22 Results
for author "Yue-wen Wang"
Figure 1.—Association of Rpc19p with Gtr1p. (a) Extracts from cultures of S. cerevisiae colonies harboring two yeast genes, GTR1 wild type, S20L or the Q65L form in the pAS404 vector, or RPC19 in the pACT2 vector as shown, were obtained. Their β-galactosidase activities were measured as described before (Sekiguchi et al. 2001) and are shown as means of triplicate values with standard deviations. A β-galactosidase filter assay was also performed (middle). Patches of blue color appearing within 30 min represent a positive signal. White patches represent a negative signal. Expression of HA-tagged Gtr1p and Rpc19p in the yeast extracts described above was shown by immunoblotting against the HA tag (right). (b) Either GST-Rpc19p (lanes 1, 3, 5, and 7) or GST (lanes 2, 4, 6, and 8) was coexpressed with HA-tagged Gtr1p in NBW5 gtr1Δ. Prepared crude extracts were mixed with glutathione Sepharose-4B beads and pulled down as described previously (Nakashima et al. 1999). Bound proteins were run on SDS-polyacrylamide gels and transferred onto nylon membranes, which were immunoblotted with either anti-HA (lanes 1–4) or anti-GST (lanes 5–8) antibodies. Lanes 1, 2, 5, and 6: total proteins (10% input). An asterisk indicates degraded protein. (c) HA-tagged Gtr1p was expressed in NBW5 gtr1Δ. Yeast cell lysate was prepared and pulled down with either E. coli-produced GST-Rpc19p (lanes 2 and 5) or GST (lanes 3 and 6), which were bound to glutathione Sepharose-4B. Bound proteins were run on SDS-polyacrylamide gels and transferred onto nylon membranes, which were immunoblotted with the anti-HA (lanes 1–3) antibody. The filters were then stained with Coomassie Brilliant Blue R-250 (lanes 4–6). Lanes 1 and 4: total proteins (10% input). (d) 35S-labeled recombinant proteins (Gtr2p, lanes 1 and 5; Gtr1p, lanes 2 and 6; Gtr1p GTP form, lanes 3 and 7; the GDP form of Gtr1p, lanes 4 and 8) were produced with the TNT-Quick Coupled reaction kit and were pulled down with either GST-Rpc19p (lanes 1–4) or GST (lanes 5–8) protein, which was bound to glutathione Sepharose-4B beads. Bound proteins were run on SDS-polyacrylamide gels and analyzed using the Fuji image analyzer. Input proteins (2%) are shown in lanes 9–12. (e) Extracts from cultures of S. cerevisiae colonies harboring two human genes—RRAG A wild type (the T21L or the Q66L form in the pAS404 vector) and RPA16 in the pACT2 vector as shown—were obtained. Their β-galactosidase activities are shown as means of triplicate values with standard deviations. β-Galactosidase filter assay using Y190 strains harboring pAS404-RRAG A, pAS404-RRAG A(T21L) (GDP form), or pAS404-RRAG A(Q66L) (GTP form) and pACT2-RPA16 was performed as described above.
Figure 2.—Mode of association of Gtr1p with Rpc19p and Nop8p. (a) Y190 strains harboring pAS404-RPC19 and pACT2-RPC19, pACT2-RPC40, pACT2-GTR1, pACT2-GTR2, pACT2-NOP8, or pACT2 vector were plated on SD −L, −T or SD −L, −T, −H, +3-AT plate at 30° for 2 or 5 days. Extracts from cultures of S. cerevisiae colonies harboring two yeast genes, RPC19 in the pAS404 vector and GTR1, GTR2, RPC19, RPC40, or NOP8 in the pACT2 vector as shown—were obtained. Their β-galactosidase activities are shown as means of triplicate values with standard deviations. A β-galactosidase filter assay was performed (bottom). (b) Y190 strains harboring pAS404-GTR1, pAS404-gtr1-11 (S20L) (GDP form), or pAS404-gtr1-13 (Q65L) (GTP form) and pACT2-NOP8 were plated on SD −L, −T or SD −L, −T, −H, +3-AT plate at 30° for 2 or 5 days.- TABLE 1Plasmids used in this study
Figure 3.—Ribosomal RNA synthesis was decreased in the gtr1Δ strain. (a) The growth curve of NBW5 gtr1Δ (•), NBW5 gtr1Δ expressing wild-type Gtr1p (pL242) (○), NBW5 gtr1Δ expressing Q65L Gtr1p (pL244) (□), NBW5 gtr1Δ expressing S20L Gtr1p (pL243) (▵), and wild-type NBW5 (+) strains at 26° (left) and at 14° (right) was studied as previously described (Zanchin et al. 1997). Decrease in the growth rate of gtr1Δ and S20L strains was significant at 14°. (b) To examine the global rRNA synthesis rate in the strains described above, exponentially growing cultures were labeled with [3H]uracil (50 μCi/ml) for 10 min at 26° or 30 min at 14° and cold excess uracil (300 μg/ml) was added and incubated to complete the processing for 1 hr at 26° or 3 hr at 14°. Extracted RNAs were run on formaldehyde agarose gel and transferred to Hybond filters. The filters were exposed to an imaging plate (TR2040, Fuji Photo film). Lanes 1 and 5, NBW5 gtr1Δ; lanes 2 and 6, NBW5 gtr1Δ (pL242); lanes 3 and 7, NBW5 gtr1Δ (pL243); lanes 4 and 8, NBW5 gtr1Δ (pL244). Relative radioactivity (R.R.) of 25S or 18S rRNA is shown at the bottom left (gtr1Δ, 26° = 100). RNA was stained with ethidium bromide showing that an equal amount of RNA was loaded on the gel (right). (c) Cells grown at 30° to midlog phase. 5′ ITS1 rRNA was localized by fluorescence in situ hybridization. Chromosomal DNA was labeled with 4′,6-diamidino-2-phenylindole-dihydrochloride to identify the location of the nucleus. (d) Total RNA was isolated from logarithmically grown gtr1Δ, gtr2Δ, and wild-type strains at either 26° or 16° for 2 hr. Total RNA (5 μg) was run on formaldehyde agarose gels and transferred onto nylon membranes and blotted with the 32P-labeled probes indicated at the right. Relative radioactivity (R.R.) of RPS3, RPL3, or ACT1 is shown at the bottom (wild type, 26° = 100). (e) rRNA processing was examined by 2-min pulse labeling with [3H]methyl methionine and chased for 2, 6, and 15 min in the presence of methionine (100 μg/ml), as described previously (Zanchin et al. 1997). Positions of 27S, 25S, 20S, and 18S are indicated.
Figure 4.—RNA pol III activity was also decreased in gtr1Δ strain. (a) Equal amounts of RNA samples from Figure 3b were separated with 10% polyacrylamide gels and transferred onto Hybond filters. Radioactivity was detected using a Fuji image analyzer. Relative radioactivity (R.R.) is shown at the bottom with the count of gtr1Δ at 26° assumed to be 100. This experiment was performed twice with similar results. (b) Northern blot analysis of precursor RNAs transcribed by RNA pol III. Total RNA from wild-type and gtr1Δ strains was analyzed for pr-tRNALeu3 or U4 RNA as indicated. R.R. is shown (NBW5, 26° = 100)
Figure 5.—Gel-filtration analysis of Gtr1p in wild-type and gtr1Δ strains. Gtr1p appears at the same location (void) with Rpc19p in addition to the smaller molecular weight fractions in the S300 gel-filtration analysis. Disruption of GTR1 leads to an accumulation of the smaller Rpc19p-containing complex. Accumulation of A135 and A190 in the same fraction as Rpc19p was not observed in the gtr1Δ strain. (Top) HA-tagged Gtr1p was detected with anti-HA antibody. Molecular mass is indicated above by arrows. Yeast strains are indicated at the left: gtr1Δ, NBW5gtr1Δ; gtr1Δ + HA-GTR1, NBW5 gtr1Δ expressing HA-tagged Gtr1p. This experiment was performed twice with similar results.- TABLE 1Genomic variation in the rice RILs based on AFLP analysis
Figure 1.—Examples of genomic variation in the three rice RILs (RZ1, RZ2, and RZ35) as compared with their rice parent (cv. Matsuame) detected by AFLP analysis with primer combinations (from left to right) EcoRI + AAG/MseI + CAA, EcoRI + AAG/MseI + CAG, and EcoRI + AAG/MseI + CAT, respectively. The arrows, arrowheads, and circles respectively refer to parental bands disappeared in one or more RILs, novel bands appeared in one or more RILs, and bands putatively from the donor species Z. latifolia.