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)

Additional parameters for the chloramphenicol acetyltransferase (CAT) activity in spores of S. griseus are substantiated. A linear increase in activity was observed with increasing spore number up to a concentration of 5 x 10(10) spores/ml. Similarly an increase of the chloramphenicol concentration up to 500 mug/ml increased the activity. However, a drastic decrease in activity was noted above this level suggesting inhibition of the enzyme by the substrate. The CAT activity in the spores was highly influenced by the pH of the medium reaching a maximum at pH 6.5. This may suggest that CAT is apparently located to the outer surface of the spores and therefore very sensitive to variations in pH of the medium. The CAT showed a marked specificity for D-threo and D-erythrochloramphenicol, while no activity was observed with L-isomers. The enzyme acetylates D,L-erythrodechlor-chloramphenicol with a yield of 45% as compared to the D-threo parent antibiotic. While the tyrosinase characteristics (melanin formation) of S. griseus was eliminated by acriflavine or ethidium bromide treatment the CAT characteristic was persistent. The melanin negative variants retained all otherproperties of the parent strain including the production of antimicrobial agents; and revertants were not detected. The results suggest that the tyrosinase determinant gene is apparently located on an extrachromosomal element (plasmid). On the other hand, the location of the gene for CAT is not assigned yet. The nature of CAT in growing cells and the spores of S. griseus was investigated. The results show that CAT accumulated during the sporulation phase or the vegetative growth is inducible in nature; therefore the morphogenetic sequence in the strain bears no influence on CAT induction.
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PMID:Biotransformation of antibiotics. II. Investigation of the chloramphenicol acetyltransferase in Streptomyces griseus. 82 95

The mouse c locus encodes tyrosinase (monophenol monooxygenase; monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1), the key enzyme in melanin synthesis, which is expressed in the pigment epithelium of the retina and in melanocytes derived from the neural crest. To define regulatory regions of the gene that are important for cell type-specific expression, a deletion series of the tyrosinase 5' region was fused to a chloramphenicol acetyltransferase (CAT) reporter gene and electroporated into tyrosinase-expressing and -nonexpressing cell lines. We show that 270 base pairs 5' of the transcriptional start site is sufficient for CAT expression in a human and a mouse melanoma cell line. This 5' flanking fragment, when cloned in the context of a tyrosinase minigene construct and injected into fertilized eggs of an albino mouse strain, is sufficient for cell type-specific expression in mice. The transgenic mice were pigmented in both skin and eyes. In situ hybridization analysis shows that the 270-base-pair regulatory region contains elements sufficient for specific expression of the transgene both in the pigmented epithelial cells of the retina, which are derived from the optic cup, and in neural crest-derived melanocytes.
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PMID:The mouse tyrosinase promoter is sufficient for expression in melanocytes and in the pigmented epithelium of the retina. 190 69

Streptococcus thermophilus (ST) chromosomal DNA (chr DNA) fragments having promoter activity were cloned and selected in Escherichia coli using a chloramphenicol acetyltransferase- (cat-) based promoter-probe vector pKK520-3. Insertion of a promoterless streptomycete melanin biosynthesis operon (melC) downstream from the promoters of the library further identified clone STP2201 as a strong promoter in E. coli. Subcloning of a STP2201-melC DNA fragment into the pMEU-series S. thermophilus-E. coli shuttle vectors yielded pEU5xML2201x plasmids that conferred Mel+ phenotype to E. coli. The pEU5aML2201a was further shown to afford a high level of tyrosinase pro-anti-tyrosinase antiserum in S. thermophilus. Substituting melC with a streptomycete cholesterol oxidase gene (choA) in the same orientation yielded pEU5aCH2201a that conferred ChoA activity to an E. coli transformant at a level of (1.06 +/- 0.15) x 10(-7) units mg-1 protein. Introduction of this plasmid into S. thermophilus by electrotransformation yielded ChoA+ transformant that produced the enzyme at about 25% of the level found in E. coli.
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PMID:Expression of Streptomyces melC and choA genes by a cloned Streptococcus thermophilus promoter STP2201. 766 96

Proacrosin, the zymogen form of the serine protease acrosin, is located within the acrosomal vesicle of mammalian spermatozoa and has been suggested to be involved in the fertilization process. In mouse and rat, expression of the proacrosin gene starts in pachytene spermatocytes and continues through the early stages of spermiogenesis. We have shown recently that 2.3 kilobase pairs of the 5'-flanking region of the rat proacrosin gene is sufficient to direct chloramphenicol acetyltransferase gene expression in a germ cell-specific and developmental stage-specific manner in the mouse. Additional transgenic lines have been generated which include two deletions in the 5'-flanking region and a tyrosinase minigene as marker for gene expression. Transgenic mice bearing these two truncated fragments showed different patterns of reporter gene expression. Transgenic lines (BM, B3, B2) harboring the 397-base pair (bp) fragment (from 45 to 442 bp upstream of ATG) showed no chloramphenicol acetyltransferase (CAT) activity in either testis or other tissues, but analysis via reverse transcription polymerase chain reaction confirmed low levels of reporter gene transcription in testis. Transgenic line TC bearing a longer fragment of 877 bp (from 45 to 922 bp upstream of ATG) showed a reporter gene expression and chloramphenicol acetyltransferase enzyme activity which was identical to that found in mice harboring the 2.3-kilobase pair 5'-flanking region. The analysis of the CAT gene expression during testicular development showed diploid transcription and haploid translation. It can be concluded that all sequences required for a basic level of testis-specific transcription of transgene are present within the 397-bp fragment, and other DNA sequences located outside of the 397-bp fragment but present within the 877-bp fragment can function as enhancer elements. Two fragments within the 877-bp region were identified by gel retardation assays as binding exclusively to nuclear factor(s) from testis protein extracts. In both fragments we identified sequence elements which are present in the promoter region of the germ cell-specific genes for histone H2B and protamine 1, respectively.
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PMID:Functional and molecular characterization of the transcriptional regulatory region of the proacrosin gene. 779 16

Tyrosinase is the principal enzyme in the biosynthesis of melanin. The expression of tyrosinase is tissue-specific and appears to be regulated by various hormonal and environmental factors. Elucidation of the genomic structure and molecular basis of control of tyrosinase gene expression will greatly enhance our understanding of the regulation of human pigmentation. To this end, we have isolated and performed restriction mapping of recombinant cosmid and lambda phage clones containing the human tyrosinase gene, sequenced a 2.2-kilobase (kb) region of its promoter, and determined the potential regions regulating the tyrosinase gene expression in transient-expression system. The human tyrosinase gene is comprised of five exons and four introns. Based on our restriction mapping studies, the gene spans a distance of over 65-kb on chromosome 11 (q14-->q21). We constructed a series of plasmids (pHTY-CAT) that contain 5' sequential deletions of the human tyrosinase 5' flanking sequence fused to the reporter gene, chloramphenicol acetyltransferase (CAT). The plasmids were used to locate promoter regions that are potential regulators of tyrosinase gene expression in a transient expression system using melanoma cell lines. In human melanoma cells, the plasmid construct with a -2020 base pair (bp) promoter yielded the highest CAT activity. When the deletions reached -1739 bp, the CAT activity was dramatically reduced, indicating that important enhancer elements for transcription control are present between -1739 and -2020 bp. Further deletions up to -550 bp also resulted in dramatic decreases of CAT activity. However, when the deletion included -550 bp of the 5' flanking sequence, there was 26 percent of the CAT activity compared to that of the -2020 bp promoter. Deletions beyond -550 bp also showed markedly decreased CAT activity. Based on our data, we suggest that human tyrosinase gene expression is governed by both tissue-specific and multiple regulatory elements.
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PMID:Structural organization of the human tyrosinase gene and sequence analysis and characterization of its promoter region. 817 57

5-Bromodeoxyuridine (BrdU), a thymidine analog, suppresses melanogenesis in Syrian hamster melanoma cells. Tyrosinase, which is the key enzyme for the synthesis of melanin, is suppressed by exposure to BrdU, and the drop in enzyme activity is correlated with a drop in tyrosinase mRNA level. In order to investigate whether suppression of tyrosinase mRNA by BrdU is due to BrdU substitution into coding sequences or upstream sequences of the tyrosinase gene, we carried out stable and transient transfection assays with constructs containing either the human tyrosinase cDNA sequence under the control of a nontyrosinase promoter or a chloramphenicol acetyltransferase (CAT) reporter gene under the control of 5' flanking sequences of the mouse tyrosinase gene. When the plasmid containing the tyrosinase cDNA was stably transfected into mouse fibroblasts, tyrosinase activity in the transfectants was not suppressed by BrdU. Since BrdU would be incorporated into the tyrosinase cDNA integrated in these transfectants, the results suggest that BrdU suppression of tyrosinase gene expression is not due to its incorporation into coding sequences of the tyrosinase gene. When plasmids with tyrosinase regulatory sequences were transfected into melanoma cells for transient expression assays, CAT gene expression was suppressed by BrdU. Because the CAT plasmids do not contain a mammalian origin of replication and should not replicate under the conditions of transient transfection, BrdU would not be incorporated into the DNA of those plasmids. Therefore, these results suggest that the suppression of tyrosinase gene expression by BrdU also is not due to the incorporation of BrdU into upstream sequences of the tyrosinase gene.
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PMID:Suppression of tyrosinase gene expression by bromodeoxyuridine in Syrian hamster melanoma cells is not due to its incorporation into upstream or coding sequences of the tyrosinase gene. 833 36

The cAMP-dependent pathway up-regulates MITF (microphthalmia-associated transcription factor), important for key melanogenic proteins such as tyrosinase, TRP-1 (tyrosinase-related protein 1) and TRP-2. We asked whether MITF is also a key transcription factor for PKC-beta (protein kinase C-beta), required to phosphorylate otherwise inactive tyrosinase. When paired cultures of human melanocytes were treated with isobutylmethylxanthine, known to increase intracellular cAMP, both protein and mRNA levels of PKC-beta were induced by 24 h. To determine whether MITF modulates PKC-beta expression, paired cultures of human melanocytes were transfected with dn-MITF (dominant-negative MITF) or empty control vector. By immunoblotting, PKC-beta protein was reduced by 63+/-3.7% within 48 h. Co-transfection of an expression vector for MITF-M, the MITF isoform specific for pigment cells, or empty control vector with a full-length PKC-beta promoter-CAT (chloramphenicol acetyltransferase) reporter construct (PKC-beta/CAT) into Cos-7 cells showed >60-fold increase in CAT activity. Melanocytes abundantly also expressed MITF-A, as well as the MITF-B and MITF-H isoforms. However, in contrast with MITF-M, MITF-A failed to transactivate co-expressed PKC-beta/CAT or CAT constructs under the control of a full-length tyrosinase promoter. Together, these results demonstrate that MITF, specifically MITF-M, is a key transcription factor for PKC-beta, linking the PKC- and cAMP-dependent pathways in regulation of melanogenesis.
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PMID:MITF mediates cAMP-induced protein kinase C-beta expression in human melanocytes. 1641 96

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