RESISTANCE GENES

Resistance to hygromycin is conferred by hygromycin B phosphotransferase (Hph). Hph catalyzes the phosphorylation of the 4-hydroxyl group on the cyclitol ring (hyosamine), thereby producing 7"-O-phosphoryl-hygromycin B which totally lacks biological activity both in vivo and in vitro (2).

Two major genes encoding Hph have been characterized. The first gene was isolated from Streptomyces hygroscopicus (1,7), the hygromycin B producer species. The second gene is a plasmid-borne resistance gene isolated from Escherichia coli (3,4) and Klebsiella pneumoniae (5). Most of hygromycin-based vectors used in gene transfer experiments harbor the E. coli hph 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 (8), and methylation on the cytosine residue of CpG (10) leading to gene silencing (9-16). 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 (11), resulting in loss of gene expression.

To avoid any hph gene silencing in eukaryotic expression vectors, due to the presence of CpG dinucleotides, a functional CpG free hph gene is available (cf. vectors). Codon usage in this synthetic gene has also been modified. The synthetic hph-DCpG gene displays higher hygromycin-resistance than its wild-type counterpart.

 

References

  1. Leboul J, Davies J. (1982) Enzymatic modification of hygromycin B in Streptomyces hygroscopicus. J Antibiot (Tokyo). 35(4):527-8.
  2. Pardo, J.M., et al. (1985) Biochemical basis of resistance to hygromycin B in Streptomyces hygroscopicus--the producing organism. J Gen Microbiol 131(Pt 6):1289-98.
  3. Rao RN, Allen NE, Hobbs JN Jr, Alborn WE Jr, Kirst HA, Paschal JW. (1983) Genetic and enzymatic basis of hygromycin B resistance in Escherichia coli. Antimicrob Agents Chemother 24(5):689-95.
  4. Kuhstoss S, Rao RN. (1983) Expression in Streptomyces ambofaciens of an Escherichia coli K-12 gene which confers resistance to hygromycin B. Gene 26(2-3):295-9.
  5. Gritz L, Davies J. (1983) Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene 25(2-3):179-88.
  6. Kaster KR, Burgett SG, Rao RN, Ingolia TD. (1983) Analysis of a bacterial hygromycin B resistance gene by transcriptional and translational fusions and by DNA sequencing. Nucleic Acids Res. 11(19):6895-911.
  7. Malpartida F, Zalacain M, Jimenez A, Davies J (1983) Molecular cloning and expression in Streptomyces lividans of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus. Biochem Biophys Res Commun 117(1):6-12.
  8. Krieg, A.M., et al. (1995) CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374(6522): 546-9.
  9. 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.
  10. Bird, A.P. (1980) DNA methylation and the frequency of CpG in animal DNA. Nucleic Acids Res 8(7): 1499-504.
  11. Bird, A.P. (1986) CpG-rich islands and the function of DNA methylation. Nature 321(6067): 209-13.
  12. Siegfried, Z and H. Cedar (1997) DNA methylation: a molecular lock. Curr.Biol. 7:305-307.
  13. 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.
  14. 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.
  15. Keshet, I., Yisraeli, J., and H. Cedar (1985) Effect of regional DNA methylation on gene expression. Proc. Natl. Acad. Sci. USA 82:2560-2564.
  16. 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.