Mol Gen Genet (1992) 233 : 337-347

© Springer-Verlag 1992

Original articles

CfT-I: an LTR-retrotransposon in Cladosporium fulvum, a fungal pathogen of tomato

Mark T. McHale 1'2, lan N. Roberts 1'3, Stuart M. Noble 1, Christine Beaumont 1, Michael P. Whitehead 1, Devanshi Seth 1'4, and Richard P. Oliver 1

i Norwich Molecular Plant Pathology Group, University of East Anglia, School of Biological Sciences, Norwich NR4 7T J, UK

Received November 6, 1991

Summary. A retrotransposon from the fungal tomato pathogen Cladosporium fulvum (syn. Fulvia fulva) has been isolated and characterised. It is 6968 bp in length and bounded by identical long terminal repeats of 427 bp; 5 bp target-site duplications were found. Puta- tive first- and second-strand primer binding sites were identified. Three long open reading frames (ORFs) are predicted from the sequence. The first has homology to retroviral gag genes. The second includes sequences ho- mologous to protease, reverse transcriptase, RNAse H and integrase, in that order. Sequence comparisons of the predicted ORFs indicate that this element is closely re- lated to the gypsy class of LTR retrotransposons. Races of the pathogen exhibit polymorphisms in their comple- ment of at least 25 copies of the sequence. Virus-like particles which co-sediment with reverse transcriptase activity were observed in homogenates of the fungus. This is the first report of an LTR retrotransposon in a filamentous fungus.

Key words: Transposable elements - Leaf mould - Virus- like particles - Reverse transcriptase - Fulvia fulva

Introduction

Despite intensive investigation of the genetic structure of filamentous fungi, knowledge of transposable elements in this group of organisms in sparse. A LINE-like trans- poson was detected in an African strain of Neurospora crassa (Kinsey and Helber 1989) and a number of mobile introns have also been found (Michel and Lang 1985). Laboratory strains of Aspergillus nidulans and N. crassa appear to lack active transposons (Kinsey and Helber

Present addresses. 2 SmithKline Beecham, The Frythe, Welwyn, Herts, UK 3 Institute of Food Research Norwich, Colney Lane, Norwich, UK 4 Biotech International, Technology Park, Bentley, Western Aus- tralia Correspondence to. R.P. Oliver

1989; J. R. Kinghorn and C. Scazzocchio, personal com- munications), a feature that doubtless aided their contri- bution to classical genetics. In contrast, many phyto- pathogenic fungi display phenotypic instability, which is characteristic of organisms harbouring transposons. For example, many pathogens spontaneously produce sterile, non-pathogenic mycelium when cultured in vitro, a situa- tion that is often cured by passaging through the plant host to restore vigour. In the field, the rapid appearance of novel, virulent forms in response to resistant varieties of crops is all too frequent (see e.g. Dinoor et al. 1988 ; Lindhout et al. 1989). The molecular basis of these phenomena is unknown, in part because detailed genetic analysis of these species is in its infancy.

Retrotransposons are an otherwise ubiquitous class of mobile genetic elements that require a reverse transcrip- tion step to undergo replicative transposition (Boeke and Corces 1989). The 'life-cycle' of a retrotransposon is similar to that of retroviruses; however, retrotrans- posons do not produce infectious virus particles, instead they produce non-infectious virus-like particles that are intermediates during transposition (Shiba and Saigo 1983; Garfinkel et al. 1985; Mellor et al. 1985; Goreleva et al. 1989). Numerous virus-like particles have been observed in preparations of filamentous fungi (Buck 1986; Bozarth 1972): functions have been ascribed to only a handful.

The integrated copy of a retrotransposon undergoes transcription via host RNA polymerase II to produce a polyadenylated mRNA, which acts as both a template for translation and as a single-stranded genomic RNA in the mature virus-like particle (Fink et al. 1986). Transla- tion of the retrotransposon mRNA produces the struc- tural proteins of the gag (group-specific antigen) gene and several enzymatic activities (acid proteases, reverse transcriptase, RNase H, integrase) encoded by the pol (polymerase) gene. A third open reading frame (ORF) may encode a protein analogous to retroviral env (en- velope) genes.

LTR retrotransposons and retroviruses possess a sim- ilar genomic organisation and in certain cases share ex-

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tensive DNA and amino acid sequence homology. LTR retrotransposons have been divided into two distinct groups on the basis of sequence comparisons (Xiong and Eickbush 1988; Doolittle et al. 1989). One such group is the gypsy group of retrotransposons. This class of retro- transposon exhibits the closer phylogenetic relationship to true retroviruses. The other group of LTR retrotrans- posons includes the eponymous copia from Drosophila melanogaster (Mount and Rubin 1985), Tyl from Sae- charomyces cerevisiae (Clare and Farabaugh 1985) and Tntl from Nicotiana tabacum (Grandbastien et al. 1988).

The gypsy group of LTR retrotransposons has a di- verse membership, comprising retroelements from Lilium henryi [del (Sentry and Smyth 1989; Smyth et al. 1989), Dictyostelium discoideum [D1RS-1] (Cappello et al. 1985), S. cerevisiae [Ty3] (Clark et al. 1988; Hansen et al. 1988), Schizosaecharomyces pombe (Levin et al. 1990) [Tfl, Tf2], several Drosophila species [gypsy, 412, 297, 17.6, TOM] (Marlor et al. 1986; Yuki et al. 1986; Inouye et al. 1986; Saigo et al 1984; Tanda et al. 1988) and a moth [TED] (Friesen and Nissen 1990). The plant viruses CAMV [cauliflower mosaic virus] (Gardner et al. 1981) and CERV [carnation etched ring virus] (Hull et al. 1986) are also related to this group. To our knowledge, LTR retrotransposons have never been reported in filamen- tous fungi.

In this paper we describe the isolation of an LTR retrotransposon from the genome of the filamentous phytopathogen Cladosporium fulvum, a biotrophic, non- obligate member of the Deuteromycetes (Fungi imper- fecti), the causative agent of tomato leaf mould. The disease is a model system for the study of plant-pathogen interactions. The pathogen exists in a number of physio- logical races, which exhibit a gene-for-gene relationship with cultivars of tomato carrying different resistance genes (de Wit 1977). Five avirulence genes have been identified (Lindhout 1989) and Avr9 has been cloned (van Kan et al. 1991). We have previously described the isola- tion of a 225 bp sequence, designated P5, from the genome of C. fulvum (McHale et al. 1989). P5 encoded a putative protein with striking homology to reverse transcriptase sequences from various retroelements. P5 was shown to be a moderately repetitive sequence, which hybridised to about six bands in pulsed field gel separa- tions of C. fulvum chromosomes (Talbot et al. 1991). In this paper we report the isolation and characterisation of cosmid clones containing DNA homologous to P5. We show that they contain full-length copies of a retrotrans- poson and suggest that these elements be designated CfT-1 (C. fulvum transposon).

ed pAN7-2, a vector based on pAN7-1 (Punt et al. 1987). Colony and Southern hybridisations used Hybond-N membranes (Amersham) and standard procedures (Hodgson and Fisk 1987; Maniatis et al. 1982).

Single- and double-stranded clones were sequenced using the Sequenase 2.0 kit (United States Biochemicals), the Pharmacia (Uppsala) T7 polymerase kit for manual electrophoresis or the Auto-read kit and an ALF-Phar- macia sequencing apparatus. Primers were synthesised on a Cyclone BioSearch synthesiser. Sequences were analysed using the DNASIS package (Pharmacia) and the NBRF and EMBL databases. Phylogenetic analysis used the PAUP 3.0 package (Swofford 1990). Alignments of amino acids were performed by eye.

For isolation of virus-like particles (VLPs), 5 x 103 condiospores per 50 ml of Czapeck's Dox liquid medium (Oxoid) were incubated at 25 ° C with rapid shaking (200 rpm) for 10 days. Cells from 10~12 g wet weight of mycelium were broken down by freezing in liquid nitro- gen and grinding in a pestle and mortar in 15 ml of extraction buffer (1 M MgSO4, 10 mM TRIS, pH 7.4, 10 mM CaCI2) with the aid of sand (E1-Sherbeini et al. 1984). The homogenate was incubated on ice for 20 rain and centrifuged at 9750 x g in a Sorvall SS34 rotor for 1 h. Three-millilitre aliquots of the supernatant were overlaid onto 1 ml of 45% w/v sucrose in PKE buffer (0.02 M NazHPO4, 0.15 M KC1, 0.01 M EDTA, pH 7.6). The samples were centrifuged in an SW41 Beckman rotor at 61000 x g overnight at 4 ° C. The supernatant was dis- carded and the pellet was resuspended in 1 ml of PKE and kept on ice. The samples were loaded onto a 10-80% w/v sucrose density gradient (made up in PKE buffer) and centrifuged in an SW41 rotor (130000 x g, 4 ° C, 4 h). One millilitre fractions were collected by pipetting gently from the top of the tube. The fractions, numbered 1 to 12, from the top to the bottom of the tube, were diluted 5 times in PKE and centrifuged at 150 000 x 9 at 4 ° C in a Beckman Ti65 rotor. The pellets were resuspended in a minimum volume of PKE and assayed for reverse transcriptase by the method of Garfinkel et al. (1985), with poly G as template and oligo dC as primer.

Aliquots from each fraction of the sucrose density gradient were extracted with one volume of chloroform to remove lipid and allowed to settle for 5 min onto carbon-coated plastic film on nickel grids. Samples were fixed for 5 min with 2.5% glutaraldehyde in phosphate buffer, pH 7.4; 2% (w/v) uranyl acetate was used for negative staining for 2 rain. The samples were rinsed in phosphate buffer in between the steps. Grids were exam- ined for VLPs in a JEOL 100C electron microscope.

Materials and methods Results and discussion

C. fulvum races were maintained on V8 juice agar (Harl- ing et al. 1988) plates at 25 ° C. Escherichia coli JM83, JM101, pUC19 and M13 mp 18 and 19 were used for subcloning and sequencing (Norrander et al. 1983). Fun- gal DNA was prepared according to Raeder and Broda (1985). The cosmid library was constructed by ligating DNA partially digested with Sau3A into BamHI-digest-

Isolation of full-length copies of CfT-1

A cosmid library was screened with the C. fulvum reverse transcriptase clone pNOMP5 (McHale et al. 1989). Nine hundred cosmid clones were screened, from which five positive clones were isolated. DNA from the clones was digested with BamHI or HindIII, and probed with

pNOMP5 DNA. BamHI digestion revealed that cosmids 691,712 and 786 each contained a 1.7 and 1.8 kb doublet of hybridising DNA. This doublet was reminiscent of BamHI-digested, C. fulvum genomic DNA probed with pNOMP5 (McHale et al. 1989). pNOMP5 hybridised to different BamHI bands in the remaining two cosmids, 3612 and 920 (data not shown). Southern hybridisation of HindIII-cut C. fulvum genomic DNA with pNOMP5 had revealed a multitude of high molecular weight bands, implying that intact copies of the P5 retrotransposon are not cut with this restriction enzyme (McHale et al. 1989). Similarly, pNOMP5 hybridised to high molecular weight bands in the HindIII-digested cosmid DNAs, indicating that they may contain full-length copies of the C. fuF rum retroelement. The cosmids which hybridised to pNOMP5 were subcloned by partially digesting with HindIII, religating and transforming into E. eoli. The smallest subclones that retained the 1.7 and 1.8 kb Barn- HI doublet were selected for detailed analysis. Subclones pNOM712:3 (15 kb) and pNOM691:125 (20 kb), were mapped with EcoRI, BamHI and HindIII.

Sequencing of CfT-1:712

The PstI, BamHI and EcoRI fragments of pNOM712:3 were subcloned into pUC18 (Norrander et al. 1983), thus generating a series of overlapping subclones that were representative of the whole of 712:3 (Fig. 1). The long terminal repeats were detected by probing digests of 712:3 with each of the subclones, searching for multiple hybridisations. This delimited the element to approxi- mately 7.4 kb. (Fig. 1). Many of the subclones were transferred to M13 mpl8. Both strands of the element were sequenced by subcloning and using specific primers (Fig. 2).

The Ion 9 terminal repeats

Examination of the LTRs revealed many features that are characteristic of retrotransposons (Fig. 3). The LTRs are 427 bp long and are identical. Sequencing the CfT-1:691 LTRs from the PstI sites outwards also gave the same identical sequences. Since LTR sequences are

l kb

P P BE X EBE PXB P X I I I I I III I11 I I

ILTRi ILTRI

I P3 I P31 I P13 I P6 [ B21 I B13 I B15 I

I E12 I I E21 P5

ORF1 ORF3 O R F 2

Fig. 1. Restriction map, subclones and open reading frames (ORFs) of the CJT- 1.712 region. Abbreviations of restriction enzymes used are as follows-PstI, P; EcoRI, E; BamHI, B; J(hoI, X. Subclones are referred to in the text with prefix pNOM. LTR, long terminal repeat

339

homogenised during replication (Boeke and Corces 1989) and would be expected to diverge once integrated into the genome, the identity of the LTR sequences places a limit on the time at which the cloned elements transposed. The presence of highly homologous se- quences as represented in the 691 and 712 subclones, at different genomic locations reinforces the view that this element has recently transposed.

Examination of the sequences flanking the LTRs in 691 and 712 did not permit an unequivocal assignment of the length of the target site duplication because of degeneracy of the sequences. However, comparison of the 691 and 712 sequences indicated that 5 bp target-site sequences were duplicated during integration. The two duplicated sequences° TATAG(712) and GTACC(691), bear no obvious relationship to each other. The first sequence is reminiscent of the TATA and ATAT target- sites of the gypsy retrotransposons, 17.6 297 and Beagle (Bingham and Zachar 1989). The ends of the LTRs exhibit a 5 bp perfect inverted repeat structure. The LTRs terminate in the characteristic sequences 5'TG...CA3'.

Transcription of retrotransposons starts and termi- nates within the LTR sequences. In some cases, se- quences within the LTRs have been shown to have the properties of enhancers (Errede et al. 1985). It is inte- resting to note that sequences in the CfT-1 LTR show striking homology to the Tyl enhancer (Xu and Boeke 1990) and also to enhancers identified in SV40 (Weber et al. 1984), yeast PH080 (Gilliquet et al. 1987) and N. crassa (Geever et al. 1983) (Fig. 3). Two sets of re- peated sequences lie on either side of the SV40 en- hancer-like region (Fig. 3).

Immediately 3' to the 5' LTR is the presumed primer binding site for first-strand reverse transcription. There is no TGG sequence, implying that an internal tRNA fragment rather than an intact tRNA is used for priming. Internal tRNA fragments are used for priming copia retrotransposition (Kikuchi et al. 1986). The CfT-1 primer binding site is complementary to conserved re- gions of tRNAs. The sequence can be imperfectly aligned with an internal region of a S. pombe serine tRNA (Kohli et al. 1984). Second-strand priming involves annealing to a polypurine tract immediately 5' to the 3' LTR. In CfT-1, the sequence A G A G A G G G G A T G G is found at this position.

Four PstI sites were found in CfT-1, one in each LTR and two in the internal, single copy region. Southern hybridisation of the subclone pNOMP5 to PstI-cut C. fulvum genomic DNA revealed just two very intense bands of 3.2 kb and 4.9 kb (Fig. 4). All other restriction enzymes, such as XhoI (lane 2), give a ladder of heteroge- neous bands in addition to the strong band predicted by the presence of two internal XhoI sites (Fig. 1) in 712, when hybridised to fragments of CfT- 1. This implies that all pNOMP5-related sequences have conserved PstI sites in the LTRs. The internal PstI sites are symmetrically arranged so either one, but not both, is absent in about half the genomic copies of the retrotransposon. The presence of conserved LTR sequences amongst all the CfT-1 sequences is evidence of an unusual degree of

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TAAGAGACGGTAATAGACTATA~AAGGAG CTTAAGATAT CGGTATACG CACT~C=ATAAACAATAG OOAG OOATA

TAC C CTTATATAq~2GTTAC C CGTAATAATATAATATA~[fG%~gACGGAT CGGACACGAC CGAAGGAGCGT CAG C CAATCAATAG C C CG C

ACTAATGACAGG CG ~ C CACAG CTAGG CC~gGCACAG C CG CTG CAGAGTG C CTGGAC4I~ CCTC~AG~fG C CTGGAGTG CCTA~OO

TAGG CACAGTAC4] CACC~ATAGGCACGGTAC4~ CA~AAAGGAATGGA;~I'I'I'C tit ±~T~IT I'AGGAACG CATCGTAC CCITATC4~TT

CGAGGCG%~f CAAOOCACTGTT CAATAT CACTG'±'±'I~GAATACACCACAAC C CAACCTCTG COO CACI]9 CTG C±'±GGTc ±'I'I'ACAAGAC

GAGACAG OTACTG CI~9 CAT CACTCTC C CT CAC CITCCAT CGGCCACGAGACAAAACCCGATI~AG~T tT±'t~fGATCI'fGTGATA

T CA~AGGTTATAC CAAAC OOAACAGc ±TIGAACGC COO CAACGATGGGAGATCGACAGAGTGAC ~AACACC CGC CC C CAGTGA

Met Leu Set Met Pro Trp Leu Leu Ile Gln Ala Thr ASn

604 AGCA~CAGAGCCCGACGACOOCAGACCGCGAACGATG CTA TCA ATG CCT

Pro Gln Gly Trp Gln Pro Glu Phe Phe T!rr Gly Asp Arg Val

677 COO CAAGGT TGG CAA CCA GAG %~fC qTgC TAT GGC GAC CGA GTC

Met Asp.Met Tyr Phe Leu Phe Asn Set Met Thr Glu Asn Leu

743 ATG GAT ATGTAC~C q*fG~CAAC TCC ATG ACT GAG AAC OOC

Leu Arg Gly Arg Ala Gln His Trp Val Lys Pro Phe Leu Arg

809 ~q~ AGA GGA CGA GCA CAG CAT q~G GTC AAA CCA ~TC CI~ AC~

Asp ASn Ala ASp Gly Val Phe Lys Ser T!rr ASn His Leu Lys

875 GAC AAT GCTGAC GGAGTC %~TCAAG TCTTACAAC CAT OOCAAG

Val Ser Ash Glu Ile Ala Thr Ala Val Arg Val Ile Gln His

875 GTC TCG AAC GAG A_q~ GCC ACT GCC C4I~ CC/f GTT ATT CAG CAC

Glu Tyr Ala Ala Lys Phe Gln Glu Tyr Ala Gln Leu Thr ASp

941 GAA TAT GOO GCC AAG qTC CAG GAA TAC GCG CAA CTC ACC GAT

Met q~rr Arg Arg Gly Leu Lys Glu His Val Lys Asp Glu Leu

1073 Aq~Z TAT CGAAGA GGA CI~ AAG GAG CAT GTC AAA GAC GAG Gly Leu Gly ASp Leu Val Gln Val Thr Ile ASp Leu Asp ASp

1139 GGC ~TA GGC GAC OOA GTC CAA GTC ACG ATC GAT CI~ GAC GAC

Arg Arg Tyr ASp Set Lys Val Ser Gly Lys Ala Gly T!rr Thr

1205 CGA CGA TAC GAC TCC AAG GTA TOO GGAAAG GCC GGA TAC ACG

Gly Phe Ash asp ASh q~yr ASn Lys Pro Lys Asp Lys Pro Tyr

1271 GGT TTC AAC GAC AAC TAC AAC AAA CCC AAG GAC AAG CCG TAC

ASp Val Thr Glu Lys Gly Arg Lys Ile Arg Asn Ser Lys Gly

1337 GAC GTT ACC GAG AAA GGT CGC AAG ATC AGG AAC AGC AAG GGG

Glu Thr Arg Thr Cys Tyr Gly Cys Gly Lys Pro Gly His Ile

1403 GAA ACA CGA ACT TGC TAT GGA TGC GGC AAA COO GGC CAC ATA

Met Val Arg Arg Glu Gln Pha ASn Met Met Gln Arg Arg Thr

1469 ATG GTG CGC CGC GAA CAG ~C AAC ATG ATG CAA CGA CGA ACT

Ser Leu Gly Asp Ile ASp Tyr Thr Arg Arg Val Glu Glu Arg

1535 TOO CTG GGA GACATC GAC TAT ACT CGGAGA GTG GAG GAG AGA

Leu leu ASp pro Ser Gln Gly Arg gly Gly Cys His Gly Gln

1601 OOC CTG GAC CCC TCA CAA GGA OGA GGA GGA TGC CAT GGG C~

Leu Gln ASn Gln Pro Arg Gln Phe Asn Met Met Ala Arg Arg

1667 CTG CAG AAC CAG CCA CGA CAG TTC AAC ATG ATG GCA CGA AGA

Set Arg Asn Gly His Gly Ser Leu His Trp Arg Phe Cys T!rr

1733 TCT CGAAAT GGT CAC GGA AGC CI~ CAT TGG C~A%~fCTGT TAC

Set Ala Lys Set Gly Ala Gly Tyr Trp Pro Gln Gln Pro Arg 1799 TCC GCG AAA TCA GGA GCA GGG TAT TC49 CCT CAG CAA CCG CGA

Gln ASp Ala Thr Leu Gln Glu Val ASp Ile Asp Glu Set Cys

1865 CAA GAC GCC ACG OOC CAA GAA GTG GAC ATC GAC GAA TCC q~C

Glu ASn Gln ASp Pro Gly Trp ASn Gly Ser Trp Ser Pro Glu

1931 GAA AAT CAG GAC CCA GGA TGG AAC GGA TCG TC49 TCA CCG GAA

Thr Gly Leu ASp Thr Ile Glu GIu Asp Gln Asp Pro Glu Glu

1997 ACC GGG CTG GAT ACC ATC GAG GAA GAC CAG GAT CCG GAA GAA

Gly Glu Glu Set Asp Thr ASp ASp Asp ASn Glu Gln Met Thr

2063 GGA GAAGAGAGC GAC AOOGAC GAC GACAAC GAACAAATG ACC

Leu Tyr ASp Met Ile Ile His Leu Gln Arg Arg His Glu Glu

2129 CTC TAC GAC ATGATA An CAC OOC CAACGA AC~ CAT GAAGAA

Arg Met Leu His Ser Ile Glu Phe Asp Lys Thr Leu Asp Thr

2195 CGA ATG OOA CAC TCC ATT GAG '~±'±' GAC AAG ACA OOC GAC ACC

Pro Leu Met Glu Thr qg~r Glu ASh Leu Ala Thr Ala Val Thr

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2327

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Fig. 2

13 TGG CTA CTC ATA CAG GCA ACG AAT

Lys Phe Asp Thr Trp Val Set Gln 35

AAG qrfC GAC ACT TGG GTC TCT CAG

Lys Pro Ile Phe Ala Ala ~hr Phe 57

AAG CCT ATC 'i'±'±' GCC ACC ACC TTC

Lys T!rr Leu Asp Set Asn Gly Glu 79

AAA TAC CTG GAT AGC AAC C~A GAA

His Ala Met Lys Ser VAI Phe Gly i01

CAT GCT ATG AAG AGC GTC '±'±',' GGT

Leu Thr Gln Lys Thr Set Thr Ala 123

ACT CAG AAG ACT TCC ACA GCC

Trp Asp Asp Glu Ala Leu Gln Val 145

TC4Z GAC GAC GAA GCA CTT CAA GTC

Met Arg ASp Gly Arg Lys Ile Asp 167

ATG AGG GAC GGT CGG AAG ATC GAT

Lys Leu T!rr Glu Arg Ala Met Glu 189

AAG CTC TAC GAG AGA GCT ATG GAA

Pro Gly Tyr Asp Asn Arg Asn Arg 211

CCA GGC TAC GAC AAC CGT AAC AGA

Tlrr Gly Pro Gln Pro Met Glu Leu 233

TAC GGA CCA CAG CCA ATG GAG CTC

Asn Arg Arg Pro Pro Set Set Arg 255

AAC AGA AGA CCC CCG A6~ TCA AGA

Ala Arg Asp Cys Arg Gly Lys Asn 277 GCA AGG GAC TGC CGC GGT AAG AAT

Set Lys Ser Glu Set Set Val Glu 299

AGT AAA TO0 GAG AGC A~T G%~f GAG Ala Set Asn Gly Set Thr GIu Pro 321

GCT AGC AAT GGA AGC ACG GAG COO

Gln Met Val Val Ile Pro Phe Glu 343 CAA ATG GTG GTC A~9 CCA TTC GAG

Val Asp Tyr Glu Ile Glu Set GIu 365

GTC GAC TAC GAA ATC GAG TCC GAA

Gln Asp Set Cys Gln Val His Tyr 387

CAA GAC TCG TGT CAG GT~ CAC TAC

Gly Thr Leu Gly Ala ~ His Arg 409

GGA ACC CTG GGA GCA ACA CAT AGG

phe Asp Asp Asp Gly Set Asp Lys 431

~C GAC GAC CAT GGA AGC GAC AAA

Pro Gln Glu Glu Set Glu Glu Thr 453

CCA CAG GAA GAA AC]r CAA GAA ACC

Set Ser Glu GIu ASp Set Ser Glu 475 TCA AGC GAA GAA GAT TCC TCC GAA

Phe Thr Val Asp Ala Pro Lys Lys 497

~C ACC GTA GAC GCA CCG AAG AAG

Phe Leu Pro Arg Ile Gly Gly Arg 519

qTC CTA CCG AGG ATA GGC GGA CGA

Leu Arg Gly Met Ala Trp Gly Tyr 541

CTA CGA GGC ATG GCC TGG GGA TAC

Glu Arg Pro Pro Ile Gly Set Arg 563

CCA CTC ATG GAA ACG ACC GAA AAC CTA GCA ACT GCT GTC ACA GAG CGA CCG CCG ATA GGA AGT AG

Met Ile Gly Thr Gly Tyr Leu Thr Pro Set Gly Thr Phe Val Ser Asn Glu Leu Arg Asn Met Val

ASh Asp Arg T!rr Arg Ile Pro His Thr Val Arg Asn Ile Arg Ile Lys Arg Ile Ala Gln His Gly

AAT GAT AGG TAC CGG ATA CCT CAC ACC GTC AGG AAC AZ~f CC]f ATC AAA CGA ~ GCG CAA CAT GGT Gln His Ala Arg Ala Leu Tyr Set Glu Thr Gln.Lys Ile Gln Glu Arg His Arg Val Gln Met Trp

Ala Ala Arg Thr Arg Thr Leu Leu Arg Ash Ala Glu Asp Pro Gly Thr Ser Ser Set q~r Asp Val

GCA GCA CGC ACG CGC ACT CTA CTC AGA AAC GCA GAA GAT CCA GGA ACG TCA TCG AGT ACA GAT GTG Thr Gln Lys Gly Set Arg Gln Val Gln Ala Glu Ala Set Arg His Glu Ala Arg Arg Set Thr Tyr

Asp Thr Glu Arg Leu Ala Pro Gly Ser Set Gly Gly Leu Glu Thr Arg Set Thr Thr Phe His Ile GAC ACA GAA AGG CTC GCG CCA GGT TCA AGC GGA GGC CTC GAG ACA CGA AGC ACG ACG q~C CAC ATA

Glu Gln Gly Lys Gly Set Tyr Pro Gln Glu Stop

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Arg Thr Gly Lys Arg Ile Asn Ser Pro Gly Met Ser Asp Arg Arg Glu Leu Cys Asn Thr Glu Lys 88

CGA ACA GGG AAA AGG ATC AAC TCC CCA GGA ATG AGC GAC CGA AGG GAG ~TPA TGC AAC ACA GAA AAG

Ala Gln Ala Set Pro Thr Thr Thr Val Phe Arg Thr Lys Ile Ile Val Ash Gly His Lys Thr Asp ii0

GCT CAA GCA TCA CCA ACG ACA ACA GTG 'i'I'I' C~A ACC AAG ATr ATC GTC AAC GGG CAC AAG ACA GAT

Ala Met Ile Asp Set Gly Ala Ser Gly Ash Phe Ala Set Glu Set Phe Val Thr Arg Ash Arg Ile 132

GCC ATG ATA GAT TCC GGA GCG AGC GGG AAT '±'±'£ GCC TCC GAA TCA 'i'±'±' GTI' ACA AGA AAC AGG ATC

Ala Thr Cys Lys Lys Lye Glu Gly Tyr Glu Leu Ile Ala Val Asp Gly Ser Ser Leu Pro Set Val 154 GCT ACC TGT AAG AAG AAA GAG GGT TAT GAA ~ ATC GCG GTA GAC GGA TCA TCC ~PA CCC AGC GTG

GIu Arg Glu Thr Ile Pro Leu Pro Leu Ala Ile Gln Arg His His Glu Glu Ile Thr Leu Asp Val 176

GAG CGC GAG ACG ATA CCG C~A CCA CTT GCf ATC CAA CGG CAC CAC GAG GAG ~ ACC ~ GAT GTT

Thr Asp Met Ala Ser His ASp Ile Val Leu Gly Met Pro Trp Leu Arg Lys His Asn Pro Val Ile 198

AC9 GAT ATG GCC AGT CAT GAC ATC GTA CTA GGT ATG CCT TGG q~G AGA AAG CAC AAC CCA GTA ATC

Asp Trp Arg Arg Gly Val Leu Thr Phe Arg Glu Cys Glu Cys Val Ile Asp Ile Gln Pro Ala Gln 220 GAC TGG AGA AGA GGA GTA C9C ACA 'i'±'±' AGG GAA TGC GAA TGC GTT ATC GAC An CAG CCT GCG CAG

Trp Gln Arg Ser Leu Ala Asp Glu Ala Arg Lys Gln Leu ASn Arg Ile Gin Leu Ala Pro Thr Arg 242

TGG CAG CC/f TCA CTG GCA GAT GAG GCA AGG AAA CAG C~C AAC AGG ATA C/~ CTT GCG CCG ACT AGA

Arg Thr Glu Glu Pro Pro Set Thr Gly Thr Asp Thr Gly Val Gly Pro Pro Gly His Glu Val Thr 264

AGG ACA GAA GAA CCA CCT TCG ACC GGG ACA GAC AC~P GGC GTG GGG CCG CC~ GGT CAC GAA GTC ACT

Gly Ser Asp Gly Ser ASn Ala Pro Ser Lys Asp Thr Ash Ile Ser GIu Leu Set Ile Pro Lys Glu 286 GGA AGT GAT GGA AGT AAC GCA CCA TOG AAG GAT ACC AAC ATC TCA GAG ~I~ AGT Aq'P CCC AAG GAG

Tyr Arg Lys Trp Set Arg Leu Phe Glu Glu Glu Arg Gly Lys Asp Ala Leu Pro Lys His Gln Pro 308

TH/T CGC ~ ~ TOG CGA CTC TI'I' GAA GAG GAA AGA GGC AAG GAC GCC ~A CCT AAG CAC CAA CCA

Trp ASp His Lys Ile ASn Ile Gln Pro Gly Lys Glu Pro Pro Trp Gly Pro Leu Tyr Gln Met Ser 330

TGG GAT CAC AAG ATA AAC A~ CAG CCA GGG AAA GAG CCT CCA TGG GGA CCC CTA TAT CAA ATG TCT

Glu Lys Glu Leu Gln Thr Leu Arg Glu Trp Leu Lys Glu Lys Leu Ala Lys Gly Trp Ile Arg Arg 352 GAG AAA GAG CTA CAG ACC CTA OGA GAA TGG CI~ AAG GAG AAG CTA GCC AAA GGA TGG ATA CGA CGA

Ser Thr Set Set Ala Gly Thr Pro Cys Met Phe Val Pro Lys Ala Asn Gly Lys Leu Arg Leu Val 374

TCC ACC TCA AGT GCA GGA ACT CCA TGC ATG TTC G~ CCA AAA GCA AAC GC~ AAA CTA CGA CTC GTA

Gln Asp Tyr Arg Lys Leu Asn Glu Ile Thr Ile Lys Asn Arg Tyr Pro Leu Pro Asn Ile Glu Glu 396

CAA GAC TAC CGA ILAG qrfG AAC GAG ATC ACG ATC AAG AAC CGA TAT CCG CTA CCC AAC ATC GAA GAA

Ala Gln ASp Arg Leu Thr Gly Set Asp Tip Tyr Thr Lys Ile Asp Leu Arg Asp Ala Phe Tyr Ala 418 GCA CAA GAC AGA qTDA ACC GGA TCA GAC TGG TAC ACG AAG ~ GAC CTA CGA GAC GCC ~ TAT GCT

Ile Arg Met Ala Glu Gly Glu Glu Tip Lys Thr Ala Phe Arg Thr Arg qhyr Gly Leu Tyr Glu Phe 440

ATC CGA ATG GCA GAA GGA GAA GAA TGG AAA ACC GCT %~fC AGG ACA AGA TAC GGA C9C TAC GAA ~C

Leu Val Met Pro Met Gly Leu Thr ASn Ala Pro Ala Set Cys Gln ASp Leu Val Asn Glu Thr Leu 462

qq~3 GTC ATG CCA ATG GGA ~ ACC AAC GCA CCC GCA TCC TGC CAG GAC CTI ~ GTC AAC GAA ACA CI~

Arg Asp Leu Leu Asp Val Cys Val Val Ala Tyr Met Asp Asp Ile Leu Val Tyr Thr Lys Gly Set 484 AGA GAC CTA CTC GAC GTG TGC (19C GTT GCT TAC ATG GAC GAC ATA CTG GTC TAC ACA AAA GGA TCC

Leu Gln Glu His Thr Lys Gln Val Gln Asp Val Phe Glu Arg Leu Thr Lys Set Gly Phe Lys Thr 506

CTC CAG GAA CAT ACC AAG CAA G%~f CAA GAT GTG TTC GAA CGA CTC ACG AAG TCC GGA TTC AAG ACA

Ala Pro Glu Lys Cys Glu Phe His Lys Lys Glu Val Lys Phe Leu Gly Phe Ile Ile Set Thr Thr 528

GCA CCC GAG AAA TC-C GAA TTC CAC AAG AAA GAA GTC AAG '±'l'±' TTA GGC qq~f ATC ATC AGT ACA ACA

Gly Ile Thr Ile Asp Pro Ala Lys Thr Gln Ser Ile Arg Glu Trp Pro Glu Pro Lys Thr Val Lys 550 GGG ATA ACG ATC GAC CCT GCA AAG ACA CAG TCA ATC AGA GAA TGG CCA GAA CCG AAG ACA GTC AAG

Asp Val Gln Set Phe Leu Gly Leu Ala Asn Tyr Asn Arg Lys Phe Ile Lys Asp Tyr Set Lys Thr 572

GAT GTA CAG TCA TTC CI~ GGA CTC GCC AAC TAC AAC CGG AAA %vfC ATT AAG GAC TAT TCA AAG ACA

Ala Ala Pro Met Thr Met Leu Thr Arg Lys Asp Val ASn Trp Lys Trp Gly Lys GIu Gln Thr Glu 594

GCA GCA CCA ATG ACG A%~ CI~ ACA AGA AAA GAC GTC AAC TGG AAA TGG GGA AAA GAA CAG ACC GAA

Ala Phe Lys Arg Leu Lys Glu Gln Cys Ala Set Ala Pro Thr Leu Arg Leu Phe Asp Gly Set Lys 616 GCG ~PC AAA AGA CTC AAG GAA CAG TGC GCT TCA GCC CCA ACG CTr CGA CTA qTfC GAT GGT AGC AAG

GIu Val His Ile Glu Thr ASp Ala Ser ASp Met Ala Ile Gly Ala Cys Leu Thr Gln Thr His ASp 638

GAA GTC CAC ATC GAG ACC GAC GCG TCT GAT ATG GCA ATA GGC GCA TGT CTA ACA CAG ACA CAC GAT

Gly Lys Arg His Pro Val Ala T!rr Tlrr Set Arg Lys Met Thr Thr Ala Glu Gln Asn Tyr Asp Ile 660

GGG AAA AGA CAC CCA GTG GCC TAT TAT TCC CGG AAA ATG ACC ACA GCG GAA CAG AAC TAC GAC ATC

His Asp Lys Glu Leu Leu Ala Ile Val Ala Ala Met Gln His Tip Arg Val Tyr Val Glu Gly Pro 682 CAT GAC AA~ GAG CTT CTA GCC ATT G~ GCC GCC ATG CAA CAT TGG AGA GTG TAC GTC GAG GGC CCA

Pro Lys Leu Thr Ile Leu Set Asp His Lys ASh Leu Thr Tyr Phe Thr Thr Thr Lys Glu Leu Thr 704

CCG AAG q~gA ACG A~ CIT TCA GAC CAC AAG AAT CTC ACG TAC ~C ACG AC~ ACG AAG GAA c±'i ~ ACC

Arg Arg Gln Ala Arg Trp Ser Glu Leu Leu Gly Gln Tyr Lys Phe Glu Ile Lys Tyr Thr Pro Gly 726

CGA AGA CAA GCC CGC TGG TCG GAG CTG C~9 GGG CAG TAC AAG q'fC GAA ATC AAA TAC ACT CCA GGA

Thr Glu Asn Gly Pro Ala Asp Ala Leu Set Arg Arg Set Asp Tyr Met Glu Gly Lys Glu Pro Val 748 ACA GAG AAC GGC CCG GCA GAT GCG CTG AGC CGA AGA AGC GAT TAC ATG GAA GGA AAA GAA CCA GTG

Gln His Lys Ile Leu Lye Thr Ash Pro Asp Gly Set Leu Set Ala Ash Thr Arg Glu Phe Asn Asn 770

CAA CAC AAA ATA Cq~ AAG ACA AAC CCC GAC GGA AGT CTT AGT GCC AAC AOG AGA GAG ~ AAC AAC

Ile Val Arg Ile Leu Set Asp Lys Glu Glu Gln Phe Pro Ile Set Gln Gly Lys Tyr Gln Val Pro 792

Aq*9 @TA CGA ATC CTA AGC GAC AAG GAA GAA CAG q~gC CCT ATA TC~ CAA GGA AAG TAC CAA GTA CCA

Lys ASp Arg Glu Glu Glu Cys Ile Arg Gln His His ASp Glu Pro Thr Tyr Gly His Pro Gly Thr 814 AAG GAT CGA GAA GAA GAA TGC ATA CGA CAA CAT CAC GAC GAG CCA ACA TAC GGA CAT CCT GGA ACT

Set Lys Thr Val Asp Leu Ile Gln Arg Set Phe Set Phe Pro Gln Met Arg Leu Lys Val Leu Arg 836

342

4770

4836

4902

4968

5034

5100

5166

5232

5298

5364

5430

5505

5582

5648

5714

5780

5846

5912

5978

6044

6126

6213

6300

6387

6474

6561

6648

6735

6822

6909

6996

7083

7170

7257

7344

TCA AAG ACA GTC GAC CfA ATA CAG CGC AGC T�C TCA ffTC CCA CAG q~rr Ile Lys Lys Cys Val His Cys Gln Gln Asn Lys Ala Ala Arg

TAC ATC AAG AAA TGC GTA CAC ff~C CAA CAA AAC AAA GCT GCA CGG

Gln Phe Arg Thr Pro Pro Thr Lys Pro Trp Asp Glu Val Thr Met

CAG ff~C AGG ACA CCA CCA ACG AAA CCA TGG GAC GAG GIfT ACG ATG

Arg Ser Lys Asp Arg Val Thr Gly Gln Ala Tyr ASp Met Ile Leu

AGG TCA AAG GAT CGA GTC ACA C43A CAA GCC TAT GAC ATG ATA CTA

Lys ff~rr Ala His Phe Ile Pro Ala Ser Glu Ile ff~agr Thr Ala Glu

AAA TAT GCA CAC ~TC ATT CCT GCA TCA GAA ATA TAC ACT GCA GAG

Asp Arg Leu Ile Arg Tyr His Gly Phe Pro Glu Val Phe Ile Thr

GAC AGA ffff~ ATC CGA TAT CAC GGA ff~fC CCG GAA GTA ~C ~ ACA

Set ASn Tyr Trp Lys Thr Leu Met Gly Thr Ile Gly Ile Lys His

TCA AAC TAC TGG AAG ACG CTC ATG GGA ACG ~ GGA ATC AAG CAC

Pro Glu Thr ASp Gly Gln Thr Glu Arg Thr Asn Gln Thr Leu Glu

CCA GAG ACG GAT GGG CAA ACG GAA AGA ACG AAC CAG ACA CTC GAG

Asn Tyr Ala Gln Asp Asn Trp Val Set Leu Leu Pro Met Ala Gln AAC TAC GCA CAA GAC AAC TGG Gffrf TCA q~fA CTG CCA ~ C-CG CAG

Set Glu Thr Thr Set Thr Thr Pro Phe Met Arg Thr Leu Ala Arg

TCA GAG ACA ACT TCG ACG ACG CCA '±'±'±' ATG CGA ACT ffff~ GC~ AGG

Leu Asp Pro Thr His Arg Pro Arg Glu Gln Ser Stop

CTG GAT CCC ACC CAC AGG CCG AGA GAG CAA TCG TGA

AGA CTT AAG GT~ CTA CGC His Ala Lys Tyr Gly His Leu

CAC GCG AAA TAC C4~T CAC CTA

ASp Phe Ile Thr Lys Leu Pro

GAC q~2C ATT ACG AAA CTC CC~

Val Met Val ASp Arg Leu Thr

GTC ATG GTC GAC AGA CTC ACA

Gln Leu Gly Tyr Leu Val Leu

CAG CI~ C~A TAC CTC GTA CTG

Asp Arg ASp Lys Leu Phe Thr

GAC AGA GAC AAG CTC %aTC ACA

Lys Leu Ser Thr Ala Tyr His

AAG TI~ TCA ACA GCA TAC CAT

Gln Tyr Leu Arg His T!rr Ile

CAA TAC CTA CGG CAC TAC ATC

Ile Ala Leu Asn Asn His Lys

ATC GCA CTG AAC AAC CAC AAA

Thr Leu Thr Tyr Pro Glu His

ACC C�A ACC TAT CCG GAA CAC

CGACGGAAACTCT CAAGG~ CACAAAGAAG CAC~CA

Met Ala Pro Leu Leu Lys Glu

AGGCGATCGAAGATGCACAAC_AAAGACTGTC �CAACGACGGCAAGA~AAAA ATG GCA CCT C~G CTA AAA GAG

Gly Asp Lys Val Tlrr Leu Leu Thr Lys Asn Leu Lys Thr Arg Arg Gln Thr Lys Lys Leu Asp His

GC49 GAT AAG GTC TAT CTC CTC ACA AAA AAC CTG AAG ACA AGA AGA

Val Lys Val Gly Pro Phe Phe Ile Asp Lys Val Val Gly Pro Val

Gffrf AAG GTC GGA CCA 'I'I'I ~ 'i'I'I' 2~_TC GAC AAA GTC GTA GGA CCA GTC

Pro Asp Ala Lys Ile His Pro Val Phe His Ile Ser Lys Leu Glu CCT GAT GCG AAA ATC CAC CCG GTC TTC CAT ATC TCC AAG TTG GAA

Cys Gln Glu Set Phe His Phe Glu Pro Glu Ala Glu Asn Glu Phe

TGC CAG GAG AGC ~fC CAT TTC GAG CCG GAA GCA GAA AAC GAA '±'±'i'

Lys Lys Gly Gln Arg %marr Leu Val Lys Trp Lys Gly q~r Asp Glu

AAG AAG GGT CAG CGA TAC CffT GTC AAA TGG AAA GGA TAC GAC GAA

Arg Ile Ash Leu Ala ASh Cys Tyr Gln Leu Leu Arg Gln Phe Gln

AGA ~ AAT CTG GCG AAC TGC TAC CAG CIrf CI~ CGA CAG TTC CAG

Lys Gln Glu Ala Gln Glu Arg Arg Ala Ser Pro ASp Gln Thr Arg

AAA CAA GAA GCT CAG GAA CGG AGA GCA AGT CCG GAT CAG ACC AGA

Ala Arg Thr Lys Stop

Fig. 2. Sequence and deduced ORFs of CJT-1.712. The target-site duplications are in bold-type. The inverted repeats at the ends of the LTRs are underlined. The numbers at the left indicate nucleotide

CAG ACC AAG AAA CTG GAT CAC

Asn Tyr Arg Leu Arg Leu Pro

AAC TAC CGA CTA CGA CTA CCA

Pro Ala Asp Ale Glu Thr Pro

CCA GCG GAC GCC GAA ACA CCT

Glu Val Glu Lys Ile Leu Asp GAA GTC GAA AAA ~ CTA GAC

Set Glu Asn Thr Trp Glu Pro

TCA GAA AAT ACC ff~G GAA CCA

Lys Trp Arg Gln Asp Set Arg

AAG TGG CGC CAA GAT TCC CGG

Ser Arg Pro Lys Tyr Pro His

AGT CGT CCG AAA TAC CCT CAC

858

88O

9O2

924

946

968

990

i012

1034

1045

7

29

51

73

95

117

139

161

165

GCA AGG ACC /LAG TAG GAGGGGAAAGAGACAGCGAGC~AC~CCACGTCI~CGTCCGGAAGCTC~CAGCCrfC

CTCCACTG CGATCGCCTCGGTCTCTCGACGCTCTAGAAGCTCCAG CTCCc±'I'I C49AGACGGGCCTTCITfCT CCCAAG CGG CGGCCTC

TTCCTCCTGTAT CC9~ CGCACGAC~ CTTCCR~TATCcI'I'CGAAG CGAAGCCTC~TATG C f f r f ~ C T C C ~ ~

T CGACGATG CT C CGACAC~-I "I "i', "I'CTCGAGAGCCrCG CGCI~ACGGCG CAGACR]CGACCA-q-~CCG CCT~AAC~ACAqrfACA

GT ~CGA~T I'i'C c± T i~ZfACAGACACCACAC CGACCAGAC C CGACATGGACACGACAGACGACffrf CGT CT CG C C a A ~ A

CAGGGACGAACCATGATI~ CGCCCAGGG CITCG~I'I'I'±'CTCGGATTGACGAATG~A~ ~ ~ ~ ~ ~ C

ARX~ffff~AAGGAAAGAAGA~AG CCATGACGACCG CT CAACC CAAGGAAAC CT CGAffWA-DaT CATAAG CT CC<'~ACGAG CTAAG CTAGAG

AGGGG~ACGGATCGGACACGACCGAAGGAGC~TCAG CCAATCAATAG CCCGCACTAATGACAGGCG CGGC CCACAG CTAG

G CC~49 CACAG CCG~AGTG C~AGTG CCR~GAGTGCCTC~AGTGCCTACGGACTT~~~AT~ CA~ T ~ ' TI'Cc±'±'I GT2~'±TAC4~AACGCATCGTACCCTP~CGAGGCGTT CAACTCA~ C-AATA

TCACI~I'I-IC~AATACACCACAAC C CAACC�CTG CCTCACI~Cff~ ~ c±'±'±'ACAAGACGAGACAG CfACTG u'IwCATCACTCT

C C CTCACCI~CCAT CC~ C CACC~ACAAAAC CCG/ZPI~AG ~ T CIr9 CleAT ~ATCAff~ACaZTTATAC CAAAC CWAAC

~ATA~k~_%AC~CTCGAGG CCTTATAGAffWCTACTTCTACC~CTCTu'±'x'±'~ACfAGAAA.'±'I'±'ATACGCAAATACATAATAC CCCT

AATCCc ±' ± • I C=AOGTAAAG C CGACGACTAATAC~fAGGAGATAGAA~AC~fACACGG C3~A-q~2ATATACG CGG C C CS~fATAGACCrf

AC�I~AT CGG C~AqTfAffWACGAGCAAA~ATACTACGT CAC CGAC CP CGAATATAAT~ c ± TI 'AG C CTAC CC�TAG CTTATAG C

TATAAC CTAAACAT CI~C~fAGGAGATAGGCGASGTGGCA~TI'/~TCI~ CA~

positions, those at the right indicate numbering of amino acid residues in the respective open reading frames. The sequence has been submitted to EMBL as accession Z 11 866

sequence conservation. This may imply relatively recent acquisition of the CfT-1 elements within the C. fulvum genome.

The open readin9 frames

Three long ORFs, all on the same strand, were found when the sequence of CfT-1 was analysed (Fig. 1). The

first starts 99 bp from the 3' end of the 5' LTR. The predicted sequence of 639 amino acids has homology to 9a9 genes of retroviruses. In particular, amino acids 260-273 (Fig. 5) have the characteristics of a zinc-finger DNA-binding domain (C-X2-C-X�-C Covey 1986).

Retroelement genomes are typically transcribed into a long polycistronic mRNA which is translated into a polyprotein and, in turn, cleaved to give the final proteins

Length 427 bp

Duplicated sequences 712 TAATAATATAATATAG(113).(7084)TATAGAAAGGCTC at the insertion sites 691 TAACGGGCCTAGTACC . . . . . . . . . GTACCTTCTAGAA

Inverted repeats ( 114)TGTTACG CCTAACA(540)

Enhancer-like sequences and repeats (210) GAGTGCCTG

(241)

(252)

GAGTGCCTG GAGTGCCTG GAGTGCCTA Repeatl

GCCTACGGACTT GCCTATGTACTT SV40enhancerregion A

TAGGCACAG TAGGCACGA TAGGCACGG TAGGCACAG Repeat2

343

(461) TTCCA TTCCA Neurospora 5' enhancer TTCCA Tyl enhancer core

(528) ACCAAACCTAACAGCTTT ACCTCACCTAATGACTTT SV40 enhancer region B

(527) TACCA TACCA pho80 enhancer core

Primer binding sites First strand (539)5'.CAGCTTTGAACGCCCTCAA

Schizo~,,~cho~omyco=- I [ I I I I l ] l l pombe tRNA-Ser GUC AAACUUG

CU GAC. 5'

Second strand (6644) AGAGAGGGATGG

Fig. 3. Features of the long terminal repeats present in the Cladosporium fulvum transposon efT-1. Numbers refer to the nucleotide sequence in Fig. 2

1 2

4 .9

3.2

2 .7

Fig. 4. Hybridisation of pNOMP5 to PstI-(lane 1) and XhoI-(lane 2) digested C. fulvum genomic DNA, demonstrating the conservation of the PstI sites in the LTRs. The numbers indicate mol. wt. markers in kb

( K i n g s m a n and K i n g s m a n 1988). The t r ans l a t ion o f ret- rovi ra l and gypsy class r e t r o t r a n s p o s o n m R N A s requires r i b o s o m a l f rameshi f t ing f rom the gag O R F to the pol O R F . In CfT-1 , the pu ta t ive pol O R F over laps the gag O R F by 229 bases. The mos t l ikely pos i t i on for f rame- switching is the run o f 4As at pos i t i on 2534. The pol O R F extends for 1045 a m i n o acids. Sequences h o m o l o g o u s to p ro tease , reverse t ranscr ip tase , R N a s e H and in tegrase

Table 1. Distance matrix ,of reverse transcriptase sequences"

1 2 3 4 5 6 7 8 9 10 11

1 17.6 2 297 10 3 GYPSY 41 36 4 412 38 42 45 5 CAMV 46 47 47 48 6 DIRS 51 52 55 55 49 7 Cfr-1 43 44 51 51 52 8 HIV1 54 56 58 56 53 9 DEL 42 42 49 48 53

10 Ty3 37 37 39 47 43 11 TED 28 27 39 46 46 12 copia 67 66 63 66 65

51 59 56 55 45 61 53 50 58 47 53 43 57 48 63 64 67 67

44 63 67

The 79 amino acids identified by Poch et al. (1989) from the gypsy class retrotransposons, HIV-1, copia and CJT-1 were aligned and analysed using the PAUP program (Swofford 1990) " The numbers of non-identical amino acids between pairs of sequences were calculated and are displayed

are f o u n d in tha t order . Thus CfT-1 m a y possess all the enzymat i c act ivi t ies r equ i red for a u t o n o m o u s t rans- pos i t ion .

In each case the s t ronges t s imilar i t ies are to sequences o f r e t r o t r a n s p o s o n s in the gypsy g roup o f L T R re t ro-

344

Orf 1 Nucleic

CfT-I 258 Copia 230 Ty3-2 265 HIV-I 390

Off-2 Reverse

CfT-I 428 TED 402 17.6 319

Acid Binding Domain Orf-2 Protease Domain

RTCYGCGKPGHIARDCRG CfT-I 112 MIDSGASGNFA VKCHHCGREGHIKKDCFH Copia 290 VLDSGASDHLI RLCFTCKKEGHRLNECRA TED 38 LIDTGSTVNMT VKCFNCGKEGHTARNCRA 17.6 28 LIDTGSTVNMT

^ ^^ ^ ^ ^

Transcriptase Domain

KTAFRTRYGLYEFLVMPMGLTNAPASCQDLVNETLRDLLDVCWAYMDDIL KTAFNVEHGHFEFLRMPMGLKNSPSTFQRVMDNVLRGLQNNICLVYLDDII KTAFSTKHGHYEYLRMPFGLKNAPATFQRCMNDILRPLLNKHCLVYLDDIV ^^^^ ^ ^ ^^ ^^ ^ ^ ^^ ^ ^ ^^^

Orf-2 RNAse H Domain

CfT-I 557 NYDIHDKELLAIVAAM CfT-I TED 630 NYSTIEKELLAIVWAT TED 17.6 545 NYSTIEKELLAIVWAT Copia Gypsy 530 NYATNERELLAIVWAL HIV-I

^^ ^^^^6^ ^

Off-2 Endonuclease Domain

' 958 GIKHLSTAYH-PETDGQTERTNQTI 1031 KIIHHKTLPHSPSDNGNIERFHSTI 568 GISYHLTVPHTPQLNGVSERMIRTI 861 GIKQEFGIPYNPQSQGVVESMNKEL

^

Fig. 5. Alignments of ORF 1 and 2 sequences to retroelement protein sequences. Sequences were taken from Mount and Rubin 1985 (co- pia), Hansen et al. 1988 (TyJ); Ratner et al. 1985 (HIV-1); Friesen and Nissen 1990 (TED); Inouye et al. 1986 (17.6) and Marlor et al. 1986 (gypsy). The circumflex symbols indicate identi- cal amino acids. Numbers refer to coordinates in the deduced amino acid sequences

transposons (Doolittle et al. 1989). Poch et al. (1989) have identified a set of amino acids that are most con- served between reverse transcriptase and RNA-depen- dent D N A polymerases. Alignments of these amino acids from various retroelements with the homologous se- quences of CfT-1 were analysed phylogenetically using a parsimony algorithm (Swofford 1990). This analysis clearly demonstrates that CfT-1 lies within the enlarged gypsy group (Table 1) but failed to identify a convincing monophyletic tree for the gypsy group. Bootstrapping with a minimum of 80% confidence co-rooted all se- quences to HIV-1 with the exception of 17.6 and 297, two very closely related Drosophila retrotransposons (Inouye et al. 1986; Saigo et al. 1984). The amino acid sequences of CfT-1 are essentially equidistant from 17.6, 297, Del, TED and Ty3.

The third OR F starts 100 bp after pol ORF and ex- tends for 165 amino acids. A database search revealed no convincing homology. The relationship of this and other retrotransposon third ORFs to retroviral env genes in unclear.

The presence of strikingly homologous sequences, particularly as found in the pol ORF, in such highly diverse organisms as fungi, plants and insects may be regarded as primafacie evidence of horizontal gene trans- fer. The degree of homology is greater than would be expected of non-essential genes in organisms separated in evolution since ancestral divergence (Doolittle et al. 1989). An examination of codon usage within the CfT-1 open reading frames provides further evidence for the recent acquisition of these genes. The three ORFs of CfT-1 comprise a total of 1777 codons. The base usage frequency at the third wobble position is non-random, with A and C found to occur at frequencies of 32% and 29%, respectively; G is found at 24% of sites and T at 15% (Table 2). This pattern is found equally in ORFs 1 and 2 and to a lesser extent in the short ORF 3, and may be compared with the codon usage of the three sequenced C. fulvum cDNAs, Avr9, P1 and P17, as examples of "host" genes (van Kan et al. 1991; P.J.G.M. de Wit, personal communication). The third position bases are dominated by C (36%) followed by T (24%), G (21%) and A (19 %). This pattern of C > T > G > A is generally seen in the consensus of all filamentous fungal genes (Gurr et

Table 2. Third codon position bias in CJT-1 genes

A C G T No. codons

CJT-10RF1 28 32 25 15 645 CJT-10RF2 33 28 25 14 1045 CJT-10RF3 35 26 19 20 165 CJT-10FRs 1-3 32 29 24 15 1855 Cladosporium fulvum 19 36 21 24 381

cDNAs

The calculated percentages are shown of each base in the third position of codons for the three predicted CJT-10RFs, individually and together, and for the sum of the Avr9, P1 and P17 cDNAs (van Kan et al. 19991); P.J.G.M. de Wit, personal communication

al. 1987). This pattern contrasts markedly with the A > C > G > T pattern in the CfT- 1 open reading frames and suggests a recent extrachromosomal origin. Similar results were reported for the copia and 17.6 retrotrans- posons (Hansen et al. 1988). It is clear that a more widespread survey of the presence of retrotransposons and their codon usage is justified.

Race-specific polymorphisms

Evidence was sought that CfT- 1 might be responsible for some of the variation in pathotype between the physio- logical races of the fungus. For this purpose, southern blots of genomic D N A of various isolates, digested with BamHI and EeoRI, were probed with pNOMP3, the PstI fragment comprising the 3' half of the 5 'LTR and 2 kb of the 5' internal domain (Fig. 6). In each track, about 25 strong bands can be counted in addition to weaker bands corresponding to hybridisation to the 3' LTR. This finding, together with the frequency of positive cosmids, is consistent with a moderate copy number of about 25.

Race 4 differs from race 0 in that the putative avir~ ulence 4 gene is not expressed in race 4. It is intriguing to note that the race 4 hybridisation pattern (lanes 3 and 7) exhibits single extra bands when compared to race 0 (lanes 4 and 8). Race 2,4,5,9,11 differs in that all of the

23.1

9 .4

6 .6

1

4.4

2 3 4 5 6 7

Fig. 6. Hybridisation of pNOMP3 to EcoRI-(lanes 1-4) and Barn- HI-(lanes 5 8) digested genomic DNA. DNA from race 0 (lanes 4 and 8) is compared with race 4 (lanes 3 and 7), race 5 (lanes 2 and 6) and race 2,4,5,9,11 (lanes 1 and 5). Numbers to the left are mol. wt. markers in kb; Arrows highlight the polymorphisms between race 0 and race 4 DNA

Fig. 7. Transmission electron micrograph of virus-like particles (VLPs) in fraction 5 of the 10-80% sucrose gradient fractionation of a C. fulvum VLP preparation

345

known avirulences are unexpressed and about 5 extra bands are seen when compared to race 0 (lanes 1 and 5).

Virus-like particles as replicative intermediates durin9 C. fulvum retrotransposition

The retrotransposons Ty and copia encapsidate their genomic RNA and enzymatic pol functions into virus- like particles (VLPs) (Garfinkel et al. 1985; Mellor et al. 1985; Goreleva et al. 1989). In order to determine wheth- er CfT-1 is also encapsidated, VLP preparations were made from lysates of C. fulvum mycelium. The VLPs were separated on a 10-80% sucrose gradient, frac- tionated and pelleted. Each of the fractions was exam- ined for the presence of VLPs by electron microscopy. VLPs of between 40 and 50 nm in size and with electron- dense cores were repeatedly found in fraction 5 of the gradient (Fig. 7). The particles are strikingly similar to Ty particles observed in galactose-induced strains of yeast, containing the Gal-Ty element (Garfinkel et al. 1985). It is worth noting that Ty particles cannot be reliably detected in uninduced cells.

Each of the fractions of the sucrose gradient was assayed for reverse transcriptase (RT) activity by incor- poration of [Gt-32p]dCTP into polynucleotide, using oligo dC as primer and poly G as template (Fig. 8a). A peak of activity was found in fraction 5; coincidence of VLPs and RT activity was found in three separate experiments. The RT activity in the peak fraction was found to be dependent on Mg 2+, primer, and template, and was not supported by Mn 2 + ions (Table 3). Reverse transcriptase activity has not been demonstrated previously in filamen- tous fungi. The VLP fractions from the sucrose gradient were phenol-extracted and dot-blotted onto nitro- cellulose. The reverse transcriptase-encoding clone pNOMP5 hybridised primarily to the nucleic acid in fraction 5, containing the VLP peak (Fig. 8b). This fur-

16

rn 0

x

¢J

a

/.a. •

10

8

6 •

• .\/'J / 2 I--I 0 T I /i~q ~,~ 0 • •

0 1 2 3 4 5 6 7 8 9 10 11 12 VLP f rec t ions

b • o O 0 • • •

Fig. 8. a Reverse transcriptase activity in sucrose gradient fractions of C. fulvum VLP. b Dot-blot hybridisation of pNOMP5 to sucrose gradient fractions

346

T a b l e 3 . Reverse transcriptase activity (cpm) of fraction 5 from C. fulvum VLP sucrose gradient centrifugation

Complete 3804

- Mg z + 469 - Mg 2+ + Mn 2+ 187 - oligo dC 340 -po ly G 219 - enzyme 350

VLP, Virus-like particle The assays were performed methods

as decribed in the Materials and

ther suppor ts the view that CJT- l - re la ted elements are t ranscribed into R N A , which is packaged in VLPs tha t conta in reverse transcriptase.

C o n c l u s i o n

The results presented in this paper indicate tha t C.fulvum contains a modera t e number o f genomic elements with m a n y o f the conserved features o f re t ro t ransposons . These elements, which are represented by the c loned and sequenced 712 clone, have been designated CJT-1 ele- ments. The element appears to be expressed and pack- aged into VLP particles, bu t there is no direct evidence that the elements are still capable o f fur ther trans- posit ion. However , the un in te r rupted open reading frames and identical L T R s suggest tha t the c loned copy has t ransposed recently. This is the first repor t o f an L T R re t ro t ransposon in a f i lamentous fungus.

The origin o f the element is an intr iguing question. Its similarity to elements in diverse organisms and the biased c o d o n usage are suggestive o f hor izon ta l transfer. The role o f the element in p r o m o t i n g var ia t ion in pa thogen popula t ions , by cont r ibut ing to the b o o m - a n d - b u s t cycle whereby pa thogens rapidly overcome the resistance in new varieties o f crop plant is u n k n o w n but is under active study. The s tudy o f genetically uncharacter ised filamen- tous fungi is current ly severely hampered by a lack o f versatile genetic tools. The ability to use t ransposons to tag genes o f interest would be a significant advance.

Acknowledgements. We are grateful to A. Coddington for construct- ing the cosmid library, L. Flegg for technical assistance, Mark Coleman for helpful discussions and M. Oliver for help with the figures. This work was supported by grants to R.P.O. from the AFRC and the Gatsby Charitable foundation and from NERC for the sequencer and a studentship to M.T.M. from SERC.

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C o m m u n i c a t e d by D.J . F i n n e g a n