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Studies of the Mechanism and Products of Transcription of the Nuclear Genome in Animal Cells


Murphy, William Ignatius, III (1974) Studies of the Mechanism and Products of Transcription of the Nuclear Genome in Animal Cells. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/r002-ng85.


Part I

Chapter 1. The metabolic stability of pulse labeled or long-term labeled mRNA from cytoplasmic free polysomes has been measured in HeLa cells, using chase conditions which do not involve inhibitors of RNA synthesis, and chromatography on benzoylated-DEAE-cellulose or poly(T)-cellulose for the isolation of mRNA. For these studies, a new chase technique has been developed which allows the analysis of the stability of mRNA labeled during a short 3H-uridine pulse. Pulse labeled and long-term labeled mRNA have been found to decay with an estimated average half-life of about 2 and 3 days, respectively, much longer than hitherto assumed.

Chapter 2. Polyadenylated messenger RNA extracted from HeLa cells was hybridized with a mass excess of HeLa DNA. The kinetics of the hybridization reaction demonstrated that most of the mRNA is transcribed from nonrepetitive DNA. The amount of mRNA hybridized to DNA was measured both with and without prior RNase treatment. Comparison of the results indicates that within the limits of detection, HeLa mRNA does not contain repetitive sequence elements covalently linked to nonrepetitive sequence transcripts. However, a small fraction of the HeLa mRNA preparation is transcribed entirely from repetitive DNA sequences. This fraction represents about 6% of the total polyadenylated mRNA preparation.

Chapter 3. The sedimentation properties of pulse-labeled and longterm labeled mRNA from HeLa cell free-polysomes, selected for poly(A) content by two successive passages through poly(T)-cellulose columns, was analyzed under native and denatured conditions. The sedimentation profile of the mRNA on both sodium dodecyl SO4-sucrose gradients and formaldehyde-sucrose gradients showed a broad distribution of components with estimated molecular weights ranging from 2 x 105 to 5.5 x 106 daltons and a weight-average molecular weight of 8.5 x 105 daltons.

Part II

The >50 S HnRNA, isolated from either HeLa cells or irranature duck erythrocytes labeled for different times with [5-3H]uridine, was examined for the presence of complementary transcripts capable of forming RNase-resistant duplexes. After extensive self-annealing of the HnRNA, carried out under conditions such that complementary RNA sequences present once or a few times in the RNA population would have formed hybrids, no evidence was found for the existence of symmetrical transcripts in either cell system. However, 2-3% and 4-5% of the purified duck and HeLa HnRNA, respectively, did form RNase-resistant hybrids. These hybrids resulted from base-pairing of complementary regions within the HnRNA molecule, as judged from the lack of concentration dependence and from the kinetics of formation of the RNA-RNA duplexes. The weight-average length of the RNase-resistant fragments from the duck HnRNA was found to be approximately 125 nucleotide pairs; however, shorter double-stranded segments as well as longer duplexes, up to 2000 nucleotide pairs, were also observed. Annealing of the duck HnRNA in the presence of an excess of 10 S hemoglobin mRNA showed that 2% of the HnRNA formed RNase-resistant hybrids in excess of those expected from intramolecular homology. The RNase-resistant complexes formed between the 10 S mRNA and HnRNA had about the same size range as the intramolecular duplexes.

The failure to detect any intermolecular hybridization in the short pulse labeled HnRNA from either actively growing cells or highly differentiated, non-dividing cells, strongly suggests that the mechanism for the synthesis of HnRNA in animal cells does not involve the production of high molecular weight complementary transcripts.

Part III

This report describes the use of purified rDNA to map by electron microscopy the relative position of the 18 S and 28 S RNA regions within the duck rRNA precursor and their relationship to the nonconserved portions of the precursor molecule. In the first part, the purification from duck erythrocytes of rDNA sequences suitable for use in the electron microscopic mapping of the rRNA precursor is discussed. By repeated fractionation of the total DNA, based on the relative reassociation rates of the DNA sequences with different degrees of repetition, a fraction of the rapidly renaturing DNA was obtained which comprised only 6% of the total DNA, but contained 71% of the rRNA cistrons. Further purification of the rDNA was achieved by saturation hybridization with rRNA and separation of the rRNA-rDNA hybrids by banding in CsCl. In this manner, an rDNA-rRNA fraction was obtained which had a buoyant density of 1.805 gm/cm3, an RNA to DNA ratio of 1.01, and a base composition for the RNA present in the hybrid identical to that of an equimolar mixture of 18 sand 28 S rRNA. The final yield of rDNA isolated by this procedure is 32%. When the purified rDNA was annealed with a mixture of 18 sand 28 S rRNA and the hybrids spread for electron microscopy, they appeared as two distinct populations with a number-average length of 0.62±0.13 μm and 1.37±0.18 μm, respectively. Likewise, hybrids between the rRNA precursor, isolated from duck embryo fibroblasts, and the rDNA appeared as structures containing two duplex regions of lengths 0.60± 0.11 μm and 1.38±0.15 μm, separated from each other by a single-stranded region appearing as a large bush: this represents the portion of the precursor molecule not conserved during processing of the parent molecule. From these observations a model of the structure of the avian rRNA precursor is proposed.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Biochemistry and Chemistry
Degree Grantor:California Institute of Technology
Major Option:Biochemistry
Minor Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Attardi, Giuseppe (advisor)
  • Owen, Ray David (co-advisor)
Thesis Committee:
  • Unknown, Unknown
Defense Date:3 April 1974
Funding AgencyGrant Number
U.S. Public Health ServiceUNSPECIFIED
Record Number:CaltechTHESIS:06042021-220829673
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:14244
Deposited By: Benjamin Perez
Deposited On:07 Jun 2021 19:13
Last Modified:07 Jun 2021 19:14

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