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.     

Structural and functional analysis of the origin of replication of mitochondrial DNA from Paramecium aurelia

Summary Replication of mitochondrial DNA in Paramecium aurelia involves the formation of a covalent crosslink at one end of this linear molecule and proceeds unidirectionally, producing a dimer consisting of two head to head monomers. The initiation regions within the dimer molecules have been sequenced and shown to be palindromic except for a central nonpalindromic A+T rich sequence, arranged in direct tandem repeats. This nonpalindromic region (see accompanying paper) has been identified as the cross-link which converts the initiation terminus into a continuous sequence. In this study, yeast transformation was used to assay the dimer initiation regions of P. aurelia mtDNA for the presence of autonomously replicating sequences. P. aurelia mtDNA fragments from species 1 and 4 were cloned into the yeast vector YIP5 and the hybrid plasmids (YPaM) were used to transform yeast. The dimer initiation regions from both species promoted high frequency transformation and extrachromosomal maintenance of YPaM plasmids. Subcloning analysis of the ARS-containing mtDNA fragments indicates, specifically, that the nonpalindrome, repetitive sequences are responsible for the autonomously replicating properties of YPaM plasmids.     

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.     

Autonomously replicating sequences in young and senescent mitochondrial DNA from Podospora anserina

Summary Senescence in the filamentous fungus Podospora anserina is characterized by the accumulation of multimeric circular mitochondrial DNA molecules, known as senDNAs. These tandemly repeated DNA sequences, which originate from broadly dispersed regions of the young mitochondrial genome, behave as independently replicating molecules. In this study, the yeast transformation system was used to assay senDNAs and their young mtDNA counterparts for the presence of autonomously replicating sequences. P. anserina mtDNA fragments were cloned into the yeast vector YIp5 and the hybrid YPM plasmids were used to transform yeast. All of the senDNAs and their homologous young mtDNAs promoted high frequency transformation and extrachromosomal maintenance of YPM plasmids. The putative origin of replication for the P. anserina mitochondrial genome was also cloned into YIp5 and shown to confer autonomously replicating properties.     

Integrative transformation of a zygomycete,Absidia glauca,with vectors containing repetitive DNA

Summary Experimental evidence for integration of transformed DNA into the genome of Absidia glauca, a member of the fungal class of zygomycetes is presented. According to the limited knowledge on the molecular biology of these fungi, autonomous replication of transformed plasmids seems to be the preferential mode of DNA propagation. By inserting fragments of highly repetitive DNA elements into an autonomously replicating vector conferring neomycin resistance, we were able to obtain integrative transformation events. With such plasmids we observed stable mitotic propagation of a selective marker gene (NPT II under the control of a homologous actin promoter). Analysis of DNA from transformants in Southern type experiments, as well as restriction analysis of retransformants into Escherichia coli, provide evidence that integration of foreign DNA into the genome of Absidia glauca is possible. These transformation events are often associated with the appearance of mutant phenotypes.     

Integrative transformation of the yeast Yarrowia lipolytica

Summary An EcoR1 shotgun of Yarrowia lipolytica DNA was inserted into the plasmid YIp333 which carries the LYS2 gene of S. cerevisiae. The resulting plasmid pool was transformed in both S. cerevisiae and Y. lipolytica. Whereas numerous replicating plasmids could be isolated from the S. cerevisiae Lys⁺ transformants, all transformants of Y. lipolytica so far analyzed were found to result from integrative transformation. This occurred at a frequency of 1 to 10 transformants per g of input DNA. Co-transformation occurred at high frequency and resulted in tandem integration of 2 to 10 copies of the incoming DNA. Structural and segregational stability of the transforming DNA were both high.     

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).     

Plasmids of mitochondrial origin in senescent mycelia of Podospora curvicolla

Summary Podospora curvicolla displays symptoms of senescence similar but not quite identical to those reported for Podospora anserina. In Podospora curvicolla single hyphae may escape from death leading to a new growth front and consequently to a mode of growth characterized by alternating phases of growth and non-growth. Restriction analyses and hybridization experiments have revealed that the Podospora curvicolla type of senescence is correlated with plasmids originating from amplification of a single distinct region of the mitochondrial DNA containing the IrRNA gene. In the yeast transformation system sequences of this region may function as autonomously replicating sequences (ARS). Plasmids (pl1, pl2 and pl3) isolated from different, independently aged mycelia are largely homologous to each other but differ in their excision/junction sites and have different sizes: 10.85 kb (pl1), 9.01 kb (p12) and 10.50 kb (pl3). The sequence of the most frequently occurring plasmid in ageing strains of Podospora anserina is absent in Podospora curvicolla either as free plasmid DNA or as an integrated part of the mtDNA. Possibly there is a correlation between the absence of this particular sequence in Podospora curvicolla and the type of senescence displayed in this organism.     

A high-frequency transformation system for the yeast Kluyveromyces lactis

Summary This communication describes conditions for Kluyveromyces lactis protoplast regeneration and transformation and shows that a high frequency of transformation can be obtained with recombinant plasmids containing one of a series of K. lactis DNA fragments that presumably carry autonomously replicating sequences (called KARS). The vector YRp7, which contains a Saccharomyces cerevisiae autonomously replicating sequence (ARS), only led to integrative transformation of K. lactis indicating substantial differences in specificity between the DNA replication mechanisms of both yeast species. It is further shown that 2-m DNA derived vectors giving high frequency transformation in S. cerevisiae can transform K. lactis only with low frequency.     

Disruption of the cyclosporin synthetase gene of Tolypocladium niveum

Cyclosporin A is a potent and clinically-important immunosuppressive drug (Sandimmun^R). It is produced by the fungus Tolypocladium niveum. A transformation system for T. niveum ATCC34921 based on hygromycin selection was established. In order to obtain a T. niveum promoter, the cyclophilin gene was isolated using the Neurospora crassa gene as probe. A plasmid vector was constructed in which the promoter region of the T. niveum cyclophilin gene was fused to a bacterial hygromycin phosphotransferase gene. Protoplasts were transformed with this plasmid and hygromycin-resistant transformants were isolated. Using this transformation system, mutants of T. niveum with disrupted versions of the cyclosporin synthetase gene (simA) were engineered by DNA-mediated transformation. Disruption of the gene resulted in loss of the ability to produce cyclosporins.     

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.     

Mitochondrial DNA of the filamentous ascomycete Cochliobolus heterostrophus

Summary The mitochondrial chromosome of Cochliobolus heterostrophus is a circle approximately 115 kb in circumference, among the largest known from fungi. A physical map of C. heterostrophus mtDNA was constructed using the restriction enzymes BamHI, EcoRI, and PvulI by DNA-DNA hybridizations with cloned or purified mtDNA BamHI fragments. The following sequences were located on the mtDNA map: (1) the large and small mitochondrial ribosomal RNA genes (identified by heterologous hybridization to cloned Neurospora crassa rRNA genes); (2) the sequence homologous to a mitochondrial plasmid present in one field isolate of C. heterostrophus; and (3) a 1.05 kb EcoRI fragment that functions as an autonomously replicating sequence in Saccharomyces cerevisiae. An examination of mtDNA from 23 isolates of C. heterostrophus collected worldwide revealed polymorphisms in restriction enzyme sites. One such polymorphism, coupled with data on a polymorphism in nuclear rDNA, suggests that there are two genetically distinct but geographically overlapping mating populations of C. heterostrophus in the world.     

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     

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     

Transformation of Aspergillus niger using the homologous orotidine-5′-phosphate-decarboxylase gene

Summary A homologous transformation system for the filamentous fungus Aspergillus niger has been developed, based on the orotidine-5-phosphate-decarboxylase gene. A. niger Pyr^– mutants have been selected from 5-fluoroorotic acid resistant mutants. These mutants were found to comprise two complementation groups, pyrA and pyrB. The A. niger OMP-decarboxylase gene was isolated from a gene library by heterologous hybridization with the Neurospora crassa pyr4 gene. The cloned gene is capable to transform A. nidulans pyrG mutants at high frequencies. Transformation of A. niger pyrA mutants occurs with moderate frequencies (about 50 transformants/g DNA) whereas the pyrB mutants cannot be complemented with the cloned OMP-decarboxylase gene. Analysis of the DNA of the A. niger PyrA⁺ transformants showed that transformation resulted in integration of the vector DNA into the genome by homologous recombination. Both gene replacements and integration of one or more copies of the complete vector have been observed.     

Identification and characterization of CEN12 in the budding yeast Saccharomyces cerevisiae

In this paper we report the cloning, sequencing and functional characterization of CEN12 and an associated autonomously replicating sequence (ARS) from the budding yeast Saccharomyces cerevisiae. In the course of studying a dynamin-related gene, DNM1, we previously physically mapped the gene to chromosome 12. Genetic mapping showed that the gene was tightly linked (0.35 cM) to the centromere. Subcloning experiments revealed that a centromerelike activity was included in a small segment of DNA immediately downstream from the DNM1 gene. Mitotic centromere activity was discerned by the ability of the region to de-stabilize a centromere-containing plasmid, and to stabilize an ARS-containing plasmid. Meiotic centromere activity was determined by the first-division segregation in crosses of ARS plasmids containing this region. The DNA sequence of this region revealed a sequence with strong homology to the consensus for yeast centromeres.     

Transformation ofNeurospora crassa with circular and linear DNA and analysis of the fate of the transforming DNA

Summary We have conducted a detailed study of 108qa-2 ⁺ Neurospora transformants which were obtained by use of circular plasmid DNAs and various linear DNAs. Parallel genetic and molecular analyses have revealed that three classes of transformants can be identified: linked transformants, in which theqa-2 gene has integrated at the resident locus, unlinked transformants, where integration has occurred at other genomic sites, and a third class designated non-transmissible which fail to pass theqa-2 gene through a cross. The non-transmissible class comprises the majority of transformants and may identify those which harbor autonomously replicating plasmids. Evidence is presented which suggests that a 1.2 kB BamHI-Bg1IIqa-2 ⁺ DNA fragment might possess anars sequence. Transformation with linear plasmid DNAs and DNA fragments carrying theqa-2 gene resulted in a demonstrable increase in transformation frequency beyond that achieved with circular plasmid DNAs, but did not permit precise targeting to the resident locus. Southern analysis showed that linked transformants have only the normal residentqa-2 band whereas the unlinked transformants always possess the resident band plus at least one additional band. Multiple integration events appear to be common and include cases where only a portion of the transforming DNA has been integrated.     

Transformation of Trichoderma viride using the Neurospora crassa pyr4 gene and its use in the expression of a Taka-amylase A gene from Aspergillus oryzae

Summary A pyrG mutant of Trichoderma viride, a very efficient cellulase producer, was isolated from among 5-fluoroorotic acid-resistant mutants. The mutation was complemented with the pyr4 gene of Neurospora crassa and used as a selection marker for the transformation of T. viride. A plasmid vector, pDJB1-Taa, carrying both the pyr4 gene and a gene encoding Taka-amylase A from Aspergillus oryzae, was constructed and introduced into protoplasts of T. viride pyrG^-. The transformation frequency was 1–10 transformants (3 on average) per g DNA. One transformant showed highly elevated -amylase production (about 17 times higher than the recipient level) and the integration of more than one copy of the Taka-amylase gene.     

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.     

Repair of ultraviolet light damage in Saccharomyces cerevisiae as studied with double- and single-stranded incoming DNAs

Summary Purified double- and single-stranded DNAs of the autonomously replicating vector M13RK9-T were irradiated with ultraviolet light (UV) in vitro and introduced into competent whole cells of Saccharomyces cerevisiae. Incoming double-stranded DNA was more sensitive to UV in excision repair-deficient rad2-1 cells than in proficient repair RAD ⁺ cells, while single-stranded DNA exhibited high sensitivity in both host cells. The results indicate that in yeast there is no effective rescue of UV-incoming single-stranded DNA by excision repair or other constitutive dark repair processes.