THE JOURNAL OF BIOLOGICAL C H E M I S T R Y 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 20, Issue of July 15, pp. 9607-9611, 1988 Printed in U.S.A.

Isolation of a Chicken Thioredoxin cDNA Clone THIOREDOXIN mRNA IS DIFFERENTIALLY EXPRESSED IN NORMAL AND ROUS SARCOMA VIRUS-TRANSFORMED CHICKEN EMBRYO FIBROBLASTS*

(Received for publication, February 19, 1988)

Steven W. Jones$ and Ka-Cheung LukP From the Department of Cellular and Developmental Biology, The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138

We have isolated a cDNA clone for chicken thiore- doxin by differential screening of a cDNA library. The protein product which this clone encodes is very simi- lar to other thioredoxins, and it displays thioredoxin activity when expressed in Escherichia coli. This clone represents the first metazoan thioredoxin for which the protein or nucleic acid sequence is known. Com- parison of the chicken thioredoxin protein sequence with those from bacteria and plants indicates struc- tural features that appear to be essential for activity. Transformation of chicken embryo fibroblasts by Rous sarcoma virus elevates the level of thioredoxin mRNA whereas the level of thioredoxin mRNA in a nonpro- liferative tissue (brain) is much lower than in chicken embryo fibroblasts.

In several instances of malignant transformation by viruses or stimulation of cell division by growth factors, phosphoryl- ated tyrosine is detected on a protein substrate now denoted calpactin I (1-6). When this protein was first purified it was found in relatively high levels in cultured chicken embryo fibroblasts (CEF)’ and in substantially lower levels in chicken embryos or chicken brain (7, 8). Therefore, a strategy for molecular cloning of mRNA for this protein was based on a differential screen of a cDNA library from CEF with poly- adenylated RNA from CEF and chicken embryos or chicken brain. Several potential calpactin I clones were identified (8). Further analysis of one of these clones showed that although it did not correspond to mRNA for calpactin I, it did represent a mRNA species that was regulated in a transformation- dependent manner. This clone was therefore likely to be of interest for the study of transformation-relatedprocesses. The nucleotide sequence of this cDNA indicated that it encoded a protein product that was very similar to previously character- ized thioredoxins.

* This research was supported by National Institutes of Health Grants CA-07562 (to S. W. J.) and CA-42580 (R. L. Erikson) and received support from the American Business Cancer Research Foun- dation (R. L. Erikson). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 503882.

$ TO whom correspondence should be addressed. § Present address: Genelabs, Inc., 505 Penobscot Dr., Redwood

City, CA 94063. ‘The abbreviations used are: CEF, chicken embryo fibroblasts;

RSV, Rous sarcoma virus; IPTG, isopropyl /3-D-thiogalactoside; SDS, sodium dodecyl sulfate.

Thioredoxins are small proteins that participate in oxida- tion-reduction reactions and are characterized by the amino acid sequence Cys-Gly-Pro-Cys in their active sites (9). The oxidation and reduction of thioredoxin occurs through the sulfhydryl groups of these two vicinal cysteines. Thioredoxin- (SH)* donates hydrogens to metabolic reactions and forms the intramolecular disulfide, thioredoxin-Sz. Thioredoxin- (SH), is regenerated by reduction of thioredoxin-Sz by NADPH and thioredoxin reductase.

Thioredoxins have been implicated in many aspects of cellular metabolism, several of which represent important enzymatic control points. They serve as a hydrogen donor for the synthesis of deoxyribonucleotides by ribonucleotide re- ductase (10). The rat liver glucocorticoid receptor appears to be activated to a steroid binding state by a reduction reaction requiring thioredoxin and NADPH (11). A requirement for thioredoxin in the initiation of protein synthesis has been demonstrated (12). Thioredoxin-(SH)z has been shown to regulate several enzymes in the photosynthetic pathway, and it has been postulated that thioredoxin may participate in the switch between the light and dark reactions of photosynthesis (13).

Thioredoxins have been isolated from bacteria, plants, yeast, and mammals and one of the bacteriophage T4 genes encodes a thioredoxin (9). The most well characterized thio- redoxins are those from bacteria, bacteriophage T4, and plants. The bacteriophage T4-encoded thioredoxin shows no sequence homology with other thioredoxins, but its three- dimensional structure is similar to that of Escherichia coli thioredoxin (9). Several bacterial thioredoxin genes have been cloned (14-17). The complete protein sequences of several purified bacterial and plant thioredoxins have been deter- mined (9,18-20). We present here our analysis of the sequence and expression of the cDNA we have isolated and identified as chicken thioredoxin.

EXPERIMENTAL PROCEDURES

RNA Analysis-Growth of CEF and CEF infected with the Schmidt Ruppin strain of Rous sarcoma virus (RSV) was as described previ- ously (7). Total RNA was isolated from subconfluent actively growing cultured cells by lithium chloride-urea extraction (21). Poly(A)+ RNA was selected from total RNA by two cycles of oligo(dT)-cellulose chromatography. Total RNA was isolated from freshly obtained chicken brains by extraction with guanidinium thiocyanate (22).

For Northern blot analysis, samples of total RNA (10 pg) were fractionated on formaldehyde-containing gels and transferred to Biodyne A nylon membranes (Pall). The RNA was fixed to the membrane by UV irradiation, and the resulting blots were hybridized to nick-translated probes at 65 “C in 0.5 M NaPO,, pH 7.2, 7% SDS, 1% bovine serum albumin for 12 h. Washes were performed at 65 “C in 0.15 M NaCI, 0.015 M sodium citrate, pH 7.0, 1% SDS.

Probes were removed from the membrane according to the manu- facturer’s instructions. All blots were rehybridized with a probe for

9607

9608 Differential Expression of Chicken Thioredoxin chicken glyceraldehyde-3-phosphate dehydrogenase (23). This hy- bridization served as a control to ensure that each sample contained approximately equal amounts of mRNA. Extensive experience in our laboratory indicates that the level of glyceraldehyde-3-phosphate dehydrogenase mRNA does not change in CEF upon transformation by RSV. Cyanobacterium rRNAs of 2.95 and 1.5 kilobases and the 2.5- and 0.5-kilobase cleavage products of the former (24) served as size markers.

cDNA Library Construction and Nucleotide Sequencing-A cDNA library was constructed according to the procedure of Heidecker and Messing (25) from 1 pg of CEF poly(A)+ RNA. The resulting library contained 10,000 independent recombinant plasmids. DNA sequenc- ing was done using the chemical method of Maxam and Gilbert (26).

Expression of Recombinant Proteins in E. coli-A plasmid was constructed in which the chicken thioredoxin is expressed under control of the lac operator in PUC 18 (27). PUC 18 was cut with AuaI, and the ends were filled in using the large subunit of E. coli DNA polymerase I. The thioredoxin coding sequence (331 nucleo- tides) was isolated from pCEF201 (see below) by digestion with NcoI, which cuts 1 nucleotide before the thioredoxin initiation codon (nu- cleotide 144, Fig. l) and 12 nucleotides after the thioredoxin termi- nation codon. The ends were filled, in, and the thioredoxin fragment was blunt end-ligated into the AuaI-cut PUC 18. Plasmids were introduced into E. coli JM107 (27). Plasmids were isolated that contained the insert in both the forward (pCHKTRX) and reverse (pCHKrTRX) orientation with respect to the p-galactosidase se- quences in PUC 18. The 5' end of the lac-thioredoxin coding sequence in pCHKTRX is depicted in Fig. 1B. This fusion gene codes for a protein composed of the 105 amino acids of the chicken thioredoxin protein with 12 additional amino acids at the amino terminus derived from the P-galactosidase sequence. The region of the plasmid pCHKTRX around the fusion site was sequenced to confirm that the construction was correct.

To detect the protein expressed by the recombinant plasmid, overnight cultures containing either pCEFTRX or pCEFrTRX were grown in MSCA medium containing 200 p M MgS04 and 50 pg/ml ampicillin. Fifty microliters of the overnight cultures were added to 1 ml of M9CA containing 40 p~ MgS04, 50 pg/ml ampicillin, and 100 pCi of [35S]HzS0,. Where indicated, 1 mM isopropyl P-D-thioga- lactoside (IPTG) was added to induce production of the fusion pro- tein. After 2.5 h of incubation at 37 'C, the bacteria were harvested by centrifugation, washed once with STE (10 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA), and resuspended in 0.1 ml of buffer A (50 mM Tris, pH 7.4, 3 mM EDTA, 3 mM dithiothreitol, 1 mM phenyl- methylsulfonyl fluoride, 1 mM tosylphenylalanine chloromethyl ke- tone, 1 mM tosyllysine chloromethyl ketone) by sonication for 5 min at setting 5 in a Heat Systems model W-375 sonicator. This solution was held at 37 "C for 1 h under nitrogen. Proteins were carboxymeth- ylated by adding 20 pl of 0.5 M iodoacetamide and incubating in the dark for 1 h at room temperature. Proteins were resolved by electro- phoresis through 11-20% polyacrylamide SDS gels using the Laemmli buffer system (28) and were visualized by fluorography (29). To determine whether the recombinant protein was soluble or mem- brane-associated, the labeled lysate was centrifuged at 100,000 X g for 30 min, and the pellet was resuspended in 100 pl of buffer A plus 1% SDS. SDS was added to the supernatant fraction to a final concentration of 1%. The solutions were then resonicated for 5 min. Reduction, carboxymethylation, and electrophoresis were as above.

For the detection of thioredoxin activity, 1-liter unlabeled cultures were grown as described above. Bacteria were harvested by centrifu- gation, washed once with STE, resuspended in 4 ml of buffer A, and lysed by two passages through a French press at 12,000 p.s.i. The lysates were sonicated for 10 min at setting 5 at 4 "C to reduce the viscosity. Insoluble material was removed by centrifugation at 100,000 X g for 30 min. Thioredoxin activity in the supernatants was assayed according to Holmgren (30). The assay (1-ml total volume) contained 0.1 M potassium phosphate, pH 7.0,0.75 pg/ml insulin prepared as in Ref. 30, and the sample (2 mg total protein). The reaction was initiated by the addition of 0.01 ml of 0.1 M dithiothreitol and the formation of the insulin precipitate monitored by light scattering at 650 nM.

RESULTS

Isolation of a cDNA Clone for Chicken Thioredoxin-The isolation of cDNA clones that represent mRNAs expressed at higher levels in CEF than in other chicken tissues has been

previously described (8). One of these, clone H1, contained an insert of approximately 400 nucleotides and hybridized to a message of less than 750 nucleotides as assayed by Northern blot analysis. A 192-base pair fragment containing only cDNA sequences was excised from the plasmid by double digestion with the restriction enzymes EcoRI and RsaI. This fragment was isolated, labeled by nick translation, and used to screen 3000 colonies from the CEF cDNA library. Five colonies gave positive hybridization signals (pCEF 201-205). The inserts in these plasmids had identical restriction maps. The inserts in pCEF 201 and 203 were in the opposite orientation from the other inserts. Two of the inserts (from pCEF 202 and 205) were sequenced on one strand, and one (from pCEF 201) was sequenced on both strands. All of the inserts had identical sequences. The nucleotide sequence of the 661-base pair insert from these clones contains a single long open reading frame that predicts a 105-amino acid protein product (Fig. 1).

The nucleotide sequence was compared against all se- quences in the GenBank nucleotide sequence data base (re-

A 1 GAGAGCGCTG AGGAGGCCCT GAGGCTGCGT GGCCGAGCTC GGTGCGGCAG AGCGATAGCG

61 GAGAGCGCAG CGGCGTGCGG GACGCGCGTG CGCGOGACGA GGCCGTTGGC CGCCTTCTTG

121 CTGTGCAGCC CAGGAGCCGC CGCC ATG GTG M G AGC GTG GGC M T CTG GCT GAT net Val Lym S ~ K Val Gly Asn Leu Ala Asp

115 TTT GAG GCA GAA CTG AAA GCT GCT GGT GAG M G CTT GTA GTA GTT GAT TTC P h e Glu Ala Glu Leu Lys Ala Ala Gly Glu LYE Leu Val Val Val Asp P h e

226 TCT GCC ACA TGG TGT GGA CCA TGT AAA ATG ATC M G CCA TTT TTC CAT K T set Ala T h r T r p c y s Gly P r o Cy- Lye net Ile LYE P r o P h e P h e His Ser

277 CTG TGT GAC M G TTI GGT GAT GTG G T G TTC ATT G M ATT GAT GTG GAT GAT Leu Cys Asp Lys P h s Gly Aap Val Val P h e I10 Glu 110 Asp Val Asp Asp

328 GCC C M GAT GTT GCT ACA CAC TGT GAT G T G M G TGC A T G CCA ACA TTC CAG Ala Gln Asp Val Ala T h r H I S Cys Asp Val LYS C y 6 net P r o T h r P h e Gln

319 TTC TAC M G M C GGA AAG M G GTG CAG G M TTC TCT GCG GCC AAT AAA GAG P h e Tyr Lys Asn Gly Lys Lys Val G l n Glu P h e ser Gly Ala Asn Lyn Glu

430 M G CTG G M GAG ACC ATT A M ACT CTA GTC T A ATCTCCCGCC AFCCAGACAT Lye Leu Glu Glu T h r Ile Lys Ser Leu Val

102 CGGAGCATGT CTGGATCGGC TAAATTATAG CCCATMCTT TTCTACTTTC MAGAAAATC

541 CMCCATTTA ACCTATCTAG CTTGGTGGM ACTATCCTCT GATCMTGTA MTGAGTACA

602 ATAAAATGTA MTTCTTCAC T A A " &"AM AAAU"

ATG ACC ATG ATT ACG M T TCG AGC TCG GTA CCC GGC ATG GTG M G + not T h r net 110 T h r Aan Ser ser Ssr Val P r o Glv net Val LYL) +

8-galactosidase ~ ~ ~ t h l o ~ d o x i n - ~~

FIG. 1. A, the nucleotide sequence of the chicken thioredoxin cDNA clone (pCEFZO1) and the protein product it predicts. The active site of thioredoxin is underlined. B, the nucleotide and amino acid sequence of the amino terminus of the lac-thioredoxin fusion protein. The 12 amino acids that are derived from /3-galactosidase are indicated.

1 10 2 0 30 40 I I I I I

a MVKSVGNLADFEAELKMGEKLVJVDFSATWCGPCKKIKPFFHSLC b ATVKVDNSN QSDVWSS P- W E A ALDEIA C SDKIIHLTDDS DTWLK DGA-IL W E d SAAAQVTDSTPKQEVLDSDVP L FW P R VA WDEIA

A ILDEIA

e KASEAVKEVQDV DSGWKFFJIASS P-SI4 W P f MMFKFALYFLNLEQPYSATI NTTDENFQADVLDA TP L W G A A A VLEE S

L A VIDE A

50 60 B O 90 100 7 0 I I I I I I

a D K F - G D W F I E I D V D D A Q D V A T H C D ~ ~ ~ ~ F Q F Y ~ G ~ Q E F S G ~ - ~ K L E E T I K S L V

c EYQ KLTVAKLNI QNPGT PKYGIRGI LLLF EVMTIW ts GQ K FLDANLA b TEMA Q KIAKVNI ENPEL AQFG RSI LIMF D ELAANUV AP SR ADW ASA

d QQYE KIKVVKVNT ENPQ SQYGIFSI IMIF G Q D W W VP TT SQ LEKHL e KEYS KIAVTKLNT E PGI QYNIRSI VL F ERKESII DVS YQ - f NEYA K KIVKV TSCE T VKYNIRNI A U F D EV AQQV APFS M F DQNI

FIG. 2. Conserved amino acids in thioredoxins. All thiore- doxin sequences that have been completely and unambiguously de- termined were compared to each other using the FastP program: a, chicken; b, C. nephridii type C1 (20); c, E. coli (14, 15); d, Cyanobac- terium anabaena 7119 (18); e, spinach chloroplast type m (19); f, C. nephridii type C2 (16). Only residues that differ from those in chicken are shown. Gaps have been introduced to allow maximum homology and are indicated by a dash.

Differential Expression of Chicken Thioredoxin 9609

a b c d e f - kd

9 7.4 -F

68.0 +

43.0 *

- kb

23.13 *

9.42-

6.56-

4.371, 25.7 I)

18.4 +

1 2 3 4 5 6 FIG. 3. Expression of the chicken thioredoxin fusion pro-

tein in E. coli. 35S-Labeled proteins from E. coli transformed with plasmid pCEFrTRX (lanes 1 and 2 ) or plasmid pCEFTRX (lanes 3- 6) were analyzed by SDS gel electrophoresis and autoradiography. The proteins in lunes 2 and 4 are from cultures grown in the presence of IPTG. The lysate analyzed in lane 4 was separated into soluble ( l a n e 5) and membrane-associated (lane 6 ) fractions. The arrow at the right indicates the position of the recombinant protein. The positions of the molecular weight markers are indicated at the left. An equal number of disintegrations/min were applied in each lane.

0.1 5.

0 v) (D n

0

0 0 15 30 45 60 75

Time (minutes)

FIG. 4. Assay for thioredoxin in E. coli transformed with pCHKTRX. A , activity observed when expression of the fusion protein was induced with IPTG; B, activity observed in the absence of induction. This level of activity is due to the endogenous E. coli thioredoxin. C, activity observed in the absence of any added sample.

lease 28.1) using the FASTN computer program (William Pearson, University of Virginia). No significant homologies were detected. The sequence of the predicted protein product was compared against all sequences in the NBRF protein sequence data base (release 6.0) using the FASTP computer program (31). Two proteins in the data base showed signifi- cant homology with the predicted protein product from this clone. The first was thioredoxin from E. coli which is 108

2.32-

2.03-

FIG. 5. Southern analysis of the chicken thioredoxin gene. CEF DNA (10 pg/lane) was digested with: a, HindIII; b, EcoRI; c, PstI; d, NcoI; e, BglII; f , XhoI and fractionated on a 1% agarose gel. DNA was transferred to Zetabond membrane (AMF CUNO) and hybridized to a probe for the entire thioredoxin coding sequence. Hybridization was at 65°C in 0.5 M NaP04, pH 7.2, 7% SDS, 1% bovine serum albumin for 12 h. Washes were performed at 65 "C in 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0, 1% SDS.

amino acids in length and shows 27.6% amino acid identity with the chicken protein (Fig. 2, sequences a and c). The second was thioredoxin from Corynebacterium nephridii which is 105 amino acids in length and shows 30.5% identity with the chicken protein (Fig. 2, sequences a and b).

Expression of Chicken Thioredoxin in E. coli-To confirm that this clone encodes the cDNA for chicken thioredoxin, it was expressed in E. coli, and lysates from these bacteria were assayed for thioredoxin activity. Plasmids pCHKTRX and pCHKrTRX were used to transform E. coli JM107. Proteins present in cultures grown in the presence and absence of IPTG were analyzed by SDS gel electrophoresis (Fig. 3). A protein of the expected size for the lac-thioredoxin protein (13,000 kDa) was present only in lysates from pCHKTRX plus IPTG (Fig. 3, lane 4 ) . The lac-thioredoxin protein was present predominantly in the supernatant fraction (Fig. 3, lane 5 ) indicating that it behaved as a soluble protein.

Lysates from bacteria containingpCEFTRX or pCEFrTRX were assayed for thioredoxin activity before and after IPTG induction (Fig. 4). Lysates from the bacteria containing pCEFTRX had a greatly increased level of thioredoxin activ- ity when the bacteria were induced with IPTG (15 min for onset of precipitation) compared with uninduced bacteria (50- 60 min for onset of precipitation). Lysates from bacteria containing the thioredoxin gene in the reverse orientation showed no increase in thioredoxin activity over that in JM107 itself whether or not IPTG was present (50-60 min for onset of precipitation, data not shown).

Analysis of the Chicken Thioredoxin Locus-Southern blot analysis of CEF DNA with a probe for chicken thioredoxin is shown in Fig. 5. Single bands are detected in digests with

9610 Differential Expression

origin

kb 2.95 2.5

-

1.5

0.5

1 2 3 1 2 3 A 6

FIG. 6. Expression of thioredoxin mRNA in normal and RSV-transformed CEF and in chicken brain. Lane 1, RNA from normal fibroblasts; lane 2, RNA from transformed fibroblasts; lune 3, RNA from chicken brain. The blot was sequentially hybridized with a probe for the entire chicken thioredoxin coding sequence (A ) and chicken glyceraldehyde-3-phosphate dehydrogenase ( B ) . The migra- tion of the RNA size markers is indicated at the left.

NcoI, BgZII, and XhoI. These data indicate that the chicken thioredoxin gene is present as a single copy gene. The locus is contained within a 4.4-kilobase NcoI fragment. There do not appear to be other genes highly homologous to thioredoxin present in chicken.

Expression of Chicken Thioredoxin mRNA in Normal CEF, RSV-transformed CEF, and Chicken Brain-Solution hybrid- ization studies indicate that transformation of CEF by RSV leads to an increase in the level of numerous mRNA species (32). However, only a few specific mRNAs that are increased in RSV-transformed cell have been identified (33-36). North- ern blot analysis of RNA from Schmidt Ruppin RSV-trans- formed and normal CEF (Fig. 6) showed that RSV-trans- formed CEF contain an elevated level of thioredoxin mRNA. The increase observed in three independent experiments was 2-4-fold (determined by densitometric scanning of the auto- radiographs). An increase in thioredoxin mRNA levels was also detected in CEF infected with temperature-sensitive mu- tants of RSV (isolates 72-4 (37) and NY-68 (38)) when main- tained at the permissive but not the nonpermissive tempera- ture (data not shown). The increases observed with tempera- ture-sensitive viruses were not as dramatic as those observed using wild type virus, being at most 2-fold.

Experiments in which the cDNA library was screened with RNA from CEF and chicken brain indicated that brain tissue contained reduced levels of thioredoxin message. To confirm this observation, chicken brain RNA was also analyzed by Northern blot analysis (Fig. 6). The level of thioredoxin

of Chicken Thioredoxin

mRNA in brain tissue was almost undetectable at the level of analysis used in these experiments.

DISCUSSION

The cDNA sequence for chicken thioredoxin predicts a protein of 105 amino acids with a molecular weight of 11,700. The sequence of the chicken thioredoxin is very similar to the sequences of other thioredoxins (Fig. 2). It contains the con- sensus sequence Cys-Gly-Pro-Cys, which is found at the active sites of thioredoxins. When the chicken thioredoxin is com- pared to other thioredoxins several residues (those corre- sponding to chicken residue numbers 25, 26, 29, 31, 40, 50, 66, 15, 81, 83, 91, and 97) in addition to those in the active site are seen to be invariant. The invariablity of these residues in thioredoxins from such diverse organisms as plants, bac- teria, and chicken indicates that they may be essential deter- minants for thioredoxin structure and function. Several other positions are noted which show little variation. The residues which show little or no variability are clustered in the car- boxyl-terminal two-thirds of the molecule. The first 20 or SO

amino acids in the proteins show very little homology and may not significantly contribute to the enzymatic activity of thioredoxin.

The serine at position 28 in the chicken protein is especially interesting. All of the other thioredoxins that have been completely sequenced have a tryptophan at this position. Based on chemical modification studies, Holmgren and col- leagues have suggested that this tryptophan may be required for the activity of E. coli thioredoxin (39). However, partial sequence data for the chloroplast type f (40) and yeast thio- redoxins (41) indicate that, like the chicken protein, they do not have a tryptophan at this position. Taken together these results show that the presence of a tryptophan at this position is not a general requirement for activity.

The increase in the level of thioredoxin mRNA seen in RSV-transformed CEF is in agreement with the observation that thioredoxin protein levels are generally higher in actively growing tissues (42). In light of the anabolic reactions in which thioredoxin participates, it is likely that this increase in mRNA levels is a secondary requirement for rapid growth. It is also possible that this increase may be specifically induced by expression of the RSV src gene product, but we do not yet have any direct evidence to support this possibility. The low level of thioredoxin mRNA seen in a nonproliferative tissue like brain is also in agreement with the observed tissue distribution of thioredoxin (42).

Transformation of CEF by RSV induces profound changes in many enzymatic pathways. These changes are mostly likely effected through a variety of control mechanisms. Some of the enzymatic reactions that appear to be modulated by thioredoxin similarly represent major control points for cel- lular metabolism. Perhaps increased thioredoxin expression detected upon transformation may also serve to mediate some features of the transformed phenotype.

The availability of a molecular clone for chicken thiore- doxin along with the ability to overexpress it in eukaryotic cells will be of use in addressing the involvement of thiore- doxin in the metabolism of normal cells and its involvement in transformation.

Acknowledgments-We would like to thank D. Chikaraishi for providing clone H1, S. Merchant for providing Cyunobacterium rRNAs, and B. Neel, E. Erikson, and R. L. Erikson for critical reading of the manuscript.

Differential Expression of Chicken Thioredoxin 9611

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15.

16.

17.

18.

REFERENCES Erikson, E., Cook, R., Miller, G. J., and Erikson, R. L. (1981)

Erikson, E., and Erikson, R. L. (1980) Cell 21,829-836 Erikson, E., Shealy, D. J., and Erikson, R. L. (1981) J. Biol.

Hunter, T., and Cooper, J. A. (1981) Cell 24, 741-752 Radke, K., Gilmore, T., and Martin, G. S. (1980) Cell 21 , 821-

Radke, K., and Martin, G. S. (1979) Proc. Nutl. Acud. Sci. U. S.

Erikson, E., Tomasiewicz, H. G., and Erikson, R. L. (1984) Mol.

Tomasiewicz, H. G., Cook-Deegan, R., and Chikaraishi, D. M.

Holmgren, A. (1985) Annu. Reu. Biochem. 54,237-231 Laurent, T. C., Moore, E. C., and Reichard, P. (1964) J. Biol.

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

Hunt, T., Herbert, P., Campbell, E. A., Delidakis, C., and Jackson,

Buchanan, B. B. (1980) Annu. Reu. Plant Physiol. 31,341-374 Hoog, J-O., Von Bahr-Lindstrom, H., Josephson, S., Wallace, B.

J., Kushner, S. R., Jornvall, J., and Holmgren, A. (1984) Biosci. Rep. 4,917-923

Wallace, B. J., and Kushner, S. R. (1984) Gene (Amst.) 32,399- 408

Lim, C.-J., Fuchs, J. A., McFarlan, S. C., and Hogenkamp, H. P. C. (1987) J. Biol. Chem. 262,12114-12119

Lim, C.-J., Gleason, F. K., and Fuchs, J. A. (1986) J. Bucteriol.

Gleason, F. K., Whittaker, M. M., Holmgren, A., and Jornvall,

Mol. Cell. Biol. 1, 43-50

Chem. 256,11381-11384

828

A. 76,5212-5216

Cell. Biol. 4, 77-85

(1984) Mol. Cell. Biol. 4, 1935-1938

Chem. 239,3436-3444

Chem. 260,93-97

R. J. (1983) Eur. J. Biochem. 131, 303-311

168,1258-1264

H. (1985) J. Biol. Chem. 260,9567-9573

22.

23.

24.

25.

26.

27.

28. 29. 30. 31.

32.

33.

34.

35.

36.

37.

38. 39.

Chirgwin, J . M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18,5294-5299

Panabieres, F., Piechaczyk, M., Rainer, B., Dani, C., Fort, P., Riaad, S., Marty, L., Imbach, J. L., Jeanteur, P., and Blanchard, J.-M. (1984) Biochem. Biophys. Res. Commun. 118 , 767-773

Mulligan, B., Schultes, N., Chen, L., and Bogorad, L. (1984) Proc. Nutl. Acud. Sci. U. S. A. 81, 2693-2697

Heidecker, G., and Messing, J. (1983) Nucleic Acids Res. 11,

Maxam, A. M., and Gilbert, W. (1980) Methods Enzymol. 65,

Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene

Laemmli, U. K. (1970) Nature 227,680-685 Chamberlain, J. P. (1979) Anal. Biochem. 98, 132-136 Holmgren, A. (1979) J. Biol. Chem. 254, 9627-9632 Lipman, D. J., and Pearson, W. R. (1985) Science 227, 1435-

Groudine, M., and Weintraub, H. (1980) Proc. Nutl. Acud. Sci. U.

Flier, J. S., Mueckler, M. M., Usher, P., and Lodish, H. F. (1987)

Matrisian, L. M., Glaichenhaus, N., Gesnel, M. C., and Breath-

Sugano, S., Stoeckle, M. Y., and Hanafusa, H. (1987) Cell 49,

Bedard, P.-A., Alcorta, D., Simmons, D. L., Luk, K.-C., and Erikson, R. L. (1987) Proc. Nutl. Acud. Sci. U. S. A. 84,6715- 6719

Mayer, B. J., Jove, R., Krane, J. F., Poirier, F., Calothy, G., and Hanafusa, H. (1986) J. Virol. 60,858-867

Kawai, S., and Hanafusa, H. (1971) Virology 46,470-479 Eklund, H., Cambillau, C., Sjoberg, B.-M., Holmgren, A., Jorn-

vall, H., Hoog, J. D., and Branden, C. I. (1984) EMBO J. 3, 1443-1449

4891-4906

499-560

(Amst.) 33 , 103-119

1441

S. A. 77, 5351-5354

Science 235,1492-1495

nach, R. (1985) EMBO J. 4, 1435-1440

321-328

19. Maeda, K., Tsugita, A., Dalzoppo, D., Vilbois, F., and Schurmann, 40. Tsugita, A., Maeda, K., and Schurman, P. (1983) Biochem. Bio-

20. Mew, M., and Hogenkamp, H. P. C. (1981) J. Biol. Chem. 256, 41. Hall, D. E., Bladesten, A,, Holmgren, A,, and Reichard, P. (1971)

21. Auffray, C., and Rougeon, F. (1980) Eur. J. Biochem. 107,303- 42. Holmgren, A., and Luthman, M. (1978) Biochemistry 19, 4071-

~~~. _~..

P. (1986) Eur. J. Biochem. 154, 197-203 phys. Res. Commun. 115 , 1-7

9174-9182 Eur. J. Biochem. 23,328-335

314 4077