D1.3 Mutation and gene editing. Practice questions with markscheme.

• B: II-2 and II-4 have the condition and carry the allele in their blood cells, so each developed from a zygote that had it, yet it is absent from both parents' blood; a mutation in an early cell of the germ line (in a testis or an ovary) is copied by mitosis into the many cells that go on to form gametes, so more than one egg or sperm can carry it; a single mutated gamete (A) forms only one zygote, so it could not give the allele to two children of different sexes; B needs the same new mutation to arise twice, independently, and early enough in each child to reach its blood, which is very unlikely; if I-2 had the allele from her own zygote (C), it would be in all her cells, so she would have the condition and it would be found in her blood cells;

• C: plants grown from pieces of one parent plant are a clone with the same base sequences, and every plant changed the shape of its new leaves when its conditions changed, so the conditions altered how the same genes were expressed, not the genes themselves; A fails because mutations are random and rare changes to the base sequence, so the same mutation would not arise in all 15 plants at once and then be reversed in each of them when it was moved; C fails because no plants were lost: each plant itself changed the shape of its new leaves; D fails because the plants are genetically identical, so the groups cannot differ in their combinations of alleles;

• A: UUA, CUU, CUC and CUG all code for leucine, so four of the substitutions are silent because the genetic code is degenerate; one of them (CUA to UUA) changes the first base, so silent substitutions are not confined to the third base (A); every change to the second base here alters the amino acid (C); a changed codon can still code for the same amino acid (D);

• A: the error bars for the parents' blood cells do not overlap (about 21 ± 2 against 9 ± 1 per 10⁶ cells), so exposure raised the rate of mutation in these somatic cells; a child's new mutations arose in the germ cells of its parents, and the two groups of children have almost the same number (about 62 ± 2 and 61 ± 2, error bars overlapping), so no rise in the mutations passed on was detected; A ignores the rise in the parents' blood cells, so radiation did cause mutations; C cannot be drawn because blood cells are somatic cells, not gametes, and the data are not split by sex; D claims more than the data show: the gametes may have been exposed and the damage repaired, or a small rise may be too small to detect;

• D: the insertion shifts the reading frame from codon 20, but the deletion in codon 23 shifts it back, so codons 24 to 300 are read as normal; only the short stretch between the two changes is read in the shifted frame (here AGA CGG UCC ACA in place of AAC GGU CCA UCA), so the polypeptide keeps its length and may keep its function; B ignores the deletion that restores the frame; codons 21 and 22 are also read in the shifted frame, so more than two amino acids change (C); the bases between the two changes are grouped into different codons, so the changes do not simply cancel (D);

• a customer may misunderstand a variant linked to increased risk as a certain diagnosis / believe that they will definitely develop the disease;
• (leading to) unnecessary anxiety / distress / drastic decisions such as unnecessary surgery or treatment;
• (conversely) a negative result may give false reassurance, because the test does not cover every variant / most cases of the disease involve other genes or environmental factors;
• the risk reported is a probability that depends on other genes, lifestyle and family history, which the customer cannot weigh without expert help;
• results have implications for relatives, who share variants, and for insurance / employment / privacy, which the customer may not appreciate, OWTTE;

Mark the first three distinct problems only. Accept other reasonable problems that follow from the absence of expert interpretation; do not accept "the test could be wrong" without explanation.

• strength: DNA (structure/replication) is universal, so a chemical that damages bacterial DNA is likely to damage human DNA by the same chemistry;
• strength: bacteria allow huge numbers, rapid generations and easy counting, so the test is fast, cheap and quantitative (a dose–response can be measured) and avoids testing on animals/humans;
• limitation: bacteria lack human metabolism, a chemical may only become mutagenic (or be detoxified) after processing by liver enzymes, so results can be false negatives/positives (unless liver extract is added);
• limitation: uptake, DNA repair and exposure levels differ between bacteria and human tissues, and mutagenicity in bacteria does not prove the chemical causes cancer/disease in humans;
• judgement: a valuable first screen for identifying probable mutagens, but positive/negative results must be confirmed with further (mammalian-cell or epidemiological) evidence, OWTTE;

NOS: evaluating an experimental model system; require at least one strength, one limitation and a judgement.

• (a) [1]
  • 6.3;

  Accept 6.

• (b) [2]
  • salt increased the mutation rate / the number of new substitutions per line (by about 50 %, from 8.4 to 12.6);
  • the ranges given by the standard errors (8.0 to 8.8 and 12.1 to 13.1) do not overlap, so the difference is likely to be significant / not due to chance, OWTTE;

  The reason must refer to the standard errors or their ranges for the second mark.

• (c) [3]
  • for the claim: salty soil did raise the number of new mutations (by about 1.5 times) / 7 substitutions in salt-tolerance genes with salt compared with 4 without;
  • against: the number in salt-tolerance genes (7) is close to the number expected from their share of the genome (6.3) / about 1 % of all substitutions under both conditions (0.95 % and 1.1 %);
  • so the extra mutations were spread across the whole genome and were not directed to the genes that would help / mutations are random with respect to what the plant needs, OWTTE;
  • the data do not show whether any of the 7 substitutions increased salt tolerance: most mutations are neutral or harmful;
  • the numbers in salt-tolerance genes are very small (4 and 7), so a difference between them could be due to chance, OWTTE;
  • judgement: the claim is not supported: salt raised the mutation rate, but not specifically in the genes that would help, OWTTE;

  Evaluate: award [3] only for an answer that uses the data on both sides and reaches a judgement.

• (d) [3]
  • mutation is the original source of all genetic variation / of new alleles;
  • mutations are random, so only occasionally does one arise that increases salt tolerance, OWTTE;
  • most mutations are neutral or harmful, so beneficial ones are rare and many generations are needed, OWTTE;
  • the mutation must arise in a cell that gives rise to gametes (in the flowers) to be inherited by the next generation, OWTTE;
  • without new mutations there would be no new variation for natural selection to act on, OWTTE;

  Beyond this subtopic. Award if given; the marks above can be earned without it.

  • D4.1 plants carrying the beneficial allele survive and reproduce more on salty soil, so the frequency of the allele rises over the generations;

  Content pivot.

• A: the mutant polypeptide is one amino acid shorter, so bases were lost, and every amino acid after Cys is the same as in the normal polypeptide, so the reading frame was not shifted: three bases were deleted; Phe and Gly have been replaced by one different amino acid (Cys), so the three bases were not one whole codon but spanned two codons (for example, mRNA UUU GGC losing UUG leaves UGC, a codon for Cys); a single-base deletion (A) would change every amino acid after it; a substitution (B) does not change the length of the polypeptide; an insertion (C) would make it longer, not shorter;

• C: a mutation present in all her tissues must have been present in the zygote, so it was inherited through a mutated egg or sperm (the germ line); a mutation that arises in a body (somatic) cell is passed by mitosis only to the descendants of that cell, so one that arose in the tumour (B) or in a blood stem cell (D) would be confined to those cells; the identical change arising separately in every tissue is extremely unlikely (C);

• A: 1.5 times the average risk is 1.5 × 8 % = 12 %, so about 88 % of people with the variant never develop the disease; the result is a probability, not a diagnosis (A); B adds 50 percentage points and D adds 1.5 percentage points instead of multiplying the risk by 1.5; misreadings like these are why results need expert interpretation;

• D: the codons for Lys (AAA, AAG) and Asn (AAU, AAC) differ only at the third base, so a substitution of the third base (A or G replaced by U or C, or the reverse) explains the difference; this changes one amino acid, so a change to the third base does not always leave the amino acid unchanged (A); changing the first base of a lysine codon gives a codon for Gln (CAA, CAG) or another amino acid or a stop codon, and changing the second base gives Thr (ACA, ACG) or another amino acid, never Asn (B); an insertion would shift the reading frame and change every amino acid after it, not just one (D);

• B: synonymous positions differ about as often in the gene for P (38 %) as in the gene for Q (41 %), so both genes have gained mutations at a similar rate, which rules out a slower mutation rate (B) or no mutation at all (D); yet the amino acid sequence of P has hardly changed (0.4 %), so mutations that altered its amino acids must have been removed by natural selection because the function of P depends on that sequence; Q changed by 22 % over the same time, so there has been time for change (C);

• because 300 is a multiple of three, the reading frame is not shifted / the codons after the deletion are read as before;
• (but) 100 amino acids are missing from the middle of the polypeptide / the polypeptide is much shorter;
• (so) this is a major deletion: the missing section is very likely to include amino acids the enzyme needs, so the enzyme is likely to cease functioning, OWTTE;

Beyond this subtopic. Award if given; the marks above can be earned without it.

• C1.1.4 the missing amino acids may include those that form the active site / that make the bonds (R-group interactions) needed for correct folding;
• B1.2.7 (so) the polypeptide folds into a different three-dimensional (tertiary) shape;
• C1.1.7 (so) the active site is lost / no longer complementary to the substrate, and the enzyme cannot catalyse its reaction, OWTTE;

Do not accept a frameshift as the reason. Accept "a major deletion" or "a large deletion" as the category of change.

• strength: DNA (structure/replication) is universal, so a chemical that damages bacterial DNA is likely to damage human DNA by the same chemistry;
• strength: bacteria allow huge numbers, rapid generations and easy counting, so the test is fast, cheap and quantitative (a dose–response can be measured) and avoids testing on animals/humans;
• limitation: bacteria lack human metabolism, a chemical may only become mutagenic (or be detoxified) after processing by liver enzymes, so results can be false negatives/positives (unless liver extract is added);
• limitation: uptake, DNA repair and exposure levels differ between bacteria and human tissues, and mutagenicity in bacteria does not prove the chemical causes cancer/disease in humans;
• judgement: a valuable first screen for identifying probable mutagens, but positive/negative results must be confirmed with further (mammalian-cell or epidemiological) evidence, OWTTE;

NOS: evaluating an experimental model system; require at least one strength, one limitation and a judgement.

• a knockout removes the product of one gene only, so a change in phenotype can be attributed to that gene, giving direct evidence of its function;
• gene editing with CRISPR-Cas9 allows a chosen sequence to be targeted precisely, and knockouts can be produced quickly in organisms with short life cycles;
• unedited organisms of the same strain provide a control, so the comparison is valid;
• however, other genes may have overlapping functions, so a knockout may produce no visible phenotype even when the gene is important (functional redundancy);
• knocking out an essential gene may kill the embryo, so any later function of the gene cannot be observed;
• loss of one gene can alter the expression of others, so the phenotype seen may be an indirect consequence rather than the normal role of the gene;
• conclusion: the approach yields useful, testable evidence but its results need confirming by other methods, such as measuring where and when the gene is normally expressed, OWTTE;

A judgement is required for the final mark. Both strengths and weaknesses must appear.

• I, Cas9 (enzyme) / (endo)nuclease (protein);
• II, guide RNA / gRNA / sgRNA;
• III, target sequence (of the gene) / DNA base sequence complementary to the guide RNA;
• IV, DNA (double helix) / double-stranded DNA (of the chromosome);

Do not accept 'CRISPR' alone for I. For II do not accept mRNA or tRNA. For III accept 'region where the guide RNA base-pairs with the DNA'; do not accept 'guide RNA' or 'PAM'.