Browsing by Author "Chávez, R"

Now showing 1 - 5 of 5
  • No Thumbnail Available
    Item
    Differences in expression of two endoxylanase genes (xynA and xynB) from Penicillium purpurogenum
    (2002) Chávez, R; Schachter, K; Navarro, C; Peirano, A; Aguirre, C; Bull, P; Eyzaguirre, J
    A number of xylanolytic microorganisms secrete to the medium several molecular forms of endoxylanases. The physiological function of these isoforms is not clears one possibility is that they are produced under different growth conditions. To study this problem, we have used two endoxylanases (XynA and XynB) produced by the fungus Penicillium purpurogenum. These enzymes have been previously purified and characterized; they belong to family 10 and 11 of the glycosyl hydrolases, respectively. The promoters of the xynA and xynB genes have been sequenced: both present consensus sequences for the binding of the carbon catabolite repressor CreA, but otherwise show substantial differences. The xynB promoter has eight boxes in tandem for the binding of the XlnR activator and lacks the consensus sequence for the PacC pH regulator. On the other hand, the xynA promoter contains one XlnR box and three PacC consensus sequences. To investigate if these differences are reflected in gene expression, Northern blot assays were carried out. The xynA gene is transiently expressed when oat spell xylan is used as carbon source, but negligible expression was observed with birchwood xylan, xylose or xylitol. In contrast, xynB is broadly induced by all these carbon sources; this may be related to the presence of several XlnR boxes. Similar results were obtained by zymogram analysis of the expressed proteins. The different induction capabilities of birchwood and oat spelt xylan may be due to differences in their composition and structure. Expression assays carried out at different pH reflects that, despite the lack of PacC binding sites in the xynB promoter, this gene is tightly regulated by pH. The findings described here illustrate new and important differences between endoxylanases from families 10 and 11 in P. purpurogenum. They may help explain the production of multiple endoxylanase forms by this organism. (C) 2002 Published by Elsevier Science B.V.
  • No Thumbnail Available
    Item
    Electrophoretic karyotype of the filamentous fungus Penicillium purpurogenum and chromosomal location of several xylanolytic genes
    (2001) Chávez, R; Fierro, F; Gordillo, F; Martín, JF; Eyzaguirre, J
    The electrophoretic karyotype of the filamentous fungus Penicillium purpurogenum has been resolved. Using contour-clamped homogeneous electric field gel electrophoresis, five chromosomal bands were separated, with estimated sizes of 7.1, 5.2, 3.7, 2.9 and 2.3 Mbp, giving a total genome size of 21.2 Mbp. To our knowledge, this is the smallest Penicillium genome determined so far. By Southern blots and using homologous probes, the chromosomal location of five xylanolytic genes from P. purpurogenum was determined: axel (acetyl xylan esterase I), xynB (endoxylanase B) and abf1 (arabinofuranosidase 1) in chromosome I, xynA (endoxylanase A) in chromosome II, and axeII (acetyl xylan esterase II) in chromosome Ill. This is the first study where the location of xylanase genes in a Penicillium genome has been established. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
  • No Thumbnail Available
    Item
    Secretion of endoxylanase A from Penicillium purpurogenum by Saccharomyces cerevisiae transformed with genomic fungal DNA
    (2002) Chávez, R; Navarro, C; Calderón, I; Peirano, A; Bull, P; Eyzaguirre, J
    Saccharomyces cerevisiae was transformed with a genomic library from Penicillium purpurogenum, and an endoxylanase-producing yeast clone (named 44A) that grows on xylose or xylan as sole carbon source was isolated. This yeast synthesizes xynA mRNA and secretes endoxylanase A to culture media when grown on xylan or xylose, but not glucose. Analysis by pulse-field gel electrophoresis and sequencing indicates that xynA, including its eight introns, has been inserted into the yeast genome. It was shown by sequencing that clone 44A is able to correctly splice xynA introns. This is the first successful attempt to express a fungal endoxylanase gene in yeast with correct intron splicing. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
  • No Thumbnail Available
    Item
    Structure analysis of the endoxylanase A gene from Penicillium purpurogenum
    (2001) Chávez, R; Almarza, C; Schachter, K; Peirano, A; Bull, P; Eyzaguirre, J
    Penicillium purpurogenum produces several endoxylanases, two of which (XynA and XynB) have been purified and characterized. XynB has been sequenced, and it belongs to glycosyl hydrolase family 11. In this publication we report the structure of the xynA gene, The amino terminal sequence of the protein was determined and this allowed the design of oligonucleotides for use in polymerase chain reactions. Different polymerase chain reaction strategies were used to amplify and sequence the entire cDNA and the gene. The gene has an open reading frame of 1450 base pairs, including 8 introns with an average length of 56 base pairs each. Only one copy of this gene is present in the P. purpurogenum genome as shown by Southern blot. The gene encodes a protein of 329 residues (including the signal peptide), and the calculated molecular mass of the mature protein is 31,668 Da. Immunodetection assays of the expressed gene positively identified it as xynA, and sequence alignments indicate a high degree of similarity with family 10 endoxylanases. It is concluded that P. purpurogenum produces endoxylanases of family 10 and 11. The complementary action of endoxylanases of both families may be important for an efficient degradation of xylan by the fungus.
  • No Thumbnail Available
    Item
    The acetyl xylan esterase II gene from Penicillium purpurogenum is differentially expressed in several carbon sources, and tightly regulated by pH
    (2004) Chávez, R; Schachter, K; Navarro, C; Peirano, A; Bull, P; Eyzaguirre, J
    The expression of the acetyl xylan esterase II (axell) gene from Penicillium purpurogenum is repressed by glucose and induced by xylan, as well as to a small degree by xylose and xylitol. This gene is expressed at neutral pH, but not tinder alkaline or acidic conditions, in agreement with previous findings for other xylanolytic genes of this organism. This is the first report showing pH regulation of an axe gene.