Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1983;2(5):637–641. doi: 10.1002/j.1460-2075.1983.tb01477.x

Microinjection of human cell extracts corrects xeroderma pigmentosum defect.

A J de Jonge, W Vermeulen, B Klein, J H Hoeijmakers
PMCID: PMC555162  PMID: 6357782

Abstract

Cultured fibroblasts of patients with the DNA repair syndrome xeroderma pigmentosum (XP) were injected with crude cell extracts from various human cells. Injected fibroblasts were then assayed for unscheduled DNA synthesis (UDS) to see whether the injected extract could complement their deficiency in the removal of u.v.-induced thymidine dimers from their DNA. Microinjection of extracts from repair-proficient cells (such as HeLa, placenta) and from cells belonging to XP complementation group C resulted in a temporary correction of the DNA repair defect in XP-A cells but not in cells from complementation groups C, D or F. Extracts prepared from XP-A cells were unable to correct the XP-A repair defect. The UDS of phenotypically corrected XP-A cells is u.v.-specific and can reach the level of normal cells. The XP-A correcting factor was found to be sensitive to the action of proteinase K, suggesting that it is a protein. It is present in normal cells in high amounts, it is stable on storage and can still be detected in the injected cells 8 h after injection. The microinjection assay described in this paper provides a useful tool for the purification of the XP-A (and possibly other) factor(s) involved in DNA repair.

Full text

PDF
637

Images in this article

638Fig. 1.
on p.638

Selected References

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

  1. Anderson W. F., Killos L., Sanders-Haigh L., Kretschmer P. J., Diacumakos E. G. Replication and expression of thymidine kinase and human globin genes microinjected into mouse fibroblasts. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5399–5403. doi: 10.1073/pnas.77.9.5399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arase S., Kozuka T., Tanaka K., Ikenaga M., Takebe H. A sixth complementation group in xeroderma pigmentosum. Mutat Res. 1979 Jan;59(1):143–146. doi: 10.1016/0027-5107(79)90202-1. [DOI] [PubMed] [Google Scholar]
  3. Bootsma D., Mulder M. P., Cohen J. A., Pot F. Different inherited levels of DNA repair replication in xeroderma pigmentosum cell strains after exposure to ultraviolet irradiation. Mutat Res. 1970 May;9(5):507–516. doi: 10.1016/0027-5107(70)90035-7. [DOI] [PubMed] [Google Scholar]
  4. Burridge K., Feramisco J. R. Microinjection and localization of a 130K protein in living fibroblasts: a relationship to actin and fibronectin. Cell. 1980 Mar;19(3):587–595. doi: 10.1016/s0092-8674(80)80035-3. [DOI] [PubMed] [Google Scholar]
  5. Capecchi M. R. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell. 1980 Nov;22(2 Pt 2):479–488. doi: 10.1016/0092-8674(80)90358-x. [DOI] [PubMed] [Google Scholar]
  6. Ciarrocchi G., Linn S. A cell-free assay measuring repair DNA synthesis in human fibroblasts. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1887–1891. doi: 10.1073/pnas.75.4.1887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cleaver J. E. Defective repair replication of DNA in xeroderma pigmentosum. Nature. 1968 May 18;218(5142):652–656. doi: 10.1038/218652a0. [DOI] [PubMed] [Google Scholar]
  8. De Weerd-Kastelein E. A., Keijzer W., Bootsma D. Genetic heterogeneity of xeroderma pigmentosum demonstrated by somatic cell hybridization. Nat New Biol. 1972 Jul 19;238(81):80–83. doi: 10.1038/newbio238080a0. [DOI] [PubMed] [Google Scholar]
  9. Dresler S. L., Roberts J. D., Lieberman M. W. Characterization of deoxyribonucleic acid repair synthesis in permeable human fibroblasts. Biochemistry. 1982 May 11;21(10):2557–2564. doi: 10.1021/bi00539a040. [DOI] [PubMed] [Google Scholar]
  10. Giannelli F., Pawsey S. A., Avery J. A. Differences in patterns of complementation of the more common groups of xeroderma pigmentosum: possible implications. Cell. 1982 Jun;29(2):451–458. doi: 10.1016/0092-8674(82)90161-1. [DOI] [PubMed] [Google Scholar]
  11. Gluzman Y., Sambrook J. F., Frisque R. J. Expression of early genes of origin-defective mutants of simian virus 40. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3898–3902. doi: 10.1073/pnas.77.7.3898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Graessmann A., Graessmann M., Mueller C. Microinjection of early SV40 DNA fragments and T antigen. Methods Enzymol. 1980;65(1):816–825. doi: 10.1016/s0076-6879(80)65076-9. [DOI] [PubMed] [Google Scholar]
  13. Graessmann A., Wolf H., Bornkamm G. W. Expression of Epstein-Barr virus genes in different cell types after microinjection of viral DNA. Proc Natl Acad Sci U S A. 1980 Jan;77(1):433–436. doi: 10.1073/pnas.77.1.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Graessmann M., Graessman A. "Early" simian-virus-40-specific RNA contains information for tumor antigen formation and chromatin replication. Proc Natl Acad Sci U S A. 1976 Feb;73(2):366–370. doi: 10.1073/pnas.73.2.366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hashem N., Bootsma D., Keijzer W., Greene A., Coriell L., Thomas G., Cleaver J. E. Clinical characteristics, DNA repair, and complementation groups in xeroderma pigmentosum patients from Egypt. Cancer Res. 1980 Jan;40(1):13–18. [PubMed] [Google Scholar]
  16. Hayakawa H., Ishizaki K., Inoue M., Yagi T., Sekiguchi M., Takebe H. Repair of ultraviolet radiation damage in xeroderma pigmentosum cells belonging to complementation group F. Mutat Res. 1981 Feb;80(2):381–388. doi: 10.1016/0027-5107(81)90110-x. [DOI] [PubMed] [Google Scholar]
  17. Jaspers N. G., Bootsma D. Abnormal levels of UV-induced unscheduled DNA synthesis in ataxia telangiectasia cells after exposure to ionizing radiation. Mutat Res. 1982 Feb 22;92(1-2):439–446. doi: 10.1016/0027-5107(82)90242-1. [DOI] [PubMed] [Google Scholar]
  18. Jaspers N. G., Jansen-van de Kuilen G., Bootsma D. Complement analysis of xeroderma pigmentosum variants. Exp Cell Res. 1981 Nov;136(1):81–90. doi: 10.1016/0014-4827(81)90039-2. [DOI] [PubMed] [Google Scholar]
  19. Jongkind J. F. The quantitative histochemistry of hypothalamus. I. Pentose shunt enzymes in the activated supraoptic nucleus of the rat. J Histochem Cytochem. 1967 Jul;15(7):394–398. doi: 10.1177/15.7.394. [DOI] [PubMed] [Google Scholar]
  20. Keijzer W., Jaspers N. G., Abrahams P. J., Taylor A. M., Arlett C. F., Zelle B., Takebe H., Kinmont P. D., Bootsma D. A seventh complementation group in excision-deficient xeroderma pigmentosum. Mutat Res. 1979 Aug;62(1):183–190. doi: 10.1016/0027-5107(79)90231-8. [DOI] [PubMed] [Google Scholar]
  21. Keijzer W., Verkerk A., Bootsma D. Phenotypic correction of the defect in xeroderma pigmentosum cells after fusion with isolated cytoplasts. Exp Cell Res. 1982 Jul;140(1):119–125. doi: 10.1016/0014-4827(82)90163-x. [DOI] [PubMed] [Google Scholar]
  22. Kleijer W. J., de Weerd-Kastelein E. A., Sluyter M. L., Keijzer W., de Wit J., Bootsma D. UV-induced DNA repair synthesis in cells of patients with different forms of xeroderma pigmentosum and of heterozygotes. Mutat Res. 1973 Dec;20(3):417–428. doi: 10.1016/0027-5107(73)90062-6. [DOI] [PubMed] [Google Scholar]
  23. Kraemer K. H., De Weerd-Kastelein E. A., Robbins J. H., Keijzer W., Barrett S. F., Petinga R. A., Bootsma D. Five complementation groups in xeroderma pigmentosum. Mutat Res. 1975 Dec;33(2-3):327–340. doi: 10.1016/0027-5107(75)90208-0. [DOI] [PubMed] [Google Scholar]
  24. Kreis T. E., Winterhalter K. H., Birchmeier W. In vivo distribution and turnover of fluorescently labeled actin microinjected into human fibroblasts. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3814–3818. doi: 10.1073/pnas.76.8.3814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Liu C. P., Slate D. L., Gravel R., Ruddle F. H. Biological detection of specific mRNA molecules by microinjection. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4503–4506. doi: 10.1073/pnas.76.9.4503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matsukuma S., Zelle B., Keijzer W., Berends F., Bootsma D. Different rates of restoration of the repair capacity in complementing xeroderma pigmentosum cells after fusion. Exp Cell Res. 1981 Jul;134(1):103–112. doi: 10.1016/0014-4827(81)90467-5. [DOI] [PubMed] [Google Scholar]
  27. Mortelmans K., Friedberg E. C., Slor H., Thomas G., Cleaver J. E. Defective thymine dimer excision by cell-free extracts of xeroderma pigmentosum cells. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2757–2761. doi: 10.1073/pnas.73.8.2757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Roberts J. D., Lieberman M. W. Deoxyribonucleic acid repair synthesis in permeable human fibroblasts exposed to ultraviolet radiation and N-acetoxy-2-(acetylamino)fluorene. Biochemistry. 1979 Oct 16;18(21):4499–4505. doi: 10.1021/bi00588a007. [DOI] [PubMed] [Google Scholar]
  29. Smith C. A., Hanawalt P. C. Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2598–2602. doi: 10.1073/pnas.75.6.2598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Takebe H., Nii S., Ishii M. I., Utsumi H. Comparative studies of host-cell reactivation, colony forming ability and excision repair after UV irradiation of xeroderma pigmentosum, normal human and some other mammalian cells. Mutat Res. 1974 Dec;25(3):383–390. doi: 10.1016/0027-5107(74)90067-0. [DOI] [PubMed] [Google Scholar]
  31. Tanaka K., Hayakawa H., Sekiguchi M., Okada Y. Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2958–2962. doi: 10.1073/pnas.74.7.2958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tanaka K., Sekiguchi M., Okada Y. Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus). Proc Natl Acad Sci U S A. 1975 Oct;72(10):4071–4075. doi: 10.1073/pnas.72.10.4071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Weerd-Kastelein E. A., Keijzer W., Sabour M., Parrington J. M., Bootsma D. A xeroderma pigmentosum patient having a high residual activity of unscheduled DNA synthesis after UV is assigned to complementation group A. Mutat Res. 1976 Nov;37(2-3):307–312. doi: 10.1016/0027-5107(76)90044-0. [DOI] [PubMed] [Google Scholar]
  34. Zelle B., Bootsma D. Repair of DNA damage after exposure to 4-nitroquinoline-1-oxide in heterokaryons derived from xeroderma pigmentosum cells. Mutat Res. 1980 May;70(3):373–381. doi: 10.1016/0027-5107(80)90027-5. [DOI] [PubMed] [Google Scholar]
  35. de Jonge A. J., Abrahams P. J., Westerveld A., Bootsma D. Expression of human hprt gene on the inactive X chromosome after DNA-mediated gene transfer. Nature. 1982 Feb 18;295(5850):624–626. doi: 10.1038/295624a0. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES