by Dalia Perez
This work was done by LaGuardia student Dalia Perez under the supervision of Dr. Claudette Davis, from the Natural Sciences department of LaGuardia Community College. The research was supported by the NIH Bridges Program.
Dalia Parez
Dalia Perez is a hardworking, passionate student with a strong interest in science. She earned an A.S. in Biology from LaGuardia Community College and is now pursuing a B.S. in Human Biology at Hunter College. Through the NIH Bridges Program at LaGuardia, she investigated the Drosophila melanogaster genes CG9222 and CG14305 under the guidance of Dr. Claudette Davis, and she presented her findings at the 2024 Annual Biomedical Research Conference for Minority Students (ABRCMS). A dedicated pre-dental student, Dalia also volunteers at a dental clinic that serves underserved New York communities. She is committed to becoming a dentist and using her skills to improve lives.
Dr. Claudette Davis
Dr. Claudette Davis is an Associate Professor in the Natural Sciences Department. She received her Ph.D. in Cell and Molecular Biology from the Graduate School & University Center, where she studied cellular senescence and apoptosis. Her post-doctoral training occurred at Howard University, where she studied the genetics of aging using Drosophila melanogaster. She began teaching at LaGuardia in Spring 2018. She currently serves as co-Director of the Biology Program and teaches Genetics, Cell Biology, and Anatomy and Physiology. In addition to STEM education research, she is interested in exploring reproduction using Drosophila melanogaster.
Geneticists use Drosophila melanogaster, the fruit fly, as a model organism. Using fruit flies to study gene function has several advantages, including their short developmental cycle, simple body system, and large numbers of progeny produced. Gene expression can be regulated by creating transgenic flies that express the UAS-GAL4 system. We present data on uncharacterized fruit fly genes CG14305 and CG9222, which contain a serine/threonine kinase and are predicted to play a role in protein phosphorylation and signal transduction. Interestingly, the gene is highly expressed in the male germline of fruit flies. This study explores whether the two genes can be used to study male reproduction and neurological behavior. Using the NCBI-BLAST program, we compared the protein sequences of CG14305 CG9222 with four different members (older and younger) of the Drosophila family. We also compared CG14305 and CG9222 with the protein sequence of human TSSK2,and TSSK4, respectively.
The fruit fly is a valuable model organism in genetic research, making it particularly well suited for studying gene function related to fertility and reproductive biology (Baenas & Wagner, 2019). Serine/threonine kinases are vital to numerous cellular processes, playing a crucial role in signaling pathways by attaching phosphate groups to substrates through a process called phosphorylation. This modification regulates a variety of cellular functions, including apoptosis, cell division, and migration (Ardito et al., 2017). In this study, we focus on two uncharacterized Drosophila genes, CG14305 and CG9222, both of which encode serine/threonine kinases. Given the critical roles of kinases, understanding the functions of these genes may offer insights into their potential involvement in male infertility and on their role in fruit fly behavior. To investigate the evolutionary and functional aspects of CG14305 and CG9222, we utilized the NCBI-BLAST program to compare their protein sequences with those of closely related Drosophila species, including D. simulans and D. sechellia, as well as more distantly related species, D. virilis and D. grimshawi. This comparative analysis aims to identify conserved functional domains, including the ATP binding site, substrate binding site, and activation loop, which are critical for kinase activity. Afterward, we compare CG14305 with human TSSK2 and CG9222 with human TSSK4 to determine if these domains are conserved across species. Investigating the molecular mechanisms associated with TSSK2 and TSSK4, along with their interactions with other proteins, may shed light on male infertility (Wang et al., 2016). Ultimately, this research aims to enhance our understanding of the roles of the CG14305 and CG9222 genes.
Flybase
• A database of genetic and molecular data regarding Drosophila. We used FlyBase (release FB2024_01) to find information on phenotypes, functions, stocks, gene expression, and sequence data
National Center for Biotechnology Information (NCBI)
• Provides access to biomedical and genomic information
Basic Local Alignment Search Tool (BLAST)
• Allows one to see whether essential domains or functional amino acids are conserved across and/or within species
Characterization and protein sequence of comparison of CG14305 and CG9222 with other species within the Drosophila melanogaster family
Flybase data shows CG14305 is orthologous to human TSSK2 and the mRNA is expressed in the adult head and testes. According to Flybase, CG9222 is predicted to have serine/threonine kinase activity and is expressed in the adult head. Given this information, a more in-depth examination of the two Drosophila melanogaster genes was done. Using BLAST, CG14305 and CG9222 protein sequences were aligned with younger and older Drosophila relatives. Figure 1 shows the conservation of essential enzyme regions, including ATP and substrate binding sites, and the activation loop regions are well-conserved in the younger Drosophila species when CG14305 was compared to D. simulans, D. sechillia, D. virilis, and D. grimshawi.
Figure 1: BLAST analysis of D. melanogaster CG14305 protein sequence compared to two younger (D. simulans and D. sechillia) and two older (D. virilis and D. grimshawi) Drosophila relatives.
When the protein sequence of CG9222 was compared to the younger Drosophila species, we found 100% conservation of all essential regions (Figure 2). BLAST analysis revealed similar results when CG9222 was compared to the older D. melanogaster family members with the exception of the activation loop. The activation loop of D. melanogaster CG9222 had a 91% and 96% sequence identity when compared with D. virilis and D. grimshawi, respectively (Figure 2).
Figure 2: BLAST analysis of D. melanogaster CG9222 protein sequence compared to two younger (D. simulans and D. sechillia) and two older (D. virilis and D. grimshawi) Drosophila relatives.
BLAST analysis of Drosophila melanogaster CG14305 and CG9222 with human TSSK2 and TSSK4
According to Flybase, CG14305 and CG9222 have human orthologues, namely, TSSK2 and TSSK4, respectively. Since we found a high level of conservation of important functional domains within the Drosophila family, we wanted to determine whether similar findings would be observed with human TSSK2 and TSSK4. Protein sequence comparisons of CG14305 and TSSK2 revealed the conservation of 11/18 of the amino acids found in the ATP binding region of the protein; 15 of 18 amino acids were conserved within the substrate binding site, and 17 of 23 amino acids were conserved in the activation loop (Figure 3). Essential sequences were found to be conserved between CG9222 and human TSSK4. Sixty-one percent of the amino acids in the ATP binding and substrate binding sites were conserved. Within the activation loop, only 57% of the amino acids were conserved.<>
Figure 3: BLAST analysis of D. mel CG14305 and Human TSSK2 and BLAST analysis of D. mel CG9222 and human TSSK4.
Overall, our study of the Drosophila genes CG14305 and CG9222 highlights their potential as valuable models for understanding male infertility. Both genes encode serine/threonine kinases, which play crucial roles in cellular processes through protein phosphorylation. The high expression of CG14305 in the male germline of fruit flies suggests it may be involved in male fertility. Through protein sequence comparisons across various Drosophila species and with their human orthologues, TSSK2 and TSSK4, we identified conserved functional domains. These conserved domains suggest that CG14305 may function similarly to their human counterparts in the regulation of male reproductive processes since it was most conserved with their human orthologues, TSSK2. In contrast, CG9222, which is expressed in the adult head, can be further investigated by knocking out the gene to explore its effects on growth, behavior and development.
In my future studies, I plan to investigate the effects of both knocked- out genes using the UAS-GAL4 system and observe the effects in their offsprings. This research is important because it provides a foundation for using Drosophila genes CG14305 and CG9222 as models to better understand the genetic and molecular mechanisms underlying male infertility and behavior.
[1] Ardito, F., Giuliani, M., Perrone, D., Troiano, G., & Muzio, L. L. (2017). The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy (Review). International Journal of Molecular Medicine, 40(2), 271–280. https://doi.org/10.3892/ijmm.2017.3036
[2] Baenas, N., & Wagner, A. E. (2019). Drosophila melanogaster as an alternative model organism in nutrigenomics. Genes & Nutrition, 14(1). https://doi.org/10.1186/s12263-019-0641-y
[3] Wang, X., Li, H., Fu, G., Wang, Y., Du, S., Yu, L., Wei, Y., & Chen, S. (2016). Testis-specific serine/threonine protein kinase 4 (Tssk4) phosphorylates Odf2 at Ser-76. Scientific Reports, 6(1). https://doi.org/10.1038/srep22861