Incorporation Of Radioactive Uridine Research Articles

Abstract 1649: Pim kinase inhibitor, SGI-1776, induces apoptosis in acute myeloid leukemia primary cells

Abstract Small molecule SGI-1776 was found to be potent and selective inhibitor of Pim family serine-threonine kinases [IC50: Pim-1 (7 nM), Pim-2 (363 nM) and Pim-3 (69 nM)] without effects on other kinases except Flt-3 and haspin. In preclinical studies in chronic lymphocytic leukemia, SGI-1776 treatment resulted in apoptosis induction that was associated with a decrease in RNA synthesis, Mcl-1 protein level, and c-Myc phosphorylation (Chen, LS et al, Blood 2009, 114: 4150). Increased levels of Pim-2 are reported in acute myeloid leukemia (AML) as well as Flt-3 activating mutation, providing a rationale to test SGI-1776 in AML. We evaluated SGI-1776 using FLT3-ITD AML cell lines (MOLM-13, MV4-11), xenograft models, and in primary cells from AML patients, and our results indicate that SGI-1776 is effective in AML. In MOLM-13 and MV4-11 cell lines, the IC50 for viability was < 10 nM after treatment with SGI-1776 for 72 h, which was associated apoptosis induction and reduction in phosphoBad (Ser112) and Bad protein. In xenograft models of both cell lines, treatment with 135mg/kg SGI-1776 resulted in a consistent decline in tumor volume after 14 days. To further these investigations in primary cells, we obtained cells from AML patients including FLT-3 positive and mutated FLT3-ITD patients, and evaluated the potential for SGI-1776 to induce cell death. In vitro incubation of AML cells (n = 4) with 1, 3, and 10 μM SGI-1776 for 24 h resulted in an average increase in apoptosis of 5%,12% and 19% respectively, measured using Annexin-staining compared with untreated cells. Apoptosis induction was verified via PARP cleavage immunoblot, and apoptosis was observed in cells from FLT3 negative or mutated FLT3-ITD patients. In the samples evaluated, there was disparity in cytogenetic factors, Flt-3 status and prior chemotherapeutic treatment history. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. There was no observed change in phosphorylation of Pim targets histone H3 (Ser10) or p21 (Thr145), however protein levels of both total and phospho-Bad(Ser112) decreased following SGI-1776 treatment. In contrast, there was no change in anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1, and levels of pro-apoptotic proteins, Bak and Bax, were also unaffected. However, there was a decrease in phospho-c-Myc(Ser 62), which is a Pim target and associated with stabilization of c-Myc protein. Consequently, we evaluated the potential inhibition of c-Myc driven transcription by SGI-1776 by measuring total RNA synthesis. Following treatment with 1, 3 or 10 μM SGI-1776, there was a decrease in total RNA synthesis to 75%, 42% and 25% of control respectively, measured using a radioactive uridine incorporation assay (n = 4). Taken together, SGI-1776 was demonstrated to be an effective agent against AML in cell lines, xenograft models, and primary cells, and may be an effective new agent in the treatment of AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1649.

Transcriptional patterns in the X chromosome of Sciara coprophila following exposure to magnetic fields.

We previously demonstrated that exposure of salivary gland cells of the dipteran, Sciara coprophila, to either asymmetrical or symmetrical changing magnetic fields results in an increase in the incorporation of radioactive uridine into RNA. The present report is an analysis of the grain count distribution over the X chromosome of Sciara in transcription autoradiograms following exposure of the salivary gland cells to two pulsed magnetic signals and a 72-Hz sine wave signal. The results show augmented uptake of 3H-uridine into nascent RNA chains following short exposures of the cells to magnetic fields. Transcription is augmented in previously active loci, as well as at chromosome regions that are not detectable as active in control cells. The quantitative pattern of RNA synthesis in transcription autoradiograms is hypothesized to be signal specific on the basis of differences in grain counts over significantly labelled chromosome sites.

Inhibitory effect of gonadotropin releasing hormone in the uterus of rat

Estradiol-induced incorporation of radioactive uridine and leucine into the uterus of rat was inhibited by simultaneous treatment with an analogue of gonadotropin releasing hormone. The inhibitory action of gonadotropin releasing hormone was not mediated through modulation of estradiol receptor content. However, estradiol-induced increase in the level of poly(A) polymerase was inhibited by gonadotropin releasing hormone analogue, indicating that the inhibitory effect might involve post-transcriptional phenomenon.

Multiplication of Cucumber Pale Fruit Viroid in Inoculated Tomato Leaf Protoplasts

SUMMARY Protoplasts were isolated from leaves of three tomato cultivars and inoculated with citrus exocortis viroid (CEV), potato spindle tuber viroid (PSTV) and cucumber pale fruit viroid (CPFV), respectively, using glycine-KOH-buffered mannitol at pH 9. CPFV multiplied detectably in protoplasts from the tomato cultivar Hilda 72 but replication of the other two viroids was rarely detectable. The minimum viroid concentration for infection was 10 µg RNA/ml using 106 protoplasts/ml. Newly synthesized viroid RNA was detected 36 h after inoculation by bioassay, and 48 h after inoculation by 3H-uridine incorporation into the viroid RNA band obtained by polyacrylamide gel electrophoresis. Incorporation of radioactive uridine into CPFV in inoculated protoplasts reached about 0.6% of that into tRNA during 72 h after inoculation. In protoplasts isolated from systemically infected leaves, however, incorporation of 3H-uridine into CPFV was only 0.1% of that into tRNA. In inoculated protoplasts, incorporation of 3H-uridine into CPFV was much more rapid at 35 °C than at 25 °C but incorporation into tRNA was similar at the two temperatures.

The Use of Glucosamine and Actinomycin D in Radioactive Labeling of RNA in Cells in Culture

Abstract Pretreatment of confluent cultures of mouse L cells or of well-differentiated nervous system cells in primary cultures with 20–120 mM glucosamine resulted in a stimulation of the uptake of tritiated uridine, but not of adenosine. A marked stimulation of the incorporation of radioactive uridine into acid-precipitable macromolecules was also obtained, while adenosine incorporation was unchanged. Cultures of L cells in log phase of growth were similarly affected by glucosamine pretreatment. Uridine and cytidine uptakes were stimulated by 50%. Tritiated uridine incorporation was stimulated in a biphasic manner, with maximal stimulation (115%) after 15–60 min of labeling and at later times an inhibition of incorporation. The stimulation of cytidine incorporation paralleled the stimulation of its uptake. The data indicate that there is: a) a glucosamine-induced stimulation of pyrimidine nucleoside uptake, b) a marked stimulation of tritiated uridine incorporation into RNA due to depletion of the cellular pools of unlabeled uridine nucleotides during glucosamine pre-treatment, and c) a decrease in the rate of RNA synthesis after several hours of glucosamine treatment, probably related to diminished intracellular supplies of uridine nucleotides. In the presence of glucosamine, high concentrations of actinomycin D could be used to increase nuclear retention of pulse-labeled nascent RNA. Cordycepin treatment did not result in similar retention of RNA. These techniques will be useful in autoradiographic and biochemical studies of nuclear RNA synthesis.

Changes in RNA and protein metabolism preceding onset of hemoglobin synthesis in cultured friend leukemia cells

We have studied the hematopoietic process which is induced by dimethyl sulfoxide (Me 2SO) in the Ostertag FSD-1 line of Friend erythroleukemia (FL) cells and have observed several changes that precede the onset of hemoglobin synthesis at 48 hr. Although cellular viability, mitotic rate, and deoxyribonucleic acid content are unaffected by our induction procedure, the induced cells become progressively smaller, and by 96 hr contain only 55% as much ribonucleic acid and 60–70% as much protein as control cells. The decline in ribonucleic acid content is significant by 24 hr and affects 4S and ribosomal ribonucleic acids in a noncoordinated manner throughout the hematopoietic process. Furthermore, incorporation of radioactive uridine into the 45S precursor of ribosomal ribonucleic acid is specifically inhibited by 1–2 hr after first treating FL cultures with 1% Me 2SO. This earliest known effect of Me 2SO on FL cells is followed by a decline in synthesis of protein. The basic sequence of macromolecular and cell size changes are similar to those that occur during normal erythropoiesis.

The incorporation of radioactive uridine into the hepatitis B antigen of a chimpanzee.

Radioactive (3H) uridine was incorporated into RNA isolated from the HBsAg of a chimpanzee carrier. HBsAg was purified by precipitation as an immune complex with the IgG fraction of chimpanzee anti-HBs. RNA was extracted from the washed complex with buffered phenol precipitated with alcohol and four nucleotides were identified by thin layer chromatography after alkali degradation.

Presence of "receptors" for growth hormone on membranes of rat thymocytes.

Growth hormone conjugated to Sepharose-4 B was found to mimic the action of the free hormone in the stimulation of the uptake and incorporation of radioactive uridine into RNA in thymocytes in vitro. The integrity of the cellular structure was essential for the stimulating effect of the hormone. The bonding of the immunologically reactive determinants of the hormone on membranes of thymocytes was demonstrated by electron micrographs of preparations incubated sequentially with rabbit antigrowth hormone globulins and monospecific sheep anti-rabbit globulins tagged with horse-radish peroxidase.

Protein and RNA Synthesis in Follicles Isolated from Rabbit Ovaries

The in vitro synthesis of protein and RNA in follicles from mated and unmated rabbits has been studied by measuring precursor incorporation. Radioactive methionine incorporation into follicular proteins showed a transitory rise following coitus but fell to precoital levels in ovulated follicles. LH, FSH, and cyclic AMP all exerted an acute stimulatory action on the incorporation of amino acids into proteins by follicles from unmated rabbits. Radioactive uridine incorporation into follicular RNA showed the same temporal pattern of postcoital rise followed by a decline in incorporation rates in the period around ovulation. In acute incubations, FSH and cyclic AMP stimulated RNA synthesis; LH was without acute action on this process. The results of these studies suggest that a wave of follicular protein and RNA synthesis precedes ovulation and may be partly related to the preovulatory surge in gonadotropins.

Characterization by selective labelling and electrophoresis of non-ribosomal polysomal proteins from C11D LM (TK-) cells maintained in exponential growth for 28 h in the presence of actinomycin D (author's transl)

Characterization by Selective Labelling and Electrophoresis of Non-Ribosomal Polysomal Proteins from Cl1D LM (TK−) Cells Maintained in Exponential Growth for 28 h in the Presence of Actinomycin D The partial characterization in vivo of all non-ribosomal polysomal proteins of the mouse cell line Cl1D LM (TK−), by specific labelling of these proteins, is described in this study. 1 We show that Cl1D cells are able to continue their exponential growth for at least 28 h, doubling the initial cell population, in a culture medium containing actinomycin D (0.04–0.06 μg/ml), without inflexion of the growth curve. 2 Under these culture conditions, the synthesis of nucleolar ribosomal particles is completely blocked in the growing cells by actinomycin D. No incorporation of radioactive uridine in the rRNA molecules extracted from the purified polysomes has been found after 8 and 28 h of culture with actinomycin D. The only labelled polysomal RNA was 10–30 S polydisperse on sucrose gradients, as polysomal mRNA would appear. The labelled mRNA · protein particles dissociated from polysomes by EDTA, extend as a heterogeneous peak on CsCl gradients, with densities from 1.42 to 1.52 g/ml. There was no accumulation of labelling at the level of the ribosomal peak. Base Composition of polysomal and 18–55 S nuclear RNA has been determined after the cells had been cultured with and without actinomycin D, and labelled with 32P for 16 h. The distribution of the labelled 2′,3′ nucleotides (after alkaline hydrolysis of RNA and Dowex 1X8 chromatography) demonstrated that no ribosomal RNA was synthesized in the growing cells cultured in the actinomycin D-containing medium: G + C content of polysomal RNA was 43.5%, and that of 18–55 S nuclear RNA was 41%; control values (without actinomycin D) were respectively 58.7% and 57.1%. Total RNA cellular content, as estimated by the orcinol method, was 52–53% of the control, after 28 h of exponential growth with actinomycin D. 3 Using these experimental conditions, long period of protein labelling (28 h) has been used, in an attempt to label only non-ribosomal polysomal proteins and characterize them. Purified polysomes of cells cultured with or without actinomycin D, have been dissociated by EDTA, and the location of labelled proteins has been compared on CsCl gradients. The results obtained indicate that actinomycin D reduces approximately 7 times the protein labelling in the ribosomal peaks, while no obvious change is observed in the labelling level in the mRNA · protein density region. The dissociating effect of 0.3 M to 0.8 M KCl on the labelled polysomal proteins from actinomycin D treated cells has been investigated on CsCl gradients to check where labelled free proteins and polypeptide chains are migrating. These experiments show that their density region is below 1.40 g/ml. The molecular weight of the labelled polysomal proteins was investigated by polyacrylamide-dodecylsulfate electrophoresis. Without actinomycin D, the great majority of labelled polysomal proteins is within a molecular-weight range from about 8000 to 50 000, the known molecular-weight range of structural ribosomal proteins. With actinomycin D, there is practically no labelled material in this molecular-weight range. The labelled proteins are found in the high molecular weight region of the gels, demonstrating that the molecular weight of non-ribosomal polysomal proteins (cytoplasmic polysomal proteins and mRNA bound proteins) are nearly all in the range of 50000 to 150000, or more.

Primary cultures of dissociated sympathetic neurons. III. Changes in metabolism with age in culture.

Several biochemical parameters of dissociated sympathetic neurons from superior cervical ganglia of the newborn rat were monitored as a function of age in culture. The neurons, which were grown in the virtual absence of non-neural cells, displayed a striking increase in their ability to synthesize and accumulate catecholamines. This capacity increased 50-fold during a 3-wk period in vitro, after which it appeared to reach a steady level. The major change took place during the second week. The time course of this change was not affected by plating the neurons at a higher cell density. The change in the catecholamine metabolism was far greater in magnitude and quite different in time course from the overall growth of the cells which was monitored by the incorporation of radioactive tyrosine into protein, lipid synthesis from radioactive choline, and incorporation of radioactive uridine into acid-precipitable material. Of the total tyrosine used by the cultures, the proportion devoted to catecholamine synthesis increased to 25% (a 10-fold rise) during the 3-wk period. This changing pattern of metabolism in the cultures suggested a process of maturation which may be similar to neuronal development in vivo.

Isolation and characterization of an organ specific ribonucleoprotein from goat brain.

AbstractAn organ specific protein (NP) has been isolated from the goat brain by salt fractionation and preparative polyacrylamide gel electrophoresis. The antiserum against this protein gave immunological cross reaction with the brain extracts of a wide variety of animals but did not react with extracts of liver, lung, kidney, spleen, heart and blood. Similar proteins have also been found to be present in monkey brain and human astrocytomas in culture. Evidence is presented to show that it is a ribonucleoprotein. This is based on the following observations: (1) its absorption characteristics before and after RNase treatment, (2) chemical analysis showing the presence of ribose, purines and pyrimidines, (3) incorporation of radioactive uridine and lysine in monkey brain and in human astrocytomas in culture into a protein band having electrophoretic mobility and immunological characteristics analogous to NP protein.The molecular weight of the NP protein has been found to be 25,120 Daltons, while the residual moiety after treatment with RNase has a molecular weight of 19,060. The RNA content of NP protein in four separate preparations was 20.65% (±0.55 S.D.) as determined by the orcinol colour reaction and 23.85% (± 0.88 S.D.) gauged by the ratio of extinctions at 260 and 280 nm. The base composition of RNA has also been determined. The nature of association of the RNA moiety with the protein is not known. The two are, however, not dissociable by SDS and high pH excluding the possibility of aminoacyl linkage and nonspecific adsorption.

Reversal learning and RNA labeling in neurological mutant mice and normal littermates

Incorporation of radioactive uridine into five brain areas, following reversal learning, was explored for neurological mutant mice with a deficit of myelin in the brain and spinal cord (quaking, qkqk) and normal littermates (?/+) from strain C57BL/6-qk (Jackson Laboratory). Both groups mastered the reversal task, although the mutants performed at a lower level. There was a Genotype × Test Conditions × Brain Area interaction with littermates exhibiting increased labeling of RNA in the amygdala and the hippocampus, and the quaking mice exhibiting no significant changes in these two areas following learning. Findings suggest that a genetic defect in the integrity of the CNS, a deficiency of myelin, affects reversal learning, and that this effect may be reflected in changes in RNA synthesis, or specific activity, for structures which are critical to reversal learning ability.

A Correlation between a Ribonucleic Acid Fraction Selectively Labeled in the Presence of Gibberellic Acid and Amylase Synthesis in Barley Aleurone Layers

The effects of gibberellic acid on the incorporation of radio-active uridine and adenosine into RNA of barley aleurone layers were investigated using a double labeling method combined with acrylamide gel electrophoresis. After 16 hours of incubation, gibberellic acid stimulated the incorporation of label into all species of RNA, but the effects were very small (0-10%) for ribosomal and transfer RNA and comparatively large (up to 300%) for RNA sedimenting between 5S and 14S. This result was obtained for both isolated aleurone layers and for layers still attached to the endosperm. A similar but less marked pattern occurred in layers incubated for 8 hours, but the effect was not observed after 4 hours. The gibberellic acid-enhanced RNA labeling was not due to micro-organisms. The following evidence was obtained for an association between the gibberellic acid-enhanced RNA synthesis and alpha-amylase synthesis: (a) synthesis of alpha-amylase took place in parallel with incorporation of label into gibberellic acid-RNA; (b) actinomycin D inhibited amylase synthesis and gibberellic acid-RNA by similar percentages; (c) 5-fluorouracil halved incorporation of label into ribosomal RNA but had no effect on amylase synthesis and gibberellic acid-RNA; and (d) abscisic acid had little effect on synthesis of RNA in the absence of gibberellic acid, but when it was included with gibberellic acid the synthesis of both enzyme and gibberellic acid-RNA was eliminated. We conclude that large changes in the synthesis of the major RNA species are not necessary for alpha-amylase synthesis to occur but that alpha-amylase synthesis does not occur without the production of gibberrellic acid-RNA. Gibberellic acid-RNA is probably less than 1% of the total tissue RNA, is polydisperse on acrylamide gels, and could be messenger species for alpha-amylase and other hydrolytic enzymes whose synthesis is under gibberellic acid control.

Synthesis of RNA by dissociated cells of the sea urchin embryo

The incorporation of radioactive uridine into RNA by micromeres, mesomeres and macromeres of sea urchin embryos was studied, employing methods for separating the cell types in pure suspension. At the 16-cell stage, the 3-cell types, on a per genome basis, synthesized RNA at approximately the same rate although on a per mg protein basis the micromere-RNA synthetic rate was considerably higher than either mesomeres or macromeres. At the 32-cell stage, incorporation of radioactive uridine by micromeres decreased relative to mesomeres and macromeres. It was demonstrated that radioactive uridine could not be effectively washed or diluted out of the cells of 16-cell stage embryos. Experiments on reaggregating cells did not detect any transfer or transport of radioactivity from micromeres to the other cells. Possible explanations for these findings versus the disparate results of previous investigators were presented.

Embryogenesis and germination in rye (Secale cereale L.)

When rye embryos imbibe water they rapidly return to a condition of biochemical and structural complexity. Three stages of imbibition can be recognised: Phase I a short period (10 min) of physical wetting; Phase II a longer period (1 h) when little further imbibition occurs, followed by Phase III a continuous phase of active water uptake. The latter coincides with an increase in respiration rate and an increase both in the number of mitochondria and of cristae within them. Changes in fine structure become evident in all organelles in Phase III, after 2 h of imbibition. In the unimbibed embryo endoplasmic reticulum is present only as short crescents associated with electron lucent bodies, but in Phase III the endoplasmic reticulum proliferates to form many surrounding cirlets. After 6 h these circlets become fewer and instead the endoplasmic reticulum is seen in close association with the nuclear membrane. Concurrently incorporation of radioactive uridine and thymidine is first detectible. This suggests that the large increase in protein synthesis occurs on new ribosomes present on the reticulum associated with the nuclear membrane. For the first 6 h protein synthesis must occur either on polysomes within the dense packing of ribosomes or on these circlets of endoplasmic reticulum associated with electron lucent bodies.

Changements de l'ultrastructure des cellules radiculaires de Zea mays au d�but de la germination

The ultrastructure of root cells of the germinating corn embryo has been studied during the first 72 hours of soaking. The most spectacular ultrastructural modifications occur in the nucleus. In the dry seed, the chromatin is heavily condensed and complete dispersion occurs during the first 8 hr of germination. The nucleolus appears as a compact structure in the dormant embryo, and as a uniform granular structure after 3 hr. At the 8th hour, large nucleolar vacuoles appear filled with material structurally similar to chromatin. Later on, the nucleolus is composed of a central, fibrillo-granular region surrounded by a thin, peripheral, granular region and fewer nucleolar vacuoles are found. In a previous autoradiographic study (Deltour, 1970), it was shown that the onset of RNA synthesis in these cells occurs 4 hr after soaking. From that time to the 8th hour, uridine-3H is incorporated exclusively into the chromatin. Incorporation of radioactive uridine into the nucleolus begins only after the 8th hour. It is interesting that the onset of RNA synthesis in the chromatin occurs simultaneously with the dispersion of this cell component, and that the appearance of vacuoles in the nucleolus is correlated with the beginning if uridine incorporation into this organelle. The following ultrastructural changes take place in the cytoplasm; (a) the lamellae system of proplastids increases slightly; (b) phytoferritin granules present in the proplastids of the dry seed disappear very rapidly; (c) polysomes appear 72 hr after soaking; (d) the spherosomes which are essentially localized in the vicinity of the wall in the dormant embryo become uniformly distributed throughout the cytoplasm at the 72nd hr.

Inhibition of RNA synthesis by actinomycin D: characteristic dose-response of different RNA species.

AbstractA quantitative dose‐response study was made of the inhibition by actinomycin D of the synthesis of various RNA species in cultured L cells. After a suitable preincubation with various concentrations of actinomycin, synthesis was measured by a brief incorporation of radioactive uridine. The 45S precursor of ribosomal RNA, 5S ribosomal RNA, transfer RNA and various sized fractions of heterogeneous nuclear RNA were extracted from the appropriate subcellular fractions and resolved by acrylamide gel electrophoresis. Polyribosomal messenger RNA was also studied. Over the range of actinomycin concentrations employed cellular uptake of the drug is proportional to external concentration, and maximal inhibition of RNA synthesis is generally achieved within one hour after exposure.For most of the RNA species studied the dose‐response data appear to follow an exponential inactivation function with some “tailing” evident at the higher doses. Sensitivities, defined as the reciprocal of the dose necessary to reduce synthesis to 1/e of the control level, were compared and examined with respect to the molecular weight of the RNA species under consideration. Among the heterogeneous nuclear RNA's there is a good correlation between RNA size and sensitivity, such that the sensitivity per unit molecular weight is relatively constant. A similar relationship is roughly applicable to the ribosomal and transfer RNA's when taken as a group, although in this case the sensitivity per unit molecular weight is 50 to 100 fold greater.A model is proposed in which this marked difference in actinomycin sensitivity arises as a consequence of the transcription of stretches of contiguous repetitive genes by RNA polymerase molecules which attach to the DNA only at a limited number of entry points, the highest probability of attachment being at the beginning of a stretch.

Altered viral ribonucleic acid synthesis by a temperature-sensitive mutant of eastern equine encephalitis virus

A temperature-sensitive mutant (Ets-4) derived from eastern equine encephalitis virus induced the synthesis of unusually large amounts of viral RNA in infected chick embryo cells. Ets-4 produced two to four times more RNA at 37 °C; however, its yield of mature virus was about 10 times less than the parent virus. Viral RNA extracted from infected chick embryo cells was fractionated on sucrose gradients. Three types of viral RNA were synthesized by both viruses: the infectious 45 s RNA, 27 s “interjacent” RNA and a 20 s ribonuclease resistant double-stranded RNA.Ets-4 produced greater amounts of each RNA species than did encephalitis virus. Production of the 45 s infectious RNA by Ets-4 as measured by plaque assay was less than that of the parent virus, but the incorporation of radioactive uridine into this RNA species was greater, suggesting that the 45 s RNA was biologically defective. On a specific infectivity basis (infectious 45 s RNA/radioactive 45 s RNA) the 45 s RNA of Eta-4 possessed only 2% of the infectivity observed for its parent. Viral protein synthesis, measured by the complement fixation and hemagglutinin tests, induced by Ets-4 was depressed below that of the parent virus indicating that the increased RNA synthesis of the mutant did not result in correspondingly increased amounts of virus protein. The apparent defect in viral protein synthesis may be the result of a defect in viral RNA made by Eta-4.On the basis of the information available, it appeared that viral RNA synthesis in Eta-4 infected cells was out of control. Control seemed to be restored to the level and pattern of the parent by superinfection with the parent encephalitis virus or Venezuelan equine encephalitis virus but not with other viruses.

Some notes on differential chromosome activity in Drosophila.

In the lethal mutantltl ofDrosophila melanogaster, a characteristic pattern of puffs on the right arm of the third chromosome is described. This pattern includes anaerobiosis puffs in some of the lethal larvae. Puffing, and incorporation of radioactive uridine was observed even at advanced stages of disintegration. From the behaviour of the chromosomes it may be concluded that the internal milieu ofltl larvae differs from that of artificially (by ligating) over-aged wild type larvae as well as from that of the imago.