Kara I. Ludlow and Dr. Randy Bennett, Zoology
My original proposal was to clone the extradenticle gene in Tribolium castaneum (red flour beetle); however, I worked in conjunction with Laura Newell who received an ORCA grant to work with Dr. Bennett cloning the Ultrabithorax gene of the red flour beetle. To further previous research, it was determined that the Ultrabithorax gene needed to be cloned first, so Laura and I worked on that project together. Due to complications and unexpected delays, we were only able to finish cloning the Ultrabithorax gene of the beetle and did not have time to clone the extradenticle gene, as well.
The genetic properties of the Drosophila fly have been thoroughly analyzed, making it a model for studies of all higher organisms. This analysis has immensely increased our understanding of genetics and the regulation of genes. However, generalizations of genetic patterns in all species cannot be made based on the study of only one organism. The genome of the red flour beetle has been mapped and cDNA libraries have been made. This makes it an excellent subject for research since much has already been discovered, and the cDNA library allows easy access to desired portions of the beetle genome for study. Tribolium DNA has also been found to have many homologies to the Drosophila genome.
In DNA, there are some genes, called homeotic genes, which act to regulate the activity of other genes. Ultrabithorax is a homeotic gene and when the sequence of Ultrabithorax is compared between Drosophila and Tribolium, slight variations are found. These differences may contribute to different patterns of gene activity in these species. To discover how Ultrabithorax controls the activity levels of other genes, we needed to clone the sequence of Ultrabithorax so that controlled mutations may be introduced into the beetle DNA. Once we had altered the sequences we would introduce them into live adult beetles. By then observing the various phenotypes of the beetle offspring, from the mutated groups, many of the genes under the control of Ultrabithorax can be found.
To clone the Ultrabithorax gene, we first collected beetle embryos and ground them under liquid nitrogen. This helped fracture the cells with little damage to the DNA and RNA. We then separated the RNA from all other cell components by various centrifugation techniques. Once we obtained pure RNA, we began making DNA copies of the beetle RNA. We did this two times to obtain a complete, double-stranded DNA copy of the RNA from the beetle embryos. This gave us a DNA set of all the active genes in the red flour beetles since only active genes are copied from the genome to be RNA.
To isolate and amplify only the Ultrabithorax gene from our DNA copies, we ordered primers designed to bind to the middle and each end of the Ultrabithorax gene. We then added polymerases, which make DNA copies, that bound to our primers and copied the Ultrabithorax gene. By performing repeated rounds of heating and cooling with our mixture of primers, DNA, and polymerases (this process is called PCR – polymerase chain reaction), we were able to significantly increase the number of copies of the Ultrabithorax gene in proportion to the other DNA. We verified that we had copied the desired gene by performing gel electrophoresis. The DNA moves in the gel according to its size, so by running size markers along with our sample, we were able to determine the approximate size of our DNA. Knowing how large the Ultrabithorax gene is (since the genome is sequenced), we knew if we had actually amplified our gene.
The final step was to bind the Ultrabithorax gene into a vector, which is a piece of circular DNA. We ordered vectors along with ligators and endonucleases. The endonucleases allowed us to cut open the circular DNA at a specified site. Once the Ultrabithorax gene was then inserted, the ligators bound the ends together to form a complete circle again. We had then cloned Ultrabithorax. To insure that we had, actually, cloned the desired gene, we had it sequenced by a DNA sequencing center.
After the project was completed, Dr. Bennett took the clones to Wisconsin to initiate experiments (explained earlier) to determine what genes are under the control of Ultrabithorax and further discover its role in Tribolium castaneum.
The research experience and knowledge I gained working on this ORCA project was very helpful while I was a teaching assistant for a molecular biology course at BYU. It was also instrume