The genes encoding subunits of ATP synthase are conserved in the reduced plastid genome of the heterotrophic alga Prototheca wickerhamii.

The heterotrophic unicellular alga Prototheca wickerhamii is closely related to the photoautotrophic Chlorella vulgaris but has a 54,100-bp plastid DNA (ptDNA) that is much smaller than the chloroplast DNA of C. vulgaris (150,613 bp). The nucleotide sequence of 28,093 bp of the Prototheca ptDNA has been determined. No genes for photosynthetic functions have been found, except for sequences encoding six subunits of the ATP synthase ( atpA, atpB, atpE, atpF, atpH, and atpI). Transcripts of these atp genes have also been detected. Whether the leucoplasts of Prototheca contain a functional ATP synthase has still to be elucidated. Identified genes further include tufA, minD, cysT, and genes coding for three rRNAs, 22 tRNAs, and 12 ribosomal proteins. The results support the idea that, in the reduced plastid genome of Prototheca, genes coding for components of the plastid translational apparatus have been preferentially retained, and might be needed for the expression of the atp genes and some unassigned ORFs.

Identification of amino acid residues of nitrite reductase from Anabaena sp. PCC 7120 involved in ferredoxin binding.

The nitrite reductase gene (nirA) from the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 (A. PCC 7120) was expressed in Escherichia coli using the pET-system. Co-expression of the cysG gene encoding siroheme synthase of Salmonella typhimurium increased the amount of soluble, active nitrite reductase four fold. Nitrite reductase was purified to homogeneity. In order to identify amino acid residues involved in ferredoxin (PetF)-nitrite reductase electron transfer in A. PCC 7120, we performed a sequence comparison between ferredoxin-dependent nitrite reductases from various species. The alignment revealed a number of conserved residues possibly involved in ferredoxin nitrite reductase interaction. The position of these residues relative to the [4Fe4S]-cluster as the primary electron acceptor was tentatively localized in a three dimensional structure of the sulfite reductase from E. coli, which is closest related to nitrite reductase among the proteins with known tertiary structure. The exchange of certain positively charged amino acid residues of the nitrite reductase with uncharged residues revealed the influence of these residues on the interaction of nitrite reductase with reduced ferredoxin. We identified at least two separate regions of nitrite reductase that contribute to the binding of ferredoxin.

Functional analysis of a nitrite reductase promoter from birch in transgenic tobacco.

Nitrate assimilation is a highly regulated process in higher plants, and the regulatory cues governing gene expression in this pathway include both external and internal factors. In birch (Betula pendula Roth) the expression of nitrate reductase (NR) and nitrite reductase (NiR) genes is co-regulated by light and nitrate at the transcriptional level. In order to identify cis-acting DNA-elements involved in light and nitrate induction of the birch NiR gene, a 0.9 kb 5' flanking region of the NiR gene was isolated, analysed on the DNA level, and the transcription start site was determined. Deletion analysis of the birch NiR promoter region fused to the GUS reporter gene (uidA) in transgenic tobacco (Nicotiana tabacum) revealed the presence of light- and nitrate-responsive promoter fragments. The responsive fragments showed different activities in leaves and roots. Further, gel mobility shift assays using nuclear proteins from leaves detected a specific DNA-binding activity to the sequence between -146 and -267 bp that was induced in darkness and disappeared in the light. The deletion analysis has shown that this region is critical for light inducibility of the birch NiR gene in leaves.

Complete gene map of the plastid genome of the nonphotosynthetic euglenoid flagellate Astasia longa.

Astasia longa is a colourless heterotrophic flagellate closely related to the photoautotrophic Euglena gracilis. A circular 73 kb plastid DNA (ptDNA) has been isolated from A. longa that is about half the size of the chloroplast DNA of E. gracilis (143 kb). We have determined the complete sequence of the ptDNA of A. longa and established a complete gene map. All chloroplast genes for photosynthesis-related proteins are completely absent from the A. longa plastid DNA except for rbcL, the gene for the ribulose-1,5-bisphosphate carboxylase large subunit. Identified genes encode components of the plastid transcriptional and translational machinery: genes for three subunits of a chloroplast RNA polymerase, 20 chloroplast ribosomal protein genes, a gene for a plastid elongation factor Tu, 27 plastidic tRNA genes and three tandemly arranged repeats of 16S, 23S and 5S rDNA. Transcripts of a number of genes were detected by Northern hybridisation. The ribulose-1,5-bisphosphate carboxylase large subunit protein has been identified by immunoblotting.

The choice of reducing substrate is altered by replacement of an alanine by a proline in the FAD domain of a bispecific NAD(P)H-nitrate reductase from birch.

Differences in the amino acid sequence between the bispecific NAD(P)H-nitrate reductase of birch (Betula pendula Roth) and the monospecific NADH-nitrate reductases of a variety of other higher plants have been found at the dinucleotide-binding site in the FAD domain. To pinpoint amino acid residues that determine the choice of reducing substrate, we introduced mutations into the cDNA coding for birch nitrate reductase. These mutations were aimed at replacing certain amino acids of the NAD(P)H-binding site by conserved amino acids located at identical positions in NADH-monospecific enzymes. The mutated cDNAs were integrated into the genome of tobacco by Agrobacterium-mediated transformation. Transgenic tobacco (Nicotiana tabacum) plants were grown on a medium containing ammonium as the sole nitrogen source to keep endogenous tobacco nitrate reductase activity low. Whereas some of the mutated enzymes showed a slight preference for NADPH, as does the nonmutated birch enzyme, the activity of some others greatly depended on the availability of NADH and was low with NADPH alone. Comparison of the mutations reveals that replacement of a single amino acid in the birch sequence (alanine871 by proline) is critical for the use of reducing substrate.

Genes for components of the chloroplast translational apparatus are conserved in the reduced 73-kb plastid DNA of the nonphotosynthetic euglenoid flagellate Astasia longa.

The colourless, nonphotosynthetic protist Astasia longa is phylogenetically related to Euglena gracilis. The 73-kb plastid DNA (ptDNA) of A. longa is about half the size of most chloroplast DNAs (cpDNAs). More than 38 kb of the Astasia ptDNA sequence has been determined. No genes for photosynthetic function have been found except for rbcL. Identified genes include rpoB, tufA, and genes coding for three rRNAs, 17 tRNAs, and 13 ribosomal proteins. Not only is the nucleotide sequence of these genes highly conserved between A. longa and E. gracilis, but a number of these genes are clustered in a similar fashion and have introns in the same positions in both species. The results further support the idea that photosynthetic genes normally encoded in cpDNA have been preferentially lost in Astasia, but that the chloroplast genes coding for components of the plastid translational apparatus have been maintained. This apparatus might be needed for the expression of rbcL and also for that of still unidentified nonphotosynthetic genes of Astasia ptDNA.

In-frame length mutations associated with short tandem repeats are located in unassigned open reading frames of Oenothera chloroplast DNA.

Chloroplast DNAs were compared between two closely related species in the subsection Munzia of the genus Oenothera. A restriction fragment length dimorphism (273 bp) within the large inverted repeats was localized to an unassigned open reading frame that is homologous to ORF 2280 of tobacco chloroplast DNA. This dimorphism is due to different copy numbers of various short tandem repeated sequences, with each repeat unit specifying an in-frame addition or deletion. Other small length mutations were detected within an unassigned reading frame that appears to be homologous to the tobacco ORF 1244, and in the non-coding sequence upstream of that frame. These insertions and/or deletions are all associated with short direct repeats that lie in tandem.

Induction of Nitrate Assimilatory Enzymes in the Tree Betula pendula.

The coordinate appearance of the bispecific NAD(P)H-nitrate reductase (NR; EC 1.6.6.2) and nitrite reductase (NiR; EC 1.7.7.1) was investigated in leaves and roots from European white birch seedlings (Betula pendula Roth). Induction by nitrate and light of both enzymes was analyzed by in vitro assays and by measuring NR- and NiR-encoding mRNA pools with homologous cDNAs as probes. When birch seedlings were grown on a medium containing ammonium as the sole nitrogen source, low constitutive expression of NR and NiR was observed in leaves, whereas only NiR was significantly expressed in roots. Upon transfer of the seedlings to a nitrate-containing medium, mRNA pools and activities of NR and NiR dramatically increased in leaves and roots, with a more rapid induction in leaves. Peak accumulations of mRNA pools preceded the maximum activities of NR and NiR, suggesting that the appearance of both activities can be mainly attributed to an increased expression of NR and NiR genes. Expression of NR was strictly light-dependent in leaves and roots and was repressed by ammonium in roots but not in leaves. In contrast with NR, constitutive expression of NiR was not affected by light, and even a slight induction following the addition of nitrate was found in the dark in roots but not in leaves. No effect of ammonium on NiR expression was detectable in both organs. In leaves as well as in roots, NiR was induced more rapidly than NR, which appears to be a safety measure to prevent nitrite accumulation.

Sequence of a cDNA encoding nitrite reductase from the tree Betula pendula and identification of conserved protein regions.

The sequence of an mRNA encoding nitrite reductase (NiR, EC 1.7.7.1.) from the tree Betula pendula was determined. A cDNA library constructed from leaf poly(A)+ mRNA was screened with an oligonucleotide probe deduced from NiR sequences from spinach and maize. A 2.5 kb cDNA was isolated that hybridized to an mRNA, the steady-state level of which increased markedly upon induction with nitrate. The nucleotide sequence of the cDNA contains a reading frame encoding a protein of 583 amino acids that reveals 79% identity with NiR from spinach. The transit peptide of the NiR precursor from birch was determined to be 22 amino acids in size by sequence comparison with NiR from spinach and maize and is the shortest transit peptide reported so far. A graphical evaluation of identities found in the NiR sequence alignment revealed nine well conserved sections each exceeding ten amino acids in size. Sequence comparisons with related redox proteins identified essential residues involved in cofactor binding. A putative binding site for ferredoxin was found in the N-terminal half of the protein.

Sequence of a cDNA encoding the bi-specific NAD(P)H-nitrate reductase from the tree Betula pendula and identification of conserved protein regions.

Nitrate reductase (NR) assays revealed a bispecific NAD(P)H-NR (EC 1.6.6.2.) to be the only nitrate-reducing enzyme in leaves of hydroponically grown birches. To obtain the primary structure of the NAD(P)H-NR, leaf poly(A)+ mRNA was used to construct a cDNA library in the lambda gt11 phage. Recombinant clones were screened with heterologous gene probes encoding NADH-NR from tobacco and squash. A 3.0 kb cDNA was isolated which hybridized to a 3.2 kb mRNA whose level was significantly higher in plants grown on nitrate than in those grown on ammonia. The nucleotide sequence of the cDNA comprises a reading frame encoding a protein of 898 amino acids which reveals 67%-77% identity with NADH-nitrate reductase sequences from higher plants. To identify conserved and variable regions of the multicentre electron-transfer protein a graphical evaluation of identities found in NR sequence alignments was carried out. Thirteen well-conserved sections exceeding a size of 10 amino acids were found in higher plant nitrate reductases. Sequence comparisons with related redox proteins indicate that about half of the conserved NR regions are involved in cofactor binding. The most striking difference in the birch NAD(P)H-NR sequence in comparison to NADH-NR sequences was found at the putative pyridine nucleotide binding site. Southern analysis indicates that the bi-specific NR is encoded by a single copy gene in birch.

Genes for ribosomal proteins are retained on the 73 kb DNA from Astasia longa that resembles Euglena chloroplast DNA.

The nucleotide sequence of a 6.7 kb segment of the circular 73 kb DNA from Astasia longa has been determined. We identified genes for a tRNA-Ile (CAU), a tRNA-Phe (GAA), a tRNA-Cys (GCA) and the ribosomal proteins CS8, CL36, CS14 and CS2, that are normally encoded by plastid genomes. In addition, a gene for the chloroplast ribosomal protein CL5 was found that is not encoded by the plastome in either higher plants or a liverwort, but has recently been identified in Euglena chloroplast DNA. Transcripts of these protein genes, and of an unidentified open reading frame (ORF50), were detected. These results support our previous suggestion that the 73 kb DNA from Astasia is a truncated form of plastid DNA. The 73 kb DNA resembles the chloroplast DNA of Euglena gracilis but contains, almost exclusively, genes for a plastid-type translational (and presumably transcriptional) apparatus.

Organization and nucleotide sequence of ribosomal RNA genes on a circular 73 kbp DNA from the colourless flagellate Astasia longa.

Three tandemly arranged repeats (A, B, C) of 16S and 23S rDNA, and one supplementary (S) 16S rDNA adjacent to the 16S rDNA of repeat A, are present within an 18 kbp segment of a circular 73 kbp DNA from the colourless flagellate Astasia longa. The repeat units are separated by a short region containing a 5S rRNA gene and a gene for tRNA-Val (UAC). Sequence comparisons reveal 78%, 81%, and 67% identical nucleotides of the 23S rDNA (A), the 16S rDNA (B), and the 5S rDNA (A), respectively, with the corresponding genes of the Euglena gracilis chloroplast genome. As in Euglena chloroplasts, the 3'-terminal portion of the 23S rDNA is homologous to the 4.5S rRNA gene of higher plant chloroplast genomes. These results are supportive of a common evolutionary origin for the Astasia 73 kbp DNA and the Euglena 145 kbp chloroplast DNA.

Structure and expression of a gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) in the colourless euglenoid flagellate Astasia longa.

A gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) was identified on a circular 73 kb DNA from the colourless euglenoid flagellate Astasia longa. The rbcL gene of Astasia extends over 3968 bp. It is a split gene interrupted by seven introns as compared to nine intervening sequences in the rbcL gene of the phylogenetically related Euglena gracilis. Coding sequences as well as the positions of the introns within this gene are highly conserved in comparison with the Euglena rbcL except that two introns are missing in Astasia. The alignment of the amino acid sequences deduced from the nucleotide sequences of rbcL of Astasia and Euglena shows 82% identical amino acids whereas 15% of the amino acids represent conservative changes. A 1.5 kb transcript of the rbcL gene was revealed by northern blot analysis of Astasia RNA. By immunoblot analysis the gene product of rbcL was detected as a 53 kDa polypeptide. Genes for components of the chloroplast transcriptional and translational systems encoded by chloroplast DNA of plants and green algae are conserved on the 73 kb DNA of Astasia [24, 25, 26]. From our finding that Astasia obviously is capable of synthesizing the Rubisco large subunit one must conclude that these genes are expressed and form functional plastid transcriptional and translational systems.

Genes for the plastid elongation factor Tu and ribosomal protein S7 and six tRNA genes on the 73 kb DNA from Astasia longa that resembles the chloroplast DNA of Euglena.

The nucleotide sequence of a 6156 bp segment of the circular 73 kb DNA from Astasia longa resembling the chloroplast DNA of Euglena was determined. The genes for the plastid elongation factor Tu (tufA) and the ribosomal protein S7 (rps7), six tRNA genes (trnQ, trnS, trnG, trnM, trnT, trnR), and three open reading frames were identified. These genes show a high degree of sequence similarity (73%-99%) to the corresponding genes on the Euglena chloroplast genome. The tufA gene contains two small AT-rich introns within its coding region. Northern analysis revealed the in vivo transcription of the tufA gene and of a reading frame of 456 codons into monocistronic mRNAs of 1.3 and 1.4 kb, respectively. The arrangement and organization of the genes on the 73 kb DNA of the colourless heterotrophic flagellate Astasia and the chloroplast DNA of autotrophic Euglena are compared.

Cytochrome c oxidase of Euglena gracilis: purification, characterization, and identification of mitochondrially synthesized subunits.

Cytochrome c oxidase was purified from mitochondria of Euglena gracilis and separated into 15 different polypeptide subunits by polyacrylamide gel electrophoresis. All 15 subunits copurify through various purification procedures, and the subunit composition of the isolated enzyme is identical to that of the immunoprecipitated one. Therefore, the 15 protein subunits represent integral components of the Euglena oxidase. In an in vitro protein-synthesizing system using isolated mitochondria, polypeptides 1-3 were radioactive labeled in the presence of [35S]methionine. This further identifies these polypeptides with the three largest subunits of cytochrome c oxidase encoded by mitochondrial DNA in other eukaryotic organisms. By subtraction, the other 12 subunits can be assigned to nuclear genes. The isolated Euglena oxidase was highly active with Euglena cytochrome c558 and has monophasic kinetics. Using horse cytochrome c550 as a substrate, activity of the isolated oxidase was rather low. These findings correlate with the oxidase activity of mitochondrial membranes. Again, reactivity was low with cytochrome c550 and 35-fold higher with the Euglena cytochrome c558. The data show that the cytochrome c oxidase of the protist Euglena is different from other eukaryotic cytochrome c oxidases in number and size of subunits, and also with regard to kinetic properties and substrate specificity.

Deletions/insertions, short inverted repeats, sequences resembling att-lambda, and frame shift mutated open reading frames are involved in chloroplast DNA differences in the genus Oenothera subsection Munzia.

A restriction fragment length mutation has been mapped in the large single copy region of the chloroplast DNA from two Munzi-Oenothera species. Fragments containing the deletion/insertion were cloned, further analysed by additional restriction enzymes, and sequenced. A deleted/inserted 136 bp sequence was identified upstream of the 5' end of a tRNA-Leu (UAA) gene and presumably is located in the spacer between this gene and a tRNA-Thr (UGU) gene. The endpoints of the 136 bp sequence are covered by short inverted repeats. Complementary inverted repeats are present in the middle of the deleted/inserted sequence. The repeats are part of sequences resembling the lambda chromosomal attachment site (att-lambda) which is essential for site specific recombination in the lambda/Escherichia coli system. Possible interactions of the repeats during the deletion/insertion process are discussed. The spacer also contains a 1 bp deletion/insertion within an open reading frame (ORF). Due to this frame shift mutation the ORF sizes are quite different between the two Oenothera species.

Chloroplast DNA differences in the genus Oenothera subsection Munzia: a short direct repeat resembling the lambda chromosomal attachment site occurs as a deletion/insertion within an intron of an NADH-dehydrogenase gene.

A small restriction fragment length mutation has been mapped in the large inverted repeats of the chloroplast (cp) DNA of Munzia-Oenothera species (vom Stein and Hachtel 1986). This mutation could be localized within the intron of a reading frame presumably coding for subunit B of an NADH-dehydrogenase (ndhB). Sequence analysis revealed a 24 bp duplication/deletion. The predicted secondary structure of the ndhB-intron is altered by this duplication/deletion. Part of the directly repeated segment shows remarkable similarity to the phage lambda attachment site. Evidence is presented for similar sequences in other plastome regions where deletions/insertions have been found. Furthermore, the locations of the genes for other components of the NADH-dehydrogenase (ndhA, ndhC, ndhD, ndhE, ndhF) were established by heterologous hybridization using gene probes from tobacco cpDNA.

Chloroplast messenger RNAs of free and thylakoid-bound polysomes from Vicia faba L.

Purified chloroplasts from developing leaves of Vicia faba L. were broken and separated into stroma and thylakoid fractions. Both fractions contained polysomes as demonstrated by analytical density gradient centrifugation and in-vitro read-out translation. Messenger RNAs of free and thylakoid-bound polysomes were isolated and analysed by hybridization with heterologous gene probes from spinach and tobacco. Transcripts of the chloroplast genes psaA, psbB, psbC, psbD and petA were found predominantly on thylakoidbound polysomes engaged in the synthesis and the contrasslational integration of membrane proteins. In contrast, transcripts of the genes rbcL, psbE, petD, atpA, atpB, atpE and atpH were found more frequently on free polysomes corresponding to a stroma-located translation of these mRNAs and a posttranslational integration of the encoded intrinsic membrane proteins. We conclude from these findings that chloroplast-encoded membrane proteins are integrated by co-and posttranslational mechanisms.

Chloroplast DNA differences between two species of Oenothera subsection Munzia: location in the chloroplast genome and relevance to possible interactions between nuclear and plastid genomes.

The chloroplast DNAs (cpDNAs) of Oenothera berteriana and Oe. odorata (subsection Munzia) were examined by restriction endonuclease analysis with Sal I, Pvu II, Kpn I, Pst I, Hind III, and Bam HI. The fragment patterns show that these cpDNAs have all 133 restriction sites in common as well as a lot of individual bands. Nevertheless the cpDNAs of the two species can be distinguished by distinct differences in size between a small number of fragments. The 42 cleavage sites produced by Sal I, Pvu II and Kpn I were mapped on the circular cpDNAs. This was achieved by an approach which combined experimental and mathematical procedures. The overall serial order of the fragments was found to be the same for both cpDNAs. The size differences of individual fragments in the Sal I, Pvu II and Kpn I patterns between Oe. berteriana and Oe. odorata cpDNA are located within five regions scattered along the plastid chromosome. Two of these regions have been localized in the larger and one in the smaller of the two single-copy parts of the cpDNA molecule. The remaining two overlap the borders between the large single-copy and each of the duplicated parts of the molecule. The positions of distinct restriction sites are altered among the two Oenothera plastome DNAs by 0.02-0.4 MDa (30-600 base pairs). These alterations probably result from insertions/deletions.