result of transcriptional downregulation of the Aanat gene, followed by reduced synthesis and activity of the encoded enzyme.

  15. Heparanase-mediated Loss of Nuclear Syndecan-1 Enhances Histone Acetyltransferase (HAT) Activity to Promote Expression of Genes That Drive an Aggressive Tumor Phenotype*

    PubMed Central

    Purushothaman, Anurag; Hurst, Douglas R.; Pisano, Claudio; Mizumoto, Shuji; Sugahara, Kazuyuki; Sanderson, Ralph D.

    2011-01-01

    Heparanase acts as a master regulator of the aggressive tumor phenotype in part by enhancing expression of proteins known to drive tumor progression (e.g. VEGF, MMP-9, hepatocyte growth factor (HGF), and RANKL). However, the mechanism whereby this enzyme regulates gene expression remains unknown. We previously reported that elevation of heparanase levels in myeloma cells causes a dramatic reduction in the amount of syndecan-1 in the nucleus. Because syndecan-1 has heparan sulfate chains and because exogenous heparan sulfate has been shown to inhibit the activity of histone acetyltransferase (HAT) enzymes in vitro, we hypothesized that the reduction in nuclear syndecan-1 in cells expressing high levels of heparanase would result in increased HAT activity leading to stimulation of protein transcription. We found that myeloma cells or tumors expressing high levels of heparanase and low levels of nuclear syndecan-1 had significantly higher levels of HAT activity when compared with cells or tumors expressing low levels of heparanase. High levels of HAT activity in heparanase-high cells were blocked by SST0001, an inhibitor of heparanase. Restoration of high syndecan-1 levels in heparanase-high cells diminished nuclear HAT activity, establishing syndecan-1 as a potent inhibitor of HAT. Exposure of heparanase-high cells to anacardic acid, an inhibitor of HAT activity, significantly suppressed their expression of VEGF and MMP-9, two genes known to be up-regulated following elevation of heparanase. These results reveal a novel mechanistic pathway driven by heparanase expression, which leads to decreased nuclear syndecan-1, increased HAT activity, and up-regulation of transcription of multiple genes that drive an aggressive tumor phenotype. PMID:21757697

  16. Differential transcription of the human spermidine/spermine N1-acetyltransferase (SSAT) gene in human lung carcinoma cells.

    PubMed Central

    Xiao, L; Casero, R A

    1996-01-01

    The expression of spermidine/spermine N1-acetyltransferase (SSAT), the rate-limiting enzyme in the catabolism of polyamines, is highly regulated by a number of factors including the natural polyamines and their analogues. The phenotype-specific cytotoxicity that occurs in response to a class of polyamine analogues, the diethylpolyamines, is associated with a phenotype-specific superinduction of SSAT in human non-small-cell lung carcinomas, whereas in non-responding cell types, including the small-cell lung carcinomas, the superinduction of SSAT does not occur. In this study, we have investigated the molecular basis of this phenotype-specific SSAT induction in human lung carcinoma cells in response to N1,N12-diethylspermine (BESpm). To facilitate the study of transcriptional regulation, we have cloned and characterized 11 kb of the human SSAT locus, including 3500 bp of the 5' promoter region. Nuclear run-on transcription studies suggest that the initial induction of SSAT results from an increase in the rate of gene transcription. Results from Northern blot analysis and ribonuclease protection assays indicate a differential expression of SSAT mRNA between the analogue-responsive H157 and non-responsive H82 cells. There is no detectable SSAT mRNA in H82 cells, even after a 24-h analogue treatment, whereas SSAT mRNA in H157 cells was detectable by Northern blot analysis and increased more than 100-fold following drug exposure. Furthermore, nuclear run-on transcription assays do not detect any active transcription of SSAT gene in either treated or untreated H82 cells. These results indicate that at least one component of the phenotype-specific induction of SSAT appears to be due to differences in transcriptional regulation of the gene. In addition, mapping of DNase I-hypersensitive sites of the SSAT gene suggest that the cell type-specific promoter/enhancer utilization may control the expression of the SSAT gene in differentially sensitive cell types in vivo. PMID

  17. Transformation of the fungus Absidia glauca by complementation of a methionine-auxotrophic strain affected in the homoserine-acetyltransferase gene

    PubMed Central

    Karimi, Sedighe; Wetzel, Jana; Wöstemeyer, Johannes; Burmester, Anke

    2012-01-01

    Transformation of fungi by complementation of auxotrophs is generally much more reliable than usage of antibiotic resistance markers. In order to establish such a system for the model zygomycete Absidia glauca, a stable methionine auxotrophic mutant was isolated after X-ray mutagenesis of the minus mating type and characterized at the molecular level. The mutant is disrupted in the coding region of the Met2-1 gene, encoding homoserine O-acetyltransferase. The corresponding wild type gene was cloned, sequenced and inserted into appropriate vector plasmids. Transformants are prototrophs and show restored methionine-independent growth, based on complementation by the autonomously replicating plasmids. PMID:23650600

  18. Cloning and characterization of the serotonin N-acetyltransferase-2 gene (SNAT2) in rice (Oryza sativa).

    PubMed

    Byeon, Yeong; Lee, Hyoung Yool; Back, Kyoungwhan

    2016-09-01

    The penultimate enzyme in melatonin synthesis is serotonin N-acetyltransferase (SNAT), which exists as a single copy in mammals and plants. Our recent studies of the Arabidopsis snat-knockout mutant and SNAT RNAi rice (Oryza sativa) plants predicted the presence of at least one other SNAT isogene in plants; that is, the snat-knockout mutant of Arabidopsis and the SNAT RNAi rice plants still produced melatonin, even in the absence or the suppression of SNAT expression. Here, we report a molecular cloning of an SNAT isogene (OsSNAT2) from rice. The mature amino acid sequences of SNAT proteins indicated that OsSNAT2 and OsSNAT1 proteins had 39% identity values and 60% similarity. The Km and Vmax values of the purified recombinant OsSNAT2 were 371 μm and 4700 pmol/min/mg protein, respectively; the enzyme's optimal activity temperature was 45°C. Confocal microscopy showed that the OsSNAT2 protein was localized to both the cytoplasm and chloroplasts. The in vitro enzyme activity of OsSNAT2 was severely inhibited by melatonin, but the activities of sheep SNAT (OaSNAT) and rice OsSNAT1 proteins were not. The enzyme activity of OsSNAT2 was threefold higher than that of OsSNAT1, but 232-fold lower than that of OaSNAT. The OsSNAT1 and OsSNAT2 transcripts were similarly suppressed in rice leaves during the melatonin induction after cadmium treatment. Phylogenetic analyses indicated that OsSNAT1 and OsSNAT2 are distantly related, suggesting that they evolved independently from Cyanobacteria prior to the endosymbiosis event. PMID:27121038

  19. Enhanced morphinan alkaloid production in hairy root cultures of Papaver bracteatum by over-expression of salutaridinol 7-o-acetyltransferase gene via Agrobacterium rhizogenes mediated transformation.

    PubMed

    Sharafi, Ali; Hashemi Sohi, Haleh; Mousavi, Amir; Azadi, Pejman; Dehsara, Bahareh; Hosseini Khalifani, Bahman

    2013-11-01

    Papaver bracteatum is an important medicinal plant valued for its high content of thebaine and an alternative to P. somniferum for benzylisoquinoline alkaloid production. Salutaridinol 7-o-acetyltransferase (SalAT) is a key gene in morphinan alkaloids biosynthesis pathway. Over expression of SalAT gene was used for metabolic engineering in P. bracteatum hairy root cultures. Transcript level of the salutaridinol 7-o-acetyltransferase gene in transgenic hairy root lines increased up to 154 and 128 % in comparison with hairy roots without SalAT over expression and wild type roots, respectively. High performance liquid chromatography analysis showed that the transgenic hairy roots relatively improved levels of thebaine (1.28 % dry weight), codeine (0.02 % dry weight) and morphine (0.03 % dry weight) compared to those hairy roots without SalAT over expression. This suggests that P. bracteatum hairy roots expressing the SalAT gene could be potentially used for the production of valuable morphinan alkaloids.

  20. Cloning, sequencing, and use as a molecular probe of a gene encoding an aminoglycoside 6'-N-acetyltransferase of broad substrate profile.

    PubMed Central

    Terán, F J; Suárez, J E; Mendoza, M C

    1991-01-01

    A gene coding for an aminoglycoside 6'-N-acetyltransferase that was able to modify amikacin was cloned from a plasmid isolated from a clinical strain of Enterobacter cloacae. Sequencing of a 955-bp segment which mediates the modifying activity revealed a single open reading frame of 432 nucleotides that predicted a polypeptide of 144 amino acid residues with a molecular weight of 16,021. Putative ribosomal binding sites and -10 and -35 sequences were located at the 5' end of the gene. The size of the polypeptide was confirmed through minicell analysis of the expression products of plasmids containing the sequence. The use of the gene as a molecular probe revealed its specificity toward strains harboring genes coding for related enzymes. This probe is therefore useful for epidemiological studies. Images PMID:2069376

  1. Characterization of the serine acetyltransferase gene family of Vitis vinifera uncovers differences in regulation of OAS synthesis in woody plants

    PubMed Central

    Tavares, Sílvia; Wirtz, Markus; Beier, Marcel P.; Bogs, Jochen; Hell, Rüdiger; Amâncio, Sara

    2015-01-01

    In higher plants cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL) and represents the last step of the assimilatory sulfate reduction pathway. It is mainly regulated by provision of O-acetylserine (OAS), the nitrogen/carbon containing backbone for fixation of reduced sulfur. OAS is synthesized by Serine acetyltransferase (SERAT), which reversibly interacts with OASTL in the cysteine synthase complex (CSC). In this study we identify and characterize the SERAT gene family of the crop plant Vitis vinifera. The identified four members of the VvSERAT protein family are assigned to three distinct groups upon their sequence similarities to Arabidopsis SERATs. Expression of fluorescently labeled VvSERAT proteins uncover that the sub-cellular localization of VvSERAT1;1 and VvSERAT3;1 is the cytosol and that VvSERAT2;1 and VvSERAT2;2 localize in addition in plastids and mitochondria, respectively. The purified VvSERATs of group 1 and 2 have higher enzymatic activity than VvSERAT3;1, which display a characteristic C-terminal extension also present in AtSERAT3;1. VvSERAT1;1 and VvSERAT2;2 are evidenced to form the CSC. CSC formation activates VvSERAT2;2, by releasing CSC-associated VvSERAT2;2 from cysteine inhibition. Thus, subcellular distribution of SERAT isoforms and CSC formation in cytosol and mitochondria is conserved between Arabidopsis and grapevine. Surprisingly, VvSERAT2;1 lack the canonical C-terminal tail of plant SERATs, does not form the CSC and is almost insensitive to cysteine inhibition (IC50 = 1.9 mM cysteine). Upon sulfate depletion VvSERAT2;1 is strongly induced at the transcriptional level, while transcription of other VvSERATs is almost unaffected in sulfate deprived grapevine cell suspension cultures. Application of abiotic stresses to soil grown grapevine plants revealed isoform-specific induction of VvSERAT2;1 in leaves upon drought, whereas high light- or temperature- stress hardly trigger VvSERAT2;1 transcription. PMID:25741355

  2. Non-syndromic retinitis pigmentosa due to mutations in the mucopolysaccharidosis type IIIC gene, heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT)

    PubMed Central

    Haer-Wigman, Lonneke; Newman, Hadas; Leibu, Rina; Bax, Nathalie M.; Baris, Hagit N; Rizel, Leah; Banin, Eyal; Massarweh, Amir; Roosing, Susanne; Lefeber, Dirk J.; Zonneveld-Vrieling, Marijke N.; Isakov, Ofer; Shomron, Noam; Sharon, Dror; Den Hollander, Anneke I.; Hoyng, Carel B.; Cremers, Frans P.M.; Ben-Yosef, Tamar

    2015-01-01

    Retinitis pigmentosa (RP), the most common form of inherited retinal degeneration, is clinically and genetically heterogeneous and can appear as syndromic or non-syndromic. Mucopolysaccharidosis type IIIC (MPS IIIC) is a lethal disorder, caused by mutations in the heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) gene and characterized by progressive neurological deterioration, with retinal degeneration as a prominent feature. We identified HGSNAT mutations in six patients with non-syndromic RP. Whole exome sequencing (WES) in an Ashkenazi Jewish Israeli RP patient revealed a novel homozygous HGSNAT variant, c.370A>T, which leads to partial skipping of exon 3. Screening of 66 Ashkenazi RP index cases revealed an additional family with two siblings homozygous for c.370A>T. WES in three Dutch siblings with RP revealed a complex HGSNAT variant, c.[398G>C; 1843G>A] on one allele, and c.1843G>A on the other allele. HGSNAT activity levels in blood leukocytes of patients were reduced compared with healthy controls, but usually higher than those in MPS IIIC patients. All patients were diagnosed with non-syndromic RP and did not exhibit neurological deterioration, or any phenotypic features consistent with MPS IIIC. Furthermore, four of the patients were over 60 years old, exceeding by far the life expectancy of MPS IIIC patients. HGSNAT is highly expressed in the mouse retina, and we hypothesize that the retina requires higher HGSNAT activity to maintain proper function, compared with other tissues associated with MPS IIIC, such as the brain. This report broadens the spectrum of phenotypes associated with HGSNAT mutations and highlights the critical function of HGSNAT in the human retina. PMID:25859010

  3. N-hydroxyarylamine O-acetyltransferase of Salmonella typhimurium: proposal for a common catalytic mechanism of arylamine acetyltransferase enzymes.

    PubMed Central

    Watanabe, M; Igarashi, T; Kaminuma, T; Sofuni, T; Nohmi, T

    1994-01-01

    Acetyl-CoA:N-hydroxyarylamine O-acetyltransferase is an enzyme involved in the metabolic activation of N-hydroxyarylamines derived from mutagenic and carcinogenic aromatic amines and nitroarenes. The O-acetyltransferase gene of Salmonella typhimurium has been cloned, and new Ames tester substrains highly sensitive to mutagenic aromatic amines and nitroarenes have been established in our laboratory. The nucleotide sequence of the O-acetyltransferase gene was determined. There was an open reading frame of 843 nucleotides coding for a protein with a calculated molecular weight of 32,177, which was close to the molecular weight of the O-acetyltransferase protein determined by using the maxicell technique. Only the residue of Cys69 in O-acetyltransferase of S. typhimurium and its corresponding residue (Cys68) in N-acetyltransferase of higher organisms were conserved in all acetyltransferase enzymes sequenced so far. The amino acid sequence Arg-Gly-Gly-X-Cys, including the Cys69, was highly conserved. A mutant O-acetyltransferase of S. typhimurium, which contained Ala69 instead of Cys69, no longer showed the activities of O- and N-acetyltransferase. These results suggest that the Cys69 of S. typhimurium and the corresponding cysteine residues of the higher organisms are essential for the enzyme activities as an acetyl-CoA binding site. We propose a new catalytic model of acetyltransferase for S. typhimurium and the higher organisms. PMID:7889864

  4. N-acetyltransferase 2 (NAT2) gene polymorphism as a predisposing factor for phenytoin intoxication in tuberculous meningitis or tuberculoma patients having seizures - A pilot study

    PubMed Central

    Adole, Prashant S.; Kharbanda, Parampreet S.; Sharma, Sadhna

    2016-01-01

    Background & objectives: Simultaneous administration of phenytoin and isoniazid (INH) in tuberculous meningitis (TBM) or tuberculoma patients with seizures results in higher plasma phenytoin level and thus phenytoin intoxication. N-acetyltransferase 2 (NAT2) enzyme catalyses two acetylation reactions in INH metabolism and NAT2 gene polymorphism leads to slow and rapid acetylators. The present study was aimed to evaluate the effect of allelic variants of N-acetyltransferase 2 (NAT2) gene as a predisposing factor for phenytoin toxicity in patients with TBM or tuberculoma having seizures, and taking INH and phenytoin simultaneously. Methods: Sixty patients with TBM or tuberculoma with seizures and taking INH and phenytoin simultaneously for a minimum period of seven days were included in study. Plasma phenytoin was measured by high performance liquid chromatography. NAT2 gene polymorphism was studied using restriction fragment length polymorphism and allele specific PCR. Results: The patients were grouped into those having phenytoin intoxication and those with normal phenytoin level, and also classified as rapid or slow acetylators by NAT2 genotyping. Genotypic analysis showed that of the seven SNPs (single nucleotide polymorphisms) of NAT2 gene studied, six mutations were found to be associated with phenytoin intoxication. For rs1041983 (C282T), rs1799929 (C481T), rs1799931 (G857A), rs1799930 (G590A), rs1208 (A803G) and rs1801280 (T341C) allelic variants, the proportion of homozygous mutant was higher in phenytoin intoxicated group than in phenytoin non-intoxicated group. Interpretation & conclusions: Homozygous mutant allele of NAT2 gene at 481site may act as a predisposing factor for phenytoin intoxication among TBM or tuberculoma patients having seizures. PMID:27488001

  5. Transgenic tobacco simultaneously overexpressing glyphosate N-acetyltransferase and 5-enolpyruvylshikimate-3-phosphate synthase are more resistant to glyphosate than those containing one gene.

    PubMed

    Liu, Yunjun; Cao, Gaoyi; Chen, Rongrong; Zhang, Shengxue; Ren, Yuan; Lu, Wei; Wang, Jianhua; Wang, Guoying

    2015-08-01

    5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) and glyphosate N-acetyltransferase (GAT) can detoxify glyphosate by alleviating the suppression of shikimate pathway. In this study, we obtained transgenic tobacco plants overexpressing AM79 aroA, GAT, and both of them, respectively, to evaluate whether overexpression of both genes could confer transgenic plants with higher glyphosate resistance. The transgenic plants harboring GAT or AM79 aroA, respectively, showed good glyphosate resistance. As expected, the hybrid plants containing both GAT and AM79 aroA exhibited improved glyphosate resistance than the transgenic plants overexpressing only a single gene. When grown on media with high concentration of glyphosate, seedlings containing a single gene were severely inhibited, whereas plants expressing both genes were affected less. When transgenic plants grown in the greenhouse were sprayed with glyphosate, less damage was observed for the plants containing both genes. Metabolomics analysis showed that transgenic plants containing two genes could maintain the metabolism balance better than those containing one gene after glyphosate treatment. Glyphosate treatment did not lead to a huge increase of shikimate contents of tobacco leaves in transgenic plants overexpressing two genes, whereas significant increase of shikimate contents in transgenic plants containing only a single gene was observed. These results demonstrated that pyramiding both aroA and GAT in transgenic plants can enhance glyphosate resistance, and this strategy can be used for the development of transgenic glyphosate-resistant crops.

  6. Isolation of Two Unknown Genes Potentially Involved in Differentiation of the Hematopoietic Pathway, and Studies of Spermidine/Spermine Acetyltransferase Regulation

    SciTech Connect

    Kubera, C.; Gavin, I.; Huberman, E.

    2002-01-01

    Differential display identified a number of candidate genes involved with growth and differentiation in the human leukemia cell lines HL-60 and HL-525. Two of these genes were previously unknown, and one is the gene for the enzyme spermidine/spermine acetyltransferase (SSAT). One of our objectives is to isolate and sequence the unknown genes, 631A1 and 510C1, in order to characterize them and determine their functions. The other is to determine how SSAT is regulated, and look at how the polyamines that SSAT regulates effect macrophage differentiation. By screening the CEM T-cell DNA library and the fetal brain library, we were able to identify clones that had inserts with homology to the 631A1 cDNA probe sequence. The insert was amplified using the polymerase chain reaction (PCR) and is currently being sent to the University of Chicago for automated sequencing. The library screens for 510C1 are currently underway, but hybridization of the 510C1 cDNA probe with nylon membranes containing CEM library phage DNA produced strong signal, indicating the gene is there. SSAT experiments identified that the rate-limiting enzyme that marks the polyamines spermidine and spermine for degradation is regulated by PKC and a transcription factor called Nrf2. The knowledge of regulation and function of these genes involved in macrophage differentiation will provide new insight into this cellular process, potentially making it possible to discover the roots of the problems that cause cancerous diseases.

  7. Expression levels of the yeast alcohol acetyltransferase genes ATF1, Lg-ATF1, and ATF2 control the formation of a broad range of volatile esters.

    PubMed

    Verstrepen, Kevin J; Van Laere, Stijn D M; Vanderhaegen, Bart M P; Derdelinckx, Guy; Dufour, Jean-Pierre; Pretorius, Isak S; Winderickx, Joris; Thevelein, Johan M; Delvaux, Freddy R

    2003-09-01

    Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the known Saccharomyces cerevisiae alcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing strain. Subsequently, the ester formation of the transformants was monitored by headspace gas chromatography and gas chromatography combined with mass spectroscopy (GC-MS). Analysis of the fermentation products confirmed that the expression levels of ATF1 and ATF2 greatly affect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsible for the formation of a broad range of less volatile esters, such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, heptyl acetate, octyl acetate, and phenyl ethyl acetate. With respect to the esters analyzed in this study, Atf2p seemed to play only a minor role compared to Atf1p. The atf1Delta atf2Delta double deletion strain did not form any isoamyl acetate, showing that together, Atf1p and Atf2p are responsible for the total cellular isoamyl alcohol acetyltransferase activity. However, the double deletion strain still produced considerable amounts of certain other esters, such as ethyl acetate (50% of the wild-type strain), propyl acetate (50%), and isobutyl acetate (40%), which provides evidence for the existence of additional, as-yet-unknown ester synthases in the yeast proteome. Interestingly, overexpression of different alleles of ATF1 and ATF2 led to different ester production rates, indicating that differences in the aroma profiles of yeast strains may be partially due to mutations in their ATF genes. PMID:12957907

  8. Molecular Characterization of a Novel N-Acetyltransferase from Chryseobacterium sp.

    PubMed Central

    Yoshida, Kenji; Tanaka, Kosei; Yoshida, Ken-ichi

    2014-01-01

    N-Acetyltransferase from Chryseobacterium sp. strain 5-3B is an acetyl coenzyme A (acetyl-CoA)-dependent enzyme that catalyzes the enantioselective transfer of an acetyl group from acetyl-CoA to the amino group of l-2-phenylglycine to produce (2S)-2-acetylamino-2-phenylacetic acid. We purified the enzyme from strain 5-3B and deduced the N-terminal amino acid sequence. The gene, designated natA, was cloned with two other hypothetical protein genes; the three genes probably form a 2.5-kb operon. The deduced amino acid sequence of NatA showed high levels of identity to sequences of putative N-acetyltransferases of Chryseobacterium spp. but not to other known arylamine and arylalkylamine N-acetyltransferases. Phylogenetic analysis indicated that NatA forms a distinct lineage from known N-acetyltransferases. We heterologously expressed recombinant NatA (rNatA) in Escherichia coli and purified it. rNatA showed high activity for l-2-phenylglycine and its chloro- and hydroxyl-derivatives. The Km and Vmax values for l-2-phenylglycine were 0.145 ± 0.026 mM and 43.6 ± 2.39 μmol · min−1 · mg protein−1, respectively. The enzyme showed low activity for 5-aminosalicylic acid and 5-hydroxytryptamine, which are reported as good substrates of a known arylamine N-acetyltransferase and an arylalkylamine N-acetyltransferase. rNatA had a comparatively broad acyl donor specificity, transferring acyl groups to l-2-phenylglycine and producing the corresponding 2-acetylamino-2-phenylacetic acids (relative activity with acetyl donors acetyl-CoA, propanoyl-CoA, butanoyl-CoA, pentanoyl-CoA, and hexanoyl-CoA, 100:108:122:10:<1). PMID:24375143

  9. Pygo2 associates with MLL2 histone methyltransferase and GCN5 histone acetyltransferase complexes to augment Wnt target gene expression and breast cancer stem-like cell expansion.

    PubMed

    Chen, Jiakun; Luo, Qicong; Yuan, Yuanyang; Huang, Xiaoli; Cai, Wangyu; Li, Chao; Wei, Tongzhen; Zhang, Ludi; Yang, Meng; Liu, Qingfeng; Ye, Guodong; Dai, Xing; Li, Boan

    2010-12-01

    Resent studies have identified Pygopus as a core component of the β-catenin/T-cell factor (TCF)/lymphoid-enhancing factor 1 (LEF) transcriptional activation complex required for the expression of canonical Wg/Wnt target genes in Drosophila. However, the biochemical involvement of mammalian Pygopus proteins in β-catenin/TCF/LEF gene activation remains controversial. In this study, we perform a series of molecular/biochemical experiments to demonstrate that Pygo2 associates with histone-modifying enzymatic complexes, specifically the MLL2 histone methyltransferase (HMT) and STAGA histone acetyltransferase (HAT) complexes, to facilitate their interaction with β-catenin and to augment Wnt1-induced, TCF/LEF-dependent transcriptional activation in breast cancer cells. We identify a critical domain in Pygo2 encompassing the first 47 amino acids that mediates its HMT/HAT interaction. We further demonstrate the importance of this domain in Pygo2's ability to transcriptionally activate both artificial and endogenous Wnt target genes and to expand breast cancer stem-like cells in culture. This work now links mechanistically Pygo2's role in histone modification to its enhancement of the Wnt-dependent transcriptional program and cancer stem-like cell expansion.

  10. CEST MRI reporter genes.

    PubMed

    Liu, Guanshu; Bulte, Jeff W M; Gilad, Assaf A

    2011-01-01

    In recent years, several reporter genes have been developed that can serve as a beacon for non-invasive magnetic resonance imaging (MRI). Here, we provide a brief summary of recent advances in MRI reporter gene technology, as well as detailed "hands-on" protocols for cloning, expression, and imaging of reporter genes based on chemical exchange saturation transfer (CEST).

  11. Promotion of Cell Viability and Histone Gene Expression by the Acetyltransferase Gcn5 and the Protein Phosphatase PP2A in Saccharomyces cerevisiae.

    PubMed

    Petty, Emily L; Lafon, Anne; Tomlinson, Shannon L; Mendelsohn, Bryce A; Pillus, Lorraine

    2016-08-01

    Histone modifications direct chromatin-templated events in the genome and regulate access to DNA sequence information. There are multiple types of modifications, and a common feature is their dynamic nature. An essential step for understanding their regulation, therefore, lies in characterizing the enzymes responsible for adding and removing histone modifications. Starting with a dosage-suppressor screen in Saccharomyces cerevisiae, we have discovered a functional interaction between the acetyltransferase Gcn5 and the protein phosphatase 2A (PP2A) complex, two factors that regulate post-translational modifications. We find that RTS1, one of two genes encoding PP2A regulatory subunits, is a robust and specific high-copy suppressor of temperature sensitivity of gcn5∆ and a subset of other gcn5∆ phenotypes. Conversely, loss of both PP2A(Rts1) and Gcn5 function in the SAGA and SLIK/SALSA complexes is lethal. RTS1 does not restore global transcriptional defects in gcn5∆; however, histone gene expression is restored, suggesting that the mechanism of RTS1 rescue includes restoration of specific cell cycle transcripts. Pointing to new mechanisms of acetylation-phosphorylation cross-talk, RTS1 high-copy rescue of gcn5∆ growth requires two residues of H2B that are phosphorylated in human cells. These data highlight the potential significance of dynamic phosphorylation and dephosphorylation of these deeply conserved histone residues for cell viability. PMID:27317677

  12. Multidrug-resistant Pseudomonas aeruginosa strain that caused an outbreak in a neurosurgery ward and its aac(6')-Iae gene cassette encoding a novel aminoglycoside acetyltransferase.

    PubMed

    Sekiguchi, Jun-ichiro; Asagi, Tsukasa; Miyoshi-Akiyama, Tohru; Fujino, Tomoko; Kobayashi, Intetsu; Morita, Koji; Kikuchi, Yoshihiro; Kuratsuji, Tadatoshi; Kirikae, Teruo

    2005-09-01

    We characterized multidrug-resistant Pseudomonas aeruginosa strains isolated from patients involved in an outbreak of catheter-associated urinary tract infections that occurred in a neurosurgery ward of a hospital in Sendai, Japan. Pulsed-field gel electrophoresis of SpeI-, XbaI-, or HpaI-digested genomic DNAs from the isolates revealed that clonal expansion of a P. aeruginosa strain designated IMCJ2.S1 had occurred in the ward. This strain possessed broad-spectrum resistance to aminoglycosides, beta-lactams, fluoroquinolones, tetracyclines, sulfonamides, and chlorhexidine. Strain IMCJ2.S1 showed a level of resistance to some kinds of disinfectants similar to that of a control strain of P. aeruginosa, ATCC 27853. IMCJ2.S1 contained a novel class 1 integron, In113, in the chromosome but not on a plasmid. In113 contains an array of three gene cassettes of bla(IMP-1), a novel aminoglycoside resistance gene, and the aadA1 gene. The aminoglycoside resistance gene, designated aac(6')-Iae, encoded a 183-amino-acid protein that shared 57.1% identity with AAC(6')-Iq. Recombinant AAC(6')-Iae protein showed aminoglycoside 6'-N-acetyltransferase activity by thin-layer chromatography. Escherichia coli expressing exogenous aac(6')-Iae showed resistance to amikacin, dibekacin, isepamicin, kanamycin, netilmicin, sisomicin, and tobramycin but not to arbekacin, gentamicins, or streptomycin. Alterations of gyrA and parC at the amino acid sequence level were detected in IMCJ2.S1, suggesting that such mutations confer the resistance to fluoroquinolones observed for this strain. These results indicate that P. aeruginosa IMCJ2.S1 has developed multidrug resistance by acquiring resistance determinants, including a novel member of the aac(6')-I family and mutations in drug resistance genes.

  13. Structure and transcriptional regulation of the Nat2 gene encoding for the drug-metabolizing enzyme arylamine N-acetyltransferase type 2 in mice.

    PubMed Central

    Boukouvala, Sotiria; Price, Naomi; Plant, Kathryn E; Sim, Edith

    2003-01-01

    Arylamine N-acetyltransferases (NATs) are polymorphic enzymes, well-known for their role in the metabolism of drugs and carcinogens. Mice have three NAT isoenzymes, of which NAT2 is postulated to be involved in endogenous, as well as xenobiotic, metabolism. To understand expression of the murine Nat2 gene, we have analysed its structure and transcriptional regulation. We have accurately mapped the transcription initiation site 6.5 kb upstream of the coding region of the gene, adjacent to a recently described non-coding exon. Transcription was demonstrated to start from this region in embryonic and adult liver, spleen, submaxillary gland, kidney, brain, thymus, lung and placenta, but not in the heart. Database searches and analyses of cDNA by PCR suggested alternative splicing of the single 6.2 kb intron of Nat2, and determined the position of the polyadenylation signal at 0.44 kb downstream of the coding region of the gene. Examination of the 13 kb sequence flanking the coding and non-coding exons of Nat2 revealed a single promoter, located close to the transcription-initiation site, and indicated regions likely to harbour control elements. The Nat2 promoter consists of an atypical TATA box and a Sp1 [SV40 (simian virus 40) protein 1] box identical with that found in many housekeeping gene promoters. Activity of the Nat2 promoter was severely reduced by deletion or mutation of either of these two elements, whereas the region of the Sp1 box bound cellular protein and resisted DNase I digestion. Finally, the ability of the promoter region to bind cellular protein was reduced by competition with oligonucleotides bearing the Sp1 consensus sequence. PMID:12904181

  14. A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

    SciTech Connect

    Tratschin, J.D.; West, M.H.P.; Sandbank, T.; Carter, B.J.

    1984-10-01

    The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/ (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

  15. Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester, methylphosphonate and phosphorothioate linkages.

    PubMed Central

    Marcus-Sekura, C J; Woerner, A M; Shinozuka, K; Zon, G; Quinnan, G V

    1987-01-01

    Several classes of oligonucleotide antisense compounds of sequence complementary to the start of the mRNA coding sequence for chloramphenicol acetyl transferase (CAT), including methylphosphonate, alkyltriester, and phosphorothioate analogues of DNA, have been compared to "normal" phosphodiester oligonucleotides for their ability to inhibit expression of plasmid-directed CAT gene activity in CV-1 cells. CAT gene expression was inhibited when transfection with plasmid DNA containing the gene for CAT coupled to simian virus 40 regulatory sequences (pSV2CAT) or the human immunodeficiency virus enhancer (pHIVCAT) was carried out in the presence of 30 microM concentrations of analogue. For the oligo-methylphosphonate analogue, inhibition was dependent on both oligomer concentration and chain length. Analogues with phosphodiester linkages that alternated with either methylphosphonate, ethyl phosphotriester, or isopropyl phosphotriester linkages were less effective inhibitors, in that order. The phosphorothioate analogue was about two-times more potent than the oligo-methylphosphonate, which was in turn approximately twice as potent as the normal oligonucleotide. Images PMID:3475677

  16. [Role of gene polymorphisms of phase II of xenobiotic biotransformation from glutathione-S-transferase and N-acetyltransferase families in susceptibility to lung cancer among Mayak workers].

    PubMed

    Rusinova, G G; Azizova, T V; Viazovskaia, N S; Glazkova, I V; Gur'ianov, M Iu; Osovets, S V

    2014-01-01

    An association between polymorphous (allelic) gene variants of phase II of enzymatic xenobiotic biotransformation (EXB) of multigene families of glutathione-S-transferase (GSTs) GSTM1*0, GSTT1*0, GSTP1*B Ile105Val, and N-acetyltransferase (NAT) NAT2*6 590G>A, NAT2*5 481C>T, as well as lung cancer in Mayak workers exposed occupationally to prolonged external γ-rays and internal α-radiation from incorporated 239Pu was studied. Analysis of the population frequency of genotypes and alleles of the studied genes in the cohort of Mayak workers revealed their compliance with the Hardy-Weinberg principle and with the corresponding frequency in the European population. The study was based on the case-control method. A case-group consisted of 49 Mayal workers with a verified diagnosis of lung cancer. The mean total absorbed dose from external γ-rays at the moment of diagnostics was 1.03 Gy; the mean total absorbed dose from internal α-radiation from incorporated 239Pu to lung was 0.35 Gy. Control consisted of 172 Mayak workers matched by the year of birth, gender, and age at the moment of employment at one of the main facilities with no lung cancer registered within the study period. No increase in the relative risk of lung cancer (odds ratio, OR) was revealed among the individuals with deletion variants of genes GSTM1*0 and GSTT1*0 (pp genotype, complete absence of gene products) as compared to the individuals with ww or wp genotype, which was determined in total for these genes (normal or partly decreased gene activity). An increase in OR of lung cancer in 1.849 times (p = 0.239) and in 2.439 times (p = 0.075) was found in the carriers with a complete absence of the product of genes GSTP1*B and NAT2*6 590G>A, correspondingly (pp genotype). A statistically significant decrease in OR of lung cancer was found in the wp genotype carriers of gene GSTP1*B (OR = 0.50, p = 0.041). Three variants of paired combinations of gene alleles were established in the carriers with a

  17. The acetyltransferase p300/CBP-associated factor is a p53 target gene in breast tumor cells.

    PubMed

    Watts, George S; Oshiro, Marc M; Junk, Damian J; Wozniak, Ryan J; Watterson, Summer; Domann, Frederick E; Futscher, Bernard W

    2004-01-01

    p300/CBP-associated factor (PCAF) is a coactivator of the tumor suppressor, p53. PCAF participates in p53's transactivation of target genes through acetylation of both bound p53 and histones within p53 target promoters. Using microarrays, we discovered that PCAF itself is induced by p53 in a panel of breast tumor cell lines. Two p53 mutant breast tumor cell lines, BT-549 and UACC-1179, were chosen for further study of PCAF induction by wild-type p53. PCAF induction following adenoviral transduction of p53 expression was confirmed with real-time polymerase chain reaction in a time course experiment. Chromatin immunoprecipitation experiments then showed that PCAF induction was associated with increased p53 binding to the PCAF promoter, which contains p53 consensus-binding sites. PCAF induction by p53 activity was further demonstrated in wild-type p53 MCF10A cells when PCAF expression was induced following activation of endogenous wild-type p53 with doxorubicin in a dose- and time-dependent manner. Furthermore, the doxorubicin-induced increase in PCAF expression was blocked by pretreatment of the MCF10A cells with siRNA (small interfering RNA) targeted against p53 mRNA. Taken together, the results show that PCAF expression can be induced by wild-type p53.

  18. Expression of a human placental alkaline phosphatase gene in transfected cells: Use as a reporter for studies of gene expression

    SciTech Connect

    Henthorn, P.; Zervos, P.; Raducha, M.; Harris, H.; Kadesch, T.

    1988-09-01

    The human placental alkaline phosphatase gene has been cloned and reintroduced into mammalian cells. When a plasmid carrying the gene under control of the simian virus 40 early promoter (pSV2Apap) is transfected into a variety of different cell types, placental alkaline phosphatase activity can readily be detected by using whole cell suspensions or cell lysates. Alkaline phosphatase activity can also be visualized directly in individual transfected cells by histochemical staining. The gene is appropriate for use as a reporter in studies of gene regulation since its expression is dependent on the presence of exogenous transcription control elements. The overall assay to detect the expression of the gene is quantitative, very rapid, and inexpensive. Cotransfections of cells with pSV2Apap and a related plasmid carrying the bacterial chloramphenicol acetyltransferase gene (pSV2Acat) indicate that transcription of these two genes is detected with roughly the same sensitivity.

  19. Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells.

    PubMed Central

    Capone, J P; Sedivy, J M; Sharp, P A; RajBhandary, U L

    1986-01-01

    We have used oligonucleotide-directed site-specific mutagenesis to convert serine codon 27 of the Escherichia coli chloramphenicol acetyltransferase (cat) gene to UAG, UAA, and UGA nonsense codons. The mutant cat genes, under transcriptional control of the Rous sarcoma virus long terminal repeat, were then introduced into mammalian cells by DNA transfection along with UAG, UAA, and UGA suppressor tRNA genes derived from a human serine tRNA. Assay for CAT enzymatic activity in extracts from such cells allowed us to detect and quantitate nonsense suppression in monkey CV-1 cells and mouse NIH3T3 cells. Using such an assay, we provide the first direct evidence that an opal suppressor tRNA gene is functional in mammalian cells. The pattern of suppression of the three cat nonsense mutations in bacteria suggests that the serine at position 27 of CAT can be replaced by a wide variety of amino acids without loss of enzymatic activity. Thus, these mutant cat genes should be generally useful for the quantitation of suppressor activity of suppressor tRNA genes introduced into cells and possibly for the detection of naturally occurring nonsense suppressors. Images PMID:3023959

  20. The enhancing effect of genistein on apoptosis induced by trichostatin A in lung cancer cells with wild type p53 genes is associated with upregulation of histone acetyltransferase.

    PubMed

    Wu, Tzu-Chin; Lin, Yi-Chin; Chen, Hsiao-Ling; Huang, Pei-Ru; Liu, Shang-Yu; Yeh, Shu-Lan

    2016-02-01

    Genistein has been shown to enhance the antitumor activity of trichostatin A (TSA) in human lung carcinoma A549 cells. However, whether the combined treatment exerts the same effect in other lung cancer cells is unclear. In the present study we first compared the enhancing effect of genistein on the antitumor effect of TSA in ABC-1, NCI-H460 (H460) and A549 cells. Second, we investigated whether the effects of genistein are associated with increased histone/non-histone protein acetylation. We found that the enhancing effect of genistein on cell-growth-arrest in ABC-1 cells (p53 mutant) was less than in A549 and H460 cells. Genistein enhanced TSA induced apoptosis in A549 and H460 cells rather than in ABC-1 cells. After silencing p53 expression in A549 and H460 cells, the enhancing effect of genistein was diminished. In addition, genistein increased TSA-induced histone H3/H4 acetylation in A549 and H460 cells. Genistein also increased p53 acetylation in H460 cells. The inhibitor of acetyltransferase, anacardic acid, diminished the enhancing effect of genistein on all TSA-induced histone/p53 acetylation and apoptosis. Genistein in combination with TSA increased the expression of p300 protein, an acetyltransferase, in A549 and NCI-H460 cells. Furthermore, we demonstrated that genistein also enhanced the antitumor effect of genistein in A549-tumor-bearing mice. Taken together, these results suggest that the enhancing effects of genistein on TSA-induced apoptosis in lung cancer cells were p53-dependent and were associated with histone/non-histone protein acetylation.

  1. Comparative genomic and phylogenetic investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family.

    PubMed

    Glenn, Anthony E; Karagianni, Eleni P; Ulndreaj, Alphantigona; Boukouvala, Sotiria

    2010-07-16

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes characterized in several bacteria and eukaryotic organisms. We report a comprehensive phylogenetic analysis employing an exhaustive dataset of NAT-homologous sequences recovered through inspection of 2445 genomes. We describe the first NAT homologues in viruses, archaea, protists, many fungi and invertebrates, providing complete annotations in line with the consensus nomenclature. Contrary to the NAT genes of vertebrates, introns are commonly found within the homologous coding regions of lower eukaryotes. The NATs of fungi and higher animals are distinctly monophyletic, but evidence supports a mixed phylogeny of NATs among bacteria, protists and possibly some invertebrates.

  2. The Multi-Copy Mouse Gene Sycp3-Like Y-Linked (Sly) Encodes an Abundant Spermatid Protein That Interacts with a Histone Acetyltransferase and an Acrosomal Protein1

    PubMed Central

    Reynard, Louise N.; Cocquet, Julie; Burgoyne, Paul S.

    2009-01-01

    Deletion analysis has established that genes on the Y chromosome are essential for normal sperm production in humans, mice, and Drosophila. In mice, long-arm deletions have an impact on spermiogenesis, with the most extensive deletions resulting in severe sperm head malformations and infertility. Intriguingly, smaller deletions are compatible with fertility but result in a distorted sex ratio in favor of females, and recently it was found that Y long-arm deletions are also associated with a marked upregulation of several X-encoded and Y-encoded spermatid-expressed genes. The mouse Y long arm encodes a number of distinct transcripts, each of which derives from multiple gene copies. Of these multicopy genes, the recently described Sly has been favored as the gene underlying the spermiogenic defects associated with Y long-arm deletions. To assess the candidacy of Sly, the expression of this gene was examined in the testis at the transcript and protein levels. Sly is transcribed after the first meiotic division in secondary spermatocytes and round spermatids and encodes two transcript variants, Sly_v1 and Sly_v2 (proteins referred to as SLY1 and SLY2). We raised an antibody against SLY1 which detected the protein in round and early elongating spermatids, where it is predominantly cytoplasmic. Yeast two-hybrid and coimmunoprecipitation studies demonstrated that SLY1 interacts with the acrosomal protein DKKL1, the histone acetyltransferase KAT5 (also known as TIP60), and the microtubule-associated protein APPBP2. Together, these data suggest SLY1 may be involved in multiple processes during spermiogenesis, including the control of gene expression and the development or function of the acrosome. PMID:19176879

  3. Virtual Ligand Screening of the p300/CBP Histone Acetyltransferase: Identification of a Selective Small Molecule Inhibitor

    PubMed Central

    Bowers, Erin M.; Yan, Gai; Mukherjee, Chandrani; Orry, Andrew; Wang, Ling; Holbert, Marc A.; Crump, Nicholas T.; Hazzalin, Catherine A.; Liszczak, Glen; Yuan, Hua; Larocca, Cecilia; Saldanha, S. Adrian; Abagyan, Ruben; Sun, Yan; Meyers, David J.; Marmorstein, Ronen; Mahadevan, Louis C.; Alani, Rhoda M.; Cole, Philip A.

    2010-01-01

    Summary The histone acetyltransferase (HAT) p300/CBP is a transcriptional coactivator implicated in many gene regulatory pathways and protein acetylation events. While p300 inhibitors have been reported, a potent, selective, and readily available active-site directed small molecule inhibitor is not yet known. Here we use a structure-based, in silico screening approach to identify a commercially available pyrazolone-containing small molecule p300 HAT inhibitor, C646. C646 is a competitive p300 inhibitor with a Ki of 400 nM and is selective versus other acetyltransferases. Studies on site-directed p300 HAT mutants and synthetic modifications of C646 confirm the importance of predicted interactions in conferring potency. Inhibition of histone acetylation and cell growth by C646 in cells validate its utility as a pharmacologic probe and suggest that p300/CBP HAT is a worthy anti-cancer target. PMID:20534345

  4. Nuclear Rho kinase, ROCK2, targets p300 acetyltransferase.

    PubMed

    Tanaka, Toru; Nishimura, Dai; Wu, Ray-Chang; Amano, Mutsuki; Iso, Tatsuya; Kedes, Larry; Nishida, Hiroshi; Kaibuchi, Kozo; Hamamori, Yasuo

    2006-06-01

    Rho-associated coiled-coil protein kinase (ROCK) is an effector for the small GTPase Rho and plays a pivotal role in diverse cellular activities, including cell adhesion, cytokinesis, and gene expression, primarily through an alteration of actin cytoskeleton dynamics. Here, we show that ROCK2 is localized in the nucleus and associates with p300 acetyltransferase both in vitro and in cells. Nuclear ROCK2 is present in a large protein complex and partially cofractionates with p300 by gel filtration analysis. By immunofluorescence, ROCK2 partially colocalizes with p300 in distinct insoluble nuclear structures. ROCK2 phosphorylates p300 in vitro, and nuclear-restricted expression of constitutively active ROCK2 induces p300 phosphorylation in cells. p300 acetyltransferase activity is dependent on its phosphorylation status in cells, and p300 phosphorylation by ROCK2 results in an increase in its acetyltransferase activity in vitro. These observations suggest that nucleus-localized ROCK2 targets p300 for phosphorylation to regulate its acetyltransferase activity.

  5. Infection by bacterial pathogens expressing type III secretion decreases luciferase activity: ramifications for reporter gene studies.

    PubMed

    Savkovic, S D; Koutsouris, A; Wu, G; Hecht, G

    2000-09-01

    Pathogenic microbes influence gene regulation in eukaryotic hosts. Reporter gene studies can define the roles of promoter regulatory sequences. The effect of pathogenic bacteria on reporter genes has not been examined. The aim of this study was to identify which reporter genes are reliable in studies concerning host gene regulation by bacterial pathogens expressing type III secretory systems. Human intestinal epithelial cells, T84, Caco-2 and HT-29, were transfected with plasmids containing luciferase (luc), chloramphenicol acetyltransferase (CAT) or beta-galactosidase (beta-gal) as reporter genes driven by the inducible interleukin-8 (IL-8) or constitutively active simian virus 40 (SV40) promoter. Cells were infected with enteropathogenic E. coli or Salmonella typhimurium, and the reporter activity was assessed. Luc activity significantly decreased following infection, regardless of the promoter. The activity of recombinant luc was nearly ablated by incubation with either EPEC or Salmonella in a cell-free system. Activity was partially preserved by protease inhibitors, and immunoblot analysis showed a decreased amount and molecular weight of recombinant luc, suggesting protein degradation. Neither beta-gal nor CAT activity was altered by infection. Disruption of type III secretion prevented the loss of luc activity. We conclude that CAT or beta-gal, but not luc, can be used as reliable reporter genes to assess the impact of pathogenic microbes, especially those expressing type III secretion on host cell gene regulation.

  6. Structure and Biochemical Characterization of Protein Acetyltransferase from Sulfolobus solfataricus

    SciTech Connect

    Brent, Michael M.; Iwata, Ayaka; Carten, Juliana; Zhao, Kehao; Marmorstein, Ronen

    2009-09-02

    The Sulfolobus solfataricus protein acetyltransferase (PAT) acetylates ALBA, an abundant nonspecific DNA-binding protein, on Lys{sup 16} to reduce its DNA affinity, and the Sir2 deacetylase reverses the modification to cause transcriptional repression. This represents a 'primitive' model for chromatin regulation analogous to histone modification in eukaryotes. We report the 1.84-{angstrom} crystal structure of PAT in complex with coenzyme A. The structure reveals homology to both prokaryotic GNAT acetyltransferases and eukaryotic histone acetyltransferases (HATs), with an additional 'bent helix' proximal to the substrate binding site that might play an autoregulatory function. Investigation of active site mutants suggests that PAT does not use a single general base or acid residue for substrate deprotonation and product reprotonation, respectively, and that a diffusional step, such as substrate binding, may be rate-limiting. The catalytic efficiency of PAT toward ALBA is low relative to other acetyltransferases, suggesting that there may be better, unidentified substrates for PAT. The structural similarity of PAT to eukaryotic HATs combined with its conserved role in chromatin regulation suggests that PAT is evolutionarily related to the eukaryotic HATs.

  7. Effect of Single Nucleotide Polymorphisms in Cytochrome P450 Isoenzyme and N-Acetyltransferase 2 Genes on the Metabolism of Artemisinin-Based Combination Therapies in Malaria Patients from Cambodia and Tanzania

    PubMed Central

    Staehli Hodel, Eva Maria; Csajka, Chantal; Ariey, Frédéric; Guidi, Monia; Kabanywanyi, Abdunoor Mulokozi; Duong, Socheat; Decosterd, Laurent Arthur; Olliaro, Piero; Genton, Blaise

    2013-01-01

    The pharmacogenetics of antimalarial agents are poorly known, although the application of pharmacogenetics might be critical in optimizing treatment. This population pharmacokinetic-pharmacogenetic study aimed at assessing the effects of single nucleotide polymorphisms (SNPs) in cytochrome P450 isoenzyme genes (CYP, namely, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) and the N-acetyltransferase 2 gene (NAT2) on the pharmacokinetics of artemisinin-based combination therapies in 150 Tanzanian patients treated with artemether-lumefantrine, 64 Cambodian patients treated with artesunate-mefloquine, and 61 Cambodian patients treated with dihydroartemisinin-piperaquine. The frequency of SNPs varied with the enzyme and the population. Higher frequencies of mutant alleles were found in Cambodians than Tanzanians for CYP2C9*3, CYP2D6*10 (100C→T), CYP3A5*3, NAT2*6, and NAT2*7. In contrast, higher frequencies of mutant alleles were found in Tanzanians for CYP2D6*17 (1023C→T and 2850C→T), CYP3A4*1B, NAT2*5, and NAT2*14. For 8 SNPs, no significant differences in frequencies were observed. In the genetic-based population pharmacokinetic analyses, none of the SNPs improved model fit. This suggests that pharmacogenetic data need not be included in appropriate first-line treatments with the current artemisinin derivatives and quinolines for uncomplicated malaria in specific populations. However, it cannot be ruled out that our results represent isolated findings, and therefore more studies in different populations, ideally with the same artemisinin-based combination therapies, are needed to evaluate the influence of pharmacogenetic factors on the clearance of antimalarials. PMID:23229480

  8. NF-κB drives the synthesis of melatonin in RAW 264.7 macrophages by inducing the transcription of the arylalkylamine-N-acetyltransferase (AA-NAT) gene.

    PubMed

    Muxel, Sandra Marcia; Pires-Lapa, Marco Antonio; Monteiro, Alex Willian Arantes; Cecon, Erika; Tamura, Eduardo Koji; Floeter-Winter, Lucile Maria; Markus, Regina P

    2012-01-01

    We demonstrate that during inflammatory responses the nuclear factor kappa B (NF-κB) induces the synthesis of melatonin by macrophages and that macrophage-synthesized melatonin modulates the function of these professional phagocytes in an autocrine manner. Expression of a DsRed2 fluorescent reporter driven by regions of the aa-nat promoter, that encodes the key enzyme involved in melatonin synthesis (arylalkylamine-N-acetyltransferase), containing one or two upstream κB binding sites in RAW 264.7 macrophage cell lines was repressed when NF-κB activity was inhibited by blocking its nuclear translocation or its DNA binding activity or by silencing the transcription of the RelA or c-Rel NF-κB subunits. Therefore, transcription of aa-nat driven by NF-κB dimers containing RelA or c-Rel subunits mediates pathogen-associated molecular patterns (PAMPs) or pro-inflammatory cytokine-induced melatonin synthesis in macrophages. Furthermore, melatonin acts in an autocrine manner to potentiate macrophage phagocytic activity, whereas luzindole, a competitive antagonist of melatonin receptors, decreases macrophage phagocytic activity. The opposing functions of NF-κB in the modulation of AA-NAT expression in pinealocytes and macrophages may represent the key mechanism for the switch in the source of melatonin from the pineal gland to immune-competent cells during the development of an inflammatory response.

  9. Inhibition of lysine acetyltransferase KAT3B/p300 activity by a naturally occurring hydroxynaphthoquinone, plumbagin.

    PubMed

    Ravindra, Kodihalli C; Selvi, B Ruthrotha; Arif, Mohammed; Reddy, B A Ashok; Thanuja, Gali R; Agrawal, Shipra; Pradhan, Suman Kalyan; Nagashayana, Natesh; Dasgupta, Dipak; Kundu, Tapas K

    2009-09-01

    Lysine acetyltransferases (KATs), p300 (KAT3B), and its close homologue CREB-binding protein (KAT3A) are probably the most widely studied KATs with well documented roles in various cellular processes. Hence, the dysfunction of p300 may result in the dysregulation of gene expression leading to the manifestation of many disorders. The acetyltransferase activity of p300/CREB-binding protein is therefore considered as a target for new generation therapeutics. We describe here a natural compound, plumbagin (RTK1), isolated from Plumbago rosea root extract, that inhibits histone acetyltransferase activity potently in vivo. Interestingly, RTK1 specifically inhibits the p300-mediated acetylation of p53 but not the acetylation by another acetyltransferase, p300/CREB-binding protein -associated factor, PCAF, in vivo. RTK1 inhibits p300 histone acetyltransferase activity in a noncompetitive manner. Docking studies and site-directed mutagenesis of the p300 histone acetyltransferase domain suggest that a single hydroxyl group of RTK1 makes a hydrogen bond with the lysine 1358 residue of this domain. In agreement with this, we found that indeed the hydroxyl group-substituted plumbagin derivatives lost the acetyltransferase inhibitory activity. This study describes for the first time the chemical entity (hydroxyl group) required for the inhibition of acetyltransferase activity.

  10. Stimulation of chloramphenicol acetyltransferase mRNA translation by reovirus capsid polypeptide sigma 3 in cotransfected COS cells.

    PubMed Central

    Giantini, M; Shatkin, A J

    1989-01-01

    The mammalian reovirus S4 gene has been implicated in the serotype-dependent inhibition of host cell protein synthesis during viral replication in mouse L cells. To examine the effect(s) of this gene on transcription or translation or both, a DNA copy of the serotype 3 S4 gene was inserted into a eucaryotic expression vector. Cotransfection of COS cells with plasmids containing S4 and the reporter gene, chloramphenicol acetyltransferase (CAT), resulted in a marked stimulation of CAT expression, predominantly at the level of translation. The significance of these findings is discussed in relation to the double-stranded-RNA-binding activity of the S4 gene product, polypeptide sigma 3. Images PMID:2724407

  11. Conserved Molecular Interactions within the HBO1 Acetyltransferase Complexes Regulate Cell Proliferation

    PubMed Central

    Avvakumov, Nikita; Lalonde, Marie-Eve; Saksouk, Nehmé; Paquet, Eric; Glass, Karen C.; Landry, Anne-Julie; Doyon, Yannick; Cayrou, Christelle; Robitaille, Geneviève A.; Richard, Darren E.; Yang, Xiang-Jiao; Kutateladze, Tatiana G.

    2012-01-01

    Acetyltransferase complexes of the MYST family with distinct substrate specificities and functions maintain a conserved association with different ING tumor suppressor proteins. ING complexes containing the HBO1 acetylase are a major source of histone H3 and H4 acetylation in vivo and play critical roles in gene regulation and DNA replication. Here, our molecular dissection of HBO1/ING complexes unravels the protein domains required for their assembly and function. Multiple PHD finger domains present in different subunits bind the histone H3 N-terminal tail with a distinct specificity toward lysine 4 methylation status. We show that natively regulated association of the ING4/5 PHD domain with HBO1-JADE determines the growth inhibitory function of the complex, linked to its tumor suppressor activity. Functional genomic analyses indicate that the p53 pathway is a main target of the complex, at least in part through direct transcription regulation at the initiation site of p21/CDKN1A. These results demonstrate the importance of ING association with MYST acetyltransferases in controlling cell proliferation, a regulated link that accounts for the reported tumor suppressor activities of these complexes. PMID:22144582

  12. Chemoproteomic Profiling of Lysine Acetyltransferases Highlights an Expanded Landscape of Catalytic Acetylation

    PubMed Central

    2015-01-01

    Lysine acetyltransferases (KATs) play a critical role in the regulation of gene expression, metabolism, and other key cellular functions. One shortcoming of traditional KAT assays is their inability to study KAT activity in complex settings, a limitation that hinders efforts at KAT discovery, characterization, and inhibitor development. To address this challenge, here we describe a suite of cofactor-based affinity probes capable of profiling KAT activity in biological contexts. Conversion of KAT bisubstrate inhibitors to clickable photoaffinity probes enables the selective covalent labeling of three phylogenetically distinct families of KAT enzymes. Cofactor-based affinity probes report on KAT activity in cell lysates, where KATs exist as multiprotein complexes. Chemical affinity purification and unbiased LC–MS/MS profiling highlights an expanded landscape of orphan lysine acetyltransferases present in the human genome and provides insight into the global selectivity and sensitivity of CoA-based proteomic probes that will guide future applications. Chemoproteomic profiling provides a powerful method to study the molecular interactions of KATs in native contexts and will aid investigations into the role of KATs in cell state and disease. PMID:24836640

  13. Flavour formation in fungi: characterisation of KlAtf, the Kluyveromyces lactis orthologue of the Saccharomyces cerevisiae alcohol acetyltransferases Atf1 and Atf2.

    PubMed

    Van Laere, Stijn D M; Saerens, Sofie M G; Verstrepen, Kevin J; Van Dijck, Patrick; Thevelein, Johan M; Delvaux, Freddy R

    2008-04-01

    Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages, such as beer and wine. In the brewers' yeast Saccharomyces cerevisiae, the major part of these esters is formed by two alcohol acetyltransferases, Atf1 and Atf2. In this paper, the existence of orthologues of these S. cerevisiae alcohol acetyltransferases in several ascomycetous fungi was investigated. Bioinformatic analysis of sequenced fungal genomes revealed the presence of multiple orthologues. The Saccharomyces sensu stricto yeasts all have two genes coding for orthologues. More distantly related fungi like Saccharomyces castelii, Candida glabrata, Kluyveromyces waltii and Kluyveromyces lactis have only one orthologue in their genome. The homology between the identified proteins and the S. cerevisiae alcohol acetyltransferases suggests a role for these orthologues in the aroma-active ester formation. To verify this, the K. lactis orthologue KlAtf was cloned and expressed in S. cerevisiae. Gas chromatographic analysis of small-scale fermentations with the transformant strains showed that, while S. cerevisiae ATF1 overexpression resulted in a substantial increase in acetate ester levels, S. cerevisiae ATF2 and K. lactis ATF overexpression only caused a moderate increase in acetate esters. This study is the first report of the presence of an ester synthesis gene in K. lactis.

  14. Association of Choline Acetyltransferase Gene Polymorphisms (SNPs rs868750G/A, rs1880676G/A, rs2177369G/A and rs3810950G/A) with Alzheimer’s Disease Risk: A Meta-Analysis

    PubMed Central

    Yuan, Hai; Xia, Qing; Ling, Kang; Wang, Xiaotong; Wang, Xiumin; Du, Xunping

    2016-01-01

    Background Epidemiological studies have investigated the role of choline acetyltransferase (ChAT) in Alzheimer’s disease (AD). ChAT gene polymorphisms (SNPs rs868750G/A, rs1880676G/A, rs2177369G/A, and rs3810950G/A) may be associated with the risk of AD. In this meta-analysis, we determined the relationship between the four polymorphisms and the risk of AD. Methods We searched MEDLINE, EMBASE, and HuGEnet databases for studies linking the four polymorphisms with AD risk. We included 16 articles in our meta-analysis to assess the association between the four polymorphisms and susceptibility to AD by calculating the pooled odds ratios (ORs) and 95% confidence intervals (CIs). Results The combined results showed no significant association with rs1880676G/A and rs2177369G/A polymorphisms. The risk of AD (GG+GA versus AA: OR = 0.01, 95%CI = 0.01–0.02, P < 0.05; GG versus GA+AA: OR = 0.85, 95%CI = 0.72–1.00, P = 0.05; GA versus AA: OR = 0.60, 95% CI = 0.37–0.98, P = 0.04) with rs868750G/A polymorphism, or the association of rs3810950G/A polymorphism with AD risk in the overall population (GA versus AA: OR = 0.64, 95% CI = 0.44–0.93, P = 0.02; GG+GA versus AA: OR = 0.62, 95% CI = 0.39–0.97, P = 0.04) or Asian group (GA versus AA: OR = 0.50, 95% CI = 0.32–0.76, P = 0.001, and GG+GA versus AA: OR = 0.46, 95% CI = 0.30–0.09, P = 0.0002) was demonstrated. Conclusions Our meta-analysis suggested that rs1880670G/A, and rs2177369 G/A polymorphisms were not risk factors for AD. However, rs3810950G/A, or rs868750G/A genetic polymorphism was a genetic risk factor for the development of AD. The rs3810950G/A polymorphism had a negative effect on the risk of AD for GA or GG+GA genotypes compared with AA in the overall population or Asians. PMID:27390868

  15. Neisseria meningitidis serogroup A capsular polysaccharide acetyltransferase, methods and compositions

    DOEpatents

    Stephens, David S.; Gudlavalleti, Seshu K.; Tzeng, Yih-Ling; Datta, Anup K.; Carlson, Russell W.

    2011-02-08

    Provided are methods for recombinant production of an O-acetyltransferase and methods for acetylating capsular polysaccharides, especially those of a Serogroup A Neisseria meningitidis using the recombinant O-acetyltransferase, and immunogenic compositions comprising the acetylated capsular polysaccharide.

  16. Oridonin, a novel lysine acetyltransferases inhibitor, inhibits proliferation and induces apoptosis in gastric cancer cells through p53- and caspase-3-mediated mechanisms

    PubMed Central

    Zhang, Juan; Diao, Hua; Li, Guangming; Xu, Ling; Wang, Ting; Wei, Jue; Meng, Wenying; Ma, Jia-Li; Yu, Heguo; Wang, Yu-Gang

    2016-01-01

    Lysine acetylation has been reported to involve in the pathogenesis of multiple diseases including cancer. In our screening study to identify natural compounds with lysine acetyltransferase inhibitor (KATi) activity, oridonin was found to possess acetyltransferase-inhibitory effects on multiple acetyltransferases including P300, GCN5, Tip60, and pCAF. In gastric cancer cells, oridonin treatment inhibited cell proliferation in a concentration-dependent manner and down-regulated the expression of p53 downstream genes, whereas p53 inhibition by PFT-α reversed the antiproliferative effects of oridonin. Moreover, oridonin treatment induced cell apoptosis, increased the levels of activated caspase-3 and caspase-9, and decreased the mitochondrial membrane potential in gastric cancer cells in a concentration-dependent manner. Caspase-3 inhibition by Ac-DEVD-CHO reversed the proapoptosis effect of oridonin. In conclusion, our study identified oridonin as a novel KATi and demonstrated its tumor suppressive effects in gastric cancer cells at least partially through p53-and caspase-3-mediated mechanisms. PMID:26980707

  17. In vitro activity assays for MYST histone acetyltransferases and adaptation for high-throughput inhibitor screening

    PubMed Central

    McCullough, Cheryl E.; Marmorstein, Ronen

    2016-01-01

    Lysine acetylation is a post-translational modification that is carried out by acetyltransferases. The MYST proteins form the largest and most diverse family of acetyltransferases, which regulate gene expression, DNA repair, and cell cycle homeostasis, among other activities, by acetylating both histone and non-histone proteins. This chapter will describe methods for the preparation and biochemical characterization of MYST family acetyltransferases, including protocols for the preparation of recombinant protein, enzyme assays for measuring steady state parameters and binding assays to measure cofactor and inhibitor binding. We also provide details on adapting these assays for high throughput screening for small molecule MYST inhibitors. This chapter seeks to prepare researchers for some hurdles that they may encounter when studying the MYST proteins so that there may be better opportunity to plan appropriate controls and obtain high quality data. PMID:27372752

  18. Comparative investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family among fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes well-characterized in several bacteria and higher eukaryotes. The role of NATs in fungal biology has only recently been investigated. The NAT1 gene of Gibberella moniliformis was the first NAT cloned and characterized from fun...

  19. Function and subcellular localization of Gcn5, a histone acetyltransferase in Candida albicans.

    PubMed

    Chang, Peng; Fan, Xueyi; Chen, Jiangye

    2015-08-01

    Candida albicans is an opportunistic fungal pathogen commonly found in humans. It has the ability to switch reversibly between three growth forms: budding yeast, pseudohypha, and hypha. The transition between yeast and hyphal growth forms is critical for the pathogenesis of C. albicans. During the yeast-to-hypha morphologic transition, gene expression is regulated by transcriptional regulators including histone modifying complexes and chromatin remodeling complexes. We previously reported that Esa1, a catalytic subunit in the histone acetyltransferase complex NuA4, is essential for the hyphal development of C. albicans. In this study, we analyzed the functional roles of Gcn5, a catalytic subunit in the histone acetyltransferase complex SAGA, in C. albicans. Gcn5 is required for the invasive and filamentous growth of C. albicans. Deletion of GCN5 impaired hyphal elongation in sensing serum and attenuated the virulence of C. albicans in a mouse systemic infection model. The C. albicans gcn5/gcn5 mutant cells also exhibited sensitivity to cell wall stress. Functional analysis showed that the HAT domain and Bromodomain in Gcn5 play distinct roles in morphogenesis and cell wall stress response of C. albicans. Our results show that the conserved residue Glu188 is crucial for the Gcn5 HAT activity and for Gcn5 function during filamentous growth. In addition, the subcellular distribution of ectopically expressed GFP-Gcn5 correlates with the different growth states of C. albicans. In stationary phase, Gcn5 accumulated in the nucleus, while during vegetative growth it localized in the cytoplasm in a morpha-independent manner. Our results suggest that the nuclear localization of Gcn5 depends on the existence of its N-terminal NLS and HAT domains.

  20. Primary structure of a chloramphenicol acetyltransferase specified by R plasmids.

    PubMed

    Shaw, W V; Packman, L C; Burleigh, B D; Dell, A; Morris, H R; Hartley, B S

    Naturally occurring isolates of chloramphenicol-resistant bacteria commonly synthesise chloramphenicol acetyltransferase (EC 2.3.28; CAT) in amounts which are sufficient to account for the resistance phenotype and often harbour plasmids which carry the structural gene for CAT. The findings of CAT in such diverse prokaryotes as Proteus mirabilis, Agrobacterium tumefaciens, Streptomyces sp., and a soil Flavobacterium has led to speculation concerning the origin and evolution of the more commonly observed CAT variants specified by plasmids in clinically important bacteria. To provide a more solid basis for studying the evolution and spread of CAT within prokaryotes we chose to determine the complete amino acid sequence of a type I variant of CAT, the variant known to be associated with most F-like plasmids conferring chloramphenicol resistance. The sequence has been determined by combining the results obtained from manual and automated sequential degradation with those obtained by mass spectrometry of peptides generated by enzymatic digestion. The directly determined primary structure is identical with that predicted by the DNA sequence analysis of the chloramphenicol resistance transponson Tn9 known to specify a type I variant of chloramphenicol acetyltransferase.

  1. Targeting of a histone acetyltransferase domain to a promoter enhances protein expression levels in mammalian cells.

    PubMed

    Kwaks, T H J; Sewalt, R G A B; van Blokland, R; Siersma, T J; Kasiem, M; Kelder, A; Otte, A P

    2005-01-12

    Silencing of transfected genes in mammalian cells is a fundamental problem that probably involves the (in)accessibility status of chromatin. A potential solution to this problem is to provide a cell with protein factors that make the chromatin of a promoter more open or accessible for transcription. We tested this by targeting such proteins to different promoters. We found that targeting the p300 histone acetyltransferase (HAT) domain to strong viral or cellular promoters is sufficient to result in higher expression levels of a reporter protein. In contrast, targeting the chromatin-remodeling factor Brahma does not result in stable, higher protein expression levels. The long-term effects of the targeted p300HAT domain on protein expression levels are positively reinforced, when also anti-repressor elements are applied to flank the reporter construct. These elements were previously shown to be potent blockers of chromatin-associated repressors. The simultaneous application of the targeted p300HAT domain and anti-repressor elements conveys long-term stability to protein expression. Whereas no copy number dependency is achieved by targeting of the p300HAT domain alone, copy number dependency is improved when anti-repressor elements are included. We conclude that targeting of protein domains such as HAT domains helps to facilitate expression of transfected genes in mammalian cells. However, the simultaneous application of other genomic elements such as the anti-repressor elements prevents silencing more efficiently.

  2. Choline Acetyltransferase-Deficient Mutants of the Nematode CAENORHABDITIS ELEGANS

    PubMed Central

    Rand, James B.; Russell, Richard L.

    1984-01-01

    We have identified five independent allelic mutations, defining the gene cha-1, that result in decreased choline acetyltransferase (ChAT) activity in Caenorhabditis elegans. Four of the mutant alleles, when homozygous, lead to ChAT reductions of>98%, as well as recessive phenotypes of uncoordinated behavior, small size, slow growth and resistance to cholinesterase inhibitors. Animals homozygous for the fifth allele retain approximately 10% of the wild-type enzyme level; purified enzyme from this mutant has altered Km values for both choline and acetyl-CoA and is more thermolabile than the wild-type enzyme. These qualitative alterations, together with gene dosage data, argue that cha-1 is the structural gene for ChAT. cha-1 has been mapped to the left arm of linkage group IV and is within 0.02 map unit of the gene unc-17, mutant alleles of which lead to all of the phenotypes of cha-1 mutants except for the ChAT deficiency. Extensive complementation studies of cha-1 and unc-17 alleles reveal a complex complementation pattern, suggesting that both loci may be part of a single complex gene. PMID:6698395

  3. Inhibition of p300 histone acetyltransferase activity in palate mesenchyme cells attenuates Wnt signaling via aberrant E-cadherin expression.

    PubMed

    Warner, Dennis R; Smith, Scott C; Smolenkova, Irina A; Pisano, M Michele; Greene, Robert M

    2016-03-01

    p300 is a multifunctional transcriptional coactivator that interacts with numerous transcription factors and exhibits protein/histone acetyltransferase activity. Loss of p300 function in humans and in mice leads to craniofacial defects. In this study, we demonstrated that inhibition of p300 histone acetyltransferase activity with the compound, C646, altered the expression of several genes, including Cdh1 (E-cadherin) in mouse maxillary mesenchyme cells, which are the cells that give rise to the secondary palate. The increased expression of plasma membrane-bound E-cadherin was associated with reduced cytosolic β-catenin, that led to attenuated signaling through the canonical Wnt pathway. Furthermore, C646 reduced both cell proliferation and the migratory ability of these cells. These results suggest that p300 histone acetyltransferase activity is critical for Wnt-dependent palate mesenchymal cell proliferation and migration, both processes that play a significant role in morphogenesis of the palate.

  4. Construction and Use of a Replication-Competent Human Immunodeficiency Virus (HIV-1) that Expresses the Chloramphenicol Acetyltransferase Enzyme

    NASA Astrophysics Data System (ADS)

    Terwilliger, E. F.; Godin, B.; Sodroski, J. G.; Haseltine, W. A.

    1989-05-01

    The construction and properties of an infectious human immunodeficiency virus (HIV) that expresses the bacterial gene chloramphenicol acetyltransferase are described. This virus can be used in vitro to screen for drugs that inhibit HIV infection. The marked virus may also be used to trace the routes of infection from the site of inoculation in animal experiments.

  5. The Candida albicans Histone Acetyltransferase Hat1 Regulates Stress Resistance and Virulence via Distinct Chromatin Assembly Pathways

    PubMed Central

    Tscherner, Michael; Zwolanek, Florian; Jenull, Sabrina; Sedlazeck, Fritz J.; Petryshyn, Andriy; Frohner, Ingrid E.; Mavrianos, John; Chauhan, Neeraj; von Haeseler, Arndt; Kuchler, Karl

    2015-01-01

    Human fungal pathogens like Candida albicans respond to host immune surveillance by rapidly adapting their transcriptional programs. Chromatin assembly factors are involved in the regulation of stress genes by modulating the histone density at these loci. Here, we report a novel role for the chromatin assembly-associated histone acetyltransferase complex NuB4 in regulating oxidative stress resistance, antifungal drug tolerance and virulence in C. albicans. Strikingly, depletion of the NuB4 catalytic subunit, the histone acetyltransferase Hat1, markedly increases resistance to oxidative stress and tolerance to azole antifungals. Hydrogen peroxide resistance in cells lacking Hat1 results from higher induction rates of oxidative stress gene expression, accompanied by reduced histone density as well as subsequent increased RNA polymerase recruitment. Furthermore, hat1Δ/Δ cells, despite showing growth defects in vitro, display reduced susceptibility to reactive oxygen-mediated killing by innate immune cells. Thus, clearance from infected mice is delayed although cells lacking Hat1 are severely compromised in killing the host. Interestingly, increased oxidative stress resistance and azole tolerance are phenocopied by the loss of histone chaperone complexes CAF-1 and HIR, respectively, suggesting a central role for NuB4 in the delivery of histones destined for chromatin assembly via distinct pathways. Remarkably, the oxidative stress phenotype of hat1Δ/Δ cells is a species-specific trait only found in C. albicans and members of the CTG clade. The reduced azole susceptibility appears to be conserved in a wider range of fungi. Thus, our work demonstrates how highly conserved chromatin assembly pathways can acquire new functions in pathogenic fungi during coevolution with the host. PMID:26473952

  6. Structure of Mesorhizobium loti arylamine N-acetyltransferase 1

    SciTech Connect

    Holton, Simon J.; Dairou, Julien; Sandy, James; Rodrigues-Lima, Fernando; Dupret, Jean-Marie; Noble, Martin E. M.; Sim, Edith

    2005-01-01

    The crystal structure of a M. loti arylamine N-acetyltransferase 1 has been determined at 2.0 Å resolution. The arylamine N-acetyltransferase (NAT) enzymes have been found in a broad range of both eukaryotic and prokaryotic organisms. The NAT enzymes catalyse the transfer of an acetyl group from acetyl Co-enzyme A onto the terminal nitrogen of a range of arylamine, hydrazine and arylhydrazine compounds. Recently, several NAT structures have been reported from different prokaryotic sources including Salmonella typhimurium, Mycobacterium smegmatis and Pseudomonas aeruginosa. Bioinformatics analysis of the Mesorhizobium loti genome revealed two NAT paralogues, the first example of multiple NAT isoenzymes in a eubacterial organism. The M. loti NAT 1 enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme was crystallized in 0.5 M Ca(OAc){sub 2}, 16% PEG 3350, 0.1 M Tris–HCl pH 8.5 using the sitting-drop vapour-diffusion method. A data set diffracting to 2.0 Å was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic spacegroup P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.2, b = 97.3, c = 114.3 Å. The structure was refined to a final free-R factor of 24.8%. The structure reveals that despite low sequence homology, M. loti NAT1 shares the common fold as reported in previous NAT structures and exhibits the same catalytic triad of residues (Cys-His-Asp) in the active site.

  7. Frequent coexpression of the vesicular glutamate transporter 1 and 2 genes, as well as coexpression with genes for choline acetyltransferase or glutamic acid decarboxylase in neurons of rat brain.

    PubMed

    Danik, Marc; Cassoly, Estelle; Manseau, Frédéric; Sotty, Florence; Mouginot, Didier; Williams, Sylvain

    2005-08-15

    It is widely believed that expression of the vesicular glutamate transporter genes VGLUT1 and VGLUT2 is restricted to glutamatergic neurons and that the two transporters segregate in different sets of neurons. Using single-cell multiplex RT-PCR (sc-RT-mPCR), we show that VGLUT1 and VGLUT2 mRNAs were coexpressed in most of the sampled neurons from the rat hippocampus, cortex, and cerebellum at postnatal Day (P)14 but not P60. In accordance, changes in VGLUT1 and VGLUT2 mRNA concentrations were found to occur in these and other brain areas between P14 and P60, as revealed by semiquantitative RT-PCR and quantitated by ribonuclease protection assay. VGLUT1 and -2 coexpression in the hippocampal formation is supported further by in situ hybridization data showing that virtually all cells in the CA1-CA3 pyramidal and granule cell layers were highly positive for both transcripts until P14. It was revealed using sc-RT-mPCR that transcripts for VGLUT1 and VGLUT2 were also present in neurons of the cerebellum, striatum, and septum that expressed markers for gamma-aminobutyric acid (GABA)ergic or cholinergic phenotypes, as well as in hippocampal cells containing transcripts for the glial fibrillary acidic protein. Our study suggests that VGLUT1 and VGLUT2 proteins may often transport glutamate into vesicles within the same neuron, especially during early postnatal development, and that they are expressed widely in presumed glutamatergic, GABAergic, and cholinergic neurons, as well as in astrocytes. Furthermore, our study shows that such coexpressing neurons remain in the adult brain and identifies several areas that contain them in both young and adult rats. PMID:15983996

  8. Effect of Increased Yeast Alcohol Acetyltransferase Activity on Flavor Profiles of Wine and Distillates

    PubMed Central

    Lilly, M.; Lambrechts, M. G.; Pretorius, I. S.

    2000-01-01

    The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid

  9. Molecular characterization of the salutaridinol 7-O-acetyltransferase involved in morphine biosynthesis in opium poppy Papaver somniferum.

    PubMed

    Grothe, T; Lenz, R; Kutchan, T M

    2001-08-17

    Salutaridinol 7-O-acetyltransferase (EC ) catalyzes the conversion of the phenanthrene alkaloid salutaridinol to salutaridinol-7-O-acetate, the immediate precursor of thebaine along the morphine biosynthetic pathway. We have isolated a cDNA clone that corresponds to the internal amino acid sequences of the native enzyme purified from a cell suspension culture of opium poppy Papaver somniferum. The recombinant enzyme acetylated the 7-hydroxyl moiety of salutaridinol in the presence of acetyl-CoA. The apparent K(m) value for salutaridinol was determined to be 9 microm and 54 microm for acetyl-CoA. The gene transcript was detected in extracts from Papaver orientale and Papaver bracteatum in addition to P. somniferum. Genomic DNA gel blot analysis indicated that there is likely a single copy of this gene in the P. somniferum genome. The amino acid sequence of salutaridinol 7-O-acetyltransferase is most similar (37% identity) to that of deacetylvindoline acetyltransferase of Catharanthus roseus. Salutaridinol 7-O-acetyltransferase is the second enzyme specific to morphine biosynthesis for which we have isolated a cDNA. Taken together with the other cDNAs cloned encoding norcoclaurine 6-O-methyltransferase, (S)-N-methylcoclaurine 3'-hydroxylase, the cytochrome P-450 reductase, and codeinone reductase, significant progress has been made toward accumulating genes of this pathway to enable the end goal of a biotechnological production of morphinan alkaloids.

  10. The Functional Analysis of Histone Acetyltransferase MOF in Tumorigenesis

    PubMed Central

    Su, Jiaming; Wang, Fei; Cai, Yong; Jin, Jingji

    2016-01-01

    Changes in chromatin structure and heritably regulating the gene expression by epigenetic mechanisms, such as histone post-translational modification, are involved in most cellular biological processes. Thus, abnormal regulation of epigenetics is implicated in the occurrence of various diseases, including cancer. Human MOF (males absent on the first) is a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs). As a catalytic subunit, MOF can form at least two distinct multiprotein complexes (MSL and NSL) in human cells. Both complexes can acetylate histone H4 at lysine 16 (H4K16); however, the NSL complex possesses broader substrate specificity and can also acetylate histone H4 at lysines 5 and 8 (H4K5 and H4K8), suggesting the complexity of the intracellular functions of MOF. Silencing of MOF in cells leads to genomic instability, inactivation of gene transcription, defective DNA damage repair and early embryonic lethality. Unbalanced MOF expression and its corresponding acetylation of H4K16 have been found in certain primary cancer tissues, including breast cancer, medulloblastoma, ovarian cancer, renal cell carcinoma, colorectal carcinoma, gastric cancer, as well as non-small cell lung cancer. In this review, we provide a brief overview of MOF and its corresponding histone acetylation, introduce recent research findings that link MOF functions to tumorigenesis and speculate on the potential role that may be relevant to tumorigenic pathways. PMID:26784169

  11. Arylamine N-acetyltransferases: a structural perspective

    PubMed Central

    Zhou, Xiaotong; Ma, Zhiguo; Dong, Dong; Wu, Baojian

    2013-01-01

    Arylamine N-acetyltransferase (NAT) plays an important role in metabolism and detoxification of many compounds including drugs and environmental carcinogens through chemical modification of the amine group with an acetyl group. Recent studies have suggested that NATs are also involved in cancer cell growth and inhibition of the enzymes may be a potential target for cancer chemotherapy. Three-dimensional (3D) structures are available for NATs from both prokaryotes and eukaryotes. These structures provide valuable insights into the acetylation mechanism, features of the active site and the structural determinants that govern substrate/inhibitor-binding specificity. Such insights allow a more precise understanding of the structure–activity relationships for NAT substrates and inhibitors. Furthermore, the structural elucidation of NATs has generated powerful tools in the design of small molecule inhibitors that should alleviate cancer, based on the important role of the enzyme in cancer biology. PMID:23517104

  12. Resistance to glufosinate is proportional to phosphinothricin acetyltransferase expression and activity in LibertyLink® and WideStrike® Cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    LibertyLink® cotton cultivars are engineered for glufosinate resistance by overexpressing the bar gene that encodes phosphinothricin acetyltransferase (PAT), whereas the insect-resistant WideStrike® cultivars were obtained by using the similar pat gene as a selectable marker. The latter cultivars ca...

  13. Histone acetyltransferase Hbo1 destabilizes estrogen receptor α by ubiquitination and modulates proliferation of breast cancers.

    PubMed

    Iizuka, Masayoshi; Susa, Takao; Takahashi, Yoshihisa; Tamamori-Adachi, Mimi; Kajitani, Takashi; Okinaga, Hiroko; Fukusato, Toshio; Okazaki, Tomoki

    2013-12-01

    The estrogen receptor (ER) is a key molecule for growth of breast cancers. It has been a successful target for treatment of breast cancers. Elucidation of the ER expression mechanism is of importance for designing therapeutics for ER-positive breast cancers. However, the detailed mechanism of ER stability is still unclear. Here, we report that histone acetyltransferase Hbo1 promotes destabilization of estrogen receptor α (ERα) in breast cancers through lysine 48-linked ubiquitination. The acetyltransferase activity of Hbo1 is linked to its activity for ERα ubiquitination. Depletion of Hbo1 and anti-estrogen treatment displayed a potent growth suppression of breast cancer cell line. Hbo1 modulated transcription by ERα. Mutually exclusive expression of Hbo1 and ERα was observed in roughly half of the human breast tumors examined in the present study. Modulation of ER stability by Hbo1 in breast cancers may provide a novel therapeutic possibility.

  14. Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells.

    PubMed

    Pazzagli, M; Devine, J H; Peterson, D O; Baldwin, T O

    1992-08-01

    The aim of this study was to compare three different luciferase genes by placing them in a single reporter vector and expressing them in the same mammalian cell type. The luciferase genes investigated were the luc genes from the fireflies Photinus pyralis (PP) and Luciola mingrelica (LM) and the lux AB5 gene, a translational fusion of the two subunits of the bacterial luciferase from Vibrio harveyi (VH). The chloramphenicol acetyltransferase (CAT) gene was also included in this study for comparison. The performances of the assay methods of the corresponding enzymes were evaluated using reference materials and the results of the expressed enzymes following transfection were calculated using calibration curves. All of the bioluminescent assays possess high reproducibility both within and between the batches (less than 15%). The comparison of the assay methods shows that firefly luciferases have the highest detection sensitivity (0.05 and 0.08 amol for PP and LM, respectively) whereas the VH bacterial luciferase has 5 amol and CAT 100 amol. On the other hand, the transfection of the various plasmids shows that the content of the expressed enzyme within the cells is much higher for CAT than for the other luciferase genes. VH luciferase is expressed at very low levels in mammalian cells due to the relatively high temperature of growing of the mammalian cells that seems to impair the correct folding of the active enzyme. PP and LM luciferases are both expressed at picomolar level but usually 10 to 70 times less in content with respect to CAT within the transfected cells. On the basis of these results the overall improvement in sensitivity related to the use of firefly luciferases as reporter genes in mammalian cells is about 30 to 50 times with respect to that of CAT. PMID:1443530

  15. Cysteine biosynthesis in Lactobacillus casei: identification and characterization of a serine acetyltransferase.

    PubMed

    Bogicevic, Biljana; Berthoud, Hélène; Portmann, Reto; Bavan, Tharmatha; Meile, Leo; Irmler, Stefan

    2016-02-01

    In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT (cysE) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase (metA). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro. Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE.

  16. Cysteine biosynthesis in Lactobacillus casei: identification and characterization of a serine acetyltransferase.

    PubMed

    Bogicevic, Biljana; Berthoud, Hélène; Portmann, Reto; Bavan, Tharmatha; Meile, Leo; Irmler, Stefan

    2016-02-01

    In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT (cysE) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase (metA). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro. Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE. PMID:26790714

  17. FOXP3 interactions with histone acetyltransferase and class II histone deacetylases are required for repression.

    PubMed

    Li, Bin; Samanta, Arabinda; Song, Xiaomin; Iacono, Kathryn T; Bembas, Kathryn; Tao, Ran; Basu, Samik; Riley, James L; Hancock, Wayne W; Shen, Yuan; Saouaf, Sandra J; Greene, Mark I

    2007-03-13

    The forkhead family protein FOXP3 acts as a repressor of transcription and is both an essential and sufficient regulator of the development and function of regulatory T cells. The molecular mechanism by which FOXP3-mediated transcriptional repression occurs remains unclear. Here, we report that transcriptional repression by FOXP3 involves a histone acetyltransferase-deacetylase complex that includes histone acetyltransferase TIP60 (Tat-interactive protein, 60 kDa) and class II histone deacetylases HDAC7 and HDAC9. The N-terminal 106-190 aa of FOXP3 are required for TIP60-FOXP3, HDAC7-FOXP3 association, as well as for the transcriptional repression of FOXP3 via its forkhead domain. FOXP3 can be acetylated in primary human regulatory T cells, and TIP60 promotes FOXP3 acetylation in vivo. Overexpression of TIP60 but not its histone acetyltransferase-deficient mutant promotes, whereas knockdown of endogenous TIP60 relieved, FOXP3-mediated transcriptional repression. A minimum FOXP3 ensemble containing native TIP60 and HDAC7 is necessary for IL-2 production regulation in T cells. Moreover, FOXP3 association with HDAC9 is antagonized by T cell stimulation and can be restored by the protein deacetylation inhibitor trichostatin A, indicating a complex dynamic aspect of T suppressor cell regulation. These findings identify a previously uncharacterized complex-based mechanism by which FOXP3 actively mediates transcriptional repression. PMID:17360565

  18. Inhibition of p300 lysine acetyltransferase activity by luteolin reduces tumor growth in head and neck squamous cell carcinoma (HNSCC) xenograft mouse model

    PubMed Central

    Selvi, Ruthrotha B.; Swaminathan, Amrutha; Chatterjee, Snehajyoti; Shanmugam, Muthu K.; Li, Feng; Ramakrishnan, Gowsica B.; Siveen, Kodappully Sivaraman; Chinnathambi, Arunachalam; Zayed, M. Emam; Alharbi, Sulaiman Ali; Basha, Jeelan; Bhat, Akshay; Vasudevan, Madavan; Dharmarajan, Arunasalam; Sethi, Gautam; Kundu, Tapas K.

    2015-01-01

    Chromatin acetylation is attributed with distinct functional relevance with respect to gene expression in normal and diseased conditions thereby leading to a topical interest in the concept of epigenetic modulators and therapy. We report here the identification and characterization of the acetylation inhibitory potential of an important dietary flavonoid, luteolin. Luteolin was found to inhibit p300 acetyltransferase with competitive binding to the acetyl CoA binding site. Luteolin treatment in a xenografted tumor model of head and neck squamous cell carcinoma (HNSCC), led to a dramatic reduction in tumor growth within 4 weeks corresponding to a decrease in histone acetylation. Cells treated with luteolin exhibit cell cycle arrest and decreased cell migration. Luteolin treatment led to an alteration in gene expression and miRNA profile including up-regulation of p53 induced miR-195/215, let7C; potentially translating into a tumor suppressor function. It also led to down-regulation of oncomiRNAs such as miR-135a, thereby reflecting global changes in the microRNA network. Furthermore, a direct correlation between the inhibition of histone acetylation and gene expression was established using chromatin immunoprecipitation on promoters of differentially expressed genes. A network of dysregulated genes and miRNAs was mapped along with the gene ontology categories, and the effects of luteolin were observed to be potentially at multiple levels: at the level of gene expression, miRNA expression and miRNA processing. PMID:26517526

  19. HAG3, a Histone Acetyltransferase, Affects UV-B Responses by Negatively Regulating the Expression of DNA Repair Enzymes and Sunscreen Content in Arabidopsis thaliana.

    PubMed

    Fina, Julieta P; Casati, Paula

    2015-07-01

    Histone acetylation is regulated by histone acetyltransferases and deacetylases. In Arabidopsis, there are 12 histone acetyltransferases and 18 deacetylases. Histone acetyltransferases are organized in four families: the GNAT/HAG, the MYST, the p300/CBP and the TAFII250 families. Previously, we demonstrated that Arabidopsis mutants in the two members of the MYST acetyltransferase family show increased DNA damage after UV-B irradiation. To investigate further the role of other histone acetyltransferases in UV-B responses, a putative role for enzymes of the GNAT family, HAG1, HAG2 and HAG3, was analyzed. HAG transcripts are not UV-B regulated; however, hag3 RNA interference (RNAi) transgenic plants show a lower inhibition of leaf and root growth by UV-B, higher levels of UV-B-absorbing compounds and less UV-B-induced DNA damage than Wassilewskija (Ws) plants, while hag1 RNAi transgenic plants and hag2 mutants do not show significant differences from wild-type plants. Transcripts for UV-B-regulated genes are highly expressed under control conditions in the absence of UV-B in hag3 RNAi transgenic plants, suggesting that the higher UV-B tolerance may be due to increased levels of proteins that participate in UV-B responses. Together, our data provide evidence that HAG3, directly or indirectly, participates in UV-B-induced DNA damage repair and signaling.

  20. Synthesis of 4′-aminopantetheine and derivatives to probe aminoglycoside N-6′-acetyltransferase

    PubMed Central

    Yan, Xuxu; Akinnusi, T. Olukayode; Larsen, Aaron T.; Auclair, Karine

    2011-01-01

    Summary A convenient synthesis of 4′-aminopantetheine from commercial D-pantethine is reported. The amino group was introduced by reductive amination in order to avoid substitution at a sterically congested position. Derivatives of 4′-aminopantetheine were also prepared to evaluate the effect of O-to-N substitution on inhibitors of the resistance-causing enzyme aminoglycoside N-6′-acetyltransferase. The biological results combined with docking studies indicate that in spite of its reported unusual flexibility and ability to adopt different folds, this enzyme is highly specific for AcCoA. PMID:21225062

  1. Histone deacetylase inhibitors modulate the transcriptional regulation of guanylyl cyclase/natriuretic peptide receptor-a gene: interactive roles of modified histones, histone acetyltransferase, p300, AND Sp1.

    PubMed

    Kumar, Prerna; Tripathi, Satyabha; Pandey, Kailash N

    2014-03-01

    Atrial natriuretic peptide (ANP) binds guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA) and produces the intracellular second messenger, cGMP, which regulates cardiovascular homeostasis. We sought to determine the function of histone deacetylases (HDACs) in regulating Npr1 (coding for GC-A/NPRA) gene transcription, using primary mouse mesangial cells treated with class-specific HDAC inhibitors (HDACi). Trichostatin A, a pan inhibitor, and mocetinostat (MGCD0103), a class I HDAC inhibitor, significantly enhanced Npr1 promoter activity (by 8- and 10-fold, respectively), mRNA levels (4- and 5.3-fold, respectively), and NPRA protein (2.7- and 3.5-fold, respectively). However, MC1568 (class II HDAC inhibitor) had no discernible effect. Overexpression of HDAC1 and HDAC2 significantly attenuated Npr1 promoter activity, whereas HDAC3 and HDAC8 had no effect. HDACi-treated cultured cells in vitro and intact animals in vivo showed significantly reduced binding of HDAC1 and -2 and increased accumulation of acetylated H3-K9/14 and H4-K12 at the Npr1 promoter. Deletional analyses of the Npr1 promoter along with ectopic overexpression and inhibition of Sp1 confirmed that HDACi-induced Npr1 gene transcription is accomplished by Sp1 activation. Furthermore, HDACi attenuated the interaction of Sp1 with HDAC1/2 and promoted Sp1 association with p300 and p300/cAMP-binding protein-associated factor; it also promoted the recruitment of p300 and p300/cAMP-binding protein-associated factor to the Npr1 promoter. Our results demonstrate that trichostatin A and MGCD0103 enhanced Npr1 gene expression through inhibition of HDAC1/2 and increased both acetylation of histones (H3-K9/14, H4-K12) and Sp1 by p300, and their recruitment to Npr1 promoter. Our findings define a novel epigenetic regulatory mechanism that governs Npr1 gene transcription.

  2. An Acetyltransferase Conferring Tolerance to Toxic Aromatic Amine Chemicals

    PubMed Central

    Martins, Marta; Rodrigues-Lima, Fernando; Dairou, Julien; Lamouri, Aazdine; Malagnac, Fabienne; Silar, Philippe; Dupret, Jean-Marie

    2009-01-01

    Aromatic amines (AA) are a major class of environmental pollutants that have been shown to have genotoxic and cytotoxic potentials toward most living organisms. Fungi are able to tolerate a diverse range of chemical compounds including certain AA and have long been used as models to understand general biological processes. Deciphering the mechanisms underlying this tolerance may improve our understanding of the adaptation of organisms to stressful environments and pave the way for novel pharmaceutical and/or biotechnological applications. We have identified and characterized two arylamine N-acetyltransferase (NAT) enzymes (PaNAT1 and PaNAT2) from the model fungus Podospora anserina that acetylate a wide range of AA. Targeted gene disruption experiments revealed that PaNAT2 was required for the growth and survival of the fungus in the presence of toxic AA. Functional studies using the knock-out strains and chemically acetylated AA indicated that tolerance of P. anserina to toxic AA was due to the N-acetylation of these chemicals by PaNAT2. Moreover, we provide proof-of-concept remediation experiments where P. anserina, through its PaNAT2 enzyme, is able to detoxify the highly toxic pesticide residue 3,4-dichloroaniline in experimentally contaminated soil samples. Overall, our data show that a single xenobiotic-metabolizing enzyme can mediate tolerance to a major class of pollutants in a eukaryotic species. These findings expand the understanding of the role of xenobiotic-metabolizing enzyme and in particular of NATs in the adaptation of organisms to their chemical environment and provide a basis for new systems for the bioremediation of contaminated soils. PMID:19416981

  3. Regulation of arylalkylamine N-acetyltransferase (AANAT) in the retina.

    PubMed

    Tosini, Gianluca; Chaurasia, Shyam S; Michael Iuvone, P

    2006-01-01

    Melatonin synthesis in retinal photoreceptors is under photic and circadian control and is regulated primarily by changes in the activity of arylalkylamine N-acetyltransferase (AANAT). Previous investigations demonstrated that Aanat transcripts are predominantly expressed in the photoreceptor cells. AANAT activity is high at night and low during the day, and illumination of the retina during the night induces rapid reduction in the activity of this enzyme. The enzyme is subject to both transcriptional and post-translational regulatory mechanisms. AANAT transcription is regulated directly by the circadian clock via the E-box present in the promoter region of the gene; the photic environment and circadian clock also influence AANAT transcription via cAMP-responsive elements. The stability of AANAT is regulated by cAMP, and light, which decreases cAMP levels in photoreceptor cells, results in rapid degradation of AANAT protein by proteasomal proteolysis. The circadian rhythm in the levels of Aanat mRNA in the rat retina persists after the suprachiasmatic nucleus (SCN) of the hypothalamus has been lesioned, indicative of its relative independence from the master clock in the brain. In non-mammalian vertebrates, the retinal clock controlling melatonin synthesis is in photoreceptor cells, but it has not been definitively localized in mammals. Several studies have also shown that dopamine plays an important role in the regulation of AANAT activity by acting via D2/D4-like receptors that are present on the photoreceptors. Finally, it is important to mention that AANAT, in addition to its role in melatonin synthesis, may play a detoxification role in the vertebrate retina by acetylating arylalkylamines that may react with retinaldehyde.

  4. A new arylalkylamine N-acetyltransferase in silkworm (Bombyx mori) affects integument pigmentation.

    PubMed

    Long, Yaohang; Li, Jiaorong; Zhao, Tianfu; Li, Guannan; Zhu, Yong

    2015-04-01

    Dopamine is a precursor for melanin synthesis. Arylalkylamine N-acetyltransferase (AANAT) is involved in the melatonin formation in insects because it could catalyze the transformation from dopamine to dopamine-N-acetyldopamine. In this study, we identified a new AANAT gene in the silkworm (Bombyx mori) and assessed its role in the silkworm. The cDNA of this gene encodes 233 amino acids that shares 57 % amino acid identity with the Bm-iAANAT protein. We thus refer to this gene as Bm-iAANAT2. To investigate the role of Bm-iAANAT2, we constructed a transgenic interference system using a 3xp3 promoter to suppress the expression of Bm-iAANAT2 in the silkworm. We observed that melanin deposition occurs in the head and integument in transgenic lines. To verify the melanism pattern, dopamine content and the enzyme activity of AANAT were determined by high-performance liquid chromatography (HPLC). We found that an increase in dopamine levels affects melanism patterns on the heads of transgenic B. mori. A reduction in the enzyme activity of AANAT leads to changes in dopamine levels. We analyzed the expression of the Bm-iAANAT2 genes by qPCR and found that the expression of Bm-iAANAT2 gene is significantly lower in transgenic lines. Our results lead us to conclude that Bm-iAANAT2 is a new arylalkylamine N-acetyltransferase gene in the silkworm and is involved in the metabolism of the dopamine to avoid the generation of melanin.

  5. N-Alpha-Acetyltransferases and Regulation of CFTR Expression.

    PubMed

    Vetter, Ali J; Karamyshev, Andrey L; Patrick, Anna E; Hudson, Henry; Thomas, Philip J

    2016-01-01

    The majority of cystic fibrosis (CF)-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to the misfolding, mistrafficking, and degradation of the mutant protein. Inhibition of degradation does not effectively increase the amount of trafficking competent CFTR, but typically leads to increased ER retention of misfolded forms. Thus, the initial off pathway steps occur early in the processing of the protein. To identify proteins that interact with these early forms of CFTR, in vitro crosslink experiments identified cotranslational partners of the nascent chain of the severe misfolded mutant, G85E CFTR. The mutant preferentially interacts with a subunit of an N-alpha-acetyltransferase A. Based on recent reports that acetylation of the N-termini of some N-end rule substrates control their ubiquitination and subsequent degradation, a potential role for this modification in regulation of CFTR expression was assessed. Knockdown experiments identified two complexes, which affect G85E CFTR proteins levels, NatA and NatB. Effects of the knockdowns on mRNA levels, translation rates, and degradation rates established that the two complexes regulate G85E CFTR through two separate mechanisms. NatA acts indirectly by regulating transcription levels and NatB acts through a previously identified, but incompletely understood posttranslational mechanism. This regulation did not effect trafficking of G85E CFTR, which remains retained in the ER, nor did it alter the degradation rate of CFTR. A mutation predicted to inhibit N-terminal acetylation of CFTR, Q2P, was without effect, suggesting neither system acts directly on CFTR. These results contradict the prediction that N-terminal acetylation of CFTR determines its fitness as a proteasome substrate, but rather NatB plays a role in the conformational maturation of CFTR in the ER through actions on an unidentified protein. PMID:27182737

  6. N-Alpha-Acetyltransferases and Regulation of CFTR Expression.

    PubMed

    Vetter, Ali J; Karamyshev, Andrey L; Patrick, Anna E; Hudson, Henry; Thomas, Philip J

    2016-01-01

    The majority of cystic fibrosis (CF)-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to the misfolding, mistrafficking, and degradation of the mutant protein. Inhibition of degradation does not effectively increase the amount of trafficking competent CFTR, but typically leads to increased ER retention of misfolded forms. Thus, the initial off pathway steps occur early in the processing of the protein. To identify proteins that interact with these early forms of CFTR, in vitro crosslink experiments identified cotranslational partners of the nascent chain of the severe misfolded mutant, G85E CFTR. The mutant preferentially interacts with a subunit of an N-alpha-acetyltransferase A. Based on recent reports that acetylation of the N-termini of some N-end rule substrates control their ubiquitination and subsequent degradation, a potential role for this modification in regulation of CFTR expression was assessed. Knockdown experiments identified two complexes, which affect G85E CFTR proteins levels, NatA and NatB. Effects of the knockdowns on mRNA levels, translation rates, and degradation rates established that the two complexes regulate G85E CFTR through two separate mechanisms. NatA acts indirectly by regulating transcription levels and NatB acts through a previously identified, but incompletely understood posttranslational mechanism. This regulation did not effect trafficking of G85E CFTR, which remains retained in the ER, nor did it alter the degradation rate of CFTR. A mutation predicted to inhibit N-terminal acetylation of CFTR, Q2P, was without effect, suggesting neither system acts directly on CFTR. These results contradict the prediction that N-terminal acetylation of CFTR determines its fitness as a proteasome substrate, but rather NatB plays a role in the conformational maturation of CFTR in the ER through actions on an unidentified protein.

  7. Human NAT10 Is an ATP-dependent RNA Acetyltransferase Responsible for N4-Acetylcytidine Formation in 18 S Ribosomal RNA (rRNA)*

    PubMed Central

    Ito, Satoshi; Horikawa, Sayuri; Suzuki, Tateki; Kawauchi, Hiroki; Tanaka, Yoshikazu; Suzuki, Takeo; Suzuki, Tsutomu

    2014-01-01

    Human N-acetyltransferase 10 (NAT10) is known to be a lysine acetyltransferase that targets microtubules and histones and plays an important role in cell division. NAT10 is highly expressed in malignant tumors, and is also a promising target for therapies against laminopathies and premature aging. Here we report that NAT10 is an ATP-dependent RNA acetyltransferase responsible for formation of N4-acetylcytidine (ac4C) at position 1842 in the terminal helix of mammalian 18 S rRNA. RNAi-mediated knockdown of NAT10 resulted in growth retardation of human cells, and this was accompanied by high-level accumulation of the 30 S precursor of 18 S rRNA, suggesting that ac4C1842 formation catalyzed by NAT10 is involved in rRNA processing and ribosome biogenesis. PMID:25411247

  8. A unique GCN5-related glucosamine N-acetyltransferase region exist in the fungal multi-domain glycoside hydrolase family 3 β-N-acetylglucosaminidase

    PubMed Central

    Qin, Zhen; Xiao, Yibei; Yang, Xinbin; Mesters, Jeroen R.; Yang, Shaoqing; Jiang, Zhengqiang

    2015-01-01

    Glycoside hydrolase (GH) family 3 β-N-acetylglucosaminidases widely exist in the filamentous fungi, which may play a key role in chitin metabolism of fungi. A multi-domain GH family 3 β-N-acetylglucosaminidase from Rhizomucor miehei (RmNag), exhibiting a potential N-acetyltransferase region, has been recently reported to show great potential in industrial applications. In this study, the crystal structure of RmNag was determined at 2.80 Å resolution. The three-dimensional structure of RmNag showed four distinctive domains, which belong to two distinguishable functional regions — a GH family 3 β-N-acetylglucosaminidase region (N-terminal) and a N-acetyltransferase region (C-terminal). From structural and functional analysis, the C-terminal region of RmNag was identified as a unique tandem array linking general control non-derepressible 5 (GCN5)-related N-acetyltransferase (GNAT), which displayed glucosamine N-acetyltransferase activity. Structural analysis of this glucosamine N-acetyltransferase region revealed that a unique glucosamine binding pocket is located in the pantetheine arm binding terminal region of the conserved CoA binding pocket, which is different from all known GNAT members. This is the first structural report of a glucosamine N-acetyltransferase, which provides novel structural information about substrate specificity of GNATs. The structural and functional features of this multi-domain β-N-acetylglucosaminidase could be useful in studying the catalytic mechanism of GH family 3 proteins. PMID:26669854

  9. Crystal structure of bacillus subtilis YdaF protein : a putative ribosomal N-acetyltransferase.

    SciTech Connect

    Brunzelle, J. S.; Wu, R.; Korolev, S. V.; Collart, F. R.; Joachimiak, A.; Anderson, W. F.; Biosciences Division; Northwestern Univ.; Saint Louis Univ. School of Medicine

    2004-12-01

    Comparative sequence analysis suggests that the ydaF gene encodes a protein (YdaF) that functions as an N-acetyltransferase, more specifically, a ribosomal N-acetyltransferase. Sequence analysis using basic local alignment search tool (BLAST) suggests that YdaF belongs to a large family of proteins (199 proteins found in 88 unique species of bacteria, archaea, and eukaryotes). YdaF also belongs to the COG1670, which includes the Escherichia coli RimL protein that is known to acetylate ribosomal protein L12. N-acetylation (NAT) has been found in all kingdoms. NAT enzymes catalyze the transfer of an acetyl group from acetyl-CoA (AcCoA) to a primary amino group. For example, NATs can acetylate the N-terminal {alpha}-amino group, the {epsilon}-amino group of lysine residues, aminoglycoside antibiotics, spermine/speridine, or arylalkylamines such as serotonin. The crystal structure of the alleged ribosomal NAT protein, YdaF, from Bacillus subtilis presented here was determined as a part of the Midwest Center for Structural Genomics. The structure maintains the conserved tertiary structure of other known NATs and a high sequence similarity in the presumed AcCoA binding pocket in spite of a very low overall level of sequence identity to other NATs of known structure.

  10. The Set3 Complex Antagonizes the MYST Acetyltransferase Esa1 in the DNA Damage Response

    PubMed Central

    Torres-Machorro, Ana Lilia; Clark, Lauren G.; Chang, Christie S.

    2015-01-01

    Acetylation is a dynamic posttranslational modification that contributes to chromatin-regulated processes, including DNA replication, repair, recombination, and gene expression. Acetylation is controlled by complexes containing opposing lysine and histone acetyltransferase (KAT and HAT) and deacetylase (KDAC and HDAC) activities. The essential MYST family Esa1 KAT acetylates core histones and many nonhistone substrates. Phenotypes of esa1 mutants include transcriptional silencing and activation defects, impaired growth at high temperatures, and sensitivity to DNA damage. The KDAC Rpd3 was previously identified as an activity opposing Esa1, as its deletion suppresses growth and silencing defects of esa1 mutants. However, loss of Rpd3 does not suppress esa1 DNA damage sensitivity. In this work, we identified Hos2 as a KDAC counteracting ESA1 in the damage response. Deletion of HOS2 resulted in changes of esa1's transcriptional response upon damage. Further, loss of HOS2 or components of the Set3 complex (Set3C) in which it acts specifically suppressed damage sensitivity and restored esa1 histone H4 acetylation. This rescue was mediated via loss of either Set3C integrity or of its binding to dimethylated histone H3K4. Our results thus add new insight into the interactions of an essential MYST acetyltransferase with diverse deacetylases to respond specifically to environmental and physiological challenges. PMID:26303527

  11. The histone acetyltransferase PsGcn5 mediates oxidative stress responses and is required for full virulence of Phytophthora sojae.

    PubMed

    Zhao, Wei; Wang, Tao; Liu, Shusen; Chen, Qingqing; Qi, Rende

    2015-10-01

    In eukaryotic organisms, histone acetyltransferase complexes are coactivators that are important for transcriptional activation by modifying chromatin. In this study, a gene (PsGcn5) from Phytophthora sojae encoding a histone acetyltransferase was identified as a homolog of one component of the histone acetyltransferase complex from yeasts to mammals. PsGcn5 was constitutively expressed in each stage tested, but had a slightly higher expression in sporulating hyphae and 3 h after infection. PsGcn5-silenced mutants were generated using polyethylene glycol-mediated protoplast stable transformation. These mutants had normal development, but compared to wild type strains they had higher sensitivity to hydrogen peroxide (H2O2) and significantly reduced virulence in soybean. Diaminobenzidine staining revealed an accumulation of H2O2 around the infection sites of PsGcn5-silenced mutants but not for wild type strains. Inhibition of the plant NADPH oxidase by diphenyleneiodonium prevented host-derived H2O2 accumulation in soybean cells and restored infectious hyphal growth of the mutants. Thus, we concluded that PsGcn5 is important for growth under conditions of oxidative stress and contributes to the full virulence of P. sojae by suppressing the host-derived reactive oxygen species.

  12. Structure of Arabidopsis thaliana At1g77540 Protein, a Minimal Acetyltransferase from the COG2388 Family †,‡

    PubMed Central

    Tyler, Robert C.; Bitto, Eduard; Berndsen, Christopher E.; Bingman, Craig A.; Singh, Shanteri; Lee, Min S.; Wesenberg, Gary E.; Denu, John M.; Phillips, George N.; Markley, John L.

    2008-01-01

    We describe X-ray crystal and NMR solution structures of the protein coded for by Arabidopsis thaliana gene At1g77540.1 (At1g77540). The crystal structure was determined to 1.15 Å with an R factor of 14.9% (Rfree = 17.0%) by multiple-wavelength anomalous diffraction using sodium bromide derivatized crystals. The ensemble of NMR conformers was determined with protein samples labeled with 15N and 13C+15N. The X-ray structure and NMR ensemble were closely similar with r.m.s.d 1.4 Å for residues 8–93. At1g77540 was found to adopt a fold similar to that of GCN5-related N-acetyltransferases. Enzymatic activity assays established that At1g77540 possesses weak acetyltransferase activity against histones H3 and H4. Chemical shift perturbations observed in 15N-HSQC spectra upon the addition of CoA indicated that the cofactor binds and identified its binding site. The molecular details of this interaction were further elucidated by solving the X-ray structure of the At1g77540–CoA complex. This work establishes that the domain family COG2388 represents a novel class of acetyltransferase and provides insight into possible mechanistic roles of the conserved Cys76 and His41 residues of this family. PMID:17128971

  13. Epigenetic change in kidney tumor: downregulation of histone acetyltransferase MYST1 in human renal cell carcinoma

    PubMed Central

    2013-01-01

    Background MYST1 (also known as hMOF), a member of the MYST family of histone acetyltransferases (HATs) as an epigenetic mark of active genes, is mainly responsible for histone H4K16 acetylation in the cells. Recent studies have shown that the abnormal gene expression of hMOF is involved in certain primary cancers. Here we examined the involvement of hMOF expression and histone H4K16 acetylation in primary renal cell carcinoma (RCC). Simultaneously, we investigated the correlation between the expression of hMOF and clear cell RCC (ccRCC) biomarker carbohydrase IX (CA9) in RCC. Materials and methods The frozen RCC tissues and RCC cell lines as materials, the reverse transcription polymerase chain reaction (RT-PCR), western blotting and immunohistochemical staining approaches were used. Results RT-PCR results indicate that hMOF gene expression levels frequently downregulated in 90.5% of patients (19/21) with RCC. The reduction of hMOF protein in both RCC tissues and RCC cell lines is tightly correlated with acetylation of histone H4K16. In addition, overexpression of CA9 was detected in 100% of ccRCC patients (21/21). However, transient transfection of hMOF in ccRCC 786–0 cells did not affect both the gene and protein expression of CA9. Conclusion hMOF as an acetyltransferase of H4K16 might be involved in the pathogenesis of kidney cancer, and this epigenetic changes might be a new CA9-independent RCC diagnostic maker. PMID:23394073

  14. Structure of a pathogen effector reveals the enzymatic mechanism of a novel acetyltransferase family.

    PubMed

    Zhang, Zhi-Min; Ma, Ka-Wai; Yuan, Shuguang; Luo, Youfu; Jiang, Shushu; Hawara, Eva; Pan, Songqin; Ma, Wenbo; Song, Jikui

    2016-09-01

    Effectors secreted by the type III secretion system are essential for bacterial pathogenesis. Members of the Yersinia outer-protein J (YopJ) family of effectors found in diverse plant and animal pathogens depend on a protease-like catalytic triad to acetylate host proteins and produce virulence. However, the structural basis for this noncanonical acetyltransferase activity remains unknown. Here, we report the crystal structures of the YopJ effector HopZ1a, produced by the phytopathogen Pseudomonas syringae, in complex with the eukaryote-specific cofactor inositol hexakisphosphate (IP6) and/or coenzyme A (CoA). Structural, computational and functional characterizations reveal a catalytic core with a fold resembling that of ubiquitin-like cysteine proteases and an acetyl-CoA-binding pocket formed after IP6-induced structural rearrangements. Modeling-guided mutagenesis further identified key IP6-interacting residues of Salmonella effector AvrA that are required for acetylating its substrate. Our study reveals the structural basis of a novel class of acetyltransferases and the conserved allosteric regulation of YopJ effectors by IP6. PMID:27525589

  15. Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity

    PubMed Central

    Lalonde, Marie-Eve; Avvakumov, Nikita; Glass, Karen C.; Joncas, France-Hélène; Saksouk, Nehmé; Holliday, Michael; Paquet, Eric; Yan, Kezhi; Tong, Qiong; Klein, Brianna J.; Tan, Song; Yang, Xiang-Jiao; Kutateladze, Tatiana G.; Côté, Jacques

    2013-01-01

    Histone acetyltransferases (HATs) assemble into multisubunit complexes in order to target distinct lysine residues on nucleosomal histones. Here, we characterize native HAT complexes assembled by the BRPF family of scaffold proteins. Their plant homeodomain (PHD)–Zn knuckle–PHD domain is essential for binding chromatin and is restricted to unmethylated H3K4, a specificity that is reversed by the associated ING subunit. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as catalytic acetyltransferase subunit. Interestingly, while the previously reported HBO1 complexes containing JADE scaffold proteins target histone H4, the HBO1–BRPF1 complex acetylates only H3 in chromatin. We mapped a small region to the N terminus of scaffold proteins responsible for histone tail selection on chromatin. Thus, alternate choice of subunits associated with HBO1 can switch its specificity between H4 and H3 tails. These results uncover a crucial new role for associated proteins within HAT complexes, previously thought to be intrinsic to the catalytic subunit. PMID:24065767

  16. Biophysical analysis of the putative acetyltransferase SACOL2570 from methicillin-resistant Staphylococcus aureus

    PubMed Central

    Luo, Hai-Bin; Knapik, Aleksandra A.; Petkowski, Janusz J.; Demas, Matthew; Shumilin, Igor A.; Zheng, Heping; Chruszcz, Maksymilian

    2013-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of a myriad of insidious and intractable infections in humans, especially in patients with compromised immune systems and children. Here, we report the apo- and CoA-bound crystal structures of a member of the galactoside acetyltransferase superfamily from methicillin-resistant S. aureus SACOL2570 which was recently shown to be down regulated in S. aureus grown in the presence of fusidic acid, an antibiotic used to treat MRSA infections. SACOL2570 forms a homotrimerin solution, as confirmed by small-angle X-ray scattering and dynamic light scattering. The protein subunit consists of an N-terminal alpha-helical domain connected to a C-terminal LβH domain. CoA binds in the active site formed by the residues from adjacent LβH domains. After determination of CoA-bound structure, molecular dynamics simulations were performed to model the binding of AcCoA. Binding of both AcCoA and CoA to SACOL2570 was verified by isothermal titration calorimetry. SACOL2570 most likely acts as an acetyltransferase, using AcCoA as an acetyl group donor and an as-yet-undetermined chemical moiety as an acceptor. SACOL2570 was recently used as a scaffold for mutations that lead the generation of cage-like assemblies, and has the potential to be used for the generation of more complex nanostructures. PMID:23963951

  17. FOXP3 interactions with histone acetyltransferase and class II histone deacetylases are required for repression

    PubMed Central

    Li, Bin; Samanta, Arabinda; Song, Xiaomin; Iacono, Kathryn T.; Bembas, Kathryn; Tao, Ran; Basu, Samik; Riley, James L.; Hancock, Wayne W.; Shen, Yuan; Saouaf, Sandra J.; Greene, Mark I.

    2007-01-01

    The forkhead family protein FOXP3 acts as a repressor of transcription and is both an essential and sufficient regulator of the development and function of regulatory T cells. The molecular mechanism by which FOXP3-mediated transcriptional repression occurs remains unclear. Here, we report that transcriptional repression by FOXP3 involves a histone acetyltransferase–deacetylase complex that includes histone acetyltransferase TIP60 (Tat-interactive protein, 60 kDa) and class II histone deacetylases HDAC7 and HDAC9. The N-terminal 106–190 aa of FOXP3 are required for TIP60–FOXP3, HDAC7–FOXP3 association, as well as for the transcriptional repression of FOXP3 via its forkhead domain. FOXP3 can be acetylated in primary human regulatory T cells, and TIP60 promotes FOXP3 acetylation in vivo. Overexpression of TIP60 but not its histone acetyltransferase-deficient mutant promotes, whereas knockdown of endogenous TIP60 relieved, FOXP3-mediated transcriptional repression. A minimum FOXP3 ensemble containing native TIP60 and HDAC7 is necessary for IL-2 production regulation in T cells. Moreover, FOXP3 association with HDAC9 is antagonized by T cell stimulation and can be restored by the protein deacetylation inhibitor trichostatin A, indicating a complex dynamic aspect of T suppressor cell regulation. These findings identify a previously uncharacterized complex-based mechanism by which FOXP3 actively mediates transcriptional repression. PMID:17360565

  18. Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity.

    PubMed

    Lalonde, Marie-Eve; Avvakumov, Nikita; Glass, Karen C; Joncas, France-Hélène; Saksouk, Nehmé; Holliday, Michael; Paquet, Eric; Yan, Kezhi; Tong, Qiong; Klein, Brianna J; Tan, Song; Yang, Xiang-Jiao; Kutateladze, Tatiana G; Côté, Jacques

    2013-09-15

    Histone acetyltransferases (HATs) assemble into multisubunit complexes in order to target distinct lysine residues on nucleosomal histones. Here, we characterize native HAT complexes assembled by the BRPF family of scaffold proteins. Their plant homeodomain (PHD)-Zn knuckle-PHD domain is essential for binding chromatin and is restricted to unmethylated H3K4, a specificity that is reversed by the associated ING subunit. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as catalytic acetyltransferase subunit. Interestingly, while the previously reported HBO1 complexes containing JADE scaffold proteins target histone H4, the HBO1-BRPF1 complex acetylates only H3 in chromatin. We mapped a small region to the N terminus of scaffold proteins responsible for histone tail selection on chromatin. Thus, alternate choice of subunits associated with HBO1 can switch its specificity between H4 and H3 tails. These results uncover a crucial new role for associated proteins within HAT complexes, previously thought to be intrinsic to the catalytic subunit.

  19. A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators.

    PubMed

    Xie, Wensheng; Silvers, Robert; Ouellette, Michael; Wu, Zining; Lu, Quinn; Li, Hu; Gallagher, Kathleen; Johnson, Kathy; Montoute, Monica

    2016-01-01

    Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic β-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase. PMID:27316998

  20. Metabolic Regulation of Histone Acetyltransferases by Endogenous Acyl-CoA Cofactors

    PubMed Central

    Guasch, Laura; Nicklaus, Marc C.; Meier, Jordan L.

    2015-01-01

    SUMMARY The finding that chromatin modifications are sensitive to changes in cellular cofactor levels potentially links altered tumor cell metabolism and gene expression. However, the specific enzymes and metabolites that connect these two processes remain obscure. Characterizing these metabolic-epigenetic axes is critical to understanding how metabolism supports signaling in cancer, and developing therapeutic strategies to disrupt this process. Here, we describe a chemical approach to define the metabolic regulation of lysine acetyltransferase (KAT) enzymes. Using a novel chemoproteomic probe, we identify a previously unreported interaction between fatty acyl-CoAs and KAT enzymes. Further analysis reveals that palmitoyl-CoA is a potent inhibitor of KAT activity and that fatty acyl-CoA precursors reduce cellular acetylation levels. These studies implicate fatty acyl-CoAs as endogenous regulators of histone acetylation, and suggest novel strategies for the investigation and metabolic modulation of epigenetic signaling. PMID:26190825

  1. The SAGA histone acetyltransferase complex regulates leucine uptake through the Agp3 permease in fission yeast.

    PubMed

    Takahashi, Hidekazu; Sun, Xiaoying; Hamamoto, Makiko; Yashiroda, Yoko; Yoshida, Minoru

    2012-11-01

    Metabolic responses of unicellular organisms are mostly acute, transient, and cell-autonomous. Regulation of nutrient uptake in yeast is one such rapid response. High quality nitrogen sources such as NH(4)(+) inhibit uptake of poor nitrogen sources, such as amino acids. Both transcriptional and posttranscriptional mechanisms operate in nutrient uptake regulation; however, many components of this system remain uncharacterized in the fission yeast, Schizosaccharomyces pombe. Here, we demonstrate that the Spt-Ada-Gcn acetyltransferase (SAGA) complex modulates leucine uptake. Initially, we noticed that a branched-chain amino acid auxotroph exhibits a peculiar adaptive growth phenotype on solid minimal media containing certain nitrogen sources. In fact, the growth of many auxotrophic strains is inhibited by excess NH(4)Cl, possibly through nitrogen-mediated uptake inhibition of the corresponding nutrients. Surprisingly, DNA microarray analysis revealed that the transcriptional reprogramming during the adaptation of the branched-chain amino acid auxotroph was highly correlated with reprogramming observed in deletions of the SAGA histone acetyltransferase module genes. Deletion of gcn5(+) increased leucine uptake in the prototrophic background and rendered the leucine auxotroph resistant to NH(4)Cl. Deletion of tra1(+) caused the opposite phenotypes. The increase in leucine uptake in the gcn5Δ mutant was dependent on an amino acid permease gene, SPCC965.11c(+). The closest budding yeast homolog of this permease is a relatively nonspecific amino acid permease AGP3, which functions in poor nutrient conditions. Our analysis identified the regulation of nutrient uptake as a physiological function for the SAGA complex, providing a potential link between cellular metabolism and chromatin regulation.

  2. Histone H3K9 acetylation level modulates gene expression and may affect parasite growth in human malaria parasite Plasmodium falciparum.

    PubMed

    Srivastava, Sandeep; Bhowmick, Krishanu; Chatterjee, Snehajyoti; Basha, Jeelan; Kundu, Tapas K; Dhar, Suman K

    2014-12-01

    Three-dimensional positioning of the nuclear genome plays an important role in the epigenetic regulation of genes. Although nucleographic domain compartmentalization in the regulation of epigenetic state and gene expression is well established in higher organisms, it remains poorly understood in the pathogenic parasite Plasmodium falciparum. In the present study, we report that two histone tail modifications, H3K9Ac and H3K14Ac, are differentially distributed in the parasite nucleus. We find colocalization of active gene promoters such as Tu1 (tubulin-1 expressed in the asexual stages) with H3K9Ac marks at the nuclear periphery. By contrast, asexual stage inactive gene promoters such as Pfg27 (gametocyte marker) and Pfs28 (ookinete marker) occupy H3K9Ac devoid zones at the nuclear periphery. The histone H3K9 is predominantly acetylated by the PCAF/GCN5 class of lysine acetyltransferases, which is well characterized in the parasite. Interestingly, embelin, a specific inhibitor of PCAF/GCN5 family histone acetyltransferase, selectively decreases total H3K9Ac acetylation levels (but not H3K14Ac levels) around the var gene promoters, leading to the downregulation of var gene expression, suggesting interplay among histone acetylation status, as well as subnuclear compartmentalization of different genes and their activation in the parasites. Finally, we found that embelin inhibited parasitic growth at the low micromolar range, raising the possibility of using histone acetyltransferases as a target for antimalarial therapy.

  3. Arylalkylamine N-acetyltransferase (AANAT) genotype as a personal trait in melatonin synthesis.

    PubMed

    Blomeke, Brunhilde; Golka, Klaus; Griefahn, Barbara; Roemer, Hermann C

    2008-01-01

    The melatonin rhythm is arguably the best marker for the phase of the endogenous "biological clock." Arylalkylamine N-acetyltransferase (AANAT) is known to catalyze the acetylation of serotonin, a rate-limiting process in melatonin synthesis. Different single-nucleotide polymorphisms (SNPs) in the AANAT gene were identified recently in the Japanese population, and one of the genes was significantly associated with the delayed sleep phase syndrome. Thus, 54 healthy Caucasian males were genotyped to investigate whether these SNPs in the AANAT gene affected melatonin levels. The endogenous melatonin levels were analyzed in saliva under standardized experimental conditions ("constant routines") by radioimmunoassay. Despite the broad temporal variation of the human nocturnal melatonin profiles, none of the investigated SNPs were found in the AANAT gene in this study. These findings point to ethnic differences with respect to these SNPs, rather than time of day termed "morningness." In summary, SNPs in the AANAT gene identified thus far cannot explain the observed interindividual differences for nocturnal melatonin profiles in the subjects investigated.

  4. Simultaneous gene inactivation and promoter reporting in cyanobacteria.

    PubMed

    Chen, Kangming; Xu, Xinyi; Gu, Liping; Hildreth, Michael; Zhou, Ruanbao

    2015-02-01

    Determining spatiotemporal gene expression and analyzing knockout mutant phenotypes have become powerful tools in elucidating the function of genes; however, genetic approaches for simultaneously inactivating a gene and monitoring its expression have not been reported in the literature. In this study, we designed a dual-functional gene knockout vector pZR606 that contains a multiple cloning site (MCS) for inserting the internal fragment of a target gene, with a gfp gene as its transcriptional marker located immediately downstream of the MCS. By using this gene knockout system, we inactivated ava_2679 from Anabaena variabilis ATCC 29413, as well as all2508, alr2887, alr3608, and all4388 from Anabaena sp. strain PCC 7120. The ava_2679 knockout mutant fails to grow diazotrophically. Morphological analysis of ava_2679 knockout mutant after nitrogen step-down revealed defective junctions between heterocysts and adjacent vegetative cells, and the heterocyst was 1.53-fold longer compared to wild-type heterocysts. The alr2887, all4388, and alr3608 mutant colonies turned yellow and showed lack of protracted growth when deprived of fixed nitrogen, consistent with the previous reports that alr2887, all4388, and alr3608 are Fox genes. The all2508 encodes a GTP-binding elongation factor (EF4/LepA), and its knockout mutant exhibited reduced diazotrophic growth. The heterocyst development of all2508 knockout was significantly delayed, and only about 4.0 % of vegetative cells differentiated to heterocysts after nitrogen deprivation for 72 h, decreased 49.6 % compared to wild-type. Thus, we discovered that All2508 may regulate heterocyst development spatiotemporally. Concurrently, the GFP reporter revealed that all five target gene expressions were up-regulated in response to nitrogen deprivation. We demonstrated that the pZR606-based specific gene knockout approach worked effectively for the five selected genes, including four previously identified Fox genes or Fox gene

  5. The histone acetyltransferase domains of CREB-binding protein (CBP) and p300/CBP-associated factor are not necessary for cooperativity with the class II transactivator.

    PubMed

    Harton, J A; Zika, E; Ting, J P

    2001-10-19

    The class II transactivator (CIITA) is a transcriptional co-activator regulating the constitutive and interferon-gamma-inducible expression of class II major histocompatibility complex (MHC) and related genes. Promoter remodeling occurs following CIITA induction, suggesting the involvement of chromatin remodeling factors. Transcription of numerous genes requires the histone acetyltransferase (HAT) activities of CREB-binding protein (CBP), p300, and/or p300/CBP-associated factor (pCAF). These co-activators cooperate with CIITA and are hypothesized to promote class II major histocompatibility complex transcription through their HAT activity. To directly test this, we used HAT-defective CBP and pCAF. We demonstrate that cooperation between CIITA and CBP is independent of CBP HAT activity. Further, although pCAF enhances CIITA-mediated transcription, pCAF HAT domain dependence appears contingent upon the concentration of available CIITA. When HAT-defective CBP and pCAF are both present, cooperativity with CIITA is maintained. Consistent with a recent report, we show that nuclear localization of CIITA is enhanced by lysine 144, an in vitro target of pCAF-mediated HAT. Yet we find that neither mutation of lysine 144 nor deletion of residues 132-209 affects transcriptional cooperation with CBP or pCAF. Thus, acetylation of this residue may not be the primary mechanism for pCAF/CBP cooperation with CIITA. In conclusion, the HAT activities of the co-activators are not necessary for cooperation with CIITA.

  6. Luciferase as a reporter of gene activity in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since their development and introduction in the early days of plant genetic engineering, reporter genes have established a proven track record as effective tools for exploring the molecular underpinnings of gene regulation. When driven by appropriate genetic control systems (e.g. transcriptional pr...

  7. Cloning-free regulated monitoring of reporter and gene expression

    PubMed Central

    al-Haj, Latifa; Al-Ahmadi, Wijdan; Al-Saif, Maher; Demirkaya, Omer; Khabar, Khalid SA

    2009-01-01

    Background The majority of the promoters, their regulatory elements, and their variations in the human genome remain unknown. Reporter gene technology for transcriptional activity is a widely used tool for the study of promoter structure, gene regulation, and signaling pathways. Construction of transcriptional reporter vectors, including use of cis-acting sequences, requires cloning and time-demanding manipulations, particularly with introduced mutations. Results In this report, we describe a cloning-free strategy to generate transcriptionally-controllable linear reporter constructs. This approach was applied in common transcriptional models of inflammatory response and the interferon system. In addition, it was used to delineate minimal transcriptional activity of selected ribosomal protein promoters. The approach was tested for conversion of genes into TetO-inducible/repressible expression cassettes. Conclusion The simple introduction and tuning of any transcriptional control in the linear DNA product renders promoter activation and regulated gene studies simple and versatile. PMID:19267938

  8. Histone acetyltransferase HAT4 modulates navigation across G2/M and re-entry into G1 in Leishmania donovani

    PubMed Central

    Yadav, Aarti; Chandra, Udita; Saha, Swati

    2016-01-01

    Histone acetyltransferases impact multiple processes. This study investigates the role of histone acetyltransferase HAT4 in Leishmania donovani. Though HAT4 was dispensable for survival, its elimination decreased cell viability and caused cell cycle defects, with HAT4-nulls experiencing an unusually long G2/M. Survival of HAT4-nulls in macrophages was also substantially compromised. DNA microarray analysis revealed that HAT4 modestly regulated the expression of only a select number of genes, thus not being a major modulator of global gene expression. Significantly, cdc20 was among the downregulated genes. To ascertain if decreased expression of cdc20 was responsible for HAT4-null growth and cell cycle defects we expressed LdCdc20 ectopically in HAT4-nulls. We found this to alleviate the aberrant growth and cell cycle progression patterns displayed by HAT4-nulls, with cells navigating G2/M phase and re-entering G1 phase smoothly. HAT4-nulls expressing LdCdc20 ectopically showed survival rates comparable to wild type within macrophages, suggesting that G2/M defects were responsible for poor survival of HAT4-nulls within host cells also. These are the first data analyzing the in vivo functional role of HAT4 in any trypanosomatid. Our results directly demonstrate for the first time a role for Cdc20 in regulating trypanosomatid G2/M events, opening avenues for further research in this area. PMID:27272906

  9. N-acetylglucosamine sensing by a GCN5-related N-acetyltransferase induces transcription via chromatin histone acetylation in fungi

    PubMed Central

    Su, Chang; Lu, Yang; Liu, Haoping

    2016-01-01

    N-acetylglucosamine (GlcNAc) exists ubiquitously as a component of the surface on a wide range of cells, from bacteria to humans. Many fungi are able to utilize environmental GlcNAc to support growth and induce cellular development, a property important for their survival in various host niches. However, how the GlcNAc signal is sensed and subsequently transduced is largely unknown. Here, we identify a gene that is essential for GlcNAc signalling (NGS1) in Candida albicans, a commensal and pathogenic yeast of humans. Ngs1 can bind GlcNAc through the N-terminal β-N-acetylglucosaminidase homology domain. This binding activates N-acetyltransferase activity in the C-terminal GCN5-related N-acetyltransferase domain, which is required for GlcNAc-induced promoter histone acetylation and transcription. Ngs1 is targeted to the promoters of GlcNAc-inducible genes constitutively by the transcription factor Rep1. Ngs1 is conserved in diverse fungi that have GlcNAc catabolic genes. Thus, fungi use Ngs1 as a GlcNAc-sensor and transducer for GlcNAc-induced transcription. PMID:27694804

  10. Reporter genes for embryogenesis research in livestock species.

    PubMed

    Habermann, F A; Wuensch, A; Sinowatz, F; Wolf, E

    2007-09-01

    Currently, our knowledge of early mammalian embryogenesis, stem cell differentiation and development is largely based on studies performed in mouse models. However, in important aspects, e.g. the timing of epigenetic reprogramming and embryonic genome activation, livestock species probably reflect far more closely the situation in men and other non-rodent mammals. A major challenge is the fact that in mammals, the development of individual zygotes is highly variable and vulnerable, and the outcome is uncertain. Valid indicators of the highly heterogeneous development and health status, and the actual developmental potential of individual oocytes, zygotes or embryos would be crucially important to tap the full power of holistic transcriptome and proteome analyses. Fluorescent reporter proteins opened new vistas for embryology and stem cell research: they can be used as reporters for the activity of gene promoters or tagged to functional proteins to study their intracellular localization in living cells, tissues and organisms. Fluorescent reporter genes may be used to microscopically observe key processes of early development. Thus, novel information related to developmental potential can be obtained from living embryos before processing them, e.g. for "-omic" studies. This review summarizes the main current reporter gene techniques and gene transfer approaches, which might be suitable for the investigation of early embryogenesis in livestock mammals. The potential of promoter reporter genes is exemplified by a bovine model system for quantitative monitoring of transcriptional reactivation of the so-called pluripotency gene POU5F1 in cloned bovine embryos.

  11. Cloning, purification, crystallization and preliminary crystallographic analysis of a hypothetical acetyltransferase from Pyrococcus furiosus

    PubMed Central

    Biarrotte-Sorin, Sabrina; Mayer, Claudine

    2005-01-01

    The GCN5-related N-acetyltransferase (GNAT) superfamily has a primordial role in cellular processes such as transcription initiation and regulation by histone acetylation, aminoglycoside resistance and melatonin metabolism. To date, no acetyltransferase from the archaeal domain of life has been studied. This paper describes the cloning, expression, purification and crystallization of a Pyrococcus furiosus hypothetical acetyltransferase PfGNAT (MW = 22 007 Da). The crystals belong to space group P622, with one molecule in the asymmetric unit and unit-cell parameters a = b = 82.6, c = 105.92 Å, α = β = 90, γ = 120°. Crystals diffract X-rays to 3.0 Å resolution on a synchrotron-radiation source. Determination of this structure will provide new insights into the substrate-specificity of this acetyltransferase and the thermal stability of the N-acetyltransferase domain. PMID:16511014

  12. A Bacterial Acetyltransferase Destroys Plant Microtubule Networks and Blocks Secretion

    PubMed Central

    Lee, Amy Huei-Yi; Hurley, Brenden; Felsensteiner, Corinna; Yea, Carmen; Ckurshumova, Wenzislava; Bartetzko, Verena; Wang, Pauline W.; Quach, Van; Lewis, Jennifer D.; Liu, Yulu C.; Börnke, Frederik; Angers, Stephane; Wilde, Andrew

    2012-01-01

    The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption. PMID:22319451

  13. Enzyme kinetics and inhibition of histone acetyltransferase KAT8

    PubMed Central

    Wapenaar, Hannah; van der Wouden, Petra E.; Groves, Matthew R.; Rotili, Dante; Mai, Antonello; Dekker, Frank J.

    2016-01-01

    Lysine acetyltransferase 8 (KAT8) is a histone acetyltransferase (HAT) responsible for acetylating lysine 16 on histone H4 (H4K16) and plays a role in cell cycle progression as well as acetylation of the tumor suppressor protein p53. Further studies on its biological function and drug discovery initiatives will benefit from the development of small molecule inhibitors for this enzyme. As a first step towards this aim we investigated the enzyme kinetics of this bi-substrate enzyme. The kinetic experiments indicate a ping-pong mechanism in which the enzyme binds Ac-CoA first, followed by binding of the histone substrate. This mechanism is supported by affinity measurements of both substrates using isothermal titration calorimetry (ITC). Using this information, the KAT8 inhibition of a focused compound collection around the non-selective HAT inhibitor anacardic acid has been investigated. Kinetic studies with anacardic acid were performed, based on which a model for the catalytic activity of KAT8 and the inhibitory action of AA was proposed. This enabled the calculation of the inhibition constant Ki of anacardic acid derivatives using an adaptation of the Cheng-Prusoff equation. The results described in this study give insight into the catalytic mechanism of KAT8 and present the first well-characterized small-molecule inhibitors for this HAT. PMID:26505788

  14. Molecular mechanism underlying promiscuous polyamine recognition by spermidine acetyltransferase.

    PubMed

    Sugiyama, Shigeru; Ishikawa, Sae; Tomitori, Hideyuki; Niiyama, Mayumi; Hirose, Mika; Miyazaki, Yuma; Higashi, Kyohei; Murata, Michio; Adachi, Hiroaki; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Kashiwagi, Keiko; Igarashi, Kazuei; Matsumura, Hiroyoshi

    2016-07-01

    Spermidine acetyltransferase (SAT) from Escherichia coli, which catalyses the transfer of acetyl groups from acetyl-CoA to spermidine, is a key enzyme in controlling polyamine levels in prokaryotic cells. In this study, we determined the crystal structure of SAT in complex with spermidine (SPD) and CoA at 2.5Å resolution. SAT is a dodecamer organized as a hexamer of dimers. The secondary structural element and folding topology of the SAT dimer resemble those of spermidine/spermine N(1)-acetyltransferase (SSAT), suggesting an evolutionary link between SAT and SSAT. However, the polyamine specificity of SAT is distinct from that of SSAT and is promiscuous. The SPD molecule is also located at the inter-dimer interface. The distance between SPD and CoA molecules is 13Å. A deep, highly acidic, water-filled cavity encompasses the SPD and CoA binding sites. Structure-based mutagenesis and in-vitro assays identified SPD-bound residues, and the acidic residues lining the walls of the cavity are mostly essential for enzymatic activities. Based on mutagenesis and structural data, we propose an acetylation mechanism underlying promiscuous polyamine recognition for SAT. PMID:27163532

  15. Cloning and analysis of a Toxoplasma gondii histone acetyltransferase: a novel chromatin remodelling factor in Apicomplexan parasites.

    PubMed

    Hettmann, C; Soldati, D

    1999-11-15

    The yeast transcriptional adaptor GCN5 functions as a histone acetyltransferase, directly linking chromatin modification to transcriptional regulation. Homologues of yeast GCN5 have been found in Tetrahymena, Drosophila, Arabidopsis and human, suggesting that this pathway of chromatin remodelling is evolutionarily conserved. Consistent with this view, we have identified the Toxoplasma gondii homologue, referred to here as TgGCN5. The gene codes for a protein of 474 amino acids with an estimated molecular mass of 53 kDa. The protein reveals two regions of close similarity with the GCN5 family members, the HAT domain and the bromodomain. Tg GCN5 occurs in a single copy in the T.gondii genome. The introduction of a second copy of TgGCN5 in T.gondii tachyzoites is toxic unless the HAT activity is disrupted by a single point mutation. Full TgGCN5 does not complement the growth defect in a yeast gcn5 (-)mutant strain, but a chimera comprising the T.gondii HAT domain fused to the remainder of yGCN5 does. These data show that T.gondii GNC5 is a histone acetyltransferase attesting to the significance of chromatin remodelling in gene regulation of Apicomplexa.

  16. Development of Plant Gene Vectors for Tissue-Specific Expression Using GFP as a Reporter Gene

    NASA Technical Reports Server (NTRS)

    Jackson, Jacquelyn; Egnin, Marceline; Xue, Qi-Han; Prakash, C. S.

    1997-01-01

    Reporter genes are widely employed in plant molecular biology research to analyze gene expression and to identify promoters. Gus (UidA) is currently the most popular reporter gene but its detection requires a destructive assay. The use of jellyfish green fluorescent protein (GFP) gene from Aequorea Victoria holds promise for noninvasive detection of in vivo gene expression. To study how various plant promoters are expressed in sweet potato (Ipomoea batatas), we are transcriptionally fusing the intron-modified (mGFP) or synthetic (modified for codon-usage) GFP coding regions to these promoters: double cauliflower mosaic virus 35S (CaMV 35S) with AMV translational enhancer, ubiquitin7-intron-ubiquitin coding region (ubi7-intron-UQ) and sporaminA. A few of these vectors have been constructed and introduced into E. coli DH5a and Agrobacterium tumefaciens EHA105. Transient expression studies are underway using protoplast-electroporation and particle bombardment of leaf tissues.

  17. Environmental History Modulates Arabidopsis Pattern-Triggered Immunity in a HISTONE ACETYLTRANSFERASE1-Dependent Manner.

    PubMed

    Singh, Prashant; Yekondi, Shweta; Chen, Po-Wen; Tsai, Chia-Hong; Yu, Chun-Wei; Wu, Keqiang; Zimmerli, Laurent

    2014-06-24

    In nature, plants are exposed to a fluctuating environment, and individuals exposed to contrasting environmental factors develop different environmental histories. Whether different environmental histories alter plant responses to a current stress remains elusive. Here, we show that environmental history modulates the plant response to microbial pathogens. Arabidopsis thaliana plants exposed to repetitive heat, cold, or salt stress were more resistant to virulent bacteria than Arabidopsis grown in a more stable environment. By contrast, long-term exposure to heat, cold, or exposure to high concentrations of NaCl did not provide enhanced protection against bacteria. Enhanced resistance occurred with priming of Arabidopsis pattern-triggered immunity (PTI)-responsive genes and the potentiation of PTI-mediated callose deposition. In repetitively stress-challenged Arabidopsis, PTI-responsive genes showed enrichment for epigenetic marks associated with transcriptional activation. Upon bacterial infection, enrichment of RNA polymerase II at primed PTI marker genes was observed in environmentally challenged Arabidopsis. Finally, repetitively stress-challenged histone acetyltransferase1-1 (hac1-1) mutants failed to demonstrate enhanced resistance to bacteria, priming of PTI, and increased open chromatin states. These findings reveal that environmental history shapes the plant response to bacteria through the development of a HAC1-dependent epigenetic mark characteristic of a primed PTI response, demonstrating a mechanistic link between the primed state in plants and epigenetics.

  18. Molecular Evolution of Aralkylamine N-Acetyltransferase in Fish: A Genomic Survey

    PubMed Central

    Li, Jia; You, Xinxin; Bian, Chao; Yu, Hui; Coon, Steven L.; Shi, Qiong

    2015-01-01

    All living organisms synchronize biological functions with environmental changes; melatonin plays a vital role in regulating daily and seasonal variations. Due to rhythmic activity of the timezyme aralkylamine N-acetyltransferase (AANAT), the blood level of melatonin increases at night and decreases during daytime. Whereas other vertebrates have a single form of AANAT, bony fishes possess various isoforms of aanat genes, though the reasons are still unclear. Here, we have taken advantage of multiple unpublished teleost aanat sequences to explore and expand our understanding of the molecular evolution of aanat in fish. Our results confirm that two rounds of whole-genome duplication (WGD) led to the existence of three fish isoforms of aanat, i.e., aanat1a, aanat1b, and aanat2; in addition, gene loss led to the absence of some forms from certain special fish species. Furthermore, we suggest the different roles of two aanat1s in amphibious mudskippers, and speculate that the loss of aanat1a, may be related to terrestrial vision change. Several important sites of AANAT proteins and regulatory elements of aanat genes were analyzed for structural comparison and functional forecasting, respectively, which provides insights into the molecular evolution of the differences between AANAT1 and AANAT2. PMID:26729109

  19. Molecular Evolution of Aralkylamine N-Acetyltransferase in Fish: A Genomic Survey.

    PubMed

    Li, Jia; You, Xinxin; Bian, Chao; Yu, Hui; Coon, Steven L; Shi, Qiong

    2016-01-01

    All living organisms synchronize biological functions with environmental changes; melatonin plays a vital role in regulating daily and seasonal variations. Due to rhythmic activity of the timezyme aralkylamine N-acetyltransferase (AANAT), the blood level of melatonin increases at night and decreases during daytime. Whereas other vertebrates have a single form of AANAT, bony fishes possess various isoforms of aanat genes, though the reasons are still unclear. Here, we have taken advantage of multiple unpublished teleost aanat sequences to explore and expand our understanding of the molecular evolution of aanat in fish. Our results confirm that two rounds of whole-genome duplication (WGD) led to the existence of three fish isoforms of aanat, i.e., aanat1a, aanat1b, and aanat2; in addition, gene loss led to the absence of some forms from certain special fish species. Furthermore, we suggest the different roles of two aanat1s in amphibious mudskippers, and speculate that the loss of aanat1a, may be related to terrestrial vision change. Several important sites of AANAT proteins and regulatory elements of aanat genes were analyzed for structural comparison and functional forecasting, respectively, which provides insights into the molecular evolution of the differences between AANAT1 and AANAT2. PMID:26729109

  20. Molecular Evolution of Aralkylamine N-Acetyltransferase in Fish: A Genomic Survey.

    PubMed

    Li, Jia; You, Xinxin; Bian, Chao; Yu, Hui; Coon, Steven L; Shi, Qiong

    2015-12-31

    All living organisms synchronize biological functions with environmental changes; melatonin plays a vital role in regulating daily and seasonal variations. Due to rhythmic activity of the timezyme aralkylamine N-acetyltransferase (AANAT), the blood level of melatonin increases at night and decreases during daytime. Whereas other vertebrates have a single form of AANAT, bony fishes possess various isoforms of aanat genes, though the reasons are still unclear. Here, we have taken advantage of multiple unpublished teleost aanat sequences to explore and expand our understanding of the molecular evolution of aanat in fish. Our results confirm that two rounds of whole-genome duplication (WGD) led to the existence of three fish isoforms of aanat, i.e., aanat1a, aanat1b, and aanat2; in addition, gene loss led to the absence of some forms from certain special fish species. Furthermore, we suggest the different roles of two aanat1s in amphibious mudskippers, and speculate that the loss of aanat1a, may be related to terrestrial vision change. Several important sites of AANAT proteins and regulatory elements of aanat genes were analyzed for structural comparison and functional forecasting, respectively, which provides insights into the molecular evolution of the differences between AANAT1 and AANAT2.

  1. Deletion of host histone acetyltransferases and deacetylases strongly affects Agrobacterium-mediated transformation of Saccharomyces cerevisiae.

    PubMed

    Soltani, Jalal; van Heusden, Gerard Paul H; Hooykaas, Paul J J

    2009-09-01

    Agrobacterium tumefaciens is a plant pathogen that genetically transforms plant cells by transferring a part of its Ti-plasmid, the T-strand, to the host cell. Under laboratory conditions, it can also transform cells from many different nonplant organisms, including the yeast Saccharomyces cerevisiae. Collections of S. cerevisiae strains have been developed with systematic deletion of all coding sequences. Here, we used these collections to identify genes involved in the Agrobacterium-mediated transformation (AMT) of S. cerevisiae. We found that deletion of genes (GCN5, NGG1, YAF9 and EAF7) encoding subunits of the SAGA, SLIK, ADA and NuA4 histone acetyltransferase complexes highly increased the efficiency of AMT, while deletion of genes (HDA2, HDA3 and HST4) encoding subunits of histone deacetylase complexes decreased AMT. These effects are specific for AMT as the efficiency of chemical (lithium acetate) transformation was not or only slightly affected by these deletions. Our data are consistent with a positive role of host histone deacetylation in AMT.

  2. Recognition of Unmodified Histone H3 by the First PHD Finger of Bromodomain-PHD Finger Protein 2 Provides Insights into the Regulation of Histone Acetyltransferases Monocytic Leukemic Zinc-finger Protein (MOZ) and MOZ-related factor (MORF)*

    PubMed Central

    Qin, Su; Jin, Lei; Zhang, Jiahai; Liu, Lei; Ji, Peng; Wu, Mian; Wu, Jihui; Shi, Yunyu

    2011-01-01

    MOZ (monocytic leukemic zinc-finger protein) and MORF (MOZ-related factor) are histone acetyltransferases important for HOX gene expression as well as embryo and postnatal development. They form complexes with other regulatory subunits through the scaffold proteins BRPF1/2/3 (bromodomain-PHD (plant homeodomain) finger proteins 1, 2, or 3). BRPF proteins have multiple domains, including two PHD fingers, for potential interactions with histones. Here we show that the first PHD finger of BRPF2 specifically recognizes the N-terminal tail of unmodified histone H3 (unH3) and report the solution structures of this PHD finger both free and in complex with the unH3 peptide. Structural analysis revealed that the unH3 peptide forms a third antiparallel β-strand that pairs with the PHD1 two-stranded antiparallel β-sheet. The binding specificity was determined primarily through the recognition of arginine 2 and lysine 4 of the unH3 by conserved aspartic acids of PHD1 and of threonine 6 of the unH3 by a conserved asparagine. Isothermal titration calorimetry and NMR assays showed that post-translational modifications such as H3R2me2as, H3T3ph, H3K4me, H3K4ac, and H3T6ph antagonized the interaction between histone H3 and PHD1. Furthermore, histone binding by PHD1 was important for BRPF2 to localize to the HOXA9 locus in vivo. PHD1 is highly conserved in yeast NuA3 and other histone acetyltransferase complexes, so the results reported here also shed light on the function and regulation of these complexes. PMID:21880731

  3. Lysine Acetyltransferase GCN5 Potentiates the Growth of Non-small Cell Lung Cancer via Promotion of E2F1, Cyclin D1, and Cyclin E1 Expression*

    PubMed Central

    Chen, Long; Wei, Tingyi; Si, Xiaoxing; Wang, Qianqian; Li, Yan; Leng, Ye; Deng, Anmei; Chen, Jie; Wang, Guiying; Zhu, Songcheng; Kang, Jiuhong

    2013-01-01

    The lysine acetyltransferases play crucial but complex roles in cancer development. GCN5 is a lysine acetyltransferase that generally regulates gene expression, but its role in cancer development remains largely unknown. In this study, we report that GCN5 is highly expressed in non-small cell lung cancer tissues and that its expression correlates with tumor size. We found that the expression of GCN5 promotes cell growth and the G1/S phase transition in multiple lung cancer cell lines. Further study revealed that GCN5 regulates the expression of E2F1, cyclin D1, and cyclin E1. Our reporter assays indicated that the expression of GCN5 enhances the activities of the E2F1, cyclin D1, and cyclin E1 promoters. ChIP experiments suggested that GCN5 binds directly to these promoters and increases the extent of histone acetylation within these regions. Mechanistic studies suggested that GCN5 interacts with E2F1 and is recruited by E2F1 to the E2F1, cyclin D1, and cyclin E1 promoters. The function of GCN5 in lung cancer cells is abrogated by the knockdown of E2F1. Finally, we confirmed that GCN5 regulates the expression of E2F1, cyclin D1, and cyclin E1 and potentiates lung cancer cell growth in a mouse tumor model. Taken together, our results demonstrate that GCN5 specifically potentiates lung cancer growth by directly promoting the expression of E2F1, cyclin D1, and cyclin E1 in an E2F1-dependent manner. Our study identifies a specific and novel function of GCN5 in lung cancer development and suggests that the GCN5-E2F1 interaction represents a potential target for lung cancer treatment. PMID:23543735

  4. Structural and Functional Role of Acetyltransferase hMOF K274 Autoacetylation.

    PubMed

    McCullough, Cheryl E; Song, Shufei; Shin, Michael H; Johnson, F Brad; Marmorstein, Ronen

    2016-08-26

    Many histone acetyltransferases undergo autoacetylation, either through chemical or enzymatic means, to potentiate enzymatic cognate substrate lysine acetylation, although the mode and molecular role of such autoacetylation is poorly understood. The MYST family of histone acetyltransferases is autoacetylated at an active site lysine residue to facilitate cognate substrate lysine binding and acetylation. Here, we report on a detailed molecular investigation of Lys-274 autoacetylation of the human MYST protein Males Absent on the First (hMOF). A mutational scan of hMOF Lys-274 reveals that all amino acid substitutions of this residue are able to bind cofactor but are significantly destabilized, both in vitro and in cells, and are catalytically inactive for cognate histone H4 peptide lysine acetylation. The x-ray crystal structure of a hMOF K274P mutant suggests that the reduced stability and catalytic activity stems from a disordering of the residue 274-harboring a α2-β7 loop. We also provide structural evidence that a C316S/E350Q mutant, which is defective for cognate substrate lysine acetylation; and biochemical evidence that a K268M mutant, which is defective for Lys-274 chemical acetylation in the context of a K274-peptide, can still undergo quantitative K274 autoacetylation. Together, these studies point to the critical and specific role of hMOF Lys-274 autoacetylation in hMOF stability and cognate substrate acetylation and argues that binding of Ac-CoA to hMOF likely drives Lys-274 autoacetylation for subsequent cognate substrate acetylation. PMID:27382063

  5. Atomic resolution structure of human α-tubulin acetyltransferase bound to acetyl-CoA

    PubMed Central

    Taschner, Michael; Vetter, Melanie; Lorentzen, Esben

    2012-01-01

    Acetylation of lysine residues is an important posttranslational modification found in all domains of life. α-tubulin is specifically acetylated on lysine 40, a modification that serves to stabilize microtubules of axons and cilia. Whereas histone acetyltransferases have been extensively studied, there is no structural and mechanistic information available on α-tubulin acetyltransferases. Here, we present the structure of the human α-tubulin acetyltransferase catalytic domain bound to its cosubstrate acetyl-CoA at 1.05 Å resolution. Compared with other lysine acetyltransferases of known structure, α-tubulin acetyltransferase displays a relatively well-conserved cosubstrate binding pocket but is unique in its active site and putative α-tubulin binding site. Using acetylation assays with structure-guided mutants, we map residues important for acetyl-CoA binding, substrate binding, and catalysis. This analysis reveals a basic patch implicated in substrate binding and a conserved glutamine residue required for catalysis, demonstrating that the family of α-tubulin acetyltransferases uses a reaction mechanism different from other lysine acetyltransferases characterized to date. PMID:23071318

  6. Analysis of promoters in Borrelia burgdorferi by use of a transiently expressed reporter gene.

    PubMed Central

    Sohaskey, C D; Arnold, C; Barbour, A G

    1997-01-01

    A transient chloramphenicol acetyltransferase (CAT) expression system was developed for Borrelia burgdorferi. An Escherichia coli vector containing a promoterless Streptococcus agalactiae cat gene was constructed. Promoters for ospA, ospC, and flaB were placed upstream of this cat gene, and CAT assays were performed in E. coli from these stably maintained plasmids. The plasmids with putative promoters ospA and flaB were found to be approximately 20-fold more active than were the plasmids with ospC or no promoter. The level of activity correlated well with the resistance to chloramphenicol that each plasmid provided. Next, the nonreplicative plasmid constructs were transformed by electroporation into B. burgdorferi. CAT assays were performed by both thin-layer chromatography and the fluor diffusion method. Measurement of CAT activity demonstrated that the ospA promoter was again about 20-fold more active than the promoterless cat gene. The flaB and ospC promoters increased the activity seven- and threefold, respectively, over that with the promoterless construct. This simple transient-expression assay was shown to be an effective method to study promoter function in B. burgdorferi in the absence of a well-developed genetic system. PMID:9352937

  7. Histone acetyltransferases and histone deacetylases in B- and T-cell development, physiology and malignancy

    PubMed Central

    Haery, Leila; Thompson, Ryan C.; Gilmore, Thomas D.

    2015-01-01

    The development of B and T cells from hematopoietic precursors and the regulation of the functions of these immune cells are complex processes that involve highly regulated signaling pathways and transcriptional control. The signaling pathways and gene expression patterns that give rise to these developmental processes are coordinated, in part, by two opposing classes of broad-based enzymatic regulators: histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs and HDACs can modulate gene transcription by altering histone acetylation to modify chromatin structure, and by regulating the activity of non-histone substrates, including an array of immune-cell transcription factors. In addition to their role in normal B and T cells, dysregulation of HAT and HDAC activity is associated with a variety of B- and T-cell malignancies. In this review, we describe the roles of HATs and HDACs in normal B- and T-cell physiology, describe mutations and dysregulation of HATs and HDACs that are implicated lymphoma and leukemia, and discuss HAT and HDAC inhibitors that have been explored as treatment options for leukemias and lymphomas. PMID:26124919

  8. The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex in Aspergillus nidulans.

    PubMed

    Georgakopoulos, Paraskevi; Lockington, Robin A; Kelly, Joan M

    2013-01-01

    A mutation screen in Aspergillus nidulans uncovered mutations in the acdX gene that led to altered repression by acetate, but not by glucose. AcdX of A. nidulans is highly conserved with Spt8p of Saccharomyces cerevisiae, and since Spt8p is a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, the SAGA complex may have a role in acetate repression in A. nidulans. We used a bioinformatic approach to identify genes encoding most members of the SAGA complex in A. nidulans, and a proteomic analysis to confirm that most protein components identified indeed exist as a complex in A. nidulans. No apparent compositional differences were detected in mycelia cultured in acetate compared to glucose medium. The methods used revealed apparent differences between Yeast and A. nidulans in the deubiquitination (DUB) module of the complex, which in S. cerevisiae consists of Sgf11p, Sus1p, and Ubp8p. Although a convincing homologue of S. cerevisiae Ubp8p was identified in the A. nidulans genome, there were no apparent homologues for Sus1p and Sgf11p. In addition, when the SAGA complex was purified from A. nidulans, members of the DUB module were not co-purified with the complex, indicating that functional homologues of Sus1p and Sgf11p were not part of the complex. Thus, deubiquitination of H2B-Ub in stress conditions is likely to be regulated differently in A. nidulans compared to S. cerevisiae.

  9. A chromosomal chloramphenicol acetyltransferase determinant from a probiotic strain of Bacillus clausii.

    PubMed

    Galopin, Sébastien; Cattoir, Vincent; Leclercq, Roland

    2009-06-01

    The mechanism of resistance to chloramphenicol was studied in four strains of Bacillus clausii included in a probiotic mixture, which is administered to humans for prevention of gastrointestinal side effects due to oral antibiotic therapy. By cloning experiments, a chloramphenicol acetyltransferase (CAT) gene, cat(Bcl), coding for a putative 228-amino acid CAT protein was identified in B. clausii SIN. The deduced amino acid sequence displayed from 31% to 85% identity with 56 CAT proteins from other Gram-positive bacterial strains. The cat(Bcl) gene was also detected by PCR in the three other B. clausii strains resistant to chloramphenicol, whereas it was absent in the three control strains susceptible to chloramphenicol. Pulse-field gel electrophoresis of total DNA digested by I-CeuI followed by hybridization with a cat-specific probe as well as unsuccessful repeated attempts of in vitro transfer of chloramphenicol resistance to various recipient cells indicated that cat(Bcl) was chromosomally located in all four resistant B. clausii strains. PMID:19459958

  10. The MOZ Histone Acetyltransferase in Epigenetic Signaling and Disease

    PubMed Central

    Carlson, Samuel; Glass, Karen C.

    2016-01-01

    The monocytic leukemic zinc finger (MOZ) histone acetyltransferase (HAT) plays a role in acute myeloid leukemia (AML). It functions as a quaternary complex with the bromodomain PHD finger protein 1 (BRPF1), the human Esa1-associated factor 6 homolog (hEAF6), and the inhibitor of growth 5 (ING5). Each of these subunits contain chromatin reader domains that recognize specific post-translational modifications (PTMs) on histone tails, and this recognition directs the MOZ HAT complex to specific chromatin substrates. The structure and function of these epigenetic reader modules has now been elucidated, and a model describing how the cooperative activity of these domains regulates HAT activity in response to the epigenetic landscape is proposed. The emerging role of epigenetic reader domains in disease, and their therapeutic potential for many types of cancer is also highlighted. PMID:24633655

  11. The polyamine N-acetyltransferase-like enzyme PmvE plays a role in the virulence of Enterococcus faecalis.

    PubMed

    Martini, Cecilia; Michaux, Charlotte; Bugli, Francesca; Arcovito, Alessandro; Iavarone, Federica; Cacaci, Margherita; Paroni Sterbini, Francesco; Hartke, Axel; Sauvageot, Nicolas; Sanguinetti, Maurizio; Posteraro, Brunella; Giard, Jean-Christophe

    2015-01-01

    We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of the slyA operon, ef_3001, renamed pmvE (for polyamine metabolism and virulence of E. faecalis). PmvE shares strong homologies with N(1)-spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used an E. faecalis strain carrying the recombinant plasmid pMSP3535-pmvE (V19/p3535-pmvE), which allows the induction of pmvE by addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence of E. faecalis in the Galleria mellonella infection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses an N-acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence of E. faecalis, likely through its involvement in the polyamine metabolism. PMID:25385793

  12. Dual-modality gene reporter for in vivo imaging.

    PubMed

    Patrick, P Stephen; Hammersley, Jayne; Loizou, Louiza; Kettunen, Mikko I; Rodrigues, Tiago B; Hu, De-En; Tee, Sui-Seng; Hesketh, Robin; Lyons, Scott K; Soloviev, Dmitry; Lewis, David Y; Aime, Silvio; Fulton, Sandra M; Brindle, Kevin M

    2014-01-01

    The ability to track cells and their patterns of gene expression in living organisms can increase our understanding of tissue development and disease. Gene reporters for bioluminescence, fluorescence, radionuclide, and magnetic resonance imaging (MRI) have been described but these suffer variously from limited depth penetration, spatial resolution, and sensitivity. We describe here a gene reporter, based on the organic anion transporting protein Oatp1a1, which mediates uptake of a clinically approved, Gd(3+)-based, hepatotrophic contrast agent (gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid). Cells expressing the reporter showed readily reversible, intense, and positive contrast (up to 7.8-fold signal enhancement) in T1-weighted magnetic resonance images acquired in vivo. The maximum signal enhancement obtained so far is more than double that produced by MRI gene reporters described previously. Exchanging the Gd(3+) ion for the radionuclide, (111)In, also allowed detection by single-photon emission computed tomography, thus combining the spatial resolution of MRI with the sensitivity of radionuclide imaging.

  13. Crystallization and preliminary X-ray characterization of arylamine N-acetyltransferase C (BanatC) from Bacillus anthracis

    SciTech Connect

    Pluvinage, Benjamin; Li de la Sierra-Gallay, Inés; Martins, Marta; Ragunathan, Nilusha; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2007-10-01

    Bacillus anthracis arylamine N-acetyltransferase C (BanatC) is an enzyme that metabolizes the drug sulfamethoxazole. Crystals of the purified enzyme that diffract at 1.95 Å are reported. The arylamine N-acetyltransferase (NAT) enzymes are xenobiotic metabolizing enzymes that have been found in a large range of eukaryotes and prokaryotes. These enzymes catalyse the acetylation of arylamine drugs and/or pollutants. Recently, a Bacillus anthracis NAT isoform (BanatC) has been cloned and shown to acetylate the sulfonamide antimicrobial sulfamethoxazole (SMX). Subsequently, it was shown that BanatC contributes to the resistance of this bacterium to SMX. Here, the crystallization and the X-ray characterization of BanatC (Y38F mutant) are reported. The crystals belong to the tetragonal space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 53.70, c = 172.40 Å, and diffract to 1.95 Å resolution on a synchrotron source.

  14. Transferring Gus gene into intact rice cells by low energy ion beam

    NASA Astrophysics Data System (ADS)

    Zengliang, Yu; Jianbo, Yang; Yuejin, Wu; Beijiu, Cheng; Jianjun, He; Yuping, Huo

    1993-06-01

    A new technique of transferring genes by low energy ion beam has been reported in this paper. The Gus and CAT (chloramphenicol acetyltransferase) genes, as "foreign" genetic materials, were introduced into the suspension cells and ripe embryos or rice by implantation of 20-30 keV Ar + at doses ranging from 1 × 10 15 to 4 × 10 15 ions/cm 2. The activities of CAT and Gus were detected in the cells and embryos after several weeks. The results indicate that the transfer was a success.

  15. Direct Introduction of Genes into Rats and Expression of the Genes

    NASA Astrophysics Data System (ADS)

    Benvenisty, Nissim; Reshef, Lea

    1986-12-01

    A method of introducing actively expressed genes into intact mammals is described. DNA precipitated with calcium phosphate has been injected intraperitoneally into newborn rats. The injected genes have been taken up and expressed by the animal tissues. To examine the generality of the method we have injected newborn rats with the chloramphenicol acetyltransferase prokaryotic gene fused with various viral and cellular gene promoters and the gene for hepatitis B surface antigen, and we observed appearance of chloramphenicol acetyltransferase activity and hepatitis B surface antigen in liver and spleen. In addition, administration of genes coding for hormones (insulin or growth hormone) resulted in their expression.

  16. Ferritin reporter used for gene expression imaging by magnetic resonance

    SciTech Connect

    Ono, Kenji; Fuma, Kazuya; Tabata, Kaori; Sawada, Makoto

    2009-10-23

    Magnetic resonance imaging (MRI) is a minimally invasive way to provide high spatial resolution tomograms. However, MRI has been considered to be useless for gene expression imaging compared to optical imaging. In this study, we used a ferritin reporter, binding with biogenic iron, to make it a powerful tool for gene expression imaging in MRI studies. GL261 mouse glioma cells were over-expressed with dual-reporter ferritin-DsRed under {beta}-actin promoter, then gene expression was observed by optical imaging and MRI in a brain tumor model. GL261 cells expressing ferritin-DsRed fusion protein showed enhanced visualizing effect by reducing T2-weighted signal intensity for in vitro and in vivo MRI studies, as well as DsRed fluorescence for optical imaging. Furthermore, a higher contrast was achieved on T2-weighted images when permeating the plasma membrane of ferritin-DsRed-expressing GL261. Thus, a ferritin expression vector can be used as an MRI reporter to monitor in vivo gene expression.

  17. Photoacoustic imaging of gene expression using tyrosinase as a reporter gene

    NASA Astrophysics Data System (ADS)

    Paproski, Robert J.; Forbrich, Alexander; Harrison, Tyler; Hitt, Mary; Zemp, Roger J.

    2011-03-01

    Optical reporter genes, such as green fluorescence protein, are powerful research tools that allow visualization of gene expression. We have successfully used tyrosinase as a reporter gene for photoacoustic imaging. Tyrosinase is the key regulatory enzyme in the production of melanin which has a broad optical absorption spectrum. MCF-7 cells were stably transfected with tyrosinase under the control of an inducible promoter. For photoacoustic experiments, MCF-7 cells were resuspended at 108 cells/mL and injected in 700 μm (inner diameter) plastic tubing. Photoacoustic signal of MCF-7 cells expressing tyrosinase were >20-fold greater than those of untransfected MCF-7 cells. Photoacoustic signal of tyrosinaseexpressing MCF-7 cells were approximately 2-fold lesser and greater than those of blood at 576 and 650 nm, respectively, suggesting that photoacoustic signal from blood and tyrosinase-expressing cells can be separated by dualwavelength analysis. Photoacoustic signal from tyrosinase-expressing MCF-7 cells covered by chicken tissue could even be detected at a laser penetration depth of 4 cm, suggesting that tyrosinase can be used to image gene expression in relatively deep tissues. The current data suggests that tyrosinase is a strong reporter gene for photoacoustic imaging.

  18. Single Cell Visualization of Yeast Gene Expression Shows Correlation of Epigenetic Switching between Multiple Heterochromatic Regions through Multiple Generations

    PubMed Central

    Mano, Yasunobu; Kobayashi, Tetsuya J.; Nakayama, Jun-ichi; Uchida, Hiroyuki; Oki, Masaya

    2013-01-01

    Differences in gene expression between individual cells can be mediated by epigenetic regulation; thus, methods that enable detailed analyses of single cells are crucial to understanding this phenomenon. In this study, genomic silencing regions of Saccharomyces cerevisiae that are subject to epigenetic regulation, including the HMR, HML, and telomere regions, were investigated using a newly developed single cell analysis method. This method uses fluorescently labeled proteins to track changes in gene expression over multiple generations of a single cell. Epigenetic control of gene expression differed depending on the specific silencing region at which the reporter gene was inserted. Correlations between gene expression at the HMR-left and HMR-right regions, as well as the HMR-right and HML-right regions, were observed in the single-cell level; however, no such correlations involving the telomere region were observed. Deletion of the histone acetyltransferase GCN5 gene from a yeast strain carrying a fluorescent reporter gene at the HMR-left region reduced the frequency of changes in gene expression over a generation. The results presented here suggest that epigenetic control within an individual cell is reversible and can be achieved via regulation of histone acetyltransferase activity. PMID:23843746

  19. Molecular basis for histone acetyltransferase regulation by binding partners, associated domains, and autoacetylation

    PubMed Central

    McCullough, Cheryl E.; Marmorstein, Ronen

    2016-01-01

    Acetylation is a post-translational modification (PTM) that regulates chromatin dynamics and function. Dysregulation of acetylation or acetyltransferase activity has been correlated with several human diseases. Many, if not all histone acetyltransferases (HATs) are regulated in part through tethered domains, association with binding partners or post-translational modification, including predominantly acetylation. This review focuses on what is currently understood at the molecular level of HAT regulation as it occurs via binding partners, associated domains, and autoacetylation. PMID:26555232

  20. Novel ligands of Choline Acetyltransferase designed by in silico molecular docking, hologram QSAR and lead optimization.

    PubMed

    Kumar, Rajnish; Långström, Bengt; Darreh-Shori, Taher

    2016-01-01

    Recent reports have brought back the acetylcholine synthesizing enzyme, choline acetyltransferase in the mainstream research in dementia and the cholinergic anti-inflammatory pathway. Here we report, a specific strategy for the design of novel ChAT ligands based on molecular docking, Hologram Quantitative Structure Activity Relationship (HQSAR) and lead optimization. Molecular docking was performed on a series of ChAT inhibitors to decipher the molecular fingerprint of their interaction with the active site of ChAT. Then robust statistical fragment HQSAR models were developed. A library of novel ligands was generated based on the pharmacophoric and shape similarity scoring function, and evaluated in silico for their molecular interactions with ChAT. Ten of the top scoring invented compounds are reported here. We confirmed the activity of α-NETA, the only commercially available ChAT inhibitor, and one of the seed compounds in our model, using a new simple colorimetric ChAT assay (IC50 ~ 88 nM). In contrast, α-NETA exhibited an IC50 of ~30 μM for the ACh-degrading cholinesterases. In conclusion, the overall results may provide useful insight for discovering novel ChAT ligands and potential positron emission tomography tracers as in vivo functional biomarkers of the health of central cholinergic system in neurodegenerative disorders, such as Alzheimer's disease.

  1. Novel ligands of Choline Acetyltransferase designed by in silico molecular docking, hologram QSAR and lead optimization

    PubMed Central

    Kumar, Rajnish; Långström, Bengt; Darreh-Shori, Taher

    2016-01-01

    Recent reports have brought back the acetylcholine synthesizing enzyme, choline acetyltransferase in the mainstream research in dementia and the cholinergic anti-inflammatory pathway. Here we report, a specific strategy for the design of novel ChAT ligands based on molecular docking, Hologram Quantitative Structure Activity Relationship (HQSAR) and lead optimization. Molecular docking was performed on a series of ChAT inhibitors to decipher the molecular fingerprint of their interaction with the active site of ChAT. Then robust statistical fragment HQSAR models were developed. A library of novel ligands was generated based on the pharmacophoric and shape similarity scoring function, and evaluated in silico for their molecular interactions with ChAT. Ten of the top scoring invented compounds are reported here. We confirmed the activity of α-NETA, the only commercially available ChAT inhibitor, and one of the seed compounds in our model, using a new simple colorimetric ChAT assay (IC50 ~ 88 nM). In contrast, α-NETA exhibited an IC50 of ~30 μM for the ACh-degrading cholinesterases. In conclusion, the overall results may provide useful insight for discovering novel ChAT ligands and potential positron emission tomography tracers as in vivo functional biomarkers of the health of central cholinergic system in neurodegenerative disorders, such as Alzheimer’s disease. PMID:27507101

  2. Novel ligands of Choline Acetyltransferase designed by in silico molecular docking, hologram QSAR and lead optimization.

    PubMed

    Kumar, Rajnish; Långström, Bengt; Darreh-Shori, Taher

    2016-01-01

    Recent reports have brought back the acetylcholine synthesizing enzyme, choline acetyltransferase in the mainstream research in dementia and the cholinergic anti-inflammatory pathway. Here we report, a specific strategy for the design of novel ChAT ligands based on molecular docking, Hologram Quantitative Structure Activity Relationship (HQSAR) and lead optimization. Molecular docking was performed on a series of ChAT inhibitors to decipher the molecular fingerprint of their interaction with the active site of ChAT. Then robust statistical fragment HQSAR models were developed. A library of novel ligands was generated based on the pharmacophoric and shape similarity scoring function, and evaluated in silico for their molecular interactions with ChAT. Ten of the top scoring invented compounds are reported here. We confirmed the activity of α-NETA, the only commercially available ChAT inhibitor, and one of the seed compounds in our model, using a new simple colorimetric ChAT assay (IC50 ~ 88 nM). In contrast, α-NETA exhibited an IC50 of ~30 μM for the ACh-degrading cholinesterases. In conclusion, the overall results may provide useful insight for discovering novel ChAT ligands and potential positron emission tomography tracers as in vivo functional biomarkers of the health of central cholinergic system in neurodegenerative disorders, such as Alzheimer's disease. PMID:27507101

  3. Epigenetic chromatin modifiers in barley: III. Isolation and characterization of the barley GNAT-MYST family of histone acetyltransferases and responses to exogenous ABA.

    PubMed

    Papaefthimiou, Dimitra; Likotrafiti, Eleni; Kapazoglou, Aliki; Bladenopoulos, Konstantinos; Tsaftaris, Athanasios

    2010-01-01

    Histone acetylation is a vital mechanism for the activation of chromatin and the corresponding expression of genes competing the action of histone deacetylation and leading to chromatin inactivation. Histone acetyltransferases (HATs) comprise a superfamily including the GNAT/MYST, CBP and TF(II)250 families. Histone acetyltransferases have been well studied in Arabidopsis but information from agronomically important crops is limited. In the present work three full-length sequences encoding members of the GNAT/MYST family, namely HvMYST, HvELP3 and HvGCN5, respectively, were isolated and characterized from barley (Hordeum vulgare L.), a crop of high economic value. Expression analysis of the barley GNAT/MYST genes revealed significant quantitative differences in different seed developmental stages and between cultivars with varying seed size and weight, suggesting an association of these genes with barley seed development. Furthermore, all three HvGNAT/MYST genes were inducible by the stress-related phytohormone abscisic acid (ABA) involved in seed maturation, dormancy and germination, implying a possible regulation of these genes by ABA, during barley seed development, germination and stress response. PMID:20117010

  4. The Aspergillus flavus Histone Acetyltransferase AflGcnE Regulates Morphogenesis, Aflatoxin Biosynthesis, and Pathogenicity.

    PubMed

    Lan, Huahui; Sun, Ruilin; Fan, Kun; Yang, Kunlong; Zhang, Feng; Nie, Xin Y; Wang, Xiunai; Zhuang, Zhenhong; Wang, Shihua

    2016-01-01

    Histone acetyltransferases (HATs) help regulate fungal development and the production of secondary metabolites. In this study, we determined that the HAT AflGcnE influenced morphogenesis and aflatoxin biosynthesis in Aspergillus flavus. We observed that AflGcnE localized to the nucleus and cytoplasm during the conidial production and germination stages, while it was located mainly in the nucleus during the hyphal development stage. Deletion of AflgcnE inhibited the growth of A. flavus and decreased the hydrophobicity of the cell surface. The ΔAflgcnE mutant exhibited a lack of asexual sporulation and was unable to generate sclerotia. Additionally, AflgcnE was required to maintain cell wall integrity and genotoxic stress responses. Importantly, the ΔAflgcnE mutant did not produce aflatoxins, which was consistent with a significant down-regulation of aflatoxin gene expression levels. Furthermore, our data revealed that AflgcnE is a pathogenicity factor required for colonizing maize seeds. In summary, we revealed that A. flavus AflGcnE is crucial for morphological development, aflatoxin biosynthesis, stress responses, and pathogenicity. Our findings help clarify the functional divergence of GcnE orthologs, and may provide a possible target for controlling A. flavus infections of agriculturally important crops. PMID:27625637

  5. P300 acetyltransferase regulates fatty acid synthase expression, lipid metabolism and prostate cancer growth.

    PubMed

    Gang, Xiaokun; Yang, Yinhui; Zhong, Jian; Jiang, Kui; Pan, Yunqian; Karnes, R Jeffrey; Zhang, Jun; Xu, Wanhai; Wang, Guixia; Huang, Haojie

    2016-03-22

    De novo fatty acid (FA) synthesis is required for prostate cancer (PCa) survival and progression. As a key enzyme for FA synthesis fatty acid synthase (FASN) is often overexpressed in human prostate cancers and its expression correlates with worse prognosis and poor survival. P300 is an acetyltransferase that acts as a transcription co-activator. Increasing evidence suggests that P300 is a major PCa promoter, although the underlying mechanism remains poorly understood. Here, we demonstrated that P300 binds to and increases histone H3 lysine 27 acetylation (H3K27Ac) in the FASN gene promoter. We provided evidence that P300 transcriptionally upregulates FASN expression and promotes lipid accumulation in human PCa cells in culture and Pten knockout prostate tumors in mice. Pharmacological inhibition of P300 decreased FASN expression and lipid droplet accumulation in PCa cells. Immunohistochemistry analysis revealed that expression of P300 protein positively correlates with FASN protein levels in a cohort of human PCa specimens. We further showed that FASN is a key mediator of P300-induced growth of PCa cells in culture and in mice. Together, our findings demonstrate P300 as a key factor that regulates FASN expression, lipid accumulation and cell growth in PCa. They also suggest that this regulatory pathway can serve as a new therapeutic target for PCa treatment. PMID:26934656

  6. Human acetyl CoA:arylamine N-acetyltransferase variants generated by random mutagenesis.

    PubMed

    Summerscales, Joanna E; Josephy, P David

    2004-01-01

    Acetyl CoA:arylamine N-acetyltransferase (NAT) enzymes catalyze the N-acetylation of aromatic amines and the O-acetylation of aryl hydroxylamines, reactions that govern the disposition and toxicity of many drugs and carcinogens. The human NAT genes and enzymes NAT1 and NAT2 are highly polymorphic and constitute one of the best studied examples of the genetic control of drug metabolism. Naturally occurring human NAT variants provide limited insight into the relationship between NAT amino acid sequence and enzyme activity. We have shown previously that the expression of recombinant NAT2 in bacterial tester strains results in greatly enhanced sensitivity to mutagenic nitroaromatic compounds (which are reduced to aryl hydroxylamines by bacterial enzymes). We hypothesized that random mutagenesis combined with rapid screening could be used to identify functionally significant amino acid residues in NAT enzymes. Pools of NAT2 variants were generated by polymerase chain reaction-mediated random mutagenesis of the complete coding sequence. Reversion induced by a NAT-dependent mutagen, 3-methyl-2-nitroimidazo[4,5-f]quinoline, was used as the basis for screening these pools to identify variants with altered enzyme activity. Eighteen variants were characterized by quantitative mutagenicity assays and enzyme kinetic measurements. This approach can provide new insight into the biochemistry of enzymes involved in the metabolic activation of mutagens. PMID:14722254

  7. P300 acetyltransferase regulates fatty acid synthase expression, lipid metabolism and prostate cancer growth.

    PubMed

    Gang, Xiaokun; Yang, Yinhui; Zhong, Jian; Jiang, Kui; Pan, Yunqian; Karnes, R Jeffrey; Zhang, Jun; Xu, Wanhai; Wang, Guixia; Huang, Haojie

    2016-03-22

    De novo fatty acid (FA) synthesis is required for prostate cancer (PCa) survival and progression. As a key enzyme for FA synthesis fatty acid synthase (FASN) is often overexpressed in human prostate cancers and its expression correlates with worse prognosis and poor survival. P300 is an acetyltransferase that acts as a transcription co-activator. Increasing evidence suggests that P300 is a major PCa promoter, although the underlying mechanism remains poorly understood. Here, we demonstrated that P300 binds to and increases histone H3 lysine 27 acetylation (H3K27Ac) in the FASN gene promoter. We provided evidence that P300 transcriptionally upregulates FASN expression and promotes lipid accumulation in human PCa cells in culture and Pten knockout prostate tumors in mice. Pharmacological inhibition of P300 decreased FASN expression and lipid droplet accumulation in PCa cells. Immunohistochemistry analysis revealed that expression of P300 protein positively correlates with FASN protein levels in a cohort of human PCa specimens. We further showed that FASN is a key mediator of P300-induced growth of PCa cells in culture and in mice. Together, our findings demonstrate P300 as a key factor that regulates FASN expression, lipid accumulation and cell growth in PCa. They also suggest that this regulatory pathway can serve as a new therapeutic target for PCa treatment.

  8. Structural Basis for Microcin C7 Inactivation by the MccE Acetyltransferase

    SciTech Connect

    Agarwal, Vinayak; Metlitskaya, Anastasiya; Severinov, Konstantin; Nair, Satish K.

    2015-10-15

    The antibiotic microcin C7 (McC) acts as a bacteriocide by inhibiting aspartyl-tRNA synthetase and stalling the protein translation machinery. McC is synthesized as a heptapeptide-nucleotide conjugate, which is processed by cellular peptidases within target strains to yield the biologically active compound. As unwanted processing of intact McC can result in self-toxicity, producing strains utilize multiple mechanisms for autoimmunity against processed McC. We have shown previously that the mccE gene within the biosynthetic cluster can inactivate processed McC by acetylating the antibiotic. Here, we present the characterization of this acetylation mechanism through biochemical and structural biological studies of the MccE acetyltransferase domain (MccE{sup AcTase}). We have also determined five crystal structures of the MccE-acetyl-CoA complex with bound substrates, inhibitor, and reaction product. The structural data reveal an unexpected mode of substrate recognition through p-stacking interactions similar to those found in cap-binding proteins and nucleotidyltransferases. These studies provide a rationale for the observation that MccE{sup AcTase} can detoxify a range of aminoacylnucleotides, including those that are structurally distinct from microcin C7.

  9. The Aspergillus flavus Histone Acetyltransferase AflGcnE Regulates Morphogenesis, Aflatoxin Biosynthesis, and Pathogenicity

    PubMed Central

    Lan, Huahui; Sun, Ruilin; Fan, Kun; Yang, Kunlong; Zhang, Feng; Nie, Xin Y.; Wang, Xiunai; Zhuang, Zhenhong; Wang, Shihua

    2016-01-01

    Histone acetyltransferases (HATs) help regulate fungal development and the production of secondary metabolites. In this study, we determined that the HAT AflGcnE influenced morphogenesis and aflatoxin biosynthesis in Aspergillus flavus. We observed that AflGcnE localized to the nucleus and cytoplasm during the conidial production and germination stages, while it was located mainly in the nucleus during the hyphal development stage. Deletion of AflgcnE inhibited the growth of A. flavus and decreased the hydrophobicity of the cell surface. The ΔAflgcnE mutant exhibited a lack of asexual sporulation and was unable to generate sclerotia. Additionally, AflgcnE was required to maintain cell wall integrity and genotoxic stress responses. Importantly, the ΔAflgcnE mutant did not produce aflatoxins, which was consistent with a significant down-regulation of aflatoxin gene expression levels. Furthermore, our data revealed that AflgcnE is a pathogenicity factor required for colonizing maize seeds. In summary, we revealed that A. flavus AflGcnE is crucial for morphological development, aflatoxin biosynthesis, stress responses, and pathogenicity. Our findings help clarify the functional divergence of GcnE orthologs, and may provide a possible target for controlling A. flavus infections of agriculturally important crops. PMID:27625637

  10. Rational design and validation of a Tip60 histone acetyltransferase inhibitor

    NASA Astrophysics Data System (ADS)

    Gao, Chunxia; Bourke, Emer; Scobie, Martin; Famme, Melina Arcos; Koolmeister, Tobias; Helleday, Thomas; Eriksson, Leif A.; Lowndes, Noel F.; Brown, James A. L.

    2014-06-01

    Histone acetylation is required for many aspects of gene regulation, genome maintenance and metabolism and dysfunctional acetylation is implicated in numerous diseases, including cancer. Acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases and currently, few general HAT inhibitors have been described. We identified the HAT Tip60 as an excellent candidate for targeted drug development, as Tip60 is a key mediator of the DNA damage response and transcriptional co-activator. Our modeling of Tip60 indicated that the active binding pocket possesses opposite charges at each end, with the positive charges attributed to two specific side chains. We used structure based drug design to develop a novel Tip60 inhibitor, TH1834, to fit this specific pocket. We demonstrate that TH1834 significantly inhibits Tip60 activity in vitro and treating cells with TH1834 results in apoptosis and increased unrepaired DNA damage (following ionizing radiation treatment) in breast cancer but not control cell lines. Furthermore, TH1834 did not affect the activity of related HAT MOF, as indicated by H4K16Ac, demonstrating specificity. The modeling and validation of the small molecule inhibitor TH1834 represents a first step towards developing additional specific, targeted inhibitors of Tip60 that may lead to further improvements in the treatment of breast cancer.

  11. The Aspergillus flavus Histone Acetyltransferase AflGcnE Regulates Morphogenesis, Aflatoxin Biosynthesis, and Pathogenicity

    PubMed Central

    Lan, Huahui; Sun, Ruilin; Fan, Kun; Yang, Kunlong; Zhang, Feng; Nie, Xin Y.; Wang, Xiunai; Zhuang, Zhenhong; Wang, Shihua

    2016-01-01

    Histone acetyltransferases (HATs) help regulate fungal development and the production of secondary metabolites. In this study, we determined that the HAT AflGcnE influenced morphogenesis and aflatoxin biosynthesis in Aspergillus flavus. We observed that AflGcnE localized to the nucleus and cytoplasm during the conidial production and germination stages, while it was located mainly in the nucleus during the hyphal development stage. Deletion of AflgcnE inhibited the growth of A. flavus and decreased the hydrophobicity of the cell surface. The ΔAflgcnE mutant exhibited a lack of asexual sporulation and was unable to generate sclerotia. Additionally, AflgcnE was required to maintain cell wall integrity and genotoxic stress responses. Importantly, the ΔAflgcnE mutant did not produce aflatoxins, which was consistent with a significant down-regulation of aflatoxin gene expression levels. Furthermore, our data revealed that AflgcnE is a pathogenicity factor required for colonizing maize seeds. In summary, we revealed that A. flavus AflGcnE is crucial for morphological development, aflatoxin biosynthesis, stress responses, and pathogenicity. Our findings help clarify the functional divergence of GcnE orthologs, and may provide a possible target for controlling A. flavus infections of agriculturally important crops.

  12. Mutant SOD1 impairs axonal transport of choline acetyltransferase and acetylcholine release by sequestering KAP3

    PubMed Central

    Tateno, Minako; Kato, Shinsuke; Sakurai, Takashi; Nukina, Nobuyuki; Takahashi, Ryosuke; Araki, Toshiyuki

    2009-01-01

    Mutations in the superoxide dismutase 1 (sod1) gene cause familial amyotrophic lateral sclerosis (FALS), likely due to the toxic properties of misfolded mutant SOD1 protein. Here we demonstrated that, starting from the pre-onset stage of FALS, misfolded SOD1 species associates specifically with kinesin-associated protein 3 (KAP3) in the ventral white matter of SOD1G93A-transgenic mouse spinal cord. KAP3 is a kinesin-2 subunit responsible for binding to cargos including choline acetyltransferase (ChAT). Motor axons in SOD1G93A-Tg mice also showed a reduction in ChAT transport from the pre-onset stage. By employing a novel FALS modeling system using NG108-15 cells, we showed that microtubule-dependent release of acetylcholine was significantly impaired by misfolded SOD1 species. Furthermore, such impairment was able to be normalized by KAP3 overexpression. KAP3 was incorporated into SOD1 aggregates in human FALS cases as well. These results suggest that KAP3 sequestration by misfolded SOD1 species and the resultant inhibition of ChAT transport play a role in the dysfunction of ALS. PMID:19088126

  13. P300 acetyltransferase regulates fatty acid synthase expression, lipid metabolism and prostate cancer growth

    PubMed Central

    Zhong, Jian; Jiang, Kui; Pan, Yunqian; Karnes, R. Jeffrey; Zhang, Jun; Xu, Wanhai; Wang, Guixia; Huang, Haojie

    2016-01-01

    De novo fatty acid (FA) synthesis is required for prostate cancer (PCa) survival and progression. As a key enzyme for FA synthesis fatty acid synthase (FASN) is often overexpressed in human prostate cancers and its expression correlates with worse prognosis and poor survival. P300 is an acetyltransferase that acts as a transcription co-activator. Increasing evidence suggests that P300 is a major PCa promoter, although the underlying mechanism remains poorly understood. Here, we demonstrated that P300 binds to and increases histone H3 lysine 27 acetylation (H3K27Ac) in the FASN gene promoter. We provided evidence that P300 transcriptionally upregulates FASN expression and promotes lipid accumulation in human PCa cells in culture and Pten knockout prostate tumors in mice. Pharmacological inhibition of P300 decreased FASN expression and lipid droplet accumulation in PCa cells. Immunohistochemistry analysis revealed that expression of P300 protein positively correlates with FASN protein levels in a cohort of human PCa specimens. We further showed that FASN is a key mediator of P300-induced growth of PCa cells in culture and in mice. Together, our findings demonstrate P300 as a key factor that regulates FASN expression, lipid accumulation and cell growth in PCa. They also suggest that this regulatory pathway can serve as a new therapeutic target for PCa treatment. PMID:26934656

  14. A novel role for the histone acetyltransferase Hat1 in the CENP-A/CID assembly pathway in Drosophila melanogaster

    PubMed Central

    Boltengagen, Mark; Huang, Anming; Boltengagen, Anastasiya; Trixl, Lukas; Lindner, Herbert; Kremser, Leopold; Offterdinger, Martin; Lusser, Alexandra

    2016-01-01

    The incorporation of CENP-A into centromeric chromatin is an essential prerequisite for kinetochore formation. Yet, the molecular mechanisms governing this process are surprisingly divergent in different organisms. While CENP-A loading mechanisms have been studied in some detail in mammals, there are still large gaps to our understanding of CENP-A/Cid loading pathways in Drosophila. Here, we report on the characterization and delineation of at least three different CENP-A preloading complexes in Drosophila. Two complexes contain the CENP-A chaperones CAL1, FACT and/or Caf1/Rbap48. Notably, we identified a novel complex consisting of the histone acetyltransferase Hat1, Caf1 and CENP-A/H4. We show that Hat1 is required for proper CENP-A loading into chromatin, since knock-down in S2 cells leads to reduced incorporation of newly synthesized CENP-A. In addition, we demonstrate that CENP-A/Cid interacts with the HAT1 complex via an N-terminal region, which is acetylated in cytoplasmic but not in nuclear CENP-A. Since Hat1 is not responsible for acetylation of CENP-A/Cid, these results suggest a histone acetyltransferase activity-independent escort function for Hat1. Thus, our results point toward intriguing analogies between the complex processing pathways of newly synthesized CENP-A and canonical histones. PMID:26586808

  15. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase.

    PubMed

    Chen, Ji-Yun; Liu, Liang; Cao, Chun-Ling; Li, Mei-Jun; Tan, Kemin; Yang, Xiaohan; Yun, Cai-Hong

    2016-01-01

    N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine N(ε)-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity. PMID:27550639

  16. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase

    PubMed Central

    Chen, Ji-Yun; Liu, Liang; Cao, Chun-Ling; Li, Mei-Jun; Tan, Kemin; Yang, Xiaohan; Yun, Cai-Hong

    2016-01-01

    N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity. PMID:27550639

  17. Biochemical characteristics of a novel vegetative tissue geraniol acetyltransferase from a monoterpene oil grass (Palmarosa, Cymbopogon martinii var. Motia) leaf.

    PubMed

    Sharma, Pankaj K; Sangwan, Neelam S; Bose, Subir K; Sangwan, Rajender S

    2013-04-01

    Plants synthesize volatile alcohol esters on environmental insult or as metabolic induction during flower/fruit development. However, essential oil plants constitutively produce them as the oil constituents. Their synthesis is catalyzed by BAHD family enzymes called alcohol acyltransferases (AATs). However, no AAT has been characterized from plant foliage synthesizing acyclic monoterpenoids containing essential oils. Therefore, we have purified and biochemically characterized a geraniol: acetyl coenzyme A acetyltransferase (GAAT) from Palmarosa aroma grass (Cymbopogon martinii) leaf. MALDI-assisted proteomic study of the 43kDa monomeric enzyme revealed its sequence motif novelties e.g. relaxed conservation at Phe and Trp in DFGWG'. This suggests permissiveness of variations in the conserved motif without loss of catalytic ability. Also, some new conserved/semi-conserved motifs of AATs were recognized. The GAAT k(cat)/K(m) values (300-700M(-1)s(-1)) were low (a generic characteristic for secondary metabolism enzyme) but higher than those of some floral AATs. Wide substrate acceptability for catalyzing acetylation of diverse primary alcohols (chain of ≥C(6)) implied its catalytic description as a 'primary aliphatic alcohol acetyltransferase'. It signifies metabolic ability to deliver diverse aroma esters, should the acceptor alcohols be available in planta. To our knowledge, this is the first report of detailed kinetics of a vegetal monoterpenol acyltransferase.

  18. 15-Deoxy-{Delta}{sup 12,14}-prostaglandin J{sub 2} impairs the functions of histone acetyltransferases through their insolubilization in cells

    SciTech Connect

    Hironaka, Asako; Morisugi, Toshiaki; Kawakami, Tetsuji; Miyagi, Ikuko; Tanaka, Yasuharu

    2009-12-11

    The cyclopentenonic prostaglandin 15-deoxy-{Delta}{sup 12,14}-PG J{sub 2} (15d-PGJ{sub 2}) is a metabolite derived from PGD{sub 2}. Although 15d-PGJ{sub 2} has been demonstrated to be a potent ligand for peroxisome proliferator activated receptor {gamma} (PPAR{gamma}), the functions are not fully understood. In order to examine the effect of 15d-PGJ{sub 2} on histone acetyltransferases (HATs), several lines of cell including mouse embryonic fibroblast (MEF) cells were exposed to 15d-PGJ{sub 2}. Three types of HAT, p300, CREB-binding protein (CBP), and p300/CBP-associated factor (PCAF), selectively disappeared from the soluble fraction in time- and dose-dependent manners. Inversely, HATs in the insoluble fraction increased, suggesting their conformational changes. The decrease in the soluble form of HATs resulted in the attenuation of NF-{kappa}B-, p53-, and heat shock factor-dependent reporter gene expressions, implying that the insoluble HATs are inactive. The resultant insoluble PCAF and p300 seemed to be digested by proteasome, because proteasome inhibitors caused the accumulation of insoluble HATs. Taken together, these results indicate that 15d-PGJ{sub 2} attenuates some gene expressions that require HATs. This inhibitory action of 15d-PGJ{sub 2} on the function of HATs was independent of PPAR{gamma}, because PPAR{gamma} agonists could not mimick 15d-PGJ{sub 2} and PPAR{gamma} antagonists did not inhibit 15d-PGJ{sub 2}.

  19. Immunoreactivity for Choline Acetyltransferase of Peripheral-Type (pChAT) in the Trigeminal Ganglion Neurons of the Non-Human Primate Macaca fascicularis

    PubMed Central

    Koga, Tsuneyuki; Bellier, Jean-Pierre; Kimura, Hiroshi; Tooyama, Ikuo

    2013-01-01

    Transcripts of the choline acetyltransferase (ChAT) gene reveal a number of different splice variants including ChAT of a peripheral type (pChAT). Immunohistochemical staining of the brain using an antibody against pChAT clearly revealed peripheral cholinergic neurons, but failed to detect cholinergic neurons in the central nervous system. In rodents, pChAT-immunoreactivity has been detected in cholinergic parasympathetic postganglionic and enteric ganglion neurons. In addition, pChAT has been observed in non-cholinergic neurons such as peripheral sensory neurons in the trigeminal and dorsal root ganglia. The common type of ChAT (cChAT) has been investigated in many parts of the brain and the spinal cord of non-human primates, but little information is available about the localization of pChAT in primate species. Here, we report the detection of pChAT immunoreactivity in trigeminal ganglion (TG) neurons and its co-localization with Substance P (SP) and/or calcitonin gene-related peptide (CGRP) in the cynomolgus monkey, Macaca fascicularis. Neurons positive for pChAT were observed in a rather uniform pattern in approximately half of the trigeminal neurons throughout the TG. Most pChAT-positive neurons had small or medium-sized cell bodies. Double-immunofluorescence staining showed that 85.1% of SP-positive cells and 74.0% of CGRP-positive cells exhibited pChAT immunoreactivity. Most pChAT-positive cells were part of a larger population of neurons that co-expressed SP and/or CGRP. PMID:23720604

  20. Robust reconstruction of gene expression profiles from reporter gene data using linear inversion

    PubMed Central

    Zulkower, Valentin; Page, Michel; Ropers, Delphine; Geiselmann, Johannes; de Jong, Hidde

    2015-01-01

    Motivation: Time-series observations from reporter gene experiments are commonly used for inferring and analyzing dynamical models of regulatory networks. The robust estimation of promoter activities and protein concentrations from primary data is a difficult problem due to measurement noise and the indirect relation between the measurements and quantities of biological interest. Results: We propose a general approach based on regularized linear inversion to solve a range of estimation problems in the analysis of reporter gene data, notably the inference of growth rate, promoter activity, and protein concentration profiles. We evaluate the validity of the approach using in silico simulation studies, and observe that the methods are more robust and less biased than indirect approaches usually encountered in the experimental literature based on smoothing and subsequent processing of the primary data. We apply the methods to the analysis of fluorescent reporter gene data acquired in kinetic experiments with Escherichia coli. The methods are capable of reliably reconstructing time-course profiles of growth rate, promoter activity and protein concentration from weak and noisy signals at low population volumes. Moreover, they capture critical features of those profiles, notably rapid changes in gene expression during growth transitions. Availability and implementation: The methods described in this article are made available as a Python package (LGPL license) and also accessible through a web interface. For more information, see https://team.inria.fr/ibis/wellinverter. Contact: Hidde.de-Jong@inria.fr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26072511

  1. Arylamine N-acetyltransferases: from drug metabolism and pharmacogenetics to drug discovery

    PubMed Central

    Sim, E; Abuhammad, A; Ryan, A

    2014-01-01

    Arylamine N-acetyltransferases (NATs) are polymorphic drug-metabolizing enzymes, acetylating arylamine carcinogens and drugs including hydralazine and sulphonamides. The slow NAT phenotype increases susceptibility to hydralazine and isoniazid toxicity and to occupational bladder cancer. The two polymorphic human NAT loci show linkage disequilibrium. All mammalian Nat genes have an intronless open reading frame and non-coding exons. The human gene products NAT1 and NAT2 have distinct substrate specificities: NAT2 acetylates hydralazine and human NAT1 acetylates p-aminosalicylate (p-AS) and the folate catabolite para-aminobenzoylglutamate (p-abaglu). Human NAT2 is mainly in liver and gut. Human NAT1 and its murine homologue are in many adult tissues and in early embryos. Human NAT1 is strongly expressed in oestrogen receptor-positive breast cancer and may contribute to folate and acetyl CoA homeostasis. NAT enzymes act through a catalytic triad of Cys, His and Asp with the architecture of the active site-modulating specificity. Polymorphisms may cause unfolded protein. The C-terminus helps bind acetyl CoA and differs among NATs including prokaryotic homologues. NAT in Salmonella typhimurium supports carcinogen activation and NAT in mycobacteria metabolizes isoniazid with polymorphism a minor factor in isoniazid resistance. Importantly, nat is in a gene cluster essential for Mycobacterium tuberculosis survival inside macrophages. NAT inhibitors are a starting point for novel anti-tuberculosis drugs. Human NAT1-specific inhibitors may act in biomarker detection in breast cancer and in cancer therapy. NAT inhibitors for co-administration with 5-aminosalicylate (5-AS) in inflammatory bowel disease has prompted ongoing investigations of azoreductases in gut bacteria which release 5-AS from prodrugs including balsalazide. PMID:24467436

  2. Carnitine Acetyltransferase Mitigates Metabolic Inertia and Muscle Fatigue during Exercise.

    PubMed

    Seiler, Sarah E; Koves, Timothy R; Gooding, Jessica R; Wong, Kari E; Stevens, Robert D; Ilkayeva, Olga R; Wittmann, April H; DeBalsi, Karen L; Davies, Michael N; Lindeboom, Lucas; Schrauwen, Patrick; Schrauwen-Hinderling, Vera B; Muoio, Deborah M

    2015-07-01

    Acylcarnitine metabolites have gained attention as biomarkers of nutrient stress, but their physiological relevance and metabolic purpose remain poorly understood. Short-chain carnitine conjugates, including acetylcarnitine, derive from their corresponding acyl-CoA precursors via the action of carnitine acetyltransferase (CrAT), a bidirectional mitochondrial matrix enzyme. We show here that contractile activity reverses acetylcarnitine flux in muscle, from net production and efflux at rest to net uptake and consumption during exercise. Disruption of this switch in mice with muscle-specific CrAT deficiency resulted in acetyl-CoA deficit, perturbed energy charge, and diminished exercise tolerance, whereas acetylcarnitine supplementation produced opposite outcomes in a CrAT-dependent manner. Likewise, in exercise-trained compared to untrained humans, post-exercise phosphocreatine recovery rates were positively associated with CrAT activity and coincided with dramatic shifts in muscle acetylcarnitine dynamics. These findings show acetylcarnitine serves as a critical acetyl buffer for working muscles and provide insight into potential therapeutic strategies for combatting exercise intolerance. PMID:26154055

  3. Inhibition of aminoglycoside acetyltransferase resistance enzymes by metal salts.

    PubMed

    Li, Yijia; Green, Keith D; Johnson, Brooke R; Garneau-Tsodikova, Sylvie

    2015-07-01

    Aminoglycosides (AGs) are clinically relevant antibiotics used to treat infections caused by both Gram-negative and Gram-positive bacteria, as well as Mycobacteria. As with all current antibacterial agents, resistance to AGs is an increasing problem. The most common mechanism of resistance to AGs is the presence of AG-modifying enzymes (AMEs) in bacterial cells, with AG acetyltransferases (AACs) being the most prevalent. Recently, it was discovered that Zn(2+) metal ions displayed an inhibitory effect on the resistance enzyme AAC(6')-Ib in Acinetobacter baumannii and Escherichia coli. In this study, we explore a wide array of metal salts (Mg(2+), Cr(3+), Cr(6+), Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), and Au(3+) with different counter ions) and their inhibitory effect on a large repertoire of AACs [AAC(2')-Ic, AAC(3)-Ia, AAC(3)-Ib, AAC(3)-IV, AAC(6')-Ib', AAC(6')-Ie, AAC(6')-IId, and Eis]. In addition, we determine the MIC values for amikacin and tobramycin in combination with a zinc pyrithione complex in clinical isolates of various bacterial strains (two strains of A. baumannii, three of Enterobacter cloacae, and four of Klebsiella pneumoniae) and one representative of each species purchased from the American Type Culture Collection. PMID:25941215

  4. Inhibition of Aminoglycoside Acetyltransferase Resistance Enzymes by Metal Salts

    PubMed Central

    Li, Yijia; Green, Keith D.; Johnson, Brooke R.

    2015-01-01

    Aminoglycosides (AGs) are clinically relevant antibiotics used to treat infections caused by both Gram-negative and Gram-positive bacteria, as well as Mycobacteria. As with all current antibacterial agents, resistance to AGs is an increasing problem. The most common mechanism of resistance to AGs is the presence of AG-modifying enzymes (AMEs) in bacterial cells, with AG acetyltransferases (AACs) being the most prevalent. Recently, it was discovered that Zn2+ metal ions displayed an inhibitory effect on the resistance enzyme AAC(6′)-Ib in Acinetobacter baumannii and Escherichia coli. In this study, we explore a wide array of metal salts (Mg2+, Cr3+, Cr6+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Au3+ with different counter ions) and their inhibitory effect on a large repertoire of AACs [AAC(2′)-Ic, AAC(3)-Ia, AAC(3)-Ib, AAC(3)-IV, AAC(6′)-Ib′, AAC(6′)-Ie, AAC(6′)-IId, and Eis]. In addition, we determine the MIC values for amikacin and tobramycin in combination with a zinc pyrithione complex in clinical isolates of various bacterial strains (two strains of A. baumannii, three of Enterobacter cloacae, and four of Klebsiella pneumoniae) and one representative of each species purchased from the American Type Culture Collection. PMID:25941215

  5. Obesity and lipid stress inhibit carnitine acetyltransferase activity[S

    PubMed Central

    Seiler, Sarah E.; Martin, Ola J.; Noland, Robert C.; Slentz, Dorothy H.; DeBalsi, Karen L.; Ilkayeva, Olga R.; An, Jie; Newgard, Christopher B.; Koves, Timothy R.; Muoio, Deborah M.

    2014-01-01

    Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that catalyzes the interconversion of acetyl-CoA and acetylcarnitine. Emerging evidence suggests that this enzyme functions as a positive regulator of total body glucose tolerance and muscle activity of pyruvate dehydrogenase (PDH), a mitochondrial enzyme complex that promotes glucose oxidation and is feedback inhibited by acetyl-CoA. Here, we used tandem mass spectrometry-based metabolic profiling to identify a negative relationship between CrAT activity and muscle content of lipid intermediates. CrAT specific activity was diminished in muscles from obese and diabetic rodents despite increased protein abundance. This reduction in enzyme activity was accompanied by muscle accumulation of long-chain acylcarnitines (LCACs) and acyl-CoAs and a decline in the acetylcarnitine/acetyl-CoA ratio. In vitro assays demonstrated that palmitoyl-CoA acts as a direct mixed-model inhibitor of CrAT. Similarly, in primary human myocytes grown in culture, nutritional and genetic manipulations that promoted mitochondrial influx of fatty acids resulted in accumulation of LCACs but a pronounced decrease of CrAT-derived short-chain acylcarnitines. These results suggest that lipid-induced antagonism of CrAT might contribute to decreased PDH activity and glucose disposal in the context of obesity and diabetes. PMID:24395925

  6. Reconstruction of N-acetyltransferase 2 haplotypes using PHASE.

    PubMed

    Golka, Klaus; Blaszkewicz, Meinolf; Samimi, Mirabutaleb; Bolt, Hermann M; Selinski, Silvia

    2008-04-01

    The genotyping of N-acetyltransferase 2 (NAT2) by PCR/RFLP methods yields in a considerable percentage ambiguous results. To resolve this methodical problem a statistical approach was applied. PHASE v2.1.1, a statistical program for haplotype reconstruction was used to estimate haplotype pairs from NAT2 genotyping data, obtained by the analysis of seven single nucleotide polymorphisms relevant for Caucasians. In 1,011 out of 2,921 (35%) subjects the haplotype pairs were clearcut by the PCR/RFLP data only. For the majority of the data the applied method resulted in a multiplicity (2-4) of possible haplotype pairs. Haplotype reconstruction using PHASE v2.1.1 cleared this ambiguity in all cases but one, where an alternative haplotype pair was considered with a probability of 0.029. The estimation of the NAT2 haplotype is important because the assignment of the NAT2 alleles *12A, *12B, *12C or *13 to the rapid or slow NAT2 genotype has been discussed controversially. A clear assignment is indispensable in surveys of human bladder cancer caused by aromatic amine exposures. In conclusion, PHASE v2.1.1 software allowed an unambiguous haplotype reconstruction in 2,920 of 2,921 cases (>99.9%).

  7. Two Proteins with Ornithine Acetyltransferase Activity Show Different Functions in Streptomyces clavuligerus: Oat2 Modulates Clavulanic Acid Biosynthesis in Response to Arginine

    PubMed Central

    de la Fuente, A.; Martín, J. F.; Rodríguez-García, A.; Liras, P.

    2004-01-01

    The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrations. PMID:15375131

  8. Expression and organization of BP74, a cyclic AMP-regulated gene expressed during Dictyostelium discoideum development.

    PubMed Central

    Hopkinson, S B; Pollenz, R S; Drummond, I; Chisholm, R L

    1989-01-01

    We have characterized a cDNA and the corresponding gene for a cyclic AMP-inducible gene expressed during Dictyostelium development. This gene, BP74, was found to be first expressed about the time of aggregate formation, approximately 6 h after starvation. Accumulation of BP74 mRNA did not occur in Dictyostelium cells that had been starved in fast-shaken suspension cultures but was induced in similar cultures to which cyclic AMP pulses had been added. The BP74 cDNA and gene were characterized by DNA sequence analysis and transcriptional mapping. When the BP74 promoter region was fused with a chloramphenicol acetyltransferase reporter gene and reintroduced into Dictyostelium cells, the transfected chloramphenicol acetyltransferase gene displayed the same developmentally regulated pattern of expression as did the endogenous BP74 gene, suggesting that all of the cis-acting elements required for regulated expression were carried by a 2-kilobase cloned genomic fragment. On the basis of sequence analysis, the gene appeared to encode a protein containing a 20-residue hydrophobic sequence at the amino-terminal end and 26 copies of a 20-amino-acid repeat. Images PMID:2555685

  9. [Construction and function identification of luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR].

    PubMed

    Yang, Shuo; Li, Jia-li; Bi, Hui-chang; Zhou, Shou-ning; Liu, Xiao-man; Zeng, Hang; Hu, Bing-fang; Huang, Min

    2016-01-01

    This study aims to investigate the function of two SNPs (rs8904C > T and rs696G >A) in 3' untranslated region (3'UTR) of NFKBIA gene by constructing luciferase reporter gene. A patient's genomic DNA with rs8904 CC and rs696 GA genotype was used as the PCR template. Full-length 3'UTR of NFKBIA gene was amplified by different primers. After sequencing validation, these fragments were inserted to the luciferase reporter vector, pGL3-promoter to construct recombinant plasmids containing four kinds of haplotypes, pGL3-rs8904C/rs696G, pGL3-rs8904C/rs696A, pGL3-rs8904T/rs696G and pGL3-rs8904T/rs696A. Then these plasmids were transfected into LS174T cells and the luciferase activity was detected. Compared with pGL3-vector transfected cells (negative control), the luciferase activity of the four kinds of recombinant plasmids was significantly decreased (P < 0.001). For rs696G > A, the luciferase activity of the recombinant plasmids containing A allele (pGL3-rs8904C/rs696A and pGL3-rs8904T/rs696A) was about 45.1% (P < 0.05) and 56.1% (P < 0.001) lower than those containing G allele (pGL3-rs8904C/rs696G and pGL3-rs8904T/rs696G), respectively. For rs8904C > T, there were no significant differences in the luciferase activity between the recombinant plasmids containing T allele and those with C allele. Together, the luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR were constructed successfully and rs696G > A could decrease the luciferase activity while rs8904C >T didn't have much effect on the luciferase activity. PMID:27405166

  10. [Construction and function identification of luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR].

    PubMed

    Yang, Shuo; Li, Jia-li; Bi, Hui-chang; Zhou, Shou-ning; Liu, Xiao-man; Zeng, Hang; Hu, Bing-fang; Huang, Min

    2016-01-01

    This study aims to investigate the function of two SNPs (rs8904C > T and rs696G >A) in 3' untranslated region (3'UTR) of NFKBIA gene by constructing luciferase reporter gene. A patient's genomic DNA with rs8904 CC and rs696 GA genotype was used as the PCR template. Full-length 3'UTR of NFKBIA gene was amplified by different primers. After sequencing validation, these fragments were inserted to the luciferase reporter vector, pGL3-promoter to construct recombinant plasmids containing four kinds of haplotypes, pGL3-rs8904C/rs696G, pGL3-rs8904C/rs696A, pGL3-rs8904T/rs696G and pGL3-rs8904T/rs696A. Then these plasmids were transfected into LS174T cells and the luciferase activity was detected. Compared with pGL3-vector transfected cells (negative control), the luciferase activity of the four kinds of recombinant plasmids was significantly decreased (P < 0.001). For rs696G > A, the luciferase activity of the recombinant plasmids containing A allele (pGL3-rs8904C/rs696A and pGL3-rs8904T/rs696A) was about 45.1% (P < 0.05) and 56.1% (P < 0.001) lower than those containing G allele (pGL3-rs8904C/rs696G and pGL3-rs8904T/rs696G), respectively. For rs8904C > T, there were no significant differences in the luciferase activity between the recombinant plasmids containing T allele and those with C allele. Together, the luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR were constructed successfully and rs696G > A could decrease the luciferase activity while rs8904C >T didn't have much effect on the luciferase activity.

  11. Intestinal lactase as an autologous beta-galactosidase reporter gene for in vivo gene expression studies.

    PubMed

    Salehi, Siamak; Eckley, Lorna; Sawyer, Greta J; Zhang, Xiaohong; Dong, Xuebin; Freund, Jean-Noel; Fabre, John W

    2009-01-01

    Intestinal lactase has potential as an autologous beta-galactosidase reporter gene for long-term gene expression studies in vivo, using chromogenic, luminescent, and fluorogenic substrates developed for Escherichia coli beta-galactosidase. In normal rat tissues, reactivity with a chromogenic fucopyranoside (X-Fuc, the preferred substrate of lactase) was present only at the lumenal surface of small intestine epithelial cells. Full-length lactase (domains I-IV), mature lactase (domains III and IV), and a cytosolic form of mature lactase (domains III and IV, without the signal sequence or transmembrane region) were evaluated. Transfection of HuH-7 cells in vitro, and hydrodynamic gene delivery to the liver in vivo, resulted in excellent gene expression. The full-length and mature (homodimeric, membrane-bound) forms reacted strongly with X-Fuc but not with the corresponding galactopyranoside (X-Gal). However, the presumptively monomeric cytosolic lactase unexpectedly reacted equally well with both substrates. The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was cleaved by cytosolic lactase, but not by full-length or mature lactase. Full-length lactase, when expressed ectopically in hepatocytes in vivo, localized exclusively to the bile canalicular membrane. Intestinal lactase is highly homologous in mice, rats, and humans and has considerable potential for evaluating long-term gene expression in experimental animals and the clinic.

  12. Replication-Competent Influenza A Viruses Expressing Reporter Genes

    PubMed Central

    Breen, Michael; Nogales, Aitor; Baker, Steven F.; Martínez-Sobrido, Luis

    2016-01-01

    Influenza A viruses (IAV) cause annual seasonal human respiratory disease epidemics. In addition, IAV have been implicated in occasional pandemics with inordinate health and economic consequences. Studying IAV, in vitro or in vivo, requires the use of laborious secondary methodologies to identify virus-infected cells. To circumvent this requirement, replication-competent IAV expressing an easily traceable reporter protein can be used. Here we discuss the development and applications of recombinant replication-competent IAV harboring diverse fluorescent or bioluminescent reporter genes in different locations of the viral genome. These viruses have been employed for in vitro and in vivo studies, such as the screening of neutralizing antibodies or antiviral compounds, the identification of host factors involved in viral replication, cell tropism, the development of vaccines, or the assessment of viral infection dynamics. In summary, reporter-expressing, replicating-competent IAV represent a powerful tool for the study of IAV both in vitro and in vivo. PMID:27347991

  13. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    DOEpatents

    Gambhir, Sanjiv; Pritha, Ray

    2011-06-07

    Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  14. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    DOEpatents

    Gambhir; Sanjiv , Pritha; Ray

    2009-04-28

    Novel double and triple fusion reporter gene constructs harboring distinct imageable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  15. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    DOEpatents

    Gambhir, Sanjiv; Pritha, Ray

    2015-07-14

    Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  16. Effect of maternal deprivation on N-acetyltransferase activity rhythm in blinded rat pups.

    PubMed

    Katoh, Y; Takeuchi, Y; Yamazaki, K; Takahashi, K

    1998-02-15

    It has been reported that the rhythms of infant rats synchronize with the mother's rhythm until the light-dark cycle comes and has strong effects on their endogenous clocks. We found that periodic maternal deprivation (PMD) was able to cause a phase shift of serotonin N-acetyltransferase (NAT) in neonatal blinded rat pups. PMD in which contact with the mother was allowed for only 4 h caused a phase shift of NAT rhythm, irrespective of the timing of contact with the mother in a day. Acute single mother deprivation caused an excess of NAT activity for more hours than usual and contact with the mother prevented such an excessive response. Mother deprivation may act as a cold stress, since artificial warming of pups gave the same results as contact with the mother. When the pups were artificially warmed by a heater during a 1-week deprivation period, a flat 24-h pattern of NAT was observed. The mechanism causing a phase shift of NAT activity rhythm of rat pups may be complicated. PMID:9523895

  17. Retinal, pineal and diencephalic expression of frog arylalkylamine N-acetyltransferase-1.

    PubMed

    Isorna, Esther; Besseau, Laurence; Boeuf, Gilles; Desdevises, Yves; Vuilleumier, Robin; Alonso-Gómez, Angel L; Delgado, María J; Falcón, Jack

    2006-06-27

    The arylalkylamine N-acetyltransferase (AANAT) is a key enzyme in the rhythmic production of melatonin. Two Aanats are expressed in Teleost fish (Aanat1 in the retina and Aanat2 in the pineal organ) but only Aanat1 is found in tetrapods. This study reports the cloning of Aanat1 from R. perezi. Transcripts were mainly expressed in the retina, diencephalon, intestine and testis. In the retina and pineal organ, Aanat1 expression was in the photoreceptor cells. Expression was also seen in ependymal cells of the 3rd ventricle and discrete cells of the suprachiasmatic area. The expression of Aanat1 in both the retina and pineal organ, and the absence of Aanat2 suggests that green frog resembles more to birds and mammals than to Teleost fish, as far as Aanat is concerned. The significance of Aanat1 in extra-pineal and extra-retinal tissues remains to be elucidated; in the diencephalon, it might be associated to the so-called deep brain photoreceptor cells.

  18. Fungal Rtt109 Histone Acetyltransferase is an Unexpected Structural Homolog of Metazoan p300/CBP

    SciTech Connect

    Tang,Y.; Holbert, M.; Wurtele, H.; Meeth, K.; Rocha, W.; Gharib, M.; Jiang, E.; Thibault, P.; Verreault, A.; et al

    2008-01-01

    Rtt109, also known as KAT11, is a recently characterized fungal-specific histone acetyltransferase (HAT) that modifies histone H3 lysine 56 (H3K56) to promote genome stability. Rtt109 does not show sequence conservation with other known HATs and depends on association with either of two histone chaperones, Asf1 or Vps75, for HAT activity. Here we report the X-ray crystal structure of an Rtt109-acetyl coenzyme A complex and carry out structure-based mutagenesis, combined with in vitro biochemical studies of the Rtt109-Vps75 complex and studies of Rtt109 function in vivo. The Rtt109 structure reveals noteworthy homology to the metazoan p300/CBP HAT domain but exhibits functional divergence, including atypical catalytic properties and mode of cofactor regulation. The structure reveals a buried autoacetylated lysine residue that we show is also acetylated in the Rtt109 protein purified from yeast cells. Implications for understanding histone substrate and chaperone binding by Rtt109 are discussed.

  19. Crystal Structures of Murine Carnitine Acetyltransferase in Ternary Complexes with Its Substrates

    SciTech Connect

    Hsiao,Y.; Jogl, G.; Tong, L.

    2006-01-01

    Carnitine acyltransferases catalyze the reversible exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids in the mitochondria for energy production, and are attractive targets for drug discovery against diabetes and obesity. To help define in molecular detail the catalytic mechanism of these enzymes, we report here the high resolution crystal structure of wild-type murine carnitine acetyltransferase (CrAT) in a ternary complex with its substrates acetyl-CoA and carnitine, and the structure of the S554A/M564G double mutant in a ternary complex with the substrates CoA and hexanoylcarnitine. Detailed analyses suggest that these structures may be good mimics for the Michaelis complexes for the forward and reverse reactions of the enzyme, representing the first time that such complexes of CrAT have been studied in molecular detail. The structural information provides significant new insights into the catalytic mechanism of CrAT and possibly carnitine acyltransferases in general.

  20. N-acetyltransferase 2 genetic polymorphism: Effects of carcinogen and haplotype on urinary bladder cancer risk

    PubMed Central

    Hein, David W.

    2006-01-01

    A role for the N-acetyltransferase 2 (NAT2) genetic polymorphism in cancer risk has been the subject of numerous studies. Although comprehensive reviews of the NAT2 acetylation polymorphism have been published elsewhere, the objective of this paper is to briefly highlight some important features of the NAT2 acetylation polymorphism that are not universally accepted to better understand the role of NAT2 polymorphism in carcinogenic risk assessment. NAT2 slow acetylator phenotype(s) infer a consistent and robust increase in urinary bladder cancer risk following exposures to aromatic amine carcinogens. However, identification of specific carcinogens is important as the effect of NAT2 polymorphism on urinary bladder cancer differs dramatically between monoarylamines and aryldiamines. Misclassifications of carcinogen exposure and NAT2 genotype/phenotype confound evidence for a real biological effect. Functional understanding of the effects of NAT2 genetic polymorphisms on metabolism and genotoxicity, tissue-specific expression and the elucidation of the molecular mechanisms responsible are critical for interpretation of previous and future human molecular epidemiology investigations into the role of NAT2 polymorphism on cancer risk. Although associations have been reported for various cancers, this paper focuses on urinary bladder cancer, a cancer in which a role for NAT2 polymorphism was first proposed and for which evidence is accumulating that the effect is biologically significant with important public health implications. PMID:16550165

  1. N-Acetyltransferase 2 genotype, exfoliated urothelial cells and benzidine exposure.

    PubMed

    Ma, Qing-wen; Lin, Guo-fang; Chen, Ji-gang; Guo, Wei-Chao; Qin, Yi-qiu; Golka, Klaus; Shen, Jian-hua

    2012-01-01

    Most studies report an association of the slow N-acetyltransferase 2 (NAT2) status with elevated bladder cancer risk. In this study, NAT2 genotypes and the decades-long records of Papanicolaou's grading of exfoliated urothelial cells in a former benzidine-exposed cohort of the Shanghai dyestuff industry (29 bladder cancer patients; 307 non-cancer cohort members, some of them presenting different grades of pre-malignant alterations of exfoliated urothelial cells) were investigated. The cohort members had been enrolled in regular medical surveillance since mid-1980s. No overall increase of slow NAT2 genotypes in the former benzidine-exposed bladder cancer patients was found, compared with non-diseased members of the same cohort. A lower presentation of the homozygous wild genotype NAT2 4/4 was observed in bladder cancer patients, compared with non-diseased members with averaged Papanicolaou's grading (APG)3 II (OR=0.31, 95 percent CI 0.10-0.96, p=0.034) or with APG less than II (OR=0.36,95 percent CI 0.12-1.10, p=0.063). Nevertheless, neither a protective influence of rapid NAT2 genotypes on bladder cancer risk nor on pre-malignant cytological alterations could be confirmed by the present data.

  2. Photoacoustic molecular imaging of ferritin as a reporter gene

    NASA Astrophysics Data System (ADS)

    Ha, S.; Carson, A.; Kim, K.

    2012-02-01

    Spectral analysis of photoacoustic (PA) molecular imaging (PMI) of ferritin expressed in human melanoma cells (SK-24) was performed in vitro. Ferritin is a ubiquitously expressed protein which stores iron that can be detected by PA imaging, allowing ferritin to act as a reporter gene. To over-express ferritin, SK-24 cells were co-transfected with plasmid expressing Heavy chain ferritin (H-FT) and plasmid expressing enhanced green fluorescent protein (pEGFP-C1) using LipofectamineTM 2000. Non-transfected SK-24 cells served as a negative control. Fluorescent imaging of EGFP confirmed transfection and transgene expression in co-transfected cells. To detect iron accumulation in SK-24 cells, a focused high frequency ultrasonic transducer (60 MHz, f/1.5), synchronized to a pulsed laser (<20mJ/cm2), was used to scan the PA signal from 680 nm to 950 nm (in 10 nm increments) from the surface of the 6-well culturing plate. PA signal intensity from H-FT transfected SK-24 cells was not different from that of non-transfected SK-24 cells at wavelengths less than 770 nm, but was over 4 dB higher than non-transfected SK-24 cells at 850 ~ 950 nm. Fluorescent microscopy indicates significant accumulation of ferritin in H-FT transfected SK-24 cells, with little ferritin expression in non-transfected SK-24 cells. The PA spectral analysis clearly differentiates transfected SK-24 cells from nontransfected SK-24 cells with significantly increased iron signal at 850 ~ 950 nm, and these increased signals were associated with transfection of H-FT plasmid. As such, the feasibility of ferritin as a reporter gene for PMI has been demonstrated in vitro. The use of ferritin as a reporter gene represents a new concept for PA imaging, and may provide various opportunities for molecular imaging and basic science research.

  3. Structural Studies on a Glucosamine/Glucosaminide N-Acetyltransferase.

    PubMed

    Dopkins, Brandon J; Tipton, Peter A; Thoden, James B; Holden, Hazel M

    2016-08-16

    Glucosamine/glucosaminide N-acetyltransferase or GlmA catalyzes the transfer of an acetyl group from acetyl CoA to the primary amino group of glucosamine. The enzyme from Clostridium acetobutylicum is thought to be involved in cell wall rescue. In addition to glucosamine, GlmA has been shown to function on di- and trisaccharides of glucosamine as well. Here we present a structural and kinetic analysis of the enzyme. For this investigation, eight structures were determined to resolutions of 2.0 Å or better. The overall three-dimensional fold of GlmA places it into the tandem GNAT superfamily. Each subunit of the dimer folds into two distinct domains which exhibit high three-dimensional structural similarity. Whereas both domains bind acetyl CoA, it is the C-terminal domain that is catalytically competent. On the basis of the various structures determined in this investigation, two amino acid residues were targeted for further study: Asp 287 and Tyr 297. Although their positions in the active site suggested that they may play key roles in catalysis by functioning as active site bases and acids, respectively, this was not borne out by characterization of the D287N and Y297F variants. The kinetic properties revealed that both residues were important for substrate binding but had no critical roles as acid/base catalysts. Kinetic analyses also indicated that GlmA follows an ordered mechanism with acetyl CoA binding first followed by glucosamine. The product N-acetylglucosamine is then released prior to CoA. The investigation described herein provides significantly new information on enzymes belonging to the tandem GNAT superfamily.

  4. Structural Studies on a Glucosamine/Glucosaminide N-Acetyltransferase.

    PubMed

    Dopkins, Brandon J; Tipton, Peter A; Thoden, James B; Holden, Hazel M

    2016-08-16

    Glucosamine/glucosaminide N-acetyltransferase or GlmA catalyzes the transfer of an acetyl group from acetyl CoA to the primary amino group of glucosamine. The enzyme from Clostridium acetobutylicum is thought to be involved in cell wall rescue. In addition to glucosamine, GlmA has been shown to function on di- and trisaccharides of glucosamine as well. Here we present a structural and kinetic analysis of the enzyme. For this investigation, eight structures were determined to resolutions of 2.0 Å or better. The overall three-dimensional fold of GlmA places it into the tandem GNAT superfamily. Each subunit of the dimer folds into two distinct domains which exhibit high three-dimensional structural similarity. Whereas both domains bind acetyl CoA, it is the C-terminal domain that is catalytically competent. On the basis of the various structures determined in this investigation, two amino acid residues were targeted for further study: Asp 287 and Tyr 297. Although their positions in the active site suggested that they may play key roles in catalysis by functioning as active site bases and acids, respectively, this was not borne out by characterization of the D287N and Y297F variants. The kinetic properties revealed that both residues were important for substrate binding but had no critical roles as acid/base catalysts. Kinetic analyses also indicated that GlmA follows an ordered mechanism with acetyl CoA binding first followed by glucosamine. The product N-acetylglucosamine is then released prior to CoA. The investigation described herein provides significantly new information on enzymes belonging to the tandem GNAT superfamily. PMID:27348258

  5. Insertion of a GFP reporter gene in influenza virus.

    PubMed

    Perez, Jasmine T; García-Sastre, Adolfo; Manicassamy, Balaji

    2013-01-01

    The incorporation of a fluorescent reporter gene into a replication-competent influenza A virus (IAV) has made it possible to trace IAV infection in vivo. This protocol describes the process of inserting a green fluorescent protein (GFP) reporter into the IAV genome using the established reverse genetics system. The strategy begins with the reorganization of segment eight of the IAV genome, during which the open reading frames of nonstructural protein 1 (NS1) and the nuclear export protein (NEP) are separated to allow for GFP fusion to the NS1 protein. The NS1, GFP, and NEP open reading frames (ORF) are then cloned into the IAV rescue system backbone. Upon construction of the GFP-encoding segment eight rescue plasmid, recombinant NS1-GFP influenza virus can be rescued via co-transfection with the remaining seven rescue plasmids. The generated NS1-GFP IAV can subsequently be used to visualize infected cells, both in vitro and in vivo.

  6. Elongator subunit 3 positively regulates plant immunity through its histone acetyltransferase and radical S-adenosylmethionine domains

    PubMed Central

    2013-01-01

    Background Pathogen infection triggers a large-scale transcriptional reprogramming in plants, and the speed of this reprogramming affects the outcome of the infection. Our understanding of this process has significantly benefited from mutants that display either delayed or accelerated defense gene induction. In our previous work we demonstrated that the Arabidopsis Elongator complex subunit 2 (AtELP2) plays an important role in both basal immunity and effector-triggered immunity (ETI), and more recently showed that AtELP2 is involved in dynamic changes in histone acetylation and DNA methylation at several defense genes. However, the function of other Elongator subunits in plant immunity has not been characterized. Results In the same genetic screen used to identify Atelp2, we found another Elongator mutant, Atelp3-10, which mimics Atelp2 in that it exhibits a delay in defense gene induction following salicylic acid treatment or pathogen infection. Similarly to AtELP2, AtELP3 is required for basal immunity and ETI, but not for systemic acquired resistance (SAR). Furthermore, we demonstrate that both the histone acetyltransferase and radical S-adenosylmethionine domains of AtELP3 are essential for its function in plant immunity. Conclusion Our results indicate that the entire Elongator complex is involved in basal immunity and ETI, but not in SAR, and support that Elongator may play a role in facilitating the transcriptional induction of defense genes through alterations to their chromatin. PMID:23856002

  7. Mutation of the CH1 Domain in the Histone Acetyltransferase CREBBP Results in Autism-Relevant Behaviors in Mice

    PubMed Central

    Zheng, Fei; Kasper, Lawryn H.; Bedford, David C.; Lerach, Stephanie; Teubner, Brett J. W.; Brindle, Paul K.

    2016-01-01

    Autism spectrum disorders (ASDs) are a group of neurodevelopmental afflictions characterized by repetitive behaviors, deficits in social interaction, and impaired communication skills. For most ASD patients, the underlying causes are unknown. Genetic mutations have been identified in about 25 percent of ASD cases, including mutations in epigenetic regulators, suggesting that dysregulated chromatin or DNA function is a critical component of ASD. Mutations in the histone acetyltransferase CREB binding protein (CBP, CREBBP) cause Rubinstein-Taybi Syndrome (RTS), a developmental disorder that includes ASD-like symptoms. Recently, genomic studies involving large numbers of ASD patient families have theoretically modeled CBP and its paralog p300 (EP300) as critical hubs in ASD-associated protein and gene interaction networks, and have identified de novo missense mutations in highly conserved residues of the CBP acetyltransferase and CH1 domains. Here we provide animal model evidence that supports this notion that CBP and its CH1 domain are relevant to autism. We show that mice with a deletion mutation in the CBP CH1 (TAZ1) domain (CBPΔCH1/ΔCH1) have an RTS-like phenotype that includes ASD-relevant repetitive behaviors, hyperactivity, social interaction deficits, motor dysfunction, impaired recognition memory, and abnormal synaptic plasticity. Our results therefore indicate that loss of CBP CH1 domain function contributes to RTS, and possibly ASD, and that this domain plays an essential role in normal motor function, cognition and social behavior. Although the key physiological functions affected by ASD-associated mutation of epigenetic regulators have been enigmatic, our findings are consistent with theoretical models involving CBP and p300 in ASD, and with a causative role for recently described ASD-associated CBP mutations. PMID:26730956

  8. Mutation of the CH1 Domain in the Histone Acetyltransferase CREBBP Results in Autism-Relevant Behaviors in Mice.

    PubMed

    Zheng, Fei; Kasper, Lawryn H; Bedford, David C; Lerach, Stephanie; Teubner, Brett J W; Brindle, Paul K

    2016-01-01

    Autism spectrum disorders (ASDs) are a group of neurodevelopmental afflictions characterized by repetitive behaviors, deficits in social interaction, and impaired communication skills. For most ASD patients, the underlying causes are unknown. Genetic mutations have been identified in about 25 percent of ASD cases, including mutations in epigenetic regulators, suggesting that dysregulated chromatin or DNA function is a critical component of ASD. Mutations in the histone acetyltransferase CREB binding protein (CBP, CREBBP) cause Rubinstein-Taybi Syndrome (RTS), a developmental disorder that includes ASD-like symptoms. Recently, genomic studies involving large numbers of ASD patient families have theoretically modeled CBP and its paralog p300 (EP300) as critical hubs in ASD-associated protein and gene interaction networks, and have identified de novo missense mutations in highly conserved residues of the CBP acetyltransferase and CH1 domains. Here we provide animal model evidence that supports this notion that CBP and its CH1 domain are relevant to autism. We show that mice with a deletion mutation in the CBP CH1 (TAZ1) domain (CBPΔCH1/ΔCH1) have an RTS-like phenotype that includes ASD-relevant repetitive behaviors, hyperactivity, social interaction deficits, motor dysfunction, impaired recognition memory, and abnormal synaptic plasticity. Our results therefore indicate that loss of CBP CH1 domain function contributes to RTS, and possibly ASD, and that this domain plays an essential role in normal motor function, cognition and social behavior. Although the key physiological functions affected by ASD-associated mutation of epigenetic regulators have been enigmatic, our findings are consistent with theoretical models involving CBP and p300 in ASD, and with a causative role for recently described ASD-associated CBP mutations.

  9. Amidoligases with ATP-grasp, glutamine synthetase-like and acetyltransferase-like domains: synthesis of novel metabolites and peptide modifications of proteins

    PubMed Central

    Iyer, Lakshminarayan M.; Abhiman, Saraswathi; Burroughs, A. Maxwell; Aravind, L.

    2011-01-01

    Recent studies have shown that the ubiquitin system had its origins in ancient cofactor/amino acid biosynthesis pathways. Preliminary studies also indicated that conjugation systems for other peptide tags on proteins, such as pupylation, have evolutionary links to cofactor/amino acid biosynthesis pathways. Following up on these observations, we systematically investigated the non-ribosomal amidoligases of the ATP-grasp, glutamine synthetase-like and acetyltransferase folds by classifying the known members and identifying novel versions. We then established their contextual connections using information from domain architectures and conserved gene neighborhoods. This showed remarkable, previously uncharacterized functional links between diverse peptide ligases, several peptidases of unrelated folds and enzymes involved in synthesis of modified amino acids. Using the network of contextual connections we were able to predict numerous novel pathways for peptide synthesis and modification, amine-utilization, secondary metabolite synthesis and potential peptide-tagging systems. One potential peptide-tagging system, which is widely distributed in bacteria, involves an ATP-grasp domain and a glutamine synthetase-like ligase, both of which are circularly permuted, an NTN hydrolase fold peptidase and a novel alpha helical domain. Our analysis also elucidates key steps in the biosynthesis of antibiotics such as friulimicin, butirosin and bacilysin and cell surface structures such as capsular polymers and teichuronopeptides. We also report the discovery of several novel ribosomally synthesized bacterial peptide metabolites that are cyclized via amide and lactone linkages formed by ATP-grasp enzymes. We present an evolutionary scenario for the multiple convergent origins of peptide ligases in various folds and clarify the bacterial origin of eukaryotic peptide-tagging enzymes of the TTL family. PMID:20023723

  10. Genetic engineering with a gene encoding a soybean storage protein. Progress report

    SciTech Connect

    Beachy, R.N.

    1983-01-01

    Progress is reported in gene transfer experiments using the soybean seed storage protein gene. The sequencing of gene Gmg ..cap alpha..' 17.1 has been completed. Several deletion mutants of this gene are being prepared for experiments to transfer the gene into the Ti-plasmid of Agrobacterium tumefaciens. The purpose is to determine which, if any, of the upstream sequences are those which regulate the developmental expression of the gene. (ACR)

  11. Single neuron transcriptomics identify SRSF/SR protein B52 as a regulator of axon growth and Choline acetyltransferase splicing

    PubMed Central

    Liu, Boyin; Bossing, Torsten

    2016-01-01

    We removed single identified neurons from living Drosophila embryos to gain insight into the transcriptional control of developing neuronal networks. The microarray analysis of the transcriptome of two sibling neurons revealed seven differentially expressed transcripts between both neurons (threshold: log21.4). One transcript encodes the RNA splicing factor B52. Loss of B52 increases growth of axon branches. B52 function is also required for Choline acetyltransferase (ChAT ) splicing. At the end of embryogenesis, loss of B52 function impedes splicing of ChAT, reduces acetylcholine synthesis, and extends the period of uncoordinated muscle twitches during larval hatching. ChAT regulation by SRSF proteins may be a conserved feature since changes in SRSF5 expression and increased acetylcholine levels in brains of bipolar disease patients have been reported recently. PMID:27725692

  12. Hormonal activity of polycyclic musks evaluated by reporter gene assay.

    PubMed

    Mori, Taiki; Iida, Mitsuru; Ishibashi, Hiroshi; Kohra, Shinya; Takao, Yuji; Takemasa, Takehiro; Arizono, Koji

    2007-01-01

    Synthetic musk fragrance compounds, such as polycyclic musks (PCMs), are a group of chemicals used extensively as personal care products, and can be found in the environment and the human body. PCMs, such as 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexa-methylcyclopenta-gamma-2-benzopyran (HHCB) and 7-acetyl-1,1,3,4,4,6-hexamethyltetralin (AHTN), are known to have agonistic activities toward human estrogen receptor alpha (hERalpha) and hERbeta, and have antagonistic activity toward the human androgen receptor (hAR), as shown in several reporter gene assays. However, little is known about the interaction of PCMs with the human thyroid hormone receptor (hTR), and the hormonal effects of other PCMs except for HHCB and AHTN. In this study, we focus on the interactions of six PCMs, namely, HHCB, AHTN, 4-acetyl-1,1-dimethyl-6-tert-butyl-indan (ADBI), 6-acetyl-1,1,2,3,3,5-hexamethylindan (AHMI), 6,7-dihydro-1,1,2,3,3-pentamethyl-4(5H)-indanone (DPMI), and 5-acetyl-1,1,2,6-tetramethyl-3-isopropy-lindan (ATII) with hERalpha, hAR, and hTRbeta by in vitro reporter gene assay using Chinese hamster ovary cells. All the samples were found to be agonists toward hERalpha, whereas no agonistic activities of these PCMs for hAR and hTRbeta were observed. No antagonistic activities for hERalpha and hTRbeta were observed at the concentrations tested. However, several PCMs, namely, HHCB, AHTN, ATII, ADBI, and AHMI, showed dose-dependent antagonistic activities for hAR, and the IC50 values of these compounds were estimated to be 1.0 x 10(-7), 1.5 x 10(-7), 1.4 x 10(-7), 9.8 x 10(-6), and 1.4 x 10(-7) M, respectively. The results suggest that these PCMs interact with hERalpha and hAR but have no hormonal effect on hTRbeta. This is the first report on the agonistic and antagonistic activities of ATII, ADBI, AHMI, and DPMI for hERalpha and hAR as determined by in vitro reporter gene assay using stably transfected Chinese hamster ovary cells.

  13. Immunochemical detection of arylamine N-acetyltransferase during mouse embryonic development and in adult mouse brain.

    PubMed

    Stanley, L A; Copp, A J; Pope, J; Rolls, S; Smelt, V; Perry, V H; Sim, E

    1998-11-01

    Arylamine N-acetyltransferases (NATs) are important in susceptibility to xenobiotic-induced disorders (e.g., drug-induced autoimmune disease, bladder cancer), but their role in endogenous metabolism is yet to be elucidated. The discovery that human NAT1 acts upon p-aminobenzoylgluatamate (p-ABG) to generate p-acetamidobenzoylglutamate (p-AABG), a major urinary metabolite of folic acid, suggests that human NAT1 may play a role in folic acid metabolism and hence in the normal development of the neural tube. In this study we examined the distribution of NAT in neuronal tissue from adult mice and embryos. Immunohistochemical staining of the adult mouse cerebellum revealed NAT2 (the mouse homologue of human NAT1) expression in the cell bodies and dendrites of Purkinje cells and in the neuroglia of the molecular layer. In embryos, NAT2 was detected in developing neuronal tissue on days 9.5, 11.5, and 13.5. It was expressed intensely in the nerual tube around the time of closure. The level of expression subsequently declined in the neuroepithelium but increased in glial cells. In addition, NAT2 was detected in the developing heart and gut. These findings demonstrate that the embryo itself expresses an enzyme which is involved in the metabolism of folic acid, so that the role played by both mother and embryo must be considered when examining the role of folic acid in embryonic development. These findings imply that polymorphisms in NAT genes could play a role in determining susceptibility to neural tube defects (NTD) and orofacial clefting, developmental disorders which can be prevented by dietary administration of folic acid. PMID:9839355

  14. Functional properties of an alternative, tissue-specific promoter for human arylamine N-acetyltransferase 1

    PubMed Central

    Barker, David F.; Husain, Anwar; Neale, Jason R.; Martini, Benjamin D.; Zhang, Xiaoyan; Doll, Mark A.; Christopher States, J.; Hein, David W.

    2007-01-01

    Variable expression of human arylamine N-acetyltransferase 1 (NAT1) due to genetic polymorphism, gene regulation or environmental influences is associated with individual susceptibility to various cancers. Recent studies of NAT1 transcription showed that most mRNAs originate at a promoter, P1, located 11.8 kb upstream of the single open reading frame (ORF) exon. We have now characterized an alternative NAT1 promoter lying 51.5 kb upstream of the NAT1 ORF. In the present study, analysis of human RNAs representing 27 tissue types by RT-PCR and quantitative RT-PCR showed the upstream 51.5 kb promoter, designated P3, to be most active in specific tissues, including kidney, liver, lung, and trachea. All NAT1 P3 mRNAs included 5’-untranslated region (5’-UTR) internal exons of 61 and 175 nucleotides in addition to the 79 nucleotide 5’-UTR exon present in P1 mRNA. CAP-dependent amplification of 5’ P3 mRNA termini defined an 84 bp transcription start region in which most start sites are centrally clustered. The hepatoma-derived HepG2 cell line expressed a high level of P3 mRNA with the same spliced structure and start site pattern as found in normal tissues. A 435 bp minimal promoter was defined by transfection of HepG2 with luciferase expression constructs containing genomic fragments from the P3 start region. These findings imply a fundamental role for P3 in NAT1 regulation and define additional regions for genetic polymorphisms associated with enhanced cancer risk. PMID:16788383

  15. Arylamine N-acetyltransferase 2 (NAT2) genetic diversity and traditional subsistence: a worldwide population survey.

    PubMed

    Sabbagh, Audrey; Darlu, Pierre; Crouau-Roy, Brigitte; Poloni, Estella S

    2011-01-01

    Arylamine N-acetyltransferase 2 (NAT2) is involved in human physiological responses to a variety of xenobiotic compounds, including common therapeutic drugs and exogenous chemicals present in the diet and the environment. Many questions remain about the evolutionary mechanisms that have led to the high prevalence of slow acetylators in the human species. Evidence from recent surveys of NAT2 gene variation suggests that NAT2 slow-causing variants might have become targets of positive selection as a consequence of the shift in modes of subsistence and lifestyle in human populations in the last 10,000 years. We aimed to test more extensively the hypothesis that slow acetylation prevalence in humans is related to the subsistence strategy adopted by the past populations. To this end, published frequency data on the most relevant genetic variants of NAT2 were collected from 128 population samples (14,679 individuals) representing different subsistence modes and dietary habits, allowing a thorough analysis at both a worldwide and continent scale. A significantly higher prevalence of the slow acetylation phenotype was observed in populations practicing farming (45.4%) and herding (48.2%) as compared to populations mostly relying on hunting and gathering (22.4%) (P = 0.0007). This was closely mirrored by the frequency of the slow 590A variant that was found to occur at a three-fold higher frequency in food producers (25%) as compared to hunter-gatherers (8%). These findings are consistent with the hypothesis that the Neolithic transition to subsistence economies based on agricultural and pastoral resources modified the selective regime affecting the NAT2 acetylation pathway. Furthermore, the vast amount of data collected enabled us to provide a comprehensive and up-to-date description of NAT2 worldwide genetic diversity, thus building up a useful resource of frequency data for further studies interested in epidemiological or anthropological research questions involving

  16. Arylamine N-Acetyltransferase 2 (NAT2) Genetic Diversity and Traditional Subsistence: A Worldwide Population Survey

    PubMed Central

    Sabbagh, Audrey; Darlu, Pierre; Crouau-Roy, Brigitte; Poloni, Estella S.

    2011-01-01

    Arylamine N-acetyltransferase 2 (NAT2) is involved in human physiological responses to a variety of xenobiotic compounds, including common therapeutic drugs and exogenous chemicals present in the diet and the environment. Many questions remain about the evolutionary mechanisms that have led to the high prevalence of slow acetylators in the human species. Evidence from recent surveys of NAT2 gene variation suggests that NAT2 slow-causing variants might have become targets of positive selection as a consequence of the shift in modes of subsistence and lifestyle in human populations in the last 10,000 years. We aimed to test more extensively the hypothesis that slow acetylation prevalence in humans is related to the subsistence strategy adopted by the past populations. To this end, published frequency data on the most relevant genetic variants of NAT2 were collected from 128 population samples (14,679 individuals) representing different subsistence modes and dietary habits, allowing a thorough analysis at both a worldwide and continent scale. A significantly higher prevalence of the slow acetylation phenotype was observed in populations practicing farming (45.4%) and herding (48.2%) as compared to populations mostly relying on hunting and gathering (22.4%) (P = 0.0007). This was closely mirrored by the frequency of the slow 590A variant that was found to occur at a three-fold higher frequency in food producers (25%) as compared to hunter-gatherers (8%). These findings are consistent with the hypothesis that the Neolithic transition to subsistence economies based on agricultural and pastoral resources modified the selective regime affecting the NAT2 acetylation pathway. Furthermore, the vast amount of data collected enabled us to provide a comprehensive and up-to-date description of NAT2 worldwide genetic diversity, thus building up a useful resource of frequency data for further studies interested in epidemiological or anthropological research questions involving

  17. Osteoblast-specific gene expression after transplantation of marrow cells: Implications for skeletal gene therapy

    PubMed Central

    Hou, Zhen; Nguyen, Que; Frenkel, Baruch; Nilsson, Susan K.; Milne, Moira; van Wijnen, André J.; Stein, Janet L.; Quesenberry, Peter; Lian, Jane B.; Stein, Gary S.

    1999-01-01

    Somatic gene therapies require targeted transfer of the therapeutic gene(s) into stem cells that proliferate and then differentiate and express the gene in a tissue-restricted manner. We have developed an approach for gene therapy using marrow cells that takes advantage of the osteoblast specificity of the osteocalcin promoter to confine expression of chimeric genes to bone. Adherent marrow cells, carrying a reporter gene [chloramphenicol acetyltransferase (CAT)] under the control of a 1.7-kilobase rat osteocalcin gene promoter, were expanded ex vivo. After transplantation by intravenous infusion, engrafted donor cells in recipient mice were detected by the presence of the transgene in a broad spectrum of tissues. However, expression of the transgene was restricted to osteoblasts and osteocytes, as established by biochemical analysis of CAT activity and immunohistochemical analysis of CAT expression at the single cell level. Our data indicate that donor cells achieved long-term engraftment in various tissues of the recipients and that the CAT gene under control of the osteocalcin promoter is expressed specifically in bone. Thus, transplantation of multipotential marrow cells containing the osteocalcin promoter-controlled transgene provides an efficacious approach to deliver therapeutic gene expression to osteoblasts for treatment of bone disorders or tumor metastasis to the skeleton. PMID:10377408

  18. The Methionine Transamination Pathway Controls Hepatic Glucose Metabolism through Regulation of the GCN5 Acetyltransferase and the PGC-1α Transcriptional Coactivator.

    PubMed

    Tavares, Clint D J; Sharabi, Kfir; Dominy, John E; Lee, Yoonjin; Isasa, Marta; Orozco, Jose M; Jedrychowski, Mark P; Kamenecka, Theodore M; Griffin, Patrick R; Gygi, Steven P; Puigserver, Pere

    2016-05-13

    Methionine is an essential sulfur amino acid that is engaged in key cellular functions such as protein synthesis and is a precursor for critical metabolites involved in maintaining cellular homeostasis. In mammals, in response to nutrient conditions, the liver plays a significant role in regulating methionine concentrations by altering its flux through the transmethylation, transsulfuration, and transamination metabolic pathways. A comprehensive understanding of how hepatic methionine metabolism intersects with other regulatory nutrient signaling and transcriptional events is, however, lacking. Here, we show that methionine and derived-sulfur metabolites in the transamination pathway activate the GCN5 acetyltransferase promoting acetylation of the transcriptional coactivator PGC-1α to control hepatic gluconeogenesis. Methionine was the only essential amino acid that rapidly induced PGC-1α acetylation through activating the GCN5 acetyltransferase. Experiments employing metabolic pathway intermediates revealed that methionine transamination, and not the transmethylation or transsulfuration pathways, contributed to methionine-induced PGC-1α acetylation. Moreover, aminooxyacetic acid, a transaminase inhibitor, was able to potently suppress PGC-1α acetylation stimulated by methionine, which was accompanied by predicted alterations in PGC-1α-mediated gluconeogenic gene expression and glucose production in primary murine hepatocytes. Methionine administration in mice likewise induced hepatic PGC-1α acetylation, suppressed the gluconeogenic gene program, and lowered glycemia, indicating that a similar phenomenon occurs in vivo These results highlight a communication between methionine metabolism and PGC-1α-mediated hepatic gluconeogenesis, suggesting that influencing methionine metabolic flux has the potential to be therapeutically exploited for diabetes treatment.

  19. PET/CT imaging of human somatostatin receptor 2 (hsstr2) as reporter gene for gene therapy

    NASA Astrophysics Data System (ADS)

    Hofmann, M.; Gazdhar, A.; Weitzel, T.; Schmid, R.; Krause, T.

    2006-12-01

    Localized information on region-selective gene expression in small animals is widely obtained by use of reporter genes inducing light emission. Using these reporter genes for imaging deep inside the human body fluorescent probes are hindered by attenuation, scattering and possible fluorescence quenching. This can be overcome by use of radio-peptide receptors as reporter genes. Therefore, the feasibility of the somatostatin receptor 2 expression vector system for expression imaging was checked against a control vector containing luciferase gene. For in vivo transduction of vector DNA into the rat forelimb muscles the in vivo electroporation technique was chosen because of its high regio-selectivity. The gene expression was imaged by high-sensitive CCD camera (luciferase activity) and by PET/CT using a Ga-68-DOTATOC as radio peptide probe. The relative sstr2 expression was enhanced by gene transduction at maximum to a factor of 15. The PET/CT images could be fully quantified. The above demonstrated feasibility of radio-peptide PET/CT reporter gene imaging may serve in the future as a tool for full quantitative understanding of regional gene expression, especially in large animals and humans.

  20. Gemini, a Bifunctional Enzymatic and Fluorescent Reporter of Gene Expression

    PubMed Central

    Endy, Drew

    2009-01-01

    Background The development of collections of quantitatively characterized standard biological parts should facilitate the engineering of increasingly complex and novel biological systems. The existing enzymatic and fluorescent reporters that are used to characterize biological part functions exhibit strengths and limitations. Combining both enzymatic and fluorescence activities within a single reporter protein would provide a useful tool for biological part characterization. Methodology/Principal Findings Here, we describe the construction and quantitative characterization of Gemini, a fusion between the β-galactosidase (β-gal) α-fragment and the N-terminus of full-length green fluorescent protein (GFP). We show that Gemini exhibits functional β-gal activity, which we assay with plates and fluorometry, and functional GFP activity, which we assay with fluorometry and microscopy. We show that the protein fusion increases the sensitivity of β-gal activity and decreases the sensitivity of GFP. Conclusions/Significance Gemini is therefore a bifunctional reporter with a wider dynamic range than the β-gal α-fragment or GFP alone. Gemini enables the characterization of gene expression, screening assays via enzymatic activity, and quantitative single-cell microscopy or FACS via fluorescence activity. The analytical flexibility afforded by Gemini will likely increase the efficiency of research, particularly for screening and characterization of libraries of standard biological parts. PMID:19888458

  1. Expression of phosphinothricin N-acetyltransferase in Escherichia coli and Pseudomonas fluorescens: influence of mRNA secondary structure, host, and other physiological conditions.

    PubMed

    Madduri, Krishna M; Snodderley, Erika M

    2007-10-01

    Expression of a plant codon optimized pat gene encoding phosphinothricin acetyltransferase (PAT) in bacterial expression systems required modification of the 5' end of the pat ORF. Modifications necessary for improving the expression were identified by a coupled in vitro transcription and translation process. The dramatic improvement in the expression of PAT was due to the removal of a potential secondary structure that could have resulted in the inhibition of translational initiation. Therefore, in vitro transcription and translation is a versatile tool to optimize gene sequence for protein overexpression. Additionally, this method was shown to be successful in both Escherichia coli and Pseudomonas fluorescens. Gene sequence optimization and choice of host along with cultivation conditions also had major impact on PAT expression. P. fluorescens was a better host than E. coli resulting in 30-fold more expression of PAT. We were able to recover approximately 95mg of purified PAT from P. fluorescens using a three step chromatographic process.

  2. Cohesin recruits the Esco1 acetyltransferase genome wide to repress transcription and promote cohesion in somatic cells

    PubMed Central

    Rahman, Sadia; Jones, Mathew J. K.; Jallepalli, Prasad V.

    2015-01-01

    The cohesin complex links DNA molecules and plays key roles in the organization, expression, repair, and segregation of eukaryotic genomes. In vertebrates the Esco1 and Esco2 acetyltransferases both modify cohesin’s Smc3 subunit to establish sister chromatid cohesion during S phase, but differ in their N-terminal domains and expression during development and across the cell cycle. Here we show that Esco1 and Esco2 also differ dramatically in their interaction with chromatin, as Esco1 is recruited by cohesin to over 11,000 sites, whereas Esco2 is infrequently enriched at REST/NRSF target genes. Esco1’s colocalization with cohesin occurs throughout the cell cycle and depends on two short motifs (the A-box and B-box) present in and unique to all Esco1 orthologs. Deleting either motif led to the derepression of Esco1-proximal genes and functional uncoupling of cohesion from Smc3 acetylation. In contrast, other mutations that preserved Esco1’s recruitment separated its roles in cohesion establishment and gene silencing. We conclude that Esco1 uses cohesin as both a substrate and a scaffold for coordinating multiple chromatin-based transactions in somatic cells. PMID:26305936

  3. Cohesin recruits the Esco1 acetyltransferase genome wide to repress transcription and promote cohesion in somatic cells.

    PubMed

    Rahman, Sadia; Jones, Mathew J K; Jallepalli, Prasad V

    2015-09-01

    The cohesin complex links DNA molecules and plays key roles in the organization, expression, repair, and segregation of eukaryotic genomes. In vertebrates the Esco1 and Esco2 acetyltransferases both modify cohesin's Smc3 subunit to establish sister chromatid cohesion during S phase, but differ in their N-terminal domains and expression during development and across the cell cycle. Here we show that Esco1 and Esco2 also differ dramatically in their interaction with chromatin, as Esco1 is recruited by cohesin to over 11,000 sites, whereas Esco2 is infrequently enriched at REST/NRSF target genes. Esco1's colocalization with cohesin occurs throughout the cell cycle and depends on two short motifs (the A-box and B-box) present in and unique to all Esco1 orthologs. Deleting either motif led to the derepression of Esco1-proximal genes and functional uncoupling of cohesion from Smc3 acetylation. In contrast, other mutations that preserved Esco1's recruitment separated its roles in cohesion establishment and gene silencing. We conclude that Esco1 uses cohesin as both a substrate and a scaffold for coordinating multiple chromatin-based transactions in somatic cells. PMID:26305936

  4. Characterization of the human p53 gene promoter

    SciTech Connect

    Tuck, S.P.; Crawford, L.

    1989-05-01

    Transcriptional deregulation of the p53 gene may play an important part in the genesis of some tumors. The authors report here an accurate determination of the transcriptional start sites of the human p53 gene and show that the majority of p53 mRNA molecules do not contain a postulated stem-loop structure at their 5' ends. Recombinant plasmids of the human p53 promoter-leader region fused to the bacterial chloramphenicol acetyltransferase gene (cat) were constructed. After transfection into rodent or human cells, a 350-base-pair fragment spanning the promoter region conferred 4% of the CAT activity mediated by the simian virus 40 early promoter/enhancer. They monitored the efficiency with which 15 3' and 5' promoter deletion constructs initiated transcription. Their results show that an 85-base-pair fragment, previously thought to have resided in exon 1, is that is required for full promoter activity.

  5. Characterization of the mammalian DNA polymerase gene(s) and enzyme(s). Annual progress report

    SciTech Connect

    Mishra, N.C.

    1995-01-01

    Two Genes for DNA polymerase delta were identified from the wild type Chinese hamster ovary cells. These genes were cloned via RT-PCR from mRNA prepared the Chinese hamster ovary cells using primers specific to conserved sequences of the DNA polymerase {delta} gene. The first gene encodes a PCNA dependent DNA polymerase {delta} gene whereas the second gene encodes a PCNA independent DNA polymerase {delta} gene. Methods were developed to clone these genes in expression vector and host systems. The role of the two genes in DNA replication and repair was determined.

  6. MRI reporter genes: applications for imaging of cell survival, proliferation, migration and differentiation.

    PubMed

    Vandsburger, Moriel H; Radoul, Marina; Cohen, Batya; Neeman, Michal

    2013-07-01

    Molecular imaging strives to detect molecular events at the level of the whole organism. In some cases, the molecule of interest can be detected either directly or with targeted contrast media. However many genes and proteins and particularly those located in intracellular compartments are not accessible for targeted agents. The transcriptional regulation of these genes can nevertheless be detected, although indirectly, using reporter gene encoding for readily detectable proteins. Such reporter proteins can be expressed in the tissue of interest by genetically introducing the reporter gene in the target cells. Imaging of reporter genes has become a powerful tool in modern biomedical research. Typically, expression of fluorescent and bioluminescent proteins and the reaction product of expressed enzymes and exogenous substrates were examined using in vitro histological methods and in vivo whole body imaging methods. Recent advances in MRI reporter gene methods raised the possibility that MRI could become a powerful tool for concomitant high-resolution anatomical and functional imaging and for imaging of reporter gene activity. An immediate application of MRI reporter gene methods was by monitoring gene expression patterns in gene therapy and in vivo imaging of the survival, proliferation, migration and differentiation of pluripotent and multipotent cells used in cell-based regenerative therapies for cancer, myocardial infarction and neural degeneration. In this review, we characterized a variety of MRI reporter gene methods based on their applicability to report cell survival/proliferation, migration and differentiation. In particular, we discussed which methods were best suited for translation to clinical use in regenerative therapies.

  7. Photoacoustic microscopy of tyrosinase reporter gene in vivo

    NASA Astrophysics Data System (ADS)

    Krumholz, Arie; Vanvickle-Chavez, Sarah J.; Yao, Junjie; Fleming, Timothy P.; Gillanders, William E.; Wang, Lihong V.

    2011-08-01

    Photoacoustic tomography is a hybrid modality based on optical absorption excitation and ultrasonic detection. It is sensitive to melanin, one of the primary absorbers in skin. For cells that do not naturally contain melanin, melanin production can be induced by introducing the gene for tyrosinase, the primary enzyme responsible for expression of melanin in melanogenic cells. Optical resolution photoacoustic microscopy was used in the ex vivo study reported here, where the signal from transfected cells increased by more than 10 times over wild-type cells. A subsequent in vivo experiment was conducted to demonstrate the capability of photoacoustic microscopy to spectrally differentiate between tyrosinase-catalyzed melanin and various other absorbers in tissue.

  8. [Construction and specificity of porcine bmp15 gene reporter vector].

    PubMed

    Qin, Mingming; Wei, Jianghua; Yu, Xiaoli; Zhang, Jinglong; Liu, Xiaopeng; Ma, Xiaoling; Wang, Huayan

    2014-02-01

    The aim of this study is to identify the express specificity of bone morphogenetic protein 15 (Bmp15) in porcine. The pBMP15-EGFP reporter vector was constructed from the 2.2 kb fragment of porcine bmp15 promoter to trace the differentiation process of stem cells into oocyte-like cells. We used porcine ovary and Chinese Hamster Ovary cell line (CHO), mouse myoblast cell line (C2C12) and porcine amniotic fluid stem cell (pAFSC) to investigate the expression and regulation of this gene via RT-PCR, immunofluorescence, cell transfection, and microinjection methods. We also used single layer cell differentiation to detect the application potential of bmp15. The results show that bmp15 gene was specifically expressed in the porcine ovary and CHO rather than in C2C12 and pAFSC. In addition, the characteristic of tissue-specific of Bmp15 was detected on CHO instead of other cell lines by transient transfection. We also detected the expression of Bmp15 in oocyte at different development stages by immunofluorescence of fixed paraffin-embedded ovary sections. Furthermore, microinjection results show that bmp15 expressed in oocytes at 18 h of maturation in vitro, and continued up to 4-cell stage embryos. Most importantly, we found that the expression of Bmp15 started at day 12 after inducing pAFSC into oocyte-like cells by transfection; green fluorescent was visible in round cell masses. It indicated that bmp15 has the expression specificity and the pBMP15-EGFP reporter vector can be used to trace Bmp15 action in the differentiation of stem cells into germ cells.

  9. Testosterone locally increases vasopressin content but fails to restore choline acetyltransferase activity in other regions in the senescent male rat brain.

    PubMed

    Goudsmit, E; Luine, V N; Swaab, D F

    1990-05-01

    Age-related decreases have been reported in both vasopressinergic and cholinergic innervation in the rat brain. Since both systems are also sensitive to sex steroids, the effect of testosterone supplementation on vasopressin (AVP) levels and on choline acetyltransferase (ChAT) activity was investigated in the brains of young, middle-aged and aged male rats. Although no age-related changes in AVP levels were observed in the lateral septum or the medial amygdala (MA), peripheral testosterone administration raised AVP levels in the MA in all age groups. ChAT activity decreased with age in the medial preoptic area and was not restored by testosterone.

  10. (Genetic engineering with a gene encoding a soybean storage protein). Progress report

    SciTech Connect

    Beachy, R.N.

    1985-01-01

    Progress is reported on research directed toward introducing a gene (Gmg 17.1) encoding the ..cap alpha..'-subunit of ..beta..-conglycinin, a soybean seed protein, into petunia plants using gene transfer mechanisms. (ACR)

  11. Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B).

    PubMed

    Haigney, Allison; Ricketts, M Daniel; Marmorstein, Ronen

    2015-12-18

    The HAT-B enzyme complex is responsible for acetylating newly synthesized histone H4 on lysines K5 and K12. HAT-B is a multisubunit complex composed of the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46) histone chaperone. Hat1 is predominantly localized in the nucleus as a member of a trimeric NuB4 complex containing Hat1, Hat2, and a histone H3-H4 specific histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1 interacts with Asf1 (anti-silencing function 1), a histone chaperone that has been reported to be involved in both replication-dependent and -independent chromatin assembly. To elucidate the molecular roles of the Hif1 and Asf1 histone chaperones in HAT-B histone binding and acetyltransferase activity, we have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B and the effect of this binding on the enzymatic activity of HAT-B. We find that Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby Hif1 binds directly to Hat2, and Asf1 is only capable of interactions with HAT-B through contacts with histones H3-H4. We also demonstrate that HAT-B is significantly more active against an intact H3-H4 heterodimer over a histone H4 peptide, independent of either Hif1 or Asf1 binding. Mutational studies further demonstrate that HAT-B binding to the histone tail regions is not sufficient for this enhanced activity. Based on these data, we propose a model for HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes.

  12. Structure of soybean serine acetyltransferase and formation of the cysteine regulatory complex as a molecular chaperone

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase (OASS). Formation of the cysteine regulatory complex (CRC) is a critical biochem...

  13. Identification of genes in anonymous DNA sequences. Final report: Report period, 15 April 1993--15 April 1994

    SciTech Connect

    Fields, C.A.

    1994-09-01

    This Report concludes the DOE Human Genome Program project, ``Identification of Genes in Anonymous DNA Sequence.`` The central goals of this project have been (1) understanding the problem of identifying genes in anonymous sequences, and (2) development of tools, primarily the automated identification system gm, for identifying genes. The activities supported under the previous award are summarized here to provide a single complete report on the activities supported as part of the project from its inception to its completion.

  14. Retinoic acid-mediated gene expression in transgenic reporter zebrafish.

    PubMed

    Perz-Edwards, A; Hardison, N L; Linney, E

    2001-01-01

    Retinoic acid-mediated gene activation is important for normal vertebrate development. The size and nature of retinoic acid make it difficult to identify the precise cellular location of this signaling molecule throughout an embryo. Additionally, retinoic acid (RA) signaling is regulated by a complex combination of receptors, coactivators, and antagonizing proteins. Thus, in order to integrate these signals and identify regions within a whole developing embryo where cells can respond transcriptionally to retinoic acid, we have used a reporter transgenic approach. We have generated several stable lines of transgenic zebrafish which use retinoic acid response elements to drive fluorescent protein expression. In these zebrafish lines, transgene expression is localized to regions of the neural tube, retina, notochord, somites, heart, pronephric ducts, branchial arches, and jaw muscles in embryos and larvae. Transgene expression can be induced in additional regions of the neural tube and retina as well as the immature notochord, hatching gland, enveloping cell layer, and fin by exposing embryos to retinoic acid. Treatment with retinoic acid synthase inhibitors, citral and diethylaminobenzaldehyde (DEAB), during neurulation, greatly reduces transgene expression. DEAB treatment of embryos at gastrulation phenocopies the embryonic effects of vitamin A deprivation or targeted disruption of the RA synthase retinaldehyde dehydrogenase-2 in other vertebrates. Together these data suggest that the reporter expression we see in zebrafish is dependent upon conserved vertebrate pathways of RA synthesis.

  15. [Clinical relevance of N-acetyltransferase type 2 (NAT2) genetic polymorphism].

    PubMed

    Furet, Y; Bechtel, Y; Le Guellec, C; Bechtel, P R; Autret-Leca, E; Paintaud, G

    2002-01-01

    Polymorphic N-acetyltransferase (NAT2) is involved in the metabolism of several compounds relevant in pharmacology or toxicology, with diverse clinical consequences. Inter-ethnic variations in distribution of the acetylation phenotype are significant. The caffeine test is most often used to assess the acetylation phenotype and to identify rapid and slow acetylators. The NAT2 phenotype could account for the increased risk of certain side effects in slow acetylators treated with isoniazid (particularly peripheral neuropathies and lupus erythematosus), although therapeutic efficacy seems to be independent of the acetylation status. Hypersensibility reactions with sulfonamides (including Lyell and Stevens-Johnson syndromes) are more frequent in slow acetylators, who also show poor tolerance to sulfasalazine and dapsone. In contrast, myelotoxicity induced by amonafide is more frequent in rapid acetylators, probably because of increased production of a toxic metabolite of the drug. In carcinogenesis, NAT2 may play a protective role against bladder cancer, although studies have shown contradictory results. Slow acetylators may have a risk of developing primitive liver cancer. For lung cancer, data are not conclusive, but slow acetylation status may predispose to mesothelioma in subjects exposed to asbestos. No relation has been found between acetylation phenotype and breast cancer. Contradictory results were reported on its role in colorectal cancer. Non-smoking type 1 diabetics may be at increased risk of nephropathy if they are rapid acetylators. Parkinson's disease may be more frequent among slow acetylators, but again, data have shown contradictory results. Finally, a poor acetylator phenotype may predispose to atopic diseases. PMID:12611196

  16. Reporter gene expression in dendritic cells after gene gun administration of plasmid DNA.

    PubMed

    Watkins, Craig; Hopkins, John; Harkiss, Gordon

    2005-07-21

    Dendritic cells (DC) play an integral role in plasmid DNA vaccination. However, the interaction between plasmid DNA and DC in vivo is incompletely understood. In this report, we utilise the sheep pseudoafferent cannulation model to examine the interaction between plasmid DNA encoding enhanced green fluorescent protein (pEGFP) and afferent lymph DC (ALDC) following gene gun administration. The results show that peaks of fluorescent ALDC tended to appear around days 1-4 and 9-13, then erratically thereafter for up to 2 months. Phenotypic analysis showed that EGFP+ ALDC expressed MHC class II, WC6, CD1b, and SIRPalpha markers. Plasmid, detected by PCR, was found in lymph cells and cell-free plasma on a daily basis, and was present variably for up to 2 months. Plasmid was also detected in purified CD1b+ ALDC, but the presence of plasmid did not correlate with EGFP expression by ALDC. Free EGFP in afferent lymph plasma was detectable by luminometry only after three administrations of the plasmid. The results show that gene gun administered pEGFP persisted for extended periods after a single administration, leeching out of skin on a daily basis. The plasmid was associated with both the cellular and fluid components of afferent lymph. EGFP protein appeared in afferent lymph in a pulsatile manner, but associated only with ALDC.

  17. Regulation of a Protein Acetyltransferase in Myxococcus xanthus by the Coenzyme NADP+

    PubMed Central

    Liu, Xin-Xin

    2015-01-01

    ABSTRACT NADP+ is a vital cofactor involved in a wide variety of activities, such as redox potential and cell death. Here, we show that NADP+ negatively regulates an acetyltransferase from Myxococcus xanthus, Mxan_3215 (MxKat), at physiologic concentrations. MxKat possesses an NAD(P)-binding domain fused to the Gcn5-type N-acetyltransferase (GNAT) domain. We used isothermal titration calorimetry (ITC) and a coupled enzyme assay to show that NADP+ bound to MxKat and that the binding had strong effects on enzyme activity. The Gly11 residue of MxKat was confirmed to play an important role in NADP+ binding using site-directed mutagenesis and circular dichroism spectrometry. In addition, using mass spectrometry, site-directed mutagenesis, and a coupling enzymatic assay, we demonstrated that MxKat acetylates acetyl coenzyme A (acetyl-CoA) synthetase (Mxan_2570) at Lys622 in response to changes in NADP+ concentration. Collectively, our results uncovered a mechanism of protein acetyltransferase regulation by the coenzyme NADP+ at physiological concentrations, suggesting a novel signaling pathway for the regulation of cellular protein acetylation. IMPORTANCE Microorganisms have developed various protein posttranslational modifications (PTMs), which enable cells to respond quickly to changes in the intracellular and extracellular milieus. This work provides the first biochemical characterization of a protein acetyltransferase (MxKat) that contains a fusion between a GNAT domain and NADP+-binding domain with Rossmann folds, and it demonstrates a novel signaling pathway for regulating cellular protein acetylation in M. xanthus. We found that NADP+ specifically binds to the Rossmann fold of MxKat and negatively regulates its acetyltransferase activity. This finding provides novel insight for connecting cellular metabolic status (NADP+ metabolism) with levels of protein acetylation, and it extends our understanding of the regulatory mechanisms underlying PTMs. PMID:26598367

  18. Development of Timd2 as a reporter gene for MRI

    PubMed Central

    Patrick, P. Stephen; Rodrigues, Tiago B.; Kettunen, Mikko I.; Lyons, Scott K.; Neves, André A.

    2015-01-01

    Purpose To assess the potential of an MRI gene reporter based on the ferritin receptor Timd2 (T‐cell immunoglobulin and mucin domain containing protein 2), using T1‐ and T2‐weighted imaging. Methods Pellets of cells that had been modified to express the Timd2 transgene, and incubated with either iron‐loaded or manganese‐loaded ferritin, were imaged using T1‐ and T2‐weighted MRI. Mice were also implanted subcutaneously with Timd2‐expressing cells and the resulting xenograft tissue imaged following intravenous injection of ferritin using T2‐weighted imaging. Results Timd2‐expressing cells, but not control cells, showed a large increase in both R2 and R1 in vitro following incubation with iron‐loaded and manganese‐loaded ferritin, respectively. Expression of Timd2 had no effect on cell viability or proliferation; however, manganese‐loaded ferritin, but not iron‐loaded ferritin, was toxic to Timd2‐expressing cells. Timd2‐expressing xenografts in vivo showed much smaller changes in R2 following injection of iron‐loaded ferritin than the same cells incubated in vitro with iron‐loaded ferritin. Conclusion Timd2 has demonstrated potential as an MRI reporter gene, producing large increases in R2 and R1 with ferritin and manganese‐loaded ferritin respectively in vitro, although more modest changes in R2 in vivo. Manganese‐loaded apoferritin was not used in vivo due to the toxicity observed in vitro. Magn Reson Med, 2015. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Magn Reson Med 75:1697–1707, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society

  19. Anti-histone acetyltransferase activity from allspice extracts inhibits androgen receptor-dependent prostate cancer cell growth.

    PubMed

    Lee, Yoo-Hyun; Hong, Soon Won; Jun, Woojin; Cho, Hong Yon; Kim, Han-Cheon; Jung, Myung Gu; Wong, Jiemin; Kim, Ha-Il; Kim, Chang-Hoon; Yoon, Ho-Geun

    2007-11-01

    Histone acetylation depends on the activity of two enzyme families, histone acetyltransferase (HAT) and deacetylase (HDAC). In this study, we screened various plant extracts to find potent HAT inhibitors. Hot water extracts of allspice inhibited HAT activity, especially p300 and CBP (40% at 100 microg/ml). The mRNA levels of two androgen receptor (AR) regulated genes, PSA and TSC22, decreased with allspice treatment (100 microg/ml). Importantly, in IP western analysis, AR acetylation was dramatically decreased by allspice treatment.Furthermore, chromatin immunoprecipitation indicated that the acetylation of histone H3 in the PSA and B2M promoter regions was also repressed. Finally, allspice treatment reduced the growth of human prostate cancer cells, LNCaP (50% growth inhibition at 200 microg/ml). Taken together, our data indicate that the potent HAT inhibitory activity of allspice reduced AR and histone acetylation and led to decreased transcription of AR target genes, resulting in inhibition of prostate cancer cell growth.

  20. Absence of Rtt109p, a fungal-specific histone acetyltransferase, results in improved acetic acid tolerance of Saccharomyces cerevisiae.

    PubMed

    Cheng, Cheng; Zhao, Xinqing; Zhang, Mingming; Bai, Fengwu

    2016-03-01

    RTT109 is a histone acetyltransferase for the acetylation of histone H3. It is still not clear whether RTT109 plays a role in regulation of gene expression under environmental stresses. In this study, the involvement of RTT109 in acetic acid stress tolerance of Saccharomyces cerevisiae was investigated. It was revealed that the absence of RTT109 enhanced resistance to 5.5 g L(-1) acetic acid, which was indicated by improved growth of RTT109Δ mutant compared with that of the wild-type BY4741 strain. Meanwhile, the lag phase was shortened for 48 h and glucose consumption completed 36 h in advance for RTT109Δ mutant compared to the wild-type strain, with ethanol production rate increased from 0.39 to 0.60 g L(-1) h(-1). Significantly, elevated transcription levels of HSP12, CTT1 and GSH1, as well as increased activities of antioxidant enzymes were observed in RTT109Δ under acetic acid stress. Improved flocculation of RTT109Δ compared to that of the control strain BY4741 under the acetic acid stress was also observed. These results suggest that the absence of RTT109 not only activates transcription of stress responsive genes, but also improves resistance to oxidative stress, which ultimately contributes to improved acetic acid tolerance in S. cerevisiae.

  1. Absence of Rtt109p, a fungal-specific histone acetyltransferase, results in improved acetic acid tolerance of Saccharomyces cerevisiae.

    PubMed

    Cheng, Cheng; Zhao, Xinqing; Zhang, Mingming; Bai, Fengwu

    2016-03-01

    RTT109 is a histone acetyltransferase for the acetylation of histone H3. It is still not clear whether RTT109 plays a role in regulation of gene expression under environmental stresses. In this study, the involvement of RTT109 in acetic acid stress tolerance of Saccharomyces cerevisiae was investigated. It was revealed that the absence of RTT109 enhanced resistance to 5.5 g L(-1) acetic acid, which was indicated by improved growth of RTT109Δ mutant compared with that of the wild-type BY4741 strain. Meanwhile, the lag phase was shortened for 48 h and glucose consumption completed 36 h in advance for RTT109Δ mutant compared to the wild-type strain, with ethanol production rate increased from 0.39 to 0.60 g L(-1) h(-1). Significantly, elevated transcription levels of HSP12, CTT1 and GSH1, as well as increased activities of antioxidant enzymes were observed in RTT109Δ under acetic acid stress. Improved flocculation of RTT109Δ compared to that of the control strain BY4741 under the acetic acid stress was also observed. These results suggest that the absence of RTT109 not only activates transcription of stress responsive genes, but also improves resistance to oxidative stress, which ultimately contributes to improved acetic acid tolerance in S. cerevisiae. PMID:26851403

  2. N-Acetyltransferase 1 (NAT1) Genotype: A Risk Factor for Urinary Bladder Cancer in a Lebanese Population

    PubMed Central

    Yassine, Ibrahim A.; Kobeissi, Loulou; Jabbour, Michel E.; Dhaini, Hassan R.

    2012-01-01

    In Lebanon, bladder cancer is the second most incident cancer among men. This study investigates a possible association between N-acetyltransferase 1 (NAT1) genotype, a drug-metabolizing enzyme coding gene, and bladder cancer in Lebanese men. A case-control study (54 cases and 105 hospital-based controls) was conducted in two major hospitals in Beirut. Cases were randomly selected from patients diagnosed in the period of 2002–2008. Controls were conveniently identified and selected from the same settings. Data was collected using interview questionnaire and blood analysis. NAT1 genotypes were determined by PCR-RFLP. Statistical analysis revolved around univariate, bivariate, and multivariate logistic regression models, along with checks for effect modification. Results showed NAT1∗14A allele, smoking, occupational exposure to combustion fumes, and prostate-related symptoms, to be risk factors for bladder cancer. The odds of carrying at least one NAT1∗14A allele are 7 times higher in cases compared to controls (OR = 7.86, 95% CI: 1.53–40.39). A gene-environment interaction was identified for NAT1∗14A allele with occupational exposure to combustion fumes. Among carriers of NAT1∗14A allele, the odds of bladder cancer dropped to 2.03 from 3.72. Our study suggests NAT1∗14A allele as a possible biomarker for bladder cancer. Further research is recommended to confirm this association. PMID:22956951

  3. Serine O-acetyltransferase is important, but not essential for cysteine-methionine synthesis in Fusarium graminearum.

    PubMed

    Fu, Jing; Zhang, Xiaoping; Chen, Xiang; Yin, Yanni; Ma, Zhonghua

    2014-04-01

    O-acetyltransferase (SAT) is a key enzyme converting serine into O-acetylserine in the synthesis of sulphur-containing amino acids. To characterize the function of FgSAT in Fusarium graminearum, three deletion mutants of FgSAT (ΔFgSAT-1, -2 and -18) were obtained using a gene replacement strategy. The three mutants did not show recognizable phenotypic changes on potato dextrose agar medium, but exhibited a very weak growth on fructose gelatin agar (FGA) medium containing SO₄²⁻ as sole sulfur source. Supplementation of O-acetylserine, cysteine, or methionine, but not serine, rescued the defect of mycelial growth in FgSAT deletion mutants, indicating that FgSAT is involved in conversion of serine into O-acetylserine. The three mutants had a decrease in conidiation in mung bean liquid, but not in carboxymethyl cellulose. Virulence, deoxynivalenol production and fungicide sensitivity assays found that the three mutants showed no significant difference from wild-type progenitor PH-1. Real-time PCR assays detected an increase in expression levels of FgOAHS, FgCBS and FgCGL genes involved in the alternative pathway in FgSAT deletion mutants, suggesting that the alternative pathway in F. graminearum is present and can operate. Addition of homoserine, the upstream substrate of the alternative pathway, also restored the normal mycelial growth of FgSAT deletion mutants on FGA, indicating that the alternative pathway in F. graminearum might be positively regulated by homoserine.

  4. Delphinidin, a specific inhibitor of histone acetyltransferase, suppresses inflammatory signaling via prevention of NF-{kappa}B acetylation in fibroblast-like synoviocyte MH7A cells

    SciTech Connect

    Seong, Ah-Reum; Yoo, Jung-Yoon; Choi, KyungChul; Lee, Mee-Hee; Lee, Yoo-Hyun; Lee, Jeongmin; Jun, Woojin; Kim, Sunoh; Yoon, Ho-Geun

    2011-07-08

    Highlights: {yields} Delphinidin is a novel inhibitor of p300/CBP histone acetyltransferase. {yields} Delphinidin prevents the hyperacetylation of p65 by inhibiting the HAT activity of p300/CBP. {yields} Delphinidin efficiently suppresses the expression of inflammatory cytokines in MH7A cells via hypoacetylation of NF-{kappa}B. {yields} Delphinidin inhibits cytokine release in the Jurkat T lymphocyte cell line. -- Abstract: Histone acetyltransferase (HAT) inhibitors (HATi) isolated from dietary compounds have been shown to suppress inflammatory signaling, which contributes to rheumatoid arthritis. Here, we identified a novel HATi in Punica granatum L. known as delphinidin (DP). DP did not affect the activity of other epigenetic enzymes (histone deacetylase, histone methyltransferase, or sirtuin1). DP specifically inhibited the HAT activities of p300/CBP. It also inhibited p65 acetylation in MH7A cells, a human rheumatoid arthritis synovial cell line. DP-induced hypoacetylation was accompanied by cytosolic accumulation of p65 and nuclear localization of IKB{alpha}. Accordingly, DP treatment inhibited TNF{alpha}-stimulated increases in NF-{kappa}B function and expression of NF-{kappa}B target genes in these cells. Importantly, DP suppressed lipopolysaccharide-induced pro-inflammatory cytokine expression in Jurkat T lymphocytes, demonstrating that HATi efficiently suppresses cytokine-mediated immune responses. Together, these results show that the HATi activity of DP counters anti-inflammatory signaling by blocking p65 acetylation and that this compound may be useful in preventing inflammatory arthritis.

  5. The Saccharomyces cerevisiae Piccolo NuA4 histone acetyltransferase complex requires the Enhancer of Polycomb A domain and chromodomain to acetylate nucleosomes.

    PubMed

    Selleck, William; Fortin, Israël; Sermwittayawong, Decha; Côté, Jacques; Tan, Song

    2005-07-01

    Chromatin modification complexes are key gene regulatory factors which posttranslationally modify the histone component of chromatin with epigenetic marks. To address what features of chromatin modification complexes are responsible for the specific recognition of nucleosomes compared to naked histones, we have performed a functional dissection of the Esa1-containing Saccharomyces cerevisiae Piccolo NuA4 histone acetyltransferase complex. Our studies define the Piccolo determinants sufficient to assemble its three subunits into a complex as well as Piccolo determinants sufficient to specifically acetylate a chromatin template. We find that the conserved Enhancer of Polycomb A (EPcA) homology region of the Epl1 component and the N-terminal 165 amino acids of the Yng2 component of Piccolo are sufficient with Esa1 to specifically act on nucleosomes. We also find that the Esa1 chromodomain plays a critical role in Piccolo's ability to distinguish between histones and nucleosomes. In particular, specific point mutations in the chromodomain putative hydrophobic cage which strongly hinder growth in yeast greatly reduce histone acetyltransferase activity on nucleosome substrates, independent of histone methylation or other modifications. However, the chromodomain is not required for Piccolo to bind to nucleosomes, suggesting a role for the chromodomain in a catalysis step after nucleosome binding.

  6. The Saccharomyces cerevisiae Piccolo NuA4 Histone Acetyltransferase Complex Requires the Enhancer of Polycomb A Domain and Chromodomain To Acetylate Nucleosomes

    PubMed Central

    Selleck, William; Fortin, Israël; Sermwittayawong, Decha; Côté, Jacques; Tan, Song

    2005-01-01

    Chromatin modification complexes are key gene regulatory factors which posttranslationally modify the histone component of chromatin with epigenetic marks. To address what features of chromatin modification complexes are responsible for the specific recognition of nucleosomes compared to naked histones, we have performed a functional dissection of the Esa1-containing Saccharomyces cerevisiae Piccolo NuA4 histone acetyltransferase complex. Our studies define the Piccolo determinants sufficient to assemble its three subunits into a complex as well as Piccolo determinants sufficient to specifically acetylate a chromatin template. We find that the conserved Enhancer of Polycomb A (EPcA) homology region of the Epl1 component and the N-terminal 165 amino acids of the Yng2 component of Piccolo are sufficient with Esa1 to specifically act on nucleosomes. We also find that the Esa1 chromodomain plays a critical role in Piccolo's ability to distinguish between histones and nucleosomes. In particular, specific point mutations in the chromodomain putative hydrophobic cage which strongly hinder growth in yeast greatly reduce histone acetyltransferase activity on nucleosome substrates, independent of histone methylation or other modifications. However, the chromodomain is not required for Piccolo to bind to nucleosomes, suggesting a role for the chromodomain in a catalysis step after nucleosome binding. PMID:15964809

  7. Spt-Ada-Gcn5-Acetyltransferase (SAGA) Complex in Plants: Genome Wide Identification, Evolutionary Conservation and Functional Determination.

    PubMed

    Srivastava, Rakesh; Rai, Krishan Mohan; Pandey, Bindu; Singh, Sudhir P; Sawant, Samir V

    2015-01-01

    The recruitment of RNA polymerase II on a promoter is assisted by the assembly of basal transcriptional machinery in eukaryotes. The Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex plays an important role in transcription regulation in eukaryotes. However, even in the advent of genome sequencing of various plants, SAGA complex has been poorly defined for their components and roles in plant development and physiological functions. Computational analysis of Arabidopsis thaliana and Oryza sativa genomes for SAGA complex resulted in the identification of 17 to 18 potential candidates for SAGA subunits. We have further classified the SAGA complex based on the conserved domains. Phylogenetic analysis revealed that the SAGA complex proteins are evolutionary conserved between plants, yeast and mammals. Functional annotation showed that they participate not only in chromatin remodeling and gene regulation, but also in different biological processes, which could be indirect and possibly mediated via the regulation of gene expression. The in silico expression analysis of the SAGA components in Arabidopsis and O. sativa clearly indicates that its components have a distinct expression profile at different developmental stages. The co-expression analysis of the SAGA components suggests that many of these subunits co-express at different developmental stages, during hormonal interaction and in response to stress conditions. Quantitative real-time PCR analysis of SAGA component genes further confirmed their expression in different plant tissues and stresses. The expression of representative salt, heat and light inducible genes were affected in mutant lines of SAGA subunits in Arabidopsis. Altogether, the present study reveals expedient evidences of involvement of the SAGA complex in plant gene regulation and stress responses.

  8. The histone acetyltransferase GcnE (GCN5) plays a central role in the regulation of Aspergillus asexual development.

    PubMed

    Cánovas, David; Marcos, Ana T; Gacek, Agnieszka; Ramos, María S; Gutiérrez, Gabriel; Reyes-Domínguez, Yazmid; Strauss, Joseph

    2014-08-01

    Acetylation of histones is a key regulatory mechanism of gene expression in eukaryotes. GcnE is an acetyltransferase of Aspergillus nidulans involved in the acetylation of histone H3 at lysine 9 and lysine 14. Previous works have demonstrated that deletion of gcnE results in defects in primary and secondary metabolism. Here we unveil the role of GcnE in development and show that a ∆gcnE mutant strain has minor growth defects but is impaired in normal conidiophore development. No signs of conidiation were found after 3 days of incubation, and immature and aberrant conidiophores were found after 1 week of incubation. Centroid linkage clustering and principal component (PC) analysis of transcriptomic data suggest that GcnE occupies a central position in Aspergillus developmental regulation and that it is essential for inducing conidiation genes. GcnE function was found to be required for the acetylation of histone H3K9/K14 at the promoter of the master regulator of conidiation, brlA, as well as at the promoters of the upstream developmental regulators of conidiation flbA, flbB, flbC, and flbD (fluffy genes). However, analysis of the gene expression of brlA and the fluffy genes revealed that the lack of conidiation originated in a complete absence of brlA expression in the ∆gcnE strain. Ectopic induction of brlA from a heterologous alcA promoter did not remediate the conidiation defects in the ∆gcnE strain, suggesting that additional GcnE-mediated mechanisms must operate. Therefore, we conclude that GcnE is the only nonessential histone modifier with a strong role in fungal development found so far.

  9. Optical imaging of reporter gene expression using a positron-emission-tomography probe

    NASA Astrophysics Data System (ADS)

    Liu, Hongguang; Ren, Gang; Liu, Shuanglong; Zhang, Xiaofen; Chen, Luxi; Han, Peizhen; Cheng, Zhen

    2010-11-01

    Reporter gene/reporter probe technology is one of the most important techniques in molecular imaging. Lately, many reporter gene/reporter probe systems have been coupled to different imaging modalities such as positron emission tomography (PET) and optical imaging (OI). It has been recently found that OI techniques could be used to monitor radioactive tracers in vitro and in living subjects. In this study, we further demonstrate that a reporter gene/nuclear reporter probe system [herpes simplex virus type-1 thymidine kinase (HSV1-tk) and 9-(4-18F-fluoro-3-[hydroxymethyl] butyl) guanine ([18F]FHBG)] could be successfully imaged by OI in vitro and in vivo. OI with radioactive reporter probes will facilitate and broaden the applications of reporter gene/reporter probe techniques in medical research.

  10. Key gene regulating cell wall biosynthesis and recalcitrance in Populus, gene Y

    DOEpatents

    Chen, Jay; Engle, Nancy; Gunter, Lee E.; Jawdy, Sara; Tschaplinski, Timothy J.; Tuskan, Gerald A.

    2015-12-08

    This disclosure provides methods and transgenic plants for improved production of renewable biofuels and other plant-derived biomaterials by altering the expression and/or activity of Gene Y, an O-acetyltransferase. This disclosure also provides expression vectors containing a nucleic acid (Gene Y) which encodes the polypeptide of SEQ ID NO: 1 and is operably linked to a heterologous promoter.

  11. Salmonella enterica Serovar Typhimurium blaPER-1-Carrying Plasmid pSTI1 Encodes an Extended-Spectrum Aminoglycoside 6′-N-Acetyltransferase of Type Ib

    PubMed Central

    Casin, Isabelle; Hanau-Berçot, Beatrice; Podglajen, Isabelle; Vahaboglu, Haluk; Collatz, Ekkehard

    2003-01-01

    We have studied the aminoglycoside resistance gene, which confers high levels of resistance to both amikacin and gentamicin, that is carried by plasmid pSTI1 in the PER-1 β-lactamase-producing strain of Salmonella enterica serovar Typhimurium previously isolated in Turkey. This gene, called aac(6′)-Ib11, was found in a class 1 integron and codes for a protein of 188 amino acids, a fusion product between the N-terminal moiety (8 amino acids) of the signal peptide of the β-lactamase OXA-1 and the acetyltransferase. The gene lacked a plausible Shine-Dalgarno (SD) sequence and was located 45 nucleotides downstream from a small open reading frame, ORF-18, with a coding capacity of 18 amino acids and a properly spaced SD sequence likely to direct the initiation of aac(6′)-Ib11 translation. AAC(6′)-Ib11 had Leu118 and Ser119 as opposed to Gln and Leu or Gln and Ser, respectively, which were observed in all previously described enzymes of this type. We have evaluated the effect of Leu or Gln at position 118 by site-directed mutagenesis of aac(6′)-Ib11 and two other acetyltransferase gene variants, aac(6′)-Ib7 and -Ib8, which naturally encode Gln118. Our results show that the combination of Leu118 and Ser119 confers an extended-spectrum aminoglycoside resistance, with the MICs of all aminoglycosides in clinical use, including gentamicin, being two to eight times higher for strains with Leu118 and Ser119 than for those with Gln118 and Ser119. PMID:12543680

  12. Comparative genomic survey of microbial arylamine N-acetyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: Microorganisms are constantly exposed to exogenous chemical influences. Our previous genomic surveys have identified putative NAT genes across a phylogenetic spectrum of prokaryotic and eukaryotic microorganisms. We are currently pursuing two lines of investigation: The first looks int...

  13. Nickel and cobalt resistance engineered in Escherichia coli by overexpression of serine acetyltransferase from the nickel hyperaccumulator plant Thlaspi goesingense.

    PubMed

    Freeman, John L; Persans, Michael W; Nieman, Ken; Salt, David E

    2005-12-01

    The overexpression of serine acetyltransferase from the Ni-hyperaccumulating plant Thlaspi goesingense causes enhanced nickel and cobalt resistance in Escherichia coli. Furthermore, overexpression of T. goesingense serine acetyltransferase results in enhanced sensitivity to cadmium and has no significant effect on resistance to zinc. Enhanced nickel resistance is directly related to the constitutive overactivation of sulfur assimilation and glutathione biosynthesis, driven by the overproduction of O-acetyl-L-serine, the product of serine acetyltransferase and a positive regulator of the cysteine regulon. Nickel in the serine acetyltransferase-overexpressing strains is not detoxified by coordination or precipitation with sulfur, suggesting that glutathione is involved in reducing the oxidative damage imposed by nickel. PMID:16332856

  14. Nickel and Cobalt Resistance Engineered in Escherichia coli by Overexpression of Serine Acetyltransferase from the Nickel Hyperaccumulator Plant Thlaspi goesingense

    PubMed Central

    Freeman, John L.; Persans, Michael W.; Nieman, Ken; Salt, David E.

    2005-01-01

    The overexpression of serine acetyltransferase from the Ni-hyperaccumulating plant Thlaspi goesingense causes enhanced nickel and cobalt resistance in Escherichia coli. Furthermore, overexpression of T. goesingense serine acetyltransferase results in enhanced sensitivity to cadmium and has no significant effect on resistance to zinc. Enhanced nickel resistance is directly related to the constitutive overactivation of sulfur assimilation and glutathione biosynthesis, driven by the overproduction of O-acetyl-l-serine, the product of serine acetyltransferase and a positive regulator of the cysteine regulon. Nickel in the serine acetyltransferase-overexpressing strains is not detoxified by coordination or precipitation with sulfur, suggesting that glutathione is involved in reducing the oxidative damage imposed by nickel. PMID:16332856

  15. Reporter enzyme inhibitor study to aid assembly of orthogonal reporter gene assays.

    PubMed

    Ho, Pei-i; Yue, Kimberley; Pandey, Pramod; Breault, Lyne; Harbinski, Fred; McBride, Aaron J; Webb, Brian; Narahari, Janaki; Karassina, Natasha; Wood, Keith V; Hill, Adam; Auld, Douglas S

    2013-05-17

    Reporter gene assays (RGAs) are commonly used to measure biological pathway modulation by small molecules. Understanding how such compounds interact with the reporter enzyme is critical to accurately interpret RGA results. To improve our understanding of reporter enzymes and to develop optimal RGA systems, we investigated eight reporter enzymes differing in brightness, emission spectrum, stability, and substrate requirements. These included common reporter enzymes such as firefly luciferase (Photinus pyralis), Renilla reniformis luciferase, and β-lactamase, as well as mutated forms of R. reniformis luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and a proprietary luciferase termed "NanoLuc" derived from the luminescent sea shrimp Oplophorus gracilirostris. To determine hit rates and structure-activity relationships, we screened a collection of 42,460 PubChem compounds at 10 μM using purified enzyme preparations. We then compared hit rates and chemotypes of actives for each enzyme. The hit rates ranged from <0.1% for β-lactamase to as high as 10% for mutated forms of Renilla luciferase. Related luciferases such as Renilla luciferase mutants showed high degrees of inhibitor overlap (40-70%), while unrelated luciferases such as firefly luciferases, Gaussia luciferase, and NanoLuc showed <10% overlap. Examination of representative inhibitors in cell-based assays revealed that inhibitor-based enzyme stabilization can lead to increases in bioluminescent signal for firefly luciferase, Renilla luciferase, and NanoLuc, with shorter half-life reporters showing increased activation responses. From this study we suggest strategies to improve the construction and interpretation of assays employing these reporter enzymes.

  16. System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveals Substrate Specificity of Protein Acetyltransferases

    SciTech Connect

    Crosby, Heidi A; Pelletier, Dale A; Hurst, Gregory {Greg} B; Escalante-Semerena, Jorge C

    2012-01-01

    Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.

  17. [Histochemistry and choline acetyltransferase in cat spinal cord and spinal ganglia].

    PubMed

    Motavkin, P A; Okhotin, V E

    1978-09-01

    Cytochemical activity of choline acetyltransferase has been studied in the pericaryon of motor neurons of the spinal enlargement and sensitive neurocytes of the intervertebral ganglia in the cat by means of Burt's method. It has been demonstrated that cytoplasm of all motor neurons positively reacts with acetyl KoA. According to the activity of choline acetyltransferase, four groups of neurons have been determined. In cerebrospinal ganglia, the enzyme is present in 58% of pseudounipolar cells, which seem to be cholinergic neurocytes. It has been stated that for all nonspecific reactions the presence of massive and dense residue in all the neurons, walls of small blood vessels and sometimes in astrocytes is a characteristic feature. PMID:718431

  18. Use of Reporter Genes in the Generation of Vaccinia Virus-Derived Vectors

    PubMed Central

    Al Ali, Sally; Baldanta, Sara; Fernández-Escobar, Mercedes; Guerra, Susana

    2016-01-01

    Vaccinia virus (VACV) is one of the most extensively-studied viruses of the Poxviridae family. It is easy to genetically modify, so it has become a key tool for many applications. In this context, reporter genes facilitate the study of the role of foreign genes introduced into the genome of VACV. In this review, we describe the type of reporter genes that have been used to generate reporter-expressing VACV and the applications of the recombinant viruses obtained. Reporter-expressing VACV are currently employed in basic and immunology research, in the development of vaccines and cancer treatment. PMID:27213433

  19. Use of Reporter Genes in the Generation of Vaccinia Virus-Derived Vectors.

    PubMed

    Al Ali, Sally; Baldanta, Sara; Fernández-Escobar, Mercedes; Guerra, Susana

    2016-01-01

    Vaccinia virus (VACV) is one of the most extensively-studied viruses of the Poxviridae family. It is easy to genetically modify, so it has become a key tool for many applications. In this context, reporter genes facilitate the study of the role of foreign genes introduced into the genome of VACV. In this review, we describe the type of reporter genes that have been used to generate reporter-expressing VACV and the applications of the recombinant viruses obtained. Reporter-expressing VACV are currently employed in basic and immunology research, in the development of vaccines and cancer treatment. PMID:27213433

  20. Gallic Acid Decreases Inflammatory Cytokine Secretion Through Histone Acetyltransferase/Histone Deacetylase Regulation in High Glucose-Induced Human Monocytes.

    PubMed

    Lee, Wooje; Lee, Sang Yeol; Son, Young-Jin; Yun, Jung-Mi

    2015-07-01

    Hyperglycemia contributes to diabetes and several diabetes-related complications. Gallic acid is a polyhydroxy phenolic compound found in various natural products. In this study, we investigated the effects and mechanism of gallic acid on proinflammatory cytokine secretion in high glucose-induced human monocytes (THP-1 cells). THP-1 cells were cultured under normoglycemic or hyperglycemic conditions, in the absence or presence of gallic acid. Hyperglycemic conditions significantly induced histone acetylation, nuclear factor-κB (NF-κB) activation, and proinflammatory cytokine release from THP-1 cells, whereas gallic acid suppressed NF-κB activity and cytokine release. It also significantly reduced CREB-binding protein/p300 (CBP/p300, a NF-κB coactivator) gene expression, acetylation levels, and CBP/p300 histone acetyltransferase (HAT) activity. In addition, histone deacetylase 2 (HDAC2) expression was significantly induced. These results suggest that gallic acid inhibits hyperglycemic-induced cytokine production in monocytes through epigenetic changes involving NF-κB. Therefore, gallic acid may have potential for the treatment and prevention of diabetes and its complications.

  1. Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase.

    PubMed

    Byeon, Yeong; Back, Kyoungwhan

    2016-08-01

    Melatonin is a well-known bioactive molecule produced in animals and plants and a well-studied natural compound. Two enzymatic steps are required for the biosynthesis of melatonin from serotonin. First, serotonin N-acetyltransferase (SNAT) catalyzes serotonin to N-acetylserotonin (NAS) followed by the action of N-acetylserotonin O-methyltransferase (ASMT), resulting in the synthesis of O-methylated NAS, also known as melatonin. Attempts to document melatonin production in Escherichia coli have been unsuccessful to date due to either low enzyme activity or inactive ASMT expression. Here, we employed caffeic acid O-methyltransferase (COMT) instead of ASMT, as COMT is a multifunctional enzyme that has ASMT activity as well. Among several combinations of dual expression cassettes, recombinant E. coli that expressed sheep SNAT with rice COMT produced a high quantity of melatonin, which was measured in a culture medium (1.46 mg/L in response to 1 mM serotonin). This level was several orders of magnitude higher than that produced in transgenic rice and tomato overexpressing sheep SNAT and ASMT, respectively. This heterologous expression system can be widely employed to screen various putative SNAT or ASMT genes from animals and plants as well as to overproduce melatonin in various useful microorganisms. PMID:27005412

  2. Rare allele of a previously unidentified histone H4 acetyltransferase enhances grain weight, yield, and plant biomass in rice

    PubMed Central

    Song, Xian Jun; Kuroha, Takeshi; Ayano, Madoka; Furuta, Tomoyuki; Nagai, Keisuke; Komeda, Norio; Segami, Shuhei; Miura, Kotaro; Ogawa, Daisuke; Kamura, Takumi; Suzuki, Takamasa; Higashiyama, Tetsuya; Yamasaki, Masanori; Mori, Hitoshi; Inukai, Yoshiaki; Wu, Jianzhong; Kitano, Hidemi; Sakakibara, Hitoshi; Jacobsen, Steven E.; Ashikari, Motoyuki

    2015-01-01

    Grain weight is an important crop yield component; however, its underlying regulatory mechanisms are largely unknown. Here, we identify a grain-weight quantitative trait locus (QTL) encoding a new-type GNAT-like protein that harbors intrinsic histone acetyltransferase activity (OsglHAT1). Our genetic and molecular evidences pinpointed the QTL-OsglHAT1’s allelic variations to a 1.2-kb region upstream of the gene body, which is consistent with its function as a positive regulator of the traits. Elevated OsglHAT1 expression enhances grain weight and yield by enlarging spikelet hulls via increasing cell number and accelerating grain filling, and increases global acetylation levels of histone H4. OsglHAT1 localizes to the nucleus, where it likely functions through the regulation of transcription. Despite its positive agronomical effects on grain weight, yield, and plant biomass, the rare allele elevating OsglHAT1 expression has so far escaped human selection. Our findings reveal the first example, to our knowledge, of a QTL for a yield component trait being due to a chromatin modifier that has the potential to improve crop high-yield breeding. PMID:25535376

  3. Stem Cell Gene Therapy for Fanconi Anemia: Report from the 1st International Fanconi Anemia Gene Therapy Working Group Meeting

    PubMed Central

    Tolar, Jakub; Adair, Jennifer E; Antoniou, Michael; Bartholomae, Cynthia C; Becker, Pamela S; Blazar, Bruce R; Bueren, Juan; Carroll, Thomas; Cavazzana-Calvo, Marina; Clapp, D Wade; Dalgleish, Robert; Galy, Anne; Gaspar, H Bobby; Hanenberg, Helmut; Von Kalle, Christof; Kiem, Hans-Peter; Lindeman, Dirk; Naldini, Luigi; Navarro, Susana; Renella, Raffaele; Rio, Paula; Sevilla, Julián; Schmidt, Manfred; Verhoeyen, Els; Wagner, John E; Williams, David A; Thrasher, Adrian J

    2011-01-01

    Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA. PMID:21540837

  4. Mechanism of the lysosomal membrane enzyme acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase

    SciTech Connect

    Bame, K.J.

    1986-01-01

    Acetyl-CoA:..cap alpha..-glucosaminide N-acetyltransferase is a lysosomal membrane enzyme, deficient in the genetic disease Sanfilippo C syndrome. The enzyme catalyzes the transfer of an acetyl group from cytoplasmic acetyl-CoA to terminal ..cap alpha..-glucosamine residues of heparan sulfate within the organelle. The reaction mechanism was examined using high purified lysosomal membranes from rat liver and human fibroblasts. The N-acetyltransferase reaction is optimal above pH 5.5 and a 2-3 fold stimulation of activity is observed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicate that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. The binding of acetyl-CoA to the enzyme is measured by exchange label from (/sup 3/H)CoA to acetyl-CoA, and is optimal at pH's above 7.0. The acetyl-enzyme intermediate is formed by incubating membranes with (/sup 3/H)acetyl-CoA. The acetyl group can be transferred to glucosamine, forming (/sup 3/H)N-acetylglucosamine; the transfer is optimal between pH 4 and 5. Lysosomal membranes from Sanfilippo C fibroblasts confirm that these half reactions carried out by the N-acetyltransferase. The enzyme is inactivated by N-bromosuccinimide and diethylpyrocarbonate, indicating that a histidine is involved in the reaction. These results suggest that the histidine residue is at the active site of the enzyme. The properties of the N-acetyltransferase in the membrane, the characterization of the enzyme kinetics, the chemistry of a histidine mediated acetylation and the pH difference across the lysosomal membrane all support a transmembrane acetylation mechanism.

  5. Metabolism of triethylenetetramine and 1,12-diamino-3,6,9-triazadodecane by the spermidine/spermine-N(1)-acetyltransferase and thialysine acetyltransferase.

    PubMed

    Hyvönen, Mervi T; Weisell, Janne; Khomutov, Alex R; Alhonen, Leena; Vepsäläinen, Jouko; Keinänen, Tuomo A

    2013-01-01

    Triethylenetetramine (TETA; Syprine; Merck Rahway, NJ), a drug for Wilson's disease, is a copper chelator and a charge-deficient analog of polyamine spermidine. We recently showed that TETA is metabolized in vitro by polyamine catabolic enzyme spermidine/spermine-N(1)-acetyltransferase (SSAT1) and by thialysine acetyltransferase (SSAT2) to its monoacetylated derivative (MAT). The acetylation of TETA is increased in SSAT1-overexpressing mice compared with wild-type mice. However, SSAT1-deficient mice metabolize TETA at the same rate as the wild-type mice, indicating the existence of another N-acetylase respons 2ible for its metabolism in mice. Here, we show that siRNA-mediated knockdown of SSAT2 in HEPG2 cells and in primary hepatocytes from the SSAT1-deficient or wild-type mice reduced the metabolism of TETA to MAT. By contrast, 1,12-diamino-3,6,9-triazadodecane(SpmTrien), a charge-deficient spermine analog, was an extremely poor substrate of human recombinant SSAT2 and was metabolized by SSAT1 in HEPG2 cells and in wild-type primary hepatocytes. Thus, despite the similar structures of TETA and SpmTrien, SSAT2 is the main acetylator of TETA, whereas SpmTrien is primarily acetylated by SSAT1.

  6. Structure-based molecular design for thermostabilization of N-acetyltransferase Mpr1 involved in a novel pathway of L-arginine synthesis in yeast.

    PubMed

    Nasuno, Ryo; Hirase, Saeka; Norifune, Saki; Watanabe, Daisuke; Takagi, Hiroshi

    2016-02-01

    Previously, N-Acetyltransferase Mpr1 was suggested to be involved in a novel pathway of L-arginine biosynthesis in yeast. Our recent crystallographic analysis demonstrated that the overall structure of Mpr1 is a typical folding among proteins in the Gcn5-related N-acetyltransferase superfamily, and also provided clues to the design of mutations for improvement of the enzymatic functions. Here, we constructed new stable variants, Asn203Lys- and Asn203Arg-Mpr1, which exhibited 2.4-fold and 2.2-fold longer activity half-lives than wild-type Mpr1, respectively, by structure-based molecular design. The replacement of Asn203 with a basic amino acid was suggested to stabilize α-helix 2, which is important for the Mpr1 structure, probably by neutralizing its dipole. In addition, the combination of two amino acid substitutions at positions 65 and 203 in Mpr1, Phe65Leu, which was previously isolated by the screening from PCR random mutagenesis library of MPR1, and Asn203Lys or Asn203Arg, led to further stabilization of Mpr1. Our growth assay suggests that overexpression of the stable Mpr1 variants increase L-arginine synthesis in yeast cells. Our finding is the first report on the rational engineering of Mpr1 for thermostabilization and could be useful in the construction of new yeast strains with higher L-arginine synthetic activity and also improved fermentation ability.

  7. Conformational flexibility and subunit arrangement of the modular yeast Spt-Ada-Gcn5 acetyltransferase complex.

    PubMed

    Setiaputra, Dheva; Ross, James D; Lu, Shan; Cheng, Derrick T; Dong, Meng-Qiu; Yip, Calvin K

    2015-04-17

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility.

  8. Signal transduction pathways that regulate CAB gene expression. Progress report

    SciTech Connect

    Chory, J.

    1993-12-31

    We have completed the initial genetic and phenotypic characterization of several classes of new mutants that affect CAB gene expression. The doc mutants (for dark overexpression of cab) are characterized by elevated levels of CAB gene expression in the dark; however, unlike the previously isolated de-etiolated mutants (also isolated in my lab), the doc mutants still appear etiolated. The doc alleles define 3 loci, each of which maps to a separate chromosome. The details of the mutant isolation scheme and the genetic and phenotypic description of these new mutants are described. The second class of mutants, the gun mutants (for genomes uncoupled) show accumulation of CAB mRNA in the absence of chloroplast gene expression and development. Thus, the normally tightly coordinated expression between the chloroplast and nuclear genes that encode chloroplast-destined proteins has been uncoupled. We have shown that the Arabidopsis HY3 locus encodes the type B phytochrome apoprotein gene and have characterized the phenotypes of null hy3 alleles to ascertain a role for this phytochrome in Arabidopsis development. We have also isolated and characterized a number of alleles of the phytochrome A gene.

  9. Structural characterization of Streptococcus pneumoniae serotype 9A capsule polysaccharide reveals role of glycosyl 6-O-acetyltransferase wcjE in serotype 9V capsule biosynthesis and immunogenicity.

    PubMed

    Calix, Juan J; Saad, Jamil S; Brady, Allison M; Nahm, Moon H

    2012-04-20

    The putative capsule O-acetyltransferase gene wcjE is highly conserved across various Streptococcus pneumoniae serotypes, but the role of the gene in capsule biosynthesis and bacterial fitness remains largely unclear. Isolates expressing pneumococcal serotype 9A arise from precursors expressing wcjE-associated serotype 9V through loss-of-function mutation to wcjE. To define the biosynthetic role of 9V wcjE, we characterized the structure and serological properties of serotype 9V and 9A capsule polysaccharide (PS). NMR data revealed that both 9V and 9A PS are composed of an identical pentasaccharide repeat unit, as reported previously. However, in sharp contrast to previous studies on 9A PS being devoid of any O-acetylation, we identified O-acetylation of α-glucuronic acid and α-glucose in 9A PS. In addition, 9V PS also contained -CH(2) O-acetylation of β-N-acetylmannosamine, a modification that disappeared following in vitro recombinatorial deletion of wcjE. We also show that serotyping sera and monoclonal antibodies specific for 9V and 9A bound capsule PS in an O-acetate-dependent manner. Furthermore, IgG and to a lesser extent IgM from human donors immunized with serotype 9V PS displayed stronger binding to 9V compared with 9A PS. We conclude that serotype 9V wcjE mediates 6-O-acetylation of β-N-acetylmannosamine. This PS modification can be selectively targeted by antibodies in immunized individuals, identifying a potential selective advantage for wcjE inactivation and serotype 9A emergence.

  10. Interactions between the Class II Transactivator and CREB Binding Protein Increase Transcription of Major Histocompatibility Complex Class II Genes

    PubMed Central

    Fontes, Joseph D.; Kanazawa, Satoshi; Jean, Dickson; Peterlin, B. Matija

    1999-01-01

    Class II major histocompatibility (class II) genes are regulated in a B-cell-specific and gamma interferon-inducible fashion. The master switch for the expression of these genes is the class II transactivator (CIITA). In this report, we demonstrate that one of the functions of CIITA is to recruit the CREB binding protein (CBP) to class II promoters. Not only functional but also specific binding interactions between CIITA and CBP were demonstrated. Moreover, a dominant negative form of CBP decreased the activity of class II promoters and levels of class II determinants on the surface of cells. Finally, the inhibition of class II gene expression by the glucocorticoid hormone could be attributed to the squelching of CBP by the glucocorticoid receptor. We conclude that CBP, a histone acetyltransferase, plays an important role in the transcription of class II genes. PMID:9858618

  11. 82-kDa choline acetyltransferase and SATB1 localize to β-amyloid induced matrix attachment regions.

    PubMed

    Winick-Ng, Warren; Caetano, Fabiana A; Winick-Ng, Jennifer; Morey, Trevor M; Heit, Bryan; Rylett, R Jane

    2016-01-01

    The M-transcript of human choline acetyltransferase (ChAT) produces an 82-kDa protein (82-kDa ChAT) that concentrates in nuclei of cholinergic neurons. We assessed the effects of acute exposure to oligomeric amyloid-β1-42 (Aβ1-42) on 82-kDa ChAT disposition in SH-SY5Y neural cells, finding that acute exposure to Aβ1-42 results in increased association of 82-kDa ChAT with chromatin and formation of 82-kDa ChAT aggregates in nuclei. When measured by chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we identified that Aβ1-42-exposure increases 82-kDa ChAT association with gene promoters and introns. The Aβ1-42-induced 82-kDa ChAT aggregates co-localize with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 had a similar genomic association as 82-kDa ChAT, with both proteins associating with synapse and cell stress genes. After Aβ1-42 -exposure, both SATB1 and 82-kDa ChAT are enriched at the same S/MAR on the APP gene, with 82-kDa ChAT expression attenuating an increase in an isoform-specific APP mRNA transcript. Finally, 82-kDa ChAT and SATB1 have patterned genomic association at regions enriched with S/MAR binding motifs. These results demonstrate that 82-kDa ChAT and SATB1 play critical roles in the response of neural cells to acute Aβ-exposure. PMID:27052102

  12. 82-kDa choline acetyltransferase and SATB1 localize to β-amyloid induced matrix attachment regions

    PubMed Central

    Winick-Ng, Warren; Caetano, Fabiana A.; Winick-Ng, Jennifer; Morey, Trevor M.; Heit, Bryan; Rylett, R. Jane

    2016-01-01

    The M-transcript of human choline acetyltransferase (ChAT) produces an 82-kDa protein (82-kDa ChAT) that concentrates in nuclei of cholinergic neurons. We assessed the effects of acute exposure to oligomeric amyloid-β1–42 (Aβ1–42) on 82-kDa ChAT disposition in SH-SY5Y neural cells, finding that acute exposure to Aβ1–42 results in increased association of 82-kDa ChAT with chromatin and formation of 82-kDa ChAT aggregates in nuclei. When measured by chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we identified that Aβ1–42 -exposure increases 82-kDa ChAT association with gene promoters and introns. The Aβ1–42 -induced 82-kDa ChAT aggregates co-localize with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 had a similar genomic association as 82-kDa ChAT, with both proteins associating with synapse and cell stress genes. After Aβ1–42 -exposure, both SATB1 and 82-kDa ChAT are enriched at the same S/MAR on the APP gene, with 82-kDa ChAT expression attenuating an increase in an isoform-specific APP mRNA transcript. Finally, 82-kDa ChAT and SATB1 have patterned genomic association at regions enriched with S/MAR binding motifs. These results demonstrate that 82-kDa ChAT and SATB1 play critical roles in the response of neural cells to acute Aβ -exposure. PMID:27052102

  13. Reporter gene technologies for imaging cell fates in hematopoiesis.

    PubMed

    Kusy, Sophie; Contag, Christopher H

    2014-01-01

    Advances in noninvasive imaging technologies that allow for in vivo dynamic monitoring of cells and cellular function in living research subjects have revealed new insights into cell biology in the context of intact organs and their native environment. In the field of hematopoiesis and stem cell research, studies of cell trafficking involved in injury repair and hematopoietic engraftment have made great progress using these new tools. Stem cells present unique challenges for imaging since after transplantation, they proliferate dramatically and differentiate. Therefore, the imaging modality used needs to have a large dynamic range, and the genetic regulatory elements used need to be stably expressed during differentiation. Multiple imaging technologies using different modalities are available, and each varies in sensitivity, ease of data acquisition, signal to noise ratios (SNR), substrate availability, and other parameters that affect utility for monitoring cell fates and function. For a given application, there may be several different approaches that can be used. For mouse models, clinically validated technologies such as magnetic resonance imaging (MRI) and positron emission tomography (PET) have been joined by optical imaging techniques such as in vivo bioluminescence imaging (BLI) and fluorescence imaging (FLI), and all have been used to monitor bone marrow and stem cells after transplantation into mice. Photoacoustic imaging that utilizes the sound created by the thermal expansion of absorbed light to generate an image best represents hybrid technologies. Each modality requires that the cells of interest be marked with a genetic reporter that acts as a label making them uniquely visible using that technology. For each modality, there are several labels to choose from. Multiple methods for applying these different labels are available. This chapter provides an overview of the imaging technologies and commonly used labels for each, as well as detailed

  14. Terminator Operon Reporter: combining a transcription termination switch with reporter technology for improved gene synthesis and synthetic biology applications

    PubMed Central

    Zampini, Massimiliano; Mur, Luis A. J.; Rees Stevens, Pauline; Pachebat, Justin A.; Newbold, C. James; Hayes, Finbarr; Kingston-Smith, Alison

    2016-01-01

    Synthetic biology is characterized by the development of novel and powerful DNA fabrication methods and by the application of engineering principles to biology. The current study describes Terminator Operon Reporter (TOR), a new gene assembly technology based on the conditional activation of a reporter gene in response to sequence errors occurring at the assembly stage of the synthetic element. These errors are monitored by a transcription terminator that is placed between the synthetic gene and reporter gene. Switching of this terminator between active and inactive states dictates the transcription status of the downstream reporter gene to provide a rapid and facile readout of the accuracy of synthetic assembly. Designed specifically and uniquely for the synthesis of protein coding genes in bacteria, TOR allows the rapid and cost-effective fabrication of synthetic constructs by employing oligonucleotides at the most basic purification level (desalted) and without the need for costly and time-consuming post-synthesis correction methods. Thus, TOR streamlines gene assembly approaches, which are central to the future development of synthetic biology. PMID:27220405

  15. Terminator Operon Reporter: combining a transcription termination switch with reporter technology for improved gene synthesis and synthetic biology applications.

    PubMed

    Zampini, Massimiliano; Mur, Luis A J; Rees Stevens, Pauline; Pachebat, Justin A; Newbold, C James; Hayes, Finbarr; Kingston-Smith, Alison

    2016-01-01

    Synthetic biology is characterized by the development of novel and powerful DNA fabrication methods and by the application of engineering principles to biology. The current study describes Terminator Operon Reporter (TOR), a new gene assembly technology based on the conditional activation of a reporter gene in response to sequence errors occurring at the assembly stage of the synthetic element. These errors are monitored by a transcription terminator that is placed between the synthetic gene and reporter gene. Switching of this terminator between active and inactive states dictates the transcription status of the downstream reporter gene to provide a rapid and facile readout of the accuracy of synthetic assembly. Designed specifically and uniquely for the synthesis of protein coding genes in bacteria, TOR allows the rapid and cost-effective fabrication of synthetic constructs by employing oligonucleotides at the most basic purification level (desalted) and without the need for costly and time-consuming post-synthesis correction methods. Thus, TOR streamlines gene assembly approaches, which are central to the future development of synthetic biology. PMID:27220405

  16. Sex-dependent differences in estrogen regulation of choline acetyltransferase are altered by neonatal treatments.

    PubMed

    Luine, V N; Renner, K J; McEwen, B S

    1986-08-01

    We investigated whether estrogenic actions of testosterone during development which mediate the suppression of feminine reproductive behavior and cyclic gonadotropin secretion also contribute to reported sex differences in the induction of choline acetyltransferase (ChAT) after estrogen priming in the diagonal band region of the preoptic area. Newborn female rats received estradiol (E2 females); newborn males received 1,4,6-androstatrien-3,17-dione (ATD), an inhibitor of aromatase (ATD males); and some of both sexes received vehicle treatment (control). In adulthood, feminine sexual behavior (lordosis) was tested after E2 plus progesterone priming. The neonatal treatments reversed the sex-specific response pattern; E2 females were defeminized and displayed minimal lordosis, as did control males, while ATD males showed maximal lordosis, as did control females. E2 was then administered, and ChAT activity was measured in the horizontal and vertical nuclei of the diagonal bands (hDB and vDB, respectively). Controls exhibited the normal sex-specific response to E2. Females showed increased ChAT activity in the hDB and unaltered activity in the vDB: males had unaltered ChAT activity in the hDB and decreased activity in the vDB. In neonatally treated males and females, ChAT activity after E2 administration was not altered from the normal sex-specific pattern in the hDB, i.e. all females showed increased hDB ChAT after E2, and no male responded. In the vDB, groups defeminized in terms of lordosis (E2 females and control males) showed higher ChAT activity in the absence of E2 priming, and E2 treatment decreased vDB ChAT in these groups. In addition, ATD males showed a unique response to E2 in the vDB, namely increased ChAT activity. Although neonatal E2 and ATD treatments did not completely reverse the sex-specific pattern of E2 priming on ChAT activity, the results obtained suggest that a net increase in diagonal band cholinergic function, as indexed by increased Ch

  17. The nucleosome remodeling complex, Snf/Swi, is required for the maintenance of transcription in vivo and is partially redundant with the histone acetyltransferase, Gcn5.

    PubMed Central

    Sudarsanam, P; Cao, Y; Wu, L; Laurent, B C; Winston, F

    1999-01-01

    Snf/Swi, a nucleosome remodeling complex, is important for overcoming nucleosome-mediated repression of transcription in Saccharomyces cerevisiae. We have addressed the mechanism by which Snf/Swi controls transcription in vivo of an Snf/Swi-dependent promoter, that of the SUC2 gene. By single-cell analysis, our results show that Snf/Swi is required for activated levels of SUC2 expression in every cell of a population. In addition, Snf/Swi is required for maintenance of SUC2 transcription, suggesting that continuous chromatin remodeling is necessary to maintain an active transcriptional state. Finally, Snf/Swi and Gcn5, a histone acetyltransferase, have partially redundant roles in the control of SUC2 transcription, suggesting a functional overlap between two different mechanisms believed to overcome repression by nucleosomes, nucleosome remodeling and histone acetylation. PMID:10357821

  18. Inflammatory bowel disease gene discovery. CRADA final report

    SciTech Connect

    1997-09-09

    The ultimate goal of this project is to identify the human gene(s) responsible for the disorder known as IBD. The work was planned in two phases. The desired products resulting from Phase 1 were BAC clone(s) containing the genetic marker(s) identified by gene/Networks, Inc. as potentially linked to IBD, plasmid subclones of those BAC(s), and new genetic markers developed from these plasmid subclones. The newly developed markers would be genotyped by gene/Networks, Inc. to ascertain evidence for linkage or non-linkage of IBD to this region. If non-linkage was indicated, the project would move to investigation of other candidate chromosomal regions. Where linkage was indicated, the project would move to Phase 2, in which a physical map of the candidate region(s) would be developed. The products of this phase would be contig(s) of BAC clones in the region exhibiting linkage to IBD, as well as plasmic subclones of the BACs and further genetic marker development. There would also be continued genotyping with new polymorphic markers during this phase. It was anticipated that clones identified and developed during these two phases would provide the physical resources for eventual disease gene discovery.

  19. Bioluminescent reporters for catabolic gene expression and pollutant bioavailability

    SciTech Connect

    Heitzer, A.; DiGrazia, P.M.; Sayler, G.S. . Center for Environmental Biotechnology); Burlage, R.S. )

    1991-01-01

    The application of visualized catabolic nah-gene expression using a luxCDABE gene fusion provides a valuable method to measure quantitatively and specifically naphthalene and salicylate bioavailability. It has been demonstrated that the physiological state of the test culture together with the intrinsic regulation mechanisms of the naphthalene degradation pathway as well as the physiological aspects of the lux gene fusion have to be taken into account. The method presented provides a high potential for in situ bioprocess monitoring. In addition, the results obtained with immobilized cells provide a basis for the development of biosensors for environmental applications in specific pollutant monitoring in waste streams and soil slurry systems but, as a general method, also for more conventional biotechnological process control. 8 refs., 2 figs., 1 tab.

  20. The plant mitochondrial mat-r gene/nad1 gene complex. Progress report

    SciTech Connect

    Wolstenholme, D.R.

    1994-06-01

    The authors have completed sequencing the segments (totalling 19 kb, both complementary strands) of the maize mtDNA molecule that encode the entire NADH dehydrogenase subunit (nadl) gene. They have identified nucleotides in mature transcripts of the nadl gene that are edited and have generated clones of cDNAs of entire mature (fully spliced) nadl transcripts. They have examined the relative rates of splicing in transcripts of the four nadl gene group II introns and begun examining nadl intron cDNAs to determine the extent and distribution of RNA edits in introns, in order to evaluate the possibility that intron excision and exon splicing might be editing independent.

  1. Far-red fluorescence gene reporter tomography for determination of placement and viability of cell-based gene therapies.

    PubMed

    Lu, Yujie; Darne, Chinmay D; Tan, I-Chih; Zhu, Banghe; Hall, Mary A; Lazard, Zawaunyka W; Davis, Alan R; Simpson, Lashan; Sevick-Muraca, Eva M; Olmsted-Davis, Elizabeth A

    2013-10-01

    Non-invasive injectable cellular therapeutic strategies based on sustained delivery of physiological levels of BMP-2 for spinal fusion are emerging as promising alternatives, which could provide sufficient fusion without the associated surgical risks. However, these injectable therapies are dependent on bone formation occurring only at the specific target region. In this study, we developed and deployed fluorescence gene reporter tomography (FGRT) to provide information on in vivo cell localization and viability. This information is sought to confirm the ideal placement of the materials with respect to the area where early bone reaction is required, ultimately providing three dimensional data about the future fusion. However, because almost all conventional fluorescence gene reporters require visible excitation wavelengths, current in vivo imaging of fluorescent proteins is limited by high tissue absorption and confounding autofluorescence. We previously administered fibroblasts engineered to produce BMP-2, but is difficult to determine 3-D information of placement prior to bone formation. Herein we used the far-red fluorescence gene reporter, IFP1.4 to report the position and viability of fibroblasts and developed 3-D tomography to provide placement information. A custom small animal, far-red fluorescence tomography system integrated into a commercial CT scanner was used to assess IFP1.4 fluorescence and to demark 3-D placement of encapsulated fibroblasts with respect to the vertebrae and early bone formation as assessed from CT. The results from three experiments showed that the placement of the materials within the spine could be detected. This work shows that in vivo fluorescence gene reporter tomography of cell-based gene therapy is feasible and could help guide cell-based therapies in preclinical models.

  2. Antioxidant N-acetyltransferase Mpr1/2 of industrial baker's yeast enhances fermentation ability after air-drying stress in bread dough.

    PubMed

    Sasano, Yu; Takahashi, Shunsuke; Shima, Jun; Takagi, Hiroshi

    2010-03-31

    During bread-making processes, yeast cells are exposed to multiple stresses. Air-drying stress is one of the most harmful stresses by generation of reactive oxygen species (ROS). Previously, we discovered that the novel N-acetyltransferase Mpr1/2 confers oxidative stress tolerance by reducing intracellular ROS level in Saccharomyces cerevisiae Sigma1278b strain. In this study, we revealed that Japanese industrial baker's yeast possesses one MPR gene. The nucleotide sequence of the MPR gene in industrial baker's yeast was identical to the MPR2 gene in Sigma1278b strain. Gene disruption analysis showed that the MPR2 gene in industrial baker's yeast is involved in air-drying stress tolerance by reducing the intracellular oxidation levels. We also found that expression of the Lys63Arg and Phe65Leu variants with enhanced enzymatic activity and stability, respectively, increased the fermentation ability of bread dough after exposure to air-drying stress compared with the wild-type Mpr1. In addition, our recent study showed that industrial baker's yeast cells accumulating proline exhibited enhanced freeze tolerance in bread dough. Proline accumulation also enhanced the fermentation ability after air-drying stress treatment in industrial baker's yeast. Hence, the antioxidant enzyme Mpr1/2 could be promising for breeding novel yeast strains that are tolerant to air-drying stress. PMID:20096471

  3. Arabidopsis serotonin N-acetyltransferase knockout mutant plants exhibit decreased melatonin and salicylic acid levels resulting in susceptibility to an avirulent pathogen.

    PubMed

    Lee, Hyoung Yool; Byeon, Yeong; Tan, Dun-Xian; Reiter, Russel J; Back, Kyoungwhan

    2015-04-01

    Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in the melatonin biosynthesis pathway in plants. We examined the effects of SNAT gene inactivation in two Arabidopsis T-DNA insertion mutant lines. After inoculation with the avirulent pathogen Pseudomonas syringe pv. tomato DC3000 harboring the elicitor avrRpt2 (Pst-avrRpt2), melatonin levels in the snat knockout mutant lines were 50% less than in wild-type Arabidopsis Col-0 plants. The snat knockout mutant lines exhibited susceptibility to pathogen infection that coincided with decreased induction of defense genes including PR1, ICS1, and PDF1.2. Because melatonin acts upstream of salicylic acid (SA) synthesis, the reduced melatonin levels in the snat mutant lines led to decreased SA levels compared to wild-type, suggesting that the increased pathogen susceptibility of the snat mutant lines could be attributed to decreased SA levels and subsequent attenuation of defense gene induction. Exogenous melatonin treatment failed to induce defense gene expression in nahG Arabidopsis plants, but restored the induction of defense gene expression in the snat mutant lines. In addition, melatonin caused translocation of NPR1 (nonexpressor of PR1) protein from the cytoplasm into the nucleus indicating that melatonin-elicited pathogen resistance in response to avirulent pathogen attack is SA-dependent in Arabidopsis.

  4. Epigenetic regulation of proliferation and invasion in hepatocellular carcinoma cells by CBP/p300 histone acetyltransferase activity.

    PubMed

    Inagaki, Yuji; Shiraki, Katsuya; Sugimoto, Kazushi; Yada, Takazumi; Tameda, Masahiko; Ogura, Suguru; Yamamoto, Norihiko; Takei, Yoshiyuki; Ito, Masaaki

    2016-02-01

    Altered epigenetic control of gene expression plays a substantial role in tumor development and progression. Accumulating studies suggest that somatic mutations of CREB binding proteins (CBP)/p300 occur in some cancer cells. CBP/p300 possess histone acetyltransferase (HAT) activity, and are involved in many cellular processes. In this study, we investigated the expression and functional role of CBP/p300 in hepatocellular carcinoma (HCC) using the specific inhibitor C646 of CBP/p300 HAT activity. We examined its effect on several apoptosis-related proteins and invasion-related genes. The results showed that CBP/p300 were highly expressed in HCC tissues and that expression of p300, but not of CBP, was strongly correlated with the malignant character of HCC. C646 inhibited proliferation of HCC cell lines in a dose dependent manner. C646 significantly augmented TRAIL-induced apoptotic sensitivity, which was accompanied by reduced levels of survivin, in HepG2, HLE and SK-HEP1 cells. C646 significantly inhibited invasion of Huh7, HLE and SK-HEP1 cells. The level of matrix metallopeptidase 15 (MMP15) mRNA expression was significantly reduced, whereas the level of laminin alpha 3 (LAMA3) and secreted phosphoprotein 1 (SPP1) mRNA expression was significantly increased in Huh7 cells following exposure to C646. In conclusion, our results suggest that CBP/p300 HAT activity has an important role in malignant transformation, proliferation, apoptotic sensitivity and invasion in HCC. CBP/p300 could be a promising therapeutic target in HCC. PMID:26676548

  5. Arylamine N-acetyltransferase (NAT2) mutations and their allelic linkage in unrelated caucasian individuals: Correlation with phenotypic activity

    SciTech Connect

    Cascorbi, I.; Drakoulis, N.; Brockmoeller, J.

    1995-09-01

    The polymorphic arylamine N-acetyltransferase (NAT2; EC2.3.1.5) is supposed to be a susceptibility factor for several drug side effects and certain malignancies. A group of 844 unrelated German subjects was genotyped for their acetylation type, and 563 of them were also phenotyped. Seven mutations of the NAT2 gene were evaluated by allele-specific PCR (mutation 341C to T) and PCR-RFLP for mutations at nt positions 191, 282, 481, 590, 803, and 857. From the mutation pattern eight different alleles, including the wild type coding for rapid acetylation and seven alleles coding for slow phenotype, were determined. Four hundred ninety-seven subjects had a genotype of slow acetylation (58.9%; 95% confidence limits 55.5%-62.2%). Phenotypic acetylation capacity was expressed as the ratio of 5-acetylamino-6-formylamino-3-methyluracil and 1-methylxanthine in urine after caffeine intake. Some 6.7% of the cases deviated in genotype and phenotype, but sequencing DNA of these probands revealed no new mutations. Furthermore, linkage pattern of the mutations was always confirmed, as tested in 533 subjects. In vivo acetylation capacity of homozygous wild-type subjects (NAT2{sup *}4/{sup *}4) was significantly higher than in heterozygous genotypes (P = .001). All mutant alleles showed low in vivo acetylation capacities, including the previously not-yet-defined alleles {sup *}5A, {sup *}5C, and {sup *}13. Moreover, distinct slow genotypes differed significantly among each other, as reflected in lower acetylation capacity of {sup *}6A, {sup *}7B, and {sup *}13 alleles than the group of {sup *}5 alleles. The study demonstrated differential phenotypic activity of various NAT2 genes and gives a solid basis for clinical and molecular-epidemiological investigations. 34 refs., 4 figs., 7 tabs.

  6. NAT8L (N-Acetyltransferase 8-Like) Accelerates Lipid Turnover and Increases Energy Expenditure in Brown Adipocytes*

    PubMed Central

    Pessentheiner, Ariane R.; Pelzmann, Helmut J.; Walenta, Evelyn; Schweiger, Martina; Groschner, Lukas N.; Graier, Wolfgang F.; Kolb, Dagmar; Uno, Kyosuke; Miyazaki, Toh; Nitta, Atsumi; Rieder, Dietmar; Prokesch, Andreas; Bogner-Strauss, Juliane G.

    2013-01-01

    NAT8L (N-acetyltransferase 8-like) catalyzes the formation of N-acetylaspartate (NAA) from acetyl-CoA and aspartate. In the brain, NAA delivers the acetate moiety for synthesis of acetyl-CoA that is further used for fatty acid generation. However, its function in other tissues remained elusive. Here, we show for the first time that Nat8l is highly expressed in adipose tissues and murine and human adipogenic cell lines and is localized in the mitochondria of brown adipocytes. Stable overexpression of Nat8l in immortalized brown adipogenic cells strongly increases glucose incorporation into neutral lipids, accompanied by increased lipolysis, indicating an accelerated lipid turnover. Additionally, mitochondrial mass and number as well as oxygen consumption are elevated upon Nat8l overexpression. Concordantly, expression levels of brown marker genes, such as Prdm16, Cidea, Pgc1α, Pparα, and particularly UCP1, are markedly elevated in these cells. Treatment with a PPARα antagonist indicates that the increase in UCP1 expression and oxygen consumption is PPARα-dependent. Nat8l knockdown in brown adipocytes has no impact on cellular triglyceride content, lipogenesis, or oxygen consumption, but lipolysis and brown marker gene expression are increased; the latter is also observed in BAT of Nat8l-KO mice. Interestingly, the expression of ATP-citrate lyase is increased in Nat8l-silenced adipocytes and BAT of Nat8l-KO mice, indicating a compensatory mechanism to sustain the acetyl-CoA pool once Nat8l levels are reduced. Taken together, our data show that Nat8l impacts on the brown adipogenic phenotype and suggests the existence of the NAT8L-driven NAA metabolism as a novel pathway to provide cytosolic acetyl-CoA for lipid synthesis in adipocytes. PMID:24155240

  7. Overexpression of DYRK1A inhibits choline acetyltransferase induction by oleic acid in cellular models of Down syndrome.

    PubMed

    Hijazi, Maruan; Fillat, Cristina; Medina, José M; Velasco, Ana

    2013-01-01

    Histological brain studies of individuals with DS have revealed an aberrant formation of the cerebral cortex. Previous work from our laboratory has shown that oleic acid acts as a neurotrophic factor and induces neuronal differentiation. In order to characterize the effects of oleic acid in a cellular model of DS, immortalized cell lines derived from the cortex of trisomy Ts16 (CTb) and normal mice (CNh) were incubated in the absence or presence of oleic acid. Oleic acid increased choline acetyltransferase expression (ChAT), a marker of cholinergic differentiation in CNh cells. However, in trisomic cells (CTb line) oleic acid failed to increase ChAT expression. These results suggest that the overdose of specific genes in trisomic lines delays differentiation in the presence of oleic acid by inhibiting acetylcholine production mediated by ChAT. The dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) gene is located on human chromosome 21 and encodes a proline-directed protein kinase. It has been proposed that DYRK1A plays a prominent role in several biological functions, leading to mental retardation in DS patients. Here we explored the potential role of DYRK1A in the modulation of ChAT expression in trisomic cells and in the signaling pathways of oleic acid. Down-regulation of DYRK1A by siRNA in trisomic CTb cells rescued ChAT expression up to levels similar to those of normal cells in the presence of oleic acid. In agreement with these results, oleic acid was unable to increase ChAT expression in neuronal cultures of transgenic mice overexpressing DYRK1A. In summary, our results highlight the role played by DYRK1A in brain development through the control of ChAT expression. In addition, the overexpression of DYRK1A in DS models prevented the neurotrophic effect of oleic acid, a fact that may account for mental retardation in DS patients. PMID:23124096

  8. Characterization of Arabidopsis Genes Involved in Gene Silencing. Final Progress Report

    SciTech Connect

    Grant, S. R.

    1999-02-05

    Enhancer of gene silencing 1 (egs1) is an Arabidopsis mutant that enhances post-transcriptional gene silencing of the rolB gene introduced by genetic engineering (transgene). The goal of our proposal was cloning EGS1 based on its map position. Although we screened more than 2000 chromosomes for recombination, we were unable to get closer than 2 cM to the gene. We experienced an unexpected tendency of the post-transcriptionally silenced transgene to switch to a more stable silenced state. This made it impossible to select egs1 homozygotes for map based cloning. This forced us to reconsider our cloning strategy. One possibility would have been to use a different transgene as the target of gene silencing. We tested two other transgenes. Both encoded proteins unrelated to the first but they were all expressed from the same type of promoter and they all had a similar tendency to become post-transcriptionally silenced. After screening over 80 F2 segregants from each cross between our egs1 mutant and Arabidopsis of the same ecotype homozygous for the new transgene, we were disappointed to find that the egs1 mutation did not enhance post-transcription silencing of the two new genes. In 80 plants we expected to have between 4 and 6 plants that were homozygous for the transgene and for the mutant egs1 allele. If egs1 mutations could enhance gene silencing of the new transgene, these plants would not express it. However all the double homozygotes still expressed the transgene. Therefore, we could not change the target transgene for mapping. This was the state of the cloning at the time for renewal of the grant in 1999. Because the selection of new meaningful recombinant plants had become extremely inefficient using the original rolB transgene, we abandoned the attempt at map based cloning and did not apply for further funding.

  9. Analysis of Gene Targeting & Nonhomologous End-joining. Final Report

    SciTech Connect

    Haber, J. E.

    2002-11-30

    Overall, we identified a number of new proteins that participate in nonhomologous end-joining and also in telomere addition to the ends of broken chromosomes. We showed that NHEJ is severely reduced in cells expressing both yeast mating-type genes and then went on to identify the NEJ1 gene that was under this control. We showed the epistasis relations among a set of mutations that impair telomere addition and we showed that there are in fact two pathways to repair broken chromosomes in the absence of telomerase. We characterized the DNA damage checkpoint pathway in response to a single broken chromosome and characterized especially the adaptation of cells arrested by an unrepaired DSB. We demonstrated that the DNA damage response is nuclear-limited. We showed adaptation defects for Tid1and Srs2 proteins and showed that Srs2 was also recovery-defective, even when DNA was repaired.

  10. N-acetyltransferase-2 and medical history in bladder cancer cases with a suspected occupational disease (BK 1301) in Germany.

    PubMed

    Weistenhofer, Wobbeke; Blaszkewicz, Meinolf; Bolt, Hermann M; Golka, Klaus

    2008-01-01

    In 187 bladder cancer cases reported to the employers' liability insurance association in Germany as suspected cases of an occupational disease produced by aromatic amines, N- acetyltransferase-2 (NAT2) activity status, occupational exposure data, period of latency, and clinical parameters were determined. In 83 out of 187 cases surveyed within the period 1991-1999, the NAT2 acetylator status was investigated by determining the molar ratio of an acetylated and a nonacetylated caffeine metabolite in urine (phenotyping) and/or by NAT2 genotyping according to standard polymerase chain reaction (PCR) protocol. The proportion of slow NAT2 acetylators in the surveyed 83 bladder cancer cases was 67%. In the entire group of surveyed 187 cases, mean duration of exposure was 17.6 yr and mean period of latency was 34.7 yr. Occupational exposures to potential bladder carcinogens were observed in 73 occupations, including chemical industry (25%), and occupations as a painter and/or varnisher (23%) were most often encountered. In 12% of the surveyed bladder cancer cases, a second primary malignancy was observed. The NAT2 distribution observed in the 83 cases is comparable to the proportion in 40 occupationally exposed bladder cancer cases in a Department of Urology located close to a former German production site of benzidine-based azo dyes, but higher than in most studies involving NAT2 genetic status in bladder cancer cases.

  11. New N-Acetyltransferase Fold in the Structure and Mechanism of the Phosphonate Biosynthetic Enzyme FrbF

    SciTech Connect

    Bae, Brian; Cobb, Ryan E.; DeSieno, Matthew A.; Zhao, Huimin; Nair, Satish K.

    2015-10-15

    The enzyme FrbF from Streptomyces rubellomurinus has attracted significant attention due to its role in the biosynthesis of the antimalarial phosphonate FR-900098. The enzyme catalyzes acetyl transfer onto the hydroxamate of the FR-900098 precursors cytidine 5'-monophosphate-3-aminopropylphosphonate and cytidine 5'-monophosphate-N-hydroxy-3-aminopropylphosphonate. Despite the established function as a bona fide N-acetyltransferase, FrbF shows no sequence similarity to any member of the GCN5-like N-acetyltransferase (GNAT) superfamily. Here, we present the 2.0 {angstrom} resolution crystal structure of FrbF in complex with acetyl-CoA, which demonstrates a unique architecture that is distinct from those of canonical GNAT-like acetyltransferases. We also utilized the co-crystal structure to guide structure-function studies that identified the roles of putative active site residues in the acetyltransferase mechanism. The combined biochemical and structural analyses of FrbF provide insights into this previously uncharacterized family of N-acetyltransferases and also provide a molecular framework toward the production of novel N-acyl derivatives of FR-900098.

  12. Consensus: a framework for evaluation of uncertain gene variants in laboratory test reporting

    PubMed Central

    2012-01-01

    Accurate interpretation of gene testing is a key component in customizing patient therapy. Where confirming evidence for a gene variant is lacking, computational prediction may be employed. A standardized framework, however, does not yet exist for quantitative evaluation of disease association for uncertain or novel gene variants in an objective manner. Here, complementary predictors for missense gene variants were incorporated into a weighted Consensus framework that includes calculated reference intervals from known disease outcomes. Data visualization for clinical reporting is also discussed. PMID:22640420

  13. A specific library of randomly integrated reporter genes for the isolation of inducible functions by cell sorting

    SciTech Connect

    Lapeyre, J.N.; Marini, F.; Gratzner, H.G. AMC ImmunoDiagnostics, Houston, TX )

    1993-01-01

    A library of cells containing randomly integrated reporter genes has been constructed. The purpose of this library is to enable the isolation of genes of interest which are inducible by radiation, biological response modifiers, cytokines, or other agents. These genes are located near reporter genes which can be induced by the upstream promoter of the gene of interest. The reporter gene, Lac Z, was randomly inserted into the genome by retroviral transduction and subsequent selection of the neo[sup r] gene with gentamycin. Studies of radiation inducible genes were undertaken, whereby cells with the radiation sensitive function were isolated by sorting the cells fluorescent after staining with the beta gal substrate, fluorescein digalactoside (FDG). This gene-tagging approach is an improvement over the cDNA library subtraction protocol in that a single library of cells with random marker gene integration can be repeatedly and sequentially probed by sorting under different, selective conditions, dependent upon the genes to be characterized.

  14. Identification and analysis of aarP, a transcriptional activator of the 2'-N-acetyltransferase in Providencia stuartii.

    PubMed Central

    Macinga, D R; Parojcic, M M; Rather, P N

    1995-01-01

    The aarP gene has been identified in a search for activators of the 2-N-acetyltransferase [encoded by aac(2')-Ia] in Providencia stuartii. Introduction of aarP into P. stuartii on a multicopy plasmid resulted in a 9.9-fold increase in the accumulation of beta-galactosidase from an aac(2')-lacZ fusion. Northern (RNA) blot analysis demonstrated that this increased aac(2')-Ia expression occurred at the level of mRNA accumulation. The deduced AarP protein was 15,898 Da in size and exhibited significant homology to a number of transcriptional activators in the AraC/XyIS family, including TetD,Rob, MarA, and SoxS. The similarity of AarP to the MarA and SoxS proteins prompted an investigation to determine whether AarP is involved in activation of genes in either the multiple antibiotic resistance (Mar) phenotype or redox stress (SoxRS) system. Introduction of aarP on a multicopy plasmid into either P. stuartii or Escherichia coli conferred a Mar phenotype with higher levels of resistance to tetracycline, chloramphenicol, and ciprofloxacin. Multiple copies of aarP in E. coli also resulted in activation of the endonuclease IV gene (nfo), a gene in the SoxRS regulon of E. coli. The function of aarP in its single-copy state was addressed by using allelic replacement to construct an aarP::Cm disruption, which resulted in a fivefold reduction in the accumulation of aac(2')-Ia mRNA. Analysis of aarP regulation showed that aarP mRNA accumulation was slightly increased by exposure to tetracycline and dramatically increased in cells containing the aarB3 (aar3) mutation, which was previously shown to increase transcription of the aac(2')-Ia gene. (P.N. Rather, E. Oroz, K.J. Shaw, R. Hare, and G. Miller, J. Bacteriol. 175:6492-6498). PMID:7768849

  15. Lysine Acetyltransferase GCN5b Interacts with AP2 Factors and Is Required for Toxoplasma gondii Proliferation

    PubMed Central

    Wang, Jiachen; Dixon, Stacy E.; Ting, Li-Min; Liu, Ting-Kai; Jeffers, Victoria; Croken, Matthew M.; Calloway, Myrasol; Cannella, Dominique; Ali Hakimi, Mohamed; Kim, Kami; Sullivan, William J.

    2014-01-01

    Histone acetylation has been linked to developmental changes in gene expression and is a validated drug target of apicomplexan parasites, but little is known about the roles of individual histone modifying enzymes and how they are recruited to target genes. The protozoan parasite Toxoplasma gondii (phylum Apicomplexa) is unusual among invertebrates in possessing two GCN5-family lysine acetyltransferases (KATs). While GCN5a is required for gene expression in response to alkaline stress, this KAT is dispensable for parasite proliferation in normal culture conditions. In contrast, GCN5b cannot be disrupted, suggesting it is essential for Toxoplasma viability. To further explore the function of GCN5b, we generated clonal parasites expressing an inducible HA-tagged dominant-negative form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative GCN5b was mediated through ligand-binding to a destabilization domain (dd) fused to the protein. Induced accumulation of the ddHAGCN5b(E703G) protein led to a rapid arrest in parasite replication. Growth arrest was accompanied by a decrease in histone H3 acetylation at specific lysine residues as well as reduced expression of GCN5b target genes in GCN5b(E703G) parasites, which were identified using chromatin immunoprecipitation coupled with microarray hybridization (ChIP-chip). Proteomics studies revealed that GCN5b interacts with AP2-domain proteins, apicomplexan plant-like transcription factors, as well as a “core complex” that includes the co-activator ADA2-A, TFIID subunits, LEO1 polymerase-associated factor (Paf1) subunit, and RRM proteins. The dominant-negative phenotype of ddHAGCN5b(E703G) parasites, considered with the proteomics and ChIP-chip data, indicate that GCN5b plays a central role in transcriptional and chromatin remodeling complexes. We conclude that GCN5b has a non-redundant and indispensable role in regulating gene expression required during the

  16. New ANTXR1 Gene Mutation for GAPO Syndrome: A Case Report.

    PubMed

    Salas-Alanís, Julio C; Scott, Claire A; Fajardo-Ramírez, Oscar R; Duran, Carola; Moreno-Treviño, María G; Kelsell, David P

    2016-07-01

    GAPO syndrome is a very rare genetic disorder characterized by growth retardation, alopecia, pseudoanodontia and progressive optic atrophy (GAPO). To date, only 30 cases have been described worldwide. Recently, gene alterations in the ANTXR1 gene have been reported to be causative of this disorder, and an autosomal recessive pattern has been observed. This gene encodes a matrix-interacting protein that works as an adhesion molecule. In this report, we describe 2 homozygous siblings diagnosed with GAPO syndrome carrying a new missense mutation. This mutation produces the substitution of a glutamine in position 137 for a leucine (c.410A>T, p.Q137L). PMID:27587992

  17. A Novel Binary T-Vector with the GFP Reporter Gene for Promoter Characterization

    PubMed Central

    Jiang, Shu-Ye; Vanitha, Jeevanandam; Bai, Yanan; Ramachandran, Srinivasan

    2014-01-01

    Several strategies have been developed to clone PCR fragments into desired vectors. However, most of commercially available T-vectors are not binary vectors and cannot be directly used for Agrobacterium-mediated plant genetic transformation. In this study, a novel binary T-vector was constructed by integrating two AhdI restriction sites into the backbone vector pCAMBIA 1300. The T-vector also contains a GFP reporter gene and thus, can be used to analyze promoter activity by monitoring the reporter gene. On the other hand, identification and characterization of various promoters not only benefit the functional annotation of their genes but also provide alternative candidates to be used to drive interesting genes for plant genetic improvement by transgenesis. More than 1,000 putative pollen-specific rice genes have been identified in a genome-wide level. Among them, 67 highly expressed genes were further characterized. One of the pollen-specific genes LOC_Os10g35930 was further surveyed in its expression patterns with more details by quantitative real-time reverse-transcription PCR (qRT-PCR) analysis. Finally, its promoter activity was further investigated by analyzing transgenic rice plants carrying the promoter::GFP cassette, which was constructed from the newly developed T-vector. The reporter GFP gene expression in these transgenic plants showed that the promoter was active only in mature but not in germinated pollens. PMID:25197968

  18. Dual-therapeutic reporter genes fusion for enhanced cancer gene therapy and imaging.

    PubMed

    Sekar, T V; Foygel, K; Willmann, J K; Paulmurugan, R

    2013-05-01

    Two of the successful gene-directed enzyme prodrug therapies include herpes simplex virus-thymidine kinase (HSV1-TK) enzyme-ganciclovir prodrug and the Escherichia coli nitroreductase (NTR) enzyme-CB1954 prodrug strategies; these enzyme-prodrug combinations produce activated cytotoxic metabolites of the prodrugs capable of tumor cell death by inhibiting DNA synthesis and killing quiescent cells, respectively. Both these strategies also affect significant bystander cell killing of neighboring tumor cells that do not express these enzymes. We have developed a dual-combination gene strategy, where we identified HSV1-TK and NTR fused in a particular orientation can effectively kill tumor cells when the tumor cells are treated with a fusion HSV1-TK-NTR gene- along with a prodrug combination of GCV and CB1954. In order to determine whether the dual-system demonstrate superior therapeutic efficacy than either HSV1-TK or NTR systems alone, we conducted both in vitro and in vivo tumor xenograft studies using triple negative SUM159 breast cancer cells, by evaluating the efficacy of cell death by apoptosis and necrosis upon treatment with the dual HSV1-TK genes-GCV-CB1954 prodrugs system, and compared the efficiency to HSV1-TK-GCV and NTR-CB1954. Our cell-based studies, tumor regression studies in xenograft mice, histological analyses of treated tumors and bystander studies indicate that the dual HSV1-TK-NTR-prodrug system is two times more efficient even with half the doses of both prodrugs than the respective single gene-prodrug system, as evidenced by enhanced apoptosis and necrosis of tumor cells in vitro in culture and xenograft of tumor tissues in animals.

  19. MS-1 magA: Revisiting Its Efficacy as a Reporter Gene for MRI.

    PubMed

    Pereira, Sofia M; Williams, Steve R; Murray, Patricia; Taylor, Arthur

    2016-01-01

    Bacterial genes involved in the biomineralization of magnetic nanoparticles in magnetotactic bacteria have recently been proposed as reporters for magnetic resonance imaging (MRI). In such systems, the expression of the bacterial genes in mammalian cells purportedly leads to greater concentrations of intracellular iron or the biomineralization of iron oxides, thus leading to an enhancement in relaxation rate that is detectable via MRI. Here, we show that the constitutive expression of the magA gene from Magnetospirillum magnetotacticum is tolerated by human embryonic kidney (HEK) cells but induces a strong toxic effect in murine mesenchymal/stromal cells and kidney-derived stem cells, severely restricting its effective use as a reporter gene for stem cells. Although it has been suggested that magA is involved in iron transport, when expressed in HEK cells, it does not affect the transcription of endogenous genes related to iron homeostasis. Furthermore, the magA-induced enhancement in iron uptake in HEK cells is insignificant, suggesting this gene is a poor reporter even for cell types that can tolerate its expression. We suggest that the use of magA for stem cells should be approached with caution, and its efficacy as a reporter gene requires a careful assessment on a cell-by-cell basis. PMID:27118760

  20. Development of a gene reporter system in moderately halophilic bacteria by employing the ice nucleation gene of Pseudomonas syringae.

    PubMed Central

    Arvanitis, N; Vargas, C; Tegos, G; Perysinakis, A; Nieto, J J; Ventosa, A; Drainas, C

    1995-01-01

    The expression of the ice nucleation gene inaZ of Pseudomonas syringae in several moderate halophiles was investigated to establish its utility as a reporter for promoter activity and gene expression studies in these biotechnologically and environmentally important bacteria. A promoterless version of inaZ was introduced in two different restriction sites and at both orientations in a recombinant plasmid able to replicate in moderate halophiles and, in particular, within the sequence of its pHE1 part, a native plasmid of Halomonas elongata. One orientation of both recombinant constructs expressed high levels of ice nucleation activity in H. elongata and Volcaniella eurihalina cells, indicating that inaZ was probably introduced in the correct orientation downstream of putative native promoters. A recombinant construct carrying a tandem duplication of inaZ at the same orientation gave significantly higher ice nucleation activity, showing that inaZ is appropriate for gene dosage studies. The ice nucleation gene was also expressed in H. elongata and V. eurihalina under the control of Pbla (the promoter of the beta-lactamase gene of Escherichia coli) and Ppdc (the promoter of the pyruvate decarboxylase gene of Zymomonas mobilis). One of the inaZ reporter plasmids expressing high levels of ice nucleation activity under the control of a native putative promoter was also transferred in Halomonas subglaciescola, Halomonas meridiana, Halomonas halodurans, and Deleya halophila. In all cases, Ice+ transconjugants were successfully isolated, demonstrating that inaZ is expressed in a wide spectrum of moderately halophilic species. PMID:8526492

  1. Hypoxia-induced protein binding to O2-responsive sequences on the tyrosine hydroxylase gene.

    PubMed

    Norris, M L; Millhorn, D E

    1995-10-01

    We reported recently that the gene that encodes tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, is regulated by hypoxia in the dopaminergic cells of the mammalian carotid body (Czyzyk-Krzeska, M. F., Bayliss, D. A., Lawson, E. E. & Millhorn, D. E. (1992) J. Neurochem. 58, 1538-1546) and in pheochromocytoma (PC12) cells (Czyzyk-Krzeska, M. F., Furnari, B. A., Lawson, E. E. & Millhorn, D. E. (1994) J. Biol. Chem. 269, 760-764). Regulation of this gene during low O2 conditions occurs at both the level of transcription and RNA stability. Increased transcription during hypoxia is regulated by a region of the proximal promoter that extends from -284 to + 27 bases, relative to transcription start site. The present study was undertaken to further characterize the sequences that confer O2 responsiveness of the TH gene and to identify hypoxia-induced protein interactions with these sequences. Results from chloramphenicol acetyltransferase assays identified a region between bases -284 and -150 that contains the essential sequences for O2 regulation. This region contains a number of regulatory elements including AP1, AP2, and HIF-1. Gel shift assays revealed enhanced protein interactions at the AP1 and HIF-1 elements of the native gene. Further investigations using supershift and shift-Western analysis showed that c-Fos and JunB bind to the AP1 element during hypoxia and that these protein levels are stimulated by hypoxia. Mutation of the AP1 sequence prevented stimulation of transcription of the TH-chloramphenicol acetyltransferase reporter gene by hypoxia. PMID:7559551

  2. Role of starvation genes in the survival of deep subsurface bacterial communities. Final report

    SciTech Connect

    Matin, A.; Schmidt, T.; Caldwell, D.

    1998-11-01

    The investigation dealt with several aspects of subsurface bacterial survival and their nature. Mutants of Pseudomonas putida, a common environmental bacterium with counterparts in the subsurface, were isolated by transposon mutagenesis. These mutants were highly sensitive to starvation stress. Reporter gene fusions also showed that these genes were starvation genes since they were induced several fold when the cultures were started. Since the regulatory religions (promoters) of starvation genes are of interest in bioremediation and in experiments designed to understand the roles of starvation genes in the maintenance of microbial community structure, the promoter of one of these genes (pstarv1, contained in strain MK107) was characterized in detail. As a preliminary to these studies, the growth characteristics of Pseudomonas putida MK1 and MK107 were compared for cells growing in batch cultures or as an attached monolayer in microstat cultures.

  3. Novel SOST gene mutation in a sclerosteosis patient from Morocco: a case report.

    PubMed

    Belkhribchia, Mohamed Reda; Collet, Corinne; Laplanche, Jean-Louis; Hassani, Redouane

    2014-03-01

    Sclerosteosis (OMIM 269500) is a rare autosomal recessive condition characterized by increased bone density associated with syndactyly. It is linked to a genetic defect in the SOST gene coding for sclerostin. So far, seven different loss-of-function mutations in SOST have been reported in patients with sclerosteosis. Recently, two mutations in LRP4 gene underlying sclerosteosis were identified, reflecting the genetic heterogeneity of this disease. We report here a 30-years-old Moroccan man presented with typical clinical and radiological features of sclerosteosis who carries a novel homozygous mutation in the SOST gene, characterized as a nonsense mutation (c.79C > T; p.Gln27∗) in exon 1 of the SOST gene. This is to our knowledge the first case of sclerosteosis reported from Morocco and North Africa.

  4. Repression of the interleukin 6 gene promoter by p53 and the retinoblastoma susceptibility gene product

    SciTech Connect

    Santhanam, U.; Ray, A.; Sehgal, P.B. )

    1991-09-01

    The aberrant overexpression of interleukin 6 (IL-6) is implicated as an autocrine mechanism in the enhanced proliferation of the neoplastic cell elements in various B- and T-cell malignancies and in some carcinomas and sarcomas; many of these neoplasms have been shown to be associated with a mutated p53 gene. The possibility that wild-type (wt) p53, a nuclear tumor-suppressor protein, but not its transforming mutants might serve to repress IL-6 gene expression was investigated in HeLa cells. The authors transiently cotransfected these cells with constitutive cytomegalovirus (CMV) enhancer/promoter expression plasmids overproducing wt or mutant human or murine p53 and with appropriate chloramphenicol acetyltransferase (CAT) reporter plasmids containing the promoter elements of human IL-6, c-fos, or {beta}-actin genes or of porcine major histocompatibility complex (MHC) class I gene in pN-38 to evaluate the effect of the various p53 species on these promoters. These observations identify transcriptional repression as a property of p53 and suggest that p53 and RB may be involved as transcriptional repressors in modulating IL-6 gene expression during cellular differentiation and oncogenesis.

  5. Space experiment "Rad Gene"-report 1; p53-Dependent gene expression in human cultured cells exposed to space environment

    NASA Astrophysics Data System (ADS)

    Takahashi, Akihisa; Ohnishi, Takeo; Suzuki, Hiromi; Omori, Katsunori; Seki, Masaya; Hashizume, Toko; Shimazu, Toru; Ishioka, Noriaki

    The space environment contains two major biologically significant influences: space radiations and microgravity. A p53 tumor suppressor protein plays a role as a guardian of the genome through the activity of p53-centered signal transduction pathways. The aim of this study was to clarify the biological effects of space radiations, microgravity and a space environment on the gene and protein expression of p53-dependent regulated genes. Space experiments were performed with two human cultured lymphoblastoid cell lines: one cells line (TSCE5) bears a wild-type p53 gene status, and another cells line (WTK1) bears a mutated p53 gene status. Un-der one gravity or microgravity condition, the cells were grown in the cell biology experimental facility (CBEF) of the International Space Station (ISS) for 8 days without experiencing the stress during launching and landing because the cells were frozen during these periods. Ground control samples also were cultured for 8 days in the CBEF on the ground during the same periods as space flight. Gene and protein expression was analyzed by using DNA chip (a 44k whole human genome microarray, Agilent Technologies Inc.) and protein chip (PanoramaTM Ab MicroArray, Sigma-Aldrich Co.), respectively. In addition, we analyzed the gene expression in cultured cells after space flight during 133 days with frozen condition. We report the results and discussion from the viewpoint of the functions of the up-regulated and down-regulated genes after an exposure to space radiations and/or microgravity. The initial goal of this space experiment was completely achieved. It is expected that data from this type of work will be helpful in designing physical protection from the deleterious effects of space radiations during long term stays in space.

  6. Transient expression of choline acetyltransferase-like immunoreactivity in Purkinje cells of the developing rat cerebellum.

    PubMed

    Gould, E; Butcher, L L

    1987-08-01

    The expression of choline acetyltransferase (ChAT)-like immunoreactivity was studied immunohistochemically in the cerebelli of developing rats. Brains were examined from the day of birth (postnatal day 1: P1) until adulthood. From P4 through P21, several Purkinje cells in the uvula, nodule, and flocculus of the cerebellum demonstrated ChAT-like immunoreactivity. After P23, no ChAT-positive neurons were observed in any region of the cerebellum. This finding paralleled the transient expression of acetylcholinesterase in Purkinje cells of these same cerebellar areas during development.

  7. One-step purification of phosphinothricin acetyltransferase using reactive dye-affinity chromatography.

    PubMed

    Wang, Cunxi; Lee, Thomas C; Crowley, Kathleen S; Bell, Erin

    2015-01-01

    Reactive dye purification is an affinity purification technique offering unique selectivity and high purification potential. Historically, purification of phosphinothricin acetyltransferase (PAT) has involved several steps of precipitation and column chromatography. Here, we describe a novel purification method that is simple, time-saving, inexpensive, and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein can be purified to homogeneity from E. coli or plant tissue with high recovery efficiency. PMID:25749943

  8. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products. Progress report, June 1, 1991--May 31, 1992

    SciTech Connect

    Kuchka, M.R.

    1992-05-01

    The following is a review of research accomplished in the first two years of funding for the above mentioned project. The work performed is a molecular characterization of nuclear mutants of Chlamydomonas reinhardtii which are deficient in different stages in the post-transcriptional expression of a single chloroplast encoded polypeptide, the D2 protein of Photosystem II. Our long-term goals are to understand the molecular mechanisms by which nuclear gene products affect the expression of chloroplast genes. Specifically, we which to understand how specific nuclear gene products affect the turnover rate of the D2 encoding mRNA (psbD), how other nuclear encoded factors work to promote the translation of psbD mRNA and/or stabilize the D2 protein, and what the role of the D2 protein itself is in Photosystem II assembly and in the control of expression of other chloroplast genes. This progress report will be organized into four major sections concerning (I) The characterization of nuclear mutants affected in D2 translation/turnover, (II) The study of trans-acting factors which associate with the 5{prime} end of the psbD mRNA, (III) In vitro mutagenesis of the psbD gene, and (IV) Additional studies.

  9. Inhibitors of acetyltransferase domain of N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). Part 1: Hit to lead evaluation of a novel arylsulfonamide series.

    PubMed

    Green, Oluyinka M; McKenzie, Andrew R; Shapiro, Adam B; Otterbein, Ludovic; Ni, Haihong; Patten, Arthur; Stokes, Suzanne; Albert, Robert; Kawatkar, Sameer; Breed, Jason

    2012-02-15

    A novel arylsulfonamide-containing series of compounds represented by 1, discovered by highthroughput screening, inhibit the acetyltransferase domain of N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). X-ray structure determination confirmed that inhibitor binds at the site occupied by acetyl-CoA, indicating that series is competitive with this substrate. This letter documents our early hit-to-lead evaluation of the chemical series and some of the findings that led to improvement in in-vitro potency against Gram-negative and Gram-positive bacterial isozymes, exemplified by compound 40.

  10. Differences in Enzymatic Properties of the Saccharomyces kudriavzevii and Saccharomyces uvarum Alcohol Acetyltransferases and Their Impact on Aroma-Active Compounds Production.

    PubMed

    Stribny, Jiri; Querol, Amparo; Pérez-Torrado, Roberto

    2016-01-01

    Higher alcohols and acetate esters belong to the most important yeast secondary metabolites that significantly contribute to the overall flavor and aroma profile of fermented products. In Saccharomyces cerevisiae, esterification of higher alcohols is catalyzed mainly by the alcohol acetyltransferases encoded by genes ATF1 and ATF2. Previous investigation has shown other Saccharomyces species, e.g., S. kudriavzevii and S. uvarum, to vary in aroma-active higher alcohols and acetate esters formation when compared to S. cerevisiae. Here, we aimed to analyze the enzymes encoded by the ATF1 and ATF2 genes from S. kudriavzevii (SkATF1, SkATF2) and S. uvarum (SuATF1, SuATF2). The heterologous expression of the individual ATF1 and ATF2 genes in a host S. cerevisiae resulted in the enhanced production of several higher alcohols and acetate esters. Particularly, an increase of 2-phenylethyl acetate production by the strains that harbored ATF1 and ATF2 genes from S. kudriavzevii and S. uvarum was observed. When grown with individual amino acids as the nitrogen source, the strain that harbored SkATF1 showed particularly high 2-phenylethyl acetate production and the strains with introduced SkATF2 or SuATF2 revealed increased production of isobutyl acetate, isoamyl acetate, and 2-phenylethyl acetate compared to the reference strains with endogenous ATF genes. The alcohol acetyltransferase activities of the individual Atf1 and Atf2 enzymes measured in the cell extracts of the S. cerevisiae atf1 atf2 iah1 triple-null strain were detected for all the measured substrates. This indicated that S. kudriavzevii and S. uvarum Atf enzymes had broad range substrate specificity as S. cerevisiae Atf enzymes. Individual Atf1 enzymes exhibited markedly different kinetic properties since SkAtf1p showed c. twofold higher and SuAtf1p c. threefold higher K m for isoamyl alcohol than ScAtf1p. Together these results indicated that the differences found among the three Saccharomyces species during the

  11. Differences in Enzymatic Properties of the Saccharomyces kudriavzevii and Saccharomyces uvarum Alcohol Acetyltransferases and Their Impact on Aroma-Active Compounds Production

    PubMed Central

    Stribny, Jiri; Querol, Amparo; Pérez-Torrado, Roberto

    2016-01-01

    Higher alcohols and acetate esters belong to the most important yeast secondary metabolites that significantly contribute to the overall flavor and aroma profile of fermented products. In Saccharomyces cerevisiae, esterification of higher alcohols is catalyzed mainly by the alcohol acetyltransferases encoded by genes ATF1 and ATF2. Previous investigation has shown other Saccharomyces species, e.g., S. kudriavzevii and S. uvarum, to vary in aroma-active higher alcohols and acetate esters formation when compared to S. cerevisiae. Here, we aimed to analyze the enzymes encoded by the ATF1 and ATF2 genes from S. kudriavzevii (SkATF1, SkATF2) and S. uvarum (SuATF1, SuATF2). The heterologous expression of the individual ATF1 and ATF2 genes in a host S. cerevisiae resulted in the enhanced production of several higher alcohols and acetate esters. Particularly, an increase of 2-phenylethyl acetate production by the strains that harbored ATF1 and ATF2 genes from S. kudriavzevii and S. uvarum was observed. When grown with individual amino acids as the nitrogen source, the strain that harbored SkATF1 showed particularly high 2-phenylethyl acetate production and the strains with introduced SkATF2 or SuATF2 revealed increased production of isobutyl acetate, isoamyl acetate, and 2-phenylethyl acetate compared to the reference strains with endogenous ATF genes. The alcohol acetyltransferase activities of the individual Atf1 and Atf2 enzymes measured in the cell extracts of the S. cerevisiae atf1 atf2 iah1 triple-null strain were detected for all the measured substrates. This indicated that S. kudriavzevii and S. uvarum Atf enzymes had broad range substrate specificity as S. cerevisiae Atf enzymes. Individual Atf1 enzymes exhibited markedly different kinetic properties since SkAtf1p showed c. twofold higher and SuAtf1p c. threefold higher Km for isoamyl alcohol than ScAtf1p. Together these results indicated that the differences found among the three Saccharomyces species during the

  12. Utility of an appropriate reporter assay: Heliotrine interferes with GAL4/upstream activation sequence-driven reporter gene systems.

    PubMed

    Luckert, Claudia; Hessel, Stefanie; Lampen, Alfonso; Braeuning, Albert

    2015-10-15

    Reporter gene assays are widely used for the assessment of transcription factor activation following xenobiotic exposure of cells. A critical issue with such assays is the possibility of interference of test compounds with the test system, for example, by direct inhibition of the reporter enzyme. Here we show that the pyrrolizidine alkaloid heliotrine interferes with reporter signals derived from GAL4-based nuclear receptor transactivation assays by a mechanism independent of luciferase enzyme inhibition. These data highlight the necessity to conduct proper control experiments in order to avoid perturbation of reporter assays by test chemicals.

  13. Biochemical analysis and structure determination of bacterial acetyltransferases responsible for the biosynthesis of UDP-N,N'-diacetylbacillosamine.

    PubMed

    Morrison, Michael J; Imperiali, Barbara

    2013-11-01

    UDP-N,N'-diacetylbacillosamine (UDP-diNAcBac) is a unique carbohydrate produced by a number of bacterial species and has been implicated in pathogenesis. The terminal step in the formation of this important bacterial sugar is catalyzed by an acetyl-CoA (AcCoA)-dependent acetyltransferase in both N- and O-linked protein glycosylation pathways. This bacterial acetyltransferase is a member of the left-handed β-helix family and forms a homotrimer as the functional unit. Whereas previous endeavors have focused on the Campylobacter jejuni acetyltransferase (PglD) from the N-linked glycosylation pathway, structural characterization of the homologous enzymes in the O-linked glycosylation pathways is lacking. Herein, we present the apo-crystal structures of the acetyltransferase domain (ATD) from the bifunctional enzyme PglB (Neisseria gonorrhoeae) and the full-length acetyltransferase WeeI (Acinetobacter baumannii). Additionally, a PglB-ATD structure was solved in complex with AcCoA. Surprisingly, this structure reveals a contrasting binding mechanism for this substrate when compared with the AcCoA-bound PglD structure. A comparison between these findings and the previously solved PglD crystal structures illustrates a dichotomy among N- and O-linked glycosylation pathway enzymes. Based upon these structures, key residues in the UDP-4-amino and AcCoA binding pockets were mutated to determine their effect on binding and catalysis in PglD, PglB-ATD, and WeeI. Last, a phylogenetic analysis of the aforementioned acetyltransferases was employed to illuminate the diversity among N- and O-linked glycosylation pathway enzymes. PMID:24064219

  14. Multimodality Imaging of Gene Transfer with a Receptor-Based Reporter Gene

    PubMed Central

    Chen, Ron; Parry, Jesse J.; Akers, Walter J.; Berezin, Mikhail Y.; El Naqa, Issam M.; Achilefu, Samuel; Edwards, W. Barry; Rogers, Buck E.

    2010-01-01

    Gene therapy trials have traditionally used tumor and tissue biopsies for assessing the efficacy of gene transfer. Non-invasive imaging techniques offer a distinct advantage over tissue biopsies in that the magnitude and duration of gene transfer can be monitored repeatedly. Human somatostatin receptor subtype 2 (SSTR2) has been used for the nuclear imaging of gene transfer. To extend this concept, we have developed a somatostatin receptor–enhanced green fluorescent protein fusion construct (SSTR2-EGFP) for nuclear and fluorescent multimodality imaging. Methods An adenovirus containing SSTR2-EGFP (AdSSTR2-EGFP) was constructed and evaluated in vitro and in vivo. SCC-9 human squamous cell carcinoma cells were infected with AdEGFP, AdSSTR2, or AdSSTR2-EGFP for in vitro evaluation by saturation binding, internalization, and fluorescence spectroscopy assays. In vivo biodistribution and nano-SPECT imaging studies were conducted with mice bearing SCC-9 tumor xenografts directly injected with AdSSTR2-EGFP or AdSSTR2 to determine the tumor localization of 111In-diethylenetriaminepentaacetic acid (DTPA)-Tyr3-octreotate. Fluorescence imaging was conducted in vivo with mice receiving intratumoral injections of AdSSTR2, AdSSTR2-EGFP, or AdEGFP as well as ex vivo with tissues extracted from mice. Results The similarity between AdSSTR2-EGFP and wild-type AdSSTR2 was demonstrated in vitro by the saturation binding and internalization assays, and the fluorescence emission spectra of cells infected with AdSSTR2-EGFP was almost identical to the spectra of cells infected with wild-type AdEGFP. Biodistribution studies demonstrated that the tumor uptake of 111In-DTPA-Tyr3-octreotate was not significantly different (P > 0.05) when tumors (n = 5) were injected with AdSSTR2 or AdSSTR2-EGFP but was significantly greater than the uptake in control tumors. Fluorescence was observed in tumors injected with AdSSTR2-EGFP and AdEGFP in vivo and ex vivo but not in tumors injected with AdSSTR2

  15. Tyrosinase as a multifunctional reporter gene for Photoacoustic/MRI/PET triple modality molecular imaging

    PubMed Central

    Qin, Chunxia; Cheng, Kai; Chen, Kai; Hu, Xiang; Liu, Yang; Lan, Xiaoli; Zhang, Yongxue; Liu, Hongguang; Xu, Yingding; Bu, Lihong; Su, Xinhui; Zhu, Xiaohua; Meng, Shuxian; Cheng, Zhen

    2013-01-01

    Development of reporter genes for multimodality molecular imaging is highly important. In contrast to the conventional strategies which have focused on fusing several reporter genes together to serve as multimodal reporters, human tyrosinase (TYR) – the key enzyme in melanin production – was evaluated in this study as a stand-alone reporter gene for in vitro and in vivo photoacoustic imaging (PAI), magnetic resonance imaging (MRI) and positron emission tomography (PET). Human breast cancer cells MCF-7 transfected with a plasmid that encodes TYR (named as MCF-7-TYR) and non-transfected MCF-7 cells were used as positive and negative controls, respectively. Melanin targeted N-(2-(diethylamino)ethyl)-18F-5-fluoropicolinamide was used as a PET reporter probe. In vivo PAI/MRI/PET imaging studies showed that MCF-7-TYR tumors achieved significant higher signals and tumor-to-background contrasts than those of MCF-7 tumor. Our study demonstrates that TYR gene can be utilized as a multifunctional reporter gene for PAI/MRI/PET both in vitro and in vivo. PMID:23508226

  16. First report of the blaVIM gene in environmental isolates of Buttiauxella sp.

    PubMed

    Pitondo-Silva, André; Martins, Vinicius Vicente; Stehling, Eliana Guedes

    2015-04-01

    Several works have demonstrated the presence of metallo-β-lactamases (MBLs) in clinical bacteria. However, in environmental isolates, few works have reported on these enzymes. In this study, we report for the first time two environmental isolates of Buttiauxella sp. recovered from chrysanthemum plantations in Brazil containing blaVIM gene and producing MBLs.

  17. Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation

    PubMed Central

    Kirov, Julia V.; Adkisson, Michael; Nava, A. J.; Cipollone, Andreana; Willis, Brandon; Engelhard, Eric K.; Lloyd, K. C. Kent; de Jong, Pieter; West, David B.

    2015-01-01

    Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown. PMID:26275310

  18. Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases.

    PubMed

    Dahlin, Jayme L; Chen, Xiaoyue; Walters, Michael A; Zhang, Zhiguo

    2015-01-01

    During DNA replication, nucleosomes ahead of replication forks are disassembled to accommodate replication machinery. Following DNA replication, nucleosomes are then reassembled onto replicated DNA using both parental and newly synthesized histones. This process, termed DNA replication-coupled nucleosome assembly (RCNA), is critical for maintaining genome integrity and for the propagation of epigenetic information, dysfunctions of which have been implicated in cancers and aging. In recent years, it has been shown that RCNA is carefully orchestrated by a series of histone modifications, histone chaperones and histone-modifying enzymes. Interestingly, many features of RCNA are also found in processes involving DNA replication-independent nucleosome assembly like histone exchange and gene transcription. In yeast, histone H3 lysine K56 acetylation (H3K56ac) is found in newly synthesized histone H3 and is critical for proper nucleosome assembly and for maintaining genomic stability. The histone acetyltransferase (HAT) regulator of Ty1 transposition 109 (Rtt109) is the sole enzyme responsible for H3K56ac in yeast. Much research has centered on this particular histone modification and histone-modifying enzyme. This Critical Review summarizes much of our current understanding of nucleosome assembly and highlights many important insights learned from studying Rtt109 HATs in fungi. We highlight some seminal features in nucleosome assembly conserved in mammalian systems and describe some of the lingering questions in the field. Further studying fungal and mammalian chromatin assembly may have important public health implications, including deeper understandings of human cancers and aging as well as the pursuit of novel anti-fungal therapies.

  19. Structural and Functional Conservation of the NuA4 Histone Acetyltransferase Complex from Yeast to Humans

    PubMed Central

    Doyon, Yannick; Selleck, William; Lane, William S.; Tan, Song; Côté, Jacques

    2004-01-01

    The NuA4 histone acetyltransferase (HAT) multisubunit complex is responsible for acetylation of histone H4 and H2A N-terminal tails in yeast. Its catalytic component, Esa1, is essential for cell cycle progression, gene-specific regulation and has been implicated in DNA repair. Almost all NuA4 subunits have clear homologues in higher eukaryotes, suggesting that the complex is conserved throughout evolution to metazoans. We demonstrate here that NuA4 complexes are indeed present in human cells. Tip60 and its splice variant Tip60b/PLIP were purified as stable HAT complexes associated with identical polypeptides, with 11 of the 12 proteins being homologs of yeast NuA4 subunits. This indicates a highly conserved subunit composition and the identified human proteins underline the role of NuA4 in the control of mammalian cell proliferation. ING3, a member of the ING family of growth regulators, links NuA4 to p53 function which we confirmed in vivo. Proteins specific to the human NuA4 complexes include ruvB-like helicases and a bromodomain-containing subunit linked to ligand-dependent transcription activation by the thyroid hormone receptor. We also demonstrate that subunits MRG15 and DMAP1 are present in distinct protein complexes harboring histone deacetylase and SWI2-related ATPase activities, respectively. Finally, analogous to yeast, a recombinant trimeric complex formed by Tip60, EPC1, and ING3 is sufficient to reconstitute robust nucleosomal HAT activity in vitro. In conclusion, the NuA4 HAT complex is highly conserved in eukaryotes, in which it plays primary roles in transcription, cellular response to DNA damage, and cell cycle control. PMID:14966270

  20. Enteric plexuses of two choline-acetyltransferase transgenic mouse lines: chemical neuroanatomy of the fluorescent protein-expressing nerve cells.

    PubMed

    Wilhelm, Márta; Lawrence, J Josh; Gábriel, Robert

    2015-02-01

    We studied cholinergic circuit elements in the enteric nervous system (ENS) of two distinct transgenic mouse lines in which fluorescent protein expression was driven by the choline-acetyltransferase (ChAT) promoter. In the first mouse line, green fluorescent protein was fused to the tau gene. This construct allowed the visualization of the fiber tracts and ganglia, however the nerve cells were poorly resolved. In the second mouse line (ChATcre-YFP), CRE/loxP recombination yielded cytosolic expression of yellow fluorescent protein (YFP). In these preparations the morphology of enteric neurons could be well studied. We also determined the neurochemical identity of ENS neurons in muscular and submucous layers using antibodies against YFP, calretinin (CALR), calbindin (CALB), and vasoactive intestinal peptide (VIP). Confocal microscopic imaging was used to visualize fluorescently-conjugated secondary antibodies. In ChATcre-YFP preparations, YFP was readily apparent in somatodendritic regions of ENS neurons. In the myenteric plexus, YFP/CALR/VIP staining revealed that 34% of cholinergic cells co-labeled with CALR. Few single-stained CR-positive cells were observed. Neither YFP nor CALR co-localized with VIP. In GFP/CALB/CALR staining, all co-localization combinations were represented. In the submucosal plexus, YFP/CALR/VIP staining revealed discrete neuronal populations. However, in separate preparations, double labeling was observed for YFP/CALR and CALR/VIP. In YFP/CALR/CALB staining, all combinations of double staining and triple labeling were verified. In conclusion, the neurochemical coding of ENS neurons in these mouse lines is consistent with many observations in non-transgenic animals. Thus, they provide useful tools for physiological and pharmacological studies on distinct neurochemical subtypes of ENS neurons.

  1. Characterization of the mammalian DNA polymerase gene(s) and enzyme(s). Annual progress report

    SciTech Connect

    Mishra, N.C.

    1994-01-01

    Consistent with the long term goal of our research to understand the nature of the key enzymes in eukaryotic DNA replication we have characterized the properties of the wild type DNA polymerases of the {alpha}-family and their mutants. We have also provided evidence for the role of aphidicolin in the elongation process of the in vivo DNA replication in eukaryotic cells. We also developed a technology for planned prep from a large numbers of clones for direct screening by size or restriction digestion in order to facilitate our goals to clone the DNA polymerase gene.

  2. Report of a chimeric origin of transposable elements in a bovine-coding gene.

    PubMed

    Almeida, L M; Amaral, M E J; Silva, I T; Silva, W A; Riggs, P K; Carareto, C M

    2008-02-01

    Despite the wide distribution of transposable elements (TEs) in mammalian genomes, part of their evolutionary significance remains to be discovered. Today there is a substantial amount of evidence showing that TEs are involved in the generation of new exons in different species. In the present study, we searched 22,805 genes and reported the occurrence of TE-cassettes in coding sequences of 542 cow genes using the RepeatMasker program. Despite the significant number (542) of genes with TE insertions in exons only 14 (2.6%) of them were translated into protein, which we characterized as chimeric genes. From these chimeric genes, only the FAST kinase domains 3 (FASTKD3) gene, present on chromosome BTA 20, is a functional gene and showed evidence of the exaptation event. The genome sequence analysis showed that the last exon coding sequence of bovine FASTKD3 is approximately 85% similar to the ART2A retrotransposon sequence. In addition, comparison among FASTKD3 proteins shows that the last exon is very divergent from those of Homo sapiens, Pan troglodytes and Canis familiares. We suggest that the gene structure of bovine FASTKD3 gene could have originated by several ectopic recombinations between TE copies. Additionally, the absence of TE sequences in all other species analyzed suggests that the TE insertion is clade-specific, mainly in the ruminant lineage.

  3. Immunolocalization of choline acetyltransferase of common type in the central brain mass of Octopus vulgaris

    PubMed Central

    Casini, A.; Vaccaro, R.; D'Este, L.; Sakaue, Y.; Bellier, J.P.; Kimura, H.; Renda, T.G.

    2012-01-01

    Acetylcholine, the first neurotransmitter to be identified in the vertebrate frog, is widely distributed among the animal kingdom. The presence of a large amount of acetylcholine in the nervous system of cephalopods is well known from several biochemical and physiological studies. However, little is known about the precise distribution of cholinergic structures due to a lack of a suitable histochemical technique for detecting acetylcholine. The most reliable method to visualize the cholinergic neurons is the immunohistochemical localization of the enzyme choline acetyltransferase, the synthetic enzyme of acetylcholine. Following our previous study on the distribution patterns of cholinergic neurons in the Octopus vulgaris visual system, using a novel antibody that recognizes choline acetyltransferase of the common type (cChAT), now we extend our investigation on the octopus central brain mass. When applied on sections of octopus central ganglia, immunoreactivity for cChAT was detected in cell bodies of all central brain mass lobes with the notable exception of the subfrontal and subvertical lobes. Positive varicosed nerves fibers where observed in the neuropil of all central brain mass lobes. PMID:23027350

  4. Photoaffinity labelling of carnitine acetyltransferase with S-(p-azidophenacyl)thiocarnitine.

    PubMed Central

    Mauro, J M; Lewis, R V; Barden, R E

    1986-01-01

    A photolabile reagent, p-azidophenacyl-DL-thiocarnitine, was synthesized and tested as a photoaffinity label for carnitine acetyltransferase (EC 2.3.1.7) from pigeon breast. p-Azidophenacyl-DL-thiocarnitine is an active-site-directed reagent for this acetyltransferase, since it is a competitive inhibitor (Ki 10 microM) versus carnitine. U.v. irradiation of a mixture of p-azidophenacyl-DL-thiocarnitine and enzyme produces irreversible inhibition. Acetyl-DL-carnitine protects the enzyme from inhibition by photoactivated p-azidophenacyl-DL-thiocarnitine. In the presence of 30 mM-2-mercaptoethanol as a scavenger, the relationship between loss of activity and photoincorporation of reagent suggests that one molecule of reagent is incorporated per molecule of inhibited enzyme. However, peptide maps of enzyme labelled with p-azidophenacyl[14C]thiocarnitine indicate that several (about six) tryptic peptides (of a possible 60-65) are modified. The presence of 5 mM-acetyl-DL-carnitine significantly decreases the incorporation of reagent in each labelled tryptic peptide. PMID:3800901

  5. Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT)

    PubMed Central

    Salah Ud-Din, Abu Iftiaf Md; Tikhomirova, Alexandra; Roujeinikova, Anna

    2016-01-01

    General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) catalyze the transfer of an acyl moiety from acyl coenzyme A (acyl-CoA) to a diverse group of substrates and are widely distributed in all domains of life. This review of the currently available data acquired on GNAT enzymes by a combination of structural, mutagenesis and kinetic methods summarizes the key similarities and differences between several distinctly different families within the GNAT superfamily, with an emphasis on the mechanistic insights obtained from the analysis of the complexes with substrates or inhibitors. It discusses the structural basis for the common acetyltransferase mechanism, outlines the factors important for the substrate recognition, and describes the mechanism of action of inhibitors of these enzymes. It is anticipated that understanding of the structural basis behind the reaction and substrate specificity of the enzymes from this superfamily can be exploited in the development of novel therapeutics to treat human diseases and combat emerging multidrug-resistant microbial infections. PMID:27367672

  6. Crystal structure analysis of the polysialic acid specific O-acetyltransferase NeuO.

    PubMed

    Schulz, Eike C; Bergfeld, Anne K; Ficner, Ralf; Mühlenhoff, Martina

    2011-01-01

    The major virulence factor of the neuroinvasive pathogen Escherichia coli K1 is the K1 capsule composed of α2,8-linked polysialic acid (polySia). K1 strains harboring the CUS-3 prophage modify their capsular polysaccharide by phase-variable O-acetylation, a step that is associated with increased virulence. Here we present the crystal structure of the prophage-encoded polysialate O-acetyltransferase NeuO. The homotrimeric enzyme belongs to the left-handed β-helix (LβH) family of acyltransferases and is characterized by an unusual funnel-shaped outline. Comparison with other members of the LβH family allowed the identification of active site residues and proposal of a catalytic mechanism and highlighted structural characteristics of polySia specific O-acetyltransferases. As a unique feature of NeuO, the enzymatic activity linearly increases with the length of the N-terminal poly-ψ-domain which is composed of a variable number of tandem copies of an RLKTQDS heptad. Since the poly-ψ-domain was not resolved in the crystal structure it is assumed to be unfolded in the apo-enzyme.

  7. Crystal Structure Analysis of the Polysialic Acid Specific O-Acetyltransferase NeuO

    PubMed Central

    Schulz, Eike C.; Bergfeld, Anne K.; Ficner, Ralf; Mühlenhoff, Martina

    2011-01-01

    The major virulence factor of the neuroinvasive pathogen Escherichia coli K1 is the K1 capsule composed of α2,8-linked polysialic acid (polySia). K1 strains harboring the CUS-3 prophage modify their capsular polysaccharide by phase-variable O-acetlyation, a step that is associated with increased virulence. Here we present the crystal structure of the prophage-encoded polysialate O-acetyltransferase NeuO. The homotrimeric enzyme belongs to the left-handed β-helix (LβH) family of acyltransferases and is characterized by an unusual funnel-shaped outline. Comparison with other members of the LβH family allowed the identification of active site residues and proposal of a catalytic mechanism and highlighted structural characteristics of polySia specific O-acetyltransferases. As a unique feature of NeuO, the enzymatic activity linearly increases with the length of the N-terminal poly-ψ-domain which is composed of a variable number of tandem copies of an RLKTQDS heptad. Since the poly-ψ-domain was not resolved in the crystal structure it is assumed to be unfolded in the apo-enyzme. PMID:21390252

  8. Biochemical and structural analysis of an Eis family aminoglycoside acetyltransferase from bacillus anthracis.

    PubMed

    Green, Keith D; Biswas, Tapan; Chang, Changsoo; Wu, Ruiying; Chen, Wenjing; Janes, Brian K; Chalupska, Dominika; Gornicki, Piotr; Hanna, Philip C; Tsodikov, Oleg V; Joachimiak, Andrzej; Garneau-Tsodikova, Sylvie

    2015-05-26

    Proteins from the enhanced intracellular survival (Eis) family are versatile acetyltransferases that acetylate amines at multiple positions of several aminoglycosides (AGs). Their upregulation confers drug resistance. Homologues of Eis are present in diverse bacteria, including many pathogens. Eis from Mycobacterium tuberculosis (Eis_Mtb) has been well characterized. In this study, we explored the AG specificity and catalytic efficiency of the Eis family protein from Bacillus anthracis (Eis_Ban). Kinetic analysis of specificity and catalytic efficiency of acetylation of six AGs indicates that Eis_Ban displays significant differences from Eis_Mtb in both substrate binding and catalytic efficiency. The number of acetylated amines was also different for several AGs, indicating a distinct regiospecificity of Eis_Ban. Furthermore, most recently identified inhibitors of Eis_Mtb did not inhibit Eis_Ban, underscoring the differences between these two enzymes. To explain these differences, we determined an Eis_Ban crystal structure. The comparison of the crystal structures of Eis_Ban and Eis_Mtb demonstrates that critical residues lining their respective substrate binding pockets differ substantially, explaining their distinct specificities. Our results suggest that acetyltransferases of the Eis family evolved divergently to garner distinct specificities while conserving catalytic efficiency, possibly to counter distinct chemical challenges. The unique specificity features of these enzymes can be utilized as tools for developing AGs with novel modifications and help guide specific AG treatments to avoid Eis-mediated resistance. PMID:25928210

  9. RNA helicase module in an acetyltransferase that modifies a specific tRNA anticodon

    PubMed Central

    Chimnaronk, Sarin; Suzuki, Tateki; Manita, Tetsuhiro; Ikeuchi, Yoshiho; Yao, Min; Suzuki, Tsutomu; Tanaka, Isao

    2009-01-01

    Post-transcriptional RNA modifications in the anticodon of transfer RNAs frequently contribute to the high fidelity of protein synthesis. In eubacteria, two genome-encoded transfer RNA (tRNA) species bear the same CAU sequence as the anticodons, which are differentiated by modified cytidines at the wobble positions. The elongator tRNAMet accepts an acetyl moiety at the wobble base to form N4-acetylcytidine (ac4C): an inherent modification ensures precise decoding of the AUG codon by strengthening C−G base-pair interaction and concurrently preventing misreading of the near cognate AUA codon. We have determined the crystal structure of tRNAMet cytidine acetyltransferase (TmcA) from Escherichia coli complexed with two natural ligands, acetyl-CoA and ADP, at 2.35 Å resolution. The structure unexpectedly reveals an idiosyncratic RNA helicase module fused with a GCN5-related N-acetyltransferase (GNAT) fold, which intimately cross-interact. Taken together with the biochemical evidence, we further unravelled the function of acetyl-CoA as an enzyme-activating switch, and propose that an RNA helicase motor driven by ATP hydrolysis is used to deliver the wobble base to the active centre of the GNAT domain. PMID:19322199

  10. Immunolocalization of choline acetyltransferase of common type in the central brain mass of Octopus vulgaris.

    PubMed

    Casini, A; Vaccaro, R; D'Este, L; Sakaue, Y; Bellier, J P; Kimura, H; Renda, T G

    2012-07-19

    Acetylcholine, the first neurotransmitter to be identified in the vertebrate frog, is widely distributed among the animal kingdom. The presence of a large amount of acetylcholine in the nervous system of cephalopods is well known from several biochemical and physiological studies. However, little is known about the precise distribution of cholinergic structures due to a lack of a suitable histochemical technique for detecting acetylcholine. The most reliable method to visualize the cholinergic neurons is the immunohistochemical localization of the enzyme choline acetyltransferase, the synthetic enzyme of acetylcholine. Following our previous study on the distribution patterns of cholinergic neurons in the Octopus vulgaris visual system, using a novel antibody that recognizes choline acetyltransferase of the common type (cChAT), now we extend our investigation on the octopus central brain mass. When applied on sections of octopus central ganglia, immunoreactivity for cChAT was detected in cell bodies of all central brain mass lobes with the notable exception of the subfrontal and subvertical lobes. Positive varicosed nerves fibers where observed in the neuropil of all central brain mass lobes.

  11. Intracellular localization of α-tubulin acetyltransferase ATAT1 in rat ciliated cells.

    PubMed

    Nakakura, Takashi; Suzuki, Takeshi; Nemoto, Takahiro; Tanaka, Hideyuki; Asano-Hoshino, Anshin; Arisawa, Kenjiro; Nishijima, Yoshimi; Kiuchi, Yoshiko; Hagiwara, Haruo

    2016-09-01

    Cilia are microtubule-based hair-like organelles on basal bodies located beneath the cell membrane in various tissues of multicellular animals, and are usually classified into motile cilia and primary cilia. Microtubules are assembled from the heterodimers of α- and β-tubulin. The lysine residue at position 40 (K40) of α-tubulin is an important site for acetylation, and this site is acetylated in the cilium. α-Tubulin N-acetyltransferase 1 (ATAT1) is an acetyltransferase specific to the K40 residue of α-tubulin; however, its intracellular distribution in mammalian tissues remains unclear. In this study, we analyzed ATAT1 localization in rat trachea, oviduct, kidney, retina, testis and the third ventricle of the brain by immunohistochemical techniques using a specific antibody against ATAT1. ATAT1 was distributed to the motile cilia of multiciliated cells of the trachea, third ventricle of the brain and oviduct, and in the primary cilia of the renal medullary collecting duct. ATAT1 also localized to the primary cilia, inner and outer segments of retinal photoreceptor cells, and at the Golgi apparatus of spermatocytes and spermatids of testis. These results indicated that α-tubulin acetylation by ATAT1 at distinct subcellular positions may influence the functional regulation of microtubules and cilia in a variety of ciliated cells. PMID:26700226

  12. The Protein Acetyltransferase PatZ from Escherichia coli Is Regulated by Autoacetylation-induced Oligomerization*

    PubMed Central

    de Diego Puente, Teresa; Gallego-Jara, Julia; Castaño-Cerezo, Sara; Bernal Sánchez, Vicente; Fernández Espín, Vanesa; García de la Torre, José; Manjón Rubio, Arturo; Cánovas Díaz, Manuel

    2015-01-01

    Lysine acetylation is an important post-translational modification in the metabolic regulation of both prokaryotes and eukaryotes. In Escherichia coli, PatZ (formerly YfiQ) is the only known acetyltransferase protein and is responsible for acetyl-CoA synthetase acetylation. In this study, we demonstrated PatZ-positive cooperativity in response to acetyl-CoA and the regulation of acetyl-CoA synthetase activity by the acetylation level. Furthermore, functional analysis of an E809A mutant showed that the conserved glutamate residue is not relevant for the PatZ catalytic mechanism. Biophysical studies demonstrated that PatZ is a stable tetramer in solution and is transformed to its octameric form by autoacetylation. Moreover, this modification is reversed by the sirtuin CobB. Finally, an in silico PatZ tetramerization model based on hydrophobic and electrostatic interactions is proposed and validated by three-dimensional hydrodynamic analysis. These data reveal, for the first time, the structural regulation of an acetyltransferase by autoacetylation in a prokaryotic organism. PMID:26251518

  13. Acetyltransferase SAS2 and sirtuin SIR2, respectively, control flocculation and biofilm formation in wine yeast.

    PubMed

    Rodriguez, María E; Orozco, Helena; Cantoral, Jesús M; Matallana, Emilia; Aranda, Agustín

    2014-09-01

    Cell-to-cell and cell-to-environment interactions of microorganisms are of substantial relevance for their biotechnological use. In the yeast Saccharomyces cerevisiae, flocculation can be an advantage to clarify final liquid products after fermentation, and biofilm formation may be relevant for the encapsulation of strains of interest. The adhesion properties of wine yeast strains can be modified by the genetic manipulation of transcriptional regulatory proteins, such as histone deacetylases, and acetylases. Sirtuin SIR2 is essential for the formation of mat structures, a kind of biofilm that requires the expression of cell-wall protein FLO11 as its deletion reduces FLO11 expression, and adhesion of cells to themselves and to agar in a commercial wine strain. Deletion of acetyltransferase GCN5 leads to a similar phenotype. A naturally flocculant wine yeast strain called P2 was characterized. Its flocculation happens only during grape juice fermentation and is due to the presence of a highly transcribed version of flocculin FLO5, linked to the presence of a δ sequence in the promoter. Deletion of acetyltransferase SAS2 enhances this phenotype and maltose fermentation even more. Therefore, the manipulation of acetylation/deacetylation machinery members is a valid way to alter the interaction of industrial yeast to their environment.

  14. The N-terminal acetyltransferase Naa10 is essential for zebrafish development

    PubMed Central

    Ree, Rasmus; Myklebust, Line M.; Thiel, Puja; Foyn, Håvard; Fladmark, Kari E.; Arnesen, Thomas

    2015-01-01

    N-terminal acetylation, catalysed by N-terminal acetyltransferases (NATs), is among the most common protein modifications in eukaryotes and involves the transfer of an acetyl group from acetyl-CoA to the α-amino group of the first amino acid. Functions of N-terminal acetylation include protein degradation and sub-cellular targeting. Recent findings in humans indicate that a dysfunctional Nα-acetyltransferase (Naa) 10, the catalytic subunit of NatA, the major NAT, is associated with lethality during infancy. In the present study, we identified the Danio rerio orthologue zebrafish Naa 10 (zNaa10). In vitro N-terminal acetylation assays revealed that zNaa10 has NAT activity with substrate specificity highly similar to that of human Naa10. Spatiotemporal expression pattern was determined by in situ hybridization, showing ubiquitous expression with especially strong staining in brain and eye. By morpholino-mediated knockdown, we demonstrated that naa10 morphants displayed increased lethality, growth retardation and developmental abnormalities like bent axis, abnormal eyes and bent tails. In conclusion, we identified the zebrafish Naa10 orthologue and revealed that it is essential for normal development and viability of zebrafish. PMID:26251455

  15. The Protein Acetyltransferase PatZ from Escherichia coli Is Regulated by Autoacetylation-induced Oligomerization.

    PubMed

    de Diego Puente, Teresa; Gallego-Jara, Julia; Castaño-Cerezo, Sara; Bernal Sánchez, Vicente; Fernández Espín, Vanesa; García de la Torre, José; Manjón Rubio, Arturo; Cánovas Díaz, Manuel

    2015-09-18

    Lysine acetylation is an important post-translational modification in the metabolic regulation of both prokaryotes and eukaryotes. In Escherichia coli, PatZ (formerly YfiQ) is the only known acetyltransferase protein and is responsible for acetyl-CoA synthetase acetylation. In this study, we demonstrated PatZ-positive cooperativity in response to acetyl-CoA and the regulation of acetyl-CoA synthetase activity by the acetylation level. Furthermore, functional analysis of an E809A mutant showed that the conserved glutamate residue is not relevant for the PatZ catalytic mechanism. Biophysical studies demonstrated that PatZ is a stable tetramer in solution and is transformed to its octameric form by autoacetylation. Moreover, this modification is reversed by the sirtuin CobB. Finally, an in silico PatZ tetramerization model based on hydrophobic and electrostatic interactions is proposed and validated by three-dimensional hydrodynamic analysis. These data reveal, for the first time, the structural regulation of an acetyltransferase by autoacetylation in a prokaryotic organism.

  16. Alpha-fetoprotein-targeted reporter gene expression imaging in hepatocellular carcinoma.

    PubMed

    Kim, Kwang Il; Chung, Hye Kyung; Park, Ju Hui; Lee, Yong Jin; Kang, Joo Hyun

    2016-07-21

    Hepatocellular carcinoma (HCC) is one of the most common cancers in Eastern Asia, and its incidence is increasing globally. Numerous experimental models have been developed to better our understanding of the pathogenic mechanism of HCC and to evaluate novel therapeutic approaches. Molecular imaging is a convenient and up-to-date biomedical tool that enables the visualization, characterization and quantification of biologic processes in a living subject. Molecular imaging based on reporter gene expression, in particular, can elucidate tumor-specific events or processes by acquiring images of a reporter gene's expression driven by tumor-specific enhancers/promoters. In this review, we discuss the advantages and disadvantages of various experimental HCC mouse models and we present in vivo images of tumor-specific reporter gene expression driven by an alpha-fetoprotein (AFP) enhancer/promoter system in a mouse model of HCC. The current mouse models of HCC development are established by xenograft, carcinogen induction and genetic engineering, representing the spectrum of tumor-inducing factors and tumor locations. The imaging analysis approach of reporter genes driven by AFP enhancer/promoter is presented for these different HCC mouse models. Such molecular imaging can provide longitudinal information about carcinogenesis and tumor progression. We expect that clinical application of AFP-targeted reporter gene expression imaging systems will be useful for the detection of AFP-expressing HCC tumors and screening of increased/decreased AFP levels due to disease or drug treatment. PMID:27468205

  17. No evidence for role of extracellular choline-acetyltransferase in generation of gamma oscillations in rat hippocampal slices in vitro.

    PubMed

    Hollnagel, J O; ul Haq, R; Behrens, C J; Maslarova, A; Mody, I; Heinemann, U

    2015-01-22

    Acetylcholine (ACh) is well known to induce persistent γ-oscillations in the hippocampus when applied together with physostigmine, an inhibitor of the ACh degrading enzyme acetylcholinesterase (AChE). Here we report that physostigmine alone can also dose-dependently induce γ-oscillations in rat hippocampal slices. We hypothesized that this effect was due to the presence of choline in the extracellular space and that this choline is taken up into cholinergic fibers where it is converted to ACh by the enzyme choline-acetyltransferase (ChAT). Release of ACh from cholinergic fibers in turn may then induce γ-oscillations. We therefore tested the effects of the choline uptake inhibitor hemicholinium-3 (HC-3) on persistent γ-oscillations either induced by physostigmine alone or by co-application of ACh and physostigmine. We found that HC-3 itself did not induce γ-oscillations and also did not prevent physostigmine-induced γ-oscillation while washout of physostigmine and ACh-induced γ-oscillations was accelerated. It was recently reported that ChAT might also be present in the extracellular space (Vijayaraghavan et al., 2013). Here we show that the effect of physostigmine was prevented by the ChAT inhibitor (2-benzoylethyl)-trimethylammonium iodide (BETA) which could indicate extracellular synthesis of ACh. However, when we tested for effects of extracellularly applied acetyl-CoA, a substrate of ChAT for synthesis of ACh, physostigmine-induced γ-oscillations were attenuated. Together, these findings do not support the idea that ACh can be synthesized by an extracellularly located ChAT. PMID:25453770

  18. DNA binding by Sgf11 protein affects histone H2B deubiquitination by Spt-Ada-Gcn5-acetyltransferase (SAGA).

    PubMed

    Koehler, Christian; Bonnet, Jacques; Stierle, Matthieu; Romier, Christophe; Devys, Didier; Kieffer, Bruno

    2014-03-28

    The yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex is a transcription coactivator that contains a histone H2B deubiquitination activity mediated by its Ubp8 subunit. Full enzymatic activity requires the formation of a quaternary complex, the deubiquitination module (DUBm) of SAGA, which is composed of Ubp8, Sus1, Sgf11, and Sgf73. The crystal structures of the DUBm have shed light on the structure/function relationship of this complex. Specifically, both Sgf11 and Sgf73 contain zinc finger domains (ZnF) that appear essential for the DUBm activity. Whereas Sgf73 N-terminal ZnF is important for DUBm stability, Sgf11 C-terminal ZnF appears to be involved in DUBm function. To further characterize the role of these two zinc fingers, we have solved their structure by NMR. We show that, contrary to the previously reported structures, Sgf73 ZnF adopts a C2H2 coordination with unusual tautomeric forms for the coordinating histidines. We further report that the Sgf11 ZnF, but not the Sgf73 ZnF, binds to nucleosomal DNA with a binding interface composed of arginine residues located within the ZnF α-helix. Mutational analyses both in vitro and in vivo provide evidence for the functional relevance of our structural observations. The combined interpretation of our results leads to an uncommon ZnF-DNA interaction between the SAGA DUBm and nucleosomes, thus providing further functional insights into SAGA's epigenetic modulation of the chromatin structure.

  19. Identification and characterization of rabbit ROSA26 for gene knock-in and stable reporter gene expression

    PubMed Central

    Yang, Dongshan; Song, Jun; Zhang, Jifeng; Xu, Jie; Zhu, Tianqing; Wang, Zhong; Lai, Liangxue; Chen, Y. Eugene

    2016-01-01

    The laboratory rabbit has been a valuable model system for human disease studies. To make the rabbit model more amendable to targeted gene knockin and stable gene over-expression, we identified a rabbit orthologue of the mouse Rosa26 locus through genomic sequence homology analysis. Real-time PCR and 5′ RACE and 3′ RACE experiments revealed that this locus encodes two transcript variants of a long noncoding RNA (lncRNA) (rbRosaV1 and rbRosaV2). Both variants are expressed ubiquitously and stably in different tissues. We next targeted the rabbit Rosa26 (rbRosa26) locus using CRISPR/Cas9 and produced two lines of knock-in rabbits (rbRosa26-EGFP, and rbRosa26-Cre-reporter). In both lines, all the founders and their offspring appear healthy and reproduce normally. In F1 generation animals, the rbRosa26-EGFP rabbits express EGFP, and the rbRosa26-Cre-reporter rabbits express tdTomato ubiquitously in all the tissues examined. Furthermore, disruption of rbRosa26 locus does not adversely impact the animal health and reproduction. Therefore, our work establishes rbRosa26 as a safe harbor suitable for nuclease mediated gene targeting. The addition of rbRosa26 to the tool box of transgenic research is expected to allow diverse genetic manipulations, including gain-of function, conditional knock out and lineage-tracing studies in rabbits. PMID:27117226

  20. Identification of a cyclic-AMP-responsive element within the rat somatostatin gene.

    PubMed Central

    Montminy, M R; Sevarino, K A; Wagner, J A; Mandel, G; Goodman, R H

    1986-01-01

    We have examined the regulation of somatostatin gene expression by cAMP in PC12 rat pheochromocytoma cells transfected with the rat somatostatin gene. Forskolin at 10 microM caused a 4-fold increase in somatostatin mRNA levels within 4 hr of treatment in stably transfected cells. Chimeric genes containing the somatostatin gene promoter fused to the bacterial reporter gene encoding chloramphenicol acetyltransferase were also induced by cAMP in PC12 cells. To delineate the sequences required for response to cAMP, we constructed a series of promoter deletion mutants. Our studies defined a region between 60 and 29 base pairs upstream from the transcriptional initiation site that conferred cAMP responsiveness when placed adjacent to the simian virus 40 promoter. Within the cAMP-responsive element of the somatostatin gene, we observed an 8-base palindrome, 5'-TGACGTCA-3', which is highly conserved in many other genes whose expression is regulated by cAMP. cAMP responsiveness was greatly reduced when the somatostatin fusion genes were transfected into the mutant PC12 line A126-1B2, which is deficient in cAMP-dependent protein kinase 2. Our studies indicate that transcriptional regulation of the somatostatin gene by cAMP requires protein kinase 2 activity and may depend upon a highly conserved promoter element. Images PMID:2875459

  1. Gene transcription and electromagnetic fields. Final progress report

    SciTech Connect

    Henderson, A.S.

    1992-12-31

    Our overall aim is to obtain sufficient information to allow us to ultimately determine whether ELF EM field exposure is an initiating factor in neoplastic transformation and/or if exposure can mimic characteristics of the second-step counterpart in neoplastic disease. This aim is based on our previous findings that levels of some transcripts are increased in cells exposed to EM fields. While the research is basic in nature, the ramifications have bearing on the general safety of exposure to EM fields in industrial and everyday life. A large array of diverse biological effects are reported to occur as the result of exposure to elf EM fields, suggesting that the cell response to EM fields is at a basic level, presumably initiated by molecular and/or biophysical events at the cell membrane. The hypothesized route is a signal transduction pathway involving membrane calcium fluxes. Information flow resulting from signal transduction can mediate the induction of regulatory factors in the cell, and directly affect how transcription is regulated.

  2. Genetics and molecular biology of methanogen genes. Final report

    SciTech Connect

    Konisky, J.

    1997-10-07

    Adenylate kinase has been isolated from four related methanogenic members of the Archaea. For each the optimum temperature for enzyme activity was similar to the temperature for optimal microbial growth and was approximately 30 C for Methanococcus voltage, 70 C for Methanococcus thermolithotrophicus, 80 C for Methanococcus igneus and 80--90 C for Methanococcus jannaschii. The enzymes were sensitive to the adenylate kinase inhibitor, Ap{sub 5}A [P{sup 1}, P{sup 5}-di(adenosine-5{prime}) pentaphosphate], a property that was exploited to purify the enzymes by CIBACRON Blue affinity chromatography. The enzymes had an estimated molecular weight (approximately 23--25 kDa) in the range common for adenylate kinases. Each of the enzymes had a region of amino acid sequence close to its N-terminus that was similar to the canonical P-loop sequence reported for all adenylate kinases. However, the methanogen sequences lacked a lysine residue that has previously been found to be invariant in adenylate kinases including an enzyme isolated from the Archeon, Sulfolobus acidocaldarius. If verified as a nucleotide binding domain, the methanogen sequence would represent a novel nucleotide binding motif. There was no correlation between amino acid abundance and the optimal temperature for enzyme activity.

  3. High-throughput screening identifies novel inhibitors of the acetyltransferase activity of Escherichia coli GlmU.

    PubMed

    Pereira, Mark P; Blanchard, Jan E; Murphy, Cecilia; Roderick, Steven L; Brown, Eric D

    2009-06-01

    The bifunctional GlmU protein catalyzes the formation of UDP-N-acetylglucosamine in a two-step reaction using the substrates glucosamine-1-phosphate, acetyl coenzyme A, and UTP. This metabolite is a common precursor to the synthesis of bacterial cell surface carbohydrate polymers, such as peptidoglycan, lipopolysaccharide, and wall teichoic acid that are involved in the maintenance of cell shape, permeability, and virulence. The C-terminal acetyltransferase domain of GlmU exhibits structural and mechanistic features unique to bacterial UDP-N-acetylglucosamine synthases, making it an excellent target for antibacterial design. In the work described here, we have developed an absorbance-based assay to screen diverse chemical libraries in high throughput for inhibitors to the acetyltransferase reaction of Escherichia coli GlmU. The primary screen of 50,000 drug-like small molecules identified 63 hits, 37 of which were specific to acetyltransferase activity of GlmU. Secondary screening and mode-of-inhibition studies identified potent inhibitors where compound binding within the acetyltransferase active site was requisite on the presence of glucosamine-1-phosphate and were competitive with the substrate acetyl coenzyme A. These molecules may represent novel chemical scaffolds for future antimicrobial drug discovery. In addition, this work outlines the utility of catalytic variants in targeting specific activities of bifunctional enzymes in high-throughput screens.

  4. Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane

    PubMed Central

    2014-01-01

    Background Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene. Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. Results The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences. Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures. Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes. In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. Conclusions We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred

  5. A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines

    PubMed Central

    Pasumarthi, Ravi K.; Baridon, Brian; Djan, Esi; Trainor, Amanda; Griffey, Stephen M.; Engelhard, Eric K.; Rapp, Jared; Li, Bowen; de Jong, Pieter J.; Lloyd, K.C. Kent

    2015-01-01

    Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ∼80% of mutants showed specific staining in one or more tissues, while ∼20% showed no specific staining, ∼13% had staining in only one tissue, and ∼25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (∼50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known. PMID:25591789

  6. Role of human N-acetyltransferases, NAT1 or NAT2, in genotoxicity of nitroarenes and aromatic amines in Salmonella typhimurium NM6001 and NM6002.

    PubMed

    Oda, Y; Yamazaki, H; Shimada, T

    1999-06-01

    Human NAT1 and NAT2 genes were subcloned into pACYC184 vector and the plasmids thus obtained were introduced into Salmonella typhimurium O-acetyltransferase-deficient strain NM6000 (TA1538/1, 8-DNP/pSK1002), establishing new strains NM6001 and NM6002, respectively. We compared the sensitivities of these two strains with those of NM6000 towards carcinogenic nitroarenes and aromatic amines in the SOS/umu response. The induction of umuC gene expression by these chemicals in the presence and absence of the S9 fraction was assayed by measuring the cellular beta-galactosidase activity expressed by the umuC"lacZ fusion gene in the tester strains. 2-Nitrofluorene and 2-aminofluorene induced umuC gene expression more strongly in the NM6001 strain than in the NM6002 strain. In contrast, induction of umuC gene expression by 1, 8-dinitropyrene, 6-aminochrysene and 2-amino-3,5-dimethylimidazo[4, 5-f]quinoline was weaker in the NM6001 strain than in the NM6002 strain. 1-Nitropyrene, 2-amino-6-methyl-dipyrido[1,2-a:3', 2'-d]imidazole, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3-methyl-9H-pyrido[2,3-b]indole were found to induce umuC gene expression at similar extents in both strains. These results suggest that the newly developed strains can be employed for the studies on mechanisms of genotoxicity of a variety of nitroarenes and aromatic amines, along with the assessment of cancer risk to humans. PMID:10357791

  7. Characterization of tissue-specific transcription by the human synapsin I gene promoter

    SciTech Connect

    Thiel, G. Univ. of Texas, Dallas ); Greengard, P. ); Suedhof, T.C. )

    1991-04-15

    Synapsin Ia and synapsin Ib are abundant synaptic vesicle proteins that are derived by differential splicing from a single gene. To identify control elements directing the neuronal expression of synapsins Ia/b, the authors functionally analyzed the promoter region of the human synapsin I gene. A hybrid gene was constructed containing 2 kilobases of 5{prime} flanking sequence from the synapsin I gene fused to the bacterial gene chloramphenicol acetyltransferase and transfected into 12 different neuronal and nonneuronal cell lines. In general, expression of the chimeric reporter gene showed excellent correlation with endogenous expression of synapsin I in different neuronal cell lines, whereas transcription was low in all nonneuronal cell lines examined. The addition of the simian virus 40 enhancer promoted non-tissue-specific expression. Deletion mutagenesis of the synapsin I promoter revealed the presence of positive and negative sequence elements. A basal (constitutive) promoter that directs reporter gene expression in neuronal and nonneuronal cell lines was mapped to the region {minus}115 to +47. The promoter region from {minus}422 to {minus}22 contains positive elements that upon fusion with the herpes simplex virus thymidine kinase promoter potentiate its transcription in PC12 and neuroblastoma cells but not in Chinese hamster ovary cells.

  8. Alpha-fetoprotein-targeted reporter gene expression imaging in hepatocellular carcinoma

    PubMed Central

    Kim, Kwang Il; Chung, Hye Kyung; Park, Ju Hui; Lee, Yong Jin; Kang, Joo Hyun

    2016-01-01

    Hepatocellular carcinoma (HCC) is one of the most common cancers in Eastern Asia, and its incidence is increasing globally. Numerous experimental models have been developed to better our understanding of the pathogenic mechanism of HCC and to evaluate novel therapeutic approaches. Molecular imaging is a convenient and up-to-date biomedical tool that enables the visualization, characterization and quantification of biologic processes in a living subject. Molecular imaging based on reporter gene expression, in particular, can elucidate tumor-specific events or processes by acquiring images of a reporter gene’s expression driven by tumor-specific enhancers/promoters. In this review, we discuss the advantages and disadvantages of various experimental HCC mouse models and we present in vivo images of tumor-specific reporter gene expression driven by an alpha-fetoprotein (AFP) enhancer/promoter system in a mouse model of HCC. The current mouse models of HCC development are established by xenograft, carcinogen induction and genetic engineering, representing the spectrum of tumor-inducing factors and tumor locations. The imaging analysis approach of reporter genes driven by AFP enhancer/promoter is presented for these different HCC mouse models. Such molecular imaging can provide longitudinal information about carcinogenesis and tumor progression. We expect that clinical application of AFP-targeted reporter gene expression imaging systems will be useful for the detection of AFP-expressing HCC tumors and screening of increased/decreased AFP levels due to disease or drug treatment. PMID:27468205

  9. Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines

    PubMed Central

    Sandy, James; Mushtaq, Adeel; Holton, Simon J.; Schartau, Pamela; Noble, Martin E. M.; Sim, Edith

    2005-01-01

    The NATs (arylamine N-acetyltransferases) are a well documented family of enzymes found in both prokaryotes and eukaryotes. NATs are responsible for the acetylation of a range of arylamine, arylhydrazine and hydrazine compounds. We present here an investigation into the catalytic triad of residues (Cys-His-Asp) and other structural features of NATs using a variety of methods, including site-directed mutagenesis, X-ray crystallography and bioinformatics analysis, in order to investigate whether each of the residues of the catalytic triad is essential for catalytic activity. The catalytic triad of residues, Cys-His-Asp, is a well defined motif present in several families of enzymes. We mutated each of the catalytic residues in turn to investigate the role they play in catalysis. We also mutated a key residue, Gly126, implicated in acetyl-CoA binding, to examine the effects on acetylation activity. In addition, we have solved the structure of a C70Q mutant of Mycobacterium smegmatis NAT to a resolution of 1.45 Å (where 1 Å=0.1 nm). This structure confirms that the mutated protein is correctly folded, and provides a structural model for an acetylated NAT intermediate. Our bioinformatics investigation analysed the extent of sequence conservation between all eukaryotic and prokaryotic NAT enzymes for which sequence data are available. This revealed several new sequences, not yet reported, of NAT paralogues. Together, these studies have provided insight into the fundamental core of NAT enzymes, and the regions where sequence differences account for the functional diversity of this family. We have confirmed that each of the three residues of the triad is essential for acetylation activity. PMID:15869465

  10. FOXP2 gene deletion and infant feeding difficulties: a case report.

    PubMed

    Zimmerman, Emily; Maron, Jill L

    2016-01-01

    Forkhead box protein P2 (FOXP2) is a well-studied gene known to play an essential role in normal speech development. Deletions in the gene have been shown to result in developmental speech disorders and regulatory disruption of downstream gene targets associated with common forms of language impairments. Despite similarities in motor planning and execution between speech development and oral feeding competence, there have been no reports to date linking deletions within the FOXP2 gene to oral feeding impairments in the newborn. The patient was a nondysmorphic, appropriately and symmetrically grown male infant born at 35-wk gestational age. He had a prolonged neonatal intensive care unit stay because of persistent oral feeding incoordination requiring gastrostomy tube placement. Cardiac and neurological imagings were within normal limits. A microarray analysis found an ∼9-kb loss within chromosome band 7q3.1 that contains exon 2 of FOXP2, demonstrating a single copy of this region instead of the normal two copies per diploid gene. This case study expands our current understanding of the role FOXP2 exerts on motor planning and coordination necessary for both oral feeding success and speech-language development. This case report has important consequences for future diagnosis and treatment for infants with FOXP2 deletions, mutations, and varying levels of gene expression.

  11. FOXP2 gene deletion and infant feeding difficulties: a case report

    PubMed Central

    Zimmerman, Emily; Maron, Jill L.

    2016-01-01

    Forkhead box protein P2 (FOXP2) is a well-studied gene known to play an essential role in normal speech development. Deletions in the gene have been shown to result in developmental speech disorders and regulatory disruption of downstream gene targets associated with common forms of language impairments. Despite similarities in motor planning and execution between speech development and oral feeding competence, there have been no reports to date linking deletions within the FOXP2 gene to oral feeding impairments in the newborn. The patient was a nondysmorphic, appropriately and symmetrically grown male infant born at 35-wk gestational age. He had a prolonged neonatal intensive care unit stay because of persistent oral feeding incoordination requiring gastrostomy tube placement. Cardiac and neurological imagings were within normal limits. A microarray analysis found an ∼9-kb loss within chromosome band 7q3.1 that contains exon 2 of FOXP2, demonstrating a single copy of this region instead of the normal two copies per diploid gene. This case study expands our current understanding of the role FOXP2 exerts on motor planning and coordination necessary for both oral feeding success and speech–language development. This case report has important consequences for future diagnosis and treatment for infants with FOXP2 deletions, mutations, and varying levels of gene expression. PMID:27148578

  12. Mesoscopic tomography imaging of reporter genes in thick printed tissue constructs

    NASA Astrophysics Data System (ADS)

    Ozturk, Mehmet S.; Lee, Vivian K.; Zhao, Lingling; Dai, Guohoa; Intes, Xavier

    2013-06-01

    We report an application of Mesoscopic Fluorescence Molecular Tomography to 3D tissue engineering construct. Engineered thick tissue was hosting two 3D printed vasculatures. The channels were formed by live cells, expressing GFP and mCherry reporter genes, embedded in 3mm turbid media. Tissue and cells kept in a 3mm thick perfusion chamber during the entire imaging process which took less than 5 minutes.

  13. AMINOGLYCOSIDE RESISTANCE GENES IN Pseudomonas aeruginosa ISOLATES FROM CUMANA, VENEZUELA

    PubMed Central

    TEIXEIRA, Bertinellys; RODULFO, Hectorina; CARREÑO, Numirin; GUZMÁN, Militza; SALAZAR, Elsa; DONATO, Marcos DE

    2016-01-01

    The enzymatic modification of aminoglycosides by aminoglycoside-acetyltransferases (AAC), aminoglycoside-adenyltransferases (AAD), and aminoglycoside-phosphotransferases (APH), is the most common resistance mechanism in P. aeruginosa and these enzymes can be coded on mobile genetic elements that contribute to their dispersion. One hundred and thirty seven P. aeruginosa isolates from the University Hospital, Cumana, Venezuela (HUAPA) were evaluated. Antimicrobial susceptibility was determined by the disk diffusion method and theaac, aadB and aph genes were detected by PCR. Most of the P. aeruginosa isolates (33/137) were identified from the Intensive Care Unit (ICU), mainly from discharges (96/137). The frequency of resistant P. aeruginosaisolates was found to be higher for the aminoglycosides tobramycin and amikacin (30.7 and 29.9%, respectively). Phenotype VI, resistant to these antibiotics, was the most frequent (14/49), followed by phenotype I, resistant to all the aminoglycosides tested (12/49). The aac(6´)-Ib,aphA1 and aadB genes were the most frequently detected, and the simultaneous presence of several resistance genes in the same isolate was demonstrated. Aminoglycoside resistance in isolates ofP. aeruginosa at the HUAPA is partly due to the presence of the aac(6´)-Ib, aphA1 andaadB genes, but the high rates of antimicrobial resistance suggest the existence of several mechanisms acting together. This is the first report of aminoglycoside resistance genes in Venezuela and one of the few in Latin America. PMID:27007556

  14. AMINOGLYCOSIDE RESISTANCE GENES IN Pseudomonas aeruginosa ISOLATES FROM CUMANA, VENEZUELA.

    PubMed

    Teixeira, Bertinellys; Rodulfo, Hectorina; Carreño, Numirin; Guzmán, Militza; Salazar, Elsa; De Donato, Marcos

    2016-01-01

    The enzymatic modification of aminoglycosides by aminoglycoside-acetyltransferases (AAC), aminoglycoside-adenyltransferases (AAD), and aminoglycoside-phosphotransferases (APH), is the most common resistance mechanism in P. aeruginosa and these enzymes can be coded on mobile genetic elements that contribute to their dispersion. One hundred and thirty seven P. aeruginosa isolates from the University Hospital, Cumana, Venezuela (HUAPA) were evaluated. Antimicrobial susceptibility was determined by the disk diffusion method and theaac, aadB and aph genes were detected by PCR. Most of the P. aeruginosa isolates (33/137) were identified from the Intensive Care Unit (ICU), mainly from discharges (96/137). The frequency of resistant P. aeruginosaisolates was found to be higher for the aminoglycosides tobramycin and amikacin (30.7 and 29.9%, respectively). Phenotype VI, resistant to these antibiotics, was the most frequent (14/49), followed by phenotype I, resistant to all the aminoglycosides tested (12/49). The aac(6´)-Ib,aphA1 and aadB genes were the most frequently detected, and the simultaneous presence of several resistance genes in the same isolate was demonstrated. Aminoglycoside resistance in isolates ofP. aeruginosa at the HUAPA is partly due to the presence of the aac(6´)-Ib, aphA1 andaadB genes, but the high rates of antimicrobial resistance suggest the existence of several mechanisms acting together. This is the first report of aminoglycoside resistance genes in Venezuela and one of the few in Latin America. PMID:27007556

  15. AMINOGLYCOSIDE RESISTANCE GENES IN Pseudomonas aeruginosa ISOLATES FROM CUMANA, VENEZUELA.

    PubMed

    Teixeira, Bertinellys; Rodulfo, Hectorina; Carreño, Numirin; Guzmán, Militza; Salazar, Elsa; De Donato, Marcos

    2016-01-01

    The enzymatic modification of aminoglycosides by aminoglycoside-acetyltransferases (AAC), aminoglycoside-adenyltransferases (AAD), and aminoglycoside-phosphotransferases (APH), is the most common resistance mechanism in P. aeruginosa and these enzymes can be coded on mobile genetic elements that contribute to their dispersion. One hundred and thirty seven P. aeruginosa isolates from the University Hospital, Cumana, Venezuela (HUAPA) were evaluated. Antimicrobial susceptibility was determined by the disk diffusion method and theaac, aadB and aph genes were detected by PCR. Most of the P. aeruginosa isolates (33/137) were identified from the Intensive Care Unit (ICU), mainly from discharges (96/137). The frequency of resistant P. aeruginosaisolates was found to be higher for the aminoglycosides tobramycin and amikacin (30.7 and 29.9%, respectively). Phenotype VI, resistant to these antibiotics, was the most frequent (14/49), followed by phenotype I, resistant to all the aminoglycosides tested (12/49). The aac(6´)-Ib,aphA1 and aadB genes were the most frequently detected, and the simultaneous presence of several resistance genes in the same isolate was demonstrated. Aminoglycoside resistance in isolates ofP. aeruginosa at the HUAPA is partly due to the presence of the aac(6´)-Ib, aphA1 andaadB genes, but the high rates of antimicrobial resistance suggest the existence of several mechanisms acting together. This is the first report of aminoglycoside resistance genes in Venezuela and one of the few in Latin America.

  16. Circadian dynamics of the cone-rod homeobox (CRX) transcription factor in the rat pineal gland and its role in regulation of arylalkylamine N-acetyltransferase (AANAT).

    PubMed

    Rohde, Kristian; Rovsing, Louise; Ho, Anthony K; Møller, Morten; Rath, Martin F

    2014-08-01

    The cone-rod homeobox (Crx) gene encodes a transcription factor in the retina and pineal gland. Crx deficiency influences the pineal transcriptome, including a reduced expression of arylalkylamine N-acetyltransferase (Aanat), a key enzyme in nocturnal pineal melatonin production. However, previous functional studies on pineal Crx have been performed in melatonin-deficient mice. In this study, we have investigated the role of Crx in the melatonin-proficient rat pineal gland. The current study shows that pineal Crx transcript levels exhibit a circadian rhythm with a peak in the middle of the night, which is transferred into daily changes in CRX protein. The study further shows that the sympathetic innervation of the pineal gland controls the Crx rhythm. By use of adenovirus-mediated short hairpin RNA gene knockdown targeting Crx mRNA in primary rat pinealocyte cell culture, we here show that intact levels of Crx mRNA are required to obtain high levels of Aanat expression, whereas overexpression of Crx induces Aanat transcription in vitro. This regulatory function of Crx is further supported by circadian analysis of Aanat in the pineal gland of the Crx-knockout mouse. Our data indicate that the rhythmic nature of pineal CRX protein may directly modulate the daily profile of Aanat expression by inducing nighttime expression of this enzyme, thus facilitating nocturnal melatonin synthesis in addition to its role in ensuring a correct tissue distribution of Aanat expression.

  17. Snf1p-dependent Spt-Ada-Gcn5-acetyltransferase (SAGA) recruitment and chromatin remodeling activities on the HXT2 and HXT4 promoters.

    PubMed

    van Oevelen, Chris J C; van Teeffelen, Hetty A A M; van Werven, Folkert J; Timmers, H Th Marc

    2006-02-17

    We previously showed that the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex is recruited to the activated HXT2 and HXT4 genes and plays a role in the association of TBP-associated factors. Using the HXT2 and HXT4 genes, we now present evidence for a functional link between Snf1p-dependent activation, recruitment of the SAGA complex, histone H3 removal, and H3 acetylation. Recruitment of the SAGA complex is dependent on the release of Ssn6p-Tup1p repression by Snf1p. In addition, we found that the Gcn5p subunit of the SAGA complex preferentially acetylates histone H3K18 on the HXT2 and HXT4 promoters and that Gcn5p activity is required for removal of histone H3 from the HXT4 promoter TATA region. In contrast, histone H3 removal from the HXT2 promoter does not require Gcn5p. In conclusion, although similar protein complexes are involved, induction of HXT2 and HXT4 displays important mechanistic differences.

  18. Safety evaluation of the phosphinothricin acetyltransferase proteins encoded by the pat and bar sequences that confer tolerance to glufosinate-ammonium herbicide in transgenic plants.

    PubMed

    Hérouet, Corinne; Esdaile, David J; Mallyon, Bryan A; Debruyne, Eric; Schulz, Arno; Currier, Thomas; Hendrickx, Koen; van der Klis, Robert-Jan; Rouan, Dominique

    2005-03-01

    Transgenic plant varieties, which are tolerant to glufosinate-ammonium, were developed. The herbicide tolerance is based upon the presence of either the bar or the pat gene, which encode for two homologous phosphinothricin acetyltransferases (PAT), in the plant genome. Based on both a review of published literature and experimental studies, the safety assessment reviews the first step of a two-step-approach for the evaluation of the safety of the proteins expressed in plants. It can be used to support the safety of food or feed products derived from any crop that contains and expresses these PAT proteins. The safety evaluation supports the conclusion that the genes and the donor microorganisms (Streptomyces) are innocuous. The PAT enzymes are highly specific and do not possess the characteristics associated with food toxins or allergens, i.e., they have no sequence homology with any known allergens or toxins, they have no N-glycosylation sites, they are rapidly degraded in gastric and intestinal fluids, and they are devoid of adverse effects in mice after intravenous administration at a high dose level. In conclusion, there is a reasonable certainty of no harm resulting from the inclusion of the PAT proteins in human food or in animal feed.

  19. Unusual regioversatility of acetyltransferase Eis, a cause of drug resistance in XDR-TB

    SciTech Connect

    Chen, Wenjing; Biswas, Tapan; Porter, Vanessa R.; Tsodikov, Oleg V.; Garneau-Tsodikova, Sylvie

    2011-09-06

    The emergence of multidrug-resistant and extensively drug-resistant (XDR) tuberculosis (TB) is a serious global threat. Aminoglycoside antibiotics are used as a last resort to treat XDR-TB. Resistance to the aminoglycoside kanamycin is a hallmark of XDR-TB. Here, we reveal the function and structure of the mycobacterial protein Eis responsible for resistance to kanamycin in a significant fraction of kanamycin-resistant Mycobacterium tuberculosis clinical isolates. We demonstrate that Eis has an unprecedented ability to acetylate multiple amines of many aminoglycosides. Structural and mutagenesis studies of Eis indicate that its acetylation mechanism is enabled by a complex tripartite fold that includes two general control non-derepressible 5 (GCN5)-related N-acetyltransferase regions. An intricate negatively charged substrate-binding pocket of Eis is a potential target of new antitubercular drugs expected to overcome aminoglycoside resistance.

  20. Comparison of Protein Acetyltransferase Action of CRTAase with the Prototypes of HAT

    PubMed Central

    Ponnan, Prija; Kumar, Ajit; Singh, Prabhjot; Gupta, Prachi; Joshi, Rini; Saso, Luciano; Prasad, Ashok K.; Rastogi, Ramesh C.; Parmar, Virinder S.; Raj, Hanumantharao G.

    2014-01-01

    Our laboratory is credited for the discovery of enzymatic acetylation of protein, a phenomenon unknown till we identified an enzyme termed acetoxy drug: protein transacetylase (TAase), catalyzing the transfer of acetyl group from polyphenolic acetates to receptor proteins (RP). Later, TAase was identified as calreticulin (CR), an endoplasmic reticulum luminal protein. CR was termed calreticulin transacetylase (CRTAase). Our persistent study revealed that CR like other families of histone acetyltransferases (HATs) such as p300, Rtt109, PCAF, and ESA1, undergoes autoacetylation. The autoacetylated CR was characterized as a stable intermediate in CRTAase catalyzed protein acetylation, and similar was the case with ESA1. The autoacetylation of CR like that of HATs was found to enhance protein-protein interaction. CR like HAT-1, CBP, and p300 mediated the acylation of RP utilizing acetyl CoA and propionyl CoA as the substrates. The similarities between CRTAase and HATs in mediating protein acylation are highlighted in this review. PMID:24688408

  1. Effects of acute ethanol administration on nocturnal pineal serotonin N-acetyltransferase activity

    SciTech Connect

    Creighton, J.A.; Rudeen, P.K.

    1988-01-01

    The effect of acute ethanol administration on pineal serotonin N-acetyltransferase (NAT) activity, norepinephrine and indoleamine content was examined in male rats. When ethanol was administered in two equal doses (2 g/kg body weight) over a 4 hour period during the light phase, the nocturnal rise in NAT activity was delayed by seven hours. The nocturnal pineal norepinephrine content was not altered by ethanol except for a delay in the reduction of NE with the onset of the following light phase. Although ethanol treatment led to a significant reduction in nocturnal levels of pineal serotonin content, there was no significant effect upon pineal content of 5-hydroxyindoleacetic acid (5-HIAA). The data indicate that ethanol delays the onset of the rise of nocturnal pineal NAT activity.

  2. Structure of homoserine O-acetyltransferase from Staphylococcus aureus: the first Gram-positive ortholog structure

    PubMed Central

    Thangavelu, Bharani; Pavlovsky, Alexander G.; Viola, Ronald

    2014-01-01

    Homoserine O-acetyltransferase (HTA) catalyzes the formation of l-O-acetyl-homoserine from l-homoserine through the transfer of an acetyl group from acetyl-CoA. This is the first committed step required for the biosynthesis of methionine in many fungi, Gram-positive bacteria and some Gram-negative bacteria. The structure of HTA from Staphylococcus aureus (SaHTA) has been determined to a resolution of 2.45 Å. The structure belongs to the α/β-hydrolase superfamily, consisting of two distinct domains: a core α/β-domain containing the catalytic site and a lid domain assembled into a helical bundle. The active site consists of a classical catalytic triad located at the end of a deep tunnel. Structure analysis revealed some important differences for SaHTA compared with the few known structures of HTA. PMID:25286936

  3. Structure of homoserine O-acetyltransferase from Staphylococcus aureus: the first Gram-positive ortholog structure.

    PubMed

    Thangavelu, Bharani; Pavlovsky, Alexander G; Viola, Ronald

    2014-10-01

    Homoserine O-acetyltransferase (HTA) catalyzes the formation of L-O-acetyl-homoserine from L-homoserine through the transfer of an acetyl group from acetyl-CoA. This is the first committed step required for the biosynthesis of methionine in many fungi, Gram-positive bacteria and some Gram-negative bacteria. The structure of HTA from Staphylococcus aureus (SaHTA) has been determined to a resolution of 2.45 Å. The structure belongs to the α/β-hydrolase superfamily, consisting of two distinct domains: a core α/β-domain containing the catalytic site and a lid domain assembled into a helical bundle. The active site consists of a classical catalytic triad located at the end of a deep tunnel. Structure analysis revealed some important differences for SaHTA compared with the few known structures of HTA.

  4. Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organisms.

    PubMed

    Rathore, Om Singh; Faustino, Alexandra; Prudêncio, Pedro; Van Damme, Petra; Cox, Cymon J; Martinho, Rui Gonçalo

    2016-02-10

    Protein N-terminal acetylation is an ancient and ubiquitous co-translational modification catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). Prokaryotes have at least 3 NATs, whereas humans have six distinct but highly conserved NATs, suggesting an increase in regulatory complexity of this modification during eukaryotic evolution. Despite this, and against our initial expectations, we determined that NAT diversification did not occur in the eukaryotes, as all six major human NATs were most likely present in the Last Eukaryotic Common Ancestor (LECA). Furthermore, we also observed that some NATs were actually secondarily lost during evolution of major eukaryotic lineages; therefore, the increased complexity of the higher eukaryotic proteome occurred without a concomitant diversification of NAT complexes.

  5. Coenzyme A Binding to the Aminoglycoside Acetyltransferase (3)-IIIb Increases Conformational Sampling of Antibiotic Binding Site

    SciTech Connect

    Hu, Xiaohu; Norris, Adrianne; Baudry, Jerome Y; Serpersu, Engin H

    2011-01-01

    NMR spectroscopy experiments and molecular dynamics simulations were performed to describe the dynamic properties of the aminoglycoside acetyltransferase (3)-IIIb (AAC) in its apo and coenzyme A (CoASH) bound forms. The {sup 15}N-{sup 1}H HSQC spectra indicate a partial structural change and coupling of the CoASH binding site with another region in the protein upon the CoASH titration into the apo enzyme. Molecular dynamics simulations indicate a significant structural and dynamic variation of the long loop in the antibiotic binding domain in the form of a relatively slow (250 ns), concerted opening motion in the CoASH enzyme complex and that binding of the CoASH increases the structural flexibility of the loop, leading to an interchange between several similar equally populated conformations.

  6. Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organisms

    PubMed Central

    Rathore, Om Singh; Faustino, Alexandra; Prudêncio, Pedro; Van Damme, Petra; Cox, Cymon J.; Martinho, Rui Gonçalo

    2016-01-01

    Protein N-terminal acetylation is an ancient and ubiquitous co-translational modification catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). Prokaryotes have at least 3 NATs, whereas humans have six distinct but highly conserved NATs, suggesting an increase in regulatory complexity of this modification during eukaryotic evolution. Despite this, and against our initial expectations, we determined that NAT diversification did not occur in the eukaryotes, as all six major human NATs were most likely present in the Last Eukaryotic Common Ancestor (LECA). Furthermore, we also observed that some NATs were actually secondarily lost during evolution of major eukaryotic lineages; therefore, the increased complexity of the higher eukaryotic proteome occurred without a concomitant diversification of NAT complexes. PMID:26861501

  7. Interferon-Induced Spermidine-Spermine Acetyltransferase and Polyamine Depletion Restrict Zika and Chikungunya Viruses.

    PubMed

    Mounce, Bryan C; Poirier, Enzo Z; Passoni, Gabriella; Simon-Loriere, Etienne; Cesaro, Teresa; Prot, Matthieu; Stapleford, Kenneth A; Moratorio, Gonzalo; Sakuntabhai, Anavaj; Levraud, Jean-Pierre; Vignuzzi, Marco

    2016-08-10

    Polyamines are small, positively charged molecules derived from ornithine and synthesized through an intricately regulated enzymatic pathway. Within cells, they are abundant and play several roles in diverse processes. We find that polyamines are required for the life cycle of the RNA viruses chikungunya virus (CHIKV) and Zika virus (ZIKV). Depletion of spermidine and spermine via type I interferon signaling-mediated induction of spermidine/spermine N1-acetyltransferase (SAT1), a key catabolic enzyme in the polyamine pathway, restricts CHIKV and ZIKV replication. Polyamine depletion restricts these viruses in vitro and in vivo, due to impairment of viral translation and RNA replication. The restriction is released by exogenous replenishment of polyamines, further supporting a role for these molecules in virus replication. Thus, SAT1 and, more broadly, polyamine depletion restrict viral replication and suggest promising avenues for antiviral therapies.

  8. Primary structure of the human M2 mitochondrial autoantigen of primary biliary cirrhosis: Dihydrolipoamide acetyltransferase

    SciTech Connect

    Coppel, R.L.; McNeilage, L.J.; Surh, C.D.; Van De Water, J.; Spithill, T.W.; Whittingham, S.; Gershwin, M.E. )

    1988-10-01

    Primary biliary cirrhosis is a chronic, destructive autoimmune liver disease of humans. Patient sera are characterized by a high frequency of autoantibodies to a M{sub r} 70,000 mitochondrial antigen a component of the M2 antigen complex. The authors have identified a human cDNA clone encoding the complete amino acid sequence of this autoantigen. The predicted structure has significant similarity with the dihydrolipoamide acetyltransferase of the Escherichia coli pyruvate dehydrogenase multienzyme complex. The human sequence preserves the Glu-Thr-Asp-Lys-Ala motif of the lipoyl-binding site and has two potential binding sites. Expressed fragments of the cDNA react strongly with sera from patients with primary biliary cirrhosis but not with sera from patients with autoimmune chronic active hepatitis or sera from healthy subjects.

  9. Mapping Lysine Acetyltransferase-Ligand Interactions by Activity-Based Capture.

    PubMed

    Montgomery, D C; Meier, J L

    2016-01-01

    Changes in reversible protein acetylation mediate many key aspects of genomic regulation and enzyme function. The catalysts for this posttranslational modification, lysine acetyltransferases (KATs), have been difficult targets for characterization due to their complex architecture and challenging reconstitution. To address this challenge, here we describe methods to profile endogenous KAT activities using activity-based probes. This method facilitates the targeted analysis of several cellular KATs and can be used to study their interactions with many different types of ligands, including acyl-CoA metabolites. This competitive activity-based capture approach provides a method to assess the selectivity of ligands for different KAT families in complex proteomic settings, and thus has the potential to offer substantial insights into the regulation of cellular KAT function. PMID:27423859

  10. WUS and STM-based reporter genes for studying meristem development in poplar

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5’ flanking regions of close homologs were used to drive expression o...

  11. Structural and functional characterization of TRI3 trichothecene 15-O-acetyltransferase from Fusarium sporotrichioides

    PubMed Central

    Garvey, Graeme S; McCormick, Susan P; Alexander, Nancy J; Rayment, Ivan

    2009-01-01

    Fusarium head blight is a devastating disease of cereal crops whose worldwide incidence is increasing and at present there is no satisfactory way of combating this pathogen or its associated toxins. There is a wide variety of trichothecene mycotoxins and they all contain a 12,13-epoxytrichothecene skeleton but differ in their substitutions. Indeed, there is considerable variation in the toxin profile across the numerous Fusarium species that has been ascribed to differences in the presence or absence of biosynthetic enzymes and their relative activity. This article addresses the source of differences in acetylation at the C15 position of the trichothecene molecule. Here, we present the in vitro structural and biochemical characterization of TRI3, a 15-O-trichothecene acetyltransferase isolated from F. sporotrichioides and the “in vivo” characterization of Δtri3 mutants of deoxynivalenol (DON) producing F. graminearum strains. A kinetic analysis shows that TRI3 is an efficient enzyme with the native substrate, 15-decalonectrin, but is inactive with either DON or nivalenol. The structure of TRI3 complexed with 15-decalonectrin provides an explanation for this specificity and shows that Tri3 and Tri101 (3-O-trichothecene acetyltransferase) are evolutionarily related. The active site residues are conserved across all sequences for TRI3 orthologs, suggesting that differences in acetylation at C15 are not due to differences in Tri3. The tri3 deletion mutant shows that acetylation at C15 is required for DON biosynthesis even though DON lacks a C15 acetyl group. The enzyme(s) responsible for deacetylation at the 15 position of the trichothecene mycotoxins have not been identified. PMID:19319932

  12. Histone acetyltransferase inhibitor CPTH6 preferentially targets lung cancer stem-like cells

    PubMed Central

    Di Martile, Marta; Desideri, Marianna; De Luca, Teresa; Gabellini, Chiara; Buglioni, Simonetta; Eramo, Adriana; Sette, Giovanni; Milella, Michele; Rotili, Dante; Mai, Antonello; Carradori, Simone; Secci, Daniela; De Maria, Ruggero; Del Bufalo, Donatella; Trisciuoglio, Daniela

    2016-01-01

    Cancer stem cells (CSCs) play an important role in tumor initiation, progression, therapeutic failure and tumor relapse. In this study, we evaluated the efficacy of the thiazole derivative 3-methylcyclopentylidene-[4-(4′-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6), a novel pCAF and Gcn5 histone acetyltransferase inhibitor, as a small molecule that preferentially targets lung cancer stem-like cells (LCSCs) derived from non-small cell lung cancer (NSCLC) patients. Notably, although CPTH6 inhibits the growth of both LCSC and NSCLC cell lines, LCSCs exhibit greater growth inhibition than established NSCLC cells. Growth inhibitory effect of CPTH6 in LCSC lines is primarily due to apoptosis induction. Of note, differentiated progeny of LCSC lines is more resistant to CPTH6 in terms of loss of cell viability and reduction of protein acetylation, when compared to their undifferentiated counterparts. Interestingly, in LCSC lines CPTH6 treatment is also associated with a reduction of stemness markers. By using different HAT inhibitors we provide clear evidence that inhibition of HAT confers a strong preferential inhibitory effect on cell viability of undifferentiated LCSC lines when compared to their differentiated progeny. In vivo, CPTH6 is able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Strikingly, the ability of CPTH6 to inhibit tubulin acetylation is also confirmed in vivo. Overall, our studies propose histone acetyltransferase inhibition as an attractive target for cancer therapy of NSCLC. PMID:26870991

  13. Structural and functional characterization of TRI3 trichothecene 15-O-acetyltransferase from Fusarium sporotrichioides

    SciTech Connect

    Garvey, Graeme S.; McCormick, Susan P.; Alexander, Nancy J.; Rayment, Ivan

    2009-08-14

    Fusarium head blight is a devastating disease of cereal crops whose worldwide incidence is increasing and at present there is no satisfactory way of combating this pathogen or its associated toxins. There is a wide variety of trichothecene mycotoxins and they all contain a 12,13-epoxytrichothecene skeleton but differ in their substitutions. Indeed, there is considerable variation in the toxin profile across the numerous Fusarium species that has been ascribed to differences in the presence or absence of biosynthetic enzymes and their relative activity. This article addresses the source of differences in acetylation at the C15 position of the trichothecene molecule. Here, we present the in vitro structural and biochemical characterization of TRI3, a 15-O-trichothecene acetyltransferase isolated from F. sporotrichioides and the 'in vivo' characterization of Deltatri3 mutants of deoxynivalenol (DON) producing F. graminearum strains. A kinetic analysis shows that TRI3 is an efficient enzyme with the native substrate, 15-decalonectrin, but is inactive with either DON or nivalenol. The structure of TRI3 complexed with 15-decalonectrin provides an explanation for this specificity and shows that Tri3 and Tri101 (3-O-trichothecene acetyltransferase) are evolutionarily related. The active site residues are conserved across all sequences for TRI3 orthologs, suggesting that differences in acetylation at C15 are not due to differences in Tri3. The tri3 deletion mutant shows that acetylation at C15 is required for DON biosynthesis even though DON lacks a C15 acetyl group. The enzyme(s) responsible for deacetylation at the 15 position of the trichothecene mycotoxins have not been identified.

  14. Relation of pontine choline acetyltransferase immunoreactive neurons with cells which increase discharge during REM sleep.

    PubMed

    Shiromani, P J; Armstrong, D M; Bruce, G; Hersh, L B; Groves, P M; Gillin, J C

    1987-03-01

    The purpose of this study was to determine whether neurons in the medial pontine reticular formation with high discharge rates during REM sleep could be localized in regions of the brainstem having neurons displaying choline acetyltransferase immunoreactivity. Six cats were implanted with sleep recording electrodes and microwires to record extracellular potentials of neurons in the pontine reticular formation. Single-units with a S:N ratio greater than 2:1 were recorded for at least two REM sleep cycles. A total of 49 units was recorded from the pontine reticular formation at medial-lateral planes ranging from 0.8 to 3.7 mm. The greatest proportion of the units (28.6%) showed highest discharge during active waking and phasic REM sleep compared to quiet waking, non-REM sleep, transition into REM sleep or quiet REM sleep periods. A percentage (20.4%) of the cells had high discharge associated with phasic REM sleep periods while 8.2% of the cells showed a progressive increase in discharge from waking to REM sleep. Subsequent examination of the distribution of choline acetyltransferase immunoreactive cells in the PRF revealed that cells showing high discharge during REM sleep were not localized near presumed cholinergic neurons. Indeed, we did not find any ChAT immunoreactive somata in the medial PRF, an area which has traditionally been implicated in the generation of REM sleep. These results suggest that while increased discharge of PRF cells may be instrumental to REM sleep generation, these cells are not cholinergic.

  15. Highly sensitive umu test system for the detection of mutagenic nitroarenes in Salmonella typhimurium NM3009 having high O-acetyltransferase and nitroreductase activities

    SciTech Connect

    Oda, Yoshimitsu; Yamazaki, Hiroshi; Watanabe, Masahiko; Nohmi, Takehiko; Shimada, Tsutomu )

    1993-01-01

    A highly sensitive umu test system for the detection of genotoxic activities of a variety of mutagenic nitroarenes has been developed using a new tester strain, Salmonella typhimurium NM3009 having high O-acetyltransferase (O-AT) and nitroreductase (NR) activities. The NM3009 was constructed by subcloning both the O-AT and NR genes into plasmid vector pACYC184, and the resulting plasmid was introduced into the parent tester strain S. typhimurium TA1535/pSK1002 harboring an umuC[prime]-[prime]lacZ fusion gene. The induction of umuC gene expression could be monitored by measuring the cellular [beta]-galactosidase activity produced by fusion gene. The purpose of the study was to evaluate whether the newly developed strain NM3009 is highly sensitive toward nitroarene compounds. The sensitivity of the strain NM3009 was compared with those of the parent TA1535/pSK1002 strain, the NR-overexpressing strain NM1011, the NR-deficient strain NM1000, the O-AT-overexpressing strain NM2009, and the O-AT-defective strain NM2000. The newly developed NM3009 strain had about 13-fold and 3-fold higher activities for N-AT and NR, respectively, than the original S. typhimurium TA1535/pSK1002 strain. Among six strains tested, NM3009 showed the highest sensitivity toward such chemicals as 1-nitronaphthalene, 2-nitrofluorene, 3,7-dinitro-fluoranthene, 3-nitrofluoranthene, 5-nitroacenaphthene, 2-nitronaphthalene, 1-nitropyrene, 1,6-dinitropyrene, 3,9-dinitrofluoranthene, 4,4[prime]-dinitrobiphenyl, 1,8-dinitropyrene, m-dinitrobenzene, 2,4-dinitrotoluene, and 1,3-dinitropyrene. The authors have also found that the order of sensitivities to induce umuC gene expression toward a variety of nitroarenes was NM3009 > NM2009 > NM1011 > TA1535/pSK1002 > NM2000 > NM1000. 40 refs., 3 figs., 3 tabs.

  16. Engineering an enhanced, thermostable, monomeric bacterial luciferase gene as a reporter in plant protoplasts.

    PubMed

    Cui, Boyu; Zhang, Lifeng; Song, Yunhong; Wei, Jinsong; Li, Changfu; Wang, Tietao; Wang, Yao; Zhao, Tianyong; Shen, Xihui

    2014-01-01

    The application of the luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens as a reporter has been published for bacteria, yeast and mammalian cells. We report here the optimization of fused luxAB (the bacterial luciferase heterodimeric enzyme) expression, quantum yield and its application as a reporter gene in plant protoplasts. The fused luxAB gene was mutated by error prone PCR or chemical mutagenesis and screened for enhanced luciferase activity utilizing decanal as substrate. Positive luxAB mutants with superior quantum yield were subsequently shuffled by DNase I digestion and PCR assembly for generation of recombinants with additional increases in luciferase activity in bacteria. The coding sequence of the best recombinant, called eluxAB, was then optimized further to conform to Arabidopsis (Arabidopsis thaliana) codon usage. A plant expression vector of the final, optimized eluxAB gene (opt-eluxAB) was constructed and transformed into protoplasts of Arabidopsis and maize (Zea mays). Luciferase activity was dramatically increased for opt-eluxAB compared to the original luxAB in Arabidopsis and maize cells. The opt-eluxAB driven by two copies of the 35S promoter expresses significantly higher than that driven by a single copy. These results indicate that the eluxAB gene can be used as a reporter in plant protoplasts. To our knowledge, this is the first report to engineer the bacterium Photorhabdus luminescens luciferase luxAB as a reporter by directed evolution which paved the way for further improving the luxAB reporter in the future.

  17. Exploration of new perspectives and limitations in Agrobacterium mediated gene transfer technology. Progress report, [June 1, 1992-- May 31, 1994

    SciTech Connect

    Marton, L.

    1994-12-31

    This report describes progress aimed at constructing gene-transfer technology for Nicotiana plumbaginifolia. Most actual effort as described herein has so far been directed at exploring new perspectives and limitations in Agrobacterium mediated gene transfer. Accomplishments are described using a core homologous gene targeting vector.

  18. Comparison and Calibration of Different Reporters for Quantitative Analysis of Gene Expression

    PubMed Central

    Garcia, Hernan G.; Lee, Heun Jin; Boedicker, James Q.; Phillips, Rob

    2011-01-01

    Absolute levels of gene expression in bacteria are observed to vary over as much as six orders of magnitude. Thermodynamic models have been proposed as a tool to describe the expression levels of a given transcriptional circuit. In this context, it is essential to understand both the limitations and linear range of the different methods for measuring gene expression and to determine to what extent measurements from different reporters can be directly compared with one aim being the stringent testing of theoretical descriptions of gene expression. In this article, we compare two protein reporters by measuring both the absolute level of expression and fold-change in expression using the fluorescent protein EYFP and the enzymatic reporter β-galactosidase. We determine their dynamic and linear range and show that they are interchangeable for measuring mean levels of expression over four orders of magnitude. By calibrating these reporters such that they can be interpreted in terms of absolute molecular counts, we establish limits for their applicability: autofluorescence on the lower end of expression for EYFP (at ∼10 molecules per cell) and interference with cellular growth on the high end for β-galactosidase (at ∼20,000 molecules per cell). These qualities make the reporters complementary and necessary when trying to experimentally verify the predictions from the theoretical models. PMID:21806921

  19. Multi-wavelength photoacoustic imaging of inducible tyrosinase reporter gene expression in xenograft tumors

    PubMed Central

    Paproski, Robert J.; Heinmiller, Andrew; Wachowicz, Keith; Zemp, Roger J.

    2014-01-01

    Photoacoustic imaging is an emerging hybrid imaging technology capable of breaking through resolution limits of pure optical imaging technologies imposed by optical-scattering to provide fine-resolution optical contrast information in deep tissues. We demonstrate the ability of multi-wavelength photoacoustic imaging to estimate relative gene expression distributions using an inducible expression system and co-register images with hemoglobin oxygen saturation estimates and micro-ultrasound data. Tyrosinase, the rate-limiting enzyme in melanin production, is used as a reporter gene owing to its strong optical absorption and enzymatic amplification mechanism. Tetracycline-inducible melanin expression is turned on via doxycycline treatment in vivo. Serial multi-wavelength imaging reveals very low estimated melanin expression in tumors prior to doxycycline treatment or in tumors with no tyrosinase gene present, but strong signals after melanin induction in tumors tagged with the tyrosinase reporter. The combination of new inducible reporters and high-resolution photoacoustic and micro-ultrasound technology is poised to bring a new dimension to the study of gene expression in vivo. PMID:24936769

  20. Molecular characterization of a maize regulatory gene. Progress report, July 1989--March 1990

    SciTech Connect

    Wessler, S.

    1990-12-31

    This progress report contains information concerning the characterization of the Maize regulatory gene. The findings of this research program have immediate significance. Firstly, it provides support for the notion that R proteins, produced by the regulatory gene, are functionally equivalent. Secondly, the success of these experiments provides a simple transient assay for either natural or constructed R protein mutations. The relative ease of this assay coupled with overnight results are important prerequisites to the proposed experiments involving a structure-function analysis of the R protein.

  1. Rapid, specific detection of alphaviruses from tissue cultures using a replicon-defective reporter gene assay.

    PubMed

    Li, Jiangjiao; Zhu, Wuyang; Wang, Huanqin; Li, Jiandong; Zhang, Quanfu; He, Ying; Li, Jia; Fu, Juanjuan; Li, Dexin; Liang, Guodong

    2012-01-01

    We established a rapid, specific technique for detecting alphaviruses using a replicon-defective reporter gene assay derived from the Sindbis virus XJ-160. The pVaXJ expression vector containing the XJ-160 genome was engineered to form the expression vectors pVaXJ-EGFP expressing enhanced green fluorescence protein (EGFP) or pVaXJ-GLuc expressing Gaussia luciferase (GLuc). The replicon-defective reporter plasmids pVaXJ-EGFPΔnsp4 and pVaXJ-GLucΔnsp4 were constructed by deleting 1139 bp in the non-structural protein 4 (nsP4) gene. The deletion in the nsP4 gene prevented the defective replicons from replicating and expressing reporter genes in transfected BHK-21 cells. However, when these transfected cells were infected with an alphavirus, the non-structural proteins expressed by the alphavirus could act on the defective replicons in trans and induce the expression of the reporter genes. The replicon-defective plasmids were used to visualize the presence of alphavirus qualitatively or detect it quantitatively. Specificity tests showed that this assay could detect a variety of alphaviruses from tissue cultures, while other RNA viruses, such as Japanese encephalitis virus and Tahyna virus, gave negative results with this system. Sensitivity tests showed that the limit of detection (LOD) of this replicon-defective assay is between 1 and 10 PFU for Sindbis viruses. These results indicate that, with the help of the replicon-defective alphavirus detection technique, we can specifically, sensitively, and rapidly detect alphaviruses in tissue cultures. The detection technique constructed here may be well suited for use in clinical examination and epidemiological surveillance, as well as for rapid screening of potential viral biological warfare agents.

  2. An infectious West Nile virus that expresses a GFP reporter gene.

    PubMed

    Pierson, Theodore C; Diamond, Michael S; Ahmed, Asim A; Valentine, Laura E; Davis, Carl W; Samuel, Melanie A; Hanna, Sheri L; Puffer, Bridget A; Doms, Robert W

    2005-03-30

    West Nile virus is a mosquito-borne, neurotropic flavivirus that causes encephalitis in humans and animals. Since being introduced into the Western hemisphere in 1999, WNV has spread rapidly across North America, identifying this virus as an important emerging pathogen. In this study, we developed a DNA-launched infectious molecular clone of WNV that encodes a GFP reporter gene. Transfection of cells with the plasmid encoding this recombinant virus (pWNII-GFP) resulted in the production of infectious WNV capable of expressing GFP at high levels shortly after infection of a variety of cell types, including primary neurons and dendritic cells. Infection of cells with WNII-GFP virus was productive, and could be inhibited with both monoclonal antibodies and interferon-beta, highlighting the potential of this system in the development and characterization of novel inhibitors and therapeutics for WNV infection. As expected, insertion of the reporter gene into the viral genome was associated with a reduced rate of viral replication, providing the selective pressure for the development of variants that no longer encoded the full-length reporter gene cassette. We anticipate this DNA-based, infectious WNV reporter virus will allow novel approaches for the study of WNV infection and its inhibition both in vitro and in vivo.

  3. Principal components analysis and the reported low intrinsic dimensionality of gene expression microarray data.

    PubMed

    Lenz, Michael; Müller, Franz-Josef; Zenke, Martin; Schuppert, Andreas

    2016-01-01

    Principal components analysis (PCA) is a common unsupervised method for the analysis of gene expression microarray data, providing information on the overall structure of the analyzed dataset. In the recent years, it has been applied to very large datasets involving many different tissues and cell types, in order to create a low dimensional global map of human gene expression. Here, we reevaluate this approach and show that the linear intrinsic dimensionality of this global map is higher than previously reported. Furthermore, we analyze in which cases PCA fails to detect biologically relevant information and point the reader to methods that overcome these limitations. Our results refine the current understanding of the overall structure of gene expression spaces and show that PCA critically depends on the effect size of the biological signal as well as on the fraction of samples containing this signal. PMID:27254731

  4. Principal components analysis and the reported low intrinsic dimensionality of gene expression microarray data

    PubMed Central

    Lenz, Michael; Müller, Franz-Josef; Zenke, Martin; Schuppert, Andreas

    2016-01-01

    Principal components analysis (PCA) is a common unsupervised method for the analysis of gene expression microarray data, providing information on the overall structure of the analyzed dataset. In the recent years, it has been applied to very large datasets involving many different tissues and cell types, in order to create a low dimensional global map of human gene expression. Here, we reevaluate this approach and show that the linear intrinsic dimensionality of this global map is higher than previously reported. Furthermore, we analyze in which cases PCA fails to detect biologically relevant information and point the reader to methods that overcome these limitations. Our results refine the current understanding of the overall structure of gene expression spaces and show that PCA critically depends on the effect size of the biological signal as well as on the fraction of samples containing this signal. PMID:27254731

  5. Expression pattern of STOP lacZ reporter gene in adult and developing mouse brain.

    PubMed

    Couégnas, Alice; Schweitzer, Annie; Andrieux, Annie; Ghandour, M Said; Boehm, Nelly

    2007-05-15

    Stable tubulin-only polypeptide (STOP) proteins are microtubule-associated proteins responsible for microtubule stabilization in neurons. STOP null mice show apparently normal cerebral anatomy but display synaptic defects associated with neuroleptic-sensitive behavioral disorders. STOP null mice have therefore been proposed as an animal model for the study of schizophrenia. In the present study, the expression pattern of STOP gene in developing and adult brain has been examined by using lacZ gene inserted in the STOP locus, as a reporter gene. beta-Galactosidase (beta-gal) immunostaining was confined to neuronal cells and projections. Strong labeling was observed in the whole olfactory system, cortical layer VII, hippocampus, hypothalamus, cerebellum, habenula, fasciculus retroflexus, and interpeduncular nucleus in adults. Additionally, ventral thalamic nucleus, clusters of positive cells in striatum, and Cajal-Retzius cells of cortical layer I were labeled in young mice. The strong expression of STOP lacZ reporter gene observed in brain is confined to areas that may be involved in the schizophrenia-related symptoms observed in STOP-deficient mice.

  6. [Cloning of mouse adam10 gene promoter and construction and identification of dual luciferase reporter system].

    PubMed

    Chen, Wei; Chen, Chong; Zhang, Huan-Xin; Cao, Jiang; Sang, Wei; Wu, Qing-Yun; Zhao, Kai; Zang, Yu; Zeng, Ling-Yu; Xu, Kai-Lin

    2012-06-01

    This study was aimed to clone mouse adam10 gene promoter and construct its dual luciferase report vector, and to investigate its transcriptional activity. Total DNA was extracted from mouse brain and used for amplifying the fragment containing adam10 gene promoter by PCR. The amplified product was inserted into pGL-4.10 vector to construct pGL4.10-adam10. The pGL4.10-adam10 and control plasmid pGL4.74 were co-transfected into HEK293 FT cells by lipofectamine 2000. The activity of adam10 gene promoter was assayed by luciferase system. The results showed that the recombinant plasmid pGL4.10-adam10 containing promoter of mouse adam10 was correctly constructed. The method was optimized by changing ratio of two plasmids. Moreover, the transcriptional activity of pGL4.10-adam10 stimulated by ionomycin increased. It is concluded that the dual luciferase reporter system is successfully established, which is useful in bioluminescence imaging technology in vitro. The effect of ionomycin can enhance the transcriptional activity of adam10 gene promoter.

  7. Bortezomib and ixazomib protect firefly luciferase from degradation and can flaw respective reporter gene assays.

    PubMed

    Becker, Jonas Philipp; Clemens, Jannick Robert; Theile, Dirk; Weiss, Johanna

    2016-09-15

    Firefly luciferase-based reporter gene assays are the most commonly used assays to investigate the transcriptional regulation of gene expression. However, direct interaction of tested compounds with the firefly luciferase leading to altered enzymatic activity may lead to misinterpretation of experimental data. When investigating the proteasome inhibitors bortezomib, carfilzomib, and ixazomib, we observed increased luminescence for bortezomib and ixazomib, but not for carfilzomib, in a pregnane-X-receptor (PXR) reporter gene assay, which was inconsistent with the mRNA expression levels of the main PXR target gene CYP3A4. To further scrutinize this phenomenon, we performed experiments with constitutively expressed firefly luciferase and demonstrated that the increase in cellular firefly luciferase activity is independent from PXR activation or CYP3A4 promoter. Using cell-free assays with recombinant firefly luciferase enzyme, we made the counterintuitive observation that firefly luciferase activity is inhibited by bortezomib and ixazomib in a reversible and competitive manner. This inhibition stabilizes the firefly luciferase enzyme against proteolytic degradation (e.g., toward trypsin), thereby increasing its half-life with subsequent enhancement of total cellular luminescence that eventually mimicked PXR-driven luciferase induction. These data show that particular compounds can strikingly interfere with firefly luciferase and once more illustrate the importance of careful interpretation of data obtained from luciferase-based assays. PMID:27325500

  8. Bortezomib and ixazomib protect firefly luciferase from degradation and can flaw respective reporter gene assays.

    PubMed

    Becker, Jonas Philipp; Clemens, Jannick Robert; Theile, Dirk; Weiss, Johanna

    2016-09-15

    Firefly luciferase-based reporter gene assays are the most commonly used assays to investigate the transcriptional regulation of gene expression. However, direct interaction of tested compounds with the firefly luciferase leading to altered enzymatic activity may lead to misinterpretation of experimental data. When investigating the proteasome inhibitors bortezomib, carfilzomib, and ixazomib, we observed increased luminescence for bortezomib and ixazomib, but not for carfilzomib, in a pregnane-X-receptor (PXR) reporter gene assay, which was inconsistent with the mRNA expression levels of the main PXR target gene CYP3A4. To further scrutinize this phenomenon, we performed experiments with constitutively expressed firefly luciferase and demonstrated that the increase in cellular firefly luciferase activity is independent from PXR activation or CYP3A4 promoter. Using cell-free assays with recombinant firefly luciferase enzyme, we made the counterintuitive observation that firefly luciferase activity is inhibited by bortezomib and ixazomib in a reversible and competitive manner. This inhibition stabilizes the firefly luciferase enzyme against proteolytic degradation (e.g., toward trypsin), thereby increasing its half-life with subsequent enhancement of total cellular luminescence that eventually mimicked PXR-driven luciferase induction. These data show that particular compounds can strikingly interfere with firefly luciferase and once more illustrate the importance of careful interpretation of data obtained from luciferase-based assays.

  9. A beta-glucuronidase reporter gene construct for monitoring aflatoxin biosynthesis in Aspergillus flavus.

    PubMed Central

    Flaherty, J E; Weaver, M A; Payne, G A; Woloshuk, C P

    1995-01-01

    Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and A. parasiticus. Current research is directed at the elimination of these compounds in important food sources. The objective of this research was to develop a method to study the induction and regulation of aflatoxin biosynthesis by examining the expression of one aflatoxin pathway gene, ver1. The promoter region of ver1 was fused to the beta-glucuronidase (GUS) gene (uidA) from Escherichia coli to form the reporter construct, GAP13. A. flavus 656-2 was transformed with this construct. Aflatoxin production, GUS activity, and transcript accumulation were determined in transformants after shifting the cultures from a nonconducive medium to a medium conducive to aflatoxin biosynthesis. Transformants harboring GAP13 displayed GUS expression only when aflatoxin was detected in culture. Further, the transcription of the uidA gene driven by the ver1 promoter followed the same profile as for the ver1 genes. The results show that the GAP13 construct may be useful as a genetic tool to study the induction of aflatoxin in situ and to identify substances that affect the expression of genes involved in aflatoxin biosynthesis. The utility of this construct to detect inducers of aflatoxin biosynthesis in maize kernels was tested in a bioassay. A heat-stable inducer of aflatoxin with a molecular size of less than 10 kDa was detected in extracts from maize kernels colonized by A. flavus. PMID:7618859

  10. Use of the Aspergillus oryzae actin gene promoter in a novel reporter system for exploring antifungal compounds and their target genes.

    PubMed

    Marui, Junichiro; Yoshimi, Akira; Hagiwara, Daisuke; Fujii-Watanabe, Yoshimi; Oda, Ken; Koike, Hideaki; Tamano, Koichi; Ishii, Tomoko; Sano, Motoaki; Machida, Masayuki; Abe, Keietsu

    2010-08-01

    Demand for novel antifungal drugs for medical and agricultural uses has been increasing because of the diversity of pathogenic fungi and the emergence of drug-resistant strains. Genomic resources for various living species, including pathogenic fungi, can be utilized to develop novel and effective antifungal compounds. We used Aspergillus oryzae as a model to construct a reporter system for exploring novel antifungal compounds and their target genes. The comprehensive gene expression analysis showed that the actin-encoding actB gene was transcriptionally highly induced by benomyl treatment. We therefore used the actB gene to construct a novel reporter system for monitoring responses to cytoskeletal stress in A. oryzae by introducing the actB promoter::EGFP fusion gene. Distinct fluorescence was observed in the reporter strain with minimum background noise in response to not only benomyl but also compounds inhibiting lipid metabolism that is closely related to cell membrane integrity. The fluorescent responses indicated that the reporter strain can be used to screen for lead compounds affecting fungal microtubule and cell membrane integrity, both of which are attractive antifungal targets. Furthermore, the reporter strain was shown to be technically applicable for identifying novel target genes of antifungal drugs triggering perturbation of fungal microtubules or membrane integrity.

  11. Evaluation of a GFP Report Gene Construct for Environmental Arsenic Detection

    SciTech Connect

    Roberto, F.F.; Barnes, J.M.; Bruhn, D.F.

    2002-03-28

    Detection of arsenic and other heavy metal contaminants in the environment is critical to ensuring safe drinking water and effective cleanup of historic activities that have led to widespread contamination of soil and groundwater. Biosensors have the potential to significantly reduce the costs associated with site characterization and long term environmental monitoring. By exploiting the highly selective and sensitive natural mechanisms by which bacteria and other living organisms respond to heavy metals, and fusing transcriptionally active components of these mechanisms to reporter genes, such as B-galactosidase, bacterial luciferase (lux), or green fluorescent protein (GFP) from marine jellyfish, it is possible to produce inexpensive, yet effective biosensors. This article describes the response to submicrogram quantities of arsenite and arsenate of a whole cell arsenic biosensor utilizing a GFP reporter gene.

  12. Reporter Gene Imaging of Immune Responses to Cancer: Progress and Challenges

    PubMed Central

    Dubey, Purnima

    2012-01-01

    Immune responses to cancer are dynamic processes which take place through the concerted activity of innate and adaptive cell populations. In order to fully understand the efficacy of immune therapies for cancer, it is critical to understand how the treatment modulates the function of each cell type involved in the anti-tumor immune response. Molecular imaging is a versatile method for longitudinal studies of cellular localization and function. The development of reporter genes for tracking cell movement and function was a powerful addition to the immunologist's toolbox. This review will highlight the advances and challenges in the use of reporter gene imaging to track immune cell localization and function in cancer. PMID:22509199

  13. Evaluation of a GFP reporter gene construct for environmental arsenic detection.

    PubMed

    Roberto, Francisco F; Barnes, Joni M; Bruhn, Debby F

    2002-08-16

    Detection of arsenic and other heavy metal contaminants in the environment is critical to ensuring safe drinking water and effective cleanup of historic activities that have led to widespread contamination of soil and groundwater. Biosensors have the potential to significantly reduce the costs associated with site characterization and long term environmental monitoring. By exploiting the highly selective and sensitive natural mechanisms by which bacteria and other living organisms respond to heavy metals, and fusing transcriptionally active components of these mechanisms to reporter genes, such as beta-galactosidase, bacterial luciferase (lux), or green fluorescent protein (GFP) from marine jellyfish, it is possible to produce inexpensive, yet effective biosensors. This article describes the response to submicrogram quantities of arsenite and arsenate of a whole cell arsenic biosensor utilizing a GFP reporter gene.

  14. Application of immunoaffinity column as cleanup tool for an enzyme linked immunosorbent assay of phosphinothricin-N-acetyltransferase detection in genetically modified maize and rape.

    PubMed

    Xu, Wentao; Huang, Kunlun; Zhao, Heng; Luo, Yunbo

    2005-06-01

    We have developed a new immunoassay method to detect genetically modified (GM) maize and rape containing phosphinothricin-N-acetyltransferase (PAT). PAT encoded by Bialaphos resistance gene (bar) was highly expressed in soluble form in Escherichia coli BL21(DE3) and purified to homogeneity by Ni2+ affinity chromatography. A simple and efficient extraction and purification procedure of PAT from GM maize and rape was developed by means of the immunoaffinity column (IAC) as a cleanup tool. Purified polyclonal antibodies against PAT was produced and coupled covalently to CNBr-activated Sepharose 4B. Both the binding conditions and elution protocols were optimized. The IAC was successfully employed to isolate and purify the PAT from the various tissues of GM maize (Bt11 and Bt176) and rapes (MS1/RF1 and MS8/RF3). Enzyme linked immunosorbent assay (ELISA) procedures were established further on to measure the PAT protein. GM maize cannot be differentiated from non-GM maize by ELISA. But IAC-ELISA allowed 0.5% GMOs to be detected in MS1/RF1 and MS8/RF3 and 10% GMOs to be detected in Bt11 and Bt176, which makes this method an acceptable method to access PAT protein in GM rapes and maize.

  15. Characterization, Localization, Essentiality, and High-Resolution Crystal Structure of Glucosamine 6-Phosphate N-Acetyltransferase from Trypanosoma brucei ▿ ‡ §

    PubMed Central

    Mariño, Karina; Güther, M. Lucia Sampaio; Wernimont, Amy K.; Qiu, Wei; Hui, Raymond; Ferguson, Michael A. J.

    2011-01-01

    A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N-acetyltransferase (TbGNA1; EC 2.3.1.4) was cloned and expressed in Escherichia coli. The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form T. brucei GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under nonpermissive conditions revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed. PMID:21531872

  16. rs1495741 as a tag single nucleotide polymorphism of N-acetyltransferase 2 acetylator phenotype associates bladder cancer risk and interacts with smoking

    PubMed Central

    Ma, Chong; Gu, Liyan; Yang, Mingyuan; Zhang, Zhensheng; Zeng, Shuxiong; Song, Ruixiang; Xu, Chuanliang; Sun, Yinghao

    2016-01-01

    Abstract Rs1495741 has been identified to infer N-acetyltransferase 2 (NAT2) acetylator phenotype, and to decrease the risk of bladder cancer. However, a number of studies conducted in various regions showed controversial results. To quantify the association between rs1495741 and the risk of bladder cancer and to estimate the interaction effect of this genetic variant with smoking, we performed a systematic literature review and meta-analysis involving 14,815 cases and 58,282 controls from 29 studies. Our results indicates rs1495741 significantly associated with bladder cancer risk (OR = 0.85, 95% CI = 0.82–0.89, test for heterogeneity P = 0.36, I2 = 7.0%). And we verified this association in populations from Europe, America, and Asia. Further, our stratified meta-analysis showed rs1495741's role is typically evident only in ever smokers, which suggests its interaction with smoking. This study may provide new insight into gene-environment study on bladder cancer. PMID:27495060

  17. The Transcriptional Histone Acetyltransferase Cofactor TRRAP Associates with the MRN Repair Complex and Plays a Role in DNA Double-Strand Break Repair†

    PubMed Central

    Robert, Flavie; Hardy, Sara; Nagy, Zita; Baldeyron, Céline; Murr, Rabih; Déry, Ugo; Masson, Jean-Yves; Papadopoulo, Dora; Herceg, Zdenko; Tora, Làszlò

    2006-01-01

    Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling. PMID:16382133

  18. Gene Disruption by Homologous Recombination in the Xylella fastidiosa Citrus Variegated Chlorosis Strain

    PubMed Central

    Gaurivaud, Patrice; Souza, Leonardo C. A.; Virgílio, Andrea C. D.; Mariano, Anelise G.; Palma, Renê R.; Monteiro, Patrícia B.

    2002-01-01

    Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for β-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to β-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant. PMID:12200328

  19. Final Report [Function of the Arabidopsis TIR1 gene in auxin response

    SciTech Connect

    Estelle, Mark

    2000-12-18

    During this grant period substantial progress was made in the characterization of the TIR1 gene in Arabidopsis. Studies showed that the TIR1 protein is part of a protein complex that includes AtCUL1, ASK1 and RBX1. This complex, called SCF-TIR1, functions in the ubiquitin-mediated protein degradation pathway. Our work is the first report of an SCF complex in a plant system. The results of our studies are described in more detail in the report together with a publication resulting from this study.

  20. Mosaic 18q21.2 deletions including the TCF4 gene: a clinical report.

    PubMed

    Rossi, Massimiliano; Labalme, Audrey; Cordier, Marie-Pierre; Till, Marianne; Blanchard, Gaëlle; Dubois, Remi; Guibaud, Laurent; Heissat, Sophie; Javouhey, Etienne; Lachaux, Alain; Mure, Pierre-Yves; Ville, Dorothée; Edery, Patrick; Sanlaville, Damien

    2012-12-01

    Pitt-Hopkins syndrome (PTHS) is characterized by distinctive facial dysmorphism, profound intellectual disability, and the possible occurrence of epilepsy and breathing anomalies. It is caused by haploinsufficiency of the TCF4 gene. No significant difference in clinical severity has been reported to date between PTHS patients carrying 18q21 deletions including the TCF4 gene, and those harboring TCF4 point mutations, suggesting a lack of genotype/phenotype correlation. Moreover, the size of 18q21 deletions including the TCF4 gene does not appear to have a significant effect on the phenotypic severity, suggesting that TCF4 haploinsufficiency is the most important prognostic factor in 18q deletions. We describe two unrelated patients presenting with clinical features reminiscent of PTHS and carrying mosaic interstitial 18q21 deletions characterized by array comparative genomic hybridization. One of the patients presented the lowest level of mosaic 18q21 deletion reported to date (5-10%). Our report and a review of the literature show that the mosaic status does not appear to have a significant effect on the clinical severity of 18q21 deletions, which are associated with a poor neurological outcome, whereas a mosaic TCF4 point mutation can result in a significantly milder phenotype. Malformations of internal organs are currently considered to be rare in PTHS. The patients described here had visceral anomalies, suggesting that a full morphological assessment, including heart and abdominal ultrasound scans, should be performed systematically in PTHS patients. PMID:23165966

  1. A specific mechanism for nonspecific activation in reporter-gene assays.

    PubMed

    Auld, Douglas S; Thorne, Natasha; Nguyen, Dac-Trung; Inglese, James

    2008-08-15

    The importance of bioluminescence in enabling a broad range of high-throughput screening (HTS) assay formats is evidenced by widespread use in industry and academia. Therefore, understanding the mechanisms by which reporter enzyme activity can be modulated by small molecules is critical to the interpretation of HTS data. In this Perspective, we provide evidence for stabilization of luciferase by inhibitors in cell-based luciferase reporter-gene assays resulting in the counterintuitive phenomenon of signal activation. These data were derived from our analysis of luciferase inhibitor compound structures and their prevalence in the Molecular Libraries Small Molecule Repository using 100 HTS experiments available in PubChem. Accordingly, we found an enrichment of luciferase inhibitors in luciferase reporter-gene activation assays but not in assays using other reporters. In addition, for several luciferase inhibitor chemotypes, we measured reporter stabilization and signal activation in cells that paralleled the inhibition determined using purified luciferase to provide further experimental support for these contrasting effects.

  2. Two N-Terminal Acetyltransferases Antagonistically Regulate the Stability of a Nod-Like Receptor in Arabidopsis

    PubMed Central

    Li, Lin; Gannon, Patrick; Linster, Eric; Huber, Monika; Kapos, Paul; Bienvenut, Willy; Giglione, Carmela; Zhang, Yuelin; Chen, She

    2015-01-01

    Nod-like receptors (NLRs) serve as immune receptors in plants and animals. The stability of NLRs is tightly regulated, though its mechanism is not well understood. Here, we show the crucial impact of N-terminal acetylation on the turnover of one plant NLR, Suppressor of NPR1, Constitutive 1 (SNC1), in Arabidopsis thaliana. Genetic and biochemical analyses of SNC1 uncovered its multilayered regulation by different N-terminal acetyltransferase (Nat) complexes. SNC1 exhibits a few distinct N-terminal isoforms generated through alternative initiation and N-terminal acetylation. Its first Met is acetylated by N-terminal acetyltransferase complex A (NatA), while the second Met is acetylated by N-terminal acetyltransferase complex B (NatB). Unexpectedly, the NatA-mediated acetylation serves as a degradation signal, while NatB-mediated acetylation stabilizes the NLR protein, thus revealing antagonistic N-terminal acetylation of a single protein substrate. Moreover, NatA also contributes to the turnover of another NLR, RESISTANCE TO P. syringae pv maculicola 1. The intricate regulation of protein stability by Nats is speculated to provide flexibility for the target protein in maintaining its homeostasis. PMID:25966763

  3. Acetyl Coenzyme A Acetyltransferase of Rhizobium sp. (Cicer) Strain CC 1192.

    PubMed

    Kim, S A; Copeland, L

    1997-09-01

    To investigate why Rhizobium sp. (Cicer) strain CC 1192 cells accumulate poly-R-3-hydroxybutyrate in the free-living state but not as bacteroids in nodules on chickpea (Cicer arietinum L.) plants, we have examined the kinetic properties of acetyl coenzyme A (acetyl-CoA) acetyltransferase (also known as acetoacetyl-CoA thiolase and 3-ketothiolase [EC 2.3.1.9]) from both types of cells. The enzyme had a native molecular mass of 180 (plusmn) 4 kDa, and the subunit molecular mass was 44 (plusmn) 1 kDa. The seven amino acids from the N terminus were Lys-Ala-Ser-Ile-Val-Ile-Ala. Thiolysis and condensation activity of the enzyme from free-living CC 1192 cells were optimal at pHs 7.8 and 8.1, respectively. The relationship between substrate concentrations and initial velocity for the thiolysis reaction were hyperbolic and gave K(infm) values for acetoacetyl-CoA and CoA of 42 and 56 (mu)M, respectively. The maximum velocity in the condensation direction was approximately 10% of that of the thiolysis reaction. With highly purified preparations of the enzyme, a value of approximately 1 mM was determined for the apparent K(infm) for acetyl-CoA. However, with partially purified enzyme preparations or when N-ethylmaleimide was included in reaction mixtures the apparent K(infm) for acetyl-CoA was close to 0.3 mM. In the condensation direction, CoA was a potent linear competitive inhibitor with an inhibition constant of 11 (mu)M. The much higher affinity of the enzyme for the product CoA than the substrate acetyl-CoA could have significance in view of metabolic differences between bacteroid and free-living cells of CC 1192. We propose that in free-living CC 1192 cells, the acetyl-CoA/CoA ratio reaches a value that allows condensation activity of acetyl-CoA acetyltransferase, but that in CC 1192 bacteroids, the ratio is poised so that the formation of acetoacetyl-CoA is not favored.

  4. Biological Sensor for Sucrose Availability: Relative Sensitivities of Various Reporter Genes

    PubMed Central

    Miller, William G.; Brandl, Maria T.; Quiñones, Beatriz; Lindow, Steven E.

    2001-01-01

    A set of three sucrose-regulated transcriptional fusions was constructed. Fusions p61RYTIR, p61RYlac, and p61RYice contain the scrR sucrose repressor gene and the promoterless gfp, lacZ, and inaZ reporter genes, respectively, fused to the scrY promoter from Salmonella enterica serovar Typhimurium. Cells of Erwinia herbicola containing these fusions are induced only in media amended with sucrose, fructose, or sorbose. While a large variation in sucrose-dependent reporter gene activity was observed in cells harboring all gene fusions, fusions to the inaZ reporter gene yielded a much wider range of activity and were responsive to lower levels of sucrose than either lacZ or gfp. The lacZ reporter gene was found to be more efficient than gfp, requiring approximately 300-fold fewer cells for a detectable response over all concentrations of sucrose. Similarly, inaZ was found to be more efficient than lacZ, requiring 30-fold fewer cells at 1.45 μM sucrose and 6,100-fold fewer cells at 29 mM sucrose for a quantifiable response. The fluorescence of individual cells containing p61RYTIR was quantified following epifluorescence microscopy in order to relate the fluorescence exhibited by populations of cells in batch cultures with that of individual cells in such cultures. While the mean fluorescence intensity of a population of individual cells increased with increasing concentrations of sucrose, a wide range of fluorescence intensity was seen among individual cells. For most cultures the distribution of fluorescence intensity among individual cells was log-normally distributed, but cells grown in intermediate concentrations of sucrose exhibited two distinct populations of cells, one having relatively low fluorescence and another with much higher fluorescence. When cells were inoculated onto bean leaves, whole-cell ice nucleation and gfp-based biological sensors for sucrose each indicated that the average concentration of sucrose on moist leaf surfaces was about 20

  5. Phytoalexin detoxification genes and gene products: Implication for the evolution of host specific traits for pathogenicity. Final report

    SciTech Connect

    VanEtten, H.

    1997-06-01

    The overall objectives of this research were to determine which differences among PDA genes were associated with different levels of virulence on pea and to clone and characterize a MAK gene. The authors also proposed to characterize the pisatin detoxifying system in pea pathogens in addition to N. haematococca to assess whether pathogens of a common host had evolved similar pathogenicity genes.

  6. Vesicular stomatitis virus matrix protein inhibits host cell-directed transcription of target genes in vivo.

    PubMed Central

    Black, B L; Lyles, D S

    1992-01-01

    Infection by vesicular stomatitis virus (VSV) results in a rapid inhibition of host cell transcription and translation. To determine whether the viral matrix (M) protein was involved in this inhibition of host cell gene expression, an M protein expression vector was cotransfected with a target gene vector, encoding the target gene, encoding chloramphenicol acetyltransferase (CAT). Expression of M protein caused a decrease in CAT activity in a gene dosage-dependent manner, and inhibition was apparent by 12 h posttransfection. The inhibitory effect of M protein was quite potent. The level of M protein required for a 10-fold inhibition of CAT activity was less than 1% of the level of M protein produced during the sixth hour of VSV infection. Northern (RNA) analysis of cotransfected cells showed that expression of M protein caused a reduction in the steady-state level of the vector-encoded mRNAs. Expression of both CAT and M mRNAs was reduced in cells cotransfected with a plasmid encoding M protein, indicating that expression of small amounts of M protein from plasmid DNA inhibits further expression of both M and CAT mRNAs. Nuclear runoff transcription analysis demonstrated that expression of M protein inhibited transcription of the target genes. This is the first report of a viral gene product which is capable of inhibiting transcription in vivo in the absence of any other viral component. Images PMID:1318397

  7. Extinction of expression of the genes encoding haematopoietic cell-restricted transcription factors in T-lymphoma × fibroblast cell hybrids

    PubMed Central

    Oikawa, Tsuneyuki; Yamada, Toshiyuki; Kondoh, Nobuo; Negishi-Kihara, Fumiko; Hitomi, Yoshiaki; Suzuki, Mitsuhiro; Teramoto, Sayaka

    2001-01-01

    We previously reported that expression of the T-cell receptor (TCR) α and lck genes is extinguished in hybrids between mouse T-lymphoma EL4 cells and mouse fibroblast B82 cells. In the present study, we found that the activities of the TCRα minimum enhancer and the lck promoter monitored by the luciferase or chloramphenicol acetyltransferase (CAT) assays were markedly inhibited in the hybrids. Expression of the TCF-1, LEF-1, GATA-3, Ikaros, c-myb and Fli-1 genes, which encode the haematopoietic cell-restricted transcription factors that appear to be responsible for the activities of the enhancer and the promoter, was fully extinguished or markedly suppressed in the hybrids. On the other hand, expression of the transcription factor genes observed in both parental cells, such as the AML1 and c-ets-1 genes, and that of the genes encoding ubiquitously expressed transcription factors, such as the E2A, CREB and c-ets-2 genes, was not significantly suppressed in the hybrids. These results suggest that the genes encoding haematopoietic cell-restricted transcription factors are targets for negative regulation in fibroblastic background and that the repression of these genes may consequently lead to suppression of the promoter and/or enhancer activities of several T-cell-specific structural genes in T-lymphoma × fibroblast cell hybrids. PMID:11683956

  8. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae

    PubMed Central

    Masser, Anna E.; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan

    2016-01-01

    Abstract Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon‐optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half‐lives of 40 and 5 min, respectively. The commercial substrate Nano‐Glo® is compatible with detection of yNluc bioluminescence in < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat‐shock promoter (PCYC1–HSE), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C‐terminus of a temperature‐sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:26860732

  9. Comparative Analysis of T Cell Imaging with Human Nuclear Reporter Genes

    PubMed Central

    Moroz, Maxim A.; Zhang, Hanwen; Lee, Jason; Moroz, Ekaterina; Zurita, Juan; Shenker, Larissa; Serganova, Inna; Blasberg, Ronald; Ponomarev, Vladimir

    2015-01-01

    Monitoring genetically altered T cells is an important component of adoptive T cell therapy in patients, and the ability to visualize their trafficking/targeting, proliferation/expansion, and retention/death using highly sensitive reporter systems that do not induce an immunologic response would provide useful information. Therefore, we focused on human reporter gene systems that have the potential for translation to clinical studies. The objective of the in vivo imaging studies was to determine the minimum number of T cells that could be visualized with the different nuclear reporter systems. We determined the imaging sensitivity (lower limit of T cell detection) of each reporter using appropriate radiolabeled probes for PET or SPECT imaging. Methods Human T cells were transduced with retroviral vectors encoding for the human norepinephrine transporter (hNET), human sodiumiodide symporter (hNIS), a human deoxycytidine kinase double mutant (hdCKDM), and herpes simplex virus type 1 thymidine kinase (hsvTK) reporter genes. After viability and growth were assessed, 105 to 3 × 106 reporter T cells were injected subcutaneously on the shoulder area. The corresponding radiolabeled probe was injected intravenously 30 min later, followed by sequential PET or SPECT imaging. Radioactivity at the T cell injection sites and in the thigh (back-ground) was measured. Results The viability and growth of experimental cells were unaffected by transduction. The hNET/meta-18F-fluorobenzylguanidine (18F-MFBG) reporter system could detect less than 1 × 105 T cells because of its high uptake in the transduced T cells and low background activity. The hNIS/124I-iodide reporter system could detect approximately 1 × 106 T cells; 124I-iodide uptake at the T cell injection site was time-dependent and associated with high background. The hdCKDM/2′-18F-fluoro-5-ethyl-1-β-D-arabinofuranosyluracil (18F-FEAU) and hsvTK/18F-FEAU reporter systems detected approximately 3 × 105 T cells

  10. The first report of the vanC₁ gene in Enterococcus faecium isolated from a human clinical specimen.

    PubMed

    Sun, Mingyue; Wang, Yue; Chen, Zhongju; Zhu, Xuhui; Tian, Lei; Sun, Ziyong

    2014-09-01

    The vanC₁ gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC₁gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC₁ and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC₁ gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC₁gene. However, this study is the first to report the presence of the vanC₁gene in E. faecium of human origin. Additionally, our research showed the vanC₁gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC₁gene from different species.

  11. Response of ATP sulfurylase and serine acetyltransferase towards cadmium in hyperaccumulator Sedum alfredii Hance*

    PubMed Central

    Guo, Wei-dong; Liang, Jun; Yang, Xiao-e; Chao, Yue-en; Feng, Ying

    2009-01-01

    We studied the responses of the activities of adenosine-triphosphate (ATP) sulfurylase (ATPS) and serine acetyltransferase (SAT) to cadmium (Cd) levels and treatment time in hyperaccumulating ecotype (HE) Sedum alfredii Hance, as compared with its non-hyperaccumulating ecotype (NHE). The results show that plant growth was inhibited in NHE but promoted in HE when exposed to high Cd level. Cd concentrations in leaves and shoots rapidly increased in HE rather than in NHE, and they became much higher in HE than in NHE along with increasing treatment time and Cd supply levels. ATPS activity was higher in HE than in NHE in all Cd treatments, and increased with increasing Cd supply levels in both HE and NHE when exposed to Cd treatment within 8 h. However, a marked difference of ATPS activity between HE and NHE was found with Cd treatment for 168 h, where ATPS activity increased in HE but decreased in NHE. Similarly, SAT activity was higher in HE than in NHE at all Cd treatments, but was more sensitive in NHE than in HE. Both ATPS and SAT activities in NHE leaves tended to decrease with increasing treatment time after 8 h at all Cd levels. The results reveal the different responses in sulfur assimilation enzymes and Cd accumulation between HE and NHE. With increasing Cd stress, the activities of sulfur assimilation enzymes (ATPS and SAT) were induced in HE, which may contribute to Cd accumulation in the hyperaccumulator Sedum alfredii Hance. PMID:19353742

  12. Response of ATP sulfurylase and serine acetyltransferase towards cadmium in hyperaccumulator Sedum alfredii Hance.

    PubMed

    Guo, Wei-dong; Liang, Jun; Yang, Xiao-e; Chao, Yue-en; Feng, Ying

    2009-04-01

    We studied the responses of the activities of adenosine-triphosphate (ATP) sulfurylase (ATPS) and serine acetyltransferase (SAT) to cadmium (Cd) levels and treatment time in hyperaccumulating ecotype (HE) Sedum alfredii Hance, as compared with its non-hyperaccumulating ecotype (NHE). The results show that plant growth was inhibited in NHE but promoted in HE when exposed to high Cd level. Cd concentrations in leaves and shoots rapidly increased in HE rather than in NHE, and they became much higher in HE than in NHE along with increasing treatment time and Cd supply levels. ATPS activity was higher in HE than in NHE in all Cd treatments, and increased with increasing Cd supply levels in both HE and NHE when exposed to Cd treatment within 8 h. However, a marked difference of ATPS activity between HE and NHE was found with Cd treatment for 168 h, where ATPS activity increased in HE but decreased in NHE. Similarly, SAT activity was higher in HE than in NHE at all Cd treatments, but was more sensitive in NHE than in HE. Both ATPS and SAT activities in NHE leaves tended to decrease with increasing treatment time after 8 h at all Cd levels. The results reveal the different responses in sulfur assimilation enzymes and Cd accumulation between HE and NHE. With increasing Cd stress, the activities of sulfur assimilation enzymes (ATPS and SAT) were induced in HE, which may contribute to Cd accumulation in the hyperaccumulator Sedum alfredii Hance. PMID:19353742

  13. Salt-induced changes in the subunit structure of the Bacillus stearothermophilus lipoate acetyltransferase.

    PubMed

    Shigeoka, Yuichi; Fujisawa, Tetsuro; Teshiba, Satoshi; Fukumori, Hisayoshi; Yamamoto, Kohji; Banno, Yutaka; Aso, Yoichi

    2013-01-01

    The Bacillus stearothermophilus lipoate acetyltransferase (E2), composed of sixty identical, subunits is the core component of the pyruvate dehydrogenase complex (PDC). E2 polypeptide is composed of LD, PSBD, and CD domains. Most studies had focused on a truncated E2 that is deficient in LD and PSBD, because CD mainly contributes to maintaining the multimeric structure. We examined salt-induced changes in E2 without truncation and constructed reaction models. We speculate that in the presence of KCl, E2 is dissociated into a monomer and then assembled into an aggregative complex (C(A)) and a quasi-stable complex (C(Q)). C(A) was larger than C(Q), but smaller than intact E2. C(A) and C(Q), were dominant complexes at about neutral pH and at basic pH respectively. PDC, in which PSBD is occupied by other components, and a truncated E2 undergo dissociation only. LD-PSBD region besides CD might then contribute to the partial association of dissociated E2. PMID:23924725

  14. A direct, ratiometric, and quantitative MALDI–MS assay for protein methyltransferases and acetyltransferases

    PubMed Central

    Richardson, Stacie L.; Hanjra, Pahul; Zhang, Gang; Mackie, Brianna D.; Peterson, Darrell L.; Huang, Rong

    2016-01-01

    Protein methylation and acetylation play important roles in biological processes, and misregulation of these modifications is involved in various diseases. Therefore, it is critical to understand the activities of the enzymes responsible for these modifications. Herein we describe a sensitive method for ratiometric quantification of methylated and acetylated peptides via MALDI-MS by direct spotting of enzymatic methylation and acetylation reaction mixtures without tedious purification procedures. The quantifiable detection limit for peptides with our method is approximately 10 fmol. This is achieved by increasing the signal-to-noise ratio through the addition of NH4H2PO4 to the matrix solution and reduction of the matrix α-cyanohydroxycinnamic acid concentration to 2 mg/ml. We have demonstrated the application of this method in enzyme kinetic analysis and inhibition studies. The unique feature of this method is the simultaneous quantification of multiple peptide species for investigation of processivity mechanisms. Its wide buffer compatibility makes it possible to be adapted to investigate the activity of any protein methyltransferase or acetyltransferase. PMID:25778392

  15. The Role of Sas2, an Acetyltransferase Homologue of Saccharomyces Cerevisiae, in Silencing and Orc Function

    PubMed Central

    Ehrenhofer-Murray, A. E.; Rivier, D. H.; Rine, J.

    1997-01-01

    Silencing at the cryptic mating-type loci HML and HMR of Saccharomyces cerevisiae requires regulatory sites called silencers. Mutations in the Rap1 and Abf1 binding sites of the HMR-E silencer (HMRa-e**) cause the silencer to be nonfunctional, and hence, cause derepression of HMR. Here, we have isolated and characterized mutations in SAS2 as second-site suppressors of the silencing defect of HMRa-e**. Silencing conferred by the removal of SAS2 (sas2Δ) depended upon the integrity of the ARS consensus sequence of the HMR-E silencer, thus arguing for an involvement of the origin recognition complex (ORC). Restoration of silencing by sas2Δ required ORC2 and ORC5, but not SIR1 or RAP1. Furthermore, sas2Δ suppressed the temperature sensitivity, but not the silencing defect of orc2-1 and orc5-1. Moreover, sas2Δ had opposing effects on silencing of HML and HMR. The putative Sas2 protein bears similarities to known protein acetyltransferases. Several models for the role of Sas2 in silencing are discussed. PMID:9093847

  16. Muscle-specific deletion of carnitine acetyltransferase compromises glucose tolerance and metabolic flexibility.

    PubMed

    Muoio, Deborah M; Noland, Robert C; Kovalik, Jean-Paul; Seiler, Sarah E; Davies, Michael N; DeBalsi, Karen L; Ilkayeva, Olga R; Stevens, Robert D; Kheterpal, Indu; Zhang, Jingying; Covington, Jeffrey D; Bajpeyi, Sudip; Ravussin, Eric; Kraus, William; Koves, Timothy R; Mynatt, Randall L

    2012-05-01

    The concept of "metabolic inflexibility" was first introduced to describe the failure of insulin-resistant human subjects to appropriately adjust mitochondrial fuel selection in response to nutritional cues. This phenomenon has since gained increasing recognition as a core component of the metabolic syndrome, but the underlying mechanisms have remained elusive. Here, we identify an essential role for the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT), in regulating substrate switching and glucose tolerance. By converting acetyl-CoA to its membrane permeant acetylcarnitine ester, CrAT regulates mitochondrial and intracellular carbon trafficking. Studies in muscle-specific Crat knockout mice, primary human skeletal myocytes, and human subjects undergoing L-carnitine supplementation support a model wherein CrAT combats nutrient stress, promotes metabolic flexibility, and enhances insulin action by permitting mitochondrial efflux of excess acetyl moieties that otherwise inhibit key regulatory enzymes such as pyruvate dehydrogenase. These findings offer therapeutically relevant insights into the molecular basis of metabolic inflexibility. PMID:22560225

  17. Human Neural Stem Cells Overexpressing Choline Acetyltransferase Restore Unconditioned Fear in Rats with Amygdala Injury

    PubMed Central

    Shin, Kyungha; Cha, Yeseul; Kim, Kwang Sei; Choi, Ehn-Kyoung; Choi, Youngjin; Guo, Haiyu; Ban, Young-Hwan; Kim, Jong-Choon; Park, Dongsun; Kim, Yun-Bae

    2016-01-01

    Amygdala is involved in the fear memory that recognizes certain environmental cues predicting threatening events. Manipulation of neurotransmission within the amygdala affects the expression of conditioned and unconditioned emotional memories such as fear freezing behaviour. We previously demonstrated that F3.ChAT human neural stem cells (NSCs) overexpressing choline acetyltransferase (ChAT) improve cognitive function of Alzheimer's disease model rats with hippocampal or cholinergic nerve injuries by increasing acetylcholine (ACh) level. In the present study, we examined the effect of F3.ChAT cells on the deficit of unconditioned fear freezing. Rats given N-methyl-d-aspartate (NMDA) in their amygdala 2 weeks prior to cat odor exposure displayed very short resting (freezing) time compared to normal animals. NMDA induced neuronal degeneration in the amygdala, leading to a decreased ACh concentration in cerebrospinal fluid. However, intracerebroventricular transplantation of F3.ChAT cells attenuated amygdala lesions 4 weeks after transplantation. The transplanted cells were found in the NMDA-injury sites and produced ChAT protein. In addition, F3.ChAT-receiving rats recuperated freezing time staying remote from the cat odor source, according to the recovery of brain ACh concentration. The results indicate that human NSCs overexpressing ChAT may facilitate retrieval of unconditioned fear memory by increasing ACh level. PMID:27087745

  18. Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure

    PubMed Central

    Howes, Stuart C.; Alushin, Gregory M.; Shida, Toshinobu; Nachury, Maxence V.; Nogales, Eva

    2014-01-01

    Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments. PMID:24227885

  19. Structural Analysis of a Putative Aminoglycoside N-Acetyltransferase from Bacillus anthracis

    SciTech Connect

    Klimecka, Maria M.; Chruszcz, Maksymilian; Font, Jose; Skarina, Tatiana; Shumilin, Igor; Onopryienko, Olena; Porebski, Przemyslaw J.; Cymborowski, Marcin; Zimmerman, Matthew D.; Hasseman, Jeremy; Glomski, Ian J.; Lebioda, Lukasz; Savchenko, Alexei; Edwards, Aled; Minor, Wladek

    2012-02-15

    For the last decade, worldwide efforts for the treatment of anthrax infection have focused on developing effective vaccines. Patients that are already infected are still treated traditionally using different types of standard antimicrobial agents. The most popular are antibiotics such as tetracyclines and fluoroquinolones. While aminoglycosides appear to be less effective antimicrobial agents than other antibiotics, synthetic aminoglycosides have been shown to act as potent inhibitors of anthrax lethal factor and may have potential application as antitoxins. Here, we present a structural analysis of the BA2930 protein, a putative aminoglycoside acetyltransferase, which may be a component of the bacterium's aminoglycoside resistance mechanism. The determined structures revealed details of a fold characteristic only for one other protein structure in the Protein Data Bank, namely, YokD from Bacillus subtilis. Both BA2930 and YokD are members of the Antibiotic-NAT superfamily (PF02522). Sequential and structural analyses showed that residues conserved throughout the Antibiotic-NAT superfamily are responsible for the binding of the cofactor acetyl coenzyme A. The interaction of BA2930 with cofactors was characterized by both crystallographic and binding studies.

  20. The dietary compound curcumin inhibits p300 histone acetyltransferase activity and prevents heart failure in rats

    PubMed Central

    Morimoto, Tatsuya; Sunagawa, Yoichi; Kawamura, Teruhisa; Takaya, Tomohide; Wada, Hiromichi; Nagasawa, Atsushi; Komeda, Masashi; Fujita, Masatoshi; Shimatsu, Akira; Kita, Toru; Hasegawa, Koji

    2008-01-01

    Hemodynamic overload in the heart can trigger maladaptive hypertrophy of cardiomyocytes. A key signaling event in this process is nuclear acetylation by histone deacetylases and p300, an intrinsic histone acetyltransferase (HAT). It has been previously shown that curcumin, a polyphenol responsible for the yellow color of the spice turmeric, possesses HAT inhibitory activity with specificity for the p300/CREB-binding protein. We found that curcumin inhibited the hypertrophy-induced acetylation and DNA-binding abilities of GATA4, a hypertrophy-responsive transcription factor, in rat cardiomyocytes. Curcumin also disrupted the p300/GATA4 complex and repressed agonist- and p300-induced hypertrophic responses in these cells. Both the acetylated form of GATA4 and the relative levels of the p300/GATA4 complex markedly increased in rat hypertensive hearts in vivo. The effects of curcumin were examined in vivo in 2 different heart failure models: hypertensive heart disease in salt-sensitive Dahl rats and surgically induced myocardial infarction in rats. In both models, curcumin prevented deterioration of systolic function and heart failure–induced increases in both myocardial wall thickness and diameter. From these results, we conclude that inhibition of p300 HAT activity by the nontoxic dietary compound curcumin may provide a novel therapeutic strategy for heart failure in humans. PMID:18292809

  1. Structure of the E. Coli Bifunctional GlmU Acetyltransferase Active Site with Substrates and Products

    SciTech Connect

    Olsen,L.; Vetting, M.; Roderick, S.

    2007-01-01

    The biosynthesis of UDP-GlcNAc in bacteria is carried out by GlmU, an essential bifunctional uridyltransferase that catalyzes the CoA-dependent acetylation of GlcN-1-PO{sub 4} to form GlcNAc-1-PO{sub 4} and its subsequent condensation with UTP to form pyrophosphate and UDP-GlcNAc. As a metabolite, UDP-GlcNAc is situated at a branch point leading to the biosynthesis of lipopolysaccharide and peptidoglycan. Consequently, GlmU is regarded as an important target for potential antibacterial agents. The crystal structure of the Escherichia coli GlmU acetyltransferase active site has been determined in complexes with acetyl-CoA, CoA/GlcN-1-PO{sub 4}, and desulpho-CoA/GlcNAc-1-PO{sub 4}. These structures reveal the enzyme groups responsible for binding the substrates. A superposition of these complex structures suggests that the 2-amino group of GlcN-1-PO{sub 4} is positioned in proximity to the acetyl-CoA to facilitate direct attack on its thioester by a ternary complex mechanism.

  2. Genetically based N-acetyltransferase metabolic polymorphism and low-level environmental exposure to carcinogens.

    PubMed

    Vineis, P; Bartsch, H; Caporaso, N; Harrington, A M; Kadlubar, F F; Landi, M T; Malaveille, C; Shields, P G; Skipper, P; Talaska, G

    1994-05-12

    The metabolic activation or inactivation of carcinogens varies considerably in human populations, and is partly genetically determined. Inter-individual variability in the susceptibility to carcinogens may be particularly important at low degrees of environmental exposure. Examples of probable human carcinogens that present widespread low-dose exposures are environmental tobacco smoke and diesel exhaust. We have determined levels of DNA adducts in bladder cells and of 4-aminobiphenyl-haemoglobin adducts in 97 volunteers, together with the N-acetylation non-inducible phenotype, the corresponding genotype, and the levels of nicotine-cotinine in the urine. We find that among the slow acetylators, 4-aminobiphenyl adducts were higher than in rapid acetylators at low or null nicotine-cotinine levels, whereas the difference between slow and rapid acetylators was less evident at increasing nicotine-cotinine levels. The N-acetyltransferase genotype is highly predictive of the acetylation phenotype. Our results indicate that the clearance of low-dose carcinogens is decreased in the genetically based slow-acetylator phenotype. Such genetic modulation of low-dose environmental risks is relevant to 'risk assessment' procedures. PMID:7909916

  3. Exogenous nerve growth factor stimulates choline acetyltransferase activity in aging Fischer 344 male rats.

    PubMed

    Williams, L R

    1991-01-01

    The effect of age and exogenous nerve growth factor (NGF) infusion on choline acetyltransferase (ChAT) specific activity is examined in microdissections of cerebral and hippocampal cortices, and the cholinergic nuclei of the medial septum and diagonal band of Broca (MS/DB), the nucleus basalis magnocellularis (NBM), and striatum of Fischer 344 male rats. Significant, 20% losses in ChAT activity are found in the MS/DB and striatum of 24-month-old rats (n = 21) compared to 4-month-old animals, but there is no apparent loss of enzyme activity in the NBM. Loss of ChAT activity in the MS/DB is only observed in animals older than 19 months of age, while a striatal deficit is found in animals older than 7 months. Treatment for 2 weeks with NGF at 1.2 micrograms/day results in significant 70% increases of ChAT activity in the MS/DB and striatum of 24-month-old rats compared to untreated and vehicle-treated 4-month-old rats, but does not stimulate activity in the NBM. Sensitivity of ChAT activity in the MS/DB and striatum to exogenous NGF increases with age. These experiments indicate that in the MS/DB, NBM, and striatum of Fischer 344 male rat there is an age-associated, differential regulation of ChAT enzyme activity and sensitivity to exogenous NGF.

  4. Effects of sex hormones, forskolin, and nicotine on choline acetyltransferase activity in human isolated placenta.

    PubMed

    Wessler, Ignaz; Schwarze, Sören; Brockerhoff, Peter; Bittinger, Fernando; Kirkpatrick, Charles James; Kilbinger, Heinz

    2003-04-01

    The activity of choline acetyltransferase (ChAT) was investigated in the human placenta before and after long-term incubation (24 h) to test the effects of sex hormones, nicotine and forskolin. ChAT activity differed considerably between the amnion (0.03 micromol/mg protein/h) and the villus (0.56). After long-term incubation, ChAT activity persisted in the latter but declined in the amnion. Neither sex hormones (beta-estradiol, testosterone, progesterone; 10 or 100 nM each) nor follicle stimulating hormone and luteinizing hormone (FSH/LH; 8.4 U/ml each) modified ChAT activity. Also nicotine (1 nM-100 microM) did not affect ChAT activity. Forskolin, an activitor of adenylyl cyclase, reduced ChAT activity in the villus but not in amnion. The present model offers the possibility to investigate ChAT regulation in intact tissue under long-term incubation. The risks of maternal smoking during pregnancy cannot be attributed to an effect of nicotine on placental ChAT activity. Differences in the regulation of ChAT appear to exist between neuronal and nonneuronal cells.

  5. Histone acetyltransferase mediated regulation of FOXP3 acetylation and Treg function

    PubMed Central

    Xiao, Yan; Li, Bin; Zhou, Zhaocai; Hancock, Wayne W.; Zhang, Hongtao; Greene, Mark I.

    2010-01-01

    Regulatory T cells (Tregs) are required for the maintenance of immune homeostasis as first clearly described by Herman Waldmann’s laboratory. Dysfunction of Treg cells also leads to fatal autoimmunity in humans and mice. Conversely, the activation of different classes of Tregs operative systemically and within the cancer microenvironment can suppress host anti-tumor immune responses and promote tumor progression. Therefore, the development of new therapeutic approaches to regulate the activity of Treg cells may have considerable clinical potential. FOXP3 is the key transcriptional regulator of Treg development and function. The activity of FOXP3 is regulated by acetylation, a process catalyzed by distinct types of histone/protein acetyltransferases (HATs) that regulate the functions of many transcription factors, independently of FOXP3, as well as non-histone proteins, in addition to their effects on chromatin accessibility. Interactions between FOXP3 and these enzymes determine the suppressive function of FOXP3. Clearly, small molecules targeting these enzymes are candidates for the regulation of Treg function in vaccines and tumor therapies. PMID:20869864

  6. Human Neural Stem Cells Overexpressing Choline Acetyltransferase Restore Unconditioned Fear in Rats with Amygdala Injury.

    PubMed

    Shin, Kyungha; Cha, Yeseul; Kim, Kwang Sei; Choi, Ehn-Kyoung; Choi, Youngjin; Guo, Haiyu; Ban, Young-Hwan; Kim, Jong-Choon; Park, Dongsun; Kim, Yun-Bae

    2016-01-01

    Amygdala is involved in the fear memory that recognizes certain environmental cues predicting threatening events. Manipulation of neurotransmission within the amygdala affects the expression of conditioned and unconditioned emotional memories such as fear freezing behaviour. We previously demonstrated that F3.ChAT human neural stem cells (NSCs) overexpressing choline acetyltransferase (ChAT) improve cognitive function of Alzheimer's disease model rats with hippocampal or cholinergic nerve injuries by increasing acetylcholine (ACh) level. In the present study, we examined the effect of F3.ChAT cells on the deficit of unconditioned fear freezing. Rats given N-methyl-d-aspartate (NMDA) in their amygdala 2 weeks prior to cat odor exposure displayed very short resting (freezing) time compared to normal animals. NMDA induced neuronal degeneration in the amygdala, leading to a decreased ACh concentration in cerebrospinal fluid. However, intracerebroventricular transplantation of F3.ChAT cells attenuated amygdala lesions 4 weeks after transplantation. The transplanted cells were found in the NMDA-injury sites and produced ChAT protein. In addition, F3.ChAT-receiving rats recuperated freezing time staying remote from the cat odor source, according to the recovery of brain ACh concentration. The results indicate that human NSCs overexpressing ChAT may facilitate retrieval of unconditioned fear memory by increasing ACh level. PMID:27087745

  7. Effects of humic acid-metal complexes on hepatic carnitine palmitoyltransferase, carnitine acetyltransferase and catalase activities

    SciTech Connect

    Fungjou Lu; Youngshin Chen . Dept. of Biochemistry); Tienshang Huang . Dept. of Medicine)

    1994-03-01

    A significant increase in activities of hepatic carnitine palmitoyltransferase and carnitine acetyltransferase was observed in male Balb/c mice intraperitoneally injected for 40 d with 0.125 mg/0.1 ml/d humic acid-metal complexes. Among these complexes, the humic acid-As complex was relatively effective, whereas humic acid-25 metal complex was more effective, and humic acid-26 metal complex was most effective. However, humic acid or metal mixtures, or metal such as As alone, was not effective. Humic acid-metal complexes also significantly decreased hepatic catalase activity. A marked decrease of 60-kDa polypeptide in liver cytoplasm was also observed on SDS-polyacrylamide gel electrophoresis after the mice had been injected with the complexes. Morphological analysis of a histopathological biopsy of such treated mice revealed several changes in hepatocytes, including focal necrosis and cell infiltration, mild fatty changes, reactive nuclei, and hypertrophy. Humic acid-metal complexes affect activities of metabolic enzymes of fatty acids, and this results in accumulation of hydrogen peroxide and increase of the lipid peroxidation. The products of lipid peroxidation may be responsible for liver damage and possible carcinogenesis. Previous studies in this laboratory had shown that humic acid-metal complex altered the coagulation system and that humic acid, per se, caused vasculopathy. Therefore, humic acid-metal complexes may be main causal factors of not only so-called blackfoot disease, but also the liver cancer prevailing on the southwestern coast of Taiwan.

  8. Perturbation of Mitosis through Inhibition of Histone Acetyltransferases: The Key to Ochratoxin A Toxicity and Carcinogenicity?

    PubMed Central

    Czakai, Kristin; Müller, Katja; Mosesso, Pasquale; Pepe, Gaetano; Schulze, Markus; Gohla, Antje; Patnaik, Debasis; Dekant, Wolfgang; Higgins, Jonathan M.G.; Mally, Angela

    2011-01-01

    Ochratoxin A (OTA) is one of the most potent rodent renal carcinogens studied to date. Although controversial results regarding OTA genotoxicity have been published, it is now widely accepted that OTA is not a mutagenic, DNA-reactive carcinogen. Instead, increasing evidence from both in vivo and in vitro studies suggests that OTA may promote genomic instability and tumorigenesis through interference with cell division. The aim of the present study was to provide further support for disruption of mitosis as a key event in OTA toxicity and to understand how OTA mediates these effects. Immortalized human kidney epithelial cells (IHKE) were treated with OTA and monitored by differential interference contrast microscopy for 15 h. Image analysis confirmed that OTA at concentrations ≥ 5μM, which correlate with plasma concentrations in rats under conditions of carcinogenesis, causes sustained mitotic arrest and exit from mitosis without nuclear or cellular division. Mitotic chromosomes were characterized by aberrant condensation and premature sister chromatid separation associated with altered phosphorylation and acetylation of core histones. To test if OTA directly interferes with histone acetyltransferases (HATs) which regulate lysine acetylation of histones and nonhistone proteins, a cell-free HAT activity assay was conducted using total nuclear extracts of IHKE cells. In this assay, OTA significantly blocked HAT activity in a concentration-dependent manner Overall, results from this study provide further support for a mechanism of OTA carcinogenicity involving interference with the mitotic machinery and suggest HATs as a primary cellular target of OTA. PMID:21551354

  9. Catalytic mechanism of bleomycin N-acetyltransferase proposed on the basis of its crystal structure.

    PubMed

    Oda, Kosuke; Matoba, Yasuyuki; Noda, Masafumi; Kumagai, Takanori; Sugiyama, Masanori

    2010-01-01

    Bleomycin (Bm) N-acetyltransferase, BAT, is a self-resistance determinant in Bm-producing Streptomyces verticillus ATCC15003. In our present study, we crystallized BAT under both a terrestrial and a microgravity environment in the International Space Station. In addition to substrate-free BAT, the crystal structures of BAT in a binary complex with CoA and in a ternary complex with Bm and CoA were determined. BAT forms a dimer structure via interaction of its C-terminal domains in the monomers. However, each N-terminal domain in the dimer is positioned without mutual interaction. The tunnel observed in the N-terminal domain of BAT has two entrances: one that adopts a wide funnel-like structure necessary to accommodate the metal-binding domain of Bm, and another narrow entrance that accommodates acetyl-CoA (AcCoA). A groove formed on the dimer interface of two BAT C-terminal domains accommodates the DNA-binding domain of Bm. In a ternary complex of BAT, BmA(2), and CoA, a thiol group of CoA is positioned near the primary amine of Bm at the midpoint of the tunnel. This proximity ensures efficient transfer of an acetyl group from AcCoA to the primary amine of Bm. Based on the BAT crystal structure and the enzymatic kinetic study, we propose that the catalytic mode of BAT takes an ordered-like mechanism. PMID:19889644

  10. Choline acetyltransferase in the hippocampus is associated with learning strategy preference in adult male rats.

    PubMed

    Hawley, Wayne R; Witty, Christine F; Daniel, Jill M; Dohanich, Gary P

    2015-08-01

    One principle of the multiple memory systems hypothesis posits that the hippocampus-based and striatum-based memory systems compete for control over learning. Consistent with this notion, previous research indicates that the cholinergic system of the hippocampus plays a role in modulating the preference for a hippocampus-based place learning strategy over a striatum-based stimulus--response learning strategy. Interestingly, in the hippocampus, greater activity and higher protein levels of choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine, are associated with better performance on hippocampus-based learning and memory tasks. With this in mind, the primary aim of the current study was to determine if higher levels of ChAT and the high-affinity choline uptake transporter (CHT) in the hippocampus were associated with a preference for a hippocampus-based place learning strategy on a task that also could be solved by relying on a striatum-based stimulus--response learning strategy. Results confirmed that levels of ChAT in the dorsal region of the hippocampus were associated with a preference for a place learning strategy on a water maze task that could also be solved by adopting a stimulus-response learning strategy. Consistent with previous studies, the current results support the hypothesis that the cholinergic system of the hippocampus plays a role in balancing competition between memory systems that modulate learning strategy preference.

  11. Characterization of two metagenome-derived esterases that reactivate chloramphenicol by counteracting chloramphenicol acetyltransferase.

    PubMed

    Tao, Weixin; Lee, Myung Hwan; Yoon, Mi-Young; Kim, Jin-Cheol; Malhotra, Shweta; Wu, Jing; Hwang, Eul Chul; Lee, Seon-Woo

    2011-12-01

    Function-driven metagenomic analysis is a powerful approach to screening for novel biocatalysts. In this study, we investigated lipolytic enzymes selected from an alluvial soil metagenomic library, and identified two novel esterases, EstDL26 and EstDL136. EstDL26 and EstDL136 reactivated chloramphenicol from its acetyl derivates by counteracting the chloramphenicol acetyltransferase (CAT) activity in Escherichia coli. These two enzymes showed only 27% identity in amino acid sequence to each other; however both preferentially hydrolyzed short-chain p-nitrophenyl esters (< or =C5) and showed mesophilic properties. In vitro, EstDL136 catalyzed the deacetylation of 1- and 3- acetyl and 1,3-diacetyl derivates; in contrast, EstDL26 was not capable of the deacetylation at C1, indicating a potential regioselectivity. EstDL26 and EstDL136 were similar to microbial hormone-sensitive lipase (HSL), and since chloramphenicol acetate esterase (CAE) activity was detected from two other soil esterases in the HSL family, this suggests a distribution of CAE among the soil microorganisms. The isolation and characterization of EstDL26 and EstDL136 in this study may be helpful in understanding the diversity of CAE enzymes and their potential role in releasing active chloramphenicol in the producing bacteria. PMID:22210605

  12. Purification of phosphinothricin acetyltransferase using Reactive brown 10 affinity in a single chromatography step.

    PubMed

    Wang, Cunxi; Lee, Thomas C; Crowley, Kathleen S; Bell, Erin

    2013-08-01

    The expression of phosphinothricin N-acetyltransferase (PAT) protein in transgenic plants confers tolerance to the herbicide glufosinate. To enable the characterization of PAT protein expressed in plants, it is necessary to obtain high purity PAT protein from the transgenic grain. Because transgenically expressed proteins are typical present at very low levels (i.e. 0.1-50 μg protein/g grain), a highly specific and efficient purification protocol is required to purify them. Based on the physicochemical properties of PAT, we developed a novel purification method that is simple, time-saving, inexpensive and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein was purified to homogeneity from cottonseed with high recovery efficiency. As expected, the Reactive brown 10-produced PAT was enzymatically active. Other applications of the method on protein expression and purification, and development of PAT enzymatic inhibitors were also discussed. PMID:23748142

  13. Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75

    SciTech Connect

    Berndsen, Christopher E; Tsubota, Toshiaki; Lindner, Scott E; Lee, Susan; Holton, James M; Kaufman, Paul D; Keck, James L; Denu, John M

    2010-01-12

    Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by {approx}100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the in vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rtt109. Together, these data provide evidence for a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.

  14. Mechanistic and Structural Analysis of Drosophila melanogaster Arylalkylamine N-Acetyltransferases

    PubMed Central

    2015-01-01

    Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and other N-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a critical step in Drosophila melanogaster for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two AANAT variants (AANATA and AANATB) have been identified in D. melanogaster, in which AANATA differs from AANATB by the truncation of 35 amino acids from the N-terminus. We have expressed and purified both D. melanogaster AANAT variants (AANATA and AANATB) in Escherichia coli and used the purified enzymes to demonstrate that this N-terminal truncation does not affect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identified an ordered sequential mechanism, with acetyl-CoA binding first, followed by tyramine. We used a combination of pH–activity profiling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to an assignment of Glu-47 as the general base in catalysis with an apparent pKa of 7.0. Using the data generated for the kinetic mechanism, structure–function relationships, pH–rate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA. PMID:25406072

  15. The NatA Acetyltransferase Couples Sup35 Prion Complexes to the [PSI+] Phenotype

    PubMed Central

    Pezza, John A.; Langseth, Sara X.; Raupp Yamamoto, Rochele; Doris, Stephen M.; Ulin, Samuel P.; Salomon, Arthur R.

    2009-01-01

    Protein-only (prion) epigenetic elements confer unique phenotypes by adopting alternate conformations that specify new traits. Given the conformational flexibility of prion proteins, protein-only inheritance requires efficient self-replication of the underlying conformation. To explore the cellular regulation of conformational self-replication and its phenotypic effects, we analyzed genetic interactions between [PSI+], a prion form of the S. cerevisiae Sup35 protein (Sup35[PSI+]), and the three Nα-acetyltransferases, NatA, NatB, and NatC, which collectively modify ∼50% of yeast proteins. Although prion propagation proceeds normally in the absence of NatB or NatC, the [PSI+] phenotype is reversed in strains lacking NatA. Despite this change in phenotype, [PSI+] NatA mutants continue to propagate heritable Sup35[PSI+]. This uncoupling of protein state and phenotype does not arise through a decrease in the number or activity of prion templates (propagons) or through an increase in soluble Sup35. Rather, NatA null strains are specifically impaired in establishing the translation termination defect that normally accompanies Sup35 incorporation into prion complexes. The NatA effect cannot be explained by the modification of known components of the [PSI+] prion cycle including Sup35; thus, novel acetylated cellular factors must act to establish and maintain the tight link between Sup35[PSI+] complexes and their phenotypic effects. PMID:19073888

  16. Identification of AAAS gene mutation in Allgrove syndrome: A report of three cases

    PubMed Central

    LI, WENJING; GONG, CHUNXIU; QI, ZHAN; WU, DI; CAO, BINGYAN

    2015-01-01

    Allgrove syndrome (AS) is an autosomal recessive congenital disease, caused by mutations in the AAAS gene, and is characterized by the triad of Addison's disease, achalasia and alacrima. The present study describes three newly diagnosed cases of AS, in which genetic analysis of the AAAS gene was used to identify AAAS gene mutations, to enhance the understanding of the pathogenesis and clinical manifestations of AS in the Chinese population. Two of the cases exhibited homozygous mutations of c.771delG (p.Arg258GlyfsX33) in exon 8 and one case exhibited a homozygous mutation of c.1366C>T (p.Q456X) in exon 15. A review of the current literature suggests that the AAAS c.771delG mutation has only been reported in the Chinese population. Genetic analysis of the AAAS gene in Chinese AS patients at a young age may facilitate an earlier diagnosis and the timely initiation of the appropriate treatment, ultimately improving the patient outcome. PMID:26622478

  17. Regulation of Sinorhizobium meliloti 1021 rrnA-reporter gene fusions in response to cold shock.

    PubMed

    Gustafson, Ann M; O'Connell, Kevin P; Thomashow, Michael F

    2002-09-01

    We previously reported that mutants of Sinorhizobium meliloti 1021 carrying luxAB insertions in each of the three 16S rRNA genes exhibited a dramatic (> or = 28-fold) increase in luminescence following a temperature downshift from 30 to 15 degrees C. These results raised the possibility that the rRNA operons (rrn) of S. meliloti were cold shock loci. In testing this possibility, we found that fusion of the S. meliloti 1021 rrnA promoter to two different reporter genes, luxAB and uidA, resulted in hybrid genes that were transiently upregulated (as measured by transcript accumulation) about four- to sixfold in response to a temperature downshift. These results are consistent with the hypothesis that the rrn promoters are transiently upregulated in response to cold shock. However, much of the apparent cold shock regulation of the initial luxAB insertions was due to an unexpected mechanism: an apparent temperature-dependent inhibition of translation. Specifically, the rrnA sequences from +1 to +172 (relative to the start of transcription) were found to greatly decrease the ability of S. meliloti to translate hybrid rrn-luxAB transcripts into active protein at 30 degrees C. This effect, however, was largely eliminated at 15 degrees C. Possible mechanisms for the apparent transient increase in rrnA promoter activity and temperature-dependent inhibition of translation are discussed.

  18. A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

    PubMed

    Marc; Granger; Brincat; Fisher; Kao; McCubbin; Cyr

    1998-11-01

    Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation. PMID:9811799

  19. Validation of Mitochondrial Gene Delivery in Liver and Skeletal Muscle via Hydrodynamic Injection Using an Artificial Mitochondrial Reporter DNA Vector.

    PubMed

    Yasuzaki, Yukari; Yamada, Yuma; Ishikawa, Takuya; Harashima, Hideyoshi

    2015-12-01

    For successful mitochondrial transgene expression, two independent processes, i.e., developing a mitochondrial gene delivery system and construction of DNA vector to achieve mitochondrial gene expression, are required. To date, very few studies dealing with mitochondrial gene delivery have been reported and, in most cases, transgene expression was not validated, because the construction of a reporter DNA vector for mitochondrial gene expression is the bottleneck. In this study, mitochondrial transgene expression by the in vivo mitochondrial gene delivery of an artificial mitochondrial reporter DNA vector via hydrodynamic injection is demonstrated. In the procedure, a large volume of naked plasmid DNA (pDNA) is rapidly injected. We designed and constructed pHSP-mtLuc (CGG) as a mitochondrial reporter DNA vector that possesses a mitochondrial heavy strand promoter (HSP) and an artificial mitochondrial genome with the reporter NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. We delivered the pDNA into mouse liver mitochondria by hydrodynamic injection, and detected exogenous mRNA in the liver using reverse transcription PCR analysis. The hydrodynamic injection of pHSP-mtLuc (CGG) resulted in the expression of the Nluc luciferase protein in liver and skeletal muscle. Our mitochondrial transgene expression reporter system would contribute to mitochondrial gene therapy and further studies directed at mitochondrial molecular biology.

  20. c-Ha-ras down regulates the alpha-fetoprotein gene but not the albumin gene in human hepatoma cells.

    PubMed Central

    Nakao, K; Lawless, D; Ohe, Y; Miyao, Y; Nakabayashi, H; Kamiya, H; Miura, K; Ohtsuka, E; Tamaoki, T

    1990-01-01

    We studied the effects of transfection of the normal c-Ha-ras gene, rasGly-12, and its oncogenic mutant, rasVal-12, on expression of the alpha-fetoprotein (AFP) and albumin genes in a human hepatoma cell line, HuH-7. The mutant and, to a lesser extent, the normal ras gene caused reduction of the AFP mRNA but not the albumin mRNA level in transfected HuH-7 cells. Cotransfection experiments with a rasVal-12 expression plasmid and a chloramphenicol acetyltransferase reporter gene fused to AFP regulatory sequences showed that rasVal-12 suppressed the activity of enhancer and promoter regions containing A + T-rich sequences (AT motif). In contrast, rasVal-12 did not affect the promoter activity of the albumin and human hepatitis B virus pre-S1 genes even though these promoters contain homologous A + T-rich elements. ras transfection appeared to induce phosphorylation of nuclear proteins that interact with the AFP AT motif, since gel mobility analysis revealed the formation of slow-moving complexes which was reversed by phosphatase treatment. However, similar changes in complex formation were observed with the albumin and hepatitis B surface antigen pre-S1 promoters. Therefore, this effect alone cannot explain the specific down regulation of the AFP promoter and enhancer activity. ras-mediated suppression of the AFP gene may reflect the process of developmental gene regulation in which AFP gene transcription is controlled by a G-protein-linked signal transduction cascade triggered by external growth stimuli. Images PMID:1690841

  1. Quantitative analysis of gene expression in preimplantation mouse embryos using green fluorescent protein reporter.

    PubMed

    Medvedev, Serguei Yuri; Tokunaga, Tomoyuki; Schultz, Richard M; Furukawa, Tsutomu; Nagai, Takashi; Yamaguchi, Manabu; Hosoe, Misa; Yakovlev, Alexander F; Takahashi, Seiya; Izaike, Yoshiaki

    2002-07-01

    We have developed a method to monitor noninvasively, quantitatively, and in real-time transcription in living preimplantation mouse embryos by measuring expression of a short half-life form of enhanced green fluorescent protein (EGFP) following microinjection of a plasmid-borne EGFP reporter gene. A standard curve was established by injecting known amounts of recombinant green fluorescent protein, and transcriptional activity was then determined by interpolating the amount of fluorescence in the DNA-injected embryos. This approach permitted multiple measurements in single embryos with no significant detrimental effect on embryonic development as long as light exposure was brief (<30 sec) and no more than two measurements were made each day. This method should facilitate analysis of the regulation of gene expression in preimplantation embryos; in particular, during the maternal-to-zygotic transition, and in other species in which limited numbers of embryos are available. PMID:12080029

  2. Gene-Environment Interactions in Cancer Epidemiology: A National Cancer Institute Think Tank Report

    PubMed Central

    Hutter, Carolyn M.; Mechanic, Leah E.; Chatterjee, Nilanjan; Kraft, Peter; Gillander, Elizabeth M.

    2014-01-01

    Cancer risk is determined by a complex interplay of genetic and environmental factors. Genome-wide association studies (GWAS) have identified hundreds of common (minor allele frequency [MAF]>0.05) and less common (0.01genes and environment, including gene-environment interactions, into epidemiologic studies of cancer. To help address these questions, and to better inform research priorities and allocation of resources, the National Cancer Institute sponsored a “Gene-Environment Think Tank” on January 10th–011th, 2012. The objective of the Think Tank was to facilitate discussions on: 1) the state of the science; 2) the goals of gene-environment interaction studies in cancer epidemiology; and 3) opportunities for developing novel study designs and analysis tools. This report summarizes the Think Tank discussion, with a focus on contemporary approaches to the analysis of gene-environment interactions. Selecting the appropriate methods requires first identifying the relevant scientific question and rationale, with an important distinction made between analyses aiming to characterize the joint effects of putative or established genetic and environmental factors and analyses aiming to discover novel risk factors or novel interaction effects. Other discussion items include measurement error, statistical power, significance and replication. Additional designs, exposure assessments, and analytical approaches need to be considered as we move from the current small number of success stories to a fuller understanding of the interplay of genetic and environmental factors. PMID:24123198

  3. First report of multiresistance gene cfr in Enterococcus species casseliflavus and gallinarum of swine origin.

    PubMed

    Liu, Yang; Wang, Yang; Dai, Lei; Wu, Congming; Shen, Jianzhong

    2014-06-01

    The aim of this study was to investigate the presence and genetic environment of the multiresistance gene cfr in Enterococcus species of swine origin. Twenty-five cfr-carrying Enterococcus isolates were collected from swine in Beijing, Guangzhou, and Shandong, China. The isolates consist of 24 Enterococcus casseliflavus and one Enterococcus gallinarum isolate, and exhibited six SmaI PFGE patterns. The cfr gene was located on plasmids in all isolates except E. casseliflavus En83, in which cfr was located on the chromosomal DNA. The cfr gene environments in most of these isolates contain DNA sequences similar to pEF-01, which was first found in Enterococcus. However, inverse PCR analysis suggested that the cfr-carrying circular forms might be different from pEF-01. The circular forms in Eg51 and its transconjugant, and En23, En10, and En94 are similar to the circular form in pEF-01, except for the truncated IS1216, which is replaced by a transposase of the IS256 family in En24. The cfr circular form could not be detected in either En77 or En83, and the same cfr-carrying segments of ∼ 10 kb had only 3500bp of sequence similar to pEF-01. This is the first report of cfr gene in E. casseliflavus and E. gallinarum. The potential dissemination of the multidrug resistance gene amongst different bacterial species, especially in enterococci of human and animal origins, is concerning and should be closely monitored.

  4. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products. Progress report, June 1, 1990--June 30, 1992

    SciTech Connect

    Kuchka, M.R.

    1992-08-01

    Many individual chloroplast genes require the products of a collection of nuclear genes for their successful expression. These nuclear gene products apparently work with great specificity, each committed to the expression of a single chloroplast gene. We have chosen as a model nuclear mutants of Chlamydomonas affected in different stages in the expression of the chloroplast encoded Photosystem II polypeptide, D2. We have made the progress in understanding how nuclear gene products affect the translation of the D2 encoding MRNA. Two nuclear genes are required for this process which have been mapped genetically. In contrast to other examples of nuclear control of translation in the chloroplast, these nuclear gene products appear to be required either for specific stages in translation elongation or for the post-translational stabilization of the nascent D2 protein. Pseudoreversion analysis has led us to a locus which may be directly involved in D2 expression. We have made considerable progress in pursuing the molecular basis of psbd MRNA stabilization. psbD 5` UTR specific transcripts have been synthesized in vitro and used in gel mobility shift assays. UV-crosslinking studies are underway to identify the transacting factors which bind to these sequences. The continued examination of these mutants will help us to understand how nuclear gene products work in this specific case of chloroplast gene expression, and will elucidate how two distinct genomes can interact generally.

  5. Arsenic Trioxide Reduces Global Histone H4 Acetylation at Lysine 16 through Direct Binding to Histone Acetyltransferase hMOF in Human Cells

    PubMed Central

    Liu, Da; Wu, Donglu; Zhao, Linhong; Yang, Yang; Ding, Jian; Dong, Liguo; Hu, Lianghai; Wang, Fei; Zhao, Xiaoming; Cai, Yong; Jin, Jingji

    2015-01-01

    Histone post-translational modification heritably regulates gene expression involved in most cellular biological processes. Experimental studies suggest that alteration of histone modifications affects gene expression by changing chromatin structure, causing various cellular responses to environmental influences. Arsenic (As), a naturally occurring element and environmental pollutant, is an established human carcinogen. Recently, increasing evidence suggests that As-mediated epigenetic mechanisms may be involved in its toxicity and carcinogenicity, but how this occurs is still unclear. Here we present evidence that suggests As-induced global histone H4K16 acetylation (H4K16ac) partly due to the direct physical interaction between As and histone acetyltransferase (HAT) hMOF (human male absent on first) protein, leading to the loss of hMOF HAT activity. Our data show that decreased global H4K16ac and increased deacetyltransferase HDAC4 expression occurred in arsenic trioxide (As2O3)-exposed HeLa or HEK293T cells. However, depletion of HDAC4 did not affect global H4K16ac, and it could not raise H4K16ac in cells exposed to As2O3, suggesting that HDAC4 might not directly be involved in histone H4K16 de-acetylation. Using As-immobilized agarose, we confirmed that As binds directly to hMOF, and that this interaction was competitively inhibited by free As2O3. Also, the direct interaction of As and C2CH zinc finger peptide was verified by MAIDI-TOF mass and UV absorption. In an in vitro HAT assay, As2O3 directly inhibited hMOF activity. hMOF over-expression not only increased resistance to As and caused less toxicity, but also effectively reversed reduced H4K16ac caused by As exposure. These data suggest a theoretical basis for elucidating the mechanism of As toxicity. PMID:26473953

  6. Search for major genes with progeny test data to accelerate the development of genetically superior loblolly pine. Technical progress report

    SciTech Connect

    2000-02-15

    This report details the progress of the three tasks of this project. The tasks are: (1) develop genetic models and analytical methods; (2) molecular confirmation of major gene segregation; and (3) develop strategies for marker-assisted breeding.

  7. Acrolein, an α,β-unsaturated aldehyde, irreversibly inhibits the acetylation of aromatic amine xenobiotics by human arylamine N-acetyltransferase 1.

    PubMed

    Bui, Linh C; Manaa, Amine; Xu, Ximing; Duval, Romain; Busi, Florent; Dupret, Jean-Marie; Rodrigues-Lima, Fernando; Dairou, Julien

    2013-07-01

    Acrolein is an electrophilic α,β-unsaturated aldehyde of industrial, pharmaceutic, and toxicologic importance to which we are exposed in environmental, occupational, and therapeutic situations. Acrolein is known to exert different biologic effects through reactions with cellular macromolecules such as DNA, certain proteins, or glutathione. In many situations (such as in tobacco smoke or other fumes), exposure to acrolein occurs concomitantly with other compounds such as aromatic amine chemicals. Interestingly, it has been shown that acrolein could impact the cellular metabolism of aromatic xenobiotics through an indirect mechanism based on the transcriptional induction of phase II xenobiotic-metabolizing enzymes. Here we report a novel mechanism by which acrolein acts on the metabolism of aromatic foreign chemicals. We provide molecular, kinetic, and cellular evidence that acrolein can react directly and irreversibly with arylamine N-acetyltransferases, a major family of xenobiotic-metabolizing enzymes involved in the metabolization of aromatic amine chemicals. Formation of an acrolein adduct with a catalytic cysteine residue in the active site is responsible for the impairment of aromatic amine acetylation by the enzyme. This biochemical process may represent an additional mechanism by which acrolein impacts the metabolism and fate of aromatic amine drugs and pollutants.

  8. Genetic variation in aryl N-acetyltransferase results in significant differences in the pharmacokinetic and safety profiles of amifampridine (3,4-diaminopyridine) phosphate

    PubMed Central

    Haroldsen, Peter E; Garovoy, Marvin R; Musson, Donald G; Zhou, Huiyu; Tsuruda, Laurie; Hanson, Boyd; O’Neill, Charles A

    2015-01-01

    The clinical use of amifampridine phosphate for neuromuscular junction disorders is increasing. The metabolism of amifampridine occurs via polymorphic aryl N-acetyltransferase (NAT), yet its pharmacokinetic (PK) and safety profiles, as influenced by this enzyme system, have not been investigated. The objective of this study was to assess the effect of NAT phenotype and genotype on the PK and safety profiles of amifampridine in healthy volunteers (N = 26). A caffeine challenge test and NAT2 genotyping were used to delineate subjects into slow and fast acetylators for PK and tolerability assessment of single, escalating doses of amifampridine (up to 30 mg) and in multiple daily doses (20 mg QID) of amifampridine. The results showed that fast acetylator phenotypes displayed significantly lower Cmax, AUC, and shorter t1/2 for amifampridine than slow acetylators. Plasma concentrations of the N-acetyl metabolite were approximately twofold higher in fast acetylators. Gender differences were not observed. Single doses of amifampridine demonstrated dose linear PKs. Amifampridine achieved steady state plasma levels within 1 day of dosing four times daily. No accumulation or time-dependent changes in amifampridine PK parameters occurred. Overall, slow acetylators reported 73 drug-related treatment-emergent adverse events versus 6 in fast acetylators. Variations in polymorphic NAT corresponding with fast and slow acetylator phenotypes significantly affects the PK and safety profiles of amifampridine. PMID:25692017

  9. Genetic analysis of the regulation of TCH gene expression, Final Report

    SciTech Connect

    Braam, Janet

    2008-10-28

    The Arabidopsis TCH genes, originally isolated as a consequence of their upregulation in response to the mechanical stimulus of touch, are also upregulated by a variety of seemingly disparate environmental and hormonal stimuli. To gain insight into the complexities of TCH gene regulation, a number of approaches were taken. Regulatory elements responsible for regulation were identified and characteristics of the regulation were evaluated. Reporter genes were used to monitor expression localization and dynamics. Microarray analyses of genome-wide expression behavior indicated that touch-inducible gene expression is more widespread than generally appreciated. Identification of all touch-regulated genes shed light on the types of cellular processes that may be altered in response to mechanical stress perturbations. Expression of the TCH2 gene, also called CML24, encoding a calmodulin (CaM)-like (CML) protein, was evaluated. CML24 shares over 40% amino acid sequence identity with CaM, has 4 EF hands and undergoes a Ca2+-dependent change in migration rate through denaturing gel electrophoresis, indicating that CML24 binds Ca2+ and, as a consequence, undergoes conformational changes. CML24 expression occurs in all major organs and is induced from 2- to 15-fold in plants subjected to touch, darkness, heat, cold, hydrogen peroxide, abscisic acid (ABA) and indole-3-acetic acid. The putative CML24 regulatory region confers reporter expression at sites of predicted mechanical stress, in regions undergoing growth, in vascular tissues and various floral organs and in stomata, trichomes and hydathodes. CML24 underexpressing transgenics are resistant to ABA inhibition of germination and seedling growth, defective in long-day induction of flowering, and have enhanced tolerance to CoCl2, molybdic acid, ZnSO4 and MgCl2. These data present evidence that CML24 encodes a potential Ca2+ sensor that may function to enable responses to ABA, day length and presence of various salts. Further

  10. Self-Immolative Polycations as Gene Delivery Vectors and Prodrugs Targeting Polyamine Metabolism in Cancer

    PubMed Central

    2015-01-01

    Polycations are explored as carriers to deliver therapeutic nucleic acids. Polycations are conventionally pharmacological inert with the sole function of delivering therapeutic cargo. This study reports synthesis of a self-immolative polycation (DSS-BEN) based on a polyamine analogue drug N1,N11-bisethylnorspermine (BENSpm). The polycation was designed to function dually as a gene delivery carrier and a prodrug targeting dysregulated polyamine metabolism in cancer. Using a combination of NMR and HPLC, we confirm that the self-immolative polycation undergoes intracellular degradation into the parent drug BENSpm. The released BENSpm depletes cellular levels of spermidine and spermine and upregulates polyamine catabolic enzymes spermine/spermidine N1-acetyltransferase (SSAT) and spermine oxidase (SMO). The synthesized polycations form polyplexes with DNA and facilitate efficient transfection. Taking advantage of the ability of BENSpm to sensitize cancer cells to TNFα-induced apoptosis, we show that DSS-BEN enhances the cell killing activity of TNFα gene therapy. The reported findings validate DSS-BEN as a dual-function delivery system that can deliver a therapeutic gene and improve the outcome of gene therapy as a result of the intracellular degradation of DSS-BEN to BENSpm and the subsequent beneficial effect of BENSpm on dysregulated polyamine metabolism in cancer. PMID:25153488

  11. Functional activation of the egr-1 (early growth response-1) gene by hydrogen peroxide.

    PubMed

    Nose, K; Ohba, M

    1996-06-01

    The redox-based regulation of gene expression is one of the fundamental mechanisms of cellular functions, and hydrogen peroxide seems to act as an intracellular second messenger of signal transduction of cytokines. Hydrogen peroxide at non-toxic doses induced the accumulation of mRNA for the early growth response-1 (egr-1) gene in mouse osteoblastic cells. The Egr-1 protein is a transcription factor that binds the GCGGGGGCG sequence and contains a zinc-finger structure that is essential for DNA binding. Egr-1 protein is sensitive to oxidative stress and loses specific DNA-binding activity when exposed to high levels of oxidative stress. Incubating cells with hydrogen peroxide at about 50 microM, however, increased the accumulation of Egr-1 protein, and the Egr-1 product seemed to be functional, judging by its binding activity to the GCGGGGGCG sequence and its ability to activate the chloramphenicol acetyltransferase reporter gene under the control of the human thymidine kinase enhancer containing the Egr-1 binding sequence. It was reported that the activity of Egr-1 protein as a transcription factor was negatively regulated by active oxygens. However, with appropriate concentrations of active oxygen, its capacity to bind a specific DNA sequence and to enhance the transcriptional activity of target genes is thought to be elevated.

  12. Retinoid-mediated transcriptional regulaton of keratin genes in human epidermal and squamous cell carcinoma cells

    SciTech Connect

    Stellmach, V.; Leask, A.; Fuchs, E. )

    1991-06-01

    Vitamin A and other retinoids profoundly inhibit morphological and biochemical heatures of epidermal differentiation in vivo and in vitro. To elucidate the molecular mechanisms underlying the differential expression of epidermal keratins and their regulation by retinoids, the authors retinoid-mediated changes in total protein expression, protein synthesis, mRNA expression, and transcription in cultured human keratinocytes and in squamous cell carcinoma (SCC-13) cells of epidermal origin. The studies revealed that the epidermal keratins, K5, K6, K14, and K16, their mRNAs, and their transcripts were diminished relative to actin as a consequence of retinoic acid (RA) treatment. The effects were most pronounced in SCC-13 and were detected as early as 6 hr post-RA treatment, with enhancement over an additional 24-48 hr. Repression was also observed when 5{prime} upstream sequences of K14 or K5 genes were used to drive expression of a chloramphenicol acetyltransferase reporter gene in SCC-13 keratinocytes. Both cell types were found to express mRNAs for the RA receptors {alpha} and {gamma}, which may be involved in the RA-mediated transcriptional changes in these cells. The rapid transcriptional changes in epidermal keratin genes were in striking contrast to the previously reported slow transcriptional changes in simple epithelial keratin genes.

  13. Brief Report: Aggression and Stereotypic Behavior in Males with Fragile X Syndrome-- Moderating Secondary Genes in a "Single Gene" Disorder

    ERIC Educational Resources Information Center

    Hessl, David; Tassone, Flora; Cordeiro, Lisa; Koldewyn, Kami; McCormick, Carolyn; Green, Cherie; Wegelin, Jacob; Yuhas, Jennifer; Hagerman, Randi J.

    2008-01-01

    Although fragile X syndrome (FXS) is a single gene disorder with a well-described phenotype, it is not known why some individuals develop more significant maladaptive behaviors such as aggression or autistic symptoms. Here, we studied two candidate genes known to affect mood and aggression, the serotonin transporter (5-HTTLPR) and monoamine…

  14. Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage

    PubMed Central

    Balic, Adam; Garcia-Morales, Carla; Vervelde, Lonneke; Gilhooley, Hazel; Sherman, Adrian; Garceau, Valerie; Gutowska, Maria W.; Burt, David W.; Kaiser, Pete; Hume, David A.; Sang, Helen M.

    2014-01-01

    We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens. PMID:25063453

  15. Detection of transformed cells in crown gall tumors using the GUS reporter gene and correlation of GUS stained cells with T-DNA gene activity

    SciTech Connect

    Black, R.C. ); Labriola, J.; Binns, A.N. )

    1990-05-01

    Crown gall tumors are a mixture of transformed hormone producing cells and normal cells. Until now it has not been possible to directly visualize these cell types in situ. We have constructed strains of Agrobacterium tumefaciens that carry the 35S-{beta}-glucuronidase (GUS) reporter gene in either wild type or mutant Ti plasmids. Using histochemical staining for GUS activity, blue (GUS positive) sectors are observed in tumor sections. In order to demonstrate that the blue sectors actually represent cells expressing other T-DNA genes, we have looked for T-DNA gene encoded enzyme activity in the stained and unstained sectors. The blue sectors accumulate octopine (a product of the octopine synthase gene on the T-DNA) while the white (GUS negative) sectors do not. We conclude that the use of the GUS reporter gene provides a sensitive and reliable method for visualizing transformation events in plant tissues. A comparison of the proportion of transformed and nontransformed cells in wild type tumors vs. tumors deficient in auxin or cytokinin encoding genes will be discussed.

  16. Identification of genes in anonymous DNA sequences. Annual performance report, February 1, 1991--January 31, 1992

    SciTech Connect

    Fields, C.A.

    1996-06-01

    The objective of this project is the development of practical software to automate the identification of genes in anonymous DNA sequences from the human, and other higher eukaryotic genomes. A software system for automated sequence analysis, gm (gene modeler) has been designed, implemented, tested, and distributed to several dozen laboratories worldwide. A significantly faster, more robust, and more flexible version of this software, gm 2.0 has now been completed, and is being tested by operational use to analyze human cosmid sequence data. A range of efforts to further understand the features of eukaryoyic gene sequences are also underway. This progress report also contains papers coming out of the project including the following: gm: a Tool for Exploratory Analysis of DNA Sequence Data; The Human THE-LTR(O) and MstII Interspersed Repeats are subfamilies of a single widely distruted highly variable repeat family; Information contents and dinucleotide compostions of plant intron sequences vary with evolutionary origin; Splicing signals in Drosophila: intron size, information content, and consensus sequences; Integration of automated sequence analysis into mapping and sequencing projects; Software for the C. elegans genome project.

  17. A Novel Homozygous Mutation in the Transient Receptor Potential Melastatin 6 Gene: A Case Report

    PubMed Central

    Altıncık, Ayça; Schlingmann, Karl Peter; Tosun, Mahya Sultan

    2016-01-01

    Hereditary hypomagnesemia with secondary hypocalcemia (HSH) is a rare autosomal recessive disease caused by mutations in the transient receptor potential melastatin 6 (TRPM6) gene. Affected individuals present in early infancy with seizures caused by the severe hypocalcemia and hypomagnesemia. By presenting this case report, we also aimed to highlight the need for molecular genetic analysis in inbred or familial cases with hypomagnesemia. A Turkish inbred girl, now aged six years, had presented to another hospital at age two months with seizures diagnosed to be due to hypomagnesemia. She was on magnesium replacement therapy when she was admitted to our clinic with complaints of chronic diarrhea at age 3.6 years. During her follow-up in our clinic, she showed an age-appropriate physical and neurological development. In molecular genetic analysis, a novel homozygous frame-shift mutation (c.3447delT>p.F1149fs) was identified in the TRPM6 gene. This mutation leads to a truncation of the TRPM6 protein, thereby complete loss of function. We present the clinical follow-up findings of a pediatric HSH case due to a novel mutation in the TRPM6 gene and highlight the need for molecular genetic analysis in inbred or familial cases with hypomagnesemia. PMID:26759217

  18. Cytochrome P450 2C9 gene polymorphism in phenytoin induced gingival enlargement: A case report.

    PubMed

    Babu, S P K Kennedy; Ramesh, V; Samidorai, Agila; Charles, N S C

    2013-07-01

    Gingival enlargement comprises any clinical condition in which an increase in the size of the gingiva is observed. Among the drugs that induce gingival enlargement, the antiepileptic agent phenytoin has been widely related to this condition. The Cytochrome P450(CYP) superfamily is the most commonly involved enzymes in metabolism of drugs. Common coding region CYP variants that affects drug elimination and response has been studied in great detail. Pharmacogenetic influences on drug metabolism have been widely reviewed and gene polymorphism of cytochrome P450 2C9 appeared to be responsible for much of the interindividual variability on drug elimination. Genetic variation in the CYP2C9 gene can affect metabolism, leading to altered phenotypes. Individuals with poor metaboliser alleles of CYP2C9 gene were shown to have a reduced metabolism of phenytoin compared with wild-type alleles. Thus identification of patients genotype prior to anti-epileptic drug administration could potentially prevent higher serum drug concentrations leading to adverse side effects such as gingival enlargement. This case report addresses the influence of CYP2C9 genetic polymorphism on Phenytoin drug metabolism thereby causing gingival enlargement. PMID:24082701

  19. Gonadotropic pituitary carcinoma: HER-2/neu expression and gene amplification. Report of two cases.

    PubMed

    Roncaroli, Federico; Nosé, Vania; Scheithauer, Bernd W; Kovacs, Kalman; Horvath, Eva; Young, William F; Lloyd, Ricardo V; Bishop, Mari C; Hsi, Bradley; Fletcher, Jonathan A

    2003-08-01

    The authors report on two gonadotropic carcinomas of the adenohypophysis that occurred in a55-year-old man (Case 1) and a 53-year-old woman (Case 2), with signs of mass effect and amenorrhea, respectively. Both lesions were macroadenomas. The tumor in Case 1 metastasized to dura mater, skull, nasal sinus, and larynx 2 years after patient presentation, whereas that in Case 2 spread to vertebral bodies and ribs after a 19-year latency. Histologically, the primary, recurrent, and metastatic lesions in Case 1 featured brisk mitotic activity and high MIB-1 levels as well as p53 labeling indices. Immunoreactivity for HER-2/neu was assessable only in rare neoplastic cells of the second recurrence and in 80% of cells of the dural metastasis. Low-level HER-2/neu gene amplification was evident in the recurrent tumors and metastasis. The sellar and metastatic tumors in Case 2 resembled benign gonadotropic adenoma with oncocytic change; p53 accumulation, HER-2/neu overexpression, and HER-2/neu gene amplification were not present. The results indicate that low-level amplification of the HER-2/neu gene might be associated with pituitary carcinomas in which more aggressive behavior is seen. Further studies are needed to determine whether HER-2/neu plays a role in the pathogenesis of pituitary carcinoma.

  20. Benzodiazepines: rat pinealocyte binding sites and augmentation of norepinephrine-stimulated N-acetyltransferase activity

    SciTech Connect

    Matthew, E.; Parfitt, A.G.; Sugden, D.; Engelhardt, D.L.; Zimmerman, E.A.; Klein, D.C.

    1984-02-01

    Studies of (/sup 3/H)diazepam binding to intact rat pineal cells were carried out in tissue culture preparations. The binding was saturable, reversible and proportional to the number of cells used. Scatchard analysis resulted in a linear plot (Kd . 23 nM, maximum binding sites (Bmax) . 1.56 pmol/mg of protein for cells in monolayer culture; Kd . 7 nM, Bmax . 1.3 pmol/mg of protein for cells in suspension culture). Inhibition constants (Ki) for clonazepam (500 nM), flunitrazepam (38 nM) and Ro-5-4864 (5 nM) indicated that the binding sites were probably of the ''peripheral'' type. In addition, the effects of diazepam on norepinephrine-stimulated N-acetyltransferase (NAT) activity were studied in organ culture and dissociated cell culture. Diazepam (10-50 microM) both prolonged and increased the magnitude of the norepinephrine-induced increase in NAT activity but did not affect the initial rate of rise of enzyme activity. The effect was dose-dependent and was also seen with clonazepam, flunitrazepam and Ro-5-4864, but not with Ro-15-1788. Diazepam, by itself, at these concentrations, had no effect on NAT, but enzyme activity was increased by higher concentrations (0.1-1 mM). Although a relationship between the (/sup 3/H)diazepam binding sites described here and the effect of benzodiazepines on NAT cannot be established from these studies, the data suggest that the benzodiazepines may alter melatonin levels through their action on NAT.

  1. Choline acetyltransferase mutations causing congenital myasthenic syndrome: molecular findings and genotype-phenotype correlations

    PubMed Central

    Arredondo, Juan; Lara, Marian; Gospe, Sídney M.; Mazia, Claudio G.; Vaccarezza, Maria; Garcia-Erro, Marcela; Bowe, Constance; Chang, Celia; Mezei, Michelle; Maselli, Ricardo A.

    2015-01-01

    Choline acetyltransferase catalyzes the synthesis of acetylcholine at cholinergic nerves. Mutations in human CHAT cause a congenital myasthenic syndrome (CMS) due to impaired synthesis of ACh; this severe variant of the disease is frequently associated with unexpected episodes of potentially fatal apnea. The severity of this condition varies remarkably, and the molecular factors determining this variability are poorly understood. Furthermore, genotype–phenotype correlations have been difficult to establish in patients with biallelic mutations. We analyzed the protein expression of seven ChAT mutations, p.Val136Met, p.Arg207His, p.Arg186Trp, p.Val194Leu, p.Pro211Ala, p.Arg566Cys and p.Ser694Cys, in HEK-293 cells to phosphorylated ChAT, determined their enzyme kinetics and thermal instability, and examined their structural changes. Three mutations, p.Arg207His, p.Arg186Trp and p.Arg566Cys, are novel, and p.Val136Met and p.Arg207His are homozygous in three families and associated with severe disease. The characterization of mutants showed a decrease in the overall catalytic efficiency of ChAT; in particular, those located near the active-site tunnel produced the most seriously disruptive phenotypic effects. On the other hand, p.Val136Met is located far from both active and substrate-binding sites produced the most drastic reduction of ChAT expression. Overall, CHAT mutations producing low enzyme expression and severe kinetic effects are associated with the most severe phenotypes. PMID:26080897

  2. Origins of spinal cholinergic pathways in amphibians demonstrated by retrograde transport and choline acetyltransferase immunohistochemistry.

    PubMed

    López, Jesús M; Morona, Ruth; Moreno, Nerea; Domínguez, Laura; González, Agustín

    2007-09-25

    The existence of propriospinal cholinergic pathways and the origin of supraspinal cholinergic descending projections have been investigated in anuran and urodele amphibians. Retrograde tract tracing techniques with dextran amines injected in the spinal cord at different levels were combined with immunohistochemistry for choline acetyltransferase (ChAT). The analysis of the brachial, thoracic and lumbar spinal cord demonstrated that doubly labeled cells were present only close to the injection site. Thus, the participation of the spinal cholinergic cells in distant intersegmental connections is not present, or is very limited, in amphibians. In anurans, tracer applications to the brachial cord revealed cholinergic cells of origin of spinal projections located in four distinct brain nuclei. The most rostrally located cells were found bilaterally in the preoptic area, among the magnocellular cells. In the ipsilateral isthmic region, the laterodorsal tegmental nucleus also showed doubly labeled cells. Throughout the brainstem, abundant codistribution was observed but actual coexistence of the tracer and ChAT was only found in the nucleus of the solitary tract and the inferior reticular nucleus. In the case of the urodele, abundant codistribution between retrogradely labeled cells and ChAT-positive neurons in zones like the suprachiasmatic nucleus, the isthmic region and the rhombencephalic reticular formation was observed, but the only doubly labeled cells were the Mauthner neurons. The present results in amphibians contrast with previous data in mammals in which is striking the presence of a widespread intrinsic cholinergic innervation of the spinal cord and the virtual absence of cholinergic projections descending from the brainstem.

  3. Functional Consequences and Structural Interpretation of Mutations of Human Choline Acetyltransferase

    PubMed Central

    Shen, Xin-Ming; Crawford, Thomas O.; Brengman, Joan; Acsadi, Gyula; Iannaconne, Susan; Karaca, Emin; Khoury, Chaouky; Mah, Jean K.; Edvardson, Shimon; Bajzer, Zeljko; Rodgers, David; Engel, Andrew G.

    2011-01-01

    Choline acetyltransferase (ChAT; EC 2.3.1.6) catalyzes synthesis of acetylcholine from acetyl-CoA and choline in cholinergic neurons. Mutations in CHAT (MIM # 118490) cause potentially lethal congenital myasthenic syndromes associated with episodic apnea (ChAT-CMS) (MIM # 254210). Here we analyze the functional consequences of 12 missense and 1 nonsense mutations of CHAT in 11 patients. Nine of the mutations are novel. We examine expression of the recombinant missense mutants in Bosc 23 cells, determine their kinetic properties and thermal stability, and interpret the functional effects of 11 mutations in the context of the atomic structural model of human ChAT. Five mutations (p.Trp421Ser, p.Ser498Pro, p.Thr553Asn, p.Ala557Thr, p.Ser572Trp) reduce enzyme expression to <50% of wild-type. Mutations with severe kinetic effects are located in the active-site tunnel (p.Met202Arg, p.Thr553Asn and p.Ala557Thr) or adjacent to the substrate binding site (p.Ser572Trp), or exert their effect allosterically (p.Trp421Ser and p.Ile689Ser). Two mutations with milder kinetic effects (p.Val136Met, p.Ala235Thr) are also predicted to act allosterically. One mutation (p.Thr608Asn) below the nucleotide binding site of CoA enhances dissociation of AcCoA from the enzyme-substrate complex. Two mutations introducing a proline residue into an α-helix (p.Ser498Pro and p.Ser704Pro) impair the thermal stability of ChAT. PMID:21786365

  4. N-acetyltransferase 1 in colon and rectal cancer cases from an industrialized area.

    PubMed

    Roemer, Hermann C; Weistenhofer, Wobbeke; Lohlein, Dietrich; Geller, Frank; Blomeke, Brunhilde; Golka, Klaus

    2008-01-01

    Colon and rectal cancers are both associated with genetic as well as nutritional, occupational, and environmental factors. Aromatic amines and heterocyclic amines are established colorectal carcinogens. The polymorphic enzyme N-acetyltransferase 1 (NAT1) contributes to heterocyclic amine metabolism in the human colon. Thereby, NAT1 may influence the risk for development of colorectal cancer. The distribution of NAT1 genotypes was determined in 107 colon cancer cases, 77 rectal cancer cases, and 185 controls (suffering from nonmalignant diseases) by standard methods. In addition, possible occupational and nonoccupational risk factors were determined by a personal interview. Cancer cases and controls were derived from an area of former coal, iron, and steel industries, which is known for elevated colon cancer mortality. The proportions of NAT1*4/*4 genotype were 72% in controls, 75% in rectal cancer cases, and 72% in colon cancer cases. The proportions of the NAT1*4/*10 genotype were 17.8% in controls, 12.9% in rectal cancer cases, and 14% in colon cancer cases. Combinations of the determined NAT1 alleles *3/*3, *3/*10, *4/*3, *4/*11, *10/*10 and *11/*11 contributed to 10.2% of the genotypes in controls, 12.1% in rectal cancer cases, and 14% in colon cancer cases. In contrast to another study on healthy German volunteers, the NAT1*4/*4 genotype (wild type) is overrepresented. This might be due to the variation in the proportion of NAT1 alleles in the general population. The present study does not support a relevant impact of the NAT1 genotype on colorectal cancer risk development in the study area.

  5. Garcinol, a Histone Acetyltransferase Inhibitor, Radiosensitizes Cancer Cells by Inhibiting Non-Homologous End Joining

    SciTech Connect

    Oike, Takahiro; Ogiwara, Hideaki; Torikai, Kohta; Nakano, Takashi; Yokota, Jun; Kohno, Takashi

    2012-11-01

    Purpose: Non-homologous end joining (NHEJ), a major pathway used to repair DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), requires chromatin remodeling at DSB sites through the acetylation of histones by histone acetyltransferases (HATs). However, the effect of compounds with HAT inhibitory activities on the DNA damage response (DDR), including the NHEJ and cell cycle checkpoint, as well as on the radiosensitivity of cancer cells, remains largely unclear. Here, we investigated whether garcinol, a HAT inhibitor found in the rinds of Garcinia indica fruit (called mangosteens), has effects on DDR, and whether it can be used for radiosensitization. Methods and Materials: The following assays were used to examine the effect of garcinol on the inhibition of DSB repair, including the following: a conventional neutral comet assay; a cell-based assay recently developed by us, in which NHEJ repair of DSBs on chromosomal DNA was evaluated; the micrococcal nuclease sensitivity assay; and immunoblotting for autophosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We assessed the effect of garcinol on the cell cycle checkpoint after IR treatment by analyzing the phosphorylation levels of checkpoint kinases CHK1 and CHK2 and histone H3, and by cell cycle profile analysis using flow cytometry. The radiosensitizing effect of garcinol was assessed by a clonogenic survival assay, whereas its effects on apoptosis and senescence were examined by annexin V and senescence-associated {beta}-galactosidase (SA-{beta}-Gal) staining, respectively. Results: We found that garcinol inhibits DSB repair, including NHEJ, without affecting cell cycle checkpoint. Garcinol radiosensitized A549 lung and HeLa cervical carcinoma cells with dose enhancement ratios (at 10% surviving fraction) of 1.6 and 1.5, respectively. Cellular senescence induced by IR was enhanced by garcinol. Conclusion: These results suggest that garcinol is a radiosensitizer that

  6. A robust method for the rapid generation of recombinant Zika virus expressing the GFP reporter gene.

    PubMed

    Gadea, Gilles; Bos, Sandra; Krejbich-Trotot, Pascale; Clain, Elodie; Viranaicken, Wildriss; El-Kalamouni, Chaker; Mavingui, Patrick; Desprès, Philippe

    2016-10-01

    Zika virus (ZIKV) infection is a major public health problem with severe human congenital and neurological anomalies. The screening of anti-ZIKV compounds and neutralizing antibodies needs reliable and rapid virus-based assays. Here, we described a convenient method leading to the rapid production of molecular clones of ZIKV. To generate a molecular clone of ZIKV strain MR766(NIID), the viral genome was directly assembled into Vero cells after introduction of four overlapping synthetic fragments that cover the full-length genomic RNA sequence. Such strategy has allowed the production of a recombinant ZIKV expressing the GFP reporter gene that is stable over two culturing rounds on Vero cells. Our data demonstrate that the ZIKV reporter virus is a very reliable GFP-based tool for analyzing viral growth and measuring the neutralizing antibody as well as rapid screening of antiviral effect of different classes of inhibitors.

  7. MEGDEL Syndrome in a Child From Palestine: Report of a Novel Mutation in SERAC1 Gene.

    PubMed

    Dweikat, Imad M; Abdelrazeq, Samer; Ayesh, Suhail; Jundi, Tawfeeq

    2015-07-01

    We report the first Palestinian child manifesting with 3-methylglutaconic aciduria psychomotor delay, muscle hypotonia, sensori-neural deafness, and Leigh-like lesions on brain magnetic resonance imaging (MRI), a clinical phenotype that is characteristic of MEGDEL syndrome. MEGDEL syndrome was recently found to be caused by mutations in SERAC1, encoding a protein essential for mitochondrial function, phospholipid remodeling, and intracellular cholesterol trafficking. We identified a novel homozygous mutation in SERAC1 gene (c.1018delT) that generates frame shift and premature termination of protein translation. Plasma and cerebrospinal fluid lactate, plasma alanine, and respiratory chain complexes in fresh muscle were normal. This report further expands the genetic spectrum of MEGDEL syndrome and adds to the evidence that it is associated with variable patterns of respiratory chain abnormalities.

  8. Tumor-selective gene transduction and cell killing with an oncotropic autonomous parvovirus-based vector.

    PubMed

    Dupont, F; Avalosse, B; Karim, A; Mine, N; Bosseler, M; Maron, A; Van den Broeke, A V; Ghanem, G E; Burny, A; Zeicher, M

    2000-05-01

    A recombinant MVMp of the fibrotropic strain of minute virus of mice (MVMp) expressing the chloramphenicol acetyltransferase reporter gene was used to infect a series of biologically relevant cultured cells, normal or tumor-derived, including normal melanocytes versus melanoma cells, normal mammary epithelial cells versus breast adenocarcinoma cells, and normal neurons or astrocytes versus glioma cells. As a reference cell system we used normal human fibroblasts versus the SV40-transformed fibroblast cell line NB324K. After infection, we observed good expression of the reporter gene in the different tumor cell types, but only poor expression if any in the corresponding normal cells. We also constructed a recombinant MVMp expressing the green fluorescent protein reporter gene and assessed by flow cytometry the efficiency of gene transduction into the different target cells. At a multiplicity of infection of 30, we observed substantial transduction of the gene into most of the tumor cell types tested, but only marginal transduction into normal cells under the same experimental conditions. Finally, we demonstrated that a recombinant MVMp expressing the herpes simplex virus thymidine kinase gene can, in vitro, cause efficient killing of most tumor cell types in the presence of ganciclovir, whilst affecting normal proliferating cells only marginally if at all. However, in the same experimental condition, breast tumor cells appeared to be resistant to GCV-mediated cytotoxicity, possibly because these cells are not susceptible to the bystander effect. Our data suggest that MVMp-based vectors could prove useful as selective vehicles for anticancer gene therapy, particularly for in vivo delivery of cytotoxic effector genes into tumor cells.

  9. The use of the NIS reporter gene for optimizing oncolytic virotherapy

    PubMed Central

    Miller, Amber; Russell, Stephen J

    2016-01-01

    Introduction: Oncolytic viruses are experimental cancer therapies being translated to the clinic. They are unique in their ability to amplify within the body, therefore requiring careful monitoring of viral replication and biodistribution. Traditional monitoring strategies fail to recapitulate the dynamic nature of oncolytic virotherapy. Consequently, clinically relevant, noninvasive, high resolution strategies are needed to effectively track virotherapy in real time. Areas covered: The expression of the sodium iodide symporter (NIS) reporter gene is tightly coupled to viral genome replication and mediates radioisotope concentration, allowing noninvasive molecular nuclear imaging of active viral infection with high resolution. This provides insight into replication kinetics, biodistribution, the impact of vector design, administration, and dosing on therapeutic outcomes, and highlights the heterogeneity of spatial distribution and temporal evolution of infection. NIS-mediated imaging in clinical trials confirms the feasibility of this technology to noninvasively and longitudinally observe oncolytic virus infection, replication, and distribution. Expert opinion: NIS-mediated imaging provides detailed functional and molecular information on the evolution of oncolytic virus infection in living animals. The use of NIS reporter gene imaging has rapidly advanced to provide unparalleled insight into the spatial and temporal context of oncolytic infection which will be integral to optimization of oncolytic treatment strategies. PMID:26457362

  10. Mutations of SCN4A gene cause different diseases: 2 case reports and literature review

    PubMed Central

    Liu, Xiao-li; Huang, Xiao-jun; Luan, Xing-hua; Zhou, Hai-yan; Wang, Tian; Wang, Jing-yi; Chen, Sheng-di; Tang, Hui-dong; Cao, Li

    2015-01-01

    SCN4A encodes the Nav1.4 channel and mutations in SCN4A lead to different ionic channelopathies. In this study, one sporadic individual of periodic paralysis, one paramyotonia family and 200 normal healthy controls are enrolled. Genomic DNA was extracted from peripheral blood leukocytes, followed by polymerase chain reaction and DNA sequencing of candidate genes, including SCN4A and CACNA1S. As a result, heterozygous mutations c.2024G>A (R675Q) and c.1333G>A (V445M) of gene SCN4A were identified in the hypokalemic periodic paralysis patient and the paramyotonia congenita family respectively. Both mutations were not detected in healthy controls. Compared with reported cases, patients with mutation R675Q usually do not present hypokalemic periodic paralysis but hyperkalemic or normokalemic periodic paralysis. The mutation V445M was first reported in Chinese patients with nondystrophic myotonias. In addition, we carried out literature review by summarizing clinical features of the 2 mutations and establish the genotype–phenotype correlations to provide guidance for diagnosis. PMID:25839108

  11. Development of tyrosinase-based reporter genes for preclinical photoacoustic imaging of mesenchymal stem cells

    NASA Astrophysics Data System (ADS)

    Märk, Julia; Ruschke, Karen; Dortay, Hakan; Schreiber, Isabelle; Sass, Andrea; Qazi, Taimoor; Pumberger, Matthias; Laufer, Jan

    2014-03-01

    The capability to image stem cells in vivo in small animal models over extended periods of time is important to furthering our understanding of the processes involved in tissue regeneration. Photoacoustic imaging is suited to this application as it can provide high resolution (tens of microns) absorption-based images of superficial tissues (cm depths). However, stem cells are rare, highly migratory, and can divide into more specialised cells. Genetic labelling strategies are therefore advantageous for their visualisation. In this study, methods for the transfection and viral transduction of mesenchymal stem cells with reporter genes for the co-expression of tyrosinase and a fluorescent protein (mCherry). Initial photoacoustic imaging experiments of tyrosinase expressing cells in small animal models of tissue regeneration were also conducted. Lentiviral transduction methods were shown to result in stable expression of tyrosinase and mCherry in mesenchymal stem cells. The results suggest that photoacoustic imaging using reporter genes is suitable for the study of stem cell driven tissue regeneration in small animals.

  12. Testotoxicosis: Report of Two Cases, One with a Novel Mutation in LHCGR Gene

    PubMed Central

    Özcabı, Bahar; Tahmiscioğlu Bucak, Feride; Ceylaner, Serdar; Özcan, Rahşan; Büyükünal, Cenk; Ercan, Oya; Tüysüz, Beyhan; Evliyaoğlu, Olcay

    2015-01-01

    Testotoxicosis is a rare disorder which presents as isosexual peripheral precocious puberty in males. Despite the pattern of autosomal dominant inheritance, sporadic cases also may occur. Due to activating mutation in luteinizing hormone (LH))/choriogonadotropin receptor (LHCGR) gene, early virilization and advancement in bone age are common with increased serum testosterone levels above adult ranges, despite low LH and follicular-stimulating hormone (FSH) levels. There are different treatment regimens, such as combination of bicalutamide (antiandrogen agent) and a third-generation aromatase inhibitor, that are reported to be well-tolerated and successful in slowing bone age advancement and preventing progression of virilization. We report here two patients who presented with peripheral precocious puberty and an activating mutation in the LHCGR gene: one with a family history and previously determined mutation and the other without family history and with a novel mutation (c.830G>T). Combination of bicalutamide+anastrozole was ineffective in slowing pubertal progression and bone age. Short-term results were better with ketoconazole. PMID:26831561

  13. Use of reporter-gene based bacteria to quantify phenanthrene biodegradation and toxicity in soil.

    PubMed

    Shin, Doyun; Moon, Hee Sun; Lin, Chu-Ching; Barkay, Tamar; Nam, Kyoungphile

    2011-02-01

    A phenanthrene-degrading bacterium, Sphingomonas paucimobilis EPA505 was used to construct two fluorescence-based reporter strains. Strain D harboring gfp gene was constructed to generate green fluorescence when the strain started to biodegrade phenanthrene. Strain S possessing gef gene was designed to die once phenanthrene biodegradation was initiated and thus to lose green fluorescence when visualized by a live/dead cell staining. Confocal laser scanning microscopic observation followed by image analysis demonstrates that the fluorescence intensity generated by strain D increased and the intensity by strain S decreased linearly at the phenanthrene concentration of up to 200 mg/L. Such quantitative increase and decrease of fluorescence intensity in strain D (i.e., from 1 to 11.90 ± 0.72) and strain S (from 1 to 0.40 ± 0.07) were also evident in the presence of Ottawa sand spiked with the phenanthrene up to 1000 mg/kg. The potential use of the reporter strains in quantitatively determining biodegradable or toxic phenanthrene was discussed.

  14. Enterococcus gallinarum carrying the vanA gene cluster: first report in Brazil.

    PubMed

    Camargo, I L B C; Barth, A L; Pilger, K; Seligman, B G S; Machado, A R L; Darini, A L C

    2004-11-01

    In 2000, Enterococcus faecalis resistant to vancomycin was first reported at a tertiary hospital in Porto Alegre, southern Brazil. The resistance spread to other hospitals and surveillance programs were established by hospital infection committees to prevent the spread of vancomycin-resistant enterococci. In February 2002, an isolate initially identified at the genus level as Enterococcus was obtained by surveillance culture (rectal swab) from a patient admitted to a hospital for treatment of septic arthritis in the shoulder. The isolate proved to be resistant to vancomycin by the disc diffusion method and confirmed by an E-test resulting in a minimal inhibitory concentration of > or = 256 microg/ml. This isolate was sent to a reference laboratory (Laboratorio Especial de Bacteriologia e Epidemiologia Molecular, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, USP) for further study and proved to be an E. gallinarum by the polymerase chain reaction (PCR) using specific primers for the species. Due to the phenotype of unusually high vancomycin resistance, the isolate presumably had the resistance genes (vanA and vanB) and this was confirmed by PCR, which indicated the presence of the vanA gene. A 10.8-kb Tn1546-related transposon was also identified by long-PCR. Interspecies transfer of the vancomycin-resistance gene from the donor E. gallinarum was performed in a successful conjugation experiment in vitro, using E. faecium GE-1 and E. faecalis JH22 as receptors. This is the first report of the detection of a vanA determinant naturally acquired by E. gallinarum in Brazil, indicating the importance of characterizing VRE by both phenotype and genotype methods.

  15. Structural, Functional, and Inhibition Studies of a Gcn5-related N-Acetyltransferase (GNAT) Superfamily Protein PA4794

    PubMed Central

    Majorek, Karolina A.; Kuhn, Misty L.; Chruszcz, Maksymilian; Anderson, Wayne F.; Minor, Wladek

    2013-01-01

    The Gcn5-related N-acetyltransferase (GNAT) superfamily is a large group of evolutionarily related acetyltransferases, with multiple paralogs in organisms from all kingdoms of life. The functionally characterized GNATs have been shown to catalyze the transfer of an acetyl group from acetyl-coenzyme A (Ac-CoA) to the amine of a wide range of substrates, including small molecules and proteins. GNATs are prevalent and implicated in a myriad of aspects of eukaryotic and prokaryotic physiology, but functions of many GNATs remain unknown. In this work, we used a multi-pronged approach of x-ray crystallography and biochemical characterization to elucidate the sequence-structure-function relationship of the GNAT superfamily member PA4794 from Pseudomonas aeruginosa. We determined that PA4794 acetylates the Nϵ amine of a C-terminal lysine residue of a peptide, suggesting it is a protein acetyltransferase specific for a C-terminal lysine of a substrate protein or proteins. Furthermore, we identified a number of molecules, including cephalosporin antibiotics, which are inhibitors of PA4794 and bind in its substrate-binding site. Often, these molecules mimic the conformation of the acetylated peptide product. We have determined structures of PA4794 in the apo-form, in complexes with Ac-CoA, CoA, several antibiotics and other small molecules, and a ternary complex with the products of the reaction: CoA and acetylated peptide. Also, we analyzed PA4794 mutants to identify residues important for substrate binding and catalysis. PMID:24003232

  16. Genes and gene expression: Localization, damage and control -- A multi-level and interdisciplinary study. Progress report, February 1, 1992--January 31, 1993

    SciTech Connect

    Ts`o, P.O.P.

    1992-08-01

    This progress report describes gains made in three projects entitled (1) 3-Dimensional nuclear topography of genes and chromosomes in interphase nuclei, (2) Sequence specific identification and perturbation of the genomic DNA in living cells by nonionic oligonucleotide analogs (Matagen), and Resolution and isolation of specific DNA restriction fragments.(DT)

  17. Validating tyrosinase homologue melA as a photoacoustic reporter gene for imaging Escherichia coli

    NASA Astrophysics Data System (ADS)

    Paproski, Robert J.; Li, Yan; Barber, Quinn; Lewis, John D.; Campbell, Robert E.; Zemp, Roger

    2015-10-01

    To understand the pathogenic processes for infectious bacteria, appropriate research tools are required for replicating and characterizing infections. Fluorescence and bioluminescence imaging have primarily been used to image infections in animal models, but optical scattering in tissue significantly limits imaging depth and resolution. Photoacoustic imaging, which has improved depth-to-resolution ratio compared to conventional optical imaging, could be useful for visualizing melA-expressing bacteria since melA is a bacterial tyrosinase homologue which produces melanin. Escherichia coli-expressing melA was visibly dark in liquid culture. When melA-expressing bacteria in tubes were imaged with a VisualSonics Vevo LAZR system, the signal-to-noise ratio of a 9× dilution sample was 55, suggesting that ˜20 bacteria cells could be detected with our system. Multispectral (680, 700, 750, 800, 850, and 900 nm) analysis of the photoacoustic signal allowed unmixing of melA-expressing bacteria from blood. To compare photoacoustic reporter gene melA (using Vevo system) with luminescent and fluorescent reporter gene Nano-lantern (using Bruker Xtreme In-Vivo system), tubes of bacteria expressing melA or Nano-lantern were submerged 10 mm in 1% Intralipid, spaced between <1 and 20 mm apart from each other, and imaged with the appropriate imaging modality. Photoacoustic imaging could resolve the two tubes of melA-expressing bacteria even when the tubes were less than 1 mm from each other, while bioluminescence and fluorescence imaging could not resolve the two tubes of Nano-lantern-expressing bacteria even when the tubes were spaced 10 mm from each other. After injecting 100-μL of melA-expressing bacteria in the back flank of a chicken embryo, photoacoustic imaging allowed visualization of melA-expressing bacteria up to 10-mm deep into the embryo. Photoacoustic signal from melA could also be separated from deoxy- and oxy-hemoglobin signal observed within the embryo and

  18. Validating tyrosinase homologue melA as a photoacoustic reporter gene for imaging Escherichia coli.

    PubMed

    Paproski, Robert J; Li, Yan; Barber, Quinn; Lewis, John D; Campbell, Robert E; Zemp, Roger

    2015-10-01

    To understand the pathogenic processes for infectious bacteria, appropriate research tools are required for replicating and characterizing infections. Fluorescence and bioluminescence imaging have primarily been used to image infections in animal models, but optical scattering in tissue significantly limits imaging depth and resolution. Photoacoustic imaging, which has improved depth-to-resolution ratio compared to conventional optical imaging, could be useful for visualizing melA-expressing bacteria since melA is a bacterial tyrosinase homologue which produces melanin. Escherichia coli-expressing melA was visibly dark in liquid culture. When melA-expressing bacteria in tubes were imaged with a VisualSonics Vevo LAZR system, the signal-to-noise ratio of a 9×dilution sample was 55, suggesting that ∼20 bacteria cells could be detected with our system. Multispectral (680, 700, 750, 800, 850, and 900 nm) analysis of the photoacoustic signal allowed unmixing of melA-expressing bacteria from blood. To compare photoacoustic reporter gene melA (using Vevo system) with luminescent and fluorescent reporter gene Nano-lantern (using Bruker Xtreme In-Vivo system), tubes of bacteria expressing melA or Nano-lantern were submerged 10 mm in 1% Intralipid, spaced between <1 and 20 mm apart from each other, and imaged with the appropriate imaging modality. Photoacoustic imaging could resolve the two tubes of melA-expressing bacteria even when the tubes were less than 1 mm from each other, while bioluminescence and fluorescence imaging could not resolve the two tubes of Nano-lantern-expressing bacteria even when the tubes were spaced 10 mm from each other. After injecting 100-μL of melA-expressing bacteria in the back flank of a chicken embryo, photoacoustic imaging allowed visualization of melA-expressing bacteria up to 10-mm deep into the embryo. Photoacoustic signal from melA could also be separated from deoxy- and oxy-hemoglobin signal observed within the embryo and

  19. Epitope-Tagged Autotransporters as Single-Cell Reporters for Gene Expression by a Salmonella Typhimurium wbaP Mutant

    PubMed Central

    Curkić, Ismeta; Schütz, Monika; Oberhettinger, Philipp; Diard, Médéric; Claassen, Manfred; Linke, Dirk; Hardt, Wolf-Dietrich

    2016-01-01

    Phenotypic diversity is an important trait of bacterial populations and can enhance fitness of the existing genotype in a given environment. To characterize different subpopulations, several studies have analyzed differential gene expression using fluorescent reporters. These studies visualized either single or multiple genes within single cells using different fluorescent proteins. However, variable maturation and folding kinetics of different fluorophores complicate the study of dynamics of gene expression. Here, we present a proof-of-principle study for an alternative gene expression system in a wbaP mutant of Salmonella Typhimurium (S. Tm) lacking the O-sidechain of the lipopolysaccharide. We employed the hemagglutinin (HA)-tagged inverse autotransporter invasin (invAHA) as a transcriptional reporter for the expression of the type three secretion system 1 (T1) in S. Tm. Using a two-reporter approach with GFP and the InvAHA in single cells, we verify that this reporter system can be used for T1 gene expression analysis, at least in strains lacking the O-antigen (wbaP), which are permissive for detection of the surface-exposed HA-epitope. When we placed the two reporters gfp and invAHA under the control of either one or two different promoters of the T1 regulon, we were able to show correlative expression of both reporters. We conclude that the invAHA reporter system is a suitable tool to analyze T1gene expression in S. Tm and propose its applicability as molecular tool for gene expression studies within single cells. PMID:27149272

  20. Superinduction of estrogen receptor mediated gene expression in luciferase based reporter gene assays is mediated by a post-transcriptional mechanism.

    PubMed

    Sotoca, A M; Bovee, T F H; Brand, W; Velikova, N; Boeren, S; Murk, A J; Vervoort, J; Rietjens, I M C M

    2010-10-01

    Several estrogenic compounds including the isoflavonoid genistein have been reported to induce a higher maximal response than the natural estrogen 17β-estradiol in in vitro luciferase based reporter gene bioassays for testing estrogenicity. The phenomenon has been referred to as superinduction. The mechanism underlying this effect and thus also its biological relevance remain to be elucidated. In the present study several hypotheses for the possible mechanisms underlying this superinduction were investigated using genistein as the model compound. These hypotheses included (i) a non-estrogen receptor (ER)-mediated mechanism, (ii) a role for an ER activating genistein metabolite with higher ER inducing activity than genistein itself, and (iii) a post-transcriptional mechanism that is not biologically relevant but specific for the luciferase based reporter gene assays. The data presented in this study indicate that induction and also superinduction of the reporter gene is ER-mediated, and that superinduction by genistein could be ascribed to stabilization of the firefly luciferase reporter enzyme increasing the bioluminescent signal during the cell-based assay. This indicates that the phenomenon of superinduction may not be biologically relevant but may rather represent a post-transcriptional effect on enzyme stability.