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A dicistronic construct for the expression of functional spinach chloroplast ferredoxin:thioredoxin reductase in Escherichia coli
Ferredoxin:thioredoxin reductase (FTR) is a heterodimeric Fe---S containing disulfide reductase involved in the light-dependent activation of photosynthetic enzymes. We have designed a dicistronic construct for the heterologous expression of this nucleus encoded chloroplast protein in Escherichia coli. The coding sequences for the two mature subunits have been inserted in tandem into the expression vector pET-3d. This dicistronic construct is correctly translated yielding soluble, perfectly functional FTR. The recombinant enzyme is composed of both subunits, contains the correctly inserted Fe---S cluster as evidenced by its spectral properties and is indistinguishable from the enzyme isolated from leaves in its capacity to activate chloroplast fructose-1,6-bisphosphatase, one of the well known light activated enzymes of the Calvin cycle.
Analysis of the 22 kbp long psbD-psbC gene cluster of Euglena gracilis chloroplast DNA : Evidence for overlapping transcription units undergoing differential processing
The clustered genes psbD and psbC covering together close to 22000 nucleotides contain ten and eleven exons, respectively. The corresponding translation products, i.e, Photosystem II core 34 kDa (D2) protein and the CP43 chlorophyll binding protein are highly conserved. Introns vary in lenght from 305 to 4144 nucleotides. The two genes have about 900 nucleotides in common including an intron. To obtain stable mRNAs of about 1400 (psbD) and 1500 (psbC) nucleotides the pre-transcripts must undergo differential processing and/or splicing events within the overlapping region.
Structure et fonction des gènes pour l'ARNr 16S et le facteur d'élongation Tu engagés dans la protéosynthèse de E.coli et du chloroplaste de soja
Analysis of the Euglena gracilis chloroplast genome: Fragment Eco-I encodes the gene for the Mr 32 000–33 000 thylakoid protein of photosystem II reaction center
The DNA fragment Eco-I (4700 kilobasepairs) from the circular chloroplast genome of Euglena gracilis is shown to be transcribed in etioplasts at all stages of light-induced plastid development and in fully differentiated chloroplasts. Major stable transcription products are mRNAs of 14S and 17S. Using a rabbit reticulocyte lysate translation system we can show that the fragment Eco-I selects a mRNA which directs the synthesis of a Mr 32 000–33 000 polypeptide. Eco-I also hybridizes with a 330 basepair DNA probe cut from within the spinach chloroplast gene encoding the Mr 32 000 thylakoid membrane protein of the photosystem II reaction center. We conclude that the fragment Eco-I carries the corresponding Euglena gracilis chloroplast gene.
Modification of the reactivity of spinach chloroplast thioredoxin f by site-directed mutagenesis
Spinach chloroplast thioredoxin f
Complete sequence of Euglena gracilis chloroplast DNA
We report the complete DNA sequence of the Euglena gracllis, Pringsheim strain Z chloroplast genome. This circular DNA is 143,170 bp, counting only one copy of a 54 bp tandem repeat sequence that is present in variable copy number within a single culture. The overall organization of the genome involves a tandem array of three complete and one partial ribosomal RNA operons, and a large single copy region. There are genes for the 16S, 5S, and 23S rRNAs of the 70S chloroplast ribosomes, 27 different tRNA species, 21 ribosomal proteins plus the gene for elongation factor EF-Tu, three RNA polymerase subunits, and 27 known photosynthesis-related polypeptides. Several putative genes of unknown function have also been identified, including five within large introns, and five with amino acid sequence similarity to genes in other organisms. This genome contains at least 149 introns. There are 72 individual group II introns, 46 individual group III introns, 10 group II introns and 18 group III introns that are components of twintrons (introns-within-introns), and three additional introns suspected to be twintrons composed of multiple group II and/or group III introns, but not yet characterized. At least 54,804 bp, or 38.3% of the total DNA content is represented by introns.
Structure of the Euglena gracilis chloroplast gene (psbA) coding for the 32-kDa protein of Photosystem II
An EcoRI fragment from Euglena gracilis chloroplast DNA, called Eco.I (4.9 kbp), contains the gene for the ‘32-kDa’ thylakoid membrane protein of Photosystem II (psb A gene), the tRNA2Leu gene and additional sequences which possibly code for two proteins of unknown function. The transcription polarity is the same for all these coding sequences. The psb A gene is split: 4 introns (size range, 433–616 bp) separate 5 exons (size range, 39–579 bp) which code for a protein of Mr 38380 (345 amino acids). The Euglena protein is about 87% homologous with the higher plant counterparts but it contains 5 lysine residues.
The tuf gene family of soybean : structure and differential transcription
The nuclear genome of soybean (Glycine max L.) contains a family of four tuf genes coding for the chloroplast translation elongation factor EF-Tu. Genomic and cDNA sequencing reveal that the four tuf genes belong to two subfamilies (type A and type B). Tuf genes A1/A2 are highly expressed in green organs (leaves and stems) while tuf genes B1/B2 are poorly expressed in leaves and stems. Both types of genes are about equally expressed in roots albeit at a low level. The 5′ flanking regions of tufA1 and B1 are tested for promoter activities (GUS) in transformed green tobacco. The tufA1 promoter is much stronger than the B1 promoter. Promoter deletion studies with tufA1-derived constructs allowed identification of a DNA segment essential for gene transcription.
Mapping and sequencing of an actively transcribed Euglena gracilis chloroplast gene (ccsA) homologous to the Arabidopsis thaliana nuclear gene cs(ch-42)
We mapped and sequenced a novel chloroplast gene encoding a protein (348 amino acids) which shows a high sequence identity with both the decoded nuclear cs(ch-42) gene product of Arabidopsis thaliana, and the C-terminal half of the decoded ‘crtA’ gene product of Rhodobacter capsulatus. The chloroplast gene (ccsA) is split (two exons) and transcribed into a stable mRNA of about 1200 nucleotides. The putative protein may be involved in the biosynthesis of photosynthetic pigments.
The Euglena gracilis chloroplast genome : structural features of a DNA region possibly carrying the single origin of DNA replication
We sequenced a Bg1II-HindIII DNA fragment of the Euglena gracilis chloroplast genome which most likely carries the single origin of DNA replication according to recent electronmicroscopic mapping studies (Koller and Delius 1982a; Ravel-Chapuis et al. 1982). This DNA fragment contains a polymorphic region (Schlunegger et al. 1983) which is composed, as will be shown, of multiple tandem repeats (54 bp, 87% A+T). Furthermore we located on this DNA fragment a short inverted repeat element (96 positions) observed in the electronmicroscopic studies (Koller and Delius 1982b). Between the borders of the polymorphic region and the nearby inverted repeat (distance of 179 positions) we retrieved an exact copy of parts of the rDNA leader (105 positions) including 49 positions of the chloroplast trnW gene. A computer search for bacterial type Ori-regions did not reveal any significant sequence homology. However, the polymorphic region and its immediate vicinity have the capacity to form multiple stem and loop structures which may be involved in DNA replication initiation.