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. 1992 May;76(1):95–102.

Molecular cloning, reconstruction and expression of the gene encoding the alpha-chain of the bovine CD8--definition of three peptide regions conserved across species.

P Lalor 1, C Bucci 1, M Fornaro 1, M C Rattazzi 1, H Nakauchi 1, L A Herzenberg 1, S Alberti 1
PMCID: PMC1421739  PMID: 1628904

Abstract

We report the cloning of a cDNA encoding the alpha-chain of the bovine CD8 (BoCD8 alpha). A bovine thymus cDNA library was hybridized at low stringency with a human CD8 alpha cDNA clone. The first round of screening of 5 x 10(4) independent colonies yielded 12 clones containing incomplete BoCD8 alpha genes. Two further rounds of colony hybridization were conducted, each using as a probe the 5' fragment from the longest BoCD8 alpha clone previously isolated. The final screening yielded a clone containing a 2 kilobase (kb) insert. We mapped and sequenced the 2 kb BoCD8 alpha clone and compared it with the published sequences of the genes encoding the human, mouse and rat CD8 alpha. Sequence analysis confirmed that the clone under study encoded the BoCD8 alpha. The overall similarity of the BoCD8 alpha coding region with the human CD8 alpha coding sequence is 74.7% at the nucleotide level and 62.1% at the protein level. Lower levels of similarity are found with the mouse and rat CD8 alpha. Interestingly, three separate highly homologous regions are clearly defined at the peptide level in bovine versus human and mouse versus rat comparisons. Two of the regions are highly conserved among all species analysed, while the most 5' region is not. We speculate that the latter region may contain the binding site of CD8 alpha to the alpha 3 domain of major histocompatibility complex (MHC) class I molecules. Sequence analysis showed that the 2 kb BoCD8 alpha clone contains an incomplete coding region, i.e. lacks six bases corresponding to the first two amino acids of the leader region. To allow efficient translation and processing of the BoCD8 alpha gene, we constructed a chimeric gene containing the coding sequence of the BoCD8 alpha clone and synthetic sequences corresponding to the first two amino acids of the human CD8 alpha leader sequence. The chimeric gene was subcloned in the pKSV10 expression vector. The pKSV10-BoCD8 alpha construct is efficiently expressed both transiently in COS cells and stably in L cells, as determined by Northern blot and by FACS analysis, using the ILA-51 monoclonal antibody to BoCD8 alpha. The latter result formally proves that the ILA-51 antibody does indeed recognize the product of the BoCD8 alpha gene, as previously suggested on serological grounds.

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Selected References

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  1. Alberti S., Fornaro M. Higher transfection efficiency of genomic DNA purified with a guanidinium thiocyanate-based procedure. Nucleic Acids Res. 1990 Jan 25;18(2):351–353. doi: 10.1093/nar/18.2.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alberti S., Herzenberg L. A. DNA methylation prevents transfection of genes for specific surface antigens. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8391–8394. doi: 10.1073/pnas.85.22.8391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alberti S., Parks D. R., Herzenberg L. A. A single laser method for subtraction of cell autofluorescence in flow cytometry. Cytometry. 1987 Mar;8(2):114–119. doi: 10.1002/cyto.990080203. [DOI] [PubMed] [Google Scholar]
  4. Cantor H., Boyse E. A. Lymphocytes as models for the study of mammalian cellular differentiation. Immunol Rev. 1977 Jan;33:105–124. doi: 10.1111/j.1600-065x.1977.tb00364.x. [DOI] [PubMed] [Google Scholar]
  5. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ellis J. A., Baldwin C. L., MacHugh N. D., Bensaid A., Teale A. J., Goddeeris B. M., Morrison W. I. Characterization by a monoclonal antibody and functional analysis of a subset of bovine T lymphocytes that express BoT8, a molecule analogous to human CD8. Immunology. 1986 Jul;58(3):351–358. [PMC free article] [PubMed] [Google Scholar]
  7. Irwin M. J., Heath W. R., Sherman L. A. Species-restricted interactions between CD8 and the alpha 3 domain of class I influence the magnitude of the xenogeneic response. J Exp Med. 1989 Oct 1;170(4):1091–1101. doi: 10.1084/jem.170.4.1091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Johnson P., Gagnon J., Barclay A. N., Williams A. F. Purification, chain separation and sequence of the MRC OX-8 antigen, a marker of rat cytotoxic T lymphocytes. EMBO J. 1985 Oct;4(10):2539–2545. doi: 10.1002/j.1460-2075.1985.tb03968.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kavathas P., Sukhatme V. P., Herzenberg L. A., Parnes J. R. Isolation of the gene encoding the human T-lymphocyte differentiation antigen Leu-2 (T8) by gene transfer and cDNA subtraction. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7688–7692. doi: 10.1073/pnas.81.24.7688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  11. Littman D. R. The structure of the CD4 and CD8 genes. Annu Rev Immunol. 1987;5:561–584. doi: 10.1146/annurev.iy.05.040187.003021. [DOI] [PubMed] [Google Scholar]
  12. Littman D. R., Thomas Y., Maddon P. J., Chess L., Axel R. The isolation and sequence of the gene encoding T8: a molecule defining functional classes of T lymphocytes. Cell. 1985 Feb;40(2):237–246. doi: 10.1016/0092-8674(85)90138-2. [DOI] [PubMed] [Google Scholar]
  13. Nakauchi H., Nolan G. P., Hsu C., Huang H. S., Kavathas P., Herzenberg L. A. Molecular cloning of Lyt-2, a membrane glycoprotein marking a subset of mouse T lymphocytes: molecular homology to its human counterpart, Leu-2/T8, and to immunoglobulin variable regions. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5126–5130. doi: 10.1073/pnas.82.15.5126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nakayama K., Tokito S., Okumura K., Nakauchi H. Structure and expression of the gene encoding CD8 alpha chain (Leu-2/T8). Immunogenetics. 1989;30(5):393–397. doi: 10.1007/BF02425282. [DOI] [PubMed] [Google Scholar]
  15. Shiue L., Gorman S. D., Parnes J. R. A second chain of human CD8 is expressed on peripheral blood lymphocytes. J Exp Med. 1988 Dec 1;168(6):1993–2005. doi: 10.1084/jem.168.6.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Snow P. M., Keizer G., Coligan J. E., Terhorst C. Purification and N-terminal amino acid sequence of the human T cell surface antigen T8. J Immunol. 1984 Oct;133(4):2058–2066. [PubMed] [Google Scholar]
  17. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  18. Sukhatme V. P., Sizer K. C., Vollmer A. C., Hunkapiller T., Parnes J. R. The T cell differentiation antigen Leu-2/T8 is homologous to immunoglobulin and T cell receptor variable regions. Cell. 1985 Mar;40(3):591–597. doi: 10.1016/0092-8674(85)90207-7. [DOI] [PubMed] [Google Scholar]
  19. Tagawa M., Nakauchi H., Herzenberg L. A., Nolan G. P. Formal proof that different-size Lyt-2 polypeptides arise from differential splicing and post-transcriptional regulation. Proc Natl Acad Sci U S A. 1986 May;83(10):3422–3426. doi: 10.1073/pnas.83.10.3422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Takada S., Engleman E. G. Evidence for an association between CD8 molecules and the T cell receptor complex on cytotoxic T cells. J Immunol. 1987 Nov 15;139(10):3231–3235. [PubMed] [Google Scholar]
  21. Turner J. M., Brodsky M. H., Irving B. A., Levin S. D., Perlmutter R. M., Littman D. R. Interaction of the unique N-terminal region of tyrosine kinase p56lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine motifs. Cell. 1990 Mar 9;60(5):755–765. doi: 10.1016/0092-8674(90)90090-2. [DOI] [PubMed] [Google Scholar]
  22. Van Seventer G. A., Van Lier R. A., Spits H., Ivanyi P., Melief C. J. Evidence for a regulatory role of the T8 (CD8) antigen in antigen-specific and anti-T3-(CD3)-induced lytic activity of allospecific cytotoxic T lymphocyte clones. Eur J Immunol. 1986 Nov;16(11):1363–1371. doi: 10.1002/eji.1830161109. [DOI] [PubMed] [Google Scholar]
  23. Walker I. D., Hogarth P. M., Murray B. J., Lovering K. E., Classon B. J., Chambers G. W., McKenzie I. F. Ly antigens associated with T cell recognition and effector function. Immunol Rev. 1984 Dec;82:47–77. doi: 10.1111/j.1600-065x.1984.tb01117.x. [DOI] [PubMed] [Google Scholar]
  24. Wigler M., Pellicer A., Silverstein S., Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. doi: 10.1016/0092-8674(78)90254-4. [DOI] [PubMed] [Google Scholar]

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