RESISTANCE GENES

The pac gene encoding a Puromycin N-acetyl-tranferase (PAC) has been isolated from a Streptomyces producing strain (1,2). It is located in a region of the pur cluster linked to the other genes determining the puromycin biosynthetic pathway.
The expression of pac gene confers puromycin resistance to transfected mammalians cells (3) expressing it. In some particular conditions puromycin can also be used for selection of E. coli strains transformed with plasmids carrying the pac gene.

 

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 (4), and methylation on the cytosine residue of CpG (6) leading to gene silencing (5-12). 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 (7), resulting in loss of gene expression.

To avoid pac gene silencing in eukaryotic expression vectors, due to the presence of CpG dinucleotides, one can use an engineered pac gene where most (77) CpG motifs have been removed and codon usage has been optimized for mammals (cf. vectors).

 

References

  1. Vara J., Perez-Gonzalez J.and A.Jimenez (1985) Biosynthesis of puromycin by Streptomyces alboniger. Characterization of puromycin N-acetyltransferase. Biochemistry 24: 8074-8081
  2. Lacalle R.A.,Pulido D., Vara J., Zalacain M. and A. Jimenez (1989) Molecular analysis of the pac gene encoding a puromycin-N-acetyl transferase from Streptomyces alboniger. Gene 79: 375-380
  3. De La Luna S. and J.Ortin (1992) Pac gene as efficient dominant marker and reporter gene in mammalian cells. Methods In Enzymology 216:376-385
  4. Krieg, A.M., et al. (1995) CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374(6522): 546-9.
  5. 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.
  6. Bird, A.P. (1980) DNA methylation and the frequency of CpG in animal DNA. Nucleic Acids Res 8(7): 1499-504.
  7. Bird, A.P. (1986) CpG-rich islands and the function of DNA methylation. Nature 321(6067): 209-13.
  8. Siegfried, Z and H. Cedar (1997) DNA mathylation: a molecular lock. Curr.Biol. 7:305-307.
  9. 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.
  10. 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.
  11. Keshet, I., Yisraeli, J., and H. Cedar (1985) Effect of regional DNA methylation on gene expression. Proc. Natl. Acad. Sci. USA 82:2560-2564.
  12. 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.