Hygromycin Resistance Vectors for Chlamydomonas reinhardtii

We have developed a dominant selectable marker for the green alga Chlamydomonas reinhardtii using the Streptomyces hygroscopicus aminoglycoside phosphotransferase (aph7") gene mediating resistance against hygromycin B (Berthold et al. 2002. Protist 153:401-412).  This marker is contained in  two plasmids, pHyg3 and pHyg4, respectively. In both plasmids the aph7" gene is expressed from the C. reinhardtii beta 2 tubulin promoter and terminated by the C. reinhardtii rbcS2 gene 3' untranslated region. In addition, pHyg3 contains the first intron of the C. reinhardtii rbcS2 gene inserted into the coding region of the aph7" gene (pHyg4 does not contain this intron).

Plasmid pHyg3 has been used in routine application in three different laboratories for several years now with good success. In addition, pHyg3 has meanwhile been distributed to more than 80 laboratories worldwide. We are happy to share this marker with everyone who is interested and who wants to use it for non-commercial applications.

Requests for the pHyg3 plasmid or questions concerning this transformation system should be directed to:

wolfgang.mages@biologie.uni-regensburg.de

The plasmids pHyg3 and pHyg4 are also available through the Chlamydomonas Resource Center at the University of Minnesota: http://www.chlamy.org/

Transformation of the HygR marker

To introduce the HgyR marker into Chlamydomonas cells we are using the glass bead method of Karen Kindle. Both pHyg3 and pHyg4 have been used to transform both cell wall deficient (cw2, cw15) and wild type (CC124) strains, pHyg4 being somewhat less efficient in terms of transformation rates (probably due to the lack of an intron). Transformants can be selected easily on TAP plates supplemented with 10 g hygromycinB per ml.  We buy "our" hygromycinB from Roche (order no. 843 555).

Plasmid Construction

Details of the construction and transformation protocols can be found in our pertinent publication (see above). You can also get some additional information that may be useful following this link:

pHyg3 annotation update-10-2009.pdf

Here you can go directly to a *.html and a *.txt Version of the Hyg Sequence
 

Physical map and sequence

 The following figure provides:
a) a schematic representation of the two slightly different marker constructs in plasmids pHyg3 and pHyg4
and  
b) the sequence of the marker cassette in plasmid pHyg3.
   
 

Please note:
The labeling of the rbcS2 intron in our respective publication (Berthold et al. 2002. Protist, Vol. 153, 401412) is wrong. Four bases (418-421) in the following figure which is taken from our publication in fact do belong to the rbcS2 intron. The box for the intron was inadvertently misplaced in the published figure (using the wrong GT as the intron 5' border).

   


 
 

Insert of pHyg3 as text-file

 
Complete pHyg3 as text-file

 

Please direct questions to:     wolfgang.mages@biologie.uni-regensburg.de