19.20 Past Paper Practise
Q1
Read through the following account of genetic engineering and then fill in the spaces with the most appropriate
word or words.
During the process of hormone manufacture by genetic engineering, human RNA is extracted and converted to single stranded DNA by treatment with ……………………………………………. . This is then treated with …………………………………………… to produce double stranded (double helix) DNA. Plasmid DNA is also extracted from suitable bacteria for use as a ………………………………. .The human and plasmid DNAs are then treated separately with ………………………………………. which cuts them into fragments which have the same complementary ………………………………………… .The fragments of human and plasmid DNA are then mixed with
…………………………………………… . This joins the two types of DNA together as ……………………………… DNA which will hopefully contain the gene required for hormone synthesis. The plasmids are then mixed with host bacterial cells, such as cells of ……………………………….. . The presence of the chemical …………………………………………. aids the plasmid uptake by the bacteria. The bacteria can then undergo large scale culture and should produce suitable quantities of the required hormones. Hormones made in this way are……………………………………………and …………………………………. .
reverse transcriptase;
DNA polymerase;
vector;
restriction endonuclease;
sticky ends;
DNA ligase;
recombinant;
E. coli/any correct example;
calcium chloride/any appropriate salt;
insulin; (or somatotropin/growth hormone;)
somatotropin/growth hormone; (or insulin;)
Q2
The diagram shows the results of a DNA fingerprint analysis using a microscopic blood sample found at the scene of a crime. The DNA profile of the blood is shown on the left and labelled ‘scene of crime’. DNA profiles produced using blood samples from four suspects are shown on the right.
(a) (i) Which suspect has been incriminated by the DNA analysis? [1]
suspect 3;
(ii) Give a reason for your answer. [1]
because the bands match closely to the DNA at the scene of the crime/other individuals’ bands don’t match;
(b)Briefly describe the part played by each of the following in the production of the DNA profiles:
(i) polymerase chain reaction. [1]
increase quantity/number of copies of DNA (under investigation)/amplification of DNA;
(ii) restriction endonucleases. [2]
used to cut DNA into fragments;
if use the same restriction endonuclease samples from all suspects are cut at the same/similar places;
(iii) gel electrophoresis. [2]
separates cut fragments of DNA;
according to size;
(iv) radioactive DNA probes. [3]
probes are DNA strands with complementary sequence to cut fragments;
labelled using phosphorus–32;
probes bind to the complementary sequence;
fragments/radiation detected by autoradiography/using X-ray film;
specific sequences show up as dark bands;
Q3
Recombinant DNA products can be made either from genetically modified cloned cells such as bacteria or from
genetically modified mammalian cells in tissue culture.
(a) Name three recombinant DNA products that are manufactured for medical use. [3]
any three of:
insulin/somatotropin (accept growth hormone)/alpha-1 antitrypsin/interferon/any other correct examples;;;
(b) Suggest three advantages of producing genetically modified products from cloned bacterial cells rather than
from tissue cultures of mammalian cells. [3]
bacteria are very easily cultured in bulk but tissue culture cells usually remain as sheets of cells (which limits their culture size);
bacterial cultures are quick growing but tissue culture is a slow process, (thus bacterial cultures produce the product more quickly);
bacterial cultures usually give higher yields of products (than tissue cultures);
tissue cultures require more complex growth media/are more prone to contamination/infection/more difficult to manage (than
bacterial cultures);
max
(c) Recombinant DNA products can also be produced in transgenic animals. For instance, alpha-1 antitrypsin
can be produced by transgenic sheep. Suggest an advantage of producing a recombinant DNA product from
a sheep rather than from a bacterial culture. [2]
a bacterial culture may only survive for a short time/few weeks;
a sheep may survive for several years continually making the product/sheep can make the product for several years;
Q4
A genetic disorder in humans is alpha-1 antitrypsin deficiency. The gene coding for alpha-1 antitrypsin mutates with the result that the liver fails to manufacture this enzyme. The function of alpha 1-trypsin is to destroy any unwanted protease enzymes in body tissues. If the enzyme is missing then the main symptom is degeneration of alveolar tissue in the lungs due to attack by proteases. This leads to the condition of ‘inherited emphysema’.
The disorder can be treated either by gene therapy or by dosage with alpha-1 antitrypsin. It is possible to use transgenic sheep to provide adequate quantities of alpha-1 antitrypsin to enable patients to be treated.
DNA from sheep mammary gland cells, human DNA containing the alpha-1 antitrypsin gene and DNA containing a gene for neomycin resistance are combined as recombinant DNA. This DNA construct is placed into sheep cells which are then cultured in large numbers. The cells are then treated with neomycin and the surviving cells are used as nuclear donors to transfer the human alpha-1 antitrypsin gene into transgenic sheep.
(a) (i) How is human DNA usually obtained for use in gene technology? [2]
messenger RNA is extracted (from human cells);
treated with reverse transcriptase to make copy DNA/cDNA
(ii) How is recombinant DNA made from the sheep and human DNA? [3]
treat human DNA with restriction endonuclease to produce sticky ends;
treat sheep DNA with same restriction endonuclease (to obtain complementary sticky ends);
mix two sets of DNA fragments together;
treat with DNA ligase to seal fragments together;
(iii) How is the DNA construct placed into sheep cells. [2]
DNA construct mixed with sheep cells in tissue culture;
exposed to calcium phosphate/heat shock to make cells take up DNA;
(iv) Why are the cells treated with neomycin? [2]
neomycin will kill any sheep cells that have not taken in the (recombinant) DNA with the neomycin resistant gene;
remaining cells should contain the alpha-1 antitrypsin gene (and can be cultured in large numbers);
(b)If the insertion of the recombinant DNA into the transgenic sheep is successful then the sheep should start to
secrete large quantities of human alpha-1 antitrypsin from its liver.
(i) How may alpha-1 antitrypsin be obtained from the sheep? [1]
it could be collected from blood/plasma of the sheep/ it could be obtained from the sheep’s milk;
(ii) How could the alpha-1 antitrypsin be introduced to the lungs during treatment? [1]
by aerosol/inhalation;
Q5
The two commonest hereditary lung diseases in individuals of European descent are alpha-1 antitrypsin deficiency and cystic fibrosis. Both of the human genes involved have been cloned and gene therapy is of potential use in the treatment of both diseases.
Cystic fibrosis is due to a mutation in a gene that codes for a chloride channel protein in the cell membranes of epithelial cells. This protein regulates the secretion of chloride ions from the epithelial cells. If the secretion of chloride ions is reduced then the membrane resting potentials are raised, the surfaces are inadequately moistened with tissue fluid and mucus accumulates. This becomes infected and inflammation occurs. The symptoms affect the lungs, liver, gastrointestinal tract and pancreas. Since 90% of deaths due to cystic fibrosis are because of respiratory failure, gene therapy has focussed on correcting the genetic defect in the lungs.
(a) (i) What is meant by the phrase ‘the human genes have been cloned’ (line 2)? [2]
the genes have been isolated and inserted into recombinant DNA;
and multiplied many times to produce identical copies/ref to polymerase chain reaction;
(ii) List the sequence of steps involved in cloning, naming any enzymes that are used. [5]
DNA is copied from human RNA using reverse transcriptase;
treated with restriction endonuclease to produce DNA fragments with sticky ends;
bacterial plasmid /viral DNA treated with same restriction endonuclease;
to produce DNA fragments with complementary sticky ends;
DNA fragments mixed together and sealed/joined to make recombinant DNA using DNA ligase;
many copies made by polymerase chain reaction/amplification;
(b) In gene therapy the cloned genes must be delivered to the body organs that need them. In the case of cystic fibrosis this is the lungs. The genes must be inserted into suitable vectors for delivery.
(i) Suggest two suitable vectors that are used to transport cloned genes to lung cells. [2]
viruses/adenoviruses;
plasmid-liposome complexes;
(ii) Suggest two ways to introduce the vectors to the lungs. [2]
using an aerosol/inhaler;
intravenous injection;
(c) Alpha-1 antitrypsin is an enzyme that destroys protease enzymes in body tissues. It is manufactured by the liver cells but its most important site of operation is in the lungs. If alpha-1 antitrypsin is deficient then lung tissue is damaged by protease activity and hereditary emphysema develops. In gene therapy of this disease which would be the target organ for the gene vectors? Give a reason for your answer. [2]
liver;
this is where the gene operates to make alpha-1 antitrypsin;
Q6
Is genetical modification of crop plants for insect and pesticide resistance the answer to the world’s food problems or could it lead to a potential ecological disaster?
The European Corn Borer insect costs American farmers more than $1,000,000 in losses per year.
The first gene transplant into a plant by recombinant DNA technology was made over 15 years ago. Recombinant DNA technology enables geneticists to introduce new variations into a wide range of organisms.
Many transgenic plants planted in American corn and cotton fields were supposed to give the plants ‘do-it yourself’ resistance to insect attack. The implanted gene was taken from a soil bacterium, Bacillus thuringiensis, which is a pathogen to many species of insects. The gene manufactures a protein (referred to as Bt) which damages the gut of chewing insects, resulting in their death. The transgenic corn and cotton plants produce Bt in adequate quantities to kill insects that eat the crop. Mammals, fish and beneficial insects that eat crop pests are unharmed.
For several years in America, farmers have bought and planted genetically modified Bt corn and cotton seed, and
as a result have obtained greatly increased yields, and saved large amounts of money by not using insecticides.
However, in 1996 more than 18,000 acres of Bt crops were overwhelmed and devastated by insects.
In 1998 Bt modified corn was found to be releasing pollen which contained the Bt protein. When this pollen landed on milkweed plants, the food plant of the Monarch Butterfly caterpillar, it was found that caterpillars which had eaten the contaminated milkweed died within four days.
(a) Outline the steps which would be used to transfer the Bt gene from Bacillus thuringiensis into cotton plants. [4]
extract DNA from B thuringiensis and cut with (restriction) endonuclease;
extract DNA from (suitable) vector and cut with the same (restriction) endonuclease;
example of vector/virus/Agrobacterium tumefaciens (crown gall disease);
pool two DNA samples and DNA ligase to make recombinant DNA;
insert rDNA into vector using heat treatment/calcium ions to aid uptake;
mass culture vector and then infect cotton plants;
(b) Outline the steps by which the Bt gene enables the cotton plant to synthesize the Bt protein. [4]
Bt gene is present in cotton plant DNA/genome;
copied onto messenger RNA by transcription;
attached to ribosomes which enable translation;
transfer RNA brings specific amino acids to ribosomes;
reference to use of codons and anticodons to assemble polypeptide;
final assembly of Bt protein in Golgi body;
(c) Some insects have already developed resistance to the Bt protein since Bt crop damage due to insect attack
is occuring. Suggest a mechanism by which Bt resistance may have developed in insects. [4]
gene mutation/point mutation of gene/ref base substitution or equivalent;
to produce a gene which gives resistance to effects of Bt protein/protects insect gut from Bt protein;
these insects survive and reproduce, passing on the resistant gene;
not selected against/less competition since susceptible insects have died;
thus population can grow at a fast rate/huge population develops, (because crop provides almost unlimited food);
(d) Suggest two advantages and two possible disadvantages of introducing insecticide resistance into crop plants.
Advantages: [2]
Advantages:
greater yield/more profit/more food for people;
better quality food since no insect damage;
no need to use chemical insecticides/less expense/less pollution;
Bt insecticide (hopefully) only kills insect pest and no other organisms; max 2
Disadvantages: [2]
Disadvantages:
pollen may carry Bt protein and could kill (chewing) insects on contaminated plants;
reference to disruption of food chains (because of insect links being destroyed);
pollen may hybridise into other plants/weeds giving them insecticide resistance/selective advantage;
will probably cause the selection (and flourishing) of resistant populations of the insects it was developed to kill; max 2
(e) Comment on the ethical issues involved in the introduction of new varieties of crop plants by gene technology. [2]
it is important for humans to produce enough food to feed the whole human population;
so if GM crops help to do this in a safe way they should be developed;
there is a risk that GM crops may cause damage to ecological systems/cause serious risks to the survival of other organisms/lead to massive resistant insect/weed population explosions/be harmful to humans;
thus scientists should proceed with caution/employ stringent testing procedures/not be governed by short term
financial gain;
Reject:
vague non-scientific statements, such as ‘man should not play at being God/man should not interfere with God’s
creation’.
Q7
Restriction enzymes are so-called because they cut DNA at specific ‘restricted’ sites known as recognition sites.
The recognition site and the cleavage products of the restriction enzyme EcoRL are shown below.
(a) (i) The recognition site of the restriction enzyme Hind III is shown below. Write down the cleavage products in the space provided.
(ii) Using the example given in (a) (i) explain what is meant by the term ‘sticky ends’ [3]
fragments of DNA formed by a restriction enzyme;
which have dangling/exposed nucleotides
e.g. —TTCGA and AGCTT— ;
which are complementary to each other;
(iii) Outline the significance of sticky ends in genetic engineering. [2]
a particular restriction enzyme will cut any type of DNA in exactly the same position;
sticky ends will be complementary so DNA from any organism can be joined to DNA from any other organism;
Q8
The diagram below shows an outline of the processes involved in the production of useful genes and proteins by
genetic engineering.
(a) (i) Name enzyme X. [1]
restriction enzyme/restriction endonuclease;
(ii) Name the enzyme which will be used to open the vector. [1]
enzyme X/same restriction enzyme/same restriction endonuclease;
(b) What is the likely nature of the vector? [1]
plasmid/virus;
(c) Outline process B. [3]
plasmid/vector and bacterium suspended in (cool) calcium chloride solution;
then heated to 420C;
plasmid/vector enters bacterium;
(d) Outline the process which takes place inside the bacterium. [2]
plasmid/vector replicates;
if plasmid/vector contains gene, gene also replicates;
protein coded for by the gene may be synthesised;
Q9
Bacillus thuringiensis (Bt) is a bacterium that lives naturally in the soil. Bt produces a toxic protein known as T toxin which is poisonous to caterpillars and some other insects. Genetic engineers have inserted the gene for the production of T toxin into tomato plants. An outline of the procedure which they used is given below.
(a) What is the purpose of inserting the T toxin into tomato plants? [1]
make them toxic to pests/insects/caterpillars/give them a natural insecticide/reduce use of artificial pesticide;
(b) Explain how scientists could have identified the T toxin gene. [3]
identify sequence of amino acids in toxin;
use genetic code to identify the codons/base sequences;
make complementary radioactive gene probe;
to find those codons in the bacterial DNA;
(c) Name one enzyme which would have been used during cloning at stage 2. [1]
restriction enzyme/restriction endonuclease;
(d) The scientists were not sure that all of the leaves of the tomato plants would produce the toxin. Outline how this could be investigated in a laboratory. [3]
put genetically engineered plant/leaves from a genetically engineered plant into a container;
set up similar container using ordinary plant/leaves;
expose each to similar number of insects/caterpillar
record leaf damage/consumption/count dead insects (after a few hours);
Q10
In an effort to mass produce a useful protein, scientists extracted a precise section of mRNA from the cytoplasm of cells which naturally produced the protein.
The mRNA strand was then used as a template to enzymatically synthesise a strand of complementary DNA (cDNA).
A 2nd enzyme was then used synthesise a strand of DNA complementary to the first DNA strand.
The cDNA fragment was then inserted into a plasmid. To do this the plasmid was opened using enzymes and the sticky ends of the plasmid were joined to the complimentary bases on the cDNA.
(a) Identify the enzymes A B and C. [3]
A: reverse transcriptase;
B: DNA polymerase;
C: restriction enzyme/restriction endonuclease;
(b) Identify the vector which was used in this investigation. [1]
the plasmid;
(c) Name one other type of vector. [1]
viral DNA/phage DNA/liposomes/Agrobacterium tumefaciens;
(d)The vector was then inserted into the bacterium E.coli for mass production of the protein.
(i) How could the vector be inserted into the bacterium? [2]
mix vector and bacteria together;
treat with calcium chloride solution/give heat shock;
(ii) How could the protein be mass produced? [2]
culture bacteria on large scale/in industrial fermenter;
in continuous culture if protein is a primary metabolite/in batch culture if protein is a secondary metabolite;
Q11
Genetic engineering is used to ‘improve’ many crop plants. One example is the introduction of a gene into potato plants which gives them resistance to infection by potato leafroll virus (PLRV). The tumour inducing (Ti) plasmid of Agrobacterium tumefaciens is used as a vector. The diagrams below show the Ti plasmid in its natural state and in its recombinant state.
(a) (i) How could large quantities of DNA fragments which contain the required gene be obtained? [6]
extract DNA from potato plant which has shown resistance to (potato leafroll) virus (infection);
cut DNA into sections with sticky ends using a restriction endonuclease enzyme;
separate DNA fragments by (gel) electrophoresis;
treat with alkali to split double helix to single strands;
blot DNA onto a (nylon) membrane and treat with radioactive gene probe;
to recognise specific base sequences/thus locating DNA fragments with the required gene;
locate DNA fragments with X ray film;
collect by washing from nylon;
amplify/multiply fragments using the polymerase chain reaction;
(ii) How could Ti plasmid fragments with the tumour causing genes cut out be obtained? [4]
extract DNA from bacterium and separate plasmid DNA;
by (ultra)centrifugation/(gel) electrophoresis;
treat with same restriction endonuclease to obtain complementary sticky ends;
separate fragments by gel electrophoresis;
use gene probe to identify and discard fragments containing tumour gene;
use polymerase chain reaction to multiply/amplify remaining plasmid fragments;
(iii) How could recombinant Ti plasmids be obtained? [2]
mix plasmid DNA with sticky ends and potato DNA with sticky ends together;
treat with DNA ligase to seal ends together;
(iv) How could the plasmids be introduced into Agrobacterium tumefaciens? [2]
mix plasmids in growing culture of the bacterium;
in presence of calcium ions/apply heat shock;
(v) How could the spliced genes be inserted into potato plants? [3]
culture transformed bacteria and potato tissue/callus together;
bacteria infect potato tissue and plasmids incorporate into potato cells;
using plant cell attachment gene;
callus/culture differentiates into new resistant potato plants;
can be recognised by effects of marker gene;
(b) The potato plants modified above are known as transgenic plants. State two other applications of transgenic plants. [2]
insect resistant plants;
fungal resistant plants;
pesticide resistant plants;
improved flavour tomatoes;
increased shelf life tomatoes; (any correct examples)
Q12
Briefly describe the following:
(a) a gene probe: [4]
gene probe is a length of single stranded DNA used to locate a gene;
is tagged with a radioactive tracer/fluorescent dye so that it can be located;
probe contains base sequences complementary to those in the gene/either side of the gene;
target DNA containing the required gene must be in single stranded state;
thus when probe and target DNA are placed together they bind (and the gene is marked);
(b) the polymerase chain reaction: [6]
target DNA containing required gene is located using a gene probe;
short nucleotide sequences either side of the gene are determined and complementary lengths of DNA (oligonucleotides) are
chemically synthesised;
target DNA is heated/heated to 930C which causes double helix to unwind;
cooled/cooled to 550C, oligonucleotides added which bind to complementary sequences (either side of gene);
DNA polymerase added and temperature raised slightly/to 720C;
two new copies of the gene are then made;
process then repeated, the number of copies doubling each time;
ref to use of automated process/machine which can produce millions of copies in a few hours;
(c) DNA fingerprinting: [5]
DNA extracted from blood/semen/any biological material;
cut into lengths using a restriction endonuclease;
fragments separated by gel electrophoresis;
transferred/blotted onto a nylon membrane/ref Southern blotting;
radioactive DNA probe then applied and attaches to specific base sequences;
nylon membrane placed in contact with X-ray film to locate radioactive regions;
banding patterns on different DNA samples can thus be compared for similarity;
Q13
The table below refers to the uses of several enzymes which are associated with genetic engineering. Complete the table by writing the names of an appropriate enzyme in each empty box.
| Action of enzyme: | Named enzyme: |
| Enables transcription of DNA from mRNA | |
| Enables transcription of mRNA from DNA | |
| Cuts DNA at specific base sequences | |
| Binds DNA fragments of different origin together | |
| Enables polypeptide synthesis from amino acids in the ribosomes | |
| Enables DNA replication in the cell cycle | |
| Used to make multiple copies of DNA in genetic engineering |
| Action of enzyme: | Named enzyme: |
| Enables transcription of DNA from mRNA | reverse transcriptase; |
| Enables transcription of mRNA from DNA | RNA polymerase; |
| Cuts DNA at specific base sequences | restriction endonuclease; |
| Binds DNA fragments of different origin together | DNA ligase; |
| Enables polypeptide synthesis from amino acids in the ribosomes | peptide synthetase; |
| Enables DNA replication in the cell cycle | DNA polymerase; |
| Used to make multiple copies of DNA in genetic engineering | DNA polymerase; |
Q14
Biologists have traditionally recognised three different subspecies of chimpanzee based on physical characteristics, genetics and geography. Pan troglodytes troglodytes lives in Central Africa, Pan troglodytes schweinfurthii lives in East Africa and Pan troglodytes verus lives in West Africa. Recently, a small new population of chimpanzees living in southeastern Nigeria has come to the attention of biologists. It is suspected that this small population make up a new subspecies. Genetic analysis of animals for taxonomic purposes is usually carried out on mitochondrial DNA.
(a) (i) Name a technique which could be used to compare the mitochondrial DNA of the different chimpanzee subspecies. [1]
DNA fingerprinting;
(ii) Briefly describe the technique you have named. [5]
collect chimpanzee mitochondria by cell disruption/(ultra)centrifugation;
extract mitochondrial DNA and split with restriction endonucleases;
separate DNA fragments by gel electrophoresis;
blot onto nylon membrane/Southern blotting;
treat with radioactive/fluorescent gene probe to recognise and label specific base sequences;
overlay with X-ray film to detect labelled sequences;
(iii) What results would you expect to see if the Nigerian chimpanzees were a distinct subspecies? [2]
labelled areas of DNA show up as dark bands;
if (many) bands do not match those of other subspecies/are different from other subspecies, then they are probably an
independent subspecies;
(b) Name two other applications of the technique you have described. [2]
forensic science/murder/rape;
paternity disputes;
confirming animal pedigrees;
identification of human remains;
locating genes causing inherited disease;
locating genes for animal/plant breeding;