Comparative study of nuclear RNA of the liver and experimental hepatoma by the method of DNA-RNA molecular hybridization]

DNA:RNA molecular hybridization of rat liver and hepatoma nyclear RNAs was carried out under controlled conditions as to nucleotide composition and quantitative ratios of competing RNAs and the time of labelling. These factors are shown to influence the results of competition hybridization experiments. For instance a lower competitive ability of rat liver nuclear RNA as compared to that of hepatoma nuclear RNA stems to certain from a relatively higher GC-content of the former. However differences in the competitive efficiency of nuclear RNAs studied could be revealed even with preparations of equal nucleotide composition, these differences being but of quantitative character. The results of the experiments suggest that hepatoma nuclear RNAs are relatively rich in the fast-hybridizable fraction which does not differ qualitatively from the corresponding fraction is characterized by a high metabolic activity and certain tissue specifity.     

The appearance of new polyadenylated nuclear RNA sequences during rat-liver regeneration

Polyadenylated RNA populations from normal and 16-hour regenerating rat-liver nuclei were compared by heterologous hybridisation reactions with cDNA and unique DNA probes. Whereas unique DNA hybridisations did not show differences between the RNA populations, comparisons by cDNA hybridisation showed that about 10--15% by weight of polyadenylated sequences present in the nuclei of 16-hour regenerating rat livers were not found in the polyadenylated nuclear RNA of normal rat livers. These regenerating-specific nuclear cDNA sequences were isolated and characterised; the experiments showed that the complexity of the new sequences was 1-2 x 10(7) nucleotides (equivalent to 5,000--10,000 RNA sequences of 2,000 nucleotides in length) and that they were probably not potential messenger RNA sequences.     

Changes in nuclear and polysomal polyadenylated RNA sequences during rat-liver regeneration.

Nuclear and polysomal polyadenylated RNA populations of normal and 16 hour regenerating rat liver have been compared by mRNA-cDNA hybridisations and by unique DNA saturation experiments. It was found that nuclear polyadenylated RNA hybridises to 6.8% of unique DNA in both normal and 16 hour regenerating rat liver. However, cross-hybridisation experiments using cDNA have shown that 10-15% by weight of nuclear polyadenylated RNA sequences are specific to 16 hour regenerating rat-liver. Since both unique DNA and cDNA hybridisation have shown that normal and 16 hour regenerating rat-liver polysomal polyadenylated RNA populations are qualitatively very similar sequences specific to 16 hour regenerating rat-liver nuclear polyadenylated RNA are nucleus confined. Polysomal RNA sequences which were abundant in normal rat-liver have become less abundant in regenerating rat liver.     

Comparison between chromosomal and nuclear sap RNA from Chironomus tentans salivary gland cells by RNA/DNA hybridization

Newly-synthesized, high molecular weight RNA from salivary gland polytene chromosomes and from the nuclear sap was investigated by RNA/DNA hybridization. Salivary glands were incubated for 90 min with radioactive nucleosides and afterwards fixed. Chromosomes and nuclear sap were subsequently isolated by microdissection. Labelled RNA, extracted from three different chromosomal fractions and from the nuclear sap, was subjected to different hybridization procedures under conditions which primarily allow repeated nucleotide sequences to interact.In one type of experiments RNA was hybridized by a microtechnique to filter-bound DNA at increasing RNA/DNA input ratios. Nuclear sap RNA saturated 0.25−0.30% of the DNA, while the chromosomal RNA fractions had not reached a plateau even after hybridization with 0.5−1% of the DNA. Thus chromosomal RNA appears to contain sequences which are absent from, or present in only low concentration in, the nuclear sap. Nuclear sap RNA hybrids also showed a higher thermal stability than chromosomal RNA hybrids, which may reflect a higher precision of base-pairing in hybrids formed by nuclear sap RNA.In a second type of experiments the time dependence of hybrid formation was investigated. The hybridization rate for nuclear sap RNA was about three times as high as the corresponding rate for chromosomal RNA. This result indicates a relative enrichment of rapidly hybridizing RNA sequences in the nuclear sap.The difference in hybridization properties between chromosomal and nuclear sap RNA may be due to a predominance in the nuclear sap of RNA from a special chromosomal puff, the Balbiani Ring 2 (BR2), which has been shown to contain highly repeated DNA sequences. A comparison between the hybridization properties of nuclear sap RNA and BR2 RNA indicated that 55–70% of nuclear sap RNA may be derived from BR2.The specific hybridization rate of chromosomal RNA points to an average multiplicity of about 30 for its complementary DNA sequences. On the basis of the present and previous results it is suggested that the repeated DNA is arranged in families of related sequences and that sequences belonging to a particular family are distributed in different chromosomes.     

Differences in sequence content of nuclear and cytoplasmic polyribosomal RNA from adenovirus-infected cells.

RNA molecules from nuclear and cytoplasmic polyribosomes of adenovirus-infected HeLa cells were compared by hybridization to analyse the sequence content. Nuclear polyribosomes were released by exposure of intact detergent-washed nuclei to poly(U) and purified. Cytoplasmic polyribosomes were also purified from the same cells. To show that nuclear polyribosomes contain ribosomes linked by mRNA, polyribosomes were labelled with methionine and uridine in the presence of actinomycin D in adenovirus-infected cells. Purified nuclear polyribosomes were treated with EDTA under conditions which dissociate polyribosomes into ribosomes and subunits with a simultaneous release of mRNA, and sedimented. The treatment dissociated these polyribosomes, releasing the mRNA from them. Radiolabelled total RNA from each polyribosome population was fractionated in sucrose gradients into several pools or hybridized to intact adenovirus DNA to select virus-specific RNA. Sucrose-gradient-fractionated pool-3 RNA (about 28S) and virus-specific RNA were then hybridized to fragments of adenovirus DNA cleaved by restriction endonucleases SmaI, HindIII and EcoRI by the Southern-blot technique and by filter hybridization. The results showed that nuclear RNA contained sequences, from about 0 to 18 map units, which were essentially absent from cytoplasmic RNA. Furthermore, the amount of virus-specific RNA for a particular sequence was also different in the two populations.     

Preferential expression of unique sequences adjacent to middle repetitive sequences in mouse cytoplasmic RNA.

Total single-copy DNA and single-copy DNA contiguous to middle repetitive sequences were isolated from mouse brain by successive hydroxylapatite column chromatographies. These DNAs, termed repeat-contiguous single-copy DNA, were found to constitute 48% of the total single-copy DNA. The saturation hybridization values of these two DNA probes to nuclear RNA and cytoplasmic RNA containing polyA of mouse brain and liver were measured. The saturation hybridization levels of total single-copy DNA to brain and liver nuclear RNA were 13.5% and 8.8%, respectively, and those of repeat-contiguous single-copy DNA to the same RNA samples were 13.3% and 8.5%, respectively. On the contrary, the saturation hybridization levels of single-copy DNA to cytoplasmic RNA containing polyA of brain and liver were 3.8% and 2.0%, respectively, and those of repeat-contiguous single-copy DNA to the same RNA samples were 5.8% and 4.0%, respectively. Similar results were obtained with total cytoplasmic RNA. These results indicate that about half the steady state nuclear RNA is transcribed from repeat-contiguous single-copy DNA, and that cytoplasmic RNA containing polyA is mainly derived from repeat-contiguous single-copy DNA.     

A procedure for repeated usage of 33P-labeled DNA probes in hybridization experiments.

We describe a technique for repeated use of 33P-labeled DNA probes in Southern hybridization experiments. A nick-translated 33P-labeled DNA probe in a volume of 0.5-1.0 ml of hybridization mixture (final concentration, 10-100 ng/ml) is used to wet a sheet of filter paper (approx 10 microliters/cm2), which covers a nylon membrane with DNA transferred by Southern blotting, and both are set between two washed X-ray films. The "sandwich" is placed in a plastic bag for hybridization for 16-24 h at 42 degrees C. This very simple procedure using 33P-labeled DNA probes has a number of advantages over the standard method using 32P-labeled probes: (a) a significantly lower biohazard (body/arms exposure); (b) a very small volume of hybridization mixture in contact with a DNA-containing membrane and the higher probe concentrations attainable, causing some increase in sensitivity, and, finally, (c) repeated use of the probe-containing filter (over approx 3 days for unique sequences and up to 2 weeks for reiterated sequences) due to a relatively long 33P half-life (25.3 days).     

Relative stabilities of RNA/DNA hybrids: effect of RNA chain length in competitive hybrization

Escherichia coli DNA and fragmented rRNA were used as a model system to study the effect of RNA fragment size in hybridization-competition experiments. Though no difference in hybridization rates was observed, the relative stabilities of the RNA/DNA hybrids were found to be largely affected by the fragment size of the RNA molecule. Intact rRNA was shown to replace shorter homologous rRNA sequences in their hybrids, the rate of the displacement being dependent on the molecular size of the RNA fragments. Hybridization-competition experiments between molecules of different lengths are expected to be complicated by the displacement reaction. The synthesis of tRNA^Tyr-like sequences transcribed in vitro on φ80psu₃⁺ bacteriophage DNA was measured by hybridization competition assays. Indirect competition with labelled E. coli tRNA^Tyr hybridization revealed that the in vitro-synthesized RNA contained significant amounts of tRNA^Tyr; these sequences could not, however, be detected by the direct competition method in which labelled in vitro-synthesized RNA competes with E. coli tRNA^Tyr for hybridization to φ80psu₃⁺ DNA. These contradictory results can be traced to the differences in size of the competing molecules in the hybridization-competition reaction. Indeed, in vitro-transcribed tRNA^Tyr-like sequences, longer than mature tRNA, were found to displace efficiently E. coli tRNA^Tyr from its hybrids with φ80psu₃⁺ DNA. These findings explain why such sequences could not be detected by direct competition with E. coli tRNA^Tyr.