Ars region in TL-DNA on octopine type Ti plasmids

Summary In the TL-DNA region of the octopine type Ti plasmids, an ars region was assigned as the DNA segment conferring the replicational ability to YIp5 in Saccharomyces cerevisiae. T-DNA:YIp5 hybrid plasmids containing a particular T-DNA region could transform yeast cells at a frequency of 10³–10⁴ transformants per g plasmid DNA and they were rescued in Escherichia coli, although the transformed phenotype was mitotically unstable. The instability was inferred to be caused by segregation of the plasmids due to their low efficiency of replication. The ars region was mapped on the noncoding region between the coding regions corresponding to no. 5 and no. 7 mRNA, and its minimal length determined in this experiment was about 150 bp.Abbreviations Ti plasmid tumor inducing plasmid - T-DNA transferred DNA or tumor DNA - TL-DNA left T-DNA - ars autonomously replicating sequences     

Replicative transformation of the filamentous fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae ARS elements.

We have developed a transformation system for the filamentous ascomycete fungus Ashbya gossypii. Mycelial protoplasts were transformed to geneticin-resistance with plasmids containing the Escherichia coli kanamycin-resistance gene as a selectable marker and autonomously replicating sequences (ARS) from Saccharomyces cerevisiae (ARS1, 2 mu ARS). Transformation frequencies of up to 63 transformants per microgram of plasmid DNA were obtained. The transformants were unstable under nonselective conditions. Southern analysis of DNA separated by conventional and pulsed-field-gel electrophoresis showed that the transforming DNA was present as autonomously replicating plasmid. Plasmid integration into chromosomal DNA was not detected. We concluded that the S. cerevisiae ARS elements are functional in A. gossypii, since vectors lacking such elements did not yield transformants.     

Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors

Summary A high frequency transformation system for the methylotrophic yeast Hansenula polymorpha has been developed. This system depends on complementation of isolated uracil auxotrophs by the URA3 gene of Saccharomyces cerevisiae. Maintenance of the uracil prototrophy is based on integration of plasmid YIp5 at random sites within the H. polymorpha genome and on autonomously replicating plasmids containing ARS1 of S. cerevisiae or related sequences cloned from the host DNA. The sequence of one autonomously replicating sequence (HARS1) from H. polymorpha has been determined showing an AT-rich region of 9 bp with some similarity to the consensus sequence of known eukaryotic replication origins. Mitotic loss of autonomously replicating sequences is high; selection for stable uracil prototrophs yields multiple tandem arrangement of the transformed DNA with no detectable loss of the phenotype on non-selective medium. These features offer the possibility for extensive gene expression in H. polymorpha.     

Isolation of physarum DNA segments that support autonomous replication in yeast

Summary Hybrid plasmids containing the bacterial resistance-transfer factor pBR322 and the yeast leu2 ⁺gene have been used to isolate DNA fragments of Physarum that are capable of initiating DNA replication in a yeast host. Five of forty hybrid plasmids containing Physarum sequences transform leu2 ^-yeast to Leu⁺ at high frequency. The resulting Leu⁺ transformants are characterized by phenotypic instability. Supercoiled plasmid molecules containing pBR322 sequences can be detected in the transformed yeast, indicating that the transforming DNA replicates autonomously. Plasmid DNA isolated from Leu⁺ yeast can transform leuB bacteria. The hybrid plasmid recovered from the Leu⁺ bacterial transformants is identical to the original plasmid, indicating structural integrity is maintained during passage through the yeast host. These hybrid plasmids containing Physarum sequences have the same characteristics as those containing autonomously replicating yeast chromosomal sequences. As the temporal sequence of DNA replication is particularly accessible to study in Physarum plasmodia, the functional significance of these segments should be amenable to study.     

Integrative transformation by homologous recombination in the zygomycete Mucor circinelloides

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.     

Retention of autonomous replicating plasmids in cultured Drosophila cells

Summary Six kinds of autonomously replicating sequences (ARSs) derived from Drosophila or tobacco were inserted into the vector pDSV, constructed with pSV2-gpt and the copia long terminal repeat (LTR). The resulting ARS-containing plasmids, pDSV-ARSs, were transfected into the cultured Drosophila cells of GM1 S1cl1. Most of the plasmids remained for about 2 weeks and some for about 1 month in these cells. The retention time of the plasmid was not directly correlated with autonomously replicating activity of ARSs detected in the yeast. Two plasmids, one carrying ARS of Drosophila nuclear DNA and the other carrying tobacco DNA, showed the longest retention time in transformed cells and replication was confirmed in these cells. Some of these long lived plasmids were recovered, however, as modified forms. Other plasmids had disappeared 1 month after transfection. Two months following transfection, none of plasmids were recovered but they were detected in nuclear DNA as the integrated form. The integration patterns in all the cells transformed by different kinds of ARS-containing plasmids were similar to each other, and to the distribution pattern of copia LTR in the genome. These results suggest that copia LTR sequences contained in the pDSV-ARSs may participate in the integration process of these plasmids into Drosophila DNA.     

Transformation of Mucor circinelloides with Autoreplicative Vectors Containing Homologous and Heterologous ARS Elements and the Dominant Cbx r Carboxine-Resistance Gene

Mucor circinelloides transformants prototrophic to leucine and resistant to carboxine (Leu⁺ Cbx^r) have been obtained by treatment of protoplasts with plasmid constructs containing homologous leuA gene and adjacent autonomously replicating sequences (ARS) element combined with the Cbx^r(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.     

Conjugative transfer and autonomous replication of a promiscuous IncQ plasmid in the cyanobacterium Synechocystis PCC 6803

Summary The promiscuous IncQ plasmid pKT210 (Cm^r, Sm^r) is efficiently transferred by transpecific conjugation from Escherichia coli to the facultatively heterotrophic cyanobacterium Synechocystis PCC6803 when mobilized by a helper plasmid coding for IncP transfer functions. The IncQ plasmid is stably maintained in the cyanobacterium as an autonomously replicating multicopy plasmid with no detectable structural alterations and can be recovered by transformation back to E. coli when using a mcrA mcrB host. Thus, the replicative host-range of IncQ plasmids extends beyond purple bacteria to the distinct procaryotic taxon of cyanobacteria, allowing the use of these small plasmids as convenient cloning vectors in Synechocystis PCC6803 and presumably also in cyanobacteria that are not amenable to genetic transformation. In contrast, an IncQ plasmid bearing the TRP1 gene of Saccharomyces cerevisiae failed to replicate when transferred to that yeast by transformation.     

Construction of Plasmid Vectors and Transformation of the Marine Yeast Debaryomyces hansenii

We have constructed two plasmid vectors (pMR95 and pMR96) with selectable markers for the marine yeast Debaryomyces hansenii. Plasmid pMR95 contains an autonomously replicating sequence previously isolated from Debaryomyces and a hygromycin B resistance gene from the plasmid pLG90 under the control of the isocytochrome C1 promoter and terminator sequences, while pMR96 has, in addition, the Saccharomyces URA3 gene. Transformation in Debaryomyces was accomplished by electroporation. Plasmid pMR95 was capable of transforming both Saccharomyces cerevisiae and D. hansenii to hygromycin resistance at low frequencies; pMR96 transformed both yeasts at low frequencies when selected for hygromycin B resistance and at very high efficiencies when selected for uracil prototrophy. The presence of the plasmids in the transformed yeast was confirmed by polymerase chain reaction. The plasmids could be recovered back in Escherichia coli when transformed with total DNA from the yeast transformants, indicating at least a partial autonomous existence of the plasmids in the marine yeast. To our knowledge this is the first successful attempt to transform D. hansenii. Received April 16, 1998; accepted June 30, 1998.     

Efficient gene targeting in the filamentous fungusAlternaria alternata

To characterize homologous recombination of transforming DNA in the filamentous fungusAlternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. TheA. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus.BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internalBRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Me1^–) transformants accounted for 30 to 80% of hygromycin B-resistant (Hy^R) transformants, correlating closely with the size of theBRM1 segment in the transforming DNA. Restriction enzyme digestion within theBRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of theBRM1 segments. Plasmids carrying bothBRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Me1^– transformants accounted for only 5% of Hy^R transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within theBRM1 segment was used, almost all transformants were Me1^–. These results indicate that homologous integration of circular molecules inA. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.     

In vivo linearization and autonomous replication of plasmids containing human telomeric DNA in Aspergillus nidulans

Plasmids containing two inverted 0.6-kb stretches of human telomeric repeats transform Aspergillus nidulans at frequencies characteristic of autonomously replicating vectors. Transformation frequency is not affected when the plasmids are linearized in vitro prior to transformation by cutting between the inverted repeats. Southern analysis reveals the presence of a homogeneous pool of linear plasmid molecules in mycelium of transformants. Addition of the AMA1 plasmid replicator to the telomere-containing plasmids has only a minor effect on transformation. The phenotypic stability of the transformants is low. However, unlike conventional replicative transformants containing AMA1-bearing plasmids, these transformants are prone to spontaneous stabilization which occurs predominantly by conversion of the mutant chromosomal allele of the marker gene to the plasmid-borne allele. The data strongly suggest that telomeric DNA can act as a plasmid replicator. An alternative interpretation is that autonomous replication of linear DNA fragments, in contrast to covalently closed supercoiled molecules, does not require any special replicator sequences. Received: 13 January 1998 / Accepted: 10 June 1998     

Illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae

 We studied illegitimate recombination by transforming yeast with a single-stranded (ss) non-replicative plasmid. Plasmid pCW12, containing the ARG4gene, was used for transformation of yeast strains deleted for the ARG4, either in native (circular) form or after linearization within the vector sequence by the restriction enzyme ScaI. Both circular and linearized ss plasmids were shown to be much more efficient in illegitimate integration than their double-stranded (ds) counterparts and more than two-thirds of the transformants analysed contained multiple tandem integrations of the plasmid. Pulsed-field gel electrophoresis of genomic DNA revealed significant changes in the karyotype of some transformants. Plasmid DNA was frequently detected on more than one chromosome and on mitotically unstable, autonomously replicating elements. Our results show that the introduction of nonhomologous ss DNA into yeast cells can lead to different types of alterations in the yeast genome. Received: 9 February 1996/Accepted: 7 July 1996     

Development of an autonomously replicating linear vector of the yeast Cryptococcus humicola by using telomere-like sequence repeats

The yeast Cryptococcus humicola has several attractive properties for practical applications such as in bioremediation and as a source of industrially useful enzymes and compounds. We have developed an autonomously replicating vector of C. humicola to improve its properties. We initially tried to isolate an autonomously replicating sequence (ARS) from genomic DNA by transformation using a genomic DNA library. We obtained a candidate plasmid vector harboring an ARS that gave high transformation efficiency. Southern blot analysis of transformants revealed the autonomous replication of the introduced vector in some transformants. However, the vector was not only variously altered in length but also linearized. PCR analysis indicated that a telomere-like sequence repeat (TTAGGGGG) _n was added to the termini of linearized vector. Thus, we constructed an autonomously replicating linear vector having ten repeats of the telomere-like sequence at both ends. The vector transformed the yeast cells with high transformation efficiency (3230 CFU/μg of DNA), which was approximately 25-fold higher than that of a control vector lacking the repeats, and was autonomously replicated at a roughly constant size. The copy number was estimated to be less than one copy, and Ura⁺ mitotic stability varied widely among the transformants and was related to plasmid segregation efficiency.     

Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors

We have used vectors derived from avian leukosis viruses to transduce exogenous genes into early somatic stem cells of chicken embryos. The ecotropic helper cell line, Isolde, was used to generate stocks of NL-B vector carrying theNeo ^r selectable marker and theEscherichia coli lacZ gene. Microinjection of the NL-B vector directly beneath unincubated chicken embryo blastoderms resulted in infection of germline stem cells. One of the 16 male birds hatched (6.25%) from the injected embryos contained vector DNA sequences in its semen. Vector sequences were transmitted to G₁ progeny at a frequency of 2.7%.Neo ^r andlacZ genes were transcribedin vitro in chicken embryo fibroblast cultures from transgenic embryos of the G₂ progeny.     

Intermolecular recE4-independent recombination in Bacillus subtilis: formation of plasmid pKBT1

Summary The plasmid pKBT1 was derived by in vivo recE4-independent recombinational event(s) yielding a structure containing regions of plasmid and chromosomal origin. BamHI digests of plasmid pUB110 (Kan^r/Neo^r) and Bg/II digests of pTL12 (Tmp^r, leuA) were mixed, ligated and used to transform competent cells of a recE4 strain of Bacillus subtilis. Kanamycin-resistant transformants were electrophoretically screened for hybrid plasmids. Plasmid pKBT1 (8.0 kb) was smaller than pTL12 (10.4 kb) but larger than monomeric pUB110 (4.5 kb). Plasmid PKBT1 was stably maintained in recE4 strains of B. subtilis and conferred kanamycin resistance but did not specify trimethoprim resistance or leucine prototrophy. At least 86% of the pUB110 monomer length was present in pKBT1 and was completely contained within a single 5.58 kb HindIII fragment. The other segment of pKBT1 was of chromosomal origin as evidenced by lack of homology to pTL12 and strong hybridization to B. subtilis chromosomal DNA. At least one of the in vivo recE4-independent event(s) which produced pKBT1 must have involved intermolecular recombination between transforming and chromosomal DNA. This finding differs from previous reports in which recE4-independent recombination involving pUB110 sequences was a strictly intramolecular event.     

Construction of a Genetic Transformation System for a Freeze-tolerant Yeast Kluyveromyces thermotolerans

We have developed a genetic transformation system for a freeze-tolerant yeast Kluyveromyces thermotolerans. K. thermotolerans spheroplasts could be transformed with a YRp-type vector containing an autonomously replicating sequence (ARS) of Saccharomyces cerevisiae. However, transformation with a YEp-type vector containing a replication origin of S. cerevisiae 2 μM DNA was not successful. The cycloheximide resistance gene (RIM-C) of Candida maltosa and the URA3 gene of S. cerevisiae were successfully used to transform a prototrophic strain of K. thermotolerans and an Ura^? mutant of this yeast isolated in this study, respectively. Transformation was also possible by using intact cells treated with lithium salts or thiol compounds. The YRp-type vectors were maintained as plasmids in the transformants under selective conditions. This is the first report of successful transformation of K. thermotolerans.     

Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha

A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.     

The plasmids of the morels: characterization and prerequisites for vector development

Summary In several Morchella species linear dsDNA plasmids have been discovered and analyzed. Two linear plasmids of the strain Morchella conica 3 analyzed in detail are associated with the mitochondria but not as an integrative part of the high molecular weight mt DNA. In addition, homologies between different plasmids from different species could be detected. These homologies extend to all parts of the plasmids. Differences in size are due to different lengths of nonhomologous inner parts rather than to differences at the termini. Fragments comprising the entire plasmids were cloned into bacterial vectors and a selectable marker gene for eukaryotes was added to the resulting hybrid plasmids. Transformation of yeast led to the identification of autonomously replicating sequences, this being a prerequisite for the development of autonomously replicating vectors for Morchella.Dedicated to Prof. Dr. H.-J. Rehm on the occasion of his 60th birthday     

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

Summary A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu^–) was transformed by pRC2312 to Leu^– at a frequency of 1.41 × 10⁵ colonies per g DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura⁺ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 × 10³ per g DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per g DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15±3 per haploid genome in S. cerevisiae and 2–3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7–12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade^– strains of both C. albicans and S. cerevisiae to Ade⁺. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.     

Transformation of Phycomyces with a bacterial gene for kanamycin resistance

Summary Phycomyces protoplasts transformed with a plasmid containing the bacterial gene for kanamycin resistance grow in the presence of G418, a kanamycin analogue. The plasmid also contains a Phycomyces DNA sequence that supports autonomous replication in yeast. We obtained about 250 transformants per microgram DNA or one per 5000 viable protoplasts. The transformant phenotype is retained under selective conditions and lost in the majority of the vegetative spores. Recovered plasmids and Southern analysis indicate that the plasmid probably replicates autonomously in Phycomyces.