| Co-Investigator(Kenkyū-buntansha) |
SHIBA Yuichi Osaka University Hospital., Clinical Fellow, 医学部附属病院, 医員
KAJIMOTO Yoshitaka Osaka University Hospital., Clinical Fellow, 医学部附属病院, 医員
岩間 令道 大阪大学, 医学部・附属病院, 医員
山崎 義光 大阪大学, 医学部, 助手 (40201834)
河盛 隆造 大阪大学, 医学部, 講師 (00116021)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1993: ¥800,000 (Direct Cost: ¥800,000)
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| Research Abstract |
Two basic helix-loop-helix (bHLH) transcription factors, E47 and E12, are involved in the insulin geneexpression as part of dimeric complexes which interact with the cis-acting motif, E-box. Although both generated from a single gene (E2A) by means of alternative splicing, the structural difference in these bHLH regions between the two suggests that two bHLH proteins may differ in some of their functions. We reccuntly indentified an amino acid (aa) polymorphism in the highly conserved region within the leucine zipper (LZ) motif. Interestingly, the location and pattern of aa delections are identical to those previously described for the aa polymorphism within the human counterpart, E47 (E12) [Cell60 (1990) 547-555], which was insertion or deletion of glutamine.
To explore the possible physiological meaning of the polymorphism, we surveyed seven mouse strains (C57BL/6 ob/ob, C57BL/6J,C57BL/KsJ,ICR,BALB/c ByJ,DBA/2J and C3H/HeJ) in terms of the type of this polymorphism. The LZ regions of
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the AI cDNAs (nt31-401) were cloned and DNA-sequenced. Among the seven strains that we examined, six had the glutamine (-)-type A1, suggesting that this type of A1 is rather prevalent in terms of numbers of strains. Only the C3H strain had the glutamine (+)-type A1. Because the C3H strain is known to be relatively hyperinsulinemic, the possible physiological significance was suggested. However, the two types of A1, when expressed in CHO cells, revealed the same DNA-binding activity, suggesting that they retains the same DNA-binding activity at least in CHO cells.
To elucidate the individual implications of E47 and E12, we also investigated the mRNA expression ratios of their homologues (A1 and KA1, respectively) in mouse tissues and cell lines. Measurement using reverse-transcription polymerase chain reaction and a specific oligodeoxyribonucleotide hybridization technique revealed that both the A1 and kA1 mRNAs were ubiquitously expressed in all tissues examined. However, their ratios varied : e.g.skeletal muscle, 2.2<plus-minus>0.3 (mean<plus-minus>SE), spleen, 2.0<plus-minus>0.2, pancreatic islet cells, 1.2<plus-minus>0.2. The A1 kA1 ratios in the cell lines investigated were similar to those of their original tissues. The ubiquity in mRNA expression observed for both the E47 and E12 homologues in mouse provides support for their involvement in a broad range of transcriptional regulation. The variation in the A1/kA1 expression ratios, on the other hand, supports the idea that A1 (E47) and KA1 (E12) each have some unique roles in the functions of these E2A geneencoded bHLH p Less
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