Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Several genes homologous to the methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli have been cloned and characterized from the Gram-positive bacterium, Bacillus subtilis. Sequence analysis reveals four large open reading frames, designated mcpA, mcpB, tlpA, and tlpB, each encoding a predicted 72-kDa protein. These proteins exhibit strong homology to chemoreceptors from several organisms, although similarity is limited to the C-terminal domain. These transducer genes were mapped to a chromosomal position of 279 degrees, which is distant from previously identified fla, mot, or che loci. Each gene was inactivated by insertion of a nonpolar chloramphenicol acetyltransferase cassette in the N-terminal region. In vivo methylation of the bacterial strain deficient in mcpA revealed the loss of several methylated bands in the range of the MCP previously designated as H1, and greatly reduced methylation of the MCP designated as H2. Furthermore, this bacterial strain exhibited a chemotaxis deficiency toward glucose and alpha-methyl-glucoside. Inactivation of mcpB caused a reduction in methylation of the MCP designated as H3, while chemotaxis toward asparagine, aspartate, glutamine, and histidine was significantly impaired in this strain. Despite strong homology, inactivation of tlpA and tlpB did not result in an observed deficiency in chemotaxis. Most unusually, these mutant strains exhibited a striking tendency to adhere together and resisted disaggregation.
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PMID:Cloning and characterization of genes encoding methyl-accepting chemotaxis proteins in Bacillus subtilis. 818 84

We have analyzed the nucleotide sequence of complementary and genomic DNAs of the human androgen receptor (AR) gene in two siblings (patients 9006 and 9030) with receptor-positive complete androgen insensitivity (Rec(+)-CAI). Northern analysis indicated that mRNA of the AR was normal in size. However, its expression was relatively reduced in both patients. Consistent with the normal androgen-binding capacity (496 and 552 fmol/mg DNA for patients 9006 and 9030, respectively) but decreased DNA-binding ability (168 fmol/mg DNA) measured in genital skin fibroblasts, no mutation was found in both N-terminal and ligand-binding domains of the AR. However, a single base substitution (G-->A) was found in the second zinc finger of the DNA-binding domain at nucleotide 2372 of the AR cDNA in both cases. This resulted in the replacement of a highly conserved arginine residue (amino acid 614) by a histidine. When the mutated receptor plasmid was cotransfected into PC-3 cells together with the reporter chloramphenicol acetyltransferase gene, chloramphenicol acetyltransferase activity was not induced by 5 alpha-dihydrotestosterone treatment, confirming that the mutation renders the AR nonfunctional and can, therefore, be held responsible for the clinical features in these patients. These results highlight the importance of Arginine-614 in the second zinc finger of the DNA-binding domain of the AR in the protein-DNA interaction.
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PMID:A point mutation in the second zinc finger of the DNA-binding domain of the androgen receptor gene causes complete androgen insensitivity in two siblings with receptor-positive androgen resistance. 841 10

A catalytically essential histidine residue (His-195) of chloramphenicol acetyltransferase (CAT) acts as a general base in catalysis, abstracting a proton from the primary hydroxy group of chloramphenicol. The pKa of His-195 has been determined from the pH-dependence of chemical modification. Both methyl 4-nitrobenzenesulphonate and iodoacetamide inactivate CAT by irreversible modification of His-195. The kinetics of inactivation by methyl 4-nitrobenzenesulphonate are pseudo-first-order, and the pH-dependence of inactivation yields a pKa value of 6.60. Iodoacetamide inactivation proceeds with second-order kinetics and a pKa value of 6.80. An alternative site of modification at the active site of CAT is the thiol group of Cys-31, a residue which has no catalytic role. On replacement of Cys-31 with alanine (Ala-31 CAT), the pH-dependence of iodoacetamide inactivation gives a pKa value of 6.66. The pKa values derived from chemical-modification experiments directed at His-195 are in agreement with the pKa values of 6.62 and 6.61 determined for wild-type and Ala-31 CAT respectively from the pH-dependence of kcat/Km.
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PMID:The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase. 843 83

Peptide synthetases are multienzymatic complexes that synthesize bioactive peptides molecules by the thiotemplate mechanism. Comparison of the known sequences of peptide synthetases led us to the identification of a 350 amino acids domain catalysing elongation and containing the motif HHxxxDG. This motif is present as many times as acyltransfer or epimerisation reactions occur during biosynthesis of the peptide. The distance between this motif and the phosphopantetheinyl attachment site is nearly invariant. An identical motif is found in other enzymes effecting acyl transfer such as chloramphenicol acetyltransferase from Tn9 and dihydrolipoamide acyltransferase. Altogether, the HHxxxDG motif may constitute the signature of a superfamily sharing a common catalytic mechanism based on the acid-base properties of the second histidine for effecting acyl transfer or peptide epimerisation.
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PMID:Multienzymatic non ribosomal peptide biosynthesis: identification of the functional domains catalysing peptide elongation and epimerisation. 852 Oct 76

Cancer-related mutations of the p53 tumor suppressor gene are clustered in the four so-called 'hot spots', codons 175, 248, 273 and 281/282. By using recombination PCR in vitro mutagenesis, we introduced point mutations into the codon 273 of wild-type (wt) p53 (pC53-SN3) from Arg to His (pC53-273H [273H]), Asp (273D), Pro (273P), Lys (273K), Leu (273L) or Thr (273T), and compared their biological and biochemical activities with wt p53 and cancer-derived 175H, 248W and 273H/309S. Among them, 273H/309S, 273H and 273D as well as wt p53 transactivated the chloramphenicol acetyltransferase (CAT) gene placed downstream of the p53 binding consensus, while none of the other mutants including 273L did. Transcriptions from human c-fos and rat PCNA promoters were suppressed by wt p53 and 273D, while they were enhanced variously by all other mutants in Saos-2 and/or NIH3T3 cells. On the other hand, growth of human squamous carcinoma cell lines measured by the plating efficiency of G418-resistant colonies was enhanced by transfection of 175H, 248W, 273H/309S and 273P, while suppressed by not only wt p53, 273D and 273H but also 273L. Thus, 273H/309S enhanced cell growth in spite of its p53-specific transactivation activity, while 273L suppressed cell growth in spite of its complete loss of the p53-specific transactivation. We concluded that the sequence-specific transactivation of p53 is not always correlated with its growth inhibitory activity.
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PMID:The 273rd codon mutants of p53 show growth modulation activities not correlated with p53-specific transactivation activity. 864 76

Simple tandem repeats of the trinucleotide sequence CAG encode homopolymeric stretches of glutamine. Although polyglutamine has been identified in diverse proteins, it is present predominantly in transcription factors. We observed that oncogene-immortalized mouse macrophages express several genes that contain a CAG repeat motif. Therefore, we attempted to clone a novel gene that contains a CAG repeat and is associated with cytokine activation of macrophages. Screening of a mouse macrophage cDNA library with a probe comprising 12 consecutive CAG triplets identified at least one unique clone. The cDNA encodes a protein (named GRP-1 or glutamine repeat protein-1) with 171 amino acids, a calculated molecular mass of 21.6 kDa, and a predicted pI of 10.67. Greater than two-thirds of GRP-1 are only two amino acids, namely glutamine (50%) and histidine (18%). There are four polyglutamine motifs interspersed with histidine-rich regions. There is also a putative nuclear localization signal flanked by sites for possible serine phosphorylation. GRP-1 mRNA was expressed constitutively in some macrophage cell lines and B and T cell lines. Interferon-gamma or lipopolysaccharide augmented GRP-1 mRNA expression in the mouse macrophage cell line ANA-1. Western blot analyses using an antipeptide serum revealed that GRP-1 was localized in the nucleus of ANA-1 macrophages and transfected 3T3 fibroblasts. Overexpression of GRP-1 decreased Sp1-driven chloramphenicol acetyltransferase gene expression in transient cotransfection experiments. Because polyglutamine motifs can cause protein oligomerization and can function as transcriptional activation domains, we suggest that GRP-1 may be a transcription factor associated with interferon-gamma- or lipopolysaccharide-induced activation of macrophages.
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PMID:Molecular cloning and characterization of a novel mouse macrophage gene that encodes a nuclear protein comprising polyglutamine repeats and interspersing histidines. 881 Mar 23

Leukemia-inhibitory factor (LIF) is a neuropoietin able to regulate the differentiation and the survival of many cell types, which include some neuronal populations. The present study describes the genetic construction, expression, purification and properties of a diphtheria-toxin-related LIF gene fusion in which the native receptor-binding domain of diphtheria toxin was replaced with a gene encoding human LIF. The fusion protein expressed from the chimeric tox gene was designated DT-(1-389)-LIF-(2-184)-peptide. This fusion protein has a deduced molecular mass of 65980 Da and is formed by fusion of the first 389 amino acids of diphtheria toxin to amino acids 2-184 of mature human LIF, using a linker of 34 amino acids that includes six consecutive histidine residues. The latter span allows for single-step purification of the fusion protein by Ni(2+)-resin affinity chromatography. This linker provides a high degree of flexibility between the diphtheria toxin and LIF domains, thereby permitting aggregation-free refolding of the chimeric protein while bound to the affinity column. Both LIF and DT-(1-389)-LIF-(2-184)-peptide induced the phosphorylation of CLIP1 and CLIP2 in LIF-responsive neuroblastoma SH-N-BE cells. DT-(1-389)-LIF-(2-184)-peptide was selectively cytotoxic for cultured neuroblastoma cells bearing the LIF receptor, and for sympathetic neurons. The cytotoxic action of DT-(1-389)-LIF-(2-184)-peptide, like that of native diphtheria toxin, required receptor-mediated endocytosis, passage through an acidic compartment, and delivery of an ADP-ribosyltransferase to the cytosol of target cells. The latter point was confirmed by the fact that, while both LIF and DT-(1-389)-LIF-(2-184)-peptide increased c-fos mRNA expression in SH-N-BE cells, only LIF induced proenkephalin and c-fos promoter activities in cells transiently transfected with c-fos-chloramphenicol acetyltransferase and proenkephalin-chloramphenicol acetyltransferase fusion genes. Mutational analysis suggested that the C-terminal helix (helix D) of human LIF may, in part, constitute or contribute to the active site for LIF receptor binding and cell activation. The cytotoxic properties of DT-(1-389)-LIF-(2-184)-peptide may be useful in selectively depleting neuronal and immune cell populations that express the LIF beta receptor.
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PMID:Synthesis, cytotoxic properties and effects on early and late gene induction of a chimeric diphtheria toxin-leukemia-inhibitory factor protein. 891 49

The molecular signal for targeting catalases to peroxisomes has not been defined. In this study, a plant in vivo import system (tobacco BY-2 suspension culture cells) was used to test the current postulate that the peroxisome targeting signal (PTS) for mammalian catalases is the internal Ser-Lys-Leu (SKL) motif found approximately eight amino acid residues from the C-terminus. Elucidation of the catalase PTS has been hampered previously by the ubiquitous presence of catalase in peroxisomes. The current study was possible because antibodies to mammalian catalases did not recognize endogenous, tobacco peroxisome catalase. Rat and mouse liver catalases (Rcat and Mcat), with an internal Ser-His-Ile (SHI) and Ser-His-Met (SHM), respectively, and both with a C-terminal Ala-Asn-Leu (ANL), were expressed transiently in BY-2 cells and targeted to the peroxisomes. Sorting was demonstrated by double-label immunofluorescence colocalization of these catalases with tobacco catalase. Peroxisome targeting of Rcat was abolished as expected when the internal SHI residues were removed by deletion of three C-terminal portions (28, 16, or 11 residues). Surprisingly, peroxisome targeting was still abolished when SHI (or SHL produced by site-directed mutagenesis) were at the extreme C-terminus as a consequence of deleting eight residues. However, when SHL was at the C-terminus in full-sized Rcat via a mutation of ANL-COOH, the enzyme sorted to peroxisomes indicating that the position of the PTS is significant in Rcat. The importance of the internal context of the SHI (or SHL) was examined further by changing ANL-COOH to a non-SKL motif, AGS-COOH. This Rcat did not sort to the peroxisomes, nor did Rcat with its ANL-COOH deleted; these data indicated the necessity of the C-terminal tripeptide. Sufficiency of ANL was demonstrated when chloramphenicol acetyltransferase with an appended ANL-COOH was redirected from the cytosol to peroxisomes. Collectively, these results do not support the internal PTS hypothesis, but indicate that a type 1 PTS slightly divergent from the typical SKL motif serves as the necessary and sufficient PTS for rat liver and probably other eukaryotic catalases.
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PMID:Rat liver catalase is sorted to peroxisomes by its C-terminal tripeptide Ala-Asn-Leu, not by the internal Ser-Lys-Leu motif. 892 63

The bacteriocin haemocin is produced by most type b strains of Haemophilus influenzae, including strains of diverse genetic lineage, and is toxic to virtually all nontypeable H. influenzae strains. An H. influenzae transformant bearing a plasmid with a 1.5-kbp chromosomal fragment capable of conferring haemocin immunity on a haemocin-susceptible H. influenzae mutant was selected by using partially purified haemocin. Deletional and site-directed mutagenesis localized the haemocin immunity gene to the 3' open reading frame (ORF) within this chromosomal fragment. Subcloning of this ORF demonstrated that it was sufficient to confer haemocin immunity on wild-type haemocin-susceptible H. influenzae strains as well as haemocin-susceptible strains of Escherichia coli. This ORF, designated hmcl, encodes a 105-amino-acid protein with an estimated molecular mass of 12.6 kDa. Primer extension analysis revealed a putative transcriptional start site 34 bp upstream of the start codon, and the presence of a promoter immediately upstream of hmcI was confirmed by cloning the gene into a promoterless chloramphenicol acetyltransferase vector. To characterize the hmcI gene product, a His-HmcI fusion protein was constructed.
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PMID:Cloning and characterization of the haemocin immunity gene of Haemophilus influenzae. 904 29

We have developed plasmid-based expression systems that encode modified forms of T7 RNA polymerase (RNAP) having 6-12 histidine residues fused to the amino terminus. The histidine-tagged RNAPs (His-T7 RNAPS) are indistinguishable from the wild-type (WT) enzyme in nearly all biochemical assays. Similar plasmids that encode His-tagged T3 and SP6 RNAPs have also been constructed. To facilitate site-directed mutagenesis of the RNAP gene, the size of the target plasmid was minimized by using T7 RNAP itself as a selectable marker. BL21 (DCAT4) cells (which carry a chromosomal copy of the chloramphenicol acetyltransferase cat gene under control of a T7 promoter) are resistant to chloramphenicol when functional T7 RNAP is expressed, thus allowing the selection and maintenance of the target plasmid in these cells. Mutagenesis is accomplished by denaturing the plasmid, annealing mutagenic DNA primers, and repairing the plasmid with T4 DNA polymerase. Two DNA primers are used: one corrects a defect in the bla gene, the other introduces the desired mutation into the RNAP gene; 30-85% of the ampicillin-resistant transformants carry the desired mutation in the RNAP gene. By using BL21 (DCAT4) cells as a recipient for transformation the functional integrity of the RNAP gene may conveniently be monitored by assessing the level of chloramphenicol resistance in vivo. Methods for rapid, simultaneous purification of multiple samples of modified (His-tagged) and conventional RNAPs are described. Together, these developments greatly enhance our ability to characterize this important class of enzymes.
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PMID:Rapid mutagenesis and purification of phage RNA polymerases. 911 96


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