Chyan Y J, Strauss P R, Wood T G, Wilson S H
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-1068, USA.
DNA Cell Biol. 1996 Aug;15(8):653-9. doi: 10.1089/dna.1996.15.653.
Recently, we reported the organization of the thirteen exons of the human DNA polymerase beta (beta-pol) gene and the sequences of the exon-intron junctions. Splice variants of human beta-pol mRNA have been postulated to be related to cancer development. Here, we report the characterization of isoforms of human beta-pol mRNA in different cells by reverse transcription polymerase chain reaction (RT-PCR). DNA sequence analysis of RT-PCR products revealed eight alternative splicing mRNA isoforms in the brain cancer cell line, SK-N-MC. These various isoforms were consistent with alternative splicing of four exons (II, IV, V, and VI) and with a 105-nucleotide insertion (exon alpha) between exons VI and VII. We also found an isoform with a 19-nucleotide sequence inserted into the exon IV and V junction, which resulted from usage of a different 3' splice site. Seven of the isoforms resulted in truncated open reading frame (ORF); five corresponded to deduced peptide of amino acids 1-20 of beta-pol and two corresponded to amino acids 1-60 of beta-pol. Only one of the right mRNA isoforms, that with the exon alpha insertion, was in-frame with the entire wild-type ORF resulting in a deduced protein of 370 residues, compared with the wild-type protein of 335 residues and 39 kD. This longer ORF was shown to be capable of encoding a beta-pol protein, larger than wild-type beta-pol, that cross-reacted with beta-pol antibody and exhibited beta-pol enzymatic activity. The mRNA isoform with the exon alpha insertion was not tumor specific because it as detected in low abundance in all cells tested, except the colon cell line CCD18 Co where the isoform was absent. The genomic location of exon alpha is in intron VI, 990 bp upstream of exon VII and flanked by consensus splice sites. Thus, this 105-bp genomic sequence is a beta-pol exon present in a low-abundance beta-pol mRNA isoform capable of encoding an approximately 42-kD beta-pol.
最近，我们报道了人类DNA聚合酶β（β-pol）基因13个外显子的组织情况以及外显子-内含子连接区的序列。据推测，人类β-pol mRNA的剪接变体与癌症发展有关。在此，我们通过逆转录聚合酶链反应（RT-PCR）报道了不同细胞中人类β-pol mRNA异构体的特征。RT-PCR产物的DNA序列分析揭示了脑癌细胞系SK-N-MC中有8种可变剪接的mRNA异构体。这些不同的异构体与4个外显子（II、IV、V和VI）的可变剪接以及外显子VI和VII之间105个核苷酸的插入片段（外显子α）一致。我们还发现一种异构体，其在第IV和第V外显子连接处插入了19个核苷酸的序列，这是由于使用了不同的3'剪接位点所致。其中7种异构体导致截短的开放阅读框（ORF）；5种对应于β-pol第1至20位氨基酸的推导肽段，2种对应于β-pol第1至60位氨基酸的推导肽段。只有一种正确的mRNA异构体，即插入了外显子α的异构体，与整个野生型ORF读框一致，推导的蛋白质有370个残基，而野生型蛋白质有335个残基，分子量为39 kD。这种较长的ORF能够编码一种比野生型β-pol更大的β-pol蛋白，该蛋白与β-pol抗体发生交叉反应并表现出β-pol酶活性。插入了外显子α的mRNA异构体并非肿瘤特异性的，因为除了结肠癌细胞系CCD18 Co中未检测到该异构体外，在所有测试细胞中其丰度都很低。外显子α的基因组位置在内含子VI中，位于外显子VII上游990 bp处，两侧为共有剪接位点。因此，这段105 bp的基因组序列是β-pol外显子，存在于一种低丰度的β-pol mRNA异构体中，能够编码一种约42 kD的β-pol。
