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J F Ernst

Publications and source records attributed to J F Ernst.

At least 19 recordsLinked to original sources
PubMed1992

Specific in vitro O-glycosylation of human granulocyte-macrophage colony-stimulating-factor-derived peptides by O-glycosyltransferases of yeast and rat liver cells.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is O-glycosylated at residues Ser9 and Thr10 during secretion by yeast and COS-1 cells [Ernst, J.F., Mermod, J.-J. and Richman, L.I. (1992) Eur. J. Biochem. 203, 663-667]. Two types of octapeptides encompassing residues 4-11 (peptide 4-11) and variants thereof, or residues 8-15 (peptide 8-15) of hGM-CSF were tested as substrates for in vitro O-glycosylation using dolichyl-phosphate- D-mannose: protein O-D-mannosyltransferase (Man-transferase) of the yeast Saccharomyces cerevisiae, or UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) of rat liver cells. Peptide 8-15 was found to be O-glycosylated at residues Ser9 and Thr10 by GalNAc-transferase and, with reduced efficiency, also by Man-transferase. Peptide 4-11 was a good substrate for yeast Man-transferase, leading to mannosylation of only Thr10, whereas it was very poorly O-glycosylated at positions Ser5 and Ser7 by GalNAc-transferase. The observed differences in peptide-acceptor activities indicate that the site of O-glycosylation depends on similar, but not identical protein structural features in yeast and mammalian cells.

Amino Acid Sequence↗
PubMed1992

Site-specific O-glycosylation of human granulocyte/macrophage colony-stimulating factor secreted by yeast and animal cells.

To compare the site specificity of O-glycosylation in lower and higher eukaryotes, we expressed human granulocyte/macrophage colony-stimulating factor (hGM-CSF) in the yeast Saccharomyces cerevisiae and in COS-1 cells. Analyses of specific hGM-CSF mutants secreted by yeast led to the conclusion that efficient O-glycosylation in yeast requires residues S9 and T10. However, only S9 is used as an attachment point for an extended O-glycosyl chain in a 15.5-kDa hGM-CSF form. A 14.5-kDa hGM-CSF form, secreted by yeast, appears substituted by single mannosyl residues at both positions S9 and T10, indicating that O-glycosylation at T10 inhibits extension of the O-glycosyl chain attached to S9. As in yeast cells, the addition of O-glycosyl chains to hGM-CSF secreted by COS-1 cells requires the presence of S9 and T10 residues. These results demonstrate that, inspite of different biosynthetic routes, the selection of O-glycosylation sites is similar between lower and higher eukaryotes.

Amino Acid Sequence↗
PubMed1992

beta-Galactosidase of Kluyveromyces lactis (Lac4p) as reporter of gene expression in Candida albicans and C. tropicalis.

Vectors containing fusions of the Candida albicans ACT promoter to heterologous genes were constructed and transformed into a C. albicans host strain. beta-Galactosidase (Lac4p) activity was detected in transformants carrying an ACT fusion to the Kluyveromyces lactis LAC4 gene, while fusions to the Escherichia coli lacZ gene and to other heterologous genes were not expressed. Lac4p was also produced by C. tropicalis transformants carrying the ACT/LAC4 fusion. Plasmids in transformed C. albicans strains were present either as free multimers in high copy number or, more frequently, integrated into the genome in low copy number yielding high and low LAC4 mRNA and Lac4p expression levels, respectively. Lac4p-expressing transformants of C. tropicalis, but not of C. albicans, were able to utilize lactose as sole carbon source. An ACT/LAC4 fusion was not differentially expressed during the yeast and hyphal growth phases of C. albicans, indicating that the ACT promoter is not regulated during morphogenesis. These results define the first reporter gene system for convenient monitoring of gene expression in Candida species.

Base Sequence↗
PubMed1990

Molecular genetics of pathogenic fungi: some recent developments and perspectives.

The diagnosis and the treatment of fungal diseases remains problematic in many cases. Difficulties in diagnosis are due (1) to the ubiquitous presence of fungal pathogens that may lead to false positive test results and (2) to difficulties in the evaluation of the aetiological significance of these pathogens. The relatively small number of effective antifungal agents reflects to a large extent on the fact that many aspects of fungal physiology and virulence are not well understood. The methods of molecular genetics provide effective tools for the diagnosis of mycoses and may also contribute to the identification of new targets for antifungals by genetic analyses of fungal virulence. During the last 3 years molecular genetic methods have been developed for the asexual pathogen Candida albicans that may be used for strain identification. This success indicates a general use of molecular genetics for the analysis of fungal pathogenesis.

Candida albicans↗
PubMed1989

Sequence and transcript analysis of the C. albicans URA3 gene encoding orotidine-5'-phosphate decarboxylase.

The human pathogen Candida albicans grows either as a yeast or in filamentous form. We have determined the sequence of a 1.365 kb genomic C. albicans fragment that complements Saccharomyces cerevisiae ura3 and Escherichia coli pyrF mutations. An open reading frame within this fragment corresponds to a protein of 270 amino acids that shows homology to orotidine-5'-phosphate decarboxylases (ODCases) of other fungal species. The C. albicans ODCase is most closely related to the ODCases of the budding yeasts Kluyveromyces lactis and S. cerevisiae (74% and 71% homology, respectively). Most 5' ends of URA3 transcripts in the authentic host and in the heterologous host S. cerevisiae were found to be identical. These results demonstrate a close taxonomic relationship between non-pathogenic budding yeasts and C. albicans.

Amino Acid Sequence↗
PubMed1988

Coupling of heme attachment to import of cytochrome c into yeast mitochondria. Studies with heme lyase-deficient mitochondria and altered apocytochromes c.

Cytochrome c is synthesized in the cytoplasm as apocytochrome c, lacking heme, and then imported into mitochondria. The relationship between attachment of heme to the apoprotein and its import into mitochondria was examined using an in vitro system. Apocytochrome c transcribed and translated in vitro could be imported with high efficiency into mitochondria isolated from normal yeast strains. However, no import of apocytochrome c occurred with mitochondria isolated from cyc3- strains, which lack cytochrome c heme lyase, the enzyme catalyzing covalent attachment of heme to apocytochrome c. In addition, amino acid substitutions in apocytochrome c at either of the 2 cysteine residues that are the sites of the thioether linkages to heme, or at an immediately adjacent histidine that serves as a ligand of the heme iron, resulted in a substantial reduction in the ability of the precursor to be translocated into mitochondria. Replacement of the methionine serving as the other iron ligand, on the other hand, had no detectable effect on import of apocytochrome c in this system. Thus, covalent heme attachment is a required step for import of cytochrome c into mitochondria. Heme attachment, however, can occur in the absence of mitochondrial import since we have detected CYC3-encoded heme lyase activity in solubilized yeast extracts and in an Escherichia coli expression system. These results suggest that protein folding triggered by heme attachment to apocytochrome c is required for import into mitochondria.

Apoproteins↗
PubMed1988

Multiple base-pair mutations in yeast.

The nucleotide changes associated with both forward and reverse mutations at the CYC1 locus in the yeast Saccharomyces cerevisiae have been investigated by sequencing the mutated gene product, iso-1-cytochrome c and, more directly, by sequencing appropriate DNA segments. Although the majority of these mutations are the result of single base-pair changes, approximately 10% are the result of multiple mutations and these occur predominantly at certain sites and with certain patterns. Most multiple base-pair changes occur within 20 nucleotides of each other and are generally within six nucleotides. On the basis of the frequencies and patterns of mutations, these nucleotide changes are considered to have occurred as single, concerted events, rather than as multiple independent mutations. Analysis of these mutations indicates that multiple base-pair changes can arise by widely differing mechanisms. We have recognized the following classes of mutations: multiple base-pair changes that yield (1) direct repeats or (2) inverted repeats of local DNA sequences; (3) substitutions of two tandem base-pairs; (4) frameshift and contiguous single base-pair substitutions; and (5) recombination of the CYC1 gene with a non-allelic gene, resulting in alterations within contiguous segments that can be over 150 nucleotides in length. Some of the multiple base-pair changes do not fall into any of these categories. We suggest mechanisms to account for each of these five classes.

Amino Acid Sequence↗
PubMed1988

Efficient secretion and processing of heterologous proteins in Saccharomyces cerevisiae is mediated solely by the pre-segment of alpha-factor precursor.

A novel processing site was identified in fusions of the alpha-factor precursor of Saccharomyces cerevisiae following its 19 amino-terminal residues (pre-segment). Fusions of the pre-segment to heterologous proteins, including aminoglycoside phosphotransferase (APH) and human granulocyte-macrophage colony stimulating factor (hGM-CSF), were as efficiently secreted and processed as corresponding pre-pro fusions. Pre- and pre-pro fusions to hGM-CSF were identically N- and O-glycosylated. While pre-pro fusions to interleukin-1 beta were not cleaved, pre-fusions were correctly processed during secretion. The high secretion efficiency of pre-fusions suggests that the pro-segment of the alpha-factor precursor is not required for efficient secretion and processing of protein fusions.

Genetic Vectors↗
PubMed1988

Differential mismatch repair can explain the disproportionalities between physical distances and recombination frequencies of cyc1 mutations in yeast.

Recombination rates have been examined in two-point crosses of various defined cyc1 mutations that cause the loss or nonfunction of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. Recombinants arising by three different means were investigated, including X-ray induced mitotic recombination, spontaneous mitotic recombination, and meiotic recombination. Heteroallelic diploid strains were derived by crossing cyc1 mutants containing a series of alterations at or near the same site to cyc1 mutants containing alterations at various distances. Marked disproportionalities between physical distances and recombination frequencies were observed with certain cyc1 mutations, indicating that certain mismatched bases can significantly affect recombination. The marker effects were more pronounced when the two mutational sites of the heteroalleles were within about 20 base pairs, but separated by at least 4 base pairs. Two alleles, cyc1-163 and cyc1-166, which arose by G.C----C.G transversions at nucleotide positions 3 and 194, respectively, gave rise to especially high rates of recombination. Other mutations having different substitutions at the same nucleotide positions were not associated with abnormally high recombination frequencies. We suggest that these marker effects are due to the lack of repair of either G/G or C/C mismatched base pairs, while the other mismatched base pair of the heteroallele undergoes substantial repair. Furthermore, we suggest that diminished recombination frequencies are due to the concomitant repair of both mismatches within the same DNA tract.

Alleles↗
PubMed1987

Identification and sequence of the gene encoding cytochrome c heme lyase in the yeast Saccharomyces cerevisiae.

Mitochondrial cytochrome c contains a heme group covalently attached through thioether linkages to two cysteinyl residues of the protein. We demonstrate here that the nuclear gene, CYC3, in the yeast Saccharomyces cerevisiae, encodes cytochrome c heme lyase (CCHL), the enzyme catalyzing the attachment of heme to apocytochrome c. Mitochondrial extracts from cyc3- mutants are deficient in CCHL activity compared with extracts from normal strains, whereas strains carrying multiple copies of the CYC3 gene exhibit high levels of the activity. The CYC3 gene was cloned by functional complementation of a cyc3- mutant using a previously isolated plasmid containing the gene PYK1, which is tightly linked to CYC3. An open reading frame encoding a protein of 269 amino acids was identified from the DNA sequence of a fragment encompassing the CYC3 gene, and the corresponding transcript shown to be approximately 0.9 kb in length. CCHL appears to be a single polypeptide chain which acts specifically on the two forms of cytochrome c, but not on cytochrome c1.

Amino Acid Sequence↗
PubMed1987

Increased biological activity of deglycosylated recombinant human granulocyte/macrophage colony-stimulating factor produced by yeast or animal cells.

Human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced by several recombinant sources including Escherichia coli, yeast, and animal cells was studied. Recombinant animal cells produced hGM-CSF in low quantities and in multiple forms of varying size. Mammalian hGM-CSF was purified 200,000-fold using immunoaffinity and lectin chromatography. Partially purified proteins produced in yeast and mammalian cells were assayed for the effects of deglycosylation. Following enzymatic deglycosylation, immunoreactivity was measured by radioimmunoassay and biological activity was measured in vitro on responsive human primary cells. Removal of N-linked oligosaccharides from both proteins increased their immunoreactivities by 4- to 8-fold. Removal of these oligosaccharides also increased their specific biological activities about 20-fold, to reach approximately the specific activity of recombinant hGM-CSF from E. coli. The E. coli produced-protein--lacking any carbohydrate--had by far the highest specific activity observed for the recombinant hGM-CSFs.

Animals↗
PubMed1986

Improved secretion of heterologous proteins by Saccharomyces cerevisiae: effects of promoter substitution in alpha-factor fusions.

The effects of promoter strength on secretion of a heterologous protein, somatomedin-C (SMC), by the yeast Saccharomyces cerevisiae were studied by using the promoters of the MF alpha 1, ACT, and CYC1 genes to control expression of alpha-factor/SMC gene fusions. When a low-copy centromere vector was used to carry the gene fusions in yeast transformants, the greatest secretion was obtained with the MF alpha 1 promoter construction and the least with the CYC1 promoter construction. Unexpectedly, using two types of multicopy vectors, the greatest secretion was obtained with the CYC1 promoter construction and the least with the MF alpha 1 promoter construction. The decrease in secretion by the strongest promoter construction (MF alpha 1 promoter) on multicopy vectors was associated with a decrease in SMC mRNA during growth, a decrease in vector copy number, a decrease in vector stability, and a decrease in transformation frequency. The results demonstrate that, unlike in intracellular expression, promoter strength is not simply related to secretion expression levels. Selection against oversecreting cells during growth may explain the reduced secretion efficiency of strong promoter constructions.

Actins↗
PubMed1985

Substitutions of proline 76 in yeast iso-1-cytochrome c. Analysis of residues compatible and incompatible with folding requirements.

Fine-structure genetic mapping previously revealed numerous nonfunctional cyc1 mutations having alterations at or near the site corresponding to amino acid position 76 of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae. DNA sequencing of the alterations in four of these cyc1 mutations indicated that the normal Pro-76 was replaced by Leu-76. Revertants containing at least partially functional iso-1-cytochromes c were isolated, and the alterations were analyzed by DNA sequencing and protein analysis. Specific activities of the altered iso-1-cytochromes c were estimated in vivo by growth of the strains in lactate medium; compared to normal iso-1-cytochrome c with Pro-76, the following activities were associated with the following replacements: approximately 90% for Val-76, approximately 60% for Thr-76, approximately 30% for Ser-76, approximately 20% for Ile-76, and 0% for Leu-76. In order to develop an understanding of the factors that determine whether or not an altered iso-1-cytochrome c will function, we undertook a theoretical analysis which led to the conclusion that the activity of the proteins was dependent on both short- and long-range interactions. Short-range interactions were estimated from studies on known protein structures which gave the likelihood that various amino acids would be found in a local backbone configuration similar to the native protein; long-range interactions with the rest of the molecule were analyzed by considering the size of the side chain. We believe this approach can be used to analyze a wide variety of mutant proteins.

Amino Acid Sequence↗
PubMed1985

DNA sequences of frameshift and other mutations induced by ICR-170 in yeast.

ICR-170-induced mutations in the CYC1 gene of the yeast Saccharomyces cerevisiae were investigated by genetic and DNA sequence analyses. Genetic analysis of 33 cyc1 mutations induced by ICR-170 and sequence analysis of eight representatives demonstrated that over one-third were frameshift mutations that occurred at one site corresponding to amino acid positions 29-30, whereas the remaining mutations were distributed more-or-less randomly, and a few of these were not frameshift mutations. The sequence results indicate that ICR-170 primarily induces G.C additions at sites containing monotonous runs of three G.C base pairs. However, some (Formula: see text) sites within the CYC1 gene were not mutated by ICR-170. Thus, ICR-170 is a relatively specific mutagen that preferentially acts on certain sites with monotonous runs of G.C base pairs.

Amino Acid Sequence↗
PubMed1985

Characterization of Saccharomyces cerevisiae mutants supersensitive to aminoglycoside antibiotics.

We describe mutants of Saccharomyces cerevisiae that are more sensitive than the wild type to the aminoglycoside antibiotics G418, hygromycin B, destomycin A, and gentamicin X2. In addition, the mutants are sensitive to apramycin, kanamycin B, lividomycin A, neamine, neomycin, paromomycin, and tobramycin--antibiotics which do not inhibit wild-type strains. Mapping studies suggest that supersensitivity is caused by mutations in at least three genes, denoted AGS1, AGS2, and AGS3 (for aminoglycoside antibiotic sensitivity). Mutations in all three genes are required for highest antibiotic sensitivity; ags1 ags2 double mutants have intermediate antibiotic sensitivity. AGS1 was mapped 8 centimorgans distal from LEU2 on chromosome III. Analyses of yeast strains transformed with vectors carrying antibiotic resistance genes revealed that G418, gentamicin X2, kanamycin B, lividomycin A, neamine, and paromomycin are inactivated by the Tn903 phosphotransferase and that destomycin A is inactivated by the hygromycin B phosphotransferase. ags strains are improved host strains for vectors carrying the phosphotransferase genes because a wide spectrum of aminoglycoside antibiotics can be used to select for plasmid maintenance.

Aminoglycosides↗
PubMed1981

The cyc1-11 mutation in yeast reverts by recombination with a nonallelic gene: composite genes determining the iso-cytochromes c.

DNA sequence analysis of a cloned fragment directly established that the cyc1-11 mutation of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae is a two-base-pair substitution that changes the CCA proline codon at amino acid position 76 to a UAA nonsense codon. Analysis of 11 revertant proteins and one cloned revertant gene showed that reversion of the cyc1-11 mutation can occur in three ways: a single base-pair substitution, which produces a serine replacement at position 76; recombination with the nonallelic CYC7 gene of iso-2-cytochrome c, which causes replacement of a segment in the cyc1-11 gene by the corresponding segment of the CYC7 gene; and either a two-base-pair substitution or recombination with the CYC7 gene, which causes the formation of the normal iso-1-cytochrome c sequence. These results demonstrate the occurrence of low frequencies of recombination between nonallelic genes having extensive but not complete homology. The formation of composite genes that share sequences from nonallelic genes may be an evolutionary mechanism for producing protein diversities and for maintaining identical sequences at different loci.

Amino Acid Sequence↗