LECTURE 5 Flashcards
DNMT controlled reaction?
Methylation of cytosine
What supplies the methyl group for the methylation of Cytosine
SAM-ch3
Methylation causes
deactivation of genes and a decreased gene expression. this is due to a reduced Transcription factors binding.
Methylated DNA is also?
more condensed =less accessible for Transcription factors
Methylation heritability ?
yes heritable, when DNA synthesis occurs, methylation each strand (hemimethylated)DNMTI enzyme will come and methylate the other strand
Requirement for methylation to occur
Short palindromic sequence
function of NP65
ensures correct methylation on the unmethylated strand of the hemimethylated DNA
Enzyme which controls new gene methylation?
DNMT3A and “3B
TET1-3 enzymes enable
demethylaiton
In meiosis methylation
characteristic
genome wide demethylation
paternal methylation rate is greater than the maternal
Methylation during development
Determine cell fate
- As cells migrate from the neural layer they receive different methylation signals
- deffuenbaiation into different cell types
theory to do with 5-Hydroxyl-methyl-cytosine?
Present in bacteria could it be to present in humans ?
Method to test presence of of 5-hm-cytosine in mammals
1- Took cells and some from the brain (purkinje)
2- Extract the DNA
3- Into single nucleotides (digest)
4- Thin layer chromatography (to separate them)
5- A T G C and another spot
did the same with bacteria and received data in which the unknown peak and the peak in the bacteria known to be 5HM-C aligned
What conclusion was lead to about why 5HMC is present in small volumes in the cell?
Intermediate In converting back into cytosine (5methylcytosine)
Conclusion which lead scientists to believe that 5hmc has a function role as well as being an intermediate ?
Mutation in MeCP2 gene are known to cause rent syndrome
MeCP2 function? Previously
Bind to 5-mC in order to promote chromatin condensing
MeCP2 also known to ?
Bind to 5HMC to cause the chromosome to be less condense and activation of genes and their expression.
Determining the locations of 5MC and 5HMC ?
Genetic map
Why can’t we use standard sequencing approaches?
they are undetected. resemble cytosine in PCR Reactions
Bisulphite sequencing?
Cytosine converted to uracil compare to original 5mc not converted PCR - Sanger or illumina sequencing
TAB-sequencing
Ability to distinguish between 5mc and 5hmc
- bisulfite sequencing
- Tet assisted: in which Glucose-Transferase adds a glucose group to HMC only
- TET enzyme does not work on glucose addition groups (5hmc) but does on mc to form carboxyl group.
- Carboxylated groups (5mc)are read as Thymines and glucose addition groups (hmc) are read as cytosine
- compare the bisulfites to the Tet assised, when there is a t instead of a C (in bisulfite only) presence of 5mc
