The heterologous protein showed the highest activity and affinity with l-rhamnose and a lower activity and affinity with l-mannose and l-lyxose.. The cells were harvested before the sugars
Trang 1L-rhamnose-1-dehydrogenase gene
Outi M Koivistoinen, Satu Hilditch, Sanni P Voutilainen, Harry Boer, Merja Penttila¨ and
Peter Richard
VTT Technical Research Centre of Finland, Espoo, Finland
l-Rhamnose (l-6-deoxy-mannose) is a C6 sugar that is
enriched in some fractions of plant biomass, such as
hemicellulose and pectin Several microorganisms
liv-ing on decayliv-ing plant material are able to use
l-rham-nose as a source of carbon and energy There are at
least two pathways for the catabolism of l-rhamnose,
one with phosphorylated intermediates and one
with-out The path with the phosphorylated intermediates
was described in bacteria, and involves the following
intermediates: l-rhamnulose, l-rhamnulose
1-phos-phate, dihydroxyacetone phos1-phos-phate, and
l-lactalde-hyde The corresponding enzymes are l-rhamnose
isomerase (EC 5.3.1.14) [1,2], rhamnulokinase
(EC 2.7.1.5) [3,4] and rhamnulose-1-phosphate aldolase
(EC 4.1.2.19) [5] l-Lactaldehyde can then be reduced
to 1,2-propenediol or oxidized to lactate by lactalde-hyde reductase (EC 1.1.1.77) or lactaldelactalde-hyde dehydro-genase (EC 1.2.1.22) respectively, depending on the redox conditions [6] Gene sequences for all these enzymes have been described [7]
The pathway without phosphorylated intermediates
is distinctly different It has been described in different yeast species [8,9] The intermediates in this pathway are l-rhamnono-1,4-lactone, l-rhamnonate, l-erythro-3,6-dideoxyhexulosonate, pyruvate and l-lactaldehyde The corresponding enzymes are NAD-utilizing
l-rhamnose-1-dehydrogenase (EC 1.1.1.173) [10,11],
l-rhamnono-1,4-lactonase (EC 3.1.1.65), l-rhamnonate dehydratase (EC 4.2.1.90), and l -erythro-3,6-dide-oxyhexulosonate aldolase (EC 4.1.2.-) (Fig 1) The
Keywords
L -rhamnonate; L -rhamnose catabolism;
L -rhamnose dehydrogenase;
MALDI-TOF MS; Pichia stipitis
Correspondence
P Richard, Technical Research Centre of
Finland, Tietotie 2, Espoo, PO Box 1000,
02044 VTT, Finland
Fax: +358 20 722 7071
Tel: +358 20 722 7190
E-mail: peter.richard@vtt.fi
(Received 4 January 2008, revised 10 March
2008, accepted 11 March 2008)
doi:10.1111/j.1742-4658.2008.06392.x
There are two distinctly different pathways for the catabolism of l-rham-nose in microorganisms One pathway with phosphorylated intermediates was described in bacteria; here the enzymes and the corresponding gene sequences are known The other pathway has no phosphorylated intermedi-ates and has only been described in eukaryotic microorganisms For this pathway, the enzyme activities have been described but not the correspond-ing gene sequences The first enzyme in this catabolic pathway is the NAD-utilizing l-rhamnose 1-dehydrogenase The enzyme was purified from the yeast Pichia stipitis, and the mass of its tryptic peptides was determined using MALDI-TOF MS This enabled the identification of the correspond-ing gene, RHA1 It codes for a protein with 258 amino acids belongcorrespond-ing to the protein family of short-chain alcohol dehydrogenases The ORF was expressed in Saccharomyces cerevisiae As the gene contained a CUG codon that codes for serine in P stipitis but for leucine in S cerevisiae, this codon has changed so that the same amino acid was expressed in
S cerevisiae The heterologous protein showed the highest activity and affinity with l-rhamnose and a lower activity and affinity with l-mannose and l-lyxose The enzyme was specific for NAD A northern blot analysis revealed that transcription in P stipitis is induced during growth on
l-rhamnose but not on other carbon sources
Abbreviation
YNB, yeast nitrogen base.
Trang 2l-lactaldehyde is oxidized to l-lactate in an
NAD-cou-pled reaction, as in the pathway with the
phosphory-lated intermediates For this pathway, only the enzyme
activities have been described; the corresponding genes
have not been identified in any yeast or in any other
organism
l-Rhamnose dehydrogenase activity was described
in Pichia stipitis NRC5568 This enzyme used NAD as
a cofactor The enzyme activity was
l-rhamnose-induced and d-glucose-repressed With the crude cell
extract, an activity of about 0.1 lmolÆmin)1Æmg)1 was
observed It was suggested that the reaction product
was the l-rhamnono-d-lactone and not the more stable
l-rhamnono-c-lactone, as the c-lactone could not be
identified as a reaction product [9]
In the present work, we identified the gene coding
for the l-rhamnose dehydrogenase in P stipitis We
expressed it in the heterologous host
Saccharomy-ces cerevisiae and characterized the enzyme kinetic
properties
Results
The P stipitis strain CBS 6054 was grown on yeast
nitrogen base (YNB) supplemented with 2%
l-rham-nose, 1% d-glucose and 1% l-rhamnose or 2%
d-glucose The cells were harvested before the sugars were utilized, and the crude cell extract was analyzed for l-rhamnose dehydrogenase activity Cells grown on
l-rhamnose as a sole carbon source had an l-rhamnose dehydrogenase activity of 14 nkatÆmg)1 of extracted protein Cells grown on the d-glucose⁄ l-rhamnose mixture had an activity of 2 nkatÆmg)1, and the cells grown on d-glucose did not show any l-rhamnose dehydrogenase activity
We used the cell extract of the l-rhamnose-grown cells to purify the protein The purification included three steps: a DEAE column, native PAGE, and SDS⁄ PAGE From the DEAE column, which was eluted with a salt gradient, the activity eluted as a sin-gle peak with a specific activity of 10 nkatÆmg)1 The fractions around this activity peak were analyzed by SDS⁄ PAGE, and showed about 20 different proteins (Fig 2) The active fraction was then concentrated and separated by native PAGE, and a single band with
l-rhamnose dehydrogenase activity was identified using zymogram staining This active band from the native PAGE was cut out from the gel, and the partially puri-fied protein was eluted It was then applied to an SDS⁄ PAGE gel, and this revealed four proteins with estimated sizes of 30, 35, 52 and 70 kDa (Fig 2) The
30 kDa protein was preliminarily identified as the
COOH
C O
CH3
HC
C H
CH3 HO O pyruvic acid
L-lactaldehyde
L-rhamnose
L -rhamnose dehydrogenase
EC 1.1.1.173
L -erythro-3,6-dideoxy hexulosonic acid aldolase
EC 4.1.2.
L -lactaldehyde dehydrogenase
EC 1.2.1.22
L-lactic acid
COOH
C OH C C
H OH
C
HO H
HO H
CH 3
H
COOH
C O C C
H H
C
HO H
HO H
CH3
L-rhamnonic acid
L -rhamnonic ac id dehydratase
EC 4.2.1.90
L-erythro-3,6-dideoxy hexulosonic acid
COOH
C H
CH3 HO
NAD
H2O NADH
NADH NAD
OH
H H
HO OH
H H O
H3C
HO
H
(OH)
(H)
H
HO OH
O
H3C HO
H
O
L-rhamnonic acid-1,4-lactone
L-rhamnonic acid lactonase
EC 3.1.1.65
Fig 1 Fungal path for L -rhamnose catabolism The enzyme activities but not the corresponding gene sequences of this pathway have been described previously The identification of a gene coding for the L -rhamnose dehydrogenase is the subject of the present article.
Trang 3l-rhamnose dehydrogenase This was done by
compar-ing the results of the SDS⁄ PAGE that was done after
the zymogram staining with those of the SDS⁄ PAGE
of the active fractions after the DEAE column and
correlating them with the enzyme activity of that
frac-tion The 30 kDa protein matched with l-rhamnose
dehydrogenase activity
After trypsination, the peptide masses of the 30 kDa
protein were determined by MALDI-TOF MS As the
genome sequence of P stipitis is known [12], these
masses allowed the identification of the protein on the
basis of matching peptide sequences The masses
555.247, 900.475, 1199.639, 1761.782, 1872.708 and
2552.586 were identified as tryptic peptides of a protein
with the GenBank identifier ABN68405 This protein
had been annotated as a putative
d-glucose-1-dehydro-genase II The protein has 258 amino acids and a
calculated molecular mass of 27.102 Da, and belongs
to the family of short-chain alcohol dehydrogenases
We called the gene for the first gene in the l-rhamnose
catabolic pathway RHA1
To verify that we had indeed identified the
l-rham-nose dehydrogenase, we expressed the protein in S
ce-revisiae P stipitis is known to translate CTG to serine
and not to leucine [13] The l-rhamnose dehydrogenase
contained one such codon at bp 166–168 of the ORF,
which we changed to TCG In this way, we ensured
that a protein with the same amino acid sequence was
expressed in S cerevisiae In S cerevisiae, the
l-rham-nose dehydrogenase was expressed from a multicopy plasmid with the S cerevisiae PGK1 promoter, which
is a strong and constitutive promoter In the crude extract of S cerevisiae, we found an l-rhamnose dehy-drogenase activity of about 200 nkatÆmg)1 of protein
In the control strain, which contained the empty vec-tor, no activity was observed
In order to facilitate the purification, we expressed the Rha1 protein in S cerevisiae with an N-terminal
or with a C-terminal histidine-tag The histidine-tags were introduced by adding the additional nucleotide sequence by PCR as specified in Experimental proce-dures Both constructs were expressed with the same vector in the same yeast strain When testing the two modified proteins in the crude extract of
S cerevisiae, we observed that the N-terminally tagged enzyme did not exhibit any activity The C-terminally tagged enzyme showed activity in the crude extract; however, the activity was reduced by about 80% when compared to the activity of the nontagged enzyme in the crude extract As the tag-ging of the enzyme had such a strong effect on the activity, we did not proceed to purify the enzyme Instead, we used the crude cell extract of the S cere-visiae strain expressing the untagged enzyme for the kinetic characterization
We observed activity with l-rhamnose, l-lyxose, and
l-mannose No activities were observed with d-eryth-rose, d-allose, d-ribose, d-arabinose, d-tagatose, d-glu-cose, d-galactose, d-xylose and l-arabinose, and none
of the sugars showed activity in the control strain with the empty vector The highest activity, Vmax about
200 ± 20 nkatÆmg)1 of protein in the crude extract, was observed with l-rhamnose With l-lyxose and
l-mannose, the activities were lower; the Vmax values were 170 ± 20 nkatÆmg)1 and 75 ± 10 nkatÆmg)1 respectively The highest affinity was towards l-rham-nose, the Km being 1.5 ± 0.025 mm Lower affinities were obtained with l-lyxose and l-mannose; here, the
Km values were 5 ± 0.5 mm and 25 ± 5 mm respec-tively The enzyme showed activity with NAD as a cofactor; the Vmax was 200 ± 20 nkatÆmg)1, and the
Km was 0.2 ± 0.03 mm (Fig 3) No activity was observed with NADP as a cofactor (Fig 4)
The activity was pH-dependent At pH 6.8, the activity was about 100 ± 10 nkatÆmg)1, at pH 8.0 it was 200 ± 20 nkatÆmg)1, and at pH 9.5 it was
240 ± 25 nkatÆmg)1 To test the activity in the reverse direction, we incubated the enzyme preparation with
l-rhamnonate and NADH at pH 8.0 No activity was observed under these conditions At this pH, l-rhamn-onate is expected to be in the linear and not in the c-lactone or d-lactone form
Fig 2 Coomassie-stained SDS ⁄ PAGE gel of the protein fractions
after the different purification steps Lane A contains the molecular
mass markers (masses in kDa are indicated) Lane B contains the
combined active fractions after the DEAE column separation.
Lane C shows the protein eluted from the excised band of the
native PAGE gel after zymogram staining.
Trang 4Northern analysis
To study the role of the l-rhamnonate dehydrogenase
in P stipitis, the transcription of RHA1 with different
carbon sources was studied by northern analysis The
P stipitis strain CBS 6054 was grown on l-rhamnose,
d-glucose, maltose, d-galactose, d-xylose and a
glyc-erol⁄ ethanol mixture as carbon sources The results
are shown in Fig 5 We observed transcription only
on l-rhamnose, suggesting that RHA1 expression is
l-rhamnose-induced
Discussion
There are at least two different catabolic pathways for
l-rhamnose One pathway has phosphorylated
inter-mediates; the enzyme activities and the corresponding
genes have been well described, and it has only been observed in prokaryotic microorganisms The other pathway has no phosphorylated intermediates, and has
so far been described only in yeast For this pathway, the enzyme activities have been described; however, none of the corresponding genes had been identified
In this work, we identified the gene coding for the first enzyme in this pathway, an NAD:l-rhamnose-1-dehy-drogenase
Twerdochlib et al [9] had reported previously that
l-rhamnose dehydrogenase activity was present in
P stipitis when the yeast was grown on a mixture of
d-glucose and l-rhamnose, and absent when grown on
d-glucose as a sole carbon source We confirmed this, and also noticed that the activity was increased sev-eral-fold when the yeast was grown on l-rhamnose as
a sole carbon source The enzymatic activity that was induced in this way was then purified During the puri-fication, the activity always appeared as a single peak, indicating that only one enzyme is responsible for this activity The purified enzyme was then digested with trypsin, and the masses of the peptides were identified using MALDI-TOF MS The genome sequence of
P stipitis is available, and this enabled the identifica-tion of the corresponding ORF, which was then called RHA1 RHA1 was induced when the yeast was grown
on l-rhamnose, but not when it was grown on any other carbon sources, as shown by the northern blot analysis (Fig 5) This suggests that the induction of the l-rhamnose dehydrogenase activity is the result of induction of transcription of RHA1
To characterize the enzyme’s kinetic properties, we expressed RHA1 in the heterologous host S cerevisiae This resulted in l-rhamnose-1-dehydrogenase activity, showing that this gene does indeed code for a protein with this activity We also added a histidine-tag to the N-terminus or to the C-terminus of the protein in order to facilitate the purification However, the tagged proteins showed no or very much reduced activity in the crude extract As this might be an indication that the tag is interfering with the catalytic activity, we did not use any of the tagged proteins for the kinetic char-acterization, but used the crude cell extract of the
S cerevisiaestrain expressing RHA1
The Rha1 protein was specific for NAD as a cofac-tor, which is in agreement with earlier observations [9] The sugars that were accepted in the catalytic reaction were l-rhamnose, l-lyxose, and l-mannose (Figs 3 and 4) C1 to C4 in these sugars share the same configura-tion When the hydroxyl group at C4 was in the oppo-site configuration, as in d-ribose, no activity was observed Also, no activity was found with any other C1–C4 configuration, and when the C5 was missing, as
0 10 20 30 40 50 60
0
50
100
150
200
A
B
L-rhamnose
L-lyxose
L-mannose
0.0 0.5 1.0 1.5 2.0
0
50
100
150
200
NA D
Fig 3 Kinetic properties of the L -rhamnose-1-dehydrogenase The
heterologously expressed protein was analyzed in a crude cell
extract at pH 8.0 (A) The NAD concentration is 1.5 m M The curves
are calculated assuming a Michaelis–Menten kinetic model: L
-rham-nose, Km= 1.5 m M , Vmax= 200 nkatÆmg)1; L -lyxose, Km= 5 m M ,
V max = 170 nkatÆmg)1; L -mannose, K m = 25 m M , V max = 75 nkatÆ
mg)1 (B) The L -rhamnose concentration is 60 m M The curve is
cal-culated assuming a Michaelis–Menten kinetic model: Km= 0.2 m M ,
V max = 200 nkatÆmg)1.
Trang 5in d-erythrose This indicates that the C1–C4
stereo-chemical configuration is essential for recognition by
the enzyme, and that an additional carbon atom must
be attached to C4 We cannot say whether a hydroxyl
group on C5 is required, as a sugar without a hydroxyl
at C5 was not tested Among the three sugars that
showed activity, the highest activity and affinity were
observed with l-rhamnose, indicating that this enzyme
is indeed an l-rhamnose dehydrogenase The
possibil-ity that the enzyme is a glucose-1-dehydrogenase, as suggested in the first annotation based on sequence similarity, can be excluded, as no activity was observed with glucose
Twerdochlib reported that the l-rhamnonse dehy-drogenase from P stipitis did not produce any detect-able amounts of l-rhamnono-c-lactone, suggesting that this enzyme produced the more unstable l-rhamnono-d-lactone [9] If the l-rhamnono-l-rhamnono-d-lactone was in a rapid equilibrium with the l-rhamnonic acid at neutral
pH, one might expect to see some reverse activity with
l-rhamnonic acid and NADH We tested the reverse reaction but could not observe any, indicating that the intermediate is a lactone that, at neutral pH, is present
in too low concentrations for the reverse reaction to occur
Sugar dehydrogenases that oxidize the sugar to a sugar acid are not very common in eukaryotic micro-organisms S cerevisiae has NADP-requiring [14] and NAD-requiring d-arabinose dehydrogenases [15], ARA1 and ARA2, which contribute to erythroascorbic acid production These proteins belong to the family
of aldo⁄ keto reductases In the mold Hypocrea
jecori-na, an NADP-requiring d-xylose dehydrogenase was described that belonged to the GFO⁄ IDH ⁄ MOCA protein family [16]
There are also other reports of eukaryotic sugar dehydrogenases, such as an NADP-utilizing d-glucose dehydrogenase in Schizosaccharomyces pombe [17], an NAD-utilizing d-glucose dehydrogenase in Aspergil-lus niger [18], and an NADP-utilizing d-xylose dehy-drogenase in Pichia quercuum [19] However, for these proteins, the corresponding sequences are not known, and it is not clear to what protein family they belong The protein described in this article belongs to the pro-tein family of short-chain dehydrogenases, and has the conserved domain of a fabG [3-ketoacyl-(acyl-carrier protein) reductase] The sugar dehydrogenases in eukary-otic microorganisms belong to very different protein families, although the catalytic reaction is very similar
Experimental procedures
Enzyme assays
If not otherwise specified, the enzyme activity was measured
in a reaction mixture containing 100 mm Tris⁄ HCl (pH 8.0), 1 mm NAD, and the crude cell extract or a pro-tein preparation The reaction was started by the addition
of 10 mm l-rhamnose or other sugars when specified The formation of NADH was followed by measuring the absorbance at 340 nm To assay the enzyme activity in the reverse direction, the crude cell extract was incubated in
CHO
C
C
C
CH3
H
CHO
C C
C
CH2OH
H
CHO
C C
CH2OH
H
Fig 4 Fischer projection of the sugars that showed activity with
the L -rhamnose dehydrogenase.
Fig 5 Northern blot analysis of RHA1 expression The expression
of RHA1 in P stipitis on L -rhamnose (lane A), D -glucose (lane B),
maltose (lane C), D -galactose (lane D), D -xylose (lane E) and a
glyc-erol ⁄ ethanol mixture (lane F) The lower panel shows the total RNA
in the gel after staining with the SYBR Green II RNA gel stain.
Trang 6100 mm Tris⁄ HCl (pH 8.0), 200 lm NADH and 100 mm
l-rhamnonate The disappearance of NADH was followed
by measuring the absorbance at 340 nm l-Rhamnonate was
synthesized from l-rhamnose by oxidation with bromine,
and purified by ion exchange chromatography as described
by Yew et al [20] The analysis was done in a Cobas Mira
automated analyzer (Roche, Basel, Switzerland) at 30C
Enzyme purification
The P stipitis strain CBS 6054 was grown in 500 mL of
medium containing YNB without amino acids (BD,
Rock-ville, MD, USA) and 2% l-rhamnose as a carbon source in
shake flasks under aerobic conditions for about 2 days The
yeast was collected by centrifugation at 3000 g for 15 mins,
washed, and resuspended in 40 mL of 5 mm sodium
phos-phate (pH 7.0) supplemented with Complete medium
with-out EDTA (Roche) protease inhibitor Equal amounts of
glass beads (0.4 mm diameter), fresh cell cake and
resuspen-sion buffer were extracted in a Mini-Bead Beater (Biospec
Products, Bartlesville, OK, USA) two times for 1 min each
The mixture was then centrifuged in an Eppendorf
micro-centrifuge at full speed for 20 min at 4C The supernatant
was desalted with a PD10 column (GE Healthcare,
Amer-sham, UK) equilibrated with 5 mm sodium phosphate
(pH 7.0) and subsequently loaded onto a 10 mL DEAE
column (Merck, Darmstadt, Germany) The protein amount
loaded onto the column was about 16 mg The column was
then eluted with 200 mL of a linear gradient from 0 to
200 mm NaCl in the same buffer Fractions of 2.5 mL were
collected and analyzed for l-rhamnose dehydrogenase
activ-ity The fractions in which activity was observed were then
concentrated using Vivaspin 2 10 000 MWCO PES
centrifu-gation columns (Vivascience Satorius group) and analyzed
by SDS⁄ PAGE The concentrated protein was then
sepa-rated by native PAGE (12% acrylamide) The gel was then
stained in a zymogram staining solution similar to what has
been described previously [21] The zymogram staining
solu-tion contained 200 mm Tris⁄ HCl (pH 8.0), 100 mm
l-rham-nose, 0.25 mm nitroblue tetrazolium, 0.06 mm phenazine
methosulfate, and 0.5 mm NAD The only band that
appeared was cut out and eluted by overnight incubation in
100 mm Tris⁄ HCl (pH 8.0) and 0.1% SDS The protein was
again concentrated, and separated by SDS⁄ PAGE Of the
four proteins that were detected, the 30 kDa protein
coin-cided with the l-rhamnose dehydrogenase activity as judged
by the previous SDS⁄ PAGE gel of the active fractions
In-gel digestion and MALDI-TOF MS
The 30 kDa protein observed in the SDS⁄ PAGE gel was
in-gel digested with trypsin, and the peptides were extracted
essentially according to the method of Rosenfeld et al [22]
The samples were desalted using a C-18 matrix (Eppendorf
Perfect Pure C-18 Tip) The saturated matrix solution was
prepared by dissolving recrystallized a-cyano-4-hydroxycin-namic acid (CCA; Bruker Daltonics, Bremen, Germany) in
a 50% acetonitrile⁄ 0.1% trifluoroacetic acid solution Equal volumes of purified peptide sample or calibration standard (peptide calibration mixture II; Bruker Daltonics) were mixed with the saturated matrix solution One microliter of this matrix⁄ sample mixture was applied to the target (target plate ground steel TF; Bruker Daltonics) and allowed to dry at room temperature The peptide masses were then determined by MALDI-TOF MS using a Bruker Auto-flex II mass spectrometer flexanalysis software (Bruker Daltronics) was used for the data analysis
Cloning of the ORF, site-directed mutagenesis and heterologous expression
The ORF was amplified from the genomic DNA by PCR using primers 5¢-GGATCCATCATGACTGGATTGTTGA ATGG-3¢ and 5¢-GGATCCCTATTGTAAATTGACGAA CAATCCTC-3¢, and the DynazymeEXT DNA polymerase (Finnzymes, Espoo, Finland) The primers contained BamHI restriction sites (underlined) to facilitate further plasmid constructions The PCR product was then ligated
to the pCR2.1 TOPO vector (Invitrogen, Carlsbad, CA, USA) and cloned Nucleotides 166–168 of the ORF were changed from CTG to TCG with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) After the site-directed mutagenesis, the ORF was released
as a BamHI fragment and ligated to the BglII site of p1181, which is a multicopy yeast expression vector based
on YEplac195, containing URA3 for selection, where the PGK1 promoter and terminator were introduced [23] The
S cerevisiae strain CEN.PK2-1D was then transformed with the resulting plasmid and grown on selective medium
A control strain contained p1181 S cerevisiae strains expressing C-terminally or N-terminally histidine-tagged enzymes were generated in a similar way The coding sequences for six histidines were introduced by PCR either
at the N-terminus or at the C-terminus To introduce the histidine-tag at the N-terminus, we introduced a coding sequence for MHHHHHHGG before the original start codon To introduce the histidine-tag at the C-terminus, we introduced the coding sequence for GGHHHHHH before the stop codon The template for the PCR was the vector where the CTG was changed to TCG For the expression
of the histidine-tagged proteins, the same plasmid was used
A crude cell extract was made by vortexing with glass beads
as described above for P stipitis
Northern analysis The P stipitis strain CBS 6054 was grown in YNB medium supplemented with 20 gÆL)1 of the following carbon sources: l-rhamnose, d-glucose, maltose, d-galactose,
d-xylose, and an ethanol⁄ glycerol mixture The RNA was
Trang 7extracted from the yeast cells with the Trizol reagent kit
(Life Technologies Inc.); about 5 lg of the total RNA per
sample was used in the analysis The RNA amount was
checked by staining (Fig 4, lower panel) with the SYBR
Green II RNA gel stain (Lonza, Rockland, ME, USA)
Northern hybridization was carried out using standard
methods As a probe for the hybridization, the ORF,
released as a BamHI fragment from the TOPO vector, was
used The probe was labeled with [32P]dCTP[aP] (GE
Healthcare) using the randomly primed DNA labeling kit
(Roche)
Acknowledgements
This research was supported by an Academy Research
Fellowship for P Richard from the Academy of
Finland We thank Outi Ko¨no¨nen for excellent
techni-cal assistance
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