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Proteoglycans: Gene Cloning

  • Mauricio Cortes
  • James R. Mensch
  • Miriam Domowicz
  • Nancy B. SchwartzEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 836)

Abstract

Aggrecan is a large proteoglycan that plays roles in numerous tissues during vertebrate development and adult life. The 6,327-nt chick aggrecan coding sequence had been determined from overlapping clones, but a full-length cDNA, needed for use in transgenic expression studies, had not been constructed. The strategy employed to do so was to generate two overlapping cDNA subfragments that shared a unique restriction site in the overlap and then join them at that site. These subfragments were obtained and cloned into the TOPO-TA vector pCR2.1. Digestion of the two constructs with the shared-site enzyme, XbaI, produced vector/5′-cDNA and 3′-cDNA fragments with XbaI-ends; these were ligated to produce the final full-length cDNA.

Key words

Proteoglycan Aggrecan Cloning Strategy Full-length cDNA 

Notes

Acknowledgments

The authors would like to thank Alex Baria and Danijela Maric for technical assistance.

This work was supported by R01 HD17732 (N.B.S.), and T32.G M 008720 and G M 007381 (M.C.).

References

  1. 1.
    Schwartz, N. B., Domowicz, M., Krueger, R. K., Li, H., and Mangoura, D. (1996) Brain Aggrecan, Perspectives in Dev. Neurobiol. 3, 291–306.Google Scholar
  2. 2.
    Schwartz, N. B., Pirok, E. W., III, Mensch, J. R., Jr., and Domowicz, M. S. (1999) Domain organization, genomic structure, evolution, and regulation of expression of the aggrecan gene family, Prog Nucleic Acid Res Mol Biol 62, 177–225.PubMedCrossRefGoogle Scholar
  3. 3.
    Cortes, M., Baria, A. T., and Schwartz, N. B. (2009) Sulfation of chondroitin sulfate proteoglycans is necessary for proper Indian hedgehog signaling in the developing growth plate, Development 136, 1697–1706.PubMedCrossRefGoogle Scholar
  4. 4.
    Domowicz, M. S., Cortes, M., Henry, J. G., and Schwartz, N. B. (2009) Aggrecan modulation of growth plate morphogenesis, Dev Biol 329, 242–257.PubMedCrossRefGoogle Scholar
  5. 5.
    Domowicz, M. S., Sanders, T. A., Ragsdale, C. W., and Schwartz, N. B. (2008) Aggrecan is expressed by embryonic brain glia and regulates astrocyte development, Developmental Biology 315, 114–124.PubMedCrossRefGoogle Scholar
  6. 6.
    Domowicz, M. S., Mueller, M. M., Novak, T. E., Schwartz, L. E., and Schwartz, N. B. (2003) Developmental expression of the HNK-1 carbohydrate epitope on aggrecan during chondrogenesis, Dev Dyn 226, 42–50.PubMedCrossRefGoogle Scholar
  7. 7.
    Domowicz, M., Mangoura, D., and Schwartz, N. B. (2000) Cell specific-chondroitin sulfate proteoglycan expression during CNS morphogenesis in the chick embryo, Int J Dev Neurosci 18, 629–641.PubMedCrossRefGoogle Scholar
  8. 8.
    Domowicz, M. S., Pirok, E. W., III, Novak, T. E., and Schwartz, N. B. (2000) Role of the C-terminal G3 domain in sorting and secretion of aggrecan core protein and ubiquitin-mediated degradation of accumulated mutant precursors, J Biol Chem 275, 35098–35105.PubMedCrossRefGoogle Scholar
  9. 9.
    Domowicz, M. S., Krueger, R. C., Li, H., Mangoura, D., Vertel, B. M., and Schwartz, N. B. (1996) The nanomelic mutation in the aggrecan gene is expressed in chick chondrocytes and neurons, Int. J. Dev. Neurosci. 14, 191–201.PubMedCrossRefGoogle Scholar
  10. 10.
    Domowicz, M. S., Li, H., Hennig, A. K., Vertel, B., and Schwartz, N. B. (1995) The biochemically and immunologically distinct CSPG of notochord is a product of the aggrecan gene, Dev. Biol. 171, 655–664.PubMedCrossRefGoogle Scholar
  11. 11.
    Vertel, B. M., Grier, B. L., Li, H., and Schwartz, N. B. (1994) The chondrodystrophy, nanomelia: biosynthesis and processing of the defective aggrecan precursor, Biochem J. 301, 211–216.PubMedGoogle Scholar
  12. 12.
    Li, H., Schwartz, N. B., and Vertel, B. M. (1993) cDNA cloning of chick cartilage chondroitin sulfate (aggrecan) core protein and identification of a stop codon in the aggrecan gene associated with the chondrodystrophy, nanomelia, J Biol Chem 268, 23504–23511.PubMedGoogle Scholar
  13. 13.
    Vertel, B. M., Walters, L. M., Grier, B., Maine, N., and Goetinck, R. F. (1993) Nanomelic chondrocytes synthesize, but fail to translocate, a truncated aggrecan precursor, J. Cell Sci. 104, 939–948.PubMedGoogle Scholar
  14. 14.
    Kimata, K., Barrach, H.-J., Brown, K. S., and Pennypacker, J. P. (1981) Absence of proteoglycan core protein in cartilage from cmd/cmd (cartilage matrix deficiency) mice., J. Biol. Chem. 256, 6961–6968.PubMedGoogle Scholar
  15. 15.
    Krueger, R. C., Kurima, K., and Schwartz, N. B. (1999) Completion of the mouse aggrecan structure and identification of the defect in the cmd-Bc as a near complete deletion of the murine aggrecan gene., Mamm. Genome 10, 1119–1125.Google Scholar
  16. 16.
    Dorfman, A., Hall, T., Ho, P. L., and Fitch, F. (1980) Clonal antibodies for core protein of chondroitin sulfate proteoglycan, Proc. Natl. Acad. Sci. 77, 3971–3973.PubMedCrossRefGoogle Scholar
  17. 17.
    Dorfman, A. D., Vertel, B. M., and Schwartz, N. B. (1980) Immunological methods in the study of chondroitin sulfate proteoglycans., Dev. Biol. 14, 169–198.Google Scholar
  18. 18.
    Krueger, R. C., Fields, T. A., Mensch, J. R., and Schwartz, N. B. (1990) Chick cartilage chondroitin sulfate proteoglycan core protein II. Nucleotide sequence of cDNA clone and localization of the S103L epitope, J Biol Chem 265, 12088–12097.PubMedGoogle Scholar
  19. 19.
    Li, H., Domowicz, M. S., Hennig, A., and Schwartz, N. B. (1996) S103L reactive chondroitin sulfate proteoglycan (aggrecan) mRNA expressed in developing chick brain and cartilage is encoded by a single gene, Mol. Brain Res. 36, 309–321.PubMedCrossRefGoogle Scholar
  20. 20.
    Zhou, G., Garofalo, S., Mukhopadhyay, K., Lefebvre, V., Smith, C. N., Eberspaecher, H., and de Crombrugghe, B. (1995) A 182 bp fragment of the mouse pro alpha 1(II) collagen gene is sufficient to direct chondrocyte expression in transgenic mice, J Cell Sci 108, 3677–3684.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Mauricio Cortes
    • 1
  • James R. Mensch
    • 1
  • Miriam Domowicz
    • 1
  • Nancy B. Schwartz
    • 2
    Email author
  1. 1.Departments of PediatricsThe University of ChicagoChicagoUSA
  2. 2.Departments of Pediatrics, and Biochemistry and Molecular BiologyThe University of ChicagoChicagoUSA

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