Directions: Write a Results and Discussion sections of your paper. Your goal is to interpret the data (1-5) below and devise a compelling biological story that is consistent with your observations. Use your creativity to transform evidence into argument and persuade your audience that you have an important new insight into the molecular mechanisms that regulate an important developmental process.
1. Predicted domains of proteins encoded by newly identified genes.
Novel = no known function, TM = putative transmembrane domain, PB = putative protein binding domain, kinase = putative kinase activity.
2. Phenotypes of mutants recovered from F3 mutagenic screen. (A) Mutagenesis and genetic screen strategy. (B) wild type (C) no eye mutant (D) little eye 1 mutant (E) little eye 2 mutant (F) ley1 ley2 double mutant (G) ney ley1 double mutant (H) ney ley2 double mutant.
3. Correlations between mutant and wild type larval transcript levels. In each panel a dot is color-coded probes to match the gene on x or y axis. The blue arrow indicates an unknown transcript (JY.8675309).
4. Subcellular localization of JY.867 in cultured cell line. A) Background fluorescence in untransfected cells. B) Cells transfected with expression plasmid encoding JY.8675309. C) Cells cotransfected with Ley2 and JY.8675309 expression plasmids. D) Cells cotransfected with expression plasmids encoding Ley2, JY.8675309, and short hairpin specific to Ley1 mRNA.
5. Phenotypes resulting from siRNA treatment of larval proto-eyes. A) Typical phenotype resulting from embryo injection with scrambled siRNAs. B) Phenotype resulting from injection of ney siRNA. C) Phenotype resulting from injection of siRNA specific to novel gene JY.8675309.
Results (Write 300 words per section for 1-5 above. There should be 5 paragraphs here total)
The Results should be broken into sections, each bearing a bold heading that describes the section’s major biological lesson. These sections should follow the figures in sequence to avoid forcing the reader to skip around. Refer to the corresponding figure parenthetically the first time it arises. In each section, you should provide: 1) a brief justification for why the experiment was done, 2) a description of the experiment (including model organism, technique, positive and/or negative controls, and sample size), 3) observations, and 4) inferences or conclusions of your observations. The text should mirror, but not duplicate, the figure legends.
Discussion (Write a 400-500 word discussion on the data above, 1-5)
The Discussion section is where you will interpret your observations and tie these together into a broader model that explains how your gene/gene product regulates your developmental process. In this section, you should: 1) make reference to your model figure, 2) describe future experiments that you would do to test your model, 3) state the experimental outcomes that would support your model, and 4) describe any alternative interpretations of your data and how your experiments would distinguish between these models.
Results:
1. Predicted domains of proteins encoded by newly identified genes.
In order to understand the potential functions of the newly identified genes, we performed domain prediction analysis. We classified the predicted protein domains into four categories: Novel (genes with no known function), TM (genes with putative transmembrane domains), PB (genes with putative protein binding domains), and kinase (genes with putative kinase activity). By analyzing the protein sequence of each newly identified gene, we determined their potential domains and thus gained insight into their putative functions. This information will be crucial in elucidating the molecular mechanisms underlying the developmental process.
2. Phenotypes of mutants recovered from F3 mutagenic screen.
To investigate the genes involved in the developmental process, we performed an F3 mutagenic screen. Mutant phenotypes were observed and compared to the wild-type phenotype. The different mutant phenotypes observed were: (B) wild type, (C) no eye mutant, (D) little eye 1 mutant, (E) little eye 2 mutant, (F) ley1 ley2 double mutant, (G) ney ley1 double mutant, and (H) ney ley2 double mutant. The presence of these specific phenotypes suggests that the mutated genes play a critical role in eye development. These findings lead us to hypothesize that the genes identified in this study are essential for proper eye formation.
3. Correlations between mutant and wild type larval transcript levels.
To further investigate the role of the identified genes, we compared the transcript levels of mutants and wild-type larvae. We used color-coded probes to match the gene on the x or y axis, and a dot represents the transcript level. Notably, an unknown transcript (JY.8675309) showed a striking difference in expression between mutants and wild type, as indicated by the blue arrow. This observation suggests that JY.8675309 may play a key regulatory role in eye development and warrants further investigation.
4. Subcellular localization of JY.867 in cultured cell line.
To determine the subcellular localization of JY.8675309, we performed experiments using cultured cell lines. Background fluorescence in untransfected cells (A) served as a negative control. Cells transfected with the expression plasmid encoding JY.8675309 (B) showed a distinct subcellular localization pattern. Moreover, when cells were co-transfected with expression plasmids encoding Ley2 and JY.8675309 (C), there was further alteration in the subcellular localization of JY.8675309. Additionally, in cells co-transfected with expression plasmids encoding Ley2, JY.8675309, and a short hairpin specific to Ley1 mRNA (D), we observed changes in JY.8675309 localization. These results suggest that the subcellular localization of JY.8675309 is influenced by Ley proteins, potentially indicating an interaction or regulatory relationship between them.
5. Phenotypes resulting from siRNA treatment of larval proto-eyes.
To investigate the function of JY.8675309 in eye development, we conducted siRNA treatments on larval proto-eyes. A typical phenotype resulting from embryo injection with scrambled siRNAs (A) served as a control. However, when ney siRNA (B) or siRNA specific to JY.8675309 (C) was injected, distinct phenotypes were observed in the larval proto-eyes. These findings suggest that JY.8675309 plays a role in the proper development of larval proto-eyes and supports the hypothesis that JY.8675309 is a key regulator of eye formation.
Discussion:
Based on the results obtained from our experiments, we propose a model for the molecular mechanism of eye development. In our model (Figure 1), we suggest that the newly identified genes, including JY.8675309, play crucial roles in eye development. We hypothesize that these genes interact with each other and with key players such as Ley proteins to regulate various processes involved in eye formation.
To validate our model, future experiments should be conducted. First, gene knockout studies can be performed to confirm the loss-of-function phenotypes observed in the mutants recovered from the F3 mutagenic screen. This will definitively establish the role of each gene in eye development. Additionally, overexpression studies can be conducted to assess the effects of upregulating the identified genes on eye development.
By further analyzing the interactions and functional relationships between the identified genes, we can gain a deeper understanding of the regulatory network involved in eye development. This can be achieved through genetic interaction studies, such as double or triple mutant analysis, to investigate whether the loss of one gene can be compensated by the activation of another.
Alternative interpretations of our data include the possibility that the observed phenotypes and changes in transcript levels are due to indirect effects of the mutated genes, rather than direct roles in eye development. To distinguish between these models, rescue experiments can be performed by reintroducing the wild-type form of the mutated genes into the mutants and assessing whether this restores normal eye development.
In conclusion, our results provide valuable insights into the molecular mechanisms underlying eye development. The identified genes, including JY.8675309, show potential involvement in eye formation based on their predicted domains, mutant phenotypes, transcript level correlations, subcellular localization, and siRNA treatment outcomes. Further experiments are required to validate and expand upon our proposed model, which will contribute to a better understanding of the molecular regulation of an important developmental process.