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. 1987 Feb;84(3):764–768. doi: 10.1073/pnas.84.3.764

Introduction of the human pro alpha 1(I) collagen gene into pro alpha 1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I collagen.

A Schnieke, M Dziadek, J Bateman, T Mascara, K Harbers, R Gelinas, R Jaenisch
PMCID: PMC304296  PMID: 3468512

Abstract

The Mov-13 mouse strain carries a retroviral insertion in the pro alpha 1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of pro alpha 2 mRNA are synthesized. We have introduced genomic clones of either the human or mouse pro alpha 1(I) collagen gene into homozygous cell lines to assess whether the human or mouse pro alpha 1(I) chains can associate with the endogenous mouse pro alpha 2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human alpha 1 chains and one mouse alpha 2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both alpha 1(I) and alpha 2(I) chains in the human-mouse hybrid molecules were retarded, compared to the alpha (I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse alpha 1 and alpha 2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human alpha chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow us to study the effect specific mutations introduced in transfected pro alpha 1(I) genes have on the synthesis, assembly, and function of collagen I.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Auffray C., Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem. 1980 Jun;107(2):303–314. doi: 10.1111/j.1432-1033.1980.tb06030.x. [DOI] [PubMed] [Google Scholar]
  2. Barsh G. S., Roush C. L., Bonadio J., Byers P. H., Gelinas R. E. Intron-mediated recombination may cause a deletion in an alpha 1 type I collagen chain in a lethal form of osteogenesis imperfecta. Proc Natl Acad Sci U S A. 1985 May;82(9):2870–2874. doi: 10.1073/pnas.82.9.2870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barsh G. S., Roush C. L., Gelinas R. E. DNA and chromatin structure of the human alpha 1 (I) collagen gene. J Biol Chem. 1984 Dec 10;259(23):14906–14913. [PubMed] [Google Scholar]
  4. Bateman J. F., Mascara T., Chan D., Cole W. G. Abnormal type I collagen metabolism by cultured fibroblasts in lethal perinatal osteogenesis imperfecta. Biochem J. 1984 Jan 1;217(1):103–115. doi: 10.1042/bj2170103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bonadio J., Byers P. H. Subtle structural alterations in the chains of type I procollagen produce osteogenesis imperfecta type II. Nature. 1985 Jul 25;316(6026):363–366. doi: 10.1038/316363a0. [DOI] [PubMed] [Google Scholar]
  6. Bond J. F., Fridovich-Keil J. L., Pillus L., Mulligan R. C., Solomon F. A chicken-yeast chimeric beta-tubulin protein is incorporated into mouse microtubules in vivo. Cell. 1986 Feb 14;44(3):461–468. doi: 10.1016/0092-8674(86)90467-8. [DOI] [PubMed] [Google Scholar]
  7. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  8. Chojkier M., Peterkofsky B., Bateman J. New method for determining the extent of proline hydroxylation by measuring changes in the ratio of [4-3H]:[14C]proline in collagenase digests. Anal Biochem. 1980 Nov 1;108(2):385–393. doi: 10.1016/0003-2697(80)90603-x. [DOI] [PubMed] [Google Scholar]
  9. Chu M. L., Gargiulo V., Williams C. J., Ramirez F. Multiexon deletion in an osteogenesis imperfecta variant with increased type III collagen mRNA. J Biol Chem. 1985 Jan 25;260(2):691–694. [PubMed] [Google Scholar]
  10. Chu M. L., Myers J. C., Bernard M. P., Ding J. F., Ramirez F. Cloning and characterization of five overlapping cDNAs specific for the human pro alpha 1(I) collagen chain. Nucleic Acids Res. 1982 Oct 11;10(19):5925–5934. doi: 10.1093/nar/10.19.5925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chu M. L., de Wet W., Bernard M., Ding J. F., Morabito M., Myers J., Williams C., Ramirez F. Human pro alpha 1(I) collagen gene structure reveals evolutionary conservation of a pattern of introns and exons. 1984 Jul 26-Aug 1Nature. 310(5975):337–340. doi: 10.1038/310337a0. [DOI] [PubMed] [Google Scholar]
  12. Chu M. L., de Wet W., Bernard M., Ramirez F. Fine structural analysis of the human pro-alpha 1 (I) collagen gene. Promoter structure, AluI repeats, and polymorphic transcripts. J Biol Chem. 1985 Feb 25;260(4):2315–2320. [PubMed] [Google Scholar]
  13. Cohn D. H., Byers P. H., Steinmann B., Gelinas R. E. Lethal osteogenesis imperfecta resulting from a single nucleotide change in one human pro alpha 1(I) collagen allele. Proc Natl Acad Sci U S A. 1986 Aug;83(16):6045–6047. doi: 10.1073/pnas.83.16.6045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Colbère-Garapin F., Horodniceanu F., Kourilsky P., Garapin A. C. A new dominant hybrid selective marker for higher eukaryotic cells. J Mol Biol. 1981 Jul 25;150(1):1–14. doi: 10.1016/0022-2836(81)90321-1. [DOI] [PubMed] [Google Scholar]
  15. French B. T., Lee W. H., Maul G. G. Nucleotide sequence of a cDNA clone for mouse pro alpha 1(I) collagen protein. Gene. 1985;39(2-3):311–312. doi: 10.1016/0378-1119(85)90329-4. [DOI] [PubMed] [Google Scholar]
  16. Harbers K., Kuehn M., Delius H., Jaenisch R. Insertion of retrovirus into the first intron of alpha 1(I) collagen gene to embryonic lethal mutation in mice. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1504–1508. doi: 10.1073/pnas.81.5.1504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jaenisch R., Harbers K., Schnieke A., Löhler J., Chumakov I., Jähner D., Grotkopp D., Hoffmann E. Germline integration of moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death. Cell. 1983 Jan;32(1):209–216. doi: 10.1016/0092-8674(83)90511-1. [DOI] [PubMed] [Google Scholar]
  18. Khillan J. S., Schmidt A., Overbeek P. A., de Crombrugghe B., Westphal H. Developmental and tissue-specific expression directed by the alpha 2 type I collagen promoter in transgenic mice. Proc Natl Acad Sci U S A. 1986 Feb;83(3):725–729. doi: 10.1073/pnas.83.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lemischka I. R., Farmer S., Racaniello V. R., Sharp P. A. Nucleotide sequence and evolution of a mammalian alpha-tubulin messenger RNA. J Mol Biol. 1981 Sep 5;151(1):101–120. doi: 10.1016/0022-2836(81)90223-0. [DOI] [PubMed] [Google Scholar]
  21. Lovell-Badge R. H., Bygrave A. E., Bradley A., Robertson E., Evans M. J., Cheah K. S. Transformation of embryonic stem cells with the human type-II collagen gene and its expression in chimeric mice. Cold Spring Harb Symp Quant Biol. 1985;50:707–711. doi: 10.1101/sqb.1985.050.01.087. [DOI] [PubMed] [Google Scholar]
  22. Löhler J., Timpl R., Jaenisch R. Embryonic lethal mutation in mouse collagen I gene causes rupture of blood vessels and is associated with erythropoietic and mesenchymal cell death. Cell. 1984 Sep;38(2):597–607. doi: 10.1016/0092-8674(84)90514-2. [DOI] [PubMed] [Google Scholar]
  23. Monson J. M., Friedman J., McCarthy B. J. DNA sequence analysis of a mouse pro alpha 1 (I) procollagen gene: evidence for a mouse B1 element within the gene. Mol Cell Biol. 1982 Nov;2(11):1362–1371. doi: 10.1128/mcb.2.11.1362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Myers J. C., Chu M. L., Faro S. H., Clark W. J., Prockop D. J., Ramirez F. Cloning a cDNA for the pro-alpha 2 chain of human type I collagen. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3516–3520. doi: 10.1073/pnas.78.6.3516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Peterkofsky B., Diegelmann R. Use of a mixture of proteinase-free collagenases for the specific assay of radioactive collagen in the presence of other proteins. Biochemistry. 1971 Mar 16;10(6):988–994. doi: 10.1021/bi00782a009. [DOI] [PubMed] [Google Scholar]
  26. Prockop D. J., Kivirikko K. I. Heritable diseases of collagen. N Engl J Med. 1984 Aug 9;311(6):376–386. doi: 10.1056/NEJM198408093110606. [DOI] [PubMed] [Google Scholar]
  27. Schmidt A., Rossi P., de Crombrugghe B. Transcriptional control of the mouse alpha 2(I) collagen gene: functional deletion analysis of the promoter and evidence for cell-specific expression. Mol Cell Biol. 1986 Feb;6(2):347–354. doi: 10.1128/mcb.6.2.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schmidt A., Setoyama C., de Crombrugghe B. Regulation of a collagen gene promoter by the product of viral mos oncogene. Nature. 1985 Mar 21;314(6008):286–289. doi: 10.1038/314286a0. [DOI] [PubMed] [Google Scholar]
  29. Schnieke A., Harbers K., Jaenisch R. Embryonic lethal mutation in mice induced by retrovirus insertion into the alpha 1(I) collagen gene. 1983 Jul 28-Aug 3Nature. 304(5924):315–320. doi: 10.1038/304315a0. [DOI] [PubMed] [Google Scholar]
  30. Steinmann B., Rao V. H., Vogel A., Bruckner P., Gitzelmann R., Byers P. H. Cysteine in the triple-helical domain of one allelic product of the alpha 1(I) gene of type I collagen produces a lethal form of osteogenesis imperfecta. J Biol Chem. 1984 Sep 10;259(17):11129–11138. [PubMed] [Google Scholar]
  31. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tkocz C., Kühn K. The formation of triple-helical collagen molecules from alpha-1 or alpha-2 polypeptide chains. Eur J Biochem. 1969 Feb;7(4):454–462. doi: 10.1111/j.1432-1033.1969.tb19631.x. [DOI] [PubMed] [Google Scholar]

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