Skip to main content
SpringerLink
Log in

The ornithine aminotransferase gene in gyrate, atrophy of the retina: Analysis of expression and gross structure of this gene in cultured fibroblasts

  • Rapid Communications in Cell Biology
  • Published:
In Vitro Cellular & Developmental Biology Aims and scope Submit manuscript

Summary

Gyrate atrophy (GA), a degenerative disease of the human chorioretina, is associated with a deficiency of ornithine aminotransferase (OAT) activity, hyperornithinemia, and ornithinuria. We have characterized a cDNA clone for OAT (HLOAT) that was isolated from a cDNA library constructed from mRNA prepared from Hep G2, cells, a human hepatoma cell line. We have used HLOAT and a nearly full length OAT cDNA clone isolated from, a rat liver library (RLOAT) to examine in cultured fibroblasts from individuals with GA and control individuals, the expression of OAT mRNA and the gross structure of the OAT gene. Northern blot analyses of total cellular RNA indicated that 3 of 3 control cell lines and 5 of 6 GA cell lines are capable of expressing an OAT related mRNA of approximately 2100 bases, the size of OAT mRNA. To date, this is the only case of GA in which a complete lack of OAT mRNA has been observed. Southern blot analyses of DNA isolated from these cell lines indicated that the gross structure of the OAT gene is usually not detectably altered in individuals with GA. However, a unique pattern, of restriction fragments was observed upon digestion with Eco RI or Hind III of DNA from the GA cell line that does not express OAT mRNA. These unique Eco RI and Hind III fragments arise from the OAT structural gene and will serve as useful molecular markers that allow this particular defective OAT allele to be identified. When the cellular DNAs were digested with Hinf I and examined with a probe that corresponds to at least a portion of the active site of the enzyme, i. e., the pyridoxal phosphate binding site, identical patterns of fragments were detected in all samples. Therefore, it appears unlikely that the loss of OAT activity associated with these GA cases, 4 of which are pyridoxal phosphate responders, is the result of insertions or deletions in this region of the OAT gene. This study indicates that the lack of OAT enzyme activity associated with GA is the result of a variety of different molecular defects within the OAT gene.

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

References

  1. Barrett, D. J.; Bateman, J. B.; Sparkes, R. S., et al. Chromosomal localization of human ornithine aminotransferase gene sequences to 10q26 and Xp11.2. Invest. Opthal. Vis. Sci. 28:1037–1042; 1987.

    CAS  Google Scholar 

  2. Feinberg, A. P.; Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6–13; 1983.

    Article  PubMed  CAS  Google Scholar 

  3. Inana, G.; Totsuka, S.; Redmond, M., et al. Molecular cloning of human ornithine aminotransferase mRNA. Proc. Natl. Acad. Sci. USA 83:1203–1207; 1986.

    Article  PubMed  CAS  Google Scholar 

  4. Inana, G.; Hotta, Y.; Zintz, C., et al. Expression defect of ornithine aminotransferase gene in gyrate atrophy. Invest. Opthal. Vis. Sci. 29:1001–1005; 1988.

    CAS  Google Scholar 

  5. Kennaway, N. G.; Weleber, R. G.; Buist, N. R. M. Gyrate atrophy of choroid and retina: Deficient activity of ornithine ketoacid aminotransferase in cultured skin fibroblasts. N. Engl. J. Med. 297:1180; 1977.

    PubMed  CAS  Google Scholar 

  6. Lehrach, H.; Diamond, D.; Wozney, J. M., et al. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16:4743–4750; 1977.

    Article  PubMed  CAS  Google Scholar 

  7. Maniatis, T.; Fritsch, E. F.; Sambrook, J. Molecular Cloning: A Laboratory Manual. Gold Spring Harbor, New York: Cold Spring Harbor Laboratory; 1982.

    Google Scholar 

  8. Mitchell, G. A.; Brody, L. C.; Looney, J. E., et al. An initiator codon mutationin ornithine-σ-aminotransferase causing gyrate atrophy of the choroid and retina. J. Clin. Invest. 81:630–633; 1988.

    Article  PubMed  CAS  Google Scholar 

  9. Mitchell, G. A.; Looney, J. E.; Brody, L. C., et al. Human ornithine-σ-aminotransferase cDNA cloning and analysis of the structural gene. J. Biol. Chem. 263:14288–14295; 1988.

    PubMed  CAS  Google Scholar 

  10. Mitchell, G. A.; Brody, L. C.; Sipila, I., et al. At least two mutant alleles of ornithine-σ-aminotransferase cause gyrate atrophy of the choroid and retina in Finns. Proc. Natl. Acad. Sci. USA 86:197–201; 1989.

    Article  PubMed  CAS  Google Scholar 

  11. Mueckler, M. M.; Pitot, H. C. Sequence of the precursor to ornithine aminotransferase deduced from a cDNA clone. J. Biol. Chem. 260:12993–12997; 1985.

    PubMed  CAS  Google Scholar 

  12. O'Donnell, J. J.; Sandman, R. P.; Martin, S. R. Deficient Lornithine: 2-oxoacid aminotransferase activity in cultured fibroblasts from a patient with gyrate atrophy of the retina. Biochem. Biophys. Res. Commun. 79:396–399; 1977.

    Article  PubMed  Google Scholar 

  13. O'Donnell, J. J.; Sandman, R. P.; Martin, S. R. Gyrate atrophy of the retina: Inborn error of L-ornithine: 2-oxoacid aminitransferase. Science 200:200–201; 1978.

    Article  PubMed  Google Scholar 

  14. Ramesh, V.; Shaffer, M. M.; Allaire, J. M., et al. Investigation of gyrate atrophy using a cDNA clone for human ornithine aminotransferase. DNA 5:493–501; 1986.

    PubMed  CAS  Google Scholar 

  15. Ramesh, V.; Benoit, L. A.; Crawford, P., et al. The ornithine anminotransferase (OAT) locus: Analysis of RFLPs in gyrate atrophy. Am. J. Hum. Gen. 42:365–372; 1988.

    CAS  Google Scholar 

  16. Ramesh, V.; McClatchey, A. I.; Ramesh, N., et al. Molecular basis of ornithine aminotransferase deficiency in B-6-responsive and-nonresponsive forms of gyrate atrophy. Proc. Natl. Acad. Sci. USA 85:3777–3780; 1988.

    Article  PubMed  CAS  Google Scholar 

  17. Rigby, P. W. J.; Dieckman, M.; Rhodes, C., et al. Labeling deoxyribonucleic acid to high specific activity,in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113:237–242; 1977.

    Article  PubMed  CAS  Google Scholar 

  18. Shaw, G.; Kamen, R. A conserved AU sequence from the 3′ untranslated reion of GM-CSF mRNA mediates selective mRNA degradation. Cell 46:659–667; 1986.

    Article  PubMed  CAS  Google Scholar 

  19. Shih, V. E.; Berson, E. L.; Mandell, R., et al. Ornithine ketoacid transaminase deficiency in gyrate atrophy of the choroid and retina. Am. J. Hum. Genet. 30:174–179; 1978.

    PubMed  CAS  Google Scholar 

  20. Shull, J. D.; Pitot, H. C. Structure and expression of ornithine aminotransferase (OAT) gene and related genes in various rat tissues and fibroblasts from humans with gyrate atrophy. Fed. Proc. 46:1950; 1987.

    Google Scholar 

  21. Simell, O.; Takki, K. Raised plasma ornithine and gyrate atrophy of the choroid and retina. Lancet 1:1031; 1973.

    Article  PubMed  CAS  Google Scholar 

  22. Simmaco, M.; John, R. A.; Barra, D., et al. The primary structure of ornithine aminotransferase. FEBS 199:39–42; 1986.

    Article  CAS  Google Scholar 

  23. Valle, D.; Kaiser-Kupfer, M. I.; Del Valle, L. A. Gyrate atrophy of the choroid and retina: Deficiency of ornithine aminotransferase in transformed lymphocytes. Proc. Natl. Acad. Sci. USA 74:5159–5161; 1977.

    Article  PubMed  CAS  Google Scholar 

  24. Weleber, R. G.; Wirtz, M. K.; Kennaway, N. G. Gyrate atrophy of the choroid and retina: Clinical and biochemical heterogeneity and response to vitamin B6. Birth Defects: Original Article Series 18:219–230; 1982.

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Eppley Institute for Research in Cancer and Department of Biochemistry, University of Nebraska Medical Center, 42nd and Dewey Avenue, 68105, Omaha, Nebraska

    James D. Shull & Henry C. Pitot

  2. McArdle Laboratory for Cancer Research, University of Wisconsin, 53706, Madison, Wisconsin

    James D. Shull & Henry C. Pitot

Authors
  1. James D. Shull
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Henry C. Pitot
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This project was initiated in the laboratory of H. C. P. and was supported by grants CA07175, CA22484, and 5 T32 CA09020 from the National Cancer Institute and Postdoctoral Fellowship PF-2414 from the American Cancer Society. The continuing work in the laboratory of J. D. S. was supported by grants CA36727 and HD24189 from the National, Institutes of Health, grants SIG-16, ACS-IN165A, and a Junior Faculty Research Award (JFRA-227) from the American Cancer Society, and by University of Nebraska Medical Center Seed Research Grant 88-10.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shull, J.D., Pitot, H.C. The ornithine aminotransferase gene in gyrate, atrophy of the retina: Analysis of expression and gross structure of this gene in cultured fibroblasts. In Vitro Cell Dev Biol 25, 971–976 (1989). https://doi.org/10.1007/BF02624012

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02624012

Key words

Search

Navigation