RESISTANCE GENES

Three blasticidin-resistance genes have been cloned and sequenced: an acetyl transferase gene, bls, from Streptoverticillum sp. JCM 4673, a blasticidin producer strain (1), and two deaminase genes, bsr , from Bacillus cereus (2,3) and BSD from Aspergillus terreus (4,5). Both genes, bsr and BSD, are used as dominant selectable markers for transformation experiments in mammalian and plant cells. Although Blasticidin S was developed as a selection agent for mammalian cells, the bsr and BSD resistance genes can also be used in E. coli.

 

Optimized Resistance Gene

Exogenous DNA, such as resistance genes from bacterial origin, may be poorly suitable for expression in mammalian cells. First, codon usage in bacteria is very different from mammalian codon usage. Then, and even more crucial, the foreign (bacterial) DNA composition in CpG dinucleotides is very different from the CpG distribution in mammalian DNA. This difference elicits two phenomena which negatively affect gene expression: recognition of the bacterial DNA as foreign by the mammalian immune system (6), and methylation on the cytosine residue of CpG (8) leading to gene silencing (7-14). Presence of methylcytosine alters the binding of transcriptional factors and other proteins to DNA and also attracts methyl-DNA-binding proteins that modify chromatin structure (9), resulting in loss of gene expression.

To avoid bsr gene silencing in eukaryotic expression vectors, due to the presence of CpG dinucleotides, a functional reduced-CpG (14 to 4) bsr gene is available (cf. vectors). Codon usage in this synthetic gene has also been modified.

 

References

  1. PEREZ-GONZALEZ J.A, RUIZ D., ESTEBAN J.A. and A.JIMENEZ. 1990. Cloning and characterization of the gene encoding a blasticidin S acetyltransferase from Streptoverticillium sp. Gene.86: 129-134
  2. IZUMI M., MIYAZAWA H., KAMAKURA T.,YAMAGUCHI I., ENDO T.and F.HANAOKA. 1991. Blasticidin S-resistance gene (bsr): a novel selectable marker for mammalian cells.Exp.Cell Res.197: 229-233
  3. ITAYA M.,YAMAGUCHI I., KOBAYASHI K., ENDO T.and T.TANAKA. 1990. The blasticidin S resistance gene (bsr) selectable in a single copy state in the Bacillus subtilis chromosome. J.Biochem. 107: 799-801
  4. KIMURA M., KAMAKURA T.,TAO Q.Z., KANEKO I and I.YAMAGUCHI. 1994. Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae. Mol. Gen. Genet. 242: 121-129
  5. KIMURA M.,TAKATSUKI A and I.YAMAGUCHI. 1994. Blasticidin S deaminase gene from Aspergillus terreus (BSD) a new drug resistance gene for transfection of mammalian cells. Biochim.Biophys.Acta.1219:653-659
  6. Krieg, A.M., et al. (1995) CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374(6522): 546-9.
  7. Komura, J., T. Okada, and T. Ono (1995) Repression of transient expression by DNA methylation in transcribed regions of reporter genes introduced into cultured human cells. Biochim Biophys Acta 1260(1): 73-8.
  8. Bird, A.P. (1980) DNA methylation and the frequency of CpG in animal DNA. Nucleic Acids Res 8(7): 1499-504.
  9. Bird, A.P. (1986) CpG-rich islands and the function of DNA methylation. Nature 321(6067): 209-13.
  10. Siegfried, Z and H. Cedar (1997) DNA methylation: a molecular lock. Curr.Biol. 7:305-307.
  11. Kass, S. U., Goddard, J. P., and R. L. Adams (1993) Inactive chromatin spreads from a focus of methylation. Mol. Cell. Biol. 13:7372-7379.
  12. Kass, S. U., Landsberger, N., and A. P. Wolffe (1997) DNA methylation directs a time-dependent repression of transcription initiation. Curr. Biol. 7:157-165.
  13. Keshet, I., Yisraeli, J., and H. Cedar (1985) Effect of regional DNA methylation on gene expression. Proc. Natl. Acad. Sci. USA 82:2560-2564.
  14. Yisraeli, J., D. Frank, A. Razin and H. Cedar (1988) Effect of DNA methylation on beta globin gene expression. Proc. Natl. Acad. Sci USA 85:4638-4642.