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Vegulatory mechanisms of the papillomaviruses
WONITOR
rEGULATORYMECHANISMSofthe PAPILLOMAVIRUSES P. WARD*t, D. V. COLEMAN* AND A. D. B. MALCOLM'r *DEPARTMENT OF CYFOPATHOLOGY, ST MARY*SHOSPITAL MEDICAL SCHOOL, LONDON W2 II'G, 1"K. tDEPARTMENT OF BIOCHEMISTRY, CHARING CROSS HOSPITAL MEDICAL SCHOOL, LONDON, 17K.
apillomaviruses are small DNA proteins of the papillomaviruses 6. v~mses that infect higher verte- For example, HPV DNA has been brates, including man. Although found in all cell lines derived from they usually cause warts, these cervical carcinomas; the E6/7 ORFs viruses have recently been associat- are always conserved in these cells ed with malignant tumoursl-4; in and give rise to the most abundant humans the most common associ- mRNA transcripts~. Sequencing of ation is with cervical cancers. DNA from three such lines has The papillomavirus genome revealed a small insert of host DNA consists of a double-stranded loop (intron) in the E6 ORF, giving rise of DNA about 8000 base pairs, to a protein that has some similfunctionally divided into two long arity to epidermal growth factor domains each containing a series (EGF) 9. EGF stimulates cell growth of open reading frames (ORFs) that and division and inhibits many code for viral proteins (Fig. 1). The functions associated with differentiE (early) region contains eight ated cells. Perhaps excess levels of ORFs that code for the proteins an EGF-like protein stimulate associated with genome replication undifferentiated growth of an and control. The L (late) region infected cell 10-12. A similar intecontains two large ORFs coding for gration site is also found within the the structural proteins of the virus E6 ORFs of other HPV types associcapsid. The regulatory region (long ated with cervical cancers, but not control region, LCR) lies between in HPV types 6/11, which are the stop codon of L1 and the start found most commonly in benign codon of the E 6 0 R F . Several lesions of the cervix. promoter sites have been idenTransfection of the E6/7 prodtified throughout the genome; ucts has been shown to result in those of particular interest are cell transformation in vitro 13. found at nucleotides 89, 2443 Moreover, the E7 ORFs of the HPV and 3080 (p89, p2443 and p3080). types 16, 18, 31 and 33 (but not In benign lesions the viral DNA is found as an extrac El I chromosomal closed R I--6-I I~ I E2 II L2 I circular strand (epivP i 0 some); in malignant tumours the viral H [---g/---] I t2 E1 I DNA is often found vP F ~ I 1 [] L1 to be integrated into 6 the host chromosomal material, usually r-gcq r~ Ilm i-~-Ir-E-E~a I L1 at the sites of the v E1 I E1/2 ORFs. The biol6 ogy of the viruses has ] [] recently been reB E1 I viewed in T/Gs. vP [] I L2 II L~ 1
arm E6/70RFs For several years it has been recognized that the E6/7 ORFs code for the major transforming
,gn..
0 p89 Transforming proteins
GENOME
E 2 GENE PRODUCT The protein product of the E2 ORF is the major regulatory protein of the papillomaviruses. This protein has been sequenced in 10 papillomavirus species and although the amino acid sequences are poorly conserved, computer prediction indicates they share a similar structure is (Fig. 2). There are three domains within the protein, two of which are responsible for its biological properties, while the third (domain B) appears to act as a hinge I between the two LOB , p active parts. The 7868 amino-terminal portion of the protein (domain A) is reI LOR ! =.-quired for efficient 7e~5 trans-activation, while domain C is required I for DNA binding l~,. LCR I ~ The whole E2 gene 7902 product has been shown to act as an I enhancer of DNA ,CR transcription, whereas , P p2443 p3080 7945 a smaller protein (E2 Regulatory Capsid Tr) derived from the proteins proteins 3' end of the E2 ORF has repressor activiFIG]Ill ty 17. Both proteins act ORGANIZATION OF TYPICAL PAPILLOMAVIRUSES. by exerting control
TIG APRIL1989 VOL. 5 NO. 4 @1989 Elsevier Science Publisher,, Ltd (UK) 0168 - 952S/89/$02.00
those of types 6 and 11) can act in conjunction with an activated r a s oncogene to induce transformation of primary rat kidney cells i n vitro 1~. Surprisingly, the E7 ORF sequences of all these viruses are very similar, despite their different oncogenic properties. Their differing effects may be the result of an alteration in the structure of the protein products, but this has yet to be shown. The E6/7 transcripts are derived from the p89 promoter, which is regulated by the gene product of the E2 ORF.
WONITOR over transcription initiated at promoter sites within the genome by acting at an enhancer site in the LCR.
LONGCONTROLREGION The length of the LCR of the papillomaviruses varies between species but it has several conserved regions and maintains a similar organization. The 3' region of the LCR is the least conserved and seems to be implicated in the maintenance of the episomal form of the virus in infected cells TM. The 5' end contains several repeats of an ACCG-N<,-CGGT sequence that is c o m m o n to all papillomavirus types. Also within this region are many promoter elements (TATA and CAAT boxes) for polymerase II binding, and many repeats and inverted repeats commonly found in viral control regions. An enhancer that is tran&activated by the gene product of the E2 ORF has been identified in this region, and consists of at least two tandem repeats of the ACCG-N4-CGGT sequenceW. It is at this site that the carboxy-terminal portion of the E2 gene product binds 15A~,. E2 Tr also acts at this site, in this case to repress transcription~7. The repressot and trans-activator proteins share a c o m m o n carboxy terminus and compete for the enhancer sites in the LCR16. Although the exact
mechanism of regulation is uncertain, it may be a result of different levels of the two proteins. The levels of the RNA transcript from p3080 - believed to be the E2 Tr transcript - are at least 10 times those from P2443, which is thought to encode the whole E2 molecule ~9. If these transcripts are equally translated in vivo then the E2 repressor would be more abundant than the E2 enhancer; this ratio could account for the low levels of viral transcription in BPV-l-transformed cells. Most of the mRNA transcripts from cervical cancer cell lines are from the E6/70RFs. The viral DNA in these lines is integrated into the host cell genome at the E2 ORF, which seems to disrupt the normal function of the E2 product. Since the balance of the E2 products inhibits transcription, disruption of this activity may result in uncontrolled expression of the transforming proteins and lead to malignant change in the host tissues. In support of this hypothesis, p89 (the major promoter site for the E6/70RFs) is controlled by the E2 gene product, and is normally held in a repressed state 20. Since the promoter sites p2443 and p3080 are also regulated by this mechanism, it appears that the gene products of the E2 ORF regulate their own transcription. These
G A1 Aa
• prolines I-1 =-helices Domain B
~1 /]-sheets - - uncertain structure O conserved amino acids
V Domain C (DNA-binding domain) FIGlil
STRUCTURE OF THE PAPILLOMAVIRUS E2 GENE PRODUCT (FIGURE MODIFIED FROM REF.
TIG APRIl.1989 VOL. 5 NO. 4 9~
16).
sites are a considerable distance from the LCR enhancer; it may be that regulation is effected by an alteration of the spatial orientation of the bound protein and the DNA.
]~MORALCONTROLMECHANISMS Although papillomavirus DNA is detectable in undifferentiated epithelial cells, virus replication usually takes place within the terminally differentiated kemtinocyte. The reasons for this are unclear, but may involve interplay between host proteins and the viral genome. Evidence for a host-virus interaction was provided by experiments in which HeLa cells (which contain HPV 18 DNA) were fused to normal human fibroblasts. This results in the suppression of the malignant phenotype. In the nonmalignant hybrid, loss of chromosomal material - particularly of human chromosome 11 - from the fibroblast donor results in the reacquisition of malignant growth on transplantation into nude mice 2j (Fig. 3). Furthermore, i*z vitro HeLa cells, malignant hybrids and nonmalignant hybrids express the same levels of HPV 18 mRNA; however, on transplantation into porous chambers grafted onto nude mice, the nonmalignant hybrid cell line fails to produce any HPV 18 RNA whereas the malignant hybrids and the HeLa cell lines continue to do so. It appears therefore that a diffusible protein produced by normal host cells can suppress virus transcription in infected cells 22. This humoral control mechanism, which has yet to be fully eh_lcidated, may be involved in the prevention of malignant transformation in the infected host. Perhaps this explains why although there is a high incidence of latent infection of the female genital tract with HPV types known to be oncogenic, the actual incidence of cervical cancer within the population is less than 1%. Further evidence that humoral factors are involved in the genesis of cervical cancer c(nnes from reports that part of the LCR responds to glucocorticoid hormones 23 and progesterone 2~. HPV 16 DNA (but not HPV 11 DNA) and an activated ras oncogene cause malignant transformation of rat
]~]ONITOR HeLa cell
Normal cell
No regulation~ ofviral ('-' (,~1~ ~ transcription. Cancer inhibiting factor(CIF) gene functionally inactive.
/I ~
~
No viral DNA.
CIF gene inactive.
I FUSION
aCI~O~t~DGD~ENT$ P. Ward is coordinator of an epidemiological study of the risk associated with papillomavirus infection of the cervix and is supported by a grant from the Cancer Research Campaign.
rEFEgENCES 1 Jarrett, w. F. H. etal. (1978) Nature 244, 215-217 2 Kreider, J. w. (1982) Cold Spring Harbor Conf. Cell Proliferation 7,
INVITRO
INVIVO
' ~ CIF not activated
~ ~ g e n e Humoral activatedby HF factor(HF)
Malignantphenotype
Suppressionof malignantphenotype
I
REVERTANTS (Loss of chromosome)
INVITRO
INVlVO
Absenceof functioning CIF gene
ot ableto activateCIF gene
Malignantphenotype
Malignantgrowth FIG~
HOW HUMORAL FACTORS MAY INFLUENCE PAPILLOMAVIRUSTRANSCRIPTION. (FIGURE MODIFIED
FROMREF.22.) primary kidney cells only if the culture medium contains progesterone or dexamethasone 25. While this mechanism may be important in the genesis of cervical cancer, the significance in vivo is at present uncertain a6. I U T U ~ IRIOSI~CI~ The next steps are likely to include the identification of
associated cellular factors by the use of anti-E2 antisera, and the investigation of the E2 protein and the LCR enhancer region using the cloned E2 enhancer elements already identified. The lessons learnt from this investigation should shed light on the mechanisms of genome control n o t only of viruses but also of eukaryotes.
TIG APRIL 1 9 8 9 VOL. 5 NO. 4
99
283-299 3 zur Hausen, H. (1977) Curr. Top. Microbiol. Immunol. 78, 1-30 4 Gissman, L. et al. (1984)./. Invest. Dermatol. 83 (Suppl.), 26s-28s 5 Girl, I. and Danos, O. (1986) Trends Genet. 2, 227-232 6 Yang, Y., Okayama, H. and Howley, P. M. (1985) Proc. Natl Acad. Sci. USA 82, 1030-1034 7 Baker, C. etaL (1987)J. Virol. 61, 962-971 8 Shirasawa, H. et al. (1987)J. Gen. Virol. 68, 583-591 9 Schneider-Gaedicke, A. and Schwartz, E. (1986) F~BOJ. 5, 2285-2292 10 Carpenter, G. (1979) Annu. Rev. Biochem. 48, 193-216 11 Hendler, F. J. and Ozanne, B. (1984) J. Clin. Invest. 74, 647-651 12 Downward, J. et al. (1984) Nature 307, 521-527 13 Matlashewski, G. et al. (1987) EMBOJ. 6, 1741-1746 14 Storey, A. et al. (1988) EMBOJ. 7, 1815-1820 15 McBride, A., Schlegel, R. and HoMey, P. (1988) ~ B O J . 7, 533-539 16 Gift, I. and Yaniv, M. (1988) EMBO J. 7, 2823-2829 17 Lambert, P. E, Spalholz, B. A. and Howley, P. (1987) Cell 50, 69-78 18 Waldeck, W., Rosi, E and Zentgraf, H. (1984) EMBOJ. 3, 2173-2178 19 Baker, C. and Howley, P. M. (1987) EMBOJ. 6, 1027-1035 20 Hermonat, P. A., Spalholz, B. A. and Howley, P. M. (1988) EMBOJ. 7, 2815-2821 21 Stanbridge, E. J. et al. (1982) Science 215, 252-259 22 zur Hausen, H. (1987) Cancer 59, 1692-1696 23 Miesfield, R., Godowski., P. J., Maler, B. A. and Yamamoto, K. R. (1987) Science 236,423-427 24 Strahle, U., Klock, G. and Schutz, G. (1987) Proc. Natl Acad. Sci. USA 84, 7871-7875 25 Pater, M. M. et al. (1988) Nature 335, 832-835 26 McCance, D. (1988) Nature 335, 765-766
rEGULATORYMECHANISMSofthe PAPILLOMAVIRUSES P. WARD*t, D. V. COLEMAN* AND A. D. B. MALCOLM'r *DEPARTMENT OF CYFOPATHOLOGY, ST MARY*SHOSPITAL MEDICAL SCHOOL, LONDON W2 II'G, 1"K. tDEPARTMENT OF BIOCHEMISTRY, CHARING CROSS HOSPITAL MEDICAL SCHOOL, LONDON, 17K.
apillomaviruses are small DNA proteins of the papillomaviruses 6. v~mses that infect higher verte- For example, HPV DNA has been brates, including man. Although found in all cell lines derived from they usually cause warts, these cervical carcinomas; the E6/7 ORFs viruses have recently been associat- are always conserved in these cells ed with malignant tumoursl-4; in and give rise to the most abundant humans the most common associ- mRNA transcripts~. Sequencing of ation is with cervical cancers. DNA from three such lines has The papillomavirus genome revealed a small insert of host DNA consists of a double-stranded loop (intron) in the E6 ORF, giving rise of DNA about 8000 base pairs, to a protein that has some similfunctionally divided into two long arity to epidermal growth factor domains each containing a series (EGF) 9. EGF stimulates cell growth of open reading frames (ORFs) that and division and inhibits many code for viral proteins (Fig. 1). The functions associated with differentiE (early) region contains eight ated cells. Perhaps excess levels of ORFs that code for the proteins an EGF-like protein stimulate associated with genome replication undifferentiated growth of an and control. The L (late) region infected cell 10-12. A similar intecontains two large ORFs coding for gration site is also found within the the structural proteins of the virus E6 ORFs of other HPV types associcapsid. The regulatory region (long ated with cervical cancers, but not control region, LCR) lies between in HPV types 6/11, which are the stop codon of L1 and the start found most commonly in benign codon of the E 6 0 R F . Several lesions of the cervix. promoter sites have been idenTransfection of the E6/7 prodtified throughout the genome; ucts has been shown to result in those of particular interest are cell transformation in vitro 13. found at nucleotides 89, 2443 Moreover, the E7 ORFs of the HPV and 3080 (p89, p2443 and p3080). types 16, 18, 31 and 33 (but not In benign lesions the viral DNA is found as an extrac El I chromosomal closed R I--6-I I~ I E2 II L2 I circular strand (epivP i 0 some); in malignant tumours the viral H [---g/---] I t2 E1 I DNA is often found vP F ~ I 1 [] L1 to be integrated into 6 the host chromosomal material, usually r-gcq r~ Ilm i-~-Ir-E-E~a I L1 at the sites of the v E1 I E1/2 ORFs. The biol6 ogy of the viruses has ] [] recently been reB E1 I viewed in T/Gs. vP [] I L2 II L~ 1
arm E6/70RFs For several years it has been recognized that the E6/7 ORFs code for the major transforming
,gn..
0 p89 Transforming proteins
GENOME
E 2 GENE PRODUCT The protein product of the E2 ORF is the major regulatory protein of the papillomaviruses. This protein has been sequenced in 10 papillomavirus species and although the amino acid sequences are poorly conserved, computer prediction indicates they share a similar structure is (Fig. 2). There are three domains within the protein, two of which are responsible for its biological properties, while the third (domain B) appears to act as a hinge I between the two LOB , p active parts. The 7868 amino-terminal portion of the protein (domain A) is reI LOR ! =.-quired for efficient 7e~5 trans-activation, while domain C is required I for DNA binding l~,. LCR I ~ The whole E2 gene 7902 product has been shown to act as an I enhancer of DNA ,CR transcription, whereas , P p2443 p3080 7945 a smaller protein (E2 Regulatory Capsid Tr) derived from the proteins proteins 3' end of the E2 ORF has repressor activiFIG]Ill ty 17. Both proteins act ORGANIZATION OF TYPICAL PAPILLOMAVIRUSES. by exerting control
TIG APRIL1989 VOL. 5 NO. 4 @1989 Elsevier Science Publisher,, Ltd (UK) 0168 - 952S/89/$02.00
those of types 6 and 11) can act in conjunction with an activated r a s oncogene to induce transformation of primary rat kidney cells i n vitro 1~. Surprisingly, the E7 ORF sequences of all these viruses are very similar, despite their different oncogenic properties. Their differing effects may be the result of an alteration in the structure of the protein products, but this has yet to be shown. The E6/7 transcripts are derived from the p89 promoter, which is regulated by the gene product of the E2 ORF.
WONITOR over transcription initiated at promoter sites within the genome by acting at an enhancer site in the LCR.
LONGCONTROLREGION The length of the LCR of the papillomaviruses varies between species but it has several conserved regions and maintains a similar organization. The 3' region of the LCR is the least conserved and seems to be implicated in the maintenance of the episomal form of the virus in infected cells TM. The 5' end contains several repeats of an ACCG-N<,-CGGT sequence that is c o m m o n to all papillomavirus types. Also within this region are many promoter elements (TATA and CAAT boxes) for polymerase II binding, and many repeats and inverted repeats commonly found in viral control regions. An enhancer that is tran&activated by the gene product of the E2 ORF has been identified in this region, and consists of at least two tandem repeats of the ACCG-N4-CGGT sequenceW. It is at this site that the carboxy-terminal portion of the E2 gene product binds 15A~,. E2 Tr also acts at this site, in this case to repress transcription~7. The repressot and trans-activator proteins share a c o m m o n carboxy terminus and compete for the enhancer sites in the LCR16. Although the exact
mechanism of regulation is uncertain, it may be a result of different levels of the two proteins. The levels of the RNA transcript from p3080 - believed to be the E2 Tr transcript - are at least 10 times those from P2443, which is thought to encode the whole E2 molecule ~9. If these transcripts are equally translated in vivo then the E2 repressor would be more abundant than the E2 enhancer; this ratio could account for the low levels of viral transcription in BPV-l-transformed cells. Most of the mRNA transcripts from cervical cancer cell lines are from the E6/70RFs. The viral DNA in these lines is integrated into the host cell genome at the E2 ORF, which seems to disrupt the normal function of the E2 product. Since the balance of the E2 products inhibits transcription, disruption of this activity may result in uncontrolled expression of the transforming proteins and lead to malignant change in the host tissues. In support of this hypothesis, p89 (the major promoter site for the E6/70RFs) is controlled by the E2 gene product, and is normally held in a repressed state 20. Since the promoter sites p2443 and p3080 are also regulated by this mechanism, it appears that the gene products of the E2 ORF regulate their own transcription. These
G A1 Aa
• prolines I-1 =-helices Domain B
~1 /]-sheets - - uncertain structure O conserved amino acids
V Domain C (DNA-binding domain) FIGlil
STRUCTURE OF THE PAPILLOMAVIRUS E2 GENE PRODUCT (FIGURE MODIFIED FROM REF.
TIG APRIl.1989 VOL. 5 NO. 4 9~
16).
sites are a considerable distance from the LCR enhancer; it may be that regulation is effected by an alteration of the spatial orientation of the bound protein and the DNA.
]~MORALCONTROLMECHANISMS Although papillomavirus DNA is detectable in undifferentiated epithelial cells, virus replication usually takes place within the terminally differentiated kemtinocyte. The reasons for this are unclear, but may involve interplay between host proteins and the viral genome. Evidence for a host-virus interaction was provided by experiments in which HeLa cells (which contain HPV 18 DNA) were fused to normal human fibroblasts. This results in the suppression of the malignant phenotype. In the nonmalignant hybrid, loss of chromosomal material - particularly of human chromosome 11 - from the fibroblast donor results in the reacquisition of malignant growth on transplantation into nude mice 2j (Fig. 3). Furthermore, i*z vitro HeLa cells, malignant hybrids and nonmalignant hybrids express the same levels of HPV 18 mRNA; however, on transplantation into porous chambers grafted onto nude mice, the nonmalignant hybrid cell line fails to produce any HPV 18 RNA whereas the malignant hybrids and the HeLa cell lines continue to do so. It appears therefore that a diffusible protein produced by normal host cells can suppress virus transcription in infected cells 22. This humoral control mechanism, which has yet to be fully eh_lcidated, may be involved in the prevention of malignant transformation in the infected host. Perhaps this explains why although there is a high incidence of latent infection of the female genital tract with HPV types known to be oncogenic, the actual incidence of cervical cancer within the population is less than 1%. Further evidence that humoral factors are involved in the genesis of cervical cancer c(nnes from reports that part of the LCR responds to glucocorticoid hormones 23 and progesterone 2~. HPV 16 DNA (but not HPV 11 DNA) and an activated ras oncogene cause malignant transformation of rat
]~]ONITOR HeLa cell
Normal cell
No regulation~ ofviral ('-' (,~1~ ~ transcription. Cancer inhibiting factor(CIF) gene functionally inactive.
/I ~
~
No viral DNA.
CIF gene inactive.
I FUSION
aCI~O~t~DGD~ENT$ P. Ward is coordinator of an epidemiological study of the risk associated with papillomavirus infection of the cervix and is supported by a grant from the Cancer Research Campaign.
rEFEgENCES 1 Jarrett, w. F. H. etal. (1978) Nature 244, 215-217 2 Kreider, J. w. (1982) Cold Spring Harbor Conf. Cell Proliferation 7,
INVITRO
INVIVO
' ~ CIF not activated
~ ~ g e n e Humoral activatedby HF factor(HF)
Malignantphenotype
Suppressionof malignantphenotype
I
REVERTANTS (Loss of chromosome)
INVITRO
INVlVO
Absenceof functioning CIF gene
ot ableto activateCIF gene
Malignantphenotype
Malignantgrowth FIG~
HOW HUMORAL FACTORS MAY INFLUENCE PAPILLOMAVIRUSTRANSCRIPTION. (FIGURE MODIFIED
FROMREF.22.) primary kidney cells only if the culture medium contains progesterone or dexamethasone 25. While this mechanism may be important in the genesis of cervical cancer, the significance in vivo is at present uncertain a6. I U T U ~ IRIOSI~CI~ The next steps are likely to include the identification of
associated cellular factors by the use of anti-E2 antisera, and the investigation of the E2 protein and the LCR enhancer region using the cloned E2 enhancer elements already identified. The lessons learnt from this investigation should shed light on the mechanisms of genome control n o t only of viruses but also of eukaryotes.
TIG APRIL 1 9 8 9 VOL. 5 NO. 4
99
283-299 3 zur Hausen, H. (1977) Curr. Top. Microbiol. Immunol. 78, 1-30 4 Gissman, L. et al. (1984)./. Invest. Dermatol. 83 (Suppl.), 26s-28s 5 Girl, I. and Danos, O. (1986) Trends Genet. 2, 227-232 6 Yang, Y., Okayama, H. and Howley, P. M. (1985) Proc. Natl Acad. Sci. USA 82, 1030-1034 7 Baker, C. etaL (1987)J. Virol. 61, 962-971 8 Shirasawa, H. et al. (1987)J. Gen. Virol. 68, 583-591 9 Schneider-Gaedicke, A. and Schwartz, E. (1986) F~BOJ. 5, 2285-2292 10 Carpenter, G. (1979) Annu. Rev. Biochem. 48, 193-216 11 Hendler, F. J. and Ozanne, B. (1984) J. Clin. Invest. 74, 647-651 12 Downward, J. et al. (1984) Nature 307, 521-527 13 Matlashewski, G. et al. (1987) EMBOJ. 6, 1741-1746 14 Storey, A. et al. (1988) EMBOJ. 7, 1815-1820 15 McBride, A., Schlegel, R. and HoMey, P. (1988) ~ B O J . 7, 533-539 16 Gift, I. and Yaniv, M. (1988) EMBO J. 7, 2823-2829 17 Lambert, P. E, Spalholz, B. A. and Howley, P. (1987) Cell 50, 69-78 18 Waldeck, W., Rosi, E and Zentgraf, H. (1984) EMBOJ. 3, 2173-2178 19 Baker, C. and Howley, P. M. (1987) EMBOJ. 6, 1027-1035 20 Hermonat, P. A., Spalholz, B. A. and Howley, P. M. (1988) EMBOJ. 7, 2815-2821 21 Stanbridge, E. J. et al. (1982) Science 215, 252-259 22 zur Hausen, H. (1987) Cancer 59, 1692-1696 23 Miesfield, R., Godowski., P. J., Maler, B. A. and Yamamoto, K. R. (1987) Science 236,423-427 24 Strahle, U., Klock, G. and Schutz, G. (1987) Proc. Natl Acad. Sci. USA 84, 7871-7875 25 Pater, M. M. et al. (1988) Nature 335, 832-835 26 McCance, D. (1988) Nature 335, 765-766