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CGCC Flashcards

1
Q

Describe the four stages of mitotic cell division.

A

G1 - Signal committing the cell to replicate DNA is received. Contents of the cell is duplicated (excluding chromosomes).

S - 46 chromosomes are duplicated.

G2 - Chromosomes are checked for errors and spindles form.

Mitosis - The chromosomes are separated and the cell splits (cytokinesis).

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2
Q

What happens if a cell doesn’t receive the signal to replicate?

A

It enters the quiescent G0 phase.

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3
Q

What is semi-conservative replication?

A

Where each new DNA molecule contains one parent and one daughter strand.

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4
Q

What is an origin of replication?

A

The point on the double helix where DNA replication is initiated. This is done by initiator proteins.

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5
Q

What unwinds DNA and what prevents immediate reformation of the double helix?

A

DNA helicase and single strand binding proteins.

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6
Q

What prevents supercoiling and how does it do this?

A

Topoisomerase breaks a phosphodiester bond in one of the parental strands ahead of the replication fork.

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7
Q

Which strand of DNA is the leading strand?

A

3’ to 5’

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8
Q

How does DNA synthesis begin and why?

A

It begins with a short DNA primer (synthesised by RNA primase/polymerase).

This is because DNA polymerase is unable to initiate DNA synthesis but can initiate RNA synthesis.

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9
Q

How do Okazaki fragments join up?

A

The RNA primer that precedes a fragment is degraded by an exonuclease and the gap left is filled by DNA polymerase. The missing phosphodiester bond is put in place by DNA ligase.

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10
Q

How are single base changes removed from DNA replication?

A

An exonuclease found within DNA polymerase goes backwards and removes the mistake when the movement of DNA polymerase stops as a consequence of the mistake.

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11
Q

What is the end replication paradox?

A

Where the ends of linear chromosomes (i.e. eukaryotic) are lost after each round of replication. The free 5’ end of the template.

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12
Q

How is the end replication paradox prevented?

A

The ends of chromosomes are capped by telomeres which are repeated bases. These are formed by telomerase.

Telomerase extends DNA without using a chromosomal template because it carries its own template (a short stretch of RNA).

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13
Q

What is thought to be a significant contribution to ageing?

A

The loss of telomeric sequences due to somatic cell replication. This is because telomerase is switched off in somatic cells.

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14
Q

What do 85% of all human primary tumours test positive for?

A

Telomerase activity.

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15
Q

What are 1) Silent 2) Missense and 3) Nonsense mutations?

A

1) Where a single base is changed but the same amino acid is coded.
2) Where a point mutation leads to a different amino acid being coded for.
3) Where a stop codon is created leading to premature truncation.

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16
Q

What are indels?

A

Small scale insertions and deletions.

If this occurs in a multiple of 3, the reading frame is maintained. If this does not occur in a multiple of 3, then a frameshift occurs.

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17
Q

What are inversions and translocations?

A
  • Where a chunk of DNA removes itself, turns 180 degrees then reforms.
  • Where a piece of DNA moves from one chromosome to another.
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18
Q

What are examples of chromosomal deletions and translocations?

A

Deletion - Cri du chat syndrome.

Translocation - Chronic myelogenous leukaemia.

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19
Q

What is deamination and give examples.

A

Deamination is the removal of an amine group from a molecule.

  • C turns to U (can now pair with A)
  • A turns to Hypoxanthine (now pairs with C)
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20
Q

What are the effects of UV-C and UV-B?

A
  • UV-C is the most lethal form but the ozone layer absorbs it.
  • UV-B is the major mutagenic fraction in sunlight and induces chemical bonds between adjacent thymines distorting DNA and causing problems during DNA replication often resulting in point mutations.
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21
Q

Give examples of physical and chemical mutagens.

A

Physical: ionising radiation (single or double strand breaks).

Chemical: nitrous acid (C to U), alkylating agents (guanine modification) and free radicals (strand breaks and base modifications).

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22
Q

Describe nucleotide excision repair (NER).

A

The region where the error has occurred is unwound by DNA helicase and then removed by nuclease. The gap is then filled by DNA polymerase.

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23
Q

What is the error rate of DNA polymerase and the overall error rate of DNA replication?

A
  • 10^-7

- 10^-10

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24
Q

What is the Central Dogma?

A

An explanation of the flow of genetic information within a biological system. Information moves from nucleic acid to protein, but this flow cannot be reversed.

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25
Q

Where is the promoter region found on DNA?

A

Upstream of the transcription initiation site. Its nucleotides are marked with negative numbers.

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26
Q

How is prokaryotic transcription initiated?

A

The pribnow box upstream of the transcription start site is recognized by the Sigma factor protein. The sigma protein then allows RNA polymerase to bind to the DNA. Once transcription is initiated, sigma factor dissociates.

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27
Q

How is the 5’ end of mRNA capped?

A

GTP reacts with the 5’ end of mRNA resulting in the addition of phosphate groups. The guanine is then methylated. A 7-methylguanosine cap is formed.

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28
Q

What to RNA Polymerase I, II and III produce?

A
  • Pol I = rRNAs (long)
  • Pol II = mRNAs, microRNAs
  • Pol III = tRNAs, rRNAs (5S)
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29
Q

What turns genes on and off?

A

When genes are switched on, the chromatin is unwound, the cytosines are unmethylated and histones are acetylated.

When genes are off, chromatin is condensed, cytosines are methylated and histones are deacetylated.

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30
Q

What is Rett syndrome?

A

A developmental brain disorder that is caused by mutations in methyl CpG binding protein (MECP2). This leads to a loss or reduced levels of protein within the brain.

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31
Q

What is the mutation that causes sickle cell anaemia?

A

Glutamate (GAG) to Valine (GTG). Point/missense mutation.

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32
Q

What is ‘wobble pairing’?

A

This is pairing between two nucleotides in RNA molecules that does not follow the traditional Watson-Crick base pair rules (A-T, G-C). This occurs between mRNA and tRNA.

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33
Q

What makes up a 70S complex within an E-coli ribosome?

A

Large subunit (50S) and small subunit (30S).

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34
Q

Describe the process of initiation.

A

The initiation complex is formed comprising of a ribosome, mRNA and initiator tRNA. Initiation factor proteins IF1, 2 and 3 are required. IF2 is activated by binding to GTP.

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35
Q

What is the Shine-Dalgarno sequence?

A

A sequence upstream of AUG which binds to 16S rRNA. It ensures translation along the correct reading frame.

Found only in prokaryotes.

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36
Q

Describe the process of elongation.

A

The activated amino acid binds to elongation factor EF-Tu-GTP and enters the A site of the ribosome. Peptide bonds form between amino acids in P and A sites using the energy from EF-Tu-GTP hydrolysis. EF-G-GTP then binds and ribosome translocases using GTP hydrolysis so that the A site is free again.

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37
Q

Describe the process of termination.

A

When the stop codon of mRNA reaches the A site, a release factor (RF1 or 2) binds. This causes hydrolysis of the protein from tRNA. The ribosome complex is then disassembled using IF3, ribosomal recycling factor and GTP hydrolysis.

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38
Q

How do some antibiotics work? Give some examples.

A

They distinguish between eukaryotic and prokaryotic transcription and translation.

Examples:

  • Streptomycin
  • Tetracyclines
  • Cycloheximide
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39
Q

What is recombinant DNA technology also known as and why?

A

Gene cloning because copies (or clones) are made up of unique pieces of DNA.

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40
Q

Where are restriction enzymes derived from and why?

A

They are derived from bacteria because they use these enzymes as a defence against viral infections.

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41
Q

What is a method used to replicate and amplify DNA fragments?

A

The use of plasmid cloning vectors.

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42
Q

How can harvesting mRNA from different cells help genetic studies?

A

Because not all cells express the same genes, harvesting mRNA from different cells allows you to see which genes are expressed by which cells and how this changes in disease.

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43
Q

What is cDNA and why is it used?

A

cDNA is called complementary DNA. It is a DNA copy of mRNA made by using an enzyme called reverse transcriptase. cDNA is used because you cannot clone RNA. The introns are removed and the clone size is smaller.

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44
Q

Describe the steps needed to make cDNA.

A
  1. Add oligo(dT) primer to the poly-A tail of the mRNA to be copied.
  2. Add reverse transcriptase which extends the primer forming a complementary strand.
  3. Partially digest the RNA with RNase H leaving small sequences behind.
  4. Add DNA polymerase I which recognises the small RNA sequences and synthesises a complementary strand.
  5. Add DNA ligase to seal the gaps.
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45
Q

How do you add cDNA strands to bacterial vectors?

A
  1. Protect EcoRi sites in cDNA from digestion using EcoRi methylase.
  2. Add ligate linkers containing EcoRI sites to the ends of the strand.
  3. Digest with EcoRI and clone into the vector.
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46
Q

What is the main difference between cDNA and a genomic DNA library?

A

cDNA is copied from mRNA so only contains the DNA that the cell was expressing at that point; the genomic DNA is every gene that the organism has within its genome regardless of what is being expressed.

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47
Q

What is dideoxynucleotide chain-termination sequencing?

A

A method of DNA sequencing where DNA polymerase is used to copy single stranded DNA, but stops when it encounters a dideoxy nucleotide.

This method can be automated and allows for rapid sequencing of large amounts of DNA.

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48
Q

How do dideoxynucleotides stop DNA sequencing?

A

ddNTPs do not have an -OH on the 3’ carbon which means that DNA polymerase cannot incorporate any further nucleotides after ddNTP as there is no 3’ OH which is needed to form a phosphodiester bond.

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49
Q

What methods does automated DNA sequencing utilise?

A
  1. The use of fluorescent tags on each of the different ddNTPs. (ddATP is green, ddTTP is blue etc.).
  2. The use of a capillary gel to separate the fragments - uses a laser to stimulate the fluorescent tags to emit wavelengths.
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50
Q

What is the 100,000 Genome Project?

A

A project that is sequencing genomes of humans from various backgrounds, with rare diseases or cancer to investigate the basis of variation in humans and help to identify the genetic basis of disease, explain individual susceptibility to disease, response to drugs or reaction to environmental factors, and identify single base pair differences (SNPs) between individuals.

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51
Q

What DNA polymerases are used in PCR and why?

A

Taq of Pfu are used because they are thermostable.

Taq was isolated from bacteria that colonise hot thermal springs so they can function at the high temperatures used in PCR.

52
Q

Describe the three steps of PCR.

A
  1. Denaturation = mixture is heated to 95°C to denature the DNA into single strands.
  2. Primer annealing = temperature is reduced to 45-68°C allowing primers to anneal to their complementary sequences on the now-exposed DNA strands.
  3. Primer extension = temperature is raised to 72°C allowing Taq polymerase to synthesise the DNA.
53
Q

What are DNA microarrays?

A

These are modern devices which use nucleic acid hybridisation to rapidly measure which genes are expressed in a tissue sample.

54
Q

How do DNA microarrays work?

A

All the mRNA from a sample is converted to cDNA, fluorescently labelled, denatured into single strands and used as a probe. The probe is then applied to the array which contains ssDNA (oligonucleotides) matching all the genes in the genome.

The labelled cDNA binds to the spots on the slides that contain complementary sequences. Because the cDNA is fluorescently tagged, the pairs will be seen.

The location and brightness of the fluorescent spots on the microarray identifies the gene expression and their level of expression.

55
Q

What are polymorphisms?

A

Differences in DNA between humans. They are considered to be neutral and common in the general population.

56
Q

How often do SNPs occur in the human genome and what does this mean?

A

Once in every 300 nucleotides which means there are roughly 10,000 SNPs in the human genome.

57
Q

What is a haplotype?

A

A series of SNP alleles along a single chromosome.

58
Q

If SNPs are not independent what does this mean?

A

Means that two SNPs are located very close together and you more often than not see both of them together.

59
Q

What is linkage disequilibrium?

A

Where two SNPs are found together and are very unlikely to be found separate.

60
Q

What are tandem repeat polymorphisms?

A

Repeated sets of nucleotides in DNA. These repeats can be two or more base pairs long.

An example of this is Huntington’s disease.

61
Q

What are structural variations in DNA?

A

Where large segments of DNA can be absent in some chromosomes or present in multiple tandem (repeating) copies.

They are known as copy number variants or polymorphisms (CNV/CNP).

62
Q

What is the Hardy-Weinburg equilibrium?

A

It describes a balance between the genotypes in a population and the allele frequencies.

63
Q

What are the conditions required for the Hardy-Weinburg equilibrium?

A
  • Random mating
  • Random transmission
  • Random survival
  • Large population
  • No migration
  • No selection
64
Q

How can the two sides of the Hardy-Weinburg Equilibrium be portrayed algebraically?

A

For observed allele frequencies: p+q=1

For expected genotype frequencies: P^2+2pq+q^2 = 1

65
Q

What is genetic drift?

A

Where all genetic variants will change in their frequency over long time periods.

66
Q

What is genetic fitness?

A

Fitness measures the ability of genotypes to reproduce. It is measured on a scale between 0 (no reproduction) and 1 (full reproductive ability).

67
Q

What kind of distribution do many human traits have?

A

Bell-shaped curve.

68
Q

What are quantitative traits?

A

They are characteristics that are determined by a number different genes as well as environmental influence.

69
Q

What is heritability?

A

The proportion of the phenotypic variation that is due to genetic difference among individuals.

Heritability estimates for continuous traits and disorders.

70
Q

What defines common and rare variants?

A
  • Common = when the minor allele frequency (MAF) is greater than 1%. Also known as SNPs.
  • Rare = when MAF is less than 1%. Also known as mutations if pathogenic.
71
Q

Which chromosome is affected in Down’s syndrome?

A

Chromosome 21

72
Q

What is the difference between complex and Mendelian diseases?

A

Complex diseases are caused by both genetics and the environment whereas Mendelian diseases are caused by genetics alone.

Mendelian diseases are also more rare than complex diseases.

73
Q

What is the most common cystic fibrosis mutation?

A

ΔF508, a deletion of 3

nucleotides, resulting in the loss of the amino acid phenylalanine (F) at the 508th position on the protein.

74
Q

What causes Huntington’s disease?

A

CAG repeat expansion.

75
Q

What is an example of a complex disease?

A

Psoriasis

76
Q

What are monozygotic twins?

A

This means they are identical twins.

77
Q

Define functional genomics.

A

Where information about the function of genes is associated with the knowledge of DNA sequence. This includes systemic analysis of mRNA and protein expression as well as exploration of gene product interactions and their influence on different phenotypical traits to define gene functions.

78
Q

What is bioinformatics?

A

The organisation, visualisation, analysis and interpretation of biological data.

79
Q

What does personalised medicine require?

A

The identification of predictive biomarkers that separate patients according to their response to the drug (theranostic biomarker).

80
Q

What is cytochrome P450 and what happens when it is reduced/absent or in high amounts?

A

It is an enzyme that is predominantly made by the liver. It converts codeine to its active form morphine. When it is absent, codeine is ineffective. In high amounts, codeine can be lethal for fast metabolisers.

81
Q

What is cytogenetics?

A

The study of the genetic components of a cell through the visualisation and analysis of chromosomes.

82
Q

What are G-bands?

A

Chromosomes stained with Giemsa show alternating light and dark bands. The pattern is characteristic for each chromosome pair.

83
Q

What does metacentric, submetacentric and acrocentric describe?

A

The location of the centromere on a chromosome.

84
Q

How much of the Y chromosome confers maleness?

A

A small region at the end of the short arm of the Y chromosome called the MSY (male-specific region) is all that is needed. Some women are XY, but with the MSY on the Y chromosome missing.

85
Q

What is the only region on the Y chromosome required for male development?

A

SRY is a gene that encodes a protein that acts as a testis determining factor (TDF). SRY is the only region of the Y chromosome required for male development.

86
Q

What is aneuploidy?

A

An irregular number of chromosomes.

87
Q

What are trisomies?

A

Where you have three copies of one chromosome in the karyotype.

88
Q

What is the cytogenetic nomenclature of Down’s syndrome?

A

47, XX or XY +21

89
Q

What is Down’s syndrome also called?

A

Trisomy 21

90
Q

What is a simian crease?

A

Where there is only one obvious crease present on the palm of the hand. This is a characteristic of Down syndrome but can be present in people with a normal karyotype.

91
Q

What is Patau syndrome?

A

Also known as Trisomy 13 (47, XX or XY+13) causes heart defects and incomplete brain development. Very short life expectancy.

92
Q

What is Edward’s syndrome?

A

Also called Trisomy 18 (47, XX or XY+18) causes heart defects and intestines protruding outside the body. 95% die in utero and those that survive barely reach 1 year.

93
Q

Why do trisomy 13, 18 and 21 survive birth?

A

Because these chromosomes are small meaning they have less genes that are duplicated meaning less damage.

94
Q

What is Klinefelter syndrome?

A

Where the person has XXY sex chromosomes. Patients can appear normal, with limited development of secondary sexual characteristics (small testes).

95
Q

What is Turner syndrome?

A

This is a monosomy with only one sex chromosome X. No adolescent growth spurt is seen. Women are ova degenerate in utero (infertile), have amenorrhoea (lack of menstrual cycle) and have limited development of secondary sexual characteristics.

This is the only known example of human monosomy.

96
Q

What is euploidy?

A

A complete set of chromosomes.

97
Q

What causes aneuploidy?

A

Non-dysjunction = the failure of homologous chromosomes to separate properly during meiosis.

98
Q

How does an amniocentesis test for aneuploidy?

A

Foetal cells that have rubbed off the baby during development are extracted from amniotic fluid and amplified by PCR. They are then grown in a culture medium and the karyotype is determined.

99
Q

What is Huntington’s disease caused by?

A

Greater than 36 repeats of CAG codon - polyglutamine tract.

100
Q

What does it mean if the percentage of concordance of a disease is higher in monozygotic twins?

A

This indicates that there is a genetic contribution to the disease.

101
Q

Describe Hershey and Chase’s experiment in 1952.

A
  • They labelled proteins in phages with 35S (as there is no sulphur in DNA) and the DNA with 32P (there is no phosphorus in protein).
  • They then infected bacteria with these phages causing copies of itself to be made.
  • When the final copies were analysed, they saw that the phages with labelled DNA carried their reactivity in progeny phages, but phages with labelled proteins did not.
102
Q

What are purines?

A

Nucleic acids with two ring structures. These are guanine and adenine.

103
Q

What are pyramidines?

A

Nucleic acids with one ring structure. These are cytosine, thymine and uracil.

104
Q

What bond links the nucleic acid to its ribose?

A

Glycosydic bond

105
Q

What forms the polynucleotide chain of DNA?

A

A phosphodiester bond between the 3’ hydroxyl and the 5’ monophosphate of the next nucleotide in line.

106
Q

What are the three criteria that DNA must meet?

A

1) Must incorporate a feature that determines its role as a store of information.
2) Must suggest a replication mechanism that permits its passage across generations of progeny.
3) Must be capable of undergoing mutation such that the information is altered in a heritable manner.

107
Q

What studies did Rosalind Franklin and Maurice Wilkins undertake? What did they discover?

A

They performed X-ray diffraction studes and discovered diffraction patterns in an X-shape which is consistent with the structure being a helix.

108
Q

What are Chargaff’s rules on DNA?

A

The amount of guanine is always equal to the amount of cytosine.

The amount of adenine is always equal to the amount of thymine.

109
Q

What is the diameter of a DNA helix?

A

2nm

110
Q

What is the bonding between nucleotide pairs?

A

A-T = double hydrogen bond.

G-C = triple hydrogen bond.

111
Q

What is the proportion of our DNA that codes for genes?

A

1.1%

112
Q

What proportion of the human genome are introns?

A

23%

113
Q

What is one theory as to why introns are present in DNA?

A

DNA polymerase makes mistakes. If these mistakes happen within exons then this is potentially catastrophic (build-up of mistakes over our lifetime is what leads to cancer). If you make the genome large, with most of it removed and non-coding, then this could act as a sink to take up the mistakes.

114
Q

What are a) transposons and b) retrotransposons?

A

a) A piece of DNA that is mobile - can move from one point of the genome to another.
b) They stay where they are, are transcribed by RNA polymerase, converted to DNA by reverse transcriptase and the DNA goes and integrates itself into a different part of the genome.

115
Q

What are LINES and SINES?

A

Long Interspersed Elements and Short Interspersed Elements. They are both retrotransposons.

116
Q

What is heterochromatin and what is its clinical significance?

A

It is highly condensed DNA and never transcribed.

The number of repeats varies immensely between individuals so can provide a genetic fingerprint so is used in paternity testing and forensics.

117
Q

What does de-coding mRNA go on to form?

A

Ribosomes

118
Q

How is the second X chromosome in females silenced?

A

When the Xist gene is transcribed, it causes a spread of heterochromatin forming a ‘bar body’ across the entire chromosome preventing it from transcribing.

119
Q

What is anhidrotic ectodermal dysplasia?

A

It is an X-linked recessive disorder that reveals X-inactivation. Heterozygous females have random patterns of tissue with and without sweat glands.

120
Q

What does the binding of miRNA to mRNA cause?

A

Deadenylation (mRNA instability). This reduces the half life of mRNA causing it to degrade faster.

121
Q

What is a distinctive feature of miRNAs when they are transcribed?

A

They are given a poly-A tail and a cap that are complementary to each other causing the miRNA to bend over and bind with itself to form a hairpin loop.

122
Q

What does mitochondria encode for?

A
  • 13 polypeptides (majority of which are used in the electron transport chain.
  • rRNA
  • tRNA
123
Q

What makes mitochrondrial DNA unique?

A

It has no introns, no repetitive DNA and is inherited exclusively from the mother.

124
Q

What are the organs and tissues that are most affected in mitochondrial cytopathies?

A

Those that use high amounts of ATP for energy - muscles and nerves.

125
Q

What does matrilineal mean?

A

The gene does not follow any of the autosomal or sex-linked inheritance patterns. This relates to mitochondrial DNA.

126
Q

How does three-parent in vitro fertilisation work?

A
  • The nucleus is removed from an egg cell with faulty mitochondria (the mother).
  • The nucleus is transported to an egg cell with healthy mitochondria and the nucleus also removed (the donor).
  • The egg is then fertilised by sperm (the father).

FBS 1 (6 decks)

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