T8: gene expression Flashcards
define a gene mutation
- a change in the base sequence of DNA
- can arise during DNA replication (interphase)
define a mutagenic agent
- a factor that increases rate of mutation
- e.g. ultraviolet or alpha particles
Explain how a gene mutation can lead to th production of a non-functional protein or enzyme
- changes seqence of base triplets in DNA so changes sequence of codons on mRNA
- so changes amino acid seuqnce in the encoded polypeptide
- so changes position of hydrogen/ionic/disulphide bonds between amino acids.
- so changes tertiary structure (shape) of protein
- enzymes - active site changes shape so substrate can’t bind , enzyme substrate complexes cannot form.
Describe the different types of gene mutation
substitution: Replacement of a base by a different base (in DNA); (1)
addition: 1 or more bases/ nucleotides are added to the DNA base sequene
deletion: nucleotides are lost from the DNA base sequence ( can lead to frame shift)
duplication: a sequence of DNA bases / nucleotides is repeated.
inversion: a sequence of bases detaches from the DNA sequence then rejoins at the same position in the reverse order
translocation: a sequence of DNA bases detaches and is inserted as a different location within the same or a different chromosome
Explain why not all gene mutations affect the order of amino acids
- some substitutions change only 1 triplet codon which could still code for the same amino acid. As the code is degenerate.
- some occur in introns which do not code for amino acids.
Explain why a change in amino acid sequence is not always harmful
- may not change tertiary structure of protein ( if position of ionic disulfide and H bonds don’t change.
- may positively change the properties of the protein , giving the organnism a selective advantage
Explain what is meant by a frame shift .
- occurs when gene mutations change the number of nucelotides + bases by a number not / by 3
- this shifts the way the genetic code is read , so all the DNA triplets / mRNA codons downstream from the mutation change.
(i) Suggest how a mutation can lead to the production of a protein that has one amino acid missing. (2)
(i) Loss of 3 bases / triplet = 2 marks;;
Suggest how the production of a protein with one amino acid missing may lead to a genetic disorder such as Ellis-van Creveld syndrome.
1. Change in tertiary structure / active site;
2. (So) faulty / non-functional protein / enzyme;
Accept: reference to examples of loss of function eg fewer E-S complexes formed
A mutation in the gene coding for enzyme B could lead to the production of a non-functional enzyme. Explain how. (3)
- Change in base sequence (of DNA / gene) leading to change in amino acid sequence / primary structure (of enzyme);
- Change in hydrogen / ionic / disulphide bonds leading to change in the tertiary structure / active site (of enzyme);
3. Substrate not complementary / cannot bind (to enzyme / active site) / no enzyme-substrate complexes form;
Explain how a new form of myosin with different properties could have been produced as a result of mutation. (4)
- addition / deletion / substitution of a base in DNA of the gene which codes for myosin;
- changes in base sequence in DNA
- change in amino acid sequence / primary structure;
causes a different tertiary structure; - which alters the binding properties of myosin;
what are stem cells?
undifferentiated cells capable of :
- dividing by mitosis to replace themselves indefinitely
- differentiating into other types of specialised cells.
Describe how stem cells become specialised during development
- how do they obtain a specific function/ differentiate
- A stimulus activates specific genes through transcription factors
- transcription of mrna from these genes occurs and are translated into proteins
- which modify the cell’s structure and function, resulting in specialisation.
Describe totipotent cells
- occur for a limited time in early mammalian embryos.
- can divide and differentiate into any type of body cells ( including extra-embryonic cells) e.g. placenta.
Describe pluripotent cells
- found in mammalian embryos ( after first few cell division)
- can divide and differentiate into most cell types ( every cell type in the body but not placenta)
Describe multipotent cells
- found in mature mammals
- can divide + differentiate into a limited number of cell types
- e.g. in bone marrow which can differentiate into types of blood cells.
Describe unipotent cells , using an example
- 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
what are meristem cells
- undifferentiated cells found in the tips of roots and shoots.
- able to rapdily divide and produce a new plant
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)
- e.g. type 1 diabetes / bone marrow stem cell transplant ‘ blood cancers
Explain how induced pluripotent stem cells are produced
- use adult somatic cells from patient
- add specific transcription factors which allow for genes associated with pluripotency to be expressed
- transcription factors attach to promoter regions of DNA stimulating or inhibiting transription
- culture cells to allow them to divide by mitosis
Evaluate the use of stem cells in treating human disorders
argue for - 4
for:
- can divide and differentiate into required healthy cells, so could relieve human suffering by saving lives and improving quality of life.
- embryos are often left over IVF and so would otherwise be destroyed.
- IPS cells unlikely to be rejected by patient’s immune system as made with patient’s own cells
- IPS cells can be made without destruction of embryo and adult can permission
Evaluate the use of stem cells in treating human disorders
- ethical issues with embryonic stem cells as obtaining them require destruction of an embryo and potential life ( embryo cannot consent)
- immune system could reject cells and immunosuppresant drugs are required.
- cells could divide out of control , leading to formation of tumours/cancer
what are transcription factors
- proteins which regulate (stimulate or inhibit) transcription of specific target genes in eukaryotes
- by binding to a specific DNA base sequence on a promoter region
Describe how transcription can be regulated using transcription factors
- Transcription factors move from cytoplasm to nucleus
- Bind to DNA at a specific DNA base sequence on a promoter region ( before/upstream of target gene)
- this stimulates or inhibits transcription (production of mRNA) of target genes by helping or preventing RNA polymerase binding.
Explain how oestrogen affects transcription
- oestrogen is a lipid soluble steroid hormone so diffuses into cell across the phospholipid bilayer
- in cytoplasm, oestrogen binds to its receptor, an inactive transcription factor, forming an oestrogen-receptor complex.
- This changes the shape of the inactive transcription factor, forming an active transcription factor
- the complex difuses from cytoplasm into the nucleus
- then binds to a specific DN base sequence on the promoter region of a target gene
- stimulating transcription of target genes forming mRNA by helping RNA polymerase to bind
explain why oestrogen only affects target cells
- other cells do not have oestrogen receptors
Describe what is meant by epigenetics
- heritable changes in gene function / expression without changes to the base sequence of DNA
- caused by changes in the environment ( e.g. diet ,stress,toxins)
Describe what is meant by epigenome
- all chemical modification of DNA and histone proteins - methyl groups on DNA and acetyl groups on histones
Summarise the epigenetic control of gene expression in eukaryotes
- increased methylation of DNA :inhibits transcription
- decreased methylation of DNA: allows transcription
- increased acetylation of histones: allows transcription
- decreased acetylation of histones: inhibits transcription
Explain how methylation can inhibit transcription
- Increased methylation of DNA: methyl groups added to cytosine bases in DNA
- So nucleosomes (DNA wrapped around histone) pack more tightly together
- Preventing transcription factors and RNA polymerase binding to promoter
Explain how acetylation can inhibit transcription
- decreased acetylation of histones increases positive charge of histones
- so histones bind DNA ( negatively charged) more tightly
- preventing transcription factors and RNA polymerase bidning to promoters
Explain the relevance of epigenetics on disease development and treatment
- environmental factors (e.g. diet,stress,toxins) can lead to epigenetic changes
- these can stimulate/inhibit expression of certain genes that can lead to disease development
- diagnositc tests can be developed that detect the epigenetic changes before symptoms present
- drugs can be developed to reverse these epigenetic changes
What is RNA interference (RNAi)?
- a form of post-transcriptional modification which occurs in the cytoplasm
- where siRNA, miRNA inhibit the translation of target mRNA,
- silencing gene expression.
Describe the regulation of translation by RNA interference
- SiRNA/miRNA binds to a protein, forming an RNA induced silencing complex (RISC)
- single-stranded miRNA/siRNA binds to target mRNA with a complementary base sequence
- This leads to hydrolysis of mRNA into fragments which are then degraded / prevents ribosomes from binding
- Reducing /preventing translation of target mRNA into protein
Describe how tumours and cancers form
8.2.3: gene expression and cancer
- mutations in DNA/genes controlling mitosis can lead to uncoontrolled cell division
- tumoour formed if this results in a mass of abnormal cells
- malignant: cancerous , can spread by metastasis
- benign: non-cancerous
Compare the main characteristics of benign and malignant tumours
benign:
- grows slowly/cells fivide less often
- cells are specialised
- normal regular nuclei
- do not spread by metastisis
malignant:
- grow faster
- unspecialised
- irregular /darker nuclei
- spread by metastasis: cells break off and spread to the other parts of the body
Describe the function of tumour supressor genes
- codes for proteins that
- inhibit / slow cell cycle (e.g. if DNA damage detected)
- or cause self-destruction (Apoptosis) of potential tumour cells (e.g. if damaged DNA can’t be repaired)
Explain the role of tumour supressor genes in the development of tumours
- A mutation in DNA base sequence leads to the production of non-functional protein
- by leading to a change in amino acid sequence which changes protein tertiary structure
- decreased histone acetylation or increased DNA methylation which prevents production of protein by preventing binding of RNA polymerase to promoter region, inhibiting transcription
- both lead to uncontrolled cell division
Describe the function of proto oncongenes
- code for proteins that stimulate cell division
what are oncogenes?
- mutated /abnormally expressed form of the corresponding proto-oncogene
Explain the role of oncogenees in the development of tumours
- through epigenetics and mutation
-
mutation in DNA base sequence leads to an overproduction of protein or permanently activated protein by leading to change in amino acid sequence which chages protein tertiary structure.
OR - **decreased DNA methylation **or increased histone acetylation - increases production of protein by stimulating the bidning of RNA polymerase to promoter region
- stimulating transcription leading to uncontrolled cell division
Suggest why tumours require mutations in both alleles of a tumour supressor gene but only one allele of an oncogene
- loss-of-function mutations that occur in tumor suppressor genes are recessive and so one mutation can still produce a protein to slow the cell cycle
- whereas one mutated oncogene allele can produce enough protein to lead to rapid/uncontrolled cell division as it is dominant
Explain the relevance of epigenetics in cancer treatment
- drugs could reverse epigentic changes that caused cancer, preventing uncontrolled cell division
- increasing DNA methylation or decreasing histone acetylation of oncogene to inhibit transcription/expression
- decreasing DNA methylation or increasing histone acetylation of tumour supressor gene to stimulate transcription / expression
Explain the role of increased oestrogen concentrations in the development of some ** breast cancers
** oestrogen-receptor positive
- some breast cancer cells have oestrogn receptors , which are inactive transcription factors
- if oestrogen concentration is increased , more oestrogen binds to oestrogen receptors, forming more O-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 increasnign the rate fo cell divison
Suggest how drugs that have a similar structure to oestrogen help treat oestrogen -receptor positive breast cancers
- drugs bind to oestrogen receptors ( inactive transcription factors , preventing binding of oestrgen
- so no fewer transcription factors bind to promoter regions of genes that stimulate the cell cycle
define:
- genome
- proteome
- genome: the complete set of genes in cell
- proteome: the full range of proteins that a cell can produce coded for by the cell’s DNA / genome
What is genome sequencing and why is it important?
- identifying the DNA base sequence of an organism’s genome
- so amino acid sequences of proteins that derive from an organism’s genetic code can be determined.
Explain how determining the genome of a pathoge could allow vaccines to be developed
- could identify the pathogen’s proteome
- so could identify potential antigens ( proteins that stimulate an immune response) to use inn the vaccine
suggest some other potential applications of genome sequencing projects
- identification of genes / alleles associated with genetic diseases/ cancer
- new targeted drugs / gene therpy can be developed
- can screen patients , allowing early prevention / personalised medicine
- identification of species and evolutionary relationships
Explain why the genome cannot be directly translated into the proteome in complex organisms
- presence of non-coding DNA (e.g. introns within genes)
- presence of regulatory genes ( which regulate expression of other genes)
describe how sequencing methods have changed
- have become automated so are faster and more cost efffective
- they are continiously updated
what is recombinant DN technology?
- transfer of DNA fragments from one organism or species to another
Explain why transferred DNA can be translated within cells of recipient transgenic organisms
- genetic code is universal
- transcription and translation mechanisms are universal
Describe the three examples of recombinant DNA technonlogy
- using restriction enzymes
- using reverse transcriptase
- gene machine
Describe how DNA fragements can be produced using restriction enzymes
- restriction enzymes cut DNA at specific base recognition sequences either side of the desired gene
- shape of recognition site is complementary to active site
- Many cut in a staggered fashion forming sticky ends / single stranded overhanging bases
Describe how DNA fragments can be produced 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. RT 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
suggest two advantages of obtaining genes from mRNA rather than directly from the DNA removed from cells
- more mrna in cells making the protein then dna - so easily extracted
- in mRNA introns have been removed by splicing whereas DNA contains introns - so can be transcribed & translated by prokaryotes who can’t remoove introns by splicing
Describe how fragments of DNA can be produced using a gene machine
- determine amino acid sequence of protein
- allows base sequence to be established
- these do not contain introns so can be transcribed and translated by prokaaryotes
- synthesises fragments of DNA quickly and accurately from scratch without need for a DNA template
Name in vitro and in vivo techniques used to amplify DNA fragments
- in vitro ( outside a living organism): polymerase chain reaction
- in vivo (inside a living organism): culturing transformed host cells eg. bacteria
What is the mixture that is used in PCR?
- DNA fragments , DNA polymerase (e.g. taq polymerase) , primers and DNA nucleotides
Explain how DNA fragments can be amplified by PCR
- 95°C: this seperates DNA strands , breaking hydrogen bonds between bases
- 55°C: allows primers to bind to DNA fragment template strand by forming H bonds between complementary bases
- 72°C: nucleotides align next to complemenntary exposed bases. DNA polymerase joins adjacent DNA nucleotides , forming phosphodiester bonds
mixture heated at x
this cycle is repeated. In every cycle, the amount of DNA doubles causing an exponential increase 2n
Explain the role of primers in PCR
- Primers are short ,** single stranded** DNA fragments
- complementary to DNA base sequence at start of desired gene to be copied
- allowing DNA polymerase to bind to start synthesis
- two different primers are required as base sequences at ends are different
Suggest one reason why DNA replication eventually stops in PCR
- there are a limited number of primers and nucleotides which are eventually used up
what are promoter and terminator regions
what is a marker gene and what are they used for?
- marker genes code for fluorescent proteins which can be seen under UV light
- used to identify the successfully transformed bacteria
- those that have not taken up the desired gene can be destroyed and are not cultured
Summarise the steps involved in amplifying DNA fragments in vivo
- Isolate DNA fragment and Add promoter and terminator regions to the plasmid
- Insert DNA fragments & marker genes into vectors (eg. plasmids) using restriction enzymes and ligases
- Transform host cells (eg. bacteria) by inserting vectors
- Detect genetically modified /transformed cells by identifying those containing the marker gene
- Culture these transformed host cells, allowing them to divide and form clones
tranform: refers to modification of the host cell / plasmid
Explain why promoter and terminator regions are added to DNA fragments that are used to genetically modify organisms
Promoter regions:
- a sequence of bases that act as a binding site for RNA polymerase and transcription factors allowing transcription.
- Can be selected to ensure gene expression happens only in specific cell types
Terminator regions:
- Ensure transcription stops at the end of a gene, by stopping RNA polymerase
TF : transcription facctors
What are the role of vectors in recombinant DNA technology?
- To transfer DNA into host cells / organisms eg. plasmids or viruses (bacteriophage).
Explain the role of enzymes in in vivo cloning
- Restriction endonucleases cut vector DNA
- The same enzyme is then used to cut the target gene out
- the vector/plasmid DNA &
DNA fragment have sticky ends that can join by complementary base pairing - DNA ligase joins DNA fragments to vector DNA, Forming phosphodiester bonds between adjacent nucleotides
Describe how host cells are transformed using vectors
- Plasmids enter bacterial cell membrane through following heat shock in a calcium ion solution
Or - Viruses inject their DNA into cells which is then integrated into host DNA
Explain why marker genes are inserted into vectors
• To allow detection of genetically modified / transgenic cells / organisms
• If marker gene codes for antibiotic resistance, cells that survive antibiotic exposure = transformed
• If marker gene codes for fluorescent proteins, cells that fluoresce under UV light = transformed
• As not all cells / organisms will take up the vector and be transformed
Suggest how recombinant DNA tech can be useful in medicine
- GM bacteria produce human proteins (eg. insulin for type I diabetes) → more ethically acceptable than using animal proteins and less likely to cause allergic reactions
- GM animals / plants produce pharmaceuticals (pharming) → cheaper
• Gene therapy
Suggest how recombinant DNA technology can be useful in agriculture
- GM crops resistant to herbicides → only weeds killed when crop sprayed with herbicide
- GM crops resistant to insect attack → reduce use of insecticide
- GM crops with added nutritional value (eg. Golden rice has a precursor of vitamin A)
- GM animals with increased growth hormone production (eg. Salmon)
Suggest how recombinant DNA technology can be useful in industry
- GM bacteria produce enzymes used in industrial processes and food production
Describe gene therapy
- Introduction of new DNA into cells, often containing healthy / functional alleles
- To overcome effect of faulty / non-functional alleles in people with genetic disorders eg. cystic librosis
Suggest some issues associted with gene therapy
- effect is short lived as modifed cells (e.g. T cells) have a limited lifespan requires regular treatment
- immune response against genetically modiefied cells or viruses due to recognition of antigens
- long term effect not known: side effects can cause cancer
- DNA may be inserted into other genes , disrupting them - interfering with gene expression
Suggest why environmentalists and anti-globalisation activists might oppose recombinant DNA technology
- recombinant DNA may be transferred to other plants : potential herbicide resistant superweeds
- potential effects on food webs e.g. affect wild insects -> reduce biodiversity
- large biotech companies my control the technology and own patents
What are 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 fluoresvent 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 only likely to occur in target alleles
What is DNA hybridisation?
- binding od a single stranded DNA probe to a complementary single strand of DNA
- forming hydrogen bonds/base pairs
Extract how genetic screening can be used to locate specific alleles of genes
- extract DNA and amplify by PCR
- Cut DNA at specific base sequences ( either side of the target gene) using restriction enzymes
- Separate DNA fragments / alleles using gel electrophoresis
- Transfer to a nylon membrane and treat to form single strands with exposed bases
- Add labelled DNA probes which hybridise / bind with target alleles ( & wash to remove unbound probe)
- 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.
what is gel electrophresis?
- a method used to seperate nucleic acid (DNA/RNA) fragments or proteins
- according to length/mass ( number of bases/amino acids) and charge (DNA is negatively charged due to phosphate groups and protein charge varies based on amino acid R groups)
Explain how gel electrophoresis can be used to seperate DNA fragments
- DNA samples loaded into wells in a porous gel and covered in buffer solution ( which conducts eelctricity)
- Electrical current passed thorugh - DNA is negatively charged so moves towards positive electrode
- Shorter DNA fragments travel faster so travel further
How can one interpret data showing results of gel electrophoresis?
- run a standard with DNA fragments/proteins of known lengths under the same conditions
- compare to position of unknown DNA fragments/proteins to estimate their size
- shorter DNA fragments / proteins travel further / faster
Describe examples of the use of labelled DNA probes
- screening patients for heritable conditions (e.g. cystic fibrosis)
- screening patients for drug responses
- screening patients for health risks
Describe the role of a genetic counseller
- Explain results of genetic screening including consequences of a disease
- discuss treatments available for genetic conditiion
- Discuss lifestyle choices/precautions that might reduce risk of a genetic condition developing
- Explain probability of condition/alleles being passed onto offspring - enable patients to make informed decisions about having children
What is personalised medicine?
- medicine tailored to an individual’s genotype / DNA
- increasing effectiveness of treatment e.g, by identifying to the particular mutation / allele causing cancer and treating it with tailored drugs.
Evaluate the screening of indidviduals for genetically determined conditions and drug responses
For
- can enable people to make lifestyle choices to reduce chances of diseases developing
- allows people to make informed decisions about having their own biological children
- allows use of personalised medicisnes , increasing effectiveness of treatment
Evaluate the screening of indidviduals for genetically determined conditions and drug responses
Against
- screening for incurable diseases or diseases that develop later in life (where nothing positivve can be done in response) may lead to depression.
- could lead to discrimination by insurance companies / employers
- may cause undue stress if patient does not develop the disease
what is genetic fingerprinting
- a laboratory technique that identifies a person by their DNA.
- It’s used in forensics, medicine, and other scientific fields.
what are variable number tandem repeats (VNTRs)
- repeating sequences of nucleotides / bases
- found within non-coding sections of DNA at many sites throughout an organism’s genome
why are VNTRs useful in genetic fingerprinting
- probability of two individuals having the same VNTRs is very low
- as an organism’s genome contains many VNTRs and lengths at each loci differ between individuals
Explain how genetic fingerprinting can be used to analyse DNA fragments
- Extract from sample (e.g.blood cells) and amplify by PCR
- Cut DNA at specific base sequences/recognition sites using restriction enzymes
- Separate VNTR fragments according to length using gel electrophoresis
- Transfer to a nylon membrane and treat to form single strands with exposed bases
- Add labelled DNA probes which hybridise . bind with complementary VNTRs & wash to remove unbound probe
- to show bound probe , expose membrane to UV light if fluorescently labelled probe was used OR use autoradiography ( expose to X-ray film) if a radioactive probe was used.
Compare and contrast genetic fingerprinting with genetic screening
- both use PCR to amplify DNA sample
- Both use eelctrophoresis to seperate DNA fragments
- Both use labelled DA probes to visualise specific DNA fragments
- Genetic fingerprinting analyses VNTRs whereas geentic screening analyses specific alleles of a gene
Explain how genetic fingerprinting can 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 around 50% of VNTRs/ bads with child ( due to inheritance)
Explain how genetic fingerprinting can be used to fetermine genetic variability within a population
- differences in VNTRs arise from mutations, so more differences show greater diversity within a population
Explain the use of genetic fingerprinting in the fields of forensic science
- compare genetic fingerprinting of suspects to genetic fingerprint of DNA at a crime scene
- if many bands match , the suspect was likely present at the crime scene
Explain the use of genetic fingerprinting in the fields of medical diagnosis
- Some VNTR patterns are asssociated with an increased risk of certain genetic disorders
Explain the use of genetic fingerprinting in the fields of animal and plant breeding
- shows how closely related 2 individuals are , so that inbreeding can be avoided
- breed pairs with dissimilar genetic fingerprinting
