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BIOL 202 > Moon immunogenetics > Flashcards

Moon immunogenetics Flashcards

1
Q

What is the definition of an Antigen? an Antibody? an Autoimmune disease?

A

Antigen → any molecules (usually a protein) that elicits an immune response

Antibody → (also called immunoglobulins) proteins than bind to antigens marking them for destruction by phagocytic cells, Ab are present in the blood and other body fluids

Autoimmune disease → an immune reaction against its own antigens (proteins)

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2
Q

What are the 2 aspects of the Adaptative Immune Response?

A

Adaptative Immune Response ~ antigen-specific immune response

Humoral immunity → production and secretion of Ab by B cells (specialized lymphocytes/white cells)

Cellular immunity → T cells produce T-cell receptors that recognize and bind Ag found only on the surface of the body’s own cells

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3
Q

How does clonal selection/expansion of B-cells occur?

A
  1. In a large pool of B lymphocytes, each is specific for 1 Antigen
  2. When an antigen binds to a B cell, the B cell divides and proliferates (primary immune response)
  3. Some of these cells differentiate into Ab-secreting plasma cells → Ab are specific for that antigen
    3.5 Among the clonse, some don’t differentiate into plasma cells but remain incirculation → memory B cells
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4
Q

What is the secondary immune response?

A

Occurs when exposed to the same antigen a second time:
- Memory B cells are already circulating in the blood → Antigen binds to memory B cells → gives rise to a rapid secondary immune response

*Similar process for T cells and T cell receptors (for primary and secondary immune response)

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5
Q

Why is vaccination so efficient?
What are the characteristics of a the active agent of a vaccine

A

Because of the second immune response

The active agent (antigen) of a vaccine:
- Intact but inactivated pathogen (non-infective)
- Attenuated (reduced infectivity) forms of the pathogen
- Purified components of the pathogen found to be highly immunogenic

Vaccine →primary immune response → memory cells

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6
Q

What is the structure of immunoglobulins?

A

2 Light chains:
- 2 types (kappa and lambda) encoded on different chromosomes
- Segment V, J and C
- kappa/kappa or lambda/lambda, not a mix in the same Ab

2 Heavy chains:
- 5 types (alpha, delta, gamma, epsilon or mu)
- Segments V, D, J and C
- Each type of light chain can potentially combine with each type of heavy chain

*Unique light and heavy chain combination can recognize a specific antigen

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7
Q

What are 3 ways for generating diversity in antibodies?
Why are the mechanisms important?

A

They are important because the human genome has 20,000-25,000 protein coding genes, but human have the capacity to produce 10^11 different specific Ab

  1. Somatic recombination
  2. Junctional diversity
  3. Somatic hypermutation
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8
Q

When/How does somatic recombination occur to generate antibody specificity?

A

During lymphocyte development → Somatic recombination between V and J gene segments of kappa light chains

RAG1, RAG2 and DNA repair enzymes introduce dsDNA breaks and joint random V and J segments
*At DNA level

Germ-line DNA has 30-35 different V gene segments + 5 J gene segments + 1 C gene segment

Ex of mature B cell DNA: V1-V2-J3-J4-J5-C

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9
Q

How is the specific Antibody made from the mature B cell DNA to the acutal protein ?

A
  1. Mature B cell DNA
  2. B cell DNA is transcribed to make pre-mRNA → spliced to produce unique combination of V-J-C light chains + polyA→mature mRNA
    Mature mRNA = 1x V, 1x J, 1x C

*Immunoglobulin generated in a given B cell is always the same

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10
Q

How is the heavy chain locus different from the light chain locus?
Which parts of the heavy chains contribute to the Ab specificity?

A

Heavy chain locus has multiple D (diversity) gene segments in addition to V, J and C segments

V, D and J of the heavy chains contribute to Ab specificity, but NOT the constant region

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11
Q

How many different Antibodies are produced by VDJ somatic recombination?

A
  • Variable segments → 30 Kappa light chains + 35 Lambda light chains + 100 heavy chains
  • Diversity segments → 23 heavy chains
  • Joining segment → 5 kappa light chains + 7 lambda light chain + 5 heavy chains
    *Constant region does not contribute to diversity

Possibility of Kappa + Heavy and Lambda + Heavy

Total 4.5x10^6 different combinations

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12
Q

What is Junction diversity?

A

It is a mechanism that generates antibody diversity
Few random nucleotides are lost or gained from imprecise junction of V and J segment after dsbreak
→ In many cases results in a frameshit that produces nonfunctional gene, but when x3 nt deleted → functional Ab

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13
Q

What is a somatic hypermutation?

A

Immunoglobulin genes are subject to a high mutation rate
ex: Deamination of Cytosine → Uracil
Uracil is replaced by the DNA repair mechaisms by another base → point mutation

*Changes the AA sequence that is recongized by the Ab

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14
Q

How does T-cell receptor acquire its diversity?
What is different from B cell Ab diversity?

A

T-cell receptors are composed of alpha and beta chains (TM proteins) that have variable regions

Alpha chain → 44-46 V gene segments + 50 J gene segments + 1 C segment

Beta chain → similar to alpha chain, but contains D gene segments

Somatic recombination and junction diversity, but NO hypermutation

Difference → NO hypermutations to generate diversity

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15
Q

What is an important effect of the Src oncogene?

A

It transforms normal cells to becomes insensitive to contact inhibition
Ex: normal 3T3 fibroblast

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16
Q

What are 4 common inherited cancer syndromes for which genetic testing is available?

A
  1. Hereditary breast cancer and ovarian cancer syndrome → genes: BRCA1, BRCA2
  2. Li-Fraumeni syndrome → gene: P53
  3. Familial adenomatous polyposis → gene: APC
  4. Retinoblastoma → gene: RB1
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17
Q

Which 3 categories of genes are implicated in cancer?

A

Oncogenes → positive regulators driving tumorigenesis

Tumor supressor genes → negative regulators that are inactive in cancer

DNA repair genes → prevent mutations maintining DNA integrity (to not accumulate mutations that can affect proto-oncogenes and tumor supressor genes)

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18
Q

Which was the first oncogene discovered?

A

v-Src was isolated from Rous sarcoma virus in 1970 → Peyton Rous (RNA tumor virus)

proto-oncogene (c-Src) → required for normal cellular function but when mutated, become oncogenes and promote cancer formation

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19
Q

How do viral oncogenes function?

A
  1. retrovirus inserts its RNA into the cell
  2. Undergoes reverse transcription and inserts randomly into the host chromosome next to a proto-oncogene
  3. When the virus reproduces, the proto-oncogene is incorporated into the virus genome
  4. In repeated round of viral infection and reproduction → proto-oncogene becomes rearranged or mutated or both → producing an oncogene that is inserted back into the host chromosome (PROBLEM)
    *Promotes cancer formation whe expressed in normal cells

Ex: in v-Src, the C-terminus of the protein responsible for negative regulation is absent

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20
Q

What is an alternative way a retrovirus can infect cells?

A

If the provirus inserts near a proto-oncogene → the strong viral promotor can stimulate over-expression of the proto-oncogene

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21
Q

What are non-viral transformation of proto-oncogenes → oncogenes?

A

In human, most proto-oncogenes are activated in the absence of viral infection:
- Mutation in coding sequence (ex: constitutive Ras activation)
- Chromosome abnormalities: increased expression, fusion protein (cancer specific form) ex: Bcr-Abl

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22
Q

How are Ras and receptor tyorsine kinases related?

A

They are part of the same signaling pathways:
1. RTK (TM) binds to a GF → conformational change
2. RTK autophosphorylates Y residues
3. Adaptor molecules recruit Ras to phosphorylated RTK → Ras binds GTP and becomes active
4. Ras-GTP (not kinase) binds/activates Raf (kinase)
5. Cascade of phosphorylation events
6. Activation of TF that promote cellular growth

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23
Q

What is the importance of Sos?

A

It is a GEF → interacts with Ras to stimulate GDP→GTP exchange

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24
Q

What is the effect of a single point mutation in Ras?

A

G12V is sufficient to drive tumorigenesis

GGC → GTC
Gly → Val
*WT → oncogene (prevent Ras inactivation/ activity of GAP)

25
Q

What is the main difference between mandelian traits and cancer?

A

Cancer phenotype only has to appear in 1 single cell vs mandelian diseases have to be in the germline to be expressed in all cells

Proto-oncogenes normally produce factors that stimulate cell division
Mutant alleles → oncogenes tend to be DOMINANT because 1 copy of the mutant allele is sufficient ot induce excessive cell proliferation

26
Q

What is observed/causes Burkitt lymphoma (B-cell)?

A

Chromosomal reciproqual translocation between Ch8 and Ch14
*t(8:14)

c-MYS on chromosome 8 (tightly regulated GF) → Ch14 just under the immunoglobin locus (new regulatory element)

Translocation: Reg IG (14) - MYC (8)

Coding sequence of Myc is not changed, but Myc is overexpressed by IG regulatory element → oncogene

27
Q

What change in the genome is observed in Chronic Myelogenous Leukemia (CML)?

A

Philadelphia chromosome → t(9:22) observed in 95% of patients → c-ABL-BCR → hyperactive kinase

Ch22 → normal bcr
Ch9 → normal c-abl (c-Abl protein kinase = proto-oncogene)
philadelphia ch → bcr-abl → NEW hyperactive kinase (chimeric protein)

*Since its a novel protein, can target it with Gleevec

28
Q

What are the 2 types of Retinoblastoma?

A

Unilateral → affects 1 eye
Bilateral → affects both eyes

*40% of cases involved inherited mutation

29
Q

How was the 2 hit hypothesis discovered?

A

In 1971, Knudson oberved a difference in the kinetics of Rb development between unilateral (dev. much slower) and bilateral cases (much earlier dev.)

Hypothesis: Rb must develop as a result of 2 independent factors (2 mutations, 1 in each allele) → consistent with having recessive mutations in both alleles of a single gene

In bilateral cases, the 1st hit was inherited, making the chances of getting a second hit in both eyes much stronger

30
Q

What are chromosomal abnormalities frequently found in Retinoblastoma?

A

Chromosomal deletion on Ch 13 (13q14)

31
Q

Which type/cause of cancer is the most inherited?

A

Mutations in tumor supressor genes → they can be inherited because TMG and happlosuficient most of the time
TSG are often inherited as a DOMINANT trait → the chances of acquiring the second mutatio in a somatic cell is so high
*But it is a recessive allele
Not all carriers develop cancer → penetrance

Oncogenes are not really inherited because they are highly active and act dominantly once they are induced

32
Q

What is the role of Rb in the cell cycle?

A
  1. Rb binds E2F and keeps it inactive
  2. Increasing concentrations of cyclin-D-CDK and cyclin-E-CDK phosphorylate Rb
    *cyclins-CDK are oncogenes!!
  3. Phosphorylated Rb is inactivated and released E2F
  4. E2F binds to DNA and stimulates transcription of genes required for DNA replication
33
Q

Which protein is considered to be the guardian of the genome?

A

p53:
1. Stops cell cycle
2. Senescence
3. Regulates apoptosis

  • Acts downstream of DNA damage → sensors of DNA damage phosphorylate/activate p53 → if not able to repair, p53 induces apoptotic factors
34
Q

What is mutation is inherited in Li-Fraumeni syndrome?

A

p53 mutation → unable to bind DNA but able to form homotetramer → dominant negative allele

*p53 is mutated in > 50% of all human tumors

35
Q

What are the different domains of p53?

A
  • Transcription activation domain (TAD)
  • DNA-binding domain (DBD)
  • Oligomerization domain (OD)
36
Q

What is the effect of HPV?

A

95% of cervical cancer patients are infected with HPV → virus promotes tumor development
*DNA tumor virus

37
Q

What are viral oncoproteins?

A

They form DNA tumor virus → when infect cells, their DNA encode for viral proteins that act to inhibit tumor supressor genes

Ex: HPV encode for E6 and E7 → bind to inactivate Rb (E7) and p53 (E6)

38
Q

What are the different steps of clonal evolution of cancer cells?

A
  1. 1st mutation → A cell is predisposed to proliferate at abnormally high rate (making more clones of it)
  2. 2nd mutation → Causes cell to divide rapidly → make even more clones of it
  3. 3rd mutation → structural changes
  4. 4th mutation → cell divides uncontrolably and invades other tissues

*Cycle of acquiring mutations, clonal expansion, more mutation, more expansion, etc. → fully transformed cancer cell

39
Q

Which genetic changes are associated with specific stage of colon cancer development?

A
  1. Loss of normal tumor-supressor gene APC
    *APC gene = familial adenomatous polyposis
    → Polyp forms on the colon wall (small growth/bump)
    → A benign, precancerous tumor grows
  2. Activation of oncogene Ras
    → An adenoma (benign tumor) grows
  3. Loss of tumor-supressor gene p53
    → A carcinoma (malignant tumor develops)
  4. Other changes; loss of antimetastasis gene
    → Cancer metastasizes through the bloodstream

*Not necessarily in the same order of mutations, but multi-steps

40
Q

What mechanism do tumor cells use to supress T cell immune response?

A

PD-1 activation (on T cells) by tumor cells → prevents hyperactivation of the immune response

Cancer cells increase expression of the PD-L1 (B7-H1) → interact with PD-1 on T-cells → reduce immune response

*Tumor regression when treat patient with ANTI- CTLA-4 and PD-1 (Ab) bc block checkpoint inhibition

41
Q

What are a few envionmental factors that play a role in cancer formation?

A
  • Tobacco
  • Diet
  • Obesity
  • Alcohol
  • Occupation
  • UV radiation
  • Infections
  • Radiation (ionizing)
    etc.
42
Q

Which 2 general methods are/were used to treat/study the genome?

A

Somatic cell fusion → gene mapping (before sequencing)

Gene therapy: (when we know the gene responsible for a specific disease)
- Conventional gene therapy
- Genome editing and iPS

43
Q

How whole cell hybridization/fusion done?

A
  1. Take human cell + Rodent cell + virus particle in vitro
  2. Virus particle binds the 2 cells
  3. Viral membrane fuses with the membranes of the 2 cells → cytoplasmic bridge
  4. Forms a single cell with 2 nuclei
  5. At 1st mitotic division, human and rodent chromosomes are attached to a single spindle apparatus → hybrid nucleus in the daughter cells

Genome of fused cells are unstable → randmly keeping or losing chromosomes
In hybrids, rodent chromosomes are predominantly kept and must human chromosomes are lost (some kept at random) → used for mapping

44
Q

What is HAT medium?

A

It is a highly selectable marker to select for hybrid cells as cell fusion is highly inefficient (~ 1% of cells actually fuse, others don’t)

  • Hypoxanthine → converted to guanine (dGTP) by HPRT (nt precursor)
  • Aminopterin → blocks de Novo pathway for DNA synthesis
  • Thymidine → phosphorylated by thymine kinase → dTTP)
45
Q

What are the 2 pathways for DNA synthesis?

A

de Novo pathway: major DNA precursor → newly synthesized DNA

Salvage pathway: minor DNA precursors (recycle byproducts from DNA/RNA degradation) → newly synthesied DNA
*Requires HPRT → dGTP and TK → dTTP

*Normal cells can survive without the minor pathway, not without the major pathway

46
Q

How are hybrid cells selected using HAT medium?

A

Major DNA precursor is blocked by aminopterin
Cells can only survive with the minor pathway if they are provided with Hypoxanthine and Thymidine (from HAT) and have TK and HPRT enzymes

TK deficient cells → no dTTP → dead
HPRT deficient cells → no dGTP → dead
Fusion of the 2 lines → both TK and HPRT → survive to the HAT medium → can make newly synthesized DNA

47
Q

How was thw measles virus receptor mapped in the human genome?

A

Measle virus doesn’t infect rodents, only human and primate cells

make many hybrids between primate and rodent cells → a few random human chromosomes are randomly kept in each hybrid → identify the hybrid cells that can be infected by measles → determine which human chromosome are kept/common in those cells

*Account for sections of the chromosomes that might be translocated to another chromosome

48
Q

What is gene therapy?

A

Gene therapy involves adding a normal (WT) copy of a gene to the genome of an individual carrying defective (mutated) copies of the gene → often for recessive mutations

Only few of the 4000 inherited human diseases are currently treatable (without gene therapy)

49
Q

What are 2 types of viral vectors used in gene therapy?

A
  1. Derived from adenovirus:
    - Will infect all cells even non-dividing ones
    - The vectors will not be integrated in the genome → transgene is diluted and eventually lost
    - Safer in a way
  2. Derived from retrovirus:
    - Many will infect only dividing cells, but others (HIV) can infect without host cell division
    - The transgene and the viral vector are incorporated into the genome of the infected cell → more stable
50
Q

What are 2 types of gene therapy?

A
  1. Somatic gene editing:
    - transfer of a gene in somatic cells → not transmitted to the next generation
    - limited to the somatic tissues
    - most are approved
  2. Germline gene editing:
    - Transfer of a gene to all cells of an organism through the germline transmission
    - Inherited to the next generation
    - Low approbation for ethical issues
51
Q

What is the difference between in vivo and ex vivo gene therapy? (both somatic)

A

In vivo: inject virus into the tissue you want to change the gene in → not much control in which cells are infected bc not

In vitro: manly for hematopoietic system
- Take hematopoietic target cells → transfer the gene in culture → infuse the cells back into the patient’s blood
- Can control efficiency of gene transfer

52
Q

What is ADA-SCID?

A

Adenosine Deaminase-Severe Combined Immunodefficiency Disease

Rare autosomal disease of the immune system (bubble boy disease) → affected individuals essentially have no immune system

In absence of ADA, deoxyadenosine accumulates in T lymphocytes and eventually kills them → no T cells in these patients

Most common treatment for SCID is bone marrow transplant

53
Q

What elements are required in the construction of a retroviral vector?

A
  • LTR are needed for the vector DNA integration in the cells
  • SV40 promotor → allow transgene expression in human cells
  • NEO^R needed for selection of human cells that will produce the virus in vitro (that have integrated the virus)
  • Improved safety by stopping promotor activity at the 3’LTR to not propagate unwanted strong promotor activity

*A lot of patients treated for SCID have developed leukemia due to the integration of the retrovial vector near a proto-oncogene

54
Q

What is Retinitis Pigmentosa?
How is gene therapy useful for its treatment?

A

RP makes cells in the retina break down slowly over time → progressive vision loss (ex: tunnel vision)

Multiple different mutations can lead to RP → autosomal dominant, autosomal recessive, X-linked mutations
- About 5% of RP is caused by autosomal recessive mutation of RPE65, retinoid isomerohydrolase

→ Can inject a WT copy of the RPE65 gene direclty in the eye

55
Q

What are different issues with conventional gene therapy?
What is a potential solution?

A
  1. Integration sites cannot be controlled
  2. Expression level of rescued gene may not be optimal (ex: SV40 might be too strong of a promotor)
  3. Ex vivo experiment is limited to certain types of cells

Potential solution → direclty edit genomic sequences of stem cells from the patient instead of adding an additional cop/extra genomic information

56
Q

What is a very efficient tool to target specific sequences in the genome for direct genomic editing?

A

CRISPR → clustered regularly interspaced palindromic repeats → system uses RNA molecules to target specific DNA sequences

SLIDE 16 L34

57
Q

Chromosomal changes are frequently associated with specific types of cancer. For example, reciprocal translocation between Ch 8 and Ch 14 is frequently observed in Burkitt lymphoma. The translocation affects the genomic loci where immunoglobulin gene (Ch8) and Myc gene (Ch 14) are located.
If this translocation occurs in T-cells, would it promote cancer development the same way as it did in B-cells?

A

Immunoglobulin is expressed in B-cells, not in T-cells. Therefore, the same chromosomal translocation in T-cell is unlikely to increase Myc expression since the immunoglobulin promoter is inactive in T-cells.

58
Q

When compared to Mendelian diseases such as sickle cell anemia, cancer is not a disease that
can be easily treated by conventional gene therapy. Why?

A

Every single cancer cells have to be targeted with 100% efficiency for cancer therapy. Missing
few cells likely result in relapse. For most Mendelian disease, improving the function of any
fraction of affected Ussue/organ is likely beneficial to the patient.

BIOL 202 (7 decks)

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