Control of Gene Expression

• Types of Mutation


  • Insertion: one or more bases added, frameshift
  • Deletion: one or more bases removed, frameshift
  • Substitution: one or more bases changed, may have no effect
  • Duplication: one or more bases repeated, frameshift/increase in repeating subunits
  • Inversion: sequence of bases reversed, may have no effect
  • Translocation: sequence of bases moved from one location to another, including non-homologous
• Stem cells


  • Cells that can differentiate and become specialised into various cell types (potency/plasticity)
  • Become specialised when they can only transcribe and translate certain genes, the rest are switched off
  • Expressed genes are determined by cell conditions
• Types of stem cell

  • Totipotent: become embryonic and extraembryonic tissues, found in very early embryos, all genes activated
  • Pluripotent: become any tissue in body except embryonic, found in early embryos, most of genes active
  • Multipotent: become limited cell types, bone marrow cells, some genes switched off
  • Unipotent: become only one type of cell, cardiomyocytes, almost all genes switched off
• Embryonic stem cells

  • Taken from embryo in a very early stage of development
  • Cells must be totipotent as embryo must make new cells
  • First 4 days all cells are totipotent, 4-7 days they are pluripotent as have begun to differentiate
  • Can be used to grow new tissues and organs
  • Improves quality of life, reduce organ donation
• Stem cell research issues

  • Use is controversial
  • Unused IVF embryos can be donated but some people believe that the embryo has the right to life
  • Egg cells frozen for IVF could be used as wouldn't survive as an embryo
  • Use of stem cells is highly regulated
• Adult stem cells


  • Obtained from bone marrow in adults with a small low risk operation
  • Multipotent cells
  • Can be used to replace faulty bone marrow
  • Can also be used to treat paralysis by replacing nerve tissue
• Induced pluripotent stem cells


  • Made by reprogramming specialised adult cells to become pluripotent
  • Made to express a series of transcription factors "switching on" genes that are usually in pluripotent cells
  • Allows adult cells to be as plastic as embryo cells
  • Can be made from a patient's own cells, can grow tissues/organs that won't be rejected
• Clinical trials done to see if stem cells can be used to replace organs and tissues
• Transcription factors


  • Protein in cytoplasm that controls gene expression
  • Binds to promoter region of DNA
  • Can activate or repress transcription by controlling how RNA polymerase binds to DNA, turning genes on or off
  • Repressors bind to promoter region and prevent RNA polymerase binding, in turn stopping transcription
• Inhibitors + hormones


  • Transcription factors can be switched off by an inhibitor molecule
  • Prevent it from attaching to DNA
  • Without the (activatory) transcription factor the gene cannot be transcribed
  • Other molecules such as hormones bind to other site on transcription factor, releasing inhibitor and allowing it to bind
• Oestrogen


  • Steroid hormone (can diffuse through membrane)
  • Diffuses across cell membrane, binds to receptor on transcription factor, alters shape
  • Has to bind to oestrogen receptor, forming oestrogen-oestrogen receptor complex (not all cells have receptor)
  • Complex moves from cytoplasm to nucleus, binds to HRE in promoter region, activating or repressing transcription
• Sequencing methods


  Old:

  Labour intensive

  Expensive

  Small Scale

  New:

  Automated

  Cheaper

  Cost effective on large scale
• RNA Interference

  • Translation of mRNA can be inhibited by breaking mRNA down before its code can be translated
  • Double stranded siRNA associates with proteins in cytoplasm and unwinds, one strand binds to target mRNA, allowing associated proteins to cut mRNA into small fragments that move into a processing body to be degraded
• miRNA

  • Not fully complementary to mRNA molecules
  • Less specific so targets multiple mRNA molecules
• Potential use for 'gene silencing' of inherited disease. Patients can be treated with siRNA for faulty genes
• DNA structure

  • DNA held in nucleus surrounding proteins called histones that package and order DNA into nucleosomes
  • Histones associate with DNA and help DNA to condense into chromatin
  • Chromatin that is tightly bound to histones cannot be transcribed
• Epigenetics

  Heritable change in gene function without changes to base sequence of DNA
• Epigenome

  • DNA + histones are covered in chemical tags that form a second layer called the epigenome
  • Keeps inactive genes tightly packed and active genes exposed
  • Flexible as responds to environmental change
• Methylation of DNA

  • Methyl group added to cytosine base
  • Prevents binding of transcription factors
  • Attracts proteins that condense DNA-histone complex, making DNA inaccessible to transcription factors
• Acetylation of histones


  • Acetyl group removed from histones, increasing positive charge and attraction to phosphates
  • DNA-histone complex more condensed so less accessible to transcription factors
• • Altering the epigenetic process can cause abnormal activation or silencing of genes
  • Can be used to treat diseases as the changes are reversible with targeted drugs
• Cancer

  • Rate of cell division can depend on the environment, growth factors (hormones) and genes
  • Mutations in genes that control cell division can cause uncontrolled cell growth if pass checkpoints in cell cycle
  • Causes cells to form tumours
  • Those that invade and destroy surrounding tissues are cancers
• Proto-oncogenes


  • Can cause cancer when turned on
  • Genes that normally help cells grow
  • Can become permanently activated oncogenes when mutated
• Tumour suppressor genes


  • Can cause cancer when turned off
  • Normally help slow cell division, repair DNA mistakes or stimulate apoptosis
  • Cells grow out of control if genes don't work properly
• Cancer cells vs normal

  • Different to normal cells in structure and function
  • Usually either die during apoptosis or are destroyed by the immune system
  • Have large dark nuclei, maybe multiple
  • Irregular shape
  • Do not produce proper proteins
  • Have different antigens
  • Don't respond to usual growth regulation process
• Benign vs malignant


  • Benign: non-cancerous, slow growing, don't metastasise but can put pressure on organs or cause blockages, normal shaped cells
  • Malignant: cancerous, fast growing, invade and destroy other tissues and metastasise around the body, may have abnormal shaped cells
• Increased exposure to oestrogen over an extended period


  Can increase breast cancer risk
• Epigenetics + cancer


  • Abnormal methylation of proto-oncogenes and tumour suppressor genes can cause cancer
  • Hypermethylation of TSGs prevents them from being transcribed
  • Hypomethylation of proto-oncogenes causes them to act as oncogenes
• Genome projects use technology to determine the complete sequence of bases that make up the DNA of an organism
• • Once sequenced, genomes can be compared within particular species to highlight or identify mutations which cause diseases
  • Comparison of genomes between species can be used to explain evolutionary relationships between species and used to build phylogenetic trees
• • Sequencing of genomes can allow the proteome to be determined
  • Only easy in simple organisms with small genomes such as bacteria + viruses
  • Helpful to identify antigens of viruses
  • Can be used to produce vaccines, monitor mutations and identify antibiotic resistance
• Proteomes in eukaryotes


  • Large sections of the genome of complex organisms are made of introns
  • Contains regulatory genes, genes that code for tRNA and rRNA, and large repeating sequences from years of evolution
  • Means that proteome of an organism is hard to determine from the genome as it is unknown which sections are exons
• Methods to make DNA fragments

  • Using reverse transcriptase
  • Using restriction enzymes
  • Using a gene machine
• Using reverse transcriptase


  • Cells that contain protein made by the target gene will have lots of mRNA that is complementary to the target gene
  • Reverse transcriptase (from a retrovirus) converts RNA back into isolated DNA
  • Reverse transcriptase is added to sample and uses free nucleotides to make cDNA (complementary to mRNA) from mRNA template
• Using restriction enzymes


  • Restriction endonucleases are enzymes that recognise and hydrolyse hydrogen bonds
  • Cut at a specific recognition sequence that is complementary to their active site
  • Form sticky ends (pieces of DNA with exposed nucleotides on a single strand at each end) that allow DNA fragments to be joined by ligase enzymes
  • Restriction sequence is palindromic due to complementary base pairing
• Using gene machine


  • Amino acid sequence of protein is studied and MRNA + DNA sequences are identified
  • Typed into a computer that checks for biosafety + biosecurity
  • Desired sequence is made
  • First nucleotide is fixed to a support, further nucleotides then added one at a time
  • Short overlapping DNA sequences (oligonucleotides) are produced that then join together
• In vivo cloning

  • Relies on recombinant DNA technology or genetic modification
  • Genes are isolated from one organism and inserted into another using a vector creating new gene combinations
  • Vector is usually a plasmid but can be a virus (bacteriophage)
  • Will infect host bacterium by injecting its DNA into bacterium
  • Viral DNA (with target gene) will then integrate into bacterial DNA
• In vivo cloning process

  1. Gene isolated using endonuclease enzymes

  2. Promoter (tell RNA polymerase to start) and terminator regions (tell RNA polymerase to stop) added for correct transcription

  3. Cut a plasmid + gene with same restriction enzyme to leave sticky ends that allow them to lyse

  4. Ligase enzymes join fragments via condensation

  5. Solution added (make cell wall permeable) and heat shock/electroporation make holes in membrane
• Marker genes

  • Inserted into vector at same time as gene to identify bacteria that take up the gene
  • Can code for antibiotic resistance so only transformed bacteria will survive
  • Can code for UV fluorescence so transformed colonies show up under UV light
  • Lactase gene turns certain colourless substances blue
  • Gene inserted into DNA fragment to disrupt it, transformer bacteria can't turn substance blue
• Polymerase Chain Reaction

  • Artificial replication of short DNA sequences
  • DNA fragments undergo a series of heating and cooling in the thermocycler to replicate them many times
  • Mixture made of DNA sample, nucleotides, Taq polymerase and primers (string of complementary bases to end of DNA sequence, shows where enzyme should bind)