gene expression + DNA technology Flashcards
(108 cards)
1
Q
what are gene mutations
A
alterations in DNA’s base sequence
(of nucleotides)
may code for diff amino acid sequence in polypeptide
2
Q
what do mutagenic agents do + give examples of them
A
increase rate of mutation
e.g x-rays, UV light
3
Q
what are the types of mutations
A
- inversion
- translocation
- substitution
- addition
- deletion
- duplication
ITSADD
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
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
6
Q
tumours
A
masses of dividing cells
7
Q
cancer
A
group of diseases caused by alterations in the genes that usually regulate mitosis + cell cycle -> so uncontrolled division occurs
8
Q
what are the types of tumours
A
malignant = cancerous
benign= non- cancerous
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
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
11
Q
stem cells
A
undifferentiated cells that can divide by mitosis
and differentiate into diff types of cells
12
Q
what are the diff types of stem cells
A
- totipotent
- pluripotent
- multipotent
- unipotent
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
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
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)
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
17
Q
describe how totipotent cells eventually develop into unipotent + multipotent
A
- Totipotent cells are in the embryos at very early stages of its development and are present in the 1st few cell divisions
- eventually totipotent cells develop into pluripotent cells in the embryo
- in mature mammals : multipotent + unipotent cells are present
- multipotent stem cells e.g in bone marrow can differentiate into any type of blood cell
- unipotent e.g cardiomyocytes develop into heart muscle cells
or epithelial cells, neurone cells etc
look at diagram saved on phone
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
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
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 )
- transcription factors are proteins that control the rate of transcription of genes
- transcription factors move from cytoplasm to nucleus and attatch to a promoter region close to the target gene
- complementary transcription factor binds to a specific sequence of nucleotides in a promoter sequence
- transcription factors act as an ‘activator’ or a ‘repressor’ by either promoting or blocking the binding of RNA polymerase
- this will will either increase or decrease the rate of transcription
- genes is then expressed if RNA polymerase can bind to DNA
- mRNA can be transcribed from these genes
- this mRNA can be translated into diff specific proteins
- the expression of diff genes results in diff proteins being coded for
- resulting in diff specialised cells being produced = differentiation
21
Q
transcription factors
A
proteins found in the cytoplasm
which control rate of transcription by allowing/inhibiting expression of target genes
22
Q
promoter region
A
a region of DNA where transcription of a gene is initiated
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
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
25
hormonal regulation of gene expression
- some hormones can enter target cell
- then stimulate the expression of a specific gene in target cell
26
How does oestrogen regulate gene expression
1. oestrogens lipid soluble as its a steroid hormone
2. so it can easily diffuse across phospholipid bilayer
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
27
oestrogen causing breast cancer
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
28
treatment of breast cancer caused by high conc of oestrogen
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
29
how can benign tumours harm the body
- could put pressure on organs as it grows
- may damage organs
30
prevention of translation by siRNA
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
31
describe siRNA
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
32
epigenetics
heritable changes in gene function
WITHOUT changes to DNA base sequence
33
epigenetic changes
- 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
34
what is increased methylation of DNA
- 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)
35
acetylation of Histones in increasing/decreasing transcription
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
36
what happens when histones are less acetylated
histones- less acetylated (removal of acetyl groups)
so chromatins more condensed
so transcriptions inhibited as genes not accessible to transcription factors
37
what happens when histones are more acetylated
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
38
epigenetics + disease
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
39
how can cancers develop via methylation
abnormal changes in level of methylation
1. hypermethylation (too much methylation)
2. hypomethylation ( too little methylation)
40
how does cancer develop from hypermethylation
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
41
how does cancer develop from hypomethylation
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
42
whats the difference between a proto-oncogene and an oncogene
proto-oncogene= code for proteins that stimulate cell division
oncogene= result as a mutation in proto-oncogenes leading to rapid uncontrolled cell division
43
DNA TECHNOLOGY
44
restriction enzymes/ restriction endonucleases
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
what are sticky ends
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
blunt ends
bases are all paired
47
What is gel electrophoresis
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
describe process of gel electrophoresis
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
what are DNA ladders
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
polymerase chain reaction (PCR)
when multiple copies of identical fragments of DNA or genes are produced/amplified from a smaller sample
produces identical copies of DNA fragments
51
process of PCR
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
3. mixtures cooled at 50°C for 2 mins
4. to allow primers to bind to their specific complementary target strand/ sequence
5. free DNA nucleotides align + attach to DNA strands by complementary base-pairing
6. temps increased to 72°C (optimum temp for DNA polymerase)
7. DNA polymerase joins the individual nucleotides of a strand tg by phosphodiester bonds to form a new complementary strand
8. multiple cycles of heating + cooling produce increasing number of DNA molecules
52
how might a PCR reaction stop
if all nucleotides are used up
53
how to find the number of DNA molecules produced by PCR and why
each cycle of PCR doubles the number of DNA molecules
n.o molecules : 2 to power of n
n = n.o cycles
54
what reactants are required in PCR
- DNA to be copied
- primers
- free DNA nucleotides
- heat stable DNA polymerase
55
what are DNA primers
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
DNA probes
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
what is recombinant DNA technology
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
why can transferred DNA be translated within transgenic organisms
genetic code is universal as well as transcription + translation
59
what is a transgenic organism
organism thats recieved transferred DNA
60
methods that can be used to obtain fragments of DNA
1. using reverse transcriptase
2. using restriction endonucleases
3. using 'gene machine'
61
how is reverse transcriptase used to obtain DNA fragments
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
key tip 4
if transcription involves obtaining mRNA from DNA
reverse trancriptase will include obtaining DNA from mRNA
63
how can bacteria produce/ transcribe DNA fragments by the use of reverse transcriptase
mRNA does not have introns so fragments are able to be transcribed by bacteria
64
advantage of using reverse transcriptase to obtain DNA fragments
mRNA is present in large amounts in the cell that makes the protein/ gene
mRNA has no introns
65
how are restriction endonucleases used to obtain DNA fragments
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
how is a gene machine used to obtain DNA fragments
- 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
advantage of using a gene machine to produce DNA fragments
faster (as its automated) than all enzyme- catalysed reactions
68
how can bacteria produce/ transcribe DNA fragments by the use of a gene machine
absence of introns
so fragments can be transcribed by bacteria
69
If the source of a gene being transferred is eukaryotic, and the intended recipient is prokaryotic, what must not be present?
introns
as splicing cannot occur in prokaryotes as it does in eukaryotes
so gene wouldnt be properly expressed
70
what sections of DNA must be added to the gene/fragment for successful transcription of transferred genes
- promoter regions
- terminator regions
71
promoter regions
initiate transcription of the gene by promoting the binding of RNA polymerase
72
terminator regions
marks the end of a gene + triggers the release of the mRNA transcribed
73
what does it mean for fragments of DNA to be amplified
increased n.o of fragments by replication
74
in vivo
INSIDE organism
e.g copies of DNA made inside
75
in vitro
OUTSIDE living organism
e.g copies of DNA made outside organism , typically by PCR
76
where can the DNA fragments/ genes be transferred
fragment/ gene may be transferred into bacteria or eukaryotic cells
77
what are genes transferred with
vectors
78
what are vectors
in bacteria : usually plasmid
viruses, liposomes,
79
what are bacteria widely used for in DNA technology
- 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
how are vectors used to transfer genes / fragments
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
enzyme ligase
used to form phosphodiester bonds between sticky ends of vector + sticky ends of DNA fragment/gene so they join tg
82
2 issues that may cause vectors not to work
- 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
how can it be checked that gene/fragments been successfully transferred
by using marker genes
84
marker genes
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
.
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
benefits of recombinant technology
(GM / genetically modified organisms)
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
opposition of recombinant technology from environmentalists
- 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
what is Gene therapy
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
stages of gene therapy
-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
2 types gene therapy
- somatic
- germline therapy
91
differences between somatic + germline therapy
SOMATIC:
- involves targeting specific cells
- not heritable as its not in sex cells
GERMLINE :
- heritable as it involves altering in sex cells
- illegal
92
gene therapy for cystic fibrosis
93
DNA sequencing projects
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
how can DNA probes be used in medical diagnosis
by screening (locating) specific alleles in individual
95
what is screening
the use of DNA probes + DNA hybridisation to screen/locate specific alleles
96
process of screening
- 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
DNA hybridisation
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
what are the types of labels that DNA probes could be attached to and how are they detected
- fluorescent labels - detected from UV light as they emit fluorescence under the light
- radioactive labels- detected from
x- ray films - emit radioactivity
99
why does the patients DNA sample in screening have to be made single stranded
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
uses of DNA screening
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
uses of genetic counselling
- 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
what are VNTR's
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
what is genetic finger-printings aim
analyses VNTR's to determine relation between individuals / match identities of DNA sample to individual (e.g crime scene)
104
procedure of genetic fingerprinting
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
8. these fragments are separated by gel electrophoresis
9. fragments are treated to form single strands ( use alkali)
10. transferred single strands onto nylon membrane
11. radioactive DNA probes added
12. these r complementary to VNTR’s
13. each probe binds with VNTR’s by DNA hybridisation
14. radioactive probes allow position of fragments to be identified when membranes placed on X-ray film so genetic fingerprint can be obtained
105
what is the position of fragments dependent on
number of nucleotides present
will correspond to number of repetitive sequences in each fragment
fragments w smaller number of nucleotides will travel further
106
comparison of genetic fingerprints
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
uses of genetic fingerprinting :
- 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)
108
What is cell differentiation
development of cells into specialised cells
