Control of gene expeession Flashcards
(90 cards)
1
Q
Define gene mutation
A
Spontaneous changes in the sequence of nucleotides in DNA molecules
2
Q
What are insertion/deletion mutations?
A
One or more nucleotide is inserted or deleted from the sequence and can cause a frame shift
3
Q
What is a duplication mutation?
A
One or more bases is repeated and therefore produces a frame shift
4
Q
What is an inversion mutation?
A
Group of bases become separated form the DNA sequence then rejoin at the same position in reverse order.
5
Q
What is a translocation mutation?
A
Group of bases become separated from the DNA sequence on one chromosome and are inserted into the DNA sequence on another chromosome
6
Q
What are the 3 types of substitutions and explain them?
A
Silent- does not alter the amino acid sequence
Missense - alters a single amino acid in the polypeptide
Nonsense- creates a premature stop codon
7
Q
What are the effects of a mutation?
A
- Change amino acid
- Change in primary structure
- Change in tertiary structure
8
Q
How do mutations arise?
A
-Spontaneously during DNA replication
- Mutagenic agents
9
Q
What are the mutagenic agents?
A
- Chemical mutagens: like alcohol, tobacco
- Ionising radiation: alpha and beta, UV
- Spontaneous errors in replication
10
Q
What are stem cells?
A
Undifferentiated cells which can keep dividing by mitosis to differentiate into different cell types
11
Q
What is a totipotent stem cell?
A
Can differentiate into any stem cell
12
Q
What is a pluripotent stem cell?
A
Can differentiate to most cell types
13
Q
What is a multipotent stem cell?
A
Can differentiate to some cell types
14
Q
What is a unipotent stem cell?
A
Can differentiate to one type of stem cell
15
Q
What is a cardiomyocyte?
A
Muscle cell that makes up the heart and is responsible for contraction (unipotent)
16
Q
What are embryonic stem cells?
A
Derived form totipotent cells and pluripotent cells
17
Q
What are Adult stem cells?
A
Derived from multipotent cells
18
Q
What are iPS cells?
A
Induced pluripotent stem cells, pluripotent cells that are created by unipotent cells, via protein transcription factors
19
Q
How do cells differentiate?
A
Differences in how cells are expressed, so results in the synthesis of different polypeptide, determines the type of cell
20
Q
What is stem cell therapy?
A
When pluripotent cells are stimulated to multiply and differentiate, these can be transplanted
21
Q
How do we control transcription?
A
In eukaryotes, transcription of target genes can be stimulated or inhibited when specific transcriptional factors move form the cytoplasm to the nucleus.
-This can turn on and off genes, so only certain proteins are produced in a particular cell, this is what enables them to become specialised
22
Q
Outline transcriptional factors
A
-Transcription of a gene will only occur when a molecule from the cytoplasm enters the nucleus and binds to the DNA in the nucleus
-These molecules are proteins called transcription factors and each one can bind to different base sequences on DNA, therefore initiate transcription of genes.
-Once bound, transcription begins, creating mRNA and so on.
-Without the binding of a transcription factor, the gene is inactive, protein won’t be made.
23
Q
What hormone can initiate transcription and briefly explain how it works?
A
Oestrogen: steroid hormone that can initiate transcription
-Binds to a receptor site on the transcriptional factor
-This causes a change in shape which makes it complementary and able to bind to DNA to initiate transcription
-RNA polymerase can now attach and make mRNA
24
Q
Explain what RNA Interference is (regulation of translation)
A
Translation of mRNA produced from target genes can be inhibited by RNA interference (RNAi).
-mRNA molecule that has already been transcribed gets destroyed before its translated to a polypeptide chain.
-This is done by small interfering RNA (siRNA).
25
Explain the process of RNA interference
1. An enzyme can cut mRNA (double stranded) to siRNA. (single)
2. One strand of siRNA then combines with another enzyme
3. The siRNA-enzyme complex (RISC) will bind via complementary base pairing to another mRNA molecule.
4. Once bound, enzyme will cut up mRNA so it cannot be translated.
26
What is cancer?
A result of mutation in genes that regulate mitosis.
27
What causes a tumour?
Uncontrollable, spontaneous division of cells
28
Describe a benign tumour
-Non cancerous
-Grow at a slow rate
-Adhesion molecules stick to a particular tissue
-Remain compact so easier to remove
29
Describe a malignant tumour
-Cancerous
-Grow large rapidly
-Metastasis occurs, tumour breaks off and spreads to other parts of the body
-Not encapsulated, can grow projections into surrounding tissues and develop its own blood supply
-Need treatment radio or chemotherapy
-Recurrance is more likely
30
How does a tumour develop?
Due to a gene mutation in either the tumour suppressor gene and/or oncogene, the abnormal methylation of tumour suppressor genes and oncogenes, increased oestrogen concs.
31
What are oncogenes?
A mutated version of a proto-oncogene which creates a protein involved in the initiation of DNA replication and mitosis.
-Oncogene mutations can result in this process being permanently activated to make cells divide continually.
32
What are tumour suppressor genes?
-Produce proteins that slow down cell division and cause cell death if DNA copying errors are detected.
-A mutation may not produce this protein, cell division could continue and mutated cells would not be destroyed.
33
Describe abnormal methylation and the 2 types
Links to control of transcription- methylation can cause a gene to turn on or off.
Hypermethylated: tumour suppressor genes have an increased number of methyl groups attached to it, gene is inactivated (turned off).
Hypomethylated: oncogenes decrease the number of methyl groups attached, gene is permanently turned on.
34
Discuss increased oestrogen concentrations
-After menopause, oestrogen no longer regulates the menstrual cycle.
-Instead, fat cells in breast tissues can produce oestrogen, linked to breast cancer in women post-menopause
-This tumour results in even more oestrogen production which increases the size of tumour and attracts white blood cells, size increases.
This may be because oestrogen can activate a gene by binding to gene that initiates transcription, if a proto-oncogene the result is it is permanently turned on and activating cell division.
35
What are epigenetics?
A form of gene expression
The heritable change in gene function without changing the DNA base sequence.
36
What are epigenetics caused by?
Changes in the environment that can inhibit transcription
37
What factors can add epigenetic (chem tags) to DNA?
Diet, stress, toxins
A single layer of chemical tags on DNA is called the epigenome and impacts the the shape of DNA-histone complex, whether its tightly wound (no expression) or unwound (expression).
38
Describe methylation of DNA
-Increased methylation inhibits transcription
-When added to DNA, methyl groups attach to cytosine base
-prevents transcriptional factors from binding and attracts proteins that condense the DNA-Histone complex, so prevents a section being transcribed
39
Describe acetylation of histone proteins
-Decreased acetylation inhibits transcription
-Acetyl groups removed, histones become more positive and are attracted to phosphate group on DNA.
-Makes DNA and histones more strongly associated and hard for transcription factors to bind.
40
What is a genome?
The entire genetic material of an organism in the nucleus
41
What is sequencing of the genome?
Working out the DNA base sequence for all the DNA in a cell
42
What is an example of a genome sequencing method?
Sanger method
43
Discuss genome projects in smaller organisms
e.g prokaryotes, do not contain introns
-You can directly sequence the proteins that derive from the genetic code (proteome)
-Could identify potential antigens to use in a vaccine
44
Discuss genome projects in larger more complex organisms
Has introns, so knowledge of the genome cannot be easily translated into the proteome
45
What does gene sequencing allow?
Genome wide comparisons between individuals and species
-Find evolutionary relationships
-Develop medicine
46
What is the Human Genome Project?
International scientific project which has successfully determined the sequences of bases of a human genome
so we can:
-screen for mutated sequences
-screen fir disorder e.g Huntington's disease
47
What is a limitation of the Human Genome Project?
Ethical concerns:
-People being discriminated against
-Misuse of ownership of genetic info
48
What is recombinant DNA technology?
The combining of different organisms DNA, which could enable scientists to manipulate and alter genes to improve industrial processes or medical treatment.
49
What is the first step in recombinant DNA technology?
Produce DNA fragments to be recombined with another piece of DNA.
50
What are the 3 methods of creating DNA fragments?
1. Reverse transcription
2. Restriction endonucleases
3. Gene machine
51
Explain the 'Reverse Transcription' method of creating a DNA fragment
1. Reverse transcriptase makes DNA copies from mRNA
2. A cell that naturally produces the protein of interest is selected; these cells should have large amounts of mRNA for the protein.
3. The reverse transcriptase enzyme joins the DNA nucleotides with complementary bases to the mRNA sequence.
4. Single stranded DNA is made (cDNA)
5. To make this DNA fragment double stranded, the enzyme DNA polymerase is used.
52
Explain the 'Restriction Endonucleases' method of creating a DNA fragment
1. Restriction endonuclease are enzymes that cut up DNA (these naturally occur in bacteria as a defence mechanism)
2. Many of these enzymes have an active site complementary in shape to a range of different DNA base sequences, described as recognition sequences and therefore each enzyme cuts the DNA at a specific location.
3. Some enzymes cut at the same location in the double strand and create a blunt end, other enzymes create staggered ends and expose DNA bases.
4. The exposed staggered ends are palindromic and are 'sticky ends' because they have the ability to join to DNA with complementary base pairs.
53
Explain the 'Gene Machine' method of creating a DNA fragment
In the lab, using a computerised machine
1. Scientists examine the protein of interest to identify the amino acid sequence, and from that work out what mRNA and DNA sequence would be.
2. DNA sequence is entered into a computer, checks for biosafety and biosecurity that the DNA is safe and ethical to produce.
3. Computer can create small sections of overlapping single strands of nucleotides that make up the gene, called oligonucleotides.
4. The oligonucleotides can be joined to create DNA for the entire gene.
5. PCR can be used to amplify the quantity and make a double strand
This process is very quick and accurate and makes intron free DNA so it can be transcribed in prokaryotic cells.
54
Pros and cons of Reverse transcriptase?
+ mRNA present in the cell is from actively transcribed genes, so lots of the mRNA of interest available to make cDNA
- More steps, more time consuming and technically more difficult
55
Pros and cons of Restriction endonucleases?
+ Sticky ends on DNA fragment make it easier to insert into recombinant DNA
-Still contains introns
56
Pros and cons of Gene Machine?
+ Can design exact DNA fragment you want with sticky ends, labels and preferential codons
- Need to know the sequence of amino acids or bases.
57
What is in vivo cloning?
Within the organism, like body cells
58
Explain what promoter and terminator regions are used for in in vivo cloning
Promotor region: must be added at the start of the DNA fragment.
-This is a sequence of DNA which is the binding site for RNA polymerase to enable transcription to occur.
Terminator region: must be added to the end of the gene.
-Causes RNA polymerase to detach and stop transcription, so only one gene at a time is copied into mRNA
59
What is the first step of in vivo cloning?
Inserting DNA into a vector
60
What is a vector?
Something to carry the isolated DNA fragment into the host cell
Such as plasmids
61
Explain how a DNA fragment is inserted into a vector (step 1 in vivo)
1. The plasmid is cut open using the same restriction endonuclease
2. This creates the same sticky ends
3. The DNA fragments sticky ends are complementary to the sticky ends on the plasmid.
4. DNA fragment and cut plasmid are combined by the enzyme DNA ligase
-Ligase catalyses the condensation reaction to form phosphodiester bonds.
62
Explain the second step to in vivo cloning
Vector next needs to be inserted into the host cell, where the gene will be expressed to create the protein required.
1. Cell membrane of the host cell must be more permeable
2. To increase permeability, the host cells are mixed with Ca 2+ ions and heat shocked.
This enables the vector to enter the host cell cytoplasm.
63
What is the 3rd step in in vivo cloning?
Identifying transformed cells
-Not all host cells successfully take up a recombinant plasmid
64
What may be some issues with in vivo cloning?
-Recombinant plasmid does not get inside cell
-Plasmid re-joins before DNA fragment entered
-DNA fragment sticks to itself
65
What are marker genes?
On plasmids, can be used to identify which bacteria successfully took up the recombinant plasmid
66
What are the 3 different types of marker genes?
1. Antibiotic resistance genes
2. Genes coding for fluorescent proteins
3. Genes coding for enzymes
67
Explain the process of antibiotic resistance marker genes
Insert into plasmid: a gene that makes it resistant to the antibiotic tetracycline and a gene resistant to ampicillin.
1. DNA fragment is inserted in the middle of the resistant tetracycline gene, so it is disrupted, so can no longer create a protein that is resistant to tetracycline
2. Grow the bacteria on agar
3. Transfer bacterial colonies to a plate with ampicillin antibiotic in the agar (using a sterile velvet block), if it grows it must have the plasmid in it
4. Transfer those bacterial colonies to a plate with tetracycline antibiotic within agar. Any colonies still growing must the original plasmid that does not contain the gene of interest.
68
Explain the process of fluorescent markers
Jellyfish contain a gene coding for green fluorescent protein (GFP)
1. GFP is inserted to fragment
2. DNA fragment is inserted in the middle of the GFP gene
3. This disrupts it and prevents GFP production
4. Once grown on agar and put under UV, the ones that don't glow is the recombinant plasmid
69
Explain the process of enzyme markers
The enzyme lactase can turn a certain substance blue from colourless
1. The gene for this enzyme is inserted into the plasmid
2. DNA fragment is inserted in the middle of this gene to disrupt it
3. The bacteria are then grown on an agar plate with the colourless substance.
4. The colonies which cannot turn the colourless substance blue contain the recombinant plasmid.
70
How do you grow the host cell?
A fermenter is used to grow multiple copies of the host cell which have been identified as containing the recombinant plasmid.
-This large, cloned population of the host cell can then produce the protein coded for by the inserted DNA fragment.
71
What is in vitro cloning?
DNA fragments are amplified via PCR
-Cloning outside of the body e.g in a test tube
72
What does PCR stand for?
Polymerase chain reaction
73
What equipment do you need for PCR?
-Thermocycler
-DNA fragment
-DNA polymerase (taq polymerase from hot springs)
-Primers (Short sequence of single stranded DNA)
-DNA nucleotides
74
Explain the method of PCR
1. Denaturing: Temp (in thermocycler) is increased to 95 degrees to break the hydrogen bonds and split the DNA fragments into 2 strands
2. Annealing: temp is decreased to 50-65 degrees so that primers can attach
3. Synthesis: temp increased to 72 degrees (optimum for taq polymerase) and DNA polymerase then attaches complementary free nucleotides and makes a new strand to align next to each template.
75
What are the advantages of PCR?
-Automated: more efficient
-Rapid: 100 billion copies of DNA can be made within hours
-Doesn't require living cells: quicker and less complex techniques needed
76
What is genetic fingerprinting?
The examination of VNTRs
77
What are VNTRs?
Variable number tandem repeats
78
Discuss VNTRs
-Found in introns of human DNA
-The more closely related you are, the more similar your VNTRs are
-Analysing them can determine genetic relationships and genetic variability within a population.
79
What are the 6 steps within genetic fingerprinting?
1. Collection and extraction
2. Digestion
3. Separation (gel electrophoresis)
4. Hybridisation
5. Development
6. Analysis
80
Explain step 1 of genetic fingerprinting- collection and extraction
-Collect from blood, body cells or hair follicles
-If you only have a small sample, PCR can be used to amplify amount of DNA
81
Explain step 2 of genetic fingerprinting- digestion
-restriction endonucleases are added to cut the DNA into smaller fragments
-select enzymes that cut just before VNTRs
82
Explain step 3 of genetic fingerprinting- Separation
Gel electrophoresis:
1. DNA samples are loaded into small wells in agar gel. The gel is placed in a buffer liquid with an electrical voltage applied.
2. DNA is negatively charged (phosphate group) so DNA samples move through gel towards the positive end of the gel
3. The gel creates resistance for the moving DNA and smaller pieces can move faster and further
4. This is how different lengths of DNA (VNTRs) are separated
5. An alkaline is then added to separate the double strands of DNA
83
Explain step 4 of genetic fingerprinting- Hybridisation
-DNA probes (short single stranded pieces of DNA) are added to label the fragments, they are complementary to the VNTRs
84
Explain step 5 of genetic fingerprinting- Development
-Agar will shrink and crack as it dries so the VNTRs and DNA probes are transferred to a nylon sheets.
-The nylon sheets can be exposed to x-rays to visualise the position of the radioactive gene probes, or UV is fluorescent used
85
Explain step 6 of genetic fingerprinting- Analysis
-Position of DNA bands are compared to identify genetic relationships
e.g paternity tests
all bands will line up to mum, ones that don't line up to dads.
86
What are DNA probes?
-Short, single stranded DNA
-Labelled radioactively or fluorescently
-Used to locate specific alleles of genes and to screen patients for heritable conditions
-Created to have complementary base sequence to the allele that is being screened for
87
What is DNA hybridisation?
-Patients DNA sample is heated to make it single stranded
-Heat causes hydrogen bonds to break between bases
This is mixed with the DNA probe and cooled, complementary sequences can align and form hydrogen bonds.
88
What is genetic screening?
-Screen for potential genetic disorders or for the presence of cancer causing oncogenes.
-Using an array, you can screen for multiple diseases simultaneously.
89
What is an advantage of genetic screening?
Personal medicine:
-Certain drugs are more or less effective depending on your genotype
-Helps determine the best dose, increases effectiveness, safety and save money.
90
What is genetic counselling?
Like a type of social work where people can have their family history researched to consider the likelihood of them carrying any alleles linked to diseases before starting a family or for their general health
