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Isolation and characterization of three novel dictyostelium ras genes : evidence for a unique, developmentally regulated ras gene family

University of British Columbia

Members of the ras gene superfamily encode low molecular weight (21-23 kDa) GTP-binding proteins that are believed to play a role in signal transduction. In the cellular slime mold Dictyostelium discoideum, two ras genes, (rasD and rasG), and one ras-related gene, (rap1), have been previously isolated and characterized, but preliminary evidence suggested that there were additional ras genes in Dictyostelium. Using the differential hybridization and polymerase chain reaction (PCR) techniques, no additional rap genes were isolated, but three novel ras genes, rasB, rasC, and rasS, were cloned and characterized. The expression of all three genes is developmentally regulated; in the wild type Dictyostelium strain V12M2, maximum levels of the rasB transcript were detected during growth and early development, while maximum levels of the rasC and rasS transcripts were detected during aggregation. Consequently, during early development and aggregation, at least four ras genes (rasB, rasC, rasG, and rasS) are being expressed simultaneously in Dictyostelium. The deduced amino acid sequences of rasB, rasC, and rasS are more related to the Dictyostelium RasD and RasG proteins, and the human H-Ras protein than they are to any of the other Ras-related proteins such as Ral, R-Ras, Rho, and Rab. Like RasD, RasG, and Rap1, the RasB protein possesses a conserved effector binding domain relative to H-Ras. However, RasC and RasS have single amino acid substitutions in their effector binding domains at residues that are believed to be necessary for Ras biological activity (RasC: D38N, and RasS: 136L). RasB, RasC, and RasS also have at least three amino acid differences in their effector-proximal domain, a domain which is believed to be involved in Ras target activation and which also acts in conjunction with the effector domain. In addition, all three proteins have amino acid substitutions in the Yl3-259 epitope, a region believed to be involved in mediating Ras:GAP interactions. The RasB and RasC GST-fusion proteins, which have single amino acid substitutions in the Yl3-259 epitope, reacted with the Y13-259 monoclonal antibody but the RasS fusion protein, with three amino acid substitutions in the epitope, did not. Since RasD and RasG have identical Y13-259 epitope, effector, and effector-proximal domain sequences, the atypical variations in the corresponding domains of RasB, RasC, and RasS, suggest that RasB, RasC, and RasS may each interact with different effector and/or GAP molecules than RasD and RasG, and hence have distinct functions. Furthermore, the multiplicity of ras genes in Dictyostelium, coupled with their distinctive gene expression patterns, suggests that the distinct ras gene products may be involved in an intricate network of signal transduction pathways which regulate the Dictyostelium developmental process.

Thesis/Dissertation

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2009-04-08

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http://hdl.handle.net/2429/6960

Microbiology and Immunology

University of British Columbia

UBCV

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