Molecular Basis of Gene Expression - Workshop 1
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Questions and Answers

What is the primary function of the araBAD promoter in the context of pGLO?

  • To facilitate the transformation of E. coli cells
  • To inhibit the expression of GFP
  • To activate the expression of GFP when arabinose is present (correct)
  • To serve as a selection marker for antibiotic resistance

    • Which of the following is NOT mentioned as a characteristic of a good reporter protein?

  • Must be quantifiable
  • Must produce a distinct signal
  • Must be highly stable (correct)
  • Must be easy to visualize

    • Which reporter gene is associated with the production of light?

  • β-Glucuronidase
  • Chloramphenicol Acetyltransferase
  • β-Galactosidase
  • Luciferase (correct)

    • In the experiment with pGLO, what method was used to visualize the expression of GFP?

    <p>UV light source (C)</p> Signup and view all the answers

    What type of reporter gene is GFP classified as in the pGLO experiment?

    <p>Transcriptional reporter (D)</p> Signup and view all the answers

    What is the primary function of Green Fluorescent Protein (GFP)?

    <p>To emit green fluorescence (C)</p> Signup and view all the answers

    What is the role of competent E. coli cells in the pGLO experiment?

    <p>To take up plasmid DNA for transformation (A)</p> Signup and view all the answers

    Which of the following fluorescent proteins can be derived from Aequorea?

    <p>GFP (D)</p> Signup and view all the answers

    Which excitation wavelength is required to visualize the fluorescence emitted by GFP?

    <p>395 nm (D)</p> Signup and view all the answers

    What type of microscopy is necessary to detect GFP within living cells?

    <p>Confocal microscopy (A)</p> Signup and view all the answers

    Which of the following reporter genes can be used to study gene regulation?

    <p>All of the above (D)</p> Signup and view all the answers

    Which system is required for detecting GFP expression in whole organisms?

    <p>An imaging system linked to a camera and software (D)</p> Signup and view all the answers

    In the context of fusion proteins, where is the reporter gene typically placed?

    <p>Downstream of the protein coding region (D)</p> Signup and view all the answers

    What is a fusion protein?

    <p>A protein formed by combining multiple genes into one (D)</p> Signup and view all the answers

    Which of the following organisms is mentioned as having GFP expressed in its eyes?

    <p>Drosophila (C)</p> Signup and view all the answers

    What do transcriptional reporters typically include?

    <p>Native gene and regulatory region upstream of the reporter gene (A)</p> Signup and view all the answers

    What is the length of the regulatory region in plasmid Ptrx-1(1kb)::GFP?

    <p>1 kb (A)</p> Signup and view all the answers

    Which neurons were observed to express GFP when the Ptrx-1(1kb)::GFP plasmid was microinjected into C.elegans?

    <p>ASJ neurons (A)</p> Signup and view all the answers

    What indicates that the transgenic animals show fluorescent head neurons in the study?

    <p>A double asterisk (**) (D)</p> Signup and view all the answers

    What does plasmid Pssu-1(0.5kb)::GFP contain in terms of regulatory region?

    <p>500 bp upstream of the ssu-1 start codon (A)</p> Signup and view all the answers

    What method was used to create transgenic C.elegans with the plasmids?

    <p>Microinjection (A)</p> Signup and view all the answers

    What happens to GFP expression in ASJ neurons when mutations occur in the identified sequences?

    <p>It significantly reduces or abolishes expression (A)</p> Signup and view all the answers

    Which of the following motifs are identified as necessary for trx-1 expression in ASJ neurons?

    <p>CAACCC and AATTAA (D)</p> Signup and view all the answers

    What does the sequence logo image produced by the researchers represent?

    <p>Consensus sequences of ASJ motifs (C)</p> Signup and view all the answers

    What likely conclusion can be drawn about the trx-1 regulatory region from the constructs analyzed?

    <p>The regulatory sequences controlling gene expression are located in the upstream region (D)</p> Signup and view all the answers

    How are the two identified motifs for ssu-1 expression structured?

    <p>Separated by a 3 bp linker (D)</p> Signup and view all the answers

    What was observed when the whole Ptrx-1(1kb)::GFP plasmid was microinjected?

    <p>GFP expression was observed in the ASJ neurons (D)</p> Signup and view all the answers

    What is the role of the transcription factor SPTF-1 in relation to the motifs?

    <p>It binds to the ASJ motif and regulates expression (D)</p> Signup and view all the answers

    Why does the figure legend state that the figure is 'adapted' and not 'taken from'?

    <p>Substantial changes were made to the original data (B)</p> Signup and view all the answers

    What is a limitation of the sequence logo generated by the bioinformatics program?

    <p>It is based on a small number of sequences (D)</p> Signup and view all the answers

    How many DNA motifs were compared by the researchers to produce the sequence logo?

    <p>Fifteen different motifs (C)</p> Signup and view all the answers

    Which region can be deleted without affecting the expression of GFP in ASJ neurons?

    <p>-200 to -860 bp upstream of the trx-1 start codon (B)</p> Signup and view all the answers

    What does the deletion of the region between -103 and -180 bp indicate?

    <p>It causes a complete knock out of GFP expression. (D)</p> Signup and view all the answers

    Where is the likely regulatory region of trx-1 located?

    <p>-103 and -180 bp upstream of the trx-1 start codon (B)</p> Signup and view all the answers

    What effect does deleting the region between 0 and -84 bp have on GFP expression?

    <p>Has no effect on expression (B)</p> Signup and view all the answers

    Constructs 6 and 8 demonstrate that:

    <p>GFP expression remains unchanged despite the deletion of -84 to 0 bp. (D)</p> Signup and view all the answers

    If the trx-1 expression in ASJ neurons is disrupted, which region is most likely implicated?

    <p>Between -103 and -180 bp (D)</p> Signup and view all the answers

    What percentage of GFP expression was seen when the region between -103 and -180 bp was deleted?

    <p>0-2% (C)</p> Signup and view all the answers

    What conclusion can be drawn from the overall study regarding trx-1 regulatory sequences?

    <p>They are likely confined to a narrow range within -103 to -180 bp. (D)</p> Signup and view all the answers

    What is the likely position of the region that controls trx-1 expression in ASJ neurons in C.elegans?

    <p>-200 to -173 bp upstream of the trx-1 start codon (B)</p> Signup and view all the answers

    What technique was used to identify the cis-regulatory motif controlling trx-1 expression?

    <p>Scanning substitution mutagenesis (A)</p> Signup and view all the answers

    Which nucleotides were targeted in the scanning substitution mutagenesis experiment?

    <p>-200 to -173 bp upstream of the trx-1 start codon (B)</p> Signup and view all the answers

    What does a 0% GFP expression indicate in the context of the trx-1 gene study?

    <p>The regulatory region is ineffective in controlling expression (A)</p> Signup and view all the answers

    What could be a reason for not obtaining stably transmitting lines from the trx-1 construct?

    <p>The mutagenesis altered crucial regulatory sequences (A)</p> Signup and view all the answers

    In the experiment, what does the use of orange-highlighted nucleotides signify?

    <p>Targeted mutations made in the experimental sequence (C)</p> Signup and view all the answers

    What was one of the outcomes after the scanning substitution mutagenesis was performed?

    <p>No score of GFP expression in ASJ neurons (B)</p> Signup and view all the answers

    How did the researchers confirm the regions necessary for trx-1 expression?

    <p>Through a combination of mutagenesis and GFP expression analysis (B)</p> Signup and view all the answers

    Flashcards

    Reporter Genes

    Genes used to study gene expression. They are fused to regulatory regions and their expression is monitored.

    GFP

    Green Fluorescent Protein, a common reporter gene.

    Regulatory Region

    Part of a gene that controls when and how much of the gene is expressed, often including a promoter.

    Transcription Factors

    Proteins that bind to specific sequences in regulatory regions, influencing gene transcription.

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    Promoter

    A region within a gene that specifies where transcription begins.

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    Arabinose

    A molecule that activates the araBAD promoter, causing GFP to be expressed.

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    pGLO plasmid

    A plasmid that contains GFP and the araBAD promoter, used to study gene expression in bacteria.

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    Visualizing gene expression

    Ways to see if a gene is being switched on/off (e.g., fluorescent proteins)

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    Excitation Wavelength

    The wavelength of light needed to make GFP glow.

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    Emission Wavelength

    The wavelength of light emitted by GFP when it glows.

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    Fusion Protein

    A protein created by combining two or more different proteins.

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    Reporter gene construct

    A construct of gene with regulatory region that combined with a reporter gene

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    Core Promoter

    The part of a gene that's essential for the gene to be transcribed.

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    Ptrx-1(1kb):: GFP

    A plasmid containing the 1 kb region upstream of the trx-1 start codon fused to GFP, used to study gene expression in the ASJ neurons of C. elegans.

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    Pssu-1(0.5kb):: GFP

    Similar to Ptrx-1(1kb):: GFP, this plasmid contains the 500 bp region upstream of the ssu-1 start codon fused to GFP. It was also used in the study to analyze gene expression in ASJ neurons.

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    Transgenic Worms

    Worms that have been modified to carry foreign DNA, like the Ptrx-1(1kb):: GFP plasmid, allowing researchers to study gene expression.

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    ASJ Neurons

    Specific neurons in C.elegans that are involved in sensing chemical cues.

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    Deletion Constructs

    Modified plasmids where certain parts of the regulatory region have been removed. These are used to identify which parts of the regulatory region are important for gene expression.

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    GFP Expression in ASJ Neurons

    The amount of GFP produced in the ASJ neurons of transgenic C.elegans. This is used to understand how the regulatory region controls gene expression.

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    Finding the Regulatory Sequences

    By analyzing the results of deletion constructs, researchers can identify the specific DNA sequences in the regulatory region that control gene expression in ASJ neurons.

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    -860 to -200 bp region

    This region upstream of the trx-1 start codon can be deleted without affecting GFP expression in ASJ neurons.

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    -200 to -84 bp region

    This region upstream of the trx-1 start codon can be deleted without affecting GFP expression in ASJ neurons.

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    -103 to -180 bp region

    This region upstream of the trx-1 start codon is crucial for GFP expression in ASJ neurons. Deleting it shuts down GFP production.

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    Regulatory region of trx-1

    The specific DNA sequence within a gene that controls when and how much of the gene is expressed. For trx-1, it's likely between -103 and -180 bp upstream of the start codon.

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    trx-1

    A gene studied in C. elegans, likely involved in the development of ASJ neurons.

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    GFP in ASJ neurons

    Green Fluorescent Protein (GFP), a marker used to study gene expression, specifically in ASJ neurons.

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    Microdeletions

    Small deletions of the DNA sequence within a gene, specifically in the promoter region of trx-1.

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    Ptrx-1(1kb)::GFP plasmid

    A modified plasmid containing the trx-1 gene (1kb) with a GFP reporter gene fused to it, used to study trx-1 expression.

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    trx-1 gene

    A gene in C. elegans that is crucial for regulating the development and function of the ASJ sensory neurons.

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    Regulatory Region (trx-1)

    A specific DNA sequence located upstream of the trx-1 gene that controls when and how much the gene is expressed in ASJ neurons.

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    Scanning Substitution Mutagenesis

    A technique used to systematically change individual nucleotides within a DNA sequence, helping to identify important regulatory elements.

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    Cis-Regulatory Motif

    A specific DNA sequence within the regulatory region that binds to proteins to control gene expression.

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    Knockout

    Inactivation or deletion of a gene's function, often through mutation or targeted removal.

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    What is the ASJ motif?

    A specific DNA sequence (or motif) found in the regulatory regions of genes expressed only in ASJ neurons. It controls when and how much of these genes are expressed.

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    What are the ASJ motifs in trx-1a?

    Two 6bp sequences (CAACCC and AATTAA) separated by a 3bp linker.

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    What is the function of the ASJ motif?

    It controls the expression of genes (like trx-1a) specifically in ASJ neurons.

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    ssu-1 ASJ motif

    Two similar motifs (CTAACC and AATTAG) separated by a 3bp linker, found in the regulatory region of ssu-1.

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    How many ASJ motifs were analyzed?

    The researchers analyzed 15 different ASJ motifs.

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    What's a sequence logo?

    A graphical representation showing the most common nucleotide at each position within a set of DNA sequences.

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    What does a bigger letter in a sequence logo indicate?

    A bigger letter indicates a higher probability of that nucleotide being found at that position in the ASJ motif.

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    What does the sequence logo for the ASJ motifs suggest?

    It suggests a consensus sequence for the ASJ motif, highlighting the most common nucleotides within the motif.

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    Study Notes

    Molecular Basis of Gene Expression - Workshop 1

    • Workshop focus: Using reporter genes to study gene expression
    • Course code: 5BBG0205
    • Lecturer: Dr Shirley Coomber

    Learning Outcomes

    • Students should be able to identify different types of reporter genes
    • Students should understand how reporter genes locate regulatory regions of genes
    • Students should understand how transcription factors bind to specific sequences in regulatory regions of genes

    Revision (Practical 4BBY1070)

    • Plasmid pGLO transformed into competent E. coli cells
    • Transformed cells grown on different media agar plates
    • pGLO digested with restriction enzymes (practical 1)
    • GFP (green fluorescent protein) cloned in front of araBAD promoter

    Revision (Practical 4BBY1070 continued)

    • E. coli pGLO cells grown on two LB agar plates
      • One with ampicillin
      • One with ampicillin and arabinose
    • GFP presence in E. coli pGLO colonies detected using UV light
    • Colonies glow green in UV light when grown on LB amp agar plates with arabinose
    • Arabinose activates araBAD promoter, leading to GFP expression
    • This is an example of a transcriptional reporter system (GFP acts as a reporter gene)

    Reporter Genes

    • Various genes used as reporter genes: Chloramphenicol Acetyltransferase (CAT), Luciferase, β-Galactosidase, β-Glucuronidase (GUS), Alkaline Phosphatase (AP), β-Lactamase, Fluorescent Proteins
    • Good reporter proteins are easy to visualize/detect and quantifiable

    Fluorescent Proteins

    • Derived from Aequorea GFP or Discosoma RFP
    • Excited at specific wavelengths, emitting different wavelengths

    Methods for Detecting GFP within Living Cells (Slide 1)

    • Fluorescence microscopy (epifluorescence or confocal) needed
    • High magnification allows visualization of nucleus, mitochondria, and other cellular structures where the reporter gene is located
    • Equipment needs excitation wavelength delivery and detection mechanisms

    Methods for Detecting GFP within Living Cells (Slide 2)

    • Whole organism imaging: Imaging system (computer/software) linked to camera with appropriate lens needed
    • Example: Drosophila with GFP expressed in eyes

    Making a Reporter Gene Construct

    • The native gene, including core promoter and regulatory region, are upstream of the start codon
    • Regulatory region and core promoter placed upstream of another reporter gene (transcriptional reporter)
    • Reporter gene placed downstream from protein-coding region of native gene to create a fusion protein (translational reporter)

    Definition of Fusion Protein

    • Fusion proteins combine two or more proteins originally encoded by separate genes

    Using GFP to Identify Regulatory Regions (Study Materials Summary)

    • Study uses C. elegans
    • Focuses on two genes, trx-1 and ssu-1, expressed exclusively in ASJ neurons
    • Focus for this study is on trx-1

    Introduction (Further Details)

    • trx-1 is a gene (lower case italics)
    • TRX-1 is the protein (upper case)
    • Species names always in italics
    • Use abbreviations for second mention of species

    Introduction (Fourth Slide)

    • Two plasmids used
      • Ptrx-1 (1kb): 1kb region upstream of trx-1 start codon joined to GFP
      • Pssu-1 (0.5kb): 500bp region upstream of ssu-1 start codon joined to GFP

    Introduction (Fifth Slide)

    • Whole Ptrx-1(1Kb):GFP plasmid microinjected into wild-type C. elegans to create transgenic worms
    • GFP observed in ASJ neurons
    • Similar results with Pssu-1(0.5Kb):GFP plasmid

    Figure 4 and Questions

    • Results of microinjecting plasmids with deletions of the trx-1 regulatory region
    • Constructs 1 through 5 show that no regulatory control exists between positions -860 to -200
    • Constructs 6 and 8 show that a regulatory region exists from -200 to -84
    • Constructs 7, 9 and 10 indicate that the required regulatory region is between positions -103 and -180

    Workshop Information (First Slide)

    • Created a series of non-overlapping 20-30bp deletions of trx-1 promoter upstream region
    • Microinjected into C. elegans
    • Results shown in Figure 5

    Questions Based on Figure 5

    • Deletion of -173 and -200 bp upstream the trx-1 start codon prevents GFP expression in C. elegans
    • The regulatory sequences for trx-1 expression in the ASJ neuron of C. elegans are located within this region of DNA.

    Workshop Information (Second Slide – Continued)

    • Scanning substitution mutagenesis of the region from -200 to -173
    • Changes nucleotides in the Ptrx-1(1kb):: GFP plasmid

    Figure 6

    • Identifying the ASJ motif in the promoter of the trx-1 gene using scanning substitution mutagenesis
    • DNA changes are in orange in Figure 6
    • Wild-type DNA is in the top line
    • Mutations in the two 6 bp sequences (separated by a 3bp linker) significantly reduce or abolish GFP expression in ASJ neurons
    • CAACCC and AATTAA are likely regulatory motifs (separated by a 3bp linker)

    Workshop Information (Third Slide –Continued)

    • Experiments using ssu-1 promoter region
    • Identified two similar motifs CTAACC and AATTAG regions located -221bp from the ssu-1 start codon
    • Suggests a bipartite ASJ motif is necessary for the expression of trx-1 and ssu-1 in ASJ neurons
    • Further experiments found transcriptional factor SPTF-1 binds to the ASJ motif and controls expression of trx-1 and ssu-1 in ASJ neurons of C. elegans

    Additional Information

    • Researchers identified and compared ASJ motifs found in 15 different C. elegans genes using a bioinformatics program (Weblogo)
    • The ASJ motif is likely to be present in other C. elegans genes.

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    5BBG0205 Workshop - Reporter Genes Answers 2024-2025 PDF

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    Join Dr. Shirley Coomber in this engaging workshop focused on using reporter genes to study gene expression. Learn to identify different types of reporter genes and understand their role in locating regulatory regions and transcription factor binding. Perfect for students aiming to deepen their practical understanding of genetic studies.

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