Background do they remain free from methylation .the several

Background of the problem  

CpG islands are the region with a high
frequency of CpG site  with at least 200
base pair , a gc percent greater than 50% 
and observed to expected ratio greater than 60% .They are found
exclusively on the 5′ end of the gene (Bird 1986,1987) in mouse and man these
gene include all the sequenced housekeeping gene and many gene that show tissue
restricted pattern of expression (Antequera and Bird 1993 ) . Human genome contain
30000 CpG island .CGIs associated with promoter 
alayws remain unmethylated , many of the9000 cpg island lying with in
the gene become methylated during the development and differentiation . The CpG
dinucleotide is the target for mammalian methyltransferase  that 
methylate the cytosine residue at the carbon 5’position .as CpG island
are rich in these dinucleotide how do they remain free from methylation .the
several model has been proposed the simplest of which is that cpg island is a
poor substrate for methyltransferase . in support of this idea it has been
shown that cpg rich sequence are methylated insufficiently in vitro (carotti et
all 1989) . A second model propose that CpG island  subject to de novo methylation but the
modification is proposed by a island specific demethylating activity (frank et
all 1991) .The model is based on the finding that the partially methylated CpG
island lose methylation when introduced into fertilised mouse egg or embryonic
cell (scyzf et all 1990) A third model propose that steric hindrance by the
protein factor is responsible for excluding the methyltransferase from the CpG
island (Bird 1986)  .

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Experiment to address the problem

In order to investigate the basis of immunity to
methylation in a specific CpG island a housekeeping gene mouse aprt is chosen .
its cpg island occupy a typical location ,extending from the- 600 bp from the
promoter downstream into the transcribed region . The bidirectional HTF9
promoter and the PGK promoter are atypical inthese respect as the promoter
region is centrally located with in the island each case.

Distribution of the Cpg island: to accurately position the CpG island
with respect to methylation  the
structrure of mouse aprt gene and the distribution of CpG dinucleotide was
plotted using the published dna sequence (dush et all 1985)

To analyse the boundaries of CpG island with respect to methylation ; Bisulphite
modification technique was used (frommer et all ) the procedure change the
nonmethylated cytosine into urasil whileleaving the methylated cytosine
unchanged .subsequent PCR amplification and sequencing reveal the methylated
base .A large region of the aprt gene including the whole CpG island  was analysed in this way using liver DNA as
the starting material

Invivofootprinting across the CpG island in order to understand
whether protein factor are bound in the CpG 
island . To dectet protein DNA interaction ligation mediated polymerase
chain reaction technique is used to study the aprt promoter in F9 embryonal
carcinoma .A pair of DNA primer for LMPCR that spanned the island sequence was
used . By compairing the aprt (G ladder ) produced by DMS treated three
footprint were obtained which coincoides with three GC box identified in the
aprt promoter (Dush et all) . The GC box is the consensus binding site for the
obiquitous transcription factor sp1 ( kadonga et all)

Transcription factor are not the only protein expected to bind CpG  island nucleosome may also present . T o
determine nucleosome position F9 nuclei were incubated with micrococcal
nuclease .

Expected outcome and alternative strategy .
CpG island methylation generaly leads to gene silencing

From the above experiment it is clear that
GC box in the aprt promter which is binding site for sp1 play an important role
in preventing CpG island methylation .probably sp1 act as a potential binding
site for a demethylating enzyme(still not discovered ) which remove the
methylgroup from the gene that need to be expressed. Hypomethylation or
hypermethylation generaly leads to malignancy biological system must have
checking machinery that ensure that CpG island in gene correctly methylated.


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