Reversible inactivation of a foreign gene, hph, during the asexual cycle in Neurospora crassa transformants

Summary A plasmid construct carrying the hygromycin phosphotransferase (hph) gene fused to the expression elements of the trpC gene of Aspergillus nidulans was used to obtain hygromycin B (Hyg)-resistant transformants of Neurospora crassa. The plasmid does not have any homology with the N. crassa genome. Here we demonstrate that most of the transformants arise from integration of the transforming DNA into only one of the nuclei present in the protoplasts. Furthermore, in most of the transformants the integrated transforming DNA is physically stable after growth of the transformants for about 25 nuclear divisions without Hyg selection, in spite of being present in multiple copies. In transformants carrying only a single insertion, phenotypic expression of the hph gene remains unaltered in conidial isolates obtained withoug Hyg selection. On the other hand, about 40% of transformants harbouring plasmid DNA integrated at more than one location yield conidial isolates showing reversible inactivation of the hph genes. Interestingly, the presence of methylated cytosine residues in the integrated DNA is strongly correlated with the number of plasmid copies. The hph genes are heavily methylated in transformants harbouring multiple copies but not in those harbouring only one copy of the plasmid. Phenotypic expression of the inactive hph genes can be restored by growing the transformants either under Hyg selection pressure or in the presence of 5-azacytidine. In the first case the hph genes are again inactivated when Hyg selection pressure is removed, while the activation of the hph gene by 5-azacytidine gives stable Hyg^r strains.Dedicated to Dr. T.A. Trautner on the occasion of his 60^th birthday     

A dominant selectable marker that is meiotically stable in Neurospora crassa: the amdS gene of Aspergillus nidulans.

Summary When Neurospora crassa is transformed using a Neurospora gene as the selectable marker, the vegetatively stable transformants obtained cannot be used successfully in a cross because the selectable marker will be inactivated by the process of RIP (repeat-induced point mutation). Introduction of the acetamidase-encoding gene amdS of Aspergillus nidulans into N. crassa by transformation yielded transformants that would grow in minimal medium containing acetamide as a sole nitrogen source. In mitotically stable transformants containing a single copy of the amdS gene, the capacity to utilize acetamide as a sole nitrogen source was maintained in the progeny of a sexual cross. Therefore, the A. nidulans amdS gene is an appropriate dominant selectable marker for use in transformation analyses with N. crassa in which sexual crosses will be subsequently performed.     

Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive rDNA sequences

An attempt was made to transform Alternaria alternata protoplasts using a plasmid vector, pDH25, bearing the Escherichia coli hygromycin B (Hy) phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter. Transformants arose on a selective medium containing 100 μg Hy/ml. There were two types of transformants, forming large and small colonies on the selective medium. Transformation with one μg of the vector produced an average of 4.5 large colonies and 600 small ones. In large-colony transformants, the vector often integrated into the recipient chromosome in the form of highly rearranged tandem arrays. To increase transformation efficiency, fragments of the highly repetitive ribosomal RNA gene cluster (rDNA) of A. alternata were used to construct four new vectors for homologous recombination system. Use of these vectors gave higher transformation efficiency than the original plasmid. The best vector, pDH25r1a, gave rise to large-colony transformants at a frequency 20 times higher than pDH25. Transformation events in A. alternata with pDH25r1a occured by homologous recombination as a single crossover between the plasmid-borne rDNA segment and its homologue in the chromosome, often giving rise to tandemly repeated vector DNA.     

Integrative Transformation of Aspergillus oryzae with a Plasmid Containing the Aspergillus nidulans argB Gene

The transformation of Aspergillus oryzae has been achieved with a plasmid carrying the Aspergillus nidulans argB gene coding for ornithine carbamoyltransferase (OCTase). The frequency of transformation was relatively low (0.7 transformants/μg DNA) but the transformed phenotype was extremely stable for many generations without selective pressure.

Southern blot analysis revealed that transformation had occurred by integration of multiple tandem copies of plasmid DNA into the host genome through non-homologous recombination. There was no evidence of the existence of free plasmid in the transformants. The number of integrated copies of the plasmid ranged from 15 to 60. The specific activity of OCTase in the cell- free extract was proportional to the copy number of the plasmid, indicating that most of the integrated argB gene was expressed.     

Targeted transformation in Coprinus cinereus

Summary We examined the influence of DNA form and size on the arrangement and genomic location of transforming DNA sequences in the basidiomycete Coprinus cinereus. Protoplasts with either single or double mutations in the tryptophan synthetase (TRPI) gene were transformed with cloned copies of this gene which contained only a single DNA strand, contained a specific single nick within the C. cinereus sequences (4.8 kb), contained a specific double-strand break, or contained an additional 35 kb of flanking genomic sequences. Gene replacement events were recovered when each DNA type was used. However, none of these substrates offers a substantial improvement in transformation or targeting frequency when compared to supercoiled circular DNA, which has allowed recovery of both gene replacements as well as homologous insertions in 5 % of the transformants analyzed. The frequency of transformants carrying tandem insertions with multiple copies of the transforming DNA was reduced when single-stranded DNA was used, and increased when DNA containing double-strand breaks was used. These results have important implications for the efficient design of targeted transformation and co-transformation experiments.     

Analysis of Aspergillus niger transformants for single site integration and vector recombination

Summary In order to understand the mechanism of integrative transformation, bulk DNA from several transformants of Aspergillus niger was assaved for the presence of the heterologous vector p3SR2. In all cases vector sequences and recombinant vector fragments were found. Transformant T31-6 containing seven plasmid-probed bands was studied in detail. Recloning of six of these plasmids in Escherichia coli revealed that internal recombination events had produced deletions and insertions. Only one plasmid was isolated among 402 analysed clones that had involved A. niger DNA. Sequence analysis revealed that the integration of vector p3SR2 occurred via its A. nidulans amdS ⁺ sequence. These data support the assumption that the integration of multiple copies of p3SR2 occurred at a single genomic site.Offprint requests to: K. Esser     

Relationship of vector insert size to homologous integration during transformation of Neurospora crassa with the cloned am (GDH) gene

Summary We used lambda and plasmid vectors containing the am ⁺ gene in an insert of from 2.7 to 9.1 kb, to transform am point mutant and deletion strains. A total of 199 transformants were examined with the potential to yield am ^– transformants by homologous recombination. When we used vectors that had 9.1 kb of homology with the chromosomal DNA, 30% of the transformants obtained were the result of homologous recombination regardless of whether the vector was a lambda molecule, a circular plasmid, or a plasmid that had been linearized prior to transformation. When vectors with up to 5.1 kb of homology were used, very few transformants (1 of 89 tested) resulted from homologous recombination. Of a sample of 29 ectopic integration events obtained by transformation with the 9.1 kb fragment cloned in a vector, 18 included a major part (usually almost all) of both arms of lambda with the entire Neurospora 9.1 kb insert between them. Four included only long arm sequence together with an adjacent segment of the insert containing the am gene. The remaining seven were the result of multiple integrations. There was no evidence of circularization of the vector prior to integration. All transformants that had multiple copies of the am gene appeared to be subject to the RIP process, which causes multiple mutations in duplicated sequences during the sexual cycle.     

Heterologous insertion of transforming DNA and generation of new deletions associated with transformation in Aspergillus nidulans

Summary The analysis of four transformants for the proline catabolism (prn) gene cluster of Aspergillus nidulans is reported. Using a combination of traditional genetic methodology and Southern hybridisation we have shown that in two cases multiple copies of the transforming plasmid have been integrated into linkage groups other than VII, which contains the prn cluster. In the other two cases integration of the plasmid has probably occurred homologously. The phenotype of these transformants is broadly consistent with increased copy number resulting in increased expression. Genetic manipulation of these transformants using the sexual or parasexual cycles has shown that recombination events during and possibly also subsequent to integration of the transforming DNA can generate new mutational lesions, in particular, deletions.     

Yeast proteins can activate expression through regulatory sequences of the amdS gene of Aspergillus nidulans

The upstream regulatory region of the amdS gene of Aspergillus nidulans contains a CCAAT sequence known to be important in setting both basal and derepressed levels of expression. We have investigated whether the CCAAT-binding HAP2/3/4 complex of the yeast Saccharomyces cerevisiae can recognise this sequence in an amdS context. Sequences from the 5 region of amdS were cloned in front of the CYCI-lacZ fusion gene bearing a minimal promoter and transformed into wild-type and hap2 strains of yeast. This study has indicated that amdS sequences are capable of promoting regulated expression of the fusion gene in response to carbon limitation. The yeast HAP2/3/4 complex can recognise the amdS CCAAT sequence and activate expression from this sequence. In addition, the results indicate that other yeast proteins can also regulate expression from the A. nidulans amdS 5 sequences under carbon-limiting conditions.     

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.     

Gene Knockout of the Intracellular Amylase Gene by Homologous Recombination in Streptococcus bovis

Streptococcus bovis expresses two different amylases, one intracellular and the other secreted. A suicide vector containing part of the intracellular α-amylase gene from Streptococcus bovis WI-1 was recombined into the S. bovis WI-1 chromosome to disrupt the endogenous gene. Recombination was demonstrated by Southern blot, and zymogram analysis confirmed the loss of the intracellular amylase. Amylase activity in cell-free extracts of the recombinant grown in the presence of 1% starch was only 7% of wild type. The rate of logarithmic growth of the recombinant was 15–20% of the wild type in medium containing either 1% glucose, starch, or cellobiose. Revertants and non-amylase control recombinants had logarithmic growth rates that were the same as wild type. Plasmid transformants containing multiple copies of the cloned gene expressed up to threefold higher levels of intracellular amylase activity than wild type but did not demonstrate elevated growth rates. These results suggest that a critical level of expression of the intracellular amylase gene may be important for rapid growth of the bacterium. Received: 26 August 1996 / Accepted: 18 December 1996     

香菇印gpd-Le和ras-Le启动子的功能分析

利用从香菇菌丝体中克隆的启动子片段gpd-Le(613bp)和ras-Le(715bp)分别连接于报告基因gfp(绿色荧光蛋白基因)的上游,构建了启动子功能活性检测表达质粒pLg-gfp和pLr-gfp。采用PEG介导法把表达质粒pLg-gfp和pLr-gfp分别与辅助质粒pCc1001(含有trp1基因)共转化进色氨酸营养缺陷型的灰盖鬼伞粉孢子的原生质体中。经过选择培养基筛选、假定转化子的分子鉴定以及GFP荧光检测。结果表明:香菇gpd-Le启动子在灰盖鬼伞的菌丝中具有较强驱动外源gfp基因表达的活性,在荧光显微镜和共聚焦显微镜下观察到gfp基因表达的绿色荧光。而香菇ras-Le启动子没有检测到有驱动外源gfp基因表达的活性。     

香菇印gpd-Le和ras-Le启动子的功能分析

利用从香菇菌丝体中克隆的启动子片段gpd-Le(613bp)和ras-Le(715bp)分别连接于报告基因gfp(绿色荧光蛋白基因)的上游,构建了启动子功能活性检测表达质粒pLg-gfp和pLr-gfp。采用PEG介导法把表达质粒pLg-gfp和pLr-gfp分别与辅助质粒pCc1001(含有trp1基因)共转化进色氨酸营养缺陷型的灰盖鬼伞粉孢子的原生质体中。经过选择培养基筛选、假定转化子的分子鉴定以及GFP荧光检测。结果表明:香菇gpd-Le启动子在灰盖鬼伞的菌丝中具有较强驱动外源gfp基因表达的活性,在荧光显微镜和共聚焦显微镜下观察到gfp基因表达的绿色荧光。而香菇ras-Le启动子没有检测到有驱动外源gfp基因表达的活性。     

A bifunctional plasmid carrying the recA gene of E. coli

Summary To investigate the effect of an active, plasmid-carried recA gene on the stability and/or the expression of plasmid genes in different genetic backgrounds, we have constructed a bifunctional plasmid (able to replicate in Escherichia coli and in Bacillus subtilis). Chimeric plasmids were obtained by inserting pC194 (Ehrlich 1977) into pDR1453 (Sancar and Rupp 1979). pDR1453 is a 12.9 Kbp plasmid constructed by inserting an E. coli chromosome fragment carrying the recA gene into pBR322. The expected bifunctional recombinant (pMR22/1) (15.7 Kbp) was easily obtained but surprisingly the Cm resistance was expressed only at a very low level in E. coli (as compared, for example, to pHV14, pHV15). We attribute this effect to the presence of multiple recA genes in the cell. On the contrary, Cm^r E. coli transformants bear a recombinant plasmid (pMR22/n) containing tandemly repeated copies of pC194 in equilibrium with excised free pC194. Such amplification has never been observed in a Rec^- background and is therefore mediated by the recA genes. Growth of these clones in the absence of Cm causes the loss of the extra copies, yielding a plasmid with a single copy of pC194, indistingishable from pMR22/1. Interestingly, we have observed that deletions occur at high frequency in pC194, which drastically increase Cm^r in E. coli containing plasmids with a single copy of pC194. Two types of such deletions were detected: (a) large 1050 bp deletions covering about onethird of pC194 and (b) small 120–150 bp deletions (near the MspI site) in the region containing the replicative functions of pC194 (Horinouchi and Weisblum 1982). Both types of deletion render the recombinant plasmid unable to replicate in B. subtilis. pM22/1 replicates, although with a low copy-number, and is stable in B. subtilis wild type; the recA gene of E. coli does not complement any of the rec ^- mutations of B. subtilis. A strong instability, mainly of the E. coli and pBR322 sequences, was observed in many dna and rec mutants of B. subtilis yielding smaller plasmid with a much higher copy-number.     

Effect ofEscherichia coliIHF Mutations on Plasmid p15A Copy Number

Four isogenic strains (himAhimDdouble mutant,himAandhimDsingle mutants, and their wild type counterpart) harboringorip15A plasmid (pACYC184 or pACYC184Amp or pACYC177) show different copy numbers of that plasmid in the early stationary phase of cultivation. The copy number oforip15A plasmid increases about four times in thehimAhimDdouble (65–70 copies per cell) andhimDsingle mutant cells (50–56 copies per cell) and was almost the same inhimAmutant (17–18 copies per cell) and wild type cells (14–16 copies per cell). The results suggest that HimD can form homodimers, which are functionally competent for the regulation oforip15A plasmid copy number. Complementation experiments ofhimAhimDdouble mutant cells using plasmid carryinghimAandhimDgenes (pP_LhiphimA-5) confirm the effect of integration host factor (IHF) absence on increasing the copy number oforip15A plasmid (plasmid producing IHF complemented the defect of IHF mutant). The absence of IHF (usinghimAhimDdouble mutant as host) had no effect on the copy number of the pBR322 (oripMB1) plasmid.     

Construction of a food-grade multiple-copy integration system for Lactococcus lactis

A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori⁺) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori⁺ vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background. Single-cross-over integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions. In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5–15) × 10⁻² copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth. These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest. Received: 23 September 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997     

Properties and transforming activities of two plasmids in Streptococcus pneumoniae

Summary Two plasmids from group B streptococcus were introduced into pneumococcus (Streptococcus pneumoniae) and examined for copy number, stability, and some features of the process by which they transform pneumococcal recipients. The 3.6 Mdal pMV158 (tet) was present at a minimum of 12 to 16 copies per chromosome and was never observed to be cured. The 20 Mdal pIP501 (cat erm) had a minimum copy number of 3 to 4 per chromosome and was lost spontaneously at a frequency near 0.03 per division. The presence of novobiocin increased this frequency 2 to 3-fold. Competence for chromosomal transformation and the membrane endonuclease needed for normal DNA entry were required for plasmid transformation. Plasmid transformants segregated transformed cells one generation ahead of chromosomal transformants. Both single and multiple hit components of the transformation reaction kinetics were observed, but the latter could not be seen in the presence of competing chromosomal DNA. The majority of the transforming activity behaved as covalently closed circular DNA in dye-buoyancy gradients. Although most of the activity for both plasmids sedimented in sucrose gradients more rapidly than did monomeric closed circular DNA, a significant fraction was found at a position suggesting that it may have been due to monomeric plasmids.     

Analysis of the site of action of the amdR product for regulation of the amdS gene of Aspergillus nidulans

Summary The amdR gene of Aspergillus nidulans regulates a number of structural genes in response to omega amino acid inducers. The site of action of the amdR product on expression of the amdS gene was investigated by studying the effects of changes in the 5 region of amdS, generated in vitro, on the induction, and on responses of an amdS-lacZ fusion gene to an amdR ^c allele. A sequence was identified that is sufficient for amdR regulation and that shows identity with sequences involved in amdR regulation of the gatA and lam genes. This sequence includes a CCAAT sequence and it was shown that this sequence is an important element in setting the basal level of amdS expression.