34. Structure and distribution of Pcel elements in the white rot basidiomyce Phanerochaete chrysosporium.
Dan Cullen1, Philip Stewart1, Jill Gaskell1 and Bernard Janse 2. 1 Forest Products Laboratory, One Gifford Pinchot Dr., Madison, WI 53705 and 2 Department of Microbiology, University of Stellenbosch, 7600 Stellenbosch, South Africa.
Phanerochaete chrysosporium strain BKM-F-1767 was previously shown to harbor a 1747
bp insertional mutation within lignin peroxidase allele lipI2. Designated Pcel, the element
transcriptionally inactivated lipI2, but not lipI1. The mutant allele is inherited in a 1:1 ratio
among haploid progeny. A second element derived from BKM-F-1767 has now been
characterized. The nucleotide sequence of Pce2 is >99% identical to Pce 1. The elements are
distantly linked showing approximately 85% cosegregation. Chef gel blots localized both
elements to the same allelic homologue. Nucleotide sequence of regions adjacent to Pce2 showed
no similarities with any known fungal genes. Ongoing studies with geographically diverse P.
chrysosporium strains indicate wide distribution of elements nearly identical to Pce I /Pce2.
35. Evidence for horizontal transmission of the Fot1 element between Fusarium species and discovery of a RIP-like process.
Marie-josee Daboussi and Thierry Langin, Universite Paris-Sud, Orsay, France.
To understand the evolutionary origin of the well-characterized Fot1 mobile element
identified in the Fusarium oxysporum species, we have undertaken an extensive survey of the
Fusarium genus using Southern blot hybridization and Polymerase chain reaction (PCR)
amplification to determine its distribution. The results showed that Fotl-homologous sequences
widely distributed in the F. oxysporum species, are absent in the most closely related F.
moniliforme species but present in some strains belonging to the distant species F. solani . The
discontinuity in the distribution of the Fot1 element together with the remarkably close similarity
between Fotl sequences from distant species supports the conclusion that Fot1 elements have
been transferred horizontally among these species in the recent past. However, the fate of a Fot1
copy in these heterologous species depends on the sexual state of the strain it entered. In the
homothallic strain, Neocosmospora spp, analysis of the nucleotide divergence indicated that Fot1
copies have probably been submitted to a RIP process. This result addresses the question of the
occurrence of such phenomenon across the Fusarium genus.
36. F. oxysporum transposons as tools for the isolation of fungal genes.
Marie-Josee Daboussil, Jean-Michel Davierel, Aurelie HuaVan1, Fiona Kaperl, Thierry Langinl, Richard Laugel, Quirico Mighelil, and Christian Steinberg2, lUniversite Paris-sud, Orsay and 2INRA Dijon, France.
We have investigated the activity of the impala and Fotl transposons to develop a method
for transposon mutagenesis in Fusarium oxysporum. A number of findings about their
transposition are pertinent when designing gene tagging experiments. In general, excision is
followed by integration at a new site offering the opportunity to screen thousands of strains for
mutations in genes involved in many aspects of pathogenesis. Non-pathogenic mutants have
already been obtained and are under characterization. In order to use these elements for
transposon tagging in different strains, experiments were designed to identify autonomous copies.
Therefore, we employed a phenotypic assay for excision of the elements from the nia gene in
different genetic backgrounds. Results obtained so far indicate that these copies transpose in
different species thus providing a potential gene tagging system for filamentous fungi.
37. Distribution of the restless Transposon in Different Beauveria nivea strains.
Sabine Jacobsen, Frank Kempken, Ulrich Kuck; Lehrstuhl fur Allg. Botanik, RuhrUniversitat, D-44780 Bochum
The restless element is an Ac like class 11 transposon of 4,097 bp, which was found in the filamentous fungus Beauveria nivea (syn. Tolypocladium inflatum). This strain (ATCC 34921) has been shown previously to carry about fifteen copies of the restless element (1). As a first step in the study of the evolution and distribution of this element, three other B. nivea strains from type culture collections were chosen for a molecular characterization. The B. nivea strains were investigated by Southern hybridization and PCR for the presence of copies of the restless element. The molecular analysis of the four strains with similar morphology includes differentiation by RAPD and a partial sequence comparision of the conserved tubulin gene. In contrast to the sequence analysis, which showed a high degree of similarity between all strains, the number of restless copies was divergent in all strains investigated. In addition to strain B. nivea ATCC 34921 with about fifteen restless copies we identified strains with only a single or a partial copy. These copies were further characterized by DNA and cDNA sequencing. The integration sites have been determined by inverse PCR. Finally, we identified a strain, which did not show any sequence homology to the restless element.
(1) Kempken F, Kuck U (1996) Mol Cell Biol 16: 6563-6572
38. A retroelement from Ascobolus immersus is not subject to "methylation induced premeiotically".
Frank Kempken; Lehrstuhl fur Allgemeine Botanik, Ruhr-Universitat Bochum, Universitatsstrae 150, D-44780 Bochum, Germany
The first evidence for the presence of transposable elements in mycelial fungi came from classical genetic analysis of two genetic instable stocks (28 and 50) of Ascobolus immersus [1]. The existence of transposable elements in mycelial fungi is well established know and a number of transposons have been characterized on the molecular level [e.g. 2, 3], which includes at least one retroelement from A.immersus stock 28 [cited in ref 4]. Differential hybridization was employed to identify repetitive DNA sequences from Ascobolus immersus stock 50, which has a different system of genetic instability than stock 28 [1]. One stock 50 specific repeated DNA sequence was further analyzed. Preliminary sequence analysis indicates the presence of long terminal repeats flanking the element, which presumably represents a retrotransposon. Most interestingly, this element does not appear to be subject to "methylation induced premeiotically", a mechanism which is believed to inactivate repeated DNA sequences in A. immersus [4].
References:[1] Nicolas A, Hamza H, Mekki-Berrada A, Kalogeropoulos A, Rossignol J-L (1987)
Genetics 116:33-43- [2] Daboussi M-J, Langin T (1994) Genetica 93:49-59; [3] Kempken F,
Kuck U (1996) Mol Cell Biol 16:6563-6572; [4] Rossignol JL, Faugeron G (1994) Experientia
50:307-317
39. Regulation of Restless, a fungal member of the hAT- transposon family.
Frank Kempken, Tina Nakonz and Ulrich Kuck; Lehrstuhl fur Allgemeine Botanik, Ruhr-Universitat Bochum, Universitatsstrae 150, D-44780 Bochum, Germany.
In the past years several fungal transposable elements have been identified [1]. We have isolated and characterized Restless, a new type of fungal class II transposons from Tolypocladium inflatum ATCC34921 (synonym: Beauveria nivea) which so far has not been found in any other fungus [2]. It carries short inverted repeats and eight basepair target site duplications, and encodes a large open reading frame which is interrupted by a single intron sequence showing a rare intron consensus sequence. The predicted amino acid sequence deduced from this frame shows significant homology to transposases of the hAT transposon family, e.g. the maize Activator element. Based on cDNA sequencing, alternate RNA splicing may lead to two different proteins, both encoded by the transposon. These proteins may be involved in regulation of transposition. To proof this hypothesis, proteins were overexpressed in E. coli and employed for gel shift and footprint analysis. From these data, a model will be presented for regulation of restless transposition.
References: [1] Daboussi M-J., Langin T. (1994) Genetica 93:49-59- [2] Kempken F., Kuck U.
(1996) Mol Cell Biol 16:6563-6572
40. Promiscuous template switching by the Mauriceville plasmid reverse transcriptase and its role in senescence. John C. Kennell, Dept. of Biological Sciences, Southern Methodist University, Dallas, TX 75275
Variant forms of the Mauriceville mitochondrial (mt) plasmid of Neurospora crassa appear
to cause senescence by integrating into mtDNA or by overaccumulating in the mitochondrion.
The first step in senescence is the copying of mitochondrial transcripts by the plasmid-encoded
reverse transcriptase (RT) and the incorporation of these cDNAs into the circular DNA plasmid.
The RT can template switch directly from the 5' end to the 3' end of the plasmid transcript and,
unlike strand switching associated with retroviral RTs, this reaction is not dependent on homology
between templates. Without the specificity conferred by a template switching mechanism that
requires sequence homology between templates, the plasmid RT can copy unrelated RNAs
resulting in the production of suppressive forms of the plasmid and ultimately to senescence. In
vitro studies show that the plasmid RT can template switch at high frequencies and has less
specificity for templates than is required for primer-independent cDNA initiation.
Characterization of multimeric cDNA products showed that there are nontemplated nucleotides
inserted at the junction sites, which may provide a clue concerning the mechanism of homology-independent template switching. Studies of senescent cultures show that formation of variant
plasmids occurs at high frequencies and a new type of suppressive plasmid has been identified.
The evolutionary significance of a homology-independent mechanism of template switching will
also be discussed.
41. LINEs and SINES in Magnaporthe grisea.
Marc J. Orbach and M. Anthony Meyn. Department of Plant Pathology, University of Arizona, Tucson, AZ.
Several transposable elements have been identified in the rice blast pathogen, Magnaporthe grisea, in attempts to determine their role in the genetic instability observed in this fungus. We report the characterization of the first LINE retrotransposon found in M. grisea, that we have designated MGL, which is homologous to MGR583. This 6 kb element is highly conserved both in rice pathogens and pathogens of other grasses, although there is variability in copy number among the strains. Surprisingly, although full length transcripts have been observed, movement of MGL has not. MGL contains two open reading frames (ORFS) which overlap by a single base pair. The first ORF contains homology to retroviral gag proteins and the second to reverse transcriptase (RT) polypeptides. Most MGL copies are flanked by short, variablelength, direct repeats as is characteristic of this class of elements. The 3' end of MGL is characterized by variable numbers of a TAC repeat, a feature also observed in the SINE element, MG-SINE, of M. grisea (Kachroo et al. 1995). Sequence comparisons reveal that the last 240 nt of MG-SINE are 90% identical to MGL. This homology suggests a model for the origin of MG-SINE, and a mechanism for MG-SINE transposition that relies on the RT of MGL, and utilizes the end of MGL for initiation of reverse transcription. A revised distribution of these two elements will be presented.
Kachroo, P., Leong, S.A., Chattoo, B.B. (1995) Mg-SINE: a short interspersed nuclear element from the rice blast fungus, Magnaporthe grisea. Proc. Natl. Acad. Sci. USA 92:11125-11129