A Homologous Gene Replacement based Genetic Algorithm

2016 ◽  
Author(s):  
Sumaiya Iqbal ◽  
Md Tamjidul Hoque
Keyword(s):  
Genetic Algorithm ◽  
Gene Replacement ◽  
Homologous Gene

scholarly journals Contributions ofyap1Mutation and SubsequentatrFUpregulation to Voriconazole Resistance inAspergillus flavus

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Yuuta Ukai ◽  
Miho Kuroiwa ◽  
Naoko Kurihara ◽  
Hiroki Naruse ◽  
Tomoyuki Homma ◽  
...  
Keyword(s):  
Point Mutation ◽  
Nuclear Export ◽  
Resistance Mechanism ◽  
Resistant Mutant ◽  
Gene Replacement ◽  
Bzip Transcription Factor ◽  
Homologous Gene ◽  
Sequencing Analysis ◽  
Content Type

ABSTRACTAspergillus flavusis the second most significant pathogenic cause of invasive aspergillosis; however, its emergence risks and mechanisms of voriconazole (VRC) resistance have not yet been elucidated in detail. Here, we demonstrate that repeated exposure ofA. flavusto subinhibitory concentrations of VRCin vitrocauses the emergence of a VRC-resistant mutant with a novel resistance mechanism. The VRC-resistant mutant shows a MIC of 16 μg/ml for VRC and of 0.5 μg/ml for itraconazole (ITC). Whole-genome sequencing analysis showed that the mutant possesses a point mutation inyap1, which encodes a bZIP transcription factor working as the master regulator of the oxidative stress response, but no mutations in thecyp51genes. This point mutation inyap1caused alteration of Leu558 to Trp (Yap1Leu558Trp) in the putative nuclear export sequence in the carboxy-terminal cysteine-rich domain of Yap1. This Yap1Leu558Trpsubstitution was confirmed as being responsible for the VRC-resistant phenotype, but not for that of ITC, by the revertant to Yap1wild typewith homologous gene replacement. Furthermore, Yap1Leu558Trpcaused marked upregulation of theatrFATP-binding cassette transporter, and the deletion ofatrFrestored susceptibility to VRC inA. flavus. These findings provide new insights into VRC resistance mechanisms via a transcriptional factor mutation that is independent of thecyp51gene mutation inA. flavus.

scholarly journals Adaptation of the Yeast URA3 Selection System to Gram-Negative Bacteria and Generation of a ΔbetCDE Pseudomonas putida Strain

2005 ◽  
Vol 71 (2) ◽  
pp. 883-892 ◽  
Author(s):  
Teca Calcagno Galvão ◽  
Víctor de Lorenzo
Keyword(s):  
Pseudomonas Putida ◽  
General Procedure ◽  
Gene Replacement ◽  
Selection Marker ◽  
Homologous Gene ◽  
Gram Negative Bacteria ◽  
Selection System ◽  
Gram Negative ◽  
Acid Sensitivity ◽  
Delivery Vector

ABSTRACT A general procedure for efficient generation of gene knockouts in gram-negative bacteria by the adaptation of the Saccharomyces cerevisiae URA3 selection system is described. A Pseudomonas putida strain lacking the URA3 homolog pyrF (encoding orotidine-5′-phosphate decarboxylase) was constructed, allowing the use of a plasmid-borne copy of the gene as the target of selection. The delivery vector pTEC contains the pyrF gene and promoter, a conditional origin of replication (oriR6K), an origin of transfer (mobRK2), and an antibiotic selection marker flanked by multiple sites for cloning appropriate DNA segments. The versatility of pyrF as a selection system, allowing both positive and negative selection of the marker, and the robustness of the selection, where pyrF is associated with uracil prototrophy and fluoroorotic acid sensitivity, make this setup a powerful tool for efficient homologous gene replacement in gram-negative bacteria. The system has been instrumental for complete deletion of the P. putida choline-O-sulfate utilization operon betCDE, a mutant which could not be produced by any of the other genetic strategies available.

scholarly journals Fungal Functional Genomics: Tunable Knockout-Knock-in Expression and Tagging Strategies

2009 ◽  
Vol 8 (5) ◽  
pp. 800-804 ◽  
Author(s):  
Luis F. Larrondo ◽  
Hildur V. Colot ◽  
Christopher L. Baker ◽  
Jennifer J. Loros ◽  
Jay C. Dunlap
Keyword(s):  
Functional Genomics ◽  
Target Gene ◽  
High Efficiency ◽  
Expression System ◽  
Gene Replacement ◽  
Fungal Species ◽  
Homologous Gene ◽  
Molecular Tools ◽  
Phenotypic Information ◽  
Endogenous Loci

ABSTRACT Strategies for promoting high-efficiency homologous gene replacement have been developed and adopted for many filamentous fungal species. The next generation of analysis requires the ability to manipulate gene expression and to tag genes expressed from their endogenous loci. Here we present a suite of molecular tools that provide versatile solutions for fungal high-throughput functional genomics studies based on locus-specific modification of any target gene. Additionally, case studies illustrate caveats to presumed overexpression constructs. A tunable expression system and different tagging strategies can provide valuable phenotypic information for uncharacterized genes and facilitate the analysis of essential loci, an emerging problem in systematic deletion studies of haploid organisms.

scholarly journals Deletion of the 389 N-Terminal Residues of the Transcriptional Activator AREA Does Not Result in Nitrogen Metabolite Derepression in Aspergillus nidulans

1998 ◽  
Vol 180 (21) ◽  
pp. 5762-5764 ◽  
Author(s):  
Mark X. Caddick ◽  
Herbert N. Arst
Keyword(s):  
Amino Acids ◽  
Aspergillus Nidulans ◽  
Deletion Mutant ◽  
Transcriptional Activator ◽  
Gene Replacement ◽  
Untranslated Regions ◽  
Homologous Gene ◽  
Heterologous Promoter ◽  
Native Promoter ◽  
N Terminus

ABSTRACT Utilizing a homologous gene replacement in order to retain the native promoter and 5′ and 3′ untranslated messenger regions (and thereby ensure physiological validity), we have shown that deletion of the N-terminal 389 amino acids of the transcriptional activator AREA does not result in nitrogen metabolite derepression inAspergillus nidulans. Our results provide no evidence for a modulating interaction involving the N terminus of AREA and contrast with those of H. K. Lamb, A. L. Dodds, D. R. Swatman, E. Cairns, and A. R. Hawkins (J. Bacteriol. 179:6649–6656, 1997), who used nontargeted ectopic copies of a construct containing a heterologous promoter and untranslated regions. Results obtained with this deletion mutant, nevertheless, provide further evidence for the dispensability of large portions of AREA.

scholarly journals Homologous gene replacement in Physarum.

Genetics ◽  
1995 ◽  
Vol 139 (1) ◽  
pp. 147-158
Author(s):  
T G Burland ◽  
D Pallotta
Keyword(s):  
Plasmid Dna ◽  
Cell Types ◽  
Single Copy ◽  
Gene Replacement ◽  
Terminal Region ◽  
Homologous Gene ◽  
Cell Type ◽  
Carboxy Terminal Region ◽  
Cloned Gene ◽  
Carboxy Terminal

Abstract The protist Physarum polycephalum is useful for analysis of several aspects of cellular and developmental biology. To expand the opportunities for experimental analysis of this organism, we have developed a method for gene replacement. We transformed Physarum amoebae with plasmid DNA carrying a mutant allele, ardD delta 1, of the ardD actin gene; ardD delta 1 mutates the critical carboxy-terminal region of the gene product. Because ardD is not expressed in the amoeba, replacement of ardD+ with ardD delta 1 should not be lethal for this cell type. Transformants were obtained only when linear plasmid DNA was used. Most transformants carried one copy of ardD delta 1 in addition to ardD+, but in two (5%), ardD+ was replaced by a single copy of ardD delta 1. This is the first example of homologous gene replacement in Physarum. ardD delta 1 was stably maintained in the genome through growth, development and meiosis. We found no effect of ardD delta 1 on viability, growth, or development of any of the various cell types of Physarum. Thus, the carboxy-terminal region of the ardD product appears not to perform a unique essential role in growth or development. Nevertheless, this method for homologous gene replacement can be applied to analyze the function of any cloned gene.

scholarly journals δ-Aminolaevulinic acid synthesis is required for virulence of the wheat pathogen Stagonospora nodorum

Microbiology ◽  
2006 ◽  
Vol 152 (5) ◽  
pp. 1533-1538 ◽  
Author(s):  
Peter S. Solomon ◽  
Cordula I. Jörgens ◽  
Richard P. Oliver
Keyword(s):  
Single Gene ◽  
Acid Synthesis ◽  
Gene Replacement ◽  
Stagonospora Nodorum ◽  
Homologous Gene ◽  
In Planta ◽  
Gene Encoding ◽  
Aminolaevulinic Acid ◽  
Key Enzyme

δ-Aminolaevulinic acid (ALA) is synthesized in fungi by ALA synthase, a key enzyme in the synthesis of haem. The requirement for ALA synthase in Stagonospora nodorum to cause disease in wheat was investigated. The single gene encoding ALA synthase (Als1) was cloned and characterized. Expression analysis determined that Als1 transcription was up-regulated during germination and also towards the latter stages of the infection. The Als1 gene was further characterized by homologous gene replacement. The inactivation of Als1 resulted in strains producing severely stunted germ tubes leading quickly to death. The strains could be recovered by supplementation with 33 μM ALA. Pathogenicity assays revealed the als1 strains were essentially non-pathogenic, inferring a key role for the synthesis of ALA during in planta growth. Supplementing the strains with ALA restored growth in vitro and also pathogenicity for up to 5 days after inoculation. Further examination by inoculating the als1 strains onto wounded leaves found that pathogenicity was only partially restored, suggesting that host-derived in planta levels of ALA are not sufficient to support growth. This study has identified a key role for fungal ALA synthesis during infection and revealed its potential as an antifungal target.

scholarly journals Mating Type Protein Mat1-2 from Asexual Aspergillus fumigatus Drives Sexual Reproduction in Fertile Aspergillus nidulans

2008 ◽  
Vol 7 (6) ◽  
pp. 1029-1040 ◽  
Author(s):  
Wioletta Pyrzak ◽  
Karen Y. Miller ◽  
Bruce L. Miller
Keyword(s):  
Aspergillus Fumigatus ◽  
Aspergillus Nidulans ◽  
Mating Type ◽  
Sexual Development ◽  
Genetic System ◽  
Gene Replacement ◽  
Sexual Cycle ◽  
Homologous Gene ◽  
Gene Exchange ◽  
First Report

ABSTRACT The lack of an experimentally amenable sexual genetic system in Aspergillus fumigatus is a major limitation in the study of the organism's pathogenesis. A recent comparative genome analysis revealed evidence for potential sexuality in A. fumigatus. Homologs of mating type genes as well as other genes of the “sexual machinery” have been identified in anamorphic A. fumigatus. The mat1-2 gene encodes a homolog of MatA, an HMG box mating transcriptional factor (MatHMG) that regulates sexual development in fertile Aspergillus nidulans. In this study, the functionalities of A. fumigatus mat1-2 and the Mat1-2 protein were determined by interspecies gene exchange between sterile A. fumigatus and fertile A. nidulans. Ectopically integrated A. fumigatus mat1-2 (driven by its own promoter) was not functional in a sterile A. nidulans ΔmatA strain, and no sexual development was observed. In contrast, the A. fumigatus mat1-2 open reading frame driven by the A. nidulans matA promoter and integrated by homologous gene replacement at the matA locus was functional and conferred full fertility. This is the first report showing that cross species mating type gene exchange between closely related Ascomycetes did not function in sexual development. This is also the first report demonstrating that a MatHMG protein from an asexual species is fully functional, with viable ascospore differentiation, in a fertile homothallic species. The expression of mat1-2 was assessed in A. fumigatus and A. nidulans. Our data suggest that mat1-2 may not be properly regulated to allow sexuality in A. fumigatus. This study provides new insights about A. fumigatus asexuality and also suggests the possibility for the development of an experimentally amenable sexual cycle.

scholarly journals Analysis of the Saccharomyces cerevisiae mitochondrial COX3 mRNA 5' untranslated leader: translational activation and mRNA processing.

1995 ◽  
Vol 15 (6) ◽  
pp. 3291-3300 ◽  
Author(s):  
G Wiesenberger ◽  
M C Costanzo ◽  
T D Fox
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Replacement ◽  
Mrna Processing ◽  
Start Codon ◽  
Detectable Effect ◽  
Homologous Gene ◽  
Activator Proteins ◽  
Large Deletions ◽  
Translational Activation

We used transformation of yeast mitochondria and homologous gene replacement to study features of the 613-base COX3 mRNA 5' untranslated leader (5'-UTL) required for translational activation by the protein products of the nuclear genes PET54, PET122, and PET494 in vivo. Elimination of the single AUG triplet in the 5'-UTL had no detectable effect on expression, indicating that activator proteins do not work by allowing ribosomes to bypass that AUG. Deletion of the entire 5'-UTL completely prevented translation, suggesting that the activator proteins do not function by antagonizing any other negative element in the 5'-UTL. Removal of the 15 terminal bases from the 5' end of the 5'-UTL did not block activator-dependent translation. The largest internal deletion that did not interfere with translation removed 125 bases from the upstream portion of the leader. However, two large deletions that blocked translation could be reverted to activator-dependent expression by secondary changes in the remaining 5'-UTL sequences, indicating that the original deletions had not removed the translational activator target but only deformed it. Taken together, the deletion mutations and revertants define a region of 151 bases (between positions -480 and -330 relative to the start codon) containing sequences that are sufficient for translational activation when modified slightly. Suppression of the respiratory phenotypes of two 5'-UTL mutations by overexpression of PET54, PET122, and PET494 indicated functional interactions between the leader and the three activator proteins. The mature COX3 mRNA is cleaved from a precursor immediately downstream of the preceding tRNAVal in a fashion resembling mRNA processing in vertebrate mitochondria. Our results indicate that the site of this cleavage in Saccharomyces cerevisiae is determined solely by the position of the tRNA.

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