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Sample records for acetyltransferase cat gene

  1. Application of the chloramphenicol acetyltransferase (CAT) diffusion assay to transgenic plant tissues.

    PubMed

    Peach, C; Velten, J

    1992-02-01

    Chloramphenicol acetyltransferase (CAT) activity was quantified in crude extracts from tobacco callus tissues using a modification of a previously reported diffusion assay. We describe here the alterations necessary in applying this rapid and simple assay procedure to plant materials. Due to the high concentration of nonspecific oxidases present in most plant tissues, some type of protective agent is required to maintain enzyme activity. We have tested beta-mercaptoethanol, cysteine, dithiothreitol, ascorbic acid and polyvinyl pyrrolidone as protective agents within the initial extraction buffer. We also investigated the effect of heat (60 degrees C, 10 min) and 5 mM EDTA on CAT activity. The highest CAT activity was obtained using 5 mM cysteine plus 5 mM EDTA in 40 mM Tris-HCl (pH 7.8) as the initial extraction buffer followed by a heat treatment. Using this buffer, CAT activity was stable on ice for more than two hours. In our hands, total acetyl-coenzyme A concentration within the assay mixture was found to be saturating at 250 microM and the Km determined to be 100 microM. Assays performed using the same crude plant extract indicate that 1) duplicate assays show less than 1.5% variation in activities and 2) CAT activity increases linearly with respect to volume of extract used.

  2. In vitro expression of a Tn9-derived chloramphenicol acetyltransferase gene fusion by using a Bacillus subtilis system.

    PubMed Central

    Zaghloul, T I; Doi, R H

    1987-01-01

    A coupled in vitro protein-synthesizing system has been developed with components derived totally from Bacillus subtilis. The system synthesized specific gene products from various exogenous DNA templates, including B. subtilis phage phi 29, plasmid pUB110, and a heterologous B. subtilis-Escherichia coli gene fusion containing the transposon Tn9-derived chloramphenicol acetyltransferase (cat) gene. The gene fusion product was able to show CAT activity, bind specifically to a Sephacryl-chloramphenicol column, and react immunologically against anti-CAT antiserum. The fidelity of this in vitro system was demonstrated by the synthesis of gene products identical to that made in vivo. We suggest that this system may be used to study the regulation of gene expression in vitro. Images PMID:3102458

  3. The facC Gene of Aspergillus nidulans Encodes an Acetate-Inducible Carnitine Acetyltransferase

    PubMed Central

    Stemple, Christopher J.; Davis, Meryl A.; Hynes, Michael J.

    1998-01-01

    Mutations in the facC gene of Aspergillus nidulans result in an inability to use acetate as a sole carbon source. This gene has been cloned by complementation. The proposed translation product of the facC gene has significant similarity to carnitine acetyltransferases (CAT) from other organisms. Total CAT activity was found to be inducible by acetate and fatty acids and repressed by glucose. Acetate-inducible activity was found to be absent in facC mutants, while fatty acid-inducible activity was absent in an acuJ mutant. Acetate induction of facC expression was dependent on the facB regulatory gene, and an expressed FacB fusion protein was demonstrated to bind to 5′ facC sequences. Carbon catabolite repression of facC expression was affected by mutations in the creA gene and a CreA fusion protein bound to 5′ facC sequences. Mutations in the acuJ gene led to increased acetate induction of facC expression and also of an amdS-lacZ reporter gene, and it is proposed that this results from accumulation of acetate, as well as increased expression of facB. A model is presented in which facC encodes a cytosolic CAT enzyme, while a different CAT enzyme, which is acuJ dependent, is present in peroxisomes and mitochondria, and these activities are required for the movement of acetyl groups between intracellular compartments. PMID:9829933

  4. Molecular cloning of cDNAs encoding human carnitine acetyltransferase and mapping of the corresponding gene to chromosome 9q34.1

    SciTech Connect

    Corti, O.; Finocchiaro, G.; DiDonato, S.

    1994-09-01

    Using a combination of PCR screening of cDNA libraries and reverse transcription PCR, we have cloned three overlapping DNA fragments that encode human carnitine acetyltransferase (CAT), a key enzyme for metabolic pathways involved with the control of the acyl-Co/CoA ratio in mitochondria, peroxisomes, and endoplasmic reticulum. The resulting cDNA (2436 bp) hybridizes to a mRNA species of {approximately}2.9 kb that is particularly abundant in skeletal muscle and encodes a 68-kDa protein containing a peroxisomal targeting signal. The sequence matches those of several tryptic peptides obtained from purified human liver CAT and shows striking similarities with other members of the carnitine/choline acetyltransferase family very distant throughout evolution. CAT cDNA has also been used for fluorescence in situ hybridization on metaphase spreads of human chromosomes, and the corresponding gene, CAT1, has been mapped to chromosome 9q34.1. 29 refs., 4 figs.

  5. Pitfalls of the CAT reporter gene for analyzing translational regulation in Leishmania.

    PubMed

    Folgueira, Cristina; Requena, Jose M

    2007-10-01

    Heterologous reporter genes are widely used for the characterization of gene expression in many organisms. Particularly, constructs bearing reporter genes have greatly contributed to our understanding of gene regulation in kinetoplastids. In some specific circumstances, however, such heterologous reporter has a risk of resulting in irrelevant observations and conclusions, which are primarily due to the introduction of foreign sequence elements. This communication describes our recent experience using the chloramphenicol acetyltransferase (CAT) gene as a reporter for analysis of the translational regulation of HSP70 genes in Leishmania infantum. We show that chimeric mRNAs consisting of the CAT open reading frame (ORF) and the untranslated regions (UTRs) from HSP70-II genes behave differently as endogenous HSP70-II mRNAs and that this difference is due to the presence of CAT sequences. Thus, the main purpose of this communication is to alert researchers working in gene regulation to be cautious when interpreting results based on heterologous reporter genes.

  6. 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...

  7. 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...

  8. 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.

  9. 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

  10. The CSRP2BP histone acetyltransferase drives smooth muscle gene expression.

    PubMed

    Ma, Yanlin; Li, Qi; Li, Ankang; Wei, Yunjian; Long, Ping; Jiang, Xinxing; Sun, Fei; Weiskirchen, Ralf; Wu, Bangyong; Liang, Chao; Grötzinger, Joachim; Wei, Yanxing; Yu, Wei; Mercola, Mark; Huang, Yuanhua; Wang, Jun; Yu, Yanhong; Schwartz, Robert J

    2017-04-07

    The expression of nearly all smooth muscle genes are controlled by serum response factor binding sites in their promoter regions. However, SRF alone is not sufficient for regulating smooth muscle cell development. It associates with other cardiovascular specific cofactors to regulate smooth muscle gene expression. Previously, we showed that the transcription co-factor CRP2 was a regulator of smooth muscle gene expression. Here, we report that CSRP2BP, a coactivator for CRP2, is a histone acetyltransferase and a driver of smooth muscle gene expression. CSRP2BP directly interacted with SRF, CRP2 and myocardin. CSRP2BP synergistically activated smooth muscle gene promoters in an SRF-dependent manner. A combination of SRF, GATA6 and CRP2 required CSRP2BP for robust smooth muscle gene promoter activity. Knock-down of Csrp2bp in smooth muscle cells resulted in reduced smooth muscle gene expression. We conclude that the CSRP2BP histone acetyltransferase is a coactivator for CRP2 that works synergistically with SRF and myocardin to regulate smooth muscle gene expression. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Nucleotide sequence and phylogeny of a chloramphenicol acetyltransferase encoded by the plasmid pSCS7 from Staphylococcus aureus.

    PubMed

    Schwarz, S; Cardoso, M

    1991-08-01

    The nucleotide sequence of the chloramphenicol acetyltransferase gene (cat) and its regulatory region, encoded by the plasmid pSCS7 from Staphylococcus aureus, was determined. The structural cat gene encoded a protein of 209 amino acids, which represented one monomer of the enzyme chloramphenicol acetyltransferase (CAT). Comparisons between the amino acid sequences of the pSCS7-encoded CAT from S. aureus and the previously sequenced CAT variants from S. aureus, Staphylococcus intermedius, Staphylococcus haemolyticus, Bacillus pumilis, Clostridium difficile, Clostridium perfringens, Escherichia coli, Shigella flexneri, and Proteus mirabilis were performed. An alignment of CAT amino acid sequences demonstrated the presence of 34 conserved amino acids among all CAT variants. These conserved residues were considered for their possible roles in the structure and function of CAT. On the basis of the alignment, a phylogenetic tree was constructed. It demonstrated relatively large evolutionary distances between the CAT variants of enteric bacteria, Clostridium, Bacillus, and Staphylococcus species.

  12. Regulatory regions that control expression of two chloramphenicol-inducible cat genes cloned in Bacillus subtilis.

    PubMed

    Duvall, E J; Williams, D M; Mongkolsuk, S; Lovett, P S

    1984-06-01

    Plasmid pPL603 is a promoter cloning vector for Bacillus subtilis and consists of a 1.1-kilobase fragment of Bacillus pumilus DNA inserted between the EcoRI and BamHI sites of pUB110. The gene cat-86, specifying chloramphenicol-inducible chloramphenicol acetyltransferase, is located on the 1.1-kilobase cloned DNA. When pPL603 is present in B. subtilis, cat-86 is unexpressed during vegetative growth but expressed during sporulation. The regulation of cat-86 in pPL603 is due to sequences within two restriction fragments, designated P1 and R1, that precede the main coding portion of the gene. The P1 fragment promotes transcription of cat-86 only during sporulation, whereas the adjacent R1 fragment lacks promoter function but contains sequences essential to chloramphenicol inducibility. A second B. pumilus gene, cat-66, was cloned in B. subtilis and is expressed throughout the vegetative growth and sporulation cycle. The cat-66 coding region is preceded by two adjacent restriction fragments designated as P2 and R2. P1 and P2 are identical in size and share 95% conservation of base sequence. R1 and R2 are also identical in size and share 91% conservation of base sequence. Fragment substitution experiments demonstrate that R2 can functionally replace R1. The substitution of P2 for P1 promotes cat-86 expression throughout vegetative growth and sporulation. Analysis of a derivative of pPL603 in which P2 has replaced P1 demonstrates that P2 promotes transcription of cat-86 during vegetative growth and that P2 contains the start site for transcription of cat-86. Thus, P1 and P2 differ strikingly in vegetative promoter function, yet they differ by single-base substitutions at only 11 positions of 203.

  13. Involvement of Arabidopsis histone acetyltransferase HAC family genes in the ethylene signaling pathway.

    PubMed

    Li, Chao; Xu, Jiang; Li, Jian; Li, Qingyun; Yang, Hongchun

    2014-02-01

    Epigenetic modifications play a fundamental role in regulating chromatin dynamics and gene expression. The level of histone acetylation is controlled by two functionally antagonistic enzymes, namely histone acetyltransferase (HAT) and histone deacetylase (HDAC). CREB-binding protein (CBP)/p300 proteins, a subfamily of highly conserved HATs, are involved in various physiological events including proliferation, differentiation and apoptosis. In this work, we study the poorly known function of their homologous genes, the HAC genes, in Arabidopsis. We found that hac1-involved mutants displayed pleiotropic phenotypes, in particular hypersensitivity to ethylene both in the dark and in the light. We also found that the transcriptional levels of ethylene-responsive genes are significantly higher in the hac1hac5 double mutant than in wild-type plants. Moreover, an ethylene synthesis inhibitor cannot release the triple responses of hac mutants. These results suggest that HACs are involved in the ethylene signaling pathway.

  14. Sequence and transcriptional analysis of the nourseothricin acetyltransferase-encoding gene nat1 from Streptomyces noursei.

    PubMed

    Krügel, H; Fiedler, G; Smith, C; Baumberg, S

    1993-05-15

    We have determined the nucleotide (nt) sequence of nat1, a gene encoding nourseothricin (Nc) acetyltransferase (AT) from Streptomyces noursei, and its transcriptional start point (tsp). The nt sequence upstream from the coding region is completely different from that of the stat gene (encoding streptothricin AT) from Streptomyces lavendulae [S. Horinouchi, K. Furuya, M. Nishiyama, H. Suzuki and T. Beppu, J. Bacteriol. 169 (1987) 1929-1937], even though the nt sequences of the two genes and the deduced amino acid (aa) sequences of the two enzymes show a high degree of similarity. Another stat gene, derived from a Gram-negative plasmid, showed only deduced aa similarity, but not nt sequence similarity, to the above two. A database search for related aa sequences did not reveal any clear-cut homologies to other types of protein. A multiple aa sequence alignment of several ATs is presented.

  15. No association between apolipoprotein E or N-acetyltransferase 2 gene polymorphisms and age-related hearing loss.

    PubMed

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

    2015-01-01

    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. Candidate gene association study of a continuous phenotype. 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. 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). 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. © 2014 The American Laryngological, Rhinological and Otological Society, Inc.

  16. 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

  17. 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

  18. K-Lysine acetyltransferase 2a regulates a hippocampal gene expression network linked to memory formation

    PubMed Central

    Stilling, Roman M; Rönicke, Raik; Benito, Eva; Urbanke, Hendrik; Capece, Vincenzo; Burkhardt, Susanne; Bahari-Javan, Sanaz; Barth, Jonas; Sananbenesi, Farahnaz; Schütz, Anna L; Dyczkowski, Jerzy; Martinez-Hernandez, Ana; Kerimoglu, Cemil; Dent, Sharon YR; Bonn, Stefan; Reymann, Klaus G; Fischer, Andre

    2014-01-01

    Neuronal histone acetylation has been linked to memory consolidation, and targeting histone acetylation has emerged as a promising therapeutic strategy for neuropsychiatric diseases. However, the role of histone-modifying enzymes in the adult brain is still far from being understood. Here we use RNA sequencing to screen the levels of all known histone acetyltransferases (HATs) in the hippocampal CA1 region and find that K-acetyltransferase 2a (Kat2a)—a HAT that has not been studied for its role in memory function so far—shows highest expression. Mice that lack Kat2a show impaired hippocampal synaptic plasticity and long-term memory consolidation. We furthermore show that Kat2a regulates a highly interconnected hippocampal gene expression network linked to neuroactive receptor signaling via a mechanism that involves nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In conclusion, our data establish Kat2a as a novel and essential regulator of hippocampal memory consolidation. PMID:25024434

  19. Histone acetyltransferase p300 modulates gene expression in an epigenetic manner at high blood alcohol levels.

    PubMed

    Bardag-Gorce, Fawzia; French, Barbara A; Joyce, Michael; Baires, Mercedes; Montgomery, Rosalyn O; Li, Jun; French, Samuel

    2007-04-01

    When rats are fed ethanol intragastrically at a constant rate for 1 month, the urinary alcohol level (UAL) cycles over 7-9 day intervals. At the peak UAL, the liver is hypoxic shifting the redox state to a reduced rate. Microarray analysis done on livers at the UAL peaks shows changes in approximately 1300 gene expression compared to the pair-fed controls. To determine the mechanism of the gene expression changes, histone acetylation regulation was investigated in liver nuclear extracts at the peaks and troughs of the UAL and their pair-fed controls. No change occurred in SirT-1. P300, a histone acetyltransferase (HAT), which acetylates histone H3 on lysine 9, was increased at the peaks. Histone 3 acetylated at lysine 9 was also increased at the peaks. This indicates that the up regulated genes at the UAL peaks resulted from an increase in p300 transcription regulation, epigenetically. P300 activates transcription of numerous genes in response to signal transcription factors such as H1F 1alpha, increased in the nucleus at UAL peaks. Signal transduction pathways, such as NFkappaB, AP-1, ERK, JNK, and p38 were not increased at the peaks. beta-Catenin was increased in the nuclear extract at the UAL troughs, where increased gene expression was absent. The increase in gene expression at the peaks was due, in part, to increased acetylation of histone 3 at lysine 9.

  20. In silico identification and characterization of N-Terminal acetyltransferase genes of poplar (Populus trichocarpa).

    PubMed

    Zhu, Hang-Yong; Li, Chun-Ming; Wang, Li-Feng; Bai, Hui; Li, Yan-Ping; Yu, Wen-Xi; Xia, De-An; Liu, Chang-Cai

    2014-01-27

    N-terminal acetyltransferase (Nats) complex is responsible for protein N-terminal acetylation (Nα-acetylation), which is one of the most common covalent modifications of eukaryotic proteins. Although genome-wide investigation and characterization of Nat catalytic subunits (CS) and auxiliary subunits (AS) have been conducted in yeast and humans they remain unexplored in plants. Here we report on the identification of eleven genes encoding eleven putative Nat CS polypeptides, and five genes encoding five putative Nat AS polypeptides in Populus. We document that the expansion of Nat CS genes occurs as duplicated blocks distributed across 10 of the 19 poplar chromosomes, likely only as a result of segmental duplication events. Based on phylogenetic analysis, poplar Nat CS were assigned to six subgroups, which corresponded well to the Nat CS types (CS of Nat A-F), being consistent with previous reports in humans and yeast. In silico analysis of microarray data showed that in the process of normal development of the poplar, their Nat CS and AS genes are commonly expressed at one relatively low level but share distinct tissue-specific expression patterns. This exhaustive survey of Nat genes in poplar provides important information to assist future studies on their functional role in poplar.

  1. The MYST Family Histone Acetyltransferase Regulates Gene Expression and Cell Cycle in Malaria Parasite Plasmodium falciparum

    PubMed Central

    Miao, Jun; Fan, Qi; Cui, Long; Li, Xiaolian; Wang, Haiyan; Ning, Gang; Reese, Joseph C.; Cui, Liwang

    2010-01-01

    Summary Histone lysine acetylation, normally associated with euchromatin and active genes, is regulated by different families of histone acetyltransferases (HATs). A single Plasmodium falciparum MYST (PfMYST) HAT was expressed as a long and a short version in intraerythrocytic stages. Whereas the recombinant PfMYST expressed in prokaryotes and insect cells did not show HAT activity, recombinant PfMYST purified from the parasites exhibited a predilection to acetylate histone H4 in vitro at K5, K8, K12, and K16. Tagging PfMYST with the green fluorescent protein at the C-terminus showed that PfMYST protein was localized in both the nucleus and cytoplasm. Consistent with the importance of H4 acetylation in var gene expression, PfMYST was recruited to the active var promoter. Attempts to disrupt PfMYST were not successful, suggesting that PfMYST is essential for asexual intraerythrocytic growth. However, overexpression of the long, active or a truncated, non-active version of PfMYST by stable integration of the expression cassette in the parasite genome resulted in changes of H4 acetylation and cell cycle progression. Furthermore, parasites with PfMYST over-expression showed changes in sensitivity to DNA damaging agents. Collectively, this study showed that PfMYST plays important roles in cellular processes such as gene activation, cell cycle control, and DNA repair. PMID:20807207

  2. Vitiligo susceptibility and catalase gene (CAT) polymorphisms in sicilian population.

    PubMed

    Caputo, Valentina; Niceta, Marcello; Fiorella, Santi; La Vecchia, Marco; Bastonini, Emanuela; Bongiorno, Maria R; Pistone, Giuseppe

    2017-02-15

    Catalase gene (CAT) polymorphisms were analyzed as responsible for the deficiency of catalase enzyme activity and concomitant accumulation of excessive hydrogen peroxide in Vitiligo patients. Catalase is a well known oxidative stress regulator that could play an important role in the pathogenesis of Vitiligo. This study was conducted to evaluate three CAT gene polymorphisms (-89A/T, 389C/T, 419C/T) and their association with Vitiligo susceptibility in Sicilian population. 60 out of 73 Sicilian patients with Vitiligo were enrolled and submitted to CAT gene analysis. Contrary to the Northern part of Europe but likewise to the Mediterranean area, the frequency of the CAT genotypes in Sicily is equally distributed. Out of all CAT genotypes, only CAT -89 T/T frequency was found to be significantly higher amongst Vitiligo patients than controls. Despite the involvement of the CAT enzyme in the pathogenesis of Vitiligo, the biological significance of CAT gene polymorphisms is still controversial. With the only exception for CAT variant -89A/T, the other studied CAT gene polymorphisms (389C/T and 419C/T) might not to be associated with Vitiligo in Sicilian population.

  3. Variation of the N-acetyltransferase 2 gene in a Romanian and a Kyrgyz population.

    PubMed

    Rabstein, Sylvia; Unfried, Klaus; Ranft, Ulrich; Illig, Thomas; Kolz, Melanie; Rihs, Hans-Peter; Mambetova, Chinara; Vlad, Mariana; Brüning, Thomas; Pesch, Beate

    2006-01-01

    As part of a project on environmental disasters in minority populations, this study aimed to evaluate differences in the sequence of N-acetyltransferase 2 (NAT2) as a metabolic susceptibility gene in yet unexplored ethnicities. Eight single nucleotide polymorphisms (SNP) in the NAT2 coding region and a variant in the 3' flanking region were analyzed in 290 unrelated Kyrgyz and 140 unrelated Romanians by SNP-specific PCR analysis. The variants 341C, 481T, and 803G were less and 857A more prevalent in Kyrgyz (P < 0.0001). The variant at site 857 indicates Asian descent. 282C>T and 590G>A showed no significant variation by ethnicity. 364G>A and 411A>T turned out to be monomorphic. Database comparisons of the NAT2 minor allele frequencies support that Romanians belong to Caucasians and Kyrgyz are in between Caucasians and East Asians. The distributions of predicted haplotypes differed significantly between the two ethnicities where the Kyrgyz showed a higher genetic diversity. The haplotype without mutations was more common in Kyrgyz (40.1% in Kyrgyz, 29.3% in Romanians). Accordingly, the imputed slow acetylator phenotype was less prevalent in Kyrgyz (35.2% versus 51.4% in Romanians). We found pronounced ethnic differences in NAT2 genotypes with yet unknown effect on the health risks for environmental or occupational exposures in minority populations.

  4. 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

  5. Deciphering the Ancient and Complex Evolutionary History of Human Arylamine N-Acetyltransferase Genes

    PubMed Central

    Patin, Etienne; Barreiro, Luis B.; Sabeti, Pardis C.; Austerlitz, Frédéric; Luca, Francesca; Sajantila, Antti; Behar, Doron M.; Semino, Ornella; Sakuntabhai, Anavaj; Guiso, Nicole; Gicquel, Brigitte; McElreavey, Ken; Harding, Rosalind M.; Heyer, Evelyne; Quintana-Murci, Lluís

    2006-01-01

    The human N-acetyltransferase genes NAT1 and NAT2 encode two phase-II enzymes that metabolize various drugs and carcinogens. Functional variability at these genes has been associated with adverse drug reactions and cancer susceptibility. Mutations in NAT2 leading to the so-called slow-acetylation phenotype reach high frequencies worldwide, which questions the significance of altered acetylation in human adaptation. To investigate the role of population history and natural selection in shaping NATs variation, we characterized genetic diversity through the resequencing and genotyping of NAT1, NAT2, and the pseudogene NATP in a collection of 13 different populations with distinct ethnic backgrounds and demographic pasts. This combined study design allowed us to define a detailed map of linkage disequilibrium of the NATs region as well as to perform a number of sequence-based neutrality tests and the long-range haplotype (LRH) test. Our data revealed distinctive patterns of variability for the two genes: the reduced diversity observed at NAT1 is consistent with the action of purifying selection, whereas NAT2 functional variation contributes to high levels of diversity. In addition, the LRH test identified a particular NAT2 haplotype (NAT2*5B) under recent positive selection in western/central Eurasians. This haplotype harbors the mutation 341T→C and encodes the “slowest-acetylator” NAT2 enzyme, suggesting a general selective advantage for the slow-acetylator phenotype. Interestingly, the NAT2*5B haplotype, which seems to have conferred a selective advantage during the past ∼6,500 years, exhibits today the strongest association with susceptibility to bladder cancer and adverse drug reactions. On the whole, the patterns observed for NAT2 well illustrate how geographically and temporally fluctuating xenobiotic environments may have influenced not only our genome variability but also our present-day susceptibility to disease. PMID:16416399

  6. 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

  7. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells

    SciTech Connect

    Gorman, C.M.; Moffat, L.F.; Howard, B.H.

    1982-09-01

    The authors constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV 40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. They also constructed a recombinant, pSVO-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

  8. Horizontal gene transfer of acetyltransferases, invertases and chorismate mutases from different bacteria to diverse recipients.

    PubMed

    Noon, Jason B; Baum, Thomas J

    2016-04-12

    Hoplolaimina plant-parasitic nematodes (PPN) are a lineage of animals with many documented cases of horizontal gene transfer (HGT). In a recent study, we reported on three likely HGT candidate genes in the soybean cyst nematode Heterodera glycines, all of which encode secreted candidate effectors with putative functions in the host plant. Hg-GLAND1 is a putative GCN5-related N-acetyltransferase (GNAT), Hg-GLAND13 is a putative invertase (INV), and Hg-GLAND16 is a putative chorismate mutase (CM), and blastp searches of the non-redundant database resulted in highest similarity to bacterial sequences. Here, we searched nematode and non-nematode sequence databases to identify all the nematodes possible that contain these three genes, and to formulate hypotheses about when they most likely appeared in the phylum Nematoda. We then performed phylogenetic analyses combined with model selection tests of alternative models of sequence evolution to determine whether these genes were horizontally acquired from bacteria. Mining of nematode sequence databases determined that GNATs appeared in Hoplolaimina PPN late in evolution, while both INVs and CMs appeared before the radiation of the Hoplolaimina suborder. Also, Hoplolaimina GNATs, INVs and CMs formed well-supported clusters with different rhizosphere bacteria in the phylogenetic trees, and the model selection tests greatly supported models of HGT over descent via common ancestry. Surprisingly, the phylogenetic trees also revealed additional, well-supported clusters of bacterial GNATs, INVs and CMs with diverse eukaryotes and archaea. There were at least eleven and eight well-supported clusters of GNATs and INVs, respectively, from different bacteria with diverse eukaryotes and archaea. Though less frequent, CMs from different bacteria formed supported clusters with multiple different eukaryotes. Moreover, almost all individual clusters containing bacteria and eukaryotes or archaea contained species that inhabit very similar

  9. Constitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus species AN-22: production, purification, characterization and gene analysis of CatA, CatB and CatC.

    PubMed

    Matsumura, Eitaro; Sakai, Masashi; Hayashi, Katsuaki; Murakami, Shuichiro; Takenaka, Shinji; Aoki, Kenji

    2006-01-01

    The aniline-assimilating bacterium Rhodococcus sp. AN-22 was found to constitutively synthesize CatB (cis,cis-muconate cycloisomerase) and CatC (muconolactone isomerase) in its cells growing on non-aromatic substrates, in addition to the previously reported CatA (catechol 1,2-dioxygenase). The bacterium maintained the specific activity of the three enzymes at an almost equal level during cultivation on succinate. CatB and CatC were purified to homogeneity and characterized. CatB was a monomer with a molecular mass of 44 kDa. The enzyme was activated by Mn2+, Co2+ and Mg2+. Native CatC was a homo-octamer with a molecular mass of 100 kDa. The enzyme was stable between pH 7.0 and 10.5 and was resistant to heating up to 90 degrees C. Genes coding for CatA, CatB and CatC were cloned and named catA, catB and catC respectively. The catABC genes were transcribed as one operon. The deduced amino acid sequences of CatA, CatB and CatC showed high identities with those from other Gram-positive micro-organisms. A regulator gene such as catR encoding a regulatory protein was not observed around the cat gene cluster of Rhodococcus sp. AN-22, but a possible relic of catR was found in the upstream region of catA. Reverse transcriptase-PCR and primer extension analyses showed that the transcriptional start site of the cat gene cluster was located 891 bp upstream of the catA initiation codon in the AN-22 strain growing on both aniline and succinate. Based on these data, we concluded that the bacterium constitutively transcribed the catABC genes and translated its mRNA into CatA, CatB and CatC.

  10. Constitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus species AN-22: production, purification, characterization and gene analysis of CatA, CatB and CatC

    PubMed Central

    Matsumura, Eitaro; Sakai, Masashi; Hayashi, Katsuaki; Murakami, Shuichiro; Takenaka, Shinji; Aoki, Kenji

    2005-01-01

    The aniline-assimilating bacterium Rhodococcus sp. AN-22 was found to constitutively synthesize CatB (cis,cis-muconate cycloisomerase) and CatC (muconolactone isomerase) in its cells growing on non-aromatic substrates, in addition to the previously reported CatA (catechol 1,2-dioxygenase). The bacterium maintained the specific activity of the three enzymes at an almost equal level during cultivation on succinate. CatB and CatC were purified to homogeneity and characterized. CatB was a monomer with a molecular mass of 44 kDa. The enzyme was activated by Mn2+, Co2+ and Mg2+. Native CatC was a homo-octamer with a molecular mass of 100 kDa. The enzyme was stable between pH 7.0 and 10.5 and was resistant to heating up to 90 °C. Genes coding for CatA, CatB and CatC were cloned and named catA, catB and catC respectively. The catABC genes were transcribed as one operon. The deduced amino acid sequences of CatA, CatB and CatC showed high identities with those from other Gram-positive micro-organisms. A regulator gene such as catR encoding a regulatory protein was not observed around the cat gene cluster of Rhodococcus sp. AN-22, but a possible relic of catR was found in the upstream region of catA. Reverse transcriptase-PCR and primer extension analyses showed that the transcriptional start site of the cat gene cluster was located 891 bp upstream of the catA initiation codon in the AN-22 strain growing on both aniline and succinate. Based on these data, we concluded that the bacterium constitutively transcribed the catABC genes and translated its mRNA into CatA, CatB and CatC. PMID:16156722

  11. 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

  12. 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. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  13. Genes of the Ecdysone Biosynthesis Pathway Are Regulated by the dATAC Histone Acetyltransferase Complex in Drosophila▿

    PubMed Central

    Pankotai, T.; Popescu, C.; Martín, D.; Grau, B.; Zsindely, N.; Bodai, L.; Tora, L.; Ferrús, A.; Boros, I.

    2010-01-01

    Uncovering mechanisms that regulate ecdysone production is an important step toward understanding the regulation of insect metamorphosis and processes in steroid-related pathologies. We report here the transcriptome analysis of Drosophila melanogaster dAda2a and dAda3 mutants, in which subunits of the ATAC acetyltransferase complex are affected. In agreement with the fact that these mutations lead to lethality at the start of metamorphosis, both the ecdysone levels and the ecdysone receptor binding to polytene chromosomes are reduced in these flies. The cytochrome genes (spookier, phantom, disembodied, and shadow) involved in steroid conversion in the ring gland are downregulated, while the gene shade, which is involved in converting ecdysone into its active form in the periphery, is upregulated in these dATAC subunit mutants. Moreover, driven expression of dAda3 at the site of ecdysone synthesis partially rescues dAda3 mutants. Mutants of dAda2b, a subunit of the dSAGA histone acetyltransferase complex, do not share phenotype characteristics and RNA profile alterations with dAda2a mutants, indicating that the ecdysone biosynthesis genes are regulated by dATAC, but not by dSAGA. Thus, we provide one of the first examples of the coordinated regulation of a functionally linked set of genes by the metazoan-specific ATAC complex. PMID:20584983

  14. Nucleotide sequences of genes encoding the type II chloramphenicol acetyltransferases of Escherichia coli and Haemophilus influenzae, which are sensitive to inhibition by thiol-reactive reagents.

    PubMed Central

    Murray, I A; Martinez-Suarez, J V; Close, T J; Shaw, W V

    1990-01-01

    Sensitivity of enzymes to inhibition by thiol-reactive reagents is often presented as evidence for the possible involvement of cysteine residues in substrate binding and catalysis or to highlight possible important differences in structure and mechanism between closely related enzymes. The primary phenotypic distinction between the enterobacterial type II chloramphenicol acetyltransferase (CATII; typified by the enzyme encoded by the incW transmissible plasmid pSa) and the CATI and CATIII variants is the greatly enhanced susceptibility of CATII to inactivation by thiol-specific modifying reagents. Determination of the nucleotide sequence of the gene, catII, present on pSa and that of a related determinant, catIIH, isolated from Haemophilus influenzae indicates that sensitivity to such reagents cannot be due to the presence of additional reactive cysteine residues in CATII. Comparative analysis of the inactivation of CATII and CATIII by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), 4,4'-dithiodipyridine (DTDP) and methyl methanethiosulphonate (MMTS) suggests that (i) inactivation occurs as a result of chemical modification of the same residue (Cys-31) in each enzyme, (ii) reagents that inactivate via a pseudo-first-order process (DTNB and DTDP) appear to bind with a greater affinity to CATII, and (iii) the intrinsic reactivity of Cys-31 in CATII greatly exceeds that of the corresponding residue in CATIII. The results lead to the conclusion that a striking difference in chemical reactivity of a unique and conserved thiol group between closely related enzyme variants may not be easily explained even when a high-resolution tertiary structure is available for one of them. Plausible explanations include more favourable access of reagents to Cys-31 in CATII or an enhanced reactivity of its thiol group imposed by the side chains of residues that are not in immediate contact with it. PMID:2268278

  15. Toxoplasma gondii lysine acetyltransferase GCN5-A functions in the cellular response to alkaline stress and expression of cyst genes.

    PubMed

    Naguleswaran, Arunasalam; Elias, Eliana V; McClintick, Jeanette; Edenberg, Howard J; Sullivan, William J

    2010-12-16

    Parasitic protozoa such as the apicomplexan Toxoplasma gondii progress through their life cycle in response to stimuli in the environment or host organism. Very little is known about how proliferating tachyzoites reprogram their expressed genome in response to stresses that prompt development into latent bradyzoite cysts. We have previously linked histone acetylation with the expression of stage-specific genes, but the factors involved remain to be determined. We sought to determine if GCN5, which operates as a transcriptional co-activator by virtue of its histone acetyltransferase (HAT) activity, contributed to stress-induced changes in gene expression in Toxoplasma. In contrast to other lower eukaryotes, Toxoplasma has duplicated its GCN5 lysine acetyltransferase (KAT). Disruption of the gene encoding for TgGCN5-A in type I RH strain did not produce a severe phenotype under normal culture conditions, but here we show that the TgGCN5-A null mutant is deficient in responding to alkaline pH, a common stress used to induce bradyzoite differentiation in vitro. We performed a genome-wide analysis of the Toxoplasma transcriptional response to alkaline pH stress, finding that parasites deleted for TgGCN5-A fail to up-regulate 74% of the stress response genes that are induced 2-fold or more in wild-type. Using chromatin immunoprecipitation, we verify an enrichment of TgGCN5-A at the upstream regions of genes activated by alkaline pH exposure. The TgGCN5-A knockout is also incapable of up-regulating key marker genes expressed during development of the latent cyst form, and is impaired in its ability to recover from alkaline stress. Complementation of the TgGCN5-A knockout restores the expression of these stress-induced genes and reverses the stress recovery defect. These results establish TgGCN5-A as a major contributor to the alkaline stress response in RH strain Toxoplasma.

  16. Toxoplasma gondii Lysine Acetyltransferase GCN5-A Functions in the Cellular Response to Alkaline Stress and Expression of Cyst Genes

    PubMed Central

    Naguleswaran, Arunasalam; Elias, Eliana V.; McClintick, Jeanette; Edenberg, Howard J.; Sullivan, William J.

    2010-01-01

    Parasitic protozoa such as the apicomplexan Toxoplasma gondii progress through their life cycle in response to stimuli in the environment or host organism. Very little is known about how proliferating tachyzoites reprogram their expressed genome in response to stresses that prompt development into latent bradyzoite cysts. We have previously linked histone acetylation with the expression of stage-specific genes, but the factors involved remain to be determined. We sought to determine if GCN5, which operates as a transcriptional co-activator by virtue of its histone acetyltransferase (HAT) activity, contributed to stress-induced changes in gene expression in Toxoplasma. In contrast to other lower eukaryotes, Toxoplasma has duplicated its GCN5 lysine acetyltransferase (KAT). Disruption of the gene encoding for TgGCN5-A in type I RH strain did not produce a severe phenotype under normal culture conditions, but here we show that the TgGCN5-A null mutant is deficient in responding to alkaline pH, a common stress used to induce bradyzoite differentiation in vitro. We performed a genome-wide analysis of the Toxoplasma transcriptional response to alkaline pH stress, finding that parasites deleted for TgGCN5-A fail to up-regulate 74% of the stress response genes that are induced 2-fold or more in wild-type. Using chromatin immunoprecipitation, we verify an enrichment of TgGCN5-A at the upstream regions of genes activated by alkaline pH exposure. The TgGCN5-A knockout is also incapable of up-regulating key marker genes expressed during development of the latent cyst form, and is impaired in its ability to recover from alkaline stress. Complementation of the TgGCN5-A knockout restores the expression of these stress-induced genes and reverses the stress recovery defect. These results establish TgGCN5-A as a major contributor to the alkaline stress response in RH strain Toxoplasma. PMID:21179246

  17. HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression

    PubMed Central

    Guha, Manti; Srinivasan, Satish; Guja, Kip; Mejia, Edison; Garcia-Diaz, Miguel; Johnson, F Brad; Ruthel, Gordon; Kaufman, Brett A; Rappaport, Eric F; Glineburg, M Rebecca; Fang, Ji-Kang; Szanto, Andres Klein; Nakagawa, Hiroshi; Basha, Jeelan; Kundu, Tapas; Avadhani, Narayan G

    2016-01-01

    Reduced mitochondrial DNA copy number, mitochondrial DNA mutations or disruption of electron transfer chain complexes induce mitochondria-to-nucleus retrograde signaling, which induces global change in nuclear gene expression ultimately contributing to various human pathologies including cancer. Recent studies suggest that these mitochondrial changes cause transcriptional reprogramming of nuclear genes although the mechanism of this cross talk remains unclear. Here, we provide evidence that mitochondria-to-nucleus retrograde signaling regulates chromatin acetylation and alters nuclear gene expression through the heterogeneous ribonucleoprotein A2 (hnRNAP2). These processes are reversed when mitochondrial DNA content is restored to near normal cell levels. We show that the mitochondrial stress-induced transcription coactivator hnRNAP2 acetylates Lys 8 of H4 through an intrinsic histone lysine acetyltransferase (KAT) activity with Arg 48 and Arg 50 of hnRNAP2 being essential for acetyl-CoA binding and acetyltransferase activity. H4K8 acetylation at the mitochondrial stress-responsive promoters by hnRNAP2 is essential for transcriptional activation. We found that the previously described mitochondria-to-nucleus retrograde signaling-mediated transformation of C2C12 cells caused an increased expression of genes involved in various oncogenic processes, which is retarded in hnRNAP2 silenced or hnRNAP2 KAT mutant cells. Taken together, these data show that altered gene expression by mitochondria-to-nucleus retrograde signaling involves a novel hnRNAP2-dependent epigenetic mechanism that may have a role in cancer and other pathologies. PMID:27990297

  18. 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.

  19. Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters.

    PubMed

    Peach, C; Velten, J

    1991-07-01

    Forty-five individually transformed clonal tobacco callus lines were simultaneously assayed for both chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS) activity resulting from expression of introduced reporter genes driven by the adjacent and divergent mannopine (mas) promoters. Excluding lines in which one or both of the enzyme activities was essentially zero, the activities of the reporter genes varied by as much as a factor of 136 (CAT) and 175 (GUS) between individual transformants. Superimposed upon the high degree of inter-clonal expression variability was an intra-clonal variability of 3-4-fold. The observed degree of intra-clonal reporter gene activity may be more extreme because of the regulatory characteristics of the mannopine promoters, but must still be addressed when considering the limitations of reporter gene-based analysis of transgene function and structure. There was no consistent correlation between the expression levels of the introduced CAT and GUS genes since the ratio of GUS to CAT activities (nmol min-1 mg-1) within individual lines varied from 0.05 to 49. Even divergent transcription from two directly adjacent promoter regions (both contained within a 479 bp TR-DNA fragment) is insufficient to guarantee concurrent expression of two linked transgenes. Our quantitative data were compared to published data of transgene expression variability to examine the overall distribution of expression levels in individual transformants. The resulting frequency distribution indicates that most transformants express introduced transgenes at relatively low levels, suggesting that a potentially large number of Agrobacterium-mediated transformation events may result in silent transgenes.

  20. Activation of the 2'-N-acetyltransferase gene [aac(2')-Ia] in Providencia stuartii by an interaction of AarP with the promoter region.

    PubMed

    Macinga, D R; Paradise, M R; Parojcic, M M; Rather, P N

    1999-07-01

    The aac(2')-Ia gene in Providencia stuartii encodes a 2'-N-acetyltransferase capable of acetylating both peptidoglycan and certain aminoglycoside antibiotics. Regulation of the aac(2')-Ia gene is influenced in a positive manner by the product of the aarP gene, which encodes a small transcriptional activator of the AraC (XylS) family. In this study, we demonstrate the sequence requirements at the aac(2')-Ia promoter for AarP binding and activation.

  1. 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

  2. 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.

  3. Genes, environment, and orofacial clefting: N-acetyltransferase and folic acid.

    PubMed

    Erickson, Robert P

    2010-09-01

    Nonsyndromic orofacial clefting has been the subject of intense studies, both genetic and epidemiological. The findings have frequently been controversial because of lack of reproducibility. Mouse models provide the potential both for genetic and environmental uniformity. We have chosen to study the role of genetic susceptibility to teratogen-induced orofacial clefting, using 2 drugs (dilantin and corticosteroid) and 1 nondrug teratogen (6-aminonicotinamide). The strongest single genetic influence we have found is N-acetyltransferase 2. Our recent work and that of others suggest that the influence of this locus is mediated through alterations in folate metabolism. Our results support epidemiological findings in humans and possibly implicate altered cytosine methylation, potentially caused by environmental factors, at least in the A/J model.

  4. Histone acetyltransferase AtGCN5/HAG1 is a versatile regulator of developmental and inducible gene expression in Arabidopsis.

    PubMed

    Servet, Caroline; Conde e Silva, Natalia; Zhou, Dao-Xiu

    2010-07-01

    Histone acetylation/deacetylation is a dynamic process and plays an important role in gene regulation. Histone acetylation homeostasis is regulated by antagonist actions of histone acetyltransferases (HAT) and deacetylases (HDAC). Plant genome encodes multiple HATs and HDACs. The Arabidopsis HAT gene AtGCN5/HAG1plays an essential role in many plant development processes, such as meristem function, cell differentiation, leaf and floral organogenesis, and responses to environmental conditions such as light and cold, indicating an important role of this HAT in the regulation of both long-term developmental switches and short-term inducible gene expression. AtGCN5 targets to a large number of promoters and is required for acetylation of several histone H3 lysine residues. Recruitment of AtGCN5 to target promoters is likely to be mediated by direct or indirect interaction with DNA-binding transcription factors and/or by interaction with acetylated histone lysine residues on the targets. Interplay between AtGCN5 and other HAT and HDAC is demonstrated to control specific regulatory pathways. Analysis of the role of AtGCN5 in light-inducible gene expression suggests a function of AtGCN5 in preparing chromatin commitment for priming inducible gene activation in plants.

  5. A 252 bp upstream region of the rat spermatocyte-specific hst70 gene is sufficient to promote expression of the hst70-CAT hybrid gene in testis and brain of transgenic mice.

    PubMed

    Widłak, W; Markkula, M; Krawczyk, Z; Kananen, K; Huhtaniemi, I

    1995-11-07

    The rat hst70 gene belongs to a heat shock hsp70 multigene family and its expression has been detected so far solely in spermatocytes. To investigate the cis-elements responsible for testis-specific expression of the hst70 gene we produced several lines of transgenic mice carrying fragments of the 5'-flanking regions of the hst70 gene fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Hybrid genes of series B were constructed such that, besides the 780 bp, 343 bp and 163 bp 5'-flanking region these plasmids contained no other sequences of the hst70 gene. In hybrid genes of series D the CAT gene was ligated to 343 bp and 252 bp 5'-flanking regions together with the 57 bp of the 5'-end nontranslated (leader) sequences of the hst70 gene. We found that in 780/B, 343/B, 343/D and 252/D adult mice the transgene was specifically and highly expressed in testes. In developing testes the high CAT activity appeared in transgenic mice aged 3 weeks and older. None of the three 163/B transgenic lines exhibited CAT activity in any tissue analyzed. In all CAT expressing lines a weak but significant CAT activity (up to 5% of that in testis) was detected also in the brain. RNase protection assay confirmed that the endogenous hst70 gene transcripts are present in testis as well as in brain of nontransgenic rats and mice. Our data show that the cis-regulatory sequences responsible for testis-specific and developmentally regulated expression of the hst70 gene are localized within the 252 bp region 5' to the gene and neither the 5'-end nor 3'-end nontranslated sequences of the gene are important for this specificity.

  6. 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.

  7. 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

  8. Characterization of genetic variation and natural selection at the arylamine N-acetyltransferase genes in global human populations.

    PubMed

    Mortensen, Holly M; Froment, Alain; Lema, Godfrey; Bodo, Jean-Marie; Ibrahim, Muntaser; Nyambo, Thomas B; Omar, Sabah A; Tishkoff, Sarah A

    2011-11-01

    Functional variability at the arylamine N-acetyltransferase genes is associated with drug response in humans and may have been adaptive in the past owing to selection pressure from diet and exposure to toxins during human evolution. We have characterized nucleotide variation at the NAT1 and NAT2 genes, and at the NATP1 pseudogene in global human populations, including many previously under-represented African populations, in order to identify potential functional variants and to understand the role that natural selection has played in shaping variation at these loci in globally diverse populations. We have resequenced approximately 2800 bp for each of the NAT1 and NAT2 gene regions, as well as the pseudogene NATP1, in 197 African and 132 nonAfrican individuals. We observe a signature of balancing selection maintaining variation in the 3'-UTR of NAT1, suggesting that these variants may play a functional role that is currently undefined. In addition, we observed high levels of nonsynonymous functional variation at the NAT2 locus that differs amongst ethnically diverse populations.

  9. 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

    2014-11-27

    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.

  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. DNA damage induces N-acetyltransferase NAT10 gene expression through transcriptional activation.

    PubMed

    Liu, Haijing; Ling, Yun; Gong, Yilei; Sun, Ying; Hou, Lin; Zhang, Bo

    2007-06-01

    NAT10 (N-acetyltransferase 10) is a protein with histone acetylation activity and primarily identified to be involved in regulation of telomerase activity. The presented research shows its transcriptional activation by genotoxic agents and possible role in DNA damage. NAT10 mRNA could be markedly increased by using hydrogen peroxide (H2O2) or cisplatin in a dose- and time-dependent way, and the immunofluorescent staining revealed that the treatment of H2O2 or cisplatin induced focal accumulation of NAT10 protein in cellular nuclei. Both H2O2 and cisplatin could stimulate the transcriptional activity of the NAT10 promoter through the upstream sequences from -615 bp to +110 bp, with which some nuclear proteins interacted. Ectopic expression of NAT10 could enhance the number of survival cells in the presence of H2O2 or cisplatin. The above results suggested that NAT10 could be involved in DNA damage response and increased cellular resistance to genotoxicity.

  13. CBP and p300 histone acetyltransferases contribute to homologous recombination by transcriptionally activating the BRCA1 and RAD51 genes.

    PubMed

    Ogiwara, Hideaki; Kohno, Takashi

    2012-01-01

    Histone acetylation at DNA double-strand break (DSB) sites by CBP and p300 histone acetyltransferases (HATs) is critical for the recruitment of DSB repair proteins to chromatin. Here, we show that CBP and p300 HATs also function in DSB repair by transcriptionally activating the BRCA1 and RAD51 genes, which are involved in homologous recombination (HR), a major DSB repair system. siRNA-mediated depletion of CBP and p300 impaired HR activity and downregulated BRCA1 and RAD51 at the protein and mRNA levels. Chromatin immunoprecipitation assays showed that CBP and p300 bind to the promoter regions of the BRCA1 and RAD51 genes, and that depletion of CBP and/or p300 reduces H3 and H4 acetylation and inhibits binding of the transcription factor E2F1 to these promoters. Depletion of CBP and p300 impaired DNA damage-induced phosphorylation and chromatin binding of the single-strand DNA-binding protein RPA following BRCA1-mediated DNA end resection. Consistent with this, subsequent phosphorylation of CHK1 and activation of the G2/M damage checkpoint were also impaired. These results indicate that the HATs CBP and p300 play multiple roles in the activation of the cellular response to DSBs.

  14. Feline immunodeficiency virus env gene evolution in experimentally infected cats.

    PubMed

    Kraase, Martin; Sloan, Richard; Klein, Dieter; Logan, Nicola; McMonagle, Linda; Biek, Roman; Willett, Brian J; Hosie, Margaret J

    2010-03-15

    Feline immunodeficiency virus (FIV), an immunosuppressive lentivirus found in cats worldwide, is studied to illuminate mechanisms of lentiviral pathogenesis and to identify key components of protective immunity. During replication, lentiviruses accumulate errors of nucleotide mis-incorporation due to the low-fidelity of reverse transcriptase and recombination between viral variants, resulting in the emergence of a complex viral "quasispecies". In patients infected with HIV-1, env sequences may vary by up to 10% and the detection of quasispecies with greater heterogeneity is associated with higher viral loads and reduced CD4+ T cell numbers [1], indicating that transmission of more complex quasispecies may lead to disease progression. However, little is known about how FIV evolves as disease progresses, or why some cats develop AIDS rapidly while disease progression is slow in others. The aim of this study was to determine whether disease progression may be governed by viral evolution and to examine the diversity of viral variants emerging following infection with an infectious molecular clone. The FIV env gene encoding the envelope glycoprotein (Env) was examined at early (12 weeks) and late (322 weeks) stages of FIV infection in two groups of cats infected experimentally with the FIV-GL8 molecular clone. Viral variants were detected within quasispecies in cats in the late stages of FIV infection that contained differing amino acid compositions in several variable loops of Env, some of which were identified as determinants of receptor usage and resistance to neutralization. Therefore these results indicate that the FIV env gene evolves during the course of infection, giving rise to variants that resist neutralization and likely lead to disease progression.

  15. Daily oscillation and photoresponses of clock gene, Clock, and clock-associated gene, arylalkylamine N-acetyltransferase gene transcriptions in the rat pineal gland.

    PubMed

    Wang, Guo-Qing; Du, Yu-Zhen; Tong, Jian

    2007-01-01

    This study was conducted to investigate the circadian rhythms and light responses of Clock and arylalkylamine N-acetyltransferase (NAT) gene expressions in the rat pineal gland under the environmental conditions of a 12 h light (05:00-17:00 h): 12 h-dark (17:00-05:00 h) cycle (LD) and constant darkness (DD). The pineal gland of Sprague-Dawley rats housed under a LD regime (n=42) for four weeks and of a regime (n=42) for eight weeks were sampled at six different times, every 4 h (n=7 animals per time point), during a 24 h period. Total RNA was extracted from each sample, and the semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was used to determine temporal changes in mRNA levels of Clock and NAT genes during different circadian or zeitgeber times. The data and parameters were analyzed by the cosine function software, Clock Lab software, and the amplitude F test was used to reveal the circadian rhythm. In the DD or LD condition, both the Clock and NAT mRNA levels in the pineal gland showed robust circadian oscillation (p<0.05) with the peak at the subjective night or at nighttime. In comparison with the DD regime, the amplitudes and mRNA levels at the peaks of Clock and NAT expressions in LD in the pineal gland were significantly reduced (p<0.05). In the DD or LD condition, the circadian expressions of NAT were similar in pattern to those of Clock in the pineal gland (p>0.05). These findings indicate that the transcriptions of Clock and NAT genes in the pineal gland not only show remarkably synchronous endogenous circadian rhythmic changes, but also respond to the ambient light signal in a reduced manner.

  16. A series of shuttle vectors using chloramphenicol acetyltransferase as a reporter enzyme in yeast.

    PubMed

    Mannhaupt, G; Pilz, U; Feldmann, H

    1988-07-30

    Reports from numerous laboratories have shown that the gene coding for the bacterial enzyme chloramphenicol-3-O-acetyltransferase can be used as a reporter gene (cat) in mammalian and plant systems to analyze gene activity at the transcriptional level when combined with endogenous regulatory signals; the enzyme activity can be quantified by a chromatographic or a photometric assay. To adapt this simple and highly sensitive test for the yeast system, we constructed a series of yeast vectors containing the cat gene together with selectable markers for Escherichia coli and yeast; integrating, autonomously replicating and centromere-carrying plasmids were used. We show that the cat gene lacking the endogenous promoter is expressed at low levels in yeast transformants. To demonstrate functional expression of the cat gene placed under the control of a yeast promoter, we chose the PHO5 regulatory region. We found that cat expression was induced via the PHO5 promoter in a manner as observed for the endogenous PHO5 gene, whereas in the repressed state cat expression remained low. Using these vectors, it should be feasible to analyze other sequences conferring promoter activity or other control functions in yeast.

  17. 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

  18. Effect of monochromatic light on circadian rhythmic expression of clock genes and arylalkylamine N-acetyltransferase in chick retina.

    PubMed

    Cao, Jing; Bian, Jiang; Wang, Zixu; Dong, Yulan; Chen, Yaoxing

    2017-09-14

    Birds have more developed visual function. They not only have the ability to detect light and darkness but also have the color vision. Previous study showed that monochromatic light influenced avian physiological processes, which were controlled by clock genes. Therefore, bird's eye is a good model to studying the impact of color of light on circadian rhythms. Avian retina is one of the most important central oscillations. The study was designed to investigate the effect of color of light on the expression of clock genes and arylalkylamine N-acetyltransferase (Aanat) mRNA expression in chick retina. A total of 240 post-hatching day (P) 0 broiler chickens were exposed to blue (BL), green (GL), red (RL) and white light (WL) from a LED system under a light-dark cycle 12L:12D for 14 d. The results show that the significant daily variations existed in the gene expression of cBmal1, cBmal2, cCry1, cCry2, cPer2 and cPer3, but not for cClock under four light treatments. The genes cBmal1, cCry1, cPer2 and cPer3 presented circadian rhythmic expression under the various monochromatic lights. When compared with WL, GL elevated the expression of positive regulators of cellular clock (cBmal1, cBmal2 and cClock) and cAanat mRNA level, whereas RL increased the mRNA levels of negative regulators of cellular clock (cCry1, cCry2, cPer2 and cPer3) and decreased the cAanat mRNA expression in the retina. These results demonstrated that monochromatic light affect the periodic expression levels of the biological clock mRNA by positive and negative feedback loop interactions, GL activated the transcription of cAanat; while RL suppressed the transcription of cAanat. Thereby, color of light regulates ocular cAanat expression by affecting on expression of cellular clock regulators.

  19. Thermoadaptation-directed evolution of chloramphenicol acetyltransferase in an error-prone thermophile using improved procedures.

    PubMed

    Kobayashi, Jyumpei; Furukawa, Megumi; Ohshiro, Takashi; Suzuki, Hirokazu

    2015-07-01

    Enhancing the thermostability of thermolabile enzymes extends their practical utility. We previously demonstrated that an error-prone thermophile derived from Geobacillus kaustophilus HTA426 can generate mutant genes encoding enzyme variants that are more thermostable than the parent enzyme. Here, we used this approach, termed as thermoadaptation-directed enzyme evolution, to increase the thermostability of the chloramphenicol acetyltransferase (CAT) of Staphylococcus aureus and successfully generated a CAT variant with an A138T replacement (CAT(A138T)). This variant was heterologously produced, and its enzymatic properties were compared with those of the wild type. We found that CAT(A138T) had substantially higher thermostability than CAT but had comparable activities, showing that the A138T replacement enhanced protein thermostability without affecting the catalytic activity. Because variants CAT(A138S) and CAT(A138V), which were generated via in vitro site-directed mutagenesis, were more thermostable than CAT, the thermostability enhancement resulting from the A138T replacement can be attributed to both the presence of a hydroxyl group and the bulk of the threonine side chain. CAT(A138T) conferred chloramphenicol resistance to G. kaustophilus cells at high temperature more efficiently than CAT. Therefore, the gene encoding CAT(A138T) may be useful as a genetic marker in Geobacillus spp. Notably, CAT(A138T) generation was achieved only by implementing improved procedures (plasmid-based mutations on solid media); previous procedures (chromosome-based mutations in liquid media) were unsuccessful. This result suggests that this improved procedure is crucial for successful thermoadaptation-directed evolution in certain cases and increases the opportunities for generating thermostable enzymes.

  20. Expression of a streptomycete leaderless mRNA encoding chloramphenicol acetyltransferase in Escherichia coli.

    PubMed Central

    Wu, C J; Janssen, G R

    1997-01-01

    The chloramphenicol acetyltransferase (cat) gene from Streptomyces acrimycini encodes a leaderless mRNA. Expression of the cat coding sequence as a leaderless mRNA from a modified lac promoter resulted in chloramphenicol resistance in Escherichia coli. Transcript mapping with nuclease S1 confirmed that the 5' end of the cat message initiated at the A of the AUG translational start codon. Site-directed mutagenesis of the lac promoter or the cat start codon abolished chloramphenicol resistance, indicating that E. coli initiated translation at the 5' terminal AUG of the cat leaderless mRNA. Addition of 5'-AUGC-3' to the 5' end of the cat mRNA resulted in translation occurring also from the reading frame defined by the added AUG triplet, suggesting that a 5'-terminal start codon is an important recognition feature for initiation and establishing reading frame during translation of leaderless mRNA. Addition of an untranslated leader and Shine-Dalgarno sequence to the cat coding sequence increased cat expression in a cat:lacZ fusion; however, the level of expression was significantly lower than when a fragment of the bacteriophage lambda cI gene, also encoding a leaderless mRNA, was fused to lacZ. These results indicate that in the absence of an untranslated leader and Shine-Dalgarno sequence, the streptomycete cat mRNA is translated by E. coli; however, the cat translation signals, or other features of the cat mRNA, provide for only a low level of expression in E. coli. PMID:9352935

  1. [Circadian rhythms and light responses of clock gene and arylalkylamine N-acetyltransferase gene expressions in the pineal gland of rats].

    PubMed

    Wang, Guo-Qing; Du, Yu-Zhen; Tong, Jian

    2005-02-25

    This study was to investigate the circadian rhythms and light responses of Clock gene and arylalkylamine N-acetyltransferase (NAT) gene expressions in the rat pineal gland under the 12 h-light : 12 h-dark cycle condition (LD) and constant darkness (DD). Sprague-Dawley rats housed under the light regime of LD (n=36) for 4 weeks and of DD (n=36) for 8 weeks were sampled for the pineal gland once a group (n=6) every 4 h in a circadian day. The total RNA was extracted from each sample and the semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was used to determine the temporal changes in mRNA levels of Clock and NAT genes during different circadian times or zeitgeber times. The data were analysed by the cosine function software, Clock Lab software and the amplitude F test was used to reveal the circadian rhythm. The main results obtained are as follows. (1) In DD or LD condition, both of Clock and NAT genes mRNA levels in the pineal gland showed robust circadian oscillation (P< 0.05) with the peak at the subjective night or at night-time. (2) In comparison with DD regime, the amplitudes and the mRNA levels at peaks of Clock and NAT genes expressions in LD in the pineal gland were significantly reduced (P< 0.05). (3) In DD or LD condition, the circadian expressions of NAT gene were similar in pattern to those of Clock gene in the pineal gland (P> 0.05). These findings suggest that the expressions of Clock and NAT genes in the pineal gland not only show remarkably synchronous endogenous circadian rhythmic changes, but also response to the ambient light signal in a reduced manner.

  2. 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.

  3. Different functions of the histone acetyltransferase HAC1 gene traced in the model species Medicago truncatula, Lotus japonicus and Arabidopsis thaliana.

    PubMed

    Boycheva, Irina; Vassileva, Valya; Revalska, Miglena; Zehirov, Grigor; Iantcheva, Anelia

    2017-03-01

    In eukaryotes, histone acetyltransferases regulate the acetylation of histones and transcription factors, affecting chromatin structural organization, transcriptional regulation, and gene activation. To assess the role of HAC1, a gene encoding for a histone acetyltransferase in Medicago truncatula, stable transgenic lines with modified HAC1 expression in the model plants M. truncatula, Lotus japonicus, and Arabidopsis thaliana were generated by Agrobacterium-mediated transformation and used for functional analyses. Histochemical, transcriptional, flow cytometric, and morphological analyses demonstrated the involvement of HAC1 in plant growth and development, responses to internal stimuli, and cell cycle progression. Expression patterns of a reporter gene encoding beta-glucuronidase (GUS) fused to the HAC1 promoter sequence were associated with young tissues comprised of actively dividing cells in different plant organs. The green fluorescent protein (GFP) signal, driven by the HAC1 promoter, was detected in the nuclei and cytoplasm of root cells. Transgenic lines with HAC1 overexpression and knockdown showed a wide range of phenotypic deviations and developmental abnormalities, which provided lines of evidence for the role of HAC1 in plant development. Synchronization of A. thaliana root tips in a line with HAC1 knockdown showed the involvement of this gene in the acetylation of two core histones during S phase of the plant cell cycle.

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

    PubMed

    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.

  5. 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

  6. 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.

  7. Molecular Cloning and Expression Analysis of a Catalase Gene (NnCAT) from Nelumbo nucifera.

    PubMed

    Dong, Chen; Zheng, Xingfei; Diao, Ying; Wang, Youwei; Zhou, Mingquan; Hu, Zhongli

    2015-11-01

    Rapid amplification cDNA end (RACE) assay was established to achieve the complete cDNA sequence of a catalase gene (NnCAT) from Nelumbo nucifera. The obtained full-length cDNA was 1666 bp in size and contained a 1476-bp open reading frame. The 3D structural model of NnCAT was constructed by homology modeling. The putative NnCAT possessed all the main characteristic amino acid residues and motifs of catalase (CAT) protein family, and the phylogenetic analysis revealed that NnCAT grouped together with high plants. Moreover, recombinant NnCAT showed the CAT activity (758 U/mg) at room temperature, holding high activity during temperature range of 20-50 °C, then the optimal pH of recombinant protein was assessed from pH 4 to pH 11. Additionally, real-time PCR assay demonstrated that NnCAT mRNA was expressed in various tissues of N. nucifera, with the highest expression in young leaf and lowest level in the root, and mRNA level of NnCAT was significantly augmented in response to short-time mechanical wounding. Different expression pattern of NnCAT gene suggested that NnCAT probably played a defensive role in the initial stages of oxidative stress, regulating the level of reactive oxygen species (ROS) by extracellular stimuli such as short-time mechanical wounding.

  8. CAT8, a new zinc cluster-encoding gene necessary for derepression of gluconeogenic enzymes in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Hedges, D; Proft, M; Entian, K D

    1995-01-01

    The expression of gluconeogenic fructose-1,6-bisphosphatase (encoded by the FBP1 gene) depends on the carbon source. Analysis of the FBP1 promoter revealed two upstream activating elements, UAS1FBP1 and UAS2FBP1, which confer carbon source-dependent regulation on a heterologous reporter gene. On glucose media neither element was activated, whereas after transfer to ethanol a 100-fold derepression was observed. This gene activation depended on the previously identified derepression genes CAT1 (SNF1) (encoding a protein kinase) and CAT3 (SNF4) (probably encoding a subunit of Cat1p [Snf1p]). Screening for mutations specifically involved in UAS1FBP1 derepression revealed the new recessive derepression mutation cat8. The cat8 mutants also failed to derepress UAS2FBP1, and these mutants were unable to grow on nonfermentable carbon sources. The CAT8 gene encodes a zinc cluster protein related to Saccharomyces cerevisiae Gal4p. Deletion of CAT8 caused a defect in glucose derepression which affected all key gluconeogenic enzymes. Derepression of glucose-repressible invertase and maltase was still normally regulated. A CAT8-lacZ promoter fusion revealed that the CAT8 gene itself is repressed by Cat4p (Mig1p). These results suggest that gluconeogenic genes are derepressed upon binding of Cat8p, whose synthesis depends on the release of Cat4p (Mig1p) from the CAT8 promoter. However, gluconeogenic promoters are still glucose repressed in cat4 mutants, which indicates that in addition to its transcription, the Cat8p protein needs further activation. The observation that multicopy expression of CAT8 reverses the inability of cat1 and cat3 mutants to grow on ethanol indicates that Cat8p might be the substrate of the Cat1p/Cat3p protein kinase. PMID:7891685

  9. Nucleotide sequence and genetic analysis of the Azotobacter chroococcum nifUSVWZM gene cluster, including a new gene (nifP) which encodes a serine acetyltransferase.

    PubMed Central

    Evans, D J; Jones, R; Woodley, P R; Wilborn, J R; Robson, R L

    1991-01-01

    Nucleotide sequence was obtained for a region of 7,099 bp spanning the nifU, nifS, nifV, nifW, nifZ, and nifM genes from Azotobacter chroococcum. Chromosomal mutations constructed at several sites within the locus confirmed a requirement for this region for expression of the molybdenum nitrogenase in this organism. The genes are tightly clustered and ordered as in Klebsiella pneumoniae except for two additional open reading frames (ORFs) between nifV and nifW. The arrangement of genes in A. chroococcum closely matches that described for Azotobacter vinelandii. The polypeptide encoded by ORF4 immediately downstream from nifV is 41% identical over 186 amino acids to the product of the cysE gene from Escherichia coli, which encodes serine acetyltransferase (SAT), a key enzyme in cysteine biosynthesis. Plasmids which potentially express ORF4 complemented E. coli JM39, a cysteine auxotroph which lacks SAT. SAT activity was detected in crude extracts of one such complemented strain. A strain of A. chroococcum carrying a chromosomal disruption of ORF4 grew normally with ammonium as the N source but more slowly than the parental strain when N2 was the sole N source. These data suggest that ORF4 encodes a nif-specific SAT required for optimizing expression of nitrogenase activity. ORF4 was assigned the name nifP. nifP may be required to boost rates of synthesis or intracellular concentrations of cysteine or methionine. Sequence identity between nifV and leuA gene products suggests that nifV may catalyze a condensation reaction analogous to that carried out by isopropylmalate synthase (LEUA) but in which acetyl coenzyme and alpha-ketoglutarate are substrates for the formation of homocitrate, the proposed product of NIFV activity. PMID:1885524

  10. Nucleotide sequence analysis of the gene specifying the bifunctional 6'-aminoglycoside acetyltransferase 2"-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities.

    PubMed

    Ferretti, J J; Gilmore, K S; Courvalin, P

    1986-08-01

    The gene specifying the bifunctional 6'-aminoglycoside acetyltransferase [AAC(6')] 2"-aminoglycoside phosphotransferase [APH(2")] enzyme from the Streptococcus faecalis plasmid pIP800 was cloned in Escherichia coli. A single protein with an apparent molecular weight of 56,000 was specified by this cloned determinant as detected in minicell experiments. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 479 amino acids and with a molecular weight of 56,850. The deduced amino acid sequence of the bifunctional AAC(6')-APH(2") gene product possessed two regions of homology with other sequenced resistance proteins. The N-terminal region contained a sequence that was homologous to the chloramphenicol acetyltransferase of Bacillus pumilus, and the C-terminal region contained a sequence homologous to the aminoglycoside phosphotransferase of Streptomyces fradiae. Subcloning experiments were performed with the AAC(6')-APH(2") resistance determinant, and it was possible to obtain gene segments independently specifying the acetyltransferase and phosphotransferase activities. These data suggest that the gene specifying the AAC(6')-APH(2") resistance enzyme arose as a result of a gene fusion.

  11. 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. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  12. 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

  13. 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

  14. Pseudogenization of a sweet-receptor gene accounts for cats' indifference toward sugar.

    PubMed

    Li, Xia; Li, Weihua; Wang, Hong; Cao, Jie; Maehashi, Kenji; Huang, Liquan; Bachmanov, Alexander A; Reed, Danielle R; Legrand-Defretin, Véronique; Beauchamp, Gary K; Brand, Joseph G

    2005-07-01

    Although domestic cats (Felis silvestris catus) possess an otherwise functional sense of taste, they, unlike most mammals, do not prefer and may be unable to detect the sweetness of sugars. One possible explanation for this behavior is that cats lack the sensory system to taste sugars and therefore are indifferent to them. Drawing on work in mice, demonstrating that alleles of sweet-receptor genes predict low sugar intake, we examined the possibility that genes involved in the initial transduction of sweet perception might account for the indifference to sweet-tasting foods by cats. We characterized the sweet-receptor genes of domestic cats as well as those of other members of the Felidae family of obligate carnivores, tiger and cheetah. Because the mammalian sweet-taste receptor is formed by the dimerization of two proteins (T1R2 and T1R3; gene symbols Tas1r2 and Tas1r3), we identified and sequenced both genes in the cat by screening a feline genomic BAC library and by performing PCR with degenerate primers on cat genomic DNA. Gene expression was assessed by RT-PCR of taste tissue, in situ hybridization, and immunohistochemistry. The cat Tas1r3 gene shows high sequence similarity with functional Tas1r3 genes of other species. Message from Tas1r3 was detected by RT-PCR of taste tissue. In situ hybridization and immunohistochemical studies demonstrate that Tas1r3 is expressed, as expected, in taste buds. However, the cat Tas1r2 gene shows a 247-base pair microdeletion in exon 3 and stop codons in exons 4 and 6. There was no evidence of detectable mRNA from cat Tas1r2 by RT-PCR or in situ hybridization, and no evidence of protein expression by immunohistochemistry. Tas1r2 in tiger and cheetah and in six healthy adult domestic cats all show the similar deletion and stop codons. We conclude that cat Tas1r3 is an apparently functional and expressed receptor but that cat Tas1r2 is an unexpressed pseudogene. A functional sweet-taste receptor heteromer cannot form, and

  15. Pseudogenization of a Sweet-Receptor Gene Accounts for Cats' Indifference toward Sugar

    PubMed Central

    Li, Xia; Li, Weihua; Wang, Hong; Cao, Jie; Maehashi, Kenji; Huang, Liquan; Bachmanov, Alexander A; Reed, Danielle R; Legrand-Defretin, Véronique; Beauchamp, Gary K; Brand, Joseph G

    2005-01-01

    Although domestic cats (Felis silvestris catus) possess an otherwise functional sense of taste, they, unlike most mammals, do not prefer and may be unable to detect the sweetness of sugars. One possible explanation for this behavior is that cats lack the sensory system to taste sugars and therefore are indifferent to them. Drawing on work in mice, demonstrating that alleles of sweet-receptor genes predict low sugar intake, we examined the possibility that genes involved in the initial transduction of sweet perception might account for the indifference to sweet-tasting foods by cats. We characterized the sweet-receptor genes of domestic cats as well as those of other members of the Felidae family of obligate carnivores, tiger and cheetah. Because the mammalian sweet-taste receptor is formed by the dimerization of two proteins (T1R2 and T1R3; gene symbols Tas1r2 and Tas1r3), we identified and sequenced both genes in the cat by screening a feline genomic BAC library and by performing PCR with degenerate primers on cat genomic DNA. Gene expression was assessed by RT-PCR of taste tissue, in situ hybridization, and immunohistochemistry. The cat Tas1r3 gene shows high sequence similarity with functional Tas1r3 genes of other species. Message from Tas1r3 was detected by RT-PCR of taste tissue. In situ hybridization and immunohistochemical studies demonstrate that Tas1r3 is expressed, as expected, in taste buds. However, the cat Tas1r2 gene shows a 247-base pair microdeletion in exon 3 and stop codons in exons 4 and 6. There was no evidence of detectable mRNA from cat Tas1r2 by RT-PCR or in situ hybridization, and no evidence of protein expression by immunohistochemistry. Tas1r2 in tiger and cheetah and in six healthy adult domestic cats all show the similar deletion and stop codons. We conclude that cat Tas1r3 is an apparently functional and expressed receptor but that cat Tas1r2 is an unexpressed pseudogene. A functional sweet-taste receptor heteromer cannot form, and

  16. 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.

  17. The lac operon galactoside acetyltransferase.

    PubMed

    Roderick, Steven L

    2005-06-01

    Of the proteins encoded by the three structural genes of the lac operon, the galactoside acetyltransferase (thiogalactoside transacetylase, LacA, GAT) encoded by lacA is the only protein whose biological role remains in doubt. Here, we briefly note the classical literature that led to the identification and initial characterization of GAT, and focus on more recent results which have revealed its chemical mechanism of action and its membership in a large superfamily of structurally similar acyltransferases. The structural and sequence similarities of several members of this superfamily confirm the original claim for GAT as a CoA-dependent acetyltransferase specific for the 6-hydroxyl group of certain pyranosides, but do not yet point to the identity of the natural substrate(s) of the enzyme.

  18. Adr1 and Cat8 Mediate Coactivator Recruitment and Chromatin Remodeling at Glucose-Regulated Genes

    PubMed Central

    Biddick, Rhiannon K.; Law, G. Lynn; Young, Elton T.

    2008-01-01

    Background Adr1 and Cat8 co-regulate numerous glucose-repressed genes in S. cerevisiae, presenting a unique opportunity to explore their individual roles in coactivator recruitment, chromatin remodeling, and transcription. Methodology/Principal Findings We determined the individual contributions of Cat8 and Adr1 on the expression of a cohort of glucose-repressed genes and found three broad categories: genes that need both activators for full derepression, genes that rely mostly on Cat8 and genes that require only Adr1. Through combined expression and recruitment data, along with analysis of chromatin remodeling at two of these genes, ADH2 and FBP1, we clarified how these activators achieve this wide range of co-regulation. We find that Adr1 and Cat8 are not intrinsically different in their abilities to recruit coactivators but rather, promoter context appears to dictate which activator is responsible for recruitment to specific genes. These promoter-specific contributions are also apparent in the chromatin remodeling that accompanies derepression: ADH2 requires both Adr1 and Cat8, whereas, at FBP1, significant remodeling occurs with Cat8 alone. Although over-expression of Adr1 can compensate for loss of Cat8 at many genes in terms of both activation and chromatin remodeling, this over-expression cannot complement all of the cat8Δ phenotypes. Conclusions/Significance Thus, at many of the glucose-repressed genes, Cat8 and Adr1 appear to have interchangeable roles and promoter architecture may dictate the roles of these activators. PMID:18197247

  19. 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.

  20. Arylamine n-acetyltransferases in eukaryotic microorganisms

    USDA-ARS?s Scientific Manuscript database

    Microorganisms can survive highly toxic environments through numerous xenobiotic metabolizing enzymes, including arylamine N-acetyltransferases (NATs). NAT genes are present in bacteria, archaea, protists and fungi. In lower taxa of fungi, NAT genes are found in chytridiomycetes. In Dikarya, NAT gen...

  1. Aloe-emodin inhibited N-acetylation and DNA adduct of 2-aminofluorene and arylamine N-acetyltransferase gene expression in mouse leukemia L 1210 cells.

    PubMed

    Chung, Jing-Gung; Li, Yu-Ching; Lee, Yi-Min; Lin, Jing-Pin; Cheng, Kwork-Chui; Chang, Weng-Cheng

    2003-09-01

    N-Acetyltransferases (NATs) plays an important role in the first step of arylamine compounds metabolism. Polymorphic NAT is coded for rapid or slow acetylatoion phenotypes, which are recognized to affect cancer risk related to environmental exposure. Aloe-emodin has been shown to exit anticancer activity. The purpose of this study is to examine whether or not aloe-emodin could affect arylamine N-acetyltransferase (NAT) activity and gene expression (NAT mRNA) and DNA-2-aminofluorene (DNA-AF) adduct formation in mouse leukemia cells (L 1210). By using high performance liquid chromatography, N-acetylation and non-N-acetylation of AF were determined and quantitated. By using reverse transcriptase-polymerase chain reaction (RT-PCR) and PCR, NAT mRNA was determined and quantitated. Aloe-emodin displayed a dose-dependent inhibition to cytosolic NAT activity and intact mice leukemia cells. Time-course experiments indicated that N-acetylation of AF measured from intact mice leukemia cells were inhibited by aloe-emodin for up to 24h. Using standard steady-state kinetic analysis, it was demonstrated that aloe-emodin was a possible uncompetitive inhibitor to NAT activity in cytosols. The DNA-AF adduct formation in mouse leukemia cells were inhibited by aloe-emodin. The NAT1 mRNA in mouse leukemia cells were also inhibited by aloe-emodin. This report is the first demonstration which showed aloe-emodin affect mice leukemia cells NAT activity, gene expression (NAT1 mRNA) and DNA-AF on adduct formation.

  2. Expression Levels of the Yeast Alcohol Acetyltransferase Genes ATF1, Lg-ATF1, and ATF2 Control the Formation of a Broad Range of Volatile Esters

    PubMed Central

    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.

    2003-01-01

    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 atf1Δ atf2Δ 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

  3. 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

  4. Antimicrobial resistance and virulence gene profiles in P. multocida strains isolated from cats

    PubMed Central

    Ferreira, Thais Sebastiana Porfida; Felizardo, Maria Roberta; de Gobbi, Debora Dirani Sena; Moreno, Marina; Moreno, Andrea Micke

    2015-01-01

    Cats are often described as carriers of Pasteurella multocida in their oral microbiota. This agent is thought to cause pneumonia, conjunctivitis, rhinitis, gingivostomatitis, abscess and osteonecrosis in cats. Human infection with P. multocida has been described in several cases affecting cat owners or after cat bites. In Brazil, the cat population is approximately 21 million animals and is increasing, but there are no studies of the presence of P. multocida in the feline population or of human cases of infection associated with cats. In this study, one hundred and ninety-one healthy cats from owners and shelters in São Paulo State, Brazil, were evaluated for the presence of P. multocida in their oral cavities. Twenty animals were positive for P. multocida , and forty-one strains were selected and characterized by means of biochemical tests and PCR. The P. multocida strains were tested for capsular type, virulence genes and resistance profile. A total of 75.6% (31/41) of isolates belonged to capsular type A, and 24.4% (10/41) of the isolates were untypeable. None of the strains harboured toxA, tbpA or pfhA genes. The frequencies of the other genes tested were variable, and the data generated were used to build a dendrogram showing the relatedness of strains, which were clustered according to origin. The most common resistance profile observed was against sulfizoxazole and trimethoprim-sulphamethoxazole. PMID:26221117

  5. Antimicrobial resistance and virulence gene profiles in P. multocida strains isolated from cats.

    PubMed

    Ferreira, Thais Sebastiana Porfida; Felizardo, Maria Roberta; de Gobbi, Debora Dirani Sena; Moreno, Marina; Moreno, Andrea Micke

    2015-03-01

    Cats are often described as carriers of Pasteurella multocida in their oral microbiota. This agent is thought to cause pneumonia, conjunctivitis, rhinitis, gingivostomatitis, abscess and osteonecrosis in cats. Human infection with P. multocida has been described in several cases affecting cat owners or after cat bites. In Brazil, the cat population is approximately 21 million animals and is increasing, but there are no studies of the presence of P. multocida in the feline population or of human cases of infection associated with cats. In this study, one hundred and ninety-one healthy cats from owners and shelters in São Paulo State, Brazil, were evaluated for the presence of P. multocida in their oral cavities. Twenty animals were positive for P. multocida , and forty-one strains were selected and characterized by means of biochemical tests and PCR. The P. multocida strains were tested for capsular type, virulence genes and resistance profile. A total of 75.6% (31/41) of isolates belonged to capsular type A, and 24.4% (10/41) of the isolates were untypeable. None of the strains harboured toxA, tbpA or pfhA genes. The frequencies of the other genes tested were variable, and the data generated were used to build a dendrogram showing the relatedness of strains, which were clustered according to origin. The most common resistance profile observed was against sulfizoxazole and trimethoprim-sulphamethoxazole.

  6. The novel kasugamycin 2'-N-acetyltransferase gene aac(2')-IIa, carried by the IncP island, confers kasugamycin resistance to rice-pathogenic bacteria.

    PubMed

    Yoshii, Atsushi; Moriyama, Hiromitsu; Fukuhara, Toshiyuki

    2012-08-01

    Kasugamycin (KSM), a unique aminoglycoside antibiotic, has been used in agriculture for many years to control not only rice blast caused by the fungus Magnaporthe grisea but also rice bacterial grain and seedling rot or rice bacterial brown stripe caused by Burkholderia glumae or Acidovorax avenae subsp. avenae, respectively. Since both bacterial pathogens are seed-borne and cause serious injury to rice seedlings, the emergence of KSM-resistant B. glumae and A. avenae isolates highlights the urgent need to understand the mechanism of resistance to KSM. Here, we identified a novel gene, aac(2')-IIa, encoding a KSM 2'-N-acetyltransferase from both KSM-resistant pathogens but not from KSM-sensitive bacteria. AAC(2')-IIa inactivates KSM, although it reveals no cross-resistance to other aminoglycosides. The aac(2')-IIa gene from B. glumae strain 5091 was identified within the IncP genomic island inserted into the bacterial chromosome, indicating the acquisition of this gene by horizontal gene transfer. Although excision activity of the IncP island and conjugational gene transfer was not detected under the conditions tested, circular intermediates containing the aac(2')-IIa gene were detected. These results indicate that the aac(2')-IIa gene had been integrated into the IncP island of a donor bacterial species. Molecular detection of the aac(2')-IIa gene could distinguish whether isolates are resistant or susceptible to KSM. This may contribute to the production of uninfected rice seeds and lead to the effective control of these pathogens by KSM.

  7. Isolation and expression of the catA gene encoding the major vegetative catalase in Streptomyces coelicolor Müller.

    PubMed Central

    Cho, Y H; Roe, J H

    1997-01-01

    We isolated the catA gene for the major vegetative catalase from Streptomyces coelicolor Müller. It encodes a polypeptide of 488 residues (55,440 Da) that is highly homologous to typical monofunctional catalases. We investigated catA expression by analyzing both catA mRNA and catalase activity. catA expression was increased by H2O2 treatment but did not increase during stationary phase. A putative catalase (CatB) cross-reactive with anti-CatA antibody appeared during stationary phase and in the aerial mycelium. PMID:9190825

  8. Panax ginseng induces the expression of CatSper genes and sperm hyperactivation

    PubMed Central

    Park, Eun Hwa; Kim, Do Rim; Kim, Ha Young; Park, Seong Kyu; Chang, Mun Seog

    2014-01-01

    The cation channel of sperm (CatSper) protein family plays important roles in male reproduction and infertility. The four members of this family are expressed exclusively in the testis and are localized differently in sperm. To investigate the effects of Panax ginseng treatment on the expression of CatSper genes and sperm hyperactivation in male mice, sperm motility and CatSper gene expression were assessed using a computer-assisted semen analysis system, a Fluoroskan Ascent microplate fluorometer to assess Ca2+ influx, real-time polymerase chain reaction, Western blotting and immunofluorescence. The results suggested that the Ca2+ levels of sperm cells treated with P. ginseng were increased significantly compared with the normal group. The P. ginseng-treated groups showed increased sperm motility parameters, such as the curvilinear velocity and amplitude of lateral head displacement. Taken together, the data suggest that CatSper messenger ribonucleic acid levels were increased significantly in mouse testes in the P. ginseng-treated group, as was the protein level, with the exception of CatSper2. In conclusion, P. ginseng plays an important role in improving sperm hyperactivation via CatSper gene expression. PMID:24969054

  9. Cystinuria Associated with Different SLC7A9 Gene Variants in the Cat

    PubMed Central

    Raj, Karthik; Osborne, Carl; Giger, Urs

    2016-01-01

    Cystinuria is a classical inborn error of metabolism characterized by a selective proximal renal tubular defect affecting cystine, ornithine, lysine, and arginine (COLA) reabsorption, which can lead to uroliths and urinary obstruction. In humans, dogs and mice, cystinuria is caused by variants in one of two genes, SLC3A1 and SLC7A9, which encode the rBAT and bo,+AT subunits of the bo,+ basic amino acid transporter system, respectively. In this study, exons and flanking regions of the SLC3A1 and SLC7A9 genes were sequenced from genomic DNA of cats (Felis catus) with COLAuria and cystine calculi. Relative to the Felis catus-6.2 reference genome sequence, DNA sequences from these affected cats revealed 3 unique homozygous SLC7A9 missense variants: one in exon 5 (p.Asp236Asn) from a non-purpose-bred medium-haired cat, one in exon 7 (p.Val294Glu) in a Maine Coon and a Sphinx cat, and one in exon 10 (p.Thr392Met) from a non-purpose-bred long-haired cat. A genotyping assay subsequently identified another cystinuric domestic medium-haired cat that was homozygous for the variant originally identified in the purebred cats. These missense variants result in deleterious amino acid substitutions of highly conserved residues in the bo,+AT protein. A limited population survey supported that the variants found were likely causative. The remaining 2 sequenced domestic short-haired cats had a heterozygous variant at a splice donor site in intron 10 and a homozygous single nucleotide variant at a branchpoint in intron 11 of SLC7A9, respectively. This study identifies the first SLC7A9 variants causing feline cystinuria and reveals that, as in humans and dogs, this disease is genetically heterogeneous in cats. PMID:27404572

  10. KNOCKDOWN OF GCN5 HISTONE ACETYLTRANSFERASE by siRNA DECREASES ETHANOL INDUCED HISTONE ACETYLATION AND AFFECTS DIFFERENTIAL EXPRESSION OF GENES IN HUMAN HEPATOMA CELLS

    PubMed Central

    Choudhury, Mahua; Pandey, Ravi S.; Clemens, Dahn L.; Davis, J. Wade; Lim, Robert W.; Shukla, Shivendra D.

    2011-01-01

    We have investigated whether Gcn5, a histone acetyltransferase (HAT), is involved in ethanol induced acetylation of histone H3 at lysine 9 (H3AcK9) and has any effect on the gene expression. Human hepatoma HepG2 cells transfected with ethanol metabolizing enzyme alcohol dehydrogenase 1 (VA 13 cells) were used. Knockdown of Gcn5 by siRNA silencing decreased mRNA and protein levels of GCN5, HAT activity, and also attenuated ethanol induced H3AcK9 in VA13 cells. Illumina gene microarray analysis using total RNA showed 940 transcripts affected by GCN5 silencing or ethanol. Silencing caused differential expression of 891 transcripts (≥ 1.5 fold up- or down- regulated). Among these, 492 transcripts were up- and 399 were down- regulated compared to their respective controls. Using a more stringent threshold (≥ 2.5 fold) the array data from GCN5 silenced samples showed 57 genes differentially expressed (39 up-regulated and 18 down-regulated). Likewise, ethanol caused differential regulation of 57 transcripts with ≥ 1.5 fold change (35 gene up-regulated and 22 down-regulated). Further analysis showed that eight genes were differentially regulated that were common for both ethanol treatment and GCN5 silencing. Among these, SLC44A2 (a putative choline transporter) was strikingly up-regulated by ethanol (3 fold), and GCN5 silencing down regulated it (1.5 fold). The quantitative RT-PCR profile corroborated the array findings. This report demonstrates for the first time that (a) GCN5 differentially affects expression of multiple genes, (b) ethanol induced histone H3-lysine 9 acetylation is mediated via GCN5 and (c) that GCN5 is involved in ethanol induced expression of the putative choline transporter SLC44A2. PMID:21367571

  11. aarC, an essential gene involved in density-dependent regulation of the 2'-N-acetyltransferase in Providencia stuartii.

    PubMed

    Rather, P N; Solinsky, K A; Paradise, M R; Parojcic, M M

    1997-04-01

    The 2'-N-acetyltransferase [AAC(2')-Ia] in Providencia stuartii has a dual function where it is involved in the acetylation of peptidoglycan and certain aminoglycosides. A search for negative regulators of the aac(2')-Ia gene has resulted in the identification of aarC. A missense allele (aarC1) resulted in an 8.9-fold increase in beta-galactosidase accumulation from an aac(2')-lacZ transcriptional fusion. Northern blot analysis demonstrated an increase in aac(2')-Ia mRNA accumulation that was specific to cells at high density. In addition, the aarC1 allele also resulted in a substantial increase in the expression of aarP, a transcriptional activator of the aac(2')-Ia gene. The wild-type aarC gene was isolated by complementation and encodes a predicted protein of 365 amino acids with a molecular mass of 39,815 Da. The predicted AarC protein exhibited 88% amino acid homology to the previously identified GcpE protein of Escherichia coli and 86% homology to a gene product from Haemophilus influenzae. The E. coli gcpE gene was able to functionally complement the aarC1 allele in P. stuartii. The aarC1 allele was identified as a T to G transversion that resulted in a valine to glycine substitution at position 136 in the AarC protein. The aarC gene appears to be essential for cell viability as construction of a disrupted copy (aarC::lacZ) was possible only in cells that carried an episomal copy of aarC or gcpE.

  12. Analysis of 8 sarcomeric candidate genes for feline hypertrophic cardiomyopathy mutations in cats with hypertrophic cardiomyopathy.

    PubMed

    Meurs, K M; Norgard, M M; Kuan, M; Haggstrom, J; Kittleson, M

    2009-01-01

    Hypertrophic cardiomyopathy (HCM) is the most common heart disease in cats. Causative mutations have been identified in the Maine Coon (MC) and Ragdoll breed in the cardiac myosin binding protein C gene (MYBPC3). HCM is thought to be inherited in other breeds. That a causative mutation for HCM in the British Shorthair (BSH), Norwegian Forest (NWF), Siberian, Sphynx, or MC cats would be identified in the exonic and splice site regions of 1 of 8 genes associated with human familial HCM. Three affected BSH, NWF, Siberians, Sphynx, 2 MC (without the known MC mutation), and 2 Domestic Shorthair cats (controls) were studied. Prospective, observational study. Exonic and splice site regions of the genes encoding the proteins cardiac troponin I, troponin T, MYBPC3, cardiac essential myosin light chain, cardiac regulatory myosin light chain, alpha tropomyosin, actin, and beta-myosin heavy chain were sequenced. Sequences were compared for nucleotide changes between affected cats, the published DNA sequences, and control cats. Changes were considered to be causative for HCM if they involved a conserved amino acid and changed the amino acid to a different polarity, acid-base status, or structure. A causative mutation for HCM was not identified, although several single nucleotide polymorphisms were detected. Mutations within these cardiac genes do not appear to be the only cause of HCM in these breeds. Evaluation of additional cardiac genes is warranted to identify additional molecular causes of this feline cardiac disease.

  13. Profiling brain expression of the spermidine/spermine N1-acetyltransferase 1 (SAT1) gene in suicide.

    PubMed

    Klempan, Timothy A; Rujescu, Dan; Mérette, Chantal; Himmelman, Carla; Sequeira, Adolfo; Canetti, Lilian; Fiori, Laura M; Schneider, Barbara; Bureau, Alexandre; Turecki, Gustavo

    2009-10-05

    Altered stress reactivity is considered to be a risk factor for both major depressive disorder and suicidal behavior. The authors have sought to expand their previous findings implicating altered expression of spermidine/spermine N(1)-acetyltransferase 1 (SAT1), the rate-limiting enzyme involved in catabolism of the polyamines spermidine and spermine in the polyamine stress response (PSR), across multiple brain regions between control individuals and depressed individuals who have died by suicide. Microarray expression of probesets annotated to SAT1 were examined across 17 brain regions in 13 controls and 26 individuals who have died by suicide (16 with a diagnosis of major depression and 10 without), all of French-Canadian origin. Profiling conducted on the Affymetrix U133A/B chipset was further examined on a second chipset (U133 Plus 2.0) using RT-PCR, and analyzed in a second, independent sample. A reduction in SAT1 expression identified through multiple probesets was observed across 12 cortical regions in depressed individuals who have died by suicide compared with controls. Of these, five cortical regions showed statistically significant reductions which were supported by RT-PCR and analysis on the additional chipset. SAT1 cortical expression levels were also found to be significantly lower in an independent sample of German subjects with major depression who died by suicide in comparison with controls. These findings suggest that downregulation of SAT1 expression may play a role in depression and suicidality, possibly by impeding the normal PSR program or through compensation for the increased polyamine metabolism accompanying the psychological distress associated with depressive disorders.

  14. 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. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Whole-exome-sequencing identifies mutations in histone acetyltransferase gene KAT6B in individuals with the Say-Barber-Biesecker variant of Ohdo syndrome.

    PubMed

    Clayton-Smith, Jill; O'Sullivan, James; Daly, Sarah; Bhaskar, Sanjeev; Day, Ruth; Anderson, Beverley; Voss, Anne K; Thomas, Tim; Biesecker, Leslie G; Smith, Philip; Fryer, Alan; Chandler, Kate E; Kerr, Bronwyn; Tassabehji, May; Lynch, Sally-Ann; Krajewska-Walasek, Malgorzata; McKee, Shane; Smith, Janine; Sweeney, Elizabeth; Mansour, Sahar; Mohammed, Shehla; Donnai, Dian; Black, Graeme

    2011-11-11

    Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS or Ohdo syndrome) is a multiple anomaly syndrome characterized by severe intellectual disability, blepharophimosis, and a mask-like facial appearance. A number of individuals with SBBYSS also have thyroid abnormalities and cleft palate. The condition usually occurs sporadically and is therefore presumed to be due in most cases to new dominant mutations. In individuals with SBBYSS, a whole-exome sequencing approach was used to demonstrate de novo protein-truncating mutations in the highly conserved histone acetyltransferase gene KAT6B (MYST4/MORF)) in three out of four individuals sequenced. Sanger sequencing was used to confirm truncating mutations of KAT6B, clustering in the final exon of the gene in all four individuals and in a further nine persons with typical SBBYSS. Where parental samples were available, the mutations were shown to have occurred de novo. During mammalian development KAT6B is upregulated specifically in the developing central nervous system, facial structures, and limb buds. The phenotypic features seen in the Qkf mouse, a hypomorphic Kat6b mutant, include small eyes, ventrally placed ears and long first digits that mirror the human phenotype. This is a further example of how perturbation of a protein involved in chromatin modification might give rise to a multisystem developmental disorder.

  16. Mechanical regulation of the proangiogenic factor CCN1/CYR61 gene requires the combined activities of MRTF-A and CREB-binding protein histone acetyltransferase.

    PubMed

    Hanna, Mary; Liu, Haibo; Amir, Jawaria; Sun, Yi; Morris, Stephan W; Siddiqui, M A Q; Lau, Lester F; Chaqour, Brahim

    2009-08-21

    Smooth muscle-rich tissues respond to mechanical overload by an adaptive hypertrophic growth combined with activation of angiogenesis, which potentiates their mechanical overload-bearing capabilities. Neovascularization is associated with mechanical strain-dependent induction of angiogenic factors such as CCN1, an immediate-early gene-encoded matricellular molecule critical for vascular development and repair. Here we have demonstrated that mechanical strain-dependent induction of the CCN1 gene involves signaling cascades through RhoA-mediated actin remodeling and the p38 stress-activated protein kinase (SAPK). Actin signaling controls serum response factor (SRF) activity via SRF interaction with the myocardin-related transcriptional activator (MRTF)-A and tethering to a single CArG box sequence within the CCN1 promoter. Such activity was abolished in mechanically stimulated mouse MRTF-A(-/-) cells or upon inhibition of CREB-binding protein (CBP) histone acetyltransferase (HAT) either pharmacologically or by siRNAs. Mechanical strain induced CBP-mediated acetylation of histones 3 and 4 at the SRF-binding site and within the CCN1 gene coding region. Inhibition of p38 SAPK reduced CBP HAT activity and its recruitment to the SRF.MRTF-A complex, whereas enforced induction of p38 by upstream activators (e.g. MKK3 and MKK6) enhanced both CBP HAT and CCN1 promoter activities. Similarly, mechanical overload-induced CCN1 gene expression in vivo was associated with nuclear localization of MRTF-A and enrichment of the CCN1 promoter with both MRTF-A and acetylated histone H3. Taken together, these data suggest that signal-controlled activation of SRF, MRTF-A, and CBP provides a novel connection between mechanical stimuli and angiogenic gene expression.

  17. Sequence Variants and Haplotype Analysis of Cat ERBB2 Gene: A Survey on Spontaneous Cat Mammary Neoplastic and Non-Neoplastic Lesions

    PubMed Central

    Santos, Sara; Bastos, Estela; Baptista, Cláudia S.; Sá, Daniela; Caloustian, Christophe; Guedes-Pinto, Henrique; Gärtner, Fátima; Gut, Ivo G.; Chaves, Raquel

    2012-01-01

    The human ERBB2 proto-oncogene is widely considered a key gene involved in human breast cancer onset and progression. Among spontaneous tumors, mammary tumors are the most frequent cause of cancer death in cats and second most frequent in humans. In fact, naturally occurring tumors in domestic animals, more particularly cat mammary tumors, have been proposed as a good model for human breast cancer, but critical genetic and molecular information is still scarce. The aims of this study include the analysis of the cat ERBB2 gene partial sequences (between exon 17 and 20) in order to characterize a normal and a mammary lesion heterogeneous populations. Cat genomic DNA was extracted from normal frozen samples (n = 16) and from frozen and formalin-fixed paraffin-embedded mammary lesion samples (n = 41). We amplified and sequenced two cat ERBB2 DNA fragments comprising exons 17 to 20. It was possible to identify five sequence variants and six haplotypes in the total population. Two sequence variants and two haplotypes show to be specific for cat mammary tumor samples. Bioinformatics analysis predicts that four of the sequence variants can produce alternative transcripts or activate cryptic splicing sites. Also, a possible association was identified between clinicopathological traits and the variant haplotypes. As far as we know, this is the first attempt to examine ERBB2 genetic variations in cat mammary genome and its possible association with the onset and progression of cat mammary tumors. The demonstration of a possible association between primary tumor size (one of the two most important prognostic factors) and the number of masses with the cat ERBB2 variant haplotypes reveal the importance of the analysis of this gene in veterinary medicine. PMID:22489125

  18. Sequence variants and haplotype analysis of cat ERBB2 gene: a survey on spontaneous cat mammary neoplastic and non-neoplastic lesions.

    PubMed

    Santos, Sara; Bastos, Estela; Baptista, Cláudia S; Sá, Daniela; Caloustian, Christophe; Guedes-Pinto, Henrique; Gärtner, Fátima; Gut, Ivo G; Chaves, Raquel

    2012-01-01

    The human ERBB2 proto-oncogene is widely considered a key gene involved in human breast cancer onset and progression. Among spontaneous tumors, mammary tumors are the most frequent cause of cancer death in cats and second most frequent in humans. In fact, naturally occurring tumors in domestic animals, more particularly cat mammary tumors, have been proposed as a good model for human breast cancer, but critical genetic and molecular information is still scarce. The aims of this study include the analysis of the cat ERBB2 gene partial sequences (between exon 17 and 20) in order to characterize a normal and a mammary lesion heterogeneous populations. Cat genomic DNA was extracted from normal frozen samples (n = 16) and from frozen and formalin-fixed paraffin-embedded mammary lesion samples (n = 41). We amplified and sequenced two cat ERBB2 DNA fragments comprising exons 17 to 20. It was possible to identify five sequence variants and six haplotypes in the total population. Two sequence variants and two haplotypes show to be specific for cat mammary tumor samples. Bioinformatics analysis predicts that four of the sequence variants can produce alternative transcripts or activate cryptic splicing sites. Also, a possible association was identified between clinicopathological traits and the variant haplotypes. As far as we know, this is the first attempt to examine ERBB2 genetic variations in cat mammary genome and its possible association with the onset and progression of cat mammary tumors. The demonstration of a possible association between primary tumor size (one of the two most important prognostic factors) and the number of masses with the cat ERBB2 variant haplotypes reveal the importance of the analysis of this gene in veterinary medicine.

  19. Effect of inhibition of aloe-emodin on N-acetyltransferase activity and gene expression in human malignant melanoma cells (A375.S2).

    PubMed

    Lin, Shuw-Yuan; Yang, Jen-Hung; Hsia, Te-Chun; Lee, Jau-Hong; Chiu, Tsan-Hung; Wei, Yau-Huei; Chung, Jing-Gung

    2005-12-01

    Arylamine carcinogens and drugs are N-acetylated by cytosolic N-acetyltransferase (NAT), which uses acetyl-coenzyme A as a cofactor. NAT plays an initial role in the metabolism of these arylamine compounds. 2-Aminofluorene is one of the arylamine carcinogens which have been demonstrated to undergo N-acetylation in laboratory animals and humans. Our previous study showed that human cancer cell lines (colon cancer, colo 205; liver cancer, Hep G2; bladder cancer, T24; leukemia, HL-60; prostate cancer, LNCaP; osteogenic sarcoma, U-2 OS; malignant melanoma, A375.S2) displayed NAT activity, which was affected by aloe-emodin in human leukemia cells. The purpose of this study was to determine whether aloe-emodin could affect the enzyme activity and gene expression of NAT at the mRNA and protein levels in malignant human melanoma A375.S2 cells. The results showed that aloe-emodin inhibited NAT1 activity (decreased N-acetylation of 2-aminofluorene) in intact cells in a dose-dependent manner. The effect of aloe-emodin on NAT1 at the protein level was determined by Western blotting and the mRNA levels were examined by polymerase chain reaction (PCR) and cDNA microarray. These results clearly indicate that aloe-emodin inhibits the mRNA expression and enzyme activity of NAT1 in A375.S2 cells.

  20. Sanfilippo syndrome type C: mutation spectrum in the heparan sulfate acetyl-CoA: alpha-glucosaminide N-acetyltransferase (HGSNAT) gene.

    PubMed

    Feldhammer, Matthew; Durand, Stéphanie; Mrázová, Lenka; Boucher, Renée-Myriam; Laframboise, Rachel; Steinfeld, Robert; Wraith, James E; Michelakakis, Helen; van Diggelen, Otto P; Hrebícek, Martin; Kmoch, Stanislav; Pshezhetsky, Alexey V

    2009-06-01

    Mucopolysaccharidosis (MPS) type IIIC or Sanfilippo syndrome type C is a rare autosomal recessive disorder caused by the deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl-CoA (AcCoA): alpha-glucosaminide N-acetyltransferase (HGSNAT; EC 2.3.1.78), which catalyzes transmembrane acetylation of the terminal glucosamine residues of heparan sulfate prior to their hydrolysis by alpha-N-acetylglucosaminidase. Lysosomal storage of undegraded heparan sulfate in the cells of affected patients leads to neuronal death, causing neurodegeneration and severely impaired development accompanied by mild visceral and skeletal abnormalities, including mild dwarfism, coarse facies, and joint stiffness. To date, 50 HGSNAT mutations have been identified in MPS IIIC patients: 40 were previously published and 10 novel mutations are reported here. The mutations span the entire structure of the gene and include 13 splice-site mutations, 11 insertions and deletions, 8 nonsense mutations, and 18 missense mutations (http://chromium.liacs.nl/LOVD2/home.php?select_db=HGSNAT). In addition, four polymorphisms result in amino acid changes that do not affect activity of the enzyme. In this work we discuss the spectrum of MPS IIIC mutations, their clinical presentation and distribution within the patient population, and speculate how the mutations may affect the structure and function of HGSNAT.

  1. An approach to identify SNPs in the gene encoding acetyl-CoA acetyltransferase-2 (ACAT-2) and their proposed role in metabolic processes in pig.

    PubMed

    Sodhi, Simrinder Singh; Ghosh, Mrinmoy; Song, Ki Duk; Sharma, Neelesh; Kim, Jeong Hyun; Kim, Nam Eun; Lee, Sung Jin; Kang, Chul Woong; Oh, Sung Jong; Jeong, Dong Kee

    2014-01-01

    The novel liver protein acetyl-CoA acetyltransferase-2 (ACAT2) is involved in the beta-oxidation and lipid metabolism. Its comprehensive relative expression, in silico non-synonymous single nucleotide polymorphism (nsSNP) analysis, as well as its annotation in terms of metabolic process with another protein from the same family, namely, acetyl-CoA acyltransferase-2 (ACAA2) was performed in Sus scrofa. This investigation was conducted to understand the most important nsSNPs of ACAT2 in terms of their effects on metabolic activities and protein conformation. The two most deleterious mutations at residues 122 (I to V) and 281 (R to H) were found in ACAT2. Validation of expression of genes in the laboratory also supported the idea of differential expression of ACAT2 and ACAA2 conceived through the in silico analysis. Analysis of the relative expression of ACAT2 and ACAA2 in the liver tissue of Jeju native pig showed that the former expressed significantly higher (P<0.05). Overall, the computational prediction supported by wet laboratory analysis suggests that ACAT2 might contribute more to metabolic processes than ACAA2 in swine. Further associations of SNPs in ACAT2 with production traits might guide efforts to improve growth performance in Jeju native pigs.

  2. 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

  3. The time enzyme in melatonin biosynthesis in fish: Day/night expressions of three aralkylamine N-acetyltransferase genes in three-spined stickleback.

    PubMed

    Kulczykowska, Ewa; Kleszczyńska, Agnieszka; Gozdowska, Magdalena; Sokołowska, Ewa

    2017-03-16

    In vertebrates, aralkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) is a time-keeping enzyme in melatonin (Mel) biosynthesis. Uniquely in fish, there are several AANAT isozymes belonging to two AANAT subfamilies, AANAT1 and AANAT2, which are encoded by distinct genes. The different substrate preferences, kinetics and spatial expression patterns of isozymes indicate that they may have different functions. In the three-spined stickleback (Gasterosteus aculeatus), there are three genes encoding three AANAT isozymes. In this study, for the first time, the levels of aanat1a, aanat1b and aanat2 mRNAs are measured by absolute RT-qPCR in the brain, eye, skin, stomach, gut, heart and kidney collected at noon and midnight. Melatonin levels are analysed by HPLC with fluorescence detection in homogenates of the brain, eye, skin and kidney. The levels of aanats mRNAs differ significantly within and among organs. In the brain, eye, stomach and gut, there are day/night variations in aanats mRNAs levels. The highest levels of aanat1a and aanat1b mRNAs are in the eye. The extremely high expressions of these genes which are reflected in the highest Mel concentrations at this site at noon and midnight strongly suggest that the eye is an important source of the hormone in the three-spined sticklebacks. A very low level of aanat2 mRNA in all organs may suggest that AANAT1a and/or AANAT1b are principal isozymes in the three-spine sticklebacks. A presence of the isozymes of defined substrate preferences provides opportunity for control of acetylation of amines by modulation of individual aanat expression and permits the fine-tuning of indolethylamines and phenylethylamines metabolism to meet the particular needs of a given organ.

  4. Identifying diagnostic endocrine markers and changes in endometrial gene expressions during pyometra in cats.

    PubMed

    Jursza-Piotrowska, Ewelina; Siemieniuch, Marta J

    2016-06-01

    Pyometra is a significant reproductive problem in cats. The aims of this study were to evaluate (i) the immunological profile of queens by studying plasma concentrations of metabolites of prostacyclin I2 (6-keto-PGF1α), leukotriene B4 (LTB4) and leukotriene C4 (LTC4); and (ii) the gene transcription profiles of Toll-like receptors (TLRs) 2 and 4 (TLR2/4), PGE2-synthase (PGES), PGF2α-synthase (PGFS) and prostaglandin-endoperoxide synthase 2 (PTGS2) in the feline endometrium throughout the estrous cycle, after medroxyprogesterone acetate (MPA) treatment and during pyometra. The concentration of plasma 6-keto-PGF1α in pyometra was increased in comparison to other groups studied (p<0.01). Endometrial mRNA coding for TLR2 was up-regulated in cats suffering from pyometra compared to other groups (p<0.001). Expression of mRNA for TLR4 was up-regulated in endometria originating from MPA-treated cats, pyometra and late diestrus cats, compared with tissues from cats during estrus and anestrus (p<0.05). As expected, endometrial mRNA for PTGS2 was up-regulated only in pyometra, compared with other groups (p<0.001). Similarly, endometrial mRNA for PGFS was up-regulated in pyometra, compared with endometria from anestrus, late diestrus and from MPA-treated cats (p<0.05), or from cats during estrus (p<0.01). Overall, these results indicate that plasma concentrations of LTB4 and LTC4 are not useful diagnostic markers since they were not increased in queens with pyometra, in contrast to 6-keto-PGF1α. In addition, treatment with MPA evoked neither endocrine nor molecular changes in endometria of cats.

  5. Mutations within the FGF5 gene are associated with hair length in cats.

    PubMed

    Drögemüller, C; Rüfenacht, S; Wichert, B; Leeb, T

    2007-06-01

    Hereditary hair length variability in mice and dogs is caused by mutations within the fibroblast growth factor 5 (FGF5) gene. The aim of this study was to evaluate the feline FGF5 orthologue as a functional candidate gene for the long hair phenotype in cats, which is recessive to short hair. We amplified the feline FGF5 cDNA and characterised two alternatively spliced transcripts by RT-PCR. Comparative cDNA and genomic DNA sequencing of long- and short-haired cats revealed four non-synonymous polymorphisms in the FGF5 coding sequence. A missense mutation (AM412646:c.194C>A) was found in the homozygous state in 25 long-haired Somali, Persian, Maine Coon, Ragdoll and crossbred cats. Fifty-five short-haired cats had zero or one copy of this allele. Additionally, we found perfect co-segregation of the c.194C>A mutation within two independent pedigrees segregating for hair length. A second FGF5 exon 1 missense mutation (AM412646:c.182T>A) was found exclusively in long-haired Norwegian Forest cats. The c.182T>A mutation probably represents a second FGF5 mutation responsible for long hair in cats. In addition to the c.194C>A mutation, a frameshift mutation (AM412646:c.474delT) was found with a high frequency in the long-haired Maine Coon breed. Finally, a missense mutation (AM412646:c.475A>C) was also associated with the long-haired phenotype in some breeds. However, as one short-haired cat was homozygous for this polymorphism, it is unlikely that it has a functional role in the determination of hair length.

  6. Molecular Cloning, Characterization, and Functional Analysis of Acetyl-CoA C-Acetyltransferase and Mevalonate Kinase Genes Involved in Terpene Trilactone Biosynthesis from Ginkgo biloba.

    PubMed

    Chen, Qiangwen; Yan, Jiaping; Meng, Xiangxiang; Xu, Feng; Zhang, Weiwei; Liao, Yongling; Qu, Jinwang

    2017-01-02

    Ginkgolides and bilobalide, collectively termed terpene trilactones (TTLs), are terpenoids that form the main active substance of Ginkgo biloba. Terpenoids in the mevalonate (MVA) biosynthetic pathway include acetyl-CoA C-acetyltransferase (AACT) and mevalonate kinase (MVK) as core enzymes. In this study, two full-length (cDNAs) encoding AACT (GbAACT, GenBank Accession No. KX904942) and MVK (GbMVK, GenBank Accession No. KX904944) were cloned from G. biloba. The deduced GbAACT and GbMVK proteins contain 404 and 396 amino acids with the corresponding open-reading frame (ORF) sizes of 1215 bp and 1194 bp, respectively. Tissue expression pattern analysis revealed that GbAACT was highly expressed in ginkgo fruits and leaves, and GbMVK was highly expressed in leaves and roots. The functional complementation of GbAACT in AACT-deficient Saccharomyces cerevisiae strain Δerg10 and GbMVK in MVK-deficient strain Δerg12 confirmed that GbAACT mediated the conversion of mevalonate acetyl-CoA to acetoacetyl-CoA and GbMVK mediated the conversion of mevalonate to mevalonate phosphate. This observation indicated that GbAACT and GbMVK are functional genes in the cytosolic mevalonate (MVA) biosynthesis pathway. After G. biloba seedlings were treated with methyl jasmonate and salicylic acid, the expression levels of GbAACT and GbMVK increased, and TTL production was enhanced. The cloning, characterization, expression and functional analysis of GbAACT and GbMVK will be helpful to understand more about the role of these two genes involved in TTL biosynthesis.

  7. GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN CDNA MICROARRAY ANALYSES

    EPA Science Inventory

    GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN cDNA MICROARRAY ANALYSES

    B.S. Pukazhenthi1, J. C. Rockett2, M. Ouyang3, D.J. Dix2, J.G. Howard1, P. Georgopoulos4, W.J. J. Welsh3 and D. E. Wildt1

    1Department of Reproductiv...

  8. GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN CDNA MICROARRAY ANALYSES

    EPA Science Inventory

    GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN cDNA MICROARRAY ANALYSES

    B.S. Pukazhenthi1, J. C. Rockett2, M. Ouyang3, D.J. Dix2, J.G. Howard1, P. Georgopoulos4, W.J. J. Welsh3 and D. E. Wildt1

    1Department of Reproductiv...

  9. Characterization of the putative amino acid transporter genes AtCAT2, 3 &4: the tonoplast localized AtCAT2 regulates soluble leaf amino acids.

    PubMed

    Yang, Huaiyu; Krebs, Melanie; Stierhof, York-Dieter; Ludewig, Uwe

    2014-05-01

    The plant vacuole constitutes a large transient storage compartment for nutrients, proteins and metabolites, and is a major cellular sink for toxic waste compounds. Amino acids can cross the vacuolar membrane via specific transport proteins, which are molecularly not well characterized. Two members of a small subfamily of the cationic amino acid transporters, AtCAT2 and AtCAT4, were primarily localized at the tonoplast when tagged with GFP. The closely related AtCAT3, by contrast, was detected in the endoplasmic reticulum membrane. The exchange of a di-acidic motif at the carboxy-tail affected their sub-cellular localization, with larger effects visible in transiently transformed protoplasts compared to stably expressing plant lines. The genes have broad, partially overlapping tissue expression, with CAT2 dominating in most tissues. Loss-of-function mutants of individual CATs showed no visible phenotype under various conditions, but the overall tissue concentration of amino acids was increased in soil-grown cat2 mutants. The data suggest that CAT2 is a critical target of leaf amino acid concentrations and manipulation of this tonoplast transporter can significantly alter total tissue amino acid concentrations. Copyright © 2013 Elsevier GmbH. All rights reserved.

  10. Circadian expression of the maize catalase Cat3 gene is highly conserved among diverse maize genotypes with structurally different promoters.

    PubMed Central

    Polidoros, A N; Scandalios, J G

    1998-01-01

    The Cat3 gene of maize exhibits a transcriptionally regulated circadian rhythm. In the present study we examined the following: (1) the extent of the circadian Cat3 expression between maize genotypes of diverse origin; (2) the functional significance of a Tourist transposable element located in the Cat3 promoter of the inbred line W64A, which harbors putative regulatory elements (GATA repeat, CCAAT boxes) shown to be involved in the light induction and circadian regulation of the Arabidopsis CAB2, as well as other plant genes; and (3) aspects of the physiological role of CAT-3 in maize metabolism. Results confirm that the circadian Cat3 expression is a general phenomenon in maize. Regulation of Cat3 gene expression is not dependent on the presence of the Tourist element in the promoter of the gene nor on the presence of motifs similar to those found significant in the circadian expression of the Arabidopsis CAB2 gene. Structural diversity was revealed in the Cat3 promoters of maize genotypes of diverse origins. However, highly conserved regions with putative regulatory motifs were identified. Relevance of the conserved regions to the circadian regulation of the gene is discussed. Possible physiological roles of CAT-3 are suggested. PMID:9584112

  11. Regulation of cAMP-induced arylalkylamine N-acetyltransferase, Period1, and MKP-1 gene expression by mitogen-activated protein kinases in the rat pineal gland.

    PubMed

    Chansard, Mathieu; Iwahana, Eiko; Liang, Jian; Fukuhara, Chiaki

    2005-10-03

    In rodent pineal glands, sympathetic innervation, which leads to norepinephrine release, is a key process in the circadian regulation of physiology and certain gene expressions. It has been shown that gene expression of the rate-limiting enzyme in the melatonin synthesis arylalkylamine N-acetyltransferase (Aa-Nat), circadian clock gene Period1, and mitogen-activated protein kinase (MAPK) phosphtase-1 (MKP-1), is controlled mainly by a norepinephrine-beta-adrenergic receptor-cAMP signaling cascade in the rat pineal gland. To further dissect the signaling cascades that regulate those gene expressions, we examined whether MAPKs are involved in cAMP-induced gene expression. Western blot and immunohistochemical analyses showed that one of the three MAPKs, c-Jun N-terminal kinase (JNK), was expressed in the pineal, and was phosphorylated by cAMP analogue stimulation with a peak 20 min after start of the stimulation, in vitro. A specific JNK inhibitor SP600125 (Anthra[1,9-cd]pyrazol-6(2H)-one1,9-pyrazoloanthrone), but not its negative control (N1-Methyl-1,9-pyrazoloanthrone), significantly reduced cAMP-stimulated Aa-Nat, Period1, and MKP-1 mRNA levels. Although another MAPK, p38(MAPK), has also been shown to be activated by cAMP stimulation, a p38(MAPK) inhibitor, SB203580 (4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole, HCl), showed no effect on cAMP-induced Aa-Nat and Period1 mRNA levels; whereas SB203580, but not its negative analogue SB202474 (4-Ethyl-2(p-methoxyphenyl)-5-(4'-pyridyl)-IH-imidazole, DiHCl), significantly reduced cAMP-induced MKP-1 mRNA levels. Taken together, our data suggest that cAMP-induced Aa-Nat and Period1 are likely to be mediated by activation of JNK, whereas MKP-1 may be mediated by both p38(MAPK) and JNK activations.

  12. Does a pleiotropic gene explain deafness and blue irises in white cats?

    PubMed

    Geigy, Caroline A; Heid, Silvia; Steffen, Frank; Danielson, Kristen; Jaggy, André; Gaillard, Claude

    2007-05-01

    The prevalence of deafness is high in cat populations in which the dominant white gene is segregating. The objective of this study was to investigate whether there is a gene that is responsible for deafness as well as for blue eyes and to establish a plausible mode of inheritance. For this purpose, data from an experimental colony with deaf cats were analyzed. The hearing status was determined by acoustically evoked brain stem responses (BAER). Complex segregation analyses were conducted to find out the most probable mode of inheritance using maximum likelihood procedures. The prevalence of deafness and partial hearing in the experimental colony was 67% and 29%, respectively. The results of the bivariate segregation analysis support the hypothesis of a pleiotropic major gene segregating for deafness and blue iris colour. The high heritability coefficients for both traits, 0.55 and 0.75 respectively, indicate that beside the major gene there is an important influence of polygenic effects.

  13. Application of a novel phosphinothricin N-acetyltransferase (RePAT) gene in developing glufosinate-resistant rice

    PubMed Central

    Cui, Ying; Liu, Ziduo; Li, Yue; Zhou, Fei; Chen, Hao; Lin, Yongjun

    2016-01-01

    Currently, only few glufosinate-resistant genes are available for commercial application. Thus, developing novel glufosinate-resistant genes with commercial feasibility is extremely important and urgent for agricultural production. In this study, we transferred a newly isolated RePAT gene into a japonica rice variety Zhonghua11, resulting in a large number of independent T0 transgenic plants, most of which grew normally under high-concentration glufosinate treatment. Four transgenic plants with one intact RePAT expression cassette integrated into the intergenic region were selected. Agronomic performances of their T2 progenies were investigated, and the results suggested that the expression of RePAT had no adverse effect on the agronomic performance. Definite glufosinate resistance of the selected transgenic plants was further confirmed to be related to the expression of RePAT by assay on the medium and qRT-PCR. The inheritance and expression of RePAT in two transgenic plants were confirmed to be stable. Finally, the two-year field assay of glufosinate resistance suggested that the agronomic performance of the transgenic plant (PAT11) was not affected by high dosage of glufosinate (5000 g/ha). Collectively, our study proves the high resistance of a novel gene RePAT to glufosinate and provides a glufosiante-resistant rice variety with agricultural application potential. PMID:26879398

  14. A missense mutation in the glucosamine-6-phosphate N-acetyltransferase-encoding gene causes temperature-dependent growth defects and ectopic lignin deposition in Arabidopsis.

    PubMed

    Nozaki, Mamoru; Sugiyama, Munetaka; Duan, Jun; Uematsu, Hiroshi; Genda, Tatsuya; Sato, Yasushi

    2012-08-01

    To study the regulatory mechanisms underlying lignin biosynthesis, we isolated and characterized lignescens (lig), a previously undescribed temperature-sensitive mutant of Arabidopsis thaliana that exhibits ectopic lignin deposition and growth defects under high-temperature conditions. The lig mutation was identified as a single base transition in GNA1 encoding glucosamine-6-phosphate N-acetyltransferase (GNA), a critical enzyme of UDP-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. lig harbors a glycine-to-serine substitution at residue 68 (G68S) of GNA1. Enzyme activity assays of the mutant protein (GNA1(G68S)) showed its thermolability relative to the wild-type protein. The lig mutant exposed to the restrictive temperature contained a significantly smaller amount of UDP-GlcNAc than did the wild type. The growth defects and ectopic lignification of lig were suppressed by the addition of UDP-GlcNAc. Since UDP-GlcNAc is an initial sugar donor of N-glycan synthesis and impaired N-glycan synthesis is known to induce the unfolded protein response (UPR), we examined possible relationships between N-glycan synthesis, UPR, and the lig phenotype. N-glycans were reduced and LUMINAL BINDING PROTEIN3, a typical UPR gene, was expressed in lig at the restrictive temperature. Furthermore, treatment with UPR-inducing reagents phenocopied the lig mutant. Our data collectively suggest that impairment of N-glycan synthesis due to a shortage of UDP-GlcNAc leads to ectopic lignin accumulation, mostly through the UPR.

  15. 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

  16. Human β-NGF gene transferred to cat corneal endothelial cells.

    PubMed

    Luo, Wen-Juan; Liu, Min; Zhao, Gui-Qiu; Wang, Chuan-Fu; Hu, Li-Ting; Liu, Xiang-Ping

    2016-01-01

    To transfect the cat corneal endothelial cells (CECs) with recombinant human β-nerve growth factor gene adeno-associated virus (AAV-β-NGF) and to observe the effect of the expressed β-NGF protein on the proliferation activity of cat CECs. The endothelium of cat cornea was torn under the microscope and rapidly cultivated in Dulbecco's modified Eagle's medium (DMEM) to form single layer CECs and the passage 2 endothelial cells were used in this experiment. The recombinant human AAV-β-NGF was constructed. The recombinant human AAV-β-NGF was transferred into cat CECs directly. Three groups were as following: normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-β-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the β-NGF target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected CEC detected at 48h by MTT assay; the percentage of G1 cells among CECs after transfect was detected by flow cytometry method (FCM); cell morphology was observed under inverted phase contrast microscope. The torn endothelium culture technique rapidly cultivated single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. Forty-eight hours after transfect, the human β-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed that there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05); there was significant difference between two control groups and recombinant CEC-AAV-β-NGF group (P<0.05). MTT assay showed that transfect of recombinant AAV-β-NGF promoted the proliferation activity of cat CEC, while there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05). FCM result showed that the

  17. Human β-NGF gene transferred to cat corneal endothelial cells

    PubMed Central

    Luo, Wen-Juan; Liu, Min; Zhao, Gui-Qiu; Wang, Chuan-Fu; Hu, Li-Ting; Liu, Xiang-Ping

    2016-01-01

    AIM To transfect the cat corneal endothelial cells (CECs) with recombinant human β-nerve growth factor gene adeno-associated virus (AAV-β-NGF) and to observe the effect of the expressed β-NGF protein on the proliferation activity of cat CECs. METHODS The endothelium of cat cornea was torn under the microscope and rapidly cultivated in Dulbecco's modified Eagle's medium (DMEM) to form single layer CECs and the passage 2 endothelial cells were used in this experiment. The recombinant human AAV-β-NGF was constructed. The recombinant human AAV-β-NGF was transferred into cat CECs directly. Three groups were as following: normal CEC control group, CEC-AAV control group and recombinant CEC-AAV-β-NGF group. Forty-eight hours after transfection, the total RNA was extracted from the CEC by Trizol. The expression of the β-NGF target gene detected by fluorescence quantitative polymerase chain reaction; proliferation activity of the transfected CEC detected at 48h by MTT assay; the percentage of G1 cells among CECs after transfect was detected by flow cytometry method (FCM); cell morphology was observed under inverted phase contrast microscope. RESULTS The torn endothelium culture technique rapidly cultivated single layer cat corneal endothelial cells. The self-designed primers for the target gene and reference gene were efficient and special confirmed through electrophoresis analysis and DNA sequencing. Forty-eight hours after transfect, the human β-NGF gene mRNA detected by fluorescence quantitative polymerase chain reaction showed that there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05); there was significant difference between two control groups and recombinant CEC-AAV-β-NGF group (P<0.05). MTT assay showed that transfect of recombinant AAV-β-NGF promoted the proliferation activity of cat CEC, while there was no significant difference between normal CEC control group and CEC-AAV control group (P>0.05). FCM result

  18. Transgenic plants containing the phosphinothricin-N-acetyltransferase gene metabolize the herbicide L-phosphinothricin (glufosinate) differently from untransformed plants.

    PubMed

    Dröge, W; Broer, I; Pühler, A

    1992-04-01

    L-Phosphinothricin (L-Pt)-resistant plants were constructed by introducing a modified phosphinothricin-N-acetyl-transferase gene (pat) via Agrobacterium-mediated gene transfer into tobacco (Nicotiana tabacum L), and via direct gene transfer into carrot (Daucus carota L). The metabolism of L-Pt was studied in these transgenic, Pt-resistant plants, as well as in the untransformed species. The degradation of L-Pt, (14)C-labeled specifically at different C-atoms, was analysed by measuring the release of (14)CO2 and by separating the labeled degradation products on thin-layer-chromatography plates. In untransformed tobacco and carrot plants, L-Pt was deaminated to form its corresponding oxo acid 4-methylphosphinico-2-oxo-butanoic acid (PPO), which subsequently was decarboxylated to form 3-methylphosphinico-propanoic acid (MPP). This compound was stable in plants. A third metabolite remained unidentified. The L-Pt was rapidly N-acetylated in herbicide-resistant tobacco and carrot plants, indicating that the degradation pathway of L-Pt into PPO and MPP was blocked. The N-acetylated product, L-N-acetyl-Pt remained stable with regard to degradation, but was found to exist in a second modified form. In addition, there was a pH-dependent, reversible change in the mobility of L-N-acetyl-Pt thin-layer during chromatography.

  19. Isoform-level brain expression profiling of the spermidine/spermine N1-Acetyltransferase1 (SAT1) gene in major depression and suicide.

    PubMed

    Pantazatos, Spiro P; Andrews, Stuart J; Dunning-Broadbent, Jane; Pang, Jiuhong; Huang, Yung-Yu; Arango, Victoria; Nagy, Peter L; John Mann, J

    2015-07-01

    Low brain expression of the spermidine/spermine N-1 acetyltransferase (SAT1) gene, the rate-limiting enzyme involved in catabolism of polyamines that mediate the polyamine stress response (PSR), has been reported in depressed suicides. However, it is unknown whether this effect is associated with depression or with suicide and whether all or only specific isoforms expressed by SAT1, such as the primary 171 amino acid protein-encoding transcript (SSAT), or an alternative splice variant (SSATX) that is involved in SAT1 regulated unproductive splicing and transcription (RUST), are involved. We applied next generation sequencing (RNA-seq) to assess gene-level, isoform-level, and exon-level SAT1 expression differences between healthy controls (HC, N = 29), DSM-IV major depressive disorder suicides (MDD-S, N = 21) and MDD non-suicides (MDD, N = 9) in the dorsal lateral prefrontal cortex (Brodmann Area 9, BA9) of medication-free individuals postmortem. Using small RNA-seq, we also examined miRNA species putatively involved in SAT1 post-transcriptional regulation. A DSM-IV diagnosis was made by structured interview. Toxicology and history ruled out recent psychotropic medication. At the gene-level, we found low SAT1 expression in both MDD-S (vs. HC, p = 0.002) and MDD (vs. HC, p = 0.002). At the isoform-level, reductions in MDD-S (vs. HC) were most pronounced in four transcripts including SSAT and SSATX, while reductions in MDD (vs. HC) were pronounced in three transcripts, one of which was reduced in MDD relative to MDD-S (all p < 0.1 FDR corrected). We did not observe evidence for differential exon-usage (i.e. splicing) nor differences in miRNA expression. Results replicate the finding of low SAT1 brain expression in depressed suicides in an independent sample and implicate low SAT1 brain expression in MDD independent of suicide. Low expressions of both SSAT and SATX isoforms suggest that shared transcriptional mechanisms involved in RUST may account for low SAT1 brain

  20. Astrocyte Elevated Gene 1 Interacts with Acetyltransferase p300 and c-Jun To Promote Tumor Aggressiveness

    PubMed Central

    Liu, Liping; Guan, Hongyu; Li, Yun; Ying, Zhe; Wu, Jueheng; Zhu, Xun; Song, Libing

    2016-01-01

    ABSTRACT Astrocyte elevated gene 1 (AEG-1) is an oncoprotein that strongly promotes the development and progression of cancers. However, the detailed underlying mechanisms through which AEG-1 enhances tumor development and progression remain to be determined. In this study, we identified c-Jun and p300 to be novel interacting partners of AEG-1 in gliomas. AEG-1 promoted c-Jun transcriptional activity by interacting with the c-Jun/p300 complex and inducing c-Jun acetylation. Furthermore, the AEG-1/c-Jun/p300 complex was found to bind the promoter of c-Jun downstream targeted genes, consequently establishing an acetylated chromatin state that favors transcriptional activation. Importantly, AEG-1/p300-mediated c-Jun acetylation resulted in the development of a more aggressive malignant phenotype in gliomas through a drastic increase in glioma cell proliferation and angiogenesis in vitro and in vivo. Consistently, the AEG-1 expression levels in clinical glioma specimens correlated with the status of c-Jun activation. Taken together, our results suggest that AEG-1 mediates a novel epigenetic mechanism that enhances c-Jun transcriptional activity to induce glioma progression and that AEG-1 might be a novel, potential target for the treatment of gliomas. PMID:27956703

  1. Astrocyte Elevated Gene 1 Interacts with Acetyltransferase p300 and c-Jun To Promote Tumor Aggressiveness.

    PubMed

    Liu, Liping; Guan, Hongyu; Li, Yun; Ying, Zhe; Wu, Jueheng; Zhu, Xun; Song, Libing; Li, Jun; Li, Mengfeng

    2017-03-01

    Astrocyte elevated gene 1 (AEG-1) is an oncoprotein that strongly promotes the development and progression of cancers. However, the detailed underlying mechanisms through which AEG-1 enhances tumor development and progression remain to be determined. In this study, we identified c-Jun and p300 to be novel interacting partners of AEG-1 in gliomas. AEG-1 promoted c-Jun transcriptional activity by interacting with the c-Jun/p300 complex and inducing c-Jun acetylation. Furthermore, the AEG-1/c-Jun/p300 complex was found to bind the promoter of c-Jun downstream targeted genes, consequently establishing an acetylated chromatin state that favors transcriptional activation. Importantly, AEG-1/p300-mediated c-Jun acetylation resulted in the development of a more aggressive malignant phenotype in gliomas through a drastic increase in glioma cell proliferation and angiogenesis in vitro and in vivo Consistently, the AEG-1 expression levels in clinical glioma specimens correlated with the status of c-Jun activation. Taken together, our results suggest that AEG-1 mediates a novel epigenetic mechanism that enhances c-Jun transcriptional activity to induce glioma progression and that AEG-1 might be a novel, potential target for the treatment of gliomas.

  2. Neonatal Gene Therapy With a Gamma Retroviral Vector in Mucopolysaccharidosis VI Cats

    PubMed Central

    Ponder, Katherine P; O'Malley, Thomas M; Wang, Ping; O'Donnell, Patricia A; Traas, Anne M; Knox, Van W; Aguirre, Gustavo A; Ellinwood, N Matthew; Metcalf, Jason A; Wang, Bin; Parkinson-Lawrence, Emma J; Sleeper, Meg M; Brooks, Doug A; Hopwood, John J; Haskins, Mark E

    2012-01-01

    Mucopolysaccharidosis (MPS) VI is due to a deficiency in the activity of N-acetylgalactosamine 4-sulfatase (4S), also known as arylsulfatase B. Previously, retroviral vector (RV)-mediated neonatal gene therapy reduced the clinical manifestations of MPS I and MPS VII in mice and dogs. However, sulfatases require post-translational modification by sulfatase-modifying factors. MPS VI cats were injected intravenously (i.v.) with a gamma RV-expressing feline 4S, resulting in 5 ± 3 copies of RV per 100 cells in liver. Liver and serum 4S activity were 1,450 ± 1,720 U/mg (26-fold normal) and 107 ± 60 U/ml (13-fold normal), respectively, and were directly proportional to the liver 4S protein levels for individual cats. This study suggests that sulfatase-modifying factor (SUMF) activity in liver was sufficient to result in active enzyme despite overexpression of 4S. RV-treated MPS VI cats achieved higher body weights and longer appendicular skeleton lengths, had reduced articular cartilage erosion, and reduced aortic valve thickening and aortic dilatation compared with untreated MPS VI cats, although cervical vertebral bone lengths were not improved. This demonstrates that therapeutic expression of a functional sulfatase protein can be achieved with neonatal gene therapy using a gamma RV, but some aspects of bone disease remain difficult to treat. PMID:22395531

  3. Neonatal gene therapy with a gamma retroviral vector in mucopolysaccharidosis VI cats.

    PubMed

    Ponder, Katherine P; O'Malley, Thomas M; Wang, Ping; O'Donnell, Patricia A; Traas, Anne M; Knox, Van W; Aguirre, Gustavo A; Ellinwood, N Matthew; Metcalf, Jason A; Wang, Bin; Parkinson-Lawrence, Emma J; Sleeper, Meg M; Brooks, Doug A; Hopwood, John J; Haskins, Mark E

    2012-05-01

    Mucopolysaccharidosis (MPS) VI is due to a deficiency in the activity of N-acetylgalactosamine 4-sulfatase (4S), also known as arylsulfatase B. Previously, retroviral vector (RV)-mediated neonatal gene therapy reduced the clinical manifestations of MPS I and MPS VII in mice and dogs. However, sulfatases require post-translational modification by sulfatase-modifying factors. MPS VI cats were injected intravenously (i.v.) with a gamma RV-expressing feline 4S, resulting in 5 ± 3 copies of RV per 100 cells in liver. Liver and serum 4S activity were 1,450 ± 1,720 U/mg (26-fold normal) and 107 ± 60 U/ml (13-fold normal), respectively, and were directly proportional to the liver 4S protein levels for individual cats. This study suggests that sulfatase-modifying factor (SUMF) activity in liver was sufficient to result in active enzyme despite overexpression of 4S. RV-treated MPS VI cats achieved higher body weights and longer appendicular skeleton lengths, had reduced articular cartilage erosion, and reduced aortic valve thickening and aortic dilatation compared with untreated MPS VI cats, although cervical vertebral bone lengths were not improved. This demonstrates that therapeutic expression of a functional sulfatase protein can be achieved with neonatal gene therapy using a gamma RV, but some aspects of bone disease remain difficult to treat.

  4. Peroxisome proliferator-activated receptor gamma and spermidine/spermine N1-acetyltransferase gene expressions are significantly correlated in human colorectal cancer

    PubMed Central

    Linsalata, Michele; Giannini, Romina; Notarnicola, Maria; Cavallini, Aldo

    2006-01-01

    Background The peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that regulates adipogenic differentiation and glucose homeostasis. Spermidine/spermine N1-acetyltransferase (SSAT) and ornithine decarboxylase (ODC) are key enzymes involved in the metabolism of polyamines, compounds that play an important role in cell proliferation. While the PPARγ role in tumour growth has not been clearly defined, the involvement of the altered polyamine metabolism in colorectal carcinogenesis has been established. In this direction, we have evaluated the PPARγ expression and its relationship with polyamine metabolism in tissue samples from 40 patients operated because of colorectal carcinoma. Since it is known that the functional role of K-ras mutation in colorectal tumorigenesis is associated with cell growth and differentiation, polyamine metabolism and the PPARγ expression were also investigated in terms of K-ras mutation. Methods PPARγ, ODC and SSAT mRNA levels were evaluated by reverse transcriptase and real-time PCR. Polyamines were quantified by high performance liquid chromatography (HPLC). ODC and SSAT activity were measured by a radiometric technique. Results PPARγ expression, as well as SSAT and ODC mRNA levels were significantly higher in cancer as compared to normal mucosa. Tumour samples also showed significantly higher polyamine levels and ODC and SSAT activities in comparison to normal samples. A significant and positive correlation between PPARγ and the SSAT gene expression was observed in both normal and neoplastic tissue (r = 0.73, p < 0.0001; r = 0.65, p < 0.0001, respectively). Moreover, gene expression, polyamine levels and enzymatic activities were increased in colorectal carcinoma samples expressing K-ras mutation as compared to non mutated K-ras samples. Conclusion In conclusion, our data demonstrated a close relationship between PPARγ and SSAT in human colorectal cancer and this could represent an attempt to decrease

  5. Mutations in the 3c and 7b genes of feline coronavirus in spontaneously affected FIP cats.

    PubMed

    Borschensky, C M; Reinacher, M

    2014-10-01

    Feline infectious peritonitis (FIP) is the most frequent lethal infectious disease in cats. However, understanding of FIP pathogenesis is still incomplete. Mutations in the ORF 3c/ORF 7b genes are proposed to play a role in the occurrence of the fatal FIPV biotype. Here, we investigated 282 tissue specimens from 28 cats that succumbed to FIP. Within one cat, viral sequences from different organs were similar or identical, whereas greater discrepancies were found comparing sequences from various cats. Eleven of the cats exhibited deletions in the 3c gene, resulting in truncated amino acid sequences. The 7b gene was affected by deletions only in one cat. In three of the FIP cats, coronavirus isolates with both intact 3c genes as well as 7b genes of full length could also be detected. Thus, deletions or stop codons in the 3c sequence seem to be a frequent but not compelling feature of FIPVs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Overexpression and characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase of Providencia stuartii.

    PubMed

    Franklin, K; Clarke, A J

    2001-08-01

    The gene coding for aminoglycoside 2'-N-acetyltransferase Ia [AAC(2')-Ia] from Providencia stuartii was amplified by PCR and cloned. The resulting construct, pACKF2, was transferred into Escherichia coli for overexpression of AAC(2')-Ia as a fusion protein with an N-terminal hexa-His tag. The fusion protein was isolated and purified by affinity chromatography on Ni(2+)-nitrilotriacetic acid agarose and gel permeation chromatography on Superdex 75. Comparison of the specific activity of this enzyme with that of its enterokinase-digested derivative lacking the His tag indicated that the presence of the extra N-terminal peptide does not affect activity. The temperature and pH optima for activity of both forms of the 2'-N-acetyltransferase were 20 degrees C and pH 6.0, respectively, while the enzymes were most stable at 15 degrees C and pH 8.1. The Michaelis-Menten kinetic parameters for AAC(2')-Ia at 20 degrees C and pH 6.0 were determined using a series of aminoglycoside antibiotics possessing a 2'-amino group and a concentration of acetyl coenzyme A fixed at 10 times its K(m) value of 8.75 microM. Under these conditions, gentamicin was determined to be the best substrate for the enzyme in terms of both K(m) and k(cat)/K(m) values, whereas neomycin was the poorest. Comparison of the kinetic parameters obtained with the different aminoglycosides indicated that their hexopyranosyl residues provided the most important binding sites for AAC(2')-Ia activity, while the enzyme exhibits greater tolerance further from these sites. No correlation was found between these kinetic parameters and MICs determined for P. stuartii PR50 expressing the 2'-N-acetyltransferase, suggesting that its true in vivo function is not as a resistance factor.

  7. Evolution of the male-determining gene SRY within the cat family Felidae.

    PubMed

    King, V; Goodfellow, P N; Pearks Wilkerson, A J; Johnson, W E; O'Brien, S J; Pecon-Slattery, J

    2007-04-01

    In most placental mammals, SRY is a single-copy gene located on the Y chromosome and is the trigger for male sex determination during embryonic development. Here, we present comparative genomic analyses of SRY (705 bp) along with the adjacent noncoding 5' flank (997 bp) and 3' flank (948 bp) in 36 species of the cat family Felidae. Phylogenetic analyses indicate that the noncoding genomic flanks and SRY closely track species divergence. However, several inconsistencies are observed in SRY. Overall, the gene exhibits purifying selection to maintain function (omega = 0.815) yet SRY is under positive selection in two of the eight felid lineages. SRY has low numbers of nucleotide substitutions, yet most encode amino acid changes between species, and four different species have significantly altered SRY due to insertion/deletions. Moreover, fixation of nonsynonymous substitutions between sister taxa is not consistent and may occur rapidly, as in the case of domestic cat, or not at all over long periods of time, as observed within the Panthera lineage. The former resembles positive selection during speciation, and the latter purifying selection to maintain function. Thus, SRY evolution in cats likely reflects the different phylogeographic histories, selection pressures, and patterns of speciation in modern felids.

  8. Evolution of the Male-Determining Gene SRY Within the Cat Family Felidae

    PubMed Central

    King, V.; Goodfellow, P. N.; Wilkerson, A. J. Pearks; Johnson, W. E.; O'Brien, S. J.; Pecon-Slattery, J.

    2007-01-01

    In most placental mammals, SRY is a single-copy gene located on the Y chromosome and is the trigger for male sex determination during embryonic development. Here, we present comparative genomic analyses of SRY (705 bp) along with the adjacent noncoding 5′ flank (997 bp) and 3′ flank (948 bp) in 36 species of the cat family Felidae. Phylogenetic analyses indicate that the noncoding genomic flanks and SRY closely track species divergence. However, several inconsistencies are observed in SRY. Overall, the gene exhibits purifying selection to maintain function (ω = 0.815) yet SRY is under positive selection in two of the eight felid lineages. SRY has low numbers of nucleotide substitutions, yet most encode amino acid changes between species, and four different species have significantly altered SRY due to insertion/deletions. Moreover, fixation of nonsynonymous substitutions between sister taxa is not consistent and may occur rapidly, as in the case of domestic cat, or not at all over long periods of time, as observed within the Panthera lineage. The former resembles positive selection during speciation, and the latter purifying selection to maintain function. Thus, SRY evolution in cats likely reflects the different phylogeographic histories, selection pressures, and patterns of speciation in modern felids. PMID:17277366

  9. Molecular cloning, characterization and gene expression of an antioxidant enzyme catalase (MrCat) from Macrobrachium rosenbergii.

    PubMed

    Arockiaraj, Jesu; Easwvaran, Sarasvathi; Vanaraja, Puganeshwaran; Singh, Arun; Othman, Rofina Yasmin; Bhassu, Subha

    2012-05-01

    In this study, we reported a full length of catalase gene (designated as MrCat), identified from the transcriptome database of freshwater prawn Macrobrachium rosenbergii. The complete gene sequence of the MrCat is 2504 base pairs in length, and encodes 516 amino acids. The MrCat protein contains three domains such as catalase 1 (catalase proximal heme-ligand signature) at 350-358, catalase 2 (catalase proximal active site signature) at 60-76 and catalase 3 (catalase family profile) at 20-499. The mRNA expressions of MrCat in healthy and the infectious hypodermal and hematopoietic necrosis virus (IHHNV) challenged M. rosenbergii were examined using quantitative real time polymerase chain reaction (qRT-PCR). The MrCat is highly expressed in digestive tract and all the other tissues (walking leg, gills, muscle, hemocyte, hepatopancreas, pleopods, brain and eye stalk) of M. rosenbergii taken for analysis. The expression is strongly up-regulated in digestive tract after IHHNV challenge. To understand its biological activity, the recombinant MrCat gene was constructed and expressed in Escherichia coli BL21 (DE3). The recombinant MrCat existed in high thermal stability and broad spectrum of pH, which showed over 95% enzyme activity between pH 5 and 10.5, and was stable from 40 °C to 70 °C, and exhibited 85-100% enzyme activity from 30 °C to 40 °C.

  10. Xanthine urolithiasis in a cat: a case report and evaluation of a candidate gene for xanthine dehydrogenase.

    PubMed

    Tsuchida, Shuichi; Kagi, Akiko; Koyama, Hidekazu; Tagawa, Masahiro

    2007-12-01

    Xanthine urolithiasis was found in a 4-year-old spayed female Himalayan cat with a 10-month history of intermittent haematuria and dysuria. Ultrasonographs indicated the existence of several calculi in the bladder that were undetectable by survey radiographic examination. Four bladder stones were removed by cystotomy. The stones were spherical brownish-yellow and their surface was smooth and glossy. Quantitative mineral analysis showed a representative urolith to be composed of more than 95% xanthine. Ultrasonographic examination of the bladder 4.5 months postoperatively indicated the recurrence of urolithiasis. Analysis of purine concentration in urine and blood showed that the cat excreted excessive amounts of xanthine. In order to test the hypothesis that xanthinuria was caused by a homozygote of the inherited mutant allele of a gene responsible for deficiency of enzyme activity in purine degradation pathway, the allele composition of xanthine dehydrogenase (XDH) gene (one of the candidate genes for hereditary xanthinuria) was evaluated. The cat with xanthinuria was a heterozygote of the polymorphism. A single nucleotide polymorphism analysis of the cat XDH gene strongly indicated that the XDH gene of the patient cat was composed of two kinds of alleles and ruled out the hypothesis that the cat inherited the same recessive XDH allele suggesting no activity from a single ancestor.

  11. Gene dosage of the spermidine/spermine N(1)-acetyltransferase ( SSAT) gene with putrescine accumulation in a patient with a Xp21.1p22.12 duplication and keratosis follicularis spinulosa decalvans (KFSD).

    PubMed

    Gimelli, Giorgio; Giglio, Sabrina; Zuffardi, Orsetta; Alhonen, Leena; Suppola, Suvikki; Cusano, Roberto; Lo Nigro, Cristiana; Gatti, Rosanna; Ravazzolo, Roberto; Seri, Marco

    2002-09-01

    Keratosis follicularis spinulosa decalvans (KFSD) or Siemens-1 syndrome is a rare X-linked disease of unknown etiology affecting the skin and the eye. Although most affected families are compatible with X-linked inheritance, KFSD appears to be clinically and genetically heterogeneous. So far, the gene has been mapped to Xp22.13p22.2 in two extended KFSD families. Analysis of additional recombination events in the first Dutch pedigree located the gene to an interval covering approximately 1 Mb between markers DXS7163 and DXS7593/DXS7105, whereas haplotype reconstruction in the second German family positioned the gene outside the previously identified region, proximal to marker DXS274. We report here the molecular characterization of an Xp21.1p22.12 duplication present in a patient affected with dosage-sensitive sex reversal (DSS) and KFSD. The duplicated region includes both the DAX1 gene (previously demonstrated to be responsible for DSS) and the KFSD interval, in which the gene encoding spermidine/spermine N(1)-acetyltransferase ( SSAT) is located. This enzyme catalyzes the N(1)-acetylation of spermidine and spermine and, by the successive activity of polyamine oxidase, the spermine can be converted to spermidine and the spermidine to putrescine. Overexpression of the SSAT enzyme in a mouse model results in putrescine accumulation and a phenotype with skin and hair abnormalities reminiscent of human KFSD. Analysis of polyamine metabolism in the cells of the patient indicated that the levels of metabolites such as putrescine, spermidine and spermine were consistent with the overexpression of the SSAT gene as in the murine model. Thus, we propose that overexpression of SSAT and the consequent putrescine accumulation are involved in the KFSD phenotype, at least in our propositus.

  12. Maine Coon renal screening: ultrasonographical characterisation and preliminary genetic analysis for common genes in cats with renal cysts.

    PubMed

    Gendron, Karine; Owczarek-Lipska, Marta; Lang, Johann; Leeb, Tosso

    2013-12-01

    The objective of this study was to assess the prevalence of renal cysts and other renal abnormalities in purebred Maine Coon cats, and to characterise these through genetic typing. Voluntary pre-breeding screening programmes for polycystic kidney disease (PKD) are offered for this breed throughout Switzerland, Germany and other northern European countries. We performed a retrospective evaluation of Maine Coon screening for renal disease at one institution over an 8-year period. Renal ultrasonography was performed in 187 healthy Maine Coon cats. Renal changes were observed in 27 of these cats. Renal cysts were found in seven cats, and were mostly single and unilateral (6/7, 85.7%), small (mean 3.6 mm) and located at the corticomedullary junction (4/6, 66.7%). Sonographical changes indicating chronic kidney disease (CKD) were observed in 10/187 (5.3%) cats and changes of unknown significance were documented in 11/187 (5.9%) cats. All six cats genetically tested for PKD1 were negative for the mutation, and gene sequencing of these cats did not demonstrate any common genetic sequences. Cystic renal disease occurs with a low prevalence in Maine Coons and is unrelated to the PKD observed in Persians and related breeds. Ultrasonographical findings compatible with CKD are not uncommon in juvenile Maine Coons.

  13. Presence and diversity of the beta-lactamase gene in cat and dog staphylococci.

    PubMed

    Malik, Seidu; Christensen, Henrik; Peng, Haihong; Barton, Mary D

    2007-07-20

    Staphylococci are part of the normal microflora of humans and animals and some are potential pathogens that have become resistant to almost all known antibiotics. Despite the widespread reports of penicillin resistance in cat and dog staphylococci, the mechanism underlying penicillin resistance has not been examined. This study was aimed at investigating the molecular basis of resistance to penicillin in cat and dog staphylococcal isolates that showed phenotypic resistance to beta-lactam antibiotics. An 861 bp fragment of the structural blaZ gene which codes for beta-lactamase production in staphylococci was amplified by polymerase chain reaction (PCR) and the products were sequenced. Sequenced fragments were analysed by protein signature typing and sequences were compared to published blaZ sequences of human and bovine staphylococcal strains held in a public database. Four known protein signature types (1, 3, 5 and 6) and one new type (12) were identified in this study. When sequences were compared with published blaZ sequences, gene phylogenetic analysis revealed three major groups. The four variants of beta-lactamases types (A, B, C and D) belonged to each major group except for types A and D which were both in group II. These findings confirm that the blaZ gene is responsible for beta-lactamase production leading to subsequent resistance to beta-lactam antibiotics in feline and canine staphylococci and that the gene shows similar diversity and relatedness as found with blaZ sequences obtained from human and bovine staphylococci.

  14. Structure of a putative acetyltransferase (PA1377) from Pseudomonas aeruginosa

    SciTech Connect

    Davies, Anna M.; Tata, Renée; Chauviac, François-Xavier; Sutton, Brian J.; Brown, Paul R.

    2008-05-01

    The crystal structure of an acetyltransferase encoded by the gene PA1377 from Pseudomonas aeruginosa has been determined at 2.25 Å resolution. Comparison with a related acetyltransferase revealed a structural difference in the active site that was taken to reflect a difference in substrate binding and/or specificity between the two enzymes. Gene PA1377 from Pseudomonas aeruginosa encodes a 177-amino-acid conserved hypothetical protein of unknown function. The structure of this protein (termed pitax) has been solved in space group I222 to 2.25 Å resolution. Pitax belongs to the GCN5-related N-acetyltransferase family and contains all four sequence motifs conserved among family members. The β-strand structure in one of these motifs (motif A) is disrupted, which is believed to affect binding of the substrate that accepts the acetyl group from acetyl-CoA.

  15. Functional comparison of catalase genes in the elimination of photorespiratory H2O2 using promoter- and 3'-untranslated region exchange experiments in the Arabidopsis cat2 photorespiratory mutant.

    PubMed

    Hu, Ye-Qin; Liu, Sheng; Yuan, Hong-Mei; Li, Jing; Yan, Da-Wei; Zhang, Jian-Feng; Lu, Ying-Tang

    2010-10-01

    Photorespiration-associated production of H(2) O(2) accounts for the majority of total H(2) O(2) in leaves of C(3) plants and is mainly eliminated by catalases. In Arabidopsis, lack of CAT2, but not CAT1 or CAT3, results in growth suppression and a marked accumulation of H(2) O(2) in leaves. To evaluate the contribution of individual catalase genes and their promoters to catalase function, we investigated the growth suppression and H(2) O(2) accumulation phenotypes of Arabidopsis derivatives expressing catalase genes from heterologous CAT promoters in a cat2 mutant background. The expression of CAT2 from the CAT2 promoter restored the wild-type phenotype in a cat2-1 mutant, while CAT1 and CAT3 promoter-driven expression of CAT2 did not. Ectopic expression of CAT3 from the CAT2 promoter also restored the normal phenotype, unlike that of CAT1 which required replacement of the CAT1 3'-untranslated region (UTR) with that of CAT2. These results demonstrated that the photorespiratory role of CAT2 is determined mainly by the regulation of its promoter activity. The 3'-UTR of CAT2 was vital for controlling CAT2 protein levels under photorespiratory conditions. Identification of component of heterotetramers catalase isoforms suggested that there is some functional redundancy between CAT2 and CAT1 and CAT3. © 2010 Blackwell Publishing Ltd.

  16. Concurrence of cat and tet genes in multiple antibiotic-resistant bacteria isolated from a sea cucumber and sea urchin mariculture farm in China.

    PubMed

    Dang, Hongyue; Song, Linsheng; Chen, Mingna; Chang, Yaqing

    2006-11-01

    A basic understanding of abundance and diversity of antibiotic-resistant microbes and their genetic determinants is necessary for finding a way to prevent and control the spread of antibiotic resistance. For this purpose, chloramphenicol and multiple antibiotic-resistant bacteria were screened from a mariculture farm in northern China. Both sea cucumber and sea urchin rearing ponds were populated with abundant antibiotic-resistant bacteria, especially marine vibrios. Sixty-five percent chloramphenicol-resistant isolates from sea cucumber harbored a cat gene, either cat IV or cat II, whereas 35% sea urchin isolates harbored a cat gene, actually cat II. The predominant resistance determinant cat IV gene mainly occurred in isolates related to Vibrio tasmaniensis or Pseudoalteromonas atlantica, and the cat II gene mainly occurred in Vibrio splendidus-like isolates. All the cat-positive isolates also harbored one or two of the tet genes, tet(D), tet(B), or tet(A). As no chloramphenicol-related antibiotic was ever used, coselection of the cat genes by other antibiotics, especially oxytetracycline, might be the cause of the high incidence of cat genes in the mariculture farm studied.

  17. Morphine does not reduce the intraspinal release of calcitonin gene-related peptide in the cat.

    PubMed

    Morton, C R; Hutchison, W D

    1990-09-18

    In anaesthetised cats, antibody microprobes were used to measure the release of immunoreactive calcitonin gene-related peptide (irCGRP) in the lumbar dorsal horn during stimulation of non-nociceptive or nociceptive primary afferent fibres. Release of irCGRP was detected in the substantia gelatinosa, where immunostaining for CGRP was subsequently observed. Intravenous administration of morphine did not affect release of irCGRP detected during either non-nociceptive or nociceptive afferent stimulation. The results suggest that the analgesic action of morphine does not involve reduced release of CGRP from the central terminals of nociceptive primary afferents.

  18. Four Independent Mutations in the Feline Fibroblast Growth Factor 5 Gene Determine the Long-Haired Phenotype in Domestic Cats

    PubMed Central

    Kehler, James S.; David, Victor A.; Schäffer, Alejandro A.; Bajema, Kristina; Eizirik, Eduardo; Ryugo, David K.; Hannah, Steven S.; O’Brien, Stephen J.; Menotti-Raymond, Marilyn

    2013-01-01

    To determine the genetic regulation of hair length in the domestic cat, a whole genome scan was performed in a multi-generational pedigree in which the long-haired phenotype was segregating. The two markers that demonstrated the greatest linkage to the long-haired trait (LOD≥6), flanked an estimated 10 Mb region on cat chromosome B1 containing the Fibroblast Growth Factor 5 gene (FGF5), a candidate gene implicated in regulating hair follicle growth cycle in other species. Sequence analyses of FGF5 in 26 cat breeds and two pedigrees of non-breed cats, revealed four separate mutations predicted to disrupt the biological activity of the FGF5 protein. Pedigree analyses demonstrated that different combinations of paired mutant FGF5 alleles segregated with the long-haired phenotype in an autosomal recessive manner. Association analyses of over 380 genotyped breed and non-breed cats were consistent with mutations in the FGF5 gene causing the long-haired phenotype in an autosomal recessive manner. In combination, these genomic approaches demonstrated that FGF5 is the major genetic determinant of hair length in the domestic cat. PMID:17767004

  19. Triggering Respirofermentative Metabolism in the Crabtree-Negative Yeast Pichia guilliermondii by Disrupting the CAT8 Gene

    PubMed Central

    Qi, Kai

    2014-01-01

    Pichia guilliermondii is a Crabtree-negative yeast that does not normally exhibit respirofermentative metabolism under aerobic conditions, and methods to trigger this metabolism may have applications for physiological study and industrial applications. In the present study, CAT8, which encodes a putative global transcriptional activator, was disrupted in P. guilliermondii. This yeast's ethanol titer increased by >20-fold compared to the wild type (WT) during aerobic fermentation using glucose. A comparative transcriptional analysis indicated that the expression of genes in the tricarboxylic acid cycle and respiratory chain was repressed in the CAT8-disrupted (ΔCAT8) strain, while the fermentative pathway genes were significantly upregulated. The respiratory activities in the ΔCAT8 strain, indicated by the specific oxygen uptake rate and respiratory state value, decreased to one-half and one-third of the WT values, respectively. In addition, the expression of HAP4, a transcriptional respiratory activator, was significantly repressed in the ΔCAT8 strain. Through disruption of HAP4, the ethanol production of P. guilliermondii was also increased, but the yield and titer were lower than that in the ΔCAT8 strain. A further transcriptional comparison between ΔCAT8 and ΔHAP4 strains suggested a more comprehensive reprogramming function of Cat8 in the central metabolic pathways. These results indicated the important role of CAT8 in regulating the glucose metabolism of P. guilliermondii and that the regulation was partially mediated by repressing HAP4. The strategy proposed here might be applicable to improve the aerobic fermentation capacity of other Crabtree-negative yeasts. PMID:24747899

  20. An insight into population structure and gene flow within pure-bred cats.

    PubMed

    Leroy, G; Vernet, E; Pautet, M B; Rognon, X

    2014-02-01

    Investigation of genetic structure on the basis of pedigree information requires indicators adapted to the specific context of the populations studied. On the basis of pedigree-based estimates of diversity, we analysed genetic diversity, mating practices and gene flow among eight cat populations raised in France, five of them being single breeds and three consisting of breed groups with varieties that may interbreed. When computed on the basis of coancestry rate, effective population sizes ranged from 127 to 1406, while the contribution of founders from other breeds ranged from 0.7 to 16.4%. In the five breeds, FIS ranged between 0.96 and 1.83%, with this result being related to mating practices such as close inbreeding (on average 5% of individuals being inbred within two generations). Within the three groups of varieties studied, FIT ranged from 1.59 to 3%, while FST¯ values were estimated between 0.04 and 0.91%, which was linked to various amounts of gene exchanges between subpopulations at the parental level. The results indicate that cat breeds constitute populations submitted to low selection intensity, contrasting with relatively high individual inbreeding level caused by close inbreeding practices. © 2013 Blackwell Verlag GmbH.

  1. Molecular identification and phylogenic analysis of Bartonella henselae isolated from Iranian cats based on gltA gene

    PubMed Central

    Mazaheri Nezhad Fard, Ramin; Vahedi, Seyed Milad; Ashrafi, Iraj; Alipour, Faranak; Sharafi, Golnaz; Akbarein, Hesam; Aldavood, Seyed Javid

    2016-01-01

    One of the most important species of the Bartonella genus is B. henselae that causes a zoonotic infection, cat scratch disease (CSD). The main source of the bacteria is cat and the carrier is Ctenocephalides felis flea. One hundred and forty nail and saliva samples were collected from 70 domestic cats. Positive samples for B. henselae were characterized by polymerase chain reaction (PCR) and sequencing. Sequences of gltA gene were trimmed using BioEdit software and then compared with the sequences of the same gene from B. henselae isolated from cats and humans in GenBank database. Phylogenic tree was constructed using CLC Sequence Viewer software and unweighted pair group method with arithmetic mean (UPGMA) method. Molecular assessments showed that five samples out of 70 nail samples (7.14%) and one sample out of 70 saliva samples (1.42%) were genetically positive for B. henselae. At least an 87.00% similarity was seen between the gene sequences from the current study and the reference sequences from the GenBank database. Phylogenic analysis has shown that strains isolated in this study were grouped in a different haplo group, compared to other strains. Among the Asian countries, the prevalence of the bacteria in Iran was close to that in Japan and Turkey. In conclusion, findings of this study showed the prevalence of B. henselae in Iranian cats which is important due to its public health issues, especially for the immunocompromised pet owners. PMID:27226890

  2. Molecular identification and phylogenic analysis of Bartonella henselae isolated from Iranian cats based on gltA gene.

    PubMed

    Mazaheri Nezhad Fard, Ramin; Vahedi, Seyed Milad; Ashrafi, Iraj; Alipour, Faranak; Sharafi, Golnaz; Akbarein, Hesam; Aldavood, Seyed Javid

    2016-01-01

    One of the most important species of the Bartonella genus is B. henselae that causes a zoonotic infection, cat scratch disease (CSD). The main source of the bacteria is cat and the carrier is Ctenocephalides felis flea. One hundred and forty nail and saliva samples were collected from 70 domestic cats. Positive samples for B. henselae were characterized by polymerase chain reaction (PCR) and sequencing. Sequences of gltA gene were trimmed using BioEdit software and then compared with the sequences of the same gene from B. henselae isolated from cats and humans in GenBank database. Phylogenic tree was constructed using CLC Sequence Viewer software and unweighted pair group method with arithmetic mean (UPGMA) method. Molecular assessments showed that five samples out of 70 nail samples (7.14%) and one sample out of 70 saliva samples (1.42%) were genetically positive for B. henselae. At least an 87.00% similarity was seen between the gene sequences from the current study and the reference sequences from the GenBank database. Phylogenic analysis has shown that strains isolated in this study were grouped in a different haplo group, compared to other strains. Among the Asian countries, the prevalence of the bacteria in Iran was close to that in Japan and Turkey. In conclusion, findings of this study showed the prevalence of B. henselae in Iranian cats which is important due to its public health issues, especially for the immunocompromised pet owners.

  3. Season-dependent effects of photoperiod and temperature on circadian rhythm of arylalkylamine N-acetyltransferase2 gene expression in pineal organ of an air-breathing catfish, Clarias gariepinus.

    PubMed

    Singh, Kshetrimayum Manisana; Saha, Saurav; Gupta, Braj Bansh Prasad

    2017-08-01

    Arylalkylamine N-acetyltransferase (AANAT) activity, aanat gene expression and melatonin production have been reported to exhibit prominent circadian rhythm in the pineal organ of most species of fish. Three types of aanat genes are expressed in fish, but the fish pineal organ predominantly expresses aanat2 gene. Increase and decrease in daylength is invariably associated with increase and decrease in temperature, respectively. But so far no attempt has been made to delineate the role of photoperiod and temperature in regulation of the circadian rhythm of aanat2 gene expression in the pineal organ of any fish with special reference to seasons. Therefore, we studied effects of various lighting regimes (12L-12D, 16L-8D, 8L-16D, LL and DD) at a constant temperature (25°C) and effects of different temperatures (15°, 25° and 35°C) under a common photoperiod 12L-12D on circadian rhythm of aanat2 gene expression in the pineal organ of Clarias gariepinus during summer and winter seasons. Aanat2 gene expression in fish pineal organ was studied by measuring aanat2 mRNA levels using Real-Time PCR. Our findings indicate that the pineal organ of C. gariepinus exhibits a prominent circadian rhythm of aanat2 gene expression irrespective of photoperiods, temperatures and seasons, and the circadian rhythm of aanat2 gene expression responds differently to different photoperiods and temperatures in a season-dependent manner. Existence of circadian rhythm of aanat2 gene expression in pineal organs maintained in vitro under 12L-12D and DD conditions as well as a free running rhythm of the gene expression in pineal organ of the fish maintained under LL and DD conditions suggest that the fish pineal organ possesses an endogenous circadian oscillator, which is entrained by light-dark cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. [Preparation of monoclonal antibody against phosphinothricin acetyltransferase].

    PubMed

    Gao, Xudong; Wang, Yongzhi; Shi, Shengfeng; Li, Zhongpeng; Li, Xiaoyu; Xu, Wenjing; Zhang, Zhengkun; Lu, Yang; Zhang, Jiashi; Li, Qiyun; Wang, Jingang

    2013-05-01

    To express phosphinothricin acetyltransferase (PAT) with biological activity and prepare monoclonal antibodies against PAT. The full length bar gene was cloned by PCR and inserted into prokaryotic expression vector pET28a⁺. The recombinant plasmid pET28-bar was transformed into E.coli BL21(DE3), and under the induction of IPTG, PAT was expressed. The expressed protein was purified by Ni⁺; affinity chromatography to analyze its activity. The purified PAT was used to immunize BALB/c mice, and then the spleen cells from the immunized mice were fused with Sp2/0 cells. The hybridoma clones secreting antibodies against PAT were isolated by indirect ELISA and then subcloned. Soluble PAT was expressed in E.coli. The purified PAT had the activity of acetyltransferase. We totally prepared 9 hybridoma cell lines which secreted specific anti-PAT monoclonal antibodies. The expressed recombinant PAT can be used for biological reagent to prevent and relieve herbicide damage. Monoclonal antibodies against PAT may be used to detect the transgenic products.

  5. Arylalkylamine N-acetyltransferase 1 gene (TcAANAT1) is required for cuticle morphology and pigmentation of the adult red flour beetle, Tribolium castaneum.

    PubMed

    Noh, Mi Young; Koo, Bonwoo; Kramer, Karl J; Muthukrishnan, Subbaratnam; Arakane, Yasuyuki

    2016-12-01

    In the insect cuticle tanning pathway (sclerotization and pigmentation), the enzyme arylalkylamine N-acetyltransferase (AANAT) catalyzes the acetylation of dopamine to form N-acetyldopamine (NADA), which is one of the major precursors for quinone-mediated tanning. In this study we characterized and investigated the function of TcAANAT1 in cuticle pigmentation of the red flour beetle, Tribolium castaneum. We isolated a full length TcAANAT1 cDNA that encodes a protein of 256 amino acid residues with a predicted GCN5-related acetyltransferase domain containing an acetyl-CoA binding motif. TcAANAT1 transcripts were detected at all stages of development with lowest expressions at the embryonic and pharate pupal stages. We expressed and purified the encoded recombinant TcAANAT1 protein (rTcAANAT1) that exhibited highest activity at slightly basic pH values (for example, pH 7.5 to 8.5 using dopamine as the substrate). In addition, rTcAANAT1 acts on a wide range of substrates including tryptamine, octopamine and norepinephrine with similar substrate affinities with Km values in the range of 0.05-0.11 mM except for tyramine (Km = 0.56 mM). Loss of function of TcAANAT1 caused by RNAi had no effect on larval and pupal development. The tanning of pupal setae, gin traps and urogomphi proceeded normally. However, the resulting adults (∼70%) exhibited a roughened exoskeletal surface, separated elytra and improperly folded hindwings. The body wall, elytra and veins of the hindwing of the mature adults were significantly darker than those of control insects probably due to the accumulation of dopamine melanin. A dark pigmentation surrounding the bristles located on the inter-veins of the elytron was evident primarily because of the underlying darkly pigmented trabeculae that partition the dorsal and ventral layers of the elytron. These results support the hypothesis that TcAANAT1 acetylates dopamine and plays a role in development of the morphology and pigmentation of T

  6. The Novel Kasugamycin 2′-N-Acetyltransferase Gene aac(2′)-IIa, Carried by the IncP Island, Confers Kasugamycin Resistance to Rice-Pathogenic Bacteria

    PubMed Central

    Moriyama, Hiromitsu; Fukuhara, Toshiyuki

    2012-01-01

    Kasugamycin (KSM), a unique aminoglycoside antibiotic, has been used in agriculture for many years to control not only rice blast caused by the fungus Magnaporthe grisea but also rice bacterial grain and seedling rot or rice bacterial brown stripe caused by Burkholderia glumae or Acidovorax avenae subsp. avenae, respectively. Since both bacterial pathogens are seed-borne and cause serious injury to rice seedlings, the emergence of KSM-resistant B. glumae and A. avenae isolates highlights the urgent need to understand the mechanism of resistance to KSM. Here, we identified a novel gene, aac(2′)-IIa, encoding a KSM 2′-N-acetyltransferase from both KSM-resistant pathogens but not from KSM-sensitive bacteria. AAC(2′)-IIa inactivates KSM, although it reveals no cross-resistance to other aminoglycosides. The aac(2′)-IIa gene from B. glumae strain 5091 was identified within the IncP genomic island inserted into the bacterial chromosome, indicating the acquisition of this gene by horizontal gene transfer. Although excision activity of the IncP island and conjugational gene transfer was not detected under the conditions tested, circular intermediates containing the aac(2′)-IIa gene were detected. These results indicate that the aac(2′)-IIa gene had been integrated into the IncP island of a donor bacterial species. Molecular detection of the aac(2′)-IIa gene could distinguish whether isolates are resistant or susceptible to KSM. This may contribute to the production of uninfected rice seeds and lead to the effective control of these pathogens by KSM. PMID:22660700

  7. Gene expression profiling of pluripotency and differentiation-related markers in cat oocytes and preimplantation embryos.

    PubMed

    Filliers, Muriel; Goossens, Karen; Van Soom, Ann; Merlo, Barbara; Pope, Charles Earle; de Rooster, Hilde; Smits, Katrien; Vandaele, Leen; Peelman, Luc J

    2012-01-01

    During mammalian preimplantation development, two successive differentiation events lead to the establishment of three committed lineages with separate fates: the trophectoderm, the primitive endoderm and the pluripotent epiblast. In the mouse embryo, the molecular mechanisms underlying these two cell fate decisions have been studied extensively, leading to the identification of lineage-specific transcription factors. Species-specific differences in expression patterns of key regulatory genes have been reported, raising questions regarding their role in different species. The aim of the present study was to characterise the gene expression patterns of pluripotency (OCT4, SOX2, NANOG) and differentiation (CDX2, GATA6)-related markers during feline early development using reverse transcription-quantitative polymerase chain reaction. In addition, we assessed the impact of in vitro development on gene expression by comparing transcript levels of the genes investigated between in vitro and in vivo blastocysts. To normalise quantitative data within different preimplantation embryo stages, we first validated a set of stable reference genes. Transcript levels of all genes investigated were present and changed over the course of preimplantation development; a highly significant embryo-stage effect on gene expression was observed. Transcript levels of OCT4 were significantly reduced in in vitro blastocysts compared with their in vivo counterparts. None of the other genes investigated showed altered expression under in vitro conditions. The different gene expression patterns of OCT4, SOX2, CDX2 and GATA6 in cat embryos resembled those described in mouse embryos, indicative of a preserved role for these genes during early segregation. However, because of the absence of any upregulation of NANOG transcription levels after embryonic genome activation, it is unlikely that NANOG is a key regular of lineage segregation. Such results support the hypothesis that the behaviour of

  8. AtMEK1 mediates stress-induced gene expression of CAT1 catalase by triggering H2O2 production in Arabidopsis.

    PubMed

    Xing, Yu; Jia, Wensuo; Zhang, Jianhua

    2007-01-01

    Catalase and hydrogen peroxide (H(2)O(2)) have been extensively studied for their roles in various stress responses. However, little is known about the triggering mechanisms for stress-induced catalase gene expression or about H(2)O(2) production as a stress signal. It is reported here that ABA-, drought-, and salt stress-induced gene expression of CAT1 catalase is mediated by AtMEK1, an Arabidopsis MAPK kinase, by triggering H(2)O(2) signal production. Both CAT1 expression and AtMEK1 activity were activated by ABA, drought, and salt stresses. The mek1 mutant totally blocked stress-induced CAT1 expression and, interestingly, stress-induced H(2)O(2) production was also blocked. Over-expression of AtMEK1 significantly promoted stress-induced CAT1 expression, and also promoted H(2)O(2) production. These results conclusively indicate that stress-induced CAT1 expression is mediated by AtMEK1 and, furthermore, that the triggering of H(2)O(2) production might be involved in this process, as further proved by the observation that CAT1 expression was induced by applied H(2)O(2.) Surprisingly, the signalling mechanisms for stress-induced gene expression of CAT2 and CAT3 were very different from that of CAT1. Except for drought stress, expression of CAT2 or CAT3 was also activated by salt stress or ABA treatment, and AtMEK1 was not proved to be involved in the drought-induced expression of CAT2 or CAT3. Further studies showed that stomatal movement was much less sensitive to ABA in AtMEK1 mutant (mek1), and over-expression of AtMEK1 in Arabidopsis increased plant resistance to drought or salt stress, which further demonstrated that AtMEK1 is a crucial mediator in plant stress signal transduction.

  9. A silk peptide fraction restores cognitive function in AF64A-induced Alzheimer disease model rats by increasing expression of choline acetyltransferase gene.

    PubMed

    Cha, Yeseul; Lee, Sang Hoon; Jang, Su Kil; Guo, Haiyu; Ban, Young-Hwan; Park, Dongsun; Jang, Gwi Yeong; Yeon, Sungho; Lee, Jeong-Yong; Choi, Ehn-Kyoung; Joo, Seong Soo; Jeong, Heon-Sang; Kim, Yun-Bae

    2017-01-01

    This study investigated the effects of a silk peptide fraction obtained by incubating silk proteins with Protease N and Neutrase (SP-NN) on cognitive dysfunction of Alzheimer disease model rats. In order to elucidate underlying mechanisms, the effect of SP-NN on the expression of choline acetyltransferase (ChAT) mRNA was assessed in F3.ChAT neural stem cells and Neuro2a neuroblastoma cells; active amino acid sequence was identified using HPLC-MS. The expression of ChAT mRNA in F3.ChAT cells increased by 3.79-fold of the control level by treatment with SP-NN fraction. The active peptide in SP-NN was identified as tyrosine-glycine with 238.1 of molecular weight. Male rats were orally administered with SP-NN (50 or 300mg/kg) and challenged with a cholinotoxin AF64A. As a result of brain injury and decreased brain acetylcholine level, AF64A induced astrocytic activation, resulting in impairment of learning and memory function. Treatment with SP-NN exerted recovering activities on acetylcholine depletion and brain injury, as well as cognitive deficit induced by AF64A. The results indicate that, in addition to a neuroprotective activity, the SP-NN preparation restores cognitive function of Alzheimer disease model rats by increasing the release of acetylcholine.

  10. Hepatocyte-specific ablation of spermine/spermidine-N1-acetyltransferase gene reduces the severity of CCl4-induced acute liver injury

    PubMed Central

    Barone, Sharon L.; Xu, Jie; Steinbergs, Nora; Schuster, Rebecca; Lentsch, Alex B.; Amlal, Hassane; Wang, Jiang; Casero, Robert A.; Soleimani, Manoocher

    2012-01-01

    Activation of spermine/spermidine-N1-acetyltransferase (SSAT) leads to DNA damage and growth arrest in mammalian cells, and its ablation reduces the severity of ischemic and endotoxic injuries. Here we have examined the role of SSAT in the pathogenesis of toxic liver injury caused by carbon tetrachloride (CCl4). The expression and activity of SSAT increase in the liver subsequent to CCl4 administration. Furthermore, the early liver injury after CCl4 treatment was significantly attenuated in hepatocyte-specific SSAT knockout mice (Hep-SSAT-Cko) compared with wild-type (WT) mice as determined by the reduced serum alanine aminotransferase levels, decreased hepatic lipid peroxidation, and less severe liver damage. Cytochrome P450 2e1 levels remained comparable in both genotypes, suggesting that SSAT deficiency does not affect the metabolism of CCl4. Hepatocyte-specific deficiency of SSAT also modulated the induction of cytokines involved in inflammation and repair as well as leukocyte infiltration. In addition, Noxa and activated caspase 3 levels were elevated in the livers of WT compared with Hep-SSAT-Cko mice. Interestingly, the onset of cell proliferation was significantly more robust in the WT compared with Hep-SSAT Cko mice. The inhibition of polyamine oxidases protected the animals against CCl4-induced liver injury. Our studies suggest that while the abrogation of polyamine back conversion or inhibition of polyamine oxidation attenuate the early injury, they may delay the onset of hepatic regeneration. PMID:22723264

  11. Pronounced enhancement of glucocorticoid-induced gene expression following severe heat shock of heat-conditioned cells hints to intricate cell survival tactics.

    PubMed

    Mitsiou, Dimitra J; Florentin, Ida; Baki, Lia; Georgakopoulos, Anastasios; Alexis, Michael N

    2005-02-01

    We have previously reported that severe heat shock of HeLa cells stably transfected with a chloramphenicol acetyltransferase (CAT) gene, transcription of which is controlled by two glucocorticoid-responsive elements and a minimal promoter, pronouncedly enhanced glucocorticoid-induced CAT expression compared to that of non-heated cells, in spite of the glucocorticoid-receptor-mediated transcription of the gene being temporarily compromised by the shock. We now report that prolonged severe heat shock of properly heat-conditioned cells resulted in far more pronounced enhancement of glucocorticoid-induced CAT mRNA and protein expressions, in spite of a similar heat-induced loss of receptor-mediated CAT gene transcription. During recovery from the shock the hormonal activation of transcription exceeded that of non-heated cells. While CAT mRNA translation was restored appreciably later than CAT gene transcription, mRNA and protein expressions were thermally enhanced to a comparable extent, consistent with the integrity of CAT mRNA being preserved during recovery. CAT mRNA turnover was fully impaired during early recovery, suggesting that stabilisation of CAT mRNA as well as stimulation of the hormonal activation of CAT gene transcription account for the thermal enhancement of glucocorticoid-induced CAT expression. This data hint to cell survival tactics designed to safeguard high expression of genes of stress-enduring function.

  12. Fungalysin and dipeptidyl-peptidase gene transcription in Microsporum canis strains isolated from symptomatic and asymptomatic cats.

    PubMed

    Mathy, Anne; Baldo, Aline; Schoofs, Laura; Cambier, Ludivine; Defaweux, Valérie; Tabart, Jérémy; Maréchal, Françoise; Symoens, Françoise; Mignon, Bernard

    2010-11-20

    Microsporum canis is the main pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis in domestic carnivores. In cats, M. canis causes symptomatic or asymptomatic infection. Recent conflicting data raise the question of whether the clinical status of the infected cat (symptomatic or asymptomatic) is directly correlated to the proteolytic activity of M. canis strains. Here, the transcription of fungalysin and dipeptidyl-peptidase genes (DPP) of M. canis was compared between four strains isolated from symptomatic and asymptomatic cats during the first steps of the infection process, namely in arthroconidia, during adherence of arthroconidia to corneocytes and during early invasion of the epidermis, using a new ex vivo model made of feline epidermis. There was no detectable transcription of the fungalysin genes in arthroconidia or during the first steps of the infection process for any of the tested strains, suggesting that these proteases play a role later in the infection process. Among DPP, the DPP IV gene was the most frequently transcribed both in arthroconidia and later during infection (adherence and invasion), but no significant differences were observed between M. canis strains isolated from symptomatic and asymptomatic cats. This study shows that the clinical aspect of M. canis feline dermatophytosis depends upon factors relating to the host rather than to the proteolytic activity of the infective fungal strain.

  13. Gene frequencies in the cat population of Buenos Aires, Argentina, and the possible origin of this population.

    PubMed

    Kajon, A; Centrón García, D; Ruiz-García, M

    1992-01-01

    The phenotypes of 295 stray cats seen in the capital area of Buenos Aires, Argentina, between March and December of 1989 were recorded. The corresponding mutant allele frequencies were as follows: O = 0.28, a = 0.83, Ta = 0.01; tb = 0.31; d = 0.45; I = 0.40; S = 0.28; W = 0.02. The allele frequencies calculated at the O locus were consistent with those expected for a randomly breeding population according to the formula for the Hardy-Weinberg equilibrium. The analysis of the genetic distances between Buenos Aires and several European cat populations revealed that the Spanish and, especially, those with a proven more ancestral genetic constitution, are the most closely related. When a similar analysis was carried out, including other Latin American cat populations, those of Spanish origin were found to show the highest degree of relatedness. These findings suggest that the Buenos Aires cat population was not genetically structured following the "two-stepping-stone" model and support the hypothesis that differential gene flows play a transcendental role in understanding the genetic composition of domestic cat populations.

  14. Heart‐Specific Overexpression of Choline Acetyltransferase Gene Protects Murine Heart Against Ischemia Through Hypoxia‐Inducible Factor‐1α–Related Defense Mechanisms

    PubMed Central

    Kakinuma, Yoshihiko; Tsuda, Masayuki; Okazaki, Kayo; Akiyama, Tsuyoshi; Arikawa, Mikihiko; Noguchi, Tatsuya; Sato, Takayuki

    2013-01-01

    Background Murine and human ventricular cardiomyocytes rich in acetylcholine (Ach) receptors are poorly innervated by the vagus, compared with whole ventricular innervation by the adrenergic nerve. However, vagal nerve stimulation produces a favorable outcome even in the murine heart, despite relatively low ventricular cholinergic nerve density. Such a mismatch and missing link suggest the existence of a nonneuronal cholinergic system in ventricular myocardium. Methods and Results To examine the role of the nonneuronal cardiac cholinergic system, we generated choline acetyltransferase (ChAT)–expressing cells and heart‐specific ChAT transgenic (ChAT‐tg) mice. Compared with cardiomyocytes of wild‐type (WT) mice, those of the ChAT‐tg mice had high levels of ACh and hypoxia‐inducible factor (HIF)‐1α protein and augmented glucose uptake. These phenotypes were also reproduced by ChAT‐overexpressing cells, which utilized oxygen less. Before myocardial infarction (MI), the WT and ChAT‐tg mice showed similar hemodynamics; after MI, however, the ChAT‐tg mice had better survival than did the WT mice. In the ChAT‐tg hearts, accelerated angiogenesis at the ischemic area, and accentuated glucose utilization prevented post‐MI remodeling. The ChAT‐tg heart was more resistant to ischemia–reperfusion injury than was the WT heart. Conclusions These results suggest that the activated cardiac ACh‐HIF‐1α cascade improves survival after MI. We conclude that de novo synthesis of ACh in cardiomyocytes is a pivotal mechanism for self‐defense against ischemia. PMID:23525439

  15. Proximal Tubule Epithelial Cell Specific Ablation of the Spermidine/Spermine N1-Acetyltransferase Gene Reduces the Severity of Renal Ischemia/Reperfusion Injury

    PubMed Central

    Zahedi, Kamyar; Barone, Sharon; Wang, Yang; Murray-Stewart, Tracy; Roy-Chaudhury, Prabir; Smith, Roger D.; Casero, Robert A.; Soleimani, Manoocher

    2014-01-01

    Background Expression and activity of spermidine/spermine N1-acetyltransferase (SSAT) increases in kidneys subjected to ischemia/reperfusion (I/R) injury, while its ablation reduces the severity of such injuries. These results suggest that increased SSAT levels contribute to organ injury; however, the role of SSAT specifically expressed in proximal tubule epithelial cells, which are the primary targets of I/R injury, in the mediation of renal damage remains unresolved. Methods Severity of I/R injury in wt and renal proximal tubule specific SSAT-ko mice (PT-SSAT-Cko) subjected to bilateral renal I/R injury was assessed using cellular and molecular biological approaches. Results Severity of the loss of kidney function and tubular damage are reduced in PT-SSAT-Cko- compared to wt-mice after I/R injury. In addition, animals treated with MDL72527, an inhibitor of polyamine oxidases, had less severe renal damage than their vehicle treated counter-parts. The renal expression of HMGB 1 and Toll like receptors (TLR) 2 and 4 were also reduced in PT-SSAT-Cko- compared to wt mice after I/R injury. Furthermore, infiltration of neutrophils, as well as expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) transcripts were lower in the kidneys of PT-SSAT-Cko compared to wt mice after I/R injury. Finally, the activation of caspase3 was more pronounced in the wt compared to PT-SSAT-Cko animals. Conclusions Enhanced SSAT expression by proximal tubule epithelial cells leads to tubular damage, and its deficiency reduces the severity of renal I/R injury through reduction of cellular damage and modulation of the innate immune response. PMID:25390069

  16. Isolation and characterization of a catalase gene "HuCAT3" from pitaya (Hylocereus undatus) and its expression under abiotic stress.

    PubMed

    Nie, Qiong; Gao, Guo-Li; Fan, Qing-jie; Qiao, Guang; Wen, Xiao-Peng; Liu, Tao; Peng, Zhi-Jun; Cai, Yong-Qiang

    2015-05-25

    Abiotic stresses usually cause H2O2 accumulation, with harmful effects, in plants. Catalase may play a key protective role in plant cells by detoxifying this excess H2O2. Pitaya (Hylocereus undatus) shows broad ecological adaptation due to its high tolerance to abiotic stresses, e.g. drought, heat and poor soil. However, involvement of the pitaya catalase gene (HuCAT) in tolerance to abiotic stresses is unknown. In the present study, a full-length HuCAT3 cDNA (1870 bp) was isolated from pitaya based on our previous microarray data and RACE method. The cDNA sequence and deduced amino acid sequence shared 73-77% and 75-80% identity with other plant catalases, respectively. HuCAT3 contains conserved catalase family domain and catalytic sites. Pairwise comparison and phylogenetic analysis indicated that HuCAT3 is most similar to Eriobotrya japonica CAT, followed by Dimocarpus longan CAT and Nicotiana tabacum CAT1. Expression profile analysis demonstrated that HuCAT3 is mainly expressed in green cotyledons and mature stems, and was regulated by H2O2, drought, cold and salt stress, whereas, its expression patterns and maximum expression levels varied with stress types. HuCAT activity increased as exposure to the tested stresses, and the fluctuation of HuCAT activity was consistent with HuCAT3 mRNA abundance (except for 0.5 days upon drought stress). HuCAT3 mRNA elevations and HuCAT activities changes under cold stress were also in conformity with the cold tolerances among the four genotypes. The obtained results confirmed a major role of HuCAT3 in abiotic stress response of pitaya. This may prove useful in understanding pitaya's high tolerance to abiotic stresses at molecular level.

  17. The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat.

    PubMed

    Wang, Zi T; Verma, Shiv K; Dubey, Jitender P; Sibley, L David

    2017-03-01

    The Toxoplasma gondii genome contains two aromatic amino acid hydroxylase genes, AAH1 and AAH2 encode proteins that produce L-DOPA, which can serve as a precursor of catecholamine neurotransmitters. It has been suggested that this pathway elevates host dopamine levels thus making infected rodents less fearful of their definitive Felidae hosts. However, L-DOPA is also a structural precursor of melanins, secondary quinones, and dityrosine protein crosslinks, which are produced by many species. For example, dityrosine crosslinks are abundant in the oocyst walls of Eimeria and T. gondii, although their structural role has not been demonstrated, Here, we investigated the biology of AAH knockout parasites in the sexual reproductive cycle within cats. We found that ablation of the AAH genes resulted in reduced infection in the cat, lower oocyst yields, and decreased rates of sporulation. Our findings suggest that the AAH genes play a predominant role during infection in the gut of the definitive feline host.

  18. The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat

    PubMed Central

    Verma, Shiv K.; Dubey, Jitender P.

    2017-01-01

    The Toxoplasma gondii genome contains two aromatic amino acid hydroxylase genes, AAH1 and AAH2 encode proteins that produce L-DOPA, which can serve as a precursor of catecholamine neurotransmitters. It has been suggested that this pathway elevates host dopamine levels thus making infected rodents less fearful of their definitive Felidae hosts. However, L-DOPA is also a structural precursor of melanins, secondary quinones, and dityrosine protein crosslinks, which are produced by many species. For example, dityrosine crosslinks are abundant in the oocyst walls of Eimeria and T. gondii, although their structural role has not been demonstrated, Here, we investigated the biology of AAH knockout parasites in the sexual reproductive cycle within cats. We found that ablation of the AAH genes resulted in reduced infection in the cat, lower oocyst yields, and decreased rates of sporulation. Our findings suggest that the AAH genes play a predominant role during infection in the gut of the definitive feline host. PMID:28288194

  19. The application of the yeast N-acetyltransferase MPR1 gene and the proline analogue L-azetidine-2-carboxylic acid as a selectable marker system for plant transformation

    PubMed Central

    Tsai, Fei-Yi; Ulanov, Alexander; Widholm, Jack M.

    2010-01-01

    The yeast N-acetyltransferase MPR1 gene has previously been shown to confer resistance to the toxic proline analogue azetidine-2-carboxylic acid (A2C) in yeast and transgenic tobacco. Here experiments were carried out to determine if MPR1 and A2C can work as a selectable marker system for plant transformation. The MPR1 gene was inserted into a binary vector under the control of the cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and transformed into tobacco via the Agrobacterium tumefaciens-mediated leaf disc method. A2C was applied in the selection medium to select for putative transformants. PCR analysis showed that 28.4% and 66.7% of the plantlets selected by 250 μM and 300 μM A2C were positive for the MPR1 gene, respectively. Southern and northern blot analysis and enzyme activity assay confirmed the stable gene incorporation, transcription, and translation of the MPR1 transgene in the transgenic plants. The transgene-carrying T1 progeny could be distinguished from the recessive progeny when grown on 400, 450, or 500 μM A2C. Examination of the metabolism of 22 transgenic plants by gas chromatography–mass spectrometry profiling did not reveal any significant changes. In conclusion, the results demonstrate that MPR1/A2C is a safe and efficient selection system that does not involve microbial antibiotic or herbicide resistance genes. Recent studies showed that MPR1 can protect yeast against oxidative stresses by decreasing the accumulation of the proline catabolite Δ1-pyrroline-5-carboxylate (P5C). However, H2O2 treatment resulted in contradictory responses among the five transgenic lines tested. Further experiments are required to assess the response of MPR1 transgenic plants under oxidative stress. PMID:20430752

  20. Structure and function of histone acetyltransferase MOF.

    PubMed

    Chen, Qiao Yi; Costa, Max; Sun, Hong

    2015-01-01

    MOF was first identified in Drosophila melanogaster as an important component of the dosage compensation complex. As a member of MYST family of histone acetyltransferase, MOF specifically deposits the acetyl groups to histone H4 lysine 16. Throughout evolution, MOF and its mammalian ortholog have retained highly conserved substrate specificity and similar enzymatic activities. MOF plays important roles in dosage compensation, ESC self-renewal, DNA damage and repair, cell survival, and gene expression regulation. Dysregulation of MOF has been implicated in tumor formation and progression of many types of human cancers. This review will discuss the structure and activity of mammalian hMOF as well as its function in H4K16 acetylation, DNA damage response, stem cell pluripotency, and carcinogenesis.

  1. Structure and function of histone acetyltransferase MOF

    PubMed Central

    Chen, Qiao Yi; Costa, Max; Sun, Hong

    2016-01-01

    MOF was first identified in Drosophila melanogaster as an important component of the dosage compensation complex. As a member of MYST family of histone acetyltransferase, MOF specifically deposits the acetyl groups to histone H4 lysine 16. Throughout evolution, MOF and its mammalian ortholog have retained highly conserved substrate specificity and similar enzymatic activities. MOF plays important roles in dosage compensation, ESC self-renewal, DNA damage and repair, cell survival, and gene expression regulation. Dysregulation of MOF has been implicated in tumor formation and progression of many types of human cancers. This review will discuss the structure and activity of mammalian hMOF as well as its function in H4K16 acetylation, DNA damage response, stem cell pluripotency, and carcinogenesis. PMID:28503659

  2. Molecular characterization of the L1 gene of papillomaviruses in epithelial lesions of cats and comparative analysis with corresponding gene sequences of human and feline papillomaviruses.

    PubMed

    Anis, Eman A; O'Neill, Sarah H; Newkirk, Kim M; Brahmbhatt, Rupal A; Abd-Eldaim, Mohamed; Frank, Linda A; Kania, Stephen A

    2010-12-01

    To characterize the L1 gene of papillomaviruses detected in epithelial lesions of cats and to determine the relationship between those L1 gene nucleotide sequences and known L1 gene sequences of human and feline papillomaviruses. 10 tissue samples of epithelial lesions from 8 cats. DNA was extracted from tissue samples. Primers were designed to amplify the L1 gene of papillomaviruses. Amplicons of DNA were sequenced; nucleotide sequences were compared with known L1 gene nucleotide sequences of papillomaviruses and used for phylogenetic analysis. Tissue samples were obtained from lesions (diagnosed as dysplasia [n=1], squamous cell carcinoma in situ [3], or squamous cell carcinoma [6]) of the skin (9) and oral mucosa [1]. Two amplicons had 99% homology with the L1 gene nucleotide sequence of human papillomavirus type 38b subtype FA125. Another amplicon had 84% homology with the L1 gene nucleotide sequence of human papillomavirus type 80 and was considered to be a new type of papillomavirus. Phylogenetic tree analysis revealed that these 3 papillomaviruses were grouped into 2 clades that were not similar to the clades of Felis domesticus papillomavirus type 1 or F domesticus papillomavirus type 2 (FdPV2). The remaining 7 amplicons had 98% to 100% homology with the L1 gene nucleotide sequence of FdPV2. Phylogenetic tree analysis revealed that those 7 papillomaviruses were grouped nto a single clade with FdPV2. Results support the likelihood of transmission of papillomaviruses between humans and cats.

  3. Properties of a pentapeptide inhibitor of peptidyltransferase that is essential for cat gene regulation by translation attenuation.

    PubMed Central

    Gu, Z; Harrod, R; Rogers, E J; Lovett, P S

    1994-01-01

    Inducible chloramphenicol resistance genes cat and cmlA are regulated by translation attenuation. For both genes, the leader codons that must be translated to deliver a ribosome to the induction site specify a peptide that inhibits peptidyltransferase in vitro. The antipeptidyltransferase activity of the peptides is thought to select the site of ribosome stalling that is essential for induction. Using variations of the cat-86 leader-encoded 5-mer peptide MVKTD, we demonstrate a correlation between the in vitro antipeptidyltransferase activity and the ability of the same peptide to support induction by chloramphenicol in vivo. MVKTD footprints to nucleotides 2058, 2059, and 2060 in 23S rRNA. In vivo methylation of nucleotide 2058 by the ermC methylase interferes neither with cat-86 induction nor with peptide inhibition of peptidyltransferase. The methylation eliminates the competition that normally occurs in vitro between erythromycin and MVKTD. MVKTD inhibits the peptidyltransferase of several eubacteria, a representative Archaea species, and the eukaryote Saccharomyces cerevisiae. Bacillus stearothermophilus supports the in vivo induction of cat-86, and the RNA that is phenol extracted from the 50S ribosomes of this gram-positive thermophile is catalytically active in the peptidyltransferase assay and sensitive to peptide inhibition. Our results indicate that peptidyltransferase inhibition by a cat leader peptide is essential to induction, and this activity can be altered by minor changes in the amino acid sequence of the peptide. The broad range of organisms shown to possess peptide-inhibitable peptidyltransferase suggests that the target is a highly conserved component of the ribosome and includes 23S rRNA. Images PMID:7928994

  4. Accelerated evolution of CES7, a gene encoding a novel major urinary protein in the cat family.

    PubMed

    Li, Gang; Janecka, Jan E; Murphy, William J

    2011-02-01

    Cauxin is a novel urinary protein recently identified in the domestic cat that regulates the excretion of felinine, a pheromone precursor involved in sociochemical communication and territorial marking of domestic and wild felids. Understanding the evolutionary history of cauxin may therefore illuminate molecular adaptations involved in the evolution of pheromone-based communication, recognition, and mate selection in wild animals. We sequenced the gene encoding cauxin, CES7, in 22 species representing all major felid lineages, and multiple outgroups and showed that it has undergone rapid evolutionary change preceding and during the diversification of the cat family. A comparison between feline cauxin and orthologous carboxylesterases from other mammalian lineages revealed evidence of strong positive Darwinian selection within and between several cat lineages, enriched at functionally important sites of the protein. The higher rate of radical amino acid replacements in small felids, coupled with the lack of felinine and extremely low levels of cauxin in the urine of the great cats (Panthera), correlates with functional divergence of this gene in Panthera, and its putative loss in the snow leopard. Expression studies found evidence for several alternatively spliced transcripts in testis and brain, suggesting additional roles in male reproductive fitness and behavior. Our work presents the first report of strong positive natural selection acting on a major urinary protein of nonrodent mammals, providing evidence for parallel selection pressure on the regulation of pheromones in different mammalian lineages, despite the use of different metabolic pathways. Our results imply that natural selection may drive rapid changes in the regulation of pheromones in urine among the different cat species, which in turn may influence social behavior, such as territorial marking and conspecific recognition, therefore serving as an important mechanism for the radiation of this group

  5. Expression in Escherichia coli of a sub-gene encoding the lipoyl and peripheral subunit-binding domains of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.

    PubMed

    Hipps, D S; Perham, R N

    1992-05-01

    A sub-gene encoding the N-terminal 170 residues of the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was over-expressed in Escherichia coli. The expressed polypeptide consists of the lipoyl domain, inter-domain linker and peripheral subunit-binding domain; these were found to have folded into their native functional conformations as judged by reductive acetylation of the lipoyl domain, limited proteolysis of the linker region and ability to bind the dihydrolipoamide dehydrogenase dimer. The di-domain was largely (80%) unlipoylated; a small proportion (4%) was correctly modified with lipoic acid and the remainder (16%) was aberrantly modified with octanoic acid. A polyclonal antiserum was raised that recognized both the di-domain and the individual component domains. The 400 MHz 1H-n.m.r. spectrum of the di-domain showed resonances corresponding to those seen in spectra of the lipoyl domain, plus others characteristic of amino acid residues in the flexible linker region. Further, as yet unidentified, resonances are likely to be derived from the peripheral subunit-binding domain. The existence and independent folding of the peripheral subunit-binding domain is thus confirmed and its purification in large-scale amounts for detailed structural analysis is now possible.

  6. Expression in Escherichia coli of a sub-gene encoding the lipoyl and peripheral subunit-binding domains of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.

    PubMed Central

    Hipps, D S; Perham, R N

    1992-01-01

    A sub-gene encoding the N-terminal 170 residues of the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was over-expressed in Escherichia coli. The expressed polypeptide consists of the lipoyl domain, inter-domain linker and peripheral subunit-binding domain; these were found to have folded into their native functional conformations as judged by reductive acetylation of the lipoyl domain, limited proteolysis of the linker region and ability to bind the dihydrolipoamide dehydrogenase dimer. The di-domain was largely (80%) unlipoylated; a small proportion (4%) was correctly modified with lipoic acid and the remainder (16%) was aberrantly modified with octanoic acid. A polyclonal antiserum was raised that recognized both the di-domain and the individual component domains. The 400 MHz 1H-n.m.r. spectrum of the di-domain showed resonances corresponding to those seen in spectra of the lipoyl domain, plus others characteristic of amino acid residues in the flexible linker region. Further, as yet unidentified, resonances are likely to be derived from the peripheral subunit-binding domain. The existence and independent folding of the peripheral subunit-binding domain is thus confirmed and its purification in large-scale amounts for detailed structural analysis is now possible. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. Fig. 6. PMID:1590756

  7. 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.

  8. Absence of seasonal changes in FSHR gene expression in the cat cumulus-oocyte complex in vivo and in vitro.

    PubMed

    Hobbs, Rebecca J; Howard, JoGayle; Wildt, David E; Comizzoli, Pierre

    2012-07-01

    Domestic cat oocytes are seasonally sensitive to FSH. Compared with those collected during the breeding season, oocytes from the nonbreeding (NB) season require more FSH during in vitro maturation to achieve comparable developmental competence. This study tested the hypothesis that this seasonal variation was due to altered expression of FSH receptors (FSHR) and/or FSH-induced genes. Relative expression levels of FSHR mRNA and FSH-enhanced gene estrogen receptor β (ESR2) were measured by qPCR in whole ovaries and immature cumulus-oocyte complexes (COCs) isolated from cat ovaries during the natural breeding vs NB seasons. Expression levels of FSH-induced genes prostaglandin-endoperoxide synthase 2 (PTGS2), early growth response protein-1 (EGR1), and epidermal growth factor receptor (EGFR) were examined in mature COCs from both seasons that were a) recovered in vivo or b) matured in vitro with conventional (1 μg/ml) or high (10 μg/ml) FSH concentrations. Overall, FSHR mRNA levels were lower in whole ovaries during the NB compared with breeding season but were similar in immature COCs, whereas ESR2 levels did not differ in either group between intervals. We observed changes in PTGS2, EGR1, and EGFR mRNA expression patterns across maturation in COCs within but not between the two seasons. The lack of seasonal differentiation in FSH-related genes was not consistent with the decreased developmental capacity of oocytes fertilized during the NB season. These findings reveal that the seasonal decrease in cat oocyte sensitivity to FSH occurs both in vivo and in vitro. Furthermore, this decline is unrelated to changes in expression of FSHR mRNA or mRNA of FSH-induced genes in COCs from antral follicles.

  9. Arylamine N-Acetyltransferases in Mycobacteria

    PubMed Central

    Sim, Edith; Sandy, James; Evangelopoulos, Dimitrios; Fullam, Elizabeth; Bhakta, Sanjib; Westwood, Isaac; Krylova, Anna; Lack, Nathan; Noble, Martin

    2008-01-01

    Polymorphic Human arylamine N-acetyltransferase (NAT2) inactivates the anti-tubercular drug isoniazid by acetyltransfer from acetylCoA. There are active NAT proteins encoded by homologous genes in mycobacteria including M. tuberculosis, M. bovis BCG, M. smegmatis and M. marinum. Crystallographic structures of NATs from M. smegmatis and M. marinum, as native enzymes and with isoniazid bound share a similar fold with the first NAT structure, Salmonella typhimurium NAT. There are three approximately equal domains and an active site essential catalytic triad of cysteine, histidine and aspartate in the first two domains. An acetyl group from acetylCoA is transferred to cysteine and then to the acetyl acceptor e.g. isoniazid. M. marinum NAT binds CoA in a more open mode compared with CoA binding to human NAT2. The structure of mycobacterial NAT may promote its role in synthesis of cell wall lipids, identified through gene deletion studies. NAT protein is essential for survival of M. bovis BCG in macrophage as are the proteins encoded by other genes in the same gene cluster (hsaA-D). HsaA-D degrade cholesterol, essential for mycobacterial survival inside macrophage. Nat expression remains to be fully understood but is co-ordinated with hsaA-D and other stress response genes in mycobacteria. Amide synthase genes in the streptomyces are also nat homologues. The amide synthases are predicted to catalyse intramolecular amide bond formation and creation of cyclic molecules, e.g. geldanamycin. Lack of conservation of the CoA binding cleft residues of M. marinum NAT suggests the amide synthase reaction mechanism does not involve a soluble CoA intermediate during amide formation and ring closure. PMID:18680471

  10. Expression of the Saccharomyces cerevisiae MPR1 gene encoding N-acetyltransferase in Zygosaccharomyces rouxii confers resistance to L-azetidine-2-carboxylate.

    PubMed

    Pribylová, L; Sychrová, H

    2006-01-01

    The osmotolerant yeast Zygosaccharomyces rouxii is sensitive to the toxic L-proline analogue, L-azetidine-2-carboxylate (AZC). The possibility of use of the Saccharomyces cerevisiae MPR1 gene (ScMPR1) encoding the AZC-detoxifying enzyme as a dominant selection marker in Z. rouxii was examined. The heterologous expression of ScMPR1 in two Z. rouxii strains resulted in AZC-resistant colonies, but that of ScMPR1 as a dominant marker gene in vectors was affected by a high frequency of spontaneously resistant colonies. The same was found for an AZC-sensitive S. cerevisiae strain in which the ScMPR1 was expressed. In both yeasts, ScMPR1 can be used only as an auxiliary marker gene.

  11. Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat

    SciTech Connect

    Kenney, S.; Kamine, J.; Markovitz, D.; Fenrick, R.; Pagano, J.

    1988-03-01

    Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, the authors demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.

  12. Frequency of a FAS ligand gene variant associated with inherited feline autoimmune lymphoproliferative syndrome in British shorthair cats in New Zealand.

    PubMed

    Aberdein, D; Munday, J S; Dittmer, K E; Heathcott, R W; Lyons, L A

    2017-11-01

    AIMS To determine the frequency of the FAS-ligand gene (FASLG) variant associated with feline autoimmune lymphoproliferative syndrome (FALPS) and the proportion of carriers of the variant in three British shorthair (BSH) breeding catteries in New Zealand. METHODS Buccal swabs were collected from all cats in two BSH breeding catteries from the South Island and one from the North Island of New Zealand. DNA was extracted and was tested for the presence of the FASLG variant using PCR. Cats with the FASLG variant were identified and the frequency of the FASLG variant allele calculated. Pedigree analysis was performed and inbreeding coefficients were calculated for cats with the FASLG variant. RESULTS Of 32 BSH cats successfully tested for the presence of the FASLG variant, one kitten (3%) was homozygous (FALPS-affected), and seven (22%) cats were heterozygous (carriers) for the FASLG variant allele, and 24 (75%) cats were homozygous for the wild type allele. The overall frequency of the FASLG variant allele in these 32 cats was 0.14. Cats carrying the FASLG variant were from all three breeding catteries sampled, including two catteries that had not previously reported cases of FALPS. Pedigree analysis revealed common ancestry of FALPS-affected and carrier cats within six generations, as well as frequent inbreeding, with inbreeding coefficients >0.12 for five cats with the FASLG variant. CONCLUSIONS AND CLINICAL RELEVANCE There was a high frequency of the FASLG variant allele (0.14) in this small sample of BSH cats, with 22% of healthy cats identified as carriers of the FASLG variant. For an inherited disease, lethal at a young age, in a small population in which inbreeding is common, these results are significant. To prevent future cases of disease and stop further spread of the FASLG variant allele within the BSH population in New Zealand, it is recommended that all BSH and BSH-cross cats be tested for the presence of the FASLG variant before mating. Cats identified as

  13. Active cigarette smoking and the risk of breast cancer at the level of N-acetyltransferase 2 (NAT2) gene polymorphisms.

    PubMed

    Kasajova, Petra; Holubekova, Veronika; Mendelova, Andrea; Lasabova, Zora; Zubor, Pavol; Kudela, Erik; Biskupska-Bodova, Kristina; Danko, Jan

    2016-06-01

    The aim of our study was to assess the correlation between the tobacco exposure and NAT2 gene (rs1041983 C/T, rs1801280 T/C, rs1799930 G/A) polymorphisms in association with breast cancer development. We wanted to determine the prognostic clinical importance of these polymorphisms in association with smoking and breast cancer. For the detection of possible association between smoking, NAT2 gene polymorphisms, and the risk of breast cancer, we designed a case-controlled study with 198 patients enrolled, 98 breast cancer patients and 100 healthy controls. Ten milliliters of peripheral blood from the cubital vein was withdrawn from every patient. The HRM (high resolution melting) analysis was used for the detection of three abovementioned NAT2 gene polymorphisms. When comparing a group of women smoking more than 5 cigarettes a day with the patients smoking fewer than 5 cigarettes a day, we found out that if women were the carriers of aberrant AA genotype for rs1799930, the first group of women had higher risk of breast carcinoma than the second group. If patients were the carriers of aberrant TT genotype for rs1041983, for rs1801280CC genotype, and rs1799930AA genotype and they smoked more than 5 cigarettes a day, they had higher risk of malignant breast disease than never-smoking women. Our results confirm the hypothesis that NAT2 gene polymorphisms (rs1041983 C/T, rs1801280 T/C, and rs1799930 G/A) in association with long-period active smoking could be the possible individual risk-predicting factors for breast cancer development in the population of Slovak women.

  14. MYST protein acetyltransferase activity requires active site lysine autoacetylation.

    PubMed

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-04

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

  15. MYST protein acetyltransferase activity requires active site lysine autoacetylation

    PubMed Central

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-01

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases. PMID:22020126

  16. Defining the orphan functions of lysine acetyltransferases.

    PubMed

    Montgomery, David C; Sorum, Alexander W; Meier, Jordan L

    2015-01-16

    Long known for their role in histone acetylation, recent studies have demonstrated that lysine acetyltransferases also carry out distinct "orphan" functions. These activities impact a wide range of biological phenomena including metabolism, RNA modification, nuclear morphology, and mitochondrial function. Here, we review the discovery and characterization of orphan lysine acetyltransferase functions. In addition to highlighting the evidence and biological role for these functions in human disease, we discuss the part emerging chemical tools may play in investigating this versatile enzyme superfamily.

  17. A second chromosomal copy of the catA gene endows Pseudomonas putida mt-2 with an enzymatic safety valve for excess of catechol.

    PubMed

    Jiménez, Jose I; Pérez-Pantoja, Danilo; Chavarría, Max; Díaz, Eduardo; de Lorenzo, Víctor

    2014-06-01

    Pseudomonas putida mt-2 harbours two different routes for catabolism of catechol, namely one meta pathway encoded by the xyl genes of the TOL plasmid pWW0 and one ortho pathway determined by the chromosomal ben and cat genes. P. putida mt-2 has a second chromosomal copy of the catA gene (named catA2) located downstream of the ben operon that encodes an additional catechol-1,2-dioxygenase. The metabolic and regulatory phenotypes of strains lacking one enzyme, the other and both of them in cells with and without the TOL plasmid were evaluated. The data consistently indicated that induction of the ortho pathway by benzoate plasmid-less strain P. putida KT2440 led to catechol surplus, the toxicity of which at high concentrations being counteracted by CatA2. Cells carrying pWW0 but lacking catA2 experienced both a rapid loss of the plasmid when grown on benzoate (a substrate of the lower pathway) and a slowdown of their growth rate when cultured with benzylalcohol (a substrate converted to benzoate by the upper pathway). These data reveal the role of CatA2 as a type of metabolic safety valve for excess catechol that alleviates the metabolic conflict generated by simultaneous expression of the meta and ortho pathways, thereby facilitating their co-existence.

  18. Cloning, expression and purification of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor via a fusion product with chloramphenicol acetyltransferase.

    PubMed

    Dekeyzer, N; Engelborghs, Y; Volckaert, G

    1994-01-01

    A gene coding for the Nereis sarcoplasmic calcium-binding protein (NSCP) was synthesized and expressed in Escherichia coli. The sequence of the gene was derived from the protein sequence by reverse translation. It possesses a number of unique, regularly spaced, restriction endonuclease cleavage sites to facilitate future site-directed mutagenesis. For the cloning strategy the gene sequence was divided into four parts. Three parts were cloned by ligation of hybridized oligomers and one part by inverse PCR. The protein was expressed as a fusion protein with the bacterial chloramphenicol acetyl-transferase (CAT), which could be easily purified by affinity chromatography. At the junction of the CAT and NSCP moieties a recognition site for the proteolytic enzyme factor Xa was built in. However, the distance between the moieties appeared to be crucial to warrant cleavage. A kinetic analysis showed that NSCP prepared from the sandworm and the one expressed by E. coli behaved in the same way. This system provides a basis for site-specific mutagenesis studies, in order to elucidate the molecular mechanism of cation binding and concomitant conformational changes.

  19. Rapid evolution of the env gene leader sequence in cats naturally infected with feline immunodeficiency virus.

    PubMed

    Bęczkowski, Paweł M; Hughes, Joseph; Biek, Roman; Litster, Annette; Willett, Brian J; Hosie, Margaret J

    2015-04-01

    Analysing the evolution of feline immunodeficiency virus (FIV) at the intra-host level is important in order to address whether the diversity and composition of viral quasispecies affect disease progression. We examined the intra-host diversity and the evolutionary rates of the entire env and structural fragments of the env sequences obtained from sequential blood samples in 43 naturally infected domestic cats that displayed different clinical outcomes. We observed in the majority of cats that FIV env showed very low levels of intra-host diversity. We estimated that env evolved at a rate of 1.16×10(-3) substitutions per site per year and demonstrated that recombinant sequences evolved faster than non-recombinant sequences. It was evident that the V3-V5 fragment of FIV env displayed higher evolutionary rates in healthy cats than in those with terminal illness. Our study provided the first evidence that the leader sequence of env, rather than the V3-V5 sequence, had the highest intra-host diversity and the highest evolutionary rate of all env fragments, consistent with this region being under a strong selective pressure for genetic variation. Overall, FIV env displayed relatively low intra-host diversity and evolved slowly in naturally infected cats. The maximum evolutionary rate was observed in the leader sequence of env. Although genetic stability is not necessarily a prerequisite for clinical stability, the higher genetic stability of FIV compared with human immunodeficiency virus might explain why many naturally infected cats do not progress rapidly to AIDS. © 2015 The Authors.

  20. Rapid evolution of the env gene leader sequence in cats naturally infected with feline immunodeficiency virus

    PubMed Central

    Hughes, Joseph; Biek, Roman; Litster, Annette; Willett, Brian J.; Hosie, Margaret J.

    2015-01-01

    Analysing the evolution of feline immunodeficiency virus (FIV) at the intra-host level is important in order to address whether the diversity and composition of viral quasispecies affect disease progression. We examined the intra-host diversity and the evolutionary rates of the entire env and structural fragments of the env sequences obtained from sequential blood samples in 43 naturally infected domestic cats that displayed different clinical outcomes. We observed in the majority of cats that FIV env showed very low levels of intra-host diversity. We estimated that env evolved at a rate of 1.16×10−3 substitutions per site per year and demonstrated that recombinant sequences evolved faster than non-recombinant sequences. It was evident that the V3–V5 fragment of FIV env displayed higher evolutionary rates in healthy cats than in those with terminal illness. Our study provided the first evidence that the leader sequence of env, rather than the V3–V5 sequence, had the highest intra-host diversity and the highest evolutionary rate of all env fragments, consistent with this region being under a strong selective pressure for genetic variation. Overall, FIV env displayed relatively low intra-host diversity and evolved slowly in naturally infected cats. The maximum evolutionary rate was observed in the leader sequence of env. Although genetic stability is not necessarily a prerequisite for clinical stability, the higher genetic stability of FIV compared with human immunodeficiency virus might explain why many naturally infected cats do not progress rapidly to AIDS. PMID:25535323

  1. Structure of the lac operon galactoside acetyltransferase.

    PubMed

    Wang, Xing-Guo; Olsen, Laurence R; Roderick, Steven L

    2002-04-01

    The galactoside acetyltransferase (thiogalactoside transacetylase) of Escherichia coli (GAT, LacA, EC 2.3.1.18) is a gene product of the classical lac operon. GAT may assist cellular detoxification by acetylating nonmetabolizable pyranosides, thereby preventing their reentry into the cell. The structure of GAT has been solved in binary complexes with acetyl-CoA or CoA and in ternary complexes with CoA and the nonphysiological acceptor substrates isopropyl beta-D-thiogalactoside (IPTG) or p-nitrophenyl beta-D-galactopyranoside (PNPbetaGal). A hydrophobic cleft that binds the thioisopropyl and p-nitrophenyl aglycones of IPTG and PNPbetaGal may discriminate against substrates with hydrophilic substituents at this position, such as lactose, or inducers of the lac operon. An extended loop projecting from the left-handed parallel beta helix domain contributes His115, which is in position to facilitate attack of the C6-hydroxyl group of the substrate on the thioester.

  2. Myocyte-specific M-CAT and MEF-1 elements regulate G-protein gamma 3 gene (gamma3) expression in cardiac myocytes.

    PubMed

    McWhinney, Charlene; Robishaw, Janet D

    2008-07-01

    Little is known regarding the mechanisms that control the expression of G-protein alpha, beta, and gamma subtypes. We have previously shown that the G-protein gamma(3) gene is expressed in the heart, brain, lung, spleen, kidney, muscle, and testis in mice. We have also reported that the G-protein gamma(3) subunit is expressed in rat cardiac myocytes, but not in cardiac fibroblasts. Other studies have shown that the gamma(3) subunit couples to the angiotensin A1A receptor in portal vein myocytes, and has been shown to mediate beta-adrenergic desensitization in cardiac myocytes treated with atorvastatin. In the present study, we evaluated G-protein gamma(3) promoter-luciferase reporter constructs in primary myocytes to identify key regulatory promoter regions. We identified two important regions of the promoter (upstream promoter region [UPR] and downstream promoter region [DPR]), which are required for expression in cardiac myocytes. We observed that removal of 48 bp in the UPR diminished gene transcription by 75%, and that the UPR contains consensus elements for myocyte-specific M-CAT and myocyte enhancer factor 1 (MEF-1) elements. The UPR and DPR share transcription factor elements for myocyte-specific M-CAT element. We observed that cardiac myocyte proteins bind to gamma(3) oligonucleotides containing transcription factor elements for myocyte-specific M-CAT and MEF-1. Myocyte-specific M-CAT proteins were supershifted with transcriptional enhancer factor-1 (TEF-1) antibodies binding to the gamma(3) M-CAT element, which is in agreement with reports showing that the M-CAT element binds the TEF-1 family of transcription factors. The 150 bp DPR contains three M-CAT elements, an INR element, an upstream stimulatory factor 1 element, and the transcription start site. We have shown that myocyte gamma(3) gene expression is regulated by myocyte-specific M-CAT and MEF-1 elements.

  3. Sequence variation in the B1 gene among Toxoplasma gondii isolates from swine and cats in Italy.

    PubMed

    Santoro, Azzurra; Veronesi, Fabrizia; Milardi, Giovanni Luigi; Ranucci, David; Branciari, Raffaella; Diaferia, Manuela; Gabrielli, Simona

    2017-07-01

    The evaluation of the genetic variations of Toxoplasma gondii among isolates of a wide variety of animal hosts can provide significant information for better understanding the epidemiology and population structure of the parasite in different geographical areas. The aim of this study was to provide information on T. gondii genetic diversity in host species living in central Italy, which could act as a potential source of human infection. Seventy-seven feline faecal samples, and 36 and 20 diaphragm pillar tissue samples from pigs and wild boars were collected in Umbria (central Italy). The samples were tested by a nested-PCR protocol amplifying an informative region within the B1 gene, a multi-copy genetic target, showing a good rate of variability. Thirty-six specimens (27.07%) belonging to 10 pigs, 13 wild boars and 13 cats, tested positive to the B1 nested-PCR screening. Of these, 23 good quality sequences (8 from wild boars, 5 from pigs, and 10 from cats) were analyzed. A comparison of the B1 DNA sequences showed that a single homogeneous nucleotide substitution (C/T) was present at position 31 in the isolates from pigs and wild boars compared with the sampled cats and other hosts (including humans) available in GenBank™. The present results suggest the existence of a T. gondii genetic diversity for swine host species, based on a SNP (C/T) of the B1 gene. Further studies are needed to draw more solid conclusions on the discriminatory power of the B1 target by collecting more swine samples from much broader geographical areas. Copyright © 2017. Published by Elsevier Ltd.

  4. 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

  5. Roles of catalase (CAT) and ascorbate peroxidase (APX) genes in stress response of eggplant (Solanum melongena L.) against Cu(+2) and Zn(+2) heavy metal stresses.

    PubMed

    Soydam-Aydın, Semra; Büyük, İlker; Cansaran-Duman, Demet; Aras, Sümer

    2015-12-01

    Eggplant (Solanum melongena L.) is a good source of minerals and vitamins and this feature makes its value comparable with tomato which is economically the most important vegetable worldwide. Due to its common usage as food and in medicines, eggplant cultivation has a growing reputation worldwide. But genetic yield potential of an eggplant variety is not always attained, and it is limited by some factors such as heavy metal contaminated soils in today's world. Today, one of the main objectives of plant stress biology and agricultural biotechnology areas is to find the genes involved in antioxidant stress response and engineering the key genes to improve the plant resistance mechanisms. In this regard, the current study was conducted to gain an idea on the roles of catalase (CAT) and ascorbate peroxidase (APX) genes in defense mechanism of eggplant (S. melongena L., Pala-49 (Turkish cultivar)) treated with different concentrations of Cu(+2) and Zn(+2). For this aim, the steady-state messenger RNA (mRNA) levels of CAT and APX genes were determined by quantitative real-time PCR (qRT-PCR) in stressed eggplants. The results of the current study showed that different concentrations of Cu(+2) and Zn(+2) stresses altered the mRNA levels of CAT and APX genes in eggplants compared to the untreated control samples. When the mRNA levels of both genes were compared, it was observed that CAT gene was more active than APX gene in eggplant samples subjected to Cu(+2) contamination. The current study highlights the importance of CAT and APX genes in response to Cu(+2) and Zn(+2) heavy metal stresses in eggplant and gives an important knowledge about this complex interaction.

  6. Functional identification of the promoter for the gene encoding the alpha subunit of calcium/calmodulin-dependent protein kinase II.

    PubMed Central

    Olson, N J; Massé, T; Suzuki, T; Chen, J; Alam, D; Kelly, P T

    1995-01-01

    To examine the expression of the alpha subunit of calcium/calmodulin-dependent protein kinase II, various 5' flanking genomic sequences were inserted into a chloramphenicol acetyltransferase (CAT) reporter plasmid and CAT enzyme activities were analyzed in transfected NB2a neuroblastoma cells and mRNA transcription was analyzed by nuclease protection assays. A core promoter was identified which contained an essential TATA element located 162 nt 5' to the transcription start site. Sequences 3' to the transcription start site, as well as 5' to the TATA element, increased levels of CAT activity in transfected cells. The alpha-subunit gene promoter displayed higher CAT activities, relative to a simian virus 40 promoter, in transfected neuronal cell lines than in nonneuronal cell lines. Results also suggested that sequence surrounding the natural alpha-gene transcription initiation site may be important for targeting transcription initiation 162 nt downstream of its TATA element. Images Fig. 1 Fig. 3 PMID:7878035

  7. Crossed and uncrossed projections to the cat sacrocaudal spinal cord: III. Axons expressing calcitonin gene-related peptide immunoreactivity.

    PubMed

    Ritz, L A; Murray, C R; Foli, K

    2001-10-01

    We have investigated the projection patterns of peptidergic small-diameter primary afferent fibers to the cat sacrocaudal spinal cord, a region associated with midline structures of the lower urogenital system and of the tail. Calcitonin gene-related peptide (CGRP)-immunoreactive (CGRP-IR) primary afferent fibers were observed within the superficial laminae, rostrally as the typical inverted U-shaped band that capped the separate dorsal horns (S1 to rostral S2) and caudally as a broad band that spanned the entire mediolateral extent of the fused dorsal horns (caudal S2 and caudal). Within the dorsal gray commissure, labeling was seen as a periodic vertical, midline band. CGRP-IR labeling was prevalent in an extensive mediolateral distribution at the base of the dorsal horn, originating from both lateral and medial collateral bundles that extend from the superficial dorsal horn. Some bundles, in part traveling within the dorsal commissure, conspicuously crossed the midline. In addition to the robust projection to the superficial dorsal horn, there was a more extensive distribution of CGRP-IR fibers within the deeper portions of the cat sacrocaudal dorsal horn than has been reported for other regions of the cat spinal cord. Presumably, these deep projections convey visceral information to projection or segmental neurons at the neck of the dorsal horn and in the region of the central canal. This deep distribution overlaps the reported projections of the pelvic and pudendal nerves. In addition, the contralateral projections of CGRP-IR fibers may form an anatomical substrate of the bilateral receptive fields for selective dorsal horn neurons. The density and variety of CGRP-IR projection patterns is a reflection of the functional attributes of the innervated structures.

  8. Infection of cats with atypical feline coronaviruses harbouring a truncated form of the canine type I non-structural ORF3 gene.

    PubMed

    Le Poder, Sophie; Pham-Hung d'Alexandry d'Orangiani, Anne-Laure; Duarte, Lidia; Fournier, Annie; Horhogea, Cristina; Pinhas, Carine; Vabret, Astrid; Eloit, Marc

    2013-12-01

    Feline and canine coronaviruses (FCoV and CCoV, respectively) are common pathogens of cats and dogs sometimes leading to lethal infections named feline infectious peritonitis (FIP) and canine pantropic coronavirus infection. FCoV and CCoV are each subdivided into two serotypes, FCoV-I/II and CCoV-I/II. A phylogenetic relationship is evident between, on one hand, CCoV-I/FCoV-I, and on the other hand, CCoV-II/FCoV-II, suggesting that interspecies transmission can occur. The aim of the present study was to evaluate the prevalence of coronavirus (CoV)-infected cats according to their contact with dogs and to genetically analyse the CoV strains infecting cats. From 2003 to 2009, we collected 88 faecal samples from healthy cats and 11 ascitic fluids from FIP cats. We investigated the possible contact with dog in the household and collected dogs samples if appropriate. Out of 99 cat samples, 26 were coronavirus positive, with six cats living with at least one dog, thus showing that contact with dogs does not appear as a predisposing factor for cats CoV infections. Molecular and phylogenetic analyses of FCoV strains were conducted using partial N and S sequences. Six divergent strains were identified with the N gene clustering with CCoV-I whereas the 3' end of S was related to FCoV-I. Further analysis on those six samples was attempted by researching the presence of the ORF3 gene, the latter being peculiar to CCoV-I to date. We succeeded to amplify the ORF3 gene in five samples out of six. Thus, our data strongly suggest the circulation of atypical FCoV strains harbouring the CCoV-I ORF3 gene among cats. Moreover, the ORF3 genes recovered from the feline strains exhibited shared deletions, never described before, suggesting that these deletions could be critical in the adaptation of these strains to the feline host. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

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

    DOEpatents

    Stephens, David S [Stone Mountain, GA; Gudlavalleti, Seshu K [Kensington, MD; Tzeng, Yih-Ling [Atlanta, GA; Datta, Anup K [San Diego, CA; Carlson, Russell W [Athens, GA

    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.

  10. Insights into the Specificity of Lysine Acetyltransferases

    SciTech Connect

    Tucker, Alex C.; Taylor, Keenan C.; Rank, Katherine C.; Rayment, Ivan; Escalante-Semerena, Jorge C.

    2014-11-07

    Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. In this paper, we report the structure of a GNAT in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Finally, our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs.

  11. Genetic basis of the association of resistance genes mef(I) (macrolides) and catQ (chloramphenicol) in streptococci.

    PubMed

    Mingoia, Marina; Morici, Eleonora; Brenciani, Andrea; Giovanetti, Eleonora; Varaldo, Pietro E

    2014-01-01

    In streptococci mef(I) and catQ, two relatively uncommon macrolide and chloramphenicol resistance genes, respectively, are typically linked in a genetic module designated IQ module. Though variable, the module consistently encompasses, and is sometimes reduced to, a conserved ∼5.8-kb mef(I)-catQ fragment. The prototype IQ module was described in Streptococcus pneumoniae. IQ-like modules have subsequently been detected in Streptococcus pyogenes and in different species of viridans group streptococci, where mef(E) may be found instead of mef(I). Three genetic elements, one carrying the prototype IQ module from S. pneumoniae and two carrying different, defective IQ modules from S. pyogenes, have recently been characterized. All are integrative and conjugative elements (ICEs) belonging to the Tn5253 family, and have been designated ICESpn529IQ, ICESpy029IQ and ICESpy005IQ, respectively. ICESpy029IQ and ICESpy005IQ were the first Tn5253 family ICEs to be described in S. pyogenes. A wealth of new information has been obtained by comparing their genetic organization, chromosomal integration, and transferability. The origin of the IQ module is unknown. The mechanism by which it spreads in streptococci is discussed.

  12. [Fur color gene profiles and length of hair of several cat populations in Cuba, Costa Rica, Colombia, Paraguay, Chile and Argentina, and possible genetic origins of these cat populations].

    PubMed

    Ruiz-Garsia, M; Campos, H A; Alvaréz, D; Kajon, A; Diáz, S

    2002-02-01

    Until the present moment, only a scarce number of Latin American domestic cat populations have been studied from a population genetic standpoint. For this reason, the cat populations of La Havana (Cuba), San José (Costa Rica), Bogota and Ibagué (Colombia), Asuncion (Paraguay), Santiago (Chile) and Buenos Aires (Argentina) were sampled for several coat genes. The results obtained were as follows: (1) there was a strong genetic resemblance between several Hispanic American cat populations (especially, those of Buenos Aires, San José and the two Colombian populations studied) and those from South Western United States (California, Texas and Colorado), which adds support to the suspicion that these populations probably have a common origin; (2) The cat population of Santiago (Chile), contrarily to the other Hispanic American populations studied, showed a strong genetic resemblance with some Anglo North American populations; and (3) The l (long hair) and d (dilution) alleles showed systematic higher frequencies in the Hispanic American populations than those observed in Spain. Although the Hispanic American populations were not identical to the current Spanish populations (with the exception of Asuncion), this historic genetic experiment was very different to that found for the British populations and their overseas colonies.

  13. Feline infectious peritonitis virus with a large deletion in the 5'-terminal region of the spike gene retains its virulence for cats.

    PubMed

    Terada, Yutaka; Shiozaki, Yuto; Shimoda, Hiroshi; Mahmoud, Hassan Youssef Abdel Hamid; Noguchi, Keita; Nagao, Yumiko; Shimojima, Masayuki; Iwata, Hiroyuki; Mizuno, Takuya; Okuda, Masaru; Morimoto, Masahiro; Hayashi, Toshiharu; Tanaka, Yoshikazu; Mochizuki, Masami; Maeda, Ken

    2012-09-01

    In this study, the Japanese strain of type I feline infectious peritonitis virus (FIPV), C3663, was found to have a large deletion of 735 bp within the gene encoding the spike (S) protein, with a deduced loss of 245 aa of the N-terminal region of the S protein. This deletion is similar to that observed in porcine respiratory coronavirus (PRCoV) when compared to transmissible gastroenteritis virus, which correlates with reduced virulence. By analogy to PRCoV, we expected that the pathogenicity of C3663 may be attenuated in cats. However, two of four cats inoculated with C3663 died of FIP, and a third C3663-inoculated cat showed FIP lesions at 91 days after challenge. These results indicate that the 5'-terminal region of the S gene is not essential for the development of FIP.

  14. Determination of the 'critical region' for cat-like cry of Cri-du-chat syndrome and analysis of candidate genes by quantitative PCR.

    PubMed

    Wu, Qingfa; Niebuhr, Erik; Yang, Huanming; Hansen, Lars

    2005-04-01

    Cri-du-chat (CDC, OMIM 123450) is a chromosomal syndrome that results from partial deletions on the short arm of chromosome 5. The clinical features of CDC normally include high-pitched cat-like cry, mental retardation, microcephaly, hypertelorism and epicanthic folds. The cat-like cry is the most prominent clinical characteristic in newborn children and is usually considered as diagnostic for the CDC syndrome. Using a strategy of 'phenotype dissection', the critical region for cat-like cry was mapped to the chromosomal segment 5p15.3-5p15.2 in previous reports. In this study, the distal breakpoints of two interstitial deletions in two clinical distinctive CDC patients are analysed, one with and one without the cat-like cry. Using PCR, the critical region for the cat-like cry is mapped to a short 640 kbp region on chromosome 5p. Genome analysis of this critical region reveals a gene-rich sequence containing five known genes, five putative genes and three spliced EST sequences, altogether 71 predicted exons. Three genes, FLJ25076, a homolog to a ubiquitin-conjugating enzyme UBC-E2, FLJ20303, a nucleolar protein NOP2, which may play a role in the regulation of the cell cycle and MGC5309, a protein with similarity to Nut2, a Drosophila transcriptional coactivator, have been characterized and expression profiles determined by quantitative PCR. These results suggest that one candidate gene, FLJ25076, encodes a ubiquitin-conjugated enzyme E2 type, which is locally expressed in thoracic and scalp tissues. The other two genes are expressed uniformly in all tissues tested, which suggest that they are housekeeping genes.

  15. A polymorphism in the melanocortin 4 receptor gene (MC4R:c.92C>T) is associated with diabetes mellitus in overweight domestic shorthaired cats.

    PubMed

    Forcada, Y; Holder, A; Church, D B; Catchpole, B

    2014-01-01

    Feline diabetes mellitus (DM) shares many pathophysiologic features with human type 2 DM. Human genome-wide association studies have identified genes associated with obesity and DM, including melanocortin 4 receptor (MC4R), which plays an important role in energy balance and appetite regulation. To identify single nucleotide polymorphisms (SNPs) in the feline MC4R gene and to determine whether any SNPs are associated with DM or overweight body condition in cats. Two-hundred forty domestic shorthaired (DSH) cats were recruited for the study. Of these, 120 diabetics were selected (60 overweight, 60 lean), along with 120 nondiabetic controls (60 overweight and 60 lean). Males and females were equally represented. A prospective case-control study was performed. Genomic DNA was extracted from blood samples and used as template for PCR amplification of the feline MC4R gene. The coding region of the gene was sequenced in 10 cats to identify polymorphisms. Subsequently, genotyping by restriction fragment length polymorphism (RFLP) analysis assessed MC4R:c.92C > T allele and genotype frequencies in each group of cats. No significant differences in MC4R:c.92C>T allele or genotype frequencies were identified between nondiabetic overweight and lean cats. In the overweight diabetic group, 55% were homozygous for the MC4R:c.92C allele, compared to 33% of the lean diabetics and 30% of the nondiabetics. The differences between the overweight diabetic and the nondiabetics were significant (P < .01). We identified a polymorphism in the coding sequence of feline MC4R that is associated with DM in overweight DSH cats, similar to the situation in humans. Copyright © 2013 by the American College of Veterinary Internal Medicine.

  16. Phylogenetic and biological investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family among fungi

    USDA-ARS?s Scientific Manuscript database

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes well-characterized in several bacteria and eukaryotic organisms. The role of NATs in fungal biology has only recently been investigated. The NAT1 (FDB2) gene of Fusarium verticillioides was the first NAT cloned and character...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  18. State of cat genomics.

    PubMed

    O'Brien, Stephen J; Johnson, Warren; Driscoll, Carlos; Pontius, Joan; Pecon-Slattery, Jill; Menotti-Raymond, Marilyn

    2008-06-01

    Our knowledge of cat family biology was recently expanded to include a genomics perspective with the completion of a draft whole genome sequence of an Abyssinian cat. The utility of the new genome information has been demonstrated by applications ranging from disease gene discovery and comparative genomics to species conservation. Patterns of genomic organization among cats and inbred domestic cat breeds have illuminated our view of domestication, revealing linkage disequilibrium tracks consequent of breed formation, defining chromosome exchanges that punctuated major lineages of mammals and suggesting ancestral continental migration events that led to 37 modern species of Felidae. We review these recent advances here. As the genome resources develop, the cat is poised to make a major contribution to many areas in genetics and biology.

  19. Sequence variation in mitochondrial cox1 and nad1 genes of ascaridoid nematodes in cats and dogs from Iran.

    PubMed

    Mikaeili, F; Mirhendi, H; Mohebali, M; Hosseini, M; Sharbatkhori, M; Zarei, Z; Kia, E B

    2015-07-01

    The study was conducted to determine the sequence variation in two mitochondrial genes, namely cytochrome c oxidase 1 (pcox1) and NADH dehydrogenase 1 (pnad1) within and among isolates of Toxocara cati, Toxocara canis and Toxascaris leonina. Genomic DNA was extracted from 32 isolates of T. cati, 9 isolates of T. canis and 19 isolates of T. leonina collected from cats and dogs in different geographical areas of Iran. Mitochondrial genes were amplified by polymerase chain reaction (PCR) and sequenced. Sequence data were aligned using the BioEdit software and compared with published sequences in GenBank. Phylogenetic analysis was performed using Bayesian inference and maximum likelihood methods. Based on pairwise comparison, intra-species genetic diversity within Iranian isolates of T. cati, T. canis and T. leonina amounted to 0-2.3%, 0-1.3% and 0-1.0% for pcox1 and 0-2.0%, 0-1.7% and 0-2.6% for pnad1, respectively. Inter-species sequence variation among the three ascaridoid nematodes was significantly higher, being 9.5-16.6% for pcox1 and 11.9-26.7% for pnad1. Sequence and phylogenetic analysis of the pcox1 and pnad1 genes indicated that there is significant genetic diversity within and among isolates of T. cati, T. canis and T. leonina from different areas of Iran, and these genes can be used for studying genetic variation of ascaridoid nematodes.

  20. One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells

    SciTech Connect

    Protic-Sabljic, M.; Kraemer, K.H.

    1985-10-01

    The authors have developed a host cell reactivation assay of DNA repair utilizing UV-treated plasmid vectors. The assay primarily reflects cellular repair of transcriptional activity of damaged DNA measured indirectly as enzyme activity of the transfected genes. They studied three plasmids (pSV2cat, 5020 base pairs; pSV2catSVgpt, 7268 base pairs; and pRSVcat, 5027 base pairs) with different sizes and promoters carrying the bacterial cat gene (CAT, chloramphenicol acetyltransferase) in a construction that permits cat expression in human cells. All human simian virus 40-transformed cells studied expressed high levels of the transfected cat gene. UV treatment of the plasmids prior to transfection resulted in differential decrease in CAT activity in different cell lines. With pSV2catSVgpt, UV inactivation of CAT expression was greater in the xeroderma pigmentosum group A and D lines than in the other human cell lines tested. The D0 of the CAT inactivation curve was 50 J X m-2 for pSV2cat and for pRSVcat in the xeroderma pigmentosum group A cells. The similarity of the D0 data in the xeroderma pigmentosum group A cells for three plasmids of different size and promoters implies they all have similar UV-inactivation target size. UV-induced pyrimidine dimer formation in the plasmids was quantified by assay of the number of UV-induced T4 endonuclease V-sensitive sites. In the most sensitive xeroderma pigmentosum cells, with all three plasmids, one UV-induced pyrimidine dimer inactivates a target of about 2 kilobases, close to the size of the putative CAT mRNA.

  1. Feline infectious peritonitis: role of the feline coronavirus 3c gene in intestinal tropism and pathogenicity based upon isolates from resident and adopted shelter cats.

    PubMed

    Pedersen, Niels C; Liu, Hongwei; Scarlett, Jennifer; Leutenegger, Christian M; Golovko, Lyudmila; Kennedy, Heather; Kamal, Farina Mustaffa

    2012-04-01

    Feline infectious peritonitis virus (FIPV) was presumed to arise from mutations in the 3c of a ubiquitous and largely nonpathogenic feline enteric coronavirus (FECV). However, a recent study found that one-third of FIPV isolates have an intact 3c and suggested that it is not solely involved in FIP but is essential for intestinal replication. In order to confirm these assumptions, 27 fecal and 32 FIP coronavirus isolates were obtained from resident or adopted cats from a large metropolitan shelter during 2008-2009 and their 3a-c, E, and M genes sequenced. Forty percent of coronavirus isolates from FIP tissues had an intact 3c gene, while 60% had mutations that truncated the gene product. The 3c genes of fecal isolates from healthy cats were always intact. Coronavirus from FIP diseased tissues consistently induced FIP when given either oronasally or intraperitoneally (i.p.), regardless of the functional status of their 3c genes, thus confirming them to be FIPVs. In contrast, fecal isolates from healthy cats were infectious following oronasal infection and shed at high levels in feces without causing disease, as expected for FECVs. Only one in three cats shed FECV in the feces following i.p. infection, indicating that FECVs can replicate systemically, but with difficulty. FIPVs having a mutated 3c were not shed in the feces following either oronasal or i.p. inoculation, while FIPVs with intact 3c genes were shed in the feces following oronasal but not i.p. inoculation. Therefore, an intact 3c appears to be essential for intestinal replication. Although FIPVs with an intact 3c were shed in the feces following oronasal inoculation, fecal virus from these cats was not infectious for other cats. Attempts to identify potential FIP mutations in the 3a, 3b, E, and M were negative. However, the 3c gene of FIPVs, even though appearing intact, contained many more non-synonymous amino acid changes in the 3' one-third of the 3c protein than FECVs. An attempt to trace FIPV

  2. Phylogenetic studies of pantherine cats (Felidae) based on multiple genes, with novel application of nuclear beta-fibrinogen intron 7 to carnivores.

    PubMed

    Yu, Li; Zhang, Ya-Ping

    2005-05-01

    The pantherine lineage of the cat family Felidae (order: Carnivora) includes five big cats of genus Panthera and a great many midsized cats known worldwide. Presumably because of their recent and rapid radiation, the evolutionary relationship among pantherines remains ambiguous. We provide an independent assessment of the evolutionary history of pantherine lineage using two complete mitochondrial (mt) genes (ND2 and ND4) and the nuclear beta-fibrinogen intron 7 gene, whose utility in carnivoran phylogeny was first explored. The available four mt (ND5, cytb, 12S, and 16SrRNA) and two nuclear (IRBP and TTR) sequence loci were also combined to reconstruct phylogeny of 14 closely related cat species. Our analyses of combined mt data (six genes; approximately 3750 bp) and combined mt and nuclear data (nine genes; approximately 6500 bp) obtained identical tree topologies, which were well-resolved and strongly supported for almost all nodes. Monophyly of Panthera genus in pantherine lineage was confirmed and interspecific affinities within this genus revealed a novel branching pattern, with P. tigris diverging first in Panthera genus, followed by P. onca, P. leo, and last two sister species P. pardus and P. uncia. In addition, close association of Neofelis nebulosa to Panthera, the phylogenetic redefinition of Otocolobus manul within the domestic cat group, and the relatedness of Acinonyx jubatus and Puma concolor were all important findings in the resulting phylogenies. The potential utilities of nine different genes for phylogenetic resolution of closely related pantherine species were also evaluated, with special interest in that of the novel nuclear beta-fibrinogen intron 7.

  3. Chromosome 3p loss of heterozygosity and mutation analysis of the FHIT and beta-cat genes in squamous cell carcinoma of the head and neck.

    PubMed Central

    González, M V; Pello, M F; Ablanedo, P; Suárez, C; Alvarez, V; Coto, E

    1998-01-01

    AIMS: To study the loss of heterozygosity at the short arm of chromosome 3 in primary tumours from patients with squamous cell carcinoma of the head and neck; to determine whether the FHIT gene, mapped to 3p14.2 and the CTNNB1 (beta-cat) gene, mapped to 3p21, are deleted or mutated in these tumours. METHODS: DNA was extracted from fresh tumours. Loss of heterozygosity was assessed by microsatellite analysis of the following markers: D3S1283 and D3S1286 (3p24), D3S966 (3p21), and D3S1300 (3P14.2). Homozygous deletion was determined by radioactive multiplex polymerase chain reaction of exons 5 and 6 of the FHIT gene. The presence of mutations in FHIT exon 5 and beta-cat exon 3 was studied by single strand conformation polymorphism. RESULTS: 50% of informative cases (25/50) showed loss of heterozygosity for at least one of the 3p markers. 3p21 was the region with the highest rate of allelic deletion (63%). No point mutation was found in FHIT exon 5 or beta-cat exon 3. No case showed homozygous deletion for the FHIT (exons 5 and 6) or the beta-cat exon 3. CONCLUSIONS: The short arm of chromosome 3 is often deleted in the head and neck squamous cell carcinomas. In the remaining alleles of the FHIT or beta-cat genes, no evidence was found for point mutations or deletions, documented in other common carcinomas. Inactivation could occur by different mechanisms such as methylation, or other genes (not studied here) could be target of allelic losses in squamous cell carcinoma of the head and neck. Images PMID:9797729

  4. Scriptaid and 5-aza-2'deoxycytidine enhanced expression of pluripotent genes and in vitro developmental competence in interspecies black-footed cat cloned embryos.

    PubMed

    Gómez, M C; Biancardi, M N; Jenkins, J A; Dumas, C; Galiguis, J; Wang, G; Earle Pope, C

    2012-12-01

    Somatic cell nuclear transfer offers the possibility of preserving endangered species including the black-footed cat, which is threatened with extinction. The effectiveness and efficiency of somatic cell nuclear transfer (SCNT) depends on a variety of factors, but 'inappropriate epigenetic reprogramming of the transplanted nucleus is the primary cause of the developmental failure of cloned embryos. Abnormal epigenetic events such as DNA methylation and histone modifications during SCNT perturb the expression of imprinted and pluripotent-related genes that, consequently, may result in foetal and neonatal abnormalities. We have demonstrated that pregnancies can be established after transfer of black-footed cat cloned embryos into domestic cat recipients, but none of the implanted embryos developed to term and the foetal failure has been associated to aberrant reprogramming in cloned embryos. There is growing evidence that modifying the epigenetic pattern of the chromatin template of both donor cells and reconstructed embryos with a combination of inhibitors of histone deacetylases and DNA methyltransferases results in enhanced gene reactivation and improved in vitro and in vivo developmental competence. Epigenetic modifications of the chromatin template of black-footed cat donor cells and reconstructed embryos with epigenetic-modifying compounds enhanced in vitro development, and regulated the expression of pluripotent genes, but these epigenetic modifications did not improve in vivo developmental competence.

  5. Scriptaid and 5-aza-2'deoxycytidine enhanced expression of pluripotent genes and in vitro developmental competence in interspecies Black-footed cat cloned embryos

    USGS Publications Warehouse

    Gómez, M. C.; Biancardi, M.N.; Jenkins, J.A.; Dumas, C.; Galiguis, J.; Wang, G.; Earle Pope, C.

    2012-01-01

    Somatic cell nuclear transfer offers the possibility of preserving endangered species including the black-footed cat, which is threatened with extinction. The effectiveness and efficiency of somatic cell nuclear transfer (SCNT) depends on a variety of factors, but 'inappropriate epigenetic reprogramming of the transplanted nucleus is the primary cause of the developmental failure of cloned embryos. Abnormal epigenetic events such as DNA methylation and histone modifications during SCNT perturb the expression of imprinted and pluripotent-related genes that, consequently, may result in foetal and neonatal abnormalities. We have demonstrated that pregnancies can be established after transfer of black-footed cat cloned embryos into domestic cat recipients, but none of the implanted embryos developed to term and the foetal failure has been associated to aberrant reprogramming in cloned embryos. There is growing evidence that modifying the epigenetic pattern of the chromatin template of both donor cells and reconstructed embryos with a combination of inhibitors of histone deacetylases and DNA methyltransferases results in enhanced gene reactivation and improved in vitro and in vivo developmental competence. Epigenetic modifications of the chromatin template of black-footed cat donor cells and reconstructed embryos with epigenetic-modifying compounds enhanced in vitro development, and regulated the expression of pluripotent genes, but these epigenetic modifications did not improve in vivo developmental competence.

  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. Detection of feline calicivirus (FCV) from vaccinated cats and phylogenetic analysis of its capsid genes.

    PubMed

    Ohe, K; Sakai, S; Sunaga, F; Murakami, M; Kiuchi, A; Fukuyama, M; Furuhata, K; Hara, M; Soma, T; Ishikawa, Y; Taneno, A

    2006-04-01

    We analysed genogroups of four feline calcivirus (FCV) isolates (FCV-S, H10, Ao198-1 and ML89) obtained from cats that experienced FCV infection after having been vaccinated against FCV. New PCR primer sets (8F/8R, Ao-S/Ao-A, cp-S/cp-A) were also designed, since the conventional Seal primer failed to amplify the target sequences in two samples. The genogroups of the four isolates as well as eight global and 17 domestic strains were determined by phylogenetic analysis of their amino acid sequences. One out of the four strains (25%) isolated in this study, H10, was grouped into genogroup I, along with the vaccine strains F9 and FCV-255. The other three isolates (75%) belonged to genogroup II. Thus, there were more isolates in genogroup II than in genogroup I. However, the antibody values of the four isolates against cat anti-F9 antisera were significantly decreased. There may be no relationship between the neutralizing antibody titre and genogroup. Amino acid sequence alignment of the four isolates showed that only a single amino acid in region C, which is involved in neutralization epitopes, was different in ML89 strain from that of F9. The other three strains, H10, Ao198-1 and FCV-B, shared the same amino acid sequence with F9. Alignment of amino acids for linear epitopes in the F9 strain, which are located at regions D and E, showed variations in 5' hypervariable region (HVR) of E, whereas D and conE had only synonymous substitutions i.e. no change in the amino acid sequence. This mutation in 5' HVR of region E suggested a vaccine breakdown, as the region is known to be essential for antigenicity. The genogroup II FCV is likely to be the cause of the FCV infection in this study, while the vaccine strains belong to genogroup I. Thus, the existing vaccine may need reevaluation for its effectiveness.

  8. Structure and mechanism of non-histone protein acetyltransferase enzymes

    PubMed Central

    Friedmann, David R.

    2014-01-01

    Post translational modification (PTM) of proteins is ubiquitous and mediates many cellular processes including intracellular localization, protein-protein interactions, enzyme activity, transcriptional regulation and protein stability. While the role of phosphorylation as a key PTM has been well studied, the more evolutionarily conserved acetylation PTM has only recently attracted attention as a key regulator of cellular events. Protein acetylation has been largely studied in the context of its role in histone modification and gene regulation, where histones are modified by histone acetyltransferases (HATs) to promote transcription. However, more recent acetylomic and biochemical studies have revealed that acetylation is mediated by a broader family of protein acetyltransferases (PATs). The recent structure determination of several PATs has provided a wealth of molecular information regarding structural features of PATs, their enzymatic mechanisms, their mode of substrate-specific recognition and their regulatory elements. In this minireview, we will briefly describe what is known about non-histone protein substrates, but mainly focus on a few recent structures of PATs to compare and contrast them with HATs to better understand the molecular basis for protein recognition and modification by this burgeoning family of protein modification enzymes. PMID:23742047

  9. Chemical inhibition of the histone acetyltransferase activity in Arabidopsis thaliana.

    PubMed

    Aquea, Felipe; Timmermann, Tania; Herrera-Vásquez, Ariel

    2017-01-29

    Chemical inhibition of chromatin regulators provides an effective approach to investigate the roles of chromatin modifications in plant and animals. In this work, chemical inhibition of the Arabidopsis histone acetyltransferase activity by γ-butyrolactone (MB-3), the inhibitor of the catalytic activity of mammalian GENERAL CONTROL NON-REPRESSIBLE 5 (GCN5) is evaluated. Arabidopsis seedlings were germinated in LS medium supplemented with different concentrations of MB-3, and inhibition in the root length and yellowed leaves were observed. The yellowed leaves phenotype of the plants grown in 100 μM of MB-3 was reverted when plants were additionally treated with 1 μM of TSA, a histone deacetylase inhibitor. Using an immunoblot assay with specific antibodies revealed a reduction of H3K14 acetylation levels at 3 and 24 h post-treatment. At 24 h post-treatment a reduction of H3K9 acetylation levels was observed. Targets of GCN5 related to stress were downregulated at 3 h post-treatment but no change was observed in target genes related to developmental transition. Our results indicate that MB-3 is a chemical inhibitor of the histone acetyltransferase in Arabidopsis and suggest that this inhibitor could function in other plants species. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Gene-gene interaction of GJB2, SOD2, and CAT on occupational noise-induced hearing loss in Chinese Han population.

    PubMed

    Wang, Sheng Li; Yu, Lu Gang; Liu, Ren Ping; Zhu, Wan Zhan; Gao, Wei Min; Xue, Li Ping; Jiang, Xu; Zhang, Ya Han; Yi, Ding; Chen, Dong; Zhang, Yong Hong

    2014-12-01

    The effects of genetic factors on the noise-induced hearing loss (NIHL) are still unclear. In the present study, eight single-nucleotide polymorphisms (SNPs) included rs1227049 and rs3802711 (CDH23), rs1695 (GSTP1), rs137852540 (GJB2), rs2289274 (PMCA2), rs4880 (SOD2), rs7943316, and rs769214 within CAT that might associated with NIHL were further validated in Chinese workers. The results showed that the carriers of the T allele (AT+TT) of rs7943316 and A allele (GA+AA) of rs769214, were significantly associated with an increased risk of NIHL compared to those with AA genotype (P<0.05) and GG genotype (P<0.05). Moreover, a significant three-locus model (P=0.0107) involving rs2016520, rs9794, and rs1805192 were observed that might associated with NIHL, with 53.95% of testing accuracy. Thus, our present study provided the evidence that GJB2, SOD2, and CAT genes might account for the NIHL development in independently and/or in an interactive manner.

  11. AAC(3)-XI, a New Aminoglycoside 3-N-Acetyltransferase from Corynebacterium striatum

    PubMed Central

    Galimand, Marc; Fishovitz, Jennifer; Lambert, Thierry; Barbe, Valérie; Zajicek, Jaroslav

    2015-01-01

    Corynebacterium striatum BM4687 was resistant to gentamicin and tobramycin but susceptible to kanamycin A and amikacin, a phenotype distinct among Gram-positive bacteria. Analysis of the entire genome of this strain did not detect any genes for known aminoglycoside resistance enzymes. Yet, annotation of the coding sequences identified 12 putative acetyltransferases or GCN5-related N-acetyltransferases. A total of 11 of these coding sequences were also present in the genomes of other Corynebacterium spp. The 12th coding sequence had 55 to 60% amino acid identity with acetyltransferases in Actinomycetales. The gene was cloned in Escherichia coli, where it conferred resistance to aminoglycosides by acetylation. The protein was purified to homogeneity, and its steady-state kinetic parameters were determined for dibekacin and kanamycin B. The product of the turnover of dibekacin was purified, and its structure was elucidated by high-field nuclear magnetic resonance (NMR), indicating transfer of the acetyl group to the amine at the C-3 position. Due to the unique profile of the reaction, it was designated aminoglycoside 3-N-acetyltransferase type XI. PMID:26149994

  12. Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus.

    PubMed Central

    Mollet, B; Knol, J; Poolman, B; Marciset, O; Delley, M

    1993-01-01

    Several pGEM5- and pUC19-derived plasmids containing a selectable erythromycin resistance marker were integrated into the chromosome of Streptococcus thermophilus at the loci of the lactose-metabolizing genes. Integration occurred via homologous recombination and resulted in cointegrates between plasmid and genome, flanked by the homologous DNA used for integration. Selective pressure on the plasmid-located erythromycin resistance gene resulted in multiple amplifications of the integrated plasmid. Release of this selective pressure, however, gave way to homologous resolution of the cointegrate structures. By integration and subsequent resolution, we were able to replace the chromosomal lacZ gene with a modified copy carrying an in vitro-generated deletion. In the same way, we integrated a promoterless chloramphenicol acetyltransferase (cat) gene between the chromosomal lacS and lacZ genes of the lactose operon. The inserted cat gene became a functional part of the operon and was expressed and regulated accordingly. Selective pressure on the essential lacS and lacZ genes under normal growth conditions in milk ensures the maintenance and expression of the integrated gene. As there are only minimal repeated DNA sequences (an NdeI site) flanking the inserted cat gene, it was stably maintained even in the absence of lactose, i.e., when grown on sucrose or glucose. The methodology represents a stable system in which to express and regulate foreign genes in S. thermophilus, which could qualify in the future for an application with food. Images PMID:8331064

  13. 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.

  14. A 211-bp enhancer of the rat uncoupling protein-1 (UCP-1) gene controls specific and regulated expression in brown adipose tissue.

    PubMed Central

    Cassard-Doulcier, A M; Gelly, C; Bouillaud, F; Ricquier, D

    1998-01-01

    The uncoupling protein-1 gene is uniquely expressed in brown adipose tissue (BAT) and is positively regulated by cold exposure of animals and the sympathetic nervous system. To analyse the importance of a previously identified 211-bp enhancer [Cassard-Doulcier, Gelly, Fox, Schrementi, Raimbault, Klaus, Forest, Bouillaud and Ricquier (1993) Mol. Endocrinol. 7, 497-506] in the tissue-specific expression of this gene, transgenic mice were generated using the chloramphenicol acetyltransferase (CAT) gene as a reporter gene. One out of fourteen lines of the control transgenic mice bearing the Herpes simplex thymidine kinase (TK) promoter expressed weakly the CAT reporter gene in several tissues, whereas the other lines did not express CAT. Eight founders bearing the 211-bp enhancer-TK transgene were obtained. In six lines, no expression of CAT was detected. In one line, the expression of CAT was restricted to BAT. In another line, the expression of CAT was found in BAT and, to a lesser extent, in testis. Moreover, in these lines a marked and specific increase in the expression of the reporter gene in BAT was observed either after exposure of mice to the cold or by treating them with a beta-adrenoceptor agonist drug. These results demonstrate that the 211-bp enhancer alone is sufficient to both direct and restrict expression to BAT. This enhancer also mediates the transcriptional response of the gene to beta-adrenergic stimulation, although it does not contain conserved cAMP response element. PMID:9657961

  15. ATF-1 transcription factor regulates the expression of ccg-1 and cat-1 genes in response to fludioxonil under OS-2 MAP kinase in Neurospora crassa.

    PubMed

    Yamashita, Kazuhiro; Shiozawa, Azusa; Watanabe, Setsuko; Fukumori, Fumiyasu; Kimura, Makoto; Fujimura, Makoto

    2008-12-01

    The ATF/CREB family transcriptional factors are regulated by stress-activated MAP kinase in yeast. The disruptants of the atf-1 gene, which encodes an ATF/CREB family transcriptional factor, were isolated and characterized in Neurospora crassa. The characteristic phenotypes in the os-2 MAP kinase strain, such as osmotic sensitivity and fludioxonil resistance, were not observed in the Deltaatf-1 strain; however, like the os-2 strain, up-regulation of the catalase gene cat-1 and the clock-controlled gene ccg-1 by treatment with fludioxonil (1 microg/mL) or 4% NaCl was almost completely abolished in the Deltaatf-1 strain. A gel shift assay indicated that ATF-1 bound to the cat-1 and ccg-1 promoters probably through the CRE motifs. The enzyme activity of large-subunit catalase CAT-1, the major conidial catalase, was not detected in the Deltaatf-1 strain, suggesting that the production of CAT-1 during formation of conidia is largely dependent on ATF-1. Among 11 clock-controlled genes, the expression of ccg-1, ccg-9, ccg-13, and ccg-14 was induced by fludioxonil in an OS-2-dependent manner; however, induction of ccg-13 and ccg-14 was observed in the Deltaatf-1 strain, suggesting the existence of another transcription factor regulated by OS-2. The homozygous cross between the Deltaatf-1 strains produced perithecia and ascospores; however, their ascospores never germinated. These findings suggest that ATF-1 acts as one of the transcriptional factors downstream of the OS-2 MAP kinase and probably regulates some genes involved in conidiation, circadian rhythm, and ascospore maturation in N. crassa.

  16. Post-Weaning Diet Affects Faecal Microbial Composition but Not Selected Adipose Gene Expression in the Cat (Felis catus)

    PubMed Central

    Bermingham, Emma N.; Kittelmann, Sandra; Young, Wayne; Kerr, Katherine R.; Swanson, Kelly S.; Roy, Nicole C.; Thomas, David G.

    2013-01-01

    The effects of pre- (i.e., gestation and during lactation) and post-weaning diet on the composition of faecal bacterial communities and adipose expression of key genes in the glucose and insulin pathways were investigated in the cat. Queens were maintained on a moderate protein:fat:carbohydrate kibbled (“Diet A”; 35:20:28% DM; n  =  4) or high protein:fat:carbohydrate canned (“Diet B”; 45:37:2% DM; n = 3) diet throughout pregnancy and lactation. Offspring were weaned onto these diets in a nested design (n  =  5 per treatment). Faecal samples were collected at wk 8 and 17 of age. DNA was isolated from faeces and bacterial 16S rRNA gene amplicons were analysed by pyrosequencing. RNA was extracted from blood (wk 18) and adipose tissue and ovarian/testicular tissues (wk 24) and gene expression levels determined using RT-qPCR. Differences (P<0.05) in composition of faecal bacteria were observed between pregnant queens fed Diet A or B. However, pre-weaning diet had little effect on faecal bacterial composition in weaned kittens. In contrast, post-weaning diet altered bacterial population profiles in the kittens. Increased (P<0.05) abundance of Firmicutes (77% vs 52% of total reads) and Actinobacteria (0.8% vs 0.2% of total reads), and decreased (P<0.05) abundance of Fusobacteria (1.6% vs 18.4% of total reads) were observed for kittens fed the Diet A compared to those fed Diet B post-weaning. Feeding Diet B pre-weaning increased (P<0.05) the expression levels of INRS, LEPT, PAI-1 and tended to increase GLUT1, while the expression levels of IRS-1 in blood increased in kittens fed Diet A pre-weaning. Post-weaning diet had no effect on expression levels of target genes. Correlations between the expression levels of genes involved in glucose and insulin pathways and faecal Bacteriodetes and Firmicutes phyla were identified. The reasons for why post-weaning diet affects microbial populations and not gene expression levels are of interest. PMID:24312255

  17. Insights into the Specificity of Lysine Acetyltransferases

    DOE PAGES

    Tucker, Alex C.; Taylor, Keenan C.; Rank, Katherine C.; ...

    2014-11-07

    Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. In this paper, we report the structure of a GNATmore » in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Finally, our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs.« less

  18. Directed evolution of a histone acetyltransferase--enhancing thermostability, whilst maintaining catalytic activity and substrate specificity.

    PubMed

    Leemhuis, Hans; Nightingale, Karl P; Hollfelder, Florian

    2008-11-01

    Histone acetylation plays an integral role in the epigenetic regulation of gene expression. Transcriptional activity reflects the recruitment of opposing classes of enzymes to promoter elements; histone acetyltransferases (EC 2.3.1.48) that deposit acetyl marks at a subset of histone residues and histone deacetylases that remove them. Many histone acetyltransferases are difficult to study in solution because of their limited stability once purified. We have developed a directed evolution protocol that allows the screening of hundreds of histone acetyltransferase mutants for histone acetylating activity, and used this to enhance the thermostability of the human P/CAF histone acetyltransferase. Two rounds of directed evolution significantly stabilized the enzyme without lowering the catalytic efficiency and substrate specificity of the enzyme. Twenty-four variants with higher thermostability were identified. Detailed analysis revealed twelve single amino acid mutants that were found to possess a higher thermostability. The residues affected are scattered over the entire protein structure, and are different from mutations predicted by sequence alignment approaches, suggesting that sequence comparison and directed evolution methods are complementary strategies in engineering increased protein thermostability. The stabilizing mutations are predominately located at surface of the enzyme, suggesting that the protein's surface is important for stability. The directed evolution approach described in the present study is easily adapted to other histone modifying enzymes, requiring only appropriate peptide substrates and antibodies, which are available from commercial suppliers.

  19. [Phenotypic and genotypic characteristics of russian hairless cats].

    PubMed

    Zhigachev, A I; Vladimirova, M V; Katser, I Ia

    2000-04-01

    A novel mutation that causes the loss of hair was found in Russian cats. In contrast to hairless cats known in other countries (Sphinx cats of Canada, Great Britain, France, and Germany, etc.), in which the loss of hair is inherited as a monogenic recessive trait, in Russian hairless cats this trait is determined by a semidominant gene with the participation of other genes.

  20. Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknuellt protein

    SciTech Connect

    Yamaguchi, Masamitsu; Hirose, Fumiko; Nishida, Yasuyoshi; Matsukage, Akio )

    1991-10-01

    A 631-bp fragment containing the 5{prime}-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5{prime}-deletion derivatives revealed that the promoter function resided within a 192-bp region. Cotransfection with a zerknuellt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region fused with lacZ were established. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

  1. Identification of functional tag single nucleotide polmorphisms within the entire CAT gene and their clinical relevance in patients with noise-induced hearing loss

    PubMed Central

    Yang, Junhui; Zhang, Jieyuan; Wang, Xiaoming; Wang, Chaoyong; Chen, Jichuan; Qian, Yu; Duan, Zhaoxia

    2015-01-01

    Objectives: Noise-induced hearing loss (NIHL) is an important occupational disease which results from an interaction between genetic and environmental factors. More and more evidences suggested that Catalase (CAT) gene polymorphism plays an important role in the development of NIHL. The aim of this study was to investigate the association of CAT gene polymorphisms with NIHL in a case-control study. Design: A total of 719 unrelated adult Chinese Han population, including 225 healthy volunteers and 494 noise-exposed workers were recruited in this study. Six tag single-nucleotide polymorphisms (tSNPs) were genotyped using an improved multiplex ligation detection reaction technique. Subsequently, the interaction between noise exposure level and genotypes and their effect on NIHL were analyzed using logistic regression. Results: Among six tSNPs, two of them (rs208679 and rs769217) were significantly associated with noise exposure level. For rs208679 recessive effect, GG genotype had a significantly increased of NIHL risk in the exposure level of < 85 dB; and for rs769217 dominant effect, the combined genotypes TT/TC had a significantly increased of NIHL risk in the exposure level of 85 dB~92 dB; and the haplotype A-G-T-C-A-C had a risk effect on the NIHL in the exposure level of 85 dB~92 dB. In addition, the rs769217 polymorphism could enhance the transcription activities of the CAT gene. Conclusions: This study identified CAT is a NIHL susceptibility gene when noise exposure levels are taken into account. Rs208679 and rs769217 polymorphisms might be used as relevant risk estimates for the development of NIHL in population with different noise exposure levels. PMID:26045794

  2. Assays for mechanistic investigations of protein/histone acetyltransferases.

    PubMed

    Berndsen, Christopher E; Denu, John M

    2005-08-01

    Protein/histone acetyltransferases (PATs/HATs) have been implicated in a number of cellular functions including gene regulation, DNA synthesis, and repair. This paper reviews methods that can be used to quantitatively determine the activity and ultimately the catalytic/kinetic mechanism of PAT/HATs in vitro. Two methods will be described in detail. The first method is a filter-binding assay that measures the transfer of radiolabeled acetate from acetyl-CoA to protein. The second method is a continuous, spectroscopic, enzyme-coupled assay that links the PAT/HAT reaction to the reduction of NAD+ by pyruvate or alpha-ketoglutarate dehydrogenase. Both methods are highly applicable in determining steady-state reaction rates, and obtaining the kinetic constants Vmax, Km, and V/K from substrate saturation curves. We describe a new application of the filter-binding assay to determine the kinetic parameters for HATs using low concentrations of nucleosomal substrates.

  3. Influence of A-21T and C-262T genetic polymorphisms at the promoter region of the catalase (CAT) on gene expression.

    PubMed

    Saify, Khyber; Saadat, Iraj; Saadat, Mostafa

    2016-09-01

    Catalase (CAT, OMIM: 115500) is one of the major antioxidant enzymes, which plays an important role in the clearance of reactive oxygen species. Three genetic polymorphisms of A-21T (rs7943316), C-262T (rs1001179), and C-844T (rs769214) in the promoter region of the CAT have been reported. It has been suggested that these polymorphisms may alter the recognition sites of transcriptional factors, therefore it might be concluded that these polymorphisms may alter the expression levels of the gene. The aim of the present study is to evaluate the associations between these genetic variations and the CAT mRNA levels in human peripheral blood cells. The present study consisted of 47 healthy students of Shiraz University (south-west Iran). Genotypes of the CAT polymorphisms were determined by PCR based method. The quantitative CAT mRNA expression levels were investigated using quantitative real-time PCR. Analysis of variance revealed significant differences between the study genotypes (For A-21T polymorphism: F = 7.45; df = 2, 44; P = 0.002; For C-262T polymorphism: F = 15.17; df = 2, 44; P < 0.001). The studied polymorphisms showed linkage disequilibrium (D' = 1.0, r (2) = 0.1813, χ (2) = 17.03, P < 0.0001). The mRNA levels of CAT in the AC/TT, TC/TC, TC/TT, and TC/TC diplotypes significantly were higher than the mRNA levels in AC/AC diplotype. There was a significant difference between the study genotypes (F = 9.24; df = 5, 41; P < 0.001). The TC/TC and TT/TT diplotypes showed about 2 and 4 folds CAT mRNA levels compared with the AC/AC diplotype. The present findings indicated that these polymorphisms were significantly associated with the gene expression.

  4. Cat Batiks.

    ERIC Educational Resources Information Center

    Buban, Marcia H.

    1998-01-01

    Discusses an art activity where fourth-grade students created backgrounds using melted paraffin and a variety of paints for their cat batik/collage. Explains that after the students created their backgrounds, they assembled their paper cats for the collage using smaller shapes glued together and wax to add texture for fur. (CMK)

  5. Cat Batiks.

    ERIC Educational Resources Information Center

    Buban, Marcia H.

    1998-01-01

    Discusses an art activity where fourth-grade students created backgrounds using melted paraffin and a variety of paints for their cat batik/collage. Explains that after the students created their backgrounds, they assembled their paper cats for the collage using smaller shapes glued together and wax to add texture for fur. (CMK)

  6. Low genetic variation in the MHC class II DRB gene and MHC-linked microsatellites in endangered island populations of the leopard cat (Prionailurus bengalensis) in Japan.

    PubMed

    Saka, Toshinori; Nishita, Yoshinori; Masuda, Ryuichi

    2017-07-09

    Isolated populations of the leopard cat (Prionailurus bengalensis) on Tsushima and Iriomote islands in Japan are classified as subspecies P. b. euptilurus and P. b. iriomotensis, respectively. Because both populations have decreased to roughly 100, an understanding of their genetic diversity is essential for conservation. We genotyped MHC class II DRB exon 2 and MHC-linked microsatellite loci to evaluate the diversity of MHC genes in the Tsushima and Iriomote cat populations. We detected ten and four DRB alleles in these populations, respectively. A phylogenetic analysis showed DRB alleles from both populations to be closely related to those in other felid DRB lineages, indicating trans-species polymorphism. The MHC-linked microsatellites were more polymorphic in the Tsushima than in the Iriomote population. The MHC diversity of both leopard cat populations is much lower than in the domestic cat populations on these islands, probably due to inbreeding associated with founder effects, geographical isolation, or genetic drift. Our results predict low resistance of the two endangered populations to new pathogens introduced to the islands.

  7. Type B Chloramphenicol Acetyltransferases Are Responsible for Chloramphenicol Resistance in Riemerella anatipestifer, China

    PubMed Central

    Huang, Li; Yuan, Hui; Liu, Ma-Feng; Zhao, Xin-Xin; Wang, Ming-Shu; Jia, Ren-Yong; Chen, Shun; Sun, Kun-Feng; Yang, Qiao; Wu, Ying; Chen, Xiao-Yue; Cheng, An-Chun; Zhu, De-Kang

    2017-01-01

    Riemerella anatipestifer causes serositis and septicaemia in domestic ducks, geese, and turkeys. Traditionally, the antibiotics were used to treat this disease. Currently, our understanding of R. anatipestifer susceptibility to chloramphenicol and the underlying resistance mechanism is limited. In this study, the cat gene was identified in 69/192 (36%) R. anatipestifer isolated from different regions in China, including R. anatipestifer CH-2 that has been sequenced in previous study. Sequence analysis suggested that there are two copies of cat gene in this strain. Only both two copies of the cat mutant strain showed a significant decrease in resistance to chloramphenicol, exhibiting 4 μg/ml in the minimum inhibitory concentration for this antibiotic, but not for the single cat gene deletion strains. Functional analysis of the cat gene via expression in Escherichia coli BL21 (DE3) cells and in vitro site-directed mutagenesis indicated that His79 is the main catalytic residue of CAT in R. anatipestifer. These results suggested that chloramphenicol resistance of R. anatipestifer CH-2 is mediated by the cat genes. Finally, homology analysis of types A and B CATs indicate that R. anatipestifer comprises type B3 CATs. PMID:28298905

  8. Type B Chloramphenicol Acetyltransferases Are Responsible for Chloramphenicol Resistance in Riemerella anatipestifer, China.

    PubMed

    Huang, Li; Yuan, Hui; Liu, Ma-Feng; Zhao, Xin-Xin; Wang, Ming-Shu; Jia, Ren-Yong; Chen, Shun; Sun, Kun-Feng; Yang, Qiao; Wu, Ying; Chen, Xiao-Yue; Cheng, An-Chun; Zhu, De-Kang

    2017-01-01

    Riemerella anatipestifer causes serositis and septicaemia in domestic ducks, geese, and turkeys. Traditionally, the antibiotics were used to treat this disease. Currently, our understanding of R. anatipestifer susceptibility to chloramphenicol and the underlying resistance mechanism is limited. In this study, the cat gene was identified in 69/192 (36%) R. anatipestifer isolated from different regions in China, including R. anatipestifer CH-2 that has been sequenced in previous study. Sequence analysis suggested that there are two copies of cat gene in this strain. Only both two copies of the cat mutant strain showed a significant decrease in resistance to chloramphenicol, exhibiting 4 μg/ml in the minimum inhibitory concentration for this antibiotic, but not for the single cat gene deletion strains. Functional analysis of the cat gene via expression in Escherichia coli BL21 (DE3) cells and in vitro site-directed mutagenesis indicated that His79 is the main catalytic residue of CAT in R. anatipestifer. These results suggested that chloramphenicol resistance of R. anatipestifer CH-2 is mediated by the cat genes. Finally, homology analysis of types A and B CATs indicate that R. anatipestifer comprises type B3 CATs.

  9. Raccoonpox in a Canadian cat.

    PubMed

    Yager, Julie A; Hutchison, Lisa; Barrett, John W

    2006-12-01

    Poxvirus infections affecting the skin of cats are extremely rare in North America, in contrast to Europe where cowpox virus is well recognized as an accidental pathogen in cats that hunt small rodents. The virus or viruses responsible for the anecdotal cases in North America have never been characterized. This paper reports a case of raccoonpox infection in a Canadian cat. Biopsy of the initial ulcerative lesion on the forepaw revealed ballooning degeneration of surface and follicular keratinoctyes. Infected cells contained large eosinophilic type A inclusions. Electron microscopic examination revealed virions of an orthopoxvirus, subsequently identified as raccoonpox by polymerase chain reaction and gene sequencing. The cat made a full recovery.

  10. The Novel SLIK Histone Acetyltransferase Complex Functions in the Yeast Retrograde Response Pathway

    PubMed Central

    Pray-Grant, Marilyn G.; Schieltz, David; McMahon, Stacey J.; Wood, Jennifer M.; Kennedy, Erin L.; Cook, Richard G.; Workman, Jerry L.; Yates III, John R.; Grant, Patrick A.

    2002-01-01

    The SAGA complex is a conserved histone acetyltransferase-coactivator that regulates gene expression in Saccharomyces cerevisiae. SAGA contains a number of subunits known to function in transcription including Spt and Ada proteins, the Gcn5 acetyltransferase, a subset of TATA-binding-protein-associated factors (TAFIIs), and Tra1. Here we report the identification of SLIK (SAGA-like), a complex related in composition to SAGA. Notably SLIK uniquely contains the protein Rtg2, linking the function of SLIK to the retrograde response pathway. Yeast harboring mutations in both SAGA and SLIK complexes displays synthetic phenotypes more severe than those of yeast with mutation of either complex alone. We present data indicating that distinct forms of the SAGA complex may regulate specific subsets of genes and that SAGA and SLIK have multiple partly overlapping activities, which play a critical role in transcription by RNA polymerase II. PMID:12446794

  11. [Effects of grafting on cucumber leaf SOD and CAT gene expression and activities under low temperature stress].

    PubMed

    Gao, Jun-jie; Qin, Ai-guo; Yu, Xian-chang

    2009-01-01

    This paper studied the relative expression of Mn-SOD, Cu/Zn-SOD and CAT mRNAs and the changes of SOD, Mn-SOD, Cu/Zn-SOD and CAT activities in grafted and own-rooted cucumber leaves under low temperature stress, and their relations with the cold resistance of cucumber. For both grafted and own-rooted cucumber leaves, the relative expression of Mn-SOD and Cu/Zn-SOD mRNAs under low temperature stress was respectively accordance with the changes of Mn-SOD and Cu/Zn-SOD activities, while the expression of CAT mRNA was not accordance with the change of CAT activity. The relative expression of Mn-SOD and Cu/Zn-SOD mRNAs and the activities of SOD, Mn-SOD and Cu/Zn-SOD in grafted cucumber leaves were higher than those in own-rooted cucumber leaves, while the MDA content and electrolytic leakage were in adverse. The higher SOD activity regulated by the higher SOD mRNAs expression in grafted cucumber leaves might be the key factor of grafted cucumber having a higher cold resistance to low temperature stress than own-rooted cucumber. The relative expression of CAT mRNA was slightly higher in functional leaves but lower in young leaves of grafted cucumber, while less difference was observed in CAT activity, comparing with own-rooted cucumber, which illustrated that low temperature stress had lesser effects on the relative expression of CAT mRNA and the activity of CAT in grafted cucumber leaves.

  12. Regulation and function of histone acetyltransferase MOF.

    PubMed

    Yang, Yang; Han, Xiaofei; Guan, Jingyun; Li, Xiangzhi

    2014-03-01

    The mammalian MOF (male absent on the first), a member of the MYST (MOZ, YBF2, SAS2, and Tip60) family of histone acetyltransferases (HATs), is the major enzyme that catalyzes the acetylation of histone H4 on lysine 16. Acetylation of K16 is a prevalent mark associated with chromatin decondensation. MOF has recently been shown to play an essential role in maintaining normal cell functions. In this study, we discuss the important roles of MOF in DNA damage repair, apoptosis, and tumorigenesis. We also analyze the role of MOF as a key regulator of the core transcriptional network of embryonic stem cells.

  13. Bowenoid in situ carcinomas in two Devon Rex cats: evidence of unusually aggressive neoplasm behaviour in this breed and detection of papillomaviral gene expression in primary and metastatic lesions.

    PubMed

    Munday, John S; Benfell, Mike W; French, Adrienne; Orbell, Geoff M B; Thomson, Neroli

    2016-06-01

    Bowenoid in situ carcinomas (BISCs) are rare feline tumours that are thought to be caused by papillomavirus infection. Although they usually develop in old cats and are slowly progressive, multiple aggressive BISCs have been reported previously in a comparatively young Devon Rex cat. A 5-year-old (Case 1) and an 8-year-old (Case 2) Devon Rex cat developed numerous BISCs. Rapid progression resulted in euthanasia of both cats after 8 months. A postmortem examination was possible only for Case 2 and revealed pulmonary metastases. Consensus PCR amplified only Felis catus papillomavirus type 2 (FcaPV-2) DNA from lesions from both cats. High FcaPV-2 copy number and FcaPV-2 E6/E7 gene expression were detected in a BISC from Case 1. High FcaPV-2 copy number and FcaPV-2 gene expression were detected in a BISC, a cutaneous squamous cell carcinoma (SCC) and the pulmonary metastases from Case 2, but not in two other cutaneous SCCs. The results provide additional evidence that BISCs develop at a younger age in Devon Rex cats and that BISCs in Devon Rex cats have a more aggressive behaviour than BISCs in other cat breeds. These unusual features should be considered when evaluating and treating skin disease in Devon Rex cats. The detection of FcaPV-2 gene expression in the lung neoplasms suggests a potential role of FcaPV-2 in the development of metastatic disease. However, the absence of FcaPV-2 gene expression in two cutaneous SCCs suggests that other factors could have also promoted cancer development. © 2016 ESVD and ACVD.

  14. The histone acetyltransferase p300 promotes intrinsic axonal regeneration.

    PubMed

    Gaub, Perrine; Joshi, Yashashree; Wuttke, Anja; Naumann, Ulrike; Schnichels, Sven; Heiduschka, Peter; Di Giovanni, Simone

    2011-07-01

    Axonal regeneration and related functional recovery following axonal injury in the adult central nervous system are extremely limited, due to a lack of neuronal intrinsic competence and the presence of extrinsic inhibitory signals. As opposed to what occurs during nervous system development, a weak proregenerative gene expression programme contributes to the limited intrinsic capacity of adult injured central nervous system axons to regenerate. Here we show, in an optic nerve crush model of axonal injury, that adenoviral (cytomegalovirus promoter) overexpression of the acetyltransferase p300, which is regulated during retinal ganglion cell maturation and repressed in the adult, can promote axonal regeneration of the optic nerve beyond 0.5 mm. p300 acetylates histone H3 and the proregenerative transcription factors p53 and CCAAT-enhancer binding proteins in retinal ganglia cells. In addition, it directly occupies and acetylates the promoters of the growth-associated protein-43, coronin 1 b and Sprr1a and drives the gene expression programme of several regeneration-associated genes. On the contrary, overall increase in cellular acetylation using the histone deacetylase inhibitor trichostatin A, enhances retinal ganglion cell survival but not axonal regeneration after optic nerve crush. Therefore, p300 targets both the epigenome and transcription to unlock a post-injury silent gene expression programme that would support axonal regeneration.

  15. Astronomy CATS

    NASA Astrophysics Data System (ADS)

    Brissenden, Gina; Prather, Edward E.; Impey, Chris

    2012-08-01

    The Center for Astronomy Education's (CAE's) NSF-funded Collaboration of Astronomy Teaching Scholars (CATS) Program is a grassroots multi-institutional effort to increase the capacity for astronomy education research and improve science literacy in the United States.Our primary target population is the 500,000 college students who each year enroll in an introductory general education (a breadth requirement for non-science majors) Earth, Astronomy, and Space Science (EASS) course (Fraknoi 2001, AGI 2006).An equally important population for our efforts is the individuals who are, or will be, teaching these students. In this chapter, we will briefly discuss the goals of CAE and CATS, the varied personnel that make up the CATS collective, the diverse projects we've undertaken, and the many challenges we have had to work through to make CATS a success.

  16. NAT2*6A, a haplotype of the N-acetyltransferase 2 gene, is an important biomarker for risk of anti-tuberculosis drug-induced hepatotoxicity in Japanese patients with tuberculosis

    PubMed Central

    Higuchi, Norihide; Tahara, Naoko; Yanagihara, Katsunori; Fukushima, Kiyoyasu; Suyama, Naofumi; Inoue, Yuichi; Miyazaki, Yoshitsugu; Kobayashi, Tsutomu; Yoshiura, Koh-ichiro; Niikawa, Norio; Wen, Chun-Yang; Isomoto, Hajime; Shikuwa, Saburou; Omagari, Katsuhisa; Mizuta, Yohei; Kohno, Shigeru; Tsukamoto, Kazuhiro

    2007-01-01

    AIM: To investigate an association between N-acetyltransferase 2 (NAT2)-haplotypes/diplotypes and adverse effects in Japanese pulmonary tuberculosis patients. METHODS: We studied 100 patients with pulmonary TB treated with anti-TB drugs including INH. The frequencies and distributions of single nucleotide polymorphisms, haplotypes, and diplotypes of NAT2 were determined by the PCR-restriction fragment length polymorphism method, and the results were compared between TB patients with and without adverse effect, using multivariate logistic regression analysis. RESULTS: Statistical analysis revealed that the frequency of a variant haplotype, NAT2*6A, was significantly increased in TB patients with hepatotoxicity, compared with those without hepatotoxicity [P = 0.001, odds ratio (OR) = 3.535]. By contrast, the frequency of a wild-type (major) haplotype, "NAT2*4", was significantly lower in TB patients with hepatotoxicity than those without hepatotoxicity (P < 0.001, OR = 0.265). There was no association between NAT2-haplotypes and skin rash or eosinophilia. CONCLUSION: The present study shows that NAT2 is one of the determinants of anti-TB drug-induced hepatotoxicity. Moreover, the haplotypes, NAT2*4 and NAT2*6A, are useful new biomarkers for predicting anti-TB drug-induced hepatotoxicity. PMID:18023090

  17. Construction and use of a replication-competent human immunodeficiency virus (HIV-1) that expresses the chloramphenicol acetyltransferase enzyme.

    PubMed Central

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

    1989-01-01

    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. Images PMID:2726755

  18. Comparative genomic, phylogenetic, and functional investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family among fungi

    USDA-ARS?s Scientific Manuscript database

    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 (Glenn and Bacon, 2009; Glenn et al., 2010). The NAT1 gene of Gibberella moniliformis was the...

  19. Cat odor exposure induces distinct changes in the exploratory behavior and Wfs1 gene expression in C57Bl/6 and 129Sv mice.

    PubMed

    Raud, Sirli; Sütt, Silva; Plaas, Mario; Luuk, Hendrik; Innos, Jürgen; Philips, Mari-Anne; Kõks, Sulev; Vasar, Eero

    2007-10-16

    129Sv and C57Bl/6 (Bl6) strains are two most widely used inbred mice strains for generation of transgenic animals. The present study confirms the existence of substantial differences in the behavior of these two mice strains. The exploratory behavior of Bl6 mice in a novel environment was significantly higher compared to 129Sv mice. The exposure of mice to cat odor-induced an anxiety-like state in Bl6, but not in 129Sv mice. The levels of Wfs1 gene expression did not differ in the prefrontal cortex, mesolimbic area and temporal lobe of experimentally naive Bl6 and 129Sv mice. However, after cat odor exposure the expression of Wfs1 gene was significantly lower in the mesolimbic area and temporal lobe of Bl6 mice compared to 129Sv strain. Dynamics of Wfs1 gene expression and exploratory behavior suggest that the down-regulation of Wfs1 gene in Bl6 mice might be related to the increased anxiety. Further studies are needed to test the robustness and possible causal relationship of this finding.

  20. Determination of antibiotic resistance genes in relation to phylogenetic background in Escherichia coli isolates from fecal samples of healthy pet cats in Kerman city

    PubMed Central

    Akhtardanesh, Baharak; Ghanbarpour, Reza; Ganjalikhani, Sadaf; Gazanfari, Parisa

    2016-01-01

    The aim of this study was to determine antibiotic resistance genes, phylogenetic groups and anti-microbial resistance patterns of Escherichia coli isolates from fecal samples of healthy pet cats in Kerman city. Ninety E. coli isolates were recovered from obtained rectal swabs. Antibiotic resistance pattern of the isolates against seven selected antibiotic was determined using disc diffusion method. Phylogenetic background of the isolates was determined according to the presence of the chuA, yjaA and TspE4C2 markers. Theisolates were examined to determine a selection of antibiotic resistance genes including tetA, tetB, aadA, sulI and dhfrV by polymerase chain reaction. Forty two isolates (46.6%) were positive at least for one of the examined genes. Phylotyping revealed that the isolates are segregated in phylogenetic groups A (66.7%), B1 (1.2%), B2 (13.4%) and D (18.9%). Among 90 isolates, 26.6% were positive for tetB gene, 10.0% for cqnrS gene, 12.3% for sulI and aadA genes, 8.9% for tetA and 2.2% for dhfrVgene. None of the E. coli isolates were positive for qnrA and qnrB genes. Sixteen combination patterns of antibiotic resistance genes were identified which belonged to four phylogroups. Maximum and minimum resistant isolates were recorded against to tetracycline (82.3%) and gentamycin (1.2%), respectively. Fifteen antibiotic resistance patterns were determined in different phylo-genetic groups. In conclusion, feces of healthy pet cat in Kerman could be a source of antibiotic resistant E. coli isolates, whereas these isolates were distributed all over the main phylogroups. PMID:28144421

  1. Interspecies Transmission of Feline Immunodeficiency Virus from the Domestic Cat to the Tsushima Cat (Felis bengalensis euptilura) in the Wild

    PubMed Central

    Nishimura, Yoshiaki; Goto, Yuko; Yoneda, Kumiko; Endo, Yasuyuki; Mizuno, Takuya; Hamachi, Masaharu; Maruyama, Hiroyuki; Kinoshita, Hirotoshi; Koga, Susumu; Komori, Mitsuru; Fushuku, Seigo; Ushinohama, Kanji; Akuzawa, Masao; Watari, Toshihiro; Hasegawa, Atsuhiko; Tsujimoto, Hajime

    1999-01-01

    Feline immunodeficiency virus (FIV) was isolated from a wild-caught Tsushima cat (Felis bengalensis euptilura), an endangered Japanese nondomestic subspecies of leopard cat (F. bengalensis). Phylogenetic analysis of the env gene sequences indicated that the FIV from the Tsushima cat belonged to a cluster of subtype D FIVs from domestic cats. FIVs from both the Tsushima cat and the domestic cat showed similar levels of replication and cytopathicity in lymphoid cell lines derived from these two species. The results indicated the occurrence of interspecies transmission of FIV from the domestic cat to the Tsushima cat in the wild. PMID:10438892

  2. Interspecies transmission of feline immunodeficiency virus from the domestic cat to the Tsushima cat (Felis bengalensis euptilura) in the wild.

    PubMed

    Nishimura, Y; Goto, Y; Yoneda, K; Endo, Y; Mizuno, T; Hamachi, M; Maruyama, H; Kinoshita, H; Koga, S; Komori, M; Fushuku, S; Ushinohama, K; Akuzawa, M; Watari, T; Hasegawa, A; Tsujimoto, H

    1999-09-01

    Feline immunodeficiency virus (FIV) was isolated from a wild-caught Tsushima cat (Felis bengalensis euptilura), an endangered Japanese nondomestic subspecies of leopard cat (F. bengalensis). Phylogenetic analysis of the env gene sequences indicated that the FIV from the Tsushima cat belonged to a cluster of subtype D FIVs from domestic cats. FIVs from both the Tsushima cat and the domestic cat showed similar levels of replication and cytopathicity in lymphoid cell lines derived from these two species. The results indicated the occurrence of interspecies transmission of FIV from the domestic cat to the Tsushima cat in the wild.

  3. Multiple links between the NuA4 histone acetyltransferase complex and epigenetic control of transcription.

    PubMed

    Galarneau, L; Nourani, A; Boudreault, A A; Zhang, Y; Héliot, L; Allard, S; Savard, J; Lane, W S; Stillman, D J; Côté, J

    2000-06-01

    NuA4 is an essential histone H4/H2A acetyltransferase complex that interacts with activators and stimulates transcription in vitro. We have identified three novel NuA4 subunits: Act3/Arp4, an actin-related protein implicated in epigenetic control of transcription, Act1, and Epl1, a protein homologous to Drosophila Enhancer of Polycomb. Act3/Arp4 binds nucleosomes in vitro and is required for NuA4 integrity in vivo. Mutations in ACT3 and acetyltransferase-encoding ESA1 cause gene-specific transcription defects. Accordingly, NuA4 is localized in precise loci within the nucleus and does not overlap with the silent chromatin marker Sir3. These data along with the known epigenetic roles of Act3/Arp4 and homologs of Epl1 and Esa1 strongly support an essential role for chromatin structure modification by NuA4 in transcription regulation in vivo.

  4. The chromosomal 2'-N-acetyltransferase of Providencia stuartii: physiological functions and genetic regulation.

    PubMed

    Macinga, D R; Rather, P N

    1999-02-01

    Intrinsic chromosomal acetyltransferases involved in aminoglycoside resistance have been identified in a number of bacteria. In Providencia stuartii, a chromosomal acetyltransferase (AAC(2')-Ia) has been characterized in detail. In addition to the ability to acetylate aminoglycosides, the AAC(2')-Ia enzyme has at least one physiological function, which is the acetylation of peptidoglycan. This modification is likely to influence the autolytic system in P. stuartii. The regulation of aac(2')-Ia expression is extremely complex involving at least seven regulatory genes acting in at least two pathways. This complexity in regulation indicates that aac(2')-Ia expression must be tightly controlled in response to different environmental conditions. This presumably reflects the importance of maintaining correct levels of peptidoglycan acetylation. In this review, a summary of data will be presented involving both the physiological and genetic aspects of aac(2')-Ia in P. stuartii.

  5. Levels of Ancylostoma infections and phylogenetic analysis of cox 1 gene of A. ceylanicum in stray cat faecal samples from Guangzhou, China.

    PubMed

    Hu, W; Yu, X G; Wu, S; Tan, L P; Song, M R; Abdulahi, A Y; Wang, Z; Jiang, B; Li, G Q

    2016-07-01

    Ancylostoma ceylanicum is a common zoonotic nematode. Cats act as natural reservoirs of the hookworm and are involved in transmitting infection to humans, thus posing a potential risk to public health. The prevalence of feline A. ceylanicum in Guangzhou (South China) was surveyed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In total, 112 faecal samples were examined; 34.8% (39/112) and 43.8% (49/112) samples were positive with hookworms by microscopy and PCR method, respectively. Among them, 40.8% of samples harboured A. ceylanicum. Twelve positive A. ceylanicum samples were selected randomly and used for cox 1 sequence analysis. Sequencing results revealed that they had 97-99% similarity with A. ceylanicum cox 1 gene sequences deposited in GenBank. A phylogenetic tree showed that A. ceylanicum isolates were divided into two groups: one comprising four isolates from Guangzhou (South China), and the other comprising those from Malaysia, Cambodia and Guangzhou. In the latter group, all A. ceylanicum isolates from Guangzhou were clustered into a minor group again. The results indicate that the high prevalence of A. ceylanicum in stray cats in South China poses a potential risk of hookworm transmission from pet cats to humans, and that A. ceylanicum may be a species complex worldwide.

  6. MOZ and MORF acetyltransferases: Molecular interaction, animal development and human disease.

    PubMed

    Yang, Xiang-Jiao

    2015-08-01

    Lysine residues are subject to many forms of covalent modification and one such modification is acetylation of the ε-amino group. Initially identified on histone proteins in the 1960s, lysine acetylation is now considered as an important form of post-translational modification that rivals phosphorylation. However, only about a dozen of human lysine acetyltransferases have been identified. Among them are MOZ (monocytic leukemia zinc finger protein; a.k.a. MYST3 and KAT6A) and its paralog MORF (a.k.a. MYST4 and KAT6B). Although there is a distantly related protein in Drosophila and sea urchin, these two enzymes are vertebrate-specific. They form tetrameric complexes with BRPF1 (bromodomain- and PHD finger-containing protein 1) and two small non-catalytic subunits. These two acetyltransferases and BRPF1 play key roles in various developmental processes; for example, they are important for development of hematopoietic and neural stem cells. The human KAT6A and KAT6B genes are recurrently mutated in leukemia, non-hematologic malignancies, and multiple developmental disorders displaying intellectual disability and various other abnormalities. In addition, the BRPF1 gene is mutated in childhood leukemia and adult medulloblastoma. Therefore, these two acetyltransferases and their partner BRPF1 are important in animal development and human disease.

  7. Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates.

    PubMed

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

    2000-02-01

    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

  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. The histone acetyltransferase MOF overexpression blunts cardiac hypertrophy by targeting ROS in mice.

    PubMed

    Qiao, Weiwei; Zhang, Weili; Gai, Yusheng; Zhao, Lan; Fan, Juexin

    2014-06-13

    Imbalance between histone acetylation/deacetylation critically participates in the expression of hypertrophic fetal genes and development of cardiac hypertrophy. While histone deacetylases play dual roles in hypertrophy, current evidence reveals that histone acetyltransferase such as p300 and PCAF act as pro-hypertrophic factors. However, it remains elusive whether some histone acetyltransferases can prevent the development of hypertrophy. Males absent on the first (MOF) is a histone acetyltransferase belonging to the MYST (MOZ, Ybf2/Sas3, Sas2 and TIP60) family. Here in this study, we reported that MOF expression was down-regulated in failing human hearts and hypertrophic murine hearts at protein and mRNA levels. To evaluate the roles of MOF in cardiac hypertrophy, we generated cardiac-specific MOF transgenic mice. MOF transgenic mice did not show any differences from their wide-type littermates at baseline. However, cardiac-specific MOF overexpression protected mice from transverse aortic constriction (TAC)-induced cardiac hypertrophy, with reduced radios of heart weight (HW)/body weight (BW), lung weight/BW and HW/tibia length, decreased left ventricular wall thickness and increased fractional shortening. We also observed lower expression of hypertrophic fetal genes in TAC-challenged MOF transgenic mice compared with that of wide-type mice. Mechanically, MOF overexpression increased the expression of Catalase and MnSOD, which blocked TAC-induced ROS and ROS downstream c-Raf-MEK-ERK pathway that promotes hypertrophy. Taken together, our findings identify a novel anti-hypertrophic role of MOF, and MOF is the first reported anti-hypertrophic histone acetyltransferase. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Defining and mapping mammalian coat pattern genes: multiple genomic regions implicated in domestic cat stripes and spots.

    PubMed

    Eizirik, Eduardo; David, Victor A; Buckley-Beason, Valerie; Roelke, Melody E; Schäffer, Alejandro A; Hannah, Steven S; Narfström, Kristina; O'Brien, Stephen J; Menotti-Raymond, Marilyn

    2010-01-01

    Mammalian coat patterns (e.g., spots, stripes) are hypothesized to play important roles in camouflage and other relevant processes, yet the genetic and developmental bases for these phenotypes are completely unknown. The domestic cat, with its diversity of coat patterns, is an excellent model organism to investigate these phenomena. We have established three independent pedigrees to map the four recognized pattern variants classically considered to be specified by a single locus, Tabby; in order of dominance, these are the unpatterned agouti form called "Abyssinian" or "ticked" (T(a)), followed by Spotted (T(s)), Mackerel (T(M)), and Blotched (t(b)). We demonstrate that at least three different loci control the coat markings of the domestic cat. One locus, responsible for the Abyssinian form (herein termed the Ticked locus), maps to an approximately 3.8-Mb region on cat chromosome B1. A second locus controls the Tabby alleles T(M) and t(b), and maps to an approximately 5-Mb genomic region on cat chromosome A1. One or more additional loci act as modifiers and create a spotted coat by altering mackerel stripes. On the basis of our results and associated observations, we hypothesize that mammalian patterned coats are formed by two distinct processes: a spatially oriented developmental mechanism that lays down a species-specific pattern of skin cell differentiation and a pigmentation-oriented mechanism that uses information from the preestablished pattern to regulate the synthesis of melanin profiles.

  11. Cat scratch disease (image)

    MedlinePlus

    Cat scratch disease is an infectious illness associated with cat scratches, bites, or exposure to cat saliva, causing chronic swelling of the lymph nodes. Cat scratch disease is possibly the most common cause of chronic ...

  12. The Functional Analysis of Histone Acetyltransferase MOF in Tumorigenesis.

    PubMed

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

    2016-01-14

    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.

  13. 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

  14. Immunization of Cats against Feline Immunodeficiency Virus (FIV) Infection by Using Minimalistic Immunogenic Defined Gene Expression Vector Vaccines Expressing FIV gp140 Alone or with Feline Interleukin-12 (IL-12), IL-16, or a CpG Motif

    PubMed Central

    Leutenegger, Christian M.; Boretti, Felicitas S.; Mislin, Caroline N.; Flynn, J. Norman; Schroff, Matthias; Habel, Andre; Junghans, Claas; Koenig-Merediz, Sven A.; Sigrist, Brigitte; Aubert, Andre; Pedersen, Niels C.; Wittig, Burghardt; Lutz, Hans

    2000-01-01

    Four groups of cats, each containing four animals, were immunized at 0, 3, and 6 weeks with minimalistic immunogenic defined gene expression vector (MIDGE) vaccines containing the gene(s) for feline immunodeficiency virus (FIV) gp140, FIV gp140 and feline interleukin-12 (IL-12), FIV gp140 and feline IL-16, or FIV gp140 and a CpG motif. MIDGEs were coated onto gold beads and injected intradermally with a gene gun. A fifth group of four cats were immunized in an identical manner but with blank gold beads. All cats were challenge exposed to virulent FIV 4 weeks following the final immunization, and the course of infection was monitored. The two groups of cats immunized with the FIV gp140 gene alone or with blank gold particles became highly viremic and seroconverted as early as 4 weeks after infection. In contrast, three of four cats immunized with FIV gp140 in combination with feline IL-12 failed to become viremic or seropositive, as has been shown elsewhere (F. S. Boretti, C. M. Leutenegger, C. Mislin, et al., AIDS 14:1749–1757, 2000). Here we show the effect of IL-12 when used as an adjuvant on the viral RNA and DNA load and on the cytokine profile. In addition, the two groups of cats immunized either with gp140 and IL-16 or with gp140 and the CpG had greatly reduced viremia. Protection correlated weakly with cytotoxic T-lymphocyte activity and increased cytokine transcription of IL-12, gamma interferon, and IL-10 by peripheral blood mononuclear cells in the postchallenge period. This study extends the data on IL-12 and provides new results on CpG motifs and IL-16 used as adjuvants in the FIV cat model. PMID:11044089

  15. Lack of association between the 389C>T polymorphism (rs769217) in the catalase (CAT) gene and the risk of vitiligo: an update by meta-analysis.

    PubMed

    He, Jie; Li, Xiaoyan; Li, Yunhui; Ren, Bocheng; Sun, Jian; Zhang, Wei; Li, Wancheng

    2015-08-01

    The catalase (CAT) T/C at codon 389 in the exon 9 polymorphism has been implicated in susceptibility to vitiligo but a large number of studies have reported inconclusive results. The purpose of this study is to evaluate the association between the catalase gene polymorphism (389C>T) and the risk of vitiligo. A meta-analysis was carried out to analyse the association between 389C>T and vitiligo risk. Eight case-control studies with 2923 cases and 4237 controls were included in the meta-analysis. The results indicated that there was no association between this polymorphism and vitiligo (TT + CT versus CC: OR = 1.08, 95% CI = 0.98-1.20, P = 0.11, T versus C: OR = 1.07, 95% CI = 0.99-1.16, P = 0.092). In a subgroup analysis by ethnicity, no significant association between the CAT gene 389C>T polymorphism and vitiligo susceptibility was found in Caucasians (TT + CT versus CC: OR = 1.15, 95% CI = 0.98-1.35, P = 0.08; T versus C: OR = 1.07, 95% CI = 0.97-1.19, P = 0.173) and Asians (TT + CT versus CC: OR = 1.12, 95% CI =0.93-1.34, P = 0.23; T versus C: OR = 1.07, 95% CI = 0.94-1.21, P = 0.321). Our results suggest that 389C>T may not contribute to vitiligo susceptibility. However, larger primary studies with the consideration of gene-environment and gene-gene interactions are still required to further evaluate the interaction of CAT gene polymorphism with vitiligo susceptibility. © 2014 The Australasian College of Dermatologists.

  16. Gene-environment interaction in the onset of eczema in infancy: filaggrin loss-of-function mutations enhanced by neonatal cat exposure.

    PubMed

    Bisgaard, Hans; Simpson, Angela; Palmer, Colin N A; Bønnelykke, Klaus; McLean, Irwin; Mukhopadhyay, Somnath; Pipper, Christian B; Halkjaer, Liselotte B; Lipworth, Brian; Hankinson, Jenny; Woodcock, Ashley; Custovic, Adnan

    2008-06-24

    Loss-of-function variants in the gene encoding filaggrin (FLG) are major determinants of eczema. We hypothesized that weakening of the physical barrier in FLG-deficient individuals may potentiate the effect of environmental exposures. Therefore, we investigated whether there is an interaction between FLG loss-of-function mutations with environmental exposures (pets and dust mites) in relation to the development of eczema. We used data obtained in early life in a high-risk birth cohort in Denmark and replicated the findings in an unselected birth cohort in the United Kingdom. Primary outcome was age of onset of eczema; environmental exposures included pet ownership and mite and pet allergen levels. In Copenhagen (n = 379), FLG mutation increased the risk of eczema during the first year of life (hazard ratio [HR] 2.26, 95% confidence interval [CI] 1.27-4.00, p = 0.005), with a further increase in risk related to cat exposure at birth amongst children with FLG mutation (HR 11.11, 95% CI 3.79-32.60, p < 0.0001); dog exposure was moderately protective (HR 0.49, 95% CI 0.24-1.01, p = 0.05), but not related to FLG genotype. In Manchester (n = 503) an independent and significant association of the development of eczema by age 12 mo with FLG genotype was confirmed (HR 1.95, 95% CI 1.13-3.36, p = 0.02). In addition, the risk increased because of the interaction of cat ownership at birth and FLG genotype (HR 3.82, 95% CI 1.35-10.81, p = 0.01), with no significant effect of the interaction with dog ownership (HR 0.59, 95% CI 0.16-2.20, p = 0.43). Mite-allergen had no effects in either cohort. The observed effects were independent of sensitisation. We have demonstrated a significant interaction between FLG loss-of-function main mutations (501x and 2282del4) and cat ownership at birth on the development of early-life eczema in two independent birth cohorts. Our data suggest that cat but not dog ownership substantially increases the risk of eczema within the first year of life in

  17. Characterization of a Spontaneous Novel Mutation in the NPC2 Gene in a Cat Affected by Niemann Pick Type C Disease

    PubMed Central

    Zampieri, Stefania; Bianchi, Ezio; Cantile, Carlo; Saleri, Roberta; Bembi, Bruno; Dardis, Andrea

    2014-01-01

    Niemann-Pick C disease (NPC) is an autosomal recessive lysosomal storage disorder characterized by accumulation of unesterified cholesterol and other lipids within the lysosomes due to mutation in NPC1 or NPC2 genes. A feline model of NPC carrying a mutation in NPC1 gene has been previously described. We have identified two kittens affected by NPC disease due to a mutation in NPC2 gene. They manifested with tremors at the age of 3 months, which progressed to dystonia and severe ataxia. At 6 months of age cat 2 was unable to stand without assistance and had bilaterally reduced menace response. It died at the age of 10 months. Post-mortem histological analysis of the brain showed the presence of neurons with cytoplasmic swelling and vacuoles, gliosis of the substantia nigra and degeneration of the white matter. Spheroids with accumulation of ubiquitinated aggregates were prominent in the cerebellar cortex. Purkinje cells were markedly reduced in number and they showed prominent intracytoplasmic storage. Scattered perivascular aggregates of lymphocytes and microglial cells proliferation were present in the thalamus and midbrain. Proliferation of Bergmann glia was also observed. In the liver, hepatocytes were swollen because of accumulation of small vacuoles and foamy Kupffer cells were also detected. Foamy macrophages were observed within the pulmonary interstitium and alveoli as well. At 9 months cat 1 was unable to walk, developed seizures and it was euthanized at 21 months. Filipin staining of cultured fibroblasts showed massive storage of unesterified cholesterol. Molecular analysis of NPC1 and NPC2 genes showed the presence of a homozygous intronic mutation (c.82+5G>A) in the NPC2 gene. The subsequent analysis of the mRNA showed that the mutation causes the retention of 105 bp in the mature mRNA, which leads to the in frame insertion of 35 amino acids between residues 28 and 29 of NPC2 protein (p.G28_S29ins35). PMID:25396745

  18. Histone H3 specific acetyltransferases are essential for cell cycle progression

    PubMed Central

    Howe, LeAnn; Auston, Darryl; Grant, Patrick; John, Sam; Cook, Richard G.; Workman, Jerry L.; Pillus, Lorraine

    2001-01-01

    Longstanding observations suggest that acetylation and/or amino-terminal tail structure of histones H3 and H4 are critical for eukaryotic cells. For Saccharomyces cerevisiae, loss of a single H4-specific histone acetyltransferase (HAT), Esa1p, results in cell cycle defects and death. In contrast, although several yeast HAT complexes preferentially acetylate histone H3, the catalytic subunits of these complexes are not essential for viability. To resolve the apparent paradox between the significance of H3 versus H4 acetylation, we tested the hypothesis that H3 modification is essential, but is accomplished through combined activities of two enzymes. We observed that Sas3p and Gcn5p HAT complexes have overlapping patterns of acetylation. Simultaneous disruption of SAS3, the homolog of the MOZ leukemia gene, and GCN5, the hGCN5/PCAF homolog, is synthetically lethal due to loss of acetyltransferase activity. This key combination of activities is specific for these two HATs because neither is synthetically lethal with mutations of other MYST family or H3-specific acetyltransferases. Further, the combined loss of GCN5 and SAS3 functions results in an extensive, global loss of H3 acetylation and arrest in the G2/M phase of the cell cycle. The strikingly similar effect of loss of combined essential H3 HAT activities and the loss of a single essential H4 HAT underscores the fundamental biological significance of each of these chromatin-modifying activities. PMID:11731478

  19. Molecular analysis of the transcriptional regulatory region of an early baculovirus gene.

    PubMed Central

    Nissen, M S; Friesen, P D

    1989-01-01

    Transcription of the gene encoding a 35,000-molecular-weight protein (35K protein) from the EcoRI-S region (86.8 to 87.8 map units) of Autographa california nuclear polyhedrosis virus (AcMNPV) occurs early in infection and declines later. The region promoting the gene for the 35K protein, extending from 426 base pairs (bp) upstream to 12 bp downstream from the RNA start site, was linked to the bacterial chloramphenicol acetyltransferase gene (CAT) for analysis. CAT expression was monitored in cells that were transfected with plasmids containing the promoter-CAT fusion as well as cells infected with recombinant viruses containing the chimeric gene inserted into the AcMNPV genome. Mapping of the 5' ends of CAT-specific RNAs indicated that transcription initiated from the proper sites in both assays; moreover, the promoter fragment retained its early activity, despite an alternate location in the viral genome. The 5' boundary of upstream regulatory sequences was determined by constructing deletions of the promoter fragment extending toward the early RNA start site (position +1). In transient assays, a gradual reduction in CAT expression occurred as sequences from positions -426 to -31 were removed. In contrast, promoter deletions from positions -426 to -155 in recombinant viruses exhibited no effect on CAT expression, whereas deletions to position -55 abolished early expression but had no effect on late expression. Late CAT expression was eliminated when deletions to position -4 removed part of the late RNA start site. DNA signals potentiating early transcription were therefore located upstream (between positions -155 and -55) from those involved in late transcription of the gene encoding the 35K protein. Potential consensus sequences for early and late regulatory elements were identified. Images PMID:2642976

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

    USDA-ARS?s Scientific Manuscript database

    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...

  1. Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

    SciTech Connect

    Trempe, J.P.; Carter, B.J.

    1988-01-01

    The authors studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p/sub 40/ promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p/sub 40/. When the rep gene was present in cis or in trans, cat expression from p/sub 40/ was decreased 3- to 10-fold, but there was a 2- to 10-fold increase in the level of p/sub 40/ mRNA. Conversely, cat expression increased and the p/sub 40/ mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p/sub 40/ mRNA levels. They also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p/sub 5/ promoter in a fashion independent of rep.

  2. Spermidine/spermine-N(1)-acetyltransferase: a key metabolic regulator.

    PubMed

    Pegg, Anthony E

    2008-06-01

    Spermidine/spermine-N(1)-acetyltransferase (SSAT) regulates cellular polyamine content. Its acetylated products are either excreted from the cell or oxidized by acetylpolyamine oxidase. Since polyamines play critical roles in normal and neoplastic growth and in ion channel regulation, SSAT is a key enzyme in these processes. SSAT is very highly regulated. Its content is adjusted in response to alterations in polyamine content to maintain polyamine homeostasis. Certain polyamine analogs can mimic the induction of SSAT and cause a loss of normal polyamines. This may have utility in cancer chemotherapy. SSAT activity is also induced via a variety of other stimuli, including toxins, hormones, cytokines, nonsteroidal anti-inflammatory agents, natural products, and stress pathways, and by ischemia-reperfusion injury. These increases are initiated by alterations in Sat1 gene transcription reinforced by alterations at the other regulatory steps, including protein turnover, mRNA processing, and translation. Transgenic manipulation of SSAT activity has revealed that SSAT activity links polyamine metabolism to lipid and carbohydrate metabolism by means of alterations in the content of acetyl-CoA and ATP. A high level of SSAT stimulates flux through the polyamine biosynthetic pathway, since biosynthetic enzymes are induced in response to the fall in polyamines. This sets up a futile cycle in which ATP is used to generate S-adenosylmethionine for polyamine biosynthesis and acetyl-CoA is consumed in the acetylation reaction. A variety of other effects of increased SSAT activity include death of pancreatic cells, blockage of regenerative tissue growth, behavioral changes, keratosis follicularis spinulosa decalvans, and hair loss. These are very likely due to changes in polyamine and putrescine levels, although increased oxidative stress via the oxidation of acetylated polyamines may also contribute. Recently, it was found that the SSAT protein and/or a related protein, thialysine

  3. Gene VI of figwort mosaic virus (caulimovirus group) functions in posttranscriptional expression of genes on the full-length RNA transcript.

    PubMed

    Gowda, S; Wu, F C; Scholthof, H B; Shepherd, R J

    1989-12-01

    Experimental evidence for a molecular function for gene VI of the caulimoviruses is presented. Based on experiments with the figwort mosaic virus (FMV), it appears that gene VI has a role in the posttranscriptional expression of the closely packed genes (VII and I-V), which appear on the larger, full-length RNA transcript of this virus. Gene VI with its flanking 5'/3' expression signals included as a separate plasmid during electroporation of DNA into protoplasts of Nicotiana edwardsonii shows an unusual type of transactivation of a chloramphenicol acetyltransferase (CAT) gene fused at its 5' end to a small open reading frame (gene VII) of the long 5' leader of the full-length RNA transcript of the FMV genome. The level of activity of the CAT gene is increased up to 20-fold over the activity of control plasmids when gene VI is included in the electroporation mixture. Mutagenesis of the coding portions of gene VI of pGS1 RVI, a transactivating plasmid used in the electroporation experiments, demonstrated that it was probably the polypeptide product of gene VI that was responsible for the transactivating effect. Experiments with various portions of the 5' leader of the large, full-length RNA of FMV showed that the coding region of gene VII is necessary for the transactivation event. Clones of cauliflower mosaic virus (CaMV) or FMV with intact gene VI were found to reciprocally transactivate gene VII-CAT fusions (FMV) or gene I-CAT fusions (CaMV) located downstream of the 5' leader sequences of either viral genome.

  4. Autoacetylation of the MYST lysine acetyltransferase MOF protein.

    PubMed

    Yang, Chao; Wu, Jiang; Sinha, Sarmistha H; Neveu, John M; Zheng, Yujun George

    2012-10-12

    The MYST family of histone acetyltransferases (HATs) plays critical roles in diverse cellular processes, such as the epigenetic regulation of gene expression. Lysine autoacetylation of the MYST HATs has recently received considerable attention. Nonetheless, the mechanism and function of the autoacetylation process are not well defined. To better understand the biochemical mechanism of MYST autoacetylation and the impact of autoacetylation on the cognate histone acetylation, we carried out detailed analyses of males-absent-on-the-first (MOF), a key member of the MYST family. A number of mutant MOF proteins were produced with point mutations at several key residues near the active site of the enzyme. Autoradiography and immunoblotting data showed that mutation of these residues affects the autoacetylation activity and HAT activity of MOF by various degrees demonstrating that MOF activity is highly sensitive to the chemical changes in those residues. We produced MOF protein in the deacetylated form by using a nonspecific lysine deacetylase. Interestingly, both the autoacetylation activity and the histone acetylation activity of the deacetylated MOF were found to be very close to that of wild-type MOF, suggesting that autoacetylation of MOF only marginally modulates the enzymatic activity. Also, we found that the autoacetylation rates of MOF and deacetylated MOF were much slower than the cognate substrate acetylation. Thus, autoacetylation does not seem to contribute to the intrinsic enzymatic activity in a significant manner. These data provide new insights into the mechanism and function of MYST HAT autoacetylation.

  5. Autoacetylation of the MYST Lysine Acetyltransferase MOF Protein*

    PubMed Central

    Yang, Chao; Wu, Jiang; Sinha, Sarmistha H.; Neveu, John M.; Zheng, Yujun George

    2012-01-01

    The MYST family of histone acetyltransferases (HATs) plays critical roles in diverse cellular processes, such as the epigenetic regulation of gene expression. Lysine autoacetylation of the MYST HATs has recently received considerable attention. Nonetheless, the mechanism and function of the autoacetylation process are not well defined. To better understand the biochemical mechanism of MYST autoacetylation and the impact of autoacetylation on the cognate histone acetylation, we carried out detailed analyses of males-absent-on-the-first (MOF), a key member of the MYST family. A number of mutant MOF proteins were produced with point mutations at several key residues near the active site of the enzyme. Autoradiography and immunoblotting data showed that mutation of these residues affects the autoacetylation activity and HAT activity of MOF by various degrees demonstrating that MOF activity is highly sensitive to the chemical changes in those residues. We produced MOF protein in the deacetylated form by using a nonspecific lysine deacetylase. Interestingly, both the autoacetylation activity and the histone acetylation activity of the deacetylated MOF were found to be very close to that of wild-type MOF, suggesting that autoacetylation of MOF only marginally modulates the enzymatic activity. Also, we found that the autoacetylation rates of MOF and deacetylated MOF were much slower than the cognate substrate acetylation. Thus, autoacetylation does not seem to contribute to the intrinsic enzymatic activity in a significant manner. These data provide new insights into the mechanism and function of MYST HAT autoacetylation. PMID:22918831

  6. Blood pressure regulation by CD4+ lymphocytes expressing choline acetyltransferase

    PubMed Central

    Olofsson, Peder S.; Steinberg, Benjamin E.; Sobbi, Roozbeh; Cox, Maureen A.; Ahmed, Mohamed N.; Oswald, Michaela; Szekeres, Ferenc; Hanes, William M.; Introini, Andrea; Liu, Shu Fang; Holodick, Nichol E.; Rothstein, Thomas L.; Lövdahl, Cecilia; Chavan, Sangeeta S.; Yang, Huan; Pavlov, Valentin A.; Broliden, Kristina; Andersson, Ulf; Diamond, Betty; Miller, Edmund J.; Arner, Anders; Gregersen, Peter K.; Backx, Peter H.; Mak, Tak W.; Tracey, Kevin J.

    2017-01-01

    Blood pressure regulation is known to be maintained by a neuro-endocrine circuit, but whether immune cells contribute to blood pressure homeostasis has not been defined. We previously described that CD4+ T lymphocytes that express choline acetyltransferase (ChAT), which catalyzes the synthesis of the vasorelaxant acetylcholine, relay neural signals1. Here we show that these CD4+ CD44high CD62Llow T helper cells by gene expression are a distinct T cell population defined by ChAT (CD4 TChAT). Mice lacking ChAT expression in CD4+ cells have elevated arterial blood pressure and echocardiographic assessment consistent with increased vascular resistance as compared to littermate controls. Jurkat T cells overexpressing ChAT (JTChAT) decreased blood pressure when infused into mice. Co-incubation of JTChAT increased endothelial cell levels of phosphorylated eNOS, and of nitrates and nitrites in conditioned media, indicating increased release of the potent vasodilator nitric oxide. The isolation and characterization of CD4 TChAT cells will enable analysis of the role of these cells in hypotension and hypertension, and may suggest novel therapeutic strategies by targeting cell-mediated vasorelaxation. PMID:27617738

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

    PubMed Central

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

    2016-01-01

    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

  8. 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.

  9. Detection of Toxoplasma gondii in faeces of privately owned cats using two PCR assays targeting the B1 gene and the 529-bp repetitive element.

    PubMed

    Veronesi, Fabrizia; Santoro, Azzurra; Milardi, Giovanni L; Diaferia, Manuela; Morganti, Giulia; Ranucci, David; Gabrielli, Simona

    2017-03-01

    Toxoplasma gondii infection is a worldwide parasitic zoonosis with a high-health risk for humans. The key epidemiological role played by felids is related to oocyst shedding. The present study compared two amplification protocols for the molecular diagnosis of Toxoplasma infection in owned cats. A total of 78 owned cats referred to an Italian university-teaching hospital and exposed to various T. gondii-associated risk factors were sampled for blood and faeces. Faecal specimens were processed by flotation and tested using 2 copro-PCRs targeting the widely used B1 gene and the 529-bp repetitive element (RE). The sera were tested by the indirect immunofluorescent antibody test (IFAT) for the detection of immunoglobulins against T. gondii. Sixteen faeces (20.52%) tested positive for T. gondii DNA; 12 samples were positive only at B1-PCR, two at 529-bp RE-PCR and two at both genetic targets (overall agreement = 82.11%). The amplicons obtained were sequenced, and the Basic Local Alignment Search Tool analysis showed a high homology with the T. gondii strains available in reference databases. Two stool samples were microscopically positive for T. gondii-like oocysts and also tested positive by both B1 and 529-bp RE-PCRs. Thirty-three (42.3%) sera tested positive for antibodies; of which, seven were found to have T. gondii DNA-positive results using the B1 genetic target (overall agreement = 57.77%). The amplification sets targeting B1 and 529-bp RE showed substantially different yields. Further research is needed to better understand the significance and the sensitivities of using these multi-copy-targeted molecular methods from cat faeces before being used for routine diagnosis.

  10. Molecular Detection of Rickettsia felis in Humans, Cats, and Cat Fleas in Bangladesh, 2013-2014.

    PubMed

    Ahmed, Rajib; Paul, Shyamal Kumar; Hossain, Muhammad Akram; Ahmed, Salma; Mahmud, Muhammad Chand; Nasreen, Syeda Anjuman; Ferdouse, Faria; Sharmi, Rumana Hasan; Ahamed, Farid; Ghosh, Souvik; Urushibara, Noriko; Aung, Meiji Soe; Kobayashi, Nobumichi

    2016-05-01

    High prevalence of Rickettsia felis in patients with fever of unknown origin was revealed in the north-central Bangladesh from 2012 to 2013. Subsequently, in this study, prevalence of R. felis in cats and cat fleas (Ctenocephalides felis), together with febrile patients, was studied by PCR detection of 17 kDa antigen gene and DNA sequencing. R. felis was detected in 28% (28/100) and 21% (14/68) of cat blood and cat flea samples, respectively, whereas 42% (21/50) of patients were positive for R. felis. R. felis-positive cat fleas were detected at significantly higher rate on R. felis-positive cats. The results suggested a potential role of cats and cat fleas for transmission of R. felis to humans in Bangladesh.

  11. Production of tetraacetyl phytosphingosine (TAPS) in Wickerhamomyces ciferrii is catalyzed by acetyltransferases Sli1p and Atf2p.

    PubMed

    Ter Veld, Frank; Wolff, Daniel; Schorsch, Christoph; Köhler, Tim; Boles, Eckhard; Poetsch, Ansgar

    2013-10-01

    Wickerhamomyces ciferrii secretes tetraacetyl phytosphingosine (TAPS), and in this study, the catalyzing acetyltransferases were identified using mass spectrometry-based proteomics. The proteome of wild-type strain NRRL Y-1031 served as control and was compared to the tetraacetyl phytosphingosine defective mating type NRRL Y-1031-27. Acetylation of phytosphingosine in W. ciferrii is catalyzed by acetyltransferases Sli1p and Atf2p, encoded by genes similar to Saccharomyces cerevisiae YGR212W and YGR177C, respectively. Ablation of SLI1 resulted in an almost complete loss of tri- and tetraacetyl phytosphingosines, whereas the loss ATF2 resulted in an 15-fold increase in triacetyl phytosphingosine. Most likely, it is the concerted action of these two acetyltransferases that yields tetraacetyl phytosphingosine, in which Sli1p catalyzes initial O- and N-acetylation, producing triacetyl phytosphingosine. Finally, Atf2p catalyzes final O-acetylation to yield tetraacetyl phytosphingosine. The current study demonstrates that mass spectrometry-based proteomics can be employed to identify key steps in ill-explored metabolite biosynthesis pathways of nonconventional microorganisms. Furthermore, the identification of phytosphingosine as substrate for alcohol acetyltransferase Atf2p broadens the known substrate range of this enzyme. This interesting property of Atf2p may be exploited to enhance the secretion of heterologous compounds.

  12. Structural and Functional Evidence for Bacillus subtilis PaiA as a Novel N1-spermidine/spermine acetyltransferase (SSAT)

    SciTech Connect

    Forouhar,F.; Lee, I.; Vujcic, J.; Vujcic, S.; Shen, J.; Vorobiev, S.; Xiao, R.; Acton, T.; Montelione, G.; et al.

    2005-01-01

    Bacillus subtilis PaiA has been implicated in the negative control of sporulation as well as production of degradative enzymes. PaiA shares recognizable sequence homology with N-acetyltransferases, including those that can acetylate spermidine/spermine substrates (SSATs). We have determined the crystal structure of PaiA in complex with CoA at 1.9 Angstrom resolution and found that PaiA is a member of the N-acetyltransferase superfamily of enzymes. Unexpectedly, we observed the binding of an oxidized CoA dimer in the active site of PaiA, and the structural information suggests the substrates of the enzyme could be linear, positively charged compounds. Our biochemical characterization is also consistent with this possibility since purified PaiA possesses N1-acetyltransferase activity towards polyamine substrates including spermidine and spermine. Further, conditional over-expression of PaiA in bacteria results in increased acetylation of endogenous spermidine pools. Thus, our structural and biochemical analyses indicate that PaiA is a novel N-acetyltransferase capable of acetylating both spermidine and spermine. In this way, the pai operon may function in regulating intracellular polyamine concentrations and/or binding capabilities. In addition to preventing toxicity due to polyamine excess, this function may also serve to regulate expression of certain bacterial gene products such as those involved in sporulation.

  13. Bacterial microbiome in the nose of healthy cats and in cats with nasal disease.

    PubMed

    Dorn, Elisabeth S; Tress, Barbara; Suchodolski, Jan S; Nisar, Tariq; Ravindran, Prajesh; Weber, Karin; Hartmann, Katrin; Schulz, Bianka S

    2017-01-01

    Traditionally, changes in the microbial population of the nose have been assessed using conventional culture techniques. Sequencing of bacterial 16S rRNA genes demonstrated that the human nose is inhabited by a rich and diverse bacterial microbiome that cannot be detected using culture-based methods. The goal of this study was to describe the nasal microbiome of healthy cats, cats with nasal neoplasia, and cats with feline upper respiratory tract disease (FURTD). DNA was extracted from nasal swabs of healthy cats (n = 28), cats with nasal neoplasia (n = 16), and cats with FURTD (n = 15), and 16S rRNA genes were sequenced. High species richness was observed in all samples. Rarefaction analysis revealed that healthy cats living indoors had greater species richness (observed species p = 0.042) and Shannon diversity (p = 0.003) compared with healthy cats living outdoors. Higher species richness (observed species p = 0.001) and Shannon diversity (p<0.001) were found in middle-aged cats in comparison to healthy cats in different age groups. Principal coordinate analysis revealed separate clustering based on similarities in bacterial molecular phylogenetic trees of 16S rRNA genes for indoor and outdoor cats. In all groups examined, the most abundant phyla identified were Proteobacteria, Firmicutes, and Bacteroidetes. At the genus level, 375 operational taxonomic units (OTUs) were identified. In healthy cats and cats with FURTD, Moraxella spp. was the most common genus, while it was unclassified Bradyrhizobiaceae in cats with nasal neoplasia. High individual variability was observed. This study demonstrates that the nose of cats is inhabited by much more variable and diverse microbial communities than previously shown. Future research in this field might help to develop new diagnostic tools to easily identify nasal microbial changes, relate them to certain disease processes, and help clinicians in the decision process of antibiotic selection for individual patients.

  14. Bacterial microbiome in the nose of healthy cats and in cats with nasal disease

    PubMed Central

    Tress, Barbara; Suchodolski, Jan S.; Nisar, Tariq; Ravindran, Prajesh; Weber, Karin; Hartmann, Katrin; Schulz, Bianka S.

    2017-01-01

    Background Traditionally, changes in the microbial population of the nose have been assessed using conventional culture techniques. Sequencing of bacterial 16S rRNA genes demonstrated that the human nose is inhabited by a rich and diverse bacterial microbiome that cannot be detected using culture-based methods. The goal of this study was to describe the nasal microbiome of healthy cats, cats with nasal neoplasia, and cats with feline upper respiratory tract disease (FURTD). Methodology/Principal findings DNA was extracted from nasal swabs of healthy cats (n = 28), cats with nasal neoplasia (n = 16), and cats with FURTD (n = 15), and 16S rRNA genes were sequenced. High species richness was observed in all samples. Rarefaction analysis revealed that healthy cats living indoors had greater species richness (observed species p = 0.042) and Shannon diversity (p = 0.003) compared with healthy cats living outdoors. Higher species richness (observed species p = 0.001) and Shannon diversity (p<0.001) were found in middle-aged cats in comparison to healthy cats in different age groups. Principal coordinate analysis revealed separate clustering based on similarities in bacterial molecular phylogenetic trees of 16S rRNA genes for indoor and outdoor cats. In all groups examined, the most abundant phyla identified were Proteobacteria, Firmicutes, and Bacteroidetes. At the genus level, 375 operational taxonomic units (OTUs) were identified. In healthy cats and cats with FURTD, Moraxella spp. was the most common genus, while it was unclassified Bradyrhizobiaceae in cats with nasal neoplasia. High individual variability was observed. Conclusion This study demonstrates that the nose of cats is inhabited by much more variable and diverse microbial communities than previously shown. Future research in this field might help to develop new diagnostic tools to easily identify nasal microbial changes, relate them to certain disease processes, and help clinicians in the decision process of

  15. Evidence for arylamine N-acetyltransferase in Hymenolepis nana.

    PubMed

    Chung, J G; Kuo, H M; Wu, L T; Lai, J M; Lee, J H; Hung, C F

    1997-02-01

    N-acetyltransferase activities with p-aminobenzoic acid and 2-aminofluorene were determined in Hymenolepis nana, a cestode found in the intestine of the Sprague-Dawley rats. The N-acetyltransferase activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. The N-acetyltransferase activities from a number of Hymenolepis nana whole tissue homogenizations were found to be 2.83 +/- 0.31 nmole/min/mg for 2-aminofluorene and 2.07 +/- 0.24 nmole/min/mg for p-aminobenzoic acid. The apparent Km and Vmax were 1.06 +/- 0.38 mM and 8.92 +/- 1.46 nmol/min/mg for 2-aminofluorene, and 2.16 +/- 0.19 mM and 12.68 +/- 2.26 nmol/min/mg for p-aminobenzoic acid. The optimal pH value for the enzyme activity was pH 8.0 for both substrates tested. The optimal temperature for enzyme activity was 37 degrees C for both substrates. The N-acetyltransferase activity was inhibited by iodacetamide. At 0.25 mM iodacetamide the activity was reduced 50% and 1.0 mM iodacetamide inhibited activity more than 90%. Among a series of divalent cations and salts, Fe2+, Ca2+ and Zn2+ were demonstrated to be the most potent inhibi-tors. Among the protease inhibitors, only ethylenediaminetetraacetic acid significantly protected N-acetyltransferase. Iodoacetate, in contrast to other agents, markedly inhibited N-acetyltransferase activity. This is the first demonstration of acetyl CoA:arylamine N-acetyltransferase activity in a cestode and extends the number of phyla in which this activity has been found.

  16. Ubiquitylation of the acetyltransferase MOF in Drosophila melanogaster

    PubMed Central

    Schunter, Sarah; Villa, Raffaella; Flynn, Victoria; Heidelberger, Jan B.; Classen, Anne-Kathrin; Beli, Petra; Becker, Peter B.

    2017-01-01

    The nuclear acetyltransferase MOF (KAT8 in mammals) is a subunit of at least two multi-component complexes involved in transcription regulation. In the context of complexes of the ‘Non-Specific-Lethal’ (NSL) type it controls transcription initiation of many nuclear housekeeping genes and of mitochondrial genes. While this function is conserved in metazoans, MOF has an additional, specific function in Drosophila in the context of dosage compensation. As a subunit of the male-specific-lethal dosage compensation complex (MSL-DCC) it contributes to the doubling of transcription output from the single male X chromosome by acetylating histone H4. Proper dosage compensation requires finely tuned levels of MSL-DCC and an appropriate distribution of MOF between the regulatory complexes. The amounts of DCC formed depends directly on the levels of the male-specific MSL2, which orchestrates the assembly of the DCC, including MOF recruitment. We found earlier that MSL2 is an E3 ligase that ubiquitylates most MSL proteins, including MOF, suggesting that ubiquitylation may contribute to a quality control of MOF’s overall levels and folding state as well as its partitioning between the complex entities. We now used mass spectrometry to map the lysines in MOF that are ubiquitylated by MSL2 in vitro and identified in vivo ubiquitylation sites of MOF in male and female cells. MSL2-specific ubiquitylation in vivo could not be traced due to the dominance of other, sex-independent ubiquitylation events and conceivably may be rare or transient. Expressing appropriately mutated MOF derivatives we assessed the importance of the ubiquitylated lysines for dosage compensation by monitoring DCC formation and X chromosome targeting in cultured cells, and by genetic complementation of the male-specific-lethal mof2 allele in flies. Our study provides a comprehensive analysis of MOF ubiquitylation as a reference for future studies. PMID:28510597

  17. Ubiquitylation of the acetyltransferase MOF in Drosophila melanogaster.

    PubMed

    Schunter, Sarah; Villa, Raffaella; Flynn, Victoria; Heidelberger, Jan B; Classen, Anne-Kathrin; Beli, Petra; Becker, Peter B

    2017-01-01

    The nuclear acetyltransferase MOF (KAT8 in mammals) is a subunit of at least two multi-component complexes involved in transcription regulation. In the context of complexes of the 'Non-Specific-Lethal' (NSL) type it controls transcription initiation of many nuclear housekeeping genes and of mitochondrial genes. While this function is conserved in metazoans, MOF has an additional, specific function in Drosophila in the context of dosage compensation. As a subunit of the male-specific-lethal dosage compensation complex (MSL-DCC) it contributes to the doubling of transcription output from the single male X chromosome by acetylating histone H4. Proper dosage compensation requires finely tuned levels of MSL-DCC and an appropriate distribution of MOF between the regulatory complexes. The amounts of DCC formed depends directly on the levels of the male-specific MSL2, which orchestrates the assembly of the DCC, including MOF recruitment. We found earlier that MSL2 is an E3 ligase that ubiquitylates most MSL proteins, including MOF, suggesting that ubiquitylation may contribute to a quality control of MOF's overall levels and folding state as well as its partitioning between the complex entities. We now used mass spectrometry to map the lysines in MOF that are ubiquitylated by MSL2 in vitro and identified in vivo ubiquitylation sites of MOF in male and female cells. MSL2-specific ubiquitylation in vivo could not be traced due to the dominance of other, sex-independent ubiquitylation events and conceivably may be rare or transient. Expressing appropriately mutated MOF derivatives we assessed the importance of the ubiquitylated lysines for dosage compensation by monitoring DCC formation and X chromosome targeting in cultured cells, and by genetic complementation of the male-specific-lethal mof2 allele in flies. Our study provides a comprehensive analysis of MOF ubiquitylation as a reference for future studies.

  18. 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

  19. The Yeast ATF1 Acetyltransferase Efficiently Acetylates Insect Pheromone Alcohols: Implications for the Biological Production of Moth Pheromones.

    PubMed

    Ding, Bao-Jian; Lager, Ida; Bansal, Sunil; Durrett, Timothy P; Stymne, Sten; Löfstedt, Christer

    2016-04-01

    Many moth pheromones are composed of mixtures of acetates of long-chain (≥10 carbon) fatty alcohols. Moth pheromone precursors such as fatty acids and fatty alcohols can be produced in yeast by the heterologous expression of genes involved in insect pheromone production. Acetyltransferases that subsequently catalyze the formation of acetates by transfer of the acetate unit from acetyl-CoA to a fatty alcohol have been postulated in pheromone biosynthesis. However, so far no fatty alcohol acetyltransferases responsible for the production of straight chain alkyl acetate pheromone components in insects have been identified. In search for a non-insect acetyltransferase alternative, we expressed a plant-derived diacylglycerol acetyltransferase (EaDAcT) (EC 2.3.1.20) cloned from the seed of the burning bush (Euonymus alatus) in a yeast system. EaDAcT transformed various fatty alcohol insect pheromone precursors into acetates but we also found high background acetylation activities. Only one enzyme in yeast was shown to be responsible for the majority of that background activity, the acetyltransferase ATF1 (EC 2.3.1.84). We further investigated the usefulness of ATF1 for the conversion of moth pheromone alcohols into acetates in comparison with Ea DAcT. Overexpression of ATF1 revealed that it was capable of acetylating these fatty alcohols with chain lengths from 10 to 18 carbons with up to 27- and 10-fold higher in vivo and in vitro efficiency, respectively, compared to Ea DAcT. The ATF1 enzyme thus has the potential to serve as the missing enzyme in the reconstruction of the biosynthetic pathway of insect acetate pheromones from precursor fatty acids in yeast.

  20. Cat scratch disease

    MedlinePlus

    ... t scratch and bite. Don't allow a cat to lick your skin, eyes, mouth, or open wounds or scratches. Use flea control measures to lower the risk your cat develops the disease. Don't touch feral cats. ...

  1. The human homolog of insect-derived growth factor, CECR1, is a candidate gene for features of cat eye syndrome.

    PubMed

    Riazi, M A; Brinkman-Mills, P; Nguyen, T; Pan, H; Phan, S; Ying, F; Roe, B A; Tochigi, J; Shimizu, Y; Minoshima, S; Shimizu, N; Buchwald, M; McDermid, H E

    2000-03-15

    Cat eye syndrome (CES) is a developmental disorder with multiple organ involvement, associated with the duplication of a 2-Mb region of 22q11.2. Using exon trapping and genomic sequence analysis, we have isolated and characterized a gene, CECR1, that maps to this critical region. The protein encoded by CECR1 is similar to previously identified novel growth factors: IDGF from Sarcophaga peregrina (flesh fly) and MDGF from Aplysia californica (sea hare). The CECR1 gene is alternatively spliced and expressed in numerous tissues, with most abundant expression in human adult heart, lung, lymphoblasts, and placenta as well as fetal lung, liver, and kidney. In situ hybridization of a human embryo shows specific expression in the outflow tract and atrium of the developing heart, the VII/VIII cranial nerve ganglion, and the notochord. The location of this gene in the CES critical region and its embryonic expression suggest that the overexpression of CECR1 may be responsible for at least some features of CES, particularly the heart defects.

  2. Pre- and post-weaning diet alters the faecal metagenome in the cat with differences vitamin and carbohydrate metabolism gene abundances

    PubMed Central

    Young, Wayne; Moon, Christina D.; Thomas, David G.; Cave, Nick J.; Bermingham, Emma N.

    2016-01-01

    Dietary format, and its role in pet nutrition, is of interest to pet food manufacturers and pet owners alike. The aim of the present study was to investigate the effects of pre- and post-weaning diets (kibbled or canned) on the composition and function of faecal microbiota in the domestic cat by shotgun metagenomic sequencing and gene taxonomic and functional assignment using MG-RAST. Post-weaning diet had a dramatic effect on community composition; 147 of the 195 bacterial species identified had significantly different mean relative abundances between kittens fed kibbled and canned diets. The kittens fed kibbled diets had relatively higher abundances of Lactobacillus (>100-fold), Bifidobacterium (>100-fold), and Collinsella (>9-fold) than kittens fed canned diets. There were relatively few differences in the predicted microbiome functions associated with the pre-weaning diet. Post-weaning diet affected the abundance of functional gene groups. Genes involved in vitamin biosynthesis, metabolism, and transport, were significantly enriched in the metagenomes of kittens fed the canned diet. The impact of post-weaning diet on the metagenome in terms of vitamin biosynthesis functions suggests that modulation of the microbiome function through diet may be an important avenue for improving the nutrition of companion animals. PMID:27876765

  3. The Purification of Choline Acetyltransferase of Squid-Head Ganglia

    PubMed Central

    Husain, S. S.; Mautner, Henry G.

    1973-01-01

    Choline acetyltransferase (EC 2.3.1.6) isolated from the head ganglia of squid could be purified by use of mercurial-Sepharose columns as well as Sepharose columns to which the enzyme inhibitor p-(m-bromophenyl)vinyl pyridinium had been attached. These columns, in conjunction with 30-55% ammonium sulfate precipitation, 40-30% ammonium sulfate extraction, chromatography on sulfopropyl-Sephadex and on cellulose phosphate and hydroxylapatite columns, led to the isolation of three factions of choline acetyltransferase ranging in activity from 1000 to 4000 μmole/mg of protein/per hr. Polyacrylamide gel electrophoresis suggests that two of these fractions are homogeneous. The squid choline acetyltransferase is different from the mammalian-brain enzymes in having a larger molecular weight under the conditions used and in being relatively poorly inhibited by styryl pyridinium compounds. Images PMID:4521199

  4. 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

    2014-03-07

    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.

  5. Immediate early gene expression in cat visual cortex during and after the critical period: differences between EGR-1 and Fos proteins.

    PubMed

    Kaplan, I V; Guo, Y; Mower, G D

    1996-02-01

    Immediate early gene (IEG) expression in the cat visual cortex is highly responsive to visual input and may initiate genetic mechanisms responsible for neuronal plasticity. The present study used immunohistochemical methods to address two issues regarding IEG expression in response to visual input. One was to define the differential response of distinct IEG families by comparing EGR-1 (also termed zif-268, NGFI-A, and Krox-24) and Fos proteins. The second was to determine whether IEG expression, in addition to reflecting neural activity, is related to the state of plasticity by comparing young and adult visual cortex. Immunoreactivity of the two IEG proteins was compared between 5-week-old and adult cats under three conditions of visual input: ambient light to assess basal levels of expression, 1 week of darkness to assess the effect of reduced activity, and exposure to light after 1 week of darkness to determine rapid changes in expression as a result of visual input. At both ages, there were marked differences in the expression of the two IEG proteins. EGR-1 responded to visual input with sustained changes in its level of expression. It showed high basal levels, reduced expression in darkness, and a rapid return to high constitutive levels with the introduction of light. Fos showed a markedly different profile. It had very low basal expression which was not demonstrably affected by darkness and its principal response was a marked transient induction upon exposure to light after darkness. These unique changes in expression highlight the complex response across IEGs to environmental input and suggest a genetic "on/off' signaling mechanism. There were marked differences in the laminar distribution of EGR-1 and Fos proteins between young and adult cats. In young animals, cells in all visual cortical layers showed high levels of EGR-1 and Fos proteins. In adults, immunostaining was largely specific to cells located above and below layer IV and only very faint labeling

  6. Purification and characterization of catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 and cloning and sequencing of its catA gene.

    PubMed Central

    Strachan, P D; Freer, A A; Fewson, C A

    1998-01-01

    A method was developed for the purification of catechol 1, 2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 that had been grown in the presence of benzyl alcohol. The enzyme has very similar apparent Km (1-2 microM) and Vmax (13-19 units/mg of protein) values for the intradiol cleavage of catechol, 3-methylcatechol and 4-methylcatechol and it is optimally active at pH9. Cross-linking studies indicate that the enzyme is a homodimer. It contains 0.6 atoms of Fe per subunit. The enzyme was crystallized with 15% (w/v) poly(ethylene glycol) 4000/0.33 M CaCl2/25 mM Tris (pH7.5) by using a microseeding technique. Preliminary X-ray characterization showed that the crystals are in space group C2 with unit-cell dimensions a=111.9 A, b=78.1 A, c=134.6 A, beta=100 degrees. An oligonucleotide probe, made by hemi-nested PCR, was used to clone the gene encoding catechol 1,2-dioxygenase (catA). The deduced 282-residue sequence corresponds to a protein of molecular mass 31539 Da, close to the molecular mass of 31558 Da obtained by electrospray MS of the purified enzyme. catA was subcloned into the expression vector pTB361, allowing the production of catechol 1,2-dioxygenase to approx. 40% of the total cellular protein. The deduced amino acid sequence of the enzyme has 56% and 75% identity with the catechol 1, 2-dioxygenases of Arthrobacter mA3 and Rhodococcus erythropolis AN-13 respectively, but less than 35% identity with intradiol catechol and chlorocatechol dioxygenases of Gram-negative bacteria. PMID:9677336

  7. Genetic testing in domestic cats

    PubMed Central

    Lyons, Leslie A.

    2012-01-01

    Varieties of genetic tests are currently available for the domestic cat that support veterinary health care, breed management, species identification, and forensic investigations. Approximately thirty-five genes contain over fifty mutations that cause feline health problems or alterations in the cat’s appearance. Specific genes, such as sweet and drug receptors, have been knocked-out of Felidae during evolution and can be used along with mtDNA markers for species identification. Both STR and SNP panels differentiate cat race, breed, and individual identity, as well as gender-specific markers to determine sex of an individual. Cat genetic tests are common offerings for commercial laboratories, allowing both the veterinary clinician and the private owner to obtain DNA test results. This article will review the genetic tests for the domestic cat, and their various applications in different fields of science. Highlighted are genetic tests specific to the individual cat, which are a part of the cat’s genome. PMID:22546621

  8. The effect of synthetic surfactant Exosurf on gene transfer in mouse lung in vivo.

    PubMed

    Raczka, E; Kukowska-Latallo, J F; Rymaszewski, M; Chen, C; Baker, J R

    1998-10-01

    Gene transfer in the lung holds promise for the treatment of diseases such as pulmonary fibrosis, cystic fibrosis and asthma. Pulmonary surfactant has been reported to enhance expression from endobronchial, adenovirus-mediated gene transfer in experimental animals. This study examines the effect of exogenous synthetic surfactant (Exosurf) on gene expression from naked plasmid DNA administered endobronchially to adult mice. Transfection efficiency was evaluated by quantifying the expression of chloramphenicol acetyltransferase (CAT) and luciferase (Luc) genes in the lung. Endobronchial administration of either CAT or Luc expression plasmid DNA resulted in detectable concentrations of each reporter protein. CAT expression from plasmid DNA was monitored after endobronchial administration with the maximal expression observed at 3-5 days after administration and decreasing for 5 days thereafter. When DNA was delivered in a 50% suspension of Exosurf, the expression of either CAT or Luc was significantly reduced by 89.6 +/- 1.4% and 82.7 +/- 10.5%, respectively. The decrease in Luc expression was closely correlated (r = 0.99, P < 0.001) to log concentration of surfactant in the plasmid buffer solution (IC50 = 8.6%). CAT expression was not altered when surfactant was administered either 2 h before or after plasmid DNA instillation. Examination of the components of Exosurf revealed that two compounds, DPPC and tyloxapol, showed inhibitory effects on CAT expression. However, the inhibition caused by Exosurf appeared greater than that of either component. Our results suggest that the lung surfactant is a barrier to transfection of the endobronchial airway and may be partly responsible for the low expression of exogenous DNA in vivo in the bronchial tree.

  9. An extracellular factor regulating expression of the chromosomal aminoglycoside 2'-N-acetyltransferase of Providencia stuartii.

    PubMed

    Rather, P N; Parojcic, M M; Paradise, M R

    1997-08-01

    The chromosomal aac(2')-Ia gene in Providencia stuartii encodes a housekeeping 2'-N-acetyltransferase [AAC(2')-Ia] involved in the acetylation of peptidoglycan. In addition, the AAC(2')-Ia enzyme also acetylates and confers resistance to the clinically important aminoglycoside antibiotics gentamicin, tobramycin, and netilmicin. Expression of the aac(2')-Ia gene was found to be strongly influenced by cell density, with a sharp decrease in aac(2')-Ia mRNA accumulation as cells approached stationary phase. This decrease was mediated by the accumulation of an extracellular factor, designated AR (for acetyltransferase repressing)-factor. AR-factor was produced in both minimal and rich media and acted in a manner that was strongly dose dependent. The activity of AR-factor was also pH dependent, with optimal activity at pH 8.0 and above. Biochemical characterization of conditioned media from P. stuartii has shown that AR-factor is between 500 and 1,000 Da in molecular size and is heat stable. In addition, AR-factor was inactivated by a variety of proteases, suggesting that it may be a small peptide.

  10. Genetic heterogeneity among slow acetylator N-acetyltransferase 2 phenotypes in cryopreserved human hepatocytes.

    PubMed

    Doll, Mark A; Hein, David W

    2017-07-01

    Genetic polymorphisms in human N-acetyltransferase 2 (NAT2) modify the metabolism of numerous drugs and carcinogens. These genetic polymorphisms modify both drug efficacy and toxicity and cancer risk associated with carcinogen exposure. Previous studies have suggested phenotypic heterogeneity among different NAT2 slow acetylator genotypes. NAT2 phenotype was investigated in vitro and in situ in samples of human hepatocytes obtained from various NAT2 slow and intermediate NAT2 acetylator genotypes. NAT2 gene dose response (NAT2*5B/*5B > NAT2*5B/*6A > NAT2*6A/*6A) was observed towards the N-acetylation of the NAT2-specific drug sulfamethazine by human hepatocytes both in vitro and in situ. N-acetylation of 4-aminobiphenyl, an arylamine carcinogen substrate for both N-acetyltransferase 1 and NAT2, showed the same trend both in vitro and in situ although the differences were not significant (p > 0.05). The N-acetylation of the N-acetyltransferase 1-specific substrate p-aminobenzoic acid did not follow this trend. In comparisons of NAT2 intermediate acetylator genotypes, differences in N-acetylation between NAT2*4/*5B and NAT2*4/*6B hepatocytes were not observed in vitro or in situ towards any of these substrates. These results further support phenotypic heterogeneity among NAT2 slow acetylator genotypes, consistent with differential risks of drug failure or toxicity and cancer associated with carcinogen exposure.

  11. The gene for death agonist BID maps to the region of human 22q11.2 duplicated in cat eye syndrome chromosomes and to mouse chromosome 6.

    PubMed

    Footz, T K; Birren, B; Minoshima, S; Asakawa, S; Shimizu, N; Riazi, M A; McDermid, H E

    1998-08-01

    Cat eye syndrome (CES) is associated with a duplication of a segment of human chromosome 22q11.2. Only one gene, ATP6E, has been previously mapped to this duplicated region. We now report the mapping of the human homologue of the apoptotic agonist Bid to human chromosome 22 near locus D22S57 in the CES region. Dosage analysis demonstrated that BID is located just distal to the CES region critical for the majority of malformations associated with the syndrome (CESCR), as previously defined by a single patient with an unusual supernumerary chromosome. However, BID remains a good candidate for involvement in CES-related mental impairment, and its overexpression may subtly add to the phenotype of CES patients. Our mapping of murine Bid confirms that the synteny of the CESCR and the 22q11 deletion syndrome critical region immediately telomeric on human chromosome 22 is not conserved in mice. Bid and adjacent gene Atp6e were found to map to mousechromosome 6, while the region homologous to the DGSCR is known to map to mouse chromosome 16.

  12. Overexpression of a Maize Sulfite Oxidase Gene in Tobacco Enhances Tolerance to Sulfite Stress via Sulfite Oxidation and CAT-Mediated H2O2 Scavenging

    PubMed Central

    Xia, Zongliang; Sun, Kaile; Wang, Meiping; Wu, Ke; Zhang, Hua; Wu, Jianyu

    2012-01-01

    Sulfite oxidase (SO) plays an important role in sulfite metabolism. To date, the molecular mechanisms of sulfite metabolism in plants are largely unknown. Previously, a full-length cDNA of the putative sulfite oxidase gene from maize (ZmSO) was cloned, and its response to SO2/sulfite stress at the transcriptional level was characterized. In this study, the recombinant ZmSO protein was purified from E.coli. It exhibited sulfite-dependent activity and had strong affinity for the substrate sulfite. Over-expression (OE) of ZmSO in tobacco plants enhanced their tolerance to sulfite stress. The plants showed much less damage, less sulfite accumulation, but greater amounts of sulfate. This suggests that tolerance of transgenic plants to sulfite was enhanced by increasing SO expression levels. Interestingly, H2O2 accumulation levels by histochemical detection and quantitative determination in the OE plants were much less than those in the wild-type upon sulfite stress. Furthermore, reductions of catalase levels detected in the OE lines were considerably less than in the wild-type plants. This indicates that SO may play an important role in protecting CAT from inhibition by excess sulfite. Collectively, these data demonstrate that transgenic tobacco plants over-expressing ZmSO enhance tolerance to excess sulfite through sulfite oxidation and catalase-mediated hydrogen peroxide scavenging. This is the first SO gene from monocots to be functionally characterized. PMID:22693572

  13. 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.

  14. 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.

  15. Identification of a lens-specific regulatory region (LSR) of the murine alpha B-crystallin gene.

    PubMed

    Gopal-Srivastava, R; Piatigorsky, J

    1994-04-11

    Previous studies have shown that the -661/+44 sequence of the murine alpha B-crystallin gene contains a muscle-preferred enhancer (-426/-257) and can drive the bacterial chloramphenicol acetyltransferase (CAT) gene in the lens, skeletal muscle and heart of transgenic mice. Here we show that transgenic mice carrying a truncated -164/+44 fragment of the alpha B-crystallin gene fused to the CAT gene expressed exclusively in the lens; by contrast mice carrying a -426/+44 fragment of the alpha B gene fused to CAT expressed highly in the lens, skeletal muscle and heart, and slightly in the lung, brain, kidney, spleen and liver. DNase I protection experiments indicated that the -147/-118 sequence is protected by nuclear proteins from alpha TN4-1 lens cell line, but not by nuclear proteins from myotubes of the C2C12 cell line. Site directed mutagenesis of this sequence decreased promoter activity in transiently-transfected lens cells, consistent with this sequence being a lens-specific regulatory region (LSR). We conclude that the -426/-257 enhancer is required for expression in skeletal muscle, heart and possibly other tissues, and that the -164/+44 sequence of the alpha B-crystallin gene is sufficient for expression in the lens of transgenic mice.

  16. Age-associated mitochondrial oxidative decay: Improvement of carnitine acetyltransferase substrate-binding affinity and activity in brain by feeding old rats acetyl-l- carnitine and/or R-α-lipoic acid

    PubMed Central

    Liu, Jiankang; Killilea, David W.; Ames, Bruce N.

    2002-01-01

    We test whether the dysfunction with age of carnitine acetyltransferase (CAT), a key mitochondrial enzyme for fuel utilization, is due to decreased binding affinity for substrate and whether this substrate, fed to old rats, restores CAT activity. The kinetics of CAT were analyzed by using the brains of young and old rats and of old rats supplemented for 7 weeks with the CAT substrate acetyl-l-carnitine (ALCAR) and/or the mitochondrial antioxidant precursor R-α-lipoic acid (LA). Old rats, compared with young rats, showed a decrease in CAT activity and in CAT-binding affinity for both substrates, ALCAR and CoA. Feeding ALCAR or ALCAR plus LA to old rats significantly restored CAT-binding affinity for ALCAR and CoA, and CAT activity. To explore the underlying mechanism, lipid peroxidation and total iron and copper levels were assayed; all increased in old rats. Feeding old rats LA or LA plus ALCAR inhibited lipid peroxidation but did not decrease iron and copper levels. Ex vivo oxidation of young-rat brain with Fe(II) caused loss of CAT activity and binding affinity. In vitro oxidation of purified CAT with Fe(II) inactivated the enzyme but did not alter binding affinity. However, in vitro treatment of CAT with the lipid peroxidation products malondialdehyde or 4-hydroxy-nonenal caused a decrease in CAT-binding affinity and activity, thus mimicking age-related change. Preincubation of CAT with ALCAR or CoA prevented malondialdehyde-induced dysfunction. Thus, feeding old rats high levels of key mitochondrial metabolites can ameliorate oxidative damage, enzyme activity, substrate-binding affinity, and mitochondrial dysfunction. PMID:11854488

  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. The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat

    USDA-ARS?s Scientific Manuscript database

    The Toxoplasma gondii genome contains two aromatic amino acid hydroxylase genes, AAH1 and AAH2, which encode proteins that produce L-DOPA, which can serve as a precursor of catecholamine neurotransmitters. It has been suggested that this pathway elevates host dopamine levels thus making infected rod...

  19. 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.

  20. 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

  1. The histone acetyltransferase MOF activates hypothalamic polysialylation to prevent diet-induced obesity in mice

    PubMed Central

    Brenachot, Xavier; Rigault, Caroline; Nédélec, Emmanuelle; Laderrière, Amélie; Khanam, Tasneem; Gouazé, Alexandra; Chaudy, Sylvie; Lemoine, Aleth; Datiche, Frédérique; Gascuel, Jean; Pénicaud, Luc; Benani, Alexandre

    2014-01-01

    Overfeeding causes rapid synaptic remodeling in hypothalamus feeding circuits. Polysialylation of cell surface molecules is a key step in this neuronal rewiring and allows normalization of food intake. Here we examined the role of hypothalamic polysialylation in the long-term maintenance of body weight, and deciphered the molecular sequence underlying its nutritional regulation. We found that upon high fat diet (HFD), reduced hypothalamic polysialylation exacerbated the diet-induced obese phenotype in mice. Upon HFD, the histone acetyltransferase MOF was rapidly recruited on the St8sia4 polysialyltransferase-encoding gene. Mof silencing in the mediobasal hypothalamus of adult mice prevented activation of the St8sia4 gene transcription, reduced polysialylation, altered the acute homeostatic feeding response to HFD and increased the body weight gain. These findings indicate that impaired hypothalamic polysialylation contribute to the development of obesity, and establish a role for MOF in the brain control of energy balance. PMID:25161885

  2. Characterization of a plasmid carrying cat, ermB and tetS genes in a foodborne Listeria monocytogenes strain and uptake of the plasmid by cariogenic Streptococcus mutans.

    PubMed

    Li, Lili; Olsen, Rikke Heidemann; Shi, Lei; Ye, Lei; He, Jianhua; Meng, Hecheng

    2016-12-05

    A multi-drug resistant (MDR) Listeria monocytogenes isolate (serotype 1/2c) was recovered from a quick-frozen rice flour product collected from Langfang city in northern China. PCR screening identified the presence of cat, ermB and tetS genes. The plasmid profile of the strain showed the presence of an approximately 22.4-kb plasmid. Curing of this plasmid resulted in the loss of cat, ermB and tetS genes and increased susceptibility to several antibiotics, suggesting the involvement of the plasmid in multiple antibiotic resistances. Moreover, the plasmid was able to be uptaken by human oral pathogen Streptococcus mutans by natural transformation and resulted in the acquiring of multiple resistances in the transconjugants. This study contributes to our knowledge on acquired multi-drug resistance in foodborne pathogenic L.monocytogenes, which will add to a better understanding of effective clinical management of listeriosis.

  3. Diversity among the gram-positive acetyltransferases inactivating streptogramin A and structurally related compounds and characterization of a new staphylococcal determinant, vatB.

    PubMed

    Allignet, J; el Solh, N

    1995-09-01

    A gene encoding an acetyltransferase inactivating streptogramin A (SgA) and structurally similar compounds was isolated from a staphylococcal plasmid and sequenced. This gene, designated vatB, potentially encodes a 212-amino-acid protein, VatB, of 23,320 Da with 47.4 and 58.4% amino acid identities with two other enzymes with the same activity, Vat and SatA, respectively, which are encoded by a staphylococcal plasmid and an enterococcal plasmid, respectively. The C-terminal parts of these three enzymes share significant homology with the C-terminal parts of 10 other acetyltransferases modifying various substrates. A pair of degenerate primers representing the conserved motifs shared by VatB, Vat, and SatA was designed to detect the three genes encoding these SgA acetyltransferases. Five of 12 clinical SgAr Staphylococcus aureus isolates tested carried neither these genes nor the gene vga, which confers resistance to SgA by a different mechanism, suggesting that another gene(s) and possibly another mechanism of resistance to SgA in staphylococci remains to be characterized.

  4. Diversity among the gram-positive acetyltransferases inactivating streptogramin A and structurally related compounds and characterization of a new staphylococcal determinant, vatB.

    PubMed Central

    Allignet, J; el Solh, N

    1995-01-01

    A gene encoding an acetyltransferase inactivating streptogramin A (SgA) and structurally similar compounds was isolated from a staphylococcal plasmid and sequenced. This gene, designated vatB, potentially encodes a 212-amino-acid protein, VatB, of 23,320 Da with 47.4 and 58.4% amino acid identities with two other enzymes with the same activity, Vat and SatA, respectively, which are encoded by a staphylococcal plasmid and an enterococcal plasmid, respectively. The C-terminal parts of these three enzymes share significant homology with the C-terminal parts of 10 other acetyltransferases modifying various substrates. A pair of degenerate primers representing the conserved motifs shared by VatB, Vat, and SatA was designed to detect the three genes encoding these SgA acetyltransferases. Five of 12 clinical SgAr Staphylococcus aureus isolates tested carried neither these genes nor the gene vga, which confers resistance to SgA by a different mechanism, suggesting that another gene(s) and possibly another mechanism of resistance to SgA in staphylococci remains to be characterized. PMID:8540711

  5. Molecular cloning and characterization of cat, gpx1 and Cu/Zn-sod genes in pengze crucian carp (Carassius auratus var. Pengze) and antioxidant enzyme modulation induced by hexavalent chromium in juveniles.

    PubMed

    Li, Meng; Zheng, Yao; Liang, Hongwei; Zou, Linhu; Sun, Jiejie; Zhang, Yingying; Qin, Fang; Liu, Shaozhen; Wang, Zaizhao

    2013-04-01

    Hexavalent chromium (Cr(6+)) is a common pollutant transient metal with high toxicity in the environment. The toxicological effects partly result from oxidative damage due to the production of excessive reactive oxygen species (ROS) in the reductive process of Cr(6+). To explore the influence of ROS induced directly by Cr(6+) on the oxidative stress generation and antioxidant system, the full length cDNAs of antioxidant-related genes cat, gpx1 and Cu/Zn-sod were successfully acquired from pengze crucian carp first and analyzed. Furthermore, the mRNA expression of the antioxidant genes encompassing catalase (cat), copper/zinc superoxide dismutase (Cu/Zn-sod) and glutathione peroxidase (gpx1), antioxidant enzyme activities of CAT, SOD, and GPx and total protein content were further studied in the gill, intestine and liver of pengze crucian carp (Carassius auratus var. Pengze) juveniles upon acute exposure to Cr(6+) at concentrations of 0.1, 1.0, 10 and 100 mg/L for 4 days. Differential significant changes of the antioxidant enzymes and gene expression were observed in different tissues. The findings contribute to better understanding the antioxidant mechanisms induced by Cr(6+) and selecting the organic-specific sensitive biomarkers to monitor the safety of the aquatic ecosystem. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Study of Genotypes and virB4 Secretion Gene of Bartonella henselae Strains from Patients with Clinically Defined Cat Scratch Disease

    PubMed Central

    Woestyn, Sophie; Olivé, Nathalie; Bigaignon, Geoffroy; Avesani, Véronique; Delmée, Michel

    2004-01-01

    Bartonella henselae is the causative agent of cat scratch disease (CSD), which usually presents as a self-limiting lymphadenopathy. Occasionally, the bacteria will spread and be responsible for tissue and visceral involvement. Two B. henselae genotypes (genotypes I and II) have been described to be responsible for uncomplicated CSD on the basis of 16S rRNA sequence analysis. A type IV secretion system (T4SS) similar to the virulence-associated VirB system of Agrobacterium tumefaciens was recently identified in the B. henselae Houston-1 genotype I strain. We studied the correlations of the B. henselae genotypes with the clinical presentations and with the presence of T4SS. Isolates originated from CSD patients whose lymph nodes were prospectively analyzed. B. henselae genotype I was identified in 13 of 42 patients (30%). Among these, two teenage twins presented with hepatosplenic CSD and one immunocompetent adult presented with osteomyelitis. Genotype II was detected in 28 of 42 patients (67%), all of whom presented with uncomplicated CSD. The last patient was infected with both genotypes. T4SS was studied by PCR amplification of the virB4 gene. Amplification of virB4 codons 146 to 256, 273 to 357, and 480 to 537 enabled us to detect 66, 90, and 100% of the B. henselae isolates, respectively. Sequence analysis revealed sequence variations that correlated with genotype distribution. Our studies suggest that B. henselae genotype I strains harbor virB4 genes that are different from those harbored by genotype II strains and that genotype I strains might be more pathogenic. PMID:15070983

  7. Histological and dermatoscopic description of sphynx cat skin.

    PubMed

    Genovese, David W; Johnson, Tammy L; Lamb, Ken E; Gram, Wallace D

    2014-12-01

    Histological and hair coat abnormalities of the alopecic sphynx cat have not been described in detail. The hairless allele (hr) in sphynx cats represents a mutation in the gene for keratin 71, a protein expressed in the inner root sheath of humans and mice. To describe the histological and dermatoscopic abnormalities of sphynx cat skin. Skin biopsies were collected from 14 sphynx cats and five cats with normal coats. Dermatoscopic examinations were performed on 11 sphynx cats and six additional control cats. Vertical and horizontal sections of skin biopsy samples from sphynx and control cats were reviewed. Dermatoscopic images were compared between sphynx and control cats. Sphynx cat hair follicles were often small, curved and kinked and demonstrated infundibular hyperkeratosis and dilatation. Changes in the inner root sheath of sphynx cats included a poorly defined Henle's layer in addition to vacuolar-like changes and eosinophilic globules in Huxley's layer. Dermal papillae in sphynx cat anagen bulbs lacked the normal flame shape and were surrounded by epithelial cells arranged in a disorderly manner. The degree of follicular abnormalities varied between follicles. Follicular density was similar for both sphynx cats and control animals. Sphynx cat hair shafts were misshapen, smaller in diameter and rarely medullated. Dermatoscopy revealed similar hair coat density in sphynx and control cats. Sphynx cats demonstrated hair follicle dysplasia, with abnormal shaft production but without a decrease in follicle quantity. Abnormalities in sphynx cat follicles are similar to those in murine KRT71 mutants and suggest abnormal hair shaft keratinization. © 2014 ESVD and ACVD.

  8. 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.

  9. Cat-Scratch Disease

    MedlinePlus

    ... Spanish Announcements Connect With Us Digital Press Kit Cat-Scratch Disease Recommend on Facebook Tweet Share Compartir ( ... play and learn how to attack prey. How cats and people become infected Kitten playing with a ...

  10. Cat Scratch Disease

    MedlinePlus

    Cat scratch disease (CSD) is an illness caused by the bacterium Bartonella henselae. Almost half of all cats carry the infection ... symptoms of CSD, call your doctor. Centers for Disease Control and Prevention

  11. [Primary hyperaldosteronism in cats].

    PubMed

    Willi, B; Kook, P H; Quante, S; Boretti, F; Sieber-Ruckstuhl, N S; Grest, P; Scherrer, O; Riond, B; Hofmann-Lehmann, R; Nussberger, J; Reusch, C E

    2012-12-01

    Primary hyperaldosteronism is a clinical syndrome characterized by an elevated aldosterone secretion by the adrenals. The present case series describes 7 cats with primary hyperaldosteronism, which were presented between 2002 and 2011. Common clinical symptoms were weakness, anorexia, cervical ventroflexion and blindness. All cats showed hypokalemia. In 6 cats, blood pressure was determined: 5 cats showed hypertension, of which 4 animals exhibited retinal detachment and blindness. In the ultrasonographic examination, unilateral adrenomegaly was present in 6 cats whereas one animal showed normal adrenals. In 4 cats, the serum aldosterone concentration was above the reference range. Five cats underwent unilateral adrenalectomy, which was accomplished uneventfully and returned the electrolytes back to normal. Histopathological examination of the adrenals revealed 2 carcinomas and 4 adenomas; one cat with ultrasonographic normal adrenals exhibited bilateral nodular hyperplasia.

  12. Chronic progressive polyarthritis in a female cat.

    PubMed

    Oohashi, Eiji; Yamada, Kazutaka; Oohashi, Mirai; Ueda, Junji

    2010-04-01

    Feline chronic progressive polyarthritis is a rare immune-mediated disease that has only previously been reported in male cats. A one-year-old female cat was presented with anorexia, lassitude and lameness. The tarsal, carpal and elbow joints revealed swelling, pain, stiffness, crepitus and regional lymphadenopathy, and fever was present. The cat was clinically diagnosed with chronic progressive polyarthritis based on the fever, swelling of joints, imaging of erosive proliferative periosteal polyarthritis, positivity for antinuclear antibody, synovial fluid analyses and urinalyses. Both feline leukemia virus antigen and feline immunodeficiency virus antibody were positive. Using hair root DNA, polymerase chain reaction amplification targeting the sex-determining region on the Y chromosome gene amplified the fragment of DNA from a normal male cat, but not amplified from a normal female cat or the present cat. Accordingly, the present cat was classified as genetically female. Cyclosporine treatment was started, and the general condition and movement quickly improved and continued for 8 months post-diagnosis. This is the first report of chronic progressive polyarthritis in a female cat.

  13. New perspectives for the regulation of acetyltransferase MOF

    PubMed Central

    Li, Xiangzhi; Dou, Yali

    2012-01-01

    In higher eukaryotes, histone acetyltransferase MOF (male absent on the first) is the major enzyme that acetylates histone H4 lysine 16, a prevalent mark associated with chromatin decondensation. Recent studies show that MOF resides in two different but evolutionarily conserved complexes, MSL and MOF-MSL1v1. Although these two MOF complexes have indistinguishable activity on histone H4 K16, they differ dramatically in acetylating non-histone substrate p53. The regulation of MOF activity in these complexes remains elusive. Given the evolution conservation of MOF and the importance of H4 K16 acetylation in maintaining higher order chromatin structures, understanding the function and regulation of MOF bears great significance. Here, we discussed the key differences in two MOF complexes that may shed light on the regulation of their distinct acetyltransferase activities. We also discussed coordinated functions of two MOF complexes with different histone methyltransferase complexes in transcription regulation. PMID:20305383

  14. Radioenzymatic assays for aminoglycosides with kanamycin 6'- acetyltransferase

    SciTech Connect

    Weber, A.; Smith, A.L.; Opheim, K.E.

    1985-03-01

    To facilitate the rapid and accurate quantitation of parenterally administered aminoglycosides, the optimum conditions (pH, duration of incubation, and cofactor concentrations) were defined to permit radioenzymatic assays with kanamycin acetyltransferase. The accuracy in quantitating tobramycin, netilmicin, kanamycin, and amikacin at concentrations in the therapeutic range was greater than 90%, with a mean recovery of 102.8%. The mean of the interassay coefficient of variation was 7.8%. Typical standard curves at six different concentrations resulted in a correlation coefficient (r value) of greater than 0.99 for each aminoglycoside. The radioenzymatic assay correlates well with the bioassay (tobramycin and netilmicin) and radioimmunoassay (amikacin and kanamycin); the correlation coefficient is greater than 0.90 for all. The authors conclude that the radioenzymatic assay utilizing kanamycin 6'-acetyltransferase is feasible for all commercially available parenterally administered aminoglycosides.

  15. New perspectives for the regulation of acetyltransferase MOF.

    PubMed

    Li, Xiangzhi; Dou, Yali

    2010-04-01

    In higher eukaryotes, histone acetyltransferase MOF (male absent on the first) is the major enzyme that acetylates histone H4 lysine 16, a prevalent mark associated with chromatin decondensation. Recent studies show that MOF resides in two different but evolutionarily conserved complexes, MSL and MOF-MSL1v1. Although these two MOF complexes have indistinguishable activity on histone H4 K16, they differ dramatically in acetylating non-histone substrate p53. The regulation of MOF activity in these complexes remains elusive. Given the evolution conservation of MOF and the importance of H4 K16 acetylation in maintaining higher order chromatin structures, understanding the function and regulation of MOF bears great significance. Here, we discussed the key differences in two MOF complexes that may shed light on the regulation of their distinct acetyltransferase activities. We also discussed coordinated functions of two MOF complexes with different histone methyltransferase complexes in transcription regulation.

  16. Molecular Basis for the Cat-2 Null Phenotype in Maize

    PubMed Central

    Bethards, L. A.; Scandalios, J. G.

    1988-01-01

    Previous reports have described several maize lines whose developmental patterns of catalase gene expression vary from the ``typical'' maize line, W64A. Among these variants are the lines A16 and A338, both found to be null for the CAT-2 protein. Identification of a third CAT-2 null line, designated A340, is described. RNA blots and S1 nuclease protection analysis indicate that all three CAT-2 null lines produce a similarly shortened Cat2 transcript. The molecular basis for this aberrant Cat2 transcript is discussed. PMID:8608925

  17. Molecular basis for the CAT-2 null phenotype in maize

    SciTech Connect

    Bethards, L.A.; Scandalios, J.G.

    1988-01-01

    Previous reports have described several maize lines whose developmental patterns of catalase gene expression vary from the typical maize line, W64A. Among these variants are the lines A16 and A338, both found to be null for the CAT-2 protein. Identification of a third CAT-2 null line, designated A340, is described. RNA blots and S1 nuclease protection analysis, using (/sup 32/P)-labeled dCTP, indicate that all three CAT-2 null lines produce a similarly shortened Cat2 transcript. The molecular basis for this aberrant Cat2 transcript is discussed.

  18. Phenylpropanoid pathway intermediates regulate transient expression of a chalcone synthase gene promoter.

    PubMed Central

    Loake, G J; Choudhary, A D; Harrison, M J; Mavandad, M; Lamb, C J; Dixon, R A

    1991-01-01

    A chimeric gene construct containing a bean chalcone synthase (CHS) promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene was strongly expressed when electroporated into alfalfa protoplasts that were then exposed to a fungal elicitor. Low concentrations (5 x 10(-6) to 10(-4) M) of exogenously applied trans-cinnamic acid (CA), the first intermediate of the phenylpropanoid pathway, slightly stimulated elicitor-induced CAT expression, whereas high concentrations (greater than 10(-4) M) severely reduced expression to below the levels observed in the absence of elicitor. In contrast, trans-p-coumaric acid (4-CA, the second intermediate in the pathway) stimulated expression from the CHS promoter up to 4.5-fold at 5 x 10(-4) M. Expression of CAT driven by the promoters of other elicitor-inducible defense response genes was not markedly affected by CA or 4-CA. Stimulation of CHS promoter expression by low concentrations of CA and 4-CA was completely abolished by 5' deletion to position -130, but not -174. When the -180 to -130 region of the CHS15 promoter was coelectroporated into elicited protoplasts on a separate plasmid along with the intact -326 CHS-CAT construct, the decreased CAT expression as a function of CA or 4-CA concentration was consistent with the coelectroporated sequence competing in trans with the intact promoter for the binding of a factor(s) involved in the up regulation of CHS transcription by 4-CA and low concentrations of CA. Our data support the hypothesis that phenylpropanoid compounds may act as natural and specific regulators of plant gene expression and define the location of a cis-acting element in the CHS15 promoter involved in the induction by phenylpropanoid pathway intermediates. PMID:1820822

  19. CATS Featured Articles

    Atmospheric Science Data Center

    2017-01-31

      CATS Featured Articles       A Slice of Cirrus: Image of ... just hours before by the Cloud-Aerosol Transport System (CATS) onboard the International Space Station. Nighttime View of Raung Volcanic Plume : Natural Hazards  - The CATS instrument slices through darkness to reveal the vertical structure of a ...

  20. Ethyl acetate production by the elusive alcohol acetyltransferase from yeast.

    PubMed

    Kruis, Aleksander J; Levisson, Mark; Mars, Astrid E; van der Ploeg, Max; Garcés Daza, Fernando; Ellena, Valeria; Kengen, Servé W M; van der Oost, John; Weusthuis, Ruud A

    2017-05-01

    Ethyl acetate is an industrially relevant ester that is currently produced exclusively through unsustainable processes. Many yeasts are able to produce ethyl acetate, but the main responsible enzyme has remained elusive, hampering the engineering of novel production strains. Here we describe the discovery of a new enzyme (Eat1) from the yeast Wickerhamomyces anomalus that resulted in high ethyl acetate production when expressed in Saccharomyces cerevisiae and Escherichia coli. Purified Eat1 showed alcohol acetyltransferase activity with ethanol and acetyl-CoA. Homologs of eat1 are responsible for most ethyl acetate synthesis in known ethyl acetate-producing yeasts, including S. cerevisiae, and are only distantly related to known alcohol acetyltransferases. Eat1 is therefore proposed to compose a novel alcohol acetyltransferase family within the α/β hydrolase superfamily. The discovery of this novel enzyme family is a crucial step towards the development of biobased ethyl acetate production and will also help in selecting improved S. cerevisiae brewing strains. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  1. Generation of cloned transgenic cats expressing red fluorescence protein.

    PubMed

    Yin, Xi Jun; Lee, Hyo Sang; Yu, Xian Feng; Choi, Eugene; Koo, Bon Chul; Kwon, Mo Sun; Lee, Young S; Cho, Su Jin; Jin, Guang Zhen; Kim, Lyoung Hyo; Shin, Hyoung Doo; Kim, Teoan; Kim, Nam Hyung; Kong, Il Keun

    2008-03-01

    A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries. Donor fibroblasts were obtained from an ear skin biopsy of a white male Turkish Angora cat, cultured for one to two passages, and subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein (RFP) gene. A total of 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 +/- 7.5 per recipient). Three surrogate mothers were successfully impregnated (27.3%) and delivered two liveborn and one stillborn kitten at 65 to 66 days of gestation. Analysis of nine feline-specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole-body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cat but not in the surrogate mother cat. Red fluorescence was detected in tissue samples, including hair, muscle, brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, tongue, and even excrement of the stillborn TG cat. These results suggest that this nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.

  2. Hairless cats in Great Britain.

    PubMed

    Hendy-Ibbs, P M

    1984-01-01

    Ten hairless kittens are known to have been born in Britain since 1978. Pedigree study supports the hypothesis of a monogenic, recessive mode of inheritance proposed in previous reports. A review of the literature suggests the possibility of at least two mutations giving rise to hairless cats, one of which has normal whiskers and the other attenuated whiskers. For these, the gene symbols hi, and hr, respectively, have been proposed.

  3. Integration of Bioorthogonal Probes and Q-FRET for the Detection of Histone Acetyltransferase Activity.

    PubMed

    Han, Zhen; Luan, Yepeng; Zheng, Yujun George

    2015-12-01

    Histone acetyltransferases (HATs) are key players in the epigenetic regulation of gene function. The recent discovery of diverse HAT substrates implies a broad spectrum of cellular functions of HATs. Many pathological processes are also intimately associated with the dysregulation of HAT levels and activities. However, detecting the enzymatic activity of HATs has been challenging, and this has significantly impeded drug discovery. To advance the field, we developed a convenient one-pot, mix-and-read strategy that is capable of directly detecting the acylated histone product through a fluorescent readout. The strategy integrates three technological platforms-bioorthogonal HAT substrate labeling, alkyne-azide click chemistry, and quenching FRET-into one system for effective probing of HAT enzyme activity. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. 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

  5. Structural Basis of Substrate-Binding Specificity of Human Arylamine N-acetyltransferases

    SciTech Connect

    Wu,H.; Dombrovsky, L.; Tempel, W.; Martin, F.; Loppnau, P.; Goodfellow, G.; Grant, D.; Plotnikov, A.

    2007-01-01

    The human arylamine N-acetyltransferases NAT1 and NAT2 play an important role in the biotransformation of a plethora of aromatic amine and hydrazine drugs. They are also able to participate in the bioactivation of several known carcinogens. Each of these enzymes is genetically variable in human populations, and polymorphisms in NAT genes have been associated with various cancers. Here we have solved the high resolution crystal structures of human NAT1 and NAT2, including NAT1 in complex with the irreversible inhibitor 2-bromoacetanilide, a NAT1 active site mutant, and NAT2 in complex with CoA, and have refined them to 1.7-, 1.8-, and 1.9- Angstroms resolution, respectively. The crystal structures reveal novel structural features unique to human NATs and provide insights into the structural basis of the substrate specificity and genetic polymorphism of these enzymes.

  6. An E-box/M-CAT hybrid motif and cognate binding protein(s) regulate the basal muscle-specific and cAMP-inducible expression of the rat cardiac alpha-myosin heavy chain gene.

    PubMed

    Gupta, M P; Gupta, M; Zak, R

    1994-11-25

    Expression of the cardiac myosin heavy chain (MHC) genes is regulated developmentally and by numerous epigenetic factors. Here we report the identification of a cis-regulatory element and cognate nuclear binding protein(s) responsible for cAMP-induced expression of the rat cardiac alpha-MHC gene. By Northern blot analysis, we found that, in primary cultures of fetal rat heart myocytes, the elevation of intracellular levels of cAMP results in up-regulation of alpha-MHC and down-regulation of beta-MHC mRNA expression. This effect of cAMP was dependent upon the basal level of expression of both MHC transcripts and was sensitive to cycloheximide. In transient expression analysis employing a series of alpha-MHC/CAT constructs, we identified a 31-base pair fragment located in the immediate upstream region (-71 to -40), which confers both muscle-specific and cAMP-inducible expression of the gene. Within this 31-base pair fragment there are two regions, an AT-rich portion and a hybrid motif which contains overlapping sequences of E-box and M-CAT binding sites (GGCACGTGGAATG). By substitution mutation analysis, both elements were found important for the basal muscle-specific expression; however, the cAMP-inducible expression of the gene is conferred only by the E-box/M-CAT hybrid motif (EM element). Using mobility gel shift competition assay, immunoblotting, and UV-cross-linking analyses, we found that a protein binding to the EM element is indistinguishable from the transcription enhancer factor-1 (TEF-1) in terms of sequence recognition, molecular mass, and immunoreactivity. Methylation interference and point mutation analyses indicate that, besides M-CAT sequences, center CG dinucleotides of the E-box motif CACGTG are essential for protein binding to the EM element and for its functional activity. Furthermore, our data also show that, in addition to TEF-1, another HF-1a-related factor may be recognized by the alpha-MHC gene EM element. These results are first to

  7. Garcinol Inhibits GCN5-Mediated Lysine Acetyltransferase Activity and Prevents Replication of the Parasite Toxoplasma gondii

    PubMed Central

    Jeffers, Victoria; Gao, Hongyu; Checkley, Lisa A.; Liu, Yunlong; Ferdig, Michael T.

    2016-01-01

    Lysine acetylation is a critical posttranslational modification that influences protein activity, stability, and binding properties. The acetylation of histone proteins in particular is a well-characterized feature of gene expression regulation. In the protozoan parasite Toxoplasma gondii, a number of lysine acetyltransferases (KATs) contribute to gene expression and are essential for parasite viability. The natural product garcinol was recently reported to inhibit enzymatic activities of GCN5 and p300 family KATs in other species. Here we show that garcinol inhibits TgGCN5b, the only nuclear GCN5 family KAT known to be required for Toxoplasma tachyzoite replication. Treatment of tachyzoites with garcinol led to a reduction of global lysine acetylation, particularly on histone H3 and TgGCN5b itself. We also performed transcriptome sequencing (RNA-seq), which revealed increasing aberrant gene expression coincident with increasing concentrations of garcinol. The majority of the genes that were most significantly affected by garcinol were also associated with TgGCN5b in a previously reported chromatin immunoprecipitation assay with microarray technology (ChIP-chip) analysis. The dysregulated gene expression induced by garcinol significantly inhibits Toxoplasma tachyzoite replication, and the concentrations used exhibit no overt toxicity on human host cells. Garcinol also inhibits Plasmodium falciparum asexual replication with a 50% inhibitory concentration (IC50) similar to that for Toxoplasma. Together, these data support that pharmacological inhibition of TgGCN5b leads to a catastrophic failure in gene expression control that prevents parasite replication. PMID:26810649

  8. N-Acetyltransferase Mpr1 confers ethanol tolerance on Saccharomyces cerevisiae by reducing reactive oxygen species.

    PubMed

    Du, Xiaoyi; Takagi, Hiroshi

    2007-07-01

    N-Acetyltransferase Mpr1 of Saccharomyces cerevisiae can reduce intracellular oxidation levels and protect yeast cells under oxidative stress, including H(2)O(2), heat-shock, or freeze-thaw treatment. Unlike many antioxidant enzyme genes induced in response to oxidative stress, the MPR1 gene seems to be constitutively expressed in yeast cells. Based on a recent report that ethanol toxicity is correlated with the production of reactive oxygen species (ROS), we examined here the role of Mpr1 under ethanol stress conditions. The null mutant of the MPR1 and MPR2 genes showed hypersensitivity to ethanol stress, and the expression of the MPR1 gene conferred stress tolerance. We also found that yeast cells exhibited increased ROS levels during exposure to ethanol stress, and that Mpr1 protects yeast cells from ethanol stress by reducing intracellular ROS levels. When the MPR1 gene was overexpressed in antioxidant enzyme-deficient mutants, increased resistance to H(2)O(2) or heat shock was observed in cells lacking the CTA1, CTT1, or GPX1 gene encoding catalase A, catalase T, or glutathione peroxidase, respectively. These results suggest that Mpr1 might compensate the function of enzymes that detoxify H(2)O(2). Hence, Mpr1 has promising potential for the breeding of novel ethanol-tolerant yeast strains.

  9. Involvement of OS-2 MAP kinase in regulation of the large-subunit catalases CAT-1 and CAT-3 in Neurospora crassa.

    PubMed

    Yamashita, Kazuhiro; Shiozawa, Azusa; Banno, Shinpei; Fukumori, Fumiyasu; Ichiishi, Akihiko; Kimura, Makoto; Fujimura, Makoto

    2007-08-01

    Neurospora crassa has four catalase genes--cat-1, cat-2, cat-3, and ctt-1/cat-4. cat-1 and cat-3 encode two fungal-specific large-subunit catalases CAT-1 and CAT-3 normally produced in conidia and growing hyphae, respectively. cat-2 encodes CAT-2 catalase-peroxidase normally produced in conidia. ctt-1 (or cat-4), of which expression was controlled by OS-2 MAP kinase (Noguchi et al., Fungal Genet. Biol. 44, 208-218), encodes a small-subunit catalase with unknown function. To clarify the contribution of OS-2 on the regulation of CAT-1, CAT-2, and CAT-3, we performed quantitative RT-PCR and in-gel catalase activity analyses. When the hyphae were treated with a fungicide (1 mug/ml fludioxonil) or subjected to an osmotic stress (1 M sorbitol), cat-1 was strongly upregulated and CAT-1 was reasonably induced in the wild-type strain. Interestingly, fludioxonil caused not only the CAT-1 induction but also a remarkable CAT-3 decrease in the wild-type hyphae, implying of an abnormal stimulation of asexual differentiation. These responses were not observed in an os-2 mutant hyphae, indicating an involvement of OS-2 in the cat-1 expression; however, os-2 was dispensable for the production of CAT-1 in conidia. In contrast, the expression of cat-2 was significantly induced by heat shock (45 degrees C) and that of cat-3 was moderately stimulated by an oxidative stress (50 microg/ml methyl viologen) in both the wild-type strain and the os-2 mutant, and corresponding enzyme activities were detected after the treatments. Although basal levels of transcription of cat-1 and cat-3 in an os-2 mutant hyphae were a few-fold lower than in the wild-type hyphae, the os-2 mutant exhibited a considerably lower levels of CAT-3 activity than the wild-type strain. These findings suggest that OS-2 MAP kinase regulated the expression of cat-1 and cat-3 transcriptionally, and probably that of cat-3 posttranscriptionally, even though the presence of another regulatory system for each of these two

  10. Biochemical characterization of two cloned resistance determinants encoding a paromomycin acetyltransferase and a paromomycin phosphotransferase from Streptomyces rimosus forma paromomycinus.

    PubMed Central

    Pérez-González, J A; López-Cabrera, M; Pardo, J M; Jiménez, A

    1989-01-01

    The mechanism conferring resistance to paromomycin in Streptomyces rimosus forma paromomycinus, the producing organism, was studied at the level of both protein synthesis and drug-inactivating enzymes. Ribosomes prepared from this organism grown in either production or nonproduction medium were fully sensitive to paromomycin. A paromomycin acetyltransferase and a paromomycin phosphotransferase, both characteristic of the producer, were highly purified from extracts prepared from two Streptomyces lividans transformants harboring the relevant genes inserted in pIJ702-derived plasmids. In vitro, paromomycin was inactivated by either activity. In vivo, however, S. lividans clones containing the gene for either enzyme inserted in the low-copy-number plasmid pIJ41 were resistant to only low levels of paromomycin. In contrast, an S. lividans transformant containing both genes inserted in the same pIJ41-derived plasmid displayed high levels of resistance to paromomycin. These results indicate that both genes are required to determine the high levels of resistance to this drug in the producing organism. Paromomycin is doubly modified by the enzymes. However, whereas acetylparomomycin was a poorer substrate than paromomycin for the phosphotransferase, phosphorylparomomycin was modified more actively than was the intact drug by the acetyltransferase. These findings are discussed in terms of both a permeability barrier to paromomycin and the possible role(s) of the two enzymes in the biosynthetic pathway of this antibiotic. PMID:2536659

  11. Transcriptional regulation of the tyrosine hydroxylase gene by glucocorticoid and cyclic AMP

    SciTech Connect

    Lewis, E.J.; Harrington, C.A.; Chikaraishi, D.M.

    1987-06-01

    Glucocorticoid and cyclic AMP increase tyrosine hydroxylase (TH) activity and mRNA levels in pheochromocytoma cultures. The transcriptional activity of the TH gene, as measured by nuclear run-on assay, is also increased when cultures are treated with the synthetic glucocorticoid dexamethasone or agents that increase intracellular cyclic AMP, such as forskolin and 8-BrcAMP. Both inducers effect transcriptional changes within 10 min after treatment and are maximal after 30 min for forskolin and after 60 min for dexamethasone. The 5' flanking sequences of the TH gene were fused to the bacterial gene chloramphenicol acetyltransferase (CAT), and the hybrid gene was transfected into pheochromocytoma cultures and GH/sub 4/ pituitary cells. In both cell lines, a region of the TH gene containing bases -272 to +27 conferred induction of CAT by cyclic AMP, but not by glucocorticoid. The same results were found when a region of the TH gene containing -773 to + 27 was used. Thus, the sequences required for induction of TH by cyclic AMP are contained within 272 bases of 5' flanking sequence, but sequences sufficient for glucocorticoid regulation are not contained with 773 bases.

  12. Transcription enhancer factor 1 interacts with a basic helix-loop-helix zipper protein, Max, for positive regulation of cardiac alpha-myosin heavy-chain gene expression.

    PubMed Central

    Gupta, M P; Amin, C S; Gupta, M; Hay, N; Zak, R

    1997-01-01

    The M-CAT binding factor transcription enhancer factor 1 (TEF-1) has been implicated in the regulation of several cardiac and skeletal muscle genes. Previously, we identified an E-box-M-CAT hybrid (EM) motif that is responsible for the basal and cyclic AMP-inducible expression of the rat cardiac alpha-myosin heavy chain (alpha-MHC) gene in cardiac myocytes. In this study, we report that two factors, TEF-1 and a basic helix-loop-helix leucine zipper protein, Max, bind to the alpha-MHC EM motif. We also found that Max was a part of the cardiac troponin T M-CAT-TEF-1 complex even when the DNA template did not contain an apparent E-box binding site. In the protein-protein interaction assay, a stable association of Max with TEF-1 was observed when glutathione S-transferase (GST)-TEF-1 or GST-Max was used to pull down in vitro-translated Max or TEF-1, respectively. In addition, Max was coimmunoprecipitated with TEF-1, thus documenting an in vivo TEF-1-Max interaction. In the transient transcription assay, overexpression of either Max or TEF-1 resulted a mild activation of the alpha-MHC-chloramphenicol acetyltransferase (CAT) reporter gene at lower concentrations and repression of this gene at higher concentrations. However, when Max and TEF-1 expression plasmids were transfected together, the repression mediated by a single expression plasmid was alleviated and a three- to fourfold transactivation of the alpha-MHC-CAT reporter gene was observed. This effect was abolished once the EM motif in the promoter-reporter construct was mutated, thus suggesting that the synergistic transactivation function of the TEF-1-Max heterotypic complex is mediated through binding of the complex to the EM motif. These results demonstrate a novel association between Max and TEF-1 and indicate a positive cooperation between these two factors in alpha-MHC gene regulation. PMID:9199327

  13. Identification of cis-acting sequences responsible for phorbol ester induction of human serum amyloid A gene expression via a nuclear factor kB-like transcription factor

    SciTech Connect

    Edbrooke, M.R.; Cheshire, J.K.; Woo, P.; Burt, B.W.

    1989-05-01

    The authors have analyzed the 5'-flanking region of one of the genes coding for the human acute-phase protein, serum amyloid A (SAA). They found that SAA mRNA could be increased fivefold in transfected cells by treatment with phorbol 12-myristate 13-acetate (PMA). To analyze this observation further, they placed a 265-base-pair 5' SAA fragment upstream of the reporter chloramphenicol acetyltransferase (CAT) gene and transfected this construct into HeLa cells. PMA treatment of these transient transfectants resulted in increased CAT expression. Nuclear proteins from PMA-treated HeLa cells bound to this DNA fragment, and methylation interference analysis showed that the binding was specific to the sequence GGGACTTTCC (between -82 and -91), a sequence previously described by others as the binding site for the nuclear factor NF/kappa/B. In a cotransfection competition experiment, they could abolish PMA-induced CAT activity by using cloned human immunodeficiency virus long-terminal-repeat DNA containing the NF/kappa/B-binding sequence. The same long-terminal-repeat DNA containing mutant NF/kappa/B-binding sequences did not affect CAT expression, which suggested that binding by an NF/kappa/B-like factor is required for increased SAA transcription.

  14. Molecular detection of feline hemoplasmas in feral cats in Korea.

    PubMed

    Yu, Do-Hyeon; Kim, Hyun-Wook; Desai, Atul R; Han, In-Ae; Li, Ying-Hua; Lee, Mi-Jin; Kim, In-Shik; Chae, Joon-Seok; Park, Jinho

    2007-12-01

    The purpose of this study was to determine if Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' exist in Korea. Three hundreds and thirty one feral cats were evaluated by using PCR assay targeting 16S rRNA gene sequence. Fourteen cats (4.2%) were positive for M. haemofelis, 34 cats (10.3%) were positive for 'Candidatus M. haemominutum' and 18 cats (5.4%) were positive for both species. Partial 16S rRNA gene sequences were closely (>98%) related to those from other countries. This is the first molecular detection of feline hemoplasmas in Korea.

  15. A transcriptionally [correction of transcriptively] active complex of APP with Fe65 and histone acetyltransferase Tip60.

    PubMed

    Cao, X; Südhof, T C

    2001-07-06

    Amyloid-beta precursor protein (APP), a widely expressed cell-surface protein, is cleaved in the transmembrane region by gamma-secretase. gamma-Cleavage of APP produces the extracellular amyloid beta-peptide of Alzheimer's disease and releases an intracellular tail fragment of unknown physiological function. We now demonstrate that the cytoplasmic tail of APP forms a multimeric complex with the nuclear adaptor protein Fe65 and the histone acetyltransferase Tip60. This complex potently stimulates transcription via heterologous Gal4- or LexA-DNA binding domains, suggesting that release of the cytoplasmic tail of APP by gamma-cleavage may function in gene expression.

  16. 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. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  17. Nitric oxide regulation of gene transcription via soluble guanylate cyclase and type I cGMP-dependent protein kinase.

    PubMed

    Idriss, S D; Gudi, T; Casteel, D E; Kharitonov, V G; Pilz, R B; Boss, G R

    1999-04-02

    Nitric oxide (NO) regulates the expression of multiple genes but in most cases its precise mechanism of action is unclear. We used baby hamster kidney (BHK) cells, which have very low soluble guanylate cyclase and cGMP-dependent protein kinase (G-kinase) activity, and CS-54 arterial smooth muscle cells, which express these two enzymes, to study NO regulation of the human fos promoter. The NO-releasing agent Deta-NONOate (ethanamine-2,2'-(hydroxynitrosohydrazone)bis-) had no effect on a chloramphenicol acetyltransferase (CAT) reporter gene under control of the fos promoter in BHK cells transfected with an empty vector or in cells transfected with a G-kinase Ibeta expression vector. In BHK cells transfected with expression vectors for guanylate cyclase, Deta-NONOate markedly increased the intracellular cGMP concentration and caused a small (2-fold) increase in CAT activity; the increased CAT activity appeared to be from cGMP activation of cAMP-dependent protein kinase. In BHK cells co-transfected with guanylate cyclase and G-kinase expression vectors, CAT activity was increased 5-fold in the absence of Deta-NONOate and 7-fold in the presence of Deta-NONOate. Stimulation of CAT activity in the absence of Deta-NONOate appeared to be largely from endogenous NO since we found that: (i) BHK cells produced high amounts of NO; (ii) CAT activity was partially inhibited by a NO synthase inhibitor; and (iii) the inhibition by the NO synthase inhibitor was reversed by exogenous NO. In CS-54 cells, we found that NO increased fos promoter activity and that the increase was prevented by a guanylate cyclase inhibitor. In summary, we found that NO activates the fos promoter by a guanylate cyclase- and G-kinase-dependent mechanism.

  18. Nucleotide sequencing and characterization of Pseudomonas putida catR: a positive regulator of the catBC operon is a member of the LysR family.

    PubMed Central

    Rothmel, R K; Aldrich, T L; Houghton, J E; Coco, W M; Ornston, L N; Chakrabarty, A M

    1990-01-01

    Pseudomonas putida utilizes the catBC operon for growth on benzoate as a sole carbon source. This operon is positively regulated by the CatR protein, which is encoded from a gene divergently oriented from the catBC operon. The catR gene encodes a 32.2-kilodalton polypeptide that binds to the catBC promoter region in the presence or absence of the inducer cis-cis-muconate, as shown by gel retardation studies. However, the inducer is required for transcriptional activation of the catBC operon. The catR promoter has been localized to a 385-base-pair fragment by using the broad-host-range promoter-probe vector pKT240. This fragment also contains the catBC promoter whose -35 site is separated by only 36 nucleotides from the predicted CatR translational start. Dot blot analysis suggests that CatR binding to this dual promoter-control region, in addition to inducing the catBC operon, may also regulate its own expression. Data from a computer homology search using the predicted amino acid sequence of CatR, deduced from the DNA sequence, showed CatR to be a member of a large class of procaryotic regulatory proteins designated the LysR family. Striking homology was seen between CatR and a putative regulatory protein, TfdS. Images FIG. 4 FIG. 5 FIG. 6 PMID:1688844

  19. Thyroid hormone receptors bind to the promoter of the mouse histone H10 gene and modulate its transcription.

    PubMed Central

    Bauer-Hofmann, R; Alonso, A

    1995-01-01

    It has been shown that the mouse histone H10 promoter contains a DNA element, composed of a direct repeat of the sequence GGTGACC separated by 7 nt, which is able to bind retinoic acid receptors and to modulate transcription of reporter genes following treatment with retinoic acid. We have now investigated whether this DNA motif is also responsive to thyroid hormone. We co-transfected CV-1 monkey kidney cells with chloramphenicol acetyltransferase (CAT) expression plasmids containing either 740 bp of the H10 wild-type promoter or five copies of the repeat element cloned in front of the thymidine kinase promoter and expression vectors for human thyroid hormone receptors (TRs) alpha or beta and retinoid X receptor alpha (RXR alpha). Treatment of transfected cells with triiodothyronine led to a dose-dependent increase in CAT activity. Transfection experiments with increasing amounts of expression vectors for either TR alpha or RXR alpha resulted in up to 6-fold enhancement of CAT transcription. Furthermore, point mutations within the half-sites of the response element of the H10 promoter, as well as deletions within the interspace region, lowered CAT activity to 60-80% of that of the wild-type control. Electrophoretic mobility shift assays showed that the repeat element was able to form retarded complexes with TR alpha homodimers, as well as with TR alpha-RXR alpha heterodimers. Our results suggest that thyroid hormone receptors are involved in the regulation of mouse histone H10 expression. Images PMID:8559662

  20. Analysis of the promoter region of the gene encoding NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase.

    PubMed Central

    Bélanger, C; Peri, K G; MacKenzie, R E

    1991-01-01

    Sequence analysis of the 5'-flanking region of the gene encoding NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) revealed several putative cis-regulatory elements. To delineate the function of these regulatory elements, various deletion mutants of the 5'-flanking region were connected to the reporter gene chloramphenicol acetyltransferase (CAT) and promoter activity was measured in transient transfection assays. Transfection experiments performed with the sequence extending from -508 to +59 produced a high-level transient expression of the CAT gene in BALB/c 3T3-SV-T2 and NIH 3T3 cells. Removal of the sequence from +16 to +59 which includes the second transcription start point at +43, a TATA-like box and 5'-untranslated sequences abolished the promoter activity. Deletion analysis of 5'-upstream sequences revealed that the region from positions -55 to +59 is sufficient to mediate a high CAT activity comparable to the level obtained with the construct -508/+59. Within this region are found a CAAT box, a TATA-like box and two putative GC boxes. A functional analysis of the promoter showed that the sequence from -55 to +59 is sufficient to respond to stimulation by serum. Images PMID:1843253

  1. 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

  2. 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

  3. Cat scratch encephalopathy.

    PubMed

    Silver, B E; Bean, C S

    1991-06-01

    Cat scratch disease is usually benign, self-limited and without sequelae. Margileth has established four clinical criteria, three of which must be satisfied to make the diagnosis: 1) a history of animal exposure, usually kitten, with primary skin or ocular lesions; 2) regional chronic adenopathy without other apparent cause; 3) a positive cat scratch disease antigen skin test; and 4) lymph node biopsy demonstrating noncaseating granulomas and germinal center hyperplasia. Central nervous system involvement in cat scratch disease has been previously reported, although it is extremely uncommon. In a several-month period, we encountered two cases of cat scratch disease complicated by encephalopathy. The intents of this paper are twofold: 1) to briefly review the current literature on cat scratch disease, 2) to demonstrate that cat scratch disease complicated by encephalopathy presents acutely with seizures, posturing and coma and resolves rapidly with supportive care.

  4. Chemical biology of histone acetyltransferase natural compounds modulators.

    PubMed

    Piaz, Fabrizio Dal; Vassallo, Antonio; Rubio, Osmany Cuesta; Castellano, Sabrina; Sbardella, Gianluca; De Tommasi, Nunziatina

    2011-05-01

    Histone acetyltransferases (HATs) are a class of epigenetic enzymes crucial for chromatin restructuring and transcriptional regulation in eukaryotic cells, thus being a promising target for therapeutic development. Nonetheless, differently from histone deacetylases (HDACs) inhibitors, there is still paucity of small-molecule modulators of HAT activity. After a decline during past decade, natural products and their derivatives could be once again a valuable tool in the lead discovery process and meet such need of Novel Chemical Entities (NCEs). In this review, we will provide a comprehensive summary on the discovery of small-molecule HAT modulators from naturally occurring molecular scaffolds.

  5. Regulation of the promoter of rat apolipoprotein A-I gene in cultured cells

    SciTech Connect

    Chao, Y.; Pan, T.; Wu, T.; Hao, Q.; Yamin, T.; Kroon, P.A.

    1987-05-01

    In order to study the regulation of the promoter of apolipoprotein (apo) A-I gene, they joined the 5' end of rat apo A-I gene (1.9 Kb) to the coding region of bacterial chloramphenicol acetyltransferase (CAT) gene. The chimeric gene produced high levels of CAT activity in both mouse L cells and Hep G2 cells in transient expression assays. Ethanol increased the levels of rat apo A-I promoter activity in both cells. However, dexamethasone increased rat apo A-I promoter activity only in Hep G2 cells. Similar results were obtained in stable expression cell lines. Nucleotide deletion experiments showed DNA sequences between -149 and -469 base pairs upstream from the rat apo A-I transcription site are required for the high level of expression and that the regulatory sequences are located further upstream. These data demonstrated that the 5' end of rat apo A-I gene contains sequences which are responsible for the regulation of apo A-I expression by ethanol and dexamethasone and that the expression and regulation of rat apo A-I promoter are cell specific.

  6. An M-CAT binding factor and an RSRF-related A-rich binding factor positively regulate expression of the alpha-cardiac myosin heavy-chain gene in vivo.

    PubMed Central

    Molkentin, J D; Markham, B E

    1994-01-01

    Cardiac muscle-restricted expression of the alpha-myosin heavy-chain (alpha-MHC) gene is regulated by multiple elements in the proximal enhancer/promoter. Within this region, an M-CAT site and an A-rich site were identified as potential regulatory elements. Site-specific mutations in each site, individually, reduced activity from the wild-type promoter by approximately 85% in the adult rat heart, demonstrating that these sites were positive regulatory elements. alpha-MHC, beta-MHC, and chicken cardiac troponin T (cTnT) M-CAT sites interacted with an M-CAT-binding factor (MCBF) from rat heart nuclear extracts that was immunologically related to transcriptional enhancer factor 1, a factor that binds within the simian virus 40 enhancer. The factor that bound the A-rich region (ARF) was antigenically related to the RSRF family of proteins, ARF was distinct from myocyte-specific enhancer factor 2 (MEF-2) on the basis of DNA-binding specificity and developmental expression. Like MEF-2, ARF DNA-binding activity was present in the heart and brain; however, no ARF activity was detected in extracts from skeletal muscle or C2C12 myotubes. MCBF and ARF DNA-binding activities were developmentally regulated with peak levels in the 1- to 2-day neonatal heart. The activity of both factors increased nearly fivefold in adult rat hearts subjected to a pressure overload. By comparison, the levels of alpha-MHC binding factor 2 did not change during hypertrophy. Binding sites for MCBF and ARF are present in several genes that are upregulated during cardiac hypertrophy. Our results suggest that these factors participate in the alterations in gene expression that occur during cardiac development and hypertrophy. Images PMID:8035789

  7. 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.

  8. The ADA Complex Is a Distinct Histone Acetyltransferase Complex in Saccharomyces cerevisiae

    PubMed Central

    Eberharter, Anton; Sterner, David E.; Schieltz, David; Hassan, Ahmed; Yates, John R.; Berger, Shelley L.; Workman, Jerry L.

    1999-01-01

    We have identified two Gcn5-dependent histone acetyltransferase (HAT) complexes from Saccharomyces cerevisiae, the 0.8-MDa ADA complex and the 1.8-MDa SAGA complex. The SAGA (Spt-Ada-Gcn5-acetyltransferase) complex contains several subunits which also function as part of other protein complexes, including a subset of TATA box binding protein-associated factors (TAFIIs) and Tra1. These observations raise the question of whether the 0.8-MDa ADA complex is a subcomplex of SAGA or whether it is a distinct HAT complex that also shares subunits with SAGA. To address this issue, we sought to determine if the ADA complex contained subunits that are not present in the SAGA complex. In this study, we report the purification of the ADA complex over 10 chromatographic steps. By a combination of mass spectrometry analysis and immunoblotting, we demonstrate that the adapter proteins Ada2, Ada3, and Gcn5 are indeed integral components of ADA. Furthermore, we identify the product of the S. cerevisiae gene YOR023C as a novel subunit of the ADA complex and name it Ahc1 for ADA HAT complex component 1. Biochemical functions of YOR023C have not been reported. However, AHC1 in high copy numbers suppresses the cold sensitivity caused by particular mutations in HTA1 (I. Pinto and F. Winston, personal communication), which encodes histone H2A (J. N. Hirschhorn et al., Mol. Cell. Biol. 15:1999–2009, 1995). Deletion of AHC1 disrupted the integrity of the ADA complex but did not affect SAGA or give rise to classic Ada− phenotypes. These results indicate that Gcn5, Ada2, and Ada3 function as part of a unique HAT complex (ADA) and represent shared subunits between this complex and SAGA. PMID:10490601

  9. Volatile Ester Formation in Roses. Identification of an Acetyl-Coenzyme A. Geraniol/Citronellol Acetyltransferase in Developing Rose Petals1

    PubMed Central

    Shalit, Moshe; Guterman, Inna; Volpin, Hanne; Bar, Einat; Tamari, Tal; Menda, Naama; Adam, Zach; Zamir, Dani; Vainstein, Alexander; Weiss, David; Pichersky, Eran; Lewinsohn, Efraim

    2003-01-01

    The aroma of roses (Rosa hybrida) is due to more than 400 volatile compounds including terpenes, esters, and phenolic derivatives. 2-Phenylethyl acetate, cis-3-hexenyl acetate, geranyl acetate, and citronellyl acetate were identified as the main volatile esters emitted by the flowers of the scented rose var. “Fragrant Cloud.” Cell-free extracts of petals acetylated several alcohols, utilizing acetyl-coenzyme A, to produce the corresponding acetate esters. Screening for genes similar to known plant alcohol acetyltransferases in a rose expressed sequence tag database yielded a cDNA (RhAAT1) encoding a protein with high similarity to several members of the BAHD family of acyltransferases. This cDNA was functionally expressed in Escherichia coli, and its gene product displayed acetyl-coenzyme A:geraniol acetyltransferase enzymatic activity in vitro. The RhAAT1 protein accepted other alcohols such as citronellol and 1-octanol as substrates, but 2-phenylethyl alcohol and cis-3-hexen-1-ol were poor substrates, suggesting that additional acetyltransferases are present in rose petals. The RhAAT1 protein is a polypeptide of 458 amino acids, with a calculated molecular mass of 51.8 kD, pI of 5.45, and is active as a monomer. The RhAAT1 gene was expressed exclusively in floral tissue with maximum transcript levels occurring at stage 4 of flower development, where scent emission is at its peak. PMID:12692346

  10. Choline acetyltransferase expression does not identify early pathogenic events in fetal SMA spinal cord.

    PubMed

    Soler-Botija, Carolina; Cuscó, Ivón; López, Eva; Clua, Agustín; Gich, Ignasi; Baiget, Montserrat; Ferrer, Isidre; Tizzano, Eduardo F

    2005-03-01

    We investigated the expression of choline acetyltransferase, a specific marker for cholinergic neurons, in control and spinal muscular atrophy fetuses and newborns. By immunoblot we observed at 12 and 15 weeks a similar pattern of choline acetyltransferase expression in spinal muscular atrophy with respect to controls, although at 22 weeks this expression was reduced, probably due to a smaller number of motor neurons in the spinal muscular atrophy spinal cord. By immunohistochemistry, the counting of positive and negative motor neurons for choline acetyltransferase immunostaining in control and spinal muscular atrophy fetuses showed a similar proportion at all stages analyzed. The choline acetyltransferase-negative motor neurons were of similar appearance in both groups. After birth, chromatolytic motor neurons were detected in spinal muscular atrophy, all of which were choline acetyltransferase-negative. Our results in spinal muscular atrophy fetuses indicate that choline acetyltransferase immunostaining does not identify early events in neuronal pathogenesis and suggest that the spinal muscular atrophy surviving motor neurons may not be dysfunctional during this period. Furthermore, spinal muscular atrophy choline acetyltransferase-negative motor neurons showed detectable pathological changes only after birth, indicating that choline acetyltransferase is a late marker for motor neuron degeneration and not a primary contributing factor in this process.

  11. Cat-scratch Disease.

    PubMed

    Klotz, Stephen A; Ianas, Voichita; Elliott, Sean P

    2011-01-15

    Cat-scratch disease is a common infection that usually presents as tender lymphadenopathy. It should be included in the differential diagnosis of fever of unknown origin and any lymphadenopathy syndrome. Asymptomatic, bacteremic cats with Bartonella henselae in their saliva serve as vectors by biting and clawing the skin. Cat fleas are responsible for horizontal transmission of the disease from cat to cat, and on occasion, arthropod vectors (fleas or ticks) may transmit the disease to humans. Cat-scratch disease is commonly diagnosed in children, but adults can present with it as well. The causative microorganism, B. henselae, is difficult to culture. Diagnosis is most often arrived at by obtaining a history of exposure to cats and a serologic test with high titers (greater than 1:256) of immunoglobulin G antibody to B. henselae. Most cases of cat-scratch disease are self-limited and do not require antibiotic treatment. If an antibiotic is chosen, azithromycin has been shown in one small study to speed recovery. Infrequently, cat-scratch disease may present in a more disseminated form with hepatosplenomegaly or meningoencephalitis, or with bacillary angiomatosis in patients with AIDS.

  12. Barotrauma in a cat.

    PubMed

    Manning, M M; Brunson, D B

    1994-07-01

    A 3.5-year-old domestic long-hair cat was admitted to the veterinary hospital for routine procedures, including dental prophylaxis. The cat appeared clinically normal. General anesthesia was induced, and 30 minutes later, the pop-off valve was inadvertently left in a closed position. The cat developed pneumothorax, which was treated via thoracentesis and administration of oxygen. The condition resolved and the cat was released from the hospital to its owner. Barotrauma resulted because of high pressure in the anesthetic circuit. Barotrauma is a life-threatening complication of general anesthesia.

  13. Cloning and expression of the ccdA-associated thiol-disulfide oxidoreductase (catA) gene from Brevibacillus choshinensis: stimulation of human epidermal growth factor production.

    PubMed

    Tanaka, Ryoichi; Mizukami, Makoto; Ishibashi, Matsujiro; Tokunaga, Hiroko; Tokunaga, Masao

    2003-06-12

    Brevibacillus choshinensis (Bacillus brevis) is a protein-hyperproducing bacterium with a useful host-vector system for the production of recombinant proteins. Here, we cloned the ccdA-catA (cmacr;cdA āssociated thioredoxin-like tmacr;hiol-disulfide oxidoreductase) locus of B. choshinensis HPD31-S5. CatA protein (molecular weight, 19664) contains a thioredoxin-like motif, Cys-Gly-Pro-Cys. It was successfully expressed in B. choshinensis extracellularly ( approximately 100 microg x ml(-1) culture) using the secretion vector pNCMO2, and in Escherichia coli intracellularly ( approximately 350 microg x ml(-1) culture) with an amino-terminal His-tag. Both recombinant proteins showed thiol-disulfide oxidoreductase activity. Incubation of non-native human epidermal growth factor (hEGF) containing incorrect disulfide bonds with B. choshinensis cells secreting CatA protein resulted in the stimulation of the conversion of non-native hEGF to the native form. Furthermore, co-expression of CatA protein with recombinant hEGF in the B. choshinensis production system increased the yield of native hEGF.

  14. 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

  15. Analysis of the cat eye syndrome critical region in humans and the region of conserved synteny in mice: a search for candidate genes at or near the human chromosome 22 pericentromere.

    PubMed

    Footz, T K; Brinkman-Mills, P; Banting, G S; Maier, S A; Riazi, M A; Bridgland, L; Hu, S; Birren, B; Minoshima, S; Shimizu, N; Pan, H; Nguyen, T; Fang, F; Fu, Y; Ray, L; Wu, H; Shaull, S; Phan, S; Yao, Z; Chen, F; Huan, A; Hu, P; Wang, Q; Loh, P; Qi, S; Roe, B A; McDermid, H E

    2001-06-01

    We have sequenced a 1.1-Mb region of human chromosome 22q containing the dosage-sensitive gene(s) responsible for cat eye syndrome (CES) as well as the 450-kb homologous region on mouse chromosome 6. Fourteen putative genes were identified within or adjacent to the human CES critical region (CESCR), including three known genes (IL-17R, ATP6E, and BID) and nine novel genes, based on EST identity. Two putative genes (CECR3 and CECR9) were identified, in the absence of EST hits, by comparing segments of human and mouse genomic sequence around two solitary amplified exons, thus showing the utility of comparative genomic sequence analysis in identifying transcripts. Of the 14 genes, 10 were confirmed to be present in the mouse genomic sequence in the same order and orientation as in human. Absent from the mouse region of conserved synteny are CECR1, a promising CES candidate gene from the center of the contig, neighboring CECR4, and CECR7 and CECR8, which are located in the gene-poor proximal 400 kb of the contig. This latter proximal region, located approximately 1 Mb from the centromere, shows abundant duplicated gene fragments typical of pericentromeric DNA. The margin of this region also delineates the boundary of conserved synteny between the CESCR and mouse chromosome 6. Because the proximal CESCR appears abundant in duplicated segments and, therefore, is likely to be gene poor, we consider the putative genes identified in the distal CESCR to represent the majority of candidate genes for involvement in CES.

  16. Regulated expression of the human cytomegalovirus pp65 gene: Octamer sequence in the promoter is required for activation by viral gene products

    SciTech Connect

    Depto, A.S.; Stenberg, R.M.

    1989-03-01

    To better understand the regulation of late gene expression in human cytomegalovirus (CMV)-infected cells, the authors examined expression of the gene that codes for the 65-kilodalton lower-matrix phosphoprotein (pp65). Analysis of RNA isolated at 72 h from cells infected with CMV Towne or ts66, a DNA-negative temperature-sensitive mutant, supported the fact that pp65 is expressed at low levels prior to viral DNA replication but maximally expressed after the initiation of viral DNA replication. To investigate promoter activation in a transient expression assay, the pp65 promoter was cloned into the indicator plasmid containing the gene for chloramphenicol acetyltransferase (CAT). Transfection of the promoter-CAT construct and subsequent superinfection with CMV resulted in activation of the promoter at early times after infection. Cotransfection with plasmids capable of expressing immediate-early (IE) proteins demonstrated that the promoter was activated by IE proteins and that both IE regions 1 and 2 were necessary. These studies suggest that interactions between IE proteins and this octamer sequence may be important for the regulation and expression of this CMV gene.

  17. 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

  18. Enzyme kinetics and inhibition of histone acetyltransferase KAT8.

    PubMed

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

    2015-11-13

    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 anacardic acid (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.

  19. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. 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

  1. Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection.

    PubMed Central

    Pepin, M C; Barden, N

    1991-01-01

    Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids. Images PMID:1996114

  2. Cell-specific transcriptional regulation and reactivation of galectin-1 gene expression are controlled by DNA methylation of the promoter region.

    PubMed Central

    Benvenuto, G; Carpentieri, M L; Salvatore, P; Cindolo, L; Bruni, C B; Chiariotti, L

    1996-01-01

    The galectin-1 gene is developmentally regulated gene whose activity is strongly modulated during cell differentiation and transformation. We have previously shown that galectin-1 promoter constructs are highly active when transiently transfected in cells both expressing and not expressing the endogenous gene and that the basal activity is determined by a small region encompassing the transcription start site (from positions -50 to +50). We have now investigated the role of DNA methylation in galectin-1 gene expression. Southern blot analysis with HpaII and MspI endonucleases and sodium bisulfite analysis of genomic DNA from expressing and nonexpressing cell lines and cell hybrids showed a close correlation between gene activity and demethylation of the 5' region of the galectin-1 gene. We found that the galectin-1 promoter region is fully methylated, at every CpG site on both strands, in nonexpressing differentiated rat liver (FAO) and thyroid (PC C13) cells and unmethylated in the expressing undifferentiated liver (BRL3A) and thyroid transformed (PC myc/raf) cell lines. In addition, reactivation of the silent FAO alleles in FAO-human osteosarcoma (143tk-) hybrid cells is accompanied by a complete demethylation of the promoter region. Finally, when galectin-1 chloramphenicol acetyltransferase (CAT) promoter constructs were methylated in vitro by SssI methylase at every cytosine residue of the CpG doublets and transfected into mouse fibroblasts, the transcription of the CAT reporter gene was strongly inhibited. PMID:8649381

  3. 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.

  4. Examinee Issues in CAT.

    ERIC Educational Resources Information Center

    Wise, Steven L.

    The perspective of the examinee during the administration of a computerized adaptive test (CAT) is discussed, focusing on issues of test development. Item review is the first issue discussed. Virtually no CATs provide the opportunity for the examinee to go back and review, and possibly change, answers. There are arguments on either side of the…

  5. That Fat Cat

    ERIC Educational Resources Information Center

    Lambert, Phyllis Gilchrist

    2012-01-01

    This activity began with a picture book, Nurit Karlin's "Fat Cat On a Mat" (HarperCollins; 1998). The author and her students started their project with a 5-inch circular template for the head of their cats. They reviewed shapes as they drew the head and then added the ears and nose, which were triangles. Details to the face were added when…

  6. That Fat Cat

    ERIC Educational Resources Information Center

    Lambert, Phyllis Gilchrist

    2012-01-01

    This activity began with a picture book, Nurit Karlin's "Fat Cat On a Mat" (HarperCollins; 1998). The author and her students started their project with a 5-inch circular template for the head of their cats. They reviewed shapes as they drew the head and then added the ears and nose, which were triangles. Details to the face were added when…

  7. Do products of the myc proto-oncogene play a role in transcriptional regulation of the prothymosin alpha gene?

    PubMed Central

    Mol, P C; Wang, R H; Batey, D W; Lee, L A; Dang, C V; Berger, S L

    1995-01-01

    The Myc protein has been reported to activate transcription of the rat prothymosin alpha gene by binding to an enhancer element or E box (CACGTG) located in the first intron (S. Gaubatz et al., Mol. Cell. Biol. 14:3853-3862, 1994). The human prothymosin alpha gene contains two such motifs: in the promoter region at kb -1.2 and in intron 1, approximately 2 kb downstream of the transcriptional start site in a region which otherwise bears little homology to the rat gene. Using chloramphenicol acetyltransferase (CAT) reporter constructs driven either by the 5-kb human prothymosin alpha promoter or by a series of truncated promoters, we showed that removal of the E-box sequence had no effect on transient expression of CAT activity in mouse L cells. When intron 1 of the prothymosin alpha gene was inserted into the most extensive promoter construct downstream of the CAT coding region, a diminution in transcription, which remained virtually unchanged upon disruption of the E boxes, was observed. CAT constructs driven by the native prothymosin alpha promoter or the native promoter and intron were indifferent to Myc; equivalent CAT activity was observed in the presence of ectopic normal or mutant Myc genes. Similarly, expression of a transiently transfected wild-type prothymosin alpha gene as the reporter was not affected by a repertoire of myc-derived genes, including myc itself and dominant or recessive negative myc mutants. In COS-1 cells, equivalent amounts of the protein were produced from transfected prothymosin alpha genes regardless of whether genomic E boxes were disrupted, intron 1 was removed, or a repertoire of myc-derived genes was included in the transfection cocktail. More importantly, cotransfection of a dominant negative Max gene failed to reduce transcription of the endogenous prothymosin alpha gene in COS cells or the wild-type transfected gene in COS or L cells. Taken together, the data do not support the idea that Myc activates transcription of the

  8. Cloning and characterization of a serotonin N-acetyltransferase from a gymnosperm, loblolly pine (Pinus taeda).

    PubMed

    Park, Sangkyu; Byeon, Yeong; Lee, Hyoung Yool; Kim, Young-Soon; Ahn, Taeho; Back, Kyoungwhan

    2014-10-01

    Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis in both animals and plants. SNAT catalyzes serotonin into N-acetylserotonin, an immediate precursor for melatonin biosynthesis by N-acetylserotonin methyltransferase (ASMT). We cloned the SNAT gene from a gymnosperm loblolly pine (Pinus teada). The loblolly pine SNAT (PtSNAT) gene encodes 255 amino acids harboring a transit sequence with 67 amino acids and shows 67% amino acid identity with rice SNAT when comparing the mature polypeptide regions. Purified recombinant PtSNAT showed peak activity at 55°C with the K(m) (428 μM) and Vmax (3.9 nmol/min/mg protein) values. As predicted, PtSNAT localized to chloroplasts. The SNAT mRNA was constitutively expressed in all tissues, including leaf, bud, flower, and pinecone, whereas the corresponding protein was detected only in leaf. In accordance with the exclusive SNAT protein expression in leaf, melatonin was detected only in leaf at 0.45 ng per gram fresh weight. Sequence and phylogenetic analysis indicated that the gymnosperm PtSNAT had high homology with SNATs from all plant phyla (even with cyanobacteria), and formed a clade separated from the angiosperm SNATs, suggestive of direct gene transfer from cyanobacteria via endosymbiosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. 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.

  10. 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.

  11. Histone acetyltransferase cofactor Trrap maintains self-renewal and restricts differentiation of embryonic stem cells.

    PubMed

    Sawan, Carla; Hernandez-Vargas, Hector; Murr, Rabih; Lopez, Fabrice; Vaissière, Thomas; Ghantous, Akram Y; Cuenin, Cyrille; Imbert, Jean; Wang, Zhao-Qi; Ren, Bing; Herceg, Zdenko

    2013-05-01

    Chromatin states are believed to play a key role in distinct patterns of gene expression essential for self-renewal and pluripotency of embryonic stem cells (ESCs); however, the genes governing the establishment and propagation of the chromatin signature characteristic of pluripotent cells are poorly understood. Here, we show that conditional deletion of the histone acetyltransferase cofactor Trrap in mouse ESCs triggers unscheduled differentiation associated with loss of histone acetylation, condensation of chromatin into distinct foci (heterochromatization), and uncoupling of H3K4 dimethylation and H3K27 trimethylation. Trrap loss results in downregulation of stemness master genes Nanog, Oct4, and Sox2 and marked upregulation of specific differentiation markers from the three germ layers. Chromatin immunoprecipitation-sequencing analysis of genome-wide binding revealed a significant overlap between Oct4 and Trrap binding in ESCs but not in differentiated mouse embryonic fibroblasts, further supporting a functional interaction between Trrap and Oct4 in the maintenance of stemness. Remarkably, failure to downregulate Trrap prevents differentiation of ESCs, suggesting that downregulation of Trrap may be a critical step guiding transcriptional reprogramming and differentiation of ESCs. These findings establish Trrap as a critical part of the mechanism that restricts differentiation and promotes the maintenance of key features of ESCs. Copyright © 2013 AlphaMed Press.

  12. 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

  13. 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

    2014-06-01

    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.

  14. The Lysine Acetyltransferase Activator Brpf1 Governs Dentate Gyrus Development through Neural Stem Cells and Progenitors

    PubMed Central

    You, Linya; Yan, Kezhi; Zhou, Jinfeng; Zhao, Hong; Bertos, Nicholas R.; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

    2015-01-01

    Lysine acetylation has recently emerged as an important post-translational modification in diverse organisms, but relatively little is known about its roles in mammalian development and stem cells. Bromodomain- and PHD finger-containing protein 1 (BRPF1) is a multidomain histone binder and a master activator of three lysine acetyltransferases, MOZ, MORF and HBO1, which are also known as KAT6A, KAT6B and KAT7, respectively. While the MOZ and MORF genes are rearranged in leukemia, the MORF gene is also mutated in prostate and other cancers and in four genetic disorders with intellectual disability. Here we show that forebrain-specific inactivation of the mouse Brpf1 gene causes hypoplasia in the dentate gyrus, including underdevelopment of the suprapyramidal blade and complete loss of the infrapyramidal blade. We trace the developmental origin to compromised Sox2+ neural stem cells and Tbr2+ intermediate neuronal progenitors. We further demonstrate that Brpf1 loss deregulates neuronal migration, cell cycle progression and transcriptional control, thereby causing abnormal morphogenesis of the hippocampus. These results link histone binding and acetylation control to hippocampus development and identify an important epigenetic regulator for patterning the dentate gyrus, a brain structure critical for learning, memory and adult neurogenesis. PMID:25757017

  15. Molecular and Biochemical Analysis of a Madagascar Periwinkle Root-Specific Minovincinine-19-Hydroxy-O-Acetyltransferase1

    PubMed Central

    Laflamme, Pierre; St-Pierre, Benoit; De Luca, Vincenzo

    2001-01-01

    The terminal steps in the biosynthesis of the monoterpenoid indole alkaloids vindoline and minovincinine are catalyzed by separate acetyl coenzyme A-dependent O-acetyltransferases in Madagascar periwinkle (Catharanthus roseus G. Don). Two genes were isolated that had 63% nucleic acid identity and whose deduced amino acid sequences were 78% identical. Active enzymes that were expressed as recombinant His-tagged proteins in Escherichia coli were named minovincinine-19-O-acetyltransferase (MAT) and deacetylvindoline-4-O-acetyltransferase (DAT) because they catalyzed the 19-O-acetylation of indole alkaloids such as minovincinine and hörhammericine and the 4-O-acetylation of deacetylvindoline, respectively. Kinetic studies showed that the catalytic efficiency of recombinant MAT (rMAT) was very poor compared with that of recombinant DAT (rDAT), whose turnover rates for Acetyl-coenzyme A and deacetylvindoline were approximately 240- and 10,000-fold greater than those of rMAT. Northern-blot analyses showed that MAT is expressed in cortical cells of the root tip, whereas DAT is only expressed in specialized idioblast and laticifer cells within light exposed tissues like leaves and stems. The coincident expression of trytophan decarboxylase, strictosidine synthase, and MAT within root cortical cells suggests that the entire pathway for the biosynthesis of tabersonine and its substituted analogs occurs within these cells. The ability of MAT to catalyze the 4-O-acetylation of deacetylvindoline with low efficiency suggests that this enzyme, rather than DAT, is involved in vindoline biosynthesis within transformed cell and root cultures, which accumulate low levels of this alkaloid under certain circumstances. PMID:11154328

  16. Molecular and biochemical analysis of a Madagascar periwinkle root-specific minovincinine-19-hydroxy-O-acetyltransferase.

    PubMed

    Laflamme, P; St-Pierre, B; De Luca V

    2001-01-01

    The terminal steps in the biosynthesis of the monoterpenoid indole alkaloids vindoline and minovincinine are catalyzed by separate acetyl coenzyme A-dependent O-acetyltransferases in Madagascar periwinkle (Catharanthus roseus G. Don). Two genes were isolated that had 63% nucleic acid identity and whose deduced amino acid sequences were 78% identical. Active enzymes that were expressed as recombinant His-tagged proteins in Escherichia coli were named minovincinine-19-O-acetyltransferase (MAT) and deacetylvindoline-4-O-acetyltransferase (DAT) because they catalyzed the 19-O-acetylation of indole alkaloids such as minovincinine and hörhammericine and the 4-O-acetylation of deacetylvindoline, respectively. Kinetic studies showed that the catalytic efficiency of recombinant MAT (rMAT) was very poor compared with that of recombinant DAT (rDAT), whose turnover rates for Acetyl-coenzyme A and deacetylvindoline were approximately 240- and 10,000-fold greater than those of rMAT. Northern-blot analyses showed that MAT is expressed in cortical cells of the root tip, whereas DAT is only expressed in specialized idioblast and laticifer cells within light exposed tissues like leaves and stems. The coincident expression of trytophan decarboxylase, strictosidine synthase, and MAT within root cortical cells suggests that the entire pathway for the biosynthesis of tabersonine and its substituted analogs occurs within these cells. The ability of MAT to catalyze the 4-O-acetylation of deacetylvindoline with low efficiency suggests that this enzyme, rather than DAT, is involved in vindoline biosynthesis within transformed cell and root cultures, which accumulate low levels of this alkaloid under certain circumstances.

  17. A distinct DGAT with sn-3 acetyltransferase activity that synthesizes unusual, reduced-viscosity oils in Euonymus and transgenic seeds

    PubMed Central

    Durrett, Timothy P.; McClosky, Daniel D.; Tumaney, Ajay W.; Elzinga, Dezi A.; Ohlrogge, John; Pollard, Mike

    2010-01-01

    Endosperm and embryo tissues from the seeds of Euonymus alatus (Burning Bush) accumulate high levels of 3-acetyl-1,2-diacyl-sn-glycerols (acTAGs) as their major storage lipids. In contrast, the aril tissue surrounding the seed produces long-chain triacylglycerols (lcTAGs) typical of most other organisms. The presence of the sn-3 acetyl group imparts acTAGs with different physical and chemical properties, such as a 30% reduction in viscosity, compared to lcTAGs. Comparative transcriptome analysis of developing endosperm and aril tissues using pyrosequencing technology was performed to isolate the enzyme necessary for the synthesis of acTAGs. An uncharacterized membrane-bound O-acyltransferase (MBOAT) family member was the most abundant acyltransferase in the endosperm but was absent from the aril. Expression of this MBOAT in yeast resulted in the accumulation of acTAGs but not lcTAG; hence, the enzyme was named EaDAcT (Euonymus alatus diacylglycerol acetyltransferase). Yeast microsomes expressing EaDAcT possessed acetyl-CoA diacylglycerol acetyltransferase activity but lacked long-chain acyl-CoA diacylglycerol acyltransferase activity. Expression of EaDAcT under the control of a strong, seed-specific promoter in Arabidopsis resulted in the accumulation of acTAGs, up to 40 mol % of total TAG in the seed oil. These results demonstrate the utility of deep transcriptional profiling with multiple tissues as a gene discovery strategy for low-abundance proteins. They also show that EaDAcT is the acetyltransferase necessary and sufficient for the production of acTAGs in Euonymus seeds, and that this activity can be introduced into the seeds of other plants, allowing the evaluation of these unusual TAGs for biofuel and other applications. PMID:20439724

  18. Comparative study of aural microflora in healthy cats, allergic cats and cats with systemic disease.

    PubMed

    Pressanti, Charline; Drouet, Clémence; Cadiergues, Marie-Christine

    2014-12-01

    Twenty healthy cats (group 1) with clinically normal ears, 15 cats with systemic disease (group 2) and 15 allergic cats (group 3) were included in a prospective study. The experimental unit was the ear. A clinical score was established for each ear canal after otoscopic examination. Microbial population was assessed on cytological examination of smears performed with the cotton-tipped applicator smear technique. Fungal population was significantly more prominent in allergic cats (P <0.001) and in diseased cats compared with healthy cats (P <0.02). Bacterial population was significantly higher in allergic cats than in healthy cats (P <0.001) and cats suffering from systemic disease (P <0.001). Bacterial overgrowth was also higher in cats with systemic disease than healthy cats. In cats from group 2, only fungal overgrowth was associated with otitis severity. In group 3, only bacterial overgrowth was associated with otitis severity.

  19. Analysis of single-nucleotide polymorphisms in the APOBEC3H gene of domestic cats (Felis catus) and their association with the susceptibility to feline immunodeficiency virus and feline leukemia virus infections.

    PubMed

    de Castro, Fernanda Luz; Junqueira, Dennis Maletich; de Medeiros, Rúbia Marília; da Silva, Tailene Rabello; Costenaro, Jamile Girardi; Knak, Marcus Braga; de Matos Almeida, Sabrina Esteves; Campos, Fabrício Souza; Roehe, Paulo Michel; Franco, Ana Cláudia

    2014-10-01

    Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are widely distributed retroviruses that infect domestic cats (Felis catus). Restriction factors are proteins that have the ability to hamper retroviruses' replication and are part of the conserved mechanisms of anti-viral immunity of mammals. The APOBEC3 protein family is the most studied class of restriction factors; they are cytidine deaminases that generate hypermutations in provirus DNA during reverse transcription, thus causing hypermutations in the viral genome, hindering virus replication. One of the feline APOBEC3 genes, named APOBEC3H, encodes two proteins (APOBEC3H and APOBEC3CH). In other mammals, APOBEC3H single-nucleotide polymorphisms (SNPs) can alter the stability and cellular localization of the encoded protein, thus influencing its subcellular localization and reducing its anti-viral effect. In cats, the association of APOBEC3H SNPs with susceptibility to retroviral infections was not yet demonstrated. Therefore, this study aimed the investigation on the variability of APOBEC3H and the possible association with FIV/FeLV infections. DNA obtained from whole blood of fifty FIV- and/or FeLV-infected cats and fifty-nine FIV- and/or FeLV-uninfected cats were used as templates to amplify two different regions of the APOBEC3H, with subsequent sequencing and analysis. The first region was highly conserved among all samples, while in the second, six single-nucleotide variation points were identified. One of the SNPs, A65S (A65I), was significantly correlated with the susceptibility to FIV and/or FeLV infections. On the other hand, the haplotype analysis showed that the combination "GGGGCC" was positively correlated with the lack of FIV and/or FeLV infections. Our results indicate that, as previously shown in other mammals, variability of restriction factors may contribute to susceptibility of domestic cats to retroviral infections; however, these results should be confirmed by more

  20. Regulatory elements controlling pituitary-specific expression of the human prolactin gene.

    PubMed Central

    Peers, B; Voz, M L; Monget, P; Mathy-Hartert, M; Berwaer, M; Belayew, A; Martial, J A

    1990-01-01

    We have performed transfection and DNase I footprinting experiments to investigate pituitary-specific expression of the human prolactin (hPRL) gene. When fused to the chloramphenicol acetyltransferase (CAT) reporter gene, 5,000 base pairs of the 5'-flanking sequences of the hPRL gene were able to drive high cat gene expression in prolactin-expressing GH3B6 cells specifically. Deletion analysis indicated that this pituitary-specific expression was controlled by three main positive regulatory regions. The first was located just upstream from the TATA box between coordinates -40 and -250 (proximal region). We have previously shown that three motifs of this region bind the pituitary-specific Pit-1 factor. The second positive region was located in the vicinity of coordinates -1300 to -1750 (distal region). DNase I footprinting assays revealed that eight DNA motifs of this distal region bound protein Pit-1 and that two other motifs were recognized by ubiquitous factors, one of which seems to belong to the AP-1 (jun) family. The third positive region was located further upstream, between -3500 and -5000 (superdistal region). This region appears to enhance transcription only in the presence of the distal region. Images PMID:2388622

  1. Regulatory elements controlling pituitary-specific expression of the human prolactin gene.

    PubMed

    Peers, B; Voz, M L; Monget, P; Mathy-Hartert, M; Berwaer, M; Belayew, A; Martial, J A

    1990-09-01

    We have performed transfection and DNase I footprinting experiments to investigate pituitary-specific expression of the human prolactin (hPRL) gene. When fused to the chloramphenicol acetyltransferase (CAT) reporter gene, 5,000 base pairs of the 5'-flanking sequences of the hPRL gene were able to drive high cat gene expression in prolactin-expressing GH3B6 cells specifically. Deletion analysis indicated that this pituitary-specific expression was controlled by three main positive regulatory regions. The first was located just upstream from the TATA box between coordinates -40 and -250 (proximal region). We have previously shown that three motifs of this region bind the pituitary-specific Pit-1 factor. The second positive region was located in the vicinity of coordinates -1300 to -1750 (distal region). DNase I footprinting assays revealed that eight DNA motifs of this distal region bound protein Pit-1 and that two other motifs were recognized by ubiquitous factors, one of which seems to belong to the AP-1 (jun) family. The third positive region was located further upstream, between -3500 and -5000 (superdistal region). This region appears to enhance transcription only in the presence of the distal region.

  2. Development of a Double Nuclear Gene-Targeting Method by Two-Step Transformation Based on a Newly Established Chloramphenicol-Selection System in the Red Alga Cyanidioschyzon merolae.

    PubMed

    Fujiwara, Takayuki; Ohnuma, Mio; Kuroiwa, Tsuneyoshi; Ohbayashi, Ryudo; Hirooka, Shunsuke; Miyagishima, Shin-Ya

    2017-01-01

    The unicellular red alga Cyanidioschyzon merolae possesses a simple cellular architecture that consists of one mitochondrion, one chloroplast, one peroxisome, one Golgi apparatus, and several lysosomes. The nuclear genome content is also simple, with very little genetic redundancy (16.5 Mbp, 4,775 genes). In addition, molecular genetic tools such as gene targeting and inducible gene expression systems have been recently developed. These cytological features and genetic tractability have facilitated various omics analyses. However, only a single transformation selection marker URA has been made available and thus the application of genetic modification has been limited. Here, we report the development of a nuclear targeting method by using chloramphenicol and the chloramphenicol acetyltransferase (CAT) gene. In addition, we found that at least 200-bp homologous arms are required and 500-bp arms are sufficient for a targeted single-copy insertion of the CAT selection marker into the nuclear genome. By means of a combination of the URA and CAT transformation systems, we succeeded in producing a C. merolae strain that expresses HA-cyclin 1 and FLAG-CDKA from the chromosomal CYC1 and CDKA loci, respectively. These methods of multiple nuclear targeting will facilitate genetic manipulation of C. merolae.

  3. Development of a Double Nuclear Gene-Targeting Method by Two-Step Transformation Based on a Newly Established Chloramphenicol-Selection System in the Red Alga Cyanidioschyzon merolae

    PubMed Central

    Fujiwara, Takayuki; Ohnuma, Mio; Kuroiwa, Tsuneyoshi; Ohbayashi, Ryudo; Hirooka, Shunsuke; Miyagishima, Shin-Ya

    2017-01-01

    The unicellular red alga Cyanidioschyzon merolae possesses a simple cellular architecture that consists of one mitochondrion, one chloroplast, one peroxisome, one Golgi apparatus, and several lysosomes. The nuclear genome content is also simple, with very little genetic redundancy (16.5 Mbp, 4,775 genes). In addition, molecular genetic tools such as gene targeting and inducible gene expression systems have been recently developed. These cytological features and genetic tractability have facilitated various omics analyses. However, only a single transformation selection marker URA has been made available and thus the application of genetic modification has been limited. Here, we report the development of a nuclear targeting method by using chloramphenicol and the chloramphenicol acetyltransferase (CAT) gene. In addition, we found that at least 200-bp homologous arms are required and 500-bp arms are sufficient for a targeted single-copy insertion of the CAT selection marker into the nuclear genome. By means of a combination of the URA and CAT transformation systems, we succeeded in producing a C. merolae strain that expresses HA-cyclin 1 and FLAG-CDKA from the chromosomal CYC1 and CDKA loci, respectively. These methods of multiple nuclear targeting will facilitate genetic manipulation of C. merolae. PMID:28352279

  4. Occurrence of OXA-48 Carbapenemase and Other β-Lactamase Genes in ESBL-Producing Multidrug Resistant Escherichia coli from Dogs and Cats in the United States, 2009–2013

    PubMed Central

    Liu, Xiaoqiang; Thungrat, Kamoltip; Boothe, Dawn M.

    2016-01-01

    Objective: The aim of this study was to explore the occurrence and molecular characterization of extended-spectrum β-lactamases (ESBL), plasmid-mediated AmpC β-lactamase (pAmpC) and carbapenemases among ESBL-producing multidrug resistant (MDR) Escherichia coli from dogs and cats in the United States. Methods: Of 2443 E.coli isolated from dogs and cats collected between August 2009 and January 2013, 68 isolates were confirmed as ESBL-producing MDR ones. PCR and sequencing were performed to identify β-lactamases and plasmid-mediated quinolone resistance (PMQR) genes, and shed light on the virulence gene profiles, phylogenetic groups and ST types. Results: Phylogenic group D and B2 accounted for 69.1% of the isolates. 50 (73.5%) isolates carried CTX-M ESBL gene, and the most predominant specific CTX-M subtype identified was blaCTX−M−15 (n = 33), followed by blaCTX−M−1 (n = 32), blaCTX−M−123 (n = 27), blaCTX−M−9 (n = 19) and blaCTX−M−14 (n = 19), and blaCTX−M−123 was firstly reported in E. coli isolates in the United States alone or in association. Other β-lactamase genes blaTEM, blaSHV, blaOXA−48, and blaCMY−2 were detected in 41.2, 29.4, 19.1, and 17.6% of 68 ESBL-producing MDR isolates, respectively. The blaTEM and blaSHV genes were classfied as ESBLs with the exception of the blaTEM−1 gene. Additionally, 42.6% (29/68) of isolates co-expressed blaCTX−M−15 and PMQR gene aac(6′)-Ib-c. The overall occurrence of virulence genes ranged from 11.8 (ireA) to 88.2% (malX), and most of virulence genes were less frequent among CTX-M-producing isolates than non-CTX-M isolates with the exception of malX and iutA. The 68 isolates analyzed were assigned to 31 STs with six being novel. Three pandemic clonal lineages ST131 (n = 10), ST648 (n = 9), and ST405 (n = 9) accounted for more than 41% of the investigated isolates, and ST648 and ST405 of phylogenetic D were firstly reported in E. coli from dogs and cats in the United States. Conclusion

  5. X monosomy in a virilized female cat.

    PubMed

    Szczerbal, I; Nizanski, W; Dzimira, S; Nowacka-Woszuk, J; Ochota, M; Switonski, M

    2015-04-01

    An infertile Siamese female cat was subjected for clinical, histological, cytogenetic and molecular studies due to ambiguous external genitalia (vulva, vagina, rudimentary penis and scrotum-like structure) and masculine behaviour. An elevated oestrogen activity and a detectable level of testosterone were found. The cat underwent laparotomy. The gonads and the uterus were removed and subjected for histological studies, which showed ovaries with corpora lutea and a some primordial follicles. Chromosome studies of lymphocyte and fibroblast cultures, with the use of Giemsa staining, G-banding and whole X chromosome painting by fluorescence in situ hybridization, revealed pure X monosomy. Molecular analysis showed the absence of the SRY gene. Our study revealed for the first time that X monosomy in cats may be associated with virilization, in spite of the lack of the SRY gene.

  6. Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice.

    PubMed

    Knotts, S; Rindt, H; Robbins, J

    1995-08-25

    Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms.

  7. Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice.

    PubMed Central

    Knotts, S; Rindt, H; Robbins, J

    1995-01-01

    Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms. Images PMID:7667107

  8. 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

  9. Molecular Basis for Cohesin Acetylation by Establishment of Sister Chromatid Cohesion N-Acetyltransferase ESCO1.

    PubMed

    Rivera-Colón, Yadilette; Maguire, Andrew; Liszczak, Glen P; Olia, Adam S; Marmorstein, Ronen

    2016-12-16

    Protein acetylation is a prevalent posttranslational modification that is regulated by diverse acetyltransferase enzymes. Although histone acetyltransferases (HATs) have been well characterized both structurally and mechanistically, far less is known about non-histone acetyltransferase enzymes. The human ESCO1 and ESCO2 paralogs acetylate the cohesin complex subunit SMC3 to regulate the separation of sister chromatids during mitosis and meiosis. Missense mutations within the acetyltransferase domain of these proteins correlate with diseases, including endometrial cancers and Roberts syndrome. Despite their biological importance, the mechanisms underlying acetylation by the ESCO proteins are not understood. Here, we report the X-ray crystal structure of the highly conserved zinc finger-acetyltransferase moiety of ESCO1 with accompanying structure-based mutagenesis and biochemical characterization. We find that the ESCO1 acetyltransferase core is structurally homologous to the Gcn5 HAT, but contains unique additional features including a zinc finger and an ∼40-residue loop region that appear to play roles in protein stability and SMC3 substrate binding. We identify key residues that play roles in substrate binding and catalysis, and rationalize the functional consequences of disease-associated mutations. Together, these studies reveal the molecular basis for SMC3 acetylation by ESCO1 and have broader implications for understanding the structure/function of non-histone acetyltransferases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Faecal Microbiota of Cats with Insulin-Treated Diabetes Mellitus

    PubMed Central

    Bell, Erin T.; Suchodolski, Jan S.; Isaiah, Anitha; Fleeman, Linda M.; Cook, Audrey K.; Steiner, Jörg M.; Mansfield, Caroline S.

    2014-01-01

    Microorganisms within the gastrointestinal tract significantly influence metabolic processes within their mammalian host, and recently several groups have sought to characterise the gastrointestinal microbiota of individuals affected by metabolic disease. Differences in the composition of the gastrointestinal microbiota have been reported in mouse models of type 2 diabetes mellitus, as well as in human patients. Diabetes mellitus in cats has many similarities to type 2 diabetes in humans. No studies of the gastrointestinal microbiota of diabetic cats have been previously published. The objectives of this study were to compare the composition of the faecal microbiota of diabetic and non-diabetic cats, and secondarily to determine if host signalment and dietary factors influence the composition of the faecal microbiota in cats. Faecal samples were collected from insulin-treated diabetic and non-diabetic cats, and Illumina sequencing of the 16S rRNA gene and quantitative PCR were performed on each sample. ANOSIM based on the unweighted UniFrac distance metric identified no difference in the composition of the faecal microbiota between diabetic and non-diabetic cats, and no significant differences in the proportions of dominant bacteria by phylum, class, order, family or genus as determined by 16S rRNA gene sequencing were identified between diabetic and non-diabetic cats. qPCR identified a decrease in Faecalibacterium spp. in cats aged over ten years. Cat breed or gender, dietary carbohydrate, protein or fat content, and dietary formulation (wet versus dry food) did not affect the composition of the faecal microbiota. In conclusion, the composition of the faecal microbiota was not altered by the presence of diabetes mellitus in cats. Additional studies that compare the functional products of the microbiota in diabetic and non-diabetic cats are warranted to further investigate the potential impact of the gastrointestinal microbiota on metabolic diseases such as

  11. Faecal microbiota of cats with insulin-treated diabetes mellitus.

    PubMed

    Bell, Erin T; Suchodolski, Jan S; Isaiah, Anitha; Fleeman, Linda M; Cook, Audrey K; Steiner, Jörg M; Mansfield, Caroline S

    2014-01-01

    Microorganisms within the gastrointestinal tract significantly influence metabolic processes within their mammalian host, and recently several groups have sought to characterise the gastrointestinal microbiota of individuals affected by metabolic disease. Differences in the composition of the gastrointestinal microbiota have been reported in mouse models of type 2 diabetes mellitus, as well as in human patients. Diabetes mellitus in cats has many similarities to type 2 diabetes in humans. No studies of the gastrointestinal microbiota of diabetic cats have been previously published. The objectives of this study were to compare the composition of the faecal microbiota of diabetic and non-diabetic cats, and secondarily to determine if host signalment and dietary factors influence the composition of the faecal microbiota in cats. Faecal samples were collected from insulin-treated diabetic and non-diabetic cats, and Illumina sequencing of the 16S rRNA gene and quantitative PCR were performed on each sample. ANOSIM based on the unweighted UniFrac distance metric identified no difference in the composition of the faecal microbiota between diabetic and non-diabetic cats, and no significant differences in the proportions of dominant bacteria by phylum, class, order, family or genus as determined by 16S rRNA gene sequencing were identified between diabetic and non-diabetic cats. qPCR identified a decrease in Faecalibacterium spp. in cats aged over ten years. Cat breed or gender, dietary carbohydrate, protein or fat content, and dietary formulation (wet versus dry food) did not affect the composition of the faecal microbiota. In conclusion, the composition of the faecal microbiota was not altered by the presence of diabetes mellitus in cats. Additional studies that compare the functional products of the microbiota in diabetic and non-diabetic cats are warranted to further investigate the potential impact of the gastrointestinal microbiota on metabolic diseases such as

  12. Regulatory sequences of duck hepatitis B virus C gene transcription.

    PubMed Central

    Schneider, R; Will, H

    1991-01-01

    The regulatory elements involved in transcription of the C gene of duck hepatitis B virus (DHBV) were investigated. Several DHBV DNA fragments were assayed for C gene promoter, enhancer, and silencer activity by using a chloramphenicol acetyltransferase (CAT) reporter gene and transfection of established liver and nonliver cell lines. A major transcript initiating at nucleotide positions 2532 and 2533 and three minor transcripts initiating at positions 2453/2454 and 2461 were identified in cells containing these constructs. These positions correspond to the 5' end of the C mRNA and were close to that of the pre-C mRNAs, respectively, found in infected livers. The pre-C mRNAs were only detected when sequences located between the initiation sites of the pre-C and C mRNAs were deleted. These sequences downregulated, in an orientation-independent fashion, a heterologous promoter and were found to contain a consensus motif common to negative transcriptional regulatory elements previously characterized in other cellular and viral genes. C gene promoter activity was only observed in highly differentiated liver cells and was dependent on a short DHBV DNA fragment containing an enhancer core consensus motif. These data indicate that transcription of the DHBV C gene is regulated by positive, negative, and differentiation factor-responsive elements. Images PMID:1920612

  13. CAT — EDRN Public Portal

    Cancer.gov

    The CAT gene product, catalase, occurs in the peroxisome of almost all respiring organismÃÆ'¢â‚¬â„¢s cells. Catalase is a heme enzyme that converts the reactive oxygen species hydrogen peroxide to water and oxygen, diminishing the toxic effects of hydrogen peroxide on the cell. Catalase promotes growth of cells including T-cells, B-cells, myeloid leukemia cells, melanoma cells, mastocytoma cells and normal and transformed fibroblast cells. Polymorphisms in this gene have been associated with decreases in catalase activity but, to date, acatalasemia is the only disease known to be caused by this gene.

  14. The MOZ histone acetyltransferase in epigenetic signaling and disease.

    PubMed

    Carlson, Samuel; Glass, Karen C

    2014-11-01

    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 action 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.

  15. Melatonin production: proteasomal proteolysis in serotonin N-acetyltransferase regulation.

    PubMed

    Gastel, J A; Roseboom, P H; Rinaldi, P A; Weller, J L; Klein, D C

    1998-02-27

    The nocturnal increase in circulating melatonin in vertebrates is regulated by 10- to 100-fold increases in pineal serotonin N-acetyltransferase (AA-NAT) activity. Changes in the amount of AA-NAT protein were shown to parallel changes in AA-NAT activity. When neural stimulation was switched off by either light exposure or L-propranolol-induced beta-adrenergic blockade, both AA-NAT activity and protein decreased rapidly. Effects of L-propranolol were blocked in vitro by dibutyryl adenosine 3',5'-monophosphate (cAMP) or inhibitors of proteasomal proteolysis. This result indicates that adrenergic-cAMP regulation of AA-NAT is mediated by rapid reversible control of selective proteasomal proteolysis. Similar proteasome-based mechanisms may function widely as selective molecular switches in vertebrate neural systems.

  16. CATS EYES Adjustment Procedures

    DTIC Science & Technology

    1993-04-01

    AL-TR-1 993-0025 AD-A264 069 CATS EYES ADJUSTMENT PROCEDURES A R M Joseph C. Antonio DTIC S ELECTET University of Dayton Research Institute MAY 13...Final November 1992 - January 1993 4. TITLE AND SUBTITLE S. FUNDING NUMBERS C F33615-90-C-0005 CATS EYES Adjustment Procedures PE - 62205F 6. AUTHOR(S) PR...the loss of NVG performance resulting from improper goggle adjustments. This report describes correct adjustment procedures for the CATS EYES NVG system

  17. Computerised Axial Tomography (CAT)

    DTIC Science & Technology

    1990-06-01

    Ministry of’ Defence, Defence Research Information Centre, UK. Computerised Axial Tomography ( CAT ) Report Secufty C"uMiauion tide Onadtiicadon (U. R, Cor S...DRIC T 8485 COMPUTERISED AXIAL TOMOGRAPHY ( CAT ) F.P. GENTILE, F. SABETTA, V. TRO1* ISS R 78/4.Rome, 1.5 Mlarch 1978 (from Italian) B Distribution(f...dello Radiazioni ISSN 0390--6477 F.P. GENTILE, F. SABETTA. V. TROI Computerised Axial Tomography ( CAT ) March 15, 1978). This paper is a review of

  18. Giardia infection in cats.

    PubMed

    Janeczko, Stephanie; Griffin, Brenda

    2010-08-01

    The protozoon Giardia duodenalis is a common gastrointestinal parasite of cats. While most Giardia-infected cats are asymptomatic, acute small bowel diarrhea, occasionally with concomitant weight loss, may occur. Giardia poses a diagnostic challenge, but newer tests, including a commercially available ELISA kit, have improved clinicians' ability to obtain an accurate diagnosis. Several treatment options have been reported, and although none has been shown to be universally effective, most cases can be successfully managed with drug therapy, supportive measures, and environmental control. Current recommendations suggest that combination therapy with fenbendazole and metronidazole may be the safest, most effective treatment option for symptomatic cats.

  19. Permethrin toxicosis in cats.

    PubMed

    Linnett, P-J

    2008-01-01

    A retrospective analysis of all adverse experience reports received by the Australian Pesticides and Veterinary Medicines Authority's Adverse Experience Reporting Program for veterinary medicines since 1995, showed that permethrin toxicity in cats usually occurred after the owner applied a canine permethrin-containing product, typically a spot-on. Cats are also at risk from grooming or being in direct contact with recently treated dogs. This paper reviews permethrin toxicosis and its treatment in cats, incorporating information from the Australian and selected overseas veterinary pharmacovigilance programs.

  20. Diabetes mellitus in cats.

    PubMed

    Rand, Jacquie S; Marshall, Rhett D

    2005-01-01

    Feline diabetes is a multifactorial disease with genetic and environmental factors, including diet, excess body weight, and physical inactivity, involved in its pathogenesis. Although type 2 diabetes is most common in cats, most cats are insulin-dependent at the time of diagnosis. If good glycemic control can be achieved early after diagnosis, a substantial proportion of diabetic cats go into clinical remission. Diabetic remission may be facilitated by using a low-carbohydrate-high-protein diet combined with a long-acting insulin, such as glargine, administered twice daily. Rather than just controlling clinical signs, these new treatment modalities make curing feline diabetes a realistic goal for practitioners.

  1. Antiviral restriction factor transgenesis in the domestic cat.

    PubMed

    Wongsrikeao, Pimprapar; Saenz, Dyana; Rinkoski, Tommy; Otoi, Takeshige; Poeschla, Eric

    2011-09-11

    Studies of the domestic cat have contributed to many scientific advances, including the present understanding of the mammalian cerebral cortex. A practical capability for cat transgenesis is needed to realize the distinctive potential of research on this neurobehaviorally complex, accessible species for advancing human and feline health. For example, humans and cats are afflicted with pandemic AIDS lentiviruses that are susceptible to species-specific restriction factors. Here we introduced genes encoding such a factor, rhesus macaque TRIMCyp, and eGFP, into the cat germline. The method establishes gamete-targeted transgenesis for the first time in a carnivore. We observed uniformly transgenic outcomes, widespread expression, no mosaicism and no F1 silencing. TRIMCyp transgenic cat lymphocytes resisted feline immunodeficiency virus replication. This capability to experimentally manipulate the genome of an AIDS-susceptible species can be used to test the potential of restriction factors for HIV gene therapy and to build models of other infectious and noninfectious diseases.

  2. Puromycin-N-acetyltransferase as a selectable marker for use in Plasmodium falciparum.

    PubMed

    de Koning-Ward, T F; Waters, A P; Crabb, B S

    2001-10-01

    The limited number of selectable markers available for malaria transfection has hindered extensive manipulation of the Plasmodium falciparum genome and subsequently thorough genetic analysis of this organism. In this paper, we demonstrate that P. falciparum is highly sensitive to the drug puromycin, but that transgenic expression of the puromycin-N-acetyltransferase (PAC) gene from Streptomyces alboninger confers resistance to this drug with the IC(50) and IC(90) values increasing approximately 3- and 7-fold, respectively in PAC-expressing parasites. Despite this relatively low level of resistance, parasite populations transfected with the PAC selectable marker and selected directly on puromycin emerged at the same rate post-transfection as human dihydrofolate reductase (hDHFR)-expressing parasites, selected independently with the anti-folate drug WR99210. Transfected parasites generally maintained the PAC expression plasmid episomally at between two and six copies per parasite. We also demonstrate by cycling transfected parasites in the presence and absence of puromycin for several weeks, that the PAC selectable marker can be used for gene-targeting. Since the mode of action of puromycin is distinct from other drugs currently used for the stable transfection of P. falciparum, the PAC selectable marker should also have applicability for use in conjunction with other positive selectable markers, thereby increasing the possibilities for more complex functional studies of this organism.

  3. The acetyltransferase Tip60 contributes to mammary tumorigenesis by modulating DNA repair

    PubMed Central

    Bassi, C; Li, Y-T; Khu, K; Mateo, F; Baniasadi, P S; Elia, A; Mason, J; Stambolic, V; Pujana, M A; Mak, T W; Gorrini, C

    2016-01-01

    The acetyltransferase Tip60/Kat5 acetylates both histone and non-histone proteins, and is involved in a variety of biological processes. By acetylating p53, Tip60 controls p53-dependent transcriptional activity and so is implicated as a tumor suppressor. However, many breast cancers with low Tip60 also show p53 mutation, implying that Tip60 has a tumor suppressor function independent of its acetylation of p53. Here, we show in a p53-null mouse model of sporadic invasive breast adenocarcinoma that heterozygosity for Tip60 deletion promotes mammary tumorigenesis. Low Tip60 reduces DNA repair in normal and tumor mammary epithelial cells, both under resting conditions and following genotoxic stress. We demonstrate that Tip60 controls homologous recombination (HR)-directed DNA repair, and that Tip60 levels correlate inversely with a gene expression signature associated with defective HR-directed DNA repair. In human breast cancer data sets, Tip60 mRNA is downregulated, with low Tip60 levels correlating with p53 mutations in basal-like breast cancers. Our findings indicate that Tip60 is a novel breast tumor suppressor gene whose loss results in genomic instability leading to cancer formation. PMID:26915295

  4. Proper Gcn5 histone acetyltransferase expression is required for normal anteroposterior patterning of the mouse skeleton.

    PubMed

    Lin, Wenchu; Zhang, Zhijing; Chen, Chih-Hsin; Behringer, Richard R; Dent, Sharon Y R

    2008-06-01

    Histone acetylation plays important roles in gene regulation. However, the functions of individual histone acetyltransferases (HATs) in specific developmental transcription programs are not well defined. To define the functions of Gcn5, a prototypical HAT, during mouse development, we have created a series of mutant Gcn5 alleles. Our previous work revealed that deletion of Gcn5 leads to embryonic death soon after gastrulation. Embryos homozygous for point mutations in the catalytic center of Gcn5 survive longer, but die soon after E16.0 and exhibit defects in cranial neural tube closure. Embryos bearing a hypomorphic Gcn5(flox(neo)) allele also exhibit neural closure defects and die at or soon after birth. We report here that Gcn5(flox(neo)/flox(neo)) and Gcn5(flox(neo)/Delta) embryos exhibit anterior homeotic transformations in lower thoracic and lumbar vertebrae. These defects are accompanied by a shift in the anterior expression boundary of Hoxc8 and Hoxc9. These data provide the first evidence that Gcn5 contributes to Hox gene regulation and is required for normal anteroposterior patterning of the mouse skeleton.

  5. Basic nuclear processes affected by histone acetyltransferases and histone deacetylase inhibitors.

    PubMed

    Legartová, Soňa; Stixová, Lenka; Strnad, Hynek; Kozubek, Stanislav; Martinet, Nadine; Dekker, Frank J; Franek, Michal; Bártová, Eva

    2013-08-01

    The optimal balance between histone acetylation and deacetylation is important for proper gene function. Therefore, we addressed how inhibitors of histone-modifying enzymes can modulate nuclear events, including replication, transcription, splicing and DNA repair. Changes in cell signaling pathways upon treatment with histone acetyltransferases and/or histone deacetylase inhibitors were studied by cDNA microarrays and western blots. We analyzed the effects of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and the histone acetylase inhibitor MG149. SAHA altered the expression of factors involved in DNA replication complexes, basal transcription and the spliceosome pathway. DNA repair-related genes, including Rad51, Rad54 and BRCA2, were significantly downregulated by SAHA. However, MG149 had no effect on the investigated nuclear processes, with the exception of the spliceosome network and Sestrins, involved in DNA repair. Based on our results, we propose that the studied epigenetic drugs have the distinct potential to affect specific cell signaling pathways depending on their respective molecular targets.

  6. Polymorphisms in the Human Cytochrome P450 and Arylamine N-Acetyltransferase: Susceptibility to Head and Neck Cancers

    PubMed Central

    Khlifi, Rim; Messaoud, Olfa; Rebai, Ahmed; Hamza-Chaffai, Amel

    2013-01-01

    The occurrence of head and neck cancer (HNC) is associated with smoking and alcohol drinking. Tobacco smoking exposes smokers to a series of carcinogenic chemicals. Cytochrome P450 enzymes (CYP450s), such as CYP1A1, CYP1B1, and CYP2D6, usually metabolize carcinogens to their inactive derivatives, but they occasionally convert the chemicals to more potent carcinogens. In addition, via CYP450 (CYP2E1) oxidase, alcohol is metabolized to acetaldehyde, a highly toxic compound, which plays an important role in carcinogenesis. Furthermore, two N-acetyltransferase isozymes (NATs), NAT1 and NAT2, are polymorphic and catalyze both N-acetylation and O-acetylation of aromatic and heterocyclic amine carcinogens. Genetic polymorphisms are associated with a number of enzymes involved in the metabolism of carcinogens important in the induction of HNC. It has been suggested that such polymorphisms may be linked to cancer susceptibility. In this paper, we select four cytochrome P450 enzymes (CYP1A1, CYP1BA1, CYP2D6, and CYP2E1), and two N-acetyltransferase isozymes (NAT1 and NAT2) in order to summarize and analyze findings from the literature related to HNC risk by focusing on (i) the interaction between these genes and the environment, (ii) the impact of genetic defect on protein activity and/or expression, and (iii) the eventual involvement of race in such associations. PMID:24151610

  7. The Polyamine N-Acetyltransferase-Like Enzyme PmvE Plays a Role in the Virulence of Enterococcus faecalis

    PubMed Central

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

    2014-01-01

    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 N1-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

  8. Balance of activities of alcohol acetyltransferase and esterase in Saccharomyces cerevisiae is important for production of isoamyl acetate.

    PubMed

    Fukuda, K; Yamamoto, N; Kiyokawa, Y; Yanagiuchi, T; Wakai, Y; Kitamoto, K; Inoue, Y; Kimura, A

    1998-10-01

    Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash.

  9. Balance of Activities of Alcohol Acetyltransferase and Esterase in Saccharomyces cerevisiae Is Important for Production of Isoamyl Acetate

    PubMed Central

    Fukuda, Kiyoshi; Yamamoto, Nagi; Kiyokawa, Yoshifumi; Yanagiuchi, Toshiyasu; Wakai, Yoshinori; Kitamoto, Katsuhiko; Inoue, Yoshiharu; Kimura, Akira

    1998-01-01

    Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash. PMID:9758847

  10. Cat-Eyed Saturn

    NASA Image and Video Library

    2006-04-13

    Bright, high altitude clouds, like those imaged here, often appear more filamentary or streak-like than clouds imaged at slightly deeper levels in Saturn atmosphere. This view also shows one of the many cat eye vortices.

  11. Cat tongue Velcro

    NASA Astrophysics Data System (ADS)

    Noel, Alexis; Martinez, Andrea; Jung, Hyewon; Tsai, Ting-Wen; Hu, David

    2016-11-01

    A cat's tongue is covered in an array of spines called papillae. These spines are thought to be used in grooming and rasping meat from bones of prey, although no mechanism has been given. We use high-speed video to film a cat removing cat food deeply wedged into a 3-D printed fur mat. We show that the spines on the tongue act as Velcro for particles. The tongue itself is highly elastic. As the cat presses it against a substrate, the tongue flattens and the spines separate. When the tongue is removed from the substrate the spines come together, wedging particles between them. This elasticity-driven entrapment permits the surface of the tongue to act as a carrier for hard to reach particles, and to increase the efficacy of grooming and feeding.

  12. Promoter elements and transcriptional control of the chicken tropomyosin gene [corrected

    PubMed Central

    Toutant, M; Gauthier-Rouviere, C; Fiszman, M Y; Lemonnier, M

    1994-01-01

    The chicken beta tropomyosin (beta TM) gene has two alternative transcription start sites (sk and nmCAP sites) which are used in muscle or non muscle tissues respectively. In order to understand the mechanisms involved in the tissue-specific and developmentally-regulated expression of the beta TM gene, we have analyzed the 5' regions associated with each CAP site. Truncated regions 5' to the nmCAP site were inserted upstream to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and these constructs were transfected into avian myogenic and non myogenic cells. The maximum transcription is driven by the CAT construct (-168/ + 216 nt) in all cell types. Previous deletion analysis of the region 5' to the beta TMskCAP site has indicated that 805 nt confer myotube-specific transcription. In this work, we characterized an enhancer element (-201/-68 nt) which contains an E box (-177), a variant CArG box (-104) and a stretch of 7Cs (-147). Mutation of any of these motifs results in a decrease of the myotube-specific transcriptional activity. Electrophoretic mobility shift assays indicate that these cis-acting sequences specifically bind nuclear proteins. This enhancer functions in an orientation-dependent manner. Images PMID:8208608

  13. Fatal big cat attacks.

    PubMed

    Cohle, S D; Harlan, C W; Harlan, G

    1990-09-01

    Two cases of fatal attacks by large cats are presented. In the first case, a 30-year-old female zoo worker was attacked by a jaguar that had escaped its cage. In the second case, a 2-year-old girl was fatally injured by her father's pet leopard. The pattern of injuries in these cases is nearly identical to those of these cats' prey in the wild.

  14. CATS Installed on ISS

    NASA Image and Video Library

    2017-09-28

    On Jan. 22, 2015, robotic flight controllers successfully installed NASA’s Cloud Aerosol Transport System (CATS) onboard the International Space Station. CATS will collect data about clouds, volcanic ash plumes and tiny airborne particles that can help improve our understanding of aerosol and cloud interactions, and improve the accuracy of climate change models. CATS had been mounted inside the SpaceX Dragon cargo craft’s unpressurized trunk since it docked at the station on Jan. 12. Ground controllers at NASA’s Johnson Space Center in Houston, Texas, used one of the space station’s robotic arms, called the Special Purpose Dexterous Manipulator, to extract the instrument from the capsule. The NASA-controlled arm passed the instrument to a second robotic arm— like passing a baton in a relay race. This second arm, called the Japanese Experiment Module Remote Manipulator System, is controlled by the Japanese Aerospace Exploration Agency. The Japanese-controlled arm installed the instrument to the Space Station’s Japanese Experiment Module, making CATS the first NASA-developed payload to fly on the Japanese module. CATS is a lidar remote-sensing instrument designed to last from six months to three years. It is specifically intended to demonstrate a low-cost, streamlined approach to developing science payloads on the space station. CATS launched aboard the SpaceX Dragon spacecraft on Jan. 10 at Cape Canaveral Air Force Station in Florida. To learn more about the impact of CATS data, visit: www.nasa.gov/cats/ NASA image use policy. NASA Goddard Space Flight Center enables NASA’s mission through four scientific endeavors: Earth Science, Heliophysics, Solar System Exploration, and Astrophysics. Goddard plays a leading role in NASA’s accomplishments by contributing compelling scientific knowledge to advance the Agency’s mission. Follow us on Twitter Like us on Facebook Find us on Instagram

  15. The yeast SAS (something about silencing) protein complex contains a MYST-type putative acetyltransferase and functions with chromatin assembly factor ASF1

    PubMed Central

    Osada, Shigehiro; Sutton, Ann; Muster, Nemone; Brown, Christine E.; Yates, John R.; Sternglanz, Rolf; Workman, Jerry L.

    2001-01-01

    It is well established that acetylation of histone and nonhistone proteins is intimately linked to transcriptional activation. However, loss of acetyltransferase activity has also been shown to cause silencing defects, implicating acetylation in gene silencing. The something about silencing (Sas) 2 protein of Saccharomyces cerevisiae, a member of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, promotes silencing at HML and telomeres. Here we identify a ∼450-kD SAS complex containing Sas2p, Sas4p, and the tf2f-related Sas5 protein. Mutations in the conserved acetyl-CoA binding motif of Sas2p are shown to disrupt the ability of Sas2p to mediate the silencing at HML and telomeres, providing evidence for an important role for the acetyltransferase activity of the SAS complex in silencing. Furthermore, the SAS complex is found to interact with chromatin assembly factor Asf1p, and asf1 mutants show silencing defects similar to mutants in the SAS complex. Thus, ASF1-dependent chromatin assembly may mediate the role of the SAS complex in silencing. PMID:11731479

  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. Who's behind that mask and cape? The Asian leopard cat's Agouti (ASIP) allele likely affects coat colour phenotype in the Bengal cat breed.

    PubMed

    Gershony, L C; Penedo, M C T; Davis, B W; Murphy, W J; Helps, C R; Lyons, L A

    2014-12-01

    Coat colours and patterns are highly variable in cats and are determined mainly by several genes with Mendelian inheritance. A 2-bp deletion in agouti signalling protein (ASIP) is associated with melanism in domestic cats. Bengal cats are hybrids between domestic cats and Asian leopard cats (Prionailurus bengalensis), and the charcoal coat colouration/pattern in Bengals presents as a possible incomplete melanism. The complete coding region of ASIP was directly sequenced in Asian leopard, domestic and Bengal cats. Twenty-seven variants were identified between domestic and leopard cats and were investigated in Bengals and Savannahs, a hybrid with servals (Leptailurus serval). The leopard cat ASIP haplotype was distinguished from domestic cat by four synonymous and four non-synonymous exonic SNPs, as well as 19 intronic variants, including a 42-bp deletion in intron 4. Fifty-six of 64 reported charcoal cats were compound heterozygotes at ASIP, with leopard cat agouti (A(P) (be) ) and domestic cat non-agouti (a) haplotypes. Twenty-four Bengals had an additional unique haplotype (A2) for exon 2 that was not identified in leopard cats, servals or jungle cats (Felis chaus). The compound heterozygote state suggests the leopard cat allele, in combination with the recessive non-agouti allele, influences Bengal markings, producing a darker, yet not completely melanistic coat. This is the first validation of a leopard cat allele segregating in the Bengal breed and likely affecting their overall pelage phenotype. Genetic testing services need to be aware of the possible segregation of wild felid alleles in all assays performed on hybrid cats. © 2014 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics.

  18. Immediate-early gene region of human cytomegalovirus trans-activates the promoter of human immunodeficiency virus

    SciTech Connect

    Davis, M.G.; Kenney, S.C.; Kamine, J.; Pagano, J.S.; Huang, E.S.

    1987-12-01

    Almost all homosexual patients with acquired immunodeficiency syndrome are also actively infected with human cytomegalovirus (HCMV). The authors have hypothesized that an interaction between HCMV and human immunodeficiency virus (HIV), the agent that causes acquired immunodeficiency syndrome, may exist at a molecular level and contribute to the manifestations of HIV infection. In this report, they demonstrate that the immediate-early gene region of HCMV, in particular immediate-early region 2, trans-activates the expression of the bacterial gene chloramphenicol acetyltransferase that is fused to the HIV long terminal repeat and carried by plasmid pHIV-CAT. The HCMV immediate-early trans-activator increases the level of mRNA from the plamid pHIV-CAT. The sequences of HIV that are responsive to trans-activation by the HDMV immediate-early region are distinct from HIV sequences that are required for response to the HIV tat. The stimulation of HIV gene expression by HDMV gene functions could enhance the consequences of HIV infection in persons with previous or concurrent HCMV infection.

  19. Potential predictive biomarkers of obesity in Burmese cats.

    PubMed

    Lee, Peter; Mori, Akihiro; Coradini, Marcia; Mori, Nobuko; Sagara, Fumi; Yamamoto, Ichiro; Rand, Jacquie S; Arai, Toshiro

    2013-02-01

    Australian Burmese cats are predisposed to diabetes mellitus and, compared to other breeds, have delayed triglyceride clearance that may result in subtle changes within cells and tissues that trigger specific alterations in gene expression within peripheral blood leucocytes (PBLs). Expression of genes involved in energy metabolism (glucose-6-phosphate dehydrogenase and malate dehydrogenase), lipogenesis (ATP citrate lyase [ACL], fatty acid synthase [FAS] and sterol regulatory binding protein-1c [SREBP-1c]), and insulin signalling (insulin receptor substrates 1 and 2, and phosphatidylinositol-3 kinase), as well as cholesterol lipoprotein subfraction profiling were carried out on PBLs from lean Burmese cats and compared with similar profiles of age and gender matched lean and obese Australian domestic shorthaired cats (DSHs) in an attempt to identify possible biomarkers for assessing obesity. For the majority of the genes examined, the lean Burmese cats demonstrated similar PBL gene expression patterns as age and gender matched obese Australian DSH cats. Lean Burmese had increased expression of ACL and FAS, but not SREBP-1c, a main upstream regulator of lipid synthesis, suggesting possible aberrations in lipogenesis. Moreover, lean Burmese displayed a 3- to 4-fold increase in the very low density cholesterol fraction percentage, which was double that for obese DSH cats, indicating an increased degree of lipid dysregulation especially in relation to triglycerides. The findings suggest that Burmese cats may have a particular propensity for dysregulation in lipid metabolism.

  20. Close linkage fo the dominant cataract mutations (Cat-2) with Idh-1 and cryge on mouse Chromosome 1

    SciTech Connect

    Loester, J.; Pretsch, W.; Sandulache, R.

    1994-09-01

    The murine dominant gene Cat-2 was located on chromosome 1 between the loci of fuzzy and leaden. Subsequent linkage analysis revealed one recombinant between Cat-2{sup t} and isocitrate dehydrogenase-1, and one between Cat-2{sup t} and {gamma}E-crystallin among 338 offspring in three-point backcrosses. The resulting genetic distance between the loci is 0.3 {+-} 0.3 cM. The very close linkage between the Cat-2 and the {gamma}-crystallin gene cluster together with the finding of reduced {gamma}-strongly that the {gamma}-crystallin genes may be candidate genes for the Cat-2 mutations.

  1. Metabolic regulation of histone acetyltransferases by endogenous Acyl-CoA cofactors | Center for Cancer Research

    Cancer.gov

    Unraveling the metabolic regulation of lysine acetyltransferases (KATs). Montgomery et al. detail the application of a competitive chemoproteomic strategy to quantitatively characterize the interactions of acyl-CoA metabolites with cellular KAT enzymes.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  3. Cyclic AMP regulation of the human glycoprotein hormone. cap alpha. -subunit gene is mediated by an 18-base-pair element

    SciTech Connect

    Silver, B.J.; Bokar, J.A.; Virgin, J.B.; Vallen, E.A.; Milsted, A.; Nilson, J.H.

    1987-04-01

    cAMP regulates transcription of the gene encoding the ..cap alpha..-subunit of human chorionic gonadotropin (hCG) in the choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, the authors inserted fragments from the 5' flanking region of the ..cap alpha..-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between position -146 and -111. In the absence of cAMP, the ..cap alpha..-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. They localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the ..cap alpha..-subunit gene.

  4. Genetics of pigmentation in dogs and cats.

    PubMed

    Kaelin, Christopher B; Barsh, Gregory S

    2013-01-01

    Color variation in companion animals has long been of interest to the breeding and scientific communities. Simple traits, like black versus brown or yellow versus black, have helped to explain principles of transmission genetics and continue to serve as models for studying gene action and interaction. We present a molecular genetic review of pigmentary variation in dogs and cats using a nomenclature and logical framework established by early leaders in the field. For most loci in which molecular variants have been identified (nine in dogs and seven in cats), homologous mutations exist in laboratory mice and/or humans. Exceptions include the K locus in dogs and the Tabby locus in cats, which give rise to alternating stripes or marks of different color, and which illustrate the continued potential of coat color genetics to provide insight into areas that transcend pigment cell biology.

  5. The Feline Mystique: Dispelling the Myth of the Independent Cat.

    ERIC Educational Resources Information Center

    Soltow, Willow

    1984-01-01

    Describes learning activities about cats for primary and intermediate grades. Primary grade activity subjects include cat behavior, needs, breeds, storybook cats, and celestial cats. Intermediate grade activity subjects include cat history, care, language, literary cats, and cats in art. (BC)

  6. The Feline Mystique: Dispelling the Myth of the Independent Cat.

    ERIC Educational Resources Information Center

    Soltow, Willow

    1984-01-01

    Describes learning activities about cats for primary and intermediate grades. Primary grade activity subjects include cat behavior, needs, breeds, storybook cats, and celestial cats. Intermediate grade activity subjects include cat history, care, language, literary cats, and cats in art. (BC)

  7. Early adipogenesis is regulated through USP7-mediated deubiquitination of the histone acetyltransferase TIP60.

    PubMed

    Gao, Yuan; Koppen, Arjen; Rakhshandehroo, Maryam; Tasdelen, Ismayil; van de Graaf, Stan F; van Loosdregt, Jorg; van Beekum, Olivier; Hamers, Nicole; van Leenen, Dik; Berkers, Celia R; Berger, Ruud; Holstege, Frank C P; Coffer, Paul J; Brenkman, Arjan B; Ovaa, Huib; Kalkhoven, Eric

    2013-01-01

    Transcriptional coregulators, including the acetyltransferase Tip60, have a key role in complex cellular processes such as differentiation. Whereas post-translational modifications have emerged as an important mechanism to regulate transcriptional coregulator activity, the identification of the corresponding demodifying enzymes has remained elusive. Here we show that the expression of the Tip60 protein, which is essential for adipocyte differentiation, is regulated through polyubiquitination on multiple residues. USP7, a dominant deubiquitinating enzyme in 3T3-L1 adipocytes and mouse adipose tissue, deubiquitinates Tip60 both in intact cells and in vitro and increases Tip60 protein levels. Furthermore, inhibition of USP7 expression and activity decreases adipogenesis. Transcriptome analysis reveals several cell cycle genes to be co-regulated by both Tip60 and USP7. Knockdown of either factor results in impaired mitotic clonal expansion, an early step in adipogenesis. These results reveal deubiquitination of a transcriptional coregulator to be a key mechanism in the regulation of early adipogenesis.

  8. Role of Saccharomyces cerevisiae serine O-acetyltransferase in cysteine biosynthesis.

    PubMed

    Takagi, Hiroshi; Yoshioka, Kenji; Awano, Naoki; Nakamori, Shigeru; Ono, Bun ichiro

    2003-01-28

    Some strains of Saccharomyces cerevisiae have detectable activities of L-serine O-acetyltransferase (SATase) and O-acetyl-L-serine/O-acetyl-L-homoserine sulfhydrylase (OAS/OAH-SHLase), but synthesize L-cysteine exclusively via cystathionine by cystathionine beta-synthase and cystathionine gamma-lyase. To untangle this peculiar feature in sulfur metabolism, we introduced Escherichia coli genes encoding SATase and OAS-SHLase into S. cerevisiae L-cysteine auxotrophs. While the cells expressing SATase grew on medium lacking L-cysteine, those expressing OAS-SHLase did not grow at all. The cells expressing both enzymes grew very well without L-cysteine. These results indicate that S. cerevisiae SATase cannot support L-cysteine biosynthesis and that S. cerevisiae OAS/OAH-SHLase produces L-cysteine if enough OAS is provided by E. coli SATase. It appears as if S. cerevisiae SATase does not possess a metabolic role in vivo either because of very low activity or localization. For example, S. cerevisiae SATase may be localized in the nucleus, thus controlling the level of OAS required for regulation of sulfate assimilation, but playing no role in the direct synthesis of L-cysteine.

  9. 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.

  10. Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire

    PubMed Central

    Huang, Fu; Paulson, Ariel; Dutta, Arnob; Venkatesh, Swaminathan; Smolle, Michaela

    2014-01-01

    KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and have been shown to play important roles in transcriptional regulation. Here, we demonstrate that the Drosophila KAT6 Enok acetylates histone H3 Lys 23 (H3K23) in vitro and in vivo. Mutants lacking functional Enok exhibited defects in the localization of Oskar (Osk) to the posterior end of the oocyte, resulting in loss of germline formation and abdominal segments in the embryo. RNA sequencing (RNA-seq) analysis revealed that spire (spir) and maelstrom (mael), both required for the posterior localization of Osk in the oocyte, were down-regulated in enok mutants. Chromatin immunoprecipitation showed that Enok is localized to and acetylates H3K23 at the spir and mael genes. Furthermore, Gal4-driven expression of spir in the germline can largely rescue the defective Osk localization in enok mutant ovaries. Our results suggest that the Enok-mediated H3K23 acetylation (H3K23Ac) promotes the expression of spir, providing a specific mechanism linking oocyte polarization to histone modification. PMID:25512562

  11. Histone Acetyltransferase Complexes Can Mediate Transcriptional Activation by the Major Glucocorticoid Receptor Activation Domain

    PubMed Central

    Wallberg, Annika E.; Neely, Kristen E.; Gustafsson, Jan-Åke; Workman, Jerry L.; Wright, Anthony P. H.; Grant, Patrick A.

    1999-01-01

    Previous studies have shown that the Ada adapter proteins are important for glucocorticoid receptor (GR)-mediated gene activation in yeast. The N-terminal transactivation domain of GR, τ1, is dependent upon Ada2, Ada3, and Gcn5 for transactivation in vitro and in vivo. Using in vitro techniques, we demonstrate that the GR-τ1 interacts directly with the native Ada containing histone acetyltransferase (HAT) complex SAGA but not the related Ada complex. Mutations in τ1 that reduce τ1 transactivation activity in vivo lead to a reduced binding of τ1 to the SAGA complex and conversely, mutations increasing the transactivation activity of τ1 lead to an increased binding of τ1 to SAGA. In addition, the Ada-independent NuA4 HAT complex also interacts with τ1. GAL4-τ1-driven transcription from chromatin templates is stimulated by SAGA and NuA4 in an acetyl coenzyme A-dependent manner. Low-activity τ1 mutants reduce SAGA- and NuA4-stimulated transcription while high-activity τ1 mutants increase transcriptional activation, specifically from chromatin templates. Our results demonstrate that the targeting of native HAT complexes by the GR-τ1 activation domain mediates transcriptional stimulation from chromatin templates. PMID:10454542

  12. Epigenetic Regulation of Axonal Growth of Drosophila Pacemaker Cells by Histone Acetyltransferase Tip60 Controls Sleep

    PubMed Central

    Pirooznia, Sheila K.; Chiu, Kellie; Chan, May T.; Zimmerman, John E.; Elefant, Felice

    2012-01-01

    Tip60 is a histone acetyltransferase (HAT) enzyme that epigenetically regulates genes enriched for neuronal functions through interaction with the amyloid precursor protein (APP) intracellular domain. However, whether Tip60-mediated epigenetic dysregulation affects specific neuronal processes in vivo and contributes to neurodegeneration remains unclear. Here, we show that Tip60 HAT activity mediates axonal growth of the Drosophila pacemaker cells, termed “small ventrolateral neurons” (sLNvs), and their production of the neuropeptide pigment-dispersing factor (PDF) that functions to stabilize Drosophila sleep–wake cycles. Using genetic approaches, we show that loss of Tip60 HAT activity in the presence of the Alzheimer’s disease-associated APP affects PDF expression and causes retraction of the sLNv synaptic arbor required for presynaptic release of PDF. Functional consequence of these effects is evidenced by disruption of the sleep–wake cycle in these flies. Notably, overexpression of Tip60 in conjunction with APP rescues these sleep–wake disturbances by inducing overelaboration of the sLNv synaptic terminals and increasing PDF levels, supporting a neuroprotective role for dTip60 in sLNv growth and function under APP-induced neurodegenerative conditions. Our findings reveal a novel mechanism for Tip60 mediated sleep–wake regulation via control of axonal growth and PDF levels within the sLNv-encompassing neural network and provide insight into epigenetic-based regulation of sleep disturbances observed in neurodegenerative diseases like Alzheimer’s disease. PMID:22982579

  13. Transgenic rice plants expressing trichothecene 3-O-acetyltransferase show resistance to the Fusarium phytotoxin deoxynivalenol.

    PubMed

    Ohsato, Shuichi; Ochiai-Fukuda, Tetsuko; Nishiuchi, Takumi; Takahashi-Ando, Naoko; Koizumi, Shinzo; Hamamoto, Hiroshi; Kudo, Toshiaki; Yamaguchi, Isamu; Kimura, Makoto

    2007-04-01

    Fusarium head blight (FHB) is a devastating disease of small grain cereal crops caused by the necrotrophic pathogen Fusarium graminearum and Fusarium culmorum. These fungi produce the trichothecene mycotoxin deoxynivalenol (DON) and its derivatives, which enhance the disease development during their interactions with host plants. For the self-protection, the trichothecene producer Fusarium species have Tri101 encoding trichothecene 3-O-acetyltransferase. Although transgenic expression of Tri101 significantly reduced inhibitory action of DON on tobacco plants, there are several conflicting observations regarding the phytotoxicity of 3-acetyldeoxynivalenol (3-ADON) to cereal plants; 3-ADON was reported to be highly phytotoxic to wheat at low concentrations. To examine whether cereal plants show sufficient resistance to 3-ADON, we generated transgenic rice plants with stable expression and inheritance of Tri101. While root growth of wild-type rice plants was severely inhibited by DON in the medium, this fungal toxin was not phytotoxic to the transgenic lines that showed trichothecene 3-O-acetylation activity. This is the first report demonstrating the DON acetylase activity and DON-resistant phenotype of cereal plants expressing the fungal gene.

  14. The Histone Acetyltransferase MOF is a Key Regulator of the Embryonic Stem Cell Core Transcriptional Network

    PubMed Central

    Li, Xiangzhi; Li, Li; Pandey, Ruchi; Byun, Jung S.; Gardner, Kevin; Qin, Zhaohui; Dou, Yali

    2012-01-01

    SUMMARY Pluripotent embryonic stem cells (ESCs) maintain self-renewal and the potential for rapid response to differentiation cues. Both ESC features are subject to epigenetic regulation. Here we show that histone acetyltransferase Mof plays an essential role in the maintenance of ESC self-renewal and pluripotency. ESCs with Mof deletion lose characteristic morphology, alkaline phosphatase (AP) staining and differentiation potential. They also have aberrant expression of core transcription factors Nanog, Oct4 and Sox2. Importantly, the phenotypes of Mof null ESCs can be partially suppressed by Nanog overexpression, supporting that Mof functions as an upstream regulator of Nanog in ESCs. Genome-wide ChIP sequencing and transcriptome analyses further demonstrate that Mof is an integral component of ESC core transcription network and Mof primes genes for diverse developmental programs. Mof is also required for Wdr5 recruitment and H3 K4 methylation at key regulatory loci, highlighting complexity and interconnectivity of various chromatin regulators in ESCs. PMID:22862943

  15. Evidence of a MOF histone acetyltransferase-containing NSL complex in C. elegans

    PubMed Central

    Hoe, Matthew; Nicholas, Hannah R

    2014-01-01

    Regulation of chromatin is a key process in the developmental control of gene expression. Many multi-subunit protein complexes have been found to regulate chromatin through the modification of histone residues. One such complex is the MOF histone acetyltransferase-containing NSL complex. While the composition of the human and Drosophila NSL complexes has been determined and the functions of these complexes investigated, the existence of an equivalent complex in nematodes such as Caenorhabditis elegans has not yet been explored. Here we summarise evidence, from our own work and that of others, that homologues of NSL complex components are found in C. elegans. We review data suggesting that nematode proteins SUMV-1 and SUMV-2 are homologous to NSL2 and NSL3, respectively, and that SUMV-1 and SUMV-2 may form a complex with MYS-2, the worm homolog of MOF. We propose that these interactions suggest the existence of a nematode NSL-like complex and discuss the roles of this putative NSL complex in worms as well as exploring the possibility of crosstalk between NSL and COMPASS complexes via components that are common to both. We present the groundwork from which a full characterization of a nematode NSL complex may begin. PMID:26430553

  16. Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis.

    PubMed

    Zaunbrecher, M Analise; Sikes, R David; Metchock, Beverly; Shinnick, Thomas M; Posey, James E

    2009-11-24

    The emergence of multidrug-resistant (MDR) tuberculosis (TB) highlights the urgent need to understand the mechanisms of resistance to the drugs used to treat this disease. The aminoglycosides kanamycin and amikacin are important bactericidal drugs used to treat MDR TB, and resistance to one or both of these drugs is a defining characteristic of extensively drug-resistant TB. We identified mutations in the -10 and -35 promoter region of the eis gene, which encodes a previously uncharacterized aminoglycoside acetyltransferase. These mutations led to a 20-180-fold increase in the amount of eis leaderless mRNA transcript, with a corresponding increase in protein expression. Importantly, these promoter mutations conferred resistance to kanamycin [5 microg/mL < minimum inhibitory concentration (MIC)

  17. 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

  18. 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

  19. Function of neuromuscular synapses in the zebrafish choline-acetyltransferase mutant bajan.

    PubMed

    Wang, Meng; Wen, Hua; Brehm, Paul

    2008-10-01

    We have identified a zebrafish mutant line, bajan, in which compromised motility and fatigue result from a point mutation in the gene coding choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine (ACh) synthesis. Although the mutation predicts loss of ChAT function, bajan inexplicably retains low levels of neuromuscular transmission. We exploited this residual activity and determined the consequences for synaptic function. The attenuated synaptic responses were a direct consequence of a decrease in both resting mean quantal size and quantal content. To replicate behavioral fatigue in swimming, motorneurons were stimulated at high frequencies. A prominent reduction in quantal content, reflecting vesicle depletion, was coincident with a small additional reduction in quantal size. In humans, defective ChAT leads to episodic apnea, a form of congenital myasthenic syndrome characterized by use-dependent fatigue. In contrast to bajan, however, afflicted individuals exhibit a normal resting quantal size and quantal content. The fatigue in humans results from a pronounced long-lasting drop in quantal size with little or no change in quantal content. These differences have important implications for interpreting fatigue as well as on understanding the impact of ACh availability on vesicle filling and recycling.

  20. 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.

  1. Schizosaccharomyces pombe mst2+ Encodes a MYST Family Histone Acetyltransferase That Negatively Regulates Telomere Silencing†

    PubMed Central

    Gómez, Eliana B.; Espinosa, Joaquín M.; Forsburg, Susan L.

    2005-01-01

    Histone acetylation and deacetylation are associated with transcriptional activity and the formation of constitutively silent heterochromatin. Increasingly, histone acetylation is also implicated in other chromosome transactions, including replication and segregation. We have cloned the only Schizosaccharomyces pombe MYST family histone acetyltransferase genes, mst1+ and mst2+. Mst1p, but not Mst2p, is essential for viability. Both proteins are localized to the nucleus and bound to chromatin throughout the cell cycle. Δmst2 genetically interacts with mutants that affect heterochromatin, cohesion, and telomere structure. Mst2p is a negative regulator of silencing at the telomere but does not affect silencing in the centromere or mating type region. We generated a census of proteins and histone modifications at wild-type telomeres. A histone acetylation gradient at the telomeres is lost in Δmst2 cells without affecting the distribution of Taz1p, Swi6p, Rad21p, or Sir2p. We propose that the increased telomeric silencing is caused by histone hypoacetylation and/or an increase in the ratio of methylated to acetylated histones. Although telomere length is normal, meiosis is aberrant in Δmst2 diploid homozygote mutants, suggesting that telomeric histone acetylation contributes to normal meiotic progression. PMID:16199868

  2. Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire.

    PubMed

    Huang, Fu; Paulson, Ariel; Dutta, Arnob; Venkatesh, Swaminathan; Smolle, Michaela; Abmayr, Susan M; Workman, Jerry L

    2014-12-15

    KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and have been shown to play important roles in transcriptional regulation. Here, we demonstrate that the Drosophila KAT6 Enok acetylates histone H3 Lys 23 (H3K23) in vitro and in vivo. Mutants lacking functional Enok exhibited defects in the localization of Oskar (Osk) to the posterior end of the oocyte, resulting in loss of germline formation and abdominal segments in the embryo. RNA sequencing (RNA-seq) analysis revealed that spire (spir) and maelstrom (mael), both required for the posterior localization of Osk in the oocyte, were down-regulated in enok mutants. Chromatin immunoprecipitation showed that Enok is localized to and acetylates H3K23 at the spir and mael genes. Furthermore, Gal4-driven expression of spir in the germline can largely rescue the defective Osk localization in enok mutant ovaries. Our results suggest that the Enok-mediated H3K23 acetylation (H3K23Ac) promotes the expression of spir, providing a specific mechanism linking oocyte polarization to histone modification. © 2014 Huang et al.; Published by Cold Spring Harbor Laboratory Press.

  3. Contribution of gentamicin 2'-N-acetyltransferase to the O acetylation of peptidoglycan in Providencia stuartii.

    PubMed

    Payie, K G; Rather, P N; Clarke, A J

    1995-08-01

    A collection of Providencia stuartii mutants which either underexpress or overexpress aac(2')-Ia, the chromosomal gene coding for gentamicin 2'-N-acetyltransferase (EC 2.3.1.59), have been characterized phenotypically as possessing either lower or higher levels of peptidoglycan O acetylation, respectively, than the wild type. These mutants were subjected to both negative-staining and thin-section electron microscopy. P. stuartii PR100, with 42% O acetylation of peptidoglycan compared with 52% O acetylation in the wild type, appeared as irregular rods. In direct contrast, P. stuartii strains PR50.LM3 and PR51, with increased levels of peptidoglycan O acetylation (65 and 63%, respectively), appeared as coccobacilli and chain formers, respectively. Membrane blebbing was also observed with the chain-forming strain PR51. Thin sectioning of this mutant indicated that it was capable of proper constriction and separation. P. stuartii PM1, when grown to mid-exponential phase, did not have altered peptidoglycan O-acetylation levels, and cellular morphology remained similar to that of wild-type strains. However, continued growth into stationary phase resulted in a 15% increase in peptidoglycan O acetylation concomitant with a change of some cells from a rod-shaped to a coccobacillus-shaped morphology. The fact that these apparent morphological changes were directly related to levels of O acetylation support the view that this modification plays a role in the maintenance of peptidoglycan structure, presumably through the control of autolytic activity.

  4. Sas3 is a histone acetyltransferase and requires a zinc finger motif.

    PubMed

    Takechi, S; Nakayama, T

    1999-12-20

    SAS3 was originally isolated as a gene related to SAS2, which encodes a positive regulator of transcriptional silencing in yeast. The Sas3 protein possesses an evolutionally conserved domain that is shared by a group of SAS-like factors. This conserved domain contains an atypical zinc finger motif and a putative acetyl-CoA binding motif. We showed that recombinant Sas3 exhibits histone acetyltransferase (HAT) activity toward acetylate core histones H2A, H3, and H4. This substrate specificity is similar to those of Tip60 and Esa1. Analysis of a series of deletion mutants revealed that the minimum region required for HAT activity is located within amino acid residues 241-577, including the domain conserved in the MYST family proteins. Amino acid substitution mutant analysis showed that both the acetyl-CoA binding motif and the zinc finger motif are required for HAT activity. These results suggest that SAS3 and its family members require the zinc finger motif for their activity. Copyright 1999 Academic Press.

  5. The histone acetyltransferase MOF is a key regulator of the embryonic stem cell core transcriptional network.

    PubMed

    Li, Xiangzhi; Li, Li; Pandey, Ruchi; Byun, Jung S; Gardner, Kevin; Qin, Zhaohui; Dou, Yali

    2012-08-03

    Pluripotent embryonic stem cells (ESCs) maintain self-renewal and the potential for rapid response to differentiation cues. Both ESC features are subject to epigenetic regulation. Here we show that the histone acetyltransferase Mof plays an essential role in the maintenance of ESC self-renewal and pluripotency. ESCs with Mof deletion lose characteristic morphology, alkaline phosphatase (AP) staining, and differentiation potential. They also have aberrant expression of the core transcription factors Nanog, Oct4, and Sox2. Importantly, the phenotypes of Mof null ESCs can be partially suppressed by Nanog overexpression, supporting the idea that Mof functions as an upstream regulator of Nanog in ESCs. Genome-wide ChIP-sequencing and transcriptome analyses further demonstrate that Mof is an integral component of the ESC core transcriptional network and that Mof primes genes for diverse developmental programs. Mof is also required for Wdr5 recruitment and H3K4 methylation at key regulatory loci, highlighting the complexity and interconnectivity of various chromatin regulators in ESCs.

  6. Structural analysis and promoter characterization of the human collagenase-3 gene (MMP13)

    SciTech Connect

    Pendas, A.M.; Balbin, M.; Llano, E.

    1997-03-01

    Human collagenase-3 (MMP13) is a recently identified member of the matrix metalloproteinase (MMP) family that is expressed in breast carcinomas and in articular cartilage from arthritic patients. In this work we have isolated and characterized genomic clones coding for human collagenase-3. This gene is composed of 10 exons and 9 introns and spans over 12.5 kb. The overall organization of the collagenase-3 gene is similar to that of other MMP genes clustered at chromosome 11q22, including fibroblast collagenase (MMP-1), matrilysin (MMP-7), and macrophage metalloelastase (MMP-12), but is more distantly related to genes coding for stromelysin-3 (MMP-11), gelatinase-A (MMP-2), and gelatinase-B (MMP-9), which map outside of this gene cluster. Nucleotide sequence analysis of about 1 kb of the 5{prime}-flanking region of the collagenase-3 gene revealed the presence of a TATA box, an AP-1 motif, a PEA-3 consensus sequence, an osteoblast specific element (OSE-2), and a TGF-{beta} inhibitory element. Transient transfection experiments in HeLa and COS-1 cells with chloramphenicol acetyltransferase (CAT)-containing constructs showed that the AP-1 site is functional and responsible for the observed inducibility of the reporter gene by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). However, and in contrast to other MMP genes, no significative synergistic effect on CAT activity between the AP-1 and PEA-3 elements found in the collagenase-3 gene promoter was found. DNA binding analysis with nuclear extracts from HeLa cells revealed the formation of specific complexes between collagenase-3 promoter sequences containing the AP-1 site and nuclear proteins. The presence of this AP-1 functional site, which is able to confer responsiveness to a variety of tumor promoters and oncogene products, may contribute to explaining the high-level expression of collagenase-3 in breast carcinomas and degenerative joint diseases. 48 refs., 5 figs., 2 tabs.

  7. 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.

  8. Cats protecting birds revisited.

    PubMed

    Fan, Meng; Kuang, Yang; Feng, Zhilan

    2005-09-01

    In this paper, we revisit the dynamical interaction among prey (bird), mesopredator (rat), and superpredator (cat) discussed in [Courchamp, F., Langlais, M., Sugihara, G., 1999. Cats protecting birds: modelling the mesopredator release effect. Journal of Animal Ecology 68, 282-292]. First, we develop a prey-mesopredator-superpredator (i.e., bird-rat-cat, briefly, BRC) model, where the predator's functional responses are derived based on the classical Holling's time budget arguments. Our BRC model overcomes several model construction problems in Courchamp et al. (1999), and admits richer, reasonable and realistic dynamics. We explore the possible control strategies to save or restore the bird by controlling or eliminating the rat or the cat when the bird is endangered. We establish the existence of two types of mesopredator release phenomena: severe mesopredator release, where once superpredators are suppressed, a burst of mesopredators follows which leads their shared prey to extinction; and mild mesopredator release, where the mesopredator release could assert more negative impact on the endemic prey but does not lead the endemic prey to extinction. A sharp sufficient criterion is established for the occurrence of severe mesopredator release. We also show that, in a prey-mesopredator-superpredator trophic food web, eradication of introduced superpredators such as feral domestic cats in the BRC model, is not always the best solution to protect endemic insular prey. The presence of a superpredator may have a beneficial effect in such systems.

  9. Hearing disorders in cats.

    PubMed

    Strain, George M

    2017-03-01

    Practical relevance: Auditory function is a sense that is central to life for cats - being important in situational awareness of potential predators, pursuit of prey, and for communication with conspecifics, humans and other species. Deafness in cats is most frequently the result of a genetic disorder, strongly associated with white fur and blue eyes, but may also result from acquired causes such as advancing age, ototoxic drugs, infection, environmental noise and physical trauma. Deafness can be sensorineural, where there is loss of cochlear hair cells, or conductive, where sound is muffled on its way to the inner ear. Clinical challenges: Establishing whether a cat is deaf can be difficult as behavioral testing of hearing is subjective and does not reliably detect unilateral deafness. Brainstem auditory evoked response testing is an objective measure but is limited in its availability. Currently, sensorineural deafness is irreversible because no treatments are available to restore lost hair cells. Conductive hearing loss can usually be treated, although full hearing recovery following otitis media may take weeks as the body clears the middle ear of debris. Evidence base: The author draws on the published literature and his extensive research on clinical aspects and molecular genetics of deafness, principally in companion animals, to review types and forms of deafness in cats. He also discusses current diagnostic approaches and provides brief advice for managing cats with hearing loss.

  10. Evidence for multiple promoters of the human IL-5 receptor alpha subunit gene: a novel 6-base pair element determines cell-specific promoter function.

    PubMed

    Zhang, J; Kuvelkar, R; Cheewatrakoolpong, B; Williams, S; Egan, R W; Billah, M M

    1997-12-01

    In addition to a previously characterized promoter (P1), we now show the existence of a second promoter for the human IL-5Ralpha gene. Initially, a genomic region (P2) 5' upstream of human IL-5Ralpha exon 2 was cloned by an inverted PCR. The transcriptional start site was then mapped to a deoxycytidine (C) residue within P2 by analyzing cellular mRNA with both the 5' rapid amplification of cDNA end-PCR and S1 nuclease protection assays. Transfection of eosinophilic HL-60 cells with reporter gene constructs in which either P1 or P2 was linked to the bacterial chloramphenicol acetyltransferase (CAT) gene resulted in CAT