Characterization of isoforms and genomic organization of mouse calumenin☆
Introduction
Variety of cellular functions such as muscle contraction, neurotransmitter release and apoptosis is related with the cytosolic Ca2+ level, which is mainly regulated by the ER/SR membrane systems (Carafoli, 1987). Different types of Ca2+-binding proteins have been identified in the ER/SR membrane systems Carafoli, 1987, Honore and Vorum, 2000, Hong et al., 2001. The Ca2+-binding CREC family including Cab 45 (Scherer et al., 1996), reticulocalbin (Ozawa and Muramatsu, 1993), ERC-55 (Weis et al., 1994), calumenin Yabe et al., 1997, Vorum et al., 1998 and crocalbin (Hseu et al., 1999) has been identified to contain multiple EF-hands and a C-terminal ER retention signal. Presence of calumenin (mouse calumenin 1) in mouse cardiac ER was first reported by Yabe et al. (1997). Cloning of mouse calumenin 1 predicted that the protein consist of 315 aa containing a N-terminal signal sequence, six EF-hands and a unique C-terminal ER/SR retention signal, HDEF (Yabe et al., 1997).
Although the CREC family has been predicted to serve important roles in cellular Ca2+ signaling processes, the specific functional roles of the proteins are largely unknown. Circumstantial evidence has suggested that (1) a serum amyloid P component is a human calumenin interacting protein (Vorum et al., 2000), (2) an increased expression of calumenin is related with genetic warfarin resistance in rat (Wallin et al., 2001), and (3) a decreased expression of calumenin is associated with the metastatic head and neck cancer (Wu et al., 2002).
In the present study, we have identified and cloned a novel mouse calumenin isoform and further characterized the genomic organization of the calumenin genes. We named the new calumenin isoform as mouse calumenin 2. Both calumenin 1 (Yabe et al., 1997) and 2 isoforms encompass six exons, and among the six exons, five exons are identical and each of the neighboring two exons (Exon2-1 and Exon2-2) determines the types of isoform. We are also showing evidence that mouse and human calumenins have the same genomic organization.
Section snippets
Isolation of RNA and RT-PCR
Total RNAs from adult mouse heart (left ventricle) or skeletal (medial gastrocnemius) muscles were isolated using Invisorb Spin Tissue RNA Mini Kit (Invitek), and subsequently cDNAs were synthesized using random hexamer/oligo(dT) and Ominiscript reverse transcriptase (Qiagen). To clone mouse calumenin 2 cDNA encoding full ORF with 5′ and 3′ UTRs (1170 bp), two oligonucleotide primers were designed based on mouse calumenin 1 (GenBank accession number U81829) and mouse genome database (UCSC
cDNA cloning and sequence analysis of mouse calumenin isoforms
Mouse calumenin 1 was previously identified as Ca2+-binding ER protein having a C-terminal ER retention signal (Yabe et al., 1997). A search for the mouse genome database (UCSC Genome Bioinformatics) led us to predict that there would be the second isoform of calumenin in mouse genome. In order to isolate the second isoform of mouse calumenin (mouse calumenin 2), total RNA was purified from adult mouse heart and reverse transcribed as described in Section 2.1. Using the specific primers
Acknowledgements
This work was supported by grants from the Korea Ministry of Science and Technology (Systems Biology Research Grant, M1-0309-00-006), Korea Science and Engineering Foundation (Basic Research Program 1999-1-20700-002-5) and Ministry of Education (Brain Korea 21 Project).
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