共查询到20条相似文献,搜索用时 46 毫秒
1.
H. Yamazaki Y. Ohnishi K. Takeuchi N. Mori N. Shiraishi Y. Sakata H. Suzuki S. Horinouchi 《Applied microbiology and biotechnology》1999,52(3):401-409
Rhizomucor pusillus 1116R3 has a defect in alg2 encoding a mannosyltransferase in the asparagine (N)-linked oligosaccharide biosynthetic pathway and produces proteins in less-glycosylated forms. For development of a genetic
transformation system for this zygomycete, an uracil auxotroph (mutant 1116U17) as the host strain was derived by ultraviolet
(UV) mutagenesis as 5-fluoroorotic acid-resistant colonies and the orotidine-5′-monophosphate (OMP) decarboxylase (pyr4) gene as a selection marker was cloned from the wild-type strain R. pusillus F27 by the polymerase chain reaction with primers designed on the basis of the pyr4 sequences from other fungi. The amino acid sequence of R. pusillus Pyr4 deduced from the nucleotide sequence showed high homology with the OMP decarboxylases from various fungi. The pyr4 gene on pUC19 (plasmid pRPPyr4) was introduced into protoplasts of R. pusillus 1116U17 by polyethylene glycol-assisted transformation. Transformation under optimized conditions yielded 5 Ura+ transformants with 1 μg pRPPyr4 DNA and 1 × 107 viable protoplasts. Southern blot analysis of the genomic DNA from the transformants showed that multiple copies of the pRPPyr4
sequence were integrated into the genome by homologous recombination at the pyr4 locus. For the purpose of production of a milk-clotting aspartic proteinase (MPP) in a less-glycosylated form, mpp from the wild-type strain was cloned in pRPPyr4 and introduced into protoplasts of R. pusillus 1116U17. Transformants obtained in this way contained multiple copies of mpp at the chromosomal mpp locus and produced MPP as a mixture of molecules having no sugar chains and Man0∼1GlcNAc2 at the two N-linked glycosylation sites in an amount about 12 times larger than the parent strain. The transformation system for R. pusillus 1116U17 would be useful for production of proteins with truncated N-linked oligosaccharide chains.
Received: 1 February 1999 / Received revision: 26 February 1999 / Accepted: 20 March 1999 相似文献
2.
R. W. Herzog H. Daniell N. K. Singh P. A. Lemke 《Applied microbiology and biotechnology》1996,45(3):333-337
An Aspergillus nidulans strain, auxotrophic for pyrimidine, was transformed to prototrophy by means of microprojectile bombardment. The transformation
frequency was somewhat lower than conventional polyethyleneglycol-mediated transformation of protoplasts. However, the percentage
of stable transformants was considerably higher with the biolistic approach. Typically, integrations of several copies of
the plasmid introduced into chromosomal DNA were observed. The effect of several parameters, like the concentration of conidia,
chamber pressure during bombardment and size of microprojectiles, on transformation frequencies were investigated and compared
to previously published data on microprojectile bombardment of fungal conidia. Optimum results (6 transformants/μg plasmid
DNA) were obtained when 108 conidia were bombarded with a helium pressure of 5.5–8.3 MPa (800–1200 lb/in2). M5, M10 and M17 tungsten particles were equally efficient.
Received: 9 August 1995/Received revision: 27 September 1995/Accepted: 4 October 1995 相似文献
3.
Ruirong Yi Takashi Tachikawa Hiroyuki Mukaiyama Yusuke Mochida Mariko Ishikawa Tadanori Aimi 《Mycoscience》2009,50(2):123-129
We cloned a gene encoding the succinate dehydrogenase iron-sulfur protein subunit (sip) from a bipolar mushroom, Pholiota microspora, and introduced a point mutation that confers carboxin resistance into this gene. Using this homologous selective marker
and also a heterologous drug selective marker, the hygromycin B phosphotransferase gene (hph), we successfully constructed a DNA-mediated transformation system in P. microspora. Both these selection markers have high transformation efficiency: the efficiency of carboxin resistance transformation was
about 88.8 transformants/μg pMBsip2 DNA using 5 × 106 protoplasts in regeneration plates containing 1.0 μg/ml carboxin, and the efficiency of hygromycin B resistance transformation
was about 122.4 transformants/μg pMBhph1 DNA using 5 × 106 protoplasts in regeneration plates containing 150 μg/ml hygromycin B. Southern hybridization analysis showed that the introduced
sequence (mutant sip or hph) was integrated into the chromosomal DNA in these transformants with a copy number of one or more. 相似文献
4.
Ortiz-Alvarado R Gonzalez-Hernandez GA Torres-Guzman JC Gutierrez-Corona JF 《Current microbiology》2006,52(3):178-181
Mucor circinelloides transformants prototrophic to leucine and resistant to carboxine (Leu+ Cbxr) have been obtained by treatment of protoplasts with plasmid constructs containing homologous leuA gene and adjacent autonomously replicating sequences (ARS) element combined with the Cbxr(carboxine-resistance) gene of Ustilago maydis and ARS sequences from this basidiomycete (plasmid pGG37) or from the 2 μ plasmid of Saccharomyces cerevisiae (plasmid pGG43). The presence in the same plasmid molecule of the M. circinelloides leuA gene and adjacent ARS element together with heterologous ARS elements produced an increase in the transformation frequency
of about 65–120%. The presence of autoreplicating plasmid molecules in the transformants was demonstrated by mitotic stability
experiments, by Southern analysis, and by the rescue of plasmids from transformed bacterial cells. 相似文献
5.
Homologous recombination and allele replacement in transformants of Fusarium fujikuroi 总被引:3,自引:0,他引:3
The ascomycete Fusarium fujikuroi could be transformed stably to hygromycin resistance only when the transforming plasmid contained a fragment of DNA from
the fungus. The transformation frequencies were roughly independent of the sequence of the particular fungal DNA fragment
used, of its size (1.8 or 6 kb), and of whether this DNA was present only once in the fungal genome or about forty times (the
genes for ribosomal RNA). The plasmid was integrated into the fungal genome by homologous recombination in the eighteen transformants
tested; ectopic integration was never observed. The carB gene of F. fujikuroi was cloned and shown to complement unpigmented mutants deficient in phytoene dehydrogenase. A mutant carB allele was prepared in vitro and used to transform wild-type protoplasts; the transformants contained a genomic duplication
and were heterozygous for carB; the mutant allele replaced the original wild-type allele when this was spontaneously lost in the transformants. This loss
was due to gene conversion in some cases and to recombination between repeated sequences in others.
Received: 5 November 1999 / Accepted: 16 March 2000 相似文献
6.
Draper J.; Davey M. R.; Freeman J. P.; Cocking E. C.; Cox B. J. 《Plant & cell physiology》1982,23(3):451-458
Suspension cell protoplasts of albino Petunia hybrida have beentransformed by isolated Agrobacterium tumefaciens Ti plasmid.Uptake of octopine Ti plasmid (pTiACH5) into protoplasts wasstimulated by poly-L-ornithine and polyethylene glycol (PEG).The frequency and efficiency of transformation of protoplaststo phytohormone autotrophy was compared using the two uptakeagents with various concentrations of plasmid. Transformationwas most efficient with PEG-mediated uptake, 5 µg of Tiplasmid per 106 protoplasts giving a frequency of 6?105.Octopine was not synthesised in any of the transformants afterthe second subculture on hormone-free medium. DNA-DNA hybridisationshowed the presence of DNA homologous to the T-DNA region ofpTiACH5 in all clones analysed. (Received November 9, 1981; Accepted January 29, 1982) 相似文献
7.
Won Noh Sang-Woo Kim Bae Dong-Won Jae-Yean Kim Hyeon-Su Ro 《Journal of microbiology (Seoul, Korea)》2010,48(2):253-256
Pleurotus eryngii was transformed via restriction enzyme-mediated integration. In order to construct the transformation plasmid, the enhanced
cyan fluorescent protein (ECFP) gene was ligated next to the gpd promoter of the plasmid pAN7-1. Transformation was facilitated via the heat treatment of a transformation mixture containing
1 μg of the HindIII-digested plasmid DNA and 106 mushroom protoplasts in 40% polyethyleneglycol solution, resulting in 10–40 hygromycin-resistant transformants. Successful
transformation was evidenced by PCR, Southern blot, and confocal fluorescence microscopic analyses on the selected transformants.
To date, this is the first report on the transformation of P. eryngii by REMI technique. 相似文献
8.
Douglas J. MacNeil Linda M. Klapko 《Journal of industrial microbiology & biotechnology》1987,2(4):209-218
Summary Polyethylene glycol (PEG) efficiently mediated the transformation ofStreptomyces avermitilis protoplasts by plasmid DNA to yield 107 transformants per g of plasmid DNA. Under conditios in which the maximum transformation frequency was observed, the cotransformation frequency exceeded 10%. The number of transformants increased linearly with the amount of DNA and number ofS. avermitilis protoplasts. Relaxed and supercoiled, but not linear DNA transformed protoplasts efficiently. Dimethyl sulfoxide (DMSO)-mediated transformation of protoplasts was 1000-fold less efficient. PEG and, less efficiently, DMSO also mediated the transformation of whole cells ofS. avermitilis by DNA. 相似文献
9.
Hideo Kusaoke Yoshitaka Hayashi Yasuhiro Kadowaki Hisashi Kimoto 《Bioscience, biotechnology, and biochemistry》2013,77(9):2441-2446
It was found that plasmid DNA (pUB 110) can be introduced into not only protoplasts but also intact cells of Bacillus subtilis by electric field pulses. The transformation of, B. subtilis using protoplasts results in an efficiency of 2.5 × 104 transformants per μg of DNA, with a single pulse of 50 jisec with an initial electric field strength of 7kV/cm. Even transformation of intact B. subtilis cells results in a maximum efficiency of 1.5 × 103 transformants per μg DNA, with a single pulse of 400 μsec with an initial electric field strength of 16kV/cm. The cell survival of protoplasts and intact cells was approximately 100% and 30%, respectively, under the conditions found to be optimal for the transformation process. Plasmid DNA isolated from pUB 110 containing transformants was indistinguishable from authentic preparations of pBU 110 on gel electrophoretic analysis. 相似文献
10.
Effect of Proteolytic Enzymes on Transfection and Transformation of Streptococcus lactis Protoplasts 总被引:3,自引:2,他引:1
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With both chymotrypsin and mutanolysin used to form protoplasts, consistent transformation frequencies of 104 to 105 transformants and transfectants per μg of DNA were achieved. The procedure was used to transform protoplasts of Streptococcus cremoris CS224 at low frequency (5 transformants per μg of DNA). 相似文献
11.
We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were
used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed
the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration
of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied
depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes
investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation
with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to
the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation
efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation,
electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion
at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic
sites in a high proportion of transformants.
Received: 6 March 1998 / Accepted: 25 May 1998 相似文献
12.
Clostridium thermocellum cell extracts exhibit specific endonuclease activity with very little non-specific exonuclease activity at 55°C. The Dam
methylation system of Escherichia coli offers complete protection from digestion by C. thermocellum ATCC 27405 cell extracts for all DNA tested (totaling >100 kb, insuring that most potential restriction sequences have been
exposed). Based on both the Dam recognition sequence and the similarity of cell extract and MboI DNA digests, the C. thermocellum restriction enzyme recognition sequence appears to be 5′ GATC 3′. Cell extracts made from a second thermophile, C. thermosaccharolyticum ATCC 31960 do not exhibit specific endonuclease activity under the conditions tested. Genomic DNA from C. thermocellum exhibits a Dam+ phenotype while genomic DNA from C. thermosaccharolyticum exhibits a Dam- phenotype.
Received: 10 March 1995/Received revision: 4 September 1995/Accepted: 13 September 1995 相似文献
13.
Summary Transformation of a Mucor circinelloides Leu– strain with the plasmid pAD45, harbouring the wild-type allele (leuA+) and a chymosin gene, led to the identification of mitotically stable transformants after one to three vegetative growth cycles on non-selective medium. Southern analysis of the stable transformed strains demonstrated that the vector is integrated, as an intact molecule, into the resident Mucor leuA locus. Retransformation of Escherichia coli with genomic DNA restricted with enzymes having no or only a single recognition site within the inserted sequence did not permit isolation of plasmids or fragments carrying the leuA or chymosin gene. 相似文献
14.
S. Nakotte S. Schaffer M. Böhringer P. Dürre 《Applied microbiology and biotechnology》1998,50(5):564-567
Procedures have been developed allowing recombinant DNA work with Clostridium acetobutylicum DSM 792. Electroporation was used to introduce plasmid DNA into exponentially growing clostridial cells and 6 × 102 transformants/μg DNA could be obtained at a time constant of 5.5 ms, 1.8 kV, 50 μF, and 600 Ω. The method also allowed the
taxonomic group IV strain NI-4082 to be transformed (101 transformants/μg DNA). Plasmid preparation from recombinant clostridia was optimal when a modification of the alkaline lysis
method was employed. It was also important to use cells from the mid-logarithmic growth phase. Recombinant strains could be
easily preserved as spore suspensions; under all conditions tested plasmids were maintained.
Received: 17 March 1998 / Received revision: 17 August 1998 / Accepted: 26 August 1998 相似文献
15.
Competent cells of Bacillus subtilis were transformed with DNA from gently lysed protoplasts. Significant linkages among markers separated by distances of approximately 2.3% of the total chromosome were found, which have not been detected for conventional transformation. In comparison to previous reports, enhanced plasmid transformation was observed [4.0×107 transformants per g DNA (one transformant per 5×104 molecules added)], when competent cells were transformed with DNA from lysed protoplasts harboring pUB110. 相似文献
16.
An efficient and relatively simple procedure forMicromonospora melanosporea protoplast preparation and transformation is described. Transformation ofM. melanosporea protoplast by theStreptomyces plasmid pIJ702 was optimized by altering parameters affecting the formation, regeneration, and transformation of protoplasts. Improvement of regeneration medium resulted in relatively quick growth of transformants (only 7 days). As a result of these experiments we describe a new transformation method that has routinely yielded 106 transformants/µg plasmid DNA. 相似文献
17.
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
18.
F. W. Paradis F. Shareck C. Dupont D. Kluepfel R. Morosoli 《Applied microbiology and biotechnology》1996,45(5):646-651
Streptomyces lividans IAF18, obtained by homologous cloning, is capable of over-producing XlnA. To investigate the possibility of the expression
of foreign genes, various coding regions of the xylanase A gene (xlnA) were analysed. Expression/secretion vectors were constructed containing the regulatory elements of xlnA with the coding region of the leader peptide with or without the truncated structural gene encoding the first 310 amino acids
of the XlnA. The genes coding for the Escherichia coliβ-glucuronidase and subunit 1 of the Bordetella pertussis toxin (S1) were used and their expression analysed. S. lividans transformants where the β-glucuronidase gene was fused with the leader sequence produced up to 30 mg β-glucuronidase/culture
filtrate whereas only fused XlnA/S1 was detected and its yield was estimated to be 1 mg/l. The disappearence of the B. pertussis toxin S1 and β-glucuronidase from the culture medium was due to the concomitant appearence of secreted proteases from S. lividans.
Received: 19 July 1995/Received revision: 3 November 1995/Accepted: 20 November 1995 相似文献
19.
An efficient one-step transformation method for the dimorphic yeast Yarrowia lipolytica is described. Using cells grown overnight on agar plates, the whole process is carried out within 1 h. The transformant clones
could be recovered on selective plates as early as 36–48 h after plating. The efficiency was better than 105 transformants/μg replicative plasmid DNA. Effects of cell density, dithiothreitol, heat shock, poly(ethylene glycol) 4000
concentration and the wetness of selective plates were investigated.
Received: 17 February 1997 / Received revision: 4 April 1997 / Accepted: 19 April 1997 相似文献
20.
Transformation of Mucor circinelloides is routinely achieved by using a plasmid containing the wild-type leuA gene to complement the leucine requirement of an auxotrophic host strain. As is the case for other zygomycetes, the transforming DNA is usually not integrated into the genome of M. circinelloides, but is maintained as an autonomously replicating plasmid. However, even under selective conditions, the plasmid is segregationally unstable, resulting in a rather low number of cells carrying the plasmid. We report here on a new transformation vector based on a dominant selection marker conferring resistance to geneticin, which allows for plasmid maintenance in high copy numbers. The vector was also used to transform Mucor rouxii and Rhizomucor pusillus, and should therefore be a valuable tool for gene expression studies in zygomycetes. The functionality and regulatory properties of the promoter of the M. circinelloides gpd1 gene (which codes for glyceraldehyde-3P-dehydrogenase) were demonstrated in R. pusillus using geneticin selection. In this work, we have also determined the molecular basis of the Leu- phenotype of the M. circinelloides host strain R7B. The leucine requirement is due to a single point mutation in the leuA gene that results in the replacement of a glutamic acid by a lysine residue.Communicated by E. Cerdà-OlmedoOn 1 January, 2004, the Biotechnological Institute became Bioneer A/S () 相似文献