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1.
To investigate the correlation between mutations in promoter, attenuator, and the AmpC enzyme overproduction in Escherichia coli. ampC Promoters from 4 Escherichia coli clinical isolates were cloned upstream to the chloramphenicol acetyltransferase (CAT) gene in pCAT3 reporter plasmid. Promoter
strengths were measured by chloramphenicol MIC and gene sequencing was done on the cloned ampC promoter and attenuator. The strength of promoters from AmpC hyperproducers were 8- to 64-fold higher than those from a low-level
AmpC producers. In one of the high-strength promoters, the mutations were located at positions −32, +22, +26, +32 (attenuator),
−76, and +79. In another promoter, the mutations were located at positions −88, −82, −18, −1, and +58. In the third promoter,
mutations were found at positions −1, +58, −80, −73, −28, and +82. Mutations in Escherichia coli promoter and attenuator sequences promoted Chloramphenicol MICs, which may be the primary causal mechanism for resistance
to β-lactams antibiotics. 相似文献
2.
Miksch G Bettenworth F Friehs K Flaschel E 《Applied microbiology and biotechnology》2005,69(3):312-320
We constructed a library of synthetic stationary-phase promoters for Escherichia coli. For designing the promoters, the known −10 consensus sequence, as well as the extended −10 region, and an A/T-rich region downstream of the −10 region were kept constant, whereas sequences from −37 to −14 were partially or completely randomised. For detection and selection of stationary-phase promoters, green fluorescent protein (GFP) with enhanced fluorescence was used. To establish the library, 33 promoters were selected, which differ in strength from 670 to more than 13,000 specific fluorescence units, indicating that the strength of promoters can be modulated by the sequence upstream of the −10 region. DNA sequencing revealed a preferential insertion of nucleotides depending on the position. By expressing the promoters in an rpoS-deficient strain, a special group of stationary-phase promoters was identified, which were expressed exclusively or preferentially by RNA polymerase holoenzyme Eσs. The DNA sequence of these promoters differed significantly in the region from −25 to −16. Furthermore, it was shown that the DNA curvature of the promoter region had no effect on promoter strength. The broad range of promoter activities make these promoters very suitable for fine-tuning of gene expression and for cost-effective large-scale applications in industrial bioprocesses. 相似文献
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5.
Lingxia Zhao Liya Lu Lida Zhang Aoxue Wang Ning Wang Zhuobin Liang Xiaowen Lu Kexuan Tang 《Journal of biosciences》2009,34(1):71-83
The E8 gene is related to ethylene biosynthesis in plants. To explore the effect of the expression pattern of the E8 gene on different E8 promoters, the molecular evolution of E8 promoters was investigated. A total of 16 E8 promoters were cloned from 16 accessions of seven tomato species, and were further analysed. The results from 19 E8 promoters including three previously cloned E8 promoters (X13437, DQ317599 and AF515784) showed that the size of the E8 promoters varied from 2101 bp (LA2150) to 2256 bp (LA2192); their sequences shared 69.9% homology and the average A/T content
was 74.9%. Slide-window analysis divided E8 promoters into three regions — A, B and C — and the sequence identity in these regions was 72.5%, 41.2% and 70.8%, respectively.
By searching the cis-elements of E8 promoters in the PLACE database, mutant nucleotides were found in some functional elements, and deletions or insertions were
also found in regions responsible for ethylene biosysnthesis (−1702 to −1274) and the negative effect region (−1253 to −936).
Our results indicate that the size of the functional region for ethylene biosynthesis in the E8 promoter could be shortened from 429 bp to 113 bp (−1612 to −1500). The results of molecular evolution analysis showed that
the 19 E8 promoters could be classified into four clade groups, which is basically consistent with evolution of the tomato genome.
Southern blot analysis results showed that the copy number of E8 promoters in tomato and some other wild species changed from 1 to 4. Taken together, our study provides important information
for further elucidating the E8 gene expression pattern in tomato, analysing functional elements in the E8 promoter and reconstructing the potent E8 promoter.
Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.
Supplementary material pertaining to this article is available on the Journal of Biosciences Website at 相似文献
6.
Shu-Ming Wu Chi Feng Jin Zhong Lian-Dong Huan 《World journal of microbiology & biotechnology》2011,27(1):99-106
Nattokinase (NK) is a health product for the prevention and potential control of thrombosis diseases. To explore the possibility
of enhancing NK production in Bacillus subtilis by altering the promoter of NK gene (PaprN), we tested several methods. We substituted the wild-type −10 box (TACAAT) of PaprN with the consensus sequence (TATAAT) of σA-dependent promoters, mutated the original −35 box (TACTAA) to a partial consensus sequence (TACACA), and expressed aprN from two tandem promoters, respectively. The efficacies of these changes were monitored by fibrinolytic activity, SDS-PAGE,
and northern blotting analyses. Fibrinolytic activity analysis showed that altering the −10 region of PaprN could increase NK production by 136%. This production is significantly higher than those reported in the literatures. Similar
results were obtained in SDS-PAGE and northern blotting analyses. This engineered promoter was also able to enhance the expression
of β-glucuronidase (GUS) by 249%. Partial alteration of the −35 element could slightly improve the production of NK by 13%,
while two tandem promoters just had marginal effects on the production of NK. Our study showed that alteration of −10 or −35
elements in PaprN, especially −10 element, is an effective way to enhance the production of heterologous proteins in B. subtilis. 相似文献
7.
A promoter vector pACPR33 for Escherichia coli based on the promotorless ampicillin-resistance gene from pBR322 has been constructed. The promoter of the ampicillin-resistance
gene was deleted and replaced by a suitable multiple cloning site. Molecular cloning of promoters into the polylinker resulted
in activation of the ampicillin resistance in E. coli. The plasmid contains a functional origin of DNA replication and a tetracycline resistance gene for E. coli, and a chloramphenicol resistance gene for S. aureus. The vector permitted direct detection of promoter activity, especially strong promoters, by easy iodometric determination
of β-lactamase activity in liquid or solid media.
Received: 26 July 1999 / Accepted: 22 November 1999 相似文献
8.
A. V. Seliverstov E. A. Lysenko V. A. Lyubetsky 《Russian Journal of Plant Physiology》2009,56(6):838-845
Plastome is thought to be a very conservative part of plant genome but little is known about the evolution of plastome promoters.
It was previously shown that one light-regulated promoter (LRPpsbD) is highly conserved in different flowering plant species and in black pine. We have undertaken search and demonstrated that
gene ndhF is located in a plastome region that rarely underwent substantial rearrangements in terrestrial plants. However, alignment
of sequences upstream ndhF suggests that promoters of this gene underwent comparatively rapid evolution in flowering plants. Probably, the ancestor
of two basal Magnoliophyta branches (magnoliids and eudicotyledons) had the promoter PA-ndhF, which was substituted with other promoters—PB-ndhF and PC-ndhF—in some phylogenetic lineages of dicots. We failed to reveal conservative sequences with potential promoters of −10/−35 type
upstream ndhF genes of monocotyledonous plants, including nine representatives of the grass family (Poaceae). Multiple alignments of sequences
from related taxa showed that the predicted ndhF promoters (A–C) underwent frequent mutations and these mutations are not only nucleotide substitutions but also small insertions
and deletions. Thus, we can assume that at least some plastome promoters evolve rapidly. 相似文献
9.
Development of marker-free strains of Bacillus subtilis capable of secreting high levels of industrial enzymes 总被引:1,自引:0,他引:1
B Widner M Thomas D Sternberg D Lammon R Behr A Sloma 《Journal of industrial microbiology & biotechnology》2000,25(4):204-212
Different strategies have been employed to achieve high-level expression of single-copy genes encoding secreted enzymes in
Bacillus subtilis. A model system was developed which utilizes the aprL gene from Bacillus clausii as a reporter gene for monitoring expression levels during stationary phase. An exceptionally strong promoter was constructed
by altering the nuceotide sequence in the −10 and −35 regions of the promoter for the amyQ gene of Bacillus amyloliquefaciens. In addition, two or three tandem copies of this promoter were shown to increase expression levels substantially in comparison
to the monomer promoter alone. Finally, the promoter and mRNA stabilization sequences derived from the cry3A gene of Bacillus thuringiensis were used in combination with the mutant amyQ promoter to achieve the highest levels of aprL expression. These promoters were shown to be fully functional in a high-expressing Bacillus strain grown under industrial fermentation conditions. The ability to obtain maximum expression levels from a single copy
gene now makes it feasible to construct environmentally friendly, marker-free industrial strains of B. subtilis. Journal of Industrial Microbiology & Biotechnology (2000) 25, 204–212.
Received 05 January 2000/ Accepted in revised form 26 June 2000 相似文献
10.
There is ample evidence that the enhancers of a promoterless yellow locus in one homologous chromosome can activate the yellow promoter in the other chromosome where the enhancers are inactive or deleted, which is indicative of a high specificity of
the enhancer–promoter interaction in yellow. In this paper, we have found that the yellow sequence from −100 to −69 is essential for stimulation of the heterologous eve (TATA-containing) and white (TATA-less) promoters by the yellow enhancers from a distance. However, the presence of this sequence is not required when the yellow enhancers are directly fused to the heterologous promoters or are activated by the yeast GAL4 activator. Unexpectedly, the
same promoter proximal region defines previously described promoter-specific, long-distance repression of the yellow promoter by the gypsy insulator on the mod(mdg4)
u1
background. These finding suggest that proteins bound to the −100 to −69 sequence are essential for communication between
the yellow promoter and upstream regulatory elements. 相似文献
11.
A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin. The individual plasmid vectors consist of the
minimal essential genetic elements, including an origin of replication for Escherichia coli, a H. pylori-specific replicon originally identified on a small cryptic H. pylori plasmid, an oriT sequence and a multiple cloning site. Shuttle plasmid pHel2 carries a chloramphenicol resistance cassette (cat
GC) and pHel3 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E. coli and H. pylori. The shuttle plasmids were introduced into the H. pylori strain P1 by natural transformation. A efficiency of 7.0 × 10−7 and 4.7 × 10−7 transformants per viable recipient was achieved with pHel2 and pHel3, respectively, and both vectors showed stable, autonomous
replication in H. pylori. An approximately 100-fold higher H. pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H. pylori strain, rather than E. coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation. Interestingly,
both shuttle vectors could also be mobilized efficiently from E. coli into different H.␣pylori recipients, with pHel2 showing an efficiency of 2.0 × 10−5 transconjugants per viable H. pylori P1 recipient. Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer. The functional complementation
of a recA-deficient H. pylori mutant by the cloned H. pylorirecA
+ gene, and the expression of the heterologous green fluorescent protein (GFP) in H.␣pylori demonstrate the general usefulness of␣this system, which will significantly facilitate the molecular analysis of H. pylori virulence factors in the future.
Received: 22 April 1997 / Accepted: 4 November 1997 相似文献
12.
Li Ang Chen LiangLiang Ren HaiYun Wang XueChen Zhang HaiWen Huang Rong-Feng 《中国科学:生命科学英文版》2008,51(3):280-285
In rice, the characterization of OsEBP-89 is inducible by various stress-or hormone-stimuli, including ethylene, abscisic acid (ABA), jasmonate acid (JA), drought
and cold. Here, we report the investigation of essential DNA region within OsEBP-89 promoter for methyl jasmonic acid (MeJA) induction. PLACE analysis indicates that this promoter sequence contains multiple
potential elements in response to various stimuli. First, we fused this promoter with GUS gene and analyzed its expression
under MeJA treatment through Agrobacterium infiltration mediating transient expression in tobacco leaves. Our results revealed that this chimeric gene could be inducible
by MeJA in tobacco leaves. To further determine the crucial sequences responsible for MeJA induction, we generated a series
of deletion promoters which were fused with GUS reporter gene respectively. The results of transient expression of GUS gene
driven by these mutant promoters show that the essential region for MeJA induction is positioned in the region between −1200
and −800 in OsEBP-89 promoter containing a G-box (−1127), which is distinct from the essential region containing ERE (−562) for ACC induction.
In all, our finding is helpful in understanding the molecular mechanism of OsEBP-89 expression under different stimuli. OsEBP-89, essential DNA region, methyl jasmonic acid, transient assay, promoter, tobacco leaves
Contributed equally to this work
Supported by the National Basic Research Program of China (Grant No. 2006CB101700) and the National Natural Science Foundation
of China (Grant Nos. 30671135, 30525034 and 30730060) 相似文献
13.
GTP cyclohydrolase I (GTPCH) is a key enzyme in the de novo synthesis of tetrahydrobiopterin. Previously, the Drosophila melanogaster GTPCH gene has been shown to be expressed from two different promoters (P1 and P2). In our study, the 5′-flanking DNA regions required
for P1 and P2 promoter activities were characterized using transient expression assay. The DNA regions between −98 and +31,
and between −73 and +35 are required for efficient P1 and P2 promoter activities, respectively. The regions between −98 and
−56 and between −73 and −41 may contain critical elements required for the expression of GTPCH in Drosophila. By aligning the nucleotide sequences in the P1 and P2 promoter regions of the Drosophila melanogaster and Drosophila virilis GTPCH genes, several conserved elements including palindromic sequences in the regions critical for P1 and P2 promoter activities
were identified. Western blot analysis of transgenic flies transformed using P1 or P2 promoter-lacZ fusion plasmids further
revealed that P1 promoter expression is restricted to the late pupae and adult developmental stages but that the P2 promoter
driven expression of GTPCH is constitutive throughout fly development. In addition, X-gal staining of the embryos and imaginal discs of transgenic flies
suggests that the P2 promoter is active from stage 13 of embryo and is generally active in most regions of the imaginal discs
at the larval stages. 相似文献
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15.
The expression and application of Bacillus thuringiensis (Bt) chitinase genes have been extensively investigated. However, little information is available regarding the regulation
of chitinase gene expression in Bt. In this study, a shuttle promoter-probe vector was constructed incorporating the thermostable
β-galactosidase gene bgaB of B. stearothermophilus as the reporter for the study of Bt promoters. Using this plasmid, the activity of the chiA gene promoter in Bt was investigated. Deletion analysis of the putative chiA promoter region revealed that the sequence located ~75 bp DNA from positions −116 to −42, with respect to the translation
start site, is the core promoter of chiA gene. Furthermore, a site for chitin induction was identified near position −36. This site for negative regulation was indicated
downstream of the RNA polymerase binding sites of the promoter of chiA. The expression of chiA started in cell grown for about 6 h and reached the maximum after 60 h of incubation. Induction of chiA expression by chitin was demonstrated by an increase in β-galactosidase activity of ~2.5-fold. 相似文献
16.
We isolated the 5′ flanking region of a gene for phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Pinus taeda, PtaPAL. To investigate the tissue-specific expression of the PtaPAL promoter, histochemical assay of GUS activity was performed using the transgenic tobacco expressing the PtaPAL promoter-GUS. The region of −897 to −420 in PtaPAL promoter showed high activities in the secondary xylem and response to bending stress. To characterize the cis-regulatory functions of the promoters for enzymes in phenylpropanoid biosynthesis, we examined the activity of chimeric promoters
of PtaPAL and a 4-coumarate CoA ligase, Pta4CLα. The chimeric promoter showed similar activity as the Pta4CLα promoter. Electrophoretic mobility shift assays implicated −897 to –674 of PtaPAL promoter containing cis-elements of the expression in xylem of Pinus taeda. The results suggested that AC elements of PtaPAL have multiple functions in the expression under the various developmental stages and stress conditions in the transgenic
tobacco.
The nucleotide sequence data reported will appear in the EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under the accession
number AB449103 (PtaPAL promoter sequence). 相似文献
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19.
M. Ficker H.-H. Kirch R. Eijlander E. Jacobsen R. D. Thompson 《Molecular & general genetics : MGG》1998,257(2):132-142
A functional analysis of the promoter of the S
2
-RNase gene from potato was performed in transgenic potato and tobacco plants, using a deletion series of S
2
-RNase promoter GUS fusions. A detailed histochemical and quantitative analysis of the transgenic tobacco plants revealed that S
2
promoter fragments ranging in size from 5.6 kb in length down to 0.2 kb mediate a weak developmentally regulated expression
in the pistil, and strong ectopic expression in pollen. In the pistil, different expression patterns were seen depending on
the transformant, the predominant one being characterised by expression in the stigma and the transmitting tract of the style,
whereas a few plants showed expression exclusively either in the stigma or in the stylar transmitting tissue. All transformants
also showed GUS expression in the placental epidermis of the ovary. Two sequences that are conserved between the potato S
1
-RNase and S
2
-RNase promoters, termed motif I and motif III, are located in a fragment of the S
2
promoter extending from position −200 to bp −100, and motif II, located between bp −498 and −480, was identified on the basis
of sequence comparisons between pistil-specific promoters. Motif II was found to be dispensible for pistil-specific and for
pollen-specific expression. Two submotifs, A and B, were identified within motif I. Both were essential for expression in
the pistil but only B was necessary for expression in pollen. Although motif III has a similar bipartite structure and sequence
to motif I, it was not sufficient to confer either pollen- or pistil-specific expression. However, deletion of motif III abolished
pollen-specific expression in transient expression experiments, suggesting that an interaction between the two sequence motifs
may be needed to specify cell type-specific expression. In transgenic potato the S
2
-RNase promoter also mediates expression in pollen and in the pistil; however, significantly fewer plants showed expression than
in tobacco, with most plants also exhibiting GUS expression in other tissues.
Received: 7 August 1997 / Accepted: 8 September 1997 相似文献
20.
Hisada H Sano M Ishida H Hata Y Abe Y Machida M 《Applied microbiology and biotechnology》2006,72(5):1048-1053
The manganese superoxide dismutase gene (sodM) is very highly expressed in Aspergillus oryzae. To elucidate the basis for this high-level expression, deletion analysis of the promoter was undertaken using β-glucuronidase (GUS) as a reporter. Deletion of a 63-bp sequence from −200 to −138 in the 1,038-bp sodM promoter caused a drastic decrease in GUS activity. In addition, an electrophoretic gel mobility shift assay (EMSA) implicated a 30-bp element from −209 to −178 containing cis-element(s) in the high-level expression. The results of fine structure deletion analysis of this region were consistent with the EMSA results. To confirm these findings, we constructed enhanced sodM promoters by incorporating tandem repeats of this region, which resulted in an approximate twofold increase in expression relative to the native sodM promoter. 相似文献