Reversible impairment of the ku80 gene by a recyclable marker in Aspergillus aculeatus
Auxotrophic mutants of Aspergillus can be isolated in the presence of counter-selective compounds, but the process is laborious. We developed a method to enable reversible impairment of the ku80 gene ( Aaku80 ) in the imperfect fungus Aspergillus aculeatus . Aaku80 was replaced with a selection mark...
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Published in | AMB Express Vol. 3; no. 1; p. 4 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
Berlin/Heidelberg
Springer Berlin Heidelberg
12.01.2013
Springer Nature B.V BioMed Central Ltd Springer |
Subjects | |
Online Access | Get full text |
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Summary: | Auxotrophic mutants of
Aspergillus
can be isolated in the presence of counter-selective compounds, but the process is laborious. We developed a method to enable reversible impairment of the
ku80
gene (
Aaku80
) in the imperfect fungus
Aspergillus aculeatus
.
Aaku80
was replaced with a selection marker, orotidine 5’-phosphate decarboxylase (
pyrG
), followed by excision of
pyrG
between direct repeats (DR) to yield the
Aaku80
deletion mutant (MR12). The gene-targeting efficiency at the ornithine carbamoyltransferase (
argB
) locus was drastically elevated from 3% to 96% in MR12. The frequency of marker recycling depended on DR length. One uridine auxotroph was obtained from 3.3 × 10
5
, 1.4 × 10
5
, and 9.2 × 10
3
conidia from strains harboring 20-, 98-, and 495-bp DRs, respectively. Because these strains maintained the short DRs after 5 d of cultivation, we investigated whether
Aaku80
function was disrupted by
pyrG
insertion with the 20-bp DR and restored after excision of
pyrG
. The
Aaku80
disruption mutant (coku80) was bred by inserting
pyrG
sandwiched between 20-bp DRs into the second intron of
Aaku80
, followed by excision of
pyrG
between the DRs to yield the coku80rec strain. Analyses of homologous recombination frequency and methyl methanesulfonate sensitivity demonstrated that
Aaku80
function was disrupted in coku80 but restored in coku80rec. Furthermore,
pyrG
was maintained in coku80 at least for ten generations. These data indicated that reversible impairment of
ku80
in
A
.
aculeatus
is useful for functional genomics in cases where genetic segregation is not feasible. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 2191-0855 2191-0855 |
DOI: | 10.1186/2191-0855-3-4 |