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Gone,111 (1992) 229-233© 1992 Elsevier Science Publishers B.V. All fights reserved. 0378-1119/92/ 05.00

GE NE 06296

229

P r i m a r y s t r u c t u r e o f th e equor ea victoria r e e n - f l u o r e s c e n t p r o t e i n

(Bioluminescence; Cnidaria; aequorin; energy transfer; chro m oph ore; cloning)

Douglas C. Prasher a Virginia K. Eckenrode b Wil l iam W . W ard c Frank G. Prendergast d and M ilton J . Cormier b

Biology Department, Woo ds Ho le Oceanographic Institution, Wood s Hole, MA 02543 U. S.A .); b Biochemistry Department, UniGeorgia, Athen s, G A 30602 U.S .A.) Tel. 404)54 2-I747; c Department of Biochemistry an d Microbiology. Cook College. Rutgers UNew Brunswick, N J 08903 U.S .A.) Tel. 908)932-9562; and u Depa rtment of Biochemistry and Molecular Biology. M ayo Foundatioester. M N 55905 U.S .A.) Tel. 507)284-2065

Received by S.R. K ushner: 21 March 1991Revised/Accepted: 13 September/27 September 1991Received at publishers: 26 Nov ember 1991

S U M M R Y

M any cnidarian s utilize green-fluorescent proteins (G FP s) a s energy-Wansfer acc epte rs in bioluminescence. G FP s fluoresc e in vivo up on receiving energy from either a luciferase-oxyluciferin excited-state complex or a Ca 2 + -activated phtoprotein. Th ese highly fluorescent proteins are unique du e to th e chemical n ature of their chromopho~ e, which is com prisof modified am ino acid (aa) residues within the polypeptide. This report describes the cloning and sequencing of both cD Nand genomic clones o f G FP from the cnidarian,Aequorea victoria.The gfplOcD N A en codes a 238-aa-residue polypeptide

with a calculated Mr of 26 888. Com parison ofA. victoriaG F P genornic clones sho ws three different restriction enzymepatterns which suggests that at least three different genes are present in theA. victoriapopulat ion at Friday Harbor,Washington. The gfp gene encoded b y the AGF P2 genomic clone is comprised o f at least three exerts spread over 2.6 kThe n ucleotide sequ ences of the cD N A and the gene will aid in the elucidation of structure-function relationships in thunique c lass o f proteins.

I N T R O D U C T I O N

Lum inescenc e is com m on in a variety of marine inver-tebrates. M any cnidarians and probab ly all c tenophoresemit light when mechanically disturbed. Proteins respon-sible for bioluminescence from sev eral species of these two

Correspondence o:Dr. D .C. Prasher, Redfield Bldg., Woo ds Hole Oceano-graphic Institution, W oods H ole, MA 02543 (U .S.A.)Tel. (508)457-2000, ext. 23 i I; Fax (508)457-2195.

Abbr~.wiations:A., Aequorea; aa,amin o acid(s); bp, base pair(s); GF P,grecn-fluorescent protein; gfp, DN A or R N A encoding GF P; kb, kilo-base(s) or 1000bp; nt, nucleofide(s), oligo, oligodenxyribonucleofide;OR Fo open reading fram e(s).

phyla have been characterized . Light from luminescent cni-

daria is primarily green whereas light emitted from cteno-phore s is blue. Th e green light of cnidaria is du e to thepresenc e of a class o f proteins called green-fluorescent pro-reins (G FP s). The y are highly fluorescent and are activatedin vivo by an energy transfe r proc ess via a luciferase o r aCa2+-act ivated photoprotein, both o f which produ ce en-ergy during the ox idatio n of coelenterate-type luciferin. Inthe cnidarianAequorea thephotoprotein aequorin excitesthe G FP by an unkn own mechar6sm to release green l ight .Previous studies suggesting thatAequoreaGFP is s t imu-lated via a radiationless mechanism (M ofise etal. , 1974)

have been questioned (Ward, 1979). The G F P fromRe-nilla another cnidarian, on the other hand, clearly receivesenergy from theRenillaluciferase-oxyluciferin excited state

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complex by a radiationless energy transfe r mechan ism(Ward and Cormier, 1976).

The GFPs most thoroughly studied have been isolatedfrom Aequorea and Renilla(Ward, 1979). TheAequoreaGF P has been reported to be a 30-kDa m onomer (Pren-dergast and M ann, 1978) whereas theRenillaGFP i s a54-kDa homodim er (Ward and Cormier, 1979). The twoproteins have diffelent absorption spectra but identicalemission spectra (~o ~ = ~99 nm). Up on denaturat ion thetwo GFPs have the same absorption spectra. Ward et ai .(1980) have predicted that bo thAequorea and RenillaG F P scontain chromophores having the same structure but thatthe different absorption spectra are explained by differentapoprotein environments.

Biochem ical properties of theAequoreaGF P show i t t ohave unique structural properties. The fluorescent chro-mo phore is stable to a variety of harsh conditions includ-ing heat, extreme pH, and chemical denaturants. Fluores-

cence is lost, for example, to base or acid treatment oraddition of guanidine hydrochloride, bu t upon neutraliza-tion of the pH or removal o f the d enaturan t, fluorescencereturns with an identical emission spectrum (Bokman andW ard, 1981; W ard and B okman, 1982). The chromop horestructure is very different from those of the phycobilipro-teins which a re also highly fluorescent. The chrom oph orein the GF Ps is covalently bou nd and is formed by modi-fication of certain a a residues within the polypeptide. T hechemical structure o f theAequoreaGFP ch romophore(Fig. 1), first characterized by Shim om ura (1979), has bee n

thoroughly re-examined (W ard et al., 1989 : W.W .W., u n-published) and is shown here (Fig. 1) in its revised form.In this study, theAequoreaGFP gene and i t s eDNA havebeen isolated and characterized in pur suit of elucidating themechanism o f energy transfer betwe en aequorin and G F Pas well as addressing evolutionary relationships in coelen-terate bioluminescence.

o©c _ o

N.~ /N OH C NH CH C NH CH COOHI

. 2 . ? . c . . . c . c . s c . 3 tCH2

CH2 CH2OH [C=OI

NH

Fig. 1. The chemical structure of the chromophore inequoreaG F PW.W,W., unpublished). The cyclized chromophore is formed from the

trimer Ser-dehydroTyr-Gly within the polypeptide by an unknown mech-anism.

E X P E R IM E N TA L A N D D I S C U S S I O N

(a) Construction of cDNA librariesA n A. victoriaeDNA library, constructed in pBR322

(Prasher et ai. , 1985). was screened for the presence of ag/ p eD NA using tw o ol igo mixtures who se sequences w erebased on the aa sequences derived from GFP-derivedCNBr fragments. The oligos contained the following ntsequences: A: 5 -AA~AAA TCATG ~TGTCT TCAT ~20-mer

A A A ~T Cwith 32 redundancies) , B: 5 -TTGTAGTTGTA. f CA T(17-mar with 16 red un d~c ies) . The hybridizat ion of the32p-labeled mixtures A a nd B to replicate filters containingthis l ibrary were performed according to the method ofW oo d et ai. (1985) utilizing tetramethylam mo nium ch lorideduring the washing steps. T he temp eratures us ed duringt h ewashing steps for mixtures A and B were 55°C and 50°C,respectively.

A single ~fp eD N A wa s isolated from the library by this

method. This c lone , pGFPI , conta ined aPstI insert of511 bp having an O R F encoding 168a a. T he deducedtranslation of the nt sequ ence indicated theg/pl c D N Alacked both the 5 - and 3 -sequences o f the coding region.However, the sequence FSYGV Q wi thin the deducedtranslat ion permit ted the chromo phore structure to be de-ciphered (W .W.W ., unpublished). U po n rescreeningt h elibrary withgfpl eD NA , no addit ional cD NA s were found.

A second A.victoriaeD NA l ibrary was cons t ruc ted (Gu-bier and Ho ffman , 19 83) in Agtl0 (Hu ynh et al. , 1985). ThePstl insert fromgfpl eDNA was used as a hybridizat ion

prob e against the entire Agtl0 library of 1.4 × 106 recom -binant phage. N o g/p-related recom binants w ere identifiedupon screening the primary library. The phage remainingon the plates were extracted from the top agar and used asan am plified library (M aniatis et ai., 1982). U po n screen-ing this prepara tion of the library, four recomb inants hy-bridized to the gfpl eD N A following their purification. Thefour eD NA clones were designated gG FP 10, 11, 12, and13. All four recomb inants were shown to co ntain an insertof 1 kb u pon digestion withEcoRI.

(b) C h a r a c t e r i z a t i o n o f t h e g/plO e D N AThe entire EcoRl inser t o f ~GF P10 was sequenced

(Fig. 2). Limited nt se quen ces obtained from AG FP 11 and12 were identical with that from AGFP10 suggesting thatthey were siblings and, h ence, w ere not seq uen ced further.Even though the entire coding region appears to b e present(see below), three features of the eD N A insert of AG FPI 0suggest it is not qu ite full-length. First, the eD N A is 965 ntwhere the gfp m R N A is 1.05 kb in length as determined byNorthern analysis (Fig. 3) . Second, the 5 -unt ransl atedregion is very short. T hird, n o poly(A ) track is observ ed in

t h e gfplOeD NA sequence (F ig . 2) despi te the presence ofthe ~fp m RN A in only the poly(A)+ RN A f rac t ion ofA.vic

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T A ~ G ~ T ~ T ~ C ~ G ~ T ~ ~ C ~ A C T ~ G ~ C ~ A ~ C ~ G ~ T T A ~ T ~ T 8 5M S K G E E L F T G V V P I L V E L D G 2 0

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

D V N G H K F S V S G E ~ E G D A T Y G K L T L K

~ C A C T A C T ~ A ~ C T A C C T G ~ C C A ~ C C ~ R C A ( ? ~ G ~ A C T A C T ~ T T A T ~ T G ~ T T T ~C T T G K L P V P W P T L V T T F S Y G V Q C F S

~ A G A T C & T A ~ C A G C A T ~ T ~ G ~ T ~ A ~ C ~ T T A T G T A ~ G ~ g ~ A C T A T & ~

P D H M K Q H D F P X S A M P E G Y V Q E R T I F

~ T ~ C T & C ~ A ~ T ~ T ~ G ~ G ~ T ~ T A C C ~ G ~ T A ~ A ~ A ~D D G N Y K T R A E V K F E G D T L V H R I E L K

T T T A T T 1 6 6

F I 4 7

A G A TA C 2 4 7R Y 74

F K 1 0 1

2

3

D F K E D G N I L G H K L E Y N Y N S H N V Y Z M

UK Q K N G Z K V N Y K Z R H N / E D G S V Q L A D

~ A~ 4 9 k bG I 1 2 8

6 5 6G C A G A C 4 9 0

^ l s s 4 3 6

C AT TAT S ? lH I f 82

~ C ~ T C T C C A A T T ~ T ~ C C T G ~ C T T ~ ~ C ~ C C T T A C C ~ C ~ C ~ T ~ C C ~ G S 2 ~ . ~Q Q N T P I G D G P V L L P D N H Y L S T Q G A L 8 K 2 9 2 0 3

G A T C ~ C G ~ G ~ G A C C A C A ~ G ~ C T T C ~ G G ~ G T A A C A ~ T ~ T ~ A ~ A ~ T ~ A ~ T ~ C T K ? S SD P H E K R D f l M V L L E P V T A A G Z T H G H D E L 2 3 6

TA C A A A T ~ AT G TC C A G AC T T C C A AT T G A C A C TA R R ,G T G T C C G A R ~ R AT TA C TA R R AT C T C G G G T T C C T G G T T R R AT T C R G G C TG A G A TAT TAT T T T8 3 2¥ K 2 3 8

ATAT T TATA G AT T C AT TA A A AT T G TAT G A A T R AT T TAT T G AT G T TAT T ~ TA ~ q G G T TAT T T T C T TAT TA A A C AC ~ CTA e T T C ~ G T G TA T T C T TAAT T C 9 3 2 0 5 6

TATAT TA AT T A C A AT T T G AT T T ~ q C T T G C T C A A A 9 6 2

Fig. 2. Fig. 3.

Fig. 2. Nugleotide sequence oft h gfplOcD NA and the deduced aa sequence. Below the f irst n t o f each cod on is the single-le tter designation for taa . The h or izontal l ines under l ine those aa sequenced direct ly f rom nat ive GF P. The down ward arrows indicate the posi t ions of in t rons when co ralto the n t s equence o f thegfp2gene. Arrowhead : s tar t codon ; period: s top c odon . DIqA fragments f rom both eDN A and genomic c lones were subek ninto Ml 3m pl 8 and M 13m pl9 (Yan isch.Perron et a l ., 1985), and unidirect ional dele t ions were prepared using the method o f Dale e t a l. (1985) . Sequewas performed using e i ther the Klanow fragm ent or an a l tered T7 DH A polymerase (Sequenase Ver 2 .0 , Un i ted S tates Biochemical Corp.) in the didchain terminat ion method (Sanger e t a l ., 1977) . Both D NA strands of the sequences descr ibed in this report here have been sequenced. The G enaccession No. for theg[plOsequence is M62653.

Fig. 3 , Northe rn analysis of the A,victoria gfpmR NA . Th e poly(A) ÷ mR NA (lane 1) was denatured usingg l y o x a lprior to electrophoresis, as describby Thom as (1983). Eiectrophoresis was pe rformed for 3 h in a 1% agarose gel (pret reated with 10 mM sodium iodoac©tate) equi l ibrated in 10 mMdium p hos pha te pH 7.0 buffer. Overnigh t trans fer of the nucleic acid s to nitrocellulose was facilitated w ith 20 x SSC . Hybridization of riP-labele deDN A to the membrane-bound nuc le ic ac ids was a t 42°C fo r 28 h in 5 x SSC/5 x Den hard t 's /20 mM Na .phosph a tc pH 6 .8 /100 g pe r ml o f denaherr ing sperm D NA /10% polyethyleneglycol /50~ formamide.HindIll digestedA DN A, r iP- labeled, and t reated in paral le l wi th the R NA , wa s usedmolecular weight standards (lane 2).

toria RNA (data not shown). A typical polyadenylat ionsignal is located at nt 861-865 (Fig. 2).

The n t sequence of theg/ plOeDNA con ta in s an ORFencoding a 238-a a protein having a ca lculate d Mr of 26 888.

This compares favorably wi th 30 kD a for nat ive G FP asdetermined by denaturing electrophoresis (Prendergastand M ann, 1978). The dedu ced translat ion contains aasequences of numerous peptides isolated from native G FP(underlined in Fig. 2). W hen com par ed to thegfplOc D N Asequence (Fig. 2), thegfpl cDNA was de termined to en-code aa residues 28-195. Oligo mixture A is complimen-tary to the codons encoding aa 78-84 and mixture B iscomplimentary to the codon s encoding aa 141-146 (Fig.2 ) .The trimer Ser-Tyr-Gly, modified in the native protein toform the chromophore (W.W.W., unpublished), is located

a t aa 65-6 7 . The chrom ophore cons is t s of an imidazolonering formed b y the residues Se r-dehydroTyr-Gly within the

polypep tide (Fig. 1). Locate d 8 aa u pstream of this chrmope ptide is GF P s only Trp. The inabil ity to detect tfluorescence from this Trp makes it unusual (W.W.¢unpublished). Perh aps energy-transfer occu rs betw een

and the chromophore in the native protein preventing tTrp fluorescence (3 20 -35 0 nm). Th e Trp is flanked by s~eral Pro residues (Pro-Val-Pro-Trp-Pro). Th e significanof this pentapeptide is not unde rstood bu t a search of tprotein datab ases ( PIR ver 25; Sw iss-Prot ver 14) sho,i t to be present on ly in cytochrome P-450 proteins.

( c ) Isolation andc h a r a c t e r i z a t io n o f g J pgenomic cloneTh e gfpl eD N A wa s al~o used to ~olat~, genoraic clo~

prior to the availability of thegfplOe D N A . A nA. victogenomic library was constructed in ~2001 (Karn el;

1984) essentially as d escr ibed (M aniatis et al., 1982). Elirecombinant phages hybridizing to thegfpl cDNA wq

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232

gep9

~ t k b

RT I

B B Ri i

R R R Rg f p 3

R R R R: 2 i i

Im

II UI IV

Fig. 4. Restriction enzyme map s of threeAequorea g/ pgenes. (A) Th emap s of three representative genomie clones are compared. T he doub lelines represent those DNA fragments which hybridize toffpl e D N A .Sou thern-blot analysis indicated three other genomic clones, 2G FP I, 4and 8 (not shown ) lack the 3 ' end o f the gone. (B) The e xon/ intron ar-rangement of the gone encoded by 2G FP 2 was determined by compar-ing the nt sequences of the 5-kbE¢JRI-BamHIand the overlapping 1.8-kbHind l l l f r agments o f 2GF P2 and theEcoRli n ser t o f 2GFP I0 eDNA.The exons are represented by the blackened boxes, I , I and 1I . TheGenBank accession No. for theg[p2sequence is M62653.

purified from he genomic D N A library. B ased on restric-tion enzyme and Southern-blot analyses, they represent sixdifferent isolates having at least three different restrictionm aps (Fig. 4). W hen D N A fragments from the 5'- andY-ends of the g f p l eDNA were used as hybridizationprobes, all of the genomic clones w ere found likely to con -tain the 5 ' -en d of the gone, but onlyf l ip2 3 and 9 alsocontained the Y end. The three type s of genomi¢ clones areconsistent with the presence of multiple G F P isoforms iso-lated from A.vfctorfa(A. Roth, M. Cutler and W.W.W.,unpublished). Since theA victoriagenomie DNA used forthe genomic library was isolated from a large num ber o f

TABLE 1

Sequence differences in the coding regions o f thegfpclones

5 S p X £ ¢o S i l : o 3 S p X i c o S ~ t e

]• • ) F I N T Tt la ~ l G 't :~ , l) , . . . i n k : o n . e c c e c ~ r ' a ~ lG

• . . £n t ro n I . . . ATTCTTATATTTTTACAG Gj.98

4 1 2 c A G T A A G T G . .. i n t r o n I I . .. G A T T T T G T C T C T T T T A G A I T 9 4 7

1 2 3 8 A T T I T A T G T . , . i n t r o n I I I . . ~ A T G A T T C A A C T T T T C A G I A 23 0 8

Fig. 5 . Al ignment of the nt sequences ing] p2at the spl ice junct ions . Theintron sequences were identified b y com paring the nt seq uence sofffp2and theg/ plOeDNA (Fig. 2) . The consensus sequence is taken fromSen apath y et al. (1990).

j e ll y fi s h ( c o ll e c t e d a t F r i d a y H a r b o r, W a s h i n g t o n ) , t h e t h r e eg / p g e n e s a r e r e p r e s e n t a ti v e o f t h eA e q u o r e ap o p u l a t i o n a so p p o s e d t o i n d i v i d u a l j e ll y f is h .

T h e E c o R I - B a m H la n d a n o v e r l a p p i n g H i n d l l l f r a g -m e n t s i n t h e g e n o m i c c lo n e 2 G F P 2 ( F i g . 4 ) w e r e s e q u e n c e da n d c o m p a r e d t o t h a t o f t h eg / p l O D N A t o e x a m in e th es tr u c tu r e o f t h e g e n e . T h e g / p g o n e e n c o d e d b y ~ . G F P 2c o n t a i n s a t le a s t t h r ee e x o n s s p r e ad o v e r 2 . 6 k b o f D N A( F i g . 4 ) . T h e s e e x o n s , d e s i g n a t e d I I , II I , a n d I V, e n c o d e 6 9 ,9 8 , a n d 7 1 a a , r e s p e c t iv e l y. P r e s u m a b l y, a f o u r t h e x o n i sl o c a t ed u p s t r e a m f r o m t h e g e n o m e s i n c e t h e 1 5 n t a t t h e 5e n d o f t h eg [ p l O D N A s e q u e n c e c a n n o t b e a l ig n e d to t h e5 r e g io n o f th e D N A s e q u e n c e d e ri v ed f r o m th eg f p 2gone .T h e p o s i t i o n s o f th e i n t r o n s w i t h r e s p e c t t o t h ee D N As e q u e n c e a r e i n d i c a t e d ( F i g . 2 ) . T h e a a r e s i d u e s i n v o l v e d i nt h e c h r o m o p h o r e a r e e n c o d e d a t t h e 3 e n d o f e x o n I I. T h en t s e q u e n c e s o f t h e g f p m R N A s p l ic e j u n c t i o n s a g re e re a -s o n a b l y w e l l w i t h c o n s e n s u s s e q u e n c e s ( F i g . 5 ).

T h e g / p l Oe D N A I s n o t e n c od e d b y th eg/ p2g o n e s i n c ethe re a re seve ra l n t d i f f e re~ tces be tween the i r s equences .T h e n t d i f f e r e n c e s w i t h i n t h e p r o t e i n - c o d i n g r e g i o n s a r es u m m a r i z e d i n Ta b l e I A . F o u r o f th e 1 2 s i n g l e n t d if fe r-e n c e s r e s u l t i n c o n s e r v a t i v e a a r e p l a c e m e n t s a t p o s i t i o n s1 0 0 , 1 0 8 , 14 1 a n d 2 1 9 ( Ta b l e I B ) . T h e a a r e s i d u e s e n c o d e da t t h e s e f o u r p o s i t i o n s a r e c o n s i s t e n t w i t h t h e a a s e q u e n c e s

Nucleotide differences withrespect to thegfp2gone a

gfplOe D N Agfp] e D N A

12 (8 silent)2 (2 s i lent)

Am ino acid differences b

aa posi t ion ~fp2gene ~plOe D N A ~ p l e D N A

100 Tyr Phe Tyr1 0 8 S c r T h r S e r141 M et Leu M e t219 l i e Va l

a Toud num ber oty~erved upon com parison of the nt sequence~ of the OR Fs in the M'P cD NA s w ith the homo logous sequences in thegfp2gcne,Obse rved upon compar i son o f the t rans l a tions o f the ORFs o f bo th eD NA s and the c~;ons o f the~ p2gone. The aa numbering is the same as that usedr ig l

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observed in GFP-derived peptides w hich show ed a Tyr atposition 100 , a M et at position 14 1, but a Thr at position108. Eight additional nt differences occu r with t~hef p 2genein the Y-non-translated region of the g / p 1 0eDNA (da tanot shown). I t i s not known whether the gf 'pl eD N Arepresents an allele ofg f p 2or another ~p gene.

Th ese results w ill enable us tO ¢o,nstruct an exp ressionvector for the preparation of he~,-fluorescent apoGFP.Since no information is y et a vailable r,,.garding the biosyn-thesis of the chromophore, a recombinant form of thisprotein will be a valuable reagent with wh ich to examine thebiochemistry ofc hrom oph ore formation in this unique classof proteins and the m echanism of energy transfer betweenaequorin and GFP.

A C K N O W L E D G E M E N T S

We want to extend our thanks to Bonnie Woodward,Darlene Bianca, and R ichard McC ann for their excellenttechnical assistance. Supported in part by a Mellon A wardfrom the Wo ods Ho le Oceanographic Institution (27/50.44)and a grant from the American Cancer Society (N P640 ) toD.C .P. A paper of the journal series N ew Jersey Agricul-tural Experiment Station. T his wor k wa s performed as partof NJA ES Project No . 01102. A special thanks goes to Dr.A.O.D. Willows, Director, Friday Harbor Laboratories,fur use o f laboratory facilities.

R E F E R E N C E S

Bokman, S.H. and Ward, W.W.: Renmuration of/lequorea green-fluorescent protein. Biochem. Biophys. Res. Commun. 101 (1981)1372-1380,

Dale, R.M.K ., McClure, B.A. and H ouchins, J.P.: A rapid single-strandedcloning strategy for producing a sequential series of overlapping clonesfor use in DNA sequencing: application to sequencing the corn mi-tochondrial 18S rD NA . Plasmid 13 09 85 ) 31-40 .

Gubler, U. and Hoffman, B.J.: A simple and very efficien method forgenerating eD N A libraries. Gen e 25 (1983) 263-269.

Huynh, T.V., Young, R.A. and D avis, R.W.: Constructing and screeningeDNA l ibrar ies in ggt l0 and ) .gt l l . In: Glover, D.M. (Ed.) , DNACloning: A Practical Approach, Vol. i. IRL Press, Oxford, 1985,

pp. 49-78.

33

Karn, J. , M atthes, H .W.D ., Gait, M .J. and Brenner, S.: A new selectivephage cloning vector, ~.2001, with sites forXbaI BamHl Hindl l l ,EcoRl Sst l and Xhol .Ge ne 32 (1984) 217-224.

Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. ALaboratory Manual. Cold Spring Harbor Laboratory, Cold SpringHarbor, NY, 1982.

Morise J.G. Shimomura, O., Johnson, F.H. and Winant, J.: Intermo-lecular energy transfer in th e bioluminescent systemof Aequorea.Bio-

chemistry 13 (1974) 2656-2662.Prasher, D., McCann, R.O. and Cormier, M.J.: Cloning and expression

of the eD NA coding for aequorin, a bioluminescent calcium-activatedprotein. Biochem. Biophys. R es. Commu n. 126 (1985) 1259-126 8.

Prendergast, F.G . and M ann, K .G.: Chemical and physical properties ofaequorin and th e green-fluorescent protein isolated fromAequoreaforskalea.Biochemistry 17 (1978) 3448-3453.

Sanger, F., Nicklen, S. and Coulson, A.R.: D NA sequencing with chain-terminating inhibitors. Prec. Natl. Acad. Sci. USA 74 (1977) 5463-5467.

Senapathy, P., Shapiro, M .R and Harris, N.L.: Splice junctions, branchpoint site, and exerts: sequence statistics, identification, and applica-tions to genome project. Methods Enzymol. 183 (1990) 252-278.

Shimomura, O.: Structure of the chromophore ofAequoreagreen fluo-rescent protein. F EB S Lea . 104 (1979) 220-222.

Thomas, P.S.: Hybridization ofd enatur ed R NA transferred or dotted tonitrocellulose paper. Methods Enzym ol. lfl0B d,1983) 255-266.

Ward, W .W.: Energy transfer processes in bioluminescence. Photochem .Photobiol. Rev. 4 (1979) 1-57.

Ward, W .W. and Bokman, S.H.: Reversible denaturation ofAequoreagreen-fluorescent protein: physical separation and characterization ofthe renatured protein. Biochemistry 21 (1982) 4535-4550.

Ward, W .W. and Corm ier, M J. : In vitro energy transfer inRenillabio-luminescence. J. Phys. Chem. 80 (1976) 2289-2291.

W ard, W .W. and Cormier, M .L: A n energy transfer protein in coelenter-ate bioluminescence. J. Biol. Chem. 254 (1979) 781-788.

W ard, W.W., Cody, C.W., Hart, R.C. and Cormier, MJ .: Spectropho-

tometric identity of the energy-transfer chromop bores inRenilla andAequoreagreen-fluorescent proteins. Photochem . P hotohiol. 31 (1980)611-615.

Ward, W.W., Cody, C.W., Prasher, D.C. and Prendergast, F.G.: Se-quence of the chemical structure o f the hexapeptide chromophore ofA equoreagreen-fluorescent protein. Phu tochem. Photobiol. 49 (1989)62S.

Wood, W.I., Gitschier, J. , Lasky, L.A. and Lawn, R.M.: Basecomposition-independent hybridization in tctramethylammoniumchloride: a method for oligonucleotide screening of highly complexgene libraries. Prnc. Natl. Acad. Sci. USA 82 (1985) 1585-1588.

Yanisch-Perron, C ., Vieira, J. and Messing, J.: Im proved M i3 phagecloning vectors and hos t strains: nucleotide sequences o f the

M I3m pl8 and pU CI 9 vectors . Gene 33 (1985) 103-119.