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Graduate Research Project
I have not always thought of myself as a scientist, but nevertheless, I finished the requirements for a Master of Science degree in Plant Biology at Arizona State University in December 2003. My research project was the molecular study of arc2, a mutant line of Arabidopsis thaliana. I compared specific attributes of this mutant line, arc2, with its background ecotype (Landsberg erecta wildtype) along with comparison between a T-DNA knockout mutant line with its background ecotype (Columbia ecotype). The arc2 line was generated through EMS application, which results in point mutations in DNA. Arc2 was generated along side several other lines of plants, which were then selected in such a way that the mutation was located in the nuclear DNA (not chloroplast DNA) and there was just one point in the DNA that was affected. These EMS lines that were generated along with arc2 were classified as chloroplast mutants. These lines were examined in regards to their chloroplast volume, shape, and thylakoid stacking. What is interesting about this is that chloroplasts are thought to have evolved from an endosymbiotic event (one cell swallowed another cell whole). This means that although the chloroplast genome (DNA) has been reduced over the years, but it originally could do everything it needed to do on its own. Many of the genes that have been lost by the chloroplast have been found in the nuclear genome of the cell (DNA head quarters.) Scientists are interested in examining the molecular interactions between nuclear DNA (transcription, translation and targeting) and the chloroplast because the chloroplast is stuck between being independent of and dependent on cellular processes. Questions that scientists want to answer are along the lines of the following;
Personally, I like talking about chloroplasts a lot, for many reasons. To name just a few;
While my research did not solve any of the larger questions about chloroplasts, it did eliminate an avenue of research. The objectives of my research were the following: to quantitate relative transcript level of arc2 in regards to the putative gene, sequence the putative 2 Kb gene in arc2, and compare chloroplast morphology between a T-DNA knockout and arc2. I performed reverse transcriptase real-time polymerase chain reaction (Real Time RT-PCR) to relatively quantitate the transcript level of the putative gene in two background ecotypes and the mutant line. I sequenced the putative gene (1-8 fold coverage) in the same lines mentioned above. Finally, I examined chloroplast morphology, specifically thylakoid stacking, using transmission electron microscopy. To see my data, you can click here. |
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Definitions:
Arc2 stands for accumulation and replication of chloroplasts |
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Arabidopsis thaliana is a "model organism" used in plant biology. It is a model organism because the genome has been sequenced,
it is relatively short lived, gives rise to many seeds, and there are many, many, many researchers working on it, so resources
on the species are huge. Back to top Back to Graduate Research Project |
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T-DNA is short for Ti plasmid. This is where a plasmid (extrachromosomal DNA) found in a plant specific bacterium is engineered
in such a way as to integrate its DNA into the plant DNA. If the integrated DNA stays, and the plant line is breed over several
generations, the line is said to be a T-DNA line. The theory is that when genes are interrupted, this leads to a discernable
phenotype (what the plant looks like). This should indicate what the gene is responsible for doing when it isn't interrupted.
This process is called "reverse genetics." Back to top Back to Graduate Research Project |
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Point mutation refers to just one nucleotide being wrong in a sequence of nucleotides. DNA is a long line of nucleotides strung
together. The sequence that these nucleotides are put together create a gene (or transcription unit). Most of the time point
mutations do nothing to the organism, but sometimes something is changed. This is really fundamentally how evolution occurs, but
in the case of these Arabidopsis plants, it's mainly to see if phenotypes can change because of a single change in the
sequence. Back to top Back to Graduate Research Project |
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EMS stands for ethyl methyl sulfonate. It is a chemical that favors single point transitions.
It changes G to C and A to T. Back to top Back to Graduate Research Project |
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The EMS mutants with funky chloroplasts were identified, classified,
and studied.
The original scientists involved in the classification of this specific mutant class were;
K.A. Pyke,
R.M. Leech,
J.L. Marrison,
E.J. Robertson, and
S.M. Rutherford.
Click on the names for popular articles in PubMed citation index. Back to top Back to Graduate Research Project |
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Endosymbiosis is also called endocytobiosis . It is the theory that explains the evolutionary process by which double membrane bound
organelles came to be within eukaryote cells. It describes the engulfing of one cell by another, the adaptation of both cells to the
new enviroment, and the process by which both formerly independent organisms become symbiotic. Back to top Back to Graduate Research Project |
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There are multiple chloroplasts in a single plant cell. Each of these small, double membrane bound organelles, have their
own DNA. Chloroplasts orginate from a bacteria-like organism, so
chloroplasts have circular DNA, unlike many eukaryotes. All of the DNA in
the organelle is called the "genome". The chloroplast genome codes for
between 60-200 proteins, with the majority of them
involved in photosynthesis.
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The nuclear genome of Arabidopsis is fairly large and largely duplicative. This duplication leads many scientists to dismiss
the plant as a good model organism. There are 5 chromosomes, with a total of 12 megabase pairs
(12 million As, Ts, Gs, & Cs).
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How a chloroplast is created continues to be heavily researched, but it is generally agreed upon that chloroplasts arise from
an existing undifferentiated (not developed) plastid. Plastids are just double membrane bound organells without much function until
they are differentiated, in other words, given purpose in life. The inside membrane of the plastid begins to fold in on itself in
much the same way a mitochondrion inner membrane folds, but the inner membrane of the chloroplast eventually pinches off from itself,
creating "sacs." These sacs are stacked on top of one another to create thylakoid stacks. This becomes the really important part
of the chloroplast because all of the photosynthetic proteins are located there.
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Photosynthesis is:
H2O + CO2 → C6H12O6
+ O2 We all know that essentially, photosynthesis is the conversion of light energy into chemical energy. That is to say, the light of the sun is converted (using proteins in the thylakoid membrane) into energy molecules that facilitate the linking of carbon molecules into a stored energy molecule (starch) to be used later on for "food". The reaction of photosynthesis is subdivided into two main reactions; the light reaction (or Hill reaction) and the Calvin cycle (or dark reaction.) The light reaction requires light energy and is responsible for the generation of the oxygen molecule and energy molecules. The energy molecules are required by the Calvin cycle to "fix" carbon (in the form of carbon dioxide.) The Calvin cycle is subdivided into three components; fixation (carbon dioxide is captured by an enzyme), reduction (carbon dioxide is converted into a larger molecule, a sugar, using the energy from the light reaction) and regeneration (the enzyme that initially captures carbon dioxide is set up to receive more carbon dioxide). Back to top Back to Graduate Research Project |
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To distinguish ribosomes (which occur in eukaryotes and prokaryotes)
from other organelles (which occur only in eukaryotes), it is convention to say, "double membrane bound" organelles and "single membrane
bound" organelles. |
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My Research History: I never felt anything either way about Biology growing up. I enjoyed Mr. Greenhalgh's Biology class, while I hated Mr. Taysom's Chemistry class. I liked both the teachers though. I loved Humanities from day one. In high school, I took Chemistry at ASU, took the AP exam and received no credit for the course. I did not take any additional English classes, but took the AP exam and tested will into the 300's for college credit. Go figure. I worked as a high school intern at the USDA Western Cotton Research Laboratory in Phoenix, but entered college as a English Literature Major. Half way through college, I took a class in Plant Biology that challenged me academically beyond all other classes. The experience defined science for me; we know so much and yet, we know nothing. I changed my major to Plant Biology, but kept the Humanities minor. Joining Peace Corps, left me out of the scientific loop for a couple of years, but I got to read so many books! When I got back, I worked at a bookstore (the reading) and as a laboratory technician (the science), eventually deciding to go back to school. I finished with a Master of Science degree in Plant Science. Just goes to show you that you never can tell where you'll end up being in the end. Back to top |