Molecular Biology of the Gene & Gene Expression (Part 2) PDF

Molecular Biology of the Gene & Gene Expression (Part 2) Dr Talat Nasim [email protected] 1  LEARNING OUTCOME OF THIS SESSION  Re-inforce what we have learnt from the last session  Some application exercises  Why do we need to know...

Molecular Biology of the Gene & Gene Expression (Part 2) Dr Talat Nasim [email protected] 1  LEARNING OUTCOME OF THIS SESSION  Re-inforce what we have learnt from the last session  Some application exercises  Why do we need to know about gene expression?  What is the medical importance?  What techniques are used to detect/determine DNAs, RNAs and proteins  Can we make DNAs, RNAs and proteins in tube?  APPLICATION EXERCISE Activity 1: A. Draw a diagram of an eukaryotic gene. B. Write the sequences of that gene (just write the sequence which is around 30 base pair long) C. Make a protein molecule out of the above gene. A) Untranslated AUC /Start signal) UGA VAG VAA (Stop Signal) CUTR region 5'3 S enhancer Promotor ExoIuNiE I E Terminator enhancer 3 Silencer TATA box M Silencer B) A CGA T GG(GAATT va(vAccGCVVAA  The Genetic Code 64 codons - 20 amino acids 3rd base is degenerate Start 64 (odon 20 AA Start code AUG codes for methionine (Met) – the first amino acid in a polypeptide UGU UGC UAst codon Ava 4 met  OPEN DISCUSSION Why do we need to know about gene expression?  One gene - one protein hypothesis? Proposed by Beadle and Tatum (1941) DNA makes RNA makes Protein GENE GENE XX PROTEIN X or PROTEIN Xa PROTEIN Xb PROTEIN Xc One gene makes one protein PROTEIN Xd PROTEIN Xe 17,800 genes How can a similar number of genes give a worm and us? 20,000 genes 6  Answer 1 - alternative Splicing! During splicing the exons can be joined together in a variety of ways 7 Alternative splicing of the drosophila Dscam gene could potentially generate 38,016 isoforms Alternative mRNAof a single gene Cell 118: 619-633, 2004 8 Medical Importance Errors in gene expression! 9  ERRORS IN GENE EXPRESSION  May cause many uncommon disorders (more than 3000) such as Duchene Muscular Dystrophy, Spinal Muscular Atrophy, Cystic Fibrosis and Pulmonary Arterial Hypertension.  Can also influence the predisposition of many common diseases such as breast cancer, lung cancer, prostate cancer and 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 lead to disease  Many examples!  Over-expression of the transcription factor (TF) MYC is found in numerous cancers  If one copy of the gene encoding the TF is mutated this can also lead to disease e.g.:  Haploinsufficiency – where one copy of the gene is not enough e.g. p53 gene  Dominant mutation – where a change in the gene sequence generates change in the protein that exerts dominance over the wild-type e.g. TFCP2L3 gene and genetic deafness 12  Splicing - mRNA processing errors  In many cases this involves errors in alternative splicing  Cystic fibrosis  Mutations found in the exon 7 of SMN1 gene.  As a result, the exon 7 is skipped and the protein produced is non- functional August 3, 2001 - October 29, 2002 13  mRNA translation errors  Mutations that affect translational efficiency  However…  Also examples of mutations in key translation proteins leading to disease:  Translation initiation factors, e.g. eIF2 gene - a neurodegenerative disease called Vanishing White Matter disease (VWM)  Release factors, e.g. eRF3 gene – gastric cancer  Mutations affecting the tRNA transfer proteins or the ribosome itself (rare types of anaemia and several neurodegenerative diseases) 14  OPEN QUESTION How do we determine gene expression? What techniques do we use to detect DNAs, RNAs and proteins?  Gel Electrophoresis of DNAs/RNAs DNA is negatively charged (-): Anode (-) Cathode (+) So will move from the anode to the cathode Run at: 200 Volts 1% agarose gel 1x TAE or TBE buffer 16 Gel Electrophoresis of DNAs/RNAs 17 A gel image loaded with DNAs ladder ladder 18 Western blot to detect proteins 75 KDa p-TAK1 35 KDa TAK1 45 KDa β-actin Nasim et al., Human Molecular Genetics 2012 19 Steps involved in western blotting GBBAD 1. Gel Electrophoresis 2. Blotting (or transfer. e.g. semi-dry) 3. Blocking 4. Antibody Probing 5. Detection/Visualisation Steps involved in western blotting CAN YOU MAKE RNA IN A TUBE? Optional Reading: Protocol CAN YOU MAKE THE PRE-MRNA TO SPLICE IN A TUBE? Nasim et al., Nucleic Acids Res 2002  How can we detect gene expression? - PCR Polymerase Chain Reaction (PCR)  ‘Invented’ by Kary Mullis in the mid-1980’s  Key technique in molecular biology!  Enables the amplification of a specific region of DNA from just a single molecule of starting material  Gene expression profiling (Quantitative real-time PCR, qPCR)  Detects how many copies of a gene (genes) you have  Idea of the regulation of gene expression at a given time  Diagnostics  Gene cloning  Legal disputes 24 How can we detect gene expression? - PCR 25  SUMMARY  Application exercises- structure of an eukaryotic gene and translation of DNA/RNA to protein.  What is the medical importance- errors in gene expression can cause many diseases  Techniques that are used to detect/determine DNAs, RNAs and proteins  We can make DNAs, RNAs and proteins in tube