United States Patent (19)
`Duan et al.
`
`54 ELL2, A NEW MEMBER OF AN ELL FAMILY
`OF RNA POLYMERASE II ELONGATION
`FACTORS
`
`75 Inventors: D. Roxanne Duan, Bethesda, Md.; Ali
`Shilatifard, St. Louis, Mo.; Joan W.
`Conaway; Ronald C. Conway, both of
`Oklahoma City, Okla.
`73 Assignees: Human Genome Sciences, Inc.,
`Rockville, Md., Oklahoma Medical
`Research Foundation, Oklahoma City,
`Okla.
`
`21 Appl. No.: 09/026,343
`22 Filed:
`Feb. 19, 1998
`Related U.S. Application Data
`60 Provisional application No. 60/038.447, Feb. 19, 1997.
`51
`Int. CI.
`C12N 15/63; C12N 15/64
`nt. Cl. ...........................
`C12N 15/11; C12N 15/12
`52 U.S. Cl. ..................... 435/69.1; 435/91.41; 435/243;
`435/252.3; 435/254.11; 435/320.1; 435/325;
`435/419; 435/455; 435/468; 536/23.1; 536/23.4;
`536/23.5
`58 Field of Search .................................. 435/69.1, 91.4,
`435/320.1, 455, 325, 91.41, 243, 252.3,
`254.11, 419,468; 536/23.5, 23.1, 23.4
`
`56)
`
`References Cited
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`(45) Date of Patent:
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`
`(List continued on next page.)
`Primary Examiner John S. Brusca
`Attorney, Agent, or Firm-Sterne, Kressler, Goldstein &
`Fox, p.l.l.c.
`ABSTRACT
`57
`ELL2 polypeptides and polynucleotides and methods for
`producing Such polypeptides by recombinant techniques are
`disclosed. Also disclosed are methods for utilizing ELL2
`polypeptides and polynucleotides in the design of protocols
`for the treatment of neoplastic disorders, among others and
`diagnostic assays for Such conditions.
`
`96 Claims, 7 Drawing Sheets
`
`IPR2022-00090 - LGE
`Ex. 1007 - Page 1
`
`

`

`6,008.018
`Page 2
`
`OTHER PUBLICATIONS
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`Press, Ltd., New York, NY, pp. 263-278 (1994).
`Reines, D. et al., “The RNA polymerase II general elonga
`tion factors,” Trends in Biochemical Sci. (TiBS) 21:351–355
`(Sep. 1996).
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`88:4245-4249 (1991).
`Ruff, P. et al., “Molecular identification of a major palmi
`toylated erythrocyte membrane protein containing the Src
`homology 3 motif.” Proc. Natl. Sci. USA 88:6595-6599
`(1991).
`Shilatifard, A. et al., “An RNA Polymerase II Elongation
`Factor Encoded by the Human ELL Gene,” Science
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`Stark, M.J.R., “Multicopy expression vectors carrying the
`lac repressor gene for regulated high-level expression of
`genes in Escherichia coli,” Gene 51:255-267 (1987).
`Tan, S. et al., “A Bacteriophage Vector Suitable for Site-Di
`rected Mutagenesis and High-Level Expression of Multi
`subunit Proteins in E.coli,” BioTechniques 16:824-828
`(1994).
`Thirman, M.J. et al., “Cloning of ELL, a gene that fuses to
`MLL in a to 11:19)(q23;p13.1) in acute myeloid leukemia,”
`Proc. Natl. Acad. Sci. USA 91:12110–12114 (1994).
`Willott, E. et al., “The tight junction protein ZO-1 is
`homologous to the Drosphila discS-large tumor Suppressor
`protein of a septate junctions.” Proc. Natl. Acad. Sci. USA
`90:7834–7838 (1993).
`Woods, D.F. and P.J. Bryant, “The Discs-Large Tumor
`Suppressor Gene of Drosophila Encodes a Guanylate Kinase
`Homolog Localized at Septate Junctions,” Cell 66:451-464
`(1991).
`Yu, H. et al., “Structural Basis for the Binding of Proli
`ne-Rich Peptides to SH3 Domains,” Cell 76:933–945
`(1994).
`
`NCBI Entrez, GenBank Report, Accession No. Z20670,
`from MRC Human Genome Mapping Project Resource
`Centre (1995), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. T89063,
`from Hillier, L. et al. (1995), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. R12663,
`from Hillier, L. et al. (1995), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. R16400,
`from Hillier, L. et al. (1995), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. N39822,
`from Hillier, L. et al. (Jan. 1996), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. W92650,
`from Hillier, L. et al. (Nov. 1996), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. W94585,
`from Hillier, L. et al. (Nov. 1996), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. AA191245,
`from Hillier, L. et al. (Jan. 1997), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. AA414900,
`from Marra, M. et al. (May 1997), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. AA370180,
`from Adams, M.D. et al. (Apr. 1997), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. AA370048,
`from Adams, M.D. et al. (Apr. 1997), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. AA375277,
`from Adams, M.D. et al. (Apr. 1997), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. AAO09921,
`from Hillier, L. et al. (May 1997), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. AA545429,
`from Marra, M. et al. (Aug. 1997), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. AA252607,
`from NCI-CGAP (Aug. 1997), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. AA243384,
`from NCI-CGAP (Aug. 1997), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. AA524290,
`from NCI-CGAP (Aug. 1997), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. AA655966,
`from Marra, M. et al. (Nov. 1997), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. AA743118,
`from NCI-CGAP (Jan. 1998), with Revision History.
`NCBI Entrez, GenBank Report, Accession No. W13766,
`from Marra, M. et al. (Oct. 1997), with Revision History.
`
`IPR2022-00090 - LGE
`Ex. 1007 - Page 2
`
`

`

`U.S. Patent
`
`Dec. 28, 1999
`
`Sheet 1 of 7
`
`6,008.018
`
`CAGTGGCGGCGGGTGCAGAAGCCCAAGCAGCGCGGCCGCAGTGGAGGCTAGAGCCGGAGC
`1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - --
`
`GGCGGCGGCGGCGGCACCCCGGGGAGGTTTAAGATGGCGGCGGGGGGGACAGGGGGCCTG
`61 - - - - - - - - - + - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
`-
`--
`--
`--
`--
`M A A G G T G G L.
`
`60
`
`120
`
`121
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`. m n
`
`v
`
`180
`
`240
`
`300
`
`CGGGAGGAGCAGCGCTATGGGCTGTCGTGCGGACGGCTGGGGCAGGACAACATCACCGTA
`--
`--
`- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
`.
`R E E Q R Y G L S C G R L G Q D N I T V
`CTGCATGTGAAGCTCACCGAGACGGCGATCCGGGCGCTCGAGACTTACCAGAGCCACAAG
`181 - - - - - - - - - - - - - - - - - - - - - - - - - - - h - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`L.
`H. V K L T E T A I R A L E T Y Q S H K
`AATTTAATTCCTTTTCGACC TCAATCCAGTTCCAAGGACTCCACGGGCTTGTCAAAATT
`241 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`N
`I P F R P S I O F O G L H G L V K I
`CCCAAAAATGATCCCCTCAATGAAGTTCATAACTTAACTTTTATTTGTCAAATGTGGGC
`301 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + 360
`P K N D P L N E W H N F N F Y L S N W G
`AAAGACAACCCTCAGGGCAGCTTTGACTGCATCCAGCAAACATTCTCCAGCTCTGGAGCC
`361 - - - - - - - - - - - - - - - - - - t- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + 420
`K D N P O G S F D C I Q Q T F S S S G A
`TCCCAGCTCAATTGCCTGGGATTTATACAAGATAAAATTACAGTGTGTGCAACAAACGAC
`421 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - +
`S O L N C L G F I Q D K I T V C A T N D
`TCGTATCAGATGACACGAGAAAGAATGACCCAGGCAGAGGAGGAATCCCGCAACCGAAGC
`481 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`S Y Q M T R E R M T Q A E E E S R N R S
`ACAAAAGTTATCAAACCCGGTGGACCATATGTAGGGAAAAGAGTGCAAATTCGGAAAGCA
`541 - - - - - - - - - - - - - - - - - - hi - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`T K W I K P G G P Y W G K R V Q I R K A
`
`480
`
`540
`
`600
`
`CCTCAAGCTGTTTCAGATACAGTTCC GAGAGGAAAAGGTCAACCCCCATGAACCCTGCA
`601 - - - - - - - - - - - - - - - - - - - h - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`P Q A V S D T V P E R K R S T P M N P A
`AATACAATTCGAAAGACACATAGCAGCAGCACCATCTCTCAGAGGCCATACAGGGACAGG
`661 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + 720
`N T I R K T H S S S T I S Q R P Y R D R
`
`660
`
`721 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`W I H L L A
`K A Y K K P E L L A R L Q
`F.G. 1A
`
`78O
`
`IPR2022-00090 - LGE
`Ex. 1007 - Page 3
`
`

`

`U.S. Patent
`
`Dec. 28, 1999
`
`Sheet 2 of 7
`
`6,008.018
`
`901
`
`840
`
`900
`
`960
`
`1020
`
`1080
`
`1140
`
`AAAGATGGTGTCAATCAAAAAGACAAGAACTCCCTGGGAGCAATTCTGCAACAGGTAGCC
`781 - - - - - - - - - + - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
`--
`--
`--
`--
`--
`K D G V N 0 K D K N S L G A I
`L Q 0 W A
`AATCTGAATTCTAAGGACCTCTCATATACCTTAAAGGATTATGTTTTTAAAGAGCTTCAA
`841 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
`--
`+
`--
`--
`--
`N L N S K D L S Y T L K D Y V F K E L Q
`AGAGACTGGCCTGGATACAGTGAAATAGACAGACGGTCATTGGAGTCAGTGCTCTCTAGA
`- - - - -
`- -a- -
`- + - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -h
`R D W P G Y S E I D R R S L E S W L S R
`AAACTAAATCCGTCTCAGAATGCTACAGGCACCAGCCGTTCAGAATCTCCTGTATGTTCT
`961 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`K L N P S Q N A T G T S R S E S P W C S
`AGTAGAGATGCTGTATCTTCTCCTCAGAAACGGCTTTTGGATTCAGAGTTTATTGATCCT
`1021 - - - - - - - - - - - - - - - - - - - h - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`S R D A W S S P 0 K R L L D S E F I D P
`TTAATGAATAAAAAAGCCCGAATATCTCACCTGACGAACAGAGTACCACCAACACTAAAT
`1081 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`M N K K A R I S H L T N R W P P T L N
`GGTCATTTGAATCCCACCAGGAAAAATCGGCTGCAGGCCTCCCACTGCCCCCTGCGGCT
`1141 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + - - - - - - - - - + 1200
`G H L N P T S E K S A A G L P L P P A A
`GCTGCCATCCCCACCCCTCCACCGCTGCCTTCAACCTATCTGCCCATCTCACATCCTCCT
`1201 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - hi - - - - - - - - - - - - - - - - - - - +
`A A I
`P T P P P L P S T Y
`P I S H P P
`CAGATTGTAAATTCTAACTCCAACTCCCCTAGCACTCCAGAAGGCCGGGGGACTCAAGAC
`1261 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`Q I V N S N S N S P S T P E G R G T Q D
`CACCTGTTGACAGTTTTAGTCAAAACGATAGTATCTATGAGGACCAGCAAGACAAATAT
`1321 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`L P W D S F S Q N D S I Y E D C Q D K Y
`ACCTCTAGGACTTCTCTGGAAACCTTACCCCCTGGTTCCGTTCTACTAAAGTGTCCAAAG
`1381 - - - - - - - - - h - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`T S R T S L E T L P P G S v L L K C P K
`CCTATGGAAGAAAACCATTCAAGTCTCACAAAAAGTCCAAAAAGAAGTCTAAAAAACAT
`1441 - - - - - - - - - - - - - - - - - - -- - - - - - - - - - h - - - - - - - - - -- - - - - - - - - - - - - - - - - - - +
`P M E E N H S M S H K K S K K K S K K H
`AAGGAAAAGGACCAAATAAAAAAGCACGACATTGAGACTATTGAGGAAAAGGAGGAAGAT
`1501 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`K E K D O I K K H D I
`E T |
`E E K E E D
`CAAGAGAGAAGAGGAAATTGCCAAGCTAAATAACTCCAGTCCAAATTCCAGGGAGGA
`1561 - - - - - - - - - - - - - - - - - - - - - - - - - - - - h - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`
`1260
`
`1320
`
`1380
`
`1440
`
`1500
`
`1560
`
`1620
`
`F.G. 1B
`
`IPR2022-00090 - LGE
`Ex. 1007 - Page 4
`
`

`

`U.S. Patent
`
`Dec. 28, 1999
`
`Sheet 3 of 7
`
`6,008.018
`
`1621
`
`K R E E E J A K L N N S S P N S S G G
`GTTAAAGAGGATTGCACTGCCTCCATGGAACCTTCAGCAATTGAACTCCCAGATTATTTG
`He rom
`-
`-
`-
`- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
`V K E D C T A S M El P S A
`E L P D Y L.
`ATAAAATATATCGCTATCGTCTCCTATGAGCAACGCCAGAATTATAAGGATGACTTCAAT
`1681 - - - - - - - - - - - - - - - - - - - - - - - - - - - - h - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`I K Y I A I V S Y E Q R O N Y K D D F N
`GCAGAGTATGATGAGTACAGAGCTTTGCATGCCAGGATGGAGACTGTAGCTAGAAGATTT
`1741 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`A E Y D E Y R A L H A R M El T W A R R F
`
`ATCAAACTAGATGCACAAAGAAAGCGCCTTTCTCCAGGCT CAAAAGAGTATCAGAATGTT
`1801 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - h - - - - - - - - - +
`--
`I K L D A Q R K R L S P G S K E Y O N V
`CATGAAGAAGTCTTACAAGAATATCAGAAGAAAAGCAGTCTAGTCCCAATTACCATGAA
`1861 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`
`1921
`
`GAAAAATACAGATGTGAATATCTTCATAACAAGCTGGCTCACATCAAAAGGCTAATAGGT
`-
`-
`Wr
`- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
`E K Y R C E Y L H N K L A H I K R L I
`G
`GAATTTGACCAACAGCAAGCAGAGTCATGGTCCTAGAACTCTGCTTGGACCAGAAGATGT
`1981 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - * - - - - - - - - - +
`E F D Q Q Q A E S W S *
`GAATAAACTTAAGCTTATTTATTAAAATCCAAATGAGTTGCTCTAGATTCTAAAAAGG
`2041 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
`
`TGAAACTTTGGCTGTTGAAAGTTTCAGTATTAGTAAACT
`2101 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
`
`2139
`
`1680
`
`1740
`
`1800
`
`1860
`
`1920
`
`1980
`
`2040
`
`2100
`
`FIG. 1 C
`
`IPR2022-00090 - LGE
`Ex. 1007 - Page 5
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`

`

`U.S. Patent
`
`Dec. 28, 1999
`
`Sheet 4 of 7
`
`6,008,018
`
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`OaMSIVES
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`ella
`
`14
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`ell
`
`113
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`e114
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`
`IPR2022-00090 - LGE
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`Ex. 1007 - Page 6
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`Ex. 1007 - Page 6
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`U.S. Patent
`
`Dec. 28, 1999
`
`Sheet 5 of 7
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`6,008.018
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`TRASCRIPTION
`ELONGATION ACTIVITY
`ELL2A(1-10) O-O-O
`ELL2A(1-50) O-O-O
`ELL2A(499-640) O-O-
`ELL2A(389-640) O
`ELL2A(194-640) O
`ELL2A(100-640) O
`ELL2A(50-194) ONu-O-O-O
`TRANSCRIPTION
`VARIABLE
`ELONGATION DOMAN PRO-RCH
`R3
`R
`R2
`EL2 -O-O-O --
`K-RCH
`
`
`
`SMILAR TO OCCLUDIN
`70- BNDING DOMAIN
`
`Occludin
`
`ELL2
`Occludin
`ELL2
`Occludin
`ELL2
`
`415 DWIREYPPTSDQQRQYKRNFDTGLQEYKSLQSELDENKELSRLDKEL
`:: .
`. .
`.
`::... ::... ::.: , ; ; ;
`;
`527
`DYLIKYIAIWSYEQRONYKDDFNAEYDEYRALHARMETWARRFKLDAQR
`465 DDYREESEEWAAAD.EYNRLKOVKGSADYKSKKNHCKOLKSKLSH
`.
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`. . .
`. ::
`:::... : .
`. .
`.
`577
`KRLSPGSKEYQNWHEEVLQEYQKIKO. SSPNYHEEKYRCEYLHNKLAHI
`511 KKWGDYDROKT
`522
`:::::...
`625 KRLIGEFDOOOA
`636
`
`464
`
`576
`510
`624
`
`FIG. 3
`
`IPR2022-00090 - LGE
`Ex. 1007 - Page 7
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`U.S. Patent
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`Dec. 28, 1999
`
`Sheet 6 of 7
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`6,008.018
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`
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`Hind
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`U.S. Patent
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`Dec. 28, 1999
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`Sheet 7 of 7
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`6,008,018
`
` WWILLVOVOVOLLULVYVVVLVDDOVOLILIVWODOVLOLVOVVIZ430}D4ad9Ol-YYIVVIVOOOOVILOILOVOLILOVIVWWVWYVOOLOVYWWVVLLIOVY
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`
`IPR2022-00090 - LGE
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`

`

`1
`ELL2, A NEW MEMBER OF AN ELL FAMILY
`OF RNA POLYMERASE II ELONGATION
`FACTORS
`
`This application claims the benefit of the filing date of
`provisional application 60/038,447 filed on Feb. 19, 1997,
`which is herein incorporated by reference.
`
`5
`
`FIELD OF THE INVENTION
`This invention relates to newly identified polynucleotides,
`polypeptides encoded by them and to the use of Such
`polynucleotides and polypeptides, and to their production.
`More particularly, the polynucleotides and polypeptides of
`the present invention relate to a new member of an ELL
`15
`family of RNA polymerase II elongation factors, hereinafter
`referred to as ELL2. The invention also relates to inhibiting
`or activating the action of Such polynucleotides and
`polypeptides.
`
`BACKGROUND OF THE INVENTION
`The elongation Stage of eukaryotic messenger RNA Syn
`thesis is a major Site for the regulation of gene expression
`(Reines, D. et al., Trends. Biochem. Sci. 21:351–355 (1996),
`Bentley, D. L., Curr. Opin. Genet. Dev. 5:210–216 (1995)).
`Moreover, a growing body of evidence Suggests that mis
`regulation of elongation may be a key element in a variety
`of human diseases (ASO, T. et al., J. Clin. Invest.
`97:1561–1569 (1996)).
`To date, one virally encoded protein (Tat) and five cellular
`proteins (SII, P-TEFb, TFIIF, Elongin (SIII), and ELL) have
`been defined biochemically and shown to be capable of
`controlling the activity of the RNA polymerase II elongation
`complex. Among these elongation factors, three have been
`implicated in human disease. The HIV-1 encoded Tat protein
`is required for efficient transcription of HIV-1 genes and for
`productive infection by the virus (Jones, K. A. & Peterlin, B.
`M., Annu. Rev. Biochem. 63:717–743 (1994)). Elongin (SIII)
`is a potential target for regulation by the product of the Von
`Hippel-Lindau (VHL) tumor Suppressor gene, which is
`mutated in the majority of clear-cell renal carcinomas and in
`families with VHL disease, a rare genetic disorder that
`predisposes individuals to a variety of cancers (Duan, D. R.
`et al., Science 269:1402–1406 (1995), Kibel, A. et al.,
`Science 269: 1444–1446 (1995)). The ELL gene on chromo
`Some 19pl3.1 was originally isolated as a gene that under
`goes frequent translocations with the Drosophila trithorax
`like MLL gene on chromosome 11q23 in acute myeloid
`leukemia (Thirman, M.J. et al., Proc. Natl. Acad. Sci. U.S.A.
`91: 12110-12114 (1994), Mitani, K. et al., Blood
`85:2017-2024 (1995)).
`This indicates that these proteins have an established,
`proven history as therapeutic targets. Clearly there is a need
`for identification and characterization of further related
`proteins which can play a role in preventing, ameliorating or
`correcting dysfunctions or diseases, including, but not lim
`ited to, AIDS and neoplastic disorders, among others.
`
`25
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`35
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`40
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`45
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`50
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`55
`
`SUMMARY OF THE INVENTION
`In one aspect, the invention to ELL2 polypeptides and
`recombinant materials and methods for their production.
`Another aspect of the invention relates to methods for using
`Such ELL2 polypeptides and polynucleotides. Such uses
`includes the treatment of neoplastics disorders, among oth
`erS. In Still another aspect, the invention relates to methods
`to identify agonists and antagonists using the materials
`
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`2
`provided by the invention, and treating conditions associated
`with ELL2 imbalance with the identified compounds. Yet
`another aspect of the invention relates to diagnostics assays
`for the detection of diseases associated with inappropriate
`ELL2 activity or levels and mutations in ELL2 that might
`lead to neoplastic disorders (particularly leukemias).
`BRIEF DESCRIPTION OF THE FIGURES
`FIG. 1A-1C shows the nucleotide and deduced amino
`acid sequence of human ELL2 (SEQ ID NOS: 1 and 2,
`respectively).
`FIG. 2 shows a comparison of the deduced amino acid
`sequences of human ELL2 (SEQ ID NO:2) and ELL (SEQ
`ID NO:7). Similar amino acids (A.S.T.P.; D.E.N.O; H.R.K;
`I.L.M.V., F.Y.W) and identical amino acids are boxed.
`FIG. 3 shows the localization of the ELL2 elongation
`activation domain and a Summary of ELL2 mutants and their
`activities in transcription. Wild type ELL2 is diagramed at
`the bottom of the panel. Conserved regions 1, 2, and 3 (R1,
`R2, and R3) are indicated by the shaded boxes. The align
`ment of region 3 (SEQ ID NO:2) with the C-terminal ZO-1
`binding domain of occludin (SEQ ID NO:8) was generated
`with the BESTFIT program of the Genetics Computer
`Group package, using the Symbol comparison table of
`Gribskov and Burgess (Gribskov, M. & Burgess, R. R.,
`Nucleic. Acids. Res. 14:6745–6763 (1986)).
`FIG. 4 shows a schematic representation of the pHE4-5
`expression vector (SEQ ID NO:33) and the subcloned ELL2
`cDNA coding Sequence. The locations of the kanamycin
`resistance marker gene, the ELL2 coding Sequence, the oriC
`Sequence, and the lacq coding Sequence are indicated.
`FIG. 5 shows the nucleotide sequence of the regulatory
`elements of the pHE promoter (SEQ ID NO:34). The two lac
`operator Sequences, the Shine-Delgarno Sequence (S/D), and
`the terminal HindIII and Ndel restriction sites (italicized) are
`indicated.
`
`DETAILED DESCRIPTION OF THE
`PREFERRED EMBODIMENTS
`
`Definitions
`The following definitions are provided to facilitate under
`Standing of certain terms used frequently herein.
`“Neoplastic disorder” refers to a disease state which is
`related to the hyperproliferation of cells. Neoplastic disor
`ders include, but are not limited to, carcinomas, Sarcomas
`and leukemias.
`“Protein Activity” or “Biological Activity of the Protein'
`refers to the metabolic or physiologic function of said ELL2
`protein including Similar activities or improved activities or
`these activities with decreased undesirable Side-effects. Also
`included are antigenic and immunogenic activities of Said
`ELL2 protein. Among the physiological or metabolic activi
`ties of Said protein are the regulation of the activity of the
`RNA polymerase II elongation complex. AS demonstrated in
`Example 1, ELL2 increases the overall rate of elongation by
`RNA polymerase II during both promoter-dependent and
`-independent transcription. Additional activities include the
`ability to bind components of the RNA polymerase II
`elongation complex and SH3 domains.
`“ELL2 polypeptides' refers to polypeptides with amino
`acid Sequences Sufficiently similar to ELL2 protein
`Sequences that they exhibit at least one biological activity of
`the protein.
`“ELL2 gene' refers to a polynucleotide having the nucle
`otide sequence set forth in SEQ ID NO:1 or the nucleotide
`
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`3
`Sequence encoding the protein as contained in the cDNA
`insert of ATCC Deposit No. 97863 or allelic variants thereof
`and/or their complements.
`“ELL2 polynucleotides” refers to a polynucleotide con
`taining a nucleotide Sequence which encodes an ELL2
`polypeptide or fragment thereof or that encodes an ELL2
`polypeptide or fragment wherein Said nucleotide Sequence
`has at least 95% identity to a nucleotide Sequence encoding
`the polypeptide of SEQ ID NO:2 or the corresponding
`fragment thereof or which has Sufficient identity to a nucle
`otide sequence contained in SEQ ID NO:1 or contained in
`the cDNA insert of ATCC Deposit No. 97863 to hybridize
`under conditions useable for amplification or for use as a
`probe or marker.
`"Antibodies' as used herein includes polyclonal and
`monoclonal antibodies, chimeric, Single chain, and human
`ized antibodies, as well as Fab fragments, including the
`products of an Fab or other immunoglobulin expression
`library.
`“Isolated” means altered “by the hand of man” from the
`natural State. If an "isolated” composition or Substance
`occurs in nature, it has been changed or removed from its
`original environment, or both. For example, a polynucle
`otide or a polypeptide naturally present in a living animal is
`not "isolated,” but the same polynucleotide or polypeptide
`Separated from the coexisting materials of its natural State is
`"isolated’, as the term is employed herein. Thus, a polypep
`tide or polynucleotide produced and/or contained within a
`recombinant host cell is considered isolated for purposes of
`the present invention. Also intended as an "isolated polypep
`tide' or an "isolated polynucleotide' are polypeptides or
`polynucleotides that have been purified, partially or
`Substantially, from a recombinant host cell or from a native
`Source. For example, a recombinantly produced version of
`the ELL2 polypeptide can be substantially purified by the
`one-step method described in Smith and Johnson, Gene
`67:31-40 (1988).
`“Polynucleotide’ generally refers to any polyribonucle
`otide or polydeoxribonucleotide, which may be unmodified
`RNA or DNA or modified RNA or DNA. “Polynucleotides”
`include, without limitation Single- and double-Stranded
`DNA, DNA that is a mixture of single- and double-stranded
`regions, single- and double-stranded RNA, and RNA that is
`mixture of Single- and double-Stranded regions, hybrid mol
`ecules comprising DNA and RNA that may be single
`Stranded or, more typically, double-Stranded or a mixture of
`Single- and double-Stranded regions. In addition, "poly
`nucleotide' refers to triple-Stranded regions comprising
`RNA or DNA or both RNA and DNA. The term polynucle
`otide also includes DNAS or RNAS containing one or more
`modified bases and DNAS or RNAS with backbones modi
`fied for stability or for other reasons. “Modified” bases
`include, for example, tritylated bases and unusual baseS Such
`as inoSine. A variety of modifications have been made to
`DNA and RNA; thus, “polynucleotide' embraces
`chemically, enzymatically or metabolically modified forms
`of polynucleotides as typically found in nature, as well as the
`chemical forms of DNA and RNA characteristic of viruses
`and cells. “Polynucleotide” also embraces relatively short
`polynucleotides, often referred to as oligonucleotides.
`"Polypeptide' refers to any peptide or protein comprising
`two or more amino acids joined to each other by peptide
`bonds or modified peptide bonds, i. e., peptide isosteres.
`“Polypeptide” refers to both short chains, commonly
`referred to as peptides, oligopeptides or oligomers, and to
`longer chains, generally referred to as proteins. Polypeptides
`
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`4
`may contain amino acids other than the 20 gene-encoded
`amino acids. "Polypeptides' include amino acid Sequences
`modified either by natural processes, Such as post
`translational processing, or by chemical modification tech
`niques which are well known in the art. Such modifications
`are well described in basic texts and in more detailed
`monographs, as well as in a Voluminous research literature.
`Modifications can occur anywhere in a polypeptide, includ
`ing the peptide backbone, the amino acid Side-chains and the
`amino or carboxyl termini. It will be appreciated that the
`Same type of modification may be present in the same or
`varying degrees at Several Sites in a given polypeptide. Also,
`a given polypeptide may contain many types of modifica
`tions. Polypeptides may be branched as a result of
`ubiquitination, and they may be cyclic, with or without
`branching. Cyclic, branched and branched cyclic polypep
`tides may result from post-translation natural processes or
`may be made by synthetic methods. Modifications include
`acetylation, acylation, ADP-ribosylation, amidation, cova
`lent attachment of flavin, covalent attachment of a heme
`moiety, covalent attachment of a nucleotide or nucleotide
`derivative, covalent attachment of a lipid or lipid derivative,
`covalent attachment of phosphotidylinositol, croSS-linking,
`cyclization, disulfide bond formation, demethylation, for
`mation of covalent cross-links, formation of cystine, forma
`tion of pyroglutamate, formylation, gamma-carboxylation,
`glycosylation, GPI anchor formation, hydroxylation,
`iodination, methylation, myristoylation, oxidation, pro
`teolytic processing, phosphorylation, prenylation,
`racemization, Selenoylation, Sulfation, transfer-RNA medi
`ated addition of amino acids to proteins Such as arginylation,
`and ubiquitination. See, for instance, PROTEINS
`STRUCTURE AND MOLECULAR PROPERTIES, 2nd
`Ed., T. E. Creighton, W. H. Freeman and Company, New
`York, 1993 and Wold, F., Posttranslational Protein Modifi
`cations: Perspectives and Prospects, pgs. 1-12 in POST
`TRANSLATTONAL