CONTOL OF GENE EXPRESSION Flashcards
TOPIC 8
GENE MUTATION
change in base sequence of DNA
occurs during DNA replication
MUTANGENIC AGENTS
chemical or radiation that increases mutation rate
.Base analogs: can substitute for base eg. 5-bromouracil can substitute for Thymine, but pairs w/Guanine instead of Alanine
.Altering bases: eg. alkylating agents add an alkyl to Guanine so changing its structure so that it pairs w/Thymine instead of Cytosine
.Altering DNA structure: eg. Ultra Violet radiation can cause adjacent Thymines to pair
ADDITION MUTATION
One extra base is added to the DNA sequence
causes all subsequent codons to be altered-frameshift
DELETION MUTATION
One base is deleted in the DNA sequence
causes all subsequent codons to be altered-frameshift
SUBSITUTION MUTATION
One base in DNA sequence is changed
no frameshift
only one codon changes
may have no impact due to degenerate genetic code
INVERTED MUTATION
section of bases detach from DNA sequence and re-join inverted
results in different amino acids being coded for in this region
DUPLICATION MUTATION
One base is duplicated at least once in sequence causes frameshift to right
TRANSLOCATION MUTATION
section of bases on one chromosome detaches and attaches to different chromosome
FRAMESHIFT
change in all the codons after point of mutation each base shifts left or right one position
IMPACT OF GENE MUTATIONS ON PROTEINS
- DNA base sequence
- mRNA codons
- amino acid sequence
- ionic/H/disulphide bonds
- tertiary structure
MUTATIONS HAVING NO EFFECT ON PROTEINS
Some mutations, such as substitutions, change only one triplet code in DNA-degenerate nature of genetic code this means that sequence of amino acids will not change
CHANGE IN AMINO ACID SEQUENCE COULD CAUSE
.May affect location of ionic/hydrogen/disulphide bond between R groups
.Change tertiary structure of protein
.May create nonfunctional protein
NON-FUNCTIONING PROTIEN
Protein w/different primary and tertiary structure- shape is changed it cannot carry out its function + prevent enzyme-substrate complexes from forming
STEM CELLS
undifferentiated cells that can continually divide and become specialised
TOTIPOTENT STEM CELLS
divide and produce any type of body cell
.Only present for limited time in first few divisions of mammalian embryo
.During development not all stem cell genes are expressed, so some are not transcribed or translated to proteins
.genes that are expressed lead to synthesis of proteins that determine cell structure and function-cell is now specialised and this is irreversible
PLURIPOTENT STEM CELL
Embryonic stem cells that can divide in unlimited numbers and develop to most of body’s cell types
can be used to treat human disorders, as they can differentiate to any body cell- get from 4-5 day old embryos that are spare from being used in IVF In vitro fertilisation treatment and rest of embryo is destroyed leads to ethical concerns regarding their use in research and treatment
MULTI-POTENT STEM CELL
Adult cells that can develop into limited number of cell types
have an operation to obtain bone marrow cells from donor that can develop to red blood cells or white blood cells
Can be used to replace variety of different cell types but it is limited-Requires donation of stem cells from genetically compatible donor – reduces chance of rejection, but rejection is still possible
Reliance on donors is an issue due to lack of suitable donors
Has less ethical issues as donors are usually consenting adults
UNIPOTENT STEM CELLS
Can only differentiate to one type of cell
E.g. Cardiomyocytes are highly specialised heart muscle cells- are very specialised, they cannot regenerate by mitosis themselves- formed from one type of unipotent stem cell that divides and differentiates to cardiomyocyte
IPS BEING USED IN RESEARCH + MEDICINE
.Take somatic adult specialised cells, and infect them w/modified virus w/genes coding for transcription factors so that cells become pluripotent
.Transcription factors attach to promoter region of DNA and stimulate RNA polymerase to stimulate transcription
.cells divide and differentiate to desired cells
IPS BEING TRANPLANTED
cells modified come from patient themselves- reduces chance of rejection when new cells are transplanted back in
EVALUATING USE OF STEM CELLS IN TREATING HUMAN DISORDER
Benefits: All stem cell treatments in medicine provide long term treatment as they continuously divide
Stem cells could be used to grow organs to save lives, or replace eye tissue
Bone marrow stem cells are already being used to treat leukaemia and have been used to cure HIV
Issues: Obtaining embryonic stem cells involves destruction of an embryo
Better to use adult stem cells e.g. iPS which could possibly be made from patient’s own cells-cells continuously dividing increases risk of uncontrollable cell division and production of tumours
nonfunctioning cells must be destroyed e.g. by chemotherapy treatment, before new functioning stem cells are injected – this can be painful and increase chance of infection
GENE EXPRESSION
ability of gene to be transcribed to mRNA and translated to polypeptide
PROMOTOR
section of DNA before gene where RNA Polymerase binds
TRANSCRIPTION FACTORS
proteins which bind to promotor region on DNA they move from cytoplasm to nucleus and stimulate RNA polymerase to produce mRNA + Inhibit binding of RNA polymerase preventing production of mRNA
ACTIVATORS
increase rate of transcription eg. Help RNA polymerase bind
REPRESSORS
decrease rate of transcription eg. Bind to start of gene and prevent RNA polymerase from binding
OESTROGEN
initiate transcription of target genes
small, hydrophobic hormone-means it is lipid soluble and can diffuse through phospholipid bilayers of membranes to all cells
Only target cells contain oestrogen receptor ERα in cytoplasm
ERα is also a transcription factor but is only ‘activated’ when oestrogen binds
When oestrogen binds to ERα oestrogen receptor in cytoplasm, it changes shape
ERα oestrogen receptor can now enter nucleus and bind to promoter region of one of its target genes, stimulating RNA polymerase to transcribe that target gene
MIRNAS
formed as hair-pin bends of RNA but processed to single strands about 22 to 26 nucleotides long- single strands become incorporated to protein-based RISC
SIRNAS
formed as long double-stranded molecule and then diced to smaller fragments about 21 to 25 base pairs long- One of their strands becomes incorporated to protein-based RISC
HOW RNAI INHIBIT TRANSLATION MRNA
1.DNA produces miRNA or siRNA in nucleus
2.RNAi moves from nucleus to cytoplasm
3.RNAi unwinds to become single stranded
4.RNAi binds to protein to form RISC
5.single stranded siRNA or miRNA binds to target mRNA molecule by complementary base pairing
6.prevents mRNA from attaching to ribosome and being translated
7.Enzymes may then hydrolyse mRNA
THERAPUTIC APPPLICATION OF SIRNAS
.siRNAs created against viral genetic material will signal for their degradation and stop the virus from using host’s cellular machinery to replicate itself
.siRNAs can be used in cancer treatment by targeting oncogenes that have been expressed or unregulated
reduces number of proteins produced that can lead to cancer or that maintain cancerous growth
EPIGENTICS
heritable change in gene function
without changing DNA base sequence
caused by changes in environment
can inhibit transcription
INCREASED METHYLATION OF DNA
Methyl groups –CH3 are attached to gene
Hypermethylation of promoter prevents transcription factors from binding
RNA Polymerase is not stimulated- gene is not transcribed, and mRNA is not made + gene is not expressed
DECREASED ACETYLATION ASSOCIATION OF HISTONES
Acetyl groups –COCH3 are removed which condenses chromatin-Transcription factors cannot bind to promoter region- RNA Polymerase is not stimulated
gene can’t be transcribed so mRNA is not made + gene is not expressed
WHY ARE EPIGENETIC CHANGES ARE GOOD TARGETS FOR DRUGS
drugs can be developed to switch DNA methylation off so can be used to treat cancers cause by increased methylation of tumour suppressor genes
TUMOUR SUPPRESSOR GENES
group of genes which prevent cell from dividing or triggers apoptosis-programmed cell death
INCREASED METHYLATION/DECREASED ACETYLATION IN TUMOUR SUPPRESSOR GENES
prevent transcription factors and RNA Polymerase from binding-mRNA cannot be made and therefore protein is not produced-results in cell going into mitosis when it normally would not and therefore causing uncontrollable cell division
PROTO-OCONGENES
group of genes which trigger cell division
genes which regulate cell cycle are not expressed ‘normally’ then cell can divide uncontrollably leading to tumour
DECREASED METHYLATION/INCREASED ACETYLATION IN PROTO-OCONGENES
allows transcription factors and RNA Polymerase to bind more readily-More mRNA can be produced, leading to more of protein to be produced and causing uncontrolled cell division
OCTOGENE
mutated version of proto-oncogene
results in constant initiation of DNA replication and mitotic cell division
causes tumour formation
BENIGN TUMPOUR
Slow growing
Non-invasive
Do not metastasise (spread) to other parts of body
MALIGNANT TUMOUR
Fast growing
Invasive
Do metastasise (spread) to other parts of body
HOW OESTROGEN INCREASES THE RISK OF BREAST CANCER
Oestrogen is steroid hormone it binds to oestrogen receptor site on transcriptional factor
causing change in shape + moves to nucleus so it can bind to promoter of proto-oncogene stimulating RNA Polymerase and causing mRNA to be created which initiates transcription result in uncontrolled cell division
THERAPEUTIC APPLICATION OF BREAST CANCER
.Prevention: Can screen for specific known cancer-causing mutations eg. BRCA1 for breast cancer- could have mastectomy if mutation is present
.Treatment: Different mutations need treating differently
.Cure: gene therapy to replace inactivated tumour suppressor genes
GENEOME
entire set of DNA including all genes of cell
PROTEOME
full number of different proteins that cell is able to produce
THERAPEUTIC APPLICATION OF GENEOMES
Knowing which gene in genome codes for which protein allows antigen producing alleles to be identified in pathogens- allows scientists to design DNA and mRNA-based vaccines which are faster to engineer than traditional antigen based vaccines
SEQUENCING PROJECTS
Reading full genome of organisms
provides opportunities to screen DNA to identify potential medical problems
GEL ELECTOPHORESIS
separate DNA fragments based upon their length and size
WHY FRAGMENTS MOVE + SEPARATE
1.DNA has a slight negative charge due to phosphate groups so is attracted towards positively charged electrode – so DNA moves
2.gel used in technique has small pores that smaller fragments can move easier through – causes smaller fragments to move further in given time than larger fragments
UNIVERSAL GENETIC CODE
codon in one species code for same amino acid in another species
RECOMBINANT DNA
combining different organisms’ DNA
enable scientists to manipulate and alter genes to improve industrial processes and medical treatment
REVERSE TRANCRIPTASE TO CREATE DNA FRAGMENT
convert mRNA to single stranded complementary DNA
1.Cells that create target protein are isolated from an organism as these will have lots of mRNA in cytoplasm
2.target mRNA is extracted, and reverse transcriptase added
3.Single stranded complementary DNA is created
4.DNA Polymerase is used to create complementary strand to produce double stranded DNA fragments
ADVANTAGES + DISADVANTAGES OF REVERSE TRANSCRIPTASE
Advantages: lots of DNA fragments can be produced as there will be multiple copies of mRNA at the start
creates DNA fragments that do not have introns
Disadvantages: relatively slow process
RESTRICTION ENDONUCLEASE TO CREATE DNA FRAGMENT
recognise specific DNA sequences called recognition sites – bind to these sites and break bonds, cutting the gene from rest of DNA
many different restriction enzymes as they are specific to one recognition site- because they are enzymes w/specific tertiary structure, resulting in specifically shaped active site, complementary to recognition sequence
ADVANTAGES + DISADVANTAGES OF RESTRICTION ENDONUCLEASE
Advantages: Many restriction enzymes naturally cut DNA in way that creates sticky ends: where one strand of DNA is longer than other w/small tail of unpaired bases
sticky ends make it easier to insert desired gene to another organism’s DNA or to vector as they can easily form hydrogen bonds w/complementary base sequences on other pieces of DNA that have been cut w/same restriction enzyme
Disadvantages: creates DNA fragments w/ introns which could not be inserted to prokaryotic organisms
maximum of 2 DNA fragments per genome could be gained
Relatively slow process
GENE MACHINE TO CREATE DNA FRAGMENT
genetic code-amino acids are required scientists use computers to generate nucleotide sequence rather than an mRNA template to produce gene
ADVANTAGES + DISADVANTAGES OF GENE MACHINE
Advantage: Faster to use gene machine than all enzyme-catalysed reactions
creates DNA fragments without introns
Disadvantages: potential harm when creating new or more vigorous pests and pathogens
PCR
1.Denaturation: Heat to 95oC breaks hydrogen bonds to separate DNA strands which become templates for new complimentary strands
2.Annealing: Cooling to 55oC causes primers to attach to DNA template strands at complimentary sequence as hydrogen bonds reform
3.Elongation - Synthesis of new DNA: Raise temperature to 70oC optimum for DNA polymerase which attaches to primer and adds new bases complementary to template
WHAT PCR REQUIRES
.Target DNA or RNA being amplified
.primers-short sequences of single-stranded DNA that have base sequences complementary to 3’ end of DNA or RNA being copied define region that is to be amplified by identifying to DNA polymerase where to begin building new strands
.DNA polymerase – enzyme used to build new DNA or RNA strand- most commonly used polymerase is Taq polymerase as it does not denature at high temperature involved during first stage of PCR reaction and secondly, its optimum temperature is high enough to prevent annealing of DNA strands that have not been copied yet
.Free nucleotides – used in construction of DNA or RNA strands
.Buffer solution –provide optimum pH for reactions to occur in
PCR GRAPH EXPLAINED
1.relatively low amounts of DNA
2.DNA is doubling
3.exponential increase larger amount DNA doubling each cycle
4.ran out of primer + DNA nucleotides
TERMINATOR REGION
DNA sequences indicating to RNA polymerase when to stop producing mRNA
VECTOR
DNA molecule used as vehicle to carry DNA fragment eg. plasmids/viruses
PALIDROMIC SEQUENCE
sequences of bases that read same forwards as they do backwards
STICKY ENDS
Exposed staggered ends of bases
palindromic base sequences created by restriction endonuclease enzymes
DNA LIGASE
used to stick DNA fragment to create recombinant DNA
STEPS TO PRODUCE PROTEINS
- promotor + terminator region to modify fragment
- plasmid DNA is cut using same restriction enzyme which was used to isolate DNA fragment
- produces sticky ends on plasmid that are complementary to sticky ends of of DNA fragment
- DNA ligase is used to ‘anneal’ donor and vector DNA-DNA is now called recombinant DNA
- Vectors are inserted into target host cells
- Marker genes are inserted to vectors at same time as DNA fragment
MARKER GENE
ensures that only transformed cells form colonies
WHAT MARKER GENES CAN CODE FOR
.antibiotic resistance
.Fluorescent proteins
.Enzymes which cause colour changes
USES OF RECOMBINANT DNA TECHNOLOGY
.Genetic engineering of microorganisms, plants and animals
.Inserting gene to egg cell of female or early animal embryo means all cells of offspring will contain gene
.Promotor regions will only be activated in specific cell types so it could be used to control exactly what cells produces protein could mean protein is harvested more easily and avoids damage to organism by avoiding producing protein in wrong cell
.Agricultural crops could be produced to give higher yield, be resistant to pests or be more nutritious.
.Used in industry e.g. cheese making
.Drug production e.g. insulin
.Gene therapy: inserting alleles into cells using vectors e.g. altered viruses, plasmids or liposomes
SOMATIC THERAPY
alters alleles of body cells e.g. epithelial cells targeted for patients w/cystic fibrosis
alters alleles in sex cells, meaning all offspring cells will be affected
GERM LINE THERAPY
alters alleles in sex cells, meaning all offspring cells will be affected
ISSUES OF USING RECOMBINANT DNA TECHNOLOGY
.Monoculture: planting one type of transformed crop – reduced biodiversity
.Transformed crops could interbreed w/wild plants forming e.g. superweeds
.Organic crops could become contaminated by wind-blown seeds and pollen from transformed crops
.Anti-globalisation activists oppose growth of large multi-national companies which may force smaller companies out of business
.Designer babies – currently illegal
HUMANITARIANS THINK RECOMBINANT DNA WILL HELP PEOPLE BY
.drought – resistant crops could reduce risk of famine
.Transformed crops could be used to make pharmaceutical products e.g. vaccines and medicines more cheaply
.Gene therapy to treat disease
DNA PROBES
short, single, strands of DNA that have specific sequence which is complementary to part of target allele
STEPS OF DNA PROBES
- DNA sample broken down to smaller fragments
- DNA fragments separated by electrophoresis
- DNA fragment treated form single strands + DNA probes added
- DNA probes allow specific base sequences of DNA to be detected
DNA HYBRIDISATION
two complementary single-stranded DNA molecules combine through base pairing to form double-stranded DNA molecule
THERAPEUTIC APPLICATION OF DNA PROBES
.diagnose genetic disorders
.Can also be used to identify health risks e.g. to cancer and determine how patients will respond to drugs and inform personalised drug treatment
.used to inform genetic counselling: advising on treatment, prevention
GENETIC FINGERPRINTING
distinguishing between individuals based on their unique DNA sequence
VNTRS
variable number tandem repeats sequences of bases in introns unique to each person + non coding sequences of DNA
CREATING A GENETIC FINGERPRINT
.sample cells contain DNA is obtained eg. blood
.use PCR make copies if sample of DNA is small
.use restrictive enzymes to hydrolyse DNA targeted to VNTR regions
.use gel electrophoresis to separate DNA fragments by size
.DNA strand transferred to nylon membrane + treated to make single stranded
.DNA probes specific to each targeted VNTR are added + bind to complementary base pairings
.DNA fragments viewed using UV or X ray to produce pattern of bands
.compare size + positions of bands
USES OF VNTRS IN PATERNITY TESTS
Individuals inherit half of their VNTRs from each parent-means that 50% of an individual’s bands in their fingerprint will match mother’s bands in their fingerprint means that remaining 50% of bands must match biological father
PCR OF VNTRS ONLY
Instead of copying entirety of DNA, primers complementary to targeted VNTRs are added to sample- means that scientists can amplify specific VNTRs, allowing forensic analysis of tiny tissue samples and partially degraded DNA
multiple VNTR copies are then separated and analysed using gel electrophoresis
STR PROFILLING
type of VNTR that occurs when short sequence of DNA is repeated many times in row –eg. triplet such as CAG
DNA LADDER
mixture of DNA fragments of known lengths-pattern it creates can then be compared to unknown fragments to determine their lengths
OTHER USES OF GENETIC FINGERPRINTING
more closely pattern of banding matches more closely related two people are
Determining genetic variability in population-more number of repeats vary, greater genetic variability could inform and prevent inbreeding between animals and plants involved in breeding programmes which could lead to genetic disorders
Medical diagnosis of genetic disorders: STR testing is used to diagnose conditions that are predominantly caused by expansions in length of specific region of genome
