Molecular Analysis of the Cryptococcus neoformans ADE2 Gene, a Selectable Marker for Transformation and Gene Disruption

1999 ◽  
Vol 27 (1) ◽  
pp. 36-48 ◽  
Author(s):  
Sunil Sudarshan ◽  
Robert C Davidson ◽  
Joseph Heitman ◽  
J.Andrew Alspaugh
Keyword(s):  
Cryptococcus Neoformans ◽  
Molecular Analysis ◽  
Gene Disruption ◽  
Selectable Marker ◽  
Ade2 Gene

scholarly journals Use of the Riboflavin Synthase Gene (ribC) as a Model for Development of an Essential Gene Disruption and Complementation System for Haemophilus influenzae

2004 ◽  
Vol 70 (7) ◽  
pp. 4136-4143 ◽  
Author(s):  
Amna Saeed-Kothe ◽  
Wei Yang ◽  
Scott D. Mills
Keyword(s):  
Haemophilus Influenzae ◽  
Gene Disruption ◽  
Biosynthetic Pathway ◽  
Selectable Marker ◽  
Essential Gene ◽  
Tetracycline Resistance ◽  
Single Copy ◽  
Multiple Cloning Site ◽  
Chloramphenicol Resistance ◽  
Reliable Assessment

ABSTRACT We have developed a system for rapid and reliable assessment of gene essentiality in Haemophilus influenzae Rd strain KW20. We constructed two “suicide” complementation vectors (pASK5 and pASK6) containing 5′ and 3′ regions of the nonessential ompP1 gene flanking a multiple cloning site and a selectable marker (a chloramphenicol resistance gene or a tetracycline resistance cassette). Transformation of H. influenzae with the complementation constructs directs chromosomal integration of a gene of interest into the ompP1 locus, where the strong, constitutive ompP1 promoter drives its expression. This single-copy, chromosome-based complementation system is useful for confirming the essentiality of disrupted genes of interest. It allows genetic analysis in a background free of interference from any upstream or downstream genetic elements and enables conclusive assignment of essentiality. We validated this system by using the riboflavin synthase gene (ribC), a component of the riboflavin biosynthetic pathway. Our results confirmed the essentiality of ribC for survival of H. influenzae Rd strain KW20 and demonstrated that a complementing copy of ribC placed under control of the ompP1 promoter reverses the lethal phenotype of a strain with ribC deleted.

A novel, negative selectable marker for gene disruption in Dictyostelium

Gene ◽  
1997 ◽  
Vol 202 (1-2) ◽  
pp. 171-176 ◽  
Author(s):  
Alastair Morrison ◽  
Rolf Marschalek ◽  
Theo Dingermann ◽  
Adrian J Harwood
Keyword(s):  
Gene Disruption ◽  
Selectable Marker ◽  
Negative Selectable Marker

An efficient gene-disruption method in Cryptococcus neoformans by double-joint PCR with NAT-split markers

2009 ◽  
Vol 390 (3) ◽  
pp. 983-988 ◽  
Author(s):  
Min Su Kim ◽  
Seo-Young Kim ◽  
Ja Kyung Yoon ◽  
Yin-Won Lee ◽  
Yong-Sun Bahn
Keyword(s):  
Cryptococcus Neoformans ◽  
Gene Disruption ◽  
Efficient Gene ◽  
Disruption Method

Gene Disruption by Biolistic Transformation in Serotype D Strains of Cryptococcus neoformans

2000 ◽  
Vol 29 (1) ◽  
pp. 38-48 ◽  
Author(s):  
Robert C. Davidson ◽  
M.Cristina Cruz ◽  
Rey A.L. Sia ◽  
Brandy Allen ◽  
J.Andrew Alspaugh ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Gene Disruption ◽  
Biolistic Transformation

scholarly journals Repeated use of GAL1 for gene disruption in Candida albicans.

Genetics ◽  
1991 ◽  
Vol 129 (1) ◽  
pp. 19-24 ◽  
Author(s):  
J A Gorman ◽  
W Chan ◽  
J W Gorman
Keyword(s):  
Candida Albicans ◽  
Gene Disruption ◽  
Selectable Marker ◽  
Direct Repeats ◽  
Wild Type ◽  
Ura3 Gene ◽  
Repeated Use ◽  
Cat Gene ◽  
Gal1 Gene ◽  
Diploid Organism

Abstract A technique which has the potential to allow repeated use of the same selectable marker to create gene disruptions in Candida albicans has been developed. In this approach, originally described for Saccharomyces cerevisiae, the selectable marker is flanked by direct repeats. Mitotic recombination between these repeats leads to elimination of the selectable marker. A module in which the GALq1 gene is flanked by direct repeats of the bacterial CAT gene was constructed and used to disrupt one copy of the URA3 gene in a gal1 mutant. Gal- revertants were selected by plating on 2-deoxy-D-galactose (2DOG). The frequency of 2DOG-resistant colonies recovered was 20 times higher than that obtained with a similar construct not flanked by direct repeats. Of these, 20% had lost the GAL1 gene by recombination between the direct repeats. The GAL1 gene was used again to disrupt the remaining wild-type copy of the URA3 gene of one of these gal1 isolates, resulting in a stable ura3 mutant. This technique should be generally applicable to derive homozygous gene disruptions in this diploid organism.

scholarly journals Rapid Hypothesis Testing with Candida albicans through Gene Disruption with Short Homology Regions

1999 ◽  
Vol 181 (6) ◽  
pp. 1868-1874 ◽  
Author(s):  
R. Bryce Wilson ◽  
Dana Davis ◽  
Aaron P. Mitchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Gene Function ◽  
Gene Disruption ◽  
Genomic Sequence ◽  
Selectable Marker ◽  
Genome Project ◽  
Gene Products ◽  
Pcr Products

ABSTRACT Disruption of newly identified genes in the pathogen Candida albicans is a vital step in determination of gene function. Several gene disruption methods described previously employ long regions of homology flanking a selectable marker. Here, we describe disruption of C. albicans genes with PCR products that have 50 to 60 bp of homology to a genomic sequence on each end of a selectable marker. We used the method to disrupt two known genes,ARG5 and ADE2, and two sequences newly identified through the Candida genome project,HRM101 and ENX3. HRM101 and ENX3are homologous to genes in the conserved RIM101 (previously called RIM1) and PacC pathways ofSaccharomyces cerevisiae and Aspergillus nidulans. We show that three independenthrm101/hrm101 mutants and two independentenx3/enx3 mutants are defective in filamentation on Spider medium. These observations argue that HRM101 andENX3 sequences are indeed portions of genes and that the respective gene products have related functions.

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