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A metabolic deficiency that occurs in roughly one out of every 45,000 newborns is transferase deficiency galactosemia, or “Classis Galactosemia.” This deficiency has been modeled in Saccharomyces cerevisiae by knocking out the gal7 gene which encodes for the yeast transferase. By mutagenizing yeast which are gal7-, it is possible to generate colonies that are able to thrive on galactose-containing media, suggesting that there are other key proteins involved in galactose sensitivity that have not yet been identified. Out of these colonies, there are strains in which these traits were previously shown to affect a single gene locus. The Gal-GalR nature of this strain, JFy4238, was confirmed by plating the yeast onto SGE complete, SGE+0.02% gal, and Sgal media. Through the use of nutritional markers, these yeast were mated with wild-type and gal7-GalSGal- strains to demonstrate the recessive nature of this trait. The yeast strain JFy4238 was further characterized through the use of enzyme activity assays.
The Leloir pathway enzymes include kinase, transferase, and epimerase. These enzymes metabolize galactose into UDP-glucose as shown in Figure 1, which is used for cell glycosylation reactions. This metabolic pathway has been conserved from bacteria to humans. The fact that yeast is eukaryotic and easily manipulated genetically allows them to be the ideal model in which to observe the effects of galactosemia.
Classic galactosemia, which was first diagnosed in 1917, is characterized by the inability to metabolize galactose due to a deficiency in the activity of the enzyme galactose-1-phosphate uridylyltransferase, thus causing an accumulation of galactose-1-phosphate. Since galactose is a component of breast milk, it is important to diagnose this disease in the newborn. Most industrialized countries currently perform newborn screenings for galactosemia.
Once a patient is determined to have this disease, they are placed on a galactose-restricted diet. Even though this restricted diet essentially eliminates the lethality of the disease, long term complications such as learning disabilities and ovarian failure in females may potentially develop. Since the mechanism for galactose toxicity is unknown, there are currently no therapeutic interventions other than dietary restriction.
Yeast Strains
All media involved in this experiment was synthetic media composed of yeast nitrogenous base, ammonium sulfate, 2% glycerol and 2% ethanol. For plates there was an addition of bacto-agar to allow solidifying of the media.
In order to confirm galactose-resistant phenotype, a random colony was picked and streaked onto plates as shown in Figure 2. To confirm the recessive nature of the galactose-resistant phenotype, JFy4238 (which is mating type alpha) was transformed so that it was autotrophic for the amino acid tryptophan. Subsequently, JFy4238 was mated with a wild-type strain and a gal7- strain, both of which were mating type A and autotrophic for uracil. Diploids were then picked and grown in SGE-trp-ura liquid media and plated as shown in Figures 3 and 4.
Enzyme Assays
Yeast was grown in 5 mL of SGE complete liquid media overnight at 30°C. They were then diluted to an optical density at 600 nm (OD600) of 0.1 and incubated for an hour before the addition of galactose. The yeast was then grown to an OD600 of 1.0, pelleted and lysed in Johnston Buffer containing proteinase inhibitors. Serial dilutions of the proteins were then made and enzyme activity assays were performed at each protein concentration to find linear range.
As shown in Figure 2, JFy4238 grew on SGE and SGE+galactose but not on Sgalactose. From Figure 3 it can be seen that the WT/JFy4238 diploid grew on all media. The GalS/JFy4238 grew only on SGE media as shown in Figure 4. The percent conversions for each of the Leloir enzymes from the wild-type strain are shown in Figure 5 and can be seen to increase roughly logarithmically. The linear range for the Leloir enzymes is shown to be between 0 and 30% conversion.
Since JFy4238 was able to grow on media which contained galactose but not on media where galactose was the sole carbon source, JFy4238 is indeed galactose resistant. Also, as shown in Figures 3 and 4, the phenotype of the diploid formed between JFy4238 and another haploid is determined by the other haploid and not JFy4238, which supports the recessive nature of the mutation.
The linear range determination shows that the ideal amount of protein to use in activity assays is 0.02 μg for epimerase, 0.2 μg for kinase and 0.25 μg for transferase. Activity assays for these enzymes are currently underway for the remaining strains.
Further experiments planned for this particular strain include verification of the gal7- genotype through PCR amplification and HPLC analysis of galactose metabolites. Also growth curves will be determined to find how JFy4238 grows in galactose containing media with higher precision instead of simple growth vs. no growth.
Once this strain has been fully characterized, there will be an effort to find what gene locus is affected by the mutation leading to the galactose-resistant phenotype.
The authors would like to acknowledge all of those special individuals without whom this project could never have been possible. Thanks to Kim Openo, Jewels Chhay, Kerry Ross, Jane Mumma, Jamie Wasilenko, Rebecca Sanders, Melissa Brykailo and Weining Tang.
This material is based upon work supported by the Howard Hughes Medical Institute under Grant No.52003727 as well as funds from the National Institutes of Health.
There is a disease in humans called galactosemia which causes people to get sick and possibly die if they consume galactose, which is in many different types of food including dairy products. This disease can also occur in yeast that has been altered so that it cannot digest galactose. This lab has found a yeast which cannot digest galactose but does not get sick. My research is trying to find out why this yeast is able to still live even though galactose is present.
HPLC analysis of metabolites, Yeast transformation, extraction of genomic DNA from yeast, enzyme assays, growth curves
metabolic disorder, nutrition, galactosemia, enzymatic disorders, yeast genetics
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