Repetitive DNA elements in fungi (Mycota): impact on ... - Springer Link

Curr Genet (2002) 41: 189–198 DOI 10.1007/s00294-002-0306-y

R EV IE W A RT I C L E

Johannes Wo¨stemeyer Æ Anne Kreibich

Repetitive DNA elements in fungi (Mycota): impact on genomic architecture and evolution

Received: 27 March 2002 / Accepted: 24 April 2002 / Published online: 21 June 2002
Springer-Verlag 2002

Abstract Repetitive DNA elements, microsatellites or simple repeats, minisatellites, mobile elements that transpose at the level of DNA, retrotransposons and various derivatives thereof are ubiquitous constituents of all fungal genomes. Many of these elements, especially the different types of transposon, have been cloned and characterised at the sequence level. Their biological role, however, has not yet been sufficiently elucidated. We are far from understanding the selection mechanisms that tend to conserve repeated DNA at defined loci. There is also little insight into the mechanisms that provide the balance between spreading repetitive elements within genomes and control of their copy number. Depending on the fungal group, this balance can be stabilised at different levels. Asco- and basidiomycetes rarely contain more than 5% repetitive DNA, whereas the phylogenetically older division Zygomycota is characterised by typically more than 30%. The effects of repetitive DNAs on the expression of adjacent genes are only rarely understood and their role for genomic plasticity on an evolutionary time scale is still especially enigmatic. This survey summarises the main characteristics of well studied experimental systems and intends to define important open questions for stimulating future research. Keywords Genome organisation Æ Microsatellite Æ Minisatellite Æ Transposon

Introduction The reason why fungi are favoured as model systems in the molecular analysis of basic biological phenomena is their

Communicated by K. Esser J. Wo¨stemeyer (&) Æ A. Kreibich FSU Jena, Institut fu¨r Mikrobiologie, Lehrstuhl fu¨r Allgemeine Mikrobiologie und Mikrobengeneti, Neugasse 24, 07743 Jena, Germany E-mail: [email protected]

small genome, which, as a rule, contains only limited amounts of repetitive DNA. Aspergillus nidulans, with a genomic complexity of 27 Mb, has only some 5% repetitive DNA, most of which is accounted for by rDNA repeats (Timberlake 1978). In other widely studied model systems, Neurospora crassa and Saccharomyces cerevisiae (Mewes et al. 1997), the situation is similar. According to reassociation analysis, N. crassa has a genomic complexity of 27 Mb and contains not more than 8% repetitive DNA, most of which is accounted for by rDNA repeats (Krumlauf and Marzluf 1980). Nevertheless, even these streamlined genomes are not free of repetitive elements. Transposons, diverse retrons, micro- and minisatellites are also normal constituents of fungal genomes. All these elements, including retrons, are also found in prokaryotic organisms and are thus considered as evolutionarily ancient genomic components of the living world. Among the Eumycota, the evolutionarily younger divisions, Ascomycota and Basidiomycota, have a strong tendency towards streamlined genomes. The situation is different for the more ancient division, Zygomycota. Mucoralean zygomycetes have total genomic sizes (including repetitive DNA) between 42 Mb (Phycomyces blakesleeanus, Phycomycetaceae; Dusenbery 1975) and 54 Mb (Absidia glauca, Absidiaceae; Wo¨stemeyer and Burmester 1986). In these zygomycetes, the repetitive DNA content ranges around 35%. The picture emerging from P. blakesleeanus and A. glauca seems to be a general one. Repetitive DNA contents between 16% (Actinomucor elegans, Mucorales) and 62% (Coemansia formosensis, Kickxellales) were measured for 21 different species from the major orders of Zygomycetes (Bru¨hschwein and Wo¨stemeyer, unpublished data). The data were determined by reassociation kinetics, which yields very precise measurements of genomic size, but tends to underestimate the amount of repetitive elements if these are short. Sequence analysis of extended chromosomal regions revealed short repetition units as major constituents of all fungal genomes.

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Interestingly, the basal fungal division Zygomycota has more complex genomes and a much higher degree of repetitivity than the younger groups, asco- and basidiomycetes. In addition to micro- and minisatellites, they contain more complex repetitive DNA elements (Avalos et al. 1996; Burmester and Wo¨stemeyer, unpublished data) that cannot be classified appropriately, due to a scarcity of sequence information. Detailed information in this fungal group is only available for the glomalean arbuscular mycorrhizal fungus Scutellospora castanea, with respect to two longer repetitive DNA elements, SC1 (Zeze et al. 1996) and MYCDIRE (Zeze et al. 1999). Unfortunately, even now there is no information on basic genomic parameters of probably the most ancient eumycotal division, the Chytridiomycota. Apart from characterising rDNA repeat units, studying repetitive DNA in fungi has never been performed on a large scale. The first recognised fungal minisatellite was sequenced in 1984 before the term minisatellite was coined (Saccharomyces cerevisiae; Horowitz and Haber 1984), followed by a minisatellite from S. carlsbergensis more than 10 years later (Andersen and Nilsson-Tillgren 1997). Even today, only six minisatellite sequences, all from ascomycetes, have been characterised in some detail. Although repetitive DNAs have been widely used for molecular taxonomy and pathogen analysis (Voigt et al. 1998; Weising et al. 1991), functional aspects have been little studied and only recently. This review deals predominantly with structural and functional aspects of three major classes of repetitive DNA elements: microsatellites, minisatellites and transposons. Applicative aspects, like using microsatellite probes for fingerprinting, molecular taxonomy and diagnosis of phytopathogens, is not covered.

Microsatellites DNA regions, usually 20–60 bp in length, with tandem repetitions of 1–5 base pairs are called microsatellites. Typical repeat units are (GT)n, (CA)n, (CAA)n or (GACA)n. They are a common feature in all eukaryotes, may occur in as many as 105 different locations (Field and Wills 1996) and are also found in prokaryotes, although at lower frequency. In the yeast S. cerevisiae, microsatellites have also been found in mitochondrial DNA (Sia et al. 2000). Microsatellites in fungi have been detected by PCR analysis of bulk DNA, based on typical microsatellite primers (Meyer et al. 1991, 1992). They have been found in all divisions of Eumycota and also in the myxomycete Physarum polycephalum. The most frequent repeat in fungi is AT. Relative repeat frequencies are similar between fungi and plants, but differ considerably in metazoa (Groppe et al. 1995). As many as 25 different microsatellites were detected by this approach in a single species, Ascochyta rabiei (Weising et al. 1991). Microsatellite regions are highly mutable and thus are able to differentiate between related taxa, even at the level of

individual isolates in a single species. Simple DNAprofiling methods based on microsatellite variability provide possibilities to identify individual genotypes for studies in population genetics, ecology and taxonomy. Few investigations have focused on characterising individual fungal microsatellites at the sequence level. Table 1 provides an overview on the most thoroughly studied microsatellites in fungi. Epichloe typhina Microsatellites in fungal genomes were used for straintyping, especially of phytopathogens before the loci where they reside were sequenced. Probably the first combination of a simple strain-typing study with detailed molecular analysis was performed for the primarily mutualistic grass endophyte E. typhina, which may develop into a castrator pathogen of grasses later in their development (Groppe et al. 1995). By cloning and sequencing segregated randomly amplified polymorphic DNA bands with similar size, a specific locus with (AAG)n repeats and n of 8–18 was identified. In addition, many microsatellites with different monomeric motifs were detected in Epichloe sp. Podospora anserina All chromosomes of P. anserina harbour the simple repeat (GT)n (Osiewacz et al. 1996). Screening a genomic library constructed in k EMBL4 for this sequence revealed that the poly(GT) microsatellite occurs in approximately 5% of all fragments. One location was analysed at the nucleotide level; and a stretch of 101 bp of alternating purine/ pyrimidine nucleotides was found to contain an uninterrupted (GT)38 microsatellite. The positions of the (GT)n microsatellites are unexpectedly constant in genetically distinguishable Podospora isolates from different geographical locations, as hybridisation studies with a labelled (GT)8 probe revealed only a few restriction-length polymorphisms. The data are not sufficient to reach conclusions on the functional characteristics of the (GT)n Table 1. Fungal microsatellites Organism

Sequence motif

Number of repetitions

Reference

Epichloe typhina

AAG

8–18

Podospora anserina

GT

38

Saccharomyces cerevisiae S. cerevisiae, mtDNA

GT

7–52

AT

Highly variable

GT

Variable, shorter than AT-tracts

Groppe et al. (1995) Osiewacz et al. (1996) Wierdl et al. (1997) Sia et al. (2000); Foury et al. (1998) Sia et al. (2000); Foury et al. (1998)

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microsatellite. The high constancy of poly(GT) locations between Podospora isolates may indeed be due to selective advantages, as the authors propose. Alternatively, if the loss rates of microsatellites are small, these loci may have arisen prior to the genetic separation of the isolates. In any case, stretches with alternating purine and pyrimidine are good candidates for regulatory function, as they may form Z-DNA structures in vivo (Nordheim and Rich 1983) and, therefore, may be targets of regulatory proteins. In this respect, poly(GT) microsatellites in the upstream region of genes deserve further study. In fungi, such a situation was found in the regulatory region of the gene MRAS3 in the mucoralean zygomycete Mucor circinelloides (Casale et al. 1990; Osiewacz et al. 1996). The frequent association of microsatellites with promoter regions and possibly other putative regulatory functions has been discussed in some detail for mammals (Kashi et al. 1997; King et al. 1997). S. cerevisiae nuclear genome The influence of tract length of a (GT)n microsatellite was studied in the nuclear genome of S. cerevisiae (Wierdl et al. 1997). Poly(GT) stretches with lengths of 15–105 bp were inserted in-frame into the coding region of a plasmid-encoded URA3 gene propagated by a ura3 host (Ura+ strains). As a consequence of microsatellite instability, most derivatives contained out-of-frame lengths of the (GT)n stretch and were phenotypically uridine auxotrophs, which could be selected by adding 5-fluoro-orotic acid to the medium. This simple genetic test system revealed several basic characteristics. 1. The instability rates between short and long microsatellites differ by a factor of 102. 2. The frequency of single-repeat additions increases continuously with microsatellite length. 3. Larger deletions (>2 repeats) are more frequent in (GT)n stretches >51 bp than in shorter ones. 4. Larger tracts (99 bp, 105 bp) have a tendency towards addition rather than deletion of single repeats. 5. Undergoing meiosis does not influence mutational rates. Most probably, length variations are consequences of DNA polymerase slippage reactions during replication. If the resulting loops are not corrected by mismatch repair, the deviations manifest themselves as deletions or insertions. The decision between this pair of possibilities depends on the position of the loop, which is either on the template strand, leading to deletions, or on the newly synthesised primer strand, leading to insertions. S. cerevisiae chondriome The yeast mitochondrial genome is rich in microsatellites. To test these sequences for stability, arg8m

derivatives with out-of-frame inserts of (AT)n or (GT)n stretches were constructed (Sia et al. 2000). ARG8 is a nuclear gene, the product of which is transported into mitochondria; and arg8m is a mutant derivative of the nuclear ARG8 gene that can be expressed directly in mitochondria. When introduced into mitochondrial DNA by transformation, arg8m is able to complement nuclear ARG8 defects. Restoring arginine prototrophy by frame-shift mutations in the repetitive inserts was used as an indicator for the mutability of mitochondrial microsatellites. The results differed from those obtained in nuclei. In contrast to the nuclear genome, mitochondrial microsatellite mutations usually involve single-repeat deletions rather than insertions. Whereas poly(AT) and poly(GC) have nearly the same stability in nuclei, poly(AT) has a considerably increased stability in mitochondria. If polymerase slippage is the reason for mutant generation, it is hard to see why slippage itself should occur more frequently in the less stable (GT)n tracts. It appears more reasonable to assume that (AT)n loops are better substrates either for mismatch repair by the proof-reading exonuclease function of DNA polymerase c, or for mitochondrial postreplicative mismatch repair. There is also a striking difference between haploid and diploid cells. Both types of microsatellite are approximately 100-fold more stable in diploids than in haploids. Why the ploidy of the nucleus influences mitochondrial mutability is not understood. A. rabiei The general picture of microsatellites as reiterations of short simple repeats must be completed by the more complex compound microsatellite ArMS1, which was detected in an extremely AT-rich chromosomal region of the chickpea (Cicer arietinum) pathogen A. rabiei. ArMS1 (Geistlinger et al. 1997) is composed of the repeated pentanucleotides (CATTT)n, (CATTA)n and (TATTT)n. It also contains repetitions of the decanucleotide (CATATCATTT)n. ArMS1 has a length of 515 bp, consists exclusively of uninterrupted stretches of the four basic repeat units and belongs to the largest microsatellites so far known. With respect to total extension, the length of the basic sequence motifs and its composite structure, ArMS1 marks the border to the more complex minisatellites.

Minisatellites Minisatellites, by definition, consist of basic sequence motifs of 10–60 bp, which are amplified to lengths of 0.1–30 kb. They are ubiquitous, rapidly evolving sequences with a high degree of length polymorphism, but their position appears to be essentially stable. They are often GC-rich; and many minisatellites share a common consensus sequence with a striking similarity to the C-sequence of the Escherichia coli bacteriophage k.

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Although suggestive, the similarity with this recombination signal from prokaryotes is probably coincidental. At least in mammals, the expected increase in meiotic recombination of regions flanking the consensus sequence could not be found (Vergnaud et al. 1991). With respect to metazoa, especially humans, the properties of minisatellites have recently been reviewed (Vergnaud and Denoeud 2000). Not many fungal minisatellites have been analysed in detail; and the few well studied systems are introduced here in some detail. Table 2 provides an overview of characterised minisatellites in fungi. S. cerevisiae Shortly before the word ‘‘minisatellite’’ was coined by Jeffreys et al. (1985), such a tandem arrangement of between 8 and 20 copies of a 36-bp basic unit was found in a subtelomeric region of S. cerevisiae (Horowitz and Haber 1984). A second minisatellite was also found subtelomerically. Here, a basic unit of 56 bp was repeated eight times (Louis et al. 1994). In both minisatellites, as in the one from S. carlsbergensis discussed below, the repeats are flanked by direct repeats of 5 bp. The authors propose that the short direct repeats, which are also found in many human minisatellites, may play a crucial role for their phylogenetic origin by polymerase slippage, followed by several rounds of unequal crossing-over or gene conversion (Haber and Louis 1998). Although this model may reflect the origin of some minisatellites, its general applicability is hard to imagine. If 5-bp flanking regions are sufficient for converting DNA with the appropriate length of approximately 100 bp between them into minisatellites, the frequency of such elements should be much higher than observed. The chance of generating identical oligonucleotide pentamers is 45 or roughly 103. Thus, minisatellites should occur after every tenth segment of the usual repeat length of 100 bp, at least in non-coding genomic regions. This exceeds by far the observed incidence of minisatellites in fungi. If the model is correct Table 2. Fungal minisatellites

Organism

Element

Saccharomyces cerevisiae S. carlsbergensis

and generally valid, additional assumptions with respect to limiting factors of minisatellite generation, frequency of slippage, efficiency of repair systems, or similar must be made. S. carlsbergensis A minisatellite from the lager-brewing yeast S. carlsbergensis has a composite structure of 13 tandem repeats with a length of 12 bp. The minisatellite is inserted in-frame in an open reading frame (ORF), which in S. cerevisiae is called YCL010c. Deletion of 12 of the 13 repeat units would restore the alignment with S. cerevisiae (Andersen and Nilsson-Tillgren 1997). This observation may be interpreted as a clue towards the phylogenetic emergence of minisatellites and suggests that they were created by expansion of pre-existing structures. P. anserina By screening a genomic cosmid library of P. anserina with a (GT)8 probe, a minisatellite with high GTcontent and a complex structure was isolated (Hamann and Osiewacz 1998). PaMin1 is composed of ten identical copies of a 16-bp basic repeat unit. Each repeat unit contains the motif (GT)5. This central region is flanked by incomplete repeat units. Downstream, the element is followed by another GT-stretch and an additional repetitive element consisting of four slightly varied dodecamers. Six alleles of PaMin1, distinguishable by variations in the number of repeat units, were found in 18 different P. anserina isolates. According to Southern blotting data, PaMin1 maps to a single locus. Whereas the immediate vicinity of the element, analysed by looking at a 1.8-kb Taq1 restriction fragment, is conserved in different strains, the sequences at larger distances are highly polymorphic. BamHI restriction fragments vary in length between 8 kb and 20 kb. Monomer length

Number of repeats

Characteristics

Reference

36

8–10

56

8

12

13

5-bp direct flanking repeats 5-bp direct flanking repeats Located in ORF

Horowitz and Haber (1984) Louis et al. (1994) Andersen and NilssonTillgren (1997) Hamann and Osiewacz (1998) Giraud et al. (1998) Attard et al. (2001)

Podospora anserina

PaMin1

16

10

Botrytis cinerea

MSB1

37

5–11

Leptosphaeria maculans

MinLm1

23

2–7

Occurring in a small microsatellite cluster Located in gene for ATP synthase 6-bp direct flanking repeats

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Botrytis cinerea An AT-rich minisatellite, MSB1, was identified in an intron of the gene for ATP synthase in the plant pathogenic ascomycete B. cinerea (Giraud et al. 1998). The basic repeat unit has a length of 37 bp; and the number of repeats from different isolates varies between 5 and 11. The repeat units differ somewhat in sequence and can, therefore, be recognised individually as variants of a common motif. These variants map in identical order in all fungal isolates. There is also no evidence for recombination in flanking regions. Both observations together suggest that the repeat variants are not generated by recombination after parent strains with differences in minisatellite sequences crossed, but rather by mistakes of the replication machinery, basemisincorporation and slippage. The minisatellite is taxon-specific and is lacking in P. anserina and even in the very closely related species Sclerotinia sclerotiorum and S. homoeocarpa. MSB1 must have been generated after the evolutionary process separating the genera Botrytis and Sclerotinia. Leptosphaeria maculans Like MSB1, MinLm1 is a single-locus minisatellite, isolated from aggressive (tox+) field isolates of the rape seed pathogenic loculoascomycete L. maculans (Attard et al. 2001). The basic repeat unit has a length of 23 bp, is highly conserved, completely strand-asymmetric for G/C distribution and flanked by 6-bp direct repeats. The polymorphism between different fungal isolates is due to variation in the number of repeat units (2–7 repeats). MinLm1 maps to a region with many different microsatellites.

at the DNA level. The basic structural characteristics of fungal transposable elements are summarised schematically in Table 3 and Fig. 1; and the categories match essentially with previous schemes (Daboussi 1996, 1997; Kempken and Ku¨ck 1998a). Classification is not always easy, for example Eg-R1 from Erysiphe graminis shares features of SINEs and LINEs. Transcript size and the lack of major ORFs are SINE-like features but, in contrast, the transcript is probably transcribed by RNA polymerase II, a typical LINE feature (Wei et al. 1996). Four different strategies were used to identify fungal transposable elements; and these aspects were reviewed in detail by Kempken and Ku¨ck (1998a). 1. The most successful approach for the isolation of active elements was transposon trapping. In almost all cases, the transposon was isolated after insertion into the nitrate reductase gene (niaD). Isolation of the Tad element from N. crassa was facilitated by spontaneous insertion into the glutamate dehydrogenase gene (am; Kinsey and Helber 1989). Ascot from Ascobolus immersus was cloned after insertion in the b2 gene specifying spore colour (Colot et al. 1995). The niaD gene had some advantages, as nitrate reductase deficient mutants were resistant to chlorate and thus could easily be selected for. 2. Transposons with high copy number were often found via cloning and sequencing of repetitive DNA. 3. Retroelements could also be detected by screening expression libraries with antibodies against reverse transcriptase or simply by PCR with oligonucleotide primers complementary to conserved RT motifs. 4. In some cases, the use of heterologous probes was successful.

Epigenetic control of repeated DNA segments

Transposons Diversity of transposable elements The analysis of repeated DNA elements at the molecular level in fungi dates back to the discovery of Ty1 in Saccharomyces cerevisiae (Cameron 1979). The interest in transposable elements increased when geneticists began to search for active elements, suitable for transposon tagging. With this aim, the Tad element from N. crassa was cloned and analysed (Kinsey and Helber 1989). Portable genetic elements are divided into two main classes, depending on their mechanism of transposition. Class I elements possess their own reverse transcriptase and transpose via RNA intermediates. Short interspersed nuclear elements (SINEs), which may be described as deletion products of autonomous long interspersed nuclear elements (LINEs) and lack the gene for reverse transcriptase, are an exception among class I elements, as they depend on complementation by intact elements. Class II elements transpose directly

Several mechanisms for inactivation (not only of transposable elements) are known in fungi. Both RIP (repeat induced point mutation; primarily: rearrangement induced premeiotically), identified in N. crassa (Selker 1990), and MIP (methylation induced premeiotically), discovered in A. immersus, depend on the sexual cycle, as they occur in the heterokaryotic stage between fertilisation and karyogamy. RIP results in C to T and G to A transitions and is often accompanied by the methylation of remaining cytosines. The system has a strong preference for CA dinucleotides (Watters et al. 1999). MIP leads to methylation of cytosine residues without mutations and, in contrast to RIP, is a reversible process. Both systems share the feature that, in duplicated sequences, either both copies or neither copy is subject to RIP or MIP. Repetitive sequences like transposons are often highly methylated. After ectopic multi-copy integration of Restless (an active class II element from Tolypocladium inflatum) in N. crassa, all transformants exhibit at

194 Table 3. Fungal transposons. Data are based on previous compilations (Daboussi 1997; Kempken and Ku¨ck 1998a) and are supplemented by novel literature. Where element lengths are not given,

data vary too much between different isolates or were measured only for parts of the element. LINE Long interspersed nuclear element, SINE short interspersed nuclear element

Organism

Element

Class

Type

Ascobolus immersus

Mars4 Mars2 Mars3 Mars1 Ascot1 Tascot Hideway Afut1 Dane1,2 Ant1 Vader Tan Hupfer Boty Flipper CfT1 Fcc1 Cgret CgT1 Crypt1 Eg-R1 EgH24 Foret1 Skippy Palm Foxy Impala Fot1 Fot2 Folyt Tfo1 Hop Maggy Grh Grasshopper Fosbury MGR583 MGSR1 Mg-SINE Pot2 MGR586 Nrs1 DAB1 Tad1 Punt Guest Pce1 Prt1 Yeti Pat Restless

I I I I II II I I I II II II II I II I II I I II I I I I I I II II II II II II I I I I I I I II II I I I II

Gypsy Copia Copia LINE Ac Ac

Aspergillus fumigatus A. nidulans A. niger Beauveria bassiana Botrytis cinerea Cladosporium fulvum Cochliobolus carbonum Colletotrichum gloeosporioides Cryphonectria parasitica Erysiphe graminis Fusarium oxysporum

Magnaporthe grisea

Nectria haematococca Neurospora crassa

Phanerochaete chrysosporium Phycomyces blakesleeanus Podospora anserina Tolypocladium inflatum

II I I II II

least partial methylation, in contrast to site-specific single copy integration transformants (Windhofer et al. 2000). Apart from mechanisms that modify repeated DNA elements during sexual differentiation pathways, functionally similar processes have been elucidated during mitotic growth. One pathway known to inactivate expression of repeated sequences is quelling. Additional copies of DNA integrated in N. crassa sometimes lead to inactivation of the homologous gene. Quelling seems to be a post-transcriptional process that operates in vege-

Gypsy Gypsy Tc/Mariner Fot1 Fot1 Tc/Mariner Gypsy Fot1 Gypsy Fot1 Gypsy LINE Ac SINE SINE Gypsy Gypsy LINE SINE Tc/Mariner Fot1 Fot1 Ac Ac Gypsy Gypsy Gypsy Gypsy LINE SINE SINE Fot1 Fot1 SINE Gypsy LINE Fot1

Gypsy Fot1 Ac

Size (kb)

3.6 2.5 6.9 4.8 0.4 1.7 3.3 1.9 7.0 1.8 7.9 5.7 3.6 0.9 7.8 0.7 1.3 1.9 2.1 2.6 2.8 3.5 5.6 8.0 0.8 0.5 1.9 1.9 0.5 6.9 1.9 0.1 1.7 4.7 6.9 1.85 4.1

Reference Goyon et al. (1996) Goyon et al. (1996) Goyon et al. (1996) Goyon et al. (1996) Colot et al. (1995) Accession number Y07695 Kempken (2001) Neuveglise et al. (1996) Nielsen et al. (2001) Glayzer et al. (1995) Amutan et al. (1996) Nyysso¨nen et al. (1996) Maurer et al. (1997) Diolez et al. (1995) Levis (1997) McHale et al. (1992) Panaccione et al. (1996) Zhu and Oudemans (2000) He et al. (1996) Linder-Basso et al. (2001) Wei et al. (1996) Rasmussen et al. (1993) Julien et al. (1992) Anaya and Roncero (1995) Moyna et al. (1996) Mes et al. (2000) Langin et al. (1995) Daboussi et al. (1992) Daboussi and Langin (1994) Go´mez-Go´mez et al. (1999) Okuda et al. (1998) Daboussi and Langin (1994) Farman et al. (1996b) Dobinson et al. (1993) Dobinson et al. (1993) Shull and Hamer (1996) Hamer et al. (1989) Sone et al. (1993) Kachroo et al. (1995) Kachroo et al. (1994) Farman et al. (1996a) Kim et al. (1995) Bibbins et al. (1998) Kinsey and Helber (1989) Margolin et al. (1998) Yeadon and Catcheside (1995) Gaskell et al. (1995) Ruiz-Pe´rez et al. (1996) Hamann et al. (2000a) Hamann et al. (2000b) Kempken and Ku¨ck (1996)

tative cells and reduces the level of mature mRNA without affecting primary transcription efficiency. Quelling appears to include a diffusible factor, as it is dominant in heterokaryons (Pandit and Russo 1992; Cogoni et al. 1996; for review see Selker 1997). The impala element impB from Fusarium oxysporum exhibits features reminiscent of RIP. impB repeats have an increased tendency towards transitions, but, in contrast to ripping, the system does not discriminate between CA and CG dinucleotides (Hua-Van et al. 1998). It is not completely clear whether these genetic

195

F. oxysporum have been encouraging. A non-autonomous imp::hph element was constructed and could be shown to be activated in trans, both in the homologous genetic background and in F. moniliforme (Hua-Van et al. 2001).

Genomic plasticity, a consequence of repeated DNA elements?

Fig. 1. Genetic organisation of major fungal transposon types. Class I elements are essentially similar and may even originate from common ancestors, including short interspersed nuclear elements, which are interpreted as deletion derivatives of long interspersed nuclear elements without open reading frames. The genetic diversity of Class II elements and their transposition mechanisms is probably much higher. env Gene for viral envelope-like protein, gag gene for structural protein probably of viral origin, pol gene for reverse transcriptase, LTR long terminal repeat, TIR terminal inverted repeat, TSD target site duplication. The pol region often carries additional domains: In integrase, Pr protease, RH RNaseH, RT reverse transcriptase

modifications, in addition to abolishing the transposase activity, actually reduce transposition frequency by in trans complementation. impala has indeed been shown to transpose by in trans expression of the transposase with a modified element, obtained by artificial integration of a HygR marker gene (Hua-Van et al. 2001). In this respect, impala bears similarities to the classic Activator/Dissociation (Ac/Ds) two-element-system in Zea mays (McClintock 1951). Gene tagging Active transposable elements deserve special interest with respect to gene tagging in homologous and even more so in heterologous systems. Provided a transposon has little sequence specificity with respect to its target site, the arbitrary hitting of genes and thus site-specific saturation mutagenesis of complete fungal genomes could be possible. In plants, the maize Ac transposon has been used successfully in Arabidopsis thaliana (Altmann et al. 1995). The autonomous element Restless from Tolypocladium inflatum, which is thought to transpose via a circular intermediate (Kempken and Ku¨ck 1998b), is one of few promising candidates (Windhofer et al. 2000), although inactivation in foreign genetic backgrounds by RIP-like processes (Windhofer et al. 2000) may pose technical problems, depending on the time-course of inactivation after introducing the element. Employing autonomous elements for gene tagging may have the disadvantage of generating unstable insertions. To cope with this problem, the use of twoelement systems, analogous to the maize Ac/Ds system, has been proposed. First experiments with genetically engineered derivatives of the impala element from

One of the most intriguing questions concerning repetitive DNA elements, be they simple repeats, minisatellites or autonomous transposable elements, is their impact on genomic architecture and evolution. This is more a field of opinion and speculation, as modelling experiments under controlled laboratory conditions do not exist. The availability of genetically engineered elements, by insertion of appropriate reporter genes, could open innovative experimental strategies. Both in bacteria and in eukaryotes, the insertion of repetitive DNA is able to modulate the transcriptional activity of adjacent genes. Frequent transposition of autonomous elements, insertion of retrons or, in the case of microsatellites, simple repeated DNA stretches could also provide preferential sites for recombination. There is, however, little experimental evidence in fungi that the presence of repetitive elements generally increases genetic instability at the loci where they reside. However, experimental indications were noticed that, for the retro-transposon skippy from F. oxysporum, environmental parameters (in this case stress induction by addition of chlorate anions) led to increased activity of the element. Several alternative recombination events were monitored: increased tandem amplifications of the element, generation of incomplete copies of the element at novel loci and also excision of the element (Anaya and Roncero 1996). With our present knowledge on the genetic consequences of DNA repetitions in fungi, there seem to be as many mechanisms to eliminate the activity of repeated elements as there are to spread them. At least for ascoand basidiomycetes, the tendency for excluding large amounts of repetition seems to predominate. In zygomycetes, with their extraordinarily high degree of repeated DNA, the situation may be different. However, even in this fungal division, there is no evidence for increased genetic instability, neither via increased transposition nor by stimulation of recombination frequency. In the highly repeated rDNA clusters in S. cerevisiae, recombination frequencies are reduced and mechanisms that prevent certain regions from undergoing crossover have been postulated. Although rDNA has been shown to be not completely free from recombination events (Kempken 2001), the reduction of recombination in essential repeats is definitely beneficial with respect to genomic stability, as it prevents these repeats from being lost by deletions via crossovers between adjacent tandem repeats and from other deleterious rearrangements.

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