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.     

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     

A new senescence-inducing mitochondrial linear plasmid in field-isolated Neurospora crassa strains from India

Summary Several field-collected strains of Neurospora crassa from the vicinity or Aarey, Bombay, India, are prone to precocious senescence and death. Analysis of one strain, Aarely-1e, demonstrated that the genetic determinants for the predisposition to senescence are maternally inherited. The senescence-prone strains contain a 7-kb, linear, mitochondrial DNA plasmid, maranhar, which is not present in long-lived isolates from the same geographical location. The maranhar plasmid has inverted terminal repeats with protein covalently bound at the 5 termini. Molecular hybridization experiments have demonstrated no substantial DNA sequence homology between the plasmid and the normal mitochondrial (mtDNA) and nuclear genomes of long-lived strains of N. crassa. Integrated maranhar sequences were detected in the mtDNAs of two cultures derived from Aarey-1e, and mtDNAs with the insertion sequences accumulated during subculturing. Nucleotide sequence analysis of cloned fragments of the two insertion sequences demonstrates that that they are flanked by long inverted repeats of mtDNA. The senescence syndrome of the maranhar strains, and the mode of integration of the plasmid, are reminiscent of those seen in the kalilo strains of N. intermedia. Nonetheless, there is no detectable nucleotide sequence homology between the maranhar and kalilo plasmids.     

Genetic organization and structural features of maranhar,a senescence-inducing linear mitochondrial plasmid of Neurospora crassa

Summary The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa, was determined. The termini of the 7-kb plasmid are 349-bp inverted repeats (TIRs). Each DNA strand contains a long open reading frame (ORF) which begins within the TIR and extends toward the centre of the plasmid. ORF-1 codes for a single-subunit RNA polymerase that is not closely related to that encoded by another Neurospora plasmid, kalilo. The ORF-2 product may be a B-type DNA polymerase resembling those encoded by terminal protein-linked linear genetic elements, including linear mitochondrial plasmids and linear bacteriophages. A separate coding sequence for the terminal protein could not be identified; however, the DNA polymerase of maranhar has an amino-terminal extension with features that are also present in the terminal proteins of linear bacteriophages. The N-terminal extensions of the DNA polymerases of other linear mitochondrial plasmids contain similar features, suggesting that the terminal proteins of linear plasmids may be comprised, at least in part, of these cryptic domains. The terminal protein-DNA bond of maranhar is resistant to mild alkaline hydrolysis, indicating that it might involve a tyrosine or a lysine residue. Although maranhar and the senescence-inducing kalilo plasmid of N. intermedia are structurally similar, and integrate into mitochondrial DNA by a mechanism thus far unique to these two plasmids, they are not closely related to each other and they do not have any nucleotide sequence features, or ORFs, that distinguish them clearly from mitochondrial plasmids which are not associated with senescence and most of which are apparently non-integrative.     

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.     

Complex terminal structure of a linear mitochondrial plasmid from Physarum polycephalum: three terminal inverted repeats and an ORF encoding DNA polymerase

The mitochondria of Physarum polycephalum have a linear plasmid (mF) which promotes mitochondrial fusion. To determine the terminal structure of the mF plasmid, restriction fragments derived from its ends were cloned and sequenced. The sequences showed that the mF plasmid has three kinds of terminal inverted repeats (TIRs). The most characteristic feature is a 144-bp repeating unit which exists between a 205-bp TIR at the extreme ends of the plasmid and another 591-bp TIR. All of the clones showed at least one of these 144-bp repeating units. The GC content of the 205-bp TIR (49%) was higher than those of the other TIRs and of another sequenced region (23%). This TIR can form three thermodynamically-stable hairpin structures based on complex internal palindromic components. Moreover, in the right terminal region of the mF plasmid, there is an open reading frame (ORF) which covers the entire 591-bp TIR and most of one of the 144-bp repeating units. This ORF encodes a 547-amino-acid polypeptide, ORF-547, and shows extensive homology with the polymerization domain of the putative DNA polymerases of linear mitochondrial plasmids from other sources.     

Structure of a Gelasinospora linear plasmid closely related to the kalilo plasmid of Neurospora intermedia

We have determined the complete nucleotide sequence of a linear mitochondrial plasmid from a natural isolate of a homothallic species ofGelasinospora. The plasmid genome is 8231 by long. It carries terminal inverted repeats of 1137 bp. Extending inwards from the terminal repeats are two long open reading frames coding for putative proteins with similarity to DNA and RNA polymerases. These are separated by a short intergenic region. The plasmid sequence shows remarkable similarity to that of theNeurospora intermedia senescence-plasmid kalilo. Overall the two plasmids have a similar genetic organization and are clearly homologous at the sequence level. The main differences are in the intergenic region and in the terminal repeats.     

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 kalilo linear senescence-inducing plasmid of Neurospora is an invertron and encodes DNA and RNA polymerases

Summary The nucleotide sequence of kalilo, a linear plasmid that induces senescence in Neurospora by intergrating into the mitochondrial chromosome, reveals structural and genetic features germane to the unique properties of this element. Prominent features include: (1) very long perfect terminal inverted repeats of nucleotide sequences which are devoid of obvious genetic functions, but are unusually GC-rich near both ends of the linear DNA; (2) small imperfect palindromes that are situated at the termini of the plasmid and are cognate with the active sites for plasmid integration into mtDNA; (3) two large, non-overlapping open-reading frames, ORF-1 and ORF-2, which are located on opposite strands of the plasmid and potentially encode RNA and DNA polymerases, respectively, and (4) a set of imperfect palindromes that coincide with similar structures that have been detected at more or less identical locations in the nucleotide sequences of other linear mitochondrial plasmids. The nucleotide sequence does not reveal a distinct gene that codes for the protein that is attached to the ends of the plasmid. However, a 335-amino acid, cryptic, N-terminal domain of the putative DNA polymersse might function as the terminal protein. Although the plasmid has been co-purifed with nuclei and mitochondria, its nucleotide composition and codon usage indicate that it is a mitochondrial genetic element.     

Mitochondrial DNA of Chlamydomonas reinhardtii: the structure of the ends of the linear 15.8-kb genome suggests mechanisms for DNA replication

The mitochondrial genome of Chlamydomonas reinhardtii is a linear double-stranded DNA of 15.8 kb. With the exception of the termini its DNA sequence has been published. Here we describe the unique structure of the two termini determined from cloned fragments or, for the very terminal sequences, by the Maxam and Gilbert method after 5 labeling of uncloned terminal fragments. The 15.8-kb DNA is characterized by terminal inverted repeats of 531 or 532 bp in length including long 3 extensions. The 3 single-stranded extensions of the left and right ends are non-complementary, identical in sequence, and comprise 39 to 41 nucleotides. Remarkably, the linear genome possesses in addition an internal 86-bp repeat of the two outermost sequences. The unusual structure of the 15.8-kb DNA termini is compared with those of other linear mitochondrial DNAs. Possible mechanisms of 15.8-kb DNA replication are discussed.Dedicated to Prof Dr. Piotr Slonimski on the occasion of this 70th birthday     

The kalilo senescence plasmid of Neurospora intermedia has covalently-linked 5′ terminal proteins

Summary Kalilo is a linear plasmid associated with senescence in Neurospora. The terminal Eco R1 restriction fragments of this element are linked to a protein component which remains bound despite denaturation with high concentrations of SDS. Following digestion with proteinase K, the 5 termini of the plasmid remain resistant to lambda exonuclease whereas the 3 termini are sensitive to exonuclease III, suggesting that the terminal protein is covalently linked. From an analysis of iodinated proteins released by nuclease digestion, the size of the terminal protein was estimated to be 120 kDa. The covalent linkage between DNA and protein was shown to be alkali-labile suggesting that it is a phosphodiester bond. Electron micrographs of the intact plasmid demonstrate that the associated proteins are terminal, and may be involved in replication.     

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

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

The linear plasmid pMC3-2 from Morchella conica is structurally related to adenoviruses

Summary pMC3-2, one of two linear plasmids localised in the mitochondria of the ascomycete Morchella conica, was completely sequenced. It is 6044 bp in size, contains terminal inverted repeats of 713 and 710 bp length and two open reading frames, ORF1 and ORF2, spanning 2706 bp and 918 bp, respectively. ORF1 probably encodes a viral B-type DNA-polymerase. Concerning ORF2, no homology to any other published protein-or DNA-sequence could be detected. According to the structure of DNA-polymerases, linear plasmids can be grouped into two classes reflecting their localisation either in the cytoplasm or within the mitochondria. In general, the structure of plasmid pMC3-2, as well as of other linear plasmids from filamentous fungi, indicates a close relationship of these genetic elements to adenoviruses.     

Structure of a linear plasmid of the yeast Kluyveromyces lactis; Compact organization of the killer genome

Summary Some strains of the yeast Kluyveromyces lactis contain a pair of linear DNA plasmids, k1 and k2, 8.8 and 13.8 kilobase pairs long, respectively. Simultaneous presence of the two plasmids confer a killer phenotype on the cell by producing a toxin which blocks the growth of sensitive yeast species. Previous genetic studies have suggested that the toxin protein is coded by the k1 plasmid. We have now determined the total nucleotide sequence of k1 DNA. The genome is 8,874 base pairs in length. It contains four protein-coding reading frames, three transcribed from one strand and the fourth transcribed from the complementary strand and has terminal inverted repeats of 202 base pairs. Nuclease S1 mapping confirmed this arrangement and showed that these genes are transcribed. The terminal repeats and the four genes form an extremely compact genome, with some overlapping of genes. All four genes use highly biased codons, 86% of them having A or T at the wobble position, reminiscent of yeast mitochondrial genes. Three genes share a very similar 5 leader sequence. The nature of gene products is discussed in the light of what is known of the excreted toxin protein.     

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

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

Regulatory region of the Aspergillus nidulans argB gene

Summary We have constructed a series of deletion plasmids which contain the Aspergillus nidulans argB gene for ornithine carbamoyltransferase (OTC). These deletions comprise the 5 upstream sequence of the argB gene. The pro^– arg^– strain of A. nidulans was transformed with the above plasmids. Several arg⁺ transformants of integration types I and II, obtained using each of the deletion plasmids, were studied, and their ability to de-repress OTC level by proline starvation was compared. It was concluded that nucleotides located between –150 and –50 by upstream of the argB gene are significant for its cross-pathway regulation. This regulatory region contains three copies of the TGACTC hexanucleotide which is a cis-acting regulatory sequence of general amino acid control in yeast.Abbreviations bp base pairs - kb 10³ base pairs - OTC ornithine carbamoyltransferase - ORF open reading frame     

Rapid identification of myxoma virus variants by long-range PCR and restriction fragment length polymorphism analysis

A long-range PCR method directed at the Myxoma virus (MV) left hand and right hand terminal inverted repeats (TIRs) for rapid amplification of genomic DNA and MV isolate differentiation by restriction fragment length polymorphism (RFLP) analysis is described. The efficacy of this method was tested by comparing the results from full genome RFLPs with those from TIRs amplified separately using reference strain Lausanne (Lu) and a field MV strain characterised previously for its virulence in rabbits. The usefulness of this method was also demonstrated by amplifying MV DNA directly from the eyelid tissue of an infected rabbit and comparative RFLP analysis with respect to Lu. The results proved the long-range PCR technique to be a simple highly efficient method for identifying mutations between MV genomes by RFLP analyses of the amplified TIRs and may be used in future studies to identify variable regions for phylogenetic studies.     

Kalilo plasmids are a family of four distinct members with individual global distributions across species

Kalilo is a linear 9-kb plasmid, isolated originally from Hawaiian strains of the heterothallic fungus Neurospora intermedia. Its properties include terminal inverted repeats, two ORFs coding for a presumptive DNA and an RNA polymerase, and the ability to cause senescence in its original host and in the closely related species Neurospora crassa. We have examined natural isolates alleged to contain plasmids homologous to kalilo. Most of these isolates do in fact contain plasmids with so close an identity to kalilo as to be certain relatives. We found a new case of kalilo in Neurospora tetrasperma from Moorea-Tahiti, and a new case of LA-kalilo (previously found only in N. tetrasperma) in N. crassa from Haiti. A previously unreported, substantially shorter, kalilo variant has been found in three geographically separate isolates of the heterothallic species Neurospora discreta. Therefore, if the previously reported kalilo variant from the genus Gelasinospora is included, in all there are four members of the kalilo plasmid family. The main differences between these plasmids are in the terminal inverted repeats (TIRs). The phylogeny of the TIR sequences is largely congruent with that of nuclear DNA in the species in which they are found, suggesting that the plasmids are related by vertical descent throughout the evolution of these species. However, there are two cases of a plasmid found in a heterothallic and a pseudohomothallic species in the same global area; these cases might have arisen from more recent horizontal transmission or introgression. Received: 14 July / 17 September 1999     

Conservation of amino-acid sequence motifs in lentivirus Vif proteins

The nonstructural/regulatory genes of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses are believed to play an important role in the replication and pathogenesis of these viruses. In HIV-1 and other lentiviruses, thevif (viral infectivity factor) open reading frame (ORF) (also termedsor orQ in some lentivirus genomes) is located in the central region, overlapping the 3 end of thepol ORF, but in a different reading frame. Among the lentiviruses, only equine infectious anemia virus lacks avif ORF. The predicted Vif protein sequences from 38 lentiviruses were analyzed for the presence of global and local sequence similarity. The Vif proteins of closely related lentiviruses are highly conserved (HIV-1_HXB2:HIV-1_mn=91% identity), while those of more distantly related lentirviruses have diverged significantly (HIV-1_HXB2: simian immunodeficiency virus_mac=30% identity). A search for local sequence similarity revealed that a unifying feature of predicted lentivirus Vif proteins is the presence of at least one of two short, highly conserved sequence motifs, SL(I/V)X₄YX₉Y and SLQXLA. SLQXLA was present in 34 of 38 lentiviruses examined, while the remaining four lentiviruses had one (three viruses) or two (one virus) substitutions in this motif (of five total substitutions, three were conservative changes). The SL(I/V)X₄YX₉Y motif was found only in primate lentiviruses and in bovine immunodeficiency-like virus. Based on these findings, we suggest that the locus designationvif be used to denote all lentivirus ORFs previously calledvif, Q, orsor.     

A restriction endonuclease map of the chloroplast genome of pearl millet

Chloroplast DNA from pearl millet (Pennisetum americanum) was used to construct recombinant plasmids. These plasmids contained 97 kilobase pairs of unique DNA sequences. The chloroplast DNA fragments in these plasmids were mapped with the restriction endonucleases SalI, SphI, XhoI, BglI and HpaI. The technique of overlapping hybridization or chromosome walking was used to orient these DNA fragments on a restriction endonuclease map of the chloroplast genome. The size of the chloroplast DNA from pearl millet was estimated in this fashion to be 127–138 kilobase pairs. Twenty one kilobase pairs of the cloned DNA fragments were represented twice on the genome as inverted repeats. Thus, the recombinant plasmids which were isolated contained approximately 86–93% of the nucleotide sequences in the chloroplast genome of pearl millet. Previously characterized cloned chloroplast DNA sequences from other plants were used as hybridization probes to locate the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase, the -coupling factor of ATPase and the 32 kilodalton polypeptide of photo system 11 on the restriction endonuclease map of the pearl millet chloroplast genome.