Radioactive RNA Research Articles

RNA synthesis and stability in UV-irradiated and nonirradiated mouse L cells.

In mouse L cells, relatively low doses of UV light (e.g., about 35 J/m2) induced the rapid breakdown of the molecules of many RNA species transcribed shortly before irradiation. This included 28S, 18S, 5.8S, and 5S rRNA, U1, U2, U3, U4, and U5 small nuclear RNA, but not the main band of transfer RNAs or 7SL RNA. At higher UV doses, an RNA band that contains tRNAleu was also degraded rapidly after UV irradiation. RNA molecules synthesized long before irradiation (e.g., 22 h for small RNAs, 4 h for large rRNAs) were not affected. Our results suggest that the maturation and/or assembly into fully mature ribonucleoprotein particles of several small RNA species is not completed 4 h after transcription. The effect of UV radiation occurred in mouse L cells, but not in human HeLa or KB cells. In a previous report, L cells were transformed by DNA transfection with two mouse U1b RNA genes, named U1.1 and U1.2. We observed now that, in L cells transformed with the U1.2 gene, the ratio of radioactivity in the apparent U1b and U1a RNA precursors after 5 min of labeling was about 20 times higher than a) this ratio in briefly labeled L cells that had been transformed with the U1.1 gene, and b) the ratio of radioactive mature U1b and U1a RNA after 20 h of chase in L cells transformed with the U1.2 gene. These results suggest that very high levels of U1b RNA are transcribed from the exogenous U1.2 gene copies, followed by the rapid degradation of most of these transcripts.

Pathology

Transfer of molecular genetic technology and knowledge from the research to the diagnostic laboratory is giving the pathologist new, powerful tools to diagnose and monitor disease. The most immediate application is the use of nucleic acid probes to detect viral genomic material in cytological smears or microscopic sections by in situ hybridization.¹Hybridization either with a radioactive recombinant DNA or RNA for autoradiography or with a nonradioactively labeled nucleic acid to produce a visible product now can be performed with commercially available reagents. This identifies genomic sequences of adenoviruses, BK and JC viruses (human polyomavirus), cytomegalovirus, herpes simplex virus-1 and -2, Epstein-Barr virus, human immunodeficiency virus, human T-cell lymphotropic virus type I, hepatitis A and B viruses, and human papillomaviruses. Unfortunately, in situ hybridization is still relatively insensitive since 100 to 200 genomic copies or more are needed in a single cell for detection so that negative or equivocal

The direct measurement of ribosomal RNA gene activity in wheat–rye hybrids

Cloned rDNA segments, from wheat (Triticum aestivum) and rye (Secale cereale), which included the entire spacer region plus parts of the flanking 18S and 26S rRNA genes, were used to assay transcripts formed in the nuclei from wheat–rye hybrids. The tetraploid triticales (× Triticosecale Wittmack) carrying a complete rye chromosome complement and a combination of wheat chromosomes, as well as wheat and rye per se, were studied. Nuclei isolated from young leaves were incubated in the presence of [32P]UTP for 10 min, after which the total RNA was isolated. Hybridization of this radioactive RNA to the cloned rDNA segments showed that the suppression of rye NOR activity, measured cytologically, can be directly related to a major reduction in rRNA synthesis.Key words: NOR suppression, RNA transcripts, isolated nuclei.

Subcellular localization of tobacco mosaic virus minus strand RNA in infected protoplasts

Radioactive RNA probes were prepared which specifically hybridize with sequences complementary to 5′ and 3′ regions of tobacco mosaic virus (TMV) RNA. These probes were used in Northern hybridization to locate TMV-RNA minus strands in the subcellular fractions of infected tobacco protoplasts. When the protoplasts were lysed with Triton X-100, full-length minus strands were present in the cytoplasmic but not in the nuclear fraction. With mechanically broken protoplasts, the crude nuclear fraction (250 g pellet) contained small amount of minus strands which appeared to derive from unbroken protoplasts, but most of minus strands were recovered in a fraction sedimented between 250 and 2500 g, little if any being found in lighter fractions. The results indicate that TMV-RNA replicates in association with an extranuclear structure.

CDNA heterogeneity suggests structural variants related to the high-affinity IgE receptor.

The high-affinity IgE receptor present on mast cells and basophils is responsible for the IgE-mediated activation of these cells. The current model for this receptor depicts a four-subunit structure, alpha beta gamma 2. A cDNA for the alpha subunit was recently cloned and predicts a structure consisting of two homologous extracellular domains, a transmembrane segment, and a cytoplasmic tail. Using a synthetic oligonucleotide corresponding to the amino-terminal sequence of the alpha subunit, we identified a number of cDNA clones from a rat basophilic leukemia cell cDNA library. Nucleotide sequencing established four different forms of cDNA: one is nearly identical to the published cDNA; the second differs from the first in the 5' untranslated sequence; the other two forms use either one or the other of the 5'-end sequences as above and lack 163 base pairs in the region coding for the second extracellular domain. RNase protection analysis with radioactive RNA probes established the heterogeneity of rat basophilic leukemia cell mRNA with regard to both the 5' and the internal sequences. Our results suggest the existence of at least four different protein forms related to the alpha subunit of the high-affinity IgE receptor.

Axoplasmic RNA species synthesized in the isolated squid giant axon

Isolated squid stellate nerves and giant fiber lobes were incubated for 8 hr in Millipore filtered sea water containing [3H]uridine. The electrophoretic patterns of radioactive RNA purified from the axoplasm of the giant axon and from the giant fiber lobe (cell bodies of the giant axon) demonstrated the presence of RNA species with mobilities corresponding to tRNA and rRNA. The presence of labeled rRNAs was confirmed by the behavior of the large rRNA component (31S) which, in the squid, readily dissociates into its two constituent moyeties (17S and 20S). Comparable results were obtained with the axonal sheath and the stellate nerve. In all the electrophoretic patterns, additional species of radioactive RNA migrated between the 4S and the 20S markers, i.e. with mobilities corresponding to presumptive mRNAs. Chromatographic analysis of the purified RNAs on oligo(dT)cellulose indicated the presence of labeled poly(A)+ RNA in all tissue samples. Radioactive poly(A)+ RNA represented approximately 1% of the total labeled RNA in the axoplasm, axonal sheath and stellate nerve, but more than 2% in the giant fiber lobe. The labeled poly(A)+ RNAs of the giant fibre lobe showed a prevalence of larger species in comparison to the axonal sheath and stellate nerve. In conclusion, the axoplasmic RNAs synthesized by the isolated squid giant axon appear to include all the major classes of axoplasmic RNAs, that is rRNA, tRNA and mRNA.

Development and characterisation of a line of bread wheat, Triticum aestivum, which lacks the short-arm satellite of chromosome 1B and the Gli-B1 locus

About 360 offspring of a tri-parental cross were screened by gel electrophoresis and unexpectedly one of them did not contain chromosome 1B ω-gliadins derived from either of the primary parents. A line disomic for the ω-gliadin null was developed from the surviving embryo half of the unique grain. Two dimensional electrophoresis revealed that all the storage protein genes at Gli-B1, coding for γ-gliadins, β-gliadins and low-molecular-weight subunits of glutenin as well as the ω-gliadin, were not expressed. The nuclei of dividing root-tip cells were shown by light microscopy to lack the normal short-arm satellites of chromosome 1B, indicating that the genes for the missing storage proteins had been lost through a terminal deletion. Using a radioactive ribosomal RNA probe, the deficient 1B chromosomes were shown to contain ribosomal RNA genes demonstrating that at least two-thirds of the short arm was still present. Examination of serial sections of chromosome 1B at metaphase by low-power electron microscopy showed that the point of scission of this chromosome was within the secondary constriction where the ribosomal RNA genes are located. The Gli-B1 locus must therefore be carried on the short-arm satellite. Transmission of the deficient chromosome from female gametes to progeny was normal (i.e., about 50%) but from pollen it was poor (8.8%). Recombination mapping indicated that the distance from the ribosomal RNA genes (Nor1) to Glu-B1 was 22 cM, equivalent to 13 cM from Nor1 to the centromere.

Screening recombinant phage M13 plaques with RNA probes; a one-step procedure which identifies clones containing either of the complementary DNA strands

We describe a method for detecting specific DNA sequences cloned in M13 phage vectors, based on the procedure of Woo (in Wu, R., Methods in Enzymology, Vol. 68, Academic Press, New York, 1979, pp. 389–395). M13 plaques are adsorbed to a nitrocellulose filter that has been pre-saturated with bacteria. The filter is incubated on an agar plate to amplify the phage; the DNA is alkali-denatured and then hybridized with a radioactive RNA probe. Unlike standard procedures, this method detects and distinguishes M13 plaques containing phage particles which harbor either the coding or non-coding (RNA-like) DNA strand, when single-stranded RNA is used as probe. We have optimized this procedure with M13 clones containing mouse histidine tRNA gene sequences and have used it to determine the sequence of both strands of a mouse glycine tRNA gene.

Incorporation of 3H-amino acids into proteins in a partially purified fraction of axoplasm: evidence for transfer RNA-mediated, post- translational protein modification in squid giant axons

Transfer RNA (tRNA) has been demonstrated to be present in axons of both invertebrates and the higher vertebrates, but nothing is known of its role in the metabolism of the axon. The present experiments were performed to determine whether tRNA functions in axons as a participant in post-translational protein modification of endogenous proteins. RNA was extracted from the axoplasm of squid giant axons and incubated with a variety of 3H-amino acids, aminoacyl-tRNA synthetases (obtained from squid optic lobe), and an appropriate reaction mixture. All of the amino acids tested were bound to an RNA fraction, but this reaction did not occur when samples were incubated in the presence of ribonuclease or in the absence of axoplasmic RNA. When radioactive RNA was chromatographed by polyacrylamide gel electrophoresis, the radioactivity comigrated with known tRNA markers, suggesting the presence of 3H-aminoacylated tRNA. Aminoacylation of RNA could also be demonstrated by incubating fresh axoplasm with labeled amino acids and a reaction mixture, minus exogenous aminoacyl-tRNA synthetases. These findings indicate the presence in axoplasm of a variety of species of aminoacyl-tRNAs as well as their corresponding synthetase enzymes. In the latter experiment no radioactivity was found associated with the protein fraction. This was also the finding when 3H-aminoacylated tRNA was either injected directly into the axon or incubated with extruded axoplasm. Thus, under the conditions described above, there is no evidence of transfer of amino acids from tRNA to proteins. In other experiments, axoplasm was pooled to a volume of 50 to 100 microliters, homogenized gently, and centrifuged at 150,000 X g for 1 hr. Some of the high speed supernatant was incubated with labeled amino acids and an appropriate reaction mixture, and the remainder was passed through an S-200 Sephacryl column before incubation with the same reaction mixture. There was no incorporation of amino acids into protein in the high speed supernatant fraction. However, in the S-200 purified fraction 3H-labeled Arg, Lys, Tyr, Leu, and Asp were all incorporated into proteins in amounts of 44, 30, 7, 5 and 3.5 times heat-inactivated controls. The reaction is not inhibited by Ca2+ or Ca2+-activated proteases, but appears to be dependent on the presence of tRNA. The addition of amino acids to protein is not protein synthesis since the reactions occurred in a partially purified fraction of the 150,000 X g supernatant, a fraction devoid of ribosomes and free amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

Origin of axoplasmic RNA in the squid giant fiber.

The origin of axoplasmic RNA in the squid giant fiber was investigated after exposure of the giant axon or of the giant fiber lobe to [3H]uridine. The occurrence of a local process of synthesis was indicated by the accumulation of labeled axoplasmic RNA in isolated axons incubated with the radioactive precursor. Similar results were obtained in vivo after injection of [3H]uridine near the stellate nerve at a sizable distance from the ganglion. Exposure of the giant fiber lobe to [3H]uridine under in vivo and in vitro conditions was followed by the appearance of labeled RNA in the axoplasm and in the axonal sheath. While the latter process is attributed to incorporation of precursor by sheath cells, a sizable fraction of the radioactive RNA accumulating in the axoplasmic is likely to originate from neuronal perikarya by a process of axonal transport.

Characterization of Viral-complementary RNA Associated with Polyribosomes from Tobacco Infected with Sonchus Yellow Net Virus

SUMMARY Polyribosomal RNA from tobacco infected with sonchus yellow net virus was resolved by electrophoresis on agarose gels, transferred to nitrocellulose and hybridized to radioactive viral RNA. Polyadenylated RNA contained four major viral complementary species, molecular weights 2.2 × 106, 0.83 × 106, 0.62 × 106 and 0.46 × 106, plus a minor species, molecular weight approx. 3 × 106. No such species could be detected in the non-polyadenylated RNA fraction. The polyadenylated RNA from membrane-bound polyribosomes contained only the 0.83 × 106 and 0.46 × 106 molecular weight RNAs. The sizes of the RNAs were consistent with the expected sizes of messages for the virus proteins, calculated from the molecular weight of these proteins.

Evidence of transcription from the late region of the integrated simian virus 40 genome in transformed cells: location of the 5' ends of late transcripts in cells abortively infected and in cells transformed by simian virus 40.

By means of S1 mapping, we observed that spliced 16S and 19S viral late mRNAs--in addition to early mRNAs--were present in cytoplasmic polyadenylated RNA preparations from simian virus 40-transformed cell lines of rat or mouse origin containing no detectable amount of free viral DNA. The amounts of early and late virus-specific mRNAs in these lines were quantified by hybridization of radioactive cytoplasmic polyadenylated RNA with cloned region-specific restriction fragments. The relative amount of late viral mRNA produced in these transformed cells was found to be of the same order as that produced in simian virus 40-infected, nonpermissive baby mouse kidney cells. Moreover, by using the S1 nuclease protection method, we compared the 5' ends of late mRNAs produced (i) in transformed cells, (ii) in abortively infected mouse cells, and (iii) in the late phase of the lytic cycle. The 5' ends of late mRNAs both in abortively infected and in transformed cells were less heterogeneous than the 5' ends of late mRNAs produced during the lytic cycle; however, they were a subset of the 5' ends of late transcripts produced in the lytic cycle.

Transcriptional control of the fibronectin gene in chick embryo fibroblasts transformed by Rous sarcoma virus.

Rous sarcoma virus transformed-chick embryo fibroblasts contain decreased amounts of the adhesive protein fibronectin that results from a lowering of fibronectin mRNA levels (Fagan, J.B., Sobel, M.E., Yamada, K.M., de Crombrugghe, B., and Pastan, I. (1981) J. Biol. Chem. 256, 520-525). In the present study, nuclei isolated from chick embryo fibroblasts (CEF) and Rous sarcoma virus transformed-chick embryo fibroblasts (RSV-CEF) were used to measure relative rates of fibronectin RNA synthesis. [32P]RNA was synthesized by preinitiated endogenous RNA polymerases in nuclei, and the radioactive RNA was subsequently hybridized to DNA fragments spanning approximately 40 kilobase pairs (approximately 85%) of the fibronectin gene. Transformation of CEF by RSV resulted in a 7- to 8-fold reduction in the amount of 32P-labeled nuclear RNA hybridizing with fibronectin DNA sequences when the nuclei were incubated for either 3.5 or 45 min. These results indicate that decreased transcription and not reduced RNA stability plays a major role in lowering fibronectin mRNA levels in RSV-CEF. When nuclear RNA was transcribed in the presence of Sarkosyl, diminished RNA synthesis was still observed suggesting that reduced fibronectin transcription in RSV-CEF is not due to a block in RNA chain elongation. Our data indicate that transcriptional regulation of the fibronectin gene is an important mechanism of lowering steady state levels of fibronectin mRNA in RSV-CEF.

18] Hybridization of denatured RNA transferred or dotted to nitrocellulose paper

The technique of E.M. Southern for transferring electrophoretically separated DNA fragments to nitrocellulose paper for subsequent hybridization with radioactive RNA or DNA probes has proved to be a powerful tool for the analysis of DNA. Until recently, the only comparable procedure for RNA has been to transfer and covalently couple it to activated cellulose paper (diazobenzyloxymethyl paper, DBM paper) according to the method of Alwine et al . Although DBM paper does offer the advantage of covalent attachment, it has several drawbacks as discussed in this chapter. The chapter also describes a related method for dotting RNA onto nitrocellulose pretreated with high salt. Thus, all the advantages that nitrocellulose blotting previously afforded for DNA analysis are now available for RNA analysis as well.

Chromatin structure in situ, as visualized in thin sections selectively stained for DNA

The ultrastructural localization of [3H]uridine-labelled RNA synthesized in the course of the cell cycle of synchronized CHO cells is studied using high resolution autoradiography combined with a differential staining for nucleoproteins. It is shown that sites of RNA transcription can already be visualized on the periphery of chromosomes of apparently late metaphase-early anaphase cells with no visible association with the reforming nuclear membrane. In interphase cells they are associated with the border of intranucleolar chromatin or condensed nucleoplasmic chromatin, wherever this localization is made possible by the degree of chromatin dispersion. In cells labeled for 1 or 3 h, the rate of RNA synthesis is higher in S and G2 than in G1. When cells fixed immediately after a [3H]uridine pulse are compared with those post-incubated for 13 h in isotope-free medium, there is a clear difference in intensity of labelling between the nucleus and the cytoplasm. However, the localization pattern of radioactive RNA in the nucleus is similar for all incubation periods as well as for all phases of interphase. The groups of interchromatin granules are generally labeled weakly with radioactivity associated rather with the periphery of their clusters, or remain unlabelled. These results are discussed in the context of other recent findings concerning the distribution of RNA and RNP-structures in the nucleus.

Nucleolus organizer regions in Drosophila species of the repleta group

The locations of nucleolus organizer regions in the genomes of D. neohydei, D. eohydei and D. repleta were studied by in situ hybridization. All three species carry one nucleolus organizer in the X heterochromatin. The Y chromosomes of D. neohydei and D. eohydei carry two nucleolus organizers in terminal positions comparable to those of D. hydei. The Y chromosome of D. repleta appears also to carry two nucleolus organizer regions, but this is difficult to establish because of the small size of this chromosome. In situ hybridization of ribosomal RNA with salivary gland chromosomes revealed the presence of additional ribosomal DNA sequence clusters in autosomes 2, 3 and 4 of D. neohydei. They form no nucleolus in salivary glands nor are they associated with the nucleolus originating from the sex chromosomal nucleolus organizers. After in vitro pulse-labelling salivary glands with radioactive RNA precursors, no autoradiographic label is associated with the autosomal ribosomal RNA genes. Transcript in situ hybridization gives no label in these autosomal loci. We conclude therefore that the autosomal rDNA sequences are inactive in salivary glands. In situ hybridization with clones of a major part of the 28S intervening sequence (IVS) of D. hydei to polytene chromosomes of D. neohydei results in intense hybridization of the 28S IVS fragment to the autosomal ribosomal DNA sequences. It is therefore suspected that most if not all of the rDNA copies in the autosomes contain an IVS. The inactivity of these ribosomal genes might be the consequence of the presence of intervening sequences in the 28S gene. These observation supply additional evidence in favour of a transcriptional inactivity of ribosomal genes with an IVS in Drosophila. Additional loci with IVS sequences are found in different positions of the genome which do not hybridize with rDNA sequences without IVS.

An improved method for preparing cell suspensions used for the cloning of human tumors.

The majority of studies dealing with cytotoxic effects concern variations in cell behavior during mitosis. The highly diverse techniques developed can be employed to determine the number of cells undergoing synthesis following incorporation of radioactive RNA or DNA precursors [l] or to detect clonogenic cells in a liquid medium or soft agar [2,3]. While all of these techniques are easy to apply to cell lines, they prove more difficult for use with cells extracted extemporaneously from solid tumors. Yet clinical applications frequently involve this latter type of cell. Tumor tissues constitute a heterogeneous entity in which the boundary between normal and pathological cells is ill-defined, and it proves difficult to extract such cells from the existing conjunctive and nutrient environment. Isolation of cells from tissue results in a mixture of cells which makes interpretation difficult if not impossible. In tests utilizing radioactive RNA or DNA precursor incorporation, the lack of cell homogeneity can in fact vitiate the results; the lymphocytes of the inflammatory stroma also incorporate these elements, and the suspension must be made as uniform as possible. As for the cloning of cancer cells in agar, colonies of more than 10 cells must be eliminated since they can be confused with clones during reading. We describe below a simple method, reducing the majority of these inconveniences, which we employ before determining the cloning efficiency of cancers using the method of Salmon [2]. Following removal of all surrounding fatty tissue, the tissue fragment is cut mechanically with small scissors in a nutrient medium. The medium employed depends on the histological type of the cancer cultivated: Eagle’s minimum essential medium (MEM) modified with Earle’s salts for undifferentiated adenocarcinomas and melanomas; Eagle’s medium modified as per Dulbecco for sarcomas; Ham F12

Cytogenetic and DNA analyses of equine abortion

Although no major structural or numerical abnormalities were found in the karyotypes of 12 aborted equine fetuses, two unrelated abortuses each carried a large polymorphism for the amount of heterochromatin in chromosome 1. In both karyotypes this chromosome was shown to be larger than its homolog. To determine the nature of the extra DNA in these chromosomes, equine DNA was isolated and characterized by buoyant density analysis. Equine mainband DNA had a buoyant density in neutral CsCl of 1.699 g/cm3, while the highly repetitive (dG+dC)-rich fraction had a buoyant density of 1.715 g/cm3. A radioactive RNA probe complementary to the purified satellite fraction was used for in situ hybridization to chromosomal spreads containing the enlarged chromosome 1. The results indicated that an increase in highly repetitive (dG+dC)-rich DNA was responsible for the increase in the size of the abnormal No. 1 chromosomes. While two of the 12 aborted fetuses exhibited marked heterochromatic dimorphisms, none of the karyotypes obtained from individuals with no family history of abortion exhibited such obvious polymorphisms.

Characterisation of the LdsRNA encoded mRNA of yeast

Radioactive messenger RNA (mRNA) was isolated from a Saccharomyces cerevisiae strain containing the L double stranded RNA (dsRNA) species. This mRNA was hybridised to LdsRNA isolated from the same strain. Analysis of the hybrid formed shows it to be of a similar size to the LdsRNA. It is concluded from this result that the mRNA complementary to the -ve strand of the LdsRNA is polycistronic.When the total mRNA is fractionated on oligo (dT) cellulose, RNA complementary to the -ve strand of LdsRNA is found only in the non-binding fraction. When LdsRNA is fractionated on oligo (dT) cellulose the majority is found in the non-binding fraction. It is concluded that neither the LdsRNA genome nor the mRNA it encodes have poly(A) tails of a significant length.

Isolation of cloned ribosomal protein genes from the yeast Saccharomyces carlsbergensis

A colony bank of yeast DNA obtained by cloning HindIII-generated fragments of total yeast nuclear DNA in Escherichia coli K-12 with the vector pBR322, was screened with a radioactive RNA probe enriched for a subset of ribosomal protein mRNAs. The selected recombinant DNA molecules were hybridized with poly(A)-containing mRNA under R-loop conditions. From the DNA-RNA hybrids the respective mRNAs were melted off and translated in vitro in a rabbit reticolocyte cell-free system. The translational products were analyzed by immunoprecipitation with antibodies raised against ribosomal proteins. The identity of the ribosomal protein gene products was further established by electrophoresis on two-dimensional gels. At least 15 recombinant DNA molecules were shown to contain ribosomal protein genes. Four of them, i.e. Y65, Y89, Y113, and Y138, have been characterized preliminarily.