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Reading Guide - A Drosophila model for LRRK2-linked parkinsonism
Hello BIOL230W Students –
Your first big experiment in BIOL230W lab is isolating and analyzing a gene from Drosophila (fruit fly) that
may have an impact in human health. In science, we use model organism like Drosophila to understand genes
with conserved functions in humans. It’s much easier to manipulate genes in a fly than in a human being. This
paper examines one such gene that plays an important role in Parkinson disease. It provides a great entry point
into the next several weeks of lab.
In this reading guide, I will explain how I approach reading a paper for the first time. Some of you may be
experienced in reading primary literature and some of you may be new to the practice. Please use this guide as
you see fit for your level of reading. And remember that one of the goals in BIOL230W is to teach you the
skills to read and interpret scientific literature. This assignment is one assessment in that process.
One of the first things I note in papers are abbreviations. Scientists love abbreviations and they are often more
confusing that just using the complete words. Therefore, I will make a list of abbreviations so I can keep track
of information as I read the paper. Here is the list of abbreviations and corresponding notes I made while
reading the paper:
Abbreviations
1. LRRK2: leucine-rich repeat kinase gene (from humans)
2. LRRK2-G2019S: mutant form of the normal LRRK2 gene that leads to Parkinson symptoms. The
G2019S notation means that a normal G (glycine) amino acid is mutated to a S (serine) at the 2019
amino acid in the polypeptide chain.
3. CG5483: the ortholog (see below) of the LRRK2 gene in Drosophila (related gene)
4. PD: Parkinson disease
5. UAS: upstream activating sequence from yeast. Part of the GAL4/UAS system (I have no idea what
this is during the first read – see below)
6. DA neurons: dopaminergic neurons
7. L-DOPA: a chemical that is a precursor to dopamine
8. TH: tyrosine hydroxylase, an enzyme involved in dopamine synthesis that is used to mark DA neurons.
In addition to creating a list of abbreviations, it is more important to me to keep a list of things I don’t
understand and had to look up. Sometimes it is just a vocab term and other times it’s a technique or process.
When I read a paper, I have Google and/or Wikipedia close by to search for answers or quick fixes to gaps in
my knowledge. So, here’s that list:
Things I didn’t know (or forgot and had to refresh my memory)
1. Pleiomorphic – referring to multiple characteristics rather than just one.
2. Loss-of-function versus gain-of-function mutation: The mutation in both cases is referring to a change
in DNA sequence. A loss-of-function mutation causes a phenotype because the new protein (transcribed
and translated from the mutant form of DNA OR not able to be transcribed because of the mutation)
fails to work. A gain-of-function mutation causes a phenotype because the new protein (transcribed and
translated from the mutant form of DNA) has additional functions compared to the wildtype (or normal)
gene. In this paper, the LRRK2-G2019S mutation is a gain-of-function mutation that causes the new
protein to have increase kinase activity compared to normal. (I didn’t really forget about this, but its
really important that you understand this concept to understand the paper).
3. The GAL4/UAS system. Clearly this was an important technique in the paper, but I haven’t heard of it
(probably because I studied plants, not flies). So, I Googled it and found a great image explaining what
the system actually does. It’s a really interesting way of controlling the expression of genes in a tissue
that you care about studying – this way a scientist can target gene expression to specific tissues rather
than have it expressed throughout the whole organism. This is really helpful if the scientist is studying
certain neurons, as in those involved in Parkinson Disease
The system relies on two
transgenes that must be
introduced into the fly.
1. An enhancer linked to a
GAL4 (common transcription
factor from yeast) gene.
2. A gene of interest linked to
the UAS (promoter region) from
yeast that binds GAL4.
The GAL4 gene is expressed
first via control of the enhancer.
The GAL4 then binds the UAS
sequence and causes expression of the gene of interest. The cool twist is that the GAL4 gene is only transcribed
in tissue that has an activator protein that binds to the enhancer. Many years of research has accumulated
known enhancers and what tissue expresses the activators. Here is a list of all the transgenes used in this system
for this paper:
Transgene 1 - Enhancers:
 GMR-GAL4 for expression in photoreceptor (eye) cells as a model for degeneration.
 ddc-GAL4 for expression in DA (dopaminergic) neurons
 elav-GAL4 for expression in all neurons (panneuronal)
Transgene 2 - Gene of interest
 UAS:LRRK2 for expression of the normal human gene (stimulating gain-of-function). The scientists
tested two different “lines” or transgenic events to make sure that the process of making the transgene
wasn’t the cause of the phenotype, but rather the transgene itself (Lines 1 and 4). A noted inaccuracy is
that the authors start using UAS:WT:LRRK2 half way through the paper to signify wildype (normal),
but it’s the same at the UAS:LRRK2 (see - abbreviations are confusing)
 UAS:LRRK2-G2019S expression of the human mutant form that is already a gain-of-function =
increased kinase activity (Lines 2 and 3).
 Drosophila with only the enhancer (as listed above) OR only the gene of interest (as listed immediately
above) are used as control. A fly only expressing the enhancer results in a GAL4 protein that has
nothing to bind to because GAL4 is a yeast transcription factor. And a gene of interest with a UAS
promoter region ONLY (no GAL4 present) will not express the gene. The final result in both controls in
NO EXPRESSION of the gene on interest. Hence, a control for expression.
Back to the list of things I didn’t know…
4. N-terminal FLAG tag: a known protein that can be added to a gene of interest to test for expression of
the gene via protein antibodies (proteins that bind other proteins, typically very specific). A western blot
is similar to a gel electrophoresis to examine amount of protein instead of PCR product. Antibodies help
visual the protein, like how you’d use ethidium bromide to visualize DNA.
5. Eclosion: emergence of an adult insect from a pupal case or larvae from an egg
6. Anti-TH staining: using antibodies that bind to TH to identify DA neurons.
7. Ortholog: I sometimes get orthlog, paralog, homolog confused so it’s good to refresh. Ortholog and
paralogs are both examples of homologs. An ortholog is a related gene (from a common ancestor) in
different species. So Drosophila gene CG5483 is the ortholog of human LRRK2.
8. I didn’t really know any of the physiology associated with the Drosophila (ommatidia eye structure,
neuronal structure of the brain), so I relied on the information provided in the paper and only worried
about the big-picture results. For example: “retinal degeneration was detectable after 3 weeks”. I don’t
really need to know all the Drosophila details to understand the final result.
Tips while you read
1. I always read the abstract and the introduction first (makes sense because they’re first). If there are big
concepts or vocab I don’t know from those sections, I will make sure to look them up right away.
2. As I read the results sections, I will always take time to read the figure captions carefully and make sure
I understand what’s going on in the figure. It can be really helpful to have a hardcopy of the paper to jot
down notes next to images or remind yourself of abbreviations.
3. Sometimes I don’t understand all the techniques used, but the technical understanding isn’t always
needed to get the big picture results. A lot of times, I won’t sweat the details of the methods as long as I
can understand what the results are. An exception to that is a process (like the GAL4 – UAS system)
that I did have to understand to make sense of the results.
4. If you read the discussion and find that you are lost, you should probably go back to the results and
make sure you understand things.
5. You might want to consider going through the paper with a study group so you can discuss the figures
and techniques.
Well, I hope that covering some of these topics helps you understand this paper about an important Drosophila
model for Parkinson Disease. Please give yourself enough time to read the paper and fill out the Sci Lit
Analysis #1 template. It definitely took me an hour or two to get through and fully understand. This is
replacing your Week 3 lab, so it is fair to think the assignment will take about 3 hours with the help of the this
reading guide. It’s definitely not something you can get through in 20 minutes before lab.
Thanks and have a good Week 3!
Dr. Malcos
An example of the
Drosophilia climbing
assay. Healthy flies in
tube 1 climb up to the
cotton plug. The flies in
tube 2 and 3 express a
mutant protein that affects
locomotion (tested at two
time points: 30 and 35
days), similar to
overexpression of LRRK2
or LRRK2 - G2019S in
the paper for lab.