Please use this identifier to cite or link to this item:

Ribozyme-mediated cleavage of a synthetic tat gene transcript

File Description SizeFormat 
uhm_phd_9429609_r.pdfVersion for non-UH users. Copying/Printing is not permitted3.74 MBAdobe PDFView/Open
uhm_phd_9429609_uh.pdfVersion for UH users3.71 MBAdobe PDFView/Open

Item Summary

Title: Ribozyme-mediated cleavage of a synthetic tat gene transcript
Authors: Carlos, Ruben
Issue Date: 1994
Abstract: HIV-1 has at least two genes, tat and rev, encoded by overlapping reading frames, involved in up-regulating HIV-1 transcription and replication. Tat encodes a nuclear protein that is a transcriptional trans-activator and rev encodes a protein regulating RNA splicing. The tat/rev RNA transcripts were examined as a target for cleavage by ribozymes, a possible means for "intracellular immunization" to treat my disease. Ribozymes are RNA molecules that cleave other RNA molecules at specific motif sequences using a conserved sequence which folds into a catalytic secondary structure and targeting sequences, which form duplexes with the sequences flanking the cleavable motif in the targeted RNA molecules. Ribozymes RZ1, RZ2, RZ310, RZ412, and RZ514 were designed to cleave the tat-s transcript. Both hammerhead ribozymes, derived from the (+)-strand satellite RNA of tobacco ringspot virus and a hairpin ribozyme, derived from the (-j-strand satellite RNA of tobacco ringspot virus were tested. These ribozymes do not cleave control actin RNA transcripts which possess sixteen cleavage motif sequences but lack flanking complementary ribozyme recognition sequences. The ribozymes cleave at the targeted site of the tat-s RNA in experiments performed under near physiological pH and temperature. Substrate cleavage rates show a dependence on both ribozyme and substrate concentrations suggesting that a bimolecular reaction with second order kinetics is the rate limiting reaction. Rot calculations of expected reactions rates at the lowest ribozyme concentrations indicate a t1/2 reaction of ribozyme with substrate of twenty-one minutes, suggesting that the bimolecular reaction should be completed quickly and not be the rate limiting step of substrate cleavage. It is proposed that the second order reaction rate is due to the continued dissociation and reassociation of the ribozyme:substrate complex before substrate cleavage. For perspective, the number of molecules required in a cell to achieve ribozyme concentrations performed in these studies were determined to be about 268 molecules/cell. From this cellular perspective, initial rate reaction experiments predict about 200 tat-s molecules/hour are cleaved. This rate may be sufficient to cleave the relatively low amounts of tat message in a quiescent HIV-infected cell.
Description: Thesis (Ph. D.)--University of Hawaii at Manoa, 1994.
Includes bibliographical references (l. 116-126)
xiii, 126 leaves, bound illus. 29 cm
Rights: All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections:Ph.D. - Biomedical Sciences (Biochemistry)

Please contact if you need this content in an ADA compliant alternative format.

Items in ScholarSpace are protected by copyright, with all rights reserved, unless otherwise indicated.