transposon methylation and gene silencing - (Apr/29/2006 )

[font=Times New Roman]Hello,
I am working with a fungal avirulence gene which is between a transposon and a teliomere, very unconfortable location, so gene is exteremly hypervariable. Recently I have found out that transposon methylation may cause to silencing of neigbouring genes. I would like to search for this possibilty for this case. Now my question is... (may sound very ignorant, I have no experince in epigenetics... ) How can I understand If transposon is methylated , is there a way to design specific primers, If you could give me some ideas I will be really thankful,
Niki

it is certainly possible if you have unique sequences flanking the transposon so primers for a PCR can be selected.

Nick

Fungi!!

without actually fully picking primers for you, I think it would be better that I point you in the right directions.

So have you got a sequence of a clone that contains both elements from which you can then use repeatmasker to identify repetitive elements so you can then pick bisulfite primers for?

So I would try and find a genome browser for your organism Magnaporthe grisea and then from there mask repeats with RepeatMasker. Then you can feed the sequences through to something like Perlprimer or Methprimer from which primers will be selected for methylation analysis.

See how you go and if you have any problems, yell out!

Nick

Fungi!!

without actually fully picking primers for you, I think it would be better that I point you in the right directions.

So have you got a sequence of a clone that contains both elements from which you can then use repeatmasker to identify repetitive elements so you can then pick bisulfite primers for?

So I would try and find a genome browser for your organism Magnaporthe grisea and then from there mask repeats with RepeatMasker. Then you can feed the sequences through to something like Perlprimer or Methprimer from which primers will be selected for methylation analysis.

See how you go and if you have any problems, yell out!

Nick

Hi,
I got these results, lower case letters should be the repeat sequences, Now How can I design a primer to undertstand if transposon is methylated ?
Thanks Methylnick
Fungi










ACGTGGTTCATCAGGACCCCGGACATATCAGGATCCCGGACATTTTTCAA
CCCAGTTTCATCCTTTATTTTCCACACGTGTTCTTTCCCCCAACAGTAAT
GGAATCATCAAATTGCGAAGCGCGAATCATTCTTGCTCTAAATGAGCTCC
GATCAAGCAAAAAGAAAAGCATACGAAAGgttgcattaatatataatgtc
ccaaaatcgacactacacgacagaataaacggcatcgcttctttggccaa
tcgtcggccaggcaaccaaaagctaacggaaagggaagaggaagtaattg
tccagtatatactggacctggattcccgagggtttccagcccagatcgct
gatgtggccgcaatggccgatcatcttctcgctgcgcgggacgcgcgacc
ggtcggcaagcaatgggcttatcgcttcgtacaacgacgcacagaattaa
aaacgcgtttttctcgcgcttacgatttccaaagagctctttgcgaagat
cctgacgcgttaaacgcgtggtttcaattggtggccaatatgagggccaa
atatggcatccaagactgcgacatgtacaacttcgacgaaaccggcttta
tgatgggccagatttgcgctggaatggtcgtaactgggtccgaaagacgc
ggaagaaggaaaaaagtacagcctggcaatcgagaatgggcgactgcaat
ttgttgcatcagtggcgacggttacgatatacccccgtatatcatcgtca
aaggcttttaccatttgtccaattggtatacagaaggcggtcttccggac
acgtggcgcctcaaacccaccgttaatggatggaccgacaacgagactgg
cctcgactgggttcagcatttcgacaaccatacaaaatcgcggacaaaag
gcgtatatcggatgttagtgctcgatgggcacggcagtcatcgatcccct
gaattcgagggctattgcaaaaatcacaatattattccactttacctgcc
tgctcattcatctcatttaacccagccacttgatgtcggagtgtttaacg
tccttaagcgggcgtacggtcaaaaaattaacgactttatccgggcccac
atcactaacatcagcaaagtcgacttctttttggcttttgcagcggctta
caaaaagtcaatgacaaaagaaaatatggccgggggttttcgaggggcgg
gaattatcccccatagcccggaaatggtcatatCTAAGCTGGATGTTAGG
CTACGGACGCCGTCACCTAAAGAGCTTGATTTTTCCAGCACCGAAACCTG
GGTCTCTCAAACACCGCACAACCCGACAGAAGCCGTTAATCAATCTACCC
TTGTTAAAAGTCGAATCAACTGTCATCAGGGAAGCTCGCCAACGCCTATT
TTTAATGCTGTAAAGCAGCTAGCAAAAGGGTTGGAGTCGATTGCTCATCG
AACCACACTTTTGGAAGCGGAGAACCACAGCCTTCGGAAGGCCAACGAAG
CGCTTAGCAAGCGGCGCAGGGCTCAAAAAACACGTATCCGCGAAGGAGGG
TCATTTACCATACAAGAGGGTCAGAATTTGCTTCAATCAAGTGGTGCCGA
TGGTCTAATATACGAAAAGAAAGATGAAAATGGGGAGGGGAGTAGTGCGC
AGCCGGCGACTAAACGACGTTGCGGCAACTGCGGTAAACCTGGACATAAT
GCACGCACCTGTCAGGAGGATGCAGAAATGTCTGATGTACATATATCCGA
TTGCATTGAGGTAAATTGAACAACGCTGGCGTTGCAATTGAAATTGGAAG
TAGTATTGTGCAGGAAAAGTGTCCGGGGTCCTGATATGTCCGGGGTCCTG
ATGAACCACGT



file name: RM2sequpload_1146455647
sequences: 1
total length: 1861 bp (1861 bp excl N/X-runs)
GC level: 48.41 %
bases masked: 1054 bp ( 56.64 %)
=====================================================
number of length percentage
elements occupied of sequence
-----------------------------------------------------
Retroelements 0 0 bp 0.00 %
SINEs: 0 0 bp 0.00 %
Penelope 0 0 bp 0.00 %
LINEs: 0 0 bp 0.00 %
CRE/SLACS 0 0 bp 0.00 %
L2/CR1/Rex 0 0 bp 0.00 %
R1/LOA/Jockey 0 0 bp 0.00 %
R2/R4/NeSL 0 0 bp 0.00 %
RTE/Bov-B 0 0 bp 0.00 %
L1/CIN4 0 0 bp 0.00 %
LTR elements: 0 0 bp 0.00 %
BEL/Pao 0 0 bp 0.00 %
Ty1/Copia 0 0 bp 0.00 %
Gypsy/DIRS1 0 0 bp 0.00 %
Retroviral 0 0 bp 0.00 %

DNA transposons 1 1054 bp 56.64 %
hobo-Activator 0 0 bp 0.00 %
Tc1-IS630-Pogo 0 0 bp 0.00 %
En-Spm 0 0 bp 0.00 %
MuDR-IS905 0 0 bp 0.00 %
PiggyBac 0 0 bp 0.00 %
Tourist/Harbinger 0 0 bp 0.00 %
Other (Mirage, 0 0 bp 0.00 %
P-element, Transib)

Rolling-circles 0 0 bp 0.00 %

Unclassified: 0 0 bp 0.00 %

Total interspersed repeats: 1054 bp 56.64 %


Small RNA: 0 0 bp 0.00 %

Satellites: 0 0 bp 0.00 %
Simple repeats: 0 0 bp 0.00 %
Low complexity: 0 0 bp 0.00 %
==================================================

that is a start Niki,

okay in repeat masker there is an option to mask repeats with N check that option and re-run the program so you get something like this:

ACGTGGTTCATCAGGACCCCGGACATATCAGGATCCCGGACATTTTTCAA
CCCAGTTTCATCCTTTATTTTCCACACGTGTTCTTTCCCCCAACAGTAAT
GGAATCATCAAATTGCGAAGCGCGAATCATTCTTGCTCTAAATGAGCTCC
GATCAAGCAAAAAGAAAAGCATACGAAAGnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
nnnnnnnnn--transposon of interest --nnnnnnnnnnnnnnnnnnnnnnnnnn
CTAAGCTGGATGTTAGG
CTACGGACGCCGTCACCTAAAGAGCTTGATTTTTCCAGCACCGAAACCTG
GGTCTCTCAAACACCGCACAACCCGACAGAAGCCGTTAATCAATCTACCC
TTGTTAAAAGTCGAATCAACTGTCATCAGGGAAGCTCGCCAACGCCTATT
TTTAATGCTGTAAAGCAGCTAGCAAAAGGGTTGGAGTCGATTGCTCATCG
AACCACACTTTTGGAAGCGGAGAACCACAGCCTTCGGAAGGCCAACGAAG
CGCTTAGCAAGCGGCGCAGGGCTCAAAAAACACGTATCCGCGAAGGAGGG
TCATTTACCATACAAGAGGGTCAGAATTTGCTTCAATCAAGTGGTGCCGA
TGGTCTAATATACGAAAAGAAAGATGAAAATGGGGAGGGGAGTAGTGCGC
AGCCGGCGACTAAACGACGTTGCGGCAACTGCGGTAAACCTGGACATAAT
GCACGCACCTGTCAGGAGGATGCAGAAATGTCTGATGTACATATATCCGA
TTGCATTGAGGTAAATTGAACAACGCTGGCGTTGCAATTGAAATTGGAAG
TAGTATTGTGCAGGAAAAGTGTCCGGGGTCCTGATATGTCCGGGGTCCTG
ATGAACCACGT

you can then feed this sequence into perlprimer to pick BSP primers, or methprimer for both BSP and <SP primers, i trust perlprimer more but you can pick several to do bisulfite pcr and sequencing, you could of course design a southern blot assay to detect methylation by methylation sensitive restriction digest and probing with a unique flanking sequence, does the same trick as well

Nick

Hi Nick,
I followed all the steps and pick this primer

Forward Primer Pos Len Tm Reverse Primer Pos Len Tm Amp dG
TAATTGTTATTAGGGAAGTTYGTTA 1366 25 56.56 CTCCTAACAAATRCATRCATTATATCCAAA 1717 30 59.91 351 0


What should be the conditions for a bisulphite PCR, what should I do different than regular PCR?

and my other question how does methly sensitive restriction digestion works I already cloned this gene I can label it with HPR but how can I understand whether it is methylated?

I am affraid I will be kicked out of this forum after all this questions

Niki,

for bisulfite PCR it's best to have hemi or fully nested primer sets so two rounds of PCR can be performed, this will also enhance specificity.

PCR conditions are very similar to normal PCRs, i typically perform 30 cycles for each round.

Methylation sensitive restriction digests enable you to detect DNA methylation if you use a methyl-sensitive enzyme and a methyl insensitive isochizhemer on gDNA transfer onto a membrane and probe with a unique DNA probe that flanks your transposon of interest. If it is methylated then you will see less bands in the methyl-sensitive lane when compared with the methyl-insensitive lane. Thus you are able to "infer" DNA methylation.

Bisulfite is more superior as you are able to measure DNA methylation at every potential methylation site, ie: you can see which parts of your transposon, if any, are methylated or not.

Good luck, and you won't be kick out of this forum, it's for questions like these!!!

Thanks Nick,
Hope, can make it work