The entire DNA sequence of chromosome III of the yeast Saccharomyces cerevisiae has been determined. This is the first complete sequence analysis of an entire chromosome from any organism. The 315-kilobase sequence reveals 182 open reading frames for proteins longer than 100 amino acids, of which 37 correspond to known genes and 29 more show some similarity to sequences in databases. Of 55 new open reading frames analysed by gene disruption, three are essential genes; of 42 non-essential genes that were tested, 14 show some discernible effect on phenotype and the remaining 28 have no overt function.
We have cloned cDNAs of the tobacco homolog of the luminal binding protein (BiP) that has been described in other higher eukaryotes. In contrast to the mammalian and yeast protein, tobacco BiP is encoded by a multigene family. The gene products of all the cloned members of this family contain a carboxy-terminal His-Asp-Glu-Leu peptide that may form the signal for retention in the endoplasmic reticulum. Analysis of expression patterns revealed that BiP transcripts are predominantly present in tissues with high rates of cell divisions, in secretory tissues, and in cells treated with tunicamycin. We also show that a chimeric gene containing the coding region of one of the tobacco BiP genes is able to complement a mutation in the Saccharomyces cerevisiae BiP gene.
The coding region of the human interleukin-6 (hIL6) gene was fused to the prepro secretion signal of the amating factor gene in several yeast host strains. It was found that the KEX-2 protease was unable to cleave the prepro-Lys-Arg-Pro-IL6 sequence, but that unspecific cleavage of the precursor protein had occurred. The preproLys-Arg-Ala-Pro-IL6 sequence, however, was correctly recognized and cleaved by the KEX-2 protease, and IL6 was efficiently secreted into the culture medium. The N-terminal Ala-Pro peptide was removed during processing by wild-type yeast strains, but was retained in a stel3 mutant. IL6 as well as the aberrant proteins were not glycosylated. The transformed cells could secrete up to 30 pg/ml IL6. The protein was purified from the medium to homogeneity by ion-exchange chromatography and gel filtration, and had a specific activity of about 2 x 10' IU/ mg in a proliferation assay.Human interleukin-6 (hIL6), a protein with a variety of biological activities, affects B-cells and induces the production of acute-phase proteins by hepatocytes/hepatoma cells [1 -31. Further detailed investigation of these and other functions [4, 51 depends on the availability of a pure and biologically active product. When the hIL6 gene is expressed in Escherichia coli, the unfolded product precipitates in the form of inclusion bodies, creating problems of solubilization, refolding and purification [6]. Therefore, we preferred to produce hIL6 in a fully functional form, which we achieved using an efficient secretion system in Saccharomyces cerevisiae.Several plasmid constructions were made and transformed into different yeast strains. A plasmid containing the highly efficient, inducible GAL1 promoter was chosen for further use. This promoter was followed by the a-mating factor (MF) secretion signal, a system frequently used for the expression of mammalian immunoregulating factors [7 -lo], fused to mature hIL6. The yeast genes involved in the processing of the protein precursors are well known, and mutants in these functions are available [ll -181. MATERIALS AND METHODSStrains, culture media and fermentation E. coli strains HBlOl [19] and MC1061 [20] were used for production of plasmid constructions. The S. cerevisiae strains used are shown in Table 1.The yeast cells were grown at 28 "C in one of the following media, made up with distilled water. Rich yeast/peptane/dextrose (used for routine growth of untransformed cells) was prepared as follows: A solution of 1% Bacto yeast extract (Difco) and 2% Bacto peptone (Difco), supplemented with 4% Bacto casamino acids (Difco) and autoclaved; dextrose was added from a sterile stock solution to a final concentration of 2%.After culture, the yeast cells were transformed by the lithium acetate method [23]. The transformed yeast cells were plated, using a mixture of 3% agar, 0.67% Bacto yeast nitrogen base (Difco), 0.2% Bacto casamino acids (Difco) and 2% glucose was autoclaved for maximum 15 min and used as a solid culture medium in Petri dishes. Plusmid constructions and prepa...
The nucleotide sequence of a 1558 bp DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae contains an open reading frame of 954 nucleotides with coding potential for a protein with high similarity to the ubiquitous cyclophilins which are both peptidyl-prolyl cis-trans isomerases and cyclosporin A-binding proteins. It should, therefore, represent the third gene (SCC3) of this kind from S. cerevisiae. SCC3 is present in a single copy in the genome of S. cerevisiae and results in a constitutively expressed 1.2 kb transcript during cell growth. Its putative protein product (Scc3) contains two hydrophobic cores, one at the amino terminal, 20 amino acids long, which could serve as a signal peptide, and the other one at the carboxyl end with a structure similar to a transmembrane helix. These findings suggest that Scc3 could be a secretory or, more likely, a transmembrane protein. The only cyclophilin with similar structure to that of Scc3 is ninaA from Drosophila melanogaster, a transmembrane protein which seems to be implicated in the correct folding and/or intercalation of rhodopsin in the endoplasmic reticulum of the fly photoreceptors (Stamnes, M.A. et al., Cell 65, 219-227, 1991). In addition, the amino and the carboxy regions of Scc3 and ninaA share a significant level of homology, which suggests that they have a similar function, albeit for different target proteins.
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