INTRODUCTION

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Transposable elements constitute up to 50% of the eukaryotic genome (51, 53). Though they can act as positive forces in the evolution of an organism, both by providing part of the chromosomal architecture (30) and by providing a source of mutagens (1), they also represent a burden on the host cell genome and a potential threat to host cell viability, should they "jump" into an essential region of the genome or mediate a rearrangement thereof. Different transposable elements have developed different means to balance their survival with that of the host cell by controlling their spread within the genome. Most balancing mechanisms described thus far appear to involve control of either transposon expression or target site selection (9). These mechanisms often rely on host-specific factors, such as transcription factors (10, 27), splicing factors (47), and chromatin organization (58). Characterization of different transposable elements can therefore reveal both new mechanisms for control of element mobility and new host-element interactions.

Long terminal repeat (LTR)-type retrotransposons (hereafter referred to simply as retrotransposons) have been isolated from many different eukaryotic organisms, including fruit flies, maize, and the yeast Saccharomyces cerevisiae (6, 23, 48-50). Retrotransposons resemble retroviruses in both genome structure and replication mechanism (9). Like retroviruses, they possess terminally redundant ends (LTRs), a primer binding site for the initiation of reverse transcription, and a polypurine tract that serves as the primer in second-strand synthesis of the cDNA copy of the element. A single mRNA encodes proteins homologous to the retroviral structural proteins capsid (CA) and, sometimes, nucleocapsid (NC) and to the retroviral enzymes protease (PR), reverse transcriptase-RNase H (RT), and integrase (IN). These proteins coassemble to form retrovirus-like particles in which reverse transcription of an RNA intermediate takes place. The resulting cDNA is then typically integrated into the host cell genome by the element-encoded IN, generating 4- to 6-bp target site duplications (TSDs).

Retrotransposition has been extensively studied in yeast. Several yeast retrotransposons exhibit target site bias during transposition. The Ty1 and Ty3 elements of Saccharomyces cerevisiae target integration to regions upstream of RNA polymerase III-transcribed genes (13, 16), while Ty5 exclusively targets telomeres and silenced chromatin (58). This targeting appears to rely on interactions with host cell factors such as RNA polymerase III, transcription factors, and chromatin components. Since the targeted regions lack open reading frames (ORFs) and elements for controlling expression of downstream genes, they represent one mechanism for balancing retrotransposon and host survival: preferential mobilization into a "safe haven" in the host cell genome (14, 55).

Retrotransposons are typically found in multiple copies in a host cell genome. In yeast, this has resulted in very-low-frequency homologous recombination of retrotransposon cDNA with preexisting genomic copies of its parent element, in addition to normal transposition (39). This integrase-independent pathway relies on host cell recombination machinery (43, 52). Because of these two different modes of entry into the host genome, we use the term mobilization to refer to total element movement when the use of the two pathways cannot be distinguished. Though recombination occurs at different percentages of total mobilization for each element examined (less than 10%), all yeast retrotransposons studied to date mobilize primarily through the integrase-dependent pathway (7, 21, 31, 58).

The Tf2 element is a retrotransposon isolated from the fission yeast Schizosaccharomyces pombe (35). It is closely related to the well-characterized S. pombe Tf1 element. Both are 4.9-kbp elements with a single ORF encoding the CA-, PR-, RT-, and IN-like domains. The cloned elements Tf2-1 and Tf1-107 are almost identical in nucleotide and amino acid sequence in the RT and IN regions and are identical in the extreme carboxyl terminus of the PR region; however, they are highly divergent in the 5' untranslated region (5' UTR) between the 5' LTR and the ORF, which in retroviruses comprises all or part of a specific RNA packaging signal (for a review, see reference 46), as well as in the DNA encoding CA and about two-thirds of PR (56) (Fig. 1A). In addition, Tf2-1 and Tf1-107 are quite divergent in the U3 region of the LTR, a region containing cis-acting sequences involved in transcriptional regulation (5, 12, 18, 54). Only the Tf2 element is found in the form of full-length copies in the commonly used lab strains 972 and 975 (35).

View larger version (26K): [in this window] [in a new window]   FIG. 1.   (A) Tf2 differs from Tf1 in both translated and untranslated sequences. Comparisons of Tf2 and Tf1 nucleotide and amino acid sequences revealed discrete regions of difference (35, 56), represented here by differently shaded boxes. Filled triangle, LTR; AUG, start of ORF; UGA, end of ORF; CA, capsid-like protein; ppt, polypurine tract (100% identity [id] between Tf2 and Tf1); thickly outlined filled boxes, nucleotide sequence; thinly outlined filled boxes, amino acid sequence. (B) Tf2-neo expression plasmids. Tf2 was placed under the control of the repressible nmt (no message in thiamine) promoter in a plasmid construct similar to that previously described for Tf1-neo (pHL414-2). Expression was induced by transferring cells from medium containing 10 µM thiamine to medium lacking thiamine, allowing intracellular levels of thiamine to drop below the repression threshold (50 pmol/107 cells). The neo gene, inserted in the 3' end of the retrotransposon between the ORF and the 3' LTR, in the orientation opposite that of nmt1-directed transcription, confers resistance to the drug G418 (Geneticin). This plasmid allows for selection of mobilized copies of the marked retrotransposon once a cycle of expression and transposition has been induced. tsp, transcription start point.

Until now, analysis of the Tf2 element had been limited (35, 56), whereas the Tf1 element had been shown to be a functional retrotransposon, capable of generating true transposition events at a high frequency (31, 33). The presence of a Tf1-like ORF in Tf2 indicated that it too might be functional. Furthermore, based on Tf2's homology to Tf1, it appeared likely that the former shares Tf1's polyprotein processing (3, 34) and novel self-priming (31, 36) pathways. However, the differences between Tf2 and Tf1, particularly those in their 5' UTR and the CA regions, raised the possibility that the Tf2 and Tf1 elements mobilize differently, in terms of either regulation or efficiency; these differences could occur at transcriptional, translational, or posttranslational steps. Our characterization of Tf2 has revealed that its mobilization is significantly different from that of Tf1. Most importantly, for its propagation, it relies heavily on homologous recombination of its cDNA. This mobilization phenotype, which we have termed integration site recycling, may represent a unique means of balancing retrotransposon survival with that of the host cell.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Growth media. For nonselective growth, S. pombe was cultured in rich medium, YEC (0.5% yeast extract-0.2% Casamino Acids) plus uracil (250 µg/ml), adenine (500 µg/ml), and glucose (3%). For selective growth, S. pombe strains were grown in Edinburgh minimal medium (EMM), prepared according to the manufacturer's instructions (BIO 101, Vista, Calif.), supplemented with amino acids and pyrimidines, either individually or as part of a 5S dropout mix (with complete 5S containing Lys, His, Leu, uracil [U], and adenine [A], each at 250 µg/ml [final concentration]). Thiamine (10 µM) was added to the media when indicated. For selection against URA3-based plasmids, 5-fluoroorotic acid (FOA) was added to a final concentration of 0.1% to plates containing 250 µg of uracil per ml. For selection for the presence of the neo gene, Geneticin (G418) was added where indicated to a final actual concentration of 500 µg/ml.

Strains and plasmids. The strains used or constructed in this study are described in Table 1. Strains carrying a plasmid were grown and then transformed by the lithium acetate-Tris-EDTA method as described previously (25), with minor modifications as necessary. Plasmid pEH143-1 (Tf2-neo with an nmt promoter [pnmt]) was constructed in five steps. First, the 3' end of Tf2 was subcloned as a 3.9-kbp PvuII-XbaI fragment from pHL325-1 into EcoRV- and XbaI-cut pBSIIKS. It was then subjected to site-specific mutagenesis with JB112 (5'-TACATAGAAGATCTTGGGGAGGG-3'), as described previously (29), to create a unique BglII site in the noncoding region between the end of the ORF and the beginning of the 3' LTR, creating plasmid pEH129-1. The mutagenized Tf2 sequence was then subcloned as a 1.6-kbp BsrGI-EheI fragment into the 10.5-kbp BsrGI-BamHI site of pHL411 (34), creating intermediate plasmid pEH130 and eliminating the BamHI site. pEH130 also lacks the 2.2 kbp of S. pombe genomic sequence flanking the 3' end of Tf2 in pEH125-1 and pHL416-38. Complete Tf2 with a BglII site and under the control of the nmt promoter was made by replacing the Tf1 sequence in pEH130 with Tf2 sequence (a 3.2-kbp XhoI-BsrGI fragment) from pHL416-38, resulting in pEH133-1. pEH134-1 was created simultaneously by cloning the Tf2 sequence-containing XhoI-BsrGI fragment from pEH128-1 (pHL416-38 cut with BamHI, filled in, and religated) into XhoI- and BsrGI-cut pEH130, resulting in a version of pnmtTf2 with a BglII site and with a frameshift in the Gag (CA) region. pEH143-1 and pEH145-1 were generated by cloning the neo gene of Tn903 as a BamHI fragment from pGH54 into the BglII site of pEH133-1 or pEH134-1, respectively; in these plasmids, the orientation of neo is the reverse of pnmt-directed transcription (33). pnmtTf2-neo with a frameshift in the IN domain was created by cutting pEH143-1 with BsrGI, filling in with the Klenow fragment of DNA polymerase, and religating, resulting in pEH546-3.

RNA isolation and blotting. Total cellular RNA was typically isolated from 10 ml of an S. pombe culture grown to log phase (an optical density at 600 nm [OD₆₀₀] of 1.0) at 30°C and harvested by centrifugation. Cell pellets were disrupted by freeze-thawing and resuspended in 0.3 ml of RNA extraction buffer (0.1 M NaCl, 0.1 M Tris-Cl [pH 7.5], 0.03 M EDTA, and 1% Sarkosyl). Cold glass beads were added to the meniscus, 0.3 ml of PCIA (phenol-chloroform-isoamyl alcohol, 50:48:2) was added, and the mixture was vortexed for 10 s. After centrifugation in an Eppendorf microcentrifuge for 4 min at 4°C and 14,000 rpm, the aqueous phase was reextracted once with PCIA and once with chloroform-isoamyl alcohol (24:1), and then the RNA was precipitated. RNA pellets were washed with 70% ethanol and resuspended in 100 µl of diethyl pyrocarbonate-treated deionized water (DEPC-dH₂O) or DEPC-dH₂O containing 0.1 M EDTA.

Probes. The DNA probes used were a 0.27-kbp Tf2-specific EcoRI-BamHI probe, a 0.96-kbp neo fragment (a BamHI fragment from pGH54), a 0.75-kbp BstXI-BglII fragment from the 3' end of Tf2 (taken from pEH129-1), and a 0.7-kbp BamHI-HindIII fragment corresponding to part of the act1 ORF (taken from pHL859). Probes were prepared by labelling the fragments with Redivue [-³²P]dGTP (Amersham), by using the random hexamer labeling method of Feinberg and Vogelstein (17).

Protein preparations and blotting. Total soluble protein from cells expressing the Tf elements from the nmt promoter was prepared by growing a preculture of cells in EMM plus 5S and lacking U (EMM+5S-U) overnight at 30°C and then inoculating 10 ml of fresh EMM+5S-U to an OD₆₀₀ of 0.2 to 0.3. Each culture was grown to an OD₆₀₀ of 1.0 and harvested by centrifugation, and the pellets were subjected to freezing before extraction (5 min on dry ice-ethanol, or 70°C storage). Extraction was performed by resuspending each pellet in 0.1 ml of YLB (0.05 M Tris-Cl [pH 7.5], 1% SDS) plus additives (leupeptin, antipain, benzamidine, chymostatin, pepstatin A, each at 1 µg/ml; 10 U of aprotinin/ml; 0.014 M -mercaptoethanol; 1 mM phenylmethylsulfonyl fluoride), adding cold glass beads to the meniscus, and vortexing the mixture at 4°C for 10 min. At 80% cell lysis (monitored by phase microscopy), the mixture was then boiled for 3 min, the supernatant was transferred to a new tube, and the beads were then washed with 50 µl of YLB plus additives. Pooled supernatants were microcentrifuged for 20 min at 4°C, the new supernatant was transferred to a new tube and respun for 5 min, and then the approximate protein concentration was measured by reading the A₂₈₀ of a 1:200 dilution. On the basis of this measurement, about 150 to 300 µg (100 to 200 A₂₈₀ units/ml) of each sample was mixed with 2× Laemmli buffer (20% glycerol, 125 mM Tris [pH 6.8], 5% SDS, 1.4 M -mercaptoethanol), and the mixture was boiled for 1 min, loaded onto an SDS-10% polyacrylamide gel, and run in Tris (25 mM)-glycine (192 mM)-0.1% SDS buffer; 50 to 100 µg of high-molecular-weight prestained markers (Gibco-BRL) was also loaded for molecular sizing purposes. The proteins were then electroblotted onto an Immobilon-P (millipore) polyvinylidene difluoride membrane (60 min at 300 to 500 mA in Tris-glycine-15% methanol).

Mobilization assay. To detect the Tf element expression-dependent movement of neo information from the pnmtTf-neo plasmids into the S. pombe chromosomes, a mobilization assay was used. In the qualitative assay, cells from pertinent strains were patched in approximately 2-cm²-sized patches on thiamine-containing medium (EMM+5S-U with 10 µM thiamine) and grown for 2 days at 32°C. They were then replica printed to nonrepressive medium and induced for 2 to 4 days at 32°C; in parallel, the thiamine dependence of the G418^r phenotype was tested by repressing on medium containing thiamine. The plates were then replica plated to EMM-5S-FOA-thiamine medium to select for the loss of the plasmid (2 days, 32°C) and then printed to YEC plus U-A-FOA-G418 to select for chromosomal Tf-neo mobilization events (i.e., growth). After 2 days as well as 3 days of incubation, growth on the G418 plates was scored and the plates were photographed. (Multiple transformants for each plasmid were initially tested to establish consistency of phenotype.)

Genomic-DNA preparation and blotting. Total DNA was typically prepared from a 10-ml culture grown in rich medium (YEC-U-A plus 3% glucose; see above) for 24 to 36 h at 30°C, as previously described (35). For Southern analysis, 1 to 2 µg of DNA was digested with the appropriate restriction enzyme for at least 12 h, electrophoresed on a 1% agarose gel in 1× Tris-taurine-EDTA, and subjected to capillary transfer to a Genescreen Plus nylon membrane in 10× SSC after denaturation and neutralization were performed by standard methods.

Hybridization and washing. Filters were prehybridized in 4× Denhardt's solution-3× SSC for at least 3 h at 65°C. Hybridization was carried out with random-hexamer-labeled probes added at 5 × 10⁵ to 1 × 10⁶ cpm/ml of hybridization solution (6× SSC-4× Denhardt's solution-10 mM EDTA-0.5% [wt/vol] SDS) at 65°C for 16 h. Washes were performed in 200 ml of 2× SSC-1% (wt/vol) SDS, once for 30 min and once for 15 to 30 min, at 65°C. The filters were then subjected to autoradiography or phosphorimaging, using a STORM scanner and ImageQuant software.

Cloning of transposition sites. Putative Tf2-neo transpositions were cloned out of genomic DNA by doubly digesting DNA preparations with XbaI-SpeI or XbaI-NheI, electrophoresing the digests on an 0.8% agarose gel, and dividing the >4.4-kbp fraction into two pools; the DNA was then purified by using a GeneClean kit (Bio 101), and the fragments were ligated into XbaI-cut pBSIISK. The ligation products were transformed into Electromax DH10b cells (Gibco-BRL) by electroporation, plated on Luria-Bertani medium containing carbenicillin at 50 µg/ml, and then replica printed to Luria-Bertani medium containing kanamycin at 25 µg/ml. DNA from Carb^R) Kan^R clones was analyzed by restriction digestion to determine if it contained Tf2-neo. DNA flanking Tf2-neo was sequenced by using primer JB236 (5'-AGAGTTCAGTTATTGTA-3'), which lies 3' of the 5' LTR, as well as the T3 and T7 primers. Flanking sequence was then used to search the Sanger Centre S. pombe sequence database, so that recombination into a preexisting Tf2 or LTR could be differentiated from an actual novel transposition event. Flanking sequence was also used to prepare primers for PCR of the empty site in the parent strain YHL912.

Analysis of gap-repaired plasmids by sampling genomic Tf2 elements. Colonies of strain YHL912 transformed with the 10.5-kbp BamHI-BsrGI fragment of pEH143-1 were tested for being simultaneously U⁺ and G418^r while being separately Foa^R, indicating linkage of episomal Ura⁺ phenotype to the neo gene. Plasmids were then rescued into Escherichia coli DH5 cells from several candidates satisfying these criteria, by using the STET/glass bead method of Robzyk and Kassir (48a). The rescued plasmids were then analyzed by digestion with BamHI and BsrGI and were shown to have the same overall structure as pEH143-1. A minimum of two bacterial transformants per original yeast DNA preparation were analyzed.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

The Tf2-neo transcript is stably expressed from the nmt promoter. To characterize the expression and mobilization competence of Tf2, the neo gene from Tn903 was inserted at a BglII site introduced downstream of the ORF; neo allows for selection of resistance to Geneticin (G418) in S. pombe in single copy in the Tf context (33). To control its expression, the Tf2-neo construct was cloned under the control of the nmt1 promoter (pnmt) on an S. cerevisiae URA3-based plasmid (Fig. 1B, pEH143-1); S. cerevisiae URA3 complements S. pombe ura4. The S. pombe nmt1 promoter allows high-level expression of genes cloned downstream of it when cells are grown in the absence of thiamine but can be repressed by 80-fold or more in the presence of 2 µM thiamine (4, 38). By using a similar strategy, Tf1-107 marked with neo (Tf1-neo) had previously been cloned under the control of the nmt1 promoter on a URA3-marked plasmid and was shown to exhibit high-level expression of proteins and a high mobilization efficiency (34). Two mutant constructs, one with a frameshift mutation in the Tf2 Gag region (pEH145-1; GagFS) and the other with a frameshift mutation in IN (pEH546-3, INFS), were made as controls for experiments described below.

View larger version (48K): [in this window] [in a new window]   FIG. 2.   RNA expression from pnmtTf2-neo plasmids. (A) Tf2-neo RNA is stable and of the correct size. Expression of Tf2-neo in strains SZP57-1, 59-1, and 73-1 was compared to endogenous Tf2 RNA expression by RNA blot analysis with a 32P-labelled Tf2-specific probe; the lanes are labeled with the Tf2-neo species. The signal intensity in this blot is not indicative of relative expression, since less sample was loaded for SZP57-1 (Tf2-neo). A single major band corresponding to Tf2-neo migrated to a position corresponding to a size about 1 kb larger than that of the endogenous Tf2 signal (seen in both the vector-only and Tf2-neo lanes). The secondary bands that comigrated with rRNA bands (marked "r") probably represent degraded RNAs. A quantitative comparison of the observed Tf2 and Tf2-neo signals shows that the Tf2-neo signal represents a 10-fold increase in total Tf2 message. (B and C) Tf2-neo RNA is expressed at levels similar to those of Tf1-neo RNA. (B) An RNA blot of samples from induced cultures of strains SZP57-1, 59-1, 73-1, 66-1, 67-1, 68-1, and 64-1, probed with neo and the S. pombe act1 gene (41) simultaneously. The lanes are labeled with the Tf-neo species. The RNA load in the SZP57-1 (Tf2-neo) sample was much lower than those of the others; hence, the signal was less intense. (C) Tf-neo signals and act1 signals from induced and repressed (grown in the presence of thiamine) samples were then quantitated by phosphorimaging; the Tf-neo signal was normalized to the act1 signal, and normalized signals (measured in arbitrary units that are proportional to signal intensity) show that there is a similar level of expression. The act1 probe detects three actin RNAs (41); the smallest one (*) was used for normalization. This blot was stripped and reprobed for use in panel A (controlling appropriately for residual signal); thus, the smallest actin band is still apparent in panel A in an eightfold-longer exposure than that used for panel B. I, induced; R, repressed.

Tf2-neo is competent for mobilization. Since the Tf2-neo element mRNA could be stably expressed in S. pombe, the mobilization phenotype of this element was assayed. Strains harboring the pnmtTf2-neo plasmids were assayed for mobilization of neo information from the plasmid to the chromosome, after induction or repression of Tf2-neo expression, by selection for chromosomal acquisition of neo on medium containing FOA and G418. The FOA selects against the Tf2-neo plasmid, since URA3 confers FOA sensitivity (8); thus, only cells that have both lost the plasmid copy of neo and gained a chromosomal copy should be able to grow on medium containing both FOA and G418. Since acquisition of the chromosomal copy of neo should be dependent on mobilization of the Tf2-neo element, growth should also be observed only when an active copy of Tf2-neo has been expressed.

View larger version (81K): [in this window] [in a new window]   FIG. 3.   Tf2-neo mobilization. (A) Tf2-neo mobilization depends on expression of an intact Tf2-neo element. Results of a qualitative mobilization assay using patches of cells from SZP57-1,-2, 59-1,-2, and 64-1 are shown. Cells were induced in the mobilization assay for 3 days, then printed to minimal medium containing uracil and FOA and grown for 2 days; finally, they were transferred to rich YEC-glucose medium containing both FOA and G418 (YEC-FOA/G418). Growth on YEC-FOA/G418 medium is indicative of mobilization. Tf2-neo expression (induction [I]) generates clones that are resistant to G418 in the absence of the plasmid, as shown by papillation on YEC-FOA/G418. Induction of the negative control, Tf2GagFS-neo, generates FoaR G418r clones at a frequency no higher than that seen for patches of cells mock induced on repression-inducing medium containing thiamine (R). (B) Tf2-neo mobilization is not integrase dependent. Shown are the results of a mobilization assay comparing strains SZP57-1, 59-1, and 73-1, containing nmt plasmids with Tf2-neo, Tf2GagFS-neo, and Tf2INFS-neo, respectively (indicated in the figure). The frameshift in IN was introduced by filling in the BsrGI site (Fig. 1B). Growth on G418-containing medium represents the relative mobilization frequency. Tf2INFS-neo mobilizes neo nearly as frequently as Tf2-neo. (a) The Tf2INFS-neo mobilization phenotype is not like the Tf1INFS-neo mobilization phenotype. I, induced; R, repressed. (b) Mobilization assay comparing IN frameshifts (INFS) of Tf2 and Tf1 to wt cognates.

Tf2-neo mobilization is mostly IN independent. The majority (about 94%) of Tf1-neo mobilization events are IN dependent (31), indicating the occurrence of true retrotransposition. This phenotype was observed when a Tf1-neo element with a frameshift in the IN domain was used. To determine genetically the percentage of Tf2-neo mobilization events that are IN dependent, a similar frameshift was introduced into the IN of Tf2, and the mobilization of this construct (Tf2INFS-neo) was compared to those of Tf2-neo, Tf1-neo, and Tf1INFS-neo (Fig. 3B). Surprisingly, the mobilization frequency of the Tf2INFS-neo mutant was about 70% of the wild-type (wt) Tf2-neo mobilization frequency whereas Tf1INFS-neo mobilized at only 8.2% of the wt Tf1-neo frequency (Table 2). Both the wt Tf2-neo and the Tf2INFS-neo mobilization frequencies were within twofold of that of the Tf1-INFS-neo construct also assayed in these experiments (Table 2), suggesting that these three Tf-neo elements follow similar mobilization pathways.

Tf2-neo targets endogenous Tf2 elements for recombination. The results obtained in these mobilization assays suggested that Tf2-neo might mobilize mostly, or even entirely, by an IN-independent pathway, presumably homologous recombination. Thus, it was important to determine the molecular nature of the Tf2-neo mobilization events. To facilitate the molecular analysis of the Tf2-neo elements mobilized into the YHL912 parent strain genome, a determination of endogenous Tf2 copy number and distribution in YHL912 was performed. This determination was performed by blot analysis of YHL912 genomic DNA digested with enzymes that cut Tf2-1 one or more times upstream of a 3'-end probe (Fig. 4A).

View larger version (31K): [in this window] [in a new window]   FIG. 4.   DNA blot analysis of endogenous Tf2 elements. (A) Map of Tf2-1 showing minimum lengths of 3'-end fragments generated by cleavage at conserved restriction sites. Assuming a low degree of polymorphism, endogenous Tf2 elements cut with a particular restriction enzyme should generate fragments, plus flanking sequence, of a minimum predictable size that can be detected with the Tf2 3'-end probe. Dark box, Tf2 3'-end-specific fragment; hatched box, Tf2 5'-end fragment. (B and C) DNA blot analysis of digests of YHL912 DNA, using the Tf2 3'-end probe (B) and the 5'-end probe (C). (B) DNA from parent strain YHL912 was digested with the enzymes indicated in panel A, subjected to agarose gel electrophoresis, transferred to a Genescreen Plus membrane, and blotted with a 32P-labeled Tf2 3'-end-specific probe. Five different fragment patterns are evident. A unit-sized band (T) of about 5.0 kb is detectable in YHL912 digests prepared with enzymes cutting once in Tf2 (PstI, BsrGI, and AccI). (C) This band is also present when the PstI digest is probed with the 32P-labelled Tf2 5'-end-specific probe.

View larger version (44K): [in this window] [in a new window]   FIG. 5.   DNA blot analysis of Tf2-neo and Tf2INFS-neo mobilization candidates: identification of putative transposition events. (A) Map of Tf2-neo depicting the BsrGI site and 5'-end and 3'-end probes. Hatched box, 5'-end probe; dark box, 3'-end probe. The neo probe encompasses the entire neo gene (open box). (B) DNA blot analysis of parent strain YHL912, using Tf2 probes. YHL912 was digested with BsrGI and hybridized with 32P-labeled Tf2 5'-end probe (lanes 1 and 2) or 3'-end probe (lane 3). Hybridizing bands in the blot probed with the 5'-end probe are assigned letters a to l, with numbers being used when doublets are present. Hybridizing bands in the blot probed with the 3'-end probe are assigned numbers 1 to 12, with letters being used when doublets are present. T, tandem band (see the legend to Fig. 4). (C) DNA blot analysis comparing YHL912 and two Tf2-neo mobilization events, using the Tf2 3'-end probe (a) or the neo probe (b). Event 1-1 has been identified as a recombination event (REC) because it shifts an endogenous band (band 2a or b); event 1-2 has been identified as a possible transposition event (TXP) because it generates a new band with no shifts. The neo probe hybridized to the same bands.

Tf2-neo can generate transposition events. What appeared in genomic DNA blot analyses to be Tf2-neo transposition events might actually have represented Tf2-neo cDNA recombination into endogenous solo LTRs or other sites in an IN-independent fashion, events that would have a restriction pattern indistinguishable from that of a real transposition event. To determine whether any of the candidates identified by DNA blot analysis were the result of true transposition eventsthat is, that they represented the integration of a Tf2-neo sequence into a naive site (one lacking an endogenous Tf2 sequence), with hallmark TSDs flanking the element LTRscandidate transposition events were cloned out. The genomic DNA flanking the element was sequenced to look for TSDs and to determine whether a corresponding empty site existed in the parent strain YHL912 (Fig. 6). Two of the seven candidate transposition events identified by Southern analysis, WT3 and WT22, were recovered as full-length clones with 5'-end and 3'-end flanking sequences. Restriction analysis diagnosed the presence of the Tf2-neo sequence, and sequence analysis with T3, T7, and/or Tf2-neo-specific primers directly revealed hallmark TSDs for WT22 and a presumed TSD (from one flanking sequence) for WT3. Subsequent BLAST searches of the Sanger Centre S. pombe sequence database, using the flanking DNA sequence, revealed the empty sites: a WT3 event flanking sequence is found on cosmid c9G1 from chromosome I (Chr I), and a WT22 event sequence is found on cosmid c17H9 (also from Chr I) (Fig. 6A); both sequences lack any Tf2 homology at the point of integration (Fig. 6B). The absence of preexisting LTRs or full-length elements at the point of integration in parent strain YHL912 (a derivative of strain 972) was directly confirmed for both events by PCR analysis of genomic DNA from this strain, using primers derived from the genomic sequence flanking each cloned Tf2-neo insertion; the products were of the expected size for a sequence lacking a preexisting Tf2 insertion (data not shown). Thus, Tf2-1 is capable of retrotransposition, albeit at a lower frequency than Tf1.

View larger version (13K): [in this window] [in a new window]   FIG. 6.   Cloned Tf2-neo transpositions. (A) Sequences of two transpositions with flanking genomic DNA. Tf2-neo candidate transpositions were cloned into pBSIISK, and DNA flanking the event was sequenced, by using both the Tf2 and pBSII primers. WT22 shows the sequence obtained directly flanking either end of Tf2-neo, including TSDs (underlined). For WT3, the sequence flanking the 5' LTR, including the putative TSD, was obtained; however, the sequence immediately flanking the 3' LTR actually represents the predicted sequence at the site of insertion, based on identity of the 5' flanking sequence to an S. pombe sequence in the Sanger Centre database. Thus, the indicated TSD and 3' flanking sequence is predicted. (B) YHL912 sequences at Tf2-neo insertion sites. By using the S. pombe sequence database, the target site for each transposition event was found to lack Tf2 homology, having neither a Tf2 LTR nor a full-length Tf2; this was confirmed by PCR analysis of the genomic site of insertion (data not shown). Thus, WT22 and WT3 represent true transposition events.

Analysis of mobilization steps: protein expression. Although Tf2-1 is capable of mobilization, its ability to generate true retrotransposition events was greatly reduced compared to that of Tf1 under the conditions tested. Thus, we attempted to elucidate the steps at which the Tf2 retrotransposition pathway is inhibited and to determine what directs Tf2 to an IN-independent pathway for mobilization. Since mRNA expression was similar for Tf2-neo and Tf1-neo, protein expression was examined.

View larger version (51K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   FIG. 7.   Tf2 protein production and processing. (A) Tf2 produces and processes a polyprotein. Shown are the results of an immunoblot analysis of total soluble protein from strains expressing Tf2 or Tf1 from the nmt promoter. Tf1 and vector samples were taken from gradient-purified protein. Induced Tf2 samples show both a high-molecular-weight intermediate and a Tf1-like IN species (Tf2 IN) when blotted with antibody to Tf1 IN (-[Tf1 IN]). Panel a was blotted with anti-Tf1 IN; panel b was blotted with anti-Tf1 Gag polyclonal Ab. (-[Tf1 Gag]). I, induced, R, repressed. (B) Tf2-neo has a proteolytic processing defect. An immunoblot analysis of total soluble protein from strains expressing Tf2-neo or Tf1-neo constructs, using anti-Tf1 IN (a) or anti-Tf1 RT (b), is shown. Tf2 produces mature IN and PR-RT-IN but does not produce a detectable RT species or RT-IN species. A Tf2-neo species corresponding to the predicted size for a PR-RT intermediate is detectable in the RT panel; this same species is detectable in Tf1-neo protein samples as well, but at a much lower intensity than that for mature RT. Tf1 "VLPs", transposition intermediates (putative VLPs) isolated as described by Levin et al. (34). (C) Polyprotein processing pathways for Tf elements. Based on the protein species detected in panel B, two alternative pathways for Tf polyprotein processing can be envisioned. Both involve initial cleavage of the CA from the polyprotein, but they differ in the order of the second and third cleavages. Values in parentheses indicate molecular masses observed in this study, versus the published sizes (3).

Tf2 produces a PR-RT fusion protein. The use of an antiserum raised against Tf1 RT, which is greater than 99% identical to Tf2 RT, revealed differences in Tf2 and Tf1 protein processing. In the first direct detection of a Tf RT species, immunoblotting of Tf2-neo and Tf1-neo protein samples with anti-Tf1 RT antiserum demonstrated the presence of a mature Tf1 RT species of the expected size, 60 kDa (Fig. 7B, panel b). However, both this blot and a duplicate blot incubated with anti-Tf1 IN (Fig. 7B) showed that Tf2 does not make a detectable mature RT or RT-IN intermediate, molecules readily observed at 60 and 120 kDa in the Tf1 lanes (Fig. 7B, panel b). Instead, a PR-RT intermediate of 72 kDa (panel b) was observed. The 72-kDa species was also observed in the Tf1 lanes, but its intensity was lower than that of the mature RT. These data suggest that Tf2 uses a proteolytic processing pathway different from that used by Tf1 (Fig. 7C).

Tf2-neo produces fourfold less cDNA than Tf1-neo. Another crucial step in the retrotransposon life cycle that was examined with regard to phenotypic differences between Tf2 and Tf1 is the production of the full-length double-stranded cDNA genome. It has been demonstrated that overexpression of Tf1-neo from the nmt promoter produces easily detectable levels of full-length Tf1-neo cDNA intermediates (3). Detection and comparison of the levels of cDNA intermediates produced by Tf2-neo and Tf1-neo might therefore also offer insight into the reduced mobilization frequency of Tf2-neo.

View larger version (29K): [in this window] [in a new window]   FIG. 8.   Tf2-neo produces a steady-state level of cDNA about fourfold lower than that of Tf1-neo in log-phase cells. (A) Scheme of cDNA analysis by DNA blotting. Previously described by Atwood et al. (3), this scheme relies on easy detectability of cDNA in total-DNA preparations from induced cells harvested in log phase. The diagram shows the Tf-neo cDNA genome with BsrGI and BstXI sites; cutting with these enzymes produces a 2.5- or 2.0-kbp cDNA-specific fragment detectable with the neo probe. Boxed triangles represent LTRs. (B) DNA blotting of Tf2-neo and Tf1-neo samples cut with BstXI. DNA preparations from cells expressing Tf2-neo, Tf2GagFS-neo, Tf1-neo, or Tf1PRFS-neo were cut with BstXI, resolved by agarose gel electrophoresis, subjected to blotting, and then hybridized with the neo probe. Linear (L) and circular (C) cDNA species are apparent only when functional constructs are induced. P, plasmid band. pEH143-1 BstXI and pEH143-1 XhoI/BamHI are size standards. (C) Quantitative comparison of cDNA species, normalized to plasmid levels. neo-hybridizing species were quantitated by phosphorimage analysis, and the values obtained for the cDNA (L and C) species were normalized to those of the plasmid species (P) to enable the comparison of samples on the same blot. Normalized values are presented as a ratio of Tf1-neo to Tf2-neo for the linear cDNA and circular cDNA species.

Comparison of Tf2-1 with other Tf2 elements. The differences in the Tf2-1 and Tf1-107 mobilization phenotypes led us to question whether the Tf2-1 clone is typical of the Tf2 group. The other element analyzed in the original sequence analysis of Tf2, Tf2-43, showed only a 1-base difference in the region of overlap, occurring in the U3 region of the LTR (56). The 1,673 bp of Tf2-43 sequenced covers the entire region exhibiting the greatest degree of diversity between Tf2 and Tf1, suggesting that the Tf2 elements are conserved. However, given the estimated 15 Tf2 elements in strain YHL912, it was also possible that a subclass of Tf2 elements possessed a high transposition efficiency; if so, mobilization differences could be linked to crucial differences between Tf2-1 and one of these elements in either the coding or noncoding sequences.

View larger version (42K): [in this window] [in a new window]   FIG. 9.   Functional assays of other Tf2 elements. (A) Scheme used to assay for activity of other Tf2 elements. Either the Tf2-neo plasmid was cut with BamHI and BsrGI (arrowheads) and transformed directly into YHL912, to allow for homologous repair of the sequence between the BamHI and BsrGI sites by endogenous Tf2 elements (gray shading), or Tf2 sequences from library plasmid clones were subcloned into the plasmid's BamHI-BstXI sites (arrows). Jagged lines are used for gap-repaired Tf2 to indicate that we do not know the points of crossover in the repaired plasmids. (B) Mobilization assay of candidates from the gap repair transformation. Candidates were chosen for being Ura+, G418r, and also FoaR in the absence of selection for a Ura+ phenotype; a subset is shown (see text). Most candidates did not show an improved mobilization phenotype when compared to Tf2-1 (box), as judged by the degree of papillation on G418-containing medium. Two of three candidates isolated that were identified as having an apparently better mobilization phenotype than Tf2-1 are shown on this plate (arrows). In all three cases, this phenotype was subsequently found to be independent of the URA3 plasmid. (C) Mobilization assay of strains transformed with plasmids carrying Tf2 sequences from random Tf2-containing library plasmids. Neither of the library clones tested (no. 13 and no. 15) exhibited an increased mobilization frequency associated with the sequence between the Tf2 BamHI and BstXI sites.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

This study was undertaken to determine whether Tf2 is a functional retrotransposon and to compare and contrast its transposition capability with that of the related element Tf1. We have shown that Tf2 can be stably expressed from the nmt promoter and that it can mediate the occurrence of true transposition events in the host cell chromosome. However, the frequency of Tf2-neo mobilization is about 20-fold lower than the Tf1-neo mobilization frequency. Furthermore, unlike what is observed with Tf1-neo or any other wt retrotransposon, the majority of Tf2-neo mobilization events result from homologous recombination between the Tf2-neo cDNA and endogenous Tf2 elements rather than from true transposition.

Tf2-neo mobilizes at a level similar to that seen with the Tf1-neo IN mutant. The Tf2-neo IN mutant, however, mobilizes at about the same frequency as wt Tf2-neo rather than dropping 15- to 100-fold, as has been observed for the corresponding Tf1-neo constructs (31, 37). The majority (70%) of the wt Tf2 mobilization events are homologous recombination events, indicating that integration is somehow blocked. Attempts to elucidate the reasons for Tf2's lower level of mobilization by analyzing mobilization components revealed that Tf2 has a different mode of proteolytic processing that results in accumulation of a PR-RT species and no detectable mature RT. Tf2 produces steady-state levels of IN that are two- to fourfold lower than those observed for Tf1, and it appears that its CA species is maintained at a similarly low level. Finally, Tf2 also produces fourfold less cDNA than Tf1. Based on both functional and sequence analyses of other Tf2 elements, we believe that these phenotypes are typical of these elements.

Tf2 proteolytic processing. Tf2 processes its polyprotein differently than Tf1. The Tf2 PR produces CA, IN, and a PR-RT species but does not release detectable mature RT. Tf1 makes the PR-RT species seen in Tf2 protein samples, but at a much lower level than it makes mature RT. Building on the previous model for Tf1 polyprotein cleavage (3), these data suggest that there are two alternative pathways for Tf polyprotein proteolytic processing: one in which the PR processes the CA-PR junction, the PR-RT junction, and then the RT-IN junction (favored by Tf1) and one in which the second cleavage occurs at the RT-IN junction (favored by Tf2). A key unknown is whether the PR-RT species produced in the second pathway is intrinsically refractory to cleavage by either Tf element PR or the Tf2 PR is simply unable to cleave the PR-RT junction in any context. It is also formally possible that Tf2 PR cleaves the PR-RT junction but an aberrant N terminus is generated and the resulting Tf2 RT and RT-IN proteins are highly unstable and, therefore, undetectable.

Tf2 recombination phenotype. Tf2-neo not only has a decreased mobilization frequency but also moves mostly by recombination of Tf2-neo cDNA with endogenous Tf2 elements. This recombination of cDNA with endogenous elements has been observed for other yeast retrotransposons (2, 24, 31, 39, 52), but never as the major mobilization pathway for the wt element in question. It is formally possible that the high proportion of cDNA recombination events occurs as a result of overexpression of the Tf2-neo element; however, yeast retrotransposon studies using overexpression systems have generally reflected the biology of the endogenous elements (15, 26).

Conclusions. The Tf2-1 element from Leupold strain 972, when marked and overexpressed in this strain background, exhibits reduced protein expression, cDNA generation, and mobilization compared with those of its sister element, Tf1. The majority of mobilization events that Tf2-1 can mediate involve homologous recombination into preexisting elements. The use of such a propagation pathway may aptly be named integration site recycling. This route of mobilization saves the host cell genome from a potentially lethal mutation resulting from fresh Tf2 integrations in or near an essential gene(s) while still enabling the Tf2 elements to evolve. As such, it represents a highly adapted relationship between the Tf2 retrotransposon and its fungal host, S. pombe, similar to what is seen with the Ty elements in Saccharomyces cerevisiae.