5S RNA Synthesis Research Articles

Control of 5S RNA synthesis during early development of anucleolate and partial nucleolate mutants of Xenopus laevis.

Ribosomes of all eukaryotes contain a single molecule of 5S, 18S, and 28S RNA. In the frog Xenopus laevis the genes which code for 18S and 28S RNA are located in the nucleolar organizer, but these genes are not linked to the 5S RNA genes. Therefore the synthesis of the three ribosomal RNAs provides a model system for studying interchromosomal aspects of gene regulation. In order to determine if the synthesis of the three ribosomal RNAs are interdependent, the relative rate of 5S RNA synthesis was measured in anucleolate mutants (o/o), which do not synthesize any 18S or 28S RNA, and in partial nucleolate mutants (p(l-1)/o), which synthesize 18S and 28S RNA at 25% of the normal rate. Since the o/o and p(l-1)/o mutants have a complete and partial deletion of 18S and 28S RNA genes respectively, but the normal number of 5S RNA genes, they provide a unique system in which to study the dependence of 5S RNA synthesis on the synthesis of 18S and 28S RNA. Total RNA was extracted from embryos labeled during different stages of development and analyzed by polyacrylamide gel electrophoresis. Quite unexpectedly it was found that 5S RNA synthesis in o/o and p(l-1)/o mutants proceeds at the same rate as it does in normal embryos. Furthermore, 5S RNA synthesis is initiated normally at gastrulation in o/o mutants in the complete absence of 18S and 28S RNA synthesis.

Experimental studies on the mechanism of action of both developmental-physiologically active fractions of the brain hormones of insects (activation factor I and II) of the prothoracic gland

2 prothoracotropical factors (activation factor I and II) have been obtained by gel filtration techniques from brains and corpora cardiaca of the cockroachPeriplaneta americana. In contrast to activation factor II, activation factor I caused significant influence of RNA synthesis. The RNA pattern of prothoracic glands stimulated by activation factor I as demonstrated by polyacrylamide gel electrophoresis consists of different kinds of RNA. Short time incubation revealed effects on sRNA synthesis, while long time incubation demonstrated predominantly increase of ribosomal RNA synthesis. Measurements of the membrane potential of the prothoracic gland cells of the wax mothGalleria mellonella indicated an increase by activation factor II; activation factor I was without any visible effect. Our results demonstrate for the first time that the two activation factors induce different effects at cellular level.

Regulation of ribosomal RNA and 5s RNA synthesis in Drosophila melanogaster. I. Bobbed mutants.

Analysis of the rates and amounts of rRNA and 5s RNA synthesized in Drosophila melanogaster bobbed mutants was done by using acrylamide-gel electrophoresis. The results show that the amounts of rRNA synthesized are constant, although the rates of rRNA synthesis in bb's are reduced to 30% of the wild-type level. The rates of synthesis of 5s RNA were constant. The rate of synthesis of the two kinds of molecules that enter in equimolar amounts into the mature ribosome is non-coordinated.-The rates of rRNA synthesis were shown to be proportional to the length of the scutellar bristles, supporting the notion that in trichogen cells there is no developmental delay, but the size of the bristle depends directly on the rate of rRNA synthesis.

Selective interruption of high molecular weight RNA synthesis in HeLa cells by camptothecin.

THE study of macromolecular metabolism in eukaryotic cells has depended to a large extent on the use of selective inhibitors. Camptothecin is a potent, rapidly acting inhibitor of both DNA and RNA synthesis1–3 which first came to attention as a potential anti-tumour agent4 and which has since been shown to have the remarkable property of inducing breaks in cellular DNA5; it also has unusual effects on RNA synthesis possibly as a result of DNA breakage. We show that the drug causes aberrant synthesis of high molecular weight nuclear RNA, but has a much smaller effect on the labelling of 4S RNA and does not affect 5S RNA synthesis. If the effect on RNA synthesis is due to DNA breakage, the results suggest that the breaks are induced at specific points.

Synthesis of 5S and 4S RNA in metaphase-arrested HeLa cells.

The continued synthesis of both 5S and 4S RNA in metaphase-arrested HeLa cells is demonstrated; 5S RNA is apparently synthesized at approximately 74 percent of the interphase rate, while 4S RNA is synthesized at approximately one-third the rate. The ratio of uridine incorporation to RNA methylation is used to correct for the alteration in the specific activity of the pyrimidine pool during metaphase arrest.

Differential inhibition of 28S-18S RNA and 5S RNA synthesis in the cycloheximide-treated [formula omitted] embryonic cells

28S-18S RNA and 5S RNA synthesis were measured in the isolated cells from Xenopus laevis embryos whose protein synthesis was inhibited by cycloheximide. The inhibition of 5S RNA synthesis increased linearly with time,while that of 28S-18S RNA synthesis was greater in early phase but slowly increased later. Eventually, both were inhibited to a 10 per cent level of control after 5 hours. These results indicate that the sensitivity of 5S RNA synthesis toward cycloheximide is definitely different from that of 28S-18S RNA synthesis and might not be in favor of the co-ordination between them.

The utilization of genes for ribosomal RNA, 5S RNA, and transfer RNA in liver cells of adult rats.

The rates of synthesis of ribosomes, 5S RNA, and tRNA necessary to maintain the steady-state concentrations of these entities in liver cytoplasm of adult rats were determined. On the average, each liver cell in the adult rat synthesizes 650 ribosomes, 650 molecules of 5S RNA, and 11,000 molecules of tRNA each minute. The numbers of genes per liver cell for rRNA, 5S RNA, and tRNA were 330, 1660, and 13,000, respectively, as determined by RNA: DNA hybridization experiments. Thus, on the average, individual genes for rRNA, tRNA, and 5S RNA are transcribed twice a minute, once a minute, and once every 2.5 minutes, respectively, in the adult rat liver.

Persistent synthesis of 5S RNA when production of 28S and 18S ribosomal RNA is inhibited by low doses of actinomycin D.

AbstractTo determine whether transcription of the genes coding for 5S ribosomal RNA depends upon the concurrent activity of the genes for 28S and 18S ribosomal RNA we studied the synthesis of 5S RNA in cultured L cells when the synthesis of the 45S precursor of 28 and 18S RNA was inhibited with low doses of actinomycin D. Synthesis of 5S RNA was measured by following the incorporation of 3H uridine into components which, upon acrylamide gel electrophoresis, co‐migrated with markers of 5S RNA obtained from 32P labeled ribosomes. We observed that the synthesis of 5S RNA persists in the absence of detectable 28S and 18S RNA synthesis, continuing at the normal rate for several hours and at a reduced rate for at least a generation time. This strongly supports the concept that 45S RNA is not a precursor of 5S RNA, and indicates furthermore that neither the 45S component, nor any of the intermediates involved in its transition to 28S and 18S RNA, are involved in controlling the output of the 5S genes The 5S RNA which is made during actinomycin treatment is retained predominantly in the nucleoplasm and undergoes a turnover of about 3.5% per hour. It apparently cannot be utilized when ribosome synthesis resumes following removal of the actinomycin.

Synthesis of soluble ribonucleates in Escherichia coli infected with bacteriophage R17.

Measurements were made on the level of synthesis of soluble ribonucleates (sRNA) in Escherichia coli K-12 infected with the ribonucleic acid (RNA)-containing bacteriophage R17. Little or no decrease in sRNA synthesis was found to occur under conditions of infection which result in a 70 to 80% inhibition of ribosomal RNA synthesis.

Biochemical studies of radiation damage. I. Inactivation of the pH5 fraction in amino acyl sRNA synthesis in vitro and the binding of amino acids with protein and nucleic acid by gamma-ray irradiation.

SummaryThe ph 5 enzyme fraction obtained from yeast cells was irradiated alone with gamma-rays. After the addition to a reaction mixture and incubation, the inactivation mode of the enzymatic activity of phenylalanyl sRNA synthesis in vitro was shown to be sigmoidal as a function of log dose. When the enzyme fraction plus the reaction mixture was irradiated, unusual bindings of amino acid with protein and sRNA occurred. The amino acids bound abnormally by irradiation were specifically limited to those having a resonance ring, or those containing sulphur atoms.

SEQUENTIAL SYNTHESES OF VARIOUS MOLECULAR SPECIES OF RIBONUCLEIC ACID IN THE EARLY PHASE OF CONIDIA GERMINATION IN ASPERGILLUS ORYZAE

Ribonucleic acid synthesis in early stages of germination of Aspergillus oryzae conidia was studied using orthophosphate 32P or 14CO2 as precursor. RNA was fractionated by the methylated albumin-coated kieselguhr column chromatography or by the sucrose density gradient centrifugation analysis.RNA synthesis occurred at any time of germination so far observed and rRNA or its precursor was synthesized at the initial step of germination. sRNA synthesis occurred a little latter than the onset of germination and mRNA synthesis became apparent after 60min of incubation. Even in non-germinating medium (phosphate buffer), conidia could synthesize rRNA. It was suggested that the synthesis of rRNA or its precursor may be one of the starting reactions being driven at the very initial step of germination.

Liver sRNA synthesis in actinomycin D-treated rats

Liver sRNA synthesis in actinomycin D-treated rats

S-RNA synthesis V. Reconstitution of a biological property of phosphodiesterase-treated s-RNA

Brief treatment of soluble ribonucleic acid with snake venom phosphodiesterase results in the liveration of approximately 10% of the ribonucleotides by a process of random degradation. Phosphodiesterase-treated soluble ribonucleic acid shows a greatly enhanced capacity to incorporate cytidylic acid and uridylic acid but a decreased capacity to accept adenylic acid. The presence of the three complementary ribonucleoside 5′-triphosphates enhances the capacity of the degraded solbule ribonucleic acid to incorporate adenylic acid and cytidylic acid but inhibits the capacity of this material to accept uridylic acid. The latter effects are not observed with the untreated soluble ribonucleic acid. The capacity of soluble ribonucleic acid to accept threonine and valine is greatly reduced after the phosphodiesterase treatment. The threonine acceptor capacity of the treated soluble ribonucleic acid is almost completely restored by incubation with all 4 ribonucleoside triphosphates and the nucleotide incorporating enzyme system. Under similar conditions, only partial reconstitution of the ability of soluble ribonucleic acid to accept radioactuve valine occurs. The capacity of phosphodiesterase-treated soluble ribonucleic acid to accept a mixture of 5 amino acids is maximal after pre-incubation with adenosine triphosphate, cytidine triphosphate and uridine triphosphate and the nucleotide incorporating enzyme system.

S-RNA synthesis IV. The effect of inorganic pyrophosphate and a method for the extraction of s-RNA

The effect of inorganic pyrophosphate on the incorporation of adenosine triphosphate and of cytidine triphosphate into soluble ribonucleic acid has been studied. It has been found that inorganic pyrophosphate will pyrophosphorolyze soluble ribonucleic acid liberating ribonucleoside 5′-triphosphates. This effect is predominantly on the end of the soluble ribonucleic acid molecule. Pyrophosphorolysis of the soluble ribonucleic acid in this manner has made possible tentative identification of adenylic acid as the fourth ribonucleotide of the terminal sequence of soluble ribonucleic acid. A method for the salt-alcohol extraction of soluble ribonucleic acid also is presented.

Studies on s-RNA synthesis: II. Assay and method of purification of s-RNA α, β and γ

Methods are described for the assay and for the purification of components of the enzymic system from rat liver which incorporates ribonucleotide units derived from ribonucleoside triphosphates into a soluble form of ribonucleic acid.