Gene Expression and Protein Synthesis Quiz

Learning Outcomes

• Reinforce knowledge from a previous session.
• Apply knowledge through exercises.
• Understand why studying gene expression is important.
• Recognize the medical significance.
• Identify techniques used to study DNA, RNA, and proteins.
• Explore the possibility of synthesizing DNA, RNA, and proteins in vitro.

The Genetic Code

• There are 64 codons that code for 20 amino acids.
• Third base in a codon is degenerate meaning multiple codons can code for the same amino acid.
• Start codon is AUG, which codes for methionine, the first amino acid in a polypeptide.

One Gene - One Protein Hypothesis

• Proposed by Beadle and Tatum in 1941.
• Not always true as some genes can make multiple proteins through alternative splicing.

Alternative Splicing

• Process where exons are joined together in different combinations to produce various mRNA isoforms.
• Example: Drosophila Dscam gene can produce up to 38,016 isoforms through alternative splicing.

Medical Importance of Gene Expression Errors

• Errors in gene expression can lead to various disorders, including:
  • Duchenne Muscular Dystrophy
  • Spinal Muscular Atrophy
  • Cystic Fibrosis
  • Pulmonary Arterial Hypertension
• Can also influence the predisposition to common diseases, such as:
  • Breast cancer
  • Lung cancer
  • Prostate cancer
  • Blood cancer

Errors in Genes Causing Diseases

• Duchene Muscular Dystrophy: mutation in the DMD gene
• Spinal Muscular Atrophy: mutation in the SMN1 gene
• Cystic Fibrosis: mutation in the CFTR gene
• Pulmonary Arterial Hypertension: mutation in the BMPR2 gene
• Breast cancer: mutation in the BRCA1 and BRCA2 genes.

Transcription Errors

• Over-expression of the transcription factor (TF) MYC is found in various cancers.
• Mutations in one copy of the gene encoding the TF can also lead to diseases:
  • Haploinsufficiency: one copy of the gene is not enough (e.g., p53 gene).
  • Dominant mutation: change in the gene sequence alters protein function and dominates over the wild-type (e.g., TFCP2L3 gene and genetic deafness).

Splicing Errors

• Errors in alternative splicing are common.
• In Cystic Fibrosis: mutation in exon 7 of the SMN1 gene leads to its skipping and a non-functional protein.

Translation Errors

• Mutations can affect translational efficiency.
• Mutations in key translation proteins can lead to disease, including:
  • Translation initiation factors (e.g., eIF2 gene - Vanishing White Matter disease).
  • Release factors (e.g., eRF3 gene - gastric cancer).
  • tRNA transfer proteins or the ribosome itself (rare anemias and several neurodegenerative diseases).

Techniques for Detecting Gene Expression

• Gel Electrophoresis of DNA/RNA:
  • DNA is negatively charged and moves towards the cathode in an electric field.
  • Used to separate DNA or RNA fragments based on size.
• Western blotting:
  • Used to detect specific proteins.
  • Steps:
    • Gel Electrophoresis
    • Blotting
    • Blocking
    • Antibody Probing
    • Detection/Visualisation
• PCR: polymerase chain reaction
  • Amplifies specific regions of DNA.
  • Used for:
    • Gene expression profiling (qPCR)
    • Diagnostics
    • Gene cloning
    • Legal disputes

Making DNA, RNA, and Protein in a Tube

• There are protocols available for synthesizing RNA in vitro.
• Researchers have successfully created pre-mRNA for splicing in a tube.