the control of gene expression

mutation

• change in the base sequence of DNA

• can arise spontaneously during interphase

mutagenic agent

a factor which increases the rate of gene mutation

gene mutations → non-functional protein/enzyme

1. changes position of base triplets in DNA, so changes sequence of codons on mRNA

2. changes sequence of AAs in polypeptide

3. changes position of hydrogen/ionic/disfulfide bonds

4. changes tertiary structure of protein

5. enzymes → changes shape of AS, no longer complementary to substrate = no E-S complexes

gene mutations do not always affect order of AAs

• genetic code is degenerate

• mutations can occur in introns

AA changes are not always harmful

• may not change 3 structure of protein

• may positively change the properties of the protein, giving the organism a selective advantage

frameshift

• mutation changes number of nucleotides to a number not divisible by three

• shifts the way the genetic code is read, so all the DNA triplets/mRNA codons downstream from the mutation change

• sequence of AAs change = significant change to polypeptide

• could also lead to the production of a stop codon → shorter polypeptide

stem cells

undifferentiated cells capable of dividing by mitosis to replace themselves indefinitely and capable of differentiating into other types of specialised cells

stem cell specialisation

• stimuli lead to activation of some genes (due to transcription factors)

• mRNA is transcribed only from the activated genes and then translated to form proteins

• these proteins modify cells permanently and determine cell structure/function

totipotent cells

• occur for a limited time in early mammalian embryos

• can divide and differentiate into any type of cell

pluripotent cells

• found in mammalian embryos (after first few cell division)

• can divide and differentiate into most cell types except placental cells

multipotent cells

• found in mature mammals

• can divide and differentiate into a limited number of cells

• e.g. multipotent cells in bone marrow → different types of blood cells

unipotent cells

• found in mature mammals

• can divide and differentiate into one cell type

• e.g. unipotent cells in the heart → only cardiomyocytes

stem cell treatment

• transplanted into cells to divide in unlimited numbers

• then differentiate into required healthy cells

• to replace faulty/damaged cells

iPS cells

1. obtain adult somatic cells from patient

2. add specific protein transcription factors associated with pluripotency to cells so that they express genes associated with pluripotency (reprogramming)

   • transcription factors attach to promoter regions of DNA, stimulating or inhibiting transcription

3. culture cells to allow them to divide by mitosis

once made, iPS cells can divide and differentiate into healthy cells to be transplanted into the same patient

arguments for the use of stem cells

• improve quality of life and relieve human suffering → by dividing into required healthy cells

• embryos are often left over from IVF and would otherwise be destroyed

• iPS cells unlikely to be rejected by the patient’s immune system as made with the patient’s own cells

• iPS cells can be made without destruction of embryo + patient can give consent

arguments against the use of stem cells

• ethical issues with embryonic stem cells as obtaining them requires the destruction of an embryo + potential life (embryo cannot consent)

• immune system could reject cells and immunosuppressant drugs are required

• cells could divide uncontrollably → cancer/tumours

transcription factors

• proteins which regulate (stimulate/inhibit) transcription of specific target genes in eukaryotes

• by binding to a specific DNA base sequence on a promoter region

regulation of transcription

1. TCFs move from cytoplasm to nucleus

2. bind to DNA at a specific DNA base sequence on a promoter region (upstream of target gene)

3. this stimulates of inhibits transcription (mRNA production) by aiding or preventing the binding of RNA polymerase

oestrogen + transcription

1. oestrogen = lipid soluble so diffuses into cell across PPLB

2. in cytoplasm, oestrogen binds to its complementary receptor (inactive TCF) → oestrogen-receptor complex

3. changes shape of the inactive TCF → becomes active

4. complex diffuses from cytoplasm → nucleus

5. complex binds to specific DNA base sequence or promoter region of a target gene

6. stimulates transcription of target gene → mRNA formed by helping RNA polymerase to bind

why does oestrogen only affect target cells

other cells do not have oestrogen receptors

epigenetics

• heritable changes in gene function/expression without changes to the base sequence of DNA

• caused by changes in the environment

epigenome

a chemical modification of DNA and histone proteins

• methyl groups of DNA

• acetyl groups on histones

epigenetic control of gene expression

inhibition of transcription:

• increased methylation of DNA

• decreased acetylation of histones

encouragement of transcription:

• decreased methylation of DNA

• increased acetylation of histones

how does methylation inhibit transcription

1. increased methylation of DNA → methyl groups added to cytosine bases in DNA

2. nucleosomes pack more tightly together

3. prevent binding of TCFs and RNA polymerase bind to promoter

how does acetylation inhibit transcription

1. decreased acetylation of histones → increases positive charge of histones

2. +ve histones bind -ve DNA more tightly

3. preventing TCFs and RNA polymerase binding to promoter

epigenetics in disease development and treatment

• environmental factors → epigenetic changes

• stimulate/inhibit expression of certain genes that lead to disease development

  • increased methylation/decreased acetylation → inhibit transcription

  • decreased methylation/increased acetylation → encourage transcription

• diagnostic tests can be developed that detect these epigenetic changes before symptoms present

• drugs can be developed to reverse these epigenetic changes

RNA interference

• inhibition of translation of mRNA produced from target cells, by RNA molecules

• inhibits expression of a target gene

regulation of translation by RNAi

• small interfering RNA (siRNA) or micro-RNA (miRNA) is incorporated into/binds to a protein → forms RNA-induced silencing complex (RISC)

• single stranded miRNA/siRNA within RISC binds to target mRNA with complementary base sequence

• leads to hydrolysis of mRNA into fragments which are then degraded OR prevents ribosomes binding

• reducing/preventing translation of target mRNA into protein

binding of siRNA to protein

synthesised as double-stranded RNA → 1 strand incorporated

binding of miRNA to protien

synthesised as a double-stranded hairpin bend of RNA → both strands incorporated

tumour

mass of abnormal cells

benign tumours

slower growth

cells are specialised

normal, regular nuclei

well defined borders → often surrounded by a capsule so do not invade surrounding tissue

do not spread by metastasis (as cell adhesion molecules → stick cells together

can normally be removed by surgery → rarely return

malignant tumours

faster growth

unspecialised cells

larger, darker, irregular nuclei

poorly defined borders → not encapsulated so can invade surrounding tissues (growing projections)

spread by metastasis → cells break off and spread to other parts of the body forming secondary tumours (due to lack of adhesion molecules)

can normally be removed by surgery combined with radiotherapy/chemotherapy but the often return

function of tumour suppressor genes

code for proteins that:

• inhibit/slow cell cycle

• OR cause self-destruction of potential tumour cells

role of tumour suppressor genes in the development of tumours

mutation in DNA base sequence → production on non-functional protein

• due to change in AA sequence and therefore 3 structure

decreased histone acetylation/increased DNA methylation → inhibits production of protein

• by preventing binding of RNA polymerase to promoter region → inhibiting transcription

both non-functional protein and no protein at all → uncontrollable cell division

proto-oncogenes

code for proteins that stimulate cell division

role of oncogenes in the development of tumours

oncogene = mutated form of proto-oncogene

• mutation in DNA base sequence → overproduction of protein/permanently activated protein

  • by leading to change in AA sequence therefore protein 3 structure

• decreased DNA methylation/increased histone acetylation → increase in protein production

  • increased binding of RNA polymerase to promoter region = transcription

• both lead to uncontrolled cell division (cell division is permanently stimulated)

why do tumours require both alleles of TSG to be mutated while one one of an oncogene

• one functional allele of a TSG can produce enough protein to slow the cell cycle OR cause self destruction of potential tumours cells, controlling cell division

• one mutates oncogene allele can produce enough protein to lead to rapid/uncontrolled cell division

epigenetics in cancer treatment

drugs = reverse epigenetic changes that caused cancer, preventing uncontrolled cell division

• increasing DNA methylation/decreasing histone acetylation of oncogene → decreases expression

  • by inhibiting the binding of RNA polymerase to the promoter region → less transcription

• decreasing DNA methylation/increasing histone acetylation of TSG → increases expression

  • by allowing the binding of RNA polymerase to the promoter region → more transcription

[oestrogen] in breast cancer

• some BC cells have oestrogen receptors (inactive TCFs)

• if [oestrogen] increases, more oestrogen bids to oestrogen receptors, forming more oestrogen-receptor complexes, which are active TCFs

• bind to promoter regions of genes that code for proteins stimulating cell division

• increases transcription/expression of these genes, increasing the rate of cell division

drugs treating oestrogen receptor-positive breast cancers

• drugs bind to oestrogen receptors (inactive TCFs), preventing the binding of oestrogen (therefore no oestrogen-receptors complexes form)

• therefore less bind is complexes to promoter regions of genes which stimulate the cell cycle

genome sequencing

• identifying the DNA base sequence of an organism’s genome

• so AA sequences of proteins that derive from an organism’s genetic code can be determined

how can determining the genome of a pathogen aid vaccine production

• could identify the proteome of the pathogen

• so could identify potential antigens to use in the vaccine

applications of genome sequencing projects

• identification of genes/alleles associated with genetic diseases/cancers

  • new targeted drugs/gene therapy can be developed

  • can screen patients, allowing for early prevention/personalised medicine

• identification of species and evolutionary relationships

why can the genome not directly be translated into the proteome in complex organisms

• presence of non-coding DNA

• presence of regulatory genes (which regulate the expression of other genes e.g. by coding for miRNA and siRNA

how are sequencing methods changing

• have become automated (so are faster, more cost effective and can be done on a larger scale)

• are continuously updated

recombinant DNA technology

transfer of DNA fragments from one organism or species to another

why can transferred DNA be translated within cells of recipient (transgenic) organisms

• genetic code is universal

• transcription and translation mechanisms are also universal

how can restriction enzymes produce DNA fragments

1. restriction enzymes cut at specific base recognition sequences either side of the desired gene

2. many cut in a staggered fashion forming ‘sticky ends’ (single stranded overhang)

DNA fragments from mRNA

• isolate mRNA from a cell that readily synthesises the protein coded for by the desired gene

• mix mRNA with DNA nucleotides and reverse transcriptase

• reverse transcriptase uses mRNA as a template to synthesise a single strand of complementary DNA (cDNA)

• DNA polymerase can form a second strand of DNA using cDNA as a template

mRNA genes > genes from DNA removed from cells

• much mRNA than DNA → easily extracted

• in mRNA, introns have been removed by splicing, while DNA contains introns

  • therefore it can be transcribed/translated by prokaryotes who cannot remove introns

gene machine → DNA fragments

• synthesises DNA fragments quickly and accurately from scratch without a need for a DNA template

  • AA sequence of protein determined, allowing base sequence to be established

• fragments do not contain introns so can be transcribed/translated by prokaryotes

in vitro vs in vivo methods of amplifying DNA fragments

• in vitro → PCR (polymerase chain reaction)

• in vivo → culturing transformed host cells e.g. bacteria

PCR mixture

• DNA fragment

• DNA polymerase

• primers

• DNA nucleotides

how are DNA fragments amplified by PCR

1. mixture heated to 95 C

   • separates DNA strands by breaking H-bonds between bases

2. mixture cooled to 55 C

   • allows primers to bind to DNA fragment template strand

   • by forming H-bonds between complementary bases

3. mixture heated to 72 C

   • nucleotides align next to complementary exposed bases

   • DNA polymerase joins adjacent DNA nucleotides, forming phosphodiester bonds

cycle is repeated → following each, amount of DNA doubles (exponential increase)

role of primers in PCR

• short, single stranded DNA fragments

• complementary to base sequence at edges of region to be copies/start of desired gene

• allowing DNA polymerase to bind to start synthesis (can only ass nucleotides to pre-existing 3’ end)

• two different primers are required (as base sequences at ends of gene are different)

why does DNA replication eventually stop in PCR

there are a limited number of primers and nucleotides which are eventually used up

in vivo amplification

1. add promoter and terminator regions to DNA fragments

2. insert DNA fragments and marker genes into vectors (e.g. plasmids) using restriction enzymes and ligases

3. transform host cells (e.g. bacteria) by inserting these vectors

4. detect genetically modified organisms by identifying those containing the marker gene (e.g. one which codes for a fluorescent protein)

5. culture the transformed host cells → allowing them to divide and form clones

why are promoter regions added to DNA fragments that are used to GM organisms

allow transcription to start by allowing RNA polymerase to bind to DNA

can be selected to ensure genetic expression happens only in specific cell types

• e.g. in the glands of a mammal so that the protein can be easily harvested

why are terminator regions added to DNA fragments that are used to GM organisms

ensure transcription stops at the end of a gene by stopping RNA polymerase

role of vectors in recombinant DNA technology

to transfer DNA into host cells/organisms e.g. plasmids or viruses

role of enzymes in inserting DNA fragments into vectors

1. restriction endonucleases/enzymes cut vector DNA

   • same enzyme used that cut the gene out so that vector DNA and fragments have sticky ends that can join together by complementary base pairing

2. DNA ligase joins DNA fragment to vector DNA

   • forming phosphodiester bonds between adjacent nucleotides

how are host cells transformed using vectors

• plasmids enter cells

• viruses inject their DNA into cells which is then integrated into host DNA

why are marker genes inserted into vectors

allow detection of GM/transgenic cells/organisms

• if marker gene codes for antibiotic resistance, cells that survive antibiotic exposure = transformed

• if marker gene codes for fluorescent protein, calls that fluoresce under UV light = transformed

as not all cells/organisms will take up the vector and be transformed

recombinant DNA in medicine

• GM bacteria produce human proteins (e.g. insulin) → more ethically acceptable than using animal proteins + less likely to cause an allergic reaction

• GM animals/plants produce pharmaceuticals → cheaper

• gene therapy

recombinant DNA in agriculture

• GM crops resistant to herbicides → only weeds killed when crop is sprayed with herbicide

• GM crops resistant to insect attack → reduce use of insecticide

• GM crops with added nutritional value (e.g. golden rice and vitamin A)

• GM animals with increased growth hormone production

recombinant DNA in industry

• GM bacteria produce enzymes used in industrial processes and food production

gene therapy

• introduction of new DNA into host cells, often containing healthy/functional alleles

• to overcome effect of faulty/non-functional alleles in people with genetic disorders (e.g. cystic fibrosis)

issues with gene therapy

• effect is short lived as modified cells (e.g. T cells) have a limited lifespan → required regular treatment

• immune response against GM cells or viruses due to recognition of antigens

• long term effect not known → side effects e.g. could cause cancer

  • DNA may be inserted into other genes → interfering with gene expression

support for recombinant gene technology

• GM crops increase yields → increased global food production → reduced risk of famine/malnutrition

• GM has the potential to cure many genetic disorders

• pharming (production of pharmaceuticals by GM animals/plants) makes medicines available to more people as they become cheaper

opposition to recombinant DNA technology

• recombinant DNA may be transferred to other plants → potential herbicide resistant ‘super-weeds’

• potential effects on food webs e.g. effect wild insects → reduce biodiversity

• large biotech companies may control the technology and own patents

DNA probes

• short single-stranded pieces of DNA

• with a base sequence complementary to bases on part of a target allele/region

• usually labelled with a fluorescent or radioactive tag for identification

suggest why DNA probes are longer than just a few bases

• a sequence of a few bases would occur at many places throughout the genome

• longer sequences are likely to only occur in the target allele

DNA hybridisation

• binding of single-stranded DNA probe to a complementary strand of DNA

• forming H-bonds/base pairs

how can genetic screening be used to locate specific alleles of genes

1. extract DNA and amplify by PCR

2. cut DNA at specific base sequences (either side of target gene) using restriction enzymes

3. separate DNA fragments/alleles using gel electrophoresis

4. transfer to a nylon membrane and treat to form single strands with exposed bases

5. add labelled DNA probes which hybridise/bind with target alleles (wash to remove unbound probe)

6. to show bound probe, expose membrane to UV light if a fluorescently labelled probe was used OR use autoradiography is a radioactive probe was used

gel electrophoresis

• method used to separate nucleic acid fragments OR proteins

• according to length/mass and charge (DNA is negatively charged due to P and charge of protein varies based on AA R groups)

GE method

1. DNA samples loaded into wells in a porous gel and covered in buffer solution (which conducts electricity)

2. electrical current passed through → DNA is negatively charged due to P so moves towards positive electrode

3. shorter DNA moves faster so travel further

interpretation of GE results

• run a standard with DNA fragments/proteins of unknown lengths under the same conditions

• compare to position of unknown DNA fragments/proteins to estimate their size

• shorter DNA fragments/proteins travel further/faster

uses of labelled DNA probes

• screening patients for heritable conditions

• screening patients for drug responses

• screening patients for health risks

role of genetic counsellor

• explain results of genetic screening

• discuss treatments available for genetic condition

• discuss lifestyle choices/precautions that might reduce risk of a genetic conditions developing e.g. regular screening

• explain probability of condition/alleles being passed onto offspring → enable patients to make informed decisions about having children

personalised medicine

• medicine tailored to an individual’s DNA/genotype

• increasing effectiveness of treatment e.g. by identifying the particular mutation/allele causing cancer and treating it with tailored drugs

arguments for genetic screening

• can enable people to make certain lifestyle choices to reduce chances of diseases developing

• allows people to make informed decisions about having their own biological children

• allows use of personalised medicines, increasing effectiveness of treatment

arguments against genetic screening

• screening for incurable diseases or diseases that develop in later life → cause depression

• could lead to discrimination by insurance companies/employers

• may cause undue stress if patient does not develop the disease

what are variable number tandem repeats (VNTRs)

• repeating sequences of nucleotides/bases (e.g. GATA)

• found within non-coding sections of DNA at many sites throughout an organism’s genome

usefulness of VNTRs

• probability of two individuals having same VNTRs is very low

• as an organism’s genome contains many VNTRs and lengths at each loci differ between individuals

genetic fingerprinting in analysis of DNA fragments

1. extract DNA from sample (eg. blood cells) and amplify by PCR

2. cut DNA at specific base sequences / recognition sites (either side of VNTRs) using restriction enzymes

3. separate VNTR fragments according to length using gel electrophoresis (shorter ones travel further)

4. transfer to a nylon membrane and treat to form single strands with exposed bases

5. add labelled DNA probes which hybridise / bind with complementary VNTRs (& wash to remove unbound probe)

6. to show bound probe, expose membrane to UV light if a fluorescently labelled probe was used OR use autoradiography (expose to X-ray film) if a radioactive probe was used

genetic fingerprinting vs screening

• both use PCR to amplify DNA sample

• both use electrophoresis to separate DNA fragments

• both use labelled DNA probes to visualise specific DNA fragments

• genetic fingerprinting analyses VNTRs whereas genetic screening analyses specific alleles of a gene

how can genetic fingerprinting be used to determine genetic relationships

• more closely related organisms have more similar VNTRs, so more similarities in genetic fingerprints

• paternity testing → father should share ~50% of VNTRs with child (due to inheritance)

how can genetic fingerprinting be used to determine genetic variability within a population

differences in VNTRs arise as a result of mutations

more mutations = greater diversity

genetic fingerprinting in forensics

• compare genetic fingerprints of suspects to genetic fingerprint of DNA at crime scene

• if many bands match, the suspect was likely present at the crime scene

genetic fingerprinting in medical diagnosis

some VNTR patterns are associated with an increased risk of certain genetic disorders e.g. Huntington’s

genetic fingerprinting in animal and plant breeding

• shows how closely related 2 individuals are, so interbreeding can be avoided

• breed pairs with dissimilar genetic fingerprints