Giles 23 April 09

Following to be done for ArdC, ProP and AltB at

mid and late G2

◦ Quantification of transcription

◦ Quantification of PolII occupancy

◦ Quantification of H3/H4 replacement

Perform high resolution mapping at mid and late

G2 for AltB

Showed change in

AltB expression as

expected

Showed possible

enrichment of PolII on

AltB in late G2

0

1000

2000

3000

4000

5000

6000

ArdC ProP AltB

Mid G2

Late G2

-20

0

20

40

60

80

100

120

140

ArdC ProP AltB

Mid G2

Late G2

Late G2 Mid G2

Imp Un IP Imp Un IP

Following to be done for ArdC, ProP and AltB at

mid and late G2

◦ Quantification of transcription

◦ Quantification of PolII occupancy

◦ Quantification of H3/H4 replacement

Perform high resolution mapping at mid and late

G2 for AltB

-230 -156

Comparison of mononucleosomes from control and flag incorporated cells

-8

-6

-4

-2

0

2

4

6

8

10

1 2 3 4 5 6 6 7

8a

8b 9

10

11a

11B

12

13

14

15

16

17

18

19

20

Ard

C

Pro

P

DC

t

MC-C MF-F

Determine relative amplification from DNA cut 5 min and 15 min compared to uncut

0

5

10

15

20

25

1 2 3 4 5 6 7 8A 8B 9 10 11A 11B 12 13 14 15 17 19 ArdC

dCt5 dCt15

-4

-3

-2

-1

0

1

2

3

4

1 2 3 4 5 6 7 8a 8b 9 10 11a 11b 12 13 14 15 16 17 18 19 20

Mid G2 Late G2

100bp

-230 -156

Primer extension assay

-230 -156

Tried to quantify/amplify of different products in primer extension reaction

-230 -156

Mid G2 Late G2

PE Q1 Q2 Q3 Q4 Q5 PE Q1 Q2 Q3 Q4 Q5

P1/P2 P1 P2P1/P2 P1 P2 P1/P2 P1 P2P1/P2 P1 P2

Neat 1:100 Neat 1:100

Mid G2 Late G2

-230 -156

Use reconstituted 5s nucleosomes

Confirm position ◦ restriction digest

◦ New method

Apply new method to map completely AltB