Alicia González Manjarrez

Intereses de Investigación

LA DUPLICACION GENOMICA DE S. cerevisiae Y LA RETENCION SELECTIVA DE GENES DUPLICADOS DIO LUGAR A UN NUEVO MODO DE VIDA:
METABOLISMO FACULTATIVO

La redundancia génica es una característica de todos los seres vivos. La duplicación de genes funcionales representa una fuente de material genético para el origen de capacidades nuevas o especializadas. Una duplicación genómica ofrece la posibilidad de que todas las proteínas codificadas por el ADN de un organismo evolucionen y que potencialmenteéste se adapte a un “nuevo” modo de vida. La redundancia génica: ¿Es real o aparente? ¿Cómo diverge la función de genes duplicados? ¿Cómo se adquieren funciones nuevas?.

Origen de los Genes Duplicados
Una de las preguntas más importantes que ha planteado la genética molecular es:¿cómo surgen nuevos genes y qué repercusión tienen en la fisiología de los organismos? La respuesta a esta interrogante puede encontrarse en el estudio de los genes duplicados, o parálogos, los cuales pueden originarse a través de los siguientes procesos: a) entrecruzamiento de regiones homólogas del genoma (proceso también denominado crossing over), b) retrotranscripción y c) duplicaciones cromosómicas o genómicas

Estudio de la función de genes duplicados en la levadura Saccharomyces cerevisiae(S. cerevisiae).
El análisis de la secuencia genómica de la levadura S. cerevisiae puso de manifiesto la existencia y la magnitud de la redundancia génica; alrededor del 30% de su genoma está constituido por genes presentes en más de una copia. Estos genes parálogos son el resultado tanto de una duplicación total del genoma que involucró la pérdida y retención selectiva de algunos de ellos, así como de eventos independientes de duplicación génica a menor escala. Estos hallazgos permitieron identificar un gran número de genes parálogos que codifican para enzimas implicadas en el metabolismo del carbono y nitrógeno. Nuestro grupo de investigación inició el estudio de la diversificación de genes y proteínas parálogas, utilizando como modelo algunos pares de genes cuyos productos participan en la biosíntesis de aminoácidos de S. cerevisiae. La selección de estos genes está relacionada con enzimas que participan en la biosíntesis de amino ácidos y que utilizan esqueletos de carbono producidos durante la glicólisis o durante el ciclo de los ácidos tricarboxílicos, y por tanto su especialización podría resultar en que estos desarrollaran un papel importante en la coordinación de la utilización de esqueletos de carbono para la biosíntesis de intermediarios o para la generación de energía (ATP).
La levadura S. cerevisiae tiene la capacidad de crecer y generar energía en condiciones fermentativas o respiratorias (metabolismo facultativo). Dado que se ha propuesto que la retención selectiva de genes parálogos facilitó la adquisición del metabolismo facultativo, nuestro grupo se ha centrado en el estudio de la diversificación o redundancia de la función de genes parálogos, analizando el papel de cada uno de los dos genes cuando la levadura se cultiva en condiciones fermentativas, fermento-respiratorias y respiratorias. Así mismo hemos comparado la función de los parálogos de S. cerevisiae con la de los ortólogos de especies denominadas “tipo ancestral”
Algunas de las preguntas que nos ha interesado contestar al estudiar la función de los parálogos de la levadura son: i) La duplicación de genes constituye una redundancia funcional? ii) ¿Cómo diversifican los genes parálogos a lo largo de la evolución?, iii) ¿Qué mecanismos moleculares están involucrados en la diversificación funcional?, iv) ¿Cómo se diversifican las enzimas parálogas? v) ¿Qué ventajas adaptativas y fisiológicas representa el preservar estos genes duplicados? y vi) ¿La conservación de genes parálogos constituyó una adaptación al metabolismo facultativo?
Conclusiones
El estudio del destino evolutivo de algunos genes duplicados presentes en S. cerevisiae, cuyos productos participan en la biosíntesis de aminoácidos, indica que la retención y diversificación de los mismos ha tenido un profundo impacto en la adaptación de la levadura S. cerevisiae al metabolismo facultativo, jugando un papel importante en la adquisición de un “nuevo” modo de vida, que depende de la capacidad de crecer y obtener energía en condiciones fermentativas o respiratorias.

Lineas de Investigación

1.- Duplicación Génica y Evolución de la Levadura Saccharomyces cerevisiae
2.- Diversificación Funcional de Genes y Proteínas Parálogas: Papel de las Propiedades Bioquímicas y la Localización Subcelular de las Proteínas Parálogas y de la Regulación Transcripcional y Organización de la Cromatina de Genes Parálogos. (GDH1-GDH3, LYS20-LYS21, LEU4-LEU9, BAT1-BAT2, ALT1-ALT2).
3.- Reguladores Transcripcionales Híbridos, Su Organización y Identificación de las Redes de Genes Regulados por Cada Modulador Híbrido.
4.- La localización Subcelular Influye en la función de las proteínas parálogas?
5.- Caracterización de Genes Tipo Ancestral en Kluyveromyces lactis y Lacchancea kluyveri
6.-Organización de proteínas parálogas hetero-oligoméricas:una instancia de diversificación functional
7.- Organiuzación hetero-oligomérica: ¿Porque se privilegia, sobre la homo-oligomerización?

Investigaciones en Curso

1.- Papel de la localización sub-celular de enzimas parálogas en su diversificación functional. Transaminasas de amino ácidos de cadena ramificada: Bat1 (mitochondrial) / Bat2 (citoplásmica).
2.- La transaminasa de alanina Alt1, participa en la biosíntesis de alanina, es la única enzima capaz de degradar alanina. Adicionalmente, hemos encontrado que mutantes carentes de ALT1 (alt1Δ) presentan un fenotipo “petite” y no son capaces de crecer en etanol como única fuente de carbono. ¿Se trata de una proteína moonlight?
3.-Actualmente estudiamos el papel del regulador híbrido Nrg1-Rtg3. Nuestros resultados demuestran que el complejo Nrg1-Rtg3 determina la estabilidad e integridad del DNA mitochondrial. Se trata del primer regulador transcripcional que determina la estabilidad del DNA mitochondrial. En su ausencia, S. cerevisiae adopta esclusivamente el metabolismo fermentativo

Trayectoria Profesional

Investigadora Titular C

Formación Académica

Doctorado en Investigación Biomédica Básica, IBB UACPyP, Universidad NAcional Autónoma de México, México ()

Líneas de Investigación

Subfunctionalization of paralogous genes in Saccharomyces cerevisiae and their role in the acquisition of fermentative metabolism: WHOLE-GENOME DUPLICATION AND YEAST´S FRUITFUL WAY OF LIFE Only when a redundant gene locus is created by duplication is it permitted to accumulate formerly forbidden mutations and emerge as a new gene locus with hitherto unknown function Ohno 1970 Gene redundancy is a major feature of living beings. Duplication of functional genes represents the source of genetic material for the origin of new or specialized functions. Genome duplication can represent the possibility of a given organism to experiment the evolution of all proteins encoded in its DNA, and the potentiality to adapt to a “new” way of life. Why has been redundancy conserved? Is it real or apparent? How do duplicate genes functionally diverge? How are new functions acquired? S. cerevisiae genome sequence revealed the presence of 26% duplicated genes in this genome, suggesting that this lineage arose from Whole Genome Duplication (WGD), making this organism a suitable model to study diversification of duplicated genes. In fact further phylogenetic studies have found compelling evidence indicating that the S. cerevisiae lineage arose from an interspecies hybridization between one strain related to the Kluyveromyces, Lachancea and Eremothecium (KLE) clade and another one related to Zygosaccharomyces rouxii and Torulaspora delbrueckii (ZT) clade. After the allotetraploid was formed, intragenic recombinations, full gene conversion, differential gene loss and selection pressures shaped S. cerevisiae genome to the one we observe today, harboring conserved blocks of duplicated genes. After duplication, functional normal ploidy was recovered because of the massive loss of 90% of duplicated genes. Selective retention of paralogous genes was accompanied by subfunctionalization, determining the distribution of the ancestral function, so that in the contemporaneous yeast both paralogous genes are required to fulfill the function, which in the ancestor was carried out by a single gene copy. Particullarly important has been the study of the effect of paralogous gene retention in the acquisition of fermentative metabolism. It has been proposed that the selective conservation of duplicated genes in S. cerevisiae has been essential for the acquisition of a predominantly fermentative lifestyle, also termed the ‘make-accumulate-consume’ strategy (i.e., upon sugar availability, ethanol is produced, accumulated, and finally consumed). This strategy largely relies on the Crabtree effect that allows yeast to out-compete other microorganisms. The majority of cells only use fermentation when respiration is impaired, for example, when oxygen concentration is low (Pasteur effect). However, in the presence of high glucose concentrations and regardless of oxygen availability, Saccharomyces cerevisiae prefers to ferment glucose to ethanol. This physiological phenomenon is named the Crabtree effect. In this metabolic state, the cytoplasmic pyruvate produced by glycolysis is transformed to acetaldehyde and then to ethanol, which diffuses to the extracellular medium The retention of 551 WGD gene pairs involved in different cellular processes suggested that selective gene retention could have influenced the acquisition of fermentative metabolism and a new way of life. S. cerevisiae is presently the best suited model to study the acquisition and development of fermentative metabolism, due to the vast amount of technical resources and information available for this organism. In addition, the advancement of whole-genome sequencing and phenotyping of 1011 S. cerevisiae isolates from diverse geographical locations has provided valuable information into the ecological and evolutive niche of this yeast . Our laboratory has addressed the role of WGD in the acquisition of fermentative metabolism in S. cerevisiae. Our group has selected specific of paralogous gene pairs to study the mechanisms involved in subfunctionalization and selective gene retention of particular gene pairs. Detailed analysis has been necessary to identify the molecular mechanisms involved in functional diversification and the roles of specific duplicate genes in the acquisition of fermentative metabolism. Whole Genome Duplication and the acquisition of a new way of life: fermento-respiratory metabolism. 1.- What has been the role of WGD and selective retention of paralogous genes in the acquisition of fermento-respiratory metabolism. We have analyzed the role of gene expression, kinetic properties, homo or heteromeric organization and sub-cellular localization of paralogous genes/products in their function and role in fermentative and respiratory metabolism. a) Transcriptional regulation determines functional diversification of paralogous genes. For example in the case of BAT1 and BAT2 which codify for branched chain amino acid aminotransferases, BAT1 is preferentially expressed under biosynthetic conditions, while BAT2 is mainly expressed under catabolic conditions, thus, Bat1 will play a biosynthetic function, while Bat2 a catabolic role in accordance with the expression profile of their encoding genes. b) The kinetic properties of the encoded paralogous proteins can vary. Gdh1 has higher affinity for a-ketoglutarate than Gdh3, this has an important physiological repercussion, differentially modulating fermentative and respiratory metabolism. c) Heteroligomerization of paralogous dimeric proteins render homodimeric and heterodimeric proteins, with various sensitivity to regulators, providing a mechanism, which differentially control the use of intermediates and product biosynthesis under fermentative and respiratory conditions. d) Role of sub-cellular localization on the functional role of paralogous proteins. 2.- Characterization of the ortologous ancestral type genes in Kluyveromyces lactis and Lacchancea kluyveri Gene 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. We have investigated whether the single genes which were retained in double copy after WGD in S. cerevisiae and which originated from a single copy ancestral type gene displaying a bifunctional character, have distributed bifunctionality in the two retained paralogous genes, and whether this conservation has impacted S. cerevisiae metabolism. Our study shows that BAT1 and BAT2 differential expression and sub-cellular 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. 3.- Organization and regulatory role of hybrid transcriptional complexes. Transcriptional responses rely on a repertoire of modulators, which decipher regulatory information through their specific binding to cognate sequences, and their capacity to selectively recruit the components that constitute a given transcriptional complex. These modulators are endowed with three basic functions: a) site-specific DNA binding, b) transcriptional activation and c) ability to respond to regulatory signals. In the case of modulators composed of a single protein, the three. Adaptation to new environments, nutrient utilization and various stresses, result in intricate changes in the physiology of cells, allowing them to turn on peculiar response pathways in order to survive and adapt to the new conditions. The response of S. cerevisiae to various nutrient conditions constitutes a model whose study has most importantly contributed to our knowledge of the mechanisms determining transcriptional regulation. Nutrient limitation results in the modification of the regulatory pattern of particular sets of genes, whose products are needed to provoke an appropriate physiological response. Transcriptional modulators are composed of a DNA-binding domain, which targets these proteins to specialized binding sites, and an activation domain that mediates transcription initiation. In the yeast S. cerevisiae, these two domains are contained in a single polypeptide. There is a single exception to this rule, constituted by the HAP complex, which is organized in four polypeptides: Hap2-Hap3 and Hap5 constitute the DNA binding domain, while the fourth polypeptide Hap4 constitutes the activation domain. This finding opened the possibility for combinatorial activation, proposed by Guarente. The proposition suggested that, for example, a given activator A1 could borrow the DNA Hap2-3-5 binding domain and use the A1 native activation domain forming what we have named “hybrid transcriptional activator” (Hap2-3-5-A1). Although all organisms possess a vast array of DNA-binding regulatory proteins which play crucial roles in the specificity of transcriptional responses, the possibility of enlarging the repertoire of modulators through the combined action of DNA-binding and activation domains, generating hybrid activators, could result in novel responses. In 2011, our studies led to the finding of the first hybrid transcriptional modulator composed of Hap2-3-5-Gln3: Hap as DNA binding domain and Gln3 as activation domain . 5.- Characterization of the Novel Nrg1-Rtg3 Chimeric regulatory Complex In Saccharomyces cerevisiae, the transcriptional repressor Nrg1 (Negative Regulator of Glucose-repressed genes) and the β-Zip transcription factor Rtg3 (ReTroGrade regulation) mediate glucose repression and signalling from the mitochondria to the nucleus, respectively. Here, we show a novel function of these two proteins, in which alanine promotes the formation of a chimeric Nrg1/Rtg3 regulator that represses the ALT2 gene (encoding an alanine transaminase paralogue of unknown function). An NRG1/NRG2 paralogous pair, resulting from a post-wide genome small-scale duplication event, is present in the Saccharomyces genus. Neo-functionalization of only one paralogue resulted in the ability of Nrg1 to interact with Rtg3. Both nrg1Δ and rtg3Δ single mutant strains were unable to use ethanol and showed a typical petite (small) phenotype on glucose. Neither of the wild-type genes complemented the petite phenotype, suggesting irreversible mitochondrial DNA damage in these mutants. Neither nrg1Δ nor rtg3Δ mutant strains expressed genes encoded by any of the five polycistronic units transcribed from mitochondrial DNA in S. cerevisiae. This, and the direct measurement of the mitochondrial DNA gene complement, confirmed that irreversible damage of the mitochondrial DNA occurred in both mutant strains, which is consistent with the essential role of the chimeric Nrg1/Rtg3 regulator in mitochondrial DNA maintenance. 6.- Does Sub-cellular Localization of Paralogous Proteins Affect Their Physiological Role? The case of Bat1/Bat2 BAT1 and BAT2 are paralogous genes that codify for branched-chain aminotransferase enzymes in S. cerevisiae (Bat1 and Bat2). Utilization of branched chain amino acids (valine, leucine and isoleucine) as nitrogen source and the synthesis of these amino acids are functions, which are exclusively carried out by these two enzymes. Bat1 and Bat2 have a 73.6% of aminoacidic identity and similar kinetic profiles. However, BAT1 and BAT2 have opposite expression profiles: BAT1 is preferentially expressed when the cell has a primary nitrogen source (NH4) (biosynthetic conditions) and BAT2 expression is mainly achieved when the amino acidic concentration in the medium increases (catabolic conditions). This transcriptional divergence correlates with studies showing that Bat1 has a biosynthetic profile, whereas Bat2 has a catabolic one. In biosynthetic conditions, a bat1∆ mutant shows a reduced growth rate compared to a wild type strain or the bat2∆ mutant, while in catabolic conditions a bat2∆ mutant has a deficient growth rate compared to the wild type strain and the bat1∆ mutant. The opposite expression profiles of the enzymes Bat1 and Bat2 can be explained by the expression regulation of BAT1 and BAT2. Moreover, these enzymes have also diversified in their sub-cellular localization: Bat1 is a mitochondrial enzyme while Bat2 is cytosolic. Is Bat1 and Bat2 functional role dependent on their sub-cellular localization? A fundamental goal of cell biology is to define the functions of proteins in the context of compartments that organize them in the cellular environment. Here we describe the construction and analysis of a collection of yeast strains in which, Bat1 and Bat2 have been relocalized from the mitochondria to the cytosol or viceversa with or without the paralogous present, or both proteins present in the same compartment. Obtention of relocalyzed mutants and analysis of their growth phenotype on biosynthetic (glucose+NH4) or catabolic conditions (glucose+VIL), suggest that differential localization of Bat1 and Bat2 could also play a role determining their biological function. Suggested Literature Force, A. et al. 1999 Preservation of duplicate genes by complementary, degenerative mutations, en Genetics, vol. 151, num 4, pp. 15331-1545 Gu, Z. et al. 20002. Extent of gene duplication in the genomes of Drosophila, nematode and yeast, en Molecular Biology and Evolution, vol. 19, num,3, pp, 256-262. Levasseur, Anthony y Pierre Pontarotti. 2011. The role of duplications in the evolution of genomes highlights the need for evolutionary-based approaches in comparative genomics, en Biology Direct, vol, 6, pp. 1-12. Lilly, M etal, 2006. The effect of increased branched chain amino acid transaminase activity in yeast on the production of higher alcohols and on the flavour profiles of wine and distillates, en FEMS Yeast Research, num. 5, pp. 726-7433 Lynch, M. y J. S. Conery. 2003. The origins of genome complexity, en Science, num. 302, pp. 1401-1404. Lynch, M. y J. S. Conery. 2000. The evolutionary fate and consequences of duplicate genes, en Science, num. 290, pp. 1151-1155. Mombaerts, Peter. 2001. The human repertoire of odorant receptor genes and pseudogenes, en Annual Review of Genomics and Human Genetics, vol 2, pp. 493-510. Ohno, Susumo, 1967. Sex chromosomes and sex-linked genes. Springer, Berlin. Wolfe, K. H. y D. C. Shields. 1997. Molecular evidence for an ancient duplication of the entire yeast genome. en Nature, num. 387, pp. 708-713. Zhang, J.2003. Evolution by gene duplication an update, Trends in Ecology and Evolution, num. 188, pp 292-298.

Integrantes del Laboratorio

José Carlos Campero Basaldua

Estudiantes