| Human genes typically contain multiple introns, and in many cases the exons can be joined in more than one way to generate multiple mRNAs, encoding distinct protein isoforms. This process - alternative splicing - is a major mechanism for modulating the expression of cellular genes and enables a single gene to increase its coding capacity, allowing the synthesis of several structurally and functionally distinct protein isoforms.The CYP2D subfamily comprises the CYP2D6 gene and 4 psudogenes, CYP2D7P1 and 2 (or CYP2D7AP and CYP2D7BP) and CYP2D8P1 and 2 (or CYP2D8AP and CYP2D8BP) . Six mRNA splice variants of CYP2D have been identified in human. Varients a and b retaining the intron 5 and 6, respectively; varient b' has the 3' 91 bp portion of exon 6 deleted; varient c has exon 6 deleted; varient d retaining 57 bp portion of intron 6; varient e has the 3' portion of intron 6 included and the 61 bp fragment at the 3' end of exon 6 deleted.The human CYP2C subfamily comprises four members, CYP2C8, CYP2C9, CYP2C18 and CYP2C19. CYP2C18 mRNA was expressed majorly in human epidermis. Transcripts that have skipped CYP2C18 exon 5, exon 4, exon 4, 5 and 6, or exon 4, 5, 6, and 7 were also identified in epidermis.In order to study the metabolic functions of human CYP2D6 and CYP2C18, we cloned CYP2D6 and CYP2C18 cDNA from Chinese liver tissue by reverse transcription-polymerase chain reaction (RT-PCR). We found a new type of pre-mRNA alternative splicing of human CYP2D6 by DNA sequencing. Then we identified another two new types of pre-mRNA alternative splicing of human CYP2D6 variants in other liver tissue. A CYP2C18 variant with exon 5 skipped beidentified in human liver tissue, and its metabolic function were studied.Part 1 Identification of three new types of CYP2D6 alternative splicing variantsMethods: CYP2D6 cDNA was cloned by RT-PCR from human liver tissue and ligated with pGEM-T. pGEM-CYP2D6 was constructed and sequenced. The transcripts of CYP2D6 exon 1 to 4 were analysed by RT-PCR in 17 liver tissue. Several RT-PCR products of CYP2D6 exon 1 to exon 4 that shorter than the anticipated total length of 700 bp were sequenced. Genomic DN A was analysed by sequencing the PCR products amplified using primers covering exon 1 to exon 4 of CYP2D6 gene.Results: A much shorter RT-PCR product of CYP2D6 cDNA about 1.1 kb in length was found. This cDNA was ligated with pGEM-T and sequenced. Sequence analysis indicated the cDNA clone has 470 nucleotides from 79 to 548, or the 3' end portion 102-bp of exon 1 (180 bp), exon2, exon 3, and the 5' end portion 43-bp of exon 4 (161 bp) been skipped, and 13 base pairs substitution and 1 base pair insertion and 1 base pair deletion, e.g. 620A→G, 712G→T, 1196T→G, 1401G→C, 1405C→G, 1408A→G, 1410T→C, 1432C→T, 1433A→C, 1435G→C, 1440 insG, 1442T→C, 1443T→A, 1449Cdel, 1457G→C. This cDNA has premature stop codon at codon 96. This variant was named variant f. Then the transcripts of CYP2D6 exon 1 to 4 were assayed with RT-PCR using 17 liver tissue. 4 samples expressed both wild type and skipped mRNA, 5 samples expressed only wild type mRNA, and 8 samples expressed only skipped mRNA. There are two types of short in length of RT-PCR products, e.g. about 300 and 550 bp. Two more alternative splicing mRNA been identified by sequencing the 300 and 550 bp RT-PCR products. One variant losing 411 nucleotides from 175 to 585 (137 amino acid residue from 58 to 194), or the 3' end portion 6 bp of exon 1 (180 bp), exon 2, exon 3, and the 5' end portion 81 bp of exon 4 (161 bp). This variant was found in 4 different liver tissue by sequencing RT-PCR products. Another variant has exon 3 been skipped, losing 153 bp (51 amino acid residue), and has two base pair substitution 100 C→T and 336C→T. Resulting in an amino acid substitute: Pro34Ser and112Phe, respectively. Genomic DNA from liver tissue expressing only wild type mRNA(3 samples), only skipped mRNA(2 samples) and both wild type and skipped mRNA(3 samples) were analysed by PCR amplified using primers covering exo... |
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