Download Name: Date: Title: Nucleosomes and Chromatin Structure

Name:
Date:
Title: Nucleosomes and Chromatin Structure.
Introduction.
A diploid human cell contains about 6.4 x 109 nucleotide pairs of DNA. This corresponds to
about two metres of double helix. This DNA has to fit inside the cell nucleus which, in an average human cell, is about 5μm in diameter. This is roughly equivalent to packing sixty miles of
fine thread inside a basketball. In the cell nucleus the packing problem is compounded because
the DNA has to be both accessible for transcription and replication and yet capable of being segregated during cell division. The solution is a hierarchy of coiling and folding patterns that can
result in either interphase chromatin or metaphase chromosomes.
It is now known that the basic repeating unit of chromatin is the nucleosome. A string of nuclesomes forms 10nm fibre, which is folded to give 30nm fibre. Loops of 30nm fibre are attached to
the matrix (or scaffold), which can be coiled in different ways to make interphase or metaphase
chromosomes. Several approaches contributed to the development of this understanding (see figure 1). Biochemical analysis showed that chromatin contains histone proteins, with H1, H2A,
H2B, H3, and H4 being present in a molar ratio of 1:2:2:2:2. Electron microscopy revealed the
existence of nucleosomes, 10nm fibre, and 30nm fibre. Nuclease digestion uncovered a 200np
repeat and a 146np limit product, contributing to our understanding of the path of DNA around
and between nucleosomes. In addition, nuclease sensitivity studies demonstrated structural differences between active and inactive chromatin. X-ray crystallography provided a detailed picture of the relationship between the histone core and the DNA wrapped around it.
Figure 1: Steps in elucidation of nucleosome structure. SDS polyacrylamide gel showing histone proteins (a). Electron microscopy showing 10nm fibre (b) and 30nm fibre (c).
Nuclease digestion study showing 200np ladder (d). X-ray structure (e).
Genetics Laboratory 16.1
In this laboratory exercise you will isolate nuclei from wheat germ and use nuclease digestion to
release and characterize nucleosomes.
Methods.
Day One
In this laboratory session we will isolate nuclei from wheat germ. We will incubate the nuclei
with micrococcal nuclease to release fragments of chromatin, and with Sarkosyl and ribonuclease to remove proteins and RNA.
IMPORTANT
Do not expose sterile materials to the air for longer than absolutely
necessary. Handle sterile materials as little as possible, particularly
pipette tips.
Keep DNA and enzyme solutions on ice as much as possible.
(1) Weigh 1g of wheat germ into a beaker. Add 10ml nuclear incubation buffer. Swirl gently on
ice for five minutes.
The buffer contains NP-40, a detergent that will lyse the cells, releasing the nuclei.
(2)
Filter the material through three layers of cheesecloth into a 50ml centrifuge tube, squeezing to ensure that all liquid is collected.
(3)
Centrifuge at 4°C for five minutes at 5krpm.
This step pellets nuclei. The next step washes away some cellular debris.
(4)
Discard the supernatant. Add 5ml of nuclear incubation buffer to the pellet. Resuspend the
pellet by gently pipetting the liquid up and down several times using a sterile transfer
pipette. Avoid making bubbles as much as possible. Centrifuge at 4°C for five minutes at
5krpm.
(5)
Discard the supernatant. Add 1ml of nuclear incubation buffer. Resuspend the pellet by
gently pipetting the liquid up and down several times using a sterile transfer pipette. Avoid
making bubbles as much as possible.
(6)
Label three microfuge tubes “1”, “2”, and “3”. Aliquot the nuclear suspension into the
tubes. Place on ice.
(7)
Add 5μl of micrococcal nuclease to tube 2 and 30μl of micrococcal nuclease to tube 3. Incubate these tubes in a 37°C heating block for 10 minutes.
Genetics Laboratory 16.2
(8)
Centrifuge all three tubes at room temperature for five minutes at 5krpm.
This step pellets nuclei but any small chromatin fragments released by micrococcal nuclease will remain in the supernatant.
(9)
Label three microfuge tubes “1”, “2”, and “3” and add your initials. Carefully transfer 30μl
of each supernatant into the corresponding tubes.
(10) Add 30μl of sample buffer to each tube. Incubate the three tubes in a 55°C heating block
for 15 minutes.
The sample buffer contains Sarkosyl, a detergent which will remove histones from the
DNA, and ribonuclease, an enzyme that will degrade any contaminating RNA.
(11) Place your three tubes in the box provided. Your samples will be stored at -20°C until the
next laboratory session.
Genetics Laboratory 16.3
Day Two
In this laboratory session we will separate any DNA fragments produced in the previous session
by agarose gel electrophoresis.
IMPORTANT
Do not expose sterile materials to the air for longer than absolutely
necessary. Handle sterile materials as little as possible, particularly
pipette tips.
Keep DNA solutions on ice as much as possible.
(1)
Your instructor will load a molecular weight standard and three example reactions (untreated and treated with 5μl and 30μl of micrococcal nuclease).
(2)
Add 60μl of distilled water to each sample and mix gently. (Experience has shown that
loading undiluted sample overloads the gel, which causes distortion of the tracks.)
(3)
Load 20μl of each diluted sample onto the gel. Record the track order for all samples and
molecular weight standards on the gel.
(4)
Connect the gel apparatus to the power supply, set the voltage to 60V, and electrophorese
for 30 minutes.
IMPORTANT
The ultra-violet light source used in the next step will cause severe
burns with very short exposure times. Wear the protective glasses
and face shield provided. All exposed skin should be covered.
(5)
After electrophoresis the gel will be placed on a UV transilluminator and photographed.
You may observe this procedure if you wish.
Genetics Laboratory 16.4
Report.
Your report should be typed or neatly written. Marks may be deducted for illegibility and/or poor
grammar and/or poor spelling. If loose sheets are used, they must be stapled or in some form of
binder. Paper clips are not acceptable.
Your report should be in the following format:
Name and date.
Title of experiment.
Introduction to experiment.
Description of methods and materials used.
The above information is in this manual. You can attach the manual pages to your report in
place of this information.
Results obtained.
Include a picture of the gel. Clearly label all tracks on the picture.
Analysis of results obtained and conclusions.
Carefully examine the tracks of the gel that contained the molecular weight standard. Measure the distance that each fragment migrated from the well. Generate a standard curve by
graphing the log of the fragment size versus the distance migrated.
Carefully examine the other tracks of the gel. For each sample measure the distance that
each fragment migrated from the well. Use the standard curve to determine their molecular
weights. Present the results in table format.
Discussion.
From our current understanding of chromatin structure, what result would you expect to see
after treatment of chromatin with increasing amounts of a non-specific nuclease such as
micrococcal nuclease? Explain your answer thoroughly.
Are the results observed with the three chromatin samples loaded by the instructor in
agreement with expectations? If not, suggest possible explanations for any discrepancies.
Are the results observed with the three chromatin samples that you prepared in agreement
with expectations? If not, suggest possible explanations for any discrepancies.
How would your expectations be changed were there 250np of DNA tightly wrapped
around each nucleosome instead of 146np? Assume that nucleosomes are distributed evenly
along the DNA and that the linker is still 50np long.
Genetics Laboratory 16.5
How would your expectations be changed were there 250np of DNA tightly wrapped
around each nucleosome instead of 146np and nucleosomes were distributed unevenly
along the DNA (i.e., the length of the linker is variable)?
Nuclei from brain cells and nuclei from liver cells are treated with increasing amounts of micrococcal nuclease. DNA is isolated and the fragments are separated by gel electrophoresis. Complete the diagram below to show the pattern of DNA fragments that would be expected from a
gene that is not transcribed in brain but is transcribed in liver.
Chromatin
(Brain)
Micrococcal
nuclease
0
Chromatin
(Liver)
0
10,000np
gene
fragment
7,500np
5,000np
2,500np
A due date for your report will be announced. Late reports will be penalized.
You should each write your own report, although you may discuss your results with each other. If
you require any help with your report, please come and see me. There is no penalty attached to
this help.
Genetics Laboratory 16.6