Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain

Uridine, the major circulating pyrimidine nucleoside, participating in the regulation of a number of physiological processes, is readily uptaken into mammalian cells. The balance between anabolism and catabolism of intracellular uridine is maintained by uridine kinase, catalyzing the first step of UTP and CTP salvage synthesis, and uridine phosphorylase, catalyzing the first step of uridine degradation to β-alanine in liver. In the present study we report that the two enzymes have an additional role in the homeostatic regulation of purine and pyrimidine metabolism in brain, which relies on the salvage synthesis of nucleotides from preformed nucleosides and nucleobases, rather than on the de novo synthesis from simple precursors. The experiments were performed in rat brain extracts and cultured human astrocytoma cells. The rationale of the reciprocal regulation of purine and pyrimidine salvage synthesis in brain stands (i) on the inhibition exerted by UTP and CTP, the final products of the pyrimidine salvage pathway, on uridine kinase and (ii) on the widely accepted idea that pyrimidine salvage occurs at the nucleoside level (mostly uridine), while purine salvage is a 5-phosphoribosyl-1-pyrophosphate (PRPP)-mediated process, occurring at the nucleobase level. Thus, at relatively low UTP and CTP level, uptaken uridine is mainly anabolized to uridine nucleotides. On the contrary, at relatively high UTP and CTP levels the inhibition of uridine kinase channels uridine towards phosphorolysis. The ribose-1-phosphate is then transformed into PRPP, which is used for purine salvage synthesis.     

Ribose 1-phosphate and inosine activate uracil salvage in rat brain

The purpose of this study was to determine the mechanism by which inosine activates pyrimidine salvage in CNS. The levels of cerebral inosine, hypoxanthine, uridine, uracil, ribose 1-phosphate and inorganic phosphate were determined, to evaluate the Gibbs free energy changes (deltaG) of the reactions catalyzed by purine nucleoside phosphorylase and uridine phosphorylase, respectively. A deltaG value of 0.59 kcal/mol for the combined reaction inosine+uracil <==> uridine+hypoxanthine was obtained, suggesting that at least in anoxic brain the system may readily respond to metabolite fluctuations. If purine nucleoside phosphorolysis and uridine phosphorolysis are coupled to uridine phosphorylation, catalyzed by uridine kinase, whose activity is relatively high in brain, the three enzyme activities will constitute a pyrimidine salvage pathway in which ribose 1-phosphate plays a pivotal role. CTP, presumably the last product of the pathway, and, to a lesser extent, UTP, exert inhibition on rat brain uridine nucleotides salvage synthesis, most likely at the level of the kinase reaction. On the contrary ATP and GTP are specific phosphate donors.     

Synthesis of pyrimidine nucleotides in the heart: uridine and cytidine kinase activity

These experiments were designed to determine through the study of uridine and cytidine kinase activity, the precise mechanisms of plasma nucleoside salvage leading to pyrimidine nucleotide synthesis in the rat heart. The kinetic parameters were: Km = 10 microM, V = 4 nmol g-1 min-1 for cytidine kinase activity and Km = 43 microM and V = 18 nmol g-1 min-1 for uridine kinase activity. Competing activity as concerns the two nucleosides was shown to occur, suggesting that in the rat myocardium as in other cells, one and the same enzyme phosphorylates both uridine and cytidine. UTP and CTP were shown to exert a potent inhibitory action on nucleoside phosphorylation; two factors thus exert a joint influence on the control of pyrimidine nucleotide synthesis in the rat heart: the extracellular concentration of precursor and the intracellular level of UTP and CTP. The kinetic parameters for kinase activities are discussed, taking into account the actual concentration of plasmatic nucleosides. Comparison of these data with respectively those for incorporation of nucleosides into the pyrimidine nucleotides of isolated rat heart and with nucleotide turnover rates in vivo suggests that, under physiological conditions, the utilization of plasma cytidine is crucial to the synthesis of myocardial pyrimidine synthesis.     

Metabolic interplay between intra- and extra-cellular uridine metabolism via an ATP driven uridine–UTP cycle in brain

Uridine, a pyrimidine nucleoside essential for the synthesis of RNA and biomembranes, has several trophic functions in the central nervous system, that involve a physiological regulation of pyrimidine nucleotides and phospholipids content, and a maintenance of brain metabolism under ischemia, or pathological situations. The understanding of uridine production in the brain is therefore of fundamental importance. Brain has a limited capacity to synthesize ex novo the pyrimidine ring, and a reasonable source of brain uridine is UTP. The kinetics of UTP breakdown, as catalysed by post-mitochondrial brain extracts and membrane preparations reported herein suggests that in normoxic conditions uridine is locally generated in brain exclusively in the extracellular space, and that any uptaken uridine is salvaged to UTP. It is now well established that cytosolic UTP can be released to interact with a subset of P2Y receptors, inducing a variety of molecular and cellular effects, leading to neuroprotection, while uridine is uptaken via an equilibrative or a Na⁺-dependent transport system, to exert its trophic effects in the cytosol. An ATP driven uridine–UTP cycle can be envisaged, based on the strictly compartmentalized processes of uridine salvage to UTP and uridine generation from UTP, in which uptaken uridine is anabolised to UTP in the cytosol, and converted back to uridine in extracellular space.     

Pyrimidine Nucleotide Metabolism and Pathways of Thymidine Triphosphate Biosynthesis in Salmonella typhimurium

The nucleoside triphosphate pools of two cytidine auxotrophic mutants of Salmonella typhimurium LT-2 were studied under different conditions of pyrimidine starvation. Both mutants, DP-45 and DP-55, are defective in cytidine deaminase and cytidine triphosphate (CTP) synthase. In addition, DP-55 has a requirement for uracil (uridine). Cytidine starvation of the mutants results in accumulation of high concentrations of uridine triphosphate (UTP) in the cells, while the pools of CTP and deoxy-CTP drop to undetectable levels within a few minutes. Addition of deoxycytidine to such cells does not restore the dCTP pool, indicating that S. typhimurium has no deoxycytidine kinase. From the kinetics of UTP accumulation during cytidine starvation, it is concluded that only cytidine nucleotides participate in the feedback regulation of de novo synthesis of UTP; both uridine and cytidine nucleotides participate in the regulation of UTP synthesis from exogenously supplied uracil or uridine. Uracil starvation of DP-55 in presence of cytidine results in extensive accumulation of CTP, suggesting that CTP does not regulate its own synthesis from exogenous cytidine. Analysis of the thymidine triphosphate (dTTP) pool of DP-55 labeled for several generations with (32)P-orthophosphate and (3)H-uracil in presence of (12)C-cytidine shows that only 20% of the dTTP pool is derived from uracil (via the methylation of deoxyuridine monophosphate); 80% is apparently synthesized from a cytidine nucleotide.     

The PRPP synthetase spectrum: what does it demonstrate about nucleotide syndromes?

Defects in X-linked phosphoribosylpyrophosphate synthetase 1 (PRPS1) manifest as follows: (1) PRS-I enzyme "superactivity" (gain-of-function mutations affecting allosteric regions); (2) PRS-I overexpression (which may be linked to miRNA mutation); (3) severe PRS-I deficiency/Arts syndrome (missense mutations producing loss-of-function); (4) moderate PRS-I deficiency/Charcot-Marie-Tooth disease-5 (less severe loss-of-function mutations); and (5) mild PRS-I deficiency/Deafness-2 (mutations producing slight destabilization). Similar to Lesch-Nyhan disease, PRPS1-related disorders arise from phosphoribosyl-pyrophosphate (PRPP)-dependent nucleotide "depletion" of purine nucleotides (e.g., ATP, GTP). S-adenosylmethionine (SAMe) appears to partially alleviate purine depletion via a PRPP-independent path. Synthesis of pyrimidine nucleotides is PRPP dependent, with uridine monophosphate synthase deficiency producing pyrimidine nucleotide depletion. But pyrimidine salvage from uridine does not require PRPP, and this nucleoside is transported freely to pyrimidine-depleted tissues. Regulation of nicotinamide nucleotides is less clear; synthesis from pyridine nucleobases is PRPP dependent. Nucleotide "depletion" contrasts with nucleotide "toxicity," exemplified by the purine disorders adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiencies or by pyrimidine nucleotidase deficiency. These are characterized by the accumulation of one or more abnormal nucleotides such as succinyl- or deoxy-nucleotides or their metabolites, which interrupt other nucleotide or related pathways or are toxic to specific cell types. Theoretically, purine toxicity disorders would not be ameliorated by SAMe therapy, and this was confirmed for one adenylosuccinate lyase-deficient child. Nucleotide defects may also be seen as an aspect of mitochondrial disease, with SAMe-based mitochondrial therapy perhaps meriting further investigation.     

Molecular mechanisms of nucleoside recycling in the brain

A major role of plasma membrane bound ectonucleotidases is the modulation of ATP, ADP, adenosine (the purinergic agonists), UTP, and UDP (the pyrimidinergic agonists) availability in the extracellular space at their respective receptors. We have recently shown that an ATP driven uridine-UTP cycle is operative in the brain, based on the strictly compartmentalized processes of uridine salvage to UTP and uridine generation from UTP, in which uptaken uridine is anabolized to UTP in the cytosol, and converted back to uridine in the extracellular space by the action of ectonucleotidases (Ipata et al. Int J Biochem Cell Biol 2010;42:932-7). In this paper we show that a similar cytidine-CTP cycle exists in rat brain. Since (i) brain relies on imported preformed nucleosides for the synthesis of nucleotides, RNA, nuclear and mitochondrial DNA, coenzymes, pyrimidine sugar- and lipid-conjugates and (ii) no specific pyrimidinergic receptors have been identified for cytidine and their nucleotides, our results, taken together with previous studies on the intra- and extracellular metabolic network of ATP, GTP, UTP, and their nucleosides in the brain (Barsotti and Ipata. Int J Biochem Cell Biol 2004;36:2214-25; Balestri et al. Neurochem Int 2007;50:517-23), strongly suggest that, apart from the modulation of ligand availability, ectonucleotidases may serve the process of local nucleoside recycling in the brain.     

Regulation of uridine kinase. Evidence for a regulatory site

Uridine kinase from mouse Ehrlich ascites tumor cells may exist at 4 degrees C in multiple aggregation states that only slowly equilibrate with one another. Increasing the temperature leads to dissociation, and the appearance of a single predominant species: at 22 degrees C the enzyme exists as a tetramer. There is also a break in the dependence of enzyme activity on temperature as measured in an Arrhenius plot. The feedback inhibitors CTP and UTP cause the enzyme to dissociate to the monomer, whereas the substrate ATP reverses this process. Kinetic studies show that the monomer has little or no activity. Studies of the reaction mechanism show that binding of substrates is ordered, leading to a ternary complex, and release of products is ordered: uridine is the first substrate bound, ADP the first product released. Except for the inhibitors UTP and CTP, all other nucleoside triphosphates, whether purine or pyrimidine, or containing ribose or deoxyribose, act as phosphate donor. Especially interesting are the opposite effects of CTP and dCTP on uridine kinase: unlike CTP, dCTP does not dissociate the enzyme and is competent as a phosphate donor. We propose that the various effects of different ligands are best explained by the existence of a regulatory site (with more stringent specificity than the catalytic site) that controls dissociation of uridine kinase to the inactive monomer.     

Evidence for compartmentation of uridine nucleotide pools in rat hepatoma cells

Interaction between the de novo and salvage pathways of pyrimidine metabolism was studied in a line of rat hepatoma cells by co-labelling with [14C]-uridine and [3H]orotate. A difference in the ratio of 14C/3H between CTP and UTP in acid-soluble nucleotide pool was reflected in the corresponding ratios in CMP and UMP in RNA, with uridine labelling cytidine nucleotides relatively more effectively than orotate. These results are not compatible with the concept of a single UTP pool, and a new model for pyrimidine anabolic pathways, based on compartmentation of de novo from salvage pathways, is proposed.     

Brain nucleoside recycling

The rate limiting reactions of nucleotide synthesis are modulated by intracellular fluctuations of nucleoside triphosphate concentrations. This topic has been mostly studied at the level of the de novo nucleotide synthesis from simple precursors. However, there are districts, such as brain, which rely more heavily on the salvage of preformed purine and pyrimidine rings, mainly in the form of nucleosides. This raises the following question: how do these districts maintain the right balance between the purine and pyrimidine pools? We believe that it is now safe to state that a cross talk exists between the extra- and intracellular metabolism of purine and pyrimidine nucleosides in the brain. The extracellular space is the major site of nucleoside generation through successive dephosphorylations of released triphosphates, whereas brain cytosol is the major site of multiple phosphorylations of uptaken nucleosides at their 5′-position. Modulation of both extracellular nucleoside generation by membrane bound ectonucleotidases, and intracellular nucleoside phosphorylation by cytosolic kinases might contribute to maintain the right extra- and intracellular purine and pyrimidine nucleotide balance in the brain.     

Effect of D-galactosamine on nucleotides in the rat heart. Effects of cytidine and uridine administration

The treatment of rats by galactosamine (2 mmol/kg i.p.), which dramatically alters the metabolism of pyrimidine nucleotides in the liver, has been used to investigate the dynamics of pyrimidine nucleotides in the rat heart. Six hours after administration of the drug, the UTP and UDPG myocardial contents were decreased by respectively 40 and 52% while the sum of uracil nucleotides was increased by 66% and that of cytosine nucleotides by 15%. When administered 5 h after galactosamine treatment, cytidine (750 nmol/rat i.v.) induced a further increase in cytosine nucleotides (46% above control value 1 h later) without however effect on uracil nucleotides. On the other hand, the administration of uridine (250 nmol/rat, i.v. 5 h after galactosamine), while restoring UTP, UDPG and the pool of uracil nucleotides, provoked a decrease in cytosine nucleotide level (-17%). In the absence of galactosamine treatment, the administration of uridine and cytidine did not induce changes in nucleotide levels despite a rise in blood cytidine concentration. All these observations support the hypothesis that: 1. the pathway for cytosine nucleotide synthesis predominant in the heart is that utilizing preformed exogenous cytidine and 2. this pathway is mainly controlled by the intracellular concentration of UTP rather than that of CTP.     

Subcellular compartmentation of uridine nucleotides and nucleoside-5' -diphosphate kinase in leaves

The subcellular compartmentation of nucleoside diphosphate kinase (EC 2.7.4.6) and the uridine nucleotides has been studied in leaves. Membrane filtration of barley (Hordeum vulgare L.) leaf mesophyll protoplasts and differential centrifugation of spinach (Spinacia oleracea L.) leaf extracts showed that about half the nucleoside diphosphate kinase is present in the cytosol. The activity is adequate to account for the turnover of UTP and UDP during photosynthetic sucrose synthesis. Nonaqueous density gradient centrifugation of freeze-stopped, lyophilized spinach leaf material showed that the uridine nucleotides are predominantly located in the cytosol and that the cytosolic UDP-glucose pool is considerably larger than the UTP or UDP pools.     

Neuroblastoma Growth Factors Derived from Neurofibroma (NF1): Participation of Uridine in a Neuroblastoma Growth

Abstract: Human glioma cell extracts were found to elicit a marked growth-promoting activity on human neuroblastoma cells. This activity was also detected in the extracts of neurofibroma type 1 (NF1; von Recklinghausen neurofibromatosis) comprising aberrant Schwann cell growth. The purified substance from the NF1 extracts by HPLC on ODS columns was identical to a pyrimidine nucleoside, uridine, the chemical structure of which was identified by gas chromatography-mass spectrometry. The authentic uridine showed a strong growth-promoting activity on human neuroblastoma cells. Other purine or pyrimidine nucleotides, their derivatives, and ribose sources for their syntheses were employed to test the activity; a purine nucleoside, adenosine, showed a stronger activity than uridine. The current study raises the possibility that human neuroblastoma cells may be affected by dysfunctions of the de novo pathway of both purine and pyrimidine nucleotide biosyntheses.     

In vitro recycling of alpha-D-ribose 1-phosphate for the salvage of purine bases

In this paper, we extend our previous observation on the mobilization of the ribose moiety from a purine nucleoside to a pyrimidine base, with subsequent pyrimidine nucleotides formation (Cappiello et al., Biochim. Biophys. Acta 1425 (1998) 273-281). The data show that, at least in vitro, also the reverse process is possible. In rat brain extracts, the activated ribose, stemming from uridine as ribose 1-phosphate, can be used to salvage adenine and hypoxanthine to their respective nucleotides. Since the salvage of purine bases is a 5-phosphoribosyl 1-pyrophosphate-dependent process, catalyzed by adenine phosphoribosyltransferase and hypoxanthine guanine phosphoribosyltransferase, our results imply that Rib-1P must be transformed into 5-phosphoribosyl 1-pyrophosphate, via the successive action of phosphopentomutase and 5-phosphoribosyl 1-pyrophosphate synthetase; and,in fact, no adenosine could be found as an intermediate when rat brain extracts were incubated with adenine, Rib-1P and ATP, showing that adenine salvage does not imply adenine ribosylation, followed by adenosine phosphorylation. Taken together with our previous results on the Rib-1P-dependent salvage of pyrimidine nucleotides, our results give a clear picture of the in vitro Rib-1P recycling, for both purine and pyrimidine salvage.     

Aminoimidazole carboxamide ribonucleoside toxicity: a model for study of pyrimidine starvation

Aminoimidazole carboxamide ribonucleoside (AIC-R), a purine precursor, has biphasic effects on the growth of Chinese hamster fibroblasts. At 200 microM AIC-R cell growth is almost completely arrested, while at 50 and 700 microM AIC-R cell growth is comparable to that observed in the absence of nucleoside. The growth inhibition produced by AIC-R is the consequence of inhibition of the orotate phosphoribosyltransferase-orotidylic decarboxylase (OPRT-ODC) reactions, as evidenced by a 87% reduction in the intracellular concentrations of UTP and CTP, accumulation of orotate in the medium, and restoration of normal growth by inclusion of 100 microM uridine in the medium. Inhibition of pyrimidine nucleotide synthesis at 200 microM AIC-R is associated with an 82% reduction in the intracellular concentration of PP-ribose-P and a 150% increase in the concentration of purine nucleotides. Restoration of cell growth to a normal rate at 700 microM AIC-R--a condition under which PP-ribose-P remains depressed and purine nucleotide concentrations are also depressed (40% of control)--and absence of toxicity at 50 microM AIC-R--a condition under which purine nucleotide concentrations are increased by 150% and PP-ribose-P concentration is normal--suggest that the inhibition of OPRT-ODC observed at 200 microM AIC-R is caused by the combination of the reduction in PP-ribose-P and increase in purine nucleotides. These studies provide a better understanding of the control of the OPRT-ODC reactions in the cell and provide additional insight into the basis of pyrimidine starvation induced by purine nucleosides.     

Pyrimidine synthesis in Burkholderia cepacia ATCC 25416

K. LI AND T.P. WEST. 1995. Pyrimidine synthesis in the food spoilage agent Burkholderia cepacia ATCC 25416 was investigated. The five de novo pathway enzymes of pyrimidine biosynthesis were found to be active in B. cepacia ATCC 25416 and growth of this strain on uracil had an effect on the de novo enzyme activities. The in vitro regulation of aspartate transcarbamoylase activity in B. cepacia ATCC 25416 was studied and its activity was inhibited by PP_i, ATP, GTP, CTP and UTP. The enzymes cytidine deaminase, uridine phosphorylase and cytosine deaminase were found to be active in the salvage of pyrimidines in ATCC 25416. Overall, de novo pyrimidine synthesis in B. cepacia ATCC 25416 was regulated at the level of enzyme activity and its pyrimidine salvage enzymes differed from those found in B. cepacia ATCC 17759.     

Evaluation of uridine metabolism in human and animal spermatozoa

The objective of this study was to elucidate the role of uridine for spermatozoa, since this pyrimidine nucleoside was found in millimolar concentration in human seminal plasma. Here, the degradative activity of uridine-phosphorylase [EC 2.4.2.3] and the salvage activity of uridine kinase [EC 2.7.1.48] were detected in human spermatozoa. HPLC analysis depicted the uptake of exogeneous 14C-labelled adenine, but not of uridine and of hypoxanthine, into nucleotide pools of boar spermatozoa. On addition of uridine, the computer-assisted semen analysis (CASA) of human cells revealed a reduction of the percentage of motile spermatozoa in contrast to an elevation of some velocity parameters. It is concluded that exogeneous uridine could function as suppressor for early capacitation and as a substrate for phosphorolysis, if ribose is needed, rather than to satisfy a demand for intracellular pyrimidine nucleotides.     

Blood uridine concentration may be an indicator of the degradation of pyrimidine nucleotides during physical exercise with increasing intensity

During prolonged maximal exercise, oxygen deficits occur in working muscles. Progressive hypoxia results in the impairment of the oxidative resynthesis of ATP and increased degradation of purine nucleotides. Moreover, ATP consumption decreases the conversion of UDP to UTP, to use ATP as a phosphate donor, resulting in an increased concentration of UDP, which enhances pyrimidine degradation. Because the metabolism of pyrimidine nucleotides is related to the metabolism of purines, in particular with the cellular concentration of ATP, we decided to investigate the impact of a standardized exercise with increasing intensity on the concentration of uridine, inosine, hypoxanthine, and uric acid. Twenty-two healthy male subjects volunteered to participate in this study. Blood concentrations of metabolites were determined at rest, immediately after exercise, and after 30 min of recovery using high-performance liquid chromatography. We also studied the relationship between the levels of uridine and indicators of myogenic purine degradation. The results showed that exercise with increasing intensity leads to increased concentrations of inosine, hypoxanthine, uric acid, and uridine. We found positive correlations between blood uridine levels and indicators of myogenic purine degradation (hypoxanthine), suggesting that the blood uridine level is related to purine metabolism in skeletal muscles.     

Non-allosteric regulation of the uridine kinase from seeds of Zea mays.

Uridine kinase (ATP: uridine 5'-phosphotransferase, EC 2.7.1.48) has been partially purified from ungerminated hybrid corn seed. It is associated with a soluble high molecular weight fraction from which it apparently cannot be dissociated without loss of activity. The stability of the enzyme is enhanced by the addition of dithiothreitol, glycerol and nucleotide substrate. The nucleoside specificity of the enzyme is limited to nucleosides containing pyrimidine and ribose moieties, such as uridine and cytidine. High concentrations of nucleosides cause substrate inhibition, however. The Km values for uridine and cytidine are 53 muM and 125 muM, respectively, and under subsaturating conditions uridine is phosphorylated about five times faster than cytidine. The reaction follows an ordered Bi Bi kinetic pattern, with ATP and ADP in competition for the free form of the enzyme. Purine, but not pyrimidine, nucleoside triphosphates serve as phosphate donors without regard to the sugar moiety. However, all of these triphosphates appear to compete for the same site on the enzyme. (Km ATP equals 590 muM, Km (app) GTP equals 61 muM, and CTP and UTP are linear competitive inhibitors against ATP, with Ki values of 60 muM and 240 muM, respectively.) Therefore, end product control of uridine kinase apparently does not involve allosteric sites, but instead is envisioned as simple competition between relatively effective or ineffective phosphate donors for a position on the enzyme.     

Utilization of L-cell nucleoside triphosphates by Chlamydia psittaci for ribonucleic acid synthesis.

Long-term, 32-P-labeled L cells were infected with the obligately intracellular parasite Chlamydia psittaci (strain 6 BC). At 20 h postinfection, [3-H]uridine was added, and the infected cells were sampled at intervals for incorporation of the labels into the uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools of the host L cell and the uridine monophosphate (UMP) and cytidine monophosphate (CMP) in 16S ribosomal ribonucleic acid (RNA) of the parasite. The specific activity of the nucleotides was calculated from the ratio of 3-H to 32-P counts in the nucleotides. The rate of approach to equilibrium labeling of UTP and CTP in L-cell pools and UMP and CMP in 16S RNA from the exogenous uridine label was determined from the increase in the ratios of the specific activities of CTP to UTP and CMP to UMP with time. The rate of approach to equilibrium CMP:UMP labeling of the 16S RNA of C. psittaci was consistent with the rate predicted from the kinetics of labeling of the CTP and UTP pools of the host L cell. In analogous experiments, the rate of approach to equilibrium guanosine monophosphate:adenosine monophosphate labeling of 16S RNA from an exogenous [14-C]adenine label was consistent with the rate predicted from the kinetics of labeling of the purine nucleoside triphosphate pool of the host cell. These results support the concept that members of the genus Chlamydia owe their obligate intracellular mode of reproduction to a requirement for energy intermediates which is fulfilled by the host cell. In addition, evidence was obtained that the total acid-soluble purine nucleoside triphosphate pool of L cells accurately represents the precursors of L-cell 18S ribosomal RNA.