Regulatory Agent Research Articles

Regulation of Sertoli cell differentiation by the testicular paracrine factor PModS: potential role of immediate-early genes.

Testicular peritubular cells produce a paracrine factor, PModS, under androgen control that modulates Sertoli cell functions that are essential for the process of spermatogenesis. PModS has a more dramatic effect on Sertoli cell differentiated functions in vitro than any regulatory agent previously shown to influence the cell, including FSH. Investigation of the actions of PModS on a molecular level have used transferrin expression as a marker of Sertoli cell differentiation. PModS was found to stimulate transferrin gene expression while having no effect on transferrin mRNA stability. The ability of PModS to elevate transferrin mRNA levels was inhibited by cycloheximide. Therefore, the actions of PModS require ongoing protein synthesis and appear to be indirectly mediated through trans-acting early event genes. PModS was found to dramatically increase mRNA levels for c-fos, but had no effect on c-jun mRNA levels. The c-fos mRNA levels increased transiently within a few minutes to a maximal level of stimulation at 1 h and returned to basal levels within 6 h. The rise in c-fos mRNA preceded the elevation in transferrin mRNA, which started to increase at 2 h to a maximum level between 6-12 h that was maintained at high levels for several days in cell culture. Treatment of Sertoli cells with an antisense c-fos oligonucleotide was found to inhibit the actions of PModS on transferrin expression. Combined results support the hypothesis that PModS acts indirectly through transcription factors (e.g. c-fos) to induce Sertoli cell differentiated functions (e.g. transferrin expression). Therefore, PModS appears to act as a differentiation-type factor to promote and maintain optimal Sertoli cell function.

Colocalization of nitric oxide synthase and NADPH-diaphorase in cultured myenteric neurones.

Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity were examined in explant culture preparations of the myenteric plexus from beneath the taenia coli of the guinea-pig caecum. Nitric oxide synthase immunoreactive neurones formed approximately one third of the total neuronal population. NADPH-diaphorase positive neurones, demonstrated histochemically, constituted a similar proportion of the total number of neurones. Immunocytochemistry and NADPH-diaphorase histochemistry performed on the same preparations revealed that all nitric oxide synthase immunoreactive neurones expressed NADPH-diaphorase activity. This histochemical evidence is consistent with the view that nitric oxide may act as a regulatory agent in the guinea-pig caecum.

Osmotic Adjustment and Growth of Salt‐stressed Cotton as Improved by a Bioregulator

AbstractFor examining links between growth of salinity‐exposed cotton and corresponding changes in physiological characters, a bioregulator was used as a tool for specific manipulations. Greenhouse‐grown cotton (Gossy‐pium birsutum L. cv. Paymaster 145) was subjected to NaCl levels up to 14.9 dS m−1. The bioregulator MCBuTTB, a cytokinin analog with known ability in improving growth and yield in several salt‐stressed crops, was applied by imbiding seed (0 or 268 ppm) plus one foliar spray in dosages equaling 0, 1, and 3 kg ha−1 at 45 DAP. MCBuTTB improved germination, growth, flowering, and finally boll weight. This result coincided with lower osmotic and higher water potentials in leaves, higher leaf turgidity, and more agilely reacting stomata. Treated plants had higher K and preferably Na concentrations in its leaves but ion accumulation was not a precondition for osmotic adjustments, because in cotton exposed to a manmtol‐simulated drought MCBuTTB triggered a similar osmotic adjustment and sustained vigour. Regression analyses indicated that osmotic adjustment alone did not cause the raised leaf turgidity. All characters which contribute to salinity resistance appeared as mutually depending and interacting but they respond to a regulatory agent of cytokinin‐like activity.

Actions of the Testicular Paracrine Factor (P-Mod-S) on Sertoli Cell Transferrin Secretion Throughout Pubertal Development*

Peritubular cells that surround the seminiferous tubules have been shown to produce a paracrine factor, termed P-Mod-S, that has dramatic effects on Sertoli cell function in vitro and is postulated to be important in the control of testicular function. The current study was designed to determine whether P-Mod-S has the ability to regulate Sertoli cell function during pubertal development. Sertoli cells were isolated from 10-, 20-, and 35-day-old rats which correspond to the prepubertal, mid-pubertal, and late pubertal stages of development. Histochemical analysis of cultured cells isolated from each age group was performed to establish the purity of the cell populations used. Testicular transferrin production by Sertoli cells was used as a marker of cellular differentiation. Basal production of transferrin by the cultured cells was found to increase during the pubertal period. P-Mod-S stimulated transferrin production by Sertoli cells isolated from 10-, 20-, and 35-day-old rats. FSH appears to enhance the ability of Sertoli cells to respond to P-Mod-S with cells obtained from 10-day-old rats. Sertoli cells from 35-day-old rats were nonresponsive to regulatory agents such as FSH. P-Mod-S alone, however, significantly stimulated transferrin production by Sertoli cells from this more adult stage of development. P-Mod-S was the only individual regulatory agent tested that could stimulate transferrin production by Sertoli cells from 35-day-old rats. Results indicate that P-Mod-S has the ability to regulate Sertoli cell function throughout pubertal development. Observations suggest that P-Mod-S and FSH may act together in the prepubertal testis to promote Sertoli cell differentiation and that P-Mod-S may act in the adult testis to maintain optimal Sertoli cell function and differentiation.

Flavonoid Evolution: An Enzymic Approach

Flavonoid evolution in land plants is discussed from an enzymic point of view, based on the present day distribution of the major subgroups of flavonoids in bryophytes, lower and higher vascular plants. The importance of varied functions in the origin of pathways with a series of sequential steps leading to end-products is considered; it is argued that the initial function is that of an internal regulatory agent, rather than as a filter against ultraviolet irradiation. The basic synthases, hydroxylases, and reductases of flavonoid pathways are presumed to have evolved from enzymes of primary metabolism. A speculative scheme is presented of flavonoid evolution within a primitive group of algae derived from a Charophycean rather than a Chlorophycean line, as a land environment was invaded. Flavonoid evolution was preceded by that of the phenylpropanoid and malonyl-coenzyme A pathways, but evolved prior to the lignin pathway.

PDGF B-chain in neurons of the central nervous system, posterior pituitary, and in a transgenic model

Platelet-derived growth factors (PDGFs) are growth-regulatory molecules that stimulate chemotaxis, proliferation, and increased metabolism of primarily connective tissue cells. In a survey of normal tissues, we found specific immunostaining for PDGF B-chain in neurons, principal dendrites, some axons, and probable terminals throughout the brain, in the dorsal horn of the spinal cord, and in the posterior pituitary of a nonhuman primate (Macaca nemestrina). PDGF activity was extracted from brain cortex and posterior pituitary, and ubiquitous expression of transcripts for the two chains of PDGF and both PDGF receptors was detected throughout the brain and posterior pituitary. A transgenic model was also evaluated in which the chioramphenicol acetyltransferase gene was placed under transcriptional control of the PDGF B-chain promoter. The transgene was preferentially expressed within neural cell bodies in the cortex, hippocampus, and cerebellum. PDGF may act as a neuronal regulatory agent. Neuronal release of PDGF could contribute to nerve regeneration and to glial proliferation that leads to gliosis and scarring.

Quantitative autoradiographic study of the postnatal development of adenosine A 1 receptors and their coupling to G proteins in the rat brain

Adenosine is now considered as a major regulatory agent in the mammalian central nervous system. Its actions are mediated by specific receptors which are coupled with an adenylate cyclase system via a G protein. The postnatal development of adenosine A 1 receptors was studied by quantitative autoradiography using [ 3H] N 6-cyclohexyladenosine, a potent receptor agonist in 42 rat brain structures. The coupling of these sites to G proteins was examined by measuring the effects of in vitro addition of guanylyl-5′-imidodiphosphate, a stable analogue of guanosine triphosphate, on N 6-cyclohexyladenosine binding. [ 3H] N 6-Cyclohexyladenosine-specific binding was quite low at birth, around 10% of adult levels, and exhibited a rather homogeneous distribution pattern, except in thalamic nuclei. Data showed a sequential development of adenosine A 1 receptors in relation to the time course of maturation of cerebral structures with a proliferation peak which paralleled rapid brain growth. The time period by which adult levels are reached differed according to the cerebral region studied. N 6-Cyclohexyladenosine-specific binding sites appeared to be functionally linked to G proteins in all structures and at all postnatal stages. However, the potency of guanylyl-5′-imidodiphosphate to displace N 6-cyclohexyladenosine binding was significantly lower before 5 days of age, suggesting functional changes during postnatal maturation in cerebral pathways modulated by adenosine.

The relationship between plasma free fatty acids and liver mitochondrial function in vivo

P O ratio, state 3 and 4 respiration rates, and acceptor control index (ACI) were assessed in rat liver mitochondria following an overnight fast and single bout of treadmill exercise of 30–180 min. P O was unaffected by fasting and 30 min of exercise; however, ACI was reduced because of an increase in state 4 respiration. Fasting, followed by running for 1 h or more decreased P O approx. 40% and ACI by 50%, an effect that could be attributed to a reduction in state 3 respiration. The decrease in P O was reversed 15 min after the cessation of exercise, whereas ACI remained depressed. All these functional alterations were mimicked by incubation of isolated mitochondria with palmitate and reversed by washing them with albumin. No direct correlation between plasma free fatty acids and the alterations in mitochondrial respiration was apparent. These data demonstrate that the decrease in the normal coupling of oxidation and phosphorylation in liver mitochondria produced by fasting /exercise is reversed rapidly in vivo. Furthermore, it is apparent that, if fatty acids act as a regulatory agent under these conditions, they do not do so solely on the basis of their plasma concentration.

Regulation of Sertoli Cell Function and Differentiation through the Actions of a Testicular Paracrine Factor P-Mod-S*

The current study was designed to investigate the actions of the testicular paracrine factor P-Mod-S on Sertoli cell function and differentiation. Transferrin production by Sertoli cells was stimulated by P-Mod-S to a greater extent than any individual regulatory agent and in a manner similar to a combination of FSH, insulin, retinol, and testosterone (FIRT). P-Mod-S had an additive response in combination with FIRT. The increase in transferrin production with a combination of P-Mod-S and FIRT is the highest level of stimulation (up to 8-fold) observed. These profound effects of P-Mod-S on Sertoli cell function implied a potential unique mechanism of action for the paracrine factor. FSH and FIRT significantly stimulated cAMP levels with both 60-min and 72-h treatments. In contrast, P-Mod-S had no effect on cAMP levels with a 60-min treatment and only a small increase with a 72-h treatment. Interestingly, P-Mod-S stimulated cGMP levels that remained above basal levels up to 72 h of treatment. FSH had no effect on cGMP levels. P-Mod-S did not affect inositol phosphate hydrolysis with treatments between 15 and 60 min. The actions of P-Mod-S on cGMP levels influenced Sertoli cell function on a molecular level. Northern blot analysis indicated that P-Mod-S and FIRT both stimulated the apparent levels of the 2.6-kilobase transcript of transferrin and the 1.7-kilobase transcript of androgen-binding protein. A solution hybridization procedure was used to quantitate the influence of P-Mod-S on Sertoli cell gene expression. P-Mod-S stimulated steady state levels of both transferrin and androgen-binding protein message approximately 2-fold, similar to the effects of FIRT. Both forms of P-Mod-S had similar biological activities and mechanisms of action. P-Mod-S (A) and P-Mod-S (B) both stimulated cGMP, altered Sertoli cell gene expression, and had profound effects on transferrin production. Although slightly different biochemically, both forms of P-Mod-S appear to be functionally similar. Combined observations indicate that the paracrine factor produced by peritubular cells, P-Mod-S, acts on Sertoli cells in part through a cGMP-mediated response to influence the expression of specific genes which subsequently have profound effects on Sertoli cell function and differentiation.

Growth factors in embryogenesis.

The growing appreciation that embryonic material is a rich source of growth factors underlies increasing interest in the role of growth factors in early development. These developments have arisen from a desire to understand fundamental embryological processes; the control of differentiation and the generation of form. One expectation of this research is the discovery of new forms of regulatory agent with novel biological and therapeutic potential. Furthermore, there is a direct relationship between growth and differentiation processes in early development and pathological processes in the adult; for example, the behaviour of tumour cells has historically been compared to that of embryonic cells and regenerative processes in the adult such as angiogenesis, wound healing and fracture repair may be considered to recapitulate events that occur in the normal course of embryogenesis. An understanding of the mechanisms that underlie these fundamental processes in normal development may therefore lead to a concomitant understanding of related pathological events in the adult.

Cloning of bovine GAP and its interaction with oncogenic ras p21.

The plasma membrane-bound mammalian ras proteins of relative molecular mass 21,000 (ras p21) share biochemical and structural properties with other guanine nucleotide-binding regulatory proteins (G-proteins). Oncogenic ras p21 variants result from amino acid substitutions at specific positions that cause p21 to occur predominantly complexed to GTP in vivo. Recently, a GTPase activating protein (GAP) with cytosolic activity has been discovered that stimulates the GTPase activity of normal but not of oncogenic ras p21. GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21. We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity. Here, using pure GAP in a kinetic competition assay, we show that GAP interacts preferentially with the active GTP complexes of both normal and oncogenic Harvey (Ha) ras p21 compared with the inactive GDP complexes. We also report the cloning and sequencing of the complementary DNA for bovine GAP. Regions of GAP share amino acid similarity with the noncatalytic domain of adenylate cyclase from the yeast Saccharomyces cerevisiae and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases.

Intraspecific interactions between adults and juveniles of the subtidal limpet, Patelloida mufria.

Regular monitoring of populations of the subtidal limpet Patelloida mufria (Hedley), which were at naturally large densities (>1000/m2), showed that at the times when there were peaks in juvenile recruitment, the mortality of adults increased greatly. This produced a cyclical pattern in the density of adults, suggesting the juveniles play a partial regulatory role. Experimental manipulations were done to test the influence of overall density and the density of juveniles on the growth and survival of adults. These indicated that an increase in overall density reduced the rate of growth of individuals and increased the rate of adult mortality during the peaks in juvenile recruitment. Similarly, removing these juveniles as they recruited averted this decline in adult abundance. The proposed mechanism to explain these results is that the juveniles have a competitive advantage over adults as they are much smaller and therefore require less energy to survive. It is hypothesisied, therefore, that there will be a threshold of adult size below which the recruits will lose their competitive advantage. Furthermore, if their density became too great, the cover of crustose algae (which is the main food of this species) would be removed resulting in a rapid decline in the numbers of P. mufria. Such an occurrence could be the ultimate regulatory agent for this species.

Purification of a paracrine factor, P-Mod-S, produced by testicular peritubular cells that modulates Sertoli cell function.

A testicular paracrine factor, P-Mod-S, was purified from conditioned medium obtained from serum-free cultures of peritubular cells. Stimulation of testicular transferrin production by cultured Sertoli cells was utilized as a bio-assay for P-Mod-S. A bioactive protein with an apparent molecular weight of 50,000 under physiological conditions was isolated by high pressure size exclusion chromatography. P-Mod-S was found to have an affinity for heparin and bound to a heparin affinity column. Two forms of P-Mod-S were purified with reverse-phase chromatography. The less hydrophobic form was referred to as P-Mod-S (A) and is a 56,000 molecular weight protein. The more hydrophobic form was referred to as P-Mod-S (B) and is a 59,000 molecular weight protein. Purification of P-Mod-S (A) and P-Mod-S (B) from peritubular cell-radiolabeled secreted proteins revealed that both proteins contain radioactivity. This result demonstrates active synthesis and secretion of P-Mod-S by peritubular cells. Although the amino acid composition of the two proteins indicates distinct differences in the content of several amino acids, the relationship of P-Mod-S (A) and P-Mod-S (B) is unknown at present. A greater than 1000-fold increase in the specific activity of P-Mod-S was achieved with the purification procedure utilized. P-Mod-S can account for essentially all the bioactivity present in crude peritubular cell-secreted protein preparations. The effects of the two forms of P-Mod-S on both transferrin and androgen-binding protein production by Sertoli cells was examined. Purified forms of P-Mod-S were found to have a greater effect on Sertoli cell function than any individual regulatory agent previously known to influence the cell, including follicle-stimulating hormone. The significance of peritubular cell-Sertoli cell interactions mediated via P-Mod-S to spermatogenesis and testicular function is discussed, as well as insight provided into general mesenchymal-epithelial cell interactions.

Effects of bone marrow ablation on compartmental prostaglandin synthesis by mononuclear phagocytes.

Mononuclear phagocyte functions were studied in mice selectively deprived of bone marrow and rendered profoundly monocytopenic by the administration of the bone seeking isotope, 89Sr. Characteristics of such mice include severe impairment of monocyte-M phi elicitation, ablation of C. parvum induction of PGSM but the persistence of resident peritoneal and pulmonary alveolar M phi populations; splenic M phi increase in number concomitantly with splenic hemopoiesis. Studies on compartmental regulation in this model suggest that the capacity of splenic M phi to synthesize and release PGE2 is dependent upon a function of the bone marrow and is not wholly determined by the local environment. The relationship of blood monocytes to PGSM is uncertain. In contrast to splenic M phi, the capacity of resident peritoneal M phi for eicosanoid synthesis appears to be independent of bone marrow function. Monocyte influx, moreover, does not appear necessary for the maintenance of the resident peritoneal and alveolar M phi populations. We do not yet know whether bone marrow ablation destroys a migratory precursor of PGSM or the source of a crucial regulatory agent. In conclusion, the observations discussed show that prostaglandin metabolism within the spleen is subject to extracompartmental influence. It is clearly important to determine the regulatory characteristics of individual M phi compartments and generalizations about functional properties of mononuclear phagocytes should be made with circumspection.

Role of Predators in Regulation of Endemic Populations of Matsucoccus matsumurae (Homoptera: Margarodidae) in Japan

Life table data for Matsucoccus matsumurae (Kuwana) were gathered from April through September 1984 from 30 ornamental and forest stands of two native hosts, Pinus densiflora Sieb. & Zucc. and P. thunbergiana Franco, and an exotic host, P. massoniana Lamb., located in central and southern Japan. The scale inhabited nearly every stand of the two Japanese pines, but its densities were always low and trees suffered no apparent injury. Trees were damaged, however, in forest stands of P. massoniana where densities were many times greater than on adjacent Japanese pines. Differences in scale abundance among host species and between forest and ornamental settings reflected differences in survivorship of first instars and fecundity of adult females. Population trend values calculated from life table data revealed that predators have an important role in the regulation of endemic populations of M. matsumurae. Harmonia axyridis Pallas, which comprised 84% of the total number of predators captured (n = 3,071), killed 97% of the scales in one heavily infested forest stand of P. massoniana in Kamigamo, Honshu in <4 weeks. When realistic densities of beetles were caged on infested pines, 81% of the small, inconspicuous stages of the scale and 98% of the larger, more conspicuous stages were consumed, which resulted in a 67-fold decrease in scale population growth compared with control cages containing no beetles. Despite prevalence of cannibalism among beetles both in cages and in the natural population, H. axyridis was clearly an effective regulatory agent for endemic populations of M. matsumurae and should be a prime candidate for biological control of exotic populations of this scale.

Allosteric regulation of glycerol kinase by enzyme IIIglc of the phosphotransferase system in Escherichia coli and Salmonella typhimurium.

The mechanism by which enzyme IIIglc of the bacterial phosphotransferase system regulates the activity of crystalline glycerol kinase from Escherichia coli has been studied, and the inhibitory effects have been compared with those produced by fructose-1,6-diphosphate. It was shown that the free, but not the phosphorylated, form of enzyme IIIglc inhibits the kinase. Mutants of Salmonella typhimurium were isolated which were resistant to inhibition by either enzyme IIIglc (glpKr mutants) or fructose-1,6-diphosphate (glpKi mutants), and each mutant type was shown to retain full sensitivity to inhibition by the other regulatory agent. Other mutants were fully or partially resistant to regulation by both agents. The two regulatory sites on the kinase are evidently distinct but must overlap or interact functionally. Kinetic analyses have revealed several mechanistic features of the regulatory interactions. (i) Inhibition by both allosteric regulatory agents is strongly pH dependent, with maximal inhibition occurring at ca. pH 6.5 under the assay conditions employed. (ii) Binding of enzyme IIIglc to glycerol kinase is also pH dependent, the Ki being near 4 microM at pH 6.0 but near 10 microM at pH 7.0. (iii) Whereas fructose-1,6-diphosphate inhibition apparently requires that the enzyme exist in a tetrameric state, both the dimer and the tetramer appear to be fully sensitive to enzyme IIIglc inhibition. (iv) Inhibition by enzyme IIIglc (like that by fructose-1,6-diphosphate) is noncompetitive with respect to both substrates. (v) The inhibitory responses of glycerol kinase to fructose-1, 6-diphosphate and enzyme IIIglc show features characteristic of positive cooperativity at low inhibitor concentration. (vi) Neither agent inhibits completely at high inhibitor concentration. (vii) Apparent negative cooperativity with respect to ATP binding is observed with purified E. coli glycerol kinase, with glycerol kinase in crude extracts of wild-type S. typhimurium cells, and with glpKr and glpKi mutant forms of glycerol kinase from S. typhimurium. These results serve to characterize the regulatory interactions which control the activity of glycerol kinase by fructose-1,6-diphosphate and by enzyme IIIglc of the phosphotransferase system.

Side Effects of Systemic Cyclosporine in Patients Not Undergoing Transplantation

Abstract Twenty-six patients with ocular inflammatory disorders of presumed autoimmune origin were treated with oral cyclosporine. Cyclosporine is a potent T cell regulatory agent that has been utilized extensively in organ transplantation. In general, the patients in this study did not have chronic debilitating illnesses that are observed in organ transplant recipients, did not receive corticosterolds in addition to cyclosporine, and did not undergo any surgical procedure during their treatment. This study describes the observed adverse reactions in this group of patients not undergoing transplantation. The reported side effects of cyclosporine in organ transplant recipients have included nephrotoxicity, hepatotoxicity, infections, lymphoma, hirsutism, gingivitis, and central nervous system toxicity. Side effects were observed that were similar to those in previous reports, but the severity of the nephrotoxicity and hepatotoxicity appeared to be less. Nephrotoxicity occurred in eight patients and hepatotoxicity occurred in one patient. No lymphomas were seen. Hypertension and anemia each were observed in six patients. In addition, previously unreported effects of hyperuricemia, elevated sedimentation rate, and hidradenits were observed. However, the overall severity of the side effects did not seriously affect the usage of cyclosporine in the patients in this study. Cyclosporine may be useful in the treatment of other autoimmune diseases.

Side effects of systemic cyclosporine in patients not undergoing transplantation

Twenty-six patients with ocular inflammatory disorders of presumed autoimmune origin were treated with oral cyclosporine. Cyclosporine is a potent T cell regulatory agent that has been utilized extensively in organ transplantation. In general, the patients in this study did not have chronic debilitating illnesses that are observed in organ transplant recipients, did not receive corticosterolds in addition to cyclosporine, and did not undergo any surgical procedure during their treatment. This study describes the observed adverse reactions in this group of patients not undergoing transplantation. The reported side effects of cyclosporine in organ transplant recipients have included nephrotoxicity, hepatotoxicity, infections, lymphoma, hirsutism, gingivitis, and central nervous system toxicity. Side effects were observed that were similar to those in previous reports, but the severity of the nephrotoxicity and hepatotoxicity appeared to be less. Nephrotoxicity occurred in eight patients and hepatotoxicity occurred in one patient. No lymphomas were seen. Hypertension and anemia each were observed in six patients. In addition, previously unreported effects of hyperuricemia, elevated sedimentation rate, and hidradenits were observed. However, the overall severity of the side effects did not seriously affect the usage of cyclosporine in the patients in this study. Cyclosporine may be useful in the treatment of other autoimmune diseases.

Bombesin-like peptides and receptors in human tumor cell lines

Human cancer cell lines were assayed for bombesin-like peptides and receptors. Acid extracts derived from small cell lung cancer, but not other types of cancer had high levels of immunoreactive bombesin. Regardless of patient treatment, site of tumor origin (bone marrow, lymph node, or pleural effusion) or culture conditions, small cell lung cancer cell lines had high levels of bombesin-like peptides. Thus, bombesin levels in small cell lung, but not other types of human cancer, are routinely elevated. Also, small cell lung cancer lines in contrast to other cell lines have a high density of binding sites for a radiolabeled bombesin analogue. The presence of high concentrations of bombesin-like peptides and receptors suggests that bombesin may function as an important regulatory agent in human small cell lung cancer.

Expression of the gene for ribosomal protein S20: effects of gene dosage.

We have exploited the properties of three different plasmids which carry the gene for Escherichia coli ribosomal protein S20 (rpsT) to test the effects of gene dosage on the expression of rpsT. Over a range of total copies of rpsT of 1 to 58 per haploid genome equivalent, the rate of incorporation of uridine during a 30-s pulse into RNA annealing to either of two specific probes for S20 mRNA increased essentially in proportion to copy number. In contrast, the rate of synthesis of S20 protein increased no more than 2.1-fold at the highest copy number. We conclude, in contrast to an earlier report (D. Geyl, and A. Böck, Mol. Gen. Genet. 154:327-334, 1977), that the synthesis of S20 is regulated at a posttranscriptional step. We propose that S20 itself is the regulatory agent and that binding of S20 to its own mRNA in regions homologous in structure with 16S rRNA can account for our results.