Cloned mitochondrial DNA from the zygomycete Absidia glauca promotes autonomous replication in Saccharomyces cerevisiae

Summary We have cloned fragments from mitochondrial and chromosomal DNA of the zygomycete Absidia glauca in Saccharomyces cerevisiae using the ARS selection vector YIp5. Though it has not been possible to select ARS elements from chromosomal DNA, we succeeded in isolating two clones of mitochondrial origin that support autonomous replication in bakers' yeast. DNA from these plasmids has been shown to hybridize with mitochondrial DNA from both mating types. Generation times of the transformed yeast strain in selective medium are around 20 h. In liquid minimal medium only 6% of the cells contain the plasmid; in complete medium a mitotic stability of 50% has been determined.     

A nucleotide sequence involved in replicative transformation of a filamentous fungus

Replicative plasmids generated through in-vivo recombination have been identified among transformants of the fungus Pleurotus ostreatus. In addition to sequences from a standard selection vector (pAN7-1), these recombinant plasmids contain recombined sequences of chromosomal origin conferring replicative potential upon the vector. One such recombined sequence, an 1148-bp insert into plasmid pP01, has been characterized. This sequence has been analyzed for secondary structural features as well as for consensus sites affiliated with origins of replication (ori) in other eukaryotic systems. The 1148-bp insert lacks an ORF and does not contain an acceptable match to the commonly identified 11-bp ars consensus sequence (A/TTTTATA/GTTTA/T) for autonomous replication in the yeast Saccharomyces cerevisiae. The analysis, however, revealed a cluster of three hairpin-loop-forming subsequences with individual G_25°C free energy values of-7.6,-6.4 and-5.2 kcal mol^-1. Also found were two 7-bp analogues to centromere-affiliated sequences recognized in other fungi, as well as several putative gyrase recognition sites comparable to the 9-bp S. cerevisiae/E. coli gyrase-binding consensus sequence. Sequences comparable to the ori of the yeast 2-m plasmid or to various sequences associated with ori of yeast/fungal mitochondrial DNAs (mtDNA) were not present in the 1148-bp insert. Replication of pP01 appears rather to involve a replication of chromosomal derivation devoid of an ars-type consensus.     

Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha

Summary For the transformation of the yeast Hansenula polymorpha we have constructed a set of hybrid plasmids carrying the LEU2 gene of Saccharomyces cerevisiae as a selective marker and fragments of mitochondrial DNA of Candida utilis and H. polymorpha or chromosomal DNA fragments of H. polymorpha as replicator sequences. The replication properties of chimeric plasmids in the yeast H. polymorpha were investigated. We showed that for plasmids propagated autonomously in this yeast the plasmid monomers could be detected in the transformants only during the immediate time after the transformation event. Further growth under selective conditions led to the selection of polymeric forms of plasmid DNA as it was clearly shown for transformants carrying cosmid pL2 with mtDNA fragment of C. utilis. Such transformants carrying polymerized plasmids showed a remarkably increased stability of the transformed phenotype. Cosmid pL2 was able to shuttle between Escherichia coli, S. cerevisiae and H. polymorpha, whereas plasmids with DNA fragments from H. polymorpha did not transform S. cerevisiae effectively.     

Titration of replication activity by increasing ARS dosage in yeast plasmids

The rep1 region of the yeast mitochondrial genome, a putative replication origin, contains a weak autonomously replicating sequence (ARS). Nucleotidesequence and deletion analyses have identified two 11-base pair ARS consensus sequences, numerous near matches to the ARS core, and a region of curvature that may contribute to ARS function. Based on the amplified nature of petite-derivative mitochondrial DNA encompassing this locus, we have constructed plasmids containing an increasing dosage of ARS elements. The rep1 ARS element can have an additive effect on plasmid stability when present either as a tandem dimer or as an unlinked pair. However, the presence of a third ARS copy does not further enhance plasmid stability. These results indicate that measurable dosage effects can be defined only in circumstances where weak ARS elements are employed, and that plasmid maintenance within yeast cells is saturable and varies among the different sequences promoting replication.     

Chloroplast origins of DNA replication are distinct from chloroplast ARS sequences in two green algae

Summary A 5.3 kb chloroplast restriction fragment of Chlamydomonas reinhardii containing an origin of DNA replication and a sequence capable of promoting autonomous replication in C. reinhardii (ARC sequence) also carries an ARS sequence (autonomous replication in yeast). The ARC and ARS elements have been physically mapped and shown to be distinct from the origin of DNA replication. Similarly, restriction fragments containing the origin of chloroplast DNA replication from Euglena gracilis are unable to promote autonomous replication in yeast.     

An improved host-vector system for Candida maltosa using a gene isolated from its genome that complements the hiss mutation of Saccharomvces cerevisiae

Summary The host-vector system of an n-lkaneassimilating-yeast, Candida maltosa, which we previously constructed using an autonomously replicating sequence (ARS) region isolated from the genome of this yeast, utilizes C. maltosa J288 (leu2 ^–) as a host. As this host had a serious growth defect on n-alkane, we developed an improved host-vector system using C. maltosa CHI (his) as host. The vectors were constructed with the Candida ARS region and a DNA fragment isolated from the genome of C. maltosa. Since this DNA fragment could complement histidine auxotrophy of both C. maltosa CH1 and S. cerevisiae (hiss ^–), we termed the gene contained in this DNA fragment C-HIS5. The vectors were characterized in terms of transformation frequency and stability, and the nucleotide sequence of C-HISS was determined. The deduced amino acid sequence (389 residues) shared 51% homology with that of HISS of S. cerevisiae (384 residues; Nishiwaki et al. 1987).     

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     

Comparison of Tetrahymena ARS sequence function in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe

Summary We have isolated several Tetrahymena thermophila chromosomal DNA fragments which function as autonomously replicating sequences (ARS) in the heterologous Saccharomyces cerevisiae and Schizosaccharomyces pombe selection systems. The Tetrahymena ARS sequences were first isolated in S. cerevisiae and were derived from non-ribosomal micro- and macronuclear DNA. Sequence analysis of the ARS elements identified either perfect or close matches with the 11 by S. cerevisiae ARS core consensus sequence. Subcloning studies of two Tetrahymena ARS elements defined functional regions ranging in size from 50 to 300 bp. Testing of the ARS elements in S. pombe revealed that most of the T. thermophila inserts confer ARS function in both yeasts, at least in the sense of promoting a high transformation frequency to plasmids which contain them. However, the actual sequences responsible for ARS activity were not always identical in the two yeasts.     

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.     

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.     

Transformation of Schizosaccharomyces pombe by non-homologous,unstable integration of plasmids in the genome

Summary In the fission yeast, Schizosaccharomyces pombe, transformation with recombinant plasmids always results in a high proportion of mitotically unstable transformants. This suggested that specialised (ARS) sequences might not be required for autonomous replication of plasmids in S. pombe, contrary to the situation in Saccharomyces cerevisiae. We have shown that specialised ARS sequences, analogous to those in S. cerevisiae, do exist in S. pombe, supporting the view that ARS elements are a general feature of eukaryotes. In addition, there is a further mechanism of plasmid maintenance which involves homologous and non-homologous integration into, and excision from the genome.     

Inhibition of DNA replication in Saccharomyces cerevisiae by araCMP

Summary Cytosine arabinoside (araC), a potent inhibitor of DNA replication in mammalian cells, was found to be completely ineffective in Saccharomyces cerevisiae. The 5 monophosphate derivative, araCMP, is toxic and effectively inhibits both nuclear and mitochondrial DNA synthesis in this organism. Although wild-type strains can be inhibited by araCMP, dTMP permeable (tup ^-) strains were found to be much more sensitive to the analogue. In vivo labelling experiments indicate that araC enters yeast cells; however, it is extensively catabolized by deamination and breakage of the glycosidic bond. In addition, the analogue is not efficiently phosphorylated in S. cerevisiae owing to an apparent lack of deoxynucleoside kinase activity. These results provide further evidence that deoxyribonucleotides can be synthesized only through de novo pathways in this organism. Finally, araCMP was found to be recombinagenic in S. cerevisiae which suggests, together with other previous studies, that, in general, inhibition of DNA synthesis in yeast promotes mitotic recombination events.     

Isolation of the TRP2 and the TRP3 genes of Saccharomyces cerevisiae by functional complementation in yeast

Summary This paper describes the isolation of the TRP2 and the TRP3 genes of Saccharomyces cerevisiae. Two pools of plasmids consisting of BamHI and Sa1GI yeast DNA inserts into the bifunctional yeast — Escherichia coli vector pLC544 (Kingsman et al. 1979) were constructed in E. coli and used for the isolation of the two genes by selection for functional complementation of trp2 and trp3 mutations, respectively, in yeast.The TRP2 gene was isolated on a 6.2 kb BamHl and a 5.8 kb Sa1GI yeast DNA fragment which shared an identical 4.5 kb BamHI-SaIGI fragment. The TRP3 gene was located on a 5.2 kb BamHl fragment.By physical, genetic and physiological experiments it could be shown that the cloned yeast DNA fragments contained the whole structural sequences as well as the regulatory regions of the TRP2 and the TRP3 genes.     

Behaviour of a replicating mitochondrial DNA sequence fromAspergillus amstelodami inSaccharomyces cerevisiae andAspergillus nidulans

Summary An amplified sequence of mitochondrial DNA from a ragged (rgd) mutant ofAspergillus amstelodami has been shown to exist in multimeric circular form, suggesting that it is excised from the genome and can exist independently of it. This sequence has replicative (ARS) activity inSaccharomyces cerevisiae, and a subfragment responsible for this activity has been identified and sequenced. A homologous sequence fromAspergillus nidulans mtDNA also has ARS activity inS. cerevisiae. BothA. amstelodami andA. nidulans ARS elements have been incorporated into the integrative transformation vector pDJBI and the derived vectors used to transformA. nidulans. Inclusion of theA. nidulans ARS element enhanced the transformation frequency 5-fold relative to pDJBI. No increase in transformation frequency was evident with the ARS element fromA. amstelodami. The stability of transformants was variable but in comparison to pDJBI, ARS-containing plasmids were mitotically unstable inA. nidulans. Although plasmid DNAs could be rescued inEscherichia coli from undigested transformant DNA, no freely replicating plasmids were detected by Southern hybridisation.     

Structure, replication efficiency and fragility of yeast ARS elements

DNA replication in eukaryotes initiates at specific sites known as origins of replication, or replicators. These replication origins occur throughout the genome, though the propensity of their occurrence depends on the type of organism. In eukaryotes, zones of initiation of replication spanning from about 100 to 50,000 base pairs have been reported. The characteristics of eukaryotic replication origins are best understood in the budding yeast Saccharomyces cerevisiae, where some autonomously replicating sequences, or ARS elements, confer origin activity. ARS elements are short DNA sequences of a few hundred base pairs, identified by their efficiency at initiating a replication event when cloned in a plasmid. ARS elements, although structurally diverse, maintain a basic structure composed of three domains, A, B and C. Domain A is comprised of a consensus sequence designated ACS (ARS consensus sequence), while the B domain has the DNA unwinding element and the C domain is important for DNA-protein interactions. Although there are ∼400 ARS elements in the yeast genome, not all of them are active origins of replication. Different groups within the genus Saccharomyces have ARS elements as components of replication origin. The present paper provides a comprehensive review of various aspects of ARSs, starting from their structural conservation to sequence thermodynamics. All significant and conserved functional sequence motifs within different types of ARS elements have been extensively described. Issues like silencing at ARSs, their inherent fragility and factors governing their replication efficiency have also been addressed. Progress in understanding crucial components associated with the replication machinery and timing at these ARS elements is discussed in the section entitled “The replicon revisited”.     

Use of the polymerase chain reaction to identify coding sequences for chitin synthase isozymes in Phialophora verrucosa

Based on conserved amino-acid regions predicted for the chitin synthases (Chs) of Saccharomyces cerevisiae, two different primer sets were synthesized and used in polymerase chain reactions (PCRs) to amplify 614-bp and 366-bp sequences from genomic DNA of the zoopathogenic fungus Phialophora verrucosa. DNA-sequencing and Southern-blotting analyses of the 614-bp DNA amplification products suggested that portions of two distinct P. verrucosa chitin synthase genes (PvCHS1, PvCHS2), coding for two different zymogenic-type PvChs isozymes, had been identified. The deduced amino-acid sequence of each fell into different Chs classes, namely class I and class II. In addition, the 366-bp DNA segment was shown to code for a conserved region having homology with the CSD2/CAL1 gene of S. cerevisiae, which encodes a nonzymogenic-type enzyme, Chs3, in that fungus. The amino-acid sequence derived from PvCHS3 exhibits 88.2% similarity and 78.4% identity to the same amino-acid region of the S. cerevisiae enzyme. These results provide a critical first step toward investigating the molecular and pathogenic importance of CHS gene regulation in this fungus and for exploring steps leading to Chs function as potential targets for the design of new therapeutic agents.     

Isolation and structure of an acetolactate synthase gene from Schizosaccharomyces pombe and complementation of the ilv2 mutation in Saccharomyces cerevisiae

A gene encoding a functional acetolactate synthase (ALS) subunit has been isolated from the fission yeast Schizosaccharomyces pombe, and has been structurally and genetically characterized. The approximate 5-kbp cloned DNA segment was found to contain a 2007-bp open reading frame capable of encoding a 669 aminoacid polypeptide which exhibited 57.1% similarity to the corresponding ALS subunit from Saccharomyces cerevisiae. The putative ilv1 isolated from S. pombe was shown to encode a functional subunit of acetolactate synthase by complementation of an S. cerevisiae strain deleted for the ILV2 locus.     

Heterologous gene expression of the glyphosate resistance marker and its application in yeast transformation

Summary The E. coli aroA gene was inserted between yeast promoter and terminator sequences in different shuttle expression plasmids and found to confer enhanced EPSP synthase activity as well as resistance to glyphosate toxicity. Subsequently, a transformation system using these newly constructed vectors in yeast was characterized. The efficiency of the glyphosate resistance marker for transformation and selection with plasmid pHR6/20-1 in S. cerevisiae laboratory strain SHY2 was found to be relatively high when compared with selection for LEU2 prototrophy. The fate of the recombinant plasmid pHR6/20-1 in the transformants, the preservation of the aroA E. coli DNA fragment in yeast, mitotic stability, EPSP synthase activity, and growth on glyphosate-containing medium have been investigated. As this plasmid also allows direct selection for glyphosate resistant transformants on rich media, the glyphosate resistance marker was used for transforming both S. cerevisiae laboratory strain SHY2 and brewer's yeast strains S. cerevisiae var. uvarum BHS5 and BHS2. In all cases, the vector pHR6/20-1 was maintained as an autonomously replicating plasmid. The resistance marker is, therefore, suitable for transforming genetically unlabeled S. cerevisiae laboratory, wild, and industrial yeast strains.Abbreviations EPSP 5-enolpyruvylshikimate 3-phosphate