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Biology Year 2 > Molecular Genetics (Katherine Ford 25-30) > Flashcards

Molecular Genetics (Katherine Ford 25-30) Flashcards

1
Q

Give 5 reasons why we use model organisms?

A

1) Easy to grow and maintain in the lab.
2) easy to manipulate.
3) short generation times.
4) small, sequenced genomes.
5) can extrapolate from simple organisms to complex ones.

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2
Q

What are a typical gram-negative bacteria used as a model organism?

A

E.coli

  • 20 min generation time!
  • can be frozen/stored
  • used to study conjugation 1946
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3
Q

What are a typical gram-positive bacteria used as a model organism?

A

Bacillus subtillis

  • found in soil & digestive systems of ruminants.
  • spores are tough
  • easily secretes proteins.
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4
Q

Why is Saccharomyces cerevisiae a good model organism?

A
  • doubles every 90 mins
  • full genome available
  • several plasmids to work with, including integrating plasmids.
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5
Q

Why are modified plasmids good cloning vectors?

A
  • easy to transfer between cells
  • easy to miniprep/ isolate DNA
  • abundant in cell
  • easy to screen for recombinants

(Note: PCRII-TOPO)

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6
Q

What are the three types of artificial chromosomes that can be used as vectors?

A
  • BACs (circular)
  • YACs (Linear/ can take the largest fragments)
  • PACs

-used for genomic libraries

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7
Q

Name an advantage and a disadvantage of using YIPs as vectors.

A
  • Yeast integrating plasmid = integrates into the chromosome.
  • produces stable transformants
  • but, transformation rate is slow.
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8
Q

How do Lambda bacteriophages help with producing transformants?

A

Lambda replacement vectors are from a bacteriophage and can complete their lifecycle even with foreign DNA inserted into them.

  • The ds linear DNA has two cohesive ends (Cos sequences), once in the cell, these are cleaved off and the DNA circularises.
  • The middle of the vector is replaced with a multiple cloning site.
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9
Q

What are Cosmid vectors and how do the work?

A

They have highly modified Lambda vectors but everything between the cos sites is replaced instead.
- DNA up to 45Kb can be inserted

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10
Q

What is Gateway cloning?

A

Exploits Cosmid vectors and can clone up to 4 DNA fragments.
- ccdB region is replaced with the gene of interest.
PCR product is taken up by the vector and clonases swaps the gene of interest into lots of destination vectors.

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11
Q

What method is useful if we want to clone more than 4 fragments?

A

Using Shuttle vectors as they have more selection markers.

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12
Q

List the steps required for transforming vectors into yeast.

A
  • LiAOc makes yeast competent.
  • Add PEG, the plasmids and PCR product.
  • culture and heat shock
  • plate out on nutritional medium (auxotrophic selection) and recover plasmids via miniprep.
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13
Q

List the steps required for transforming vectors into E.coli.

A
  • permeablise the cell wall by calcium chloride and heatshock & ice or by electroshock.
  • Blue-white screening
  • miniprep.
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14
Q

How can you transform fungi?

A

Electroshock, PEG-mediation or via Agrobacterium.

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15
Q

Which fungus is used for citric acid production?

A

Aspergillus niger produces citric acid with the same chemical composition.

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16
Q

Which microbe is often used to produce amino acids?

A

Corynebacterium glutamicum

  • accumulates glutamate under limiting conditions.
  • also, mutant forms which could withstand high lysine conditions (limiting) have been used to produce Lysine, an essential amino acid which is added to animal feed.
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17
Q

Describe how vitamin C is produced by biotechnology.

A
  • initially, Gluconobacter Oxydans was used alongside chemical synthesis.
  • Using Ewrwinia & Corynebacterium together is found to be more useful but they require different growth conditions.
  • Can GM the Corynebacterial gene into Erwinia so that all is required is acid treatment.
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18
Q

What are three disadvantages of using E.coli in recombinant technology and what can be done to overcome them?

A

1) Codon bias (AAA codes for lysine 75% of the time)
- Overcome by site-directed mutagenesis/ synthesising the DNA/ adding the missing tRNAs/ integrating relevant genes into E.coli.

2) Poor Secretion
- can be overcome by fusing protein to a bacterial protein that is usually secreted
- adding additional secretory sequences.
- expressing the protein in a gram-positive bacterium instead.
- modification of E.coli to give it better extracellular secretion.

3) Post-translational modifications are different to eukaryotes. (70% of human genes are glycosylated and add glycosyl groups to N-terminal groups; whereas prokaryotes at them to side chains.
- overcome by adding glycosylation genes to E.coli.

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19
Q

What model plant species is often used when investigating crops?

A

Arabidopsis thaliana

  • has a similar genetic response to stress and diseases.
  • crops are complex
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20
Q

What are the three types of tissue culture used when producing transgenic plants?

A

1) Plant leaf discs
2) callus culture (immature embryos or meristems-totipotent)
3) suspension culture (immature protoplasts/ pollen.)

21
Q

What pathogen is exploited as a tool in plant transgenics?

A

Agrobacterium tumefaciens

  • gram-negative
  • attracted to plant wounds
  • induces the plant to produce excess auxin and cytokinin.
  • galls contain opines synthesised by a tumour inducing (Ti) plasmid.
22
Q

How does A.tumerfaciens act as a pathogen and how is this exploited by transgenics?

A
  • wounded plant cells produce signal molecules which are recognised by receptors.
  • Agrobacterium attaches to plant cell and activates VIR proteins which produce T-DNA.
  • T-DNA complex forms and transfers into cells, where it moves into the nucleus.
  • T-DNA is then expressed by bacterial proteins.

-we utilise this by inserting a selectable marker and gene of interest into the T-DNA.

23
Q

How does Agrobacterium tumefaciens regulate VIR proteins to produce T-DNA?

A
  • VirA detects wound and autophosphorylates.
  • VirA then phosphorylates VirG (a transcription factor) which initiates transcription of genes Vir B-E.
  • VirD1 and VirD2 are DNA nickases which cut and releases ssT-DNA.
  • VirD2 attaches to the 5’ end of the T-DNA and VirE2 coast it.
  • T-DNA exits the bacterium, into the plant and is moved to the nucleus.
24
Q

How can plant transgenics be used for herbicide tolerance?

A
  • make a crop resistant to glyphosate (roundup)
  • glyphosate inhibits synthesis of some amino acids by binding to EPSPS
  • by making mutant ESPS, glyphosate can’t bind to it.
25
Q

How can plant transgenics be used for insect tolerance?

A

Bt toxin:

  • cry genes from Bacillus thuringiensis produce a toxic crystal protein which binds to the epithelial cells of the digestive tract and creates holes.
  • CRY1 proteins are effective against Lepidopteran pests.
  • CRY3 proteins are effective against Coleoptera.
  • can choose Bt toxin specific to a pest.
  • expressed Bt in Maize and reduce problems caused by corn borers. (pesticide use was able to decline 11%)
26
Q

Why is Drosophila melanogaster a good model organism?

A
  • 2-week life cycle
  • easy to maintain the lab
  • well studied (only 4 chromosomes)
  • polytene chromosomes with characteristic banding patterns - good for mar marking.
27
Q

How can insects be used as vectors?

A
  • exploitation of baculoviruses
  • insect eats contaminated leaf and the intestinal epithelial cells release single virus particles which infect adjacent cells
  • Packages of virus particles are formed called polyhedrons.
  • Polyhedron promoters are v strong
  • a sequence can be placed in AcMNPV vector.

(could also use Bacmids from E.coli.)

28
Q

How may insect vectors be able to prevent the spread of human diseases?

A

Malaria is caused by Plasmodium sp.

- can engineer mosquitoes so they are less susceptible to Plasmodium by increasing levels of defensin A.

29
Q

Why are Nematodes good model organisms?

A
  • 2 sexes
  • the generation time of 3 days (4 larval stages before adult)
  • easy to maintain in the lab
  • transparency can be useful for reporter such as GFP.
30
Q

What is special about Flavr Savr tomatoes?

A

They were modified using antisense version of a gene.
- Agrobacterium tumefaciens was used to introduce an antisense copy of the polygalacturonase gene = delayed ripening and prolonged shelf life.

31
Q

Three ways in which RNAi can silence genes.

A

1) introducing antisense versions of genes
2) include a dual promoter for positive and antisense RNA
3) hairpins can produce dsRNA and form a loop, preventing translation.

32
Q

Why do extra RNAs silence genes?

A
  • may block translation by preventing ribosomal binding

- block post-transcriptional modifications by preventing binding of spliceosomes

33
Q

How can RNAi permanently repress gene expression?

A

RISC and associated siRNAs can bind complementary DNA.

  • siRNA directs heterochromatin- forming enzymes to the target loci.
  • when ‘open’ DNA is converted to heterochromatin, no more mRNA is produced.
34
Q

Advantages and disadvantages of using mammalian cells for recombination.

A
  • effective post-translation modifications
  • accurate folding
  • codon usage
  • accurate splicing
  • little regulation needed
  • difficult to culture cells
  • expensive and slow
  • limited large-scale production
  • low yield
  • susceptible to human viruses.
35
Q

What is the difference between cloning and transgenesis?

A

Cloning = generating progeny that are identical to the parent.

Transgenesis = introducing foreign DNA

36
Q

What are pluripotent cells?

A

Cells that can differentiate into all cells inside the embryo (not extraembryonic ones)

37
Q

What are the 3 methods to create transgenic mammals?

A

1) Microinjection of DNA into fertilised eggs and then implanted into a foster mother.
- need to cross heterozygous transformants to get 25 % homozygous progeny.

2) Transforming embryonic stem cells/ iPSCs (induced pluripotent cells from adults)
- insert vector with the gene of interest and the cell will express it.

3) Retroviruses
- ‘Infect’ the developing embryo with no need for microinjection
- viral DNA is also sometimes incorporated and sequences are limited to 8kb.

38
Q

4 Examples of transgenic animals.

A

1) Rat somatotrophin gene injected into mice was the first case of stable gene inheritance and normal function
- produced larger mice
- Note: mighty mouse/ smart mouse etc

2) Bovine growth hormone injected into cows for increased milk production.
3) Glofish on sale in the US
4) AquAdvantage Atlantic salmon has a gene from Pacific chinook and promotor from ocean pout = can grow all year round and reaches market size in half the time.

39
Q

What three methods may be used in gene therapy to deliver a gene to cells?

A
  • Direct injection
  • Aerosol inhalation
  • cell removal, treatment and implantation
40
Q

How do Adenoviruses cause infection?

A
  • penton fibre tip binds to CAR receptor found in many tissues.
  • penton base binds to integrins on host cell wall/ membrane
  • the membrane forms a vesicle and the virus is released into the cytoplasm
  • Virus disassembles and viral DNA enters the nucleus.
41
Q

Advantages and disadvantages of using Adenoviruses as vector.s

A

Advantages:

  • relatively harmless
  • easy to culture in large amounts
  • the life cycle is well understood
  • we know the function of its viral genes
  • the genome is sequenced.

Disadvantages:

  • limited by size (up to 5kb)
  • short-lived (2 weeks)
  • immunity can develop = can’t reinfect
42
Q

How did Adenoviruses help Cystic fibrosis sufferers, and why are they not used anymore?

A
  • In healthy cells, the CFTR protein forms an ion channel that opens and closes as a phosphate group is attached and detached.
  • In CF sufferers, this channel is the wrong shape = thickening of mucus coating the lungs. Leads to obstruction of airways, increased pathogen growth, scar tissue and respiratory failure.
  • Gene therapy used to clone a healthy CFTR gene into an adenovirus vector and inhaled through the nose.
  • Normal channel function was restored.
  • However, effect was only temporary as immunity developed and a severe immune response caused a death in 1999,
43
Q

How may Adenoviruses help with cancer treatments?`

A
  • Direct replacement of non-functional gene with a functional one.
  • Direct attack introduces a tumour necrosis factor.
  • Suicide genes may also be used.
  • Can provoke the immune system to attack also.
44
Q

How may Retroviruses infect a host?

A

(they’re made of +ve sense ssRNA)

  • enters the cell
  • RNA is copied into DNA by reverse transcriptase
  • DNA circularises and is integrated into the host genome
  • A strong promoter in the LTR region enables transcription of viral genes
  • New virus particles assemble and are packaged (the envelope contains glycoproteins acquired from the host cell membrane.
45
Q

How may Retroviruses be used to treat Murine Leukaemia Virus (MuLV)?

A
  • vectors have all viral genes removed except for the LTR promoter and the packaging signal.
  • The virus only contains the vector with the cloned gene insert and reverse transcriptase.
  • RNA is converted to DNA and the DNA is integrated into the host genome
  • No immune response and the effect is permanent.
46
Q

Describe the success of using Retroviruses to treat SCID (defective B&T cells)?

A
  • Add a functional ADA gene into a vector with a selectable marker.
  • Isolate bone marrow cells and culture them in vitro.
  • Infect the cells with the retrovirus and transfer them back to the body.
  • Produces functional lymphocytes
  • 100% of children survived and 75% needed no further treatment!
47
Q

What are the advantages of Adeno-associated viruses (AAVs) as vectors?

A
  • No immune response
  • No inflammation
  • Affects vertebrates (multiple cell lines)
  • Works in non-dividing cells.
  • Integrates into a specific site.

-But can only hold sequences up to 5kb.
(require another viral vector for replication and capsid production.

48
Q

What are two nonviral methods of gene therapy and how do they work?

A

Liposomes:

  • add DNA into a hollow liposome, which will fuse to plasma membranes deliver DNA by lipofection.
  • non-specific delivery but can inject into target cells.

RNA:
- can use antisense RNAi (delivered by liposomes also) to prevent transcription.
(antisense IGF1 stopped tumour growth)
(Can inject siRNA directly into the eye to target age-related macular degeneration.

Biology Year 2 (10 decks)

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