Reinitiation of synthesis of small cytoplasmic RNA species K and L in isolated HeLa cell nuclei in vitro.

Isolated HeLa cell nuclei were used to synthesize low molecular weight RNA species in-vitro. The labelled RNA released from the nuclei during the incubation mainly consists of 5S RNA, pre-tRNA and small cytoplasmic RNA species K and L. All these low molecular weight RNA species are synthesized by RNA polymerase C (or III). The polyanion heparin was applied to study the reinitiation of these RNA molecules in-vitro. A comparison of the kinetics of RNA synthesis in the absence and in the presence of this inhibitor demonstrates a highly efficient in-vitro reinitiation of scRNA species K and L as well as 5S and pre-tRNA by RNA polymerase C. These results indicate a general competence of this enzyme to catalyze the de-novo formation of specific gene products in-vitro.     

Biochemical and Cytological Analyses of RNA Synthesis in Kinetin-treated Pea Root Parenchyma

Excised cortical parenchyma from the pea root (cv. Little Marvel) responds to kinetin/auxin treatment with an increased rate of RNA synthesis well before reinitiating DNA synthesis. Few cells synthesize RNA in the 1st hour of culture. In the presence of kinetin/auxin, the nuclear labeling index increases 2.5-fold as compared to control cultures. The RNA synthesis response has an apparent lag period of 2-4 hours as shown by double label ([³H]adenosine/[¹⁴C]adenosine) experiments. Qualitatively, the RNA synthesized at 4-6 hours sediments between 18S and 5S. The RNA synthesized at 14-16 hours and 24-26 hours is primarily ribosomal RNA when kinetin is present. In the absence of kinetin, no clear pattern of RNA synthesis emerges.     

Transcription of Ribosomal and Messenger RNAs in Early Wheat Embryo Germination

Germinating wheat embryos (Triticum aestivum L). synthesize both ribosomal and messenger RNA at the earliest times after the onset of germination. The rates of synthesis of these two RNAs are determined at various stages in germination by an analysis of newly synthesized radioactive RNA on oligo(dT)-cellulose. The rate of messenger RNA synthesis is essentially constant throughout 18 hours of germination, while that of ribosomal RNA synthesis increases steadily, particularly after the onset of cell expansion (6 hours), reaching at 16 to 18 hours, a rate of synthesis between 5- and 20-fold greater than that observed at the earliest stages. The net effect is a relative decrease in the fraction of transcribed high molecular weight RNA that is mRNA. Throughout the first 7 hours of germination, mRNA is 25 to 30% of the transcribed fraction, whereas by 16 to 18 hours it has declined to a level of 4 to 8%.     

In Vivo and In Vitro Synthesis of Human Rhinovirus Type 2 Ribonucleic Acid

HeLa cells infected with human rhinovirus type 2 synthesize a mixture of single-and double-stranded ribonucleic acid (RNA). The RNA synthesized by the membrane-bound RNA polymerase complex in vitro is also a mixture of single- and double-stranded RNA, whereas the deoxycholate-treated RNA polymerase complex synthesized only double-stranded RNA. Although twice as much cell-associated viral RNA is synthesized in vivo at 34 C than at 37 C, there is no difference in the rate of RNA synthesized in vitro at 34 C and 37 C by the polymerase complex. The RNA polymerase complex, after treatment with deoxycholate, sediments as a broad peak with an average sedimentation value of 120S.     

The bacteriophage P4 alpha gene is the structural gene for bacteriophage P4-induced RNA polymerase

Two temperature-sensitive mutants of satellite phage P4 which do not synthesize P4 DNA at the nonpermissive temperature have been isolated. One of these phage is mutated in the P4 alpha gene. It complements a P4 delta mutant, but not a P4 alpha amber mutant; both mutants are phenotypically identical to alpha amber mutants in all properties studied. They synthesize P4 early proteins 1 and 2 as well as two additional P4-induced early proteins, 5 and 6, which are described here. P4 late proteins are not synthesized by these mutants and cannot be transactivated by helper phage P2. The mutants are unable to transactivate P2 late proteins from a P2 AB mutant. The P4 RNA polymerase activity which has been suggested to be involved in P4 DNA synthesis is not detected at the nonpermissive temperature. The P4 polymerase activity in partially purified extracts prepared from cells infected with the mutant at the permissive temperature is temperature sensitive. Reduced activity is found in vitro when these extracts are preincubated at 41 degrees C or assayed at temperatures higher than 37 degrees C. Thus, the P4 RNA polymerase is the product of the alpha gene. Temperature shift experiments show that the alpha gene product is required until late in the P4 cycle.     

Semiconservative synthesis of single-stranded RNA by bacteriophage phi 6 RNA polymerase.

The RNA polymerase in the nucleocapsid of Pseudomonas phaseolicola bacteriophage phi 6 transcribed large, medium, and small single-stranded RNA from the viral double-stranded RNA genome by a semiconservative (displacement) mechanism. Approximately 23%, 63%, and 65% of the nucleocapsid particles in the assay mixture synthesized at least one round of large, medium, and small single-stranded RNA molecules, respectively. Some of these particles reinitiated synthesis such that an average of 1.5 large, 33 medium, and 24 small single-stranded RNAs were synthesized from each double-stranded RNA.     

THE SYNTHESIS OF 5S RIBOSOMAL RNA DURING POLLEN DEVELOPMENT

Tradescantia paludosa 5S ribosomal RNA (rRNA) has been characterized with respect to its base composition and relative electrophoretic mobility in comparison with that of E. coli . The period of 5S rRNA synthesis during pollen grain development was determined by pulse labeling the RNA synthesized during a 24 hr period of development with ³²P and then chasing in cold medium until pollen maturity. The period of highest 5S rRNA synthesis was found to occur prior to microspore mitosis. During and following mitosis over a period of 4 days there was a sharp decrease in the amount of 5S RNA synthesized and during the last 48 hr of pollen maturation, no 5S rRNA was synthesized.     

Changes in ribosomal RNA, poly(A)+ RNA, and collagen alpha 2(I) mRNA synthesis and processing during hypertonic treatment of cultured chick embryo cells

The effect of hypertonic conditions on RNA synthesis in cultured chick embryo cells was examined. The appearance of newly synthesized 28 S, 18 S, and 4 S and 5 S RNA into the cytoplasm was found to be decreased by hypertonic conditions. The appearance of newly synthesized poly(A)+ RNA into the cytoplasm was also found to be depressed. To examine the behavior of a specific mRNA, nuclear and cytoplasmic levels of procollagen alpha 2(I) mRNA were measured during high salt treatment. While nuclear levels of this mRNA were found to increase, those of the cytoplasm fell markedly. S1 nuclease digestion studies of an intron flanked by two exons revealed that the pro alpha 2(I) collagen nuclear RNA that accumulated under hypertonic conditions was spliced. The nuclear accumulation of mRNA appears therefore to be due to a hypertonic block of nuclear-cytoplasmic transport, and not to an inhibition of RNA splicing.     

RNA synthesis in isolated HeLa cell nuclei.

RNA synthesis has been studied in isolated nuclei of HeLa cells. The incubation medium has been optimized for RNA synthesis and the requirements for the presence of specific components previously used by other investigators has been examined. Nuclei isolated by centrifugation through 2 M sucrose synthesize RNA linearly for at least 1 h only at low temperature (25 degrees C). Low molecular weight RNA is found in the supernatant fraction after incubation; this RNA accounts for about 10% of the RNA synthesized. The RNA which remains within nuclei is of high molecular weight and processing of this RNA into molecules of the size of cytoplasmic mRNA does not seem to occur in isolated nuclei. We have studied the effect of an inhibitor of protein-nucleic acid interaction - aurintricarboxylic acid - on RNA synthesis by isolated nuclei. At concentrations below 0.1 mM, this drug does not inhibit RNA synthesis effectively, whereas at concentrations above 0.1 mM it inhibits RNA synthesis by about 80%. In view of the proposed mechanism of action of aurintricarboxylic acid, we suggest that completion of nucleotide chains initiated before nuclei isolation accounts for 20% of the RNA synthesized in our system by isolated nuclei, whereas nucleotide chains initiated during the in vitro incubation account for 80% of the RNA synthesized.     

Inhibition of chloroplast protein synthesis by lincomycin and 2-(4-methyl-2,6- dinitroanilino)-N-methylpropionamide

Selective effects of lincomysin and cycloheximide in detached shoots of Pisum sativum on the synthesis of photosystem I and II proteins, and a chloroplast membrane protein of molecular weight 32000, confirm results obtained from studies of protein synthesis by isolated chloroplasts. A model is proposed in which one role of chloroplast ribosomes is to synthesize membrane proteins required for the immobilization of chloroplast components, such as photosystem I protein, which are synthesized by cytoplasmic ribosomes. 2-(4-Methyl-2,6-dinitroanilino)-N-methylpropionamide rapidly inhibits the synthesis of both the large and small subunits of Fraction I protein in greening detached pea shoots. This observation can be reconciled with the site of synthesis of the large subunit being in the chloroplast by a model which proposes that the small subunit is a positive initiation factor for the synthesis or translation of the messenger RNA for the large subunit.