Expression of multiple forms of MEL1 gene products.

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Expression of multiple forms of MEL1 gene products. Expression of multiple forms of MEL1 gene products. (A) Schematic representation of the amino-terminus of MEL1, RNase protection, and primer extension mapping with 5′-rapid amplification of cDNA ends (5′-RACE) analysis. Part of the human MEL1 genomic structure is schematically shown as a bar with the predicted domain structure as indicated. The nucleotide and exon numbers, the position of 5 in-frame ATG sites, the Bgl II restriction site, and the PR domain are indicated. From the RNase protection assay (indicated as S-1), 3 transcription initiation sites were identified and are indicated by asterisks, as in panel B. From the primer extension and 5′-RACE (indicated as 5′-RACE), 3 transcription initiation sites with nucleotide numbers were identified (arrowheads). The lower 3 lanes show the 5′ end of cDNA clones (N207, N1163/N2094, and Δ13MEL1, respectively) with exon structures and the position of ATG (indicated as M). (B) RNase protection assay. An antisense MEL1 RNA probe was generated and radiolabeled with T3 RNA polymerase using the Bgl II-linearized plasmid template SK207. The RNA probe was hybridized with 30 μg yeast tRNA (lane 3), or total RNA from Kasumi-3 with t(3;7)(q26;q22) (lane 4), or t(1;3)(p36;q21)-positive leukemia cells (lane 5). The positions of major protected fragments or undigested probes (lane 6) are marked with stars and arrows, respectively. Lanes 1 and 2 are 100-bp and 1-kb ladder markers, respectively, and the numbers of base pairs are given as bp. (C) Detection of cDNA products by primer extension and 5′-RACE of MEL1. Two major bands, 450 and 200 bp, are shown in cDNA from poly(A)+ RNA from t(1;3)(p36;q21)-positive AML cells (lane 1), but no cDNA was amplified from control CD4+ lymphocytes (lane 2). The 100-bp ladder markers are presented in lane 3. (D) Nucleotide sequences of exons 1 and 2 with 5′ end of the MEL1 cDNA clones. Black flags indicate the 5′ end of 3 cDNA clones (N207, N1163, and N2094). White flags indicate the 5′ end of 5′-RACE cDNAs, including 5 clones from nt –176, 3 clones from nt 90, and 7 clones from nt 333. Ichiro Nishikata et al. Blood 2003;102:3323-3332 ©2003 by American Society of Hematology