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Lecture 5

Lecture 5.pdf

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Department
Biology (Sci)
Course Code
BIOL 301
Professor
Nam Sung Moon

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Biology 301 - Molecular Biology Lecture 5: BIOL 301 Announcements Gene expression, gene regulation & reverse genetics ➢  Study of gene function Review Session ▪  Gene expression analysis Next Friday •  Promoter-reporter gene fusions •  Dissection of gene regulation with promoter fusions Answer questions and go over old midterm •  Caveats to transcript analyses ▪  Reverse genetics question. (It will be recorded) •  Creation of gene knockouts to analyze gene function •  RNA interference Midterm on Monday Feb 17 th (myCourse) •  Over- and inappropriate gene expression Tester quiz is up and running 1 2 Promoter-reporter gene fusion -promoter is upstream region of gene where TFs and machinery bind and direct expression of RNA. Thus, if you want to study RNA expression, you can study promoter. -ppl realized that if you are interested in a particular gene, you can take the promoter and place it upstream of a reporter gene. Now, reporter gene is expressed at time and place where gene of interest is Example of fusion proteins in vivo: expressed. This is really easy to detect. However, this reporter gene This shows a GFP reporter fusion. Slide shows back of drosohpila cannot be something that is normally expressed by the species you are and shows sensory organs which consists of cells called sensory working with (ie it has to be non-endogenous). organ precursors (SOP). These cells will divide asymmetrically. -GUS is eg of reporter - it will cleave substrate to produce blue colored products. To visualize cell division of SOPs, you can use GFP fusion -another reporter is luciferase. It is an enzyme and cleaves a substrate. proteins that are in SOP cells and watch them under microscope When it cleaves, it emits light - by quantifying this, you can get idea of (dont need to kill flies!). Now, you can visualize in vivo how the the strength of the promoter (how much of the RNA is expressed). Also, cell divisions occurs. you can use this in vivo Promoter-reporter gene fusions Application of reporter genes ➢  Promoter – region upstream (5’) of gene where transcription machinery & transcriptional regulators bind ➢  If the promoter of GOI is fused to a “reporter gene”, the “reporter gene” is expressed in organisms where and eg of when the GOI is normally expressed reporter ➢  Reporter genes ▪  non-endogenous, easy to detect ▪  β-glucuronidase (GUS) & β-galactosidase (LacZ) – cleave substrates to give blue product ▪  luciferase – cleaves luciferin to give luminous product Emery et al. Curr Biol. 2005 •  can quantify •  can detect in vivo ▪  green fluorescent protein – needs no substrate! •  need fluorescence microscope •  can see in vivo 3 4 -another reporter is GFP. You don't need substrate to visualize it. You need flourescence microscope to detect reporter. It can also be used in vivo Pros: -one of main reasons why we use promoter-reporter fusion is b/c it is easier to detect Why do ppl make promoter-reporter if there are cons for it? compared to in situ hybridization. In situ techiniques are laborious (have to prepare tissue, hybridize..etc) B/c there are other purposes for them -can look in vivo and quantify (eg luciferase) 1. can use them as cell type specific markers. -one of main reason why ppl use promoter-reporter is b/c you can use them for other -Slide shows section of adult fly. If we want to study axonal purposes (we will discuss 2 today) guidance of photoreceptor and you want to identify genes Con: involved in this process, normally you would mutagenize WT -the half life of reporter protein may not reflect the endogenous transcript/protein level genes and see which ones affect axonal guidance. But this technique is laborious. (cont below) (you protein of interest may be rapidly degraded but you can't determine this with reporter protein) Other use of reporter gene – Promoter Promoter-reporter gene fusions fusions as markers PROMOTER OF G.O.I. GFP ▪  As cell type specific markers (e.g. neuron, stem cells, etc) ➢  Pros ▪  Easier to detect compared to in situ head ▪  Can look in vivo (GFP, luciferase) eye ▪  Can quantitate easily (luciferase) ▪  Can be used for other purposes ➢  Cons: ▪  Reporter protein half life or motility may differ from endogenous transcript and protein ▪  Dependent on what cis sequences actually use •  If missing key regulatory sequence! ▪  Have to construct & transform into organism Cons (continue) -Not all genes have defined promoters. Sometimes, you might miss important regulatory elements (this is a big caveat of this technique) Garrity et al. 2000. Cell 5 6 -you need to create construct of reporter-promoter. So you have to If you know a gene that is only expressed in photoreceptor, you can do cloning and transformation take promoter of that gene and fuse it to reporter (eg lacZ) --> this reporter will only be expressed in photoreceptors. To look for mutants with defects in axon guidance, you mutagenize these transgenic flies that express lacZ as cell-specific markers for photoreceptors and look for mutants. By using reporter fusions as cell type specific markers, we can look for mutants in particular cell types -bottom pics show wing imaginal disks. If you are interested in expression pattern of gene called m-beta. You want to see which portion of promoter is important for expression of m-beta --> you can fuse reporter gene to diff portions of the promoter and see if reporter is expressed 2) the other use of reporter genes is that you can actually -in slide, the numbers after m-beta represent the size of the promoter that was use this technique to study the promoter itself used.. Here, reporter was LacZ. If you take 0.8 kb of promoter, it has certain pattern of expression. If you take 0.4 kb of promoter, you see expression is -for eg, how much of the 5' upstream sequence do you need for it to actually be a promoter. See other questions you can weaker but pattern of expression is pretty similar. If you take only 018 of promoter, look at in slide you see that the expresion of the gene in the wing pouch disappears but expression at the dorsal-ventral boundary is retained (Cont. below) Other use of reporter gene – Promoter Other use of reporter gene – Promoter dissection dissection ➢  What constitutes the promoter for a gene? ➢  What portions of the promoter control what part of its wing mβ0.8 mβ0.4 mβ0.18 expression? pouch dorsal -ventral boundary ➢  Where do the transcription factors (regulators of expression) bind to drive transcription? Cooper et al. Dev. Biol. 2000 The area of promoter used in all three experiments must be the region of 7 8 promoter responsible for expression of m-beta in the dorsal-ventral boundary. A next step that can be taken is to take this 0,4 kb of promoter and do reporter fusion; but now, mutate diff sequences in the promoter and sees which one fails to expression the reporter at dorsal-ventral boundary --> this allows you to see which sequences allow you to get expression of m-beta at dorsal-ventral boundary --> then, you can look at TFs that bind there The portion of the 0.4 kb fragment of promoter that is upstream of the 0.18 fragment must be responsible for expression of m-beta in wing pouch There are things to consider when you make promoter-reporter Reasons to go beyond transcript: -one of problem with this fusion is that you don't know if you have used enough promoter sequence to recapitulate the endogenous expression pattern and level of transcript. Thus, first thing you do when you make fusion is to see if the signal you get with the reporter reflect the mRNA expression level/pattern. If you are examining expression pattern, you have to back it up with direct detection techniques (Eg in situ) cont below Where is a gene expressed? Things to consider when constructing promoter-reporter fusion Beyond transcript analysis ➢ Protein expression – Dr. Vogel ▪  Immunodetection Does the promoter have all the ▪  Protein reporter gene fusions necessary regulatory elements? ➢ Why go beyond transcript analysis? ➢  Back it up with direct detection ▪  Not all “gene expression” is regulated at transcript level techniques such as in situ. ▪  Can be multiple levels of regulation ▪  Production of mRNA doesn’t tell you: Is the detection method optimal? •  If protein made ➢  Tissue with no reporter (no •  If protein functional promoter) •  Where protein acting in cell ➢  Tissue expressing reporter with •  If protein has modifiers, regulators, partners known pattern There are controls you need with this technique: 9 just b/c you have mRNA, that doesn't tell you if 10 -First of all, you need tissue that doesn't have this reporter. For many reporters protein is made. Even if you see the protein, that (eg GUS), they are enzymatic rxns and you could have some basal level rxns if doesn't mean its functional (it can be modified by you have substrate. thus, you want control next to your experiment to see what post-translational modifications). is the basal level of the reaction. This is the negative control -the other control is that you want to have promoter of a gene that should be Expression doesn't give you gene function but it expressed in the tissue (Can use housekeeping gene). This is for in case you gives you idea about where it might function failed to get signalling, you want to ensure it is not b/c technique failed. This is the positive control Once we get our construct, we clone the promoter-reporter (for moelcular complementation, we are cloning WT gene) but afterwards, the way to transform into animal is the same If you are working with model organisms and you are interested in particular genes, there might be mutants already available if
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