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1.
A family of plasmid cloning vectors has been constructed that make use of the leftward promoter () of phage λ to provide for efficient expression of cloned genes in Escherichia coli. The promoter activity of is fully repressed at low temperature by a thermolabile repressor product of the gene, and can be activated by heat induction. Examples are given (β-lactamse, tryptophan synthetase A) where, under optimal conditions, between 30 and 40% of the total protein synthesis is directed by the cloned gene under control. 相似文献
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A Sugino 《Biochemical and biophysical research communications》1979,91(4):1321-1329
Mitochondrial DNA from contains high “A+T”-rich region. Its DNA replication starts in the “A+T”-rich region and proceeds unidirectionally around the molecule. In order to determine precise location of the DNA replication origin and elucidate unique feature of its nucleotide sequence, the “A+T”-rich region of mitochondrial DNA from has been cloned in . The chimeric plasmid DNA containing the “A+T”-rich region stimulates DNA replication system from mitochondria about ten fold higher than the parental plasmid DNA, as does native mitochondrial DNA. 相似文献
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K Shimada T Nakamura S Maeda Y Takagi 《Biochemical and biophysical research communications》1977,75(3):659-666
Ampicillin-resistant colonies that did not utilize galactose appeared sporadically in cultures of galactose genedeleted K-12 cells containing colicin E1 factor carrying genes for galactose utilization and ampicillin resistance. Most of these colonies contained small plasmid DNAs. These plasmids existed as monomer DNAs within K-12 cells and formed a series of covalently closed circular DNA molecules ranging in size from 6.3 × 106 to 15.1 × 106 daltons. The use of these plasmid DNAs was discussed. 相似文献
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The replication defective transducing phage λp3 carries a portion of the operon in the 2 region of the lambda phage. This operon segment contains the promoter, the operator, and the β-galactosidase gene, but does not contain the repressor gene. The gene can be expressed from both the inserted promoter and the phage promoter. When strain 594 (?, +) or JC6256 (Δ) is infected by λp3 in the absence of additional cyclic AMP, β-galactosidase synthesis is shown to be expressed from the phage promoter. When 594 (λ+) or JC6256 (λ+) is infected by λp3 in the presence of additional cyclic AMP and IPTG, β-galactosidase synthesis is shown to be expressed from the inserted promoter.The ability to separate the phage promoter from the inserted promoter for β-galactosidase expression will simplify the interpretation whenever λp5 is used. 相似文献
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Y.‐T. Horng K.‐C. Chang C.‐C. Chien Y.‐H. Wei Y.‐M. Sun P.‐C. Soo 《Letters in applied microbiology》2010,50(2):158-167
Aim: To develop an approach to enhance polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose PBAD promoter in Escherichia coli. Method and Results: The polyhydroxyalkanoates (PHAs) synthesis operon, (phaCAB), from Ralstonia eutropha was overexpressed under the regulation of the arabinose PBAD promoter in Escherichia coli, and the vgb gene encoding bacterial haemoglobin from Vitreoscilla stercoraria (VHb) was further cloned at downstream of phaCAB to form an artificial operon. The cell dry weight (CDW), PHB content and PHB concentration were enhanced around 1·23‐, 1·57‐, and 1·93‐fold in the engineered cell harbouring phaCAB–vgb (SY‐2) upon 1% arabinose induction compared with noninduction (0% arabinose). Furthermore, by using a recombinant strain harbouring PBAD promoter‐vgb along with native promoter‐phaCAB construction, the effect of vgb expression level on PHB biosynthesis was positive correlation. Conclusions: The results exploit the possibility to improve the PHB production by fusing the genes phaCAB–vgb from different species under the arabinose regulation system in E. coli. It also demonstrates that increase in VHb level enhances the PHB production. Significance and Impact of the Study: We were successful in providing a new coexpressed system for PHB synthesis in E. coli. This coexpressed system could be regulated by arabinose inducer, and is more stable and cheaper than other induced systems (e.g. IPTG). Furthermore, it could be applied in many biotechnology or fermentation processes. 相似文献
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《Gene》1998,208(1):43-50
We have developed a method for the identification of promoters recognized by a particular sigma factor of RNA polymerase, based on a two-compatible plasmid system in Escherichia coli (Ec). Using the method, a DNA fragment containing the promoter, PREN40, recognized by sporulation-specific Streptomyces aureofaciens (Sa) sigma factor RpoZ, was cloned. High-resolution S1 nuclease mapping using RNA prepared from Ec, and Sa from various developmental stages has shown a high degree of similarity of PREN40 to consensus sequence of flagellar and chemotaxis promoters. The promoter was induced at the time of aerial mycelium formation, and was off in the Sa strain with the rpoZ-disrupted gene. A promoter-bearing DNA fragment was inserted into the promoter-probe plasmid pARC1 to give expression patterns consistent with the results of direct RNA analysis. The region downstream of the promoter was cloned in Sa. Sequence analysis revealed an open reading frame (ORF) of 283 amino acids (Mr 30 006), encoding a highly basic (pI 12.35) protein with high percentage of serine, threonine and alanine (41.8%). 相似文献
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During the process of transformation cells bind its own DNA but little or no foreign DNA. This specificity for recognition of DNA was studied by cloning Haemophilus DNA in . Haemophilus DNA fragments were cloned using plasmid pBR322 as a vector. The fragment cH7 cloned in pBR322 was found to be homologous to Haemophilus DNA and shown to bind irreversibly to competent Haemophilus cells. The fact that cH7 isolated from lacks Haemophilus modification leads to the conclusion that modification does not play a role in the uptake mechanism. Uptake specificity is a function of recognition sequences that reside in DNA itself. 相似文献
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Hybrids were constructed between K12 ? mutants defective in nitrate respiration and an F′ plasmid carrying nitrogen fixation genes from . Examination of these hybrids showed that expression of Kp+ genes does not require a functional nitrate respiratory system, but that nitrate reductase and nitrogenase do share some Mo-processing functions. For nitrate repression of nitrogenase activity, reduction of nitrate to nitrite is not necessary, but the Mo-X cofactor encoded by genes is essential. Nitrate probably inhibits nitrogen fixation by affecting the membrane relationship of the nitrate and fumarate reduction systems such that the membrane cannot be energized for nitrogenase activity. 相似文献
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Y Terawaki Y Kobayashi H Matsumoto Y Kamio 《Biochemical and biophysical research communications》1980,97(2):694-699
A deletion mutant was isolated from a kanamycin resistance R plasmid Rtsl. This mutant plasmid, pTW20, was found to enhance the lethal effect of UV irradiation on host, especially at 42°C. A cloning experiment with pTW20 DNA demonstrated that the gene, , being responsible for the UV sensitivity was located on the kanamycin resistance gene containing H1 fragment of pTW20. This fragment conferred a sensitivity to methyl methane sulfonate on its host along with the sensitivity to UV, suggesting that a reapir process of the host chromosome is impaired by the presence of . 相似文献
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Sonia M. De Toledo Arnaldo Zaha Nelson Durán 《Biochemical and biophysical research communications》1982,104(3):990-995
containing pAT 153 plasmid undergoes strand scission when exposed to the indole-3-acetic acid/peroxidase/D2 system. Neither the initial components of this reaction nor the final stable products are responsible for this effect. Indole-3-aldehyde in its triplet state and singlet oxygen have been recently identified in this system. That singlet oxygen is one of the species acting on the plasmid in cells was suggested by protective effect of histidine and guanosine which are singlet oxygen quenchers. Similar effect on plasmid with malonaldehyde/peroxidase/O2 system was observed, which is an excellent singlet oxygen generator. This is the first report of a biological system where it is possible to detect a DNA scission in the intact cell by a bioenergized process. This presumably is related to spontaneous mutagenesis. 相似文献
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Genetic and enzymatic characterization of a conditional lethal mutant of Escherichia coli K12 with a temperature-sensitive DNA ligase 总被引:28,自引:0,他引:28
ts7 an Escherichia coli 15 strain with a thermolabile DNA ligase, has previously been shown to be a temperature-sensitive conditional lethal mutant that is sensitive to methyl methane sulfonate and to ultraviolet irradiation; it also accumulates 10 S DNA fragments to an abnormal extent. When the ligase mutation is transferred to a wild-type E. coli K12 strain, the strain becomes temperature sensitive for growth and displays the same characteristics as ts7. These findings show that a functional DNA ligase is essential for normal DNA replication and repair in E. coli. 相似文献
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《Journal of Fermentation and Bioengineering》1990,69(1):63-66
Among the polyamines tested, spermine strongly inhibited the growth of Saccharomyces cerevisiae DKD-5D-H. Two kinds of DNA fragments that confer strong and weak resistances to spermine were cloned onto a vector plasmid, YEp13. The restriction map of the DNA fragment conferring strong resistance was the same as that of a DNA fragment responsible for ethionine resistance in the same yeast cells. (Shiomi et al., Appl. Microbiol. Biotechnol., 29, 302–304, 1988) The yeast cells with the DNA fragment conferring strong resistance to spermine were resistant to ethionine and accumulated intracellularly. 相似文献
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Jeffrey K. Griffith Judith M. Buckingham John L. Hanners Carl E. Hildebrand Ronald A. Walters 《Plasmid》1982,8(1):86-88
E. coli HB101 cells transformed to tetracycline resistance with the plasmids pMB9 or pBR322 display a 105–106-fold lower plating efficiency on agar containing 440 μm CdCl2 than nontransformed cells. When DNA is inserted into the BamH1 site of the plasmid tet gene, or when DNA spanning the BamH1 site is deleted, tetracycline resistance and cadmium hypersensitivity are both lost. In contrast, insertion of DNA into the ampicillin resistance gene does not affect cadmium hypersensitivity. 相似文献
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Hybrids were constructed between K12 ? mutants uncoupled in oxidative phosphorylation, and thus defective in ATP biosynthesis, and an F′ plasmid carrying nitrogen fixation genes from . Examination of these hybrids showed that expression of +Kp genes in K12 does not require coupling of oxidative phosphorylation but needs the contribution of an anaerobic electron transport system involving fumarate reduction. The Kp cluster of genes does not contain functions able to complement a defective Mg2+-ATPase aggregate but does contain a function(s) which appears to interact with the B? mutant over the formation of a redox system. 相似文献
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Diffusion of tetracycline across the outer membrane of Escherichia coli K-12: involvement of protein Ia. 总被引:4,自引:0,他引:4
The possibility that passage of tetracycline across the outer membrane of K-12 is controlled by one or more of the proteins Ia, Ib and II1 (Henning's nomenclature) was investigated. A mutant lacking protein Ia (obtained by selection for resistance to phage TuIa) was more resistant to tetracycline than wild-type strains or those lacking only proteins Ib or II1. The envelope protein composition of a tetracycline-resistant mutant () was altered in several respects, but the major change involved loss of protein Ia. These data support our previous suggestion [12] that tetracycline diffuses across the outer membrane through hydrophilic regions. Furthermore, they imply that only protein Ia plays a significant role in the passage of this antibiotic across the outer membrane. 相似文献
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