Good afternoon, everyone.
My name is Bea Middleton, and I'm
a senior and neuroscience major here at Mount Holyoke
Like Pho, I spent my summer studying proteins.
But instead of studying them computationally
with a computer, I was working in a wet lab--
so studying actual proteins in living cells.
I was a summer student fellow at Woods Hole Oceanographic
Institution, working in the Hahn Lab,
studying how animals sense and adapt
to the different chemicals in their environment.
And this ties in with my title, "When
Poisons Overtake Your Home--
How the Atlantic Killifish Adapted
Resistance to the Chemicals in its Environment.
So what are some of these chemicals or toxicants
that I'm talking about?
PCBs, or Polychlorinated Biphenyls,
are a toxic waste product of the electrical industry,
and they currently pollute New Bedford Harbor, Massachusetts.
Here, there's a population of killifish
that, over many generations, have developed a resistance
to these PCBs.
And this is a resistance that the fish of the clean reference
site, Scorton Creek, Massachusetts, do not have.
So how is this possible?
What makes the New Bedford Harbor population resistant?
This is our question.
The Hahn Lab believes it has a lot
to do with the Aryl Hydrocarbon Receptor,
or AHR, because this protein mediates the toxic response.
So in other words, it binds to the chemical, like a PCB,
and, in its now activated form, becomes a transcription factor.
So it goes into the nucleus, binds to the DNA,
and alters gene regulation, leading
to toxic effects on the cell.
But this still doesn't really answer our question
because if both the resistant and the non-resistant fish
have this protein, what makes them respond to the chemicals
differently?
Well, it turns out there's a lot of variation among AHRs.
There are actually four different AHR genes or types
of AHRs.
And for any given type, there's a number of variants,
structural variants.
Interestingly, two variants of AHR2a
are found more frequently among the resistant population
of New Bedford Harbor.
So that's the orange and the blue
there that I'm referring to.
And another two, the light pink and the light blue,
are found more frequently in the sensitive population
of Scorton Creek.
So this should made you wonder, is this structural difference
between the variants of the two different populations
underlying a functional difference
that could explain why one population's resistant
while the other one is sensitive?
And this ties in with my job for the summer, which
is to measure the activity of the different AHR
variants in response to treatment with a given
toxicant, like a PCB.
To do this, well, if you remember,
the AHR is a transcription factor.
So if I want to measure its activity,
I'm going to measure the amount of transcription that's
occurring in the cells.
To measure the amount of transcription,
I use a luciferase assay, which essentially lets me use light
as a proxy for AHR activity.
I'm going to walk you through some
of the basic molecular biology behind this technique
just so you can appreciate just how cool this technique is,
and it'll also give you a better picture
of what I was doing on a day-to-day basis in lab.
So I start out with a cell culture,
and I transpect with the AHR variant of interest,
where I put into the cell that protein that I'm interested in.
And along with it, I put in this piece of DNA
so that when the AHR is activated
when I treat with the toxicant, it actually binds to the DNA
and turns on transcription of this luciferase gene,
producing luciferase protein in the cell.