Unit 8: Gene expression and DNA technology Flashcards
What is a gene mutation?
changes in sequence of nucleotide bases in DNA
What genes control the rate of cell division?
Proto-oncgenes
Tumour suppressor genes
What is the function of a Proto-oncogene
genes that code for proteins that stimulate cell division
What is the function of Tumour suppressor genes?
genes code for proteins that slow cell devision
What can happen if mutations occur in proto-oncogenes and tumour suppressor genes?
mutations can lead to rapid uncontrolled cell division (by mitosis) leading to the development of a tumour
How do mutations in proto-oncogenes occur?
a mutated version called a oncogene stimulates cells to divide too quickly
resulting in tapid uncontrollable cell division
How do mutations in Tumour suppressor genes occur?
a mutation leads to the tumour suppressor protein not being made of being non functional*
results in rapid uncontrollable cell division
What is cancer?
a group of diseases caused by alterations in the genes that regulate mitosis and the cell cycle
What is a tumour?
masses of dividing cells
What are the two types if tumours
Benign
Malignant
Describe a benign tumour
- grow slower than malignant
- non cancerous, they dont spread to other tissues bc the tumour is enclosed by fibrous tissue
- cells remain differentiated (specialised)
- nucleus has a normal appearance
Describe a Malignant Tumour
- grow faster than benign
- cancerous cells break off and spread to other parts of the body bc tumour isn’t enclosed
- cells become undifferentiated (not specialised)
- nucleus is larger and darker
What are stem cells?
undifferentiated cells that can divide by mitosis and differentiate into different types of cells
What are Totipotent stem cells?
occur for a limited time in early manmalian embryos
can differentiate into any type of cell
What are pluripotent stem cells?
- found in embryos and develop from totipotent stem cells
- can differentiate into almost any type of cell
- cant produce cells of embryonic tissue
What ate the two embryonic stem cells
Totipotent
Pluripotent
What are multi-potent stem cells?
adult stem cells
found in mature aminals
differentiate into few limited types of specialised cells
What are unipotent stem cells?
found in mature animals
can only differentiate into one type of cell
What are induced pluripotent stem cells (iPS)?
type of pluripotent cell produced from unipotent stem cells
appropriate transcription factors make the unipotent cell pluripotent
What is the function of iPS cells?
develop into a wide range of different types of tissue which could be used to treat people with certain diseases
What is the function of a transcription factors?
proteins that bring about expressions of some genes and inhibit other genes so that these cells differentiate a particular cell
Describe the role of oestrogen and gene expression
- oestrogen is lipid soluble and diffuses across the cell membrane
- oestrogen specifically bind to a receptor protein that is part of a transcription factor
- transcription factor enters nucleus
- binding change of shape of TF and allows it to bind to promoter sequence of a gene
- allows RNA polymerase to attach to gene and catalyse transcription
- mRNA then subscribed then translated into a protein
how can oestrogen lead to cancer?
In some tissues oestrogen increases the expressions of genes
so high concentrations can increase uncontrollable cell division
How does Tamoxifen treat some breast cancer?
it is converted into endoxifen which is a molecule of a similar structure to oestrogen
It competes with oestrogen for biding to an oestrogen receptor
what is siRNA?
short double stranded sections of RNA
Usually 20 to 25 base pairs long
what is the function of siRNA?
Regulates gene expression by causing mRNA to be broken down after transcription thus preventing translation
How does siRNA prevent translation?
- double trended RNA is hydrolysed into short molecules
- RNA becomes single stranded siRNA
- siRNA binds to an enzyme that hydrolyses mRNA
- it then binds to a specific molecule of mRNA by complementary base pairing and guides to hydrolytic enzyme to a target molecule of mRNA
- The enzyme hydrolyses the mRNA this prevents the translation
What is epigenetics?
change in gene function without changes in the base sequence DNA
heritable
What causes epigenetics?
aspects if the environment
e.g stress diet
What are. two epigenetic changes?
increased methylation of DNA
decreased acetylation of associated histones
How does increased methylation occur?
- methyl group attaches to the DNA sequence of a gene
- attaches to CpG site
- prevents binding of transcription factors to promoter sequence so gene isn’t expressed
- thus preventing transcription
How does acetylation increase transcription?
histones are more acetylated meaning the chromatin is less condense
so transcription is more likely as genes are more accessible to transcription factors
How does acetylation decrease transcription?
histones are less acetylated so the chromatin is more condensed
This inhibits transcription as genes are not accessible to transcription factors
how can epigenetic changes lead to disease?
By causing abnormal activation/inhibition of genes
How can cancer develop from hypermethylation?
too much methylation of tumour suppressor genes so they are not transcribed
the proteins that slow down cell division are not produces causing rapid cell division
How can Hypomethylation cause cancer?
too little methylation of proto-oncogenes so they’re continually transcribed
this increases production of proteins involved in stimulating cell division causing rapid cell division and to development
What are the function of restriction endonucleases?
hydrolyse phosphodiester bonds in DNA/RNA producing smaller fragments
Where do restriction enzymes hydrolyse DNA/RNA?
at specific base sequences AKA recognition sequences
How are sticky ends made
when restriction enzymes hydrolyse DNA at different locations other then the recognition sites
How are blunt ends made?
When restriction enzymes hydrolyse DNA at the same position in both strands
What is the function of sticky ends?
enable DNA to be joined or spliced onto a different piece of DNA more easily
because complementary base pairing occurs between sticky ends
What is gel electrophoresis?
Separates DNA/RNA fragments from
- small fragments will travel faster/further thru the gel when an electric charge is applied
Where do fragments move in gel electrophoresis?
Negatively charged DNA fragments move towards positively charged terminals
Outline gel electrophoresis
- DNA samples placed in well at top of gel
- DNA fragments in the each sample separate according to size
- fragments are then transferred to a nylon membrane then radioactively labelled probes are added
- nylon membrane is placed on X-ray and position of radioactivity labelled fragments are revealed as dark bands (autoradiography)
What are DNA ladders?
has DNA fragments of known size and are used to calculate the size of unknown samples
What is a polymerase chain reaction?
enables multiple copies of identical fragments of DNA/genes to be produced from a small sample
outline a polymer chain reaction
- Primers and DNA are mixed and heated at 95°/ 5 minutes (breaks hydrogen bonds s)
- cool to 55°/2 minutes allows primers to anneal to specific target sequence
- free DNA nucleotide align to DNA strands by CBP
- temperature is increased to 72° (optimum for DNA polymerase) enzyme joins nucleotides to form new complementary strand
how do you calculate the number of molecules produced in PCR?
2^n
n= number of cycles
What is a DNA primer?
short single standard molecules of DNA
Provide starting sequence for DNA polymerase
- Important because DNA polymerase can’t begin at a single stranded starting point
Prevent original DNA strands joining back together
What is a DNA probe?
short single standard molecules of DNA that are radioactively/fluorescently labelled
Used to identify/locate known sequences of DNA
What is recombinant DNA technology?
transfer of fragments of DNA from one organism/species to another
What is a transgenic organism?
organism that has received transferred DNA
how can you obtain a required fragment/gene using reverse transcriptase?
- mRNA uses as a template to produce required chain/fragment of DNA
- mRNA is mixed with free nucleotides and reverse transcriptase
- free DNA nucleotides align next to
complementary bases on mRNA - reverse transcription joints, DNA nucleotide together triple produce a fragment/gene
- DNA strand produced by this is called complementary DNA
- Double stranded DNA is produced from cDNA using DNA nucleotides and DNA polymerase
- No introns
How can you obtain a fragment using restriction enzymes?
required gene/fragment can be removed from the DNA using restriction endonuclease enzymes
-Contains introns
How can you produce fragments using a gene machine?
- doesn’t need pre-existing DNA or mRNA as a template
- amino acid sequence of a protein is used as a template to determine the sequence of DNA nucleotides for a specific gene
- automated process — required nucleotide sequence is programmed into the gene machine
No introns
Why must introns not be present in gene transfers?
If the source of the gene is eukaryotic and the recipient is prokaryotic introns can’t be present
what are promoter and terminator regions?
sections of DNA which must be added to the gene/fragment of DNA for successful transcription of the transferred genes in the recipient cells
What is a promoter region?
initiate transcription by promoting the binding of RNA polymerase
What is a terminator region?
marks the end of a gene and triggers release of the mRNA transcribed
What does it mean to amplify fragments of DNA?
Increase the number by replication
what is invivo?
copies are made inside the living organism
What is invitro?
copies are made outside a living organism
Usually by PCR
What are the two techniques of amplification?
in vivo
In vitro
What is a vector used for?
Transfers genes
What vector is used in bacteria?
plasmid
What are some other vectors?
Viruses and liposomes (phospholipid sack)
What is bacteria used for?
- producing a protein code for by a transferred gene
- clone genes/fragments (invivo cloning)
How does bacteria clone gene/fragments?
in vivo cloning
the rapid reproduction rate of bacteria allows a transferred gene to be quickly copied so a large amount of gene product can be obtained
How is the plasmid used to transfer a fragment/gene?
- plasma is caught using the same restriction endonuclease that cut the gene
- plasma DNA and foreign DNA join by base pairing
as they have complementary sticky ends - ligase is used to form the phosphodiester bonds
- the plasma with the phone DNA is referred to as a recombinant plasmid
What is transformation?
process in which bacteria take up the recombinant plasmid
This allows these plasma vectors to be added to a culture of bacteria
Why may the use of vectors not work?
- cells may not take up the vector
- Cells may take up the vector with the vector might not contain the gene
(plasma may have joined back together without the foreign DNA being taken up)
What is marker gene?
Allows successfully transformed bacteria/eukaryotic cells to be detected and isolated for subsequent culturing
How does GFP gene detect bacteria/eukaryotes?
GFP gene codes for the production of green fluorescent protein
GFP gene added to the gene being transferred
bacterial/eukaryotes can be identified as they fluorescence when viewed with UV light under a microscope
What are some humanitarian benefits of using recombinant DNA technology?
- Reducing famine and malnutrition by developing GM plants/animals which produce high yields and are resistant to disease
- producing vaccines and drugs
- Treating genetic diseases by gene therapy
what are some drawbacks of using recombinant DNA that come from environmentalists and anti globalisation activists?
- possible transfer of foreign genes to non target organisms
- An irreversible process with no certainty of economic benefits
- Ethical considerations with regard to permanently altering the genome of animals
- Long-term ecological and evolutionary consequences are unknown
what is gene therapy?
Uses recombinant DNA technology for the treatment of genetic diseases
How does gene therapy work?
Involves the introduction of functional copies of an allele into an organism which possesses defective alleles of the same gene
What are the stages of gene therapy?
• identify gene causing the disease
• obtaining and cloning copies of the functional allele
• transferring these functional values into the patient (by use of a vector)
• ensuring that the values reached the target cells and function normally
What is DNA sequencing?
involves technique to determine the sequence of DNA nucleotide bases (genome)
This allows the sequence of proteins (protein) to be determined
what are applications of DNA sequencing?
Can identify potential antigens for use vaccines
Why can the genome not be translated into proteom in some organisms?
these organisms are more complex
presence of non coding DNA and regulatory genes means the genome can’t easily be translated into the proteome
What is used to screen people for specific alleles
DNA probes and DNA hybridisation
What is the procedure for screening?
- a DNA probe is made that is complementary to the DNA sequence of the allele being investigated
- multiple copies of the DNA probe are made using PCR
- the sample of DNA is obtained from the person/organism being tested
Outline screening
- The DNA that’s being tested is fragmented by restriction endonucleases
- Fragments are separated by gel electrophoresis
- Fragments are treated and split into single strands
- Single strands are transferred to nylon membrane and probes are added
- Membrane is washed remove unattached probes
- If allele is present labelled DNA probe will bind to a complementary base on one of its strands
presents for as position of probe can be identified by the radioactivity/fluorescence it admits
what can patients be screened for?
- Heritable conditions
- individual drug responses
people respond differently to particular drugs due to differences in their alleles — this leads to personalised medicine - Health risks
what is genetic counselling?
uses information concerning the presence of identified mutant alleles
What is genetic counselling used for?
- understand the probability of people developing a disease
- Advise parents who may be carriers of a disease causing allele
- decide the best course of drug treatment for genetic diseases
What is a variable number Tandem Repeat
many repetitive non-coding sequences of nucleotide bases in a genome
what are VNTRs used for?
Analysis of these can be used to:
- determine the relatedness between individuals
- match the identity of a DNA sample to an individual
Outline the procedure of genetic fingerprinting
- PCR is used to amplify the sample DNA
- It’s then cut into fragments using restriction endonucleases
cut DNA at sites close to, but not within the VTNRs giving a large number of DNA fragments
(some fragments will be the same length others different) - fragments are separated by gel electro forces.
- Fragments are treated using alkali to form single strands
- Singles strands are transferred to a nylon membrane
- Radioactive probes are added that are complimentary to the repeated sequences.
Many probes are used and each bind with the VNTRs by DNA hybridisation - Radioactive probes allowed the position of fragments to be identified when the membrane is placed onto an x-ray film (the genetic fingerprint is obtained).
How can genetic fingerprints be compared?
if fingerprints have bands at the same position on the gel it means they have the same number of nucleotides and repetitive sequences
How is genetic fingerprint used in forensic science?
By comparing DNA samples from crime scene with DNA of suspects
How is genetic fingerprinting used to diagnose?
Certain diseases involve unique patterns of several areas and can be identified by fingerprinting
How is genetic fingerprinting used to determine genetic relationships?
it can determine genetic relationship relationships and genetic variability in a population as more closely related species have more similar VNTRs
how is genetic fingerprinting used in animal and plant breeding?
ensuring genetic diversity is maintained by screening organisms to prevent inbreeding between closely related individuals
