DNA & Gene Expression - CFrench.pdf

Medical Genetics DNA and gene expression The field of Genetics has always been important in Medicine....

Medical Genetics DNA and gene expression The field of Genetics has always been important in Medicine. Genetics is now relevant to all areas of medicine. So it is important to have a solid understanding of basic genetic concepts. Improved technology which has led to improved diagnostic ability the importance has expanded. Precision medicine: Tailoring specific treatment regimes to genetic profiles Curtis R. French Genome editing: Making specific changes in the genome to treat or cure Associate Professor disease – RECENTLY APPROVED SICKLE-CELL ANEMIA CURE (CASGEVY) INTRODUCED A NEW Biomedical Sciences MUTATION THAT COMPENSATES FOR THE MUTATION IN THE Beta-GLOBIN GENE 1 2 DNA and Health Nature versus Nurture Variations in our DNA can impact our health in a variety of ways. Here are a few examples: Both arguments are true DNA is inherited. – Single Gene Genetic Syndromes Hereditary deafness DNA is methylated. Hereditary blindness – The methylation pattern of DNA influences gene Hereditary Cancer Syndromes expression Cardiomyopathies Epilepsy – Environmental factors influence methylation and therefore nurture (environment) plays a role in – Multifactorial, complex Disorders gene expression Intellectual disability Epilepsy – DNA methylation patters may be heritable 3 4  Objectives Replication outline the central dogma of molecular 6407 biology (DNA > RNA > protein) summarize how DNA is able to act as the 6408 store of genetic information describe the structure and organization of 6409 genes describe the synthesis of RNA and how it 6410 is regulated (transcription) explain the concept of differential gene 6411 expression 6412 explain the importance of RNA processing summarize the process of protein 6413 synthesis including post-translational modifications 5 6 DNA- Chromosomes and Histones What is DNA Deoxyribonucleic Acid Three basic components – Deoxyribose (Pentose Sugar) – Phosphate Group – 4 nitrogenous bases A,G,T,C Adenine and guanine are purines (double carbon nitrogen rings) Thymine and cytosine are pyrimidines (single carbon nitrogen rings) Adenine pairs with thymine and Guanine pairs with Cytosine NHS – genetics education 7 8  DNA – is a double helix – Each strand is composed of a polymer of nucleotides arranged with a sugar-phosphate backbone – The phosphate entity joins the 3'-carbon of one deoxyribose with the 5'- carbon of the next. – The DNA molecule has polarity, each chain has a 5' end and a 3' end. Watson, Crick and Wilkins won the Nobel Peace Price for deciphering the double helix nature of DNA in 1962. Rosalind Franklin was also 9 instrumental. She died in 1958 at the age of 37 10 DNA Replication- -enzymes and accessory proteins: DNA Replication The two original (parental) strands are separated (denatured) Helicases – unwind the DNA – a new daughter strand is synthesized for each DNA molecule DNA polymerase – adds nucleotides and also The synthesis of new strands occurs at replication forks along the chromosome. proofreads Primase – an RNA polymerase Is unidirectional… – New synthesis occurs in the 5’-3’ initiates the synthesis of each direction. Okazaki fragment – The DNA polymer grows by adding bases to 3’ end Ligase – joins the Okazaki – One strand grows by a series of short fragments daughter strands, called Okazaki fragments, 11 12  Case 1 Werner Syndrome 41 year old woman who presented with a Premature aging syndrome constellation of concerns: – Early greying (age 28) Caused by mutations in the WRN gene – Bilateral cataracts (age 38) – Type 2 diabetes (age 32) The WRN gene encodes a helicase involved in DNA – Premature menopause (age 36) replication, repair, and transcription – osteoporosis 13 14 Summary 1 Genes Objective 6407- Central Dogma of Genetics DNA sequences that encode polypeptides (proteins) DNA RNA Protein The “expression” of the genetic information is a two step process: Objective 6408- DNA as the store of genetic information – Transcription (DNA RNA) ….nucleus 4 nitrogen bases Base pairing rules allow for copying of DNA to daughter cells – Translation (RNA Protein)…cytoplasm 15  Gene Transcription (DNA) Genetic code is “transcribed” by new RNA synthesis. The final product in the expression of most genes is messenger RNA (mRNA). – RNA is a polymer molecule similar to DNA..except the sugar is ribose (vs. deoxyribose)..Uracil(U) replaces Thymine (T)..and it is usually single-stranded Messenger RNA Important proteins necessary for transcription include: (mRNA) – RNA polymerase – Transcription Factors Copyrights apply 18 Transcription Transcription Factors (TF) Specialised proteins required for DNA RNA: General transcription factors – Necessary for transcription of most genes Specific transcription factors – Initiate transcription of specific genes at specific times (eg. Tissue specific; precise developmental stage). – These specific transcription factors play an important role in Gene Expression TFs bind to specific DNA sequences in the regulatory region of genes. – Upstream, withing introns, can act over long ranges 19 20  Epigenetic methylation affects Non-coding RNAs and gene expression transcription Several types of RNA can be transcribed and but don’t encode proteins. They can affect gene expression (mRNA levels) MicroRNAs (miRNA) – Bind in the 3’ untranslated region of a mRNA, facilitate their degredation (or their trnsaltion into proteins) Long non-coding RNAs (LncRNA) – Can regulate epigenetic changes DNMT: Dinucleotide methyl transferases Wang et al., 2017 21 22 Differential gene expression Summary 2 Objective 6410- Synthesis of RNA Different cells have different levels of gene expression – Different transcription factors, methylation patterns, non-coidng RNAs – Requires transcription factors, RNA polymerase. – Synthesized using the antisense strand as template. Changes in gene expression can lead to disease states – Bases have a ribose sugar ring – Uricil replaced thymidine “Omics Technology” Can sequence the transcription of a single cell Objective 6411- Differential gene expression – Different cells express different genes Identify differentially expressed genes – Controlled by cell/tissue specific transcription factors, promoter Cancer “transcriptomics” methylation, interactions with non-coding RNAs – Normal cancer tissue – Changes in gene expression can lead to diseases – Aid in prognosis – Changes in gene expression can be used as prognostic markers – Personalized treatments 23 24  RNA Processing RNA Processing RNA transcript (primary RNA) Coding sequences are “Interrupted” by non- coding sequences – Exons represent protein coding sequences post-transcriptional – Introns represent non-coding sequences modifications (intervening sequences). mRNA (Messenger RNA) NHS – genetics education 25 Post-transcriptional Modifications CASE Series 2 – Splicing (remove introns from primary transcript) – 5’ capping (5’ end) – Poly-adenylation (3’ end) Colorectal cancer pedigrees from Newfoundland 27 28  Case Study 2 Summary 3 Research testing revealed a Objective 1402- Importance of RNA processing mutation in the Adenomatous polyposis coli (APC) gene. An identical 5´splice site acceptor Introns must be spliced out to obtain the correct mutation was found in five reading frame for polypeptide synthesis Newfoundland families with colorectal cancer. (Hum Genet, 1999, Spirio et al) A cap 5’ (m7G), and a 3’ PolyA tail is required for The mutation results in abnormal splicing leading to an abnormal stability and subsequent translation. RNA without exon 4. APC gene is a tumor suppressor gene. 29 30 31 Copyrights apply  Translation (RNA Protein) mRNA is transported to the cytoplasm The amino acid sequence of the protein is encoded in the mRNA by units of three RNA bases, termed codons. The initiator codon (AUG) establishes the reading frame of the mRNA. The nascent string of amino acids is called a polypeptide 33 34 The genetic code is degenerate (20 amino acids are encoded by 64 Translation 01_22.jpg codons (61 code for amino acids, 3 code for stops) Transfer RNAs (tRNAs) – provide the molecular link between the coded base sequence on the mRNA and the amino acid sequence of the protein. – The tRNA molecule has a 3-base sequence called the anticodon. – This anticodon is complementary to the 3-base codon of the mRNA. – Complementary base pairing between the tRNA and the mRNA delivers the encoded amino acid to the growing polypeptide. – Each mRNA has a 5’ and a 3’ untranslated region The four codons (in red) are interpreted differently in the nucleus (in purple) and mitochondria (in green) of mammalian cells 35 36  Errors in translation (examples) A mutation that changes the codon AAA to TAA – will introduce a stop codon. – This is considered a nonsense mutation and will cause the protein to be truncated The Central Dogma: (pathogenic) A mutation that does not change the amino acid is called a silent mutation and is Post-translational modification usually not pathogenic. (example CCG-CCA both encode the amino acid Proline) DNA RNA Protein Mature Protein A mutation that changes AAA to AAC will cause a change in the amino acid. LncRNA – This is considered a missence mutation (more difficult to predict if the mutation is pathogenic it will depend on how the amino acid change miRNA alters the protein function) rRNA tRNA INDEL mutation- insertion or deletion of nucleotides. This can lead to a shift in the open reading frame of a gene, and are usually pathogenic, especially when the insertion or deletion is not a multiple of 3. Post-translational modifications Osteogenesis Imperfecta Type IX Changes that occur to the nascent polypeptide chain. Smaller, less mineralized May involve: bones, shape deformities – cleavage of the polypeptide (eg. Proinsulin>insulin subunits) – formation of disulfide bridges Can range from mild to severe – attachment of functional groups: Carbohydrate (sugars) Mutation in PPIB gene Phosphate (required to fold collagen Lipids into triple helix formation. Acetate Collagen is a major If the post-translational modification does not occur properly due to a component of bone mutation – the protein may not function correctly or at all. 39 40  Summary 4 Objective 6413- Protein synthesis and post- translation modifications – Polypeptide synthesis occurs in the cytoplasm using ribosomes. – tRNA molecules build growing polypeptide chains based on three anticodons that recognize 3 base pair codons in the mRNA – Translation begins at the start codon (AUG) and ends when the ribosome reached a Stop codon (UAA, UAG, UGA) – Post translationsal modifications include folding with other polypeptides, cleavage of the polypeptides, addition of sugar and lipids (and other small molecules) 41