Annotation Conf. Call 2016-02-08: Difference between revisions
Jump to navigation
Jump to search
Line 66: | Line 66: | ||
'''Co-transfection experiments''' | '''Co-transfection experiments''' | ||
Co-transfection experiments include those experiments where two or more gene products are expressed in a heterologous system such | Co-transfection experiments include those experiments where two or more gene products are expressed in a heterologous system, such | ||
a cell line for the purposes of interrogating a functional interaction between them. | as a cell line, for the purposes of interrogating a functional interaction between them. | ||
In ''C. elegans'', the response to dauer pheromone, a mixture of small molecules, is mediated by G protein-coupled receptors | In ''C. elegans'', the response to dauer pheromone, a mixture of small molecules, is mediated by G protein-coupled receptors |
Revision as of 15:02, 8 February 2016
Agenda
Bluejeans Conference Line
- Recurring meeting URL: https://bluejeans.com/993661940
Review New IGI Documentation
- Following on from the 2016-01-12 call where we discussed how to annotate co-transfection experiments, below is a draft of new IGI documentation for curation.
- For this call, I'd like to review the new documentation, and solicit input for examples.
- For reference, here is the link to the existing documentation on the web site: http://geneontology.org/page/igi-inferred-genetic-interaction
IGI: Inferred from Genetic Interaction
• Genetic interactions such as suppression, enhancement, synergistic (synthetic) interaction, etc. • Co-transfection experiments • Functional complementation • Phenotypic rescue?? Formerly ‘Rescue experiments’. Does this refer to multicopy suppressors? Something else? • Inference about one gene drawn from the phenotype of a mutation in a different gene??
The IGI evidence code is used for annotations based on experiments reporting the effects of perturbations in the sequence or expression of one or more genes or gene products. IGI is also used for experiments that interrogate functional interactions between two or more genes or gene products when co-expressed, for example, in a cell line. Additional uses of IGI include functional complementation experiments, genetic redundancy, phenotypic rescue (?), and inferences made about one gene drawn from the phenotype of mutations in a different gene (?).
Key to deciding whether or not to use the IGI or IMP (Inferred from Mutant Phenotype) evidence code is consideration of the point of reference (i.e., what is being compared) to determine a possible interaction. If experiments interrogate the effects of multiple mutations or differences from the control, then use IGI. If experiments interrogate the effects of a single mutation or difference from the control, then use IMP.
The IGI evidence code requires curators enter a stable database identifier for the interacting entity in the With/From field of the Gene Association File (GAF). Independent interactors may be captured in the With/From field by separating each entry with a pipe. If the interaction experiment involves multiple perturbations simultaneously, e.g. triply mutant strains, then the respective interactors are separated with a comma.
Genetic interactions such as suppression, enhancement, synergistic (synthetic) interactions, etc.
This use of the IGI evidence code refers to the more “traditional” genetic interaction experiments performed in model organisms, such as Saccharomyces cerevisiae, as well as more recent approaches adopted in a number of different systems such as RNA-mediated knockdown or genome editing techniques. Note that genetic interaction experiments may be performed with both loss- and gain-of-function mutations. Consequently, curators will need to use their expertise to determine whether interaction phenotypes resulting from gain-of-function mutations are informative about the normal, wild type role of a gene or gene product.
Example 1: Loss-of-function mutations Need example
Example 2: Gain-of-function mutations The response to axonal injury requires the activities of MAP kinase and cAMP signaling pathways that are required, for example, for signaling growth cone formation. In C. elegans, the activity of the upstream-most kinase in one of the MAPK signaling pathways, DLK-1, is stimulated by Ca2+ influx mediated by the EGL-19 voltage-gated calcium channel. EGL-19’s regulatory role in the MAPK-mediated axon regeneration pathway was determined, in part, through doubly mutant animals containing an egl-19 gain-of- function mutation and a dlk-1 loss-of-function mutation that showed a reduced axon regenerative response when compared to egl-19(gf) alone.
EGL-19 positive regulation of MAPK cascade involved in axon regeneration (GO:1904922) PMID:20203177 IGI DLK-1
Note that in this example, reciprocal IGI annotations are not made, as the GO term selected for EGL-19 does not make sense for DLK-1.
Example 3: Synergistic (synthetic) interactions
Disruption of the MSB2 gene in S. cerevisiae has no appreciable effects on the cell's ability to activate the High-Osmolarity Glycerol (HOG) pathway upon osmotic stress, or on cellular growth on high-osmolarity media. To identify potential osmosensors in the SHO1 branch of the HOG pathway, the authors screened for a mutant that is osmosensitive only in an msb2Δ background and recovered mutations in the HKR1 gene. Like MSB2, mutations in HRK1 alone confer no osmosensitivity to the cells.
MSB2 hyperosmotic response (GO:0006972) PMID:17627274 IGI HKR1 HKR1 hyperosmotic response (GO:0006972) PMID:17627274 IGI MSB2
Co-transfection experiments
Co-transfection experiments include those experiments where two or more gene products are expressed in a heterologous system, such as a cell line, for the purposes of interrogating a functional interaction between them.
In C. elegans, the response to dauer pheromone, a mixture of small molecules, is mediated by G protein-coupled receptors (GPCRs). Genetic analysis has implicated two GPCRs, SRBC-64 and SRBC-66, in a signaling pathway that responds to specific components of dauer pheromone. To assess the biochemical role of SRBC-64 and SRBC-66, the gene products were expressed singly or in combination in HEK293 cells. Only when expressed in combination were the GPCRs able to enhance forskolin-stimulated cAMP production.
SRBC-64 G-protein coupled receptor signaling pathway (GO:0007186) PMID:19797623 IGI SRBC-66 SRBC-66 G-protein coupled receptor signaling pathway (GO:0007186) PMID:19797623 IGI SRBC-64
Functional complementation Functional complementation refers to experiments in which a gene from one species complements a mutation in another. For these annotations, the With/From column should list the identifier for the endogenous gene that is complemented by the heterologously expressed gene being annotated. In annotations from cross-species functional complementation experiments, the gene referred to in the With/From column will thus be from a different species than the gene being annotated.