5BBG0205 Workshop - Reporter Genes Answers 2024-2025 PDF

5BBG0205 Molecular basis of gene expression Workshop 1. Using reporter genes to study gene expression For KEATS Dr Shirley Coomber Learning outcomes After this workshop students should 1. Be able to name different types of reporter genes. 2....

5BBG0205 Molecular basis of gene expression Workshop 1. Using reporter genes to study gene expression For KEATS Dr Shirley Coomber Learning outcomes After this workshop students should 1. Be able to name different types of reporter genes. 2. Understand how reporter genes can be used to identify regulatory regions of genes. 3. Understand that transcription factors bind to specific sequences in regulatory regions of genes.  Revision In the 4BBY1070 Genetics and Molecular Biology practical 2 the plasmid pGLO was transformed into competent E. coli cells and the transformed cells grown on different media agar plates. N.B. pGLO was digested with restriction enzymes in 4BBY1070 practical 1. LO has green fluorescent protein (GFP) cloned in front of the araBAD prom Revision (continued) The E. coli pGLO cells were grown on two different LB agar plates, one containing ampicillin (amp) and one containing ampicillin and arabinose (ara), for 24 hrs at 37°C. The presence of GFP in the E. coli pGLO colonies was studied using a UV light source. GFP was present (colonies glow green in UV light) when the colonies were grown on LB amp agar plates in the presence of arabinose. Arabinose activates the araBAD promoter so that GFP is expressed. This is an example of a transcriptional reporter because GFP is cloned in front of the araBAD promoter and the araBAD promoter is controlling expression of GFP.  Reporter genes A number of genes can be used as reporter genes including; Chloramphenicol Acetyltransferase (CAT) Luciferase β-Galactosidase A good reporter protein β-Glucuronidase (GUS) must be easy to Alkaline Phosphatase (AP) visualise/detect and be β-Lactamase quantifiable. Various Fluorescent Proteins Fluorescent proteins derived from Aequorea GFP or Discosoma RFP, expressed in bacteria. Each different protein is excited (Exc) at different wavelengths and then emits (EM) light in the visible spectrum. This picture was also in 4BBY1070 background information to practicals.  Additional information for Green Fluorescence protein (GFP) Gene from the jellyfish Aequorea Victoria Green Fluorescent protein (27 kDa) The excitation wavelength is 395 nm (UV range), and fluorescence is emitted at 509 nm (green). Need to have UV light source to create green fluorescence. Methods for detecting GFP within living cells (slide 1) 1. Within cells (intracellular). Need fluorescence microscope (epifluorescence microscope or confocal microscope). High magnification will allows you to see nucleus, mitochondria and see which area of the cell the reporter was located in. Equipment needs ability to deliver a excitation wavelength and detect a specified emission wavelength. Methods for detecting GFP within living cells (slide 2) 2. In whole organisms. Need imaging system (computer/software) linked to camera with suitable lens. Drosophila with GFP expressed in eyes Figure 1. Making a reporter gene construct A. The native gene showing the core promoter and regulatory region upstream of the start codon. B. Transcriptional reporter. The regulatory region and core promoter are placed upstream of a reporter gene. C. Translation reporter. The reporter is typically placed downstream of the protein coding region of the native gene, leading to synthesis of a fusion protein composed of the native and reporter polypeptides. upstream of ATG downstream of ATG Definition of term fusion protein Fusion proteins (sometimes called chimeric proteins) are proteins created through the joining of two or more genes that originally coded for separate proteins to create a single protein. Transcription and slicing of introns (etc.) Mature mRNA Translation Protein of Reporter interest e.g. (red) GFP(green) Fusion protein COOH end NH2 end Using GFP to identify a regulatory region in two genes that are expressed only in the bilateral sensory neurons of C. elegans. The following parts of the workshop draw on information from González-Barrios, M., Fierro-González, J. C., Krpelanova, E., Mora- Lorca, J. A., Pedrajas, J. R., Peñate, X., Chavez, S., Swoboda, P., Jansen, G., & Miranda-Vizuete, A. (2015). Cis- and trans-regulatory mechanisms of gene expression in the ASJ sensory neuron of Caenorhabditis elegans. Genetics, 200(1), 123–134. https://doi.org/10.1534/genetics.115.176172 http://www.wormatlas.org/neurons/Individual%20Neurons/ASJframe set.html Information about the ASJ neurons (accessed September 2022) http://www.wormbook.org/chapters/www_transformationmicroinject ion/transformationmicroinjection.html Information about the method to create transgenic worms (accessed September 2022) You should not need to read these sources of information to answer Introduction Caenorhabditis elegans is a good model organism to identify key genes and proteins involved in the nervous system. C. elegans ASJ neurons are a bilaterally symmetrical pair of cilliated sensory neurons located at the anterior (head) of the nematode in the chemosensory organ. Figure 2 shows the location of the ASJ neurons in C. elegans. The ASJ neurons control the entry of the worm into the dauer stage (a form in which the worm can survive harsh conditions) and have a role in the regulation of aging and Introduction (second slide) In previous work (in other research papers) only two genes trx-1 (thioredoxin-1) and ssu-1 (alcohol sulfotransferase-1) have been reported to be expressed exclusively in ASJ neurons. TRX-1 belongs to the thioredoxin family of redox proteins, and plays a role in regulating worm longevity and dauer formation. SSU-1 is a sulfotransferase whose function is required for the response to volatile anaesthetics. This workshop mainly focuses on the work done on trx-1. Introduction (third slide) Note. In C. elegans trx-1 is the gene (lower case italics) Note. In C. elegans TRX-1 is the protein (UPPER CASE) Each species has it own agreed form of gene/protein nomenclature (different in different species). https://en.wikipedia.org/wiki/Gene_nomenclature Note. Species names are always in italics. The first time you mention a species name you should write in full in italics e.g. Caenorhabditis elegans. The second time you can use standard abbreviation genus = capital letter full stop species = full name e.g. C. elegans You don’t need to put abbreviation in brackets after the full species name. Introduction (fourth slide) Two plasmids were used to identify the regulatory regions of the trx-1 and ssu-1 genes. a. Plasmid Ptrx-1(1kb):: GFP contains the 1 kb region upstream of the trx-1 start codon joined to GFP. b. Plasmid Pssu-1(0.5kb):: GFP contains the 500 bp region upstream of the ssu-1 start codon joined to GFP. Regulatory DNA cloned here Note. You have to go to previous papers cited in the paper to work out which plasmid the authors used. Introduction (fifth slide) When the whole Ptrx-1(1kb):: GFP plasmid is microinjected into wild type C. elegans to create transgenic worms, GFP is observed in both the ASJ neurons as shown in Figure 3. A similar result was found also with plasmid Pssu-1(0.5kb):: GFP (not shown). Figure 3. Brightfield (top) and fluorescence (bottom) images of Ptrx-1(1kb):: GFP transgenic C. elegans. Bar, 20 µm. Taken from González-Barrios et al. 2015. Figure 4 (next slide) shows the results of microinjecting plasmids containing different deletions of the regulatory region of the Ptrx-1(1kb):: GFP plasmid into wild type C. elegans. 50 worms from 2 or 3 different transgenic lines were analysed for GFP expression in the ASJ region of each Figure 4 Figure 4. Results from transgenic C. elegans that contain different deletions of the trx-1 regulatory region in the Ptrx- 1(1kb):: GFP plasmid. Adapted from González-Barrios et al. 2015. Why does this figure legend say adapted (and not just taken from)? Questions based on Figure 4 a. What are the results obtained from constructs 1, 2, 3, 4 and 5 indicating? b. What are the results from constructs 6 and 8 indicating? c. What are the results from constructs 7, 9 and 10 indicating? d. Taking into account your answers to a, b and c where is the most likely place to look for the regulatory sequences in the trx-1 upstream region that control the expression of this gene in ASJ neurons?  a. What are the results obtained from constructs 1, 2, 3, 4 and 5 indicating? You can delete regions between -200 and -860 bp upstream of the trx-1 start codon and it does not affect the expression of GFP in the ASJ neurons. Therefore this region (-200 and -860 bp upstream of start codon) is not required for normal expression of trx-1 in ASJ neurons in C. elegans. The regulatory region of trx-1 is likely to be located between 0 and -200 bp. 100% GFP b. What are the results from constructs 6 and 8 indicating? You can delete the region between 0 and -84 bp upstream of the trx-1 start codon without affecting the expression of GFP in the ASJ neurons in C. elegans. The regulatory region is between -84 bp and -200 bp 100% GFP c. What are the results from constructs 7, 9 and 10 indicating? You can delete region between -103 and -180 bp upstream of the trx-1 start codon and it WILL knock out expression of GFP in the ASJ neurons in C. elegans. Therefore this region is likely to be required for normal expression of trx-1 in ASJ neurons in C elegans. Therefore the regulatory region of trx-1 is likely to be located between -103 and -180 bp upstream of the trx-1 start codon. 0-2% GFP d. Taking into account your answers to a, b and c where is the most likely place to look for the regulatory sequences in the trx-1 upstream region that control the expression of this gene in ASJ neurons? Answer - The regulatory region is likely to be between -103 and -180 bp upstream of the trx-1 start codon (so the region between the blue dotted lines on Figure 4). So ~80 bp region. Workshop information (first slide) The researchers then created a series of non-overlapping 20 to 30 bp deletions of the minimal promoter region upstream of the trx-1 start codon in the Ptrx-1(1kb):: GFP plasmid and microinjected each of these plasmids into C. elegans. The results are shown in Figure 5. Figure 5. Results from transgenic C. elegans that contain different microdeletions of the minimal promoter region of the Ptrx-1(1kb):: GFP plasmid. Adapted from González-Barrios et al. 2015. Questions based on Figure 5 a. What do the results in figure 5 tell you about the likely position of the region that controls trx-1 expression in ASJ neurons in C. elegans? The deletion that knocks out expression of GFP in C. elegans is -173 and -200 bp upstream of the trx-1 start codon. Therefore it is likely that the regulatory sequences that control trx-1 expression in ASJ neurons in C. elegans are in this region of DNA. 0% GFP Workshop information continued (second slide) To further identify the cis-regulatory motif that controls trx-1 expression in C. elegans ASJ neurons the researchers performed scanning substitution mutagenesis of the region - 200 bp and -173 bp upstream of the trx-1 start codon. In scanning substitution mutagenesis nucleotides are targeted specifically to change them to another nucleotide in the target DNA – in this case the Ptrx-1(1kb):: GFP plasmid. Figure 6 shows the result of this experiment. Figure 6 Figure 6. Identification of the ASJ motif in the promoter of the trx-1 gene. Scanning substitution mutagenesis was used to change nucleotides in the Ptrx-1(1kb):: GFP plasmid within the sequence in identified as necessary for trx-1 expression in ASJ neurons. The changed nucleotides are in orange. The top line is the wild type DNA sequence. Adapted from González- Barrios et al. 2015. From figure legend in actual paper A single asterisk (*) indicates that we have been unable to get stably transmitting lines from this construct; however, 10 F1 transgenic animals were scored and no GFP expression in ASJ was observed. A double asterisk (**) indicates that these transgenic animals show several fluorescent head neurons including ASJ. 0-6% GFP Questions based on Figure 6 a. What do the results in figure 6 tell you about the DNA sequences (motifs) most likely to control trx-1a expression in ASJ neurons? Mutations in two 6 bp sequences, separated by a 3 bp linker, significantly reduce or abolish GFP expression in ASJ neurons in C. elegans. CAACCC and AATTAA, separated by a 3 bp linker, are most likely to be the motifs that are required to control trx-1 expression in ASJ neurons in C. elegans. N.B. The two 6 bp sequences are shown in green and light blue on Figure 6. Workshop information (third slide) The researchers performed a similar set of experiments using the promoter region of ssu-1 (the second gene known to be expressed only in ASJ neurons). These experiments identified two similar motifs CTAACC and AATTAG (separated by a 3 bp linker) -221 bp from the ssu-1 start codon which were responsible for ssu-1 expression in ASJ neurons. These results suggest a bipartite ASJ motif are necessary for expression of trx-1 and ssu-1 in ASJ neurons. Further experiments found transcription factor SPTF-1 binds to the ASJ motif and regulates expression of trx-1 and ssu-1 in ASJ neurons (González-Barrios et al. 2015). a. In González-Barrios et al. 2015, the researchers compared the DNA sequences of fifteen different ASJ motifs using a bioinformatics program and produced the image shown below. What does this image show?  a. In González-Barrios et al. 2015, the researchers compared the DNA sequences of fifteen different ASJ motifs using a bioinformatics program and produced the image shown below. What does this image show? This is one way of showing a consensus sequence. Known as a sequence logo. The bigger the letter the more likely this nucleotide will be found in a putative ASJ motif. It is probably not very accurate as only 15 sequences where used to generate it only from Caenorhabditis sp. (see next slide) Sequence alignment of the predicted ASJ motif found in the promoters of the trx-1, ssu-1 and sptf-1 genes of several Caenorhabditis species define the ASJ motif, which is composed of two well-conserved 6 bp elements separated by a 3 bp more divergent linker sequence. Taken from González- Barrios et al. 2015. González-Barrios et al. 2015 used on-line software known as Weblogo. http://weblogo.threeplusone.com/ (accessed September 2022) They chose to present the sequence logo in what is known as a flat sequence logo (the size of the letters adds up to 1). Most sequence logos in publications are presented another way shown below (this is one of the options within Weblogo). b. The ASJ motif is responsible for the expression on trx-1 and ssu-1 in ASJ neurons in C. elegans. Would you expect to find the ASJ motif in other C. elegans genes? The simplest answer is yes. You might expect some genes that are also expressed in the ASJ neurons (and somewhere else in C. elegans) also have a ASJ motif in their regulatory region and SPTF-1 is involved in their expression in ASJ neurons. And other transcription factors would be expected to regulate the expression of these genes elsewhere. In González-Barrios et al. 2015 they use bioinformatics to screen the upstream region of a number of genes that are known expressed in ASJ neurons (and somewhere else in C. elegans) and identified ASJ like motifs in 53 of these genes. N.B. You would have determine experimentally if all these putative ASJ motifs in the 53 genes were real.