Germination of Phaseolus vulgaris III. The role of nucleic acid and protein synthesis in the initiation of axis elongation

Excised embryonic axes of Phaseolus vulgaris L. (var. WhiteMarrowfat) begin cell elongation after approximately 4 hr ofincubation at 26°C. The incorporation of ³²P into nucleicacids and phenylalanine-l-¹⁴C into protein markedly increasesduring the 4th hr of incubation, prior to initiation of cellelongation. CH, which inhibits incorporation of phenylalanine-l-¹⁴C intoprotein by 93% during the 2nd hr after its addition, completelyprevents the initiation of axis elongation if added up to 2hr after the beginning of imbibition. Actinomycin D reducesthe fresh weight increase of the axes, and inhibits both ³²Pincorporation into nucleic acids and phenylalanine-l-¹⁴C incorporationinto protein. 5-FU inhibits ³²P incorporation into nucleic acidsbut not phenylalanine-l-¹⁴C incorporation into protein or thefresh weight increase of the axes. MAK column chromatography indicates that actinomycin D inhibitsthe synthesis of all types of nucleic acids to about the sameextent, while 5-FU almost completely inhibits the accumulationof ³²P in ribosomal RNA with lesser but significant inhibitoryeffects on accumulation of ³²P in tRNA. The results suggest an absolute requirement for protein synthesisprior to initiation of cell elongation and at least a partialrequirement for synthesis of nucleic acid species other thanribosomal RNA, tRNA and DNA. The kinetic data suggest that theaxes develop a greatly increased capacity for nucleic acid andprotein synthesis prior to initiation of axis elongation. ¹This research was supported by NSF grant GB 4145 and a grantfrom the U. S. Forest Service. (Received December 16, 1968; )     

Cytokinin-Mediated Translational Control of Protein Synthesis in Cultured Cells of Glycine max

Polyribosome formation was stimulated by cytokinin treatmentof cultured cells of Glycine max cv. Funk Delicious. When suspensioncultures were given 0·5 µM zeatin after 24 h inculture in medium lacking a cytokinin, a nearly 2-fold increasein the polyribosome/monoribosome ratio occurred over the subsequent3 h. The effect of actinomycin D and of 5-fluorouridine on RNAsynthesis and on the polyribosome/monoribosome ratios of thesecells was examined. Actinomycin D at 5 and 20 µg/ml^–1inhibitedtotal RNA synthesis by 39 and 60%, respectively, as measuredby [³H]uridine incorporation into acid-precipitable material.The degree of inhibition of precursor incorporation into polyribosomalRNA was similar. At 0·1 mM, 5-fluorouridine inhibited[³H]uridine incorporation by 76%, and [³H]guanosine incorporationby 66% into polyribosomal RNA after 3 h of treatment. Fractionationof the polyribosomal RNA by oligo(dT)-cellulose chromatographydemonstrated that low concentrations of both actinomycin D (5µg ml^–1) and 5-fluorouridine (0·1 mM) inhibitedthe synthesis of ribosomal RNA to a greater extent than thepoly(A)-containing fraction of the messenger RNA. Synthesisof the poly(A)-containing RNA was inhibited by 24% with 5µgml^–1 actinomycin D and by 30% with 0·1 mM 5-fluorouridine.At the above concentrations, these two inhibitors reduced thepolyribosome/monoribosome ratio of the cytokinin-deprived cellsover a 3 h period, but they did not prevent cytokinin-inducedpolyribosome formation. These results provide further evidencethat cytokinin regulates polyribosome levels through an effecton protein synthesis at the translational level     

Diurnal rhythm of uridine incorporation into RNA regulated by two light-perceiving systems in a long-day duckweed, Lemna gibba G3

A long-day duckweed, Lemna gibba G3, was found to be controlledby two lightperceiving systems; a system perceiving a prolonged,high-intensity white light and the phytochrome system, withrespect to the incorporation of radioactive uridine into RNA.When the duckweed was exposed to short or long days, the uridineincorporating activity into RNA changed diurnally reaching itshighest level at 18 hr and its lowest one at 6 hr after thebeginning of a light period. The level of maximum activity rosein proportion to an increase in the length of the light periodup to 12 hr or in light intensity up to 3000 ergs/cm²sec. Thefar-red light termination of the light period resulted in adecrease in uridine incorporation, the extent of which was constantirrespective of the length of the light period. The uridine incorporating activity changed diurnally when theduckweed was exposed to continuous light. The period lengthof the rhythm was circadian and was constant over a temperaturerange of 16° to 30°C. (Received September 1, 1975; )     

RNA synthesis required for DNA replication in Vicia seed embryos

The synthesis of DNA and RNA during germination of Vicia seedswas examined. Incorporation of ³H-thymidine into DNA reacheda maximum at about 32 hr after the beginning of imbibition,and RNA synthesis was shown to precede DNA replication. Sedimentationanalyses of ³H-uridine-labeled RNAs indicated that the embryossynthesize all types of rRNA, heterodisperse RNA and 4–5SRNA before and also during the phase of DNA replication. Actinomycin-treatments at lower concentrations (50 or 100 µg/ml)resulted in the specific inhibition of rRNA synthesis. Suchinhibition did not lead to a large reduction in ³H-thymidineincorporation during the replication phase. However, DNA synthesiswas drastically inhibited by a higher level (200 µg/ml)of actinomycin D. The results strongly suggest the involvementof synthesis of heterodisperse RNA in DNA replication. (Received May 28, 1976; )     

Inhibitors and the Dependence of Kinetin-induced Expansion on RNA Synthesis in Fenugreek Cotyledons: ETHIONINE AS AN INHIBITOR

The effect of a range of inhibitors on kinetin-induced increasein fresh weight (expansion) and in RNA content of isolated cotyledonsof fenugreek (Trigonella foenum-graecum L.) has been measuredover incubation periods of up to 48 h in darkness. Some compounds inhibited both expansion and net RNA increase:2, 4-dinitrophenol, cycloheximide, L-azetidine-2-carboxylicacid, 6-methylpurine, thiouracil, and actinomycin D. Other compoundsinhibited net RNA increase but not expansion: 5-fluorouracil,2, 6-diamino-purine, 5-azacytidine, and L-ethionine. Ethionine stimulated the induction of nitrate reductase. Effectsby ethionine on RNA content were reversed by methionine, butnot by adenosine. Inhibitory interactions between ethionineand guanosine, hypoxanthine and especially some 6-substitutedadenines were observed. Ethionine, apart from inhibiting uptakeof labelled uridine, also inhibited its incorporation into rRNAbut not that into tRNA. Results confirm that kinetin-induced expansion in cotyledonsis dependent on mRNA synthesis and suggest that the inhibitoryeffect of ethionine on kinetin-induced RNA increase is not dueto ATP trapping or inhibition of protein synthesis or reducedmethylation of tRNA, but to interference with the metabolismof rRNA.     

AUXIN-INDUCED GROWTH OF TUBER TISSUE OF JERUSALEM ARTICHOKE: II. THE RELATION TO PROTEIN AND NUCLEIC ACID METABOLISM

The following results were obtained using tissue slices excisedfrom cold-stored Jerusalem artichoke tuber. 1. Increase in protein content of the tissue was small duringthe washing (i.e. "aging"), and great in the growth phase, particularlyin washed tissue. 2. RNA content of tissue increased during the growth periodsimilarly in non-growing tissue (in water) and actively growingtissue (in 2,4-D plus KIN). 3. Both RNA and DNA increased during the washing, the increasebeing greater in RNA than in DNA. This RNA increase was enhancedby gibberellic acid. 4. 2-Thiouracil, 8-azaguanine, puromycin, and mitomycin C givenat the washing inhibited the subsequent growth. The effect ofthese inhibitors was not significant when they were given inthe growth period. 5. Mitomycin C reduced the basophilia of nuclei and made themswell, as did deoxyribonuclease. 6. The effect of inhibitors of nucleic acid metabolism was reversedto some extent by gibberellic acid and by kinetin. 7. Chloramphenicol inhibited the growth strongly if given inthe growing period, but not so strongly if given during thewashing. 8. An autoradiographic study using ³H-cytidine suggested thatRNA is synthesized in nucleus during the period of washing andis transferred to cytoplasm via nucleolus. It is conjectured that the RNA synthesized during the agingis responsible for the expansion growth to be caused later byauxin or auxin plus kinetin. (Received September 4, 1965; )     

Low Temperature Treatments Induce an Increase in the Relative Content of Both Linolenic and {lambda}3-Hexadecenoic Acids in Thylakoid Membrane Phosphatidylglycerol of Squash Cotyledons

The effect of low temperatures on the fatty acid compositionof phosphatidylglycerol (PG) in thylakoid membranes, in particularon the ratios of nmol% 16:1(3t) (mg fresh weight)^–1 ofcotyledons and nmol 16:1(3t) (mg chlo rophyll)^–1 weremeasured during squash seedling growth. Plants were germinatedand grown for one day at 30°C then were either kept at 30°C(control plants) or trans ferred to low temperatures (18, 14or 10°C). When plant were transferred from 30°C to lowtemperatures, the increase in fresh weight was gradually limited.The lowe the temperature, the smaller was the fresh weight.In contrast, the relative content of 16:1(3t) and 18:3, as wella the ratios of nmol 16:1(3t) (mg chlorophyll)^–1 and mol%16:1(3t) (mg cotyledon fresh weight)^–1 increased indicatingthat the increase of fresh weight and chlorophyll was mor sensitiveto low temperature than PG desaturation in thyla-koid membranes.Furthermore, low temperatures inducei an increase in 16:1(3t)and 18:3 (the final products of PC synthesis) at the expenseof 16:0 and 18:1 (the initial products of PG synthesis). However,within a range of temperature from 10 to 18°C, the extentof these changes (nmol% of 18:3 or 16:1(3t) per day) was graduallylimited by lower temperatures. We therefore propose that lowtemperature inhibit both fatty acid synthesis and desaturationactivities. However, at low temperatures the fatty acid synthesisis likely to be more strongly inhibited than the desaturationactivities, thus explaining the observed increase in the relativecontent of PG-18:3 and PG-16:l(3t). Results an discussed interms of the mechanism which could be in volved in the metabolismof PG in squash cotyledons. (Received July 5, 1996; Accepted March 10, 1997)     

PHYSIOLOGICAL AND BIOCHEMICAL CHANGES OF CARROT ROOT CALLUS DURING SUCCESSIVE CULTURES

1. The longer the period of stock culture, the more remarkableis the growth inhibition by 8-azaguanine in callus.
2. Chloramphenicol,5-methyltryptophane and mitomycin C exert greaterinhibitionon growth in CCL than in CCS.
3. Bud formation is inhibited bysome concentrations of chloramphenicolwithout accompanyinginhibition of the growth.
4. Cell size and the contents of RNA,DNA, protein and lipid percell of CCL are greater than thoseof CCS, respectively. Thecontents per cell of RNA and lipidin "mitochondrial fraction"are higher in CCL than in CCS.
5. Incorporationof guanine-8-¹⁴C into RNA of CCS occurs rapidlyin the first12 hr and slows down thereafter, but that in CCL-RNAincreasessteadily for 16 hr. This difference in rate of theincorporationafter 12 hr between CCS and CCL is principallydue to the differencein rate of the incorporation into RNAof nuclear, mitochondrialand soluble fractions.

1. The rate of RNA breakdown in CCL wasnot so great as the rateof synthesis.
2. 8-azaguanine (10^–3and 10^–4M) inhibits incorporationof guanine-8.¹⁴C intoRNA of both CCS and CCL during 14 hr,but thereafter (up to25 hr) it inhibits the incorporation intoCCL-RNA alone leavingthat into CCS-RNA unaffected.

1. In CCL 510^–5M 8.azaguaninedoes not affect total radioactivityincorporated into bulk RNA,but inhibits incorporation intoRNA of "mitochondrial fraction".

(Received December 23, 1964; )     

Stability of Polysome-Associated mRNA in Potato Tuber Cells during Aging of Tissue Discs

The stability of polysome-associated mRNA in potato tuber discsin the early stage of aging was examined by pulse-chase labelingexperiments and the change in the translational capacity ofthe RNA was studied using a wheat germ translation system. Theincorporation of pulse-fed ³H-uridine into polysomal RNA wasnot arrested immediately after the addition of actinomycin Dto the tissue, but increased by 25% during 4 hr of chasing.The radioactivity in the polysomal RNA then decreased by only30% of the value at the 4th hr during the next 9 hr in the presenceof actinomycin D. The remaining radioactivity in the polysomalRNA was stable at least for 18 hr. The proportion of radioactivityin polyadenylated RNA to that in non-polyadenylated RNA didnot vary appreciably during the chasing period. Non-polyadenylatedRNA of high molecular weight degraded faster than that of lowmolecular weight, but polyadenylated RNA did not show such size-selectivedegradation. The translational capacity of the polysomal RNAalso decreased by about 23% within 9 hr during the period ofinhibited RNA synthesis. In vivo experiments of ¹⁴C-leucineincorporation into proteins in the absence of RNA synthesissuggested that stable polysome-associated mRNA was actuallyfunctioning in the cells. SDS-polyacrylamide gel electrophoresisof the in vitro translation products indicated that mRNA codingfor polypeptides with relatively high molecular weights turnedover slightly faster than those for low molecular weight polypeptides. ¹Present address: Department of Agricultural Chemistry, Facultyof Horticulture, Chiba University, Matsudo 271, Japan. (Received May 12, 1982; Accepted August 26, 1982)     

Effects of Temperature on Growth and Protein and RNA Syntheses of Excised Vigna mungo Embryonic Axis

Embryonic axes excised from dry Vigna mungo seeds cultivatedon wet filter paper grew well at 27°C, but did poorly below15°C. The increase in fresh weight at 27°C was completelyinhibited by both cycloheximide and a-amanitin. Excised axescould grow well at 15°C only if cultivated at 27°C forthe first 12 h. Also, excised axes cultivated at 15°C forseveral days grew well when transferred to 27°G. These resultssuggest that the ability of the axes to grow is retained duringcultivation at 15°C, even when no growth occurs, and thatonly the initial stage of cultivation requires high temperaturesfor growth. The incorporation of ³H-leucine into protein in the excisedaxes during the first 6 h of cultivation at 15°C was muchslower than that at 27°C. The incorporation rate at 15°Gduring the second 6 h was, however, considerably high. In contrast,the incorporation of ³H-uridine into the total RNA fractionat 15°C was much slower during the first and the second6 h as compared to that at 27°C, suggesting a differencein the response to temperature between protein and RNA synthesesin the axes. (Received February 9, 1983; Accepted August 4, 1983)     

SOME OBSERVATIONS OF ABNORMAL EMBRYOS INDUCED BY SHORT-PERIOD TREATMENT WITH CHLORAMPHENICOL DURING EARLY DEVELOPMENT OF SEA URCHIN

Sea urchin embryos, which were treated with 5 × 10⁻³ M chloramphenicol for 1 to 4 hr at certain stages before hatching, developed to several types of abnormal embryos. No significant effect on the shape of the embryo was observed when the concentrations of chloramphenicol used in the short-period treatment were lower than 2 × 10⁻³ M. Embryos up to the 2-cell stage, treated with 5 × 10⁻³ M chloramphenicol for a short period, became small blastulae filled with mesenchyme-like cells (type A). A similar effect of puromycin (2 μg/ml) was also observed at this stage. When the chloramphenicol treatment (for 1 to 4 hr) was applied at 8 ∽ 32-cell stages, vegetalized larvae were produced (type B). Embryos treated with chloramphenicol at 7 hr after insemination at 20°C, developed to another type of abnormal larva different from the previous types (type C). A concentration of puromycin (2 μg/ml) which inhibited protein synthesis to the same degree as 5 × 10⁻³ M chloramphenicol, induced only type A. Between these chloramphenicol-sensitive stages, there were chloramphenicol-insensitive stages for forming abnormal embryos.     

An Analysis of Seed Development in Pisum sativum: IV. COTYLEDON CELL POPULATIONS IN VIVO AND IN VITRO

A method for quantifying changes in the cell population of Pisumsativum cotyledons during development is described. The methodis based on determining the frequency distribution for cellarea following the random sampling of a single-cell suspensionof cotyledon cells. The population profile of these cells changedprogressively and systematically from a single population, similarin size to meristematic cells, found in embryos less than 3.0mg in fresh weight, to a bimodal population in embryos greaterthan 100 mg fresh weight. This method was used to compare embryosof similar size from two genotypes near-isogenic except forgenes at the r locus. No significant differences were foundbetween the cell population profiles of embryos up to 30 mgfresh weight. However, a significant difference was found betweenembryos with fresh weights of 100 mg, the wrinkled (rr) linehaving a higher mean and maximum cell area (2 951 µm²and 9 240 µm² respectively) than the round (RR) line (2591µm²and 6470 µm²respectively). Comparisons were alsomade between cotyledon cell populations from round (RR) embryosgrown in vivo and in vitro. The most obvious differences werethe higher mean and maximum cell size of the large cell populationof in vitro grown embryos which were twice those found in vivo.Embryos grown to either 30 mg or 100 mg fresh weight in vitrohad a much greater proportion of large cells in the populationwith a corresponding reduction in total cotyledon cell number,compared with similar sized embryos grown in vivo. These data suggest that comparisons between different genotypes,or, between cultured and in vivoembryos, based on morphologicalsimilarities between embryos, may be invalid and subject tomisinterpretation. Key words: Pea, seed development, cell population     

Germination of phaseolus vulgaris I. Resumption of axis growth

Growth of the excised axis of Phaseolus vulgaris L. (var. White Marrowfat) begins after a 7-hour incubation in buffer or water at 26°. Growth, as measured by axis elongation or fresh weight increase, is linear for at least 8 hours with a resultant fresh weight increase of approximately 65%. Cell elongation begins 4 or 5 hours prior to cell division and 5 or 6 hours prior to radicle protrusion in the intact seed.

The initiation of axis elongation is apparently dependent on synthesis of RNA and protein. Both actinomycin D and puromycin inhibit the initiation of elongation. Actinomycin I) inhibits the incorporation of ATP-8-C¹⁴ into axis RNA and C¹⁴-leucine into protein, while puromycin inhibits the incorporation of C¹⁴-leucine into axis protein.

The respiratory rate of the axes increases sharply at about the time of initiation of cell elongation. Dinitrophenol initially increases O₂ uptake by the axes, but at the end of 15 hours the rates of O₂ uptake by control or dinitrophenol-treated axes are approximately the same.

     

RNA metabolism in apices of Pharbitis nil daring floral induction

RNA metabolism was studied in apices of Pharbitis nil duringand after floral induction. In continuous light ³H-uridine accumulatedin RNA at a constant rate over an 18 hr period. In darkness,however, the rate of accumulation of label into RNA was constantuntil the 10th hour at which time a rapid burst of accumulationoccurred, peaking at the 14th hour of darkness and followedby a net loss of label. The RNA involved in this burst is probablymRNA due to its size and poly(A) content. This phenomenon doesnot seem to be associated with floral induction, since the siteof perception is the apex, and it also occurs under conditionswhere floral initiation is inhibited by a brief light interruptionof the dark period. Immediately after floral induction by a16-hr dark period the rate of RNA synthesis was suppressed about14%. This suppression lasts for about 12 hr and was followedby a twofold increase in the rate of RNA synthesis, comparedto non-induced apices, at 64 hr after the beginning of the inductivedark period. These post-induction changes were found to occurin all RNA fractions. ¹Present address: Department of Radiation Biology and Biophysics,University of Rochester School of Medicine and Dentistry, Rochester,N.Y. 14642, U.S.A. (Received March 15, 1976; )     

RNA Metabolism During Breakage of Seed Dormancy by Low Temperature Treatment of Fruits of Acer plataanoides(Norway Maple)

Stratification of Acer platanoides fruits at 4 °C led toan accumulation of RNA in the embryo axis and to breakage ofseed dormancy. The accumulated RNA was mainly rRNA. Storageof fruits at 17 °C led neither to an accumulation of RNAnor to breakage of dormancy. The proportion of embryo axis mRNA,as measured by poly(A) content, decreased during both fruitstorage and stratification, although levels of poly(A) wereconsistently lower in embryo axes from stored seeds. Isolatedembryos from both stored and stratified fruits were capableof incorporating [³H]uridine into embryo axis RNA. When assayedat 17–20 °C, however, this incorporation was significantlylower in embryos of stored fruits. The distribution of radioactivitybetween the different RNA species was similar in both storedand statified seeds. Acer platanoides, Norway Maple, dormancy, fruit, seed, ribonucleic acid, stratification, nucleic acid metabolism     

RNA SYNTHESIS IN THE DIFFERENT PHASES OF CELL CYCLE OF CHICK EMBRYO CELLS

RNA synthesis was studied at different phases of the cell cycle of chick embryo fibroblasts, which were synchronized by medium replacement in the confluent phase. The synthesis of DNA started at 4 hr and continued for 8 hr. RNA synthesis increased with time after medium change. The ratio of total amount of radioactivity in nuclear RNA prepared at 0, 2 and 8 hr was 1.0:1.03:5.05. The distribution of radioactive RNA in the sedimentation pattern was similar, showing remarkable incorporation in 45S region of ribosomal precursor RNA. The base composition of newly synthesized RNA, however, varied at different time intervals after medium replacement. Even within the G₁ phase, the molar percentage of G and C was quite different. Treatment with actinomycin D at a concentration of 0.02 μg/ml for 1 hr specifically inhibited ribosomal RNA synthesis. At 2 hr after medium change, ribosomal and AU-rich RNA including larger than 28S were synthesized in about equal amounts.     

In vivo Studies on the Occurrence of Stored mRNA in Embryonic Axes of Vigna unguiculata Seeds

-Amanitin and cordycepin at various concentrations were testedfor their inhibitory effect on the fresh weight increase ofVigna unguiculata embryonic axes after the onset of imbibitionand on the incorporation rate of ³H-labeled leucine into proteinin axes of the 36–38 h stage. -Amanitin at 0.5–5µ/Kg/ml clearly exerted an inhibitory effect on both thefresh weight increase and the protein synthesis. This drug at1 µg/ml, however, showed no significant effect on theprotein synthesis at an early stage of imbibition (4 h), whereascycloheximide was a very potent inhibitor. By experiments inwhich ‘dry’ axes were allowed to imbibe 3H-lebeledadenosine solution for 4 and 12 h in the presence of -amanitin,it was found that poly A⁺RNA was newly synthesized to some extentin axes as early as 4 h after the onset of imbibition and thatthe drug effectively inhibited the poly A⁺RNA synthesis. Theresults may indicate the occurrence of stored mRNA in embryonicaxes of V. unguiculata seeds. (Received June 11, 1983; Accepted August 16, 1983)     

STUDIES ON THE PRODUCTION AND PHYSIOLOGICAL EFFECTS OF ASYMMETRIN

In shake-flask culture asymmetrin was produced by PenicIlliumthomiiaud Byssochlamys niveaduring autolysis. A bioassay wasdevised to estimate relative concentrations of this compound.Culture filtrates of P. thomiicontaining asymmetrin caused adecrease in wet weight, dry weight, and total nitrogen of Phaseolusvulgarisup to 18 days after treatment. At certain intervalsafter treatment with culture filtrate of B. nivea,respirationwas inhibited, and changes were observed in isotope ratio valuesof plants supplied with glucose-¹⁴ C. Plants treated previouslywith culture filtrates of B. nivearesponded but little to gibberelhcacid. High concentrations of mdoleacetic acid inhibited growthof control plants but stimulated growth of plants treated withculture filtrate of B. nivea. ¹Present address: United Fruit Company, Norwood, Massachusetts.Supported in part by a Public Health Service Fellowship No.GF 13,776 from the division of General Medical Sciences, PublicHealth Service, National Institutes of Health, and Grant G 20989from the National Science Foundation. ²Journal Paper No. 2170 of the Purdue Agricultural ExperimentStation.     

AUXIN-INDUCED GROWTH OP TUBER TISSUE OP JERUSALEM ARTICHOKE: III. EFFECT OF ACTINOMYCIN D ON AUXIN-INDUCED EXPANSION GROWTH AND RNA SYNTHESIS

1. The following results were obtained using tissue slices excisedfrom cold-stored Jerusalem artichoke tubers.
2. Actinomycin Dat the concentration of 20 µg/ml given duringthe agingperiod did not affect the subsequent expansion growthcausedby auxin or auxin plus kinetin.
3. Actinomycin D given in thegrowth period, on the other hand,strongly inhibited the expansiongrowth of tissue slices agedin the absence of the antibiotic.
4. In the growth period, auxin or auxin plus kinetin promotedtheincorporation of uracil-2-¹⁴C into RNA fraction.
5. ActinomycinD inhibited the incorporation of ³²P orthophosphateinto ribosomalRNA during the aging period.
6. In the growth period, the incorporationof ³²P into RNA wasenhanced by auxin and was inhibited by actinomycinD, more remarkablyin ribosomal RNA than in lighter RNA.

¹A part of this paper was presented at the Conference on PlantGrowth Regulators held by the New York Academy of Sciences onMay 16, 1966.     

The effect of inhibitors in vivo on protein synthesis and the amino acid pool in the sheep blowfly, Lucilia cuprina.

1. The rates of detoxification of cycloheximide (33 mug/g fresh wt.), puromycin (167 mug/g fresh wt.) and actinomycin D (1 mug/g fresh wt.) were assessed in vivo on the basis of acid-insoluble [14C]leucine incorporation in the sheep blowfly, Lucilla cuprina; these were compared with quantitative estimates which took account not only of incorporation data but also of leucine pool size and turnover. Quantitatively, cycloheximide and puromycin were still at least 50% effective in inhibiting protein synthesis after 6.5 and 24.5h of exposure respectively, whereas values based only on incorporation data suggested that cycloheximide was 83% effective and puromycin completely ineffective after the respective periods. Quantitative estimates also showed that actinomycin D effectiveness increased with increasing exposure over 24.5h, in contrast with values based only on incorporation data, which suggested that it was completely ineffective after 24h.2. All inhibitors affected the dynamic state of the amino acid pool; there was a marked decrease in the rate of leucine-pool turnover as well as an increase in the half-life of leucine in the pool. 3. Inhibition of protein synthesis resulted in changes in leucine-pool size; the most pronounced increase occurred with cycloheximide and puromycin and the most pronounced decreases with actinomycin D. 4. Evidence is presented which suggests that proteolysis is functionally linked to protein synthesis, which determines its rate indirectly.