Cloning and characterization of the rat glutathione peroxidase gene

FEBS 10904 Volume 301, number I. 5-9 0 1992 Fcder~~tiun ol’ European Birlchsmicul Socictios 00145793/9!/S5.01~

Cloning and characterization

April

of the rat glutathione

1992

peroxidase gene

Ye-Shill Ho and Gdriann J. Hc;ward

Rccciwd

27 January

1992:

revised version received 21 February

1992

The incrcused aclivily orglututhionc psroxidasc (GSHPs) in rili lungs is ;wxiaicd with the dwclopmcnt of tolcranw of the animals IO hvpcroxh. To undcrslund rurlhcr the regulation or cxprwsian ol’tliis cneymc. Ihc molcculsr structure or the corrcspondinp rat gcnc was chnractchzcd. The rilt GSHPx gent consists of two axons inkrrupicd by u singlc inwon ol’?17 base pairs. The sumc initialion silts for tranrripiion wcrc found 10 bu ulilizcd in bolh lung and liver. The promowr ol’ 11x GSHPx gcnc coniuins nciihcr ;I ‘TATA’ box nor ;I ‘CAAT’ box. lnstc;id. ii comprisct two copies of Spl binding niolil’ :rnd one copy 01’ AP-2 binding molil’, Thcsc fcnlurcs ol‘ the promoter may offer U clue IO the mcchunisms by which llic cxprcssion ol’ this gcnc is controlled. Gcnomic

cloning:

SI

IlUClCilSC

lllilppillg:

1. 1NTRODUCTION Glutilthione pcroxidLsc (GSHPx) plays an important role in cellultir antioxidant del’cnsc by reducing hydrogen peroxidasc or various hydroperoxides using glutathione as a reducing agent to form water or corresponding alcohols, respectively (H,O, + 2GSH + 2HZ0 + GSSG or ROOH + 2GSH + GSSG + ROH + H20) [I]. This enzyme, which contuins selcnocystcine at the active site, is present on both cystosol and mitochondria in eukaryotic cells [l-5]. Molecular cloning data have revealed that selenocysteine is encoded by an opal nonsense codon (UGA) in mouse, rat. bovine and human genes [G-13]. However, the mechanisms by which selenocysteinc is incorporated into this enzyme arc less conclusive [14-161. The recent discovery of a unique species of opal suppressor selenocystcyl-tRNA”” in rat mummary tumor cells suggests selenocysteine is inserted cotranslationally during the synthesis of this enzyme [17]. It has also been demonstrated by Berry et al. [I81 that the 3’ untranslated sequence of rat glutathione pcroxidase cDNA can substitute the 3’ untranslated sequence of Type I iodothyronine 5’ deiodinase cDNA (which also contains an opal stop codon coding for selenocysteine) in directing insertion of selenccysteine in response to UGA codon [l8]. Their results further substantiate the co-translational mechanism for usage of opal codon in selenium incorporation into this enzyme. Exposure of mammals to hyperoxia can cause extensive lung injury [ 191.This type of pulmonary damage is Co~~es~or~r/~~r?~~ rnflrcus: Y 3. Ho. Room 4.000. Inshulc ofChcn$zal Toxicology. Wnync Stute Universily. 2727 Socorid Awiuc. Detroit. MI 482012654, USA, Fax: (I) (313) 577-0082.

Primer

cxlcnsion;

Rcgultitory

scqucncc

due to the overproduction of reduced oxygen species in the lung. Rcsistancc to a lethal concentration of oxygen (100%) does occur in adult rats after exposing them to a sublethal concentration of oxy~;n (85%) for 5-7 days, and this resistance is associated with an increase in the activities of lung antioxidant enzymes including glutathione peroxidasc [20,21]. To elucidate the molecular mechanisms that regulate the expression of this enzyme in rat lungs during hyperoxia, we chose to first undcrstand the structure of the corrcspondirig ge~~c.3n this report, we describe the isolation and characterization of the rat GSHPx gcnc, and the nature of its promoter sequence which may play ti role in regulating its exprcssion under normal physiologic and hyperoxic conditions.

2. MATERIALS AND METHODS A kmulc Sprague-Lkwlcy r;+I gcnornic DNA libtxry. constructed in vccior ,I Churon 4A with il~~t~lII pariiully digest cd gcnomic DNA (Clonlcch Lnborutorics Inc.. Palo Alto. CA). Wils scrccncd wiih the 3’ Ecr~Rl frugmcm (from bdscs G2S LO 1I61 ) of I~C rm gltnulhionc pcroxidusc cDNA according LO 11x proccdurcs dcscribcd by Bcmon and Davis [22]. Two poshivcty hybridized clotxy., clones I2 and 18. wcrc isolated und subscqucmly dcicrmincd 10 bc idcniicul by rcstriclion digestion anulysis. The 3’ end gcnomic DNA frugmcnt was cxciscd from clone I! by ,&~RI dig&on. and ihcn ligakd into plusmid pKS (Sirutagcnc. Lulollii, CA) for DNA scqucncc analysis [23]. The 5’ end gcnomic DNA frapmcm which was found noI IO bc rclcuscd by LkoRI dig&on. presumably due to loss of 5’ EtaRI cloning site during the library conarucGon. was cxciscd from the phugc arm by K~III und EtvRl digestion. Sin&-stranded plasmid DNAs wcrc gcncrated by infecting baclcriu hurboring various plasmids with hclpr M I3 baacriophagc VCS [24]. and then scqucnccd by Sangcr’s didcouy chain-tcrminution mclhod using univcr%ul and complcmcniury ohgonuclcotidc primers [25].

5

Volume 301,

imtber I

FEBS LETTERS

DNA blot analysis of rat genomic DNA, and RNA blot amllysis were pcrformcd according Lothe methods dcscribcd by Southern [26] and Thomas [27], respeckly. One antiscnse oliyonuclcotidc with sequence 5’ CAGCGGGCGCGCGGAGAAGGCATACACGGT 3’ complcmcntary to nuclrotidc residues +7$ IO +I01 was used in thcsc experiments. For SI nuclcasc mappinguxperimcnt, the oligonucleotidc was initially labeled at 5’ end with y-[JzP]ATP und polynucleotidc kinusc, and then annealed to a sense, single-strunded plasmid containing the 5’ end BrrtrlHI-&oRl fragment of the rdt GSHPx gent. lbllowed by synthesis of the comple. mentary strand of DNA using the Klenow fragment of E. co/i DNA polymerasc I. The rcsultunt double-stranded DNA was digested with restriction enzyme BorrtHI and then the labeled single-stranded DNA was purified after separation on a polyacrylumide-urea gel. Fifty micrograms of tota! rat lung and liver RNA were used for 51 nuclcase mupping according to the procedures dcscribcd by Grccnc and Strubl [28]. The 5’ end “zP-l;lbclcd oligonucleotidc WIS ulso used in primer oxtension cxpcrimcnts. Fifty micrograms of total lung und liver RNA were used in primrr cxlcnsion experiments i’ollowing the methods described by Kingston [a].

3. RESULTS AND DISCUSSION gene

Two identical rat gcnomic clones were isolated from a Sprague-Dawley rat genomic library using the 3’ EcoRI rat GSHPx cDNA fragment as a probe [9], and the nucleotide sequence of the DNA containing the entire GSHPx gene was completely determined (Fig. 1). By comparing the genomic and cDNA sequences, the rat GSHPx gene is divided into two exons interrupted by a single intron of 217 base pairs. There is no sequence homology found between the first 3 16 base pairs of the cDNA sequence determined by our laboratory [9] and

April 1993

the genozlic DNA. Furthermore, the sequence of the 3’ E=coRIcDNA fragment for rat GSHPx c!oned hy Reddy et al. [7] was also not found in the genomic clone. These portions of cDNAs isolated by us and Dr. Reddy and colleagues are presumably derived from cloning artifacts, since no potential splicing donor and acceptor sites cnpab!e of generating those non-homologous cDNA sequences are found at the 5’ and 3’ end of the genoinic sequence. 3.2. DNA md RNA blot rrnalysisof the I’ULGSHPx gerw In order to understand the complexity of the GSHPx gene in rat genomc, DNA blot analysis of total rat genomic DNA digested with various restriction enzymes was performed using the cDNA (from bases 3 18 to 1161, generated by the polymerase chain reaction [30]) as a probe. Fig. 2a shows that two PstI genomic DNA fragments of 1.2 and 0,8 kilobases (kb), which are identical to the sizes predicted from genomic cloning data, hybridized with the probe. The numbers of other hybridized restriction genomic DNA fragments were also consistent to those predicted from the cIoning result. These observations strollgly suggest that a single gene coding for this enzyme is present per haploid rat genome. Expression of GSHPx in rat tissues was also examined by RNA blot analysis (Fig. 2b). A single species of 1.3 kb mRNA was found to hybridize with the probe. The rat GSHPx gene expresses at a higher level in the liver than does in the lung, 3.3, Mopping qf rrunsfcuiorrtr/skirt site(s) The transcriptional initiation site(s) in rat lung and liver wad determined by both Sl nuclease mapping and

a

__.

clone 12

7

I__

Fig. I. (a) Restriction mapandorganizution of the rat GSHPx gcnc. E. B, H, Pand X reprcscnt restriction sites for enzymes k&RI, kt11HI, f?it~dlll. and Xlrc~l,respectively. Solid and open boxes represent trans!:+ted and untranslured regions of the mRNA. rcspcctively. The mapofrccombinunt phagc clone I2 is shown at the lop. Em denotes EcwRI restriction silt artificiully gcneratcd during library conslruction.

Purl

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Volume 301, number I

b

April 1992

FEB.5 LETTERS

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. AQTACATCATT~GGTCCCCOG~GTOCCGCAACGAChTTT~CTGG~CT~TG~G~G~T~CTGGT~GGTCCAGACGGTGTTCCAGTGCGCA ysTyrIleIloTrpSorProv5lcytAr~AanA5pIloserTcpA~nPhoGluLysPhoL5uValGlyProA5pOlyVAlProVA~Ar0A . . . . . OATACAGCAGGCGCTTTCGCACCATCGACATCGAACeCaATATAGhaGeeCTGCTGTCC~GCAGCCTAGC~CCCCT~GGCATTCCTG r~TyrS5rhtgAcgPheArgThrIl5~5pIleGluPcOAspIloGluAl5LouL5U55rLySGlflPrOSOrAs~pcOE~d . OTATCTGGGCT~GOTGATGG~~GG~TG~~~T~~GGGGGGAG~TTTTTCCAT~ACGOTOTTT~CTCTAAATTTACATGGA4AAACACCTGA . . . . . . TTTCCAGAAAAATCCCCTCAGATGGGCGCTGGTCTCGTCCATTCCCGATGCCTTTACGCCT~G~GGCGGT~TCACCACT~G~~A ~OTG~TGAAT~.~AMG~~~~TGTTTGTGTG~~~~GT~T~~TGTCT . . . . TCCCTTTGOACGGCACCTTACCCATATCTCCCCTGTCTCGGGACATGCTT~GGGTAGTCCCGA~TCGCTGTTAC~CAG~CCCTTACTC . . . . . CAGAAAGAGGAAGAChGTAGACMGGGCTGACAGTTAAGOCCACCCTAACCCC~OC(iTGAACTTCTCCTTTCTGAGTTTTTTG~TGG~ . . . 1386 GAGGAAGTGACAGTGTGCCTCTOCTCACACAGGACTCGAGAATCTAGGTCTGTTTTGCA~AGC~OCAO

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Fig. I. (b) Nuclcotidc scqucnce and deduced amino ncid scquuncc of the rtil GSHPx gcnc. The transcriptional starl site is numbered BS nrfclcotidc rcsiduc I of lhc gcnc. The sequences homologous IO the known cuktiryoiic rcguhltory elcmcnls and polyadcnylalion site arc undcrhnd.

primer extension experiments (Fig. 3). A singlestranded DNA probe encompassing nucleotides -260 to +I01 of the genomic sequence was used in Sl nuclease analysis. As shown in Fig. 3, multip!c Sl nucleaseresistant fmgments approximately 100 nucleotides long were observed. The amounts of the protected fragments derived from the protection of RNA isolated from lung or liver were proportional to the levels of the GSHPx RNA in each tissue revealed by RNA blot analysis. The same size, multiple primer- extended DNA fragments were also obtained from primer extension experiments. Furthermore, identical SI nuclease protected and primer-extended fragments were derived from lung and liver RNAs. These results indicated that the same mul-

tiple transcriptional initiation sites were used in rat lung and liver. The most prominent band revealed by both mapping experiments corresponds to the transcript initiated at 35 base pairs upstream from the first AUG codon of the gene. This transcriptional start site was then designated as nucleotide residue 1 of the rat GSHPx gene. Chwu&v~i:utiort of 5’ jhkittg wgiort of lltc rat GSHPX gurc The nucleotide sequence immediately upstream from the transcriptional start site contains neither a ‘TATA nor a ‘CAAT’ box. However, several potcntia.1 regulatory elements were found within the promoter region

3.4.

7

Volume ,301. number

FEBS LETTERS

I

April start i

b

a

EXON 1 m

probe

3131nt-

protected traemente

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Palmer extenelon

1

I

1992

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Fig. 2. DNA and RNA blot ;in;\lyscs of the ritt GSHPX gene. (u) DNA blol an;dysis or the rut GSHPx gone, Ten micrograms or gcnomic DNA isolvtcd liom Sprilguc-Dtiwlcy rat liver wcrc diycstcd with various roshction cnzyms. und used for blot nniilysis. The restriction C~ZY~XS used ilrc shown ill the top ol’thu autorudiogtxph, DNA sire niarkcrs are Hirldlll-d&stud i DNA. (b) RNA blot an;llysis 01 GSHPx mRNA in rat lung rind liver. Filiy microgrnms 01’ total rat lung and liver RKAs wcrc used in blot nnalysis. The positions ol’ RNA size murkcrs urc shown on ilw MI.

(Fig. 1b). Two stretches of DNA sequence homologous binding scqucncc, consensus the SPl ~~T)(GA)GGcG(GT)(cIA)(GA)(cT), iire present at positions -312 and -104 [31]. One copy of AP-2 binding motif conferring activation of gene expression in response to phorbol esters of CAMP locates at position -181 [32,33]. Since the regulatory sequences for transcription of a particular gene are often conserved across species during the evolution of mammals, we further compared the promoter sequences of the GSHPx genes among rat, mouse and human [6,13]. The promoters of rat and mouse GSHPx genes share a high degree of homology (Fig. 4). Nonetheless, there is virtually no homology found between the rodent and the published, short sequence of human promoter ( I34 base pairs) [ 131. The human GSHPx promoter was originally defined by comparing the cloned genomic and cDNA sequences, rather than by SI nuclease mapping and primer extension experiments. It is possible that the cDNA for human enzyme might be derived from a rare species of transcript initiated 5’ to the normal transcriptional start site. WC, therefore, further aligned the human sequence upstream from the first AUG to the promoters cf rat and mouse genes (Fig. 4). Interestingly, several regions of homologous sequence were found among these specks. The Spl binding motif present at position - 104 of the rat promoter apparently is conserved in all species. The AP-2 binding site was found to be unique in rat 8

Fig. 3. Mupping or the trunscriptionul initktion site(s) of the rat GSHPx ycnc. The Itxnscription;ll initiation sit&) wns dctcrrnincd by both S I I~ICIC~SCmuppinp (lunes l-3) und prinxr cxicnsion (hcs 1-6) cxpcrimcnts, Filiy microyrums ol’ ye;ist tRNA (Iuncs I und 4). tot;11 rat lung RNA (1~~s 2 ;md 5) und totnl rut liver RNA (Iirncs 3 und 6) wcrc used in each cxpcrinxnt. A didcoxynuclcoiidc sequencing luddor. ohtuincd wilh the s;unc primer und sin&-sirandcd DNA icmplatc used to prcp:lrc the probe for SI nuclCiIsC niupping. w:is gcncr;ltcd and used Ibr rslinxltion 01’ the sizes 01’the rcsuliunt GNA litlgmcnis. The idcn\ical prinicr cslcndcd lixgnicnts derived lioni lung RNA 10 those I’rom liver RNA C;III bc SCCII ;tlicr il lonyur cxposurc 01’ ihc auioradiopraph. The arrow indicalcs ~hc major SI 11uEICilsC.rCsislilnt und primer-cxtcndcd lixgmcnis.

promoter. Additional three regions of highly homologous DNA sequence between the second Spl binding site and mRNA start site were also evident. Furthel experiments are required LOdissect the function of each of the conserved DNA elements in regulating the expression of the GSHPx gene. A~/i,~r~~~~~~,~~~~~/t~c/tI.r: This rcscurch \V;IS supported by Grants HL-39585 and HL-43571 from the National Instituics of Hwlth. WC gratcl’ully ackno*.vlcdgc Ms. Lind;l Crisp for her cxccllcnt sccrciariul ussist;incc UKI ;jr. Ron Thomas Ibr critical rclrdinp 01’ the manuscript.

REFERENCES [I] Wcndcl. A. ( 1980) in: Enzynxaic

Basis ol‘ Dcloxificaiion. vol. I (W. Jakoby. cd,) pp, 333-353, Academic Press. New York. [I] Epp. D.. L;idcnstcin. R. and Wcndcl. A. (19%) Eur. J. Biochcm. 133. 51-69. [3] Tuppcl. A.L. (19RJ) Curr. Top. Cell. Rcgul. 24, 87-97,

Volume 3@!, number 1

FEBS LETTERS

April 1991

RAT

-277

GT~GCc^AGAGCTGGA~ATCCCAGCTTTGGCCAGAGAGC~~GC~GCAG....CPTCCA

MOUSE

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GTAAGACAGAGCTGGAAGATCCC. . . . . . . . . . ..CGCCTAAGCAAGCAGCTTCCTTCCA

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Met Fig, 4. Comparison ol’ promoter scqucnccs 01’ r;ll. IIIOLISCund human CSHPx gcncs. Solid lint bows indicate positions scqucnccs urc present. The posilions or Spl biuding and AP-2 inolil’s ure ulso ovcrlincd,

I41 Zurowski, J. and Tuppcl. A.L. (197X) Biochim. Biophys. Acta 526, 65-76 151Timccnko-Yousscf, L.. Yamaznki. R.K. und Kimiiril. T. (1985) 1. Biol. Chcm. 260. 13355-13359, [61 Chumbers. 1.. Framplon. J.. Goldlhrb. P.. Allhru, N.. McBuin. W. und Hurrison, P.R, (1986) EMBO J. 5. 1211-1227. t71 Rcddy, A.P.. Hsu. B.L.. Rcddy. P.S. Li. N.-q.. Thyugurrju. K.. Rcddy, C.C.. Turn, M.F. and Tu. C.-P.D. (1988) Nucleic Acids Rcs. 16. 5557-5568. PI Yoshimuru, S.. Takekoshi. S., Wanatabc. K. und Fujii- Kuriyumu. Y, (1988) Biochcm. Bioplrys, Rcs. Commun. 154. IOXI OS.?. PI Ho, Y,-S., Howard, A,J. and Cmpo. J.D. (19Y8) Nucleic Acids Rcs. 16. 5207. PUI Mullenbach. G.T.. Tubriz. A., Irvine, B.D.. Bull. G.I.. Truincr. J.A. and Hallewell. R.A. (1988) Prolein Eng. 2. 239-246. II 11Multcnbach. G.T.. Tabriz, A.. Irvine. B.D.. Bell, G,I. iind Hallewcll, R.A, (1987) Nucleic Acids Rcs. IS. 5484. [1A Sukenaga, Y.. Ishida, K.. Takcda. T. and Takagi. K. (1987) Nucleic Acids Rcs. 15, 7 178. [l31 Ishidu, K., Morino, T., Tdkagi, K. snd Sukenagtl. Y. (1987) Nucleic Acids Rcs. IS, 10051. [l41 Hawkcs, W.C, und Tappcl. A.L. (1983) Biochim. Biophys. Acm 739. X5-234. 1151Hawkcs, W.C,, Lyons, D.E. and Tuppel, A.L. (1982) Biochim. Biophys. Acts 699. 183-191. 1161Sunde, R.A. and Evenson, J.K. (1987) J. Biol. Chcm. 261, 933937. 1171Lee. B.J., Worlund. P.J.. Davis. J.N.. Stadtmun. T.C. 2nd Hulfield, D,L. ( 1989).I. Biol. Chcm. 264, 972rC9727. [l81 Berry. M-J.. Banu. L.. Chen. Y.. Mundcl. S.J., Kicller. J.D..

at

which homologous

Hurncy. J.W. nnd Lurscn. P.R. (1991) Nature 353.273-276. [I‘)] Smith. J.L. (1899) J. Dhysiol. 24. 19-35. [20] Crupo. J.D.. Burry. B-E.. Foscuc. HA. und Shclburnc, J. (1980) Am. Rev. Rcspir. Dis. 122. 123-143. [$I] Kimbull. R-E.. Rcddy. K,. Picrcc, T.H.. Schwartz. L.W.. Mus~111. M.G. iind Cross. C.E. (1976) Am. 1. Physiol. 130. 14251431. 1221Bcnton. W.D. und Diivis. W. (!977) Scicacc 196. 180-182. [23] Sumbrook. J.. Frhsch. E.F. and Manialis, T. (1989) Molecular Cloning: A Luboralory Manual, Cold Spring Harbor Liiboratory. Cold Spring Harbor. NY. [24] Lcvinson. A,. Silver. D. und Seed. B. (1985) J. Mol. Appl. Gcncl. 2. 507-S 17. [25] Sangcr. F.. Nicklcn. S. and Coulson. A.R. (1977) Proc. NatI. Acad. Sci. USA. 74. 5463-5467. [36] Solrlhcrn. E. (1975) J.Mol. Biol. 98. 503-517. [27] Thomas. P.S. (19X0) Proc. Null. Acad. Sci. USA 77. 5201-5205. [‘I(] Grconc. J.M. and Wruhl. K. (1988) in: Current Protocols in MoIcculur Biology, vol. I. (Ausubcl. F.M.. Brent. R.. Kingston. R.E.. Moore. D.D.. Scidmun. J.G.. Smim. J.A. imd Slruhl. K. cds.) pp. 4.6.1-4.6.13. New York. [?9] Kingston. R.E. (1987) in: Currcnt Protocols in Molecular Biology. vol. I. (Ausubcl. F.M.. Brent. R.. Kingston. RX.. Mom. D.D.. Scidman. J.G.. Stnhh. J.A. and Slruhl. K. cds.) pp. 4X14,.8.3. New Yor!:. [30] %iiki. R.K., Scharl’. S.. Faloona. F.. Mullis. K.B.. Horn. G.T.. Erlich. HA, and Arnhcim. N. (1985) Scirncc 130. 1350-1354. [3l] Briggs, M.R.. KadonuS;i. J.T.. Bell. S.P. and Tjhn. R. (1986) Scicncc 234 47-51. [31] Mitchell. P.J.. Wang. C. und Tjiun. R. (1987) Cell 50. 847-X61. [33] Imaguw M.. Chiu. R. and Knrin. M. (1987) Cell 51. 251-160.