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Model Organisms
| Question | Answer |
|---|---|
| How does reverse genetics work? | instead of observing a phenotype and mapping it to the causal mutations, a gene is manipulated to determine its function - then compares to homologous genes and identified for functional domains. |
| Why do model organisms help in studying complex genetic pathways | Reverse genetics is not always possible/affordable. model organisms provide centralised mutants and transgenic fly collects, as well as a vast array of different plasmid constructs. |
| What is MARRVEL? | a pool of info, searchable for a patient variant or gene of interest. |
| WHy are drosophila used? | fast life cycle, small and easy to rear in the lab in high numbers - techniques are well established. |
| Give some examples of genetic tools co-opted from different organism systems: | transposase from DNA transposons, plasmids and LacZ from Bacteria, Gal4 from Yeast, GFP from Jellyfish. Plus many more tools exist - CRISPR, CAS9. |
| What are some reporters and what do they do? | Fluorescent proteins and Enzymes such as LacZ = Beta-galactosidase from E.coli. They track/visualise experimental results. |
| Fusion of a reporter to a DNA sequence.... | can allow you to determine the pattern of expression it directs. |
| Why do plasmids require antibiotic selection for cloning? | only cells hwich have integrated the plasmid will survive. |
| What does integration of reporter into Drosophila genome require/ | p-element transposons. Naturally found in fly genomes, integrate into fly genome by terminal inverted repeats, mediated by transposase. |
| Where must the Multiple Cloning Site be situated? | Upstream of the reporter gene of choice. |
| What are the steps of engineering a transgenic fly? | 1 - clone fragment of DNA into plasmid construct. 2 - microinjection of construct into Drosophila embryos with transposase. 3 - Rear survivors and backcross adult flies that emerge to white eyed flies. 4 - screen flies from this cross for eye colour |
| When must injection of the construct be done? | early <30 mins into development. |
| What is a syncytium/ | embry of drosophila where nuclei are not separated by cell membranes yet but share a common cytoplasm. |
| Why must flies have a mutation in the marker gene? | to allow us to detect inseriton events. |
| What will the cross show? | integration events if varying colours of eyes - depends on where plasmid integrate dinto genome. |
| WHat are some common genes of interest for drosophila? | Lac Z, GFP, Wilt-type for structural genes. |
| what is the overall effect of GAL4>UAS in a cell | drive expression of a gene o I. |
| How do regulators in developmental genes work? | can act as respressors or activator - expressed in specific regions of developing embryo. Control each others expression by binding cis-regulatory sequences. |
| What are maternal effect genes? | the maternal genome determines whether the molecule is functional. Example of one is Egg-polarity genes. |
| Describe the inheritance pattern of maternal effect genes: | if a mutation is maternal effect recessive, then a female homozygous for the mutation may appear phenotypically normal, however her offspring will show the mutant phenotype, even if they are heterozygous for the mutation. |
| What is sterility of Mater/- vertebrates? | Males are fertile, females are sterile. Development is blocked at two-cell stage of embryogenesis. |
| What is Bicoid | Bicoid mRNA deposited by mother in embryo in early oogenesis, sets up a morphogen gradient, more concentrated in anterior pole. |
| What does the concentration of Bicoid influence? | expression of Gap genes e.g. Hunchback. |
| Is bicoid cis or trans acting? | trans that binds to specific site wihtin cis-regulatory DNA elements. |
| What does an analysis of flied carrying reporter constructs (e.g. GFP) show? | That the expression level of hunchback is proportional to the # of Bicoid sites occupied in the enhancer region. |
| What is the hierarchy of developmental genes? | egg-polarity genes, gap genes, pair-rule genes, segment-polarity genes, hox genes. In short, maternally deposited>segmentation genes/zygotic expression. |
| What's an example of a zygotic segmentation gene? | Krüppel - gap gene. |
| Enhancers in zygotic segmentation genes are: | modular, meaning different cis-acting regions of DNA control gene expression for different stripes. |
| Describe the eve reporter construct: | cis-regulatory element controlling expresion of Eve in stripe 2. $ transcriptional regulators bind to these sites. There can be multiple sites present in the region. Some repressor sites overlap activator sites. |
| What is the genetic evidence for the roles of transcriptional regulators? | 1 - protein staining - Hunchback and Bicoid in stripe 2, while Giant and Krüppel not detected here. 2 - flies unable to express bicoid or hunchback also unable to express Eve in stripe 2. |
| What is the genetic evidence for the roles of transcriptional regulators? | 3 - mutants in krüppel gene have a posterior expansion of Eve expression. 4 - disruption of Krüppel sites in the cis-regulatory element also lead to a posterior expansion of expression. |
| what does the hox gene do? | defines segment identity |
| What do the phenotypes of hox mutants indicate? | segmental control. |
| What are the 2 main hox complexes? | Antennapedia and bithorax on chro. 3 |
| What is the homeobox? | 180bp region of DNA coding sequence similar enough to allow cross hybridisation b/w 8 other hox genes. |
| How many AAs are conserved between the 8 genes? | 60 |
| What is the homeodomain protein? | 3 alpha-helices make specific contacts with bases in the major groove . |
| What structure do helices 2 and 3 make? | helix-turn-helix structure. |
| Are there additional interactions? | Yes, in minor groove and backbone. |
| How many genes are conserved in developmental pathways for mammals and drosophila? | mammals - 39. Drosophila - 8. |
| From where did the ancestral complex arise? | from serial duplications of a single homeotic selector gene. |
| What did the mammalian lineage experience? | complex was repeatedly duplicated in its entirety with individual gene losses and duplicates within these new complexes. |
| Give example of hox gene defect: | HoxA10 normally expressed in lumbar region. Gain function: artificially expressed in developing vertebral tissue all along body. Loss function: vertebrae taking on thoracic character instead of lumbar or sacral. |
| Give another example of hox gene defect: | HoxA2 autosomal recessive microtic ears. Q186K substitution mutation. Glutamine at position 186 is evolutionarily conserved. Affects position 44 of homeodomain, which normally functions in recognition helix. |
Created by:
lmdavis97
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