Browsing by Author "Gonzalez, Francisco J."
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- HE-TAIL PCR, a powerfull tool for identification of Trichoderma harzianum lip1 genePublication . Jorge, Lurdes; Gonzalez, Francisco J.; Monte, Enrique; Choupina, AltinoTrichoderma harzianum is a widespread soil fungus, known as a biocontrol agent against soilborne plant pathogens. Species of Trichoderma are commercially applied as biological control agents against plant fungal pathogens based on different mechanisms, such as antibiosis, competition for nutrients and mycoparasitism. One of the mechanisms involved is the production of several lytic enzymes. In T. harzianum has been identified several glucanases, celulases, chitinases and proteases, but nothing are already known about its lipolytic system. In this work we described the identification of lip1 from T. harzianum, a gene encoding the first extracellular triacylglicerol lipase known in this specie. A cDNA library of T. harzianum CECT 2413 cloned in pBluescript SK+ was screened, and the expressed sequence tags (ESTs) with more similarity against lipase gene sequences in international databases selected and sequenced. Elucidation of complete gene nucleotide sequence of lip1, including 52 bp of the open reading frame at the N-terminal region and 693 bp of the promoter region were achieved by high-efficiency thermal asymmetric interlaced (HE-TAIL) PCR (Michiels et al., 2003), which confirmed to be a powerful tool to identify flanking regions from short known sequences. Lip1 codifies a 558 amino acids protein, with 59322,6 KDa and a calculated global iso-electric point value of 4,56. The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AM18087.
- Identification of Trichoderma harzianum lip2 gene by HE-TAIL PCRPublication . Jorge, Lurdes; Monte, Enrique; Gonzalez, Francisco J.; Choupina, AltinoTrichoderma harzianum is a widespread soil fungus, known as a biocontrol agent against soil borne plant pathogens. Its biological control against plant fungi pathogens is based on different mechanisms, namely the production of several lytic enzymes. In T. harzianum several glucanases, cellulases, chitinases and proteases, has been identified but little is known about its lipolytic system. The aim of this work was to achieve the complete elucidation of T. harzianum lip2 by HE-TAIL PCR (High-Efficiency Thermal Asymetric Interlaced PCR), a method described as efficient to identify flanking regions from short known DNA sequences. From a cDNA library of T. harzianum CECT 2413, obtained by NewBiotechnic, it has been selected an EST who showed lipase homology in agreement with the program FASTA: EST-1279. DNA sequencing was performed using an ABI 373 automated sequencer. After two sequencing rounds, EST-1279 had 1168bp, and a great homology (1,8e-80 ) with a hypothetical lipase of Fusarium graminearum. Comparison between two sequences, suggested that still are lacking 300bp of the beginning of the ORF. In order to determine the lacking bases, it was used an HE-TAIL PCR, that seemed suitable to the problem resolution. Degenerated 16bp primers R1 (5'- NGTCGASWGAMAWGAA-3'), R2 (5'-GTNCGASWCANAWGTT-3'), R3 (5'- WGTGNAGWANCANAGA-3') and R4 (5'-NCAGCTWSCTMTSCTT-3'), were used. Gene-specific primers, lip2a (5'-CTGGCAGAACCGATTCCCGAGCGC TT-3'), lip2b (5'-ACGCAACTACGATGGCGCCTTGCTCG-3'), lip2c (5'-TGC GATGAACCCACAGCTATCGCCGA-3') and lip2d (5'-GAGAAAGCCTGTACT CCACGTAGAGG-3') with 26bp and melting temperatures of 70-72ºC were designed in the incomplete ORF of lip2. From genomic DNA three rounds of PCR were performed on a MyCycler Thermal Cycler (BIORAD), using the product of the previous PCR as a template for the next. In primary and secondary PCR reactions, a single-step annealing-extension at 62ºC-66ºC was used. Separation and identification of DNA products of the tertiary reaction, including the controls, were made by agarose gel electrophoresis. At tertiary PCR reaction none amplification products were obtained with degenerate primers R1, R2 or R3. Bands only appear in the combinations R4+lip2c and R4+lip2d. A 2000bp band was selected, purified by geneclean and partially sequenced, who allowed the elucidation of lip2 ORF beginning and part of the promoter region. In the end, lip2 sequence had a total of 1992bp, including 548bp of the promoter, 1215bp of ORF and 227bp of terminator. The complete gene sequence was submitted to EMBL databases (Accession number AM774154). Nucleotide and deduced amino acid sequences were analyzed using FASTA programs from EMBL databases. Lip2 codifies a 404 amino acids protein, with 44.6KDa. The UniProt accession number for the amino-acid sequence reported in this paper is B7ZET5_TRIHA. Elucidation of complete gene nucleotide sequence of lip2, including part of the open reading frame at the N-terminal region and 548bp of the promoter region was achieved by HE-TAIL PCR, which confirmed to be a powerful tool to identify flanking regions from previous known ones.
- Identification of Trichoderma harzianum lip2 gene by HE-TAIL PCRPublication . Jorge, Lurdes; Gonzalez, Francisco J.; Monte, Enrique; Choupina, AltinoTrichoderma harzianum is a widespread soil fungus, known as a biocontrol agent against soilborne plant pathogens. Its biological control against plant fungi pathogens is based on different mechanisms, namely the production of several lytic enzymes. In T. harzianum several glucanases, celulases, chitinases and proteases has been identified but little is known about its lipolytic system. The aim of this work was to achieve the complete elucidation of T. harzianum lip2 by HE-TAIL PCR (High-Efficiency Thermal Asymetric Interlaced PCR), a method described as efficient to identify flanking regions from short known DNA sequences
- Isolation and analysis of lip2 gene from Trichoderma harzianumPublication . Vaz, Madalena; Belo, Hélio; Jorge, Lurdes; Gonzalez, Francisco J.; Monte, Enrique; Choupina, AltinoTrichodorma spp. covers a group of fungi extremely common in soils of all climatic areas. These fungi arc efficient enzyme producers, with industrial applications, or in nature, involved in the degradation of the coli wall of the phytopathogens as well as in the degradation of other fungi, organic matter and nutrients secreted by roots.
- Isolation and analysis of lip2 gene from Trichoderma harzianumPublication . Vaz, Madalena; Belo, Hélio; Jorge, Lurdes; Gonzalez, Francisco J.; Monte, Enrique; Choupina, AltinoThe genus Trichoderma is cosmopolitan in soils, wood decomposition and plant material. Species of Trichoderma are often dominant components of the soil microflora in various habitats. This is due to different metabolic capacity of the Trichoderma species and its aggressive competitiveness in nature. The genus Trichoderma are frequently used in biological control because of its antagonist ability of phytopathogenic fungi. The mechanisms employed by Trichoderma spp. to antagonize other fungi are competition (for space and / or nutrients), antibiosis and microparasites, while in the latter case, involved lytic enzymes such as proteases, glucanases, chitinases and lipases. Some of these proteins have a large agricultural potential, especially as active components of new formulations of fungicides. Trichoderma harzianum Rifai (Ascomycota, Hypocreales, Hypocreaceae) is a filamentous fungus, asexual, commonly isolated of tropical soil of plant material, rhizosphere ecosystems and decomposing organic material a ratio of 101-103 spores per gram of soil (Figure 1).
- Isolation, characterization and expression of genes encoding enzymes with lipase activity in Yarrowia lipolyticaPublication . Choupina, Altino; Gonzalez, Francisco J.; Fermiñán, Encarnación; Burguillo, Francisco J.; Dominguez, ÁngelYarrowia lipolytica is a dimophic yeast with very high secretory capacities and able to grow in alkanes and fatty acids as the sole carbon source. Using PCR, we have isolated two DNA fragments that after sequencing show homology with fungal lipases. By genomic DNA subcloning, we have isolated a gene nominated YlLIP1 located on Y. lipolytica chromosome IV. The gene presents an ORF of 1458 bp which encodes a putative protein of 486 amino acids with an apparent molecular weight of 55.4 kDa. The nucleotide sequence of the 5' and 3' ends shows the consensus signals for translation initiation and transcription termination motifs in the yeast. A disruption of YlLIP1 has been performed. The deleted ΔYllip1 strain shows lower lipase activity in comparison to the wild type. The DNA encoding YlLip1p was expressed in Saccharomyces cerevisiae and Kluyveromyces lactis. Both yeast species are able to synthesize a functional enzyme under the control of GAL1 promoter. We are currently isolating the second gene, both in the wild type and in the ΔYllip1 strain.
- Lipase assay in yarrowia lipolyticaPublication . Morín, M.; Gonzalez, Francisco J.; Choupina, Altino; Burguillo, Francisco J.; Dominguez, ÁngelLipases (triacylglycerol acyl hydrolases, EC 3. 1.1.3) are key enzymes in fat metabolism produced by microorganisms, plants, and animals that catalyze the breakdown of triacylglycerols to free fatty adds and glycerol. In Y. lipolytica severaJ genes encoding lipases have been isoiated,LlP2 coding for a secreted lipase (Pignede et al. 1000) and LlPl and LIP3 encoding two lipase genes of the carboxylesrerase family (Choupina et al. 1999). Lipase expression was repressed by glucose and induced by fatty acids and olive oil.
- Producción de lipasas por la levadura Yarrowia lipolyticaPublication . Morín, M.; Choupina, Altino; Fermiñán, Encarnación; Burguillo, Francisco J.; Gonzalez, Francisco J.; Ruiz, L.; Dominguez, ÁngelYarrowia lipolytica es una levadura dimórfica que secreta gran cantidad de proteínas al medio de cultivo, entre ellas: proteasas - alcalinas, neutras y ácidas -, ribonucleasas, fosfatasas y lipasas. Las lipasas son enzimas importantes en el metabolismo de ácidos grasos, catalizando la hidrolisis de triglicéridos a ácidos grasos y glicerol.
- The lipase system of Yarrowia lipolyticaPublication . Choupina, Altino; Gonzalez, Francisco J.; Morín, M.; Burguillo, Francisco J.; Fermiñán, Encarnación; Dominguez, ÁngelAmong yeast species, Yarrowia lipolytica is one of the highest producers of extracellular proteins ( acid, neutral and alkaline proteases, ácid phosphatase, ribonucleases and lipases). Lipases ( triacylglycerol hydrolases) are important enzymes in fat metabolism, catalyzing the breakdown of triacilglycerols to free fatty acids and glycerol. Owing to the very low solubility of ther natural substrats, this hydrolysis is catalysed at the interfase beteween an insoluble substrat and the aqueous phase in which the enzyme is solubilized. This feature distinguishes them from esterases, which preferentially catalyze the hydrolisys of soluble esters in water.