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|a Genetic Screening to Find Novel Regulators of Tumor Suppressor Homolog Kinase Responsive to Stress B (KrsB) |h [electronic resource]. |
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|a [S.l.] : |b SUNY Oswego, |c 2020. |
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|a Genetic Screening to Find Novel Regulators of Tumor Suppressor Homolog Kinase Responsive to Stress B (KrsB)
Emily Fingar, Ali Khan, Swin Ratnayake, Yulia Artemenko
Dictyostelium discoideum social amoeba is a well-established model organism for the
study of amoeboid-type migration, which is the type of movement seen in neutrophils and
metastatic cancer cells. Cycling between active and inactive forms of the serine/threonine kinase
responsive to stress B (KrsB), a homolog of mammalian tumor suppressor MST1/2 and
Drosophila Hippo, contributes to the dynamic regulation of cell adhesion that is needed for
proper cell adhesion and chemotaxis in D. discoideum. However, the exact mechanism by which
KrsB affects the cell’s ability to adhere is unclear. The goal of this project is to find new
regulators or effectors of KrsB using a genetic suppressor screen. Cells lacking KrsB were
transformed with a cDNA library and mutants that exhibited either a rescue or an enhancement
of the original phenotype were isolated. Cells lacking KrsB have a distinct phenotype when they
form plaques on a bacterial lawn, with an enlarged region of cells in streams and an uneven
expanding front of vegetative cells, which makes krsB ̄ plaques appear to have rough edges.
A transformation was done using 3μg of cDNA library and 3μg of pKF3 showing the most prominent results and PKF3-KrsB served as a positive control. Later on, the cells were enriched in media so floating cells can be plated separately from adherent ones. On average, there were more adherent cells than floating cells. In total, 29 plaques were identified with either a more severe phenotype or a rescue phenotype, with 21 plaques originating from the floating cell fraction and 8 from the adherent cell fraction. These mutants were either characterized as a more severe “s” phenotype in which they looked much smaller and underdeveloped in comparison to wild-type or KrsB-null plaques, there were seven of these. Rescue-like plaques, which were denoted as “r”, had smooth round edges or more developed fruiting bodies than KrsB-null plaques, there were 22 identified in total. A secondary screen was done in which cells from these 29 plaques were collected and grown in media so their morphology can be analyzed further. After clonally plating these cells on bacteria again, five plaques exhibited a rescue-like phenotype and five exhibited a more severe one, all consistent with the identified morphology in the initial screen. These ten will be examined further by isolation of plasmids with the cDNA library inserts and determining the identity of the cDNA insert by sequencing, which will aid in identifying the genes responsible for the rescue of the krsB ̄ phenotype or for making the phenotype more severe. Identification of these genes will give us a better understanding of the molecular mechanism of KrsB function in cell adhesion and migration. |
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|a Electronic reproduction. |c SUNY Oswego Institutional Repository, |d 2020. |f (Oswego Digital Library) |n Mode of access: World Wide Web. |n System requirements: Internet connectivity; Web browser software. |
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|a Collected for SUNY Oswego Institutional Repository by the online self-submittal tool. Submitted by Emily Fingar. |
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|a Dictyostelium discoideum. |
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|a Oswego Digital Library. |
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|a SUNY Oswego Scholarly and Creative Works. |
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|a Summer 2020 Scholarly and Creative Activities Symposium. |
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|a OswegoDL |c SUNY Oswego Scholarly and Creative Works |
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|u https://digitallibrary.oswego.edu/SUOS000905/00001 |y Electronic Resource |
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|a SUNY Oswego Scholarly and Creative Works |