Cloning and characterization of industrially important alpha-galactosidase genes from the human pathogen aspergillus fumigatus

Download

index.pdf

Date

2004

Author

Söyler, U. Betül

Metadata

Show full item record

Item Usage Stats

312
views

111
downloads

In this study, molecular cloning studies were performed on the a-galactosidase genes of Aspergillus fumigatus IMI 385708. This organism is an opportunistic saprophytic fungus and a human pathogen, mainly affecting immunocompromised patients. A. fumigatus is a thermotolerant fungus and can efficiently produce thermostable a-galactosidase. Two different cloning strategies were undertaken in this study. A. fumigatus cDNA library, prepared previously, was screened with three different probes. No net results were obtained from these screenings. However, the DNA probes used were shown to be homologous to the a-galactosidase gene (agl1) of Trichoderma reesei. After the completion of the genome project of A. fumigatus, regions with homology to a-galactosidase genes were searched on the genome of A. fumigatus. PCR-based cloning studies were performed by designing specific primers for these regions. Two a-galactosidase genes, namely aglA and aglB were amplified with these specific primers, sequenced, and ligated to vector pUC19. The recombinant plasmid was then used to transform E. coli XL1 Blue MRF̕ cells. aglB gene consists of an open reading frame of 1684 bp containing six introns. The gene encodes a protein of 447 amino acids with a signal sequence of 22 amino acids and four N-glycosylation sites. aglA gene has an open reading frame of 1599 bp without introns. The gene encodes a protein of 532 amino acids with a putative signal sequence of 21 amino acids and four putative N-glycosylation sites. Cloning of a-galactosidase genes represents a first step towards the high level expression of these enzymes in a GRAS host.

Subject Keywords

Genetics.

URI

http://etd.lib.metu.edu.tr/upload/12605106/index.pdf
https://hdl.handle.net/11511/14329

Collections

Graduate School of Natural and Applied Sciences, Thesis

Suggestions

OpenMETU
Core

Development of a genetic material transfer approach for gene therapy Ayaz, Şerife; Hasırcı, Vasıf Nejat; Department of Biotechnology (2005) This thesis is focused on the development of a gene delivery system, especially for the purpose of DNA vaccination. DNA expression vectors have the potential to be useful therapeutics for a wide variety of applications. A carrier system was designed to realize the delivery of genes to cells and the promotion of controlled adequate expression in the target cells. The low gene delivery efficiency observed with systems composed of polyplexes is mainly due to low stability of polycation e.g polyethylenimine-DNA...
PCR cloning and heterologous expression of Scytalidium thermophilum laccase gene in Aspergillus sojae Koçlar, Gülden; Ögel, Zümrüt Begüm; Department of Biotechnology (2005) In this study, Scytalidium thermophilum laccase gene was first cloned into E. coli and then heterologously expressed in A. sojae. S. thermophilum is a thermophilic fungus with an important role in determining selectivity of compost produced for growing Agaricus bisporus. S. thermophilum laccase gene was first cloned by Novo Nordisk Bio Tech, Inc. in 1998. This laccase gene (lccS) has an open reading frame of 2092bp. It is composed of five exons punctuated by four small introns. The coding region, excluding ...
Identification and analysis of genomic regions with large between-population differentiation in humans Myles, S.; Tang, K.; Somel, Mehmet; Green, R. E.; Kelso, J.; Stoneking, M. (Wiley, 2008-01-01) The primary aim of genetic association and linkage studies is to identify genetic variants that contribute to phenotypic variation within human populations. Since the overwhelming majority of human genetic variation is found within populations, these methods are expected to be effective and can likely be extrapolated from one human population to another. However, they may lack power in detecting the genetic variants that contribute to phenotypes that differ greatly between human populations. Phenotypes that...
Analysis of self-processing mechanism of galactose oxidase by site-directed mutagenesis and heterologous expression in Escherichia Coli Gençer, Burçak; Ögel, Zümrüt Begüm; Department of Biotechnology (2005) In this study, self-catalytic maturation of heterologously expressed pro-galactose oxidase was analysed in E.coli by altering some amino acids which were supposed to play a crucial role in pro-peptide removal. Galactose oxidase (GOase; EC 1.1.3.9) from Fusarium graminearum; having a molecular mass of 68kDa, is a monomeric, copper containing enzyme with an unusual thioether bond. The enzyme is produced as a precursor with an additional 8 amino acid pre- and a 17- amino acid pro-sequence at the N terminus. Pr...
A novel approach for small sample size family-based association studies: sequential tests İlk Dağ, Özlem; Dungul, Dilay Ciglidag; ÖZDAĞ, Hilal; İLK, HAKKI GÖKHAN (Springer Science and Business Media LLC, 2011-08-01) In this paper, we propose a sequential probability ratio test (SPRT) to overcome the problem of limited samples in studies related to complex genetic diseases. The results of this novel approach are compared with the ones obtained from the traditional transmission disequilibrium test (TDT) on simulated data. Although TDT classifies single-nucleotide polymorphisms (SNPs) to only two groups (SNPs associated with the disease and the others), SPRT has the flexibility of assigning SNPs to a third group, that is,...

Citation Formats

U. B. Söyler, “Cloning and characterization of industrially important alpha-galactosidase genes from the human pathogen aspergillus fumigatus,” M.S. - Master of Science, Middle East Technical University, 2004.