Erika Darrah et al. (2003) International Worm Meeting "Expression analysis of unc-13 and identification of proteins interacting with its products"

Erika Darrah, Bethany Stitt, Cristina Polinsky, Rebecca Kohn

[International Worm Meeting, 2003]

We are analyzing the expression of different forms of UNC-13 protein and are identifying other proteins that interact with UNC-13. Some forms of UNC-13 protein are involved in regulating neurotransmitter release. The most abundant protein product expressed from this gene, UNC-13 LR, localizes to synapses (Kohn, 2000). UNC-13 interacts with other presynaptic proteins and affects vesicle priming (Richmond, 2001). The C. elegans unc-13 gene is predicted to express several different protein products including UNC-13 LR, UNC-13 LMR, and UNC-13 MR. L represents the left region of the protein, M represents the middle, and R represents the right. Mutations in the unc-13 L region of the gene alter expression of some protein products and result in uncoordinated movement. A large deletion in the unc-13 R region eliminates all protein products and results in lethality (Kohn, 2000). We are analyzing regulation of expression of the different protein products. Specifically, we are exploring the possibility that an internal promoter is present in the gene that regulates expression of the UNC-13 MR product. A large 7.5 kb intron separates the unc-13 L region of the gene from the unc-13 M region. A cosmid including most of the 7.5 kb intron as well as the unc-13 M and unc-13 R regions can partially rescue deletion mutants. We are fusing part of this intron to GFP in order to determine if a promoter within the intron can drive expression of the gene and to identify cells in which the promoter is active. We are also using a two hybrid screen to identify proteins that interact with the different UNC-13 protein products.

Theresa Moser et al. (2004) East Coast Worm Meeting "Analysis of an UNC-13 protein expressed from an internal promoter"

Brooke Swalm, Monique Spencer, Theresa Moser, Rebecca Kohn, Lydia Sanchez, Bethany Stitt, Kristin Servent

[East Coast Worm Meeting, 2004]

Several protein products are expressed from unc-13 . The predominant product localizes to synapses (Kohn, 2000) and is important for vesicle priming (Richmond, 2001). We are interested in understanding the roles of the other proteins expressed from the gene and are examining their expression patterns. Previous cosmid rescue experiments indicated that an internal promoter is used for expression of an UNC-13 protein product. We constructed a fusion of the putative promoter with the GFP gene and injected it into worms. We also developed antibodies to recognize the protein product expressed from the internal promoter. These antibodies bind UNC-13 proteins on Western transfers. GFP expression and immunohistochemical studies will determine the localization patterns of the less abundant form of UNC-13.

Theresa Moser et al. (2005) International Worm Meeting "Analysis of different forms of UNC-13 proteins in the Caenorhabditis elegans nervous system"

Bethany Stitt, Lydia Sanchez, Chanelle Houston, Cristina Polinsky, Erika Darrah, Graciela Gallo, Monique Spencer, Nancy Fernandez, Dana Francis, Theresa Moser

[International Worm Meeting, 2005]

We are studying the expression and function of unc-13 protein products in the Caenorhabditis elegans nervous system. The predominant protein expressed from unc-13 interacts with syntaxin and affects SNARE complex formation. This form of UNC-13 localizes to presynaptic plasma membranes and is important in priming synaptic vesicles (Richmond, 2001). Expression and function of other UNC-13 protein products are not completely characterized. We are analyzing regions of unc-13 for internal promoters regulating expression of shorter UNC-13 protein products. We fused regions of unc-13 including possible promoters to GFP and injected constructs into C. elegans. Progeny were analyzed for GFP expression. A construct incorporating 2.5kb of a 7.5kb intron did not express GFP. A construct including a larger region of this intron and a construct including a different intron are being tested for promoter activity. To identify additional proteins that interact with UNC-13, we used a yeast two-hybrid screen and analyzed a suppressor mutant. The yeast two-hybrid screen identified three candidates for proteins interacting with the predominant form of UNC-13. Five candidates for proteins interacting with a short form of UNC-13 were found. Eight candidates for proteins that may interact with all forms of UNC-13 were identified. A mutation that partially suppresses paralysis in an unc-13 missense mutant was analyzed. Genetic crosses showed that the suppressor is recessive and unlinked to unc-13. The mutation also suppresses an unc-13 allele that does not express the predominant unc-13 protein product. We are mapping the suppressor gene using snip-SNP mapping.