http://www.abbs.info E-mail: [email protected]
ISSN
1672-9145
Acta Biochim Biophys Sin
2005, 37(1): 11–18
CN 31-1940/Q
Cloning, Characterization and Primary Function Study of a Novel Gene, Cymg1, Related to Family 2 Cystatins
Yang XIANG, Dong-Song NIE, Jian WANG, Xiao-Jun TAN, Yun DENG, Shu-Wei LUO, and Guang-Xiu LU*
Human Reproductive and Stem Cell Engineering Institute, Central South University, Changsha 410078, China
Abstract
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, Cymg1
(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 cDNA
library. The gene is located in the 2G3 area of chromosome 2. The full cDNA
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 Cymg1 is specifically expressed in adult mouse testes. Cell
location studies showed that the GFP-tagged CYMG1 protein was localized in the
cytoplasm of HeLa cells. Immunohistochemistry revealed that the CYMG1 protein
was expressed in mouse testes spermatogonium, spermatocytes, round spermatids,
elongating spermatids and spermatozoa. RT-PCR results also showed that Cymg1
was expressed in mouse testes and spermatogonium. The Cymg1 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 maintained during 13 to 57 weeks
postpartum. The Cymg1 expression level in the testes over different
developmental stages correlates with the mouse spermatogenesis and sexual
maturation process. All these indicate that Cymg1 might play an
important role in mouse spermatogenesis and sexual maturation.
Key words
cystatins; gene cloning; testis; spermatogenesis; sexual maturation
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Received: August 6, 2004 Accepted: December 9, 2004
This work was supported by the grants from the National Natural Science Foundation of China (No. 30371493) and the Major State Basic Research Development Program of China (No. G1999055901)
*Corresponding author: Tel, 86-731-4805319; Fax, 86-731-4497661; E-mail, [email protected]