Effect of CDP-choline on the biosynthesis of nucleic acids and proteins in brain regions during hypoxia

The effect of CDP-choline on the in vivo incorporation of labeled precursors into DNA, RNA, and proteins in cerebral hemispheres, cerebellum, and brainstem of guinea pigs after hypoxic treatment was studied. The labeling of macromolecules extracted from the various subcellular fractions of these brain regions was also determined. Hypoxic treatment affected macromolecular labeling to a different extent in the three brain regions examined. CDP-choline treatment was not able to reverse the effect of hypoxia on DNA labeling, but it was able to remove the effect of hypoxia on RNA and protein labeling. The action of CDP-choline was particularly evident on the labeling of RNA in nuclei and mitochondria of the cerebellum and on the labeling of proteins in microsomes of the three brain regions examined.     

Effect of undernutrition on DNA and RNA synthesis in subcellular fractions from different regions of the developing rat brain

The effect of undernutrition on the incorporation of [methyl-³H]thymidine into DNA and of 5-[³H]uridine into RNA of cerebral hemispheres, cerebellum, and brain stem was studied in vivo and in vitro in rats. The labeling of DNA from nuclei and mitochondria and of RNA from nuclei, mitochondria, microsomes, and soluble fractions, was also measured in vitro. The results demonstrate that nucleic acid synthesis is impaired and delayed during undernutrition. Specific effects were observed for the different brain regions and subcellular fractions: at 10 days nuclear and mitochondrial DNA and RNA synthesis was impaired, whereas at 30 days only the mitochondrial nucleic acid synthesis was affected.The delay of DNA and RNA labeling, caused by undernutrition, was most evident in the cerebellum, probably due to its intense cell proliferation during postnatal development. The specific sensitivity of mitochondria as compared to other subcellular fractions, may be due to the intense biogenesis and/or turnover of nucleic acids in brain mitochondria not only during postnatal development, but also in the adult animal.     

Mitochondrial DNA,RNA, and protein synthesis in different regions of developing rat brain

In vivo and in vitro (tissue slices) incorporation of labeled precursors into DNA, RNA, and proteins was measured in mitochondria obtained from cerebral hemispheres, cerebellum, and brain stem of rats at different days of postnatal development. To compare the synthesis of macromolecules in mitochondria with that in other subcellular fractions, the incorporation of labeled precursors into DNA, RNA, and proteins extracted from nuclei and into RNA and proteins extracted from microsomes and cytoplasmic soluble fractions was also measured.The results obtained showed that the incorporation of [³H]thymidine into DNA and of [¹⁴C]leucine into proteins of nuclei and mitochondria from the various brain regions examined decreased during postnatal development, however, at 30 days of age the specific radioactivity of mitochondrial DNA was higher than that of nuclear DNA. [³H]Uridine incorporation into RNA decreased from 10 to 30 days of age in nuclei while in mitochondria it was quite similar at both ages. This result may be due to a faster turnover of mitochondrial RNA compared to that of mitochondrial DNA and proteins. The results obtained suggest an active biosynthesis of macromolecules in brain mitochondria and might indicate an intense biogenesis of these organelles in rat brain during postnatal development.Preliminary reports of these results were presented at the XI FEBS Meeting, Copenhagen, August 14–19, 1977, Poster number A2-2-155-3, and at III Meeting of Italian Biochem. Soc., Siena, October 3–5, 1977, Abstract C6.     

Effects of electroconvulsive shock and cycloheximide on the incorporation of amino acids into proteins of mouse brain subcellular fractions.

—The incorporation of [4,5-³H]lysine and [1-¹⁴C]leucine into the proteins of subcellular fractions of mouse brain was examined following a single electroconvulsive shock (ECS) or following cycloheximide injections. When the [³H]lysine was injected intraperitoneally immediately after the ECS the incorporation into total brain proteins was decreased by more than 50% as compared to sham controls. The proportion of lysine incorporated into the microsomal fraction was increased, but no changes were observed in the other subcellular fractions including the synaptosomal fraction. With extended pulses administered at various times after the ECS there was no change in total incorporation nor were selective effects seen in any subcellular fractions. With intracranial injections of both [³H]lysine and [¹⁴C]leucine the decreased incorporation caused by ECS was not observed, neither were there selective changes in any subcellular fraction. This lack of inhibition occurred because the intracranial injection itself severely inhibited [³H]lysine incorporation. Cycloheximide (30 mg/kg) which depressed [³H]lysine incorporation into brain proteins by 84% caused a selective depression of the incorporation into the cell-sap fraction and selective elevations into the microsomal and synaptosomal fractions. Similar changes were seen with a higher (amnestic) dose of cycloheximide (150 mg/kg) which inhibited incorporation by 94%. These data are interpreted in terms of the diverse mechanisms by which ECS and cycloheximide inhibit protein synthesis.     

Spatial and Temporal Variations in Bacterial Macromolecule Labeling with [methyl-3H]Thymidine in a Hypertrophic Lake

The incorporation of [methyl-³H]thymidine into three macromolecular fractions, designated as DNA, RNA, and protein, by bacteria from Hartbeespoort Dam, South Africa, was measured over 1 year by acid-base hydrolysis procedures. Samples were collected at 10 m, which was at least 5 m beneath the euphotic zone. On four occasions, samples were concurrently collected at the surface. Approximately 80% of the label was incorporated into bacterial DNA in surface samples. At 10 m, total incorporation of label into bacterial macromolecules was correlated to bacterial utilization of glucose (r = 0.913, n = 13, P < 0.001). The labeling of DNA, which ranged between 0 and 78% of total macromolecule incorporation, was inversely related to glucose uptake (r = -0.823), total thymidine incorporation (r = -0.737), and euphotic zone algal production (r = -0.732, n = 13, P < 0.005). With decreased DNA labeling, increasing proportions of label were found in the RNA fraction and proteins. Enzymatic digestion followed by chromatographic separation of macromolecule fragments indicated that DNA and proteins were labeled while RNA was not. The RNA fraction may represent labeled lipids or other macromolecules or both. The data demonstrated a close coupling between phytoplankton production and heterotrophic bacterial activity in this hypertrophic lake but also confirmed the need for the routine extraction and purification of DNA during [methyl-³H]thymidine studies of aquatic bacterial production.     

Effects of acute hypoxia on incorporation of [1-14C]arachidonic acid into glycerolipids of rat brain

The effects of 1 min of acute hypoxic treatment (1% O₂ in N₂) on incorporation of [1-¹⁴C]arachidonic acid into brain lipids of 16-day-old rats were investigated at 3, 6, and 12 min after intracerebral injection of the labeled fatty acid. The hypoxic-hypoxia condition associated with convulsive seizures caused a decrease in the conversion of labeled arachidonate to its acyl-CoA as well as incorporation of the label into the brain phospholipids. Among the phospholipids, there was a specific decrease in the labeling of diacylglycerophosphoinositol (GPI), and this change was accompanied by an increase in labeling of the diacylglycerols. These results indicate that metabolism of the long-chain fatty acids and some glycero-lipids in brain are vulnerable to acute hypoxic treatment.     

Effect of 5-Fluoro-2′-Deoxyuridine on [3H]Thymidine Incorporation by Bacterioplankton in the Waters of Southwest Florida

The effect of 5-fluoro-2′-deoxyuridine (FdUrd) on [methyl-³H] thymidine incorporation by bacterioplankton populations in subtropical freshwater, estuarine, and oceanic environments was examined. In estuarine waters, intracellular isotope dilution was inhibited by FdUrd, which enabled us to estimate both intracellular and extracellular isotope dilution. In 2 of 10 cases, extracellular isotope dilution was significant. At low concentrations of [methyl-³H]thymidine or [6-³H]thymidine, FdUrd completely inhibited incorporation of radioactivity into protein and RNA. At high concentrations of [³H]thymidine, however, FdUrd had little effect on labeling patterns. The dihydrofolate reductase inhibitors amethopterin and trimethoprim had no effect on macromolecular labeling patterns. These results suggest that thymidylate synthase is not involved in nonspecific labeling and that FdUrd inhibits nonspecific labeling by blocking some other enzyme involved in thymidine catabolism. In oligotrophic oceanic and freshwater samples, FdUrd did not inhibit intracellular isotope dilution or [³H]thymidine labeling of protein and RNA, but caused some inhibition of [³H]thymidine incorporation into DNA. The ability of FdUrd to inhibit nonspecific macromolecular labeling during [³H]thymidine incorporation was significantly correlated (r = 0.84) with total thymidine incorporation (in picomoles per liter per hour). The results are discussed in terms of applications of FdUrd to routine bacterial production measurements and the general assumptions of [³H]thymidine incorporation.     

Catabolism of Tritiated Thymidine by Aquatic Microbial Communities and Incorporation of Tritium into RNA and Protein

The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited significant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which ≥95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-³H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [³H]thymidine experiments and compared the degree of nonspecific labeling by [³H]adenine with that derived from [³H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.     

Selected Nucleic Acid Precursors in Studies of Aquatic Microbial Ecology

The use of radiolabeled nucleosides and nucleic acid bases to estimate the rates of RNA and DNA synthesis in naturally occurring microbial assemblages requires numerous assumptions, several of which are evaluated herein. Comparative time series analyses of the uptake and incorporation, labeling specificity, and extent of catabolism of [2-³H]adenine, [methyl-³H]thymidine, and [5-³H]uridine were performed with pure bacterial and algal cultures, as well as with environmental samples. [³H]thymidine yielded the most variable results, especially with regard to the extent of nonspecific macromolecular labeling. The pathways of [³H]thymidine and [³H]adenine metabolism were further evaluated by isotope dilution methods and by comparing incorporation patterns of thymidine labeled at different sites of the molecule. The advantages, uncertainties, and limitations of the use of radiolabeled nucleic acid precursors in studies of aquatic microbial ecology are discussed and a prospectus for future studies presented.     

Evidence for the Involvement of Docosahexaenoic Acid in Cholinergic Stimulated Signal Transduction at the Synapse

[4,5-³H]Docosahexaenoic acid ([³H]DHA) or [9,10-³H]palmitic acid ([³H]PAM) was infused intravenously for 5 min to awake, adult male rats before and after treatment with arecoline (15 mg/kg, i.p.), a cholinergic agonist. Animals were killed 15 min post-infusion, the brains were rapidly removed and subcellular fractions were obtained after sucrose density centrifugation. In control animals, [³H]DHA and [³H]PAM were incorporated into the synaptosomal fractions, representing 50%–60% of total membrane label. Most remaining membrane label (30%–40%) was in the microsomal fraction. Both fractions contained the synaptic marker synaptophysin. The remaining 10% of radioactivity was in the myelin and mitochondrial fractions. Arecoline significantly increased [³H]DHA entry into the synaptosomal fractions by 100% and into the microsomal fraction by 50%. In these fractions 60%–65% of the [³H]DHA was in phospholipid, the rest corresponding to free fatty acid and diacylglycerol. In contrast, arecoline did not change [³H]PAM incorporation into any brain fraction. These results demonstrate that plasma [³H]DHA incorporation is selectively increased into synaptic membrane phospholipids of the rat brain in response to cholinergic activation. The increased incorporation of DHA but not of PAM into synaptic membranes in response to cholinergic stimulation indicates a primary role for DHA in phospholipid mediated signal transduction at the synapse involving activation of phospholipase A₂ and/or C.     

The rat retina is a useful in vivo model to study membrane lipid synthesis: Rates of biosynthesis of neutral glycerides and phospholipids

The phospholipid composition was studied in the whole rat retina, as well as in its subcellular fractions. A relative enrichment of phosphatidic acid, phosphatidylethanolamine, and phosphatidylserine was observed in rod outer segments (ROS) in comparison with entire retina: nuclear-photoreceptor inner segmentssynaptic bodies (P₁) and synaptosomal-mitochondrial (P₂) fractions. Phosphatidylcholine was the predominant phospholipid class found in all subcellular fractions analyzed. The microsomal fraction was relatively enriched in phosphatidic acid and in phosphatidylinositol. In addition, the rat eye has been used as an in vivo system to study membrane lipid synthesis. After intravitreal injections of [2-³H]glycerol a rapid labeling of retinal glycerolipids took place. Up to 120 min after injection only the glycerol backbone of lipids was labeled. Phosphatidic acid and diacylglycerol displayed rapid rates of synthesis and breakdown. Fastest rates of labeling were attained by phosphatidylcholine followed by phosphatidylinositol. Differences were found when in vitro labeling by [2-³H]glycerol was compared with intravitreal injections. Labeling of phospholipids of subcellular fractions by intravitreally injected [2-³H]glycerol showed that most of the label accumulated in microsomal phosphatidylcholine and phosphatidylinositol. Diacylglycerols and phosphatidylethanolamine also took up 10 and 20% respectively of the precursor. It is concluded that the rat eye is a useful experimental model to study synthesis and metabolism of membrane lipids in the retina.     

The labelling of nucleic acids by radioactive precursors in the blue-green algae Anacystis nidulans and Synechocystis aquatilis Sanv.

Summary The labelling of nucleic acids of growing cells of the blue-green algae Anacystis nidulans and Synechocystis aquatilis by radioactive precursors has been studies. A. nidulans cells most actively incorporate radioactivity from [2-¹⁴C]uracil into both RNA and DNA, while S. aquatilis cells incorporate most effectively [2-¹⁴C]uracil and [2-¹⁴C]thymine.Deoxyadenosine does not affect incorporation of label from [2-¹⁴C]thymidine into DNA, but weakly inhibits [2-¹⁴C]thymine incorporation into both nucleic acids and significantly suppresses the incorporation of [2-¹⁴C]uracil.The radioactivity from [2-¹⁴C]uracil and [2-¹⁴C]thymine is found in RNA uracil and cytosine and DNA thymine and cytosine. The radioactivity of [2-¹⁴C]thymidine is incorporated into DNA thymine and cytosine. These results and data of comparative studies of nucleic acid labelling by [2-¹⁴C]thymine and [5-methyl-¹⁴C]thymine suggest that the incorporation of thymine and thymidine into nucleic acids of A. nidulans and S. aquatilis is accompanied by demethylation of these precursors. In this respect blue-green algae resemble fungi and certain green algae.     

Age-dependent changes of nucleic acid labeling in different rat brain regions

The effects of aging on in vivo DNA and RNA labeling and on RNA content in various brain regions of 4-, 12-, and 24-month-old rats were investigated. No difference in [methyl-¹⁴C]thymidine incorporation into DNA of cerebral cortex and cerebelllum during aging was observed.The ratio of RNA/DNA content significantly decreased from 4 to 24 months of age in cerebral cortex, cerebellum and striatum. RNA labeling decreased by 15% in cerebral cortex of 24-month-old animals while in the other brain areas examined (cerebellum, hippocampus, hypothalamus, brainstem, striatum) did not change during aging.In the cerebral cortex, the ratio of the specific radioactivity of microsomal RNA to that of nuclear RNA, determined by in vivo experiments, was not affected by the aging process. A significant decrease of total, poly(A)+ RNA and poly(A)- RNA content was observed in the same brain area of 24-month-old rats compared to 4-month-old ones. Moreover, densitometric and radioactivity patterns obtained by gel electrophoresis of labeled RNA after in vitro experiments (tissue slices of cerebral cortex) showed a different ribosomal RNA processing during aging. In vivo chronic treatment with CDP-choline was able to increase RNA labeling in corpus striatum of 24-month-old animals.     

Effect of experimental colchicine encephalopathy on brain protein synthesis and tubulin metabolism

Colchicine blocks axoplasmic flow and produces neurofibrillary degeneration. Brain slices from mice injected intracerebrally with colchicine incorporated more [¹⁴C]leucine into protein and had a decreased uptake of [¹⁴C]leucine into the perchloric acid-soluble pool than did their controls. Brain RNA content was decreased and free leucine increased by colchicine-induced encephalopathy. The specific activities of proteins from subcellular fractions of colchicine-injected brain were increased in the nuclear fraction, the 100,000-g supernatant, and its vinblastine-precipitable tubulin. The ratio of the specific activity of the crude mitochondrial fraction to that of the total homogenate was decreased, as would consistent with impaired movement of newly labeled protein into synaptosomes. Colchicine-injected brain extracts contained one or more cytosol fractions that stimulated ribosomal incorporation of [¹⁴C]leucine into protein in a cell-free system. Colchicine-binding-activity measurements indicated loss of soluble and particulate tubulin in colchicine-injected brains; the decrease of soluble tubulin was verified by its selective precipitation with vinblastine. Colchicine encephalopathy did not affect the rate of spontaneous breakdown of in vitro colchicine binding activity. Similarities of colchicine encephalopathy to the neuron's response to axonal damage suggest that colchicine-induced increase in protein synthesis may, in part, reflect a neuronal response to blockage of neuroplasmic transport.     

The effects of phenylpyruvate and hyperphenylalaninemia on incorporation of (6- 3 H)glucose into macromolecules of slices of rat cerebral cortex

The effects of phenylpyruvate and hyperphenylalaninemia on the incorporation of [6-³H]glucose into lipids, proteins and nucleic acids were examined in differentiating and adult rat brain. Foetal brain was most sensitive to inhibition by phenylpyruvate in vitro, with significant effects occurring at 2·5 mM for labelling of lipids and proteins and at 5 mM for labelling RNA and DNA. Older age groups were less affected, and cortical slices from adult brain were slightly or not at all affected by phenylpyruvate. The inhibition by phenylpyruvate of incorporation of [6-³H]glucose into nucleic acids, proteins, and lipids could be further distinguished by the reversibility of the effect on nucleic acid and protein synthesis at high levels of glucose and the irreversibility of the effect on lipid synthesis. Lipid synthesis was most sensitive to inhibition by phenylpyruvate at the stage of fatty acid synthesis, with lesser effect on the formation of glyceride glycerol. Exposure in utero of the foetal brain to maternal hyperphenylalaninemia resulted in reduction of 26–38 per cent in the subsequent incorporation in vitro of [6-³H]glucose into lipids, proteins, RNA and DNA of brain slices from foetal animals. Feeding hyperphenylalaninemic pregnant rats a high-glucose diet significantly protected the foetal brain from the neurotoxicity accompanying the hyperphenylalanemia.     

PROVENANCE OF THE ACETYL GROUP OF ACETYLCHOLINE AND COMPARTMENTATION OF ACETYL-CoA AND KREBS CYCLE INTERMEDIATES IN THE BRAIN IN VIVO

—The origin of the acetyl group in acetyl-CoA which is used for the synthesis of ACh in the brain and the relationship of the cholinergic nerve endings to the biochemically defined cerebral compartments of the Krebs cycle intermediates and amino acids were studied by comparing the transfer of radioactivity from intracisternally injected labelled precursors into the acetyl moiety of ACh, glutamate, glutamine, ‘citrate’(= citrate +cis-aconitate + isocitrate), and lipids in the brain of rats. The substrates used for injections were [1-¹⁴C]acetate, [2-¹⁴C]acetate, [4-¹⁴C]acetoacetate, [1-¹⁴C]butyrate, [1, 5-¹⁴C]citrate, [2-¹⁴C]glucose, [5-¹⁴C]glutamate, 3-hydroxy[3-¹⁴C]butyrate, [2-¹⁴C]lactate, [U-¹⁴C]leucine, [2-¹⁴C]pyruvate and [³H]acetylaspartate. The highest specific radioactivity of the acetyl group of ACh was observed 4 min after the injection of [2-¹⁴C]pyruvate. The contribution of pyruvate, lactate and glucose to the biosynthesis of ACh is considerably higher than the contribution of acetoacetate, 3-hydroxybutyrate and acetate; that of citrate and leucine is very low. No incorporation of label from [5-¹⁴C]glutamate into ACh was observed. Pyruvate appears to be the most important precursor of the acetyl group of ACh. The incorporation of label from [1, 5-¹⁴C]citrate into ACh was very low although citrate did enter the cells, was metabolized rapidly, did not interfere with the metabolism of ACh and the distribution of radioactivity from it in subcellular fractions of the brain was exactly the same as from [2-¹⁴C]pyruvate. It appears unlikely that citrate, glutamate or acetate act as transporters of intramitochondrially generated acetyl groups for the biosynthesis of ACh. Carnitine increased the incorporation of label from [1-¹⁴C]acetate into brain lipids and lowered its incorporation into ACh. Differences in the degree of labelling which various radioactive precursors produce in brain glutamine as compared to glutamate, previously described after intravenous, intra-arterial, or intraperitoneal administration, were confirmed using direct administration into the cerebrospinal fluid. Specific radioactivities of brain glutamine were higher than those of glutamate after injections of [1-¹⁴C]acetate, [2-¹⁴C]acetate, [1-¹⁴C]butyrate, [1,5-¹⁴C]citrate, [³H]acetylaspartate, [U-¹⁴C]leucine, and also after [2-¹⁴C]pyruvate and [4-¹⁴C]acetoacetate. The intracisternal route possibly favours the entry of substrates into the glutamine-synthesizing (‘small’) compartment. Increasing the amount of injected [2-¹⁴C]pyruvate lowered the glutamine/glutamate specific radioactivity ratio. The incorporation of ¹⁴C from [1-¹⁴C]acetate into brain lipids was several times higher than that from other compounds. By the extent of incorporation into brain lipids the substrates formed four groups: acetate > butyrate, acetoacetate, 3-hydroxybutyrate, citrate > pyruvate, lactate, acetylaspartate > glucose, glutamate. The ratios of specific radioactivity of ‘citrate’ over that of ACh and of glutamine over that of ACh were significantly higher after the administration of [1-¹⁴C]acetate than after [2-¹⁴C]pyruvate. The results indicate that the [1-¹⁴C]acetyl-CoA arising from [1-¹⁴C]acetate does not enter the same pool as the [1-¹⁴C]acetyl-CoA arising from [2-¹⁴C]pyruvate, and that the cholinergic nerve endings do not form a part of the acetate-utilizing and glutamine-synthesizing (‘small’) metabolic compartment in the brain. The distribution of radioactivity in subcellular fractions of the brain after the injection of [1-¹⁴C]acetate was different from that after [1, 5-¹⁴C]citrate. This suggests that [1-¹⁴C]acetate and [1, 5-¹⁴C]citrate are utilized in different subdivisions of the ‘;small’ compartment.     

Neonatal malnutrition in the rat affects the delivery of sulfatides from microsomes and their entry into myelin

Brain slices from 18 day old normal and malnourished rats were incubated in the presence of [³⁵S]sulfate to explore its incorporation into sulfatides of a total brain homogenate and the appearance of labeled sulfatides in different subcellular fractions. While the incorporation of label into sulfatides of the total homogenate was similar in both groups of animals, in subcellular fractions separated on a linear sucrose density gradient, labeling of sulfatides in malnourished animals was relatively higher in the region corresponding to the microsomal fraction. Time course incorporation and pulse-chase experiments were carried out to explore the kinetics of labeling of microsomal and myelin sulfatides. In pulse-chase experiments, normal controls showed a decrease in the specific radioactivity of sulfatides in the microsomal fraction after the chase, which was not observed in malnourished animals, while the appearance of labeled sulfatides in the myelin fraction of the latter group of animals was found to be lower than in normals. These results suggest that in neonatal malnutrition there is a defect in the transport of de novo synthesized sulfatides towards myelin or/and a problem in the assembly of these lipids into the myelin membrane.     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.