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IN TER A C TIO N OF D N A WIT H RIBOSOMES 321 Interaction of D N A with ribosomes in cell-free protein synthetizing systems of Chlorella pyrenoidosa G. G a l l in g Pflanzenphysiologisches Institut der Universität Göttingen (Z. Naturforschg. 24 b , 321— 327 [1969] ; ein g eg a n g en am 4. Septem ber 1968) 1. In cell-free protein synthetizing systems of the green alga Chlorella pyrenoidosa, DNA from various sources enhances the amino acid incorporation. 2. This stimulation is neither inhibited by actinomycin D nor by chloramphenicol or cycloheximide (actidione). 3. In the presence of ribonuclease, some precipitable polypeptide is formed with DNA, although the endogenous incorporation is completely inhibited by ribonuclease. 4. After sucrose density gradient centrifugation, polysomal aggregates of ribosomes with DNA are found. Electron micrographs of such polysomes show a direct association of the DNA molecule with several ribosomes. 5. In Chlorella, direct translation of DNA can be obtained also in the presence of neomycin. The kinetics of this reaction are different from those of endogenous m-RNA mediated and of DNA stimulated polypeptide synthesis. Cell-free protein synthetizing systems are shown to decode usually messenger RNA during the for mation of polypeptides. From the green alga C h lo rella, cell-free extracts were shown to incorporate amino acids into polypeptides by an energy depen dent, GTP stimulated, ribonuclease sensitive reac tion *. This endogenous incorporation can be sti mulated by the addition of RNA from several cell fractions 2> 3. Recently, a direct interaction of DNA with ribo somes was described in cell-free systems obtained from rat liver nuclei 4. This stimulation was inter preted as direct translation of DNA. It could not be obtained with extracts from bacterial cells, in which DNA exhibits an inhibitory effect5. With contrast to this, bacterial cell-free systems are able to translate directly DNA of various sources in the presence of n eom ycin6-8. This effect of neomycin could not be observed in cell-free systems of rat liver nuclei 5. In C hlorella , DNA enhances the amino acid incorpora tion in vitro 9 while systems of the blue-green alga A n a cystis nidulans are not stimulated by the addi tion of DNA 2. The results of experiments reported here indi cate, that DNA of various sources enhances the Z. Naturforschg. 2 1 b , 993 [1966], Z. Naturforschg. 2 2 b , 687 [1967]. 3 G . G a l l i n g , Planta 79, 44 [1968]. 4 H. N a o r a , Biochim. biophysica Acta [Amsterdam] 123, 151 [1966]. 5 H. N a o r a and K . K o d a i r a , Biochim. biophysica Acta [Amsterdam] 1 2 3 ,4 2 5 [1966]. 1 G. G a llin g , 2 G . G a llin g , amino acid incorporation in cell-free systems from Chlorella. This stimulation also takes place in the presence of various antibiotics like actinomycin D and chloramphenicol. Ribonuclease does not com ple tely inhibit the formation of polypeptides in the pre sence of DNA. Electron micrographs show that the ribosomes form complexes with single DNA m ole cules in vitro. These complexes are also formed in the presence of actinomycin D. Furthermore, ribo som es of Chlorella are also able to translate directly the DNA in the presence of neomycin. M aterials and Methods Materials Chlorella pyrenoidosa, strain 211/8 b was obtained from the Algensammlung des Pflanzenphysiologischen Instituts der Universität Göttingen. Salmon sperm DNA, calf thymus DNA and neomycin sulphate were obtained from Serva, Heidelberg. (2-14C) thymine came from The Radiochemical Centre, Amersham. The amino acid mixture, uniformly 14C-labelled, was from New England Nuclear, Inc. Transfer RNA (yeast), the nucleoside triphosphates, and chloramphenicol (paraxin) were obtained from Boehringer, Mannheim. Cycloheximide (actidione) came from The Upjohn Comp., Kalamazoo. Actinomycin D was a gift from Merck, Sharp and Dohme, New York. 8 J. J. H o l l a n d and B. J. M c C a r t h y , Proc. nat. Acad. Sei. USA 52, 880 [1965]. 7 B . J. M c C a r t h y , J. J. H o l l a n d , and C . A. B u c k , Cold Spring Harbor Sympos. quantitat. B i o l . vol. XXXI, 683 [1967]. 8 A. R. M o r g a n , R. D. W e l l s , and H. G. K h o r a n a , J. molecular Biol. 26, 477 [1967]. 9 G . G a l l i n g , Z. Naturforschg. 22 b, 348 [1967]. Unauthenticated Download Date | 6/18/17 1:12 AM 322 G. GALLING Methods Electron microscopy Preparation of cell-free systems Aliquots of the polysomal fractions were plated on carbon coated formvar films. After drying, the pre parations were stained with uranyl acetate overnight, washed several times in bidist. water, and dried. Elec tron microscopy was performed in a Philips EM 200 electron microscope equipped with a cooling device. Chlorella pyrenoidosa was growrn in sterile, axenic culture in inorganic salt medium 10 under continuous illumination at 30 °C. The single steps of the prepara tion of ribosomes and supernatant enzymes have been previously described *. After disruption of the cells with glass beads, the homogenate was precentrifuged 30 min at 30 000 g. From this supernatant, the ribo somes were pelleted after 90 min at 105 000 g 0 . The protein of the supernatant was precipitated with am monium sulphate at full saturation in the cold and dialyzed overnight against buffer. The ribosome pellet was resuspended and centrifuged 180 min at 105 000 g 0 . From this pellet, the upper layer consisting of small chloroplast particles was discarded and the clear opalescent ribosome pellet was resuspendet and cleared by centrifugation in 20 min at 20 000 g 0 . All steps were performed near 0 °C. R e su lts In cell-free amino acid incorporating systems of consisting of ribosomes and enzymes, D NA of various sources enhances the incorporation of amino acids into peptides (table 1 ). The require ments of this reaction are the same as in endo genous mRNA mediated protein synthesis. Trans fer RNA, nucleoside triphosphates, and an energy regenerating system as phosphoenolpyruvate and Chlorella, Preparation of labelled and unlabelled DNA from Chlorella Expt. To obtain radioactive DNA from Chlorella, the al gae were grown 5 days in the presence of 0.2 /^C/ml of (2-14C) thymine. After preparation of the nucleic acids with the phenol method n , DNA was purified by chromatography on MAK-columns. The DNA-containing fractions were collected, and the nucleic acids precipitated with ethanol. After dialysis, the volume was decreased by dialysis against a 30 percent dextrane solution in buffer 12. Unlabelled DNA from Chlo rella and also from the blue-green alga Anacystis nidulans were prepared and purified by direct isolation of macromolecular DNA after deproteinization with isopropanol and chloroform 13. 1 2 Incubation and analysis of polysomal fractions The incubation mixture of the cell-free system of Chlorella has been previously described 1. For precipi tation and purification of the polypeptides formed in vitro, the paper filter disc method was used 14. In some experiments, aliquots of the samples were layered after 10 min of incubation on top of a 10 through 40 per cent linear sucrose gradient in tris buffer, supplied with Mg20 and K®, and centrifuged 60 min in the SW 651 rotor of the Spinco ultracentrifuge writh 65 000 rev/min at 0 °C. After puncturing the bottom of the tubes, the optical density of the effluent at 260 m/t was recorded automatically in a Zeiss PM Q II spectrophotometer equipped with continuous flow quartz cell. 10 A. K u h l , Beitr. Physiol, u. Morphol. d. Algen, G. Fischer, Stuttgart, 1962, p. 157. 11 G . G a l l i n g and G. R i c h t e r , Biochim. biophysica Acta [Amsterdam] 123, 613 [1966]. 3 conditions counts/min.mg ribosomes basic system 1,530 t= 0 320 510 500 390 4,860 5.260 3.260 2,970 — transfer RNA — enzymes + ribonuclease (5 /ug) + 100 fig thymus DNA + 100 fig salmon sperm DNA + 100 fig Anacystis DNA + 100 fig Chlorella DNA basic system + 100 fig salmon sperm DNA t= 0 — ribosomes, — enzymes — transfer RNA — ATP, — phosphoenolpyruvate, — kinase + ribonuclease (5 fig) basic system - CTP, - UTP + 100 fig salmon sperm DNA + 100 fig salmon sperm DNA, - CTP, - UTP 4.030 625 50 1,225 1,285 1,550 1,860 1,740 3,040 2,980 Table 1. Stimulation of amino acid incorporation by DNA in cell-free systems of Chlorella pyrenoidosa. The incubation mixture contained in a total volume of 0.4 ml (in /mmoles) : tris 10, Mg-Acetate 4. KC1 12, CaCl2 0.1, Z nS04 0.02, ATP 0.4, GTP 0.2, CTP 0.1, UTP 0.1, phosphoenolpyruvate 1, pyruvate kinase 20 u g, transfer RNA (yeast) 0.2 mg, ribo somes 1 mg, supernatant protein 1 mg, and 0.5 /uC of a r e labelled amino acid mixture, specific activity 40 mC/milliatom C. Incubation: 30 min at 38 °C, pH 7,6. 12 J. C. D r a c h and J. B. L i n g r e l , Biochim. biophysica Acta [Amsterdam] 123, 345 [1966]. 13 J. M a r m u r , J. molecular Biol. 3, 208 [1961]. 14 R. M a n s and D. G. N o v e l l i , Arch. Biochemistry 94, 48 [1961]. Unauthenticated Download Date | 6/18/17 1:12 AM IN TER A C TIO N OF D N A WIT H RIBOSOMES pyruvate-kinase are needed. The stimulatory effect of DNA on the system can be shown also when CTP and UTP are ommited. In the presence of DNA, the incorporation is not completely inhibited by the ad dition of ribonuclease. The same concentration of ribonuclease completely inhibits the endogenous mRNA mediated reaction. To obtain more information on the role of DNA in polypeptide formation in vitro, two concentrations of actinomycin D were added to cell-free systems (table 2 ). With 5 fig actinomycin, the endogenous incorporation is not affected. In the presence of DNA and the antibiotic, the same stimulation as in untreated controls is observed. U sing 10 fig of acti nomycin D, the endogenous incorporation is strongly affected, while in mixtures with DNA, the incorpora tion nearly reaches the value of the untreated con trol. Other antibiotics were added to the D NA stimu lated reaction mixture to study the type of ribo somes involved in this reaction. Chloramphenicol only slightly inhibits the endogenous mRNA mediated amino acid incorporation in the cell-free system of Chlorella. In the presence of DNA and chloramphe nicol, only a little more polypeptide is formed. The stimulation is strongly reduced in the presence of the antibiotic (table 2 ) . The other antibiotic used, actidione, strongly affects the endogenous incorpora tion. Only thirty percent of the control without acti dione is reached. If DNA and actidione are present in the reaction mixture, the incorporation comes up E xpt. conditions to 77 percent as compared with the control in the presence of DNA. Here, the DNA stimulated incor poration is affected comparatively little by actidione. Ribosomes of liver and thymus glands have been shown to form electrophoretically stable complexes with DNA in vitro lo. This complex is stable during centrifugation. Ribosomes of Chlorella were incu bated with labelled DNA of the same organism in the presence of all cofactors required for cell-free protein synthesis except labelled amino acids. After incubation, the mixture was centrifuged under con ditions known to be sufficient only for sedimentation of ribosomes but not of DNA. As shown in table 3, an average of 4 0 percent of the labelled D NA can be found in the fraction of the ribosomes. ribosome fraction supernatant fraction total counts/m in. ml percent o f total 4,765 6,960 11,725 40,5 59.5 100,0 Table 3. Association of labelled DNA from Chlorella with ribosomes after incubation with cofactors and centrifugationIncubation mixture as in table 1. Instead of 14C-labelled amino acids, the mixture contained 4,4 OD260 of (2 —14C) thymine —labelle d DNA from Chlorella, specific activity: 3000 counts min. OD26o • Öfter incubation, the ribosomes were pelleted by centrifugation in 60 min at 105 000 g, 0 °C, and aliquots of the suspended ribosome fraction and of the supernatant were counted in liquid scintillation fluid. counts/m in.m g ribosomes 1 basic system + 5 f i g actinom ycin D + 100 f i g D N A + 100 fi g D N A + 5 fig actinom ycin D 3.710 3,700 6.920 2 basic system -j- 10 jug actinom ycin D -j- 100 f i g D N A + 100 fi g D N A + 10 fig actinom ycin D 3,875 710 5,000 3 basic + 10 + 10 basic system + + 10 -j- 10 3,875 2,755 1,225 5,000 3,115 3,885 system jug chloramphenicol jug actidione 100 f i g D N A jug chloramphenicol f i g actidione 323 7,010 2,525 Table 2. Effect of various antibiotics on the stimulation of amino acid incorporation by DNA in cell-free systems of Chlorella. Experimental conditions: see table 1. DNA = sal mon sperm DNA. bottom dr° P n r *■ top Fig. 1. Sucrose density gradient pattern of freshly prepared Chlorella ribosomes. The ribosomes were layered directly after preparation on top of a 10 to 40 percent linear sucrose gradient and centrifuged 60 min at 65 000 rev/min, 0 °C in the SW 65 1 rotor of the spinco ultracentrifuge. The tubes were punctured and the optical density of the effluent was measured and recorded automatically. The arrow indicates the monomer position (80 s ) . Unauthenticated Download Date | 6/18/17 1:12 AM 324 G. GALLING To study the role of DNA — ribosome complexes formed in vitro during the incubation with cofactors, required for polypeptide synthesis, sucrose density gradient centrifugation and electron microscopy were performed. Ribosomes prepared from Chlorella were centrifuged in density gradients before and after incubation with DNA. Fig. 1 shows the den sity gradient pattern of unincubated ribosomes di rectly after the preparation from freshly harvested cells. Most of the ribosomes from a single peak con sisting of monomers (fraction 80 to 8 8 ). Ahead of this peak, several smaller peaks of polysome frac tions are seen. Part of the ribosomes are disinte grated forming two peaks of subunits, visible in frac tions 92 and 102. Possibly they come from chloroplasmic ribosom es wich easily disintegrate as was recently shown for C h la m y d o m o n a s 16. In fig. 2 the density gradient pattern of DNA alone and of ribo somes after incubation with DNA and all cofactors are shown. The DNA used in these experiments is unable to migrate through the sucrose gradient of 10 to 40 percent. The DNA therefore only can be found on top of the gradient. The ribosom es, centri fuged after incubation with all cofactors required for cell-free protein synthesis and with DNA, still show some clear polysom e peaks as seen in fractions 52, 62, and 76. From such a gradient centrifugation, samples of the different peaks were taken for elec tron m icroscopie studies. The arrow in fig. 2 indi cates the fraction from which the electron micro graph, shown in fig. 3 * was taken. In this figure, the direct association of a DNA molecule with several minutes — ► Fig. 2. Sucrose density gradient pattern of ribosomes of Chlorella after incubation with DNA and cofactors, and of DNA alone. The incubation mixture as in table 1 was layered after 10 min of incubation on top of a 10 to 40 percent linear sucrose gradient, and centrifuged and measured as in fig. 1. One gradient tube contained only 100 jug of salmon sperm DNA, dissolved in bidist. H20 . Solid line: gradient pattern of the incubation mixture. The arrow indicates the fraction from which the electron micrograph, fig. 3., is taken. Broken line: salmon sperm DNA. 15 H. N a o r a and H. N a o r a , Biochim. biophysica Acta [Am sterdam] 134, 277 [1967]. Fig. 4. Kinetics of the amino acid incorporation by cell-free systems of Chlorella. Two times the reaction mixture (see table 1) was incubated a) without further additions O—O, b) with 100 /ag salmon sperm DNA • — # . c) with 1 0 0 /<g salmon sperm DNA and 50 /ug neomycin A - A- Aliquots were taken to the given time and peptides were precipitated and purified 14. ribosomes is shown. On an unbranched fiber, which is an average of 24 Ä in thickness, four ribosomes are located directly. The ribosomes are 180 to 200 Ä in diameter. Many pictures of such associations have been obtained in different preparations of ribosomes 16 R. S a g e r and M. G. H a m i l t o n , Science [Washington] 157, 709 [1967]. * Fig. 3 s. Tafel S. 326 a. Unauthenticated Download Date | 6/18/17 1:12 AM IN TERA CTION OF D N A WITH RIBOSOMES after the incubation with DNA. After density gra dient centrifugation of unincubated ribosomes or from incubation mictures without DNA, clusters of ribosomes have been found in the position of poly somes. These clusters are very sim ilar to polysomes seen in electron m icrographs from other organisms. We have never seen interaction of ribosomes with DNA fibers in fresh preparations of cell-free systems incubated without D N A. The ribosomal fraction of Chlorella itself contains no D N A. Also in the enzyme preparation from the ribosome-free supernatant, only trace amounts of DNA are found. In bacterial cell-free extracts, a direct translation of D NA by ribosomes resulting polypeptides has been reported in the presence of neomycin and other streptomycines. This was also tested with ribosomes and supernatant enzymes of Chlorella. Table 4 shows, that neom ycin stimulates the rate of polypeptide syn thesis in the presence of D NA to a high extent. The 325 reaction rate is linear for at last 40 min and only decreases slightly during the 60 min incubation ex periment. Discussion endogenous mRNA mediated amino acid incorpora tion is not affected by neom ycin alone, while in the presence of DNA and neom ycin up to 28-fold in crease is reached. The table shows also that the reac tion needs both, ribosomes and enzymes. Attempts to obtain electron micrographs of the direct interaction of DNA with ribosomes in the presence of neomycin have not been successfull. In fig. 4, the kinetics of the amino acid incorpo ration under the three conditions described above are shown. The endogenous m RNA mediated incor poration levels off after little more than ten minutes. In the presence of D N A, the reaction proceeds at a linear rate for 10 min, then levels off slowly during another 30 minutes. With neom ycin and DNA, the The concept of protein synthesis of the living cell has been formed and proven after experiments on different types of cells, organs, and organelles. It includes the transcription of the genetic message from the DNA to a messenger RNA and the trans lation of the mRNA to polypeptides by the protein synthetizing apparatus consisting of ribosomes, amino acyl transfer RNA, and different enzymes. The process of transcription and translation may occur in vitro in a complex formed of D N A, newly synthetized mRNA, ribosom e clusters, and enzymes as found in cell-free extracts from Escherichia coli 17>18. However, recently another type of expression of the genetic message has been proposed for some cellfree systems of higher organism s. In extracts from liver nuclei, DNA effects a strong stimulation of the amino acid incorporation in vitro 4’ 5. A lso in cellfree systems of Chlorella, such a stimulation by DNA extracted from various organisms was sh ow n 9, while in extracts from the blue-green alga A n a cystis nidulans, which does not possess a true nucleus, no stimulation was observed 2. An involvement of newly synthetized mRNA in the stimulation by D NA in the Chlorella system may be excluded on the basis of the following arguments. The direct stimulation of DNA is found also in the presence of actinomycin D. This antibiotic is well known to inhibit the formation of mRNA by the DNA dependent RNA polymerase re action. Furthermore, ribonuclease of the same con centration which inhibits the formation of poly peptides by endogenous mRNA, does not inhibit completely the DNA stimulated amino acid incor poration. An interaction of preformed mRNA and part of the DNA in the incubation mixture cannot be completely excluded. However, a complex for mation of Chlorella m RNA with DNA from various other organisms would be an unexpected result. Studies with antibiotics other than actinomycin gave no clear answer to the question as to wich type of ribosomes is involved in the D NA stimulated system. In the presence of DNA, more polypeptide 17 R. 18 H. A. E xpt. conditions basic system 1 t = 0 + 2 + + -f- + 100 f i g D N A + 50 n g neom ycin 100 fi g D N A + 50 f i g neom ycin basic system + 50 fi g neom ycin 100 fi g D N A + 50 f i g neom ycin D N A , + neom ycin, — ribosomes D N A , + neom ycin, — enzym es counts/m in.m g ribosomes 790 140 1,380 750 19,520 2,240 2,000 13,500 2,550 170 Table 4. Effect of neomycin on the amino incorporation in cell-free systems of Chlorella. Experimental conditions: see table 1. DNA = salmon sperm DNA. J. G. L e v i n , H. A. B l a d e n and M. W. Proc. nat. Acad. Sei. USA 52, 140 [1964], B yrne, berg , N ir e n - R. B y r n e , J. G. L e v t n and M. W. J. molecular Biol. 11, 78 [1965]. B laden, berg, N ir e n - Unauthenticated Download Date | 6/18/17 1:12 AM 326 G. GALLING is formed with ribosom es treated with chlorampheni col and also with actidione, even though the effect of chloramphenicol is clearly weaker. Chloramphenicol in the concentration used here is known to inhibit the cell-free protein synthesis only in bacterial and chloroplasmic ribosom e systems 19. In Chlorella, the endogenous mRNA mediated incorporation is only slightly affected by chloramphenicol. In the presence of chloramphenicol and DNA, the incorporation is a little higher than without DNA, but the initial rate of the control is not reached. In contrast to this, actidione is strongly affecting the endogenous incor poration in the Chlorella system. Actidione is an in hibitor of protein synthesis in cell-free systems of higher organisms. In plant cells, cytoplasmic ribo somes are strongly affected by actidione. With con trast to the endogenous incorporation, the stim ula tion of the system by DNA also takes place in the presence of actidione, although the rate of the con trol with DNA is not reached. Higher concentrations of both antibiotics, than used here, did not lower more the amino acid incorporation in vitro. With the electron microscope, a direct association of D N A with ribosom es was observed after incuba tion of the protein synthetizing system with DNA. This association is interpreted as direct interaction of DNA molecules with ribosomes. The DNA m ole cules used here are not able to m igrate alone through the sucrose gradient used. Therefore, they cannot be together with ribosom es by chance in the polysome region of the gradient from which the electron m icro scopic preparations were taken. Specific interaction must have taken place. Ribosomes of thymus glands and of liver have been shown to possess specific binding sites for D N A , leading to electrophoretically stable complexes after incubation 15. To this time it is not possible to decide if the complexes shown here are due to the involvement of the D N A in polypeptide formation or to a binding to other than the messen ger site of the ribosome. Many electron micrographs of the kind shown have been obtained with several preparations of cellfree systems after incubation with DNA. The elec tron micrograph given here looks somewhat similar to pictures of aggregations of D N A , mRNA, poly some clusters, and enzymes, shown in cell-free incu bation m ixtures from Escherichia coli 17’ 18. B ut there are some striking differences. In the cell-free ex tracts of E. coli, several clusters of ribosom es are aggregated to a DNA fiber which is branched by molecules of newly synthetized m R N A . Such a branching of the DNA fiber was never seen in p re parations from the Chlorella system . F u rth e rm o re , in the latter system, only single ribosom es are a tta ched to the DNA m olecule. T his com plex of DNA and ribosom es is form ed also in the presence of ac ti nom ycin D, under conditions which should suppress the form ation of mRNA by the polym erase reaction. The DNA fiber show n in the electron m icro g rap h does not exceed 24 Ä in thickness. T his value r e presents the thickness of a double stran d ed DNA molecule after uranyl acetate staining. The fiber therefore cannot be an ag g reg atio n by chance of several DNA molecules. Recently, a direct in te r action of double stranded RN A m olecules w ith rib o somes form ing ribonuclease stable clusters of p o ly somes w ith 5 and 11 to 14 ribosom es has been re ported after phage infection of b acterial cells 20, 21. However, any mechanism of tran slatio n of double stranded molecules of nucleic acids by ribosom es is still unknow n. The direct interaction of DNA w ith ribosom es in the presence of neom ycin has yet been described only in bacterial cell-free system s 6 > 8 : 22. In m ost of the cases it is in terp reted as a direct tran slatio n of the genetic m essage of the D N A, b ut som etim es, neom ycin leads perhaps to unspecific m isread in g of artificial m essenger polynucleotides, w hen DNA is present in the reaction m ix tu re 22. O ur results in d i cate, th at ribosom es of Chlorella are also able to translate DNA in the presence of neom ycin. It is p o s sible that only the b acterial type of ribosom es is capable to this reaction, w hile cytoplasm ic ribosom es of higher organism s are not. Chlorella seems to p o s ses two types of ribosom es, one of them com ing from the chloroplast, the other fro m the cytoplasm , as des cribed for other algae and h ig h er p la n ts 16. T he nuclear ribosom e system, obtain ed from ra t liver, which is stim ulated directly by DNA, shows not a fu rth er stim ulation by the ad d itio n of n e o m y c in 5. T his system contains only ribosom es of the cytoplasm atic type. 19 J. M. E i s e n s t a d t a n d G. B r a w e r m a n , J. m o le c u la r 10. 392 [1964]. 20 B. H o t h a m -I g l e w s k i and R. M. F r a n k l i n . P r o c . Acad. Sei. USA 58. 743 [1967]. 21 G . B io l. nat. N. G o d s o n and R. 23. 495 [1967]. 22 T. E. L ik o v e r and C. L . S in s h e im e r , G. K u r l a n d , J. molecular Biol. Proc. nat. Acad. Sei. USA 58, 2385 [1967]. Unauthenticated Download Date | 6/18/17 1:12 AM G . G a l l i n g , I n te rac ti on of D N A w it h r i b o s o m e s in c e l l-fr e e - p r o te in s y n t h e t i z i n g s y s t e m s of C h lo r e l la p y r e n o i d o s a (S . 3 2 1 ) Fig. 3. Electron micrograph of polysomes formed from ribo somes incubated with DNA and purified by sucrose density gradient centrifugation. For preparation details see Materials and Methodes. Magnification = 304 000 times. Electron micrograph : Prof. Dr. F. A m e l u n x e n . Zeitschrift für Xaturforschung 24 b. S e ite 326 a. Unauthenticated Download Date | 6/18/17 1:12 AM C hr. F. B a rd e le . U ltras truktur <lcr „Körnchen“ auf den A xo po dien von Raph id io p h rys (Cen trohelida, Hetiozoa) (S. 362 ) fr Abb. 2. Raph idiophry s ambigua. Abkürzungen s. Abb. 1. Die Pfeile weisen auf die kontrastarme Aussparung im Yorderstück des Organells, die Pfeilspitzen auf die helle Linie unterhalb der Oberfläche desselben. Abbildungsmaßstab 2a 50000 : 1. 2b 75000 : 1, 2c 60000 : 1. Zeitschrift für Xaturforschung 21 b. Seite 326 b. Unauthenticated Download Date | 6/18/17 1:12 AM IN TERA CTIO N OF D N A WITH RIBOSOMES The kinetics of the amino acid incorporation with neomycin and DNA in the cell-free system of Chlorella are similar to those of polypeptide syn thesis with artificial messenger polynucleotides like poly (U) in bacterial systems. In this respect, the reaction is completely different from the DNA sti mulated amino acid incorporation without neomycin and from the endogenous mRNA mediated poly peptide synthesis. The direct stimulation of polypeptide synthesis by DNA may be discussed with regard to its possible biological role in vivo. From the green alga C h lo rella ellipsoidea and also from a number of higher organisms, a rapidly labelled fraction of D N A has been described which differs from the bulk D NA in base composition and sedimentation behaviour 23-25. 23 T. I w a m u r a and S . K u w a s h i t a , Biochim. biophysica [Amsterdam] 80, 678 [1964]. 24 M. S a m p s o n , A. K a t o h , Y. H o t t a and H . S t e r n , Proc. nat. Acad. S e i . USA 50, 459 [1963]. 327 On behalf of the relatively large amount of DNA needed for stimulation of the amino acid incorpora tion in vitro, it is possible, that only part of the DNA is involved in the stimulatory effect. The direct stimulation of protein synthesis in vitro by DNA has been yet found only in systems from higher orga nisms. Therefore, the localization of the reaction in the nucleus is believed. However, any quantitative relation between mRNA mediated protein synthesis and direct involvement of DNA as messenger cannot yet be given. I wish to thank Professor Dr. F. A m e l u n x e n for performing electron microscopy, and Dr. J. G r a e b e for helpfull discussions. This work was supported by the Deutsche Forschungsgemeinschaft. 25 V. H e m l e b e n - V i e l h a b e n , [1966], Z. Naturforschg. 21 b, 983 Unauthenticated Download Date | 6/18/17 1:12 AM