My name is Will Ueckermann, and I have spent the past nine weeks working in Dr. Hiraizumi’s lab. In our lab we work with Drosophila melanogaster, the common fruit fly. The comparatively small genome of D. melanogaster has been sequenced, which makes it a great genetic model organism. Our research is focused on dipeptidase-B (DIP-B), an enzyme that is important in the final stages of protein digestion. Dipeptidases are found in all organisms, from bacteria to humans, which makes it a good protein-gene model system for study. More specifically, we are studying the genetic basis for why two strains, NC25III and CL55, differ in DIP-B enzyme activity. NC25III has unusually low DIP-B activity, while CL55 has typical wild-type level of enzyme activity.
This is how we maintain most of our D. melanogaster cultures.
There are several explanations as to how these two strains differ in enzyme activity levels. One possibility is that they differ in either the composition or quantity of mRNA isoforms that contain the coding sequence for the DIP-B protein. There are four reported mRNA isoforms for the D. melanogaster Dip-B gene, and they are known to differ only in the 5’ untranslated region (5’ UTR) which exist before the coding sequence. Isoforms A and C share the same initiation site for transcription and differ in how their 5’ UTR is spliced. Isoforms B and D each have their own sites of transcription initiation that differ from that of isoforms A and C.
Why are there different mRNA isoforms when each codes for the same protein? What are the regulatory DNA sequences that potentially signal initiation of transcription? Are the different isoforms equally likely to be transcribed and expressed? I searched for as many reported promoter sequences for other D. melanogaster genes as I could find to see if they were present in the Dip-B mRNA isoforms. There are three main classes of DNA sequences in D. melanogaster that promote initiation of transcription: TATA boxes, downstream core promoter elements (DPEs), and initiators. After many hours of comparative sequence analysis using bioinformatics applications, I found that isoforms A and C had only an initiator sequence, isoform B had only a TATA box, and isoform D had only a DPE sequence. I do not know which, if any sequence, is the strongest signal for initiation of transcription, but it was interesting to find these differences.
Results from Mariesa Slaughter’s (class of ’12) experiments and work from this summer suggest that there are not major differences between CL55 and NC25III adult male D. melanogaster in composition of Dip-B mRNA isoforms or overall quantity of Dip-B mRNA. Because of this, we are now hypothesizing that the differences in DIP-B activity between the two strains may be the result of differences in the coding sequence for the protein. We are currently in the process of isolating the coding regions of each strain using RT-PCR of mRNA, PCR of genomic DNA, and gel electrophoresis, with the goal of having the exons sequenced. This has presented some challenges. The first set of primers were designed to make cDNA from the Dip-B mRNA isoforms then to amplify the entire coding sequence all at once. Unfortunately, the designs of the primers were not my best work (my bad) and it led to multiple cDNA products with no consistency between replicates. The next set of primers was designed to amplify segments of the coding sequence, which produced much more meaningful outcomes. Although the first set of primers produced predicted sizes of PCR products of the 5’ end of the coding sequence, the second set of primers that were designed to cover the 3’ end of the coding sequence resulted in many non-specific PCR amplicons. I then designed another set of primers with the objective of isolating smaller segments of the 3’ end of the gene and these will be tested before the end of my research internship. In parallel with the RT-PCR experiments, I have recently isolated genomic DNA from the two strains, which will be used for PCR experiments for comparison against the RT-PCR products.
One of the many electrophoretic agarose gels that I ran this summer.
Another ongoing project was stimulated by two mysterious RT-PCR products that were generated from Chelsea Loughner’s (class of ’15) experiment to detect Dip-B mRNA isoforms A and C. The largest RT-PCR product matched both genomic DNA and unspliced RNA in size. When RNA samples were treated with DNAse to digest any DNA contaminants, this large RT-PCR product could still be detected, which indicates that it is likely unspliced RNA, or pre-isoform A and pre-isoform C. However, the second-largest RT-PCR product did not correspond to any reported Drosophila gene in the entire NCBI database. We hypothesize that it may be an as-yet unidentified Dip-B mRNA isoform, which we are tentatively calling isoform E. It is also possible that this putative isoform E might have the same 5’ splice junction within the 5’ UTR as isoforms A and C. To determine this, we identified four possible sequences that have a splice junction in the area of the 5’ UTR sequence that match this second RT-PCR product in size. I then designed sets of primers that were specific to each predicted sequence. Two of the primer combinations generated detectable RT-PCR products, as well as some unintended byproducts. So, once again I have gone back to the drawing board to design better primers, which will be tested soon. Could we have discovered Dip-B mRNA isoform E? Only future experiments will tell.
One final ongoing project has been an attempt to determine just how many mRNA isoforms of Dip-B exist. The idea was to generate Dip-B cDNA from RNA samples with a reverse primer that binds to the beginning of the coding region. The first attempt was a beautiful failure (see gel image and you can be the judge). There was no detectable product at all in the entire gel (the bands in the gel belong exclusively to standard ladder markers used to compare size). We tried the experiment again, using a more robust reverse transcriptase and a historically successful protocol, and we managed to get “promising” smudges. It still does not provide a satisfactory answer to the question of the number of Dip-B mRNA isoforms, but at least the reaction worked. Future plans include the use of a different reverse primer for cDNA generation and different electrophoresis techniques to improve both separation and resolution of cDNA products.
Images of agarose electrophoretic gels to detect Dip-B mRNA isoforms. Left: What a gel should not look like. The very faint bands at the bottom of each lane are the primers. Right: The more successful gel, with detectable cDNA products.
Finally, I learned some Python with the initial objective of writing a computer program to generate a list of predicted Dip-B mRNA isoforms that incorporates the various sequences associated with initiation points for transcription. This would supplement Chelsea Loughner’s Python program that used splice junction rules to predict potential Dip-B mRNA isoforms. This line of in silico research will remain a future endeavor.