Showing papers on "RNA published in 1968"

Nitrosamine-induced carcinogenesis. The alkylation of nucleic acids of the rat by N-methyl-N-nitrosourea, dimethylnitrosamine, dimethyl sulphate and methyl methanesulphonate

Abstract: 1. N[(14)C]-Methyl-N-nitrosourea, [(14)C]dimethylnitrosamine, [(14)C]dimethyl sulphate and [(14)C]methyl methanesulphonate were injected into rats, and nucleic acids were isolated from several organs after various time-intervals. Radioactivity was detected in DNA and RNA, partly in major base components and partly as the methylated base, 7-methylguanine. 2. No 7-methylguanine was detected in liver DNA from normal untreated rats. 3. The specific radioactivity of 7-methylguanine isolated from DNA prepared from rats treated with [(14)C]dimethylnitrosamine was virtually the same as that of the dimethylnitrosamine injected. 4. The degree of methylation of RNA and DNA produced in various organs by each compound was determined, and expressed as a percentage of guanine residues converted into 7-methylguanine. With dimethylnitrosamine both nucleic acids were considerably more highly methylated in the liver (RNA, about 1% of guanine residues methylated; DNA, about 0.6% of guanine residues methylated) than in the other organs. Kidney nucleic acids were methylated to about one-tenth of the extent of those in the liver, lung showed slightly lower values and the other organs only very low values. N-Methyl-N-nitrosourea methylated nucleic acids to about the same extent in all the organs studied, the amount being about the same as that in the kidney after treatment with dimethylnitrosamine. In each case the RNA was more highly methylated than the DNA. Methyl methanesulphonate methylated the nucleic acids in several organs to about the same extent as N-methyl-N-nitrosourea, but the DNA was more highly methylated than the RNA. Dimethyl sulphate, even in toxic doses, gave considerably less methylation than N-methyl-N-nitrosourea in all the organs studied, the greatest methylation being in the brain. 5. The rate of removal of 7-methylguanine from DNA of kidneys from rats treated with dimethylnitrosamine was compared with the rate after treatment of rats with methyl methanesulphonate. No striking difference was found. 6. The results are discussed in connexion with the organ distribution of tumours induced by the compounds under study and in relation to the possible importance of alkylation of cellular components for the induction of cancer.

Factor Fraction required for the Synthesis of Bacteriophage Qβ-RNA

Abstract: A factor fraction essential for the in vitro synthesis of RNA bacteriophage Qβ-RNA has been isolated from both infected and uninfected E. coli. This indicates that the contribution of the last bacterium to the replication of Qβ-RNA is more extensive than was earlier thought.

Mutants that make more lac repressor.

Abstract: The gene that makes the lac repressor functions at an extremely low rate. It synthesizes only a few thousandths of a per cent of the total protein of an E. coli cell: about 5-10 molecules each generation.1 In order to study this molecule more easily, we have sought to enhance the amount present in a cell. Two approaches have been successful. The repressor gene, the i gene, itself can be mutated to a form that produces more repressor, or the number of copies of the gene can be drastically increased by incorporating the gene into a phage chromosome which will multiply in the cell. The combination of these two approaches yields a cell strain which can make 0.5 per cent of its protein lac repressor. The ij Mutant.-Several mutants that make about tenfold more repressor than the wild type have been found. These if mutants (q for quantity) were selected by forcing temperature-sensitive repressor mutants, strains constitutive at 430 and inducible at 300, to revert to an inducible phenotype at 430C. At the high temperature, the temperature-sensitive mutants make too little repressor, either because the protein itself is unstable (iTL) or because the final assembly of active repressor is blocked (iTSS).2 Although the reversion could simply correct the original defect, the temperature effects can also be overcome by producing more of the protein (or a more active protein: so that the small amount left at the high temperature will suffice). However, the ability of the lac repressor to bind IPTG (isopropyl-l-thio-f-D-galactopyranoside) gives one a direct, quantitative measure of the number of molecules present.1' By screening extracts from \"revertant\" strains, we could identify those mutants which, in contrast to the wild type, gave an easily detectable binding in the crude extract. The revertants were isolated using TONPG (o-nitrophenyl-1-thio-f3-D-galactopyranoside), which will inhibit the growth of lac constitutive cells that have a functioning lac permease. y(permease-) mutants will arise and can be coullterselected against by growth on lactose in the -presence of IPTG. Both iTSS and iTL strains have yielded overproducing derivatives. Table 1 shows the IPTG-binding data for some of these strains; the data suggest that there is about ten times more repressor in the ij strains. One of these mutants, a derivative of an iTSS strain, has been studied in detail, and was used to produce pure repressor for physical characterization (studies that will be reported elsewhere). The affinity of this ij repressor for IPTG has been checked and is the same as the wild type; therefore, the increase in the binding truly means an increase in amount of repressor. Dominance and Complementation.-To study further the properties of this i\" mutant, we constructed heterozygous diploids with a variety of i and o (operator)

Differential synthesis of the genes for ribosomal RNA during amphibian oögenesis.

Abstract: Molecular hybridization experiments have shown that the genes for ribosomal RNA are located in or near the nucleolus organizer in Drosophila melanogasterl' 2 and in the toad Xenopus laevis.3' 4 Under normal circumstances the number of organizers per genome will be characteristic of the organism, and DNA from various tissues should contain the same proportion of ribosomal genes. This conclusion has been confirmed for several tissues of the chicken.2 However, cytological evidence has long suggested that the nucleolus organizer undergoes a differential replication in oocytes of certain animals. Data supporting this view will be summarized later in this article. If such a differential replication occurs, oocyte DNA should be enriched with respect to the ribosomal genes. The cytological picture is particularly striking in ovaries of the toads Bufo and Xenopus, in which the differential synthesis occurs during pachytene. In recently metamorphosed toads the ovary contains a sufficiently high proportion of pachytene o6cytes to permit detection of the differential synthesis by biochemical means.5 This communication describes the ovarian DNA of Xenopus and demonstrates that it contains an excess of sequences coding for rRNA. Recently Brown and co-workers6' 7 have studied the DNA from older oocytes of Xenopus and have reached similar conclusions.

Polypeptide cleavages in the formation of poliovirus proteins

Abstract: The final step in poliovirus morphogenesis appears to be the combination of viral RNA with a protein shell called the procapsid.(1) Concomitant with the union of the RNA and the procapsid there is a cleavage of one of the procapsid proteins, producing two of the four proteins of the virion. We have now found that cleavages play an important role in the formation of most if not all poliovirus-specific proteins. Although most mammalian messenger RNA's appear to be monocistronic, it seems possible that a cleavage mechanism may function in the synthesis of some mammalian cell proteins.

The Integrated State of Viral DNA in SV40-Transformed Cells

Abstract: It has been shown by a modified DNA-RNA hybridization technique that the nuclei of cells transformed by either polyoma virus or SV40 contain viral DNA.1 This method employs RNA synthesized in vitro with form I viral DNA2–4 and E. coli RNA polymerase. The number of viral DNA equivalents per cell varies from 5 to 60, depending on the cell line. In this communication, we will report on the physical state of the viral DNA in SV3T3 cells, an SV40-transformed cell line that contains 20 SV40 DNA equivalents per cell.

Observations on the nucleolar and total cell body nucleic acid of injured nerve cells

Abstract: 1. The nucleic acid content of neuronal nucleoli and the total cell body nucleic acid content of neurones of the hypoglossal nucleus were measured by ultraviolet absorption microspectrography. 2. After nerve injury both the nucleolar nucleic acid and the total cell body nucleic acid increased: nucleolar changes preceded those of the cell body. 3. The closer to the nerve cell body that the axon was injured the earlier was the onset and the decline of the nucleolar response. 4. Actinomycin D was given to prevent DNA-primed RNA synthesis, and the rate of `decay' of nucleolar RNA was measured. This rate varied after nerve injury and was closely related to the nucleolar nucleic acid content. 5. The apparent rate of transfer of labelled RNA from the neuronal nucleus into the cytoplasm changed after nerve injury in a manner closely related to the changes in nucleolar nucleic acid content. 6. It was demonstrated by making consecutive nerve injuries or by preventing or delaying nerve regeneration, that the nucleic acid changes were not induced by removal of contact between the neurone and its motor end-plate, and were not repressed by the restoration of such contact. 7. When regeneration was prevented the nucleolar nucleic acid content and the total cell body nucleic acid ultimately decreased to values less than normal: this decrease was greater when more of the axon was initially removed. 8. The results are discussed in relation to the factor responsible for derepression and repression of DNA cistrons for ribosome synthesis in injured nerve cells.

Nucleotide Sequence of N-Formyl-methionyl-transfer RNA

Abstract: N-Formyl-methionyl-transfer RNA is thought to be the chain-initiating tRNA of protein synthesis in bacterial systems. The complete nucleotide sequence of this tRNA from Escherichia coli has now been worked out.

Polar granules of Drosophila. II. Ultrastructural changes during early embryogenesis

Abstract: Polar granules of Drosophila show distinctive variations in structure at various stages of early embryogenesis and in different species. In most species they are attached to mitochondria at fertilization, then they become free and fragment during the cleavage divisions and during the time of pole cell formation. After pole cells have formed they again coalesce. At the time of migration of the pole cells to the embryonic gonad, the polar granules again fragment and become attached to the outer membrane of the nuclear envelope. Ribosomes are attached to the periphery of polar granules at the time they are fragmented prior to and during pole cell formation. An hypothesis is presented on the basis of published reports of RNA present in the granules and the ultrastructural observations reported here. This hypothesis suggests that the RNA in polar granules is m-RNA that directs the synthesis of those proteins necessary for the determination of the pole cells to become germ cells.

The cytoplasmic control of nuclear activity in animal development.

Abstract: Summary 1.This article reviews the occurrence, mechanism, and functional significance of the cytoplasmic regulation of nuclear activity during cell differentiation and especially during early animal development. 2.Nuclei from brain, and from other kinds of adult cell normally inactive in DNA synthesis, are rapidly induced to commence DNA synthesis by components or properties of intact egg cytoplasm. The components of egg cytoplasm which induce DNA synthesis are not species-specific and they are likely to include DNA polymerase. It is known that DNA polymerase exists in egg cytoplasm before it becomes associated with nuclei in which it is effective. The induction of DNA synthesis in brain nuclei by living egg cytoplasm is always preceded by a pronounced nuclear swelling, a dispersion of chromosomes or chromatin, and the entry of cytoplasmic protein into the nucleus. 3.RNA synthesis can be experimentally induced or repressed by living cytoplasm. The cytoplasm of unfertilized and fertilized eggs appears to contain components which can reversibly and independently repress the synthesis of ribosomal RNA, transfer RNA, and heterogeneous RNA. RNA synthesis can be induced by introducing nuclei inactive in this respect into the cytoplasm of cells very active in RNA synthesis. The induction and repression of RNA synthesis is preceded by a marked swelling of the nucleus and the dispersion of its chromosome material. 4.The cytoplasmic control of chromosome condensation before division has been demonstrated by introducing sperm or adult brain nuclei into the cytoplasm of oocytes undergoing meiotic maturation. 5.The evidence that regional differences in the composition of eggs and other cells are associated with changes in nuclear and gene activity is reviewed in Section 111. While it is certain that these regional differences are of great importance in cell differentiation, evidence that they have a direct effect on nuclear activity has been obtained in a few instances only. In some species it has been shown that the cytoplasmic components related to germ-cell differentiation include RNA and, frequently, granules. 6.It is concluded that whenever nuclei are introduced experimentally into the cytoplasm of another cell, they very quickly assume, in nearly every respect, the nuclear activity characteristic of the host cell. In many instances, altered function has been demonstrated in nuclei which subsequently support normal development. The induced nuclear changes are therefore regarded as normal and it is believed that they are achieved through the same mechanism as that by which the host cell nucleus originally came to function in its characteristic way. Examples are cited to show that changes in gene activity very frequently arise immediately after mitosis. The changes induced experimentally in transplanted nuclei resemble in very many respects those undergone by nuclei which are naturally reconstituted after mitosis, and it is argued that the two processes are functionally equivalent, It is suggested that during telophase of mitosis, chromosomes are reprogrammed in respect of potential gene activity by association with cytoplasmic proteins. Inter-phase nuclei seem not to show changes of gene activity except when they undergo a pronounced enlargement after entering a new cytoplasmic environment.

[98] Isolation of nucleic acids with phenolic solvents

Abstract: Publisher Summary Phenol was first used to extract RNA from mammalian tissues and tobacco mosaic virus, but it became clear that although phenol extracted and inhibited ribonuclease, subsequent purification resulted in degradation of the RNA. The ethylenediamine tetraacetate may inhibit ribonuclease, but this is not generally the case. Naphthalene 1,5-disulfonate is a hydrophilic salt, does not release DNA at 0.015 M concentration, and was found to lessen the amount of degradation in RNA from chicken liver. The residual protein was 1.5–2.0%. The m -Cresol is added to the phenol to serve two purposes—(1) the mixture can be cooled to 5° without the phenol crystallizing and (2) the phenol–cresol mixture is a better deproteinizing agent than phenol alone. Ribosomal RNA can be isolated without associated rapidly labeled RNA if the original homogenization is carried out in the presence of naphthalene 1,5-disulfonate and with the rapidly labeled RNA if 4-aminosalicylate is used. A method, which also yields the rapidly labeled RNA associated with the ribosomal RNA, is described in this chapter. DNA is released with ribosomal RNA when the tissues are extracted with 4-aminosalicylate, NaC1, and the phenol-cresol mixture, and it can be separated by extraction with 3 M sodium acetate (pH 6).

Biochemical Correlates of Cardiac Hypertrophy: I. Experimental Model; Changes in Heart Weight, RNA Content, and Nuclear RNA Polymerase Activity

Abstract: Cardiac hypertrophy occurred in mature rats after producing supravalvular aortic stenosis with a specially designed silver clip. For 2 weeks following this procedure, heart weight, body weight, and RNA content of the myocardium were serially determined. Heart weight and RNA content increased within 24 hours of aortic banding, reaching a maximal level in 2 days and remaining elevated during the 2 weeks of observation. Nuclei were isolated and purified from heart muscle homogenates, and changes in RNA polymerase activity following aortic banding were determined. The nearest neighbor frequency of the bases of the RNA synthesized by the polymerase from nuclear preparations was identical in both the banded animals and the sham-operated controls. Both groups could thus be compared on the basis of the enzyme assay. RNA polymerase activity in nuclei from the hearts of banded rats rose rapidly when compared with the activity in sham-operated rats; peak values were reached on the second day, the earliest detectable change being around 12 hours. The increase in RNA polymerase activity represents one of the earliest biochemical events that take place in the myocardium following aortic banding.

The turnover of nuclear DNA-like RNA in HeLa cells.

Abstract: The subcellular distribution of various types of RNA in HeLa cells is described. In addition, the relative rate of synthesis of the major classes of nuclear RNA has been determined. From these experiments it can be deduced that the heterogeneous nuclear RNA fraction is rapidly synthesized and degraded within the cell nucleus.