Yeast Metal Tolerance Protocol YEAST CADMIUM TOLERANCE FROM PLANT PCS GENES INTRODUCTION Heavy metal toxicity poses major environmental and health problems Cadmium for example is a non essential heavy metal that is toxic to living cells at very low concentrations 2 2 2 2 Cd ions displace Ca or Zn in proteins and can cause oxidative stress In humans Cd is a suspected carcinogen Furthermore the concentration of essential but at high concentrations 2 2 2 toxic metals such as Cu Zn and Fe is tightly controlled Several mechanisms are known that allow plants and other organisms to tolerate the presence of toxic non essential metal ions inside the cell In eukaryotic cells toxic ions appear to be removed from the cytosol mainly by chelation and sequestration Schizosaccharomyces pombe and other fungi as well as plants 2 2 synthesize phytochelatins PCs which chelate Cd Cu and possibly other heavy metal ions Phytochelatins are thiolate peptides with the primary structure Glu Cys n Gly which are nontranslationally synthesized from glutathione Phytochelatins are synthesized by enzymes called phytochelatin synthases Phytochelatin deficient Arabidopsis and S pombe mutants are 2 hypersensitive to Cd thereby demonstrating the importance of phytochelatins for plant and fungal metal tolerance The gene encoding phytochelatin synthase PCS has recently been cloned from plants wheat and Arabidopsis fungi S pombe and C elegans Clemens et al 1999 Ha et al 1999 Vatamaniuk et al 1999 When expressed in a species of yeast S cerevisiae that normally lacks the ability to synthesize phytochelatins these PCS genes confer significant tolerance to toxic metals allowing it to grow on much higher concentrations of these metals than wild type yeast In this lab you will confirm the ability of the PCS genes to provide increased metal tolerance to S cerevisiae by plating out different cells transformed with the wheat PCS TaPCS1 gene the Arabidopsis gene AtPCS1 and an empty vector pYES2 as a control on plates containing high levels of cadmium By observing which cells grow on the toxic media you will be able to determine which yeast cells were transformed with the PCS genes and which one was the empty vector control You will not be able to differentiate between cells transformed with AtPCS1 and TaPCS1 from the assays on cadmium containing plates so concurrently you will be performing restriction mapping on the transforming vectors By examining the vector maps you can find polymorphisms in the wheat and Arabidopsis PCS sequences which produce different restriction patterns when cut with specific restriction enzymes After extracting the vector DNA from overnight cultures and using the restriction enzymes which cut the PCS genes differently you will able to determine which vector contains which gene Yeast Metal Tolerance Protocol PLATE THE YEAST STRAINS ON CADMIUM CONTAINING MEDIA This part of the experiment will be performed on Wednesday April 26 so that the yeast cells will grow sufficiently in order to assay them on the day of the experiment The yeast strain INV does not grow in media containing toxic levels of cadmium the untransformed yeast strain requires uracil in the media for growth Yeast cells transformed with AtPCS1 TaPCS1 and pYES allowing them to grow without uracil will be provided and growth in media with different cadmium concentrations will be tested Prior to this class the lab manager will prepare sterile plates supplemented with 0 50 M and 100 M cadmium Different strains will be plated in these media by using a sterile tooth pick and streaking out over the surface of the agar Each group will obtain 3 numbered yeast strains numbered 1 through 3 transformed with unidentified plant clones Each strain should be streaked on the 3 provided plates which should be divided into three sections one for each of the strains Streak and label the plates in such a way that you can identify which yeast strains grows on each plate Plates will be incubated at 30 C during 5 days and analyzed in the next experiment DAY 1 DETERMINE THE IDENTITY OF THE STRAINS BY RESTRICTION MAPPING AtPCS1 and TaPCS1 encode phytochelatin synthase genes from two different plants so their DNA sequence are slightly different These polymorphisms give rise to different restriction patterns which can be checked by DNA electrophoresis in agarose gels Using the enclosed restriction maps choose the appropriate enzymes to distinguish between the three different plasmids choose among HindIII BamHI or XbaI DNA should be submitted to digestion with restriction enzymes for one hour During the DNA digestion an agarose gel 1 in TBE buffer should be poured Digested DNA will be electrophoresed in the gel containing Ethidium bromide and DNA fragments will be visualized using UV light Different patterns of digestion should be observed DNA Digestion Yeast Metal Tolerance Protocol The lab manager will supply plasmid preps of the three plasmids used to construct the yeast strains streaked out the previous week Each group should take an aliquot of each plasmid to digest for analysis 1 Looking at the restriction maps provided of each of the plasmids select one of the provided enzymes Xba Hind III or BamHI that will allow you to differentiate between the three plasmids 2 Use 5 l of each DNA to digest with the appropriate enzyme and incubate the reaction at 37 degrees for 1 hour Digest mixture 20 l DNA 3 l 10X Buffer 6 l distilled Water 1 l Enzyme total volume 30 l 3 While digesting the DNA make up and pour a 1 0 agarose gel with ethidium bromide 4 Add 2 l Loading Buffer to the digests and load the complete digestion reaction or as much as possible on the gel and run it for 1 hour Place the gel under UV light and make a diagram of the DNA fragments you observe From this restriction pattern you should be able to determine the identities of each unknown plasmid Yeast Metal Tolerance Protocol INTERPRETATION OF THE GROWTH ASSAYS ON THE CADMIUM CONTAINING PLATES Examining the cadmium plates you streaked out during the last class and summarize the ability of the different strains to grow on the plates by using a table and including or for strong growth and or for weak or no growth growth strong growth weak growth no growth Media Cd M 0 50 100 Yeast Strain 1 2 3 In your final report please use your results from the restriction digests to determine which strains have been transformed with which construct and to interpret the growth of yeast strains on the media containing