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.     

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.     

A recombinant plasmid carrying the mitochondrial plasmid sequence of Neurospora intermedia LaBelle yields new plasmid derivatives in Neurospora crassa transformants

Summary We have studied the efficacy of transformation of Neurospora crassa with a chimaeric plasmid. We constructed a recombinant plasmid, pMK2, consisting of the mitochondrial plasmid of N. intermedia LaBelle, a part of the qa gene cluster of Neurospora and the Escherichia coli plasmid pBR322. Compared to plasmid pVK88, not containing the LaBelle sequence, the pMK2 plasmid gives a five-fold increase in transformation of the qa2⁺ gene. Analysis of the DNA from Neurospora transformants revealed that the pMK2 plasmid is not stable. The qa insert as well as the LaBelle part of pMK2 are rapidly lost from the plasmid. In most cases the qa insert integrates into the nuclear DNA of the host. Plasmids recovered from Neurospora transformants are rearranged and show insertions or deletions. Some of these plasmids are described here. In most cases the qa insert and the LaBelle sequence of plasmid pMK2 have been deleted. Frequently plasmid dimers, carrying an insertion of mitochondrial DNA, are recovered.     

Constitutive homologous recombination between mitochondrial DNA and a linear mitochondrial plasmid in Physarum polycephalum

Summary In one particular myxamoebal strain (NG7; mF⁺) of Physarum polycephalum, a linear mitochondrial plasmid (mF plasmid) which promotes mitochondrial fusion has been identified. A mating between mF^- strains, that do not carry the mF plasmid, resulted in uniparental inheritance of the mtDNA. In matings between mF⁺ and mF^- strains a recombination occurred between the mtDNA and the mF plasmid, and recombinant mtDNA was generated with the end of the mF plasmid as its ends. The DNA sequences of the recombination site in the mtDNA and the mF plasmid, and of the recombinant mtDNA, revealed that the mF plasmid had a 473-bp sequence that was identical to, but slightly shorter than, a 477-bp sequence of the mtDNA. This so-called identical sequence was found at the junction between unique sequences of the mF plasmid and the mtDNA in the recombinant mtDNA. Thus, the recombination between the mtDNA and the mF plasmid was due to reciprocal crossing-over at the identical sequence.     

Unstable cytoplasms in Hawaiian strains of Neurospora intermedia

Summary By subjecting a large sample of natural isolates of N. intermedia to prolonged serial subculturing, 26 cytoplasmic variants have been identified. These variants show senescence, and finally death at some strain-specific point in the subculture series. All senescent strains are from the Hawaiian archipelago, where their incidence in natural populations is high. Senescent cultures can be female-fertile. Random ascospore analyses show that (i) senescence is maternally inherited; (ii) different stages of senescence give different proportions of senescent progeny; and (iii) ascospores from one cross show different degrees of senescence. These results indicate that senescence is determined by a genetic factor which re sides in the cytoplasm. This factor promotes instability of the cytoplasm, resulting initially in cytoplasmic heterogeneity shown by ascus and conidium sampling, and finally in death. Molecular studies to be published elsewhere show that the progression through senescence to death is correlated with the occurence of abnormalities in cytochrome content and mitochondrial DNA. The Hawaiian word kalilo (dying), symbolised [kal], is proposed to denote these cytoplasms.     

Inactivation of the Neurospora crassa mitochondrial outer membrane protein TOM70 by repeat-induced point mutation (RIP) causes defects in mitochondrial protein import and morphology

Mitochondrial biogenesis requires the efficient import of hundreds of different cytosolically translated preproteins into existing organelles. Recognition and translocation of preproteins at the mitochondrial outer membrane is achieved by the TOM complex (translocase of the outer mitochondrial membrane). The largest component of this complex is TOM70, an integral outer membrane protein with a large cytosolic domain thought to serve as a receptor for a specific group of preproteins. To investigate the functional role of TOM70 in Neurospora crassa the tom70 gene was inactivated using the natural phenomenon of repeat-induced point mutation (RIP). Mutant strains were identified that harbored RIPed tom70 alleles and contained no immunologically detectable TOM70. Strains that lack TOM70 grow more slowly than wild-type strains, conidiate poorly, and contain enlarged mitochondria. In vitro preprotein import studies using TOM70-deficient mitochondria revealed a defect in the uptake of the ADP/ATP carrier. Other preproteins tested were imported at wild-type rates with the exception of the precursor of the mitochondrial-processing peptidase (MPP) which was imported more efficiently by TOM70-deficient mitochondria. These data support the view that TOM70 plays a role as a specific receptor for carrier proteins in mitochondrial-preprotein import. The presence of tetratricopeptide repeats (TPRs) in the TOM70 sequence and the enlarged shape of mitochondria lacking TOM70 raise the possibility that the protein also plays a role in the maintenance of mitochondrial morphology. Received: 25 March 1999 / 18 May 1999     

The dynamics of mitochondrial plasmids in a Hawaiian population of Neurospora intermedia

We analysed the distribution of mitochondrial plasmids among 82 Neurospora intermedia isolates from Hawaii; 74% of the isolates carried the neutral circular plasmid Han-2, whereas 38% contained the linear senescence-causing plasmid kalDNA. The distributions of the two plasmids are independent. There is no significant difference between the Kauaian population of 1972 and that of 1976. To further examine the reasons for this frequency distribution we studied the transmission of both Hawaiian plasmids through the maternal parent in a large series of crosses using non-Kalilo isolates as conidial parents. Plasmids can be lost during the sexual cycle. The Han-2 plasmid is transmitted more efficiently than kalDNA. No clear cases of autonomous or non-autonomous plasmid suppression were observed, so loss can be considered accidental. One Kalilo strain proved to be ineffectual as a maternal parent, and this reduced its ability to transmit kalDNA to the next generation. The dynamic balance of plasmids in natural populations over time is probably a result of the interplay of many forces, including those described in this work and those from several other studies on Neurospora plasmids.     

Characterization and prevalence of a circular mitochondrial plasmid in senescence-prone isolates of Neurospora intermedia

Genetic and molecular analyses of the phenomenon of senescence—i.e., irreversible loss of growth and reproductive potential upon subculturing—in Neurospora intermedia strain M1991-60A, collected from Maddur in southern India, showed the presence of plasmid pMaddur1, which is homologous to the senescence-inducing circular mitochondrial plasmid, pVarkud. Maternal inheritance of senescence in M1991-60A correlated to the formation of variant pMaddur1, its subsequent insertion into mitochondrial (mt)DNA and the accumulation of defective mtDNA with the pMaddur1insert. PCR-based analyses for similar plasmids in 147 natural isolates of Neurospora from Maddur showed that nearly 40% of the strains had pMaddur1 or pMaddur2 that shared 97–98% sequence homology with pVarkud and pMauriceville. Nearly 50% of the strains that harbored either pMaddur1 or pMaddur2, also contained a circular Varkud satellite plasmid (pVS). Size polymorphism maps to the cluster of PstI sites in the non-coding region. Whereas senescence of nearly 40% of N. intermedia strains may be due to pMaddur, the presence in seven strains of pVS but not pMaddur and the absence of either of these two plasmids in other senescence-prone isolates suggests yet undiscovered mechanisms of senescence in the Maddur strains.     

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.     

Association between variant plasmid formation and senescence in retroplasmid-containing strains of Neurospora spp.

Serial transfer of Neurospora strains harboring the Mauriceville and Varkud mitochondrial retroplasmids frequently displays erratic growth and senescence. Growth impairment is associated with the formation of variant forms of the retroplasmids that can integrate into the mitochondrial genome, resulting in mtDNA rearrangements and eventual loss of respiratory function. Here, we evaluate the rate at which variant plasmids arise in subcultures of the Mauriceville strain of N. crassa and their association with the senescent phenotype. Although variant plasmid formation preceded senescence, subcultures were found to tolerate variant plasmids for variable lengths of time and no correlation could be made between the specific sequence inserted in the plasmids and the rate or frequency of senescence. In addition, many cultures were found to contain more than one variant plasmid. The lack of concordance between the timing of variant plasmid formation and growth cessation distinguishes these two events, and provides additional insight into the etiology of senescence. We also detected differences in the frequency of senescence between retroplasmid-containing strains of N. crassa and N. intermedia and report the isolation of a strain in which senescence occurs in the absence of variant plasmid formation or detectable alterations in mtDNA. Our findings indicate there are multiple pathways that lead to senescence and suggest there are host-specific mechanisms that suppress the deleterious effects of the variant plasmids. Received: 8 August 2000 / Accepted: 17 November 2000     

Expression of ORF1 of the linear 2.3 kg plasmid of maize mitochondria: product localization and similarities to the 130 kDa protein encoded by the S2 episome

Summary ORF1 of the ubiquitous 2.3 kb linear plasmid of maize mitochondria encodes a 39 kDa protein detected with polyclonal antibodies raised to a -galactosidase: ORF1 fusion protein. Almost half of this protein is similar to a domain of the 130 kDa protein encoded by the S2 episome of mitochondria from cytoplasmic malesterile lines. Antisera raised to the ORF1 2.3 kb plasmid product cross-reacts with the ORF1, 130 kDa protein from the S2 episome. Despite the shared domain, the proteins are differentially localized: the 130 kDa protein is membrane-associated while the ORF1 protein is found in the matrix. We discuss possible functions of the ORF1 protein.     

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.     

Isolation and characterization of a methylammonium resistant mutant of Neurospora crassa

Summary A mutant of Neurospora crassa has been isolated which is resistant to methylammonium, a structural analog of ammonium. In contrast to wild type, this mutant, mea-1, has derepressed nitrate reductase and nitrite reductase activities in the presence of ammonium. However, glutamine still represses these nitrate assimilation enzymes in mea-1. The nit-2 mutant was epistatic to mea-1 since the mea-1; nit-2 double mutant has the nit-2 mutant phenotype. In addition, mea-1; nit-2 double mutants cannot utilize ammonium as a nitrogen source. We suggest therefore that nit-2 and mea-1 loci play a role in ammonia/methylamine uptake.     

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.     

Preparation of a cell-free translation system from a wild-type strain of Neurospora crassa

Summary We describe the preparation of an in vitro translation system from a wild-type strain of Neurospora crassa. The system is capable of supporting efficient and faithful translation of native and in vitro transcribed eukaryotic messages. The translation products have minimal background and can be clearly analyzed by SDS-polyacrylamide gel electrophoresis. The method of preparation of the lysate is simple, fast and reproducible. The procedure should be readily applicable to other filamentous fungi.     

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     

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.