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Random exploration of the Kluyveromyces lactis genome and comparison with that of Saccharomyces cerevisiae.

ABSTRACT: The genome of the yeast Kluyveromyces lactis was explored by sequencing 588 short tags from two random genomic libraries (random sequenced tags, or RSTs), representing altogether 1.3% of the K. lactis genome. After systematic translation of the RSTs in all six possible frames and comparison with the complete set of proteins predicted from the Saccharomyces cerevisiae genomic sequence using an internally standardized threshold, 296 K.lactis genes were identified of which 292 are new. This corresponds to approximately 5% of the estimated genes of this organism and triples the total number of identified genes in this species. Of the novel K.lactis genes, 169 (58%) are homologous to S.cerevisiae genes of known or assigned functions, allowing tentative functional assignment, but 59 others (20%) correspond to S.cerevisiae genes of unknown function and previously without homolog among all completely sequenced genomes. Interestingly, a lower degree of sequence conservation is observed in this latter class. In nearly all instances in which the novel K.lactis genes have homologs in different species, sequence conservation is higher with their S.cerevisiae counterparts than with any of the other organisms examined. Conserved gene order relationships (synteny) between the two yeast species are also observed for half of the cases studied.

SUBMITTER: Ozier-Kalogeropoulos O

PROVIDER: S-EPMC148010 | biostudies-other | 1998 Dec

REPOSITORIES: biostudies-other

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Random exploration of the Kluyveromyces lactis genome and comparison with that of Saccharomyces cerevisiae.

Ozier-Kalogeropoulos O O Malpertuy A A Boyer J J Tekaia F F Dujon B B

Nucleic acids research 19981201 23

The genome of the yeast Kluyveromyces lactis was explored by sequencing 588 short tags from two random genomic libraries (random sequenced tags, or RSTs), representing altogether 1.3% of the K. lactis genome. After systematic translation of the RSTs in all six possible frames and comparison with the complete set of proteins predicted from the Saccharomyces cerevisiae genomic sequence using an internally standardized threshold, 296 K.lactis genes were identified of which 292 are new. This corresp ...[more]

PMID: 9826779

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Project description:BackgroundGene duplication is a key evolutionary mechanism providing material for the generation of genes with new or modified functions. The fate of duplicated gene copies has been amply discussed and several models have been put forward to account for duplicate conservation. The specialization model considers that duplication of a bifunctional ancestral gene could result in the preservation of both copies through subfunctionalization, resulting in the distribution of the two ancestral functions between the gene duplicates. Here we investigate whether the presumed bifunctional character displayed by the single branched chain amino acid aminotransferase present in K. lactis has been distributed in the two paralogous genes present in S. cerevisiae, and whether this conservation has impacted S. cerevisiae metabolism.Principal findingsOur results show that the KlBat1 orthologous BCAT is a bifunctional enzyme, which participates in the biosynthesis and catabolism of branched chain aminoacids (BCAAs). This dual role has been distributed in S. cerevisiae Bat1 and Bat2 paralogous proteins, supporting the specialization model posed to explain the evolution of gene duplications. BAT1 is highly expressed under biosynthetic conditions, while BAT2 expression is highest under catabolic conditions. Bat1 and Bat2 differential relocalization has favored their physiological function, since biosynthetic precursors are generated in the mitochondria (Bat1), while catabolic substrates are accumulated in the cytosol (Bat2). Under respiratory conditions, in the presence of ammonium and BCAAs the bat1Δ bat2Δ double mutant shows impaired growth, indicating that Bat1 and Bat2 could play redundant roles. In K. lactis wild type growth is independent of BCAA degradation, since a Klbat1Δ mutant grows under this condition.ConclusionsOur study shows that BAT1 and BAT2 differential expression and subcellular relocalization has resulted in the distribution of the biosynthetic and catabolic roles of the ancestral BCAT in two isozymes improving BCAAs metabolism and constituting an adaptation to facultative metabolism.

Project description:BackgroundPhaseolamin or α-amylase inhibitor 1 (αAI) is a glycoprotein from common beans (Phaseolus vulgaris L.) that inhibits some insect and mammalian α-amylases. Several clinical studies support the beneficial use of bean αAI for control of diabetes and obesity. Commercial extracts of P. vulgaris are available but their efficacy is still under question, mainly because some of these extracts contain antinutritional impurities naturally present in bean seeds and also exhibit a lower specific activity αAI. The production of recombinant αAI allows to overcome these disadvantages and provides a platform for the large-scale production of pure and functional αAI protein for biotechnological and pharmaceutical applications.ResultsA synthetic gene encoding αAI from the common bean (Phaseolus vulgaris cv. Pinto) was codon-optimised for expression in yeasts (αAI-OPT) and cloned into the protein expression vectors pKLAC2 and pYES2. The yeasts Kluyveromyces lactis GG799 (and protease deficient derivatives such as YCT390) and Saccharomyces cerevisiae YPH499 were transformed with the optimised genes and transformants were screened for expression by antibody dot blot. Recombinant colonies of K. lactis YCT390 that expressed and secreted functional αAI into the culture supernatants were selected for further analyses. Recombinant αAI from K. lactis YCT390 was purified using anion-exchange and affinity resins leading to the recovery of a functional inhibitor. The identity of the purified αAI was confirmed by mass spectrometry. Recombinant clones of S. cerevisiae YPH499 expressed functional αAI intracellularly, but did not secrete the protein.ConclusionsThis is the first report describing the heterologous expression of the α-amylase inhibitor 1 (αAI) from P. vulgaris in yeasts. We demonstrated that recombinant strains of K. lactis and S. cerevisiae expressed and processed the αAI precursor into mature and active protein and also showed that K. lactis secretes functional αAI.

Dataset Information

Random exploration of the Kluyveromyces lactis genome and comparison with that of Saccharomyces cerevisiae.

Publications

Random exploration of the Kluyveromyces lactis genome and comparison with that of Saccharomyces cerevisiae.

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