Banner
Current IssueEditorial BoardOnline First ArticlesArchive
Current IssueEditorial BoardOnline First ArticlesArchive

volume 49 issue 4 (august 2015) : 442-447,   Doi: 10.5958/0976-0555.2015.00071.0

Molecular cloning and expression of buffalo ESX-1 gene: A marker for X-chromosome bearing spermatozoa

N
N. Vijay
P
P. Harikrishna
S
S.K. Ghosh1
S
S.K. Bhure
1Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, Bareilly- 243 122, India.
Cite article:- Vijay N., Harikrishna P., Ghosh1 S.K., Bhure S.K. (2025). Molecular cloning and expression of buffalo ESX-1 gene: A marker for X-chromosome bearing spermatozoa. Indian Journal of Animal Research. 49(4): 442-447. doi: 10.5958/0976-0555.2015.00071.0.

ABSTRACT

Extra-embryonic tissue-spermatogenesis homeobox gene 1 (ESX-1), an X-linked gene that encodes homeobox protein, expresses specifically in X-chromosome bearing spermatozoa. The antibodies against this recombinant protein will be useful to assess the efficiency of sex sorted semen in buffalo. Therefore, the present study has been undertaken to clone and express the ESX-1 gene and then to study the characteristics of the expressed protein. In this investigation, the cDNA of ESX-1 was cloned, sequenced and expressed in E. coli. Predicted amino acid sequence of ESX-1 had shown highest homology with Bos taurus (91.6%). The homeobox region had shown the identity of 98.4%, 70.5%, 70.5%, 69.4% and 69.4% respectively with Bos taurus, Homo sapiens, Pan troglodytes, Macaca mulatta and Mus musculus. Expressed protein was purified using Cobalt-Agarose affinity column. SDS-PAGE showed a product ~27kDa recombinant fusion protein. The purified product was confirmed by Western blot analysis using Ni-NTA anti hisitidine HRPase conjugate. The antibodies against Esx-1 protein can be used for evaluation of sex sorted semen.

REFERENCES

  1. Blecher Stan, and Detmar Jacqueline, (November 15, 2001). Sex-chromosome-specific proteins, species specific and sperm specific proteins and methods for their identification and isolation US20010041348,.
  2. Branford, W.W.; Zhao, G.Q.; Valerius, M.T.; Weinstein, M.; Birkenmeier, E.H.; Rowe, L.B. and Potter S.S. (1997). Spx 1, a novel X-linked homeobox gene expressed during spermatogenesis. Mech .Dev., 65: 87-98
  3. Capel, B.; Swain, B.; Nicolis, S.; Hacker, A.; Walter, M.; Koopman, P.; Goodfellow, P. and Lovell-Badge, R. (1993). Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell, 73: 1019-1030.
  4. Conway, S.J.; Mahadevaiah, S.K.; Darling, S.M.; Capel, B.; Rattigan, A.M. and Burgoyne, P.S. (1994). Y353ÍB-. a candidate multiple copy spermiogenesis gene on the mouse Y chromosome. Mammalian Genome, 5: 203-210.
  5. Hendriksen, P.J.M.; Hoogerbrugge, J.W.; Themmen, A.P.N.; Koken, M.H.M.; Hoeijmakers, J.H.J.; Oostra, B.A.; van der, L. and Grootegoed, J.A. (1995). Post-meiotic transcription of X and Y chromosomal genes during spermatogenesis in the mouse. Developmental Biology, 170: 730-735.
  6. Li, Y.; Lemaire, P. and Behring, R.R. (1997). Esx1,a novel X chromosome-linked homeobox gene expressed in mouse extra- embryonic tissues and male germ cells. Dev.Biol., 188: 85-95.
  7. Nagamine, C.M.; Chan, K.; Hake, L.E. and Lau, Y.C. (1990). The two candidate testisdetermining Y genes (Zfy-1 and Zfy- 2) are differentially expressed in fetal and adult mouse tissues. Genes and Development, 4: 63-74.
  8. Sambrook, J and Russell, D.W. (2001). Molecular cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York.
  9. Shannon, M. and Handel, M.A. (1993). Expression of the Hprt gene during spermatogenesis: implications for sex-chromosome inactivation. Biology of Reproduction, 49: 770- 777.
  10. Shende, A. M.; Kutty, V. H. M.; Ramteke, S. S.; Ghosh S. K.; Prasad J. K. and Bhure, S. K. (2014). Molecular cloning and expression of buffalo SRY gene: a mammalian sex determination protein. Indian journal of biotechnology (Accepted).
  11. Spaulding and Glenn, F. (1991). Sex-associated membrane antibodies and their use for increasing the probability that offspring will be of a desired sex.
  12. Sophie, D.D.; Villeneuve, A. and Lue, L.R. (2009). Antigenic Surface structure of sperm cells associated with the Y chromosome US20090305270.
  13. Towbin, H.T. and Staehelint, J.G. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications, Proc. Natl. Acad. Sci., 76(9): 4350-4354.
  14. Yeuh, C. Y.; Vie, C. Y.; Shig, C. H. and Neng, W. L. (2005). Stage dependent expression of extra-embryonic tissue- spermatogenesis-homeobox gene 1 (ESX 1) protein a candidate marker for X chromosome-bearing sperm. Reproduction, Fertility and Development, 17: 447-455.
Published In
Indian Journal of Animal Research
Article Metrics
Metrics Icon
0
Views
0
Citations
Reviewed By
Share Article
Download Article
In this Article
    Contact us
    Follow us

    volume 49 issue 4 (august 2015) : 442-447,   Doi: 10.5958/0976-0555.2015.00071.0

    Molecular cloning and expression of buffalo ESX-1 gene: A marker for X-chromosome bearing spermatozoa

    N
    N. Vijay
    P
    P. Harikrishna
    S
    S.K. Ghosh1
    S
    S.K. Bhure
    1Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, Bareilly- 243 122, India.
    Cite article:- Vijay N., Harikrishna P., Ghosh1 S.K., Bhure S.K. (2025). Molecular cloning and expression of buffalo ESX-1 gene: A marker for X-chromosome bearing spermatozoa. Indian Journal of Animal Research. 49(4): 442-447. doi: 10.5958/0976-0555.2015.00071.0.

    ABSTRACT

    Extra-embryonic tissue-spermatogenesis homeobox gene 1 (ESX-1), an X-linked gene that encodes homeobox protein, expresses specifically in X-chromosome bearing spermatozoa. The antibodies against this recombinant protein will be useful to assess the efficiency of sex sorted semen in buffalo. Therefore, the present study has been undertaken to clone and express the ESX-1 gene and then to study the characteristics of the expressed protein. In this investigation, the cDNA of ESX-1 was cloned, sequenced and expressed in E. coli. Predicted amino acid sequence of ESX-1 had shown highest homology with Bos taurus (91.6%). The homeobox region had shown the identity of 98.4%, 70.5%, 70.5%, 69.4% and 69.4% respectively with Bos taurus, Homo sapiens, Pan troglodytes, Macaca mulatta and Mus musculus. Expressed protein was purified using Cobalt-Agarose affinity column. SDS-PAGE showed a product ~27kDa recombinant fusion protein. The purified product was confirmed by Western blot analysis using Ni-NTA anti hisitidine HRPase conjugate. The antibodies against Esx-1 protein can be used for evaluation of sex sorted semen.

    REFERENCES

    1. Blecher Stan, and Detmar Jacqueline, (November 15, 2001). Sex-chromosome-specific proteins, species specific and sperm specific proteins and methods for their identification and isolation US20010041348,.
    2. Branford, W.W.; Zhao, G.Q.; Valerius, M.T.; Weinstein, M.; Birkenmeier, E.H.; Rowe, L.B. and Potter S.S. (1997). Spx 1, a novel X-linked homeobox gene expressed during spermatogenesis. Mech .Dev., 65: 87-98
    3. Capel, B.; Swain, B.; Nicolis, S.; Hacker, A.; Walter, M.; Koopman, P.; Goodfellow, P. and Lovell-Badge, R. (1993). Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell, 73: 1019-1030.
    4. Conway, S.J.; Mahadevaiah, S.K.; Darling, S.M.; Capel, B.; Rattigan, A.M. and Burgoyne, P.S. (1994). Y353ÍB-. a candidate multiple copy spermiogenesis gene on the mouse Y chromosome. Mammalian Genome, 5: 203-210.
    5. Hendriksen, P.J.M.; Hoogerbrugge, J.W.; Themmen, A.P.N.; Koken, M.H.M.; Hoeijmakers, J.H.J.; Oostra, B.A.; van der, L. and Grootegoed, J.A. (1995). Post-meiotic transcription of X and Y chromosomal genes during spermatogenesis in the mouse. Developmental Biology, 170: 730-735.
    6. Li, Y.; Lemaire, P. and Behring, R.R. (1997). Esx1,a novel X chromosome-linked homeobox gene expressed in mouse extra- embryonic tissues and male germ cells. Dev.Biol., 188: 85-95.
    7. Nagamine, C.M.; Chan, K.; Hake, L.E. and Lau, Y.C. (1990). The two candidate testisdetermining Y genes (Zfy-1 and Zfy- 2) are differentially expressed in fetal and adult mouse tissues. Genes and Development, 4: 63-74.
    8. Sambrook, J and Russell, D.W. (2001). Molecular cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York.
    9. Shannon, M. and Handel, M.A. (1993). Expression of the Hprt gene during spermatogenesis: implications for sex-chromosome inactivation. Biology of Reproduction, 49: 770- 777.
    10. Shende, A. M.; Kutty, V. H. M.; Ramteke, S. S.; Ghosh S. K.; Prasad J. K. and Bhure, S. K. (2014). Molecular cloning and expression of buffalo SRY gene: a mammalian sex determination protein. Indian journal of biotechnology (Accepted).
    11. Spaulding and Glenn, F. (1991). Sex-associated membrane antibodies and their use for increasing the probability that offspring will be of a desired sex.
    12. Sophie, D.D.; Villeneuve, A. and Lue, L.R. (2009). Antigenic Surface structure of sperm cells associated with the Y chromosome US20090305270.
    13. Towbin, H.T. and Staehelint, J.G. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications, Proc. Natl. Acad. Sci., 76(9): 4350-4354.
    14. Yeuh, C. Y.; Vie, C. Y.; Shig, C. H. and Neng, W. L. (2005). Stage dependent expression of extra-embryonic tissue- spermatogenesis-homeobox gene 1 (ESX 1) protein a candidate marker for X chromosome-bearing sperm. Reproduction, Fertility and Development, 17: 447-455.
    In this Article
      Contact us
      Follow us
      Published In
      Indian Journal of Animal Research

      Editorial Board

      View all (0)