Stable nuclear RNA returns to post-division nuclei following release to the cytoplasm during mitosis

When nuclei from ³H-RNA-containing amebae (A. proteus), chased for as many as 8 cell generations, are implanted into unlabeled enucleate cells, the nuclei retain 30% or more of the cellular ³H-RNA (or at least 15 times the cytoplasmic concentration of ³H-RNA). After such cells divide, the daughter nuclei retain approximately the same proportion of total cellular ³H-RNA—although all (or almost all) of the nuclear RNA is liberated to the cytoplasm during mitosis. Thus, we conclude that RNA stably associated with the interphase nucleus has a particular affinity for the nucleus despite the fact it is in the cytoplasm when the chromosomes are condensed and the nuclear envelope is not intact.     

Studies of interaction of immune RNA with normal spleen cells

Summary Normal mouse spleen cells incorporate in vitro radioactively labeled immune RNA. At saturation, 10⁶ cells incorporate about 6×10¹⁶ daltons of this RNA. Immune RNA is incorporated in two times greater amounts than control RNA. Maximum incorporation is observed in the first few minutes of incubation. Immune RNA incorporated by spleen cells is present both in cytoplasm and nuclei. Protamine sulphate has stimulatory effect on immune RNA incorporation. Actinomycin D does not affect incorporation of immune RNA.This work was supported by the Polish Academy of Sciences within the project 09.7.4.1.1.     

Toxoplasma gondii: Purine synthesis and salvage in mutant host cells and parasites

Toxoplasma gondii, growing exponentially in heavily infected mutant Chinese hamster ovary cells that had a defined defect in purine biosynthesis, did not incorporate [U-¹⁴C]glucose or [¹⁴C]formate into the guanine or adenine of nucleic acids. Intracellular parasites therefore must be incapable of synthesizing purines and depend on their host cells for them. Extracellular parasites, which are capable of limited DNA and RNA synthesis, efficiently incorporated adenosine nucleotides, adenosine, inosine, and hypoxanthine into their nucleic acids; adenosine 5′-monophosphate was the best utilized precursor. Extracellular parasites incubated with ATP labeled with ³H in the purine base and ³²P in the α-phosphate incorporated the purine ring 50-fold more efficiently than they did the α-phosphate. Thus, ATP is largely degraded to adenosine before it can be used by T. gondii for nucleic acid synthesis. Two pathways for the conversion of adenosine to nucleotides appear to exist, one involving adenosine kinase, the other hypoxanthine—guanine phosphoribosyl transferase. In adenosine kinase-less mutant parasites, the efficiency of incorporation of ATP or adenosine was reduced by 75%, which indicates the adenosine kinase pathway was predominant. Extracellular parasites incorporated ATP into both the adenine and the guanine of their nucleic acids, so ATP from the host cell could supply the entire purine requirement of T. gondii. However, ATP generated by oxidative phosphorylation in the host cell is not essential for parasites because they grew normally in a cell mutant that was deficient in aerobic respiration and almost completely dependent upon glycolysis.     

Cellular distribution of RNA populations in 16-cell stage embryos of the sand dollar, Dendraster excentricus

RNA was extracted from pure preparations of micromeres and meso-plus macromeres isolated from 16-cell stage embryos of Dendraster excentricus. Molecular hybridization-competition experiments disclosed that the binding of 16-cell stage labeled RNA to denatured sperm DNA was competed equally well by micromere RNA, meso-plus macromere RNA, total 16-cell RNA and unfertilized egg RNA, indicating the egg-type populations were distributed almost equally in the different blastomeres. In contrast, experiments with ³H-RNA extracted from micromeres obtained from pulse-labeled 16-cell stage embryos showed qualitative differences when unfertilized egg RNA and total 16-cell stage RNA were used as competitors. Such differences in RNA populations could not be detected in ³H-RNA isolated from the meso-plus macromere fraction.     

Chicken erythrocyte membranes: comparison of nuclear and plasma membranes from adults and embryos

These experiments were designed to determine whether the migration of RNA molecules from an implanted nucleus to the host cytoplasm and from there into the host cell nucleus against a concentration gradient might reflect an artefact induced by the process of nuclear transplantation. That is, are RNA molecules, as previously shown for certain nuclear proteins, caused to artefactually leave a manipulated nucleus and then move into the host cell nucleus (as well as return to the grafted nucleus) during the recovery period?A variety of experiments involving different kinds of manipulative sequences and different numbers of nuclear transplantations suggest—but do not prove—that no artefact is involved in the migration of RNA from one nucleus to another but two experiments strongly support the view that the shuttling activity is a normal physiological process. One of the latter involved a determination of the rate of egress of ³H-RNA from an implanted nucleus and reveals that that rate, in contrast with the equivalent rate of egress for labeled proteins which is clearly abnormal after micromanipulation, is totally consonant with the rate of movement of RNA from nucleus to cytoplasm established from experiments that do not involve micromanipulation. The other experiment involves comparison of (1) the amount of radioactivity acquired by an unlabeled nucleus present in the cell at the time a labeled nucleus is implanted with (2) the amount of radioactivity acquired by an unlabeled nucleus implanted after a labeled nucleus had been implanted and had time to recover from any possible operation-induced trauma. With ³H-protein nuclei the host nuclei of (1) acquired much more label than the host nuclei of (2) because in (1) the host nuclei were able to acquire much of the artefactually-released ³H-protein. For the ³H-RNA experiments, however, little difference was found between (1) and (2) in the amount of label acquired by the host cell nuclei. It can be concluded that little, if any, of the non-random shuttling activity of RNA molecules can be a reflection of an artefact.     

Enrichment for temperature-sensitive and auxotrophic mutants in Saccharomyces cerevisiae by tritium suicide

Tritium suicide is shown to be an efficient technique for mutant enrichment in Saccharomyces cerevisiae. Decays from incorporated [5-³H]uridine and tritiated amino acids proved equally effective in inducing suicide; in cultures labeled to a specific activity of 50 dpm/cell, the viability fell to 2% after 12 days' storage at 4°. Mutagenized cultures were labeled with either [5-³H]uridine or a mixture of tritiated amino acids under conditions where auxotrophic mutants and temperature-sensitive mutants in RNA or protein synthesis would not incorporate a significant amount of the tritiated percursor. When survival fell to 2%, the percentages of both auxotrophic and temperature-sensitive mutants were 10-fold higher among these survivors than in the original mutagenized culture, regardless of the radioactive precursor used.     

RNA metabolism during light-induced chloroplast development in euglena

Methods are described which provide good recoveries of non-degraded chloroplast and non-chloroplast RNAs from Euglena gracilis var. bacillaris. These have been characterized by comparing the RNA from W₃BUL (an aplastidic mutant of Euglena), with that of wild-type cells which have been resolved into chloroplast and non-chloroplast fractions. Using E. coli RNA as a standard, the RNAs from W₃BUL and from the non-chloroplast fraction of green cells exhibit optical density peaks, upon sucrose gradient centrifugation, at 4S, 10S, and 19S. The chloroplast fraction exhibits optical density peaks at 19S and 14S with the 19S component predominating. Application of various techniques for the separation of RNAs to the problem of separating the chloroplast and non-chloroplast RNAs, without prior separation of the organelle, have not proven successful.

³²P_i is readily incorporated into RNA by cells undergoing light-induced chloroplast development, and fractionation at the end of development reveals that although chloroplast RNAs have a higher specific activity, the other RNAs of the cells are significantly labeled as well. The succession of labeling patterns of total cellular RNA as light-induced chloroplast development proceeds are displayed and reveal that all RNA species mentioned above eventually become labeled. In contrast, cells kept in darkness during this period incorporate little ³²P_i into any RNA fraction. In addition, a heavy RNA component, designated as 28S, while representing a negligible fraction of the total RNA, becomes significantly labeled during the first 24 hours of illumination. While there is light stimulated uptake of ³²P_i into the cells, this uptake is never limiting in the light or dark, for RNA labeling.

On the basis of these findings, we suggest that extensive activation of non-chloroplast RNA labeling during chloroplast development is the result of the activation of the cellular synthetic machinery external to the chloroplast necessary to provide metabolic precursors for plastid development. Thus the plastid is viewed as an auxotrophic resident within the cell during development. Other possibilities for interaction at this and other levels are also discussed.

     

Uptake of beta-ecdysone by the fat body and membrane vesicles of fat body cells of Sarcophaga peregrina larvae.

The fat body of Sarcophaga peregrina larvae was shown to incorporate ³H-β-ecdysone when it was incubated with the hormone in vitro. Most of the incorporated radioactivity was found in the cytoplasmic fraction as free β-ecdysone, not as a protein-β-ecdysone complex.Rapid uptake and accumulation of β-ecdysone was observed in the membrane vesicles of fat body cells in vitro. The apparent K_m value for uptake was estimated to be 1·25 × 10^?7 M. The β-ecdysone in the membrane vesicles was rapidly released when 2,4-dinitrophenol was added. These results suggest that β-ecdysone was incorporated into the membrane vesicles by active transport and not by free diffusion. The hormone is probably incorporated into larval tissues by the same mechanism as it is incorporated into the membrane vesicles of fat body cells.     

Influence of mycoplasma infection on the incorporation of different precursors into RNA components of tissue culture cells

Seven different tissue culture cells have been cultured with and without mycoplasma (M. hyorhinis) in the presence of various precursors of RNA. Total cellular RNA was isolated and analysed by electrophoresis on polyacrylamide gels. The results obtained with mycoplasma-infected cells can be summarized as follows:

1. 1. When cells are labelled with [8-³H]guanosine or [5-³H]uridine there is some incorporation into host cell 28S and 18S rRNA, but it is less than into mycoplasma 23S and 16S rRNA. [8-³H]guanosine or [5-³H]uridine are also incorporated into host cell and mycoplasma tRNA and mycoplasma 4.7S RNA, but the incorporation into host cell 5S rRNA and low molecular weight RNA components (LMW RNA) is reduced.

2. 2. [5-³H]uracil is not incorporated into host cell RNA but into mycoplasma tRNA, 4.7S RNA, a mycoplasma low molecular weight RNA component M₁ and 23S and 16S rRNA.

3. 3. [³H]methyl groups are incorporated into mycoplasma tRNA, 23S and 16S rRNA, but not into host cell 28S, 18S, 5S rRNA nor into mycoplasma 4.7S RNA.

4. 4. With [³²P]orthophosphate or [³H]adenosine as precursors, the labelling is primarily in the host RNA.

Mycoplasma infection influences the labelling of RNA primarily by an effect on the utilization of the exogenously added radioactive RNA precursors, since the generation time of mycoplasma infected cells is about the same as that of uninfected cells. Mycoplasma infection may completely prevent the identification of LMW RNA components.     

Intracellular degradation of glycoproteins in Acer pseudoplatanus L. cells

The degradation of endogenously labelled glycoproteins was studied in Acer pseudoplatanus L. cell suspension cultures in experiments using a dual-label with [¹⁴C]mannose and [³H]leucine.After harvesting the cells, protoplasts were prepared and vacuoles isolated. More than 30% of both total newly synthesized proteins (³H radioactivity) and glycoproteins (¹⁴C radioactivity) were recovered inside the vacuoles, the lytic compartment of plant cells. Half of these proteins were degraded when isolated vacuoles were incubated for 6 h at 20°C. So, the vacuolar compartment appears to be a major site of glycoprotein degradation in the cell.The glycoproteins were degraded at the same rate as the total newly synthesized proteins. However, some vacuolar hydrolytic enzymes were found to be glycoproteins and resistant to proteolytic attack. The biochemical explanation for such a resistance is not clear at this time, but in Acer cells the presence of covalently bound carbohydrates in proteins does not seem to be involved in the selectivity of protein turnover.     

Ribonucleic acid-permeable mutant of Escherichia coli

An RNA-permeable mutant was isolated from a tryptophan amber auxotrophic strain of Escherichia coli after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The rationale of the isolation was based on the suppression of an amber mutation. A strain was selected, which could grow in minimal medium supplemented with transfer RNA prepared from an suI-carrying strain but not from an su⁻ strain. This mutant incorporated³H-labeled bulk RNA into the cells at a rate 40 times higher than did the parent strain. The level of tryptophan requirement, susceptibility to the lytic action of lysozyme and RNase activity in the culture medium of the mutant strain did not differ from those of the parent strain. The mutant strain incorporated ³H-labeled ribosomal RNA equally as well as it incorporated ³H-labeled transfer RNA and the incorporation was competitively inhibited by any species of cold RNA. However, the fate of ³H-labeled rRNA after incorporation resulted in degradation to yield acid-soluble fragments whereas tRNA after incorporation remained intact in the cell.     

Effects of Actinomycin D and Ultraviolet and Ionizing Radiation on Pichinde Virus

Actinomycin D (0.05 μg/ml) suppresses the synthesis of ribosomal RNA of baby hamster kidney (BHK21) cells. The production of infectious Pichinde virus was enhanced in the presence of actinomycin D, although the production of virus particles was not substantially different from cultures inoculated in the absence of the drug. By prelabeling BHK21 cells with ³H-uridine and then allowing the virus to replicate in the presence of actinomycin D, it was possible to show that ribosomal RNA synthesized prior to infection was incorporated into the virion. A single-hit kinetics of inactivation of Pichinde virus was observed with ultraviolet light, suggesting that the virus contains only a single copy of genome per virion. Comparison of the inactivation kinetics by gamma irradiation of Pichinde virus with Sindbis and rubella virus indicated that the radiosensitive genome of Pichinde virus was about 6 × 10⁶ to 8 × 10⁶ daltons. This value is greater than the 3.2 × 10⁶ daltons which was estimated by biochemical analysis. One possible explanation considered is that the ribosomal RNA of host cell origin is functional and accounts for the differences in genome size estimated by the two methods.     

Frequency of N-benzyladenine incorporation into major RNA fractions of tobacco cell suspensions grown at stimulatory or cytostatic cytokinin concentration

The frequency of incorporation of the cytokinin N⁶-[p-³H]benzyladenine into major RNA species of tobacco (Nicotiana tabacum cv W 38) cells steadily increased as a function of its concentration in the culture medium, up to a 10 micromolar cytostatic overdose. During a 55-hour incubation of cells with 0.4 micromolar benzyladenine (BA), which is the optimal concentration for cell division, the incorporation frequency increased to one BA per 1.5 to 2.0 × 10⁴ conventional bases in total RNA. Frequencies of BA incorporation into 18S and 25S rRNA and into RNA precursors were very similar, 2- to 3-fold higher than the frequency of BA incorporation into the 4S + 5S RNA fraction. In cells incubated with 10 micromolar BA, the rate of RNA synthesis between 24 and 55 hours was lower than at optimal growth conditions; 18S and 25S rRNA synthesis was depressed more than the synthesis of 4S + 5S RNA. At 55 hours, BA was incorporated into total RNA at the steady state frequency of one per 1,300 conventional bases. All major RNA species were BA-labeled to approximately the same level, except that the labeling of the RNA precursors was 2-fold higher than the labeling of mature RNA species. These results may reflect an alteration in the processing of the RNA precursors at supra-optimal cytokinin concentration.     

Further studies on the uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice

Irvin A. D. and Young E. R. 1979. Further studies on the uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice. International Journal for Parasitology9: 109–114. An in vitro culture technique developed earlier was used to study the metabolism of nucleic acid precursors by Babesia microti and B. rodhaini of mice and by B. divergens and B. major of cattle. [³H]Hypoxanthine was readily incorporated by all species of parasite, and the presence of leucocytes did not affect this uptake. When parasites were maintained in culture their ability to incorporate [³H]hypoxanthine fell rapidly after 24 h, but when B. major was maintained at 4°C its subsequent ability to incorporate [³H]hypoxanthine persisted for at least 3 days. This finding could be of practical value in assessing infectivity of stored blood in vitro.On autoradiography, [³H]hypoxanthine appeared to be incorporated into both DNA and RNA of parasites. Salvage pathways for purine metabolism appeared to be important in all species of Babesia whereas for pyrimidine metabolism salvage pathways were more important for murine babesias and the de novo pathway more important for bovine species. This difference may relate to different permeabilities of bovine and murine erythrocyte membranes or may be a more fundamental species difference.     

Ueber den abbau von riboflavin in pflanzlichen zellsuspensionskulturen

The degradation of (2-¹⁴C)-riboflavin was investigated in plant cell suspension cultures of Cicer arietinum, Glycine max, Phaseolus aureus, Nicotiana glauca, Petroselinum crispum und Triticum aestivum. All cultures showed degradation of the isoalloxazine system with decreasing magnitude in the order shown above. Riboflavin is not degraded via lumichrome. (2-¹⁴C)lumichrome was not degraded but rather excreted from the cells into the nutrient medium after conversion to a water soluble compound. Riboflavin, but not lumichrome, was incorporated into the coenzymes FMN and FAD.     

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. II. Independent snucleotide pools for nucleic acid synthesis

Novikoff rat hepatoma cells (subline NlSl-67) in suspension culture incorporate ³H-5-uridine into the acid-soluble nucleotide pool more rapidly than into RNA, resulting in the accumulation of labeled UTP in the cells. When labeled uridine is removed from the medium after 20 minutes or 4.75 hours of labeling, the rate of incorporation of label from the nucleotide pool into RNA decreases to less than 10% of the original rate within five to ten minutes, in spite of the presence of a large pool of labeled UTP in the cells, and incorporation ceases completely if an excess of unlabeled uridine is present during the chase. Upon addition of ¹⁴C-uridine to ³H-uridine pulse-labeled, chased cells, the ¹⁴C begins to be incorporated into RNA without delay and at a rate predetermined by the concentration of ¹⁴C-uridine in the medium and without affecting the fate of the free ³H-nucleotides labeled during the pulse-period. The results are interpreted to indicate that uridine is incorporated into at least two different pools, only one of which serves as primary source of nucleotides for RNA synthesis. During active synthesis of RNA, the latter pool of free nucleotides is very small and rapidly exhausted when uridine is removed from the medium. However, UTP accumulates in this pool when cells are labeled at 4–6°, since at this temperature RNA synthesis is blocked while uridine is still phosphorylated by the cells, and the UTP is rapidly incorporated into RNA during a subsequent ten-minute chase at 37°. From these types of experiments it is estimated that only 20–25% of the total uridine nucleotides formed in the cells from uridine in the medium is directly available for RNA synthesis and that the remainder becomes available only at a slow rate. Evidence is presented which suggests that one uridine nucleotide pool is located in the cytoplasm and another in the nucleus and that mainly the nuclear pool supplies nucleotides for RNA synthesis. The size of the latter pool is under strict regulatory control, since preincubation of the cells with 0.5 mM unlabeled uridine has little or no effect on the subsequent incorporation of ³H-uridine, although it results in an increase of the overall cellular uridine nucleotide content to at least 5 mM. Other results indicate that adenosine is also incorporated into two independent nucleotide pools, whereas the cells normally appear to possess a single thymidine nucleotide pool.     

Deoxyribonucleic Acid and Ribonucleic Acid Synthesis during the Cell Expansion Phase of Cotyledon Development in Vicia faba L

In Vicia faba L., the tissue specific proteins, legumin and vicilin, are synthesized during the cell expansion phase of cotyledon development. During this growth period, RNA and nuclear DNA increase 8- to 10-fold. ³H-Uridine and ³H-adenosine are incorporated into ribosomal RNA, both 25S and 18S, and into transfer RNA. DNA isolated from cotyledons in the cell division phase of growth has been compared with DNA isolated from cotyledons undergoing expansion growth. Results indicate that the DNA increase involves replication of the whole genome (endoreduplication).     

Uridine transport and RNA synthesis in growing and in density-inhibited animal cells

Both chick embryo fibroblasts and mouse 3T3 cells reduce the rate at which they incorporate H³ uridine into RNA as their growth becomes inhibited at high cell density. This reduction occurs as a function of the cell population density, and with chick embryo cells (in contrast to 3T3 cells) it is not accompanied by significant medium alterations. This indicates the importance of the cell population density in the control of cellular metabolism. The decline in H³ uridine incorporation is paralleled by a decline in the rate of uptake of the isotope into the acid-soluble pool, suggesting that decreased entry of H³ uridine into the cell, rather than a decreased rate of RNA synthesis, is responsible for the reduced rate of incorporation into RNA of density-inhibited cells. This suggestion was confirmed by finding that when the restriction on uridine uptake was overcome by increasing the concentration of uridine in the medium, the density-dependent inhibition of uridine incorporation was largely reversed. We conclude that, even though the rate of H³ uridine incorporation into RNA is reduced three- to five-fold in density-inhibited cells, the rate of synthesis of pulse-labeled RNA continues at 70 to 85% of the rapidly-growing rate.