scholarly journals A bacterial haloalkane dehalogenase gene as a negative selectable marker in Arabidopsis

1999 ◽  
Vol 18 (5) ◽  
pp. 571-576 ◽  
Author(s):  
Henrik Naested ◽  
Marko Fennema ◽  
Lin Hao ◽  
Mathias Andersen ◽  
Dick B. Janssen ◽  
...  
Author(s):  
Mayuresh M Abhyankar ◽  
Sarah M Haviland ◽  
Carol A Gilchrist ◽  
William A Petri, Jr.

Microbiology ◽  
1997 ◽  
Vol 143 (1) ◽  
pp. 109-115 ◽  
Author(s):  
A. N. Kulakova ◽  
M. J. Larkin ◽  
L. A. Kulakov

2014 ◽  
Vol 14 (2) ◽  
pp. 140-148 ◽  
Author(s):  
Michael S. Behnke ◽  
Asis Khan ◽  
L. David Sibley

ABSTRACTQuantitative trait locus (QTL) mapping studies have been integral in identifying and understanding virulence mechanisms in the parasiteToxoplasma gondii. In this study, we interrogated a different phenotype by mapping sinefungin (SNF) resistance in the genetic cross between type 2 ME49-FUDRrand type 10 VAND-SNFr. The genetic map of this cross was generated by whole-genome sequencing of the progeny and subsequent identification of single nucleotide polymorphisms (SNPs) inherited from the parents. Based on this high-density genetic map, we were able to pinpoint the sinefungin resistance phenotype to one significant locus on chromosome IX. Within this locus, a single nonsynonymous SNP (nsSNP) resulting in an early stop codon in the TGVAND_290860 gene was identified, occurring only in the sinefungin-resistant progeny. Using CRISPR/CAS9, we were able to confirm that targeted disruption of TGVAND_290860 renders parasites sinefungin resistant. Because disruption of theSNR1gene confers resistance, we also show that it can be used as a negative selectable marker to insert either a positive drug selection cassette or a heterologous reporter. These data demonstrate the power of combining classical genetic mapping, whole-genome sequencing, and CRISPR-mediated gene disruption for combined forward and reverse genetic strategies inT. gondii.


1999 ◽  
Vol 181 (7) ◽  
pp. 2050-2058 ◽  
Author(s):  
Gerrit J. Poelarends ◽  
Johan E. T. van Hylckama Vlieg ◽  
Julian R. Marchesi ◽  
Luisa M. Freitas Dos Santos ◽  
Dick B. Janssen

ABSTRACT The newly isolated bacterial strain GP1 can utilize 1,2-dibromoethane as the sole carbon and energy source. On the basis of 16S rRNA gene sequence analysis, the organism was identified as a member of the subgroup which contains the fast-growing mycobacteria. The first step in 1,2-dibromoethane metabolism is catalyzed by a hydrolytic haloalkane dehalogenase. The resulting 2-bromoethanol is rapidly converted to ethylene oxide by a haloalcohol dehalogenase, in this way preventing the accumulation of 2-bromoethanol and 2-bromoacetaldehyde as toxic intermediates. Ethylene oxide can serve as a growth substrate for strain GP1, but the pathway(s) by which it is further metabolized is still unclear. Strain GP1 can also utilize 1-chloropropane, 1-bromopropane, 2-bromoethanol, and 2-chloroethanol as growth substrates. 2-Chloroethanol and 2-bromoethanol are metabolized via ethylene oxide, which for both haloalcohols is a novel way to remove the halide without going through the corresponding acetaldehyde intermediate. The haloalkane dehalogenase gene was cloned and sequenced. The dehalogenase (DhaAf) encoded by this gene is identical to the haloalkane dehalogenase (DhaA) of Rhodococcus rhodochrous NCIMB 13064, except for three amino acid substitutions and a 14-amino-acid extension at the C terminus. Alignments of the complete dehalogenase gene region of strain GP1 with DNA sequences in different databases showed that a large part of adhaA gene region, which is also present in R. rhodochrous NCIMB 13064, was fused to a fragment of a haloalcohol dehalogenase gene that was identical to the last 42 nucleotides of thehheB gene found in Corynebacterium sp. strain N-1074.


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

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