DNA synthesis in the giant salivary chromosomes of Drosophila virilis prior to pupation

Both two-wavelength microspectrophotometry of Feulgen-stained whole nuclei and autoradiography of H³-thymidine incorporation by giant salivary chromosomes in Drosophila virilis demonstrate a net decrease in the relative rate of salivary DNA synthesis during the late third instar and prepupal stages of development. Amounts of DNA-Feulgen per nucleus were distributed into several classes, the means of which closely approximated values projected as geometric multiples of the basic somatic DNA level estimated from hemocyte nuclei of the same larvae. Comparison of DNA polytene class frequencies showed no statistical difference between male larvae of different development stages, although female prepupae showed a greater frequency of nuclei in higher polytene classes when compared to male prepupae of the same age. Comparison of chromosomal H³-thymidine incorporation with previously described H³-histidine incorporation suggests that the amino acid labeling, which reaches a maximum during the prepupal period, has a physiological significance distinct from chromosomal endoreplication.     

STRUCTURAL AND FUNCTIONAL PROPERTIES OF POLYTENE NUCLEI ISOLATED FROM SALIVARY GLANDS OF DROSOPHILA HYDEI

Salivary gland nuclei of Drosophila hydei, isolated by a modification of the procedure described by Boyd et al. (9), retain their normal morphology during the isolation and subsequent incubation procedure. RNA synthesis was studied in isolated nuclei by biochemical and cytological techniques. In radioautographs 70% of the nuclei displayed a distribution of labeled RNA over the nuclear constituents similar to the distribution obtained after in vivo incorporation of radioactive precursor. Chromosome puffs and the nucleoli were specifically labeled. The remaining 30% of the nuclei showed a weak to very weak incorporation of radioactive precursor. In these nuclei most of the radioautographic grains were concentrated over the nucleolus, and a few grains were randomly distributed over the chromosomes. Actinomycin D and the absence of ATP, GTP, and CTP in the medium inhibited incorporation of radioactive precursor. The radioactive product was sensitive to combined pronase and RNase digestion. Addition of E. coli RNA polymerase to the incubation medium enhanced the specific labeling over the puffed regions. The sedimentation behavior of the RNA synthesized in isolated nuclei was different from that of RNA synthesized during a 20 min pulse of radioactive precursor administered to whole glands in vivo and in vitro. Neither the steroid ecdysterone nor a temperature treatment was effective in inducing new puffs in isolated nuclei.     

The replicative organization of DNA in polytene chromosomes ofDrosophila hydei

Salivary-gland nuclei ofDrosophila hydei were pulse-labeledin vitro with³H-thymidine and studied autoradiographically in squash preparations. The distribution of radioactive label over the length of the polytene chromosomes was discontinuous in most of the labeled nuclei; in some nuclei the pattern of incorporation was continuous. Comparison of the various labeling patterns of homologous chromosome regions in different nuclei showed that specific replicating units are replicated in a specific order. By combining autoradiography with cytophotometry of Feulgen-stained chromosomes, it was possible to correlate thymidine labeling of specific bands with their DNA content. The resulting data indicate that during the S-period many or perhaps all of the replicating units in a salivary-gland nucleus start DNA synthesis simultaneously but complete it at different times. Furthermore, the data support the hypothesis that the chromomere is a unit of replication or replicon. The DNA content of haploid chromomeres was found to be about 5×10^-4 pg for the largest bands inDrosophila hydei. From the results of H³-thymidine autoradiography and Feulgen-cytophotometry on neuroblast and anlage nuclei it was concluded that during growth of the polytenic nucleus heterochromatin is for the most part excluded from duplication. The results of DNA measurements in interbands of polytene chromosomes do not agree with a multistrand structure for the haploid chromatid. A chromosome model is proposed which is in accordance with the reported results and with current views concerning the replicative organization of chromosomes.     

Activation of the Cardiac Ryanodine Receptor by Sulfhydryl Oxidation is Modified by Mg2+ and ATP

The reactive disulfide 4,4′-dithiodipyridine (4,4′DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca²⁺] of 10⁻⁷ m (in the absence of Mg²⁺ and ATP), increased within ∼1 min of addition of 1 mm 4,4′-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mm cis Mg²⁺ (10⁻⁷ m cis Ca²⁺) or by 10 mm cis Mg²⁺ (10⁻³ m cis Ca²⁺), or activated by 4 mm ATP (10⁻⁷ m cis Ca²⁺), also responded to 1 mm cis 4,4′-DTDP with activation and then loss of activity. P _o and mean open time (T _o ) of the maximally activated channels were lower in the presence of Mg²⁺ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mm 4,4′-DTDP in channels inhibited by Mg²⁺, and the previously reported enhanced activation by 4,4′-DTDP in channels activated by Ca²⁺ or caffeine (Eager et al., 1997), the activation produced by 1 mm cis 4,4′-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation. Received: 8 September 1997/Revised: 20 January 1998     

Effects of inhibition of basement membrane collagen deposition on rat mammary gland development

DNA biosynthesis by a system containing giant nuclei isolated from rat trophoblast cells at Day 13 of pregnancy has been studied. A method for the isolation of giant nuclei in good yield has been described. These nuclei were capable of incorporating [³H]dTTP into DNA for 2 hr and the incorporation was proportional to the amount of DNA template (nuclei). The system was highly dependent on the four deoxyribonucleoside triphosphates, ATP, and Mg²⁺ and was stimulated by monovalent ions such as K⁺. The optimum pH was 8.6. The product of the reaction was insensitive to RNase, sensitive to DNase, and banded at 1.710 g/ml in neutral CsCl together with bulk rat trophoblast DNA. Pulse-chase and density labeling experiments utilizing bromodeoxyuridine have indicated that replicative, discontinuous synthesis was taking place at sites previously active in vivo. DNA polymerases α, β, and γ were shown to be present in the nuclei. Experiments utilizing selective inhibitors of polymerases have demonstrated that DNA replication by trophoblast nuclei in vitro was insensitive to the specific α-polymerase inhibitor, aphidicolin, but almost completely inhibited by 2′, 3′-dideoxythymidine 5′-triphosphate as well as by N-ethylmaleimide suggesting that DNA replication observed in these trophoblast nuclei in vitro may be carried out by DNA polymerase γ.     

Sorbitol Uptake in Plasma Membrane Vesicles Isolated from Immortalized Rabbit TALH Cells: Activation by a Ca2+/Calmodulin-dependent Protein Kinase

Apical plasma membrane vesicles were isolated from cultures of immortalized thick ascending limb of Henle's loop (TALH) cells and sorbitol uptake was investigated using a rapid filtration technique. In the presence of Mg²⁺, Ca²⁺, ATP, and GTP sorbitol equilibrated within three minutes with the intravesicular space; this uptake was reduced by 75% when the incubation temperature was decreased from 37°C to 4°C. A lower level of uptake was also observed in the presence of 100 μm quinidine and when Ca²⁺ or ATP were omitted from the medium. Membranes preincubated with Mg²⁺, Ca²⁺, ATP, and GTP showed, however, a high sorbitol uptake in ATP-free medium. Staurosporine, but only at high concentrations of 200 nm, inhibited sorbitol uptake when present during the transport experiments or during the preincubation with ATP. Similar results were obtained with 1 μm trifluoperazine. Protein kinase C inhibitory peptide was ineffective whereas 20 nm KT 5926, at low concentrations a specific inhibitor of Ca²⁺/calmodulin-dependent kinase, attenuated the activation. On the basis of these data we suggest that a Ca²⁺/calmodulin-dependent kinase is a mediator of regulation of sorbitol plasma membrane permeability in renal medullary cells. Received: 31 March 1997/Revised: 11 June 1997     

Sodium transport through the amiloride-sensitive Na-Mg pathway of hamster red cells

Previous work showed that in hamster red cells the amiloride-sensitive (AS) Na⁺ influx of 0.8 mmol/liter cells/hr is not mediated by Na-H exchange as in other red cells, but depends upon intracellular Mg²⁺ and can be increased by 40-fold by loading cells with Mg²⁺ to 10 mm. The purpose of this study was to verify the connection of AS Na⁺ influx with Na-dependent, amiloride-sensitive Mg²⁺ efflux and to utilize AS Na⁺ influx to explore that pathway.Determination of unidirectional influx of Na⁺ and net loss of Mg²⁺ in parallel sets of cells showed that activation by extracellular [Na⁺] follows a simple Michaelis-Menten relationship for both processes with a K _m of 105–107 mm and that activation of both processes is sigmoidally dependent upon cytoplasmic [Mg²⁺] with a [Mg²⁺]_0.5 of 2.1–2.3 mm and a Hill coefficient of 1.8. Comparison of V_max for both sets of experiments indicated a stoichiometry of 2 Na: l Mg. Amiloride inhibits Na⁺ influx and Mg²⁺ extrusion in parallel (K _i = 0.3 mm). Like Mg²⁺ extrusion, amiloride-sensitive Na⁺ influx shows an absolute requirement for cytoplasmic ATP and is increased by cell swelling. Hence, amiloride-sensitive Na⁺ influx in hamster red cells appears to be through the Na-Mg exchange pathway.There was no amiloride-sensitive Na⁺ efflux in hamster red cells loaded with Na⁺ and incubated with high [Mg²⁺] in the medium with or without external Na⁺, nor with ATP depletion. Hence, this is not a simple Na-Mg exchange carrier.     

Synthesis of herpes simplex virus DNA in a soluble nuclear extract.

A soluble extract prepared from nuclei of HeLa cells infected with herpes simplex virus type 1 has been found to synthesize herpes DNA in a process comparable to that observed in intact nuclei. The incorporation of [³H]dTTP has an absolute requirement for Mg⁺⁺ and for the other three deoxyribonucleoside triphosphates, and is relatively independent of added ATP. The reaction product, although of relatively short chain length, bands in CsCl density gradients at the density of herpes DNA and is essentially free of labeled cell DNA. Incorporation of BrdUTP results in a density shift suggesting extensive replication of endogenous DNA sequences.     

Proteins of microdissected polytenic cells

Proteins extracted from unfixed and ethanol-acetic acid fixed salivary glands were compared by SDS-polyacrylamide gel electrophoresis. The electropherograms were nearly identical when care was taken to prevent degradation in the unfixed glands. Steady state proteins from microdissected nuclei and cytoplasms showed approximately the same number of species but displayed fundamentally different electrophoretic distributions. It was calculated that the maximum number of copies for individual polypeptide species ranged from 3.8×10⁸ to 1.8×10¹⁰ in large polytene nuclei. A comparison of electropherograms from steady state nuclear proteins and nuclear proteins labeled with ³H-leucine after various periods of in vitro incubation of the glands, may suggest different turnover rates for individual protein species. An unexpected effect of incubation of the explanted salivary glands in a synthetic medium was observed. There are differences, on a quantitative level, between certain labeled proteins which accumulate in the nucleus at the beginning and at the end of a relatively long period of incubation.     

Intrasynaptosomal Free Mg2+ Concentration Measured with the Fluorescent Indicator Mag-Fura-2: Modulation by Na+ Gradient and by Extrasynaptosomal ATP

Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg²⁺ concentration ([Mg²⁺]_i) was found to be dependent on external Mg²⁺ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg²⁺ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na⁺ gradient across the plasma membrane of synaptosomes by treatment with the Na⁺ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg²⁺]_i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg²⁺. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg²⁺]_i. Reversal of the Na⁺ gradient by incubation of synaptosomes in a medium in which external Na⁺ was replaced by choline increased [Mg²⁺]_i up to 1.6 and 2.2 mM for extrasynaptosomal Mg²⁺ concentrations of 1 and 8 mM, respectively. We conclude that a Na⁺/Mg²⁺ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg²⁺ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg²⁺]_i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg²⁺]_i. It is concluded that Mg²⁺ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.     

Role of magnesium in the (Ca2++Mg2+)-stimulated membrane ATPase of human red blood cells

Summary (Ca²⁺+Mg²⁺)-stimulated ATPase of human red cell membranes as a function of ATP concentration was measured at fixed Ca²⁺ concentration and at two different but constant Mg²⁺ concentrations. Under the assumption that free ATP rather than Mg-ATP is the substrate, a value forK _m (for ATP) of 1–2m is found which is in good agreement with the value obtained in the phosphorylation reaction by A.F. Rega and P.J. Garrahan (1975.J. Membrane Biol. 22:313). Mg²⁺ increases both the maximal rate and the affinity for ATP, whereas Ca²⁺ increases the maximal rate without affectingK _m for ATP.As a by-product of these experiments, it was shown that after thorough removal of intracellular proteins the adenylate kinase reaction at approximately 1mm substrate concentration is several times faster than maximal rate of (Ca²⁺+Mg²⁺)-ATPase in red cell membranes.     

Ionic Permeability on Isolated Mouse Liver Nuclei: Influence of ATP and Ca2+

Patch-clamp experiments on isolated nuclei revealed the existence of ionic channels on the nuclear envelope, but their exact localization and function are still unknown. Recent studies have demonstrated that ATP and calcium ions play an important role in nucleocytoplasmic protein traffic. ATP is essential to allow big molecules in and out of the nucleus. However, a cytoplasmic rise of calcium ions above 300 nm decreases both ATP-dependent transport and passive diffusion through the nuclear envelope. The use of isolated nuclei placed in a saline solution provides the possibility for testing only the compounds added in the bath or in the recording pipette. In the present study, we show that ATP is responsible for an increase of nuclear ionic permeability on isolated nuclei. This result not only confirms data previously reported in in situ nuclei, but also suggests that ATP is directly involved in the modulation of passive ionic permeability. In these particular experimental conditions, calcium ions decrease the channel current starting from a concentration of 1 μm. The parallelism in the modulation action of ATP and Ca⁺⁺ between nuclear pores and ionic channels present on the nuclear envelope contributes to the support of the idea that an ionic pathway is associated with the pore complex. Received: 5 September 1996/Revised: 13 January 1997     

Effect of oligomycin on NADH oxidation and its coupled phosphorylation with the particulate fraction from dark aerobically grown Rhodospirillum rubrum

Summary NADH oxidation with the particulate fraction from dark aerobically grown Rhodospirillum rubrum is significantly stimulated by the addition of phosphate (Pi) and Mg⁺⁺, or Pi, Mg⁺⁺, ATP and the hexokinase-glucose system. K _m values for Pi in NADH oxidation and phosphorylation are 10^–3 m and 8×10^–4 m, respectively. These K _m values are almost the same as in corresponding photophosphorylation and oxidative phosphorylation catalyzed with chromatophores. As in the case of NADH oxidation with chromatophores, NADH oxidation with the particulate fraction has an optimal pH at 7.5 without additions, which is shifted to 6.9 by the addition of Pi and Mg⁺⁺, or Pi, Mg⁺⁺, ATP and the hexokinase-glucose system. The optimal pH for coupled phosphorylation is 6.9. 10 g per ml of oligomycin can suppress stimulation of NADH oxidation by Pi, or by the energy trapping system, and prevent the shift of optimal pH. The particulate fraction can catalyze Pi-incorporation into glucose-6-phosphate without externally added ATP, so that Pi-incorporation is inhibited by oligomycin. From these findings, it is concluded that NADH oxidation in the particulate fraction is tightly coupled to phosphorylation.     

Comparison of homologous polytene chromosomes in Phaseolus coccineus embryo suspensor cells: morphological,autoradiographic and cytophotometric analyses

The functional behaviour of unpaired homologous polytene chromosomes (2n=22), was investigated in nuclei of Phaseolus coccineus embryo suspensor cells. Observations were carried out on the morphological level and after ³H-thymidine and ³H-uridine autoradiography. Histone and total protein contents in the chromatin were also investigated. It was shown that corresponding regions of homologous chromosomes may show different functional structures. ³H-thymidine incorporation demonstrated differences between homologues in both DNA synthesis leading to chromosome endoreduplication (polytenization) and DNA amplification (extra DNA synthesis). ³H-uridine autoradiography showed that homologous regions in a given chromosome pair may display three labeling patterns: i) both regions labeled; ii) both regions unlabeled; iii) one region labeled and the other unlabeled. These three states are found to occur in different cells of one and the same embryo suspensor. Differences between homologous chromosome regions were also found in the ratios between DNA and protein contents in their chromatin. These results, which show that the functional activity of homologous chromosomes of the same complement may greatly differ, are discussed in relation to the characteristics of the system investigated.     

Membranous localization and properties of ATPase of rat liver lysosomes

Summary Lysosomes isolated from rat liver were found to have ATPase activity (EC No. 3.6.1.3). Subfractionation of the lysosomes revealed a membranous localization of ATPase activity. The enzyme has half maximal activity at 0.2mm ATP and is inhibited by high concentrations of ATP. The apparentK _m for divalent metal is 0.2mm, and either ca²⁺ or Mg²⁺ give maximal activity.The ATPase activity has latency when lysosomes are isolated from rats treated with Triton WR-1339. This latency may be due to the presence of internalized sucrose because the activity ofL fraction lysosomes is much less latent and Triton WR-1339 itself is not inhibitory. The latency of glucosamindase, a marker enzyme for lysosomes, contrasts with the low latency of the ATPase and points to an ATPase with an exposed active site in intact lysosomes.     

MASS PREPARATION OF NUCLEI FROM THE LARVAL SALIVARY GLANDS OF DROSOPHILA HYDEI

A method has been developed for isolating gram quantities of salivary glands from late third instar larvae of Drosophila hydei. The isolated glands have a normal appearance and incorporate RNA and DNA precursors normally. Nuclei can be isolated from these glands in 90% yield with the use of detergents. These nuclei contain morphologically normal giant polytene chromosomes.     

Mechanism of action ofVibrio cholerae enterotoxin

Summary The characteristics of the cholera toxin-stimulated adenylate cyclase of toad (Bufus marinus) and rat erythrocyte plasma membranes have been examined, with special emphasis on the response to purine nucleotides, fluoride, magnesium and catecholamine hormones. Toad erythrocytes briefly exposed to low concentrations of cholera toxin (40,000 to 60,000 molecules per cell) and incubated 2 to 4 hr at 30°C exhibit dramatic alterations in the kinetic and regulatory properties of adenylate cyclase. The approximateK _m for ATP, Mg⁺⁺ increases from about 1.8 to 3.4mm in the toxinstimulated enzyme. The stimulation by cholera toxin increases with increasing ATP, Mg⁺⁺ concentrations, from 20% at low levels (0.2mm) to 500% at high concentrations (greater than 3mm). Addition of GTP, Mg⁺⁺ (0.2mm) restores normal kinetic properties to the toxin-modified enzyme, such that stimulation is most simply explained by an elevation ofV _max. GTP enhances the toxin-treated enzyme activity two-to fourfold at low ATP concentrations, but this effect disappears at high levels of the substrate. At 0.6mm ATP and 5mm MgCl₂ the apparentK _a for GTP, Mg⁺⁺ is 5 to 10m. The control (unstimulated) enzyme demonstrates a very small response to the guanyl nucleotide. 5-ITP also stimulates the toxin-treated enzyme but cGMP, guanine, and the pyrimidine nucleotides have no effect. Cholera toxin also alters the activation of adenylate cyclase by free Mg⁺⁺, decreasing the apparentK _a from about 25 to 5mm. (–)-Epinephrine sensitizes the toad erythrocyte adenylate cyclase to GTP and also decreases the apparentK _a for free metal. Sodium fluoride, which cause a 70- to 100-fold activation of enzyme activity, has little effect on sensitivity to GTP, and does not change the apparentK _a for Mg⁺⁺; moreover, it prevents modulation of these parameters by cholera toxin. Conversely, cholera toxin severely inhibits NaF activation, and in the presence of fluoride ion the usual three- to fivefold stimulation by toxin becomes a 30 to 60% inhibition of activity. The toxin-stimulated enzyme can be further activated by catecholamines; in the presence of GTP the (–)-epinephrine stimulation is enhanced by two- to threefold. The increased catecholamine stimulation of toad erythrocyte adenylate cyclase induced by cholera toxin is explained primarily by an increase in the maximal extent of activation by the hormones. Rat erythrocyte adenylate cyclase is also modified by cholera toxin. In the mammalian system the apparent affinity for the hormone appears to be increased. Cholera toxin thus induces profound and nearly permanent changes in adenylate cyclase by a unique process which mimics the stimulation by hormones in important ways, and which also accentuates the normal hormonal response. The relevance of these findings to the mechanism of action of cholera toxin is considered.Part of this work was reported at the 1974 meeting of the Federation of American Societies for Experimental Biology (Bennett & Cuatrecasas, 1974).     

Effect of hypoxia on nucleic acid and protein synthesis in different brain regions

The incorporation of [methyl-³H]thymidine into DNA, of [5-³H]uridine into RNA, and of [1-¹⁴C]leucine into proteins of cerebral hemispheres, cerebellum, and brainstem of guinea pigs after 80 hr of hypoxic treatment was measured. Both in vivo (intraventricular administration of labeled precursors) and in vitro (tissue slices incubation) experiments were performed. The labeling of macromolecules extracted from the various subcellular fractions of the above-mentioned brain regions was also determined. After hypoxic treatment the incorporation of the labeled precursors into DNA, RNA, and proteins was impaired to a different extent in the three brain regions and in the various subcellular fractions examined; DNA and RNA labeling in cerebellar mitochondria and protein labeling in microsomes of the three brain regions examined were particularly affected.     

Effects of cations on Methanosarcina thermophila TM-1 growing on moderate concentrations of acetate: production of single cells

Summary The effects of different concentrations of Mg²⁺, Ca²⁺, or Na⁺ on the morphology and growth of Methanosarcina thermophila TM-1 growing on acetate at concentrations comparable with those found in anaerobic digestors was studied. At 30 mm Mg²⁺ or less, M. thermophila grew as large aggregates that settled rapidly. At 100 mm Mg²⁺ or more, the bacteria grew as single cells or a mixture of single cells and small aggregates is suspended culture. Mg²⁺ was necessary for growth and could not be substituted by addition of either Ca²⁺ or Na⁺. The optimal Mg²⁺ concentration was 30 mm and no growth was observed at 400 mm Mg²⁺. Cultures could be adapted to 300 mm Mg²⁺ without a change in growth rate. Added Ca²⁺ was not required for growth and had no effect on cell morphology. Inhibition by Na⁺ was directly related to the Mg²⁺ concentration. When the Mg²⁺ was 0.05 mm or less, 0.35 m Na⁺ completely inhibited growth. However, more Na⁺ was required for inhibition at higher Mg²⁺ concentrations. The same inhibitory effect of Na⁺ was observed when the temperature was 52°C or 45°C. The potential for disaggregation of Methanosarcina aggregates in anaerobic digestor environments was discussed. Offprint requests to: B. K. Ahring     

Influence of bromodeoxyuridine on the stability and function of polytene chromosomes

Polytene chromosomes of Drosophila hydei have been employed to study the effect of bromodeoxyuridine (BrdU) on chromosome function and structure. Two media have been tested for their capacity to permit substitution of thymidine by BrdU in larval salivary glands. In medium L up to 80% of the polytene DNA is replicated with maximum substitution of 80% of the thymidine in one strand by BrdU. The degree of substitution achieved with this medium is uniform within individual nuclei but varies between nuclei. An alternate medium (S) results in more complete substitution but less total DNA synthesis. Autoradiographic analyses reveal that extensive BrdU substitution is correlated with an inhibition of chromosomal RNA synthesis. Nucleolar RNA synthesis is almost totally suppressed by exposure to BrdU. Chromosomes which exhibit extensive BrdU incorporation produce puffs in response to a temperature shock or in vitro incubation. Preliminary studies suggest that puffing at some loci is anomalous. The data, therefore, indicate that moderate BrdU incorporation completely suppresses nucleolar RNA synthesis and reduces chromosomal RNA synthesis but does not generally prevent the induction of new puffs. — Exposure of larvae to BrdU also stabilizes the morphology of their polytene chromosomes in the presence of high pH and high ionic strength. Chromosome morphology that is lost at 0.6 M NaCl is regained at 0.15 M NaCl in isolated nuclei derived from BrdU-fed larvae. No recovery is observed in chromosomes of unsubstituted control larvae.