Carlsberg Research Communications最新文献

The 9-kDa thylakoid polypeptide which in vivo carries the iron-sulfur centers A and B of photosystem I was isolated from barley (Hordeum vulgare L.) and the complete amino acid sequence determined. The polypeptide shows a very high degree of homology with the corresponding polypeptides in other plant species. The polypeptide is not post-translationally processed except for the removal of the N-terminal formyl-methionine and the insertion of the iron-sulfur centers.

Proteinase yscA is an intracellular aspartic proteinase located in the lysosome-like vacuole of the yeast cell. The specificity towards denatured protein substrates was determined by separation and identification of cleavage products after digestions with proteinase yscA, and compared to that obtained with pepsin used under similar conditions. Proteinase yscA is more selective towards the peptide bonds it cleaves than pepsin, but shows the same preference for large hydrophobic residues on both sides of the cleaved bond as pepsin and lysosomal cathepsin D. Phe, Leu and Glu are favoured in substrate subsite P1 and Phe, Ile, Leu and Ala in P'1, whereas Val is unfavoured in P'1. The implications for the role of proteinase yscA as hydrolase maturase are discussed.

The two cytochrome b-559 apoproteins of 9.4 kD and 4.5 kD molecular weight have been expressed in vitro using DNA templates containing either the two genes psbE and psbF in tandem or the individual genes. Transcription with E. coli RNA-polymerase or SP6 RNA-polymerase has been followed by translation in E. coli derived lysates. Simultaneous as well as independent synthesis of the apoproteins is possible. A 9.4 kD in vitro translation product has been identified as apoprotein I by immunoprecipitation with a monoclonal antibody specific for the C-terminal part of the 9.4 kD apoprotein of cytochrome b-559. The isolated psbF gene directs the synthesis of a translation product with a molecular weight of 4.5 kD corresponding to apoprotein II. Expression of the psbE gene requires the presence of endogenous regulatory sequences 5' upstream of psbE, while this is not the case for psbF. Additional in vitro translation products of 5.7 and 2.4 kD molecular weights are synthesized and probably translated from two reading frames starting with two different out-of-phase ATG codons in the nucleotide sequence of the psbE gene.

The fabB gene of E. coli encoding beta-ketoacyl-ACP synthase I has been isolated by complementation and sequenced. The enzyme has been purified and its NH2-terminal residues sequenced. Identification of the active site was accomplished by tagging with 3H-cerulenin and radio sequencing of the region. Comparison of the deduced primary structures of the fabB gene product with the FAS2 gene product of Saccharomyces cerevisiae revealed the probable active site in chalcone synthases of higher plants.

Saccharomyces carlsbergensis strains used in the production of lager beer are structurally heterozygous in most genetic loci studied to date. Previous studies have shown that the genotype of lager yeast contains two types of genomes, one of which is derived from S. cerevisiae and the other reveals similarities to the genomes of S. bayanus and S. monacensis. Genes of homeologous chromosomes can be distinguished by characteristic restriction fragment patterns. This is true also for the LEU2 genes which encode the beta-isopropylmalate dehydrogenase and are located on chromosomes III. In the present work a LEU2 gene from S. carlsbergensis has been cloned and characterized. The cloned 2.6 kb LEU2 region complements the S. cerevisiae leu2-3 leu2-112 double mutation. The restriction endonuclease site map of the isolated S. carlsbergensis LEU2 gene is different from that of the S. cerevisiae LEU2 gene. Electrophoretic chromosome separation, as well as karl mediated transfer of single chromosomes into S. cerevisiae strains, has shown that the S. carlsbergensis specific LEU2 gene is located on a chromosome III which carries the carlsbergensis specific HIS4 gene. The cloned LEU2 gene shows preferential molecular hybridization to one of the two LEU2 structural alleles present in lager strains, an allele which is also present in type strains of S. bayanus, S. carlsbergensis, S. monacensis and S. uvarum.

The psbA, psbD and psbC genes encoding the quinone binding D-1 and D-2 apoproteins and the 44 kD chlorophyll a-apoprotein 3 have been located in the chloroplast genome of barley. They are found on a 23 kbp SalI restriction endonuclease fragment in the large single copy region of the chloroplast DNA adjacent to the inverted repeat. As in other species the psbD and psbC genes have reading frames which overlap by 53 bp. They are transcribed in the same direction but translated with a frameshift of one nucleotide. Ten amino acid substitutions are found among the 18 N-terminal residues of the D-2 polypeptide if barley, spinach, tobacco, pea and the liverwort Marchantia are compared. Only 8 substitutions are present among the 335 other residues of the D-2 polypeptide. The amino acid residues located in the putative binding site for the special pair reaction center chlorophyll and the residues probably serving as ligands to non-heme iron in the D-1 and D-2 proteins of barley are strictly conserved when compared to those of purple bacteria and of other higher plants. Identity is also observed for the residues of importance in the binding of quinones.