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BIOC practice Q's > Molecular genetics and biotechnology > Flashcards

Molecular genetics and biotechnology Flashcards

1
Q

Every cell in your body contains the same genome. With this in mind, how is a brain cell different to a liver cell?

A

only about 1/2 to 2/3 of the genes in the genome get transcribed in each cell.

Approximately 10,000 of
the same genes are expressed.

These genes are needed for basic cellular functions.

1000 to 2000 expressed genes are unique to a specific cell type,

The different cell types express different transcription factors to ensure regulation of gene expression,

where the liver (for example) will have the appropriate transcription factors to transcribe glycogen synthase, but the brain will lack this transcription factor and therefore not make glycogen synthase.

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2
Q

What would be the consequence of a failure to control gene expression?

A

Every cell would express all 21,000 protein coding genes.

No difference between different cell types.

This would not be compatible to life as a human. It would require far too much energy to be a viable process.

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3
Q

Briefly outline the key regulatory elements of a gene.

A

The regulatory elements of a gene are found upstream of the coding region.

  • contains important binding sites for key regulatory proteins.
  • contains a promotor sequence; this is where the polymerase will bind.
- contains binding sites for
transcription factors (which act to activate gene expression)
  • sites for repressor proteins to bind (which act to repress gene expression).
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4
Q

Promoter sequence in regulatory elements of a gene

A

where the polymerase will bind.

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5
Q

transcription factors in regulatory elements of a gene

A

act to activate gene expression

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6
Q

repressor protein in regulatory elements of a gene

A

act to repress gene expression

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7
Q

what is an activator?

A

a protein that increases gene transcription of a gene or set of genes.

Most activators are DNA-binding proteins

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8
Q

what is a repressor?

A

a DNA- or RNA-binding protein that inhibits the expression of one or more
genes by binding to the operator sequence or associated silencers.

A DNA-binding repressor blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes into messenger RNA.

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9
Q

what is a transcription factor?

A

a protein that controls the rate
of transcription of genetic info from DNA to messenger RNA, by binding to a specific DNA sequence.

The function of TFs is to regulate—turn on and off—genes in order to make sure that they are expressed in the right cell at the right time and in the right amount throughout the life of the cell and the organism.

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10
Q

What is an initiation complex and what is the importance of this complex?

A

a group of proteins that bind to the DNA regulatory sequences, forming a complex which is able to recruit RNA polymerase. This complex is only able to form if nothing is bound at the repressor site. This complex is important as without it,
RNA polymerase would not be able to bind to the promoter sequence and therefore, transcription would not be initiated.

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11
Q

What is a signal transduction pathway?

A

transmission of molecular signals from a cell’s exterior to its interior.

Signals received by cells must be transmitted effectively into the cell to ensure an appropriate response. This step is initiated by cell-surface receptors.

The physical signal is
transmitted through a cell as a series of molecular events, most commonly protein
phosphorylation catalysed by protein kinases, which ultimately results in a cellular response.

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12
Q

How does Leptin function to turn off insulin production and why is this important?

A

Leptin binds to leptin receptors on the surface of pancreatic b cells. This binding initiates a
cell signalling cascade, ultimately leading to the dephosphorylation of a repressor protein.

Once dephosphorylated, this repressor protein is able to bind to regulatory elements on the insulin gene, thus repressing transcription.

This is important to ensure the insulin is not produced in the fasting state, when it is not required. This ensures that adequate blood glucose concentration is maintained between meals.

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13
Q

What are Mendel’s Laws of Inheritance?

A

segregation
independent assortment
dominance

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14
Q

law of segregation

A

when gametes form. alleles separate so that each gamete carries only 1 allele for each gene.

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15
Q

law of independent assortment

A

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

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16
Q

law of dominance

A

some alleles are dominant while others are recessive; an organism with at least 1 dominant allele will display the effect of the dominant allele

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17
Q

What are alleles?

A

alternative form of a gene (one member of a pair) that is located at the same place on a chromosome.

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18
Q

how are alleles inherited? And how are they important in making each individual unique?

A

Each gamete gets one copy of the chromosome, each with a unique combination of alleles.

Different alleles can result in different observable phenotypic traits, such as different pigmentation.

A notable example of this trait of colour variation is Gregor
Mendel’s discovery that the white and purple flower colours in pea plants were the result of “pure line” traits which could be used as a control for future experiments.

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19
Q

What is the exception to Mendel’s law of independent assortment? What is the
consequence of this?

A

When 2 genes are close together on a chromosome (genetically linked)

as the alleles will often be inherited together.

Genetic linkage is a key principle in understanding the inheritance of traits. It explains why some characteristics always seem to occur together.

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20
Q

what does homozygous mean?

A

both copies of a gene or locus match.

Two dominant alleles (AA) or two recessive alleles (aa) are
homozygous.

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21
Q

what does heterozygous mean?

A

that the copies do not match.

One dominant allele and one recessive allele (Aa) is heterozygous.

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22
Q

What is the importance of the phenylalanine breakdown pathway?

A

Phenylalanine (dietary protein) metabolic pathway yields tyrosine (Produce melanin pigments)

Defects of enzymes responsible for interconversion of metabolites in the pathway are the cause of single-gene Inborn Errors of Metabolism: Phenylketonuria (PKU), Albinism (Melanin deficiency), and Alkaptonuria (excess HA).

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23
Q

what is phenylalanine?

A

primary amino acid that is abundant in dietary protein.

yield tyrosine

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24
Q

what is tyrosine?

A

production of Melanin pigments.

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25
Q

what is PKU?

A

caused by a defect in the gene that helps create the enzyme needed to break down phenylalanine.

Without the enzyme necessary to process phenylalanine, a dangerous build- up can develop when a person with PKU eats foods that contain protein or eats aspartame, an artificial sweetener.

This can eventually lead to serious health problems.

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26
Q

For the rest of their lives, people with PKU — babies, children and adults — need to follow a diet that

A

limits phenylalanine, which is found mostly in foods that contain protein.

Babies are screened for PKU soon after birth. Recognizing PKU right away can help prevent major health problems.

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27
Q

What factors can alter an individual’s phenotype?

A

Genotype

Environment

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28
Q

What factors can alter an individual’s phenotype?

For example, if someone has the genotype for PKU, they can avoid the phenotype through

A

dietary modification. So, although they still do not have the enzyme allowing for phenylalanine to be broken down to tyrosine, due to diet modification, the disease phenotype can be avoided.

The phenotype depends upon the genotype but can also be influenced by environmental
factors.

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29
Q

What is translation?

A

process in which ribosomes in the cytoplasm or ER synthesize proteins after the process of transcription of DNA to RNA in the cell’s nucleus.

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30
Q

why is translation important?

A

way in which a messenger RNA is decoded into proteins.

Without translation, gene expression would not occur and no proteins would be made.

This would not be conducive to life.

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31
Q

What are the three phases of translation?

A

Initiation
Elingation
Termination

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32
Q

what is Initiation in Translation?

A

The ribosome assembles around the target mRNA.
The first tRNA is attached
at the start codon.

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33
Q

what is Elongation in Translation?

A

The tRNA transfers an amino acid to the tRNA corresponding to the next
codon.
The ribosome then moves (translocates) to the next mRNA codon to continue the
process, creating an amino acid chain.

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34
Q

what is Termination in Translation?

A

When a peptidyl tRNA encounters a stop codon, then the ribosome folds
the polypeptide into its final structure.

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35
Q

Translate this section of mRNA into its sequence of amino acids:
GCCCAATACCTTGGCCCTTCTAGTT

A

Ala-Gln-Tyr-Leu-Gly-Pro-Ser-Ser

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36
Q

What types of mutation may occur in DNA, leading to an alteration in the amino
acid sequence?

A

Point mutation - Change of 1 letter

Frameshift mutation - loss of 1 letter

Frameshift mutation - gain of 1 letter

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37
Q

What is MODY?

A

Maturity onset diabetes of the young and refers to any of

several hereditary forms of diabetes mellitus

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38
Q

what types if mutation may occur to cause MODY?

A

mutations in an autosomal dominant gene disrupting insulin production.

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39
Q

MODY is often referred to as

A

monogenic diabetes to
distinguish it from the more common types of diabetes (type 1 and type 2), which involve more complex combinations of causes involving multiple genes and environmental factors.
MODY 2 and MODY 3 are the most common forms.

40
Q

Some examples of genes that be mutated to cause MODY:

MODY1 -
MODY2 -
MODY4 -

A

mutation in the gene hepatocyte nuclear factor 4a

  • mutation in the gene glucokinase
  • mutation in the gene for inulin promotor factor 1
41
Q

How can PCR be used in genetic testing, and how are the results analysed?
Testing for MODY2

A

Glucokinase gene will be amplified from the patient. The DNA can then be run on agarose DNA gels, in which an electric current is passed through the gel.
As DNA is negatively charged, it will run towards the positive electrode. The distance through the gel that the DNA runs is a function of its size.

Small bits of DNA will run further through the gel than large pieces of DNA.

42
Q

PCR can be used to

A

amplify a specific DNA sequence, based on the design of highly specific
primers (for example, primers that bind to the start of the glucokinase gene). This allows for a specific amplification of a short DNA sequence in questions.

43
Q

How can PCR be used in genetic testing, and how are the results analysed?
testing for huntingtons disease

A

If a genetic disorder is caused by a very large insertion into the gene (for example in
Huntington’s disease), then

when the DNA of a patient with the disease is run on the gel, it will not migrate as far through the gel as someone without disease.

44
Q

PCR and hows its used in the case of MODY2?

A

the specific mutation in the gene introduces a restriction enzyme site that is recognised by the restriction enzyme HINDIII.
So, if the glucokinase gene of a patient with MODY2 is incubated with HINDIII, then it will be cut into two pieces.

So, when run on an agarose gel alongside the DNA of someone without the mutation, there will now be two distinct bands instead of one, thereby confirming the diagnosis of MODY2.

45
Q

what is Germline mutation?

A

If a mutation occurs in a cell that go on to make gametes (egg or sperm cells), the mutation can be passed on to the next generation (e.g. in born errors of metabolism).

46
Q

what is Somatic mutation?

A

occur in other cells and cannot be passed on – can result in caner.

47
Q

What is cancer?

A
  • collection of related diseases.
  • some body’s cells begin to divide without stopping and spread into surrounding tissues.
  • start almost anywhere in the human body, which is made up of trillions of cells.
  • When cancer develops, cells become more abnormal, old or damaged
  • cells survive when they should die, and new cells form when they are not needed.
  • extra cells can divide without stopping and may form growths called tumours.
48
Q

Generally, is cancer a result of a single mutation or an accumulation of many
mutations?

A

An accumulation of many mutations.

49
Q

why is cancer a result of An accumulation of many mutations?

A

In order for cancer to develop, must have accumulated a mutation in an
- oncogene (just one copy needs to be mutated, i.e., oncogene mutations are dominant as they lead to a gain of function)

  • and mutations in both copies of a tumour suppressor gene (both copies of the gene need to be mutated i.e., tumour suppressor gene mutations are recessive as they lead to loss of function).

These mutations must be in
the same cell. This is to say that if you have a cancer contributing mutation to an oncogene in the liver, and a cancer contributing mutation in a tumour suppressor gene in the heart, you will not get cancer. The mutations both need to be in the liver, for example.

50
Q

oncogenes mutations

A

dominant

gain of function

51
Q

tumour suppressor gene mutations

A

recessive

loss of function

52
Q

What is a carcinogen?

A

any substance, radionuclide, or radiation that promotes carcinogenesis, the
formation of cancer.

This may be due to the ability to damage the genome or to the disruption of cellular metabolic processes.

53
Q

What does it mean if someone has a genetic predisposition (genetic susceptibility) to cancer?

A

is an increased likelihood of developing a particular disease based on a person’s genetic makeup.

results from specific genetic variations that are often inherited from a parent.
These genetic changes contribute to the development of a disease but do not directly cause it.

Some people with a predisposing genetic variation will never get the disease while others will, even within the same family.

54
Q

Some people with a predisposing genetic variation will never get the disease while others will, even within the same family.

Example

A

certain mutations in the BRCA1 or BRCA2 genes greatly increase a person’s risk of developing breast cancer and ovarian cancer.

So, in this instance, these people have inherited the mutation in the oncogene, so now only require a recessive mutation to the tumour suppressor gene. Thus greatly increasing their likelihood of developing cancer.

55
Q

What are tumour suppressor genes?

A

are genes whose protein products are involved in the inhibition of cell proliferation.

56
Q

What are tumour suppressor genes normal role example?

A

ensure that transcription

factors involved in the transcription of proliferation genes are held inactive in the cytoplasm of the cell.

57
Q

what is the

consequence of mutation to tumour suppressor genes?

A

cause a loss or reduction in its function, the cell can

progress to cancer, usually in combination with other genetic changes.

58
Q

What are oncogenes?

A

often code for proteins that stimulate cell division.

59
Q

what are oncogenes normal role?

A

Essential for normal growth, development and the maintenance of healthy organs and tissues.

function in a very tightly regulated fashion, only active in their ability to upregulate cell division when the cell receives the proper signals.

60
Q

what is the consequence of

mutation to oncogenes?

A

leads to a gain of function,

oncogene now upregulates cell division much more than it should and that it’s activity in doing so is now unregulated.

contributes to the development of cancer, when such mutations are found in combination with mutations to tumour suppressor genes.

61
Q

Chronic myeloid leukaemia (CML)

A

cancer of blood cells, characterized by replacement
of the bone marrow with malignant, leukemic cells. Many of these leukemic cells can be found circulating in the blood and can cause enlargement of the spleen, liver, and other organs.

62
Q

The most common oncogene mutation

A

Philadelphia chromosome. The Ph chromosome is the result of a translocation—or exchange of genetic material—between the long arms of chromosomes 9 and 22 . This exchange brings together two genes:

the BCR (breakpoint cluster region) gene on chromosome 22 and the proto-oncogene ABL (Ableson leukaemia virus) on chromosome 9. The resulting hybrid gene BCR-ABL codes for a fusion protein with tyrosine kinase activity, which activates signal transduction pathways, leading to uncontrolled cell growth.

63
Q

BCR-ABL codes for a

A

fusion protein with tyrosine kinase activity, which activates signal transduction pathways, leading to uncontrolled cell growth.

64
Q

There are many different tumour suppressor gene mutations that may contribute to CML, including mutations to

A

p53, RB, or p16.

65
Q

the RB protein functions

A

constrain a transcription factor for cell division genes in the cytosol of the cell.

If the RB gene mutates that reduce or abolish its ability to do this, then the transcription
factor has unregulated access to the nucleus, and an unregulated ability to
transcribe the cell division genes.

66
Q

What methods are currently used to treat cancer?

A

Surgery, radiation therapy, chemotherapy.

67
Q

What are recombinant DNA technologies?

A

joining together of DNA molecules from two
different species.

These are then inserted into an organism to produce (express) a useful protein.

68
Q

Describe the use of plasmids in recombinant DNA technologies?

A

Plasmids are used in genetic engineering to reproduce recombinant genetic material.

When a plasmid is inserted into a bacterium, the bacterium is encouraged to multiply, creating more copies of the recombinant DNA.

69
Q

The key components of the plasmid are

A

Origin of replication (ORI)
- allows initiation of replication using host DNA polymerase

Antibiotic resistance gene
- allows selection of cells containing plasmid

promoter
- drives expression of your favour gene (eg insulin or GFP) in cells with appropriate transcription factor machinery.

70
Q

Origin of replication (ORI)

A

allows initiation of replication using host DNA polymerase

71
Q

Antibiotic resistance gene

A

allows selection of cells containing plasmid

72
Q

promoter

A

drives expression of your favour gene (eg insulin or GFP) in cells with appropriate transcription factor machinery.

73
Q

what are restriction enzymes?

A

a protein produced by bacteria that cleaves DNA at specific sites along the DNA molecule.

In the bacterial cells, restriction enzymes cleave foreign DNA, thus eliminating infecting viruses (for example).

In the laboratory they are used to manipulate fragments of DNA, and are indispensable tools in recombinant DNA technologies.

74
Q

what are ligase?

A

– a specific type of enzyme that facilitates the joining of DNA strands together by
catalysing the formation of a phosphodiester bond.

75
Q

what are restriction enzymes and ligases uses in recombinant DNA technologies?

A

cutting and pasting our gene of interest into the bacterial plasmid.

76
Q

What is transformation?

A

Transfer of plasmids into bacteria.

77
Q

What are the main issues likely to arise when using a prokaryotic gene system to
clone eukaryotic genes?

A

Prokaryotes

  • don’t have enzymes to do post-translational modifications
  • incorrect protein folding
  • unable to splice introns

Prokaryotes do not have the
enzymes to do the types of post-translational modifications required for human proteins.

By using eukaryotic cells, the appropriate modifications can be added to the protein, ensuring optimum biological function.

In prokaryotic systems, protein
folding isn’t ideal, and correct folding can be improved by using eukaryotic cells.

Prokaryotic cellular machinery is also unable to splice introns out of a gene sequence, so, if the gene of interest contains introns, these will be incorporated into the final protein product, thereby
producing a non-functional protein. This can be overcome by using cDNA, which does not contain introns as it has been reverse transcribed from mRNA.

78
Q

Outline the key steps in producing a recombinant protein.

A
  1. Isolate gene of interest
  2. clone into expression plasmid
  3. Transform into bacteria for expression or isolation of more DNA for use in another expression system
  4. Grow cells expressing protein of interest
  5. Isolate and purify the protein
79
Q

Using the steps outlined above, briefly discuss the production of recombinant
human insulin.

Isolate gene of interest

A

Isolate human insulin mRNA and reverse transcribe into cDNA. Cleave the A chain
and B chain sequences from each other.

80
Q

Using the steps outlined above, briefly discuss the production of recombinant
human insulin.

clone into expression plasmid

A

Clone the A chain into one plasmid and the B chain into another plasmid. The
plasmids will both contain the lac Z gene upstream of the insulin chain sequence, to
act as a reporter gene.

81
Q

Using the steps outlined above, briefly discuss the production of recombinant
human insulin.

Transform into bacteria for expression or isolation of more DNA for use in another expression system

A

Transform these two plasmids into different cultures of bacteria.

82
Q

Using the steps outlined above, briefly discuss the production of recombinant
human insulin.

Grow cells expressing protein of interest

A

Grow the two populations of cells, allowing them to express lac Z/insulin A fusion
protein or the lac Z/insulin B fusion protein.

83
Q

Using the steps outlined above, briefly discuss the production of recombinant
human insulin.

Isolate and purify the protein

A

Isolate the expressed proteins from the cultures,

treat with cyanogen bromide to cleave the A and B chains off the lac Z protein.

Purify, and mix the two purified protein samples together, place under oxidising conditions, allowing active insulin to be formed.

84
Q

What are the advantages of using a prokaryotic system?

A

low cost, high yield, pathogen free

85
Q

What are the disadvantages of using a prokaryotic system?

A

proteins often partially folded, inability to perform post-translational modifications.

86
Q

What are the benefits of using eukaryotic cells instead of prokaryotic cells for the
production of therapeutic proteins?

A

Prokaryotes do not have the
enzymes to do the types of post-translational modifications required for human proteins.
- using eukaryotic cells, the appropriate modifications can be added to the protein, ensuring optimum biological function.

Another advantage is that often in prokaryotic systems, protein folding isn’t ideal, and
- correct folding can be improved by using eukaryotic cells.

87
Q

Erythropoietin (EPO)

A

hormone produced by the kidney that promotes the formation of RBC by the bone marrow.

The kidney cells that make erythropoietin are sensitive to low oxygen levels in the blood that travels through the kidney. These cells make
and release erythropoietin when the oxygen level is too low.

88
Q

why is a recombinant EPO produced?

A

use as a therapeutic agent by recombinant DNA technology in CHO cell culture, they are used in treating anaemia resulting from chronic kidney disease, chemotherapy induced anaemia in patients with cancer, inflammatory bowel disease

89
Q

How is this recombinant EPO

misused?

A

blood doping agent by endurance athletes, as a

means by which to increase RBC production, thereby increasing the oxygen carrying capacity of the blood.

90
Q

What is the concept of “Pharming”?

A

use of animals to make recombinant human proteins for the treatment of human disorders.

Using such systems has the benefit of ensuring that the
proper post-translational modifications are able to occur.

91
Q

What is gene therapy?

A

when DNA is introduced into a patient through the use of viral vectors, to treat a genetic disease.

The newly introduced DNA usually contains a functioning copy of the gene to correct for the effects of a disease-causing mutation.

Gene therapy has the potential to cure disease in a one off treatment.

92
Q

Type I diabetes

A

occurs as the beta cells of the pancreas as destroyed and can no longer produce insulin.

93
Q

How could gene therapy be used as a potential cure for Type I diabetes?

A

take the human insulin gene, and with the use of a viral vector, insert this gene into the liver to turn the liver
into an “insulin making machine”

94
Q

The problem is that we do not want insulin to be produced in the liver constantly,
why is this a problem? How to solve problem?

A

lead to type II diabetes.

Design the vector, we can fuse the pre-pro insulin gene to a glucose sensitive promotor, so that insulin
will only be produced when glucose levels rise. As is the case with pancreatic insulin
production.

95
Q

Define CRISPR-Cas, including where this system

originates, and outline how this system works.

A

CRISPR-Cas is a technology to edit parts of the genome by removing, adding, or otherwise
altering sections of DNA

(for example, a mutation may be removed, and the correct sequence of DNA put in its place).

This system was discovered in bacteria, where it is used as
a defence system against bacteriophages.

BIOC practice Q's (8 decks)

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