USOO6159729A United States Patent (19) 11 Patent Number: 6,159,729 Hofmann et al. (45) Date of Patent: Dec. 12, 2000 54 SYNTHETIC HPV6/11 HYBRID L1 DNA Russo, P.; Saccharomyces cerevisiae mRNA3 End Forming ENCODING HUMAN PAPLLOMAVIRUS Signals are also Involved in Transcription Termination; TYPE 11 L1 PROTEIN Yeast; vol. 11: (1995); pp. 447-453. 75 Inventors: Kathryn J. Hofmann, Collegeville; Thalenfeld, B. E. et al.; olil Transcripts in Wild Type and in Kathrin U. Jansen, Fort Washington; a Cytoplasmic “Petite” Mutant of Yeast; the Journal of Michael P. Neeper, Collegeville; Biological Chemistry; vol. 258; No. 23; Issue of Dec. 10; Joseph G. Joyce, Lansdale, Hugh A. (1983); pp. 14065–14068. George, Schwenksville; E. Dale Lang, W. H. et al.; A Model for Transcription Termination by Lehman, Lansdale, all of Pa. RNA Polymerase I; Cell; vol. 79; (1994); pp. 527–534. 73 Assignee: Merck & Co., Ltd., Rahway, N.J. Heidmann, S. et al.; Flexibility and Interchangeability of Polyadenylation Signals in Saccharomyces cerevisiae, Mol. 21 Appl. No.: 08/913,462 Cell. Biology; vol. 14; No. 7; (1994); pp. 4633-4642. 22 PCT Filed: Mar. 26, 1996 Brambilla, A. et al., A Simple signal element mediates transcription termination and mRNA 3’ end formation in the 86 PCT No.: PCT/US96/04117 DEG1 gene of Saccharomyces cerevisiae, Mol. Gen. Genet; S371 Date: Jan. 26, 1998 vol. 254; (1997); pp. 681–688. S 102(e) Date: Jan. 26, 1998 Henikoff, S. et al.; Transcription Terminates in Yeast Distal to a Control Sequence; Cell; vol. 33; (1983); pp. 607-614. 87 PCT Pub. No.: WO96/30520 Zaret, K. S. et al.; DNA Sequence Required for Efficient PCT Pub. Date: Oct. 3, 1996 Transcription Termination in Yeast; Cell; vol. 28; (1982); pp. 563-573. Related U.S. Application Data Rose et al. (1993) Expression of human papillomavirus type 63 Continuation of application No. 08/413,572, Mar. 30, 1995, 11 L1 protein in insect cells: in Vivo and in vitro assembly abandoned, which is a continuation of application No. of viruslike particles. J. Virol. 67: 1936–1944, Apr. 1993. 08/413,571, Mar. 30, 1995, abandoned. 51) Int. Cl." ............................ C12N 15/11; C12N 15/81 52 U.S. Cl. ...................................... 435/320.1; 536/23.72 Primary Examiner Nancy Degen 58 Field of Search .............................. 424/186.1, 204.1; ASSistant Examiner Robert Schwartzman 435/69.1, 69.3, 235.1, 320.1; 514/2, 44; Attorney, Agent, or Firm Joanne M. Giesser; Jack L. 530/387.9, 388.3, 389.4; 536/23.1, 23.72 Tribble 57 ABSTRACT 56) References Cited The present invention is directed to a synthetic DNA mol PUBLICATIONS ecule encoding purified human papillomavirus type 11 L1 Zaret, K. S. et al.; Mutationally Altered 3 Ends of Yeast protein and derivatives thereof. CYC1 mRNA Affect Transcript Stability and Translational Efficiency; J. Mol. Biol.; vol. 176; (1984); pp. 107-135. 7 Claims, 12 Drawing Sheets U.S. Patent Dec. 12, 2000 Sheet 1 of 12 6,159,729 OLIGOMER DESIGNATIONS: (#241–1) Bg II Sph I Kpn XhoI Bgill 2 3 4 5 6 7 8 9 1 0 1 1 12 24 23 22 21 20 19 18 17 6 5 4 13 FRAGMENT ASSEMBLY: OLIGOMER ANNEAL ING LIGATION A B C D SUBCLONE A,B,C,D SEPARATELY INTO pSP72 SEQUENCE RECLEAVE TO RELEASE INSERTS LIGATE C AND D INTO pSP72 A B CD RELEASE CD FROM pSP72 WITH Kpn I Bg II LIGATE WITH Bgill, Sphi-DIGESTED A AND Sph I, Kpn I-DIGESTED B ISOLATE 1.5 kb Bg III FRAGMENT LIGATE WITH Bomhi DIGESTED pCo. 1-10 Bom/Bg II Bom/Bg II PLASM ID #D361-1 FIG.1 U.S. Patent Dec. 12, 2000 Sheet 2 of 12 6,159,729 10 20 50 40 50 lowy-16Ll ATCTCTCTTT CCCTCCCTAC TGACGCCACT CTCTACTTGC CTCCTCTCCC 50 hpv161.gb ATCTCTCTTT CGCTCCCTAC. TCACCCCACT CTCTACTTCC CTCCTCTCCC 50 hpv161.mr 1 ATCTCTCTTT CGCTCCCTAG TGACCCCACT GTCTACTTCC CTCCTCTCCC 50 60 70 80 90 100 Lowy-16Ll 5 AGTATCTAAG GTTGTAAGCA CGGATGAATA TGTTGCACCC ACAAACATAT 100 hpv1611.gb 5 ACTATCTAAG GTTGTAAGCA CCGATGAATA TGTTGCACGC ACAAACAAT 100 hpv 161.mri 5 AGTATCTAAG GTTGTAAGCA CCGATGAATA TGTTGCACCC ACAAACATAT 100 110 120 130 40 150 Lowy-16Ll 101 ATTATCATCC AGGAACATCC AGACTACTTG CAGTTGGACA TCCCTATTTT 150 hpv61.gb 101 ATTATCATGC AGGAACATCC AGACTACTTG CAGTTCGACA TCCCTATTTT 150 hpv161.mr 10 ATTATCATCC ACGAACATCC AGACTACTTC CAGTTCGACA TCCCTATTTT 150 160 170 180 190 200 Lowy-16L1 151 CCTATTAAAA AACCTAACAATAACAAAATA TTAGTTCCTA AAGTATCACG 200 hpv 161.gb 151 CCTATTAAAA AACCTAACAATAACAAAATA TTAGTTCCTA AACTATCAGG 200 hpv 161.mr 151 CCTATTAAAA AACCTAACAATAACAAAATA TTAGTTCCTA AAGTATCACG 200 210 220 230 240 250 Lowy-16Ll 201 ATTACAATAC AGGGTATTTA GAATACATTT ACCTGACCCC AATAAGTTTC 250 hpv161.gb 201 ATTACAATAC ACCGTATTTA GAATACATTT ACCTGACCCC AATAACTTTC 250 hpv161.mr 20 ATTACAATAC ACCGTATTTA GAATACATTT ACCTCACCCC AATAACTTTC 250 260 270 280 290 300 Lowy-16L 25 GTTTTCCTGA CACCTCATTT TATAATCCAG ATACACACCC GCTCGTTTCC 500 hpv161.gb 25 GTTTTCCTGA CACCTCATTT TATAATCCAC ATACACACCC CCTGGTTTCC 300 hpv16.mri 25 GTTTTCCTCA CACCTCATTT TATAATCCAG ATACACACCC CCTGGTTTCC 500 30 520 50 340 350 Lowy-16L 301 GCCTGTGTAG GTGTTGACGT ACGTCGTGGT CAGCCATTAG GTGTGGGCAT 350 hpv161.gb 301 GCCTGTGTAG GTGTTGACGT ACGTCGTGGT CAGCCATTAG GTGTGGGCAT 350 hpv161.mri 501 GCCTGTGTAC GTGTTGACGT ACGTCGTCGT CAGCCATTAC GTGTGGGCAT 350 360 370 380 590 400 Lowy-16L 35 TAGTCCCCAT CCTTTATTAAATAAATTCGA TGACACAGAA AATCCTACTG 400 hpv161.gb 351 TAGTGGCCAT CCTTTATTAA ATAAATTCGA TGACACAGAAAATCCTACTG 400 hpv1611.mri 551 TAGTGGCCAT CCTTTATTAA ATAAATTGGA TGACACAGAA AATGCTAGTG 400 40 420 450 440 450 Lowy-16L 401 CTTATGCAGC AAATGCAGGT GTGGATAAA GAGAATGTAT ATCTATGGAT 450 hpv1611.gb 401 CTATCCACC AAATCCACGT GTGGATAATA GAGAATGTAT ATCTATGGAT 450 hpv6.mr 401 CTTATCCAGC AAATCCACGT CTCGATAATA CAGAATGTAT ATCTATCGAT 450 FIG.2A U.S. Patent Dec. 12, 2000 Sheet 3 of 12 6,159,729 460 470 480 490 500 Lowy-16Ll 451 TACAAACAAA CACAATTGTG TTTAATTCGT TCCAAACCAC CTATAGGGGA 500 hpv 161.gb 45 TACAAACAAA CACAATTGTG TTTAATTGGT TCCAAACCAC CTATAGGGGA 500 hpv 1611.mr 451 TACAAACAAA CACAATTGTG TTTAATTCGT TCCAAACCAC CTATAGGGGA 500 50 520 530 540 550 Lowy-16Ll 501 ACACTCGGGC AAAGGATCCC CATGTACCAA TGTTCCAGTA AATCCACGTG 550 hpv1611.gb 50 ACACTGGGCC AAACGATCCC CATGTACCAA TGTTCCAGTA AATCCACCTG 550 hpv161.mr 501 ACACTGGCGC AAAGGATCCC CATGTACCAA TGTTCCAGTA AATCCACGTG 550 560 570 580 590 600 Lowy-16L1 551 ATTCTCCACC ATTAGACTTA ATAAACACAG TTATTCACGA TCGTGATATG 600 hpv 1611.gb 551 ATTGTCCACC ATTAGAGTTA ATAAACACAG TTATTCACGA TCGTGATATG 600 hpv161.mri 551 ATTCTCCACC ATTAGAGTTA ATAAACACAG TTATTCACGA TCGTGATATG 600 610 620 630 640 650 Lowy-16Ll 601 CTTGATACTG GCTTTCCTCC TATCGACTTT ACTACATTAC ACCCTAACAA 650 hpv 1611.gb 601 GTTOATACTC CCTTTCCTCC TATCGACTTT ACTACATTACAGCCTAACAA 650 hpv1611, mri 60 GTTGATACTG CCTTTCGTGC TATCGACTTT ACTACATTAC ACGCTAACAA 650 660 670 680 690 700 Lowy-16Ll 651 AACTGAACTT CCACTCGATA TTTGTACATC TATTTCCAAA TATCCAGATT 700 hpv1611.gb 65 AACTGAACTT CCACTCGATA TTTCTACATC TATTTCCAAA TATCCAGATT 700 hpv161.mri 65 AACTGAACTT CCACTCCATA TTTGTACATC TATTTCCAAA TATCCACATT 700 710 720 730 740 750 Lowy-16L1 70 ATATTAAAAT GCTCTCAGAA CCATATCCCG ACACCTTATT TTTTTATTTA 750 hpv 1611.gb 701 ATATTAAAAT CCTGTCAGAA CCATATCGCG ACACCTTATT TTTTTATTTA 750 hpv161.mri 701 ATATTAAAAT CGTGTCAGAA CCATATCCCG ACACCTTATT TTTTTATTTA 750 760 770 780 790 800 Lowy-16L1 751 CCAACCCAAC AAATGTTTCT TAGACATTTA TTTAATACCC CTCCTACTGT 800 hpv 1611.gb 751 CGAAGGGAAC AAATGTTTGT TAGACATTTA TTTAATAGCC CTCCTACTGT 800 hpv61.mr 75 CGAAGGGAAC AAATGTTTGT TAGACATTTA TTTAATACGG CTCGTGCTGT 800 810 820 830 840 850 lowy-16L1 801 TCGTGAAAAT GTACCAGACC ATTTATACAT TAAACGCTCT GCCTCTACTG 850 hpv 1611.gb 80 TCGTGAAAAT GTACCAGACG ATTTATACAT TAAAGGCTCT GGGTCTACTG 850 hpv1611 mri 80 TCCTGAAAAT GTACCAGACC ATTTATACAT TAAACGCTCT CCGTCTACTG 850 860 870 880 890 900 Lowy-16Ll 851 CAAATTTAGC CACTTCAAAT TATTTTCCTA CACCTAGTCG TTCTATGGTT 900 hpv161.gb 851 CAAATTTACC CACTTCAAAT TATTTTCCTA CACCAGTGG TTCTATGGTT 900 hpv1611.mri 851 CAAATTTAGC CAGTTCAAAT TATTTTCCTA CACCTAGTGG TTCTATGGTT 900 FIG.2B U.S. Patent Dec. 12, 2000 Sheet 4 of 12 6,159,729 90 920 950 940 950 Lowy-16L 901 ACCTCTGATG CCCAAATATT CAATAAACCT TATTGGTTAC AACGAGCACA 950 hpv161.gb 901 ACCTCTGATG CCCAAATATT CAATAAACCT TATTGGTTAC AACGAGCACA 950 hpv61.mr 901 ACCTCTCATG CCCAAATATT CAATAAACCT TATTGGTTAC AACGAGCACA 950 960 970 980 990 1000 Lowy-16L1 951 GGGCCACAAT AATCGCATTT GTTGGGGTAA CCAACTATTT GTTACTGTTG 1000 hpv1611.gb 951 GGGCCACAAT AATGGCATTT GTTGGGGTAA CCAACTATTT GTTACTGTTG 1000 hpv161, mri 951 GGGCCACAAT AATCGCATTT GTTCGGGTAA CCAACTATTT GTTACTGTTG 1000 1010 1020 1030 1040 1050 Lowy-16L1 1001 TTGATACTAC ACGCAGTACA AATATGTCAT TATGTGCTCC CATATCTACT 1050 hpv1611.gb 1001 TTGATACTAC ACGCAGTACA AATATGTCAT TATGTGCTGC CATATCTACT 1050 hpv161.mr 1001 TTGATACTAC ACGCAGTACA AATATGTCAT TATCTCCTGC CATATCTACT 1050 1060 1070 1080 1090 1100 Lowy-16L 1051 TCAGAAACTA CATATAAAAA TACTAACTTT AAGGAGTACC TACGACATGG 1100 hpv1611.gb 1051 TCAGAAACTA CATATAAAAA TACTAACTTT AACGAGTACC TACGACATGG 1100 hpv1611.mri 1051 TCAGAAACTA CATATAAAAA TACTAACTTT AAGGAGTACC TACGACATGG 1100 1110 120 1130 140 150 Lowy-16L1 1101 GGAGGAATAT GATTTACAGT TTATTTTTCA ACTGTCCAAA ATAACCTTAA 1150 hpv1611.gb 1101 CGAGGAATAT GATTTACACT TTATTTTTCA ACTCTCCAAA ATAACCTTAA 1150 hpv161.mr 1101 CGACCAATAT GATTTACAGT TTATTTTTCA ACTGTCCAAAATAACCTTAA 1150 1160 170 1180 1190 1200 Lowy-16L1 1151 CTCCAGACGT TATGACATAC ATACATTCTA TGAATTCCAC TATTTTGGAG 1200 hpv161.gb 1151 CTCCAGACGT TATGACATAC ATACATTCTA TGAATTCCAC TATTTTGGAG 1200 hpv1611.mr 1151 CTCCAGACGT TATGACATAC ATACATTCTA TGAATTCCAC TATTTTGGAG 1200 1210 1220 1230 1240 1250 Lowy-16L1 1201 GACTGGAATT TTGGTCTACA ACCTCCCCCA GGAGCCACAC TAGAAGATAC 1250 hpv161.gb 1201 CACTCCAATT TTGGTCTACA ACCICCCCCA CGAGGCACAC TAGAAGATAC 1250 hpv1611.mr 1201 GACTGGAATT TTGGTCTACA ACdCCCCCA CGAGCCACAC