3784 Nucleic Acids Research, Vol. 20, No. 14
The nucleotide sequence of a mouse cDNA encoding the 80 kDa subunit of the Ku (p701p80) autoantigen Miriam Falzon and Edward L.Kuff Laboratory of Biochemistry, National Cancer Institute, Building 37, Room 4C03, Bethesda, MD 20892, USA Submitted May 29, 1992 The human Ku autoantigen is a heterodimeric nuclear protein first detected by antibodies from autoimmune patients (1) and proposed to play a role in DNA repair and/or transposition (2). Human Ku preparations contain equimolar amounts of 70 and 80 kDa polypeptides. The cDNA's encoding both human subunits, as well as that derived from the murine 70 kDa polypeptide, have been cloned and sequenced (3-7). Here we report the cloning of a cDNA encoding the murine 80 kDa subunit. We used a cDNA library derived from the mouse myeloma cell line MPC-1 1 constructed in XgtlO (kindly provided by Michael W.Kuehl, NCI). This library (500,000 pfu) was screened with a human 80k cDNA probe. Six hybridizing plaques were identified; these were characterized further by PCR using XgtlO primers. The PCR product from one of the clones, a 2.5 kb fragment, was inserted in the plasmid pCR2000 using the TA cloning system (Invitrogen). This clone, which contained the entire coding sequence, was sequenced on both strands by the chain termination method. The cDNA contains an open reading frame of 2199 bp, the amino acid sequence of which is shown in Figure 1 (upper line). It has strong homology (77.6%) with the human 80 kDa subunit (4, 5) (Figure 1, lower line). Most of the differences are conservative. Homology between the mouse (7) and human (3, 6) sequences (82.9%) was also observed for the 70 kDa subunit.
REFERENCES 1. Mimon,T., Akizuld,M., Yamagata,H., Inada,S., Yoshida,S. and Honma,M. (1981) J. Clin. Invest. 68, 611-620. 2. Mimori,T. and Hardin,J.A. (1986) J. Biol. Chem. 261, 10375-10379. 3. Chan,J.Y.C., Lerman,M.I., Prabhakar,B.S., Isozaki,O., Santisteban,P., Kuppers,R.C., Oates,E.L., Notkins,A.L. and Kohn,L.D. (1989) J. Biol. Chem. 264, 3651-3654. 4. Reeves,W.H. and Sthoeger,Z.M. (1989) J. Biol. Chem. 264, 5047-5052. 5. Yaneva,M., Wen,J., Ayala,A. and Cook,R. (1989) J. Biol. Chem. 264, 13407-13411. 6. Mimori,T., Ohosone,Y., Hama,N., Suwa,A., Akizuki,M., Homma,M., Griffith,A.J. and Hardin,J.A. (1990) Proc. Natl. Acad. Sci. USA 87, 1777-1781. 7. Porges,AJ., Ng,T. and Reeves,W.H. (1990) J. Inununol. 145, 4222 -4228.
EMBL accession no. X66323 10 MU
HU
20
30
40
50
60
KAWSVNKAWVLCVDVGVAMGNSIPGEESPIEQAJJVMTHFVQRQVFSESKDEIALALYG 1 111111111:111 :1:11111I 111:1 1111 1:111111111:1:111111:1:1
MVRSGNKAAVVLCMDVGFTMSNSIPGIESPFEQAKVITMFVQRQVFAENKDEIALVLFG 10
20
30
40
50
60
70
80
90
100
110
120
TDGTDMPLSGKDQYQNITVCRHLMLPDFDLLEDIGNKIQPSSQQADFLDALIVCMDLIQR 11111 1111 11111111 I 111111111111t:3111:1liii III ll:11:11: TDGTDNPLSGGDQYQNITVHRHLKLPDFDLLEDIESKIQPGSQQADFLDALIVSMDVIQH 70
80
90
100
110
120
130
140
150
160
170
180
ETIGKKFGKKHIEEVFTDLRSPFSKDQLDVIICNLKKCGISLQFFLPFPIDKNGEPGERGD ETIGKKFEKRH IEIFTDLSSRFSKSQLDI IIHSLKKCDISLQFFLPFSLGKEDGSGDRGD 130 140 150 160 170 180 190
200
210
220
230
240
LDSGLDHLKPSFPQKGLTEQQKEGIRHVTRVMLSLEGEDGLDEIYSFSESLRQLCVFXKI 1: 1111 11:1111111: I:1:31 :1 111111111111111111:lllli GPFRLGGHGPSFPLKGITEQQKEGLEIVKMVMISLEGEDGLDEIYSFSESLRKLCVFKKI 190
200
210
220
230
240
250
260
270
280
290
300
ERRSMPWPCQLTIGPNLSIKIVAYKSIVQEKFKKSWVVVDARTLKKEDIQKETVYCLNDD ERHSIHWPCRLTIGSNLSIR1AAYKSILQERVKKTWTVVDAKTLKKEDIQKETVYCLN DD 250
260
270
280
290
300
310
320
330
340
350
360
DETEVSKEDTIQGYRYGSDIIPFSKVDEEQMKYKSEGKCFSVLGFCKSSQVHRRFFMGHQ 11111 111:111:11111:llIIIIIItI1111111111111111111111:111111:1 DETEVLKEDI IQGFRYGSDIVPFSKVDEEQHKYKSEGKCFSVLGFCKSSQVQRRFFMGNQ 310
320
330
340
350
360
370
380
390
400
410
420
VLKVFAAKDDEIAAVALSSLVHLDELMNVAIVRYAYDIRSNPQVGVASLYIKDAYERLV
1111111:111111IIl:llIlIl:l:IIIIIIIII :11III
:11::11 11
VLKVFAARDDEAVALSSSLIHALDDLDg4VAIVRYAYDKRANPQVGVFPHIKHNYECLV 370
380
390
400
410
420
430
440
450
460
470
480
YVQLPFMEDLRQYMFSSLKNNKKCTPTEAQLSAIDDLIDSISLVK)NEEEDIVEDLFPAS tiltIlI 111 1111Il:II::IlIIII:I:I:IIII 111:1I:I::Is::I 111:: YVQLPFMEDLRQYHFSSLKNSKKYAPTEQLAVDALIDSSLAKKDEK "LEDLFPT 430
440
450
460
470
480
490
500
510
520
530
540
KIPNPEFQRLYQCLLHRALHLQERLPPIQQHI LNIWDPPTEJQKCESPPLKVKTLFPLT
KI PNPRFQRLFQCLLHRALHPREPLPPIQQHIWNMLNPPAEVTKSQIPLSKIKTLFPLI 490 500 510 520 530 540 550
560
570
580
590
600
EVIKKKNQVTAQDVFQDNHEEGPAJKYKTEKEEDHISISSLJEGNITKVGSVNPVENFR EA- KKCDQVTAQEI FQDNHEDGPTXKTEQGGAFSVSSLAEGSVTSVGSVNPAEN FR 550 560 570 580 590 610
620
630
640
650
660
FLVRQKIASFEEASLQLI SH IEQFLDTNETLY F1XSMCI XAFREE,kIQFSEEQRFNWSFL
11:11 1111111 111:1111 1111111 11111:111:1111I111;1111 1111:11 VLVKQK}(ASFEEASNQLINHI EQFLTNETPY FXS IDCIRAFREEAIKXFS EEQRFNNFL 600 610 620 630 640 650 670
680
690
700
710
EALREKVEIKQLNHFWIvWvErITKDGTSSITKDEEPSFIAPKDKPXEDMP:11:1111111111111111111:111 11:11:1 11:11111111111111:::11::
KALQEKVEIKQLNHFWEIVVQDG ITLITKEEAGSSVTAEAKFLPXDX-PSGDTAVF
660
670
720
730
680
690
700
710
EEAGDVDDLLDMIX
EEGGDVDDLLDHIX
720
730
Figure 1. Comparison of the amino acid sequences of the murine and human (4, 5) 80 kDa Ku subunits. Solid lines indiate homology; dots indicate conservative changes.
The nucleotide sequence of a mouse cDNA encoding the 80 kDa subunit of the Ku (p701p80) autoantigen Miriam Falzon and Edward L.Kuff Laboratory of Biochemistry, National Cancer Institute, Building 37, Room 4C03, Bethesda, MD 20892, USA Submitted May 29, 1992 The human Ku autoantigen is a heterodimeric nuclear protein first detected by antibodies from autoimmune patients (1) and proposed to play a role in DNA repair and/or transposition (2). Human Ku preparations contain equimolar amounts of 70 and 80 kDa polypeptides. The cDNA's encoding both human subunits, as well as that derived from the murine 70 kDa polypeptide, have been cloned and sequenced (3-7). Here we report the cloning of a cDNA encoding the murine 80 kDa subunit. We used a cDNA library derived from the mouse myeloma cell line MPC-1 1 constructed in XgtlO (kindly provided by Michael W.Kuehl, NCI). This library (500,000 pfu) was screened with a human 80k cDNA probe. Six hybridizing plaques were identified; these were characterized further by PCR using XgtlO primers. The PCR product from one of the clones, a 2.5 kb fragment, was inserted in the plasmid pCR2000 using the TA cloning system (Invitrogen). This clone, which contained the entire coding sequence, was sequenced on both strands by the chain termination method. The cDNA contains an open reading frame of 2199 bp, the amino acid sequence of which is shown in Figure 1 (upper line). It has strong homology (77.6%) with the human 80 kDa subunit (4, 5) (Figure 1, lower line). Most of the differences are conservative. Homology between the mouse (7) and human (3, 6) sequences (82.9%) was also observed for the 70 kDa subunit.
REFERENCES 1. Mimon,T., Akizuld,M., Yamagata,H., Inada,S., Yoshida,S. and Honma,M. (1981) J. Clin. Invest. 68, 611-620. 2. Mimori,T. and Hardin,J.A. (1986) J. Biol. Chem. 261, 10375-10379. 3. Chan,J.Y.C., Lerman,M.I., Prabhakar,B.S., Isozaki,O., Santisteban,P., Kuppers,R.C., Oates,E.L., Notkins,A.L. and Kohn,L.D. (1989) J. Biol. Chem. 264, 3651-3654. 4. Reeves,W.H. and Sthoeger,Z.M. (1989) J. Biol. Chem. 264, 5047-5052. 5. Yaneva,M., Wen,J., Ayala,A. and Cook,R. (1989) J. Biol. Chem. 264, 13407-13411. 6. Mimori,T., Ohosone,Y., Hama,N., Suwa,A., Akizuki,M., Homma,M., Griffith,A.J. and Hardin,J.A. (1990) Proc. Natl. Acad. Sci. USA 87, 1777-1781. 7. Porges,AJ., Ng,T. and Reeves,W.H. (1990) J. Inununol. 145, 4222 -4228.
EMBL accession no. X66323 10 MU
HU
20
30
40
50
60
KAWSVNKAWVLCVDVGVAMGNSIPGEESPIEQAJJVMTHFVQRQVFSESKDEIALALYG 1 111111111:111 :1:11111I 111:1 1111 1:111111111:1:111111:1:1
MVRSGNKAAVVLCMDVGFTMSNSIPGIESPFEQAKVITMFVQRQVFAENKDEIALVLFG 10
20
30
40
50
60
70
80
90
100
110
120
TDGTDMPLSGKDQYQNITVCRHLMLPDFDLLEDIGNKIQPSSQQADFLDALIVCMDLIQR 11111 1111 11111111 I 111111111111t:3111:1liii III ll:11:11: TDGTDNPLSGGDQYQNITVHRHLKLPDFDLLEDIESKIQPGSQQADFLDALIVSMDVIQH 70
80
90
100
110
120
130
140
150
160
170
180
ETIGKKFGKKHIEEVFTDLRSPFSKDQLDVIICNLKKCGISLQFFLPFPIDKNGEPGERGD ETIGKKFEKRH IEIFTDLSSRFSKSQLDI IIHSLKKCDISLQFFLPFSLGKEDGSGDRGD 130 140 150 160 170 180 190
200
210
220
230
240
LDSGLDHLKPSFPQKGLTEQQKEGIRHVTRVMLSLEGEDGLDEIYSFSESLRQLCVFXKI 1: 1111 11:1111111: I:1:31 :1 111111111111111111:lllli GPFRLGGHGPSFPLKGITEQQKEGLEIVKMVMISLEGEDGLDEIYSFSESLRKLCVFKKI 190
200
210
220
230
240
250
260
270
280
290
300
ERRSMPWPCQLTIGPNLSIKIVAYKSIVQEKFKKSWVVVDARTLKKEDIQKETVYCLNDD ERHSIHWPCRLTIGSNLSIR1AAYKSILQERVKKTWTVVDAKTLKKEDIQKETVYCLN DD 250
260
270
280
290
300
310
320
330
340
350
360
DETEVSKEDTIQGYRYGSDIIPFSKVDEEQMKYKSEGKCFSVLGFCKSSQVHRRFFMGHQ 11111 111:111:11111:llIIIIIItI1111111111111111111111:111111:1 DETEVLKEDI IQGFRYGSDIVPFSKVDEEQHKYKSEGKCFSVLGFCKSSQVQRRFFMGNQ 310
320
330
340
350
360
370
380
390
400
410
420
VLKVFAAKDDEIAAVALSSLVHLDELMNVAIVRYAYDIRSNPQVGVASLYIKDAYERLV
1111111:111111IIl:llIlIl:l:IIIIIIIII :11III
:11::11 11
VLKVFAARDDEAVALSSSLIHALDDLDg4VAIVRYAYDKRANPQVGVFPHIKHNYECLV 370
380
390
400
410
420
430
440
450
460
470
480
YVQLPFMEDLRQYMFSSLKNNKKCTPTEAQLSAIDDLIDSISLVK)NEEEDIVEDLFPAS tiltIlI 111 1111Il:II::IlIIII:I:I:IIII 111:1I:I::Is::I 111:: YVQLPFMEDLRQYHFSSLKNSKKYAPTEQLAVDALIDSSLAKKDEK "LEDLFPT 430
440
450
460
470
480
490
500
510
520
530
540
KIPNPEFQRLYQCLLHRALHLQERLPPIQQHI LNIWDPPTEJQKCESPPLKVKTLFPLT
KI PNPRFQRLFQCLLHRALHPREPLPPIQQHIWNMLNPPAEVTKSQIPLSKIKTLFPLI 490 500 510 520 530 540 550
560
570
580
590
600
EVIKKKNQVTAQDVFQDNHEEGPAJKYKTEKEEDHISISSLJEGNITKVGSVNPVENFR EA- KKCDQVTAQEI FQDNHEDGPTXKTEQGGAFSVSSLAEGSVTSVGSVNPAEN FR 550 560 570 580 590 610
620
630
640
650
660
FLVRQKIASFEEASLQLI SH IEQFLDTNETLY F1XSMCI XAFREE,kIQFSEEQRFNWSFL
11:11 1111111 111:1111 1111111 11111:111:1111I111;1111 1111:11 VLVKQK}(ASFEEASNQLINHI EQFLTNETPY FXS IDCIRAFREEAIKXFS EEQRFNNFL 600 610 620 630 640 650 670
680
690
700
710
EALREKVEIKQLNHFWIvWvErITKDGTSSITKDEEPSFIAPKDKPXEDMP:11:1111111111111111111:111 11:11:1 11:11111111111111:::11::
KALQEKVEIKQLNHFWEIVVQDG ITLITKEEAGSSVTAEAKFLPXDX-PSGDTAVF
660
670
720
730
680
690
700
710
EEAGDVDDLLDMIX
EEGGDVDDLLDHIX
720
730
Figure 1. Comparison of the amino acid sequences of the murine and human (4, 5) 80 kDa Ku subunits. Solid lines indiate homology; dots indicate conservative changes.
