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Are you interested in cloning a gene? Dr. Quarmby’s lab at Emory University is trying to clone the gene for katanin. Katanin is a protein that requires an energy source called adenosine triphosphate (ATP) in order to severe or cut microtubules. Microtubules are present in cells and are comprised of tubulin proteins. Katanin is found in human cells and severs microtubules during cell division. The exact role katanin plays in mitosis is not yet known. Dr. Quarmby’s lab studied the biflagellate alga Chlamydomonas reinhardtii katanin.
C. reinhardtii is an excellent organism to study the role of katanin. Katanin is known to be located in the flagellar basal body complexes within C. reinhardtii. The flagellar basal body complexes are where the two flagella made of microtubule tubulin units originate in C. reinhardtii. The flagella extend from these complexes out of the C. reinhardtii organism into the environment. The flagella are comprised of microtubules in the same arrangement as in human cells. Therefore, the biochemical process of deflagellation and the role katanin plays in C. reinhardtii can give great insight into the role katanin plays in cell division in humans.
Cloning is a useful technique; it allows you to gain the DNA, deoxyribonucleic acid, sequence for the gene that encodes a protein of particular interest. In order to start to clone a gene, the DNA of the organism being studied must be obtained. Every cell in any living organism contains the organisms entire DNA sequence. Therefore, DNA can be isolate from one individual cell. This DNA is called genomic DNA because it contains all the DNA sequences of the organism. However, many organisms, especially humans, have a lot of DNA sequences within their DNA that are not used for anything. These DNA sequences that are just "extra" are called introns. For this reason, scientists use complementary DNA also known as cDNA. cDNA is copied from messenger ribonucleic acid (mRNA) and only contains the DNA that is used as "blue prints" for cellular functions.
From the cDNA, a library of all the different cDNA clones from that organism can be formed. First, the mRNA isolated from a cell can be copied into single stranded DNA using the complementary bases. This DNA is cDNA because it is lacking all of the introns. The single stranded DNA makes a complementary stand of DNA using the base pairs adenine (A), cytosine (C), guanine (G), and thymine (T). Note that A only binds to T and G only binds to C. Now, a double stranded cDNA molecule is formed. This double stranded DNA is then ligated or inserted into a phage. A phage acts as a host for the cDNA.
Dr. Quarmby's lab members used lambda phage to take up the cDNA. Lambda phage is a specific phage that have had most of their DNA extracted leaving only the essential parts needed for replication. This allows the phage to have extra room to take up the cDNA inserted experimentally. Next, these phage are exposed to E. coli which act as a reproducing mechanism to replicate the cDNA. The nutrient broth they are placed in overnight is optimum for the E. coli cells to divide and multiply. Therefore, when the E. coli bacteria replicates its own DNA, the cDNA inserted in to the cells is also replicated. The E. coli cells then are spread out on agar plates that contain nutrients for them to survive. This plate then contains all of the cDNA of the organism and will form plaques. Plaques represent individual clones from the original organism. A plaque contains all the E. coli with the same DNA.
This agar plate contains individual clones for every gene in C. reinhardtii. This is called a cDNA library of C. reinhardtii. Think of each plaque on the plate as a different book in a library. Each book is important but no one will ever have the time to search through every book to find the one particular book they want. Therefore, just as the library has a card catalog, molecular biologist use a radioactive DNA probe to search for the clone they want. Dr. Quarmby's lab used a radioactive probe made from a fragment of the cDNA known to be a piece of the katanin gene. Filters are then placed over the agar plates previously prepared. The DNA on the plates will bind to the filters. These filters will then represent every clone in the cDNA library. The cDNA probe will hybridize or bind to any spot on the filter that contains the complementary sequence to the probe.
Once a clone is identified, it can be isolated by picking it off the plate. Then the cDNA within the clone and be used in Polymerase Chain Reactions (PCR) to make more of the DNA. PCR is an excellent technique used to amplify a piece of DNA. The DNA obtained from PCR was sent for sequencing. These steps were followed for the katanin cDNA library and two clones were isolated by the Quarmby lab. In the future, they will try to figure out the exact biochemical pathway of deflagellation and the role that katanin plays in deflagellation to help cell biologists understand better the process of cell division in humans.
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