Bifunctional Plasmid Research Articles

Regulation of human GDNF gene expression in nigral dopaminergic neurons using a new doxycycline-regulated NTS-polyplex nanoparticle system

The human glial-cell derived neurotrophic factor (hGDNF) gene transfer by neurotensin (NTS)-polyplex nanoparticles functionally restores the dopamine nigrostriatal system in experimental Parkinson's disease models. However, high levels of sustained expression of GDNF eventually can cause harmful effects. Herein, we report an improved NTS-polyplex nanoparticle system that enables regulation of hGDNF expression within dopaminergic neurons. We constructed NTS-polyplex nanoparticles containing a single bifunctional plasmid that codes for the reverse tetracycline-controlled transactivator advanced (rtTA-Adv) under the control of NBRE3x promoter, and for hGDNF under the control of tetracycline-response element (TRE). Another bifunctional plasmid contained the enhanced green fluorescent protein (GFP) gene. Transient transfection experiments in N1E-115-Nurr1 cells showed that doxycycline (100 ng/mL) activates hGDNF and GFP expression. Doxycycline (5 mg/kg, i.p.) administration in rats activated hGDNF expression only in transfected dopaminergic neurons, whereas doxycycline withdrawal silenced transgene expression. Our results offer a specific doxycycline-regulated system suitable for nanomedicine-based treatment of Parkinson's disease.

Construction of Plasmid Vector for Expression of Bacteriocin N15-Encoding Gene and Effect of Engineered Bacteria on Enterococcus faecalis

A 6.09-kb plasmids vector pOri253 was constructed from the plasmid pIL253 (5.2 kb) and a 0.89-kb fragment of oriColE1 from pBluescript II KS. The bifunctional plasmid pOri253 conferred erythromycin resistance in both Escherichia coli and Enterococcus faecalis. It has unique sites for EcoRI, BamHI, SalI, and PstI derived from pIL253 and was lost at a low rate in E. faecalis JCM8726 when cultured in Man, Rogosa, & Sharpe broth without antibiotic. The lactococcal promoter P23 was inserted at one end of the pOri253 multicloning site. Gene expression was assessed by an entAI gene, which produced bacteriocin N15. The E. faecalis harboring constructed plasmid carrying P23 (pOrient23) had more antibacterial activity than parental E. faecalis JCM8726 and its clone harboring non-P23-containing plasmid (pOrient), as determined by means of an overlay method.

Improved shuttle vector for expression of chitinase gene in Bacillus thuringiensis.

A 6.96-kbp plasmid vector pBCX was constructed from the plasmid pBC16 (4.4 kbp) and a 2.56-kbp fragment of pBluescript II KS. The bifunctional plasmid pBCX conferred ampicillin and tetracycline resistance in Escherichia coli but only tetracycline resistance in Bacillus thuringiensis. It has unique sites for BamHI, SmaI, PstI, HindIII, SalI, XhoI, DraII, ApaI and KpnI derived from pBluescript II KS and was lost at a low rate in B. thuringiensis subsp. israelensis when cultured in Luria-Bertani broth without antibiotic. The chitinase gene from B. circulans number 4.1 (pCHIB1) was subcloned into the HindIII sites of this vector and designated as pBX43 (9.56 kbp). This plasmid produced three times as much chitinase in B. thuringiensis subsp. israelensis strain c4Q272 as pHYB43, which comprises the commercial shuttle vector pHY300PLK plus the chitinase gene.

Cloning and Characterization of the Replicon of the Nocardia italica Plasmid, pNI100

A 19-kb plasmid, pNI100, was isolated from Nocardia italica CCRC12359; its replicon was cloned and characterized as having a single open reading frame (ORF) of 1188 bp specifying 396 amino acids (aa). Analyses of the deduced aa sequence of the Rep protein indicated that characteristics of three consensus sequences and a P-loop-like motif in the Rep protein of plasmid pSG5, a conjugative plasmid involving a rolling-circle replication mechanism, were conserved in those of plasmid pNI100. Phenotypically, a pock structure was produced in the regenerated mycelium by introducing pNI100 DNA into the Streptomyces lividans protoplast. This result strongly suggests that pNI100 is a conjugative plasmid and probably replicates by a rolling-circle replication mechanism. By using the replicon of pNI100, a bifunctional plasmid pNI105 that could replicate in both Escherichia coli and S. lividans was constructed and found to be a useful cloning shuttle vector.

Tandem integration of multiple ILV5 copies and elevated transcription in polyploid yeast.

An industrial yeast strain was modified by introducing DNA into brewing yeast such that the derived cells contain only yeast DNA. Thus selectable markers and bacterial sequences are not present in the final strain, making this procedure attractive for the development of generally acceptable brewing yeast. Linear DNA containing the cloned ILV5 gene was introduced into lager yeast along with an unlinked circular bifunctional plasmid containing a dominant resistance marker. Resistant colonies were screened for site-directed integration of the ILV5 DNA. Candidates were examined by several methods including Southern transfer and polymerase chain reaction. In this way, a strain WM56 was identified containing three tandem copies of ILV5. The amplified ILV5 region is stable during repeated subculturing in the absence of selective pressure. Correspondingly elevated levels of ILV5 transcript in strain WM56 compared to the control (i.e. non-tandem) parental strain led to increased amounts of encoded acetohydroxyacid reductoisomerase as evidenced by significantly lower diacetyl production. WM56 appears to be identical to the parental strain judged by CHEF, total restriction digestion patterns, and probing, but differs in the ILV5 region of the chromosome. The method is generally applicable to other yeast strains, and if desired, is amenable to iterated cycles of integration to increase the number of copies.

A Streptomyces PLASMID pHZ65 AND ITS DEVELOPMENT INTO CLONING VECTORS

FR--0001 and FR--005 are two fusants obtained through protoplast fusion between differ- ently marked derivatives of Streptomyces hygroscopicus 5102, which produce three agricul- turally important antibiotics. Although they show distinct differences in morphological and cultural characteristics, antimicrobial activity and metabolites, they all carry a 20.1 kb plasmid (pHZ65) with complete DNA homology. A detailed restriction map with 29 sites for restriction enzymes BamHI, BclI, EcoRV, KpnI, PstI, SphI, SstI and XhoI has been deter- mined. Four Strepiomyces-E. coli bifunctional plasmids, pHZ200, pHZ201, pHZ206 and pHZ207, have been constructed by the in vitro insertion of an E. coli plasmid pIJ2703 which carries the tsr gene (expressible in Streptomyces) and amp gene (expressible in E. coli) into the BclI or BglII sites of pHZ65. The replicability of a series of plasmids obtained by the cloning of DNA fragments from pHZ65 into the BclI site of pIJ2703 in S. lividans shows that the essential replication region of pHZ65 is not more than 4.96 kb in size within two clustered BglII fragments. Bifunctional plasmids derived from pHZ65 can be transformed into S. hygroscopicus var. yingchengensis (the fusion parent of FR-001 and FR--005), whereas many other plasmid vectors developed so far are unable to do so.

Development of Thermus-Escherichia shuttle vectors and their use for expression of the Clostridium thermocellum celA gene in Thermus thermophilus.

We describe the self-selection of replication origins of undescribed cryptic plasmids from Thermus aquaticus Y-VII-51B (ATCC 25105) and a Thermus sp. strain (ATCC 27737) by random insertion of a thermostable kanamycin adenyltransferase cartridge. Once selected, these autonomous replication origins were cloned into the Escherichia coli vector pUC9 or pUC19. The bifunctional plasmids were analyzed for their sizes, relationships, and properties as shuttle vectors for Thermus-Escherichia cloning. Seven different vectors with diverse kanamycin resistance levels, stabilities, transformation efficiencies, and copy numbers were obtained. As a general rule, those from T. aquaticus (pLU1 to pLU4) were more stable than those from the Thermus sp. (pMY1 to pMY3). To probe their usefulness, we used one of the plasmids (pMY1) to clone in E. coli a modified form of the cellulase gene (celA) from Clostridium thermocellum in which the native signal peptide was replaced in vitro by that from the S-layer gene of T. thermophilus HB8. The hybrid product was expressed and exported by E. coli. When the gene was transferred by transformation into T. thermophilus, the cellulase protein was also expressed and secreted at 70 degrees C.

Construction of two stable bifunctional plasmids for Streptomyces spp. and Escherichia coli

Two bifunctional plasmid vectors pZG5 (7.45 kb) and pZG6 (6.95 kb), for gene transfer between Streptomyces spp. and Escherichia coli have been constructed by fusion of the multicopy broad-host-range Streptomyces plasmid pIJ350 with E. coli plasmids Bluescribe M13- (pZG5) or pUC18 (pZG6). Both plasmids possess several unique restriction sites suitable for DNA cloning. Stable transformants of Streptomyces rimosus R6 and S. lividans 66 were obtained, harboring intact plasmids regardless of colony age or multiple subculturing. Moreover, pZG5 and pZG6 were successfully used to introduce several homologous transfer RNA genes into S. rimosus.

Construction of two stable bifunctional plasmids forStreptomycesspp. andEscherichia coli

Two bifunctional plasmid vectors pZG5 (7.45 kb) and pZG6 (6.95 kb), for gene transfer between Streptomyces spp. and Escherichia coli have been constructed by fusion of the multicopy broad-host-range Streptomyces plasmid pIJ350 with E. coli plasmids Bluescribe M13- (pZG5) or pUC18 (pZG6). Both plasmids possess several unique restriction sites suitable for DNA cloning. Stable transformants of Streptomyces rimosus R6 and S. lividans 66 were obtained, harboring intact plasmids regardless of colony age or multiple subculturing. Moreover, pZG5 and pZG6 were successfully used to introduce several homologous transfer RNA genes into S. rimosus.

Cloning and characterization of an extracellular temperature-labile serine protease gene fromAeromonas hydrophila

Aeromonas virulence is thought to depend on multigenic functions. The gene for an extracellular protease from Aeromonas hydrophila SO2/2 was cloned in Escherichia coli C600-1 by using pIJ860, bifunctional plasmid, as a vector. The gene encodes for a temperature-labile serine protease (P2) with a molecular mass of approx. 68 kDa which is highly inhibited by PMSF. The gene was expressed in Streptomyces lividans 1326 by transforming protoplasts with the original clone pPA2. We were also able to transfer and express the prt P2 gene in Pseudomonas putida by mating experiments. The protein P2 was secreted into the periplasms of both P. putida and E. coli C600-1 being identical in properties to one of the proteases secreted into the culture supernatant by A. hydrophila SO2/2.

Cloning and characterization of an extracellular temperature-labile serine protease gene from Aeromonas hydrophila

Aeromonas virulence is thought to depend on multigenic functions. The gene for an extracellular protease from Aeromonas hydrophila SO2/2 was cloned in Escherichia coli C600-1 by using pIJ860, bifunctional plasmid, as a vector. The gene encodes for a temperature-labile serine protease (P2) with a molecular mass of approx. 68 kDa which is highly inhibited by PMSF. The gene was expressed in Streptomyces lividans 1326 by transforming protoplasts with the original clone pPA2. We were also able to transfer and express the prt P2 gene in Pseudomonas putida by mating experiments. The protein P2 was secreted into the periplasms of both P. putida and E. coli C600-1 being identical in properties to one of the proteases secreted into the culture supernatant by A. hydrophila SO2/2.

Transfection of a rat cytochrome P450b cDNA into C3H10T1/2CL8 mouse embryo fibroblasts.

A cDNA clone of a rat cytochrome P450b gene was used to construct an expression vector driven by an SV40 promoter and containing a G418-resistance selectable marker. This bifunctional plasmid (pJRSL100) was transfected into the C3H 10T1/2CL8 mouse embryo fibroblast cell line. G418-resistant clones were selected and tested for enhanced sensitivity to the carcinogen 2-acetylaminofluorene (2-AAF), a compound that does not normally induce cytotoxicity or morphological transformation in these cells. One subclone, 19P450b-4, exhibited an increased cytotoxic response to 2-AAF compared to the parental C3H10T1/2CL8 cells. DNA analyses of this subclone showed increased number of copies of the cytochrome P450b and the appearance of unique restriction fragment bands relative to parental and control transfected cells. This subclone also exhibited increased levels of mRNA complementary to the P450b cDNA. Metabolism studies of 2-AAF in this subclone demonstrated an increase in the C-hydroxylated metabolites 1-, 3-, 5/9-, and 7-hydroxy-AAF compared with parental C3H 10T1/2CL8 cells. The results indicate that C3H 10T1/2CL8 cells can be transfected with gene/cDNAs to increase their metabolic competency and that such transfection may enhance the usefulness of the C3H 10T1/2CL8 cells in studies on chemically induced cytotoxicity and morphological transformation.

Structural instability of a bifunctional plasmid pZG1 and single-stranded DNA formation in Streptomyces

Structural instability of a hybrid plasmid pZG1, consisting of Escherichia coli pBR322 and Streptomyces pIJ350 plasmids, has been studied in Streptomyces. After transformation of S. lividans 1326 and S. rimosus R6 protoplasts with pZG1, transformants harbored the intact pZG1 and various deleted plasmid forms. The pattern of deleted plasmids varied with the transformant colony age and changed upon subcultivation. The presence of intact pZG1 in at least a part of the Streptomyces colonies indicated that the plasmid was capable of replicating in the transformants and that deletion events occurred after at least one round of replication. Less instability of pZG1 in S. rimosus R6 was observed when this strain was transformed with the DNA isolated from the same strain. pZG1 and its various derivatives were found in S. lividans 1326 and S. rimosus R6 as double- and single-stranded DNA molecules. Structural instability of pZG1 could therefore be due at least in part to the presence of single-stranded DNA.

The minimal replicon of the Streptomyces ghanaensis plasmid pSG5 identified by subcloning and Tn5 mutagenesis

The cryptic plasmid pSG5 of Streptomyces ghanaensis 5/1B (DSM 2932) was characterized to have a molecular size of 12.7 kb and approximate copy number of 20-50 per chromosome. A bifunctional derivative, designated pSW344E, consisting of pSG5 and an Escherichia coli vector plasmid was constructed. Following Tn5 mutagenesis in E. coli, the replication functions of the mutagenized pSW344E plasmids were analysed in S.lividans. A 2 kb DNA fragment of the pSG5 replicon was found to carry replication functions. Subcloning of pSG5 DNA into various replication probe vectors resulted in the identification of the pSG5 minimal replicon, identical to the above mentioned 2 kb DNA region. Several small bifunctional plasmids, able to replicate in E. coli as well as in Streptomyces, were generated during subcloning. Some of these plasmids were found to be useful shuttle vectors.

16] A new and simple rapid method for sequencing DNA

Publisher Summary This chapter describes a current and simple rapid method for sequencing long fragments of deoxyribonucleic acid (DNA) by combining the advantages of the two systems: (1) a systematic shortening of double-stranded DNA and (2) subcloning. In this method, a fragment of DNA carrying the origin of replication from a single-stranded phage, F1 or M13, is inserted into a plasmid to make a single-stranded:double-stranded bifunctional plasmid of the shortened fragments into sequencing vectors. The new family of bifunctional plasmid constructed is of pAA-PZ1, pAA-PZ2, pAA-PZ3, and pAA-PZ4. The chapter presents the methods in a simplified way for selecting the transposon-generated deletion plasmids of different sizes and for isolating the single-stranded plasmid DNA for DNA sequence analysis. The chapter discusses solutions, buffers, media, and sources of enzymes for the method. The chapter also discusses construction and properties of pAA-PZ plasmids, construction and property of E. coli strain AA102F', cloning of foreign DNA into pAA-PZ plasmids, and several related concepts and procedures.

Cloning and expression of the [formula omitted][formula omitted] D-xylose isomerase gene in [formula omitted][formula omitted

A DNA fragment containing the Escheria coli D-xylose isomerase gene and D-xylulokinase gene had been isolated from an E. coli genomic bank constructed by Clarke and Carbon. The D-xylose isomerase gene coding for the synthesis of an important industrial enzyme, xylose isomerase, was subcloned into a Bacillus - E. coli bifunctional plasmid. It was found that the intact E. coli gene was not expressed in B. subtilis , a host traditionally used to produce industrial enzymes. An attempt was then made to express the E. coli gene in B. subtilis by fusion of the E. coli xylose isomerase structural gene down-stream to the promoter of the penicillinase gene isolated from Bacillus licheniformis . Two such fused genes were constructed and they were found able to be expressed in both B. subtilis and E. coli .

New shuttle vectors for Escherichia coli and Bacillus subtilis I. Construction and characterization of plasmid pHY460 with twelve unique cloning sites

We have constructed chimeric plasmid vectors, pHY460 and pHY310, from the streptococcal tetracycline resistance (Tc R) plasmid pAMα1 (9.2kb) and the Escherichia coli vector pACYC177 (3.7kb). These bifunctional plasmids can replicate and express the Tc R gene in both E. coli and Bacillus subtilis. Plasmids pHY460 (7.0 kb) and pHY310 (4.8 kb) contain the Tc R gene of pAMα1 and the ampicillin resistance (Ap R) gene of pACYC177. Both plasmids showed high transformation efficiency in both host cells. pHY460 was maintained stably in B. subtilis and, thus, is a useful shuttle vector functioning in E. coli and B. subtilis. The PvuI, PstI, BglI and BanI sites in the Ap R gene and the HpaI, BalI and EcoRV sites in the Tc R gene can be used for selection of recombinant plasmids by insertional inactivation. In addition, plasmid pHY460 has unique sites for SacII, BstEII, XbaI, AvaI and BamHI.

Expression of human dihydrofolate reductase cDNA and its induction by chloramphenicol in Bacillus subtilis

A bifunctional plasmid (pMP358) able to replicate and to express cloned human dihydrofolate reductase cDNA (cDHFR) in both Escherichia coli and Bacillus subtilis was constructed. The expression of cDHFR in B. subtilis was the result of a deletion that placed the cDNA fragment under the control of the chloramphenicol acetyltransferase (CAT) gene promoter of Staphylococcus aureus plasmid pC194. By sequence analysis of plasmid pMP358, we observed a gene fusion occurring between the cDHFR and the 32nd codon of the CAT gene. We report that such a “hybrid” gene is able to direct the synthesis of a 25-kDal “hybrid” protein, which was found to be inducible by supplementing B. subtilis cells with sublethal doses of chloramphenicol.

Hygromycin B resistance as dominant selectable marker in yeast

Saccharomyces cerevisiae is normally sensitive to the drug hygromycin B; a hygromycin B concentration of 200 µg/ml in agar plates is sufficient to completely inhibit growth. We constructed yeast-E. coli bifunctional plasmids which confer hygromycin B resistance to Saccharomyces cerevisiae. Promoters and amino terminal coding regions of a heat shock gene, a heat shock cognate gene, and the phosphoglycerate kinase gene from yeast were fused to a bacterial hygromycin B resistance gene. In all three cases, yeast cells containing plasmids with the hybrid hygromycin B resistance gene were resistant to high levels of the drug. Yeast cells containing these plasmids can also be directly selected after transformation by using hygromycin B. The intact bacterial hygromycin B resistance gene and the kanamycin resistance gene from Tn903 were also tested in yeast for their ability to confer resistance to hygromycin B and G418. The intact bacterial genes were not effective in conferring drug resistance to yeast cells.

Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis. I. Construction and analysis of B. subtilis clone banks in Escherichia coli.

Cloning in Escherichia coli and Bacillus subtilis was carried out using the bifunctional plasmid pDH5060. B. subtilis chromosomal DNA and pDH5060 DNA were digested with either BamHI or SalI, then annealed, ligated, and transformed into E. coli SK2267. Transformants containing sequences ligated into the BamHI or SalI sites in the Tcr gene of pDH5060 were selected directly using a modification of the fusaric acid technique. The BamHI and SalI clone banks contain about 250 and 140 B. subtilis fragments, respectively, with an average insert size of 8-9 Kbp in the BamHI and 4-5 Kbp in the SalI bank. The inserts ranged in size from 0.3 Kbp to greater than 20 Kbp. The vector used here therefore accepts inserts which are significantly larger than previously reported for other B. subtilis cloning systems. All individual cloned B. subtilis sequences examined were stably propagated in E. coli SK2267. Eight of eighteen B. subtilis auxotrophic markers tested (aroG, gltA, glyB, ilvA, metC, purA, pyrD, and thrA) were transformed to prototrophy with BamHI or SalI clone bank DNA. All or part of the hybrid plasmid DNA recombined at the sites of homology in the chromosome of these Rec+ recipients. Loss of sequences from hybrid plasmids was not prevented in a r- m- recE4 recipient strain of B. subtilis. Although the recE4 background prevented recombination between homologous chromosomal DNA, a variety of cloned fragments were shown to be unstable and undergo deletions of both insert and plasmid sequences. In addition, B. subtilis sequences propagated in E. coli transformed B. subtilis recE4 recipients with a 500-1,000-fold reduced efficiency.