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.