Comunicación de los funcionarios públicos con los usuarios: modalidades

Shan ZHONG.- Nigel K. SPURR,t John 0. HAYES* and C. Roland WOLF'S

ICAF Molecular Pharmacology Group. Hugh Robson Building. George Square. Edinburgh EH8 9XD. Scotland. tiC R F Human Genetic Resources Laboratory. Clare Hall Laboratories. Blanche Lane. Potters Bar. Herts. EN6 3LD. and tU niversily Department of Clinical Biochemistry. Royal Infirmary. Edinburgh EH3 9YW , Scotland. U.K.

T he M u -C la s s g lu ta th io n e 5 -tra n sfcra se s (G S T s) a rc subject to m arke d in te r- in d iv id u a l v a ria tio n in m a n , o w in g to the fa ct th a t 4 0 - 5 0 “ ., o f the p o p u la tio n fa il to express M I su b u n its. M u -C la ss G S T fro m tw o ly m p h o b la s to id ce ll lin e s (expressing M I su b u nits and the o th e r ' n u lle d ' fo r M l ) have been stu d ie d . B o th cell lines were fo u n d to express a M u -C la s s G S T th a t has n o t been described p re v io u s ly . T h e c D N A e n c o d in g th is n o ve l transferase, designated 'G S T M 4 ', has been iso la te d and the enzym e show n to be co m p rise d o f 218 a m in o acids (in c lu d in g the in itia to r m e th io n in e residue) w ith an A /, o f a p p ro x . 25.5 k D a . M o le c u la r c lo n in g d e m o n stra te d th a t the ly m p h o b la s to id cell lin e w hich

expressed G S T M 1 possessed th e b a lle lic v a ria n t (i.e. th a t w ith an asparagine residue a t p o s itio n 173). T h e genes fo r G S T M 4 and G S T M lb have been clo n e d a n d fo u n d to c o n ta in seven in tro n s and eig h t exons. T h e c o d in g re g io n o f the G S T M 4 gene, in c lu d in g the seven in tro n s . encompasses 5.0 kb . whereas the same region o f G S T M lb is 5.5 k b ; the difTercnce in the size o f the tw o genes is due to the le n g th o f in tr o n 7. D N A sequencing a llo w e d a G S T M 4 -g e n c -s p e c ific o lig o -p r im c r to be designed w h ich has been u tiliz e d in a PC R -bascd assay to de term in e th a t the G S T M 4 gene is located on c h ro m o so m e I

IN TR O D U C T IO N

The g lu ta th io n e S -transferascs (G S T ), th ro u g h th e ir a b ility to catalyse the c o n ju g a tio n o f G S H w ith e le c tro p h ilic x c n o b io tic s , p ro v id e p ro te c tio n ag a in st carcino g e n s, e n v iro n m e n ta l p o llu ta n ts and ch e m o th e ra p e u tic d ru g s (C hasseaud. 1979; Hayes et al.. 1990). Such co m p o u n d s in c lu d e insecticides, herbicides, a n titu m o u r agents and e p o x id e -c o n ta in in g m e ta b o lite s o f p o ly c y c lic a ro m a tic h y d ro c a rb o n s (H a yes and W o lf, 1988).

M a m m a lia n G S T c o m p ris e five fa m ilie s. F o u r o f these are c y to s o lic and have been desig n ate d A lp h a . "M u . Pi and T he ta , whereas the fifth is m e m b ra n e -b o u n d and is s im p ly referred to as m ic ro s o m a l G S T (M a n n e rv ik et a l.. 1992). W ith in each gene fa m ily the G S T share at least 55 “ o a m in o a cid sequence id e n tity , b u t between fa m ilie s id e n tity is less th a n 25 Present evidence suggests th a t the A lp h a a n d M u fa m ilie s are the m ost co m p le x, b o th p o ssib ly c o m p ris in g a t least six genes, whereas the Pi and m ic ro s o m a l fa m ilie s co m p ris e o n ly one o r tw o genes (B o a rd et al.. 1990). F u rth e r c o m p le x ity in the n u m b e r o f isoenzym es arises because G S T s are d im e ric and because su b u n its th a t are m embers o f the same fa m ily can h e te ro d im e riz e (H a yes et a l., 1981 : S to c k m a n et al.. 1985). A large n u m b e r o f h e te ro d im e rs are fo u n d in m ost tissues (H u ssey et a l., 1991).

In m a n , the G S T s w h ic h c o m p ris e the M u e v o lu tio n a ry class have a ttra c te d p a rtic u la r in te re st because they a rc subject to m a rke d in te r-in d iv id u a l d ifferences (W a rh o lm et a l., 1980; B oard, 1981; S tra n ge et a l., 1984; H ussey et a l., 1986). Z y m o g ra m a n a lysis has sh o w n th a t a p p ro x . 4 5 % o f E u ro p ea n s fa il to express a transferase encoded a t the G S T M l locus (B o a rd , 1981 ; L a isn e y ct al., 1984; S tra n g e et a l., 1984). T h e n u ll p h e n o typ e is due to gene d e le tio n (S eidegârd et a l., 1988). A m o n g those in d iv id u a ls th a t express G S T M l , tw o c o m m o n a lle lic va rian ts. G S T M M a and G S T M 1 * b , exist. These alleles encode G S T M la - la and G S T M l b - lb , p re v io u s ly celled // and i/r respectively.

w h ic h are fu n c tio n a lly id e n tic a l (W id e rste n et a l., 1991), b ut d iffe r in a single a m in o acid ; G S T // c o n ta in s lysine at p o s itio n no. l7 2 (D e J o n g e ta l.. 1988), whereas G S T ^ c o n ta in s asparagine a t th is p o s itio n (Seidegârd et a l., 1988).

Since M u -C la s s G S T s are p a rtic u la r ly effective at d e a c tiv a tin g m u ta g e n ic and c a rcin o g e n ic ep o xid es (e.g. styrene oxide, tran.s-

s tilb e n c o xid e , I-n itro p y re n c o xid e , b cn zo [a ]p yre n e -7 ,8 -d io l 9,10- c p o x id e ). it is reasonable to p o stu la te th a t the p o ly m o rp h ic e xpression o f th is fa m ily results in a s ig n ific a n t re d u c tio n in the d e to x ific a tio n c a p a c ity o f n u lle d in d iv id u a ls . Seidegârd et al. (1986, 1990), w h o m o n ito re d expression at the G S T M l locus by m ea su rin g leucocyte transferase a c tiv ity to w a rd s rrr/m -s tilb c n e o x id e , re p o rte d th a t fa ilu re to express G S T M I is associated w ith an increased s u s c e p tib ility to lu n g cancer in cig a re tte sm okers. In a subsequent s tu d y we have fo u n d a sm all p o sitive c o rre la tio n between sq u am ous c a rc in o m a o f the lu n g and the n u ll g enotype ( Z h o n g et al., 1991). S u rp ris in g ly , a negative c o rre la tio n between the n u ll g e n otyp e and a d e n o c a rc in o m a o f the lu n g was also observed. S trange et al. (1991), using a starch-gel z ym o g ra m m e th o d , have fo u n d an increased fre q u e n cy o f the n u ll p h e n o typ e in p a tie n ts w ith a d e n o ca rcin o m a o f the stom ach o r co lo n .

M u c h rem a in s to be le a rn t a b o u t the m o le c u la r genetics o f the h u m a n M u -C la s s G STs. Besides the m o le c u la r c lo n in g o f G S T M I a and G S T M 1 b, tw o fu rth e r c D N A s have been re p o rte d th a t encode the 'm u s c le -s p e c ific ' transferase, G S T M 2 (V o ra c h e k et a l., 1991) and a ‘ b ra in /te s tis -s p e c ific ’ transferase, G S T M 3 (C a m p b e ll et a l., 1990). H o w e v e r, it is ce rta in th a t a d d itio n a l M u -C la s s G S T s exist, since z y m o g ra m analysis o f hum a n tissue e xtra cts suggests th a t this fa m ily is encoded a t m o re th a n three lo c i (S u zu ki et a l., 1987), a n d sequence analysis o f a ge n om ic clo n e (C O S H l- 1 0 ) , fro m a h u m a n c o sm id lib r a ry has revealed a M u -C la s s gene d is tin c t fr o m those e n co d in g G S T M l, G S T M 2 o r G S T M 3 ( T a y lo r et al., 1990). T h e co sm id clo n e isolated by T a y lo r et al. (1990) c o n ta in s p a rtia l sequences fro m tw o M u -

A b b re via iio n s used: G ST. g lu ta th io n e S -lra n s fe ra s e: D M S O . dimethyl sulphoxide. ÿ To w h o m c o rre s p o n d e n c e should be sent

The n u cleo tid e seq uences rep orted will a p p e a r in the EM BL. G e n B a n k a n d DDBJ N ucleotid e S e q uence D a ta b a s e s under the accession num bers G S T M 16. X68676. G S T M 4 a n d X68677.

42 S. Zhong and others

Class G S T genes liia l these w orkers designated ‘ G S T in u 2 ' and ‘ G S T n n i3 '; a m ino acid sequencing o f the m uscic-spccific tra n s­ ferase suggests that G STm u3 is e quivalent to G S T M 2 (see Hussey et al.. 1991), but the id e n tity o f G S T m u 2 is uncertain.

H u m an M u-C lass genes have been reported to be located on bo th chrom osom e 1 and chrom osom e 3. De Jong et al. (1988) carried o u t in siin h y b rid iz a tio n using a c D N A fo r G S T M la -1 a and showed that hum an M u-C lass genes were clustered in region p3 1 o f chrom osom e 1. T his assignment was co n firm e d by PCR m ap p in g (Z h o n g et al.. 1993). By co n tra st, using a panel o f hum an x rodent som atic-cell h yb rid s and tw o diffe re n t rat M u - Class c D N A probes. Islam et al. (1989) assigned M u-C la ss genes to chrom osom e 3. The second g ro u p o f w orkers postulated that they had m apped G S T M 2 and G S T M 3 . H ow ever, as T a y lo r et al. (1990) have fo u nd tw o separate M u-C lass genes w ith in a single cosm id clone, it remains unclear whether a ll the M u-C lass G S T genes are on the same chrom osom e.

F u rth e r m olecular in fo rm a tio n ab o ut M u-C la ss G S T genes is required to a llo w a better u n derstanding o f the m u ltip lic ity o f this fa m ily in M an and their in vo lve m e n t in p ro te ctio n against carcinogenesis T o this end. we describe the iso la tio n o f a c D N A encoding hum an G S T M 4 . The gene structu re o f G S T M 4 has been determ ined, and its ch rom osom al lo ca liza tio n indicates that at least three o f the characterized G S T M genes are fo u nd on chrom osom e 1.

— 6 5 7 b p

Figure 1 A m plification ol G STf41b and G S TM 4 cDNAs from lym phoblastoid vril lines

M phage </tXUA/H3eU\ m aiK ei lia g m e n is lanes I and 2. m e 6 5 / Dp D N A lia g m e n i lio m G S T M l c D N A Irp m a G S T M l positive cell line and G S T M 4 cO N A lio m a G S T M l null cell Ime lespeciiveiy

M A TERIALS AND METHODS Nom enclature

The nom enclature used in this study is that recom m ended fo r G S T by M a n n e rv ik et al. (1992).

Cell lines and preparation of DNA and RNA

The lym p h o b la sto id cell lines G S T 5 and G S T 6 were obtained by E p s tc in -B a rr-v iru s im m o rta liz a tio n o f n o rm a l hum an lym phocytes Cells were grow n in R P M l 1640 and 10 fo ci a 1- c a lf serum These lines have been phenotyped and genotyped fo r h G S T M l (Z h o n g et al., 1991), and the som atic h yb rid s have been described previously (Z h o n g el al., 1993). The hum an ch rom osom e content o f the h yb rid s were checked by a c o m ­ b in a tio n o f isoenzyme and D N A m a rk e r analysis and k a ry o ty p ic analysis (Evans et al., 1971 ; B o b ro w and Cross, 1974; H a rris and