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Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113-8613, Japan
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Introduction |
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-NH2 group of Lys of the acceptor substrate. A polyubiquitin chain is formed by repeated reactions through which another ubiquitin links a Lys residue at position 48 within one ubiquitin associated with the target protein, which becomes a marker for proteasome degradation (Glickman & Ciechanover 2002; Pickart 2004). In addition to aberrant proteins, many regulatory proteins in a diverse array of biologically important processes, such as mitotic cell cycle, signal transduction, or developmental programs, are known to be substrates of this protein modifying system. In addition, it has also become clear that ubiquitylation has various cellular roles other than proteolysis, such as vesicular transport and gene silencing (Glickman & Ciechanover 2002; Pickart 2004). The conjugation of ubiquitin to the substrates is carried out by three types of enzymes; E1 (ubiquitin activating enzyme), E2 (ubiquitin conjugation enzyme) and E3 (ubiquitin ligating enzyme). E1 catalyses the formation of a high-energy thioester bond between the C-terminal Gly residue of ubiquitin and a specific Cys residue in itself. E2 receives the activated ubiquitin molecules from E1, forming a thioester linkage like E1. E3 recognizes the substrates, and attaches ubiquitin transferred by E2 to the substrates or helps E2 to transfer ubiquitin to the substrates (Glickman & Ciechanover 2002; Pickart 2004). E3 plays a critical role in the selection of target proteins, because each distinct E3 usually binds a protein substrate with a degree of selectivity for ubiquitylation in a temporally and spatially regulated fashion.
Deubiquitinating enzymes (DUBs) or ubiquitin-specific processing proteases are required to generate functional ubiquitin monomers, because ubiquitin is translated as fusion proteins with certain ribosomal proteins or a polyubiquitin in which several ubiquitin moieties link tandemly. DUBs process these ubiquitin precursors at the C-terminus of the ubiquitin segment(s). After this processing, Gly76 appears at the C-terminus of ubiquitin. Since the conjugation of ubiquitin is formed via this Gly76, exposure of Gly76 is essential for the protein-modifying function of ubiquitin (Fischer 2003; Kim et al. 2003).
Various ubiquitin-like (UBL) modifiers have been identified so far, including SUMO, NEDD8 and UCRP/ISG15, which are structurally similar to ubiquitin (Tanaka et al. 1998; Jentsch & Pyrowolakis 2000; Schwartz & Hochstrasser 2003). They are covalently linked to target proteins via the C-terminal Gly residue in a manner analogous to ubiquitylation, where the reaction is catalysed by the E1-, E2- and E3-like enzymes. It is of note that most DUB-like enzymes are also necessary to remove the C-terminal extension for their conjugation except UBL(s) synthesized with Gly in the C-terminal end (Li & Hochstrasser 1999; Gan-Erdene et al. 2003; Mendoza et al. 2003; Wu et al. 2003; Hemelaar et al. 2004).
Recent studies described a novel UBL protein, Hub1 (Dittmar et al. 2002; Lüders et al. 2003; Ramelot et al. 2003). Hub1 is a UBL protein with apparently similar tertiary structure (Ramelot et al. 2003) despite the low (23%) residue identity to ubiquitin. However, whether Hub1 is a modifier protein is a matter of controversy. There is a characteristic difference between Hub1 and ubiquitin; i.e. Gly is not evolutionarily conserved at the C-terminus, but the di-Tyr sequence is conserved at the C-terminus, followed by one more extra non-conserved residue. Dittmar et al. (2002) reported that Hub1 in Saccharomyces cerevisiae was processed at the C-terminus similar to ubiquitin, and functioned as a modifier using the conserved Tyr residue, but Lüders et al. (2003) raised an objection to this conclusion using the same budding yeast. Hub1 is not essential for viability in S. cerevisiae, but deletion mutant shows the defect in cell polarization during the formation of mating projections (Dittmar et al. 2002), and slow growth phenotype in a specific strain background (Lüders et al. 2003). Hub1 is also identified as Ubl5 or Beacon in other organisms (Friedman et al. 2001; Brailoiu et al. 2003; Kantham et al. 2003; McNally et al. 2003), but it remains unresolved whether Ubl5/Beacon is a modifier protein. In the present study, we address this issue by working on Hub1 in Schizosaccharomyces pombe. We found that Hub1 is essential and functions without processing of its C-terminus to expose the conserved residue unlike other UBL modifiers. In addition, we discuss the biological function of Hub1 by isolating and examining hub1-1 mutation and its multicopy suppressor genes.
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Results |
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From the database, we identified a new UBL protein in S. pombe. This protein is highly conserved among eukaryotes, displaying 64–75% amino acid identity ranging from yeast to human (Fig. 1A). In S. cerevisiae, this gene is named HUB1 (Dittmar et al. 2002; Lüders et al. 2003), so we followed this nomenclature and named S. pombe gene hub1+. Though Hub1 is a UBL protein, there is a characteristic difference between Hub1 and ubiquitin. It has no conserved Gly residue at the C-terminus, which is essential for the covalent attachment of ubiquitin and other known UBL modifiers to other proteins. On the other hand, di-Tyr sequence is conserved among all Hub1 homologues, and it seems to be equivalent to Gly for ubiquitin (Fig. 1A).
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To explore the function of Hub1 in S. pombe, we disrupted the hub1+ gene by replacing the entire ORF with the Aureobasidine A resistant gene (aur1R.) in diploid strain (Fig. 1C, left panel). Heterozygous diploids were sporulated and dissected. From each tetrad, only two viable spore clones could be obtained (Fig. 1C, right panel), and all of them had Aureobasidine A sensitive phenotype. Thus, it is clear that hub1+ is essential for mitotic growth in S. pombe. Microscopic analysis revealed that hub1+ deleted cells ceased their growth with an elongated cell shape after 2 or 3 cycles of cell division (data not shown). To confirm the effect of lack of Hub1, we constructed the strain whose growth is dependent of hub1+ under the thiamine repressive promoter. At 12 h after repression of Hub1 by addition of thiamine, cells started to elongate, and to slightly enlarge in size (Fig. 1D).
Localization of Hub1
To determine the localization of Hub1 in living cells, GFP-Hub1 was constructed and introduced into the 
hub1 strain. The fusion gene complemented the deletion strain, so the essential function of Hub1 was not compromised by fusion to GFP. As shown in Fig. 2, GFP-Hub1 was observed in both the nucleus and the cytoplasm.
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We examined the importance of the conserved Tyr residues in Hub1. Our initial working hypothesis was that Hub1 is an essential modifier and the conserved Tyr is used for its modification. If this model is correct, we can expect the Tyr residue to be essential for Hub1 function and growth of S. pombe. A hub1::aur1R/hub1+ heterozygous diploid strain (YHY22P) was transformed with pHY58 (pUR19 (ura4+)-hub1+) and a hub1 haploid strain whose growth depended on pHY58 was obtained. This strain (YHY23P) was further transformed with pALSK (LEU2), pHY59 (pALSK-hub1+) or pHY60 (pALSK-hub1YY) in which conserved di-Tyr was deleted. Each transformant was streaked on a SD-Leu plate containing 0.5 mg/mL 5-FOA. 5-FOA is a toxic reagent for uracil autotrophic cells, so cells should dispense with pHY58 (pUR19-hub1+) to grow on 5-FOA-containing media. Of the three types of transformants, only the transformant with pALSK failed to grow on this medium while the transformant with pHY60 (pALSK-hub1YY) could grow as well as one with pHY59 (pALSK-hub1+) (Fig. 3). Thus, pHY60 (pALSK-hub1YY) could replace pHY58 (pUR19-hub1+), indicating that the di-Tyr motif is not essential for the Hub1 function and for the growth of S. pombe. Moreover, cells bearing the Hub1YY showed no growth defect even at higher temperature (data not shown).
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Ubiquitin and other UBL modifiers, such as NEDD8 or SUMO are processed to have conserved Gly exposed at the C-terminus, which is used for their covalent attachment to their substrate proteins. Alternatively, in Hub1, Tyr is conserved just before the last non-conserved residue. It is also possible that one of the other conserved residues located the 5' upside of Tyr is used for its conjugation. In either case, if Hub1 acts as a modifier protein, it should be processed to remove its non-conserved residue(s) at the C-terminus, because it is unlikely that non-conserved residue is used for its conjugation. To examine whether processing of Hub1 occur, a strain expressing Hub1–3 x Flag was obtained by chromosomal tagging of the hub1+ gene in JY741. It is known that the C-terminal ß-galactosidase fused with ubiquitin and the natural C-terminal tripeptide Ala-Thr-Tyr of SUMO are removed co-translationally and their preprocessed forms are barely observed (Bachmair et al. 1986; Li & Hochstrasser 2003). However, in the case of Hub1, we could easily detect Hub1 with C-terminal Flag tag using anti-Flag antibody (Fig. 4). On the contrary, we could not detect the Hub1 molecules without C-terminal Flag tag, judging from the Western blot analyses with anti-Flag and anti-Hub1 antibodies (Fig. 4). This result indicated that the processing at the C-terminus of Hub1 did not occur. In comparison with ubiquitin and other UBL modifiers, this feature is unique to Hub1.
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As a useful tool for characterization of essential genes, we generated one temperature-sensitive allele of hub1+ by random mutagenesis. Sequence analysis revealed that Ile42 changed to Ser42. Ile42 is conserved among Hub1 proteins (Fig. 1A), and it is located as a first residue of the third strands (Ramelot et al. 2003), so Ile42 is expected to be important for the maintenance of the UBL structure. Under restrictive temperature, hub1-1 cells were elongated similar to the Hub1-depleted cells. DAPI staining showed that cytokinesis might be defective in hub1-1 (Fig. 5A).
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Next, we tried to isolate the multicopy suppressors of hub1-1. The hub1-1 cells were transformed with a multicopy pALSK vector based S. pombe genomic library and plated at 34 °C. Plasmids were rescued from transformants that grew at this temperature. Among 77 positive clones excluding hub1+ itself, 53 clones included rpb10+. Rpb10 is a common subunit in all three forms of the RNA polymerase (Lalo et al. 1993; Gadal et al. 1999; Kimura et al. 2001). To confirm that rpb10+ is a suppressor gene of hub1-1, only rpb10+ ORF region was subcloned into the expression vector, pREP81. hub1-1 transformed with pHY63 (pREP81-rpb10+ ORF) could grow at the restricted temperature as those with the original clones (Fig. 6A, upper), so we could confirm that rpb10+ was truly a multicopy suppressor of hub1-1.
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The genetic interaction between Snu66 and Hub1 suggested that hub1+ might be involved in pre-mRNA splicing. To verify whether or not Hub1 is required for splicing in S. pombe, we used RT-PCR to measure the steady state levels of premRNA and mRNA of rpl7+ or cdc16+ in hub1-1 cells. After 3 h incubation at 37 °C, significant accumulation of premRNA of both genes was observed in the hub1-1 mutant (Fig. 7). From this result, we conclude that Hub1 plays a role for the efficient splicing of premRNA.
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Discussion |
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It is highly improbable that the conjugation sites vary from species to species, but it is possible that there are some other conserved conjugation sites in addition to Tyr72. In either case, the C-terminal processing of Hub1 to remove the non-conserved residue(s) at the C-terminus should be required to have Tyr72 or another hypothetical conjugation site exposed at the C-terminus. We constructed the Hub1 tagged with 3 x Flag C-terminally and examined whether the processing of Hub1 occurred at the C-terminus. However, we could not detect cleavage of the Flag tag, namely the C-terminal processing of Hub1 (Fig. 4), though we could not exclude the possibility that very little amount of Hub1 is processed. Considering that ubiquitin and other modifiers like NEDD8 and SUMO are processed co-translationally (Bachmair et al. 1986; Gan-Erdene et al. 2003; Li & Hochstrasser 2003; Mendoza et al. 2003; Wu et al. 2003; Hemelaar et al. 2004), the processing of Hub1 is not likely to happen. This means that Hub1 has a native C-terminal residue encoded by genome, which is different in different species (Fig. 1A). This result is consistent with the finding of Lüders et al. (2003), in which C-terminally tagged Hub1 existed stably. Thus, all our results indicate that Hub1 is unlikely to be a typical modifier protein like ubiquitin and other UBL proteins.
To date, a set of novel molecules called ubiquitin-like proteins (UBLs) that have structural similarity to ubiquitin, has been identified (Jentsch & Pyrowolakis 2000). Currently, these molecules are divided into two subclasses; type-1 UBLs, which ligate to target proteins in a manner similar, but not identical, to the ubiquitylation pathway, such as SUMO, NEDD8 and UCRP/ISG15, and type-2 UBLs (or called as UDPs, ubiquitin-domain proteins) that contain UBL structure embedded in a variety of different classes of large proteins with apparently distinct functions, such as Rad23, Elongin B, Scythe, Parkin and HOIL-1 (Tanaka et al. 1998; Jentsch & Pyrowolakis 2000; Yeh et al. 2000; Schwartz & Hochstrasser 2003). However, Hub1 obviously differs from these two-types of UBL family, based on the following two reasons. One is that the size of Hub1 is similar to that of type-1 UBLs, but it is not likely to be a covalent modifier protein. The other is that Hub1 does not form the domain structure in the large protein unlike type-2 UBL(s). Considered together, we propose that Hub1 is defined as a novel or third subgroup of UBL family proteins.
Though the precise mechanism of the biological function of Hub1 remains to be elucidated, we speculate that Hub1 is involved in the RNA metabolism, because we isolated rpb10+ and snu66+ as multicopy suppressors of the hub1-1 mutation (Fig. 6), and hub1-1 showed the pre-mRNA splicing defect (Fig. 7).
Rpb10 is one of the shared subunits among three forms of the RNA polymerase. In S. pombe, the RNA polymerase II is composed of 12 different subunits. Among them, Rpb5, 6, 8, 10 and Rpb12 are common subunits of RNA polymerases (Kimura et al. 2001). All these subunits are essential for growth, but the function of each subunit is still totally unknown. Furthermore, each subunit may have unknown functions independent of RNA polymerases, because the presence of intracellular pools of unassembled subunits are indicated (Kimura et al. 2001). To date, there is no report about rpb10+ as a multicopy suppressor gene in S. pombe, and in S. cerevisiae, the only known fact is that overproduction of Rpb10 can suppress assembly mutants of Rpc19 and Rpc40, both of which are components of RNA polymerase I and III (Gadal et al. 1999). We tried to find some genetic interactions between hub1+ and the homologues of these genes in S. pombe, but at least, overproduction of the counterparts of Rpc19 and Rpc40 in S. pombe could not work as multicopy suppressors (data not shown). We also checked the sensitivity of hub1-1 to 6-Azauracil (6-AU), but hub1-1 grew normally on 6-AU containing media (data not shown). Sensitivity to 6-AU often correlates with defects in the elongation phase of transcription by RNA polymerase II (Hampsey 1997; Ishiguro et al. 2000), so Hub1 is unlikely to be involved in the elongation phase of transcription by the RNA polymerase II.
Another suppressor gene product, Snu66 is one of the components of the 25S [U4/U6·U5] tri-snRNP (small nuclear ribonucleoprotein), which is a subcomplex of the spliceosome (Kaufer & Potashkin 2000; Jurica & Moore 2003). Over 100 spliceosomal protein components have been identified so far, but the function of many of these proteins remains unknown. Snu66 was shown to play an important role in pre-mRNA splicing in vitro (Gottschalk et al. 1999), but SNU66 is not an essential gene in S. cerevisiae, and its in vivo function is not still apparent. Hub1 has not been reported as a component of the spliceosome and the localization of Hub1 was not restricted to the nucleus (Fig. 2), so Hub1 is not likely to be the core and stable component of the spliceosome. However, pre-mRNA splicing was impaired in hub1-1 (Fig. 7), and it is possible that Hub1 has some regulatory functions to pre-mRNA splicing. It is conceivable that a defect in making proper mRNAs causes various phenotypes of hub1-1. We may also explain the phenotypic difference of the deletion mutants between S. cerevisiae and S. pombe; the former has no obvious phenotype, and the latter is lethal, because there are few genes that require premRNA splicing in S. cerevisiae.
Along with multicopy suppressors, osmotic stabilizer could suppress the hub1-1 mutation (Fig. 5B,C). In S. pombe, it is known that Spc1/Sty1 MAP kinase cascade is activated by various stress signals, including hyperosmotic shock (Hohmann 2002). Hub1 may work in cooperation with this MAP kinase cascade.
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Experimental procedures |
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Escherichia coli strain DH5
was used for propagating plasmids, and BL21 (DE3) was used for expression and purification of recombinant proteins. The following yeast strains were used in this study: JY741 (h–leu1 ura4-D18 ade6-M216), JY746 (h+leu1 ura4-D18 ade6-M210), YHY22P (h–/h+leu1/leu1 ura4-D18/ura4-D18 ade6-M216/ade6-M210hub1::aur1R/hub1+), YHY23P (hub1::aur1R pHY58), YHY24P (h–leu1 ura4-D18 ade6-M216 hub1-1-ura4+), YHY25P (h–leu1 ura4-D18 ade6-M216 hub1–3 x Flag-kanMX6), and YHY26P (hub1::aur1R pHY57) YHY25P was made by using pHY68. Media and methods for mating, sporulation, tetrad analysis, and transformation were previously described (Moreno et al. 1991; Alfa et al. 1993; Guthrie & Fink 2002). To regulate the expression of genes under the thiamine-repressible promoter (nmt1 promoter), cells were grown in minimal medium with or without 10 µM thiamine. For gene disruption, a diploid strain crossed between JY741 and JY746 was used. Correct disruption was confirmed by polymerase chain reaction (PCR) analysis.
Plasmids
Plasmids pREP1, pREP81 and pUR19, were previously described (Moreno et al. 1991; Barbet et al. 1992). Multicopy vector for S. pombe, pALSK (LEU2) and pRSC81 (pREP81-GFP) were kind gifts from Dr A. Toh-e (The University of Tokyo) and Dr A. Matsuyama (Chemical Genetics Laboratory, Riken), respectively. The constructed plasmids were as follows: pHY56 (pREP81-Flag-His6-hub1+ ORF), pHY57 (pREP81-GFP- Flag-His6-hub1+ ORF), pHY58 (pUR19-hub1+), pHY59 (pALSK-hub1+), pHY60 (pALSK-hub1YY), pHY63 (pREP81-rpb10+ ORF), pHY64 (pREP1-snu66+ ORF), pHY67 (pET15b-hub1c) and pHY68 (pBluescript II KS–PFT cassette-kanMX6). PFT cassette was from pYS419 (Saeki et al. 2002). Gene name followed by open reading frame (ORF) means the DNA fragment from the ATG codon to the terminal codon of its gene. Gene name only means the DNA fragment with the 5' and 3' regions of its gene. hub1c means cDNA of hub1+ gene. For pHY56, hub1+ ORF was amplified by PCR using primers tagubl4N (5'-GGAATTCCATATGGACTACAAGGACGACGATGACAAGCATCATCATCATCATCACATGATCGAAGTTTTATGGTAT-3') and ubl4wt c (5'-GCGCGGATCCTTAAGAATAATACATCTC-3'), and cloned into NdeI/BamHI sites of pREP81. For pHY57, DNA fragments containing Flag-His6-hub1+ ORF were excised from pHY56 with NdeI and BamHI, and cloned into NdeI/BamHI sites of pRSC81. hub1+ DNA fragments with the 5' and 3' regions were obtained by PCR with the primers ubl5 A (5'-GGCCCTGCAGGGCGAAGGACGACGCTTC-3') and ubl5 D (5'-GGCCCTGCAGGTAACCAATACATATCTG-3'), and genomic DNA as a template. Amplified fragments were digested with PstI and cloned into pUR19 for pHY58 and pALSK for pHY59. pHY60 was constructed from the pHY59 by using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) and the primers, ubl4sens (5'-GGAATGAGCTTGGAGATGTCTTAAAAAGCGAAAC-3') and ubl4anti (5'-GTTTCGCTTTTTAAGACATCTCCAAGCTCATTCC-3'). Hub1 cDNA fragments were amplified from the S. pombe cDNA library using two primers, cUBL4N (5'-GCGCGGATCCTATGATCGAAGTTTTATGTAACGAT-3') and cUbl4 sC (5'-GCGCAGATCTTTAAGAATAATACATCTCCAAGCTC-3'). Amplified fragments were cloned into BamHI site of pET15b (Novagen, Madison, WI, USA) for pHY67. pHY62 (pALSK-rpb10+) was isolated from the S. pombe genomic library.
Immunoblot analysis
For Fig. 4, cells were suspended in lysis buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-KOH, pH 7.6, 100 mMß-glycerolphosphate, 50 mM NaF, 1 mM MgCl2, 1 mM ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 5% glycerol, 0.25% Triton X-100 containing complete protease inhibitors ethylenediaminetetraacetic acid (EDTA) free (Roche)), broken by vortexing with glass beads at 4 °C for 5 min, and the cleared lysates were prepared by centrifugation at 20 000 g at 4 °C for 10 min. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were performed with the NuPAGE system (Invitrogen, San Diego, CA, USA) as per instructions provided by the manufacturer. As primary antibodies, anti-Flag M2 antibody (Sigma Chemical Co., St. Louis, MO, USA) or anti-Hub1 antibody were used, and anti-mouse or anti-rabbit IgG HRP conjugate (Promega, Madison, WI, USA) were used as secondary antibodies.
Purification of recombinant Hub1 protein, and antibody preparation
pHY67 was expressed in BL21 (DE3) at 37 °C. The cell pellet from a two little culture was suspended in 40 mL of sonication buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM dithiothreitol (DTT)). Then, 10% Triton X-100 was added to the crude lysate to a final concentration of 0.5%. The crude lysate was centrifuged at 13 000 g for 20 min at 4 °C. The pellet was resuspended in 30 mL sonication buffer with 4% Triton X-100. Hub1 inclusion bodies were pelleted by centrifugation at 13 000 g for 20 min. This wash step was repeated twice. After that, inclusion bodies were washed with distilled water twice. The purified inclusion bodies were suspended in denature buffer (100 mM NaH2PO4, 10 mM Tris-HCl, 6 M guanidine hydrochloride (GuHCl), pH 8.0). The suspension was held at room temperature for 1 h. Any precipitate was removed by centrifugation at 35 000 g for 20 min. Chelating Sepharose fast flow beads (Amersham Biosciences) charged with Ni were added to the supernatant and rotated for 30 min at 4 °C. Refolding was performed on the Ni beads using a linear 6 M– 0 M GuHCl gradient in 100 mM NaH2PO4, 10 mM Tris-HCl, pH 8.0 by Äkta FPLC equipment (Amersham Biosciences). After renaturation, proteins were eluted by the addition of 250 mM imidazole. Anti-Hub1 polyclonal antibody was prepared using purified recombinant His6-Hub1 as antigen.
Microscopic analysis
Living cells or fixed cells were soaked into 0.5 µg/mL DAPI solution for observing chromatin. Cell fixation was done with 70% ethanol. For observation of GFP-Hub1, YHY22P was transformed with pHY57, and then sporulated. The resultant tetrads were dissected, and haploid cells (YHY26P) containing both the hub1 and pHY57 were selected. YHY26P was cultured in EMM-Leu media at 26 °C. Photographs were taken by a cooled CCD camera using IP lab software equipped with model AX70 Olympus microscope using the UPlanApo x100 objective lens.
Isolation of temperature-sensitive alleles of hub1+
We used the GeneMorph kit (Stratagene) for introducing mutations into hub1+ ORF. Random mutagenized hub1+ ORFs were cloned into pREP81. This mutagenized hub1 library was used for transformation of YHY23P (hub1::aur1R. pUR19-hub1+), and transformed cells were spread on to the EMM-Leu +5-FOA media. The growth rate of the obtained 264 colonies was checked at 26 °C or 37.2 °C. Cells that could not grow at 37.2 °C were selected, and from those cells, plasmids were retrieved and the mutation sites were determined by sequence analysis. The same mutations were introduced into pHY59 (pALSK-hub1+) with the QuickChange site-directed mutagenesis kit (Stratagene), and then the ura4+ maker excised from pUR19 was inserted into the HindIII site located to the 3' region of hub1+ ORF (Fig. 1C, left panel). The wild-type strain, JY741, was transformed with the excised DNA fragments containing the mutagenized hub1+ ORF and ura4+. The growth of transformants was checked for whether it was temperature-sensitive or not. One of the transformants showed clear temperature-sensitive growth, so we designated this allele hub1-1.
RT-PCR analysis
RT-PCR analysis was carried out as previously described (Oltra et al. 2004; Webb & Wise 2004). Total RNA was isolated using RNeasy Mini Kit (Qiagen). RNA was converted to cDNA using the SuperScriptTM II Reverse Transcriptase (Invitrogen). Primers used for RT-PCR amplification were 5'-GAAGACTAAGGCTCAGAAACAATC-3' and 5'-GGACCAACAGTGTAAATTTCATG-3' (for rpl7 intron) or 5'-TTATTGGATAAATGCCTTGAAGTCC-3' and 5'-TCGGAAAATAAACATTTGTGCAATC-3' (for cdc16 intron 2). PCR products were resolved on 1% or 4% agarose gels and stained with ethidium bromide.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: hyashiro{at}rinshoken.or.jp; tanakak{at}rinshoken.or.jp
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Received: 1 May 2004
Accepted: 22 September 2004
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