Biochemical and Biophysical Research Communications
Genomic organization, chromosomal localization, and alternative splicing of the human phosphodiesterase 8B gene☆,☆☆
Section snippets
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.