Studies on Nuclear RNA

Citation

Holmes, David Salway (1973) Studies on Nuclear RNA. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/XDYW-S154. https://resolver.caltech.edu/CaltechTHESIS:07192018-114123511

Abstract

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°, T_m - 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 T_ms 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 T_m of cRNA hybrids (73.5°) and HnRNA hybrids (75.5°) are considerably lower than the T_m of native rat DNA (85.5°). This lowering of T_m 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.

Thesis Files