8. The Control Of Gene Expression Flashcards
What are the 6 types of gene mutation
Substitution of bases
Addition of bases
Deletion of bases
Duplication of bases
Inversion of bases
Translocation of bases
What are the 3 possible consequences of base substitution ?
- Formation of one of the 3 stop codons ie. production of polypeptide would be stopped prematurely
- Formation of a codon for a different amino acid ie. shape change
- Formation of a different codon but one that produces the same amino acid as before ie. mutation will have no effect
What is the consequence of a base deletion ?
Creates a frame shift to the left
Gene is read in the wrong codons and so the coded information is altered
Polypeptide will likely lead to the production of a non-functioning protein that could considerably alter the phenotype
What is the effect of the addition of bases
Usually similar effect to base deletion as there is usually a frame shift (however to the right this time)
However if 3 (or any multiple of 3) bases are added there will not be a frame shift and so the resulting polypeptide will be different but to a lesser extent than if there was a frame shift
What is the consequence of the duplication of bases
One of more of the bases are repeated- produces a frame shift to the right
What is inversion of bases and what is the consequence ?
A group of bases becomes separated from the DNA sequence and rejoin at the same position but in the inverse order
The base sequence of this portion is therefore reversed and affects the amino acid sequence that results
What is the translocation of bases and what is its effect?
A group of bases become separated from the DNA sequence on one chromosome and reinserted into the DNA sequence of a different chromosome
Leads often to abnormal phenotype, including the development of certain forms of cancer and reduced fertility
What is included under the term mutagenic agents
High energy ionising radiation - can disrupt the structure of DNA
Chemicals - alters the structure of DNA or interferes with transcription
What is cell differentiation?
The process by which each cell develops into a specialised structure suited to the role it will carry out
What are stem cells
Undifferentiated dividing cells that occur in adult animal tissue and need to be constantly replaced. They have the ability to perform self-renewal
What are totipotent stem cells
Found in the early embryo and can differentiate into any type of cell
What are pluripotent stem cells
Found in embryos and can differentiate into almost any type of cell
What are multipotent stem cells
Found in adults and can differentiate into a limited number of specialised cells
What are unipotent stem cells
Can only differentiate into a single type of cell.
Derived from multipotent stem cells and are made in the adult tissue
What are induced pluripotent stem cells (iPS cells) ?
A type of pluripotent cell that is produced from unipotent stem cells.
These cells are genetically altered in a lab to make them acquire the characteristics of embryonic stem cells (involves inducing genes and transcriptional factors within the cells to express themselves)
What are transcription factors?
Proteins that control the rate of protein synthesis by switching some genes on and some off
What is a promoter region
The short sequence of DNA at the start of a gene (where dna/rna polymerase attaches)
Give the steps in controlling gene expression by controlling transcription
Transcriptional factors move from the cytoplasm into the nucleus
Each transcriptional factor has a promoter region
When it binds, it causes this region to begin the process of transcription
mRNA is produced and the info it carries is then translated into a polypeptide
What is the effect of hormones (such as oestrogen) on gene transcription
- oestrogen is lipid soluble & therefore diffuses easily across the phospholipid bilayer
- oestrogen then binds with a site on the receptor molecule of the transcription factor by complementary pairing
- the binding of the oestrogen changes the shape of the DNA binding site of the transcription factor which can now bind to the DNA
- the transcription factor enters the nucleus through the nuclear pores and binds to the specific base sequence on DNA (promoter region)
- transcription starts
What is epigenetics
Provides explanations as to how environmental influences such as diet, stress, toxins etc can alter genetic inheritance of an organisms offspring
What is the epigenome
Both DNA and histones are covered in chemicals sometimes called tags.
These tags form the epigenome
What is the role of the epigenome
Determines the shape of the DNA-histone complex
Keeps genes that are inactive in a tightly packed arrangement, ensuring they cannot be read (switches the gene off)
Unwraps active genes so the DNA is exposed and can easily be transcribed (switches the gene on)
What is epigenetic silencing referring to
The action of switching genes off
What is the epigenome referred to as ‘flexible’
Because the chemicals tags respond to environmental changes
Factors such as diet and stress can cause the chemical tags to adjust the wrapping and unwrapping of DNA
What are the 2 processes that inhibit transcription
Decreased acetylation of histones
Methylation of DNA
When the association of histones with DNA is weak…
DNA-histone complex is less condensed
Therefore accessible to transcription factors which can initiate production of mRNA (switch the gene on)
When the association of DNA with histones is strong…
DNA-histone complex is more condensed
Therefore DNA is not accessible to transcription factors which therefore cannot initiate mRNA production (gene is switched off)
How does decreased acetylation inhibit transcription
Decreased acetylation increases the positive charges on histones and therefore increases their attraction to the phosphate groups of DNA
Association between DNA and histones is stronger so DNA is not accessible to transcription factors = mRNA production is not initiated
Gene is switched off
How does methylation of DNA inhibit transcription ?
2 ways:
- prevents the binding of transcriptional factors to DNA
- attracts proteins that condense the DNA-histone complex making the DNA inaccessible to transcription factors
Give the step by step process of siRNA operation
- An enzyme cuts large double stranded molecules of RNA (dsRNA) into smaller sections (siRNA)
- One of the 2 siRNA strands combines with an enzyme to form a siRNA-protein complex
- The siRNA molecule guides the enzyme to a mRNA by pairing its bases with the complementary ones on the mRNA molecule
- The enzyme then cuts the mRNA into smaller sections
- The mRNA is no longer capable of being translated into a polypeptide
- Gene has been blocked
What are cancerous tumours referred to as ?
Malignant
(non-cancerous tumours are referred to as Benign)
What are the differences between benign and malignant tumours
- benign grow slowly, malignant grow rapidly
- benign: cells well differentiated, malignant: cells become unspecialised
- benign : cells produce adhesion molecules, malignant: cells do not so tend to spread to other regions of body
- benign: tumours are surrounded by capsule of dense tissue, malignant: no capsule so can grow finger-like projections into surrounding tissue
- benign : localised effects, malignant : systematic effects
- benign: rarely reoccur after treatment, malignant : more frequently reoccur
What are the 2 main types of genes that play a role in cancer
Tumour supressor genes
Oncogenes
What do proto-oncogenes do ?
Stimulate a cell to divide when growth factors attach to a protein receptor on its cell surface membrane.
This then activates genes that cause DNA to replicate & the cell to divide
Most oncogenes are mutations of…
Proto-oncogenes
What may happen if a proto-oncogene mutates into an oncogene ?
What are the 2 reasons for this ?
It can become permanently activated (switched on)
2 reasons :
- receptor protein on the csm can be permanently activated so that cell divide on is switched on even in absence of growth factors
- oncogene may code for a growth factor that is then produced in excessive amounts, again stimulating excessive cell division
What is the role of a normal tumour supressor gene ?
Slow down cell division, repair mistakes in DNA, tell cells when to die (apoptosis).
Prevents formation of tumours by maintaining normal rates of cell division
What happens if a tumour suppressor gene is mutated ?
It is inactivated (switched off)
As a result stops inhibiting cell division & so cells can grow out of control
Mutated cells are often structurally and functionally different from normal cells, and while most die, those that survive can make clones of themselves and form tumours
More than half of human cancers display abnormalities in the …
TP53 gene
What is the importance of the p53 protein and how can it cause cancer if not functioning correctly ?
Involved in the process of apoptosis (programmed cell death), a process which is activated when a cell is unable to repair DNA.
If the gene for p53 is not functioning correctly, cells with damaged DNA continue to divide leading to cancer
How might hypermethylation of tumour suppressor genes lead to cancer?
- hypermethylation occurs in the promoter region of tumour suppressor genes
- leads to the tumour suppressor genes being inactivated
- therefore, transcription of the promoter regions of the tumour suppressor genes is inhibited
- tumour suppressor gene is silenced
- increased cell division as a result of inactivity of tumour suppressor genes = formation of tumour
Where is hypomethylation found to occur ?
Oncogenes where it leads to their inactivation and hence formation of tumours
Why after menopause does a woman’s risk of developing breast cancer increase ?
Increased oestrogen concentrations
Fat cells of breasts produce more oestrogen after menopause
Locally produced oestrogens trigger breast cancer and once the tumour has developed it further increases oestrogen concentration leading to increased development of the tumour
Also said that white blood cells drawn to the tumour increase oestrogen production further
How can oestrogen cause a tumour to develop?
- Oestrogen has the ability to activate a gene by binding to a gene which promotes transcription
- If the gene that oestrogen acts on is one that controls cell division & growth, then it will be activated and its continued cell division can produce a tumour
- oestrogen also causes proto-oncogenes to develop into oncogenes
What are some of the specific lifestyle factors that contribute to cancer ?
- smoking
- diet
- obesity
- physical activity
- sunlight
Why is determining the proteome of prokaryotic organisms like bacteria relatively easy ? (2 reasons)
- the vast majority of prokaryotes have just one circular piece of DNA which isn’t associated with proteins
- there aren’t any non-coding portions of DNA typical in eukaryotic cells
Why is knowledge of the proteome of organisms such as bacteria of particular interest ?
Identification of proteins that act as antigens on the surface of human pathogens.
Can be used in vaccines
Why is determining the proteome from the genome of more complex organisms difficult ?
The genome of complex organisms contains many non coding regions as well as others that have a role in regulating other genes
What is recombinant DNA
DNA of 2 different organisms that has been combined
What are the stages of protein synthesis through DNA technology
Isolation of the DNA fragments that have the gene for the desired protein
Insertion of the DNA fragment into a vector
Transformation: transfer of DNA into suitable host cells
Identification of the host cells that have successfully taken up the gene by the use of gene markers
Growth/cloning of the population inside host cells
What are the 3 methods of producing DNA fragments ?
- conversion of mRNA to cDNA using reverse transcriptase
- using restriction endonucleases to cut fragments containing desired gene from DNA
- creating the gene in a gene machine
Explain the steps in how reverse transcriptase used to produce DNA fragments
- a cell that readily produces the protein is selected
- these cells have large quantities of relevant mRNA which is extracted
- reverse transcriptase is used to make cDNA from mRNA using complimentary nucleotides
- to make the other strand of DNA, DNA polymerase is used to build up the complimentary nucleotides to cDNA.
What is the recognition sequence
Where a restriction endonuclease cuts a DNA double strand at a specific sequence of bases leaving blunt ends
Can form sticky ends with different recognition sequence
Give the outline of steps of making a gene in a gene machine
Desired nucleotide sequence is fed into a computer
Synthesis of oligonucleotides
Assembly of a gene (oligonucleotides are overlapped and then join together and made double stranded using the polymerase chain reaction)
Gene cloning - gene is inserted into bacterial plasmid
Genes are sequenced and those with errors are rejected
Gene is usually delivered incorporated into a plasmid
What are the advantages of using a gene machine to produce DNA fragments
Sequence of nucleotides can be produced in a very short period of time
Artificial genes are free of introns and other non-coding DNA so can be transcribed and translated by prokaryotic cells
What are the 2 ways in which the DNA fragment can be cloned after it has been obtained
- in vivo by transferring the fragments into a host cell by a vector
- in vitro using PCR
What needs to happen before a DNA fragment can be inserted and why?
If we want the DNA fragment to transcribe mRNA in order to make a protein, we must attach it to the necessary promoter region to start the process.
This means that both RNA polymerase and transcription factors can bind and begin transcription
We also need to add a terminator region to the other end of the DNA fragment in order to stop transcription at an appropriate point
Describe the process of the insertion of a DNA fragment into a vector
A vector is a carrying unit used to transport the DNA into a host cell (most commonly used type of vector is a plasmid)
Plasmids almost always contain gene for antibiotic resistance and the same restriction endonucleases that were used to cut DNA fragment are used at one of these genes to break plasmid loop
Sticky ends of DNA fragment and opened up plasmid are complementary so DNA fragment is incorporated using the enzyme ligase
Plasmids now have recombinant DNA
Give the process in introduction of DNA into host cells (DNA technology)
- Once DNA has been incorporated into at least some of the plasmids they must then be reintroduced into bacterial cells (transformation)
- Involves the plasmids and bacterial cells being mixed together in a medium containing Ca2+ ions
- The Ca2+ ions and changes in temp make the bacterial membrane permeable allowing plasmids to pass through the csm into the cytoplasm
What are some of the reasons for why not all bacterial cells will possess the DNA fragments with the desired gene for the desired protein ?
- only a few bacterial cells take up the plasmids when the two are mixed together
- some plasmids will have closed up before incorporating the DNA fragment
- sometimes the DNA fragment ends join together to form its own plasmid
What are the 3 types of marker genes ?
Antibiotic resistance marker genes
Fluorescent marker genes
Enzyme markers
What do marker genes involve ?
They all involve using a second, separate gene on the plasmid which is identifiable for a reason
Eg. may be resistant to an antibiotic, may make a fluorescent protein that can easily be seen, may produce an enzyme whose action can be identified
Describe and explain how antibiotic-resistance marker genes are used to identify those cells with plasmids that have taken up a new gene
Uses a 2nd antibiotic resistance gene in the plasmid (the one cut in order to incorporate the required gene)
As this gene has been cut it will no longer produce the enzyme that breaks down the antibiotic it is resistant to (no longer resistant)
We can identify these bacteria by growing them on a culture of that antibiotic
What is a problem with the use of antibiotic resistance marker genes ?
What is done about this ?
Identifying the bacteria that have taken up the required gene by growing them on a culture of antibiotic that they are no longer resistant to means that the antibiotic will destroy they very cells that contain the required gene.
Replica plating is used on order to identify living colonies of bacteria containing the required gene
Describe and explain how fluorescent marker genes are used to identify those cells with plasmids that have taken up a new gene
Transfer of a gene which produces a fluorescent protein (GFP) from a jellyfish into a plasmid
The DNA fragment is inserted into the centre of the GFP gene
Therefore any bacterial cell that has taken up the plasmid with the DNA fragment will not be able to produce GFP - won’t fluoresce
Those that do fluoresce contain the desired gene
Describe and explain how enzyme marker genes are used to identify those cells with plasmids that have taken up a new gene
- the gene that produces the enzyme lactase will turn a particular colourless substrate blue
- the required gene is transplanted into the gene that makes lactase
- if a plasmid with the required gene is present in a bacterial cell the colonies from it wont produce lactase
- therefore when these bacterial cells are grown on the colourless substrate, they will be unable to change its colour, those that do can be discounted
What does PCR require ?
- the DNA fragment
- DNA polymerase
- primers
- nucleotides
- thermocycler: a computer controlled machine that varies temps over a period of time
What are the 3 stages of PCR
- Separation of the DNA strand
- Addition (annealing) of primers
- Synthesis of DNA
What happens during the 1st step of PCR (separation of the DNA strand)
The DNA fragments,
primers and DNA polymerase are placed in a vessel in the thermocycler
Temp is raised to 95 °C causing the 2 strands of DNA fragments to separate due to the breaking of the hydrogen bonds
What happens during the 2nd step of PCR (addition/annealing of the primers)
Mixture is cooled to 55 °C causing the primers to join to their complementary bases at the end of the DNA fragment
Primers provide the starting sequences for DNA polymerase to being copying DNA
Primers also prevent the 2 separate strands from rejoining
What happens during the 3rd stage of PCR (synthesis of DNA)
Temp is increased to 72 °C (optimum temp for DNA polymerase to begin adding complimentary nucleotides along each of the separated DNA strands
Results in 2 new copies of original DNA fragment
Process is repeated many many times over
What are the advantages of in vitro cloning
Extremely fast
Doesn’t require living cells
What are the advantages of in vivo cloning
- particularly useful where we wish to introduce a gene into another organism
- almost no risk of contamination
- very accurate
- cuts out specific genes
- it produces transformed bacteria that can be used to produce large quantities of gene products
What is a DNA probe?
A short single stranded length of DNA that has some sort of label attached that makes it easily identifiable
What are the 2 most commonly used probes ?
- radioactively labelled probes : made up of nucleotides with the 32P isotope identified using an x-ray film exposed by radioactivity
- fluorescently labelled probes: emit fluorescence under certain conditions, for instance when the probe has bound to the DNA target sequence
How are dna probes used to identify particular alleles of genes?
- a DNA probe is made that has base sequences that are complementary to part of the base sequence of the DNA that makes up the allele of the gene we want to find
- the double stranded DNA is treated so it separates
- mixed with probe which binds to the complementary sequence on one of these strands (DNA hybridisation)
- site at which it binds can be identified by radioactivity or fluorescence that the probe emits
What is DNA hybridisation
Takes place when a section of DNA /RNA is combined with a single section of DNA which has complementary bases
Give the steps in locating specific alleles of genes
- determine the sequence of nucleotide bases of the mutant allele we are trying to locate (achieved using DNA sequencing techniques)
- a fragment of DNA is produced that has a complementary sequence of bases to the mutant allele we are trying to locate
- multiple copies of the DNA probe are formed using PCR
- DNA probe is made by attaching a marker to the DNA fragment
- DNA from person suspected to have mutant allele is heated (separates the strands)
- cooled in a mixture containing many DNA probes
- If the DNA contains the mutant allele the probe will bind by complementary base pairing
- DNA is washed clean of any unattached probes
- remaining hybridised DNA will now be fluorescently/radioactively labelled and can be detected
What has to happen before DNA hybridisation can take place
DNA strands of a molecule must be separated.
Achieved by heating (denaturation)
When cooled the complementary bases of each strand recombine (anneal)
What does genetic screening allow ?
- Screening can determine the probability of a couple having offspring with a genetic disorder
- Screening allows simultaneous testing for many different genetic disorders
- Detection of oncogenes responsible for cancer by preventing tumour suppressor genes from inhibiting cell division
- Allows informed decisions about lifestyle and future treatment to be made
Describe and explain the advantage of personalised medicine
- an advantage of genetic screening allows doctors to provide healthcare based on someones genotype
- some peoples genes mean that a particular drug or treatment is more/less effective in treating a condition
- knowing this allows more accurate drug dosages to be prescribed and money to be saved
Describe and explain how genetic counselling is an advantage of genetic screening
- involves discussions around the research into the family history of an inherited disease
- advises parents on the likelihood of it arising in their children
- discuss best course of treatment and survival prospects
What is genetic fingerprinting
Diagnostic tool that relies on the fact that the genome of most eukaryotic organisms constrains many non-repetitive, non-coding bases of DNA (VNTRs)
In every individual the number and length of the VNTRs has a unique pattern closely More closely related = more similar VNTRs
What is gel electrophoresis
- Used to separated DNA fragments according to their size
- The DNA fragments are placed onto an agar gel and a voltage is applied across it
- The resistance of the gel me and that the larger fragments move slower, therefore over a fixed period the smaller fragments move further than the larger ones
- If the DNA fragments are labelled with probes their final positions can be determined
Only DNA fragments up to around …. bases can be sequenced using gel electrophoresis
500
Larger genes and whole genomes must be cut using restriction endonucleases into smaller fragments
What are the 5 main stages to make a genetic fingerprint
- Extraction
- Digestion
- Separation
- Hybridisation
- Development
Give the step by step process of making a DNA fingerprint
- the DNA is extracted from the sample of tissue
- as the amount of DNA is usually small its quantity can be increased by using PCR
- the DNA is then cut into fragments using restriction endonucleases
- the DNA fragments are separated according to size using gel electrophoresis - the gel is then immersed in alkali in order to separate the double strands into single strands
- radioactive or fluorescent probes are now used to bind with VNTRs and bind to them under specific conditions such as temp and pH
- an x-ray film is put over the nylon membrane which is exposed to the radiation of the probes. Because these points correspond to the position of DNA fragments separated during electrophoresis a series of bars is revealed
How are the results from genetic fingerprints interpreted
- The DNA fingerprints from 2 samples are visually checked
- If there appears to be a match the pattern of bars in each fingerprint is passed through an automated scanning machine which calculates the length of the DNA fragments from the bands by comparing distances travelled during electrophoresis with known lengths of DNA
- the odds are calculated of someone else’s having an identical fingerprint
- the closer the match between the 2 patterns the greater probability that the 2 sets of DNA have come from the same person
What are the 4 uses of genetic fingerprinting
- genetic relationships and variability
- forensic science
- medical diagnosis
- plant and animal breeding
Describe and explain how genetic fingerprints are used for determining genetic relationships and variability
- can help resolve questions of paternity
- each band on a DNA fingerprint of an individual should have a corresponding band in one of the parents’ genetic fingerprint
- a population whose members have very similar genetic fingerprints has little genetic diversity
Describe and explain how genetic fingerprints are used in forensic science
Genetic fingerprinting can establish whether a person is likely to have been present at a crime scene
Describe and explain how genetic fingerprints are used in medical diagnosis
- can help diagnosing diseases such as Huntingtons
- a sample of DNA from a person with the allele for a disease can be cut with restriction endonucelases and a DNA fingerprint is prepared
- this can then be matched with fingerprints of people with various forms of the disease in order to determine the probability of developing symptoms
Describe and explain how genetic fingerprints are used in plant and animal breeding
- used to prevent undesirable interbreeding during breeding programmes
- also used to identify plants/animals with a particular desired gene (selective breeding)
- determining paternity and therefore the pedigree (family tree) of an individual
