Elsevier

Gene

Volume 327, Issue 2, 3 March 2004, Pages 185-194
Gene

Characterization of isoforms and genomic organization of mouse calumenin

https://doi.org/10.1016/j.gene.2003.10.014Get rights and content

Abstract

Calumenin is a multiple EF-hand protein located in endo/sarcoplasmic reticulum of mammalian heart and other tissues [J. Biol. Chem. 272 (1997) 18232; Genomics 49 (1998) 331; Biochim. Biophys. Acta 1386 (1998) 121]. In the present study, a new isoform of mouse calumenin (mouse calumenin 2) was cloned by RT-PCR and genomic DNA PCR. The deduced amino acid sequence of mouse calumenin 2 is 315 aa long with the calculated MW of 37,064 and pI of 4.26. It has 92% aa sequence identity to previously identified mouse calumenin [J. Biol. Chem. 272 (1997) 18232] (mouse calumenin 1). The difference in the aa sequence was restricted to the first two EF-hand regions (residues 74–138). Northern blot analysis shows that mouse calumenin 2 is highly expressed in heart, lung, testis and unpregnant uterus. The expression of mouse calumenin 2 appears to decrease when fetal development is progressed. Genomic DNA PCR, sequencing and data mining of mouse genome database were utilized to examine the exon–intron boundaries of mouse calumenin genes. Both mouse calumenin 1 and 2 genes encompass six exons, and five of them (Exon1, 3, 4, 5 and 6) are identical. However, mouse calumenin 1 contains Exon2-1, whereas mouse calumenin 2 contains a neighboring Exon2-2. The calumenin genes are localized on mouse chromosome 6 having conserved synteny with human chromosome 7q32. For comparison, the genomic organization of human calumenin was also examined using the published human genome database (UCSC Genome Bioinformatics at http://genome.ucsc.edu/). Like mouse calumenin genes, two human calumenin genes also consist of five identical exons (Exon1, 3, 4, 5 and 6) and a different Exon2. The present study suggests that the genomic organization of calumenin genes is well conserved between human and mouse.

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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An accession number for the sequence reported in this paper is AY225334.

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