Question

You are using a ChIP assay to study the positioning of a DNA-binding transcription factor

(Dbp1p) on transcribed genes. You fragment DNA from wild type cells and immunoprecipitate

Dbp1p with an antibody to it. Then you PCR amplify the DNA associated with Dbp1p using

different sets of primers for a particular gene (G3PD). One set of primers was specific for the

TATA box region of this gene, (lane 1) another pair of primers amplified the 3’ end of the open

reading frame for G3PD(lane 2); and a 3rd set of primers amplified the region just beyond the

polyadenylation site downstream of the open reading frame (lane 3). PCR products from

chromatin immunoprecipitation (right gel below) were compared to PCR reactions using input

chromatin without immunoprecipitating (left gel below). You also included a PCR reaction with

primers specific for a nontranscribed region (lowest band on all the gels below). Results are

shown.

A) Using a standard line drawing, show the G3PD gene (TATA, Trxn start, AUG, Stop

codon, polyA site) and draw the 4 primer pairs from above at the respective positions that

they amplify (to scale approximately).

B) For the input PCR reaction (left), explain what each of the bands represents. Why are the

bands different sizes (different mobilities) in each lane?

C) Explain why all the bands are of the same intensity for the input PCR reactions

D) What is the conclusion from the ChIP? Explain. What lanes/controls support your ideas?

Input Immunoprecipitation Lane 1 Lane 1 2 3

Answer 1

1.

Non-transcolbed Region start codon stepledon , Poly Ata LANE 2 UAA al Polyft ANI - Poly Atal ANE-3 G3PD gene

2.The band in LANE 1 represents TATA ( promoter region) may be having the low moleculat weight having much mobility than the remaining two, LANE 2 represents 3' end of open reading frame having more molecular weight than the remaining two and LANE 3 represnts next to 3' end of open reading frame i.e before polyadenylation tail having the medium molecular weight than the remaining two electrophoresed inbetween the two bands. Difference in molecular weights caused them to mobilize in three different patterns. The bands in the lower panel are from untranscribed region having low molecular weight than the three and was a positive control.

3. All bands in the input reaction are having same intensity because all of them are having high expression levels more of less equal so they are having same intensity.

4. The transcription factor of 3' end of open reading frame is expressed in high levels compared with the remaining. Equal expression in the lower panel ( in the left and right pcr reactions) indicate equal amounts of loaded DNA (like house keeping gene to determine the equal amounts of loaded DNA).