Chloroplast promoter driven expression of the chloramphenicol acetyl transferase gene in a cyanobacterium (original) (raw)
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Chloroplast transformation by Agrobacterium tumefaciens
The EMBO Journal, 1985
A chimeric gene consisting of the promoter region of the nopaline synthase gene (Pnos) fused to the coding sequence of the chloramphenicol acetyltransferase gene (cat gene) of Tn9 was introduced by co-cultivation in tobacco protoplasts followed by selection with 10 ,g/ml chloramphenicol. The chloramphenicol-resistant plants derived from these selected calli were unable to transmit the CmR phenotype through pollen. A typically maternal inheritance pattern was observed. Southern blot analysis showed that the chimeric Pnos-cat gene was present in the chloroplasts of these resistant plants. Furthermore, the chloramphenicol acetyltransferase activity was shown to be associated with the chloroplast fraction. These observations are the first proof that the Agrobacterium Tiplasmid vectors can be used to introduce genes in chloroplasts.
Assessment of Chloroplast Expression Factors in Escherichia coli
Jundishapur Journal of Microbiology, 2013
Background: Genetic manipulation of chloroplast in higher plants offers a number of unique prerogatives, including; undesirable of pleiotropic genome and gene silencing effects and also use as an important agronomic trait for producing essential biomaterials and industrial enzymes. In order to manipulate chloroplast genome, specific vectors are required. These vectors can be transformed and expressed in Escherichia coli due to the same evolutionary origin of bacteria and chloroplasts. Objectives: The aim of the present study was to construct chloroplast vector specified for spinach and assessing the chloroplast regulatory elements in a prokaryotic expression host, E. coli. Materials and Methods: Flanking sequences (INSL+INSR) were isolated by PCR from the spinach chloroplast genome and blunt-end ligated into the PvuII site of pUC19 vector to form an intermediate vector, pUCINS. Then the selectable marker cassette (including aadA gene, Prrn promoter and rbcL terminator) was isolated via PCR and blunt-end cloned into the unique PvuII site of pUCINS to make the final chloroplast vector, named pCSI. Results: The constructed vector transformed to E.coli strain DH5α and several procedures such as colony PCR, digestion and sequencing were assigned to confirm the consequence of the construct. Conclusions: The appearance of bacterial colonies on the plate containing different concentrations of streptomycin indicated the strength of resistance to streptomycin which showed the bacterial cells capability to express aadA gene under the controls of chloroplast regulatory elements.
The Plant Cell, 1990
Transcription from modified chloroplast genes has been studied in vitro, but only with the recently developed ability to stably introduce foreign DNA into Chlamydomonas reinhardtii chloroplast chromosomes in situ has it become possible to do so in vivo. Cloned chloroplast DNA sequences, into which had been inserted chimeric genes composed of the GUS coding sequence reporter under transcriptional control of chloroplast promoters for the C. reinhardtii atpA, atpB, and rbcL genes, were introduced into the cells on microprojectiles. These constructs become integrated into chloroplast chromosomes by homologous recombination. RNA gel blot analyses demonstrated that a single major 8-glucuronidase (GUS)-hybridizing transcript accumulates in each chloroplast transformant. We have found that: (1) Transcription of the chimeric gene begins at the same site as in the corresponding endogenous chloroplast gene; (2) the rates of transcription in vivo of the atpA:GUS and atpB:GUS genes relative to one another and to other genes are the same as those for the endogenous atpA and atpB genes, respectively, indicating that these promoters are fully functional despite being fused to a foreign gene and being at an alien location on the chloroplast chromosome; (3) in contrast to the atpA and atpB promoters, the rbcL promoter directs transcription of the rbcL:GUS gene at only 1% of the expected rate, suggesting that other features are required for optimal activity of this promoter; and (4) 22 base pairs upstream of the 5' end of the atpB:GUS transcript in the atpB promoter element is sufficient to confer wild-type levels of promoter activity.
Journal of bacteriology, 1982
The nucleotide sequence of pC194, a small plasmid from Staphylococcus aureus which is capable of replication in Bacillus subtilis, has been determined. The genetic determinant of chloramphenicol (CAM) resistance, which includes the chloramphenicol acetyl transferase (CAT) structural gene, the putative promoter and controlling element of this determinant, have been mapped functionally by subcloning a 1,035-nucleotide fragment which specifies the resistance phenotype using plasmid pBR322 as vector. Expression of CAM resistance is autogenously regulated since the 1,035-nucleotide fragment containing the CAT gene sequence and its promoter cloned into pBR322 expresses resistance inducibly in the Escherichia coli host. A presumed controlling element of CAT expression consists of a 37-nucleotide inverted complementary repeat sequence that is located between the -10 and ribosome-loading sequences of the CAT structural gene. Whereas the composite plasmid containing the minimal CAT determinan...
The EMBO journal, 1986
The genome of the cyanobacterium Anacystis nidulans R2 contains three genes (psbA) for the QB protein of photosystem II. This protein is essential for oxygenic photosynthetic electron tansport, and is the target for several herbicides which act by binding directly to the photosynthetic apparatus. Transcripts from the three Anacystis psbA genes are present in wild-type cells at different steady-state levels. The nucleotide sequences of two of the genes, psbAII and psbAIII, predict a protein having the same amino acid sequence which differs from that of the psbAI gene by 25 (out of 360) residues. Inactivation of each of the psbA genes in the Anacystis chromosome, singly or in pairs, shows that each of the genes is capable of producing sufficient functional QB protein to support normal photoautotrophic growth.
The Chloroplast-Located Homolog of Bacterial DNA Recombinase
Plant and Cell Physiology, 1997
The cONA for the chloroplast-located homolog of bacterial RecA protein, designated recA-AT, was placed in a plasmid appropriate for in vitro transcription and translation. Translation with ^S-labeled Met permitted demonstration of uptake of the protein product into isolated pea chloroplasts, and processing to a mature size. Preliminary evidence for the first amino acid was estimated from results using both 35 S-Met and 3 H-Leu for in vitro transcription and translation, followed by uptake into chloroplasts and processing. The labeled protein was subject to sequential amino acid hydrolyses, and radioactivity was measured in each round. Induction of gene transcription in leaves infiltrated with the DNA-damaging agent, methyl methanesulfonate was shown by Northern blot analysis. Further constructs were made for over-expression of the gene in E. coli; and one out of many tried permitted production of some soluble protein. Extracts from transformed bacteria were shown to have RecA activity using the "POM" assay [Bertrand et al. (1993) Nucl. Acids Res. 21: 3653] for DNA strand transfer. The protein was purified to close to homogeneity using methods developed for E. coli RecA isolation.
Maintenance and expression of heterologous genes in chloroplast of Chlamydomonas reinhardtii
Journal of Applied Phycology, 1994
The chloroplast genome ofChlamydomonas reinhardtii has been transformed with a chimeric gene consisting of the chloroplastatpA promoter and the bacterial gene for aminoglycoside adenine transferase (aadA). TheatpA-aadA cassette has been placed within the chloroplast DNAEcoRI restriction enzyme fragment 14, or within the chloroplastBamH1 fragment 10. The chimeric constructs were introduced into the chloroplast by particle bombardment. Integration of the cassette
Plant Molecular Biology, 2018
Key message Two intercistronic regions were identified as functional intercistronic expression elements (IEE) for the simultaneous expression of aphA-6 and gfp in a synthetic operon in the chloroplast of C. reinhardtii. Abstract Chlamydomonas reinhardtii, a biflagellate photosynthetic microalga, has been widely used in basic and applied science. Already three decades ago, Chlamydomonas had its chloroplast genome transformed and to this day constitutes the only alga routinely used in transplastomic technology. Despite the fact that over a 100 foreign genes have been expressed from the chloroplast genome, little has been done to address the challenge of expressing multiple genes in the form of operons, a development that is needed and crucial to push forward metabolic engineering and synthetic biology in this organism. Here, we studied five intercistronic regions and investigated if they can be used as intercistronic expression elements (IEE) in synthetic operons to drive the expression of foreign genes in the chloroplast of C. reinhardtii. The intercistronic regions were those from the psbB-psbT, psbN-psbH, psaC-petL, petL-trnN and tscA-chlN chloroplast operons, and the foreign genes were the aminoglycoside 3′-phosphotransferase (aphA-6), which confers resistance to kanamycin, and the green fluorescent protein gene (gfp). While all the intercistronic regions yielded lines that were resistant to kanamycin, only two (obtained with intercistronic regions from psbN-psbH and tscA-chlN) were identified as functional IEEs, yielding lines in which the second cistron (gfp) was translated and generated GFP. The IEEs we have identified could be useful for the stacking of genes for metabolic engineering or synthetic biology circuits in the chloroplast of C. reinhardtii.