Incorporation Of 3H-uridine Research Articles

Bovine rotavirus-cell interactions: Effect of virus infection on cellular integrity and macromolecular synthesis

The kinetics of rotavirus infection were examined at the ultrastructural and molecular level. As early as 6 hr postinfection, with a cytolytic bovine rotavirus (C486), particles could be seen in the cytoplasm. At times later than 6 hr postinfection (p.i.) the number of virus particles rapidly increased such that at 12–18 hr p.i. large numbers of particles were present in rough endoplasmic cysternae (RERc) and some particles were budding through the RERc. Concomitant with virus replication, as determined by electron microscopy and labeling of viral products, there was generalized destruction of all cytoplasmic organelles as well as the nucleus and inhibition of host cell macromolecular synthesis as measured by decreased incorporation of 3H-thymidine, 3H-uridine, and 3H-amino acids. In addition to inhibition of host cell DNA synthesis, there was also breakage of host cell DNA. The rapidity with which the virus shuts off host cell function and causes cell death is discussed in relationship to the speed with which rotavirus-induced diarrhea occurs in animals following infection with rotavirus.

DNA-damage in human cells treated with the closely related alkylating agents peptichemio, m-l-sarcolysin and melphalan*

In vitro experiments were performed on human peripheral leukocytes to compare DNA repair synthesis induced by peptichemio (a multipeptide complex containing m-l-sarcolysin), m-l-sarcolysin and melphalan. The peptide bound m-l-sarcolysin gave rise to maximum DNA repair synthesis at a concentration interval between 1 × 10−5 and 3 × 10−5 M as opposed to a concentration interval of 3 × 10−4 to 1 + 10−3 M for melphalan. The peptide bound m-l-sarcolysin induced DNA repair synthesis at similar molar concentrations to m-l-sarcolysin alone. The difference between melphalan on one hand, and m-l-sarcolysin and peptichemio on the other hand, may reflect a more rapid uptake of the latter substances. Inhibition of 3H-uridine incorporation in human peripheral lymphocytes supports-this hypothesis. The frequency of sister chromatid exchanges in human fibroblasts was significantly higher after treatment with m-L-sarcolysin compared to melphalan, a finding which also indicates that these chemically closely related substances modify the cellular DNA differently. When DNA repair synthesis was induced by u.v.-light and methyl-methane-sulphonate (a monofunctional alkylating agent), no additional DNA repair synthesis was induced, if peptichemio, m-L-sarcolysin or melphalan was added. These results indicate that the repair of lesions induced by melphalan peptichemio, u.v.-light and methylmethane sulphonate is mediated by at least one common controlling enzyme.

Effects of DMSO on the structure and function of polytene chromosomes of Chironomus.

Dimethylsulfoxide (DMSO) controlled puff induction and repression (or non-induction) in larval polytene chromosomes of Chironomus tentans were studied for the case of the Balbiani rings (BR). A characteristic reaction pattern, involving BR 1, BR 2, and BR 3, all in salivary gland chromosome IV was found. In vivo exposure of 4th instar larvae (not prepupae) to 10%n DMSO at 18 degrees C first evokes an over-stimulation of BR 3 while DMSO-stimulation of puffing at BR 1 and BR 2 always follows that of BR 3. After removal of the drug, a rapid uniform collapse of all puffs occurs, thus more or less restoring the banding pattern of all previously decondensed chromosome segments. Recovery proceeds as BR's and other puffs reappear. By observing the restoration, one can locate the site from which a BR (puff) originates. BR 2, which is normally the most active non-ribosomal gene locus in untreated larvae, here serves as an example. As the sizes of BR 3, BR 1 and BR 2 change, so do the quantities of the transcriptional products in these gene loci (and vice versa), as estimated electron-microscopically in ultrathin sections and autoradiographically in squash preparations. In autoradiograms, the DMSO-stimulated BRs exhibit the most dense concentration of silver grains and therefore the highest rate of transcriptional activity. In DMSO-repressed BRs (and other puffs) the transcription of the locus specific genes is not completely shut off. In chromosomes from nuclei with high labelling intensities the repressed BRs (and other puffs) always exhibit a low level of 3H-uridine incorporation in vivo. The absence of cytologically visible BR (puff) formation therefore does not necessarily indicate complete transcriptional inactivity. Typically, before the stage of puff formation the 3H-uridine labelling first appears in the interband-like regions.

Characterization of the female sterile (1) 1304 mutant of Drosophila melanogaster: pattern of RNA metabolism in the ovary.

The female sterile mutant of Drosophila melanogaster, fs(1)1304 (1-19 +/- 2), has been characterized. Our studies show that the mutation affects the organization of nucleolar material in the ovarian nurse cells and the pattern of RNA metabolism in the ovary. Autoradiographic analysis of incorporation of 3H-uridine in vivo and analysis of 3H-uridine incorporation into high molecular weight RNA in vitro suggest that RNA from the ovaries of homozygous fs flies is degraded at a higher rate than that from heterozygous fs and wild-type ovaries. It is likely that the RNA class affected is ribosomal RNA. These data are discussed in the context of the functional role for the wild-type gene allelic to fs(1)1304, and it is suggested that one of the effects of the mutation may be on the biogenesis of ribosomes that are to be stored in the oocyte.

Inhibition of stable RNA synthesis and production of a novel RNA in heat-stressed plants

In tobacco and cowpea leaves incubated at elevated temperatures, incorporation of 32P or 3H-uridine into cytoplasmic rRNA and 5S RNA was inhibited while that into tRNA continued and additionally a novel RNA species of approximately 0.49 × 10 6 daltous was produced. This RNA was single stranded with a base ratio of 33/29/23/14 (A/U/C/U) and appears not to possess a poly (A) sequence. It hybridized to total plant DNA with kinetics suggesting a unique sequence origin and synthesis was suppressed by actinomycin D. Hybridization was competed by RNA from plants maintained only at 25°C, suggesting that low levels of this RNA occur in plants at normal temperatures even though it is not labeled detectably.

An ultrastructural and autoradiographic study of the immune response in Hyalophora cecropia pupae.

Three types of hemocytes are found in the Cecropia pupa, plasmatocytes, granular cells, and spherule cells. The granular cells are the major phagocytic blood cells, taking up the bacterium Enterobacter cloacae when this is injected into the pupae. Disintegrating blood cells are observed near the pericardial tissue. No other changes in ultrastructure are noted in hemocytes and pericardial cells which could be correlated with the immune response in these pupae. The fat body cells from pupae injected with bacteria contain abundant RER and Golgi bodies, whereas those from wounded (saline injected) and untreated controls do not. The fat body is the only tissue that responds to bacterial injection by increased incorporation of 3H-uridine into RNA. These findings support the idea that the fat body is the main site of synthesis of the immune proteins.

Chromatin function in developing brain: Acetylation of chromosomal proteins and RNA synthesis

In vitro acetylation of chromosomal proteins and RNA synthesis, and their modulation by spermine were studied using slices of cerebral cortex of 3 – 30 day old developing male rats. The degree of acetylation of both histones and nonhistone chromosomal (NHC) proteins, and RNA synthesis are high at day 3 and decrease rapidly as development progresses. Spermine stimulates acetylation of histones and NHC proteins. It also stimulates incorporation of 3H-Uridine both into nuclear and cytoplasmic RNAs. These stimulatory effects decrease rapidly up to day 9 and then decrease more slowly. Such chemical and functional changes in chromatin may be necessary for the terminal differentiation of neurons, and contribute to differential gene expression during development.

The link between dosage compensation and sex differentiation in Drosophila melanogaster.

The rate of 3H-uridine incorporation into X-chromosome and autosomal RNA was measured as an indicator of relative transcription activity in larvae carrying various Sxl mutant alleles. Hyperactivity of X chromosomes was found in heteroallelic Sxlf#1/Sxlfhv#1 and homozygous Sxlf#2 female larvae. Sxlfhv#1 homozygotes. Sxlf#1/Sxl+ heterozygotes, heteroallelic X chromosome transcription. Except for Sxlf#ba, there is a correlation between the viability of the mutants and the degree to which X-chromosomes activity is elevated. Male larvae carrying the dominant male-specific lethal mutation SxlM#1 displayed X chromosomes only half as wide as those of control larvae. However, it could not be determined whether this property is the result of a lower transcription rate of underreplication of the mutated X chromosomes. The results demonstrate that the Sxl gene plays an important role in controlling X-chromosome activity. The relationship among the various genes known to act in sex differentiation and dosage compensation is discussed.

The paradoxical effect of cycloheximide on 3H-uridine incorporation by histidine-starved concanavalin A-treated lymphocytes

Cycloheximide causes a paradoxical increase in uridine incorporation by histidine-starved lymphocytes treated with concanavalin A in a serum-free medium. The mechanism of this effect is indirect, depending on tRNA aminoacylation.

A terminal labelling assay using 3H-uridine to measure cell-mediated cytotoxic responses against parainfluenza type 3 infected target cells

The standardisation of a terminal labelling cytotoxicity assay which used the incorporation of 3H-uridine to measure target cell survival of the effector phase is described. This assay was applied to a preliminary study of the cell-mediated responses of sheep following intravenous inoculation of Pi-3 virus. The assay provided a simple, accurate, and reproducible method to measure cytotoxic responses against Pi-3 infected target cells. The experimental data did not require mathematical corrections for non-specific effects as in the 51 Cr-release assay. When cytotoxicity could be detected in uninfected target cell cultures it was low and statistically insignificant

Human monocytes and lymphocytes differ in the rate of DNA repair synthesis and viability after exposure to nitrogen mustard.

Unscheduled DNA synthesis was studied in human lymphocytes and monocytes after treatment with nitrogen mustard, methyl-methane-sulphonate and ultraviolet irradiation. The unscheduled DNA synthesis was about 4 times higher in monocytes than in lymphocytes after treatment with nitrogen mustard. Treatment with methyl-methane-sulphonate or ultraviolet irradiation confirmed the higher capacity for unscheduled DNA synthesis in the monocytes. When Ficoll-Isopaque-isolated mononuclear cells were exposed to various doses of nitrogen mustard and incubated for different periods, a dose- and time-dependent increase in trypan blue uptake was found. Furthermore, a corresponding dose- and time-dependent decrease was noted in 3H-uridine incorporation. Since trypan blue uptake will not enable differentiation between monocytes and lymphocytes appearing as a mixed cell population, 3H-uridine flash labeling index was studied after exposure to nitrogen mustard. There was a dose- and time-dependent decrease in 3H-uridine labeling index, suggesting a correlation between the absence of 3H-uridine incorporation and increase in trypan blue uptake. When compared to monocytes, the lymphocytes were more affected in this respect after exposure to nitrogen mustard. The possibility that differences in cytotoxic effect may be related to DNA repair mechanisms is discussed.

Intact testosterone receptor complex does not induce RNA synthesis of Tfm-nuclei in multinucleated urethral muscle fibres of mosaic mice.

The X-linked testicular feminization mutation (Tfm) of the mouse leads to androgen insensitivity of target cells. Through the autosomal sex reversed (Sxr) factor we have converted female heterozygotes into males. Due to X-inactivation, mosaic animals arise which are composed of androgen sensitive wild-type and androgen insensitive Tfm cells. In the androgen dependent striated urethral muscle, Tfm and wild-type cells fuse and form multinucleated muscle fibres. In the muscle fibres the Tfm nuclei are exposed to the intact cytoplasmic testosterone receptor complex coded for by the wild-type nuclei. We ask the question whether under these conditions RNA synthesis can be stimulated in the Tfm nuclei. Castrated mosaic animals were injected with testosterone, and incorporation of 3H-uridine was studied by autoradiography. We found two classes of muscle cell nuclei, those with low grain counts corresponding to the Tfm controls and those with high grain counts corresponding to the stimulated male controls. The results indicate that the Tfm nuclei are not stimulated by the intact testosterone receptor complex.

Ribosomal RNA transcription during macronuclear development ofTetrahymena thermophila.

Conjugation inTetrahymena, as in other ciliated protozoa is both a necessary tool for genetic studies and a potential model system in development. Conjugation is an ordered sequence of events which involves pair formation between two cells of different mating types followed by a precise sequence of nuclear events leading to the establishment of a new recombinant germinal nucleus (micronucleus) and then to the development of a new somatic nucleus (macronnucleus) from the germinal nucleus. The whole process takes about 20 h at 30°C and can be performed with large volumes of cells.The synthesis of ribosomal RNA during macronuclear development was studied in cultures of conjugatingTetrahymena thermophila by following the incorporation of3H-uridine into whole cells and purified ribosomal and pre-ribosomal RNA as well as by measuring bulk-RNA accumulation. In starving cultures and conjugating cultures refed with growth medium during late conjugation, some (background) ribosomal RNA synthesis was detectable 11-12 h after mixing the cells, which is the time when conjugating cells come apart but the macronnucleus is still developing. However, the major burst of rRNA accumulation occurred 13-18 h in refed conjugants.Observation of the conjugating cells by transmission electron microscopy showed that development of nucleoli took place in the macronuclear analagen concomitantly with the major burst of ribosomal RNA synthesis (13-18 h). A nucleolar organization similar to that found in vegetative cells was attained in the macronuclear anlagen 18 h after mixing of the cells.

Analysis of hippocampal RNA in rats with genetically determined differences in learning ability.

1. Breeding for speed of formation of a food-getting motor conditioned reflex in the course of 12 generations led to an increase in the intensity of synthesis of nuclear and cytoplasmic fractions of hippocampal RNA in quick-learning animals compared with slow-learning. 2. Electrophoretic analysis of nuclear and cytoplasmic fractions of hippocampal RNA revealed the presence of a similar series of classes of RNA in the initial and trained groups of specially bred animals. 3. In the region of high-molecular-weight classes of nRNA incorporation of radioactive precursor was at a higher level in quick-learning animals than in slow-learning. 4. In connection with training an increase was observed in the incorporation of radio-active precursors in the region of high-molecular-weight classes of nRNA and in the 18S-4S RNA region of cRNA. 5. Incorporation of3H-uridine into polyA-RNA and changes in incorporation of label connected with training were significantly greater in quick-learning animals.

Effect of malathion on nucleic acid synthesis in phytohemagglutinin-stimulated human lymphocytes.

The effect of malathion, an organophosphorus insecticide, on DNA and RNA synthesis was investigated by measuring the rate of incorporation of 3H thymidine and 3H uridine, respectively, into human lymphocytes stimulated by phytohemagglutinin (PHA). Increasing concentrations of malathion, from 10 to 70 micrograms/ml, were added to human lymphocyte cultures at different times in relation to PHA introduction. The lowest applied dose of malathion (10 micrograms/ml) in most cases led to increased incorporation of both 3H thymidine and 3H uridine. Higher concentrations of malathion (30, 50, 70 micrograms/ml) caused a time- and dose-dependent decrease of radioisotope incorporation.

Viral Erythrocytic Necrosis (VEN) in Atlantic Cod (Gadus morhua): In Vitro Studies

The effect of natural VEN infection on cod erythrocytes in vitro was studied using several methods. It was found that VEN-infected cells were more fragile in isotonic media than uninfected cells, but there was no difference in their susceptibility to hypotonic lysis. The proportion of VEN-infected cells was reduced in an inverse relationship to the time in culture. Incorporation of 3H-labeled precursors into protein, RNA, and DNA occurred in both infected and uninfected cells during the first 6 d of incubation and declined thereafter. There was a greater incorporation of 3H-amino acids and a tenfold greater incorporation of 3H-thymidine into VEN-infected cells than in normal cells, while there was no difference in the incorporation of 3H uridine by either type of cell. These results indicated that the virus of VEN was replicating in the erythrocytes and that the infection was detrimental to the cells.Key words: viral erythrocytic necrosis, diseases, Gadus morhua

The effect of age on the ability of oocytes to synthesize RNA and proteins during in vitro maturation

The effect of age on the ability of the oocyte to resume meiosis in vitro and to incorporate 3H-uridine and 3H-leucine into RNA and protein, respectively, was examined in the rat. In comparison with the mature control oocyte, the nucleolus of the aged oocyte tends to be retained although the rate of germinal vesicle breakdown is not altered. The incorporation of 3H-uridine is reduced, while 3H-leucine incorporation is not impaired. It is concluded that the inability of the aged oocyte to synthesize RNA may be responsible for its inability to complete meiotic maturation in vitro.

Importance of RNA synthesis whthin the lag phase preceding benzyladenine-induced growth of cucumber cotyledons in the dark.

Changes in the rate of synthesis of cellular constituents were studied in detached cucumber cotyledons (Cucumis sativus L. cv. Ohio) after treatment with benzyladenine (BA). BA treatment stimulated RNA and DNA syntheses. The stimulation of RNA synthesis occurred within the lag phase preceding BA-induced growth, while that of DNA synthesis came during the growth. On the other hand, the stimulation by BA was not detected for protein and lipid syntheses. 5-Fluorouracil (FU, 10(-3) m) plus thymidine (T, 10(-4) m) did not inhibit the fresh weight increase in both water- and BA-treated cotyledons, while α-amanitin (AM, 10 µg/ml) inhibited it in both. AM added during the first 4 hr effectively inhibited the growth stimulation by BA, but AM added 6 hr or more after BA had almost no effect. FU+T strongly inhibited the incorporation of (3)H-uridine into cytoplasmic ribosomal RNA (rRNA) and brought about labelling in regions other than those of the regular rRNA species on gel electrophoresis. On the other hand, AM had less influence on the synthesis of cytoplasmic rRNA than FU+T. These results indicate that BA stimulates cotyledon growth in the dark through its effect on messenger RNA synthesis within the lag phase of growth and the synthesis of cytoplasmic rRNA is not always necessary for cotyledon growth.

Investigations on the turnover of adrenocortical mitochondria. XIV. A correlated biochemical and stereological study of the effects of chronic treatment with ethidium bromide on the growth maintenance of the rat zona fasciculata mitochondria.

The effects of ethidium bromide (EB) on rat adrenocortical cells were investigated by biochemical and stereological methods. It was found that a treatment with ip. injections of 10 microgram/gm of EB every 12 hours induced a persistent inhibition of the incorporation of 3H-thymidine and 3H-uridine into the mitochondrial fraction, but not into the nuclear fraction. Chronic treatment with this dose of EB provoked in zona fasciculata cells of rats treated with maintenance doses of ACTH a noticeable decrease in the volume of the mitochondrial compartment (due to the decrease both in the number per cell and in the average volume of the organelles) and in the surface area of the mitochondrial cristae. These findings lend support to the hypothesis that the mechanism underlying the ACTH-induced maintenance of the growth of adrenocortical mitochondria involves mitochondrial DNA reduplication and transcription.

In vitro adriamycin sensitivity test and hormonal receptors in primary breast cancer

Cytoplasmatic estrogen (ER) and progesterone (PgR) receptors were determined by the dextran coated charcoal method in 90 primary breast cancer tissues. In parallel common intrinsic chemoresistance to cytotoxic drugs was examined, using a simple standardized in vitro short term predictive test ( 3 H-uridine incorporation adriamycin inhibition test). The in vitro response rate to adriamycin is negatively correlated to steroid receptor content, which was classified receptor-negative, -poor, and -rich. Estrogen receptor-negative cancers demonstrate an increased incidence of in vitro responses to adriamycin ( 23 38 compared to receptor-rich cancers ( 7 41 ). The same hold true for the relationship progesterone receptor content/ in vitro sensitivity to chemotherapy. Results are most clearcut in cases of identical (ER, PgR) receptor content, where response to adriamycin was observed in 17 26 receptor negative cancers against 1 12 with receptor positive specimens. These tools may predict prognosis of early recurrence following mastectomy, and may be useful in stratifying patients for individual adjuvant hormone and/or chemotherapy regimens.