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.     

Cryptic DNA plasmids of the heterothallic yeast Saccharomycopsis crataegensis

Summary Three DNA plasmids, designated pScrl-1, pScrl-2, and pScrl-3 have been found in a strain of the heterothallic yeast Saccharomycopsis crataegensis (NRRL Y-5902). pScrl-l, -2 and -3 are, respectively, 15, 7, and 5 kilobase pairs (kbp) in size. Based on the results of exonuclease digestions, all three plasmids appear to be linear molecules with blocked 5 ends. All three plasmids also have a lower buoyant density than does nuclear DNA of S. crataegensis. The two lower molecular weight plasmids hybridize strongly with one another, but only weakly to the higher molecular weight plasmid. Two of four related S. crataegensis strains surveyed were found to contain two plasmids that are of the same size as the two larger plasmids of Y-5902. Evidence is presented indicating that the plasmids in strain Y-5902 reside in the cytosol since they were found not to be located within the major organelles (mitochondria and nuclei).     

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.     

De-novo generation of mitochondrial DNA plasmids following cytoplasmic transmission of a degenerative disease in Ophiostoma novo-ulmi

A mitochondrial DNA plasmid was detected in an isolate of Ophiostoma novo-ulmi with a degenerative disease. The DNA plasmid was shown to be derived from the mitochondrial DNA and to map to a region corresponding to the large ribosomal RNA coding region. The DNA plasmid was not transmitted into sexual (ascospore) progeny, irrespective of whether the diseased isolate acted as the female or male parent. Transmission of the disease to healthy, plasmid-free, recipient isolates by hyphal anastomosis was not accompanied by transfer of mitochondrial DNA or DNA plasmid from the diseased donor isolate, but resulted in de-novo generation of different plasmids, derived from the recipient's mitochondrial DNA.     

Osmophilic linear plasmids from the salt-tolerant yeast Debaryomyces hansenii

Three novel linear plasmids, pDHL1 (8.4 kb), pDHL2 (9.2 kb) and pDHL3 (15.0 kb), were discovered in the halophilic (salt-tolerant) yeast Debaryomyces hansenii. Exonuclease treatment indicated that all three plasmids were blocked at their 5 ends, presumably, by analogy with most other eukaryotic linear plasmids which involved protein attachment. The Debaryomyces plasmids were entirely cured simply by growing cells in normal culture medium, but were stably maintained in culture medium containing salts, sorbitol or glycerol at suitable concentrations. This suggested that the pDHL plasmids required an osmotic pressure for stable replication and maintenance. The Debaryomyces yeast secreted a killer toxin against various yeasts species. Toxin activity was demonstrated only in the presence of salts such as NaCl or KCl, but this killer phenotype was not associated with the pDHL plasmids. Analysis of the plasmid-curing pattern suggested that pDHL3 may play a key role in the replication of the Debaryomyces plasmids. Southern hybridization showed that an extensive homology exists between specific regions of pDHL1 and pDHL2, whereas pDHL3 is unique.     

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     

Extrachromosomal plasmids in the plant pathogenic fungus Rhizoctonia solani

Extrachromosomal DNA elements were found in field isolates of Rhizoctonia solani belonging to anastomosis groups (AG) 1–5. An isolate of AG-5 (Rh41) contains a 3.6-kbp plasmid (pRS188) which has a similar A+T content to mitochondrial DNA. pRS188 is linear and has knob structures at its ends, as revealed by electron microscopy. Exonuclease digestions show that the linear ends of pRS188 are protected, and remain protected even after proteinase K digestion. pRS188 does not hybridise to nuclear or mitochondrial DNAs of its host isolate (Rh41), to total DNAs of other plasmid-less AG-5 isolates, or to total DNA of plasmid-harbouring isolates belonging to different AGs. Cellular-fractionation experiments suggest that pRS188 is associated with mitochondria, but it remains undecided whether this occurs inside or outside of the organelles. The nucleotide sequence of about 60% of the plasmid has been determined, revealing no open reading frame longer than 91 amino acids, and no known gene or genetic element is detected in the sequence contigs of 300–1572 bp length. Similar studies were performed with the plasmid pRS104 present in an isolate of AG-4 (Rh36), the sequence of which exhibits essentially the same features as pRS188 except that its A+T content resembles that of nuclear DNA. Pathogenicity tests reveal that the isolates Rh41 and R36 are as virulent as the plasmid-less isolates of AG-4 and-5, indicating that the plasmids do not play any role in pathogenicity.     

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.     

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.     

The linear mitochondrial plasmid pAL2-1 of a long-lived Podospora anserina mutant is an invertron encoding a DNA and RNA polymerase

The molecular characterization of an additional DNA species (pAL2-1) which was identified previously in a long-lived extrachromosomal mutant (AL2) of Podospora anserina revealed that this element is a mitochondrial linear plasmid. pAL2-1 is absent from the corresponding wild-type strain, has a size of 8395 bp and contains perfect long terminal inverted repeats (TIRs) of 975 bp. Exonuclease digestion experiments indicated that proteins are covalently bound at the 5 termini of the plasmid. Two long, non-overlapping open reading frames, ORF1 (3,594 bp) and ORF2 (2847 bp), have been identified, which are located on opposite strands and potentially encode a DNA and an RNA polymerase, respectively. The ORF1-encoded polypeptide contains three conserved regions which may be responsible for a 3–5 exonuclease activity and the typical consensus sequences for DNA polymerases of the D type. In addition, an amino-acid sequence motif (YSRLRT), recently shown to be conserved in terminal proteins from various bacteriophages, has been identified in the amino-terminal part of the putative protein. According to these properties, this first linear plasmid identified in P. anserina shares all characteristics with invertrons, a group of linear mobile genetic elements.     

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.     

Characterization of viral DNAs from cells infected with chicken anaemia agent: sequence analysis of the cloned replicative form and transfection capabilities of cloned genome fragments

Summary The viral DNAs induced by the unclassified animal virus, chicken anaemia agent (CAA), during replication in MDCC-MSB 1 cells have been investigated. Analyses after S₁ nuclease, restriction endonuclease and denaturation treatments indicated that infected cell extracts contained genome-size, single-stranded DNA (M_r 2.3 kb), closed and open circular, double-stranded replicative form (RF) DNAs (M_r 2.3 kbp) and a population of smaller doublestranded DNAs (M_r 0.8 kbp). Recombinant plasmids containing 2.3 kbp CAA RF fragments cloned at the PstI, BamHI and EcoRI sites failed to transfect MDCC-MSB 1 cells. However, one plasmid, which contained two 2.3 kbp CAA RF fragments ligated in tandem at the PstI site, and cloned 2.3 kbp PstI, BamHI and EcoRI fragments, excised from their respective plasmids by restriction endonuclease digestion, were capable of transfection.The nucleotide sequence of the circular genome (2298 bp) of the Cux-1 isolate of CAA has indicated the presence of three overlapping open reading frames (ORFs) of 52 kDa, 24 kDa and 13 kDa on one strand. The existence of these ORFs was corroborated by analyses of partial sequences from three other isolates. The non-coding region of the CAA genome contained sequences with putative regulatory function. These results are discussed in relation to the rolling circle model of DNA replication.     

Physical maps of the two circular plastid DNA molecules of the brown algaPylaiella littoralis (L.) Kjellm

Summary Two circular molecules of different sizes, both belonging to the chloroplast DNA of the brown algaPylaiella littoralis, have been observed by electron microscopy (Dalmon et al. 1983). Clone banks representing 86% of the small chloroplast circular DNA molecule (58 kbp) and 69% of the large circular DNA molecule (133 kbp) have been established and used as tools in the construction of physical maps. Two rDNA operons have been mapped in a very small inverted repeat on the large circular molecule. One 16S rRNA pseudogene and one split 23S rRNA gene have been mapped on the small DNA molecule, far apart from each other. Using heterologous probes, genes for ten different proteins have also been located on these maps. Their arrangement on the large molecule is different from that found in higher plants and algae. Probes fromrbcL, psbA andrps19 genes hybridize to several separated fragments. Two of them (psbA andrps19) hybridize to both types of molecules.     

UV hypersensitivity of yeast linear plasmids

The Kluyveromyces linear plasmids pGKL 1 and pGKL2, encoding killer activity, were efficiently cured by UV irradiation. This event was investigated in more detail by the use of the terminal protein (TP)-associated cytoplasmic linear plasmids, pJKL1 and pRKL2, with a selectable marker LEU2. This observation was compared with the UV effect on the nuclear plasmids pLS1 (telomere-associated linear form) and YCp121 (centromere-integrated circular form), indicating that the UV hypersensitivity was specific to the cytoplasmic plasmids. Using rad4 and wildtype strains of S. cerevisiae, both pJKL1 and the nuclear plasmids were found to respond not only to photoreactivation repair but also to excision repair of UV-induced DNA damage. Thus these DNA repair systems were functional for both the nuclear and cytoplasmic plasmids in yeast, and it was suggested that the UV hypersensitivity of cytoplasmic plasmids might have been caused by a defect in other repair systems or in the TP-primed replication. Possibly TP-associated Debaryomyces linear plasmids were also UV hypersensitive.This paper is dedicated to Prof. Dr. Dr. h.c. mult. Karl Esser on the occasion of the 70th anniversary (March 19, 1994) of his birthday     

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.     

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.     

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     

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.     

Distribution of seven homology groups of mitochondrial plasmids in Neurospora: evidence for widespread mobility between species in nature

A survey of mitochondrial DNAs from over 225 Neurospora and related fungal isolates from around the world uncovered three new homology groups of mitochondrial plasmids, two divergent subgroups of the Fiji plasmid family, and extended previous data about plasmid distribution patterns. Newly-discovered circular plasmids, Java and MB1, and the linear Moorea plasmids, were found in relatively-few isolates. A large proportion of isolates (51%) were found to have these or previously-discovered plasmids in the Varkud, kalilo, LaBelle, or Fiji families. Plasmids in most families were found in isolates world-wide and distributed nearly randomly with respect to species. As many as three types of plasmids were found in single isolates, and plasmids typically were found alone or in pairs in a random, independent pattern. The regional clustering of some plasmids was independent of species. providing a strong argument that horizontal transfer of plasmids occurs frequently in nature. Some plasmid families were much more diverse than others. The Fiji plasmids are a superfamily composed of distinct subgroups defined by degrees of cross-hybridization. Between some subgroups there were large regions of non-homology.     

DNA synthesis in purified maize mitochondria

Summary We have developed an in organelle DNA synthesis system using isolated mitochondria from maize. The organelles used in this assay are shown to be intact by a number of criteria. Both the high molecular weight components and the smaller plasmid-like components of the mitochondrial genome are used as templates; however, the plasmid-like elements are relatively more active as templates. The termini of the linear plasmids — S-1, S-2 and the 2.3 kbp plasmids — are more highly labelled than internal regions, probably as a result of filling in of gaps at the termini mediated by a DNA polymerase or to exonuclease degradation of the 3 OH termini, with subsequent filling in. Although most of the DNA synthesis observed in this system most likely results from this type of synthesis at DNA nicks or termini, a small amount of specific, potentially replication-associated, synthesis is also detected.