Gene Expression and DNA Technology Flashcards
what are stem cells?
- undifferentiated cells that can divide by mitosis and differentiate and specialise into different types of cells
what is a totipotent stem cell and where is it found?
- cells that can differentiate into any type of cell
- found 0for a limited time in early mammalian embryos
what is a pluripotent stem cell and where is it found?
- cells that can differentiate into almost any type of cell except placental cells
- found in embryos and develop from totipotent cells
what is a multipotent stem cell and where is it found?
- cells that can differentiate into a few, limited types of specialised cells
- found in mature mammals
what is a unipotent stem cell and where is it found?
- cells that can differentiate into one type of specialised cell
- found in mature mammals
what is an example of a unipotent stem cell?
- cardiomyocyte stem cells can only differentiate into heart muscle cells
what are induced pluripotent stem cells (iPS cells) ?
- type of stem cell produced from unipotent stem cells
- specific transcription factors are used to make a unipotent stem cell pluripotent by bringing about the expression of some genes and inhibiting others
why are iPS cells useful?
- they can develop into a wide range of different types of tissue which could potentially be used to treat people with certain diseases
what are transcription factors?
- proteins which control the expression or inhibition of certain genes by promoting or inhibiting the binding of RNA polymerase
what is a promotor region
- a region of DNA where transcription of a gene is initiated
how do transcription factors work?
- they move into the nucleus and attach to a promoter region close to the target genes that it affects
- part of each transcription factor is complementary in shape to a particular sequence of nucleotides in a promotor sequence = specificity
- the binding of the transcription factor to the promotor region either promotes or blocks the recruitment of RNA polymerase resulting in either gene expression or inhibition
- the expression of different genes results in different proteins being coded for resulting in specialised cells being produced
describe how oestrogen acts as an activator
- oestrogen = lipid soluble therefore can easily pass through cell membrane
- it binds specifically to a receptor protein that is part of a transcription factor
- this changes the shape of the transcription factor and allows it to bind specifically to the promoter sequence of a particular gene
- this allows RNA polymerase to attach to the gene and catalyse the transcription of the gene
- mRNA is then transcribed from the gene and translated into protein.
How does Endoxifen reduce the growth rate of breast tumours
- it has a similar shape to oestrogen
- Endoxifen binds to receptor on transcription factor preventing Oestrogen from binding
- therefore receptor not activated and so cannot attach to promotor region
- therefore transcription is not initiated
what is the rate of cell division controlled by?
- proto-oncogenes - code for proteins which stimulate cell division
- tumour suppressor genes- these genes code for protein that slow cell division
what causes rapid, uncontrolled cell division?
- a mutated proto-oncogene called an oncogene stimulates cells to divide too quickly = rapid cell division
- a mutation in a tumour suppressor gene. The tumour suppressor protein is not made or is non functional = rapid cell division
what are the differences between a malignant and benign tumour?
- benign tumours grow slower than malignant tumours
- benign tumours don’t break off and spread to other tissues (non cancerous) whereas malignant tumours do (cancerous)
- the cells in benign tumours often remain differentiated whereas cells in malignant tumours often become undifferentiated
- in benign tumours , cell nucleus is relatively normal in comparison to malignant tumours where the nucleus is larger and darker
define epigenetics?
changes in gene function without changes in the DNA base sequence
what may cause epigenetic changes to gene function
aspects of environment - e.g. stress, diet, exposure to toxins
what is increased methylation of DNA and its effect
- methyl group attaches to DNA sequence of a gene (CpG site)
- increased methylation inhibits transcription by preventing the binding of transcription factors to the promotor sequence so that the gene is not expressed
what is decreased acetylation of Histones and its effect
- histones are less acetylated so the chromatin is more condensensed.
- this results in transcription being inhibited as the genes are not accessible to transcription factors
how can hypermethylation of tumour-suppressor genes cause cancer
- hypermethylation of tumour suppressor genes = non-transcription of the genes therefore proteins that slow cell division are not produced = rapid uncontrollable cell division
how can hypomethylation of proto-oncogenes cause cancer
- hypomethylation of proto-oncogenes so they are transcribed more than usual = increase in cell-division stimulating proteins = rapid uncontrollable cell division
what is recombinant DNA technology
- the transfer of fragments of DNA from one organism to another
why does recombinant DNA technology work?
- the genetic code is universal
- the transcription mechanism is universal
- the translation mechanism is universal
what is a transgenic organism
- an organism that has received transferred DNA
what methods are used to obtain the fragments of DNA (genes) used in RDT
- Using Reverse Transcriptase
- Using a Gene Machine
- Using Restriction Endonucleases
Describe how reverse transcriptase is used to obtain DNA fragments used in RDT
- the mRNA transcribed from the gene is used as a template
- mRNA is mixed with free DNA nucleotides and reverse transcriptase
- the free DNA nucleotides line up next to their complementary bases on the mRNA template
- reverse transcriptase then joins the DNA nucleotides together to produce the required fragment of DNA called complementary DNA (cDNA)
- double stranded DNA is produced from this cDNA using DNA nucleotides and DNA polymerase
Describe how restrictive endonuclease is used to obtain DNA fragments used in RDT
- restrictive endonucleases hydrolyse the DNA at specific recognition sites cutting out the required DNA fragment.
Describe how a gene machine is used to obtain DNA fragments used in RDT
- enables the production of the fragment of DNA without needing pre-existing DNA or mRNA as a template
- amino acid sequence is used as a template to determine the sequence of DNA nucleotides for a specific gene
what are the advantages of using reverse transcriptase for RDT
- mRNA is present in large amounts in protein-making cells
- Absence of Introns
what are the advantages of using a gene machine for RDT
- the process is automated
- faster than other methods as its not enzyme controlled
when must introns not be present during RDT
- if the source of a gene being transferred is eukaryotic and the intended recipient is prokaryotic
describe how recombinant plasmids are made?
- a plasmid is cut using the same restriction endonucleases used to cut the gene
- the plasmid DNA and foreign DNA join by complementary base pairing due to their complementary sticky ends
- the enzyme ligase is used to form phosphodiester bonds
what sections of the DNA must be added to the fragment of DNA during RDT for successful transcription of the transferred genes
- promotor regions which initiate transcription of the gene by promoting the binding of RNA polymerase
- Terminator regions which mark the end of a gene and trigger the release if the mRNA transcribed
how can the amplification (increase in number via replication) of the fragments of DNA be achieved?
- in vivo - the copies are made inside a living organism
- in vitro - the copies are made outside of a living organism
what are sticky ends and blunt ends?
sticky ends - DNA hydrolysed at different locations resulting in exposed bases
blunt ends - DNA hydrolysed at the same location resulting in no exposed bases
explain how siRNA prevents translation
- longer double stranded molecules of RNA are hydrolysed into shorter molecules
- RNA becomes single stranded siRNA
- siRNA binds to an enzyme that hydrolyses mRNA
- the siRNA binds to a specific molecule of mRNA by complementary base pairing. Thus siRNA guides the hydrolytic enzyme to a target molecule of mRNA
- the enzyme hydrolyses the mRNA molecule preventing the translation of mRNA into protein
where do enzymes hydrolyse RNA/DNA
- at specific recognition sequences/sites
describe the process of Gel Electrophoresis
- DNA samples are placed in separate wells at the top of the gel
- the DNA fragments in each sample are separated according to size (smaller moves further and faster)
describe the process of genetic fingerprinting
- PCR used to amplify the sample DNA and restriction endonucleases are used to cut amplified DNA into fragments
- DNA fragments are then separated using Gel Electrophoresis
- DNA fragments are then treated (using alkali) to form single strands and then are transferred to a nylon membrane
- radioactively labelled DNA probes are added which are complementary to the VNTRs and therefore bind to them via DNA hybridisation
- nylon membrane is placed on X-ray or photographic film and the position of the radioactively labelled fragments is revealed therefore obtaining the genetic fingerprint
what is a DNA ladder
a lane of DNA fragments of known sizes used to calculate the size of DNA fragments in the unknown samples
what are DNA primers
- short single stranded molecules of DNA which provide a starting sequence for DNA polymerase
why are DNA primers important
- DNA polymerase cannot begin at a single stranded starting point
what are DNA probes
- short single stranded molecules of DNA that are radioactively or fluorescently labelled that are used to identify or locate known sequences of DNA
what is DNA hybridisation
- a process in which two complementary single stranded DNA molecules bond together to form a double stranded molecule
describe the process of the Polymerase Chain Reaction
stage 1
- sample DNA, DNA primers, free DNA nucleotides and DNA polymerase are mixed together and heated at 95°C for 5 minutes breaking the hydrogen bonds in DNA
stage 2
- mixture is cooled to 50-60°C allowing the primers to join to their specific complementary target sequence
- free DNA nucleotides align to the DNA strands by complementary base pairing
stage 3
- the temperature is increased to 72°C=optimum for DNA polymerase
- enzyme joins the individual nucleotides of a strand together to form a new complementary strand
how to work out number of DNA molecules formed via PCR
2ⁿ , n = number of cycles
what are vectors
- how genes are transferred into different cells
what are the two types of vectors
- plasmids
- bacteriophages
define transformation
- the taking up of recombinant plasmids by a culture of bacteria
why are vectors not guaranteed to work
- the cells may not take up the vector at all
- the plasmid may have joined back together without the gene being taken up
- the DNA fragment may have annealed to itself
what are VNTRs ( variable number tandem repeats )
- many repetitive, non coding sequences of nucleotide bases
How do VNTRs correlate with how closely an organism is related to another
- the more related two organisms are , the more similar their VNTRs will be
name 3 uses of genetic fingerprinting
- forensic science - can be used to compare DNA samples form the crime scene and a suspect
- medical diagnosis - certain diseases involve unique patterns of several alleles and can be identified more readily by genetic fingerprinting
- determining genetic relationships
- the closer related organisms are, the more similar their VNTRs will be
what is gene therapy
- the introduction of functional copies of an allele into an organism which possesses defective alleles of the same gene
what is the difference between somatic and germline therapy
somatic therapy - not heritable as it is not in the gametes
germline therapy - heritable as it is in the gametes (currently illegal)
what is DNA sequencing
- the determining of the sequence of DNA nucleotide bases in an organism (genome)
why is DNA sequencing important?
- can be used to identify the proteins that the DNA codes for (proteome) which can help identify potential antigens for use in a vaccine
why is the genome not always reliable for finding out the proteome
- in more complex organisms the DNA base sequence may contain introns or regulatory genes that don’t code for proteins
what are marker genes
- genes that enable successfully transformed bacteria or eukaryotic cells to be detected and isolated
one example of a marker gene is…
- GFP gene which codes for the production of a green fluorescent protein causing successfully transformed cells to be identified as they fluoresce when viewed with UV light under a microscope
what are the pros of the use of Recombinant DNA Technology
- it can be used to reduce famine and malnutrition by developing genetically modified plants or animals which produce high yields and are resistant to disease
- used to produce vaccines and drugs
- used to treat genetic diseases by gene therapy
what are the cons of the use of Recombinant DNA Technology
- can lead to possible transfer of foreign genes to non-target organisms
- it is an irreversible process
- there are ethical considerations with regards to permanently altering the genome of animals
- long term ecological and evolutionary consequences are unknown
