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Studies on Nuclear RNA


Holmes, David Salway (1973) Studies on Nuclear RNA. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/XDYW-S154.


The isolation of giant nuclear RNA (HnRNA) from rat ascites cells is described. By the criteria of sedimentation through sucrose, formaldehyde and dimethyl sulfoxide, it is estimated that the majority of the radioactivity of giant HnRNA after a 30 minute pulse of 3H-uridine is associated with molecules in the range 5-10 x 106 daltons. In the electron microscope, under denaturing conditions, 84% (mass %) of giant HnRNA has a contour length of 4-9µ corresponding to a molecular weight of about 5-10 x 106 daltons.

Giant HnRNA has a "DNA-like" base composition (G+C = 46-54%) and has considerable secondary structure (ca. 60% helix conformation) as judged by its melting profile and reactivity with formaldehyde.

Rat nuclear DNA is characterized by its reassociation profile ((Na+) = 0.18 at 62°, Tm - 23°) as judged by chromatography on hydroxyapatite. Single-copy DNA (Cot 1/2 observed = 1.5 x 103) comprises 65% of the genome and 19% of the genome consists of sequences repeated an average 1,800 times (middle repetitive DNA, Cot 1/2 observed = 1.0). 9% of the genome (highly repetitive DNA) reassociates faster than is measured in these experiments (Cot 1/2 observed < 2 x 10-2).

Middle repetitive and single-copy DNA are isolated and characterized with respect to their reassociation kinetics and melting profiles. They reassociate with kinetics similar to the kinetics describing these components when they are present in total DNA. The reassociated single-copy DNA has a high thermal stability indicative of fidelity of base pairing; the reassociated middle repetitive DNA has a lower thermal stability which is probably attributable, in part, to base-pair mismatch.

Rat giant nuclear RNA (HnRNA, 5-10 x 106 daltons) is hybridized to isolated single copy or middle repetitive DNA ((Na+) = 0.18 at 62°) HnRNA hydbridizes to about 4.5% of the single-copy and 9.4% of the middle repetitive DNA. The Tms of single-copy and middle repetitive hybrids are 1-2° lower than those of the reassociated single-copy and middle repetitive DNA respectively. The DNA isolated from the single-copy or middle repetitive hybrids reassociates with kinetics similar to the input single-copy or middle repetitive DNA respectively. HnRNA is hybridized to total genomic DNA present in excess. 37% of the HnRNA hybridizes with kinetics (Cot 1/2 = 2.0 x 103) similar to single-copy DNa and 12% hybridizes with kinetics (Cot 1/2 = 5.6), a little more slowly than the major reassociating component of middle repetitive DNA.

A chromatin-associated RNA (cRNA) prepared from rat ascites cells hybridizes to about 16% of isolated middle repetitive and 1% of isolated single copy rat DNA. In a hybridization reaction to total DNA, present in excess, at least 50% of the cRNA hybridizes at an average rate similar to the major component of the middle repetitive DNA. These experiments indicate that the majority of cRNA consists of repetitive transcripts. Under conditions which assay essentially only repetitive transcripts cRNA hybridizes to about 4.7% and giant nuclear RNA (HnRNA) hybridizes to about 4.6% of total nuclear rat DNA immobilized on filters. The Tm of cRNA hybrids (73.5°) and HnRNA hybrids (75.5°) are considerably lower than the Tm of native rat DNA (85.5°). This lowering of Tm is probably attributable, at least in part, to base-pair mismatch. Under the same conditions of hybridization there is some hybridization competition for complementary DNA sites between cRNA and HnRNA, presumably between repetitive transcripts. Due to probable base-pair mismatch it is possible to infer only that there is a similarity between HnRNA and cRNA transcripts and not necessarily an identity.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Biology; Biochemistry
Degree Grantor:California Institute of Technology
Major Option:Biology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Bonner, James Frederick
Thesis Committee:
  • Davidson, Norman R.
  • Attardi, Giuseppe
  • Russell, Richard L.
  • Davidson, Eric H.
Defense Date:2 April 1973
Funding AgencyGrant Number
Record Number:CaltechTHESIS:07192018-114123511
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11126
Deposited By: Benjamin Perez
Deposited On:19 Jul 2018 20:53
Last Modified:21 Dec 2019 03:06

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