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
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
3
Q
3 chemical components of DNA
A
- Sugar
- Nitrogenous base
- Phosphate group
4
Q
Nucleotide structure in DNA
A
- Sugar is a pentose - Deoxyribose
- Phosphate group attached to C5
- Nitrogenous base attached to C1
5
Q
Nucleotide structure in RNA
A
- Sugar is a pentose - Ribose
- Phosphate group attached to C5
- Nitrogenous base attached to C1
6
Q
Purines
A
- 2 rings
- Adenine and Guanine
7
Q
Pyrimidine
A
- 1 ring
- Cytosine
- Thymine
- Uracil
8
Q
Hydrogen bonds between Adenine and Guanine
A
2 hydrogen bonds
9
Q
Hydrogen bonds between Cytosine and Thymine (Uracil in RNA)
A
3 hydrogen bonds
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
11
Q
5 prime end of polynucleotide chain
A
Phosphate group on C5
12
Q
3 prime end of polynucleotide chain
A
Hydroxyl group on C3
13
Q
Structure of DNA
A
- Right-handed Double helix
- Sugar-Phosphate backbone with nitrogenous bases facing inwards
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
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’
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
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
18
Q
What enzyme synthesises new strands of DNA?
A
DNA Polymerase
19
Q
What direction does DNA synthesis only occur in?
A
5’ to 3’ direction
20
Q
Antiparallel orientation of parental strands means that DNA synthesis cannot..
A
Be continuous in both strands
21
Q
DNA daughter strand that is synthesised continuously is called the..
A
The leading strand
22
Q
DNA daughter strand that is synthesised discontinuously is called the..
A
The lagging strand
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
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
25
Which enzyme produces RNA primers using RNA nucleotides?
DNA Primase
26
Single stranded binding proteins (SSBPs)?
Maintain unwound parental DNA strands in a single stranded conformation to ease replication fork progression
27
2 major steps of gene expression
Transcription and Translation
28
How does DNA encode biological form and function?
Different genes are selectively expressed in different cell types i.e. they can be switched on or off depending on if they are required
29
Process of transcription of prokaryotic gene
- 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
30
What direction does transcription occur in?
5' to 3' direction
31
Process of transcription of eukaryotic gene
- 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
32
What removes introns from immature mRNA?
Splisosome removes introns by identifying recognition sequences in exons
33
Genome
The entire DNA sequence of its chromosomes
34
Transcriptome and Proteome
- Transcriptome the entire total of mRNA expressed from genes
- Proteome the entire total of proteins expressed in a cell
35
Human genome consists of linear chromosomes..
22 autosomes and X and Y sex chromosomes
36
Diploid cells
Have 2 copies of each autosome and either XX or XY sex chromosomes - 46 chromosomes in total
37
3 main components of Chromosome structure
- Centromere
- Replication Origins
- Telomere
38
Centromeres
Repetitive DNA sequence that allows chromosome attachment to mitotic spindle via the kinetochore
39
Origins of Replication
- 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
40
Telomeres
- 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
41
DNA Replication in E. Coli
- 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
42
Genome sizes are...
Larger in more complex organisms
43
Gene numbers tend to increase as...
Organism complexity increases
44
Genome size increases as...
Number of genes increases
45
Gene density decreases as...
Organism complexity increases
46
Exons and Introns in more complex organisms...
- Exons are similar sizes
| - Introns are larger
47
Genes are bigger in more complex organisms because...
They have larger introns
48
Why do genome sizes and intron sizes increase in more complex organisms?
- Larger genomes have more repetitive DNA elements (transposons and retrotransposons)
- Larger genomes have more complex transcriptional control elements spread over large regions of DNA
49
Genotype
Genetic makeup of an organism
50
Phenotype
Observable characteristics of an organism
51
Genotype-Phenotype relationship
Phenotype is the result of interaction of the genotype with the environment
52
Dominant phenotype
Phenotype of a genotype containing the dominant allele, expressed in the heterozygote
53
Recessive phenotype
Phenotype of a genotype containing the recessive allele, expressed in the homozygote only
54
Mendel's First experiment
- 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)
55
Mendel's First Law
Principle of Segregation - 2 alleles of a gene segregate from each other during gamete formation
56
Mendel's Second Experiment
- 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
57
Mendel's Second Law
Principle of Independent Assortment - genes controlling different characteristics assort into gametes independently of one another
58
Incomplete dominance
Heterozygote phenotype is intermediate between two homozygous (parental) phenotypes e.g. RR x rr = Rr
59
Co-dominance
Simultaneous expression of two phenotypes determined by alternative alleles of a single gene e.g. ABO blood groups
60
Morgan's Experiment
- 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
61
Genetic linkage
The tendency of genes that are close together on a chromosome to be inherited together during gamete formation
62
Genetic recombination
The process of forming new allelic combination in gametes by exchanging genetic material between chromosomes
63
Genetic screens
- Identify mutations that change gene functions
| - Most mutations identified in genetic screens inactivate gene function and cause recessive phenotypes
64
Applications of classical genetic analysis
- 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
65
3 techniques for engineering mutations in specific genes?
1. Gene replacement
2. Gene knockout
3. Gene addition
66
Genetic complementation test tell us...
Whether two mutations with similar phenotypes lie in the same or different gene
67
3 major genes required for larval segment formation and patterning?
- Gap gene
- Pair-rule gene
- Segment polarity gene
68
6 essentials of genetic code?
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
69
Start codon...
AUG - codes for methionine
70
Stop codons...
- UAG
- UGA
- UAA
71
Genetic code said to be degenerate because...
Most amino acids are encoded by more than one codon
72
Amino acid attached by carboxyl group to ribose of last ribonucleatide on tRNA by...
Aminoacyl-tRNA synthetase
73
Each codon on mRNA has a corresponding..
Anticodon on a tRNA molecule
74
Wobble base pairing
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
75
3 sites of a ribosome?
1. A - aminoacyl-tRNA
2. P - peptidyl-tRNA
3. E - exit
76
Process of protein translation from mRNA
- 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
77
Which end of the growing polypeptide chain is the new amino acid attached to?
C terminus of growing polypeptide chain
78
Peptide bond
- Condensation reaction catalysed by Peptidyl Transferase
| - No rotation around C-N
79
End of translation
Binding of release factor to A site on ribosome when stop codon is reached caused disassembly of ribosome
80
Polyribosome
Complex of an mRNA molecule and two or more ribosomes that act to form polypeptide chains during active translation
81
Peptide backbone
- Same for all proteins
- Repeating amino acid units
- H2N-C-C-N-C-C-N-C-COOH
82
4 types of amino acid?
- Acidic - COOH side chains
- Basic - NH2 side chains
- Polar
- Non-polar
83
Protein primary structure
- Order of amino acids in polypeptide chain
| - Side chains of amino acids drive the folding
84
Protein secondary structure
Folding of the primary chain into an alpha helix, beta sheet or random coil
85
Protein tertiary structure
- The way in which the secondary structure packs together
| - There can be sub domains which are folding within proteins with separate functions
86
Protein quaternary structure
Relationship between proteins in a multimeric complex e.g. Haemoglobin
87
If mutations lie in the same gene they are...
Non-complementary
88
If mutations lie in a different gene they are...
Complementary
89
Folding of protein in aqueous environment?
Protein folds to put hydrophobic non-polar side chains on inside and puts hydrophilic polar side chains on outside
90
3 types of attraction in secondary structure?
- Electrostatic between +ve and -ve charged ions
- van der Waals attractive or repulsive between molecules
- Hydrogen bonds between C and N atoms
91
3 main secondary structures of proteins?
- Alpha helix
- Beta sheet
- Random coil
92
Alpha helix (intrachain) and Beta sheet (interchain) formed by...
Hydrogen bonding
93
Protein domains
Distinct functional/structural units in a protein tertiary structure that are responsible for their own function
94
3 types of protein-protein interaction?
- Domain-loop interaction
- 2 alpha helices to form a coiled coil
- Domain-domain interaction
95
Coiled coil structures are driven by...
Hydrophobic interactions between amino acids in 2 proteins
96
5 post-translational protein modifications?
- Disulphide bonds
- Proteolysis
- Glycosylation
- Lipid modification
- Phosphorylation
97
Disulphide bonds in proteins form between...
- Cysteine amino acids that have sulphide side groups
| - Can be intrachain (same protein) or interchain (different protein)
98
Disulphide bonds provide...
Stability in proteins exposed to extreme environments e.g. antibodies and enzymes outside of the cell
99
Proteolytic processing of a precursor protein?
A process where the active protein is post-translationally cleaved by proteases to yield a more functional molecule
100
Glycosylation are principally a modification of...
Extracellular proteins
101
Lipid modifications in proteins are used to...
Bind proteins to membranes
102
Protein phosphorylation/dephosphorylation is used as a major...
Regulative modification
103
4 structural types of transmembrane proteins?
Lipid membrane is hydrophobic, so parts of proteins that cross membrane must also be hydrophobic
- Single pass
- Multipass
- Beta-barrell (pore)
- Amphipathic helix
104
Single pass type 1 membrane protein?
- 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
Single pass type 2 membrane protein?
- 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
Beta barrels contain different numbers of beta strands depending on...
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
Peripheral membrane proteins attach to membrane via..
- Lipid anchors
| - Other transmembrane proteins
108
What type of lipid modifications bind proteins to cytosolic side of membrane?
- Acylation
| - Prenylation
109
What type of lipid modifications bind proteins to extracellular side of membrane?
Glycophosphatidylinositol (GPI) anchors
110
Cycling of the GTPase RAS between membrane is regulated by...
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
Differential lipid modifications allows targeting to...
Different membranes
112
2 types of glycosylation modifications?
- N-linked attachment via asparagine
| - O-linked attachment via hydroxyl group of serine/threonine
113
Glycosylation provides...
- Cellular protection via mucins
- Adhesive properties
- Set of complex surfaces for interactions with other molecules
114
Glycosylation forms the basis of...
Blood groups
115
Phosphorylation occurs on...
Serine, Theorine and Tyrosine by converting a hydroxyl group to a charged phosphate group
116
Phosphorylation acts as a switch for enzymes and activating them by...
- Physically changing protein conformation
| - Altering the nature of binding surface
117
Enzymes that phosphorylate/dephosphorylate other proteins are...
Phosphorylate - kinases
| Dephosphorylate - phosphatases
118
Linear signal transduction...
Stimulus activates linear pathway of proteins to produce a single response
119
Cascade signal transduction...
Stimulus activates protein that then activates several other proteins in a cascade to produce multiple responses
120
GTP-binding proteins (G-porteins) act as switches by...
- 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
Ubiquitination
Addition of ubiquitin chain onto target proteins catalysed by ubiquitin ligases
122
Types of ubiquitination
- Mono-ubiquitylation - histone regulation
- Multi-ubiquitylation - endocytosis
- Poly-ubiquitylation - proteasomal degradation
123
Which transmembrane protein is affected in Cystic Fibrosis?
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
124
Somatic mutations
- Affect cells and tissues of the body
| - Not passed onto progeny
125
Germline mutations
- Affect cells of reproductive tissues
| - Passed onto progeny so progeny have mutations in germline AND somatic cells
126
Gain of function mutations are...
Dominant traits
127
Loss of function mutations are...
Recessive traits
128
Loss of function mutations effects on proteins?
- Complete loss of the protein - amorph
| - Reduction of proteins function - hypomorph
129
Gain of function mutations effects on proteins?
- Increase in the proteins functions - hypermorph
- Protein that interferes with wild-type proteins function - antimorph
- Protein acquires new function - Neomorph
130
Transition mutation
Changes a purine to purine (e.g. A to G) or a pyrimidine to pyrimidine (e.g. C to T)
131
Transversion mutation
Changes a purine to pyrimidine (e.g. C to G) or a pyrimidine to purine (e.g. G to C)
132
Missense mutation
Change from one amino acid to another amino acid (e.g. AAA to GAA changes codon from lysine to glutamic acid)
133
Non-sense mutation
Change from an amino acid to a premature stop codon (e.g. AAA to TAA changes codon from lysine to UAA stop codon)
134
Non-sense mediated decay
Cells detect mutated, incomplete protein and cause mRNA coding for incomplete protein to decay
135
Neutral mutation
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
Silent mutation
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
Frameshift mutation
Addition or deletion of one or two base pairs leads to a change in reading frame and changes polypeptide sequence completely
138
Spontaneous mutation due to wobble base pairing
- 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
Spontaneous mutations due to looping out errors in DNA replication
- 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
Spontaneous mutations due to depurination (chemical change)
Common; A or G are removed and replaced with a random base
141
Spontaneous mutations due to deamination (chemical change)
- 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
Induced mutations due to radiation
- 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
Induced mutations due to chemical intercalating agents
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
Induced mutations due to chemical base modifiers
- Alter the chemical structure of bases e.g. Alkylating agents that form permanent covalent bonds with nucleophilic substances in DNA
145
Sickle Cell Anaemia
Recessive mutation in haemoglobin and when at low oxygen tension the RBC loses its concave disc shape and becomes sickle shaped
146
Sickle cell anaemia caused by...
Single missense mutation that changes glutamate to valine
147
Cystic Fibrosis carriers have a selective advantage of...
Resistance to infection by some infective agents
148
Chromosome duplication mutation
Produced when extra copies of genes are generated on a chromosome
149
Chromosome deletion mutation
Produced by the breakage of a chromosome in which the genetic material becomes lost during cell division
150
Chromsome inversion mutation
Produced when a broken chromosome segment is reversed and inserted back into the chromosome
151
Chromosome translocation mutation
Produced by the joining of a fragmented chromosome to a non-homologous chromosome
152
Principle role of the cell cycle
To produce 2 genetically identical daughter cells
153
Phases of the cell cycle
- 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
G1, S and G2
Interphase
155
Stages of mitosis
- End of G2 phase
- Prophase
- Prometaphase
- Metaphase
- Anaphase
- Telophase
- Cytokinesis
156
End of G2 phase
- DNA has been replicated
| - Centrosomes have been duplicated
157
Prophase
- Chromosome condensation - DNA wraps around histones and then supercoils into chromosomes
- Mitotic spindle assembles between 2 centrosomes at each pole
158
Prometaphase
- Nuclear envelope breaks down
- Chromosomes attach to spindle microtubules by kinetochores
- Movement of chromosomes begins
159
3 classes of microtubules in mitotic spindle?
- 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
Metaphase
- 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
Anaphase
- Sister chromatids separate to from 2 daughter chromosomes
| - Kinetochore MTs shorten by depolymerising and spindle poles move apart
162
Telophase
- 2 sets of daughter chromosomes arrive at spindle pole and decondense
- New nuclear envelope forms around each set of chromosomes
163
Cytokinesis
Cytoplasm of 2 daughter cells is separated by contractile ring to produce 2 identical daughter cells
164
Cell cycle control is regulated by...
Cyclin-dependant kinases (CDK)
165
What does CDK require for activity
Cyclin
166
Is the level of cyclin constant throughout the cell cycle?
No
167
Is the level of CDK constant throughout the cell cycle?
Yes
168
What triggers entry into mitosis?
M-cyclin-CDK complexes that are activated by phosphorylation by phosphatases
169
What regulates cyclin levels throughout mitosis?
Anaphase promoting complex
| - When activated causes ubiquitylation of cyclin that tags it for degradation by proteasome
170
Spindle assembly checkpoint of metaphase-anaphase transition
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
What does a minimal medium contain?
| What grows on it?
- Sugar
- Salts
- Trace minerals
- Wild Type bacteria will grow on this as they can synthesise all other materials needed
172
What does a complete medium contain?
| What will grow on it?
- Amino acids
- Vitamins
- Nucleotides
- Mutated bacteria that have inactivated biosynthetic pathways will grow on this
173
Can a mutated bacteria grow on a minimal medium?
Yes, but only if the biosynthetic product that they cannot synthesise is added
174
Auxotroph
Mutant bacteria that can't synthesise nutrients
175
Prototroph
Wild type bacteria for a biosynthetic process
176
Genetic transfer in bacteria requires...
Cell-cell contact
177
Transfer of F plasmid in bacteria
- 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
Transfer of host chromosomal genes through F plasmid
- 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
2 principles of metabolic gene regulation in bacteria?
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
What is an operon?
A cluster of adjacent genes with closely related biochemical functions
181
The Lac operon
- 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
The Trp operon
- 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
What are lac and trp operon examples of?
Negative feedback
184
Function of RNA Polymerase I and II?
I - Transcribes genes coding for production of ribosomal subunits
II - Transcribes all protein coding genes and a few non-protein coding genes
185
Housekeeping genes
Genes that are expressed in all cell types and encode proteins required for universal cell functions
186
How is eukaryotic transcriptional control exerted?
By binding of trans-acting transcription factor proteins to cis-acting DNA sequences next to a gene
187
How is eukaryotic transcription initiated?
- 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
Transcription repressors regulate Transcription activators factors by...
- Competing with Transcription activators for interactions with RNA polymerases
- Masking the activation surface of Transcription activators
- Direct interaction with general transcription factors
189
3 components of a Transcription factor modular structure?
- 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
Zinc finger domain
- 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
6 Regulations of transcription factor activity?
- Ligand binding
- Protein phosphorylation
- Addition of subunits
- Unmasking
- Stimulation of nuclear entry
- Release from membrane
192
What type of transcription factors do steroid hormones bind to that regulate specific gene regulation?
Nuclear hormone receptors
193
Trans-acting transcription factors bind to cis-acting DNA sequences in...
Chromatin
194
Chromatin structure - building blocks?
- A nucleosome is DNA wound around an octomer of histones
| - Histones coil within themselves allowing DNA to be complexed into chromosomes
195
Chromatin structure makes it difficult for transcription factors to find target genes, how is this resolved?
- Chromatin remodelling factors
| - Covalent modifications of DNA and histones
196
Linker Histone H1
- Fix nucleosomes into position within chromatin for compaction
- Limits accessibility of genes for transcription
197
Chromatin Remodelling Factors
Displace linker and core histones from stable interactions with DNA to facilitate transcription factor access to gene
198
Acylation of core histones induces transcriptional activation by...
Acting as a fly that recruits
- Chromatin Remodelling Factors to open up chromatin
- Transcription activators to promote gene transcription
199
Methylation of DNA on cytosine bases in CpG dinucleotides helps to repress transcription by...
CpG dinucleotides = -C-G-
-G-C-
Recruiting transcription repressors
200
Epigenetic changes
Changes to chromatin structure that affect the expression of genes within the chromatin but do not alter the nucleotide sequence of DNA
201
RNA Splicing
- 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
The 5' Cap
- Required for translation initiation
| - Is a methylated guanine residue
203
Regulation of translation of mRNA coding for Ferritin (iron storage)
- 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
Detection of DNA in homogenate and in situ?
- Homogenate - Southern blotting
| - In situ - Chromosomal painting
205
Detection of RNA in homogenate and in situ?
- Homogenate - Northern blotting
| - In situ - In situ hybridisation
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Detection of protein in homogenate and in situ?
- Homogenate - Western blotting
| - In situ - Immunocytochemistry
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Advantages and disadvantages of detection of DNA, RNA and proteins in the homogenate?
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Adv.
- Quantification
- Size
- Isolation
Disadv.
- Require large quantity of tissue for sampling
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Advantages and disadvantages of detection of DNA, RNA and proteins in situ?
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Adv.
- Tissue distribution of DNA, RNA or protein
- Function depending on location
Disadv.
- Requires tissue processing
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Steps for detection of DNA, RNA or protein in homogenate?
- 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
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Gel Electrophoresis of DNA
- 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
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4 factors affecting DNA migration in gel electrophoresis?
- 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
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Agarose or Polyacrylamide gel?
- 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
Blotting for DNA and RNA
- 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
Blotting for Proteins
- 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
In situ hybridisation importance?
- Important to use as all tissues have unique subsets of RNA
| - Used to detect and quantify mRNA sequences
216
Chromosome painting
- Locates specific genes on the chromosome
| - Probes labelled with fluorescent colours allows simultaneous viewing of different genes
217
Immunocytochemistry
- Relies on the principle of antigen-antibody interaction
| - Same method as western blotting
218
DNA Sequencing process?
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
Restriction enzymes
- 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
Restriction sites for enzymes
- 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
Sticky ends after restriction enzyme cut
- 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
Why are terminator nucleotides added in small numbers during DNA sequencing?
- 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
Polymerase Chain Reaction (PCR)
- 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
What do you need for a PCR?
- 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
3 steps in the PCR?
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
How can PCR be used to manipulate DNA sequences?
- 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
DNA Cloning
- 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
Vector
A DNA molecule that is maintained and replicated naturally by a host organism e.g. a plasmid
229
3 essential properties of cloning plasmid?
- 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
Expression plasmids must contain what to initiate transcription of the insert?
A promoter region
231
Genomic libraries
Population of identical vectors containing different inserts and used to clone all DNA sequences from a cell
232
cDNA libraries
- 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
Microarrays
- 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
siRNA for Gene Knockdown
- 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
2 methods to generate transgenic mice?
- Pronuclear injection
| - Gene targeting
236
Pronuclear injection to generate transgenic mice
- 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
Gene targeting to generate transgenic mice
- 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
Yeast 2 Hybrid Screen
- 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
REFLP
- 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
DNA Fingerprinting
- 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
