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BIOC192 > Molecular Genetics and Biotechnology > Flashcards

Molecular Genetics and Biotechnology Flashcards

1
Q

Define gene.

A

A sequence of DNA that encodes a gene product (RNA or protein).

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2
Q

Describe the central dogma of molecular biology.

A 
DNA- information
RNA- messenger
Protein- worker
Transcription from DNA to RNA
Translation from RNA to protein
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3
Q

Name five ways of regulating the pathway of DNA to protein.

A 
Transcriptional control
RNA processing and stability
Translational control
Protein processing
Protein activity and stability
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4
Q

Define transcriptional control.

A

Determining when and in what cells a gene is transcribed to produce mRNA. (first step in determining how many individual proteins are produced in a cell)

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5
Q

How many protein coding genes are unique to a specific cell type?

A

1000-2000

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6
Q

How many genes are expressed in any cell type?

A

11,000-17,000 (half to two thirds of total genes)

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7
Q

Define transcription factors.

A

Proteins that bind to a specific DNA sequence and control the rate of transcription.

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8
Q

How do transcription factors bind to the right sequence of DNA?

A

Their structure allows them to make contact with the DNA bases, and bind to the correct ones.

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9
Q

Define promoter sequence.

A

The DNA sequence of a gene at which transcription factors bind.

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10
Q

What do transcription factors do once they have bound to the promoter sequence?

A

Recruit RNA polymerase to form a stable transcription initiation complex, which can start transcription.

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11
Q

Name the roles of the two types of transcription factors.

A

Activators- turn gene on
Repressors- turn gene off

The gene is only expressed when all activator and no repressor transcription factors are present.

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12
Q

How are transcription factors regulated?

A

By external signals e.g. hormones starting a signal transduction pathway.

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13
Q

Explain Mendel’s law of segregation.

A

Alleles are separated during meiosis, so each gamete carries only one allele for each gene.

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14
Q

Explain Mendel’s law of independent assortment.

A

The segregation of alleles for a gene occurs independently to that of any other gene.

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15
Q

Explain Mendel’s law of dominance.

A

An organism with at least one dominant allele with display the effect of the dominant allele.

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16
Q

What is the exception to the law of independent assortment?

A

Genetic linkage- when two genes are close together on a chromosome, the assortment of one gene will most likely affect the assortment of the other.

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17
Q

What causes black urine?

A

Accumulation and oxidation of homogentisate, a compound of the phenylalanine metabolism pathway. Caused by a gene defect leading to an absence of enzyme.

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18
Q

Describe the role of phenylalanine hydroxylase.

A

Breaks phenylalanine down to tyrosine to start phenylalanine metabolism.

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19
Q

Describe phenylketonuria PKU.

A

Defect in the phenylalanine hydroxylase enzyme, which leads to accumulation of phenylalanine. Can lead to intellectual disability, seizures, behavioural problems and mental disorders.

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20
Q

An accumulation of phenylalanine causes the increase and decrease of which chemicals?

A

Buildup of precursors: phenylpyruvate, phenylethylamine

Decrease in tyrosine products: dopamine, adrenaline

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21
Q

How is the effect of environment used to decrease the effects of phenylketonuria PKU?

A

A low phenylalanine diet counteracts the phenylalanine buildup.

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22
Q

How can genetics decrease the effects of phenylketonuria PKU?

A

Genes leading to a less efficient Phe transporter across the blood-brain barrier.

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23
Q

What is the mutation causing PKU?

A

R408W (codon for arginine is mutated into a codon for tryptophan- A to a T) in the gene coding phenylalanine hydroxylase.

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24
Q

How does the R408W mutation in the phenylalanine hydroxylase gene result in an absence of the enzyme?

A

Due to its position in the gene sequence, the mutation results in incorrect protein folding, which forms an aggregate that enzymes in the cell degrade.

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25
Q

What does glucokinase do?

A

As an enzyme in the glucose sensing pathway, it determines how much glucose is broken down, and therefore how much insulin is produced.

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26
Q

What is the result of one mutated gene (heterozygous) and two mutated genes (homozygous) for glucokinase?

A

Heterozygous- persistent mild hyperglycaemia/ MODY 2
- because the amount of enzyme produced determines how much glucose is sensed, and how much insulin is produced

Homozygous- severe diabetes and very high blood glucose levels

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27
Q

What is the mutation that causes MODY2?

A

Glutamate to a lysine in the gene for glucokinase.

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28
Q

Name the four steps of genetic testing.

A

Obtain cells (cheek)
Isolate DNA
PCR amplify specific gene sequence
Detect genetic difference of interest

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29
Q

Name the three steps of PCR.

A

Heat DNA to 95 degrees to break H-bonds between strands.
Cool to ~60 degrees to anneal/ base pair a DNA primer.
Heat DNA to 72 degrees to allow Taq DNA polymerase to copy the DNA.

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30
Q

How is the MODY2 gene detected in PCR?

A

HindIII (restriction enzyme) cuts the specific sequence belonging to MODY2 into two fragments.

When put through agarose gel, the wild type will stay in one band, while the MODY2 fragments will separate.

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31
Q

Define germline mutation.

A

A mutation that occurs in a cell which undergoes meiosis, and is passed on to the next generation.

32
Q

Define somatic mutation.

A

A mutation that occurs in non-meiosis cells and cannot be passed on. Can result in cancer.

33
Q

Describe the basis of cancer.

A

Accumulation of mutations which leads to uncontrolled growth of cells that no longer respond to many of the signals that control cellular growth, and they evade programmed cell death.

34
Q

Why does the risk of cancer increase with age?

A

The more cell divisions undergone in your body, the more likely it is to gain a mutation which could cause cancer.

35
Q

Name two ways a somatic mutation can occur.

A

By chance/ spontaneous, or from exposure to a mutagen/ carcinogen.

36
Q

Why is skin cancer more common than bone cancer?

A

Because skin cells divide much more frequently than bone cells, so there is more chance for mutation.

37
Q

What contributes to a third of cancer risk?

A

Environment and genetics.

38
Q

Name and describe the two types of genes that can cause cancer when mutated.

A

Tumor suppressor genes

Oncogenes

39
Q

Describe a tumor suppressor gene.

A

Encodes proteins that normally prevent uncontrolled cell growth. Recessive mutations promote cancer.

40
Q

Describe an oncogene.

A

Encodes proteins that promote cell growth. Dominant mutations promote cancer.

41
Q

Describe the tumor suppressor gene mutation leading to chronic myeloid leukaemia.

A

Loss of function in the RB gene that binds E2F transcription factor in the cytoplasm.

Needs to occur in both copies of the gene (recessive) to lead to cancer.

42
Q

What are two ways oncogene mutations can increase gene activity? Give an example for each.

A

Increasing transcription- translocation of a very active promoter region with a coding sequence for cell division cause the cell to divide more frequently.
Increasing protein activity- gene duplications by increased copy number produces more proteins.

43
Q

What is an example of oncogene and tumor suppressor mutations leading to cancer?

A

Chronic myeloid leukaemia. Uncontrolled growth and accumulation of leukemic blast cells (undifferentiated) and lack of differentiated ones.

44
Q

Describe the the oncogene mutation leading to chronic myeloid leukaemia.

A

Translocation forming a Philadelphia chromosome- BCR promoter is fused to the coding sequence of the ABL gene, forming BCR-ABL gene.

BCR-ABL gene is always active and phosphorylating the transcription factor for cell division without a signal.

45
Q

How can someone’s genetics increase their chance of chronic myeloid leukaemia?

A

They inherit a germline mutation of a tumour suppressor gene- increases their chance but they need two copies to develop cancer.

46
Q

Name and describe four cancer treatments.

A

Surgery- remove cancer cells
Radiation therapy- kill cancer cells with targeted radiation
Chemotherapy- use drugs to target dividing cells
Targeted therapy- use drugs that target changes in cancer cells specifically that allow them to divide

47
Q

How does Gleevac target chronic myeloid leukaemia cancer cells?

A

Binds to the active site of tyrosine kinase that binds the BCR-ABL protein, inhibiting it so it can’t phosphorylate the transcription factor anymore.

48
Q

Define recombinant DNA technologies.

A

Process of joining segments of DNA together and inserting them into an organism to produce a useful protein.

49
Q

What is a reporter gene?

A

Gene (e.g. green fluorescent protein GFP gene) that investigates the regulation of a gene of interest (its promoter).

50
Q

Define plasmids.

A

Circular pieces of non-chromosomal ds DNA. Common in bacteria but also found in eukaryotes.

51
Q

Name the key components of a recombinant DNA plasmid.

A

ORI origin of replication- initiates replication using DNA polymerase of host cell

Antibiotic resistance gene- allows selection of cells containing plasmid- the rest die

Promoter- drives expression of the reporter gene using transcription factors from host cell

52
Q

How are some promoters only active in some cell types?

A

They can vary to only bind prokaryotic or eukaryotic transcription factors, or only those present in a specific cell type (e.g. neurons).

53
Q

Name two biological tools used to insert DNA into plasmids.

A

Restriction enzymes

DNA ligase

54
Q

Describe how DNA is inserted into plasmids.

A

Restriction enzymes cut ds DNA of plasmid at a specific sequenced, and cut the DNA insert at the same specific sequence. This allows them to complementary base pair. DNA ligase catalyses the formation of phosphodiester bonds to join the backbone together.

55
Q

Describe transformation (plasmids).

A

Transfer of plasmids into bacteria, then bacteria onto agar antibiotic plate. The antibiotic resistance gene ensures only transformed bacterial cells are selected. Plasmid gene is expressed.

56
Q

What is the significance of the universal genetic code?

A

We can transform a human gene into bacteria and it will still make the same protein.

57
Q

What must we consider when transforming eukaryotic genes into prokaryotes?

A

Prokaryotic genes have no introns, and prokaryotes don’t have the machinery to process eukaryotic introns. So we must use the coding sequence only.

58
Q

Name the five key steps in producing a recombinant protein using prokaryotic cells.

A

Isolate gene of interest (and amplify using PCR)
Clone into expression plasmid
Transform into bacteria for expression
Grow cells expressing protein of interest
Isolate and purify the protein

59
Q

How do we isolate the insulin gene?

A

Take mRNA from beta-cells in the pancreas and use reverse transcriptase to make cDNA (contains only the coding sequence CDS).

60
Q

Describe the cloning of the insulin gene in bacteria. Why do we need to express chains A and B separately?

A

Cloned into an expression plasmid with a bacterial promoter region, and supply restriction enzyme EchoR1 and DNA ligase.

Express chains A and B separately. This needs to happen because bacteria don’t have the Golgi that would usually complete processing of the protein in mammalian cells.

61
Q

Which bacteria is insulin transformed into and how?

A

E.coli
They are heat-shocked so they can take up the expression plasmid. Select the E.coli that are producing the plasmid by adding an antibiotic.

62
Q

What occurs after insulin is extracted from expressing cells and purified?

A

Extract A and B subunits.
Treat with cyanogen bromide to cleave the A and B chains.
Incubate under conditions that allow the disulfide bridges to form between them.

63
Q

What are the advantages and disadvantages of using prokaryotes to make recombinant proteins?

A

Cheap, high yield, pathogen free (can treat with antibiotic)

Proteins often not properly folded, can’t perform post-translational modifications

64
Q

What are the advantages and disadvantages of using mammalian cells to make recombinant insulin?

A

Protein can be produced as a preproprotein (protein predecessor) and processed efficiently (through the Golgi)

Using a signal peptide, the insulin will be secreted from the cells- allows for easier purification

More expensive to produce

65
Q

Name the five steps in producing recombinant insulin in eukaryotic cells.

A

Isolate cDNA for insulin
Clone into eukaryotic expression plasmid
Transform bacteria to produce more plasmid DNA
Transfect eukaryotic cells with the plasmid DNA
Extract and purify recombinant insulin

66
Q

Why does recombinant erythropoietin production require mammalian cells?

A

EPO is only active once glycosylated, and bacteria cannot perform post-translational modifications.

67
Q

What is EPO function? Which diseases is it used for?

A

Signals stem cells to produce more RBCs.

Anaemia- caused by chronic renal failure or chemotherapy

68
Q

What is glycosylation?

A

Type of post-translational modification that involves addition of carbohydrates to asparagine, serine or threonine residues of a protein. Important for biological function.

69
Q

Which cells are used for recombinant EPO production?

A

Chinese hamster ovary cells

70
Q

Name the components of the expression plasmid used for recombinant EPO production.

A

Human EPO gene (doesn’t have to be cDNA)
Promoter for transcription in mammalian cells
Antibiotic resistance gene (for transforming to bacteria and amplifying)

71
Q

Why is recombinant anti-thrombin AT produced in goats?

A

Because it requires gamma-carboxylation of glutamate, one of the post-translational modifications the cells in culture cannot perform.

72
Q

How and why is the AT gene expressed in goats?

A

In the lactating cells, by using a milk-specific promoter, ensuring only these cells have the specific transcription factors required for expression.

This is so the goats aren’t harmed by AT in their tissues, and it’s easily purified from the milk.

73
Q

What influences the choice in which host to use for protein production?

A

Financial reasons: speed and cost

Biochemical reasons: glycosylation and folding

74
Q

Describe optogenetics in the brain.

A

Use a plasmid expressing the transgene for channelrhodopsin (ion channel sensitive to light), and a promoter specific to a type of neuron.

Channelrhodopsin activates neurons by opening to let ions into the cell and starting an action potential.

75
Q

What is degenerate PCR?

A

Amplifying DNA using imperfect enzymes that create all sorts of mutations in the DNA copies. The idea is that some mutations will translate to proteins with an improved function.

76
Q

How can we provide a one-off, permanent treatment for disease (instead of regular drugs)?

A

Gene therapy: use patients as the host for protein production. Inject patient with viral vector (specific to the cell we want) that contains a cell-specific plasmid expressing the functional copy of the gene.

77
Q

Describe gene therapy to treat type I diabetes.

A

Turn liver cells into insulin-making cells (since pancreatic beta-cells are destroyed). Inject plasmid-containing viral vector into liver.

  • use a liver specific, ‘glucose responsive’ promoter (only expressed when glucose is high)
  • transgene = pre-pro insulin cDNA

BIOC192 (3 decks)

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