topic 8 gene expression

• mutations

  • gene mutation is the change in the DNA base sequence of a gene
  • mainly occurs during DNA replication
  • occurs spontaneously but the frequency is increased by exposure to mutagenic agents
  • this can result in different amino acid sequence, therefore different hydrogen and ionic bonds In different locations
  • this results in a different tertiary structure therefore a different 3D shaped protein
  • a different shape results in a different function or non-functioning protein
• addition mutations

  • one extra base is added to the sequence
  • causes the codons to be altered
  • this is know as a frame shift
  • this is dangerous as altered codons could potentially code for a different Amino acid
• types of mutations

  • deletion mutation - the deletion of a base in a sequence. causes a frame shift to the left
  • substitution mutation - one base is changed for a different base as genetic code is degenerate it may still code for same amino acid
  • inversion mutation - section of bases detach from the dna but they rejoin but they are inverted
  • duplication - one particular base is duplicated at least once In the sequence
  • translocation - section of bases on one chromosome detaches and attaches onto a different chromosome can majorly impact gene expression and the phenotype
• stem cells

  • stem cells are undifferentiated cells that can continually divide and become specialised
• totipotent stem cells

  • these stem cells can divide and produce ant type of body cell
  • occur for a limited time in the early embryonic stage
• pluripotent stem cells

  • found in embryos
  • can divide into unlimited numbers and can be used in treating human disorders
• multipotent and unipotent stem cells

  • stem cells found in mature mammals and can divide to form a limited number of different cell types
• induced pluripotent stem cells

  • can be produced from adult somatic cells using appropriate protein transcription factors to overcome the ethics of using embryonic stem cells
• control of transcription

  • transcription of target genes can be stimulated or inhibited when specific transcriptional factors move from the cytoplasm into the nucleus
  • this can turn on/off genes so only certain proteins are produced in a cell
  • turning on/off particular genes in a cell is what enables them to become specialised
• transcription factors

  • transcription of a gene will only occur when a molecule from the cytoplasm enters the nucleus and binds to the DNA in the nucleus
  • these proteins can bind to different base sequences on DNA and therefore initiate transcription of genes
  • once bound transcription begins creating the mRNA molecule for the gene which can then be translated into the cytoplasm to create the protein
  • without the binding of the transcription factor the gene is inactive
• oestrogen

  • Oestrogen is a steroid hormone
  • It is lipid soluble so can diffuse through the  cell membrane
  • Oestrogen binds to a complementary shaped receptor on a transcription factor
  • The change in tertiary structure removes the inhibitor
  • The transcription factor diffuses into the nucleus and binds to DNA promotor region
  • Transcription begins (because RNA polymerase binds)
• epigenetic

  • another way to control gene expression
  • epigenetic is the change in gene function without changing the DNA base sequences
  • these changes are caused by the changes in the environment and can inhibit transcription
• The ‘epigenome’


  DNA wraps around proteins called histones, to form chromatin

  Chemical groups (acetyl or methyl groups) can bind to

  DNA or histones

  These chemicals can influence how tightly or loosely the

  DNA wraps around the histones
• methylation

  • increased methylation of DNA inhibits transcription
  • when methyl groups (ch3) are added to dna they attach to the cytosine base
  • this prevents transcriptional factors from binding and attracts proteins that condense the dan-histone complex. causing dna to become more tightly wrapped around the histone
  • methylation prevents a section of DNA from being transcribed
• acetylation

  • Adding an acetyl group CH3CO 
  • (neutralises charge on the amino acid lysine so DNA is less attracted)
  • and means the histones become more positive and are attracted more to the phosphate group on dna
  • this makes the dna and histones more tightly wrapped creating a stronger association and hard for transcription to bind
• deacteylation

  • Removing acetyl makes histones more positive
  • So they attract DNA (negative)
  • means the histones become less positive and are less attracted to the phosphate group on the DNA
  • this makes DNA and histones less strongly associated meaning they are less strongly wrapped
  • meaning it's easier for transcription factors to bind
• RNA interference

  • translation of the mRNA produced from target genes can be inhibited by RNA interference
  • this is when an mRNA molecule that has already been transcribed gets destroyed before it's translated to create a polypeptide chain
  • this is done by a small interfering RNA (siRNA)
• siRNA

  • an enzyme can cut the mRNA into siRNA
  • one strand of the siRNA then combines with another enzyme
  • the siRNA-enzyme complex will bind via complementary base pairing to another mRNA molecule
  • one bound, the enzyme will cut up the mRNA so it cannot be translated
• cancer

  • can result from mutations in genes that regulate mitosis
  • if these genes aren't regulated then it can result in uncontrollable division of cells and create a tumour
  • This is controlled by two genes :

  Tumour Suppressor Genes (suppress cell division) BRAKE

  • Proto-oncogenes (stimulate cell division) ACCELERATOR
  • cancer can also occur with abnormal methylation of tumour suppressor genes or oncogenes
  • or increased oestrogen
• Benign tumours

  • Slow Growing
  • Encapsulated
  • Cells adhere to each other
  • Treated by surgery
  • Cells show differentiation
  • Does not metastasise
• malignant tumours

  • Fast Growing
  • Cells do not show differentiation
  • Not encapsulated
  • Treated by radiotherapy, chemotherapy and surgery
  • CanMetastasise
  • Cells do not adhere to each other
• oncogenes

  • oncogenes are the mutated version of a photo-oncogoene which creates a protein involved in the initiation of dna replication and mitosis when the body needs new cells
  • oncogenes can result in this process being permanently activated to make cells divide continuously
• tumour supressor gene

  • these genes produce proteins to slow cell division and to cause cell death if DNA copying errors are detected
  • if a mutation results in the tumour suppressor gene not producing the proteins to carry out the function. then cell division could continue and nutated cells would not be identified and destroyed
• epigenetic and cancer

  • methylation can cause a gene to turn on or off
  • tumour supressor genes could become hypermethylated meaning an increased number of methyl groups attached to it
  • this results in the gene being inactive and turned off
  • the opposite could occur in oncogenes as they may be hypomethylated reducing the number of methyl groups attached
  • this results in the gene being permanently switched on
• increased oestrogen concentration

  • increased oestrogen results in more fat cells in breast tissue which can cause breast cancer
  • this happens as oestrogen can activate a gene by binding to a gene that initiates transcription and if it's a proto-oncogene this can result in permanent turn on which results in uncontrolled cell division
• the genome

  • the genome is the entire genetic material of an organism in the nucleus of a cell
  • sequencing a genome means working out the dna base sequences for all the DNA in a cell
• genome - prokaryotic cells

  • prokaryotes don't contain introns in their DNA so that means the genome can be used directly to sequence the proteins that derive from the genetic code
  • this is useful as you can identify potential antigens to use in a vaccine
• recombinant DNA technologies

  1. creating DNA fragments
  2. genetic fingerprinting
  3. genetic screening counselling and location genes
• what are the ways to create dna fragments

  • gene machine
  • reverse transcriptase
  • restriction endonuclease
• reverse transcriptase - making DNA FRAGEMENTS

  1. this enzyme makes DNA copies from mRNA
  2. which is naturally occurring in viruses such as HIV
  3. a cell that naturally produces the protein of interest is selected
  4. these cells should have a large amount of mRNA for the protein
  5. the reverse transcriptase enzyme joins the DNA nucleotides with complementary bases to the mRNA sequence
  6. single stranded DNA is made- cDNA
  7. to make the DNA fragments double stranded the enzyme DNA polymerase is used
• restriction endonuclease - making DNA FRAGEMENTS

  • enzymes that cut up DNA
  • these occur naturally in bacteria as a defence mechanism
  • there are many restriction enzymes that have an active site complementary in shape to a range of different DNA base sequences described as recognition sequences
  • therefore enzyme cuts the dna at a specific location
  • the enzyme may cut in the same location in the double strand and create a blunt end or
  • create sticky ends which are staggered ends and exposed DNA bases
  • there is chance it can still complementary base pair
• gene machine - making DNA FRAGEMENTS

  • dan fragments created in a lab using a machine
  • scientist examine the protein of interest to identify the amino acid sequence and then from that mRNA and DNA sequence can be worked out
  • the DNA sequence is entered into a computer
  • the computer can create small sections of overlapping single strands of nucleotides that made up the gene called oligonucleotides
  • PCR can be used to ampifly the quality and make it double stranded
  • process is quick, accurate and makes intron free dna
• in vivo cloning - preparing the gene

  • restriction endonuclease cut sticky ends
  • the dna fragment must be modified to ensure transcription of these genes can occur
  • a promoter region must be added, adding it at the start of dna fragment, this means there's a binding site for RNA polymerase to attach and transcription to occur
  • a terminator region must be added so the RNA polymerase knows when to stop transcribing so only one gene is copied at a time
• in vivo cloning - inserting the gene (step 2)

  • plasmid is cut open using the same restriction endonuclease
  • this creates the same sticky ends
  • there the DNA fragment sticky ends are complementary to the sticky ends on the plasmid
  • then they're combined and enzyme ligase sticks then together through catalysing condensation reactions to form phosphodiester bonds between the nucleotides
  • when inserting the vector into host cell the membrane of the host cell must be permeable
  • to increase this you can mix host cells with ca2+ and heat shock
• how to check in-vivo cloning was successful

  • marker genes to identify whether the bacteria has successfully taken up the recombinant plasmid
  • antibiotic resistance genes
  • genes that code for fluorescent proteins
  • genes coding for enzymes
• fluorescent markers

  1. jellyfish contain a gene which codes to creat GFP
  2. PROCESS
  3. plasmid with GFP gene in it, DNA fragment is inserted in the middle of GFP, this disrupts it and prevents GFP production
  4. only non-glowing colonies contain the recombinant plasmid
• issue that can occur when transferring recombinant DNA

  1. the recombinant plasmid doesn't get in the cell
  2. the plasmid re-joins before DNA fragments entered
  3. the DNA fragment sticks to itself rather than inserting into the plasmid
• enzyme markers

  • the enzyme lactase can turn a certain substances blue from colourless
  • the gene for this enzyme is inserted into the plasmid
  • the DNA fragment is inserted in the middle of this gene to disrupt it
  • the bacteria are then grown on an agar plate with the colourless substance
• in vitro cloning - PCR

  • amplify the DNA fragments
  • once the DNA fragments have been isolated they need to be cloned to create large quantities
  • PCR ampiflies the DNA fragments through the polyermase chain reaction
• PCR method

  1. the temperature is increased to 95 degrees to break the hydrogen bonds and split the DNA into single strands
  2. temperature then is decreased to 55 degrees so that primers can attach
  3. DNA polymerase then attaches complementary free nucleotides and makes a new strand to align next to each template. temperature increased to 72 degrees which is the optimum for taq DNA polymerase