Synthesis Of Polyadenylated RNA Research Articles

Inhibition of RNA synthesis in Neurospora crassa hyphae treated with picolinic acid.

Picolinic acid, a metal-chelating agent, blocks the nuclei of germinating conidia of Neurospora crassa in the G1 phase. The addition of picolinic acid to exponentially growing cultures of N. crassa in glucose medium causes an immediate inhibition of growth. Concentrations higher than 50 mM completely inhibit growth of mycelia, whereas lower concentrations (20-30 mM) give only a partial inhibition. Picolinic acid, added at the concentration of 20 mM, immediately blocks the accumulation of stable RNA, while protein accumulation continues at an unchanged rate for at least 30 min and only thereafter is slightly inhibited. DNA accumulation rapidly slows down after the addition of the drug. The level of polyadenylated RNA decreases quickly after the addition of picolinic acid, and the rate of its synthesis also appears to be inhibited. Picolinic acid greatly affects the metabolism of stable RNA in N. crassa: both processing and transcription of rRNA are sequentially inhibited with a moderate accumulation of precursor rRNA. It is concluded that the effects of picolinic acid on the nuclear division cycle in Neurospora may be related to the requirement of specific gene products to enter the S phase, whose synthesis is inhibited following the reduction of polyadenylated RNA synthesis.

Selective inhibition of avian sarcoma virus protein synthesis in 3-deazaadenosine-treated infected chicken embryo fibroblasts.

The production of B77 avian sarcoma virions was inhibited more than 90% in infected chicken embryo fibroblasts that were treated with 100 microM 3-deazaadenosine, an inhibitor of adenosylhomocysteine hydrolase and, for this reason, an inhibitor of methylation reactions. This nucleoside analog at a concentration of 100 microM inhibited the rates of overall cellular protein synthesis and polyadenylated RNA synthesis by 40 to 50%. Rates of viral protein synthesis were compared, and the results indicated that in infected cells treated with 3-deazaadenosine syntheses of both the precursor of the gag proteins (pr76gag) and the precursor of the reverse transcriptase (pr180gag pol) were inhibited. Synthesis of the precursor of the viral envelope glycoproteins (pr92env) appeared to be affected less by the analog treatment. Most of the host polypeptides also continued to be synthesized in 3-deazaadenosine-treated cells. The fraction of the total RNA represented by virus-specific RNA in the 3-deazaadenosine-treated cells was approximately 40% of the fraction of the total RNA represented by viral RNA in control cells, as determined by hybridization kinetics. Therefore, there was a selective inhibition of viral RNA accumulation in the presence of 3-deazaadenosine. The amounts of genome-sized 35S and 38S RNAs were reduced compared with the amounts of 28S and 21S viral mRNA's. These results suggest that selective inhibition of the synthesis of viral proteins is due to selective decreases in the amounts of the mRNA's for these polypeptides.

Level and Turnover of Polyadenylate‐Containing Ribonucleic Acid in Neurospora crassa in Different Steady States of Growth

Mycelia of Neurospora crassa in a steady state of growth in different media have a ribosomal content proportional to the rate of growth. Moreover, both the percentage of polysomes and the average ribosomal activity are about the same at all different growth rates. The content of polyadenylated RNA was determined in three different conditions of exponential growth, which allowed growth rates that ranged from 0.26 to 0.51 duplications/h, and was found to constitute about the same fraction of total RNA (4.5--5.2%). Using a kinetic approach, an equation was derived which allowed determination of the average half-lives of polyadenylated RNA: in each medium the cultures were labeled from the moment of the inoculation with [32P]orthophosphate and were then given a 10-min pulse with [5-3H]uridine when they were in the exponential phase. It was found that the determined half-lives of polyadenylated RNA vary, depending on the growth medium, between 30 and 60 min, but with no direct correlation with the growth rate. Moreover, the rate of synthesis of polyadenylated RNA relative to that of stable RNA decreased with the growth rate. On the basis of previous data on the rates of synthesis of stable RNA, it was possible to make an evaluation of the absolute rate of synthesis of polyadenylated RNA. Whereas the rate of synthesis of stable ribosomal RNA increases as a function of the square of the number of duplications per hour, the increase in the rate of synthesis of polyadenylated RNA with the growth rate is much less consistent. It is concluded that in Neurospora the growth rate does not depend on the rate of synthesis of mRNA but rather on the rate of synthesis of rRNA, which sets both the ribosomal level and the steady-state level of mRNA.

Effect of the protein-synthesis-initiation inhibitor 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide on ribonucleic acid synthesis in radish seedlings.

2-(4-Methyl-2,6-dinitroanilino)-N-methylpropionamide, an inhibitor of initiation of protein synthesis in plants, selectively alters rRNA synthesis. Ribosomal genes are transcribed in the absence of protein synthesis, but processing of the transcripts is impaired. This results in an accumulation of the 2.3 X 10(6)-dalton pre-rRNA. Synthesis of polyadenylated RNA is not affected by the drug.

Poly(A) and synthesis of polyadenylated RNA in the preimplantation mouse embryo

Investigations were conducted to quantitate polyadenylic acid and estimate the synthesis of polyadenylated RNA in mouse embryos at several stages of preimplantation development. Poly(A) was assayed by molecular hybridization of total embryonic RNA with [ 3H]polyuridylic acid. The mean values of poly(A) in the ovulated oocytes and in the one-cell, two-cell, and blastocyst stages of the embryo were 1.9, 1.6, 0.68, and 3.8 pg, respectively. Synthesis of polyadenylated RNA was estimated by affinity chromatography of [ 3H]uridine-labeled embryo RNA on oligo(dT)-cellulose. The proportions of newly synthesized RNA bound by oligo(dT)-cellulose at the 2-cell, 8- to 16-cell, and blastocyst stages were 6.7, 3.5, and 3.3%, respectively. These results suggest that significant quantities of maternal mRNA are present during early development of the mouse, but that polyadenylation of RNA transcribed from the embryonic genome occurs as early as the two-cell stage.

Effects of alpha-amanitin on RNA synthesis by mouse embryos in culture.

Investigations were conducted to test the effects of alpha-amanitin on RNA synthesis in preimplantation mouse embryos. Exposure of embryos in culture to 1-100 microgram/ml alpha-amanitin produced a dose- and time-dependence suppression of total RNA synthesis as measured by incorporation of [3H]uridine. Synthesis of polyadenylated RNA in blastocyst-stage embryos was abolished by alpha-amanitin-treatment at concentrations and exposure times that suppressed total RNA synthesis by less than 15%. DNA-dependent RNA polymerase activity was measured in lysates of embryos at several stages of preimplantation development. alpha-Amanitin suppressed total polymerase activity assayed under ionic conditions favorable to the detection of RNA polymerase II. Electrophoretic analyses revealed that preincubation of blastocysts in 100 microgram/ml alpha-amanitin reduced labelling of cytoplasmic 28S and 18S RNA by inhibition of both synthesis and maturation of nucleolar 45SrRNA-precursor. This action of alpha-amanitin on nucleolar RNA synthesis cannot be correlated with the minimal suppression of nucleolar RNA polymerase activity and suggests that the synthesis and processing of rRNA may be under control of nucleoplasmic gene products.

The synthesis of polyadenylated RNA and other RNA species in synchronised CHK cells

Chinese hamster kidney cells have been synchronised by the double thymidine block technique. The rate of incorporation of [5- 3H]uridine into the polyadenylated RNA fraction has been analysed throughout two complete cell cycles and compared with the rate of incorporation into other RNA species and the UTP pool. In contrast with other reports, we have found that synthesis of 18 S rRNA continues steadily throughout the cell cycle, as does synthesis of 5 S RNA and tRNA. In the case of the polyadenylated RNA, however, our data indicate a major peak of synthesis during S phase and a smaller peak coinciding with G 1.

Polyadenylated RNA from Acetabularia

Polyadenylated RNA was isolated from whole cells and from anucleate cytoplasm of Acetabularia mediterranea. It is synthesised in nucleate but not in anucleate cells. This RNA has a high molecular weight ranging from 0.5 to 3.0 × 106 daltons. In contrast to the RNA of 80 S ribosomes the synthesis of polyadenylated RNA is only slightly enhanced in nucleated cell fragments during the initial phase of regeneration. Newly synthesised polyadenylated RNA migrates from the nucleus through the stalk at about 5 mm a day. The data suggest that polyadenylated RNA migrates independently of 80 S ribosomes.