topic 8: control of gene expression Flashcards
why does each type of antibody bind to specific to one protein
sepcific teritary structure
affects variable region
only binds to complementary protein
suggest how a mutation can lead to the production of a protein that has 1 amino acid missing
loss of 3 bases (Triplet)
suggest how the spread of mutation may have occured
reproduction of individuals between different populations (interbreeding)
gene mutation
-gene mutation = change in base sequence of DNA which results in the formation of a new allele
causes of gene mutation
-causes of genetic mutation = ionising radiation, spontaneous erroes during interphase, mutagenic agents
-some mutations occur in introns (non-coding) and DNA is degenerate
frame shift
-change in hydrogen bonds in secondary structure
-change in ionic bonds between R groups in teritary structure
-protein no longer functions
explain why addition mutation can lead to the production of a non-functional protein
-addition mutation = addition of base in sequence of DNA = change in codons downstream
-no longer code for the same amino acid due to frame shift
-change in ionic bondig in teritary structure
-protein is no longer complementary
inheret mutations
Gene mutations in body cells can disrupt normal functions like cell division potentially causing cancer
Mutations in gametes can be inherited by offspring
effects of subsitution mutations
-occur when one DNA base is rplaced by another within the genetic sequence
-can create one of three stop codons = terminates polypeptide synthsis = non-functional protein
-may create a codon for a different amino acid = change in shape and function
-create condon for same amino acid = degenerate nature of the genetic code
inversion mutation
-inversions = a segement of DNA becomes detached and then reattaches in the opposite direction, inverting sequence of codons and amino acid sequence
duplication mutation
-duplications = when one or more DNA bases is repeated altering the reading frame of codons causing frame shift to the right
translocation mutation
-translocations = segment of DNA is moved from one chromosome to another = lead to cancer and significant changes in the phenotype
addition mutation
-addition = extra base inserted in DNA sequence = frame shift to right not the left
transcription factors
Transcription factors can stimulate or inhibit the transcription of particular genes
Transcription factors bind to DNA as they have binding sites complementary to the base sequence in DNA
Trascription factors can be used to turn unipotent cells into induced pluripotent cells
epigenetics
-it is possible for peoples DNA to be 100% accurate but different genes can be activated
-inside the nucelus DNA is wrapped around proteins called histones
-less histone proteins = greatest number of active genes (more loosely packed)
-tightly packed arrangement of histones makes it less likely for transcription to take place as it is harder for trasncrption factors and RNA polmyerase to reach
-gene experession is determined by the shape of DNA-histone complexes
-chemical tags on the complex are known as epigenome
=how can the epigneome be changed = diet, stress, smoking etc
acetylation of histones
-acetyl group = O=C-CH3
-acetylation of histones determines the trasncription of genes
-increase in acetylation = transcription is stimulated = DNA histone complex loosens
-DNA = negative charge phosphate group
-Histones = positive R group
-acetyl groups bind to R group in histones and when they bind they remove the positive charge = reduces attraction between phosphate backbone and DNA, loosening from the histone
methylation of DNA
-increase in methylation inhibitis transcription = causes DNA histone complex to tighten
-methyl group binds to DNA (specifically cytoseine)
-First, the methyl groups attract proteins that encourage the DNA-histone complex to become more tightly packed. This prevents genes from being accessible, inhibiting transcription.
For example, we’ve seen that when acetylation decreases, the DNA-histone complex tightens. Methyl groups attract proteins which remove acetyl groups from the complex, thereby causing it to become more tightly packed.
define epigenetics
Epigenetics = heritable changes in gene function without changes to the base sequence of DNA
treatments with epigenetics
Uncontrolled cell growth = development of tumor cells
Trasncription of gene that controls cell growth may be inhibited due to increased methylation
Treating diseases using epigenetics:
-create drug that affects enzymes involved in methylation or acetylation
adult vs embryonic stem cells
-adult stem cells are multipotent (capable of becoming more than 1 type of specialised cell) whereas embryonic stem cells are pluripotent (capable of becoming almost any type of specialised cells)
-adult stem cells are found in bone marrow while embryonic stem cells are found in human embryos
-embryos are discarded after 4-5 days and come with ethical implications
-adult stem cells have a more limited potency
why not all cells need to be tonipotent
-totipotent stem cells are entirely capable to becoming any type of specialised cell
-not all cells need to be totipotent because their location means they only need to be able to produce specific specialised cells
-g stem cells in the bone marrow need to be able to differentiate into white and red blood cells
A skin cell can produce the protein keratin but not myosin. Why?
-all cells in our body contain the same set of genes
-however there are different due to different proteins being made and the activation of different genes
-certain genes will be activated while others not related to the function will remain inactive
-only activated genes are transcribed into mRNA which is translated into proteins
stem cells
-Stem cells = undifferentiated cells of an organism that become specialised. Used for growth, development and tissue repair
The more a stem cell can differentiate, the greater its potency
types of stem cells
-multi = more than one (capable of becoming more than one specialised cell e.g blood cells)
-pluri = entirely capable of becoming almost any type of specialised cell e.g umbillical cord
-uni = capable of becoming one type of specialised cell e.g cardiomyocytes
-toti = capable of becoming any type of cell e.g embyronic stem cell
stem cells in medicine
-stem cell transplants are given to patients with leukaemia (a type of cancer which destroys stem cells)
-stem cells can be used for testing the toxicity and side effects of drugs before human testing
-stem cells can be used for studying the development of organisms and identifyin the causes of a disorder
induced pluripotent stem cells
-body cells are genetically altered in a lab to make them acquire the characteristics of embryonic stem cells
Unipotent body cell is obtained
The cell is reprogrammed to become pluripotent
Selected genes are induced to switch on
The cells are capable of dividng indefinetly
advantages of IPS
-self-renew imdeifently
-differentiate into various cell types
-avoid ethical issues associated with the use of embyronic stem cells
-could provide an ulimited source of stem cells
growth of plant tissue cultures
-plants have totipotent cells in the meritsem tissue found in the tips of roots and shoots
-new cells added to the tips of shoots allows it to grow in specific directions this mans that it can grown in certain conditions
transcription factors
-transcription factors = proteins that regulate the transcription of genes. They bind to a specific sequence of DNA on promoter region which is upstream of the coding region to be transcribed
-RNA polymerase will detach at the terminator region. None of this can occur without transcription factors
stimulation and inhibtiion
-In eukaryotes, transcription of target genes can be stimulated or inhibited when specific transcriptonal factors move from the cytoplasm into the nucelus through nuclear pores
action of transcription factors
-Transcription factors bind to complementary sequence of DNA on a promoter region which is upstream of the coding region to be transcribed
-RNA polymerase allows for the synthesis of an mRNA strand and detaches at the terminator region
-the transcription of target genes can be stimulated or inhibited when specific transcriptional factors move from the cytoplasm to the nucleus
-Transcription factors need to be a specific shape, to be complementary to the DNA base nuelotide and bind to the promoter region
activated
-hormones like oestrogen act as transcription factors to increase the rate of transcription
-transcription factor binds to promoter region upstream of DNA
inactivated
-inactive genes = not expressed
-when a gene is switched of, transcription factors bind to DNA
-prevent transcription process = no synthesis of polypeptides
process of transcription
-DNA helicase unwinds the DNA at the target gene by hydrolysing hydrogen bonds complementary base pairs
-free nucelotides bind to the exposed bases on the template strand through complementary base pairing
-adenine and uracil / cyosteine and guanine
-uracil replaced thymine
-RNA polymerase catalyses condensation reaction = formation of phosphodietser bonds between adjacent nucelotides synthesising the sugar-phosphate backbone
-pre-mRNA is then spliced to remove introns before leaving the nuclear pore
role of oestrogen
-while oestrogen is best known for their effects on female reproduction, these steroid hormones also have important physiological functions e.g cardiovascular system, immune system etc
-the biological actions of oestrogen are only found in cells expressing oestrogen receptors = depends on the interactons of these receptors with several differennt proteins
-oestrogen receptors act as transcription factors, either activating or inhibiting the expression of a wide array of genes, recptor alpha / receptor b
how oestrogen activates DNA transcription
1) oestrogen enters the cell
2) oestrogen then binds with a site on the transcription factors. By binding oestrogen changes the shape of the DNA binding site on the transcription factor by making it complementary to the promoter region of DNA
3) the transcription factor is now able to enter the nucelus and bind to a specific base sequence of DNA
4) the presence of the transcription factor allows RNA polymerase to bind to the DNA
5) transcription of the target gene can now take place
oestrogen = lipid soluble
Hormones like oestrogen can switch on a gene and thus start transcription by combining with a receptor site on the transcriptional factor. This activates the DNA binding site by causing it to change
Shape.
Oestrogen = lipid soluble molecule so diffuses easily through phospholipid membrane
define what is meant by epigenetics
heritable changes in the environment that alter gene function without changing the base sequence of DNA
where does oestrogen bind
protein
explain how increased methylation could lead to cancer
-methyl groups added to tumor suppressor gene
-transcription of tumour suppressor gene is inhibited
-leads to uncontrolled cell division
how to bengin tumours differ to maligant tumors
benign = cannot metastise / spread to other parts of the body
oestrogen = lipid soluble
non polar = easily diffuse
explain how an activiated oestrogen receptor affects the target cell
-receptor binds to promoter region which stimulates RNA polymerase
-increases trasncription
oestrogen activation
-oestrogen enters the cytoplasm through phospholipid bilayer as it is lipid soluble
-binding to oestrogen receptor changes tertiary structure meaning inhibitory molecule in transcription factor is released
-complex can now move through nucelar pore and bind to promoter region
why does oestrogen not affect other cells in the body
other cells dont have oestrogen receptors
name 2 transcription factors in the diagram
- IRF protein
- phosphorylated STAT 1
conversion of cells
Trascription factors can be used to turn unipotent cells into induced pluripotent cells
A skin cell can produce the protein keratin but not myosin. Why?
-all cells in our body contain the same set of genes
-however there are different due to different proteins being made and the activation of different genes
-certain genes will be activated while others not related to the function will remain inactive
-only activated genes are transcribed into mRNA which is translated into proteins
Why not all stem cells need to be totipotent:
-totipotent stem cells are entirely capable to becoming any type of specialised cell
-not all cells need to be totipotent because their location means they only need to be able to produce specific specialised cells
-g stem cells in the bone marrow need to be able to differentiate into white and red blood cells
types of stem cells
-multi = more than one (capable of becoming more than one specialised cell e.g blood cells)
-pluri = entirely capable of becoming almost any type of specialised cell e.g umbillical cord
-uni = capable of becoming one type of specialised cell e.g cardiomyocytes
-toti = capable of becoming any type of cell e.g embyronic stem cell
stem cells in medicine
Stem cells in medicine:
-stem cell transplants are given to patients with leukaemia (a type of cancer which destroys stem cells)
-stem cells can be used for testing the toxicity and side effects of drugs before human testing
-stem cells can be used for studying the development of organisms and identifyin the causes of a disorder
IPS
nduced pluripotent stem cells (IPS):
-body cells are genetically altered in a lab to make them acquire the characteristics of embryonic stem cells
Unipotent body cell is obtained
The cell is reprogrammed to become pluripotent
Selected genes are induced to switch on
The cells are capable of dividng indefinetly
transcription factors
-transcription factors = proteins that regulate the transcription of genes. They bind to a specific sequence of DNA on promoter region which is upstream of the coding region to be transcribed
RNA polymerase
-RNA polymerase will detach at the terminator region. None of this can occur without transcription factors
-In eukaryotes, transcription of target genes can be stimulated or inhibited when specific transcriptonal factors move from the cytoplasm into the nucelus through nuclear pores
action of transcription factors
Transcription factors bind to complementary sequence of DNA on a promoter region which is upstream of the coding region to be transcribed
-RNA polymerase allows for the synthesis of an mRNA strand and detaches at the terminator region
-the transcription of target genes can be stimulated or inhibited when specific transcriptional factors move from the cytoplasm to the nucleus
-Transcription factors need to be a specific shape, to be complementary to the DNA base nuelotide and bind to the promoter region
how oestrogen activates DNA transcription
1) oestrogen enters the cell
2) oestrogen then binds with a site on the transcription factors. By binding oestrogen changes the shape of the DNA binding site on the transcription factor by making it complementary to the promoter region of DNA
3) the transcription factor is now able to enter the nucelus and bind to a specific base sequence of DNA
4) the presence of the transcription factor allows RNA polymerase to bind to the DNA
5) transcription of the target gene can now take place
RNA interference
-prevents the translation of mRNA = occurs within the cytoplasm that silences gene expression
-occurs in prokaryotes and eukaryotes
-bind to protein complexes which use energy from ATP to separate the 2 strands of siRNA = exposed nucelotide bases
epigenetics
Epigenetics = environmental factors that cause heritable changes in gene function without changing the base sequence of DNA. Changes can be passed onto future generations
relationship between DNA and histones
Relationship between histones and DNA = DNA is negatively charged due to the phosphate group which wraps around the positive histone proteins to form chromatin
epigenome
Epigenome = layer of chemical tags called epigenetic markers that cover the DNA-histone complex e.g acetyl or methyl groups
-when there is less association with histones DNA is more available to transcription factors and so genes are more likely to be switched on
-chemical tags on the epigenome respond to environmental changes e.g diet/stress etc
methyl groups
-only bind to DNA (cytosiene on DNA)
-methyl groups are positive
-when methyl groups vind the complex causes the DNA to tightly coil
-less transcription
-increased methylation = inhibits transcription as transcription factors cannot bind
-decreased methylation = stimulates transcription
acetyl groups
-can only bind to histones
-acetyl groups are negative
-when acetyl groups bind to histones, DNA repels them causing it to become more loosely coiled
-increased acetylation = increased transcription
-decreased acetylation = decreased transcription
interfering RNA
Small interfering RNA (siRNA) also called silencing RNA is used for short term switching off of
genes. The siRNA binds to a complementary sequence of mRNA. As mRNA is usually single
stranded and the cell therefore detects the double stranded form on mRNA and views it as
abnormal. Therefore the mRNA is broken down by enzymes preventing translation.
exposure
less of promotor region is exposed for transcription factors to bind
permanent activation of oncogenes =
leads to uncontrollable cell division due to the constant transcription of proto-oncogenes
cancer
Cancer can arise as a result of mutation. Uncontrolled cell division in cancer leads to the
formation of a tumour. There are two types of tumours, benign which do not tend to cause
much harm apart from light mechanical damage caused by pressing against blood vessels or
other cells. Benign tumours grow slowly and do not spread, whereas malignant tumours grow
rapidly and can spread to the neighbouring cells via metastasis (through the blood stream or
lymphatic system) thus causing damage by disrupting the running of important processes.
Malignant tumours are difficult to treat in comparison to benign tumours.
abnormal methylation
- Abnormal methylation of tumour suppressor genes and oncogenes - increased
methylation also called hyper-methylation plays an important role in controlling tumour
suppressor genes and oncogenes. The hyper-methylation of a tumour suppressor gene
called BRAC1 can lead to breast cancer.
increased oestrogen concentrations
- Increased oestrogen concentrations can be linked to breast cancer development. These
elevated levels are found in fatty tissues called adipose tissue in the breast of postmenopausal women. Oestrogen binds to the transcription factor which activates the
genes promoting cell division, leading to tumour formation.
tumors
-Tumors can develop when the cell cycle goes unchecked. It results in uncontrolled cell division
-Benign tumor = non-cancerous growth of cells that can be removed. They cannot metatisize
-Malignant tumors = cancerous= are able to spread to other tissues which makes them harder to remove
comparison between benign and malignant tumours
-benign tumors grow slowly while malignant tumors grow rapidly
-benign tumors have a relatively normal cell nucelus while malignant tumors nucelus is larger and darker due to the abundance of DNA
-benign tumors form primary tumors (adhesion of molecules) whilst malignant tumors spread to other regions forming secondary tumors
-benign tumours can be removed by surgery while malignant tumours need surgery and chemotherapy
-benign = specialised malignant = unspecialised
calculating tumor growth
1) calculate total hours
2) calculate how many cell divisions occur
3) 2^n (n = nunber of cell divisions)
4) convert to standard form
A tumor cell divides every 9 hours. How many tumor cells after 2 week.
14 days x 24 hours = 336
336 / 9 days = 37.3
2^37.3
= 1.73 x 10^11
Cancer originates when mutations occur in genes that regulate cell division
Two types of genes involved in this process:
Proto-onocogenes
Tumor suppressor genes
oncogenes
Oncogenes = mutated gene that has the potential to cause cancer
Proto-oncogenes –> oncogenes
Proto-oncogenes = genes that regulate normal cell division whilst onocgenes are overexpressed proto-oncogenes leading to unconctrolled cell division
normal cells and growth factors
–> in normal cells growth factors attach to the cell surface membrane and proto-oncogenes are transcribed into proteins which activate genes that cause DNA to replicate and divide
-if a mutation occurs on the protooncogene it can be permantely activated, becoming an oncogene. Results in rapid DNA replication, and uncontrollable formation of a tumour
Suggest how the attachment of a growth factors triggers the transcription of proto-onocgenes?
-growth factors bind to receptors = activate transcription factors
-this makes the transcription factor binding site complementary to the promoter region upstream of the proto-onocgene
tumor suppressor gene
-code for proteins that slow down cell division, repair mistakes in DNA and trigger apoptosis
-a normal tumour suppressor gene maintains normal rates of cell division so prevents the formation of tumors
-if the tumor suppressor gene is inactivated it causes cells to grow out of control (p53 gene)
Explain how abnormal methylation of DNA could result in tumor suppressor genes being switched off?
-increased methylation = more methyl groups bind to cytosiene on DNA due to negative phosphate group
-increased coiling of DNA-histone complex
-less promoter region exposed, transcription factors cannot bind so the tumor suppressor gene is not transcribed upstream and RNA polymerase doesn’t bind. Protein for apoptosis not made.
oestrogen in post-menoapusal women
-post menopausal women stop producing so much oestrogen
-but fat cells in their breasts start to produce oestrogen more locally (oestrogen –> transcription factor)
-oestrogen can bind to transcription factors and cause changes to transcription of certain genes
-oestrogen = protein
-oestrogen receptor = transcription factor
2 ways a gene is switched off
increased methylation
decreased acetylation
RNA interference
regulates translation (occurs between the conversion of mRNA to a polypeptide)
-in eukaryotes and some prokaryotes translation of mRNA produced from target genes can be inhibited by RNA inference (RNAi)
-this is when an mRNA molecule that has already been transcribed gets destroyed before it is translated to create a polypeptide chain. This is don’t by siRNA (small intefering RNA)
siRNA
SiRNA binds to a complementary sequence of mRNA bringing with it an enzyme which can cut up the mRNA into smaller sections before it can be translated
how siRNA interference occurs
1) Double stranded RNA molecule in the cytoplasm is cut up into smaller sections of siRNA
2) the double stranded siRNA separates into single stranded siRNA (21 base pairs long) which then pairs with an enzyme
3) the single stranded siRNA guides the enzyme to a secton on an mRNA molecule and binds via complementary base pairing
4) The enzyme cuts the mRNA into smaller sections
5) the mRNA can no longer be translated and the gene is not expressed
advantage of siRNA
Advantage of siRNA = prevent translation of oncogenes + translation of viral mRNA
SiRNA strand must be single stranded to bind to complementary base pairs
suggest how the detergent releases CENP-W from cells
-cell surface membrane is made from a phospholipid bilayer
-detergent dissolves phospholipid
explain how ultracentrificgation separates CENP-W from other molecules
-spin supernatent at high speeds
-denser molecules are released first
suggest how siRNA produced these results
siRNA reduces the amount of tubulin formed
-reduces translation
-siRNA binds to mRNA of CENP-W
when siRNA binds to mRNA name the complementary base pairs
cytosiene and guanine
adenine and uracil
2 reasons why siRNA would only affect gene expression in cells infected with HIV
-only infected cells containing mRNA of HIV
-only injected cells have HIV on protein surface
outline 2 reasons why identifying people at risk might be of benefit to the ppl who arent tested
- target drug treatment
- to warm people to make lifestyle changes
SANGER method of sequencing
-gel electropheroesis
-random incoroporation of chain terminating deoxynucelotides by DNA polymerase during in vitro DNA replication
PCR –> gel –> detection by marker
discuss how advancements in biology have allowed us to understand and slow the spread of COVID (25 marks)
-topic 2 (insertion of viruses)
-human genome project (topic 8)
-vaccinations + importance of these programmes
-dihybrid inheritance
-epigenetics
-topic 1 = biological molecules
true or false = not all DNA codes for proteins
true
describe how a gene could be removed from the cells of an amaranth plant and inserted into the cells of a potato plant
-cut out gene using restriction endonucelase from plant
-this creates sticky ends
-using the same enzyme to cut potato DNA and ligase
-inject the vector (virus infects DNA)
name the type of enzyme used to cut open the plasmid
restriction endonuclase
name the type of enzyme to insert the gene into the plasmid
ligase
why was the plant able the synthesise the insect protein
-DNA is universal in all organisms
-transcription and translation are universal processes in all organisms
-insect DNA can be transcribed
describe how enzymes could be used to insert the GH gene into a plasmid
-restriction endonuclease cuts plasmid
-produces sticky ends that are complementary to the gene
-DNA ligase creates phosphodiester bonds via complementary base pairing
what features of sticky ends allows them to join
sticky ends are compelemtary to the gene
using genome projects
determing the genome of simpler organisms allows the sequences of proteins that derive from the genetic code
-in more complex organisms the presence of non-coding DNA and of regulatory genes means that the knowledge of the genome cannot easily be translated into the proteome
-introns + non-coding DNA
human genome project
-sequence is a compostie derived from several individuals
-scientists have to break DNA into smaller pieces then separated and sequenced individually
-DNA binds to another DNA if the sequences are exactly opposite from eachother
aims of the human geneome project
-identify all the genes in the human genome
-find the location of all the genes
-determine the sequences of base pairs that make up the human DNA
-find the functions of different genes
-publish the results in a public database
simple vs complex
Simple:
-specific pathogens + explore genes
Complex:
-prevent, detect, treat genetic conditions
ethical implications of human genome projects
-may lead to discrimination
-increases pressure for germ line therapy
-data protection issues
-may lead to designer babies
-people may be put uner pressure to terminate pregnancies
-cause psychological stress
recombinant DNA technology
-recombinant DNA technology involves the process of transfer DNA from one organism or species to another (this is possible since the genetic code is unverisal and transcription/translation are all universal processes)
-transferred DNA can be translated into the cells of the transgenic organism
3 methods by which the desire gene can be isolated
-using reverse transcriptase
-using restriction endonucelase
-using the gene machine
using reverse transcriptase
-extract mature mRNA (only contains exons) and introduce the enzyme reverse transcriptase
-reverse transcriptase enzyme joins the DNA nucelotide with complementary bases to the mRNA sequence
-this creates a single stranded cDNA (copy DNA) of the mature mRNA. To make double stranded add DNA polymerase
-CDNA = thymine instead of uracil
reverse transcriptase
Reverse transcriptase = produces single stranded DNA from an mRNA template
restriction endonuclease
restriction endonucelase are enzymes that cut up doulle stranded DNA at specific sequences
-they naturally occur as a defence mechanism e.g it would cut up foregin DNA in bacteria that can cause harm
1) a striction enzyme is engineered to cut at a specific pallindromic sequence and is used to cut the desire DNA fragment from an oragnism’s genome. This produces a DNA fragment with sticky ends
2) a promoter and terminator region are often added to the fragment (binding site for RNA polymerase) to attach transcription factors.
3) the same restriction enzyme is then used to cut the bacterial plasmid, making it easier for the DNA fragment to anneal with the plasmid by the enzyme ligase
using the gene machine
-the desired DNA base sequence is typed into a computer
-the system creates that the DNA sequence will not make a protein that is a hazard and is fate
-the compouter creates dignucelotides. These can be joined to create DNA for the entire gene
-very quick, accurate, makes intron free DNA
insertion and transforming
-sticky ends increase the chance of the DNA fragment annealing with the vector plasmid and complementary base pairing
-after insertion the plasmid is described as a recombinant vector
-recombinant = contains DNA from more than one organism
-Vector –> can be used to transfer DNA into another organism
ligase
Ligase = catalyse the formation of phosphodiester bond
steps for in vivo cloning
1) isolation –> production of DNA fragment with desired gene
2) insertion –> placement of DNA fragment into vector e.g using ligase
3) transformation –> introduction of DNA fragment to a suitable host
4) identification –> recognition of host cells that have taken up the DNA using gene markers
5) amplification/cloning –> cutting of host cells containing the DNA to produce the protein on a large scale
transformation
-once DNA has been incorporated into the plamids and the plasmid needs to be reintroducted into the bacterial cells
1)the plasmids and bacterial cells are mixed together in a medium containing calcium ions and are gently heated to prevent the enzyme denaturing
2)Ca2+ ions and heat cause the plasma membrane of the bacteria to become more permeable, allowing the plasmid to pass through the cell surface membrane into the cytoplasm
->as few as 1% of bacterial cells end successfully in taking up the plasmid
–> some plasmids would have closed up without the DNA fragment or the DNA fragment joins together to form its own plasmid
identification
-2 processes we have to check:
-DNA fragment has been successfully incorporated into the plasmid
-see if the plasmid has successfully entered the bacteria cell
Antibiotic resistance gene = used as a marker = bacterial cell has been transerred with plasmid
-target gene inserted in markr gene = disrupting the marker gene allows scientists to identify if the plasmid has successfully combined with the DNA fragment
-problem with inserting target gene in middle of a gene for antiboitc resistance as a test
in vivo gene cloning
Isolated DNA fragments can be placed in plasmids in a
following way:
* Plasmid and gene are cut with the same restriction
enzyme to create complementary ends sticky ends.
This means that they inserted DNA and vector are
complementary and can be joined.
* The fragments are incubated with the plasmids. If a
plasmid takes up the insert, base pairing takes place
between the complementary ends which are then
sealed with the use of DNA ligase which forms
phosphodiester linkages.
* A recombinant DNA molecule is created
formation of transgenic microorganisms
In the formation of transgenic microorganisms, electroporation is used to stimulate bacterial
cells to take up plasmids. Electroporation facilitates the process by increasing the permeability
of bacterial membranes thus increasing the chance of success. This is achieved via the use of
calcium salts and rapid temperature change from 0 to 40 degrees.
gene markers
In order to check whether the DNA has been taken up by the bacteria gene markers are used.
There are different types of gene markers, these are antibiotic restraint genes, fluorescent
markers and enzyme markers. These genes are incorporated into the plasmid so that those
who have the plasmid can be separated from the bacteria that do not.
They are also used to determine whether the desired DNA has enter the plasmid, as the marker
gene will become inactivated.
describe how alterations to the tumour suppressor gene leads to the development of tumors
tumour suppressor gene not transcribed
leads to uncontrollable cell division
why can faulty receptor proteins be killed by the immune system
faulty receptor protein recognised as an antigen
t cells bind to faulty receptor proteins
stimulate clonal b cells to divide by mitosis and produce antibodies
apart from age suggest factors that the scientists should have considered when choosing volunteers
sex
lifestyle
body mass
genetic factors
2 features of the design of the investigating which would support its reliability
large sample
over long time period
true or false = age affects cancer
true
transgenic organism
Since the genetic code is universal, as are transcription and translation mechanisms, the transferred DNA can be translated within cells of the recipient (transgenic) organism.
notes from specification
he principles of the polymerase chain reaction (PCR) as an in vitro method to amplify DNA fragments.
The culture of transformed host cells as an in vivo method to amplify DNA fragments.
The addition of promoter and terminator regions to the fragments of DNA.
The use of restriction endonucleases and ligases to insert fragments of DNA into vectors. Transformation of host cells using these vectors.
The use of marker genes to detect genetically modified (GM) cells or organisms
process of b cells
virus contains the antigen
-phagocyte engulfts the pathogen and presents its antigens on its cell surface membrane
-this activates b cells to divide by mitotis to form clones
-plasma cells secrete antibodies that are complementary to the shape of the antigen
passive immunity =
long term use of antibodies
describe how the PCR is carried out
–DNA strand is separated at 95 degrees to break hydrogen bonds and split the DNA into single strands
-at lower temperatures (55 degrees) primers bind to specific starting points on each separated DNA strand
-free nucelotides attach here
-DNA is then synthesised using DNA polymerase which forms bonds between complementary base pairs joining nucelotides together
-cycle is repeated again
why is DNA heated to 95 degrees
form single stranded DNA
why is DNA polymerase heat stable
so it doesnt denature at 95 degrees
why is the reactin mixture cooled to 40 degrees
for the binding of primers to DNA strands
what would the scientists have inserted into the plasmid along with the spider gene
promoter region
2 ways why it was important that the spider gene was expressed only in the silk glands of silkworms
-so that the protein can be harvested
-fibres in other cells may cause harm
what is an antibody
a protein that is complementary to an antigen
sticky bends on cut DNA are
complementary to unpaired bases on the plasmid
what is meant by a vector
carrier of foreign DNA into a host cell
why do molecular biologists often use plasmids which contain antiobitic resistance genes
-acts as a marker gene
-allows the detection of cells containing plasmids
for recombinant technology
-increases plant availability = survival and growth / increasing plant yields
-reduced allergency
-healthier farm animals = increase livelistock
-improved nutrition
against recombinant technology
-lack of full understanding of science involved in recombinant technology
-reduced access to the private sector
-transfer of genes for antibiotic resistance
-contamination of organisms/crops
summary of in vivo
-desired gene is isolated from the genome using restriction enzymes
-the desired gene is inserted into the vector plasmid which has been cut using the same enzyme. This means that the sticky ends are complementary so are able to anneal
-the recombinant plasmid is then re-inserted into the host e.g bacteria forming a recombinant, This is called transformation
-in order for this to occur, calcium ions and heat are used to make the cell wall more permeable
-identification of successful recombinants then needs to occur. This is carried out using flourescent markers, lactose genes or antibiotic resitance genes located on other parts of the plasmid
-once successful recombinants have been identified they need to be amplified to clone many copies
flourescent marker
-GFP (green flourescent protein). Target gene is inserted into the GFP gene. This disrupts the gene and prevents flourescence
-the colonies that do not flourescene have the recombinant plasmid as the GFP gene is disrupted
enzyme markers (lactase)
-lactase can turn certain substances from colourless to blue
-target gene is inserted into the lactase gene, this disrupts the gene and prevents lactase production
-bacteria are grown on an agar plate with the colourless substance
-if the combinant plasmid has been taken up the colour remains colourless
in vivo vs in vitro
In vivo = living + inside
In vitro = non-living and outside
In-vitro - this gene cloning can be done
with PCR. This is fast, automated and
reliable once conditions are established.
This does not require living cells and can
have problems such as contamination and
errors.
In-vivo - gene cloning that can be done
using recombinant plasmids in bacteria.
This is accurate and useful as the gene is
placed in cells where it can be expressed.
The disadvantage though is it is very time
consuming and requires monitoring of
cell growth.
PCR
PCR = automated technique that doesn’t require living cells to rapidly replicate specific DNA fragments. It is sometimes called in vitro-cloning (polymerase chain reaction)
requirements for PCR
-desired DNA fragment
-DNA polymerase
-primers
-free nucelotides
-thermocycler
Primer = short sequence of DNA
temperature for PCR
95 degrees –> 55 degrees —> 72 degrees
quantity of DNA doubles
Each time one cycle of PCR is completed the quantity of DNA doubles
e.g copies after 16 cycles = 2^16
taq polymerase
Taq polymerase originates from extremophiles
stages of PCR
1) separation of DNA strand –> temperature is first increased to 95 degrres to break hydrogen bonds and split the DNA into single strands
2) Annealing of the primers –> temperature is then decreased to 55 degrees so primers can bind. The primers attach to the specific starting points on each of the separated DNA strands
3) DNA synthesis (extension) –> taq polymerase then attaches to complementary free nucelotides and makes the new strand align to each template. Temp is increased to 72 degrees
what is a DNA probe
a short single stranded length of DNA that is complementary to a known base sequence of DNA
why radioactive DNA probes are used to locate specific DNA fragments
DNA is invisible on gel
DNA probes allow detection
name the type of enzyme to produce cDNA
reverse transcriptase
steps for DNA probes
1) A cell sample is taken from a patient and the DNA is extracted
2) Denaturation = An individual’s DNA strands are heated causing them to separate into 2 single strands
3) Fragmentation = cut the DNA sample into fragments using restriction enzymes
4) Mixing = add flourescently or radiactively labelled DNA probes
5) Hybridisation = DNA probes bind with the complementary target sequence
6) Identification = visualise any DNA probe that has been hybridised to DNA fragments under UV light for flourescently labelled DNA probes or x-ray for radioactive probes to signal whether the allele is present
what are DNA probes
DNA probes allow us to locate a specific gene in an organisms genome e.g to screen patients with genetic disorder
Short and single stranded length of DNA that can be used to identify an allele. The probe binds to the complementary DNA sequence
types of DNA probes
-radioactively labelled isotopes e.g autoradiography
-flourescently labelled isotopes e.g UV light
DNA hybridisation
-DNA is heated to break the hydrogen bonds between complementary bases and separate the 2 strands
-mixture containing the DNA sample and labelled probes is collected, allowing the probe to anneal to the DNA strand via complementary base pairing
A DNA probe is a short, single stranded
DNA molecule that is designed to
complementary to a sequence to be
detected. DNA probes are made in smaller
quantities and then amplified using PCR.
The DNA labelling of the fragments either
uses radioactive isotopes or a fluorescent
d y e w h i c h g l o w s u n d e r c e r t a i n
wavelengths of light.
DNA probes can be used in order to detect
heritable conditions of health risks. The
diagrams shows the process.
notes from spec = genetic fingerprinting
An organism’s genome contains many variable number tandem repeats (VNTRs). The probability of two individuals having the same VNTRs is very low.
The technique of genetic fingerprinting in analysing DNA fragments that have been cloned by PCR, and its use in determining genetic relationships and in determining the genetic variability within a population.
The use of genetic fingerprinting in the fields of forensic science, medical diagnosis, animal and plant breeding.
advantage of gene therapy (scenario specific)
-non-invasive
-targeted
disadvantage of gene therapy (scenario specifc)
-long term effects are unknown
-may result in an immune response
describe how genetic fingerprinting may be carried out on a sample of panda DNA
-DNA is cut using restriction enzyme
-use electrophoresis to separate fragments according to mass
-southern blotting to make single stranded
-apply probe
-radioactive probe
-VNTRS
determine father DNA
-all bands in cub which dont come from mother must be in father’s DNA fingerprint
VNTR’s
-VNTR’s = variable number tandem repeats (found in introns)
-VNTR’s are short DNA sequences repeated a variable number of times without any spaces at a single location
-every individual has a unique pattern of VNTR’s in terms of the number and length. They are different in all individuals except twins
genetic counselling
-Genetic counselling uses the outcomes of genetic screening tests and research into family history to provide advice on the implications of genetic disorders in individuals and the risks of them being inherited to offspring
process of genetic counselling
-discussing the significance and risks of screening tests
-clarifying the results and the liklihood of associated diseases developing in indivduals and their offspring
-offering information on preventative measures or treatments if mutations are detected
Describe how the presence of the BCRA gene mutation in Melanie’s DNA would have been detected:
-DNA probe would be complementary to the base sequence of DNA for the BRCA gene
-many copies of the probe would be added to amplify the sample using PCR
-DNA hybridisation
personalised medicine
Genetic makeup influences how a person reacts to medications. Personalised medicine tailors healthcare for optimal treatment based on an individuals genetic makeup ensuring the most effective drugs are chosen
Silencing defective dominant alleles that could lead to dominant genetic disorders
Adding a working domaint allele to silence defective recessive alleles that could cause recessive genetic disorder e.g cystic fibrosis
Using vectors such as viruses or liposomes to transport health alleles into cells
Focusing on somatic (Body) cells for treating individuals to prevent passing disease to offspring
length vs number of VNRTs
-The length of VNTRs varies between different people. The probability that 2 individuals would have the same VNTR is extremely small
-As a result VNTR’s can be used to identify the source of DNA from tissue samples
-The number of VNTRs a person has is inherited (this can be used to identify biological parents)
problems with interbreeding
-reduces genetic diversity
-low genetic diversity increases the chance of genetic disorders and extinction
process of genetic fingerprinting
1) Extraction –> DNA is extracted from the sample
2) Digestion –> DNA is cut into segments using restriction endonucleases. This is cut close the the VNTR sequence
3) Separation –> DNA fragments separated using gel electrophoresis e.g put mixture into wells on gel and an electric current is passed through. DNA is negatively charged so DNA samples move through the gel towards the positive end. Immerse gel in alkaline solution/two strands of DNA separated. Gel is rinsed to remove unbound DNA probes (cover with nylon to absorb DNA). They are denatured with an alkaline to produce a single strand
4) Hybridisation –> radioactive marker/probe added that is complementary to VNTRs
5) Probes identified using x-ray film/autoradiography
movement of fragments in gel electrophoresis
-Smaller fragments of DNA move further along the gel
-position of the DNA bands are compared to control bands
one benefit and 2 concerns of recombinant plant technology
benefit = increase crop yield = less eaten by insects
concerns = harm insects which arent predators
insects may develop toxin resistance overtime
why might anti globalisation activists be against large numbers of recombinant plants
large companies in control so small companies go out of business
3 ways scientists could imrpove their methods of data collection
-sample at multiple sites
-sample at different sites = behaviour of fish change across diff sites
-sample more times before calculating mean = sample is more reliable
-sample across different seasons = different breeding
describe how siRNA can reduce the expression of a specific gene
siRNA is complementary to mRNA so binds
-enzyme with siRNA hydrolyses the mRNA so cannot be translated
2 features of DNA fragments that allow them to be separated by gel electophoresis
-DNA is negative charge
-different lengths/masses
