DNA & Gene Expression Flashcards
1
Q
Give four adaptations of DNA.
A
- Very stable - doesn’t change
- Double helix & backbone - protect bases from damage
- Weak H-bonds - strands are easily separated
- Very large - carries lots of information
2
Q
What are the two experiments for proving DNA is hereditary material?
A
- Pneumonia in mice experiment
- Virus in bacteria with radioactively labelling using sulphur/phosphorus
3
Q
State the components of a DNA nucleotide.
A
- Deoxyribose
- Phosphate group
- Organic base
4
Q
What are the four DNA bases?
A
- Adenine
- Cytosine
- Guanine
- Thymine
5
Q
What are the base pairs?
How many H-bonds are there between the bases in each pair?
A
- Adenine & Thymine - 2 bonds
- Cytosine & Guanine - 3 bonds
6
Q
Which bases are:
- Single ring
- Double ring
A
- Cytosine & Thymine
- Adenine & Guanine
7
Q
What are genes?
A
Sections of DNA that code for a specific polypeptide.
8
Q
What are alleles?
A
Different forms of a gene.
9
Q
What are the names of the:
- Coding sections of a gene
- Non-coding sections of a gene
A
- Exons
- Introns
10
Q
What is the structure of a chromosome?
A
A single chromatid (before the DNA is replicated)
OR
Two chromatids joined at a centromere (after DNA replication)
11
Q
What holds the DNA in place in a chromosome?
A
Histone proteins
12
Q
What solution is used to find out the densities of DNA relative to each other?
A
Caesium chloride (CsCl)
13
Q
Why DNA replication is semi-conservative?
A
- Each strand acts a template
- Each new helix contains one “new” strand and one “parental” strand
14
Q
What process ensures exact DNA replication?
A
Complementary base pairing
15
Q
What is the role of DNA helicase?
A
Breaks the H-bonds and separates the two strands
16
Q
What is the role of DNA polymerase?
A
Joins the activated nucleotides together, forming the second polynucleotide strand
17
Q
What are the steps of DNA hybridisation?
A
- Extract & purify DNA from two species
- Cut DNA into short pieces
- Label the DNA of one species
- Mix the DNA and heat
- All DNA to cool (reanneal)
- Separate hybrid DNA
- Heat gently
- Measure the temperature at which the strands separate
18
Q
What is a codon?
A
A base triplet on mRNA which codes for an amino acid
19
Q
What is meant by the degenerative code of DNA?
A
An amino acid can be coded for by more than one triplet
20
Q
What are the stages of protein synthesis?
A
- Transcription
- Splicing
- Translation
21
Q
What happens during transcription?
A
Occurs in the nucleus
- DNA helicase separates the two strands (breaks H-bonds)
- Free RNA nuceotides bind to the template strand by complementary base pairing
-
RNA polymerase joins the free nucleotides
- Forms phosphodiester bonds
- When a terminator region is reached, transcription ends
22
Q
What happens during splicing?
A
Spliceosomes splice out (remove) the introns from the pre-mRNA to form mature mRNA
23
Q
What happens during translation?
A
Occurs at the ribosome
- Amino acid carried to mRNA by tRNA
- Each tRNA carries a specific amino acid
- The anticodon is attracted to its complementary codon on the mRNA
- The amino acids are joined by a peptide bond
- The tRNA detaches and collects another amino acid
- The ribosome moves along the mRNA
- Continues until a stop codon is reached
24
Q
What is a mutation? What are the types?
A
A change in the DNA base sequence resulting from:
- Substitution of a base
- Deletion of a base
25
What can happen when a base is substituted?
1. Nonsense: results in a stop codon
2. Mis-sense: results in a different amino acid
3. Silent: results in no change in the amino acid sequence
26
What happens when a base is deleted?
Results in a **frame-shift** which affects all the amino acids **after** the deletion
27
What is a mutagenic agent? What are some examples?
Anything which increases the frequency at which mutations occur. For example:
1. High energy radiation (X-ray, UV, gamma rays)
2. High energy particles
3. Tobacco **tar**
4. Nitrous acid
5. Benzene
28
What genes regulate cell division?
Proto-oncogenes: stimulate cell division
Tumour suppressor gene: slow (inhibit) cell division
29
What happens when the genes regulating cell division mutate?
Proto-oncogenes:
* The pathway is activated without the relevant signalling molecule
Tumour-suppressor genes:
* The gene becomes inactivated so cell division isn't inhibited
30
What differs between specialised cells?
The genes which are expressed
31
What is a stem cell?
Unspecialised cells which can differentiate into other specialised cells
32
What are the types of stem cell?
* Totipotent: can develop into **any** cell (embryonic stem cells)
* Multipotent: can develop into **some** cells (adult stem cells)
33
What are meristem cells?
Totipotent stem cells found in plants
Found where the plant is growing (shoots/roots)
They can be used to grow a whole plant in vitro
34
How is gene expression regulated by oestrogen?
1. Oestrogen diffuses across phospholipid bilayer into cell
2. Binds to a transcription factor causing an inhibitor to be released: binding site exposed
3. This allows the transcription factor to bind the specific sequence of bases on the DNA
4. This initiates transcription of mRNA (the gene is expressed)
35
What is siRNA?
**_S_**mall **_i_**nterfering RNA: short, double stranded pieces of RNA
36
How is gene expression regulated by siRNA?
* One strand binds to an enzyme
* The strand then binds to the complementary bases on the mRNA
* The enzyme breaks up the mRNA into small pieces
* Hence, the protein isn't produced (gene isn't expressed)
37
How can DNA fragments be made?
Reverse transcriptase
Restriction enzymes
38
What is reverse transcriptase? How does it work?
* Viral enzyme, catalyses "reverse transcription"
* mRNA is used as a template to produce **cDNA** (complementary DNA)
* Produces a copy of a gene without **introns**
39
What to restrictin enzymes do?
Cut DNA at specific **recognition sites**
Forms sticky ends
40
What are the types gene cloning?
In vitro - in test tubes
In vivo - in living cells
41
How are genes cloned in vitro?
Done by PCR (polymerase chain reaction):
1. DNA sample mixed with:
* DNA polymerase
* Free nucleotides (dNTPs)
* Primers
2. Sample heated to 95ºC
* Separate strands
3. Cool to 55ºC
* Allow primers to anneal
4. Heat to 72ºC
* Allow DNA polymerase to work
42
How are genes cloned in vivo?
Done by transforming bacteria:
1. DNA and plasmid cut with the **same** restriction enzyme
* To produce same sticky ends
2. Mixed with **DNA ligase** which joins the two pieces of DNA
3. Plasmids introduced into bacteria by shock treatment
4. Identification 1 - bacteria with a plasmid
* Ampicillin resistance gene
5. Identification 2 - bacteria with plasmid with the gene
* Tetracylin resistance gene or GFP
6. Desired bacteria grown in vats at optimal conditions
7. Protein/gene/DNA fragment is extracted and purified
43
What is gene therapy? What are types?
Using cloned genes to treat genetic diseases. Types:
1. Somatic - inserted into body cells
2. Germ line - inserted into sex cells
44
What vectors are used to insert a gene into a cell?
Viruses
Liposomes
45
What is the cause of the symptoms of cystic fibrosis?
1. Caused by deletion three bases in the **CFTR gene**
* Results in non-functional Cl- channel
2. Cl- ions cannot leave cells
* Water does not follow by osmosis
3. Leads to thick and sticky mucus
46
How can cystic fibrosis be treated?
Normal CFTR gene inserted into a virus/liposome
Administered by a nasal spray
47
What are DNA probes?
Short strands of DNA that can be used to identify specific base sequences
Radioactively/fluorescently labelled
48
How can DNA be sequenced?
Sanger method
Restriction mapping
49
What is the Sanger method for determining the DNA base sequence?
1. In a test tube:
* DNA strand
* DNA polymerase
* Primers
* Free nucleotides
* Modified nucleotides (ddNTPs) one of A\*, C\*, G\* or T\*
2. The modified nucleotides cause DNA replication to terminate
3. The four tubes are run on an electrophoresis gel, the bands are read from the furthest nucleotide first to determine the base sequence
50
How can the base sequence be determined using restriction mapping?
1. DNA is digested using restriction enzymes
2. Fragments are separated by gel electrophoresis
3. Done with multiple enzymes to determine the order of the fragments
51
What does gel electropheresis do? How does it work?
Separates DNA fragements **by size**
1. The DNA is digested with restriction enzymes
2. The fragments are placed in wells of an agarose gels
3. A current is passed through the gel
* The smallest fragments move the furthest
4. The fragments are compared to fragments of known size
52
How does genetic fingerprinting work?
Uses STRs/**minisatellites** - found in everyone's DNA
Inherited from parents and unique to a person
53
What is the process of genetic fingerprinting?
1. Extract DNA and purify
2. Digest with restriction enzymes
3. Separate by gel electrophoresis
4. Heat to separate strands
5. Transfer to a nylon membrane
6. Add fluorescent/radioactive probes
7. Autoradiography - expose x-ray film (if using radioactive)
54
What are the uses of genetic fingerprinting
Paternity testing
CSI
