Virus-Specific Ribonucleic Acid in the Nucleus and Cytoplasm of Rat Embryo Cells Transformed by Adenovirus Type 2

Nuclei were isolated from rat embryo cells transformed by adenovirus type 2. Nuclear and cytoplasmic virus-specific ribonucleic acids (RNA) were characterized and quantitated by deoxyribonucleic acid (DNA)-RNA hybrid formation with adenovirus DNA. The results indicate that most, if not all, virus-specific RNA molecules are synthesized in the cell nucleus and subsequently transported into cytoplasm where they degrade with a half-life of 1 to 2 hr. No difference in base sequences between nuclear and cytoplasmic virus-specific RNA species can be detected by hybridization competition experiment with viral DNA.     

Use of actinomycin D for the specific quenching of fluorescence of deoxyribonucleic acid in cells stained with acridine aminoderivatives.

Actinomycin D specifically quenches the fluorescence of acridine orange and quinacrine bound to deoxyribonucleic acid in cytologic preparations, but does not change the fluorescence of these fluorochromes bound to RNA. The following fluorescence-cytochemical applications of techniques based on these findings can be suggested: (a) distinction between deoxyribonucleic acid and ribonucleic acid; (b) detection of double-stranded virus ribonucleic acid; (c) approximate estimation of the lengths of A-T sequences in deoxyribonucleic acid molecules.     

Mutations affecting the cAMP transduction pathway modify olfaction in Drosophila

The rutabaga and dunce genes, encode two enzymes of the cyclic adenosine monophosphate transduction pathway in Drosophila, adenylyl cyclase and cyclic adenosine monophosphate phosphodiesterase, respectively. Two main second messenger systems, depending on inositol 1,4,5-triphosphate and cyclic adenosine monophosphate, have been associated with olfaction in vertebrates as well as invertebrates. A relationship between the cyclic adenosine monophosphate signaling pathway and olfactory reception in Drosophila is suggested by the presence of cyclic nucleotide gated channels and cyclic-nucleotide modulated K+ channels in the antennae, the main olfactory organs. In this report, molecular, electrophysiological and behavioral data support the role of cyclic adenosine monophosphate in olfactory function for this species. Expression of both genes in the antennae has been shown by messenger ribonucleic acid analysis. Changes in the electroantennogram kinetics have been observed specifically on the slope of the initial rising phase, as predicted for processes that affect cyclic adenosine monophosphate concentration. Olfactory behavior changes due to both mutations were coherent with a functional meaning of the reported electrophysiological phenotype in olfactory perception. Sensitivity level increases or decreases for the mutants compared to the control line depending on the odorant. These results are compatible with some olfactory coding at the reception level by differential activation of a dual transduction system involving the inositol 1,4,5-triphosphate and cyclic adenosine monophosphate cascades.     

Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins.

We analyzed the multiplicity, heterogeneity, and organization of the genes encoding the alpha and beta tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. alpha- and beta-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The alpha cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of alpha tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The beta cDNA insertion contains the coding sequence for the 100-C terminal amino acids of beta tubulin and 83 pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous alpha- and beta-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of alpha-tubulin genes with beta-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.     

Reversion of the Frameshift hisD3018 of Salmonella typhimurium

Previous studies on histidinol dehydrogenase from His(+) revertants have shown that the frameshift mutation hisD3018 is a +1 type, resulting from inclusion of an extra cytidylate residue in messenger ribonucleic acid. Histidinol dehydrogenase from newly isolated spontaneous and N-methyl-N'-nitro-N-nitrosoguanidine (NG)-induced intragenic revertants has been examined for amino acid replacements. The results provide additional evidence that NG can delete guanine plus cytosine base pairs from deoxyribonucleic acid. One spontaneous revertant was found to result from a +2 addition of approximately 16 nucleotide residues before the +1 parent frameshift, and another by a -4 deletion about six residues before the same. Circumstantial evidence suggests the in vivo codon assignment GAG for glutamic acid. A region of histidinol dehydrogenase highly permissive of amino acid changes encoded in the minus (-) phase is now apparent.     

Regulation of tropomyosin gene expression during myogenesis.

In skeletal muscle, tropomyosin has a critical role in transduction of calcium-induced contraction. Presently, little is known about the regulation of tropomyosin gene expression during myogenesis. In the present study, qualitative and quantitative changes in the nucleic acid populations of differentiating chicken embryo muscle cells in culture have been examined. Total nucleic acid content per nucleus increased about fivefold in fully developed myotubes as compared to mononucleated myoblasts. The contribution of deoxyribonucleic acid to the total nucleic acid population decreased from 24% in myoblasts to 5% of total nucleic acid in myotubes. Concomitant with the decrement in deoxyribonucleic acid contribution to total nucleic acid was an increase in polyadenylated ribonucleic acid (RNA) content per cell which reached levels in myotubes that were 17-fold higher than those of myoblasts. Specific changes in the RNA population during myogenesis were further investigated by quantitation of the synthetic capacity (messenger RNA levels) per cell for alpha- and beta-tropomyosin. Cell-free translation and immunoprecipitation demonstrated an approximately 40-fold increase in messenger RNA levels per nucleus for alpha- and beta-tropomyosin after fusion in the terminally differentiated myotubes. Indirect immunofluorescence with affinity-purified tropomyosin antibodies demonstrated the presence of tropomyosin-containing filaments in cells throughout myogenesis. Thus, the tropomyosin genes are constitutively expressed during muscle differentiation through the production of tropomyosin messenger RNA and translation into tropomyosin protein.     

Evidence for insulin synthesis in normal mouse seminal vesicle based on in situ RNA-DNA hybridization

Cultured seminal vesicle epithelial cells exhibited cytoplasmic immunoreactivity following treatment with anti-insulin antisera. In addition, these cultured epithelial cells were found, by in situ hybridization with a radiolabeled insulin complementary deoxyribonucleic acid (cDNA) probe, to contain an insulin or insulin-like messenger ribonucleic acid (mRNA). Autoradiograms of the hybridized cells exhibited heavy labeling over the cytoplasm and minimal distribution of grains over the nuclei and background areas. These observations indicate that cultured mouse seminal vesicle epithelium contains an insulin or insulin-like peptide as well as the mRNA that is required for its synthesis.     

Nuclear-cytoplasmic relationships in human cells in tissue culture. III. Auto-radiographic study of interrelation of nuclear and cytoplasmic ribonucleic acid

The movement of ribonucleic acid (RNA) from nucleus to cytoplasm has been studied, by autoradiographic techniques, in cells of the human amnion grown in tissue culture. Cells were exposed to cytidine-H(3) for 1 hour after which time only the RNA of the nuclei was labelled. After this 1 hour exposure the cells were placed in a medium containing an excess amount of unlabelled cytidine. Periodically, cells from this medium were fixed. Autoradiographs showed that there was a progressive movement of the label from nucleus to cytoplasm, such that after 24 hours essentially all the label was in the RNA of the cytoplasm. A study of the incorporation of the cytidine-H(3) in deoxyribonucleic acid (DNA), in the same population of cells at the same times, indicated that the presence of excess amounts of unlabelled cytidine almost instantaneously inhibited further utilization of cytidine-H(3). It is concluded that RNA moves from nucleus to cytoplasm as a complex polynucleotide structure.     

Biochemical properties of Clostridium bifermentans spores.

As previously found for spores of Bacillus species, dormant spores of Clostridium bifermentans contained essentially no adenosine triphosphate, a high level of adenosine monophosphate, a high level of 3-phosphoglyceric acid, and much transfer ribonucleic acid lacking a 3'-terminal adenosine monophosphate residue. As in spores of Bacillus species, germination of C. bifermentans spores was accompanied by utilization of the 3-phosphoglyceric acid, a large increase in the adenosine triphosphate level, and the disappearance of defective transfer ribonucleic acid. In contrast to spores of Bacillus species, dormant spores of C. bifermentans contained little free amino acid.     

Nuclear-Cytoplasmic Relationships in Human Cells in Tissue Culture : III. Autoradiographic Study of Interrelation of Nuclear and Cytoplasmic Ribonucleic Acid

The movement of ribonucleic acid (RNA) from nucleus to cytoplasm has been studied, by autoradiographic techniques, in cells of the human amnion grown in tissue culture. Cells were exposed to cytidine-H³ for 1 hour after which time only the RNA of the nuclei was labelled. After this 1 hour exposure the cells were placed in a medium containing an excess amount of unlabelled cytidine. Periodically, cells from this medium were fixed. Autoradiographs showed that there was a progressive movement of the label from nucleus to cytoplasm, such that after 24 hours essentially all the label was in the RNA of the cytoplasm. A study of the incorporation of the cytidine-H³ in deoxyribonucleic acid (DNA), in the same population of cells at the same times, indicated that the presence of excess amounts of unlabelled cytidine almost instantaneously inhibited further utilization of cytidine-H³. It is concluded that RNA moves from nucleus to cytoplasm as a complex polynucleotide structure.     

Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli

Luzzati, Denise (Institut de Biologie Physico-Chimique, Paris, France). Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli. J. Bacteriol. 92:1435-1446. 1966.-During the course of thymine starvation, the rate of synthesis of messenger ribonucleic acid (mRNA, the rapidly labeled fraction of the RNA which decays in the presence of dinitrophenol or which hybridizes with deoxyribonucleic acid) decreases exponentially, in parallel with the viability of the thymine-starved bacteria. The ability of cell-free extracts of starved bacteria to incorporate ribonucleoside triphosphates into RNA was determined; it was found to be inferior to that of extracts from control cells. The analysis of the properties of cell-free extracts of starved cells shows that their decreased RNA polymerase activity is the consequence of a modification of their deoxyribonucleic acid, the ability of which to serve as a template for RNA polymerase decreases during starvation.     

ISOLATION AND CHARACTERIZATION OF DEOXYRIBONUCLEIC ACID FROM A STRAIN OF STAPHYLOCOCCUS AUREUS.

Blobel, Hans (University of Wisconsin, Madison). Isolation and characterization of deoxyribonucleic acid from a strain of Staphylococcus aureus. J. Bacteriol. 82:425-429. 1961.-Highly polymerized deoxyribonucleic acid was extracted from washed cells of Staphylococcus aureus with a mixture of equal parts of phenol and 2 m NaCl at pH 7.4. The aqueous phase was treated twice with phenol and the deoxyribonucleic acid precipitated with an equal volume of 2-ethoxyethanol. Residual ribonucleic acid was removed by treatment with ribonuclease and subsequent dialysis. Deoxyribonucleic acid was reprecipitated with 2-ethoxyethanol. The final product contained less than 1% protein. The deoxyribonucleic acid obtained from S. aureus strain S(44) had a (adenosine + thymine)/(guanine + cytosine) base ratio of 1.98. Intrinsic viscosity in 0.15 m NaCl + 0.015 m sodium citrate was approximately 76 dl/g. The sedimentation coefficient, S(20)w, was close to 25 S.     

Regulated transport of messenger ribonucleic acid from isolated liver nuclei by nucleic acid binding proteins

Rat liver nucleocytosolic messenger ribonucleic acid (mRNA) transport is shown to be regulated by proteins with a high affinity for nucleic acids. In the cell-free system described, the energy-dependent transport of all RNA classes [transfer RNA (tRNA), mRNA, and ribosomal RNA (rRNA)] exhibited a dependence upon the availability of discrete minor sets of cytosol proteins. In addition to having a different level of saturation, only the mRNA "transport protein" activities are increased by adenosine cyclic 3',5'-phosphate (cAMP), an effect most likely mediated by a cAMP-dependent protein kinase. The mRNA transport proteins were isolated from cytosol by precipitation with streptomycin sulfate followed by deoxyribonucleic acid (DNA)-cellulose affinity chromatography, or from oligo-(thymidylate)-cellulose bound cytoplasmic messenger ribonucleoprotein (mRNP) particles by high-salt extraction. Either method yielded a protein fraction which exhibited a 1000-fold increase in mRNA transport activity as compared to cytosol. Over one-half of the mRNA transport activity is associated with the mRNP of the cell. A partial homology between the cytosol and mRNP-derived proteins was demonstrated by polyacrylamide gel electrophoresis. One major (20 000 daltons) and several minor proteins (23 000, 52 000, 54 000, and 72 000 daltons) were in common. Nuclear 4-5S exited from in vitro incubated nuclei in three phases, according to their differential in vivo rates of labeling and intranuclear pool sizes. The amount of nuclear RNA transported in vitro as mRNA (about 1.0%) agrees wtih the in vivo estimates. Additional evidence for in vivo equivalence was provided by the physicochemical characterization and bioassay of the RNA. The transported mRNA sedimented in urea-sucrose gradients as an 8-18S heterodisperse product. This RNA initiated cell-free translation with the synthesis of precursor peptides as diverse in size as those for albumin and alpha 2U-globulin. The relative abundancies of various transported mRNAs were different than the corresponding abundancies of liver cytoplasmic mRNAs.     

Genetic recombination in Nocardia mediterranei.

The regulation of macromolecular biosynthesis was studied in a temperature-sensitive mutant of Escherichia coli previously identified as containing a single mutation causing a thermolabile sn-glycerol-3-phosphate acyltransferase, the first enzyme of the pathway for phospholipid biosynthesis. When this mutant was shifted to a nonpermissive temperature, phospholipid synthesis, as well as ribonucleic acid, deoxyribonucleic acid, and protein synthesis, decreased in a coordinate manner, suggesting the existence of a common regulatory mechanism. During the same time that the rate of macromolecular synthesis was decreasing at the nonpermissive temperature, the intracellular concentration of adenosine 5'-triphosphate dropped dramatically and the concentration of adenosine monophosphate increased. The concentration of adenosine 5'-diphosphate dropped, but not as markedly. The decrease in macromolecular synthesis and the changes in the adenine nucleotide concentrations can now be attributed to a thermolabile adenylate kinase. The inactivation of adenylate kinase prevented the cell from converting adenosine 5'-monophosphate to adenosine 5'-diphosphate and consequently from making adenosine 5'-triphosphate. This in turn caused a decrease in the rate of macromolecular synthesis and cell growth. Adenylate kinase, therefore, is a key enzyme in controlling the rate of cell growth. The nature of the possible relationship between adenylate kinase and glycerol-3-phosphate acyltransferase is discussed.     

Cellular Origin of a Mouse Leukemia Viral Ribonucleic Acid

Mouse erythroblastosis virus, a member of the mouse leukemia virus group, was obtained from chronically infected C(3)H mouse embryo cells and purified on sucrose gradients. The ribonucleic acid (RNA) extracted from ribonuclease-treated virus consisted of a rapidly sedimenting (72S) species and a more slowly sedimenting component (4 to 30S). The 72S RNA did not contain base sequences homologous to deoxyribonucleic acid (DNA) from infected cells as determined by hybridization studies. In contrast, the slowly sedimenting RNA enclosed within the virus had base sequences homologous to DNA from infected and uninfected C(3)H mouse embryo cells.     

Ribosomal Ribonucleic Acid Maturation During Bacterial Spore Germination

All the ribosomal ribonucleic acid made during the early stages of germination of spores of Bacillus subtilis is of the "precursor" type, i.e., that type appearing in the incomplete forms of the ribosome. Shortly before the onset of deoxyribonucleic acid synthesis in germination, this precursor ribonucleic acid changed to the mature ribosomal ribonucleic acid characteristic of the 30S and 50S ribosomal subunits.     

Virus-Specific Ribonucleic Acid in Cells Producing Rous Sarcoma Virus: Detection and Characterization

Cells producing Rous sarcoma virus contain virus-specific ribonucleic acid (RNA) which can be identified by hybridization to single-stranded deoxyribonucleic acid (DNA) synthesized with RNA-directed DNA polymerase. Hybridization was detected by either fractionation on hydroxyapatite or hydrolysis with single strand-specific nucleases. Similar results were obtained with both procedures. The hybrids formed between enzymatically synthesized DNA and viral RNA have a high order of thermal stability, with only minor evidence of mismatched nucleotide sequences. Virus-specific RNA is present in both nuclei and cytoplasm of infected cells. This RNA is remarkably heterogeneous in size, including molecules which are probably restricted to the nucleus and which sediment in their native state more rapidly than the viral genome. The nature of the RNA found in cytoplasmic fractions varies from preparation to preparation, but heterogeneous RNA (ca. 4-50S), smaller than the viral genome, is always present in substantial amounts.