Vol. 187, No. 2, 1992 September
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 949-955
16, 1992
A GENE
CLONING ENCODING
AND SEQUENCING OF YEAST THIOLTRANSFER.ASE Zhong-Ru
Gan
Department of Biological Chemistry, Merck Research Laboratory West Point, PA 19486 Received
July
31,
W26-431
1992
A 69 bp yeast genomic DNA fragment encoding yeast thioltransferase was amplified by PCR technique. A yeast genomic DNA library was screened by a specific probe obtained from the PCR product. An 718 bp DNA fragment was found to encode yeast thioltransferase and its flanking sequence. The deduced amino acid sequence of the gene, designated ‘ITR, agrees with that derived from conventional amino acid sequence analysis except two extra ammo acids on the Cterminus. In contrast to yeast thioredoxin, Southern blot analysis of total yeast genomic DNA indicated that there was only one copy of gene encoding yeast thiotransferase. A putative TATA box was found at 109 bp from the starting codon. However, no polyadenylation signal sequence was identified on the DNA sequence downstream the 3’ end of the gene. 0 1992Academic Press, Inc.
Thioltransferase thiol-disulfide reduction glutathione pyruvate (58),
(glutaredoxin)
oxidoreduction
of low molecular
presumably
mammalian
papain,
weight and protein regulates
by changing
thioltransferase
thioltransferase
in the
in the presence of
other enzyme activities, and ornithine
such as decarboxylase
the redox status of these enzymes. Recently,
was identified
The active site center of thioltransferase sequence -Cys-Pro-Phe(Tyr)-Cys-
in many cellular
participates
disulfides
iodothyronine-5’-deiodonase
the active site of thioredoxin.
involved
processes (1, 2). It directly
(3, 4). Thioltransferase kinase,
is a small protein
as a dehydroascorbate contains a dithiol
(10-14). A similar
dithiol
reductase
within
(9).
the consensus
structure
is observed in
However the pKa of the active site cysteine of
is 3 to 4 pH units lower than that of thioredoxin
(12, 15, 16), 0006-291X/92
Copyright
949
AU
rights
0
1992
of reproduction
by Academic in any
$4.00
Press, j&m
Inc.
reserved.
Vol.
187,
No.
suggesting transfer
that thioltransferase of reduction
equivalents
equivalents.
from NADPH,
purpose. The relative cellular
BIOCHEMICAL
2, 1992
redox potential
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
is much more potent than thioredoxin Whereas thioredoxin
thioltransferase
contributions
receives reduction
uses reduced glutathione
of thioltransferase
in the
and thioredoxin
for this to the
are obscure.
In the present studies, the yeast thioltransferase
gene, TTR, and its flanking
sequences were cloned and sequenced. In contrast to yeast thioredoxin, encoded by at least two copies of different of the thioltransferase
genes (17), yeast contains
which is only one copy
gene.
Experimental
Procedures
Mat&ah-Yeast genomic lambda Dash library was from Stratagene. Restriction endonucleases, polynucleotide kinase, and T4 DNA ligase were purchased fi-om New England BioLabs. Taq DNA polymerase and DNA Thermal Cycler PCR apparatus were from Perkin-Elmer Cetus. DNA sequencing kits (Sequenase Version 2) were from United States Biochemical Corp. 35S-a-dATP and 32P-a-ATP were purchased from Amersham and Du Pont NEN Products. Preparation of yeast genomic DNA-Genomic DNA from Saccharomyces cerevisiae DMY6 (Leu’) was isolated as described previously (17). The isolated DNA was pure enough to carry out polymerase chain reaction and endonuclease digestion. Polymerase chain reaction-Two primers with mixed degenerate codons were synthesized according the following sequences: 5 ’
GCGAATTCATATTGTCCATATTGTAA TCCTCC G G C C
5 ’
ATTTGAATTCCATTAGACATTTCATC C G TCT G C
C
0
Both primers contain EcoRI sites at the 5’ ends of the oligos. The PCR reaction was carried out in an automated DNA Thermal Cycler as described earlier (17). Screening of yeast genomic library-A yeast genomic lambda library (Stratagene) containing inserts with an average size larger than 15 kb was screened by a 61 mer synthetic deoxyribonucleotide probe derived from the PCR product with a sequence of GCTA CTTI’GTCTACCCTCTTCCAGAACG’M’CCCMATCCAAGGCCC~GTGT TGG. Nitrocellulose filters from 150 mm plates containing 12,000 phage were prepared as duplicates. Baked filters were washed with 6x SSC in the presence of 4x Denhardts and 0.1% SDS at 60 “C for 1 h with two changes and prehybridized at 45 “C for 1 h in the same solution with calf thymus DNA (75 pg/ml). The hybridization was carried out at 45 “C overnight in 6x SSC, 4x Denhardts, 50 pg/ml of yeast tRNA, and 32P labelled probe (10’ cpmlml). The filters were washed with 6x SSC at 55 “C for 15 min after the hybridization. After the secondary screening, 4 positive clones were obtained from 12,000 independent clones. 950
Vol.
187,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Subcloning and sequencing of thioltransferase gene-Southern blotting analysis showed that all 4 positive clones contained a 3.8 kb EcoRI fragment which hybridized with the 61 bp PCR product. Attempts to directly clone this fragment into pUC13 were unsuccessful. Thus this fragment was isolated from agarose gel and used as starting material for constructing yeast thioltransferase gene libraries. The 3.8 kb DNA fragment was partially digested with HueIII, AM, and Sau3AI respectively. Then the partially digested DNA fragments were cloned into BanHIor SmaI-cleaved pUC13. The libraries were screened by synthetic oligos derived from the PCR product. The positive clones were sequenced. A 718 bp DNA fragment containing full length coding sequence of the thioltransferase was obtained and overlapped. The DNA sequence of the coding region of the gene was determined on both strands. Southern blot analysis-Yeast genomic DNA was digested with restriction enzymes and fractionated on 1% agarose gel. The DNA was transferred to a (18). The 61 mer yeast nitrocellulose membrane as described by Sambrook, et thioltransferase gene probe with nucleotide sequence given in the section of genomic library screening was then hybridized with the bound genomic DNA at 55” C for 14 h in the presence of 6x SSC, 4x Denhardt, and 50 pg/ml yeast tRNA. The filter was washed at 65” C in 6x SSC for 15 min before autoradiography. Results According degenerate genomic
to the protein
oligo nucleotide
DNA fragment.
and Discussion
sequence of yeast thioltransferase primers
were synthesized
In order to subsequently
(14), two
to amplify
a 69 bp yeast
clone the fragment
into a
bp
4
603
4
310
4
194
4 118 * 72
Fig. 1. Amplification of a fragment of yeast thioltransferase amplified fragment from 200 pl of PCR reaction was fractionated gel. The DNA was visualized by ethidium bromide staining. Lane amplified from 2.5 pg of yeast genomic DNA. Lane B; PCR product 0.8 pg of yeast genomic DNA. Lane C: HueIII-digested 4X174 RF details, see “Experimental Procedures”. 951
gene. The on 2% agarose A: PCR product amplified from DNA. For
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
GGCCCGAAGC
ACTTTCTTCA
TGAATTTTCA
TATAMAAGT
CCCAGGACGC
CAAGAAAAGG
60
TGCCCTCTTG
ATTAACGGAC
ACTCCAACTA
CTGTTATATA
TTGTTTCATG
GAGACCAATT
120
TTTCCTTCGA
CTCGAATTTA
ATTGTTATTA
TCATTATCAC
GTTGTTTGCC
ACAAGAATTA
180
TTGCTAAAAG
ATTTTTATCT
ACTCCAAAA
ATG Met
GTA val
TCC Ser
CAG Gln
GAA Glu
ACA Thr
GTT Val
GCT Ala
CAC His
236
GTA Val
AAG Lys
GAT Asp
CTG Leu
ATT Ile
GGC Gly
CAA Gln
AAG Lys
GAA Glu
GTG Val
TTT Phe
GTT Val
GCA Ala
GCA Ala
AAG Lys
ACA Thr
TAC Tyr
201
TGC Cys
CCT Pro
TAC Tyr
TGT Cys
AAA Lys
GCT Ala
ACT Thr
TTG Leu
TCT Ser
ACC Thr
CTC Leu
TTC Phe
CAA Gln
GAA Glu
TTG Leu
AAC Asn
GTT Val
338
CCC Pro
AAA Lye
TCC Ser
AAG Lys
GCC Ala
CTT Leu
GTG Val
TTG Leu
GAA Glu
TTA Leu
GAT Asp
GAA Glu
ATG Met
AGC Ser
AAT Asn
GGC Gly
TCA Ser
389
GAG Glu
ATT Ile
CAA Gln
GAC Asp
GCT Ala
TTA Leu
GAA Glu
GAA Glu
ATC Ile
TCG Ser
GGC Gly
CAA Gln
AAA Lys
ACT Thr
GTA Val
CCT Pro
AAC Asn
440
GTA Val
TAC
ATC Ile
AAT Asn
GGC Gly
AAG Lys
CAC ATT His Ile
GGT Gly
GGT Gly
AAC Asn
AGC Ser
GAT Asp
TTG Leu
GAA Glu
ACT Thr
TTG Leu
491
Tyr
AAG Lys
AAA Lys
AAT Asn
GGC Gly
AAG Lys
TTA Leu
GCT Ala
ATA Ile
TTG Leu
AAG Lys
CCG Pro
GTA Val
TTT Phe
CAA Gln
TAG ***
539
GAA Glu
TTTAAATTAC
GCTAATATCC
CTTCATAATA
TTTACATATA
TATATCTTTT
ATTATCATTA
599
TCTTAAATCA
CATATACCTA
CAAAACTTCT
AGCTAGATTC
ACACAAGAGT
ACTTTTGAAG
659
GCTGCGCGTG
CATTTTGAAA
CCAATGAGAC
CCGCTAAATT
CCGTCTGAGG
TACAAGATC
718
Fig. 2. Nucleotide sequence of yeast thioltransferase deduced amino acid sequence. The nucleotide sequence mdmwl
from
indicated
plasmid,
EcoRI
the
5' to the
gene and the ofthe
gene
3’ end. The stop codon of the reading
is
frame is
by asterisks.
sites were designed on the 5’ ends of the primers.
A PCR fragment
with expected size was observed on the agarose gel (Fig. 1). This fragment then excised, digested with EcoRI, and cloned into pUC13 plasmid sequence analysis.
Thus an 61 bp unique probe was synthesized
used to screen a yeast genomic library
containing
The full length yeast thioltransferase
nucleotide
&er
from 12,000 recombinant
gene and its flanking
they were subcloned into pUC13.
frame of 108 amino acid residues.
TTR contains no introns,
sequence
Figure 2 shows the
sequence of the cloned yeast genomic DNA fragment
open reading
and
inserts with an average size
larger than 15 kb. Four positive clones were obtained
were obtained
for DNA
All of three clones sequenced were shown to encode 23 amino
acids of yeast thioltransferase.
phages.
was
which contains
The sequence indicates
an
that yeast
a feature common to most yeast genes. A possible TATA 952
Vol.
187, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
ABCDEFGH
RESEARCH COMMUNICATIONS
Kb
-
0.5
Fig. 3. Southern blot analysis of the yeast thioltransferase gene. Total yeast genomic DNA (3 pg per lane) was digested by Sal1 (Lane A), X&I (Lane B), BamHl (Lane C), Hind111(Lane D), Sau3AI (Lane E), PstI (Lane F), BgZII (Lane G), and EcoRI (Lane H) respectively. The arrows indicate the migration positions of DNA molecular weight marker.
box sequence can be identified at nucleotide 97 to 100, 109 bp upstream of the ATG start of translation.
However, no polyadenylation
signal sequence, AATAAA,
was found at the sequence downstream from the stop codon. Data base searches failed to identify
any other yeast genes with significant sequence similarity
to
yeast TTR at either the DNA or the protein level. Compared with the amino acid sequence obtained by direct protein sequencing (14), the amino acid sequence deduced from DNA sequence has two extra amino acids at the C-terminus of the protein. This discrepancy suggests one possibility that the two extra C-terminal amino acid are cleaved post translationally
in the cell or in the course of the
protein purification. In contrast to yeast thioredoxins which are encoded by at least two different genes (17), Southern blot analysis of yeast genomic DNA digested with 8 different enzymes showed that there was one copy of the gene encoding yeast 953
Vol.
187,
No.
2, 1992
thioltransferase
BIOCHEMICAL
(Fig. 3). Although
AND
BIOPHYSICAL
double deletion
RESEARCH
COMMUNICATIONS
of the two yeast thioredoxin
genes affected its cell cycle and resulted in auxotrophy
for methionine,
the cells
survived in the absence of both genes (19). It remains
to be established
whether
deficiency lethal
of both thioredoxin
and thioltransferase
(glutaredoxin)
genes will be
to yeast as is the case in E. coli. (20, 21). Acknowledgments
I wish to thank Dr. Carl D. Bennett for synthesizing Montgomery for kindly offering yeast strain DMYG.
oligos, and Dr. Donna L.
References 1. Axelsson, K., Eriksson, -2984.
S., and Mannervik,
B. (1978) Biochemistry
17, 2978
2. Wells, W.W., Yang, Y., Gan, Z.-R., and Deits, T.L. (1992) Advanced Enzymology, in press. 3. Mannervik, B., Axelsson, K., Sundewall, Bichem. J. 213, 519-523. 4. Mannervik,
A.-C., and Holmgren,
B., and Axelsson, K. (1975) Biochem.
5. Axelsson, K., and Mannervik,
A. (1983)
J. 149, 785-788.
B. (1983) FEBS Lett. 152, 114-118.
6. Hatakeyama, M., Lee, C., Chon, C., Hayashi, M., and Mizoguchi, Biochem. Biophys. Res. Commu. 127, 458-463. 7. Goswami,
A., and Rosenberg,
8. Flamgni, F., Marmiroli, J. 259, 111-115.
IN.
T. (1985)
(1985) J. Biol. Chem. 260, 6012-6019.
S., Caldarera,
C.M., and Guarnieri,
C. (1989) Biochem.
9. Wells, W.W., Xu, D.P., Yang, Y., and Rocque, P.A. (1990) J. Biol. Chem. 265, 15361-15364. 10. Hiiog, J.-O., Jornvall, H., Holmgren, Eur. J. Biochem. 136, 223-232.
A., Carlquist,
11. Khntrot, I.-M., Hoiig, J.-O., Jornvall, (1984) Eur. J. Biochem. 144,417-423.
H., Holmgren,
M., and Persson, M. (1983) A., and Luthman,
M.
12. Gan, Z.-R, Wells, W.W. (1987) J. Biol. Chem. 262, 6704-6707. 13. Hopper,
S., Johnson,
R.S., Vath, J.E., and Biemarm,
264, 20438-20447. 954
K. (1989) J. Biol. Chem.
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
14. Gan, Z.-R., Polokoff, M. A., Jacobs, J.W., and Sardana, Biophys. Res. Commu. 168, 944-951.
M.K. (1990) Biochem.
15. Gan, Z.-R., Sardana, M.K., Jacobs, J. W., and Polokoff, Biochem. Biophys. 282, 110-115.
M.A. (1990) Arch.
16. Kallis,
G.-B., and Holmgren,
A. (1980) J. Biol. Chem. 255, 10261-10265.
17. Gan, Z.-R. (1991) J. Biol. Chem. 266, 1692-1696. 18. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, pp9.31-9.40, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 19. Muller,
E.G.D. (1991) J. Biol. Chem. 266, 9194-9202.
20. Russel, M., and Holmgren,
A. (1988) Proc. Natl. Acad. Sci. USA 85, 990-994.
21. Russel, M., Model, P., and Holmgren,
A. (1990) J. Bacterial.
955
172, 1923-1929.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 949-955
16, 1992
A GENE
CLONING ENCODING
AND SEQUENCING OF YEAST THIOLTRANSFER.ASE Zhong-Ru
Gan
Department of Biological Chemistry, Merck Research Laboratory West Point, PA 19486 Received
July
31,
W26-431
1992
A 69 bp yeast genomic DNA fragment encoding yeast thioltransferase was amplified by PCR technique. A yeast genomic DNA library was screened by a specific probe obtained from the PCR product. An 718 bp DNA fragment was found to encode yeast thioltransferase and its flanking sequence. The deduced amino acid sequence of the gene, designated ‘ITR, agrees with that derived from conventional amino acid sequence analysis except two extra ammo acids on the Cterminus. In contrast to yeast thioredoxin, Southern blot analysis of total yeast genomic DNA indicated that there was only one copy of gene encoding yeast thiotransferase. A putative TATA box was found at 109 bp from the starting codon. However, no polyadenylation signal sequence was identified on the DNA sequence downstream the 3’ end of the gene. 0 1992Academic Press, Inc.
Thioltransferase thiol-disulfide reduction glutathione pyruvate (58),
(glutaredoxin)
oxidoreduction
of low molecular
presumably
mammalian
papain,
weight and protein regulates
by changing
thioltransferase
thioltransferase
in the
in the presence of
other enzyme activities, and ornithine
such as decarboxylase
the redox status of these enzymes. Recently,
was identified
The active site center of thioltransferase sequence -Cys-Pro-Phe(Tyr)-Cys-
in many cellular
participates
disulfides
iodothyronine-5’-deiodonase
the active site of thioredoxin.
involved
processes (1, 2). It directly
(3, 4). Thioltransferase kinase,
is a small protein
as a dehydroascorbate contains a dithiol
(10-14). A similar
dithiol
reductase
within
(9).
the consensus
structure
is observed in
However the pKa of the active site cysteine of
is 3 to 4 pH units lower than that of thioredoxin
(12, 15, 16), 0006-291X/92
Copyright
949
AU
rights
0
1992
of reproduction
by Academic in any
$4.00
Press, j&m
Inc.
reserved.
Vol.
187,
No.
suggesting transfer
that thioltransferase of reduction
equivalents
equivalents.
from NADPH,
purpose. The relative cellular
BIOCHEMICAL
2, 1992
redox potential
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
is much more potent than thioredoxin Whereas thioredoxin
thioltransferase
contributions
receives reduction
uses reduced glutathione
of thioltransferase
in the
and thioredoxin
for this to the
are obscure.
In the present studies, the yeast thioltransferase
gene, TTR, and its flanking
sequences were cloned and sequenced. In contrast to yeast thioredoxin, encoded by at least two copies of different of the thioltransferase
genes (17), yeast contains
which is only one copy
gene.
Experimental
Procedures
Mat&ah-Yeast genomic lambda Dash library was from Stratagene. Restriction endonucleases, polynucleotide kinase, and T4 DNA ligase were purchased fi-om New England BioLabs. Taq DNA polymerase and DNA Thermal Cycler PCR apparatus were from Perkin-Elmer Cetus. DNA sequencing kits (Sequenase Version 2) were from United States Biochemical Corp. 35S-a-dATP and 32P-a-ATP were purchased from Amersham and Du Pont NEN Products. Preparation of yeast genomic DNA-Genomic DNA from Saccharomyces cerevisiae DMY6 (Leu’) was isolated as described previously (17). The isolated DNA was pure enough to carry out polymerase chain reaction and endonuclease digestion. Polymerase chain reaction-Two primers with mixed degenerate codons were synthesized according the following sequences: 5 ’
GCGAATTCATATTGTCCATATTGTAA TCCTCC G G C C
5 ’
ATTTGAATTCCATTAGACATTTCATC C G TCT G C
C
0
Both primers contain EcoRI sites at the 5’ ends of the oligos. The PCR reaction was carried out in an automated DNA Thermal Cycler as described earlier (17). Screening of yeast genomic library-A yeast genomic lambda library (Stratagene) containing inserts with an average size larger than 15 kb was screened by a 61 mer synthetic deoxyribonucleotide probe derived from the PCR product with a sequence of GCTA CTTI’GTCTACCCTCTTCCAGAACG’M’CCCMATCCAAGGCCC~GTGT TGG. Nitrocellulose filters from 150 mm plates containing 12,000 phage were prepared as duplicates. Baked filters were washed with 6x SSC in the presence of 4x Denhardts and 0.1% SDS at 60 “C for 1 h with two changes and prehybridized at 45 “C for 1 h in the same solution with calf thymus DNA (75 pg/ml). The hybridization was carried out at 45 “C overnight in 6x SSC, 4x Denhardts, 50 pg/ml of yeast tRNA, and 32P labelled probe (10’ cpmlml). The filters were washed with 6x SSC at 55 “C for 15 min after the hybridization. After the secondary screening, 4 positive clones were obtained from 12,000 independent clones. 950
Vol.
187,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Subcloning and sequencing of thioltransferase gene-Southern blotting analysis showed that all 4 positive clones contained a 3.8 kb EcoRI fragment which hybridized with the 61 bp PCR product. Attempts to directly clone this fragment into pUC13 were unsuccessful. Thus this fragment was isolated from agarose gel and used as starting material for constructing yeast thioltransferase gene libraries. The 3.8 kb DNA fragment was partially digested with HueIII, AM, and Sau3AI respectively. Then the partially digested DNA fragments were cloned into BanHIor SmaI-cleaved pUC13. The libraries were screened by synthetic oligos derived from the PCR product. The positive clones were sequenced. A 718 bp DNA fragment containing full length coding sequence of the thioltransferase was obtained and overlapped. The DNA sequence of the coding region of the gene was determined on both strands. Southern blot analysis-Yeast genomic DNA was digested with restriction enzymes and fractionated on 1% agarose gel. The DNA was transferred to a (18). The 61 mer yeast nitrocellulose membrane as described by Sambrook, et thioltransferase gene probe with nucleotide sequence given in the section of genomic library screening was then hybridized with the bound genomic DNA at 55” C for 14 h in the presence of 6x SSC, 4x Denhardt, and 50 pg/ml yeast tRNA. The filter was washed at 65” C in 6x SSC for 15 min before autoradiography. Results According degenerate genomic
to the protein
oligo nucleotide
DNA fragment.
and Discussion
sequence of yeast thioltransferase primers
were synthesized
In order to subsequently
(14), two
to amplify
a 69 bp yeast
clone the fragment
into a
bp
4
603
4
310
4
194
4 118 * 72
Fig. 1. Amplification of a fragment of yeast thioltransferase amplified fragment from 200 pl of PCR reaction was fractionated gel. The DNA was visualized by ethidium bromide staining. Lane amplified from 2.5 pg of yeast genomic DNA. Lane B; PCR product 0.8 pg of yeast genomic DNA. Lane C: HueIII-digested 4X174 RF details, see “Experimental Procedures”. 951
gene. The on 2% agarose A: PCR product amplified from DNA. For
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
GGCCCGAAGC
ACTTTCTTCA
TGAATTTTCA
TATAMAAGT
CCCAGGACGC
CAAGAAAAGG
60
TGCCCTCTTG
ATTAACGGAC
ACTCCAACTA
CTGTTATATA
TTGTTTCATG
GAGACCAATT
120
TTTCCTTCGA
CTCGAATTTA
ATTGTTATTA
TCATTATCAC
GTTGTTTGCC
ACAAGAATTA
180
TTGCTAAAAG
ATTTTTATCT
ACTCCAAAA
ATG Met
GTA val
TCC Ser
CAG Gln
GAA Glu
ACA Thr
GTT Val
GCT Ala
CAC His
236
GTA Val
AAG Lys
GAT Asp
CTG Leu
ATT Ile
GGC Gly
CAA Gln
AAG Lys
GAA Glu
GTG Val
TTT Phe
GTT Val
GCA Ala
GCA Ala
AAG Lys
ACA Thr
TAC Tyr
201
TGC Cys
CCT Pro
TAC Tyr
TGT Cys
AAA Lys
GCT Ala
ACT Thr
TTG Leu
TCT Ser
ACC Thr
CTC Leu
TTC Phe
CAA Gln
GAA Glu
TTG Leu
AAC Asn
GTT Val
338
CCC Pro
AAA Lye
TCC Ser
AAG Lys
GCC Ala
CTT Leu
GTG Val
TTG Leu
GAA Glu
TTA Leu
GAT Asp
GAA Glu
ATG Met
AGC Ser
AAT Asn
GGC Gly
TCA Ser
389
GAG Glu
ATT Ile
CAA Gln
GAC Asp
GCT Ala
TTA Leu
GAA Glu
GAA Glu
ATC Ile
TCG Ser
GGC Gly
CAA Gln
AAA Lys
ACT Thr
GTA Val
CCT Pro
AAC Asn
440
GTA Val
TAC
ATC Ile
AAT Asn
GGC Gly
AAG Lys
CAC ATT His Ile
GGT Gly
GGT Gly
AAC Asn
AGC Ser
GAT Asp
TTG Leu
GAA Glu
ACT Thr
TTG Leu
491
Tyr
AAG Lys
AAA Lys
AAT Asn
GGC Gly
AAG Lys
TTA Leu
GCT Ala
ATA Ile
TTG Leu
AAG Lys
CCG Pro
GTA Val
TTT Phe
CAA Gln
TAG ***
539
GAA Glu
TTTAAATTAC
GCTAATATCC
CTTCATAATA
TTTACATATA
TATATCTTTT
ATTATCATTA
599
TCTTAAATCA
CATATACCTA
CAAAACTTCT
AGCTAGATTC
ACACAAGAGT
ACTTTTGAAG
659
GCTGCGCGTG
CATTTTGAAA
CCAATGAGAC
CCGCTAAATT
CCGTCTGAGG
TACAAGATC
718
Fig. 2. Nucleotide sequence of yeast thioltransferase deduced amino acid sequence. The nucleotide sequence mdmwl
from
indicated
plasmid,
EcoRI
the
5' to the
gene and the ofthe
gene
3’ end. The stop codon of the reading
is
frame is
by asterisks.
sites were designed on the 5’ ends of the primers.
A PCR fragment
with expected size was observed on the agarose gel (Fig. 1). This fragment then excised, digested with EcoRI, and cloned into pUC13 plasmid sequence analysis.
Thus an 61 bp unique probe was synthesized
used to screen a yeast genomic library
containing
The full length yeast thioltransferase
nucleotide
&er
from 12,000 recombinant
gene and its flanking
they were subcloned into pUC13.
frame of 108 amino acid residues.
TTR contains no introns,
sequence
Figure 2 shows the
sequence of the cloned yeast genomic DNA fragment
open reading
and
inserts with an average size
larger than 15 kb. Four positive clones were obtained
were obtained
for DNA
All of three clones sequenced were shown to encode 23 amino
acids of yeast thioltransferase.
phages.
was
which contains
The sequence indicates
an
that yeast
a feature common to most yeast genes. A possible TATA 952
Vol.
187, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
ABCDEFGH
RESEARCH COMMUNICATIONS
Kb
-
0.5
Fig. 3. Southern blot analysis of the yeast thioltransferase gene. Total yeast genomic DNA (3 pg per lane) was digested by Sal1 (Lane A), X&I (Lane B), BamHl (Lane C), Hind111(Lane D), Sau3AI (Lane E), PstI (Lane F), BgZII (Lane G), and EcoRI (Lane H) respectively. The arrows indicate the migration positions of DNA molecular weight marker.
box sequence can be identified at nucleotide 97 to 100, 109 bp upstream of the ATG start of translation.
However, no polyadenylation
signal sequence, AATAAA,
was found at the sequence downstream from the stop codon. Data base searches failed to identify
any other yeast genes with significant sequence similarity
to
yeast TTR at either the DNA or the protein level. Compared with the amino acid sequence obtained by direct protein sequencing (14), the amino acid sequence deduced from DNA sequence has two extra amino acids at the C-terminus of the protein. This discrepancy suggests one possibility that the two extra C-terminal amino acid are cleaved post translationally
in the cell or in the course of the
protein purification. In contrast to yeast thioredoxins which are encoded by at least two different genes (17), Southern blot analysis of yeast genomic DNA digested with 8 different enzymes showed that there was one copy of the gene encoding yeast 953
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187,
No.
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thioltransferase
BIOCHEMICAL
(Fig. 3). Although
AND
BIOPHYSICAL
double deletion
RESEARCH
COMMUNICATIONS
of the two yeast thioredoxin
genes affected its cell cycle and resulted in auxotrophy
for methionine,
the cells
survived in the absence of both genes (19). It remains
to be established
whether
deficiency lethal
of both thioredoxin
and thioltransferase
(glutaredoxin)
genes will be
to yeast as is the case in E. coli. (20, 21). Acknowledgments
I wish to thank Dr. Carl D. Bennett for synthesizing Montgomery for kindly offering yeast strain DMYG.
oligos, and Dr. Donna L.
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