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C Palleschi

Publications and source records attributed to C Palleschi.

At least 19 recordsLinked to original sources
PubMed2004

KlPMR1 inactivation and calcium addition enhance secretion of non-hyperglycosylated heterologous proteins in Kluyveromyces lactis.

The Kluyveromyces lactis KlPMR1 gene is the functional homologue of Saccharomyces cerevisiae PMR1 which encodes a Ca(2+)-ATPase localized in the Golgi apparatus. We studied the effects of KlPMR1 inactivation on the glycosylation and secretion of native and heterologous proteins in K. lactis. We used acid phosphatase, recombinant human serum albumin and alpha-glucoamylase from Arxula adeninivorans as reporter proteins. The Klpmr1Delta strain showed enhanced secretion of the heterologous proteins analyzed; the improved rHSA production did not result from enhanced transcription but rather involved increased translation and/or secretion efficiency. The growth rate of mutant cells resulted slower as compared to that of wild-type strain. The addition of 10mM calcium to the culture medium, however, not only completely relieved the growth defect of the mutant cells but also improved the rate of heterologous proteins production. Moreover, the addition of this ion in the culture medium of K. lactis did not suppress the glycosylation defects; this is an important difference with respect to S. cerevisiae where the glycosylation is partially restored by Ca(2+) addition. The Klpmr1Delta strain as a host offers thus an additional advantage for those cases requiring that the produced recombinant protein would not result hyperglycosylated.

Calcium↗
PubMed2000

vga Mutants of Kluyveromyces lactis show cell integrity defects.

We studied the cell wall alterations that occur in mutants of Kluyveromyces lactis impaired in glycosylation. The mutants belong to four complementation groups named vga1 to vga4 (vanadate glycosylation affected), characterized by sodium orthovanadate resistance and alteration of the glycosylation profile of native invertase. A drastic reduction of the alkali-soluble fraction of the beta-D-glucan was observed in vga1, vga2 and vga3 cells, accompanied by an increase in the chitin content of the cell wall. In vga4 cells, both beta-D-glucan fractions (alkali-soluble and alkali-insoluble) were reduced to about half of the corresponding wild-type value but the chitin content was normal. A protein related to Fks1p, the catalytic subunit of the major 1,3-beta-D-glucan synthase of S. cerevisiae, was detected in K. lactis. The amount of this Fks1p-like protein increased 7-10 times in vga1, vga2 and vga3 mutants as compared to wild-type cells; the same strains released significant amounts of beta-D-glucan in the culture supernatant. These mutations also resulted in abnormally thick cell walls with conspicuous irregularities in the structure, as revealed by electron microscopy and by an altered resistance to Zymolyase. The observed high responsiveness of cell wall phenotypes to alterations of glycosylation make K. lactis an attractive system for studying the interconnections between these processes.

Aminoglycosides↗
PubMed2000

Disruption of six novel genes from chromosome VII of Saccharomyces cerevisiae reveals one essential gene and one gene which affects the growth rate.

Six ORFs of unknown function located on chromosome VII of Saccharomyces cerevisiae were disrupted in two different genetic backgrounds, and the phenotype of the generated mutants was analysed. Disruptions of ORFs YGR256w, YGR272c, YGR273c, YGR275w and YGR276c were carried out using the disruption marker kanMX4 flanked by short homology regions, whereas ORF YGR255c was inactivated with a long flanking homology (LFH) disruption cassette (Wach et al., 1994). Tetrad analysis of the heterozygous disruptants revealed that ORF YGR255c, previously identified as COQ6 and encoding a protein involved in the biosynthesis of coenzime Q (Tzagoloff and Dieckmann, 1990), is an essential gene. The same analysis also revealed that sporulation of the ygr272cDelta heterozygous diploid produced two small colonies per ascus that were also G418-resistant, indicating that the inactivation of ORF YGR272c could result in a slower growth rate. This result was confirmed by growth tests of the haploid disruptants and by complementation of the phenotype after transformation with a plasmid carrying the cognate gene. No phenotypes could be associated to the inactivation of ORFs YGR256w, YGR273c, YGR275w and YGR276c. Two of these genes have recently been further characterized: ORF YGR255w, renamed RTT102, encodes a regulator of the Ty1-element transposition, whereas ORF YGR276c was found to encode the 70 kDa RNase H activity and was renamed RNH70 (Frank et al., 1999).

Chromosomes, Fungal↗
PubMed1999

The KlPMR1 gene of Kluyveromyces lactis encodes for a P-type Ca(2+)-ATPase.

A novel P-type Ca(2+)-ATPase gene has been cloned and sequenced in the yeast Kluyveromyces lactis. The gene has been named KlPMR1 and is localized on chromosome I. The putative gene product contains 936 residues and has a calculated molecular weight of 102,437 Da. Analysis of deduced amino acid sequence (KlPmr1p) indicated that the encoded protein retains all the highly conserved domains characterizing the P-type ATPases. KlPmr1p shares 71% amino acid identity with Pmr1p of S. cerevisiae, 62% with HpPmr1p of Hansenula polymorpha, 56% with Y1Pmr1p of Yarrowia lipolytica and 52% with the Ca(2+)-ATPase encoded for by the SPCA1 gene of Rattus norvegicus; these similarities place KlPmr1p in the SPCA group (secretory pathway Ca(2+)-ATPase) of the P-type ATPases. The K. lactis strain harbouring the Klpmr1 disrupted gene is not able to grow in presence of low calcium concentrations and shows hypersensitivity to high concentrations of EGTA in the medium. These defects are relieved by PMR1 of S. cerevisiae on a centromeric plasmid, demonstrating that KlPMR1 encodes for a functional Pmr1p homologue.

Animals↗
PubMed1999

Isolation and characterisation of a ropy Lactobacillus strain producing the exopolysaccharide kefiran.

A capsular-polysaccharide-producing strain, LM-17, was isolated from kefir grains and was identified as a slime-forming, rod-shaped Lactobacillus. According to 1H- and 13C-NMR spectral data, the exopolysaccharide produced by the isolated bacterial strain is identical to the glucogalactan extracted from kefir grains and therefore known as kefiran. The kefiran produced was characterised by means of viscosity, optical rotatory power, circular dichroism and IR spectral measurements. A batch procedure was set up for the culture and extraction of the exopolysaccharide in laboratory conditions, resulting in a yield of 2 g/l purified kefiran from the culture supernatant of the LM-17 strain.

Lactobacillus↗
PubMed1991

Incipient mitochondrial evolution in yeasts. I. The physical map and gene order of Saccharomyces douglasii mitochondrial DNA discloses a translocation of a segment of 15,000 base-pairs and the presence of new introns in comparison with Saccharomyces cerevisiae.

We have determined the physical and genetic map of the 73,000 base-pair mitochondrial genome of a novel yeast species Saccharomyces douglasii. Most of the protein and RNA-coding genes known to be present in the mitochondrial DNA of Saccharomyces cerevisiae have been identified and located on the S. douglasii mitochondrial genome. The nuclear genomes of the two species are thought to have diverged some 50 to 80 million years ago and their nucleo-mitochondrial hybrids are viable but respiratorily deficient. The mitochondrial genome of S. douglasii displays many interesting features in comparison with that of S. cerevisiae. The three mosaic genes present in both genomes are quite different with regard to their structure. The S. douglasii COXI gene has two new introns and is missing the five introns of the S. cerevisiae gene. The S. douglasii cytochrome b gene has one new intron and lacks two introns of the S. cerevisiae gene. Finally, the L-rRNA gene of S. douglasii, like that of S. cerevisiae, has one intron of which the structure is different. Another salient feature of the S. douglasii mitochondrial genome reported here is that the gene order is different in comparison with S. cerevisiae mitochondrial DNA. In particular, a segment of approximately 15,000 base-pairs including the genes coding for COXIII and S-rRNA has been translocated to a position between the genes coding for varl and L-rRNA.

Adenosine Triphosphatases↗
PubMed1991

Mitochondrial genome of Saccharomyces douglasii: genes coding for components of the protein synthetic apparatus.

Mitochondrial genes coding for some components of the protein synthetic apparatus in S. douglasii have been studies in detail. A region containing stretches of high homology to the S. cerevisiae tRNA synthesis locus (TSL) and the tRNA(fmet) gene has been identified and sequenced. The organization of this region was very similar to that present in S. cerevisiae, including the presence of a possible transcription starting signal. The S. douglasii TSL gene is shorter due to several deletions which, however, do not involve the regions coding for RNA domains know to be required for the catalytic activity of mitochondrial RNAse P. The S. douglasii LSU rRNA gene has been shown to contain a typical group I intron highly homologous to its S. cerevisiae counterpart, except for the absence of the open reading frame which in S. cerevisiae codes for I-SceI endonuclease.

Base Sequence↗
PubMed1987

Analysis of transcripts of the major cluster of tRNA genes in the mitochondrial genome of S. cerevisiae.

The transcripts of a 6Kbp region of the mitochondrial genome of S. cerevisiae, localized in the 21S rRNA-OXI1 span and including 12 tRNA genes (from tRNA(thr) to tRNA(ala)) and several G+C clusters, have been studied by analysis of in vitro capped primary transcripts and by fine mapping of the 5' ends of transcripts. The study was performed in the w.t. strain D273-10B and in several rho- mutants retaining different, partially overlapping portions of the studied region; the mutants accumulate incompletely-processed precursors of tRNAs due to the absence of the tRNA synthesis locus. Results show the presence in the region of four sites at which initiation occurs at a consensus nonanucleotide ATTATAAGTA (or a minor variant of the same); however different initiation sites are used in different strains, and several differences as compared to initiation in vitro can also be observed. Termini arising by processing are often localized at AATATAA or AATATATTTT sequences localized immediately adjacent to a G+C cluster or a tRNA sequence.

Base Sequence↗
PubMed1984

Initiation of transcription of a mitochondrial tRNA gene cluster in S. cerevisiae.

In Saccharomyces cerevisiae most mitochondrial tRNA genes are clustered in a 9 kbp region between the cap and oxil genes. Polygenic transcripts of this region have been previously identified. A transcriptional initiation site at a TTATAAGTA box, located upstream from the tRNAcys gene, has now been detected by S1 mapping experiments and by the capping of primary transcripts. Results are consistent with the hypothesis that this box represents the initiation site for transcription of a cluster of tRNA genes, while the adjacent tRNA2thr is cotranscribed with the 21S rRNA. Results obtained with various strains are compared, and the efficiency of this sequence as a transcriptional initiation site in different mitochondrial contexts is discussed.

Base Sequence↗
PubMed1984

Expression of the clustered mitochondrial tRNA genes in Saccharomyces cerevisiae: transcription and processing of transcripts.

The transcripts of a cluster of eight tRNA genes localized in the Cap-oxiI region of the mitochondrial genome of Saccharomyces cerevisiae were investigated by hybridization of gene-specific probes on Northern blots of mitochondrial RNA and by S1 mapping of the 5' termini of the transcripts. Two rho- mutants that lack mature tRNA species and accumulate precursors have been used to detect transcripts that are not detectable in wild-type (w.t.) mitochondria. The results have shown the existence of polygenic transcripts carrying at least 5-7 tRNA sequences, both in w.t. and in rho- strains. The existence of several alternative processing pathways, which involve cleavage at the 3' and 5' ends of the tRNA sequences and in the long intergenic regions (possibly at GC clusters), is suggested. Cleavage at the 5' ends of tRNA sequences is defective in the mutant strains. The transcripts of the genes for tRNAThrACN and tRNACys (the tRNA genes immediately downstream from the 21S rRNA gene) have been analyzed; the possibility that these species represent primary transcripts is considered, and potential sites for initiation of transcription of the clustered tRNA genes are discussed.

Base Sequence↗
PubMed1983

Role of nuclear genes in expression of a mitochondrial tRNA gene in Saccharomyces cerevisiae.

In yeast mitochondria, most of the isoaccepting species of tyrosyl tRNA are coded by a mitochondrial gene, tyrA. A particular isoaccepting species is coded by a second mitochondrial gene, tyrB. This gene is not expressed in certain strains of yeast which show no deficient phenotype. Genetic crosses between strains expressing or not expressing the tyrB gene demonstrate that expression is controlled by specific nuclear genes and that a mutation of the tyrA gene can be bypassed when the tyrB gene is operative.

Cell Nucleus↗
PubMed1982

Transcripts of mitochondrial tRNA genes in Saccharomyces cerevisiae.

The transcription of a group of tRNA genes from the large tRNA gene cluster of mitochondrial DNA from Saccharomyces cerevisiae has been investigated by hybridization with DNA probes carrying tRNA coding sequences and small portions of the A + T rich intergenic regions. Results have shown that in some rho- mutants (DS502, F11) mature tRNA was absent, but a few transcripts could be detected. Some high molecular weight species actually hybridized with DNA probes carrying different tRNA coding sequences. Low molecular weight transcripts (100-150 nucleotides, carrying one tRNA sequence) were also present in these mutants. A high molecular weight transcript was also observed in the wild type, though in much more limited amount. The low molecular weight transcripts were analysed by the S1 mapping technique and found to include both a tRNA sequence and the upstream 5' flanking region extending as far as the 3' end of the preceding tRNA gene. The results suggest the existence of a common transcript bearing several tRNA sequences and indicate a possible mechanism of processing, which might be defective in mutants.

Base Sequence↗
PubMed1979

Two isoaccepting seryl tRNAs coded by separate mitochondrial genes in yeast.

In S. cerevisiae four isoacceptor mitochondrial tRNAs for serine have been separated by reversed phase chromatography. At least two of these species are products of different genes. In this work the deletion mapping technique has been used to locate two genes for tRNAser. The gene for tRNAser previously localized in the oli I region of the mitochondrial genome has been found to code for tRNAser2, and another gene coding for tRNAser1 has been detected in the region where most of other tRNA genes are found. Results of fine mapping experiments allowed to localize this gene in the proximity of the gene for tRNAarg.

Chromatography↗
PubMed1979

Mitochondrial protein-synthesizing machinery in Saccharomyces cerevisiae grown in different metabolic conditions. Variability of seryl-tRNA and alanyl-tRNA isoacceptor patterns.

1. The isoacceptor patterns of mitochondrial seryl and alanyl-tRNAs from Saccharomyces cerevisiae grown under different conditions and committed to fermentative or respiratory metabolism have been studied by reversed-phase chromatography. 2. An extensive variability of the chromatographic patterns of the four isoacceptors for serine and of the three isoacceptors for alanine has been observed as a function of carbon source and stage of growth, but the main differences were observed in the different stages of growth on glucose. 3. In order to distinguish the effects due to stage of growth from those due to relief from glucose repression, the isoacceptor patterns of mitochondrial tRNA were compared in repressed and derepressed resting cells; results show that some of the mitochondrial species are almost undetectable in resting repressed cells, but are the major ones after recovery of respiration. 4. Two of the serine isoacceptors (species 1 and 2), one of which is absent or not acylable in repressed resting cells, are different gene products.

Alanine↗
PubMed1975

Heterogeneity of mitochondrial DNA from Saccharomyces cerevisiae and genetic information for tRNA.

Mitochondrial DNA from wild-type Saccharomyces cerevisiae and from an "extreme" petite mutant were analyzed by hybridization of several tRNAs on DNA fragments of different buoyant density, obtained by sonication and fractionation on a CsCl gradient. The hybridization patterns show that the genes for tRNAser, tRNAphe, tRNAhis, tRNAval, tRNAileu are present on wild-type mitochondrial DNA, while only genes for tRNAser and tRNAhis are present on petite mitochondrial DNA; moreover the hybridization patterns indicate that these genes are not clustered and suggest that more than one gene might exist for tRNAser and tRNAhis.

Centrifugation, Density Gradient↗