ATG

1
Single crossover (homologous recombination)
loops the circular plasmid into the
chromosomal snoArif1 locus
AmpR
argB
ATG
alcA
Exon 1
X
Plasmidloop-in
TAG
ATG
Exon 1
Exon 2
ATG
TAG
ATG
argB
AmpR
alcA
Exon 1
Exon 2
Exon 1
3 truncated snoArif1 allele
alcA-driven wild-type snoArif1 allele
2
A typical example of alcA-regulated gene
expression in an Aspergillus nimO18 mutant
3
When the alcArif1OE strains are grown on
inducing carbon sources (glycerol, EtOH,
Threonine), wild type snoA is overexpressed,
killing the cells in a dose dependent manner
snoA- (3 truncation)
alcAsnoA
alcA(p)
argB
Exon 1 (854)
Exon 2 (854)
Exon 1 (854)
Exon 1 (3-1)
Exon 1 (3-1)
snoA31 nimO18
alcAsnoArif1 nimO18
wt
nimO18
Rich medium 2 glucose
Minimal medum 1 glucose
Temp 29ºC
Minimal medium 50mM glycerol
Minimal medium 200mM EtOH
4
DAPI staining showed that ethanol-induced
snoArif1 overexpression causes cell cycle arrest
during interphase
Ethanol, 18 hours growth, 29oC alcArif1
overexpression causes cell cycle arrest
Glucose, 8 hours growth, 29oC Normal, wt growth
5
Integration at
Mapping functional domains of snoArif1 by using
C-terminal truncations driven by the alcA
promoter
argB snoArif1
37C Gluc 29C EtOH
37C Gluc 29C EtOH
aa1764 TAG
snoA31 W407C
snoA215 E586X
ATG
- - - -
alcA
aa1702 AatII
- - - -
ATG
alcA
TAA
aa1460 PvuII
- - - -
ATG
alcA
TAA
alcA
aa1280 HpaI
- - () -
TAA
aa971 XbaI
- () -
ATG
TAA
6
Continued truncation experiments to map snoArif1
domains
Minimum length for normal gene function
Minimum length for lethal overexpression
aa1044 HindIII
aa1236 EagI
aa1764 TAG
aa1386 SmaI
aa1409 BstZ17I
aa1048 NgoMIV
aa1196 SnaBI
snoA31 W407C
snoA215 E586X
ATG
alcA
aa1702 AatII
aa1460 PvuII
aa1280 HpaI
aa971 XbaI