Large circular and linear rDNA plasmids in Candida albicans

Although plasmids containing rRNA genes (rDNA) are commonly found in fungi, they have not been reported in Candida. We discovered that the yeast opportunistic pathogen Candida albicans contains two types of rDNA plasmids which differ in their structure and number of rDNA repeats. A large circular plasmid of unknown size consists of multiple rDNA repeats, each of which includes an associated autonomously replicating sequence (ARS). In contrast, a linear plasmid, which is represented by a series of molecules with a spread of sizes ranging from 50-150 kbp, carries a limited number of rDNA units and associated ARSs, as well as telomeres. The number of linear plasmids per cell is growth cycle-dependent, accumulating in abundance in actively growing cells. We suggest that the total copy number of rDNA is better controlled when a portion of copies are on a linear extrachromosomal plasmid, thus allowing a rapid shift in the number of corresponding genes and, as a result, better adaptation to the environment. This is the first report of a linear rDNA plasmid in yeast, as well as of the coexistence of circular and linear plasmids. In addition, this is a first report of naturally occurring plasmids in C. albicans.     

Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus

Schizosaccharomyces japonicus is a fission yeast for which new genetic tools have recently been developed. Here, we report novel plasmid vectors with high transformation efficiency and an electroporation method for Sz. japonicus. We isolated 44 replicating segments from 12 166 transformants of Sz. japonicus genomic fragments and found a chromosomal fragment, RS1, as a new replicating sequence that conferred high transformation activity to Sz. japonicus cells. This sequence was cloned into a pUC19 vector with ura4⁺ of Sz. pombe (pSJU11) or the kan gene on the kanMX6 module (pSJK11) as selection markers. These plasmids transformed Sz. japonicus cells in the early‐log phase by electroporation at a frequency of 123 cfu/µg for pSJK11 and 301 cfu/µg for pSJU11, which were higher than previously reported autonomously replicating sequences. Although a portion of plasmids remained in host cells by integration into the chromosome via RS1 segment, the plasmids could be recovered from transformants. The plasmid copy number was estimated to be 1.88 copies per cell by Southern blot analysis using a Sz. pombe ura4⁺ probe. The plasmid containing ade6⁺ suppressed the auxotrophic growth of the ade6‐domE mutant, indicating that the plasmid would be useful for suppressor screening and complementation assays in Sz. japonicus. Furthermore, pSJU11 transformed Sz. pombe cells with the same frequency as the pREP2 plasmid. This study is a report to demonstrate practical use of episomal plasmid vectors for genetic research in Sz. japonicus. RS1 has been submitted to the DDBJ/EMBL/GenBank database (Accession No. AB547343). Copyright © 2010 John Wiley & Sons, Ltd.     

Molecular cloning and analysis of autonomous replicating sequence of Candida maltosa.

A Candida maltosa chromosomal DNA fragment which confers high frequency transformation of C. maltosa and autonomous replication of recombinant plasmids was cloned and sequenced. Analysis of the nucleotide sequence of the cloned DNA revealed a sequence homologous for C. maltosa autonomously replicating sequence (ARS) elements. Vector pRJ1 for C. maltosa was constructed, which contained a 1.3 kb ARS sequence, pICEM-19H and the ADE1 gene of C. maltosa. Southern blot analysis suggested that the copy number of pRJ1 in C. maltosa was approximately 20 per genome. The sequence analysis also revealed an open reading frame, encoding a polypeptide with high homology (70%) to the RS15 protein of Brugia pagangi. This open reading frame has an intron with canonical sites for correct splicing in Saccharomyces cerevisiae.     

A shuttle vector series for precise genetic engineering of Saccharomyces cerevisiae

Shuttle vectors allow for an efficient transfer of recombinant DNA into yeast cells and are widely used in fundamental research and biotechnology. While available shuttle vectors are applicable in many experimental settings, their use in quantitative biology is hampered by insufficient copy number control. Moreover, they often have practical constraints, such as limited modularity and few unique restriction sites. We constructed the pRG shuttle vector series, consisting of single‐ and multi‐copy integrative, centromeric and episomal plasmids with marker genes for the selection in all commonly used auxotrophic yeast strains. The vectors feature a modular design and a large number of unique restriction sites, enabling an efficient exchange of every vector part and expansion of the series. Integration into the host genome is achieved using a double‐crossover recombination mechanism, resulting in stable single‐ and multi‐copy modifications. As centromeric and episomal plasmids give rise to a heterogeneous cell population, an analysis of their copy number distribution and loss behaviour was performed. Overall, the shuttle vector series supports the efficient cloning of genes and their maintenance in yeast cells with improved copy number control. Copyright © 2015 John Wiley & Sons, Ltd.     

Yeast/E. coli shuttle vectors with multiple unique restriction sites

Two yeast/E. coli shuttle vectors have been constructed. The two vectors, YEp351 and YEp352, have the following properties: (1) they can replicate autonomously in Saccharomyces cerevisiae and in E. coli; (2) they contain the beta-lactamase gene and confer ampicillin resistance to E. coli; (3) they contain the entire sequence of pUC18; (4) all ten restriction sites of the multiple cloning region of pUC18 including EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI and HindIII are unique in YEp352; these sites are also unique in YEp351 except for EcoRI and KpnI, which occur twice; (5) recombinant plasmids with DNA inserts in the multiple cloning region of YEp351 and YEp352 can be recognised by loss of beta-galactosidase function in appropriate E. coli hosts; (6) YEp351 and YEp352 contain the yeast LEU2 and URA3 genes, respectively, allowing for selection of these auxotrophic markers in yeast and E. coli; (7) both plasmids are retained with high frequency in yeast grown under non-selective conditions indicative of high plasmid copy number. The above properties make the shuttle vectors suitable for construction of yeast genomic libraries and for cloning of DNA fragments defined by a large number of different restriction sites. The two vectors have been further modified by deletion of the sequences necessary for autonomous replication in yeast. The derivative plasmids YIp351 and YIp352 can therefore be used to integrate specific sequences into yeast chromosomal DNA.     

pDUAL, a multipurpose, multicopy vector capable of chromosomal integration in fission yeast

A novel series of plasmid vectors named pDUAL have been developed. These vectors enable one to introduce not only multicopies of genes with episomal maintenance but also a single copy with chromosomal integration into the fission yeast, Schizosaccharomyces pombe. The multicopy plasmids can be easily converted to fragments for chromosomal integration by digestion of the plasmids with a certain restriction endonuclease before transformation of the yeast cells. The resultant fragments, lacking the autonomously replicating sequence, are designed for targeting into the chromosomal leu1 locus by homologous recombination. Whether the transformants are the results of episomal maintenance of the plasmid or homologous gene targeting can be readily checked by their requirement for uracil or leucine, or by the PCR diagnostic analysis. Furthermore, we propose the use of pDUAL derivatives for PCR-based chromosomal tagging of a gene to introduce several tags into 5'-terminus of a gene, employing a set of primers. Using these all-in-one vectors, a suitable mode of expression of a cloned gene can be selected for individual analysis without any complicated subcloning processes.     

A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors.

We describe a set of replicative, integrative and single-stranded shuttle vectors constructed from the pUC19 plasmid that we use routinely in our experiments. They bear a yeast selectable marker: URA3, TRP1 or LEU2. Replicative vectors carrying different yeast replication origins have been constructed in order to have plasmids based on the same construction with a high or low copy number per cell and with different mitotic stabilities. All the vectors are small in size, provide a high yield in Escherichia coli and efficiently transform Saccharomyces cerevisiae. These plasmids have many of the unique sites of the pUC19 multicloning region and many of them allow for the screening of plasmids with an insert by alpha-complementation. The nucleotide sequence of each of them is completely known.     

Scarcity of ars sequences isolated in a morphogenesis mutant of the yeast Yarrowia lipolytica

Previous attempts is isolate autonomously replicating sequences (ars) from the dimorphic yeast Yarrowia lipolytica have been unsuccessful. We isolated a Fil- mutant unable to produce hyphae and growing only in a yeast form to facilitate ars isolation. This mutant was transformed with a Y. lipolytica DNA bank and several unstable clones were obtained. Extrachromosomal plasmids were evidenced in yeast, recovered in Escherichia coli and characterized by restriction mapping. They were able to retransform Fil- and Fil+ yeast strains at high frequency and transformants displayed a slightly unstable phenotype. The detailed analysis of the plasmids showed that only two different ars sequences had been isolated, each of them corresponding to a unique sequence in the Y. lipolytica genome. We concluded that functional ars sequences that can be cloned on plasmids are rare in this yeast.     

Integrating after CEN Excision (ICE) Plasmids: Combining the ease of yeast recombination cloning with the stability of genomic integration

Yeast recombination cloning is a straightforward and powerful method for recombining a plasmid backbone with a specific DNA fragment. However, the utility of yeast recombination cloning is limited by the requirement for the backbone to contain an CEN/ARS element, which allows for the recombined plasmids to propagate. Although yeast CEN/ARS plasmids are often suitable for further studies, we demonstrate here that they can vary considerably in copy number from cell to cell and from colony to colony. Variation in plasmid copy number can pose an unacceptable and often unacknowledged source of phenotypic variation. If expression levels are critical to experimentation, then constructs generated with yeast recombination cloning must be subcloned into integrating plasmids, a step that often abrogates the utility of recombination cloning. Accordingly, we have designed a vector that can be used for yeast recombination cloning but can be converted into the integrating version of the resulting vector without an additional subcloning. We call these “ICE” vectors, for “Integrating after CEN Excision.” The ICE series was created by introducing a “rare-cutter” NotI-flanked CEN/ARS element into the multiple cloning sites of the pRS series yeast integration plasmids. Upon recovery from yeast, the CEN/ARS is excised by NotI digest and subsequently religated without need for purification or transfer to new conditions. Excision by this approach takes ~3 hr, allowing this refinement in the same time frame as standard recombination cloning.     

粪肠球菌内源性质粒的序列分析及其穿梭载体的构建

为了对粪肠球菌EXW27的内源性质粒pXW进行DNA序列的测定和分析,并基于其最小复制子构建大肠杆菌-粪肠球菌穿梭载体。本研究从具有优良益生特性的粪肠球菌EXW27中分离得到了内源性质粒pXW,对其进行了DNA序列的测定及分析,然后利用质粒pXW的复制子构建大肠杆菌-乳酸菌穿梭载体,并研究大肠杆菌-乳酸菌穿梭载体的宿主范围、转化效率和稳定性。结果表明该质粒大小为8617 bp,GC含量为33.29%,包含8个ORF,推定为θ型复制质粒。质粒pXW在粪肠球菌EXW27中拷贝数最高可达32.09±0.93,表明质粒pXW属于高拷贝数质粒。本研究确定了质粒pXW的最小复制子,并基于该复制子构建了大肠杆菌-粪肠球菌穿梭载体pXWM1。该穿梭载体具有较宽的宿主范围,可成功转化至不同类型乳酸菌,转化效率介于1.96×102~8.96×104 CFU/μg （质粒DNA）之间,质粒丢失率介于28.54%~54.17%之间。本研究成功构建了一个具有宽宿主范围、高转化效率以及高稳定性的大肠杆菌-粪肠球菌穿梭载体,为乳酸菌基因操作提供了新工具。     

Mutations in ARS1 increase the rate of simple loss of plasmids in Saccharomyces cerevisiae

Autonomously replicating sequence (ARS) elements are DNA sequences that promote extrachromosomal maintenance of plasmids in yeast. Mutations generated in vitro in the ARS1 region were examined for their effect on plasmid maintenance in a yeast centromeric plasmid. Our data show that mutations in the regions surrounding the ARS1 consensus sequence cause increases in the frequency of simple loss (1:0) events without affecting the rate of nondisjunction (2:0). Removal of the consensus sequence itself causes a drastic increase in the rate of simple loss. Sequences sensitive to mutagenesis were identified in each flanking region and differ with respect to their location and importance to ARS function. These results suggest that the role ARS1 plays in plasmid maintenance deals with the replication and/or localization of the plasmid in yeast.     

A series of yeast shuttle vectors for expression of cDNAs and other DNA sequences

Expression/shuttle vectors for the yeast Saccharomyces cerevisiae have usually been large plasmids with only one or a small number of sites that are suitable for cloning and expression. We report here the construction and properties of a series of 12 expression vectors with multiple (four to eight) unique sites in their polylinkers which allow directional cloning and expression of DNA sequences under four different promoters. Eleven of these plasmids replicate at high copy number in Escherichia coli, and all have the yeast TRP1 gene, and the 2 μm origin including REP3 sequence, allowing selection and high copy number replication in yeast. Six of the plasmids are designed for the construction and selection of cDNA libraries from various eukaryotic organisms, allowing directional cloning and expression of cDNAs. All of these six have similar polylinkers containing a unique promoter proximal EcoRI site and a unique promoter distal XhoI site, allowing for directional cloning and expression of ‘ZAP’-type cDNAs. cDNAs that complement a wide variety of yeast mutants can be selected from libraries constructed in this way. The four alternative promoters, ADH2, PGK, GAL10 and SV40 were compared for their relative activity, both in E. coli and in yeast. All yeast promoters showed substantial activity in E. coli with ADH2 showing the highest activity. ADH2 also was well-regulated in yeast, showing very high relative activity under derepressing conditions. cDNAs selected by genetic complementation from libraries constructed in these vectors should be easily subclonable into other vectors, allowing expression in different eukaryotic organisms, DNA sequencing or site-directed mutagenesis.     

基于低拷贝质粒的高产紫色杆菌素谷氨酸棒杆菌的构建和发酵

该研究以公认安全(Generally Recognized as Safe,GRAS)的谷氨酸棒杆菌(Corynebacterium glutamicum)为宿主,构建高产紫色杆菌素的重组菌株。利用谷氨酸棒杆菌天然大质粒pTET3的复制与分配元件,构建了低拷贝质粒pOK12CG1,该质粒在谷氨酸棒杆菌中的拷贝数约为6拷贝/基因组,且与谷氨酸棒杆菌常用质粒pEC-XK99E和pXMJ19兼容。以低拷贝质粒pOK12CG1为骨架构建了携带紫色杆菌素合成操纵子(vioABCDE)的质粒pCGvio,并分别以谷氨酸棒杆菌标准株ATCC 13032和插入序列(Insertion Sequence,IS)元件删除株为宿主,构建了7株合成紫色杆菌素的重组菌株。通过初步筛选,发现基于低拷贝质粒的重组菌株ATCC13032/p CGvio,其紫色杆菌素产量(508.24 mg/L)高于基于中高拷贝质粒的重组菌株ATCC 13032/pECvio(376.16 mg/L),而基于低拷贝质粒的IS元件删除重组菌株ISDM023/pCGvio紫色杆菌素产量达到了610.13mg/L。进一步采用正交实验设计对重...     

Applications of the Long and Accurate Polymerase Chain Reaction Method in Yeast Molecular Biology: Direct Sequencing of the Amplified DNA and Its Introduction into Yeast

A DNA fragment longer than 10 kb can be amplified by the long and accurate polymerase chain reaction (LA-PCR) method. We demonstrate here applications of this technique in molecular biological studies of Saccharomyces cerevisiae. We have shown that DNA fragments amplified by LA-PCR can be directly used as a template in the chain-termination sequencing protocol, making it possible to quickly identify the DNA insert of yeast genomic library clones. We have also shown that the amplified yeast DNA can easily be introduced into yeast by co-transformation with linearized vector DNA. Overlapping DNA between the amplified yeast fragment and the vector must be more than 20 bp long in order to obtain 90% or more correct recombinant plasmids. These results suggest that simple amplification of yeast clones by LA-PCR can replace the previous procedures of yeast clone recovery, consisting of transformation of Escherichia coli, propagation of plasmids in E. coli and preparation of plasmid DNA. © 1997 John Wiley & Sons, Ltd.     

Efficient electropulse transformation of intact Candida maltosa cells by different homologous vector plasmids.

Conditions for efficient and quick transformation by electroporation were developed in Candida maltosa. To investigate the efficiency of transformation with integrative as well as with autonomously replicating plasmids, a series of vectors was constructed for homologous transformation of this species. Transformants were obtained with different plasmids as covalently closed circular molecules and as linearized DNA. The influence of recipient strain and plasmid type as well as of cell number and parameters of the supplied electrical pulse on the transformation efficiency have been investigated. A maximum of 7000 transformants per 100 ng of plasmid DNA was reached. The efficiency of transformation was compared with that of the LiCl method.     

A new family of yeast vectors and S288C-derived strains for the systematic analysis of gene function

The yeast genome has been shown to contain a significant number of gene families with more than three members. In order to study these families it is often necessary to generate strains carrying deletions of all members of the family, which can require a wide range of auxotrophic markers. To facilitate such studies, we have generated yeast strains containing deletions of a selection of nutritional marker genes (ade2, ade4, ade8, met3 and met14). We have also cloned the corresponding cognate genes, allowing their use in PCR-based gene disruptions. Two new pRS family Saccharomyces cerevisiae-Escherichia coli shuttle vectors containing ADE8 (one low-copy, pRS4110, and one high-copy, pRS4210) have been produced for use in conjunction with the new strains. A system for easier synthetic lethal screening using one of these new markers is also presented. The ADE8 and HIS3 genes have been cloned together on a high-copy vector (pRS4213), providing a plasmid for red-white colour screening in the ade2 Delta 0 ade8 Delta 0 strains we have generated. In contrast to some conventional systems, this plasmid allows for screening using gene libraries constructed in URA3 plasmids.     

Construction of an autonomously replicating plasmid in n-alkane-assimilating yeast, Candida tropicalis

A transformation system using the autonomously replicating plasmid in the n-alkane-assimilating and asporogenic diploid yeast, Candida tropicalis, was developed. For the cloning of a DNA fragment containing a potential autonomously replicating sequence (ARS) from the genomic DNA of C. tropicalis, the ura3 mutant obtained using ethylmethane sulfonate as the host and the URA3 gene amplified by PCR using the C. tropicalis genomic DNA as a selectable marker were prepared. Comparison of ARSs among yeasts revealed that the consensus sequence found in S. cerevisiae was also present in C. tropicalis. The autonomously replicating plasmid containing the putative ARS as the shuttle vector, capable of replicating in both E. coli and C. tropicalis, was first constructed. The transformation system using this plasmid, in addition to the integrative transformation system, will be applicable to genetic studies of C. tropicalis.     

Saccharomyces cerevisiae Shuttle vectors

Yeast shuttle vectors are indispensable tools in yeast research. They enable cloning of defined DNA sequences in Escherichia coli and their direct transfer into Saccharomyces cerevisiae cells. There are three types of commonly used yeast shuttle vectors: centromeric plasmids, episomal plasmids and integrating plasmids. In this review, we discuss the different plasmid systems and their characteristic features. We focus on their segregational stability and copy number and indicate how to modify these properties. Copyright © 2017 John Wiley & Sons, Ltd.