Proc. Nail. Acad. Sci. USA Vol. 89, pp. 3957-3961, May 1992 Medical Sciences in the bovine virus Tax protein can abrogate the long terminal repeat-directed transactivating activity without concomitant loss of transforming potential (leukemogenesis/transactivation/zinc finger/) Luc WILLEMS*, CATHERINE GRIMONPONT*, HUBERTINE HEREMANSt, NICOLE REBEYROTTOE, GAO CHEN*, DANIEL PORTETELLE*, ARStNE BURNY*§, AND RICHARD KETTMANN* *Department of Molecular Biology, Faculty of Agronomy, B5030 Gembloux, Belgium; tREGA Institute, B3000 Leuven, Belgium; tInstitut National de la Sante et de la Recherche Mddicale, F33076 Bordeaux, France; and §Department of Molecular Biology, University of Brussels, B1640 Rhode-St-Genese, Belgium Communicated by Marc Van Montagu, January 27, 1992

ABSTRACT The Tax protein trans- In the human T-cell lymphotropic virus (HTLV) system, the activates gene expression directed by the viral long terminal Tax-I protein activates of the genes encoding repeat (LTR) and contributes to immortalization of primary interleukin 2 and its receptor, allowing preferential prolifer- cells. Theoretical analysis of the protein sequence revealed the ation of the HTLV-infected T cells by an autocrine stimula- presence of a putative zinc ringer structure at its amino end. tion process (6). (ii) The Tax protein forms a complex with a Selected mutations in that region completely abolished trans- putative tumor-suppressor gene. Such interactions are in- activation, demonstrating its importance for LTR-directed volved in tumor induction by DNA tumor viruses (adenovi- gene regulation. However, these mutations did not interfere rus, papillomavirus). A role in regulating the expression of with the ability of tax to bind zinc or to contribute to immor- genes involved in cell proliferation, in differentiation, or in talization of primary cells. Thus, transactivation of bovine controlling the initiation ofDNA synthesis may be envisaged. leukemia virus LTR and target cell transformation are inde- According to the first hypothesis, transactivation and pendent functions of Tax and involve different functional transformation by Tax seem to be tightly correlated. In domains of the protein. contrast, the interaction of Tax with a putative tumor- suppressor gene could well involve sequences of the Tax The bovine leukemia virus (BLV) tax gene product (the Tax protein different from those implicated in transactivation. In protein, p34tax) is involved in viral replication and probably this case, it should be possible to isolate transactivation- in leukemogenesis. Tax is a nuclear phosphoprotein trans- deficient tax mutants that are still transformation-competent. lated from the 3' long open reading frame ofthe BLV provirus In this report, we demonstrate that Tax mutated in its and is able to increase long terminal repeat (LTR)-directed putative zinc finger structure may completely lose its trans- gene expression without direct binding to the LTR sequences activating activity while maintaining its transforming capac- (1, 2). As such, Tax acts as a positive regulator of viral ity. transcription. Another property of Tax is its ability to im- mortalize rat embryo fibroblast (REF) cells in culture (3). Furthermore, the Tax protein in cooperation with the Ha-ras MATERIALS AND METHODS oncogene induces foci in vitro that develop into tumors in Plasmids. Plasmids pSV2neo (7), pSV2neoEJ (8), and vivo. BLV tax can thus be classified in the immortalizing pLTRCAT (3) have been described. oncogene subgroup including the adenovirus ElA, simian with the virus 40 T-antigen, and myc genes. These data emphasize the Site-directed mutagenesis was performed pSGtax causal role of tax in BLV-induced tumorigenesis. plasmid (3). Single-stranded DNA was produced by using Previous direct mutagenesis of the tax gene failed to M13K07 helper phage and was extracted as described by identify the regions implicated in its functional activity (4). Promega. Mutants 1, 3, 4, and 5 (pSGtaxMl, -3, 4, and -5) All modifications (amino acid deletions or insertions) abro- were obtained by the Eckstein procedure using the Amer- gated transactivation, suggesting that the present BLV p341 sham kit and the oligonucleotides 5'-ATGCCTGGGGC- structure results from heavy evolutionary constraints. How- CCCCTCT-3', 5'-GCGGGCCCGCTGAGCGAC-3', 5'- ever, by taking advantage of hybrids between well-defined CGCTCACCGGCGAGACCC-3', and 5'-GCGAGACCGC- regions of Tax and the yeast GAL4 transactivator protein, a TCGTATCA-3', respectively. Mutant 2 (pSGtaxM2) was specific domain (amino acids 157-197) was identified as an constructed by the polymerase chain reaction (PCR) using activating region (5). This segment is located approximately two overlapping oligonucleotides containing the desired mu- in the middle of p34tax and is globally neutral (net charge of tation (5'-GCCCCCTCGGCGGGCCCC-3' and 5'-GGGGC- zero). This Tax domain contains 24% of the leucine residues CCGCCGAGGGGGC-3'). All the mutants were sequenced possibly involved in heterologous protein interactions. in order to verify the presence ofthe desired and the The molecular mechanisms by which Tax induces cell absence of other modifications. transformation are not known. However, two main hypoth- The pTITtax+ vector is a prokaryotic expression plasmid eses (that are not mutually exclusive) can be put forward. (i) containing the tax gene cloned downstream of the T7 RNA The Tax protein transactivates transcription ofa cellular gene polymerase promoter (N.R. and R. Mamoun, unpublished involved in cell division or differentiation. The identification work). The Cla I-Nde I fragment of the pSGtaxMl-5 plas- of these genes and their mode of action remains conjectural. mids (these unique sites flank the sequence encoding the zinc

The publication costs of this article were defrayed in part by page charge Abbreviations: BLV, bovine leukemia virus; CAT, chloramphenicol payment. This article must therefore be hereby marked "advertisement" acetyltransferase; HTLV, human T-cell lymphotropic virus; LTR, in accordance with 18 U.S.C. §1734 solely to indicate this fact. long terminal repeat; REF, rat embryo fibroblast.

3957 Downloaded by guest on September 24, 2021 3958 Medical Sciences: Willems et al. Proc. Natl. Acad. Sci. USA 89 (1992) finger) was introduced into the same sites of the pTITtax+ vector, giving rise to pTITtaxM1-5. Expression in Bacteria, Zinc Column Chromatography, and Western Blotting. HMS174 bacteria were transformed with the pTITtax+ and pTITtaxM1-5 vectors, cultivated in M9 medium containing ampicillin, and infected with bacterio- phage CE6 as described by Studier et al. (9). After 2 hr of M3 incubation at 370C, bacteria were pelleted and resuspended in ;ALA36 -.4-HIS36 /,, phosphate-buffered saline. M4 For the zinc column purification, the bacterial pellets were M2 GLY 33 -- CYS33 CYS s _GLY50 resuspended in 20 mM Hepes, pH 7.9/20% (vol/vol) glycer- -Zn ol/50 mM KCl/1 mM dithiothreitol/0.5 mM phenylmethyl- ml 30-.-Q-CYS30 HIS53 ----ALA53 sulfonyl fluoride/6 M guanidinium chloride and incubated for IGLY 1.5 hr at room temperature. The sample was then progres- M5 sively diluted with 20 mM sodium phosphate, pH 7.5/0.5% MET 1 PHE309 Tween 20/0.5 M NaCl to decrease the guanidinium chloride FIG. 1. Schematic organization of the Tax protein [from the concentration to 1 M. The samples were loaded on a 2-ml initiator methionine (MET 1) to the last amino acid (PHE 309)]. The fast-flow Sepharose column (Pharmacia) saturated with theoretical tetrahedral structure ofthe finger is coordinated by a zinc ZnCl2. After extensive washing (five column volumes) with ion (Zn) interacting with three cysteines (positions 30, 33, and 50) and 20 mM sodium phosphate, pH 7.5/0.5% Tween 20/0.5 M one histidine (position 53). Amino acid modifications in the mutants NaCl, bound proteins were eluted with the same buffer (Ml to M5) are indicated. containing 250 mM EDTA. Bacterial lysate proteins were separated in an SDS/10% and histidine residues were modified to either alanine or polyacrylamide gel. Proteins were transferred onto nylon glycine codons (Fig. 1). After mutagenesis, the nucleotide membranes and incubated with a mixture of anti-p34t1 sequences of the mutants were checked for the presence of monoclonal antibodies (dilution 1:200) (4). Immune com- the expected mutations and for the lack of nondirected plexes were revealed by the phosphatase colorimetric pro- modification. cedure (Promega). Interactions Between Zn2+ and Wild-Type or Mutant Tax. Chloramphenicol Acetyltransferase (CAT) Assays. D17 cells The tax gene was cloned into the pTIT prokaryotic expres- (from canine osteosarcoma) or primary REFs were trans- sion vector downstream ofthe 17 RNA polymerase promoter fected by the calcium phosphate coprecipitation method (10). (N.R. and R. Mamoun, unpublished work). After transfor- In short, 3 ,ug of pLTRCAT (which contains the BLV LTR mation of Escherichia coli HMS174 cells and infection with cloned upstream of the CAT gene) and 2 ,g of effector bacteriophage CE6 (which codes for the T7 RNA polymer- plasmid (pSGtax or pSGtaxMl-5) were used to transfect 3 x ase), p341 was correctly expressed as demonstrated by 105 cells. Cytoplasmic extracts were prepared 48 hr after Western blot analysis using a mixture of monoclonal anti- transfection and CAT activity was determined (11). bodies directed against the Tax protein (Fig. 2A, lane a). Transformation and Tumorigenicity Assays. REFs were In order to gain insight into the interaction between p34tax obtained by mincing 14-day Fischer rat embryos (Iffa Credo, and the Zn2+ ion, the bacterial lysate containing the Tax Brussels) in trypsin solution and were cultured in Opti-MEM protein was denatured with 6 M guanidinium chloride and (GIBCO) supplemented with 10% heat-inactivated fetal bo- chromatographed on a fast-flow Sepharose column primed vine serum. One million cells were transfected with 10 ,ug of with Zn2+. After washing, p34tax was absent from the eluate plasmid DNA by calcium phosphate coprecipitation (10). [Fig. 2A, lane b(+)]. p34tax was then eluted in the presence Ability of immortal cells to grow in the presence of 1% or of EDTA [Fig. 2A, lane c(+)]. However, when the column 10% fetal bovine serum was determined from pools of cells. was not primed with Zn2+, no p34t could be recovered, Analysis ofimmortal cells for anchorage-independent growth demonstrating the importance of Zn2+ to mediate the binding was performed in 35-mm culture dishes by overlaying 104 [Fig. 2A, lanes b(-) and c(-)]. cells in Opti-MEM containing 10% fetal bovine serum and To evaluate the ability of the different mutant proteins to 0.3% agar onto a base of 0.5% agar prepared in the same bind Zn2+, the corresponding open reading frames were medium. Colonies were allowed to form at 37°C for 14 days. excised from the recombinant pSG plasmids and introduced Colony formation was determined from triplicate cultures. into the pTIT vector. The mutated Tax proteins were then The tumorigenicity assay was performed 48 hr after trans- expressed in bacteria and chromatographed on a fast-flow fection. REFs were harvested, washed twice with phosphate- Sepharose column primed with Zn2+. Western blot analysis buffered saline, and injected subcutaneously into 5-week-old using a mixture of monoclonal antibodies directed against nude mice (REGA Institute). Tax revealed that all the Tax mutants were able to interact with Zn2+ (Fig. 2B). Transactivation of the BLV LTR by the Mutated Tax RESULTS Proteins. p34w is a transactivator protein able to increase Site-Directed Mutagenesis of the tax Gene. Theoretical LTR-directed gene expression (1, 2). The functional activity analysis ofTax revealed a putative zinc finger structure at the of the Tax protein can be measured by using the pLTRCAT amino-terminal end of the protein. Five amino acids (three reporter, which contains the CAT gene cloned downstream of cysteines at positions 30, 33, and 50 and two histidines at the BLV LTR. When pLTRCAT and pSGtax (which contains positions 36 and 53) could be involved in a tetrahedrical the tax gene cloned downstream of the simian virus 40 complex able to bind divalent cations, allowing the formation promoter) were transfected into D17 or REF cells, the of a finger (amino acids 34 or 37 to 49) (Fig. 1). wild-type Tax protein increased LTR-directed CAT gene To evaluate the role ofthat theoretical zinc finger structure expression (Fig. 3). The vectors expressing the mutated Tax in the functional activity of Tax, five mutants were con- proteins (pSGtaxM1-5) were also cotransfected into D17 or structed by site-directed mutagenesis. Single point mutations REF cells with the pLTRCAT reporter. Expression of the were introduced into the tax gene cloned in the pSG5 vector various Tax proteins in D17 cells was verified by immuno- (plasmid pSGtax; ref. 3). In order to interfere with metal-zinc precipitation (data not shown). Only pSGtaxM3 was able to finger interaction, the codons corresponding to the cysteine direct the synthesis ofa protein able to transactivate the LTR Downloaded by guest on September 24, 2021 Medical Sciences: Willems et al. Proc. Natl. Acad. Sci. USA 89 (1992) 3959

A

a b c b c (-) (+) B

a b c a c b a b c a b c a b c I 2 3 5

FIG. 2. Interaction between Tax and Zn2+. After denaturation with guanidinium chloride, the samples were loaded on a fast-flow Sepharose column (Pharmacia) saturated with ZnC12. Samples were collected before loading (lanes a), at the last wash (lanes b), and after elution (lanes c). The aliquots were then analyzed by Western blotting using a mixture of anti-p34t' monoclonal antibodies. A ZnC12-containing (+) or a ZnCl2-free (-) solution was loaded on the column prior to Tax protein binding. Arrowheads indicate p34,ax. (A) Wild-type Tax. (B) Mutants Ml to M5.

promoter above background level. All the other mutations that Cys30, Cys33, Cys50, and His53 of Tax are essential for tested abolished the Tax-induced transactivation. It appears transactivation of the BLV LTR. Transformation of Primary Cells by the Mutated Tax Pro- 120 teins. The tax gene acts as an immortalizing oncogene in culture and is able to cooperate with the Ha-ras oncogene to D17 IIREF induce tumors in vivo (3). When the pSGtax plasmid (together with pSV2neo, which allows selection of colonies with the 100 neomycin analogue G418) was transfected into primary REFs, immortal clones were isolated in culture (Table 1), confirming previous results (3). However, none of them exhibited a transformed phenotype according to two trans- formation criteria: growth in soft agar and growth in medium 80i - with low serum. Furthermore, nude mice injected with these transfected cells did not develop tumors. In contrast, when the pSGtax vector was cotransfected with pSV2neoEJ 60 (which corresponds to pSV2neo and contains the Ha-ras oncogene), transformation of the REFs occurred allowing 40 selection offoci in vitro and tumor induction in vivo (Table 1). Surprisingly, when the mutants were used in this transfor- mation assay, similar results were obtained except for mu- tants 2 and 4. Since mutants 1, 2, 4, and 5 (in contrast to Table 1. Transformation of primary REFs by wild-type and mutant Tax proteins 2040 No. of Trans- Tumorigenicity Gene(s) foci formation* Tumors PCR tax 0 -/- 0/6 ND tax + ras 51 +/+ 6/6 + WT M 1 M2 M3 M4 M5 M1 + ras 9 +/+ 6/6 + M2 + ras 34 +/+ 5/6 + FIG. 3. Transactivation of the BLV LTR by wild-type (WT) and M3 + ras 20 +/+ 6/6 + mutant (M1-5) Tax proteins. D17 or REF cells were transfected with M4+ras 5 +/+ 5/6 + 3 g.g of pLTRCAT (which contains the BLV LTR cloned upstream M5 + ras 5 +/+ 4/6 + of the CAT gene) and 2 Asg of an effector plasmid (pSGtax or -/- ND pSGtaxM1-5). Forty-eight hours after transfection, CAT activity ras 0 0/6 was determined. The data were normalized to the CAT activity ND, not done. obtained after transfection of the pSGtax and pLTRCAT plasmids. *Growth in soft agar/growth in low-serum medium. Downloaded by guest on September 24, 2021 3960 Medical Sciences: Willems et al. Proc. Natl. Acad. Sci. USA 89 (1992) mutant 3) are unable to transactivate LTR-directed gene behavior: zinc finger, ATPase, origin binding, nuclear local- expression, it appears that transformation can be indepen- ization, host range, transactivation, and transformation. The dent of LTR transactivation. As a control, the ras oncogene molecular mechanism by which the T antigen induces cellular alone was unable to induce tumors in vivo (Table 1). transformation is unknown. However, two cellular proteins To verify that the sequences corresponding to the different [the retinoblastoma-susceptibility gene product (pl05RB) and mutants were indeed present in the induced tumors, chro- ] form complexes with the T antigen (17, 18). The T mosomal DNA was extracted and PCR amplifications were antigen encodes at least two distinct activities capable of conducted using two primers flanking the zinc finger. Nu- influencing transformation (19). The first domain, located in cleotide sequence demonstrated that the tumors originated the amino-terminal end of the protein, is responsible for the from REF cells transfected by the respective mutants (Table immortalizing activity as well as its ability to cooperate with 1, PCR+; data not shown). the ras oncogene. The transformation activity ofthis domain may involve complex formation with the retinomablastoma DISCUSSION gene product. The transactivation function of T antigen also resides within this domain, and thus this activity may also Site-directed mutagenesis ofthe putative zinc finger structure play a role in transformation. Another region, located in the of the BLV Tax protein demonstrated that this domain is central portion of the protein, is also involved in transfor- absolutely required for the transactivation function of Tax. Four amino acids were implicated in this structure: Cys30, mation, probably by complex formation with p53 (19). A Cys33, Cys50, and His53. His36 does not seem to be essential putative tumor-suppressor gene corresponding to the BLV to the transactivation function. Furthermore, another cys- Tax protein is still unknown. Cotransfection of p105RB and teine in the vicinity of the zinc finger, Cys64, does not seem p53 expression vectors into primary cells failed to interfere to exert a crucial role since it is not conserved between two with transformation by tax and ras (data not shown). BLV isolates (12, 13). Although we favor the tumor-suppressor gene hypothesis, Consequently, at least two domains are required for trans- we cannot exclude the possibility that transformation in- activation by Tax. The activation region (amino acids 157- volves the transcriptional activation of a cellular gene by a 197; ref. 5) and the putative zinc finger structure most different region of the Tax protein. Smith and Greene (16) probably are involved in protein-protein interactions since reported the identification of missense mutations in the Tax does not bind DNA. HTLV-I Tax protein that functionally segregate transactiva- It is of particular interest that the transactivation function tion ofthe LTR and transactivation the interleukin 2 receptor does not correlate with the ability of Tax to bind Zn2 . Point gene promoter. Furthermore, recent experiments suggested mutations of single cysteine or histidine residues do not that transactivation through the CREB (cAMP response abolish the ability ofTax to interact with the Zn2+ ion. Similar element binding) pathway plays a central role in transforma- results were obtained for the human immunodeficiency virus tion of rat fibroblasts (20). A mutant lacking the ability to Tat protein (14). We hypothesize that a single point mutation transactivate CRE-dependent promoters (but still inducing may be complemented by dimer formation. Ifso, the Zn2+ ion NF-KB) did not transform Rat-2 cells. Our data in the BLV could form a tetrahedric structure between two Tax mole- system support but do not prove the idea that transformation cules and allow binding of this complex on a fast-flow may involve an alternative mechanism to the direct transac- Sepharose column. Dimerization of the HTLV-I Tax protein tivation of cellular genes involved in growth regulation. was recently suggested by crosslinking experiments with The segregation of the transactivation and transformation glutaraldehyde (15). Alternatively, a trimeric structure could activities may have important implications in vivo. Indeed, be sufficient to stabilize Zn2+. transactivation of the LTR by Tax stimulates viral gene It has been suggested that the zinc finger structure of expression and induces a strong immunological response HTLV-I Tax constitutes a nuclear localization domain: de- destroying the BLV-infected cells. In this case, the leukemia letion mutants, predominantly located in the cytoplasm, would never arise. Therefore, nonexpression of the provirus constitute transdominant inhibitors by sequestering the wild- (and thus inhibition of the transactivation function of Tax) type Tax protein outside of the nucleus (15, 16). In the BLV seems to be absolutely required. Since the transformation system, our Tax mutants were unable to act as transdominant and transactivation activities are separated, it is reasonable to repressors of transactivation. Furthermore, indirect immu- hypothesize that the domains could also act independently. nofluorescence analysis showed that the zinc finger mutant In this model, the Tax transformation function would induce proteins (M1-5) were still located in the nucleus (data not leukemogenesis without transactivation of the viral LTR. shown). These observations suggest that the lack of trans- In conclusion, our data demonstrate that the transactiva- activation of the BLV LTR is not due to the absence of Tax tion function of Tax can be completely independent of its in the nucleus and that transactivation requires a complete immortalization property and its ability to cooperate with the finger structure to allow protein-protein interactions in the ras oncogene to induce transformation. transcription complex. The identities of the putative cellular Tumorigenicity assays were performed in Prof. Billiau's labora- proteins binding to Tax through its zinc finger domain are tory at the REGA Institute. We are grateful to R. Martin and C. Dillen unknown. for skillful technical help and to G. Buchet for photographic illus- The most striking point of this report is that the four trations. The phage CE6 and the pTITtax+ vector were kindly mutants in the zinc finger structure (Ml, M2, M4, and M5) provided by Drs. D. Derse and R. Mamoun, respectively. R.K. and that have lost their transactivating capacity are still able to act L.W. are, respectively, Directeur de Recherches and Chercheur as immortalizing oncoproteins (although the transformation qualifie of the Fonds National Belge de la Recherche Scientifique. potential of some mutants is decreased in comparison to the C.G. is supported by a Televie grant. We thank the Caisse Generale wild-type Tax protein). Transactivation and transformation d'Epargne et de Retraite for financial support. are consequently due to different activities of the protein. Such a modular organization ofthe Tax 1. Derse, D. (1987) J. Virol. 61, 2462-2471. transcriptional factor 2. Willems, L., Gegonne, A., Chen, G., Burny, A., Kettmann, R. has some homologies with tumor antigens of the DNA & Ghysdael, J. (1987) EMBO J. 6, 3385-3389. viruses. As such, T antigen of simian virus 40 is a multifunc- 3. Willems, L., Heremans, H., Chen, G., Portetelle, D., Billiau, tional protein. The single polypeptide chain carries multiple A., Burny, A. & Kettmann, R. (1990) EMBO J. 9, 1577-1581. biochemical domains that act alone or in a concerted manner 4. Willems, L., Chen, G., Portetelle, D., Mamoun, R., Burny, A. to control various aspects of viral infection and cellular & Kettmann, R. (1989) Virology 171, 615-618. Downloaded by guest on September 24, 2021 Medical Sciences: Willems et al. Proc. Natl. Acad. Sci. USA 89 (1992) 3961

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