Hygromycin resistance as a eukaryotic marker.

[Brian Foley]

------- The Hygromycin phosphotransferase gene ----------------

Gritz,L. and Davies,J. (1983)
Gene 25:  179-188
Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B 
phosphotransferase gene and its expression in Eschericia coli and 
Saccharomyces cerevisiae

Kaster,K.R., Burgett,S.G., Rao,R.N. and Ingolia,T.D.  (1983)
Nucleic Acids Research 11:  6895-6911
Analysis of a bacterial hygromycin resistance gene by transcriptional and 
translational fusions and by DNA sequencing.

Both papers report the same gene sequence from an E. coli plasmid.

 ------- Assays for hygromycin resistance in mammalian cells ---

Santerre,R.F., Allen,N.E., Hobbs,J.N., Rao, R.N. and Schmidt,R.J. (1984)
Gene 30, 147-156
Expression of prokaryotic genes for hygromycin B and G418 resistance
as dominant-selection markers in mouse L cells.

This paper reports assays for hygromycin phosphotransferase activity
as well as showing dose-response assays on whole cells.  They also
did Southern blots and other assays to determine copy number of
integrated DNA.

Note that the Hygromycin phosphotransferase gene used in this study
was directly from an E. coli plasmid.  It contains upstream start
codons that can interfere with mammalian cell expression.  Giordano, T.J.
and McAllister (1990)  Gene 88: 285-288  "Optimization of the hygromycin
B resistance-conferring gene as a dominant selectable marker in mammalian 
cells"  They report elimination of the upstream start codons in order
to get better expression in mammalian cells.

The plasmid I use is pCEP4 from Invitrogen.  The sequence they supplied 
to me shows that they did not remove the upstream start codon.  
McAllister reports that he got a 5-fold increase in protein production by
mutating this upstream ATG and altering region just upstream of the correct
ATG start codon to a consensus Kazak ribosome binding site.

--------------- Mode of action of Hygromycin ----------------

Gonzalez,A., Jimenez,A., Vazquez,D., Davies,J.E. and Schindler,D.  (1978)
Biochimica et Biophysica Acta.  521: 459-469
Studies on the mode of action of hygromycin B, and inhibitor of 
translocation in eukaryotes.

Cabanas,M.J., Vazquez,D. and Modelell,J.  (1978)
Eur. J. Biochem.  87: 21-27
Dual interference of hygromycin B with ribosomal translocation and with 
aminoacyl-tRNA recognition

Although the inhibition of protein synthesis occurs at the elongation 
step, it is independent of interference with elongation factor 2 
(eukaryotes and archaebacteria) or elongation factor G (eubacteria).
Hygromycin inhibits poly-U directed synthesis of polyphenylalanine which 
occurs in the absence of elongation factors and GTP.  It also inhibits 
elongation even when an excess of EF-G is added to cell-free translation 
assays.

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*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
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