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.



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