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Lent Flashcards

1
Q

What was Hippocrates belief on inheritance?

A

Characteristics come from both parents and mixes
The ‘material’ comes from all parts of the body

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2
Q

When was Hippocrates around?

A

460-377 BC

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3
Q

What was Aristotle’s belief on inheritance?

A

Mixing of blood
Males responsible for active element that gives life to a male/female
Female provides nutrients

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4
Q

When was Aristotle around?

A

384-322BC

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5
Q

When was Gregor Mendel around?

A

1856-1870

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6
Q

What is a true breeding strain?

A

Where a parent would produce offspring with same genotype (homozygous)

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7
Q

What did Sutton and Boveri discover in 1903?

A

The chromosome structure (maternal and paternal)
Independent distribution

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8
Q

What did Bateson, Punnet and Saunders discover in 1905-1908?

A

Breeding of sweat peas didn’t show a Mendelian inheritance

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9
Q

What did Thomas Hunt Morgan discover in 1909?

A

Studied Drosophila
Suggested that genes could be found on the same chromosome
Can be linked
Chromosomes are located in the nucleus like beads on a string

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10
Q

What are the advantages of using Drosophila?

A

Short life cycle
Easy to keep in lab
Female lay many eggs

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11
Q

What is Intra-chromosomal recombination?

A

Crossing over of chromosomes, can lead to genes commonly on the same chromosome being separated

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12
Q

What is chromosome mapping?

A

Units of centimorgans
Calculated by looking at fraction of crossover
Created by Arthur Sturtevant

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13
Q

Why might the distances in the fractional crossover not add up?

A

Crossing over occurred more than once
Crossing over is less frequent near centromere
There are areas prone to crossing over (recombination hotspots)

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14
Q

What is incomplete dominance?

A

Where there is a mix in phenotype and no one clear phenotype
e.g. pink flowers instead of white or red

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15
Q

What example shows dominance can vary when looking at different phenotypes?

A

Sickle Cell anemia
Complete dominance- for clinal phenotype
Incomplete- for RBC sickling as it can occur at low [O2]
Co-dominance- of protein forms

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16
Q

What are sex-linked diseases?

A

Diseases where the likelihood of having diseases varies significantly with gender
Male are often more likely to express disease traits than female

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17
Q

What makes fungi good for genetic analysis?

A

Produce a tetrad of spores
Haploid- so no dominance relationship
Easy to grow
S.cerevisiae- can be synchronised to start at same stage in cell cycle, depending on conditions

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18
Q

What is Epistasy?

A

Where the effect of one gene masks another one. The masking one is know as epistatic
e.g. gene for presence of eye and gene for eye colour

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19
Q

What are 2 mechanisms of Non-mendelian inheritance?

A

Cytoplasmic genes
Prokaryote genetics

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20
Q

What are cytoplasmic genes?

A

Genes contained within the mitochondria or chloroplasts

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21
Q

What are the advantages and disadvantages of using bacteria?

A

Adv-Haploid, quick regeneration, small genome size
Disadv- restricted phenotype range, don’t carry out meiosis

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22
Q

What is the life cycle of a T4 bacteriophage?

A

Lytic- eventually causes cells to burst
Multiply, assemble inside cell

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23
Q

What evidence is there to show that viruses pass on DNA and not proteins that are pathogenic?

A

Radio labellilng of P (DNA) and S (Protein) into phage
Phage infects bacteria
Centrifuge
for P(DNA) shows passing on, no passing on of S (proteins)

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24
Q

What is complementation and non-complementation? And an example

A

Complementation- where mutations are found on different genes so ‘healthy’
Non-complementation- where mutations are found on the same gene, so show disease phenotype
Deafness caused by mutations on different genes

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25
Q

How has bacteria evolved to fight back against viruses?

A

CRISPR- endonuclease to cleave out viral DNA
But not cut own by methylating a base of own bacterial DNA

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26
Q

What are the 3 ways horizontal gene transfer can occur?

A

Conjugation- pili connect
Transformation- uptake of free DNA
Transduction- infection of non-pathogenic DNA from phage

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27
Q

Who and when first discovered gene cloning?

A

1970s
Cohen and Boyer

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28
Q

How are phages used as cloning vectors?

A

Restriction enzymes cleave DNA
Electrophoresis to separate
in vitro put desired into phage
Phage can insert into bacterial DNA

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29
Q

What is the central dogma?

A

The relationship which shows how information is passed on and the forms they are in. DNA, mRNA, tRNA and proteins

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30
Q

What is the key property of histone proteins?

A

Slight positive charge due to Arg and Lys residue
Can attact -ve phosphate present in backbone of DNA

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31
Q

How is genetic information arranged from DNA to chromosome?

A

DNA
Extended chromatin
Condensed chromatin
Scaffold associated chromatin
Condensed Scaffold associated chromatin
Chromosome

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32
Q

How are the histone octomers arranged?

A

4 different type of proteins, 2x of each.
H1 present act as a clamp keeping everything wound up
Contain Arg and Lys (+vely charged)

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33
Q

How long on average is DNA found in a cell?

A

2.2m

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34
Q

What are the 3 experiments that have been carried out to find out the nucleosome size?

A

1) Limited digestion- Microccocal nucleases, removes proteins, then run electrophoresis
2) Extensive digestion- Cleave at many points, run electrophoresis
3) X-ray crystallography- unable to see histone tails, so can’t see all of DNA wrapping around histone

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35
Q

What are the 3 stages of DNA replication?

A

Initiation
Elongation
Termination

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36
Q

What are the 7 enzymes/proteins involved in prokaryotic DNA replication?

A

Topoisomerase
DNA Pol I
DNA Pol III
Helicase
Primase
DNA ligase
Single stranded binding protein

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37
Q

What is the function of Topoisomerase?

A

Unwind, reduce torsional strain exerted

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38
Q

What is the function of the single stranded binding protein?

A

Protect bases that have been exposed

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39
Q

What is the function of DNA Pol I?

A

RNA primer removal
Proof reading

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40
Q

What is the function of DNA Polll?

A

Synthesis 5’ to 3’
Exonuclease for proofreading

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41
Q

What are the 5 subunit of DNA Pol III and their function?

A

Alpha- synthesis
Beta - sliding clamp
Tao- dimerise alpha subunits
Epsilon & Theta- exonuclease

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42
Q

What does the formation of Okazaki fragments mean?

A

DNA replication is discontinuous on the lagging strand

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43
Q

How were Okazaki fragments discovered?

A

Pulse chase experiment
thymine Radiolabelling H added
Halted and centrifuged at different time
Shows presence of short DNA strands
Which then disappear and only long strands present

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44
Q

What is the trombone model?

A

Model for how DNA Pol III reads the DNA strand
Created by Alberts

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45
Q

How do DNA Pol I and DNA ligase work together?

A

DNA Pol I- binds, removes primer, synthesis new DNA, leaves nick
DNA ligase- recognizes nick, forms a new bond

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46
Q

What happens in the termination stage of circular DNA?

A

Form catennes, new loops still linked
Require Topoisomerase IV- breaks double bond, then reseals it

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47
Q

What are the similarities between eukaryotic and prokaryotic DNA replication?

A

1) contain Origin elements
2)Bi-directional
3) Synthesis 5’-3’
4) Semi-conservative
5) Semi-discontinuous
6) Multi-protein nature

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48
Q

What are the difference between eukaryotic and prokaryotic DNA replication?

A

1) Eukaryotic has many origin elements
2) Replication occurs at defined point in cell cycle
3) DNA polymerases are different
4) RNA primer removal is different
5) End replication problem (linear DNA)

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49
Q

Why does eukaryotic DNA replication contain may origin elements?

A

Long DNA
Polymerases are slow

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50
Q

Why does eukaryotic DNA replication occur at a specific point in the cell cycle?

A

Needs to be controlled
Only occurs in S phase
Controlled by the formation of Pre replication complex
Helicase requires activation

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51
Q

What are the 3 different polymerases in eukaryotic DNA replication?

A

DNA Pol alpha- synthesises primers
DNA Pol delta- synthesises lagging strand
DNA Pol epsilon- syntehsises leading strand

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52
Q

How does RNA primer removal work in eukaryotic DNA replication?

A

FEN-1 instead of DNA Pol I
Recognises primer and cleaves

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53
Q

How does eukaryotic DNA replication overcome the end replication problem?

A

Telomerases- has integral RNA seq, uses reverse transcriptase
Synthesises a sequence of bases to add on to the end to stop the continual shortening deleting key bases.

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54
Q

How can DNA damage occur?

A

UV light
Alkylation
Ionisation

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55
Q

What are the 4 DNA repair mechanisms?

A

1) Mismatch repair
2) Photoreactivation (prokaryotes)
3) Nucleotide excision repair
4) Base Excision repair

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56
Q

What does mismatch repair work, and how?

A

Repairs wrong base pairing
Recruits MutL, H and S to act as exonucleases
Then uses DNA Pol III and DNA ligase to repair

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57
Q

How does photoreactivation work?

A

Photolyases
Pyrimidine separation, from dimer formed by UV
Absorbs light , moves e- around

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58
Q

What is nucleotide excision repair?

A

Removal of pyrimidine dimer (like in photoreactivation)
1) Recognition of abnormality by damage recognition complex
2) Unwinding by helicase
3) Cleaving of abnormality by exonuclease
Uses Uvr A, B and C along with DNA Pol I

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59
Q

What is Base excision repair?

A

Swaps a single base
Damage recognised by a glycosylase enzyme
Glycosylase cleaves
Endonuclease cleaves the rest
DNA Pol and ligase fix

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60
Q

What are the differences between transcription and DNA replication?

A

Transcription- RNA polymerase, no primer needed, produces one strand, mRNA strand
DNA rep- DNA polymerases, primers needed, produces 2 strands, DNA strands

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61
Q

What are the 2 components for Bacterial transcription?

A

Sigma- specificity
Core factor- for elongation

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62
Q

Where does transcription start? Where are the promoters usually located?

A

Start at TSS (transcription start site)
Promoters at -10 and -35

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63
Q

What are the 2 mechanisms of transcription termination in bacteria?

A

Rho dependent- Rho causes dissociation
Rho independent- RNA forms a stem loop due to GCs and then U present, to weaken interaction

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64
Q

What are the differences in transcription in bacteria and eukaryotes?

A

Different RNA polymerases
mRNA later processed before exported out of nucleus
TFs present
Impact of chromatin (DNA + proteins)

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65
Q

What are the 3 types of RNA polymerases and what do they transcribe?

A

RNA Pol I- rRNA
RNA Pol II- mRNA
RNA Pol III- tRNA

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66
Q

What are 3 pre-mRNA processes that need to occur before exporting the mRNA?

A

5’ capping- protect from degradation, promotes splicing, recruits ribosome
Poly(A) tail- promote export, efficient translation
Splicing- remove introns, leave useful coding exons

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67
Q

What enzyme is involved in splicing?

A

Spliceosome- type of ribozyme

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68
Q

What is the genomic library?

A

Collection of clones of DNA fragments representing entire genome of an organism

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69
Q

What are ways DNA can be analysed?

A

Restriction Enzyme Mapping
Southern Blotting (hybridisation, autoradiography)

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70
Q

What polymerase is often used in PCR and why?

A

Taq Polymerase
Can withstand high temperatures

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71
Q

What is Sanger Sequencing?

A

uses ddNTPs- which change a OH on C3 to H, so can no longer bond
Different ddNTPs for the 4 bases
All used and then fluorescent label to then read off the order of bases, as the strands will be of different lengths

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72
Q

What is the timeline for the human genome project?

A

1990s Began
1997 Freely accessible
2003 Sequencing complete
2006 Genome finished (not introns)
2022 No gaps 99.99% accurate

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73
Q

What are the ethical issues with sequencing genomes?

A

Insurance companies discriminating
Lose privacy
Cost - wealth divide

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74
Q

Why is annotating the genome difficult?

A

Contains introns and exons, which are spliced and so difficult to analyse what codes for what

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75
Q

What is G-banding and what can it be used for?

A

Dyeing with Giemsa dye
Different dying patterns, to analyse which chromosome it is
Used to show translocation

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76
Q

What are autonomously replicating seqences (ARS)?

A

Area on the chromosome which contain the ORC, to begin DNA replication

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77
Q

What are the kinetochores?

A

Protein complex that attaches to centromere of chromosome and spindle fibres, during mitosis

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78
Q

What are transposable elements and the 2 types?

A

Sequences of bases/ genes that move around in the chromosome
Can insert themselves into genes and cause mutations/diseases
DNA transpons (cut-paste) relocated
Retrotransposons (copy-paste) transcribed to RNA, reversed back to DNA and integrated back in

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79
Q

What percent of the genome is protein coding?

A

1.5%

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80
Q

What are LINEs? + Medical example of its effect

A

Long Interspersed Nuclear Elements
Retrotransposons, replicate and insert themselves into genome
LINE 1 most common and can insert into a gene leading to hemophilia, Hunter Syndrome, cancer, neurological disorders (schizophrenia)

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81
Q

What is a pseudogene?

A

sequence of DNA which could be a gene, however due to mutations is no longer functioning

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82
Q

What are simple sequence repeats and their use?

A

SSRs/ microsatellites/ STRs
Short stretches of repeating DNA
Use to study genetic diversity, evolutionary relationships, forensics
Expansion of STRs can lead to Huntingdon’s disease

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83
Q

What is aneuploidy?

A

Abnormal number of chromosomes

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84
Q

How does Down Syndrome occur?

A

Three chromosomes on 21

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85
Q

What does the Y factor contain, that the X chromosome doesn’t?

A

Testes determining factor

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86
Q

What 2 problems arise from the Y chromosome?

A

Y chromosome shorter, for recombination PAR
Dosage compensation- technically aneuploidy, so in humans inactivate X chromosome

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87
Q

What are examples of recessive X- linked disease?

A

Duchenne Muscular Dystrophy DMD
Haemophilia
Colour Blindness

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88
Q

What are some examples of autosomal recessive diseases?

A

Sickle cell Anaemia
Spinal Muscular Atrophy
Cystic Fibrosis

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89
Q

What is the cause of Huntington’s disease?

A

Expansion of CAG repeat in ORF
Repeated sequencing of glutamine
late onset of neurodegenerative disease

90
Q

What are SNPs and how are they detected?

A

Single Nucleotide Polymorphisms
Occur in coding and non-coding genome
There are around 6 mill nucleotide differences between 2 people
Detected by NGS, like PacBio and Illumnia

91
Q

What is chromosome walking?

A

Method to identify genes located near each other
walking- because it’s a step by step process where overlapping ends are used to make a continuous stretch

92
Q

What causes CF?

A

Mutation of CFTR protein
Disrupting movement of Cl- effecting movement of water and so mucus consistency in airways, reproductive system and digestive system

93
Q

How are genetic maps created?

A

Linkage analysis- look at genetic markers that follow inheritance
- then used to show location of disease on chromosome
GWAS
- large unrelated population, look at association studies
- genotyping SNPs and using stats to identify associations between SNPs and disease

94
Q

What are 3 key genetic markers?

A

Restriction Fragment Length Polymorphisms (RFLPs)
SNPs
Microsatellites/SSRs/ STRs

95
Q

What are RFLPs?

A

Restriction Fragment Length Polymorphism
Technique to detect differences in DNA, used for sickle cell anaemia & CF
Differences in lengths of DNA strands after use of restriction enzyme, due to mutations in the restriction enzyme recognition site
Run through a gel to compare

96
Q

What is the international HapMap project?

A

Project from 2002-2011
Aimed to identify SNPs and haplotypes in different populations
Use to look at common genetic variation as well as variation linked to diseases

97
Q

What is the GWAS?

A

Genome Wide association studies
Identify SNPs and genetic variation that is linked to diseases such as cancer, diabetes, Alzheimer’s
Used an ideal population, which is large (genetic diversity), isolated (some new influx of genes) such as FINLAND

98
Q

What are 4 ways the genome can be sequenced?

A

Next Gen sequencing (PacBio, Illumina)
Sanger sequencing
Top down sequencing
Shotgun sequencing

99
Q

What does DNA fingerprinting require?

A

SSRs combinations
which vary between people

100
Q

What is a microarray?

A

tool used to detect thousands of gene expressions, by using a printing robot

101
Q

What is the DNA microarray technique?

A

RNA is extracted from cell
Converted to DNA (cDNA) and fluorescent tag added
Hybridised to DNA microarray, complementary bind
Intensity and presence of fluorescence shows if gene is expressed and how much

102
Q

What does diagnostic and prognostic mean?

A

Diagnostic- characteristic of gene, upregulation or downregulation of gene
Prognostic- signatures of treatment outcomes

103
Q

Describe the chromatin immunoprecipitation and sequencing (ChIP-seq) technique and what it is used for?

A

-Used to find location of transcription factor binding site
Crosslinking to keep proteins and DNA together
Cell lysed, DNA sheared
Antibodies added for immunoprecipitation
Reverse crosslinking with Proteinase K
Purified by RNase A
Analysed using qPCR

104
Q

What are the difficulties with proteomics?

A

DNA can yield many types of proteins, due to splicing
No method to amplify proteins to then analyse

105
Q

How did proteins used to be analysed? Describe the process.

A

2D PAGE (Polyacrylamide Gel Electrophoresis)
separated based on size and charge
2 gels run

106
Q

How was proteomics revolutionised?

A

High Throughput Ultrasensitive Mass Specs
1) Peptide Mass fingerprinting
2) de novo protein sequencing (TMS)

107
Q

Describe Peptide mass fingerprinting

A

Trypsin- cleaves enzyme into peptide
Peptides ionised and vaporized then in mass spec
Mass of each peptide determined
Sequence of protein interpreted from known peptides

108
Q

Describe Tandem Mass spec (de novo) protein sequencing

A

Uses 2 mass spectrometers
1st) Ionise and separate based on mass and size in Quadropole Analyser
- Fragmented further by collision-induced dissociation
2nd) Resulting fragments analysed by second mass spec for Time of Flight (TOF)

109
Q

What is systems biology?

A

Integration of genome sequencing, with proteomics to find solutions to medical problems
Involves engineers, biologists, chemists etc.

110
Q

Who described the first cell and when?

A

Robert Hooke in Micrographia in 1665

111
Q

What are the 4 phases of bacterial replication?

A

Lag- adjust to environment
Log- exponential growth
Stationary- lack of nutrients
Death- build up of toxic chemicals

112
Q

How are bacterial replication cycles so fast?

A

Formation of replication fork, already formed when cell divides
Begins synthesis already
Method for separation is different, quicker and regulated by reaching threshold length

113
Q

What happens in prophase?

A

Chromatin condenses into chromosomes
Centromeres migrate to poles
Interphase microtubules disassemble

114
Q

What happens in prometaphase?

A

Nuclear envelope breakdown
MT capture kinetochores

115
Q

What happens in metaphase?

A

Chromosomes are lined up in the middle

116
Q

What happens in anaphase?

A

Loss of cohesion- separation of sister chromatids
By shortening of MTs

117
Q

What happens in telophase?

A

Chromosomes decondense
Nuclear envelope reforms

118
Q

What happens in cytokinesis?

A

Powered by actomyosin ring
Constriction, until membrane separates

119
Q

What are the 3 types of microtubules (MT)?

A

Kinetochore- cause separation, attach to kinetochore
Interpolar- crosslink in the middle, don’t attach to chromosomes
Astral- make sure centrosomes are at pole

120
Q

What are the 3 ways asymmetric division can occur?

A

Alignment of chromosomes not in the middle
Cells face different environments
Cell moves out of ‘layer’ different environment

121
Q

What is an example of alignment of chromosomes leading to asymmetric division?

A

Drosophila neuroblasts
One retains identity the other differentiates

122
Q

What is an example of cells facing different environemnts, leading to asymmetric division?

A

Drosophila testes
Cell remaining in contact with HUB has stem cell identity
The other is distal from HUB so differentiates

123
Q

What is an example of cells moving layers leading to asymmetric division?

A

Mammalian brain development
Cell remaining in ventricular zone, retains progenitor identity
Outside ventricular zone starts to differentiate

124
Q

How is the number of cells controlled?

A

Programmed cell death
Quiescence- dormant, able to proliferate but not activated to do so
Proliferation v cell death

125
Q

What is haematopoiesis?

A

Formation of blood components, different cells
Has multipotent progenitors

126
Q

How does stem cell differentiation occur in the small intestine epithelium?

A

Stem cells located in the base of the crypt
Transit amplifying cells, dividing progenitors that are partially differentiated
Below +4 still stem cells
Above +4 (4 cells) post-mitotic and differentiated

127
Q

What is apoptosis and how does it occur?

A

Apoptosis is programmed cell death
Carried out by caspases
C= cysteine in active site
Asp= aspartic acid

128
Q

How is apoptosis controlled?

A

Extrinsic- binding of ligand, triggers initiator caspase-8, then triggering cascade response
Intrinsic- CytC released from mitochondria, interacts with Apaf-1, which activates caspase-9, initiating cascade response

129
Q

What are 3 types of proteins in the Bcl-2 family that control the release of CytC?

A

Bax (proapoptotic)- bind to mem, cause release
Bcl-2 (antiapoptotic)- binds to stop
Bad- (proapoptotic)- inhibits Bcl-2, stimulates Bax

130
Q

What are the 4 phases in the eukaryotic cell cycle?

A

G1 phase
S phase
G2 phase
M phase

131
Q

What cell fusion experiment suggested the presence of control and checkpoints in the cell cycle?

A

Fusion of cells with nuclei in different phases
1) Any with M phase- will follow into M phase
2) G1+S- will follow into S phase
3) G2+S- won’t replicate (Sphase)
Meaning M is dominant, G2 has a block to re-replication

132
Q

Compare the embryonic cell cycle and the somatic cell cycle

A

Embryonic- progressive replication leads to smaller cells, natural stop points can be exploited, e.g. frogs eggs, missing G1 and G2 phase, more rapid
Somatic cell- divide mitotically, control points can be synchronised by drugs

133
Q

What are the 2 approaches to find master regulators of the cell cycle?

A

Biochemical approach- cells with rapid synchronous cell division, analyse extracts, e.g. frog eggs
Genetic approach- genetically traceable systems, screen for mutations that effect the cycle, e.g. bakers yeast

134
Q

What did the biochemical & genetic approach discover (Hunt and Nurse)?

A

The presence of MPF (maturating promoting factor) controlling movement from G2 to M phase
Biochemical approach to extract, then genetic approach to sequence
Found that it was formed by 2 proteins, cyclin and cdc2

135
Q

What does cyclin do?

A

Bind to CDK which then usually promotes entry into the next phase
Cyclin A in S phase
Cyclin B in G2/M
Cyclin D in G1 phase
Cyclin E in G1/S
Breakdown of cyclin causes exit of M phase
Show periodic accumulation and degradation during cell cycle

136
Q

How can budding yeast be a good model for the somatic cell cycle?

A

Size correlates with the phase of the cycle
Easy to isolate mutants

137
Q

How do haploid and diploid budding yeast vary?

A

Haploid & Diploid when nutrients available divide mitotically
Nutrients not available
Haploid- stop and mate
Diploid- sporulate

138
Q

What is the human equivalent of the cdc-2 gene, and what does it do?

A

Cyclin A, B/ CDK-1
Transition to M phase

139
Q

How do fission yeast compare with budding yeast (bakers)?

A

Fission grow in length, along rod
Reach a critical size before entering M phase
Length correlates to position in cycle
Divide symmetrically

140
Q

What mutant can be present in fission yeast and what is its effect?

A

wee1- shorter
Inhibits cdc2, so can divide at smaller lengths

141
Q

What causes irreversibility in the eukarytoic cell cycle?

A

Ubiquitin proteolysis- breakdown of enzymes
Ubiquitin transferred, then becomes target of proteasomes

142
Q

How does ubiquitination occur?

A

1) E1= ubiquitin activation
2) E2= ubiquitin conjugating
3) E3= ubiquitin ligase
Transfers ubiquitin onto target

143
Q

What does SCF do?

A

Version of E3 protein
Contains 4 subunits: Skp1, Cul1,Rbx1 and F-box
Targets CDK inhibitors, so leads to cell cycle progression

144
Q

What is APC/C?

A

Promotes Metaphase to anaphase
Type of E3 ligase
Causes degradation of SECURIN and cyclin B1
So chromosomes can split (via SEPARASES) and enter anaphase

145
Q

Describe the Spindle assembly checkpoint (SAC)

A

Check all chromosomes and spindle fibres are attached
1) Unattached kinetochores, relay signal to stop breakdown of securin
2) Tension- Phosphorylation of kinase Aurora B to destabilise kinetochore attachment

146
Q

What are the structures of a viroin in detail?

A

Protein capsid- rodshaped, or icosahedron, may also contain host envelope
viral genome- ss or ds, RNA or DNA

147
Q

How does genome from HIV get turned into mRNA?

A

Retrovirus so ssRNA to ssDNA (reverse transcriptase)
Then ssDNA to dsDNA
then dsDNA to mRNA

148
Q

Describe the lytic pathway of the bacteriophage T4

A

adsorption onto host membrane
Insert genome
T+T of genome
Assembly of virus
Many lead to lysis

149
Q

What do RNA viruses require?

A

RNA dependent RNA polymerase to form mRNA
or reverse transcriptase (retrovirus)

150
Q

What are 4 examples of viral takeovers?

A

Polio- shuts down host transcription
SARS- can mutate spike protein, not get caught
HIV- retrovirus, integrates its genome into host
Influenza A- cap snatching from host genome

151
Q

Describe the process of HIV genome integration

A

Bind to CD4 receptors on T helper cells
Insert, reverse transcription of ssRNA
Integrate DNA into the host, Integrase
T+T of polyprotein
polyprotein cleaved by protease to yield many proteins, economical to have many genes on genome

152
Q

How can re-assortment lead to a pandemic?

A

Mix between viruses from different animals, lead to a harder virus to beat
e.g. Spanish flu reassortment of human and bird genome

153
Q

Describe the HPV mechanism

A

Human Papillomavirus
Infects epithelial cells, and can lead to cancer
Codes for E6 and E7 (Ubiquitin ligases)
Inhibit p53 and Rb (retinoblastoma)
p53- response to DNA damage
Rb- inhibits progression of cell cycle

154
Q

What is p53?

A

Tumour surpressing gene
Usually in low conc as its broken down by ubiquitin-dependent pathway
If damage is severe p53 can cause apoptosis

155
Q

What is SRC?

A

First oncogene discovered
non-receptor tyrosine kinase
Phosphorylates tyrosine on a protein, either leading to activation or inhibition

156
Q

What is an oncogene?

A

Gene that has potential to cause cancer
Normally promote cell division and growth

157
Q

What is the mutation in Chronic Myeloid leukaemia (CML)?

A

Philadelphia chromosome= translocation between chromosome 9 and 22

158
Q

How is a transfection assay carried out?

A

Infect healthy cell with disease DNA
Infect commonly by using a viral vector
If oncogene is present, phenotype will show

159
Q

What is the result of the transfection assay of normal mouse cell and human tumour cell?

A

Ras gene turned oncogene
Hyperactive
Continually switched on due to mutation in RasGTPase
Leading to cell proliferation

160
Q

What is the role of retinoblastoma (Rb)?

A

Stop progression from G1 to S phase
Bind to E2F (an Sphase TF)
Stop progression of cell cycle
Also acts as substrate for G1/S- CDK

161
Q

What does genetic instability mean?

A

Increased rate of point mutations

162
Q

What does chromosomal instability mean?

A

Tendency for cells to lose, gain or rearrange chromosomes

163
Q

How can apoptosis be detected?

A

1) Western blotting- Presence of caspases as proteases, ready for the breakdown
2) Changes in membrane composition, can be detected by adding fluorescent markers to specific proteins and monitoring change in fluorescence using a fluorescence microscope
3) Morphological changes- apoptotic cells begin to shrink

164
Q

What are restriction endonucleases and why are they produced by bacteria? Explain briefly how they have been exploited in the biosciences

A

Produced in bacteria to cleave out virus DNA
Exploit:
DNA analysis- restriction enzyme, create fragments to analyse
DNA manipulation- cleave/ insert DNA in specific places

165
Q

Cite two pieces of experimental evidence, obtained after the rediscovery of Mendel’s work, contradicting Mendel’s assertion that reciprocal crosses give the same result.

A

1902= T.H Morgan, sex linked gene for eye colour on X chromosome. 2 different crosses with w male r female and vice versa
1920s- Carl Correns, cytoplasmic inheritance (mitochondria/ chloroplasts) Mirabilis plant colour instead of 3:1 showed 9:7 pink:white

166
Q

How does DNA exemplify the properties required of any molecule used to store genetic information in a biological system?

A

1) Stable- stable molecule, secure way to store
2) Replication- can be well replicated
3) Mutation- can be mutated and changed
4) Compact store 2.2m into 5-20micrometre diameter nucleus
5) Info retrieval by conversion to RNA then proteins

167
Q

What are the properties of the transition-state complex of an enzyme-catalysed reaction?

A

1) neither a product or reactant
2) Induced fit in the enzyme, specific shape
3) High energy intermediate, highest energy point in reaction
4) Short lifespan

168
Q

Briefly describe why fatty acids provide more energy per mole than glucose under oxidative conditions.

A

-C in fat is more reduced than in glucose
- Produces more reducing equivalents, via beta oxidation
-So provide more energy
- Glucose has equal C=O, however fatty acids have more C to O
- Higher C:O in fatty acids, means more C for respiration
-Beta oxidation produces more acetyl CoA than the amount of acetyl CoA produced by glycolysis

169
Q

Explain how experiments performed by Gregor Mendel refute each of the following assertions:
(i) in reproduction the male parent provides the information and the female the
raw materials (Aristotle),
(ii) reproduction involves the irreversible mixing of characters from the two
parents (Hippocrates).

A

i) female homozygous dominant crossed with male homozygous recessive, offspring showed dominant phenotypes, meaning genetic info not all from male

ii) Having two distinct colours, green and yellow peas

170
Q

What has changed in genome sequencing technologies since the human genome sequence was published? How is this impacting on the understanding of human biology?

A

1) Cost reduction- more efficient so cheaper, now used in a variety of labs and in basic research
2) High throughput and speed- generate lots of data in a small amount of time, e.g. PacBio and Illumina technology
3) Longer reads- Sanger sequencing only allowed short reads of DNA, but with new technology longer sequences can be read
4) Epigenomic profiling- map genetic markers, and locate areas where gene expression is controlled

171
Q

How do viruses suppress the translation of host cell mRNAs and enable viral mRNAs to be translated instead?

A

1) Interfere with signalling for translation- inhibit, or activate mTOR pathway (regulates mRNA translation)
2) Interfere with ribosomes
3) Interfere with translation initiation factors
4) IRES (internal ribosome entry sites)- to synthesise their own, even when host can’t translate their own mRNA

172
Q

In protein structure, why are β -sheets made of antiparallel β -strands more stable than those of parallel strands? Why therefore is a βαβ super-secondary structure common?

A

In antiparallel the H bonds are shorter and line up better, so are stronger
βαβ common, as β is for rigidity and α is for flexibility, due to its helix nature

173
Q

When mapping two genes that are far apart on the same Drosophila chromosome, the maximum recombination frequency is 50%. With the aid of
diagrams explain why this is the case

A

Mapped using recombination frequency
Recombination can occur, max in half of the gametes produced
So max 50 centimorgans distance, if they’re on the same chromosome
More than 50% would suggest they aren’t on the same chromosome so aren’t linked
Less than 50% would suggest they are linked

174
Q

Briefly discuss why the conversion of glucose to glucose 6-phosphate is an important regulatory step in glycolysis

A

1) Leads to glucose trapping in a specific cell- where it will then go through glycolysis, or glycogen synthesis
2) Conversion, commits the molecule to glycolysis
3) G-6-P can also act as an allosteric inhibitor of hexokinase in muscles
4) G-6-P also used for other metabolic reactions, pentose phosphate pathway and glycogen synthesis

175
Q

How does a eukaryotic cell pack 2metres of DNA into a nucleus?

A
  • Wind DNA around positively charged histone proteins (octamer 2x (H2A, H2B, H3, H4)
    Metaphase is when chromatin condensed most tightly
    Form chromatin and chromosomes become ‘visible’
176
Q

What is the topological problem with DNA replication? How is this problem solved by topoisomerases?

A

Problem is the helicase when unwinding the DNA strand increases torsional stress
If it continued it would break the strand
Topoisomerases breaks loops and re-binds sections to reduce the torsional stress

177
Q

What is the structure of a nucleosome?

A

DNA wrapped around 8 histone proteins
H2A, H2B, H3 and H4
H1 acts as a clamp
Histone have Arg and Lys to be +ve and attract the -ve DNA

178
Q

Distinguish between the mode of action of Type I and type II Topoisomerases

A

Type I cleaves phosphodiester bond in one strand, Type II both strands cleaved
Type I doesn’t require ATP, Type II does
In prokaryotes Type I changes linking in circular DNA by 1 nucleotide and type 2 by 2 units

179
Q

Distinguish between the roles of DNA polymerase alpha and DNA polymerase delta in eukaryotes

A

Polymerase alpha synthesises the primer
Polymerase delta synthesises the lagging strand
Polymerase alpha acts on both leading and lagging strand
Polymerase delta replace Pol alpha as it’s faster due to clamp PCNA

180
Q

How does RNA differ from DNA? How do these properties contribute to their suitability as short or long term information storage?

A

DNA lacks OH at C2 only has H
DNA prefers double strand, making it harder to be accessible and so a long-term info storage
RNA opposite, single strand, easy to access
RNA has Uracil instead of thymine, Thymine has extra methyl so more resistant to modification

181
Q

Mammalian blood pressure is regulated by nitric oxide. Describe two mechanisms that lead to the formation of nitric oxide in endothelial cells

A
  • NOS (nitric oxide synthase) located on the endothelium
  • Nitrate- Nitrite- Nitric oxide pathway
  • Shear stress- can cause a cascade of reactions and activation of eNOS
182
Q

How can modern genome sequencing be useful in the analysis of pathogens?

A

1) Identify the pathogen, by comparing genome of pathogen to known genomes
2) Help understand evolution of the pathogen
3) Vaccine development
4) Analyse changes to pathogen e.g. different variant of SARS-Cov-2

183
Q

What is the function of a ribosome? How could the ability for a single RNA molecule to bind to several ribosomes be demonstrated?

A

Ribosome- carries out translation. Turns mRNA code into an amino acid sequence which folds into a protein
Polysome profiling used to detect several
Cross-linker used to link DNA and protein, before DNA shearing
Lysate layered on to sucrose density gradient
Centrifuged
Gradient which separates by size, can see how many proteins, size etc.

184
Q

How does p53 function as a tumour suppressor?

A

Triggered when DNA damage occurs
Stimulates apoptosis and cell death
Increase in PUMA and so increase in BAX
Cytochrome C released, binds to Apaf-1 causing activation of caspases cleaving proteins

185
Q

Explain what you could use a sucrose density gradient for, giving an example

A
  • Use to separate molecules based on density
  • Can be used to identify polysomes (multiple ribosomes bind to the mRNA strand)
    1) Cross link protein and strand
    2) Lyse cell
    3) Load onto the gradient
    4) Centrifuge to separate
    5) Analyse the fractions by enzyme assays or Western blotting
186
Q

How and why do proline and glycine differ from other amino acids in a Ramachandran plot?

A

Glycine has just H- so very flexible
- found everywhere on the Ramachandran plot
Proline- R group forms a cyclical chain
- Restricted flexibility so only found in LH helix region

187
Q

Suggest an experiment that could be used to detect lateral mobility of an integral protein in the cell membrane of a eukaryotic cell

A

FRAP (fuorescent recovery after photobleaching)
Label the integral protein with GFP
Photobleach the area
Monitor the recovery of fluorescence
Faster the recovery, the faster the movement of the proteins

188
Q

Briefly describe the major differences between photosynthesis and respiration

A

Photosynthesis takes up CO2, respiration gives out CO2
Photosynthesis makes glucose, respiration breaks down glucose to make energy. Anabolic v Catabolic
Photosynthesis is not present in humans only plants, however respiration is present in both
Photosynthesis involves NADPH electron carrier, however respiration used NADH

189
Q

Discuss the statement that “in mammals glucose cannot be synthesised from fat “

A

Fatty acids cannot be truned into glucose via gluconeogenesis
Fatty acids can be broken down to acetyl CoA and involved in Krebs Cycle to produce ATP, however no enzyme to use Acetyl CoA into oxaloacetate to make glucose

190
Q

Give TWO examples of how the study of bacteriophages contributed to our understanding of genetics in the second half of the twentieth century

A

Discovery of DNA as genetic material (Hershey- Chase)
- radioactive P and S included into phages, progeny had radioactive phosphate present because it was passed on, however sulfur (proteins) was not
Genetic recombination
- resistance to phage by bacteria causing mutations in the genome
- CRISPR in bacterial response to phages, or the act of integration of DNA via integrase by the phage

191
Q

How are hybridisation techniques used in the analysis of nucleic acids?

A

Southern blotting (DNA) Northern blotting (RNA)
DNA fragmented by restriction enzyme
Transferred onto a nylon membrane
Exposed to DNA probe (attached to dye)
If complementary sequences present, then will bind and fluoresce

192
Q

How is entry into mitosis regulated in the eukaryotic cell?

A

G2 phase
Controlled by CDK and cyclins
Cyclin builds up, activating CDK
CDK activates MPF
MPF causes nuclear envelope breakdown and spindle fibre assembly

193
Q

How do genomes of animal and plant viruses encode enough information to make virus particles and damage the host cell?

A

-Genomes (RNA/ DNA) are multifunctional
-Code for multiple proteins to regulate gene expression and impact immune response
-Small genome so less likely to be recognised
-Ability to hijack host cell machinery
- Viral evolution, high rates of replication so continually changing e.g. SARS-Cov2 changing spike protein

194
Q

Describe how to construct a genomic library of DNA from a higher eukaryote in bacteriophage lambda

A

1) Isolate “foreign” DNA and digest by partial digestion
2) isolate the 15kb fragments
3) Digest lambda DNA (with BamH1) and discard 15kb region
4) Lysate the two ends and 15kb together
5) Use spontaneous in vitro packaging
6) Infect a lawn of E.coli with the new phage
7) Store the plaques in large number

195
Q

Name the cyclin/ CDK complexes involved in cell cycle progression in humans and state which stage they control

A

Commitment to cycle= Cyclin D/ CDK4,6
Entry into S= Cyclin E/ CDK2
Entry into M = Cyclin B/ CDK1

196
Q

What is quorum sensing?

A
  • stimuli and response correlated to population density
  • phenotype is only expressed at critical density
  • In Vibrio fischeri when critical density is reached OHHL binds to LuxR
  • LuxR activates LuxI (more OHHL) and LuxAB (light emission)
197
Q

Briefly describe an experiment that would address whether or not information, in the form of DNA, is lost as an embryo develops

A

1) Take a nucleus from a differentiated cell
2) Insert into enucleated egg cell
3) Grow cells
4) See that a whole organism can form
- Information is not lost
Experiments by John Gurdon

198
Q

By reference to the results of Mendel’s single factor crosses, justify his assertion that adult plants carry two copies of each gene

A

Start with homozygous parents
F1 will show only 1 phenotype, and all be heterozygous
F2 how recessive:phenotype, 1:3
Showing that two copies are carried because the recessive is kept and this can be seen in the F2 generation

199
Q

What are restriction endonucleases and why are they produced by bacteria? Explain briefly how they have been exploited in the biosciences

A

Enzymes that makes specific double stranded cuts
To protect from bacteriophage DNA invasion
Protects own DNA by methylating it
Exploited (CRIPSR), for specific genetic modification

200
Q

Cite two pieces of evidence obtained after the rediscovery of Mendel’s work contradicting his assertion that reciprocal crosses give the same result

A

Cytoplasmic (chloroplast) inheritance (Carl Correns)
Mitochondrial inheritance in petite mutants of budding yeast
Sex linkage (Morgan) working on Drosophila

201
Q

What are microarrays ,and how can they be used to improve our understanding of biology and disease?

A
  • thousands of DNA sequences printed by a robot
    -reverse transcribe RNA and attach to fluorescent label
  • Hybridise
  • Scan and quantify gene expression
202
Q

What are the main differences in the transmission of human dominant and recessive mutations that are both X-linked?

A

Dominant mutation will show in both females and males
Frequently less severe in females than males
Recessive mutation, less likely to show in females than in males
In females only homozygous recessive will show the phenotype
However if males receive an X chromosome with the recessive mutation, they will express the mutated phenotype

203
Q

Describe 3 types of genetic alterations that can create oncogenes. Give an example of each alteration

A

Oncogene- mutated gene that promotes cancer development
1) Specific non synonymous point mutations
2) Amplifications - increased level of expression e.g. cyclin D (commitment to cell cycle)
3) Translocation- fusion of genes (Chromosome 9 and 12 to make philadelphia chromosome

204
Q

Petite mutants in yeast can be nuclear or cytoplasmic. What pattern of inheritance would you expect in the case of (i) nuclear and (ii) cytoplasmic mutations?

A

i) nuclear would expect Mendelian inheritance, both parents need to have at least 1 recessive allele for the offspring to express the phenotype
ii) cytoplasmic doesn’t show Mendelian inheritance, inheritance is through the mother. So if the mother has the mutant then it may be passed on to the offspring, however if the father has the allele it won’t be passed on

205
Q

Describe the role of sigma factors in promoter recognition in prokaryotes

A

1) Aids binding to promoter region
2) Helps formation of a closed complex around the double helix
3) Helps initiate transcription
sigma-70 for transcription of growth genes
sigma- 32 for heat shock proteins

206
Q

Explain how DNA fingerprinting works

A

1) DNA collected
2) Amplified using PCR
3) Analysed using gel electrophoresis
- can then be compared against other sources of DNA and used in forensics for example
- can also be used to look at familial connections

207
Q

What experiments would you do to test for cytoplasmic inheritance?

A

1) Track inheritance of phenotype between generations
If cytoplasmic then the allele must be passed on by the mother and not the father
2) Fusion of cytoplasm, if the traits change then may suggest cytoplasmic inheritance

208
Q

Giving examples, describe the central role of Ca2+ in cell communication

A

1) Causes the contraction of muscles when released into the cytosol from the SR. Ca2+ binds to troponin so that actin-myosin cross-bridges can form
2) Neuronal signalling, presence of AP leads to influx of Ca2+ which binds to synaptotagmin to allow the exocytosis of vesicles containing neurotransmitter
3) Intracellular signalling, elevated concentrations of Ca2+ can cause the activation of kinases and TFs
4) Gap junctions- can involve the movement of Ca2+ across these junctions to pass on signal

209
Q

What is a restriction fragment length polymorphism (RFLP)? Briefly describe
two applications of RFLPs in human genetics.

A

RFLPs- difference in homologous DNA sequences due to presence of fragments of different lengths after digestion by specific endonucleases
1) Forensic DNA analysis compare DNA at crime scene and suspect
2) Paternity testing
3) Genetic mapping, looking at disorders and what genes cause it

210
Q

What strategies are used for replication of viral RNA?

A

1) Host machinery
- integration of viral RNA into the hosts DNA by integrase e.g. in HIV which targets T-helper cells
2) RNA dependent RNA polymerases
- encoded in its own viral RNA

211
Q

What are the elements of an intercellular signalling pathway? Illustrate your answer with 3 examples

A

1) Amplification of signal
- production of secondary messengers e.g. cAMP
2) Signalling molecules (ECF)
- hormones for example, that can travel long distances e.g. adrenaline from adrenal gland
3) Receptors
- receptors on cell membrane to detect signalling e.g. GPCRs, adrenergic receptors
4) Intracellular signalling molecules
- molecules produced due to detection of signal, eg. GsPCR activates cAMP pathway

212
Q

What are the steps that may occur in the conversion of a normal cell to a tumour cell?

A

Initiated by mutations in genes
Oncogenes- mutated genes that have the potential to cause tumours
Mutation of tumour supressor genes, leading to rapid cell division and lack of control of the cell cycle e.g. mutations in p53
Angiogenesis- where the tumour forms vessels to get a blood supply to stay alive

213
Q

Name and briefly contrast two alternative cycles of viral replication

A

Lytic v lysogenic
Lytic burst the cell due to may virions, lysogenic will eventually become lytic
Retrovirus- HIV, CD4 on T- helper cells. Reverse transcriptase to get DNA, integrase to integrate into host’s DNA
HPV- direct insertion of viral DNA into hosts DNA, uncouples differentiation from proliferation = tumour, viral protein transcribed by host

214
Q

Explain the principles of the screen for cell division cycle (cdc) mutations in yeast. Provide two examples of classes of proteins that were identified in this screen.

A

Cdc control the movement from on phase to the next in the cell cycle
Budding yeast = cdc28 progression from G1 to S phase and G2 to M phase
Cdc2 entry into M phase, can be inhibited by Wee1

215
Q

Name the cyclin/CDK complexes involved in cell cycle progression in humans and state which stage they control

A

Cyclin D/ CDK4,CDK6= G1 to S phase
Cyclin E/ CDK2 = DNA replication and G1 to S phase
Cyclin A/ CDK1 = G2 to M phase
Cyclin B/ CDK1 = entry into M

216
Q

What is the role of a telomerase?

A

Combat the end replication problem by:
- Adding DNA to the end of the chromosome to reduce the amount loss on the lagging strand
- Reducing chromosome shortening
- Achieved by having an intrinsic RNA template and a reverse transcriptase

217
Q

What are the three main RNA processing steps that are needed to generate mRNA from RNA Polymerase II primary transcripts, and why are they needed?

A

1) 5’ capping - protection and recognition by ribosome
2) Splicing- removal of non coding introns
3) Poly(A) tail- increase stability, help export and efficient translation

218
Q

Discuss the contribution of studies in model organisms for understanding fundamental controls of the human cell cycle

A

Yeast used to to identify CDKs and presence of cyclins
C.elegans- research how mutations can lead to changes in cell cycle regulation
Model organisms used to identify conservation of cell cycle regulators

219
Q

Compare and contrast SCF and APC

A
  • Both ubiquitin ligases
    -Add ubiquitin and cause the degradation of a protein
  • Both large multisubunit proteins
  • APC larger than SCF
  • SCF does G1/S, APC does Metaphase to anaphase
  • SCF activate by Pi, APC activated by activator subunits
220
Q

What are the differences between the genomic DNA library and the cDNA library?

A

cDNA- only shows expressed sequences, so libraries from different tissues in an organism are different
Genomic- shows introns and exons, shows all the genetic material for mapping of coding and non-coding

221
Q

Distinguish between the direct, excision, and mismatch systems for DNA repair.

A

Direct repair acts directly on the damaged DNA however excision and mismatch excise the segment and replace it with the correct sequence.
- Mismatch is a type of excision repair, which deals with replication mistakes
Base excision- change of a single base, by action of glycosylases, exonucleases and polymerases
Nucleotide excision- changes due to UV e.g. pyrimidine

BoC (4 decks)

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