The dominant mutation Suppressor of black indicates that de novo pyrimidine biosynthesis is involved in the Drosophila tan pigmentation pathway

A deficiency in the production of -alanine causes the black (b) phenotype of Drosophila melanogaster. This phenotype is normalized by a semi-dominant mutant gene Su(b) shown previously to be located adjacent to or within the rudimentary (r) locus. The r gene codes for three enzyme activities involved in de novo pyrimidine biosynthesis. Pyrimidines are known to give rise to -alanine. However, until recently it has been unclear whether de novo pyrimidine biosynthesis is directly coupled to -alanine synthesis during the tanning process. In this report we show that flies carrying Su(b) can exhibit an additional phenotype, resistance to toxic pyrimidine analogs (5-fluorouracil, 6-azathymine and 6-azauracil). Our interpretation of this observation is that the pyrimidine pool is elevated in the mutant flies. However, enzyme assays indicate that r enzyme activities are not increased in Su(b) flies. Genetic mapping of the Su(b) gene now places the mutation within the r gene, possibly in the carbamyl phosphate synthetase (CPSase) domain. The kinetics of CPSase activity in crude extracts has been studied in the presence of uridine triphosphate (UTP). While CPSase from wild-type flies was strongly inhibited by the end-product, UTP, CPSase from Su(b) was inhibited to a lesser extent. We propose that diminished end-product inhibition of de novo pyrimidine biosynthesis in Su(b) flies increases available pyrimidine and consequently the -alanine pool. Normalization of the black phenotype results.     

Adenine nucleotide metabolism in Azotobacter vinelandii. Two metabolic pathways of AMP degradation

AMP-degrading pathways in Azotobacter vinelandii cells were investigated. AMP nucleosidase (EC 3.2.2.4) was rapidly synthesized and reached a maximum at 24 h, while the activity of 5-nucleotidase (EC 3.1.3.5) specific for AMP, which was negligible during the logarithmic phase of the growth, first appeared in 24 h-cultures, and reached a maximum after complete exhaustion of sucrose from the growth medium (70 h).Cell-free extracts of A. vinelandii of 48 h-cultures hydrolyzed AMP to ribose 5-phosphate and adenine in the presence of ATP, and adenine was deaminated to hypoxanthine. When ATP was excluded, AMP was dephosphorylated to adenosine, which was further metabolized to inosine, and finally to hypoxanthine. Hypoxanthine thus formed was reutilized for the salvage synthesis of IMP under the conditions where 5-phosphoribosyl 1-pyrophosphate was able to be supplied. These results suggest that the levels of ATP can determine the rate of AMP degradation by the AMP nucleosidase- and 5-nucleotidase-pathways. The role of ATP in the AMP degradation was discussed in relation to the regulatory properties of AMP nucleosidase, inosine nucleosidase (EC 3.2.2.2) and adenosine deaminase (EC 3.5.4.4).     

Role of cytosine deaminase and β-alanine-pyruvate transaminase in pyrimidine base catabolism by Burkholderia cepacia

A determination of the possible role of the salvage enzyme cytosine deaminase or -alanine-pyruvate transaminase in the catabolism of the pyrimidine bases uracil and thymine by the opportunistic pathogen Burkholderia cepacia ATCC 25416 was undertaken. It was of interest to learn whether these enzymes were influenced by cell growth on pyrimidine bases and their respective catabolic products to the same degree as the pyrimidine reductive catabolic enzymes were. It was found that cytosine deaminase activity was influenced very little by cell growth on the pyrimidines tested. Using glucose as the carbon source, only B. cepacia growth on 5-methylcytosine as a nitrogen source increased deaminase activity by about three-fold relative to (NH₄)₂SO₄-grown cells. In contrast, the activity of –alanine-pyruvate transaminase was observed to be at least double in glucose-grown ATCC 25416 cells when pyrimidine bases and catabolic products served as nitrogen sources instead of (NH₄)₂SO₄. Transaminase activity in the B. cepacia glucose-grown cells was maximal after the strain was grown on either uracil or 5-methylcytosine as a nitrogen source compared to (NH₄)₂SO₄-grown cells. A possible role for -alanine-pyruvate transaminase in pyrimidine base catabolism by B. cepacia would seem to be suggested from the similarity in how its enzyme activity responded to cell growth on pyrimidine bases and catabolic products when compared to the response of the three reductive catabolic enzymes.     

Lysine inhibition of Saccharomyces cerevisiae: role of repressible L-lysine ε-aminotransferase

Lysine added to grain mashes under nitrogen-limiting conditions (as in most industrial fermentations) inhibited growth of Saccharomyces cerevisiae. This inhibition was relieved by raising the assimilable nitrogen content. Lysine-induced inhibition is not mediated through accumulation of -oxoadipic acid, an intermediate of lysine metabolism which accumulates by a back up of intermediates in de novo synthesis. Lysine degradation is regulated by the synthesis of L-lysine -aminotransferase, an enzyme that catalyses the first step in one of three possible routes of lysine degradation (not previously reported in S. cerevisiae). Synthesis is repressed under nitrogenlimiting conditions, but derepressed when excess assimilable nitrogen is available. Derepression results in degradation of lysine and decreases inhibitory effects on growth. The toxic compound appears to be lysine itself.The authors are with the Department of Applied Microbiology and Food Science, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 0W0. Canada     

Phytohormone-regulated β-amylase gene expression in rice

The expression of -amylase genes in rice (Oryza sativa) and its regulation by phytohormones gibberellic acid (GA) and abscisic acid (ABA) were examined. Upon germination -amylase is synthesizedde novo in aleurone cells and (GA) is not required. Exogenous addition of GA does not enhance the -amylase activity, while ABA inhibits the -amylase activity, mRNA accumulation, and the germination of rice seeds. GA can reverse ABA inhibition of -amylase expression, but not ABA inhibition of seed germination. Such regulation represents a new interaction of ABA and GA.     

Effects of the plant alkaloid castanospermine as an antimetabolite of storage pests

The secondary plant compound castanospermine is toxic to the larvae of the bruchid beetle Callosobruchus maculatus F. and the flour beetle Tribolium confusum J. & V. when incorporated into the diet. The larval alimentary tract -D-glucosidase and -D-glucosidase activities of C. maculatus were strongly inhibited by castanospermine in a non-competitive and competitive manner respectively. The larval alimentary tract -D-glucosidase activity of T. confusum was strongly inhibited in a competitive manner, but the -D-glucosidase activity was not markedly inhibited; however, the -D-galactosidase activity exhibited strong uncompetitive inhibition.

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Résumé La castanospermine, substance secondaire végétale, incorporée à l'aliment est toxique pour les larves de Callosobruchus maculatus F. et de Tribolium confusum J. & V. Les activités -D-glucosidase et -D-glucosidase du tube digestif de C. maculatus ont été fortement inhibées par la castanospermine respectivement de façon non-compétitive et compétitive. L'activité -D-glucosidase du tube digestif de T. confusum était, fortement inhibée d'une façon compétitive, mais l'activité -D-glucosidase n'était pas nettement inhibée; cependant, l'activité -D-galactodase ne présentait pas une forte inhibition compétitive.

     

Expression of a cloned beta-glucanase gene from Bacillus amyloliquefaciens in an Escherichia coli relA strain after plasmid amplification

Summary Amino acid starvation of cells of the Escherichia coli relA strain, CP79, which cannot accumulate guanosine tetraphosphate (ppGpp) in response to amino acid limitation, increased the pEG1 plasmid content about 5- to 7-fold in comparison with exponentially growing cells (pEG1: pBR322 with an insertion of Bacillus amyloliquefaciens DNA coding for -glucanase). In contrast, no pEG1 amplification occurred in E. coli CP78, the stringently controlled counterpart, after amino acid starvation. In order to verify these results, the plasmid DNA content was monitored by measuring the expression of pEG1-encoded -glucanase from B. amyloliquefaciens both before and after plasmid amplification. When amino acid starved CP79 cells were given an additional dose of amino acids, a more than 10-fold increase in pEG1-encoded -glucanase activity (per cell mass) was measured. This increase in enzyme activity correlates with pEG1 amplification during amino acid limitation. Under comparable conditions the activity of -glucanase was not increased in strain CP78, which did not amplify the plasmid. We suggest that the replication of pEG1 in amino acid starved E. coli cells is somehow under negative control by ppGpp. Moreover, we found the Bacillus -glucanase in E. coli relA cells to be excreted into the growth medium after starvation and overexpression.     

Immunohistochemical study of the pars tuberalis of the adenohypophysis in the monkey,Macaca irus

Summary The pars tuberalis of the hypophysis in the monkey Macaca irus encompasses the hypophysial stem up to the median eminence. Histologically, it consists of several layers of chromophobic cells. A few PAS¹-positive cells also stainable with Alcian blue (pH 3.0) can be observed among the unstained elements. Using the indirect immunofluorescence antibody technique, scattered immunoreactive cells were revealed with the anti-oLH antibody; these cells did not react with the anti-hFSH antibody. In contrast, the immunoreactions to anti-hGH, anti-hPRL, anti-ACTH, anti-MSH, anti-LPH and anti-endorphin sera were completely negative. Single cells reacting with the anti-hTSH serum were observed at the inferior end of the hypophysial stalk (zona tuberalis), i.e., beyond the pars tuberalis proper. These results are compared with data reported in the literature.

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Résumé La pars tuberalis de l'hypophyse du Singe Macacus irus entoure la tige infundibulaire jusqu'à l'éminence médiane. En techniques histologiques, elle apparaît constituée de plusieurs assises cellulaires d'aspect chromophobe. On y observe quelques cellules PAS-positives réagissant simultanément avec le bleu Alcian (pH3.0). En technique d'immunofluorescence indirecte, des cellules dispersées sont mises en évidence uniquement avec un anticorps anti-oLH; ces cellules ne réagissent pas avec un anticorps anti-hFSH. L'utilisation d'anticorps anti-hGH, anti-hPRL, anti-ACTH, anti-MSH, anti-LPH et antiendorphines ne permet pas de révéler des cellules immunoréactives. Quelques cellules réagissant avec un anticorps anti-hTSH s'observent à la base de la tige hypophysaire (zona tuberalis), c'est-à-dire au-delà de la pars tuberalis proprement dite. Ces résultats sont confrontés à ceux rapportés dans la littérature.

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Abbreviations used in this Article PAS periodic acid Schiff - oLH ovine luteinizing hormone - hFSH human follicle stimulating hormone - hGH human growth hormone - hPRL human prolactin - ACTH corticotropin - MSH melanotropin - LPH lipotropin - hTSH human thyrotropin - BSA and HSA bovine and human serum albumin     

Regulation of the Pyrimidine Biosynthetic Pathway in <Emphasis Type="Italic">Pseudomonas mucidolens</Emphasis>

Control of pyrimidine biosynthesis was examined in Pseudomonas mucidolens ATCC 4685 and the five de novo pyrimidine biosynthetic enzyme activities unique to this pathway were influenced by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. When uracil was supplemented to glucose-grown ATCC 4685 cells, activities of four de novo enzymes were depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the glucose-grown mutant strain cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase activities to increase by more than 3-fold while OMP decarboxylase activity increased by 2.7-fold. The syntheses of the de novo enzymes appeared to be regulated by pyrimidines. At the level of enzyme activity, aspartate transcarbamoylase activity in P. mucidolens ATCC 4685 was subject to inhibition at saturating substrate concentrations. Transcarbamoylase activity was strongly inhibited by UTP, ADP, ATP, GTP and pyrophosphate.     

Purification and characterization of developmentally regulated AMP deaminase from Dictyostelium discoideum

AMP deaminase, the enzyme that catalyzes the conversion of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia, was purified from the cellular slime mold, Dictyostelium discoideum in the nutrient-deprived state. The native enzyme had an apparent molecular weight of 199,000 daltons. Its apparent Km was 1.6 mM and its Vmax was 1.0 mumol min-1 mg-1, as measured by the release of IMP From AMP. The enzyme, like other AMP deaminases, was found to be activated by ATP, and inhibited either by GTP or inorganic phosphate. It was also specific for the deamination of AMP. Deaminase activity was increased either when vegetative cells were placed in a nutrient-deprived medium (for up to 6 h) or when vegetative cells were treated with the drug hadacidin. In cells actively growing in complete media, enzyme activity was more non-specific, hydrolyzing adenosine as well as AMP. AMP deaminase in D. discoideum appears to be stage-specific and developmentally regulated, possibly serving to regulate the adenylated nucleotide pool and the interconversion to guanylated nucleotides during early morphodifferentiation.     

Intracellular calcium activity in split frog skin epithelium: Effect of cAMP

Summary Measurement of intracellular calcium activity (a _Ca ^c ) by ion-selective microelectrodes has previously been technically limited to relatively large cells (20 m). We now report results obtained with this technique in the small epithelial cells (10 m) of split frog skin using microelectrodes having an outer tip diameter of <0.2 m. The basolateral membrane potential was measured with Ca²⁺-selective microelectrodes (E _Ca ^sc ) and with reference micropipettes ( ^sc ) either sequentially or simultaneously in 15 successful experiments. Under baseline conditions,a _Ca ^c was measured to be 215±39nm (mean±se), in close agreement with the mean values estimated from published data obtained withNecturus proximal tubule. Stimulation of Na⁺ transport across six skins with 1mm serosal 8p-chlorophenylthio-3,5 cyclic AMP (CPTcAMP) increaseda _Ca ^c by a factor of 2.6±0.6. The increase ina _Ca ^c preceded the CPTcAMP-induced increase inI _sc. The results of the present study indicate that electrometric determination of intracellular calcium activity is now feasible in a much wider range of cell systems than heretofore possible. CPT cAMP elevates intracellular Ca²⁺ activity; this phenomenon is an early event, preceding the natriferic effect of CPTcAMP.     

Inhibition by Hadacidin,Duazomycin A,and Other Amino Acid Derivatives of de novo Starch Synthesis

Two amino acid derivative antibiotics, hadacidin and duazomycin A, were isolated as inhibitors of de novo starch synthesis in excised leaf segments from culture filtrates of Penicillium No. 467 and Streptomyces No. 317, respectively. In addition, azaserine, 6-diazo-5-oxo-l-norleucine (DON), and trifluoro-dl-methionine were found to be potent inhibitors among about 70 kinds of commercial amino acid derivatives. These amino acid derivatives inhibited de novo starch synthesis at concentrations ranging from 1 to 10ppm but did not inhibit photosynthetic oxygen evolution at a concentration of 100ppm. The inhibition caused by these diazo compounds was reversed by a supplement of l-glutamine. With hadacidin and trifluoro-dl-methionine, however, the reversal was not observed upon the addition of l-aspartic acid or l-methionine, respectively. Among these active compounds, hadacidin was herbicidal against lettuce and barnyard millet by foliar treatment at a concentration of more than 1000 ppm.     

Synergistic effects of TGIF and interferonγ (IFN-γ) on tumor cell growthTGIF and IFN-γ

Interferon- (IFN-) and tumor growth inhibitory factor (TGIF) were inducedin vitro in the supernatant from mixed culture of human peripheral blood mononuclear cells (PBMC) and OK-432. TGIF activity was determined by growth inhibition of a human gastric adenocarcinoma cell line, MK-1 cells, and IFN- activity was measured by radioimmunoassay. The production of TGIF and IFN- was time-dependent, reaching its maximum around 48 hrs. Although there was no significant correlation between TGIF production and IFN- production, combination of a subthreshold concentration of recombinant IFN- (rIFN-) and TGIF induced significant growth inhibition of MK-1 cells. This fact indicates that the effects of rIFN- and TGIF are synergistic. The antiproliferative effect of these cytokines are highly species-specific, and their synergistic effects were also species-specific. rIFN--sensitive and -resistant clones were successfully established from the original MK-1 cell line; those clones are both sensitive to TGIF. Synergistic antiproliferative effects were found when the rIFN--sensitive clone, but not the resistant clone, was used as a target, suggesting that the synergistic effects require the target cells' sensitivity to IFN-. These results indicate that the synergistic effects of TGIF and IFN- may produce a clinical antitumor action in cancer patients receiving OK-432 administration.     

Changes in ectonucleotidase activities in rat Sertoli cells during sexual maturation

Sertoli cell maturation is a complex process involving both morphological and biochemical changes. These cells have previously been shown to be targets for extracellular purine structures such as ATP and adenosine. These compounds evoke responses in rat Sertoli cells through the purinoceptor families, P₂X and P₂Y and PA₁. The signals to purinoceptors are usually terminated by the action of ectonucleotidases. In a previous work, we demonstrated that rat Sertoli cells have ecto-ATPdiphosphohydrolase (EC 3.6.1.5), ecto-5-nucleotidase (EC 3.1.3.5) and ecto-adenosine deaminase (ecto-ADA) (EC 3.5.4.4) activities. Here we investigated whether some changes occur during rat Sertoli cell maturation in these activities. Rat Sertoli cells obtained from rats of different ages representing the pre pubertal, mid pubertal and young adult (10-, 18- and 35-day-old, respectively) were cultured and used for different assays. The nucleotide hydrolysis was estimated by measuring the Pi released using a colorimetric method and by HPLC analysis. ATP and ADP hydrolysis was increased 3-fold during sexual maturation. AMP hydrolysis increased 4-fold in 10- to 35-day-old Sertoli cells. Similar results were obtained when we used other substrates to measure the extracellular hydrolysis of nucleotides (GTP, GDP, GMP and IMP). The ecto-ADA activity showed a 2-fold increase in the specific activity (18- to 35-day-old Sertoli cells). The termination of the purine cascade by adenosine degradation was faster in the 35- than in 18-day-old Sertoli cells. Follicle Stimulating Hormone (FSH) influences on the ectonucleotidase activities were investigated in 10- and 18-day-old Sertoli cells and a significant increase in the ATP and ADP hydrolysis was observed. Our results show an increase in the extracellular purine cascade during the Sertoli cell development, indicating a rise in the purine communication inside the seminiferous tubules with rat sexual maturation.     

Further studies of the mechanism(s) of polyunsaturated-fatty-acid-mediated increases in intracellular cAMP formation in N1E-115 neuroblastoma cells

Following earlier observations that increasing the polyunsaturated fatty-acid (PUFA) content of N1E-115 neuroblastoma cells elevated basal and adenosine (Ado)-stimulated intracellular cyclic AMP (cAMP) formation, we carried out studies to determine the mechanism(s) by which PUFA exerted their modulatory effects. Basal increases in cAMP in the PUFA-enriched (PUFA⁺) cells were evident with short (60 sec) exposure to a phosphodiesterase inhibitor (Ro 20-1724), and increased to a maximum at 20 min; they were not observed in the absence of Ro 20-1724. Forskolinstimulated cAMP formation in the presence of the Ro compound was 2- to 3-fold higher in the PUFA⁺ cells. Basal elevations in cAMP were reduced by 70% by exposing the PUFA⁺ cells to Ado deaminase (ADA) or to an Ado antagonist, and were further increased by inhibiting ADA, which suggested that they could be producing endogenous Ado that activated stimulatory Ado receptors. However, this did not appear to involve PUFA-mediated stimulation of 5-nucleotidase activity or inhibition of [³H]Ado uptake. Overall, the results of this study indicated that multiple mechanisms are involved in PUFA modulation of cAMP formation.Abbreviations used PUFA polyunsaturated fatty acid(s) - ACase adenylate cyclase - PDE phosphodiesterase - ADA adenosine deaminase - 2-DCF 2-deoxycoformycin - 8-PT 8-phenyltheophylline - DPR dipyridamole - CPA cyclopentyladenosine - DMEM Dulbecco's modified Eagle's medium - Ro 20-1724 4-(3-butoxy-4-methoxybenzyl) imiazolidin-2-one - BSA bovine serum albumin     

Sequential control of phytochrome-mediated synthesis de novo of β-amylase in the cotyledons of mustard (Sinapis alba L.) seedlings

In the cotyledons of mustard (Sinapis alba L.) seedlings irradiated from the time of sowing with continuous red light, the photoreversibility of the phytochrome-mediated increase in -amylase activity (EC 3.2.1.2) is lost 36 h after sowing (coupling point). However, the induced increase of -amylase activity cannot be detected before 46 h after sowing (starting point). Density labeling with deuterium oxide shows that the increase of enzyme activity in light and darkness coincides precisely with the synthesis of -amylase protein. Thus, phytochrome mediates an increase of -amylase synthesis de novo. Since there is no turnover detectable by density labeling, it is concluded that -amylase of mustard cotyledons is a physiologically stable enzyme (half-life >5 d). The 10-h time gap between loss of photoreversibility and onset of light-induced -amylase synthesis points to a relatively stable regulatory element within the signal chain (transmitter) which links -amylase synthesis to the primary action of phytochrome. A 12-h lag between the cessation of phytochrome action and the cessation of induced -amylase synthesis indicates a limited lifetime of the transmitter (about 12 h). The effect of this result on the interpretation of the coupling point is discussed.Abbreviations P_r, P_fr red and far-red absorbing forms of phytochrome     

Are active oxygen species involved in induction of β-carotene in Dunaliella bardawil?

The purpose of this work was to test whether induction of massive -carotene synthesis in the alga Dunaliella bardawil is triggered by oxygen radicals. The following results were obtained: (i) The induction of -carotene synthesis is preceded by a lag period of about 4 h during which the cells swell and photosynthesis is partially inhibited, (ii) Addition of promoters of oxygen radicals or of azide (an inhibitor of catalase and superoxide dismutase) during the induction period, under conditions which are suboptimal for massive -carotene accumulation, greatly enhances -carotene synthesis, photodegradation of chlorophyll and inhibition of photosynthesis, (iii) High irradiance, which induces massive -carotene accumulation, also induces a high catalase activity. It is suggested that photosynthetically produced oxygen radicals are involved in triggering massive -carotene accumulation in D. bardawil.     

Influence of cyclic AMP on the growth response and anaerobic metabolism of carbon monoxide in Rhodocyclus gelatinosus

Rhodocyclus gelatinosus strain 1 (str. 1), a photoheterotrophic bacterium, used CO as an energy substrate under anaerobic CO/light conditions, and exhibited a diauxic growth response when CO was removed from the culture. Changes in the level of cyclic AMP which occurred in cells during diauxie suggested that the cyclic nucleotide operated as an intracellular control molecule. During CO/light-phase growth, intracellular cyclic AMP was 30 pmol/mg protein, and, as str. 1 adapted for photosynthetic growth after removal of CO, intracellular cyclic AMP levels decreased to 9 pmol/mg protein. Reexposure of a light culture to CO induced synthesis of CO oxidation activity (measured as CO:MV oxidoreductase). If 10 mM cyclic AMP was added with CO, the rate of synthesis of CO:MV oxidoreductase activity increased 25-fold, and str. 1 produced 1,230 units of activity (nmol CO oxidized min^-1 mg^-1 protein) after only 1 h. With cyclic AMP and no CO, no incerease in CO oxidation activity was seen. Appearance of CO oxidation activity in str. 1 represented de novo protein synthesis and was blocked with chloramphenicol. In addition to stimulating formation of CO oxidative activity, a high level of cyclic AMP in str. 1 during growth with CO appeared to influence photometabolism negatively by repressing bacteriochlorophyll formation.Abbreviations Bchl a bacteriochlorophyll a - MV methyl viologen - CO MV oxidoreductase, carbon monoxide: methyl viologen oxidoreductase     

Influence of several nucleotides on the competence development of Bacillus subtilis

The influence of adenosine-3,5-cyclic monophosphate (cAMP) and other nucleotides on the competence development of Bacillus subtilis was studied. The stimulation of competence which can be achieved by exposing physiologically low-competent cells to supernatants from highly competent cultures can be inhibited with different cAMP doses. When the same cells were suspended in a minimal medium with cAMP, varying degrees of stimulation of competence were observed depending on the time of addition of the drug. This effect is not specific for cAMP. It appears to be correlated to an increase of the amount of DNA bound to the competent cells. cAMP activities were antagonized by equimolar doses of adenosine-triphosphate (ATP) and guanosine-triphosphate (GTP).List of Abbreviations ATP adenosine triphosphate - AMP adenosine monophosphoric acid - GTP guanosine-triphosphate - cGMP guanosine 3,5-cyclic-monophosphoric acid - PLC physiologically low-competent cells - TY triptone yeast - CSA competence-stimulating activity - SF filtered supernatants - NCS non-competent supernatants - MBW minimal Bott and Wilson