Cloning,Characterization and Primary Function Study of a Novel Gene,Cymg1,Related to Family 2 Cystatins

Cystatins are cysteine proteinase inhibitors,We found two expression sequence tags (ESTs), CA463109 and AV042522,from a mouse testis library using Digital differential display (DDD).By electrical hybridization,a novel gene,Cymgl(GenBank accession No.AY600990),which has a full length of 0.78 kb, and contains four exons and three introns,was cloned from a mouse testis eDNA library.The gene is located in the 2G3 area of chromosome 2.The full eDNA encompasses the entire open reading frame,encoding 141 amino acid residues.The protein has a cysteine protease inhibitor domain that is related to the family 2 cystatins but lacks critical consensus sites important for cysteine protease inhibition.These characteristics are seen in the CRES subfamily,which are related to the family 2 cystatins and are expressed specifically in the male reproductive tract.CYMG1 has a 44%(48/108)identity with mouse CRES and 30%(42/140) identity with mouse cystatin C.Northern blot analysis showed that the Cymgl is specifically expressed in adult mouse testes.Cell location studies showed that the GFP-tagged CYMG 1 protein was localized in the cytoplasm of HeLa cells,lmmunohistochemistry revealed that the CYMG1 protein was expressed in mouse testes spermatogonium,spermatocytes,round spermatids,elongating spermatids and spermatozoa.RT-PCR results also showed that Cymgl was expressed in mouse testes and spermatogonium.The Cymgl expression level varied in different developmental stages:it was low 1 week postpartum,steadily increased 2 to 5 weeks postpartum,and was highest 7 weeks postpartum.The expression level at 5 weeks postpartum was main- tained during 13 to 57 weeks postpartum.The Cymgl expression level in the testes over different develop- mental stages correlates with the mouse spermatogenesis and sexual maturation process.All these indicate that Cymgl might play an important role in mouse spermatogenesis and sexual maturation. Cystatins are cysteine proteinase inhibitors,We found two expression sequence tags(ESTs), CA463109 and AV042522,from a mouse testis library using Digital differential display (DDD).By electrical hybridization,a novel gene,Cymgl(GenBank accession No.AY600990),which has a full length of 0.78 kb, and contains four exons and three introns,was cloned from a mouse testis eDNA library.The gene is located in the 2G3 area of chromosome 2.The full eDNA encompasses the entire open reading frame,encoding 141 amino acid residues.The protein has a cysteine protease inhibitor domain that is related to the family 2 cystatins but lacks critical consensus sites important for cysteine protease inhibition.These characteristics are seen in the CRES subfamily,which are related to the family 2 cystatins and are expressed specifically in the male reproductive tract.CYMG1 has a 44%(48/108)identity with mouse CRES and 30%(42/140) identity with mouse cystatin C.Northern blot analysis showed that the Cymgl is specifically expressed in adult mouse testes.Cell location studies showed that the GFP-tagged CYMG 1 protein was localized in the cytoplasm of HeLa cells,lmmunohistochemistry revealed that the CYMG1 protein was expressed in mouse testes spermatogonium,spermatocytes,round spermatids,elongating spermatids and spermatozoa.RT-PCR results also showed that Cymgl was expressed in mouse testes and spermatogonium.The Cymgl expression level varied in different developmental stages:it was low 1 week postpartum,steadily increased 2 to 5 weeks postpartum,and was highest 7 weeks postpartum.The expression level at 5 weeks postpartum was main- tained during 13 to 57 weeks postpartum.The Cymgl expression level in the testes over different develop- mental stages correlates with the mouse spermatogenesis and sexual maturation process.All these indicate that Cymgl might play an important role in mouse spermatogenesis and sexual maturation.