Two haplotypes of 5' untranslated region in L-myc gene with different internal ribosome entry site activity.
We found two polymorphisms, A or C, at position 63 from the L-myc transcription start site, and 8- or 9-base Gs stretch between positions 82 and 89 or 90, in L-myc 5' UTR, which exhibited a strong linkage disequilibrium as two haplotypes, A-9G and C-8G. We analyzed whether both haplotypes of 5' UTR of the L-myc gene, each of which is approximately 200 nt long, are involved in translational regulation of the L-myc gene. Each haplotype of 5' UTR was inserted between Renilla luciferase and firefly luciferase genes to construct a bicistronic vector which was transcribed as a single mRNA. A translation of the downstream cistron was increased 70-100-fold in cos 7 cells, and 7-10-fold in HeLa S3 cells compared with a control vector without insertion. However, in this bicistronic assay system, both haplotypes of the L-myc 5' UTR showed similar internal ribosome entry site (IRES) activity. When a stable hairpin loop with a palindromic sequence was inserted into a 5'-end of bicistronic mRNA, the hairpin loop decreased only the activity of an upstream luciferase in both cos 7 and HeLa S3 cells, supporting the presence of an IRES. We also inserted 5' UTR of the L-myc gene into the 5'-end of reporter mRNA. C-8G showed approximately 2-fold higher IRES activity than A-9G in both cos 7 and HeLa S3 cells, suggesting that two polymorphisms in the L-myc exon 1 might be involved in regulation of L-myc protein expression.
Kumimoto
Hiroshi
H
Central Laboratory and Radiation Biology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan. kumimoto@aichi-cc.jp
Yamane
Yoshihiro
Y
Ishizaki
Kanji
K
D020121:5' Untranslated Regions / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D005091:Exons / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D016259:Genes, myc / Q000235:genetics*; D006239:Haplotypes / Q000235:genetics; D006801:Humans; D015810:Linkage Disequilibrium; D049410:Luciferases, Renilla / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational / Q000235:genetics* / Q000502:physiology; D011110:Polymorphism, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012270:Ribosomes / Q000502:physiology*; D024363:Transcription Initiation Site
D020121:5' Untranslated Regions; D012333:RNA, Messenger; D049410:Luciferases, Renilla