Isolation of new mutations in genes of the JAK signaling pathway
Nimoh Boateng (Kentucky State University), Leslie Denhard (Berea College) & Claudia Rodriguez (Bellarmine University)
Mentor: Douglas Harrison
The Janus kinase (JAK) signaling pathway has many essential roles in development and homeostasis in animals from flies to humans. In Drosophila melanogaster, there are homologues of all the primary components for both transduction and regulation of the pathway. There is a family of three proteins, Unpaired and two Unpaired-likes, that are believed to be the secreted ligands that activate the JAK pathway. Although mutations in the unpaired gene have been isolated and characterized, there are no known mutations in the unpaired-like genes. This summer, the, participating students conducted a chemical mutagenesis screen to generate and recover potential mutations in the unpaired-like genes. Approximately 35 candidate mutations were recovered and are now being genetically tested for allelism with the unpaired-like genes. In addition, there is a suppressor of cytokine signaling (SaCS) homologue in flies that is very similar to a class of mammalian regulators of the pathway. No mutations previously existed for this gene. To generate mutant alleles, a Ptransposable element inserted near the fly sacs gene was mobilized and assayed for imprecise excision, a process that deletes flanking genomic DNA. More than 40 imprecise excisions were identified in this screen and are being analyzed molecularly to determine the extent of deleted DNA. These genetic reagents will be invaluable in the continuing characterization of the JAK signaling pathway in Drosophila.
Genetic Screen for Cell Cycle Mutants in Aspergillus nidulans.
Rui Chen (Berea College), Becky Ford (Transylvania University) & Stephanie Jenkins (Bellarmine University)
Mentor: Peter Mirabito
Control of the cell division cycle is required for the growth, development, and health of all organisms. In order to understand the mechanisms of cell cycle control, the genes/proteins involved in cell cycle control must be identified and their actions and interactions studied. Fungi have long been used in genetic analysis of cell cycle control and the information gleaned from fungi has proven fundamental to our understanding of the cell cycle in all organisms, including man. A genetic screen was carried out for cell cycle genes in Aspergillus nidu/ans, a filamentous fungus which has long been used to study cell cycle control. The goal was to make new cell cycle mutants and to use those mutants to identify new cell cycle genes. Each student isolated several mutants and cloned at least one gene. The DNA sequence of those genes have not yet been determined. This project provided a collection of new cell cycle mutants which will be the subject of future, more intensive studies of cell cycle control in Aspergillus nidulans.
Sequencing the Ambystoma mitochondrial genome
Katie France (Bellarmine University)
Mentor: Randall Voss
Salamanders of the genus Ambystoma are models in biomedical and life history research. A mitochondrial genome project was initiated for two ambystomatid salamanders that exhibit considerable morphological variation: A. andersoniand A. dumerilii. Long PCR, DNA-cloning, and DNA sequence analysis was used to obtain approximately 30% of the A. andersoni genome and 15% of the A. dumeriliigenome. Comparative DNA sequence analyses indicated low (1-4%) nucleotide sequence variation between these and two other species with nearly complete mitochondrial DNA sequence. Overall, these data support the idea that ambystomatid salamanders are recently derived yet exhibit considerable morphological and life history variation.
Mutations of pyrimidine degradation in Drosophila.
Lindsay Poling (Transylvania University), Emily Steinmetz (Bellarmine University) & Alice Wright (Western Kentucky University)
Mentor: John Rawls
Degradation of pyrimidines produces ~-alanine, a compound implicated in a wide range of biochemical processes (cuticle pigmentation in insects, CoA biosynthesis, neurotransmitter agents); thus, this pathway performs both degradative and biosynthetic roles. A variety of loss-of-function mutations of the first pathway enzyme have been discovered recently among patients exhibiting toxic reactions to pyrimidine analog chemotherapy. To examine the role of this pathway genetically, a variety of screens were carried out to create mutations of the first two enzymes in Drosophila melanogaster. Mobilization of a transposon near the pydl gene generated 31 strains exhibit suppression of the phenotypes exhibited by mutants of pyrimidine biosynthesis. DNA sequence analysis of a subset of these mutations show that they are partial deletions of the pydl gene. Making use of this suppression phenotype, mobilization of a transposon near the pyd2 gene produced 6 mutant strains, at least two of which are deletions of the 5' end of the pyd2 gene. EMS mutagenesis screens for pyd2 mutants recovered 7 viable, suppressor alleles. Another EMSscreen for recessive lethals inthe pyd2 region was also performed, yielding 18 lethaIs,two ofwhich arealleles of pyd2. DNA sequence analysis of these mutations are underway. Data so far indicate that, in flies as in humans, pydl is required for pyrimidine degradation, but is not an essential gene (null mutations develop essentially normally). On the other hand, pyd2 is required in flies for pyrimidine degradation but is also required for normal development. Thus, pyd2 is probably involved in more than pyrimidine degradation.