Transposable Elements are the Factors Involved in ... - Springer Link

Russian Journal of Genetics, Vol. 39, No. 5, 2003, pp. 505–518. Translated from Genetika, Vol. 39, No. 5, 2003, pp. 621–636. Original Russian Text Copyright © 2003 by Shnyreva.

Transposable Elements are the Factors Involved in Various Rearrangements and Modifications of the Fungal Genomes A. V. Shnyreva Department of Mycology and Algology, Moscow State University, Moscow, 119992 Russia; e-mail: shnyreva@herba.msu.ru Received June 21, 2002

Abstract—Data on transposable elements in fungal genomes are reviewed. Possible role of transposons in the pathogenetic processes and regulation of mating compatibility are discussed. The transposition-inducing factors and mechanisms responsible for transposition within the genome are considered.

Transposable genetic elements (TEs), transposons, are DNA sequences that can be excised and integrated into new loci of the genome; some of them replicate autonomously. In recent 15 years, transposons were identified in a large variety of fungal species from different taxonomic groups, and their list has been constantly growing (table). Recent computer-aided analysis of several sequenced genomes of eukaryotes, such as human, nematode Caenorhabditis elegans, fruit fly Drosophila melanogaster, plant Arabidopsis thaliana, and yeast Saccharomyces cerevisiae, showed that in these organisms, most of repeated DNA sequences are represented by active or degenerate transposon copies [1–3]. In humans, they occupy at least 40% of the total genome. Transposons found in fungal genomes reflect the entire spectrum of their diversity among eukaryotes. Like in other eukaryotes, the fungal transposable elements belong to two classes that are fundamentally different in structure and mechanisms of transposition [4]. The class I mobile elements are transposed via an RNA-mediator by means of the reverse transcription that involves integration enzymes including reverse transcriptases: DNA RNA DNA. The class II mobile elements are transposed directly, DNA DNA, which is provided by transposases encoded by the transposons themselves. Terminal repeats flanking the transposon sequence (long direct terminal repeats, LTRs, and short inverted terminal repeats, ITRs, in class I and class II transposon, respectively) are directly involved in transposition of the mobile elements to new sites, because they serve as specific targets for integration. Several methods can be used to real active transpositions. (1) Cloning and analysis of the repeated DNA sequences. In this way, the MGR586 and grasshopper transposable elements of the Magnaporthe grisea [5, 6], as well as the Foret element of the Fusarium oxysporum [7] and CFT-1 transposon of Cladosporium fulvum [8] were identified. (2) Transposon trapping, a technique based on disrupting a known and character-

ized gene by an insertion of active transposon (gene disruption). For example, the Folyt1 transposon of Fusarium oxysporum caused unstable mutations when inserted in and excised from the nit1 gene [9]; the Fot1 and impala fungal elements caused unstable mutations in the nitrate reductase gene niaD [10, 11]. (3) Using spontaneous insertions into a gene. For example, the restless transposon of Tolypocladium inflatum disturbed functioning of the tnir gene involved into nitrogen metabolism, though it had not been characterized earlier [12]. (4) On the basis of high frequencies of reversion of mutant phenotype to the wild type. The transposon excision in this case may lead to restoration of the gene function (for example, the Ty1 element inserted into the HIS4C locus of the S. cerevisiae [13]). No wonder that most transposons were identified and studied in the genomes of classical genetic model objects (Saccharomyces, Neurospora, Podospora, and Ascobolus) and in the genomes of phytopathogenic fungi (Fisarium, Magnaporthe, Botrytis, and Cladosporium) because of commercial value of the latter as they are causative agents of different diseases in cultivated plants (table). The phytopathogenic fungi exhibit extremely high variation of traits, plasticity, and high adaptivity. In recent years, the attention of researchers is focused on such commercially important fungal species as Agaricus, Tolypocladium, and Phanerochaete. Class I transposons. Class I transposons are, as a rule, actively transcribed and translated; they carry the open reading frames (ORFs) of reverse transcriptases and integrases. Apart from the reverse transcriptase, the central segment of their sequences, which is several kb long, encodes some other proteins involved in integration (the gag and pol genes typical of animal retroviruses) [3, 14–16]. The class I transposons are often referred to as retrotransposons or retroposones, because of their structural similarity to the retroviruses. The pol gene encodes a multifunctional protein containing as

1022-7954/03/3905-0505$25.00 © 2003 MAIK “Nauka /Interperiodica”

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SHNYREVA

Class I and II transposons in fungal genomes Characteristics Name and group/family

Ty1/Ty2, copia Ty3, gypsy Ty4, copia Tad1 Tcen, copia Tgl1/Tg2, gypsy Dab1 Skippy, gypsy Foret, gypsy MAGGY, gypsy Grasshopper MGRL3 Fosbury MgSINE, SINE MGR583, LINE Afut1 CFT1 Tab1/Tab2

PuntRIP1/dPunt Fot1, Tc1/mariner Impala, Tc1/mariner Folyt1, hAT Tfo1, hAT

length

5.9 kb 4.748 kb 6.2 kb 7 kb ? ? ? 7.846 kb

terminal repeats (LTRs/ITRs)

ORF included and copy number

target site duplications (TSD)

Class I transposons Saccharomyces cerevisiae 330 bp LTRs 5 bp 340 bp LTRs 5 bp 350 bp LTRs 5 bp Neurospora crassa Unavailable (non-LTR) LTRs 5 bp ? Fusarium oxysporum f.sp. 429 bp LTRs LTRs 5 bp

Reference

RT, gag, pol RT, gag, pol RT, gag, pol

[22] [15] [76]

RT

[77]

RH ?

[53] [53] [40]

RT, gag, pol PR, RT, RH

[64] [7]

Magnaporthe grisea (Pyricularia oryzae) LTRs 5 bp gag, pol 198 bp LTRs RT, RH, INT 6 kb LTRs ? LTRs ? 470 kb Unavailable ? Unavailable Aspergillus fumigatus 6.914 kb 282 bp LTRs RT, RH, INT Cladosporium fulvum 6.968 kb 427 bp LTRs RT, gag, pol Agaricus bisporus ? 850 bp LTRs >50 copies Class II transposons Neurospora crassa 1.9 kb ? ? Fusarium oxysporum f.sp. 1.9 kb 44 bp ITRs TPase, 1–100 copies 1.28 kb 27 bp ITRs TPase 2.615 kb 9 bp ITRs 8 bp TPase 2.763 kb 15 bp ITRs TPase Nectria haematococca (Fusarium solani f.sp. pisi) 5.638 kb

Nht1, Tc1/mariner

2.198 kb

Pot2 MGR586

1.857 kb 1.86 kb

[78] [6] [52] [60] [59] [57] [79] [8] [30]

[45] [10] [11] [9] [31]

ITRs TPase, 1–100 copies Magnaporthe grisea (Pyricularia oryzae) 43 bp ITRs TPase, 100 copies 42 bp ITRs TPase RUSSIAN JOURNAL OF GENETICS

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TRANSPOSABLE ELEMENTS ARE THE FACTORS

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Table. (Contd.) Name and group/family

Ant1, Tc1/mariner Tan1 Vader

Characteristics length

4.798 kb 1.668 kb 437 bp

Flipper, Tc1/mariner

1.842 kb

Restless, hAT

4.097 kb

Hupfer, Tc1/mariner

3.336 kb

Ascot1, hAT Pat

409 bp 1.856 kb

Abr1, hAT

350 bp

Scooter, hAT

647 bp

Pce1

1.747 kb

terminal repeats (LTRs/ITRs)

target site duplications (TSD)

Aspergillus niger 37 bp ITRs Aspergillus niger var. awamori ITRs 44 bp ITRs Botrytis cinerea 48 bp ITRs Tolypocladium inflatum 20 bp ITRs Beauveria bassiana 30 bp ITRs Ascobolus immersus 25 bp ITRs Podospora anserina 53 bp ITRs Agaricus bisporus ITRs 7 bp Schizophyllum commune 32 bp ITRs 8 bp Phanerochaete chrysosporium ITRs

ORF included and copy number

Reference

TPase

[26]

TPase, 1 copy 15 copies

[34] [34]

TPase, 0–20 copies

[27]

TPase, 15 copies

[12]

>5 copies

[28]

Unavailable

[33]

20–25 copies

[42]

Unavailable, 15 copies

[30]

Unavailable

[32]

?, 4 copies

[70]

Note: Abbreviations (ORF) are explained in the text; ? or blanks in columns, no data.

domains protease (PR), integrase (INT), reverse transcriptase (RT), and RNAse H (RH). First, the entire DNA sequence of the transposon is transcribed to generate the general protein, which is cleaved by protease so that the separate enzymes are produced [2, 17]. The long terminal repeats (LTRs) may be either present or absent like in the LINE and SINE elements, one of the ends of which is represented by the poly(A) tract. The LTR retrotransposons are often involved into both transcription and integration. The LINE and SINE element integration may also be RNA-mediated [14]. The Ty1 elements of yeast S. cerevisiae belong to the class I (gypsy-like) transposons; they are the first TEs identified in the fungal kingdom (Fig. 1). The central Ty1 part termed ε (about 5.9 kb) is flanked by a 330-bp region of the long direct terminal repeats (σ-sequences) [2, 17]. Each of them contains, in turn, inverted repeats only 2 bp in size. Another specific feature of the Ty1 structure is the presence of the 5-bp direct repeats at the junctions of the ε and σ sequences. Insertion of the Ty1 elements is always accompanied by duplications of the 5-bp target sites [13]. The mechanism of the class I transposon integration was first demonstrated for the RUSSIAN JOURNAL OF GENETICS

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Ty1 elements of S. cerevisiae (Fig. 2) [17, 18]. The RNA copy with short terminal repeats is transcribed from the genomic DNA of the element. Reverse transcription of the RNA copy yields a DNA copy containing the long terminal repeats. This DNA copy is inserted into the new genome locus due to integrase activity. Insertion of retrotransposons into new loci generates target site duplications (TSDs), which typically have the same size [17, 19]. Class II transposons are characterized by direct (DNA DNA) transposition by means of the socalled cut-and-paste mechanism. These transposons are several kb in length and contain the short inverted repeats (ITRs) at their ends [4, 17]. Insertion of these elements also generates TSDs, the length of which varies in different transposons. The central portion of the transposon sequence encodes transposases. The DNAmediated integration of the class II TEs can be described as follows (Fig. 3) [17, 18]. The transposase generates stepwise single-strand breaks at the 3' end of the transposon sequence, as well as the single-strand breaks at the integration site of the target DNA so that the 5' end of the latter is exposed. The breaks are gen2003

508

SHNYREVA gag

pol:

PR

INT

RT

RH

LTR

5'-TGAGAT ...δ... CCAACATACCA

LTR

...

ε

...

TACCATGAGAT ...δ... CCAACA-3'

Fig. 1. Structural organization of the Ty1 yeast transposon. Black rectangles with arrows indicate long direct terminal repeats (δ-sequences); the unhatched arrows adjacent to the latter indicate direct repeats of the target DNA. The arrow direction indicates orientation of the correspondent repeats. The inverted 2-bp sequences at each end of the δ-elements are hatched. The arrow above the mobile element indicates direction of gene transcription. The figure is not to scale. The Ty1 is about 5.9 kb, direct termimal repeats are of 330 bp each, and direct repeats of the DNA target are of 5 bp each in size. An unbroken sequence is transcribed from the central portion of the element including significant fragments of its terminal repeats. The gag gene encodes a structural RNAbinding protein. The pol gene encodes a single protein, the domains of which are formed by protease (PR), integrase (INT), reverse transcriptase (RT), and RNAase H (RH) [17, 18].

erated at both 3' ends of the donor element and at the target site of the recipient molecule, which leads to formation of the 5' staggered ends. This is followed by strand transfer reaction, i.e., a strand exchange, which leads to recombination between the mobile element and the target DNA. The gap repair by replication at the 5' end of the target DNA template produces target site duplications. Note that the break at the transposon 5'end does not occur exactly at the terminal nucleotide but can vary in different families leaving gaps from two–three to seventeen nucleotides. This also results in an additional mutagenic effect induced by the excision of the transposable element, because the gap repair generally fails to restore the original sequence. Target site duplications. Integration of most TEs leads to a duplication of a short DNA region within the integration site (target site). Because of this, each TE is flanked by target site duplications (TSDs) of a conserved length; sometimes, the target site sequences are conserved. The almost obligatory duplication of the target sites suggests that most transpositions are accompanied by staggered single-strand breaks in the potential target sites, though different mechanisms underlies transposition [17]. Transposition of different TEs generates duplicated sequences of different length. The class I transposable elements induce target site duplications of a definite size (four to eight bp). At the same time, integration of class II transposons induces duplications varying in size, which may depend on specificity of enzymes catalyzing formation of the staggered single-strand breaks in the target sites [14, 17]. The invariable size of duplications induced by the class I transposons may be related to the fact that the DNA template is cleaved with endonucleases specific for each transposon.

GENOME STRUCTURAL REARRANGEMENTS AND MODIFICATIONS In most cases, each type of TEs occurs multiply in the genome, i.e., the elements form families of dispersed repeated sequences. Apart from transposon-specific mechanisms of transposition, which are typical only of these elements, they may insert into new loci by common homologous recombination, e.g., at the terminal repeats. Thus, the repeat families promote further genome rearrangements by creating the sites of ectotopic recombination, which leads to deletions, duplications, inversions, and translocations [17, 20]. The mobile element capability to induce genome rearrangements was repeatedly demonstrated for Ty elements of yeast [2, 21, 22] and the F. oxysporum transposons [23, 24]. If such rearrangements occur in individual somatic cells, their influence on the functioning of the entire organism is insignificant. But in germline cells they may cause severe mutational changes and chromosomal aberrations. When fixed in a population by natural selection or genetic drift, these rearrangements may have serious evolutionary consequences. Based on the extensive experimental evidence, the following modifications in fungal genomes related to TE transpositions can be listed [2, 23, 25]: chromosome rearrangements, or extensive mutations (deletions, duplications, inversions, and translocations), which appear due to homologous recombination between the members of the same TE family; the new transposon copies in the genome provide additional sites of homology for unequal crossing-over; transfer of long DNA segments by transposition; change in gene transcription; transposon excision results in either an altered gene product or the reading frame shift mutation; change in gene expression: transposon integration into the structural or promoter regions mostly inhibits

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RNA copy (a) Reverse transcription 5'

3'

3'

5'

DNA copy (b) Cytoplasm

5'

5' 3'

3'

Nucleus

3' 5'

(c)

+

Target DNA

5' 3'

3'

5'

5' 3'

Integration

(d)

5' 3'

(e)

5' 3'

Transcription

RNA copy Fig. 2. A simplified scheme of transposition of the class I retrotransposon. Rectangles, short direct terminal repeats of the RNA copy. Open rectangles, double-stranded DNA copy of the retrotransposon with long direct terminal repeats. The flanking rectangles adjacent to the retrotransposon in the integration sites show direct 5-bp repeats of the target DNA. The figure is not to scale. See text for further details [18].

gene expression, though gene regulation may be also altered; amplification of the DNA-sequences producing pseudogenes. One of the first comprehensive genetic analyses of unstable alleles was carried out for the HIS4 gene of S. cerevisiae in the late 1970s [13]. The HIS4 locus located on chromosome III contains three complementation groups, the A, B, and C genes. These genes control enzymes catalyzing successive stages of histidine biosynthesis. The HIS4C sequence was experimentally shown to transpose from chromosome III to chromosome IV, which induced lethal recessive mutations at the integration sites. The HIS4C locus was unstable: its excision occurred at a frequency of 1% and was followed by restoration of the activity of the gene into RUSSIAN JOURNAL OF GENETICS

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Fig. 3. A general scheme of recombinations occurring during transpositions of the class II mobile elements. Lines indicate DNA strands. In (a), solid arrows indicate the DNA-strand breaks that occur exactly at the ends of the transposons; dotted arrows indicate the breaks that occur not necessarily at the terminal nucleotide. In (d) indicates dotted line, a repair replication of the target DNA gaps, which results in formation of direct repeats from the target DNA nucleotides on both sides of the inserted element. These repeats are indicated in (e) with solid lines. See text for further explanation [18].

which the HIS4C locus has been integrated. The HIS4 locus contained Ty1 (a transposable yeast element). However, the revertant strains HIS4+ differed from the wild-type strain HIS4+ in phenotype and were cold sensitive. The fact is that the Ty1 was not excised as a whole, but a single σ sequence remained in the HIS4 locus (an insertion trace), which accounted for the revertant phenotype. Thus, a 300-bp σ insertion was almost of no importance for the HIS4+ locus functioning. This excision might have occurred during reciprocal homologous recombination at the direct repeats of the σ segments flanking the Ty1 element. 2003

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Many fungal class II transposons described in literature belong to the hAT and Fot1/pogo families. Those from the Fot1 family were identified as insertions into the coding sequences of the nitrate reductase gene niaD (for example, the Ant1 element of the Aspergillus niger [26]), flipper DNA-transposon of Botrytis cinerea [27], hupfer element of the entomopathogenic fungus Beauveria bassiana [28], and impala transposons of F. oxysporum [29]. The ORFs of these transposons are homologous to transposases of the Tc1/mariner family typical of the TEs from the animal kingdom (nematodes, insects) (table). The members of the hAT (hobo/Ac) families have specific 8-bp target site duplications (TSDs) in the genome integration sites and the transposase-homologous sequences. The hAT family includes the Abr1 element of Agaricus bisporus [30], Folyt1 and Tfo1 elements of the Fusarium oxysporum [9, 31], restless element of Tolypocladium inflatum [12], scooter element of Schizophyllum commune [32], and Ascot-1 of Ascoblus immersus [33]. The Ascot-1 transposon was isolated from the mutant allele of the b2 ascospore color locus. Unlike the typical representatives of the hAT family, the Abr1 transposon does not encode its own transposase and has atypical 7-bp TSD. This transposon is believed to be inactive (i.e., unable transpose autonomously), though it is permanently inherited in the cultivated strains of champignon [30]. An elegant study by American authors from the Vermont University on the scooter transposon [32] testify to a role of transposons in regulation of mating compatibility. Scooter is a 647-bp element from the hAT family, which has typical 8-bp TSD and inverted 32-bp terminal repeats. It was the first transposon identified in a basidiomycete fungus Schizophyllum commune. Scooter was identified as an insertion into the genes that disturb sexual mating. In all strains analyzed, the two almost identical copies, scooter-1 and scooter-2, capable of active transposition were found. Scooter-1 has been first described as an insertion into the mating locus Çβ2 (namely, into the bbr2 pheromone receptor gene), which affected sexual mating, because the pheromone receptor ability to control recognition of nuclei from the compatible mate during nuclear migration was disturbed. This mutation was previously described as the def phenotype. The excision of scooter-1 from the bbr2 pheromone receptor locus restored the Çβ2 receptor function. Transposable element scooter-2 spontaneously disrupted the thn1 gene, which phenotypically resulted in morphological mutation thin (fast growing mycelium segments at the background of normal colony growth). The thn1 gene encodes the regulator of G-protein signaling protein (RGS protein) and controls transmembrane signal transduction associated with G-proteins, which in turn regulates the expression of the hydrophobin genes. The thin mutants either lack the hydrophobin-encoding mRNA or its content in cytoplasm is decreased. The thn1 gene disruption has a pleiotropic effect related to the fungal vegetative growth and sexual development. The scooter transposons have an effect

on the signaling system activity dependent on the protein interactions, which regulates the pathways of asexual development and basidium formation in S. commune. These TEs are relatively short and thus they lack long ORFs capable of encoding their own transposases. Fowler and Mitton [32] suggest that transposition of these elements depends on transposases encoded by some other elements in the S. commune genome. A similar situation was described for class II transposons from the Fot1 family in fungus Aspergillus niger var. awamori [34]. In mutants for the niaD gene of nitrate reductase, the transposon-like 437-bp elements Vader were identified, which contained the inverted 44-bp terminal repeats. The fungal genome contained about 15 mobile copies of this element. However, the Vader sequences lack any ORFs, and their transposition depends on transposase activity of a transposase-encoding 1668-bp Tan1 sequence, the single copy of which in the genome ensures the Vader element transpositions. The Vader and Tan1 form a common cluster, though several mobile Vader copies are dispersed throughout the genome. About 30% of the Tan1 nucleotide sequence is homologous to those of transposons Pot2 of M. grisea and the Fot1 of F. oxysporum (table). The restless TE belonging to the hAT family of transposons was identified in the Tolypocladium inflatum fungus, a producer of the immunosuppressor cyclic protein cyclosporin [12]. This 4.097-kb transposon has inverted 20-bp terminal repeats and 8-bp target site duplications typical of the hAT family. The open reading frame in this transposon is disrupted with an intron sequence and, therefore, the two polypeptides (of 157 and 803 amino acids) are translated; one of them (containing 803 amino acids) is homologous to transposase. All seven fungal chromosomes separated by the pulsed field electrophoresis carry the copies of this transposon, i.e., 15 copies are dispersed throughout the entire haploid genome. As shown using the transposon trap technique and mutants for the nitrate reductase gene (niaD), this active transposon can be excised and integrated into the new loci of the genome. The strains incapable of producing cyclosporin lack the restless transposon. Analysis of the Ascot1 transposon from the Ascobolus immersus, which is similar to Ds mobile elements of plants, suggests that excision of transposons that belong to the hAT family is related to the V(D)J-recombination [33]. High DNA sequence variation in excision sites of class II transposons is due to the fact that a break at the 5' end of the mobile element does not occur strictly at the terminal nucleotide, and the original sequence is rarely restored after the gap repair. Various phenotypic and genetic effects caused by the Ascot1 excision were demonstrated in unstable ascospore color locus b2. A transposon excision produced 48 various molecular products, which differed in frequency of their appearance. Most of them contained additional


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short palindrome sequences in sites of gap formation (single-strand gaps), which were adjacent to the 5' end of the transposon. These products were not synthesized on a template of the complementary chain in a gap. The authors explained this by the fact that excision and repair of the single-strand gaps involved the V(D)Jrecombination (a variety of the ectotopic recombination), rather than a simple end-joining reaction. The V(D)J-recombination generated intermediate hairpin structures, which is confirmed by the presence of palindromes at the ends. The palindrome sequences serve as an additional source of variation to ensure a broad spectrum of spontaneous mutations [20, 25]. Interestingly, various molecular products appeared at different frequencies, which suggests that the further fate of the transposons is related to processing, i.e., specific rearrangements, at the excision site. The V(D)J recombination occurs in the Ascobolus somatic cells mostly during the last mitotic cycle before the meiosis. Sequence duplications. As shown on the example of the Fusarium oxysporum, the class II transposable elements are mostly packaged together with the repeated DNA sequences [35]. Analysis of three chromosome regions showed that the repeats are clusters of TEs of several types. Some repeats are disrupted by TEs, whereas the others themselves form insertions into the TEs. Some of these regions contain duplications. The long repeats may be considered hotspots for rapid reorganization and genomic rearrangements. Daviere et al. [24] reported high variation of the F. oxysporum electrophoretic karyotypes, which differed in chromosome number and size. The authors showed that the karyotype variation was a consequence of chromosome translocations, large deletions, and complex rearrangements. Many chromosome regions were duplicated and contained clusters, though transpositions caused no chromosome breaks. The high level of chromosome polymorphism was correlated with the transposon concentration. Daviere et al. hypothesized that polymorphism observed along the chromosome length may be a result of ectotopic recombination between transposons, and the latter themselves serve as substrates for the changes. If sequence duplications induced by mobile element transposition are considered to be a source of material for evolution, the presence of duplicated gene copies must reduce the effect of selection and promote thereby the sequence divergence. However, an excessive accumulation of duplications in the genome may be harmful, because the duplications dispersed throughout the genome may lead to significant ectotopic recombination (deletions and translocations). Specific mechanisms should protect the genetic integrity of an organism to balance the benefit of duplications against the harm they do [36]. RUSSIAN JOURNAL OF GENETICS

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THE MECHANISMS REGULATING TRANSPOSITIONS TO MAINTAIN THE GENOME INTEGRITY Many species gain advantage in survival and adaptation to the changing environments due to mobile element transpositions. Transpositions disrupt the encoding gene sequences, change the expression of the neighboring genes, and promote major chromosome rearrangements [25]. However, extremely high levels of variation and mutability may be deleterious. Too frequent transpositions may result in the genome disintegration. In view of all negative consequences of transposition, it is no wonder that the mechanisms that control transposon activity have been evolved in concert with the transposon evolution. RIP process. The repeat induced point mutation (RIP) is one of the mechanisms that control transposition of active mobile elements. These are point mutations induced in the repeated DNA sequences. The duplicated DNA sequences that are readily detectable in the Neurospora crassa were shown to be modified during the sexual cycle (at the moment of karyogamy) with involvement of the RIP process. RIP attacks both the original and duplicated copies, which are subjected to multiple GC-to-AT mutations [37]. Note that in Neurospora, RIP is observed in at least 400-bp sequences, i.e., the long duplications are mostly affected. The RIP action actually decreases homology between the duplications, i.e., results in a lower accumulation of the latter. As a consequence, the probability of homologous exchanges and major chromosome rearrangements is reduced. As shown by tetrade analysis, RIP occurs before or during premeiotic DNA synthesis and may affect both DNA strands of a duplex [37]. The results of this process are mostly detected as silent mutations, though both nonsense and missense mutations may appear as well as the functionally mutant alleles. Some authors think that RIP mostly affects duplicated sequences of the length comparable with that of a gene. Thus, the actively transcribed transposable Tad elements from the LINE group were identified only in the Adiopodoume strain of the ascomycete Neurospora crassa, whereas in other natural strains, almost all Tad copies were inactive [38]. This retrotransposon was identified when unstable alleles of the glutamate dehydrogenase gene (am) were studied; the retrotransposon integrated at the 5'-flanking noncoding region of this gene. It is experimentally shown that the active Tad transposons were rapidly eliminated during the sexual cycle and a loss of activity was a consequence of the repeat-induced point mutations, mostly the GC-to-AT nucleotide transitions [39]. These very transitions, which were identified in inactive Tad copies, testify to the RIP process occurring during the sexual cycle. The authors concluded that this mechanism controls the number of active transposons in the genome. While the Adiopodoume strain lacked the RIP mutations, the other strains contained numerous defective Tad copies. 2003

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Another type of repeated sequences, the dab1 (dead and buried) transposon was found in the Neurospora [40]. It belongs to a family of class I degenerate retroelements with a distinct homology of their sequences to the encoding sequences of transcriptases, RNases H, and endonucleases of the pol genes. The RIP process accounts for a loss of the dab1 activity (a capacity to mobilize its own transposition), which is confirmed by such a diagnostic sign of the RIP as the presence of numerous GC-to-AT transitions. The degenerate copies of the RIP-affected MAGGY elements were identified in the Pyricularia oryzae isolates [41]. In the wild-type strains of the Podospora anserina, a degenerate 1856-bp transposon Pat was found, which has inverted imperfect 53-bp repeats and the ORF homologous to the members of the Fot1 transposon family [42]. About 20–25 Pat copies are identified per haploid genome, however, none of them was shown to actively transpose under both normal and stressful environmental conditions. This transposon seems to be inactive because it contains numerous stop codons. The molecular analysis of the progeny yielded by crosses between the wild-type and transgenic strains carrying the duplicated gene copies showed that the RIP-like process proceeded actively in the P. anserina genome [42]. DNA methylation. Methylation is another molecular mechanism that controls distribution of the active (capable to transpose) mobile elements. In the ascomycetes Neurospora crassa and Ascobolus immersus, a special methylation mechanism was shown to mark duplicated sequences. Methylation inhibits transcription (transcriptional silencing), increases mutability, and inhibits recombination of the nucleotide sequences [25, 43]. This seems to be a mechanism of “noise reduction” that leads to silencing the redundant copies in the genome. In fungi and plants, the mechanism of transcription inhibition depending on the presence of homologous sequences (homology-dependent gene silencing) controls transposon expansion and protects from the viral infection. Thus, cytosine methylation was shown to suppress the Tad element ability to be actively transposed in the Neurospora [44]. The RIPmodified sequences serve usually as signals for further DNA methylation, because transcripts abnormal in length continue to be synthesized on the RIP-affected sequences [45]. The excess of useless transcription may lead to hazardous consequences in Neurospora, but DNA methylation coupled with RIP inhibits further transcription of the RIP-affected genes [36, 46]. However, this is only a hypothesis that methylation of the Neurospora genome aims at suppressing transcription of the RIP-modified genes. The experimental evidence showed that methylation of the Tad transposon suppressed both the Tad expression itself and its capacity to be transposed. Note that the reversion of the mutant gene am to the wild type depended on the degree of methylation of the Tad DNA integrated into the gene as well as on the methylation of the URSam enhancer-like

sequence. In other words, the epigenetic control of the transposon-inactivated glutamate dehydrogenase gene depended on the degree of DNA methylation [47]. Later, the Tad element in 5'-noncoding gene region was shown to carry de novo signal of cytosine methylation, which induces the reversible methylation of both Tad and the upstream sequence [48]. Thus, DNA methylation underlies the mechanism of transcriptional TE silencing. About 1.5% of the cytosine residues are methylated in the Neurospora genome (4 × 107 bp in size) and the most methylated regions form a relatively short highly methylated clusters up to 500 in number [45]. Only four of these clusters have been characterized. A segment containing 5S rRNA gene represents one of these regions. Another one is adjacent to the 5' end of the Tad element, which is inserted upstream of the am gene. The third element was identified in a tandem repeat of the ribDNA in a nucleosome. The fourth highly methylated region was found at the site of the PuntRIP1 transposon integration into the 5S rRNA pseudogene. This 1.9-kb transposon homologous to the class II Fot1 element was RIP-inactivated. The similar transposon sequences with deletions affecting transposition but without the RIP signs termed dPunt were also identified [45]. The telomeric and centromeric chromosome regions abound with repeats and transposon-like elements. In the fungal genomes, the regions of preferential location of inactive transposons have been identified. For example, the yeast Ty elements are concentrated at the silenced chromosome regions and many of them in the telomeric regions [2, 49]. Like the degenerate copia elements of Drosophila, defective Tad copies frequently occur in the centromeric and telomeric regions, which contain many different repeats. The class I transposons affected by the RIP process are localized to the centromere of chromosome VII in the Neurospora [47]. Thus, the TE location in telomeric and centromeric regions seems to be a strategy acquired in evolution by many transposon families in order to minimize abnormal gene functioning (genome disruption). That is a specific and simple mechanism, which protects from excessive ectotopic variation and maintains genome integrity. The subtelomeric and telomeric chromosome regions serve presumably as niches occupied by transposons to reduce the adverse effect of the latter on the genome. Thus, retroelements of the LINE family are mostly accumulated within the telomeric repeats in a green algae Chlorella [50]; the similar phenomenon is also observed in the higher plants [51]. The three types of transposons were found on a specific telomere of the rice pathogen Magnaporthe grisea [52]. Deletions were typical of these telomeric regions in strains rendered virulent spontaneously. Various transposons were identified at this chromosome end: Pot2, Mg-SINE and a new 6-kb transposon MGLR3 with long terminal repeats (table). The MGRL3 is highly homologous to the gypsy-class retroelements and occurs frequently in different fungal races specific for various hosts.


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Recombinations for the telomeric transposon-carrying regions may lead to chromosome breaks and loss of large fragments. However, such protection mechanisms as RIP and methylation reduce homology in potential sites of homologous recombination. In Neurospora crassa, a region of chromosome VII is represented by the clusters of inactive transposonlike elements and simple DNA repeats [53]. Some repeats are copia-like retroelements referred to as Tcen, which are localized only to the centromeric regions. Several other degenerate transposons in the centromeric region of chromosome VII are the following: gypsy-like Tgl1 disrupted with the two large insertions homologous to the incomplete copies of the Tad element. One of these insertions, the dTad1 is of 3475 bp in length, whereas another one, dTad2, is shorter (1404 bp) and contains 14-bp TSDs typical of the Tad. The degenerate copies of the Tad element were also identified in centromere on chromosome III and in subtelomeric region of chromosome IV. This localization seems to be nonrandom, because the centromeric regions are less subjected to recombination, and, hence, rare common homologous recombination may be expected between the transposon sequences. THE ROLE OF TRANSPOSONS IN PATHOGENESIS In plants, some transposons have been shown to accumulate in genomes in response to a fungal pathogen invasion. For example, the Rim2 transcripts accumulates in rice plants in case of both compatible and incompatible interactions between the plant and phytopathogenic fungus Magnaporthe grisea, as well as after plant treatment with fungal elicitors [54]. The Rim2 transcript exhibits homology to the CACTA transposons of other plants. In fungus Nectria haematococca pathogenic for pea (an anamorph of Fusarium solani f.sp. pisi), the B chromosomes unstable during sexual reproduction were identified [55]. Unlike the rice pathogen Magnaporthe grisea, these B chromosomes are mostly inert, i.e., their sequences are not transcribed. However, in some cases, the genes located on B chromosomes are directly involved into pathogenesis [56]. In a pea pathogen N. haematococca, the B chromosomes carry genes phytoalexins responsible for inactivation of the host plant. These genes, Pda6 and Mak1, are directly involved in pathogenesis and significantly increase fungal pathogenicity. The Pda6 gene belonging to a family of the cytochrome genes P450 increases virulence of the fungal strains on pea, because it controls inactivation of the major protective pea phytoalexin, pisatin. The Mak1 gene product inactivates another phytoalexin, medicarpin. One of these B chromosomes, contained the DNA repeats carrying a transposon-like class II element Nht1 of 2.198 kb in length and with inverted terminal repeats [55]. The Nht1 element contains two introns and encodes a 550-amino acid protein highly homologous to transRUSSIAN JOURNAL OF GENETICS

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posase of the Fot1 elements. In the North American population of the fungus Nht1, the distribution of this transposon is uneven. Its copy number ranges from 0 to 100 per genome in various natural isolates. The strain, in which this transposon was identified, contained six Nht1 copies on an unstable B chromosome. The genome of an extremely aggressive phytopathogenic fungus Pyricularia oryzae (a telomorph of the Magnaporthe grisea), which is a causative agent of rice blast disease, is literally packed with various transposons [57]. The MAGGY retrotransposon [41] and a class I LTR retroelement, grasshopper (grh), have been described. The latter TE predominantly occurs in the pathogen populations on wild cereals [6]. Transposition of the MAGGY retrotransposon is RNA-mediated and this mobile element has two ORFs of the gag and pol genes. The experimental evidence shows that the MAGGY can be transposed to a heterologous host, i.e., to other fungal species (Colletotrichum lagenarium and Pyricularia zingiberi); the transposition frequencies differ in different species [41]. The grh and MAGGY retroelements, which are sporadically encountered in the Pyricularia isolates, are assumed to be a relatively recent acquisition of the fungal genome, which appeared due to horizontal transfer [57]. The 1857-bp Pot2 element first identified in Discomycetes and designated flipper has the inverted terminal 43-bp repeats and the transposase ORF, which is highly identical to that of the Fot1 element of the F. oxysporum [58]. About 100 copies of the Pot2 element were identified per haploid genome. Sequence analysis of the Pot2 showed that some of them contained short 470-bp sequences (Mg-SINE) with properties typical of the SINE elements (short interspersed repeat elements) [59]. The Mg-SINE were identified not only in the Pot2 sequences, but also in other genome regions on all chromosomes, including the B chromosome. These particular dispersed repeats characteristic of both rice and nonrice strains have no distinct open reading frames and their RNA transcripts in the cytoplasm have a tRNAlike maple leaf structure with some modifications of the coupled nucleotides. The SINE element transposition is assumed to occur due to either RNA polymerase III activity in reverse transcription or the reverse transcriptases of other retroelements are used. Since the Pot2 and Mg-SINE elements are encountered in both rice and nonrice strains, these ancestral elements probably appeared in the fungal genome before its specialization of the with regard to the host plants. Recently, LINE-like retroelements MGR583 have been identified [57]. All of the above transposable elements, Pot2, MGR583, Mg-SINE, occur most frequently in the natural populations of the phytopathogenic fungus Pyricularia ((Magnaporthe), which is parasitic on the cultivated rice, rather than on the wild cereals; all these elements are represented by multiple copies in the genome. Another family of the repeated sequences MGR586 dispersed throughout the genome of the M. grisea 2003

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induce significant polymorphism of the DNA restricts (RFLPs). These sequences are represented by the class II 1.86-kb transposons with 42-bp inverted terminal repeats and ORF homologous to transposases of the Fot1 and Pot2 elements [5]. Segregation analysis of the MGR586-carrying DNA restricts showed that these transposon-like elements induce nonrandom chromosome rearrangements resulting in the development of the heterokaryotic mycelium. The fosbury is another group of the retrotransposonlike elements with long terminal repeats, which is mostly characteristic of the P. oryzae strains invading rice plants [60]. Like MGR586, these retroelements confer to the rice fungal strains clear distinctions from the strains specialized on other host plants (wild cereals). Interestingly, these two transposon-like elements are mutually associated in the fungal genome. The presence of transposons in genomes of the phytopathogenic fungi was assumed to account for the fungal strain pathogenicity, for example, in case of helminthosporiosis of maize Cochliobolus carbonum [61]. Fungal strain pathogenicity is caused by the HC toxin, a cyclic polypeptide, the secretion of which is controlled by a duplicated DNA sequence within the 22-kb Tox2 locus. The latter is flanked from both sides with the repeated elements homologous to Fot1 of the F. oxysporum and to Pot2 transposon of the M. grisea. The resistant plants synthesize HC-toxin reductase (HCTR, a product of the Hm1 gene), which inactivates HC toxin. Various class II transposons were identified in a causative agent of wilt, Fusarium oxysporum, which induces fusariosis wilt of many cultivated plants (cotton, flax, and vegetables). The Fot1 transposon was identified as an insertion into the niaD gene of nitrate reductase. This element is 1.9 kb in length including the 44-bp inverted terminal repeats; it contains a transposase-encoding ORF [10]. Fot1 has been identified in various fungal races and forma speciales. Note that nucleotide sequence divergence between the f. sp. is less than 1%, which testifies to the fact that this element appeared in the ancestral isolates before their specialization on various host plants. The genomes of various fungal races contain from 1 to 100 Fot1 copies. The presence of Fot1 may serve as a diagnostic trait in analysis of the natural phytopathogen populations [62]. The transposon-like elements impala (class II), a fungal member of the Tc1/mariner superfamily, was isolated from unstable mutants for the niaD locus [11]. This 1280-bp transposon has 27-bp inverted terminal repeats and encodes a transposase, though not all copies of the genome can replicate autonomously. Some of them are defective, because they are disrupted with several stop codons. The impala elements are grouped into a whole family, which is divided into four subfamilies differing in the degree of divergence. The nucleotide sequence polymorphism ranges from 9 to 30% in different subfamilies. These elements are identified in almost all

members of f. sp. Fusarium. Both impala and Fot1 are capable of transposing at a high frequency. The Tfo1 transposon of F. oxysporum, which is 2763 bp long and has inverted terminal 15-bp repeats, also encodes a transposase providing transposition of this element from DNA to DNA [31]. Factors activating TE transposition. Some evidence suggests the existence of factors capable of mobilizing fungal transposons [63]. Thus, the skippy (skp) element of F. oxysporum f. sp. lycopersici, which is 7846 bp in length, has long repeats of 429 bp, and belongs to the class I gypsy retroelements, is transposed under stressful environmental conditions [64]. During the fungal growth on the chlorate-containing medium, the chlorate-resistant mutants were selected. By comparing the original wild phenotype with the three chlorate-resistant mutant defective for the niaD nitrate reductase gene, these mutants were shown to contain tandem copies of the skp sequences in the new genome regions. Thus, the skp elements integrated into the new sites and, simultaneously, the resident skp copies characteristic of the wild type proved to be lost. By example of the impala elements of F. oxysporum, some active copies of these DNA transposons were shown to activate mobility of the inactive copies, which led to a so-called aberrant transposition [29, 65]. The latter was accompanied by deletions and inversions as well as by a substitution of the promoter regions. For example, the niaD gene promoter was replaced by an unknown promoter region. Note that in sites of these transpositions, multiple terminal repeats were found in direct rather than inverted orientation typical of transposons of this class [65]. Stressful environments are assumed to activate TE transpositions and thereby affect genetic variation of an organism. The French researchers Capy et al. [66] hypothesized that special promoters or binding sites for transcription activators are located in the untranslated TE regions. Thus, about 85% of all Ty element insertions in genome of yeast S. cerevisiae were shown to be insertions of direct terminal repeats (LTRs) into tRNA genes and other transcribed genes [3]. A group of the Japanese researchers showed that the promoter region of the MAGGY retrotransposon in the Magnaporthe grisea can be activated by heat shock, Cu2+, and oxidative stress to increase significantly the amount of MAGGY-RNA transcripts in the cytoplasm [67]. However, the fungal strains studied were unsusceptible to such stressful factors as UV-radiation and a fungicide paracoumaric acid, i.e., the fungal strains exhibited a selective response to environmental stresses. CHANGES IN GENE EXPRESSION The fungal mobile elements (transposons) may function as promoters, i.e., they may have an effect on regulation of the cell genes. Yeast Ty elements inserted into promoter regions were shown to modulate gene


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expression. For example, in S. cerevisiae, a so-called ROAM mutations (regulated overproducing alleles responding to mating type) are related to an increase in gene expression by tens of times [68]. In hyperproducing strains, the Ty1 transposon integrated into the 5'flanking region of the iso-2-cytochrome c gene (CYC7) is transcribed in the direction opposite to that of the adjacent gene transcription. However, hyperproduction of iso-2-cytochrome c is observed only in haploid cells, i.e, the Ty1 transcription activity is sensitive to a cell status for the mating type: in diploids (MATa/α), this activity is five to twenty times lower than in the haploid cells MATa or MATα. Transcription of the alcohol dehydrogenase gene ADHII in S. cerevisiae, which is also dependent on the Ty insertions, occurs as follows. The alcohol dehydrogenase gene is normally repressible (it is suppressed by an excess of glucose in the medium). The Ty1 insertion into the 5'-flanking region render this gene constitutive, whereas after the excision of the Ty1, the σ sequence remaining in the promoter region inactivates the ADHII gene completely [69]. Regulation of the nitrate reductase gene niaD in F. oxysporum is also shown to depend on the Fot1 insertion and orientation of the inserted transposon relative to the niaD gene [70]. The novel promoter associated with the 3'-end of the Fot1 directs transcription of the adjacent encoding niaD sequence. Gene families. The transposon involvement in the formation of the gene families in fungi is indirectly confirmed. The families of genes are described, which encode many secreted enzymes, such as laccase, cellulase, polygalacturonase and others, involved in substrate utilization by the higher fungi. In the bracket fungus Phanerochaete chrysosporium, a family the lip lignin peroxidase gene is formed, presumably, due to the transposon-induced duplications. In the lipI1 gene, the first of the family of ten genes, a transposon-like Pce1 element was identified [71]. The ten lip genes are located on the three chromosomes; eight of them are closely linked. The Pce1 transposon is identified as a 1747-bp insertion with inverted terminal repeats, which is adjacent to the fourth intron of the gene. The lipI1 gene transcription is inactivated by this transposon, though the latter is inactive with respect to an allelic homolog of this gene lipI1 in the diploid cells. The Pce1 elements contains no ORF. As shown by segregation analysis and Southern blotting, the fungal genome contains four copies of the Pce1-element, which are 98% homologous (it is not surprising because none of the copies contains extended reading frames) [72]. All Pce1 copies are located on the same chromosome, and a cluster including eight genes also occupies the latter. The three Pce1 copies are linked to each other, and only one Pce1 copy is directly bound to the gene involved in lignin substrate degradation. Various fungal lip genes differ in their regulation in response to environmental RUSSIAN JOURNAL OF GENETICS

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stress. For example, the lip2 and lip5 genes are activated on the nitrogen- and carbon-deficient media. PERSPECTIVES IN THE STUDY AND USE OF TRANSPOSONS The reports have appeared very recently on a new class of the eukaryotic transposons, which differ from those of the classes II and I in their mechanism of transposition. These are so-called RC transposons (rolling cycle transposons), or helitrons, which are transposed due to the rolling circle mechanism inherent in the IS prokaryotic elements [1]. This mechanism is similar to replication of some plasmids, single-stranded bacteriophages, and plant heminoviruses. A preliminary estimate on genomes of the C. elegans nematodes and A. thaliana plant shows that the RC transposons constitute as much as about 2% of a genome. Nevertheless, these elements are difficult to identify so far, because, unlike all other eukaryotic transposons, they induce no target site duplications after integration into new loci. The dinucleotide AT is a target site for the helitrons and insertion of the latter does not result in a duplication of this sequence. In addition, helitrons have no inverted terminal repeats typical of class II transposons. The helitron sequence begins at the 5' end with the TC nucleotide and ends at the 3' end with the CTRR sequence. The structure (but not the nucleotide sequence itself) of a 16- to 20-bp palindrome located leftwards of the CTRR sequence, may serve as consensus sequence for recognizing the helitrons in computer analysis and search. By analogy with prokaryotes, these and other structural characteristics are assumed to play an important role in the rolling circle transposition [1]. The transposon-oriented capabilities in the functional genome analysis [73], identification of the expressed genes, transformant development and study [21, 63, 74], and insertion-mediated mutagenesis [75] has been currently actively discussed in the literature. In mutagenesis, a gene is disrupted by transposon insertions and the phenotypes of the resultant disruption mutants are studied. Then, the transposon sequence can be truncated by the site-specific recombination to at most 300 bp, though the insertion epitopic tag of the truncated transposon sequence is preserved within the coding sequence of the gene studied (gene tagging). With this approach, an expressible gene can be localized to study the structure and function of the latter. This was illustrated on the examples of several S. cerevisiae genes, such as the SPA2 gene of the spindle polar body, which controls the polarized vegetative growth; the SER1 gene of 3-phosphoserine aminotransferase, and the gene of the chromatin-associated protein BDF1 [74]. The gene studied can be also disrupted by a series of insertion mutations of various regions differing in modulation of the gene expression [72]. This approach is referred to as TAGKO (transposon arrayed gene knockout) or transposon-induced gene disruption. 2003

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In conclusion, further exploration of the fungal and other eukaryotic transposons may provide new evidence of various genome modifications caused by these elements and give an insight into their biological functions as well as provide new opportunities for the use of transposons in gene engineering.

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