Oligonucleotide templates coding for the (+) and (-) strands of both the ribozyme and IRBP mRNA substrate fragment with appropriate restriction sites were synthesized on an Applied Biosystems model 395A DNA synthesizer (Fig. la). The strands were annealed, restriction digested, and directionally cloned into pBluescript KS II(+) plasmid vector (Fig. lb). The constructs were transfected into supercompetent cells (X-L-1 blue). Candidate clones were isolated and sequenced. A plasmid clone with the correct insert sequence for the ribozyme targeted to the 58th nucleotide from the 5' end of the murine IRBP mRNA was purified and digested with either PvuII or ScaI to yield linearized DNA templates coding for RNA transcripts of either 262 or 1226 nt in length when transcribed with T3 RNA polymerase (Boehringer Mannheim; Fig. lb). A candidate clone encoding the first 104 bp of the IRBP mRNA template was purified and linearized with PvuII, and then transcribed with T3 RNA polymerase in the presence of [33P]UTP (DuPont/NEN) to yield a labeled RNA substrate sequence 314 nt in length (Fig. lb). RNA transcription reactions were then digested sequentially with DNase I and Proteinase K (Life Technologies, Gaithersburg, MD), extracted with phenol and chloroform, and precipitated. Following in vitro transcription and extraction, ribozymes were resuspended in Tris HCl buffer (pH 8.0) at 3 mg/ml and subfrozen.
This concentration was chosen because of the enhanced radiation sensitivity of proteins at very low concentrations (
Ribozyme Densitometric Analysis. Following irradiation, 15 ,ug ribozyme samples were added to formamide to 75% vol/vol, heated to 70°C for 2 min and snap-cooled on ice. RNA loading buffer (1 ,l) was added and RNA samples, along with appropriate RNA size standards, were electrophoresed on 6-10% polyacrylamide/25% formamide, 6 M urea, lx TBE gels. The gels were rinsed in RNase-free water, stained with ethidium bromide, and photographed. RNA concentrations in the bands were determined by densitometric analysis of ethidium-stained RNA bands as well as by autoradiography and 3-scanning of 33P-labeled control and irradiated ribozyme samples.
Radiation Target Analysis. The measured parameters in irradiated samples were analyzed as P = Po e-kD, where Po is the value in the unirradiated sample and P is the measured property in samples exposed to a dose D in Mrads (1). When the data are plotted as an inactivation curve (In (P/Po) versus D), a straight line is obtained. A least-squares fit yields the slope, k. The radiation target mass (in kDa) is calculated from m = 1792 k.
Answers to all your Biology Questions
