HMGB proteins involved in TOR signaling as general regulators of cell growth by controlling ribosome biogenesis.

The number of ribosomes and their activity need to be highly regulated because their function is crucial for the cell. Ribosome biogenesis is necessary for cell growth and proliferation in accordance with nutrient availability and other external and intracellular signals. High-mobility group B (HMGB) proteins are conserved from yeasts to human and are decisive in cellular fate. These proteins play critical functions, from the maintenance of chromatin structure, DNA repair, or transcriptional regulation, to facilitation of ribosome biogenesis. They are also involved in cancer and other pathologies. In this review, we summarize evidence of how HMGB proteins contribute to ribosome-biogenesis control, with special emphasis on a common nexus to the target of rapamycin (TOR) pathway, a signaling cascade essential for cell growth and proliferation from yeast to human. Perspectives in this field are also discussed.

Proteomic analysis of the oxidative stress response in Kluyveromyces lactis and effect of glutathione reductase depletion.

Yeasts are unicellular eukaryotes that provide useful models for studying the oxidative stress (OS) response. Most investigations to date have been performed on the fermentative Saccharomyces cerevisiae. The respiratory Kluyveromyces lactis is emerging as an alternative model. Our previous studies showed that glutathione reductase (Glr1) is an interesting point of difference in the OS response between the two yeasts. In the present study, using extensive proteomic analyses, the response to H(2)O(2) and its relationship to Glr1 were investigated in wild-type and glr1-deletion mutant K. lactis strains. We identified 46 proteins that showed modified expression after H(2)O(2) addition and 42 for which the change was Glr1-dependent. As expected, these proteins include a variety of antioxidant enzymes, chaperones, and oxidoreductases related to defense against OS and damage repair. They also include a number of proteins necessary for energy production and carbohydrate and amino acid metabolism. H(2)O(2) addition causes down-regulation of enzymes from the glycolytic pathway and Krebs cycle in wild-type K. lactis, whereas glr1-deletion prevents this effect and actually causes up-regulation of the glycolytic, Krebs cycle, and oxidative pentose phosphate pathways. To our knowledge, this is the first global proteomic analysis performed on K. lactis.

A functional analysis of Kluyveromyces lactis glutathione reductase.

Glutathione reductase (GLR) null mutants of the yeast Kluyveromyces lactis, a model eukaryotic respiratory cell, were created and phenotypically analysed. We found that cells lacking GLR show decreased resistance to oxidative stress and higher levels of reactive oxygen species and catalase activity than the wild-type strain. However, glutathione redox levels (GSH : GSSG ratio) were similar in the DeltaKlglr1 mutant and wild-type strains. The thioredoxin-thioredoxin reductase system is proposed as an alternative system for maintaining the GSH : GSSG ratio in the DeltaKlglr1 mutant. The involvement of GLR in glucose metabolism in K. lactis is suggested by the improved growth on glucose caused by the DeltaKlglr1 mutation and by the increased GLR activity in the DeltaKlgcr1 strain, KlGcr1 being a transcriptional activator of glycolytic genes. We also studied the subcellular location of GLR in K. lactis, showing that it is present in mitochondria; however, the DeltaKlglr1 mutation does not affect mitochondrial morphology. Genomic DNA integrity and life span are also unaffected by the DeltaKlglr1 mutation, at least under the conditions tested in this study.

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.

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.

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.

Genome-wide analysis of the yeast transcriptome upon heat and cold shock.

DNA arrays were used to measure changes in transcript levels as yeast cells responded to temperature shocks. The number of genes upregulated by temperature shifts from 30 to 37 or 45 was correlated with the severity of the stress. Pre-adaptation of cells, by growth at 37 previous to the 45 shift, caused a decrease in the number of genes related to this response. Heat shock also caused downregulation of a set of genes related to metabolism, cell growth and division, transcription, ribosomal proteins, protein synthesis and destination. Probably all of these responses combine to slow down cell growth and division during heat shock, thus saving energy for cell rescue. The presence of putative binding sites for Xbp1p in the promoters of these genes suggests a hypothetical role for this transcriptional repressor, although other mechanisms may be considered. The response to cold shock (4) affected a small number of genes, but the vast majority of those genes induced by exposure to 4 were also induced during heat shock; these genes share in their promoters cis-regulatory elements previously related to other stress responses.

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.

Dealing with different methods for Kluyveromyces lactis beta-galactosidase purification.

Several micro-scale chromatography-based procedures for purification of the beta-galactosidase from the yeast Kluyveromyces lactis were assayed. Purified enzyme was suitable to be used as antigen to induce polyclonal antibodies production. Specific staining of non-denaturing PAGE gels with chromogenic substrates allowed the determination of the number of subunits forming the native enzyme.

Heterologous Kluyveromyces lactis beta-galactosidase production and release by Saccharomyces cerevisiae osmotic-remedial thermosensitive autolytic mutants.

The beta-galactosidase from Kluyveromyces lactis is a high molecular weight protein with commercial interest. A major drawback of its industrial production is the high cost associated with extraction and downstream processing due to its intracellular nature. In this work, the effectiveness of the utilization of Saccharomyces cerevisiae LD1 and LHDP1 strains, osmotic-remedial mutants which lyse at 37 degrees C, for the heterologous production and release into the extracellular medium of this protein has been proved. The highest absolute values of released beta-galactosidase have been obtained with the protease-deficient strain LHDP1 by osmotic shock.

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.

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.

Chromosomal mapping of the KlCYC1 gene from Kluyveromyces lactis.

Chromosomal assignment of the KlCYC1 gene from Kluyveromyces lactis has been performed by hybridization of the labelled probe to a DNA blot of isolated chromosomes. A clear hybridization signal to chromosome VI is reported.

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.