Short sequence-paper

Characterization and regulation of a second gene encoding thioredoxin

from the fission yeast

Yoon-Jong Lee a, Young-Wook Cho a, Daemyung Kim b, Eun-Hee Park c,James A. Fuchs d, Chang-Jin Lim a,*

aDivision of Life Sciences, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, South KoreabDepartment of Genetic Engineering, Chongju University, Chongju 360-764, South Korea

cCollege of Pharmacy, Sookmyung Women’s University, Seoul 140-742, South KoreadDepartment of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, MN 55108, USA

Received 11 September 2001; received in revised form 15 January 2002; accepted 17 January 2002

Abstract

A genomic DNA encoding a second thioredoxin (TRX2) was isolated from the chromosomal DNA of the fission yeast

Schizosaccharomyces pombe. The cloned sequence contains 1823 bp and encodes a protein of 121 amino acids. It has extra N-terminal

17 amino acid residues compared to previously identified thioredoxin (TRX1), which are positively charged and hydrophobic amino acids.

The additional N-terminal region contains a plausible prepeptidase cleavage site, indicating that the TRX2 protein exists in mitochondria. The

cloned TRX2 gene produced functional TRX estimated with insulin reduction assay. The upstream region of the TRX2 gene was fused into the

promoterless h-galactosidase gene of the shuttle vector YEp357R. The 782 bp sequence in the region further upstream of the TRX2 gene was

found to be inhibitory in its expression. Synthesis of h-galactosidase from the fusion plasmid pYFX135-HRL was enhanced by the addition

of aluminum chloride and ferrous chloride, indicating that the TRX2 protein is involved in stress response. D 2002 Elsevier Science B.V. All

rights reserved.

Keywords: Fission yeast; Genomic DNA; Regulation; Stress response; Thioredoxin; Schizosaccharomyces pombe

Thioredoxin (TRX) is a small, ubiquitous and multifunc-

tional protein that has a redox-active disulfide/dithiol within

the conserved active site sequence -Trp-Cys-Gly-Pro-Cys-.

It participates in the reduction of sulfate, methionine sulf-

oxide, and protein disulfide bonds [1]. In Escherichia coli,

TRX functions as an essential subunit of phage T7 DNA

polymerase [2] and is required for the assembly of the fila-

mentous phages M13 and f1 [3]. Furthermore, TRX is an

efficient antioxidant able to reduce hydrogen peroxide [4]

and to scavenge free radicals [5]. Eukaryotic TRX has been

implicated in various physiological functions. It can modu-

late the DNA binding activity of some transcription factors

such as TFIIIC [6], NF-nB [7] and AP-1 [8,9]. TRX was

reported to have preventive capacities towards NO-mediated

cellular injury in monocytic macrophage cells [10], and also

to be a putative oncogene conferring both growth and

survival advantage to tumor cells [11].

There are a few regulatory mechanisms identified in the

expression of TRX genes. Transcription of the Rhodobacter

sphaeroides Y TRX gene is regulated by oxygen tension

[12]. The expression of a human TRX gene is increased by

oxidative agents [13]. This type of regulation supports the

fact that TRX is involved in the regeneration of proteins

inactivated by oxidative stress [14]. Transcript levels of

Dictyostelium TRXs are regulated during the development

cycle, which are low in the growth phase and maximally

high during development [15]. TRX gene expression was

also reported to be transcriptionally up-regulated by retinol

in monkey epithelial cells [16].

More than one TRXs exist in some eukaryotes, such as

yeast [17,18] and rat [19,20]. Mitochondrial TRX has been

identified but its biological functions are not clearly known.

Reported mitochondrial TRXs are found to contain mito-

chondrial translocational signals [18,20]. The first TRX

gene was previously isolated from the fission yeast Schiz-

osaccharomyces pombe [21]. Here, we describe cloning,

characterization, and regulation of genomic DNA encoding

putative mitochondrial TRX from S. pombe.

0167-4781/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0167 -4781 (02 )00246 -4

* Corresponding author. Tel.: +82-33-250-8514; fax: +82-33-242-0459.

E-mail address: cjlim@kangwon.ac.kr (C.-J. Lim).

www.bba-direct.com

Biochimica et Biophysica Acta 1575 (2002) 143–147

The chromosomal DNAwas isolated from S. pombe cells

according to the procedure previously described [22]. PCR

was performed as described in the user’s sheet offered by

Roche Molecular Biochemicals. The PCR conditions used

in this study were 94 jC (1 min), 57 jC (1 min) and 72 jC(2 min) for 30 cycles. Thioredoxin-catalyzed reduction of

insulin by dithiothreitol was monitored as a turbidity in-

crease at 650 nm [23]. This method was used to demonstrate

the ability of thioredoxin to function as a protein disulfide

reductase. h-Galactosidase activity in extract was measured

by spectrophotometric method using o-nitrophenyl h-D-galactopyranoside (ONPG) as a substrate [24]. Protein

contents in extracts were measured by the Bradford method

[25] using bovine serum albumin as a standard.

Previously, the first genomic DNA encoding thioredoxin

(later named TRX1) was isolated and characterized from the

fission yeast S. pombe [21]. In addition to the first TRX gene, a

nucleotide sequence homologous to those of TRX genes was

identified on the genomic DNA sequence of S. pombe, which

is known to be similar to higher eukaryotes in aspects of its

physiology and genetics. To understand physiological roles

and regulation of a second thioredoxin gene, the TRX2 gene

was isolated and characterized from S. pombe. Based on the

sequence stored in the GenBank database (AL035085), two

synthetic primers (5V-AACAATT GCTTGCGGATCCC-

Fig. 1. Construction of the three subclones, pRTX602, pUTX118 and

pUTX119, used for sequencing the nucleotide sequence of the insert DNA

contained in the original clone pRTX601.

Fig. 2. The nucleotide sequence and deduced amino acid sequence of S. pombe TRX2 gene. An active site is boxed. A short bar indicates the stop codon.

Y.-J. Lee et al. / Biochimica et Biophysica Acta 1575 (2002) 143–147144

CATCTG-3V and 5V-TTAATCACCTCATAGAATTCCT-

CACGTTGTT AT-3V) were used for PCR amplification by

PyrobestR DNA polymerase. The two primers contain

BamHI and EcoRI restriction sites, respectively, which can

be used for ligation. They were also designed to amplify the

plausible TRX coding region as well as 1090 bp upstream

sequence which should contain a sufficient region for tran-

scriptional regulation. The amplified DNA product was

identified and purified from agarose gel electrophoresis,

and then, completely digested with BamHI and EcoRI. The

BamHI/EcoRI-digested PCR product was ligated into the

BamHI/EcoRI site of the E. coli–yeast shuttle vector

pRS316. The ligation mixture was used to transform the E.

coli strain MV1184 according to the CaCl2 procedure [26].

After isolating plasmid DNA from transformed E. coli cells,

the plasmid DNA was confirmed by restriction mapping as

having the desired size. Plasmid pRTX601 contain a 1823 bp

insert. The PCR product and the insert DNA fragment were

confirmed to have the same size on agarose gel electro-

phoresis (data not shown). To determine the nucleotide

sequence, three subclones were constructed using the unique

HindIII site within the insert of pRTX601. Plasmid pRTX602

contains the BamHI–HindIII fragment, whereas plasmids

pUTX118 and pUTX119 contain the HindIII–EcoRI frag-

ment in vectors pUC118 and pUC119, respectively (Fig. 1).

The original clone and the three subclones were used for

automated sequencing in Bionex Inc., Korea. The nucleotide

sequence of the BamHI–EcoRI insert of the original plasmid

pRTX601was determined by completely overlapping the two

DNA strands. The nucleotide sequence of the S. pombe TRX2

gene was submitted to the GenBank under the accession

number AY034142.

The 1823 bp sequence of the cloned TRX2 gene, shown in

Fig. 2, is identical with the stored GenBank sequence except

for three positions. The coding region contains an intron. The

unique open-reading frame encodes a protein of 121 amino

acids with a calculated mass of 13.7 kDa (Fig. 2). The

determined sequence contains an upstream sequence of

1090 bp and a downstream of 285 bp. Analysis of amino

acid composition indicates that it doesn’t contain histidine

and is rich in alanine, leucine, lysine and serine (Fig. 2). Its

isoelectric point is estimated to be 9.45. By analyzing with

computer programs, it is assumed to have six a-helix regions

and two turns, and to be relatively hydrophobic. The amino

acid sequence of the TRX2 protein was compared with that of

the TRX1 protein (Fig. 3). They have 30.6% identity, which

is scattered over the whole region. However, TRX2 has an

Fig. 3. Sequence comparison (A) and schematic alignment (B) of S. pombe

TRX1 and TRX2. The extra N-terminal region of TRX2 contains a high

ratio of positively charged and hydrophobic amino acid residues. A possible

mitochondrial prepeptidase cleavage site is indicated by an arrow, and MTS

indicates mitochondrial translocation signal. Asterisks represent identical

amino acid residues in S. pombe TRX1 and TRX2.

Fig. 4. Amino acid sequence alignments of the amino acid sequence of S. pombe TRX2 and those of other mitochondrial thioredoxins. The asterisks are

identical amino acids. The amino acid sequence of mitochondrial TRXs were collected from some organisms such as human (Accession No. XP 038644) and

rat [20].

Y.-J. Lee et al. / Biochimica et Biophysica Acta 1575 (2002) 143–147 145

extra N-terminal region, which does not exist in the TRX1

protein (Fig. 3A). The N-terminal region of the TRX2 has a

high content of positively charged and hydrophobic amino

acid residues (Fig. 3B). A possible mitochondrial prepepti-

dase cleavage site is in the N-terminal region (Fig. 3B). These

support that the TRX2 protein exists in the mitochondria. A

sequence alignment of the S. pombe TRX2 with mitochon-

drial TRXs from human and rat is shown in Fig. 4. The amino

acid sequence of the S. pombe TRX2 has 35.5% and 34.7%

homologies with those of human and rat counterparts,

respectively. All three TRXs share very similar mitochondrial

translocational signals (Fig. 4). However, the S. pombe TRX2

appears to be very dissimilar with the corresponding proteins

of the budding yeast S. cerevisiae (data not shown). It might

give one evidence that S. pombe is closer to mammalian cells

than S. cerevisiae.

To examine whether the cloned S. pombe TRX2 gene

produces a functional TRX, plasmid pRTX601 was intro-

duced into the wild-type S. pombe KP1. Then, it was grown

up to mid-exponential phase, and harvested. Cell extract

was used for insulin reduction assay. The S. pombe cells

harboring pRTX601 showed higher specific activity com-

pared with that of vector alone (Fig. 5). The HindIII/EcoRI

insert fragment of pRTX601 was transferred into the shuttle

vector pYES2 to generate the plasmid pYEX-HRL. The S.

pombe cells harboring pYEX-HRL also gave higher specific

activity (data not shown). The assay results with the two

plasmid constructs definitely support that the cloned TRX2

gene is functional in S. pombe cells.

Fig. 5. Expression of the cloned TRX2 gene in S. pombe with insulin

reduction assay. The experiment was carried out as described in the main

text. Absorbance at 650 nm was measured as an indicator of turbidity

produced from the reduction of insulin. Specific activity is represented as

DOD650/min/mg protein.

Fig. 6. Construction of TRX2– lacZ fusions and synthesis in S. pombe of h-galactosidase from the fusion plasmids. The fusion plasmid pYFX135–

HRL lacks 782 bp BamHI/HindIII fragment of the insert DNA contained in

the fusion plasmid pYFX135. Plasmid YEp357R carries 2 A origin and

promoterless h-galactosidase gene. The specific activity is expressed as

DOD420/min/Ag protein.

Fig. 7. Effect of aluminum chloride (A) and ferrous chloride (B) on the

synthesis of h-galactosidase from the fusion plasmid pYFX135–HRL in

S. pombe cells. The yeast cells harboring the fusion plasmid were grown

in minimal medium, and split at the early exponential phase. Aluminum

chloride or ferrous chloride was added to individual culture flask and con-

tinued to be shaken. The h-galactosidase activity was determined at 25 jCby the spectrophotometric assay using ONPG as a substrate. The specific

activity is expressed in DOD420/min/Ag protein.

Y.-J. Lee et al. / Biochimica et Biophysica Acta 1575 (2002) 143–147146

Considering the multiple functions of TRX in living cells,

the regulation of TRX gene may play an important role in its

functioning. To independently monitor the expression of the

S. pombe TRX2 genomic DNA, the upstream region of

pRTX601 was fused into the promoterless h-galactosidasegene of the fusion vector YEp357R. Before the ligation, the

appropriate upstream region was amplified by PCR. After

transformation into E. coli strain MV1184, the desired sub-

clone was confirmed by blue colony formation and restric-

tion mapping. The resultant fusion plasmids are pYFX135

and pYFX135-HRL. These plasmids do not contain original

N-terminal region of the TRX2 protein with the exception of

methionine. Plasmid pYFX135 contains 1090 bp BamHI/

EcoRI upstream region, whereas plasmid pYFX135-HRL

contains 308 bp HindIII/EcoRI upstream region. The S.

pombe cells harboring the two fusion plasmids were found

to contain h-galactosidase activities (Fig. 6). The S. pombe

cells containing plasmid pYFX135-HRL contains higher h-galactosidase activities. This strongly suggests that 782 bp

BamHI/HindIII upstream region is inhibitory in the expres-

sion of the TRX2 gene. The 782 bp upstream region is found

to be very rich in A/T pairs (72.0%).

According to the growth curve of the S. pombe cells

harboring plasmid pYFX135-HRL, the synthesis of h-gal-actosidase was determined. h-Galactosidase activity was

higher in the exponential phase than in the stationary phase

(data not shown). This indicates that the TRX2 gene is

expressed in a higher degree at the exponential phase. The

effect of the addition of aluminum chloride (5 mM) was

examined using exponential S. pombe cells (Fig. 7A). Its

addition significantly increased the synthesis of h-galactosi-dase. Addition of ferrous chloride to the culture also induced

h-galactosidase from the fusion plasmid (Fig. 7B). These

suggest that the TRX2 gene is involved in the response to

oxidative stress. The precise patterns of regulation of the

TRX2 gene should be approached further, which would give

important information on its physiological roles in mitochon-

dria.

Acknowledgements

This work was supported by a grant (No. R01 2000 00133)

from the Basic Research Program of the Korea Science of

Engineering Foundation.

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