Biomedical subjects

M E Cerdán

Publications and source records attributed to M E Cerdán.

At least 19 recordsLinked to original sources

PubMed2005

The nuclear genes encoding the internal (KlNDI1) and external (KlNDE1) alternative NAD(P)H:ubiquinone oxidoreductases of mitochondria from Kluyveromyces lactis.

Cloning, sequence and functional analyses of the Kluyveromyces lactis genes KlNDI1 and KlNDE1 are reported. These genes encode for proteins with high homology to the mitochondrial internal (Ndi1p) and external (Nde1p) alternative NADH:ubiquinone oxidoreductases from Saccharomyces cerevisiae and complement the respective mutations. Analysis of KlNDI1 transcriptional regulation showed that expression of this gene is lower in 2% glucose than in 0.5% glucose or non-fermentable carbon sources. Beta-galactosidase activity values, shown by lacZ fusions of KlNDI1 promoter deletions, suggested that two Adr1p binding sites mediate this carbon source regulation of KlNDI1. The expression of the KlNDE1 gene in S. cerevisiae mutant strains and measurement of respiration with isolated mitochondria showed that the protein encoded by KlNDE1 oxidizes NADPH, this being an important difference with respect to the conventional yeast S. cerevisiae. Moreover, Northern blot experiments using a phosphoglucose isomerase mutant showed that KlNDE1 gene transcription increases with glucose metabolism through the pentose phosphate pathway.

Amino Acid Sequence↗

PubMed2004

Characterization of a gene similar to BIK1 in the yeast Kluyveromyces lactis.

In Saccharomyces cerevisiae, Bik1p is a microtubule plus-end-tracking protein that plays several roles in mitosis and ploidy. KlBik1p (from Kluyveromyces lactis) maintains the same structural-domain organization as does S. cerevisiae Bik1p. As part of its characterization, we constructed a stable klbik1 mutant which is sensitive to benomyl only at 14 degrees C and has a higher frequency of crescent-shaped nuclei than S. cerevisiae bik1 mutants. This phenotype is partially rescued by S. cerevisiae BIK1. Other phenotypes associated with bik1 are not present in the K. lactis mutant. By fusion to GFP we were able to show the functionality of the KlBik1p CAP-Gly domain and found that the fusion protein changes its cellular location during the cell cycle.

Amino Acid Sequence↗

PubMed2004

Isolation and characterization of two nuclear genes encoding glutathione and thioredoxin reductases from the yeast Kluyveromyces lactis.

Response to oxidative stress has been hitherto scarcely studied in the respiratory yeast Kluyveromyces lactis. The genes coding for reductases of glutathione and thioredoxin, KlGLR1 and KlTRR1, respectively, have been cloned and characterized in this work. H(2)O(2) treatment increased transcription and enzyme activity of KlTRR1 but not of KlGLR1, suggesting a different situation from that reported for the fermentative yeast Saccharomyces cerevisiae. A consensus for Yap1p binding is functional in the KlTRR1 promoter.

Amino Acid Sequence↗

PubMed2004

Cloning genes from a library using a clustering strategy and PCR.

A new polymerase chain reaction (PCR)-based method is described for the isolation of clones of interest from a library when only part of a sequence is available. In actuality, this occurs with many genomes that have been partially sequenced using a random strategy. The method presented here, discriminating clusters by PCR (DCbyPCR), is a nonradioactive and improved alternative to colony hybridization.

Cloning, Molecular↗

PubMed2002

Genome-wide analysis of yeast transcription upon calcium shortage.

Several regulatory circuits related to important functions, like membrane excitation, immunoresponse, replication, control of the cell cycle and differentiation, among others, cause an increase in intracellular calcium level that finally has a consequence upon transcription of specific genes. The sequencing of the whole genome of eukaryotic cells enables genome-wide analysis of gene expression under many conditions not yet assessed by conventional methods. Using the array technology, the effect of calcium shortage in yeast cells was studied. Correspondence analysis of data showed that there is a response in transcription that is correlated to calcium shortage. The distribution of up-regulated-genes in functional categories suggests a regulatory connection between the cell-cycle progression and the energetic metabolic requirements for growth and division. In silico analysis of promoters reveals the frequent appearance of the Mlu I cell cycle box (MCB) cis element that binds the transcriptional regulatory factor Mcm1.

Calcium↗

PubMed2001

Haem regulation of the mitochondrial import of the Kluyveromyces lactis 5-aminolaevulinate synthase: an organelle approach.

The enzyme 5-aminolaevulinate acid synthase (ALAS) catalyses the first reaction in the haem biosynthetic pathway. In eukaryotes this protein is translated by cytosolic ribosomes and then targeted to the mitochondria. We present evidence that in the yeast Kluyveromyces lactis haem exerts a feedback control upon the import of the ALAS into mitochondria. The ALAS from K. lactis (KlALAS) contains two haem regulatory motifs (HRM) in the mitochondrial targeting signal. Mutagenesis experiments reveal the involvement of these HRM in the response of the KlALAS to haem.

5-Aminolevulinate Synthetase↗

PubMed2001

The KlCYC1 gene, a downstream region for two differentially regulated transcripts.

KlCYC1 encodes for cytochrome c in the yeast Kluyveromyces lactis and is transcribed in two mRNAs with different 3'-processing points. This is an uncommon transcription mechanism in yeast mRNAs. The 3' sequence encompassing the whole region that is needed to produce both mRNAs is analysed. We have determined identical processing points in K.lactis and in Saccharomyces cerevisiae cells transformed with KlCYC1; positions 698 and 1092 (with respect to the TAA) are the major polyadenylation points. This shows that the cis-elements present in the KlCYC1 3'-untranslated region (3'-UTR) direct a processing mechanism that has been conserved in yeast. In K. lactis there is a high predominance of the shorter transcript (1.14 kb) only at the initial logarithmic growth phase. Interestingly, this growth phase-dependent regulation of 3'-UTR processing is lost when the gene is expressed in S. cerevisiae.

3' Untranslated Regions↗

PubMed2001

New secretory strategies for Kluyveromyces lactis beta-galactosidase.

We examined several strategies for the secretion of Kluyveromyces lactis beta-galactosidase into the culture medium, in order to facilitate the downstream processing and purification of this intracellular enzyme of great industrial interest. We constructed plasmids by fusing the LAC4 gene or engineered variants to the secretion signal of the K.lactis killer toxin or to the secretion signal of the Saccharomyces cerevisiae alpha-factor. With these plasmids we transformed strains of the yeasts K.lactis and S.cerevisiae, respectively and tested beta-galactosidase extracellular activity in different culture media. We achieved partial secretion of beta-galactosidase in the culture medium since the high molecular weight and oligomeric nature of the enzyme, among other factors, preclude full secretion. The percentage of secretion was improved by directed mutagenesis of the N-terminus of the protein. We developed several deletion mutants which helped us to propose structure-function relationships by comparison with the available data on the homologous Escherichia coli beta-galactosidase. The influence of the culture conditions on heterologous beta-galactosidase secretion was also studied.

Amino Acid Sequence↗

PubMed2000

Heme-mediated transcriptional control in Kluyveromyces lactis.

Heme is of great importance in oxygen-dependent biological functions, since it serves as a prosthetic group for many proteins related to oxygen-binding, oxidative damage prevention and electron transport. It also regulates gene expression through the action of specific transcriptional regulatory factors. In this paper, we present an analysis of heme-dependent transcriptional regulation of several respiration-related genes in an aerobic respiratory yeast, Kluyveromyces lactis. We also report that the KlHEM13 gene, encoding the heme biosynthetic enzyme coproporphyrinogen oxidase, is under heme and oxygen transcriptional regulation, thereby controlling the synthesis of the effector, heme. KlHEM13 is induced during hypoxia, which represents the first report of a transcriptionally regulated gene with this behaviour in K. lactis.

Base Sequence↗

PubMed2000

Transcript analysis of 203 novel genes from Saccharomyces cerevisiae in hap1 and rox1 mutant backgrounds.

Hap1 and Rox1 are transcriptional regulators that bind regulatory sites in the promoters of oxygen-regulated genes in Saccharomyces cerevisiae. Hap1 is a heme-responsive activator of genes induced in aerobic conditions and Rox1 is a repressor of hypoxic genes in aerobic conditions. We have studied transcriptional regulation of a pool of 203 open reading frames (ORFs) from chromosomes IV, VII, and XIV in wild-type, hap1, and rox1 mutant genetic backgrounds in an attempt to extend the family of oxygen and heme regulated genes. Only three ORFs are significantly repressed by Rox1 but they cannot be considered as typical hypoxic genes because they are not overexpressed during hypoxia.

Carbon-Oxygen Lyases↗

PubMed1999

Kluyveromyces lactis HIS4 transcriptional regulation: similarities and differences to Saccharomyces cerevisiae HIS4 gene.

Sequence analysis of the Kluyveromyces lactis HIS4 (KlHIS4) gene promoter reveals relevant differences in comparison to the Saccharomyces cerevisiae HIS4 homologous gene. Among them are the absence of a Rap1 binding site and the presence of only three putative Gcn4 binding consensus sites instead of the five described in the S. cerevisiae promoter. Since these factors are implicated in the general control, we investigated the transcriptional regulation of the KlHIS4 gene under conditions of amino acid starvation and discovered that the mechanisms previously described for S. cerevisiae HIS4 regulation and related to general control are not functional in K. lactis. The expression analysis of the KlHIS4 gene under phosphate starvation or high adenine supply shows that factors, such as Bas1 or Bas2, involved in the basal control may also operate in a different way in K. lactis. Interestingly, and also in contrast to the HIS4 regulation in S. cerevisiae, we found domains for Nit2-like and yeast-Ap1-like binding sequences. Northern analyses showed transcriptional activation under ammonia starvation and oxidative stress.

Alcohol Oxidoreductases↗

PubMed1999

Disruption of six novel Saccharomyces cerevisiae genes reveals that YGL129c is necessary for growth in non-fermentable carbon sources, YGL128c for growth at low or high temperatures and YGL125w is implicated in the biosynthesis of methionine.

Six open reading frames (ORFs) from chromosome, VII, YGL131c, YGL129c, YGL128c, YGL125w, YGL124c and YGL121c, were disrupted by deletion cassettes with short flanking regions homologous to the target locus (SFH). YGL129c is necessary for growth in non-fermentable carbon sources, YGL128c for growth at low or high temperatures and YGL125w is implicated in the biosynthesis of methionine. With regard to the other ORFs, basic phenotypic analyses did not reveal any significant clues about their function.

Carbon↗

PubMed1998

Characterization of promoter regions involved in high expression of KlCYC1.

Functional analysis of the KlCYC1 promoter reveals that sequences located upstream to those already published [Freire-Picos, M. A., Rodríguez-Torres, A. M., Ramil, E., Cerdán, M. E., Breuning, K. D., Hollenberg, C. P. & Zitomer, R. S. (1993) Sequence of a cytochrome c from Kluyveromyces lactis and its upstream region, Yeast 9, 201-204] and extending from positions -780 to -371 are important for maintaining high levels of expression, although this region contains both negative and positive elements. A consensus sequence for interaction with KlCpf1p is present at position -492, into the negative site, and specific protein binding to KlCpf1p has been demonstrated. Deletion of the sequences from positions -413 to -338 diminishes KlCYC1 transcription; protein binding to two sequences included in this activator region is detected and several points of evidence indicate that the complex observed is different from the Hap2/3/4/5p complex. Binding of KlCpf1p and the activator complex to the promoter is constitutive in different carbon sources. Although the promoter contains CCAAT boxes, directed mutagenesis has revealed that they are not related to the moderate de-repression observed in glycerol media.

Base Sequence↗

PubMed1998

The HIS4 gene from the yeast Kluyveromyces lactis.

The Kluyveromyces lactis HIS4 gene was cloned by complementation of a Saccharomyces cerevisiae his4 mutant. Sequence analysis revealed a 2388 bp open reading frame encoding a single polypeptide predicted to encompass three distinct enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase). This structural organization is strikingly similar to that of the His4 proteins from S. cerevisiae and Pichia pastoris. Transcript analysis detected a single mRNA species of 2.5 kb.

Alcohol Oxidoreductases↗

PubMed1997

Isolation and characterization of the KlHEM1 gene in Kluyveromyces lactis.

The KlHEM1 gene from Kluyveromyces lactis encodes a functional 5-aminolevulinate synthase (deltaALA synthase), as confirmed by complementation of a hem1 mutant Saccharomyces cerevisiae strain, homology search, and detection of a 2.3 kb transcript. The gene is highly homologous to the ScHEM1 gene, and the sequence of the promoter region contains a complex combination of putative regulatory signals. Some of them are related to phospholipid biosynthesis, glycolytic metabolism, and regulation by carbon source. Transcription of KlHEM1 increased significantly in response to limited oxygen, and only slightly with the change from repressed (glucose) to derepressed conditions (glycerol). The deltaALA synthase from K. lactis contains, in the amino-terminal region, two heme-responsive elements that are not present in the protein from Saccharomyces cerevisiae.

5-Aminolevulinate Synthetase↗

PubMed1996

Reoxidation of the NADPH produced by the pentose phosphate pathway is necessary for the utilization of glucose by Kluyveromyces lactis rag2 mutants.

Kluyveromyces lactis mutants defective in the glycolytic enzyme phosphoglucose isomerase are able to grow in glucose media and to produce ethanol, but they depend on a functional respiratory chain and do not grow in glucose-antimycin media. We postulate that this is due to the necessity of reoxidizing, in the mitochondria, the NADPH produced by the pentose phosphate pathway, which may be highly active in these mutants in order to bypass the blockade in the phosphoglucose isomerase step. This oxidation would be mediated by a cytoplasmic-side mitochondrial NAD(P)H dehydrogenase that would pass the electrons to ubiquinone. Data supporting this hypothesis are provided.

Antimycin A↗

PubMed1996

PICDI, a simple program for codon bias calculation.

PICDI is a very simple program designed to calculate the Intrinsic Codon Deviation Index (ICDI). The program is available in Macintosh as well a PC format. Requirements for correct input of the sequences have been kept to a minimum and the analysis of sequences up to 2000 codons is very quick. The ICDI is very useful for estimation of codon bias of genes from species in which optimal codons are not known. The availability of a computer program for its calculation will increase its usefulness in the fields of Molecular Biology and Biotechnology.

Codon↗

PubMed1994

Codon usage in Kluyveromyces lactis and in yeast cytochrome c-encoding genes.

Codon usage (CU) in Kluyveromyces lactis has been studied. Comparison of CU in highly and lowly expressed genes reveals the existence of 21 optimal codons; 18 of them are also optimal in other yeasts like Saccharomyces cerevisiae or Candida albicans. Codon bias index (CBI) values have been recalculated with reference to the assignment of optimal codons in K. lactis and compared to those previously reported in the literature taking as reference the optimal codons from S. cerevisiae. A new index, the intrinsic codon deviation index (ICDI), is proposed to estimate codon bias of genes from species in which optimal codons are not known; its correlation with other index values, like CBI or effective number of codons (Nc), is high. A comparative analysis of CU in six cytochrome-c-encoding genes (CYC) from five yeasts is also presented and the differences found in the codon bias of these genes are discussed in relation to the metabolic type to which the corresponding yeasts belong. Codon bias in the CYC from K. lactis and S. cerevisiae is correlated to mRNA levels.

Amino Acids↗