Topic 8 - Control of gene expression Flashcards
define gene mutation
a change in the sequence of bases in a DNA molecule that may result in an altered polypeptide
how can mutations occur
spontaneously and continously
when do mutations usually occur
interphase - DNA replication
why do most mutations have little or no effect
due to degeneracy of genetic code
how can mutations arise from
nucleotide changes: insertion, deletion, substitution
structural changes: inversion, duplication, translocation
give 2 examples of mutagenic agents
- high energy ionising radiation such as alpha/beta/gamma radiation
- chemicals such as NO2 from tobacco smoke
what three forms can substitution mutations take
silent, nonsense, missense
define silent mutation
no change to the amino acid sequence due to the degeneracy of the genetic code
define missense mutations
alters a single amino acid potentially affecting protein function
give an example of missense mutation
sickle cell anemia
define nonsense mutations
creates a premature stop codon, producing an incomplete non-functional protein
give an example of nonsense mutation
cystic fibrosis
when does inversion mutations usually occur
during crossing over in meiosis
what happens in inversion mutations
DNA of a single gene is cut into two places. the cut portion is inverted 180 then rejoined to the same place
results in a large section of gene is backwards and multiple amino acids are affected as they are being read incorrectly
what usually results in inversion mutations
non-functional protein, or in some cases a completely new protein
what do duplication mutations result in
a gene/section being copied on the same chromosome, original gene remains intact so mutation is not harmful
how is duplication mutation important in source of evolutionary change
the duplicate can mutate over time, developing new functions
what happens in translocation
a gene is cut into two places and the section of gene cut off attaches to a seperate gene
what is a stem cell
an undifferentiated cell of an organism that is capable of self-renewal and differentiation into specialised cells
what are the 4 types of stem cells
- totipotent
- pluripotent
- multipotent
- unipotent
what types of cells can totipotent stem cells form
all cell types including extra-embryonicc tissues
where can totipotent stem cells be found
in the zygote and the first few divisions of the early embryo
what are pluripotent stem cells
they are embryonic stem cells that can differentiate into any cell type found in an embryo but are not able to differentiate into extra embryonic cells
what can pluripotent stem cells be used for
they can be used in treating human disorders
what can pluripotent cells do
they can divide in unlimited numbers and keep replacing themselves
give 2 examples of multipotent stem cells
- haematopoietic stem cells [produce all types of blood cells]
- neural stem cells [form neurons and glial cells]
describe multipotent stem cells
they are found in mature mammals and they can divide and differentiate into a limited number of cell types
describe unipotent cells
they are found in mature mammals and can divide and differentiate into just one cell type
how can stem cells be used in the treatment of human
transplated into patients to divide in unlimted numbers then differentiate into required healthy cells
explain how induced pluripotent stem cells [IPS] cells are produced
- obtain adult somatic cells from patient
- add specific protein transcription factors associated with pluripotency to cells so they express genes associated with pluripotency to cells = transcription factors attach to promoter regions of DNA, stimulating or inhibiting transcription
- culture cells to allow them to divide by mitosis
what is meant by epigenetics
heritable changes in gene function without changes to the base sequence of DNA caused by changes in the environment that inhibit transcription
what is an epigenome
all chemical modifications of DNA and histone proteins - methyl groups on DNA and acetyl groups on histones
what is a nucleosome
eukaryotic DNA wrapped around histone proteins
state arguments for using stem cells in treating human disorders
- can divide and differentiate into required healthy cells so could relieve human suffering by saving lives and imporving quality of life
- embryos are often left over from IVF and so otherwise would be destroyed
- iPS cells are unlikely to be rejected by patient’s immune system as made with patient’s own cells
- IPS cells can be made without destruction of embryo and adult can give permission
what is an acetyl group
CoCh3
describe what happens in acetylation
histone molecules contain lysine, an amino acids. the acetyl groups can add to the lysine, replacing a H+
the removal of the +ve ion can cause histone proteins to be less tightly wrapped around DNA so RNA polymerase and transcripition factos bind to DNA more easily
state arguments against using stem cells for treating human disorders
- ethical issues with emrbyonic stem cells as obtaining them requires destruction of embryo and potential life
- immune system could reject cells and immunosuppressant drugs are required
- cells could divide out of control leading to formation of tumours/cancers
what does adding acetyl groups to histone proteins result in
stimulating transcription
what does removing acetyl groups from histone proteins result in
surpressing transcription
what happens in DNA methylation
- methyl groups are added to cytosine bases (when C base is next to a G base) in DNA
- nucleosomes pack more tightly together
- preventing transcription factors and RNA polymerase binding to promotor
what does adding methyl groups result in
supressing transcription
what are transcription factors
proteins which regulate transcription of specific target genes in eukaryotes by binding to a specific DNA base sequence on a promotor region
why does DNA methylation and acetylation occur
- envrionmental factors such as diet, excercising, stress, smoking
- result of signals from neighbouring cells or even within the same cell
why is DNA methylation important in embryonic development
they influence which genes switch on and ooff when cells are differntiating
what does RNAi stand for
RNA interference
what is RNAi
the inhibition of translation of mRNA produced from target genes by RNA molecules e.g.siRNA miRNA, which inhibits the expression of a target gene
where does RNAi happeen
in the cytoplasm of eukaryotes and some prokaryotes
what is siRNA
- short double stranded RNA that can interfere with the expression of specific genes
- bases are complementary to specific sections of the target gene and the mRNA is produces
- 20 to 23 nucleotides long
describe the RNAi pathway
- double stranded RNA (dsRNA) is produced by RNA-dependent RNA polymerases
- dsRNA is hydrolysed into smaller fragments roughly 23 nucleotides long called siRNA
- in the cytoplasm siRNA binds to protein complexes which use energy from ATP to separate the two strands of siRNA, exposing the nucleotide bases so they can pair with bases from an mRNA molecule
- once the target mrRNA leaves the nucleus and enters the cytoplasm, single stranded siRNA binds to the target mRNA through complementary base papring
- mRNA molecule is cut into fragments by the protein complex associated with siRNA so this results it in being degraded, preventing ribosomes from binding
- reduces translation of target mRNA into protein
what type of tumour is cancerous
malignant
what type of tumour isn’t cancerous
benign
how does malignant tumours grow
rapidly and they invade and destroy surrounding tissues
how do benign tumours grow
slower than malignant tumours
what type of tumours does metastasis produce
secondary
describe metastasis
malignant tumour cells do not produce adhesion molecules so they can break off and spread to other parts of the body via the bloodstream and lymphatic system
what are primary tumours
when cells produce adhesion molecules that makes them stick together and so they remain within the tissue from which they arise
why do benign tumours remain as a compact structure
surrounded by a capsule of dense tissue
what could benign tumours cause
blockages or put pressure on organs
what two types of gene control cell division
- tumour rupressor genes
- proto-oncogenes
describe the function of tumour suppressor genes
codes for proteins that
- inhibit cell cycle
- cause self destructuion of potential tumour cells
describe the function of proto-oncogenes
code for proteins
- stimulate cell division
explain the tumour suppressor genes in the development of tumours
- mutation in DNA base sequence = production of non-functional protein
- decreased histone acylation/increased DNA methylation = prevents production of protein as RNA polymerase can’t bind, inhibits transcription
leads to uncontrollable cell division
explain the role of oncogenes in the development of tumours
- mutation in DNA base sequence = overproduction of protein/permanently activated protein = change in amino acid sequence, 3 structure
- decreased DNA methylation/increased histone acylation = increased production of protein due to stimulating binding of RNA polymerase, stimulates transcription
- leads to uncontrolled cell division
define oncogene
a mutated/abnormally expressed form of the corresponding proto-oncogene
what does hypermethylation cause
when the tumour supressor genes or hypermethylated = genes are not transcribed. proteins produced to slow cell division are not made = cells divide uncontrollably by mitosis = tumour development
what does hypomethylation of proto-oncogenes result in
behaving as oncogenes, increasing production of proteins that stimulate cell division = cells divide uncontrollably = formation of tumours
why do tumours require mutations in both alleles of a tumour suppressor gene but only one allele of an oncogene
- one functional allele of a tumour suppressor gene can produce enough protein to slow the cell cycle/ self destruction of potential tumour cells = cell division is controlled
- one mutated oncogene allele can produce enough protein to lead to rapid/uncontrolled cell division
explain the relevance of epigenetics in cancer treatment
- increases DNA methylation/decreases histone acylation of oncogene = inhibit transcription
- decrease DNA methylation/increase histone acylation of tumour suppressor gene = stimulate transcription
explain the role of increased oestrogen conc in the development of some oestrogen receptor positive breat cancer
- some breast cancer cells have oestrogen receptors which are inactive transcription factors
- oestrogen concentration is increased, more oestrogen binds to oestrogen receptors = more oestrogen-receptor complexes which are active transcription factors
- bind to promoter regions of genes that code for proteins stimulating cell division
- increases transcripition of genes increasing rate of cell division
suggest how drugs that have a similar structure to oestrogen help treat oestrogen receptor-positive breast cancers
- drugs bind to oestrogen receptors prventing binding of oestrogen
- fewer transcription factors bind to promoter regions of genes that stimulate the cell cycle
what can drugs do in cancer regarding epigentics
they can reverse epigenetic changes prventing uncontrolled cell division
define oncogene
a mutated protooncogene that results in uncontrollable cell division
describe how alterations to tumour suppressor genes can lead to the development of tumours
increased methylation
= mutations = tumour suppressor genes are not transcribed = uncontrollable cell division
define sequencing
the process used to determine the precise sequence of nucleotides in a length of DNA
what does whole genome sequencing involve
- cutting genome into smaller fragments
- fragments are individually sequenced
- computers align overlapping sections of DNA that reassemble the genome
why do we sequence DNA
- compare genomes of different species: determines evolutionary relationships + beneficial to medical research
- compare genomes of individuals: identifies differences for development of personalised medicine + study diseases
- research the proteins different genes code for
define the proteome
all the proteins a genome can code for
what is cellular proteome
all the proteins produced in a particular cell
what is complete proteome
all proteins produced in an organism
give 2 advantages of sequencing prokaryotes and single celled eukaryotes
cheap and easy
why is it cheap and easy to sequence the genome of simple organisms
- genomes are shorter + not associated w/ histone proteins
- prokaryotes do not possess non-coding regions of DNA
- single-celled eukaryotes have fewer genes in their genomes
state 2 uses of sequencing genomes of simple organisms
- proteome of pathogens can help scientists identify antigens = develop vaccinations against diseases
- organisms can withstand extreme environmental conditions and have potential uses in cleaning up pollutatns or manufacturing biofuels
why is determining the proteome of complex multicellular eukaryotes more difficult
- only a small percentage of DNA in human genome codes for proteins
- can be difficult to correctly identify coding regions of DNA from non-coding regions
- many of our genes have regulatory roles and affect expression of other genes
what are the roles of transcription factors in gene expression
it is responsible for allowing or preventing transcription to occur
how does RNA interference regulate gene expression
inhibits translation from occuring by destroying mRNA
what does recombinant DNA technology involve
transferring a fragment of DNA from one organism to another
what are organisms that contain tranferred DNA known as
- transgenic
- genetically modified organisms
what are the 3 ways to produce DNA fragments
- extract the gene from the DNA of a donor organism using enzymes [restriction endonucleases]
- using reverse transcriptase to synthesise a single strand of complementary DNA from the mRNA of a donor organism
- synthesising the gene artificially using nucleotides in a gene machine
what are restriction endonucleases
enzymes found in bacteria that act as a defense mechanism against bacteriophages [viruses that infect bacteria]
what do restriction endonucleases do
they cut viral genetic material at specific palindromic sequence known as recognition sequences and cut the DNA at these places
when restriction endonucleases cut the sugar phosphate backbone what can this create
- sticky ends: one DNA strand is longer than the other making it easier to insert into another organisms DNA
- blunt ends: both strands are cut at the same point
why are sticky ends preferred to blunt ends
sticky ends can form hydrogen bonds with complementary sequences that have been cut with the same restriction enzyme
blunt ends are harder to join as phosphodiester bonds are harder to form. nucleotides can be added to create sticky endsd
describe how reverse transcriptase can be used to isolate the desired gene
isolate mRNA then combine it with a reverse transcriptase enzymes and nucleotides to create a single strand of complementary DNA [cDNA]
DNA polymerase is then used to make ssingle strand of cDNA into a double stranded DNA which contains the desired code
where can you source reverse transcriptase enzymes
from retroviruses and catalyse the reaction which reverse transcription. mRNA is used as a template to make cDNA
why is it advantageous to use reverse transcriptase
easier for scientists to find the gene because specialised cells will make specific typs of mRNA, and mRNA do not contain introns
how does a gene machine synthesise DNA
- first nucleotide in sequence is fixed to some support
- nucleotides are added step by step in the correct order. protecting groups are added to ensure the sequence is linear and in the right order
- short sequences of DNA [oligonucleotides] are produced spproximately 20 nucleotides long
- once complete they are broken off from the support and protecting groups are removes
- longer DNA fragments can be made
how does reverse transcriptase help produce a gene copy from mRNA
creates cDNA
what are the advantages of using a gene machine instead of isolating genes from cells
faster, no introns present, more precise
describe one application of using reverse transcriptse in producing a desired gene
insulin being produced
what is polymerase chain reaction [PCR]
a common molecular biology technique used in most applications of gene technology
give an example of PCR used in the world
DNA profiling or genetic engineering
how can you describe pcr
in vitro method of DNA amplification
what is PCR used for
to produce large quantities of specific fragments of DNA/RNA from very small quantities, allowing for billions of identical copies within a few hours
what does each PCR reaction require
- target DNA/RNA being amplified
- primers that allow replication to start
- DNA polymerase, most commonly used is Taq polymerase [no denaturing at high temp]
- free nucleotides
- buffer solution
state the three stages of PCR
denaturation, annealing, elongation/extension
what happens in denaturation in PCR
dsDNA is heated to 95 deg, breaking hydrogen bonds that bond the two DNA strangs together
what happens in annnealing in PCR
temperature is decreased to approx 55 deg so that primers can anneal to ends of single strands of DNA
what happens in elongation/extension
temperature increased to 72 deg as this is the optimum temp for taq polymerase to build complimentary strands of DNA to produce new identical dsDNA
what are DNA probes
short single stranded pieces of DNA with a base sequence complementary to bases on target regions
labelled radioactively or fluorescently so that they can be identified
what is the function of DNA probes
to locate specific alleles of genes and to screen patients for heritable conditions, drug responses or health risks
state 3 ways to identify and diagnose heritable diseases
- labelled DNA probes + DNA hybridisation
- labelled DNA probes to screen patients for heritable conditions
- genetic counselling and personalised medicine
what is DNA hybridisation
binding of a single stranded DNA probe to a complementary single strand of DNA = forming hydrogen bonds/base pairs
explain how genetic screening can be used to locate specific alleles of genes
- extract DNA and amplify by PCR
- cut DNA at specific base sequences using restriction enzymes
- separate DNA fragments using gel electrophoresis
- transfer to a nylon membrane and treat to form single strands with exposed bases
- add labelled DNA probes which hybridise/bind with target alleles
- show bound probe, expose membrane to UV light = fluorescently labelled probe| autoradiography = radioactive probe
what is the role of a genetic counsellor
- explains the results of genetic screening including consequences of a disease
- discuss treatments available for genetic condition
- discuss lifestyle choices/precautions that may reduce risk of genetic condition developing
- explain probability of condition/alleles being passed onto offspring, enabling them to make informed decisions about having children
what is personalised medicine
- medicine tailored to an individuals genotype/DNA
- increase effectiveness of treatment
state arguments for screening individuals for genetically determined conditions and drug responses
- enable people to make lifestyle choices to reduce chance of diseases developing
- allows people to make informed decisions on having biological children
- allows use of personalised medicine = increase effectiveness of treatment
state arguments against screening individuals for genetically determined conditions and drug responses
- screening for incurable diseases/diseases which develop later in life which may lead to depression
- lead to discrimination by insurance companies/employers
- may cause undue stress if patient does not develop the disease
