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 themajority of the radioactivity of giant HnRNA after a 30 minute pulseof 3H-uridine is associated with molecules in the range 5-10 x 106daltons. In the electron microscope, under denaturing conditions,84% (mass %) of giant HnRNA has a contour length of 4-9µ correspondingto a molecular weight of about 5-10 x 106 daltons.
Giant HnRNA has a "DNA-like" base composition (G+C = 46-54%) andhas considerable secondary structure (ca. 60% helix conformation) asjudged 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 onhydroxyapatite. Single-copy DNA (Cot 1/2 observed = 1.5 x 103)comprises 65% of the genome and 19% of the genome consists of sequencesrepeated an average 1,800 times (middle repetitive DNA, Cot 1/2observed = 1.0). 9% of the genome (highly repetitive DNA)reassociates faster than is measured in these experiments (Cot 1/2observed < 2 x 10-2).
Middle repetitive and single-copy DNA are isolated and characterized with respect to their reassociation kinetics and meltingprofiles. They reassociate with kinetics similar to the kineticsdescribing these components when they are present in totalDNA. The reassociated single-copy DNA has a high thermal stabilityindicative of fidelity of base pairing; the reassociated middlerepetitive DNA has a lower thermal stability which is probablyattributable, in part, to base-pair mismatch.
Rat giant nuclear RNA (HnRNA, 5-10 x 106 daltons) is hybridizedto 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 themiddle repetitive DNA. The Tms of single-copy and middle repetitivehybrids are 1-2° lower than those of the reassociated single-copyand middle repetitive DNA respectively. The DNA isolated from thesingle-copy or middle repetitive hybrids reassociates with kineticssimilar to the input single-copy or middle repetitive DNA respectively.HnRNA is hybridized to total genomic DNA present in excess. 37% of theHnRNA hybridizes with kinetics (Cot 1/2 = 2.0 x 103) similar tosingle-copy DNa and 12% hybridizes with kinetics (Cot 1/2 = 5.6), alittle more slowly than the major reassociating component of middlerepetitive DNA.
A chromatin-associated RNA (cRNA) prepared from rat ascites cellshybridizes to about 16% of isolated middle repetitive and 1% ofisolated single copy rat DNA. In a hybridization reaction to totalDNA, present in excess, at least 50% of the cRNA hybridizes at anaverage rate similar to the major component of the middle repetitiveDNA. These experiments indicate that the majority of cRNA consistsof repetitive transcripts. Under conditions which assay essentiallyonly repetitive transcripts cRNA hybridizes to about 4.7% and giantnuclear RNA (HnRNA) hybridizes to about 4.6% of total nuclear ratDNA immobilized on filters. The Tm of cRNA hybrids (73.5°) and HnRNAhybrids (75.5°) are considerably lower than the Tm of native rat DNA(85.5°). This lowering of Tm is probably attributable, at least inpart, to base-pair mismatch. Under the same conditions of hybridizationthere is some hybridization competition for complementary DNA sitesbetween cRNA and HnRNA, presumably between repetitive transcripts.Due to probable base-pair mismatch it is possible to infer only thatthere is a similarity between HnRNA and cRNA transcripts and notnecessarily an identity.
