Genomic organization, chromosomal localization, and alternative splicing of the human phosphodiesterase 8B gene,☆☆

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Abstract

We have characterized the gene for human phosphodiesterase 8B, PDE8B, and cloned the full-length cDNA for human PDE8B (PDE8B1) and two splice variants (PDE8B2 and PDE8B3). The PDE8B gene is mapped to the long arm of chromosome 5 (5q13) and is composed of 22 exons spanning over ∼200 kb. The donor and acceptor splice site sequences match the consensus sequences for the exon–intron boundaries of most eukaryotic genes. PDE8B1 encodes an 885 amino acid enzyme, containing an N-terminal REC domain, a PAS domain, and a C-terminal catalytic domain. PDE8B2 and PDE8B3 both have deletion in the PAS domain and encode 838 and 788 amino acid proteins, respectively. RT-PCR analysis revealed that while PDE8B1 is the most abundant variant in thyroid gland, PDE8B3, but not PDE8B1, is the most abundant form in brain. These findings suggest that selective usage of exons produces three different PDE8B variants that exhibit a tissue-specific expression pattern.

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Materials and methods

Genomic cloning and sequencing of the human PDE8B gene. The genomic clones of human PDE8B gene were isolated using PCR-based technology. The down-to-the-well genomic library, which contains human BAC genomic clones, was purchased from Genomesystems and used as PCR template. For amplification of introns, we used long-range (Expand High Fidelity, Boehringer) with primers corresponding to the 5 and 3 flanking exonic sequence and 100 ng template BAC DNA. We sized the products by agarose gel

Isolation and analysis of the human PDE8B gene

Utilizing the BAC human genomic library, we obtained three clones containing the PDE8B gene. PCR was performed to amplify introns using these BAC clones as template. All introns, except introns 1, 2, and 10, were amplified and fully sequenced using primer-walking method from both neighboring exons. A database search revealed two sequences, AC022414 and AC022422, including the sequences for exons 1 and 2–22, respectively. A physical map of the human PDE8B gene is illustrated in Fig. 1A. The

Acknowledgements

This work was supported in part by grants-in-aid for Scientific Research on Priority Areas (C) from the Ministry of Education, Culture, Sports and Technology, Scientific Research (B) and (C), and Exploratory Research from the Japanese Society for the Promotion of Science, and a Grant for Pediatric Diseases from Ministry of Health Welfare, Japan. It was also supported by the program for promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research.

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The nucleotide sequences reported in this paper have been deposited with the GenBank/EMBL database. Accession Nos. are as follows: PDE8B1, AY129947; PDE8B2, AY129948; PDE8B3, AY129949; human PDE8B gene, AY129950.

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Abbreviations: PDE, 3,5-cyclic nucleotide phosphodiesterase; cAMP, adenosine 3,5-cyclic monophosphate; cGMP, guanosine 3,5-cyclic monophosphate; BAC, bacterial artificial chromosome; RT-PCR, reverse transcription-polymerase chain reaction; RACE, rapid amplification of cDNA ends; nt, nucleotide; bp, base pair; kb, kilobase pairs; IBMX, 3-isobutyl-1-methylxanthine; UTR, untranslated region; ORF, open reading frame.

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