More on chromosomes

1
More on chromosomes
September 28, 2007 Path 303 lab session 1
2
Who am I?

• iwilson_at_bccrc.ca

3
Today

• Review chromosome structure and nomenclature
• Discuss different ways to visualize chromosomes
• Take a look at online resources
• Human Molecular Genetics 2
• http//www.ncbi.nlm.nih.gov/books/

4
Objectives

• Be able to classify different types of repetitive
  DNA, and explain differences between types
• Be able to explain how giemsa banding works, how
  FISH works, how SKY works, be able to describe
  strengths/weaknesses of the various methods
• Be able to draw representative FISH or SKY images
  for your exams

5
Chromosome structure

• Histones (2x H2A, H2B, H3, H4) non-histone
  proteins DNA Chromatin
• Single copy and repetitive (most of genome is
  repetitive)
• Of single copy DNA, very little is actually
  coding DNA!

6
Repetitive DNA
Tandemly repeated
Interspersed repeats
Longer
satellite
centromeres
LINE
LINE-1
minisatellite
telomeres
Shorter
microsatellite
SINE
Alu
7
Repetitive DNA

• Tandem repeats
• Satellite DNA - longest these can be up to a
  few of total DNA, centromeres
• Minisatellites - medium, 0.1-20kb - telomeres,
  or near telomeres - DNA fingerprinting
• Microsatellites - shortest ,lt150bp more
  dispersed

8
Repetitive DNA

• Interspersed repetitive DNA
• LINE family - long intersperesed nuclear elements
• L1 subclass - some are actively transposing!!
• Found in some other mamals
• SINE family - short interspersed nuclear elements
  
• Alu repeats - 300bp all over the place, have
  internal promoter - only us primates have this
• MIR repeats - all mamals have this one

9
Repetetive DNA ctd

• Sometimes coding (but mostly not)
• rRNA, tRNA, snRNA
• MUC1 - (minisatellite length variation)

10
Repetitive DNA Ctd
Figure 7.14
11
How to visualize?!?!?

• Staining!
• Giemsa stain is the most common
• What would you need to do?
• Think about what state chromosomes are in most of
  the time

12
Giemsa staining

• Need them all to be in metaphase!
• Use colchicine which is a mitotic spindle
  inhibitor therefore no mitosis
• Ready to stain?
• Nope, need to use trypsin
• Now we stain!

13
Disordered karyogram
14
Light bands euchromatin Dark bands
heterochromatin
G-banding - the chromosomes are subjected to
controlled digestion with trypsin before staining
with Giemsa, a DNA-binding chemical dye. Dark
bands are known as G bands. pale bands are G
negative
15
Ideogram
16
Figure 2.16.
Figure 2.17.
17
Properties of Geimsa bands
18
Chromosomal nomenclature

• Two arms are designated pand q
• Petite and queue
• Bands are numbered from the centromere out (1p1,
  1p2, 1p3)
• What are the designations for centromere
  position?

19
Centromere position
20
Any other ways to visualize?
Quinacrine
Q-banding - the chromosomes are stained with a
fluorescent dye which binds preferentially to
AT-rich DNA, such as Quinacrine, DAPI
(4',6-diamidino-2-phenylindole) or Hoechst 33258,
and viewed by UV fluorescence. Fluorescing bands
are called Q bands and mark the same chromosomal
segments as G bands.
21
Drawbacks of these stains?

• Lack of specificity
• Low resolution - can only look at very big areas
  of the genome

22
Fluoresence in situ hybridization

• Specific bits of DNA (called probes) are made and
  labeled fluorescently
• Probes are then allowed to wash over chromosomes
  and they bind where they find complimentary DNA
  (hybridization)
• Visualize with fluorescent microscope and bam!
  Talk about cool!

23
FISH

• Decide what you want to probe
• Make probes for that region
• Label the probes fluorescently
• Denature the target DNA (make it single stranded)
• Let the labeled probes find homologous sequences
  on chromosomes (hybridization)
• Visualize with fluorescent microscope

24
EGFREpidermal growth factor receptor
25
Cancer
26
Compare to Giemsa

• Things to consider
• Specificity - probes can be as big or small as
  you like
• Time - probes can take a while to make, Geimsa is
  quick!
• Cost - FISH is decidedly more expensive

27
Lets compare some more

• Equipment
• FISH needs fluorescent scope and camera ususally
• More cell types, stages?
• Sometimes easier to do FISH on tissue sections,
  and interphase nuclei!!

28
The really cool stuff

• FISH probes can be made out of whole chromosomes!

29
Spectral Karyotyping
30
SKY
31
Does this look normal to you?
32
Considerations for SKY

• What can you think of in the context of
  repetetive DNA?
• Big money big money!!!
• Definitely up there on the cool factor though.

33
Switcing gears

• A number of sites exist online to view or browse
  the genome
• http//genome.ucsc.edu
• http//www.ensembl.org

34
Reading

• 2.3.5. Heterochromatin and euchromatin
• 2.5.2. Chromosomes are identified by their size,
  centromere position and banding pattern7.4.
  Extragenic repeated DNA sequences and
  transposable elements
• 10.1.4. Chromosomal in situ hybridization has
  been revolutionized by fluorescence in situ
  hybridization techniques10.2. Chromosome painting

35
Question 1

• If you used a FISH probe for a unique gene
  sequence, how many spots would you see (and where
  would they be located) at the following phases of
  the cell cycle and why?
• Interphase
• Metaphase

36
Question 2

• How would you use FISH to look for a
  translocation between chromosome 1 and chromosome
  2? (hint fluorescent microscopes can detect
  more than one colour)

37
Question 3

• You are doing FISH looking for the RB1 gene. The
  cells you are looking at only show one signal.
  What is the explanation for this?

38
Question 4

• As a graduate student, your supervisor has asked
  you to do Giemsa staining to look for a
  microsattellite. You give them two reasons why
  this will not work, what are they?

39
Question 5

• You are setting up a SKY experiment. You make
  metaphase spreads on glass slides, digest the
  proteins away, and put your multicolor probes
  (repeats are blocked) onto the slides to
  hybridize. You are disappointed to see that none
  of your chromosomes are painted. Why is this?

40
Bonuswhat created this image?