Incorporation Of Lysine Research Articles

Stimulation of retinal capillary pericyte protein and collagen synthesis in culture by high-glucose concentration.

The influence of glucose concentration on cell multiplication and protein synthesis was studied in synchronized, long-term cultures of bovine retinal microvessel pericytes. The cell multiplication rate and the mitotic rate were reduced in media containing 20 mM glucose to 57% and 54%, respectively, of that obtained in media containing 5 mM glucose. Elevated glucose, however, did not change the DNA content of individual cells. Protein and collagen synthesis were measured by the incorporation of radioactive proline and lysine, or the posttranslational production of hydroxyproline and hydroxylysine, respectively. High glucose stimulated protein and collagen synthesis per cell 2.2 +/- 0.10 (SD) and 2.1 +/- 0.06 times, respectively. Aspirin (0.5 mM), an inhibitor of nonenzymatic glycosylation, did not alter the effect of elevated glucose concentration on protein and collagen synthesis.

Mechanisms of action of aminoglycoside antibiotics in eucaryotic protein synthesis.

Tetrahymena thermophila is a eucaryotic organism that is highly susceptible to growth inhibition by aminoglycoside antibiotics. Concentrations of paromomycin, gentamicin G418, and hygromycin B at 22, 10, and 17 microM, respectively, inhibited growth by 50%. A combination of in vitro and in vivo methods was used to determine the mechanisms of action of these aminoglycoside antibiotics on protein synthesis in T. thermophila. Analysis of polysome profiles from paromomycin- and gentamicin G418-treated cells showed clear, progressive depletions of polysomes concomitant with an inhibition of in vivo [14C] lysine incorporation. In vitro, paromomycin and gentamicin G418, which are disubstituted 2-deoxystreptamine-containing molecules, were not very effective inhibitors of either the translocation of peptidyl-tRNA or the elongation of nascent polypeptide chains on polysomes. In contrast, we found that the translocation of phe-tRNA on polyuridylate programmed ribosomes was susceptible to inhibition by paromomycin. We conclude that the primary inhibitory action of paromomycin and gentamicin G418 was at (i) an early stage of elongation after initiation, (ii) the initiation stage of translation, or (iii) a stage of translation before initiation. Hygromycin B, which is a monosubstituted 2-deoxystreptamine-containing aminoglycoside, potently inhibited the elongation of nascent chains during the translation of polysomes. In addition, the in vitro translation of polysomes from two hygromycin B-resistant mutants was resistant to the inhibition of elongation caused by hygromycin B.

The role of lysine-rich proteins in the acute steroidogenic response of rat adrenal cells to ACTH

The possibility that a lysine-rich protein was involved in the acute stimulation of steroidogenesis by ACTH was investigated using [ 3H] labelled lysine and isolated adrenal cells. The results demonstrated that cycloheximide inhibited steroidogenesis in a dose-dependent, rapid fashion and inhibited the incorporation of radioactive lysine into protein. However cells incubated in a lysine-free medium showed the same response to ACTH as cells incubated in a lysine-containing medium. It was also demonstrated that ACTH had no effect on the incorporation of tritiated lysine into the protein or small peptide fractions. These observations suggest that a rapidly synthesised, lysine-rich protein is not involved in the acute response to ACTH.

Identification of a Ca2+ requirement for protein synthesis in eukaryotic cells.

The incorporation of methionine, lysine, and leucine into protein was studied in Ca2+-depleted and Ca2+-restored preparations of C-6 glial tumor cells in minimal medium. Although incorporation proceeded at linear rates in both preparations for more than 1 h and into the same spectrum of proteins, Ca2+-restored cells incorporated amino acid 5- to 10-fold more rapidly than Ca2+-depleted cells. Addition of approximately 200 microM Ca2+ in excess of chelator was required to achieve maximal rates of incorporation in Ca2+-depleted preparations. Stimulation by Ca2+ was rapid in onset (several minutes) and slowly reversible by chelator. Ca2+ was uniquely potent and specific among physiologically occurring cations in conferring such stimulation. Stimulation of amino acid incorporation by Ca2+ occurred over a broad range of pH and osmolarities and was facilitated by Mg2+. The effects of Ca2+ in stimulating amino acid incorporation were not traceable to changes in cAMP metabolism, amino acid uptake, protein catabolism, cell ATP or GTP content, or aminoacylation of transfer RNA. Actinomycin D (1 microgram/ml) did not block the stimulatory effects of Ca2+ although puromycin and cycloheximide did. The stimulatory effects of Ca2+ on protein synthesis were not restricted to C-6 in minimal medium. Protein synthesis was reduced by ethylene glycol bis(B-aminoethyl ether)-N,N,N',N'-tetraacetic acid 40 to 75% in C-6 glioma, GH3 pituitary tumor, PC-12 adrenal tumor, N2A neuroblastoma, and HeLa cells incubated under simulated growth conditions with various enriched media and sera. Ca2+-depleted S49 lymphoma, CHO ovarian tumor, and normal, dispersed chicken embryo cells in enriched medium responded to Ca2+ restoration with increased rates of protein synthesis as did collagenase-dispersed normal rat liver cells in minimal medium. Protein synthesis in rabbit reticulocyte lysates was also inhibited by Ca2+-selective chelators or by Ca2+ removal by parvalbumin affinity chromatography and the inhibition was reversed by Ca2+. These findings are consistent with the existence of a Ca2+ requirement in the translational phase of protein synthesis in eukaryotic cells.

Analogues radioactifs de la neurotensine. II. Préparation de neurotensine tritiée à partir d'un dérivé multi-insaturé de synthèse

In this second paper on the synthesis of neurotensin analogues as precursors for radiolabelling, solid phase synthesis of two polyunsaturated peptides, [Dah6, delta Pro7,10]-neurotensin and acetyl-[delta Pro10]-neurotensin-(8-13), are described. The first one contains one triple bond and two double bonds susceptible to tritiation in the same molecule, the second one contains one double bond in the shortest sequence having neurotensin activity. The C-terminal residue, Boc-Leu, was esterified on the chloromethyl-resin by its cesium salt. For the other amino acids a double coupling was carried out, the first one with dicyclohexylcarbodimide and the second one with the amino acid hydroxybenzotriazole ester. Acylation of the second amino acid, on the resin, presented some difficulties to achieve completeness and several acetylations and benzoylations had to be performed in order to block the last 4 per cent of free amines. It seems that these difficulties are related to some batches of chloromethyl-resin. Incorporation of both acetylenic lysine, N alpha-Boc-N epsilon-Z-L-2,6-diamino-4-hexynoic acid, whose synthesis is described, and N alpha-Boc-L-3,4-dehydroproline was without problems in this synthesis. After cleavage by hydrofluoric acid the crude peptides were purified by gel filtration on Bio-Gel P2 and ion exchange chromatography on carboxymethylcellulose (CM 52). [Dah6, delta Pro7,10]-neurotensin so obtained (51 per cent compared to starting Boc-Leu-resin) was in homogeneous form as characterized by amino acid analysis, thin layer chromatography in different systems and high performance liquid chromatography. The hydrogenation or tritiation product was identical with native neurotensin. Unsaturated derivative and neurotensin obtained after catalytic hydrogenation were as active as native neurotensin in inhibition of 125I-[Trp11]-neurotensin binding to rat brain synaptic membranes and in guinea pig ileum contractility test. Substitution of proline and lysine by their dehydro-derivatives did not affect the biological properties of neurotensin. The tritiated neurotensin (160-180 Ci/mmol) should be a good agent for biological characterization of neurotensin receptors and for investigation of the peptide metabolism.

3H]Lysine incorporation into protein of hemisected, cycloheximide-treated spinal cord

A single application of a protein synthesis inhibitor, puromycin, in the lesion site of a spinal hemisection transiently decreases protein incorporation for less than 24 h and results in axonal sprouting and dendritic and synaptic stability for at least 60 days. In this study we characterized the protein-altering properties of single applications of a different protein synthesis inhibitor, cycloheximide, for comparison. The protein incorporation of [ 3H] lysine into hemisected rat spinal cord (left side, T2) treated with cycloheximide (100 μg/ml in Gelfoam sponge placed in wound at time of lesion) was tested 3 h, 6 h, 12 h, 1, 3, 7, and 14 days later and compared with that in 0.9% saline in Gelfoam implants as normal controls ( N = 54). One hour prior to utilization, the animals were injected subcutaneously with 200 μCi l-[4,5(n)- 3H]lysine monohydrochloride. No treatment-related differences in brain radioactivity were noted at any time postoperation. In spinal cord, inhibition of amino acid uptake by cycloheximide was not demonstrable at 3 h after hemisection, but could be detected from 6 h to 1 day after implantation. During that time, the TCA-soluble radioactivity at the lesion site was greater than in control animals ( P < 0.05). These results suggest that as with puromycin, morphologic effects produced by cycloheximide on spinal cord regeneration may correspond to chemical events which occurred within the first 24 h after cord hemisection and cycloheximide treatment.

Studies on large mobile protein appearing in submandibular glands of isoproterenol-treated rats. V. Further studies on some aspects of its formation.

Experiments on in vivo (3H)-lysine incorporation into rat submandibular glands after isoproterenol (IPR) treatment were performed. In the IPR-treated group, incorporation of radioactivity into the trichloroacetic acid (TCA)-insoluble protein fraction was 42% higher than in the control group. The specific radioactivity of large mobile (LM) protein was 4.5-fold higher than that of the TCA-insoluble protein fraction. The stimulatory effect of IPR was markedly suppressed by the pretreatment of rats with actinomycin D. Actinomycin D inhibited the increase of LM protein concentration in submandibular glands by more than 90%, and markedly suppressed the incorporations of radioactivity into TCA-insoluble protein fraction and LM protein, indicating that the LM protein was not derived from other proteins, but was newly biosynthesized after IPR administration. The amount of LM protein in submandibular glands reached 27.6% of total soluble protein, and the steady-state level was achieved at the 11th day upon chronic IPR administration. The rate constant of disappearance of LM protein was estimated to be 0.23/d. The halflife and the rate constant for its biosynthesis were calculated to be 3.0 d and 410 μg·100mg gland-1·d-1, respectively.

The synthesis and deposition of the prolamin storage proteins (secalins) of rye

The synthesis and deposition of the endosperm storage proteins of rye, usually termed secalins, has been studied. The rate of accumulation of secalin in developing rye grain was at a maximum between 3 and 5 weeks after anthesis. Some changes in the proportions of the four major groups of secalin polypeptides were observed during maturation, notably an increase in γ-secalins of Mr 75k and a decrease in ω-secalins. In-vitro translation of mRNA fractions prepared from 4-week-old endosperms showed that secalin polypeptides were synthesised on membrane-bound polysomes. The secalin products were identified by their mobilities on SDS-PAGE and their relative incorporation of radioactive lysine, glycine, proline, leucine and methionine. Protein bodies prepared by sucrose density ultracentrifugation contained reduced amounts of γ-secalins of Mr 40k and ω-secalins compared with the total secalin fraction, but these components were present in the expected amounts when 1.0 M NaCl was added to the buffers. Treatment of the protein bodies with proteinase-k resulted in the digestion of their contents regardless of the presence of NaCl, indicating that the surrounding membrane was incomplete. It was concluded that the NaCl reduced the loss of secalins from the protein bodies by decreasing secalin solubility rather than by affecting the integrity of the protein body membrane. The results reported for the synthesis and deposition of secalins are consistent with the results of previous studies on the prolamins of wheat and barley.

Lens capsule basement membrane synthesis. Stimulation by glucose in vitro.

To examine the question of whether hyperglycemia per se can affect basement membrane synthesis, intact rat lenses, which produce basement membrane in vitro, were incubated for 24 h with radioactive proline or lysine and varying concentrations of glucose. Lens capsule basement membrane (LCBM) was subsequently purified and analyzed for radiolabel incorporation and for specific activities of proline, hydroxyproline, lysine, and hydroxylysine. [14C]-proline and lysine incorporation into LCBM was increasingly stimulated in incubations performed with 10 and 20 mM compared with 5 mM glucose. High glucose concentration increased the specific activity of proline and lysine but not hydroxyproline or hydroxylysine, and decreased the ratio of radioactive hydroxyproline to proline. Gel electrophoresis of radiolabeled LCBM prepared after high glucose incubation revealed increased radioactivity in serveral high-molecular-weight components corresponding with the major Coomassie-blue peptide bands. The results indicate that glucose stimulates LCBM synthesis, and suggest that this effect derives from increased production of noncollagenous components.

Evidence for a humoral factor mediating the effect of a pressure load on lysine incorporation in rabbit heart

Mechanisms of an increase in protein synthesis in pressure-loaded heart were studied by measuring rates of 3H-lysine incorporation into cardiac myosin B in a perfused rabbit heart. When a pressure load had been applied to the right ventricle, a significant increase in the incorporation was found not only in the right but also in the left ventricle where pressure load was not applied. Rates of the incorporation in hearts without pressure load increased significantly in both ventricles when they were co-perfused with a heart with a pressure load on the right ventricle. These results suggest that humoral factor(s) is released from the pressure-loaded ventricle which accelerate amino acid incorporation in unloaded ventricles.

Betaine Synthesis and Accumulation in Barley During Field Water‐Stress 1

The timing and extent of betaine accumulation by mature leaves of barley (Hordeum vulgare L.) were followed in irrigated (I) and non-irrigated (N-I) plots under rain-shelters. In the N-I crop, leaf water potential (/sup psi/leaf) began to fall at the five-leaf stage, continued to drop steadily until maturity, and reached a minimum of about -35 bars. Betaine accumulation started in the N-I crop about a week after the decline in /sup psi/leaf began and continued until about 10 days post-anthesis. The maximum betaine concentration attained by N-I leaves (100 ..mu..mol/g dry wt) was three times that in I leaves. Betaine accumulation by upper leaves was due mainly to de novo synthesis in these leaves, because: (1) there was little /sup 14/C-import into upper leaves when (/sup 14/C)betaine was applied to lower leaves, and (2) attached upper leaves of N-I plants rapidly converted supplied (/sup 14/C)ethanolamine to (/sup 14/C)betaine during the peak period of betaine accumulation. Phosphatidylcholine (PC) behaved as an intermediate in the conversion of (/sup 14/C)ethanolamine to betaine. The estimated peak metabolic cost of betaine biosynthesis via PC by stressed leaves (about 2 mg hexose/g dry wt/day) approached the cost of protein turnover in the same leaves (3 tomore » 5 mg hexose/g dry wt/day) as estimated from (/sup 3/H) lysine incorporation. In N-I plants, cessation of betaine synthesis preceded the onset of senescence by several days, indicating that continuous betaine production is not mandatory for leaf function at lowered /sup psi/leaf. These field results are consistent with an adaptive value for betaine accumulation in barley during prolonged water stress. A search for genetic variation in betaine-accumulating potential in barley is now warranted.« less

Lysine tRNAs from Bacillus subtilis 168: function of the isoacceptors in a rabbit reticulocyte cell-free protein-synthesizing system.

The two principal tRNA Lys isoaccepting species of Bacillus subtilis were compared in their functional activity in translating rabbit globin. Although neither species demonstrates any preference in reading either of the lysine codons, there is an overall preference for tRNa Lys 3 in lysine incorporation. The ratios of lysine incorporated by the two species into the different lysine-containing sites in the globin subunits vary over a more than two-fold range. As described in the accompanying paper, tRNA Lys 1 is a hypomodified form of tRNA Lys 3. Consistent with studies on other rRNA species, the fully modified isoacceptor functions preferentially. In contrast to these results, however, the fully modified isoacceptor (tRNA Lys 3) is found predominantly in rapidly dividing cells while the hypomodified isoacceptor (tRNA Lys 1) predominates in the stationary cells and spores of B.l subtilis.

Enhanced protein synthesis of heart in adrenal-regeneration hypertension and its reduction following antihypertensive treatment.

After unilateral nephrectomy and bilateral adrenal enucleation at 5 weeks of age, salt-loaded rats developed hypertension (ARH) at 6 weeks. Fibrous vascular protein and in vivo incorporation of 3H-lysine into this protein fraction were measured at 15 weeks of age in these animals. This study demonstrates: (1) that incorporation rates of 3H-lysine into cardiac collagen and elastin in ARH rats were greater than in control rats (p less than 0.001, respectively), and (2) that administration of phenoxy-benzamine hydrochloride decreased the incorporation of tritiated lysine into cardiac collagen in ARH rats, concomitant with a reduction in blood pressure. Based on these findings, increased synthesis of cardiac collagen and elastin appears to play an important role in the development of hypertension in ARH.

34] Biosynthesis of insoluble elastin in cell ang organ cultures

Publisher Summary The formation of insoluble elastin in in vitro systems such as cell and organ cultures has been the focus of several laboratories in recent years. There are three criteria that can be used in assessing the presence of elastin in cell cultures. These include (a) evaluation of the culture by ultrastructural analyses; (b) evaluation of the insoluble elastin formed by amino acid composition of the product obtained after purification of the elastin; and (c) detection and possible quantification of the formation of the unique lysine-derived cross-links. It is suggested that the cell cultures to be studied should be derived, if possible, from a primary culture that has undergone at least two subcultivations. Studies of elastin synthesis in organ culture systems have therefore been limited to the use of radioactive precursors, such as valine or lysine. Lysine incorporation experiments focus on the formation of cross-links in much the same manner described in the cell culture section. Radiolabeled valine is also commonly used in elastin studies because this protein has an unusually high content of valine. To perform rapid organ culture experiments on insoluble elastin accumulation one can simply employ methods that are quite similar to the preparation of lysyl oxidase or prolyl hydroxylase substrates from aortic explants.

Glucose-stimulated protein synthesis in rat testis slices: substrate specificity and effects of insulin and substrate analogs.

The effects of substrate, substrate concentration, insulin, and substrate analogs on the incorporation of lysine into rat testis slices were studied. Incorporation was stimulated by 10 mM glucose and 10 mM mannose, to a lesser extent by 10 mM fructose, but not by galactose, glucitol, or xylitol. Half-maximal stimulation occurred at 2 mM glucose or 2 mM mannose and was maximal at concentrations above 4 mM. Fructose at a concentration of 20 mM was about as effective as 2 mM glucose. The glucose-stimulated incorporation was not affected by insulin. Several analogs were tested at a final concentration of 10 mM as inhibitors of glucose -and fructose-stimulated lysine incorporation. At a glucose concentration of 4 mM, only 2-deoxyglucose and 2-deoxygalactose were inhibitory, reducing incorporation to levels not significantly different from basal. When the glucose concentration was reduced to 2 mM, other analogs were also inhibitory; the most effective of these were 5-thioglucose, 3-0-methylglucose, 4,6-ethylideneglucose, and galactosamine. When 10 mM fructose was used as a substrate, the same analogs plus the fructose analogs mannoheptulose, 1-deoxyfructose, 2,5-anhydromannose, and 2,5-anhydromannitol were inhibitory. Differences in analog inhibitions against fructose and glucose suggest different rate-limiting steps for the stimulation by these two sugars. It is proposed that the response to glucose and mannose, but not fructose, is mediated by an uptake system which is limiting at low substrate concentrations. Inhibition by various sugar analogs generally results from uptake system which is limiting at low substrate concentrations. For most of the analogs this occurs under conditions in which a step subsequent to the hexose uptake system, possibly a phosphorylation step, is not saturated.

The effects of a post-transcriptional modification on the function of tRNALys isoaccepting species in translation.

Isoacceptors of rabbit liver tRNALys which preferentially translate the codon AAG were compared for their function in several aspects of translation. As shown in other laboratories, Lys-tRNALys1,2 are two isoacceptors which differ from each other by a single base pair and are fully modified with N6-threonyl-adenosine adjacent to the anticodon. Lys-tRNALys4, which occurs commonly in rapidly dividing mammalian cells and tissues, is hypomodified at several bases and contains a precursor of N6-threonyl-adenosine next to its anticodon. These isoacceptors were incubated in cell-free protein synthesizing systems which contain rabbit globin mRNA. (Lys-tRNALys3 which translates AAA was also included.) The resulting globin was isolated and digested with trypsin, and the relative incorporation of lysine from Lys-tRNALys1,2 and from Lys-tRNALys4 into lysine-containing sites in the globin peptides as determined. Lys-tRNALys1,2 and Lys-tRNALys4 translate AAG preferentially, but Lys-tRNALys4 wobbles more than the former and translates AAA codons more efficiently. Overall, Lys-tRNALys1,2 is preferred in globin synthesis by about 30% compared to Lys-tRNALys4, and with one exception, the incorporation of lysine into the individual AAG lysine-containing sites in globin occurs more efficiently from Lys-tRNALys1,2. There is, however, considerable variation from site to site in the relative efficiencies of the Lys-tRNAs in incorporation.

Influence of prenatal hypoxia on brain development: effects on body weight, brain weight, DNA, protein, acetylcholinesterase, 3-quinuclidinyl benzilate binding, and in vivo incorporation of [14C]lysine into subcellular fractions.

The influence of prenatal hypoxia on subsequent brain development in the young rat was investigated by examining body and brain weight, cerebral cortex wet weight, protein and DNA concentrations, acetylcholinesterase (AChE) activity, 3-quinuclidinyl benzilate (QNB)-binding levels, the relative amounts of protein in various subcellular fractions, and the in vivo incorporation of [14C]lysine into the protein of homogenate and subcellular fractions. Exposure of pregnant females to a mild hypoxia (9.1% O2, 10 h per day for the 9--11 days preceding birth) resulted in a reduced body weight in the pups and days 1 and 5 after birth; total cortical DNA was reduced but brain weight and protein content were unaffected, leading to a higher protein/DNA ratio in prenatally hypoxic pups. By 10 days of age these differences between prenatally hypoxic and control animals were no longer apparent. There were no differences between prenatally hypoxic and control animals in AChE and QNB binding per milligram cortex protein. The relative amount of synaptic membrane protein from the cerebral cortex was reduced at day 1 in prenatally hypoxic animals and the synaptic membrane fraction showed a higher level of incorporation of [14C]lysine on days 1, 5, and 10. The developmental profile of [14C]lysine incorporation showed a peak on day 10 which was higher in prenatally hypoxic rats. By 46 days after birth little difference could be found between prenatally hypoxic and control animals.

Effect of Rate of Catabolism of DL-[1-14C]Lysine on Estimation of Whole Body Protein Synthesis in Rats

We investigated the effect of rate of DL-[1-14C]lysine metabolism on the estimation of whole body protein synthesis in rats. Growing rats were given single injections of DL-[1-14C]lysine via a tail vein and cumulative expiration of 14CO2 was measured after 3, 6 and 9 hours. Total 14C in protein-bound lysine, body lipids, perchloric acid-soluble material, urine, feces and gastrointestinal contents was recovered after 9 hours. After correcting for radiochemical purity and stereoisomerism of DL-[1-14C]lysine, 95.1% of injected L-[1-14C]lysine was found in expired air and protein-bound [14C]lysine. Daily nitrogen retention of rats was 150 mg and daily incorporation of lysine into body protein was 119% of that absorbed from the gastrointestinal tract, from which rates of whole body protein synthesis, accretion and degradation were estimated to be 2.58, 0.76 and 1.83 g/day, respectively. Expressed per unit of body weight the rate of protein synthesis was not significantly different from previous estimates using L-[1-14C]leucine, which indicated that differences in catabolic pathways of leucine and lysine as well as differences in intracellular specific activities of these [14C]amino acids had a negligible effect on the estimation of protein synthesis.protein synthesis lysine metabolism

Changes in [ 3H]lysine incorporation into protein in a seizure focus in rat isocortex

A focus of epileptiform discharge was induced in rat isocortex by subpial injection of 5 microliters of 100 mM FeCl3. Control animals were prepared with saline injections. Protein synthesis was estimated by uptake of [3H]lysine and its incorporation into protein at the site of iron injection, in the contralateral homotopic isocortex, and in the midline cerebellum. We found diminished uptake of [3H]lysine into all regions of rat brain in the interictal or nonseizing iron-injected animals, whereas the corrected rate of incorporation of [3H]lysine into protein was not significantly different from control rates. Actively seizing animals showed no inhibition of uptake of [3H]lysine, but [3H]lysine incorporation into protein relative to the uptake was significantly inhibited within the epileptic focus but not in the other areas examined. This decreased incorporation of amino acids into protein parallels that found in animals convulsing after electroshock or pentylenetetrazol injection.

DNA replication, chromatin structure, and histone phosphorylation altered by theophylline in synchronized HeLa S3 cells.

The onset of DNA replication normally is coincident with an increase in histone 1 phosphorylation and a relaxation in chromatin structure. In this paper we show that 5 mM theophylline, added 2 h after selective detachment to synchronized HeLa-S-3 cells, delays the onset and reduces the rate of DNA synthesis while theophylline treatment beginning at 8 h has no effect on subsequent DNA synthesis. These actions of theophylline are accompanied by an inhibition of histone 1 phosphorylation and a prevention of the normal relaxation in chromatin structure between G1 and S phases as revealed by image analysis of Feulgen-stained nuclei. The time courses of intracellular cyclic AMP levels, nonhistone protein phosphorylation, and [3H]lysine incorporation are also compared in the same treated and untreated synchronized HeLa cells. Comparison with experiments using 1-beta-D-arabinofuranosylcytosine (Ara-C) shows that the above phenomena are not a direct result of inhibition of DNA synthesis. We interpret our results as evidence that the associations between histone 1 phosphorylation, chromatin relaxation, and the onset of DNA synthesis are temporally and causally related.