3.8 The Control Of Gene Expression Flashcards
What is inversion mutation?
A section of bases detach from the DNA sequence but rejoin inverted (back to front) = different as coded in this region
E.g TTC = CTT
What is translocation mutation?
A section of bases on one chromosome detached and attached onto a different chromosome which causes sig. alterations e.g to phenotype
What is duplication mutation?
Section of bases are duplicated in the DNA sequence
What is a stem cell?
Undifferentiated cells that can continually divide and become specialised into any cell type
What are totipotent stem cells?
•can divide and produce any type of body cell
•during development they only translate part of their DNA causing cell specialisation
•occur for limited time in early mammalian embryos
What are pluripotent stem cells? (Problems with its use in research?)
•can divide into most cell types
•found in embryo
•being used in research to treat disease
Issues:
•continually divide to create tumours
•ethically is it right to make a therapeutic clone and then destroy embryo?
What are multipotent stem cells?
•can differentiate into a limited no. of cells
•found in e.g bone marrow in adults
What are unipotent stem cells?
•can only differentiate into one type of cell e.g formation of cardiomyocytes
What is an induced pluripotent stem cell? (IPS) (advantages?)
A pluripotent stem cell produced from an adult somatic (body) cell that are unipotent by using appropriate protein transcription factors that will switch on the genes switched off to make the cell specialised
Ad:
•repair and replace damaged tissues
•less risk of rejection
•don’t have to destroy embryos= unethical
How are plant stem cells different?
•plants retain many totipotent cells
•this allows for tissue cultures = cloned plants can form from a few cells
•meristems in the tip of roots/shoots
What is a transcription factor?
Molecules that can move from the cytoplasm to the DNA and bind to a specific gene (promoter region), leading to or blocking mRNA production (activate or inhibit RNA polymerase)
What is the control of transcription?
•transcription factor protein will bind to the promoter region (from cytoplasm to DNA) which will stimulate RNA polymerase to transcribe the gene (form mRNA)
•when a gene is not being expressed,site in the transcription factor that binds to DNA is blocked by an inhibitor which prevents transcription
What is the role of oestrogen in initiating transcription?
1.oestrogen diffuses through the cell membrane (lipid soluble)
2.oestrogen diffuses through the nuclear membrane
3.oestrogen attaches to oestrogen receptors (ERalpha) which was initially inhibited as part of a protein complex and activates ERalpha
4.ERalpha changes shape (complementary) and leaves the protein complex to bind to the promoter region of DNA and RNA polymerase is activated which will begin transcription of a gene
What is meant by epigenetics?
heritable change in the gene function without changing the DNA base sequence = can be caused by changes in the environment and can inhibit transcription
What is methylation of DNA?
methyl groups are attached to DNA which will cause chromatin structure to be very tightly coiled so transcriptional factors cannot bind and transcription will be inhibited
What is acetylation of histones?
acetyl groups binds to histones which causes DNA chromatin structure to be more loosely packed so transcriptional factors can bind to DNA
How can abnormal methylation lead to cancer?
•tumour suppressor genes could become hypermethylated (more methyl groups added to DNA) so transcription of this gene is inhibited (these genes won’t produce the proteins that slow cell division/cell death during DNA copying error)
•oncogenes could be hypomethylated (less methyl groups) so gene is permanently switched on (continual triggering of mitosis)
What occurs during RNA interference?
•
•RNA dependent RNA polymerases (RDRs) catalyse formation of double stranded RNA (dsRNA)
•dsRNA is hydrolysed to siRNA which will form a protein complex
•this protein complex will bind to mRNA via complementary base pairing
•mRNA will be cut up into pieces so it cannot be translated to make the gene product
What is a benign tumour?
•grow large but slow rate
•produce adhesion molecules so stick together and to specific tissues (localised)
•often in capsule so compact and easy removal
What is a malignant tumour?
•cancerous and grow large rapidly/cell can become unspecialised again
•metastasis can occur= tumour can break off and spread to form secondary tumours
•can grow projections and develop own blood supply
How can mutation in proto-oncogenes cause cancer?
•oncogenes (mutated) can form protein that is involved in initiation of DNA replication
•mutation can result in process permanently activated to make cells divide continually
How can mutation in tumour suppressor gene cause cancer?
•tumour suppressor genes produce proteins that slow down cell division and cause cell death if DNA copying error
•mutation will prevent protein production so cell division continues/mutated cells not destroyed
How can increased oestrogen concentration cause cancer?
•oestrogen can activate gene by initiating transcription = could permanently turn proto-oncogene on and activate continual cell division
•excess of oestrogen in breast tissue after menopause causing tumour growth
What is occurring in the methods used to sequence genomes?
•continually being improved
•now automated
How are DNA fragments formed using reverse transcriptase?
1.identify cell that produces protein of interest
2.reverse transcriptase is used to convert the mRNA sequence (template) into a complementary DNA sequence by adding DNA nucleotides (cDNA) - no introns so can do genetic engineering with prokaryotes
3.DNA polymerase uses cDNA as a template to form double stranded DNA
4.copy of gene for protein is formed
How are DNA fragments formed using restriction endonucleases?
•restriction enzymes have active site complementary to range of DNA base sequences (recognition sequences) so each cuts DNA at specific location
•some cut at same location so blunt ends
•some cut to create staggered ends and exposed DNA bases (sticky ends=can join to DNA with complementary base pairs)
How are DNA fragments formed using a gene machine?
•examine protein and identify specific amino acid sequence = mRNA and DNA sequence worked out
•enter into computer (checks for biosafety/ethical)
•forms DNA fragments
What occurs when DNA is inserted into a vector?
•plasmid cut using same restriction endonuclease used to isolate human gene= complementary sticky ends
•DNA ligase used to join open plasmid with human DNA = recombinant plasmid
•promoter region added to allow transcription
•terminator region added to end of gene so RNA polymerase can detach and stop transcription (one gene at a time)
What is a vector?
Something to carry desired gene into host cell e.g plasmid
How do we insert the vector into a host cell?
Transformation=
•host cells mixed with Ca2+ and heat shocked so this increases permeability of cell membrane and vector taken up
•if plant cell, 1st break cell wall
Why won’t all host cells take up a recombinant plasmid?
•recombinant plasmid doesn’t get inside the cell
•the plasmid rejoins before the DNA fragment entered
•DNA fragment sticks to itself, rather than plasmid
How does replica plating occur?
•copy of bacteria colonies made using a velvet surface = allows transfer of bacteria as it allows easy sticking
How do antibiotic marker genes work?
•2 plasmids = both resistant to ampicillin/recombinant plasmids killed by tetracycline
1)add ampicillin which will identify bacteria that have taken up plasmid/without plasmid killed
2)add tetracycline= identifies recombinant plasmid as they will die as gene for resistance has been destroyed/can’t be expressed
3)use replica plate to identify the colonies
What happens once recombinant plasmid colonies have been identified?
•bacteria cultured on growth medium (agar) = O2, glucose, moisture, warmth and aa
How is a control group formed?
•provide placebo or inactive drug
•otherwise treated exactly the same way
What is the method for PCR?
1.95•C= break hydrogen bonds and split DNA into single strands/separate strands (denaturing)
2.55•C=primers can attach at the ends of the strands (annealing) and will prevent DNA strands rejoining
3.72•C= DNA polymerase attaches free nucleotides and makes strand to align next to each template (synthesis) = heat resistant so doesn’t get denatured
What are advantages of PCR?
•automated- so more efficient
•rapid- many copies are made where doubling of DNA at each cycle
•doesn’t need living cells- quicker and less complex techniques
What is electrophoresis?
•separates DNA fragments by supplying voltage
•the -ve charged DNA fragments move towards the +ve anode
•they will move at different rates and distances based on size, e.g the smaller the fragment, the faster it moves
What are DNA probes?
Short, single stranded section of DNA that has radioactive or fluorescent label, that will complementary base pair to a target gene
What are the for and against of GM crops?
For=
•save lives as more food
•tested but no toxicity found
•cheaper/ more profit for farmer
Against=
•public lack knowledge and may disprove GM crops
•reluctant to eat it as different
•GM seeds/genes may escape into the wild and cross pollinate with other species so affect ecosystem = super weeds
•may have negative long term effects on human body/allergic
•genes can mutate and become toxic
Why is it easier to obtain genes from mRNA, rather than DNA from cell?
•mRNA is present in larger amounts
•mRNA has no introns
•mRNA codes for a single protein
How is it possible for an organism getting a desired gene from another organism to synthesise its protein?
•genetic code is universal
•so it’s DNA can be transcribed and can be translated
How can a recombinant plasmid be formed and enter into host cell?
•restriction endonuclease used to cut desired gene at recognition site= forms sticky ends
•use same enzyme to cut plasmid to form complementary sticky ends
•DNA ligase used to join the DNA and plasmid= recombinant
•Ca2+ and heat shock to enter into cell. If host cell = plant you have to remove cell wall
•marker genes used to identify which host cells have plasmid and recombinant plasmid
Why does PCR produce a limited no. of DNA? (Curve levels off)
•nucleotides used up so no complementary strands can form
•primers used up so cannot start synthesis of complementary strand
Why might 2 different primers be needed in some PCR?
Sequences at the ends of the target sequences are different (one at start and one at end)
How can DNA probe be used to identify cells that contain a specific allele?
•extract DNA and add restriction endonuclease to cut the DNA at specific base sequences
•separate DNA fragments using gel electrophoresis
•treat DNA to form single strands
•probe will bind to specific allele
•use autoradiography to show bound probe
How is a lot of DNA probe formed for a mutated gene?
1.find the order of nucleotides in the mutated gene via DNA sequencing
2.produce a fragment of DNA with complementary bases to mutated gene
3.label fragment = DNA probe
4.amplify the probe via PCR
What are DNA probes used for?
•screen patients for heritable conditions (genetic counselling)
•drug responses and health risks (personalised medicine)
What is Gene Therapy and what are the 2 ways?
•Introducing genes into an individual with abnormal genes, so they can produce functional proteins
1.germ line gene therapy=
•replacing/adding genes into fertilised eggs so all cells will have it = mitosis (moral and social issues)
2.somatic cell gene therapy=
•target affected tissues, but as cells replace themselves it needs to be repeated = limited success
What are VNTRs and what can they be used to determine?
•short, repeating sequences of DNA in non-coding regions
•in every individual, they vary in length + no. of repeats so probability of having the same VNTRs is very low (unless identical twins)
What is the process of genetic fingerprinting?
1.DNA is cut + amplify by PCR
2.Restriction endonuclease is used to cut around the VNTRs
3.Fragments are placed on gel electrophoresis (mix put in wells and electric current passed) to separate based on different lengths/mass of VNTRs
4.DNA made is single stranded by immersing the gel in alkaline solution
5.southern blotting is used to transfer it to a nylon membrane
6.radioactive probe is added to hybridise VNTRs which can be seen with X-ray and can be visualised using autoradiography
7.pattern is unique to every individual
What is genetic fingerprinting used for?
forensic science= crime scene
•for medical diagnosis
•ensure animals and plants are not closely related before breeding = prevent passing harmful genetic conditions
•paternity test
How can a mutation that doesn’t occur in the coding gene occur?
•mutation in the promoter for the transcription factor
•gene continues or stops being transcribed
