No abstract

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. Photosystem I; 16 kDa polypeptide; cDNA sequence; Transit peptide; (Barley)

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.

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 membranespanning region with a predicted alpha-helix secondary structure. The gene for the 9.5 kDa polypeptide has been designated PsaH.

A simple deletion of the URA3 promoter from a Saccharomyces cerevisiae expression plasmid was performed. The promoter-deleted plasmid is shown to have an increased expression level of a fungal lipase gene. The deletion probably causes a poor expression of the URA3 selection marker, probably resulting in a higher copy number per cell of the plasmid. This higher copy number can increase the transcript level per cell and there by the expression level. In the case of the fungal lipase gene, the expression level with defined inoculum is increased at least three times. The principle is most likely similar to the LEU2d plasmids described previously. A part of the 2-micron origin of the pYES type plasmid was also deleted by the URA3 promoter deletion without affecting transformation frequency. The URA3 promoter can easily be deleted from most pYES type plasmids by the described method.