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.     

Discontinuous replication of colicin E1 plasmid DNA in a cell extract containing thermolabile DNA ligase.

A cell extract prepared from the lig-ts7 mutant of Escherichia coli is able to carry out a complete round of DNA replication of colicin E1 plasmid at 25 °C. However, the apparent rate of elongation of the progeny strands at this temperature is much smaller than in an extract from the thermoresistant revertant cells. Chain elongation in the lig-ts extract is depressed by raising the incubation temperature from 25 °C to 32 °C, whereas that in the lig⁺ revertant extract is not. The rate of closure of the progeny strands of newly formed open circular molecules is also reduced in the lig-ts extract, even at 25 °C.The DNA pulse-labelled with the lig-ts extract for 30 seconds at 32 °C contains a large amount of short DNA fragments of approximately 7 S, in addition to DNA chains of various sizes between 7 S and 17 S (unit length). Most of these replicating molecules are converted to completely replicated closed circular molecules upon chasing with a lig⁺ extract. DNA-DNA hybridization experiments show that molecules replicated to various extents contain 7 S DNA fragments of both strands, but more of the L-strand component, whose 5′-to-3′ direction corresponds to the overall direction of unidirectional replication. The longer DNA chains are enriched in the H-strand component.The cell extracts used for the plasmid DNA replication have an activity which converts alkali-labile closed circular plasmid DNA containing apurinic sites to alkali-stable closed circular molecules. Addition of nicotinamide mononucleotide leads to conversion of the alkali-labile DNA to open circular molecules. In the replication system with the cell extract, however, the compound does not interfere with elongation of progeny strands. Chain elongation in the lig-ts extract at 25 °C is not significantly affected by nicotinamide mononucleotide. Thus, the 7 S DNA fragments formed with the lig-ts extract are unlikely to be generated as a result of incomplete repair of misincorporated nucleotides. We conclude that both strands of colicin E1 plasmid DNA replicate discontinuously.     

Dual labeling in standard DNA-RNA hybridization studies using 125 I-labeled nuclear RNA and 3H-labeled DNA.

Standard DNA-RNA hybridization studies, using nucleic acids isolated from mammalian tissues, are frequently hindered by relatively low levels of radioactivity in pulse-labeled RNA and in an inability to reliably estimate the amount of DNA present in the hybrid. In the method described here nuclear RNA is labeled in vitro with 125I to 400 000- 800 000 cpm/mug and DNA is obtained from a rat glial tumor line grown in culture and labeled to specific activities of 42 000-79 000 cpm/mug. DNA-RNA hybridization is conducted in an all solution system at RNA:DNA ratios of 3.5:1 to 18:1. Assay background is controlled by pretreatment of the hybrid and free RNA at the conclusion of the annealing study with RNase, then isolation of the hybrid together with a small fraction of free RNA oligonucleotides on hydroxyapatite. The partially purified hybrids are then trapped on Millipore filters. Assay background id 0.004% of total counts present in the annealing reaction. Comparison of the annealing reactions of pulse-labeled liver nuclear RNA and in vitro 125I-labeled nuclear RNA in saturation, kinetic, and competitive hybridization studies shows them to be essentially the same. Nuclear RNA labeled by either tritium or iodine shows a 10-20-fold greater concentration of the annealing sequences over that found in the microsomal RNA. Minor differences are noted between the nuclear RNAs in the initial rates of reaction and in the magnitude of the decrease in percent hybridization at low levels of unlabeled competitor RNA. This may be due to preferential labeling in pulse-labeled RNA of molecules which are present in lower concentrations or are transcribed from more frequently repeated DNA sequences than the average population of annealing RNA molecules. The technique has application in systems where the amount of tissue for RNA extraction is small or where the system does not permit the obtaining of pulse-labeled RNA, as in experimental rodent skin carcinogenesis or in dealing with RNA from the tissues of large mammals or humans.     

Quantitative in situ hybridization of ribosomal RNA species to polytene chromosomes of Drosophila melanogaster.

In situ hybridization of ¹²⁵I-labelled 5 S and 18 + 28 S ribosomal RNAs to the salivary polytene chromosomes of Drosophila melanogaster was successfully quantitated. Although the precision of the data is low, it is possible to compare the hybridization reaction between an RNA sample and chromosomes in situ with the reaction between the same RNA sample and Drosophila DNA immobilized on nitrocellulose filters. The in situ hybrid dissociates over a narrow temperature range with a midpoint similar to the value expected for the filter hybrid. The kinetics of the in situ hybridization reaction can be fit with a single first-order rate constant that has a value from three to five times smaller than the corresponding filter hybridization reaction. Although the reaction saturates at longer times or higher RNA concentrations, the saturation value does not correspond to an RNA molecule bound to every available DNA sequence. With the acid denaturation procedure most commonly used to preserve cytological quality, only 5 to 10% of the complementary DNA in the chromosomes is available to form hybrids in situ. This hybridization efficiency is a function of how the slides are prepared and the conditions of annealing, but is approximately constant with a given procedure for both 5 S RNA and 18 + 28 S RNA over a number of different cell types with different DNA contents. The results provide further evidence that the formation of RNA-DNA hybrids is the sole basis of in situ hybridization, and show that the properties of the in situ hybrids are remarkably similar to those of filter hybrids. It is also suggested that for reliable chromosomal localization using the in situ hybridization technique, the kinetics of the reaction should be followed to ensure that the correct rate constant is obtained for the major RNA species in the sample and an impurity in the sample is not localized instead.     

New Method of Studying the Precursor-product Relationship between High Molecular Weight RNA and Messenger RNA

THREE principal methods have been used to test whether giant heterogeneous RNA is a precursor to cytoplasmic messenger RNA in animal cells. First, competition-hybridization to DNA has shown that there is certainly a degree of similarity between the two types of molecule^1–5. The conditions used in most of these experiments allowed only the hybridization of the reiterated fraction of DNA or RNA. It is possible therefore that the competing sequences are similar, but not identical and in any case may represent only a small fraction of the total sequences. Scherrer et al.⁶, using conditions which might allow the hybridization of some of the slow sequences of DNA and which would give meaningful competition, conclude that there is a precursor-product relationship between giant heterogeneous RNA and cytoplasmic messenger RNA. Darnell, following the uptake of labelled nucleotides into the two fractions⁷, has shown that a precursor product relationship is possible although not proven. Third, the differential inhibition of synthesis of the two fractions by cordycepin⁸ has been taken to show that the giant heterogeneous RNA cannot be a precursor to the cytoplasmic messenger RNAs. Although these data would confirm previous observations on the rate of uptake and decay of label in nucleoplasmic and cytoplasmic RNA in the presence of actinomycin⁹, this type of experiment is rather indirect and open to other interpretations because the inhibitors might not act exclusively and directly on one step of the overall metabolic pathway. A relationship has been clearly established between high molecular weight precursors and the final RNA produced only for SV40 transformed cells¹⁰. In this case the hybridization experiments were much simplified because SV40 DNA, which is very small in comparison with animal DNA, is readily available. In this communication we describe a direct and general way of approaching the problem which makes novel use of molecular hybridization and can be applied to messenger RNAs which are not easily labelled. The method also overcomes the difficulty of hybridizing to the extremely complex DNA of higher organisms.     

Optimal conditions for supercoil DNA sequencing with the Escherichia coli DNA polymerase I large fragment

Sequencing ladders produced from supercoiled DNA templates with the Escherichia coli DNA polymerase Klenow fragment are often unreadable because of a high background and misincorporated nucleotides. This study showed that contaminating RNA molecules can interfere with template: primer hybridization. Procedures are provided for the purification of template DNA and stringent conditions for primer-template hybridization that overcome these problems.     

Replication of polyoma DNA in isolated nuclei. VII. Initiator RNA synthesis during nucleotide depletion.

Earlier experiments demonstrated that the Okazaki fragments synthesized during discontinuous polyoma DNA synthesis in isolated nuclei at their 5′ ends contained structural elements consisting of polyribonucleotides starting with ATP or GTP (Reichard et al., 1974). These structures could be released by digestion with pancreatic DNAase and were named initiator RNA. They consist of a large family of polyribonucleotides differing in base sequence but having a common size of about a decanucleotide. We now demonstrate that limitation of DNA synthesis by low concentrations of deoxyribonucleoside triphosphates in parallel limits the synthesis of initiator RNA. This is additional evidence for the primer function of initiator RNA. When ribonucleoside triphosphates other than ATP were deleted from the incubation medium only a small decrease of DNA and initiator RNA synthesis occurred. Under those conditions deoxyribonucleotides substituted for ribonucleotides and were incorporated internally into the primer. From this result as well as the insensitivity of initiator RNA synthesis to α-amanitin (Reichard &; Eliasson, 1979) we suggest that a mammalian counterpart to primase, the dnaG gene product of Escherichia coli(Rowen &; Kornberg, 1978a), catalyzes the synthesis of initiator RNA.     

Fine structure of ribosomal RNA: I. Conservation of homologous regions within ribosomal RNA of eukaryotes

By molecular hybridization experiments the homologies between ribosomal RNAs from a unicellular organism (Gyrodinium cohnii), three invertebrates (Drosophila hydei, Chironomus thummi, Sciara coprophila), an amphibian (Xenopus laevis), and a mammal (mouse) were determined. Competition hybridization experiments demonstrated that portions of these homologous regions are the same in all the ribosomal RNAs tested, regardless of animal species. This conclusion based on hybridization data was confirmed by comparative fingerprint analysis. The ribosomal RNA sequences involved in heterologous hybridization have a higher A + T composition than the bulk ribosomal RNA. It appears from competition experiments of a heterologous hybridization that two thirds of the conserved similar regions are present in 18 S ribosomal RNA, and the remaining one third in 28 S ribosomal RNA. It is argued that these similar regions have been conserved during evolution due to their structural and/or functional role in ribosomal RNA.     

Hybridization properties of nucleolar ribonucleic acid.

Rat liver nuclei were fractionated into chromatin and nucleolar fractions. Chromatin DNA, which does not form hybrids with rRNA, was, nevertheless, able to hybridize with 32P-labelled total nucleolar RNA. The optimal temperature for this hybridization was 55 degrees C when the reaction was carried out in 2 X SSC (0.3 MnaCl + 0.3 M-sodium citrate). The hybrids formed were specific, as judged by analysis of thermal elution profiles. The low Tm (73 degreesC) observed could be explained by the low amount of DNA in the filters. The lenth of the hybridized sequences was extimated as 54 mucleotide pairs. Contamination to nucleolar RNA by nucleoplasmic RNA was ruled out by showing the former was able to form more hybrids than the latter. Competition experiments showed that hybridization of nucleolar RNA, although not competed with by rRNA, suffered pronounced competition from total microsomal RNA, even though the levels of competition obtained did not equal thsoe with cold nucleolar RNA as competitor.     

Non-parallel kinetics and the role of tissue-specific factors in the secretion of chicken ovalbumin and lysozyme from Xenopus oocytes

Ovalbumin and lysozyme made in Xenopus oocytes under the direction of injected chicken oviduct messenger RNA accumulate at different rates in the surrounding culture medium. Pulse-chase experiments confirm that the intrinsic rate of lysozyme secretion from oocytes is 12 times that of ovalbumin. This slower rate of ovalbumin export is maintained following injection of either diluted oviduct RNA or purified ovalbumin messenger, the latter having been obtained by hybridization to cloned ovalbumin complementary DNA. These results suggest that the differential rates of transport observed in oocytes are not the consequence of competition for amphibian or avian factors and show that oviduct-specific proteins are not required for ovalbumin secretion.     

Comparison of an Avian Osteopetrosis Virus with an Avian Lymphomatosis Virus by RNA-DNA Hybridization

Myeloblastosis-associated virus (MAV)-2(0), a virus which was derived from avian myeloblastosis virus and induced a high incidence of osteopetrosis, was compared with avian lymphomatosis virus 5938, a recent field isolate which induced a high incidence of lymphomatosis. The following information was obtained. (i) MAV-2(0) induced osteopetrosis, nephroblastoma, and a very low incidence of hepatocellular carcinoma. No difference was seen in the oncogenic spectrum of end point and plaque-purified MAV-2(0). (ii) ¹²⁵I-labeled RNA sequences from MAV-2(0) formed hybrids with DNA extracted from osteopetrotic bone at a rate suggesting five proviral copies per haploid cell genome. The extent of hybridization of MAV-2(0) RNA with DNA from osteopetrotic tissue was more extensive (87%) than was observed in reactions with DNA from uninfected chicken embryos (52%). (iii) Competition of unlabeled viral RNA in hybridization reactions between the radioactive RNA from the two viruses and their respective proviral sequences present in tumor tissues showed that 15 to 20% of the viral sequences detected in these reactions were unshared. In contrast, no differences were detected in competition analyses of RNA sequences from the two viruses detected in DNA of normal chicken cells. (iv) MAV-2(0) 35S RNA was indistinguishable in size from avian lymphomatosis virus 5938 35S RNA by polyacrylamide gel electrophoresis.     

Secondary structure maps of ribosomal RNA and DNA: I. Processing of Xenopus laevis ribosomal RNA and structure of single-stranded ribosomal DNA

Precursor and mature ribosomal RNA molecules from Xenopus laevis were examined by electron microscopy. A reproducible arrangement of hairpin loops was observed in these molecules. Maps based on this secondary structure were used to determine the arrangement of sequences in precursor RNA molecules and to identify the position of mature rRNAs within the precursors. A processing scheme was derived in which the 40 S rRNA is cleaved to 38 S RNA, which then yields 34 S plus 18 S RNA. The 34 S RNA is processed to 30 S, and finally to 28 S rRNA. The pathway is analogous to that of L-cell rRNA but differs from HeLa rRNA in that no 20 S rRNA intermediate was found. X. laevis 40 S rRNA (M_r = 2.7 × 10⁶) is much smaller than HeLa or L-cell 45 8 rRNA (M_r = 4.7 × 10⁶), but the arrangement of mature rRNA sequences in all precursors is very similar. Experiments with ascites cell 3′-exonuclease show that the 28 S region is located at or close to the 5′-end of the 40 S rRNA.Secondary structure maps were obtained also for single-stranded molecules of ribosomal DNA. The region in the DNA coding for the 40 S rRNA could be identified by its regular structure, which closely resembles that of the RNA. Regions corresponding to the 40 S RNA gene alternate with non-transcribed spacer regions along strands of rDNA. The latter have a large amount of irregular secondary structure and vary in length between different repeating units. A detailed map of the rDNA repeating unit was derived from these experiments.Optical melting studies are presented, showing that rRNAs with a high (G + C) content exhibit significant hypochromicity in the formamide/urea-containing solution that was used for spreading.     

Comparative Study of Ribosomal Ribonucleic Acid Cistrons in Enterobacteria and Myxobacteria

Deoxyribonucleic acid (DNA)-ribonucleic acid (RNA) hybrids are formed by Escherichia coli 16S or 23S ribosomal RNA or pulse-labeled RNA with the DNA of various species of the Enterobacteriaceae. The relative extent of hybrid formation is always greater for ribosomal RNA. These DNA-RNA hybrids have been further characterized by their stability to increasing temperature, and, in every case, the stability of pulse-labeled RNA hybrids was lower than that of the corresponding ribosomal RNA hybrids, although 16S and 23S ribosomal RNA hybrids had very similar stabilities. Therefore, ribosomal RNA showed a greater degree of apparent conservation in base sequence than pulse-labeled or messenger RNA both in the extent of cross-reaction and in the stability of hybrid structures. Similar results were obtained with Myxococcus xanthus RNA. Since in this case the base composition of the pulse-labeled or messenger RNA is richer in guanine plus cytosine than ribosomal RNA, the higher cross-reaction of ribosomal RNA is more readily attributable to conservation of base sequence in these cistrons than to its base composition. Thus, the base sequence of ribosomal RNA cistrons of bacilli, enteric bacteria, and myxobacteria is conserved relative to those of the rest of the genomes. This conservation is, however, not absolute since the stability of heterologous ribosomal RNA hybrids is always lower than that of homologous hybrids.     

Template recognition and chain elongation in DNA synthesis in vitro.

DNA polymerase from Escherichia coli (Pol I) and from avian myeloblastosis virus (AMV polymerase) were compared for the manner in which they catalyze the polymerization of deoxynucleotides upon a variety of synthetic and natural templates. It was found that the rates of nucleotide incorporation with different natural RNAs were similar. Both polymerases have an associated RNA endonuclease which hydrolyses RNA templates containing double-stranded regions. This activity depends on the presence of the complementary deoxynucleoside triphosphates, and/or polymerization. Both enzymes copy natural DNA, which has been sonicated and treated with E. coli exonuclease III, at the same rate. However, avian myeloblastosis virus DNA polymerase, which has no associated DNA exonuclease activity, is unable to copy double-stranded DNA and copies DNAase-treated DNA only 10% as well as Pol I. Pol I copied all the homopolymers investigated at a greater rate than AMV polymerase with the exception of poly(C) · oligo(dG). However, the initial rate of chain elongation, as measured by gel electrophoresis, was the same for the two polymerases, approximately 300 nucleotides incorporated per minute. Template saturation experiments show a stoichiometric relationship between template and enzyme at optimal rates of nucleotide incorporation which suggests that all enzyme molecules are potential catalysts. Enzyme saturation experiments indicate that not all enzyme molecules are “effectively” bound to a template. Fewer AMV polymerase than Pol I molecules are functionally bound to a particular template. From these data, it is concluded that the two polymerases elongate DNA chains in a similar way and that the manner in which the polymerases bind to a particular template accounts for the discrepancies found in their turnover numbers.     

Coding of stable 4S RNA molecules by the resistance factor R1

Summary With the aid of DNA-RNA hybridization experiments it was shown that an E. coli strain harbouring the resistance factor R1 contains small, stable RNA species which are capable of annealing with the DNA of this R-factor. At a higher hybridization temperature RNA molecules from the isogenic, sensitive strain could also hybridize to the R1-DNA. These RNA-species were again detected if the RNA was extracted from P. mirabilis carrying this plasmid. The fact that sulfur-labelled RNA also hybridized to the plasmid DNA seems to indicate that some of these molecules are tRNAs.This work is in part a fulfillment of the requirements for a doctoral thesis of Franz Kricek