Topic 7

• The ethics of embryonic stem cells involve the potential benefits of producing various cell types and reducing animal testing, but also raise concerns about the beginning of human life, consent, and the killing of embryos.
• Genome refers to all of the DNA, including mitochondrial and chloroplast DNA.
• PCR can be used to amplify samples by denaturing, annealing, and synthesizing a complimentary DNA strand using TAQ polymerases.
• Gene sequencing involves replicating single stranded DNA using radioactive terminator bases, scanning fragments, and deducing the gene sequence.
• DNA profiling uses restriction enzymes to cut DNA into unique fragments, which are then separated and combined with other samples.
• Transcription factors are proteins that bind to DNA and can change the rate at which mRNA is produced.
• RNA splicing can result in different products by splicing and combining exons and organizing them into different orders.
• Epigenetic modifications, such as ncRNA, histone modification, and DNA methylation, can control gene expression.
• Epigenetic modification is important for ensuring cell differentiation.
• Stem cells have the ability to differentiate into other cell types, with totipotent, pluripotent, and multipotent stem cells having different capabilities.
• The ethics of embryonic stem cells involve benefits such as the potential to grow new organs and drawbacks such as religious and ethical concerns.
• Genome


  All genes inside an organism, including chromosomal, chloroplast, mitochondrial and ribosomal DNA
• PCR (polymerase chain reaction)


  A tool used to amplify a DNA sample using taq polymerases
• PCR process


  1. Denaturation: the hydrogen bonds between the template DNA are broken (90-95c)

  2. Annealing: primers and taq polymerase binds to the single strands of DNA (50-55c)

  3. Synthesis: nucleotides are added to create two double DNA strands (72c)
• Human genome project: a project aiming to sequence the whole human genome and identify all disease causing genes
• DNA profiling


  Analysis of unique patterns of DNA sequences in STR (short tandem repeat) patterns of a specific locus to identify individuals. Used in forensic studies and paternity testing.
• DNA sequencing


  Determination of nucleotide sequences of DNA fragment. Used to study genomes and identifying alleles in medicine.
• DNA fingerprinting


  1. Flanking regions cut into fragments by restriction enzymes

  2. DNA fragments separated via gel electrophoresis

  3. DNA band pattern transferred to nylon membrane (southern blotting)

  4. Radioactive DNA probe binds to specific DNA sequences on the nylon

  5. X-ray film placed next to membrane to detect radioactive pattern

  6. Film developed to make pattern visible. Pattern is a DNA fingerprint
• Gene probe


  Single stranded nucleic acid fragment that interacts with complementary sequence of target nucleic acid
• Transcription factors


  1. Activator proteins bind to enhancers. Binding causes DNA to bend, bringing them near a gene promoter

  2. Other transcription factor proteins join activator proteins, forming protein complex that binds to gene promoter

  3. Protein complex makes it easier for RNA polymerase to attach to promoter and transcribe gene

  4. An insulator can stop enhancers from binding to promoter and prevent the gene turning off

  5. Methylation allows the enhancer to bind to the promoter
• 4 ways for genes to be altered

  • At transcriptional level during mRNA production (turning genes on/off)
  • At post-transcriptional level after mRNA production (editing RNA)
  • At translational level during protein production (turning translation on/off)
  • At post-translational level after protein production (stopping protein function)
• Acetylation


  Change in Histone charge causing DNA to be less tightly wrapped around the histone, allowing for easier access of transcription enzymes
• Methylation


  Addition of methyl to Cytosine bases that binds DNA more tightly around histones, making gene transcription harder
• Splicing


  Different exons can be spliced together to form a different series of codons that code for different proteins
• Transcription factors


  Proteins which bind to DNA in the nucleus and increase/decrease the transcription of genes
• Silencer sequences


  Turn off transcription by binding repressor proteins
• Mutation


  Change in DNA base sequence caused by mutagens
• Mutation types


  • Point mutations: mutations that affect 1 base (substitutions)
  • Frameshift mutations: mutations that affect the entire gene (insertion/deletion)
• Mutation effects


  • Neutral/silent: no effect occurs. A base substituted could code for the same amino acid
  • Beneficial: enhanced protein function
  • Damaging: malfunctioning protein (e.g. a stop codon inserted too early)
• Transcription


  1. In the nucleus

  2. DNA helicase breaks hydrogen bonds between DNA complimentary strands

  3. Free RNA nucleotides align with their complimentary base pair and phosphodiester bonds catalysed by RNA polymerase

  4. RNA breaks away, H bonds for DNA reform
• Translation


  1. In the cytoplasm or on the RER

  2. mRNA attaches to a ribosome

  3. tRNA have an anticodon and an amino acid representative of the anticodon

  4. tRNA bind with the codon on the mRNA that matches its anticodon

  5. Peptide bond formed between the amino acids on the tRNA forming a polypeptide
• Transcriptional epigenetic modification


  Alter conditions to allow RNA polymerase to bind
• Operon


  Group of genes controlled by the regulatory mechanism and expressed at the same time
• Lac operon


  1. When lactose is absent, LAC repressor binds tightly to the operator, preventing RNA polymerase from binding and causing transcription

  2. When lactose is present, LAC repressor lets go of the operator, allows RNA polymerase to transcribe gene

  3. When glucose is present, cAMP attaches to promoter increasing rate of transcription

  4. When glucose is not present, cAMP is not made so transcription occurs at a slower rate
• RNA splicing


  Removes introns, joins exons together in any order to produce different protein sequences
• Post-translational modification


  Affects protein folding in tertiary and quaternary structure, adds non-protein groups (lipo/glycoproteins), modify amino acids to change bonds
• Nucleosome


  Eukaryotic DNA complex made of 8 histone proteins with 2 DNA loops
• Epigenetics


  Changes of gene expression caused by environmental factors
• Promoters


  Short base sequences that lie close to their target genes and initiate transcription by enabling RNA polymerase to bind to the gene they regulate
• Enhancers


  Short base sequences that lie some distance from their target genes and stimulate promoters causing an increase in the rate of transcription of the genes they regulate