Fidelity of ribosomal ribonucleic acid synthesis by nucleoli and nucleolar chromatin.

Isolated nucleoli, nucleolar chromatin, and nucleolar DNA were used as templates for DNA synthesis in appropriately supplemented systems in which RNA polymerases other than RNA polymerase I were blocked by alpha-amanitin. With the aid of nucleotide analysis, DNA-RNA hybridization, and homochromatography fingerprinting, it was found that isolated nucleoli and nucleolar chromatin serve primarily as templates for synthesis of rRNA. However, the products formed with purified nucleolar DNA as a template do not contain the specific rRNA oligonucleotides nor are they appreciably hybridized to the rDNA region on cesium chloride gradients. These results indicate that whole nucleoli and nucleolar chromatin contain control mechanisms that restrict readouts by RNA polymerase I of nucleolar DNA to rDNA.     

Quantitative micro-assay for RNA/DNA hybrids in the study of nucleolar RNA from Chironomus tentans salivary gland cells

A microassay for RNA/DNA hybrids has been designed for the study of RNA from different nuclear components of Chironomus tentans salivary gland cells. The procedure comprises a scale reduction of the conventional filter method for hybridization, using ultraviolet microphotometry for quantitation of RNA and DNA. Hybridization is performed in 0.3 μl of 2 × SSC containing 1–2 × 10^-2 μg DNA, immobilized on a 0.2 mm² ‘micro-filter’, and 0.5–5 × 10⁻² μg RNA, with a specific activity of more than 10⁶ cpm/μg. Results obtained by the microtechnique are found to agree with results obtained by a large-scale, standard procedure. The applicability of the microtechnique is demonstrated in saturation and presaturation-competition experiments. RNA from micro-isolated nucleoli hybridizes a maximum of 0.22% of Chironomus tentans DNA, which corresponds to about 100 cistrons for the 38S ribosomal precursor in the haploid genome. The hybrids show a steep thermal dissociation profile with a T_m of 79 °C, close to the value expected for hybrids with a G + C content of 42%. Presaturation of filter-bound DNA by total unlabelled nucleolar RNA prevents 80% of the subsequent hybridization by labeled nucleolar Presaturation by RNA from one of the two nucleolar organizers prevents to a similar degree the subsequent hybridization by RNA from the other nucleolar organizer. This result indicates a sequence similarity of RNA transcribed in different nucleolar organizers. Further applications of the microtechnique are presented in the accompanying paper where the hybridization properties of chromosomal and nuclear sap RNA are investigated.     

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.     

The number and location of genes for 5S ribonucleic acid within the genome of Drosophila melanogaster

DNA was prepared from wild-type and two mutant stocks of Drosophila melanogaster that differed in their dosage of the nucleolar organizer region. The relative amounts of DNA from the nucleolar organizer region in these preparations of DNA were determined by hybridization with (3)H-labelled 28S rRNA. As expected, the amount of (3)H-labelled 28S rRNA that hybridized was directly related to the dosage of nucleolar organizer region. No positive correlation was observed between the amount of (3)H-labelled 5S RNA that hybridized and the dosage of nucleolar organizer region. Thus genes for 5S RNA are located primarily, if not exclusively, outside the nucleolar organizer region. The haploid genome of the wild-type D. melanogaster used in this work has 106 genes for 28S rRNA and 96-105 genes for 5S RNA.     

Ribosomal RNA Cistron Multiplicity and Nucleolar Organizers in Hexaploid Wheat

The ribosomal DNA (DNA complementary to ribosomal RNA) content of twelve different wheat genotypes has been studied. Some of these genotypes are aneuploids with deletions or additions of chromosomes bearing nucleolar organisers. The rDNA contents of these genotypes provide several examples of a clear departure from a correlation between the number of rRNA cistrons and the number of nucleolar organisers. Thus the number of rRNA cistrons per nucleolar organiser is not constant in wheat. Wheat DNA was found to have a mean buoyant density of approximately 1.702 g/cc for all genotypes studied and rRNA hybridized selectively to DNA of buoyant density approximately 1.710 g/cc. The thermal stabilities of all the rRNA/DNA hybrids were essentially identical.     

Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix.

Mouse L-cell nucleoli were isolated from sonicated nuclei by centrifugation and extensively treated with pancreatic DNase or micrococcal nuclease to obtain "core nucleoli." Core nucleoli still contained the precursors to rRNA and about 1% of the total nuclear DNA, which remained tightly bound even after the removal of some chromatin proteins with 2 M NaCl. The core nucleolar DNA electrophoresed in a series of discrete bands, 20 to about 200 base pairs in length. Hybridization tests with specific DNA probes showed that the DNA was devoid of sequences complementary to mouse satellite, mouse Alu-like, and 5S RNA sequences. It also lacked sequences coding for cytoplasmic rRNA species, since it did not hybridize to the 18S to 28S portion of rDNA in Northern blot analyses and none of it was protected by hybridization to a 100-fold excess of total cytoplasmic RNA in S1 nuclease assays. However, the core nucleolar DNA did hybridize to nontranscribed and external transcribed spacer rDNA sequences. We infer that specific portions of rDNA are protected from DNase action by a tight association with nucleolar structural proteins.     

An analysis of the ribosomal ribonucleic acids of Escherichia coli by hybridization techniques

From analyses of the hybridization of Escherichia coli rRNA (ribosomal RNA) to homologous denatured DNA, the following conclusions were drawn. (1) When a fixed amount of DNA was hybridized with increasing amounts of RNA, only 0.35+/-0.02% of E. coli DNA was capable of binding (16s+23s) rRNA. Although preparations of 16s and 23s rRNA were virtually free from cross-contamination, the hybridization curves for purified 16s or 23s rRNA were almost identical with that of the parent specimen containing 1 weight unit of 16s rRNA mixed with 2 weight units of 23s rRNA. The 16s and 23s rRNA also competed effectively for the same specific DNA sites. It appears that these RNA species each possess all hybridizing species typical of the parent (16s+23s) rRNA specimen, though probably in different relative amounts. (2) By using hybridization-efficiency analysis of DNA-RNA hybridization curves (Avery & Midgley, 1969) it was found that (a) 0.45% of the DNA would hybridize total rRNA and (b) when so little RNA was added to unit weight of DNA that the DNA sites were not saturated, only 70-75% of the input RNA would form hybrids. The reasons for the discrepancy between the results obtained by the two alternative analytical approaches were discussed. (3) For either 16s or 23s rRNA, hybridization analysis indicated that two principal weight fractions of rRNA may exist, hybridizing to two distinct groups of DNA sites. However, these groups seem to be incompletely divided between the 16s and 23s fractions. Analysis suggested that (a) 85% of the 16s rRNA was hybridized to about half the DNA that specifically binds rRNA (0.23% of the total DNA). (b) 70% of the 23s rRNA hybridized to a further 0.23% of the DNA and (c) the minor fraction (15%) of 16s rRNA may be competitive with the major fraction (70%) of 23s rRNA. Conversely, the minor fraction (30%) of the 23s rRNA may compete with the major fraction (85%) of 16s rRNA. Models were proposed to explain the apparent lack of segregation of distinct RNA species in the two subfractions of rRNA. (4) If protein synthesis and ribosome maturation were inhibited in cells of an RC(rel) mutant, E. coli W 1665, by depriving them of an amino acid (methionine) essential for growth, the inhibition had no discernible effect on the relative rates of synthesis of rRNA species. The rRNA that accumulates in RC(rel) strains of E. coli after amino acid deprivation is apparently identical in its content of RNA species with that of the pre-existing mature RNA in the ribosomes. On the other hand, the messenger RNA is stabilized, and accumulates as about 15% of the RNA formed after withdrawal of the amino acid.     

Use of a DNA probe for mapping by electron microscopy the ribosomal sequences in ribosomal RNA precursors from duck cells

This report describes the use of purified ribosomal DNA to map by electron microscopy the relative positions of the 18 S and 28 S RNA regions within the duck rRNA precursor and their relationship to the non-conserved portions of the precursor molecule. By repeated fractionation of the total DNA, based on the relative reassociation rates of the DNA sequences with different degrees of repetition, a fraction of the rapidly renaturing DNA was obtained which comprised only 6% of the total DNA, but contained 71% of the rRNA cistrons. Further purification of the rDNA was achieved by saturation hybridization with rRNA and separation of the rRNA-rDNA hybrids by banding in CsCl. In this manner, an rDNA-rRNA fraction was obtained which had a buoyant density of 1.805 g/cm³, an RNA to DNA ratio of 1.01, and a base composition for the RNA present in the hybrid identical to that of an equimolar mixture of 18 S and 28 S rRNA. The final yield of rDNA isolated by this procedure is 32%. When the purified rDNA was annealed with a mixture of 18 S and 28 S rRNA and the hybrids spread for electron microscopy, they appeared as two distinct populations with a number-average length of 0.62 ± 0.13 μm and 1.37 ± 0.18 μm, respectively. Likewise, hybrids between the rRNA precursor, isolated from duck embryo fibroblasts, and the rDNA appeared as structures containing two duplex regions of lengths 0.60 ± 0.11 μm and 1.38 ± 0.15 μm, separated from each other by a single-stranded region appearing as a large bush: this represents a portion of the precursor molecule not conserved during processing of the parent molecule. From these observations a model of the structure of the duck rRNA precursor is proposed.     

Conserved portion in bacterial ribosomal RNA

The conserved portion in bacterial ribosomal RNA was studied by the DNA-RNA hybridization method. The hybridization percentages were as follows: Bacillus subtilis DNA and B. subtilis 23S rRNA, 0.16; Escherichia coli DNA and E. coli 23S rRNA, 0.15; B. subtilis DNA and E. coli 23S rRNA, 0.03; E. coli DNA and B. subtilis 23S rRNA, 0.04. The RNA's extracted from the heterologous hybrids could be rehybridized with DNA's of B. subtilis and E. coli. The average chain lengths of the RNA's were estimated by sucrose density gradient centrifugation and Sephadex gel filtration. The results suggested that the size might be larger than 30 nucleotides. Nucleotide compositions of the RNA's in the hybrids were also studied. Both RNA's contained higher molar percentages of guanylic acid and cytidylic acid than the whole rRNA's.     

Microbial identification by immunohybridization assay of artificial RNA labels

Ribosomal RNA (rRNA) and engineered stable artificial RNAs (aRNAs) are frequently used to monitor bacteria in complex ecosystems. In this work, we describe a solid-phase immunocapture hybridization assay that can be used with low molecular weight RNA targets. A biotinylated DNA probe is efficiently hybridized in solution with the target RNA, and the DNA-RNA hybrids are captured on streptavidin-coated plates and quantified using a DNA-RNA heteroduplex-specific antibody conjugated to alkaline phosphatase. The assay was shown to be specific for both 5S rRNA and low molecular weight (LMW) artificial RNAs and highly sensitive, allowing detection of as little as 5.2 ng (0.15 pmol) in the case of 5S rRNA. Target RNAs were readily detected even in the presence of excess nontarget RNA. Detection using DNA probes as small as 17 bases targeting a repetitive artificial RNA sequence in an engineered RNA was more efficient than the detection of a unique sequence.     

Human glutamate tRNA forms stable hybrids in vitro with 28S ribosomal RNA.

A human glutamate tRNA has been shown to form stable hybrids with 28S ribosomal RNA. This tRNA was purified from HeLa cell cytoplasmic RNA by RNA-RNA solution hybridization followed by the isolation of tRNA-28S rRNA complexes by hybridization-selection with ribosomal DNA or by recovery of the 28S peak from formamide-sucrose gradients. The single hybridizing tRNA species was identified as tRNAGluCUC by sequencing: pU-C-C-C-U-G-G-U-G-m2G-U-C-phi-A-G-U-G-G-D-phi-A-G-G-A-U-U- C-G-G-C-G-C-U-C-U-C-A-C-C-G-C-G-G-C-m5C-m5C-G-G-G-Tm-phi-C-G-A- U-U-C-C-C-G-G-U-C-A-G-G-G-A-A-C-C-AOH. Computer analysis located a nucleotide sequence near the middle of human 28S rRNA which is complementary to 15-26 nucleotides between residues 20 and 50 of this tRNA. An interaction between this tRNA and 28S rRNA suggests that tRNAGluCUC may have functions in the cell in addition to translation.     

Isolated HeLa cell nuclei synthesize meaningful DNA.

DNA replicated at the beginning of S phase was labelled by incubating nuclei isolated from cells arrested at the G/S border with radioactive deoxyribonucleoside triphosphate in a reaction mixture sustaining DNA synthesis. By hybridization against ribosomal RNA bound to nitrocellulose, the fraction of the labelled DNA which was complementary to rRNA could be quantified, and the stability of the RNA-DNA hybrids could be estimated by sequential elution of DNA at increasing temperatures. The results obtained indicate that the isolated nuclei make "meaningful" DNA, as judged by the melting characteristics of the hybrids between rRNA and the in vitro replicated DNA. Hybridization of the labelled DNA against rRNA fractionated by electrophoresis and blotted onto nitrocellulose verified the presence of sequences complementary to 18 S and 28 S rRNA.