Precursor Pool Size Research Articles

Biosynthesis of natural products.

The experimental verification of a proposed biosynthetic pathway for a given natural product is often difficult to obtain with the use of the whole organism (permeability factors) or, in the case of higher plants, a cell-free system. Until the purified enzyme for each step of biosynthesis is available, biosynthetic studies can, however, be carried out, albeit with modest incorporation values, by means of either hydroponic or injection methodology. Where viable seed sources are available it is suggested that improvements of several orders of magnitude in incorporation can be achieved by a short-term incubation (small pool size of precursors; trapping of reactive intermediates) or a long-term feeding (equilibration of precursor with the compartmentalized or induced synthetases). In bacterial, fungal, mammalian, and plant systems, where incorporation efficiencies provide the opportunity to study (13)C enrichment (at least equal to natural abundance of the isotope), we can expect a rapid expansion since the method removes the tedium of carbon-by-carbon degradation. For the next few years, however, the prognosis would seem to favor parallel studies of (13)C and (2)H, and of (14)C/(3)H ratio techniques since the last-mentioned method provides more information concerning the stereoselectivity of labeling processes on the microgram scale.

Effect of cycloheximide on pools of deoxyribonucleoside triphosphates

Inhibition of protein synthesis by cycloheximide blocks DNA replication in many eukaryotic cells. To test whether this effect was mediated through enzymes furnishing DNA precursors, pool sizes of deoxyribonucleoside triphosphates were measured following cycloheximide treatment in the synchronous mitotic cycle of Physarum. It was found that cycloheximide either did not affect the pool size of DNA precursors (dATP and dGTP) or it led to a pool expansion (dCTP and dTTP). It is concluded that the arrest of DNA replication by inhibitors of protein synthesis is not due to a lack of precursors.

Investigation of early mammalian development using interspecific chimaeras between rat and mouse.

THE analysis of genetic mosaics has allowed inferences to be made about several otherwise elusive aspects of development including cell determination, division rates, lineage and deployment and the sizes of pools of precursor cells destined to form particular organs and tissues. The most significant results have been achieved using insect mosaics arising in early development from spontaneous or induced genetic modification of one cell1, 2. Extensive genetic mosaicism may also occur in mammals either arising spontaneously during development as a result of random × chromosome inactivation in female embryos3 or by the experimental combination of embryos of different genotype to form chimaeras4–6. Human or mouse mosaics carrying enzymic, chromosomal or pigmentation markers have been used in several investigations into cell lineage and the size of precursor pools of cells. Use of these markers often, however, involves analysis long after the critical developmental events have taken place and so the results are likely to be distorted by differential proliferation, migration and death of cells. Furthermore. many of the available markers have only a limited tissue distribution and, with the exception of those involving pigmentation, differentiation of photoreceptors and isoenzymes of β-glucuronidase7–9, only yield estimates of the ratio of cells in mosaic tissues and provide no information about their spatial arrangement. Therefore some of the conclusions drawn from analyses of mammalian mosaics have been based on assumptions of questionable validity10–13.

Analysis of precursor pool labeling kinetics without pool size measurements: A computer-assisted approach

A general method is described which permits the correction of macromolecular radioactive labeling data for precursor pool specific activity variation during a labeling period. Measurement of the precursor pool size is made unnecessary. The method involves: 1. (1) Determination of radioactivity in the precursor pool at various times during an extended labeling period. 2. (2) Derivation of a theoretical equation describing the kinetics of labeling of the precursor pool. 3. (3) Computer-assisted fitting of the theoretical equation to the observed labeling data. 4. (4) Use of the parameters obtained from the curve-fitting to calculate the time course of saturation of the precursor pool with exogenous radioactive precursor . The derived saturation curves may be used to correct macromolecular labeling data, on a relative basis, for precursor pool saturation. The method may also be extended to provide absolute specific activity estimates. The application of the method is shown for the UTP and S-adenosyl methionine pools of cultured lymphocytes.

Control of ovarian cholesterol ester biosynthesis

1. Experimental evidence is presented for a role of progesterone and 20alpha-hydroxypregn-4-en-3-one as inhibitors of cholesterol ester synthetase in the acute depletion of ovarian cholesterol ester after trophic stimulation. 2. Luteinizing hormone in vitro decreased by 84% the rate of esterification of cholesterol with added [(14)C]oleate by slices of rabbit ovarian interstitial tissue; this effect was mimicked by cyclic AMP (adenosine 3':5'-cyclic monophosphate) in vitro, and occurred without large changes in precursor pool sizes or membrane permeability. 3. Cyclic AMP was shown to have no direct effect on cholesterol ester synthetase or cholesterol esterase in cell-free extracts of rabbit ovarian interstitial tissue, but decreased the activity of cholesterol ester synthetase (not that of cholesterol esterase) in extracts prepared from slices previously incubated with it. 4. The inhibitory effect of cyclic AMP on esterification of cholesterol with added [(14)C]-oleate was prevented by both cycloheximide and aminoglutethimide phosphate (which also inhibited steroid synthesis in response to cyclic AMP). 5. Cyclic AMP raised the intracellular concentrations of progesterone and 20alpha-hydroxypregn-4-en-3-one in incubated slices by factors of 2.8 and 3.9 respectively. 6. Cycloheximide and aminoglutethimide phosphate administered in vivo blocked cholesterol ester depletion in response to luteinizing hormone in rats; in these ovaries cycloheximide and aminoglutethimide phosphate decreased the concentrations of progesterone and 20alpha-hydroxypregn-4-en-3-one and luteinizing hormone raised them. 7. Progesterone and 20alpha-hydroxypregn-4-en-3-one added to cell-free extracts of rabbit ovarian interstitial tissue in vitro (at concentrations comparable with those found in incubated slices) inhibited cholesterol ester synthetase by up to 85%. 8. The results are discussed with reference to the acute control of cholesterol ester concentrations in the ovary and adrenal cortex.

The effect of temperature and substrate concentration on the kinetics of [6- 3H]uridine incorporation into RNA of toad bladder epithelial cells: The role of aldosterone

1. The effects of temperature and uridine substrate concentration during incorporation of [6- 3H]uridine into RNA were studied in epithelial cells of the isolated urinary bladder of the toad, Bufo marinus, harvested after incubation. 2. Gross accumulation of newly synthesized RNA in the acid-insoluble fraction of whole epithelial cells was maximal at 33°C., even though net RNA synthesis, i.e. RNA specific activity, was significantly reduced compared to 24°C. Cold exposure at 8°C decreased accumulation of newly synthesized RNA when compared to warmer temperatures. 3. A uridine substrate concentration constant ( K′ m ), derived in a manner similar to the Michaelis-Menten constant ( K m ), was determined to be K′ m = 2.13 · 10 −5 mole/l at 24°C for non-hormonally treated urinary bladder. 4. The in vitro effect of the hormone, aldosterone, on RNA synthesis at 24°C was evaluated by quantitating the aldosterone-control difference of both RNA and RNA specific activity in paired samples of scraped epithelial cells. By minimizing dilution of the [6- 3H]uridine due to variations in the endogenous uridine RNA precursor pool size, through the technique of providing a constant excess of exogenous uridine with the radioisotope, aldosterone stimulation of RNA specific activity can be reproducibly demonstrated. 5. Aldosterone significantly increased RNA specific activity but not bulk RNA, per se, even after 20 h of hormone exposure. 6. The mean weight ratio of epithelial cell RNA/DNA determined in 116 different bladder parts derived from 30 toads, was 2.5 ± 0.32 S.E. 7. A 1.5-h exposure to aldosterone produced a significant increase (31.5 %) in the incorporation of [6- 3H]uridine into heterogenous RNA extracted from purified nuclei, but not in RNA extracted from either cytoplasm or whole cell. This primary nuclear response to aldosterone was followed by a significant increase in aldosterone-stimulated specific activity of whole cell RNA at 3 h (74 %) and 20 h (200 %).

Animalizing ability of Evans Blue in embryos of Arbacia punctulata: Effect on Ribosomal RNA Synthesis

Two-cell embryos of Arbacia punctulata were animalized by continuous exposure to Evans Blue at a concentration of 1 25 000 [32]. Mid-gastrulae and experimental embryos were incubated for 3 h with 3H-5-uridine or with 3H-methyl- l-methionine. The time course of uptake of these isotopes into the acid-soluble fraction was found to be similar in both control and experimental embryos, thus negating the possibility that permeability differences might interfere with the analysis of the rates of ribosomal RNA synthesis. The total RNA content/10 6 embryos was the same in control and experimental groups. Animalized larvae synthesized only half as much RNA per μg total RNA and per 10 6 embryos as control larvae. Fractionation of purified cytoplasmic RNA by sucrose density gradient centrifugation and by disc electrophoresis on polyacrylamide gels revealed that Evans Blue-animalized larvae incorporated both 3H-5-uridine and 3H-methyl- l-methionine into 26S and 18S ribosomal RNA at about 50% of the rate found in control larvae. As in all animal cells examined [1, 47] the 18S molecule was more highly methylated than the 26S in both control and experimental embryos. The fact that similar decreases in the rate of synthesis of ribosomal RNA were detected using two different precursors speaks against the possibility that this reduction in rate merely reflects a modification of the size of internal precursor pools. These results suggest that part of the lesion of animalization may be due to a reduction in the rate of transcription of the genes for ribosomal RNA.

Protein turnover during maturation of mouse brain tissue.

The measurement of protein turnover involves the product of the rates of protein synthesis and degradation. It is the dynamic balance between these two components that determines the measured net rate of protein synthesis. The data reported here show that brain cells from newborn animals incorporate arginine-(14)C into acid-insoluble protein at a rate 10-fold greater than the rate for brain cells obtained from 15-day old animals. This difference in incorporation occurred even though the rate of arginine accumulation and the resulting pool size of radioactive precursor were similar for both ages. The measurement of protein turnover in brain cell suspensions prepared from 1-day old animals was shown to be complex and to exhibit a cyclic phenomenon in regard to arginine-(14)C incorporation into and release from protein. The variation in half-life calculations (0.5-3.5 hr) due to this cyclic phenomenon is discussed. Although puromycin was added in an attempt to amplify the rate of degradation by preventing the synthesis of new protein, it was found that degradation was inhibited as well, suggesting a relationship between protein synthesis and degradation.

Dependence of the Putrescine Content of Escherichia coli on the Osmotic Strength of the Medium

Putrescine is the polyamine present in highest concentration in Escherichia coli; however, few specific functions for it are known. We found that cells growing in nutrient broth supplemented with high concentrations of NaCl, KCl, MgCl2, or sucrose had greatly reduced levels of cellular putrescine. On the basis of osmotic strength, all four solutes produced similar decreases in putrescine content. In contrast, glycerol had little effect on the amount of cellular putrescine. Cellular spermidine content was not affected by any of the additives. When cells were grown in high NaCl, pools for most amino acids increased; a few remained the same or decreased slightly. A sudden increase in the osmolarity of the medium led to a rapid excretion of cellular putrescine while there was no decrease in spermidine or free amino acids. This loss of putrescine could be blocked by sodium azide or sodium arsenate. The mechanism of putrescine excretion has two components; one is dependent on high concentrations of potassium ion in the medium, and the other is not. Cells grown in the presence of 11 mm potassium and then put into high osmolarity medium lacking potassium failed to excrete [14C]putrescine rapidly. Replacing the potassium greatly stimulated putrescine excretion within 1 min. The rate of [14C]putrescine excretion was half-maximal at a potassium concentration of 0.69 mm. E. coli which were grown in medium containing 0.1 mm potassium and resuspended in 11 mm potassium initially could excrete [14C]putrescine only slowly but regained the ability to excrete putrescine maximally after 15 min. Sodium, magnesium, ammonium, and rubidium ions and pH from 6.4 to 7.3 did not affect the rate of [14C]putrescine excretion. A sudden increase in the osmolarity of the medium produces a rapid increase in the cellular potassium content. However, this rapid uptake of potassium was not dependent on excretion of large quantities of cellular putrescine. When cells growing in high salt medium were transferred to low salt medium, the putrescine content approached the low salt value only after 140 min. However, the ability of cells to take up [14C]putrescine from the medium did increase 6-fold within 2 min after resuspension in low salt medium. [14C]Spermidine was not metabolized to a measurable extent over 70 min in either low salt or high salt nutrient broth cultures. Because the cellular spermidine contents were similar in the two cultures and because [14C]spermidine was not catabolized in either culture, the rates of spermidine synthesis must also be similar, even though the precursor (putrescine) pool sizes are quite different.

Effects of fasting and feeding on protein synthesis by the rat pancreas.

These experiments were designed to determine whether fasting and feeding were associated with differing rates of protein synthesis in the rat pancreas. It has been established that feeding, acetylcholine, or cholecystokinin-pancreozymin administration was associated with enhanced rates of digestive enzyme secretion; however, the literature is unclear as to effects of such stimulation on enzyme synthesis. Rats fed ad lib. or fasted 24, 48, or 72 hr were used for this study. Pancreases were removed and incubated in tissue culture medium with l-phenylalanine-(14)C, and incorporation into TCA-insoluble material as well as purified amylase was measured. Compared with fed controls, fasting 24, 48, and 72 hr was associated with 29%, 39%, and 35% decreases in incorporation of l-phenylalanine-(14)C into protein. Decreases of similar magnitudes were apparent whether the data were expressed in terms of protein or DNA. Pancreatic amylase isolated from rats fasted 48 hr contained 57% fewer counts of l-phenylalanine-(14)C than amylase isolated from fed rats. Moreover, rats fasted for 24 hr and given bethanechol chloride incorporated greater amounts of l-phenylalanine-(14)C into protein than fasted controls. Studies were performed to exclude changes in pool size of precursor (l-phenylalanine-(14)C) or product (amylase) in accounting for decreases associated with fasting. These studies demonstrate that fasting was associated with decreased rates of pancreatic amylase and protein synthesis in rats.

Total Uptake and Incorporation into DNA of3H-Thymidine,3H-Deoxyuridine and3H-Thymine in the Primary Root ofVicia fabaL.

Total uptake of 3H-thymidine, 3H-thymine, and 3H-deoxyuridine and incorporation of these substances into DNA have been investigated in excised roots in Vicia faba. Total uptake was found to be higher in excised roots than in intact ones. This was a consequence of exogenous 3H-DNA precursors entering the excised roots through the cut surface. Where the cut surface was not immersed in the 3H-thymidine solution 3H uptake was also higher than in intact roots. In this case 3H uptake seemed to be correlated with water-loss from the cut surface. Incorporation of 3H-thymidine, 3H-thymine, and 3H-deoxyuridine into DNA was found to be higher in excised roots than in intact ones, probably as a result of a decrease in the size of the relevant endogenous precursor pools. It is suggested that this decrease resulted from the cessation of the supply of DNA precursors to these roots from the cotyledons.

Insulin stimulation of uridine incorporation into acid-soluble and acid-insoluble ribonucleotides of mammary gland explants.

It has been shown that insulin stimulates the incorporation of radioactive precursors into the RNA of mouse mammary explants (Mayne, Barry & Rivera, 1966; Mayne, Forsyth & Barry, 1968; Palmiter, 1969; Turkington & Ward, 1969; Green & Topper, 1970; El-Darwish & Rivera, 1971). This observation has usually been interpreted to mean an increase in the rate of RNA synthesis, but it can also be accounted for by such factors as changes in the pool sizes of endogenous precursors or changes in the rates of transport of radioactive precursors into the mammary cells. The results of the present study indicate that the stimulatory effect of insulin on the incorporation of uridine into RNA is reflected in a corresponding stimulation of the radioactivity of the acid-soluble ribonucleotide pool. Mammary tissues from 13-day pregnant mice were prepared for organ culture as described previously (Mayne et al. 1966; El-Darwish & Rivera, 1971). Cultures were

Age-dependent regulation of mammalian DNA synthesis and cell proliferation In vivo

That progressive increases in the time required to initiate certain enzyme inductions in rat liver in vivo may be used as a biochemical parameter of ageing was reported previously. The present paper indicates that a second biochemical manifestation of mammalian senescence may be a modification in the regulation of DNA synthesis and cell division in vivo. Administration of isoproterenol to 2-month-old rats initiates a single, tissue-specific burst of DNA synthesis, mitosis and division of certain salivary gland cells. Upon administration of identical body weight dosages of isoproterenol to older rats, it is evident that: 1. the time required to initiate DNA synthesis increases progressively and is directly proportional to the chronological age of rats from 2 to at least 24 months; 2. the delayed onset of DNA synthesis may be accompanied by a decreased magnitude of response; and 3. the ability to stimulate cell division may be abolished. These impairments probably are not attributable to changes in either the transport or pool size of deoxynucleotide precursor or in the immediate binding of isoproterenol. It seems likely, therefore, that age-dependent changes in the metabolism of RNA, protein and/or isoproterenol are responsible.

Incorporation of radioactive macromolecular precursors into intact cells and osmotically stabilized "protoplasts" of Streptococcus faecalis.

Growing "protoplasts" of Streptococcus faecalis were shown to incorporate newly administered radioactive precursors in the same manner as growing intact streptococci. No observable differences could be found between the size of the leucine precursor pools of the two cultures. The extent of turnover of protein and ribonucleic acid in both "protoplast" and streptococcal cultures appeared to be identical. Finally, the absolute rate of macromolecular biosynthesis was found to be equivalent whether determined on the basis of "new" or "old" label incorporation.

The Turnover of Nucleic Acids in Lemna minor

A method is described for measuring the rate constants of both synthesis and degradation of nucleic acids in sterile growing cultures of Lemna minor which avoids the difficulties of environmental changes in isotope uptake and precursor pool size. In fast growing cultures the half-life of ribosomal RNA has been estimated to be between 5 and 8 days.This half-life has been shown to consist of two components, cytoplasmic ribosomal RNA with a half-life of about 4 days and chloroplast ribosomal RNA with a half-life of about 15 days. The possible interference of recycling has been checked, and the evidence indicates its likely insignificance. "Heavy" labeling of Lemna with D(2)O and (15)NO(-) (3) has provided evidence for the conservation of ribosomal RNA in fast growing cultures and has also provided an alternative assessment of recycling. When Lemna is placed on water, the rate of degradation of ribosomal RNA is increased and that of synthesis is decreased. Under partial "step down" conditions it has been found that omission of either nitrate or phosphate, or calcium or magnesium leads to an increase in the rate of degradation of ribosomal RNA. In Lemna grown on water, benzyladenine increases both the synthetic and degradative rates of nucleic acid metabolism. Abscisic acid, on the other hand, markedly reduces the rate of synthesis of ribosomal RNA but leaves the degradative rate unaltered.

Effects of cortisone on ribosomal RNA synthesis in rat liver.

The early and long-term effects of cortisone on rat liver ribosomal RNA synthesis have been studied. Because of difficulties in estimating precursor pool sizes, methods have been employed which do not require estimation of rates of synthesis from rates of radioactive precursor incorporation. It is shown that a single large dose of cortisone—even in animals with intact adrenal glands—results in at least a doubling of the rate of ribosomal RNA synthesis over the ensuing 16 hr. In contrast to the effects of a single dose, repeated administration of the hormone does not result in a sustained stimulation of ribosomal RNA synthesis; the rates of turnover of liver ribosomal RNA in normal animals and in animals receiving large doses of hormone daily over a 2-week period are not appreciably different. (Endocrinology 86: 1033, 1970)

Studies on Protein Synthesis by Senescing and Kinetin‐treated Barley Leaves

AbstractUsing sterile conditions, changes in total protein synthesis were followed. over an 8 day incubation period, in detached first seedling leaves of barley from 8 day old plants during senescence and after kinetin treatment. In senescing leaves, total 14C‐alanine incorporation was enhanced by nearly 20% within 6 h of leaf detachment and by about 30 % after 24 h. Kinetin treatment stimulated protein synthesis even more, for total incorporation was promoted ca. 50 % after 6 h and by ca. 60 % after 24 h incubation. The leaf supernatant (30,000 ×g for 30 min) proteins were separated on DEAE‐Sephadex (A‐50) columns into approximately 14 fractions and changes in 14C labelling of these fractions were studied following leaf detachment and on incubation on water or kinetin for 6 days. In senescing leaves, 14C‐incorporation into supernatant proteins was sustained, even as protein levels declined rapidly The varied stabilities of the different leaf proteins was suggested by the characteristically changing specific activities of the different protein fractions. Although kinetin greatly promoted incorporation into all protein fractions, no evidence was surmised of specific effects on individual leaf proteins.Studies of changes in total protein synthesis in attached senescing first seedling leaves taken from plants aged 7 to 27 days revealed a relatively small increase in 14C‐incorporation. However, incorporation could be greatly increased in leaves up to 15 days old by detaching and preincubating such leaves for up to 2 days on water, prior to measurement. The promotion of 14C‐incorporation into protcins follwing leaf excision could result from early changes in permeability and precursor pool size.

Ribonucleic acid synthesis in relation to precursor pools in regenerating rat liver

The rate of RNA synthesis during the early stages of hepatic regeneration has been evaluated with radioactive precursor techniques. Since RNA labeling is influenced by alterations in effective liver mass, size of endogenous precursor pools, and possibly other factors as well as by regenerative activity, the experimental procedures were modified to permit estimation of RNA biosynthetic rates in terms of mμmoles of nucleoside triphosphate incorporated — conditions which permitted more meaningful comparisons than heretofore among rats differing in nutritional status, size of liver cell complement, or dose of labeled precursor. When regenerating liver was examined under these new conditions, we found that the endogenous UTP and CTP pools expanded by 50% or more within 3 h after partial hepatectomy, and the rate of RNA synthesis, after a negligible depression at time zero, rose by 50–100% at 3–6 h and then remained level for an additional 6 h. RNA content followed similar trends. Sham operation caused minor transient rises in pool size, biosynthetic rate and RNA content.

XV.—The metabolism of DNA in the liver during precocious induction of metamorphosis inRana catesbeiana

SynopsisStudies have been made on the incorporation of [8H]-deoxythymidine into the DNA of the livers ofRana catesbeianatadpoles. When metamorphosis was induced with tri-iodothyronine, the specific activity of the nuclear DNA rose 5 days after administration of the hormone. In contrast, the specific activity of the DNA from the mitochondrial fraction rose grave–2 days after hormone administration.In order to determine whether thein vivochange was due to alterations in the pool sizes of the DNA precursors,in vitrostudies on DNA polymerase were carried out. It was found that under conditions where the enzyme activity was not limited by availability of template or substrates, there was a rise in the DNA polymerase activity in crude cell extracts from the tadpole liver. Fractionation of the cell components showed that little of this increment in activity appeared to be located in the nucleus, but that a large percentage alteration in activity occurred in the mitochondrial and cell sap fractions.A possible interpretation of these results is that an increase in the mitochondrial DNA polymerase is one of the early effects of thyroid hormone. This possibility is discussed in relation to the other known effects of thyroid hormones in tadpoles, with particular reference to nucleic acid metabolism and also to mitochondrial hyperplasia.

Response of rat thymic nuclear RNA polymerase to cortisol injection.

Properties of the RNA polymerase of rat thymic nuclei have been examined with the view of assessing alterations in activity of this enzyme in response to cortisol injection. The polymerase resembled closely that described for other mammalian tissues. The presence of all 4 nucleoside triphosphates was necessary for maximal enzymatic activity. The polymerase was inhibited by actinomycin D or RNase. Injection of cortisol (5 mg/100 g body weight) into rats 3 hr prior to sacrifice resulted in a significant lowering of the rate of incorporation of triphosphatesinto nuclear RNA. Thymic nuclei also incorporated nucleoside mono- and diphosphates but not uridine into RNA; cortisol injection inhibited the rate of incorporation of these nucleoside phosphates. RNA polymerase activity was also decreased in aggregate enzyme preparations obtained from nuclei of thymic tissue from cortisol injected rats. The effect of the steroid appears to be independent of intranuclear pool sizes of RNA precursors or of nuclear transpor...