Molecular cloning of MSRG-11 gene related to apoptosis of mouse spermatogenic cells.
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.
Deng
Yun
Y
Human Reproductive and Stem Cell Engineering Institute, Central South University, Changsha, China.
Nie
Dong-Song
DS
Wang
Jian
J
Tan
Xiao-Jun
XJ
Nie
Zhao-Yan
ZY
Yang
Hong-Mei
HM
Hu
Liang-Sha
LS
Lu
Guang-Xiu
GX
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D019556:COS Cells; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods*; D020869:Gene Expression Profiling; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity; D011994:Recombinant Proteins / Q000378:metabolism; D013094:Spermatozoa / Q000378:metabolism*; D013737:Testis / Q000378:metabolism*; D014018:Tissue Distribution
D002148:Calmodulin-Binding Proteins; D011994:Recombinant Proteins