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1 Department of Biological Chemistry, School of Medicine, University of California, Irvine, California 92697-1700, USA
2 Department of Molecular and Cellular Biology, and Howard Hughes Medical Institute, University of Arizona, Tucson, Arizona 85721, USA
3 Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA
Reprint requests to: Suzanne Sandmeyer, Department of Biological Chemistry, School of Medicine, University of California, Irvine, CA 92697-1700, USA; e-mail: sbsandme{at}uci.edu; fax: (949) 824-2688.
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Keywords: Ty3; virus assembly; retrotransposon; retrovirus; mRNA localization; P-body
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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; Hansen et al. 1992; Coffin et al. 1997; Linial and Eastman 2003; Sfakianos et al. 2003; Teysset et al. 2003). An important aspect of the retrovirus life cycle is how the RNA genome and particle proteins are brought together during assembly to allow orderly progression of the life cycle. For some viruses, there is evidence that packaged and translated RNAs are segregated from early times; for others, it appears that genomic RNAs are translated and subsequently packaged (Butsch and Boris-Lawrie 2002). Recent experiments with Moloney murine leukemia virus and Mason-Pfizer monkey virus indicate that nascent viral proteins can facilitate delivery of the RNA to an assembly site (Basyuk et al. 2003; Sfakianos et al. 2003).
To gain insight into the retrovirus-like assembly process, we examined localization of the protein and RNA components of the yeast retrovirus-like element Ty3 (Sandmeyer et al. 2002). Ty3 is 5.4 kbp in length and is comprised of long terminal repeats (LTRs) of 340 bp flanking an internal domain of two overlapping ORFs, GAG3 and POL3. GAG3 encodes the major structural proteins, capsid (CA) and nucleocapsid (NC); POL3 encodes the aspartyl protease (PR), reverse transcriptase (RT), and integrase (IN) of Ty3. Proteins are synthesized initially as Gag3 and Gag3-Pol3 precursor polyproteins, which associate through Gag3 interactions into virus-like particles (VLPs) of ~50 nm (Hansen et al. 1992). The zinc finger of Ty3 NC is critical for proper assembly; by analogy with retroviruses, it is required for packaging of the genomic RNA (Orlinsky and Sandmeyer 1994). Subsequent to assembly, the polyprotein precursors are processed by PR, resulting in mature VLPs, which are analogous to retrovirus cores. Reverse transcription of the RNA yields cDNA, which is translocated into the nucleus and integrated into chromosomal DNA. Thus, although Ty3 lacks an envelope and extracellular phase of the cell cycle, it is similar in structure and function to an animal retrovirus.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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), which is induced by growth in galactose. Western analysis showed that Ty3-GFP/RFP was expressed and that both Gag3 and Gag3-Pol3-GFP/RFP proteins were properly processed (Fig. 1B). Because proper processing requires assembly into the VLP, these results suggest the fusion proteins are fully functional and able to assemble within a VLP, a conclusion also supported by localization of these fusion proteins to VLP by electron microscopy (EM) (see below).
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). Moreover, by indirect immunofluorescence, the CA and NC proteins colocalized with the Ty3-GFP, indicating that the accumulation of Ty3-GFP in foci was not restricted just to the fusion protein (data not shown). In addition, the pattern of Ty3-GFP fluorescence was similar in mating populations of cells in which Ty3 was expressed under its native pheromone-inducible promoter from a low-copy plasmid (Fig. 2). This latter result demonstrates that the accumulation of the Ty3-GFP protein in foci is not an artifact of expression of the Ty3 element from the GAL1-10 UAS. These results indicate that Ty3 proteins accumulate in specific cytoplasmic foci during the Ty3 life cycle, which could be sites of VLP assembly.
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). Cells expressing MS2-GFP and Ty3-MS2 showed multiple fluorescent foci, first faintly visible at 1 h after induction, that continued to enlarge with a concomitant reduction in number up to 6 h (Fig. 3A, upper panel; 3B). In contrast, in cells expressing only MS2-GFP, fluorescence was diffuse throughout this time course (Fig. 3A, lower panel). Moreover, Ty3-MS2 and MS2-GFP fluorescence analyzed together with indirect immunofluorescence using antibodies specific for Ty3 CA showed that the Ty3 mRNA and CA colocalized (Fig. 3C). Thus, Ty3 proteins and RNA are localized together to specific cytoplasmic foci.
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; Fig. 1D), and immunoEM using anti-GFP showed that GFP was associated with VLPs (Fig. 1E). In cells expressing Ty3 for 1 or 1.5 h, individual VLPs or clusters were rarely observed by EM (data not shown). After 3 h, however, consistent with observations using Ty3-GFP, small clusters were observed and larger clusters were apparent at 6 h. In combination, these results indicate that Ty3 proteins, mRNA, and VLPs all accumulate in cytoplasmic foci, which might be specific sites of VLP assembly.
Several observations led us to hypothesize that Ty3 foci might be associated with cytoplasmic P-bodies. First, P-bodies are sites of accumulation of nontranslating mRNAs, which can then be subject to degradation, recycling to translation, or storage for later reuse (Sheth and Parker 2003; Cougot et al. 2004; Brengues et al. 2005; Coller and Parker 2005). Thus, if VLP assembly initiated or occurred within P-bodies, competition between assembly and translation would be avoided. Moreover, P-body components were identified in genomic screens for mutations affecting retrotransposition of both Ty3 (dhh1
and xrn1/kem1) (Irwin et al. 2005) and Ty1 (pat1 and lsm1) (Griffith et al. 2003). In isolated testing, lsm1 and pat1 strains also show reduced Ty3 retrotransposition (data not shown).
To test whether Ty3 VLP clusters were physically associated with P-bodies, we expressed the Ty3-RFP fusion protein in strains in which the individual P-body proteins Xrn1/Kem1, Dhh1, Pat1, Dcp2, and Edc3 were fused to GFP (Huh et al. 2003). Strikingly, we observed that in each case, Ty3-RFP colocalized with or near the P-body protein (Fig. 4). Careful inspection of images suggested that the individual RFP and GFP foci were overlapping but not perfectly congruent. Deconvolution of Z stack images for Dhh1-GFP and Xrn1/Kem1-GFP supported this interpretation (data not shown). Furthermore, immunoEM using anti-GFP in strains expressing Ty3 and Dhh1-GFP or Xrn1/Kem1-GFP showed colloidal gold labeling of GFP overlapping or adjacent to the assemblies of VLPs (Fig. 4B; data not shown). Inspection of the GFP fluorescence showed that individual P-body proteins appear in differing numbers of foci (Fig. 4A, cf. Dhh1p and Dcp2p). Moreover, for Dcp2p, which occurred in multiple foci, Ty3-RFP colocalized with only a subset of foci (Fig. 4A). These results indicate that Ty3 proteins, RNAs, and VLPs accumulate in association with P-bodies, although it may be that only a subset of P-bodies is associated with Ty3 assembly.
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), where P-body components are generally more distributed throughout the cytoplasm (Teixeira et al. 2005).
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). Moreover, dhh1 strains showed more diffuse fluorescence of Ty3 proteins, and twice as many cells displayed multiple, small foci compared with wild-type cells (Fig. 6A,B; data not shown). Xrn1/kem1 cells contain increased P-bodies (Sheth and Parker 2003), and the Ty3 protein in this mutant accumulates in association with large P-bodies (Fig. 6A; data not shown).
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and xrn1/kem1 strains, which have defects in P-body function, show alterations in retrotrans-position (Irwin et al. 2005) and the morphology of Ty3 foci (Fig. 6). The simplest explanation for these observations is that Ty3 VLP assembly occurs in association with P-bodies. We cannot rigorously rule out the formal possibility that Ty3 VLPs assemble elsewhere in the cell and only subsequently aggregate in association with P-bodies. However, the absence of dispersed VLPs by EM and the nascent accumulation of Ty3-GFP in foci at 1 h, which is before we observe significant numbers of VLPs, suggests this is an unlikely possibility.
Interestingly, because translation is restricted from yeast P-bodies (Teixeira et al. 2005), these observations suggest that translation of the Ty3 proteins could occur in a diffuse manner followed by accumulation of the mRNA and Ty3 proteins in association with P-bodies. Assembly of VLPs in P-bodies might be advantageous because it would increase the local concentration of Ty3 RNA for packaging. Sequestering a nontranslating pool of mRNAs would also serve to restrict the disruption of packaging by translating ribosomes. Nonetheless, the specific molecular mechanistic linkage between the single wild-type P-body/Ty3 VLP cluster in most cells and the efficiency of transposition remains to be determined. Indeed, it is even possible that association of Ty3 VLPs with P-bodies affects steps in the life cycle after assembly.
Retroviruses assemble at characteristic cytoplasmic or membranous sites (Coffin et al. 1997). For example, in the case of Mason-Pfizer monkey virus, a ß retrovirus, which assembles in pericentriolar clusters, specific chaperones have been implicated in the protein assembly process (Hong et al. 2001; Sfakianos et al. 2003) and in the case of human immunodeficiency virus, RNase L inhibitor is associated with protein assembly intermediates (Zimmerman et al. 2002). Nonetheless, relatively little is known about which, if any, host factors might play a specific role in directing genomic RNA into the assembly rather than translation pathway. Because P-bodies in mammalian cells are similar in composition and function to yeast P-bodies (Bashkirov et al. 1997; Ingelfinger et al. 2002; Lykke-Andersen 2002; Cougot et al. 2004; Andrei et al. 2005; Ferraiuolo et al. 2005; Kedersha et al. 2005), and in some cases P-bodies can associate with membranes (C. Beckham and R. Parker, unpubl.), a clear implication of our results is that P-body functions might also participate in core particle assembly for at least some other retroviruses/retrotransposons in eukaryotes, including mammals.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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; Burke et al. 2000). Bacterial strain HB101 (F– hsd-20 [rB–mB–] recA13 leuB6 ara14 proA2 lacY1 galK2 rpsL20 [Smr] xyl-5 myl-1 supE44 
) was used for all plasmid preparations. Ty3-GFP/RFP expression was in the BY4741 background (MATa his31 leu20 met150 ura30) (Open Biosystems) (Winzeler et al. 1999). Expression of Ty3-MS2 was in strain YTM443 (MATa trp1-H3 ura3-52 his3-200 ade2-101 lys2-1 leu1-12 can1-100 bar1::hisG GAL+ Ty3) (Menees and Sandmeyer 1994). P-body–GFP-tagged strains (Huh et al. 2003) and deletion strains (Winzeler et al. 1999) were in the BY4741 background. Deletion strains were purchased from Open Biosystems. The plasmid from which MS2-GFP was expressed under control of the MET25 promoter was described previously (Beach et al. 1999). For Ty3 expression, cells were grown to logarithmic phase in synthetic raffinose (SR)–ura medium and induced by the addition of galactose to 2% (SG). Cultures were maintained at 30°C, except for those used for EM, which were maintained at 25°C.
Recombinant DNA constructions
The Ty3-GFP plasmid was produced by first introducing the XmaI site downstream of Ty3 POL3 at nucleotide 5057 in pDLC201 (Hansen et al. 1988) (pNB2114). A PCR fragment containing the coding region for GFP (S65T) was ligated into a XmaI site so that GFP was expressed in frame with POL3 (pNB2127). The Ty3-RFP expression plasmid was created by cloning yeast codon-usage and context-optimized DsRed Monomer sequence (Clontech) into this XmaI site of pNB2114 (pNB2266). For the negative control, the same DsRed sequence was expressed from the HindIII/BamHI site of pYES2.0 (pNB2289). The DsRed Monomer DNA was assembled with a CODA Express kit (CODA Genomics Inc.) The Ty3-MS2 expression plasmid (pTy3-MS2) was created by ligating a duplex oligonucleotide containing a tandem repeat of the MS2 CA protein recognition sequence into the XmaI site of pNB2114.
Fluorescence microscopy
Yeast fluorescence microscopy was performed essentially as previously described (Oakes et al. 1998). The IgG fraction of rabbit polyclonal anti-Ty3 CA (Menees and Sandmeyer 1994) was affinity purified on a protein A–Sepharose CL-4B column (Amersham Biosciences). The resulting anti-CA (6.2 mg/mL) IgG stock was diluted 1:1000 before use in phosphate-buffered saline (PBS) containing BSA (20 mg/mL). Rabbit IgG was detected with TRITC-conjugated goat anti-rabbit IgG fraction (14 mg/mL) (Sigma) in PBS-BSA at a 1:5000 dilution. In order to detect DNA, living cells were grown in DAPI at a concentration of 1.25 µg/mL for several hours; fixed cells were stained with 1 µg/mL of DAPI in PBS for 5 min and washed with PBS three times.
Fluorescence microscopy was performed by using a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss Inc.) equipped with a 100x NA 1.3 objective. Fluors were detected by using Chroma filter sets: RFP and TRITC, 41004 (exciter, 560/555; emitter, 645/675); GFP, 41001 (exciter, 480/440; emitter, 535/550); DAPI, CZ 902 wide UV excitation with long-pass emission (exciter, 340/360; beam splitter, 400). Images were captured by using an AxioCam MRm camera with Axiovision 3.1 software. Images were processed and pseudocolored by using Adobe Photoshop 6.0 (Adobe Systems Inc.).
Confocal microscopy was performed with a Zeiss LSM50 Meta confocal fluorescence microscope equipped with Argon/2 laser (wavelength, 488 nm), He-Ne laser (wavelength, 543 nm), and two photon Ti:Sapphire femtosecond laser (wavelength 790 nm) using a 100x 1.3 NA objective and LSM510 Software. The multitrack option was used for image acquisition. Images were processed as described for conventional fluorescence microscopy.
Electron microscopy
Cells for EM were prepared as described previously (Giddings et al. 2001). Thin sections were viewed on a Philips CM10 electron microscope (Philips Electronic Instruments Co.) and recorded by using a Gatan digital camera and Digital Micrograph Software package (Gatan Inc.). For immunoEM, thin sections were similarly prepared and floated for 2 h on rabbit anti-CA diluted 1:800 or affinity purified rabbit anti-GFP (provided by J. Kahana and P. Silver, Harvard University, Cambridge, MA) diluted in 2% nonfat dry milk in PBS-Tween, followed by floating for 1 h on 15-nm gold-conjugated anti-rabbit IgG diluted 1:20 in the same blocking solution.
Immunoblot analysis
For immunoblot analysis of Gag3, IN, and IN-GFP/RFP fusion proteins from whole cell extracts, cultures of BY4741 and dhh1 and xrn1/kem1 derivatives were transformed with pDLC201, pNB2127, or pNB2266, induced as described above for microscopy. Immunoblot analysis was performed as described previously (Irwin et al. 2005).
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ACKNOWLEDGMENTS |
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Footnotes |
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Article and publication are at http://www.rnajournal.org/cgi/doi/10.1261/rna.2264806.
Received September 30, 2005; accepted October 14, 2005.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Andrei, M.A., Ingelfinger, D., Heintzmann, R., Achsel, T., Rivera-Pomar, R., and Luhrmann, R. 2005. A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies. RNA 11: 717–727.
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. 1999. Current protocols in molecular biology. Greene Publishing Associates/Wiley-Interscience, New York.
Bashkirov, V.I., Scherthan, H., Solinger, J.A., Buerstedde, J.M., and Heyer, W.D. 1997. A mouse cytoplasmic exoribonuclease (mXRN1p) with preference for G4 tetraplex substrates. J. Cell Biol. 136: 761–773.
Basyuk, E., Galli, T., Mougel, M., Blanchard, J.M., Sitbon, M., and Bertrand, E. 2003. Retroviral genomic RNAs are transported to the plasma membrane by endosomal vesicles. Dev. Cell 5: 161–174.[CrossRef][Medline]
Beach, D.L., Salmon, E.D., and Bloom, K. 1999. Localization and anchoring of mRNA in budding yeast. Curr. Biol. 9: 569–578.[CrossRef][Medline]
Bertrand, E., Chartrand, P., Schaefer, M., Shenoy, S.M., Singer, R.H., and Long, R.M. 1998. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2: 437–445.[CrossRef][Medline]
Brengues, M., Teixeira, D., and Parker, R. 2005. Movement of eukaryotic mRNAs between polysomes and cytoplasmic processing bodies. Science 310: 486–489.
Burke, D., Dawson, D., and Stearns, T. 2000. Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Butsch, M. and Boris-Lawrie, K. 2002. Destiny of unspliced retroviral RNA: Ribosome and/or virion? J. Virol. 76: 3089–3094.
Coffin, J.M., Hughes, S.H., and Varmus, H.E., eds. 1997. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Coller, J. and Parker, R. 2005. General translation repression by activators of mRNA uncapping. Cell 122: 875–886.[CrossRef][Medline]
Cougot, N., Babajko, S., and Seraphin, B. 2004. Cytoplasmic foci are sites of mRNA decay in human cells. J. Cell Biol. 165: 31–40.
Ferraiuolo, M.A., Basak, S., Dostie, J., Murray, E.L., and Schoer, R.A. 2005. A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay. J. Cell Biol. 170: 913–924.
Garfinkel, D.J., Boeke, J.D., and Fink, G.R. 1985. Ty element transposition: Reverse transcriptase and virus-like particles. Cell 42: 507–517.[CrossRef][Medline]
Giddings, T.H., O’Toole, E.T., Morphew, M., Mastronarde, D.N., McIntosh, J.R., and Winey, M. 2001. Using rapid freeze and freeze-substitution for the preparation of yeast cells for electron microscopy and three-dimensional analysis. Methods Cell Biol. 67: 27–42.[Medline]
Griffith, J.L., Coleman, L.E., Raymond, A.S., Goodson, S.G., Pittard, W.S., Tsui, C., and Devine, S.E. 2003. Functional genomics reveals relationships between the retrovirus-like Ty1 element and its host Saccharomyces cerevisiae. Genetics 164: 867–879.
Hansen, L.J., Chalker, D.L., and Sandmeyer, S.B. 1988. Ty3, a yeast retrotransposon associated with tRNA genes, has homology to animal retroviruses. Mol. Cell. Biol. 8: 5245–5256.
Hansen, L.J., Chalker, D.L., Orlinsky, K.J., and Sandmeyer, S.B. 1992. Ty3 GAG3 and POL3 genes encode the components of intracellular particles. J. Virol. 66: 1414–1424.
Hong, S., Choi, G., Park, S., Chung, A.S., Hunter, E., and Rhee, S.S. 2001. Type-D retrovirus Gag polyprotein interacts with the cytosolic chaperonin TRiC. J. Virol. 75: 2526–2534.
Huh, W.K., Falvo, J.V., Gerke, L.C., Carroll, A.S., Howson, R.W., Weissman, J.S., and O’Shea, E.K. 2003. Global analysis of protein localization in budding yeast. Nature 425: 686–691.[CrossRef][Medline]
Ingelfinger, D., Arndt-Jovin, D.J., Luhrmann, R., and Achsel, T. 2002. The human LSm1-7 proteins colocalize with the mRNA-degrading enzymes Dcp1/2 and Xrn1 in distinct cytoplasmic foci. RNA 8: 1489–1501.[Abstract]
Irwin, B., Aye, M.S., Baldi, P., Beliakova-Bethell, N., Cheng, H., Dou, Y., Liou, W., and Sandmeyer, S. 2005. Retroviruses and yeast retrotransposons use overlapping sets of host genes. Genome Res. 15: 641–654.
Kedersha, N., Stoecklin, G., Ayodele, M., Yacono, P., Lykke-Andersen, J., Fitzler, M.J., Scheuner, D., Kaufman, R.J., Golan, D.E., and Anderson, P. 2005. Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J. Cell Biol. 169: 871–884.
Linial, M.L. and Eastman, S.W. 2003. Particle assembly and genome packaging. Curr. Top. Microbiol. Immunol. 277: 89–110.
Lykke-Andersen, J. 2002. Identification of a human decapping complex associated with hUpf proteins in nonsense-mediated decay. Mol. Cell. Biol. 22: 8114–8121.
Menees, T.M. and Sandmeyer, S.B. 1994. Transposition of the yeast retroviruslike element Ty3 is dependent on the cell cycle. Mol. Cell. Biol. 14: 8229–8240.
Oakes, M., Aris, J.P., Brockenbrough, J.S., Wai, H., Vu, L., and Nomura, M. 1998. Mutational analysis of the structure and localization of the nucleolus in the yeast Saccharomyces cerevisiae. J. Cell Biol. 143: 23–34.
Orlinsky, K.J. and Sandmeyer, S.B. 1994. The Cys-His motif of Ty3 NC can be contributed by Gag3 or Gag3-Pol3 polyproteins. J. Virol. 68: 4152–4166.
Sandmeyer, S.B., Aye, M., and Menees, T.M. 2002. Ty3: A position-specific, gypsylike element in Saccharomyces cerevisiae. In Mobile DNA II (eds. N.L. Craig et al.), pp. 663–682. ASM Press, Washington, DC.
Sfakianos, J.N., LaCasse, R.A., and Hunter, E. 2003. The M-PMV cytoplasmic targeting-retention signal directs nascent Gag poly-peptides to a pericentriolar region of the cell. Traffic 4: 660–670.[CrossRef][Medline]
Sheth, U. and Parker, R. 2003. Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300: 805–808.
Teixeira, D., Sheth, U., Valencia-Sanchez, M.A., Brengues, M., and Parker, R. 2005. Processing bodies require RNA for assembly and contain nontranslating mRNAs. RNA 11: 371–382.
Teysset, L., Dang, V.D., Kim, M.K., and Levin, H.L. 2003. A long terminal repeat-containing retrotransposon of Schizosaccharomyces pombe expresses a Gag-like protein that assembles into virus-like particles which mediate reverse transcription. J. Virol. 77: 5451–5463.
Winzeler, E.A., Shoemaker, D.D., Astromoff, A., Liang, H., Anderson, K., Andre, B., Bangham, R., Benito, R., Boeke, J.D., Bussey, H., et al.1999. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285: 901–906.
Zimmerman, C., Klein, K.C., Kiser, P.K., Singh, A.R., Firestein, B.L., Riba, S.C., and Lingappa, J.R. 2002. Identification of a host protein essential for assembly of immature HIV-1 capsids. Nature 415: 88–92.[CrossRef][Medline]
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) cells transformed with the high-copy vector pYES2.0 were mixed on solid SD-ura and allowed to undergo mating to induce Ty3-GFP expression. (A) Ty3-GFP clusters form in mating cells. Formation of zygotes at 0, 1, 2, 3, 4, and 6 h after mixing. (Top, upper panels) Nuclei and Ty3 clusters (DAPI, red; Ty3-GFP, green). (Top, lower panels) Differential interference contrast (DIC) micrographs of whole cells. Scale bar, 5.0 µm. (Bottom) Cartoon of mating cells. Cells form shmoo projections that fuse to give a zygote dumbbell which then buds. (B) Mating populations were monitored for percentage of cells in zygotes and percentage of zygotes with clusters.
