Target Discovery and Validation Reviews and Protocols

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Harnessing the Human Genome 21 Fig. 4. Structure of human SIM2. (A) Location of the SIM2 gene on a partial map (36,778,564–37,287,771 bp) of chromosome 21. HLCS, holocarboxylase synthetase; PSMD15, proteasome (prosome, macropain) 26S subunit, non-ATPase, 15; LOC388823, novel gene supported by EST alignment; MRPL20P1, mitochondrial ribosomal protein L20 pseudogene 1. (B) mRNAs for both isoforms of SIM2 (SIM2-l and SIM2-s) are shown. Boxes represent exons and lines represent introns. The unique 3′ sequences for each isoform are described. bp, base pair. (C) Schematic diagram of the conserved domains found in both SIM2 proteins. bHLH, basic helix-loop-helix; PAS1 and 2, PER/ARNT/SIM domains; HST, HIF1-α/SIM/TRH domain; S/T, Ser/Thr-rich region; NLS, nuclear localization signal; P/S RICH, Pro/Ser-rich region; P/A, Pro/Ala-rich region 5. Identification of Single-Minded 2 Gene as a Drug Therapy Target for Solid Tumors Another EST predicted by the DDD tool to be upregulated in colon tumors belonged to UniGene cluster Hs. 146186, which was homologous to the Down syndrome-associated SIM2 gene. Members of the human SIM gene family include SIM1 and SIM2, which map to 6q16.3-q21 and 21q22.2, respectively (26,27). The SIM2 locus spans over a 365-kilobase region on chromosome 21, as shown in Fig. 4A (28). In contrast to other species, the human SIM2 gene exists in two distinct forms, the long and short forms (SIM2-l and SIM2-s) as shown in Fig. 4B (26). The original cDNA clone identified was SIM2-s, whereas genomic
22 Narayanan sequencing subsequently identified SIM2-l. The SIM2-s mRNA includes a unique 3′ end, encoded by part of intron 10. This unique region, starting at the last nucleotide of exon 10 and including 1191 base pairs (bp) of intron 10, encompasses both coding and 3′ untranslated sequences. The 3′ end of SIM2-l mRNA is encoded by exon 11, which is separated from exon 10 by the complete intron 10 sequence (2528 bp). It is unclear whether these two isoforms are a consequence of an alternative use of the 3′ untranslated region contained within intron 10 of SIM2 or are due to mispriming by using an A-rich sequence within this intron (26). The SIM proteins belong to a family of transcription factors characterized by the basic helix-loop-helix (bHLH) and PAS (PER/ARNT/SIM) protein motifs (29). Regulation of transcription involves the dimerization of two bHLH-PAS proteins within the nucleus, DNA binding by the basic region of the bHLH, and interaction with the transcriptional machinery to modulate gene expression (reviewed in ref. 29). The N-terminal bHLH is the key motif for primary dimerization of the two proteins, whereas the PAS domain acts as the interface for selective protein partnering (29,30). The heterodimerizing partner choice of PAS proteins is highly specific and determines target gene regulation (31–33). The human SIM proteins are highly homologous to Drosophila and murine SIMs in their N-terminal domains, which contain a bHLH motif, two PAS domains, and the HST (HIF1-α/SIM/TRH) domain (Fig. 4C). In the mouse, mSIM1 and mSIM2 can heterodimerize with ARNT, ARNT-2 or BMAL1 (31,32,34–36), whereas in humans, hSIM1 and hSIM2 can dimerize with either ARNT or ARNT-2 (37). A novel 23-amino acid nuclear localization signal was recently identified between residues 367 and 389 (38). This signal is in a region found in both forms of the human SIM2 protein. The C-terminal part of the human proteins is considerably divergent from other family members; however, there is still high conservation between hSIM1 and hSIM2 compared with mSIM1 and mSIM2, respectively (26). Both forms of human SIM2 contain a Ser/Thr-rich region (S348–T366), a Pro/Ser-rich region (P385–S503), a Pro/Ala-rich region (P504–P544) and a positively charged region between R367 and R382. In addition, the long-form protein contains two additional Pro/Ala-rich regions (P533–P596 and P611–P644) and a positively charged region (K559–R575). Domains rich in Ser/Thr and proline residues are present in both transcriptional repressors (39) and transcriptional activators (40,41), whereas Pro/Ser and Pro/Ala domains are characteristic of repressor motifs (reviewed in ref. 42). At the time of discovery, the SIM2 gene has not been linked to any cancer types. Reasoning that if the DDD prediction of specificity of SIM2-s could be validated in a cancer model, we would be able to link the Down syndrome gene SIM2 with cancer and hence derive a novel cancer utility for an already known gene, further studies were undertaken.
Page 1 and 2: METHODS IN MOLECULAR BIOLOGY 360 T
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Page 7 and 8: vi Preface get. Approaches to targe
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Page 11 and 12: x Contributors SAHOHIME MATSUMOTO
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88 Beaty et al. 34. Peters, D. G.,
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5 Molecular Classification of Breas
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Molecular Profiling of Breast Cance
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Fig. 2. (Continued) the right ident
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6 Discovery of Differentially Expre
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Gene Target Discovery 117 In the fi
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Gene Target Discovery 119 sequences
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Gene Target Discovery 121 There is
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Gene Target Discovery 123 digestion
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Gene Target Discovery 125 Fig. 1. P
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Gene Target Discovery 127 5. Sargen
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Gene Target Discovery 129 41. Berry
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132 Matsumoto, Miyagishi, and Taira
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144 Fig. 1. The ribozyme-expression
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146 Sano and Taira 5. 10X L (low-sa
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148 Sano and Taira ribozymes may cl
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150 Fig. 3. A system for the identi
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152 Sano and Taira 4. Notes 1. We r
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9 Production of siRNA- and cDNA-Tra
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Transfected Cell Arrays 157 Fig. 1.
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Transfected Cell Arrays 159 2. The
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Transfected Cell Arrays 161 5. Simp
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164 Houdebine Fig. 1. Different met
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166 Houdebine concentrations become
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168 Houdebine Fig. 2. Major methods
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170 Houdebine integrate into the me
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172 Houdebine 2.2.3. Knock-In Into
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174 Houdebine as insulators. The co
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176 Houdebine Fig. 5. Different met
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178 Houdebine systems. Moreover, st
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180 Houdebine contribute to generat
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184 Houdebine more extensively. Tra
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186 Houdebine and stroma. Several o
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188 Houdebine explained at the mole
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190 Houdebine of the intestinal epi
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194 Houdebine 17. Whitelaw, C. B. A
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196 Houdebine 51. Bell, A. C., West
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198 Houdebine 86. Carmichael, G. G.
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200 Houdebine 121. Pailhoux, E., Vi
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202 Houdebine 156. Auerbach, A. B.,
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204 Vijayaraj, Söhl, and Magin 1.
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206 Vijayaraj, Söhl, and Magin Fig
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208 Vijayaraj, Söhl, and Magin fil
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210 Table 1 Orthologous Human and M
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212 Table 2 Orthologous Human and M
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234 Vijayaraj, Söhl, and Magin b.
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240 Vijayaraj, Söhl, and Magin alt
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242 Table 3 Time Schedule For Aggre
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250 Vijayaraj, Söhl, and Magin 101
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12 The HUVEC/Matrigel Assay An In V
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256 Skovseth, Küchler, and Haralds
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270 Iversen and Sørensen witnessed
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274 Iversen and Sørensen the core
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14 An Overview of the Immune System
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Overview of Immune System 279 diffe
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Overview of Immune System 299 (44).
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Overview of Immune System 301 demon
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Overview of Immune System 307 some
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Overview of Immune System 313 measu
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Overview of Immune System 315 42. H
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Overview of Immune System 317 74. D
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15 Potential Target Antigens for Im
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Tumor Antigen Discovery 321 develop
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Tumor Antigen Discovery 323 panel b
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Tumor Antigen Discovery 325 16. Joc
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16 Identification of Tumor Antigens
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Immunoproteomics 329 by “shot gun
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Immunoproteomics 331 isoelectric fo
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Immunoproteomics 333 3. 2D-PAGE is
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17 Protein Arrays A Versatile Toolb
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Protein Arrays 337 Table 1 Classifi
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Protein Arrays 339 fractions from c
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Protein Arrays 341 combinatorial sc
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Protein Arrays 343 The ideal proteo
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Protein Arrays 345 18. Cekaite, H.
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Protein Arrays 347 49. Yuko Kawahas
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Index 349 Index A Acetyl-CoA carbox
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Index 351 conditional by using FRT
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Index 353 Recombinant tumor antigen
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