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Structural and biochemical studies of alcohol dehydrogenases isozymes from Kluyveromyces lactis.
No full textTwo genes encoding putative mitochondrial alcohol dehydrogenases are present in the yeast Kluyveromyces lactis.
No full textFour structural genes encoding isozymes of the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis have been identified by hybridization to ADH2 DNA probes from Saccharomyces cerevisiae. In this paper we report on the isolation of KlADH4 and the complete sequencing of KlADH3 and KlADH4, two genes which show high homology to KlADH1, the ADH gene previously isolated in K. lactis, and to the ADH genes of S. cerevisiae. When compared with KlADH1, both KlADH3 and KlADH4 encode amino-terminal extensions which show the characteristics of the mitochondrial targeting sequences. These extensions are poorly conserved both at the nucleotide and the amino acid level. Surprisingly, the KlADH4 extension shows a higher identity at the amino acid level to the one encoded by ADH3 of S. cerevisiae than to the KlADH3 presequence. KlADH3 and KlADH4, in contrast to the ADH3 gene of S. cerevisiae, show a strong bias in the choice of codons
A new regulatory element mediates ethanol repression of KlADH3, a Kluyveromyces lactis gene coding for a mitochondrial alcohol dehydrogenase.
No full textKlADH3 is a Kluyveromyces lactis alcohol dehydrogenase gene induced in the presence of all respiratory carbon sources except ethanol, which specifically represses this gene. Deletion analysis of the KlADH3 promoter revealed the presence of both positive and negative elements. However, by site-directed mutagenesis and gel retardation experiments, we identified a 15-bp element responsible for the transcriptional repression of this gene by ethanol. In particular, this element showed putative sites required for the sequential binding of ethanol-induced factors responsible for the repressed conditions, and the binding of additional factors relieved repression. In addition, we showed that the ethanol element was required for in vivo repression of KlAdh3 activity
Molecular analysis of UASE, a cis-element containing stress response elements responsible for ethanol induction of the KlADH4 gene of Kluyveromyces lactis.
No full textKlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity, which is specifically induced by ethanol and insensitive to glucose repression. In this work, we report the molecular analysis of UAS(E), an element of the KlADH4 promoter which is essential for the induction of KlADH4 in the presence of ethanol. UAS(E) contains five stress response elements (STREs), which have been found in many genes of Saccharomyces cerevisiae involved in the response of cells to conditions of stress. Whereas KlADH4 is not responsive to stress conditions, the STREs present in UAS(E) seem to play a key role in the induction of the gene by ethanol, a situation that has not been observed in the related yeast S. cerevisiae. Gel retardation experiments showed that STREs in the KlADH4 promoter can bind factor(s) under non-inducing conditions. Moreover, we observed that the RAP1 binding site present in UAS(E) binds KlRap1p
Ethanol-induced and glucose-insensitive alcohol dehydrogenase activity in the yeast Kluyveromyces lactis.
No full textThe alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis is encoded by four ADH genes. In this paper we report evidence that at least three of these genes are transcribed and translated into protein. KIADH1 and KIADH2, which encode cytoplasmic activities, are preferentially expressed in glucose-grown cells with respect to ethanol-grown cells. KIADH4, which encodes one of the two activities localized within mitochondria, is induced at the transcriptional level in the presence of ethanol as is the ADH2 gene in Saccharomyces cerevisiae. However the regulation of the expression of the K. lactis gene is completely different from that of ADH2 and of other known ADH genes in that KIADH4 is insensitive to glucose repression and is not expressed on non-fermentable carbon sources other than ethanol. This kind of regulation can be clearly observed in non-fermenting strains, where the induction of KIADH4 is dependent on the addition of ethanol to the medium. On the contrary, in fermenting strains KIADH4 is always induced by ethanol or acetaldehyde produced endocellularly and this results in constitutive expression of the gene also in the presence of glucose. The mitochondrial localization of the activity encoded by KIADH4 and the peculiar regulation of this gene could be related to the fact that K. lactis is a petite negative yeast in which some mitochondrial functions seem to be essential for cell viability
Isolation and molecular characterisation of KlCOX14, a gene of Kluyveromyces lactis encoding a protein necessary for the assembly of the cytochrome oxidase complex.
No full textThe yeast Kluyveromyces lactis was mutagenized with ethyl methane sulphonate and mutants unable to grow on respiratory carbon sources were isolated. Functional complementation of one of these mutants led to the isolation of KICOX14, a gene encoding a 64 amino acid protein which is the functional homologue of Saccharomyces cerevisiae Cox14p, a protein necessary for the assembly of the cytochrome oxidase holoenzyme (Glerum et al., 1995). The disruption of KlCOX14 resulted in the absence of the absorption bands relative to cytochromes a and a(3) and in the complete loss of respiratory activity. Klcox14 mutants display the typical phenotype of pet mutants and have a reduced growth rate. In addition, unlike the wild-type, Klcox14 mutants are able to grow by fermentation also in the presence of low glucose. The nucleotide sequence of KlCOX14 has been deposited in the EMBL databank with Accession No. AJ238801. Copyright (C) 2000 John Wiley & Sons, Ltd.status: Publishe
Characterization of KlGUT2, a gene of the glycerol-3-phosphate shuttle, in Kluyveromyces lactis
No full textKlGUT2 encodes the mitochondrial component of the glycerol-3-phosphate shuttle in Kluyveromyces lactis, a dehydrogenase involved in the maintenance of the NADH redox balance and in glycerol utilization. Deletion of KlGUT2 led to glycerol accumulation during growth in glucose and growth retardation in ethanol. In addition, KlGUT2 deletion altered the expression of other mitochondrial dehydrogenases that contribute to the maintenance of the intracellular redox balance, suggesting a rerouting of ethanol oxidation from the cytoplasm to the mitochondria. Finally, Northern analysis showed that KlGUT2 has two transcripts: one constitutively expressed and dependent on HGT1, the high-affinity hexose transporter gene, and the other induced under respiratory conditions
Spettroscopia NMR multinucleare e analisi metabolomica applicata alla caratterizzazione del fenotipo metabolico di sistemi complessi
No full textLa metabolomica, negli ultimi anni, è diventata un approccio versatile, largamente impiegato nell’industria e nella ricerca nel campo delle scienze mediche, biologiche, ambientali e alimentari [1,2,3].
In ambito microbiologico la metabolomica viene spesso utilizzata per la caratterizzazione del fenotipo di microrganismi, volta all’ottimizzazione della produzione biotecnologia di sostanze come vitamine, enzimi e antibiotici. In seguito all’avvento della tecnologia del DNA ricombinante è stata applicata nell’ingegneria metabolica per la produzione di metaboliti primari e secondari [4].
Nel presente lavoro e’ stata applicata la metabolomica, basata su spettroscopia di risonanza magnetica multinucleare ad alta risoluzione e modelli di analisi multivariata, per caratterizzare le variazioni del fenotipo metabolico di un sistema complesso dipendente da una mutazione genica e per valutare il suo adattamento in funzione di variazioni ambientali.
Lo studio è stato condotto su un sistema biologico costituito da cellule eucariote di lieviti kluyveromyces lactis, wild type e geneticamente modificate, fornite dai laboratori del Dipartimento di Biologia e Biotecnologie.
La strategia di metabolic profiling adottata risponde alla necessità di una metodologia che possa caratterizzare organismi complessi descrivendone il fenotipo metabolico in diverse condizioni e consiste nell’identificazione e quantificazione dei metaboliti coinvolti nei flussi attraverso e tra le varie vie metaboliche per la produzione di energia e di materia [5].
Le variazioni nel metaboloma sono state valutate tramite spettroscopia NMR multinucleare, mappe di correlazione e analisi multivariata dei dati.
L’analisi NMR ha il vantaggio di non richiedere una preparazione dei campioni particolarmente impegnativa; si è quindi eseguita una estrazione tramite il metodo Bligh and Dyer [6] modificato dal nostro laboratorio [7] ottenendo degli estratti idro-alcolici ed organici, sui quali sono stati effettuati spettri monodimensionali di 1H e spettri bidimensionali (omonucleari TOCSY ed eteronucleari HSQC e HMBC). Si è rivolta particolare attenzione agli aspetti metodologici ottimizzando, per ogni esperimento, la risoluzione e la sensibilità al fine di assicurare la riproducibilità richiesta da un approccio di tipo metabolomico.
L’analisi metabolomica ha consentito, quindi, la valutazione delle variazioni nell’intero metaboloma, dipendenti da una singola mutazione genica.
I risultati hanno portato alla costruzione di un modello di rete metabolica per il ceppo wild type e per il rispettivo mutante.
[1] Powers R, Magnetic Resonance in Chemistry 2009, 47, S2-S11.
[2] Sobolev AP, Brosio E, Gianferri R, Segre AL, Magn. Reson. Chem. 2005, 43, 625–638.
[3] Mannina L, Cristinzio M, Sobolev AP, Ragni P, Segre AL, J. Agric. Food Chem. 2004, 52, 7988–7996.
[4] Mashego MR, Rumbold K, De Mey M, Vandamme E, Soetaert W, Heijnen JJ, Biotechnol. Lett. 2007, 29, 1-16.
[5] Miccheli A, Tomassini A, Puccetti C, Valerio M, Peluso G, Tuccillo F, Calvani M, Manetti C, Conti F, Biochimie 2006, 88, 437-448.
[6] Bligh EG, Dyer WJ, Can. J. Biochem. Physiol. 1959, 37, 911-917.
[7] Miccheli A, Aureli T, Delfini M, Di Cocco ME, Viola P, Gobetto R, Conti F, Cell. Mol. Biol. 1998, 34, 591–603
The sequence of a 8kb segment on the right arm of yeast chromosome VII identifies four new open reading frames and the gene for yTAFII 145.
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