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)

Achieving co-ordinate, high-level and stable expression of multiple transgenes in plants is currently difficult. Expression levels are notoriously variable and influenced by factors that act independently on transgenes at different genetic loci. Instability of expression due to loss, re-arrangement or silencing of transgenes may occur, and is exacerbated by increasing numbers of transgenic loci and repeated use of homologous sequences. Even linking two or more genes within a T-DNA does not necessarily result in co-ordinate expression. Linking proteins in a single open reading frame--a polyprotein--is a strategy for co-ordinate expression used by many viruses. After translation, polyproteins are processed into constituent polypeptides, usually by proteinases encoded within the polyprotein itself. However, in foot-and-mouth disease virus (FMDV), a sequence (2A) of just 16-20 amino acids appears to have the unique capability to mediate cleavage at its own C-terminus by an apparently enzyme-independent, novel type of reaction. This sequence can also mediate cleavage in a heterologous protein context in a range of eukaryotic expression systems. We have constructed a plasmid in which the 2A sequence is inserted between the reporter genes chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS), maintaining a single open reading frame. Here we report that expression of this construct in wheatgerm lysate and transgenic plants results in efficient cleavage of the polyprotein and co-ordinate expression of active CAT and GUS. Self-processing polyproteins using the FMDV 2A sequence could therefore provide a system for ensuring co-ordinated, stable expression of multiple introduced proteins in plant cells.
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PMID:Self-processing 2A-polyproteins--a system for co-ordinate expression of multiple proteins in transgenic plants. 1020 2

The use of lactobacilli as starter organisms in food fermentation processes requires thorough knowledge of their reaction to the multitude of ecological factors including their response to stress. We have characterised the dnaK gene region of Lactobacillus sakei LTH681. Two chromosomal EcoRI fragments of 2.5 and 4.0 kb were identified using a homologous dnaK probe generated by PCR. The sequence analysis of the cloned fragments showed that the dnaK gene region consists of four heat shock genes with the organisation hrcA-grpE-dnaK-dnaJ. Comparison of the deduced amino acid sequences revealed high similarity to the corresponding heat shock proteins of Gram-positive bacteria. An upstream located orfY was found which exhibited substantial similarity (41.5%) to the chloramphenicol acetyltransferase of Enterobacter aerogenes. Northern hybridisation analysis revealed that the transcription of the genes is induced by heat shock (42 degrees C) as well as salt (6%) or ethanol (10%) stress. Several transcripts were detected including a polycistronic mRNA of 4.9 kb which represents the transcript of the complete dnaK gene region indicating a tetracistronic organisation of the dnaK operon. The other RNA fragments were identified as shorter transcripts (3.7 and 1.3 kb) or cleavage products of the polycistronic mRNAs. The transcription start sites of the dnaK operon were determined under inducing and non-inducing conditions. The site varied with the applied stress condition. A regulatory CIRCE element was identified located between the transcription and translation start site. The promoter region including CIRCE was transcriptionally fused to the beta-glucuronidase reporter gene gusA and expressed in L. sakei LTH681. The kinetics of transcriptional induction of gusA by heat shocking were identical to those of the dnaK operon confirming the involvement of the CIRCE element in regulation of gene expression.
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PMID:Molecular characterisation of the dnaK operon of Lactobacillus sakei LTH681. 1055 84

In this paper we report on searching for suitable reporters to monitor gene expression and protein secretion in the amylolytic yeast Schwanniomyces occidentalis. Several potential reporter and marker genes, formerly shown to be functional in other yeasts, were cloned downstream from the homologous invertase gene (INV) promoter and their activity was followed in conditions of repression and derepression of the INV promoter. However, neither beta-glucuronidase nor beta-lactamase nor phleomycin resistance-conferring gene, all originating from E. coli, were expressed in S. occidentalis cells to such a level to allow for monitoring of their activity. All the reporter genes tested have a higher percentage of GC (47-62%) in their DNA compared to the DNA composition of S. occidentalis genes that are more AT-rich (36% GC). The codon usage of all the reporter genes also varies from that of 16 so far sequenced S. occidentalis genes. This suggests that an appropriate composition of DNA and a codon usage similar to S. occidentalis genes might be very important parameters for an efficient expression of a heterologous gene in Schwanniomyces occidentalis. Indeed, two genes originating from Staphylococcus aureus, with an AT-content in their DNA similar to that of S. occidentalis, were functionally expressed in S. occidentalis cells. Both a phleomycin resistance-conferring gene and a chloramphenicol acetyltransferase-encoding gene thus represent suitable reporters of gene expression and protein secretion in S. occidentalis. Additionally, we show in this work that the transcription-regulating region and the signal peptide sequence of the S. occidentalis invertase gene were efficient to direct gene expression and subsequent protein secretion in Saccharomyces cerevisiae.
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PMID:Development of a reporter system for the yeast Schwanniomyces occidentalis: influence of DNA composition and codon usage. 1279 30

The antisense orientation of the Peanut chlorotic streak virus (PClSV) open reading frame (ORF) VII (denoted as p7R), in conjunction with the sense orientation of the PClSV leader sequence, acts as an intron and enhances the expression of a reporter gene, analyzed in protoplasts and transgenic plants of tobacco ( Nicotiana tabacum L.). Correct 5' and 3' splicing sites were determined for intron removal from the chimeric constructs using either beta-glucuronidase (GUS) or chloramphenicol acetyltransferase (CAT) as a reporter gene. In this splicing process, the active consensus 5' splicing donor site (AG/GTATA) is located at position +283 to +289 from the transcription start site (TSS) of the PClSV full-length transcript (FLt). The 3' splice site (TAG/GATT) is located on the p7R sequence at position +785 to +791 from the TSS. The combination of PClSV FLt leader and p7R enhanced the expression of reporter genes (CAT and GUS) by as much as 2-fold compared to the strong constitutive PClSV FLt promoter without an interfering leader sequence and about 30- to 800-fold compared to constructs containing the sense orientation of PClSV ORF VII (p7) in both protoplast transient-expression experiments and stably transformed transgenic plants. An increased level of mature transcripts accompanied this. This suggests that this combination of elements can mediate the intron-mediated enhancement (IME) phenomenon. We also demonstrated comparative IME with other heterologous promoters from caulimoviruses.
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PMID:Intron-mediated enhancement of gene expression in transgenic plants using chimeric constructs composed of the Peanut chlorotic streak virus (PClSV) promoter-leader and the antisense orientation of PClSV ORF VII (p7R). 1288 84

The efficacy of the ethanol-inducible alc transgene expression system, derived from the filamentous fungus Aspergillus nidulans, has been demonstrated in transgenic tomato. Two direct comparisons have been made. First, this study has utilized two transgenic lines carrying distinct reporter genes (chloramphenicol acetyltransferase and beta-glucuronidase) to distinguish aspects of induction determined by the nature of the gene/gene product rather than that of the plant. Second, comparisons have been made to data generated in other species in order to identify any species-specific effects. The induction profiles for different genes in different species have shown remarkable similarity indicating the broad applicability of this gene switch. While there are minor differences observed between species, these probably arise from diversity in their metabolism. A series of potential alternative inducers have also been tested, revealing that ethanol (through metabolism to acetaldehyde) is better than other alcohols and ketones included in this study. Expression driven by alc was demonstrated to vary spatially, the upper younger leaves having higher activity than the lower older leaves; this will be important for some applications, and for experimental design. The highest levels of activity from ethanol-inducible transgene expression were determined to be the equivalent of those from the constitutive Cauliflower Mosaic Virus 35S promoter. This suggests that the alc system could be an important tool for plant functional genomics.
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PMID:Characterization of the ethanol-inducible alc gene expression system in tomato. 1585 14


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