Paper 2

What is a gene mutation? (2 marks)

A change in the DNA base sequences. Which can arise spontaneously during DNA replication.

What is a mutagenic agent (1 mark)

A factor that increases rate of mutation.

Explain how a gene mutation can lead to the production of a non-functional protein or enzyme (5 marks)

Changes sequence of base triplets in DNA so changes sequence of codons on mRNA. So changes sequence of amino acids 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 complex can’t form

Describe a duplication mutation (1 mark)

A sequence of DNA bases/nucleotides is repeated/copied.

Describe a inversion mutation (1 mark)

A sequence of bases / nucleotides detaches from the DNA sequence, then rejoins at the same position in the reverse order.

Describe a translocation mutation (1 mark)

A sequence of DNA bases / nucleotides detaches and is inserted at a different location within the same or a different chromosome

Explain why not all gene mutations affect the order of amino acids (2 marks)

Some substitutions change only 1 triplet code / codon which could still code for the same amino acid (As the genetic code is degenerate (an amino acid can be coded for by more than one triplet)). Some occur in introns which do not code for amino acids as they are removed during splicing.

Explain why a change in amino acid sequence is not always harmful (2 marks)

May not change tertiary structure of protein (if position of ionic / disulphide / H bonds don’t change). May positively change the properties of the protein, giving the organism a selective advantage.

Explain what is meant by a frameshift (2 marks)

Occurs when mutations change the number of nucleotides/bases by a number not divisible by 3. This shifts the way the genetic code is read, so all the DNA triplets/mRNA codons downstream from the mutation change.

Explain how mutations can lead to production of shorter polypeptides (2 marks)

Deletion or translocation causes codons/triplets to become missing so amino acids are missing. Substitution, addition, deletion, duplication, inversion or translocation can cause a premature stop codon, so amino acids are missing at the end of a polypeptide.

What are stem cells and what are they capable of?

Undifferentiated/unspecialised cells capable of: dividing by mitosis or differentiating into other types of cells.

Describe how stem cells become specialised during development (3 marks)

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.

Describe totipotent cells (2 marks)

Occur for a limited time in early mammalian embryos. Can divide and differentiate into any type of body cell.

Describe pluripotent cells (2 marks)

Found in mammalian embryos (after first few cell divisions). Can divide and differentiate into most cell types.

Describe multipotent cells (2 marks)

Found in mature mammals. Can divide and differentiate into a limited number of cell types (e.g., multipotent cells in bone marrow can divide and differentiate into different types of blood cell).

Describe unipotent cells, using an example (2 marks)

Found in mature mammals. Can divide and differentiate into just one cell type (e.g., cardiomyocytes- cardiac muscle cells).

Explain how stem cells can be used in the treatment of human disorders (2 marks)

Transplanted into patients to divide in unlimited numbers. Then differentiate into required healthy cells (to replace faulty / damaged cells).

Example of stem cell treatments of human disorders (2 marks)

Potential treatment of Type 1 diabetes, by creating healthy islet cells that produce insulin. Bone marrow stem cell transplant for sickle cell disease/blood cancers (either destroying a patients bone marrow before treatment, or transplanting stem cells from a healthy person)

Explain how induced pluripotent stem (iPS) cells are produced (3 marks)

Obtain adult somatic (body) cells from patient. Add specific protein transcription factors (which bind to promoter regions of DNA) associated with pluripotency so they express genes associated with pluripotency. Culture cells to allow them to divide by mitosis.

Evaluate the use of stem cells in treating human disorders (6 marks)

For: Can divide and differentiate into required healthy cells, so could relieve human suffering by saving lives and improving quality of life. Embyros 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 patients own cells. Against: Ethical issues with embryonic stem cells as obtaining them requires destruction of an embryo (a potential destruction of life). Immune system could reject cells and immunosuppressant drugs are required. Cells could divide out of control, leading to tumour formation.

What are transcription factors? (2 marks)

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 (3 marks)

Transcription factors move from cytoplasm to nucleus. Bind to DNA at specific DNA base sequence on promoter region. This stimulates or inhibits transcription of target gene, by helping or preventing RNA polymerase binding.

Explain how oestrogen effects transcription (6 marks)

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 diffuses from cytoplasm into the nucleus. Then binds to a specific DNA 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 (1 mark)

Other cells do not have oestrogen receptors.

Describe what is meant by epigenetic (2 marks)

Heritable changes in gene function / expression without changes to the base sequence of DNA. Caused by changes in the environment (eg. diet, stress, toxins).

What is the effect of increased methylation of DNA? (1 mark)

Inhibiting transcription

What is the effect of increased Acetylation of histones? (1 mark)

Promoting transcription

Explain how methylation can inhibit transcription (3 marks)

Increased methylation of DNA- methyl groups added to cytosine bases in DNA. So nucleosomes pack more tightly together. Preventing transcription factors and RNA polymerase binding to promoter.

Explain how acetylation can inhibit transcription (3 marks)

Decreased acetylation of histones increases positive charge of histones. So histones bind DNA (which is negatively charged) more tightly. Preventing transcription factors and RNA polymerase binding to promoter.

Explain the relevance of epigenetics on disease development and treatment (4 marks)

Environmental factors can lead to epigenetic changes. These can stimulate/inhibit gene expression that can lead to disease development. Diagnosis tests can be developed that detect these epigenetic changes. Drugs can be developed to reverse these epigenetic changes.

What is RNA interference (RNAi)

Inhibition of translation of mRNA produced from target genes, by RNA molecules eg. siRNA, miRNA. This inhibits expression of (silencing) a target gene.

Describe the regulation of translation by RNA interference (4 marks)

Small interfering RNA (siRNA) or micro RNA (miRNA) binds to a protein, forming an RNA induced silencing complex. Single-stranded miRNA / siRNA within RISC binds to target mRNA with a complementary base sequence. This leads to hydrolysis of mRNA into fragments which are then degraded OR prevents ribosomes binding. Reducing / preventing translation of target mRNA into protein.

Describe how tumours and cancers form (2 marks)

Mutations in DNA controlling mitosis can lead to uncontrolled cell division. Tumour formation results in mass of abnormal body cells.

Contrast benign and malignant tumours (6 marks)

Benign tumours grow slower. In benign tumours cells are well differentiated/specialised whereas cells in malignant tumours are poorly differentiated/unspecialised. In benign tumours, cells have normal, regular nuclei, whereas in malignant tumours, cells have irregular nuclei. In benign tumours, they often have well defined borders, often surrounded by a capsule so not invade surrounding tissue, whereas in malignant tissues, they can invade surround tissues. In benign tumours, they do not spread by metastasis, whereas they do in malignant tumours. Benign tumours rarely return but malignant tumours often return.

Describe the function of tumour suppressor genes (2 marks)

Code for proteins that: inhibit/slow cell cycle OR cause cell destruction.

Explain the role of tumour suppressor genes in the development of tumours (3 marks)

● Mutation in DNA base sequence → production of non-functional protein
○ By leading to change in amino acid sequence which changes protein tertiary structure

● Decreased histone acetylation OR increased DNA methylation → prevents production of protein ○ By preventing binding of RNA polymerase to promoter region, inhibiting transcription

● Both lead to uncontrolled cell division (cell division cannot be slowed)

Describe the function of protooncogenes (1 mark)

Code for proteins that stimulate cell division.

Explain the role of oncogenes in the development of tumours (3 marks)

● Mutation in DNA base sequence → overproduction of protein OR permanently activated protein ○ By leading to change in amino acid sequence which changes protein tertiary structure

● Decreased DNA methylation OR increased histone acetylation → increases production of protein ○ By stimulating binding of RNA polymerase to promoter region, stimulating transcription

● Both lead to uncontrolled cell division (cell division is permanently stimulated)

Explain the relevance of epigenetics in cancer treatment (2 marks)

Increasing DNA methylation OR decreasing histone acetylation of oncogene (to inhibit transcription/gene expression). Decreasing DNA methylation OR increasing histone acetylation of tumour suppressor gene (to stimulate transcription/gene expression).

Explain the role of increased oestrogen concentrations in the development of some breast cancers (4 marks)

Some breast cancers may 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, stimulating cell division. This increases transcription/expression go these genes, increasing rate of cell division.

Suggest how drugs that have a similar structure to oestrogen help treat oestrogen receptor-positive breast cancers (2 marks)

Drugs bind to oestrogen receptors, preventing binding of oestrogen. So no/few transcription factors bind to promoter regions of genes that stimulate the cell cycle.

Define genome (1 mark)

The complete set of genes in a cell.

Define proteome (1 mark)

The full range of proteins that a cell can produce.

What is genome sequencing and why is it important (2 marks)

Identifying the DNA base sequences. So amino acid sequences of proteins can be determined.

Potential applications of genome sequencing projects (2 marks)

Identification of genes/alleles associated with genetic diseases. Identification of species and evolutionary relationships.

Explain why the genome cannot be directly translated into the proteome in complex organisms (2 marks)

Presences of non coding DNA. Presence of regulatory genes.

Describe how sequencing methods are changing (2 marks)

They have become automated. They are continuously updated.

Describe how DNA fragments can be produced using restriction enzymes (2 marks)

Restriction enzymes cut DNA at specific base ‘recognition sequences’ either side of the desired gene(Shape of recognition site complementary to active site). Many cut in a staggered fashion forming ‘sticky ends’ (single stranded overhang).

Suggest two advantages of obtaining genes from mRNA rather than directly from the DNA removed from cells

Much more mRNA in cells making the protein than DNA. In mRNA, introns have been removed by splicing.

Describe how fragments of DNA can be produced using a gene machine (2 marks)

Synthesis of DNA fragments quickly and accurately from scratch. Which don’t contain introns.

Name an in vitro and in vivo technique 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

Explain how DNA fragments can be amplified by PCR

1. Mixture heated to 95oC	●  This separates DNA strands ●  Breaking hydrogen bonds between bases
2. Mixture cooled to 55oC	●  This allows primers to bind to DNA fragment template strand ●  By forming hydrogen bonds between complementary bases
3. Mixture heated to 72oC	●  Nucleotides align next to complementary exposed bases ●  DNA polymerase joins adjacent DNA nucleotides, forming phosphodiester bonds

Explain the role of primers in PCR (4 marks)

• ●  Primers are short, single stranded DNA fragments

• ●  Complementary to DNA base sequence at edges of region to be copied / start of desired gene

• ●  Allowing DNA polymerase to bind to start synthesis (can only add nucleotides onto pre-existing 3’ end)

• ●  Two different primers (forward and reverse) are required (as base sequences at ends are different)

Summarise the steps involved in amplifying DNA fragments in vivo (5 marks)

1. Add promoter and terminator regions to DNA fragments

2. Insert DNA fragments & marker genes into vectors (eg. plasmids) using restriction enzymes and ligases

3. Transform host cells (eg. bacteria) by inserting these vectors

4. Detect genetically modified (GM) / transformed cells / organisms by identifying those containing the marker gene (eg. that codes for a fluorescent protein)

5. Culture these transformed host cells, allowing them to divide and form clones

Explain why promoter and terminator regions are added to DNA fragments that are used to genetically modify organisms.

Promoter regions	●  Allow transcription to start by allowing RNA polymerase to bind to DNA ●  Can be selected to ensure gene expression happens only in specific cell types ○ Eg. in gland cells of a mammal so the protein can be easily harvested
Terminator regions	● Ensure transcription stops at the end of a gene, by stopping RNA polymerase

Explain the 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 vector DNA & fragments have ‘sticky ends’ that can join by complementary base pairing.

2. DNA ligase joins DNA fragment to vector DNA ○ Forming phosphodiester bonds between adjacent nucleotides.

Describe how host cells are transformed using vectors (2 marks)

Plasmids enter cells. Viruses inject their DNA into cell which is integrated into host cell.

Explain why marker genes are inserted into vectors

To allow detection of genetically modified cells

Suggest how Recombinant DNA technology can be useful

Medicine	●  GM bacteria produce human proteins (eg. insulin for type 1 diabetes) → more ethically acceptable than using animal proteins and less likely to cause allergic reactions ●  GM animals / plants produce pharmaceuticals (‘pharming’) → cheaper ●  Gene therapy (see below)
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)
Industry	● GM bacteria produce enzymes used in industrial processes and food production

Describe gene therapy (2 marks)

Introduction of new DNA with healthy alleles. To overcome effect of faulty alleles.

What are DNA probes? (3 marks)

• ●  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

What is DNA hybridisation? (2 marks)

Binding of a single stranded DNA probe to a complementary single strand of DNA. Forming hydrogen bonds/base pairs.

Explain how genetic screening can be used to locate specific alleles of gene (6 marks)

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 (according to length) 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 (expose to X-ray film) if a radioactive probe was used

What is gel electrophoresis (2 marks)

A method used to separate DNA/RNA according to length/mass.

Explain how gel electrophoresis can be used to separate DNA fragment (3 marks)

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 so moves towards positive electrode

3. Shorter DNA fragments travel faster so travel further

3 examples of the use of labelled DNA probes

Screening patients for: heritable conditions, drug responses and health risks.

Evaluate the screening of individuals for genetically determined conditions and drug responses.

For	✓  Some people could be heterozygous / carriers (eg. in families with a history of a disease) ✓  Can enable these people to make lifestyle choices to reduce chances of diseases developing, to prevent suffering / death ✓  Allows people to make informed decisions about having their own biological children ✓  Allows use of personalised medicines, increasing effectiveness of treatment
Against	𝖷  Screening for incurable diseases or diseases that develop later in life (where nothing positive can be done in response) may lead to depression 𝖷  May cause undue stress if patient does not develop the disease 𝖷  Could lead to discrimination by insurance companies / employers 𝖷  Many diseases are rare 𝖷  Many are caused by many genes so would need too many probes (expensive)

What are variable number tandem repeats (VNTRs)? (2 marks)

Repeating sequences of nucleotides / bases (eg. GATA). Found within non-coding sections of DNA at many sites throughout an organism’s genome.

Explain how genetic fingerprinting can be used to analyse DNA fragments (6 marks)

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

Compare and contrast genetic fingerprinting with genetic screening (4 marks)

Both use PCR to amplify DNA sample, use electrophoresis to separate DNA fragments, use labelled DNA probes to visualise specific DNA fragments. Genetic fingerprinting analyses VNTRs whereas genetic screening analyses specific alleles of a gene

Explain how genetic fingerprinting can be used to determine genetic relationships (2 marks)

More closely related organisms have more similar VNTRs. Can be used in paternity testing.

Explain how genetic fingerprinting can be used to determine genetic variability within a population (1 mark)

Differences in VNTRs arise from mutations, so more differences show greater diversity within a population.