the control of gene expression Flashcards
what is a mutation?
change in the base sequence of DNA that often arises spontaneously during replication
types of mutation
addition, deletion, substitution, inversion, duplication and translocation
addition mutation
base added to sequence causing frame shift
deletion mutation
base removed causing frame shift
substitution mutation
one base changed for a different base (silent, nonsense or missense)
inversion mutation
section of bases detaches from the DNA sequence and rejoins inverted
duplication mutation
a whole gene or a section of a gene is duplicated so two copies appear on the same chromosome (mutation is not harmful but second copy can mutate)
translocation mutation
series of bases on one chromosome detaches and attaches onto a different chromosome
mutagenic agent
factors that increase the rate of gene mutation e.g high energy and ionising radiation and carcinogens
stem cell definition
undiffrentiated cells that can continually divide and become specialised
types of stem cells
totipotent, pluripotent, multipotent and unipotent
totipotent stem cells
- stem cells that can divide and produce any type of body cell
- occur for a limited time in early mammilian embryos (e.g zygote)
pluripotent stem cells
- stem cells that are found in embryos and produce almost any type of cell apart from placenta and embryo
multipotent stem cells
- diffrentiate into a limited number of cells (e.g blood cells)
- found in bone marrow
unipotent stem cells
- can only diffrentiate into only one type of cell
- e.g epidermal stem cell or cadiomycocytes
induced pluripotent stem cells (iPS)
stem cells produced from adult somatic cells using transcription to help overcome some issues of embryonic stem cells
iPS process
- created from adult unipotent cells and are altered to return them to a state of pluripotency
- to do this, genes that were switched off to make the cell specialised must be switched back on
- this is done using transcription factors
- the iPS are very similar to embryonic pluripotent stem cells
unique feature of iPS
they can divide in unlimited numbers and can therefore be used to repair or replace damaged tissue
what is a transcriptor factor?
a protein that controls the transcription by binding to complementary promoter so that only certain parts of the DNA are expressed using DNA polymerase
how do transcription factors work?
- move to cytoplasm into nucleus
- bind to promoter region upstream of target gene
- makes it easier/more difficult for RNA polymerase to bind to gene
how does oestrogen affect transcription?
- oestrogen is lipid soluble so it can diffuse through the cell-surface membrane
- oestrogen binds to a specific shaped transcription factor
- oestrogen causes the transcription factor to change shape
- the DNA binding site of the transcription factor is now complementary to the shape of the promoter region of the target gene
- transcription factor complex diffuses into the nucleus through a nuclear pore
- the transcription factor binds to promoter region of a gene and stimulates transcription
what is RNA interference (RNAi)?
when the translation of mRNA is prevented
what organisms does RNAi occur in?
eukaryotes and some prokaryotes
what RNA inhibits translation?
siRNA
process of RNAi
- siRNA complementary to mRNA
- siRNA bind to mRNA
- ribosomes cannot bind to mRNA as it is double stranded
- prevents translation of mRNA
- no protein formed
what is cancer the result of?
mutations in genes that regulate mitosis, resulting in the uncontrollable divisions of cells and the creation of a tumour
characteristics of benign tumour
- non cancerous
- slow growth
- defined by clear boundary due to adhesion molecules
- impact is localised so does not spread
- easy to treat
- cells retain function and normal shape
characteristics of malignant tumours
- cancerous
- rapid, uncontrollable growth
- difficult to treat
- metastitise
- do not produce adhesion
- cell becomes unspecialised
how does chemotherapy work?
- prevents DNA replication in interphase
- prevents the formation of spindle fibres in prophase
what genes control cell division?
tumour-suppressor genes and proto-oncogenes
role of tumour-suppressor genes
code for proteins that control cell division when damage is detected (e.g DNA copying errors). help programme apoptosis. (stop cell division)
how can proto-oncogenes be involved in developing cancer?
mutation causes oncogenes to permanetly actiavte cell division
how can tumour-suppressor genes be involved in developing cancer?
hypomethylation/mutation results in coding a non-functional protein which does not inhibit cell division so mutations are not destroyed
role of proto-oncogenes
create proteins involved in the initiation of DNA replication and mitosis cell division when the body needs new cells (stimulate cell division)
how is abnormal methylation associted with cancer?
hypermethylation of tumour-suppressor genes (switched off) and hypomethylation of oncogenes (switched on)
what is meant by epigenetics?
a heritable change in gene function without change to the base sequence of DNA
how can increased oestrogen concentration be related to cancer?
- oestrogen activates transcription factors to bind to activate RNA polymerase
- cell division can therefore become uncontrolled
- tumours results in even more oestrogen being produced
what factors can leave epigentic tags?
diet, stress and toxins
effect of DNA methylation
- methylation of cytosine so promoter region methylates (+ve)
- transcription factor cannot bind
- because DNA-histone complex (-ve) is so tightly coiled because of attraction
- transcription inhibited
- uncontrolled cell division
effect of DNA acetylation
- acetylation (-ve) of histones cause DNA (-ve) to be repelled
- causes DNA to be less tightly bound to histones
- makes it easier for transcription factors to bind
- increases transcription
does an increase or decrease of methylation inhibit transcription?
increase
does an increase or decrease of acetylation inhibit transcription?
decrease
what is heterochromatin?
tightly coiled DNA caused by increased methylation and decreased acetylation
what is the genome?
entire genetic material of an organism in a cell
what is genome sequencing?
identifying the DNA base sequence for all DNA in a cell, this allows us to identify the amino acid sequence of the polypetides coded for by that DNA
what is the proteome?
complete set of proteins that can be produced by a cell
can we directly translate the genome into the proteome?
only in simple organisms like prokaryotes because they do not contain introns or regulatory genes
application of proteome sequencing
identifying potential antigens to use in a vaccine
applications of genome sequencing
- comparing genomes to work out evolutionary relationships
- personalised medicine
- genetic matching
- synthetic biology
how have sequecning methods changed over time?
used to be a manual process however it is now automated as a machine reads the base sequence
what is recombinant DNA technology?
the transfer of DNA fragments from one organism to another
why does recombinant DNA technology work?
because the genetic code is universal and therefore transcription and translation occur by the same mechanism and result in the same amino acid sequence across organisms
what three methods are used to create DNA fragments?
reverse transcriptase, restriction endonucleases and the gene machine
reverse transcriptase process
- RT makes DNA copies from mRNA
- pure mRNA is isolated from target cell
- RT joins free DNA nucleotides with comp bases to mRNA sequence
- creates cDNA
- DNA polymerase is used to make this double stranded
advantage of cDNA
it is intron free because it is from the mRNA sequence, prokaryotes cannot remove introns
restriction endonuclease process
- hydrolyse DNA at specific recognition sites
- cuts staggered ends to expose DNA bases
- sticky ends are palindromic so can join by comp base pairing easily
gene machine process
- DNA fragments created in lab using gene machine
- DNA sequence of protein is entered to computer
- creates oligonucleotides
- oligonucleotides join together to create single strand of DNA
what is an oligonucleotide?
short overlapping single strands of nucleotide
advantage and disadvantage of RT
- lots of mRNA to make cDNA
- more steps so more difficult
advantage and disadvantage of RE
- sticky ends makes it easier to insert DNA fragment
- still contains introns
advantage and disadvantage of gene machine
- can design exact DNA fragment with sticky ends, labels and prefernetial codons
- needs to know amino acid sequence
two ways in which DNA can be amplified
in vitro- polymerase chain reaction
in vivo- using host cells
stages of in vivo gene cloning
- creating DNA fragments
- inserting DNA fragments into a vector
- transformation of host cell
- identifying transformed clones
- growing the host cell
inserting DNA fragment process
- RE are used to cut out gene to make DNA fragments
- promoter and terminator region are added
- same RE is used to cut the DNA of a plasmid
- this creates complementary sticky ends 0f the DNA fragments and the plasmid
- pair are joined by ligase
- ligase catalyses the condensation reaction to form PPD bonds between nucleotides
transformation of host cell process
- cell membrane of host cell is made more permeable
- mixed with calcium ions and then heat shocked
- enables vector to enter host cells cytoplasm
what issues can arise with host cells taking up recombinant plasmid?
- recombinant plasmid does not get inside the cell
- plasmid rejoins before DNA fragment has entered
- DNA fragment sticks to itself
ways of identifying transformed cells
- antibiotic resistance genes
- fluorescent proteins genes
- enzyme coding genes
antiobiotic resistance process
- insert gene for antibiotic resistance 1 and 2 into plasmid
- insert DNA fragment into plasmid in the middle of antibiotic 1 gene
- grow bacteria on agar
- transfer bacteria colonies to a plate with antibiotic 2
- any colonies that grow have the plasmid
- transfer to a plate with antibiotic 1
- colonies that don’t grow contain the DNA fragment
fluorescent marker process
- insert DNA fragment into middle of fluorescent protein gene in plasmid
- use UV light
- any non-glowing colonies contain recombinant plasmid
enzyme markers process
- plasmid with lactase enzyme has DNA fragment inserted into middle
- prevents production of lactase
- colonies which cannot turn the colourless substance blue contain the recombinant plasmid
how to grow host cell for in vivo gene cloning?
use a fermenter which grows multiple copies of the host cell
what is used for in vitro cloning?
PCR
equipment and their uses for PCR
- thermocycler (temp change)
- DNA fragments
- DNA polymerase (Taq polymerase which makes new polymer chains of DNA)
- primers (complementary to start and end of DNA fragment)
- DNA nucleotides
what is a primer?
short sequence of single stranded DNA that are complementary to the start and end of a DNA fragment
PCR process
- separation: temp increases to 95c to break H+ bonds and split DNA fragment into single strands
- annealing of primers: temperature decreased to 55c so that primers can attach and form H+ bonds
- extension: DNA polymerase binds to primer and attaches comp free nucleotides and makes a new strand to align next to template, temp increased to 72c
advantages of PCR
- automated so more efficient
- rapid (100 billion copies of DNA can be made in hours)
- does not require living cells
what are VNTRs?
variable number tandem repeats in human DNA introns
why are VNTRs compared?
the probability of two individuals having the same VNTRs is very low however the more closely related you are, the more similiar the VNTR
what is genetic fingerprinting?
analysis of VNTR DNA fragments to determine genetic relationships and genetic variability
steps of genetic fingerprinting
- collection
- extraction
- digestion
- separation
- hybridisation
- development
- analysis
collection and extraction in genetic fingerprinting
- small sample of DNA can be collected for genetic fingerprinting
- e.g blood, body cells and hair follicles
- small samples can be identified using PCR
digestion in genetic fingerprinting
restriction endonucleases are used to cut VNTRs out
separation in genetic fingerprinting
- DNA samples loaded with small wells in agar gel
- gel is placed in a buffer liquid with electrical voltage applied
- DNA has -ve charge so move through towards other end of the gel (gel electrophoresis)
- agar gel creates resistance so short fragments move faster and further
- alkanine added
- creates patterns of bands
hybridisation in genetic fingerprinting
- DNA probes are complementary in base sequence to VNTRs
- the probes are radioactively or fluorescently labelled
- probes mixed with VNTR
development in genetic fingerprinting
- DNA probes and VNTRs are transferred to a nylon sheet
- can be exposed to x-ray or UV lights
analysis in genetic fingerprinting
- position of DNA bands are compared with known VNTR lengths to identify genetic relationships
applications of genetic fingerprinting
- paternity test
- forensic science
- ensure animals are not closely related before breeding
- medical diagnosis
how can genetic fingerprinting be used to measure paternity?
compare the bands of VNTRs in the mother, child and potential fathers. all of the child’s VNTRs must have been inherited from either the mother or father.
what is a DNA probe?
short, single stranded pieces of DNA that can be labelled to locate specific alleles of genes
how can DNA probes be used to locate specific alleles?
created to have a complementary base sequence to the allele being screened for. they are labelled, amplified using PCR and added to a sample of single stranded DNA, binding if the allele is present
process of genetic screening
- DNA probe made
- fragment of DNA made using PCR
- label is then added
- after hybridisation the DNA is washed so any unbound DNA probes are removed
- presence of label indicated allele of interest is present
process of DNA hybridisation
- DNA sample is heated to make it single stranded
- single stranded DNA sample is mixed with DNA probe and cooled
what can genetic screening be used for?
- can identify heritable dieases early
- personalised treatment
personalised medicine application
some drugs such as painkillers are more or less effective depending on your genotype. it can aslo be used to identify the best dose, increasing effectiveness, safety and can save money.
genetic counselling
type of social work where people can have their family history researched to consider the likelihood of them carrying any alleles linked to diseases before starting a family
