Proc. Nati. Acad. Sci. USAVol. 83, pp. 7598-7602, October 1986Biochemistry

Developmental and tissue-specific regulation of the Q10 class Igene by DNA methylation

(secreted histocompatibility antigen/liver-specific activation)

KENICHI TANAKA*, YVES BARRA*, KURT J. ISSELBACHERt, GEORGE KHOURY*, AND GILBERT JAY*t*Laboratory of Molecular Virology, National Cancer Institute, National Institutes of Health, Building 41, Room A101, Bethesda, MD 20892; and tDepartmentof Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114

Contributed by Kurt J. Isselbacher, June 12, 1986

ABSTRACT The H-2 class I genes encode cell-surfaceglycoproteins that play a critical role in the immune presen-tation of aberrant cells. The Q10 class I gene, however, encodesa secreted glycoprotein that is highly homologous to themembrane-bound molecules. While the H-2 genes are activatedin all tissue types, the expression of the Q10 gene is restrictedto only the liver. Analysis of DNA from different tissuesrevealed a unique methylation profile for the Q10 gene in liver.Developmental activation of this gene in newborn mice is alsoreflected by a coordinated temporal change in DNA methyla-tion. By comparing the methylation profiles between congenicmice, which differed in their levels of expression of the Q10gene, it is observed that methylation at the 3'-flanking regioncorrelates with expression. Methylations were at both CG andCC sequences. Since treatment of newborns with 5-azacytidine,which led to inhibition of methylation, resulted in the suppres-sion of Q10, we conclude that hypermethylation in the 3'-flanking region is responsible, at least in part if not in full, forthe activation of the Q10 gene in the liver.

The major histocompatibility complex class I antigens arecell-surface recognition elements required for the presenta-tion of virally-infected cells and tumor cells to the immunesystem (1). Suppression of the class I antigens in transformedcells may lead to the derivation of the malignant phenotype(2-5).The identification of a class I gene that encodes a secreted

molecule, designated the Q10 antigen, is intriguing (6-8).Because the secreted and the cell-surface class I moleculesare highly homologous in sequence (6, 7), it is tempting topropose that both types of molecules can be recognized bythe same macromolecular structure in the immunoreactivecells. If correct, the Q10 antigen could play the role of animmune modulator that regulates cell-cell interaction.

Unlike the membrane-bound class I antigens that areexpressed in virtually all cells in the body, the secreted classI antigen is synthesized only in the liver (9, 10). In view of thesuspected function of the Q10 antigen, a molecular under-standing of the restricted expression of this gene in the liveris of importance. In the present study, we show that activa-tion of the Q10 gene during development is regulated by DNAmethylation.

MATERIALS AND METHODSMice. SWR/J, BALB/c, B10 (C57BL/10), and B10.M

mice were obtained from The Jackson Laboratory.DNA Blot Analysis. Southern blot analysis was performed

as described (11). High molecular weight DNA was digestedwith the appropriate restriction endonuclease and applied toa 1.0% agarose gel. After electrophoresis, the DNA frag-ments were transferred from the gel to a nitrocellulose

membrane and hybridized to a 32P-labeled single-strandedDNA probe.RNA Blot Analysis. RNA gel blot analysis was carried out

as described (10). Polyadenylylated RNA was fractionated byelectrophoresis in a 1.2% agarose gel in the presence offormaldehyde. After electrophoresis, the RNA was trans-ferred from the gel to a nitrocellulose membrane and hybrid-ized to a 32P-labeled single-stranded DNA probe.

RESULTSIdentification of a Tissue-Speciflic DNA Methylation Profile.

To compare the DNA methylation profile in the vicinity oftheQ10 gene, between active and inactive tissues, we have madeuse of two restriction endonucleases, Msp I and Hpa II,which recognize the same CCGG sequence but are otherwisedifferentially sensitive to methylation (12). If the secondcytosine residue within the CCGG recognition site is methyl-ated (CG methylation), Msp I will cleave the DNA but notHpa II. However, if the first cytosine residue is methylated(CC methylation), Msp I will not cleave but Hpa II will. Bycomparing the cleavage products in a Southern blot, using theQ10-specific cDNA probe, one should be able to determinewhether a unique DNA methylation profile exists in liverwhere the Q10 gene is selectively expressed.We have demonstrated that a Q10-specific cDNA probe

can be derived from the distal half of exon 7 that does notcross-hybridize with other class I genes (Fig. 1) (10). Hy-bridization of this probe to a Southern blot containing DNAfrom SWR/J spleen (Fig. 2A) and kidney (Fig. 2B), which hadbeen digested with Msp I (lanes M), revealed a single majorcomponent of 1.9 kilobases (kb). This observation bothconfirms that the cDNA probe used detects only a single copygene and shows that CC methylation does not occur at eitherofthe CCGG sites located on each side ofthe region identifiedby the Q10 probe. These two sites, designated Ml and M2(see Fig. 1), have been mapped by DNA sequencing of a Q10genomic clone and are separated by 1.9 kb (7).

Analysis ofSWR/J liverDNA digested by Msp I, however,gave different results. The 1.9-kb fragment, while detectable,was reduced by about 50% and appeared to be accompaniedby several larger fragments (Fig. 2C, lane M). Since SWR/Jmice are inbred and should be homozygous at the Q10 locus,it appears that about half of the cells in the liver have adistinctive DNA methylation profile as detected by Msp Idigestion. This observation suggests that either the Ml, or theM2, or both the Ml and M2 recognition site(s) are methylatedat the first cytosine residue in a subset of liver cells. It shouldbe mentioned that CC methylation is rare and accounts forless than 5% of the methylation in mammalian cells (13).

Abbreviations: kb, kilobase(s); CC methylation, methylation at thefirst cytosine residue in the CCGG endonuclease recognition site; CGmethylation, methylation at the second cytosine residue in the CCGGendonuclease recognition site.tTo whom all correspondence should be addressed.

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Proc. Natl. Acad. Sci. USA 83 (1986) 7599

1 2 3

B B11 E1l IN

M3

Hpall + BamHI

Hpall + BstEIl

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FIG. 1. Schematic diagram of the Q10 geneSWR/J mice. The exon-intron organization of the lcomparing genomic and cDNA sequences (6, 7).boxes indicate translated and untranslated sequenThe locations of BamHI (B), BstEII (S), and MspMl, M2, and M3 cleavage sites are shown. Restrgenerated by combined digestion with the indicadetected by hybridization to the Q10-specific 3'-(stippled boxes), are also included. Their lengths arHpa II-sensitive (o) and Hpa II-resistant (e) CCGC

While digestion ofDNA from SWR/J spleekidney (Fig. 2B) by Msp I failed to reveal Caround the region detected by the Q10-specifition of the same DNA samples by Hpa II (laextensive CG methylation. The 1.9-kb fragmeientirely by four larger components, present in

A B

23.1 -

9.4 -

6.6 -

exons equimolar ratios. Since the same four components are de-tected in all nonexpressing tissues tested, it is likely that

B S nonactive cells have one of four methylation profiles regard-Mspl less of tissue derivation.

Analysis of SWR/J liver DNA after digestion with Hpa IIH revealed, in addition to the four fragments detected in all

nonexpressing tissues, three other components with molec-ular sizes greater than 1.9 kb (Fig. 2C). These results are

H again consistent with about 50% of the liver cells havingeither one of the three CG methylation profiles that were notdetectable in tissues other than liver. It, therefore, appearedthat the restricted expression of the Q10 antigen in liver iscorrelated with distinctive methylation profiles present in asubset of liver cells, but not detected in any other tissues

derived from the analyzed. Such modifications are in the form of both CG andgene is defined by CC methylations in the vicinity where the Q10-specific cDNAClosed and open probe hybridizes.ces, respectively. Mapping ofLiver-Specific Methylation Sites. To map the CCI (vertical lines); and CG methylation sites, two reference restriction enzymesiction fragments, were used. The cleavage sites for both BamHI and BstEIIted enzymes and have been determined (14). Digestion with these enzymes-noncoding probere indicated in kb. yielded a 3.0-kb and a 4.8-kb fragment, respectively, that3 sites are shown. hybridized to the Q10-specific probe (see Fig. 1). Since both

components include the Ml and M2 sites, they are particu-larly useful for determining whether the Ml and/or M2

n (Fig. 2A) and sequences are methylated.XC methylation As shown in Fig. 2, digestion of liver DNA with Msp Iic probe, diges- revealed the presence of CC methylation that is not detect-nes H) showed able in spleen and kidney. To identify whether Ml, M2, ornt was replaced both Ml and M2 sites are methylated, Msp I digests ofDNAapproximately from liver (lanes 1) and from kidney (lanes 2) (Fig. 3A) were

further cleaved with BamHI (Fig. 3B). The detection of aC 3.0-kb fragment in liver by hybridization would suggest

methylation at both Ml and M2 sites, the presence of a 2.5-kbfragment would imply methylation only at the M2 sites, andidentification of a 2.3-kb component would suggest modifi-cation at only the Ml site (see Fig. 1). The presence of a2.3-kb band is, therefore, consistent with specific CCmethylation in the liver at the Ml but not the M2 site. Thisconclusion is further confirmed by the use of BstEII insteadofBamHI (Fig. 3C) that only gave rise to a 3.6-kb component

*~q4 that could have been derived only by methylation at the Ml4 site (see Fig. 1). It is concluded, therefore, that expression of

A B C D E F

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FIG. 2. Comparision of the state of methylation of the Q10 genein expressing and nonexpressing tissues. High molecular weightDNA was obtained from SWR/J spleen (A), kidney (B), and liver (C).After cleavage with Msp I (M) or Hpa 11 (H), the DNA digests werefractionated by electrophoresis in a 1.0% agarose gel, transferred toa nitrocellulose membrane, and hybridized to the Q10-specific cDNAprobe (9). The molecular size markers, in kb, are indicated on the left.The arrowheads identify the liver-specific Hpa II components.

1 2 1 2 1 2 1 2 1 2 1 2

FIG. 3. Physical mapping of the methylation sites around the Q10gene. Southern blot analysis, using the Q10-specific cDNA probe, ofSWR/J DNA from liver (lanes 1) and kidney (lanes 2) that have beendigested by the following different combinations of restrictionenzymes: Msp I alone (A), Msp I/BamHI (B), Msp I/BstEII (C), HpaII alone (D), Hpa II/BamHI (E), Hpa II/BstEII (F). The molecularsize markers, in kb, are indicated on the left. The arrowheadsindicate the liver-specific fragments of 3.6 kb and 2.3 kb.

Biochemistry: Tanaka et al.

Proc. Natl. Acad. Sci. USA 83 (1986)

the Q10 gene in liver is correlated with specific CC methyla-tion at the Ml and adjacent downstream CCGG sites.The sites for CG methylation were next investigated. Hpa

II digests ofDNA from liver and from kidney (Fig. 3D) werefurther incubated with eitherBamHI (Fig. 3E) or BstEII (Fig.3F). The data showed clearly that a subset of cells in the liverhas a pattern of CG methylation not detected in kidney.Exactly analogous to the pattern ofCC methylation describedabove, these liver cells showed CG methylation at the Ml andadjacent downstream CCGG sites but not at the M2 site. Thepresence of this subset of methylation correlates with theexpression of the Q10 gene in liver. Because the Q10 gene ishypermethylated at CG sites even in nonactive tissues, it isdifficult to conclude whether the liver-specific componentswere derived by hypomethylation at the M2 site or byhypermethylation at the Ml and adjacent downstream sites.With CC methylation, however, expression of the Q10 geneunambiguously correlated wih hypermethylation in the 3'-flanking region.

Developmental Methylation in the Newborn Liver. While theQ10 transcript could be detected in the fetal liver at late stagesof gestation (Fig. 4B, lane 1), its accumulation in the newbornliver increased dramatically after birth (Fig. 4B, lanes 2-5) toa level comparable to that observed in adults (Fig. 4B, lane6). This is in contrast to the level of actin mRNA, whichremained relatively constant during the same period ofdevelopment (Fig. 4C, lanes 1-6).Hpa II digests of liver DNA at different days after birth

were compared (Fig. 4A). At approximately 1 day before(lane 1) and 1 day after birth (lane 2), when there were low butdetectable levels of Q10 RNA, the three liver-specific bands(cf. Fig. 2C) were barely discernible (Fig. 4A, filled arrow-heads). The other bands, which were also detected in non-expressing tissues, predominate. These latter Hpa II frag-ments are not present in equimolar ratios as seen in inactivetissues (cf. Fig. 2 A and B); three of the bands wereoveremphasized in liver from 1-day-old animals (Fig. 4A,open arrowheads). Interestingly, as the newborn animalsdeveloped, these three components decreased in relativemolar representation while the three liver-specific fragments

A B

"low.

increased accordingly. In each case, it appeared that asmaller Hpa II component was replaced by a larger Hpa IIcomponent. It appeared, therefore, that developmental acti-vation of the Q10 gene in liver, as evidenced by an increasein accumulation ofmRNA, is accompanied by hypermethyla-tion of DNA.Comparison Between Congenic Mouse Strains. As a first

step towards confirming the importance ofDNA methylationin the activation of the Q10 gene in liver, we analyzed DNAfrom the congenic BlO.M mouse strain. Unlike all inbred andwild mice studied, BlO.M mice express a greatly reducedlevel ofthe Q10 antigen (6). Since the steady-state level of theQ10 transcript in the liver of BlO.M mice (Fig. SB, lane 2) isalso less than 5% that detected in the background B10 strain(Fig. SB, lane 1), it is suspected that this altered expressionis regulated at the transcriptional level. If DNA methylationplays a role in controlling Q10 expression, it seemed possiblethat the methylation profile would be perturbed in BlO.Mmice.Comparison of the methylation profile of DNA from B10

mice to that of SWR/J mice showed extensive conservation.Digestion of B10 kidney DNA with Hpa II (Fig. 5A, lane 3)gave rise to three fragments that are identical in size to threeof four fragments observed for SWR/J spleen and kidneyDNA (see Fig. 2 A and B). These results are consistent withat most a single polymorphic Hpa II methylation site betweenthe two mouse strains in inactive tissues. Digestion of B10liver DNA with Hpa II also yielded tissue-specific compo-nents (Fig. SA, lane 1) very similar in size to those observedin SWR/J liver (see Fig. 2C). In fact, the digestion patternsdetected between SWR/J and BALB/c mice are also virtu-ally identical. These observations suggested that the DNAmethylation profile, including the liver-specific subset ofbands, is highly conserved among different inbred mousestrains.Hpa II-digested kidney and spleen DNA from the congenic

BlO.M mice (Fig. 5A, lanes 4 and 6) also showed remarkableconservation with those from the background B10 mice (Fig.5A, lanes 3 and 5), except for an additional fragment thatshowed tissue-specific polymorphism. Liver DNA from thesame two mouse strains, however, showed complete diver-

A

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B

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6.6 -

C C

2.3 -2.0 -

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

FIG. 4. Developmental activation of the Q10 gene in newbornmice. DNA and RNA were extracted from the liver of SWR/J miceat -1 (lanes 1), 1 (lanes 2), 3 (lanes 3), 7 (lanes 4), 12 (lanes 5), and35 (lanes 6) days after birth. (A) Southern blot analysis, using theQ10-specific cDNA probe, of Hpa II digests of liver DNA. Those

components that decreased (open arrowheads) and those that in-creased (filled arrowheads) in relative molar amounts during devel-opment are indicated. (B) RNA gel blot analysis, using the Q10-specific cDNA probe, of poly(A)+ RNA from the liver. The arrow

indicates the position of 18S rRNA. (C) RNA gel blot analysis withthe ,-actin DNA probe of poly(A)+ RNA from the liver.

FIG. 5. Comparison of the state of methylation of the Q10 genein congenic mice. DNA and RNA were extracted from the liver ofB10 (lanes 1, 3, and 5) and B1O.M (lanes 2, 4, and 6) mice. (A)Southern blot analysis, using the Q10-specific cDNA probe, of HpaII digests of DNA from liver (lanes 1 and 2), kidney (lanes 3 and 4),and spleen (lanes 5 and 6). The arrowheads indicate the liver-specificcomponents in B10 mice. (B) RNA gel blot analysis, using theQ10-specific cDNA probe (9), of poly(A)+ RNA from the liver. Thearrow indicates the position of 18S rRNA. (C) RNA gel blot analysiswith the H-2K synthetic oligonucleotide probe (15) of poly(A)+ RNAfrom the liver.

1 2

7600 Biochemistry: Tanaka et A

Proc. Natl. Acad. Sci. USA 83 (1986) 7601

B

Om

c

ia w- sm _og

1 2 3 4 1 2 3 4

FIG. 6. 5-Azacytidine treatment of newborn mice. DNA andRNA were extracted from individual control (lanes 1) and 5-azacytidine-treated (lanes 2-4) mice. (A) Southern blot analysis,using the Q10-specific cDNA probe, of Hpa II digests of liver DNA.The liver-specific fragments (filled arrowhead) and the unmethylated1.9-kb component (open arrowhead) are indicated. (B) RNA gel blotanalysis with the Q10-specific cDNA of poly(A)+ liver RNA. (C)RNA gel blot analysis of poly(A)+ RNA from liver using the 3-actinDNA probe. The arrow indicates the position of 18S rRNA.

gence with regard to the tissue-specific components (Fig. 5A,lanes 1 and 2). The fragments detected in BlO.M mice are

considerably smaller in size than those observed in B10 mice.Combined digestion with BamHI or BstEII, showed thatthese liver-specific components were not modified at the M2site but were quantitatively methylated at the Ml site in bothmouse strains (data not shown). Together, these results showthat the liver DNA of BlO.M mice is hypomethylated in the3'-flanking region of the Q10 gene and that this hypomethyla-tion correlates with nonexpression of Q10 transcripts.Treatment of Newborn Mice with 5-Azacytidine. Because

much of the liver-specific methylation occurs after birth (Fig.4) and because hypermethylation is implicated in the activa-tion of the Q10 gene (Fig. 5), treatment of newborn mice with5-azacytidine should block its methylation and its expression.All but one member of a litter ofSWR/J mice were each givena 25 ,ug dose of 5-azacytidine on day 4 and a second dose of50 ,4g on day 8 after birth. Liver DNA and liver RNA were

obtained from each surviving member of the litter on day 12.Hpa II digestions of liver DNA from each individual animalwere compared (Fig. 6A). Unlike the control littermate (lane1), the three treated mice all showed a nonmethylated 1.9-kbfragment confirming that 5-azacytidine did partially blockDNA methylation (lanes 2-4). RNA gel blot analysis of liverRNA from these same mice (Fig. 6B) showed significantreduction in steady-state Q10 RNA in the three treated mice(lanes 2-4), as compared to the control littermate (1). Thiswas not a reflection of the metabolic state of the animalsbecause hybridization of the same RNA blot to a 83-actinprobe revealed no difference between treated and untreatedmice (Fig. 6C). The results, therefore, are consistent with themodel that DNA nethylation is required for the activation ofthe Q10 gene.

DISCUSSIONIt has been noted, for reasons that are obscure at the presenttime, that CG sequences occur much less frequently in class

I genes than expected (11, 16). In the case of the Q10 class Igene, the paucity ofMsp I (CCGG) sites between exons 3 and7 is evident (see Fig. 1). It is possible that there is a highselective pressure against adventitious CG sequences be-cause DNA methylation at the appropriate sites in thevicinity of the class I gene is important for their expression.Indeed, we have previously suggested that the H-2K class Igene is regulated by DNA methylation (11).

In the present study, we have investigated the basis for thedevelopmental and tissue-specific regulation of the Q10 gene.The results obtained showed that about 50% of cells in theliver, where the Q10 gene is expressed, has a distinct DNAmethylation profile that is not detected in tissues where thisgene is not expressed. This subset of cells is not modified atthe M2 site, located in the intron between exons 3 and 4, butis methylated at the Ml and adjacent downstream sites,situated at the 3'-flanking region of the gene (see Fig. 1).Interestingly, these methylations were not found only at CGsequences but were also present at CC sequences.We have observed that developmental activation of the

Q10 gene in the liver of newborn mice is closely correlatedwith a temporal increase in DNA fragment size in Hpa IIdigests. Since these liver-specific components are notmethylated at the M2 site but are methylated at the Ml andadjacent downstream sites (see Fig. 1), either hypomethyla-tion at M2 or hypermethylation at Ml and downstream sitesappears to be important for expression. However, from ouranalysis of congenic mice expressing various levels of Q10transcripts, we conclude that hypermethylation of 3'-flankingsites is important for gene expression. In addition, partialinhibition ofDNA methylation by treatment ofnewborn micewith 5-azacytidine led to greatly reduced levels of Q10transcripts in liver.Our findings are not inconsistent with a report that the M2

site of Q10 is not methylated in liver DNA (17). While we dofind that a subset of cells in the liver has the M2 sitehypomethylated, our study with congenic mice suggests thathypermethylation at 3'-flanking sites, but not hypomethyla-tion at M2, is responsible for Q10 expression. It is interestingto note that hypermethylation at the 3'-flanking region of themurine hprt locus also correlates with gene expression (18).The suggestion of a control element at the 3' side of the Q10

gene that is regulated by DNA methylation may appearcomplex in light of the finding that expression of class I genesare controlled, at least in part, by transcriptional enhancersthat map to the 5'-flanking regions. However, there isprecedence for regulatory elements flanking both sides of thesame structural gene. The chicken p-globin gene has aninducible and hypersensitive site for a promoter element onits 5' side and a tissue-restricted transcriptional enhancer onits 3' end (19). A similar arrangement of control sequences isalso seen with the human RAS gene (20, 21).

1. Zinkernagel, R. M. & Doherty, P. C. (1979) Adv. Immunol. 27,51-77.

2. Tanaka, K., Isselbacher, K. J., Khoury, G. & Jay, G. (1985)Science 228, 26-30.

3. Hayashi, H., Tanaka, K., Jay, F., Khoury, G. & Jay, G. (1985)Cell 43, 263-267.

4. Hui, K., Grosveld, F. & Festenstein, H. (1984) Nature (Lon-don) 311, 750-752.

5. Wallich, R., Bulbuc, N., Hammerling, G. J., Katzav, S.,Segal, S. & Feldman, M. (1985) Nature (London) 315, 301-305.

6. Kress, M., Cosman, D., Dhoury, G. & Jay, G. (1983) Cell 34,189-196.

7. Mellor, A. L., Weiss, E. H., Kress, M., Jay, G. & Flavell,R. A. (1984) Cell 36, 139-144.

8. Maloy, W. L., Coligan, J. E., Barra, Y. & Jay, G. (1984) Proc.Natl. Acad. Sci. USA 81, 1216-1220.

9. Cosman, D., Khoury, G. & Jay, G. (1982) Nature (London)295, 73-76.

10. Cosman, D., Kress, M., Khoury, G. & Jay, G. (1983) Proc.

A

....:.mo ..

-,.

Biochemistry: Tanaka et al.

7602 Biochemistry: Tanaka et al.

Nati. Acad. Sci. USA 79, 4947-4951.11. Tanaka, K., AppeUa, E. & Jay, G. (1983) Cell 35, 457-465.12. Van der Ploeg, L. H. T. & FlaveU, R. A. (1980) Cell 19,947-958.13. Doerfler, W. (1981) J. Gen. Virol. 57, 1-20.14. Barra, Y., Tanaka, K., Isselbacher, K. J., Khoury, G. & Jay,

G. (1985) Mol. Cell. Biol. 5, 1295-1300.15. Kress, M., Liu, W.-Y., Jay, E., Khoury, G. & Jay, G. (1983)

J. Biol. Chem. 258, 13929-13936.16. Tykocinski, M. L. & Max, E. E. (1984) Nucleic Acids Res. 12,

4385-4396.

Proc. Nati. Acad. Sci. USA 83 (1986)

17. Miyada, C. G. & Wallace, R. B. (1986) Mol. Cell. Biol. 6,315-317.

18. Lock, L. F., Melton, D. W., Caskey, C. T. & Martin, G. R.(1986) Mol. Cell. Biol. 6, 914-924.

19. Hesse, J. E., Nickol, J. M., Lieber, M. R. & Felsenfeld, G.(1986) Proc. Natl. Acad. Sci. USA 83, 4312-4316.

20. Ishii, S., Kadonaga, J., Tjian, R., Brady, J. N., Merlino, G. T.& Pastan, I. (1986) Science 232, 1410-1413.

21. Capon, D. J., Chen, E. Y., Levinson, A. D., Seeburg, P. H. &Goeddel, D. V. (1983) Nature (London) 302, 33-37.