Wolfgang Hachtel

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.

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

SummaryThe nucleotide sequence of a 6156 by 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.

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.

A circular 73 kb DNA from the colourless flagellate Astasia longa that resembles the chloroplast DNA of Euglena: restriction and gene map

SummaryA 73 kbp circular DNA was isolated from the colourless euglenoid flagellate Astasia longa. Restriction sites of 12 restriction endonucleases were mapped on this DNA. Southern hybridization using plastid gene probes from Euglena, spinach and tobacco revealed sequence homologies to the genes coding for 16S and 23S ribosomal RNAs, elongation factor Tu (tufA) and the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL). The locations of these sequences on the restriction map were determined. Sequences homologous to chloroplast genes psaA, psbA, psbD, psbE and atpA are not present. The ribosomal RNA genes are organized in three tandem repeats, each containing one 23S and one 16S rRNA gene. In addition, there is one extra 16S rRNA gene. These results indicate the presence of a truncated form of a plastid DNA in Astasia.

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

SummaryThe 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 treeBetula pendula and identification of conserved protein regions

SummaryNitrate reductase (NR) assays revealed a bi-specific 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.

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.

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

SummaryA 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.

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

SummaryThree 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 protion 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.

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.