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DNA and Cell Structures (*Lab Techniques (Radiolabelled Assays fix…

- - - - Clinical Case
      - Notes:
        
        note the main reason why the Blood BB is able to keep molecules out is through tight junctions
        
        these tight junctions are also supported by astrocytes cells that surround the cappilaries
        
        there are no fenestrations = opennings in the Blood BB capillaries either
- - - - post-transcriptional modification
        
        Notes:
        
        3 main post-transcriptional modifications
        
        5' methyl-guanosine capping
        --> helps stabilize the 5' end of mRNA from being degraded in the cytosol
        
        3' Poly-A tail capping
        --> helps stabilize the 3' end of mRNA from being degraded in the cytosol
        
        removal of introns from the mRNA by snRNPs = small nuclear ribonucleotide proteins
        --> exons are "actual DNA", "introns are out"
      - Poly A tail and complement
        
        Notes:
        
        note always think of the 3 major post transcriptional changes to mRNA and their complements
      - *Translation by rRNA and tRNA
        
        mRNA to polypeptides (amino acid sequences --> folded into proteins)
        
        transfer RNA = *tRNA
        
        carries AAs tot he ribosome to add to the polypeptide chain
        
        Clinical Cases
        
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        Notes:
        
        note that
        
        Reading mRNA Practical Example
        
        mRNA is read in the 5' --> 3' direction
        
        opposite to DNA reading
        
        think the AAs need to match the original DNA so the mRNA is read in the opposite direction to the DNA
        
        Clinical Case
        
        Notes:
        
        note that tRNA has a CCA end at the 3' end
        --> CCA has OH group attached to it where the AAs bind
        
        tRNA is held together by hydrogen bonds of complement sequences
        
        has a DAVENPORT clover LOOP structure
        
        D = D loop
        
        A = anticodon loop
        
        V = small V loop
        T = T loop
        
        Charged vs. Uncharged tRNA
        
        Uncharged tRNA goes out to bind to AAs
        
        once tRNA binds to AAs it becomes charged and can bind to the A site of the Ribosome for giving its AA
        
        ribosomal RNA = *rRNA
        
        60S and 40S
        
        releasing factors bind to the ribosome and casue the release of the polypeptide when the mRNA reaches one of the 3 STOP codons
        --> UAA
        --> UGA
        --> UAG
        
        8releasing factors
        
        bind to the ribosome and casue the release of the polypeptide when the mRNA reaches one of the 3 STOP codons
        --> UAA
        --> UGA
        --> UAG
        
        *Ribosomes - Cytosol Ribosomes vs. RER Ribosomes
        
        RER ribosomes
        --> send proteins for cell membrane, secretory, nucleus (NOT the nucloelus), Golgi and lysosomes
        
        Modified Translation during Apoptosis
        
        use of the IRES = internal ribosomal entry site
        
        this starts translation
        
        instead of the normal recognition of the 5'prime cap
        
        Notes:
        
        note that in apoptosis, can't use the normal recognition of the 5'prime cap
        
        in normal translation, the small unit of the ribosome binds to the 5' cap
        
        the 5' cap is found by eIFs = eukaryotic initiation factors
        
        instead during apoptosis, these eIFs are degraded and need to use the IRES = internal ribosomal entry site to start translation
        
        note also here that normally during eukaryotic translation, there are not multiple open reading frames
        --> there are multiple reading frames in prokaryotes, but NOT eukaryotes
        --> not sure why I thought this was true for eukaryotes?
        
        Clinical Case
        
        Reading mRNA: Translation
        
        mRNA read in the 5' to 3' direction
        
        opposite to DNA reading
        
        Reading mRNA Practical Example
        
        mRNA is read in the 5' --> 3' direction
        
        opposite to DNA reading
        
        think the AAs need to match the original DNA so the mRNA is read in the opposite direction to the DNA
        
        Notes:
        
        here first look for the stop codons
        
        remember to read mRNA in the 5' to 3' direction
        
        find UAA UGA UAG
        
        then it is asking for the codon before this
        
        make sure to put the 3' and 5' in the right place, they switch them sometimes
        
        Clinical Case
      - NUCLEAR pot transcription modification of pre mRNA
        
        3 major steps of postranscription modification
        
        *5' Cap
        
        1/3 nuclear step of Post-transcriptional mRNA modification
        
        stops the mRNA degraded in the nucleus
        
        *3' Poly A Tail
        
        2/3 nuclear step of pre mRNA modification
        
        stops the mRNA degraded in the cytoplasm
        --> keep losing the 3' tail
        --> this makes sense as you need a STOP at the end of RNA, this is more important
        
        *Splicing out of introns
        
        LASTS = 3/3 nuclear step of pre mRNA modification
        
        *Introns and exons
        
        EXONS = Actual mRNA that get EXPRESS
        
        INTRON = just there to INTERRUPT the ACTUAL mRNA
        
        Mechanics of splicing
        
        "Get U!" -- INTRONS -- "All GONE!"
        --> GU -- INTRONS -- AG
        
        GU marks the 5' end of introns to START the cut by snRNPs and the splicosome
        --> snRNPs = small nuclear ribonucleic Proteins
        
        AG marks the 3' end of introns to END the cut by snRNPs and the splicosome
        
        1 more item...
        
        CYTOPLASM *PBODY modification
        
        use microRNA to degrade or change the mRNA
    - - Enzymes
        
        Helicase = stairCASE unwinder
        
        ssBPs = single strand binding proteins
        --> holds the unwound DNA open
        
        gyrase = topoisomerase II = stops supercoiling
        
        RNA Primase = lays the initial primer for the DNA Pol 3 on both leading and lagging strands
        --> note that RNA primase is the ONLY enzyme in DNA replication that makes RNA and not DNA
        --> doesn't need to be DNA as it is removed by DNA Pol 1 anyways
        
        DNA Pol III = think DNA read in 3' --> 5' direction = DNA POL 3 at
        --> starts after primase lays first
        --> reads DNA in 3' --> 5'
        --> makes DNA in 5' --> 3' and proof reading 3' --> 5'
        --> proof reads in BACKWARDS direction ONLY
        --> can't take out bits and add at the same time like DNA POL 1
        
        DNA Pol 1 = the ONE DNA Pol that has both 5' --> 3' synthesis and exonuclease in the same direction
        (DNA pol 3 ONLY has backwards = 3'-->5' exonuclease backwards proof reading)
        
        Notes:
        
        note that the exonuclease and DNA/mRNA synthesis 5' --> 3' always refers to the strand being made
        
        ex - DNA is read in the 3' --> 5' direction, so DNA Pol 3 has 5' --> 3' direction synthesis
      - Pre Replication Fork Factors for Transcription
        
        Transcription Enhancers = several base pair upstream ? how many
        
        CAT TATA TAT = initiating and TATA box promotor base pair upstream ? how many
        --> right before the genes to be encoded
        
        Transcription Enhancer
        
        located even more upstream from the CAAT initiater sequence
        
        creates a loop in the DNA after DNA POL has started and makes it go faster
        
        CAAT and TATA box
        
        Song = "CATS comin down the track... "
        .- "TATA TAT"
        
        CAAT = initiating sequence for DNA transcription
        
        TATA box = promoter region after CAAT
        
        CAAT and TATA box example
        
        Notes:
        
        note that
        
        Clinical Case
      - Practical Transcription - Practise Qs
        
        coding strand / sense strand = actual DNA that is the same as the mRNA made
        --> remember the protein is the important thing and ultimate goal
        unwinder
        
        template strand / nonsense strand = template DNA for the mRNA to be made from
        --> think it would not make the protein
        --> it is nonsense
    - - Notes:
        
        coding strand = sense strand
        --> identical to mRNA
        
        template strand
        --> is the template for RNA to copy as a template
    - - *leucine zipper - Transcription factors
        
        leucine zipper transcription factor
- - - - Clinical Cases
        
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        Notes:
        
        note that
- - - - Western Blot example 1
        
        Notes:
        
        Example:
      - Western Blot example 1
        
        Example:
        
        Notes:
  - - - Notes:
        
        "PCR = 95.7 is a TAQy station that PRIMES DNA for NEW TRIAL songs"
        
        PCR 95.7
        --> 95 degrees C = denature the original
        --> 55 degrees C = cools down for primer to attach to the needed sequence
        --> 72 degrees C = TAQ polymerase elongates the new DNA strand
        
        TAQy station
        --> TAQ polymerase = thermos aquatic polymerase is able to work at high temps of 72 degrees
        
        PRIME DNA for NEW TRIALS
        --> 3 ingredients needed for PCR are: primer, DNA original, nucleotide triphosphates
      - *Flanking region
        
        note you need to know the nucleotide sequence of the 2 flanking regions on either side of the exon or gene you want to copy and amplify
        
        the flanking region = DNA primer binding region
  - - - Example:
      - Notes:
        
        to identify whether someone has been exposed to a certain virus and has antibodies / immunoglobulins to that virus
        
        step 1 - line wells with viral antigen and add patient serum to the wells
        --> if they have antibodies to the virus they will bind to the antigens of the well
        
        step 2 - wash the plate
        --> this removes any other proteins etc. that have not binded to the specific antigens
        
        step 3 - add the substrate-modifying E = EL part of ELISA
        --> the enzyme that is linked to a specific substrate that will activate the enzyme is key to the whole process of the ELISA kit
        --> the enzyme here is attached to an anti-human immunoglobulin antibody
        --> this attaches to the patient's human antibody
        
        step 4 wash again
        
        step 5 - add the substrate to the wells to activate the enzyme
        
        the degree of colour change or whatever the enzyme does will tell you how much of the antibody is present in the well
        --> the substrate linked enzyme = substrate-EL is usually peroxidase
- - - - Kinesin Transport Example
        
        Clinical Case
        
        Notes:
        
        note that kinesins transport secretory vessicles towards the growing end = positive end of microtubules = AWAY from the centre of the cell and towards the periphery
        --> classic example would be ACh NTs that are brought to loading zones at the cell membrane near Ca++ channels to be ready for release
      - "DYNesins DINE with MINE at HOME,
        Kinesins are KIND and POSITIVE and dine away"
        
        Dynesins are proteins that transport vessicles along microtubules
        --> towards negative (-) end
        --> towards the cell centre
        
        Kinesins are proteins that transport vessicles along microtubules
        --> towards positive + end
        --> towards the periphery
  - - - Notes:
        
        note the period 2 wks --> 2 months post MI your heart starts laying down fibrotic scar tissue on the damage myocytes of the heart
        
        recall that myocytes can't regenerate so your body has to replace them with collagen type 1 scarring
        
        type 1 collagen is the same that is in bones, tendons, think the starting materials for tissue
      - Example:
    - - Triple alpha helix in CAG COLL - A - GEN
        
        CAG COLL - A - GEN
        --> C = Vitamin C neded for collagen synthesis
        --> A = alpha triple helices
        --> G = glycine every 3 AAs gives the flexible structure for collagen / with prolines in between that give the rigidity needed
        
        note the triple helix structure is ONLY in the PROCOLLAGEN polypeptide that has to be cleaved
        
        PROCOLLAGEN is then cleaved into collagen fibrils that all CROSS LINKED to each other by LYSYL OXIDASE
        --> think of collagen as being strong like a bacterial cell wall because it has individual building blocks = collagen fibrils that are crosslinked to gether to make it strong
        --> just like in bacterial cell wall the D-ALA - D ALA are crosslinked to make a strong bacterial cell wall
- - - - Mitochondria
        example
        
        Example:
        
        Notes:
        
        note when looking for mitochondria on electron microscope, first identify is there is a nucleus and a nucleosome present, then find the mitochondria which have lines that make up their matrix
    - - *Peroxisome Disorders
        
        can't break down VLCFA = very long chain fatty acid oxidation in the peroxisome
    - - Mitochondria
        example
        
        Example:
        
        Notes:
        
        note
    - - *Rough ER
        
        has ribosomes and makes proteins
        
        *Smooth ER
        
        has NO ribsomes and is used for making steroid hormones
        
        thus high in androgen making cells, gonads, etc.
        
        *Golgi Apparatus
        
        has no ribosomes and is used to packege proteins into secretory vessicles for transport from the cell
- - - - Clinical Cases
        
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        Notes:
        
        note that
        
        Telomeres and telomerase in Malignancy
        
        Notes:
        
        note that only germline cells have telomerase naturally since they are dividing so often and need telomerase to make and replace the telomere
        
        other than this telomerase activity is pathogenic and is present in 90% of cancers since telomeres normally signal through the p53 tumor supressor gene to start apoptosis when DNA has undergone too many divisions
        
        Case example:
    - - Clinical Cases
        
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        Notes:
        
        note that
  - - - Case example:
      - Notes:
        
        note
- - - - Notes:
        
        note that
      - Clinical Case
- - - - Case presentation:
      - Notes:
        
        note that most hormones are lipophilic and bind inside the cell
        
        think of hormones as being POWERFUL, but they take time for them to work and LASTING
        --> hence bind INSIDE the cell
        --> transcription changes genes
        
        things like insulin and glycogen bind outside the cell
- - - - Notes:
        
        note that
      - Clinical Case
- - - - Pathophys of HD
        
        CAG repeats --> cause deacetylation --> trasncription silencing