One-step gene disruption by cotransformation to isolate double auxotrophs in Candida albicans

Summary The Candida albicans LEU2 gene was disrupted by substituting lambda DNA for a small deletion within the LEU2 gene. Cotransformation with a selectable URA3 ARS vector was used to introduce a linear fragment containing the disruption into the genome of a C. albicans ura3 deletion mutant. Cotransformants containing the lambda DNA were identified by colony hybridization and the URA3 plasmid was subsequently cured. Leu2 disrupted heterozygotes were detected by Southern hybridization and one disruptant was subsequently treated with UV irradiation. Only one leu2 ura3 mutant (SGY-484) was isolated out of 11,000 mutagenized cells. SGY-484 was transformed to Leu⁺ with either the C. albicans or Saccharomyces cerevisiae LEU2 gene. Southern hybridization analysis revealed that the mutant is not homozygous for the disruption; the leu2 mutation reverts and is most likely a point mutation. Unexpectedly, an ade2 ura3 mutant was isolated from the same mutagenesis.     

Gene-directed mutagenesis on the chromosome of Bacillus subtilis 168

Summary We have devised a method whereby any mutagenized cloned DNA from Bacillus subtilis can be reinserted at the original site on the B. subtilis chromosome. The procedure depends on the accuracy and high frequency of homologous recombination between the B. subtilis chromosome and the DNA taken up by the cell. The method makes use of two drug resistance selection markers (the chloramphenicol resistance gene and the neomycin resistance gene) and a marker gene which functions as a catalyst. The utility of the method has been demonstrated using leuB and pro of B. subtilis as target gene and catalyst, respectively, and mutations such as leuB: : cat, leuB ^–, and pro: : neo constructed in vitro on the cloned DNA fragments. Transformation in sequential steps as (leuB ⁺ pro⁺)(leuB: : cat pro ⁺) (leuB ^– pro: : neo)(leuB ^– pro ⁺) resulted in a leuB ^– single mutant without affecting other regions of the B. subtilis chromosome (gene-directed mutagenesis). We also demonstrate that other single mutations such as metD ^– and pro ^–, as well as the double mutation leuB ^– pro ^– can be introduced by the same procedure. In principle, true isogenies with multiple mutations can be constructed by the method described in this paper. Furthermore, the procedure should be generally applicable to any organisms in which homologous recombination is proficient.     

Cotransformation and differential expression of introduced genes into potato (Solanum tuberosum L.) cv Bintje

Summary The Dutch potato cultivar Bintje has been transformed by Agrobacterium strain LBA1060KG, which contains two plasmids carrying three different DNAs (TL- and TR-DNA on the Agrobacterium rhizogenes plasmid and TKG-DNA on the pBI121 plasmid). Several transformed root clones were obtained after transformation of leaf, stem, and tuber segments, and plants were then regenerated from these root clones. The expression of the various marker genes [rol, opine, -glucuronidase (GUS), and neomycin phosphotransferase (NPTII)] was determined in several root clones and in regenerated plants. The selection of vigorously growing root clones was as efficient as selection for kanamycin resistance. In spite of the location of NPTII and GUS genes on the same T-DNA, 17% of the root clones did not show GUS activity. Nevertheless, Southern blot analysis showed that these root clones contained at least three copies of the GUS gene. Sixty-four per cent of the root clones contained opines. The expression of these genes, however, was negatively correlated with plant regeneration capacity and normal plant development. The differential expression of the marker genes in the transgenic potato tissues is discussed.     

Transformation of Aspergillus oryzae using the A. niger pyrG gene

Summary A transformation system for Aspergillus oryzae based on the orotidine-5-phosphate decarboxylase gene (pyrG) was developed. Transformation frequencies of up to 16 transformants per g of DNA were obtained with the vector pAB4-1, which carries the pyrG gene of A. niger. Southern blotting analysis showed that vector DNA sequences were integrated into the chromosomal DNA, in various copy numbers and presumably at different sites. Efficient cotransformation of an unselectable gene was also shown. Under the conditions used no transformants were obtained with the equivalent pyr4 gene of Neurospora crassa.     

利用不相容质粒共转化大肠杆菌对Cre重组酶体内重组活性的可视检测

源自噬菌体P1的Cre重组酶可以识别 34bp的靶DNA序列loxP ,进行位点特异性的重组反应。为了简便地检测Cre酶在大肠杆菌中的重组活性 ,分别将cre基因和上下游带有loxP的绿色荧光蛋白基因 (gfp)克隆到具有不同抗性的两种不相容质粒中 ,然后将构建的原核表达载体pET30a Cre和pET2 3b loxGFP电击共转化大肠杆菌BL2 1(DE3) ,利用卡那霉素和氨苄青霉素双抗生素抗性进行筛选。通过直接观察转化子的绿色荧光 ,便可以显示Cre酶的体内重组活性 ,并进一步通过SDS PAGE分析、质粒酶切鉴定进行了验证。结果表明 :以gfp为报告基因、通过两种不相容质粒共转化大肠杆菌可以为研究和改进Cre loxP重组系统提供一种简便直观的检测方法     

adw与adr亚型乙型肝炎表面抗原基因在哺乳动物细胞中的表达

以pSV2-dhfr DNA为载体,在其单一酶切点Bg1Ⅱ处分别插入adw或adr亚型的HBVDNA Bg1 ⅡA片段(包括完整的pre-s及s基因),构成PSDHB_(r-1)和PSDHBw重组质粒。将其分别与pX1 DNA用磷酸钙沉淀法共转化LTK-细胞,在HAT培基选择压力下,挑选LTK~ 细胞,这些细胞均能有效表达HBsAg。再改用MTX选择培基,并逐渐增加其药量,从MTX抗性细胞中获得稳定表达HBsAg的Mwc-1和Mrm细胞系。     

Development of a homologous transformation system for Aspergillus niger based on the pyrG gene

Summary The development of a homologous transformation system for Aspergillus niger is described. The system is based on the use of an orotidine-5-phosphate decarboxylase deficient mutant (pyrG) and a vector, pAB4-1, which contains the functional A. niger pyrG gene as a selection marker. Transformation of the A. niger pyrG mutant with pAB4-1 resulted in the appearance of stable Pyr⁺ transformants at a frequency of 40 transformants per g of DNA. In 90% of these transformants integration had occurred at the resident pyrG locus, resulting either in replacement of the mutant allele by the wild-type allele (60%) or in insertion of one or two copies of the vector (40%). The A. niger pyrG mutant could also be transformed with the vector pDJB2 containing the pyr4 gene of Neurospora crassa, at a frequency of 2 transformants per g of DNA. Integration at the resident pyrG locus was not found with this vector. The vector pAB4-1 is also capable of transforming an Aspergillus nidulans pyrG mutant to Pyr⁺. The pyrG transformation system was used for the introduction of a non-selectable gene into A. niger.     

Gene cloning and transformation in the basidiomycete fungus Coprinus cinereus: Isolation and expression of the isocitrate lyase gene (acu-7)

Summary The cloned isocitrate lyase structural gene of Aspergillus nidulans (acuD) was shown to hybridize under reduced stringency conditions to unique sequences in genomic DNA digests of the basidiomycete fungus Coprinus cinereus. A gene library of C. cinereus was constructed in the lambda replacement vector L47 and screened for sequences hybridizing to the A. nidulans gene. A recombinant phage was isolated which contained the hybridizing sequence on a 5.6-kb BamHI fragment. This fragment was subcloned into pUC13 to give plasmid pHIONA1 and shown to contain a functional C. cinereus isocitrate lyase gene (acu-7) by transformation of an acu-7 mutant. Direct selection for Acu⁺ transformants was not possible because of the toxicity of the acetate selection medium. Acu⁺ transformants were obtained as cotransformants by transforming an acu-7 trp-1 double mutant, having mutations in both the isocitrate lyase and tryptophan synthetase structural genes, with a plasmid containing the trp-1 gene and either pHIONA1 or the original lambda clone. Up to 47.5% of the selected Trp⁺ transformants were cotransformed to Acu⁺. A physical analysis of 40 Acu⁺ transformants showed that the acu-7 gene had integrated at non-homologous and often multiple sites in the genome. Meiotic stability of the integrated gene was demonstrated by genetic crosses.     

Transformation ofStreptomyces avermitilis by plasmid DNA

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.