CHARACTERIZATION OF HIDDEN BREAKS IN THE HIGH MOLECULAR WEIGHT RIBOSOMAL-RNA MOLECULES OF THE CILIATE, TETRAHYMENA PYRIFORMIS AND THE DIPTERAN, RHYNCHOSCIARA HOLLAENDERI.
Item
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Title
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CHARACTERIZATION OF HIDDEN BREAKS IN THE HIGH MOLECULAR WEIGHT RIBOSOMAL-RNA MOLECULES OF THE CILIATE, TETRAHYMENA PYRIFORMIS AND THE DIPTERAN, RHYNCHOSCIARA HOLLAENDERI.
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Identifier
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AAI8112762
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identifier
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8112762
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Creator
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RUBIN, NORMAN ARNOLD.
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Contributor
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Ronald A. Eckhardt
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Date
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1980
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, General
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Abstract
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The selective heat lability of Tetrahymena and Rhynchosciara HrRNA was shown be due to the presence of a hidden break in the polynucleotide chain. In vivo and under favorable conditions in vitro the two halves of the molecules remain together but can be separated by agents that disrupt secondary structure.;The kinetis of the in vitro dissociation of the HrRNA molecules were determined by estimating the percent dissociation of samples fractionated by polyacrylamide gel electrophoresis after incubation in 0.10 M sodium acetate buffer, pH 5.0 which also contained 10('-4) M EDTA and either 0 M, 0.05 M or 0.10 M NaCl at 0(DEGREES)C, 25(DEGREES)C, 37(DEGREES)C or 50(DEGREES)C for varying lengths of time. Both Tetrahymena and Rhynchosciara HrRNA remain intact for at least twentyfour hours at 0(DEGREES)C even in the low salt buffer. At 25(DEGREES)C inclusion of 0.05 M or 0.10 M NaCl is sufficient to stabilize the Rhynchosciara HrRNA for one hour but approximately 20% of the Tetrahymena HrRNA molecules dissociated in the buffer containing 0.05 M NaCl. Dissociation was complete for both Tetrahymena and Rhynchosciara within one hour of incubation in buffer lacking NaCl. Complete dissociation also occurred within five minutes of incubation at 37(DEGREES)C in the buffer lacking NaCl while 75% of the molecules dissociated in the buffer containing 0.05 M NaCl. Inclusion of 0.10 M NaCl was sufficient to stabilize the molecules for at least five minutes. At 50(DEGREES)C dissociation was complete within one minute even in the high salt buffer. These results are consistent with but not necessarily proof of the hypothesis that the secondary structure of the HrRNA molecules in the vicinity of the hidden break has been evolutionarily conserved.;The sedimentation coefficients of Tetrahymena ribosomal RNAs were determined to be 26S and 17S and of Rhynchosciara 27S and 18S. The molecular weights, which were estimated by polyacrylamide gel electrophoresis, were 1.32 and 0.70 x 10('6) for the Tetrahymenar RNAs and 1.43 and 0.70 x 10('6) daltons for the Rhynchosciara rRNAs. These values are typical for a protozoan and a dipteran respectively. Heating or incubation in 80% DMSO caused the dissociation of the Tetrahymena HrRNA into fragments of 0.69 and 0.63 x 10('6) and the Rhynchosciara HrRNA into fragments of 0.75 and 0.68 x 10('6) daltons. The reproducibility of these results is indicative of a precise location for the hidden break. However, the limit of resolution of the gel system used dos not allow for the localization of the hidden break beyond approximately (+OR-)130 nucleotides. Note that the sum of the molecular weights of the fragments is equal to the molecular weight of the HrRNA from which they were derived but a more sensitive technique will be required to determine if the hidden break is actually a gap and hence correponds to the site of removal of an intervening sequence.;The appearance of a reproducible pattern of fragments of 0.52, 0.47, 0.39 and 0.20 was dependent on the isolation procedure used. This verifies Ishikawa's hypothesis that these 'secondary fragments' are artifacts.;Although these hidden breaks are present in most animal species, its physiological function, if any, remains unknown.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biology
