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(12) Patent Application Publication (10) Pub. No.: US 2009/0023592 A1 Abu-Khabar Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2009/0023592 A1 Abu-Khabar Et Al US 20090023592A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0023592 A1 Abu-Khabar et al. (43) Pub. Date: Jan. 22, 2009 (54) SYSTEM FOR IDENTIFYING AND (57) ABSTRACT ANALYZNG EXPRESSION OF The present invention relates to a gene discovery system and ARE-CONTAINING GENES gene expression systems specific for genes encoding ARE containing mRNAS. In one aspect, the present invention (76) Inventors: Khalid S. Abu-Khabar, Riyadh relates to computational methods of selecting coding (SA); Bryan R.G. Williams, sequences of ARE-genes from databases using aone or more Cleveland, OH (US); Mathias ARE search sequences. The ARE Search sequences are from Frevel, Wellington (NZ); Robert H. 10 to 80 nucleotides in length and comprise a sequence which Silverman, Beachwood, OH (US) is encompassed by one of the following two sequences: (a) WU/T(AU/TU/TU/TA)TWWW, SEQ ID NO. 1, wherein none or one of the nucleotides outside of the parenthesis is Correspondence Address: replaced by a different nucleotide, and wherein W represents CALFEE HALTER & GRISWOLD, LLP A, U. or T; and (b) U/T(AU/TU/T/U/T)n, SEQ ID NO. 2, 800 SUPERIORAVENUE, SUITE 1400 wherein n indicates that the search sequence comprises from CLEVELAND, OH 44114 (US) 3 to 12 of the tetrameric sequences contained within the parenthesis. The method comprises extracting from the data (21) Appl. No.: 11/774,296 bases, those nucleic acids whose protein coding sequences are upstream and contiguous with a 3"untranslated region (22) Filed: Jul. 6, 2007 (UTR) that comprises one of the ARE search sequences. The present invention also relates to methods of selectively ampli Related U.S. Application Data fying RNA and cDNA molecules using primers derived from and complementary to the consensus 5' sequence motifs and (62) Division of application No. 10/257.294, filed on Jul. primers derived from and complementary to the ARE search 14, 2003, filed as application No. PCT/US01/11993 on sequence. The present invention also relates to methods of Apr. 12, 2001. selectively amplifying ARE genes which employ a 3' primer which is from 15 to 50 nucleotides and length and comprises from 2 to 10 pentamers having the sequence TAAAT. The (60) Provisional application No. 60/196.870, filed on Apr. pentameric sequences in the primers are either overlapping or 12, 2000. non-overlapping. The 3' primers are used in the reverse tran Scription step of the methods, the polymerase chain reaction Publication Classification (PCR) amplification step of the methods, or in both the reverse transcription step and the PCR amplification step of (51) Int. C. the methods. The present invention also relates to methods of C4OB 30/04 (2006.01) making libraries which comprise portions of the ARE genes CI2P 19/34 (2006.01) that are selectively amplified by the present methods and to C4OB 50/00 (2006.01) methods of making microarrays which comprise probes that C4OB 50/4 (2006.01) hybridize under Stringent conditions to portions of the protein C40B 40/08 (2006.01) coding sequences of the ARE genes that are selectively ampli fied by the present methods. The present invention also relates (52) U.S. Cl. ................ 506/9:435/91.2: 506/23: 506/30; to libraries and the microarrays that are made by such meth 506/17 ods. Aggress as a N area. als Patent Application Publication Jan. 22, 2009 Sheet 1 of 16 US 2009/0023592 A1 Fig. 1. 1320 bp 289 bp 1612 bp 838 bp Patent Application Publication Jan. 22, 2009 Sheet 2 of 16 US 2009/0023592 A1 Fig. 2. L-8 B-actin MW 1 2 3 4 5 1 2 3 4 5 600. Trehalose - - - - - - - - - - Patent Application Publication Jan. 22, 2009 Sheet 3 of 16 US 2009/0023592 A1 Fig.3. 32.5°C 35C 40°C 32.5°C 35c 37.5°C 40°C 1 2 3 4 1 2 3 4 2 3 4. f-actin Patent Application Publication Jan. 22, 2009 Sheet 4 of 16 US 2009/0023592 A1 Fig. 4. Patent Application Publication Jan. 22, 2009 Sheet 5 of 16 US 2009/0023592 A1 Fig. 5, 42OC 52°C 1929 CC2CSIS 3-actin --> 10 uM, dNTPs -------- -- - - -- 40 uM, dNTPs -- - - -- -------- 1/10 ARE-cDNA + i + + + + i + /50 ARE-cDNA -- - - - - - - -- Patent Application Publication Jan. 22, 2009 Sheet 6 of 16 US 2009/0023592 A1 sis is is s is is {&################ º.2#####*&##*/ *################# *################ !##*&######## ·jeggae Figure 6A 60 120 180 Durat iOn Of Treatment , min Figure 6B Patent Application Publication Jan. 22, 2009 Sheet 7 of 16 US 2009/0023592 A1 Super Induction by LPS+ Cycloheximide % ARE Genes Time induced repressed maximum > 1.5 > 15 induction 2O 33.3 1.6 25.82 40 28.0 2.3 52.08 6O 31.5 3.3 292.06 120 21.3 12.8 890.03 180 12.7 37.9 302.07 Figure 6C Induction by LPS % ARE Genes Time induced repressed maximum > 1.5 > 15 induction 2O 2O.O O.1 3.98 40 46 O4 5.46 6O 15.O O2 7.41 12O 28.O 1.O 103.62 18O 11.9 8.7 71.86 Patent Application Publication Jan. 22, 2009 Sheet 8 of 16 US 2009/0023592 A1 Fig. 7 GACTCCACAACCACGACACACACATACACACACACACACACACACACACACACA CACACACACACACACACNATTTCCTGGGGGCTCTGGAGCTCGGCAA ACCTCCCAT CCCAGCTCTACTACCATTTAGCTGTGTGACCTTTAGCAGGTTGTTTCACATCTCTG AGCCTCCATTTCNCCCATTAGGCACAATGAAGACTGATATNGTTGCAAGAATAAA TTAACTTGCACACCGGTATGAAATGGCCGCCTTAATACCTTAGANGGGAGA ACA CCCTCCCCCCCCCCCAAGCCTCTNGGGTTCTCCCCCTGTTCCTTTATTAATCTTTAT GGCCCCTGTTGTTTCTTGGGTTTGTTGGGAATTTCGGGGNTTTCTCCCTCCAAAAA CTTGCCAGAAAGCCNTTTATCGTTTTTCGGGATTTCCTTTATTTTAGGGTTGGACC CCTATT GACTCCACA ACCACGACACACAGTAGGCACTCAATAAATCCTTGAATGAATTTTA AAAATGAATGTGTATCACTAGTGCCTGATTCTTAATCTGATCAGTA ACTGCTTGA TGACTGAATCAATCGCTGATGGAAGGAACAATTATGCCTCATTCACTGCCGTAAC ATGGGAACCTTGCATACAGCAGGTGCTTAATAAATGCTGGGGCACATGAA ACAG ATGAAGAGGGCTTGGTTCCTCCCAATTCCCCAGGGCCAAGCCCTGTTGCTGGCAT GCAGTAGGCACTCCATTAATGCTGCCTGTGTGCACGTGATTGCTTAGCAAGAGCT GGCTGGACACCACCCCACACGAGAGCCCTCTGTGGCCTGCCAAGGGGTCCAGAG GTGGGGGCTTCTCCTCAAAACTGCAGACGCGTTACGTATCGGTTCCTTATTTAGG TGACCACTATAGAATTCCGATATGTGGTCGACAAGCTTCTCGAGCCTAGGCTAGC TCTAGACCACACT R8 GACTCCACAACCACGACACAGGATGTGAAGGAGGTCATTGAATACGCACGGCTC CGGGGTATCCGTGTGCTTTGCAGAGTTTGACACTCCTGGCCACCCTTTGTCCTGG GGACCAGGTATCCCTGGATTACTGACTCCTGCTAATCTGGGTCTGAGCCCTCTGG CACCTTTGGACCAGTGAATCCCAGTCTCAATAATACCTATGAGTTCATGAACACA TTCTTCTTAAAAATCAGCTTCTGTCTTCCCAGATTTTTATCTTCATCTTGGAGGAG ATGAGGTTGATTTCACCTGCTGGAAGTCCCAACCCAGAAGATCCCAGGACTTTAT GAGGAAGAAAGGCTTCGGTGAGGACTTCAAGCAGCGGGAGTCCTTTACATCCAG ACGCTGCTGGACATCGTCTCTTCTTATGGCAAGGGCTATGTGTCGTGGTTGTGGA GTCGGGCTCTTGTCGTCGTCATCGTGCCACCGGTACCGCTGTCGATGTGCTGGCG TTCGAATTTAGCAGCAGCGGTTTCTTTGGTAACACCCATGGTGGCCAAAGTTGAG CGTTTATTCTGAGCTTCTGC R7 GACTCCACAACCACGACACAGGATGTGAAGGAGGTCATTGAATACGCACGGCTC CGGGGTATCCGTGTGCTTTGCAGAGTTTGACACTCCTGGCCACCCTTTGTCCTGG Patent Application Publication Jan. 22, 2009 Sheet 9 of 16 US 2009/0023592 A1 GGACCAGGTATCCCTGGATTACTGACTCCTGCTAATCTGGGTCTGAGCCCTCTGG CACCTTTGGACCAGTGAATCCCAGTCTCAATAATACCTATGAGTTCATGAACACA TTCTTCTTAAAAATCAGCTTCTGTCTTCCCAGATTTTTATCTTCATCTTGGAGGAG ATGAGGTTGATTTCACCTGCTGGAAGTCCCAACCCAGAAGATCCCAGGACTTTAT GAGGAAGAAAGGCTTCGGTGAGGACTTCAAGCAGCGGGAGTCCTTTACATCCAG ACGCTGCTGGACATCGTCTCTTCTTATGGCAAGGGCTATGTGTCGTGGTTGTGGA GTCGGGCTCTTGTCGTCGTCATCGTGCCACCGGTACCGCTGTCGATGTGCTGGCG TTCGAATTTAGCAGCAGCGGTTTCTTTGGTAACACCCATGGTGGCCAAAGTTGAG CGTTTATTCTGAGCTTCTGC R6 GACTCCACAACCACGACACATTCTGGTGTAATTGCTCTGGAGCAGGACTTGGATG TCAGTGTAGTACAGCTTCTCCAGGTGATTCTAATGTGTGTCGTGGTTGTGGAGTC GGGCTCTTGTCGTCGTCATCGCTGCCACCGGTACCGCTGTCGATGTGCTGGCGTT CGAATTTAGCAGCAGCGGTTTCTTTGGTA ACACCCATGGTGGGCCAAAGTTGAAC CGTTTTATTCCTGAGCCTTCTGCAAAAAGAACAACGAG R4 GACTCCACAACCACGACACATTCTGGTGTAATTGCTCTGGAGCAGGACTTGGATG TCAGTGTAGTACAGCTTCTCCAGGTGATTCTAATGTGTGTCGTGGTTGTGGAGTC GGGCTCTTGTCGTCGTCATCGCTGCCACCGGTACCGCTGTCGATGTGCTGGCGTT CGAATTTAGCAGCAGCGGTTTCTTTGGTA ACACCCATGGTGGGCCAA AGTTGAAC CGTTTTATTCCTGAGCCTTCTGCAAAAAGAACAACGAG R3 GACTCCACA ACCACGACACACTCAGAAAACCTCCTCCATAAAGACACTGGCCTC AAAAAAAGAACCCCTGTCCCTTTCAAATTAGAAGAGAAAAGGACACGAAGATAA GTCAGCAGACTTGGCTTACCCTGCCCAGAGCGCGCGCCTCAA ACCTCCTTCAGTG AGAACTGCCCTTTTTCCAGACAGAATTCCTGACGTGCACCCTGGGCTCAAGGCTG GACAAATTCCTCCTGGAGCCTCCTGACGGAAGATGTGATGTTCACCGTGGGGGCC TCCCTTTGCTCCCTTAAAAAGGGCATTTCACACCAAAGAAAGGAATTTTA ACCTC CTTTCGGAAAAGGCCTGGCCCCCTCCCCTGGCTTGGGCCGGAACTTGGAAGGGGC ACCGGGATTTCCCCAGCCAGAAACAGGGCCCGGCTTGCTCCGTGTCACGGTTCAA GGGCTGAGCAGGGGAGGCAAGGAAGCTGGCGGGTTTAAAGGTGAATCAGTTAGC ATTGATGTGAATCAGTTACCACTGTCTTTTTCGGCAATTCAGAGGTGGGCCCTGG GACAAGCAGGGAATTGATTCCCCCGGGGTATTAAGGCAGCAAAAAATCTGAAAG ACCCTGGAGAAATCTCCCGTTAGACAGACTTTTCAGGACAGCCTGTGTCGTGGTT GTGGAGTCGGGCTTCTCCTCAA AACTGCAGACGCGTTACGTATCGGATCCTTATT TAGGTGACACTATAGAATTCCGATATTGGTCGACAAGCTTCTCGAGCGTAGGCTA GCTCTAGACCACACGTGTGGGGGCCCGACTC Patent Application Publication Jan. 22, 2009 Sheet 10 of 16 US 2009/0023592 A1 R12 GACTCCACAACCACGACACACTCAGAAAACCTCCTCCATAAAGACACTGGCCTC AAAAAAAGAACCCCTGTCCCTTTCAAATTAGA AGAGAAAAGGACACGA AGATAA GTCAGCAGACTTGGCTTACCCTGCCCAGAGCGCGCGCCTCAA ACCTCCTTCAGTG AGAACTGCCCTTTTTCCAGACAGAATTCCTGACGTGCACCCTGGGCTCAAGGCTG GACAAATTCCTCCTGGAGCCTCCTGACGGAAGATGTGATGTTCACCGTGGGGGCC TCCCTTTGCTCCCTTAAAAAGGGCATTTCACACCAAAGAAAGGAATTTTAACCTC CTTTCGGAAAAGGCCTGGCCCCCTCCCCTGGCTTGGGCCGGAACTTGGAAGGGGC ACCGGGATTTCCCCAGCCAGAAACAGGGCCCGGCTTGCTCCGTGTCACGGTTCAA GGGCTGAGCAGGGGAGGCAAGGAAGCTGGCGGGTTTAAAGGTGAATCAGTTAGC ATTGATGTGAATCAGTTACCACTGTCTTTTTCGGCAATTCAGAGGTGGGCCCTGG GACAAGCAGGGAATTGATTCCCCCGGGGTATTAAGGCAGCAAAAAATCTGAAAG ACCCTGGAGAAATCTCCCGTTAGACAGACTTTTCAGGACAGCCTGTGTCGTGGTT GTGGAGTCGGGCTTCTCCTCAA AACTGCAGACGCGTTACGTATCGGATCCTTATT TAGGTGACACTATAGAATTCCGATATTGGTCGACAAGCTTCTCGAGCGTAGGCTA GCTCTAGACCACACGTGTGGGGGCCCGACTC R11 GACTCCACAACCACGACACACTCAGAAAACCTCCTCCATAAAGACACTGGCCTC AAAAAAAGAACCCCTGTCCCTTTCAAATTAGA AGAGAAAAGGACACGA AGATAA GTCAGCAGACTTGGCTTACCCTGCCCAGAGCGCGCGCCTCAA ACCTCCTTCAGTG AGAACTGCCCTTTTTCCAGACAGAATTCCTGACGTGCACCCTGGGCTCAAGGCTG GACAAATTCCTCCTGGAGCCTCCTGACGGAAGATGTGATGTTCACCGTGGGGGCC
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