Jens Sigurd Okkels

A cDNA clone encoding a 10.8 kDa photosystem I polypeptide of barley

A cDNA clone encoding the barley photosystem I polypeptide which migrates with an apparent molecular mass of 16 kDa on SDS‐polyacrylamide gels has been isolated. The 634 bp sequence of this clone has been determined and contains one large open reading frame coding for a 15 457 Da precursor polypeptide. The molecular mass of the mature polypeptide is 10 821 Da. The amino acid sequence of the transit peptide indicates that the polypeptide is routed towards the stroma side of the thylakoid membrane. The hydropathy plot of the polypeptide shows no membrane‐spanning regions.

A cDNA clone encoding the precursor for a 10.2 kDa photosystem I polypeptide of barley

Two cDNA clones for the barley photosystem I polypeptide which migrates with an apparent molecular mass of 9.5 kDa on SDS‐polyacrylamide gels have been isolated using antibodies and an oligonucleotide probe. The determined N‐terminal amino acid sequence for the mature polypeptide confirms the identification of the clones. The 644 base‐pair sequence of one of the clones contains one large open reading frame coding for a 14 882 Da precursor polypeptide. The molecular mass of the mature polypeptide is 10 193 Da. The hydropathy plot of the polypeptide shows one membrane‐spanning region with a predicted α‐helix secondary structure. The gene for the 9.5 kDa polypeptide has been designated PsaH.

A cDNA clone from barley encoding the precursor from the photosystem I polypeptide PSI-G: sequence similarity to PSI-K.

A cDNA clone encoding the photosystem I subunit, PSI-G was isolated from barley using an oligonucleotide specifying a partial amino acid sequence from a 9 kDa polypeptide of barley photosystem I. The 724 bp sequence contains an open reading frame encoding a precursor polypeptide of 15 107 kDa. Import studies using the in vitro expressed barley PsaG cDNA clone demonstrate that PSI-G migrates with an apparent molecular mass of 9 kDa on SDS-polyacrylamide gels together with PSI-C (subunit-VII). The previous assignment of the gene product of PsaG from spinach as subunit V (Steppuhn J, Hermans J, Nechushtai R, Ljungberg U, Thümmler F, Lottspeich F, Herrmann RG, FEBS Lett 237: 218–224, 1988) needs to be re-examined. The expression of the psaG gene is light-induced similar to other barley photosystem I genes. A significant sequence similarity to PSI-K from Chlamydomonas reinhardtii was discovered when a gene database was searched with the barley PSI-G amino acid sequence. Extensive sequence similarity between the nuclear-encoded photosystem I subunits has not previously been found. The observed sequence similarity between PSI-G and PSI-K suggests a symmetric location of these subunits in the photosystem I complex. The hydropathy plot of the barley PSI-G polypeptide indicates two membrane-spanning regions which are also found at the corresponding locations in the PSI-K polypeptide. PSI-G and PSI-K probably have evolved from a gene duplication of an ancestral gene.

Highly efficient integration of foreign DNA into the genome of the green sulfur bacterium,Chlorobium vibrioforme by homologous recombination

Highly efficient and reproducible transformation ofChlorobium vibrioforme with plasmid DNA has been achieved by electroporation. Specific parameters have been optimized for the electrotransformation procedure. The method was developed using a construct containing a full copy of thepscC gene encoding the cytochromec551 subunit of the photosynthetic reaction center complex and theaadA gene encoding streptomycin resistance as selectable marker. Southern blotting analysis showed that the tested colonies were true transformants with the plasmid integrated into the genome by single homologous recombination. No transformants were obtained using the vector without thepscC gene showing that this vector does not replicate inC. vibrioforme. Thus transformation is possible only by homologous recombination. When using constructs designed to inactivate thepscC gene by insertion no transformants were obtained, indicating that the gene is indispensable for growth. The vector pVS2 carrying genes for erythromycin and chloramphenicol resistance was shown to replicate inC. vibrioforme. The two transformations shown here, provide an important genetical tool in the further analysis of structure and function of the photosynthetic apparatus in green sulfur bacteria.

Nearest Neighbour Analysis of the Photosystem I Subunits in Barley and Their Binding of Ferredoxin

Photosystem I (PS I) mediates electron transfer from plastocyanin to ferredoxin through the reaction center P700 and the five electron acceptors A0, A1, X, A and B (1). PS I preparations from barley contain the reaction center protein P700-chlorophyll a-protein 1 (CP1) and smaller polypeptides with apparent molecular masses of 18- (PSI-D), 16- (PSI-E), 14-, 9.5- (PSI-H), 9- (PSI-C), 4- and 1.5-kDa (PSI-I) (2). CP1 is a heterodimer of the two homologous approximately 82-kDa polypeptides PSI-A and PSI-B (2) and binds the reaction center P700 and the iron-sulfur center X (3). The iron-sulfur centers A and B are located on the PSI-C subunit (4). Iron-sulfur centers A and/or B donate electrons to ferredoxin on the stromal side of the thylakoid membrane.

Chloroplast Encoded Photosystem I Polypeptides of Barley

In higher plants, photochemical transfer of electrons from plastocyanin to ferredoxin is catalyzed by photosystem I (PS I), a membrane-bound protein complex binding the reaction center (P700), the photoreducible electron acceptors (A0, A1, X, A and B) as well as antennae pigments (1). The core complex of PS I is defined as the simplest pigment-protein complex which is able to photoreduce NADP+ in the presence of added plastocyanin, ferredoxin and ferredoxin-NADP+ oxidoreductase. The core complex isolated from barley (Hordeum vulgare L.) photoreduces 80–340 µmol NADP+(h x mg chl) (2). The barley core complex is composed of polypeptides of apparent molecular masses of 82-, 18-, 16-, 14-, 9.5-, 9-, 4- and 1.5-kDa as determined from their electrophoretic mobility in SDS-polyacrylamide gels (3,4,5). The PS I core complex contains one copy of each of the two different 82-kDa polypeptides and one copy of the 18-, 16-, 9,5- and 9-kDa polypeptides per P700 (5). The stoichiometry of the remaining polypeptides of the core complex has not yet been determined. Variable amounts of a 15-kDa polypeptide have also been detected in barley but did not correlate with the activity of the preparation (2). The subunits of photosystem I are of dual genetic origin.

Characterization of a cDNA Clone for the PsaE Gene from Barley and Plasma Desorption Mass Spectrometry of the Corresponding Photosystem I Polypeptide PSI-E

Photosystem I (PS I) preparations from barley contain polypeptides with apparent molecular masses of 82 (PSI-A and PSI-B), 18 (PSI-D), 16 (PSI-E), 14, 9.5 (PSI-H), 9 (PSI-C), 4, and 1.5 kDa (PSI-I) as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (1, 2). The nomenclature used for the subunits is described in more detail by Moller et al. (3). In this paper we report the characterization of a cDNA clone for the PsaE gene encoding the 16-kDa polypeptide PSI-E. The molecular mass of the mature polypeptide is 10,821 Da when deduced from the nucleotide sequence. To test whether the discrepancy between the molecular mass determinations could be due to post-translational modification of the polypeptide, the isolated polypeptide was analyzed by plasma desorption mass spectrometry. It has previously been established that the N- and C-terminal amino acid residues of the PSI-E polypeptide are not modified (4)

Characterization of a cDNA Clone for the PsaH Gene from Barley and mRNA Level of PS I Genes in Light-Induced Barley Seedlings

Photosystem I (PS I) catalyzes the coupled photoreduction of ferredoxin and photooxidation of plastocyanin. PS I core preparations from plants and cyanobacteria has been shown to contain a number of polypeptides encoded by both chloroplast and nuclear genes. So far four different chloroplast genes, designated psaA, psaB, psaC, and psaI, and cDNA clones for four different nuclear genes, designated PsaD, PsaE, PsaF, and PsaG have been characterized from various plants and cyanobacteria. The composition of PS I and the nomenclature used is discussed in detail by Moller et al. (1). A cDNA clone encoding a fifth nuclear encoded PS I polypeptide, designated PSI-H, has now been identified. We here report the nucleotide sequence of the cDNA clone and show that the corresponding gene PsaH and two other PS I genes are induced by light.