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J L Rossignol

Publications and source records attributed to J L Rossignol.

At least 19 recordsLinked to original sources
PubMed2000

Histone H1 is dispensable for methylation-associated gene silencing in Ascobolus immersus and essential for long life span.

A gene encoding a protein that shows sequence similarity with the histone H1 family only was cloned in Ascobolus immersus. The deduced peptide sequence presents the characteristic three-domain structure of metazoan linker histones, with a central globular region, an N-terminal tail, and a long positively charged C-terminal tail. By constructing an artificial duplication of this gene, named H1, it was possible to methylate and silence it by the MIP (methylation induced premeiotically) process. This resulted in the complete loss of the Ascobolus H1 histone. Mutant strains lacking H1 displayed normal methylation-associated gene silencing, underwent MIP, and showed the same methylation-associated chromatin modifications as did wild-type strains. However, they displayed an increased accessibility of micrococcal nuclease to chromatin, whether DNA was methylated or not, and exhibited a hypermethylation of the methylated genome compartment. These features are taken to imply that Ascobolus H1 histone is a ubiquitous component of chromatin which plays no role in methylation-associated gene silencing. Mutant strains lacking histone H1 reproduced normally through sexual crosses and displayed normal early vegetative growth. However, between 6 and 13 days after germination, they abruptly and consistently stopped growing, indicating that Ascobolus H1 histone is necessary for long life span. This constitutes the first observation of a physiologically important phenotype associated with the loss of H1.

Amino Acid Sequence↗
PubMed1999

Eukaryotic DNA methylation as an evolutionary device.

DNA methylation is catalyzed by a family of conserved DNA methyltransferases and is widespread among protists, plants, fungi and animals. It is however absent in some species and its genomic distribution varies among organisms. Sequence comparisons suggest that known and putative eukaryotic DNA methyltransferases fall into at least five structurally distinct subfamilies. Furthermore, it is now clear that DNA methylation can be involved in several functions, some of which may coexist within the same organism. It can inhibit transcription initiation, arrest transcript elongation, act as an imprinting signal, and suppress homologous recombination. On the basis of these observations, we argue that DNA methylation has been conserved during evolution because it provides unique possibilities for setting up functions of various types.

Animals↗
PubMed1999

Masc2, a gene from Ascobolus encoding a protein with a DNA-methyltransferase activity in vitro, is dispensable for in vivo methylation.

We have shown previously that masc1, a gene encoding a putative C5-DNA-methyltransferase (MTase), was necessary for the de novo 'Methylation Induced Premeiotically' (MIP) process and sexual reproduction in Ascobolus, whereas it was dispensable for maintenance methylation. A second MTase gene from Ascobolus, masc2, encodes a protein, Masc2, which possesses the large amino-terminal part characteristic of eukaryotic maintenance MTases. In vitro assays have shown that Masc2 displays a methylation activity, suggesting that it might be the MTase responsible for maintenance methylation. To check its function in vivo, we engineered a disruption of the masc2 gene. The resulting mutant strains did not exhibit any particular phenotype during either vegetative growth or sexual reproduction. Neither the masc2 mutation nor the double masc1 masc2 mutation had any detectable effect upon the maintenance of the pre-existing methylation of single gene copies previously subjected to MIP, natural retroelement-like repeats and tandemly repeated rDNA. The masc2 mutation did not alter either MIP or the other de novo methylation process that operates in vegetatives cells. Nor did it impair the meiotic process of methylation transfer. These results suggest that at least a third MTase gene responsible for maintenance and vegetative de novo methylation is present in Ascobolus.

Ascomycota↗
PubMed1999

[DNA repeats and homologous recombination: a probable role for DNA methylation in genome stability of eukaryotic cells].

Homologous recombination between DNA repeats directly threatens the intact transmission of repeat-rich eukaryotic genomes through mitotic and meiotic cell divisions. Besides several other factors already known, DNA methylation might contribute, in some eukaryotes, to the limitation of crossover events between repeats. A strong inhibitory effect of DNA methylation has now been directly demonstrated, in the filamentous fungus Ascobolus. This therefore reinforces the question of the biological impact of this DNA modification on the recombinational stability of repeat-rich genomes, such as those of mammals.

Animals↗
PubMed1998

Suppression of crossing-over by DNA methylation in Ascobolus.

Homologous recombination between dispersed DNA repeats creates chromosomal rearrangements that are deleterious to the genome. The methylation associated with DNA repeats in many eukaryotes might serve to inhibit homologous recombination and play a role in preserving genome integrity. We have tested the hypothesis that DNA methylation suppresses meiotic recombination in the fungus Ascobolus immersus. The natural process of methylation-induced premeiotically (MIP) was used to methylate the b2 spore color gene, a 7.5-kb chromosomal recombination hot spot. The frequency of crossing-over between two markers flanking b2 was reduced several hundredfold when b2 was methylated on the two homologs. This demonstrates that DNA methylation strongly inhibits homologous recombination. When b2 was methylated on one homolog only, crossing-over was still reduced 50-fold, indicating that the effect of methylation cannot be limited to the blocking of initiation of recombination on the methylated homolog. On the basis of these and other observations, we propose that DNA methylation perturbs pairing between the two intact homologs before recombination initiation and/or impairs the normal processing of recombination intermediates.

Alleles↗
PubMed1998

Extensive, nonrandom diversity of excision footprints generated by Ds-like transposon Ascot-1 suggests new parallels with V(D)J recombination.

Upon insertion, transposable elements can disrupt or alter gene function in various ways. Transposons moving through a cut-and-paste mechanism are in addition often mutagenic when excising because repair of the empty site seldom restores the original sequence. The characterization of numerous excision events in many eukaryotes indicates that transposon excision from a given site can generate a high degree of DNA sequence and phenotypic variation. Whether such variation is generated randomly remains largely to be determined. To this end, we have exploited a well-characterized system of genetic instability in the fungus Ascobolus immersus to perform an extensive study of excision events. We show that this system, which produces many phenotypically and genetically distinct derivatives, results from the excision of a novel Ds-like transposon, Ascot-1, from the spore color gene b2. A unique set of 48 molecularly distinct excision products were readily identified from a representative sample of excision derivatives. Products varied in their frequency of occurrence over 4 orders of magnitude, yet most showed small palindromic nucleotide additions. Based on these and other observations, compelling evidence was obtained for intermediate hairpin formation during the excision reaction and for strong biases in the subsequent processing steps at the empty site. Factors likely to be involved in these biases suggest new parallels between the excision reaction performed by transposons of the hAT family and V(D)J recombination. An evaluation of the contribution of small palindromic nucleotide additions produced by transposon excision to the spectrum of spontaneous mutations is also presented.

Ascomycota↗
PubMed1997

A gene essential for de novo methylation and development in Ascobolus reveals a novel type of eukaryotic DNA methyltransferase structure.

Molecular mechanisms determining methylation patterns in eukaryotic genomes still remain unresolved. We have characterized, in Ascobolus, a gene for de novo methylation. This novel eukaryotic gene, masc1, encodes a protein that has all motifs of the catalytic domain of eukaryotic C5-DNA-methyltransferases but is unique in that it lacks a regulatory N-terminal domain. The disruption of masc1 has no effect on viability or methylation maintenance but prevents the de novo methylation of DNA repeats, which takes place after fertilization, through the methylation induced premeiotically (MIP) process. Crosses between parents harboring the masc1 disruption are arrested at an early stage of sexual reproduction, indicating that the activity of Masc1, the product of the gene, is crucial in this developmental process.

Amino Acid Sequence↗
PubMed1996

Interchromosomal transfer of epigenetic states in Ascobolus: transfer of DNA methylation is mechanistically related to homologous recombination.

The transfer of methylation between alleles represents a plausible epigenetic mutational mechanism to explain loss of imprinting in mammals and paramutation in plants. Here, we have exploited advantages unique to the fungus Ascobolus immersus to obtain direct experimental evidence that methylation transfer can occur between homologous chromosomes. A methylated allele and an unmethylated allele of the Ascobolus b2 spore color gene were brought together in individual meiotic cells. Frequent transfer of methylation to the unmethylated allele was observed. This transfer was polarized 5' to 3' along the b2 gene, as is gene conversion, and always accompanied the latter process when tested in the same cross. These and other observations strongly suggest that methylation transfer and recombination are mechanistically related.

Alleles↗
PubMed1996

Native DNA repeats and methylation in Ascobolus.

We identified two classes of native dispersed DNA repeats in the Ascobolus genome. The first class consisted of several kilobase long, methylated repeats. These repeats, named Mars (methylated Ascobolus repeated sequences), fell in one family of LINE-like elements and in three families of LTR-containing retrotransposable elements. The methylation features of Mars elements were those expected if they were natural targets for the MIP (methylation induced premeiotically) previously discovered in Ascobolus. The second class consisted of short repeats, approximately 100 bp long, corresponding to 5S rRNA and tRNA genes. As expected from their size, which was too small to allow MIP to occur, they were unmethylated, as were 26 kb of unique sequences tested. These observations are consistent with the hypothesis that MIP is targeted at natural DNA repeats and constitutes a defensive process against the detrimental consequences of the spreading of mobile elements throughout the genome. The 9 kb tandem repeats harbouring the 28S, 18S and 5.8S rRNA genes displayed methylation features suggesting that rDNA methylation proceeds through a process other than MIP.

Amino Acid Sequence↗
PubMed1996

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.

Ascomycota↗
PubMed1995

Isolation of the Ascobolus immersus spore color gene b2 and study in single cells of gene silencing by methylation induced premeiotically.

The ascomycete Ascobolus immersus has been extensively used as a model system for the genetic study of meiotic recombination. More recently, an epigenetic process, known as methylation induced premeiotically (MIP), that acts on duplicated sequences has been discovered in A. immersus and has raised a new interest in this fungus. To try and extend these studies, we have now cloned the A. immersus spore color gene b2, a well characterized recombination hot-spot. Isolation of the whole gene was verified by physical mapping of four large b2 alterations, followed by transformation and mutant rescue of a null b2 allele. Transformation was also used to duplicate b2 and subject it to MIP. As a result, we were able for the first time to observe gene silencing as early as just after meiosis and in single cells. Furthermore, we have found evidence for a modulating effect of MIP on b2 expression, depending on the region of the gene that is duplicated and hence subjected to MIP.

Ascomycota↗
PubMed1994

Gene inactivation triggered by recognition between DNA repeats.

This chapter focuses on phenomena of gene inactivation resulting from the presence of repeated gene copies within the genome of plants and fungi, and on their possible relationships to homologous DNA-DNA interactions. Emphasis is given to two related premeiotic processes: Methylation Induced Premeiotically (MIP) and Repeat-Induced Point mutation (RIP) which take place in the fungi Ascobolus immersus and Neurospora crassa, respectively. The relationships between these processes and genetic recombination are discussed.

DNA↗
PubMed1993

Methylation induced premeiotically in Ascobolus: coextension with DNA repeat lengths and effect on transcript elongation.

The effect of duplications of gene fragments on the triggering of DNA methylation induced premeiotically (MIP) was studied in the sexual progeny of strains harboring, in addition to the resident met2 gene, a fragment of this gene inserted at an ectopic position. Cytosine methylation of the resident gene was checked for each of the eight duplications tested. Methylation was always found and it was coextensive with the length of the duplications. Silencing of the resident gene was triggered by duplications of segments corresponding to the region 5' to the open reading frame, to only the open reading frame, or to segments beginning 0.87-1.2 kb downstream from the transcription start. Silencing was accompanied by either the absence of transcripts or the presence of truncated transcripts, which suggests that methylation acts on transcript elongation.

5-Methylcytosine↗
PubMed1992

Epimutation of repeated genes in Ascobolus immersus.

Ascobolus immersus artificial gene repeats were shown previously to be subject premeiotically to both cytosine methylation and inactivation. We studied sexual progenies of strains harbouring two wild type copies of the endogenous met2 gene lying either in tandem array or at ectopic unlinked positions, by (i) investigating the methylation status, (ii) searching for mutations and (iii) analysing the inheritance of inactivation both in mitotic and sexual offspring. 100% of the 'tandem' progeny and 64% of the 'ectopic' progeny had methylated repeats and displayed gene inactivation. Similar methylation patterns involving all or most of the cytosine residues within the repeats were observed in both arrangements. The inactivated met2 copies were totally devoid of mutation, as deduced from: (i) extensive restriction site analysis and DNA sequencing; (ii) the finding that all the Met- derivatives tested reverted to prototrophy in selective conditions; and (iii) the finding that an inactivated copy of met2 stripped of its methylation through amplification in Escherichia coli regained activity when reintroduced in A.immersus. In the absence of selection, gene silencing and methylation were faithfully maintained through mitotic divisions and through five successive sexual cycles. Altogether, these data show the epimutational nature of this methylation induced premeiotically (MIP) process.

Acetyltransferases↗
PubMed1990

How does the cell count the number of ectopic copies of a gene in the premeiotic inactivation process acting in Ascobolus immersus?

Repeated genes, artificially introduced in Ascobolus immersus by integrative transformation, are frequently inactivated during the sexual phase. Inactivation is observed in about 50% of meioses if duplicated genes are at ectopic chromosomal locations, and in 90% of meioses if genes are tandemly repeated. Inactivation is associated with extensive methylation of the cytosine residues of the duplicated sequences and is induced in the still haploid nuclei of the dikaryotic cell which will undergo karyogamy and subsequent meiosis. Only repeated sequences become methylated. This raises the intriguing question of how the premeiotic inactivation machinery is informed that a nucleus contains multiple copies of a gene. By using in crosses recombinant strains of A. immersus in which either one, two or three genetically independent copies of the exogenous amdS gene from Aspergillus nidulans had been introduced, we could follow the premeiotic inactivation of each one of the ectopic amdS copies. This led us to propose that a prerequisite for inactivation is a premeiotic pairing of repeated sequences and that each copy can undergo successive cycles of pairing. In fact, once methylated, a copy can pair with a still unmethylated copy, so that an uneven number of copies can be subject to inactivation.

Ascomycota↗
PubMed1988

Molecular cloning and characterization of the met2 gene from Ascobolus immersus.

We have cloned the met2 gene from Ascobolus immersus by heterologous hybridization with the MET2 gene of Saccharomyces cerevisiae. This gene codes for the homoserine O-transacetylase, one of the methionine biosynthetic enzymes. The complete nucleotide sequence of a 2910-bp DNA fragment carrying the met2 gene has been determined. The gene contains a 165-bp intron which is similar in structure to other fungal introns. The deduced amino acid (aa) sequence (518 aa residues; Mr of 57726) shows three domains with a significant level of homology with the corresponding yeast protein. Northern-blot analysis reveals at least two transcripts (2.4 and 2.1 kb) probably due to transcription termination heterogeneity, as suggested by S1-mapping experiments. Polymorphism has been observed in the met2 gene flanking regions of Ascobolus strains from two different stocks.

Acetyltransferases↗
PubMed1988

Hybrid DNA tracts may start at different sites during meiotic recombination in gene b2 of Ascobolus.

The initiation of hybrid DNA in the b2 spore colour gene was investigated by the analysis of non-Mendelian segregation asci in the progeny of crosses involving several allelic b2 mutations. These analyses showed that, instead of the unique initiation region of hybrid DNA in b2, assumed previously by Paquette and Rossignol (1978), there are multiple sites for hybrid DNA starting. They produce major and minor fractions of hybrid DNA. The major fraction starts upstream of the left end of the gene, propagates rightwards and can cover its entire length. The minor fractions do not span the left b2 end. One of them is detected within the left half of the gene, the others within its right half.

Journal Article↗