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.

Gene replacement with one-sided homologous recombination

1992 ◽  
Vol 12 (1) ◽  
pp. 360-367
Author(s):  
N Berinstein ◽  
N Pennell ◽  
C A Ottaway ◽  
M J Shulman
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Target Gene ◽  
Gene Replacement ◽  
Immunoglobulin Genes ◽  
Nonhomologous Recombination ◽  
Immunoglobulin Locus ◽  
Chromosomal Sequences ◽  
Dna Vectors

Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.

A high efficiency cloning and expression system for proteomic analysis

PROTEOMICS ◽  
2006 ◽  
Vol 6 (14) ◽  
pp. 4038-4046 ◽  
Author(s):  
Xuan Z. Ding ◽  
Ian T. Paulsen ◽  
Apurba K. Bhattacharjee ◽  
Mikeljon P. Nikolich ◽  
Gary Myers ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
High Efficiency ◽  
Expression System ◽  
Cloning And Expression

scholarly journals Rapid, high efficiency virus-mediated mutant complementation and gene silencing in Antirrhinum

Plant Methods ◽  
2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Ying Tan ◽  
Alfredas Bukys ◽  
Attila Molnár ◽  
Andrew Hudson
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
High Efficiency ◽  
Tobacco Rattle Virus ◽  
Transient Gene Expression ◽  
Mutant Phenotype ◽  
Homologous Sequence ◽  
Homologous Gene ◽  
Loss Of Function ◽  
H Protein

Abstract Background Antirrhinum (snapdragon) species are models for genetic and evolutionary research but recalcitrant to genetic transformation, limiting use of transgenic methods for functional genomics. Transient gene expression from viral vectors and virus-induced gene silencing (VIGS) offer transformation-free alternatives. Here we investigate the utility of Tobacco rattle virus (TRV) for homologous gene expression in Antirrhinum and VIGS in Antirrhinum and its relative Misopates. Results A. majus proved highly susceptible to systemic TRV infection. TRV carrying part of the Phytoene Desaturase (PDS) gene triggered efficient PDS silencing, visible as tissue bleaching, providing a reporter for the extent and location of VIGS. VIGS was initiated most frequently in young seedlings, persisted into inflorescences and flowers and was not significantly affected by the orientation of the homologous sequence within the TRV genome. Its utility was further demonstrated by reducing expression of two developmental regulators that act either in the protoderm of young leaf primordia or in developing flowers. The effects of co-silencing PDS and the trichome-suppressing Hairy (H) gene from the same TRV genome showed that tissue bleaching provides a useful marker for VIGS of a second target gene acting in a different cell layer. The ability of TRV-encoded H protein to complement the h mutant phenotype was also tested. TRV carrying the native H coding sequence with PDS to report infection failed to complement h mutations and triggered VIGS of H in wild-type plants. However, a sequence with 43% synonymous substitutions encoding H protein, was able to complement the h mutant phenotype when expressed without a PDS VIGS reporter. Conclusions We demonstrate an effective method for VIGS in the model genus Antirrhinum and its relative Misopates that works in vegetative and reproductive tissues. We also show that TRV can be used for complementation of a loss-of-function mutation in Antirrhinum. These methods make rapid tests of gene function possible in these species, which are difficult to transform genetically, and opens up the possibility of using additional cell biological and biochemical techniques that depend on transgene expression.

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 Optimization of an In Vitro Transcription/Translation System Based on Sulfolobus solfataricus Cell Lysate

Archaea ◽  
2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Giada Lo Gullo ◽  
Rosanna Mattossovich ◽  
Giuseppe Perugino ◽  
Anna La Teana ◽  
Paola Londei ◽  
...  
Keyword(s):  
High Efficiency ◽  
Specific Activity ◽  
Expression System ◽  
Sulfolobus Solfataricus ◽  
Essential Element ◽  
23S Rrna ◽  
Translation System ◽  
Cell Lysate ◽  
Cell Extracts

A system is described which permits the efficient synthesis of proteins in vitro at high temperature. It is based on the use of an unfractionated cell lysate (S30) from Sulfolobus solfataricus previously well characterized in our laboratory for translation of pretranscribed mRNAs, and now adapted to perform coupled transcription and translation. The essential element in this expression system is a strong promoter derived from the S. solfataricus 16S/23S rRNA-encoding gene, from which specific mRNAs may be transcribed with high efficiency. The synthesis of two different proteins is reported, including the S. solfataricus DNA-alkylguanine-DNA-alkyl-transferase protein (SsOGT), which is shown to be successfully labeled with appropriate fluorescent substrates and visualized in cell extracts. The simplicity of the experimental procedure and specific activity of the proteins offer a number of possibilities for the study of structure-function relationships of proteins.

scholarly journals Genetic Analysis of Azole Resistance in the Darlington Strain of Candida albicans

2000 ◽  
Vol 44 (11) ◽  
pp. 2985-2990 ◽  
Author(s):  
Hiroshi Kakeya ◽  
Yoshitsugu Miyazaki ◽  
Haruko Miyazaki ◽  
Katherine Nyswaner ◽  
Brian Grimberg ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Candida Albicans ◽  
Amino Acid ◽  
Genetic Analysis ◽  
Expression System ◽  
Single Copy ◽  
Gene Replacement ◽  
Azole Resistance ◽  
High Level

ABSTRACT High-level azole resistance in the Darlington strain ofCandida albicans was investigated by gene replacement inC. albicans and expression in Saccharomyces cerevisiae. We sequenced the ERG11 gene, which encodes the sterol C14α-demethylase, from our copy of the Darlington strain. Both alleles contained the histidine for tyrosine substitution at position 132 (Y132H) reported in Darlington by others, but we also found a threonine-for-isoleucine substitution (I471T) not previously reported in the C. albicans ERG11. The encoded I471T change in amino acids conferred azole resistance when overexpressed alone and increased azole resistance when added to the Y132H amino acid sequence in an S. cerevisiae expression system. Replacement of one copy of ERG11 in an azole-susceptible strain of C. albicans with a single copy of the Darlington ERG11 resulted in expression of the integrated copy and a modest increase in azole resistance. The profound azole resistance of the Darlington strain is the result of multiple mutations.

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