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ISSN 1672-9145                                                Acta Biochim Biophys Sin 2005, 37(3): 159–166                                                   CN 31-1940/Q

Molecular Cloning of MSRG-11 Gene Related to Apoptosis of Mouse Spermatogenic Cells

Yun DENG, Dong-Song NIE, Jian WANG, Xiao-Jun TAN, Zhao-Yan NIE, Hong-Mei YANG, Liang-Sha HU, and Guang-Xiu LU*

 

Human Reproductive and Stem Cell Engineering Institute, Central South University, Changsha 410078, China

 

Abstract        Beginning with a new contig of the expressed sequence tags (Mm.63892) obtained by comparing testis libraries with other tissue and cell line libraries using the digital differential display program, we cloned a new gene which is related to the apoptosis of mouse spermatogenic cells using the Genscan program and polymerase chain reaction (PCR) technology. The sequence data have been submitted to the GenBank database under accession number AY747687. The full cDNA length is 1074 bp, and the gene with 7 exons and 6 introns is located in mouse chromosome 1 H5. The protein is recognized as a new member of calmodulin (CaM) binding protein family because the sequence contains three short calmodulin-binding ­mo­tifs containing conserved Ile and Gln residues (IQ motif) and is considered to play a critical role in interactions of IQ motif-containing proteins with CaM proteins. The putative protein encoded by this gene has 192 amino acid residues with a theoretical molecular mass of 23.7 kDa and a calculated isoelectric point of 9.71. The sequence shares no significant homology with any known protein in databases. RT-PCR and Northern blot analyses revealed that 1.3 kb MSRG-11 transcript was strongly expressed in adult mouse testis but weakly expressed in the spleen and thymus. The MSRG-11 gene was expressed at various levels, faintly at two weeks postpartum and strongly from three weeks postpartum in adult testes. The green fluorescence produced by pEGFP-C2/MSRG-11 was detected in the cytoplasm of COS7 cells 24 h post-transfection. The pcDNA3.1(-)/MSRG-11 plasmid was constructed and introduced into COS7 cells using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, USA). MSRG-11 can accelerate COS7 cell apoptosis, which suggests that this gene may play an important role in the development of mouse testes and is a candidate gene of testis-specific apoptosis. Based on these observations, it was considered that we cloned a new gene which probably accelerates spermatogenetic cell apoptosis in mouse.

 

Key words        MSRG-11 gene; testis-specific expression; spermatogenetic cell apoptosis; IQ motif

 

 

 

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Received: November 18, 2004        Accepted: January 12, 2005

This work was supported by a grant from the Special Fund of ­National Basic Research Program of China (No. G1999055901)

*Corresponding author: Tel, 86-731-4373187; Fax, 86-731-4497661; E-mail, [email protected]