Skip to main content
SpringerLink
Log in

Molecular cloning and expression patterns of two tumor necrosis factor alpha genes in crucian carp (Carassius carassius)

  • Cell Molecular Biology
  • Published:
Molecular Biology Aims and scope Submit manuscript

Abstract

Tumor necrosis factor (TNF[31]) is a potent inflammatory cytokine produced during inflammation. In this study, two crucian carp TNF-α genes, TNFα-1 and TNFα-2, were cloned and sequenced. The TNFα-1 is 720 bp long and consists of a 699 bp opening reading frame (ORF) encoding 232 amino acids and TNFα-2 is 793 bp long and contains an ORF of 687 bp, which encodes 228 amino acids. The genomic structure of both genes consists of 4 exons and 3 introns similar to the other known TNF-α genes. The amino acid sequences of the crucian carp TNFα-1 and TNFα-2 share the highest identity with common carp, goldfish and the lowest with flounder and humans. The phylogenetic analysis grouped crucian carp TNFα-1 and TNFα-2 with other cypriniformes, which are more closely related to goldfish and common carp TNFα-1 and TNFα-2. Real time PCR analysis showed a constitutive expression of crucian carp TNFα-1 and TNFα-2 in all seven tissues examined. The TNFα-1 mRNA was expressed significantly higher in the liver and kidney than those of TNFα-2, while TNFα-2 in turn was expressed significantly higher in the muscle. The Aeromonas hydrophila BSK-10 strain upregulated the expression level of both TNFα-1 and TNFα-2 in all of the tested tissues. At 6 h, the expression levels of TNFα-1 were increased significantly higher in the muscles, skin, and liver, while the expression levels of TNFα-2 were increased significantly higher in the muscles and gills. TNFα-1 was expressed much stronger than TNFα-2. At 12 h the expression levels started to decline and were even lower at 24 h. These results imply that both TNFα-1 and TNFα-2 mRNA are distributed differently in tissues and are implicated in the immune response to bacterial infection.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Vilcek J., Lee T.H. 1991. Tumor necrosis factor. J. Biol. Chem. 266, 7313–7316

    CAS  PubMed  Google Scholar 

  2. Cerami A. 1992. Inflammatory cytokines. Clin. Immunol. Immunopathol. 62, S3–S10.

    Article  CAS  PubMed  Google Scholar 

  3. Wang H., Czura C.J., Tracey K.J. 2003. Tumor Necrosis Factor: The Cytokine Handbook. N.Y: Academic Press.

    Google Scholar 

  4. Pfeffer K. 2003. Biological functions of tumor necrosis factor cytokines and their receptors. Cytokine Growth Factor Rev. 14, 185–191.

    Article  CAS  PubMed  Google Scholar 

  5. Ma X. 2001. TNF-alpha and IL-12: A balancing act in macrophage functioning. Microbes Infection. 3, 121–129.

    Article  CAS  PubMed  Google Scholar 

  6. Kisich K.O., Higgins M., Diamond G., Heifets L. 2002. Tumor necrosis factor alpha stimulates killing of Mycobacterium tuberculosis by human neutrophils. Infection Immunity. 70, 4591–4599.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Goetz F.W., Planas J.V., MacKenzie S. 2004. Tumor necrosis factors. Dev. Comp. Immunol. 28, 487–450.

    Article  CAS  PubMed  Google Scholar 

  8. Hirono I., Nam B.H., Kurobe T., Aoki T. 2000. Molecular cloning, characterization, and expression of TNF cDNA and gene from Japanese flounder Paralychthys olivaceus. J. Immunol. 165, 4423–4427.

    Article  CAS  PubMed  Google Scholar 

  9. Zou J., Wang T., Hirono I., Aoki, T., Inagawa H., Honda T., Soma G., Ototake M., Nakanishi T., Ellis B.E., Secombes C.J. 2002. Differential expression of two tumor necrosis factor genes in rainbow trout, Oncorhynchus mykiss. Dev. Comp. Immunol. 26, 161–172.

    Article  CAS  PubMed  Google Scholar 

  10. Laing K.J., Wang T., Zou J., Holland J., Hong S., Bols N., Hirono I., Aoki T., Secombes C.J. 2001. Cloning and expression analysis of rainbow trout Oncorhynchus mykiss tumor necrosis factor alpha. Eur. J. Biochem. 268, 1315–1322.

    Article  CAS  PubMed  Google Scholar 

  11. Garcia-Castillo J., Pelegrin P., Mulero V., Meseguer J. 2002. Molecular cloning and expression analysis of tumor necrosis factor α from a marine fish reveal its constitutive expression and ubiquitous nature. Immunogenetics. 54, 2000–2007.

    Google Scholar 

  12. Cai Z.H., Song L.S., Gao C.P., Wu L.T., Qiu L.H., Xiang J.H. 2003. Cloning and expression of tumor necrosis factor (TNF alpha) cDNA from red seabream Pagrus major. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 35, 1111–1116.

    CAS  Google Scholar 

  13. Saeij J.P., Stet R.J., de Vries B.J., van Muiswinkel W.B., Wiegertjes G.F. 2003 Molecular and functional characterization of carp TNF: A link between TNF polymorphism and trypanotolerance? Dev. Comp. Immunol. 27, 29–41.

    Article  CAS  PubMed  Google Scholar 

  14. Savan R., Sakai M. 2004 Presence of multiple isoforms of TNF alpha in carp (Cyprinus carpio L.): Genomic and expression analysis. Fish Shellfish Immunol. 17, 87–94.

    Article  CAS  PubMed  Google Scholar 

  15. Zhao X., Duan D., Feng X., Chen Y., Sun Z., Jia S., He J., Wang B., Li W., Zhang, J., Wang W., Yang Z., Lu Q. 2012 Molecular cloning and expression analysis of common carp Cyprinus carpio tumor necrosis factor-α. Fisheries Sci. 78, 1229–1236

    Article  CAS  Google Scholar 

  16. Zou J., Secombes C.J., Long S., Miller N., Clem L.W., Chinchar V.G. 2003. Molecular identification and expression analysis of tumor necrosis factor in channel catfish (Ictalurus punctatus). Dev. Comp. Immunol. 27, 845–858.

    Article  CAS  PubMed  Google Scholar 

  17. Praveen K., Evans D.L., Jaso-Friedmann L. 2006. Constitutive expression of tumor necrosis factor-alpha in cytotoxic cells of teleosts and its role in regulation of cell-mediated cytotoxicity. Mol. Immunol. 43, 279–291.

    Article  CAS  PubMed  Google Scholar 

  18. Ordas M.C., Costa M. M., Roca F. J., Lopez-Castejon G., Mulero V., Meseguer J., Figueras A., Novoa B. 2007. Turbot TNFα gene: molecular characterization and biological activity of the recombinant protein. Mol. Immunol. 44, 389–400.

    Article  CAS  PubMed  Google Scholar 

  19. Xiao J., Zhou Z.C., Chen C., Huo W.L., Yin Z.X., Weng S.P., Chan S.M., Yu X.Q., He J.G. 2007 Tumor necrosis factor-alpha gene from mandarin fish, Siniperca chuatsi: Molecular cloning, cytotoxicity analysis and expression profile. Mol. Immunol. 44, 3615–3622.

    Article  CAS  PubMed  Google Scholar 

  20. Nascimento D.S., Pereira P.J., Reis M.I., do Vale A., Zou J., Silva M.T., Secombes C.J., dos Santos N.M.S. 2007. Molecular cloning and expression analysis of sea bass (Dicentrarchus labrax L.) tumor necrosis factor-alpha (TNF-alpha). Fish Shellfish Immunol. 23, 701–710.

    Article  CAS  PubMed  Google Scholar 

  21. Morrison R.N., Zou J., Secombes C.J., Scapigliati G., Adams M.B., Nowak B.F. 2007. Molecular cloning and expression analysis of tumour necrosis factor-alpha in amoebic gill disease (AGD)-affected Atlantic salmon (Salmo salar L.). Fish Shellfish Immunol. 23, 1015–1031.

    Article  CAS  PubMed  Google Scholar 

  22. Uenobe M., Kohchi C., Yoshioka N., Yuasa A., Inagawa H., Morii K., Nishizawa T., Takahashi Y., Soma G. 2007. Cloning and characterization of a TNF-like protein of Plecoglossus altivelis (ayu fish). Mol. Immunol. 44, 1115–1122.

    Article  CAS  PubMed  Google Scholar 

  23. Grayfer L., Walsh J.G., Belosevic M. 2008. Characterization and functional analysis of goldfish (Carassius auratus L.) tumor necrosis factor-alpha. Dev. Comp. Immunol. 32, 532–543.

    Article  CAS  PubMed  Google Scholar 

  24. Kadowaki T., Harada H., Sawada Y., Kohchi C., Soma G., Takahashi Y., Inagawa H. 2009. Two types of tumor necrosis factor-alpha in bluefin tuna (Thunnus orientalis) genes: Molecular cloning and expression profile in response to several immunological stimulants. Fish Shellfish Immunol. 27, 585–594.

    Article  CAS  PubMed  Google Scholar 

  25. Nedwin G.E., Naylor S.L., Sakaguchi A.Y., Smith D., Jarrett-Nedwin J., Pennica D., Goeddel D.V., Gray P.W. 1985. Human lymphotoxin and tumor necrosis factor genes: Structure, homology and chromosomal localization. Nucleic Acids Res. 13, 6361.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Zou J., Peddie S., Scapigliati G., Zhang Y., Bols N.C. Ellis A.E., Secombes C.J. 2003. Functional characterization of the recombinant tumor necrosis factors in rainbow trout, Oncorhynchus mykiss. Dev. Comp. Immunol. 27, 813–822.

    Article  CAS  PubMed  Google Scholar 

  27. Haugland Ø., Mercy I.S., Romøren K., Torgersen J., Evensen Ø. 2007. Differential expression profiles of two tumor necrosis factor-α variants in Atlantic salmon (Salmo salar L.). Mol. Immunol. 44, 1652–1663.

    Article  CAS  PubMed  Google Scholar 

  28. Yoon J.M., Park H.Y. 2002. Genetic similarity in the cultured and wild Crucian carp (Carassius carassius) estimated with random amplified polymorphic DNA. Asian-Australasian J. Animal Sci. 15, 470–476.

    Article  CAS  Google Scholar 

  29. Kesarcodi-Watson A., Kaspar H., Lategan M.J., Gibson L. 2008. Probiotics in aquaculture: The need, principles and mechanisms of action and screening processes. Aquaculture. 274, 1–14.

    Article  Google Scholar 

  30. Sahoo P.K., Rauta P.R., Mohanty B.R., Mahapatra K.D., Saha J.N., Rye M., Eknath A.E. 2011. Selection for improved resistance to Aeromonas hydrophila in Indian major carp Labeo rohita: Survival and innate immune responses in first generation of resistant and susceptible lines. Fish Shellfish Immunol. 31, 432–438.

    Article  CAS  PubMed  Google Scholar 

  31. Köchl S., Niederstätter H., Parson W. 2005. DNA extraction and quantitation of forensic samples using the phenol-chloroform method and real-time PCR. Methods Mol. Biol. 297, 13–30.

    PubMed  Google Scholar 

  32. Hofmann K., Stoffel W. 1993. TM base: A database of membrane spanning proteins segments. Biol. Chem. Hoppe-Seyler. 374, 166–170.

    Google Scholar 

  33. Hulo N., Bairoch A., Bulliard V., Cerutti L., De Castro E., Langendijk-Genevaux P.S., Pagni M., Sigrist C.J.A. 2006. The PROSITE database. Nucleic Acids Res. 34, D227–D230.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Livak K.J., Schmittgen T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and 2(-Delta Delta C(T)) method. Methods. 25, 402–408.

    Article  CAS  PubMed  Google Scholar 

  35. Kim M.S., Hwang Y.J., Yoon K.J., Zenke K., Nam Y.K., Kim S.K., Kim K.H. 2009. Molecular cloning of rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha and its effect on the respiratory burst activity of phagocytes. Fish Shellfish Immunol. 27, 618–624.

    Article  CAS  PubMed  Google Scholar 

  36. Savan R., Sakai M. 2006. Genomics of fish cytokines. Comp. Biochem. Physiol. D: Genomics Proteomics. 1, 89–101.

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. College of Marine Sciences, Laboratory of Molecular Biology, Ningbo University, Ningbo, Zhejiang, 315211, P.R. China

    R. A. Kajungiro, L. Xue & M. Aynealem

Authors
  1. R. A. Kajungiro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. L. Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Aynealem
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to L. Xue.

Additional information

Published in Russian in Molekulyarnaya Biologiya, 2015, Vol. 49, No. 1, pp. 138–148.

The article is published in the original.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kajungiro, R.A., Xue, L. & Aynealem, M. Molecular cloning and expression patterns of two tumor necrosis factor alpha genes in crucian carp (Carassius carassius). Mol Biol 49, 120–129 (2015). https://doi.org/10.1134/S0026893315010021

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0026893315010021

Keywords

Search

Navigation