Elijah H. Martin, Matt Rich, Stanley Fields
Isolation and characterization of ethanol-tolerant yeast strains
Biofuels derived from ethanol are used worldwide, driving research efforts to
increase ethanol production from sustainable sources, such as yeast. In the
yeast model Saccharomyces cerevisiae, increased ethanol levels are toxic and
can lead to cell death. The goal of this project is to isolate and characterize yeast
strains that are resistant to high levels of ethanol. We mutagenized the lab strain
BY4741 using ethyl methanesulfonate (EMS) and then cultured the population in
a non-permissive, high-ethanol environment. We sequenced the genomes of
ethanol-tolerant mutants in order to identify novel mutations linked to the
phenotype. We expect that we will be able to identify the specific mutations that
confer ethanol resistance. The expected results will not only provide new
industry-relevant strains but will also provide their genetic annotation, a
component that is commonly missing in yeast ethanol resistance studies.
EVOLUTIONARY PROTEIN PHOSPHORYLATION IN YEAST SPECIES
Lindsay Stanford, Kelsey Haas, Joanne Hsu, Danielle Swaney, Ph.D, Judit Villen, Ph.D
Protein phosphorylation is a key mechanism used in cell signaling. Once a protein is
phosphorylated, its function is modified and the signal that was originally being
transmitted is changed. Our study focuses on examining protein phosphorylation events
in 10 different yeast species. Our ultimate goal is to see if phosphorylation is
evolutionarily conserved throughout the yeast species. For our project, we obtained and
analyzed yeast peptides to identify proteins and then we enriched for phosphopeptides to
identify phosphorylation sites. Yeast peptides and phosphopeptides were detected via
mass spectrometry and analyzed using MaxQuant software. We believe that studying
protein phosphorylation in different yeast species provides insight to similar
phosphorylation events that may occur in humans. We expect to see that yeast species
sharing a common ancestry will have similar phosphorylation sites, whereas yeast species
that are not highly related will show different sites of phosphorylation. From the data
collected, we will be able to better understand protein phosphorylation cascades and
potentially apply our newfound knowledge to other biological topics.
Joseph DeAguero
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Related individuals share more alleles in common than unrelated individuals, and
population genetics theory predicts how much allele sharing is expected for
different types of relatives. To date, however, it has not been possible to
empirically test theoretical expectations on patterns of allele sharing across the
genomes of related individuals. Advances in DNA sequencing technology now
allow whole-genome sequences to be generated quickly and inexpensively. The
goal of this project is to determine relatedness between sib pairs in a large
pedigree. These sibling pairs have whole-genome sequences that have been
determined by analyzing patterns of identical-by-descent (IBD) across the
genomes of different pairs of siblings. We used PLINK to analyze our data and
sort individuals based on their proportion of alleles shared IBD, and the
programming language R to graphically display our results. We found that even
though siblings are supposed to share half of their genes from their parents,
there is significant variation among this value. We also see that in some cases
there are individuals who are more similar to each other than expected. These
data will provide accurate information about which regions in the genome are
shared between siblings, how much variation in allele sharing exists between
different sib pairs, and whether this variation could contribute to different
probabilities of disease risk. From these results, future projects will make
statistical conclusions on whether a disease which is common in all family
members in this region is caused by a particular part of a chromosome.
Kelsey Kaeding
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In Drosophila melanogaster, disruptions in the gene tramtrack have been shown to
cause stunted dorsal appendage morphogenesis, resulting in a mutant known as
twin peaks. Homologous genes in other organisms create tubes in similar ways,
allowing dorsal appendage formation to provide insight into processes such as
human neural tube growth. Mistakes in this development can lead to serious birth
defects such as spina bifida. Tramtrack produces a transcription factor, Tramtrack
69 that regulates a number of other genes. Genes differentially expressed between
twin peaks and the wild type have been identified, though their role in tube
morphogenesis has not been characterized. Four genes were examined through the
use of in situ hybridizations, and GAL4-UAS RNAi systems. Here we show where
each of the genes is present in the egg chambers, and in what quantities, as well as
the effects of disrupting their function on dorsal appendage development. Results
may range from no visible effect, to fully stunted dorsal appendages. Any visible
effect on the appendages would indicate that the gene in question does indeed play
a critical role in tube morphogenesis.
Bianca Garcia
This project focuses on the gram negative bacterium Salmonella enterica serovar
Typhimurim which causes gastroenteritis and infects animals and humans. In
order to defend itself from attack, by our immune system, Salmonella has a
couple of regulating systems that aide in its protection from the macrophages in
our body which are the PhoP-Q and Rcsf systems. These systems modify the
outer membrane in response to outer membrane damage by antimicrobial
peptides. However, the factors and genes involved in maintaining Salmonella’s
membrane integrity are uncharacterized and therefore unknown. This project is
looking to characterize these factors and find these essential genes in order to be
able manipulate the Salmonella’s ability to modify itself. Thus making it more
susceptible to the human immune system and antibiotics. To begin searching for
these factors the wild type Salmonella strain was mutated through transposon
mutagenesis. This mutagenesis allowed the strain to be mutated through
transduction by bacteriophage. A number of these bacteriophage contained
transposon insertions. Some colonies that came from this mutagenesis were
mutated due to the insertions and were then isolated from the rest of the colonies
through the use of beta-galactosidase markers. From this original mutagenesis
many other screens have been done on different types of media to narrow down
the colonies to select for those most affected by the mutations. Soon these
colonies will be sequenced in order to determine where these mutations are
located in the Salmonella chromosome and help us determine the genes that
responsible for their survival in our bodies.
Ashley DeAguero
Amplification of SUL1 in diploid yeast cells grown in sulfate-limited conditions.
Exposure to environmental pressures, such as a limited nutrient supply, selects
for rare events that give some cells a selective advantage. When grown in a
chemostatic environment that contains a limited amount of sulfate, the yeast
Saccharomyces cerevisiae is found to amplify copies of SUL1, a high affinity
sulfur transporter gene. Recent work suggests that, through a simple error in
replication, haploid strains produce inversions, duplications, and translocations of
regions that contain amplified copies of SUL1. The evolution of diploid strains,
however, has not been characterized. To better understand this process, as it
occurs in diploid yeast cells, regions thought to contain SUL1 amplicons will be
analyzed through arrayCGH, and further probed for the definitive presence of
SUL1 by restriction digests, Southern blots, and chromosome (CHEF) gels. We
expect to fully characterize diploid yeast strains, compare our findings to those
seen in haploid strains, and also establish a time point in evolution that clearly
defines when different amplification events occur. Overall, these genomic
rearrangements that occur as a result of environmental selective pressure may
provide insight into such events that occur in higher organisms and might lead to
the discovery of novel rearrangements never before seen in the yeast genome.