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Supplementary Information
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Construction of H. polymorpha strains harboring lacZ expression plasmids under
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control of the HpMET3 promoter with various lengths
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To construct the lacZ expression plasmids under control of the HpMET3 promoter with
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various length, the 1 kb, 0.8 kb, 0.6 kb, 0.4 kb and 0.2 kb DNA fragments corresponding to
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the promoter region of HpMET3, respectively, were obtained by PCR amplification with the
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primers MET3p1000_1F, MET3p800_2F, MET3p600_3F, MET3p400_4F, MET3p200_5F
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and MET3p2B_RNC from the genomic DNA of the DL1-L strain. The resulting HpMET3
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promoter fragments were digested with ClaI and SalI and inserted into the ClaI/SalI-digested
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pDLMOX-yEGFPm expression vector, which contains the selectable marker HpLEU2, to
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generate pDLM3P1000, pDLM3P800, pDLM3P600, pDLM3P400, and pDLM3P200. The
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EcoRI/NsiI fragment of lacZ, encoding β-galactosidase, generated from pZAM522, was
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introduced into pDLM3P600, yielding pDLM3P600-lacZ. The 1 kb, 0.8 kb, 0.4 kb and 0.2 kb
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of the HpMET3 promoter fragments were digested with SalI and EcoRI from the plasmids
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above and re-inserted between the SalI/EcoRI sites of pDLM3P600-lacZ, generating
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pDLM3P1000-lacZ,
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respectively. The H. polymorpha wild-type (WT) DL1-L strain was transformed with the
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individual plasmids above and selected on SC-Leu medium.
pDLM3P800-lacZ,
pDLM3P400-lacZ,
and
pDLM3P200-lacZ,
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Construction of H. polymorpha strains harboring lacZ expression plasmids under
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control of the ScMET3 promoter
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To construct the lacZ expression plasmid under control of the ScMET3 promoter, the
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BglII/EcoRI-digested 0.6 kb ScMET3 promoter, obtained from PScMET3-ScERG7(L), was used
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to replace the HpMET3 promoter region of pDLM3P600-lacZ, resulting in pSM3P600-lacZ.
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The H. polymorpha wild-type (WT) DL1-L strain was transformed with the plasmid
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pSM3P600-lacZ and selected on SC-Leu medium.
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Supplementary Table 1. Plasmids used in this study.
Plasmid name
Description
Reference
pDLMOX-yEGFPm
H. polymorpha expression vector containing the
enhanced GFP gene under the control of the
MOX promoter, an ampicillin resistance gene,
HpLEU2, and HARS36
(Park et al. 2007)
pZAM522
H. polymorpha expression vector containing an
zeocin resistance gene, the lacZ gene, and the
TEF1 promoter
(Agaphonov,
unpublished)
pDLM3
H. polymorpha expression vector containing
HpLEU2
This study
pDLM3P1000-lacZ
pDLM3 containing the lacZ gene under the
control of the 1.0 kb HpMET3 promoter
This study
pDLM3P800-lacZ
pDLM3 containing the lacZ gene under the
control of the 0.8 kb HpMET3 promoter
This study
pDLM3P600-lacZ
pDLM3 containing the lacZ gene under the
control of the 0.6 kb HpMET3 promoter
This study
pDLM3P400-lacZ
pDLM3 containing the lacZ gene under the
control of the 0.4 kb HpMET3 promoter
This study
pDLM3P200-lacZ
pDLM3 containing the lacZ gene under the
control of the 0.2 kb HpMET3 promoter
This study
pDLMP600-PMT2
pDLM3 containing the PMT2 gene under the
control of 0.6 kb of the HpMET3 promoter and
HpLEU2
This study
pBluescript SK (+)
Bacterial expression vector containing an
ampicillin resistance gene
Stratagene
Cloning Systems
YEp351GAPII
S. cerevisiae expression vector containing 2 μ,
LEU2, and the TDH3 promoter and terminator
(Abe et al. 2003)
PScMET3-ScERG7(L)
pBluescript SK (+) containing ScLEU2 and the
0.8 kb ScERG7 fragment under the control of 0.6
kb ScMET3 promoter
This study
PHpMET3-ScERG7(L)
pBluescript SK (+) containing ScLEU2 and the
0.8 kb ScERG7 fragment under the control of 0.6
kb HpMET3 promoter
This study
pSM3P600-lacZ
H. polymorpha expression vector containing
HpLEU2 and the lacZ gene under the control of
This study
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the 0.6 kb ScMET3 promoter
YCpU
S. cerevisiae centromeric expression vector
containing ScURA3
Moon, in press
YCpU-SM3P600-lacZ
YCpU containing the lacZ gene under the control
of the 0.6 kb ScMET3 promoter
This study
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Supplementary Table 2. Oligonucleotide primers used in this study.
Primer Name
Sequences (5′–3 ′)
MET3p1000_1F
tgaggtcgaccttcagatgctcggctgga
MET3p800_2F
tgaggtcgacccagatgtgcaaggccgtca
MET3p600_3F
tgaggtcgactggccaccttgcacacaga
MET3p400_4F
tgaggtcgaccgacctcttttctgcgctg
MET3p200_5F
tgaggtcgacgtgcagagacctgtaccac
MET3p2B_RNC
gtatcgatgcattcagaattccattttgggaggagagtcg
HpPMT2_11F_RI
gtggaattcatggtgcctacgcaatctaa
HpPMT2_12B_StuI
ttaggcctgtccaccatggtgtagatacc
HpPMT2_2B_AscI_50
tatggcgcgccgtcgtctgcaactctcca
HpACT1F-RT
tccaggctgtgctgtcgttg
HpACT1B-RT
ccggccaagtcgattctcaa
HpPMT2F-RT
gacaagcggacctggtggaa
HpPMT2B-RT
tcatggcgatgttgagctgg
SspI BglII pScMET3 1F
cgcgaatattagatctgatgctcagaatacccgt
pScMET3 ATG EcoRI 2B
gcgcgaattccatgttaattatactttattcttg
EcoRI ScERG7 1F
gcgcgaattcacagaattttattctgacacaatc
ScERG7 SalI 2B
gcgcgtcgaccaaagtagtagaatgggggt
pSM3-SE7integ2B
gtggaccatggaacaatg
pSM3-SE7integ4B
tcttcgctattacgccag
pSM3-SE7integ5F
cattgagcacaagcctct
ScACT1 RTPCR fw 22mer
attgccgaaagaatgcaaaagg
ScACT1 RTPCR rv 22mer
gaaccaccaatccagacggagt
ScERG7_RT_1F
gatgaaggatggcggttggg
ScERG7_RT_2B
ggtcaataccgcggtcgatg
*Recognition sites for restriction enzymes are underlined
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Supplementary References
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Abe H, Shimma Y, Jigami Y (2003) In vitro oligosaccharide synthesis using intact yeast cells
that display glycosyltransferases at the cell surface through cell wall-anchored protein
Pir. Glycobiology 13:87-95
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Park JN et al. (2007) Identification of the cadmium-inducible Hansenula polymorpha SEO1
gene promoter by transcriptome analysis and its application to whole-cell heavy-metal
detection systems Appl Environ Microbiol 73:5990-6000
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