- Email: [email protected]
The Central PPT of the Yeast Retrotransposon Ty1 is not Essential for Transposition
doi:10.1016/S0022-2836(03)00812-X
J. Mol. Biol. (2003) 331, 315–320
COMMUNICATION
The Central PPT of the Yeast Retrotransposon Ty1 is not Essential for Transposition T. Heyman, M. Wilhelm and F.-X. Wilhelm* Institut de Biologie Mole´culaire et Cellulaire, CNRS UPR9002 15 rue Rene´ Descartes 67084 Strasbourg, France
The yeast retrotransposon Ty1 has structural and functional similarities to retroviruses. We report here that, as in retroviruses, the plus-strand DNA of Ty1 is synthesized as two segments. A central DNA flap is formed during reverse transcription consecutive to elongation (with strand displacement) of the upstream segment beyond the central polypurine tract (cPPT) until the replication machinery is stopped at the central termination sequence. Comparison of wild-type and cPPT-mutant Ty1 elements shows that the mutant element lacking the central DNA flap is only twofold defective in transposition. q 2003 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: Ty1; retrotransposon; polypurine tract; transposition; yeast
The yeast retrotransposon Ty1 has structural and functional similarities to retroviruses.1 Like retroviruses it replicates via reverse transcription of its genomic RNA.2,3 Reverse transcriptase (RT), which copies the genomic RNA of retroelements into linear double-stranded cDNA, has an absolute requirement for primers to initiate DNA synthesis. In most retroviruses and LTR-retrotransposons specific host tRNAs are used as primers for minus-strand DNA synthesis.4 – 7 The plus-strand DNA primers are purine-rich fragments of RNA, the polypurine tracts (PPTs), which persist after the RNase H activity of RT has degraded most of the genomic RNA from the RNA –DNA duplex formed during minus-strand synthesis.8 Plusstrand DNA synthesis proceeds from this primer using the minus-strand DNA as a template. In several lentiviruses including Visna virus,9 HIV-1,10 – 12 HIV-2,13,14 Human and Simian Spumaviruses,15 EIAV,16 FIV17 and in the yeast retrotransposons Ty15,18,19 and Ty320 (T.H., M.W. & F.-X., unpublished results) two PPTs located just upstream of the 30 LTR boundary and near the center of the genome are preferentially used to initiate plus-strand synthesis. Thus, plus-strand DNA is initially synthesized as two segments, the so-called downstream segment initiated at the central PPT (cPPT) and the plus-strand strong-stop DNA (þ sss DNA) initiated at the 30 PPT. An upstream segment, which results from elongation of the Abbreviations used: RT, reverse transcriptase. E-mail address of the corresponding author: [email protected]
þ sss DNA after it has been transferred to the 30 end of the minus-strand DNA is then synthesized. 30 end mapping of the upstream fragment of Ty1 has revealed that it is elongated, with strand displacement, beyond the cPPT and stops in an A rich region < 40 nt downstream of the cPPT.18 As a result, a < 40 nt plus-strand overlap is created9 (Figure 1(B)). It has been suggested that strand displacement synthesis is not sufficient to stop plusstrand synthesis but that A-tract induced minor groove narrowing could account for pausing and termination of DNA synthesis.21,22 In yeast cells containing the wild-type Ty1 element, two major fragments of 0.345 kb and 3.0 kb corresponding respectively to the þ sss DNA and to the downstream plus-strand fragment are readily detected on a Southern blot (Figure 2(C)).18,19 Mutations replacing some purines by pyrimidines in the cPPT or 30 PPT sequences (Figure 2(A) and (B)) abolish initiation of plusstrand DNA synthesis (Figure 2(C)). Using yeast cells transformed by multicopy plasmids bearing wild-type or PPT-mutant elements, we previously showed that mutations in the 30 PPT abolished transposition, whereas mutations in the cPPT did not affect transposition.18 As overproduction of a gene product (in this work, overproduction of double-stranded Ty1 preintegrative cDNA) can have biological consequences due to the altered balance between the gene product and factors within the cell (for a review see Ref. 23) it is possible that the use of multicopy plasmids did not allow to detect subtle differences of transposition between wild-type and cPPT-mutant elements.
0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
Figure 1. A. Positions and sequences of the cPPT and 30 PPT in the DNA genome of Ty1-neo with respect to the TyA (gag), TyB (pol) and neo sequences. The 50 nucleotides of the cPPT and 30 PPT correspond to positions 3783 and 5577 of the standard Ty-H3 sequence.30 B. Nucleotide sequence of the central region of Ty1 and predicted structure of the plus and minus-strand DNAs in the central region of wild-type Ty1 element. CTS, central termination sequence.
Central PPT of Ty1
317
Figure 2. Wild-type and mutant sequences of the cPPT (A) and 30 PPT (B). The wild-type 30 PPT and cPPT have an identical sequence GGGTGGTA. Three nucleotide changes (highlighted by black boxes) were introduced into the PPT sequence to form the PPT mutant. The polypurine tract is preceded by an upstream uridine-rich sequence that is essential for replication of Ty1. The amino acid sequence of the cPPT region is indicated. In cPPT/Mut74, a GGT codon is changed into GCT leading to the replacement of a glycine by an alanine residue. The 30 PPT is in the 30 non-coding region of the Ty1 sequence. C. Southern blot analysis of plus-strand DNA extracted from wild-type and PPT mutant Ty1 virus like particles. The DNA was separated by electrophoresis on 1% (w/v) agarose gel, blotted and detected with a radiolabelled minus-strand probe specific for the R region.
Here we have used low copy number centromeric plasmids to reassess the influence of cPPT mutations on Ty1 transposition. Wild-type and cPPT-mutant Ty1 elements marked with the neomycin gene (Ty1-neo) were subcloned into the centromeric plasmids pFL38 and pFL3924 bearing the selectable markers URA3 and TRP1, respectively. In both Ty1-neo elements the GAL1 promoter replaces a portion of the 50 LTR of Ty1 so that the Ty1-neo element is driven by the GAL1 promoter and can be induced in a medium containing galactose. Two host strains were used in this study: AGY9 (MATa leu2D1 ura3-52 trp1D63 his4-539 lys2801 spt3-202), a spt3 strain which fails to express endogenous Ty1 transposons25 and a rad52 mutant of AGY9 which minimizes recombination between the plasmid-based Ty1 elements.26 The yeast strains were transformed to uracil and tryptophan prototrophy with the plasmids described above. Transposition was induced by growth for five days at 22 8C on galactose-SC medium lacking uracil and tryptophan. After this step, the relative copy
number of the two plasmids present in the yeast cells was determined by restriction enzyme digestion analysis of the plasmids recovered from the galactose-induced cells in order to check that the two plasmids were maintained in equimolar amount and did not recombine during induction of transposition. The result of this experiment showed that the ratio of the URA3 and TRP1 plasmids was indeed comprised between one and 1.5 and that no recombination had occurred in the rad52 as well as in the wild-type strain. The yeast cells were then submitted to different growth steps to allow loss of the donor plasmids: the cells from the galactose plates were grown in non-selective YPD medium and then plated onto glucose-SC medium containing 5-fluoroorotic acid (5-FOA) to select cells that had lost the donor plasmid bearing the URA3 gene. To allow loss of the plasmid bearing the TRP gene, the cells from the 5-FOA plates were replica-plated several times on non-selective YPD medium. The cells were finally replica-plated onto YPD medium containing G418, glucose-SC
318
medium lacking uracil and glucose-SC medium lacking tryptophan to identify the colonies that had lost the two donor plasmids and acquired genomic copies of the Ty1-neo element. Thirty-two such colonies obtained in the AGY9 strain and 16 colonies obtained in the rad52 AGY9 strain were grown in YPD medium. The DNA was extracted from these cultures and digested with the appropriate restriction enzymes to identify the transposed element. The wild-type and cPPT mutant elements could be distinguished because mutations creating new restriction enzyme sites without changing the coding sequence of the integrase gene had been introduced upstream and downstream of the cPPT (Figure 3(A)). In the wild-type element, a Pvu1 site was created 212 nt upstream of the cPPT and a Bgl2 site was created 43 nt downstream of the cPPT. The cPPT-mutant element was marked by a Nsi1 site 17 nt downstream of the cPPT. In this way, a 261 bp band is revealed on a Southern blot upon digestion of the wild-type element with Pvu1 and Bgl2 and a 451 bp band is revealed upon digestion of the cPPT mutant element with Nsi1. A Southern blot of the digested DNA extracted from some colonies that had lost the two plasmids and acquired a genomic copy of the Ty1neo element is shown in Figure 3. Analysis of all the DNA samples revealed that, in a total of 48
Central PPT of Ty1
transposed elements, 16 (33%) had a mutant PPT sequence and 32 (67%) a wild-type PPT sequence (20 wild-type elements and 12 cPPT mutant elements in the wild-type AGY9 strain and 12 wild-type and four cPPT mutant elements in the rad52 mutant of the AGY9 strain). Thus, the cPPT affects transposition only twofold, indicating that it is not required absolutely for transposition of the Ty1 element. In Saccharomyces cerevisiae the nuclei are not dissociated during mitosis thus, in contrast to the previous suggestion that the central flap is a cis-acting determinant for nuclear localization of preintegrative cDNA,27 our results indicate that the central flap of Ty1 is not necessary for an active transport mechanism of Ty1 PIC through the nuclear pores. Our findings are consistent with recent observations of Dvorin et al.28 and Limo´n et al.29 showing that disruption of the cPPT does not affect significantly spreading of HIV-1 in a variety of cell types. The presence of a central flap in Ty1 elements which are distantly related to retroviruses suggests that it is widely represented among long terminal repeat containing retroelements. Indeed, an 88 nt flap has been identified in FIV17 and it has been demonstrated in vitro that an A-tract located downstream of the cPPT of EIAV efficiently halts the replication machinery in the context of strand
Figure 3. A. Restriction site map of the central region of wild-type and cPPT mutant Ty1-neo element. Numbering is based on the standard Ty-H3 sequence.30 The thick line indicates the position of the single-stranded oligodeoxynucleotide spanning nucleotides 3663– 3691 used to probe the Southern blots. B. Southern blot analysis of integrated Ty1 elements. Total yeast nucleic acids prepared from cells that had integrated a Ty1 element were digested with Nsi1, Pvu1 and Bgl2 and electrophoretically separated on a 1% agarose gel, blotted and probed with the 32P-labeled oligodeoxynucleotide spanning nucleotides 3663– 3691.
319
Central PPT of Ty1
displacement synthesis.16 The evolutionary advantage conferred by the cPPT on retroelements is not understood. Observation that the cPPT imparts a slight transposition advantage to Ty1 suggests that it could play a regulatory role which could be mediated by its interaction with proteins in the preintegrative complex. The structure of the Ty1 PIC and the mechanism of its nuclear import have not been investigated in yeast. Experiments aimed at identifying the component of the Ty1 PIC could shed some light on this mechanism and help to understand the regulatory role of the cPPT.
Acknowledgements
12.
13. 14. 15.
16.
We thank R. Chanet (Institut Curie, Orsay) for helpful discussions. This work was supported in part by grant 9589 from the Association pour la Recherche contre le Cancer (ARC). 17.
References 1. Boeke, J. D. & Sandmeyer, S. (1991). Yeast transposable elements. In The Molecular and Cellular Biology of the Yeast Saccharomyces cerevisiae (Broach, J. R., Jones, E. W. & Pringle, J., eds), pp. 193, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2. Boeke, J. D., Garfinkel, D. J., Styles, C. A. & Fink, G. R. (1985). Ty elements transpose through an RNA intermediate. Cell, 40, 491– 500. 3. Varmus, H. (1988). Retroviruses. Science, 240, 1427–1435. 4. Chapman, K. B., Bystrom, A. S. & Boeke, J. D. (1992). Initiator methionine tRNA is essential for Ty1 transposition in yeast. Proc. Natl Acad. Sci. USA, 89, 3236–3240. 5. Friant, S., Heyman, T., Bystrom, A. S., Wilhelm, M. & Wilhelm, F. X. (1998). Interactions between Ty1 retrotransposon RNA and the T and D regions of the tRNA(i)(Met) primer are required for initiation of reverse transcription in vivo. Mol. Cell. Biol. 18, 799–806. 6. Mak, J. & Kleiman, L. (1997). Primer tRNAs for reverse transcription. J. Virol. 71, 8087– 8095. 7. Marquet, R., Isel, C., Ehresmann, C. & Ehresmann, B. (1995). tRNAs as primer of reverse transcriptases. Biochimie, 77, 113 –124. 8. Champoux, J. J. (1993). Roles of ribonuclease H in reverse transcription. In Reverse Transcriptase (Skalka, A. M. & Goff, S. P., eds), pp. 103, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 9. Blum, H. E., Harris, J. D., Ventura, P., Walker, D., Staskus, K., Retzel, E. & Haase, A. T. (1985). Synthesis in cell culture of the gapped linear duplex DNA of the slow virus visna. Virology, 142, 270– 277. 10. Charneau, P., Alizon, M. & Clavel, F. (1992). A second origin of DNA plus-strand synthesis is required for optimal immunodeficiency virus replication. J. Virol. 66, 2814 –2820. 11. Charneau, P. & Clavel, F. (1991). A single-stranded gap in human immunodeficiency virus unintegrated
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linear DNA defined by a central copy of the polypurine tract. J. Virol. 65, 2415– 2421. Charneau, P., Mirambeau, G., Roux, P., Paulous, S., Buc, H. & Clavel, F. (1994). HIV-1 reverse transcription: a termination step at the center of the genome. J. Mol. Biol. 241, 651– 662. Hungnes, O., Tjotta, E. & Grinde, B. (1992). Mutations in the central polypurine tract of HIV-1 result in delayed replication. Virology, 190, 440– 442. Hungnes, O., Tjotta, E. & Grinde, B. (1991). The plus strand is discontinuous in a subpopulation of unintegrated HIV- 1 DNA. Arch. Virol. 116, 133– 141. Tobaly-Tapiero, J., Kupiec, J. J., Santillana-Hayat, M., Canivet, M., Peries, J. & Emanoil-Ravier, R. (1991). Further characterization of the gapped DNA intermediates of human spumavirus: evidence for a dual initiation of plus-strand DNA synthesis. J. Gen. Virol. 72, 605– 608. Stetor, S. R., Rausch, J. W., Guo, M. J., Burnham, J. P., Boone, L. R., Waring, M. J. & Le Grice, S. F. (1999). Characterization of (þ ) strand initiation and termination sequences located at the center of the equine infectious anemia virus genome. Biochemistry, 38, 3656 –3667. Whitwam, T., Peretz, M. & Poeschla, E. (2001). Identification of a central DNA flap in feline immunodeficiency virus. J. Virol. 75, 9407– 9414. Heyman, T., Agoutin, B., Friant, S., Wilhelm, F. X. & Wilhelm, M. L. (1995). Plus-strand DNA synthesis of the yeast retrotransposon Ty1 is initiated at two sites, PPT1 next to the 30 LTR and PPT2 within the pol gene. PPT1 is sufficient for Ty1 transposition. J. Mol. Biol. 253, 291– 303. Pochart, P., Agoutin, B., Rousset, S., Chanet, R., Doroszkiewicz, V. & Heyman, T. (1993). Biochemical and electron microscope analyses of the DNA reverse transcripts present in the virus-like particles of the yeast transposon Ty1. Identification of a second origin of Ty1 DNA plus-strand synthesis. Nucl. Acids Res. 21, 3513– 3520. Hansen, J. L., Chalker, D. L. & Sandmeyer, S. B. (1988). Ty3, a yeast retrotransposon associated with tRNA genes, has homology to animal retroviruses. Mol. Cell. Biol. 8, 5245– 5256. Lavigne, M. & Buc, H. (1999). Compression of the DNA minor groove is responsible for termination of RNA synthesis by HIV-1 reverse transcriptase. J. Mol. Biol. 285, 977– 995. Lavigne, M., Polomack, L. & Buc, H. (2001). DNA synthesis by HIV-1 reverse transcriptase at the central termination site: a kinetic study. J. Biol. Chem. 276, 31429– 31438. Rine, J. (1991). Gene overexpression in studies of Saccharomyces cerevisiae. In Guide to Yeast Genetics and Molecular Biology (Guthrie, C. & Fink, G. R., eds), pp. 239, Academic Press, New York. Bonneaud, N., Ozier-Kalogeropoulos, O., Li, G., Labouesse, M., Minvielle-Sebastia, L. & Lacroute, F. (1991). A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast, 7, 609– 615. Mathias, S. L., Scott, A. F., Kazazian, H. J., Boeke, J. D. & Gabriel, A. (1991). Reverse transcriptase encoded by a human transposable element. Science, 254, 1808 –1810. Wilhelm, M., Boutabout, M., Heyman, T. & Wilhelm, F. X. (1999). Reverse transcription of the yeast Ty1 retrotransposon: the mode of first strand transfer is
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either intermolecular or intramolecular. J. Mol. Biol. 288, 505– 510. 27. Zennou, V., Petit, C., Guetard, D., Nerhbass, U., Montagnier, L. & Charneau, P. (2000). HIV-1 genome nuclear import is mediated by a central DNA flap. Cell, 101, 173– 185. 28. Dvorin, J. D., Bell, P., Maul, G. G., Yamashita, M., Emerman, M. & Malim, M. H. (2002). Reassessment of the roles of integrase and the central DNA flap in human immunodeficiency virus type 1 nuclear import. J. Virol. 76, 12087– 12096.
29. Limo´n, A., Nakajima, N., Lu, R., Ghory, H. Z. & Engelman, A. (2002). Wild-type levels of nuclear localization and human immunodeficiency virus type 1 replication in the absence of the central DNA flap. J. Virol. 76, 12078– 12086. 30. Boeke, J. D., Eichinger, D., Castrillon, D. & Fink, G. R. (1988). The Saccharomyces cerevisiae genome contains functional and nonfunctional copies of transposon Ty1. Mol. Cell. Biol. 8, 1432 –1442.
Edited by M. Yaniv (Received 19 February 2003; received in revised form 8 April 2003; accepted 15 April 2003)
J. Mol. Biol. (2003) 331, 315–320
COMMUNICATION
The Central PPT of the Yeast Retrotransposon Ty1 is not Essential for Transposition T. Heyman, M. Wilhelm and F.-X. Wilhelm* Institut de Biologie Mole´culaire et Cellulaire, CNRS UPR9002 15 rue Rene´ Descartes 67084 Strasbourg, France
The yeast retrotransposon Ty1 has structural and functional similarities to retroviruses. We report here that, as in retroviruses, the plus-strand DNA of Ty1 is synthesized as two segments. A central DNA flap is formed during reverse transcription consecutive to elongation (with strand displacement) of the upstream segment beyond the central polypurine tract (cPPT) until the replication machinery is stopped at the central termination sequence. Comparison of wild-type and cPPT-mutant Ty1 elements shows that the mutant element lacking the central DNA flap is only twofold defective in transposition. q 2003 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: Ty1; retrotransposon; polypurine tract; transposition; yeast
The yeast retrotransposon Ty1 has structural and functional similarities to retroviruses.1 Like retroviruses it replicates via reverse transcription of its genomic RNA.2,3 Reverse transcriptase (RT), which copies the genomic RNA of retroelements into linear double-stranded cDNA, has an absolute requirement for primers to initiate DNA synthesis. In most retroviruses and LTR-retrotransposons specific host tRNAs are used as primers for minus-strand DNA synthesis.4 – 7 The plus-strand DNA primers are purine-rich fragments of RNA, the polypurine tracts (PPTs), which persist after the RNase H activity of RT has degraded most of the genomic RNA from the RNA –DNA duplex formed during minus-strand synthesis.8 Plusstrand DNA synthesis proceeds from this primer using the minus-strand DNA as a template. In several lentiviruses including Visna virus,9 HIV-1,10 – 12 HIV-2,13,14 Human and Simian Spumaviruses,15 EIAV,16 FIV17 and in the yeast retrotransposons Ty15,18,19 and Ty320 (T.H., M.W. & F.-X., unpublished results) two PPTs located just upstream of the 30 LTR boundary and near the center of the genome are preferentially used to initiate plus-strand synthesis. Thus, plus-strand DNA is initially synthesized as two segments, the so-called downstream segment initiated at the central PPT (cPPT) and the plus-strand strong-stop DNA (þ sss DNA) initiated at the 30 PPT. An upstream segment, which results from elongation of the Abbreviations used: RT, reverse transcriptase. E-mail address of the corresponding author: [email protected]
þ sss DNA after it has been transferred to the 30 end of the minus-strand DNA is then synthesized. 30 end mapping of the upstream fragment of Ty1 has revealed that it is elongated, with strand displacement, beyond the cPPT and stops in an A rich region < 40 nt downstream of the cPPT.18 As a result, a < 40 nt plus-strand overlap is created9 (Figure 1(B)). It has been suggested that strand displacement synthesis is not sufficient to stop plusstrand synthesis but that A-tract induced minor groove narrowing could account for pausing and termination of DNA synthesis.21,22 In yeast cells containing the wild-type Ty1 element, two major fragments of 0.345 kb and 3.0 kb corresponding respectively to the þ sss DNA and to the downstream plus-strand fragment are readily detected on a Southern blot (Figure 2(C)).18,19 Mutations replacing some purines by pyrimidines in the cPPT or 30 PPT sequences (Figure 2(A) and (B)) abolish initiation of plusstrand DNA synthesis (Figure 2(C)). Using yeast cells transformed by multicopy plasmids bearing wild-type or PPT-mutant elements, we previously showed that mutations in the 30 PPT abolished transposition, whereas mutations in the cPPT did not affect transposition.18 As overproduction of a gene product (in this work, overproduction of double-stranded Ty1 preintegrative cDNA) can have biological consequences due to the altered balance between the gene product and factors within the cell (for a review see Ref. 23) it is possible that the use of multicopy plasmids did not allow to detect subtle differences of transposition between wild-type and cPPT-mutant elements.
0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
Figure 1. A. Positions and sequences of the cPPT and 30 PPT in the DNA genome of Ty1-neo with respect to the TyA (gag), TyB (pol) and neo sequences. The 50 nucleotides of the cPPT and 30 PPT correspond to positions 3783 and 5577 of the standard Ty-H3 sequence.30 B. Nucleotide sequence of the central region of Ty1 and predicted structure of the plus and minus-strand DNAs in the central region of wild-type Ty1 element. CTS, central termination sequence.
Central PPT of Ty1
317
Figure 2. Wild-type and mutant sequences of the cPPT (A) and 30 PPT (B). The wild-type 30 PPT and cPPT have an identical sequence GGGTGGTA. Three nucleotide changes (highlighted by black boxes) were introduced into the PPT sequence to form the PPT mutant. The polypurine tract is preceded by an upstream uridine-rich sequence that is essential for replication of Ty1. The amino acid sequence of the cPPT region is indicated. In cPPT/Mut74, a GGT codon is changed into GCT leading to the replacement of a glycine by an alanine residue. The 30 PPT is in the 30 non-coding region of the Ty1 sequence. C. Southern blot analysis of plus-strand DNA extracted from wild-type and PPT mutant Ty1 virus like particles. The DNA was separated by electrophoresis on 1% (w/v) agarose gel, blotted and detected with a radiolabelled minus-strand probe specific for the R region.
Here we have used low copy number centromeric plasmids to reassess the influence of cPPT mutations on Ty1 transposition. Wild-type and cPPT-mutant Ty1 elements marked with the neomycin gene (Ty1-neo) were subcloned into the centromeric plasmids pFL38 and pFL3924 bearing the selectable markers URA3 and TRP1, respectively. In both Ty1-neo elements the GAL1 promoter replaces a portion of the 50 LTR of Ty1 so that the Ty1-neo element is driven by the GAL1 promoter and can be induced in a medium containing galactose. Two host strains were used in this study: AGY9 (MATa leu2D1 ura3-52 trp1D63 his4-539 lys2801 spt3-202), a spt3 strain which fails to express endogenous Ty1 transposons25 and a rad52 mutant of AGY9 which minimizes recombination between the plasmid-based Ty1 elements.26 The yeast strains were transformed to uracil and tryptophan prototrophy with the plasmids described above. Transposition was induced by growth for five days at 22 8C on galactose-SC medium lacking uracil and tryptophan. After this step, the relative copy
number of the two plasmids present in the yeast cells was determined by restriction enzyme digestion analysis of the plasmids recovered from the galactose-induced cells in order to check that the two plasmids were maintained in equimolar amount and did not recombine during induction of transposition. The result of this experiment showed that the ratio of the URA3 and TRP1 plasmids was indeed comprised between one and 1.5 and that no recombination had occurred in the rad52 as well as in the wild-type strain. The yeast cells were then submitted to different growth steps to allow loss of the donor plasmids: the cells from the galactose plates were grown in non-selective YPD medium and then plated onto glucose-SC medium containing 5-fluoroorotic acid (5-FOA) to select cells that had lost the donor plasmid bearing the URA3 gene. To allow loss of the plasmid bearing the TRP gene, the cells from the 5-FOA plates were replica-plated several times on non-selective YPD medium. The cells were finally replica-plated onto YPD medium containing G418, glucose-SC
318
medium lacking uracil and glucose-SC medium lacking tryptophan to identify the colonies that had lost the two donor plasmids and acquired genomic copies of the Ty1-neo element. Thirty-two such colonies obtained in the AGY9 strain and 16 colonies obtained in the rad52 AGY9 strain were grown in YPD medium. The DNA was extracted from these cultures and digested with the appropriate restriction enzymes to identify the transposed element. The wild-type and cPPT mutant elements could be distinguished because mutations creating new restriction enzyme sites without changing the coding sequence of the integrase gene had been introduced upstream and downstream of the cPPT (Figure 3(A)). In the wild-type element, a Pvu1 site was created 212 nt upstream of the cPPT and a Bgl2 site was created 43 nt downstream of the cPPT. The cPPT-mutant element was marked by a Nsi1 site 17 nt downstream of the cPPT. In this way, a 261 bp band is revealed on a Southern blot upon digestion of the wild-type element with Pvu1 and Bgl2 and a 451 bp band is revealed upon digestion of the cPPT mutant element with Nsi1. A Southern blot of the digested DNA extracted from some colonies that had lost the two plasmids and acquired a genomic copy of the Ty1neo element is shown in Figure 3. Analysis of all the DNA samples revealed that, in a total of 48
Central PPT of Ty1
transposed elements, 16 (33%) had a mutant PPT sequence and 32 (67%) a wild-type PPT sequence (20 wild-type elements and 12 cPPT mutant elements in the wild-type AGY9 strain and 12 wild-type and four cPPT mutant elements in the rad52 mutant of the AGY9 strain). Thus, the cPPT affects transposition only twofold, indicating that it is not required absolutely for transposition of the Ty1 element. In Saccharomyces cerevisiae the nuclei are not dissociated during mitosis thus, in contrast to the previous suggestion that the central flap is a cis-acting determinant for nuclear localization of preintegrative cDNA,27 our results indicate that the central flap of Ty1 is not necessary for an active transport mechanism of Ty1 PIC through the nuclear pores. Our findings are consistent with recent observations of Dvorin et al.28 and Limo´n et al.29 showing that disruption of the cPPT does not affect significantly spreading of HIV-1 in a variety of cell types. The presence of a central flap in Ty1 elements which are distantly related to retroviruses suggests that it is widely represented among long terminal repeat containing retroelements. Indeed, an 88 nt flap has been identified in FIV17 and it has been demonstrated in vitro that an A-tract located downstream of the cPPT of EIAV efficiently halts the replication machinery in the context of strand
Figure 3. A. Restriction site map of the central region of wild-type and cPPT mutant Ty1-neo element. Numbering is based on the standard Ty-H3 sequence.30 The thick line indicates the position of the single-stranded oligodeoxynucleotide spanning nucleotides 3663– 3691 used to probe the Southern blots. B. Southern blot analysis of integrated Ty1 elements. Total yeast nucleic acids prepared from cells that had integrated a Ty1 element were digested with Nsi1, Pvu1 and Bgl2 and electrophoretically separated on a 1% agarose gel, blotted and probed with the 32P-labeled oligodeoxynucleotide spanning nucleotides 3663– 3691.
319
Central PPT of Ty1
displacement synthesis.16 The evolutionary advantage conferred by the cPPT on retroelements is not understood. Observation that the cPPT imparts a slight transposition advantage to Ty1 suggests that it could play a regulatory role which could be mediated by its interaction with proteins in the preintegrative complex. The structure of the Ty1 PIC and the mechanism of its nuclear import have not been investigated in yeast. Experiments aimed at identifying the component of the Ty1 PIC could shed some light on this mechanism and help to understand the regulatory role of the cPPT.
Acknowledgements
12.
13. 14. 15.
16.
We thank R. Chanet (Institut Curie, Orsay) for helpful discussions. This work was supported in part by grant 9589 from the Association pour la Recherche contre le Cancer (ARC). 17.
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Edited by M. Yaniv (Received 19 February 2003; received in revised form 8 April 2003; accepted 15 April 2003)