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hello my name is avery fulford and i

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recently graduated with a bachelor of

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science from duke university

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i'm currently working at nasa with dr

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heather graham before i apply to phd

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programs in microbiology this fall

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today i'll be discussing my senior

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thesis entitled

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a comparative genomic analysis of the

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250

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million year old halophilic archaeon

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halo simplex carlsbadency with related

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species

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my mentor for this project is dr amy

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schmidt

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so in the year 2000 microbiologist

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vreeland at all extracted salt crystals

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from a halite formation

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deep underground in new mexico these

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salt crystals contained pockets of fluid

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which are believed to be

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the rare liquid remnants of a permian

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ocean which evaporated 250 million years

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ago

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as seen in figure 1 these halite

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crystals are pictured

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and also the fluid inclusions are marked

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with the letter i

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using extensive methods to prevent

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sample contamination

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vreeland at all swabbed petri dishes

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with these brines

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and found that four microorganisms which

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were living within the brines were able

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to grow and form colonies

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because the microbes lived in brines

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enclosed in salt crystals for these 250

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million years

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they are quite possibly the longest

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living organisms study to date

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while there has been some controversy

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surrounding one of the bacterial samples

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called sample 293 another microbe of the

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four extracted has been widely accepted

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as a new species and genus named halo

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simplex carlsbadency

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this organism is a halophilic archaeon

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so to define these terms a halophila is

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an organism adapted to highly saline

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conditions

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and an archaeon is a microorganism in

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the third domain of life

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the archaea as opposed to the bacteria

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or the eukaryota

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the overall goal of this study was to

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identify the physiological adaptations

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that enabled h-crossbarency

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to survive in the harsh environment of

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the salt crystal

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over 250 million years

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we achieved this by aligning its genome

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to the genomes of

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17 other halophiles to identify

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homologous genes

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defined as genes inherited by two

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species from their common

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homologous genes that are highly similar

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between two different species

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are said to be highly conserved which

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suggests that they encode an

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to move on to a description of our

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methodology we used several

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computational tools

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to align and compare the genome of h

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cosby density

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to the genomes of three groups of

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organisms

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the first comparison group included two

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recently discovered species within the

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halo simplex genus

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h rubrum and h pelagicum the second

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comparison group was composed of the 13

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most closely related species of archaea

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to h crossbedensi which were identified

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in a phylogenetic tree created by becker

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at all in 2014

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this tree is replicated at left in

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figure 2 in which you can see h cars by

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density highlighted in yellow

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each node or fork in the branches

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represents the last common ancestor

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shared by

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two groups of species before they

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diverged confidence values for each node

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are written below

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these are called bootstrap values which

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indicate how many times out of 100

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repetitions

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the same node was recorded when

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repeating the phylogenetic construction

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on a re-sampled set of the same data

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the third comparison group we chose

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included sample 293

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a bacterium which is another of the four

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samples extracted by relin

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at all in 2000 we assembled its genome

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from short nucleotide sequences

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using spades software

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okay for my results section i'm first

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going to talk about the genomic

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alignment and comparison

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of h cos badensi and the two other halo

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simplex species

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in this alignment we found that there

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was one long homologous sequence which

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was highly conserved among all three

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halo simplex species

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this was much longer than the other

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homologous sequences identified

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this long homologous region encodes 70

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labeled genes of known function

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as well as 93 hypothetical genes whose

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function has not been categorized yet

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we grouped the 70 labeled genes into

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categories based on gene function

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and compared the number of genes in each

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functional group

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in the longest homologous region between

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halo simplex species

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to the number of genes in each

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functional group in the entire h

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carlsbad c genome this can be seen in

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figure 3.

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in other words the green bars represent

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the percentage of all genes in the h

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cosmetic genome which are involved

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in stress response nucleotides so

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nucleotide synthesis dna repair and

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sometimes dna replication etc

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and membrane transport the blue bars

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represent the percentage of all genes in

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the longest homologous region among halo

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simplex species

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which are involved in the same three

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categories

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while there are many more variables we

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studied i've included the three with

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the greatest difference between the

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percentage of genes involved in each

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functional category

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in the entire halo simplex genome and

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the longest homologous region

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to summarize figure 2 suggests that the

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longest homologous region between the

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halo simplex species

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is enriched in stress response

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nucleotides

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and membrane transport mechanisms which

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could mean that these functions are

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important for survival

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in hypersaline conditions this is

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because these organisms are highly

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now i'm going to address the second

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genomic alignment and comparison we

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performed

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between h carlsbademc and its 13 most

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closely related species of halophilic

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archaea

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this alignment pinpointed 32 homologous

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genes within 20 clusters

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which encode highly conserved proteins

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involved in oxidative stress response

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dna replication and respiration

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oxidative stress is caused by

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hypersaline conditions

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it's defined as a disturbance in the

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balance between the production of

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reactive

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oxygen radicals and the cell's

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antioxidant defenses

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now for the sake of time i'm not going

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to elaborate on all 32 genes involved in

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this alignment

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however two interesting genes are long b

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and sophie because of their function

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which is highly specialized in

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halophilic archaea

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lawn b removes misfolded proteins from

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cells

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which are damaged in hypersaline

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conditions due to

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oxidative stress suffie repairs

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cellular iron sulfur clusters which are

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also damaged by the oxidative stress of

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highly saline brines

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these iron sulfur clusters are

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incredibly important for the cell

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because they are required for the

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function of proteins involved in a wide

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range of activities

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such as electron transport in

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respiratory chain complexes

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regulatory sensing and most importantly

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for this

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analysis dna repair

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finally the third genomic comparison we

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performed was between

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hrasbadensi and bacterial sample 293

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which again was another of the four

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ancient samples extracted

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from salt crystals by relent at all in

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2000.

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surprisingly we found a 2545 base pair

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long

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homologous region between the genomes as

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well as seven

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isolated homologous genes this is a very

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high number of homologous regions for

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two species from separate domains of

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life

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the archaea and the bacteria this is

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suggestive of horizontal gene transfer

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which is the transfer of dna from one

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organism to another

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without inheritance from a shared parent

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it has been demonstrated before in the

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literature that hypersaline environments

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make some species more likely

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to accept dna from other organisms even

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if that organism is from another domain

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of life

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this is because of the broad scale

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competency exhibited by stressed

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cells the homologous genes between h

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crossbodency and sample 293 are

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represented

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though not to scale in figure four the

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blue lines in this figure represent the

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genomes of the two species

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and the colored boxes indicate

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homologous regions which are connected

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between the genomes by a line of the

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same color

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some homologous genes of interest are

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the five dna repair proteins in one

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cluster

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as shown in the teal box there's also

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the abc

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transporter glts and duf domain protein

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which all encode inter membrane

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transporters

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there's also ace f and pep f genes

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in conclusion gene conservation across

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this halo simplex clade

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closely related halophilic archaea and

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the halophilic sample 293

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suggests that dna repair membrane

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transport

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and oxidative stress response mechanisms

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are required

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for the survival of organisms entrapped

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in hypersaline

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conditions over long periods of time

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dna repair for instance can combat the

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damage that hypersailing conditions

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wreak on dna

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particularly through the rearrangement

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of bonds in adjacent pyrimidines

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membrane transport proteins such as

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potassium and sodium transporters

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protect against salty hypertonic

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conditions which can cause

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a cell to shrivel up and die through

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desiccation

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finally oxidative stress response

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mechanisms prevent protein and membrane

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we've identified three important avenues

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for future research on this topic

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the first is the identification of

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genomic sequences in h carlsbadency

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that did not align to related species

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because genes that are unique to this

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ancient organism

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could encode novel physiological traits

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future research could also study two

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hypothetical proteins that we identified

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on in our alignment of the 14 closely

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related species of halophilic archaea

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it is rare for a hypothetical protein to

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be so highly conserved amongst a large

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group

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which suggests that these proteins could

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encode an important physiological

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function

295
00:10:26,550 --> 00:10:24,640
that is not yet characterized finally

296
00:10:29,110 --> 00:10:26,560
future researchers could investigate the

297
00:10:32,150 --> 00:10:29,120
possibility of horizontal gene transfer

298
00:10:34,949 --> 00:10:32,160
between h crossbodency and sample 293

299
00:10:36,550 --> 00:10:34,959
using two methods of species specific

300
00:10:39,509 --> 00:10:36,560
genomic metrics

301
00:10:40,069 --> 00:10:39,519
these are species-specific codon use and

302
00:10:43,350 --> 00:10:40,079
gc

303
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to conclude i'd like to thank dr amy

304
00:10:50,389 --> 00:10:48,399
schmid and riley hackley my advisors

305
00:10:52,389 --> 00:10:50,399
as well as my honors thesis faculty

306
00:10:53,990 --> 00:10:52,399
reader dr tom mitchell olds

307
00:10:56,550 --> 00:10:54,000
for their invaluable feedback and

308
00:10:57,350 --> 00:10:56,560
support i'd also like to thank dr lauren

309
00:10:59,350 --> 00:10:57,360
luger

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00:11:02,710 --> 00:10:59,360
and joey prinz and thank you for

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if you have any questions feel free to

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email me