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1.
Narrow-host-range vectors, based on an indigenous replicon and containing a multiple cloning site, have been constructed in aPseudomonas host capable of growth on unusual substrates. The new cloning vectors yield sufficient amounts of DNA for preparative purposes and belong to an incompatibility group different from that of the incP and incQ broad-host-range vectors. One of these vectors, named pDB47F, was used to clone, directly inPseudomonas, DNA fragments fromAgrobacterium, Pseudomonas, andRhizobium. A clone containingAgrobacterium and KmR gene sequences was transformed with a higher efficiency than an RSF1010-derived vector (by as much as 1250-fold) in four out of fivePseudomonas strains tested. The considerable efficiency obtained with this system makes possible the direct cloning and phenotypic selection of foreign DNA inPseudomonas.  相似文献   

2.
A secretory high-level expression cloning vector designated as pSBC-20 was constructed by inserting a DNA fragment encoding the signal peptide of ompA protein into pBV 220 vector. Any foreign DNA fragment can be inserted into the polylinker cloning sites located after the secretion signal sequence. The cloned foreign gene is under the control of the P R -P L promoter while the expression of the gene is regulated by the cI-gene product. The products are secreted into the periplasmic space of bacteria or into the medium. A recombinant plasmid (pRSD-220) was constructed by inserting the 210 bp from RSD-2, a cDNA encoding a peptide fragment of human sperm protein, into the EcoRI site of pSBC-20. TheE. coli cells transformed with pRSD-220 were propagated at 30 °C, then incubated at 42 °C for several hrs. The cloned gene product was secreted into the culture medium at a high rate. The yield was about 60 mg of gene product per liter of cultured medium.  相似文献   

3.
The Escherichia coli sodA gene encoding the antioxidant enzyme Mn-containing superoxide dismutase (MnSOD), was cloned in the expression vector pMG36e. This vector has a multiple cloning site down-stream of a promoter and Shine-Dalgarno sequences derived from Lactococcus. The protein-coding region of sodA from E. coli was amplified by the polymerase chain reaction, using a thermocycler and Taq DNA polymerase before cloning into pMG36e. When introduced into E. coli, the recombinant plasmid expressed the predicted fusion protein, both in the presence and absence of oxygen. The expression of the fusion protein in E. coli was verified by SOD assays, activity gels and Western blots. The recombinant plasmid was also introduced into Lactococcus lactis, which contains a resident SOD, and into Lactobacillus gasseri, which is devoid of SOD. Transformed lactococci expressed an active SodA fusion protein plus an active hybrid protein composed of subunits of the Lactococcus and the recombinant E. coli enzymes. Transformants of L. gasseri expressed only the fusion SodA protein, which was enzymatically active.  相似文献   

4.
The isolation of a small plasmid from Pasteurella multocida has enabled to construction of a shuttle vector for use between P. multocida and Escherichia coli. The vector pBAC64 contains the origin of replication from P. multocida, an antibiotic resistance gene which functions in P. multocida, and the E. coli vector pUC18. The presence of the pUC18 multiple cloning site together with the lacZ′ gene provides a screening method and allows cloning and manipulation in E. coli as well as cloning in P. multocida.  相似文献   

5.
A T-DNA vector for plant transformation has been constructed in which the cloning site is located 9 bp from the right-border (RB) end and 27 bp from the left-border (LB) end. In this vector cloned DNA homologous to plant chromosomal sequences is located at the T-DNA termini, and will thus be exposed by even limited exonucleolysis in planta. The arabidopsis ADH (alcohol dehydrogenase) locus was mobilized from Agrobacterium, and integration into the recipient genome was studied. Despite the terminal location of ADH homology in this vector, the T-DNA integrated essentially at random in the Arabidopsis genome rather than at the endogenous ADH locus. T-DNA integration was blocked, however, when Arabidopsis telomeric sequences were added to the construct at each end of the ADH homology. Thus the predominant mode by which incoming T-DNA is integrated into the continuity of chromosomal DNA involves free DNA ends, but, in contrast to modes of recombination such as gap repair, does not involve extensive terminal DNA sequence homology.  相似文献   

6.
A modified low copy number plant binary vector pUN has been constructed and successfully used to clone unstable DNA sequences. The vector pUN comprises of low copy number, broad host range RK2 replicon from pBin19 and of multiple cloning site (MCS) and T-DNA region, both from a pBINPLUS-derived pLV06 vector. The absence of the ColE1 replicon in the backbone of the binary vector significantly contributed to stability of hardly clonable DNA sequences and enabled their transfer into the tobacco plants through Agrobacterium-mediated transformation.  相似文献   

7.
The alkaline protease genes (cDNAALP2 gene and ALP2 gene) were amplified from complementary DNA (cDNA) and genomic DNA of the marine yeast Aureobasidium pullulans HN2-3, respectively. An open reading frame of 1,248 bp encoding a 415-amino acid protein with a calculated molecular weight of 42.9 kDa was characterized. The ALP2 gene contained two introns, which had 54 and 52 bp, respectively. When the cDNAALP2 gene was cloned into the multiple cloning sites of the surface display vector pINA1317-YlCWP110 and expressed in cells of Yarrowia lipolytica, the cells displaying protease could form a clear zone on the double plate containing milk protein and had protease activity. The cells displaying alkaline protease were also found to be able to produce bioactive peptides from different sources of proteins. The peptides produced from single-cell protein of marine yeast strain G7a had the highest angiotensin-converting enzyme inhibitory activity, while the peptides produced from spirulina protein had the highest antioxidant activity. This is the first report that the yeast cells displaying alkaline protease were used to produce bioactive peptides.  相似文献   

8.
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   

9.
Chalcone synthase is the key enzyme in biosynthesis of flavonoids, which play roles in pigmentation of flowers and protection against ultraviolet and pathogens. Inverse polymerase chain reaction (IPCR) is a method for the rapid in vitro amplification of DNA sequences that flank a region of known sequence. In this study, IPCR united with nested PCR was successfully applied in cloning full-length sequences of three Phalaenopsis chalcone synthase genes (phchs3, phchs4, and phchs5, respectively). Firstly, routine PCR with homologous primers were performed, and gene fragments of phchs3 (1 kb), phchs4 (1.2 kb), and phchs5 (800 bp) were obtained and then sequenced. Then, inverse PCR were carried out for cloning full-length sequence of each gene. Because products were not unique in single round inverse PCR, nested PCR were performed, and the specificity was much enhanced. At last, full-length sequences of 2,499 bp for phchs3, 2,502 bp for phchs4, and 1,855 bp for phchs5 were obtained. This study proved that IPCR could be more efficient if being united with nested PCR.  相似文献   

10.
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