RNA synthesis in isolated nuclei of the dinoflagellate Crypthecodinium cohnii

Atypical eukaryotic RNA polymerase activity was demonstrated in nuclei of Crypthecodinium cohnii, a eukaryote devoid of histones. Nuclei were isolated from growing cultures of this dinoflagellate and assayed for endogenous RNA polymerase (EC 2.7.7.6) activity. There was a biphasic response to Mg2+ with optima at approximately 0.01 and 0.02 M MgCl2, but in contrast to other eukaryotic RNA polymerases, this enzyme activity was inhibited by low MnCl2 concentrations. In the presence of 0.01 M MgCL2 the optimum (NH4)2SO4 concentration was 0.025 M, a concentration at which the nuclei were lysed. Incorporation of [3H]UMP into RNA was inhibited by actinomycin D and dependent on the presence of undergraded DNA, and the reaction product was sensitive to ribonuclease and KOH digestion. Omission of one or more ribonucleoside triphosphates greatly reduced the incorporation. Only a slight enhancement of RNA polymerase activity resulted from the addition of various amounts of native and denatured calf thymus DNA. Spermine caused a marked inhibition while spermidine had little effect on RNA synthesis in the nuclei. Under the optimum conditions described in the present paper the nuclei incorporated approximately 3 pmoles of [3H]UMP/microgram DNA at 25 C for 15 min, and approximately 80% of this activity was inhibited by the eukaryotic RNA polymerase II inhibitor, alpha-amanitin (20 micrograms/ml). A unique situation therefore exists in C. cohnii nuclei, in which absence of histones (a prokaryotic trait) is combined with alpha-amanitin-sensitive RNA polymerase activity (a eukaryotic trait).     

The presence of transfer RNA in the axoplasm of the squid giant axon

Previous work has revealed that 4S RNA is the primary species of RNA in the axoplasm from the giant axons of the squid and Myxicola. This study shows that axoplasmic 4S RNA from the squid giant axon has the functional properties of tRNA. Axoplasmic RNA was charged with amino acids by aminoacyl-tRNA synthetases prepared from squid brain. The aminoacylation was prevented by incubating the RNA with RNase prior to running the reaction. The amino acid-RNA complex was labile at pH 9, which is characteristic of the acyl linkage between an amino acid and its tRNA. Aminoacyl-tRNA synthetase activity was also present in the axoplasm, primarily in the soluble fraction.     

The effects of oestradiol-17beta on the ribonucleic acid polymerases of immature rabbit uterus.

Measurements of the endogenous RNA polymerase activities of nuclei isolated from immature rabbit uteri have shown that prior treatment of the animals with oestradiol-17beta has a profound effect on the apparent activities of both RNA polymerases A and B. Within 1 h of hormone treatment, the activity of RNA polymerase A is increased and continues to rise until about 4h when it reaches a plateau and remains steady until at least 8h. The activity of RNA polymerase B increases sharply after oestradiol treatment reaching an early maximum at 30-45 min. Thereafter this activity declines until by 1-2h it approaches control values but a second increase in activity then occurs with a maximum at 3-4h. Treatment of the rabbits with alpha-amanitin before the administration of oestradiol inhibits the hormone-induced stimulation of RNA polymerase A activity in isolated nuclei but when the administration of alpha-amanitin is delayed until after the early rise of RNA polymerase B activity, the oestradiol-induced stimulation of RNA polymerase A is retained. Similar results have been obtained in experiments with cycloheximide suggesting that the stimulation of RNA polymerase A activity by oestradiol is dependent on the hormone-induced stimulation of RNA polymerase B and the subsequent synthesis of protein using the RNA product of the early increase in RNA polymerase B activity. Measurement of the activities of RNA polymerases A and B after isolation of the enzymes from immature rabbit uterine nuclei before and after oestradiol treatment failed to show any differences. Therefore it would appear that the changes in the observed activities of RNA polymerases A and B in isolated nuclei are consequences of changes in the structure and function of chromatin rather than the results of modifications in the RNA polymerases themselves.     

RNA Synthesis in Isolated Nuclei of the Dinoflagellate Crypthecodinium cohnii

Atypical eukaryotic RNA polymerase activity was demonstrated in nuclei of Crypthecodinium cohnii, a eukaryote devoid of histones. Nuclei were isolated from growing cultures of this dinoflagellate and assayed for endogenous RNA polymerase (EC 2.7.7.6) activity. There was a biphasic response to Mg²⁺ with optima at ? 0.01 and 0.02 M MgCl₂, but in contrast to other eukaryotic RNA polymerases, this enzyme activity was inhibited by low MnCl₂ concentrations. In the presence of 0.01 M MgCl₂ the optimum (NH₄)₂SO₄ concentration was 0.025 M, a concentration at which the nuclei were lysed. Incorporation of [₃H]UMP into RNA was inhibited by actinomycin D and dependent on the presence of undegraded DNA, and the reaction product was sensitive to ribonuclease and KOH digestion. Omission of one or more ribonucleoside triphosphates greatly reduced the incorporation. Only a slight enhancement of RNA polymerase activity resulted from the addition of various amounts of native and denatured calf thymus DNA. Spermine caused a marked inhibition while spermidine had little effect on RNA synthesis in the nuclei. Under the optimum conditions described in the present paper the nuclei incorporated ? 3 pmoles of [₃H]UMP/muml; DNA at 25 C for 15 min, and ? 80% of this activity was inhibited by the eukaryotic RNA polymerase II inhibitor, α-amanitin (20 m?/ml). A unique situation therefore exists in C. cohnii nuclei, in which absence of histones (a prokaryotic trait) is combined with α-amanitin-sensitive RNA polymerase activity (a eukaryotic trait).     

RNA synthesis and RNA polymerase activity in hepatic nuclei isolated from rats fed the carcinogen 2-acetylaminofluorene.

An assessment was made of the activity of RNA polymerase I and the capacity for RNA synthesis, under conditions optimized for RNA polymerase I activity, in hepatic nuclei isolated from rats fed a diet containing the hepatic carcinogen AAF. Animals were maintained on the carcinogenic diet for either 4, 7 or 14 days. RNA polymerase activity progressively increased with time on the carcinogenic diet. However, the capacity for RNA synthesis remained quite constant. These results are suggestive of a progressive inhibition of DNA template activity during the early stages of AAF-induced hepatocarcinogenesis. The “permanance” of the increase in polymerase activity was examined by switching carcinogen fed animals to a control diet for either 2 or 5 days prior to making an assessment of the above parameters.     

Some effects of testosterone on the rat ventral prostate

1. Labelled testosterone, injected directly into the ventral prostate of castrated rats became associated, in part, with a cytoplasmic high-molecular-weight fraction, fraction ;A'. 2. The label present in fraction ;A' was found to be mainly associated with dihydrotestosterone. 3. Unlike fraction ;A' from testosterone-pelleted castrated rats, fraction ;A' obtained from untreated castrated rats, 48h or more after castration, was strongly inhibitory towards Escherichia coli RNA polymerase in vitro. 4. The inhibition of RNA polymerase by fraction ;A' from castrated rats was not changed by the addition of testosterone or dihydrotestosterone in vitro, but pre-heating it to 80 degrees C resulted in a loss of its inhibitory capacity. 5. Fraction ;A' from castrated rats contained ribonuclease activity. The elution profile of ribonuclease activity from Sephadex columns indicated that this activity was responsible for the inhibitory effect on the RNA polymerase assays. 6. It is concluded that, unlike the inhibitor present in the uterus of ovariectomized rats (Talwar, Segal, Evans & Davidson, 1964), no direct connexion exists between the steroid-binding capacity of prostatic fraction ;A' and its effect on E. coli RNA polymerase activity in vitro.     

In Vitro Replication of Cowpea Mosaic Virus RNA I. Isolation and Properties of the Membrane-Bound Replicase

A fraction which contained the membrane-bound cowpea mosaic virus RNA replicase was isolated from cowpea mosaic virus-infected cowpea leaves. The replicase activity appeared on day 1 after inoculation, then increased to reach a maximal on day 4. The increase in enzyme activity preceded the most-rapid virus multiplication. The membrane-bound replicase activity was almost completely insensitive to actinomycin D and DNase. The corresponding fraction from healthy leaves had no RNA-dependent RNA polymerase activity. The viral RNA synthesis in vitro proceeded linearly for 20 min and required all four ribonucleoside triphosphates and Mg(2+) ions. Mn(2+) was a poor substitute for Mg(2+). The reaction was optimal at pH 8.2. During the whole period of RNA synthesis the in vitro synthesized RNA was at least 70% resistant against RNase in 2 x SSC (0.15 M NaCl plus 0.015 M sodium citrate), but completely digestable by RNase in 0.1 x SSC. Analysis of the products by sucrose gradient centrifugation followed by treatment of separate fractions with RNase demonstrated that both single-and double-stranded RNA were present. Double-stranded RNA sedimented at about 20S, with a shoulder at 16S to 17S. A minor part of the double-stranded RNA sedimented below 10S. Single-stranded RNA sedimented with the same rate as the two viral RNAs, 26S and 34S.     

Regulation of casein messenger RNA during the development of the rat mammary gland.

Casein mRNA was isolated and partially purified from RNA extracts of rat lactating mammary glands and translated in a teterologous cell-free protein synthesizing system derived from wheat germ. Casein mRNA activity was assayed by immunoprecipitation using a specific antiserum prepared against a mixture of the purified rat caseins. Properties of rat casein mRNA were examined using a variety of sizing techniques, including chromatography on Sepharose 4B, sedimentation on sucrose gradients after heat denaturation, and electrophoresis on 2.5% agarose gels in 6 M urea. Casein mRNA activity was found in an 8-16S region after gradient centrifugation with the peak occurring at 10.5 S. In addition, the binding of rat casein mRNA to dT-cellulose was examined. Only 40% of the total casein mRNA activity was selectively retained. A partial purification of casein mRNA was accomplished by a combination of these sizing and affinity chromatography techniques. In the purified preparations casein mRNA activity comprises approximately 90% of the total mRNA activity. Characterization of this material by agarose gel electrophoresis revealed two main bands of RNA at approximately 12 and 16 S, both containing casein mRNA activity. These mRNAs were of the correct size to code for two of the principal rat caseins of approximately 25,000 and 42,000 molecular weights. Casein mRNA and total mRNA activities were then compared in total RNA extracts at various stages of normal mammary gland development in the rat, i.e. during pregnancy, lactation, and involution following weaning. A selective induction of casein mRNA activity compared to total mRNA activity was found to occur during pregnancy and lactation. Moreover, a selective loss of activity was also observed during mammary gland involution. A surprisingly high level of casein mRNA activity was found in RNA extracts from early and midpregnant mammary glands.     

Role of Subunits of 60 to 70S Avian Tumor Virus Ribonucleic Acid in Its Template Activity for the Viral Deoxyribonucleic Acid Polymerase

Heating the 60 to 70S ribonucleic acid (RNA) of Rous sarcoma virus (RSV) destroys both its subunit structure and its high template activity for RSV deoxyribonucleic acid (DNA) polymerase. In comparative analyses, it was found that the template activity of the RNA has a thermal transition of 70 C, whereas the 60 to 70S structure dissociates into 30 to 40S and several distinct small subunits with a T(m) of 55 C. Analysis by velocity sedimentation and isopycnic centrifugation of the primary DNA product obtained by incubation of 60 to 70S RSV RNA with RSV DNA polymerase indicated that most, but perhaps not all, DNA was linked to small (<10S) RSV RNA primer. Sixty percent of the high template activity of 60 to 70S RSV RNA lost after heat dissociation could be recovered by incubation of the total RNA under annealing conditions. The template activity of purified 30 to 40S subunits isolated from 60 to 70S RSV RNA was not enhanced significantly by annealing. However, in the presence of small (<10S) subunits also isolated from 60 to 70S RNA, the template activity of 30 to 40S RNA subunits was increased to the same level as that of reannealed total 60 to 70S RNA. It was concluded that neither the 30 to 40S subunits nor most of the 4S subunits of 60 to 70S RSV RNA contribute much as primers to the template activity of 60 to 70S RSV RNA. The predominant primer molecule appears to be a minor component of the <10S subunit fraction of 60 to 70S RSV RNA. Its electrophoretic mobility is similar to, and its dissociation temperature from 60 to 70S RSV RNA is higher than that of the bulk of 60 to 70S RSV RNA-associated 4S RNA. The role of primers in DNA synthesis by RSV DNA polymerase is discussed.     

Polyadenylation of pea seed RNA at the early stages of germination.

1. The RNA polyadenylating activity was found in embryo axes of dry, as well as imbibed and germinated pea seeds, both in nucleus and cytoplasm. 2. The total enzymatic activity remains unchanged during germination, but the intracellular distribution is altered; the activity in nuclei is increased about four-fold at the expense of the postmitochondrial fraction. 3. Specificity towards RNA primers of the polyadenylating system from pea embryo axes is low. 4. Cordycepin inhibits RNA polyadenylation only when [14C]ATP is used as a nucleotide donor, and has no visible influence on the activity of the system utilizing [14C]oligo(A)-nucleotides. 5. It seems that RNA in the pea embryo axes is polyadenylated by a two-step mechanism: synthesis of oligo(A)-nucleotides, and their addition to RNA.     

RNA polymerase activity in isolated nuclei ofNicotiana sanderae callus: Characteristics and modulation during differentiation

Isolated nuclei from differentiating cultures ofNicotiana sanderae showed increased levels of RNA polymerase activity as compared to the nuclei from callus cultures. The RNA synthetic activity was dependent on nucleotide triphosphates and Mg²⁺ and was destroyed by RNase. Maximum activity was obtained in the presence of 50 mM (NH₄)₂ SO₄ and α-amanitin inhibited 40% and 55% of the activity in the nuclei from callus and differentiating tissue respectively. The nuclei from differentiating tissue elicited a 3-fold increase in RNA polymerase I and a 4-fold augmentation in RNA polymerase II activities.     

Template-Dependent Initiation of Sindbis Virus RNA Replication In Vitro

Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123_C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23_C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123_C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.     

tRNA''s associated with the 70S RNA of avian myeloblastosis virus.

The distribtuion of various amino acid tRNA's in the 4S RNA components of avian myeloblastosis virus (AMV) and in 4S RNA prepared from chicken cmbryo cells, chicken myeloblasts, and chicken livers was determined. This was done by aminoacylating the 4S RNA samples with a mixture of 17 radioactive amino acids and subsequently identifying the tRNA-accepted amino acids on an amino acid analyzer after deacylation. In embryo cells, myeloblasts, and liver, tRNA's accepting all 1m amino acids were demonstrated. "Free" AMV 4S RNA was characterized by very low quantities of glutamate, valine, and tyrosine tRNA's. RNAs accepting all 17 amino acids, with the exception of tyrosine, were shown to be present in the "70S-associated" 4S RNA which dissociates at 60 C. The bulk of the 70S-associated 4S RNA was dissociated at 60 C at low ionic strength with a concomitant conversion of 70S RNA to 35S RNA. The residual associated 4S RNA was dissociated by further heating of the 35S RNA to 80 C; tryptophan tRNA accounted for greater than 90% of the total amino acid accepting activity in this fraction. The results support other studies in suggesting that tryptophan tRNA may serve as a primer for DNA synthesis in AMV, as has been shown in Rous sarcoma virus.