THE JOURNAL OF BIOLOGICAL CHEMISTRY (!I 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Yeast Thioredoxin Genes*

Val. 266, No. 3, Iasue of January 25, pp. 1692-1696,1991 Printed in U.S.A.

(Received for publication, June 14, 1990)

Zhong-Ru Gan From the Department of Biological Chemistry, Merck Sharp & Dohme Research Laboratory, West Point, Pennsylvania 19486

Based on the conserved protein sequence of thiore- doxins from yeast and other organisms, two primers were synthesized for polymerase chain reaction of yeast genomic DNA. A 34-base pair (bp) sequence around the active site of yeast thioredoxin was ob- tained from the polymerase chain reaction product. This specific sequence was used as a probe in Southern blot analysis of total yeast genomic DNA digested with various restriction enzymes. Under conditions of high stringency, more than one DNA species hybridized with the probe, suggesting that more than one gene encodes yeast thioredoxin. The probe was used to screen a yeast genomic library. Two Sau3Al frag- ments, 825 and 2045 bp, respectively, from two dif- ferent clones were cloned into pUC13. Sequence analy- sis of these fragments gave two different open reading frames without introns. The 825-bp Sau3Al fragment encodes a 103-amino acid residue protein named thio- redoxin I. The 2045-bp Sau3Al fragment contains a sequence encoding thioredoxin I1 which has 102 amino acid residues. This is the first report of the cloning and sequencing of eukaryotic thioredoxin genes from any source. Both yeast thioredoxins contain a dithiol active site sequence, Cys-Gly-Pro-Cys. Thioredoxins I and I1 show 78% amino acid sequence identity. They display more amino acid sequence similarity with mammalian thioredoxin than with Escherichia coli and plant chlo- roplast thioredoxins.

Thioredoxins are a group of small proteins widely distrib- uted in both prokaryotic and eukaryotic cells. They all contain a dithiol active site sequence of Cys-Gly-Pro-Cys (1). Thio- redoxin was first identified in yeast as a reducing agent in the reduction of methionine sulfoxide and sulfate (2, 3). Subse- quently, thioredoxin has been found to be a versatile protein, which functions as a hydrogen donor of ribonucleotide reduc- tase (4), as a regulator for a variety of enzymes and receptors such as several photosynthetic enzymes and the glucocorticoid receptor (5-7), as a subunit of phage T7 polymerase and an essential component of filamentous phages f l and m13 (8,9), and as a general thiol-disulfide oxidoreductase (10). Recently, a protein called adult T cell leukemia-derived factor was discovered in human T lymphotropic virus I transformed T cells (11). This protein, which induces interleukin-2 receptor expression, is apparently identical to human thioredoxin.

The thioredoxin gene has been studied in some prokaryotic cells. Nucleotide sequence analysis of the Escherichia coli

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "acluertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s)J05730 and 505731.

thioredoxin gene has revealed that a putative 18-amino acid leader peptide might be translated and that gene expression might be under the control of the cyclic AMP receptor protein (12). Multiple genes for thioredoxin have been identified in Cyanobacterium anabaena sp. and Corynebacterium nephridii (13, 14). The amino acid sequences deduced from nucleotide sequences show a highly conserved region around their dithiol active site. Unlike multiple species of plant thioredoxins, which can be distinguished by their specific functions on activation of different photosynthesis enzymes (5, 6, 15), physiological roles of individual thioredoxin in C. anabaena sp. and C. nephridii are not known.

During the course of purification of yeast thioredoxin, two forms of thioredoxin were detected. Partial amino acid se- quence analysis has revealed one amino acid difference around the active site of these two forms (16, 17). Yeast is the only non-photosynthetic eukaryote which contains more than one thioredoxin. However, no further structural and functional properties of the two thioredoxins have been described since they were reported two decades ago.

Here I describe the cloning and sequencing of two genes encoding yeast thioredoxins. The results reported here dem- onstrate that multiple genes encode yeast thioredoxins. This is the first genomic cloning of thioredoxin gene from eukary- otic cells.

EXPERIMENTAL PROCEDURES

Materials-Yeast genomic X DashTM library was from Stratagene. Restriction endonucleases, polynucleotide kinase, and T4 DNA ligase were purchased from New England BioLabs. Taq DNA polymerase and DNA thermal cycler PCR' apparatus were from Perkin-Elmer Cetus Instruments. DNA sequencing kits (Sequenase version 2) were from U. S. Biochemical Corp. ["SIdATP and ["PIATP were pur- chased from Amersham Corp. and Du Pont-New England Nuclear.

Preparation of Yeast Genomic DNA-Genomic DNA from Saccha- romyces cereuisiae DMY6 (Leu-) was isolated as described by Treco (18) with some modification. Briefly, yeast pellets (6 g) were sus- pended in 20 ml of sorbitol solution (0.9 M sorbitol, 0.1 M EDTA, and 0.1 M Tris-HC1, pH 8.0). To make yeast spheroplasts, 0.25 ml of zymolase (4 mg/ml) and 2 ml of B-mercaptoethanol (2.9 M ) were added to the suspension. The suspension was incubated at 37 "C for 1 h in a shaking incubator with a speed of 250 rpm. The spheroplasts were spun down by centrifugation for 5 min at 1700 X g a t room temperature. The pellet was resuspended in 20 ml of Tris/EDTA solution (50 mM Tris-HCI, pH 8.0, 20 mM EDTA), and 1.2 ml of 20% SDS was added to lyse the cells. The reaction proceeded for 20 min at 65 "C. The lysate was mixed with 10 ml of 5 M potassium acetate and the mixture was set on ice for 30 min. The cell debris was precipitated by centrifugation for 10 min a t 3000 X g. The DNA was then precipitated in 70% ethanol. RNA in the preparation was removed by RNase digestion and polyethylene glycol 8000 precipita- tion. The yield of yeast DNA was approximately 1 mg, and the DNA was pure enough to carry out polymerase chain reaction and endo- nuclease digestion.

Southern Blotting Analysis-Yeast genomic DNA was digested

' The abbreviations used are: PCR, polymerase chain reaction; TR- I, thioredoxin I; TR-11, thioredoxin 11; bp, base pair(s); kb, kilobase pair(s); SDS, sodium dodecyl sulfate.

1692

Gene Cloning, Multiple Genes, Thioredoxin 1693 A. FIG. 1. Strategy of synthesizing a

specific probe for yeast thioredoxin gene. A, mixed oligo primers for I'CH reaction. The amino acid sequence is from the active site of yeast thioredoxin (17) except that the two flanking resi- dues underlined were deduced from the conserved region of mammalian thiore- doxins. The corresponding codons used for the PCR primers are given above the amino acids.. EcoRI restriction sites a t 5' ends of the primers are boxed. Direc- tions of polymerase reaction are indi- cated by orrotus. H, nucleotide sequence B. of the prohe derived from PCR product.

Primer 1 - A A T T

271

Primer 2 - A T A G T T T

3 ' GGCTACTAGCTCTTCTA E l TT

FIG. 2. Amplification of yeast thioredoxin gene fragment around its active site. The amplified I'ragment from 200 pI of J'CH reaction was fractionated on 2.2"im agarose gel. DNA was visualized hy ethidium hromide staining. Imne 1, HaellI-digested 4x174 RF DNA; lane 2 and 3, PCR products amplified from 0.8 and 2.5 pg of yeast genomic template DNA, respectively. For details, see "Experi- mental Procedures."

with restriction enzymes and fractionated on 1% agarose gel. The DNA was transferred to a nitrocellulose memhrane as described by Samhrook et a/. (19). The memhrane was then hyhridized to thiore- doxin gene probe a t 55 "C for 14 h in the presence of 6 X SSC, 4 X Denhardt's, and 50 pg/ml yeast tRNA. The filter was washed a t 65 'C in 6 X SSC for 15 min.

I'o1.vmera.w Chain Reaction-The PCR reaction was carried out in an automated DNA thermal cycler. The reaction contained 200 pmol of each primer, 2.5 pg of yeast genomic template DNA, 10 mM KCI, 10 mM Tris-HCI, pH 8.3, 15 mM M&I,, 0.0170 gelatin, 200 p M each dATP, dTTP, dGTI', and dCTI', and 3 units of Tag DNA polymerase in a total volume of 100 pl. The DNA was amplified for 30 cycles a t 94 "C for 1 min, 46 "C for 2 min, and 70 "C for 2 min. The PCR reaction mixture was concentrated hy lyophilization. Salt and excess dNTP were removed hy a Sephadex G-25 spin column. The concen- trated product was fractionated hy 2.2% agarose gel electrophoresis. The expected hand of 7'L-hp DNA fragment containing EcoRl sites at the 5' and 3' ends was excised and the DNA was electroeluted. The DNA was then digested with I;coHI and cloned into pUCl3 plasmid for sequence analysis.

Screening o/ Yeast Genomic Library-A yeast genomic X library (Stratagene) with inserts larger than 15 kilohase pairs (kh) was screened hy a %mer deoxyrihonucleotide prohe derived from the sequence of the PCR product. Nitrocelluose filters from 150-mm plates containing 12,000 phage were prepared as duplicates. Raked lilters were washed with 6 X SSC in the presence of 4 X Denhardt's and 0.1% SDS at 60 "C for 1 h with two changes and prehyhridized at 45 "C for 1 h in the same solution with calf thymus DNA (75 pg/ m l ) . The hybridization was carried out at 45 "C overnight in 6 X SSC, 4 X Denhardt's, 50 pg/ml yeast tRNA, and '"P-labeled prohe (10' cpm/ml). The filters were washed with 6 X SSC at 55 "C for 15 min after the hyhridization. The secondary screening of the gene was carried out under the same conditions.

Subcloning and Sequencing of Thioredoxin (;ene.s-Southern hlot- ting analysis of the positive clones revealed that each clone contained

5 ' TGGTGTGGGCCATGTAAAATGATTGCACCAATGAT

either a 0.8-kb Sau3Al fragment or a 2-kh Sau3Al fragment, which hyhridized with the thioredoxin gene prohe. The 0.8-kh fragment from clone TRR and 2-kh fragment from clone TRIO were suhcloned into respective pUCI3 plasmids for sequence analysis. The DNA sequence was determined using Sequenase enzyme (C. S. Biochemical Corp.). The DNA sequences of the coding regions of the genes were determined on hoth strands.

RESULTS

Isolation of Yeast Thioredoxin Gene-A 17-amino acid se- quence around the active site of yeast thioredoxin was deter- mined 20 years ago (17). Alignment of this 17-amino acid sequence with thioredoxins from E. coli (20), spinach chloro- plasts (21), C. nephridii (22), rabbit bone marrow (23), and human lymphoblastoid B cell (24) revealed two additional conserved amino acids flanking its C terminus and N terminus (Fig. 1). The sequence of the 19-amino acid residues was used to clone the yeast thioredoxin gene. Two degenerate oligo primers were synthesized to amplify a 57-bp yeast genomic DNA fragment. In order to subsequently clone the fragment into a plasmid, EcoRI sites were designed on the 5' ends of the primers. After 30 cycles of amplification. an expected 72- bp fragment was observed on the 2.2% agarose gel (Fig. 2). This fragment was then excised, digested with EcoRI, and cloned into pUC13 plasmid. Three different clones from this PCR fragment were subjected to DNA sequence analysis. All three clones were shown to encode 17 amino acids of yeast thioredoxin. A 35-bp unique probe for thioredoxin coding sequence was synthesized according to this sequence (Fig. 1R). The probe was used to screen a yeast genomic X library containing inserts larger than 15 kb. Fifteen positive clones were obtained from 12,000 recombinant phages. The positive clones were analyzed by Sau3Al digestion and Southern blotting. All the clones gave either a 0.8- or 2-kb Sau3Al fragment that hybridized with the probe at high stringency. The 0.8- and 2-kb Sau3Al fragments were cloned into pUCl3 plasmid and sequenced. Fig. 3 shows the nucleotide sequences of the two Sau3Al fragments, which contained two different open reading frames encoding thioredoxin I (TR-I) and thio- redoxin I1 (TR-11). Both yeast thioredoxin genes contain no introns, a feature common to most yeast genes. A dithiol active site sequence, Cys-Gly-Pro-Cys, was located at amino acid residue 30-33 of TR-I and 29-32 of TR-11. In TR-I gene. a possible TATA box sequence can be identified, nucleotide 154-158, approximately 130 bp from the ATG start of trans- lation. Near the 3' end of the coding sequence of TR-I gene, a typical polyadenylation signal sequence, AATAAA, was found 48 bp downstream from the stop codon (Fig. 3, p a n d A ) . However, no such typical sequences could be identified in the TR-I1 gene.

Data base searches failed to identify any other yeast genes with significant sequence similarity to yeast thioredoxina at either the DNA or protein level. Yeast TR-I and TR-I1

1694

FIG. 3. Nucleotide sequences of yeast thioredoxin genes and de- duced amino acid sequences. The nu- cleotide sequences of the genes are num- bered from the 5’ to the 3’ end. The two Sau3.41 restriction sites at the 5’ and 3’ ends of each gene are indicated by ar- rows. The asterisks represent the stop codons. Panel A, TR-I gene. The puta- tive promoter sequence and polyadenyl- ation signal sequence are boxed. Panel B, TR-I1 gene.

AACGTGGCTCTTTTCTTACTAAGCGCGTTCAGTTTCCAGCCAGCCG~GAGGGATATCAGTA TATAA GAAAGCCATTCCGGCCATGA 178 L! AAAGCTGACAAGAGAATAACGAGGACCAGTTTTTATTTGTTGTCTAGCAACAATTATACACCCACACATACACGACAGTCTACCATATCT 268

TTAAATAACACATCAATA ATG GTC ACT CAA TTA AAA TCC GCT TCT GAA TAC GAC AGT GCT TTA GCA TCT GGC 340 Met Val Thr Gln Leu Lys Ser Ala Ser Clu tyr Asp SeK Ala Leu Ala Ser ClY

L~~ Leu Val Val Val Asp Phe Phe Ala Thr Trp Cys Gly Pro Cys Lys Met Ile Ala Pro Met Ile GAC AAG TTA GTC GTT GTT GAC TTT TTT GCC ACA TGG TGT GGG CCA TGT AAA ATG ATT GCA CCA ATG ATT 409

GAA AAC TTT GCA GAA CAA TAT TCT GAC GCT GCT TTT TAC AAG TTG GAT GTT GAT CAA CTC TCA GAT GTT 478 ~ l u LYS Phe Ala Glu Gln TYK Ser Asp Ala Ala Phe Tyr Lys Leu Asp Val Asp Glu Val Ser ASP Val

Ala Gln Lys Ala Glu Val Ser Ser Met Pro Thr Leu Ile Phe TYK Lys Gly ClY LYS Glu Val Thr Ar?, GCT CAA AAA GCT GAA GTT TCT TCC ATG CCT ACC CTA ATC TTC TAC AAG CGC GGT AAC GAG GTT ACC AGA 547

Val Val Gly Ala Asn Pro Ala Ala Ile Lys Gln Ala Ile Ala SeK Asn Val *** TACGTAAAGTACATCATGTTTACCAGTTTA AATAAA CAATTTTAAAAAGAAACTCTATTACATCTATCTATCATTATTTTCTTCATTC 707

TCTATTGTATATTTCATCATCGGTGTAACCAAGAATGTAT~TGTCAGTCATGCTCTTGGTATTCAACTTACAAGGTCCAGCTTTCTG 797

8 2 5

GTC GTC GGT GCC AAC CCA GCT GCT ATC AAG CAA GCT ATT GCT TCC AAC GTA TAG TTGCCGCTATATTAACGC 619

I CACCTTTGGCTTGGCGTTCCATGCGATC

4 Sau3A I

B

CATCAGAATGATTCAAATCATGTTGCCAGTCTTCGATGCTCCACACTTGGTTGAACAACCTAAGTTGAC‘~GCTGCTACCAACCCTAA 90 1 Sau3A

G C A A T A A G C G A T T T A A T C T C T A A T T A T T A G T T T T T T G C T T C A T A T T A T T A C T G C A 180

CTCTCACTTACCATGGAAAGACCAGACAACAAGTTGCCGACACTCTGTTGAATTGGCCTGGTTAGCCTTAACTCTGGCTCCCCTTCTTTA 270

CAAATTTCGAGAATTTCTCTTAAACGATATGTATATTCTTTTCGTTGG~GATGTCTTCC~CCGATGAATTAGTGGMC 360

CAAGGAAAAAAAAAGAGGTATCCTTGATTAAGGAACACTGTTTAAACAGTCTCGTTTCC~CCCTCAAACTCCATTAGTGTAATACM 450

GACTAGACACCTCGATACATA ATC GTr ACT CAA TTC AAA ACT GCC AGC CAA TTC CAC TCT GCA ATT CCT CAA 524 Met Val Thr Gln Phe Lys Thr Ala Ser Glu Phe Asp Scr A l a Ile Ala Gln

GAC AAG CTA GTT GTC GTA GAT TTC TAC CCC ACT TGG TCC GCT CCA TGT AAA ATG ATT GCT CCA ATC ATT 593 Asp Lys Leu Val Val Val Asp Phe TYK Ala Thr Trp Cys Gly P r o Cys Lys Met I l e Ala Pro Met Ile

GAA AAA TTC TCT GAA CAA TAC CCA CAA GCT GAT TTC TAT AAA TTG CAT CTC CAT C,AA TTC GGT CAT CTT 662 Glu Lys Phe Ser Glu Gln Tyr Pro Gln Ala Asp Phe Tyr Lys Leu Asp Val Asp Glu Leu Cly Asp Val

GCA CAA AAG AAT GAA GTT TCC CCT ATG CCA ACT TTG CTT CTA TTC AAG PAC GGT AAG CAA GTT GCA AAG 731 Ala Cln Lys Asn Glu Val Ser Ala Met Pro Thr Leu Leu Leu Phe Lys Asn Cly Lys Glu Val Ala Lys

CTT GTT GGT GCC AAC CCA GCG GCT ATT AAG CAA GCC ATT GCT CCT AAT GCT TAA ACTCACCCAATGACCGAT 803 Val Val Gly Ala Asn Pro Ala Ala Ile Lys Gln Ala lle Ala Ala Asn A l a *-;<*

ATATTGTGTTTCTATACTGTGTTTGTTATATATAGTTTACCTTTTTTAGAC~CAGAATTATATATTATCCTTATGTTTTGTTAT 893

T T A C T C C G A A G C A C A C A A T T G C C C A C C A A A G G A G G T T 983

G A A G A A T G A G A T A C T T T T C T T C C G A T T A C T T A C G T 1073

ATTTCGCAACCTTTCAGTTGGGCTTTGTTTAAGAACTGG~TACTTTTGCTTGAGTTGTTTAGTTTTATTrTATCCACTCTTGTCTTAAC 1163

A A A T A T T T T C A A G A C C G G T A G C C G A A C A T G A A A A A A T C A T T A T T ~ C T C A T T T T T T C A A C ~ T A T ~ C ~ ~ C ~ G G C A A C G C A 1253

C A A T T T T A G A G A T A C A T A C G C A G T G C A T G T T A A A A A T T T 1343

TGAAACCGAACGCAATGTGCCAAGAAATGTAAACACACTATAG~TAGAACCGTGCACATTGTGCTAGCATATCTCCTTCGTTCT 1433

GAACAAGAAGCACCTCGCCACTTTCTCCTAGCCCAATTCTTGCCAAGTTTTGAACCGCAATCTTTTGTGT~rGAACAAGCATGTATGACG 1523

GGTCAAAATTTAGTGGAGGCCGCTTACAATCCTTCTATTTCCTCTGGACCTCATTAGCCGTCTCGCCAGACCTAAGCGTCAT~TCTGGA 1613

CAATTTCATTGCATGCGAGAATATGATAACTAAGAACTTCTTTATTTATAC~GTTCCACCCACTCATACACGGCTACAATTATGACGTA 1703

TAATAACGTTTCGTCTAGCCCACCTTTTTTACTTTTGACGTTTTATTTCTTTCGAGGATTTCGCCAACAATCCCCCGAACAGC~G~ 1793

ATGGCGTCGCAGTTTCAGATGTATAGACTCATCTTGTAG~GAATGCAAGAATGAAGTCTTTTCGTGGTGTTTTG~CACTATA 1883

AACAAACCCTCAACAAACATTTTGTATAAATATTTAGCTATATATTGAATATCTTGACCAGTAAAGCACCTTGACAAATTCTAAGCTTGA 1973 L Sau3A I

AGAACGTACTTTGATATCCCTCCGTTTCATCATCCTATACCTCGTCAACAAATC~TATGAAGATC 2045

displayed 78% amino acid sequence identity (Fig. 4, panel A ) . The amino acid sequence of TR-I was compared with E. coli (20), spinach chloroplast (21), rabbit bone marrow (23), and human (24) thioredoxins (Fig. 4, panel B) . Since less struc- tural similarity outside the active region was observed be- tween mammalian, bacterial, and plant thioredoxins, the only regions of identical amino acid sequence between yeast TR-I and other thioredoxins are boxed. The amino acid identities

of yeast TR-I with human, rabbit bone marrow, spinach chloroplast, and E. coli thioredoxins are 47, 46, 31, and 27%, respectively. On the other hand, rabbit and human thioredox- ins showed 88% amino acid sequence identity, and E. coli and spinach chloroplast thioredoxins showed 54% similarity. Se- quence identities between TR-I1 and thioredoxins from other species were similar to that observed in TR-I (data not shown).

Gene Cloning, Multiple Genes, Thioredoxin 1695

7.1- 4.1- 3.1-

2.0-

1.6-

1.0-

0.5-

FIG. 5. Southern analysis of yeast thioredoxin gene. Total yeast genomic DNA (3 pg/lane) digested with: EcoRI (lane I ) ; HindIII ( l o n e 2); PstI ( l o n e 3); RamHI (lane 4 ) ; and RglII ( l o n e 5) was fractionated on 1% agarose gel. The DNA was transferred to nitro- cellulose membrane and hybridized with the R'P-labeled probe. 1-kb ladder (Bethesda Research Laboratories) was used as molecular size standards. For details, see "Experimental Procedures."

Multiple Genes Encoding Yeast Thwredoxin-Fig. 5 shows the Southern blot analysis of yeast genomic DNA with the 35-bp specific probe. Blotting of the genomic DNA digested with restriction enzyme EcoRI, PstI, and BglII all showed two bands, whereas three bands were detected on the HindIII digestion (Fig. 5). One 9-kb band was observed on the BamHI digestion. The BarnHI band is darker than the others, which implies that more than one copy of thioredoxin genes hybrid- ized with the probe because the same amount of yeast genomic DNA was applied in the Southern blot analysis. The results suggest that yeast contains more than one thioredoxin gene. The observation was supported by Southern blot analysis of the positive thioredoxin clones obtained from the X genomic

library. The 15 positive clones were subjected to EcoRI diges- tion and Southern blot analysis. Since the insert sizes of the library are above 15 kb, the majority of the clones should give EcoRI fragments with the same sizes in Southern blots if there is only one thioredoxin gene. However, the sizes of the EcoRI fragments hybridized with the gene probe showed considerable diversity, most of them having a size of 2.4, 3.8, or 6.5 kb (data not shown).

DISCUSSION

Two yeast thioredoxin genes were cloned and sequenced. Yeast TR-I and TR-I1 showed 78% amino acid sequence identity. The amino acid sequence of TR-I displayed 47% identity to human thioredoxin, 46% to rabbit bone marrow thioredoxin, 31% to spinach chloroplast thioredoxin m, and 27% to E. coli thioredoxin. Both yeast thioredoxins contained an active site dithiol, Cys-Gly-Pro-Cys, which is a consensus sequence of most thioredoxins. In addition to this consensus sequence, a tryptophan and a lysine flanking this active site dithiol are conserved in all species. It has been postulated that a positively charged amino acid residue adjacent to the active site cysteines of thioredoxin and glutaredoxin (thiol- transferase) may account for the low pK. of the sulfhydryl groups of the cysteines (25, 26). In contrast to yeast and E. coli thioredoxins, which have only two cysteines, spinach chloroplast thioredoxin f and mammalian thioredoxins con- tain 3 and 5 cysteines, respectively. It is not clear whether these extra cysteines play functional or regulatory roles. In order to study the structures and functions of yeast thiore- doxin, I have tried to express the TR-I gene in E. coli using both its native promoter and the E. coli Trp promoter. Neither expression system produced detectable thioredoxin protein as judged by sodium dodecyl sulfate polyacrylamide gel (data not shown). It may result from stability of yeast thioredoxin in E. coli.

Multiple thioredoxin genes have been observed in plants, Cyarwbacterium anabaena sp. and Corynebacterium nephridii (5, 6, 13-15). Yeast is the only non-photosynthetic eukaryote containing more than one thioredoxin. Two thioredoxin ac- tivity peaks have been observed in the course of purification of yeast thioredoxin (16). Partial amino acid sequences of yeast thioredoxin I and I1 were reported in 1971 (17). The 17- amino acid residues around the active site of thioredoxin I1 are identical to amino acid residues 26-42 of the thioredoxin sequence deduced from TR-I gene (Fig. 3). However, the C- terminal sequence of thioredoxin I1 determined by the con- ventional method was reported to be Glu-Ala-Ile-Ala-Ser- Asn-Val, which is different from the C terminus of TR-I reported here (Fig. 3). There is only a 9-amino acid sequence of thioredoxin I available (17). This sequence, Tyr-Ala-Thr- Trp-Cys-Gly-Pro-Cys-Lys, is identical to amino acid residues 25-33 of TR-I1 (Fig. 3, panel A ) . The current data suggest that there are at least two different genes encoding yeast thioredoxins. It has been well established that two types of plant chloroplast thioredoxins can be distinguished by their specific functions. The f-type thioredoxin is capable of acti- vating chloroplast fructose 1,6-bisphosphatase and other key enzymes of CO, assimilation, while the m-type thioredoxin activates NADP-dependent malate dehydrogenase (5, 6). Whether the yeast thioredoxins have different physiological roles is unknown.

Acknowledgments-I wish to thank Dr. Carl D. Bennett for syn- thesizing oligos, Dr. Donna L. Montgomery for kindly offering yeast strain DMYG, and Dr. Mark A. Polokoff for critical reading of this manuscript.

1696 Gene Cloning, Multiple Genes, Thioredoxin REFERENCES J. B., and Fuchs, J. A. (1989) Eur. J . Biochem. 179, 389-398

1. 2.

3.

8.

9.

10. 11.

12.

13.

14.

Holmgren, A. (1989) J. Biol. Chem. 264, 13963-13966 Black, S., Harte, E. M., Hudson, B., and Wartofsky, L. (1960) J.

Wilson, L. G., Asahi, T., and Bandurski, R. S. (1961) J. Bid.

Laurent, T. C . , Moore, E. C., and Reichard, P. (1964) J . Biol.

Buchanan, B. B. (1980) Annu. Rev. Plant Physiol. 31,341-374 Cseke, C., and Buchanan, B. B. (1986) Biochim. Biophys. Acta

Grippo, J. F., Holmgren, A., and Pratt, W. B. (1985) J. Biol.

Mark, D. F., and Richardson, C. C. (1976) Proc. Natl. Acad. Sci.

Russel, M., and Model, P. (1985) Proc. Natl. Acad. Sci. U. S. A.

Holmgren, A. (1985) Annu. Rev. Biochem. 54, 237-271 Tagaya, Y., Maeda, Y., Mitsui, A., Kondo, N., Matsui, H., Ha-

muro, J., Brown, N., Arai, K.-I., Yokota, T., Wakasugi, H., and Yodoi, J . (1989) E M B O J. 8, 757-764

Wallace, B. J., Zownir, O., and Kushner, S. (1986) in Thioredoxin and Glutaredonin Systems: Structure and Function (Holmgren, A,, Brandkn, C.-I., Jornvall, H., and Sjoherg, B.-M., eds) pp. 11-19, Raven Press, New York

Alam, J., Curtis, S., Gleason, F. K., Gerami-Nejad, M., and Fuchs, J. A. (1989) J. Bacteriol. 171, 162-171

Mcfarlan, S. C., Hogenkamp, H. P. C., Eccleston, E. D., Howard,

Biol. Chem. 235, 2910-2916

Chem. 236,1822-1829

Chem. 239,3436-3444

853,43-63

Chem. 260, 93-97

U. S. A . 73, 780-784

82,29-33

15.

16.

17.

18.

19.

20. 21.

22.

23.

24.

25.

26.

Florencio, F. J., Yee, B. C., Johnson, T. C., and Buchanan, B. B. (1988) Arch. Biochem. Biophys. 266, 496-507

Gonzalez Porquk, P., Baldesten, A., and Reichard, P. (1970) J . Biol. Chem. 245,2363-2370

Hall, D. E., Baldesten, A., Holmgren, A., and Reichard, P. (1971) Eur. J. Biochem. 23,328-335

Treco, D. A. (1989) in Current Protocols i n Molecular Biology (Ausuhel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J . G., Smith, J. A,, Struhl, K., eds) pp. 13.11.1- 13.11.5, John Wiley and Sons, New York

Samhrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, pp. 9.31-9.40, Cold Spring Har- bor Laboratory, Cold Spring Harbor, NY

Holmgren, A. (1968) Eur. J. Biochem. 6,475-484 Maeda, K., Tsugita, A., Dalzoppo, D., Vilbois, F., and Schurmann,

P. (1986) Eur. J . Biochem. 154, 197-203 Meng, M., and Hogenkamp, H. P. C. (1981) J. Biol. Chem. 256,

9174-9182 Johnson, R. S., Mathews, W. R., Biemann, K., and Hopper, S.

(1988) J. Biol. Chem. 263,9589-9597 Wollman, E. E., d’Auriol, L., Rimsky, L., Shaw, A,, Jacquot, J.-

P., Wingfield, P., Graher, P., Dessarps, F., Robin, P., Galihert, F., Bertoglio, J., and Fradelizi, D. (1988) J. Biol. Chem. 263, 15506-15512

Kallis, G.-B., and Holmgren, A. (1980) J. Biol. Chem. 255,10261- 10265

Gan, Z.-R., and Wells, W. W. (1987) J. Biol. Chem. 262, 6704- 6707