Sequence analysis of a Papaver somniferum L. mitochondrial DNA fragment promoting autonomous plasmid replication in Saccharomyces cerevisiae and Kluyveromyces lactis

The minimal fragment of mitochondrial DNA from Papaver somniferum L. (poppy) able to promote autonomous plasmid replication in the yeast Saccharomyces cerevisiae was sequenced. Sequence analysis of the 917-bp MK4/8 DNA fragment revealed a high AT content, and the presence of two 12-bp sequences differing from the ARS core consensus of S. cerevisiae only by a T and C insertion, respectively. The mitochondrial insert contains a further six 11-bp sequences with one mismatch to the S. cerevisiae core consensus, more then 20 related sequences with two base pair exchanges, numerous direct and inverted repeats, and many copies of a sequence motif called the ARS box. The original 4.2-kb mitochondrial DNA fragment, as well as the minimal 917-bp subfragment in vector pFL1-E (a variant of YIP5, lacking an origin of replication in yeast), were then tested for their ability to replicate autonomously in another fungus, Kluyveromyces lactis.     

Presence of a P 1 bacteriophage sequence in transforming plasmids of Pleurotus ostreatus

Replicative plasmids pP01 and pP02, recovered from Pleurotus ostreatus transformants, contain an insert of bacteriophage origin. These plasmids have been amplified by the polymerase chain reaction (PCR) and have been shown to represent a low-grade component in the initial preparation of the vector pAN7-1. The pP01 and pP02 plasmids share an insert (P01A) of virtual identity with a SmaI-BamHI genomic fragment of P 1 bacteriophage and retain remnants of a polylinker at the 5 end of this fragment. Such an insert undoubtedly represents an in vitro-generated event and did not arise, as suggested previously, by recombination of pAN7-1 with the P. ostreatus genome. The P. ostreatus transformants, however, do select for the minority pP0 plasmid, apparently recognizing the P01A insert as a heterologous or surrogate replicon.     

DNA integration into recipient yeast chromosomes by trans-kingdom conjugation between Escherichia coli and Saccharomyces cerevisiae

Summary IncQ-derived conjugative shuttle vectors, which carried the yeast gene URA3 and/or the yeast autonomously replicating sequence (ARS1), were constructed. Both the ars-plus plasmid pAY205 and the ars-less plasmid pAY201 were successfully transmitted from E. coli to S. cerevisiae by the action of mob and tra. In this trans-kingdom conjugation, plasmid pAY205 could replicate and be retained in transconjugants. Plasmid pAY201 caused the formation of micro-colonies of abortive transconjugants due to its transient expression and rapid disappearance. Nevertheless, one per about 10³ colonies caused by transmitted pAY201 plasmids were uncurable by integration into the homologous region of a yeast chromosome. Analyses by restriction enzyme mapping and Southern hybridization indicate that this integration is primarily caused by a double crossover during conjugation and not by a single reciprocal recombination.     

Construction of stable episomes in Cryptococcus neoformans

We report the generation of stable plasmids constructed by inserting specific DNA sequences into previously known unstable vectors. These sequences were obtained from a DNA library recovered from a previously reported stable minichromosome created by electroporative transformation in Cryptococcus neoformans (Varma and Kwon-Chung 1994). A 6-kb insert from this minichromosome significantly enhanced both the frequencies at which URA5 transformants were obtained as well as the stability of their uracil prototrophy on non-selective media. A 1.5-kb sequence of this insert contained telomeric sequence repeats which when introduced into plasmids resulted in significant increases in transformation frequency. A 1081-bp sequence (STAB), present in the remainder of the insert, had an ARS-like function enhancing the episomal maintenance of plasmids in the transformants regardless of the gene (ADE2/URA5) used as a selection marker. Received: 4 November 1997 / 11 May 1998     

A homologous and self-replicating system for efficient transformation ofFusarium oxysporum

A highly efficient transformation system has been developed forFusarium oxysporum f. sp.lycopersici based on the complementation of a nitrate-reductase mutant with the homologousnitI gene and on the presence ofARS and telomeric sequences in the vector. Preliminary transformation experiments with theniaD gene fromAspergillus niger generated self-replicating plasmids within the transformed entity that contained extra-fungal DNA. A fragment of the extra DNA was inserted into pUC19 together with theF. oxysporum nitl gene, resulting in plasmid pFNit-Lam. This allowed the isolation of a new linear plasmid within self-replicativeF. oxysporum transformants (pFNit-Lam-TLam, linear). The circular form of this vector yielded 5600 fungal transformants per g of DNA. All of the transformants contained autonomous linear plasmids harboring direct repeats of fungal DNA at both ends. The sequence of the 1.2-kb fragment fromF. oxysporum responsible for autonomous replication, and maintenance as linear plasmid molecules, has been determined. Comparison analysis with theARS from different organisms has shown that this fragment contained the commonly identifiedARS consensus sequence, 5A/TTTTATA/GTTTA/T3 and, in addition to this core, ten copies of theARS-box, TNTA/GAA3. Adjacent to this presumedARS, the telomeric hexanucleotide sequence (TTAGGG)_n was present in six tandem copies followed by 18 copies of its complementary sequence.     

Active recombination of pKD1-derived vectors with resident pKD1 in Kluyveromyces lactis transformation

Summary The host specificity of the 2-like circular plasmid pKD 1 is such that this plasmid replicates stably in several species of Kluyveromyces yeasts, but not in Saccharomyces cerevisiae. pKD 1-derived plasmids containing various parts of the pKD 1 sequence were capable of transforming Kluyveromyees lactis with high efficiency. When such vectors were introduced into host strains that contained resident pKD1 plasmid, the input DNA frequently recombined with it to produce high proportions of additive recombinant molecules that replicate stably. Recombination events were shown to occur with vectors differing for the presence or absence of the putative origin of replication and of the inverted repeats. Structure, stability and copy number of the recombination products were analyzed for various types of vectors.     

Yeast centromere sequences do not confer mitotic stability on circular plasmids containing ARS elements of Tetrahymena thermophila rDNA

Summary We have previously demonstrated that a 657 bp TaqI-XbaI and a 427 by XbaI-XbaI fragment from the 5 non-transcribed spacer of the extrachromosomal ribosomal DNA of Tetrahymena thermophila function as autonomously replicating sequences (ARS) in Saccharomyces cerevisiae. These fragments are adjacent to each other in a region that encompasses the in vivo origin of bidirectional replication of rDNA. The presence of a yeast centromere (CEN) fragment does not confer mitotic stability on these plasmids. A sensitive yeast colony colour assay (Hieter et al. 1985a) has been used to evaluate the cis-acting effect of each ARS segment on the pattern of inheritance of a plasmid containing CEN5:URA3:SUP4. Colonies of transformed cells obtained both in the presence and absence of selection were red with no detectable white or pink sectors. The lack of sectoring indicates that both plasmids are lost at an extremely high rate, likely due to 1:0 segregation events. We conclude that while these ARS elements confer a high frequency transformation phenotype, they lack a function which is required in cis for the maintenance of mitotic stability in the presence of a centromere. This missing cis-acting function may result in the inability of the plasmids to be brought under the control of cell-cycle regulated replication.     

Characterization of the termini of linear plasmids from Nectria haematococca and their use in construction of an autonomously replicating transformation vector

Summary The mitochondria of isolate FS37 from Nectria haematococca mating population I (Fusarium solani f. sp. cucurbitae) contain two linear plasmids, pFSCI and pFSC2, of 9.2 and 8.3 kbp, respectively. Evidence for a protein blocking the 5 termini of these plasmids was obtained from exonuclease digestion experiments. A single protein band with an apparent M_r of 80 K was labeled when the DNA-protein complex of either plasmid was reacted with [¹²⁵I] Bolton-Hunter reagent and then digested with DNase I. DNA sequence analysis of the termini of both plasmids revealed long inverted repeats of 1,211 by (pFSC1) and 1,027bp (pFSC2). No sequence similarity was found between the terminal inverted repeats (TIRs) of pFSC1 and pFSC2, nor was any similarity identified between the TIRs of the these plasmids and sequences of TIRs from other linear DNAs. A restriction fragment containing the TIR of pFSCI conferred autonomous replication when incorporated into an integrative transformation vector of Ustilago maydis.     

ARS activity along the yeast mitochondrial apocytochrome b region: correlation with the location of petite genomes and consensus sequences

Summary Seven MboI fragments spanning the mitochondrial apocytochrome b gene in Saccharomyces cerevisiae strain D273-10B were cloned in the BamHI site of the integrative yeast vector YIp5 and the capacity for autonomous replication was subsequently assayed in yeast. The positive correlation found between the ars-like activity in four fragments and the presence of regions common to multiple ethidium bromide-induced petite (rho^–) genomes suggests that the mitochondrial sequences possibly active as origins of replication in low-complexity neutral or weakly suppressive rho^– mutants could be functionally related to the yeast nuclear replicator 11 nucleotide motif defined by Broach et al. (1983).Abbreviations mtDNA mitochondrial DNA - bp base pairs - kbp kilobase pairs     

Plasmid cloning and expression of the E. coli polA + gene in S. cerevisiae

Summary The E. coli polA ⁺ gene has been subcloned from a specialised transducing phage onto a low copy number plasmid. Plasmid-encoded DNA polymerase I was synthesised at 2 to 3 times the wild-type E. coli level, and was biochemically indistinguishable from chromosomally-encoded protein. It was able to counteract the radio sensitivity of polA1, polAex1, polAex2 and polA12 mutants, but no complementation of polA107 mutants occurred, even though the plasmid polA⁺ gene was expressed. S. cerevisiae ars-1 or 2 replicative sequences were introduced into the polA⁺ plasmid. Transformation of yeast with these constructs increased total DNA polymerase levels 2–20 times, depending upon assay conditions. The additional activity was discriminated from yeast DNA polymerases by its ability to use low concentrations of substrate, by its resistance to chemical inhibition, and by co-electrophoresis with pure DNA polymerase I and its proteolytic fragments. The polA⁺ gene was expressed in yeast without the aid of yeast promotor sequences. However, deletion of cloned DNA more than 99 base pairs in front of the structural gene prevented expression in yeast but not in E. coli, indicating that the two organisms use different sequences for expression of the plasmid polA⁺ gene.     

Recovery of recombinant plasmids from Pleurotus ostreatus transformants

Summary A transformation system employing selectable resistance to hygromycin B has been developed for the mushroom-forming fungus, Pleurotus ostreatus. Vector pAN7-1, a commonly used non-replicative vector for integrative transformation in fungi, yielded 5–46 resistant colonies per g of DNA per 10⁷ viable protoplasts. Southern blot analysis of certain transformants revealed unexpected replicative plasmids containing pAN7-1 sequences, but modified for size, methylation and restriction enzyme pattern when compared to the initial transforming vector. Two such replicative derivatives of pAN7-1 have been rescued from P. ostreatus by cloning into Escherichia coli. Rescued plasmids have been used to probe DNA from untransformed P. ostreatus in an effort to identify fungal sequences that recombined in vivo with pAN7-1 to form replicative plasmids. Such replicative sequences have been localized in high molecular weight (chromosomal) DNA of wild-type P. ostreatus. Transformation has been obtained for P. ostreatus using a rescued plasmid, thereby confirming the role of this recombinant plasmid as a shuttle vector.     

Cloning and sequencing of cellulase cDNA from Aspergillus kawachii and its expression in Saccharomyces cerevisiae

The cDNA encoding the endo--1,4-glucanase (carboxymethylcellulase; CMCase-I) from Aspergillus kawachii IFO 4308 was cloned. Nucleotide-sequence analysis of the cloned cDNA insert showed a 717-bp open reading frame that encoded a protein of 239 amino-acid residues. The predicted amino-acid sequence of the mature protein had considerable homology with the protein sequence of the FI-CMCase of Aspergillus aculeatus. The cDNA was introduced into Saccharomyces cerevisiae. The expressed enzyme had carboxylmethylcellulase acitivity, identified by clear zones on a CMC-agar plate after Congo Red staining.     

The CDC8 gene product is required for transformation with episomal and integrative plasmids in Saccharomyces cerevisiae

Summary The product of the yeast CDC8 gene (thymidylate kinase), which is required for chromosomal, mitochondrial and 2 plasmid replication, also participates in plasmid transformation processes in S. cerevisiae. The thermosensitive cdc8-1 mutant strain was transformed with episomal pDQ9 and integrative pDQ9-1 plasmids both of which carry the CDC8 gene. The results suggest that thymidylate kinase is essential for the expression of genes carried on transforming episomal plasmid DNA (probably through its replication) and is also essential for homologous recombination between chromosomal and linearized integrative plasmid DNA.     

Behaviour of recombinant plasmids in Aspergillus nidulans: structure and stability

Summary A pyrG ^– Aspergillus strain was transformed with plasmid pDJB-1, derived from pBR325 by insertion of the Neurospora crassa pyr4 gene (orotidine 5-phosphate carboxylase), giving mitotically unstable transformants. Aspergillus DNA which acted as an autonomously replicating sequence (ARS) in yeast was inserted into pDJB-1 and the resulting construct, pDJB12.1, gave mitotically stable transformants when introduced into Aspergillus. Transformants obtained with pDJB-1 and pDJB12.1 gave few pyr ^– progeny in crosses to a pyrG ⁺ strain. Southern hybridisation analysis of pyr ⁺ transformants obtained with pDJB-1 revealed restriction fragments expected for integrated plasmid but transformants obtained with pDJB12-1 showed only bands derived from free plasmid. pDJB-1 and derivatives of pDJB12.1 could be recovered from transformants. These derivatives could not be explained by straightforward excision of integrated pDJB12.1 sequences but could result from recombination between plasmid molecules. Hybridisation of undigested transformant DNAs showed that the transforming DNA was present in a high molecular weight form. These results suggest: (1) pDJB12.1 derivatives and possibly pDJB-1 can replicate autonomously in Aspergillus; (2) A. nidulans DNA acting as an ARS in yeast enhances replication and/or segregation of transforming plasmids in Aspergillus; and (3) recombinant plasmids may undergo rearrangements when introduced into Aspergillus.Abbreviations PABA para-amino benzoic acid - EDTA disodium salt of ethylene diamine tetra-acetic acid - SDS sodium dodecyl sulphate - DTT dithiothreitol - UV ultra violet - SSC standard saline citrate; 0.15 M sodium chloride, 0.015 M trisodium citrate pH 7. - ARS('s) autonomously replicating sequence(s) - kb kilobase pairs     

Plasmid recovery from transformants and the isolation of chromosomal DNA segments improving plasmid replication in Neurospora crassa

Summary The efficient recovery of plasmid DNA from Neurospora crassa transformants is described. Lithium acetate-treated spores were transformed with plasmid DNA and grown in mass in liquid culture. The resulting mycelial growth was harvested and plasmid DNA was extracted and used to transform E. coli to ampicillin resistance. Although at low frequency, routine recovery of plasmid pSD3 which carries the Neurospora qa-2 ⁺ gene and pBR322 sequences has been demonstrated. About 10% of the recovered plasmids carried deletions and transformed Neurospora at a higher frequency. The liquid culture procedure was also used in attempts to isolate autonomously replicating sequences (ars). In order to select for a stable vector which contains an ars sequence, a clone bank containing a selectable marker (qa-2 ⁺) and Neurospora chromosomal BamHI fragments was constructed and used to transform Neurospora. Several plasmid isolates resulting from a screening of the clone bank showed an improvement in the efficiency of recovery from Neurospora transformants. The properties of one such isolated plasmid, pJP102, suggest that it may contain an ars sequence. Some potential applications of these results for cloning in Neurospora and other filamentous fungi are discussed.     

The Location and Sequence Composition of the Murine Cytomegalovirus Replicator (oriLyt)

DNA replication during human or simian cytomegalovirus (CMV) infection has been shown to be under control of a replicator region referred to asoriLyt. The murine CMVoriLyt has been mapped to a region of the genome located upstream of the gene encoding the herpesvirus-conserved single-stranded DNA binding protein, analogous to human and simian CMVoriLyts. A minimaloriLyt of approximately 1.7 kbp has been identified using a transient replication system. Like occurs with human and simian CMV counterparts, addition of flanking sequences to this minimal origin-stimulated replication efficiency. Analysis of the DNA sequence in this region shows that murine CMVoriLyt is complex and exhibits an asymmetric distribution of nucleotides as well as many repeat sequence elements, including distinct AT- and GC-rich regions and region with arrays of closely spaced direct repeats. Despite similarities in organization of all three CMVoriLyts, no sequence identity and only limited DNA sequence similarity was detectable. Consistent with this sequence divergence, the human and murine CMVoriLyts were unable to substitute for one another in transient replication assays.     

Identification of an autonomously replicating sequence near a histone gene of Physarum polycephalum

Summary Fragments of DNA with function as autonomous replication sequences in yeast were cloned from Physarum polycephalum. The ars activity is located in a 1.2 kbp fragment extanding 1.5 kbp to 2.7 kbp upstream of the 5 end of a histone H4 gene. Our recent finding that a replication origin is located at a distance less than 3 kbp of this histone gene suggests that the ars element identified coincides with a specialized replication origin and can be used to direct chromosome replication in Physarum polycephalum.     

Polymorphic variations in the ori sequences from the mitochondrial genomes of different wild-type yeast strains

Summary We determined the restriction maps and primary structures of two as yet poorly characterized regions of the mitochondrial genomes of different wildtype strains of Saccharomyces cerevisiae. These regions respectively comprised the ori1 sequence and the newly identified ori8 sequence. Ori1 and ori8, together with their flanking sequences, exhibit a large polymorphism, resulting from specific variations due to insertions or deletions of optional GC clusters at different locations. The mechanisms underlying such sequence rearrangements are discussed.     

High frequency recombination and the expression of genes cloned on chimeric yeast plasmids: Identification of a fragment of 2-μm circle essential for transformation

Summary We have carried out experiments aimed at explaining the observed variations in transformation frequencies when Saccharomyces cerevisiae or Saccharomyces carlbergensis are transformed with chimeric plasmids that contain one of 4 possible EcoRI fragments of the yeast 2-m circle. These plasmids fall into 2 classes when used to transform 2 different yeast his3 auxotrophs, one (strain LL20) harbours indigenous 2-m circle, and the other (strain YF233) is devoid of this plasmid. Hybrid plasmids containing either the 2.4 mega-dalton (mD) R-form EcoRI fragment (pYF88) or the l.4 mD L-form EcoRI fragment (pYF177) of 2-m circle transform either of the two hosts at a high frequency (50,000 colonies per Mg in LL20 and 10,000 colonies per g in YF233). Hybrid plasmids containing the 1.5 mD R-form EcoRI fragment (pYF87) or the 2.5 mD L-form EcoRI fragment (pYF178) of the 2-m circle transform LL20 at a reduced frequency (6,000–16,000 colonies per g) and YF233 at extremely low frequencies (1–5 colonies per g). All plasmids retrieved from strain YF233 that had been transformed with pYF88 or pYF177 were identical to the original transforming plasmid. Of the plasmids retrieved from strain LL20 that had been transformed with pYF87 and pYF178, approximately half had acquired an extra copy of the 2-m circle. Of the plasmids retrieved from strain LL20 that had been transformed with pYF88 and pYF177, an average of only approximately 13% had acquired an extra copy of 2-m circle. Taken together, these observations indicate that the transformation of yeast by a plasmid lacking the ability to replicate (pYF87 and pYF1780) occurs by the recombinational acquisition of 1 copy of the host 2-m circle, which serves to supply the incoming plasmid with missing essential sequences. A comparison of 2-m circle DNA fragments carried by pYF88 and pYF177 indicates that the region of 2-m circle required for high frequency transformation is a 1.2 mD segment that is common to the 2.4 mD R-form and 1.4 ml) L-form EcoRI fragments. This region extends from the EcoRI cut site adjacent to the PstI site, through to the end of the inverted repeat. However, the inverted repeat sequence alone is not sufficient to bestow high frequency transformation of yeast.