Molecular Biology Flashcards
1
Q
What is the structure if DNA?
A
- 2 helical antiparallel polynucleotide chains
- 4 nucleotide bases
- The molecular struture males replication very reliable
- Structural errors are very rare
2
Q
What can form if structural errors in DNA occur?
A
Although very rare, if DNA structural errors occur can cause mutations in the neurofibramin tumour mutations in the neurofibramin tumour suppressant gene causing neurofibromatosis
3
Q
What is semi conservative replication?
A
How DNA replicates - each double helix is formed from one parental strand and one newly synthesised one
4
Q
What are the steps in semi conservative replication?
A
- Strands are separated by DNA helicase forming a replication fork
- DNA polymerase synthesises new strands but can only occur in the 5’ to 3’ to ensure that the stands are antiparallel
- The leading strand can be synthesised as it goes from the 5’ to 3’ end. However, the lagging strand cannot as it goes fro 3’ to 5’. Other fragments called Okazaki fragments are used to fill the gaps
- DNA ligase covalently links fragments together
5
Q
What is the role of RNA in DNA replication?
A
- DNA polymerase can not synthesise a new chain from scratch
- So a RNA polymerase called DNA primes is used to synthesis a RNA primer
- Each primer is then extended by DNA polymerase
- Primer removed by ribonuclease H which results in a gap at the 3’ end - filled in by DNA polymerase
6
Q
What is the role of single stranded binding proteins (SSBs)?
A
Maintains the unwound parental DNA strands in a single-stranded conformation to ease replication fork progression
7
Q
What are the essential features of the prokaryotic gene transcription process?
A
- RNA polymerase binds to the DNA sequences in the promotor region
- Transcript elongation - formation of mRNA
- RNA polymerase encounters terminator region - dissociates and transcription stops
8
Q
What is the limiting factor of transcription?
A
Limited by the frequency of initiation
9
Q
What are the essential features of the eukaryotic gene transcription process?
A
- RNA polymerase binds to the promotor region
- Elongation - transcripts of eukaryotic genes have exons (present in mRNA) and introns (spliced out) and does not appear in mature RNA
- Termination
10
Q
What is a genome, transcriptome and proteome?
A
Genome - the entire DNA sequence in an organisms chromosome
Transcriptome - Entire set of RNA in a particular cell type
Proteome - Entire proteins in a particular cell type
11
Q
What is a centromere?
A
- Repetitive DNA sequence to which mitotic spindles attactches via the kinetochore (provides insertion points for microtubules )
- Complex array of repeated DNA sequences
12
Q
What is the replication origin?
A
DNA sequence where DNA replication is initiated
- Does not occur at the end - occurs in the middle and forms 2 replication forks
13
Q
What is a telomere?
A
- Specialised DNA sequences at the end of chromosomes which maintains its integrity
- Telomerase creates a long 3’ overhang which is repeated cases - protects the ends of DNA sequences
14
Q
How does the E.coli genome compare to more complex organisms?
A
- E.coli - 4000 genes compressed into a singular circular DNA molecule
- Human genome is 5 times larger
- The more complex the organism the less densely packed they are but the larger the genome
15
Q
What is different about the genomes of complex organisms in eukaryotic cells?
A
The more complex the organism the larger the introns (exons are the same)
- eg. The human Huntingtin gene is 7.5 times larger than in the pufferfish due only yo the increased size of introns
16
Q
What percentages of sequences are up the human genome?
A
- 1.5% exons
- 50% repeated sequences
- 30% unique sequences - cause genes to become active
17
Q
What is Mendel’s definition of genes?
A
Discrete physical entities that fuction independently of one another to determine physical characteristics
18
Q
Outline Mendel’s experiment for his first law
A
- Pea plants that grew from smooth peas always produced smooth peas
- He cross breaded smooth pea plant with wrinkled and all offspring were smooth
- Offspring were self fertilised and there was a 3:1 of wrinkled and smooth peas
- Concluded there must be two copies of each gene - alleles
- and that smooth was dominant and wrinkled was recessive
19
Q
What is Mendel’s first law?
A
Principle of segregation
- Two alleles of a gene segregate from each other during gamete formation
- Had the gametes carry one allele and the other half carry the other
20
Q
What is Mendel’s second law?
A
The principle of independent assortment
- Genes controlling different characteristics assort into gametes independently to each other
21
Q
Outline Mendel’s second experiment
A
He asked if two different factors inherit differently
- He tested wrinkled/smooth peas and also their colour - yellow/green
- He asked if R/r Y/y move into gametes together or are there the combinations that are combined randomly
- He found 4 phenotypic classes in equal proportions - random
22
Q
What did Mendel miss?
A
- Many genes have may alleles - not just two
- Not all alleles exhibit complete dominance or recessive
- Many genes fail to exhibit independent assortment - when lie on the same chromosome
23
Q
What is incomplete dominance?
A
Phenotypes is intermediate between 2 parental phenotypes
eg. white and red carnation —> pink flowered offspring
24
Q
What is co dominance?
A
Simultaneous expression of two phenotypes
e.g. blood groups - A and B antigens are present on AB blood type
25
Outline Morgans genetic linkage ad recombination experiment in drosophila
- He crossed grey bodies drosophila with normal wings with a black bodies with vegetal wing
- F1 phenotype: grey with normal wings ---> dominant phenotypes
- He back crossed the F1 progeny with parental black/vegistal
- All 4 possible phenotypes shown but in unequal proportions
- He concluded that the backcross led to a majority of parental phenotype combinations because the genes were located on the same chromosome
26
How are non - parental combinations in Morgans experiment explained?
Explained by genetic crossovers in the formation of some gametes - genetic recombination
- Number of recombination Is proportional to physical distance between linked genes - allows gene mapping
27
How can genes be found to analyse?
- Human genetics - natural variation
- Induced mutations - chemicals or radiation that alter DNA sequences randomly
- Engineered changes - Removing gene sequences (targeted mutations) adding new gene sequences (transgene)
28
How can you find genes require for a particular biological process?
Genetic screen
- Identifies mutations that change gene function
- Usually inactivates mutations leading to a recessive phenotype
29
How do you design a genetic screen?
- Choose an organism that can be grown easily and observed
- Short generation time
- Can be mutanised easily using chemicals or x rays
30
How do we know when mutations causing similar phenotypes lie in the same or different genes?
- Genetic complementation test
| - Cross mutants and see of any offspring are wild type. If yes the different genes
31
What is the initiation codon?
AUG
32
What are the stop codons?
UAA, UAG, UGA
33
Define degenerative and explain why it provides an advantage
More codons then amino acids
| - Provides tolerance to mutations as a codon could change but still code for the same amino acid (silent mutations)
34
How is the abundance of the amino acid related to the its number of codons?
More abundant amino acids tend to have more codons
| Arginine is an exception as it is has lots of codons but itsn't that common - suggests some soy of evolutionary factor
35
What is the role of tRNA?
It is the adapter - carries specific amino acid -complementary to anticodon
36
What is wobble base pairing?
- tRNA molecule can recognise more than one codon
| - tRNA molecules read first two bases but there is a leniency with the third base - tolerance to mutations
37
What is the structure of ribosomes?
Catalyses translation
- 3 binding sites - P, A, E
- Large ribosome subunit moves along the mRNA chain with the tRNA binding to the codons of A or P and exiting at E . Small subunit follows
- Empty tRNA falls off
38
How are amino acids joined together?
- Addition of amino acid to the C-terminus of growing polypeptide chain
- The peptide bond is formed by a condensation reaction and is catalysed by peptide transferase (part of the ribosome)
39
What is the primary structure of proteins?
- Chain of amino acids joined by a peptide bond - condensation reaction
- Determines how protein folds (hydrophobic side chains)
- Held together by non covalent interactions (van Der Waals)
40
What is the secondary structure of proteins?
- Distinct structures are formed by H-bondinf within and between peptide chains
- alpha helix - 7 amino acids per 3 turns, H bonds on every 4th residue
- beta sheets - Adjacent peptide chains. Inner - chain H bonds between strands of the beta sheet
41
What are the types of beta sheets?
Can have parallel or antiparallel beta sheets
42
What is the tertiary structure of proteins?
The folding of secondary structure into domains and proteins
- Common tertiary structure often indicates a common function
- Protein domains - functional modules
43
Give the features of protein domains
- Compact, stable, hydrophobic core, fold independently
- More domains in complex organisms --> increases function
- Addition of domains brings specificity and regulation
44
What is the quaternary structure of proteins?
Multimeric protein complexes
- Relationship between individual proteins in a multimeric complex
- e.g. haemoglobin
-
45
Where are disulphide bridges found?
- Typically found on proteins that are exposed to extreme environment
- Used in antibodies to hold chains together
46
What is proteolytic processing of a precursor protein?
A process common to many enzymes and hormones where the active hormone or enzyme is post translationally cleaved to yield a mature fractional molecule
47
Give sone examples of post-translational protein modifications?
Glycosylation - carbohydrate modifications
- Modification od extracellular proteins
Lipid modifications
- A variety of lipophilic covalent attachments that help to bind proteins to membranes
Protein phosphoylation
- Major regulatory modification
48
What is an integrated membrane protein?
- Protein in the lipid biker
- HAs a transmembrane domain - passes through membrane
- If passes once than single pass and if more than a multi pass
49
What is hydropathy plot used for?
Each peak above 0 indicates a transmembrane domain
50
What is the difference between single pass type 1 and type 2 integral proteins?
Single pass type 1:
- Protein spanning membrane one with it's N terminus on extracellular side of the membrane
Single pass type 2:
- Protein spanned membrane once with its N terminus on cytoplasmic side - functions as an anchor
51
Where are beta barrels mainly found?
Mitochondrial and bacterial membranes
52
What are peripheral membrane proteins?
- Does not penetrate the lipid bilayer
- Associate with integral membrane proteins
- Contains lipid anchors
53
Give some examples of lipid modifications
```
Intracellular
- Acylation -addition of myristyl groups
Extracellular
- GPI anchors
They regulate the cycling of small GTPase ras between membranes
```
54
Give the characteristics of carbohydrate modifications - glycosylation
- Principally a modification of extracellular proteins
- N- linked - attach via asparagine
- O-linked - hydroxyl group of serine
- Cellular protection
- Provide adhesive properties
- Blood groups - defined as patterns of glycocylation
55
What is protein phosphorylation?
- Major regulatory modification
- Phosphorylated by Kinase using ATP
- Dephosphotylated by phosphatases
- Acts as a switch
- Occurs in serine
56
What is ubiquitination?
- Addition of a chain of ubiquitin to lysine resides on target proteins
- Catalysed by ubiquitin ligases
- Different types of ubiquitination have different functions
57
How does myosin and actin act as a molecular motor?
- Myosin head bound to actin
- ATP binds to head causing a conformational change in binding site (actin)
- Cleft closes around ATP causing myosin was to move - ADP remains bound
- Myosin head weakly binds with actin and ADP released - causing power stroke
58
What is the difference between somatic and germ line mutations?
Somatic mutations affect cells and tissues of the body - not inherited
Germ line affects cells of reproductive tissue - progeny will have mutations in somatic and germ line
59
What type of mutations are dominant and recessive traits?
Dominant - gain of function (hypermorph)
| Recessive - loss of function (hypomorph) - can be conditional e.g. only at certain temperatures
60
What types of mutations are there?
- Non sense - change to stop codon (leads to incomplete polypeptide formed)
- Neutral mutation - amino acid changes to another with similar properties
- Silent mutation - amino acids doesn't change
- Frameshift - changes the reading frame so all amino acids after are affected
61
What is the difference between transition and transversion?
- Transition - purine purine or pyramidine pyramidine
| - Transversion - purine pyrimidine
62
How does looping out errors lead to mutation?
The looping out of a template strand can lead to an insertion or deletion of a base
63
How can spontaneous chemical changes lead to mutation?
```
Depurination
- Adenine/guanine lost from backbone - random base inserted to replace it during replication
Deamination
- Cytosine is deaminated to uracil
- Meaning CG will go to TA
```
64
How can mutations be induced?
Radiation
- Ionising - can break covalent bonds leading to point mutations
- Ultravoilet - cause replication problems
- Chemical - base analogues - tradition mutations
Alkylating agents
Intercalating agents
- Froms gaps in DNA strands
65
Outline the characteristics of sickle cell-anaemia
- Affects he ability of haemoglobin to bind with oxygen
- Loses concave shape - sicks shaped
- Glutamate ---> valine
- Heterozygous carriers have resistance to malaria
66
What are the features of the cystic fibrosis transmembrane regulator (CFTR)?
- Function - chloride transport in membrane of epithelial cells
- Muttaions in CFTR are unable to osmotically decrease the viscosity of mucous secretion - leads to build up of mucus in lungs causes chronic infections
- CF carriers are suggested to have resistance to TB
67
What happens in a gain of function mutation in G - protein?
- G-protein activating mutations are missense mutations resulting in loss of function of GTPase
- Ras mutations found in cancer cells cause amino acid substitutions that loses infection of GTPase - causing Res proteins to accumulate - causes cancer
68
What is the Philadelphia chromosome?
- Translocation of genetic material between chromosomes - causes formation of an oncogenic BCR-ABL fusion protein
- Causes increase tyrosine kinase activity
- Resulting in leukaemia
69
Why do the daughter cells of cell cycle have to increase in size?
Need to increase in size between divisions so that they don't keep getting smaller - need nutrients
70
What are the principle phases of the cell cycle?
G1 - environmental checking and cell growth
S - DNA replication
G2 - more growth/checks
M - nuclear/cytoplasmic divisons
71
What occurs in interphase?
- G1 - maintains cell size
- S Phase - takes 10-12 hours - DNA duplicates
- G2
Interphase takes 16+ hours
72
How can determine DNA content of cells?
Fluorescent dies can be used to determine the DNA content of large numbers of cells I a flow cytometer of fluorescence
73
How long does mitosis take?
Less than an hour
74
What happens during prophase?
```
Sister chromatids condense
Mitotic spindle assembles between two centrosomes
- Mitotic spindle made of microtubules
- Assemble at centrosome
- An aster extends from each centrosome
```
75
What happens during prometaphase?
- Nuclear envelope breaks down
- Chromosomes attached to spindle microtubules via their kinetochores
- Movement of chromosomes begin
76
What are the three types of microtubule involved in the cell cycle?
- Astral
- Kinetochore
- Interpolar
77
What happens during metaphase?
- Chromosomes aligned at equator
| - Kinetochore microtubules attach sister chromatids to opposite poles of the spindle
78
What happens during anaphase?
- Sister chromatids separate --> two daughter chromosomes
- Pulled towards their pole
- Kinetochore microtubules shorten by depolymerisation- spindle poles move apart
79
What happens during telophase?
- Two sets of daughter chromosomes at pole and they condense
| - Nuclear envelope reforms - two nuclei
80
What happens during cytokinesis?
- Cytoplasm decided into two by a contractile ring formed of actin and myosin filaments
81
What are the cell cycle checkpoints?
Operates though negative signalling
- Signal produced when if all chromosomes are not attached, signal inhibits moving to the next step
- It is not a positive signalling because the signal intensity between 45 chromosomes and 46 chromosomes would be very little and could lead to errors
- Signals are phosphorylation and proteolysis
82
How is cell cycle control regulated?
Regulated by cyclically activated kinases
- CDK - requires cyclin for activity
- CDK - regulated by phosphorylation - if not all chromosomes are attached then CDK will be phosphorylated and cell cycle will freeze
- CDK levels are constant throughout the cycle
83
How is G2/M change checked in the cell cycle?
- M-cyclin levels increase through G2 and M to create a poll of inactive M-Cdk complex
- In late G2 the cdc25 phosphatase is triggered to activate a poitive feedback loop rapidly activating mitosis
84
What happens is cell cycle goes wrong?
- DNA damage, lagging, chromosomes - mutations
- Cell death, cancer
- Withdrawal from the cycle
85
What are the good qualities of bacteria for genetic analysis?
- Bacteria are haploid
- Easy to maintain
- Isolation of mutants is straight forward
- Genetics is still possible because genes can be transferred between bacteria
86
What is a minimal medium?
A medium containing simples carbon source, salts and water
| Wild type bacteria can grow on this
87
What is a complete medium?
A medium containing all amino acids and nutrients
| Mutant bacteria that have an inactivated biosynthetic pathway needs this as they cant produce they're own amino acids
88
What are auxotrophs and prototrophs?
Auxotrophs - cannot synthesise essential nutrients
| Prototrophs - Can synthesis (wild type)
89
What is the F plasmid (the fertility factor)?
Gene transfer between bacteria
- F plasmid is a circular DNA molecule that can conjugate with f- recipient cells
- DNA transfer is unidirectional - from donor to recipient
- A F-pilus bridge is formes from donor (F+) to recipient (F-) and transfers a copy of the plasmid
- Can also cause a transfer of host chromosomal gene
90
What is the lac operon?
It is the gene cluster responsible for breaking down lactose
- Multiple proteins synthesises from one mRNA
- In the absence of lactose, the lac repressor binds to the operator region blocking transcription
- Lactose binds to lac repressor - dissociates
- Transcription by RNA polymerase
- The production of glucose from lactose inhibits transcription by inactivating the RNA polymerase co-factor CAP
91
What is the Trp Operon?
- Tryptophan limits Trp operon transcription through feedback inhibition
- Low tryptophan concentrations cause trp repressor to become inactive - transcription initiated
92
What are the three types of RNA polymerases?
RNA polymerase 1 - transcribes genes encoding ribosomal RNA genes
RNA polymerase 2 - transcribes all protein coding genes and a few non-protein (RNA) genes
RNA polymerase 3 - transcribes all tRNA genes and ribosomal RNA genes
93
What are housekeeping genes?
Ubiqitously expressed housekeeping genes
- Genes expressed in all cell types and encode for proteins that carry out universal cell functions e.g. metabolic pathways, protein synthesis, DNA repair
- Absolute levels of housekeeping gene expression may change, depending on nutrient availability and rate of tissue growth
94
What differentially expressed proteins make cells different?
- Cell adhesion proteins (mediate physical interactions between cells)
- Secreted extracellular signals (peptide hormones)
- Cell surface receptors (for extracellular signals)
- Ion channels
- Transcription factors
- Contractile proteins
95
What is the major mechanise where differential gene expression is achieved?
Cell-type specific gene transcription
96
How does transcription begin?
- The binding of general transcription factors (GTGs) to the TATA box in the promoter DNA sequence
- GTF's distort the promotor DNA sequence, which allows RNA polymerase 2 to bind th the initiation site
- This protein assemble on DNA is known as the transcription initiation complex (TIC) - inactive until contacted by transcription activator proteins
97
What is required for transcriptional activation of a target gene in vivo?
Transcription activators
Auxiliary proteins
TIC
98
What is the role of antennapedia transcription factor?
- Promotes the development of antennae
- Sequence specific DNA binding protein that regulated transcription via protein - protein interactions affecting TIC activity
- Mutations in Antp can cause legs on the head
99
What is the role of cycloid transcriptional factor?
Promotes bilateral symmetry snapdragon flowers
| - Binds to DNA sequences in target gene promotes and regulates transcription
100
What is the role of Gli3 transcription factor?
Regulates digit number in humans and mice
| - Component of the hedgehog signalling pathway and regulates transcription of hedgehog pathway target genes
101
What are the structures of transcription factor proteins?
- A sequence specific DNA binding domain
- A domain that promotes or prevent recruitment of the transcription initiation complex - an activation to repression domain
- Other domains may regulate other domains (e.g. Zinc finger domain - a protein. module that recognises 3 base-pairs of double stranded DNA and requires a zinc ion to stabilise the structure)
102
How do transcriptional repressor antagonise transcriptional activator functions?
- Compete with DNA binding
- Masking the activation surface
- Direct interaction with the general transcription factors
103
How do steroids hormones act as transcription factor?
- Each steroid binds specifically to a different nuclear hormone receptor
- Different NHRs regulate the expression o different sets of target genes
104
What does DNA coil around a histone molecule?
The net positive charge allows them to react with negative charged backbone of DNA. DNA coils around histone molecule
105
How many base pairs of DNA wrap around 1 histone molecule?
146
106
Why is DNA packaged in the way it is?
Permits efficient nd ordered packaging of large volumes of DNA into small volume of the nucleus
107
Some parts of DNA are hidden in the nucleosome, how is this overcome?
- Chromatin remodelling factorase - coil and uncoil chromatin fibres - moves histone alone DNA to access specific DNA sequences
- Covalent modification of DNA and histones - specific regions are 'flagged' which directs transcription activators and repressors to target DNA sequences
108
What is the role of linker histone 1?
Fixes nucleosomes into position
- Facilitates chromatin compaction and organisation
- Limits accessibility to genes for transcription
109
What affect does the methylation of DNA on cytosine bases in CpG dinucleotides have?
It is a hallmark of transcriptionally inactive chromatin - switches genes off
110
What are epigenetic genes?
Changes in chromatin structure that affects gene expression but not nucleotide sequence
- e.g. moving of histone molecules in tortoiseshell cats - random epigenetic silencing of a coat colour gene
111
Name a post transcriptional control of gene expression
RNA splicing
- Removal of introns and ligation of consecutive exons
- Happens by splisosome
Regulated RNA splicing controls sex determination in drosophila
112
How does RNA splicing in the drosophila determine the sex?
In males
- Splice site inactive meaning the intons remain leading to the production of a sex determining protein
In females
- Splice site active - transformer protein binds which is a splicing activator
113
How is the translation of the iron storing protein Ferritin regulated?
- Produced in response to availability of free iron
- Low iron - aconitase binds to ferritin mRNA and inhibits translation
- High iron - actonotase dissociates from ferritin mRNA and translation occurs
- Aconitase has iron binding site
114
How would you detect DNA in a homogenate?
Southern blotting
115
How would you detect RNA in a homogenate?
Northern blotting
116
How would you detect proteins in a homogenate?
Western blotting
117
How would you detect DNA in situ?
Chromosome painting
| Chromosomal spreads
118
How would you detect RNA in situ?
In situ hybridization
119
How would you detect proteins in situ?
Immunocytochemistry
| Immunohistochemistry
120
What are the advantages and disadvantages of detecting DNA, RNA and proteins in a homogenate?
```
Advantages
- Quantification
- Size
- Isolation
Disadvantages
- Requires larger quantity of tissue
```
121
What are the advantages and disadvantages of detecting DNA, RNA and proteins in situ?
```
Advantages
- Tissue/subcellular distribution
- Function
- Changes in above
Disadvantages
- Requires tissue processing
- Limited and comported by reagents and resolution
```
122
What are the stages in blotting?
- Gel electrophoresis
- Transfer out of a gel to a membrane
- Detection on membrane by a labelled probe
123
How does electrophoresis occur?
- DNA is loaded into a pre-caste gel
- The gel is a porous matrix that acts like a sieve
- DNA is negatively charged so under the force of an electric field moves towards the positive anode through the gel
- DNA is assorted in size as it moves larger molecule move slower as dont fit though pores in the gel
- Same for RNA and protein
124
What factors affect DNA migration in gel electrophoresis?
- DNA size
- Gel concentration
- DNA shape - compact moves faster then linear
- Gel type - agarose best for 100-20000 bases, polyacrylamide for 10 -700 bases
125
What techniques can be used to transfer the gel on to a membrane (blotting)?
Blotting by capillary action
- DNA fragments blotted onto nitrocellulose paper with something heavy on top it
Blotting by an electric field
- Current moving up transferring it onto a membrane
126
What do the different types of blotting rely on in order for it to work?
Northern and southern - specific base pairing of two complementary single stranded molecules
Western - Antigen-antibody interactions
127
Why is the in situ detection of RNA important?
Because all cells have the same DNA but only certain cells express certain types of RNA
128
How does in situ hybridisation work?
Relies on hybridisation od complementari DNA/RNA sequences
- Can detect and locate expression of genes
- The intensity of the signal correlates wth the level of expression
129
What is the difference between immunocytochemistry and immunohistochemistry?
Immunocytochemistry - within a cell
| Immunohistochemistry - within a tissue
130
What is the purpose of DNA sequencing?
- To predict the function of a DNA sequence
- Identify differences between mutated and normal genes
- Confirm sequence of engineered DNA
131
What is the concept of the Sanger or chain termination method?
The DNA to be sequenced is used as a template for DNA synthesis in vitro. The product betrays the template sequence
132
What is the method of the Sanger or chai termination method?
- A primer is made from a known sequence
- DNA polymerase extends the primer
- Some of the nucleotides in the solution are tagged with coloured terminator nucleotides so that once it is added to the chain, no more bases will be added
- The chains will be different lengths
- The chains are separated by gel electrophoresis - put in size order
- Chromatogrophy is then used to identify the tag on each base
- The DNA sequence will be each terminator nucleotide in size order (smallest first)
133
What is the purpose of DNA fragmentation by restricted enzymes?
- Enable analysis of DNA (southern blotting)
| - Enable DNA engineering
134
What are restriction enzymes?
Precise cutters of DNA - recognise specified DNA sequences and cut to that point
- isolated from bacteria
- Produce 5' or 3' overhang - sticky ends
135
What is the role of restriction enzymes in bacteria?
- Thought to protect bacteria from bacteriophages
| - Bacteria protect their own DNA from restriction enzymes by methylation of their DNA
136
How long are restriction sites?
4-8 bases
- 4 base site occur once in 256 bases - common
- 8 base site occur once in 65536 bases - rare
137
What does palindromic mean?
If you cut both strands of DNA at the same place as the site, the DNA sequence would read the same from both orientations
138
What is the purpose of the polymerase chain reaction (PCR)?
- Used to amplify small amounts of DNA by repeated cycles of in vitro DNA replication
- Amplification occurs exponentially
139
Why does DNA need to be amplified?
- For detection eg. diseases
- Cloning
- Modification of DNA
140
What is required for PCR?
- 1 copy of DNA - template strand
- Known DNA sequence at start and end so primers can be made
- Primers at 5' of sense strand and 3' of antisense stand
- DNA polymerase
- Nucleotides
- Thermal cycler - different temps
141
What are the steps in PCR?
- 95ºC - denaturation of template DNA
- 50-65ºC - annealing of primers to template DNA
- 72ºC - Elongation of primers
142
How is PCR used to insert modifications?
- Add desired mutation to the prier
- Can be used to add sites for restriction enzymes
- Primer mutation is then copied into all future strands
143
What host organisms can be used in DNA cloning?
- Bacteria/viruses
| - Need short life spans, cheap and easy to remove DNA from them
144
What is a vector?
- A host DNA molecule that has a piece of DNA inserted into
| - A vector is maintained and replicated by its host
145
What are the characteristics of plasmids as a vector?
- Most common the of vector
- Needs to contain an origin of DNA replication
- Contain antibiotic resistance gene
- Have restriction enzyme sites
146
What are the steps in cloning?
- Generation of compatible cohesive ends in both insert and plasmid by same restriction enzyme
- Fusion of insert to plasmid using DNA ligase
- Introduction of recombinant DNA molecules into bacteria
- Antibiotic selection for bacteria containing plasmids
- Large scale propagation of bacteria
147
What are expression plasmids?
- Must contain a promotor to initiate transcription of the insert
- Bacterial expression plasmids contain bacterial promoters for expression in bacterial cells
- Mammalian expression plasmids contain eukaryotic promotors for expression in mammalian cells
148
What are DNA libraries?
Population of identical vectors each containing a different insert
- Used to clone all DNA sequences from an organism - available for further studies
149
What are the types of DNA libraries?
Genomic libraires
- Contain the complete DN sequence of an organism
- cDNA libraries - derived from mRNA so represent the part of the genome made into mRNA
150
What can microarrays to be used for?
- Can be used to tell if some neurones are damages or not by comparison of DNA, mRNA, proteins and post-translational modification
151
How do microarrays work?
- Performed in a cell free system using isolated mRNA
- Monitors the expression of 1000's of genes at once
- Compares the transcribed genes in two tissues (e.g. heart vs brain) or conditions of the same tissue (e.g. healthy vs diseased)
152
What is a microarray chip?
Contains thousands of probe cells
| Each probe cells contains many copies of 25 base sequence that matches a particular gene
153
How are microarrays carried out?
- Take mRNA from from two tissues
- Convert the mRNA to ssDNA
- Hybrisise with bases from probe cell
- If non complementary - labelled single stranded DNA fro both samples will hybridise to other probe cells or be washed away
- If complementary it will hybridise with probe cell with matching sequence
- ssDNA is labelled
- If gene up-regulatied then appears red
- If not that yellow
- If down regulated then green
154
What can the yeast-2-hybrid screen be used for?
Can be used to find out what proteins can interact with a protein of interest
155
What does siRNA stand for?
Small interfering RNA
156
What is siRNA used for?
To reduce the expression of a specific gene whether in a single cell or in a whole organism (gene knockdown)
157
How does siRNA cause gene knockdown?
- siRNA uses the RNA interference pathway in cells
- RNA interference pathway regulates gene expression using endogenously expressed siRNA (called microRNA)
- Foreign double stranded RNA is introduced in cells
- Dicer cleaves to produce 21-22 long fragment
- RISC degrades target mRNA
- --> Gene expression reduced
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What are the two method of generation of transgenic mice?
- Pronuclear injection
| - Gene targeting
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How does prenuclear injection produce transgenic mice?
- Foreign DNA introduced into nucleus of fertilised ova
- Foreign DNA is integrated at random sites in the genome
- Several copies are uusually inserted
- Expression of transgender can e modulated by insertion site
- Each transgenic line is unique
- Used to generate GM crops and animals
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How is gene targeting used to produce transgenic mice?
- Foreign DNA introduced into cultured mouse stem cells
- Foreign DNA integrates at specific sites in the genome
- Used to generate insertions or deletions
- Copy number is unchanged
- Modified gene remains in native genetic environment
- Expression is not modified
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What are the steps in gene targeting to produce a transgenic mouse?
1. In vitro modification od embryonic stem cells
2. Injection of modified embryonic stem cells in host blastocysts
3. Implantation of injection blastocysts in foster mothers
4. Foster mothers Gove birth to chimeric mice
5. Chimeric mice produce two types pf gametes. One derived from host blastocyst and other from injected ES cells
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What are the clinical uses of genetic screening?
Diagnostic - detection of invading pathogenic organisms to screen blood for HIV or endogenous genetic mutation (sickle-cell anaemia)
Predicitve - BRCA1 and BRCA2 genes help control cell division in breast and ovarian tissues. Several variants exist for both which are associated with variance risk of developing breast cancer
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What is REFLP?
Restriction fragment length polymorphism
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How can a new restriction site be formed?
A point mutation could lead to an abolition/ creation of a restriction enzyme site. This may create a polymorphism in the number/sizes of DNA fragment produced by a particular restriction enzyme
- This change becomes a marker for the mutation
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How can the sickle-cell gene be detected by REFLD?
- Change in restriction enzyme site and therefore restriction enzyme by point mutation ---> polymorphism
- Will produce a different size fragment as enzyme cuts at different place to normal
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Are all REFLP's linked to a mutation in protein sequence?
No - due to the redudancy in the generic code some polymorphisms are silent
- Silent polymorphism lead to changes in restriction sites - without a change in protein sequence
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What is DNA fingerprinting/DNA profiling?
- Based on profiling specific regions in our genomes
- These regions contain repeats of certain short sequences
- The number of repeats within each region varies between individuals
- Named Variable Number of Tandem Repeats (VNTR)
- Uses more than one VNTR to generate a DNA profile
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What is VNTR?
- The number of repeats that caries between individuals and this can be used as a distinguishing feature
- The probability that four different VNTR sites are the same in different individuals is very small
