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Promotion, termination, and anti-termination in the rpsU-dnaG-rpoD macromolecular synthesis operon of E. coli K-12 总被引:18,自引:0,他引:18
Summary The regulatory regions for the rpsU-dnaG-rpoD macromolecular synthesis operon have been fused to a structural gene whose product is readily assayed (the Cmr structural gene coding for chloramphenicol acetyl transferase, CAT). The promoters (P1, P2, P3, Pa, Pb, Phs) for the macromolecular synthesis operon have different strengths as shown by their relative abilities to drive expression of the CAT gene. Promoter occlusion by P1 can be demonstrated within this operon. Regions 5kb upstream have a profound effect on operon gene expression. There is a thermoinducible promoter located within the dnaG structural gene. One of the macromolecular synthesis operon promoters is under lexA control. Although the operon structure allows coordinate expression of rpsU, dnaG and rpoD these additional features suggest that expression of individual genes can be independently regulated in response to altered growth conditions.Abbreviations Apr
ampicillin resistance
- CAT
chloramphenicol acetyl transferase
- Cmr
chloramphenicol resistance
- kb
kilobase pair
- orf
open reading frame
- P
promoter
- T
terminator
- Tcr
tetracycline resistance 相似文献
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Electroporation and PEG delivery of DNA into maize microspores 总被引:4,自引:0,他引:4
Summary The ability to deliver and detect reporter gene activity in maize microspores was tested. Tested expression vectors contained the chloramphenicol acetyl transferase (CAT) gene and one of the following promoter-intron combinations: 1) cauliflower mosaic virus (CaMV 35S), 2) CaMV 35S + maize alcohol dehydrogenase 1 intron 6 (Adh1-I6), 3) maize alcohol dehydrogenase 1 + intron 1 (Adh1-I1), or 4) maize ubiquitin 1 + intron 1 (Ubiq 1-I1) promoter + intron. The expression vectors were delivered into maize microspores using electroporation or polyethylene glycol (PEG). Both methods were effective for delivering free DNA into microspores. Although all four promoters were active in maize protoplasts, only two promoters were active in maize microspores. The CaMV 35S and the Adh1 promoters did not promote gene expression in maize microspore. The CaMV 35S + Adh1-I6 and Ubiq1-I1 promoters produced high levels of CAT activity in maize microspores. 相似文献
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F. Bekkaoui R. S. S. Datla M. Pilon T. E. Tautorus W. L. Crosby D. I. Dunstan 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1990,79(3):353-359
Summary Protoplasts from suspension cultures of somatic embryos of white spruce (Picea glauca Moench Voss) were electroporated with plasmids containing the chimeric genes for chloramphenicol acetyl transferase (CAT) or -glucuronidase (GUS), under control of one of three promoters. Transient CAT gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the cauliflower mosaic virus (CaMV) 35S promoter or the nopaline synthase (NOS) promoter. When the CAT gene was fused to a tandem repeat CaMV 35S promoter (pPBI-363), CAT enzyme activity compared to NOS or 35S promoters increased up to eightfold (cell line WS-34), and were up to 100-fold greater than control (electroporated without plasmid). Comparatively, protoplasts of black spruce (Picea mariana Mill) and jack pine (Pinus banksiana Lamb.), electroporated with pPBI-363, produced increases in CAT activity compared to control of 90-fold and 70-fold, respectively. White spruce (WS-34) protoplasts were subsequently electroporated with the GUS gene fused to the tandem repeat CaMV 35S promoter. Comparatively, GUS enzyme activity increased up to tenfold compared to GUS fused to a CaMV 35S promoter. The results indicated that transient expression of the CAT and GUS genes was influenced by the type of promoter and cell line used, as well as by electroporation conditions.NRCC No. 30498 相似文献
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We devised a molecular evolution procedure to evolve E. coli promoter sequences and applied it to observe an arbitrary, nonfunctional sequence evolving into functional promoters. In
the experiments, DNA sequence variations were generated with error-prone PCR and were inserted in the promoter region of the
cat (chloramphenicol acetyl transferase) gene on a plasmid. Upon transforming the cells, functional promoters on the plasmid
were selected according to the chloramphenicol resistance. Within a few cycles of mutation-selection, promoters emerged, and
the sequences converged into a small number of groups. In the process, the extended minus 10 type of promoters emerged quickly,
and small deletions were often involved in adjusting the length between the −35 and the −10 elements. Our results also suggest
a possible selection for promoter stability against mutation.
[Reviewing Editior: Dr. Laura Landweber] 相似文献
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Summary Flow cytometry was used to provide a rapid and accurate assessment of electroporation-induced uptake of macromolecules into plant protoplasts. Rice protoplasts were electroporated in the presence of fluorescein isothiocyanate-conjugated dextran (FITC-dextran). After washing, the protoplasts were resuspended in a solution containing propidium iodide which intercalates with DNA, but which is excluded by an intact plasma membrane. Electroporation in the presence of FITC-dextran gave rise to populations of protoplasts that fluoresced green or yellow due to the presence of non-conjugated FITC. Non-viable protoplasts fluoresced red because of their inability to exclude propidium iodide molecules. Flow cytometry was used to resolve and quantify these protoplast populations and thus identify optimal conditions for macromolecule uptake. A direct relationship was observed between FITC-dextran uptake and transient gene expression following plasmid uptake. Thus, simultaneous electroporation of protoplasts with foreign DNA and FITC-dextran followed by fluorescence activated cell sorting may permit partial selection of transformed cells and so reduce the need for a selectable marker.Abbreviations ADC
analogue to digital converter
- CAT
chloramphenicol acetyl transferase (enzyme)
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cat
chloramphenicol acetyl transferase (gene)
- CPW
solution cell and protoplast wash solution
- DC
direct current
- EF
electrofusion
- FALS
forward angle light scatter
- FITC
fluorescein isothiocyanate
- FITC-dextran
fluorescein isothiocyanate conjugated dextran
- PI
propidium iodide
- PMT
photomultipliertube
- TLC
thin layer chromatography 相似文献
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Electroporation was used to evaluate parameters affecting transient gene expression in Glycine max protoplasts. Protoplast viability and reporter enzyme activity for chloramphenicol acetyl transferase (CAT) and ß-glucuronidase (GUS) depended on the field strength employed. Maximum CAT and GUS activity was obtained when a field strength of 500 V/cm at 1000 F and a protoplast concentration of 1–3 × 106/ml was used. Transformation efficiencies up to approximately 1.6% GUS positive protoplasts were obtained. Transient gene expression increased with increasing plasmid DNA concentration and with the time after electroporation, reaching a maximum after 48 hr. Addition of polyethylene glycol at 5.6% and heat shock (5 rain at 45 °C) given to the protoplasts before adding DNA further enhanced the transformation efficiency. Under the optimized experimental conditions, CAT and GUS activity increased simultaneously, thereby indicating that the increased expression is caused by DNA uptake by more cells rather than greater DNA uptake by the same cells. Our results demonstrate that both GUS and CAT can be used as efficient screenable markers for transformation studies in soybean.Abbreviations CAT
chloramphenicol acetyl transferase
- GUS
ß-glucuronidase
- PEG
polyethylene glycol 相似文献
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Summary A protocol was developed for the preparation of Cucumis sativus var Straight 8 protoplasts that incorporates a two-step Ficoll® gradient and results in a high percentage of viable, debris-free protoplasts suitable for the transient expression of foreign genes. Polyethylene glycol and electroporation were compared for their effect on protoplast transfection with commonly used reporter genes. Using a polyethylene glycol method, cucumber protoplasts transfected with a plasmid containing the -glucuronidase gene showed high expression levels, while protoplasts transfected with a plasmid containing the chloramphenicol acetyl transferase gene showed levels of activity that were barely distinguishable from mock-transfected controls. Tomato ringspot virus genomic RNA was also transfected into the protoplasts, and the assembly of viral particles was confirmed. 相似文献
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P. Christou W. F. Swain 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1990,79(3):337-341
Summary Through the use of electroporation and a soybean (Glycine max L.) protoplast system, we generated stably transformed cell lines expressing a number of foreign genes (neomycin phosphotransferase,-glucuronidase, chloramphenicol acetyl transferase, and phosphinothricin acetyl transferase). Selected and unselected marker genes were cointroduced either linked on a single plasmid or as separate plasmids. Calli expressing multiple genes were recovered, and Cotransformation frequencies were established for both cases. Our results show a 50% cotransformation frequency in the case of linked genes. In situations in which two genes are introduced on independent plasmids, cotransformation frequencies are 18%–27%. Similar rates of cotransformation were observed among various marker pairs. 相似文献
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Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells 总被引:6,自引:0,他引:6
Robin Martin Anne E. Mogg Louise A. Heywood Lars Nitschke Julian F. Burke 《Molecular & general genetics : MGG》1989,217(2-3):411-418
Summary We have compared the suppression of nonsense mutations by aminoglycoside antibiotics inEscherichia coli and in human 293 cells. Six nonsense alleles of the chloramphenicol acetyl transferase (cat) gene, in the vector pRSVcat, were suppressed by growth in G418 and paromomycin. Readthrough at UAG, UAA and UGA codons was monitored with enzyme assays
for chloramphenicol acetyl transferase (CAT), in stably transformed bacteria and during transient expression from the same
plasmid in human 293 tissue culture cells. We have found significant differences in the degree of suppression amongst three
UAG codons and two UAA codons in different mRNA contexts. However, the pattern of these effects are not the same in the two
organisms. Our data suggest that context effects of nonsense suppression may operate under different rules inE. coli and human cells. 相似文献
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《Plant science》1987,50(1):65-68
Upon discovery that Brassica campestris leaf extracts harbour some chloramphenicol acetyl transferase (CAT) activity, a systematic screening of plant tissue for this activity, so far only reported for prokaryotic microorganism, has been conducted. Results were negative for three solanaceous plants as well as for the Cruciferae Arabidopsis thaliana and Orychophragmus violaceus. By contrast, the three tested species of the Cruciferae genus Brassica exhibit significant CAT activity. The Brassica CAT activity is much more heat labile than the enzyme encoded by the bacterial transposon, Tn9, that is commonly used as a reporter in gene fusion experiments. 相似文献
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P. Arokiaraj H. Jones K. F. Cheong S. Coomber B. V. Charlwood 《Plant cell reports》1994,13(8):425-431
A transformation system has been developed for Hevea brasiliensis using the particle gun method. Anther derived calluses were transformed with vectors harbouring the ß-glucuronidase (gus) gene, the neomycin phosphotransferase (nptII) gene, and the chloramphenicol acetyl transferase (cat) gene. Gene transfer was determined by histochemical staining and fluorometric assay for ß-glucuronidase activity, enzyme linked immunosorbent assay for detecting neomycin phosphotransferase II gene and direct enzyme assay for detection of expression of the cat gene. These independent assays all showed a several-fold increase, compared to control values, in gene product level and enzyme activity in extracts from transformed callus and embryoids of Hevea. These results were confirmed using polymerase chain reaction with primers designed to amplify an internal gus fragment. Together, the results show the feasibility of the particle gun method for the introduction of foreign genes into Hevea.Abbreviations BSA
bovine serine albumin
- CAT
chloramphenicol acetyl transferase
- ELISA
enzyme-linked immunosorbent assay
- GUS
ß-glucuronidase
- kb
kilobase
- MU
4-methyl-umbelliferyl-ß-D-glucuronide
- NPT-II
neomycin phosphotransferase II
- PCR
polymerase chain reaction
- Tris
Trizma base
- X-gluc
5-bromo-4-chloro-3-indolyl glucuronide 相似文献
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The effectiveness of different promoters for use in transgenic tobacco was compared using a reporter gene expressing chloramphenicol acetyl transferase (CAT). Plasmids with CAT gene controlled by cauliflower mosaic virus 35S (CaMV 35S), rice actin1 (Ract1) and tobacco polyubiquitin (Tubi.u4) promoters were delivered into tobacco plants by Agrobacterium-mediated transformation. The Ract1 promoter, previously shown to be a strong promoter in rice and other monocots, failed to promote strong expression in tobacco. CAT expression was greatest from the vector carrying Tubi.u4 with a 5'UTR and leader intron without a ubiquitin monomer. In transgenic plants harboring the Tubi.u4 promoter, CAT expression was approximately twice that of the CaMV 35S promoter. Our results suggest that foreign genes under the control of a ubiquitin promoter devoid of monomer will be useful for high-level gene expression in tobacco. 相似文献
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Expression of DNA coding for diphtheria toxin chain a is toxic to plant cells 总被引:6,自引:2,他引:4
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DNA coding for the enzymatically active subunit A of diphtheria toxin was placed under the control of the cauliflower mosaic virus 35S promoter and the Agrobacterium left transfer-DNA gene 7 polyadenylation signal. Agrobacteria carrying a binary plant vector with the chimeric diphtheria toxin A gene had very low transforming activity in tobacco (Nicotiana tabacum L.), and greatly diminished the recovery of stable transformants when mixed together with agrobacteria which alone transformed plant cells well. The introduction of this chimeric molecule into tobacco cells by electroporation lowered the level of the transient expression of the coelectroporated chloramphenicol acetyltransferase reporter gene indicating that expression of diphtheria toxin chain A in plant cells is toxic. We have developed a binary vector pGA987 which can be used for probing a variety of plant promoters. 相似文献
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Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors 总被引:12,自引:7,他引:5
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The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 105/μg of DNA to 107/μg of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination. 相似文献