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Ribozymes are polynucleotide molecules with intrinsic catalytic activity, capable of cleaving nucleic …


Biology Articles » Biophysics » Medical Biophysics » Radiation target analysis of RNA » Figures

Figures
- Radiation target analysis of RNA

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FIG. 1. Schematic of ribozyme and substrate structure, plasmid construction and transcription, and ribozyme specific cleavage. (a) Murine IRBP mRNA substrate oligonucleotide sequence and synthesized target ribozyme. Only 30 nt of the murine IRBP mRNAsubstrate are shown. Total length of the substrate fragment was 314 nt. The active ribozyme structure is shown. Both long and short ribozyme transcripts contained the active ribozyme sequence. The asterisk refers to the site of substrate cleavage by the ribozyme. (b) Plasmid restriction digestion and in vitro transcription of ribozymes and IRBP mRNA substrate. Oligonucleotide templates were directionally cloned into pBluescript II KS(+) as described. Plasmids containing the desired sequence were restricted with either PvuII (262-nt ribozyme and 314-nt IRBP mRNA substrate) or Scal (1226-nt ribozyme). The linearized plasmid had a T3 RNA polymerase promoter upstream of the transcription site. This plasmid was then transcribed as described. T3, T3 RNA polymerase transcription initiation site; RZM, ribozyme; MCS, polycloning site used for insertion of ribozyme and substrate templates. The numbers seen on the plasmid construct represent the distance from the transcription start site. (c) Cleavage patterns predicted for murine IRBP substrate by either the 262 nt (small) or 1226 nt (large) ribozyme. Ribozymes cleave the IRBP mRNA substra into new fragments of 100 nt and 214 nt. Thes'e fragments appear asbands on PAGE after incubation with active ribozyme moieties.

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FIG. 2. (a) Photograph of an ethidium bromide-stained denaturing 10% polyacrylamide gel of the 262-nt ribozyme exposed to increasing doses of radiation (0-48 Mrads). Equal amounts of irradiated RNA were loaded onto 10% denaturing polyacrylamide gel as described. Bands were analyzed by densitometry as described. The size of the transcript was 262 nt as determined by comparison with known standards. Mrad, dose of radiation (in megarads). (b) Autoradiograph of denaturing 10% polyacrylamide gel loaded with equal amounts of reaction mixture [33P-labeled substrate plus irradiated small (262 nt) ribozyme (10:1)]. The same amount of ribozyme irradiated with 0-120 Mrads was used in each assay. Electrophoresis was for 17 hr at 90 V. Arrows show the substrate (314 nt) and digestion products (214 nt and 100 nt). S, 33P-labeled substrate alone. Gels were analyzed as described in Materials and Methods. (c) Photograph of ethidium-stained denaturing 10% polyacrylamide gel with equal loadings of the large (1226 nt) ribozyme following irradiation. Electrophoresis was for 17 hr at 100 V. Gels were analyzed by densitometry as described in Materials and Methods. (d) Autoradiograph of denaturing 10% polyacrylamide loaded with equal amounts of reaction mixture [33P-labeled substrate plus irradiated large (1226 nt) ribozyme (10:1)]. The same amount of ribozyme irradiated with 0-120 Mrads was used in each assay. Electrophoresis was for 17 hr at 90 V. Arrows refer to the substrate (314 nt) and digestion products (214 nt and 100 nt). Gels were analyzed as described in Materials and Methods.

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FIG. 3. Inactivation of 262-nt ribozyme by high energy electrons. After radiation exposure at - 135°C, samples were thawed and assayed for enzymatic activity () and also for surviving intact molecules () as described. Data are from four or five independent experiments. Error bars represent the SD of the average fraction surviving after exposure to comparable doses of radiation.

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FIG. 4. Experiments similar to those shown in Fig. 3, except that the large (1226 nt) ribozyme was used. Surviving activity () and structure () are shown. Data are from three independent experiments. Error bars are as in Fig. 3.

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