Identification of novel mRNA isoforms for human DNA polymerase beta.
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.
Chyan
Y J
YJ
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-1068, USA.
Strauss
P R
PR
Wood
T G
TG
Wilson
S H
SH
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003429:Cross Reactions; D004256:DNA Polymerase I / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005091:Exons / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D009928:Organ Specificity; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D004256:DNA Polymerase I