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Nuclear genome codes for chloroplast ribosomal proteins in Acetabularia
Copyright (9 1973 by Academic Press, Inc. AN rights of reproduction in any form reserved
Experimental Cell Research 80 (1973) 69-78
NUCLEAR GENOME CODES FOR CHLOROPLAST RIBOSOMAL PROTEINS IN ACETABULARZA II. Nuclear Transplantation
Experiments
K. KLOPPSTECH and H. G. SCHWEIGER Max-Planck-Institut fir Zellbiologie, D 2940 Wilhelmshaven, BRD
SUMMARY The protein patterns of chloroplast ribosomes of Acetubuluriu have been established by means of polyacrylamide gel electrophoresis. The protein patterns of the faster sedimenting 44s ribosomal subunit of A. mediterranea, A, cliftonii, and A. crenulata have been compared and species specific differences are described. The protein patterns of hybrid cells consisting of a host cytoplasm from one species and a nucleus from another species is changed to that of the nucleus donor species after some weeks. The results indicate that at least part of the chloroplast ribosomal proteins are coded by the nuclear genome.
Chloroplasts contain DNA and all the other components of an autonomous molecular genetic apparatus. As far as the chloroplast DNA is concerned it is known that this DNA contains the genetic information for the RNA moiety of the chloroplast ribosomes [15, 181and most probably also that for chloroplast tRNA [20]. On the other hand there is no chloroplast protein which has been proven to be coded by the chloroplast genome. However, for a number of chloroplast proteins it has already been shown that the genetic information comes rather from the nuclear than from the chloroplast genome [I, 5, 8, 14, 161. These results and the fact that the genetic information for the RNA of chloroplast ribosomes is located in the chloroplast genome raise the question whether or not the proteins of the chloroplast ribosomes are coded by the chloroplast genome. In this paper evidence is presented that in Acetabu-
laria at least some of the ribosomal proteins
of the chloroplasts are coded by the nuclear genome. This evidence is based on species specific differences in the electrophoretic protein patterns of the heavy ribosomal subunit between different speciesand on the fact that after implantation of a heterologous nucleus the protein pattern is changed into that of the nucleus donor species. Preliminary results have ben presented elsewhere t5, 61. In [7] it has been shown that besidesthe 70s ribosomes 56S, 44S, and 30s particles can be separated from Acetabularia chloroplasts. The material of the most prominent peak served as ribosomal material. It sediments with 44s and represents the heavy subunit of the chloroplast ribosome. MATERIAL
AND METHODS
Plants and ribosomes. The culture conditions for A. mediterranea, A. crenulata, and A. (Polyphysa) cliftonii Exptl Cell Res 80 (1973)
IO
K. Kloppstech
& H. G. Schweiger
and the procedures for isolating chloroplast ribosomes were the same as described in the preceding paper [7]. The nucleus implantation and transplantation techniques were essentially those of Hammerling [3, 41. The intermediate caps of the transplants were cut off after 14 and 28 days, the transplants were used about 6 weeks after the beginning of the experiment. Isolation of ribosomal proteins. Ribosomal material sedimentinn with 44s at a Mg*+ concentration of 2 x 10-a M was cut and pooled from up to five gradients, diluted with gradient buffer (0.1 M KCI, 0.01 M Tris, pH 7.4, and 0.002 M Mg (CH,COO),) and collected by centrifugation for 3 h, at 190 000 g in an SW 50.1 Ti rotor. The pellets from several thousand cells (5 000 to 10000 plants for monoribosomes; 2 000 for 56s particles; 1 000 for 44 S particles) were suspended in 10 ul of a solution which contained 2.5 ul of samole gel buffer (0.48 M KOH, 0.48 M CH&OOH, pH 6.7, and 0.03 M N,N,N’N’-tetramethylethylenediamine) [13] and 7.5 ~1 of 6 M urea. To this suspension 5 ul of RNase mixture were added which was diluted 114 with 6 M urea. This mixture contained 0.4 pg of pancreatic ribonuclease, 0.3 pugof T, ribonuclease, 0.04 pmoles of EDTA, and 1 pl of sample gel buffer. The rRNA was digested by incubation of the suspension for 6 h at 37°C and the resulting protein mixture was stored at - 18°C. Electrophoresis. Gel electrophoresis was carried out between two glass plates forming a vertical chamber of 1 x 25 mm* and 100 mm height. Over a small pore gel [21] of 75 mm height a portion of spacer gel [21] was layered to fill the rest of the cuvette. Prior to polymerisation two strips of acrylglass (0.5 x 2 mm2) were inserted into the spacer gel to form two identical channels seoarated from one another by 7 mm of gel. To each channel a mixture of 15 pg ribosomal protein in 15 ~1 of diluted sample gel buffer and 15 /-cl of gel solution [13] was added and polymerized. Electrophoresis was run for 195 min at 190 V with a current of about 5 mA per chamber at a room temperature of 18°C. The electrode buffer was that described by Reisfeld [13]. The gel was stained according to Meyer & Lamberts [9]. The gels were scanned at 550 nm using a Leitz microspectograph t241.
RESULTS Separation
of ribosomal
proteins
In order to facilitate the comparison between different electrophoretic patterns the different ribosomal protein preparations of Acetabularia were run in parallel with ribosomal proteins from E. coli as reference. This method allows comparison of different runs by referring to the bands of the E. coli control. Such a protein pattern of the 44s ribosomal particles from the chloroplasts of A. Exptl Cell Res 80 (1973)
mediterranea is given in fig. 1. At least 20 bands can be detected. As was shown in a previous paper the 44s particles represent the faster sedimenting subunit of the 70s chloroplast ribosomes [7]. One therefore should expect that all of the bands of the 44s subunit are contained in the protein pattern of the 70s ribosome. A comparison of the two patterns reveals a close similarity. It is possible to find the corresponding band in the 70s pattern for all 44s bands but not vice versa. This holds especially for some weak bands. The fact that only some weak bands of the 70s pattern are missing in the 44s pattern might be due rather to different amounts of protein which could be obtained from the large 44 S and the small 70s peaks than to differences in the protein composition. On the other hand it is obvious that there are differences in the intensities of several bands. When compared with the 44s pattern the proteins of the 70S particle are distributed more regularly throughout the profile. This gives some indication in which region of the 70s pattern the bands of the 30s subunit might be located. For example, the bands of the 70s profile which correspond to the E. coli bands 10, 27, and 32 seem to be dominant bands of the 30s subunit. However, it was impossible to establish the electrophoretic protein pattern from isolated 30s particles because the amount of material which could be obtained from 10 000 cells was too small. The low yield of 30s material seemsto reflect a high instability of the 30s particles, the reason for which is not yet known. The protein pattern of the 56s particles of A. mediterranea is closely similar to that of the 70s ribosomes as can be seen from the densitometer tracings in fig. 1b, c. A pronounced similarity between the protein patterns of 56s particles and 70s ribo-
Nuclear genome and chloroplast ribosomal proteins. II 25
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somes has also been revealed in A. crenulata and in A. clijtonii. This similarity indicates a close relationship between these two types of particles. Since it is known from Phaseolus aureus that the ribosomal proteins from cytosol, from mitochondria, and from chloroplasts are different [23] it is probable that the
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Fig. 1. Ribosomal proteins isolated from the chloroplast ribosomes of A. mediterranea. A, 44.3; B, 56s; C, 70s particles. ----, E. coli control, visible bands numbered beginning from the origin. The profiles correspond to about 1000 cells in A, 2 000 cells in B and 5 000 cells in C. The bars indicate the positions of visible protein bands.
56 S particle derives from the chloroplast and not, as might be suggested from the sedimentation coefficient, either from the cytosol (larger subunit) or from the mitochondria [l 11. Since the 44 S particles contain a smaller number of different proteins and are more dominant than the 30s peak in the presence Exptl Cell Res 80 (1973)
72 K. Kloppstech & H. G. Schweiger
Fig. 2. Comparison between the proteins of the 44s ribosomal subunits isolated from the chloroplasts of three species of Acetabularia. A, A. cliftonii; B, A. mediterranea: C. A. crenulata. ----, E. coli control, bands numbered from the origin. The profiles correspond to 1 000 cells. Speciesspecific differences are indicated by the vertical dashed lines.
of 2 x 1O-3 M Mg2+ as was shown in [7] the 44s subunit was chosen for the further studies. Species-specific proteins of the 44 S subunits The protein patterns of the 44s particles from A. mediterranea, A. crenulata, and A. cliftonii are compared in fig. 2. Although the pattern of A. cZiftonii looks similar to that of A. mediterranea there are at least two bands by which the two species are distinguished. As is indicated by the dotted line there is a well separated and prominent band in A. Exptl Cell Res 80 (1973)
cliftonii corresponding to E. co& band 30, while there is only a shoulder if at all in the same position of the A. mediterranea pattern. A second difference between the two species can be detected in the position 27 of E. coli. In this case a band could be detected in the A. mediterranea but not in the A. cliftonii pattern. The differences between the two species become even more obvious when instead of using E. coli material as reference the two species undc consideration were directly compared (fig. 3). The dotted lines indicate
Nuclear genome and chloroplast ribosomal proteins. II the positions 27 and 30 of E. coli and reveal two of the differences existing between the two species. Striking differences were found for the protein patterns of the 44s particles from A. mediterranea and A. cliftonii on the one hand and from A. crenulata on the other hand (fig. 2). While there are bands at the positions 7 and 23 in the A. crenulata pattern no comparable bands can be detected in the corresponding regions of the two other species. Moreover, other differences can be demonstrated between A. crenulata and the other two species in the region between 26 and 32. The most prominent band of A. crenulata in this region is found in position 27 whereas the maximum peaks of the two other species correspond to the bands 28 to 29. The differences between A. mediterranea and A. crenulata are also clearly shown in fig. 4, where the proteins of the two species were directly compared. The lines between the two profiles indicate that there are 4 peaks in A. crenulata and 5 peaks in A. mediterranea in the region 27 to 32. The patterns of all three species are highly reproducible so that it is possible to dis-
*tar,
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Fig. 3. Direct comparisonbetweenthe proteins of the 44s subunits of A. cliftonii (upper curve) and A. mediterranea.The vertical dashed lines indicate the prominent differencesbetweenthe two species.
32
27
13
7
23
Fig. 4. Direct comparisonbetweenthe proteins of the 44s subunits of A. mediterranea(upper curve) and A. crenulata. The vertical dashed lines indicate
differencesbetweenthe species. tinguish definitely three species.
the protein patterns of all
Influence of heterologous nuclei on the chloroplast riboAoma1 protein patterns In order to elucidate a possible role of the cell nucleus in coding for the proteins of the chloroplast ribosomes heterologous nuclear transplantation experiments were performed and the influence of the implanted nucleus on the ribosomal protein pattern of the chloroplasts was investigated. A major problem was to find a method by which it was possible to obtain sufficient amounts of material. We worked with unlabelled proteins by which method a number of difficulties were avoided which might interfere with the preparation and separation of labelled proteins in Acetabularia. One such difficulty is the incorporation of sufficient amounts of label. Another one is a possible contamination by microorganisms which might become labelled very quickly. In order to obtain sufficient amounts of material it was not the hybridized cells Exptl Cell Res 80 (1973)
74
K. Kloppstech
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themselves from which the ribosomal proteins were prepared but rather cells of the F, generation of the hybrids. If the ribosomal proteins of the chloroplasts are coded by the chloroplast genome the pattern of the ribosomal proteins should remain unchanged in the F, generation. If the genetic information for the proteins comes from the nucleus the protein pattern of the F, generation should be that of the nucleus donor species. Exptl
Cell Res 80 (1973)
Fig. 5. Proteins of the 44s ribosomal subunit isolated from the chloroplasts of the F,-generation of med,clifr, ceils. Ribosomal protein pattern of the 44s subunits of FL-generation cells of: A, A. mediterranea: B, medI-clifto hybrids (the nucleus donor was A. mediterranea, enucleated cells derived from A. cliftonii); C, A. cliftonii. The vertical dashed lines indicate the species-specific bands at the positions 27 and 30.
The hybrids were prepared by implanting an isolated nucleus of one species into a basal fragment (10 to 15 mm) of the stalk of another species. The hybrids of the types used for this work would behave like normal cells and undergo the different stages of the cell cycle, that means the nucleus divides into secondary nuclei, cysts and gametes are formed, and after copulation zygotes grow out. Up to 10 000 hybrid cells could be ob-
Nuclear genome and chloroplast ribosomal proteins. II
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tained by this method from only a limited number of implants. The result of such a hybridisation experiment is shown in fig. 5. In this experiment the nucleus donor species was A. mediterranea and the acceptor species was A. cliftonii. This type of heterologous combination and its ability to generate an F, generation has early been described [17]. From cells of such an F, generation chloroplast ribosomes were isolated and the protein pattern of the
6. Proteins of the 44s ribosomal subunit isolated from the chloroplasts of cells. the F,-generation of cum,-clift, Ribosomal protein pattern of the 44s subunits of: A, A. crenulata: B, F,-generation cells of cren,-cliftn hybrids: C, A. cliftonii. The vertical dashed lines.indicate the species-specific bands at the positions 7, 23, and 27. Fig.
;““”
‘. 3
44s ribosomes were compared with the protein patterns of A. mediterranea and that of A. cliftonii. As revealed in fig. 5 the protein pattern of the F, generation of medl-cZift,, cells obviously is similar to that of the nucleus donor species, namely A. mediterranea. This is demonstrated e.g. by the presence of the band in position 27 and the absence of the predominant band in position 30. A comparable result was obtained in two other experiments of the same type. A result which Exptl Cell Res 80 (1973)
76 K. Kloppstech & H. G. Schweiger DISCUSSION
Fig. 7. Proteins of the 44s ribosomal subunit isolated from crenl-cl(ftO hybrid cells. -, cren,-c&f&, grafts; ....... E. coli control. The vertical dashed lines indicate the bands 7,23, and 27 which are absent in A. cliftonii.
was obtained with cren,-clift, hybrids is shown in fig. 6. In this case the pattern of the F, generation of the hybrid cells is similar to that of A. cremdata. This can be learned from the presence of the bands at the positions 7, 23, 27 which are all absent in A. cliftonii. This experiment shows again that the protein pattern of the daughter cells of the hybrids is nuclear-dependent. Finally a rather direct approach was used. In this experiment the changes in the protein pattern were investigated 6 weeks after transplanting a rhizoid containing a nucleus onto an anucleate stalk. This experiment was of the cren,-clift, type. About one thousand transplants served as starting material [17]. The chloroplast ribosomes were isolated 6 weeks after the transplantation and the proteins of the 44s particles were separated electrophoretically as shown in fig. 7. Although the amount of protein which was obtained from these thousand hybridized cells was very small it is obvious that the pattern of the ribosomal proteins again is quite similar to that of the donor species of the nuclei. This is demonstrated by the presence of the bands in positions 7, 23, and 27. Exptl Cell Res 80 (1973)
The proteins of the 44s ribosomal subunit of Acetabuluria chloroplasts were separated into about 25 bands and the patterns of A. mediterranea, A. cliftonii, and A. crenulata were compared. Species specific differences were found between the three species under consideration. The differences between A. mediterranea and A. cliftonii comprise at least 2 bands and between A. crenulata and the two other species at least 4 bands were different. The rather small number of species specific bands in these closely related species is striking and in accordance with results of other authors [25]. By means of the transplantation technique by which nucleate rhizoids of one species were transplanted onto anucleate fragments of another species it was shown that the species specific protein pattern changes under the influence of the transplanted cell nucleus. In a similar way after implantation the isolated heterologous nucleus caused the acceptor cytoplasm to change the ribosomal protein pattern to that of the nucleus donor species. In the latter case the pattern was determined in F, generation cells. Such changes suggest that the ribosomal proteins of the chloroplasts are coded by the nuclear genome. However, it cannot be excluded completely that nuclear-dependent changes in the secondary structure are involved. Anyway, it must be kept in mind that the nuclear coding capability is proven only for those proteins of the 44s subunits which are species-specific. Special attention should be focused on the fact that the change in the protein patern under the influence of the implanted nucleus means not only the appearance of new bands but also the disappearance of old ones, so that after an appropriate time a pure pattern of the nucleus donor species was observed rather than an
Nuclear genome and chloroplast ribosomal proteins. II
intermediate form which still resembled the host cytoplasm species. The simultaneous appearance of nucleus donor species bands and the disappearance of host cytoplasm species bands under the influence of an implanted heterologous nucleus has already been observed and discussed in the case of two enzymes.This disappearanceis rather striking since in this case it could be shown that the isozyme patterns remained the same for periods of up to at least 8 weeks after removal of the cell nucleus. So the disappearance obviously is induced by the implanted nucleus. A possible explanation for this phenomenon might be some sort of a replacement of the mRNA at the site of protein synthesis which results in an increased instability of the mRNA of the host cell. Experimental evidence from bacteria indicates that the genetic information for the ribosomal protein is organized in a single operon [lo]. If the same holds for the ribosomal proteins of the chloroplast, one might assume that those ribosomal proteins of the chloroplast for which no species specificity could be demonstrated are also located in the nuclear genome. The question of how far a limited number of heterologous chloroplasts which were transferred into the anucleate cytoplasm together with the heterologous nucleus could be bIamed for the change of the protein pattern deservesspecial attention. In nucleate cells chloroplasts have been found to undergo a divisision every 10 days [2, 191.Assuming that this multiplication rate holds for the heterologous chloroplasts in the hybrid cells also one could expect that during a period of 50 days the number of chloroplasts should not have increased to more than the 50-fold. Since the acceptor cytoplasm contains about 1 x lo6 chloroplasts and even if 1 x lo3 heterologous chloroplasts are transferred with the nucleus an increase of the hetero-
77
logous chloroplasts to a 50-fold could result in not more than 5% heterologous chloroplasts with respect to the total chloroplast population. This holds true even if the homologous host chloroplasts would stop dividing after the nuclear transplantation. From our results it can be concluded that the genetic information for the proteins of the chloroplast ribosomes originates in the nuclear genome while it is generally accepted that RNA of the chloroplast ribosomes is coded by the chloroplast DNA. One may conclude that the chloroplast ribosomes in the hybrid cells are hybrids too. The possibility that such hybrid ribosomes exist and are functionahy active in heterologous combinations implies that the ribosomal RNA of the chloroplasts of different species do not differ to as great an extent as the corresponding ribosomal proteins [ 121. Indeed it has been shown in bacteria that the ribosomal RNA of different species is highly homologous. Moreover, heterologous ribosomal combinations of RNA from one bacterial species and proteins from another specieshave been produced in vitro by means of the reconstitution technique of Traub & Nomura [22]. Such heterologous combinations have been shown to be functionally active. The skilful technical assistance of Mrs H. Stelzer is gratefully acknowleged.
REFERENCES 1. Apel, K & Schweiger, H G, Eur j biochem 25 (1972) 229. 2. Clauss, H, Liittke, A, Hellmann, F & Reinert, J, Protoplasma 69 (1970) 313. 3. Hammerling, J, Biol Zentralbl 74 (1955) 545. 4. - Wilhelm Roux Arch Entwicklungsmech Org 132 (1935) 424. 5. Kloppstech, K & Schweiger, H G, Abstract commun 7th meet Europ biochem sot (1971) 155. 6. - Biology and radiobiology of anucleate systems (ed S Bonotto, R Goutier, R Kirchmann & J Maisin) p. 127. Academic Press, New York (1972). Exptl Cell Res 80 (1973)
7% K. Kloppstech h H. G. Schweiger 7. - Exptl cell res 80 (1973) 63. 8. Kung,. S D, Thornber, J P & Wildman, S G, FEBS letters 24 (1972) 185. 9. Meyer, T S & Lamberts, B L, Biochim biophys acta 107 (1965) 144. 10. Nomura,.M & Engbaek, F, Proc natl acad sci US 69 (1972) 1526. 11. O’Brien, T W, J biol them 246 (1971) 3409. 12. Pace, B & Campbell, L L, J bact 107 (1971) 543. 13. Reisfeld, R A, Lewis, U J & Williams, D E, Nature 195 (1962) 281. 14. Reuter, W & Schweiger, H G, Protoplasma 68 (1969) 357. 15. Schweiger, H G & Berger, S, Biochim biophys acta 87 (1964) 533. 16. Schweiger, H G, Master, R W P & Werz, G, Nature 216 (1967) 554.
Exptl Cell Res 80 (1973)
17. Schweiger, H G, Curr top microbial immunol 50 (1969) 1. 18. Scott, N S & Smillie, R M, Biochem biophys res comm 28 (1967) 598. 19. Shephard. D C, Exptl cell res 37 (1965) 93. 20. Tewari, K K & Wildman, S G, Symp sot expt biol (1970) 147. 21. Traub, P h Nomura, M, J mol bio134 (1968) 575. 22. - Ibid 40 (1969) 391. 23. Vasconcelos, A C L & Bogorad, L, Biochim biophys acta 228 (1971) 492. 24. Werz, G, Leitz-Mitt Wiss u Techn 11 (1963) 178. 25. Wittmann, H G, Abstract commun 7th meet Eur biochem sot (1971) 23. Received December 12, 1972 Revised version received February 5, 1973
Experimental Cell Research 80 (1973) 69-78
NUCLEAR GENOME CODES FOR CHLOROPLAST RIBOSOMAL PROTEINS IN ACETABULARZA II. Nuclear Transplantation
Experiments
K. KLOPPSTECH and H. G. SCHWEIGER Max-Planck-Institut fir Zellbiologie, D 2940 Wilhelmshaven, BRD
SUMMARY The protein patterns of chloroplast ribosomes of Acetubuluriu have been established by means of polyacrylamide gel electrophoresis. The protein patterns of the faster sedimenting 44s ribosomal subunit of A. mediterranea, A, cliftonii, and A. crenulata have been compared and species specific differences are described. The protein patterns of hybrid cells consisting of a host cytoplasm from one species and a nucleus from another species is changed to that of the nucleus donor species after some weeks. The results indicate that at least part of the chloroplast ribosomal proteins are coded by the nuclear genome.
Chloroplasts contain DNA and all the other components of an autonomous molecular genetic apparatus. As far as the chloroplast DNA is concerned it is known that this DNA contains the genetic information for the RNA moiety of the chloroplast ribosomes [15, 181and most probably also that for chloroplast tRNA [20]. On the other hand there is no chloroplast protein which has been proven to be coded by the chloroplast genome. However, for a number of chloroplast proteins it has already been shown that the genetic information comes rather from the nuclear than from the chloroplast genome [I, 5, 8, 14, 161. These results and the fact that the genetic information for the RNA of chloroplast ribosomes is located in the chloroplast genome raise the question whether or not the proteins of the chloroplast ribosomes are coded by the chloroplast genome. In this paper evidence is presented that in Acetabu-
laria at least some of the ribosomal proteins
of the chloroplasts are coded by the nuclear genome. This evidence is based on species specific differences in the electrophoretic protein patterns of the heavy ribosomal subunit between different speciesand on the fact that after implantation of a heterologous nucleus the protein pattern is changed into that of the nucleus donor species. Preliminary results have ben presented elsewhere t5, 61. In [7] it has been shown that besidesthe 70s ribosomes 56S, 44S, and 30s particles can be separated from Acetabularia chloroplasts. The material of the most prominent peak served as ribosomal material. It sediments with 44s and represents the heavy subunit of the chloroplast ribosome. MATERIAL
AND METHODS
Plants and ribosomes. The culture conditions for A. mediterranea, A. crenulata, and A. (Polyphysa) cliftonii Exptl Cell Res 80 (1973)
IO
K. Kloppstech
& H. G. Schweiger
and the procedures for isolating chloroplast ribosomes were the same as described in the preceding paper [7]. The nucleus implantation and transplantation techniques were essentially those of Hammerling [3, 41. The intermediate caps of the transplants were cut off after 14 and 28 days, the transplants were used about 6 weeks after the beginning of the experiment. Isolation of ribosomal proteins. Ribosomal material sedimentinn with 44s at a Mg*+ concentration of 2 x 10-a M was cut and pooled from up to five gradients, diluted with gradient buffer (0.1 M KCI, 0.01 M Tris, pH 7.4, and 0.002 M Mg (CH,COO),) and collected by centrifugation for 3 h, at 190 000 g in an SW 50.1 Ti rotor. The pellets from several thousand cells (5 000 to 10000 plants for monoribosomes; 2 000 for 56s particles; 1 000 for 44 S particles) were suspended in 10 ul of a solution which contained 2.5 ul of samole gel buffer (0.48 M KOH, 0.48 M CH&OOH, pH 6.7, and 0.03 M N,N,N’N’-tetramethylethylenediamine) [13] and 7.5 ~1 of 6 M urea. To this suspension 5 ul of RNase mixture were added which was diluted 114 with 6 M urea. This mixture contained 0.4 pg of pancreatic ribonuclease, 0.3 pugof T, ribonuclease, 0.04 pmoles of EDTA, and 1 pl of sample gel buffer. The rRNA was digested by incubation of the suspension for 6 h at 37°C and the resulting protein mixture was stored at - 18°C. Electrophoresis. Gel electrophoresis was carried out between two glass plates forming a vertical chamber of 1 x 25 mm* and 100 mm height. Over a small pore gel [21] of 75 mm height a portion of spacer gel [21] was layered to fill the rest of the cuvette. Prior to polymerisation two strips of acrylglass (0.5 x 2 mm2) were inserted into the spacer gel to form two identical channels seoarated from one another by 7 mm of gel. To each channel a mixture of 15 pg ribosomal protein in 15 ~1 of diluted sample gel buffer and 15 /-cl of gel solution [13] was added and polymerized. Electrophoresis was run for 195 min at 190 V with a current of about 5 mA per chamber at a room temperature of 18°C. The electrode buffer was that described by Reisfeld [13]. The gel was stained according to Meyer & Lamberts [9]. The gels were scanned at 550 nm using a Leitz microspectograph t241.
RESULTS Separation
of ribosomal
proteins
In order to facilitate the comparison between different electrophoretic patterns the different ribosomal protein preparations of Acetabularia were run in parallel with ribosomal proteins from E. coli as reference. This method allows comparison of different runs by referring to the bands of the E. coli control. Such a protein pattern of the 44s ribosomal particles from the chloroplasts of A. Exptl Cell Res 80 (1973)
mediterranea is given in fig. 1. At least 20 bands can be detected. As was shown in a previous paper the 44s particles represent the faster sedimenting subunit of the 70s chloroplast ribosomes [7]. One therefore should expect that all of the bands of the 44s subunit are contained in the protein pattern of the 70s ribosome. A comparison of the two patterns reveals a close similarity. It is possible to find the corresponding band in the 70s pattern for all 44s bands but not vice versa. This holds especially for some weak bands. The fact that only some weak bands of the 70s pattern are missing in the 44s pattern might be due rather to different amounts of protein which could be obtained from the large 44 S and the small 70s peaks than to differences in the protein composition. On the other hand it is obvious that there are differences in the intensities of several bands. When compared with the 44s pattern the proteins of the 70S particle are distributed more regularly throughout the profile. This gives some indication in which region of the 70s pattern the bands of the 30s subunit might be located. For example, the bands of the 70s profile which correspond to the E. coli bands 10, 27, and 32 seem to be dominant bands of the 30s subunit. However, it was impossible to establish the electrophoretic protein pattern from isolated 30s particles because the amount of material which could be obtained from 10 000 cells was too small. The low yield of 30s material seemsto reflect a high instability of the 30s particles, the reason for which is not yet known. The protein pattern of the 56s particles of A. mediterranea is closely similar to that of the 70s ribosomes as can be seen from the densitometer tracings in fig. 1b, c. A pronounced similarity between the protein patterns of 56s particles and 70s ribo-
Nuclear genome and chloroplast ribosomal proteins. II 25
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somes has also been revealed in A. crenulata and in A. clijtonii. This similarity indicates a close relationship between these two types of particles. Since it is known from Phaseolus aureus that the ribosomal proteins from cytosol, from mitochondria, and from chloroplasts are different [23] it is probable that the
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Fig. 1. Ribosomal proteins isolated from the chloroplast ribosomes of A. mediterranea. A, 44.3; B, 56s; C, 70s particles. ----, E. coli control, visible bands numbered beginning from the origin. The profiles correspond to about 1000 cells in A, 2 000 cells in B and 5 000 cells in C. The bars indicate the positions of visible protein bands.
56 S particle derives from the chloroplast and not, as might be suggested from the sedimentation coefficient, either from the cytosol (larger subunit) or from the mitochondria [l 11. Since the 44 S particles contain a smaller number of different proteins and are more dominant than the 30s peak in the presence Exptl Cell Res 80 (1973)
72 K. Kloppstech & H. G. Schweiger
Fig. 2. Comparison between the proteins of the 44s ribosomal subunits isolated from the chloroplasts of three species of Acetabularia. A, A. cliftonii; B, A. mediterranea: C. A. crenulata. ----, E. coli control, bands numbered from the origin. The profiles correspond to 1 000 cells. Speciesspecific differences are indicated by the vertical dashed lines.
of 2 x 1O-3 M Mg2+ as was shown in [7] the 44s subunit was chosen for the further studies. Species-specific proteins of the 44 S subunits The protein patterns of the 44s particles from A. mediterranea, A. crenulata, and A. cliftonii are compared in fig. 2. Although the pattern of A. cZiftonii looks similar to that of A. mediterranea there are at least two bands by which the two species are distinguished. As is indicated by the dotted line there is a well separated and prominent band in A. Exptl Cell Res 80 (1973)
cliftonii corresponding to E. co& band 30, while there is only a shoulder if at all in the same position of the A. mediterranea pattern. A second difference between the two species can be detected in the position 27 of E. coli. In this case a band could be detected in the A. mediterranea but not in the A. cliftonii pattern. The differences between the two species become even more obvious when instead of using E. coli material as reference the two species undc consideration were directly compared (fig. 3). The dotted lines indicate
Nuclear genome and chloroplast ribosomal proteins. II the positions 27 and 30 of E. coli and reveal two of the differences existing between the two species. Striking differences were found for the protein patterns of the 44s particles from A. mediterranea and A. cliftonii on the one hand and from A. crenulata on the other hand (fig. 2). While there are bands at the positions 7 and 23 in the A. crenulata pattern no comparable bands can be detected in the corresponding regions of the two other species. Moreover, other differences can be demonstrated between A. crenulata and the other two species in the region between 26 and 32. The most prominent band of A. crenulata in this region is found in position 27 whereas the maximum peaks of the two other species correspond to the bands 28 to 29. The differences between A. mediterranea and A. crenulata are also clearly shown in fig. 4, where the proteins of the two species were directly compared. The lines between the two profiles indicate that there are 4 peaks in A. crenulata and 5 peaks in A. mediterranea in the region 27 to 32. The patterns of all three species are highly reproducible so that it is possible to dis-
*tar,
I
Fig. 3. Direct comparisonbetweenthe proteins of the 44s subunits of A. cliftonii (upper curve) and A. mediterranea.The vertical dashed lines indicate the prominent differencesbetweenthe two species.
32
27
13
7
23
Fig. 4. Direct comparisonbetweenthe proteins of the 44s subunits of A. mediterranea(upper curve) and A. crenulata. The vertical dashed lines indicate
differencesbetweenthe species. tinguish definitely three species.
the protein patterns of all
Influence of heterologous nuclei on the chloroplast riboAoma1 protein patterns In order to elucidate a possible role of the cell nucleus in coding for the proteins of the chloroplast ribosomes heterologous nuclear transplantation experiments were performed and the influence of the implanted nucleus on the ribosomal protein pattern of the chloroplasts was investigated. A major problem was to find a method by which it was possible to obtain sufficient amounts of material. We worked with unlabelled proteins by which method a number of difficulties were avoided which might interfere with the preparation and separation of labelled proteins in Acetabularia. One such difficulty is the incorporation of sufficient amounts of label. Another one is a possible contamination by microorganisms which might become labelled very quickly. In order to obtain sufficient amounts of material it was not the hybridized cells Exptl Cell Res 80 (1973)
74
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themselves from which the ribosomal proteins were prepared but rather cells of the F, generation of the hybrids. If the ribosomal proteins of the chloroplasts are coded by the chloroplast genome the pattern of the ribosomal proteins should remain unchanged in the F, generation. If the genetic information for the proteins comes from the nucleus the protein pattern of the F, generation should be that of the nucleus donor species. Exptl
Cell Res 80 (1973)
Fig. 5. Proteins of the 44s ribosomal subunit isolated from the chloroplasts of the F,-generation of med,clifr, ceils. Ribosomal protein pattern of the 44s subunits of FL-generation cells of: A, A. mediterranea: B, medI-clifto hybrids (the nucleus donor was A. mediterranea, enucleated cells derived from A. cliftonii); C, A. cliftonii. The vertical dashed lines indicate the species-specific bands at the positions 27 and 30.
The hybrids were prepared by implanting an isolated nucleus of one species into a basal fragment (10 to 15 mm) of the stalk of another species. The hybrids of the types used for this work would behave like normal cells and undergo the different stages of the cell cycle, that means the nucleus divides into secondary nuclei, cysts and gametes are formed, and after copulation zygotes grow out. Up to 10 000 hybrid cells could be ob-
Nuclear genome and chloroplast ribosomal proteins. II
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tained by this method from only a limited number of implants. The result of such a hybridisation experiment is shown in fig. 5. In this experiment the nucleus donor species was A. mediterranea and the acceptor species was A. cliftonii. This type of heterologous combination and its ability to generate an F, generation has early been described [17]. From cells of such an F, generation chloroplast ribosomes were isolated and the protein pattern of the
6. Proteins of the 44s ribosomal subunit isolated from the chloroplasts of cells. the F,-generation of cum,-clift, Ribosomal protein pattern of the 44s subunits of: A, A. crenulata: B, F,-generation cells of cren,-cliftn hybrids: C, A. cliftonii. The vertical dashed lines.indicate the species-specific bands at the positions 7, 23, and 27. Fig.
;““”
‘. 3
44s ribosomes were compared with the protein patterns of A. mediterranea and that of A. cliftonii. As revealed in fig. 5 the protein pattern of the F, generation of medl-cZift,, cells obviously is similar to that of the nucleus donor species, namely A. mediterranea. This is demonstrated e.g. by the presence of the band in position 27 and the absence of the predominant band in position 30. A comparable result was obtained in two other experiments of the same type. A result which Exptl Cell Res 80 (1973)
76 K. Kloppstech & H. G. Schweiger DISCUSSION
Fig. 7. Proteins of the 44s ribosomal subunit isolated from crenl-cl(ftO hybrid cells. -, cren,-c&f&, grafts; ....... E. coli control. The vertical dashed lines indicate the bands 7,23, and 27 which are absent in A. cliftonii.
was obtained with cren,-clift, hybrids is shown in fig. 6. In this case the pattern of the F, generation of the hybrid cells is similar to that of A. cremdata. This can be learned from the presence of the bands at the positions 7, 23, 27 which are all absent in A. cliftonii. This experiment shows again that the protein pattern of the daughter cells of the hybrids is nuclear-dependent. Finally a rather direct approach was used. In this experiment the changes in the protein pattern were investigated 6 weeks after transplanting a rhizoid containing a nucleus onto an anucleate stalk. This experiment was of the cren,-clift, type. About one thousand transplants served as starting material [17]. The chloroplast ribosomes were isolated 6 weeks after the transplantation and the proteins of the 44s particles were separated electrophoretically as shown in fig. 7. Although the amount of protein which was obtained from these thousand hybridized cells was very small it is obvious that the pattern of the ribosomal proteins again is quite similar to that of the donor species of the nuclei. This is demonstrated by the presence of the bands in positions 7, 23, and 27. Exptl Cell Res 80 (1973)
The proteins of the 44s ribosomal subunit of Acetabuluria chloroplasts were separated into about 25 bands and the patterns of A. mediterranea, A. cliftonii, and A. crenulata were compared. Species specific differences were found between the three species under consideration. The differences between A. mediterranea and A. cliftonii comprise at least 2 bands and between A. crenulata and the two other species at least 4 bands were different. The rather small number of species specific bands in these closely related species is striking and in accordance with results of other authors [25]. By means of the transplantation technique by which nucleate rhizoids of one species were transplanted onto anucleate fragments of another species it was shown that the species specific protein pattern changes under the influence of the transplanted cell nucleus. In a similar way after implantation the isolated heterologous nucleus caused the acceptor cytoplasm to change the ribosomal protein pattern to that of the nucleus donor species. In the latter case the pattern was determined in F, generation cells. Such changes suggest that the ribosomal proteins of the chloroplasts are coded by the nuclear genome. However, it cannot be excluded completely that nuclear-dependent changes in the secondary structure are involved. Anyway, it must be kept in mind that the nuclear coding capability is proven only for those proteins of the 44s subunits which are species-specific. Special attention should be focused on the fact that the change in the protein patern under the influence of the implanted nucleus means not only the appearance of new bands but also the disappearance of old ones, so that after an appropriate time a pure pattern of the nucleus donor species was observed rather than an
Nuclear genome and chloroplast ribosomal proteins. II
intermediate form which still resembled the host cytoplasm species. The simultaneous appearance of nucleus donor species bands and the disappearance of host cytoplasm species bands under the influence of an implanted heterologous nucleus has already been observed and discussed in the case of two enzymes.This disappearanceis rather striking since in this case it could be shown that the isozyme patterns remained the same for periods of up to at least 8 weeks after removal of the cell nucleus. So the disappearance obviously is induced by the implanted nucleus. A possible explanation for this phenomenon might be some sort of a replacement of the mRNA at the site of protein synthesis which results in an increased instability of the mRNA of the host cell. Experimental evidence from bacteria indicates that the genetic information for the ribosomal protein is organized in a single operon [lo]. If the same holds for the ribosomal proteins of the chloroplast, one might assume that those ribosomal proteins of the chloroplast for which no species specificity could be demonstrated are also located in the nuclear genome. The question of how far a limited number of heterologous chloroplasts which were transferred into the anucleate cytoplasm together with the heterologous nucleus could be bIamed for the change of the protein pattern deservesspecial attention. In nucleate cells chloroplasts have been found to undergo a divisision every 10 days [2, 191.Assuming that this multiplication rate holds for the heterologous chloroplasts in the hybrid cells also one could expect that during a period of 50 days the number of chloroplasts should not have increased to more than the 50-fold. Since the acceptor cytoplasm contains about 1 x lo6 chloroplasts and even if 1 x lo3 heterologous chloroplasts are transferred with the nucleus an increase of the hetero-
77
logous chloroplasts to a 50-fold could result in not more than 5% heterologous chloroplasts with respect to the total chloroplast population. This holds true even if the homologous host chloroplasts would stop dividing after the nuclear transplantation. From our results it can be concluded that the genetic information for the proteins of the chloroplast ribosomes originates in the nuclear genome while it is generally accepted that RNA of the chloroplast ribosomes is coded by the chloroplast DNA. One may conclude that the chloroplast ribosomes in the hybrid cells are hybrids too. The possibility that such hybrid ribosomes exist and are functionahy active in heterologous combinations implies that the ribosomal RNA of the chloroplasts of different species do not differ to as great an extent as the corresponding ribosomal proteins [ 121. Indeed it has been shown in bacteria that the ribosomal RNA of different species is highly homologous. Moreover, heterologous ribosomal combinations of RNA from one bacterial species and proteins from another specieshave been produced in vitro by means of the reconstitution technique of Traub & Nomura [22]. Such heterologous combinations have been shown to be functionally active. The skilful technical assistance of Mrs H. Stelzer is gratefully acknowleged.
REFERENCES 1. Apel, K & Schweiger, H G, Eur j biochem 25 (1972) 229. 2. Clauss, H, Liittke, A, Hellmann, F & Reinert, J, Protoplasma 69 (1970) 313. 3. Hammerling, J, Biol Zentralbl 74 (1955) 545. 4. - Wilhelm Roux Arch Entwicklungsmech Org 132 (1935) 424. 5. Kloppstech, K & Schweiger, H G, Abstract commun 7th meet Europ biochem sot (1971) 155. 6. - Biology and radiobiology of anucleate systems (ed S Bonotto, R Goutier, R Kirchmann & J Maisin) p. 127. Academic Press, New York (1972). Exptl Cell Res 80 (1973)
7% K. Kloppstech h H. G. Schweiger 7. - Exptl cell res 80 (1973) 63. 8. Kung,. S D, Thornber, J P & Wildman, S G, FEBS letters 24 (1972) 185. 9. Meyer, T S & Lamberts, B L, Biochim biophys acta 107 (1965) 144. 10. Nomura,.M & Engbaek, F, Proc natl acad sci US 69 (1972) 1526. 11. O’Brien, T W, J biol them 246 (1971) 3409. 12. Pace, B & Campbell, L L, J bact 107 (1971) 543. 13. Reisfeld, R A, Lewis, U J & Williams, D E, Nature 195 (1962) 281. 14. Reuter, W & Schweiger, H G, Protoplasma 68 (1969) 357. 15. Schweiger, H G & Berger, S, Biochim biophys acta 87 (1964) 533. 16. Schweiger, H G, Master, R W P & Werz, G, Nature 216 (1967) 554.
Exptl Cell Res 80 (1973)
17. Schweiger, H G, Curr top microbial immunol 50 (1969) 1. 18. Scott, N S & Smillie, R M, Biochem biophys res comm 28 (1967) 598. 19. Shephard. D C, Exptl cell res 37 (1965) 93. 20. Tewari, K K & Wildman, S G, Symp sot expt biol (1970) 147. 21. Traub, P h Nomura, M, J mol bio134 (1968) 575. 22. - Ibid 40 (1969) 391. 23. Vasconcelos, A C L & Bogorad, L, Biochim biophys acta 228 (1971) 492. 24. Werz, G, Leitz-Mitt Wiss u Techn 11 (1963) 178. 25. Wittmann, H G, Abstract commun 7th meet Eur biochem sot (1971) 23. Received December 12, 1972 Revised version received February 5, 1973