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BMS1062 BIO > DNA AND THE IMMUNE SYSTEM > Flashcards

DNA AND THE IMMUNE SYSTEM Flashcards

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1
Q

What is recombination?

A

The process where DNA molecules are broken and fragments rejoined in new combinations

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2
Q

Where can recombination occur?

A
  • In living cells (meiosis crossing over)

- In vitro by use of DNA and restriction enzymes breaking DNA fragments and ligase rejoining

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3
Q

How can the immune system protect us from so many different pathogens?

A
  • Innate immunity (physical barriers, cellular soluble components)
  • Adaptive Immunity (cellular and humoral mechanisms)
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4
Q

What is innate immunity?

A
  • Physical aspects, phagocytic cells, enzymes, complements
  • Doesn’t adapt to target presented, based instead on inherited traits from parents
  • There are specific receptors on the surface of phagocytic cells that recognise repeating patterns called Pathogen-Associated Molecular Pattern (PAMPs) (could be viral DNA, viral RNA, pathogen specific wall components)
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5
Q

What can’t innate immunity do?

A
  • Adapt to target
  • Can’t effectively keep up with rapid pathogen change or pathogen not encountered before
  • Isn’t specific like the adaptive immune response
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6
Q

What is adaptive immunity (general) ?

A
  • Adapts “remembers” antigen

- Clonal selection etc.

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7
Q

What is immunological memory?

A
  • 2nd encounter with antigen leads to greater and more rapid immune response
  • Memory is the reason why vaccines work
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8
Q

Where do T cells develop?

A
  • In the Thymus and are cellular immunity
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9
Q

Where do B cells develop?

A
  • In the bone marrow and produces antibodies
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10
Q

Are T cell receptors membrane bound?

A

Yes! But they don’t secrete anything

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11
Q

Are the B cell receptors membrane bound?

A

Yes! AND these DO secrete their receptors as antibodies (immunoglobulins)

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12
Q

What can we have an immune response to?

A
  • Metals (Nickel)
  • Plants (peanuts)
  • Helminths (worms-tapeworm)
  • Fungi (thrush)
  • Unicellular parasites (malaria, trypanosomes)
  • transplanted tissue
  • Our own tissue (rheumatoid)
  • tumors
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13
Q

What can the immune system recognise at the molecular level?

A
  • Proteins
  • Peptides (T cells)
  • Glycoproteins
  • Glycoliids
  • Carbohydrates
  • Nucleic Acids
  • Metal ions (complexed to proteins)
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14
Q

What do B cell receptors recognise?

A
  • The whole 3D product

- Can bind to a fold in a protein

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15
Q

Do T cells “see the whole thing” like B cells?

A

NO! They only see the processed peptide in the groove of the MHC molecule that is on the surface of specialised cell (antigen presenting cell)

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16
Q

What is the rough pathway the recognition and presenting of a strain of infection/virus?

A
  • dendritic cells (like macrophages) can break down virally infected cell,
  • process it,
  • break it down (through proteosome)
  • load peptides into MHC molecules that come to the surface
  • Then antigen presenting cell “lit up like x-mas tree”
  • MHC molecules then bind peptide that has been processed inside APC
  • Presents (in groove of MHC) the peptide to the T cell
  • So sees processed peptide in the groove of the MHC
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17
Q

Do we have receptors on the lymphocytes prior to, or after the exposure to an antigen?

A
  • PRIOR to exposure to an antigen (generated during development)
  • Both B and T cells can do this
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18
Q

How do B and T cells allow for recognition of antigens?

A
  • They are CONTINUALLY circulating in the body and drop into the lymph nodes to see if there is anything the receptor each B and T cell has can recognise.
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19
Q

What is the heavy chain on a B cell receptor analogous to?

A
  • The beta chain in the T cell receptor
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20
Q

What is the light chain on the B cell receptor analogous to?

A
  • The alpha chain in the T cell receptor
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21
Q

How do we generate so many different types of receptors with not as many genes to encode each individual one?

A
  • Somatic recombination of the inherited gene segments and by somatic recombination
  • In each B cell clone, chopping up of DNA and recombination was DIFFERENT (recombination of germline DNA)
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22
Q

Is the DNA in our genes the same in the B cells and T cells?

A

NO! In B & T lymphocytes the DNA changes

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23
Q

Where does DNA recombinaton occur in terms of B and T cells?

A
  • Gene for the heavy chain B cell receptor
  • Gene for the light chain B cell receptor
  • Gene for the beta chain T cell receptor
  • Gene for the alpha chain T cell receptor
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24
Q

How many hypervariable regions does each ANTIBODY have?

A
  • 12 hypervariable ( complementity determining) regions
  • 3 per heavy chain (32=6) + 3 per light chain (32=6)
  • So 6+6=12
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25
Q

What is the MOST variable region of all the variability regions?

A
  • Region 3
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26
Q

Why is region 3 the most variable region compared to regions 1 and 2?

A
  • Because it is encoded by the germline (like 1 and 2) BUT ALSO encoded by combinational (V D and J sections recombine) and junctional diversity.
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27
Q

What is the alpha chain made up of?

A
  • ONLY V and J complemitity determining regions (not D)
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28
Q

Which regions encode the ‘loops’ in both the heavy and light chains ?

A
  • CDR 1,2,3 encode Loops in both heavy and light chains

- Region on tip of receptor site is where loops stick out

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29
Q

Where do the loops “stick out”?

A
  • On the tip of the receptor site
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30
Q

What structural feature recognises antigens?

A
  • The loops (encoded by CDR 1 2 3)
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31
Q

When do B and T cells rearragne their receptors?

A
  • Prior encounter with antigen
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32
Q

How does baby B cell in bone marrow become mature?

A
  • Rearrange genomic DNA in a clone e.g. V1D1J1
  • These then get hooked up to constant region
  • Clones have different combos
  • mRNAs then derived from rearranged DNA
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33
Q

What do V regions recognise?

A
  • Antigens

- Different for each B or T cell

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34
Q

Is CDR (complemantrity determining region) 3 in light chain still the most variable out of V1 and V2?

A
  • YES! Because still junctional diversity from V and J
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35
Q

Where does recombination occur in B cells?

A
  • In developing B cells of bone marrow
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36
Q

What does recombination involve?

A
  • Ig genes in B cells (TCR genes in T cells) involves coordianted activity of several enzymes
  • RAG 1 and 2 enzymes
  • DNA repair enzymes here (as also in many other cells)
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37
Q

What do RAG 1 and 2 stand for?

A

Recombination Activating Genes 1 &2

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38
Q

Where are RAG 1 and 2 expressed?

A

Only in DEVELOPING B AND T CELLS

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39
Q

Do you want V and J to pair (heavy chain) or J and J? (VJ or JJ)

A

NO because you want V–>D–> J

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40
Q

How is incorrect pairing of J &J or V& J prevented?

A
  • Recombination Signal Sequences (RSS)

- 12bp or 23bp

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41
Q

Can a 12bp speces (RSS) pair with another 12bp?

A

NO!! (The same goes for 23 and 23bp)

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42
Q

What recognises RSS sequences to initiate recombination?

A
  • RAG enzymes
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43
Q

Where are the RSS sequences found?

A
  • 3’ end of each V gene segment
  • 5’ end of each J segment
  • BOTH sides of the D segment
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44
Q

What do the sequences consist of (RSS)?

A
  • Conserved 7 nucleotide heptomer
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45
Q

What is the 12/23 rule?

A
  • Only a gene segment flanked by RSS with 12bp spacer can be joined to one flanked by 23bp spacer RSS
  • Prevents VV etc
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46
Q

What can a Dheavy (beta) gene segment be joined to?

A
  • Jh gene segment
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47
Q

What can Vheavy gene segment be joined to?

A

D heavy

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48
Q

Why cant Vh be joined to Jh?

A
  • Because both V nd J heavy segments are flanked by 23bp spaces and Dh gene segments have 12bp spaces on both sides
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49
Q

Which region does recombination occur in?

A
  • Only between segments flanked by 12bp spacer and 2bp spacer
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50
Q

What occurs in light chain pairing?

A
  • DNA forms a loop and is deleted

- Loop excised and V region ligated to J region (bc no D region in light chain)

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51
Q

How do do RAG enzymes mediate recombination?

A
  • Bind to RSS
  • Then complex to each other to form hairpin loop which is the excised
  • DNA is cleaved to create hairpin strucutres at end of Ig gene segments
  • Other proteins involves to clean up recombination signal sequence
  • DNA polymerase fills in overhangs
  • Addition or subtraction of nucleotides then occurs and DNA ligase joins DNA fragments to form VJ segment
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52
Q

What can you get from the action of enzymes?

A
  • Joining of V and J segments

- Addition and subtraction of bases

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53
Q

What adds ENORMOUSLY to the diversity of the VJ segment?

A
  • Addition and subtraction of bases
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54
Q

What happens in the ADDITION of nucleotides?

A
  • TdT adds nucleotides at joint (and not encoded in the genome)
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55
Q

What happens in the subtraction/deletion of nucleotides?

A
  • Catalysed by DNA exonucleases

- Removes the nucleotides before V(D) J segments are joined

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56
Q

Which creates more diversity; recombination or junctional diversity?

A

Junctional diversity

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57
Q

When a B cell encounters an antigen what changes?

A
  • the CONSTANT regions change (not the variable region;that stays the same)
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58
Q

What defines the isotype of Immunoglobulin?

A

Constant region

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59
Q

Which antibody (Ig) is C mu (constant region mu)?

A
  • IgM
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60
Q

Which antibody is C delta ?

A
  • IgD
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61
Q

Which antibody is C gamma?

A
  • IgG
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62
Q

Which antibody is C alpha?

A
  • IgA
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63
Q

Which antibody is C epsilon?

A
  • IgE
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64
Q

Why do mast cells recognise IgEs?

A

Because they have receptor for ‘stalk’ (constant region) of IgE so different isotypes can have the same antigen binding site but different constant regions (Class/isotype switching)

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65
Q

When does isotype switching occur?

A
  • Separately and AFTER the recombination process that produces the antigen receptor
  • After B cells encounter ad are activated by antigen and T cell helper
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66
Q

What are the first Igs that can be expressed by a B cell?

A
  • IgD(membrane bound) and IgM (secreted as pentamer)

- These don’t need the T helper cell to be expressed

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67
Q

What does IgM act as?

A
  • Sort of a first line of defense for the adaptive immune system
  • It is secreted FIRST because doesn’t require T cell help
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68
Q

What allows IgA, IgE and IgG to be made?

A
  • B cell encounters antigen and B cell receptor is triggered
  • Then B cell (through CD40 ligand) contacts T cell
  • T cell sends cytokine to B cell to tell it which type of switch to make (e..g make IgG–>virus)
  • Then T cell produces certain cytokine that leads to B cell B cell producing certain transcription factors that cause it to switch to needed Ig
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69
Q

What is IgG important with?

A
  • Serum and other extracellular fluids (lymph)
  • neonate
  • can neutralise viruses
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70
Q

What is IgM do to with?

A

Serum

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71
Q

What is IgA to do with?

A
  • Serum and secretions e.g. mothers milk

- Important in gut

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72
Q

What is IgE to do with?

A
  • Beneath skin surface
  • Respiratory, GI, skin
  • Allergic reactions
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73
Q

What happens if B cell receptors are activated by antigen but NOT T cell to help?

A
  • B cell will make IgM (low affinityantibody. pentomer and can crosslink)
  • IgM antibody is secreted first (as a first line of specific defence)
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74
Q

When does a gamma interferon get released?

A
  • If we have a virus by T cells (and IgG antibody)
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75
Q

Which interferon drives IgE production?

A
  • IL-4 (IgE is monomer FYI)
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76
Q

Which interferon drives IgA production?

A
  • TGF-beta

- IgA secreted as dimer FYI

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77
Q

Are enzymes involved in class switching different to those involved in the variable region rearrangement?

A
  • YES THEY ARE DIFFERENT!

- Class switching molecules signal sequences and Dna repair enzymes

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78
Q

What do cytokines do?

A
  • Tell B cell which way to switch
79
Q

What is a T cell made up of?

A
  • Alpha and beta chian (both with variable and constant regions)
  • Also have the transmembrane and short cytoplasmic regions
80
Q

Is the T cell receptor secreted?

A
  • NO!
81
Q

How many complemantity determining regions (CDTs) do T cells have?

A

6

82
Q

Which type of T cell is activated if a dendritic cell presents PEPTIDE derived from herpes virus in the groove of MHC class I?

A
  • CD8 cells (restricted to recognising peptide in groove of MHC class I
83
Q

Where is MHC class I epxressed?

A

On the surface of ALL nucleated cells!

84
Q

Which is the only type of cell that can activate a T cell?

A
  • Dendritic cell
85
Q

Where is MHC class II expressed?

A
  • On surface of ALL antigen presenting cells (dendritic cells)
86
Q

Which type of T cells recognise class II MHC and the peptides (12-14aas) ?

A
  • CD4 T cells (helper cells) (restricted to the MHC class II)
87
Q

What is the specificity of response driven by?

A
  • (Not the TCR) BUT by other molecules associated with T cell such as CD4 and CD8
88
Q

Where do CD4 T cells bind to?

A
  • Class II MHC
89
Q

Where do CD8 T cells bind to?

A
  • Class I MHC
90
Q

What do alpha and beta chains on T cell receptors contain?

A
  • Alpha and beta chains contain short stretches where the most variability between different T cell receptors is concnetrated (hypervariable or CDR)
91
Q

How many CRDs does an alpha chain have?

A

3

92
Q

How many CDRs does a beta chain have?

A

3

93
Q

What can alter the specificity of TCR

A

Small differences in individual TCR molecules

94
Q

What is the process of a T cell maturing?

A
  • When stem cell arrives at thymus (from bone marrow) it receives signals that tell it to start to arrange T cell receptor
  • 2 CDRs encoded in germline V region gene elements (fixed but some variety)
  • 3RD CDR is generated by the junction of gene elements that come together (much more potential for variability)
95
Q

What does the alpha chain contain?

A
  • V and J
96
Q

What does the beta chain contain?

A

V D J

97
Q

What do the CDR regions recognise?

A
  • BOTH the peptide and MHC (varies between unique T cell receptors)
  • Only a few amino acids of MHC bound peptide in contact with TCR
98
Q

Do T cells (like B cells) use a ‘random ‘ process to generate diversity?

A

YES!

99
Q

What is recombination in the TCR gene like?

A
  • Similar to B cell
  • TCR alpha gene has similar strucutre to Ig light chain
  • contains V and J segments
  • V pairs with J
  • Contains V D J
100
Q

What pairs in the Beta chain (heavy) ?

A
  • D and J pair first then pair with V (do this through the 12/23 rule to stop VV combination)
101
Q

Where does T cell receptor rearranging occur?

A
  • In thymus
  • Beta chain first then alpha chain (random process)
  • T cell with TCR must decide which kind of T cell it wants to be (has CD4 and CD8 molecules)
102
Q

What happens if TCR can’t find the MHC marker?

A
  • It gets killed :(
103
Q

What happens if TCR DOES find the right MHC marker (just right because it doesn’t bind too tightly)

A
  • TCR alpha and beta chain rearranged and on the surface in thymus
  • Gets survival signal and it is excited from thymus
  • It is now circulating around body waiting to drop into lymph node and recognise ( on the surface of dendritic cell) the peptide in groove of MHC that it recognises with high affinity (loop in CDR )
  • Then dendritic cell gets signal to be activated
104
Q

What happens if once alpha and beta receptors are recognised, TCR looks for MHC and it binds REALLY STRONGLY (has high affinity for it) ?

A
  • This means it recognises MHC and SELF PEPTIDE really strongly
  • The recognition of self peptide is really dangerous (autoimmune) do it gets killed
105
Q

Is recombination for T cells the same process as B cells?

A

Yes it is analogous

  • RAG 1 and 2
  • DNA repair enzymes just like B cells
106
Q

What is another name for cancer?

A
  • Tumorogenesis
107
Q

What are 3 changes that occur in tumorogenesis?

A
  • Failure to die (IMMORTALISATION)
  • TRANSFORMATION
  • METASTISIS
108
Q

What occurs in immortalisation?

A
  • Cells capable of dividing indefinitely (but remain sensitive to constraint of growth e.g. contact inhibition)
109
Q

What occurs in transformation?

A
  • Failure to observe normal constraint of growth. (NO LONGER sensitive to contact inhibition and so grows on top of each other)
110
Q

What is metastisis?

A
  • Ability to invade normal tissues
111
Q

What is ONE possible cause of cancer?

A
  • (Proto) Oncogenes
112
Q

Are the oncogenes normal?

A
  • Yes but mutations in these genes can lead to transformation
  • So can convert to cancer- promoting oncogene
  • Common (more than 100 identified)
113
Q

What do proto oncogenes encode?

A
  • Cell signalling
  • Cell cycle control
  • Transcription factor etc.
114
Q

What can some oncogenes be mutated by?

A
  • Gene rearrangements

- This results in sequence alterations or dysregulation of expression (overproduced)

115
Q

How can therebe a bad result in VDJ recombination?

A
  • After recombination, DNA may not be repaired correctly
116
Q

What is the c-myc oncogene?

A
  • Transcription factor normally associated with turning on genes required for CELL DIVISION
  • Expression is constrained to appropriate time and rate in the cell cycle
117
Q

What happens when expression pattern of c-myc is altered ?

A
  • Can result in unrestricted growth of cell
118
Q

Which tumors is c-myc involved in?

A
  • lung carcinoma
  • sarcoma
  • leukemia
  • lymphoma
  • pastomacytoma (just to give context not examinable)
119
Q

What is burkitt’s lymphoma?

A
  • tumor of mature B cells
  • Expression of the proto-oncogene normally tightly regulated
    BUT in Burkitts Lymphoma, MYC is RELOCATED next to regulatory region of Ig heavy chain gene (also can be relocated to encounter light chain gene too)
  • Hyperproliferation of B cells leads to this lymphoma
120
Q

Where abouts does VDJ recombination occur in terms of chromosomes?

A
  • WITHIN a gene on ONE chromosome
121
Q

Where does recombiation between cMYC and Ig heavy chain occur?

A
  • Between two different chromosomes (8 AND 14)
122
Q

What does the process of recombination in Burkitts lymphoma lead to?

A
  • Swapping of the gene segment between two chromosomes and fusion of it
123
Q

When does recombination occur?

A
  • In meiosis BUT ALSO IN LYMPHOCYTES
124
Q

Where is myc gene translocated to in burkitts lmyphoma?

A
  • To region where expression is driven by Ig heavy chain regulatory sequences (Ig enhancer)
125
Q

What does the c-myc gene lose in burkitts lymphoma?

A
  • Its own regulation
126
Q

What does the loss of c-mycs own regulation cause?

A
  • unregulated expression of c-myc in B cells by Ig promoter leading to unregulated proliferation of B cell clone
  • Can also involve light chain kappa locus
127
Q

What is the proof that the Ig/myc translocation causes cancer?

A
  • Make 2 lines of transgenic mice
  • Transgene 1: C-myc alone
  • Transgene 2: c-myc fused to Ig heavy chain enhancer
  • In transgene 2 mice had massive tumors
  • Can also happen in T cells and in heavy or light chain ***
128
Q

What is hyper IgM syndrome?

A
  • Patients have normal or elevated IgM but low IgG or IgA
  • Leads to recurrent infections
  • IgM the first to be secreted without CD40 (T helper cell)
  • so mutation in the CD40 ligand on T helper cell prevents class switching
  • So IgG and IgA have an essential role in fighting infection
  • Also that Cd40 ligand has essential role in promoting isotype switching
129
Q

What is a process that the B and T cell undergo to fight infection?

A
  • B cell sees the herpes virus in 3D conformation
  • Then takes it it, processes it and spits peptide on surface of MHC II groove
  • Helper T cell in lymph that is activated has T cell receptor that can see MHC II and peptide derived herpes ON SURFACE of B cell
  • They then ‘shake hands’ (CD40 and CD40 ligand)
130
Q

What is reverse genetics?

A
  • Genetic engineering used to create organisms with artificial mutations in genes
131
Q

Why is reverse genetics important?

A

30 000 genes in genome so 30 000 potential drug targets

132
Q

What is transgenic technology? -

A
  • Genetic manipulation of organisms that affects the exprression of defined products
  • understanding the FUNCTION OF GENES
133
Q

What is transgenic known as?

A
  • ADDITION of a function

- Introducing a transgene into organisms genome

134
Q

What is ‘knockout’ known as?

A
  • REMOVAL of a function
  • Removing a gene (or portion of gene) from organisms genome
  • can occur in any organism (jellyfish)
135
Q

What is an example of use of transgenics?

A
  • Vitamin A defiiciency
  • (by using transgenic mice that synthesise beta carotene)
  • so plant and bacterial genes tat encode enzymes important in beta carotene synthesis were introduced into mice
136
Q

What can a gain of function transgenics result in?

A
  • Expression of gene not normally found in nature e.g. self gene in different location
    OR introduce the foreign gene (from different organism into another organism to be expressed)
    OR Could take a gene that is normally expressed and cause it to be OVEREXPRESSED
137
Q

Is the pre CRISPR method for transgenic mice effective?

A

NO! You could have 10 pups born without the gene!

138
Q

Does genomic or cDNA work better for transgene coding region?

A

-Genomic DNA is better BUT both can be used

139
Q

What does the basic transgene require?

A
  • Promoter and coding region
  • Promoter can change based on the location you want to be expressed
  • Could also pick inducible promoter (only induced with certain drug e.g. oestrogen)
  • Promoter and coding region then ligated together
140
Q

How can you check if the transgene has been taken up?

A
  • Fluorescence or PCR
141
Q

How do we study the particular T cell Receptor that we suspect to be causing MS?

A
  • Making mice transgenic for receptor
  • Need to make 2 transgenes (one for alpha chain and one for beta chain)
  • Can take the alpha and beta chain and inject as transgenes together in eggs (but difficult becasue so many T cells)
142
Q

How do we make sure there is only one TCR for each T cell? (Or BCR for each B cell)?

A

Once functional rearrangements have occurred, signals cause RAG 1 & 2 to be shut down, so other gene is not rearranged (blocking other chromosome-allelic exclusion)

143
Q

What can happen when you inject rearranged T and B cell receptors in into mice?

A
  • When T cell goes into Thymus, it already has the rearranged alpha and beta chain
  • Because of allelic exclusion it fails to make its own arrangement
144
Q

What occurs in Immuoglobulin BCR transgenics?

A
  • Introduces rearranged Ig gene (isolated from mature B cells)
  • Nearly all B cells in “Ig gene” transgenic mice express it
145
Q

What occurs in TCR TRANSGENIC mice?

A
  • Nearly all lymphocytes in TCR transgenic mice express the SAME antigen receptor
  • possible through process of allelic exclusion
146
Q

What is allelic exclusion in more detail?

A
  • Presence of introduced “transgenic” BCR or TCR allele prevents rearrangement of endogenous “natural” alleles by switching off recombinant genes
147
Q

How do we generate TCR transgenic mouse with specificity for defined antigen?

A
  • Look in the brain of mice with EAE (model of MS) and find different lymphocytes (CD4)
  • Take out lymphocytes and feed target oligodendrocyte (antigen stimulation) to them in dish
  • Then you get build up of cells that recognise that antigen (end up with clonal population-cells all with same TCR alpha and beta chain)
  • Then isolate rearranged DNA for alpha and beta chain and make transgenic mice.
  • Inject it so it has normal promoter for TCR alpha and Beta chain (but alpha and beta chains have already been arranged)
  • They get co-injected into eggs then selection of transgenic mice occurs
  • Then follow the fate of trasngenic cells
148
Q

What do the cells of the inner cell mass of the blastocyst give rise to?

A

-The mouse (organism)

149
Q

What do the trophoblast cells of the blastocyst give rise to?

A
  • Normally make the placenta
150
Q

What occurs in gene knockouts?

A
  • Embryonic stem cells used from the inner mass of blastocyst
  • Kept in a pluripotent state
  • This allows them to be genetically modified (by knocking gene out), checking that it;s been knocked out and still have cell capable of giving rise to an ENTIRE embryo
151
Q

What would we have to do if we wanted to knockout a Beta2 microglobuline gene (only to learn process of gene knockout)?

A
  • Get copy of the gene (Beta2M) and modify it so it is defunct
  • Then get dodgy Beta2M gene into embryonic stem cell in dish so it recombines homologously with endogenous B2M gene and swaps itself over (putting dodgy one in place of endogenous one)
152
Q

What 3 things can happen when you put stretch of DNA that is known to be homologous to gene inside nucleus of embryonic stem cell?

A
  1. NOTHING- doesn’t recombine at all
  2. RANDOM INTEGRATION- DNA can integrate anywhere so exogenous gene hasn’t replaced endogenous gene
  3. Can go in SPECIFICALLY and homologous recombination allows endogenous gene to be replaced (must have this option! but…frequency is low..so must increase frequency…but how? tbc…)
153
Q

What are two ways that frequency of homologous recombination can be increased to select for embryonic stem cells?

A
  1. Make a targeting construct by having neomycin resistance (this has been put in middle of exon because we know it will screw up the function of the gene)
    - also only ones that have integratd marker will survive in presence of neomycin (G418)
  2. Selection for homologous event
154
Q

What is the process of increasing the frequency of homologous combination in 3 steps?

A
  1. Make a targeting construct (incorporating both neomycin resistance gene and thymidine kinase)
  2. Transfect targeting construct into Embryonic Stem Cells
  3. Obtain ES clones- Select ES clones and grow these on G418 and ganciclovir
    - Targeted ES clone is injected into a blastocyst
155
Q

What are the limitations of traditional gene targeting?

A
  • Difficult, slow, expensive (only does single mutations)
  • Difficult to interpret contribution of each SNP (single nucleotide polymorphism) to complex diseases given that most variants don’t act alone
  • Not feasible to test combined mutations
156
Q

What does CRISPR function to do?

A
  • Better more efficient way of
    1. Producing knockout genes
    2. Producing transgenes (addition of function)
  • there is guide RNA that locates target sequence and CAS9 to cut the DNA upstream of PAM sequences
157
Q

What is the process of CRISPR?

-

A
  • Has guide mRNA sequence
  • On the end it has a tracrRNA sequence which allows the enzyme CAS9 to come and bind
  • Lines up homologously
  • CAS9 then makes cuts in precise location
158
Q

What happens in CRISPR once CAS9 makes a cut?

A
  • You can give it no extra help and the repair can occur itself (but this is error prone so could have deletion)
  • could add in set of bases (to change amino acids) with oligo or plasmid donor (can make single base change or large sections)
  • can also switch genes on and off
159
Q

What is a parasite?

A
  • Organism which feeds off another organism (host) without producing any benefit to that host
160
Q

What is parahistology?

A
  • Study of protozoan (unicellular) or metazoan (multicellular) eukaryotes with parasitic lifestyle
161
Q

What organisms do metazoa comprise of?

A
  • Helminths (worms), microsporidia
162
Q

What is an example of a unicellular parasite?

A

Protozoa

163
Q

What are protists and some examples?

A
  • Single celled eukaryotes
  • Trypanosomes
  • Plasmodiums
164
Q

Which large group of parasites do malaria belong to?

A
  • Apicomplexa
165
Q

What are the 3 main groups of protist parasites?

A
  • Apicomplexa (malaria, taxoplasmosis)
  • Trypanosomatids (sleeping sickness-ameriacan-chagas disease)
  • Anaeorobic and others( amoebiasis, giardiasis)
166
Q

What are trypanosomes?

A
  • trypanosoma brucei
  • affect the neurons in CNS-lethargy and coma
  • Can have neurotoxic effects if not treated
  • Causes African sleeping sickness and idseases in cattle
167
Q

How are trypanosomes injected?

A

Via a tsetse fly

168
Q

What is the rough pathway of infection of trypanosomses?

A
  • Parasite migrates to fly’s salivary glands
  • Fly bites new mammal host
  • Parasite injected into blood stream
  • Parasites multiply in blood stream
  • can reach CNS to cause sleeping sickness
169
Q

Which point in the cycle of trypanosomes could the immune system respond?

A
  • proliferative stage- when it is free in th eblood because it is vulnerable
170
Q

What is the function of the spleen?

A
  • To filter the blood
171
Q

What do trypanosomes have on their surface that makes them unique?

A
  • VSG (Variant Surface Glycoprotein)
  • This is the only thing that is recognised by the antibody
  • VSG can change so antibody will no longer recognise
172
Q

Is only one VSG gene expressed at a time?

A

YES! Another example of allelic exclusion (also one telomeric site is active at one time)

173
Q

Does VSG switching occur independently of the immune system?

A

YES!

174
Q

Where are VSGs found?

A
  • Inside chromosomes
  • Some are inside telomeres (allelic exclusion mechanism to make sure each parasite only expresses one VSG (BUT VSG expressed can change-switch to another one)
175
Q

What is class I MHC involved in?

A

CD8 T cells

176
Q

What is the relationship between the CD4 T helper cells and CD8 T cells?

A
  • CD4 T cells help the CD8 T cells to become fully activated
177
Q

What are CD8 T cells good for?

A
  • Viral pathogens (cytotoxic) but not good for pathogens in blood
178
Q

Is VSG switching spontaneous?

A

YES! Not driven by pathogen

  • Occurs independently of immune system
  • Result of DNA recombination events during cell division
179
Q

What is the frequency of VSG switching controlled by?

A
  • Parasite (genetically)
180
Q

What are 3 possible mechanisms to change coats?

A
  • Gene conversion
  • Telomere exchange
  • In situ switch
181
Q

What occurs in the gene conversion method for VSG switchign?

A
  • Case for the vast majority (most common)
  • Involves DNA repair machinery of parasite
  • Replacement of expressed VSG gene by a pre existing SILENT COPY
  • Allows parasite to use silent subtelomeric VSG genes
182
Q

What occurs in the telomeric exchange mechanism for VSG switching?

A
  • Homologous recombination between telomeres

- Less common

183
Q

What occurs in In situ switching for VSG switching?

A
  • Direct recombination in situ
  • Exchanges alleles in expression sitet
  • activating another VSG expression site
184
Q

What is malaria caused by?

A
  • Plasmodium
  • 5 species infect humans
  • P.Falcipanum - is most lethal because it can stick to blood vessels (blood cells erupt at same time–> immune response kills human)
185
Q

Where is the first target of malaria (plasmodium-F.falcipanum)?

A

The liver (through Kuffer cell)

186
Q

What is the life cycle of P.Falcipanum (plasmodium)?

A
  • Sexual stage in mosquito (9-17 days)
  • About 5-20 sporozoites injected into blood and they enter hepatocytes (liver cells) within 30 minutes
  • Asexual liver sage (sits there inactively and no symptoms)
  • Thousands of merozytes then released into blood stream. Asexual blood stagecycle is shynchronous (takes 48 hours for P.Facipanum)
187
Q

Which stage of the plasmodium cycle causes disease (malaria)?

A
  • The blood stage
188
Q

Does natural immunity to P.Falcipium develop?

A
  • YES! But very slow!

- This is anti PARASITE immunity

189
Q

What does anti DISEASE immunity involve?

A
  • Ability to diminish parasite density and pro-inflammatory cytokines
  • Rapidly acquired
  • Reduced acute mortality
190
Q

What happens if someone was bitten before by infected mosquito and they have been bitten again?

A

There will be some immunity (so some old people in a malarial endemic country will have immunity)

191
Q

When does the main immune response occur?

A
  • When the merozoites leave the merozome capsule to be free in the blood (vulnerable)
192
Q

What occurs in the adaptive immune response?

A
  • RBCs (infected) are sticky due to PFEMP1

- Anti bodies are then mounted to the sexual stage

193
Q

What causes the stickiness of RBCs in malaria?

A
  • PFMP1 which is protein on infected surface
194
Q

How do B cells try and stop the action of PfEMP1

A

Recognise and neutralise PfEMP1 to prevent cytoadherence and sequestration

BMS1062 BIO (7 decks)

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