Biology (Unit 5)

Front 1

Define the genetic code.

Back 1

This consists of base triplets in RNA which code for amino acids.

Front 2

Describe the features of genetic code.

Back 2

- It is universal - the code is the same for all organisms

- Is non-overlapping - the ribosome reads each base only once in the codons, the first three bases are read first, followed by the second three etc

- Is degenerate - more than one triplet codes for an amino acid

Front 3

Define gene mutation.

Back 3

A change in the sequence of bases in a gene.

Front 4

How can a mutation occur during DNA replication?

Back 4

- Addition/deletion

- Substitution

- Inversion

- Duplication

- Translocation

Front 5

Describe addition/deletion mutations.

Back 5

- Complete loss/gain of a base in a region of DNA

- The deleted/added nucleotide results in an alteration of base triplets from the mutation onwards

- The reading frame has been shifted to the left by one letter (frame shift)

Front 6

What would happen if a addition/deletion mutation occurred earlier in the DNA sequence?

Back 6

All of the triplets coding for a polypeptide could be altered, so all the amino acids would be altered.

Front 7

What would happen if a addition/deletion mutation occurred later in the DNA sequence?

Back 7

It would occur later in the sequence so there would a lesser effect than if it had occurred earlier in the sequence, but would still alter some amino acids at the end of a gene.

Front 8

Describe substitution mutations.

Back 8

- A substitution in the third base of any triplet would not alter the amino acid coded for (silent mutation)

- A substitution in the first base of any of any triplet will change the amino acid sequence; this will alter the sequence of amino acids in the polypeptide chain so may alter its shape and function

- When a mutation causes a triplet to code for a STOP it will cause the growing polypeptide chain to terminate and would probably not be able to perform its proper function

Front 9

Describe an inversion mutation.

Back 9

When a segment of bases is reversed end to end.

Front 10

Describe a duplication mutation.

Back 10

A doubling of a part of a chromosome, of an entire chromosome or even the whole genome.

Front 11

Describe a translocation mutation.

Back 11

When groups of base pairs relocate from one area of the genome to another, usually between non-homologous chromosomes.

Front 12

Describe a mutagenic agent.

Back 12

- High energy radiation (X-rays and Gamma rays)

Front 13

Define a mutagenic agent.

Back 13

Increase the rise of mutation; high energy radiation damages the DNA molecule and chemicals that alter the DNA structure or interfere with DNA replication.

Front 14

How do mutations occur?

Back 14

- Occur spontaneously during DNA replication

- Natural and random

- Cause permanent changes to the DNA which is passed on to future generations

- Occur at a set rate (which is different for each species)

Front 15

Why are mutations necessary?

Back 15

- Responsible for the genetic diversity of populations both in the forces of natural selection and in speciation

- Often produce organisms that are less well adapted to their environment and can alter cellular activities.

Front 16

How do zygotes divide in the womb?

Back 16

Mitosis

Front 17

Define totipotent cells.

Back 17

- Not specialised

- Capable of expressing all genes

- During development they can then differentiate to specialise to any other cell type within the organism

Front 18

How do totipotent cells change during division?

Back 18

As cell differentiate to carry put a specific function most cells lose their totipotency. This process of specialisation is irreversible in most animal cells.

Front 19

Define housekeeper genes.

Back 19

During cell specialisation, only the genes required for essential processes within the cell (called houskeeper genes) and those needed to produce the proteins required for specialised function of the cell are expressed.

Front 20

Define unipotent cells.

Back 20

Cells can only differentiate into one type of cell.

Front 21

Define pluripotent cells.

Back 21

- As an embryo develops its cells begin to specialise but they still retain their ability to develop into almost all (MOST) cell types

- AKA embryonic stem cells

Front 22

Define mutlipotent cells.

Back 22

- Only a few cells within the adult mammal retain the ability to differentiate into a limited number of different (SOME) cell types

- E.g. human adult stem cells in bone marrow

Front 23

Give an example of a unipotent cell.

Back 23

Red blood cells - produced by stem cells in the bone marrow, the stem cell produces a new cell in which the genes for haemoglobin are expressed. Other genes such as those involved in removing the nucleus are expressed also

Nerve cells - produced from stem cells in the neural tube, the stem cell produces a new cell in which the genes that direct the axon and dendrites to spread out are expressed

Cardiomyocytes - cells that make up a lot of tissue in our hearts

Front 24

What could stem cells be used for?

Back 24

1. Producing tissues for skin grafts

2. Research into producing organs for transplant

3. Research into how cells become specialised

4. Research into cancer

5. Research into serious diseases and the use of stem cells to cure such diseases as Parkinson's

Front 25

Why are stem cells useful?

Back 25

- Have the capacity to develop into many types of cells

- May provide cells to form new tissue and organs for transplant

- Embryonic stem cells are more useful than adult stem cells as they have the capacity to develop into more cell types

Front 26

How can we collect stem cells?

Back 26

- Pluripotent cells can be isolated from spare embryos produced in fertility clinics

- Embryo can be grown and pluripotent cells can be isolated and the rest of the cells isolated

- The pluripotent cells can be grown and develop into tissues

- Adult stem cells can be a source of multipotent cells

Front 27

Define iPS.

Back 27

- Induced pluripotent stem cells

- Produced in a lab

- Involves programming specialised adult body cells so that they become pluripotent

- The adult stem cells are made to express a series of transcription factors that are normally associated with pluripotent stem cells

- The transcription factors cause the adult body cells to express genes that are associated with pluripotency

Front 28

How can the transcription factors be introduced to adult stem cells used in iPS?

Back 28

- Infecting them with a specially modified virus

- The virus has the genes coding for the transcription factors within its DNA

- When the virus infects the adult cell, the genes are passed into the adult cell's DNA, meaning that the cell is able to produce the transcription factors

Front 29

What are some of the ethical concerns with using stem cells?

Back 29

- Some people consider that it is not right to use stem cells from embryos as embryos have human status from the moment of conception

- Others believe that the embryo has a special status as a potential human being but atthe early stages it is not a true human being and the removal of cells from spareembryos for medicine has many benefits

- Others believe that embryos have no moral rights whatsoever

- The use of stem cells is also questioned as animals have to be used in experiments withstem cells before the stem cells can be trialled on humans

- Concerns with iPS technology also include the patient donor giving consent for theircells uses

Front 30

Define the promotor region.

Back 30

One or more base sequences found upstream of a gene that control theexpression of that gene. It is here that the transcription factor will bind.

Front 31

Describe transcription factors.

Back 31

Proteins which when activated bind to the promoter region of a genestimulating RNA polymerase to transcribe the target gene.

Front 32

In an eukaryote, where do transcription factors move from?

Back 32

Transcription factors move from the cytoplasm to the nucleus.

Front 33

Describe how oestrogen acts as a transcription factor.

Back 33

1. Oestrogen is lipid soluble and passes through the cell membrane and nuclear membraneinto the nucleus of the cell

2. Oestrogen binds to complementary receptor (ER alpha) - inactive transcription factor

3. ER alpha oestrogen receptor changes shape and is release from protein complex whichinhibits its action

4. Oestrogen receptor binds to promoter region of target gene and stimulates RNApolymerase to transcribe the target gene

Front 34

Describe an activating transcription factor.

Back 34

Activators stimulate or increasethe rate of transcription (e.g. they help RNA polymerase bind to the start of the targetgene and activate transcription)

Front 35

Describe a repressing transcription factor.

Back 35

Repressors inhibit or decrease the rate of transcription(e.g. they bind to the start of the target gene, preventing RNA polymerase from binding,stopping transcription).

Front 36

Define RNAi.

Back 36

- In eukaryotes, gene expression is also affected by RNA interference (RNAi)

- RNAi is when small, double stranded RNA molecules stop mRNA from target genes being translated into proteins

Front 37

Define siRNA.

Back 37

- The siRNA has a complementary base sequence to part of a specific mRNA molecule

- The siRNA molecule blocks gene expression by binding to the mRNA forming on RNA-induced silencing complex (RISC)

- This action allows the enzyme (RNA hydrolyse) to bind and breakdown the target mRNA molecule or prevent ribosome attachment

- Without mRNA, translation does not take place, the polypeptide is not produced

- The expression of gene has been blocked

Front 38

Describe the applications of siRNA.

Back 38

- Scientific research as siRNA can be added via viruses or liposomes into cells to switch off a target gene to allow us to study what the gene does

- In the same way disease causing genes could also be switched off by the addition of specific siRNA to cells

Front 39

Describe the gene expression experiment.

Back 39

- E.Coli produce beta galactosidase only when lactose is present in the medium

- This enzyme converts lactose into glucose and galactose

- If lactose is not present, the gene for beta galactosidase is not transcribed

- If lactose is present, the gene for beta galactosidase is transcribed

- The biochemical signal is lactose which is controlling the expression of the gene which codes for the enzyme to break it down

Front 40

Define epigenetics.

Back 40

- Attachment or removal of chemical groups known as epigenetic marks to or from DNA or histone proteins

- This then alters how easy it is for the enzymes and other proteins needed for transcription to interact with and transcribe with DNA

- Can determine whether a gene is switched on or off

Front 41

How do epigenetic changes occur?

Back 41

- In response to changes in the environment (e.g. pollution or food availability)

Front 42

Are epigenetics inherited?

Back 42

Most epigenetic marks on the DNA are removed between generations, but some can be passed on to offspring. This means that the expression of some genes may be affected by environmental conditions that affected their parents or grandparents.

Front 43

Describe how increased methylation of DNA leads to changes in gene expression.

Back 43

- This is when a methyl group (CH3) is attached to the DNA coding for a gene

- The groupalways attached at a CgG site, which is where a cytosine and guanine base are next to eachother

- Methyltransferase is the enzyme that catalyses this methylation reaction

- The DNA structure is changed so the transcriptional machinery (enzymes etc.) can’t interactwith the gene – so the gene is switched off

Front 44

Define histones.

Back 44

- Histones are proteins that DNA wraps around to form chromatin, which makes upchromosomes

- Chromatin can be highly condensed or less condensed

- How condensed it isaffects the accessibility of the DNA and whether or not it can be transcribed

Front 45

How do histones become more loosely packed?

Back 45

Histone molecules have side branches, or ‘tails’. These ‘tails’ contain the amino acid leucine.Leucine can be acetylated, i.e. an acetyl group (COCH3) is transferred to it from acetyl-coA.When acetylated, the histones become more loosely packed, the promoter and target genes are accessible by transcription factors andRNA polymerase and the target gene can be transcribed.

Front 46

Describe how decreased acetylation of histones leads to changes in gene expression.

Back 46

- Histones can be epigenetically modified by the addition or removal of acetyl groups

- Whenhistones are acetylated, the chromatin is less condensed; this means that the transcriptionalmachinery can access the DNA, allowing the genes to be transcribed

- When acetyl groups areremoved, the chromatin becomes more condensed and genes in the DNA can’t be accessedand the genes can’t be transcribed.

Front 47

Describe how inhibiting histone deacetylase (enzyme) would result in changes in gene expression.

Back 47

Histone deacetylase (HDAC) enzymes are responsible for removing the acetyl groups. If thisenzyme is inhibited, acetyl groups cannot be removed and the gene can be easily transcribed.

Front 48

Describe how oestrogen concentrations impact the development of breast cancer.

Back 48

Some types of breast cancer arehormone stimulated. The cancer cells have ERreceptor in the membranes andoestrogen stimulates them to divide.

Front 49

Define a mutagenic agent.

Back 49

Anything that increases the rate of mutations in DNA.

Front 50

Define an acquired mutation.

Back 50

Mutation that occurs after fertilisation.

Front 51

How does the rate of mutation increase?

Back 51

The rate of mutation can be increases if a mutagenic agent acts as a base, alters bases orchanges the structure of DNA.

Front 52

Define benign tumours.

Back 52

Benign tumours are capsulated by connective tissue, slower growing and do not metastasise. Usually treatable with surgery, depending on location.

Front 53

Define malignant tumours.

Back 53

Malignant tumours are fast growing,non-capsulated and they do metastasise.

Front 54

Which types of genes control rate of cell division?

Back 54

1. PROTO-ONCOGENES which stimulate cell division

2. TUMOUR SUPPRESSOR GENES which slow cell division

Front 55

Why does cell division occur?

Back 55

Genes control cell division. In fully grown organisms, cell division occurs to replace damagedor worn out tissues.

Front 56

How does a tumour develop due to a proto oncogene?

Back 56

If a mutation occurs in a proto-oncogene, it alters to become an oncogene; thisresults in an over-stimulation of cell division, so that cell division is permanentlyswitched on. This results in a mass of cells known as a tumour.

Front 57

How does a tumour develop due to a tumour suppressor gene?

Back 57

If a mutation occurs in a tumour-suppressor gene, the gene becomes inactivated, so it stops inhibiting cell division, so the rate of cell division increases.

Front 58

If a tumour suppressor gene is over methylated, how does a tumour develop?

Back 58

If tumour suppressor gene is over methylated, the gene becomes inactivated, so it stops inhibiting cell division, so the rate of cell division increases.

Front 59

If a oncogene is under methylated, how does a tumour develop?

Back 59

If an oncogene is undermethylated the gene becomes more activated, so that cell division is switched on, so the rate of cell division increases.

Front 60

Describe the difference between transfection gene therapy and transfected gene therapy.

Back 60

The process of inserting a functioning gene to replace a faulty gene - transfection.

A cell that has received a new gene - transfected.

Front 61

Define gene therapy.

Back 61

The insertion of recombinant DNA into individuals cells, tissues to treat a disease (usually hereditary). A non-functional/harmful gene is replaced with a functional one.

Front 62

Describe the procedure of gene therapy.

Back 62

1. The base sequence of the functioning allele is determined by gene sequencing

2. DNA is extracted from the donor cell and cut into fragments by restriction endonucleases

3. Fragments are separated using gel electrophoresis and the DNA fragment containing the functioning allele is identified using a complementary gene probe

4. Gene is inserted into host cells using vectors (DNA carriers)

Front 63

Describe somatic cell gene therapy.

Back 63

Copies of the corrected gene are inserted directly into somatic or body cells of the sufferers; this type of gene therapy does not prevent the disease from occurring in the next generation because it doesn't affect the sperm and egg cell. Somatic gene therapy has to be repeated many times as the effects do not last very long.

Front 64

Describe germ line gene therapy.

Back 64

The corrected gene is inserted into fertilised egg and produced in IVF, if successful all cells of the embryo will contain the corrected gene when the cell divides by mitosis. Germ cell therapy is permanent and also ensures offspring inherit correct genes.

Front 65

What is the aim of the human genome project?

Back 65

Mapping the genes of an organism and mapping these genes on individual chromosomes.

Front 66

Why is it useful to sequence a genome?

Back 66

- Single nucleotide polymorphisms (SNPs) associated with disease can be identified and used to screen for genetic disorders

- Sequencing the DNA of different organisms have made it possible to establish evolutionary links between species

Front 67

Define a genome.

Back 67

The complete set of genes required to build a functional organism.

Front 68

Define a proteome.

Back 68

All the proteins produced by the genome (not all proteins are synthesised all the time)

Front 69

Describe shotgun sequencing.

Back 69

1. Cut the DNA from many copies of entire chromosome into overlapping fragments short enough for sequencing

2. Clone the fragments into plasmid vectors

3. Sequence each fragment

4. Order the sequence into once overall sequence using computer software

Front 70

How could knowledge of the proteome of a pathogen help to control the disease it causes?

Back 70

Allows identification of proteins that act as antigens on the surfaces of pathogens. These can be used to produce vaccines against disease.

Front 71

Why is it more difficult to determine the proteome of complex eukaryotic organisms from their genome?

Back 71

They have much more DNA and this contains introns that do not code for proteins or gene that regulate other genes.

Front 72

Describe the advantages of genetic screening.

Back 72

- Once the genotype of the individual is known, personalised health and advice can be provided

- More appropriate drugs and their dosage can be prescribed (i.e. painkillers that require a specific enzyme to activate it, if the enzymes function is affected then dosage can be altered)

- Genetic counselling looks at family history and inheritance patterns to inform the chances of the genes being passed on to future offspring; could impact the decision to have children and the use of IVF with genetic screening of embryos)

Front 73

Describe how genetic screening occurs (part1).

Back 73

- The DNA from target ells are extracted and purified and this DNA is amplified using PCR

- The amplified DNA is cut using a restriction endonuclease and then separated using gel electrophoresis

- The fragments are transferred to nylon membranes then treated to separate strands (DNA fragments are not visible)

Front 74

Describe how genetic screening occurs (part2).

Back 74

- A labelled DNA probe with the complementary sequence to the harmful gene are added

- The probes will anneal to the complementary DNA (DNA hybridisation)

- The nylon membrane is washed to remove any unbound gene probes- If the harmful gene is not present, no bands will be visible and vice versa

Front 75

Describe genetic fingerprinting.

Back 75

1. VNTRS

2. Gel electrohoresis

3. Visualise the DNa

Front 76

Describe the uses of genetic fingerprinting.

Back 76

- Paternity testing (50% of band should match each parent)

- In a given population areas with low genetic diversity will have similar genetic fingerprinting

- Was the person present at the scene? (doesn't prove guilt)

- Probes that target a mutated form of the gene that increases the risk of a disease developing can be generated

- Used to prevent inbreeding in zoos as inbreeding could result in increased frequency of undesirable alleles

- Identify a desirable allele when selecting for breeding

Front 77

Describe restriction mapping.

Back 77

Restriction mapping is the mapping of a piece of DNA showing recognition sites of specific restriction endonuclease and the number of bases of a site.

Front 78

What does VNTRs stand for?

Back 78

Variable number tandem repeats

Front 79

Define VNTRs.

Back 79

- Within the non-coding regions of DNA there are repeated sequences of bases (10-100 base pairs)

- The number repeats at specific locations different for each individual; this means that the fragment length differs

Front 80

Describe gel electrophoresis.

Back 80

- Hydrolysed DNA sample placed into a well at one end of an agarose gel

- Gel is placed into a buffer and an electrical current is passed through the gel

- As DNA is negatively charged, DNA fragments migrate toward the positively charged end of the gel

- Smaller DNA fragments migrate through the gel more quickly than larger fragments

- This process separates the fragments according to their size across the gel

Front 81

How can we visualise the DNA used in gel electrophoresis?

Back 81

- DNA fragments are transferred to a nylon filter and treated to be single stranded

- Radioactive gene probe is added

- X-ray film placed on top of nylon filter and radioactivity leaves a band on the x-ray film

- This give you the position of a specific band on the gel

Front 82

What does PCR stand for?

Back 82

Polymerase Chain Reaction - used to make millions of copies of a fragment of DNA

Front 83

What ingredients are used in PCR?

Back 83

- Target DNA (gene you want to clone)

- Primers (artificial DNA strands which are 18-25 base pairs long that are complementary to the target gene)

- Taq polymerase (heat tolerant/hot springs)

- Free DNA nucleotides

Front 84

What three stages of PCR are there?

Back 84

1. Denaturation

2. Annealing

3. Extension

Front 85

Describe PCR.

Back 85

1. Heated to 94 degrees to break hydrogen bonds between two strands of DNA

2. Cooled to 50-60 degrees so that primers can bind to strands

3. Heated to 74 degrees so that heat resistant DNA polymerase can join DNA nucleotides together to form newly synthesised complementary strands for required section of DNA

4. Two new copies of the DNA fragment are formed (one cycle complete)

5. This can be repeated many times (DNA fragments double each time)

Front 86

Describe the denaturing stage of PCR.

Back 86

Heated to 95 degrees to break hydrogen bonds between DNA

Front 87

Describe the annealing stage of PCR.

Back 87

Lowered to 50-65 degrees to allow the free DNA nucleotides to bind to complementary base pairs and form hydrogen bonds.

Front 88

Describe the extension stage of PCR.

Back 88

Heated to 72 degrees as it is the optimum temperature for taq polymerase to form phosphodiester bonds for the sugar phosphate backbone.

Front 89

How much DNA is replicated during PCR?

Back 89

Each time you replicate the cycle the amount of DNA doubles (theoretically) but each time the enzymes become less efficient.

Front 90

Define gene technology.

Back 90

Using 'in vivo' gene cloning (inside cells) to make a copy of a DNA fragment

Front 91

Describe gene technology.

Back 91

1. Isolation of the DNA fragment that has the correct gene

2. Insertion of DNA fragment into a vector (forming recombinant DNA)

3. Transformation of the DNA into a suitable host cell

4. Identification of the host cells that have successfully taken up the gene by the use of gene markers

5. Growth/cloning of the population of host cells

Front 92

Why do we use bacteria as host cells in gene technology?

Back 92

DNA in a bacteria cell doesn't contain introns and reproduce quickly (every 20 minutes)

Front 93

How can you make insulin using gene technology?

Back 93

1. Cells form islets of langerhans in the human pancreas

2. mRNA coding for insulin acts as a template on which a cDNA strand is formed using reverse transcriptase

3. Heated to form single stranded DNA

4. Double stranded DNA is formed using DNA polymerase

5. Copy of human insulin gene is placed into a host cell and replicated

Front 94

Define restriction endonucleases.

Back 94

Enzymes which cut (hydrolyse) DNA at a specific base sequence (recognition sequences) and can be used at target sites at the start and end of the DNA. These recognition sequences are palindromic. These enzymes come from bacteria that defend themselves by cutting up viral DNA.

Front 95

Define sticky ends.

Back 95

- The cut made at the recognition site is staggered

- Sticky ends can be used to bind (anneal) DNA fragments together, if other DNA molecule has a complementary sticky end

Front 96

Define vector.

Back 96

Carries foreign DNA into another cell where it can be expressed.

Front 97

Define blunt ends.

Back 97

- Where the two strands of DNA are cut in the same place so all bases are complementary

- The cut made at the recognition site is straight

Front 98

Which enzyme is used to bind two sticky ends together?

Back 98

DNA ligase is used to join the phosphodiester bonds between the two sticky ends.

Front 99

Define recombinant DNA.

Back 99

Where the DNA of two different organisms is combined, the produce is recombinant.

Front 100

Define replica plating.

Back 100

The original plate is retained as a master copy/plate, any imprint of the master copy is placed onto two different agar plates;

1. One containing ampicillin

2. One containing tetracycline

Front 101

What are the possible outcomes of replica plating?

Back 101

1. Ones that did not take up the plasmid at all - all the bacteria die

2. Ones that took up the plasmid without the gene successfully inserted - all the bacteria survive

3. Ones that took up a plasmid with the gene successfully inserted - survives amplicillin but dies by tetracycline

Front 102

How can you prevent translation during gene technology?

Back 102

RNA interference - expression can also be prevented by breaking down mRNA before it ca be translated using siRNA. Small, double stranded RNA molecules hydrolyse the mRNA and prevent translation.

Front 103

Why would you prevent translation in genes?

Back 103

- Can identify the role of genes in a pathway (by observing effects of blocking certain genes)

- Could block expression of some disease-causing genes

Front 104

Describe how siRNA could prevent translation.

Back 104

- Sections of double stranded RNA are cut into small sections

- One of the single stranded RNA combines with an enzyme to form siRNA (small interferring RNA)

- The siRNA binds to complementary bases on the mRNA

- The enzyme (RNA hydrolyse) then cuts the mRNA into small sections so translation cannot occur (breaking phosphodiester bonds)

- Gene expression is blocked

Front 105

Define the gene machine.

Back 105

- Work backwards from the protein structure to amino acids to DNA triplet codes

- DNA base sequence is fed into the machine

- Overlapping short single stranded nucleotides are made one base at a time - digonucleotides

- These are assembled to make the gene

- PCR is used to make complementary strands and replicate the gene

- Sticky ends are used to insert the gene into a vector

Front 106

Describe the advantages of the gene machine.

Back 106

- Only takes a short amount of time

- Good level of accuracy

- Have no introns so can be transcribed by prokaryotes

Front 107

What are VNTRs used for?

Back 107

- Used for DNA fingerprinting

- Each individual has two copies of each VNTR, one on each homologous chromosome (one copy inherited from each parent)

Front 108

How can you extract a VNTR?

Back 108

- Extract DNA

- PCR is used to amplify the VNTR regions within the sample; if the sample size is small

- Or restriction endonucelases are used to cut DNA into fragments (must produce blunt ends and cut outside of VNTR regions)

- Separate DNA fragments according to size using gel electrophoresis

- Make fragments single stranded and add a gene probe for VNTR's to allow positions of these fragments to be visualised as bands

Front 109

Why do we use a gene machine?

Back 109

- DNA posses genes that code for proteins which determine our characteristics

- Sequence of DNA code for the sequence of amino acids in these proteins via transcription and translation during protein synthesis

- When scientists do not know the sequence of bases of the gene they are targeting, they can isolate the protein and use the sequence of amino acids to determine the sequence of bases

- Once known the sequence can be fed into the gene machine and multiple copies can be made

Front 110

Define the advantages of using a gene machine.

Back 110

DNA base sequence that has been determined does not contain non-coding regions

Front 111

Define gene probe.

Back 111

A single stranded DNA molecule with a complementary base sequence to target DNA fragments to be located which is radioactive pr labelled by a fluorescent molecule

Front 112

Describe the use of reverse transcriptase.

Back 112

- First mRNA is mixed with free DNA nucleotides and reverse transcriptase

- Free DNA nucleotides bind to single stranded mRNA via complementary base pairing

- Reverse transcriptase joins DNA nucleotides together to form a newly synthesised DNA strand with a complementary base sequence to mRNA template (cDNA - complementary DNA)

- The addition of further DNA nucleotides and DNA polymerase is then used to make cDNA double

Front 113

How can we transfer DNA after gel electrophoresis in order to visualise it?

Back 113

As the gel is fragile and breaks easily, the DNA fragments can be transferred at the same position to a nylon membrane using southern blotting and fixed using UV light.

Front 114

Why do we need to visualise DNA after gel electrophoresis?

Back 114

DNA is colourless so it must be treated to visualise it on the gel.

Front 115

Why do we add a gene probe to the DNA fragments after gel electrophoresis?

Back 115

Scientists may not want to visualise the position of all DNA fragments in the gel

Front 116

Describe DNA hybridisation.

Back 116

During the visualisation process after gel electrophoresis, gene probes with complementary base sequences to DNA fragments are mixed with the separated DNA fragments. DNA fragments are treated to break hydrogen bonds between complementary base pairs, making them single stranded so the probes can bind to any complementary DNA. Excess probes are washed away.

Front 117

If we use a radioactive gene probe, how do we visualise DNA?

Back 117

An x-ray film is placed on top of gel membrane and a band develops at the site of the gene probe, giving the location of the DNA fragments on the gel membrane.

Front 118

If we use a florescent gene probe, how do we visualise DNA?

Back 118

A UV light is shone on top of gel membrane and a band develops at the site of the gene probe, giving the location of the DNA fragments on the gel membrane.

Front 119

How can we use DNA fingerprinting in real life?

Back 119

- Paternity testing (you share 1/2 your bands within your DNA fingerprint with each parent)

- Criminal investigations (compare the DNA fingerprint with samples taken from the crime scene, an exact match in all bands within the DNA fingerprint gives evidence that suspect has been at that crime scene - does not prove guilt)

Front 120

Describe the advantages of using reverse transcriptase.

Back 120

- Cells contain a maximum of two copies of each gene; if they are expressing the gene they will contain many mRNA molecules, with a complementary base sequence to gene so mRNA is easier to obtain

- The mRNA is isolated from the cytoplasm of the cells expressing the gene, it contains no introns (whereas the gene does). DNA fragments produced using mRNA as a template does for a functioning protein without needing further modification

- As cells expressing a gene will have many copies of mRNA in their cytoplasm, you do not need to know the base sequence of the gene to copy it using this method

Front 121

Describe the use of restriction endonucleases.

Back 121

- Enzymes which cut (hydrolysis) DNA at specific base sequences (recognition sequences)

- Different restriction endonuclease cut DNA at different specific recognition sites because the shape of the site is complementary to the enzymes active site

- These recognition sequences are palindromic - base pairs which read the same in opposite directions

Front 122

What is the importance of recognition sites in using restriction endonuclease?

Back 122

- If recognition sequences for these enzymes are present at either side of the DNA fragments you want, you can use restriction endonucelases to separate it from the rest of the DNA.

- The DNA sample is incubated with the specific restriction endonuclease which cut the DNA fragment out of the DNA

- It is important that the recognition sequence for the selected restriction endonuclease does not occur within the DNA fragment you wish to isolate

Front 123

Describe the use of 'in vivo cloning'.

Back 123

- Copies of DNA fragments can be made within a living organism

- If a foreign DNA fragment is placed into the organisms DNA, as it grows and divides it replicates the foreign DNA along with its own, creating many copies of the target DNA with a living organism

Front 124

Describe the stages of 'in vivo cloning'.

Back 124

1. Insert into a vector (RE producing sticky ends + ligase)

2. Vector transfers DNA fragments into host cell

3. Identifying transformed host cells

Front 125

Describe stage 1 of 'in vivo cloning'.

Back 125

- The isolated DNA fragment is placed into the vector DNA by cutting open the vector DNA using the same restriction endonuclease that was used to isolate the DNA fragment

- This produces complementary sticky ends between the ends of DNA fragment and cut ends of vector DNA

- DNA ligase is then used to join them together by complementary base pairing at the sugar phosphate backbone (ligation)

- The new combined fragment is called recombinant DNA

Front 126

Describe stage 2 of 'in vivo cloning'.

Back 126

- The vector containing the recombinant DNA is now used to transfer the target DNA into a host cell

- This method differs depending on the vector use: plasmid vectors or bacteriophage vectors

- Host cells which take up recombinant DNA are referred to as recombinant organisms or transformed organisms

Front 127

Describe the difference between using a plasmid vector and using a bacteriophage vector.

Back 127

Plasmid vectors - plasmids are circular pieces of DNA found in bacterial cells, if bacterial cells are placed in solutions with recombinant plasmids they can be encouraged to take up plasmids from the solution under certain conditions

Bacteriophage vectors - bacteriophages are viruses which infect bacteria by injecting its DNA into them, the phage DNA then inserts into the host DNA carrying the target DNA with it

Front 128

Why don't we use other types of cells when we transfer foreign DNA?

Back 128

Getting plant and animal cells to take up foreign DNA is not easy, viruses which infect human cells can be used as vectors, but not without risk. A gene gun can be used ti fire DNA fragments into plant cell nuclei.

Front 129

Describe stage 3 of 'in vivo cloning'.

Back 129

- As not all vectors take up the target DNA and not all host cells will have taken up a recomibant vector, now the transformed host cells need to be identified and isolated to grow further to produce many copies of target DNA

- This is done using marker genes

- Identified transformed organisms are then grown, producing lots and lots of copies of target DNA

Front 130

Describe the common types of marker genes.

Back 130

- Antibiotic resistance gene; marker gene codes for antibiotic resistance, when cells are grown in the presence of this antibiotic only transformed organisms will grow

- Fluorescent gene; marker gene codes for fluorescent, when UV light is shone on cells the transformed organisms fluoresce

Front 131

Describe how identifying specific DNA fragments are used to diagnose disease.

Back 131

1. Amplify the quality of DNA using PCR

2. Separating fragments according to size using gel electrophoresis

3. Add a gene probe that is complementary to the target gene (this is the faulty gene)

4. Visualise the probe - if the individual has the faulty gene then a band will appear

Front 132

Describe the positives of screening embryos.

Back 132

- Can remove anxieties of families faced with the prospect of serious genetic disease (older age group of women)

- Eliminate the ill or disabled before they become a financial burden on society

- Prevent unnecessary suffering of the individual

Front 133

Describe the negatives of screening embryos.

Back 133

- Risk of miscarriage associated with sampling techniques - foetus may not even have disease

- Parents have no right to decide if a child has right to life

- Some people believe that the embryo is alive from the moment of conception so abortion is murder

Front 134

Describe the positives of pre-implantation genetic diagnosis (PIGD)

Back 134

- Removes the need for abortion of foetus (8-16 stage embryo

- More acceptable to people because its earlier

Front 135

Describe the negatives of pre-implantation genetic diagnosis (PIGD)

Back 135

- Hard to select people to offer procedure; it is too expensive

- Cells still develop into a foetus so not implanting/throwing away the foetus is seen as murder

Front 136

Describe the positives of screening adults.

Back 136

- Early diagnosis may improve treatment

- Allows the individual to change lifestyle to reduce risks

- Allows the individual to make informed choices regarding their reproduction

Front 137

Describe the negatives of screening adults.

Back 137

- Diagnosis may increase insurance costs

- May result in discrimination; employment and health care

- Diagnosis could cause distress to individuals as there may be no treatment

Front 138

Define cystic fibrosis.

Back 138

Inherited disease that affects the respiratory, digestive and reproductive systems. The symptoms are caused by a thick sticky mucus that clogs or blocks the bronchioles, pancreatic duct and vas deferens or cervix in the reproductive system.

Front 139

How is the mucus found in cystic fibrosis made?

Back 139

- Mucus is thick due to a faulty transmembrane protein (responsible for the transport of chloride ions across a membrane ) in the cells lining these surfaces

- In a normal cell chloride ions are actively transported out of the cell lowering the water potential; water than follows by osmosis making the mucus less sticky

- In most cases of CF, the part of the protein that is responsible for the binding of ATP is missing 1 amino acid thereby changing its structure

- ATP cannot bind and the protein is unable to actively transport the chloride ions so the water potential is unaffected

- Water remains inside the cell resulting in sticky mucus

Front 140

How does the mucus found in cystic fibrosis affect the lungs?

Back 140

The mucus results in difficulty breathing due to the narrowing of the airways and repeated infections as the body is unable to remove any micro-organisms that have adhered to the mucus. The repeated infections cause scarring of the alveoli and a reduction in its surface area.

Front 141

How does the mucus found in cystic fibrosis affect the small intestine?

Back 141

When the pancreatic duct is blocked, important digestive enzymes are unable to reach the small intestine which means proteins, carbohydrates and lipids cannot be broken down into their monomers for absorption, resulting in a small growth rate.

Front 142

How does the mucus found in cystic fibrosis affect reproduction?

Back 142

Over 90% of men who suffer from CF are infertile because of a block or absent vas deferens that is used to transport sperm from the testes to the urethra.

As the cervix is blocked by the mucus female sufferers will have difficulty conceiving.

Front 143

Why can't a plasmid be used for gene therapy?

Back 143

Plasmids cannot carry DNA into human cells so viruses or liposome are used as vectors.

Front 144

What is the difference between using a virus as a vector and a liposome?

Back 144

Viruses are unable to reproduce independently of a cell. In order to reproduce a virus must infect a cell, and then insert its genetic material in to the host cell's genetic material, the host cell then produces new viruses then it expresses its DNA.

Front 145

How is a liposome used as a vector in gene therapy?

Back 145

A liposome is a droplet of lipid which carries the DNA into a cell, as it fuses with the cell membrane the liposome releases the DNA into the cell.

Front 146

How is a virus used as a vector in gene therapy?

Back 146

If a foreign DNA fragment is introduced into the viral genetic material it will insert the foreign gene at the same time as its own genetic material. Viral DNA is cut using the same restriction endonuclease and joined to the foreign gene using DNA ligase. The virus then acts as a vector (DNA carrier).

Front 147

What are the negatives of using germ line therapy?

Back 147

- Technology is imperfect; the effects of gene transfer are unpredictable and even if the target disease was cured, further defects could be introduced to the embryo

- Denial of human rights; individuals resulting from germ line therapy would have no say in whether their genetic material should be modified

- Potential abuse; GLT could be used to enhance favourable characteristics (designer children)

Front 148

What are the negatives of using somatic gene therapy?

Back 148

- Not all cells take up new alleles

- Not all cells express inserted allele

- Only some tissue types are accessible (i.e. lungs need to be repeated as cells die replaced by cells with faulty allele)

- Multiple treatments may be needed

- Body can produce an immune response to the vector

Front 149

Describe how restriction mapping is used.

Back 149

- Restriction enzymes used to cut DNA fragments and then are separated using electrophoresis

- The pattern of the fragments allows the distance between the recognition sites to be determined

- DNA fragments are then sequenced separately; the restriction map information is then used to piece fragments back together - giving the whole base sequence of a gene or region of DNA

Front 150

Describe the uses of the human genome project.

Back 150

- Screening of genetic disorders caused by single nucleotide polymorphisms (SNPs) - this refers to a change of one base pair in the gene

- Determine evolutionary relationships between species

- Identification of proteins that act as antigens on the surfaces of pathogens to enable us to produce vaccines

Front 151

Describe automated chain termination sequencing (ACTS)

Back 151

- When a mixture of DNA nucleotides and termination nucleotides are added to a single strand of DNA to be sequenced by chance, termination nucleotides will bind

- Termination nucleotides will stop DNA polymerase from replicating DNA

- When a termination nucleotide binds at any point the replication stops which gives many chains of DNA all of varying lengths

- The DNA is separated using gel electrophoresis

- The colour/pattern is read by a detector and fed into a computer or read manually

Front 152

What products are required for ACTS?

Back 152

- Termination nucleotides; four different types, one for each base and each is labelled with a different coloured fluorescent dye

- DNA polymerase

- Primers

Front 153

Describe manual chain termination sequencing (MCTS).

Back 153

The manual method is similar to ACTS except:

- A separate mixture is made for each type of dideoxynucleotide (ddNA, ddNC, ddNG and ddNT)

- After incubation each mixture is placed into a different well for gel electrophoresis

- X-ray film is used to identify positions of fragments

Front 154

How do you read a sequence after gel electrophoresis?

Back 154

Start with the band that has moved the furthest - this is the shortest DNA fragment. This will be the fragment where replication stopped at the first base then it's the next furthest as second base etc.

Front 155

Give an example of sex linkage.

Back 155

Colour blindness

Haemophilia

Front 156

Define genotype.

Back 156

The genetic constitution of an organism (what genes it has), it includes all the alleles of an organism.

Front 157

Define phenotype.

Back 157

The observable characteristics of an organism (physical appearance). It is the result of the interaction between expression of genotype and the environment.

Front 158

Define diploid.

Back 158

Two copies of each chromosome and therefore two copies of a gene.

Front 159

If an organism is homozygous, what happens to its alleles?

Back 159

The alleles are the same.

Front 160

If an organism is heterozygous, what happens to its alleles?

Back 160

The alleles are different.

Front 161

Where are dominant alleles always expressed?

Back 161

In the phenotype

Front 162

When are recessive alleles expressed?

Back 162

Recessive alleles are only expressed when homozygous

Front 163

When are dominant alleles expressed?

Back 163

Only one dominant allele is needed to be expressed

Front 164

Describe genetic crosses.

Back 164

These diagrams show the way in which alleles are passed from one generation to the next. The allele is expressed by a letter; the letter normally refers to the phenotype of the dominant allele.

Front 165

Describe monohybrid inheritance.

Back 165

This is the inheritance of a single characteristic,coded by a single gene. The phenotype is determined by two alleles, one of which is dominant and the other recessive.

Front 166

Describe how inheritance can be shown in the form of a pedigree (family tree).

Back 166

1. If the disease is recessive it occurs in the children in the final generation but was not present in their parents

2. The two children affected must have to genotype cc (they have the recessive phenotype)

3. Their parents must have the genotype Cc (they are normal but have passed on the recessive allele)

Front 167

Define co-dominance.

Back 167

Both alleles are expressed in a heterozygous individual to give an intermediate phenotype.

Front 168

Describe how multiple alleles can affect a punnet square.

Back 168

For many genes, there are several alleles in the population (e.g. hair colour). These may show a dominance hierarchy (e.g. brown is dominant to black) but the normal rules of inheritance apply.

Front 169

Define sex linkage.

Back 169

The X chromosome carries the normal body genes and these are linked to the genes which determine sex, so these body genes are referred to as being sex-linked genes.

Front 170

Which, of the human's 23 chromosomes, are referred to as the sex chromosomes?

Back 170

One - referred to the chromosome that carries the genes which determine sex

Front 171

What are the other 22 chromosomes called?

Back 171

Autosomal chromosomes

Front 172

What are the two different types of sex chromosomes?

Back 172

Y chromosome only carries sex genes

X chromosomes carries both sex genes and genes controlling other bodily characteristics (autosomal genes)

Front 173

What type of individual has two X chromosomes?

Back 173

Female (homogametic)

Front 174

What type of individual has one X chromosome and one Y chromosome?

Back 174

Male (heterogametic)

Front 175

Why are males more likely to express recessive alleles in their phenotype?

Back 175

In the males, the alleles 'a' and'b' have no corresponding allele on the Y chromosome. These recessive alleles will always be expressed in the phenotype, as there is only one copy of the gene.

Front 176

Why are females less likely to express recessive alleles in their phenotype?

Back 176

In the female, the other X chromosome needs to be considered. If alleles on the second X chromosome are A and B then these will be expressed in the phenotype not the recessive a and b.

Front 177

Define diversity.

Back 177

Indicates how much variation exists.

Front 178

Why does genetic diversity occur?

Back 178

Genetic diversity arises because of differences in what genes are present in an organism and due to the presence of different alleles of a gene.

Front 179

How has genetic diversity occurred?

Back 179

Mutation - produces new alleles

Random assortment

Crossing over

Random fusion of gametes

Front 180

What could a mutation lead to?

Back 180

- Confer a selective advantage

- Be neutral

- Be disadvantageous

- The adaptions may be anatomical, physiological or behavioural

Front 181

Define selection.

Back 181

Process by which organisms that are better adapted to their environment survive and reproduce in greater numbers, resulting in the increase of the frequency of the advantageous allele within the population

Front 182

How does selection occur?

Back 182

- Mutations in the DNA lead to a new allele/gene; leads to variation

- Those with the new allele/gene are better adapted individuals more likely to survive and reproduce

- These organisms are more likely to pass on their allele/gene

- Those which are less well adapted fail to survive and reproduce

- These organisms are less likely to pass on their alleles/genes

- Organisms are therefore subject to selection pressure due to environment they live in

- Selection pressure determines the spread of an allele within the gene pool

Front 183

What type of selection is this?

Back 183

Stablising selection

Front 184

What type of selection is this?

Back 184

Directional selection

Front 185

What type of selection is this?

Back 185

Disruptive selection

Front 186

Describe stabilising selection.

Back 186

- Occurs in all populations where environment is stable

- Selective pressure at both ends of distribution

- Favours the average

- Tends to eliminate extremes

- Reduces variability, the size of the range within population

- Reduces opportunity for evolutionary change

Front 187

Give an example of stabilising selection.

Back 187

Birth mass in humans, babies who are very heavy or very light show higher neonatal mortality rates (die more frequently at or just after birth) than those of medium mass. Over time selection operates to reduces the number of heavy and light babies.

Front 188

Describe directional selection.

Back 188

- Mean population represents optimum for existing conditions

- Environmental change may produce new selection pressure that favours an extreme characteristics

- When conditions change optimum necessary for survival also changes

- Some organisms will possess the new optimum

- Over time selection means that these will predominate and the mean will shift

Front 189

Give an example of directional selection.

Back 189

Thicker fur in mice is an advantage in the cold climate. Over time selection operates against the disadvantaged extreme and in favour of the other extremes. The mean values and range shift toward the favoured extreme, the frequency of the alleles that cause long hair will increase.

Front 190

Describe disruptive selection.

Back 190

This is when the mean is selected against and favours the mean phenotypes. This type of selection is more likely to bring about evolutionary changes resulting in the development of two new species - speciation.

Front 191

Define speciation.

Back 191

The evolution of a new species from existing species (a group of individuals with similar characteristics and similar genes capable of interbreeding to produce fertile offspring - belong to the same gene pool).

Front 192

How can natural selection affect speciation?

Back 192

Nation selection leads to a change in the frequency of alleles in population and provides a mechanism by which new populations of species can arise.

Front 193

Define reproductive isolation.

Back 193

As long as two populations are able to interbreed, they will not evolve into two distinct species. Two populations must undergo a period of reproductive isolation (a period when they are prevented from interbreeding) to become genetically distinct via mutations and selection.

Front 194

Describe geographical isolation.

Back 194

1.This means any physical barrier which prevents the population meeting and therefore interbreeding (e.g. river, mountain)

2. Isolated population may be subject to different selection pressures and adapts due to natural selection

3. Changes in genotype and phenotype may occur

4. Eventually when two populations are reunited they are now so different that they are unable to interbreed and now reproductively isolated

5. Each population has become a separate species with its own gene pool

Front 195

What two types of speciation?

Back 195

Allopatric

Sympatric

Front 196

Describe allopatric speciation.

Back 196

- Different countries

- Any physical barrier that prevents interbreeding

- Barrier is determined by type of species

- Environmental conditions either side of the barrier differ - different selection pressures

Front 197

Describe sympatric speciation.

Back 197

- Same country

- Population in the same area that becomes reproductively isolated

- Mutations in each population have led to evolution of genetic difference

- i.e. feed in different locations, at different time of day

Front 198

Define genetic drift.

Back 198

When populations are small there is a low genetic diversity which means they have a small gene pool. Due to their small numbers there is little breeding and a restriction on the flow of alleles. In this situation, any allele that is advantageous will quickly increase in frequency and increase the rate of speciation.

Front 199

Describe the different types of isolating mechanisms.

Back 199

- Temporal

- Ecological

- Behavioural

- Mechanical

- Gametic

- Hybrid

Front 200

Describe temporal isolating mechanism.

Back 200

Organisms breed at different times of the year

Front 201

Describe ecological isolating mechanism.

Back 201

Different habitats within the same area

Front 202

Describe behavioural isolating mechanism.

Back 202

Different behaviour patterns (i.e. courtship behaviour)

Front 203

Describe mechanical isolating mechanism.

Back 203

Anatomical differences making it impossible for gametes to come together

Front 204

Describe gametic isolating mechanism.

Back 204

Incompatibility (genetic or physiological) between gametes prevent hybrid being forming

Front 205

Describe hybrid isolating mechanism.

Back 205

Organisms interbreed by offspring are infertile

e.g. horse and donkey can produce a mule but the mule is infertile

Front 206

Describe dihybrid crosses.

Back 206

Dihybrid crosses look at the inheritance of two genes at the same time. Individuals can be homozygous dominant or recessive for both genes, heterozygous for both or a mixture of two.

Front 207

What ratio is given in the F2 generation of a monohybrid crosses?

Back 207

3:1

Front 208

What ratio is given in the F2 generation of a dihybrid crosses?

Back 208

9:3:3:1

Front 209

Why is the ratio given in the F2 generation not always present in practise?

Back 209

Autosomal linkage

Front 210

Describe autosomal linkage.

Back 210

When conducting a dihybrid cross, an assumption is made to as the location of the genes. The genes will be located on different chromosomes and cross over independently in prophase 1 of meiosis. However in some cases, the genes are found on the same chromosome - autosomal linkage. In these cases the alleles present will be crossed over together and inherited together. An autosome is any chromosome not considered to be a sex chromosome. The closer they are on the chromosome the more likely they are to be crossed over together.

Front 211

Define epistasis.

Back 211

Epistasis is when a gene at one locus influences the phenotypic expression of another gene at a different location.

Front 212

Define gene pool.

Back 212

All of the alleles of all the genes of all the individuals in a population at a given time.

Front 213

Define allelic frequency.

Back 213

The number of times an allele occurs within a gene pool

Front 214

Describe the Hardy-Weinberg equation.

Back 214

p^2 + q^2 +2pq = 1.0

Front 215

What assumptions are made when using the Hardy-Weinberg equation?

Back 215

- There should be no immigration or emigration (i.e. the gene pool is isolated and there is no flow of other alleles in or out)

- There should be no mutations

- There should be no selection for or against a particular allele

- There should be a large population

- Mating within the population should be random