Topic 8 Flashcards
What are stem cells?
Unspecialised cells capable of self renewal and can differentiate into other types of cell
How a stem cell becomes specialised
Stimulus causes selective activation of genes
mRNA transcribed of active genes- translated into polypeptide
Proteins produce modify the cell permanently , determining the cells function
Totipotent
Occur in early mammalian embryos
Able to differentiate into EVERY type of cell
Pluripotent cells
Found in embryos
Able to differentiate into MOST cells
Multipotent
Found in mature mammals eg bone marrow
Able to differentiate into a select few types of cell
Unipotent
Found in mature mammals
Can differentiate into ONE type of cell
Eg cardiomyocytes cardiac muscle
IPS
Adult somatic cells
Specific protein transcriptional factors associated with pluripotency introduced
Causing the cell to express genes associated with pluripotency
Cells are cultured ( increase in number)
Epigenetics
Heritable changes in gene function without changes to the base sequence of DNA
Caused by environmental factors
Methylation
The addition of methyl groups to the cytosine DNA bases
Increases how tightly coiled the DNA-Histone complexes are
Prevents the binding of transcription factors
Inhibits transcription of the gene
Deacetlylation
Removal of acetyl groups from histones
Increases the positive charge of histones
Increases the attraction of histones to DNA
Increases how tightly coiled the DNA-histones complexes are
Prevents binding of transcriptional factors
Inhibits transcription
Treatment of cancer and epigentics
TREATMENT
Increased acetlyation or reduced methylation that increase expression of of tumour suppressor genes
Increased methylation or deacetlyation that reduce expression of oncogenes
DIAGNOSIS
Abnormal levels of acetly methyl groups be detected as sign of cancer
Possible role in treatment drugs to prevent/ encourage methylation acetlyation
What is recombinant DNA technology?
The transfer of DNA fragments from one organism/species to another
What are the 3 methods of isolating DNA fragments
Reverse transcriptase
Restriction endonuclease
Gene machine
How is restriction endonuclease used to produce DNA fragments?
Restriction endonuclease with a specific and complimentary active site to the recognition site of the target gene is introduced to an organism DNA
Bind to DNA at recognition site and cut it out producing staggered cuts - sticky ends
How is reverse transcriptase used to produce DNA fragments?
mRNA isolated from cell that produces large amounts of protein encoded for by desired gene
mRNA, free DNA nucleotides and reverse transcriptase
Produce single stand of complimentary DNA- cDNA
DNA polymerase added, produce complimentary secondary strand of DNA using cDNA as template
Benefits of using mRNA to produce DNA fragments rather than DNA
mRNA- no introns, some organisms such as bacteria cannot splice introns out
More mRNA than DNA, so more readily available
Vivo method of amplifying DNA fragments
Promotor and terminators added
Vector DNA cut with same restriction endonuclease as fragment DNA
Complimentary sticky ends allows complimentary base pairing
DNA ligase added to join adjacent nucleotides up phosphodiester bonds at sticky ends
Ice cold calcium chloride solution, vectors added, heat shock
Increase permeability of host cell’s membrane
Uptake vector
Said to be transformed
What is the purpose of the addition of promotor and terminator regions?
Tell RNA when to stop/ start transcription of the gene
Use of marker genes
Not all host cells will uptake vector
Not all vectors will uptake target gene
Marker genes such as those that code for fluorescence or antibiotic resistance allow us to identify transformed cells and the cells which have the desired gene
Why are DNA fragments able to be translated in host cells?
Genetic code is universal- same DNA triplets code for the same amino acids in all organisms
Universal mechanisms of translation and transcription
What are VNTRs? Where are they found?
Variable tangent repeats
In introns between genes
Describe the process of genetic fingerprinting step 1
Small sample of DNA Amplified by PCR Digested by restriction endonucleases to cut up into smaller fragments Gel electrophoresis Sample added to well in agar jelly Covered with buffer solution
Genetic fingerprinting step 2
Gel electrophoresis
Sample added to well in agar jelly
Covered with buffer solution
Electrical voltage applied
DNA, negatively charged, moves towards positive end
Agar creates resistance
Smaller pieces have less resistance, able to travel futher more quickly
Separates out different lengths of fragments (VNTR)
Genetic fingerprinting step 3
Alkaline solution added to make DNA single stranded
Labelled DNA probes added
DNA combined with probes
Washed to get rid of any excess
Genetic fingerprinting stage 4
Transformed onto nylon sheet
X-ray or UV dependant on type of probe used
Analysis
Position of DNA bands are compared
More Similarities the more closely related
Uses of genetic fingerprinting
Forensic science
Determine evolutionary relationships, how closely related?
Plant breeding- make sure that two plants are not too closely related
Medical diagnosis
PCR stage 1
Heat DNA to 95
Heat breaks hydrogen bonds between complementary bases, separating the two strands
PCR stage 2
Add primers
Add free DNA nucleotides
Cool to 55
allows primers to anneal to complimentary regions on DNA fragment which mark the start and end of a region which you wish to replicate
Need to have 2 different primers in PCR
one for each different strand ( top and bottom)
to mark beginning and / or ends of the part of DNA needed / for attachment of enzymes
as DNA runs anti-parallel but DNA polymerase can only function in one direction
PCR STAGE 3
Heat to 72, optimum temp for DNA ploymerase
Add DNA polymerase, which will join adjacent nucleotides together forming phosphodiester bonds
PCR last stage
repeat process many times
amount of DNA doubles in each cycle
What is the effect of RNA-i ?
RNA interference
silences a gene
prevents TRANSLATION of mRNA- polypeptide
Two types of RNA-i
si-RNA
double stranded
mi-RNA
single stranded hair-pin loop
How does RNA-i work?
single strand of is-RNA/ mi-RNA
incorporated into a RISC
binds to mRNA @ specific base sequence complimentary to its own
hydrolyses the mRNA, cutting it into fragments
preventing full transition of the mRNA- polypeptides amino acid sequence
Whole process of genetic fingerprinting
1 DNA extracted
2 DNA cut / hydrolysed into segments using restriction endonucleases;
3 must leave minisatellites / required core sequences intact;
4 DNA fragments separated using electrophoresis;
5 detail of process e.g. mixture put into wells on gel and electric
current passed through;
6 immerse gel in alkaline solution / two strands of DNA separated;
7 Southern blotting / cover with nylon / absorbent paper (to absorb DNA);
8 DNA fixed to nylon / membrane using uv light
9 radioactive marker / probe added (which is picked up by required
fragments) / complementary to minisatellites;
10 (areas with probe) identified using X-ray film / autoradiograph
What is the advantage of the enzyme used in the polymerase chain reaction being thermostable?
So do not denature
withstand range of temp from high of 95 and low of 55
What is a vector?
a carrier of DNA to another cell/ organism
Characteristics of a benign tumour
slow growing
well defined boards, cell adhesion sticks cells together
regular nuclei
easier to treat, surely usually successful
cells are well differentiated, retaining normal function
Characteristics of malignant tumours
fast growing
undefined boarders, able to metastasise- grow into other surrounding tissues
irregular darker nuclei
cells are poorly differentiated/ unspecialised
hard to treat- often require invasive methods like radiotherapy
why is it easier to determine the genome/ proteome of simple organisms?
simple= prokaryotic
therefore do not have introns and regulatory regions of DNA that do not code for amino acids
therefore less bases= easier to sequence genome
and only coding regions so all determine amino acids sequences of proteins= proteome
Usefulness of determining a simple organisms proteome
able to identify the amino acid sequences of antigens
produce antigens for use in vaccines
immunity against t pathogens
Uses of stem cells in medicine
replace cells damaged by illness or injury
creation of insulin
creation of new blood cells from bone marrow
drugs testing
Evaluating the use of stem cells to treat humans
embryos:
tiny balls of cells incapable of feeling pain
would otherwise be destroyed if not used for stem cells
potential for life- should have human rights
IPS
less chance of rejection- same antigens on cell surface of patient
cannot yet reliably reprogram cells- could lead to cancer
no destruction of potential life
human can give consent
