Molecular Basis of Gene Expression - Workshop 1

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.