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Model Organisms

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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