Immunogenetics 35: 351-353, 1992
]inlllllllO-
geneOs
© Springer-Verlag 1992
Sequence of the tumor necrosis factor/cachectin (TNF) gene from Peromyscus leucopus (family Cricetidae) Mark D. Crew and Mark E. Filipowsky Department of Pathology, UCLA School of Medicine, Center for Health Sciences, Los Angeles, CA 90024-1732, USA Received August 6, 1991
Tumor necrosis factor/cachectin (TNF) is a potent pleiotropic cytokine implicated in a number of pathological conditions including septic shock and cachexia (Beutler and Cerami 1986; Beutler 1990). In mice and humans, TNF is synthesized by activated monocytes mainly as a 26 kd propeptide which is proteolytically cleaved to a 17 kd secreted protein bearing cytolytic activity towards a host of cell-types (Kriegler et al. 1988). The leader sequence of TNF is inordinately long (76 and 79 amino acids in humans and mice, respectively) and well-conserved relative to analogous regions in other peptide hormones (Pennica et al. 1987). Perhaps related to these features of the leader sequence, TNF is biologically active both as a secreted protein and as an integral membrane protein in which the C-terminus is extracellular and theN-terminus is intracellular (Perez et al. 1990). In addition, alternative proteolytic cleavage results in different forms (active and inactive) of both membrane-bound and secreted molecules (Kriegler et al. 1990). Human and mouse TNF proteins appear to utilize different alternative proteolytic cleavage sites (Cseh and Beutler 1989; Kriegler et al. 1990) possibly due to species-specific amino acid residues in the vicinity of the normal cleavage site. Genes encoding TNF and a related cytokine, lymphotoxin (LT), reside within the major histocompatibility complex (MHC; Spies et al. 1986; Mtiller et al. 1987). Due in part to the possible species-specific regulation of TNF and owing to our interests in evolution of MHC region genes, we have undertaken an analysis of the TNF gene from Peromyscus leucopus (family Cricetidae). The
The nucIeotidesequencedata reportedin this paper havebeen submitted to the GenBanknucleotide sequence database and have been assigned the accession number M59233. Address correspondence and offprint requests to: M.D. Crew.
initial characterization of P. leucopus MHC class I and II genes has been previously reported (Crew et al. 1989, 1990). A genomic library constructed from the testes DNA of an individual P. leucopus was screened with a mouse TNF cDNA fragment (Pennica et al. 1985). A clone harboring the complete TNF gene and approximately 3 kbp upstream (including about 1 kbp of LTgene sequence) was isolated, restriction mapped (not shown), and subcloned. The complete DNA sequence of a 2.3 kbp Eco RI fragment harboring the TNF coding regions was determined from both strands. The nucleotide sequence of the P. leucopus TNF gene is shown in Figure 1. The sequence spans from approximately 400 base pair (bp) upstream of exon 1 to about 240 nucleotides downstream of the stop codon. The P. leucopus TNF gene exhibited high homology ( > 80 %) to all mouse and rat TNF exons including those encoding the 79 amino acid leader sequence (exons 1, 2, and most of 3). Rabbit and human TNF exons displayed slightly lower homology to P. leucopus TNF, especially in exon 2 (Table 1). The homology of mouse, rat, rabbit, and human TNF gene introns to P. leucopus TNF gene introns varied from 43 %-79 %. The percent divergence at synomous and nonsynomous (replacement) sites among mammalian TNF genes was determined (Table 2). This data afforded an estimate of the time of divergence of Muridae and Cricetidae rodents. Assuming a silent substitution rate of about 0.9 % per million years (Myr; Ochman and Wilson 1987), Peromyscus diverged from Rattus and Mus approximately 42-52 Myr ago. Mus and Rattus, using this data separated about 25 Myr ago. These values are in close agreement to those obtained by DNA/DNA hybridization methods (Brownell 1983). The predicted protein sequence of P. leucopus TNF is shown in Figure 2 and is compared to mouse, rat, rabbit,
352 i 81 161 241 321 401 481
M.D. Crew and M. E. Filipowsky: Peromyscus leucopus TNF gene GAATTCTGGTAGGGAGGGGGAGGAGATTCCTTGATG~CTGGGTGT~cAA~TTTc~A/~cTCTGC~CCCG~GATGGAG 80 AAGAAACCGAGAAcAGAA•GTGTAGGGCCACTACCGCTTCcTcCACATGAGATCATGGTTTTCTCCACCAAGGAAGTTTT 160 C•GCTGGTTGAATGAGAGCATTTCCCCGCCCTCTTGccCAAGGGcTATAAAGGCAGCCGTCTGCACAC•CAGccTGCAGA 240 AGCTcTCTCAGCGAGGACATCAGGGGACCAGCCTGGAGGGAGAACAGcGAcTCCAGAACACcCTGGAAATAGcTCCCAGA 320 ~AG~G~AGCCAG~AGGCAGGTTCTGTCCCTCT~ACA~A~GGCCCAAGGTTCCACAGCT~CCTCCGG`~GGA~AC 400 •ATGAGCACAGAAAGCATGAT••GCGACGTAGAACTGGCAGA•GAGGCACT••CCAAAAAGGCGTGGGG•CCCCAGAA•T 480 MetSerThrGluSerMet~leArgAspVaiGluLeuAlaGluGluAlaLeuProLysLysAlaTrpGlyProGlnAsnS cCAGT•GGTGCCTGTGCCTCAGC•TCTTCTCCTTC•TGCTCGTGGCAGGGGcCACCACGCTCTTCTGTcTGCTGAATTTT 560
erSe•ArgCy•Leu•y•LeUSerLeUPheSerPheLeuLeuva•AlaGlyA•aThrThrLeUPhecysLeuL•uAs•Phe 561
GGGGTGAT~GGCCCCCAAAGGGAAGAGGTGAGTGCCTCAGAAGCCTTCATTCTCACTCAGGAAGAAGCAGGGCAGAAGGG 640 GlyValIleGlyProGizu~rgGluGlu CGAGAGAAGGAG~M~-GTGGGCTGAGGGG~ACAGG~AGTGTGGAGAC~ATGGAGAGG~AGCCCATGTGGAGACGGTGGC~AG 720 AGAA~GACAGCGAGACAGACGGGACCTCTGACCCACGCAGTCAGTTCACT~ACTGTTCAGTGGATGCGTGGAGGAGGGA 800 TGAATGAATGAAAAAGCATGTATACATATGTAGAGATGTGGAAAGAGATTCGGGATGTGGCCAGAAGGATGGGGAAGAGG 880 CCAGCGATAACGATGGCAGAGATGAGGAGGCATGAGTGATAAGGAGAGAGATGAGGGGAGATAAAGTGAGATCGAGCACA 960 GACGACAGAAGAGATGCTGCAGGGTAGGACGAATGAATGAACAGAGGTTGAGCGATGAAAAAACCCAGACACAGAAGCTG 1040 GGGGCTTCTCCTTTGCGGGTGACTCCTCAGCTGCTGGCTGCTAACCTTGTCCTTCTCTTCCTA•ACAGAAGTTCCCCAA• 1120 LysPheProAsn 1121 A A C C T C C C C A T c A T C G G C T C C A T G G C C C A G A C C C T C A C A • T G A G T A A • T A T C C C C C A G C C T C T C T T A A T G T A G G G T G G T G 1200 AsnLeuProIleIleGlySerMetAlaGl~ThrLeuThrLeuA 1201 G C A G T T A G A C C T G G G A T G G A A G C A G C G G G G A G A A C G T A C G G C T T T G G T T T T G G A G G A A A G G G A A T G G G G T C C A A A C A G A C 1280 1281 A G A T G C T G C C T G G G A G G C C T A A G A G T C T C A T C C C T G C C C T T C T T C T C T T C C C T T C A G G A T C G T c T T C T C A A A A T T C G A G T 1360 641 721 801 881 961 1041
rgSerSerSerGlnAsnSerSer 1361 G A C A A G C c T G T A G C C C A C G T T G T A G G T A A G A G C T C T A T G T G T G C A T C C T G G G G A C G A A G G G A C G G G A T T T G G G G G T C G A A AspLysProValAlaHisValValA 1441 C C A G G C T G A G A A G A C A G C T T G T G A A A G C G T G A A A G G G A A G C A T C C A A A A G C A G G G G G C T T A G T G G G G G T A C T C A G G A C C T 1521 T A A G G C • A A T G G G A T G T G G G A A G A C A G A G G G T G C A G G A A C C G G A A G T G A A G T G T G G G T A G A A C T T G A G G • T C A G G A T G T G 1601 G G G T G T G A A C T A A C A G G G T C A C A C T G A C T C A A C T C T C C C T C C C T C A G C A A A C C A C C A A G T G G A T G A G C A G C T G G A G T G G C
1440 1520 1600 1680
laAsnHisGlnValAspGluGlnLeuGluTrpL 1681 TGAGCCGGGGTGCCAATC-CCCTCCTGGCCAACGGCATGGATCTCAAAGACAACCAGCTGGTGATTCCA~CGGGCTG euSerArgG•yA•aAsnA•aLeuLeuA•aAsnG•yMetAspLeuLysAspAsnG•nLeuVa•I•ePr•A•aAspG•yLeu 1761 T A C C T • G T • T A C T c c C A G G T G C T C T T C A A G G G c C A A G G C T G C T C c A G C T A T G T G C T G C T C A c C C A C A C G G T C A G c c G c T T
1760 1840
TyrLeuVa•TyrSerG•nva•LeuPheLysG•yG•nG•yCysSerSerTyrVa•LeuLeuThrHisThrva•SerArgPh 1841 TG•TGTCT•TTAcGAGGACAAAGTCAA•CTC•TGTCTGCCATCAAGAGCCCCTGC•cCAAGGAAACC•C•GAGGGGTCTG 1920 eA•aVa•SerTyrG•uAspLysva•AsnLeuLeuSerA•aI•eLysSerPr•cysPr•LysG•uThrPr•G•uG•ySerG 1921 AGCT~CCTGGTACGAGCCCATCTACTTGGGAGGGGTCTTCCAGCTGGAGAAGGGAGACCGACTGAGTGCTGAGGTC 2000 •uLeuLysPr•Tr•TyrG•uPr•I•eTyrLeuG•yG•yva•PheG•nLeuG•uLysG•yAspArgLeuSerA•aG•uVa• 2001 AATCTGCCCA%AATACCTAGAcTTTGCAGAATccGGGCAGGTCTACTTTGGAGTCATTGCTCTGTAAGGTAGATGGATGAC 2080 AsnLeuProLysTyrLeuA~pPheAlaGluSerGlyGlnValTyrPheGlyValIleAlaLeu 2081 CATCCAGTCTCTACCCAGCCCCCGCGTTGACCCCTTTATTGTCTACTCCTCAGAGCCCCAGTCCATCTCCTTCTGACTTA 2160 2161 GTAAGGGAGTTATGGGTCAGGGTCAGACTCTGAG~TCCAATTGTT~A~TAT~g~kCA~T~AG`a-a3k~A~AGATA~AGGGAT 2240 2241 A G C G G C C C G G A C T G T G G G G T T C T C A T G A A C C A C C A T C A A G G A T T C G C A C G G G C T T C C A G A A T T C 2304
Table 1. Percent homology of mouse, rat, rabbit, and human TNF gene sequences to P. leucopus TNF exons and introns. TNF gene region
Mouse
Rat
Rabbit
Human
Exon 1 Intron 1 Exon 2 Intron 2 Exon 3 Intron 3 Exon 4
90.3(0) 61.5(10) 81.8(0) 75.6(4) 95.8(0) 74.0(3) 89.4(0)
88.2(0) 58.8(7) 81.8(0) 79.3(3) 91.7(0) 74.1(3) 87.5(0)
82.8(0) 43.8(2) 67.3(0) 47.5(2) 77.1(0) 65.7(6) 82.7(0)
87.6(0) 57.0(9) 58.2(2) 63.2(2) 87.5(0) 74.1(3) 84.4(1)
Numbers outside parentheses are the percent homology between DNA sequences. The numbers inside parentheses are the number of gaps introduced to maximize similarity. The algorithms of Smith and coworkers ( 1981) and Needleman and Wunsch (1974) were used for these determinations. Mouse, rat, rabbit, and human TNF gene sequences were taken from Semon and co-workers (1987), Shirai and co-workers (1989), Ito and co-workers (1986), and Nedwin and co-workers (1985) respectively.
Table 2. Percent divergence at synonymous and replacement sites between mammalian TNF genes. Mouse Mouse Rat P. leucopus Rabbit Human
Rat 3.1
24.5 38.1 58.5 58.0
46.7 58.7 64.3
P. leucopus 5.4 6.1 58.7 49.0
Rabbit
Human
13.2 13.3 12.7
12.2 16.0 12.1 11.5
43.9
The percent divergence at synomous (bold, lower left) and replacement (italics, upper right) sites was calculated according to Perler and coworkers (1980).
Fig. 1. Nucleotide sequence of the P. leucopus TNF gene. The putative TATA box is Underlined. Exon sequences are in bold type and the predicted amino acids are given below each exon. Exon and intron boundaries were deduced by comparison with mouse TNF cDNA sequences.
and h u m a n T N F protein sequences. T e n residues u n i q u e to the P. leucopus T N F protein were found. Six of these were in the m a t u r e (17 kd) peptide and four were located in the leader peptide. Crystallographic analysis has revealed the T N F protein structure to be roughly a sandwich of antiparallel/3-pleated sheets (Eck and Sprang 1989). Most of the P. leucopus-specific residues found in the mature T N F protein are localized in or immediately adjacent to peptide regions looped out of the/3-pleated sheets (Fig. 2). The only exception is a conservative a m i n o acid substitution (valine to isoleucine) comprising part of a/3strand (position 50 in F i g u r e 2). A n N-linked glycosylation site present in mouse and rat 17 kd T N F proteins is conserved in the P. leucopus T N F molecule as are residues putatively involved with receptor b i n d i n g and trimerization of T N F subunits (not shown in F i g u r e 2). Two of the four P. leucopus-specific amino acid substitutions found in the 79 a m i n o acid leader peptide were located in the portion of peptide predicted to lie on the cytoplasmic side of m e m b r a n e - b o u n d T N F . Positive charges as a rule are found at the cytoplasmic ends of t r a n s m e m b r a n e spanning regions (von H e i j n e and M a n o i l 1990) and two positively charged residues flank the cytoplasmic end of the putative t r a n s m e m b r a n e spanning region of m o u s e , rat, rabbit, and h u m a n T N F proteins. P. leucopus T N F , in contrast, lacks one positively charged residue but has instead an amino acid substitution leading to the addition of a potential N-linked glycosation site. It is unclear whether this is necessary or suffices for a m e m b r a n e anchor. It should be noted that precedence for N-linked glycosation of m e m b r a n e proteins on their intracellular d o m a i n s is rare, if not nonexistant.
M.D. Crew and M.E. Filipowsky: Peromyscus leucopus TNF gene 79
-30
P ..... y . . . .
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mouse
..................
rat rabbit human
.................... MG-L---R- i .............. GP ..... G---G-K.................... TG---G-R
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-29
Peromyscus
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_
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mouse
~
D .....
rat rabbit human
=~ ...... NK ...... G--L ~-R ..... E --QS .... HL ~ ............ RD-SL
Peromyscus
VDEQLEWLSR
GANALLANGM
mouse rat rabbit human
-E ....... Q AE ....... Q -EG--Q---Q /LEG--Q--N-
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........
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21 RSSSQNSSDK
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PVAh~VVANHQ
.....................
-S ............................ VKrPV--MV .... A-RAL .... L ...... -SPL-AV .... RTP ...........
PP-
22
71 DL~NQLVIP
................. ................. ......... K-T ..... ........ V E-R .....
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V ................... v ....... I ........... V ....... I ...... S .... VSE .... I ...........
72
Peromyscus
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121
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Peromyscus
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122
157 LSAEVNLPKY
LDFAESGQV~Z
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Fig. 2. Comparison of P. leucopus TNF protein sequences to TNF protein sequences from mouse, rat, rabbit, and human (see Table 1 for source of sequences). Dashes indicate identity to the P. leucopus sequence. Spaces indicate gaps introduced to align human TNF to rodent and rabbit TNF proteins. Asterisks indicate potential N-linked glycosylation sites and the shaded region indicates the putative transmembrane spanning region of the propeptides. Hatched residues inhibit cytolytic activity of mouse TNF (Cseh and Beutler 1989). Double underlines indicate regions comprising/3-strands (Eck and Sprang 1989).
Cseh and Beutler (1989) found that an alternative cleavage site which leaves ten amino acids on the normal 17 kd murine TNF severely impairs cytolytic activity. Two P. leucopus-specific substitutions in the 79 amino acid leader sequence are found in this region (i. e., within ten amino acids upstream of the normal scission site). Though difficult to predict based on sequence data alone, it is tempting to speculate that these substitutions may affect the utilization of alternative cleavage sites or abrogation of cytolytic activity. The observed variation in the sequence of regions around the normal propeptide cleavage site suggests that the regulation of TNF activity and secretion at the level of post-translational modification (i. e., glycosylation and peptide cleavage) may differ appreciably among mammals. These hypotheses await biochemical analysis. Acknowledgments. This work was supported by U.S. Public Health Service Grants AG04419-06 and AG08936-01 from the National Institute on Aging.
References Beutler, B. and Cerami, A. : Cachectin and tumor necrosis factor as two sides of the same biological coin. Nature 320: 584-588, 1986
353 Beutler, B.: The complex biology and regulation of TNF (cachectin). Crit Rev Oncol Hematol 2: 9-18, 1990 Brownell, E.: DNA/DNA hybridization studies ofmuriod rodents: symmetry and rates of molecular evolution. Evolution 37:1034-1051, 1983 Crew, M. D., Zeller, E. C., Smith, G. S., and Walford, R. L. : Polymorphism in the major histocompatibility class II genes of Peromyscus leucopus. Immunogenetics 30: 214-217, 1989 Crew, M. D•, Filipowsky, M.F., Zeller, E.C., Smith, G.S., and Walford, R.L.: Major histocompatibility class I genes of Peromyscus leucopus. Immunogenetics 32: 371-379, 1990 Cseh, K. and Beutler, B. : Alternative cleavage of the cachectin/tumor necrosis factor propeptide results in a larger, inactive form of the secreted protein• J Biol Chem 264: 16256-16260, 1989 Eck, M. J. and Sprang, S. R. : The structure of tumor necrosis factor-~ at 2.6 A resolution. J Biol Chem 264: 17595-17605, 1989 Ito, H., Shirai, T., Yamamoto, S., Akira, M., Kawahara, S., Todd, C., and Wallace, R. B_: Molecular cloning of the gene encoding rabbit tumor necrosis factor. DNA 5: 157-165, 1986 Kriegler, M., Perez, C., DeFay, K., Albert, I., and Lu, S. D. : A novel form of TNF/cachectin is a cell surface cytotoxic transmembrane protein: Ramifications for the complex physiology of TNF. Cell 53: 45-53, 1988 Mtiller, U., Jongeneel, C. V., Nedospasov, S. A., Lindahl, K. F., and Steinmetz, M. : Turnout necrosis factor and lymphotoxin genes map close to H-2D in the mouse major histocompatibility complex. Nature 325: 265-267, 1987 Nedwin, G.E., Naylor, S.L., Sakaguchi, A.Y., Smith, D., JarrettNedwin, J., Pennica, D., Goeddel, D. V., and Gray, P. W. : Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization. Nucleic Acids Res 13:6361-6373, 1985 Needleman, S. B., and Wunsch, C. D. : A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 48: 443-453, 1974 Ochman, H. and Wilson, A. C.: Evolution in bacteria: evidence for a universal substitution rate in cellulare genomes. J Mol Evol 26." 74-86, 1987 Pennica, D., Hayflick, J. S., Bergman, T.S., Palladino, M . A . , and Goeddel, D. V. : Cloning and expression in Escherichia coli of the cDNA for murine necrosis factor. Proc Natl Acad Sci USA 82: 6060-6064, 1985 Pennica, D., Shalaby, MR., and Palladino, MA.: Tumor necrosis factors Alpha and Beta. In S. Gillis (ed.): Recombinant Lymphokines and their Receptors, pp. 301-316, Dekker, New York, 1987 Perez, C., Albert, I., DeFay, K., Zacharides, N., Gooding, L., and Kriegler, M. : A nonsecretable cell surface mutant of tumor necrosis factor (TNF) kills by cell-to-cell contact. Cell 63: 251-259, 1990 Perler, F., Efstratiadis, A., Lomedico, P., Gilbert, G., Kolodner, R., Dodgson, J.: The evolution of genes: the chicken preproinsulin gene. Cell 20: 555-566, 1980 Semon, D., Kawashima, E., Jongeneel, C.V., Shakov, A.N., and Nedospasov, S. A. : Nucleotide sequence of the TNF locus, including the TNF-alpha (tumor necrosis factor) and TNF-beta (lymphotoxin) genes. Nucleic Acids Res 15: 9083-9084, 1987 Shirai, T., Shimizu, N., Horiguchi, S., and Ito, H. : Cloning and expression in Escherichia coli of the gene for rat tumor necrosis factor. Agric Biol Chem 53: 1733-1736, 1989 Smith, T. F., Waterman, M. S., and Fitch, W. M. : Comparative biosequence metrics. J Mol Evol 18: 38-46, 1981 Spies, T., Morton, C.C., Nedospasov, S.A., Fiers, W., Pious, D., and Strominger, J. L.: Genes for the tumor necrosis factors ~ and /3 are linked to the human major histocompatibility complex. Proc Natl Acad USA 83," 8699-8702, 1986 von Heijne, G. and Manoil, C.: Membrane proteins: from sequence to structure. Protein Eng 4: 109-112, 1990
]inlllllllO-
geneOs
© Springer-Verlag 1992
Sequence of the tumor necrosis factor/cachectin (TNF) gene from Peromyscus leucopus (family Cricetidae) Mark D. Crew and Mark E. Filipowsky Department of Pathology, UCLA School of Medicine, Center for Health Sciences, Los Angeles, CA 90024-1732, USA Received August 6, 1991
Tumor necrosis factor/cachectin (TNF) is a potent pleiotropic cytokine implicated in a number of pathological conditions including septic shock and cachexia (Beutler and Cerami 1986; Beutler 1990). In mice and humans, TNF is synthesized by activated monocytes mainly as a 26 kd propeptide which is proteolytically cleaved to a 17 kd secreted protein bearing cytolytic activity towards a host of cell-types (Kriegler et al. 1988). The leader sequence of TNF is inordinately long (76 and 79 amino acids in humans and mice, respectively) and well-conserved relative to analogous regions in other peptide hormones (Pennica et al. 1987). Perhaps related to these features of the leader sequence, TNF is biologically active both as a secreted protein and as an integral membrane protein in which the C-terminus is extracellular and theN-terminus is intracellular (Perez et al. 1990). In addition, alternative proteolytic cleavage results in different forms (active and inactive) of both membrane-bound and secreted molecules (Kriegler et al. 1990). Human and mouse TNF proteins appear to utilize different alternative proteolytic cleavage sites (Cseh and Beutler 1989; Kriegler et al. 1990) possibly due to species-specific amino acid residues in the vicinity of the normal cleavage site. Genes encoding TNF and a related cytokine, lymphotoxin (LT), reside within the major histocompatibility complex (MHC; Spies et al. 1986; Mtiller et al. 1987). Due in part to the possible species-specific regulation of TNF and owing to our interests in evolution of MHC region genes, we have undertaken an analysis of the TNF gene from Peromyscus leucopus (family Cricetidae). The
The nucIeotidesequencedata reportedin this paper havebeen submitted to the GenBanknucleotide sequence database and have been assigned the accession number M59233. Address correspondence and offprint requests to: M.D. Crew.
initial characterization of P. leucopus MHC class I and II genes has been previously reported (Crew et al. 1989, 1990). A genomic library constructed from the testes DNA of an individual P. leucopus was screened with a mouse TNF cDNA fragment (Pennica et al. 1985). A clone harboring the complete TNF gene and approximately 3 kbp upstream (including about 1 kbp of LTgene sequence) was isolated, restriction mapped (not shown), and subcloned. The complete DNA sequence of a 2.3 kbp Eco RI fragment harboring the TNF coding regions was determined from both strands. The nucleotide sequence of the P. leucopus TNF gene is shown in Figure 1. The sequence spans from approximately 400 base pair (bp) upstream of exon 1 to about 240 nucleotides downstream of the stop codon. The P. leucopus TNF gene exhibited high homology ( > 80 %) to all mouse and rat TNF exons including those encoding the 79 amino acid leader sequence (exons 1, 2, and most of 3). Rabbit and human TNF exons displayed slightly lower homology to P. leucopus TNF, especially in exon 2 (Table 1). The homology of mouse, rat, rabbit, and human TNF gene introns to P. leucopus TNF gene introns varied from 43 %-79 %. The percent divergence at synomous and nonsynomous (replacement) sites among mammalian TNF genes was determined (Table 2). This data afforded an estimate of the time of divergence of Muridae and Cricetidae rodents. Assuming a silent substitution rate of about 0.9 % per million years (Myr; Ochman and Wilson 1987), Peromyscus diverged from Rattus and Mus approximately 42-52 Myr ago. Mus and Rattus, using this data separated about 25 Myr ago. These values are in close agreement to those obtained by DNA/DNA hybridization methods (Brownell 1983). The predicted protein sequence of P. leucopus TNF is shown in Figure 2 and is compared to mouse, rat, rabbit,
352 i 81 161 241 321 401 481
M.D. Crew and M. E. Filipowsky: Peromyscus leucopus TNF gene GAATTCTGGTAGGGAGGGGGAGGAGATTCCTTGATG~CTGGGTGT~cAA~TTTc~A/~cTCTGC~CCCG~GATGGAG 80 AAGAAACCGAGAAcAGAA•GTGTAGGGCCACTACCGCTTCcTcCACATGAGATCATGGTTTTCTCCACCAAGGAAGTTTT 160 C•GCTGGTTGAATGAGAGCATTTCCCCGCCCTCTTGccCAAGGGcTATAAAGGCAGCCGTCTGCACAC•CAGccTGCAGA 240 AGCTcTCTCAGCGAGGACATCAGGGGACCAGCCTGGAGGGAGAACAGcGAcTCCAGAACACcCTGGAAATAGcTCCCAGA 320 ~AG~G~AGCCAG~AGGCAGGTTCTGTCCCTCT~ACA~A~GGCCCAAGGTTCCACAGCT~CCTCCGG`~GGA~AC 400 •ATGAGCACAGAAAGCATGAT••GCGACGTAGAACTGGCAGA•GAGGCACT••CCAAAAAGGCGTGGGG•CCCCAGAA•T 480 MetSerThrGluSerMet~leArgAspVaiGluLeuAlaGluGluAlaLeuProLysLysAlaTrpGlyProGlnAsnS cCAGT•GGTGCCTGTGCCTCAGC•TCTTCTCCTTC•TGCTCGTGGCAGGGGcCACCACGCTCTTCTGTcTGCTGAATTTT 560
erSe•ArgCy•Leu•y•LeUSerLeUPheSerPheLeuLeuva•AlaGlyA•aThrThrLeUPhecysLeuL•uAs•Phe 561
GGGGTGAT~GGCCCCCAAAGGGAAGAGGTGAGTGCCTCAGAAGCCTTCATTCTCACTCAGGAAGAAGCAGGGCAGAAGGG 640 GlyValIleGlyProGizu~rgGluGlu CGAGAGAAGGAG~M~-GTGGGCTGAGGGG~ACAGG~AGTGTGGAGAC~ATGGAGAGG~AGCCCATGTGGAGACGGTGGC~AG 720 AGAA~GACAGCGAGACAGACGGGACCTCTGACCCACGCAGTCAGTTCACT~ACTGTTCAGTGGATGCGTGGAGGAGGGA 800 TGAATGAATGAAAAAGCATGTATACATATGTAGAGATGTGGAAAGAGATTCGGGATGTGGCCAGAAGGATGGGGAAGAGG 880 CCAGCGATAACGATGGCAGAGATGAGGAGGCATGAGTGATAAGGAGAGAGATGAGGGGAGATAAAGTGAGATCGAGCACA 960 GACGACAGAAGAGATGCTGCAGGGTAGGACGAATGAATGAACAGAGGTTGAGCGATGAAAAAACCCAGACACAGAAGCTG 1040 GGGGCTTCTCCTTTGCGGGTGACTCCTCAGCTGCTGGCTGCTAACCTTGTCCTTCTCTTCCTA•ACAGAAGTTCCCCAA• 1120 LysPheProAsn 1121 A A C C T C C C C A T c A T C G G C T C C A T G G C C C A G A C C C T C A C A • T G A G T A A • T A T C C C C C A G C C T C T C T T A A T G T A G G G T G G T G 1200 AsnLeuProIleIleGlySerMetAlaGl~ThrLeuThrLeuA 1201 G C A G T T A G A C C T G G G A T G G A A G C A G C G G G G A G A A C G T A C G G C T T T G G T T T T G G A G G A A A G G G A A T G G G G T C C A A A C A G A C 1280 1281 A G A T G C T G C C T G G G A G G C C T A A G A G T C T C A T C C C T G C C C T T C T T C T C T T C C C T T C A G G A T C G T c T T C T C A A A A T T C G A G T 1360 641 721 801 881 961 1041
rgSerSerSerGlnAsnSerSer 1361 G A C A A G C c T G T A G C C C A C G T T G T A G G T A A G A G C T C T A T G T G T G C A T C C T G G G G A C G A A G G G A C G G G A T T T G G G G G T C G A A AspLysProValAlaHisValValA 1441 C C A G G C T G A G A A G A C A G C T T G T G A A A G C G T G A A A G G G A A G C A T C C A A A A G C A G G G G G C T T A G T G G G G G T A C T C A G G A C C T 1521 T A A G G C • A A T G G G A T G T G G G A A G A C A G A G G G T G C A G G A A C C G G A A G T G A A G T G T G G G T A G A A C T T G A G G • T C A G G A T G T G 1601 G G G T G T G A A C T A A C A G G G T C A C A C T G A C T C A A C T C T C C C T C C C T C A G C A A A C C A C C A A G T G G A T G A G C A G C T G G A G T G G C
1440 1520 1600 1680
laAsnHisGlnValAspGluGlnLeuGluTrpL 1681 TGAGCCGGGGTGCCAATC-CCCTCCTGGCCAACGGCATGGATCTCAAAGACAACCAGCTGGTGATTCCA~CGGGCTG euSerArgG•yA•aAsnA•aLeuLeuA•aAsnG•yMetAspLeuLysAspAsnG•nLeuVa•I•ePr•A•aAspG•yLeu 1761 T A C C T • G T • T A C T c c C A G G T G C T C T T C A A G G G c C A A G G C T G C T C c A G C T A T G T G C T G C T C A c C C A C A C G G T C A G c c G c T T
1760 1840
TyrLeuVa•TyrSerG•nva•LeuPheLysG•yG•nG•yCysSerSerTyrVa•LeuLeuThrHisThrva•SerArgPh 1841 TG•TGTCT•TTAcGAGGACAAAGTCAA•CTC•TGTCTGCCATCAAGAGCCCCTGC•cCAAGGAAACC•C•GAGGGGTCTG 1920 eA•aVa•SerTyrG•uAspLysva•AsnLeuLeuSerA•aI•eLysSerPr•cysPr•LysG•uThrPr•G•uG•ySerG 1921 AGCT~CCTGGTACGAGCCCATCTACTTGGGAGGGGTCTTCCAGCTGGAGAAGGGAGACCGACTGAGTGCTGAGGTC 2000 •uLeuLysPr•Tr•TyrG•uPr•I•eTyrLeuG•yG•yva•PheG•nLeuG•uLysG•yAspArgLeuSerA•aG•uVa• 2001 AATCTGCCCA%AATACCTAGAcTTTGCAGAATccGGGCAGGTCTACTTTGGAGTCATTGCTCTGTAAGGTAGATGGATGAC 2080 AsnLeuProLysTyrLeuA~pPheAlaGluSerGlyGlnValTyrPheGlyValIleAlaLeu 2081 CATCCAGTCTCTACCCAGCCCCCGCGTTGACCCCTTTATTGTCTACTCCTCAGAGCCCCAGTCCATCTCCTTCTGACTTA 2160 2161 GTAAGGGAGTTATGGGTCAGGGTCAGACTCTGAG~TCCAATTGTT~A~TAT~g~kCA~T~AG`a-a3k~A~AGATA~AGGGAT 2240 2241 A G C G G C C C G G A C T G T G G G G T T C T C A T G A A C C A C C A T C A A G G A T T C G C A C G G G C T T C C A G A A T T C 2304
Table 1. Percent homology of mouse, rat, rabbit, and human TNF gene sequences to P. leucopus TNF exons and introns. TNF gene region
Mouse
Rat
Rabbit
Human
Exon 1 Intron 1 Exon 2 Intron 2 Exon 3 Intron 3 Exon 4
90.3(0) 61.5(10) 81.8(0) 75.6(4) 95.8(0) 74.0(3) 89.4(0)
88.2(0) 58.8(7) 81.8(0) 79.3(3) 91.7(0) 74.1(3) 87.5(0)
82.8(0) 43.8(2) 67.3(0) 47.5(2) 77.1(0) 65.7(6) 82.7(0)
87.6(0) 57.0(9) 58.2(2) 63.2(2) 87.5(0) 74.1(3) 84.4(1)
Numbers outside parentheses are the percent homology between DNA sequences. The numbers inside parentheses are the number of gaps introduced to maximize similarity. The algorithms of Smith and coworkers ( 1981) and Needleman and Wunsch (1974) were used for these determinations. Mouse, rat, rabbit, and human TNF gene sequences were taken from Semon and co-workers (1987), Shirai and co-workers (1989), Ito and co-workers (1986), and Nedwin and co-workers (1985) respectively.
Table 2. Percent divergence at synonymous and replacement sites between mammalian TNF genes. Mouse Mouse Rat P. leucopus Rabbit Human
Rat 3.1
24.5 38.1 58.5 58.0
46.7 58.7 64.3
P. leucopus 5.4 6.1 58.7 49.0
Rabbit
Human
13.2 13.3 12.7
12.2 16.0 12.1 11.5
43.9
The percent divergence at synomous (bold, lower left) and replacement (italics, upper right) sites was calculated according to Perler and coworkers (1980).
Fig. 1. Nucleotide sequence of the P. leucopus TNF gene. The putative TATA box is Underlined. Exon sequences are in bold type and the predicted amino acids are given below each exon. Exon and intron boundaries were deduced by comparison with mouse TNF cDNA sequences.
and h u m a n T N F protein sequences. T e n residues u n i q u e to the P. leucopus T N F protein were found. Six of these were in the m a t u r e (17 kd) peptide and four were located in the leader peptide. Crystallographic analysis has revealed the T N F protein structure to be roughly a sandwich of antiparallel/3-pleated sheets (Eck and Sprang 1989). Most of the P. leucopus-specific residues found in the mature T N F protein are localized in or immediately adjacent to peptide regions looped out of the/3-pleated sheets (Fig. 2). The only exception is a conservative a m i n o acid substitution (valine to isoleucine) comprising part of a/3strand (position 50 in F i g u r e 2). A n N-linked glycosylation site present in mouse and rat 17 kd T N F proteins is conserved in the P. leucopus T N F molecule as are residues putatively involved with receptor b i n d i n g and trimerization of T N F subunits (not shown in F i g u r e 2). Two of the four P. leucopus-specific amino acid substitutions found in the 79 a m i n o acid leader peptide were located in the portion of peptide predicted to lie on the cytoplasmic side of m e m b r a n e - b o u n d T N F . Positive charges as a rule are found at the cytoplasmic ends of t r a n s m e m b r a n e spanning regions (von H e i j n e and M a n o i l 1990) and two positively charged residues flank the cytoplasmic end of the putative t r a n s m e m b r a n e spanning region of m o u s e , rat, rabbit, and h u m a n T N F proteins. P. leucopus T N F , in contrast, lacks one positively charged residue but has instead an amino acid substitution leading to the addition of a potential N-linked glycosation site. It is unclear whether this is necessary or suffices for a m e m b r a n e anchor. It should be noted that precedence for N-linked glycosation of m e m b r a n e proteins on their intracellular d o m a i n s is rare, if not nonexistant.
M.D. Crew and M.E. Filipowsky: Peromyscus leucopus TNF gene 79
-30
P ..... y . . . .
MSTESMI~LDV
mouse
..................
rat rabbit human
.................... MG-L---R- i .............. GP ..... G---G-K.................... TG---G-R
ELAEE~ALPF_K Q-
-29
Peromyscus
AWGPQNSS
_
EEKFP~LPI
mouse
~
D .....
rat rabbit human
=~ ...... NK ...... G--L ~-R ..... E --QS .... HL ~ ............ RD-SL
Peromyscus
VDEQLEWLSR
GANALLANGM
mouse rat rabbit human
-E ....... Q AE ....... Q -EG--Q---Q /LEG--Q--N-
R R R R
........
~ - ' ~ i ]
MG-F---R-
-i
~FGVIGPQR
- ; ~
+I
IGSMA~TLTL
21 RSSSQNSSDK
G - - TZ/A//~S ~ F / / ~ - / ~
PVAh~VVANHQ
.....................
-S ............................ VKrPV--MV .... A-RAL .... L ...... -SPL-AV .... RTP ...........
PP-
22
71 DL~NQLVIP
................. ................. ......... K-T ..... ........ V E-R .....
ADGLYLVYSQ
V ................... v ....... I ........... V ....... I ...... S .... VSE .... I ...........
72
Peromyscus
VLFKGQGCSS PD PD RP-
121
mouse rat rabbit human
Yg-LLTHTVS RFAVSYEEKV NLLSAIKSPC PKETPEGSEL KPWYEPIYLG ............ I--Q ........ V ...... D .... A ............ ............ I--Q--S ........... D .... A ........ M--............... PN ............ HR .... EA-P MA ........ TH ...... I- -I .... QT ............ QR ..... A-A ..........
Peromyscus
GVFQLEKGDR
mouse rat rabbit human
......... Q .......................... ......... L ............ IT ............ ............ T---Q-E--L ......... I--.............. I-R-D ............. I---
122
157 LSAEVNLPKY
LDFAESGQV~Z
FGVIAL
Fig. 2. Comparison of P. leucopus TNF protein sequences to TNF protein sequences from mouse, rat, rabbit, and human (see Table 1 for source of sequences). Dashes indicate identity to the P. leucopus sequence. Spaces indicate gaps introduced to align human TNF to rodent and rabbit TNF proteins. Asterisks indicate potential N-linked glycosylation sites and the shaded region indicates the putative transmembrane spanning region of the propeptides. Hatched residues inhibit cytolytic activity of mouse TNF (Cseh and Beutler 1989). Double underlines indicate regions comprising/3-strands (Eck and Sprang 1989).
Cseh and Beutler (1989) found that an alternative cleavage site which leaves ten amino acids on the normal 17 kd murine TNF severely impairs cytolytic activity. Two P. leucopus-specific substitutions in the 79 amino acid leader sequence are found in this region (i. e., within ten amino acids upstream of the normal scission site). Though difficult to predict based on sequence data alone, it is tempting to speculate that these substitutions may affect the utilization of alternative cleavage sites or abrogation of cytolytic activity. The observed variation in the sequence of regions around the normal propeptide cleavage site suggests that the regulation of TNF activity and secretion at the level of post-translational modification (i. e., glycosylation and peptide cleavage) may differ appreciably among mammals. These hypotheses await biochemical analysis. Acknowledgments. This work was supported by U.S. Public Health Service Grants AG04419-06 and AG08936-01 from the National Institute on Aging.
References Beutler, B. and Cerami, A. : Cachectin and tumor necrosis factor as two sides of the same biological coin. Nature 320: 584-588, 1986
353 Beutler, B.: The complex biology and regulation of TNF (cachectin). Crit Rev Oncol Hematol 2: 9-18, 1990 Brownell, E.: DNA/DNA hybridization studies ofmuriod rodents: symmetry and rates of molecular evolution. Evolution 37:1034-1051, 1983 Crew, M. D., Zeller, E. C., Smith, G. S., and Walford, R. L. : Polymorphism in the major histocompatibility class II genes of Peromyscus leucopus. Immunogenetics 30: 214-217, 1989 Crew, M. D•, Filipowsky, M.F., Zeller, E.C., Smith, G.S., and Walford, R.L.: Major histocompatibility class I genes of Peromyscus leucopus. Immunogenetics 32: 371-379, 1990 Cseh, K. and Beutler, B. : Alternative cleavage of the cachectin/tumor necrosis factor propeptide results in a larger, inactive form of the secreted protein• J Biol Chem 264: 16256-16260, 1989 Eck, M. J. and Sprang, S. R. : The structure of tumor necrosis factor-~ at 2.6 A resolution. J Biol Chem 264: 17595-17605, 1989 Ito, H., Shirai, T., Yamamoto, S., Akira, M., Kawahara, S., Todd, C., and Wallace, R. B_: Molecular cloning of the gene encoding rabbit tumor necrosis factor. DNA 5: 157-165, 1986 Kriegler, M., Perez, C., DeFay, K., Albert, I., and Lu, S. D. : A novel form of TNF/cachectin is a cell surface cytotoxic transmembrane protein: Ramifications for the complex physiology of TNF. Cell 53: 45-53, 1988 Mtiller, U., Jongeneel, C. V., Nedospasov, S. A., Lindahl, K. F., and Steinmetz, M. : Turnout necrosis factor and lymphotoxin genes map close to H-2D in the mouse major histocompatibility complex. Nature 325: 265-267, 1987 Nedwin, G.E., Naylor, S.L., Sakaguchi, A.Y., Smith, D., JarrettNedwin, J., Pennica, D., Goeddel, D. V., and Gray, P. W. : Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization. Nucleic Acids Res 13:6361-6373, 1985 Needleman, S. B., and Wunsch, C. D. : A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 48: 443-453, 1974 Ochman, H. and Wilson, A. C.: Evolution in bacteria: evidence for a universal substitution rate in cellulare genomes. J Mol Evol 26." 74-86, 1987 Pennica, D., Hayflick, J. S., Bergman, T.S., Palladino, M . A . , and Goeddel, D. V. : Cloning and expression in Escherichia coli of the cDNA for murine necrosis factor. Proc Natl Acad Sci USA 82: 6060-6064, 1985 Pennica, D., Shalaby, MR., and Palladino, MA.: Tumor necrosis factors Alpha and Beta. In S. Gillis (ed.): Recombinant Lymphokines and their Receptors, pp. 301-316, Dekker, New York, 1987 Perez, C., Albert, I., DeFay, K., Zacharides, N., Gooding, L., and Kriegler, M. : A nonsecretable cell surface mutant of tumor necrosis factor (TNF) kills by cell-to-cell contact. Cell 63: 251-259, 1990 Perler, F., Efstratiadis, A., Lomedico, P., Gilbert, G., Kolodner, R., Dodgson, J.: The evolution of genes: the chicken preproinsulin gene. Cell 20: 555-566, 1980 Semon, D., Kawashima, E., Jongeneel, C.V., Shakov, A.N., and Nedospasov, S. A. : Nucleotide sequence of the TNF locus, including the TNF-alpha (tumor necrosis factor) and TNF-beta (lymphotoxin) genes. Nucleic Acids Res 15: 9083-9084, 1987 Shirai, T., Shimizu, N., Horiguchi, S., and Ito, H. : Cloning and expression in Escherichia coli of the gene for rat tumor necrosis factor. Agric Biol Chem 53: 1733-1736, 1989 Smith, T. F., Waterman, M. S., and Fitch, W. M. : Comparative biosequence metrics. J Mol Evol 18: 38-46, 1981 Spies, T., Morton, C.C., Nedospasov, S.A., Fiers, W., Pious, D., and Strominger, J. L.: Genes for the tumor necrosis factors ~ and /3 are linked to the human major histocompatibility complex. Proc Natl Acad USA 83," 8699-8702, 1986 von Heijne, G. and Manoil, C.: Membrane proteins: from sequence to structure. Protein Eng 4: 109-112, 1990
