Understand 1st year medicine

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Cell Cycle

Chromosome → Chromatin → Nucleosome


Nucleosome = 8 histones wrapped around DNA

1. Sister Chromatid, 2. Centromere, 3. Short arm (p), 4. Long foot (q)





Nucleotide: Phosphate group + Sugar + Base



Purines (bigger than pyrmidine): A and G

Pyrmidine: C(Ytosine) and T(hYmine)


Hydrogen bonds between nitrogenous bases

DNA: A -- T C --- G

RNA: A -- U C --- G (U for uracil)


Sugar-phosphate backbone of DNA formed by phosphodiester bonds



Things always happen according to its strand’s 5’ → 3’ rule


DNA Triplet → RNA codon → Polypeptide

Transcription in nucleus: Turning DNA into RNA

Gene

Gene (DNA):

Upstream enhancer → TATA box (promoter) → Sequence coding for methylated cap and 5’ UTR (UnTranslated Region) → Sequence coding start codon → Mixture of introns and exons → Sequence coding stop codon → Sequence coding 3’ UTR and Poly-A tail


Once TF binds to TATA box, RNA polymerase binds to TATA box and starts adding nucleotide triphosphate (RNA - AUCG) via rules of base pairing. As each is added to 3’ end of the growing strand, 2 phosphates are removed.



Transcription continues until ribosome reaches transcription terminator (an inverted repeat - bases in DNA that is repeated in inverted fashion).

5'.....GCCGCCAG........CTGGCGGC....3'
3'.....CGGCGGTC........GACCGCCG....5' (template strand)

The transcription terminator codes for a hairpin loop in mRNA.

Transcribed RNA: 5'.......GCCGCCAG........CTGGCGGC.....3'

This hairpin loop causes RNA polymerase and mRNA to dissociate from DNA.



RNA Splicing


  1. Pre-mRNA in nucleus: Methylated cap + 5’ UTR + Start codon + Introns/Exons + 3’ UTR + Poly-A tail
  2. Splicing out of introns by spliceosome
    1. snRNPs (made of snRNA) assemble on pre-mRNA to form spliceosome
    2. Spliceosome cleaves 5' exon-intron boundary and formation of intron-lariat (lariat = necklace) via phosphodiester bond
    3. Form phosphodiester bond to join 5’ and 3’ exon and break off the intron as lariat RNA
  3. Final mRNA in cytoplasm

Methylated cap + 5’ UTR + Start codon + Exons + 3’ UTR + Poly-A tail


Alternative splicing: Gene spliced differently
  1. Exon 1 + Exon 2 = Protein A
  2. Exon 2 + Exon 1 = Protein B

DNA: Triplet (dt)
mRNA: Codon (mc)

3 stop codons: UAA, UGA, UAG
1 start codon: AUG ( →  Methionine)

Structure of tRNA


Each tRNA holds a particular anticodon and amino acid.



Translation in nucleus: Turning RNA into Protein

80s ribosomes in humans = 40s and 60s ( → 100s)
70s ribosomes in prokaryotes = 30 and 50s ( →  80s)

Final mRNA In cytoplasm:

Methylated cap + 5’ UTR + Start codon + Exons + 3’ UTR + Poly-A tail


Smaller unit (40s) of ribosome binds to mRNA in the methylated cap first then scans in the 5’ → 3’ direction to find the 5’ UTR. Once it reaches 5’ UTR, the larger subunit of ribosome joins in -- bringing its E, P and A site.

The tRNA (carrying methionine) which recognizes the start codon AUG on mRNA binds to P site of ribosome.

The next tRNA anticodon base pair with the next codon on mRNA arrives at A site and a peptide bond is formed between this amino acid and the preceding methionine.

The previous tRNA moves to E site and exits to find new amino acid.

The ribosome moves one codon downstream -- until reaching stop codon - NO tRNA molecules.

Protein release factors recognize the stop codon by binding to A site and requiring GTP. This  releases the polypeptide from the ribosome, and also dissasembles the ribosome.

Eukaryotic cells: Polyribosome (one mRNA strand worked by many ribosomes!)

DNA Replication


  1. Helicase separates DNA double helix
  2. Single-stranded binding proteins stabalize unwound parental DNA
  3. DNA polymerase III makes leading strand UNINTERRUPTED  as DNA polymerase moves in the direction of replication fork
  4. Lagging strand made discontinuously as Okazaki fragments as DNA polymerase moevs in the opposite direction of replication fork
    1. RNA primase adds RNA primer
    2. DNA polymerase III encodes lagging strand
    3. DNA polymerase I replaces RNA primers with DNA
    4. DNA ligase links Okazaki fragment


Telomere: Region of repetitive DNA sequence (TTAGGG) at end of chromosome
  • Deter gene degradation at ends of chromosome
  • Shortens with each and every cell division

Blue = RNA primer

Telomerase in DNA replication: DNA polymerase alpha + RNA acts to lengthen telomeres
  • Uses RNA as template
  • Absent in somatic cell
  • Present in germ-line cell (sex cells) and cancer cells
  1. Telomerase attaches to telomere of leading strand
  2. DNA polymerase of telomerase lengthens the leading strand complementary to its RNA template
  3. DNA Primase makes RNA primer near 3' end of lagging strand, complement to the leading strand
  4. DNA polymerase fills in the rest on the lagging strand

Note that a short region of the 3' end will always remain single stranded



Topoisomerase

Twist = winding of DNA around each other
Writhe = coiling of the entire thing


Supercoiling: Twisting of DNA due to under- or over-twisting (youtube.com/watch?v=LXqEJ_f3_3k)
  • +'ve supercoiling: Due to extra twists (top)
  • -’ve supercoiling: Due to less twists (bottom)


Types of topoisomerase
1a - cuts one strand and relaxes only negative supercoils by breaking dna and passing the other through; does not use ATP
1 b - cuts one strand and relaxes positive AND negative supercoils by breaking and rotating the end; does not use ATP
2 - cuts both strands and uses ATP
.



Mitosis to form 2 genetically identical daughter cells
  1. Prophase
    1. Disintegration of nuclear membrane
    2. Nucleolus disappear
    3. Chromosome condense
    4. Centrosome (pairs of centrioles) separate to form centriole (9 of 3x microtubules) at poles
      1. Form kinetochore (attach to centromere of sister chromatids) and nonkinetochore (attach to centriole) microtubules
  2. Metaphase: Chromosome aligns along equator of cell (metaphase plate)
  3. Anaphase: Kinetochore microtubules contract → chromosomes
    1. While non-kinetochore microtubules pull the centrosomes apart to lengthen cell
  4. Telophase
    1. Nuclear membrane reforms
    2. Nucleoli appear
    3. Chromosomes decondense
  5. Cytokinesis
    1. Cleavage furrow form between cells (pinch containing contractile fibers around metaphase plate)

 

Cell Signalling

Receptors

Cell surface receptors: Transmembrane (e.g. GPCR g-protein coupled receptor)
Intracellular receptor: Nuclear receptor in cytosol - glucocorticoid/mineralocortoid/sex hormone/thyroid hormone which cause the ligand-nuclear receptor to bind to DNA to control DNA expression

Growth Factors

  • Stimulate cell growth/differentiation/survival

Forms of cell signalling
  • Endocrine: Secretion into blood → effector is distant cells
    • Hormones
  • Paracrine: Secretion into tissue fluid → effector is neighbouring cells
    • growth factors, cytokines
  • Autocrine: Secretion → effector is itself
    • IL1 (InterLeukin)in monocyte, IL2 in T cells (express IL2R to cause monoclonal expansion)
  • Neuronal: Neurotransmitters
  • Contact-dependent: Cell surface signalling molecule x Cell surface receptor - no need to keep making ligands → use less energy
    • Notch signalling pathway
    • Contact inhibition in normal cells
    • Pilli: Conjugation (transfer DNA from one pathogen to another)

Signalling pathways

GPCR (G Protein Coupled Receptor): 7 transmembrane receptor

G protein = α (GTPase), β and γ subunit
  1. Ligand binds to GPCR
  2. Conformational change in GPCR → associated G-protein α subunit switches its bound GDP to GTP
  3. α subunit dissociates from G protein to cause further intracellular signalling by activating effector proteins e.g.
    1. enzyme adenylyl cyclase (ATP → cAMP - 2ndary messenger) - signal amplification as one AC can convert 100x ATP into 100x cAMP cyclic AMP)
      1. Glycogenolysis:
        Glycogen (n residues) + Pi <-->  Glycogen (n-1 residues) + G1P
        1. cAMP activates PKA → PKA activates glycogen phosphorylase (GP) → GP converts glycogen to glucose-1-phosphate

Receptor tyrosine kinase and ras

  1. Ligand binds to receptor
  2. Receptor autophosphorylates tyrosine residue to create binding site for GRB2 and SOS
  3. SOS binds and activated
  4. SOS causes ras-GDP (inactive) to ras-GTP (active)
  5. Active ras activates MAP3K (Ref) →  MAP3K activates MAP2K (Mek) →  MAP2K activates MAPK (ERK) →  MAPK phosphorylates TF Transciption Factor (active)

Ras is a G protein so its α subunit (GTPase) will eventually convert ras-GTP → ras-GDP (inactive)

Sonic hedgehog pathway
  1. Hedehog ligand binds to patched receptor
  2. Patched receptor releases smoothened
  3. Smoothened inhibits PKA and slimb which together phosphorylate Ci
  4. Ci is not phosphorylated, thus, it can enter nucleus → gene transcription


If absent ligant, patched protein doesn’t release smoothened → smoothened not released → PKA and slimb phosphorylates Ci →  Ci can’t enter nucleus →  no transcription

Wnt signalling - WuB - wnt ubiquinate b-catenin
  1. Wnt binds to frizzled receptor
  2. This activates dishevelled which inhibits destruction complex (APC, axin, GSK3) which acts to ubiquinates B-catenine
  3. B-catenine not ubiquinated, thus increase in concentration in cytoplasm and acts as TF Transcription Factor to increase gene expression


No Wnt binding → Dishevelled not activated → destruction complex (i.e. removes!) removes B-catenin



Termination of cell signalling
  • Ligand goes away (e.g. ligand gated ion channel)
  • Degradation of GTP → GDP (α subunit of G protein is GTPase) in G proteins (e.g. ras)
  • Degradation of cAMP → AMP (e.g. by cAMP phosphodiesterase)
  • Desensitization
    • GPCR phosphorylated by GRK (GPCR kinase) → less active
    • GPCR’s increased affinity for arrestin → arrestin-receptor complex initiates receptor internalization via endocytosis
  • Internalization of receptor
    • Receptor x adaptin x clathrin
    • Clathrin coat stabalizes vesicle budding

 

Cancer



Checkpoints (
purpose of check point - Is cell large enough to divide? Are all replication errors repaired? )
1. G1 checkpoint: R point
  • Damaged DNA?
    • If so, inhibit cdk4/6 (e.g. by p53) and repair before replicate

2. S phase: Replication

3. G2 checkpoint
  • Healthy cell: Antiapoptotic BCl-2 displayed on mitrochondrial membrane
      • Damaged cell
        1. Bax inhibits BCl2 and axes mitochondrial membrane
        2. Cytochrome c efflux
        3. Apaf-1, procaspase 9, Cytochrome c → apoptosome
        4. Apoptosome activates caspase 3

        - Extrinsic pathway (Death receptor pathway with CTL CD8T)

        FasL from CTL
        1. CTL presents FasL
        2. FasL bind to Fas on target cell to be killed
        3. Fas trimerization
        4. Death inducing signal complex (DISC) formed: procaspase 8, Death domain, FADD (DISC ~ pdf)
        5. Caspase 8 activated
        6. Caspase 8 activates caspase 3

        Anaplasia – dedifferentiation

        Hyperplasia – physiological proliferation

        Neoplasia – abnormal proliferation

        Dysplasia – maturation abnormality

        Metaplasia – cell type conversion



        Neoplasm: Uncoordinated (i.e. abnormal!) growth (no brakes to cell cycle)
        - Benign: No capacity to invade and metastatsize
        - Malignant: Capacity to invade and metastasize

        Non-neoplastic:
        - Hamartoma

        Common sites for metastasis
        2bs, 2ls.. breast, brain, liver, lung


        Benign tumor
        1. Organized
        2. Well differentiated (resemble parent tissue) -> monomorphic (one cell type only)
        3. Circumscribed border
        4. Non-metastatic
        5. Normal N:C ratio and normal mitotic spindle
          1. Prominent nucleoli
        6. Normal mitotic activity
        7. Normal telomerase
        8. No necrosis
        Malignant tumor
        1. Unorganized (polar)
        2. Variable differentiation: Well diff (low grade) to Poorly differentiated (high grade - anaplastic) - pleomorphic (variable cell shape and size)
        3. Infiltrative
        4. Metastatic
        5. HIGH N:C Ratio and abnormal mitotic spindle
        6. Mitotic activity depends on degree of differentiation
          1. If low grade (high highly differentiated): Norma mitosisl and no necrosis
          2. If high grade (anaplastic - poor diff.): HIGH mitosis and necrotic
        7. High telomerase activity (preserve length of telomeres at ends of chromosome)
        8. Necrosis (coagulative - infarction due to ischemia!!!))



        Radiotherapy: Therapy to kill cancerous cells using radiation
        • Also affects normal cells but these cells have REPAIR mechanisms (MSH and MLH) while cancerous cells do not
        • Curative: Destroy to cure
        • Palliative: Simply to control symptoms
        • Neoadjuvant: Radiotherapy to shrink the tumor first → Surgery to remove tumor
        • Adjuvant: Surgery → Radiotherapy
        • Internal (Brachytherapy): Radioactive substance (e.g. seed) put in close to tumor
        • External: Xray beams outside body
        • Intensity-modulated:
          • Use 3d CT scan to find dose intensity pattern for
          • Vary intensity of beams to fit tumor shape thus lessen sfx to normal cells
        • Conformal
          • Use metal blocks so beam conforms more to tumor shap
    • Excision repair
      • Base excision repair
        1. Glycosylase identifies and removes damaged base (e.g. deamination or alkylation) while leaving the sugar-phosphate backbone intact (the AP site - apurinic/apyrimidinic)
        2. AP endonucleuase breaks down backbone to produce gap
        3. DNA polymerase B replaces with correct nucleotide
        4. DNA ligase ligates using ATP
      • Nucleotide excision repair
        1. Similar but instead of removing single bases it remove PATCHES
      • Msmatch repair gene
        1. MSH: recognize mismatch (see)
        2. MLH: remove mismatch (lose)
      • If problem, inhibit cdk1


      4. Spindle assembly checkpoint - are chromosomes aligned at equator?
      If not - non-disjunction: 2n and 0n daughter cells

      CyclinCDKComplex formed
      Cyclin D
      Cyclin E
      Cyclin A
      Cyclin B
      CDK 4/6
      CDK 2
      CDK 2
      CDK 1
      G1-cdk
      g1/s-cdk
      s-cdk
      m-cdk

      DEAB, 4/6, 2, 2, 1



      Cancer - failed checkpoints

      Tumor suppressor gene: Recessive
      • TP53 - Halts G1
      • Rb - Halts R point
      • APC - ubiquinates B-catenin in Wnt pathway

      RB: RB gene codes for Rb protein (Rb-E2F) and CDKI p16
      • Left: If no GF, Rb is bound to E2F (E2F not released for making cyclin E) and p16 labels G1-cdk with ubiquitin → No S phase progression
      • Right: If GF Is present in G1, G1-CDK phosphorylates Rb →  Rb is made inactive and E2F is freed →  E2F codes for cyclin E (for G1/s) →  S phase progression

      E2F codes for cyclin E

      TP53 (Gene): codes for p53 and CDKI (p21 - 21 and 53 are odd numbers...)

      Normal: mdm2 complexed with p53
      Cell damage (e.g. UV) mdm2 x p53 dissociates -> p53 goes into nucleus and causes cell cycle arrest (via p21)
      • repair DNA
      • if DNA can’t be repaired -> Apoptosis

      Normal cell - normal TP53, thus normal p53
      In most cancer cells - defective TP53 - thus p53 can’t act in nucleus propertly and no G1 halt


      HPV in p53 and Rb protein
      p53 and Rb both binds to oncogene products
      • E6: Ubiquinates p53 for proteosomal destruction
      • E7: Binds to Rb thus freeing up E2F



      Oncogene: Dominant

      Knudson’s two hit hypothesis

      Normal -> Hyperplasia -> Dysplasia -> Preinvasive (Carcinoma in situ) -> Carcinoma -> Metastatsis

      Oncogene: A gene that when mutated/expressed at high levels, contributes to cancer.
      • Ras (GTPase in receptor tyrosine kinase)
      • Erbb1 (Her1) - codes for EGFq
      • PDGF
      • myc  (transcription factors encode other transcription factors)
      • BCL2 (gene product anti-apoptosis) - B cell leukemia
      • MSH: Recognize DNA mismatch
      • MLH: Excise DNA mismatch

      Leukemias - Burkitts lymphoma t(8,14) and B cell leukemia (BCl2) t(14,18)

      Genes coding for antibodies on Chromosome 2 (kappa light chains), 22 (lambda light chains) and 14 (heavy chains).The genes are expressed only in B lympphocytes because only they have the relevant TF (Transcription Factor) for promoters and enhancers.

      Other cancers involving translocation of heavy chain gene on chromosome 14 - multiple myeloma.

      B cells - 14




      Burkitt's lymphoma is a solid tumor of B lymphocyte.

      Burkitts ~ 8

      Reciprocal translocation of c-myc (codes for TF) from chromosome 8 to the enhancers of the antibody heavy chain genes on chromosome 14 - t(8;14)  translocation
      The long (q) arm of the resulting chromosome 8 is shorter (8q−) than its normal homologue; the long arm of translocated chromosome 14 longer (14q+).

      BCl2 - gene that codes for integral membrane protein BCl2 on chromosome 18
      B ~ 8
      l ~ 1

      In B-cell leukemia, BCl-2 on chromosome 18 reciprocal translocated to chromosome 14 (codes for antibody heavy chain) - i.e. t(14;18) translocation -- BCl-2 gene place close to heavy chain gene enhancer  →  Antiapoptic B cells


      Apoptosis: Caspase 3 activation → Caspase 3 cleaves CAD (DNAse) from ICAD (inhibitor of CAD) → CAD migrates to nucleus and degrades chromatin



      - Intrinsic pathway (mitochondrial)