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Molecular and Cellular Biology > functional genomics 2 > Flashcards

functional genomics 2 Flashcards

1
Q

what is genomics?

A

The study of genes and their functions.

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2
Q

What is the genome

A

The genome is the entire DNA content that is
present within one cell of an organism

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3
Q

What does functional genomics use?

A

uses genomic data to study gene and protein expression and function on a global (genome-wide) scale

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4
Q

What is the focus of study in functional genomics?

A

uses genomic data to study gene and protein expression and function on a global (genome-wide) scale

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5
Q

What is transcriptomics?

A

the whole complement of transcripts in a tissue, organ, or organ system.

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6
Q

What is proteomics?

A

the whole complement of proteins in a tissue, organ, or organ system.

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7
Q

Function of large scale, high-throughout assays?

A

track many genes or proteins in parallel under different experimental or environmental conditions (e.g. withsamples from patients and healthy individuals)

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8
Q

Whats the genome wide approach?

A

“genome-wide” approach allows the function of different parts of the genome to be discovered by combining information from genes, transcripts and proteins

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9
Q

What techniques are used in genome wide approach?

A

Assays to measure changes insynaptic plasticity performed
alongside assays that quantifychanges in proteins involved in a range of pathways e.g.
MAP kinase signalling cascade

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10
Q

Functional genomics integrates information from various molecular methodologies to …

A

…gain an understanding of how DNA sequence is translated into complex information in a cell
(DNA → RNA → Proteins → biological process)

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11
Q

slide 6

A
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12
Q

Proteins can be separated according to…

A

… their molecular weight

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13
Q

electrophoresis is usually conducted in…

A

… a vertical electrophoresis system.

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14
Q

the matrix where proteins are separated according to their molecular weight is…

A

… polyacrylamide instead of agarose but the principle is the same.

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15
Q

in gel electrophoresis, the smaller the molecule, the…

A

…faster they will migrate.

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16
Q

DNA molecules are uniformly charged. What does this mean?

A

the sugar phosphate backbone is negatively charged regardless of DNA sequence.

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17
Q

DNA molecules are uniformly charged, so…

A

… migration is solely due to molecular weight.

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18
Q

Protein molecules consist of …

A

different amino acids that may have different charges

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19
Q

charge of DNA molecules?

A

negative because of phosphates.

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20
Q

Why are the charge differences between amino acids important?

A

The charge differences between amino acids are important in determining protein structure and function but the presence of different amino acids means that proteins do not have uniform charge

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21
Q

Proteins are denatured prior to…

A

electrophoresis

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22
Q

Proteins are denatured prior to electrophoresis by addition of …

A

…mercaptoethanol and SDS (sodium dodecyl sulphate)

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23
Q

Mercaptoethanol disrupts …

A

disulphide bridges between cysteine amino acid pairs by reducing the double bond - SDS binds all over the protein negating its charge

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24
Q

As SDS is negatively charged the presence of excess SDS molecules binding to protein results in …

A

…a uniform negative electric charge on protein molecules

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25
Q

MW = ?

A

= molecular weight marker

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26
Q

Protein gels can be…

A

…blotted

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27
Q

the principle of blotting in protein gels is the same as…

A

… DNA and RNA gels.

28
Q

Describe the process of protein gel blotting.

A

Proteins and buffer are forced to migrate through filter paper but proteins are unable to travel through a membrane (PVDF – polyvinylidine fluoride)

This produces a duplicate of the gel on a membrane

29
Q

How is blotting accomplished in protein gels?

A

The difference is that this is accomplished by electroblotting rather than capillary action

30
Q

In the case of Southern and Northern blots we use ….

A

DNA probes to detect the presence of homologous sequences of nucleic acids

31
Q

How do we detect proteins?

A

Using antibodies.

32
Q

proteins can be bound by…

A

… antibodies

33
Q

The vertebrate immune system relies on antibodies to bind to …

A

… antigens

34
Q

The vertebrate immune system relies on antibodies to bind to antigens – these complexes are then recognised by…

A

…components of the immune system where they activate an immune response.

35
Q

If we can bind protein with antibodies and label antibodies (as we label a DNA probe) then…

A

… we have a means of detecting specific proteins in a Western blot.

36
Q

We can make use of the vertebrate immune system to …

A

… generate antibodies

37
Q

The first step in creating an antibody that specifically recognises our protein is to …

A

… express the protein (creating the antigen)

38
Q

Simple cloning techniques allow us to …

A

… insert a gene we want to express into a plasmid

39
Q

If it is downstream of a strong promoter recognised by E. coli then if we transform E. coli with the plasmid…

A

… the protein will be produced.

40
Q

The protein we want antibodies generated against is…

A

… purified and injected into an animal (usually a rat or rabbit)

41
Q

The animal then …

A

… makes antibodies specific to that protein as it recognises the protein as foreign – after several weeks blood is harvested and serum produced.

42
Q

slide 14

A
43
Q

By producing antibodies specific to our protein we have solved one problem …

A

… –we have something that will bind to our protein

44
Q

By producing antibodies specific to our protein we have solved one problem – we have something that will bind to our protein – but we still have another which is…

A

how do we detect binding?

45
Q

The answer lies in using antibodies from …

A

another animal that are raised to the first antibody (if we inject IgG from a rabbit into a goat, the goat will generate antibodies to rabbit IgG)…

46
Q

The answer lies in using antibodies from another animal that are raised to the first antibody (if we inject IgG from a rabbit into a goat, the goat will generate antibodies to rabbit IgG)

If we then collect those antibodies and covalently link another protein on to it that will give us a…

A

colour reaction then we can detect the first antibody.

47
Q

As with a northern blot, spatial expression of the protein can be…

A

… studied.

48
Q

Protein is extracted from…

A

…various tissues.

49
Q

how is protein extracted?

A

this is quite a simple procedure just relying on gently lysing the cells and collecting the protein extract

50
Q

The protein extracts are separated by…

A

…SDS-PAGE and then blotted.

51
Q

As with a northern blot, spatial expression of the protein can be studied

Protein is extracted from various tissues (this is quite a simple procedure just relying on gently lysing the cells and collecting the protein extract)

The protein extracts are separated by SDS-PAGE and blotted

The western blot is then probed with …

A

… primary antibodies and finally with secondary antibodies with an enzymatic tag.

52
Q

slide 15

A
53
Q

In ELISA assays, the same principle as western blotting can be used to …

A

… make detecting protein quantitative

54
Q

Antigen is bound to a surface and then primary antibodies, secondary antibodies, and finally substrate are

A

… added to detect antigen.

55
Q

The same drawbacks for Northern blotting occur with Western blotting and ELISA –

A

it does not say in which cells the protein is expressed and is a ‘rough estimate’ of what is actually going on.

56
Q

In the same way that the presence of RNA can be detected at the cellular level by the use of RNA in situ hybridisation we can examine…

A

… the cellular distribution of proteins

Sections can be taken of an organism and these can be probed with antibodies

57
Q

Proteins are targeted to specific parts of the cell and it is important in our investigation of gene function to find the …

A

… subcellular as well as the cellular location of a protein.

58
Q

slide 17

A
59
Q

The subcellular localisation of proteins can also be

A

analysed using electron microscopy

60
Q

Rather than relying on enzymatic reactions to detect antibody in this procedure

A

small gold particles (10nm) are attached to the secondary antibody and the presence of antibody is seen because gold is ‘opaque’ to the electron microscope

61
Q

The big problem with analysing sections using immunohistochemistry is that …

A

… the cells are dead – remember that the cell is a dynamic environment, proteins don’t just stay in the same place necessarily

62
Q

The big problem with analysing sections using immunohistochemistry is that the cells are dead – remember that the cell is a dynamic environment, proteins don’t just stay in the same place necessarily

In order to analyse what our proteins might be doing in the cell…

A

…we can make use of GFP

63
Q

Protein-GFP fusions can be constructed as for …

A

… promoter-fusions – as long as you can construct this then you can transfect into cells.

64
Q

go look at slide 19

A
65
Q

By re-engineering the GFP protein it has been possible to…

A

… produce proteins that emit light at different wavelengths – red RFP, yellow YFP and blue CFP

66
Q
A

Molecular and Cellular Biology (15 decks)

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