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BMS 109 > Molecular Biology > Flashcards

Molecular Biology Flashcards

1
Q

Characteristics of Genetic Material (3 things)

A
  • Codes for all info necessary for generating an organism
  • Faithfully replicated to be passed on to progeny
  • Regulated decoding to suit organisms development and its environment
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2
Q

Hershey-Chase Experiment

A

Identified DNA as the genetic material

  • Used bacteriophages to infect bacteria to see if they passed on DNA or proteins as genetic material
  • Used radioactively labelled Phosphate in DNA and Sulphate in proteins
  • Found that progeny have radioactive Phosphate and therefore DNA must be genetic material
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3
Q

3 chemical components of DNA

A
  • Sugar
  • Nitrogenous base
  • Phosphate group
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4
Q

Nucleotide structure in DNA

A
  • Sugar is a pentose - Deoxyribose
  • Phosphate group attached to C5
  • Nitrogenous base attached to C1
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5
Q

Nucleotide structure in RNA

A
  • Sugar is a pentose - Ribose
  • Phosphate group attached to C5
  • Nitrogenous base attached to C1
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6
Q

Purines

A
  • 2 rings

- Adenine and Guanine

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7
Q

Pyrimidine

A
  • 1 ring
  • Cytosine
  • Thymine
  • Uracil
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8
Q

Hydrogen bonds between Adenine and Guanine

A

2 hydrogen bonds

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9
Q

Hydrogen bonds between Cytosine and Thymine (Uracil in RNA)

A

3 hydrogen bonds

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10
Q

Polynucleotide chain

A
  • Made by joining the sugar of one nucleotide with the phosphate of another
  • Forms the sugar phosphate backbone with nitrogenous bases facing inwards
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11
Q

5 prime end of polynucleotide chain

A

Phosphate group on C5

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12
Q

3 prime end of polynucleotide chain

A

Hydroxyl group on C3

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13
Q

Structure of DNA

A
  • Right-handed Double helix

- Sugar-Phosphate backbone with nitrogenous bases facing inwards

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14
Q

Complimentary base pairing

A
  • Nitrogenous bases of opposite strands are complimentary and bonded by hydrogen bonds
  • Sequence of one strand dictates the sequence of the other
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15
Q

DNA strands are antiparallel

A

They have opposite polarity so one strand is 5’ to 3’ and the other is 3’ to 5’

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16
Q

Semi-conservative replication

A
  • DNA replication is semi-conservative

- Each new double helix consists of one parental strand and one newly synthesised strand

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17
Q

DNA synthesis initiated by…

What enzyme is responsible for this?

A

Creation of replication fork where 2 parental strands are separated by the enzyme DNA Helicase

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18
Q

What enzyme synthesises new strands of DNA?

A

DNA Polymerase

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19
Q

What direction does DNA synthesis only occur in?

A

5’ to 3’ direction

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20
Q

Antiparallel orientation of parental strands means that DNA synthesis cannot..

A

Be continuous in both strands

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21
Q

DNA daughter strand that is synthesised continuously is called the..

A

The leading strand

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22
Q

DNA daughter strand that is synthesised discontinuously is called the..

A

The lagging strand

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23
Q

How does DNA synthesis occur in the leading strand?

A
  • 1 RNA primer joins to template and is extended by DNA polymerase using free nucleotides in a 5’ to 3’ direction
  • Ribonuclease H removes RNA primer and DNA polymerase fills in the gap with DNA nucleotides
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24
Q

How does DNA synthesis occur in the lagging strand?

A
  • Lagging strand synthesised in short Okazaki fragments that each require their own RNA primer
  • Ribonuclease H removes RNA primers and DNA polymerase fills in the gap with DNA nucleotides
  • Okazaki fragments are then covalently linked by DNA Ligase
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25
Q

Which enzyme produces RNA primers using RNA nucleotides?

A

DNA Primase

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26
Q

Single stranded binding proteins (SSBPs)?

A

Maintain unwound parental DNA strands in a single stranded conformation to ease replication fork progression

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27
Q

2 major steps of gene expression

A

Transcription and Translation

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28
Q

How does DNA encode biological form and function?

A

Different genes are selectively expressed in different cell types i.e. they can be switched on or off depending on if they are required

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29
Q

Process of transcription of prokaryotic gene

A
  • Transcription initiated by binding of RNA polymerase to promotor regions in gene
  • RNA polymerase separates and reads DNA to produce messenger RNA molecule which is a copy of DNA strand - Transcription terminated when RNA polymerase reaches terminator region which promotes dissociation of RNA polymerase
  • Transcription rate is limited by frequency of transcription initiation
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30
Q

What direction does transcription occur in?

A

5’ to 3’ direction

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31
Q

Process of transcription of eukaryotic gene

A
  • Transcription initiated by binding of RNA polymerase to promotor regions in gene
  • RNA polymerase separates and reads DNA to produce messenger RNA molecule which is a copy of DNA strand - Transcription terminated when RNA polymerase reaches terminator region which promotes dissociation of RNA polymerase
  • Primary mRNA molecules contain both exons (coding DNA) and introns (non-coding DNA) and introns are spliced out and do not appear in mature mRNA
  • Ends of mRNA are modified by capping 5’ end and polyadenylation of 3’ end
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32
Q

What removes introns from immature mRNA?

A

Splisosome removes introns by identifying recognition sequences in exons

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33
Q

Genome

A

The entire DNA sequence of its chromosomes

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34
Q

Transcriptome and Proteome

A
  • Transcriptome the entire total of mRNA expressed from genes
  • Proteome the entire total of proteins expressed in a cell
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35
Q

Human genome consists of linear chromosomes..

A

22 autosomes and X and Y sex chromosomes

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36
Q

Diploid cells

A

Have 2 copies of each autosome and either XX or XY sex chromosomes - 46 chromosomes in total

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37
Q

3 main components of Chromosome structure

A
  • Centromere
  • Replication Origins
  • Telomere
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38
Q

Centromeres

A

Repetitive DNA sequence that allows chromosome attachment to mitotic spindle via the kinetochore

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39
Q

Origins of Replication

A
  • Located within DNA molecules and create a replication bubble with 2 forks that move away from each other
  • Eukaryotic cells have many origins of replication to allow rapid replication of chromosomes
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40
Q

Telomeres

A
  • Repetitive DNA sequences at the ends of linear chromosomes
  • Maintained by enzyme Telomerase
  • Must be protected in order to prevent cell trying to repair them
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41
Q

DNA Replication in E. Coli

A
  • E. Coli has a single circular chromosome
  • Replicated in a bidirectional manner
  • Begins at a single replication origin and 2 replication forks proceed in opposite directions to produce 2 daughter circular chromosome
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42
Q

Genome sizes are…

A

Larger in more complex organisms

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43
Q

Gene numbers tend to increase as…

A

Organism complexity increases

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44
Q

Genome size increases as…

A

Number of genes increases

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45
Q

Gene density decreases as…

A

Organism complexity increases

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46
Q

Exons and Introns in more complex organisms…

A
  • Exons are similar sizes

- Introns are larger

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47
Q

Genes are bigger in more complex organisms because…

A

They have larger introns

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48
Q

Why do genome sizes and intron sizes increase in more complex organisms?

A
  • Larger genomes have more repetitive DNA elements (transposons and retrotransposons)
  • Larger genomes have more complex transcriptional control elements spread over large regions of DNA
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49
Q

Genotype

A

Genetic makeup of an organism

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50
Q

Phenotype

A

Observable characteristics of an organism

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51
Q

Genotype-Phenotype relationship

A

Phenotype is the result of interaction of the genotype with the environment

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52
Q

Dominant phenotype

A

Phenotype of a genotype containing the dominant allele, expressed in the heterozygote

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53
Q

Recessive phenotype

A

Phenotype of a genotype containing the recessive allele, expressed in the homozygote only

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54
Q

Mendel’s First experiment

A
  • Cross-bred 2 different true-breeding pea plants
  • Crossing a smooth seeded plant with a wrinkled seeded plant yielded all smooth seeds in F1 progeny
  • When F1 progeny self-fertilised the wrinkled trait re-emerged in F2 progeny and the smooth:wrinkled ratio was 3:1
  • Mendel concluded each gene exists in 2 different forms (alleles) and each contributes one allele to its progeny
  • These can be dominant (present in homozygote or heterozygote) or recessive (only present in homozygote)
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55
Q

Mendel’s First Law

A

Principle of Segregation - 2 alleles of a gene segregate from each other during gamete formation

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56
Q

Mendel’s Second Experiment

A
  • Crossed plants breeding true for 2 alleles of 2 different traits (smooth/wrinkled and green/yellow) to see if alleles of 2 genes move into gametes together
  • Found that crossing smooth yellow seeded plants with green wrinkled seeded pants produced 4 phenotypes in equal proportion:
    1. Smooth yellow
    2. Smooth green
    3. Wrinkled yellow
    4. Wrinkled green
  • Mendel concluded that alleles for different genes segregate into gametes independently
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57
Q

Mendel’s Second Law

A

Principle of Independent Assortment - genes controlling different characteristics assort into gametes independently of one another

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58
Q

Incomplete dominance

A

Heterozygote phenotype is intermediate between two homozygous (parental) phenotypes e.g. RR x rr = Rr

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59
Q

Co-dominance

A

Simultaneous expression of two phenotypes determined by alternative alleles of a single gene e.g. ABO blood groups

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60
Q

Morgan’s Experiment

A
  • Crossed grey-normal winged (BBVV) Drosophila with black-vestigial winged (bbvv) and got all grey-normal winged (BbVv) in F1 progeny
  • Back crossed F1 progeny (BbVv) with black-vestigial winged (bbvv) parent and expected to see 4 phenotypes in equal proportions
  • Instead he observed more flies in parental phenotypes (BbVv and bbvv) than non-parental phenotypes (bbVv and Bbvv)
  • Morgan concluded this was because genes were linked i.e. on the same chromosome
  • Existence of non-parental phenotypes is explained if cross-over occurs between chromosomes during formation of some gametes - genetic recombination
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61
Q

Genetic linkage

A

The tendency of genes that are close together on a chromosome to be inherited together during gamete formation

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62
Q

Genetic recombination

A

The process of forming new allelic combination in gametes by exchanging genetic material between chromosomes

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63
Q

Genetic screens

A
  • Identify mutations that change gene functions

- Most mutations identified in genetic screens inactivate gene function and cause recessive phenotypes

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64
Q

Applications of classical genetic analysis

A
  • Creating new genetic variation, with chemicals or radiation that alter DNA sequences randomly - induced mutations
  • Engineering defined changes into genomes - removing gene sequences or adding new gene sequences
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65
Q

3 techniques for engineering mutations in specific genes?

A
  1. Gene replacement
  2. Gene knockout
  3. Gene addition
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66
Q

Genetic complementation test tell us…

A

Whether two mutations with similar phenotypes lie in the same or different gene

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67
Q

3 major genes required for larval segment formation and patterning?

A
  • Gap gene
  • Pair-rule gene
  • Segment polarity gene
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68
Q

6 essentials of genetic code?

A
  1. A triplet code made up of codons
  2. Is non-overlapping
  3. Is universal
  4. Is degenerate
  5. Has start and stop signals
  6. Accommodates some mismatches
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69
Q

Start codon…

A

AUG - codes for methionine

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70
Q

Stop codons…

A
  • UAG
  • UGA
  • UAA
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71
Q

Genetic code said to be degenerate because…

A

Most amino acids are encoded by more than one codon

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72
Q

Amino acid attached by carboxyl group to ribose of last ribonucleatide on tRNA by…

A

Aminoacyl-tRNA synthetase

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73
Q

Each codon on mRNA has a corresponding..

A

Anticodon on a tRNA molecule

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74
Q

Wobble base pairing

A

A tRNA can recognise more than one codon, has a tolerance to mutations so if 3rd nucleotide of codon is mutated, tRNA can still bind to codon to prevent damage

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75
Q

3 sites of a ribosome?

A
  1. A - aminoacyl-tRNA
  2. P - peptidyl-tRNA
  3. E - exit
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76
Q

Process of protein translation from mRNA

A
  • tRNA binding of anticodon to codon on mRNA by complimentary base pairing
  • Peptide bond formation between amino acid on tRNA and growing peptide chain
  • Large subunit translocation
  • Small subunit translocation
  • Ribosome move along mRNA in a 5’ to 3’ direction one codon at a time
  • Empty tRNA exits and new tRNA is added until a stop codon is reached then ribosome disassembles
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77
Q

Which end of the growing polypeptide chain is the new amino acid attached to?

A

C terminus of growing polypeptide chain

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78
Q

Peptide bond

A
  • Condensation reaction catalysed by Peptidyl Transferase

- No rotation around C-N

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79
Q

End of translation

A

Binding of release factor to A site on ribosome when stop codon is reached caused disassembly of ribosome

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80
Q

Polyribosome

A

Complex of an mRNA molecule and two or more ribosomes that act to form polypeptide chains during active translation

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81
Q

Peptide backbone

A
  • Same for all proteins
  • Repeating amino acid units
  • H2N-C-C-N-C-C-N-C-COOH
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82
Q

4 types of amino acid?

A
  • Acidic - COOH side chains
  • Basic - NH2 side chains
  • Polar
  • Non-polar
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83
Q

Protein primary structure

A
  • Order of amino acids in polypeptide chain

- Side chains of amino acids drive the folding

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84
Q

Protein secondary structure

A

Folding of the primary chain into an alpha helix, beta sheet or random coil

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85
Q

Protein tertiary structure

A
  • The way in which the secondary structure packs together

- There can be sub domains which are folding within proteins with separate functions

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86
Q

Protein quaternary structure

A

Relationship between proteins in a multimeric complex e.g. Haemoglobin

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87
Q

If mutations lie in the same gene they are…

A

Non-complementary

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88
Q

If mutations lie in a different gene they are…

A

Complementary

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89
Q

Folding of protein in aqueous environment?

A

Protein folds to put hydrophobic non-polar side chains on inside and puts hydrophilic polar side chains on outside

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90
Q

3 types of attraction in secondary structure?

A
  • Electrostatic between +ve and -ve charged ions
  • van der Waals attractive or repulsive between molecules
  • Hydrogen bonds between C and N atoms
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91
Q

3 main secondary structures of proteins?

A
  • Alpha helix
  • Beta sheet
  • Random coil
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92
Q

Alpha helix (intrachain) and Beta sheet (interchain) formed by…

A

Hydrogen bonding

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93
Q

Protein domains

A

Distinct functional/structural units in a protein tertiary structure that are responsible for their own function

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94
Q

3 types of protein-protein interaction?

A
  • Domain-loop interaction
  • 2 alpha helices to form a coiled coil
  • Domain-domain interaction
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95
Q

Coiled coil structures are driven by…

A

Hydrophobic interactions between amino acids in 2 proteins

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96
Q

5 post-translational protein modifications?

A
  • Disulphide bonds
  • Proteolysis
  • Glycosylation
  • Lipid modification
  • Phosphorylation
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97
Q

Disulphide bonds in proteins form between…

A
  • Cysteine amino acids that have sulphide side groups

- Can be intrachain (same protein) or interchain (different protein)

98
Q

Disulphide bonds provide…

A

Stability in proteins exposed to extreme environments e.g. antibodies and enzymes outside of the cell

99
Q

Proteolytic processing of a precursor protein?

A

A process where the active protein is post-translationally cleaved by proteases to yield a more functional molecule

100
Q

Glycosylation are principally a modification of…

A

Extracellular proteins

101
Q

Lipid modifications in proteins are used to…

A

Bind proteins to membranes

102
Q

Protein phosphorylation/dephosphorylation is used as a major…

A

Regulative modification

103
Q

4 structural types of transmembrane proteins?

A

Lipid membrane is hydrophobic, so parts of proteins that cross membrane must also be hydrophobic

  • Single pass
  • Multipass
  • Beta-barrell (pore)
  • Amphipathic helix
104
Q

Single pass type 1 membrane protein?

A
  • Protein spanning the membrane once, with its N-terminus on the extracellular side of the membrane and removal of its signal sequence
  • E.g. T cell surface glycoproteins
105
Q

Single pass type 2 membrane protein?

A
  • Protein spanning the membrane once, with its N-terminus on the cytoplasmic side of the membrane
  • Transmembrane domain located close to N-terminus and it functions as an anchor
  • E.g. Tumor Necrosis Factor
106
Q

Beta barrels contain different numbers of beta strands depending on…

A

Their function e.g. if they act as receptors have fewer strands but if they act as an ion transporter they are much larger and have may strands

107
Q

Peripheral membrane proteins attach to membrane via..

A
  • Lipid anchors

- Other transmembrane proteins

108
Q

What type of lipid modifications bind proteins to cytosolic side of membrane?

A
  • Acylation

- Prenylation

109
Q

What type of lipid modifications bind proteins to extracellular side of membrane?

A

Glycophosphatidylinositol (GPI) anchors

110
Q

Cycling of the GTPase RAS between membrane is regulated by…

A

Lipid modifications

  • Ras is modified in the Golgi (adding lipid anchor) and is transported to the membrane in secretory vesicles when its action is required
  • When it is no longer required the lipid anchor is cleaved and Has is removed from the membrane and transported back to the Golgi until it is require again
  • This cycle prevents Ras leaking to other membranes
111
Q

Differential lipid modifications allows targeting to…

A

Different membranes

112
Q

2 types of glycosylation modifications?

A
  • N-linked attachment via asparagine

- O-linked attachment via hydroxyl group of serine/threonine

113
Q

Glycosylation provides…

A
  • Cellular protection via mucins
  • Adhesive properties
  • Set of complex surfaces for interactions with other molecules
114
Q

Glycosylation forms the basis of…

A

Blood groups

115
Q

Phosphorylation occurs on…

A

Serine, Theorine and Tyrosine by converting a hydroxyl group to a charged phosphate group

116
Q

Phosphorylation acts as a switch for enzymes and activating them by…

A
  • Physically changing protein conformation

- Altering the nature of binding surface

117
Q

Enzymes that phosphorylate/dephosphorylate other proteins are…

A

Phosphorylate - kinases

Dephosphorylate - phosphatases

118
Q

Linear signal transduction…

A

Stimulus activates linear pathway of proteins to produce a single response

119
Q

Cascade signal transduction…

A

Stimulus activates protein that then activates several other proteins in a cascade to produce multiple responses

120
Q

GTP-binding proteins (G-porteins) act as switches by…

A
  • Active G-protein has GTP bound
  • Hydrolysis of GTP to GDP inactivates protein and the GDP molecule is slowly released
  • Binding of a second GTP reactivates protein and cycle continues
121
Q

Ubiquitination

A

Addition of ubiquitin chain onto target proteins catalysed by ubiquitin ligases

122
Q

Types of ubiquitination

A
  • Mono-ubiquitylation - histone regulation
  • Multi-ubiquitylation - endocytosis
  • Poly-ubiquitylation - proteasomal degradation
123
Q

Which transmembrane protein is affected in Cystic Fibrosis?

A

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

124
Q

Somatic mutations

A
  • Affect cells and tissues of the body

- Not passed onto progeny

125
Q

Germline mutations

A
  • Affect cells of reproductive tissues

- Passed onto progeny so progeny have mutations in germline AND somatic cells

126
Q

Gain of function mutations are…

A

Dominant traits

127
Q

Loss of function mutations are…

A

Recessive traits

128
Q

Loss of function mutations effects on proteins?

A
  • Complete loss of the protein - amorph

- Reduction of proteins function - hypomorph

129
Q

Gain of function mutations effects on proteins?

A
  • Increase in the proteins functions - hypermorph
  • Protein that interferes with wild-type proteins function - antimorph
  • Protein acquires new function - Neomorph
130
Q

Transition mutation

A

Changes a purine to purine (e.g. A to G) or a pyrimidine to pyrimidine (e.g. C to T)

131
Q

Transversion mutation

A

Changes a purine to pyrimidine (e.g. C to G) or a pyrimidine to purine (e.g. G to C)

132
Q

Missense mutation

A

Change from one amino acid to another amino acid (e.g. AAA to GAA changes codon from lysine to glutamic acid)

133
Q

Non-sense mutation

A

Change from an amino acid to a premature stop codon (e.g. AAA to TAA changes codon from lysine to UAA stop codon)

134
Q

Non-sense mediated decay

A

Cells detect mutated, incomplete protein and cause mRNA coding for incomplete protein to decay

135
Q

Neutral mutation

A

Change from one amino acid to another amino acid with similar chemical properties (e.g. AAA to AGA changes codon from lysine to arginine)

136
Q

Silent mutation

A

Change in codon but does not change the amino acid as code is degenerate (e.g. AAA to AAG gives a codon that still gives lysine)

137
Q

Frameshift mutation

A

Addition or deletion of one or two base pairs leads to a change in reading frame and changes polypeptide sequence completely

138
Q

Spontaneous mutation due to wobble base pairing

A
  • Mis-pairing of bases
  • T-G, C-A, A-G, T-C
  • Normal pairing typically occurs in the next round of replication; frequency of mutants in F2 is 1/4
139
Q

Spontaneous mutations due to looping out errors in DNA replication

A
  • DNA loops out on template strand, DNA polymerase skips bases, and deletion occurs
  • DNA loops out on new strand, DNA polymerase adds untemplated bases
140
Q

Spontaneous mutations due to depurination (chemical change)

A

Common; A or G are removed and replaced with a random base

141
Q

Spontaneous mutations due to deamination (chemical change)

A
  • Amino group is removed from Cytosine to produce Uracil

- If not replaced U pairs with A in next round of replication (CG to TA)

142
Q

Induced mutations due to radiation

A
  • Can cause breaks in covalent bonds in DNA resulting in point mutations
  • Can cause crosslinking of pyrimidines (especially T-T dimers) which results in DNA replication problems
143
Q

Induced mutations due to chemical intercalating agents

A

E.g. Ethidium Bromide

  • Can cause addition mutations by inserting in template strand resulting in addition of random base in new strand
  • Can cause deletion mutations by inserting in new DNA strand and then is lost in next replication
144
Q

Induced mutations due to chemical base modifiers

A
  • Alter the chemical structure of bases e.g. Alkylating agents that form permanent covalent bonds with nucleophilic substances in DNA
145
Q

Sickle Cell Anaemia

A

Recessive mutation in haemoglobin and when at low oxygen tension the RBC loses its concave disc shape and becomes sickle shaped

146
Q

Sickle cell anaemia caused by…

A

Single missense mutation that changes glutamate to valine

147
Q

Cystic Fibrosis carriers have a selective advantage of…

A

Resistance to infection by some infective agents

148
Q

Chromosome duplication mutation

A

Produced when extra copies of genes are generated on a chromosome

149
Q

Chromosome deletion mutation

A

Produced by the breakage of a chromosome in which the genetic material becomes lost during cell division

150
Q

Chromsome inversion mutation

A

Produced when a broken chromosome segment is reversed and inserted back into the chromosome

151
Q

Chromosome translocation mutation

A

Produced by the joining of a fragmented chromosome to a non-homologous chromosome

152
Q

Principle role of the cell cycle

A

To produce 2 genetically identical daughter cells

153
Q

Phases of the cell cycle

A
  • G0 - dormant non-dividing state
  • G1 Phase - environmental checking and cell growth
  • S Phase - DNA and centrosome replication
  • G2 Phase - more checks and cell growth
  • M Phase - nuclear and cytoplasmic division
154
Q

G1, S and G2

A

Interphase

155
Q

Stages of mitosis

A
  • End of G2 phase
  • Prophase
  • Prometaphase
  • Metaphase
  • Anaphase
  • Telophase
  • Cytokinesis
156
Q

End of G2 phase

A
  • DNA has been replicated

- Centrosomes have been duplicated

157
Q

Prophase

A
  • Chromosome condensation - DNA wraps around histones and then supercoils into chromosomes
  • Mitotic spindle assembles between 2 centrosomes at each pole
158
Q

Prometaphase

A
  • Nuclear envelope breaks down
  • Chromosomes attach to spindle microtubules by kinetochores
  • Movement of chromosomes begins
159
Q

3 classes of microtubules in mitotic spindle?

A
  • Astral MTs - help to position the spindle correctly
  • Interpolar MTs - help to stabilise the bipolar spindle
  • Kinetochore MTs - help separate the sister chromatid pairs
160
Q

Metaphase

A
  • Chromosomes aligned at equator of the spindle
  • Metaphase plate is imaginary structure midway between 2 spindle poles
  • Kinetochore MTs attach sister chromatids to opposite poles of the spindle
161
Q

Anaphase

A
  • Sister chromatids separate to from 2 daughter chromosomes

- Kinetochore MTs shorten by depolymerising and spindle poles move apart

162
Q

Telophase

A
  • 2 sets of daughter chromosomes arrive at spindle pole and decondense
  • New nuclear envelope forms around each set of chromosomes
163
Q

Cytokinesis

A

Cytoplasm of 2 daughter cells is separated by contractile ring to produce 2 identical daughter cells

164
Q

Cell cycle control is regulated by…

A

Cyclin-dependant kinases (CDK)

165
Q

What does CDK require for activity

A

Cyclin

166
Q

Is the level of cyclin constant throughout the cell cycle?

A

No

167
Q

Is the level of CDK constant throughout the cell cycle?

A

Yes

168
Q

What triggers entry into mitosis?

A

M-cyclin-CDK complexes that are activated by phosphorylation by phosphatases

169
Q

What regulates cyclin levels throughout mitosis?

A

Anaphase promoting complex

- When activated causes ubiquitylation of cyclin that tags it for degradation by proteasome

170
Q

Spindle assembly checkpoint of metaphase-anaphase transition

A

Proteolysis

  • Unattached chromosomes leads to inhibition of the anaphase promoting complex and metaphase arrests
  • If the APC becomes inactivated, cyclin cannot be degraded and anaphase arrests
171
Q

What does a minimal medium contain?

What grows on it?

A
  • Sugar
  • Salts
  • Trace minerals
  • Wild Type bacteria will grow on this as they can synthesise all other materials needed
172
Q

What does a complete medium contain?

What will grow on it?

A
  • Amino acids
  • Vitamins
  • Nucleotides
  • Mutated bacteria that have inactivated biosynthetic pathways will grow on this
173
Q

Can a mutated bacteria grow on a minimal medium?

A

Yes, but only if the biosynthetic product that they cannot synthesise is added

174
Q

Auxotroph

A

Mutant bacteria that can’t synthesise nutrients

175
Q

Prototroph

A

Wild type bacteria for a biosynthetic process

176
Q

Genetic transfer in bacteria requires…

A

Cell-cell contact

177
Q

Transfer of F plasmid in bacteria

A
  • Genetic exchange between bacteria is unidirectional
  • Donor contains a Fertility factor and is F+
  • Copy of F plasmid is transferred through F Pilus bridge from F+ donor to F- recipient during conjugation
178
Q

Transfer of host chromosomal genes through F plasmid

A
  • Transfer of host chromosomal genes from High frequency recombination strains to F- strains
  • F factor is integrated into host chromosome and transferred to recipient cell during conjugation
  • DNA is recombined into recipients chromosome
179
Q

2 principles of metabolic gene regulation in bacteria?

A
  1. Nutrient breakdown - when breakdown product levels increase, gene expression is decreased and transcription of enzymes is repressed
  2. Biosynthetic product - when biosynthetic product levels increase, gene expression is decreased and transcription of enzymes is repressed
180
Q

What is an operon?

A

A cluster of adjacent genes with closely related biochemical functions

181
Q

The Lac operon

A
  • Gene cluster that encode proteins for lactose breakdown
  • In absence of lactose, Lac repressor binds to Operator region, blocking RNA polymerase and gene transcription
  • In presence of lactose, lactose binds to Lac repressor causing it to dissociate, allowing CAP-assisted transcription by RNA polymerase so lactose can be broken down
  • As levels of glucose rises, this inactivates RNA polymerase co-factor CAP and transcription is inhibited and Lac repressor binds to Operator region
182
Q

The Trp operon

A
  • Gene cluster that encode proteins for tryptophan synthesis
  • In high levels of tryptophan, Trp repressor binds to Operator region, blocking RNA polymerase and gene transcription
  • In low levels of tryptophan, Trp repressor dissociates, allowing transcription by RNA polymerase to synthesise tryptophan
183
Q

What are lac and trp operon examples of?

A

Negative feedback

184
Q

Function of RNA Polymerase I and II?

A

I - Transcribes genes coding for production of ribosomal subunits
II - Transcribes all protein coding genes and a few non-protein coding genes

185
Q

Housekeeping genes

A

Genes that are expressed in all cell types and encode proteins required for universal cell functions

186
Q

How is eukaryotic transcriptional control exerted?

A

By binding of trans-acting transcription factor proteins to cis-acting DNA sequences next to a gene

187
Q

How is eukaryotic transcription initiated?

A
  • Begins with the binding of general transcription factors (GTFs) to the TATA box in promoter DNA sequences
  • GTFs distort promoter DNA sequences, allowing RNA polymerase II to bind to transcription initiation site
  • This structure is known as the transcription initiation complex (TIC) and is activated by transcription activators bound to enhancer sequences
188
Q

Transcription repressors regulate Transcription activators factors by…

A
  • Competing with Transcription activators for interactions with RNA polymerases
  • Masking the activation surface of Transcription activators
  • Direct interaction with general transcription factors
189
Q

3 components of a Transcription factor modular structure?

A
  • Sequence specific DNA binding domain
  • Activation/Repression domain - promote/prevent recruitment of Transcription Initiation Complex
  • Regulatory domain - allow other molecules to regulate other domains
190
Q

Zinc finger domain

A
  • Protein module that recognises 3 base-pairs of double stranded DNA
  • Proteins with more zinc fingers can recognise longer sequences
  • Gives transcription activator/repressor DNA-binding domain more sequence specificity
191
Q

6 Regulations of transcription factor activity?

A
  • Ligand binding
  • Protein phosphorylation
  • Addition of subunits
  • Unmasking
  • Stimulation of nuclear entry
  • Release from membrane
192
Q

What type of transcription factors do steroid hormones bind to that regulate specific gene regulation?

A

Nuclear hormone receptors

193
Q

Trans-acting transcription factors bind to cis-acting DNA sequences in…

A

Chromatin

194
Q

Chromatin structure - building blocks?

A
  • A nucleosome is DNA wound around an octomer of histones

- Histones coil within themselves allowing DNA to be complexed into chromosomes

195
Q

Chromatin structure makes it difficult for transcription factors to find target genes, how is this resolved?

A
  • Chromatin remodelling factors

- Covalent modifications of DNA and histones

196
Q

Linker Histone H1

A
  • Fix nucleosomes into position within chromatin for compaction
  • Limits accessibility of genes for transcription
197
Q

Chromatin Remodelling Factors

A

Displace linker and core histones from stable interactions with DNA to facilitate transcription factor access to gene

198
Q

Acylation of core histones induces transcriptional activation by…

A

Acting as a fly that recruits

  • Chromatin Remodelling Factors to open up chromatin
  • Transcription activators to promote gene transcription
199
Q

Methylation of DNA on cytosine bases in CpG dinucleotides helps to repress transcription by…

CpG dinucleotides = -C-G-
-G-C-

A

Recruiting transcription repressors

200
Q

Epigenetic changes

A

Changes to chromatin structure that affect the expression of genes within the chromatin but do not alter the nucleotide sequence of DNA

201
Q

RNA Splicing

A
  • Removal of introns and the ligation of consecutive exons
  • Proteins bind to the intron-exon boundaries and adenine attacks the backbone causing the release of the first part of the intron
  • Another attack takes place releasing the intron (which is now called a lariat)
202
Q

The 5’ Cap

A
  • Required for translation initiation

- Is a methylated guanine residue

203
Q

Regulation of translation of mRNA coding for Ferritin (iron storage)

A
  • Translation of mRNA encoding ferritin is positively regulated by Fe
  • Low Fe = Aconitase binding to ferritin mRNA inhibiting translation
  • High Fe= Aconitase dissociates from ferritin mRNA and translation occurs
204
Q

Detection of DNA in homogenate and in situ?

A
  • Homogenate - Southern blotting

- In situ - Chromosomal painting

205
Q

Detection of RNA in homogenate and in situ?

A
  • Homogenate - Northern blotting

- In situ - In situ hybridisation

206
Q

Detection of protein in homogenate and in situ?

A
  • Homogenate - Western blotting

- In situ - Immunocytochemistry

207
Q

Advantages and disadvantages of detection of DNA, RNA and proteins in the homogenate?

A 
Adv. 
- Quantification
- Size
- Isolation
Disadv.
- Require large quantity of tissue for sampling
208
Q

Advantages and disadvantages of detection of DNA, RNA and proteins in situ?

A 
Adv. 
- Tissue distribution of DNA, RNA or protein
- Function depending on location 
Disadv. 
- Requires tissue processing
209
Q

Steps for detection of DNA, RNA or protein in homogenate?

A
  • Separate molecules in gel according to their size
  • Separated molecules transferred to membrane and probe is added to detect required molecule
  • If molecule is present probe will detect and show up
210
Q

Gel Electrophoresis of DNA

A
  • Gel is a porous matrix that acts like a sieve
  • DNA has -ve charge so moves toward +ve electrode in gel
  • As DNA migrates through gel it separates out according to the size of the molecule - the smaller the molecule the further along it travels
211
Q

4 factors affecting DNA migration in gel electrophoresis?

A
  • DNA size - smaller DNA move faster through the gel
  • Gel conc. - higher conc. results in slower DNA migration
  • DNA shape - supercoiled DNA faster than linear DNA faster than circular DNA
  • Gel type - Agarose gels used for DNA fragments of 100-20,000 BPs and polyacrylamide gels used for DNA fragments 10-700 BPs long
212
Q

Agarose or Polyacrylamide gel?

A
  • Agarose used for larger range of DNA sizes 100-20,000 BPs

- Polyacrylamide has higher resolution and used for smaller DNA fragments 100-700 BPs long

213
Q

Blotting for DNA and RNA

A
  • Relies on the principle of hybridisation
  • Hybridisation is the specific base pairing of 2 complimentary single strands to form a double stranded molecule
  • Heat is applied to break hydrogen bonds, only the most stable molecules will remain
  • Stability of hybridisation depends on the degree of match between target and probe sequence
214
Q

Blotting for Proteins

A
  • Relies on the principle of antigen-antibody interaction
  • Primary antibody binds to target protein
  • Secondary antibody is tagged and binds to primary antibody to allow localisation of target protein
215
Q

In situ hybridisation importance?

A
  • Important to use as all tissues have unique subsets of RNA

- Used to detect and quantify mRNA sequences

216
Q

Chromosome painting

A
  • Locates specific genes on the chromosome

- Probes labelled with fluorescent colours allows simultaneous viewing of different genes

217
Q

Immunocytochemistry

A
  • Relies on the principle of antigen-antibody interaction

- Same method as western blotting

218
Q

DNA Sequencing process?

A

Chain termination method

  • DNA to be sequenced used as a template for DNA synthesis in vitro
  • DNA to be sequenced needs to be proceeded with some known sequence in order to make a primer to act as a starting point for DNA synthesis
  • Terminator nucleotides are added along with normal nucleotides at ratio of 1:100, these prevent subsequent addition of further nucleotides so DNA fragments of different lengths are produced with a known end nucleotide base e.g. Primer+1, Primer+2, Primer+3 etc
  • Mixture of DNA molecules passed through polyacrylamide gel electrophoresis
  • Terminator nucleotides are tagged with a different colour depending on their base - A, G, T or C
  • A detector reads which tagged nucleotide is first to pass through the laser in each fragment
  • Used to produce a sequence for unknown DNA strand
219
Q

Restriction enzymes

A
  • Precise cutters of DNA - recognise specific DNA sequences and cleave DNA at these sites
  • Each enzyme has its own specific restriction site where it will cut
  • Isolated from bacteria
220
Q

Restriction sites for enzymes

A
  • Short, usually 4-8 BPs
  • Sequence is palindromic, the sequence of the sense strand is the same as the antisense strand when rad in the same direction e.g. 5’ to 3’
221
Q

Sticky ends after restriction enzyme cut

A
  • Restriction enzymes cut at specific position within its recognition site
  • Leaves overhangs in DNA molecules called “sticky ends”
  • These can be used to create a new recombinant DNA molecule if the sticky ends of each molecule is complimentary
222
Q

Why are terminator nucleotides added in small numbers during DNA sequencing?

A
  • To allow a chance for unknown DNA sequence to be synthesised further down strand
  • Too many would produce DNA templates that were too short
223
Q

Polymerase Chain Reaction (PCR)

A
  • Used to amplify minute amounts of DNA by repeated cycles of in vitro DNA replication
  • DNA amplification proceeds in an exponential scale 2^cycles
224
Q

What do you need for a PCR?

A
  • 1 copy of DNA sequence to be amplified - the Template
  • Exact DNA sequence at the start and end of DNA sequence to be amplified - used to make Primers
  • Primer at the 5’ end derived from sense strand and primer at the 3’ end derived from anti-sense strand
  • DNA polymerase and lots of free single nucleotides
225
Q

3 steps in the PCR?

A
  1. Heat to 95oC for Denaturation of template DNA to separate strands
  2. Cool to 50-65oC for annealing of primers to template DNA
  3. Heat to 72oC for elongation of primers till end of template
    Cycle starts again with products then acting as templates
226
Q

How can PCR be used to manipulate DNA sequences?

A
  • Primers can be used to insert single point mutations by lowering temperature for annealing to allow imperfect binding between primer and DNA sequence
  • Tailed primers can be used to insert restriction enzyme sites to produce ‘sticky ends’ for DNA engineering
227
Q

DNA Cloning

A
  • Insert DNA (with sticky ends from restriction enzymes) inserted into a vector such as a plasmid
  • Use DNA ligase to fuse the DNA and plasmid
  • Introduce recombinant DNA into the bacteria
  • Use antibiotic selection for bacteria containing plasmids and this DNA is purified
228
Q

Vector

A

A DNA molecule that is maintained and replicated naturally by a host organism e.g. a plasmid

229
Q

3 essential properties of cloning plasmid?

A
  • Contain an origin of replication to allow it to replicate independently of the bacterial chromosome
  • Contain anti-biotic resistance genes to select for bacteria containing recombined DNA
  • Restriction enzyme sites to allow insertion of insert DNA
230
Q

Expression plasmids must contain what to initiate transcription of the insert?

A

A promoter region

231
Q

Genomic libraries

A

Population of identical vectors containing different inserts and used to clone all DNA sequences from a cell

232
Q

cDNA libraries

A
  • Population of identical vectors containing different inserts and is derived from mRNA thus represents the part of the genome that is made into mRNA
  • Can be derived from different organs or stages of development
233
Q

Microarrays

A
  • Performed in a cell free system using isolated mRNA
  • Monitors expression of 1000s of genes at once
  • Compares transcribed genes in 2 tissues or conditions of same tissue
234
Q

siRNA for Gene Knockdown

A
  • Small interfering RNA
  • Interfere with expression of genes causing a decrease in expression of the target gene
  • Uses RNA interference pathway in cells, which regulates gene expression
235
Q

2 methods to generate transgenic mice?

A
  • Pronuclear injection

- Gene targeting

236
Q

Pronuclear injection to generate transgenic mice

A
  • A foreign gene is inserted into the nucleus of a fertilised ova
  • Several copies are inserted at random sites in the genome
  • Used to generate GM crops and animals
237
Q

Gene targeting to generate transgenic mice

A
  • Foreign DNA is introduced into cultured mouse stem cells
  • Foreign DNA integrates at specific sites in genome
  • Modified ES cells transferred to blastocyst and inserted into foster mother which give birth to chimeric mice
  • Chimeric mice bred with normal mice to produce some gene targeted offspring
  • This is used to generate insertions (knockins) and deletions (knockouts)
238
Q

Yeast 2 Hybrid Screen

A
  • Screens for interacting proteins, based on transcriptional activation
  • Protein of interest (bait) is bound to DNA binding domain and proteins that bind to bait (fish) are bound to activation domains
  • Any protein that binds to bait will activate expression of the reporter gene
  • Construct a bait plasmid and library of cDNA fish plasmids, with each type of plasmid containing a marker such as an essential amino acid
  • Plasmids transformed into yeast cells, which are placed in a medium lacking the essential aa marker, so only those containing both plasmids will grow
  • Remaining cells transferred to agar plate lacking product of reporter gene to isolate cells containing interacting genes
  • Binding proteins identified by sequencing DNAs of plasmids isolated from these cells
239
Q

REFLP

A
  • Restriction Enzyme Fragment Length Polymorphism
  • Point mutations can lead to abolition or addition of restriction enzyme sites
  • So a mutation may create a polymorphism in the number/sizes of DNA fragments produced by a particular restriction enzyme.
  • This change becomes a marker for the mutation
240
Q

DNA Fingerprinting

A
  • Based on profiling specific regions in our genome
  • These regions contain repeats of certain short sequences
  • The number of repeats within each region varies between individuals (Variable Number of Tandem Repeats)
  • Location of repeats does not change between individuals

BMS 109 (8 decks)

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