Unit 8: control of gene expression Flashcards
1
Q
what is a gene mutation
A
a change in the base sequence of DNA on chromosomes that can arise spontaneously during DNA replication in the interphase
2
Q
explain how a gene mutation can lead to the production of a non-functional protein or enzyme
A
- changes sequence of base triplets in DNA so changes sequence of condons on mRNA
- so changes sequence of amino acids in the encoded polypeptide
- so changes position of hydrogen/ionic/disulphide bonds between amino acids
- changes tertiary structure of protein
- enzymes active site changes shape, so substrate cant bind, ESC cannot form
3
Q
what are the different types of mutations
A
substitution
addition
deletion
duplication
inversion
translocation
4
Q
what are insertion/deletion mutations and what are the effects of this mutation
A
where one or more nucleotide pairs are inserted or deleted from the sequence. this type of mutation alters the sequence of nucleotides after the insertion/deletion point, known as frameshift
5
Q
3 effects of substitution
A
-formation of stop codon, which will stop production of the polypeptide prematurely, protein produced wont be functional
-formation of a codon for a different amino acid, polypeptide may differ in shape and be dysfunctional
-different codons produces the same amino acid because code is degenerate so the polypeptide produced is the same
6
Q
what is a duplication and what is the effect of this mutation
A
a sequence of DNA bases are repeated and therefore produces a frameshift
7
Q
what is inversion and what effect does this mutation have
A
a sequence of bases detach from the DNA sequence and then rejoin at the same position but in reverse order, this therefore affects the amino acid that is produced
8
Q
what is translocation and what effect does this mutation have
A
a sequence of DNA bases detaches and is inserted at a different location within the same or a different chromosome
this can often lead to significant effects on the phenotype
9
Q
what is the effect of translocation on bases
A
leads to an abnormal phenotype
e.g. development of some cancers or reduced fertility
10
Q
what is ‘frameshift’ mutation
A
occurs when mutations change the number of nucleotides/bases by a number not divisible by 3
-this shifts the way that the genetic code is read, so all the DNA triplets/ mRNA codons downstream from the mutation change
11
Q
explain why not all gene mutations affect the order of amino acids
A
some substitutions change only one triplet code/codon which could still code for the same amino acid as the genetic code is degenerate
-some also occur in introns which do not code for amino acids as they are removed during splicing
12
Q
explain why a change in amino acid sequence is not always harmful
A
may not change the tertiary structure of the protein and instead may positively change the properties of the protein, giving the organism a selective advantage
13
Q
what are mutagenic agents and examples
A
factors that increase the rate of mutations occurring
e.g. radiation (UV), chemicals (benzene) and some viruses (HPV)
14
Q
how can mutagenic agents increase the rate of mutations
A
deleting bases or changing their chemical structure so that they pair with bases that they wouldn’t normally do so.
-they can also change the structure of the dna itself, which can cause problems during dna replication
15
Q
what are acquired mutations
A
mutations which occur after fertilisation
-if these occur in the genes that control mitosis, can cause uncontrolled cell division and hence may result in a tumor
16
Q
describe how tumours and cancers form
A
mutations in DNA/genes controlling mitosis can lead to uncontrolled cell division
tumour formed if this results in a mass of abnormal cells
17
Q
benign tumors
A
non invasive
-usually grow slowly
-cells have normal/regular nuclei
-cells are well differentiated
18
Q
malignant tumors
A
invasive
-grow rapidly
-invade and destroy surrounding tissue
-cells can break off and travel around in the blood or lymphatic system
19
Q
function of tumour suppressor genes
A
code for proteins that:
-inhibit/slow cell cycle
-or cause self destruction of potential tumour cells
20
Q
what role do tumor suppressor genes have in the development of tumors
A
-mutation in DNA base sequence leads to the production of a non-functional polypeptide due to a change in the amino acid sequence which changes the proteins tertiary structure.
-decreased histone acetylation or increased DNA methylation prevents the production of protein as it prevents the binding of RNA polymerase to the promoter region, inhibiting transcription
-both lead to uncontrolled cell division
21
Q
function of (proto-)oncogenes
A
code for proteins that stimulate cell division
22
Q
what role do oncogenes have in the development of tumors
A
-when a mutation occurs in the DNA base sequence of a proto-oncogene, the gene can become overactive causing an overproduction of protein produced or a permanently activated protein
-decreased DNA methylation or increased histone acetylation increases the production of protein by stimulating the binding of RNA polymerase to the promoter region, stimulating transcription
-both lead to uncontrolled cell division
(an oncogene is a mutated form of the corresponding proto-oncogene)
23
Q
how can increased oestrogen concentration lead to tumors forming
A
- some breast cancers cells have oestrogen receptors, which are inactive transcription factors
- if oestrogen concentration is increased, more oestrogen binds to oestrogen receptors,
forming more oestrogen-receptor complexes which are active transcription factors - these bind to promoter regions of genes that code for proteins stimulating cell division
- this increases transcription / expression of these genes, increasing the rate of cell division
24
Q
why can it be difficult to interpret data on the risk factors of cancer
A
-some cancers are polygenic- triggered by more than one gene
-some cancers triggered by many environmental factors- difficult to know which environmental factors are having the greatest effect
-can have a control group of lab animals but not people
25
why is understanding mutations and methylation levels important in preventing and treating cancer
prevention:
possible to screen for certain cancers and look for a mutation to their DNA
new, more sensitive tests are being developed which will diagnose the disease earlier, giving a better prognosis#
treatment:
mutations to a proto-oncogene can be treated with a drug that inhibits the enzyme produced by the mutation so the cells stop expressing it and the mutation does not spread, tumor does not grow
26
what are stem cells
undifferentiated cells which can differentiate into specialised cells
27
describe how stem cells become specialised during development
stimuli lead to activation of some genes due to transcription factors. so mRNA is transcribed only from these genes and then translated to form proteins. these proteins modify cells permanently and determine cell structure and function
28
totipotent cells
occur for a limited time in early mammalian embryos and they can divide and differentiate into any type of body cell (e.g. plantecal cells)
29
pluripotent
found in mammalian embryos
-can divide and differentiate into most cell types (every cell type in the body but not placental cells)
30
multipotent cells
found in mature mammals
can divide and differentiate into a limited number of cell types
31
unipotent cells
found in mature mammals
can divide and differentiate into just one cell type
e.g. unipotent cells in the heart can divide and differentiate into cardiomyocytes
32
explain how stem cells can be used in the treatment of human disorders
-transplanted into patients to divide in unlimited numbers
-then differentiate into required healthy cells to replace faulty/damaged cells
33
induced pluripotent stem cells
(iPS cells)
-obtain adult somatic cells from patient and add specific protein transcription factors associated with pluripotency to cells so they can express genes with pluripotency. the transcription factors attach to the promoter regions of DNA, stimulating or inhibiting transcription. culture cells to allow them to divide by mitosis
34
evaluate the use of stem cells in treating human disorders
+ can divide and differentiate into required healthy cells, so could relieve human suffering by saving lives and improving quality of lives
+ embryos are often left over from IVF and so would otherwise be destroyed
+ iPS cells unlikely to be rejected by patients immune system as made with the patients own cells
X ethical issues with embryonic stem cells as obtaining them requires destruction of an embryo and potential life (embryo cannot give consent)
X immune system could reject cells and immunosuppressants are required
X cells could divide out of control, leading to the formation of tumours/cancer
35
transcription factors
proteins which regulate (stimulate or inhibit) transcription of specific target genes in eukaryotes by binding to a specific base sequence on a promoter region
36
how do transcription factors work
transcription factors move from cytoplasm to nucleus where they then bind to DNA at a specific DNA base sequence on a promoter region. this stimulates of inhibits transcription (production of mRNA) of target gene(s) by helping or preventing RNA polymerase binding
37
activators in transcription factors
activators stimulate or increase the rate of transcription by helping RNA polymerase bind to the promoter region on the target gene
38
repressors in transcription factors
repressors inhibit or decrease the rate by binding to the promoter region preventing RNA polymerase from binding
39
what happens to the transcription factor if a gene is 'switched off'
the site of the transcription factor is specific to that DNA is inactive because an inhibitor is attached
-this means it cannot cause transcription
40
how does oestrogen help with transcription
-lipid soluble nature of oestrogen means it can freely diffuse across the cell membrane where it binds to a receptor molecule on a transcription factor , forming an oestrogen-receptor complex
-the binding alters the shape of the inactive transcription factor forming an active transcription factor
-complex diffuses from cytoplasm into the nucleus via nuclear pores where it binds to a specific DNA base sequence on the the promoter region of a target gene and initiates transcription by helping RNA polymerase to bind
41
characteristics of SiRNA
-double stranded
-taken up by cells via vectors
-not in mammals, in lower animal + plant kingdoms
-binds perfectly so can only inhibit translation of specific mRNA sequences
42
characteristics of MiRNA
-single stranded
-made inside the cell within the introns of larger RNA molecules
-in all animals + plants
-pairing is imperfect so can inhibit the translation of many different mRNA sequences
43
how does SiRNA work
-unwinds in the cytoplasm, one strand is selected and the other is degraded
-the single selected strand binds to a complementary sequence of mRNA
-cell detects the double stranded form of mRNA and views it as abnormal. therefore, the enzyme is broken down by enzymes preventing translation
44
how does miRNA work in mammals
miRNA is not fully complementary to mRNA which means it can target more than one RNA molecule
-when miRNA is first transcribed, it creates a long, folded strand. which is then processed into a double strand then single by enzymes
-one strand binds to mRNA, blocks translation instead of cutting it into pieces
-the mRNA is either stored or degraded
45
define epigenetics
the study of how environmental cues can cause heritable changes to an organisms phenotype without changing the DNA/genotype
46
what is meant by epigenome
all chemical modifications of DNA and histone proteins- methyl groups on DNA and acetyl groups on histones
47
what are epigenetic marks
chemical groups or DNA or histone proteins that regulate the activation or silencing of genes through modulation of the intermolecular interactions between the DNA strands and the protein machinery
-do not alter base sequence of DNA
-they alter how easy it is for enzymes and other proteins needed for transcription to interact and transcribe the DNA
48
heritable changes of the epigenome
-epigenome is considered flexible, the tags respond to environmental cues
-the epigenome is an accumulation of these tags during a lifetime
-most tags are removed in the early foetus so do not get passed between generations
-those that escape removal are passed onto offspring. this means that the expression of some genes can be affected by environmental changes which affected their parents/grandparents
49
summarise the epigenetic control of gene expression in eukaryotes
50
explain how methylation can inhibit transcription
1. increased methylation of DNA - methyl
groups added to cytosine bases in DNA
2. so nucleosomes (DNA wrapped around
histone) pack more tightly together
3. preventing transcription factors and
RNA polymerase binding to promoter
51
explain how acetylation can inhibit transcription
1. decreased acetylation of histones
increases positive charge of histones
2. so histones bind DNA (negatively
charged) more tightly
3. preventing transcription factors and
RNA polymerase binding to promoter
52
how can knowledge of histone acetylation and DNA methylation be used to treat diseases
-use of drugs to inhibit enzymes that cause methylation, which can re-active genes that have been silenced
-genes must be specifically targeted to prevent switching on/off genes being read incorrectly which will cause a secondary cancer
-tests to identify the level of DNA methylation and histone acetylation to indicate an early stage of disease for the patients to seek early treatment and have a better chance of being cured
53
what is RNA interference
the inhibition of translation of mRNA produced from target genes, by RNA molecules e.g. siRNA and miRNA. therefore inhibiting the expression of (silencing) a target gene
54
genome
the genetic constitution of an organism, the complete set of genes
55
proteome
all the proteins a cell/the genome can produce
-protein only produced when the cell is switched on
56
what is genome sequencing and why is it important
it is the identification of the DNA base sequence of an organisms genome so amino acid sequences of proteins that derive from an organisms genetic code can be determined
57
what did the human genome project do
determined the sequence of bases of a human genome
-more samples were collected than used and no names used to remain anonymity
58
whole genome shotgun sequencing
cutting the DNA into smaller sections with overlapping ends
-use of computer programs to assemble them into an entire genome
59
genome sequencing in simple organisms
-organisms such as bacteria, have only a small number of introns so it is relatively easy to determine the proteome
-can be very useful in medical research (determining the protein antigens on a disease-causing bacteria can help to develop new vaccines)
-also allows for diseases to be monitored during outbreaks
60
genome sequencing in complex organisms
-the presence of more introns and regulatory genes makes it difficult to find the protein-coding sections among them
-this means the genome cannot be easily translated into the proteome
61
recombinant DNA
DNA from two different organisms that has been combined
-these organisms are known as transgenic/genetically modified
62
recombinant DNA technology
involves the transfer of fragments of DNA from one organism or species to another
63
how is it possible that the DNA of one organism is not only accepted by a different species but also functions normally when it is transferred
genetic code is universal (same in all organisms)
mechanisms of transcription & translation are essentially the same in all living organisms
-so transferred DNA can be transcribed & translated within the cells of the recipient organism and the proteins it codes for can be manufactured in the same way as they would be within the donor organism
64
reverse transcriptase to make DNA fragments
reverse transcriptase is an enzyme that catalyses the production of complementary DNA to form mRNA
PROCESS:
-cell that naturally produces the protein of interest is selected
-these cells have large amounts of mRNA for the protein so it is more easily extracted
-mRNA acts as a template for the reverse transcriptase enzyme
-this joins free DNA nucleotides with complementary bases to the mRNA sequence
-single stranded cDNA is isolated by hydrolysis of the mRNA with an enzyme
-double stranded DNA is then formed on the cDNA template using the enzyme DNA polymerase
-this double strand of DNA is the required gene
-the cDNA is intron free because it is based on the mRNA template
65
using restriction endonuclease enzymes
restriction endonuclease are enzymes that cut up DNA
-each restriction endonuclease cuts DNA at a specific sequence of bases (a recognition sequence) where it has an active site complementary to the recognition sequence
-the recognition sequence is often specific palindromic sequences
-the DNA is incubated with the specific restriction restriction enzyme
-the restriction enzyme hydrolyses the DNA
-this leaves sticky ends after the cut which are used to anneal the DNA fragment with other pieces of DNA with complementary base pairs
66
using a gene machine
synthesis of DNA fragments from scratch without the need for a pre-existing DNA template using a compterised machine
-desired sequence is made in the machine if it does not already exist
-the first nucleotide is fixed with support
-nucleotides are then added one by one with a protecting group to make sure they are joined at the correct place with no unwanted branching
-short sections of DN called oligonucleotides are produced by breaking off the support and supporting groups. they are then joined together to form long sections of DNA from each short section
67
advantages of using the gene machine
-any sequence of nucleotides can be produced
-in a very short time
-with great accuracy
68
advantages of using mRNA to make DNA fragments instead of restriction enzymes to cut gene from DNA
-more mRNA in cell than DNA- easily extracted
-introns removed by splicing (in eukaryotes) whereas DNA contains introns
-bacteria cant remove introns
69
what is in-vivo cloning
where copies of genes are made within a living organism
-as organism grows, it replicates the DNA
70
what is in-vitro cloning
where copies of the genes are made outside a living organism using the polymerase chain reaction
71
process of in-vivo cloning
-insert the DNA fragment into a vector
-vector DNA cut open by restriction endonuclease to ensure sticky ends will be complementary to the DNA fragments
-vector + fragment DNA are mixed together with ligase which joins the ends together via ligation
-recombinant DNA is created
-either the vector DNA or the DNA fragment must have specific promoter + terminator sequences for the desired particular protein
-the vector containing recombinant DNA transfers the gene into host cells
-if a plasmid is used, a change in temperature and use of certain chemicals will encourage the cell to take it in
-if a bacteriophage is used, it will inject recombinant DNA into the host cell so target DNA is integrated into the bacteria DNA
-the host cells take up the vector with the DNA fragment
-it will either code for antibiotic resistance or make the transformed genes fluoresce under UV light
-only those who are resistant to the specific antibody will be able to survive and replicate so those who grow are transformed
72
process of in-vitro cloning/PCR
-create a mixture containing the DNA fragment, free nucleotides, DNA polymerase and primers
-heat the DNA mixture to 95c in order to break the hydrogen bonds
-cool the mixture to 50-65c for primers to bind to the strands
-heat the mixture to 72c in order for the DNA polymerase to work
-the DNA polymerase lines up free nucleotides along each template fragment strand to create complementary strands in each cycle. 4 strands are created in each one
73
genetically modifying plants
-desired gene is inserted into a vector of either plasmid or bacteria
-the bacteria infects the plant and inserts its DNA into the genome
-the plant produces the protein if the correct promotor gene is present
-can be used for added nutrients or to cause resistance to pests
-a bacterial vector can infect the plant and cause it to develop the disease
74
genetically modifying animals
-desired gene is added into egg cell or early stage embryo
-modifying the egg means altering the genes of the germ cells which mature into GM egg and sperm
-germline editing is only of reproductive cells. this means it is possible to correct disease genes and pass them onto future generations
75
promotor gene
control what part of the body proteins are made in
-they are only activated by certain cells present in that area
76
benefits of recombinant DNA in medicine
-cheaper production of treatments
-quicker process
-larger quantities
77
benefits of recombinant DNA in agriculture
-crops larger and higher yield so prices will fall
-crops more resistant to disease
-increased shelf life
-crops can produce herbicides themselves, so cheaper
78
benefits of recombinant DNA in industry
-food production & cleaning can be done by enzymes made by recombinant DNA
-cheap production
-larger quantities made
79
risks of recombinant DNA in medicine
-companies may limit use of certain drugs by increasing charging prices because stock is limited
80
risks of recombinant DNA in agriculture
-plants could be infected with disease from vector
-decrease in biodiversity which damages food chains & ecosystem cohabitation
-seeds may blow into nearby farms and contaminate organic products
81
risks of recombinant DNA in industry
-no choice about eating GM food
-large companies control GM tech so small businesses are forced out because they cannot compete with lower prices
82
gene therapy
altering defective alleles to treat genetic disorders and cancer
83
somatic therapy
alters faulty alleles which are affected. doesn't effect sex cells, so disorder can still be inherited e.g. CF
84
germ line therapy
alters the alleles in sex cells, this means that all the cells will be altered and the individual, as well as their offspring will suffer the disease- this is currently illegal
85
ethical issues of gene therapy
-who decides what traits are normal and which constitute a disability/disorder
-will the high costs of gene therapy make it available to the wealthy
-should it be allowed to enhance basic human traits (e.g. height, intelligence)
86
DNA probes
short, single stranded DNA molecule that can be used to locate specific alleles on genes
87
production of DNA probes
sequence the allele you want to screen for and then use PCR to produce multiple complementary copies of the allele
88
what do DNA probes also contain
markers
-show where the probe has bound
-most common markers are radioactive, detected using x-ray film or a florescent maker detected with UV light
89
process of using DNA probes to screen for an allele
1. DNA sample digested into fragments using restriction enzymes. it is then separated using gel electrophoresis
2. DNA fragments are then transferred to a nylon membrane and incubated with the fluorescent marker. if the allele is present, the DNA probe will hybridise to it
3. UV light is shone on the nylon membrane, if the allele is present then the probe will have bound to the DNA fragment and will fluoresce
90
how can the use of DNA probes be helpful in genetic counselling
-used to give advice to people and their families about the options for genes to be screened
-can also advise if someone is a carrier for a mutant allele, what type of mutation it is and what treatments are an option
91
how can the use of DNA probes assist with personalised medicine
this means that it is personalised to their DNA
-doctors can predict the drugs you will respond best to and prescribe those
-this means that recovery time should be quicker and have a smaller impact on your life
92
genetic fingerprinting
technique that can detect differences in peoples DNA, it involves the use of variable number tandem repeats- which are short repeating sequences or bases
93
why are VNTRs useful in genetic fingerprinting
the probability of two individuals having identical VNTRs is extremely low therefore VNTR analysis can be used in genetic fingerprinting
94
process of making genetic fingerprints
-sample of DNA is taken and it is cut into fragments (using restriction enzymes) and PCR is used to make many copies of the areas of DNA within VNTR
-primers are attached to the sticky ends at either side of repeats and the whole repeat is amplified
-a fluorescent tag is attached, usually to the primer to be easily viewed under UV
-end result of fragments with different lengths which correspond to the number of repeats at a specific location
95
separating DNA fragments
fragments are placed into a well of agarose gel and covered in a buffer solution which conducts electricity. an electric current is passed through the gel
-DNA is negatively charged and so will move towards the positive electrode
-shorter, lighter fragments move further whilst longer, heavier wont move as far
96
analysis of separating DNA fragments
-gel is viewed under UV light and the DNA fragments are seen as bands
-2 or more individual bands can be compared, if they have the same bands then they have the same length of VNTRs at same location so its a match
97
why are ladders added to genetic fingerprinting when analaysing DNA fragments
ladder contains DNA fragments of known lengths, this allows you to calculate the length of the fragments that appear
