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gene expression + DNA technology Flashcards

1
Q

what are gene mutations

A

alterations in DNA’s base sequence
(of nucleotides)
may code for diff amino acid sequence in polypeptide

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2
Q

what do mutagenic agents do + give examples of them

A

increase rate of mutation
e.g x-rays, UV light

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3
Q

what are the types of mutations

A
  • inversion
  • translocation
  • substitution
  • addition
  • deletion
  • duplication

ITSADD

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4
Q

what are the 2 main sets of genes which control the rate of cell division and their functions

A

1) proto-oncogenes = code for proteins that stimulate cell division

2) Tumour suppressor genes = code for proteins that slow/ suppress cell division

mutations of these genes can lead to rapid uncontrolled cell division
resulting in the development of a tumour

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5
Q

how can proto-oncogenes and tumour suppressor genes lead to the development of a tumour

A
  • a mutated proto-oncogene ( known as an oncogene) - cell division is no longer regulated = rapid uncontrolled cell division
  • mutation in tumor supressor gene - means the tumour supressor proteins non-functional/ cant be produced = rapid uncontrolled cell division
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6
Q

tumours

A

masses of dividing cells

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7
Q

cancer

A

group of diseases caused by alterations in the genes that usually regulate mitosis + cell cycle -> so uncontrolled division occurs

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8
Q

what are the types of tumours

A

malignant = cancerous
benign= non- cancerous

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9
Q

describe benign tumours

A
  • grow slower than malignant
  • non-cancerous - cells don’t spread/metastasize to other tissues as the tumours enclosed by fibrous tissue
  • cells often remain differentiated (specialised)
  • cell nucleus = normal appearance
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10
Q

describe malignant tumours

A
  • grow faster than benign
  • cancerous - cells can spread/metastasize to other parts of the body as tumours not enclosed
  • cells often become undifferentiated (unspecialised)
  • cell nucleus = larger + darker
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11
Q

stem cells

A

undifferentiated cells that can divide by mitosis
and differentiate into diff types of cells

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12
Q

what are the diff types of stem cells

A
  • totipotent
  • pluripotent
  • multipotent
  • unipotent
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13
Q

describe totipotent stem cels

A
  • occur for a limited time in early mammalian embryos ( in 1st few cell divisions)
  • can differentiate into ANY TYPE of cell
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14
Q

describe pluripotent stem cells

A
  • found in embryos e.g embryo stem cells + fetal cells
  • develop from totipotent cells
  • can differentiate into most types of cells apart from cells of the placenta
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15
Q

describe multipotent stem cells

A
  • found in mature mammals
  • can differentiate into FEW, LIMITED types of specialised cells

e.g multipotent stem cells in blood marrow can produce any type of blood cell (RBC, WBC)

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16
Q

describe unipotent stem cells

A
  • found in mature mammals
  • can only differentiate into ONE type of cell
  • cardiomyocyte stem cell = unipotent
  • cardiomyocytes can only differentiate into heart muscle cells

cardio = heart
myocyte= muscle

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17
Q

describe how totipotent cells eventually develop into unipotent + multipotent

A
  1. Totipotent cells are in the embryos at very early stages of its development and are present in the 1st few cell divisions
  2. eventually totipotent cells develop into pluripotent cells in the embryo
  3. in mature mammals : multipotent + unipotent cells are present
  4. multipotent stem cells e.g in bone marrow can differentiate into any type of blood cell
  5. unipotent e.g cardiomyocytes develop into heart muscle cells

or epithelial cells, neurone cells etc

look at diagram saved on phone

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18
Q

how can stem cells that are put into body, cause more harm than good

A
  • may lead to uncontrolled cell division - causing formation of a group of abnormal cells - developing cancerous tumour
  • could be rejected by body
  • could differentiate into wrong type of cell
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19
Q

formation of induced pluripotent stem cells ( iPS cells)

A

type of pluripotent stem cells
developed from unipotent stem cells

uses transcription factors to make unipotent stem cells, pluripotent

transcription factors cause/control expression/inhibition of genes (so cant code for a protein) so these cells can differentiate into a particular type of cell

iPS cells can differentiate into a wide range of diff cells / tissues

which could be used to treat ppl with certain diseases

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20
Q

cell differentiation by regulation of transcription

A

(organism develops by mitosis from a single fertilised egg
during development of organism, some genes are transcribed and expressed + other genes are not expressed - this is controlled by transcription factors )

  1. transcription factors are proteins that control the rate of transcription of genes
  2. transcription factors move from cytoplasm to nucleus and attatch to a promoter region close to the target gene
  3. complementary transcription factor binds to a specific sequence of nucleotides in a promoter sequence
  4. transcription factors act as an ‘activator’ or a ‘repressor’ by either promoting or blocking the binding of RNA polymerase
  5. this will will either increase or decrease the rate of transcription
  6. genes is then expressed if RNA polymerase can bind to DNA
  7. mRNA can be transcribed from these genes
  8. this mRNA can be translated into diff specific proteins
  9. the expression of diff genes results in diff proteins being coded for
  10. resulting in diff specialised cells being produced = differentiation
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21
Q

transcription factors

A

proteins found in the cytoplasm
which control rate of transcription by allowing/inhibiting expression of target genes

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22
Q

promoter region

A

a region of DNA where transcription of a gene is initiated

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23
Q

summarise cell differentiation by transcription

A

transcription factor (TF)
moves from cytoplasm to nucleus
attach to promoter region close to target gene
transcription factors complementary to specific sequence of nucleotides in promoter sequence

it then either promotes or blocks the binding of RNA polymerase

activators (TF) - promote binding of RNA polymerase- increase rate of transcription - stimulating gene expression via transcription

gene is section of DNA that codes 4 protein

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24
Q

how does gene expression influence differentiation of cells

A

the expression of diff genes (allows mRNA to be transcribed) results in diff proteins being coded for / translated from this mRNA

resulting in diff specialised cells being produced = differentiation

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25
Q

hormonal regulation of gene expression

A
  • some hormones can enter target cell
  • then stimulate the expression of a specific gene in target cell
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26
Q

How does oestrogen regulate gene expression

A
  1. oestrogens lipid soluble as its a steroid hormone
  2. so it can easily diffuse across cell membrane
  3. oestrogen binds to complementary receptor protein to activate
    transcription factor
  4. transcription factor enters nucleus from cytoplasm
  5. binding of oestrogen changes shape of transcription factor
  6. allowing it to bind specifically to complementary promoter sequence of specific gene
  7. this allows RNA polymerase to attach to gene + catalyse transcription of the gene
  8. this stimulates gene expression
  9. mRNA is transcribed from the gene
  10. mRNA is translated into protein

OESTROGEN = INCREASES expression of specific genes

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27
Q

oestrogen causing breast cancer

A

oestrogen can increase expression of genes associated w cell division
(can stimulate breast cells to divide more rapidly)
high blood concs of oestrogen over period of time can increase the risk of uncontrollable cell division - causing cancer

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28
Q

treatment of breast cancer caused by high conc of oestrogen

A

drug: tamoxifen - effective in treating forms of breast cancer linked to high oestrogen concs

tamoxifen converted to endoxifen in body

endoxifen has a similar structure to oestrogen

endoxifen competes with oestrogen for binding to oestrogen receptor on transcription factor

oestrogen cannot bind to TF so transcription factors not activated to attach to promoter sequence

no transcription occurs

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29
Q

how can benign tumours harm the body

A
  • could put pressure on organs as it grows
  • may damage organs
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30
Q

prevention of translation by siRNA

A

translation of mRNA can be inhibited by RNAi (RNA interference) - this often involves siRNA
so mRNA cant be translated into proteins

  1. long, double stranded molecules of RNA are hydrolysed by an enzyme into shorter molecules
  2. RNA becomes single stranded siRNA as it unwinds
  3. siRNA binds to an enzyme that hydrolyses mRNA
  4. siRNA binds to specific molecule of mRNA by complementary base pairing
  5. siRNA guides hydrolytic enzyme to target mRNA
  6. the enzyme hydrolyses the mRNA molecule into fragments so it can no longer be translated into protein
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31
Q

describe siRNA

A

small interfering RNA
short, double stranded sections of RNA - around 20-25 base pairs long
found in cytoplasm

regulates gene expression by
causing mRNA to be broken down after transcription - PREVENTING TRANSLATION

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32
Q

epigenetics

A

heritable changes in gene function
WITHOUT changes to DNA base sequence

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33
Q

epigenetic changes

A
  • can either increase or decrease gene expression
  • changes can be caused by aspects of the environment e.g stress/diet etc
  1. methylation of DNA
  2. acetylation of Histones
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34
Q

what is increased methylation of DNA

A
  • methyl group (CH3) attaches to DNA
  • methyl group always attatches to Cytosine (C) when its next to Guanine (G) = known as CpG site
    the ‘p’ represents phosphate between the 2 bases
  • increased methylation inhibits transcription ➜ by preventing binding of transcription factors to promoter so genes not expressed ( gene cant be transcribed)
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35
Q

acetylation of Histones in increasing/decreasing transcription

A

in eukaryotes : DNA is wrapped around proteins called histones to form chromatin

addition/ removal of acetyl groups (COCH3) may occur

histones more acetylated= transcription more likely

histones less acetylated = inhibits transcription

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36
Q

what happens when histones are less acetylated

A

histones- less acetylated (removal of acetyl groups)
so chromatins more condensed
so transcriptions inhibited as genes not accessible to transcription factors

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37
Q

what happens when histones are more acetylated

A

when histones are more acetylated (more acetyl groups added) - chromatins less condensed (loosely packed) - so transcription of genes more likely as genes are now more accessible to transcription factors

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38
Q

epigenetics + disease

A

epigenetic changes can cause abnormal activation or inhibition of genes leading to disease

epigenetic changes can be reversed
through drugs designed to target specific cells in which epigenetic changes have taken place

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39
Q

how can cancers develop via methylation

A

abnormal changes in level of methylation

  1. hypermethylation (too much methylation)
  2. hypomethylation ( too little methylation)
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40
Q

how does cancer develop from hypermethylation

A

hypermethylation of tumour supressor genes means
these genes are NOT transcribed
so the protein that slows cell division is not produced
leading to uncontrolled cell division
and the development of a tumour

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41
Q

how does cancer develop from hypomethylation

A

hypomethylation of proto-oncogenes
means these genes are continually transcribed (produced)
so increased production of proteins that stimulate cell division
leading to rapid, uncontrolled cell division
and development of a tumour

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42
Q

whats the difference between a proto-oncogene and an oncogene

A

proto-oncogene= code for proteins that stimulate cell division

oncogene= result as a mutation in proto-oncogenes leading to rapid uncontrolled cell division

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43
Q

DNA TECHNOLOGY

A
44
Q

restriction enzymes/ restriction endonucleases

A

hydrolyse DNA/ RNA into fragments

by breaking phosphodiester bonds in both strands of DNA

hydrolyse DNA/RNA at specific base sequences known as: recognition sites/ sequences

some restriction enzymes hydrolyse DNA at diff locations to produce ‘sticky ends’

some hydrolyse at the same position in both strands to produce ‘blunt ends’

45
Q

what are sticky ends

A

pieces of DNA which enable DNA to be joined or spliced into diff pieces of DNA more easily due to complementary base pairing between sticky ends

have unpaired bases at each fragment

46
Q

blunt ends

A

bases are all paired

47
Q

What is gel electrophoresis

A

separates DNA/RNA fragments

smaller DNA fragments will travel faster so travels through the gel when an electric charge is applied

negatively charged DNA fragments move towards positively charged terminal

48
Q

describe process of gel electrophoresis

A
  1. DNA sample placed into a well in gel
  2. gels covered in buffer solution (that conducts electricity)
  3. electrical currents passed through gel
  4. DNA fragments are negatively charged so move towards positive electrode
  5. small DNA fragments move faster and travel further when electric charge is applied, so DNA fragments separate according to size
  6. after electrophoresis , DNA fragments transferred to nylon membrane + radioactively labelled DNA probes are added
  7. nylon membranes placed on X-ray/ photographic film + position of radioactively labelled fragments is shown as dark bands on the film
  8. the appearance of radioactively labelled compounds on the film is due to autoradiography
  9. DNA fragments can also be identified using fluorescent labelled DNA probes
49
Q

what are DNA ladders

A

used when separating DNA fragments

DNA ladder has DNA fragments of known sizes (base number)

can run alongside unknown sample to calculate size of DNA fragments in unknown sample/s

50
Q

polymerase chain reaction (PCR)

A

when multiple copies of identical fragments of DNA or genes are produced/amplified from a smaller sample

produces identical copies of DNA fragments

51
Q

process of PCR

A
  1. set up a mixture containing:
    - DNA to be copied
    - primers
    -free DNA nucelotides
    - heat stable DNA polymerase
  2. reactants mixed tg + heated at 95°C for 5 mins to break H bonds in DNA
  3. so dna is separated into single strands as DNA polymerase denatures
  4. mixtures cooled at 50°C for 2 mins
  5. to allow primers to bind to their specific complementary target strand/ sequence
  6. free DNA nucleotides align + attach to DNA strands by complementary base-pairing
  7. temps increased to 72°C (optimum temp for DNA polymerase)
  8. DNA polymerase joins the individual nucleotides of a strand tg by phosphodiester bonds to form a new complementary strand
  9. multiple cycles of heating + cooling produce increasing number of DNA molecules
52
Q

how might a PCR reaction stop

A

if all nucleotides are used up

53
Q

how to find the number of DNA molecules produced by PCR and why

A

each cycle of PCR doubles the number of DNA molecules
n.o molecules : 2 to power of n
n = n.o cycles

54
Q

what reactants are required in PCR

A
  • DNA to be copied
  • primers
  • free DNA nucleotides
  • heat stable DNA polymerase
55
Q

what are DNA primers

A

short, single stranded molecules of DNA

to mark beginnings and ends of DNA needed for attachment of enzymes/ nucleotides

they have complementary base sequences so alleles
bind by complementary base pairing

provide starting sequence for DNA polymerase (as DNA polymerase cant begin at single stranded starting point)

prevent og DNA strands joining back tg

56
Q

DNA probes

A

short, single stranded molecules of DNA that are radioactively / fluorescently labelled

used to identify or locate known sequences of DNA

as they bind by complementary base pairing and have labels so can be detected

57
Q

what is recombinant DNA technology

A

involves the transfer of fragments of DNA from one organism/species to another by translating the transferred DNA within cells of the recipient (transgenic organism) as the genetic code is universal

58
Q

why can transferred DNA be translated within transgenic organisms

A

genetic code is universal as well as transcription + translation

59
Q

what is a transgenic organism

A

organism thats recieved transferred DNA

60
Q

methods that can be used to obtain fragments of DNA

A
  1. using reverse transcriptase
  2. using restriction endonucleases
  3. using ‘gene machine’
61
Q

how is reverse transcriptase used to obtain DNA fragments

A
  1. mRNA which has been transcribed from the specific gene is removed from cells
  2. complementary mRNA’s used as a template to produce the required gene or fragment of DNA
  3. mRNA is mixed with free nucleotides and enzyme reverse transcriptase
  4. the free DNA nucleotides align next to their complementary bases on mRNA template
  5. reverse transcriptase then joins DNA nucleotides together to produce a fragment of DNA ( gene) for…
  6. this DNA strand/ fragment produced is known as ‘complementary DNA’ (cDNA)
  7. double stranded DNA produced from this cDNA using DNA nucleotides + DNA polymerase enzyme
62
Q

key tip 4

A

if transcription involves obtaining mRNA from DNA

reverse trancriptase will include obtaining DNA from mRNA

63
Q

how can bacteria produce/ transcribe DNA fragments by the use of reverse transcriptase

A

mRNA does not have introns so fragments are able to be transcribed by bacteria

64
Q

advantage of using reverse transcriptase to obtain DNA fragments

A

mRNA is present in large amounts in the cell that makes the protein/ gene

mRNA has no introns

65
Q

how are restriction endonucleases used to obtain DNA fragments

A

these enzymes can
remove the required gene/ fragment of DNA from the DNA of an organism

by cutting DNA AT specific sequences at recognition site

a gene / fragment obtained this way from eukaryotic organism will contain introns

66
Q

how is a gene machine used to obtain DNA fragments

A
  • enables production of DNA fragments without needing
    pre- existing DNA or mRNA as a template
  • doesnt require enzymes
  1. often uses the amino acid sequence of a protein
  2. uses the AA sequence as a template to determine specific DNA nucleotide sequence for a specific gene
  3. this is automated process
  4. the required nucleotide sequence is programmed into the gene machine
67
Q

advantage of using a gene machine to produce DNA fragments

A

faster (as its automated) than all enzyme- catalysed reactions

68
Q

how can bacteria produce/ transcribe DNA fragments by the use of a gene machine

A

absence of introns
so fragments can be transcribed by bacteria

69
Q

If the source of a gene being transferred is eukaryotic, and the intended recipient is prokaryotic, what must not be present?

A

introns
as splicing cannot occur in prokaryotes as it does in eukaryotes
so gene wouldnt be properly expressed

70
Q

what sections of DNA must be added to the gene/fragment for successful transcription of transferred genes

A
  • promoter regions
  • terminator regions
71
Q

promoter regions

A

initiate transcription of the gene by promoting the binding of RNA polymerase

72
Q

terminator regions

A

marks the end of a gene + triggers the release of the mRNA transcribed

73
Q

what does it mean for fragments of DNA to be amplified

A

increased n.o of fragments by replication

74
Q

in vivo

A

INSIDE organism
e.g copies of DNA made inside

75
Q

in vitro

A

OUTSIDE living organism
e.g copies of DNA made outside organism , typically by PCR

76
Q

where can the DNA fragments/ genes be transferred

A

fragment/ gene may be transferred into bacteria or eukaryotic cells

77
Q

what are genes transferred with

A

vectors

78
Q

what are vectors

A

in bacteria : usually plasmid

viruses, liposomes,

79
Q

what are bacteria widely used for in DNA technology

A
  • produce a protein coded for by transferred gene
  • clone genes or fragments = in vivo cloning - this can occur due to bacterias rapid production rate so transferred gene can be quickly copied
80
Q

how are vectors used to transfer genes / fragments

A
  1. Plasmid ( vector) is cut from __ using the same restriction endonuclease used to cut the gene
  2. plasmid/ vector DNA and ‘ foreign’ DNA join by base-pairing as they have complementary sticky ends
  3. enzyme ligase is used to form phosphodiester bonds between sticky ends of vector + sticky ends of DNA fragment/gene so they join tg
  4. plasmid with recombinant DNA referred to as “ recombinant plasmid’
  5. recombinant plasmids ( plasmid vector) taken up by bacteria in a process known as transformation

similar process to transfer genes into eukaryotes

81
Q

enzyme ligase

A

used to form phosphodiester bonds between sticky ends of vector + sticky ends of DNA fragment/gene so they join tg

82
Q

2 issues that may cause vectors not to work

A
  • cells may not take up the vector at all
  • cell may take up vector BUT vector may not contain the gene ( plasmid may have joined back tg without the ‘foreign’ DNA/ gene being taken up

(or the DNA fragment could’ve annealed/ joint to itself rather than vector)

83
Q

how can it be checked that gene/fragments been successfully transferred

A

by using marker genes

84
Q

marker genes

A

can detect + isolate the successfully transformed bacteria or eukaryotic cells for subsequent culturing

e.g GFP marker gene - codes for production of a green fluorescent protein

so we can identify successfully transferred gene/ fragments in bacteria/ eukaryotes as they give off fluorescent light when viewed with UV light under microscope

85
Q

.

A

separating DNA fragments = gel electrophoresis

amplifiying/ producing multiple copies of DNA fragments = PCR

obtaining fragments of DNA = 3 diff methods

transferring fragment/ gene= vectors

86
Q

benefits of recombinant technology
(GM / genetically modified organisms)

A

humanitarian benefits:

  1. leads to producing vaccines + drugs
  2. treating genetic diseases by gene therapy
  3. reduce famine + malnutrition
    by developing high yields of GM plants/animals that produce high yields + are resistant to disease
87
Q

opposition of recombinant technology from environmentalists

A
  • possible transfer of foreign genes to non-target organisms - dangerous 4 humans asw
  • irreversible process + no guaranteed economical benefits

-ethical issues with regards to permanently altering animals genome

  • unknown long term ecological + evolutionary effects
  • forcing smaller companies out of business
88
Q

what is Gene therapy

A

uses recombinant DNA technology to treat genetic diseases

transfers functional copies of an allele into an organism that normally has the defective alleles of the same gene

89
Q

stages of gene therapy

A

-identify gene causing the disease
- obtain + clone copies of functional allele
- transfer these functional alleles into patient e.g by vector
- ensure the alleles reach target cells + function normally

90
Q

2 types gene therapy

A
  • somatic
  • germline therapy
91
Q

differences between somatic + germline therapy

A

SOMATIC:
- involves targeting specific cells
- not heritable as its not in sex cells

GERMLINE :
- heritable as it involves altering in sex cells
- illegal

92
Q

gene therapy for cystic fibrosis

A
93
Q

DNA sequencing projects

A

determine sequence of DNA nucleotide bases in organism (genome)

use this to determine protein sequences that derive from proteome

could lead to things like identifying potential antigens to use in vaccine production

genome cant easily be translated into proteome if non-coding DNA present

94
Q

how can DNA probes be used in medical diagnosis

A

by screening (locating) specific alleles in individual

95
Q

what is screening

A

the use of DNA probes + DNA hybridisation to screen/locate specific alleles

96
Q

process of screening

A
  • make labelled DNA probe thats complementary to DNA sequence of target allele
  • make multiple copies of labelled DNA probe using PCR
  • obtain sample of DNA from organism being tested
  • DNA fragmented/ hydrolysed by restriction endonucleases at specific recognition sites breaking phosphodiester bonds
  • DNA amplified/ multiplied using PCR
  • DNA fragments split into single strands
  • these are transferred into nylon membrane
  • DNA probe/s added to nylon membrane
  • membranes washed to remove any unattached DNA probes
  • if alleles present labelled DNA probe will bind to complementary base one of alleles strands
  • the position of probe can be detected by the radioactivity/ fluorescence it emits
97
Q

DNA hybridisation

A

2 complementary single stranded DNA molecules bind tg to form double stranded molecule. ( in screening the DNA probes complementary to single stranded DNA will bind)

98
Q

what are the types of labels that DNA probes could be attached to and how are they detected

A
  • fluorescent labels - detected from UV light as they emit fluorescence under the light
  • radioactive labels- detected from
    x- ray films - emit radioactivity
99
Q

why does the patients DNA sample in screening have to be made single stranded

A

when you mix the patients DNA with the DNA probe
theyre both single stranded
so if DNA probes complementary in sequence they can then hybridise / bind

potential that DNA could just anneal with itself rather than DNA probe

100
Q

uses of DNA screening

A

patients can be screened for:
1. identifying heritable conditions e.g cystic fibrosis
2. identifying effective drugs or personalise medicine thats effective for certain individuals with specific disease
3. identifying health risks- a gene may increase risk of developing health issue

101
Q

uses of genetic counselling

A
  • help ppl understand probability of them developing disease
  • advise prospective parents who may be carriers of disease causing alleles
  • give options of prevential drug treatment causes
102
Q

what are VNTR’s

A

variable number tandem repeats

present in genome of an organism
they’re many repetitive, non-codings sequences of nucleotide bases which repeat next to each other

the more related 2 organisms are , the more similar their VNTR’s would be

103
Q

what is genetic finger-printings aim

A

analyses VNTR’s to determine relation between individuals / match identities of DNA sample to individual (e.g crime scene)

104
Q

procedure of genetic fingerprinting

A
  1. obtain DNA sample
  2. DNA’s hydrolysed by restriction enzymes
  3. PCR used to amplify (make more) of DNA sample
  4. amplified DNA cut into fragments using restriction endonucleases
  5. the endonucleases cut DNA at sites close to but not within VNTR’s
  6. giving large number of DNA fragments
  7. these fragments are separated by gel electrophoresis
  8. fragments are treated to form single strands ( use alkali)
  9. transferred single strands onto nylon membrane
  10. radioactive DNA probes added
  11. these r complementary to VNTR’s
  12. each probe binds with VNTR’s by DNA hybridisation
  13. radioactive probes allow position of fragments to be identified when membranes placed on X-ray film so genetic fingerprint can be obtained
105
Q

what is the position of fragments dependent on

A

number of nucleotides present

will correspond to number of repetitive sequences in each fragment

fragments w smaller number of nucleotides will travel further

106
Q

comparison of genetic fingerprints

A

if both fingerprints have a band at same position on the gel it means they have same number of nucleotides + so same number of repetitive sequences (VNTR’s)

107
Q

uses of genetic fingerprinting :

A
  • forensic science- compare DNA samples from crime scenes with DNA of suspects
  • medical diagnosis of genetic disorders - identify specific alleles for certain diseases
  • determining genetic relationships + genetic variability- more closely related organisms = more similar VNTR’s
  • prevent inbreeding between closely related individuals to maintain genetic organisms (screening)

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