Proc. Natl. Acad. Sci. USA Vol. 87, pp. 5440-5443, July 1990 Immunology Complementary DNA encoding the human T-cell FK506-binding , a peptidylprolyl cis-trans distinct from (cyclosporin A/immunosuppressant/T-cell activation//amino acid sequence) NOBORU MAKI, FUMIKO SEKIGUCHI, JUNICHI NISHIMAKI, KEIKO MIWA, TOSHIYA HAYANO, NOBUHIRO TAKAHASHI*, AND MASANORI SUZUKI Corporate Research and Development Laboratory, Tonen K. K., 1-3-1 Nishitsurugaoka, Ohi-machi, Iruma-gun, Saitama 354, Japan Communicated by Frank W. Putnam, April 23, 1990 (receivedfor review February 23, 1990)

ABSTRACT The recently discovered macrolide FK506 has cis-trans isomerase (PPIase) and that CsA inhibits its activity been demonstrated to have potent unosuppressive activity led to the hypothesis that the action of CsA (for example, at concentrations 100-fold lower than cyclosporin A, a cyclic immunosuppressive action in T cells) is mediated through undecapeptide that is used to prevent rejection after trans- inhibition of the PPIase activity (4). Recently a cellular plantation of bone marrow and organs, such as kidney, heart, binding protein for FK506 (FKBP) was also found to have the and liver. After the recent discovery that the cyclosporin same enzymatic activity as cyclophilin. However, cyclo- A-binding protein cyclophilin is identical to peptidylprolyl philin and FKBP are quite distinct in terms of ligand speci- cis-trans isomerase, a cellular binding protein for FKS06 was ficity; cyclophilin binds to, and is inhibited by, CsA but does found to be distinct from cyclophilin but to have the same not recognize FK506, whereas the converse holds for FKBP enzymatic activity. In this study, we isolated a cDNA coding for (5, 6). On the basis of earlier data, which indicated that CsA FK506-binding protein (FKBP) from human peripheral blood and FK506 act on T lymphocytes in an essentially equivalent T cells by using mixed 20-mer oligonucleotide probes synthe- fashion (1), these findings suggest that the two drugs act sized on the basis of the sequence, Glu-Asp-Gly-Lys-Lys- through distinct pathways but that their mode of action Phe-Asp, reported for bovine FKBP. The DNA isolated con- converges on PPIase activities. tained an open reading frame encoding 108 amino acid resi- FKBP is abundant and is found in Jurkat T cells, bovine dues. The first 40 residues of the deduced amino acid sequence thymus, and human spleen (5, 6). FK506 binds to its protein were identical to those ofthe reported amino-terminal sequence with a 1:1 stoichiometry and an affinity close to 1 nM. FKBP of bovine FKBP, indicating that the DNA sequence isolated exhibits a 15- to 20-fold lower specific PPIase activity than represents the coding for FKBP. Computer-assisted anal- that of toward a synthetic substrate (5). The ysis of the deduced amino acid sequence indicates that FKBP molecular weight of FKBP reported by two groups is some- exhibits no internal homology and does not have sgificant what at variance (Mr = 11,000 and 14,000) and differs from sequence similarity to any other amino acid sequences ofknown that of cyclophilin (Mr of pig cyclophilin = 18,000) (5, 6). , including cyclophilin. This result suggests that two However, except for the amino-terminal sequence, which catalytically similar proteins, cyclophilin and FKBP, evolved was found to be distinct from that of cyclophilin (6), little is independently. In Northern blot analysis, mRNA species of known about the chemical structure of FKBP. In this paper, -1.8 kilobases that hybridized with human FKBP cDNA were report the sequence of human FKBP.t detected in poly(A)+ RNAs from brain, lung, liver, and pla- we nucleotide cental cells and leukocytes. Induction ofJurkat leukemic T cells with phorbol 12-myristate 13-acetate and ionomycin did not MATERIALS AND METHODS affect the level of FKBP mRNA. Southern blot analysis of human genomic DNA digested with different restriction en- Screening and Sequencing of a FKBP cDNA. A cDNA zymes suggests the existence of only a few copies of the DNA library (Agtll) of phytohemagglutinin-stimulated human pe- sequence encoding FKBP. This is in contrast to the result that ripheral blood T cells was purchased from Clontech. Esch- as many as 20 copies of the cyclophilin gene and possible erichia coli strain Y1090 was transfected with the library, and may be present in the mammalian genome. a total of 2 x 104 transfectants replica-plated on nylon filters pseudogenes were screened for FKBP cDNA sequences by using a mix- ture of 20-mer oligonucleotides as a probe. The oligonucle- Cyclosporin A (CsA), a cyclic undecapeptide, and the re- otide mixture [5'-GA(A/G)GA(T/C)GG(G/A/T/C)AA(A/ cently discovered FK506, a macrolide, are powerful immu- G)AA(A/G)TT(T/C)GA-3', 128 different sequences] was nosuppressants; CsA has been used clinically in the preven- synthesized based on the sequence, Glu-Asp-Gly-Lys- tion of graft rejection following bone marrow and organ Lys-Phe-Asp, reported for bovine FKBP (6) and labeled with transplantation (1). CsA and FK506 are chemically distinct, T4 polynucleotide kinase (Takara, Kyoto) and [(-32P]ATP but the effect of the two compounds on immunosuppression (Amersham) as described by Maniatis et al. (7). Hybridiza- appears to be remarkably similar, though FK506 is said to be tion was performed overnight at 37°C in 6x SSC (lx SSC = 100 times more potent than CsA in this regard (1). Although 0.15 M NaCI/0.015 M sodium citrate, pH 7) containing 5X the binding of CsA to calmodulin has been described (2), the Denhardt's solution (1x Denhardt's = 0.02% bovine serum action of CsA is thought to be mediated through cyclophilin, albumin/0.02% Ficoll/0.02% polyvinylpyrrolidone), 0.5% an abundant cytosolic protein representing most of the CsA- NaDodSO4, and denatured salmon sperm DNA at 50 binding activity in nearly all organs and cells (3). Our previ- ,ug/ml. ous discoveries that cyclophilin is identical to peptidylprolyl Abbreviations: PPIase, peptidylprolyl cis-trans isomerase; FKBP, FK506-binding protein; CsA, cyclosporin A. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M34539). 5440 Downloaded by guest on September 26, 2021 Immunology: Maki et al. Proc. Natl. Acad. Sci. USA 87 (1990) 5441 at EcoR I -60 -30 After hybridization, the filters were washed in 2x SSC GAATTCGGGC CGCCGCCAGG TCGCTGTTGG TCCACGCCGC CCGTCGCGCC GCCCGCCCGC 370C, and autoradiography was carried out at -70TC for 10 hr 1 30 with an intensifying screen. TCAGCGTCCG CCGCCGCC ATG GGA GTG CAG GTG GAA ACC ATC TCC CCA GGA Phage DNA from a positive clone was extracted by the lot Gly Val Gin Val Glu Thr 11e Ser Pro Gly method of Maniatis et al. (7). The insert was cleaved with 80 GAC GOG CGC ACC TTC CCC AAG COC GGC CAG ACC TGC GTG GTG CAC TAC EcoRI, isolated by the glass-powder method, and recloned Asp Gly Ars Thr Phe Pro Lys Arg GIY Gin Thr Cys Vol Val His Tyr into the pUC19 vector. The recloned fragment was digested 90 120 and subcloned into M13 phage vector for nucleotide sequenc- ACC GGG ATG CTT GAA CAT GOA AAG AAA TTT GAT TCC TCC COG GAC AGA ing by the dideoxy method of Sanger et al. (8). Thr Gly Het Lou Glu Asp Gly Lys LYs Phe Asp Ser Ser Ar8 Asp Ar8 Analysis. Poly(A)+ RNAs from 150 Northern and Southern Blot AAC AAG CCC TTT AAG TTT ATG CTA GGC AAG CAG GAG GTG ATC CGA GGC human brain, liver, placenta, lung, and leukocytes were Asn LYs Pro Phe Lys Phe Het Leu Gly Lys Gin Glu Val Ile Ar8 Gly purchased from Clontech, and total RNA from Jurkat cells 180 210 was isolated by the method of Maniatis et al. (7). Jurkat cells TGG GAA GAA GGG OTT GCC CAG ATC AGT GTC GGT CAG AGA GCC AAA CTG were kindly provided by J. Minowada of Hayashibara Bio- Trp Glu Glu Gly Val Aim Gin Hot Ser Val Gly Glu Arg Ala Lys Leu 240 270 chemical Laboratory (Okayama, Japan). The RNAs were ACT ATA TCT CCA GAT TAT 0CC TAT GOT GCC ACT GGG CAC CCA GGC ATC fractionated on a 1% agarose gel containing formaldehyde (7) Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr GIy His Pro Gly Ile and transferred to a nylon membrane (Hybond N, Amer- 300 sham). The filter was processed according to the manufac- ATC CCA CCA CAT 0CC ACT CTC GTC TTC GAT GTC GAG CTT CTA AAA CTG 5 hr in Ile Pro Pro His Ala Thr Lou Val Phe Asp Val Glu Lou Leu Lys Leu turer's instructions and then prehybridized at 65TC for 330 360 lx SSC, 5x Denhardt's solution, 0.2% NaDodSO4, and GAA TGA CA GGAATGGCCT CCTCCCTTAG CTCCCTGTTC TTGGATCTGC CATGGAGGGA salmon sperm DNA at 100 ,ug/ml. It was then hybridized for Glu 16 hr in the same solution to a 32P-labeled probe prepared by 390 420 random priming of the isolated FKBP cDNA by using a kit TCTGGTGCCT CCAGACATGT GCACATGAGT CCATATGGAG CTTTTCCTGA TGTTCCACTC 450 480 from Boehringer Mannheim. The filter was subsequently CACTTTGTAT AGACATCTGC CCTGACTGAA TGTGTTCTGT CACTCAGCTT TGCTTCCGAC washed at 65TC with decreasing concentrations of salt; the 510 540 final wash was in 0.1x SSC containing 0.2% NaDodSO4. ACCTCTGTTT CCTCTTCCCC TTTCTCCTCG TATGTGTGTT TACCTAAACT ATATOCCATA High molecular weight DNA from human placenta was 570 600 AACCTCAAGT TATTCATTTT ATTTTGTTTT CATTTTCGGG TGAAGATTCA GTTTCAGTCT purchased from Pharmacia and was digested to completion (4 630 660 hr) with five different restriction (EcoRI, BamHI, TTTGGATATA GGTTTCCAAT TAACTACATG GTCAAGTATT AACAGCACAA GTGGTAGGTT HindIII, Sac I, and Xba I), separated on a 0.8% agarose gel, 690 720 SspI and transferred to a nylon membrane. It was then analyzed AACATTAGAA TAGGAATTGG TGTTGGGGGG GOGGTTTGCA AGAATATTTT ATTTTAATTT 750 780 by Southern blot analysis (9). TTTGGATGAA ATTTTTATCT ATTATATATT AAACATTCTT GCTGCTGCGC TGCAAAGCCA 810 840 TAGCAGATTT GAGOCGCTCT TGAGGACTGA ATTACTCTCC AAGTTGAGAG ATGTCTTTGG RESULTS AND DISCUSSION 870 900 Human T-Cell FKBP cDNA. A total of 2 x 104 transfor- GTTAAATTAA AAGCCCTACC TAAAACTGAG CTOGGCATGG GGAGAGCCTT TGCCTCCACC 930 960 mants of a phytohemagglutinin-stimulated human T-cell ATTCCCACCC ACCCTCCCCT TAAACCCTCT GCCTTTGAAA GTAGATCATG TTCACTGCAA cDNA library were screened with the mixture of 128 20-mer 990 1020 oligonucleotides described in Materials and Methods. Two TGCTGCACAC TACAGGTATC TGTCCCTOGG CCAGCAGGGA CCTCTGAAGC CTTCTTTGTG positive clones were detected and found to contain a similar- 1050 1080 GCCTTTTTTT TTTTTCATCC TGTGGTTTTT CTAATGGACT TTCAGGAATT TTGTAATCTC length (1.5-kilobase) EcoRI insert. They were subcloned into 1110 1140 the EcoRI site of pUC19 and were sequenced by using an ATAACTTTCC AAGCTCCACC ACTTCCTAAA TCTTAAGAAC TTTAATTGAC AGTTTCAATT M13-derived vector as described (8). 1170 1200 The restriction map and sequencing strategy for the cDNA GAAGGTGCTG TTTGTAGACT TAACACCCAG TGAAAGCCCA GCCATCATGA CAAATCCTTG clone are shown in Fig. 1. The nucleotide sequence 1230 1260 (FKBP2) AATGTTCTCT TAAGAAAATG ATGCTGGTCA TCGCAGCTTC AGCATCTCCT GTTTTTTGAT of the insert in clone FKBP2 and the deduced amino acid 1290 1320 sequence are shown in Fig. 2. The cDNA contains an open GCTTGGCTCC CTCTGCTGAT CTCAGTTTCC TGGCTTTTCC TCCCTCAGCC CCTTCTCACC reading frame of 324 nucleotides that encodes 108 amino acid 1350 1380 residues, including the initiator methionine for FKBP. The 5' CCTTTCCTGT CCTGTGTAGT GATTTGGTCA GAAATCGTTG CTGCACCCTT CCCCCAGCAC untranslated sequence extends 78 bases upstream from the 1410 1440 EcoRI first AUG codon. The UGA stop codon is followed by about CATTTATGAG TCTCAAGTTT TATTATTGCA ATAAAAGTGC TTTATGCCCG AATTC 1.1 kilobases of 3' noncoding region. However, the poly(A)+ FIG. 2. Nucleotide and predicted amino acid sequences ofhuman tail of the mRNA was not contained in the cDNA clone. FKBP. The complete nucleotide sequence of the FKBP2 insert is Another clone, FKBP1, was also sequenced, but no differ- indicated, and nucleotide residues are numbered above the sequence beginning at the initiation codon. The location of the sequence lOObp detected by the synthetic oligomeric probe is indicated by a line AflilAf above the corresponding nucleotide sequence. The residues in the 5' [EcoRI]NcoI SmaI NcoI HpaISsPI III [EcoRI] untranslated region are indicated by negative numbers. The consen- sus signal for polyadenylylation is underlined. The deduced amino acid sequence is shown below the corresponding nucleotide se- quence.

4 ence was found in the sequence that overlaps the FKBP2 insert. FIG. 1. Sequencing strategy and partial restriction map of the From the deduced amino acid sequence, the mature FKBP, FKBP2 cDNA insert. The open box represents the coding region. is a protein of 108 amino Arrows indicate the direction and extent of sequencing by the including the initiator methionine, dideoxy method. Only the restriction sites used for the subcloning acids and has a molecular weight of 11,951. The amino- are shown. The EcoRI sites in brackets were generated during the terminal 40 residues of the deduced amino acid sequence cloning procedure. bp, Base pairs. (except for the initiator methionine) are completely identical Downloaded by guest on September 26, 2021 5442 Immunology: Maki et al. Proc. Natl. Acad. Sci. USA 87 (1990)

3 *0 2 ._

1 .1~~~~~~~~~~~~~~~~~~ :: s._ x0 0

o ._ 1 :: L. 2 ::fl!i z3 3

2 0 40 60 80 1 00 Sequence number

FIG. 3. Hydrophathy profile (16) and secondary structure predictions (17) for human FKBP calculated by use of the IDEAS program. In the secondary structure prediction, the residues are represented by dots and are shown in a-helical (X, 3-sheet (M), and 1-turn (f) conformational states.

to those reported for bovine FKBP, and the molecular weight identify any significant between cyclo- calculated from the sequence is in good agreement with that philin and FKBP even in a short stretch of their amino acid estimated by NaDodSO4/PAGE ofthe protein from Jurkat T sequences. Although the E. coli PPIase is insensitive to CsA, cells reported by Siekierka et al. (5). In addition, the isoelec- its amino acid sequence is also related to those ofcyclophilins tric point (pI 8.71) ofthe protein calculated from the deduced (T.H., N.T., N.M., M.S., and S. Kato, unpublished data). sequence is also fairly consistent with the value (pI 8.8-8.9) However, FKBP and cyclophilin, which have the same reported by Harding et al. (6). Furthermore, in order to enzymatic activity, are not related in their amino acid se- confirm that the isolated cDNA encodes FKBP, the Nco I quence, suggesting the presence of at least two separate fragment of FKBP2 cDNA, which included the entire open superfamilies for PPIase. reading frame of FKBP, was translated by constructing the A hydropathy plot ofthe sequence ofFKBP does not show expression vector, pJDB207, under control of the glyceral- any striking hydrophobic region that could serve as a signal dehyde-3-phosphate dehydrogenase promoter in AH22 peptide or a transmembrane segment (Fig. 3), indicating that cells. NaDodSO4/PAGE analysis ofthe protein expressed in the FKBP is a typical intracellular, cytosolic protein. No yeast cells showed that the molecular weight of FKBP information is available from physical measurements about synthesized was in good agreement with that reported by the secondary structure of FKBP, so we used the computer Siekierka et al. (10). These results indicate that the isolated program CHOFAS to predict the secondary structure using the cDNA sequence encodes FKBP. Chou-Fasman parameters. Overall, the distribution pre- Structural Features of FKBP. To investigate the possible dicted for the 108 residues is 12% ,8-pleated sheet, 33% occurrence of sequences homologous to FKBP in other 13-turn, and 37% a-helix. 13-Turn and a-helix are predicted to proteins, we used the IDEAS program to compare the entire dominate the secondary structure of FKBP. sequence to the Protein Identification Resource, GenBank, Northern Blot Analysis. By using 32P-labeled human FKBP and EMBL data bases (October 1989) accessed through the cDNA as a probe, poly(A)+ RNAs from several different DNA Data Bank of Japan (DDBJ; National Institute of tissues were analyzed by Northern blot analysis under strin- Genetics, Center for Genetic Information Research Labora- gent conditions as described in Materials and Methods. As tory ofGenetic Information Analysis, Mishima, Sizuoka 411, shown in Fig. 4A, only one band (about 1.8 kilobases) that Japan). The programs used, IDEAS, COMPARE-DOTPLOT, and CHOFAS, were also provided by the DDBJ. The amino acid A B sequence of FKBP was found to be unique in that no protein 3 4 or any segment gave a significant score for homology. A 1 2 :) 1 2 graphic matrix plot of the FKBP sequence against itself and against the sequences of cyclophilins/PPlases from mam- mals (4, 11-13), fly (14), fungi (15), yeast (N.M., N.T., F.S., T.H., M.S., and K. Watanabe, unpublished data), and E. coli (T.H., N.T., N.M., M.S., and S. Kato, unpublished data) was prepared by the COMPARE-DOTPLOT program. This sug- gested that FKBP does not have internal homology within its own amino acid sequence and does not have sequence similarity to cyclophilins/PPIases including E. coli PPIase, which is not sensitive to CsA (T.H., N.T., N.M., M.S., and S. Kato, unpublished data). Despite the enzymatic similarity between FKBP and cyclophilins/PPIases, the highest iden- tity found in their amino acid sequences was only three consecutive residues [e.g., Phe518Met'9-Leu6W (with yeast and Neurospora crassa cyclophilins/PPlases), Ala'40-Thr141- Gly142 and Gly'38-Val'39-Ala140 (with E. coli PPIase), and (with and bovine FIG. 4. (A) Northern blot analysis of poly(A)+ RNA (5 ,.g each Lys82-Phe83-Asp84 pig cyclophilins/ liver Phe-Met-Leu was located in lane) isolated from human lung (lane 1), placenta (lane 2), (lane PPIases)]. In these sequences 3), leukocytes (lane 4), and brain (lane 5). (B) Northern blot analysis one of the two regions that were identified to be highly of total RNA (15 ,ug each) isolated from Jurkat cells induced with conserved in the amino acid sequences of cyclophilins/ phorbol 12-myristate 13-acetate and ionomycin (lane 1) or from PPIases across species (T.H., N.T., N.M., M.S., and S. uninduced Jurkat cells (lane 2). Jurkat cells were treated with phorbol Kato, unpublished data). However, we don't know yet 12-myristate 13-acetate and ionomycin for 16 hr according to the whether or not this amino acid sequence is involved in the method described by Siekierka et al. (10). The arrow indicates the catalytic sites of the two enzymes. Thus, we were not able to position of the 1.8-kilobase mRNA species. Downloaded by guest on September 26, 2021 Immunology: Maki et al. Proc. Natl. Acad. Sci. USA 87 (1990) 5443 A B FKBP2 cDNA insert (Fig. 5). For any of the restriction fragments, only a few bands were found to hybridize with the FKBP insert, suggesting that only a few copies of the FKBP gene are present in the . The multiplicity ofthe bands may be explained either by the possible split of a gene into multiple exons or by the presence of one or two other 23 -O DNA sequences that hybridized with the FKBP gene insert 9.4 .-. isolated. In any case, this finding is in contrast to the results 6.6 that the for cyclophilin and its homologue appear to be 4 .4--. present in as many as 20 copies in the mammalian genome 2.3* (12, 13). Those results imply that cyclophilin genes have 2.0 diverged extensively during evolution and that they consti- tute a large gene family but that the FKBP gene, on the other hand, has not diverged as much. The difference in genomic 0.5--. complexity between the cyclophilin and FKBP genes may also indicate that the functional diversity of FKBP is much less than that of cyclophilin. We thank Dr. J. Minowada for providing Jurkat cells and Mr. K. FIG. 5. Southern blot analysis of genomic DNA prepared from Urakami for synthesis of the DNA probe. human placenta. (A) Twenty micrograms of high molecular weight DNA digested to completion by EcoRI (lane 1), BamHI (lane 2), 1. Thomson, A. W. (1989) Immunol. Today 10, 6-9. HindIII (lane 3), Sac I (lane 4), or Xba I (lane 5) was analyzed with 2. Colombani, P. M., Robb, A. & Hess, A. D. (1985) Science 228, the FKBP cDNA probe described in the text. (B) As a control 337-339. experiment, Southern blot analysis was done under the same con- 3. Handschumacher, R. E., Harding, M. W., ditions with a human cyclophilin cDNA probe, and this showed that Rice, J., Drugge, more than 20 bands in the human genome hybridized with the probe. R. J. & Speicher, D. W. (1984) Science 226, 544-547. Molecular are 4. Takahashi, N., Hayano, T. & Suzuki, M. (1989) Nature (Lon- size markers (in kilobases) indicated at the left. don) 337, 473-475. 5. Siekierka, J. J., Hung, S. H. Y., Poe, M., Lin, C. & Sigal, hybridized with FKBP cDNA was detected in all poly(A)+ N. H. (1989) Nature (London) 341, 755-757. RNA preparations so far analyzed (e.g., RNAs from human 6. Harding, M. W., Galat, A., Uehling, D. E. & Schreiber, S. L. brain, lung, placenta, liver, leukocytes, and Jurkat cells), (1989) Nature (London) 341, 758-760. indicating that the gene for FKBP is not expressed specifi- 7. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular cally in T cells but rather is expressed in many tissues. To Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold search for the possible involvement of FKBP in the events Spring Harbor, NY). leading to T-cell activation, the effect of mitogenic stimula- 8. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. tion ofT cells on FKBP expression was examined. However, 9. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. induction of Jurkat T cells with phorbol 12-myristate 13- 10. Siekierka, J. J., Staruch, M. J., Hung, S. H. Y. & Sigal, N. H. acetate and ionomycin induction of Jurkat T cells did not (1989) J. Immunol. 143, 1580-1583. affect the level of FKBP mRNA (Fig. 4B), suggesting that 11. Harding, M. W., Handschumacher, R. E. & Speicher, D. W. FKBP is not an inducible protein in response to these (1986) J. Biol. Chem. 261, 8547-8555. stimulations. The result was very similar to that of cyclo- 12. Haendler, B., Hofer-Warbinek, R. & Hofer, E. (1987) EMBO philin [e.g., induction of Jurkat cells by phytohemagglutinin J. 6, 947-950. and ester not 13. Danielson, P. E., Forss-Petter, S., Brow, M. A., Calavetta, L., phorbol did alter the transcription level of Douglass, J., Milner, R. J. & Sutcliffe, J. G. (1988) DNA 7, cyclophilin (12)]. Nonetheless, a report that a much greater 261-267. level of cyclophilin was expressed in leukemic T cells com- 14. Schneuwly, S., Shortridge, R. D., Larrivee, D. C., Ono, T., pared to normal lymphoid tissue (18) indicates that more Ozaki, M. & Pak, W. L. (1989) Proc. Nat!. Acad. Sci. USA 86, detailed experimentation is required for clarifying the induc- 5390-5394. ibility of cyclophilin and FKBP in normal lymphoid cells. 15. Tropschung, M., Nicholson, D. W., Hartl, F.-U., Kohler, H., Southern Blot Analysis. To investigate the genomic com- Pfanner, N., Wachter, E. & Neupert, W. (1988) J. Biol. Chem. plexity of human Southern blot was per- 263, 14433-14440. FKBP, analysis 16. Kyte, J. & Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132. formed. Human genomic DNA prepared from placental cells 17. Chou, P. Y. & Fasman, G. 0. (1978) Annu. Rev. Biochem. 47, was cleaved by limited digestion with each of the five 251-276. restriction enzymes (EcoRI, BamHI, HindIll, Sac I, and Xba 18. Koletsky, A. J., Harding, M. W. & Handschumacher, R. E. I) and hybridized under highly stringent conditions with the (1986) J. Immunol. 137, 1054-1059. Downloaded by guest on September 26, 2021