Killer toxin production in Pichia acaciae is associated with linear DNA plasmids

Summary We have identified a strain of the yeast Pichia acaciae which produces a killer toxin active against the yeast Debaryomyces tamarii. The killer phenotype was associated with the presence of two DNA plasmids, pPacl-1 (13.6 kilobase pairs) and pPacl-2 (7.3 kilobase pairs). P. acaciae strains, cured of these plasmids by irradiation with ultraviolet light, lacked killer activity and were sensitive to toxin produced by the parental strain. A partially cured strain, GS-1215, missing only the smaller plasmid, pPacl-2, also exhibited loss of both toxin activity and immunity. Exonuclease studies revealed that both plasmids were linear double-stranded DNA molecules with 5 protected ends. The P. acaciae system differs from that of the well-studied Kluyveromyces lactis killer system both in the range of susceptible strains and in the sizes of the plasmids involved. Our studies contradict previous reports that Pichia killer systems are invariably chromosomal.     

Linear DNA plasmids of Pichia inositovora are associated with a novel killer toxin activity

Summary Pichia inositovora, strain NRRL Y-18709, which contains three linear double-stranded DNA plasmids, pPinl-1, pPinl-2 and pPinl-3, was cured of these plasmids both by growing the strain in the presence of 50 g/ml bisbenzimide, and by exposure to ultraviolet light. Both cured and uncured strains were tested for growth on a variety of carbon sources. No differences in growth response were detected, indicating no discernible involvement of the linear plasmids in the catabolism of these compounds. Culture supernatants of Pichia inositovora were shown to contain a substance larger than 100 kDa that is toxic to Saccharomyces cerevisiae, strain GS 1688. Toxin activity was optimal in YEPD assay plates containing 50 mM citrate buffer with a pH between 3.4 and 4.2. Culture supernatants from P. inositovora were also weakly active against Cephaloascus albidus, strain NRRL Y-18710, and Citeromyces matritensis, strain NRRL Y-18711. Concentrated supernatants from cured P. inositovora strains did not exhibit these activities, consistent with the hypothesis that this toxic activity is linear plasmid-encoded. Unlike the wellknown Kluyveromyces lactis system or the newly identified P. acaciae system, P. inositovora strains cured of their linear plasmids do not become detectably sensitive to toxin produced by the wild-type strain, suggesting a nonplasmid-encoded immunity function.     

Extrachromosomal genetics of Claviceps purpurea

Summary Claviceps purpurea strain K1 contains several linear mitochondrial plasmids comparable to those of higher plants (Tudzynski et al. 1983). Screening of ten Claviceps wild strains from different geographic origin revealed the presence of similar plasmids in at least five strains, indicating a widespread occurrence of these genetic traits. Whereas in strain K1 plasmids have no homology to high-molecular-weight mtDNA, in two of the other wild strains (W3 and W7) sequences homologous to plasmids of strain K1 are integrated in the mt genomic DNA. Physical and genetic maps of strains W3, W7 and K1 were established. Despite completely different physical maps, the relative orientation of 6 mt genes is identical. A DNA sequence homologous to plasmids of strain K1 (termed ) was found to be integrated in both W3 and W7 at the same site: at the 5-part of the 1rRNA gene. A second region () was localized near the srRNA gene of W7 only. These findings are discussed with respect to possible transposon-like properties of the Claviceps purpurea plasmids.     

Proteins are attached to the ends of a linear plasmid in the filamentous fungus Ascobolus immersus

Summary In seven out of eleven wild strains of the Ascomycete Ascobolus immersus plasmid DNA was found. There was great variability with respect to size and number of the plasmids in the strains concerned. For a further analysis two plasmids originating from one wild strain were submitted to restriction analysis and electron microscopy. Both turned out to be linear having different molecular weights (pAIl = 7.9 kb, pAI2 = 5.6 kb). Denaturation of pAI2 and subsequent renaturation revealed the presence of inverted repeats (0.7 kb) at both ends. After treatment with proteinase K and 5 and 3 specific exonucleases it became evident that the 5 ends of pAI2 are linked with proteins. In this respect it is similar in structure to other linear genetic elements such as the linear plasmids found in Zea mays and the genomes of adenoviruses.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

Integrative and replicative genetic transformation of Aureobasidium pullulans

A hybrid selectable marker for transformation was constructed by placing the promoter (TEF1p) from the gene encoding the Aureobasidium pullulans translation elongation factor 1- (TEF1) adjacent to the 5 end of the Escherichia coli hygromycin B phosphotransferase gene (HPT). Plasmids containing this hybrid gene (TEF1p/HPT) transformed A. pullulans strain R106 to a hygromycin B-resistant (HmB^R) phenotype. A PCR-generated DNA fragment consisting of the TEF1p/HPT resistance marker flanked by 41 bp of homologous DNA has also been shown to transform A. pullulans to HmB^R. Linearized plasmid DNA consistently produced more transformants than circular plasmid DNA. Analyses of 23 HmB^R transformants revealed integration of the plasmid in only eight of these transformants. In two transformants, integration into the largest chromosome (VIII) resulted in an alteration of the molecular karyotype. In four other transformants, integration occurred in chromosome VI (the chromosome containing TEF1) but only one was the result of homologous recombination with the genomic copy of the TEF1 promoter. The remainder of the transformants contained replicative plasmids that could be visualized on an agarose gel by ethidium bromide staining. These plasmids were generally 7–8 kb in size. One transformant appeared to contain four plasmids ranging in size from 4 to 8 kb, suggesting rearrangement of the transforming DNA. One plasmid obtained from a HmB^R A. pullulans transformant was able to transform E. coli to ampicillin resistance. However, after recovery from E. coli, this plasmid (approximately 4 kb) was unable to transform A. pullulans to HmB^R.     

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.     

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     

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     

Analysis of a linear plasmid isolated from the pathogenic fungus Ceratocystis fimbriata Ell. & Halst.

Summary A linear DNA plasmid, designated pCF637, was isolated from the fungus Ceratocystis fimbriata Ell. & Halst. strain CF637. It has an apparent molecular weight of 5.3 × 10⁶ daltons (8.2 kb). A restriction pattern of pCF637 using the enzymes AvaI, EcoRV and HpaII, was done. That pCF637 was sensitive to exonuclease III, but resistant to -exonuclease, suggest that there might be a protein associated with the 5 termini. The blocking action of the protein on -exonuclease was not eliminated by treatment with either pronase or proteinase K. The plasmid was also checked for homology with pFQ501, a linear plasmid found in strain CF560 of Ceratocystis fimbriata. By Southern hybridization under moderate stringency conditions, no homology was detected. The approximate copy number of the plasmid was estimated to be about 20–30 copies per cell by scanning, with a laser densitometer, a gel electrophoretic Polaroid^TM negative. No function is known for these plasmids.     

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 abnormal cell division cycle in an AIR carboxylase-deficient mutant of the fission yeast Schizosaccharomyces pombe

Summary Adenine-requiring mutant strains of S. pombe enter the stationary phase after depleting a culture medium of adenine or its analogues. Stationary phase cells of six mutants defective at different stages of the purine nucleotide synthetic pathway were examined for cell volume and DNA content, and then compared in these respects with those of a prototrophic wild-type strain. The cell cycle of the wild-type strain was arrested in the G₂ phase (2C state) in the nitrogen rich medium, as is evident from DNA content per cell (0.0425 pg) and cell volume (47.7 m³). An AIR carboxylase-deficient (ade6) mutant strain was found to have an unusual cell volume (307.4 m³) and DNA content (0.1187 pg). By DAPI fluorescence microscopy, each mutant cell was seen to contain only one enlarged nucleus, which indicates the absence of cell populations containing cells in the 4C state of the S phase following nuclear division. It then follows that in ade6 mutant cells, DNA synthesis occurs in the absence of a completed nuclear division. Thus in S. pombe cells, the completion of nuclear division is not necessarily required for the next cycle initiation of DNA synthesis under certain physiological conditions.Abbreviation AIR aminoimidazole ribonucleotide - DAPI 4,6-diamidino-2-phenylindole - PCA perchloric acid - DABA 3,5-diaminobenzoic acid     

Linear,non-mitochondrial plasmids of Alternaria alternata

Summary Three plasmids, with sizes of 7.0 kbp, 6.8 kbp, and 5.0 kbp and designated pAal-1, pAal-2 and pAal-3 respectively, have been found in a tentoxin-producing isolate of Alternaria alternata. Exonuclease digestions show these plasmids to be linear with blocked 5 ends. Plasmid pAal-1 does not hybridize to nuclear DNA, mitochondrial DNA, or double-stranded RNA from a mycovirus found in the isolate, but does hybridize weakly to a series of linear DNAs which are not visible on gels and may include pAal-2 and pAal-3. Cellular fractionation shows that, unlike other linear fungal plasmids, these plasmids are not localized in the mitochondria. Plasmids have not been found in other tentoxin-producing isolates and there is no evidence that these plasmids have any effect on the production of tentoxin.     

Trehalose and maltose metabolism in yeast transformed by a MAL4 regulatory gene cloned from a constitutive donor strain

Summary A 6.8 kb fragment of DNA containing the regulatory sequence MAL4p has been cloned from a genomic library prepared from Saccharomyces cerevisiae strain 1403-7A which ferments maltose constitutively. The library was prepared by ligation of 5–20 kb Sau3AI restriction fragments of total yeast DNA into the BamH1 restriction site of shuttle vector YEp13. A restriction map of the cloned fragment indicates that it encompasses a 2.6 kb segment which closely resembles the regulatory MAL6 gene previously identified (Needleman et al. 1984). The hybrid plasmid, p(MAL4p)4, could transform maltose-nonfermenting strains which contain cryptic -glucosidase and maltose permease genes (malp MALg), but could not transform strains containing a functional regulatory sequence and a defective maltase-permease region (MAlp malg). A correlated absence of maltase and permease DNA from the cloned fragment was indicated by the restriction map. Although the cloned DNA fragment was derived from a constitutive strain, maltose fermentation and -glucosidase formation by yeast transformed with p(MAL4p)4 was largely inducible by maltose and sensitive to catabolite repression. Moreover, the active trehalose accumulation pattern (TAC(+) phenotype) linked to the complete MAL4 locus in strain 1403-7A and other constitutive MAL strains (Oliveira et al. 1981b) was not found in p(MAL4p)4 transformants. It may be concluded that constitutivity of maltose fermentation and the associated active trehalose accumulation are not merely consequences of a cis-dominant mutation causing constitutive formation of the MALp regulatory product. Moreover, constitutivity may not be caused solely by a mutation within the structural region of the MALp gene.     

Two Extramitochondrial Circular DNA Species in the Petite Negative Yeast Schizosaccharomyces pombe: Relative abundance and size determination by electron microscopy

Summary In this paper we present the electron microscopic analysis of two distinct extramitochondrial circular DNA species in the fission yeast Schizosaccharomyces pombe (S. pombe). Both DNA species can be isolated from mitochondrial fractions, but disappear after DNase treatment of mitochondria, demonstrating their extramitochondrial location. The size of these molecular species is 3.08 ± 0.18 m and 2.00 ± 0.09 m (standard deviation). They are present in a ratio of approximately 9:1 in the DNA preparations analyzed.     

Extrachromosomal genetics of Cephalosporium acremonium

Summary In order to facilitate strain improvement by concerted breeding in the cephalosporin producing imperfect filamentous fungus Cephalo sporium acremonium, it is attempted to develop a eukaryotic vector for molecular cloning based on mitochondrial (mt) DNA.Fragments of mtDNA from C. acremonium were inserted into a yeast/bacterial hybrid plasmid (pDAM1) lacking a eukaryotic replicon. Six hybrid plasmids were obtained each containing a different mt fragment which together comprise about 60% of the total mtDNA. One of these recombinant plasmid vectors (pCP2) showed a high replication efficiency which is comparable to that of vectors containing yeast 2 m DNA. This plasmid therefore fulfills the requirements for practical application.     

Localization of three chloroplast ribosomal protein genes at the left junction of the large single copy region and the inverted repeat of Spirodela oligorhiza chloroplast DNA

Summary In order to determine the localization of ribosomal protein genes on the chloroplast genome of Spirodela, we have followed two different approaches: First, antisera were prepared against purified 30S, 50S and 70S chloroplast ribosomal proteins from Spinacia. These antisera react with about two third of the chloroplast ribosomal proteins as shown by protein blot and immunoprecipitation experiments. Recombinant plasmids carrying the Spirodela BamHI-G or PstI-I cpDNA fragment both direct the synthesis of a 15 kD chloroplast ribosomal protein in a DNA dependent E. coli cell free system. This was confirmed by molecular weight determination, immunoprecipitation and competition immunoprecipitation experiments. Second, heterologous hybridization with the rps19 gene probe from Nicotiana revealed the localization of this gene on the chloroplast DNA of Spirodela within the BamHI-G fragment at the left junction of the large single copy region and the inverted repeat. Furthermore we show that the recombinant plasmid carrying Nicotiana rps19 also directs the synthesis of another chloroplast ribosomal protein in an E. coli cell free system. The identity of this protein is discussed.Abbreviations Bis-Tris 2,2(Bis(hydroxymethyl)2,2,2 nitrilotriethanol - (k)bp (kilo)base pairs - BPB bromphenol blue - cp chloroplast - IR inverted repeat - kD kilo dalton - LSCR large single copy region - Mw molecular weight - r-protein ribosomal protein - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodiumdodecyl sulphate     

Sed1p interacts with Arn3p physically and mediates ferrioxamine B uptake in Saccharomyces cerevisiae

Two-hybrid analysis can be used to study protein function and metabolic pathways. Using yeast two-hybrid analysis to identify a siderophore uptake pathway in the yeast Saccharomyces cerevisiae, we found that the C-terminal part of the cell-wall protein Sed1p interacts with the N-terminal region of Arn3p. To confirm the physical interaction between the Sed1p C-terminal fragment and the hydrophilic N-terminal fragment of Arn3p, we used an in vitro co-immunoprecipitation assay and a growth test of the strain with bait and SED1 plasmids in quadruple amino acid-depleted medium. The expression of SED1 was upregulated by overexpression of AFT1-1^up under the control of the GAL promoter. This occurred despite the lack of an Aft1p-binding consensus region on the upstream region of SED1 or a high concentration of free iron. Although free-iron uptake activity in the sed1 strain did not differ from that in the parental strain, ferrioxamine bound-iron uptake activity was reduced in the sed1 strain. Moreover, the sed1 strain showed low viability at high iron concentrations. Taken together, these results suggest that Sed1p mediates siderophore transport and confers iron resistance in S. cerevisiae.     

R-type plasmids in mitochondria from a single source of Zea luxurians teosinte

Two linear DNA plasmids resembling the R1 and R2 plasmids that are present in the mitochondria of several South American strains of maize were found in mitochondria from a single source of Zea luxurians collected by L. Mazoti. The Mazoti mtDNA is closely related to mtDNAs of other Z. luxurians, but mitochondria derived from the other Z. luxurians sources lack the plasmids. The larger plasmid from Mazoti mitochondria, M1, was cloned and large portions of it were sequenced. Restriction mapping and sequence comparisons showed that approximately 4.9 kb is similar to the S1 plasmid of maize and an additional 2.6 kb is related to R1 sequences integrated into the main mitochondrial genome of N cytoplasm. Therefore, the M1 plasmid appears to be very similar to the R1 plasmid. The inverted repeats at the ends of the M1 plasmid are not identitical. The left end IR is similar to the S-TIRs found at the termini of the S plasmids. The right end IR more closely resembles the integrated R1 sequences, including the variant region of the TIR. Whereas the variant region contains 13 bp in the S-TIRs and 15 bp in an integrated version of R1, it is 16 bp long in M1. The region of M1 that has no homology to the S1 plasmid is expressed at very low levels in Mazoti and RU cytoplasms, but at much higher levels in CMS-S mitochondria, where part of it is present in the main mitochondrial genome.     

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.