Construction of a human cytochrome c gene and its functional expression in Saccharomyces cerevisiae

The nucleotide sequences of a partial cDNA and three pseudogenes of human cytochrome c were determined. The complete nucleotide sequences which encode human cytochrome c were constructed on the basis of one of the pseudogenes by in vitro mutagenesis. The constructed human cytochrome c was functionally expressed in Saccharomyces cerevisiae. The recombinant human cytochrome c was purified and characterized.     

Expression of the Escherichia coli recA gene in the yeast Saccharomyces cerevisiae

The isolation of the protein coding region of the recA gene from Escherichia coli by extensive Bal31 digestion is described. The structural recA gene was ligated into an extrachromosomally replicating yeast expression vector, downstream of the yeast alcohol-dehydrogenase gene promoter region, to produce pADHrecA plasmid. The pADHrecA plasmid was transformed into the wild-type and the repair deficient strains of Saccharomyces cerevisiae. The crude protein samples were extracted from the individual yeast transformants. A 38 kDa protein was present in all transformants containing the recA gene on plasmid. Thus the recA gene from E coli was successfully expressed in cells from a lower eukaryote.     

Expression and function of a human initiator tRNA gene in the yeast Saccharomyces cerevisiae.

We showed previously that the human initiator tRNA gene, in the context of its own 5'- and 3'-flanking sequences, was not expressed in Saccharomyces cerevisiae. Here we show that switching its 5'-flanking sequence with that of a yeast arginine tRNA gene allows its functional expression in yeast cells. The human initiator tRNA coding sequence was either cloned downstream of the yeast arginine tRNA gene, with various lengths of intergenic spacer separating them, or linked directly to the 5'-flanking sequence of the yeast arginine tRNA coding sequence. The human initiator tRNA made in yeast cells can be aminoacylated with methionine, and it was clearly separated from the yeast initiator and elongator methionine tRNAs by RPC-5 column chromatography. It was also functional in yeast cells. Expression of the human initiator tRNA in transformants of a slow-growing mutant yeast strain, in which three of the four endogenous initiator tRNA genes had been inactivated by gene disruption, resulted in enhancement of the growth rate. The degree of growth rate enhancement correlated with the steady-state levels of human tRNA in the transformants. Besides providing a possible assay for in vivo function of mutant human initiator tRNAs, this work represents the only example of the functional expression of a vertebrate RNA polymerase III-transcribed gene in yeast cells.     

Expression of an Escherichia coli phr gene in the yeast Saccharomyces cerevisiae

Summary A 2 kb DNA fragment, containing the photoreactivation gene phr1 from Escherichia coli, was inserted at the BamH1 site in the tet gene of the yeast — E. coli shuttle vector pJDB207. Photoreactivation — deficient Saccharomyces cerevisiae cells transformed with this plasmid showed photoreactivation of killing after UV irradiation of the cells, while extracts of transformed cells exhibited photoreactivating activity in vitro. Far more photoreactivating enzyme molecules were found when the gene was inserted in the plasmid in the opposite orientation to the tet gene as compared with a plasmid carrying the inserted gene in the same orientation. Photoreactivating enzyme encoded by the E. coli phr1 gene and produced in transformed yeast cells has characteristics of the E. coli photoreactivating enzyme (flavoprotein) as judged from the influence of ionic strength on photoreactivating activity.     

Proteases and caspase-like activity in the yeast Saccharomyces cerevisiae

A variety of proteases have been implicated in yeast PCD (programmed cell death) including the metacaspase Mca1 and the separase Esp1, the HtrA-like serine protease Nma111, the cathepsin-like serine carboxypeptideases and a range of vacuolar proteases. Proteasomal activity is also shown to have an important role in determining cell fate, with both pro- and anti-apoptotic roles. Caspase 3-, 6- and 8-like activities are detected upon stimulation of yeast PCD, but not all of this activity is associated with Mca1, implicating other proteases with caspase-like activity in the yeast cell death response. Global proteolytic events that accompany PCD are discussed alongside a consideration of the conservation of the death-related degradome (both at the level of substrate choice and cleavage site). The importance of both gain-of-function changes in the degradome as well as loss-of-function changes are highlighted. Better understanding of both death-related proteases and their substrates may facilitate the design of future antifungal drugs or the manipulation of industrial yeasts for commercial exploitation.     

Circular dichroism studies on cytochrome c peroxidase from baker's yeast (Saccharomyces cerevisiae).

Circular dichroism spectra of cytochrome c peroxidase from baker's yeast, those of the reduced enzyme, the carbonyl, cyanide and fluoride derivatives and the hydrogen peroxide compound, Compound I, have been recorded in the wavelength range 200 to 660 nm. All derivatives show negative Soret Cotton effects. The results suggest that the heme group is surrounded by tightly packed amino acid sidechains and that there is a histidine residue bound to the fifth coordination site of the heme iron. The native ferric enzyme is probably pentacoordinated. The circular dichroism spectra of the ligand compounds indicate that the ligands form a nonlinear bond to the heme iron as a result of steric hindrance in the vicinity of the heme. The spectrum of Compound I shows no perturbation of the porphyrin symmetry. The dichroic spectrum of the native enzyme in the far-ultraviolet wave-length region suggests that the secondary structure consists of roughly equal amounts of alpha-helical, beta-structure and unordered structure. After the removal of the heme group no great changes in the secondary structure can be observed.     

Expression in Escherichia coli of the Saccharomyces cerevisiae CCT gene encoding cholinephosphate cytidylyltransferase.

The coding region of the CCT gene from the yeast Saccharomyces cerevisiae was cloned into the pUC18 expression vector. The plasmid directed the synthesis of an active cholinephosphate cytidylyltransferase in Escherichia coli, confirming that CCT is the structural gene for this enzyme. The enzyme produced in E. coli efficiently utilized cholinephosphate and N,N-dimethylethanolaminephosphate, but N-methylethanolamine-phosphate and ethanolaminephosphate were poor substrates. Consistently, disruption of the CCT locus in the wild-type yeast cells resulted in a drastic decrease in activities with respect to the former two substrates. When activity was expressed in E. coli, over 90% was recovered in the cytosol, whereas most of the activity of yeast cells was associated with membranes, suggesting that yeast cells possess a mechanism that promotes membrane association of cytidylyltransferase.     

Expression of modified cytochrome c1 genes and restoration of the respiratory function in a yeast mutant lacking the nuclear cytochrome c1 gene

Yeast cytochrome c1 is a component of complex III, an oligomeric enzyme of the mitochondrial respiratory chain. In order to investigate the structural requirement of cytochrome c1 for the function and assembly of the enzyme, we used an in vivo complementation assay to determine whether or not an in vitro mutated cytochrome c1 is functional. A yeast mutant whose nuclear cytochrome c1 gene was specifically inactivated was constructed by means of a gene disruption technique. The mutant was unable to respire, and lacked spectrally and immunochemically detectable cytochrome c1. These defects disappeared on the introduction of a plasmid carrying the cytochrome c1 gene coding the wild-type molecule or one coding a mutant molecule lacking the carboxyl (C)-terminal 17 amino acid residues. On the other hand, another mutant gene with a deletion corresponding to the C-terminal 71 residues showed no such ability. These results suggest that the region between the C-terminal 17 and 71 residues is necessary for the function of cytochrome c1.     

Pet191 is a cytochrome c oxidase assembly factor in Saccharomyces cerevisiae

The twin-Cx(9)C motif protein Pet191 is essential for cytochrome c oxidase maturation. The motif Cys residues are functionally important and appear to be present in disulfide linkages within a large oligomeric complex associated with the mitochondrial inner membrane. The import of Pet191 differs from that of other twin-Cx(9)C motif class of proteins in being independent of the Mia40 pathway.     

The gene encoding the biotin-apoprotein ligase of Saccharomyces cerevisiae

Abstract We report the isolation, genomic mapping, and DNA sequence of the BPL1 gene encoding the biotin-apoprotein ligase of Saccharomyces cerevisiae . The gene was isolated by complementation of an Escherichia coli birA (biotin-apoprotein ligase) mutant indicating that the expressed yeast protein modified the essential biotinated protein of the bacterial host. The BPL1 gene encodes a protein of 690 residues ( M _r 76.4 kDa) with strong sequence similarites to the E. coli and human biotin-apoprotein ligases. BPL1 was mapped to chromosome IV, is allelic to the previously described ACC2 gene, and encodes the major (if not the only) biotin-apoprotein ligase activity of S. cerevisiae .     

Cloning of a gene encoding choline transport in Saccharomyces cerevisiae.

Sulfur isotope effects during the oxidation of thiosulfate by Thiobacillus versutus were found to be negligible. This result is considered in relation to other oxidative and reductive processes to assess which reactions are most likely to control the isotopic compositions of sulfur compounds in microbial sulfureta.