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D. degreein Botany ', Wfi )1 Major professor Coleman Peter Wolk 3f Date ”Ill/u) ? MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES m . RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. in De THE CYANELLE AND THE CYANELLE GENOME OF CYANOPHORA PARADOXA BY Catherine Clair Hasmann A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1985 Copyright by CATHERINE CLAIR HASMANN 1985 ABSTRACT THE CYANELLE AND THE CYANELLE GENOHE OF CYANOPHORA PARADOXA By Catherine Clair Hasmann The photosynthetic organelle, called a: cyanelle, of Cyanophora paradoxa has characteristics of both cyanobacteria and chloroplasts. 2n: an attempt clarify the evolutionary position of the cyanelle an investigation has been carried out to determine the sites of synthesis of, and the locations of the genes for, the cyanelle proteins. Particular emphasis has been placed on the large and small subunits of ribulose-1,S-bisphosphate carboxylase and on the 8 subunit of ATP synthase. Cyanelle proteins were labeled in lilfl in the presence of inhibitors specific for cyanelle or cytoplasmic protein synthesis. The results suggest that cyanelle proteins are synthesized both in the cyanelle and in the cytoplasm. Among those polypeptides which may be synthesized in the cyanelle are the large subunit of RUBISCO. the a and 8 subunits of the ATP synthase and the 32,000 dalton membrane protein. >4 11' 0 ~ 3:5units < ATP syntha: :55 genes seqsence c the DNA 5e Catherine Clair Wasmann The genes for the large (rbcL) and small (rch) subunits of RUBISCO and the 8 subunit (ath) of the ATP synthase have been localized in the cyanelle DNA from Cyanophora. The nucleotide sequences of the rbcl. and the £338 genes and approximately 210 nucleotides of the coding sequence of the 51228 gene have been determined. Based on the DNA sequence, the cyanelle £238 gene is located on the same DNA strand as the LESL gene 108 basepairs from the termination codon of ngt. The coding sequences of the EEEL and 3338 genes are separated by N81 basepairs and are transcribed divergently. The recognition sites of the restriction endonucleases BglII. XhoI, Pstl. BamHI, and SalI have been localized in the cyanelle DNA and a map constructed. The size of a monomer of cyanelle DNA is approximately 128 kilo-basepairs (kb). The restriction map of the cyanelle DNA is circular. A 10-kb segment of the cyanelle DNA is duplicated. The duplicated segments are arranged in inverted orientation and are separated by DNA segments of unequal size that are unique in sequence. The cyanelle ngL, £338, and 2328 genes are located in the larger single copy region of the cyanelle DNA. DEDICATION To perseverance and to Mary and Bill Nasmann ii I uoui students, p measant a: SRecia "fibers 9 2‘ LEE HA’ kintg thoughtful haVe bee“ Th13 °°ntract ACKNOWLEDGEMENTS I would like to thank all of the faculty, graduate students, post docs, and staff of the Plant Research Lab for contributing to the atmosphere that makes the PRL such a pleasant and efficient place to "do science". Special thanks to my advisor Peter Holk and to the members of my guidance committee, Barry Chelm, Norm Good, Lee McIntosh, Ken Poff, and Harold Sadoff, for their thoughtful advice. I would also like to thank Trek Bicycle Corporation for manufacturing the TREK 970 without which my sanity would have been in serious Jeopardy. This work was supported by Department of Energy contract number DE-ACOZ-76ERO-1338. iii .c .zapter I. INTRCL‘LI Intrc Surve Th 10- to B3 U H a O" '7 no 1‘ (xi/(j) TABLE OF CONTENTS Chapter Page I. INTRODUCTION AND SURVEY OF THE LITERATURE.......... 1 IntrOduction.OOOOOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOO. 1 survey or the LiteratureOOOOOOOO0.0.0000.....'... 5 The sites of synthesis of, and the locations and the structures of the genes for, the large and small subunits of RUBISCO....................................... 5 Cyanophora paradoxa........................... 13 Prospectus.................................... 28 II. LABELING OF CYANELLE PROTEINS IN VIVO.............. 31 IntrOductionOOOOOOOOOOO0....OOOOOOOOOOOOOOIOOOOOO 31 Materials and Nethods............................ NO Organism and growth........................... “0 Purification of Cyanophora.................... N1 Conditions for labeling Cyanophora............ "2 Isolation of cyanelles from radioactively labeled Cyanophora............................ N3 Determination of incorporation of radioactively labeled compounds into protein by Cyanophora................................. an SDS polyacrylamide gel electrophoresis of radioactively labeled Cyanophora.............. "5 Determination of protein and chlorophyll 3.... NS ReSUItSOOOOOOOOOOOOOOOOOOOOIOOOOOOOOOOOOO0.0.0... ”6 Characteristics of the incorporation of labeled substrates into protein by intact cells of Cyanophora..1n....................... A6 Incorporation of Na [ C]acetate.............. H7 The effect of D-cyc oheximide and D-threo- chloramphenicol on the incorporation of acetate.O....0...0....OOOOOOOOOOOOOOOOOOOOOOOO so Incorporation of amigg acids by Cyanophora.... 50 Incorporation of Na2 SO” by cyaHOEhoralOOOO0.0.0.0...OOOOOOOOOOOOIOOOOOOOO 6o SDS-PAGEBgf proteins from cyanelles labeled With "a so”aV1VOOOOOOOOOOOOOOOOOOOOOOOOOO 62 Discussion....................................... 69 Characterization of the incorporation of radioactively labeled compounds into protein by Cyanophora paradoxa ....................... 69 iv Chapter 52 Hi In Re 11L TEE NUC LARGE A BISPECS Intro Resul Or an Th an Cc Cc Th IV. 355nm CYANELL Intro Chapter Page SDS-PACE gf intact cells and cyanelles labeled 3 with Na The polfipeptides synthesized in the cyanelle.. Recommendations............................... so 0......0.0...0.000000000000000... III. THE NUCLEOTIDE SEQUENCES OF THE GENES FOR THE LARGE AND SMALL SUBUNITS OF RIBULOSE-1.5- BISPHOSPHATE CARBOXYLASEOOOOOO0.0000000000000000... IntrOductienOOO...0.0..0....OOOOOOOOOOOOOOOOOOOOO Results and Discussion........................... Organization of the cyanelle LEELv Eggs. and £228 geneSOOOOOIIOOOOOOOOOOOOOOO0.0.0.0... The coding regions of the cyanelle 523i, £238, and 2328 genes................................ Codon usage in the cyanelle of Cyanophora..... Comparison of the 5' and 3' flanking regions.. The cyanelle and the endosymbiont hypothesis.. IV. RESTRICTION ENDONUCLEASE MAPPING OF THE CYANELLE DNAOO.'......O...0....0.000000000000000... IntrOduction..0....'......‘.....OOOOOOO.'......O. "aterials and MethOdSO0.0.....OOOOOOOOOOOOOOOOOOO Strains and culture conditions................ cyanelle isolationOOOOOOOOOOOOOOOOOOOOOOOOOOOO ISOlation Of cyanelle DNAOOOOOOOOOOOOOOOOOOOOO Restriction endonuclease analysis............. Construction and isolation of recombinant plasmids containing cyanelle DNA.............. Isolation of plasmid DNA...................... Nick translation and filter hybridization..... sources or the enzymesoootOOOOOOOOOOOOOOOOOOOO ReSUItSOOOOOOOOOOO..'.....O....’......OOOOOOOOOOO Restriction endonuclease mapping of the cyanelle genome.00.000.00.000.00.000.000.00... Ordering the restriction fragments............ Ordering the restriction fragments of orientation 1.00.00.00.00.0.00.00.00.00...O... Ordering the restriction fragments of orientation II0.000000000000000000000000...... Location of the ngL, £338, and gth genes on the map of the cyanelle DNA................ D180u331°nooooooooooooooooooooooooooooooooooooooo V. SUMMARY AND GENERAL DISCUSSION..................... summary.0.000.000.0000.0......0.0000000000000000. General Discu381°n000000I.OOOOOOOOOOOOOOOOOOOOOOO 73 76 79 81 81 87 87 90 104 107 110 11H 11" 117 117 117 118 119 120 122 123 12H 12” 12" 13" 150 152 155 156 161 161 166 Chapter ' -UO Ste—42:7 C‘ U M Sc Rest; Lo A7 C1 C1 ”edit ECldl LIST OF REFE Chapter Page APPENDIX A. MOLECULAR CLONING OF THE CYANELLE gbgL AND E223 GENES AND THEIR FLANKING REGIONS... 171 Introduction..................................... 171 "ateriala and “ethOdSOOOOOOOOOOOOOOOOOOOOOOOOOOOO 172 DNAprObeSOO.IOOOOOOOOOOOOOOOOOOOOIOO00.0.0...172 Nick translation and filter hybridization..... 173 Nomencalature................................. 1?“ Isolation of cyanelle DNA fragments........... 17“ Molecular cloning............................. 175 Sources of the enzymes........................ 178 893111113.ooooooooooooooooooooooooooooooooooooooooo178 Location of the DNA fragments containing the large subunit of RUBISCO and the 8 subunit of ATP synthase.................................. 178 Cloning of the gene for the large subunit 0fRUBISCOooooooooooococooocoooooooooooooooooo188 Cloning of the gene for the small subunit 0fRUBISCOOOOOOOOOOOOO0.00.000.000.00.00......197 Discussion....................................... 198 APPENDIX B. GROHTH MEDIA FOR CYANOPHORA PARADOXA........ 201 Hedium CYBO...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 201 Bald's 3N BristOISO O O O O O O O O O O O O O O O O C O O O O O O O O O O O O O 202 LIST OF REFERENCESOOOOOOOOOO0.0.0.000...0.00.00.00.00... 203 vi LIST OF TABLES Table 1. 3. 8. 9. 10. Rates and relative rates of incorporation of [ C]leucine into protein by intact cells and cyanelles of Cyanophora paradoxa.................... The amount of radioactivity present in protein of cells and cyanelles after labeling intact cells of Cyanophora paradoxa with [ C]leucine for 90 m1UUteSocooooooocoooooooooooooooooooooooooooooooo Radioactivity present in protein of intact cells ggd cyanelles of Cyanophora paradoxa labeled with so” ..'.....OOOOOOOO..‘.....O...'.....OOOOOOOOOOOO Polypeptides ggsociated with cyanelles labeled in vivo with 80 ' in the presence of cycloheximide Or Chlorampheni-COfO0.0.0.0.0..OOOOOOOOOOOOOOOOOOOOO. Codon utilization in the cyanelle rbcL, rch and ath genes from Cyanophora paradoxa................. Sizes and stoichiometries of DNA fragments resulting from single, double, and triple digestions of cyanelle from Cyanophora paradoxa................... BamHI and SalI restriction fragments grouped according to the orientation of the cyanelle DNA.... Digestion of BglII fragments (Bg1, Bg2, Bg3, Bgu, Bg6) of cyanelle DNA with the restriction endonucleases BamHI, SalI or XhoI................... Summary of hybridization results.................... Sizes and stoichiometries resulting from single and double digestions of a mixture of BglII fragments (Bgl, Bg2, Bg3, Bgu) of cyanelle DNA from £0 ParadoanOOOOO0.0..’.....O.‘0.0000000000000000... vii Page 58 59 63 67 105 128 130 131 133 13S hble 1. DNA {rag plasmids endonucl Table Page 11. DNA fragments generated by digestion of recombinant plasmids pCpOO1, pCpOZN, and pCpOZS with restriction endonucleaseSOOOOOO00......0.00000000000000000000000196 viii LIST OF FIGURES Figure 1. 3. N. 5. 6. 8. Time courses of the incorporation of various concentrations (pH) of [ Clacetate into protein by intact cells of Cyanophora paradoxa................. Time course of the incorporation of[1uC]acetate into protein by intact cells of Cyanophora paradoxa incubated in the light (0—0) and in the dark (H)00o.oooooooooooooooooooooooooooooooooooo The effect of various concentrations of D-cycloheximide (A) and DTfihreo-chloramphenicol (B) on the incorporation of [ CJacetate into protein by intact cells of Cyanophora paradoxa.............. Time course of the incorporation of [3SSJmethionine into protein by intact cells of Cyanophora paradoxa incubated in the light «)—-—C» or in the dark (H).OOOII..00.0.0..OOOOOOOOOOOOOOOOOOOOOOOO Time course of the incorporation of [BSSchsteine into protein by intact cells of Cyanophora paradoxa incubated in the light «D—-—C» or in the dark (H)ooooooooooooooooooooooooooooooooooooooo Time course of the incorporation of [1uCJIeucine into protein by intact cells and cyanelles of Cyanthora paradoxa upon incubation of intact cells in the absence or in the presence of D-cycloheximide or D-threo-chloramphenicol......... Time coursegsof the incorporation of N9 pH, 9? pH, and 389 uM SO ' into protein by intact cells of Cyanophora paragoxa................................ Patterns of polypeptides from the cyanelles of gganophora paradoxa labeled in vivo with SO ' u 0...'......OOOOOOOOOOOO0.0.0.0000...000...... ix Page . N8 "9 52 53 SN 56 61 65 Figure L . Hucleci Physice ccntair cyanell' Compar‘ subunii (Cy). Spinac. chloroa 1;! ‘1- Upper: from tr Lower; Figure 9. 10. 11. 12. 13. 11:. 15. 16. 17. 18. 19. 20. Physical map of the region of the cyanelle DNA containing the rbcL, rch, and ath genes.......... Nucleotide sequence of the rbcL gene of the cyanelle 0f CyanophoraOOOOOOOOO000......0.00.0.0... Comparison of amino acid sequences of the large subunits of RUBISCO from the cyanelle of Cyanophora (Cy), Anabaena 7120 (A7120), Anacystis (An), spinach chloroplasts (Sp), and maize Chloroplast: (2m)..'.....OOOICOOOOCCOO.’......0.... Upper: Nucleotide sequence of the £238 gene from the cyanelle of Cyanophora. Lower: Comparison of the amino acid sequences of the small subunits of RUBISCO form the cyanelle of Cyanophora, Anabaena, Anacystis, pea, soybean, spinach, wheat, and tobacco............... Upper: Partial sequence of the 2328 gene from the cyanelle of Cyanophora. Lower: Comparison of the amino acid sequences of the first 70 amino acids of the subunit of the ATP synthase from the cyanelle of Cyanophora (Cy), E. coli (Ec), spinach chloroplasts (Sp), and maize chloroplasts (2m)..'.....OOOOOOOOOOOOOOOO..'.....OOOOOOOOOOOOOOO Agarose gel electrophoresis of the cyanelle DNA of Cyanophora paradoxa restricted with various enzymeSOOOOOOO..’.....OOOOOOOOOOOOOOOOOOOOOOCOOOOOO Restriction endonuclease map of the cyanelle DNA of cyanOEhora paradoanOOOOOOOOOOOOO.....OOOOOOOOOOOO. Restriction endonuclease map of the repeated region of the cyanelle DNA from Cyanophora paradoxa showing both orientations.......................... Hybridization of pYNNO and pZRN8 to cyanelle DNA... Hybridization of pYNNO and pZRN8 to cyanelle DNA... Hybridization of cyanelle DNA with the 3' half of the large subunit of RUBISCO from maize............ Hybridization of cyanelle DNA with pBSNO........... Page 89 92 95 99 103 126 137 139 181 183 185 187 Figure 2L A. A r restri 8. Res DNA ca pCpCZ: Figure Page 21. A. A restriction endonuclease map of the restriction fragments Ba8 and BaS of cyanelle DNA. B. Restriction endonuclease fragments of cyanelle DNA carried by the recombinant plasmids pCp001, pCpOZN, pCp025, and pCp029......................... 193 xi CHAPTER I Accorq Eukaryotic chimeras . free'liviné chlS‘ Th4 apparatUS eukarYOLQS chlorc’Plagt prekaryotes I transitiOn CHAPTER I INTRODUCTION AND SURVEY OF THE LITERATURE Introduction According to the "endosymbiont hypothesis" of eukaryotic cell evolution (11N,171). eukaryotic cells are chimeras, their various organelles derived from once free-living prokaryotes that were engulfed by heterotrophic. cells. The strong similarities between the photosynthetic apparatus and mechanisms of cyanobacteria and photosynthetic eukaryotes (82,18N) led to the generally accepted view that chloroplasts evolved from oxygen-producing photosynthetic prokaryotes resembling cyanobacteria and Prochloron (11N). Within the context of the endosymbiont hypothesis, the transition from endosymbiont to chloroplast is a process of increasing the degree of integration between the endosymbiont and its host (11N)., The distinction then between an endosymbiont and a chloroplast is primarily a matter of degree: the cell-plastid symbiosis is more highly integrated (11N). At one end of the spectrum of integration is the chloroplast, at the other end are the cyanobacteria, one of the presumed ancestors of the plastid (11N). The N photosynth Baradoxa cyanobacte (11.153) . and 3 “Mi: The c? cell are t. IntQEI‘atior “02 fixatj Chi“Opiast carbon Occl C32 “Kati. the cytopla ledial photosynthetic inclusion, or cyanelle, of Cyanophora paradoxa has characteristics of both chloroplasts and cyanobacteria. It has been considered a chloroplast (17.153). an endosymbiotic cyanobacterium (70.89.1N5,17N), and a "bridge between cyanobacteria and chloroplasts” (1). The chloroplast and other components of the green plant cell are highly integrated. One example of the multilevel integration that unites the chloroplast and the cytoplasm is CO2 fixation. Whereas the metabolic function of the chloroplast is to fix CO much of the utilization of fixed 29 carbon occurs in the cytoplasm. In leaves, substrates for 602 fixation, C02, the cytoplasm to the chloroplast stroma and the transfer of and inorganic phosphate (Pi), move from the product of CO fixation, triose phosphate, moves in the 2 reverse direction. The exchange of Pi for triose phosphate is mediated by a specific protein, the phosphate translocator, localized in the inner membrane of the chloroplast envelope (75). The phosphate translocatcr is an example of a protein serving to integrate the functions of the chloroplast and the cytoplasm, a protein that would be useless, even detrimental, in the prokaryotic ancestor of the chloroplast. Nitrate assimilation is also a cooperative process involving both the chloroplast and the cytoplasm. The second step in this pmocess, nitrite reduction, occurs in the chloroplast (102). The incorporation of the pmoduct of nitrite aspartate, | carbon c pyruvate, in the c: titochondr chlor0p1a3 True Itself a, Senomes, the chlon QY‘LOplaSm in that t; Whereas the cite; Chloropl a cytoplasm FibuloSe nitrite reduction, NH into glutamine, glutamate, 3. aspartate, and alanine, occurs in the chloroplast. The carbon compounds, oxaloacetate, a-ketoglutarate, and pyruvate, which serve as the acceptors of reduced nitrogen in the chloroplast, are synthesized in the cytosol and mitochondria from the triose phosphate exported by the chloroplast (69,102). The proteinaceous constituents of the chloroplast itself are encoded by both the chloroplast and nuclear genomes. Some chloroplast proteins are synthesized inside the chloroplast, whereas many others are synthesized in the cytoplasm (25.51). Even the chloroplast riboscme is a hybrid in that the ribosomal RNA is synthesized in the chloroplast, whereas at least some ribosomal proteins are synthesized in the cytoplasm. Although the Calvin cycle operates in the chloroplast, many of its enzymes are synthesized in the cytoplasm. The first enzyme of the Calvin cycle, ribulose-1.S-bisphosphate carboxylase (RUBISCO). about which more will be said in the paragraphs that follow, consists of large subunits synthesized in the chloroplast and of small subunits that, in higher plants and eukaryotic green algae, are synthesized in the cytoplasm. The general goal of the work presented in this thesis was to investigate the degree to which the cyanelle and nuclear genomes of Cyanophora have become integrated. Because the biosynthesis of chloroplast proteins expresses well the genomes, I‘ of the gen small sut "GIULIOna as the Ice 1. R115 the PVC be it.‘ an 2. EU Om it an 3. R“ e: e. cl 11. T D 5. —*HA5_1 well the cooperation between the chloroplast and nuclear genomes, I thought that an investigation into the locations of the genes for and the sites of synthesis of the large and small subunits of RUBISCO might help to clarify the evolutionary position of the cyanelle. RUBISCO was chosen as the focus of this study for the following reasons: 1. RUBISCO catalyzes the initial reaction in photosyn- thetic carbon assimilation by the reductive pentose cycle (Calvin cycle) and is, therefore, presumed to be metabolically important to any organism having it, regardless of whether the enzyme is located in an organelle or in an endosymbiont; 2. RUBISCO is present in Cyanophora and both its quaternary structure and the molecular weights of its subunits are very similar to that of chloroplasts and of cyanobacteria; 3. RUBISCO is perhaps the best studied example of an enzyme whose biosynthesis, in all photosynthetic eukaryotes examined to date, involves both chloroplast and nuclear genomes; N. The biosynthesis of RUBISCO is a good paradigm of plastid-nuclear integration; 5. The cyanelle has characteristics similar to cyanobacteria, in particular, the presence of peptidoglycan. It was therefore possible that the cyanelle DNA encodes, and the cyanelle synthesizes, both subunits of RUBISCO. the struc‘. snall SUDI The c competent nuclear a: (25.51). 'I by the I photosynth MEM- cata Unknown {u algae, th transcribeC (2'10’27931 transl Precursor Survey of the Literature The sites of synthesis of, and the locations and the structures of thegenes for, the large and small subunits of RUBISCO The development and maintenance of a photosynthetically competent chloroplast entails interactions between the nuclear and plastid genomes of the cells of higher plants (25.51). The cooperation between the genomes is exemplified by the biosynthesis of RUBISCO. This key enzyme in photosynthetic carbon assimilation commonly consists of eight catalytic large subunits and eight small subunits of unknown function (120). In plants and the eukaryotic green algae, the large subunit of carboxylase is encoded, transcribed, and translated in the chloroplast (2.10,27,31,3N,N1,NN,63,65,108,113,121.1N2,181,182). whereas the small subunit of RUBISCO is encoded in the nuclear DNA and translated in the cytosol on free ribosomes as a precursor polypeptide (31,33,39,NN,N6.81,88.113,152). Genetic evidence indicating that the nuclear DNA of Nicotiana contains the gene for the small subunit of RUBISCO was obtained by Kawashima and Hildman (88). They showed that specific peptides produced by digestion of the small subunit with trypsin were inherited in a Mendelian fashion in reciprocal F1 hybrids between !.tabaccum and N. glutinosa and between N.tabaccum and N.tabaccum and N.glauca. Genetic evidence suggesting that the large subunit is encoded in the chloroplas They shove Iustraliars‘ Lsuaveole American [.sylvestrl lustraliar. The peptic ”Ciprocal duonstrat Iaternally The synthesi 3 Parallel 1 3' (N) Protein-3y or the 1; experiment CYtOplasmj small SUE Orianelle ‘al‘ge Sui. U CM W reSult: . chloroplast DNA came from the work of Chan and Nildman (3N). They showed that peptide maps of the large subunit of three Australian species of Nicotiana (N.gossei, !.excelsior, and N.suaveolens) were identical. to tone another and to five American species (N.tabaccum, N.glauca, !.glutinosa, N.sy1vestris, N.rustica) with the exception that the Australian species contained an additional tryptic peptide. The peptide maps of the large subunit from F1 hybrids from reciprocal crosses between N.gossei and N.tabaccum demonstrated that the extra tryptic peptide was inherited maternally. The results of investigations into the sites of synthesis of the large and small subunits of RUBISCO parallel the results of the genetic studies. Criddle gt gl. (NN), working with intact leaves of barley, first suggested that the chloroplast and cytoplasmic protein-synthesizing systems are involved in the formation of the two subunits of RUBISCO. In a double labeling experiment they found that cycloheximide, an inhibitor of cytoplasmic protein synthesis, blocked synthesis of the small subunit, whereas chloramphenicol, an inhibitor of organelle protein synthesis, affected the formation of the large subunit. Subsequent in vivo inhibitor studies with Chlamydomonas (113) and pea (31) have yielded similar results. 81: but not the ace synthesi labeled tryptic large so! by isola1 In? and EL:-e Gray °°mplemen1 ”£2 Prot French be. 1ncorpora t antiboggies Wheat. [3 Blair and Ellis (10) first demonstrated that the large, but not the small, subunit is a major product, in terms of the amount of [35$]methionine incorporated, of protein synthesis by isolated intact pea chloroplasts. The in 31352 labeled product was shown to be identical, on the basis of tryptic peptide maps, to the native large subunit. The large subunit is also a major product of protein synthesis by isolated chloroplasts of spinach (27,121), barley (2), and Euglena (178). Gray and Kekwick (65) have described results complementary to those of Blair and Ellis (10). using an in vitro protein-synthesizing system. Using 808 polysomes from 1”Clamino acids were French bean they demonstrated that [ incorporated into a protein that was precipitated by antisera to the small subunit. Gooding at El- (63) made antibodies to the large and small subunits of RUBISCO from wheat. [3H]puromycin was used both to label and to release nascent polypeptides from 808 and 708 ribosome, which were separated by density gradient centrifugation. The 708 ribosomes were found to react only with antisera against the large subunit, whereas 808 ribosomes reacted with antisera against either subunit. Gooding st 21* (63) suggested that the association of both large and small subunits with 808 ribosomes (reflected the complexing of completed large subunits with the nascent chains of small subunit and that it did not mean that large subunits were made in both the chloroplz messenger first by chloropla found ‘tha The 52.20 than the Spinach cl SynthesiZe chloroplast and cytoplasm. The i vitro translation of messenger RNA for the large subunit of RUBISCO was obtained first by Hartley 33 _l. (72) who translated total chloroplast RNA using a cell-free extract of §.2g.. They found that a polypeptide of 52,000 daltons was synthesized. The 52,000 dalton polypeptide was ca. 1,500 daltons smaller than the large subunit that was synthesized by isolated spinach chloroplasts. Analysis of the 52,000 dalton protein synthesized by the §.g_o_l_i system showed that it contained only seven of the nine chymotryptic peptides of the chloroplast-synthesized large subunit. A polypeptide of 35,000 daltons was also synthesized from total chloroplast RNA by the §.ggli cell-free system (72). Wheeler and Hartley (181) separated spinach chloroplast RNA into polyadenylated RNA [poly(A)+RNA] and non-polyadenylated RNA [poly(A)' RNA], and then translated those fractions in_ vitro using the §.ggli cell-free system. They found that only the poly(A)' RNA programmed the synthesis of the 52,000 dalton protein. Sagher _t. gl. (1N2) translated poly(A)+ and poly(A)' RNA from Euglena chloroplasts using, a cell-free protein-synthesizing system from wheat germ. They found that messenger RNA for the large subunit was present only in the poly(A)' RNA fraction. Moreover, the product made by the wheat germ system was identical, on the basis of two-dimensional polyacrylamide gel electrophoresis, to the native large subunit. They also found that the poly(A)-RNA from a mutant, NBBUL, that lacks chloroplast DNA, does not direct the Coen evidence i chloroplas transcript fragment synthesis serologica to native Obtained (198) and 3Plnach (2 E‘Per HartIEy (. DCUbErste1: ‘ubunit 23,000 dal protei ha abunit direct the synthesis of the large subunit. Coen 33 21! (N2) presented the first direct physical evidence that the large subunit of RUBISCO is encoded by the chloroplast DNA. They used an in 31313 linked transcription-translation system to show that a cloned fragment of maize chloroplast DNA could program the synthesis of a polypeptide that corresponds in its size, serological properties, and papain and chymotryptic peptides to native RUBISCO from maize. Similar results have been obtained with cloned chloroplast DNA from Chlamydomonas (108) and spinach (182) and with total chloroplast DNA from spinach (26). Experiments complementary to those of Wheeler and Hartley (181) and Sagher gt 5;. (1N2) were performed by Dobberstein _e_t _a_l_. (N6). They translated poly(A)IRNA from Chlamydomonas in a cell-free system from wheat germ. Upon immunoprecipitation with antibody against authentic small subunit (16,500 daltons), a single polypeptide of ca. 20,000 daltons was obtained. Because the immunoprecipitated protein had a larger molecular weight than the small subunit, Dobberstein £2 21. (N6) tentatively identified it as a precursor (p8) to the small subunit. Treatment of pS with a specific soluble endoprotease found in polysomal supernatant fractions from Chlamydomonas, resulted in its cleavage to a polypeptide of the same size as authentic small subunit. Because only the 16,500 dalton form of the snail subuI g. («6Vy removed ii into the subunit a. processed synthesis the small Proteins, DObberSteir chain Hte PT'Oteins the presec 3! h ”to" tr pr“9831M additio“ ’ Iicrographs Chlorc’Plast he leChani analog“): 10 small subunit is found in the chloroplast, Dobberstein gt 2;. (N6) suggested that the small polypeptide that is removed is involved in the transport of the small subunit into the chloroplast. Though the synthesis of the small subunit as a higher molecular weight precursor that is processed to yield the mature protein is reminiscent of the synthesis of some secretory proteins (11,12), Dobberstein gt a_1_. (N6) noted that several features of the synthesis of the small subunit differ from the synthesis of secretory proteins. In the case of secretory proteins, Blobel and Dobberstein (11,12) had proposed that the‘amino terminal chain ‘extensicn ("signal sequence") found 1J1 presecretory proteins serves to facilitate the attachment of the ribosomes to the microsomal membrane. Whereas processing of the presecretory protein, i.e., removal of the signal sequence, is accomplished by a membrane-associated enzyme before translation of the polypeptide is complete, processing of the small subunit is post-translational. In addition, cytoplasmic ribosomes (H) not appear, in electron micrographs, to be attached to the outer envelope of the chloroplast (36,N6). Dobberstein _E El: (N6) suggested that the mechanism of transport of the small subunit may be analogous to that of diphtheria toxin. This toxin is synthesized as a single polypeptide that is subsequently cleaved to yield a and B chains that remain linked by a disulfide bridge (130). The precise mechanism of transfer of the toxin across the plasma membrane is uncertain, but it involves located 1 proposed functiona toxin. 5 sequence" chloro;1a H6) with and pea l 1]. involves prior binding of the E3 subunit to a receptor located in the plasma membrane. Dobberstein g_t_ §_l_. (N6) proposed that the small peptide that is cleaved from pS is functionally equivalent to the 8 subunit of diphtheria toxin. Schmidt gt 1?: (152) proposed the term ”transit sequence" for the leader sequences of transported chloroplast proteins. The results of Dobberstein _e_t _a_l. (N6) with Chlamydomonas have been confirmed in spinach (152) and pea (33.39.81.152), so that the mechanism proposed by Dobberstein 33 21° (N6) may be more generally applicable. In the cyanobacteria Anacystis 6301 (162), also called Synechococcus (139), and Anabaena 7120 (127), the gene (rch) for the small subunit of carboxylase is located 3' (mRNA sense) from the gene (rbcL) for the large subunit. The coding sequences of the rbcL and rch genes are separated by 93 basepairs (bp) in Anacystis (162) and by 5N5 bp in Anabaena (127). Most genes coding for the large (50,000-55,000 dalton) subunit of carboxylase consist of single open reading frames coding for N72-N77 amino acids (N5,N7,116,125,137,161,163,190). An exception is the large subunit gene of the eukaryotic alga, Euglena (93), which although it is chloroplast encoded, contains nine intervening sequences. The ngL gene is present in one copy per monomer of chloroplast DNA but in 15-30 copies per chloroplast in immature leaves of spinach (158). Because a leaf cell c the copy n exceed that subunits ar apparent di the ESL an and as yet slrlthesis transcript;- subunit 3e: 0" whether cyanC’bacter stoicyuoEH achieVed 1 gen" for ”Q8 Rene; heaology “a“ 1 ed hue: he”Micki a 12 leaf cell may contain several hundred chloroplasts (158). the copy number of the £23L gene is thought to greatly exceed that of the £238 gene. Because the large and small subunits are present in RUBISCO in equimolar amounts, the apparent discrepancy between the reiteration frequencies of the £23L and £238 genes in higher plants raises interesting, and as yet unsolved questions, about the regulation of the synthesis of RUBISCO. Whether there is a lower rate of transcription and/or translation of the copies of the large subunit gene than for the copies of the small subunit gene or whether the £238 gene is amplified is not known. In the cyanobacteria Anabaena 7120 and Anacystis, the synthesis of stoichiometric amounts of the two subunits of RUBISCO may be achieved in part by cotranscription of the closely linked genes for RUBISCO. The serial arrangement of the £23L and £238 genes together with the absence of a sequence with homology to promoter sequences in the spacer between these genes led Shinosaki and Sugiura (162) to suggest that these genes are cotranscribed in Anacygtis. Recently, Nierzwicki-Bauer _e__t_ 33. (127) have demonstrated that in Anabaena the rbcL and rch genes are cotranscribed. The genes for the small subunit of RUBISCO in soybean (8). wheat (29), and pea (32,N2) occur in the nucleus as small multigene families. One of the soybean £238 genes has been sequenced and consists of three open reading frames separated by two intervening sequences (8). The rch gene of flwat contai Anabaena (12 open reading coding seque umoded seal in the £235 Pans hora first descr- descrilDtion, ”Sambling ‘ deacribed’ t cyanobacter1 colorless pr Syncyanose . c ‘3 its 1! “Gently I 13 wheat contains a single intron (29). In Anacystis (162) and Anabaena (127), the small subunit genes consist of single open reading frames of 333 bp and 327 bp, respectively. The coding sequence of the transit peptide common to the nuclear encoded small subunit genes of plants (8,29), is not present in the rch genes of Anacystis (162) and Anabaena (127). Cyanophora paradoxa Cyanophora paradoxa is a small photosynthetic eukaryote first described in 192N by Korschikoff (95). In his description, Korschikoff’ noted small intracellular bodies resembling unicellular cyanobacteria. Pascher (131) had described, ten years earlier, the general phenomenon of a: cyanobacterium living in a symbiotic association with a colorless protist, a symbiosis to which he referred as a Syncyanose. Korschikoff (95) emphasized the cyanophycean nature of the blue-green inclusion and believed the cyanelle to be an organism 323 generis. In 1929, Pascher (132) in response to a growing number of reports on symbiotic cyanobacteria, some occurring intracellularly, distinguished between ”Ectocyanosen” and "Endocyanosen". It is Pascher who first used the term cyanelle (Cyanellen) to refer to the cyanobacteria-like bodies (132). As its name suggests, Cyanophora paradoxa has proven difficult to classify taxonomically (57,133). Until quite recently, the view was widely held that it is in fact two organisms, endosymbiot classify t One concep algae (YIN) the protist not relate dinoflagely Unusual 3p; attached t thr°U8hout nuCleus th “’9“- cool 14 organisms, a colorless biflagellated protist and an endosymbiotic cyanobacterium (70,89,17N). Attempts to classify the flagellate have been largely unsatisfactory. One conception is that it is related to the cryptomonad algae (17N). However, a careful electronmicroscopic study of the protist led Mignot 32 32. (119) to conclude that it is not related to the cryptomonads, but rather to primitive dinoflagellates. Dinoflagellates possess a distinctive and unusual spindle (98,101,168). The chromosomes appear to be attached to the nuclear envelope, which remains intact throughout mitosis. Microtubules penetrate the mitotic nucleus through membrane-lined channels but do not make direct contact with the dinoflagellate chromosomes. In an attempt to clarify the nature and taxonomic affinities of the host, Pickett-Heaps (133) studied cell division in the flagellate. He found that in contrast to dinoflagellates. Cyanophora has a typical mitotic spindle. Pickett-Heaps (133) concluded that Cyanophora is not related to the dinoflagellates. but agreed with Mignot 32 33. (119) that the basal body of the flagellum of Cyanophora was unlike that of any alga which had been examined previously. The unique structure of the flagellar roots was also noted by Kies (89) who compared the the morphology and ultrastructure of Cyanophora paradoxa, Gloeochaete wittrockiana and Glaucocystis nostochinearum, three algae containing cyanelles. Rica (89) found that all three of these algae have four flagellar roots, but that the flagellar roots of Cyanophor Glaucocys nicrotubu present licrotubu‘ approximaa out that roots of 3tructural “819 of Stanules. itaV‘Shape. and a 3)‘st Vesicles dISCQuhted Mature be. Want“, assigned 15 Cyanophora are much simpler than those of Gloeochaete and Glaucocystis. Each of the flagellar roots of Glaucocystis and Gloeochaete consists of approximately 20-50 microtubules, whereas the flagellar roots of Cyangphora are present in pairs, one pair having approximately 3 microtubules per root and the other pair having approximately 10 microtubules per root. Kies (89) pointed out that except for the unique structure of the flagellar roots of Cyanophora, the three organisms shared several structurally unique characters, including two flagella at an angle of 1200-180O to each other, cytoplasmic starch granules, a parabasal dictysome, flagella lacking the star-shaped pattern of microtubules in the transition zone, and a system of lacunae (intracellular flattened membrane vesicles located next to the plasma membrane). He discounted the importance of cyanelles as a taxonomic feature because they also occur in zoological organisms. He suggested that Gloeochaete, Glaucocystis, and Cyanophora be assigned to a separate algal class, Glaucophyceae, as proposed earlier by SkuJa (165). Over the years, a considerable body of evidence has accumulated that is consistent with the hypothesis that the cyanelle is an endosymbiotic cyanobacterium. The division of the cyanelles is not synchronous with that of the nucleus resulting in unequal numbers of cyanelles in the daughter cells (70,133,17N). Whether this is truly a sign of the cyanelle' that divi has not b1 Hall ultrastruc found tha1 of cyanoba leabrane. lamellar Pigments, Centrally laaenar N hal°- The) inclusions. a”Mlle: i {RelusiOn cyanobacter to be Ver “Wash {Cannes 16 cyanelle's autonomy is questionable because the possibility that division of the cyanelles is controlled by the nucleus has not been excluded. Hall and Claus (70) conducted a detailed ultrastructural study of the cyanelles of gtparadoxa. They found that the cyanelle lacks the double—layered cell wall of cyanobacteria, but is surrounded by a thin protoplasmic membrane. The cyanelle protoplasm is divided into a lamellar chromatoplasm containing the photosynthetic pigments, and a non-lamellar centroplasm with a large centrally located electron-opaque body. Between the lamellar region and the central body is a fibril-containing halo. They found, within the lamellar region, two types of inclusions, one of which resembles the polyphosphate granules found in cyanobacteria. The other type of inclusion resembles oil droplets, a feature not found in cyanobacteria. The binary fission of the cyanelle was found to be very similar to the division of cyanobacteria. Although Hall and Claus (70) noted that several of the features of the cyanelle, namely the absence of a cell wall, the large electron—opaque central body and the inclusions tentatively identified as oil droplets, are atypical of cyanobacteria, they considered that the similarities to the cyanobacteria were sufficient to merit classification of the cyanelle as a new cyanobacterium, Cyanocyta korschikoffiana, belonging to the order Chroococcales. Because of the structural necessary it. Becau endosymbio Presence 0 an import Status 0 ultrastruC foum: that dirQCtly fickfitt-He located in 31' (174 laterial SUSKQSted cyanelles 17 structural peculiarities of the cyanelle they found it necessary to create a new family, Cyanocytaceae, to include it. Because chloroplasts lack cell walls, whereas algal endosymbionts generally retain some wall structure, the presence or absence of a typical cyanobacterial cell wall is an important character in determining the evolutionary status of the cyanelle. Conclusions drawn from ultrastructural studies have varied. Hall and Claus (70) found that the cyanelle possessed a thin limiting membrane directly apposed to a protoplasmic membrane of the host. Pickett-Heaps (133) noted the presence of wall-like material located in the division furrow of the cyanelle. Trench 32 _2. (17N) found that cyanelles have a thin layer of material external to their cytoplasmic membrane. They suggested that this layer is homologous to the peptidoglycan of cyanobacteria. Giddings 32 32. (60) subjected isolated cyanelles to freeze fracture. They found, external and adjacent to the plasma membrane of the cyanelle, a 5— to 7-nanometer layer that seems to correspond to the peptidoglycan layer of cyanobacteria. External to this layer they observed a layer that exhibited freeze fracture faces similar to those of the lipopolysaccharide layer of gram negative bacteria. They concluded that the cyanelle has a wall that differs only slightly from that of free-living cyanobacteria. The presence of a peptidoglycan similar to biochemically peptidoglycar analysis I-acetylglucc dianinopimel: l:1:1.6:1:1, has yielded Stanier (1) Schenk (1N5; Osmotically treatMent Hi a peptide“: of the DYE biosynthe $1 haVQ the ( 8e described CthY‘oPI a 3 t cyanelle in the PYan The resembl e 3 Cuph p YCQC 18 similar to that of cyanobacteria has been demonstrated biochemically. Aitken and Stanier (1) purified and analyzed peptidoglycan from cyanelles. The results of the chemical analysis indicated that N-acetylmuramic acid, N-acetylglucosamine, alanine, glutamic acid and diaminopimelic acid are present in a molar ratio of 1:1:1.6:1:1. Analysis of peptidoglycan from cyanobacteria has yielded similar results. The results of Aitken and Stanier (1) are consistent with the earlier observations of Schenk (1N6) who showed that cyanelles, which are normally osmotically stable, can be rendered osmotically sensitive by treatment with lysozyme, suggesting that they are bounded by a peptidoglycan wall. Despite the evolutionary significance of the presence of peptidoglycan in the cyanelle, the biosynthesis of the peptidoglycan has not been studied nor have the genes for it been localized. Now that recent work (described below) has shown that the cyanelle has a chloroplast-like genome, the major distinction between the cyanelle and a chloroplast is the presence of peptidoglycan in the cyanelle. The photosynthetic pigment system of the cyanelle resembles closely that of cyanobacteria (35,17N). C-phycocyanin and allOphycocyanin, chlorophyll 3, B carotene, zeaxanthin, and two xanthophylls found in many cyanobacteria (35,62), echinenone and myxoxanthophyll, are present in the cyanelle. Chlorophyll 3, normally present in the or) Cyanophc C-phycoc cyanelle found tc masses, 4 (EDS-PAGE nIOPhYCc 12.600 d Iolecular ObSErved 3”Rested the ”etho 'aplOYed, or allOph (I74) tre: biliprotej dilethYlsL D!SI.°r°sa I333“, 3:10phycoc it With 19 the cryptomonad algae, is not present, indicating that Cyanophora is not a cryptomonad (35). The biliproteins C-phycocyanin and allophycocyanin have been purified from cyanelles by Trench and Ronzio (17N). C-phycocyanin was found to consist of two subunits with apparent molecular masses, determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), of 13,200 daltons and 1N,500 daltons. Allophycocyanin was found to consist of two subunits of 12,600 daltons. Trench and Ronzio (17N) noted that the molecular masses of the subunits are lower than have been observed for the biliproteins from cyanobacteria. They suggested that the differences result from differences in the methods used, in particular, the system of SDS-PAGE employed. In order to estimate the dimeric molecular masses of allophycocyanin and C-phycocyanin, Trench and Ronzio (17N) treated electrophoretically pure preparations of each biliprotein with the bifunctional reagent dimethylsuberimidate (DMSI), and then subjected the DMSI-crosslinked proteins to SDS-PAGE. The dimeric molecular masses, 28,000 daltons and 31,000 daltons, of allophycocyanin and C-phycocyanin determined in this manner, are within the range of values reported for the corresponding proteins from cyanobacteria (13,59,186). Recently, the cyanelle DNA has become a focus of attention with the result that a number of similarities to chloroplast DNA have been found. Prom measurements of kinetic co: the size oi pars (kb). endonucleas approximate tMs thesis cyanobacter Hze range circular m inverted or Unequal 31; (rRNA) Rene (1&17.123) “thin the h two for“ or the sing in he restric Men 1°Ca11 20 kinetic complexity, Herdman and Stanier (80) deduced that the size of the cyanelle DNA was equivalent to 177 kilobase pairs (kb). Estimates of the size derived from restriction endonuclease digestion of cyanelle DNA are smaller, approximately 127 kb (1u,16,17,123, and results presented in this thesis). Although too small for that of a free-living cyanobacterium (79), the cyanelle genome is well within the size range of chloroplast genomes (15). Cyanelle DNA is a circular molecule containing a 10-kb repeated unit in inverted orientation separated by two single copy regions of unequal size (16,17,123). The 168 and 238 ribosomal RNA (rRNA) genes are located in the inverted repeat regions (16,17,123). Perhaps due to intramolecular recombination within the inverted repeat regions, the cyanelle DNA exists in two forms that appear to differ only in the orientation of the single copy segments (16). The recognition sites of the restriction endonucleases BamHI, SalI, and SmaI have been localized in the cyanelle DNA (17). Sufficient homology exists between cyanelle DNA and chloroplast DNA to permit the use of cloned fragments of chloroplast DNA as probes in Southern (167) hybridizations. Heinhorst and Shively (77) demonstrated that DNA fragments containing the coding sequence of the large subunit of RUBISCO from maize and Chlamydomonas hybridize with cyanelle DNA. Bohnert and coworkers (17) reported that fragments of spinach DNA containing portions of the genes for the large subunit I chloroplas dicyclohex 32,000 dal and subun with cy; hybridizat stringency of RUBISCc There W has a bu L695 E/Cn 21 subunit of RUBISCO, the a , B , and 6 subunits of chloroplast ATP synthase, the dicyclohexylcarbodiimide-binding protein, the 32,000 dalton protein (2223 gene product), cytochrome b6, and subunit 11 of the cytochrome b6f complex all hybridize with cyanelle DNA under stringent conditions of hybridization. Under conditions of low hybridization stringency, the nuclear-encoded gene for the small subunit of RUBISCO from pea hybridizes with cyanelle DNA (17.77). There appear to be at least two distinct strains of annophora in common use (10,17). The DNA of one of these strains, the "Pringsheim" strain (UTEX LBSSS, from the culture collection of the University of Texas at' Austin), has a buoyant density in cesium chloride of 1.692 to 1.695 g/cm3 (10,17,80) and a molecular weight of 126.530.5 kb (17). The other strain, the "Kies" strain, has a buoyant density of 1.692 g/cm3 and a molecular weight of 138 kb (17). Preliminary experiments indicate that the cyanelle DNA of the Kies strain contains a repeated unit of the same size as the inverted repeat found in the cyanelle DNA from the Pringsheim strain (17). Jaynes gt _a_l. (87) have reported that the cyanelle DNA from the Pringsheim strain has a buoyant density of 1.716 g/cm3. The reason for the discrepancy between the value of the buoyant density of the cyanelle DNA reported by Herdman and Stanier (80) and Bohnert 33 21° (10,17) and that reported by Jaynes gt 5;. (57) 13 u examined strain bu‘ Where Czanophora little e} 9073101031 Host of th PhOtosynth. that 2“0‘293pmol Plants, D 3° (17a) clinen"; hs1370f mi 00113. Flt "trapolate cbl'l'ir). l the rate 6' fa cuUetive Esters 22 (87) is unclear. It was suggested (87.90) that the strain examined by Jaynes gt 5;. may have been not the Pringsheim strain but a third strain. Whereas the cytological and morphological properties of Cyanophora have been extensively investigated, relatively little experimentation has been directed towards the physiological and biochemical features of the organism. Host of the attention in this area has focused on aspects of photosynthesis and carbon metabolism. Schenk (1117) found that oxygen evolution by Cyanophora, 200-290pmol 02/(mg chl-hr) is comparable to that by other plants. Dark CO2 fixation is negligible (97). Trench e_t_ _l. (174) measured the rate of 1"002 fixation in isolated cyanelles; the highest rate attained, determined on the basis of mg of chlorophyll, was 12 S of the rate by intact cells. Floener and Bothe (53) measured NaHCO3 dependent oxygen evolution by isolated cyanelles. The rate obtained, extrapolated to one hour, was 30 pmoles 02 evolved/(mg chl-hr). Although this rate is equivalent to ca. 333 of the rate by intact cells, the isolated cyanelles continued to fix CO2 for only 3 min. The pattern of products labeled 1” by NaH CO3 is consistent with the operation of the reductive pentose phosphate (Calvin) cycle (97): phosphate esters, especially 3-phosphoglycerate, predominate among the initial products of CO assimilation (97). 2 The cycle, 2 purified (30). RUi Cyanophor cells by centrifu. consistir 3UDernat; assayed enZyme a )hTOUgh cyanelle. rixation fixed/(m: in the pr“Mal cyanelle In 9°13: purified 23 The enzyme catalysing the initial step in the Calvin cycle, ribulose 1,5-bisphosphate carboxylase, has been purified from Cyanophora (110) and from isolated cyanelles (30). RUBISCO appears to be located within the cyanelle of Cyanophora (N0). Codd and Stewart (00) lysed the "host" cells by osmotic shock and collected the cyanelles by centrifugation at 20,000xg for 5 min. Both the pellet, consisting of intact cyanelles and cell wall debris, and the supernatant, consisting of "host” cell cytoplasm were assayed for carboxylase activity. Before being assayed for enzyme activity, the cyanelles were disrupted by passage 2 through a French press at 16,000 lb/in . The disrupted cyanelles catalyzed ribulose bisphosphate-dependent 1"002 fixation with a specific activity of 0.55 pmol 1“(202 fixed/(mg protein-hr). No enzymatic activity was detected in the 20,000xg supernatant fluid at up to 6 mg protein/assay suggesting that RUBISCO is located in the cyanelle of Cyanophora. Codd and Stewart (00) isolated RUBISCO from Cyanophora and determined, by electrophoresis in polyacrylamide gels, that the molecular weight of the purified enzyme is 525,000 daltons. Electrophoresis of the purified enzyme on sodium dodecyl sulfate-containing polyacrylamide gels (10 1 acrylamide) showed that RUBISCO from Cyanophora consists of two kinds of subunits with molecular weights of 15,000 daltons (small subunit) and 51,000 daltons (large subunit). From these results Codd and Stewar-i eight (30) h Iolecul subunit acrylam dispari T8porte (30) ma employer {rem thr 12,400 c the PUri rePOrted reported 3P°¢if1c deteY‘Iliin actiyity Stewart The thOplas lag)“ CO v 3 the lab, getpound. 24 Stewart (00) concluded that the native enzyme is composed of eight small and eight large subunits. Burnap and Trench (30) have purified RUBISCO from isolated cyanelles. The molecular weights, 56,000 daltons and 12,200 daltons, of the subunits of RUBISCO were determined by SDS-PAGE on a 12.5 1 acrylamide gel using a stacking gel of 51 acrylamide. The disparity between the molecular weights of the subunits reported by Codd and Stewart (00) and by Burnap and Trench (30) may be due to the different polyacrylamide gel systems employed by these groups“ The» molecular weights deduced from the DNA sequences (results presented in chapter 3) are 12,000 daltons and 52,800 daltons. The specific activity of the purified enzyme, 0.08 pmoles CO fixed/(mg protein-min), 2 reported by Burnap and Trench (30) was much higher than that reported by Codd and Stewart (00). However, the value of the specific activity reported by Codd and Stewart (00) was determined on a crude cyanelle lysate. The specific activity of purified RUBISCO was not reported by Codd and Stewart (00). The transfer of fixed carbon from the cyanelle to the cytoplasm has been studied by two groups of investigators. Trench st 31. (170) labeled cyanelles in vitro with NaHWCO3 and then subjected extracts of the cyanelles and of the labeling medium to thin layer chromatography. They found that the cyanelles released principally two organic compounds, glucose and a disaccharide, into the medium. They d it was unable Glucose no evic labeled 3330013 fumarat tricarb Either In comPOUn found t aerObic ¢°mb1ne anaerOb li‘ture 3.n-d1c the aerObic conCIUd‘ nit,ogel photOSy, 25 They did not identify the disaccharide, but suggested that it was probably sucrose. Kremer and coworkers (97) were unable to label isolated cyanelles. Instead, they examined the distribution of assimilates by labeling intact cells of Cyanophora with H1u003 and then by isolating the cyanelles. Glucose and maltose were the predominant sugars. They found no evidence for sucrose among the assimilates. Host of the labeled glucose, but very little of the maltose, was associated with the cyanelles. Organic acids, e.g, malate, fumarate, citrate and other intermediates of the tricarboxylic acid cycle, were barely detectably labeled in either cells or cyanelles. Information on the assimilation and metabolism of compounds other than CO is scanty. Bothe and Floener (23) 2 found that cultures of Cyanophora grown on nitrate either aerobically or anaerobically for 3-6 days in the absence of combined nitrogen were unable to reduce acetylene. The anaerobic cultures had been flushed continuously with a mixture of either 20% H / 751 N / 51 CO or 95% N l 51 CO 2 2 2 2 2‘ 3,0-dichloropheny1-N,N-dimethylurea (DCHU) had not included in the medium. From this result and the failure of Cyanophora to grow in the absence of nitrate under either aerobic or anaerobic conditions Bothe and Floener (23) concluded that Cyanophora is "unable to synthesize nitrogenase". However, because DCHU, which inhibits photosynthetic 0 evolution, was not added to cultures grown 2 under accura sensit: uncerta grown and Flc that £1 grown HdeOge Presenc nine :1 the ab. hydrogei hidroger was Peq 'Xperim, 9033“)“ anarchici requiren the 3Yni 26 under "anaerobic" conditions, those conditions may be more accurately described as microaerobic. The extreme sensitivity of nitrogenase to 02 together with the uncertainty regarding the concentration of 02 in cultures grown microaerobically, suggest that the results of Bothe and Floener (23) should not be regarded as definitive proof that Cyanophora does not synthesize nitrogenase. Cyanophora grown anaerobically does synthesize hydrogenase (23). Hydrogen consumption by cells grown anaerobically in the presence of molecular H (201 H / 752 N2/ 51 002) was almost 2 2 nine times greater than in cultures grown anaerobically in the absence of H (951 N / 51 C0 ) suggesting that 2 2 2 hydrogenase is- inducible. Whereas the formation of hydrogenase appeared to be completely inhibited by 02, 02 was required for activity of the hydrogenase (23). The experiments of Bothe and Floener (23) do not exclude the possibility that the absence of hydrogenase activity in aerobically grown cultures of Cyanophora reflects a requirement for induction by H2 rather than an inhibition of the synthesis of hydrogenase by 02. Cyanophora is capable of growth on N03- as sole source of nitrogen implying that it is able to form assimilatory nitrate and nitrite reductases. Bothe and Floener (23) found that the rate of nitrate reduction was two fold greater in the light than in the dark. In contrast to nitrate, nitrite reduction did not occur at all in the dark 3Ynthet; found Propose: 27 (23). More recently, Floener gt 2.1: (50) and Bottcher e_t 21° (20) have shown that a NADH-dependent nitrate reductase is located exclusively in the cytoplasm. Other aspects of nitrate assimilation are controversial. Floener _t' gl. (50) reported that nitrite reductase is ferredoxin-dependent and is bound to the thylakoid membranes. Glutamine synthetase and ferredoxin-dependent glutamate synthase were found in both the cyanelles and the cytoplasm. They proposed that the pathway of assimilatory nitrate reduction in Cyanophora is similar to that of photosynthetic eukaryotes and unlike that of cyanobacteria. In contrast to the results of Floener 32 El. (50), Bottcher £3 21' (20) found that most (751) of the nitrite reductase activity was associated with the cytoplasm and suggested that the cyanelle does not play a major role in nitrate assimilation. In plants, SO“: incorporation occurs in the chloroplast (157). The more than 50 species of higher plants and the eukaryotic algae, other than Cyanophora, that have been examined, have only adenosine-S'-phosphosulfate (APS) sulfotransferases, whereas both APS and 3'-phosphoadenosine-5'-phosphosulfate (PAPS) sulfotransferases are found in cyanobacteria (109,150,157.177). Schmidt and Christen (151) have partially purified a PAPS sulfotransferase from cyanelles. The enzyme is inhibited by 5'-ANP, 5'-ADP, and especially by APS. Although i 1.6-fold b; activity sulfotrans: is the or therefore assimilator W This into the 3 for the 1’B‘bis‘Phos‘. initial app in the “We (chlm'amphe 8ertilt-“Ms. 28 Although the activity of this enzyme is stimulated ca. 1.6-fold by thioredoxin, thioredoxin is not required for its activity (151). It is not certain that the cyanelle sulfotransferase characterized by Schmidt and Christen (151) is the only sulfotransferase in Cyanophora and it is therefore not certain what role the cyanelle may play in assimilatory sulfate reduction. Prospectus This thesis contains the results of an investigation into the sites of synthesis and the locations of the genes for the large and small subunits of ribulose 1,5-bisphosphate carboxylase. Chapter 2 describes the initial approach, the labeling of cyanelle proteins in 1112 in the absence or in the presence of inhibitors of cyanelle (chloramphenicol) or cytoplasmic (cycloheximide) protein synthesis. The preliminary results suggested that the large subunit of RUBISCO, and several other proteins, may be synthesized in the cyanelle. The results also suggest that many cyanelle proteins are synthesized in the cytoplasm. Although it had been known since 1977 (80) that the cyanelle genome was substantially smaller than that of free-living cyanobacteria, it was not known what genes are encoded in the cyanelle DNA. A reasonable approach to the study of the evolutionary status of the cyanelle is to elucidate the structure of its genome and to identify the genes th in the chloropl The reSL Appendix of 00815 near eac Telative Striking chloropl (28,55,9 hybridiz °°ntain1 the are deter-min hybridi; heated large Becau39 {rame “bun 1 t identif: 29 genes that it contains. I began to look for specific genes in the cyanelle DNA by using cloned genes from maize chloroplast DNA as probes in heterologous hybridizations. The results of those hybridization experiments, shown in Appendix A, suggested that the 5' ends of the large subunit of RUBISCO and the 6 subunit of the ATP synthase are located near each other in the cyanelle DNA. If true, then the relative locations of these genes in the cyanelle DNA is strikingly similar to their relative locations in the chloroplast DNAs of several plant species (28,55,96,‘l60,180). In order to verify the results of the hybridization experiments, fragments of cyanelle DNA containing the large subunit of RUBISCO and the 8 subunit of the ATP synthase were cloned, and their DNA sequences determined. The results of the DNA sequencing confirmed the hybridization results and also showed that a sequence located 108 basepairs 3' from the coding sequence of the large subunit is an open reading frame of 321 basepairs. Because the deduced amino acid sequence of this open reading frame closely matched the amino acid sequence of the small subunit from Anacystis (162), that open reading frame was identified as the structural gene for small subunit of RUBISCO. Chapter 3 contains the results of the DNA sequencing of the genes for the large and small subunits of RUBISCO and of their flanking regions. In of the restric were lo experiml cyanellr eukaryo‘ 30 In order to gain insight into the overall organization of the cyanelle DNA, the recognition sites of the restriction endonucleases BamHI, SalI, XhoI, BglII and PstI were localized in the cyanelle DNA. The results of those experiments are presented in chapter 0 and show that the cyanelle genome is similar to the genomes of plants and eukaryotic green algae. CHAPTER II The 33 havil Progress cell. C. collabor; the PPEV: CHAPTER II LABELING OF CYANELLE PROTEINS I! VIVO Introduction The endosymbiont hypothesis (110) views modern plastids as having developed from an ancestral endosymbiont by a progressive integration of metabolic function with the host cell. Chloroplast proteins, for example, are biosynthesized collaboratively by the nuclear and chloroplast genomes. In the previous section it was suggested that the biosynthesis of one particular protein, RUBISCO, is a good paradigm for the interactions of the chloroplast and nuclear genomes. The cyanelle of Cyanophora has the photosynthetic pigments (35,175), structural appearance (60,70,170), and peptidoglycan of the cyanobacteria (1). However, the cyanelle genome is approximately 10- to 20—fold smaller (16,17.80) than the genome of free-living cyanobacteria (78). In both size (16,17,80) and the presence of an inverted repeat segment (16,17) the cyanelle DNA resembles that of the chloroplast (10). Whereas the presence of peptidoglycan suggests that the cyanelle is an endosymbiont, the structure of its genome suggests that it is a 31 chloropla: bridge be‘ Beca protein 3 in partic chloropla CVolutIOn studying that some of RUBISC evidence On the o ProteinsI substrat, “Served as an en‘ Iiltl Drev10u3: °‘a“1ned cyclohex: ribos°mai clanellez sluthQSIE Chlorampp synthesi, \u 32 chloroplast. Aitken and Stanier (1) proposed that it is a bridge between the cyanobacteria and chloroplasts. Because in higher plants and eukaryotic green algae protein synthesis in general, and the synthesis of RUBISCO in particular, illustrates well the cooperation between the chloroplast and nuclear genomes, I thought that the evolutionary position of the cyanelle might be clarified by studying the synthesis of its proteins. A demonstration that some cyanelle proteins, for example the small subunit of RUBISCO, are synthesized in the cytoplasm would be strong evidence that the cyanelle is, in essence, a chloroplast. 0n the other hand, if the cyanelle synthesizes all of its proteins, and depends upon the host only for an array of substrates (an array sufficiently large as to allow for the observed reduction in genome size), it should be thought of as an endosymbiont. Although protein synthesis has not been studied previously in Cyanophora, Trench and Siebens (176) have examined the effect of the protein synthesis inhibitors, cycloheximide and chlormphenicol, on the synthesis of ribosomal RNA (rRNA) and chlorophyll a in Cyanophora and its cyanelles. They found that cycloheximide inhibited the synthesis of cytoplasmic but not cyanelle rRNA, whereas chloramphenicol inhibited cyanelle but not cytoplasmic rRNA synthesis. Cycloheximide markedly inhibited the synthesis 33 of chlorophyll 2 whereas chloramphenicol was significantly less inhibitory. Trench _e_t gl. (170) examined the uptake of dissolved organic carbon compounds. They incubated intact cells in the light in growth medium containing radioactively labeled compounds. They found that acetate and cyanocobalamin were taken up, whereas glucose, sucrose, ribose, an amino acid mixture, and (in contrast to results described below) leucine were not taken up. Three major approaches have been used to determine the sites of synthesis of chloroplast proteins, vi in vitro, z., __ situ (i.e., in isolated chloroplasts) and in vivo. For H- H- 3 vitro determinations, there are in turn, two sub-approaches, namely, ribosome run-off and use of heterologous protein-synthesizing systems. As will be described below, each of these approaches has certain advantages and certain disadvantages. In a ribosome "run off" system polysomes are isolated and peptide chains already initiated are allowed to elongate and terminate. Alternatively, heterologous protein synthesizing systems can be utilized to synthesize chloroplast proteins 3.2112122 by translation of isolated messenger RNA. Cell-free i vitro translation systems prepared from §.coli and from rabbit reticulocytes have been used successfully to translate chloroplast mRNAs. Extacts of whe mRNAs. protein protein such ; CYtOpla Protein and of a nethc 'run of ”Miles (55) a: encount antibod lention. from th larze PhenOme summit Heterol 3inthe3 c911-fr chlorop 34 of wheat germ have been used to translate cytoplasmic mRNAs. Polysomal "run off" and heterologous systems for protein synthesis in v_it_r_o allow the study of chloroplast proteins synthesized in the cytoplasm and, separately, of such proteins synthesized in the chloroplast. Because cytoplasmic mRNA and polysomes synthesize a great number of proteins, a prerequisite for study of polysomal "run off" and of heterologous systems using total cytoplasmic mRNA is a method of selecting the product of interest. Polysomal "run off" has been used successfully to identify the site of synthesis of the small subunit of RUBISCO in French been (65) and wheat (63,101). One difficulty that has been encountered is the apparent lack of specificity of the antibodies used to select the polysomes. As already mentioned (page 8), Gooding 33 21° (63) found that polysomes from the cytoplasm reacted with antisera against both the large and the small subunit and suggested that the phenomenon was due to the complexing of completed large subunits with the nascent chains of the small subunit. Heterologous systems differ from polysomal "run off" synthesis in that the synthesis occurs in partly purified cell-free systems from heterologous sources. Some of the chloroplast proteins which have been synthesized using heterologous systems for in 11313 protein synthesis are the large subunit of RUBISCO from total spinach chloroplast RNA (72), and from non-polyadenylated RNA from the chloroplast of spinach (181), Euglena (102), and Chlamydomonas synthe chloro chloro (Virtu sinthe disadv '°ripp includ into eViden QRVelo p0lYPe this n pr°te1 chloro the Dr their "°“eth. and chloro‘ 35 (108,103): the small subunit of RUBISCO from polyadenylated RNA of spinach (33), Chlamydomonas (06), and pea (81); and the psbA gene product from chloroplast RNA from spinach (72) and maize (6). The second way to demonstrate that a protein is synthesized in the chloroplast is to label intact, isolated chloroplasts. This approach has the advantage that chloroplast protein synthesis can be examined in the (virtually) complete absence of cytoplasmic protein synthesis. An in 21.12 approach has a number of disadvantages. Chloroplast protein synthesis. may be "crippled" in the absence of the cytoplasm, and it may include synthesis from nuclear encoded mRNAs which may move into the chloroplast [although there is, in fact, no evidence for the movement of mRNA across the chloroplast envelope (51)]. Another disadvantage of an in vitro approach is that it does not provide positive information about polypeptides synthesized in the cytoplasm. In addition, this method appears to be effective primarily for those proteins which either occur in great abundance in the chloroplast, have high rates of turnover, or do not require the presence of (cytoplasmically synthesized proteins for their assembly, for example into membranes (38). Nonetheless, this approach has been widely used since Blair and Ellis (10) demonstrated that isolated intact chloroplasts of pea synthesize the large, but not the small, subuni spinac Some 0 of pro 7. a P-700- c an: 32,00E Spinac (178). dEterl t0 la On th 3Ynth, 1 . 4 Q treat! experj sites the chlofa EPeeni inhib 36 subunit of RUBISCO. Similar results have been obtained in spinach (27,121), Hordeum vulgare (2), and Euglena (178). Some of the proteins which have been identified as products of protein synthesis in isolated chloroplasts are the a, 8, y, and e. subunits of ATP synthase (126) and the P-700-chlorophyll g-binding proteins of spinach (188), the a and 8 subunits of ATP synthase of maize (68), and the 32,000 dalton membrane protein (3228 gene product) of spinach (27), maize (precursor) (68), pea (08), and Euglena (178). The third major approach that has been used to determine the sites of synthesis of chloroplast proteins is to label chloroplast proteins in livg. This method relies on the (differential sensitivity' of the ribosomes of the cytoplasm and the chloroplast to inhibitors of protein synthesis. An in viva approach has the advantage over the in vitro approaches that duplicate samples of cells may be treated with different inhibitors during the same experiment. Information can thereby be gained regarding the sites of synthesis of proteins both in the cytoplasm and in the chloroplast. A disadvantage of this approach is uncertainty regarding the specificity of the inhibitors used. Hoober and Blobel (86) examined the effect of chloramphenicol and cycloheximide on the ribosomes of greening cells of Chlamydomonas. In their experiments the "inhibitory" effect of the antibiotics was measured as the disap appea cyclo not resul‘ and E cytOpj three Protei cytopl only 3 chloro but on inhibi 37 disappearance of free ribosomes and the concurrent appearance of polysomes. Hoober and Blobel (86) found that cycloheximide severely decreased the amount of free 808 but not 708 ribosomes, whereas chloramphenicol markedly decreased the number of free 708 but not 808 ribosomes. The results of Hoober and Blobel (86) were confirmed by Avadhani and Buetow (0). They isolated intact polysomes from the cytoplasm, chloroplasts, and mitochondria of Euglena. All three preparations incorporated labeled amino acids into protein. _The incorporation of amino acids into protein by cytoplasmic ribosomes was inhibited 781 by cycloheximide but only 9% by chloramphenicol. Incorporation of amino acids by chloroplast ribosomes was inhibited 52% by chloramphenicol, but only 71 by cycloheximide. Mitochondrial ribosomes were inhibited 531 by chloramphenicol and 10! by cycloheximide. Results from labeling intact leaves of barley (00) in 1112 provided the first evidence suggesting that both cytoplasmic and chloroplast ribosomes were involved in the synthesis of RUBISCO. Criddle 32 El' (00) immunoprecipitated RUBISCO labeled in 13:19. in the absence and in the presence of cycloheximide and chloramphenicol. They found that cycloheximide preferentially inhibited the synthesis of the small subunit, whereas chloramphenicol specifically inhibited the synthesis of the large subunit. Cashmore (31) labeled green, i.e., not etiolated, pea [35 seedlings with SJmethionine and then autoradiographed the polyp sulfa found signi nost subun that synth prote Photo the 38 polypeptides following their separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PACE). He found that total protein synthesis was decreased significantly by cycloheximide and that the decrease was most pronounced for soluble leaf proteins. The large subunit of RUBISCO was unique among the soluble proteins in that its synthesis was inhibited by chloramphenicol. The synthesis of the small subunit, as well as the other soluble proteins, and the major lamellar protein associated with photosystem II were affected only by cycloheximide. Many of the lamellar proteins were sensitive to chloramphenicol suggesting that they are synthesized on chloroplast ribosomes. Chua and Gillham (38) labeled intact cells of Chlamydomonas in the absence and in the presence of inhibitors specific for chloroplast (chloramphenicol, spectinomycin) or cytoplasmic (anisomycin) protein synthesis. The labeled membrane polypeptides were fractionated by SDS-PAGE. Of the 33 polypeptides resolved, at least 9 were made on chloroplast ribosomes, that is, their synthesis was inhibited by chloramphenicol or spectinomycin, but not . by anisomycin. Two of the polypeptides synthesized on chloroplast ribosomes are associated with the reaction centers of photosystems I and II. The sites of protein synthesis in 1112 of a large number of chloroplast proteins, in addition to RUBISCO and thylakoid membrane proteins, have been studied using inhibitors. These proteins include RNA polymerase, ferre fatty ref. syntht D-thrt strepi systen cYtosc and chlora reSpec thOSe Proces 39 ferredoxin, several of the enzymes of the Calvin cycle, and fatty acid synthetase. A more complete list is presented in ref. 25. The inhibitors most frequently used to study the synthesis of chloroplast proteins in 1112 are D-threo-chloramphenicol, lincomycin, spectinomycin, or streptomycin to inhibit the chloroplast protein-synthesizing system and cycloheximide to block protein synthesis in the cytosol. The results of Hoober and Blobel (86) and Avadhani and Buetow' (0), described above, suggest that chloramphenicol and cycloheximide are quite specific with respect to the class of ribosome affected in 11252, however, those results do not exclude the possibility that cellular processes other than protein synthesis are raffected. In fact cycloheximide and chloramphenicol do inhibit, both in 2.1.1.9. and _i_n 2.1.252. processes other than protein synthesis (50,58). Chloramphenicol has been found to inhibit respiration, sulfate uptake, oxidative phosphorylation (13 vitro), and mitochondrial electron transport (1 vitro): cycloheximide inhibits asparagine synthesis, malate oxidation by mitochondria (in v_i_t_r_9_), and chloride uptake (50). Despite these shortcomings, chloramphenicol and cycloheximide have been used frequently and successfully to study the synthesis of chloroplast proteins and were chosen for use in this study. the s intact of in! cyanel polype. made 0: 0f inhj 0% c"lture ‘xehic below) fla3k3’ SYrc illuMina (Lab°rat apprOxim‘ Has he“ 5”” M. Because information regarding proteins synthesized both in an organelle and in the cytoplasm can be gained from an in vivo approach, I chose such an approach for the study of the sites of synthesis of cyanelle proteins. I labeled intact cells of Cyanophora in the absence or in the presence of inhibitors of protein synthesis and then isolated the cyanelles from those labeled cells. The labeled polypeptides were separated by SDS-PAGE. Fluorographs were made of the stained gels to establish the resulting patterns of inhibition. Materials and Methods Organism and growth Cyanophora paradoxa UTEX LB 555 was obtained from the Culture Collection, University of Texas, Austin, Texas. Axenic cultures derived from this bacterized culture (see below) were grown in 250-ml cotton stoppered Erlenmeyer flasks, under continuous fluorescent illumination at 26°C on a gyrorotatory shaker at ca. 110 rpm. Incident illumination, measured with a Kettering Radiometer Model 68 (Laboratory Data Control, Riviera Beach, FL) was approximately 5000 ergs/cmzlsec. The medium, designated CYB, was medium CYII (170) with the following modifications. 5 mM N,N-bis(2-hydroxyethyl)glycine (Bicine) was substituted for tr: of NaN( rather Appendi Purific In contami Bold's E3N, a 0? con Cells Clinic; 53" to dark f. FntEr. Sterile added a cells 10“ 41 for tris(hydroxymethyl)aminomethane (Tris). Three mM, each, of NaNO3 and KNO3 were added. The pH was adjusted to 7.8 rather than to 7.6. The composition of GTE is shown in Appendix B. Purification of Cyanophora In order to limit the growth of foreign organisms, contaminated cultures of Cyanophora were maintained on Bold's 3N Bristols medium (169, Appendix B), referred to as B3N, a medium with a low content of fixed carbon. Removal of contaminating organisms was accomplished as follows. Cells were sedimented by centrifugation at 220xg in a clinical centrifuge. The pelleted cells were resuspended in 5 B3N to a density of ca. 8x10 cells/ml and incubated in the dark for 05 minutes to reduce the growth of Cyanophora. Filter-sterilized fructose, final concentration 5 mM, and sterile ampicillin, final concentration 100 pg/ml, were added and the cells returned to the dark. After 0 hours the cells were diluted 10-fold, 100-fold, 1000-fold, and 10u-fold. One ml of each dilution was added to 1 ml of 0.5S agarose and roverlayed onto B3N containing 11 agarose in 60x15mm Petri dishes. To prevent killing the cells, the 0.5% agarose was cooled to near the gelling point. The edges of the Petri dishes were sealed with strips of Parafilm (American Can 00., Greenwich, CT.) and the Petri dishes were kept at room temperature (23-2000) under fluore appear transf Erlenm cultur follow were i nainta and r7 chloro] acids 1 with ac to be : is CYB CYB.3 1 suspen: fell0H1 Sine (792 at (302 n: radioae The Ce} Illumin 42 fluorescent lamps. After 6 days, small groups of cells that appeared to be free of contaminating organisms were transferred, using a sterile Pasteur pipette, to SO-ml Erlenmeyer flasks containing 10 ml of B3N. The resulting cultures were checked for purity by microscopic examination following growth on a variety of media. No contaminants were found. Cultures were susequently transferred to and maintained on CYB. Conditions for labelingCyanophora Axenic cultures of Cyanophora were harvested at 077xg and resuspended to a density corresponding to 10-25 pg chlorophyll g/ml. Cells that were to be labeled with amino acids were resuspended in CYB, cells that were to be labeled with acetate were resuspended in CYB-Ac, and cells that were to be labeled with sulfate were resuspended in CYB-S. CYB-Ac is CYB modified by substituting (NH for NH acetate; u’zsou u CYB-S is CYB modified by substituting Cl' for son‘. The cell suspension was added to flasks containing one of the following compounds dried on the bottom of the flask: [BSSJmethionine (>500 mCi/mmole), [asslcysteine (792 mCi/mmole). Na23580n (1028 mCi/mmole>o [1" (302 mCi/mmole) or [IHCJacetate (57 mCi/mmole). 1-2 p01 of C]leucine radioactivity were added for each ml of cell suspension. The cells were incubated at 26°C on a gyrorotatory shaker. Illumination was provided by cool white fluorescent bulbs (Gene of al netho for d a tin susper paper 43 (General Electric). Dark controls were wrapped in 2 layers of aluminum foil. Incubations were terminated by one of two methods. The first method was used when taking samples only for determination of radioactivity, as, for example, during a time course experiment. Duplicate samples of the cell suspension were spotted onto 2.3-mm circles of Hhatman 3MM paper which were placed immediately into a 0°C solution of 105 trichloracetic acid (TCA) containing 100 mM of the stable form of the substrate (113., acetate, leucine, methionine, cysteine, or sulfate) used to label the cells. The filters were processed as described below. When further manipulations of the labeled cells were required (1£§., when cyanelles were to be isolated for analysis of their proteins) the cells were collected at 077xg and resuspended in 0°C CYB containing 100 mM of the stable form of the substrate used to label the cells. The centrifugation and resuspension were repeated once. Isolation of cyanelles from radioactively labeled Cyanophora After the initial wash described above, cells were sedimented by centrifugation in 1.5-ml Eppendorf microcentrifuge tubes at 077xg for 3 min. The pellets of cells were resuspended in CYB containing 0.5 M sucrose and kept at room temperature for 10 min. The cells were then sedimented by centrifugation at 1700xg for 5 min. The cells, but not the cyanelles, were lysed by resuspending them in CYB tempe sedim twice centr SECOHI PerfOI m I 0f rac either 3POtte transf of the lethio F°llow filter abon' and th 3°1Ut1. and th 30min. re3u3p‘ 37°C f, "ith e1 44 CYB containing 0.1 M sucrose. After 5 min at room temperature cyanelles, that had been released were sedimented at 880xg for 5 min. The cyanelles were washed twice by centrifugation and resuspension in CYB. The first centrifugation was performed at 077xg for 5 min and, the second at 288xg for 5 min. All centrifugations were performed in a clinical centrifuge. Determination of incorporation of radioactively labeled compounds into protein by Cyanophora. The following process was used to determine the amount of radioactivity present ix: protein, duplicate samples of either intact cells cn' isolated cyanelles. Samples were spotted onto 2.3-cm ‘Nhatman 3MM filters which were then transferred to a 0°C solution of 10$ TCA containing 100 mM of the stable form of the substrate (115., acetate, leucine, methionine, cysteine, or sulfate) used to label the cells. Following the procedure of Mans and Novelli (112), the filters were kept in the solution of 101 TCA described above, for at least 1 hr at 0°. The solution was decanted and the filters were resuspended in 5% TCA for 15 min. The solution was decanted and the filters were washed once with and then resuspended in 5% TCA, and incubated at 90°C for 30min. The filters were then washed once with 5! TCA, resuspended in ether/ethanol (1:1, v/v) and incubated at 37°C for 30 to 60 min. The filters were then washed 2 times with ether and dried in air. Radioactivity was measured in 99011 each the l 45 5 ml of ACS (Aqueous Counting Scintillant, Amersham Corporation) with a model LSTOOC Liquid Scintillation Counter (Beckman Instruments). SDS polyacrylamide gel electrophoresis of radioactively labeled Cyanophora Radioactively labeled intact cells, or cyanelles isolated from them, were first extracted with 1001 methanol and then extracted three times with chloroform-methanol (2:1, v/v). After extraction the pellets were dried in air, suspended in the SDS lysis buffer of Laemmli (99), and boiled for 5 min. Immediately thereafter, electrophoresis was performed in SDS-containing polyacrylamide gels composed of a 51 acrylamide stacking gel and a separating gel of 10%, 12.5% or 15! acrylamide (99). Equal amounts of cyanelles, equivalent to 1.3-1.6 pg of chlorophyll _a, were loaded in each lane. Gels were prepared for fluorography according to the procedure of Bonner and Laskey (22) modified by adding 55 (v/v) glycerol to the final wash to prevent cracking of the gels during drying. Pluorographs were prepared using KODAK xn-s film exposed at -uo°c. Determination of protein and chlorophyll a. Protein was determined according to the method of Lowry t al. (106). The amount of chlorophyll a was estimated by measuring the absorbance at 665 nm of a methanol extract of 2129.2 absorp of ch Absorb Spectr Charac 0f the abilit compou inhibi clanel ribOSo Previo “001d Theref [“‘C and 3S and or 001; be 45 Cyanophora or of cyanelles. The value, 70.5, of the absorption coefficient used to calculate the concentration of chlorophyll a was obtained from Mackinney (107). Absorbance was measured with a DB-G grating spectrophotometer (Beckman Instruments). Results Characteristics of the incorporation of labeled substrates into protein by intact cells of Cyanophora paradoxa Two prerequisites of an i__n_ 3313 approach to the study of the sites of synthesis of cyanelle proteins are the ability to label cyanelle proteins with exogenously supplied compounds and the ability to differentiate, by using inhibitors of protein synthesis, between proteins made on cyanelle ribosomes and proteins made on cytoplasmic ribosomes. Because protein synthesis had not been studied previously in Cyanophora, it was not known which substances would most effectively label proteins in that organism. Therefore, several different substances were tried: 10C 10 [ Jacetate, [3581methionine and cysteine, [ C]leucine, and 35song. Moreover, because the effects of cycloheximide and chloramphenicol on protein synthesis in Cyanophora had not been determined, I examined the effects of those inhibiti protein Th! conpoun< by EZEIU cyanelle Moreover Protein: chloram; Chloran; deteg‘tat Int 10 N32[ C] 700 PM, in Figur inc°rDOr °°n°entr rate ar D’Otein. 1“Co’DOr lithe. 47 inhibitors. Concentrations that might specifically inhibit protein synthesis had to be determined. The results described below show that all of the compounds tested are taken up and incorporated into protein by Cyanophora and that protein synthesis by intact cells and cyanelles is affected by cycloheximide and chloramphenicol. Moreover,the synthesis of the cycloheximide-sensitive proteins of the cyanelle is not detectably inhibited by chloramphenicol. Conversely, the synthesis of the chloramphenicol-sensitive proteins of the cyanelle is not detectably inhibited by cycloheximide. i. Incorporation of NaZL;£C]acetate Intact cells of Cyanophora were labeled with 10 Na2[ 700 pM, or 1mM. The time course for this experiment is shown Clacetate at a concentration of 35 pM, 175 pM, 038 pM, in Figure 1. The results show that Cyanophora is capable of 10 Clacetate into protein. Increasing the 10 incorporating [ concentration of [ CJacetate resulted in an increase in the rate and magnitude of .incorporation of acetate into protein. For all concentrations of acetate used the rate of incorporation increased with time. Figure 2 shows that the incorporation of acetate was lower in the dark than in the light. 1 50.9.0 >L304 a: \ 0022005005 EQO 48 450-- C 3.". 1000 2 a. a 300-- a O -' 700 O: a \ db '0 2 2 g 438 8 150-- .5 e a. O 175 l l 1 35 I I I I o 15 so 45 time (min) Figure 1. Time coursfif of the incorporation of various concentrations (9M) of [ CJacetate into protein by intact cells of Cyanophora paradoxa. 49 x10 cpm Incorporated I up chl O 1 2 3 4 time (hr) 10 Figure 2. Time course of the incorporation of I CJacetate into protein by intact cells of Cyanophora paradoxa incubated in the light (0—0) and in the dark (H). ii.:flyi chlr Th chloran incorpo presenc cells 1 sample the in! for a cyelom approx: increa‘ 300 pg concen chlora eXPEri 50 ii. The effect of D-cycloheximide and D-threo- chloramphenicol on the incorporation of acetate. The appropriate concentrations of cycloheximide and chloramphenicol were determined by measuring the incorporation of acetate into protein by intact cells in the presence of various concentrations of these inhibitors. The cells were allowed to incorporate acetate for 15 min, a sample for the determination of radioactivity was taken, and the inhibitors were added. The incorporation was followed for a further 60 minutes. All concentrations of cycloheximide used (15-50 ug/ml) inhibited incorporation approximately 651 (Figure 3). Inhibition by chloramphenicol increased with increasing concentration, reaching 05! at 300 pg/ml (Figure 3). On the basis of these results, concentrations of 25 pg cycloheximide/ml and 300 pg chloramphenicol/ml were chosen for use in subsequent experiments. iii. Incorporation of amino acids by Cyanophora In contrast to the findings of Trench 33 21° (170), I found that Cyanophora does take up and incorporate amino acids into protein. Time courses for the incorporation of L-[BSSJmethionine, L-[BSSchsteine, and lu¢1uCJleucine are shown in figures 0, 5, and 6. In contrast to the rates of incorporation of acetate (see above) and sulfate (see below), the rates of incorporation of amino acids did not 51 Figure 3. The effect of various concentrations of D-cycloheximide (“wand D-threo-chloramphenicol (B) on the incorporation of [ CJacetate into protein by intact cells of Cyanophora paradoxa. Chloramphenicol (cm) or cycloheximide was added at 15 min. The concentrations shown are pg/ml. -3 cpm Incorporated I up chl 52 control control 50 100 200 300 cm / t / / l l l l I I I I 1 5 30 45 60 0') '0 4-1- F X C E O Ii:- 0) O 3 \ '0 a 3 O E O . .‘5’ «lb . S 0 l l J l T I I I O 1 2 3 4 time (hr) 35 Figure 0. Time course of the incorporation of [ SJmethionine into protein by intact cells of Cyanophora paradoxa incubated in the light (O—-O) or in the dark (H). 30-- -3 x10 a\- 10% cpm incorporated I up chi .0- «Im- 41 I 2 time (hr) 35 Figure 5. Time course of the incorporation of [ Schsteine into protein by intact cells of Cyanophora Eeradoxa incubated in the light (O——O) or in the dark (H). 55 10 Figure 6. Time course of the incorporation of [ C]leucine into protein by intact cells and cyanelles of Cyanophora paradoxa upon incubation of intact cells in the absence or in the presence of D-cycloheximide or D-threo-chloramphenicol. At the times shown, aliquots of cells were taken, the cyanelles were isolated, and the radioactivity in protein both in the intact cells and the isolated cyanelles was determined. Intact cells were incubated in the light withoutinhibitor (H), or plus 25 pg cycloheximide/ml (H), or plus 300 pg chloramphenicol/ml (H). (O-—O) Cyanelles isolated from cells incubated in the light without inhibitor or (D—U) plus 25 pg cycloheximide/ml or (H) plus 300 pg chloramphenicol/ml. Each time point is the average of two samples. 56 300-"- 200 " Eu 9. x 33593:. Ego . . o o 1 0 30 45 60 75 90 time (min) 15 inc 57 increase with time. Cells labeled with cysteine (Figure 5) showed an anomalous result: very high levels of apparent incorporation, 9,000-10,000 cpm/pg ch13 at the zero-time point. The initial rate of incorporation of cysteine, although relatively high (approximately 170cpm/ pg chlg-min), was not high enough to account for the label accumulated during the few minutes required to take the zero-time sample. Unlike cells labeled with other compounds, methionine-labeled cells showed a relatively high rate of dark incorporation. 35% of the rate in the light. The results described above show that Cyanophora can incorporate exogenous organic substances into protein and that their incorporation into protein is reduced by cycloheximide and chloramphenicol. In order to determine whether cyanelle proteins are labeled and their synthesis affected by the inhibitors, a time course experiment was performed in which intact cells were labeled with [10 C]leucine, in the absence of inhibitors or in the presence of 25 pg cycloheximide/ml or 300 pg chloramphenicol/ml. Cyanelles were isolated at 15 minute intervals to 1 hour, and at 1.5 hours, and the amount of radioactivity incorporated into protein determined (Figure 6). The rate and magnitude of incorporation observed for each of the treatments are presented in Table1 and Table 2, respectively. In this experiment, unlike that of In1 Cy; Ini Cy; Ini Cy; [1“ Cyanophora paradoxa. 58 Table 1. Rates and relative rates of incorporation of C]leucine into protein by intact cells and cyanelles of Rate cpm/(pg chl-hr) Relative rate Relatige rate 1 I Intact Cells 192 Cyanelles 80 Intact Cells + Cm 196 Cyanelles + Cm 60 Intact Cells + Cx 108 Cyanelles + Cx 86 100 02 100 102 31 75 56 05 108 1 Rate relative to intact cells incubated in the absence of inhibitor. 2 Rate relative to cyanelles isolated from cells incubated in the absence of inhibitor. In1 Cy; Int CI: in! CT: 59 Table 2. The amount of radioactivity present in protein of cells and cyanelles pfter labeling intact cells of Cyanophora paradoxa with [ CJ—leucine for 90 minutes. cpm/pg-chla 1 incorporation 1 incorporation 1 2 Intact cells 289 100 Cyanelles 136 07 100 Intact cells + Cm 290 100 Cyanelles +Cm 95 33 69 Intact cells +0 167 58 Cyanelles +Cx 120 02 88 1 Incorporation relative to intact cells incubated in the absence of inhibitor. . 2 Incorporation relative to cyanelles isolated from intact cells incubated in the absence of inhibitor. Figl fol int chl fro tre ani sig eye: the iv. NO! to labe 0f 1! 6O 1“C]acetate was 10 Figure 3, in which incorporation of [ followed, the rate of incorporation of [ C]leucine by intact cells was not significantly reduced by 300 pg chloramphenicol/ml. However, compared to cyanelles isolated from control cells, the cyanelles isolated from cells treated with chloramphenicol showed a 25% reduction in the rate of incorporation. Cycloheximide decreased the rate of amino acid incorporation by whole cells 061 but had no significant effect on the rate of incorporation by cyanelles. The cyanelles accounted for approximately 001 of the total incorporation by intact cells. iv. Incorporation of Na2§2§gu by Cyanophora The time course of incorporation of sulfate into protein by intact cells of Cyanophora (Figure 7) is similar to that of acetate (compare Figure 2). However, in contrast to the incorporation of acetate, which was not saturated at 1mM,' the incorporation of sulfate was saturated at the lowest concentration tested, ca. 50 pH. The steady state rate of incorporation of sulfate (50 pH) was ca. 0.1 nmoles/(pg chl-hr). The incorporation of sulfate into protein was sensitive to inhibitors of protein synthesis. Intact cells were 35 2 so” in the dark in the absence, or in the light in the presence, labeled with 50 pM Na for 00 minutes in the light or of inhibitor. At the end of the labeling period, the cells 97 SE 61 150$- x10 cpm incorporated I up chi l l l j I l I O 20 4O 60 time (min) Figure 7. Timeaqjourses of the incorporation of 09 pH, 97 pM, and 389 pM SO“: into protein by intact cells of Cyanophora paradoxa. fr Cy ra an in 3P by in 38 de by re CY CY in is 80 ii of 30: 62 from each treatment were divided into two aliquots. Cyanelles were isolated from one aliquot and the radioactivity present in protein both in the intact cells and in the isolated cyanelles was determined (Table 3). Whereas cyanelles accounted for ca. 001 of the total incorporation of [1uCJleucine, they accounted for approximately 152 of the incorporation 3580"= into protein by intact cells. Chloramphenicol inhibited the incorporation of 35$ from 35$0 = into total cell protein by u 381 and into cyanelle protein by ca. 203. Cycloheximide decreased the incorporation of 35$ from 35SO“= into protein by intact cells 83%, a reduction nearly equal to reduction resulting from incubation of the cells in the dark. Hhereas cycloheximide reduced the incorporation of leucine into cyanelle protein by only 12$ (Table 2), it decreased the incorporation of sulfate into cyanelle protein by 59%. v. SDSggAGE of proteins from cyanelles labeled with gazilggu in vivo To identify which cyanelle polypeptides are synthesized in the cyanelle and which in the cytoplasm, the protein from isolated labeled cyanelles was electrophoresed on SDS-containing polyacrylamide gels. In Figure 8 the fluorograms resulting from cyanelles labeled in the absence of inhibitor or in the presence of 25 pg cycloheximide/ml, 300 pg chloramphenicol/ml or 25 pg cycloheximide/ml plus 300 63 Table 3. Radioactivity present in protein of intact §§11s_ and cyanelles of Cyanophora paradoxa labeled with $0 ’ u Treatment Intact Cells $1 Cyanelles 12 (cpm) (cpm) light (It) 58.575 100 9,025 100 dark 7.981 10 1,262 10 It + chloramphenicol 36,688 62 6,873 76 1t + cycloheximide 10,207 17 ‘ 3.723 01 1t + chloramphenicol + 0,750 8 1,039 12 cycloheximide 1 Rate relative to intact cells incubated in the light in the absence of inhibitor. 2 Rate relative to cyanelles isolated from cells incubated in the light in the absence of inhibitor. 64 Figure 8. Patterns of polypeptides from3§he gyanelles of Cyanophora paradoxa labeled lg vivo with SO ’. Intact cells were labeled for 00 min in the light in the ibsence of inhibitor (lanes 1,2) or in the presence of 300 pg chloramphenicol/ml (lanes 3,0), 25 pg cycloheximide/ml (lanes 5,6). or 300 pg chloramphenicol/ml plus 25 pg cycloheximide/ml (lanes 7.8). The cyanelles were isolated and their proteins separated by SDS-PAGE. Fluorograms (lanes 2.0.6.8) were prepared from the Coomassie Blue-stained gels (1.3.5.7). Equal amounts of cyanelles, equivalent to 1.6 pg chlorophyll a were loaded in each lane. Opll 3) 92.93 (_H i 1 0.2.3“ um I I u ..._ _ . I“ a 3n! .1... . a. 5...) . «ml-Ill .'.-III. [I o n * a e. . I no... A v.3. An;« A 0.; Aodc A «.00 A Qua 66 pg chloramphenicol/ml have been aligned with the Coomassie Blue-stained polyacrylamide gel. A summary of the polypeptides labeled in the presence of cycloheximide and chloramphenicol is contained in Table 0. The apparent molecular weights presented in Table 0 are derived from a comparison of the fluorogram with the Coomassie Blue-stained gel. It proved helpful when making the comparisons to utilize photographic contact prints of the fluorograms. When making the contact prints several different exposures were made to compensate for the differences in the intensity of the bands in the fluorogram. This was especially useful for polypeptide bands in the molecular weight range of 16,000-20,000 daltons of cyanelles from cycloheximide-treated cells. Comparison of the fluorograms of proteins from cycloheximide-treated (Figure 8 lane 6) and chloramphenicol-treated (Figure 8 lane 0) cyanelles with the fluorogram of proteins from untreated cyanelles (Figure 8 lane 2), shows that, generally, each polypeptide is affected by only one of the antibiotics. Two closely spaced polypeptides of ca. 28,500 daltons (Figure 8 lane 3) are exceptional in that their synthesis was significantly inhibited by both chloramphenicol and cycloheximide. A greater number of polypeptides was observed in cyanelles isolated form cells treated with chloramphenicol thn in cyanelles isolated from cycloheximide-treated cells. The Table i_n vivo wi chloramphen \ + chl< H x1i 67 Table 0. £3 vivo with chloramphenicol. Rapsyopeptides associated with cyanelles labeled 800. in the presence of cycloheximide or + chloramphenicol + cyclogeximide Mr x10 daltons Mr x10 daltons 89 89 80 67 55.0-53. ' 52 51 51 50.0 00 05.7 an 00 30.7 ”207-u108 3308 01.5 23.6 39.7-01.3 23.2 38 22.7 36.5-36 21.6 30.0 20.6 33.6 19.5 31.5 19.2 30.5 18.3 28 17.6 25.5 16.7 15.5 16.5 16.3 11 10 number I chloramph presented of severe together labeled be made were vis Presence 0f Cielohex- apparent Extensiv 17.600, daltons °y°1°hex ca.1o.00 eyelohex dalton n° DrOte daltens 0a. 15’ Eel8 ( 17 68 number of proteins synthesized in the presence of chloramphenicol is not well reflected in the summary presented in Table 0 because many of the bands are composed of several closely spaced polypeptides which were considered together (see Figure 8). Whereas fewer polypeptides were labeled in the presence of cycloheximide, they appeared to be made in greater amounts. No radioactive polypeptides were visible in the fluorogram of cyanelles labeled in the presence of both inhibitors (Figure 8 lane 8). Of those polypeptides synthesized in the presence of cycloheximide, a polypeptide (or polypeptides) having an apparent molecular weight of 52,000 daltons was the most extensively labeled. Polypeptides of sizes 30,700, 22,500, 17,600, 16,700, 16,500, and a diffuse band of ca. 33.800 daltons were also extensively labeled in the presence of cycloheximide. Two additional polypeptides of ca.10,000—11,000 daltons were synthesized in the presence of cycloheximide. The molecular weights of the 10,000-11,000 dalton proteins could not be accurately determined because no protein standard of molecular weight smaller than 10,000 daltons was included and because polypeptides of less than ca. 15,000 daltons behave anomalously on SDS-polyacrylamide gels (179). The most highly labeled polypeptide in cyanelles isolated from chloramphenicol-treated cells had an apparent molecular weight of ca. 89,000 daltons. Polypeptides of f 51,000, 02‘ proninant cyanelles. The sf been Studi. Sites of Presence . cyt°PlaSmi isOlated SDS-ends, Would labi 1' 53513512 % Int; taking sulfate Rho Obs! lethiOni the Fad. 10 c ‘r-c 69 51,000, 02,500, 30,000, 30,500, and 25,500 were also prominant in the fluorogram of chloramphenicol-treated cyanelles. Discussion The sites of synthesis of cyanelle proteins have not been studied previously. I undertook to investigate their sites of synthesis by labeling proteins 22 vivo in the presence of inhibitors specific for either cyanelle or cytoplasmic protein synthesis. The cyanelles were then isolated and the labeled polypeptides were separated by SDS-PAGE. It was first necessary to identify substrates that would label cyanelle proteins 1p vivo. 1. Characterization of the incorporation of radioactively labeled compounds into protein by Cyanophora paradoxa Intact cells of Cyanophora paradoxa are capable of taking up and incorporating exogenous organic compounds and sulfate into protein. In contrast to Trench 23 El- (170), who observed no uptake of amino acids, I determined that methionine, cysteine and leucine were taken up and incorporated into protein by Cyanophora. The kinetics of the incorporation varied depending upon the radioactive compound used. The rate of incorporation of 11. f 35 C from acetate or o S from sulfate into protein by intact or incorpora increasin; may be a acids fro the incor intracellt they can concentrat Twent ”Wrating did not fu PPOtein, vas reduce the fluOro °°n°°ntrat that the my real ekllcramphe the reduc Chloramphe‘ PEdUQtiOn (381) cone: further 70 intact cells increased with time, whereas the rate of incorporation of leucine did not. The lower initial but increasing rate of incorporation from acetate and sulfate may be a consequence of the time for the synthesis of amino acids from these compounds. The absence of a lag phase in the incorporation of leucine may be an indication that intracellular pools of leucine are sufficiently small that they can be rapidly saturated even at the relatively low concentration, 3 pH, of leucine used. Twenty-five pg cycloheximide/ml appears to be a saturating level of antibiotic because higher concentrations did not further reduce the incorporation of amino acids into protein. Hhen cells were labeled with sulfate in the presence of both inhibitors the incorporation into protein was reduced 88% and no labeled polypeptides were visible in the fluorographs (Figure 8). This result suggests that the concentrations of the inhibitors used were saturating or that the presence of both inhibitors is rapidly lethal. With respect to the degree of inhibition, 300 pg chloramphenicol/ml may not have been saturating. Although the reduction in incorporation of 1“Cacetate at 300 pg chloramphenicol/ml (055) is only slightly greater than the reduction in incorporation at 200 pg chloramphenicol/ml (38$) concentrations greater than 300 pg/ml might have had a further inhibitory effect. Higher concentrations were not used because of a concern that nonspecific inhibition (52.58) m1 The sensitive these ex [MCJIeuci chloramphe [MCJleuci affected 1 there is a siinthesis rate of in "33 not c"respond Has decli‘ea PTOteins Synthesis °YClohexim leucine in '0"- of th synthesize beep.l an cyanelle pretein 71 (52,58) might ensue. The incorporation of radioactivity into protein was sensitive to the inhibitors of protein synthesis used in these experiments. The rate of incorporation‘ of [1uCJleucine into cyanelle protein was reduced 25% by chloramphenicol, whereas the rate of incorporation of [IuCJIeucine into total cell protein was not detectably affected by chloramphenicol. These results suggest that there is a compensation in the rate of cytoplasmic protein synthesis when a competing sink is shut off. Hhereas the ”C]leucine into cyanelle protein rate of incorporation of [1 was not significantly affected by cycloheximide, the corresponding rate of incorporation into total cell protein was decreased 00: by cycloheximide. SDS-PAGE of cyanelle proteins labeled with 3550”= $3 1113 shows that the synthesis of many cyanelle proteins is sensitive to cycloheximide. However, the cycloheximide-insensitivity of leucine incorporation into cyanelle protein cannot mean that most of the labeled protein associated with the cyanelle is synthesized in the cyanelle. Instead, there appears to have been an increase in the rate of protein synthesis in the cyanelle in response to the inhibition of cytoplasmic protein synthesis. The chloramphenicol-insensitivity of incorporation into total cell protein and the cycloheximide-insensitivity of incorporation into cyanelle protein may, for example, result from changes in the sizes of amino a: In cap incorporat cyanelle pi cycloheximi Cyanelles incorporat snifate Chloramphe translatio espect ch sulfate 1! TherefOre: ”11 Wei; “1615' to 72 of amino acid pools. ”C]leucine, the In contrast to the incorporation of [1 incorporation of sulfate into total cell protein and into cyanelle protein was inhibited by both chloramphenicol and cycloheximide (refer to table 3). In this experiment, cyanelles accounted for approximately 15$ of the incorporation into total cell protein. The incorporation of sulfate into cyanelle protein was reduced 30$ by chloramphenicol. If chloramphenicol were inhibiting translation on cyanelle ribosomes only, then one would expect chloramphenicol to reduce the incorporation of sulfate into total cell protein ca. 55 (0.15x 0.30 = 0.05). Therefore, the 38$ reduction in incorporation into total cell protein in the presence of chloramphenicol is not due solely to the inhibition of cyanelle protein synthesis. The incorporation of sulfate into cyanelle protein was reduced to a greater degree (59$) in the presence of cycloheximide but only 30$ in the presence of chloramphenicol. These results seem to suggest that proteins of both the cyanelle and the cytoplasm are involved in the incorporation of sulfate into protein. It is also possible that some secondary effects are observed when cells are labeled with sulfate (or other compounds) in the presence of inhibitors. That is, the inhibitor may perturb some cellular process which indirectly affects protein synthesis. Of particular interest here is the inhibit carrot and 1g13), and Whereas in Umse expe an inhib: incorporat Altho whether c directly. affecting Chloramph‘ therefore ribosomes °h1°ramph phosphOr-y var-ions t is pr°duc be °°nc1\ 73 the inhibition by chloramphenicol of sulfate uptake in beet, carrot and pea, oxidative phosphorylation in corn ($3 vitro), and photophosphorylation in spinach (lg vitro) (50). Hhereas inhibition of sulfate uptake would apply only to u an inhibition of energy supply would affect the those experiments in which 3580 was used to label cells, incorporation of all compounds. Although I have not done so, it is possible to test whether chloramphenicol inhibits protein synthesis only directly, at the ribosome level, or also indirectly by affecting the uptake of precursors or the supply of energy. Chloramphenicol has two asymmetric carbon atoms, and therefore four stereoisomers. Protein synthesis by isolated ribosomes is inhibited only by the D-threo isomer of chloramphenicol, whereas all four stereoisomers inhibit phosphorylation, photophosphorylation and ion uptake in various tissues (52). If it can be shown that the inhibition is produced specifically by the D-threo isomer, then it can be concluded that the effect is upon protein synthesis. ii. SDS-PAGEApf intact cells and cyanelles labeled a with Na2 §pu Intact cells were labeled with Na23SSOu in the absence or in the presence of chloramphenicol or cycloheximide or both, and the cyanelles then isolated. Cyanelle proteins were separated by SDS-PAGE and the labeled polypeptides 74 visualized by fluorography. The presence of chloramphenicol or cycloheximide during 13 yiyg labeling of cyanelles resulted in different patterns of labeled polypeptides on fluorograms of SDS-polyacrylamide gels (see Figure 8). For the most part, the incorporation of radioactivity into individual polypeptides was sensitive to only one of the two inhibitors. In this discussion a polypeptide will be considered to be synthesized in the cyanelle if it is labeled in the presence of cycloheximide, but not in the presence of chloramphenicol. Conversely, proteins labeled when chloramphenicol, but not cycloheximide, is present will be considered to be synthesized in the cytosol. A summary of polypeptides labeled in the presence of 25 pg cycloheximide/ml or 1J1 the presence of 300 pg chloramphenicol/ml is presented in Table 0. In the paragraphs that follow, tentative identities are suggested for some of the cyanelle polypeptides that are labeled in the presence of cycloheximide. The polypeptides that have been tentatively identified are the large subunit of RUBISCO, the a and 8 subunits of the ATP synthase and the 32,000 dalton membrane protein. The identification of these polypeptides is based on the following evidence. 1. Their apparent molecular weights by SDS-PAGE are similar to those of cyanelle proteins [RUBISCO (30,00), ATP synthase (91)] that have been purified. ii. Sequences are present in the cyanelle DNA that hybridize at high stringency with 75 the genes for the large subunit of RUBISCO from maize (77. appendix A of this thesis), spinach (17), and Chlamydomonas (77); the 32,000 dalton membrane protein from spinach (17); the a subunit of ATP synthase from spinach (17); and the 8 subunit of ATP synthase from spinach (17) and maize (appendix A of this thesis). Because the coding capacity of the cyanelle genome is approximately 10- to 20-fold less than that of free-living cyanobacteria (16.17.80.78). it is likely that some cyanelle proteins are synthesized in the cytosol. Although it would be of interest to know which cyanelle polypeptides are synthesized in the cytosol, the results presented in this chapter do not allow tentative identities to be assigned to the chloramphenicol-insensitive polypeptides. The reasons that no tentative identities are suggested for these polypeptides are the following. No cyanelle protein which is cytoplasmically synthesized in plants or eukaryotic green algae has been characterized. Thus, the identity of these polypeptides would be based solely on a comparison between the apparent molecular weights of the cyanelle polypeptides and the chloroplast polypeptides. The molecular weights for a given protein that are reported in the literature may vary significantly. The large subunit of RUBISCO from Cyanophora has been reported to haver a molecular weight of 51,000 daltons (00) and 56,000 daltons (30) whereas the reported values for the small subunit are 15,000 (00) and 12,000 76 daltons (30). Moreover, the molecular weights of other Cyanophora proteins might vary from that of their plant or algal homologues. Such appears to be the case for the 8 subunit of the ATP synthase which has a molecular weight in spinach of 55,000 daltons (118,126), whereas the cyanelle 8 subunit is 52,000 daltons (91). Finally, the fact that a polypeptide is synthesized in the cytosol in plants or algae does not necessarily mean that such will be the case in Cyanophora. For example, some of the better characterized proteins that are synthesized in the cytoplasm in plants and eukaryotic green algae are the small subunit of RUBISCO and the polypeptides of the light harvesting chlorophyllg/g protein (25). While the site of synthesis of the small subunit has not been established in Cyanophora, the presence in the cyanelle DNA of a sequence for the small subunit (see chapter 3), is consistent with its being synthesized in the cyanelle. Cyanelles lack a chlorophyllg/p protein. iii. The polypeptides synthesized in the cyanelle A polypeptide, or polypeptides, with a molecular weight of ca. 52,000 daltons appears to be a major, i.e. highly labeled, product. of' cyanelle» protein synthesis. For the following reasons, I believe that there are at least three polypeptides of ca. 52,000 daltons and that they are (or include) the large subunit of RUBISCO, and the c and 8 subunits of the ATP synthase. DNA sequences for the large 77 subunit of RUBISCO and the a and 8 subunits of the ATP synthase are present in the cyanelle DNA (17, see also chapter 3, and appendix A of this thesis). The molecular weight, 52,000 daltons, of this band is close to, the molecular weight, 52,800 daltons, of the large subunit deduced from the DNA sequence (see chapter 3) of the large subunit of RUBISCO, and within the range of the reported molecular weights of 51,000 (00) and 56,000 (30) daltons for this protein. The molecular weights, 53,000 and 52,000 daltons, respectively, of the a and 8 subunits of the ATP synthase, as determined by Klein _3. _l. (91) also closely match the size of this band. The large amount of radioactivity present in this band is consistent with the presence of more than a single polypeptide. It should be noted that the use of values of the molecular weights of the subunits of the ATP synthase complex determined by Klein 52 al. (91), may not be entirely valid because the strain of Qanophora used by Klein 313 El- (91) may differ from the strain used in these studies (17.87.90). A polypeptide present as a diffuse band with a molecular weight of 33,800 daltons and a peptide of 30,700 daltons do not correspond closely to bands in gels stained with Coomassie Blue. The most rapidly labeled product of protein synthesis by isolated spinach chloroplasts is a polypeptide of 36,000 daltons, that also fails to coincide with a stained band (27). Similar polypeptides have been 78 found among the products synthesized by isolated chloroplasts of maize (6,68). pea (10,160), Spirodela (138). and Euglena (178). This membrane protein, originally referred to as peak D (160), is now usually referred to as the 32,000 dalton protein or by the name of the gene, pgpA, coding for it. It is characterized by its abundant synthesis in green tissue (160), a high rate of turnover (83), and synthesis as a slightly larger precursor protein [30,700 daltons, in maize (68) and 33.500 daltons in Spirodela (09)]. The psbA gene is present in the cyanelle DNA (17). Two groups of polypeptides that were heavily labelled in the presence of cycloheximide had apparent molecular weights ranging from 21,600-23,600 daltons and from 16.500-19,500 daltons (see Figure 8). Several of these polypeptides (16,300, 16,500, 16,700, 17.600, 18.300, 19,200, and 19,500) have molecular weights similar to the molecular weights of the red algal and cyanobacterial pigments, C-phycocyanin and allo-phycocyanin (13,59,186). Trench and Ronzio (175) have determined the molecular weights of the two subunits of C-phycocyanin (10,500 and 13,200 daltons) and Allg-phycocyanin (12,600) of Cyanophora. As noted by Trench and Ronzio (175), the apparent molecular weights of the cyanelle biliproteins are considerably smaller (by 2,000-0,000 daltons) than the molecular weights of their homologues from cyanobacteria and red algae. No 79 polypeptides having molecular weights of 12,600, 13,200, or 10,500 were observed in these experiments. It is possible that some of the labeled polypeptides in the molecular weight range of 16,500-19,2000 dalton are the subunits of the biliproteins phycobilin and allophycocyanin but because of the uncertainty regarding their molecular weights, I have not identified specific polypeptides as biliproteins. iv. Recommendations It seems clear from these results that a means of selecting the product of interest, RUBISCO, from among the many proteins synthesized would have been advantageous. Immunoprecipitation of RUBISCO labeled lg ylyg in the presence of inhibitors followed by separation of the subunits by SDS-PAGE and quantitation of the radioactivity present in the subunits might have given more specific information on the sites of synthesis of the subunits. With the use of monoclonal antibodies highly specific antibodies can be made. Ideally, the antibodies should be made against the purified enzyme from CyanOphora. Although I could isolate RUBISCO from the cyanobacterium Anabaena 29013 by the method used by Codd and Stewart (00) to isolate RUBISCO from Cyanophora, I was unsuccessful, despite numerous attempts, in applying their method to cyanelles. Another approach to identifying which proteins are encoded in the cyanelle is to study the cyanelle DNA 80 directly. The results of experiments in which the genes encoding the large and small subunits of RUBISCO and the 8 subunit of the ATP synthase were localized in the cyanelle DNA, the DNA encoding the large and small subunits sequenced, and a restriction endonuclease map of the cyanelle DNA constructed, are presented in the remainder of this thesis. CHAPTER III CHAPTER III THE NUCLEOTIDE SEQUENCES OF THE GENES FOR THE LARGE AND SMALL SUBUNITS 0F RIBULOSE-1,S-BISPHOSPHATE CARBOXYLASE Introduction Cyanophora paradoxa is a small biflagellated eukaryote of uncertain taxonomic affinities (57.133). The photosynthetic organelle, called an cyanelle, of Cyanophora has a peptidoglycan wall layer (1). phycobilin pigments‘ (35,175). and ultrastructural resemblance to free-living cyanobacteria (70). The generally accepted viewpoint that the cyanelle is an endosymbiotic cyanobacterium was challenged in 1977 by the discovery that the kinetic complexity of the cyanelle DNA was approximately 177 kilobasepairs (kb) (80), a size 10- to 20- fold smaller than the genome of free-living cyanobacteria (78). but well within the range of values obtained for chloroplast genomes (15). The overall structure of a restriction endonuclease map of the cyanelle DNA and the localization of the 16S and 23S ribosomal RNA (rRNA) genes to a large inverted repeat on that map showed also that the organization of the cyanelle Benome (16,17) is similar to that of the chloroplast (15). 81 82 These characteristics, when considered in light of the endosymbiont hypothesis of eukaryotic cell evolution (110). suggest that the cyanelle may represent an evolutionary stage which is intermediate between cyanobacteria, one of the presumed plastidal ancestors, and chloroplasts. The development and maintenance of a photosynthetically competent chloroplast requires the interaction of the nuclear and chloroplast genomes of the plant. This intergenomic cooperation is exemplified by the biosynthesis of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO). This key enzyme in photosynthetic carbon assimilation commonly consists of eight catalytic large subunits and eight small subunits of unknown function (120). The carboxylase of Cyanophora has essentially the same subunit size and stoichiometry as the enzyme from plants, eukaryotic algae, and cyanobacteria (30,00). In plants and eukaryotic green algae, the large subunit of carboxylase is encoded, transcribed, and translated in the chloroplast (25). The small subunit is encoded in the nuclear DNA (88) and translated in the cytosol on free‘ ribosomes as a precursor polypeptide (33.39.81). 33 1&2. reconstitution experiments have demonstrated that movement of the precursor polypeptide into the chloroplast and processing of that polypeptide are Post-translational events (39.81). In the cyanobacteria Agacystis 6301 (162), also called, Synechococcus (139), and 83 Anabaena 7120 (127), the gene (rch) for the small subunit of carboxylase is located 3' (mRNA sense) from the gene (rbcL) for the large subunit. The coding sequences of the rbcL and rch genes of Anacystis (162) and Anabaena (127) are separated by 93 bp and 505 bp, respectively. Most genes encoding the large, 50- to 55-kilodaltons (kd). subunit of carboxylase consist of single open reading frames coding for 072-077 amino acids (05,07,116,125.137,161,163,190). An exception is the large subunit gene of the eukaryotic alga, Euglena (93), which although it is chloroplast encoded, contains nine intervening sequences. The gggL gene is present in one copy per chloroplast DNA but in 15-30 copies per chloroplast in immature leaves of spinach (158). Because a leaf cell may contain several hundred chloroplasts (158). the copy number of the ngL gene greatly exceeds that of the £328 gene implying a reduction in transcription and/or translation of the EEEL gene or perhaps an amplification of the £238 gene in order to maintain stoichiometric amounts of the two subunits. The genes for the small subunit of RUBISCO in soybean (8) wheat (30), and pea (32,02) occur in the nucleus as small multigene families. The soybean £333 gene that has been sequenced consists of three open reading frames Separated by two intervening sequences (8). The £238 gene of Wheat contains a single intron (29). In Anacystis (162) and 84 Anabaena (127). the small subunit genes consist of single open reading frames of 333 bp and 327 bp, respectively. The coding sequence of the transit peptide common to the nuclear encoded small subunit genes of plants (7.8.29). is not present in the rch genes of Anacystis (162) and Anabaena (127). Recently it has been reported that the genes for both the large and small subunits of carboxylase are located in the DNA of the cyanelle of £.flradoxa (17,77). However, conditions of low hybridization stringency were required to obtain hybridization between the cyanelle DNA and the probes, which were prepared from cDNA clones of the gene for the pea small subunit. No evidence was found for the presence of a small subunit gene in the nuclear DNA of Cyanophora (77). Based on the hybridization of a DNA fragment containing the 5' end of the gng coding sequence to the same 2.2 kb HindIII fragment, which hybridized with the cDNA clone» of“ the pea small subunit, Heinhorst and Shively (77) suggested that the ms gene of the cyanelle may be located near and 5' from the gng gene. If true, and not simply the result of fortuitous hybridization due to the low stringency conditions employed, then Cyanophora is the first eukaryotic organism in which the genes for both of the subunits of carboxylase are located in the same cellular compartment. In addition to reporting that the rbcL and rch genes 85 are located in the cyanelle DNA, Bohnert and co-workers (17) reported that the genes for the ti, 8, and 6, subunits and the dicyclohexylcarbodiimide-binding polypeptide of the ATP synthase complex, cytochrome b6, and subunit ll of the cytochrome b6f complex are also located in the cyanelle DNA. The localization of the gene (ath) for the 8 subunit of the ATP synthase in the cyanelle DNA is of particular interest because in all higher plants examined to date the 2328 gene is located approximately 760-820 bp 5' (mRNA sense) from the ngL gene (28.55.96,160,180). i.e., close to where Heinhorst and Shively (77) tentatively localized the rch gene. In this chapter, I describe the organization of the fl-kb region (Appendix A and Chapter A) of the cyanelle genome containing the genes for the large and small subunits and the 3 subunit of the cyanelle coupling factor. The arrangement of these genes in the cyanelle DNA of Cyanophora paradoxa strain UTEX 23““ the Pringsheim strain (17), is compared to the analogous regions in both chloroplasts and cyanobacteria. I also present the complete DNA sequences of the genes for the large and small subunits of RUBISCO. The DNA sequence of the region 5' from the large subunit, up to and including the first 210 nucleotides of the coding sequence of the 2328 gene is included. The nucleotide sequences and the deduced amino acid sequences for the proteins are compared with the corresponding sequences from plants and cyanobacteria. 86 The small subunit sequence presented in this chapter is not a sequence of the region identified by Heinhorst and Shively (77) as encoding the the gene for the small subunit. Heinhorst and Shively (77) found that a 580 bp fragment from maize chloroplast DNA containing the active site region of the large subunit of RUBISCO hybridized to an 8.2 kb BglII fragment of cyanelle DNA. This same 8.2 kb BglII fragment and a 3.9 kb BglII fragment hybridized to a cloned DNA fragment containing the 3' two-thirds of the coding region of the rbcL gene from Chlamydomonas. Only the 8.2 kb BglII fragment hybridized with a 280 bp DNA fragment of the :38 gene from pea. These results are consistent with the presence of both the small subunit gene and the S'region of the large subunit on the 8.2 kb BglII fragment and with the presence of a BglII site in the 3'region of the coding sequence of the large subunit. My results from sequencing show a BglII site 350 bp from the termination codon of the large subunit confirming the results of Heinhorst and Shively (77). The small subunit which I have sequenced is located 108 bp 3' from the termination codon of the large subunit placing it in the 3.9 kb BglII fragment, a fragment which did not hybridize with the small subunit of pea (77). My results also show that the nearest gene 5' from the rbcL gene is the 2328 gene. From these results it can 'be concluded that the putative small subunit, which Heinhorst and Shively (77) identified on the basis of its hybridization with the small subunit of pea, must be located 87 on the opposite side of the ath gene from the rbcL gene. Results and Discussion 1. Organization of the cyanelle rbcL, rch and ath genes A physical map of the region of the cyanelle genome containing the (structural genes for the large and small subunits of RUBISCO and the 8 subunit of the cyanelle coupling factor, together with the strategy employed in sequencing those genes, is shown in Figure 9. Based on the DNA sequence, the N-terminal methionine codons of the large subunit of RUBISCO and of the 8 subunit of the coupling factor are separated by 1481 basepairs and are transcribed divergently. The relative locations and directions of transcription of these two genes are strikingly similar to those found in tobacco (55.160). petunia (28), maize (96), and spinach (189). in which the corresponding intergenic distances are 817 bp, 770 bp, 759 bp, and 785 bp, respectively. Because of the similarities found in the organization of these genes in several plant species and in annophora it is of interest to know if these genes are arranged similarly in the cyanobacteria, the putative ancestors of both chloroplasts and cyanelles. Though little detailed information on the locations of the ngL and 3323 genes in the cyanobacteria is available, it appears that these genes are not located near 88 Figure 9. Physical map of the region of the cyanelle DNA containing the £221., LE3. and 2223 genes. The thick bars represent the locations of the £b_c_l., Lb_cS, and 11:18 genes, with the 5' and 3' ends indicated. The strategy used in the Haxam and Gilbert sequencing of the £231. and £238 genes and the 5' 210 nucleotides of the ath gene is indicated in the lower portion of the figure. _=mz( =33. .moom .ouo .63. .09.. .002 >¢oom .095” 3a... _IEam 50...... Sun as s l. s a a s a s s «a 89 on com _..Y I J 3 _ . . _ . A. .. .L. [I _ _ r l. . Mir 1 H o a o . o . ._ .o mm .m .m h \_ won. 409 mu? \\ // \\\ I \ I \\ I \\ l \ / \\ I, \\ l \ / \. I \\ // \\\ 9: J _\ T1 m muswwm 90 each other in Anabaena (NS, 0.0. Nasmann, unpublished data). The organization of the genes for the large and small subunits of carboxylase is consistent with the hypothesis that the cyanelle is derived from a cyanobacterium. The coding region of the cyanelle small subunit is located on the same DNA strand 108 bp 3' from the termination codon of the large subunit. This arrangement is virtually identical to that found in Anacystis in which the intergenic distance is 93 bp (162). In Anabaena the corresponding distance is 5&5 bp and the genes are cotranscribed (127). The nature of the transcription of these genes in Cyanophora has not been determined. ii. The coding regions of the cyanelle rbcL, rch, and athvgenes The sequence of the noncoding DNA strand of an open reading frame of 1925 bp located in the cyanelle DNA is shown in Figure 10. I identify this sequence as that of the large subunit of RUBISCO because it hybridizes (Appendix A) with a maize chloroplast DNA fragments containing portions of the large subunit coding sequence, and because the amino acid sequence deduced from this open reading frame closely matches the amino acid sequences of other large subunits. The correct reading frame for the large subunit of RUBISCO was determined by comparison of the predicted translation 91 Figure 10. Nucleotide sequence of the EggL gene of the cyanelle of Cyanophora. Numbering begins with the first A of the methionine codon. The first methionine codon of the 2328 gene at position -N81 and the first lenthionine of the 5333 at position 1537 are shown. The inverted repeats at positions -291 to -219 and 1u39-1u86 are underlined. -404 -303 -202 -101 92 let CATA TTTTTACCTC ATTSSSAAAA ASAAAAAATA TTTTTATAAT TTTASTSTTA AAAACACTSA TATAAAAATT TTAATS TTCT AAAATTTTTS ATACTAACAA ATATTATACS CAAATTCATS ASTSTTTSAA TATSTAAAAA CAATTTTTAT AAASCTTATA AATTCTAAAC TSTTAAS TTS TTTATCCTST TATTTATATT TTAATAAIAT AAATACTAAT AAATTTATTA AACATSTATT TAAAATATTT AATATAAATA AATACTTATA TTTTATAA TA ASTATTTTTA ATTTTTTAAA TAAAAAAAAA SAATATTTTA SATSTTAACT TAATSATAAC TTTATAAAAS AATTTTSTTT TAAAAACTCC AAAAAASAS T TTTTCCSACT ATTACTAATT CATTAAATAT AATTTAASCT ATTTSTTTAA ATTAATTTAA ATTAATSAAS TAATASCATT TAAAAASCAA SSASAAATAC ATS TCA TCA net Ser Ser 30 ACT CCA Thr Pro TAT Tyr STA Val SCA SCA Ala Ala TAT TIT SGT TTC 51y Phe STT Val ACT Thr AAC Asn CCA Pro STT Val 170 TTA Leu SST Sly TTA Lou SST Sly TTA Leu TTC Phe SAC ASP ATT lle AAS Us AAA Lys SAA Slu CST SCT Ala SCT Ala ASA Ara 310 CAC His TSS Trp ATT lle TTC Phe SST Sly SAC CST Asp Arg ACT Thr CAA SAT Sln Asp ATT Ile TTC SST Phe Sly CST Arg STT SCT Val Ala ‘50 CST TTC Arg Phe SCA Ala CAA SCT Sln Ala ASA Arg ACT Thr AAA Lys 60 SAA Slu SAA Slu TCT Ser TCC Ser 90 STT Val SAA Slu CCA Pro TTA Leu ACT Thr ATS Net TAT Tyr TCC Ser AAA Lys ATT lle TST Cys 200 ACT Thr ACT Thr AAA Lys SAT Asp 230 SCT Ala TCT Ser CAA Sln TTC SCA SCT Phe Ala Ala TST Cy: CST SAT Arg Asp CST Are 340 SCA SST ACT All Sly Thr 370 TSS SCT TCC Trp Ala Ser STA TTA Val Leo SAC SAT TCT Asp Asp Ser CTT SAA SCA Leu Slu Ala AST CCT SAA Ser Pro Slu 10 ACT CAS ACA ASA SCA SSC TTC Thr Sln Thr Arg Ala Sly Phe SAC ATT Asp lle ACT SST Thr Sly CAT His SST Sly 120 ATC lle TCC Ser TTC Phe ACT Thr AAA CCA Lys Pro SAT SAA Asp Slu SAA ACT Slu Thr 260 SAA TTA Slu Leu AAT SST Asn Sly SCT AAA Ala Lys TTA SST Leu Sly ATS CCT Met Pro 400 TST TTA Cys Leu TST STT TTA SCA Leu Ala CTA SCA Leu Ala ACT Thr TSS Trp SAA Slu SAA Slu STA Val SST Sly 150 SST Sly 180 TTA Leu Sln Lys STA Asn Val SST Sly SAA Slu SAT Asp SCT Ala 290 CTT Leu CCT Pro ACA Thr TTA Leo TTC Phe STA Val SST Sly STT Val CAA Sln TTC Phe 430 CAA SCA CST AAC SAA SST CST AAC TTA Cys Val Sln Ala Arg Asn Slu Sly Arg Asn Leu aw SCT SCA TST SAA STT TSS AAA SAS ATT AAS TTC ao SCT TTC Ala Phe ASA Ara ACA ACT STA Thr Thr Val 100 TAC Tyr AAC Asn CAA Sln STA Val TTT Phe AAC Asn CCA Pro CCT Pro His SST Sly TTA Leu TCT Ser AAC Asn TCT Ser Sln ATT lle AAA Lys SST Sly CCS Pro ATC Ile ATC lle TTA Leu CAC His 320 ASA ATS TCT Arg Net Ser ATT Ile SAC TTA Asp Leu ATS Het ATS CCA Met Pro STT Val SST SST SST 20 AAA SCA SST STA AAA SAT TAT CST TTA ACT Lys Ala Sly Val Lys Asp Tyr Arg Leu Thr ATC Net 70 TSS Trp ACT Thr ACT Thr ATT lle TST C35 SST TTC Sly Phe SST ATT Sly Ile SCT AAA Ala Lys 210 CCS Pro TTC Phe CAC His TAC Tyr ATS Net His CAC CSA SCA ATS His Arg SST SST Sly Sly Asp His 350 CST SAC Arg Asp SCT TCT Ala Ser ACA TTA SST CAC Sly Sly Sly Thr Leu Sly His CAT SAC TAC CCT CAA CCA Pro Sln Pro SAT SST TTA Asp Sly Leu TAC STA SCA Tyr Val Ala 130 AAA SCA TTA Lys Ala Leu ACT STA SAS Thr Val Slu AAC TAC SST Asn Ty? Sly ATS CST TSS let Arg Trp 240 TTA AAT Leo Asn SCA Ala 27o Asp Tyr Ile Ala let His SAC CAC TTA Leu SAT CAT Asp His ATC Ile 380 SST SST ATT Sly Sly ll! 410 CCA Pro SCA 50 SSA STA CCT Sly Val Pro AST CTT Ser Leu ACC Thr TAT Ty? CCT TTA Pro Leu CST SCA TTA Arg Ala Leu 160 SAC AAA Asp Lys CST Ara CST Arg SCA STT Ala Val CST ATS SAT cs1 Asp Ara ACA Thr CCA CCT Pro Pro ACT Thr SCT SST Ala Sly 300 SCS STA ATT Ala Val lle CAT TCT SST His Ser Sly SAA CAA SAT Slu Sln Asp CAC ATT TSS His Ile Trp TSS SST AAC Trp Sly Asn 440 CST SAA SST A70 SAA TTC CCT SAA Pro Slu SAC CST Asp Arg SAT TTA Asp Leu CST TTA Arg Leu TTA AAC Leu Asn 190 TAT SAA Tyr Slu TTC TTA Phc Leu ACT TCT Thr Ser TTC ACA Phe Thr SAC CST Asp Ar! 330 ACT STT Thr Val ASA TCT Ara 50' CAC ATS His Het SCT CCA Ala Pro AAT SAA Ala Arg Slu Sly Asn Slu SAA ACT Ala Ala Cys Slu Val Trp Lys Slu lle Lys Phe Slu Phe Slu Thr TAT Tyr TAT Ty? GAS TST Slu Cys 80 TAC TIT AAA Li‘ 110 SAA Slu TTT Phe SAT Asp SAA Slu AAA TAT L13 TTA Leo TST Cys 220 TAT STA Tyr Val SAA SAA ACT CCT SAA Thr Pro Slu SCA SCA SCA Ala Ala Ala SST ASA ASC Sly Arg Ser SAA SST TCT Slu Sly Ser 140 TTA CST ATT Leu Arg lle SST CST SCT Tyr Sly Arg Ala CST SST SST Arg Sly Sly ATS SAT SCA Het Asp Ala 250 ATS ATC AAA Slu Slu Het ll! Lys TCT AAC Ser Asn CAA AAS Sln Lys STA SST Val Sly 360 CST SST Arg Sly CCT SCS Pro Ala SST SCT Sly Ala ATT ATC ll! 11! ATT SAT lle Asp 280 ACT ACA TTA Thr Thr Leu AAC CAT SST Asn His Sly AAA TTA SAA Lys Lou Slu ATT TTC TTC lle Pha Phe 390 TTA STA SAC Lou Val Asp 420 STA SCT AAC Val Ala Asn CST SAA SCT Arg Slu Ala ACT ATC TAA 168 252 336 420 672 756 840 924 1008 1092 1176 1260 1344 1428 Thr lla ochre TTTCATTTAA TTTATTTAAT TATTTASAST TTAAAAAAAC TCTAAATAAT TAATCAAAAT SATATTACTT CAATCTATTT TTATCCTTAA AATTCSSAAT T 1529 ATAAATTAT S Met Figure 10 1539 93 products with the known sequence of the large subunit of RUBISCO from maize (116). Based on the nucleotide sequence the large subunit is comprised of “75 amino acids and has a calculated molecular weight of 52.8 kd. This value agrees well with earlier determinations, 51 kd (HO) and 56 kd (30), based on the electrophoretic mobility of the purified protein. A comparison of the derived amino acid sequences of the large subunits of cyanophora, spinach (190), maize (116), Anabaena (115), and Anacystis (Synechococcus) (137,163) is presented in Figure 11. The homology of the amino acid sequence of the Cyanophora largesubunit with the large subunit sequences from plants and cyanobacteria is greater than 80 2. Inspection of Figure 11 shows that the amino acid changes in the large subunit are not distributed randomly throughout the coding region. The first 13 amino acids of the Cyanophora protein show differences in both the kind and the number of amino acids present when compared with the corresponding regions of the other species. The high degree of homology observed for the polypeptide as a whole begins with lysine 1A of the Cyanophora protein. In barley (135). direct determination of the amino acid sequence of the large subunit polypeptide indicated that the amino terminus of the mature protein is an alanine corresponding to the alanine at position 15 of the Cyanophora sequence. Later work 94 Figure 11. Comparison of amino acid sequences of the large subunits of RUBISCO from the cyanelle of Cyanophora (Cy), Anabaena 7120 (A7120), Anacystis (An), spinach chloroplasts (Sp), and maize chloroplasts (2m). Residue numbering refers to the cyanelle sequence. Sequences other than that of the cyanelle were obtained from the following references: Anabaena (N5), Anacystis (137,162), spinach (190). and maize (116). Boxes surround positions at which at least three of the proteinws have the same amino acid. The lysine residue labeled is the site of carbonate formation during the activatfbn of the enzyme by 002. 95 1% p P p p P CO RRRRR LLLLL MAMAA KKKKK AAAAA w LLLLL 100000 wafer-Prue? AQMVV VVVVV RRRRR ZPPPPP 00K. RRRRm EEEEE 0m000 5mmmmm_OEFFFF VVVVV $5555 a flHHHHH T0000 NNNNN AKKRR 111 H 1111" VmHVV AAAA SSSSS SSSSS NNNNN HUFFF HHHHH HHHHR EEEEE F. V NNNNN m m m m .2. ..u. .u. ..w... Unmwm Unmwm Qnmwm UNMWM Figure 11 96 suggested that this result may have been an artifact resulting from the relatively slow techniques used for large scale preparation of the enzyme used in that study (116). Langridge (100) found that translation of spinach chloroplast RNA in an §.ggli cell-free system resulted in a polypeptide 1-2 kd larger than that purified from chloroplasts. Subsequent treatment of ‘the 1J1 vitro synthesized product with a soluble chloroplast extract converted it to a polypeptide indistinguishable from the in 1212 synthesized large subunit (100). It is possible, as proposed by Zurawski gt 2l° (190), that post-translational processing of the large subunit polypeptide occurs in some plant species. However, there is T") direct evidence to suggest that processing occurs in either cyanobacteria or Cyanophora. In the absence of the relevant protein sequencing data, statements that the sequence heterogeneity observed in the amino terminal portion of the polypeptide is consistent with the removal of that portion of the protein during processing (163) seem premature. The large subunit catalyses both the carboxylation and oxygenation of ribulose 1,5-bisphosphate (120). Hartman and co-workers (73,1u3) used active site-directed affinity labeling to identify two lysyl residues, positions 175 and 3311, located within the domain of the active site. The epsilon amino group of lysine 201 reacts with CO forming a 20 carbamate, during activation of the enzyme by Hg++ and 002 97 (105). Lorimer (100) has suggested that, the three acidic residues, Asp-Asp-Glu, immediately following lysine 201 may + «H be involved in binding Hg+ and, that the bound Hg coordinates to and thus stabilizes the carbamate. ‘ Not unexpectedly, the parts of the protein containing these residues are well conserved. Indeed, amino acid residues 165 to 220 comprise one of the most conserved regions of the protein having substitutions at only 7 positions out of 56. The region surrounding lysine 3311 is also well conserved. The residues surrounding cysteine “59 are less conserved and in Anacystis this cysteine is replaced by leucine, suggesting that it is not directly involved in catalysis. The sequence of the noncoding DNA strand of an open reading frame of 321 bp located 3' from the large subunit coding sequence of Cyanophora is shown in the upper part of Figure 12. I identify this sequence in the cyanelle DNA as encoding the small subunit of RUBISCO because it closely matches the known sequence of the small subunits from Anacystis (162) and Anabaena (127). A polypeptide of 107 amino acids with a calculated molecular weight of 12.11 kd may be deduced from the 321 bp which comprise the coding sequence of the cyanelle small subunit. The derived amino acid sequence of the small subunit of Cyanophora is compared with the sequences of Anabaena (127). Anacystis (162). pea (7). soybean (8). spinach (115). wheat (29). and tobacco (1211) in the lower part of Figure 98 figure 13 .lll . w STTTAAAAAi ATS CAA N ht Sln Tl CA6 ATT S Sln lle A ACA SST A Thr Sly L. SAASAAS SluSluV m “ANTS Alaphe‘ Figure 12. Upper: Nucleotide sequence of the £238 gene from n the cyanelle of Cyanophora. Numbering begins with the first ARM” A of the methionine codon. The ochre termination codon of TMNWMT the ngL gene is shown. Lower: Comparison of the amino acid sequences of the small subunits of RUBISCO from the cyanelle of Cyanophora, Anabaena, Anacystis, pea, soybean. spinach, Tnmnn wheat, and tobacco. Residue numbering refers to the cyanelle sequence. Sequences other than that of the cyanelle were obtained from the following references: CHMMm Anabaena (127). Anacystis (162). pea (7). soybean (8). Mmum, spinach (115). wheat (29). and tobacco (12A). Boxes surround ‘ positions at which at least five of the proteins have the some“; same amino acid. ‘anm 132 198 264 331 411 491 571 591 ATT CST STT STA ATC SAA TTC AST TTT Ala Leu Ser Asn Sly Tyr Ser Pro Ala lle Slu Phe Ser Phe ‘0 TBS AAA TTA CCT TTA TTT SST ACA CAA TCT CCA Slu Asp Leu Val Trp Thr Leu Trp Lys Leu Pro Leu Phe Sly Thr Sln Ser Pro ro Asn Ala Tyr Ile Arg Val Val CT AAT SCT TAC STT TAC AAA CCA TTA TAS TTTAATS Leu Val Tyr Lys Pro Leu Amber C P CT AATTTCATTT AATTTATTTA ATTATTTASA - 80 STTTAAAAAA ACTCTAAATA ATTAATCAAA ATSATATTAC TTCAATCTAT TTTTATCCTT AAAATTCSSA‘ATTATAAATT ochre C 80 TT 99 Tu Sln Phe 100 STT CAA ACT TTA ATS TTC TTA S Val Sln Thr Leu Net Phe AAA Lys SCA CTT TCC AAT SST TAT ASC CCA SCA ln 90 SCA TTT SAC TCT ATC ASA e Arg Ala Phe Asp Ser 11 TAAAAAAATA CATTTTTTTC ATAATATSAA AAATCTTTTT TATSTTTAAT AATTTATASA AAAASSTTTT AATAASAACT ASAAATAAAA AAATAATAAA SAAATAAAAA ATAAAATAAA CATTSATATT ATTATTCTSA TTTASTSTTA TTTTTATTTC ATATCAATTT TTAATTAATC TACTTAAAAC AAAATTATCT AATTATTATT AATACTTTTT TTTTATATTT ASTAATTTTC TATTSTTATT ATTATTATTA ACA SST AAA SCT SAA SAC TTA STA TSS ACT TTA Thr Sly Lys Ala Slu Slu Val Leu Ser Slu Ile Sln Ala Cys SAA SAA STA CTT ASC SAA ATT CAA SCT TST Figure 12 olll ”Gena-‘KSD SSERRVR‘ PPPPP'PP LLLLLLLL QQEEENDD "Hun" LMLHHHMP QQOEEEQE RKAKKAKL AEALALLL OVRDAEEE DDDRDTTO ”NTSTDTSS TTYFFFYU AHCAGAAA NSDENDDE PPPPPPPP KEKKRKSK LLLLLLLL SSSNNDNI FYYYYYTV TYYYYYYY QQEAAASK 55555555 055ATKKK TTTTTTTT CCCVAVVA ..EEE... SE FFFFF' KRRK“K‘ 00000555 . VL VVV ..KHDVHR EEEEEEE EEEE E S S K A. RR KKKKK EEEKKLIK‘ ..LEKKE SADKKNNA ...SSSSIS A...LLV.T .RRSSSSS ...11LEIT ASNNEEESE, VKKPPPTP FFFFFFFF PPPPPP EEEEEEEE L...- HHHH' 01V LLLLL ISSEPAPE PPPPPPP ERQASATT vn T.“ K PSPPAPAA YHHHYYFY TTTVVVVV AALCCPCC ST 00000 0 00000 _PPPPPPPP_ 0K TTTT HHHHHHHH SIHVIIVV ,TACTCSCp ..5.....1 Gimgcess ..H..... FFFFFFFF “ I. 0.1 O. 0.1 .0 recs recs raps mmu mm m mun mm o Mun mm m u” ”mun on” umbm on” SE?“ anoown‘. mmc:wn. mac:wn.. fl aaowew 1 aaoflew J aaoMew cumussmt cmmnssm: cmmnssm: 12. Comp2 small sul 575 and E the £1235 from 011 conservat for (ll7 Anacystis 76-77%, a subunit 1 53-62 of the Prote amino ter 52 of the of the pr amin° aci the cyanc 100 12. Compared with the amino acid sequence of the Cyanophora small subunit, the sequences of Anabaena and Anacystis are 571 and 50% homologous, respectively. The homology between the Cyanophora sequence and the sequences from plants ranges from "11 for pea and wheat, to an: for soybean. If conservative and compensating amino acid changes are allowed for (117), the homologies are Anabaena/cyanelle 8111, and Anacystis/cyanelle 81s, (pea, soybean, and wheat)/cyanelle 76-771. and spinach/cyanelle 791. Two regions of the small subunit (see Figure 12) corresponding to residues 9-19 and 53-62 of the Cyanophora sequence are well conserved. All of the proteins except that from Anacystis have Net-Sln at the amino terminus. The region bounded by serine A3 and valine 52 of the Cyanophora sequence is variable. In this portion of the protein, the plant sequences have an insertion of 12 amino acids not found in the sequences from Cyanophora and the cyanobacteria. At the nucleotide level the small subunit sequences of Cyanophora, Anabaena, and Anacystis are all, approximately 50: homologous, with the sequence of pea. This was somewhat unexpected because the pea sequence recognizes the cyanobacterial sequences in Southern hybridization experiments but fails to hybridize with the Cyanophora sequence, even under conditions of low hybridization stringency (17.77). An examination of the nucleotide homology between pea and Cyanophora suggests that the region 101 most likely to hybridize with the pea sequence is located between amino acids 9 and 19 of the Cyanophora sequence. Although the overall homology of this segment is 8H1 (27/32), the longest stretch of perfectly matched bases is 8. If optimal strands are chosen and G-T pairs accepted as matches, then the length of the closely matched region may be increased to 15 bases. This degree of homology together with the relatively low guanine plus cytosine (0+0) content of this region, 27$ (32% for the entire coding sequence), is unlikely to allow hybridization under the conditions employed (128). The sequence of the initial 210 nucleotides of the noncoding DNA strand of an open reading frame located in the cyanelle DNA “85 bp 5' from the coding sequence of the ngL gene is presented in Figure 13. I identify this sequence as the coding sequence of the 8 subunit of the cyanelle ATP synthase because it hybridizes (Appendix A) with a maize chloroplast DNA fragment that contains a portion of the Bsubunit of the ATP synthase and because the amino acid sequence deduced from this open reading frame is homologous with the amino acid sequences of other 8 subunits. The sequence shown includes the region lying between the N-terminal methionine codons of the _aipB and _r_b_c_L genes. The deduced amino acid sequences of cyanelle, §.ggli (1AA), spinach chloroplast (189), and maize chloroplast (96) 8 subunits are aligned for comparison in the lower portion 102 Figure 13. Upper: Partial sequence of the ath gene Numbering begins with the from the cyanelle of Cyanophora. first A of the methionine codon. The first methionine of the gggL gene is indicated. Lower: Comparison of the amino acid sequences of the first 70 amino acids of the 8 subunit of the ATP synthase from the cyanelle of Cyanophora (Cy). 5. and maize chloroplasts coli (Ec), spinach chloroplasts (Sp). (Zm). Sequences other than that of Cyanophora were obtained from the following references: E. coli (1AA). spinach (189). and maize (96). Boxes surround positions at which at least three of the proteins have the same amino acid. —- Met Figure 13 CATG TATTTCTCCT TGCTTTTTAA ATGCTATTAC -484 TTCATTA ATT TAAATTAATT TAAACAAATA GCTTAAATTA TATTrnnrnn ---— -450 103 o- OmH mu s 4 alo p m e_m—u_z _ a x a p a a m H A < "EN 5 a a p m u p > z z a a a p a a o . _ s < "am a a a p m a p 4 a m - - - - - z o z m. m A < "um all a o p mam.r;a_p_ o a unuum z x u p p < “so , cm .s < a p m x m m _ p m _ w a «_m—m—m_a z p a "EN <_H~¢ m z x x m A p m p a a a m p p a z p «_HH "am > x p < z - - - - - - - - - l - - - - - - "um p p p p z p x - - - - - - - -,m p p - - p (HUB "so as . . . pu< op< pup (pa sum pas ppm < tsp Sp. ap< =m< Csp mp. was are as; epo spa =m< era uu< pp< p-NpN N N N NNN-.-NpN N N N NNN-pNN N N NN NN<-p-ppN N N N N.N-NN< N N N NNN-NN< N N N Cgp-NN< N N NN Naz-Np< N N N NC<-_c_L genes are similar to those of several plant chloroplasts [tobacco (55,160), petunia (28), spinach (180), and maize (96)) in which the 1distance between coding sequences of these genes ranges from 759 bp to 817 bp. The locations of the r_bg_L and 11328 genes have been investigated in only 3 single cyanobacterium, Anabaena; the genes are not adjacent (“5). In general the chloroplast genomes of plants and eukaryotic green algae are circular, range in size from 85 to 195 kilobasepairs (kb), and contain two single copy regions of unequal size separated by two repeated DNA segments that are arranged in an inverted orientation (15). Each repeated segment contains one copy of the ribosomal RNA 164 genes (15). Known exceptions to this omganization are the chloroplast genomes of broad bean (92), pea (37) and Euglena (67) which vary in the copy number and the orientation of the DNA segment which contains the ribosomal RNA genes. It has been suggested (9“) that intramolecular recombination within the repeats might lead to a reversal of the polarity of the single copy segments and result in a population of chloroplast DNA molecules that differ in the relative orientation of the single copy regions. Nith the exception of the chloroplast DNA of Phaseolus vulgaris (122,129) which does exist in two conformations, the orientation of the single copy regions of chloroplast DNAs have not been determined. The cyanelle DNA was digested with the restriction endonucleases BglII, XhoI, PstI, BamHI, and 8311 and a restriction map was constructed (Chapter “). The size of a monomer of cyanelle DNA, estimated by summing the molecular weights of the fragments produced by restriction of the cyanelle DNA, is approximately 128 kb. The restriction map of the cyanelle DNA is circular. A segment of the cyanelle DNA of approximately 10 kb is present twice in the cyanelle DNA. The duplicated segments are arranged in inverted orientation and are separated by DNA segments of unequal size that are unique in sequence. The cyanelle ngL, 5333, and 3328 genes are located in the larger single copy region approximately 17 kb from one of the inverted repeats 165 (Appendix A, Chapter “). The cyanelle DNA, like the chloroplast DNA of Phaseolus vulgaris (122,129), occurs in two conformations, which differ in the relative orientation of the single copy regions. These results are in agreement with those of Bohnert and coworkers (1“,16,17,103,123). In order to identify the sites of synthesis of cyanelle proteins, cyanelle proteins were labeled $2..ll!2. in the presence of inhibitors specific for cyanelle or cytoplasmic protein synthesis. Preliminary results suggest that cyanelle proteins are synthesized both in the cyanelle and in the cytoplasm. Among those polypeptides which may be synthesized in the cyanelle are the large subunit of RUBISCO, the a and 8 subunits of the ATP synthase and the 32,000 dalton membrane protein (psbA gene product). 166 General Discussion The evolutionary status of the cyanelle has been problematic because the cyanelle has characteristics of both cyanobacteria and chloroplasts. In the introduction to this thesis it was suggested that the biosynthesis of chloroplast proteins, particularly RUBISCO, is a good paradigm for the integration of chloroplast and nuclear genome function and that an investigation into the location of the genes and the sites of synthesis of the subunits of RUBISCO might help to clarify the evolutionary position of the cyanelle. The results presented in this thesis do not prove that the large and small subunits of RUBISCO are synthesized in the cyanelle. However, in view of the presence in the cyanelle DNA of sequences for the large and small subunits of RUBISCO, the similarity of their arrangement to that of the cyanobacteria (the presumed ancestors of the cyanelle), and the synthesis by the cyanelle of a polypeptide having a molecular weight similar to that of the large subunit, it seems likely that RUBISCO is encoded in the cyanelle DNA and synthesized on cyanelle ribosomes. If the location of the biosynthesis of the subunits of RUBISCO is a good measure of the integration of chloroplast and nuclear genome function, then the results presented in this thesis do not support the idea that the cyanelle is extensively integrated into the host. However, the biosynthesis of RUBISCO may not be such a measure. That is, 167 whereas the biosynthesis of this enzyme in other photosynthetic eukaryotes is an excellent example of cooperation by the chloroplast and nuclear genomes, the synthesis of both subunits by an organelle does .not necessarily imply that the organelle is poorly integrated or semiautonomous. All photosynthetic eukaryotes studied previously have separated the genes for the large and the small subunits of RUBISCO into different cellular compartments, i.e., the chloroplast and the nucleus. However only a relatively narrow spectrum of plant and algal species has been investigated. In particular, no chloroplast which contains biliproteins (e.g., the red algal chlorOplast) has been studied. The results from comparisons of 168 ribosomal RNA (rRNA) catalogues have demonstrated that chloroplasts are polyphyletic (66), that is, chloroplasts are derived indepedently from distinct prokaryotes. The comparison of 16S rRNA catalogues of the red 3133 Porphyridium cruentum (19), Lemna (183). and Euglena (187) suggests ‘that ‘these chloroplasts have different prokaryotic ancestors (66). The chloroplast of Porphyridium was found to be especially closely related to cyanobacteria (19). Because the cyanelle has phycobilin pigments (35.17“) and peptidoglycan (1), and is similar both in ultrastructure (60,70,17“) and in the arrangement of the gbgL and £238 genes (Chapter 3) to cyanobacteria, it seems almost certain to be derived from a 168 cyanobacterium. Although data for a quantitative comparison are not yet available, the red algal chloroplast and the cyanelle are probably more closely related to each other than either is to plant (and green algal) chloroplasts and their progenitors. Because the cyanelle and plant and green algal chloroplasts are likely polyphyletic and because there is no obvious reason why the pathway from endosymbiont to organelle should be the same for organelles derived from different prokaryotes, it is perhaps not surprising that the cyanelle differs from chloroplasts in synthesizing both subunits of RUBISCO. It is likely that as additional diverse photosynthetic eukaryotes are studied, choroplasts will be found, perhaps among the red algae, in which the chloroplast DNA contains the genes for both of the subunits of RUBISCO. Can a conclusion be drawn regarding the evolutionary status of the cyanelle? In addition to the striking similarities between the organization of the cyanelle genome and the genome of chloroplasts, there is a growing body of evidence suggesting that the cyanelle is well integrated into the metabolimm of Cyanophora. The coding capacity of the cyanelle genome is equivalent to that of plant and green algal chloroplasts implying that the number of cytoplasmically-synthesized cyanelle proteins may be comparable to the number of cytoplasmically-synthesized chloroplast proteins. Whereas the results from .lfl..11!2 labeling of cyanelle proteins do not permit an accurate 169 estimate to be made of the number of cytoplasmically-synthesized cyanelle proteins, they’ do suggest that many cyanelle proteins are synthesized in the cytoplasm. Kremer _e_t El' (97) and Trench £3 31. (17“) found that carbohydrate is exported from the cyanelle to the cytoplasm. That a flow of carbon also takes place from the cytOplasm to the cyanelle is implied by the $3 1112 labeling of cyanelle proteins with exogenous carbon compounds. Floener gt g_1_. (5“) and 86ttcher gt 3i. (2“) found that nitrate reductase is localized in the cytoplasm. In addition, Floener gt 2;. (5“) found that nitrite reduction occurs in the cyanelle. On the basis of their results they suggested that the pathway of nitrate assimilation is partitioned between the cytoplasm and the cyanelle in the same manner as nitrogen assimilation is partitioned between the chloroplast and cytoplasm in plants. Thus, there is evidence suggesting that the cyanelle and the cytoplasm cooperate in some important metabolic processes. That is, despite the presence of the genes for both subunits of RUBISCO in the cyanelle DNA, I think that one must conclude that the cyanelle is a highly integrated organelle. Whether or not the cyanelle is a chloroplast is a matter of choice. The major reason for not considering the cyanelle a chloroplast is the presence of peptidoglycan. Because peptidoglycan is a component of prokaryotic cell walls, it has been assumed that it is synthesized by the 170 cyanelle. In fact, the biosynthesis of the cyanelle peptidoglycan has not been studied. The transfer of genes from an endosymbiont to the host nucleus could be a means both of integrating the partners and of eliminating biosynthetic pathways of the endosymbiont which are no longer required. Compared to free-living cyanobacteria (78), the coding capacity of the cyanelle genome is much reduced (by ca. 90%) and it is quite possible that the biosynthesis of peptidoglycan is no longer solely 3 cyanelle function. APPENDICES APPENDIX A APPENDIX A MOLECULAR CLONING OF THE CYANELLE rbcL AND rch GENES AND THEIR FLANKING REGIONS Introduction This appendix describes the molecular cloning of the genes for the large (£291.) and small (5238) subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) and of a portion of the gene (3228) for the 8 subunit of the ATP synthase from Cyanophora paradoxa. Fragments of cyanelle DNA that contain the large subunit and the 8 subunit genes were identified by heterologous hybridization with 32P-labeled fragments of maize chloroplast DNA that contain the sequences of the 3338 and ngL genes. The small subunit was identified by comparing the deduced amino acid sequenme of an open reading frame, located 108 bp 3' from the coding sequence of the large subunit, with the amino acid sequence of the small subunit of Anacystis (162). 171 172 Materials and Methods The isolation of cyanelles, the isolation of cyanelle DNA, digestion of cyanelle and plasmid DNA with restriction endonucleases, and electrophoresis were performed as described in the Materials and Methods section of chapter “. DNA probes Two cloned fragments of maize chloroplast DNA containing portions of the gene (ngL) for the large subunit RUBISCO were used as probes in heterologous hybridizations. To localize the 5' end (mRNA sense) of the large subunit, pY““O (obtained from Dr. L. McIntosh, Michigan State University) was used. This plasmid carries a ““0 basepair (bp) 8coRI fragment of maize chloroplast DNA (116) that encodes the first 1“0 amino acids of the large subunit protein. To identify cyanelle DNA fragments containing the 3' end of the large subunit coding sequence, the 8“0 bp PstI-BglII fragment (obtained from Dr. L. McIntosh) of maize chloroplast DNA (116) that encodes the 3' half of the large subunit protein and ca. 160 bp of 3' flanking sequence was used. To identify cyanelle DNA fragments that contain the 8 subunit. of the .ATP synthase, two recombinant plasmids, pZR“8 (96) and p88“0, were used. pZR“8 (obtained from Dr. L. McIntosh) carries the 3.8 kilobasepair (kb) EcoRI' 173 fragment of maize chloroplast DNA that contains the entire sequence of the 8 and 3 subunits of the ATP synthase. The plasmid p88“0 (obtained from J. Fitchen, Michigan State University) carries a 10“0 bp HincII-BamHI fragment of maize chloroplast DNA (96) that encodes the first 233 amino acids plus 335 nucleotides of 5' flanking sequence of the 8 subunit of the ATP synthase. Nick translation and filter hybridization DNA was labeled in vitro with coow =55... NN+ .83 N .2530 » Jon. 09- AN ouswam GNOQOQ mug-0n vacuum 9 cocoa 194 codon (based on the homology‘ of the derived amino acid sequence to that of maize) of the large subunit to a position 330 bp to 'the right of the HindIII site in Ec16. The coding sequences of the ZEEL genes of plants, eukaryotic green algae, and cyanobacteria range from 1“16-1“31 bp and are highly conserved (“5,“7,116,137,161,163,190). The large subunit of Cyanophora hybridized with the maize large subunit under stringent hybridization conditions suggesting that it is homologous to the large subunit of maize and to large subunits of other species. It was expected, therefore, that the coding sequence of the cyanelle large subunit gene would be approximately the same size as that of other species. A coding sequence of 1“31 bp would place the termination codon of the Cyanophora rbc approximately 0.67 kb to the right of the Bc16 fragment. It therefore seemed likely that remaining portion of the large subunit coding sequence was present on the 0.63 kb and 0.27 kb EcoRI fragments. In order to clone these fragments, the 83838 fragment of cyanelle DNA was partially digested with 8coRI and ligated to dephosphorylated pBR322. Colonies were screened by isolating plasmid from 5-ml cultures and digesting it with EcoRI. In this manner a recombinant plasmid (designated pCp02“) carrying both the 0.63 kb and the 0.27 kb EcoRI fragments was selected. Localization of the 83111 sites in 835 and 83838 (Figure 21A) indicated that a 83111 site is located ca. 195 2.6 kb from the left end of 835, i.e., ca. 0.3 kb to the right of Ec16. Restriction of pCp02“ with EcoRI generates fragments. of 0.63 kb and 0.27 kb, whereas restriction with BglII+EcoRI generates fragments of 0.31 kb, 0.32 kb, and 0.27 kb. From this result it was hypothesized that Ec16 and the 0.63 kb fragment are adjacent in the cyanelle DNA. In order to verify that these fragments are adjacent, the 2.6 kb DNA fragment that extends from the BamHI site in Ec16 to the 83111 site in the 0.63 kb EcoRI fragment was cloned in pKC7. Transformants were screened by isolating plasmid from 5-ml cultures and digesting it. with BamHI+BglII. The recombinant plasmid that carries the 2.6 kb BamHI-BglII fragment was designated pCp025. In order to verify that the DNA fragment carried by pCp025 overlaps Ec16 and the 0.63 kb EcoRI fragment of pCp02“, pCp025 was restricted with EcoRI+BglII, EcoRI+HindIII, and BamHI+HindIII, pCp001 was restricted with BamHI+HindIII and EcoRI+HindIII, and pCp02“ was restricted with EcoRI+BglII. Table 11 shows that digestion of pCp025 with BamHI+HindIII or with EcoRI+HindIII generates fragments of the same size as the fragments generated by restriction of pCp001 with BamHI+HindIIIZ or with EcoRI+HindIII. Digestion of pCp02“ with EcoRI+BglII produces fragments of 0.32 kb, 0.31 kb, and 0.27 kb, whereas digestion of pCp025 with EcoRI+BglII produces a fragment of 0.31 kb. From this result I conclude that the 0.63 kb BcoRI is adjacent to 196 Table 11. DNA fragments generated by digestion of recombinant plasmids pCp001, pCp02“, and pCp025 with restriction endonucleases. Recombinant plasmids were restricted with EcoRI, EcoRI+BglII, BamHI+HindIII, and EcoRI+HindIII. The DNA fragments were separated by electrOphoresis in a 1.5 1 agarose gel. Phage A DNA digested with HindIII and pBR322 digested with Hian served as molecular weight standards. DNA fragments that consist of vector DNA or of vector plus insert DNA are not included in the table. Molecular weights are in kilobasepairs. restriction endonuclease EcoRI EcoRI+BglII BamHI+HindIII EcoRI+HindIII plasmid PCp001 2.5 2.5 1.26 1.35, 1.12 PCp02“ 0.63, 0.27 0.31, 0.32, 0.63, 0.27 0.27 PC9025 0.31 1.26 1.12 g 197 Ec16, and that the 2.6 kb BamHI-BglII fragment overlaps Ec16 and the 0.63 kb EcoRI fragment. It could not be determined from these results if the 0.27 kb EcoRI fragment carried by pCp02“ was cloned in the same orientation, relative to the 0.63 kb fragment, as in the cyanelle DNA. iii. Cloning of the gene for the small subunit of RUBISCO DNA sequencing of the large subunit of RUBISCO (results presented in chapter 3) showed that the 0.27 kb EcoRI fragment carried by pCp02“ contains 21 bp of the 3' end of the large subunit, a spacer of 108 bp, and an open reading frame of 120 bp that is terminated by the EcoRI site at the right end of the fragment. The open reading frame was tentatively identified as part of the sequence of the small subunit of RUBISCO because its deduced amino acid sequence is 60 S homologous to the amino acid sequence of the small subunit from Anacystis (162). In order to clone a DNA fragment containing the complete coding sequence of the cyanelle 5238, 8311, the 3.6 kb BglII fragment located within 835, was isolated. 8311 was digested with EcoRI+SphI and ligated to pBR322. Ampicillin-resistant colonies were screened by isolating plasmid from 5-ml cultures and digesting it with EcoRI+SphI. Three types of recombinant plasmids were found: plasmids that contained both the 0.27 kb EcoRI and the 1.5 kb EcoRI-SphI fragments; plasmids that contained only the 1.5 kb EcoRI-SphI fragment; and 198 plasmids that contained only the 0.27 kb EcoRI fragment. A recombinant plasmid, designated pCp029, containing both the 0.27 kb EcoRI fragment and the 1.5 kb EcoRI-SphI fragment was selected for use in the DNA sequencing of the small subunit of RUBISCO. Discussion The large subunit of RUBISCO and the 8 subunit of the ATP synthase have been localized on specific restriction fragments of cyanelle DNA by heterologous hybridization with portions of the genes encoding the large subunit and the 8 subunit from the chloroplast DNA of maize. Two recombinant plasmids, pCp001 and pCp02“, which together contain the complete sequence of the large subunit of the cyanelle RUBISCO were constructed. The EcoRI fragment (Ec16) of cyanelle DNA carried by pCp001 codes for the 5' ends of the large subunit of RUBISCO and of the subunit of the ATP synthase. Pcp02“ carries two EcoRI fragments, one of 0.63 kb and one of 0.27 kb. A third recombinant plasmid, pCp025, contains a 2.6 kb fragment of cyanelle DNA that spans the junction between the EcoRI fragment carried by pCp001 and the 0.63 kb fragment carried by pCp02“. A DNA sequence identified as the sequence of the small subunit of RUBISCO has been cloned in a recombinant plasmid designated pCp029. pCp02“ and pCp029 both contain the 0.27 kb EcoRI fragment of the cyanelle DNA that contains the 3' end of the 199 coding sequence of the large subunit, a spacer of 108 bp and the 5' end of the coding sequence of the small subunit. The fragments of cyanelle DNA carried . by the recombinant plasmids pCp001, pCp02“, pCp025, and pCp029 were used in the DNA sequencing of the large and small subunits of RUBISCO and 210 nucleotides of the 5' end of the 8 subunit of the ATP synthase (results presented in chapter 3). Sequencing of Ec16 showed that it contains the 5' ends of both the 8 subunit and the large subunit corroborating the results of heterologous hybridizations between portions of the genes encoding the maize chloroplast large subunit and 8 subunit and the cyanelle DNA. The results from the heterologous hybridizations and from DNA sequencing are combined in Figure 21A which shows the locations of these genes on a restriction map of 835 and 838. The positions of the large and small subunits of RUBISCO and the 3 subunit of the ATP synthase are represented by solid bars with the 5' ends indicated. The 3' end of the 8 subunit has not been localized. pZR“8, which contains the complete sequence of the B and 3 subunits of the maize chloroplast ATP synthase, hybridized to P317 and P3“. P3“ contains the 5' end of the 8 subunit which, based on the DNA sequence, is 160 bp to the left of the HindIII site in Ec16. The coding sequence of the gene for the 8 subunit of the maize chloroplast is ca. 1.5 kb. If the coding sequence of the cyanelle 8 subunit is of ZOO comparable size to that of maize, then its 3' and would lie in P317 ca. 2“0 bp to the left of the Ec16. The position of the 3' end of the subunit as presented in Figure 21A was "localized” by assuming that its coding sequence is 1500 bp. The identity of the 3.2 kb EcoRI fragment that hybridized with pZR“8, pBS“0, and pY““0 is uncertain. When the 3.2 kb EcoRI fragment, 831“, was cloned it did not hybridize with either pY““0 or pZR“8. An examination of Figures 17 and 20 shows that the hybridization of pY““0, pZR“8 and pBS“0 to the 3.2 kb EcoRI fragment is weaker than their hybridization to 8016. It is possible that in digests of cyanelle DNA 831“ was contaminated with a small amount of Ec16 which was responsible for the hybridization. It is also possible that Ec1“ was contaminated with a partial restriction product composed of 8016 (2.5 kb) and the 0.63 kb EcoRI fragment or of Ec16 and the 0.63 kb and 0.27 kb EcoRI fragments. APPENDIX B APPENDIX 8 GROWTH MEDIA FOR CYANOPHORA PARADOXA Medium CYB for 1000 mls NH” acetate 200 mg N33++Bglycerophosphate 50 mg Fe (33 Cl) 0.“ mg Ca (33 Cl) 10 mg KCl , 30 mg NaNO3 255 m8 KNO 303 mg M383 ~73 o 100 mg vitamin 81 1 pg cxa metal 51x+ 10 ml vitamin mix 33H 5 ml Bicine buffer “20 mg pH 7.8 + CYB metal mix. 1 ml contains: H 80 1.1“ mg; FeCl '6820, 9 “8 mg; MnSO -1HZO, 112 pg; ZnSOu-7HZO, 22 pg; 0080": 820, “.8 p3: Na2 DTA, 1 mg. ++ Vitamin mix 83. 1 ml contains: thiamine, 0.05 mg; biotin, 0.1 pg; p-aminobenzoic acid, 1 pg; folic acid, 0.2 pg: nicotinic acid, 0.01 mg: thymine, 0.3 m3; inositol, 0.5 mg; Ca pantothenate 0.01 mg. 201 Bold's 3N Bristols (169) 202 for 1000 mls NaNO3 CaCl2 M380 .7820 K HP u x6 PO“ Nagl P metals+ vitamin a (150 pg/ml) soil extrlgt (169) + P metals. 1 ml contains: 0.097 mg; MnCl °“H O, 0.0“1 mg; CoC12-6H20, 0.602 g; NaZMoOu, o.oou mg. 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