DNA ???????----CsCl??

1
???????????????
? ? ?? ??????????
2

• ??????????????,?????????????????????810?(??806
  ?)?????

3
??

• ?????????
• ???????
• ??????????

4
Meselson-Stahl ??
5
DNA ???????----CsCl??
6
? ? ? ?

• ?????
• ???????
• ????????

???????
7
???? - ?????

• ?????(Differential Centrifugation)
• ????????????,??????????
• ???????????,???????????
• ?? ???

??????,?????????????????(??)?????????????????,????
??????????
8
???? (s)
9
???? - ?????
???????????
?????????? -??????????-
10
???? - ???????
(Density Gradient Centrifugation)
v ????d ????(????????)Pp ????P1
????? ????g ???

•   ???????????????
• ? ??????????????????????????? 
• ???????????????,?????0?
• ?????????????????? 
• ????????????????

11
???? - ???????
(Rate Zonal Centrifugation)

• ???,???????????????????????
• ??????????????????,????????????????????????????
• ???????????????????????????????????????

12
???? - ???????

• ???????????

?? ??????????DNA?RNA
??? (Ficoll) ??????????(?????????)
Percoll SiO2/PVP???,????????
??? (CsCl) DNA?RNA???????
??? K(Na)Br(I) ????RNA
13
???? - ????????
(Isopycnic Centrifugation)

• ?????????????
• ???,???????????
• ????,???????????,??????
• ???????????????????,??????????????

14
?????????

• CsCl?????
• ???????( Nycodenz? metrizamide)
• ???(Percoll)?????

15
??????
????????????????
?????? ???????
?????? ???????
???????? ??????
?????????????????? ??????????????????
???????????????,???,??? ??????????????,???,???
16
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? ? DNA RNA ??? ? ??? ?? ??
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???
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??????
???
??, ?, ??, ???
17
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??---???????
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????
?????
?????
????
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18
????????

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• ??
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• ???????,???????,??????????????????  
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• ???????????

19
????????

• ??????,????????????? 0???90?,??????0??
• ??
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• ?????????????????????  
• ??????????????????  
• ???DNA?????????  

20
????????

• ???????????,?????????0
• ??
• ???????  
• ??????1.7g/ml,??????????DNA????  
• ??????  
• ??????DNA????  
• ?????????????  
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• ??????????  

21
?????????

• ????????????,????????????,????????????10?
• ??
• ?????????????,?????????????????????  
• ????,??????1.7g/ml? 
• ??????  
• ??????DNA????  
• ???????????  

22
?????????????
23
?????
Type of rotor Type of separation Type of separation Type of separation
Type of rotor Pelleting Rate-zonal Isopycnic
Fixed-angle Excellent Poor Poor
Vertical Poor Good Good
Swing-out Inefficient Excellent Adequate
24
?????????

?? ???????? ?????
???? ?????
????
?? ???????? ?????
5 000 x g 10 ??
30 100 000 x g 1 6 ?? (????)
100 000 - 1 000 000 x g 2 - 24 ??
?? / ?? / ??? ???
?? / ?? ???
?????
25
??????????????
? ? ? ?
26
??????????????
???
??????
444,000 g
??? (???, 70S)
??
30,000 g
???
25-40 ???? 268,000 g
??? (?????, 30S, 50S)
27
NaBr?????????
???
??????
100g
???
??
NaBr ??????
VLDL LDL HDL
NaBr ??
??
????
28
??DNA?????
????
???????
NaOH-SDS?????????DNA
??DNA??? 10 mM Tris-HCl, 1 mM EDTA, pH 7.6?????
???????1g CsCl?0.6mg EB,??Triton X-100???????0.01
????
29
? ? ? ? ? ? ?
30
Telomere End Structure(Telomeric Overhang)
T-loop formation
Telomerase extension
31
Question Whether or not both ends of chromosome
have a long overhang
32
Determination of the Length of the Telomeric
Overhang

• Is there an overhang at each end of each
  chromosome?

33
Replication of the Chromosome End
3
TTAGGG
Lagging
5

3
Leading
5
AATCCC
5
3
AATCCC
TTAGGG
Leading
Lagging
5
3
Chromosome
34
Separation of Leading and Lagging Telomeres
3
TTAGGGTTAGGG
Lagging
5
AATCCCAATCCC
CsCl
T ? BrdU
3
TTAGGGTTAGGG
Leading
5
AATCCCAATCCC
Zhao Y et al. Nucl. Acids Res. 2008.
35
Lagging Telomeres have a Much Longer Overhang
than the Leading Telomeres
Lag
Lead
-
-


Exo I
Mean Length 30 110 nt
Zhao Y et al. Nucl. Acids Res. 2008.
36
Long overhang is present at one end of the
chromosome
37
How to study telomerase extension during one cell
cycle
38
Telomerase Extension of Telomeric Overhang
Telomere
hTERT
hTR template
39
The Strategy to Study Telomerase Extension
TTAGGG-3
Replication In BrdU
3
5
AATCCC-5
Thymidine
TTAGGG-3
CsCl
Un-extended
DSN
Extended (L)
Telomerase
ThymBrdU
DSN Duplex Specific Nuclease
40
100 Ends of Telomeres Were Extended by
Telomerase in H1299 Cells
(50/50)
BrdU
T/BrdU
Thy
Signal Intensity
Density (g/ml)
Zhao Y et al. Cell.  2009.
41
Telomere Length Maintenance in Human Cancer Cells
In the absence of telomerase
PD 0
35
19kb
7.7kb
6.2kb
4.3kb
3.5kb
2.7kb
1.9kb
1.5kb
0.9kb
Tel-shortening 55 (
bp/PD )
Zhao Y et al. Mol Cell. 2011
42
Dogma Telomerase Preferentially extend the
shortest telomere
43
Sucrose Gradient Separation of Long and Short
Telomeres
44
Telomerase Extension of Telomeres is not Length
Dependent
Long Telomeres
Short Telomeres
45
? ? !