Unit 8 - Control of Gene Expression Flashcards
what causes gene mutations?
DNA replicational errors in interphase generally by substitution, addition or deletion of bases from the normal DNA sequence
occur naturally
what increases the rate of mutations?
mutagenic agents:
UV light
ionising radiation
chemical carcinogens e.g. tobacco tar & asbestos
define mutagenic agent
a factor that increases the rate of gene mutations
most mutations have…
a negative/neutral impact (selected against) on the organism but some are selected for (as they create beneficial alleles) by natural selection
what are the 3 types of base substitution mutations?
silent mutation
mis-sense mutation
non-sense mutation
describe silent mutation
base substitution - one base substituted for another
new codon codes for same amino acid
because DNA code is degenerate
so no effect on 1y structure
so no effect on 2y or 3y structure
so protein function unaffected
describe mis-sense mutation
base substitution - one base substituted for another
new codon codes for a different amino acid
so different 1y structure
so different 2y & 3y structure due to different H bonds, ionic bonds or disulfide bonds b/w R groups
so different specific 3d shape & function
describe non-sense mutation
base substitution - one base substituted for a stop codon
causes premature translation
shorter 1y structure
so different 2y & 3y structure
different H bonds, ionic bonds & disulfide bonds formed b/w R groups
different specific 3d shape
loss of function - no ESCs formed if about enzymes
describe addition & deletion mutations
addition - a base is added to the DNA sequence
deletion - a base is removed from the DNA sequence
causes frameshift, shifting the last base of each codon into the next codon to produce a different sequence downstream of the mutation
addition causes frameshift to the right
deletion causes frameshift to the left
so different 1y structure
different 2y & 3y structure
NB addition/deletion of bases in multiples of 3 does not cause frameshift so is less detrimental to the overall protein function
describe base duplication mutation
one or more bases are repeated, causing frameshift to the right
different 1y, 2y, 3y etc.
describe inversion of bases mutation
a group of bases becomes separated from the DNA sequence & re-joins at the same position but in the inverse order
different 1y, 2y, 3y etc.
describe translocation of bases mutation
bases are separated from the DNA sequence on one chromosome & re-join on a different chromosome
affect gene expression & phenotype
can lead to cancer
summary of protein synthesis
all cells (apart from gametes & RBCs) have the same DNA/genes but they express the genes differently = control of gene expression
transcription factors control which genes are transcribed
splicing
siRNA can destroy mRNA molecules = controls how much mRNA is translated
activity of a protein can be altered by other enzymes - secondary messenger model
all cells in the body…
have the same DNA/genes but have different structures & functions
they are specialised (differentiated) for a specific function & only transcribe & translate the proteins they need
sperm + egg =
zygote
describe totipotent stem cells
a fertilised egg/zygote
early cells derived from zygote by mitosis for a limited time in a mammalian embryo
unspecialised
capable of differentiation into any specialised cell
able to divide for long periods = self-renewal
how do totipotent stem cells develop?
translate only part of their DNA, leading to cell specialisation
describe pluripotent stem cells
embryonic stem cells in humans
can give rise to most cell types needed for a foetus to develop
cannot form placental cells
what is the medical use for pluripotent stem cells?
can divide an unlimited number of times (self-renewal)
& can be used to treat a variety of human disorders e.g. genetic disorders like type 1 diabetes (B cells in pancreas) & paralysis (nerve cells)
describe multipotent stem cells
adult stem cells e.g. in bone marrow
can differentiate into a limited range of specialised cells
describe unipotent cells & e.g.
cells that can only divide to form one cell type e.g. formation of cardiomyocytes
why is specialisation irreversible?
most animal adult cells are specialised & unable to divide
(stem cells replace them by mitosis)
although cells retain all the genes of an organism, many genes are permanently switched off (not expressed)
why is it controversial to use pluripotent embryonic stem cells?
right to life
embryo cannot consent
human rights argument
describe induced pluripotent stem cells
generated by using appropriate protein transcription factors
similar to embryonic stem cells
capable of self-renewal
could replace embryonic stem cells in medical research & treatment
would not have the same ethical & rejection (iPS made from patient’s adult cells) issues associated with the use of embryonic stem cells
how is transcription of target genes controlled?
transcription of target genes can be stimulated or inhibited when specific transcription factors move from the cytoplasm to the nucleus
why might embryonic pluripotent stem cells cause harm to recipient?
might differentiate into the wrong types of cells
might divide out of control, leading to a tumour/cancer
why are embryonic pluripotent stem cells suitable to treat damage?
they divide
& differentiate into spec. cells
so can replace any type of cell
describe the control of gene expression
every somatic cell in an organism contains the same DNA & genes (same genome)
some genes are permanently expressed (switched on) in all cells e.g. genes coding for respiration enzymes
other genes are never expressed (permanently switched off) e.g. insulin gene in skin cells
some genes are switched on & off as & when needed = control of gene expression
e.g. lactase enzyme
describe human haemoglobin as an example of the control of gene expression
see booklet for graph
shift in gene expression around the time of birth from gamma globulin to beta globulin
human haemoglobin has 4 globulin polypeptide chains (4y)
adult form has 2 alpha & 2 beta polypeptides
foetal form has 2 alpha & 2 gamma polypeptides
describe & explain the structure of foetal haemoglobin
foetal form has 2 alpha & 2 gamma polypeptides
higher affinity for O2
so higher saturation of O2 in placenta
how are genes expressed (switched on & off) in a eukaryote?
by transcription factors
why don’t prokaryotic cells have regulation of transcription by transcription factors?
lack a nucleus that separates transcription & translation
define transcription factor
protein usually found in cytoplasm that stimulate transcription
how do transcription factors work?
- TF move from cytoplasm into nucleus, where they bind to specific DNA base sequence or promotor region
- RNA polymerase binds to DNA-TF complex
- RNA polymerase moves along gene, catalysing the formation of phosphodiester bonds b/w adjacent RNA nucleotides by condensation
so gene is expressed & mRNA is transcribed
describe the action of an inhibitor molecule
the inhibitor molecule binds to the transcription factor so it cannot bind to DNA
so the gene not expressed
describe how non lipid soluble hormones can control gene expression
e.g. glucagon - a protein hormone so not lipid soluble
binds to receptor at CSM - secondary messenger model (see unit 6)
describe how lipid soluble hormones can control gene expression
e.g. oestrogen - a steroid hormone
1. oestrogen is lipid-soluble so can simply diffuse through the phospholipid bilayer of CSM
2. in cytoplasm, oestrogen binds to complementary receptor on the TF
3. this binding causes a change in 3y structure of the DNA binding site on the TF
4. TF enters nucleus (via nuclear pores) & binds with the specific DNA base sequence/promotor region
5. this stimulates RNA polymerase to join & transcription begins, mRNA is made
not all cells have oestrogen receptors
how can the translation of mRNA be inhibited?
breaking mRNA down before it is translated into a polypeptide at a ribosome
RNA interference is carried out by small interfering RNA (siRNA)
how does siRNA work?
- double-stranded RNA is cut into shorter lengths by an endonuclease enzyme (cuts DNA at spec. sequences) to form siRNA
- these form single strands of siRNA & bind to an enzyme
- siRNA strand with enzyme attached pairs up with complementary bases on a section of mRNA strand
- the enzyme cuts mRNA into smaller sections (by hydrolysing phosphodiester bonds) so mRNA cannot be translated into a functioning protein
5.the gene is not expressed & the mRNA sections are hydrolysed into RNA mononucleotides
describe the importance of RNA interference
- defence mechanism against viruses (HIV) as it inhibits the translation of viral proteins by host cell’s ribosomes
- useful research tool - can be used to silence particular genes of interest to see what happens without them e.g. in biochemical pathway
- effective treatment for certain diseases e.g. Huntington’s disease involves the production of a faulty protein - RNAi can reduce the translation of this protein & delay symptoms
- if the targeted at cancer cells, it could also reduce the expression of proteins that cause rapid rate of division of tumour cells
define proteome
full range of proteins that a cell is able to produce
define genome
the complete set of genes in a cell or organism
define epigenome
the collection of chemical modifications applied to DNA & histones
(without changing DNA base sequence)
this can change over time
what is phenotype determined by?
a combination of genetic & environmental factors
environment influences gene expression, which affects phenotype
describe environmental changes in gene expression
they have, until recently, been assumed to be uninheritable
we are beginning to understand that, in some cases, environmental stimuli can cause heritable changes in gene function without changes in DNA base sequence (i.e. without mutation)
define epigenetics
heritable changes in gene function without changes in DNA base sequence
what do epigenetic changes involve?
chemical tagging of DNA or their associated histone proteins, which affects the likelihood of particular genes being transribed/expressed
what do chemical tags of the epigenome do?
affect the shape & packing density of DNA-histone complexes/chromatin (nucleosomes)
this can alter the physical accessibility of genes for TF & RNA polymerase, needed for transcription
compare tightly packed vs loosely packed nucleosomes
tightly packed: make DNA & genes less accessible - genes are inactive (switched off/not expressed)
loosely packed: make DNA & genes more accessible - genes are active (switched on/expressed)
chromatin
nucleosome
DNA + associated histone proteins
single DNA-histone complex
chromatin is a series of nucleosomes of variable packing density
what causes the chemical tags to be added?
environmental stimuli
starts in embryonic development & continues throughout life
when stem cells differentiate to become specialised cells, many genes are switched on & off by epigenetic mechanisms
stimuli can arise from internal or external environment
describe acetylation
acetyl group is transferred to a molecule
deacetylation is removal of acetyl group from a molecule
decreased acetylation of histones increases the +ve charge on histones so increases the attraction b/w histones & phosphate groups (-ve) on DNA
this makes DNA-histone complex/chromatin more tightly packed so DNA is not accessible to TF & RNA polymerase
TF cannot initiate transcription so gene is not expressed & protein is not made
describe methylation
methyl group transferred to a molecule
demethylation is removal of methyl group
methyl group is added to cytosine bases of DNA at CpG site
increased methylation of DNA inhibits transcription of genes by making the DNA-histone complex/chromatin pack more tightly together so it prevents the binding of TF & RNA polymerase to the DNA
transcription not activated so gene is not expressed (protein is not made)
draw table of effects of epigenetic factors on the DNA-histone complex
see booklet
why do tumours form?
uncontrolled cell division
genes involved in the normal regulation of the cell cycle are altered, either by mutation or dysregulation (either overexpressed or under expressed)
why are the causes of most cancers complex?
varying genetic predisposition (family history & inheritance of specific alleles/genes)
environmental & lifestyle risk factors are linked to cancer development e.g. smoking, UV radiation etc.
compare benign & malignant tumours
see table in booklet
what are most cancers initially caused by?
mutation in proto-oncogenes/oncogenes
& tumour suppressor genes
describe proto-oncogenes/oncogenes
oncogenes result from a mutation in proto-oncogenes
in normal cells, proto-oncogenes encode proteins that promote cell division
they are normally switched on in cells in response to growth factors e.g. hormones
describe what mutations in proto-oncogenes (or their promotor regions) can cause
cause proto-oncogenes to be permanently overexpressed/switched on
which causes receptors from growth factors being permanently activated (even without binding to growth factor)
excess growth factor produced, stimulating too much cell division
describe tumour suppressor genes
encode proteins that:
inhibit/slow down cell division
repair DNA replication errors
stimulate apoptosis (programmed cell death e.g. in response to DNA damage)
describe what mutations in tumour suppressor genes (or their promotor regions) can cause
cause TSG to be permanently under expressed (switched off), therefore reducing a cell’s ability to inhibit cell division
summary table of proto-oncogenes vs tumour suppressor genes:
effect on cell division
effect on apoptosis
if mutated,
see booklet
describe how epigenetic changes can cause uncontrolled cell division/cancer
- hypermethylation of DNA in tumour suppressor gene promotor regions
- too many methyl groups added
- chromatin becomes more tightly packed/condensed
- transcription is inhibited so gene is not expressed
this leads to uncontrolled cell division - hypomethylation of DNA in oncogenes
- not enough methylation
- chromatin becomes less tightly packed
- transcription is stimulated so gene is expressed
this leads to uncontrolled rapid cell division
describe the role of epigenetics in the treatment of disease
epigenetic changes are reversible
so they are good targets for therapeutic drugs
drugs that prevent methylation can cause genes to be upregulated i.e. decrease methylation on TSGs
e.g. azacitidine
or increased methylation of oncogenes
inhibitor drugs cause increased acetylation of histone proteins, which can upregulate genes i.e. increased acetylation of TSGs
what is the problem with targeting epigenetic changes to treat disease?
must target only the inappropriate epigenetic changes - must make the drug specific
describe the role of oestrogen in breast cancer
- oestrogen alters the expression of target genes as they have the oestrogen receptor
- oestrogen binds, which changes the shape of receptor, allowing the DNA binding site on TF to bind to the promotor region & stimulate transcription
- in oestrogen-receptor positive breast cancer, the cancer cells have oestrogen receptors on TFs to which the oestrogen can bind, increasing the rate of cell division –> oestrogen can cause overexpression of oncogenes in breast tissue
how do drugs against breast cancer work?
binds to the oestrogen receptor, which prevents oestrogen binding so TF cannot bind to DNA promotor region
prevents transcription (RNA pol. not stimulated) of these genes so reduces rate of cell division
aromatase inhibitors block aromatase which is enzyme that synthesises oestrogen –> decreased oestrogen production –> decreased binding of TF –> decreased transcription –> decreased rate of cell division
describe genome projects
Human Genome Project - international effort to sequence the human genome, identifying & mapping all human genes
DNA sequencing is now mainly automated & cheap
how is information about the proteome derived from the genome & why is it easier in prokaryotes & viruses?
sequencing a genome –> information about the AA sequences of protein in the proteome
prokaryotes & viruses are easier to sequence as they generally have smaller genomes than eukaryotes
deriving protein sequences from their genome is also easier because: they do not have non-coding regions of DNA within genes (introns)
the have fewer regulatory genes
how can determining the genome/proteome of a pathogen help the development of vaccines?
identification of proteins that can act as antigens for the immune system
these genes can be used to synthesis the antigens & use them in a vaccine
what does recombinant DNA technology involve?
the transfer of fragments of DNA from one organism or species to another
what does a transgenic organism contain?
DNA from a different species
why does recombinant DNA technology work?
genetic code is universal
similar transcription & translation processes means that the gene transferred can function correctly
what are the 3 steps for how a transgenic bacterium is made inc. the 3 methods for step 1?
- produce a fragment of DNA containing the gene to be transferred
a. conversion of mRNA to cDNA
b. restriction endonucleases used to cut DNA
c. make in a ‘gene machine’ - amplification/cloning of the DNA fragment
- transformation - getting the DNA fragment into the target organism
describe the conversion of mRNA to cDNA
- find a cell that expresses the gene of interest e.g. insulin gene expressed by B cells
- isolate mRNA that codes for ____ (insulin)
- add reverse transcriptase & DNA nucleotides to produce a strand of complementary DNA (cDNA) using mRNA as template
- remove/digest mRNA strand
- add DNA polymerase to produce 2nd strand of cDNA = double-stranded DNA copy of the gene of interest (insulin gene)
what are the advantages of using conversion of mRNA to cDNA?
it produces a copy of the human (insulin) gene without introns - already spliced
this is vital if gene is inserted into prokaryote as they cannot remove introns themselves
mRNA is easier to extract as there are many copies in the cytoplasm
if the gene is expressed in that cell
describe how restriction endonucleases are used to cut DNA
- isolate DNA/chromosome containing gene of interest
- restriction endonucleases cut DNA at specific DNA base sequences by breaking PDE bonds
- the 2 ends formed can be blunt or sticky (more useful)
- ligase enzymes join sticky ends of different pieces of DNA together if the same restriction endonuclease was used on both pieces
- gene will still contain introns
what are restriction endonucleases (RE)?
enzymes that cut DNA at specific DNA base sequences by breaking phosphodiester bonds
each restriction endonuclease cuts DNA at a specific restriction site
why are pieces of DNA only joined if cut by the same restriction endonuclease?
overhanging ends are complementary
so will form H bonds by complementary base pairing
what are restriction endonucleases important for?
insertion of DNA fragments into plasmid vectors
DNA tech. techniques e.g. DNA fingerprinting & electrophoresis
describe making a gene in a ‘gene machine’
machines can synthesise any sequence of DNA bases required
the DNA sequence of the gene of interest must be known - can be found by DNA sequencing or can work backwards from AA sequence of the protein
can include relevant restriction sites –> helps insert DNA section into plasmid vector
no introns included
describe in vivo cloning of the DNA fragment
- DNA fragment inserted into bacterial plasmid
- plasmid inserted into bacterium
- bacteria cultured on growth medium - divide quickly by binary fission
what is in vitro cloning by polymerase chain reaction (PCR)?
method of DNA amplification
artificial form of DNA replication
amplified DNA can be used for analytical processes
what ‘ingredients’ does PCR require?
DNA template - the sequence to be amplified
DNA primers - 2 are needed, 1 for each strand of template DNA
taq DNA polymerase - produces new DNA strands complementary to the template strands by forming PDE bonds b/w nucleotides
DNA nucleotides - joined by DNA polymerase to make new DNA strands
what are DNA primers & why are they necessary for PCR?
short, single-stranded DNA with specific, complementary sequence to the flanking regions of the DNA sequence of interest
necessary because DNA polymerase can only begin DNA synthesis from a double-stranded DNA section
primers prevent separated strands from re-joining together
what is taq DNA polymerase & why is it used in PCR?
obtained from thermophile taq bacterium that can survive at high temp.
allows PCR to be run continuously without being denatured at the high temp. used to denature DNA
= speeds up process
describe the process of PCR
- denaturation of DNA:
the mixture is heated to 95C
which breaks H bonds b/w complementary base pairs
so the 2 DNA strands separate - annealing of primers
mixture is cooled to 68C
which allows primers to anneal by forming H bonds with complementary base sequence at the end of each DNA strand
this allows DNA polymerase to work & prevents separated DNA strands from re-joining - elongation
mixture is heated to 72C
which is the optimum temp. for tag DNA polymerase, which joins free-floating DNA nucleotides to form new DNA strands
this cycle repeats to increase the # of DNA copies exponentially
PCR machines do this cycle automatically
describe the process of transformation/getting the DNA fragment into target organisms
- plasmid is removed from bacteria & many copies made using PCR
- promotor & terminator regions are added to the gene of interest
- DNA fragment & the plasmid are mixed with specific RE to produce complementary sticky ends using the same RE
- ligase is added to join the plasmid DNA & DNA sequence of interest together
- marker genes can be added
- this produces recombinant plasmid vectors which are inserted into the bacterium by transformation
what are the functions of the promotor & terminator regions?
promotor: region of DNA where RNA polymerase binds to initiate transcription once TF has bound
terminator: region of DNA that signals to RNA polymerase to stop transcription
what are common vectors for recombinant DNA?
bacterial plasmids
genetically modified viruses - can deliver DNA fragment into cells & can incorporate DNA into genome of target cells –> used in human gene therapy
what are selective marker genes?
marker genes are additional genes added to the vector/plasmids that are used to help identify host cells that have successfully taken up the vector/plasmids
describe the use of selective marker genes
often genes that provide antibiotic resistance (AR) to bacterium
cells that successfully take up the recombinant plasmid will gain both the gene of interest & the gene for AR
growing bacteria on agar containing the antibiotic kills bacteria that lack the recombinant plasmid
other marker genes include genes for fluorescent proteins that give off visible light under UV
