Drosophila melanogaster: the common fruit fly and the bane of human existence. Here in the Hiraizumi Lab, we make the best out of these pesky creatures by using them as a model system to investigate regulation of dipeptidase enzymes. But what is dipeptidase? Great question! Dipeptidase is an enzyme capable of breaking down small peptides into their amino acid components. By studying dipeptidase in D. melanogaster, we potentially can relate our findings to dipeptidase enzymatic activity in humans. Low levels of specific dipeptidase activity in humans has been associated with diseases such as Alzheimer’s Disease and Huntington’s Disease, just to name a few.
Our research focus is on the dipeptidase B gene (Dip-B) in D. melanogaster. Currently, we are using two different strains of D. melanogaster, NC25III and CL55, to study biochemical and molecular differences in DIP-B enzyme. The NC25III strain of D. melanogaster exhibits a nine-fold decrease in DIP-B enzymatic activity when compared to the CL55 strain. What causes this difference in DIP-B enzymatic activity between the two strains of D. melanogaster? We continue to address two possible explanations:
- The NC25III strain of D. melanogaster produces fewer number molecules of DIP-B enzyme than does the CL55 strain.
- The two strains of D. melanogaster produce the same quantity of DIP-B enzyme; however, the DIP-B enzyme of the NC25III strain of D. melanogaster has less catalytic activity than does the CL55 strain.
Former lab member, Rachel Wigmore, conducted western analysis the year prior to compare the relative quantity of DIP-B enzyme produced between the two strains of D. melanogaster. Western Analysis is a technique that utilises antibodies to detect specific protein of interest and their relative quantities in samples.
The quantity of protein present in each strain of D. melanogaster can be determined by the density of the protein bands (i.e., how dark the bands are). Looking at the Western blot, you can see that there is no apparent difference in the band intensity of the DIP-B enzyme between the CL55 strain of D. melanogaster and the NC25III strain. After standardizing the banding intensities using the 58kD band of the ladder, we conducted a two-way ANOVA on Rachel’s Western blot. We found no significant difference between the two strains in mean band intensity. This finding suggests that the two strains of D. melanogaster produce the same number of DIP-B enzyme; thus, the DIP-B protein of the NC25III strain of D. melanogaster appears to be less catalytically active than does the CL55 strain.
Rachel conducted her Western analysis using only male flies. We decided to expand on her study to see if there could be any difference in protein quantity between male and female D. melanogaster of the two strains. But how do you determine the sex of the flies? Another great question! Male D. melanogaster have prominent black dots, known as sex combs, on their front legs. Females do not have sex combs. Furthermore, females have an elongated abdomen (presence of ovipositor) when compared to males.
Our western analysis appeared very similar to Rachel’s, where there was no detectable difference in DIP-B enzyme between strains; furthermore, there appeared to be no detectable difference in DIP-B enzyme between the two sexes.
We conducted a three-way ANOVA to determine whether or not there were differences in band intensity. To do this, we put our computer science skills to use and wrote a program that could carry out such an analysis using the SAS Operational Quantification Tool. Our three-way ANOVA revealed that there was no significant difference in the quantity of DIP-B protein between different strains and different genders.
Another objective is the comparison of Dip-B gene sequence between CL55 and NC25III strains of D. melanogaster. Dip-B coding sequences (exons only) of both strains have been sequenced and can be compared against the National Center for Biotechnology Information (NCBI) to characterize any differences between the CL55 and NC25III strains. Differences in the coding sequence could be responsible for amino acid sequence differences associated with catalytic activity. In order to confirm the coding sequence data, we are replicating sequence analysis with more samples from each strain. Although we do have information about coding sequences, we still lack sequence information of introns and the 5’UTR. Previous lab members have designed forward and reverse primers for PCR to amplify the 5’UTR and the coding sequence of the CL55 and NC25III strains of D. melanogaster. The size of PCR products can be confirmed by gel electrophoresis.
Although the primers have been designed, the work on 5’UTR and intron sequences remains unfinished. During the summer we will be amplifying the 5’UTR of both strains from their genomic DNA and also produce cDNA from their mRNA. Once the sizes of PCR products of 5’ UTR and coding sequence are verified, the bands of correct size will be excised and extracted from the gel.
We will be designing new primers to sequence this 5’UTR and the entire Dip-B gene. The sequences of 5’UTR and coding sequence will be compared with each other to see if any difference exists. The sequence data can then be used to look for differences in coding sequence between the two strains as well as differences in signals for initiation of transcription. The amino acid sequence can be predicted based on coding sequences and the structure of protein can be predicted as well. These studies will help us to understand the biochemical and molecular basis of DIP-B enzyme activity differences.
Nicolas Stauffer (2020) & Yifei Duan (2020)