EFFECT

OF CONJUGATION ON THE INCORPORATION OF GLYCINE INTO TETRAHYMENA JONAS E. RICHMOND

Department

of Nutritional

Sciences.

University

of California.

Berkelry.

CA 94720. U.S.A.

1. Conjugation of Trfruhw~m~ enhanced the mcorporatlon of glycine into the nuclear fraction by 500’!,,. 2. Incorporation of glycine into the microsomal supcrnatant was augmented by almost 500”,, hq conjugation. 3. Mitochondrial incorporation was stimulated nearly 3-fold in the conjugating strains while thr incorporation of glycine into the microsomes was enhanced approximately 2.5 times. 4. In the whole cel]. glycine incorporation was increased nearly 2-fold by conjugation. 5. Streng nuclear involvement was indicated by elevated metabolic activity and incorporation 01 glycine into RP\A and DNA. 6. Stimulation of the metabolism of Terrah~men~~ by cell communication suggests that the contents of a cell can have a synergistic effect on another cell. 7. Augmentation of the biosynthetic capacities of cells by fusion IS a demonstration of the dommnnt role of the cel1 membrane in the regulation and control of cells. X. Enhancement of biosynthesis of nuclear proteins in conjugating strains of cells indicates that fusion gives rise to the synthesis of new protein from previously existmg proteln or protein precursors. 9 The specific activities of the subcellular fractions after the incorporation of glycine into 2 separated starved strains of Trtrahymenu followed the usual pattern of nucleus less than whole cells, whole ceiis iess ihan miiochondr-ia, mitochondria iess rhan microsomes, bur with the microsomai supernaranr belng much greater than that of the microsomes. Abstract

INTRODL-CTION Trtralz~mrw pyr(fnrnri.s has been widely used as a model system for the study of nuclear (Gorovsky, 1973). mcmbranc (Thompson, 1972; Richmond et UI.. 1968; Richmond, 1976a), and nutritional (Richmond, 1976b) characteristics of eukaryotic cells. Previous work has shown the importante of nutrients in the control of growth. protein synthesis and messenger synthesis in Tetrcrhpencr (Richmond, l976b) where it was shown that the absente of a single amino acid in the growth media inhibited protein synthesis and stopped growth. The versatility of the Tetrah~mena model was demonstrated by the finding that Tetruhywwn~~resumed messenger synthesis immediately upon restoration of the missing amino acid which was followed subsequently be recovery of protein synthesis, resumption of uridine incorporation, and growth. Because this work indicaied a pivöiai roie of ihe nuclear fraction in the recovery from the deficiency of a single amino acid, an attempt is being made to study macromolecular biosynthesis in starved cells and to assess their biosynthetic activities upon conjugation, in the absente of nutrients, a process whi&h requires protein synthesis (Flickinger & Murray, 1974) and which has been shown to stimulate the cytological properties of these cel& (Gorovsky rt LI/., 1975; Flickinger & Murray, 1974). Data are given on the biosynthetic activities of the various subcellular fractions of conjugating cells. METHODS Cultures of Trtruhywnu p~+rmis. syngen 1, mating types 1 and ll were obtaincd from Dr. Sa114 Lyman Allen. 157

Unlversity of Michigan, Ann Arbor. Axenic cultures were produced by continuous culturing in 500 units:‘ml each Tetruhymentr. penicillin. streptomycin and neomycin. syngen 1, mating types 1 and 11 were grown on 2”,, (WIV) proteose peptone buffered to pH 7.2 with KZHPO, and were subcultured m this medium weekly. In each of the experiments, cells were harvested 3 days after inoculation into fresh proteose peptone. Trtrahymena, syngen 1, mating types 1 and 11 were also grown similarly in 5g;I proteose peptone. 5 g$‘l tryptone and 0.26 g,‘l K,HPO, ?HZO adjusted to pH 7.2. Tutruh~menu pyriformis strain WH6 American Type Culture Number 30007 and strain WH,S American Type Culture Number 30008 werk obtained from American Type Culture. Rockville. MD. Cells were maintained and grown on 5 g/l proteose peptone. 5 g!l tryptone. 0.26 g,l K2HP04.3HZ0 adjusted to pH 7.2. Each of the strams of Trtruh!menu was cultured in 4, I-liter Erlenmeyer Haaks. each flask containing 350 ml media at 35 C and h;tr\ested

“Ft”.. A_..” ,.F _r^... ??l.. ‘ l,ILI 1J uL1y> “1 ~l”Wi‘ The cells were counted

and analyzed by S~,X distrrbutlon analysis by use of a Coulter particle counter. Three-day-old cultures of each mating type were centrifuged at 2009 for 5 min and the supernatant carefully poured off. The cells were washed 3 times with d dilute inorganic buITer consisting of 2 mM sodium citrate. I mM NaH,PO,, 1 mM Na2HP0, and 1.5 mM CaC12 (Dryl. 1959). The cells of each mating type at roughly the same concn ranging from 5O,ooO-300,000 cellsjml were lef1 suspended in this buffer for 2 days. After the cells were ztarbed for 2 days in the inorganic buffer. an equal amount of tracers, approximately 25 &i/5 x 10h cells. was added to each mating type in the inorganic buffer. and 1 ,i of the total volume was taken from the suspension of each of the 2 mating types and mixed to obtain equivalent populations of conjugating cells. The degree of conjugation and uptake of thc tracer wil\

determined as a functlon of time and compared 10 that observed for the 2 separate matmg typea. Cells were harvested after incubation with the tracer for a specific time interval by chilling to 0 C and centrifuged at 180 9 for 5 min. These cells were then washed 3 x with the inorganic buffer at 4’C. They were then suspended in the same butl’er at a concn of approximately 3 x IOi cells;ml. mixed with an equal bolumc of ?S”,, ethanol (Ringertz (JJ trl.. 1967) made up in thc same buffer chilled to -20 C. The mixture was subjected to I? passes with the loose pestle of the Dounce homogenizer and then with X passes of thc tight. Nuclei were ohtained by cents-lfuging at 2009 for X min. Thc nuclei were washed twicc with 0.32 M sucrosc made up in the buffer Nuclei were further purified by layering over 2.2 M sucrose (Chauveau L’I rrl.. 1956) and sedimenting the nuclei. Mitochondria wcrc obtained by centrifuging at X.000 y for X min. The mitochondrial supernatant was subjected to 12O.OOOg for 60 min to obtain a microsomal fraction and a microsomal supernatant. The various fractions, whole cells. nuclei. mltochondria. microsomes and supernatant were dialyzed against H,O at 2 C for 4%60 hr. freeze-dried. and the quantity of radioactivity determined in a Packard TriCarb scintillation spectrometer.

Tritium- and ‘“C-labelled amino acids and glucoaamine were obtained from New England Nuclear. Boston. MA. and from Schwarz-Mann. Orangeburg, N.Y. Radioactivity I./l>‘ A*t0rm;“~d ,1 tkr00_r!+sn”.J 11,LcL,”I.,;nt;llot;~” Jc,‘,I,,,aL,“,, ““U.,.U,.LIIII‘ IIIUY,i.;nn UiU. LI LLLL~~-~,I‘ ,111 . ti, Tr;r,>ri. spectrometer (Model 574) for simultaneous tritium, “C and total radmactivities with standard calibrations. Sufficient counts were recorded for a counting error of 4”,, or for 100 min.

RESULTS Data in the tables are the averages obtained in triplicate experiments expressed as counts per min per mg of solid per 5 &i of tracer per 10’ cells incubated for the time specified. Variante from experiment to experiment was < 10:~;).

,-i

I*3tinn

,-rf g*,rr*rr ol~;,~inr .l.C”.~“1U..,,,, in,.,,l-n,\r,lt;r\l, LUI.“.. \I.

;nl,\ I.,L,/ t!:c nu,;! stimulated tteat.1) 3-fold in the cotl.i,ugating strains. while the incorpcuation of glycine tnto the microsomex wu\ cnhanc~cd by I- to ?-fokt in thcsc 7 strains.

> ‘.

Fig. 1. Conjugation

Strains WH, and WH,, displaycd on14 bcr! Jtglit differences in the incorporalion of glycine-?-‘H ittto the carieus cellular cnmponents whcn these ccllk \bcr‘e maintained in the presence of glycine-2-‘H fol- 17ht(Table 1). These starved ccllh of thc separated jtr;ttn\ exhibited minima1 nuclear acti\ill as ccidenccd b\ the quantity of glycine-?-“H incorporated into tn;tC.t.omoleculcs of the nuclear fraction. Somc protein \L;Ithesis was taking place in all fractions as shoMn 13~ the incorporation of glycine into all cytoplamic ft-;Ktions. The solublc cytoplasmic fraction, tnicrosomtl supernatant. had a much greater speciíìc acti\ it! than any of the subcellular fractions. The mitochondrial fraction had a specific acti\ ttj ol’ 3 j< the value OI the nuclear fraction. and thc zpecitic acli\ tt! 01‘LIK microsomes was some 4 x the incorporatton tnt~~ !hc nuclear fraction in both WH, and WH,, htt-;tin\. Incorporation of glycine into Mhole cells of ;t conj,tgating mixture of cells from strains WH, and WH, j was augmented over ZOO”,, bl conjugation. Stimtt-

1

of strain WHó and WH,, after starvation for 48 hl- followcd to conjugate for 12 hr.

bq mixing and ollowing

Incorporation

of glycine

into Trtr~rhwww

154

Table 1. Incorporation of glycine-2-3H into strains WH, and WH ,4 of Tetrahymenu p_vrifi>rmr\ after 12 hr of incubation and the effect of conjugation on the incorporation of glycine-2-‘H

Fraction

Strain

WH,

Stram

WH,,

Conjugation (Combination of strains WH, and WH,,)

(cpm’mpl whole cells nuclei mitochondria microsomes microsomal

supernatant

DISCUSSION

Conjugation

stimulated

the

5')')

1002 313 852 1243 1X60

incorporation

of gly-

cine into the macromolecular components of the nuclear fraction more than 5OOYóover the incorporation into the nuclear fractions of the individual strains not undergoing conjugation. This degree of stimulation by conjugation is strongly indicative of the dominante of nuclear involvement in the process of conjugation even though the specific activity of the total nuclear fraction is only about 4 of the specific activity of the soluble cytoplasmic fraction. The quantity of the soluble cytoplasmic fraction is several orders of magnitude smaller than the nuclear fraction and because of the methods of isolation, must contain a goocí deai of the soiubie components that were initially in the nuclear and other subcellular fractions. However, this newly synthetized protein seems to be firmly fixed within the nucleus since it was not released by extensive washing. Some redistribution or transport must be the usual occurrence since the available data suggest that most of the nuclear proteins are synthetized in the cytoplasm and are then transported to the nucleus (Goldstein, 1958; Prescott 8~ Bender. 1963). The large 7:) increase in incorporation of glycine into the nuclear components, although the overall specific activity of the nuclear fraction is low. is largely due to the stability of the bulk of the nuclear macromolecular fraction and perhaps the synthesis of a (soluble) much smaller subfraction(s) of diffusible, more labile protein(s) upon conjugation and cel1 fusion. Although the present experiments do not permit a definitive establishment of the site of synthesis of the labelled nuclear material, cytological activity and capacity of isolated nuclei to synthesize protein and to incorporate glycine into DNA and RNA clearly establish a streng nuclear involvement in these biosynthetic processes. Gorovsky (1973), has observed active nuclear activity in Tetrahynena. The 500”,, stimulation of both nuclear and soluble protein synthesis coincidentai with enhancement in the biosynthetic activities of al1 subcellular fractions is evidente for the general involvement of the major processes of the cel1 in conjugation since it has previously been shown that protein synthesis is essential for conjugation (Flickinger & Murray, 1974). Further, the augmentation of protein synthesis in the whole cel1 and in each of the subcellular fractions in conjugating cells emphasized the importante of the synthesis of new protein. Since there were no other nutrients added other

21x 714 8 10 I3OX

1174 1604 2192

2693 6510

than tracer glycine which was also added to each of the separate strains at the same concn. stimulation of glycine incorporation in an equal mixture of the 2 strains of > 5 x that found for the separate individual strains clearly shows that this enhanced protein synthesis in the conjugating cells is primarily due to the synthesis of new protein from previously existing cellular proteins, amino acids or protein precursors to make protein persumably reflecting the genetic constitution of the conjugating cells and fused cells. This elevation of the biosynthetic capacity of cells by cell-cell communication is a vivid illustration of the synergistic action of the contents of a cel1 on another cell, thus stimulating the overall metabolic activities of thc 2 ce!!s; and therefore demonstratnn? the dominant role of the cel1 membrane in the regulation and control of cells. The roles of the newly synthetized protein in cellcel1 interaction of formation of cel1 contacts, as wel1 as the conservation and synthesis of the cel1 membranes during conjugation and cel1 fusion, are under investigation. A~knowledgrmrnr~ -1 am most grateful to Professor William Balamuth for producing the axenic cultures. The author is most grateful to Dr. Sally Lyman Allen. L’niversity of Michigan. Ann Arbor. for supplying the cultures.

REFERENCES CHACVEAC

J.. MOULE Y. & ROUILLER C. (1956) Isolation of pure and unaltered liver nuclei morphology and biochemical composition. Expl. Cd/ Rrs. 11, 317-321. DRYL S. (1959) Antigenic transformation in Puramrcium aurrlia after homologous antiserum treatment during autogamy and conjugation. J. Protoxml. Suppl 6. 25 (Abstract). FLICKINGER C. J. & MURRAY R. L. (1974) Cytoplasmic membrane changes in conjugating Trtrdyn&r. CuIl Tiss. Rrs. 153, 357-364. GOLDSTEIN L. (1958)Localization of nucleus-specitic protein as shown by transplantation experiments in .1rnoc~h~ proteus. Expl. Cr/1 Rex 15, 6355631. GOROVSKY M. A., MEPIG-CHAO Y., KEEVERr J. B. & PLE

Effect of conjugation on the incorporation of glycine into tetrahymena.

EFFECT OF CONJUGATION ON THE INCORPORATION OF GLYCINE INTO TETRAHYMENA JONAS E. RICHMOND Department of Nutritional Sciences. University of Calif...
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