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PubMed1987

Spinach chloroplast fructose-1,6-bisphosphatase: identification of the subtilisin-sensitive region and of conserved histidines.

Chloroplast fructose-1,6-bisphosphatase (FbPase) is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars. The properties of the chloroplast enzyme are clearly distinct from those of cytosolic gluconeogenic FbPases. Light-dependent activation via a ferredoxin/thioredoxin system and insensitivity to inhibition by AMP are unique characteristics of the chloroplast enzyme. However, preliminary amino acid sequence data (78 residues) have demonstrated that a significant degree of amino acid sequence similarity exists between spinach chloroplast and mammalian gluconeogenic fructose-1,6-bisphosphatase [Harrsch, P.B., Kim, Y., Fox, J.L., & Marcus, F. (1985) Biochem. Biophys. Res. Commun. 133, 520-526]. In the present study, we have identified two structural features of spinach chloroplast FbPase that appear to be common to all FbPases. These include (a) the presence of a protease-sensitive area located in a region equivalent to residues 51-71 of mammalian FbPases and (b) the recognition of two conserved histidine residues, equivalent to histidines-253 and -311 of the mammalian enzymes. In addition, we have obtained sequence information accounting for more than three-fourths of the primary structure of spinach chloroplast FbPase. The high degree of homology observed between the chloroplast enzyme and gluconeogenic FbPases suggests a common evolutionary origin for all fructose-1,6-bisphosphatases in spite of their different functions and modes of regulation.

Chloroplasts↗

PubMed1989

The relationship of a prochlorophyte Prochlorothrix hollandica to green chloroplasts.

It is generally accepted that chloroplasts arose from one or more endosymbiotic events between an ancestral cyanobacterium and a eukaryote. Such an origin fits well in the case of the chloroplasts of rhodophytes that, like cyanobacteria, contain chlorophyll a and phycobilin pigments. The green chloroplasts from higher plants, green algae, and euglenoids however, contain chlorophyll b as well as chlorophyll a, and lack phycobilins. Consequently, it has been suggested that they arose independently of the rhodophyte chloroplasts, from an ancestral prokaryote containing that complement of pigments. The 'prochlorophytes' Prochloron didemni (an exosymbiont on didemnid ascidians) and Prochlorothrix hollandica (a recently discovered, free-living, filamentous form) have been suggested to be modern counterparts of the ancestor of the green chloroplasts because they are prokaryotes that also contain both chlorophylls a and b, and lack phycobilins. We report here a 16S rRNA-based phylogenetic analysis of P. hollandica. The organism is found to fall within the cyanobacterial line of descent, as do the green chloroplasts, but it is not a specific relative of green chloroplasts. Thus, similar pigment compositions do not necessarily reflect close evolutionary relationships.

Biological Evolution↗

PubMed1968

Ferrochelatase of spinach chloroplasts.

Spinach chloroplasts catalyse the incorporation of Fe(2+) into protoporphyrin, mesoporphyrin and deuteroporphyrin to form the corresponding haems. This ferrochelatase activity was detected by pyridine haemochrome formation with acetone-dried powders of chloroplasts, or from the formation of [(59)Fe]haems by intact chloroplasts. Decreasing the mitochondrial contamination of the chloroplasts by density-gradient centrifugation did not cause any loss of activity: spinach ferrochelatase appears to be principally a chloroplast enzyme. The characteristics of the enzyme were examined by using [(59)Fe]haem assay. The activity was pH-dependent: for both mesohaem and protohaem formation there were two pH maxima, a major peak at about pH7.8 and a smaller peak at about pH9.2. Lineweaver-Burk plots showed that the K(m) for Fe(2+) incorporation into protoporphyrin was 8mum and that for Fe(2+) incorporation into mesoporphyrin was 36mum. At non-saturating Fe(2+) concentrations the K(m) for protoporphyrin was 0.2mum and that for mesoporphyrin was 0.4mum. Ferrochelatase was not solubilized by treatment of chloroplasts with ultrasound but was solubilized by stirring in 1% (w/v) Tween 20 at pH10.4. Unlike the rat liver mitochondrial enzyme, chloroplast ferrochelatase was not stimulated by treatment with selected organic solvents. The spinach enzyme was inactive in aerobic conditions and it was shown by using an oxygen electrode that under such conditions the addition of Fe(2+) to buffer solutions caused a rapid uptake of dissolved oxygen, believed to be due to the oxidation of Fe(2+) to Fe(3+); Fe(3+) is not a substrate for ferrochelatase.

Centrifugation, Density Gradient↗

PubMed1968

Studies on ferrochelatase. The enzymic formation of haem in proplastids, chloroplasts and plant mitochondria.

1. Ferrochelatase was demonstrated in the chloroplasts and proplastids isolated from the primary leaves of beans (a dicotyledon) and oats (a monocotyledon). It was also detected in chloroplasts from etiolated bean seedlings made green by illumination before being harvested. The specific activities of the three types of bean organelles are similar, as are the specific activities of the oat proplastids and chloroplasts. 2. Chloroplasts from young spinach leaves also contain ferrochelatase; these chloroplasts were tested for their ability to form magnesium tetrapyrroles and found unable to catalyse the insertion of Mg(2+) into mesoporphyrin IX. 3. Ferrochelatase was also detected in potato tuber mitochondria. 4. Ferrochelatase activity in these plant preparations is much less stable on storage than similar preparations from bacteria and animal tissues. 5. Temperature affects the activities of spinach chloroplast ferrochelatase and rat liver ferrochelatase differently. Activity of the chloroplast enzyme increases as the temperature rises from 20.6 degrees to 26 degrees , but becomes increasingly inactivated as the temperature rises further to 38 degrees . The initial velocity of the mammalian enzyme, however, increases as the temperature rises from 25.8 degrees to 65 degrees , but the enzyme is inactivated after several minutes at 65 degrees .

Cell-Free System↗

PubMed1978

Low-molecular-weight (4.5S) ribonucleic acid in higher-plant chloroplast ribosomes.

A species of RNA that migrates on 10% (w/v) polyacrylamide gels between 5S and 4S RNA was detected in spinach chloroplasts. This RNA (referred to as 4.5 S RNA) was present in amounts equimolar to the 5S RNA and its molecular weight was estimated to be approx. 33 000. Fractionation of the chloroplast components showed that the 4.5S RNA was associated with the 50 S ribosomal subunit and that it could be removed by washing the ribosomes with a buffer containing 0.01 M-EDTA and 0.5 M-KCl. It did not appear to be a cleavage product of the labile 23 S RNA of spinach chloroplast ribosomes. When 125I-labelled 4.5 S RNA was hybridized to fragments of spinach chloroplast DNA produced by SmaI restriction endonuclease, a single fragment (mol.wt. 1.15 times 10(6)) became labelled. The same DNA fragment also hybridized to chloroplast 5 S RNA and part of the 23 S RNA. It was concluded that the coding sequence for 4.5 S RNA was part of, or immediately adjacent to, the rRNA-gene region in chloroplast DNA . A comparable RNA species was observed in chloroplasts of tobacco and pea leaves.

Chloroplasts↗

PubMed1979

On the control of long-chain-fatty acid synthesis in isolated intact spinach (Spinacia oleracea) chloroplasts.

1. Chloroplasts isolated from spinach leaves by using the low-ionic-strength buffers of Nakatani & Barber [(1977) Biochim. Biophys. Acta.461, 510-512] had higher rates of HCO(3) (-)-dependent oxygen evolution (up to 369mumol/h per mg of chlorophyll) and higher rates of [1-(14)C]acetate incorporation into long-chain fatty acids (up to 1500nmol/h per mg of chlorophyll) than chloroplasts isolated by using alternative procedures. 2. Acetate appeared to be the preferred substrate for fatty acid synthesis by isolated chloroplasts, although high rates of synthesis were also measured from H(14)CO(3) (-) in assays permitting high rats of photosynthesis. Incorporation of H(14)CO(3) (-) into fatty acids was decreased by relatively low concentrations of unlabelled acetate. Acetyl-CoA synthetase activity was present 3-4 times in excess of that required to account for rates of [1-(14)C]acetate incorporation into fatty acids, but pyruvate dehydrogenase was either absent or present in very low activity in spinach chloroplasts. 3. Rates of long-chain-fatty acid synthesis from [1-(14)C]acetate in the highly active chloroplast preparations, compared with those used previously, were less dependent on added cofactors, but showed a greater response to light. The effects of added CoA plus ATP, Triton X-100 and sn-glycerol 3-phosphate on the products of [1-(14)C]acetate incorporation were similar to those reported for less active chloroplast preparations. 4. Endogenous [(14)C]acetyl-CoA plus [(14)C]malonyl-CoA was maintained at a constant low level even when fatty acid synthesis was limited by low HCO(3) (-) concentrations. Endogenous [(14)C]acyl-(acyl-carrier protein) concentrations increased with increasing HCO(3) (-) concentration and higher rates of fatty acid synthesis, but were slightly lower in the presence of Triton X-100. It is proposed that rates of long-chain-fatty acid synthesis in isolated chloroplasts at saturating [1-(14)C]acetate concentrations and optimal HCO(3) (-) concentrations may be primarily controlled by rates of removal of the products of the fatty acid synthetase.

Acetates↗

PubMed1985

Localization of chloroplastic fatty acid synthesis de novo in the stroma.

The synthesis of fatty acids de novo from [2-14C]malonyl-CoA was studied in fractions from lettuce (Lactuca sativa) and pea (Pisum sativum) chloroplasts. When lettuce chloroplasts were subjected to osmotic lysis, disintegration through a Yeda press and high-speed centrifugation, essentially all of the fatty-acid-synthetic activity was found to be soluble. The distribution of the activity in various chloroplast fractions was similar to that of soluble marker enzymes such as ribulose-1,5-bisphosphate carboxylase and NADP+-linked glyceraldehyde-3-phosphate dehydrogenase. Marked differences were apparent in the quality of products from fatty acid synthesis de novo in the various fractions of chloroplasts. Thus soluble fractions produced predominantly stearate, whereas those containing membranes produced a greater proportion of palmitate. In pea chloroplasts, osmotic lysis released almost all of the fatty acid synthetase into the stromal fraction. In this instance, no major alterations in the products of fatty acid synthesis were observed. The fatty-acid-synthetic activity of the stromal fraction was still soluble after prolonged ultracentrifugation. The results show clearly the soluble nature of fatty acid synthesis de novo in lettuce and pea chloroplasts. Thus fatty acid synthesis measured in microsomal fractions from such plant tissues is not due to the presence of chloroplastic membranes.

Chlorophyll↗

PubMed1994

Substrate- and species-specific processing enzymes for chloroplast precursor proteins.

Using different precursors of chloroplast proteins and stromal extracts from both Chlamydomonas reinhardii and pea chloroplasts, we analysed the specificity of stroma-localized processing peptidases. By gel filtration of a stromal extract from isolated Chlamydomonas chloroplasts, fractions could be separated containing enzymic activities for processing the precursors of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) and of the protein OEE1 from the photosynthetic water-splitting complex (pOEE1). The enzymes differed not only in molecular size, but also in their sensitivity to inhibitors and in their pH optima. Obviously, in the stroma of Chlamydomonas chloroplasts different peptidases exist for processing of pSS and pOEE1, the latter being converted into an intermediate-sized form, iOEE1, which was found to be further processed to mature OEE1 by a thylakoid-associated protease. To study the species-specificity of the stromal peptidases, stromal extracts from Chlamydomonas and pea chloroplasts were incubated with pSS from either of these organisms. In the heterologous combinations, the precursors were partly hydrolysed, but not to the correct size. In importation assays, pSS from pea (but also the precursor of the ribosomal protein L12 from spinach) could not enter into chloroplasts from Chlamydomonas. In contrast, the algal pSS was imported into chloroplasts from pea, although it was not processed to mature SS. Our results indicate that the importation machinery and the pSS-processing enzymes in higher plants and green algae have different specificities and that in Chlamydomonas several stromal peptidases for different precursor proteins exist.

Animals↗

PubMed2001

Micromanipulation of chloroplasts using optical tweezers.

This paper describes experiments using optical tweezers to probe chloroplast arrangement, shape and consistency in cells of living leaf tissue and in suspension. Dual optical tweezers provided two-point contact on a single chloroplast or two-point contact on two adhered chloroplasts for manipulation in suspension. Alternatively, a microstirrer consisting of a birefringent particle trapped in an elliptically polarized laser trap was used to induce motion and tumbling of a selected chloroplast suspended in a solution. We demonstrate that displacement of chloroplasts inside the cell is extremely difficult, presumably due to chloroplast adhesion to the cytoskeleton and connections between organelles. The study also confirms that the chloroplasts are very thin and extremely cup-shaped with a concave inner surface and a convex outer surface.

Chloroplasts↗

PubMed2004

Extensive chloroplast haplotype variation indicates Pleistocene hybridization and radiation of North American Arabis drummondii, A. x divaricarpa, and A. holboellii (Brassicaceae).

Arabis drummondii, A. holboellii and their hybrid A. x divaricarpa are widespread perennials of open habitats in North America. A phylogenetic analysis based on noncoding chloroplast DNA sequences (trnL intron and trnL/F intergenic spacer) resolved A. drummondii as a monophyletic taxon, but found A. holboellii to bear chloroplast haplotypes from highly diverged evolutionary lineages. This raised the question of a possible polyphyletic origin of A. holboellii. Arabis x divaricarpa was found to be of recent and polytopic origin, a result consistent with its presumed hybrid origin. One hundred and three chloroplast haplotypes were detected within 719 Arabis accessions investigated. The majority of chloroplast-types were estimated to have arisen prior to the Wisconsin glaciation. Phylogeographical analysis using nested clade analysis, suggested for A. holboellii (i). past fragmentation events, partitioning genetic variation in several instances between the Sierra Nevada, the Southern Rocky Mountains and the Colorado Plateau on the one hand and the Central to Northern Rockies of the United States and adjacent Cascades on the other; and for both parental species (ii). recolonization of major areas formerly covered by the Wisconsin glaciation by three haplotypes; and (iii). restricted gene flow indicating isolation by distance in areas south of the last glacial maximum. Arabis x divaricarpa is closely codistributed with its parental species and resampled their haplotypes. The highest genetic diversity was found in the Rocky Mountains from Idaho and Montana south to Utah and Colorado. This area was further hypothesized to have played a major role in the origin of both parental species and probably represented an important glacial refugium. However, evidence for glacial refugia was also found in arctic and boreal regions of Alaska and near the Great Lakes. In comparison to nuclear ribosomal internal transcribed spacer data, chloroplast DNA divergence was very high and evidently predated the origin of A. drummondii and possibly A. holboellii. Divergence of major chloroplast lineages dates back to the middle of the Pleistocene at least. Extensive hybridization is the most likely evolutionary factor working on A. holboellii to explain the revealed discrepancy in nuclear DNA and chloroplast DNA diversification.

Arabis↗

PubMed1996

Introduction and expression of foreign DNA in isolated spinach chloroplasts by electroporation.

An electroporation-mediated method for the study of foreign gene expression within chloroplasts has been developed. The chloroplast expression vector pHD203-GUS, which consists of coding regions for beta-glucuronidase (GUS) and chloramphenicol acetyltransferase (CAT) separated by a double psbA promoter fragment from pea (in opposite orientation) was electroporated into spinach chloroplasts and the transient gene expression was examined. Conditions for the expression of the reporter genes have been optimized. Both CAT and GUS activities were detected in chloroplasts electroporated with pHD203-GUS, but not with nuclear expression vector pBI221 or negative control pUC18. No GUS activity was detected when pHD203-GUS was electroporated into spinach protoplasts. Dot immunoblot analysis using anti-GUS antibody confirmed the existence of GUS protein in chloroplasts electroporated with chloroplast-specific vector but not the negative controls, excluding the possibilities of endogenous GUS or bacterial contamination. The expression of GUS protein in treated chloroplasts was further confirmed by Western blot analysis.

Cell Fractionation↗

PubMed2002

GLK gene pairs regulate chloroplast development in diverse plant species.

Chloroplast biogenesis is a complex process that requires close co-ordination between two genomes. Many of the proteins that accumulate in the chloroplast are encoded by the nuclear genome, and the developmental transition from proplastid to chloroplast is regulated by nuclear genes. Here we show that a pair of Golden 2-like (GLK) genes regulates chloroplast development in Arabidopsis. The GLK proteins are members of the GARP superfamily of transcription factors, and phylogenetic analysis demonstrates that the maize, rice and Arabidopsis GLK gene pairs comprise a distinct group within the GARP superfamily. Further phylogenetic analysis suggests that the gene pairs arose through separate duplication events in the monocot and dicot lineages. As in rice, AtGLK1 and AtGLK2 are expressed in partially overlapping domains in photosynthetic tissue. Insertion mutants demonstrate that this expression pattern reflects a degree of functional redundancy as single mutants display normal phenotypes in most photosynthetic tissues. However, double mutants are pale green in all photosynthetic tissues and chloroplasts exhibit a reduction in granal thylakoids. Products of several genes involved in light harvesting also accumulate at reduced levels in double mutant chloroplasts. GLK genes therefore regulate chloroplast development in diverse plant species.

Amino Acid Sequence↗

PubMed2003

The Arabidopsis thaliana CUTA gene encodes an evolutionarily conserved copper binding chloroplast protein.

The Arabidopsis thaliana CUTA gene encodes a 182-amino-acid-long putative precursor of a chloroplast protein with high sequence similarity to evolutionarily conserved prokaryotic proteins implicated in copper tolerance. Northern analysis indicates that AtCUTA mRNA is expressed in all major tissue types. Analysis of cDNA clones and RT-PCR with total mRNA revealed alternative splicing of AtCUTA by retention of an intron. The intron-containing mRNA encodes a truncated 156-amino-acid protein as a result of stop codons in the included intron. The sequence of AtCutAp encoded by the fully spliced transcript suggests that the precursor consists of three domains: an N-terminal chloroplast transit sequence of 70 residues, followed by a domain with prokaryotic signal-sequence-like characteristics and finally the most conserved C-terminal domain. The N-terminal chloroplast transit sequence was functional to route a passenger protein into isolated pea chloroplasts with possible sorting to the envelope. Chloroplast localization was confirmed by Western blot analysis of isolated and fractionated chloroplasts. Recombinant AtCutA protein was expressed in Escherichia coli without the N-terminal 70-amino-acid chloroplast transit sequence. This recombinant AtCutAp was routed to the bacterial periplasm of E. coli. Purified recombinant AtCutAp is tetrameric and selectively binds Cu(II) ions with an affinity comparable to that reported for mammalian prion proteins.

Amino Acid Sequence↗

PubMed2003

Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit.

Six ribosomal proteins are specific to higher plant chloroplast ribosomes [Subramanian, A.R. (1993) Trends Biochem. Sci.18, 177-180]. Three of them have been fully characterized [Yamaguchi, K., von Knoblauch, K. & Subramanian, A. R. (2000) J. Biol. Chem. 275, 28455-28465; Yamaguchi, K. & Subramanian, A. R. (2000) J. Biol. Chem. 275, 28466-28482]. The remaining three plastid-specific ribosomal proteins (PSRPs), all on the small subunit, have now been characterized (2D PAGE, HPLC, N-terminal/internal peptide sequencing, electrospray ionization MS, cloning/ sequencing of precursor cDNAs). PSRP-3 exists in two forms (alpha/beta, N-terminus free and blocked by post-translational modification), whereas PSRP-2 and PSRP-4 appear, from MS data, to be unmodified. PSRP-2 contains two RNA-binding domains which occur in mRNA processing/stabilizing proteins (e.g. U1A snRNP, poly(A)-binding proteins), suggesting a possible role for it in the recruiting of stored chloroplast mRNAs for active protein synthesis. PSRP-3 is the higher plant orthologue of a hypothetical protein (ycf65 gene product), first reported in the chloroplast genome of a red alga. The ycf65 gene is absent from the chloroplast genomes of higher plants. Therefore, we suggest that Psrp-3/ycf65, encoding an evolutionarily conserved chloroplast ribosomal protein, represents an example of organelle-to-nucleus gene transfer in chloroplast evolution. PSRP-4 shows strong homology with Thx, a small basic ribosomal protein of Thermus thermophilus 30S subunit (with a specific structural role in the subunit crystallographic structure), but its orthologues are absent from Escherichia coli and the photosynthetic bacterium Synechocystis. We would therefore suggest that PSRP-4 is an example of gene capture (via horizontal gene transfer) during chloro-ribosome emergence. Orthologues of all six PSRPs are identifiable in the complete genome sequence of Arabidopsis thaliana and in the higher plant expressed sequence tag database. All six PSRPs are nucleus-encoded. The cytosolic precursors of PSRP-2, PSRP-3, and PSRP-4 have average targeting peptides (62, 58, and 54 residues long), and the mature proteins are of 196, 121, and 47 residues length (molar masses, 21.7, 13.8 and 5.2 kDa), respectively. Functions of the PSRPs as active participants in translational regulation, the key feature of chloroplast protein synthesis, are discussed and a model is proposed.

Amino Acid Sequence↗

PubMed2005

Dual targeting of the protein disulfide isomerase RB60 to the chloroplast and the endoplasmic reticulum.

RB60 is an atypical protein disulfide isomerase (PDI) that functions as a member of a redox regulatory protein complex controlling translation in the chloroplast of Chlamydomonas reinhardtii, but also contains a C-terminal endoplasmic reticulum (ER) retention signal, -KDEL. Here, we show by fluorescence microscopy that RB60 resides in the chloroplast but also outside of the chloroplast colocalized with BiP, an ER marker protein. RB60 accumulates in microsomes that exhibit a typical ER magnesium-shift, and cotranslationally translocates into ER microsomes. The first 50-aa leader of RB60 is sufficient for both chloroplast and ER targeting. The leader is cleaved upon translocation into the ER, whereas it remains intact after import to the chloroplast. The leader sequence also contains an acidic domain that appears necessary for the protein's association with the thylakoid membranes. Based on these and additional results, we propose that the dual localization of RB60 occurs via the two conserved transport mechanisms, to the chloroplast and to the ER, that the chloroplast RB60 most likely carries an additional function in the ER, and that its mode of transport, including the differential cleavage of its N terminus, plays an important role in its suborganellar localization and organellar-specific function.

Amino Acid Sequence↗

PubMed2003

Protein trans-splicing in transgenic plant chloroplast: reconstruction of herbicide resistance from split genes.

Inteins are intervening protein sequences that undergo self-excision from a precursor protein with concomitant joining of the flanking sequences. Here, we demonstrate intein trans-splicing in Nicotiana tabacum chloroplasts by using the naturally split Ssp DnaE intein. Trans-splicing occurred whether both intein fragments were encoded in the chloroplast or were separated into the chloroplast and nuclear genomes. A biolistic approach was used to integrate two fusion genes, one encoding aminoglycoside-3-adenyltransferase (aadA) and the first 123 aa of the Ssp DnaE intein (In) and the other encoding 36 C-terminal amino acid residues of the Ssp DnaE intein (Ic) and soluble modified green fluorescent protein (smGFP) into N. tabacum plastids. Expression of these gene fragments in the chloroplast resulted in ligated aadA-smGFP due to In-Ic-mediated trans-splicing. Furthermore, an N-terminal portion of the herbicide resistance gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) containing a chloroplast localization signal fused to In (EPSPSn-In) was integrated into the nuclear DNA of N. tabacum by using Agrobacterium tumefaciens-mediated transformation. The remaining EPSPS gene fragment (EPSPSc) fused to Ic (Ic-EPSPSc) was integrated into the chloroplast genome by homologous recombination. Western blot analysis of cell extracts from these plants showed a full-length EPSPS, demonstrating that the EPSPSn-In gene product migrated to the chloroplast and underwent trans-splicing. Furthermore, these transgenic plants displayed improved resistance to the herbicide N-(phosphonomethyl)glycine (glyphosate) when compared with wild-type N. tabacum.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1970

Identification and origin of some chloroplast aminoacyl-tRNA synthetases and tRNAs.

Light-grown, wild type Euglena gracilis contains two aminoacyl-tRNA synthetases for both phenylalanine and isoleucine. Only one of the two synthetases for each amino acid is found in isolated chloroplasts, as are the light-induced phenylalanine and isoleucine tRNAs. In each case the light-induced chloroplast tRNAs can only be acylated by the chloroplast synthetases. The chloroplast isoleucyl-tRNA synthetase is light-inducible and cannot be detected in dark-grown cells or in cells of the bleached mutant W(3)BUL. The presence of the chloroplast phenylalanyl-tRNA synthetase in W(3)BUL, which contains no chloroplast DNA or structure, indicates that this chloroplast enzyme is synthesized in the cytoplasm and is coded by nuclear genes.

Amino Acids↗

PubMed1985

Tandemly repeated nonribosomal DNA sequences in the chloroplast genome of an Acetabularia mediterranea strain.

A purified chloroplast fraction was prepared from caps of the giant unicellular green alga Acetabularia mediterranea (strain 17). High molecular weight DNA obtained from these chloroplasts contains at least five copies of a 10-kilobase-pair (kbp) sequence tandemly arranged. This unique sequence is present in DNA from chloroplasts of all stages of the life cycle examined. A chloroplast rDNA clone from mustard hybridized with some restriction fragments from Acetabularia chloroplast DNA but not with the repeated sequence. An 8-kbp EcoRI-Pst I fragment of the repeated sequence was cloned into pBR322 and used as a hybridization probe. No homology was found between the cloned 8-kbp sequence and chloroplast DNA from related species Acetabularia crenulata or chloroplast DNA from spinach.

Acetabularia↗