Columns for rapid chromatographic separation of small amounts of tracer-labeled transfer ribonucleic acids

The essential Gcd10p–Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA

“…Northern blots of total RNA (10 µg) isolated from the strains at 26°C (0 hr at 36°C) or 36°C (4, 8, 12 hr at 36°C) with (+) or without (−) thiolutin treatment were probed with a labeled oligonucleotide that specifically hybridized to both pre-tRNA i Met and mature tRNA i Met , as described in Fig. 1 tRNAs were chromatographed on a RPC-5 column (Kelmers and Heatherly 1971). It was shown previously that this technique can resolve tRNAs that differ by only a single methyl group (Diamond et al 1993).…”

A regulatory role for Sec tRNA[Ser]Sec in selenoprotein synthesis

“…Total tRNA from approximately 1 g of CHO cells was deacylated, aminoacylated with [ 3 H]serine, which labels both serine and Sec tRNAs, and chromatographed on an RPC-5 column (Kelmers and Heatherly 1971) as described previously (Moustafa et al 1998;Carlson and Hatfield 2002). The aminoacylated tRNA was chromatographed twice on the RPC-5 column, first in the absence and then presence of Mg 2+ , as described (Hatfield et al 1979).…”

Methylation of the ribosyl moiety at position 34 of selenocysteine tRNA[Ser]Sec is governed by both primary and tertiary structure

“…Oocytes were obtained from mature Xenopus females+ Stage VI oocytes were selected and maintained in OR-2 medium (82+5 mM NaCl, 2+5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM NaHCO 3 , 5 mM HEPES, pH 7+8) at room temperature+ Forty nanoliters of the mixture containing 2-3 ϫ 10 5 cpm of 32 P-labeled tRNAs (prepared as described above) were injected into the cytoplasm of oocytes and the oocytes incubated at 20 8C for 16 h+ After incubation, tRNAs were extracted as described (Tobian et al+, 1985;Lee et al+, 1987), loaded on a RPC-5 column (Kelmers & Heatherly, 1971), and the column developed in a 0+525-0+70 M NaCl linear gradient as described (Diamond et al+, 1993;Choi et al+, 1994)+ Column fractions were counted in a liquid scintillation counter (Pharmacia), the peaks representing isoacceptors pooled, precipitated in ethanol, collected, and prepared for nuclease digestion as described (Choi et al+, 1994)+ …”