Gene, 102 (1991) 221-228 0 1991 Elsevier Science Publishers

GENE

221

B.V. 0378-l 119/91/$03.50

04077

Isolation and characterization (Recombinant

DNA;

nucleotide

of human thioredoxin-eucoding

sequences;

pseudogene;

exon; intron;

genes

promoter

analysis;

early pregnancy

factor)

Kathryn F. Tonissen and Julian R.E. Wells Department of Bioc~emi~t9, University of Adelaide, Adelaide, South Australia, 5001 ~~us~a~~a~ Received by H.G. Zachau: 20 November Received: 25 January 1991

1990

SUMMARY

Thioredoxin (Trx) has recently been demonstrated to be an essential component of the early pregnancy factor activity of pregnancy serum. Here, we report the structure and sequence of human Trx-encoding genes (Trx) by analysis of genomic clones. The Trx gene extends over 13 kb and consists of five exons encoding a 12-kDa protein. A 700-bp fragment upstream from the start codon functions as a promoter when inserted in front of a human growth hormone-encoding reporter gene in tissue-culture cells. This promoter region is very G + C rich and does not contain a classical TATA or CCAAT box, but has three consensus sequences for high-affinity Spl binding. Southern analysis demonstrated the presence of several Trx genes in the human genome. The number includes at least one inactive copy as shown by the isolation and sequencing of an inactive pseudogene.

INTRODUCTION

Thioredoxins (Trx) are small ubiquitous proteins which function in many intracellular biological pathways. The active site sequence (Cys-Gly-Pro-Cys) at residues 3 l-34 of the protein is conserved between all species and the two Cys residues can be reversibly oxidized to form a disulfide bridge. This enables Trx to participate in many redox

Corresporrdettce

to;

Dr.

J.R.E.

University

of Adelaide,

(Australia)

Tel (61-8)2285353;

Abbreviations: hGH,

human

growth

polymerase

of Biochemistry,

South Australia,

bp, base pair(s);

factor;

kilobase or 1000 bp; LTR, oligo, oligodeoxyribonucleotide; NaH,P0,/145

Department

EBV, Epstein-Barr

FCS, fetal calf serum; Gt, Geneticin

hormone;

hGW, gene encoding

RSV, Rous sarcoma

hGH;

kb,

long terminal repeat; nt, nucleotide(s); PBS, 75 mM Na,HPO,/2.5 mM

mM NaCl; PolIk, Klenow (large) fragment I; RIA,

5001

Fax (61-8)2233258.

aa, amino acid(s);

virus; EPF, early pregnancy (G418);

Wells,

GPO Box 498, Adelaide,

radioimmunoassay;

RIT,

virus;

dodecyl

SDS, sodium

rosette

ofE. c&DNA inhibition

sulfate;

test;

SSC, 0.15 M

3citrate pH 7.6; SV40. simian virus 40; Trx, thio-

NaCl/O.OlS

M Na,

redoxin(s);

Trx, gene encoding

Trx.

reactions as a general protein disuhide oxido-reductase. Once present in an oxidized form, Trx is reduced by the action of Trx reductase and NADPH (Holmgren, 1985). Trx was originally discovered as a hydrogen donor for ribonucleotide reductase in Escherichia coli (Laurent et al., 1964), but has now been implicated in a wide variety of biochemical systems. These include a role in the life cycle of bacteriophage Ml3 (Russel and Model, 1986), T4 (Thelander and Reichard, 1979) and T7 (Mark and Rich~dson, 1976), regulation of photosynthetic enzymes in plants (Cseke and Buchanan, 1986) and the reduction of various mammalian proteins including insulin (Holmgren, 1979), the glucocorticoid receptor (Grippo et al., 1985) and several proteolytic enzymes (Holmgren, 1985). Recently, three independent studies have identified Trx as a participant in extracellular processes. It has been assigned a role in inducing the expression of interIeukin-2 receptors and with stimulating the growth of human T cells (Tagaya et al., 1989). In addition, the polypeptide component of a preparation purified from the medium of an EBV-transformed cell line which displayed interleukin- 1 activity (Rimsky et al., 1986) has been identified as Trx

222 (Wollman et al., 1988). Finally, we have now implicated Trx as an important component of a system that results in the expression of an activity defined as EPF (F.M. Clarke, C. Orozco, A.V. Perkins, I. Cock, K.F.T., A.J. Robins, J.R.E.W.,

tissues, the foetus also begins production after impi~tation (Morton et al., 1982). EPF activity has therefore been postulated to have a vital role in the development and/or immune protection of the embryo. The ability of all transformed cell lines to secrete EPF (Quinn et al., 1990) adds additional interest in elucidating the mechanism and biological significance of EPF activity. Recently we have isolated a cDNA clone encoding human Trx, and expressed it in a bacterial system to obtain purified recombin~t protein. The recombinant Trx was shown to cooperate with other serum factors to result in a positive response in the RIT assay. The importance of Trx as a component of pregnancy serum was also demonstrated, since absorption with specific anti-Trx antibodies removed all traces of EPF activity (F.M. Clarke, C. Orozco, A.V. Perkins, I. Cock, K.F.T., A.J. Robins, J.R.E.W.,

1991, pers. commun,).

EPF, the earliest known detectable marker of pregnancy (Morton et al., 1974), is detected in an in vitro assay, namely the RIT (Bach and Antoine, 1968) which measures an immunosuppressive activity. A positive response can be observed within hours of fe~ilization (Morton et al., 1976). The presence of EPF activity is a true indication of a viable embryo, since removal of the embryo results in a rapid removal of the EPF response from the serum (Morton et al., 1977), while transfer of a fertilized egg to a recipient female enables EPF activity to be detected subsequent to the transfer (Chen, 1985). Initially produced by maternal

TGCGTGTTTAT~GGGGAGAGAGC~GCAGCGAG

Human Trx cDNA : Pseudogene ..-

35

TCTTGAAGCTCTGTTTGGAGCTTTGGATCCATTTCCATCGGTCCTTACAGCCGCTCG 92 51 GATCTGGAACAGTGTGGAACAAC;AGAGACGGACGAGAT~TTTTTAG~TGTTTCT A 138 103

~B~~~~~~~~~~

180 145

~]$Z$~lZ-ZY$U# TGT GGG CCT TGC AAA ATG TGT GGG CCT TGC AAA ATG

GTA GTT GAC TTC TCA GCC AC GTA GTT GAC TTC TCA G&X AC~~1

TTT CAT TCC CTC TCT GAA AAG TAT TC l%l%-Z-$I CTT CTT

~~3~ GCT GCT

TCA TCA

GAG TGT GAG TGT

TTT TTT

AAG AAG AAG AAG

TTT

CAT

TCC

CTC

TCT

GAA AAG

GAA GAA

GTA GTA

GAT GAT

GTG GAT GTG GAT 1~~1

GAA GTC GAA GTC

AAA AAA

TGC ATG TGC ATG

AX TC Kl AAC

187 264 229 306 271

CCA A CCA A $

GT GAA TTT T GAA TTT

GGA CAA AAG G GGA CA& AAG G

TAT

222

TCT TCT

348 Bf

:Ej

GGA GCC GGA GCT

310 390 352 426

!$$

;E;

ZQZZI$~l

394 482 450 537 505 566 562

AAT

565

Fig. 1. Sequence of the genomic pseudogene clone (lower lane) compared to a human Trx cDNA clone (upper lane). The nick-translated cDNA insert (600 bp) of clone cHuth1 encoding the entire human Trx protein (Clarke et al., 1990) was used to screen a human genomic DNA library (kindly provided by Dr. I. Young, Canberra) formamide/l%

SDS/lo%

constructed dextran

in the phage AEMBL3A.

sulfate/l

Membranes

M NaCi/S x Denhardt/50

(Plaquescreen,

NEN-DuPont)

were hybridized

for 16 h at 42°C in 40%

mM Tris HCl pH 7.5/100 pg salmon sperm DNA per ml. The filters were washed

to a final stringency of 2 x SSCjO.1 y0 SDS at 42°C. Several fragments were subcloned into Ml3 and sequenced using Pollk (Bresatec. Adelaide, Australia). The human Trx and pseudogene sequences are aligned to obtain maximum homology and identical nt are boxed. The IO-nt inverted repeat sequence

present

at either end of the pseudogene

sequence

is underlined.

223 The isolated clone contains two additional in-frame stop codons and the ATG has been mutated to an ATC

1991, pers. commun.). To characterize further the biological role of Trx, we have isolated the genomic copy of the human Trx gene, which is the first eukaryotic Trx gene to be

sequence. The genomic sequence does not contain any introns and has similar structural characteristics to a processed pseudogene. The homology to the cDNA clone continues at the 3’ end until the polyadenylation site used by the human liver cDNA clone, although no poly(A) tail is present. However, only limited homology is present between the 5’ end of the pseudogene and the cDNA clone.

reported.

RESULTS

AND DISCUSSION

(a) Analysis of an inactive pseudogene Screening the human genomic library with the human TRX cDNA (Tonissen, 1990) as a probe yielded approx.

Pseudogenes are often flanked by short repeats, usually 1-16 bp long which are believed to play a role in the insertion of the sequence into the genome (Wilde et al., 1982; Bernstein et al., 1983). While there is no evidence for direct repeats flanking the isolated genomic sequence, there is a short inverted repeat structure (spanning 12 bp; underlined in Fig. 1). This inverted repeat occurs extremely close to each end of the homologous sequences and 10 of the 12 nt can base-pair. It is possible that mutations altering these two nt may have occurred after the insertion event.

150 positive plaques. One of these was analyzed in more detail to characterize the nature of the hybridizing sequences. Fragments which hybridized to the TRX cDNA probe were subcloned The resulting sequence

into Ml3 vectors and sequenced. is shown in Fig. 1 compared to the

human TRX cDNA clone previously isolated from a human liver cDNA library (Ton&en, 1990). While the genomic clone shows substantial DNA homology to the cDNA sequence, it is clearly unable to encode a functional protein.

11.9

Bg P I I

I

I’ a’

#’ ,/’ 4’ #’

,/ 3.9

4.4

I

I I

‘, :, ,\ :, \, :

pB51

P

EP

I II ,

I

I I

‘\

/

‘\

/ #’ ,’

4.6

,’

,’

13.8 14.3

‘\

‘\

/

9.3

‘\

‘\

‘\

‘.

9.1 9.8

10.5

20.6

H

: ,’

‘\

16.1

, :’ ,

I

kb

P

-. l.

I . *.

l

: 81’

. l.

l

*. -.

-. ‘.

*. -.

-.

15.15

16.1

16.55

Sau

H

Sau

kb

s’ Exon 1

B

Exon 2

.6 PSI1

EglII

Exon 3

1.0

1.4

IlindIII

Hind111

Exon 4

2.2

3.1 kb

PSfI

BglII

I

I

1 Fig. 2. Organization CTGCTTCAC). SSC/O.l%

Trx genes. (A) Organization

of two human

designed to bind to DNA encoding Hybridization

of a complete

Exon 5

human

Trx gene. The human

genomic

library was screened

with an oligo

the first 15 aa ofthe human Trx protein (5’-dGCGGCGTCCAGGGCCTCCTGGAAGGCGTACTTGGACTCGAT-

was performed

SDS at 50°C. Various

fragments

as above except the formamide were subcloned

was omitted

into Ml3 and sequenced

and the filters were washed

using PolIk or

to a final stringency

Tuq DNA polymerase

(Promega,

Madison,

of 6 x WI)

according to the dideoxynucleotide chain-termination method (Sanger et al., 1977). All clones were sequenced in both directions. The upper part shows the restriction map of the gene, and the regions surrounding the axons are drawn to a larger scale in the lower part of the diagram. The coding regions are shown as shaded boxes, while open boxes indicate (B) Restriction human

map of the processed

Trx cDNA

sequence.

pseudogene.

untranslated

sequence.

This clone was isolated

B, BarnHI; as described

Bg, BglII; E,EcoRI;

H, HindIII;

in Fig. 1. A stippled

box indicates

P, PsrI; SC, SacI; Sau, Sau3AI. the region homologous

to the

224

1

AGCAGCGAGTCTTGAAGCTCTGTTTGGTGCTTTGGATCCATTTCCATCGGTCCTTACAGCCGCTCGTCAGACTCCAGCAGCC~G

met val lys gin ile glu ATG GTG AAG CAG ATC GAG

ser lys thr ala phe gln glu ala leu Intron 1 AGC AAG GTACGCGCTACCGGGGAAGGCCAGGGTGCCGC...... (4.6 kb)..... .AAGCTTTTCTTTTGCTGTTAG ACT GCT TTT CAG GAA GCC TTG 40 20 asp ala ala gly asp lys leu val val val asp phe ser ala thr trp cys gly pro cys lys met ile lys pro phe phe GAC GCT GCA GGT GAT AAA CTT GTA GTA GTT GAC TTC TCA GCC ACG TGG TGT GGG CCT TGC AAA ATG ATC AAG CCT TTC TTT

his CAT GTGAGTATTAAACAATGTCTGCTTTGTAAGAGATTTGTGTGGATTTTGGTTTCGGTGGGGG GATTTCTTTGGCTCCATCTTTGGTCTAAAAGTAGTAGTATT

TGTCTCTTAGATCTATGTCTCCAAAAGATCTAAATTTTTGGCTTGGGTATGTTGCATTGC ser leu ser glu lys tyr ser asn val ile phe leu glu val asp CATTACCTAGTTCTAAATCTTTTTGGATTTTTCATTTTAATTTTCCAG TCC CTC TCT GAA AAG TAT TCC AAC GTG ATA TTC CTT GAA GTA GAT 60 val asp asp cys gln GTG GAT GAC TGT CAG GTATGTAGCTGGRRATATGAGATACTGCTGAGCTTTTCACATTGGCCTTTTCTCTG~TT~ACAGTGCTTTTTCCAT~TATGTC~

TAATTCTAGAACTGTAATCCTATCTAAAAGTTCTATCTCAGTTAGGAGCT......

Intron 3 (5.5 kb) . .. ..GATCGCTGGAGTATCTGATGCTAGTA

asp val ala se= glu cys glu val lys cys met pro thr phe gin phe GCAGTCTTGTATTTAATCCTCTCCCTTTGCTACTTTCCCTACCAG GAT GTT GCT TCA GAG TGT GAA GTC AAA TGC ATG CCA ACA TTC CAG TTT 80 phe lys lys gly gln lys TTT AAG AAG GGA CAA AAG GTACGTACATCTGACCTTTACTCTAACTGGGCAATAGTGTTCCCTTT

ACTAGTCCTATTTATATCATAAAGCACACATTTCTTATTTGTACTTGAGCCTTTTGACTTTT

CTCTTGATATTTTTTTCTTTGGTTTATAACTTAAATGAACGATTATATACTTCTTT'rTCCCTCCACCCCTATTCTTC

100

TGCTACGTCTAATGTCAATTCAATATTCTCTTAACAG

val gly glu phe ser gly ala asn lys glu lys leu glu ala thr ile asn glu GTG GGT GM TTT TCT GGA GCC AAT AAG GM AAG CTT GAA GCC ACC ATT AAT GAA

leu val *** TTA GTC TAA TCATGTTTTCTGAAAATATACCAGCCATTGGCTATTTAACCCAGTTGC CATCTGCGTGACAATAAAACATTAATGCTAACACTTTTTATGTATTTTCCTATATTC

225 (b) Isolation of a complete human Trx gene

the distance between exons 1 and 2 is 4.6 kb and the intron separating exons 3 and 4 is 5.5 kb.

To obtain the genomic clone corresponding to the transcribed gene, the human library was rescreened with an oligo designed to bind to the 5’ region of the gene. Five plaques were found to hybridize to both the human Trx cDNA probe and the 5’ oligo. One of these clones, designated ~Huthl6, was analyzed and mapped in detail. Southern analysis was performed on ~Huthl6 to detect fragments which hybridized to the cDNA probe. These fragments were subcloned into Ml3 and sequenced, which

(d) Alternative poly(A)-addition sites Exon 5 contains all of the 3’-untranslated sequence present in a cDNA clone previously isolated from a human liver cDNA library. However, the cDNA sequence isolated by Tagaya et al. (1989) extends a further 48 bp 3’ before a poly(A) tail is added. This 48-bp sequence corresponds exactly to the genomic sequence present within exon 5. Both cDNA clones contain a polyadenylation signal sequence at the expected position relative to the poly(A) addition site. The Trx cDNA sequence shown in Fig. 1 was derived from liver tissue, while Tagaya et al. (1989) isolated their cDNA clone from a transformed cell-line library. It is therefore possible that 3’ processing may vary from one tissue to another,

confirmed that 1Huth16 contained the 5’-coding sequences of the Trx gene. Since kHuth16 did not contain the entire gene, the human genomic library was rescreened with a fragment from IHuthl6 which contained the 3’-most sequences of this clone (intron 1). Two positive phage 1 clones were analyzed in detail and were shown to overlap with ~Huthl6, but contain additional 3’ sequences. Subcloning of various fragments which could hybridize to the cDNA probe, enabled the entire coding sequences of the human Trx gene to be mapped and sequenced. The restriction map and exon/intron arrangement of the human Trx gene are shown in Fig. 2 and the sequence of the gene surrounding the coding regions is shown in Fig. 3.

(e) Analysis of the promoter The putative promoter region does not contain any classical TATA or CCAAT boxes at the expected positions for genes transcribed by RNA polymerase II (Dynan and Tjian, 1985). In fact, there are no such sequences contained within the 700-bp region shown in Fig. 3. This structure resembles the promoters of several other eukaryotic constitutive or ‘housekeeping’ genes. All of these promoters have an extremely high G+C base content and contain similar patterns of direct repeats (Ishi et al., 1985; Melton et al., 1986; Morioka et al., 1988). The G +C content of the putative Trs gene promoter region is 82% and required the use of Taq DNA polymerase and 7-deaza-GTP to obtain complete and consistent sequence data. Included in this region are three separate motifs which are consensus sequences for the transcription factor Spl to bind (Fig. 3). The consensus sequence 5’-GGGGCGGGGC is the preferred sequence to bind Spl with a strong affinity (Kadonaga et al.. 1986) and can function in either orientation (Everett et al., 1983). The Trx promoter contains two repeats of this type and one of a slight variation (5’-GGGGCGGAGC) which has also been shown to bind Spl with a high affinity (Kadonaga et al., 1986). To investigate the region of the gene capable of acting as a functional promoter, a construct designated pthioGH containing nt l-700 (Fig. 3) was inserted in front of the hGH gene. The RSV-LTR promoter region was also

(e) Organization of the human Trx gene Comp~ison of the cDNA clone with the genomic sequence revealed that the human Trx gene consists of five exons distributed over approx. 13 kb (Fig. 2). Each of the exon/intron boundaries contained donor and acceptor sites which corresponded well to the consensus sequences (Mount, 1982; Breathnach et al., 1978). All of the junctions were of type 0, that is, splicing occurs between codons (Fig. 3). All of the exons are quite small. Exon 1 contains the 5’-untranslated sequence and encodes 7 aa, exon 2 encodes 35 aa and exon 3 only 20 aa. Exon 4 encodes 22 aa, while exon 5 encodes 20 aa and contains the 3’-untranslated sequences. Exon 2 contains the well-characterized active-site domain of the Trx protein, while exon 4 contains a conserved C-terminal Cys pair which is present in all vertebrate Trx, but absent from bacterial proteins. This Cys pair has been shown to be functionally important in allowing Trx to exert its effects in the RIT assay (F.M. Clarke, C. Orozco, A.V. Perkins, I. Cock, K.F.T., A.J. Robins, J.R.E.W., 1991, pers. commun.). While exons 2 and 3 are close together and exons 4 and 5 are separated by only 560 bp,

4

Fig. 3. Sequence sequencing G+ C-rich

of the human

subclones and reliable

genomic

i’rx gene, including

in both orientations; sequence

data

were obtained

using

(5’-AATAAA) in the 3’-untranslated region are underlined. region are boxed and the BamHI site used in manipulation The sequence

data will appear

all of the exons and the 5’- and 3’-flanking

the sizes of introns

in the E~BL~GenBank

1 and 3 were determined Taq DNA

polymerase

regions.

by restriction-enzyme

to incorporate

The sequence mapping.

7-deaza-GTP.

shown was obtained

The Tr.u promoter

The two polyadenylation

by

was very signals

Asterisks indicate the stop codon. The consensus sequences for Spl binding in the promoter of promoter constructs is overlined. The deduced aa residues are numbered from the Met.

nt sequence

databank

under

accession

Nos. X54539-54541.

226

inserted in front of the hGH gene, generating a construct to act as a positive control. These constructs were co-transfected into CHO-Kl cells with pSV2neo, which contains the neomycin-resistance-encoding gene under control of the SV40 early promoter. Permanent cell lines were selected with Gt and the resulting colonies were analyzed for their ability to produce hGH using a RIA assay kit. Several hGH-positive lines were obtained and three independent cell lines were selected for an expanded study. The three cell lines containing the pthioGH construct produced hGH, although the highest producer is some 18-fold less than that obtained from colonies in which hGH expression was driven by the strong RSV-LTR promoter (Table I). A similar result was obtained when RNase protection was used to analyze RNA transcribed by these cell lines (data not shown). Thus, this 700-bp region of the Trx gene was capable of functioning as a promoter in a heterologous system. TABLE

I

Analysis

of the human

Trx promoter

hGH

Cell line :’

1

cells __

produced’

(ng hGH/ml/106 neo.

in CHO-Kl

cells/24 h)

-

thio 1.4

50

thio2.3

185

thio2.6

180

RSV-GH

3154

“ A 700-bp partial SacI-BarnHI promoter

fragment

region of the Trxgene

clone pHuthSac1. allow insertion

(Fig. 3) containing

was subcloned

This clone was then digested of a 2.2-kb BumHI-&oRI

the putative

into pBSKS + to generate with BamHI+EcoRI

fragment

containing

tural coding region of the hGH gene. These hGH sequences

contained control

the pthioGH containing

construct,

the RSV-LTR Ham’s

control

promoter.

F12

medium

(1.18 mg~ml)/gentamycin (Commonwealth Serum confluency,

washed

was

with EcoRI for transfection thioI.4, thio2.3 and thio2.6

construct.

as it contained

For transfection, (Gibco)

construct

B

kb

E

Bg

H

...

.-

while the neo. 1 cell line was a negative

only the pSV2neo

was used as a positive

Human genomic DNA from two sources was digested with BarnHI, EcoRI, BglII and Hind111 and subjected to Southern analysis using the human Trx cDNA insert as a probe. The result (Fig. 4) shows that multiple copies of Trx homologous sequences exist in the human genome. From this analysis it is not possible to determine the exact number of genes, but the number of bands in each track is greater than can be accounted for in the pseudogene and the complete Trx gene described here. This situation is in contrast to the chicken, where only a single copy of the Trx gene was detected (Everett et al., 1983). This result also influences the assignment of ‘the’ Trx gene to chromosome 3 as described by Lafagt et al. (1989). In this report, the entire human Trx cDNA clone was utilized as a probe for in situ hybridization analysis and only one significant cluster of silver grains was detected. While it is possible that all the Trx genes are clustered in one region, it is unlikely that the processed pseudogene will also be in the same region of the chromosome. Since the actively transcribed gene contains introns, it is likely that the cDNA probe will preferentially detect the processed pseudogene( This was the situation found when we used the human Trx cDNA probe to screen the human genomic library. The actively transcribed gene gives a less intense signal and therefore may map to another location in the genome. In addition, while previous studies have shown thioredoxin has a role in the RIT assay used to detect EPF activity (FM. Clarke, C. Orozco, A.V. Perkins, I. Cock, K.F.T., A.J. Robins, J.R.E.W., 1991, pers. commun.), it is possible that a different copy of the gene characterized here may be expressed during pregnancy and also by transformed cell lines. However, there is sipniti-

were derived

from the plasmid ~I#JGH (Selden et al., 1986). The resulting designated pthioGH and was linearized experiments. The permanent cell lines

to

the struc-

(f) Southern analysis

The cell line RSV-GH the hGH gene driven by

CHO-Kl

supplemented

cells were grown in

with

Na

2.6 -

bicarbonate

(50 pg/ml: Schering Corporation~/lO~,” FCS Laboratories). The cells were grown to 757;

in PBS and resuspended

cells/ml. Then 0.8 ml of the cell suspension

1.8 -

in cold PBS at 3 x lo6

0.9

(2.4 x lo6 cells) was placed

-

in electroporation cuvettes with 150 ng of BumHI-linearized pSV2neo and 3 pg linearized pthioGH constructs and left at 4°C for 20 min. Cells were electroporated at 1.5 kV, 25 PFd (time constant = 0.4) in a BioRad Genepulser, left at room temperature for 5 min and then transferred to loo-mm

culture

48 hand

Gt-resistant

dishes with Ham’s F12/100/,

FCS. Cells were grown for

cells were selected with 400 pg/ml of G418 (Gibco).

After about IO-14 days, Gt-resistant colonies were transferred trays (2-ml capacity) and allowed to reach confluency. ’ Supernatants

were assayed

RIA kit (Hybritech). used for further

Positive

analysis.

for each permanent

for hGH production clones

The amount

using a Tandem

were expanded

hGH

and subsequently

of hGH produced

cell line and expressed

to 24-well

was measured

as ng hGH/ml/lOh

cells/24 h.

Fig. 4. Southern lane contains

analysis

of 7’r.y sequences

IO pg ofgenomic

in the human

genome.

DNA digested with the restriction

Each

enzyme

indicated at the top of the figure. The positions of the size markers, in kb, are shown on the left margin. For restriction enzyme abbreviations see Fig. 2 legend. The DNA was transferred to Zetaprobe membrane (BioRad) and hybridized with “P probe, consisting ofthe 600-bp human Trx cDNA

sscjo.

insert.

After hybridization,

1-3”SDS at 42°C.

the tiiter was washed

in 0.5 x

221

cant evidence to suggest the product of the gene expressed in pregnancy is identical to thioredoxin. Sequencing of a protein preparation derived from RIT-active human placental extracts yielded an identical N-terminal sequence to that predicted from the cDNA and genomic clones. In addition, the recombinant product expressed from this gene was able to function in the RIT (in combination with other serum factors) and this ability was dependent on the presence of the C-terminal pair of Cys residues (F.M. Clarke, C. Orozco, A.V. Perkins, I. Cock, K.F.T., A.J. Robins, J.R.E.W., 1991, pers. commun.). (g) Conclusions (1) Trx is not a single-copy gene but belongs to a family of related sequences. As detailed here, at least one of these members is an inactive copy and one contains the expected coding sequences as well as a functional promoter. However, the remaining sequences have not yet been characterized. (2) The functional human Trx gene studied consists of five exons distributed unevenly over approx. 13 kb and this exon arrangement can be related to the protein structure. Exon 2 contains the active-site Cys pair, while exon 4 contains the C-terminal Cys pair shown to be important for the expression of EPF activity (F.M. Clarke, C. Orozco, A.V. Perkins, I. Cock, K.F.T., A.J. Robins, J.R.E.W., 1991, pers. commun.). Thus, the two proposed active regions of the protein are each contained within exon sequences separated by a large distance. While the bacterial protein has similarity to the sequences encoded by exon 2, it has no homology over the remainder of the protein. Therefore, it is possible that the vertebrate proteins have evolved an additional exon with a separate function. (3) Sequence analysis of the 5’ region of the Trx gene shows possible elements involved in transcriptional control. The promoter region of the Trx gene is extremely G + C rich and contains three high-affinity Spl-binding sequences. These sites are present 20 bp (two helical turns) and 12 bp apart and are thus situated on the same side of the helix. Other promoters shown to be stimulated by Spl have a similar spacing of binding sites (Kadonaga et al., 1986) and therefore, there is every indication that the Trx promoter is also responsive to Spl, although direct experiments to prove this are still required. Recently, another nuclear factor designated ETF has been characterized which binds to various G+C-rich regions (Kageyama et al., 1989). It stimulated transcription from promoters lacking TATA boxes but not from those which contain TATA boxes. Thus the Trx promoter could be activated by a combination of Spl and ETF. While the identity of the transcription factors which bind to the Trx promoter remain to be characterized, this region of the Trx gene was shown to be capable of directing expression of a heterologous gene when transfected into tissue-culture cells.

(4) The isolation

and characterization

Trx

of the human

gene provides a basis for investigations of the expression and analysis of function of this gene in the diverse biological processes with which it is involved in mammalian systems.

ACKNOWLEDGEMENTS

We would like to thank Allan Robins and Frank Clarke for many helpful discussions. This work was supported in part by a grant to the Commonwealth Special Research Centre in Gene Technology. K.F.T. was a recipient of a Commonwealth postgraduate research award.

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