In organello replication and viral affinity of linear, extrachromosomal DNA of the ascomycete Ascobolus immersus

Summary Linear, extrachromosomal DNA's of the filamentous fungus Ascobolus immersus are localized within the mitochondria. These linear plasmids have no homology to the high molecular weight mtDNA (hmw mtDNA). For analysis of plasmid replication an in organello DNA synthesis system was developed, in which radionucleotides were incorporated into intact mitochondria. Plasmid DNA is labelled preferentially in this system. From replication analysis of a specific plasmid there is evidence of a virus-like protein-primed replication. Sequence analysis of this plasmid reveals that a viral DNA polymerase is encoded. Thus, these genetic elements presumably are viral remnants rather than true plasmids.     

Methylation of DNA repeats of decreasing sizes in Ascobolus immersus.

In Ascobolus immersus, DNA duplications are subject to the process of methylation induced premeiotically (MIP), which methylates the cytosine residues within the repeats and results in reversible gene silencing. The triggering of MIP requires pairing of the repeats, and its detection requires maintenance of the resulting methylation. MIP of kilobase-size duplications occurs frequently and leads to the methylation of all C residues in the repeats, including those belonging to non-CpG sequences. Using duplications of decreasing sizes, we observed that tandem repeats never escaped MIP when larger than 630 bp and showed a sudden and drastic drop in MIP frequencies when their sizes decreased from 630 to 317 bp. This contrasted with the progressive decrease of MIP frequencies observed with ectopic repeats, in which apparently the search for homology influences the MIP triggering efficiency. The minimal size actually required for a repeat to undergo detectable MIP was found to be close to 300 bp. Genomic sequencing and Southern hybridization analyses using restriction enzymes sensitive to C methylation showed a loss of methylation at non-CpG sites in short DNA segments, methylation being restricted to a limited number of CpG dinucleotides. Our data suggest the existence of two distinct mechanisms underlying methylation maintenance, one responsible for methylation at CpG sites and the other responsible for methylation at non-CpG sites.     

Presence of several plasmids in the fungus Ascobolus immersus, and homologies with the linear plasmid pA1

DNA analysis of several genetically unstable strains of the fungus Ascobolus immersus revealed the presence of at least seven different plasmids. These plasmids ranged from 2 to 20 kb in size, and showed homology to one of them, pA1. In 18 stocks directly isolated from nature, two-thirds harboured plasmids ranging from 3 to 17 kb. Plasmids with homology to pA1 had similar molecular masses (about 8.5 kb). A possible mechanism of plasmid formation from chromosomal DNA is discussed.     

Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences.

To develop a method to modify genomic sequences in Ascobolus immersus by precisely reintroducing defined DNA segments previously manipulated in vitro, we investigated the effect of transforming DNA conformation on recombination with chromosomal sequences. Circular single-stranded DNA carrying the met2 gene and double-stranded DNA linearized by cutting within the met2 gene both transformed protoplasts of a met2 mutant strain of A. immersus to prototrophy. In contrast to the equivalent circular double-stranded DNA, which chiefly integrated at nonhomologous chromosomal sites, single-stranded and double-stranded cut DNAs recombined primarily with the homologous chromosomal met2 sequence. Of the single-stranded DNA transformants, 65% resulted from replacement of the resident met2 mutation by the exogenous wild-type allele. In 70% of the double-stranded-cut DNA transformants, one or more copies of the transforming DNA had integrated at the met2 locus, leading to tandem duplications of the met2 target region separated by plasmid DNA. These duplicated sequences could recombine, leading to progeny containing only one copy of the met2 region. This resulted in a precise gene replacement if the wild-type allele had been retained. In addition, we show that newly duplicated sequences were most often de novo methylated at the cytosine residues during the sexual phase. Cytosine methylation was associated with inactivation of the integrated met2 gene(s) in segregants of crosses. However, methylation was not accurately maintained at each DNA replication cycle, so that Met- segregants recovered a wild-type phenotype through successive mitotic divisions. This finding indicated that met2 genes were silenced by methylation alone.     

New information on the mechanism of forcible ascospore discharge from Ascobolus immersus

Many ascomycete fungi spurt their spores from asci pressurized by osmosis. This paper explores the details of this process in the coprophilous species Ascobolus immersus, through a combination of biomechanical and biochemical experiments, and mathematical modeling. A. immersus forms large asci that expel 8 spores as a single, mucilage-embedded projectile. Measurements of ascus turgor using a microprobe attached to a strain gauge showed a pressure of 0.3 MPa or 3 atm. Analysis of ascus sap using GC/MS identified glycerol as a major osmolyte, accounting for 0.1 MPa of the osmotic pressure within the ascus sap. A mathematical model indicated that a pressure of 0.2 MPa would be sufficient to propel the cluster of ascospores over the distance measured in previous studies. The difference between the measured and predicted pressures is ascribed to loss of pressure as the spores are forced through the tip of the open ascus.     

Stable allele replacement and unstable non-homologous integration events during transformation of Ascobolus immersus

An homologous transformation system for the filamentous fungus Ascobolus immersus has been developed, based on the complementation of a met2 mutation by the wild-type (wt) allele gene encoding homoserine O-transacetylase. Transformation of A. immersus met2 mutants occurs with moderate frequencies (about 50 transformants per microgram input DNA). Analysis of the DNA of the met2+ transformants showed that transformation resulted either in a single integration of the donor DNA into the genome by many different nonhomologous recombination events or in the substitution of the endogenous met2 mutation by the wt transforming allele. The relative frequencies of both events depended on the vector sequences carrying the cloned met2 gene. Whereas the substitution event led, as expected, to genetically stable transformants, the non-homologous integration was always associated with a strong instability when transformants were crossed and underwent meiosis.     

Variation of gene conversion and intragenic recombination frequencies in the genome of Ascobolus immersus.

Summary Eighty mutants in 17 ascospore character genes were studied for their conversion patterns. The correlation between conversion pattern and mutagenic origin, previously found in genes b1 and b2 was extended to all the genes studied. Aberrant 4:4 asci were found in most genes irrespective of their conversion frequency. From gene to gene, the conversion frequency showed an almost 100 times variation. The frequency of intragenic recombination also showed sharp variation from gene to gene. The mean conversion frequency and the maximal intragenic recombination frequency were shown to be highly correlated in 5 genes for which these 2 values are known. This correlation was extended to 12 other genes in other Ascomycetes: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora, and Sordaria. From this study it is concluded that, 1) the probability of hybrid DNA formation undergoes considerable changes according to the region of the genome; 2) the intragenic recombination frequency primarily reflects the frequency of hybrid DNA formation rather than the physical length of the gene; 3) for a given physical distance on the DNA, a similar fraction of the gene conversion events lead to recombination in the 5 Ascomycetes.     

Chromosomal and extrachromosomal control of senescence in the ascomycete Podospora anserina.

Summary In Podospora anserina senescence leading to cellular death occurs regularly after prolonged vegetative propagation. However, the life span of this ascomycete may be extended by various means:I. Mutations in at least 8 morphogenetic genes belonging to 4 linkage groups postpone drastically or even prevent in certain pairwise combinations (e.g. i viv) the onset of senescence. 2. Inhibitors of mt DNA and of mitochondrial protein synthesis show a life prolonging effect when added in low concentrations to the growth medium. 3. A similar effect was found when mycelia were fed exclusively on non repressive carbon sources.Whereas the anti-aging effect of specific mutated genes is rather permanent, the life prolonging action of the inhibitors and carbon sources is restricted and temporary. These substances have no long lasting effect, since after their removal from the medium aging proceeds.Physiological experiments have further shown the existence of three phases in the life span of Podospora anserina. During the juvenile phase aging is prevented by all of these compounds; during the presenescent phase aging is prevented by inhibitors of mt DNA only, and during the senescent phase aging is irreversible.Senescence may be induced in juvenile protoplasts by DNA extracted from senescent mycelia. This, together with the well known fact that senescence is extrachromosomically inherited, points to extrachromosomal DNA as the causative agent of senescence. This kind of DNA may be connected with or perhaps located in the mitochondria.Collectively, the data are consistent in showing that the syndrome of senescence in Podospora anserina is controlled by a chromosomal-extrachromosomal is controlled by a chromosomal-extrachromosomal interaction. In this system, extrachromosomal DNA, perhaps a mt DNA, is identical with the infectious principle initiating the decay of the cell, and nuclear genes supervise its expression.     

Excision and replication of extrachromosomal DNA of pea (Pisum sativum).

Experiments with cultured pea roots were conducted to determine (i) whether extrachromosomal DNA was produced by cells in the late S phase or in the G2 phase of the cell cycle, (ii) whether the maturation of nascent DNA replicated by these cells achieved chromosomal size, (iii) when extrachromosomal DNA was removed from the chromosomal duplex, and (iv) the replication of nascent chains by the extrachromosomal DNA after its release from the chromosomal duplex. Autoradiography and cytophotometry of cells of carbohydrate-starved root tips revealed that extrachromosomal DNA was produced by a small fraction of cells accumulated in the late S phase after they had replicated about 80% of their DNA. Velocity sedimentation of nascent chromosomal DNA in alkaline sucrose gradients indicated that the DNA of cells in the late S phase failed to achieve chromosomal size. After reaching sizes of 70 X 10(6) to 140 X 10(6) daltons, some of the nascent chromosomal molecules were broken, presumably releasing extrachromosomal DNA several hours later. Sedimentation of selectively extracted extrachromosomal DNA either from dividing cells or from those in the late S phase showed that it replicated two nascent chains, one of 3 X 10(6) daltons and another of 7 X 10(6) daltons. Larger molecules of extrachromosomal DNA were detectable after cells were labeled for 24 h. These two observations were compatible with the idea that the extrachromosomal DNA was first replicated as an integral part of the chromosomal duplex, was cut from the duplex, and then, once free of the chromosome, replicated two smaller chains of 3 X 10(6) and 7 X 10(6) daltons.