The Control of Gene Expression Flashcards
(135 cards)
1
Q
what is a mutation?
A
any change to the base (nucleotide) sequence of DNA
2
Q
how are mutations caused?
A
by errors during DNA replication
3
Q
how can the rate of mutations be increased?
A
by mutagenic agents
4
Q
list the 6 types of mutations that can occur
A
substitution
deletion
addition
duplication
inversion
translocation
5
Q
what is substitution?
A
one or more bases are swapped for another
6
Q
what is deletion?
A
one or more based are removed
7
Q
what is addition?
A
one or more bases are added
8
Q
what is duplication?
A
one or more bases are repeated
9
Q
what is inversion?
A
a sequence of bases is reversed
10
Q
what is translocation?
A
a sequence of bases is moved from one location in the genome to another. this could be the movement within the same chromosome or movement to a different chromosome.
11
Q
what is the result of a mutation in a polypeptide?
A
a mutation in a polypeptide that makes up an enzyme may change the shape of the enzyme’s active site. this may stop substrates from being able to bind to the active site, leaving the enzyme unable to catalyse the reaction.
12
Q
why don’t all mutations affect the order of amino acids?
A
the degenerate nature of the genetic code means that some amino acids are coded for by more than one DNA triplet. this means not all types of mutations will always result in a change to the amino acid sequence of the polypeptide.
13
Q
what mutations do change the amino acid sequence of a polypeptide and why?
A
additions, duplications and deletions.
this is because these mutations all change the number of bases in the DNA code. this causes a frameshift in the base triplets that follow, so that the triplet code is read in a different way.
14
Q
what are some examples of mutagenic agents?
A
ultraviolet radiation, ionising radiation, some chemicals and some viruses.
15
Q
what are the ways that mutagenic agents are able to increase the rate of mutations?
A
acting as a base — chemicals called base analogs can substitute for a base during DNA replication, changing the base sequence in the new DNA.
altering bases — some chemicals can delete or alter bases.
changing the structure of DNA — some types of radiation can change the structure of DNA, which causes problems during DNA replication.
16
Q
what are stem cells?
A
stem cells are unspecialised cells that can develop into other types of cell.
stem cells divide to become new cells, which then become specialised.
all multicellular organisms have some form of stem cell.
17
Q
where are stem cells found?
A
stem cells are found in the embryo and in some adult tissues.
18
Q
what are totipotent cells?
A
these are stem cells that can mature/develop into any type of body cell in an organism.
they are only present in mammals in the first few cell divisions of an embryo.
19
Q
what are multipotent stem cells?
A
stem cells present in adult mammals. these are able to differentiate into a few different types of cell.
20
Q
what are unipotent stem cells?
A
stem cells present in adult mammals. these can only differentiate into one type of cell.
21
Q
what are pluripotent stem cells?
A
stem cells that can specialise into any cell in the body. but they don’t have the ability to become the cells that make up the placenta.
22
Q
why can stems cells become specialised?
A
because different genes are expressed.
23
Q
how do stem cells become specialised?
A
- stem cells all contain the same genes — but during development, not all of them are transcribed and translated; expressed.
- under the right conditions, some genes are expressed and others are switched off.
- mRNA is only transcribed from specific genes.
- the mRNA from these genes is then translated into proteins.
- these proteins modify the cell — they determine the cell structure and control its functions.
- changes to the cell produced by these proteins cause the cell to become specialised. these changes are difficult to reverse, so once a cell has specialised it stays specialised.
24
Q
what are cardiomyocytes?
A
they are heart muscle cells that make up a lot of the tissue in our hearts.
25
what was the previous idea about cardiomyocytes from scientists?
that mature mammals are not able to regenerate their own heart cells at all. and so if their hearts become damaged or worn out due to old age this is a major problem.
26
what do scientists now think about cardiomyocytes?
if old or damaged, cardiomyocytes can be replaced by new cardiomyocytes derived from a small supply on unipotent stem cells in the heart.
27
how can stem cells be used to treat human disorders?
stem cells can be used to replace cells damaged by illness or injury.
stem cell therapies exist where bone marrow which contain stem cells can become specialised to form any type of blood cell. bone marrow transplants can be used to replace the faulty bone marrow in patients that produce abnormal blood cells. the stem cells in the transplanted bone marrow divide and specialise to produce healthy blood cells. this technique has been used to treat leukaemia and lymphoma.
28
list 3 conditions with its theoretical solutions that scientists have researched, where stem cells can be used as treatment.
spinal cord injuries — stem cells could be used to replace damaged nerve tissue.
damage caused by heart attacks — stem cells could be used to replace damaged heart tissue.
organ transplants — organs could be grown from stem cells to provide new organs for people on donor waiting lists.
29
what are the huge benefits to using stem cells in medicine?
it could save many lives — stem cells could be used to grow organs for those people awaiting transplants.
it could improve the quality of life for many people — it can replace the damaged cells in the eyes of people who are blind.
30
what are the 3 main potential sources of human stem cells?
adult stem cells
embryonic stem cells
induced pluripotent stem cells
31
what are adult stem cells?
- these are obtained from the body tissues of an adult.
- they can be obtained in a relatively simple operation with very little risk involved, but quite a lot of discomfort.
- adult stem cells aren't as flexible as embryonic stem cells — they can only specialise into a limited range of cells, not all body cell types.
- they're multipotent.
32
what are embryonic stem cells?
- these are obtained from embryos at an early stage of development.
- embryos are created in a lab using IVF — egg cells are fertilised by sperm outside the womb.
- once the embryos are approximately 4 to 5 days old, stem cells are removed from them and the rest of the embryo is destroyed.
- embryonic stem cells can divide an unlimited number of times and develop into all types of body cells.
- they're pluripotent.
33
what are induced pluripotent stem cells (iPS cells)?
- they are created by scientists in the lab. the process involves 'reprogramming' specialised adult body cells so that they become pluripotent.
- the adult cells are made to express a series of transcription factors that are normally associated with pluripotent stem cells. the transcription factors cause the adult body cells to express genes that are associated with pluripotency.
- iPS Cells could become really useful in research and medicine in the future. however more research is needed on true pluripotent embryonic stem cells before they can be properly utilised.
34
what are the ethical issues surrounding embryonic stem cell use?
- obtaining stem cells from embryos created by IVF raises ethical issues because the procedure results in the destruction of an embryo that could become a fetus if placed in the womb.
some people believe that at the moment of fertilisation an individual is formed has the right of life so they believe it is wrong to destroy embryos.
35
what are other issues surrounding stem cell use?
the use of iPS Cells can be used to grow a patients tissue or an organ for a transplant however there is a risk of rejection from the patients body as their immune system may recognise the tissue as foreign and attack it.
36
what is the transcription of genes controlled by?
protein molecules called transcription factors.
37
how do transcription factors operate?
- in eukaryotes, transcription factors move from the cytoplasm to the nucleus.
- in the nucleus they bind to specific DNA sites near the start pf their target genes — the genes they control the expression of.
- they control expression by controlling the rate of transcription.
38
what are the types of transcription factors?
activators
repressors
39
what are activators?
they stimulate or increase the rate of transcription — e.g. they help RNA polymerase bind to the start of the target gene and activate transcription.
40
what are repressors?
they inhibit or decrease the rate of transcription — e.g. they bind to the start of the target gene, preventing RNA polymerase from binding, stopping transcription.
41
what other molecule can affect the expression of genes?
oestrogen
42
how does oestrogen affect transcription?
oestrogen is a steroid hormone and it affects transcription by binding to a transcription factor called an oestrogen receptor, forming an oestrogen-oestrogen receptor complex.
the complex moves from the cytoplasm into the nucleus where it binds to specific DNA sites near the start of the target gene.
the complex can act as an activator of transcription.
43
can oestrogen act as a repressor?
yes, on some cells it can. it depends on the type of cell it is and the target gene.
44
what is RNA interference (RNAi)?
RNA interference (RNAi) is a process that affects gene expression in eukaryotes by stopping mRNA from being translated into proteins.
45
what are the molecules involved in RNAi?
the molecules involved in RNAi are called siRNA (small interfering RNA) and miRNA (microRNA).
46
how does RNAi work?
RNAi works by small, double-stranded RNA molecules binding to target mRNA, preventing its translation into proteins.
47
what happens to mRNA after transcription?
once mRNA has been transcribed, it leaves the nucleus for the cytoplasm.
48
what occurs in the cytoplasm during RNAi?
in the cytoplasm, double-stranded siRNA associates with proteins, unwinds, and a single strand binds to the target mRNA.
49
what is the role of siRNA in RNAi?
the base sequence of siRNA is complementary to sections of the target mRNA, leading to the mRNA being cut into fragments.
50
what happens to the mRNA fragments after they are cut?
the mRNA fragments move into a processing body, where they are degraded.
51
is there a similar process for miRNA in plants?
yes, a similar process occurs with miRNA in plants.
52
how does miRNA in mammals typically interact with target mRNA?
in mammals, the miRNA isn't usually fully complementary to the target mRNA, making it less specific than siRNA and allowing it to target more than one mRNA molecule.
53
what do miRNA and siRNA have in common, in mammals?
like siRNA, miRNA associates with proteins and binds to target mRNA in the cytoplasm.
54
what is the role of the miRNA-protein complex in translation, in mammals?
the miRNA-protein complex physically blocks the translation of the target mRNA.
55
what happens to the mRNA after miRNA binds to it, in mammals?
- the mRNA is moved into a processing body, where it can either be stored or degraded.
- when it's stored, it can be returned and translated at another time.
56
what is E. coli's primary energy source?
E. coli primarily respires glucose but can use lactose if glucose isn't available.
57
what enzyme does E. coli produce in the presence of lactose?
E. coli produces the enzyme B-galactosidase to digest lactose.
58
when is the gene for B-galactosidase expressed?
the gene for B-galactosidase is only expressed when lactose is present.
59
what controls the production of B-galactosidase?
the production of B-galactosidase is controlled by a transcription factor called the lac repressor.
60
what happens to the lac repressor when lactose is absent?
when lactose is absent, the lac repressor binds to the DNA at the start of the gene, stopping transcription.
61
what occurs when lactose is present?
when lactose is present, it binds to the lac repressor, preventing it from binding to the DNA, allowing the gene to be transcribed.
62
in the experiment, what was the purpose of isolating different E. coli mutants?
different E. coli mutants were isolated to study their behavior in various media, such as with lactose or glucose.
63
what determines whether a gene is expressed in eukaryotes?
epigenetic control can determine whether a gene is switched on or off.
64
how does epigenetic control work?
it works through the attachment or removal of chemical groups (epigenetic marks) to or from DNA or histone proteins.
65
do epigenetic marks alter the base sequence of DNA?
No, epigenetic marks don't alter the base sequence of DNA.
66
what do epigenetic marks alter?
They alter how easy it is for the enzymes and other proteins needed for transcription to interact with and transcribe the DNA.
67
what role do epigenetic changes play?
epigenetic changes to gene expression play a role in normal cellular processes and can occur in response to environmental changes.
68
how can epigenetic changes be inherited?
some epigenetic marks escape removal between generations and can be passed on to offspring.
69
how can environmental changes affect offspring gene expression?
the expression of some genes in the offspring can be affected by environmental changes that affected their parents or grandparents.
70
can you provide an example of inherited epigenetic changes?
epigenetic changes in some plants in response to drought have been shown to be passed on to later generations.
## Footnote
For example, changes have been passed on to three generations so far.
71
what is methylation of DNA?
methylation of DNA is when a methyl group is attached to the DNA coding for a gene.
## Footnote
A methyl group is a -CH₃ group.
72
where does methylation occur?
methylation occurs at a CpG site, where a cytosine and guanine base are next to each other in the DNA.
73
what effect does increased methylation have on gene expression?
increased methylation changes the DNA structure so that the transcriptional machinery can't interact with the gene, resulting in the gene being switched off.
74
what are histones?
histones are proteins that DNA wraps around to form chromatin, which makes up chromosomes.
75
how does chromatin structure affect gene transcription?
the condensation of chromatin affects the accessibility of DNA and whether it can be transcribed.
76
what happens when histones are acetylated?
when histones are acetylated, the chromatin is less condensed, allowing the transcriptional machinery to access the DNA and enabling gene transcription.
77
what occurs when acetyl groups are removed from histones?
when acetyl groups are removed, the chromatin becomes highly condensed, preventing the transcriptional machinery from accessing the genes.
78
what are histone deacetylase (HDAC) enzymes?
HDAC enzymes are responsible for removing acetyl groups from histones.
79
what role does epigenetics play in disease development?
epigenetics can lead to the development of various diseases, including cancer, Fragile X syndrome, Angelman's syndrome, and Prader-Willi syndrome.
80
what is Fragile X syndrome?
Fragile X syndrome is a genetic disorder that can cause learning and behavioral difficulties, as well as characteristic physical features.
81
what causes Fragile X syndrome?
it is caused by a heritable duplication mutation in the FMR1 gene on the X chromosome, resulting in the CGG DNA sequence being repeated excessively.
82
how does increased methylation affect the FMR1 gene?
Increased methylation of the FMR1 gene switches it off, preventing the production of the protein it codes for, which leads to the symptoms of Fragile X syndrome.
83
why are epigenetic changes good targets for new drugs?
epigenetic changes are reversible, making them suitable targets for drugs designed to combat the diseases they cause.
84
what is an example of a drug that treats diseases caused by increased methylation?
azacitidine is a drug used in chemotherapy for cancers caused by increased methylation of tumor suppressor genes.
85
what is the role of HDAC inhibitor drugs?
HDAC inhibitor drugs are used to treat diseases caused by decreased acetylation of histones, including some types of cancer.
86
what is the mechanism of action of HDAC inhibitors?
HDAC inhibitors work by inhibiting the activity of histone deacetylase enzymes, allowing genes to remain acetylated and be transcribed.
87
what is a challenge in developing drugs for epigenetic changes?
the challenge is ensuring that drugs are specific, as epigenetic changes occur normally in many cells, to avoid damaging normal body cells.
88
what determines the phenotype of an organism?
the phenotype is determined by the organism's genotype and the interaction of its genotype with the environment.
89
explain how overeating, something believed to be caused by environmental factors, may be influenced by genes.
- overeating was thought to be caused only by environmental factors, like an increased availability of food in developed countries.
- it was discovered that food consumption increases brain dopamine levels in animals.
- once enough dopamine is released, people would stop eating.
- researchers discovered that people with one particular allele had 30% fewer dopamine receptors.
- they found that people with this particular allele were more likely to overeat — they wouldn't stop eating when dopamine levels increased.
- scientists now believe that overeating has both genetic and environmental causes.
90
how can twin studies help determine influences on phenotype?
twin studies can help determine what's due to environmental factors and what's due to genetic factors.
91
why are identical twins useful for studying phenotype differences?
identical twins are genetically identical, so any differences in phenotype must be due to environmental factors.
92
what does it indicate if a characteristic is very similar in identical twins?
if a characteristic is very similar in identical twins, genetics probably plays a more important role.
93
what does it indicate if a characteristic is different between identical twins?
if a characteristic is different between twins, the environment must have a larger influence.
94
why is a large sample size important in twin studies?
data from twin studies involving a large sample size is better for drawing valid conclusions than data based on a small sample size.
95
what are acquired mutations?
mutations that occur in individual cells after fertilisation.
96
what happens when acquired mutations occur in the genes that control the rate of cell division by mitosis?
it can cause uncontrolled cell division.
97
what are tumours and how do they come about?
tumours are a mass of abnormal cells.
if a cell divides uncontrollably the result is a tumour.
98
what are cancers?
tumours that invade and destroy surrounding tissue.
99
what are two types of genes that control cell division.
tumour suppressor genes
proto-oncogenes
100
how can a mutation in tumour suppressor genes cause cancer?
- tumour suppressor genes can be inactivated if a mutation occurs in the DNA sequence.
- when functioning normally, tumour suppressor genes slow cell division by producing proteins that stop cells dividing or cause them to self-destruct (apoptosis).
- if a mutation occurs in a tumour suppressor gene, the protein isn't produced. the cells divide uncontrollably (the rate of division increases) resulting in a tumour.
101
how can a mutation in proto-oncogenes cause cancer?
- the effect of a proto-oncogene can be increased if a mutation occurs in the DNA sequence. a mutated proto-oncogene is called an oncogene.
- when functioning normally, proto-oncogenes stimulate cell division by producing proteins that make cells divide.
- if a mutation occurs in a proto-oncogene, the gene can become overactive. this stimulates the cells to divide uncontrollably (the rate of division increases) resulting in a tumour.
102
what are the two different types of tumours?
malignant tumours
benign tumours
103
what are malignant tumours?
- these are cancers.
- they usually grow rapidly and invade and destroy surrounding tissues.
- cells can break off the tumours and spread to other parts of the body in the bloodstream or lymphatic system.
104
what are benign tumours?
- these are not cancerous.
- they usually grow slower than malignant tumours and are often covered in fibrous tissue that stops cells invading other tissues.
- benign tumours are often harmless, but they can cause blockages and put pressure on organs.
- some benign tumours can become malignant.
105
in what ways do tumour cells differ from normal cells?
- they have an irregular shape
- the nucleus is larger and darker than those in normal cells. sometimes the cells have more than one nucleus.
- they don't produce all the proteins needed to function correctly
- they have different antigens on their surface.
- they don't respond to growth regulating processes.
- they divide by mitosis more frequently than normal cells.
106
why is methylation of DNA an important method of regulating gene expression?
it can control whether or not a gene is transcribed and translated.
107
when does methylation become a problem?
when it happens too much (hypermethylation) or when it happens too little (hypomethylation).
108
how is the growth of tumours caused by abnormal methylation of certain cancer-related genes?
- when tumour suppressor genes are hypermethylated, the genes are not transcribed — so the proteins they produce to slow cell division aren't made. this means that cells are able to divide uncontrollably by mitosis and tumours can develop.
- hypomethylation of proto-oncogenes causes them to act as oncogenes — increasing the production of the proteins that encourage cell division. this stimulates cells to divide uncontrollably, which causes the formation of tumours.
109
how is increased exposure to oestrogen thought to increase a women's risk of developing breast cancer?
- oestrogen can stimulate certain breast cells to divide and replicate. the fact that more cell divisions are taking place naturally increases the chance of mutations occurring, and so increases the chances of cells becoming cancerous.
- this ability to stimulate division could also mean that if cells do become cancerous, their rapid replication could be further assisted by oestrogen, helping tumours to form quickly.
- other research suggests that oestrogen is actually able to introduce mutations directly into the DNA of certain breast cells, again increasing the chance of these cells becoming cancerous.
110
how do genetic factors affect the risk of cancer?
some cancers are linked with specific inherited alleles. if you inherit that allele you're more likely to get that type of cancer.
111
how do environmental factors affect the risk of cancer?
exposure to radiation, lifestyle choices such as smoking, increased alcohol consumption and a high-fat diet have all been linked to an increased chance of developing some cancers.
112
explain how cancer can be prevented using an example (hint: BRCA1 tumour suppressor gene)
- if a specific cancer-causing mutation is known, then it is possible to screen for the mutation in a person's DNA. e.g. it's possible to screen for the mutated allele of the BRCA1 tumour suppressor gene, which greatly increases a woman's risk of developing breast cancer in her lifetime.
- knowing about this increased risk means that preventative steps can be taken to reduce it. e.g a woman with the BRCA1 mutation may choose to have a mastectomy (removal of one or both breasts) to significantly reduce the risk of breast cancer developing. women with this mutation may also be screened for signs of breast cancer more often than the rest of the population, as early diagnosis increases the chances of recovery.
- knowing about specific mutations also means that more sensitive tests can be developed, which can lead to earlier and more accurate diagnosis.
113
explain how cancer can be treated using an example (hint: HER2 proto-oncogene, aggressive treatment, gene therapy)
- the treatment for cancer can be different for different mutations, so knowing how specific mutations actually cause cancer can be very useful for developing drugs to effectively target them. for example, breast cancer caused by a mutation of the HER2 proto-oncogene can be treated with a drug called Herceptin. this drug binds specfically to the altered HER2 protein receptor and suppresses cell division and tumour growth. breast cancer caused by other mutations are not treated with this drug as it doesn't work.
- some cancer-causing mutations require more aggressive treatment than others, so understanding how the mutation that causes them, works, can help produce the best treatment plan. e.g if a mutation is known to cause an aggressive (fast-growing) cancer, it may be treated with higher doses of radiotherapy or by removing larger areas of the tumour and surrounding tissue during surgery.
- gene therapy (where faulty alleles in a person's cells are replaced by working versions of those alleles) may be also able to treat cancer caused by some mutations. for example, if you know that cancer is being caused by inactivated tumour suppressor genes, it's hoped that gene therapy could be used in the future to provide working versions of the genes.
114
define genome
a genome is the entire set of DNA, including all the genes in an organism
115
improvements in technology have allowed us to...
sequence the genomes of a variety of organisms, from bacteria to humans
116
what do gene sequencing methods only work on?
fragments of DNA. so if you want to sequence the entire genome of an organism, you need to chop it up into smaller pieces first. the smaller pieces are sequenced and then put back in order to give the sequence of the whole genome.
117
what is the human genome project?
this was the mapping of the entire sequence of the human genome for the first time.
it was completed in 2003.
118
what is the proteome of an organism?
the proteome of an organism is all the proteins that are made by it.
119
why is it relatively easy to determine simple organisms proteome from the DNA sequence of their genome?
simple organisms, such as bacteria, don't have much non-coding DNA (introns) which makes it easy to determine their proteome from the DNA sequence of their genome.
120
what are the benefits to studying proteomes?
there have been developments in vaccines, antibiotics, diagnoses and drug targets.
121
why is it harder to translate the genome of complex organisms?
- more complex organisms contain large sections of non-coding DNA.
- they also contain complex regulatory genes, which determine when the genes that code for particular proteins should be switched on or off.
- this makes it more difficult to translate their genome into their proteome, because it's hard to find bits that code for proteins among the non-coding and regulatory DNA.
121
how have sequencing methods been updated compared to the past?
- in the past, many sequencing methods were labour-intensive, expensive and could only be done on a small scale.
- now these techniques are often automated, more cost-effective and can be done on a large scale.
- with newer, faster techniques scientists can now sequence whole genomes much more quickly.
122
what does recombinant DNA technology involve?
it involves transferring a fragment of DNA from one organism to another.
123
explain why it doesn't matter if the recipient and donor organisms are not from the same species when partaking in recombinant DNA technology
because the genetic code is universal (the same DNA base triplets code for the same amino acids in all living things), and because transcription and translation mechanisms are pretty similar too, the transferred DNA can be used to produce a protein in the cells of the recipient organism.
124
define transgenic organisms.
organisms that contain transferred DNA
125
what are the three ways that DNA fragments can be produced?
- using reverse transcriptase
- using restriction endonucelase enzymes
- using a gene machine
126
explain, using reverse transcriptase, how DNA fragments can be produced
- most cells only contain two copies of each gene, making it difficult to obtain a DNA fragment containing the target gene. but they can contain many mRNA molecules which are complementary to the gene, so mRNA is often easier to obtain.
- the mRNA molecules can be used as templates to make lots of DNA. the enzyme, reverse transcriptase, makes DNA from an RNA template. the DNA produced is called complementary DNA (cDNA).
- to do this, mRNA is first isolated from cells. then it's mixed with free DNA nucleotides and reverse transcriptase. the reverse transcriptase uses the mRNA as a template to synthesise a new strand of cDNA.
127
explain, using a restriction endonuclease enzymes, how DNA fragments can be produced
- some sections of DNA have palindromic sequences of nucleotides. these sequences consist of antiparallel base pairs (base pairs that read the same in opposite directions).
- restriction endonucleases are enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) the DNA at these places.
- different restriction endonucelases cut at different specific recognition sequences, because the shape of the recognition sequence is complementary to the enzyme's active site.
- if recognition sequences are present at either side of the DNA fragment you want, you can use restriction endonucleases to separate it from the rest of the DNA.
- the DNA sample is incubated with the specific restriction endonuclease, which cuts the DNA fragment out via a hydrolysis reaction.
- sometimes the cut leaves sticky ends — small tails of unpaired bases at each end of the fragment. sticky ends can be used to bind (anneal) the DNA fragment to another piece of DNA fragment to another piece of DNA that has sticky ends with complementary sequences.
128
explain, using a 'gene machine', how DNA fragments can be produced
- technology has been developed so that fragments of DNA can be synthesised from scratch, without the need for a pre-existing DNA template. instead a database contains the necessary information to produce the DNA fragment. this means that the DNA sequence does not have to exist naturally — any sequence can be made.
the process of a gene machine:
- the sequence that is required is designed (if one doesn't already exist)
- the first nucleotide in the sequence is fixed to some sort of support e.g. a bead.
- nucleotides are added step by step in the correct order, in a cycle of processes that includes adding projecting groups. projecting groups make sure that the nucleotides are joined at the right points, to prevent unwanted branching.
- short sections of DNA called oligonucleotides, roughly 20 nucleotides long, are produced. once these are complete, they are broken off from the support and all the projecting groups are removed. the oligonucleotides can then be joined together to make longer DNA fragments.
129
what is 'in vivo' cloning?
once we've isolated the DNA fragment, we need to amplify it and to do this we use in vivo cloning to make copies of the DNA fragment. this is made inside a living organism.
130
explain step 1 of 'in vivo' cloning
1) the DNA fragment is inserted into vector DNA. they can be plasmids (small, circular molecules of DNA in bacteria) or bacteriophages (viruses that infect bacteria).
2) the vector DNA is cut open using the same restriction endonuclease that was used to isolate the DNA fragment containing the target gene. so the sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene.
3) the vector DNA and DNA fragment are mixed together with DNA ligase. DNA ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA. this process is called ligation.
the new combination of bases in the DNA (vector DNA + DNA fragment) is called recombinant DNA.
131
explain step 2 of 'in vivo' cloning
1) the vector with the recombinant DNA is used to transfer the gene into cells called host cells.
2) if a plasmid vector is used, host cells have to be persuaded to take in the plasmid vector and its DNA.
e.g. host bacterial cells are placed into ice-cold calcium chloride solution, to make their cell walls more permeable. the plasmids are
added and the mixture is heat shocked (heated to around 42 degrees celsius, for 1- 2 minutes), which encourages the cells to take in the plasmids.
3) with a bacteriophage vector, the bacteriophage will infect the host bacterium by injecting its DNA into it. The phage DNA (with the target gene in it) then integrates into the bacterial DNA.
4) host cells that take up the vectors containing the gene of interest are said to be transformed.
133
explain step 3 of ‘in vivo’ cloning
only around 5% of host cells will take up the vector and its DNA, so it's important to be able to identify which cells have been transformed.
Marker genes can be used to identify the transformed cells:
1) marker genes can be inserted into vectors at the same time as the gene to be cloned.
this means any transformed host cells will contain the gene to be cloned and the marker gene.
2) host cells are grown on agar plates. each cell divides and replicates its DNA, creating a colony of cloned cells. transformed cells will produce colonies where all the cells contain the cloned gene and the marker gene.
3) the marker gene can code for antibiotic resistance — host cells are grown on agar plates containing the specific antibiotic, so only transformed cells that have the marker gene will survive and grow. it can also code for fluorescence - when the agar plate is placed under a UV light, only transformed cells will fluoresce.
4) identified transformed cells are allowed to grow more, producing lots and lots of copies of the cloned gene.
134
how will the transformed host cells produce proteins?
– for the transformed host cells to produce the protein coded for by the DNA fragment, the vector needs to contain specific promoter and terminator regions.
– promoter regions are DNA sequences that tell the enzyme RNA polymerase when to start producing mRNA. terminator regions tell it when to stop. without the right promoter region, the DNA fragment won't be transcribed by the host cell and a protein won't be made.
– promoter and terminator regions may be present in the vector DNA or they may have to be added in along with the fragment.
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