Exam III Outline
1) Is interphase chromatin organized in the nucleus?
2) Are the organized territories of chromatin localized in the nucleus?
o Is DNA replication organized in discrete regions?
a) HOW to mark chromosomes from one another
FISH: Fluoresence In Situ hybridization
using immobilized, denatured DNA with complementary and detectable single
stranded DNA molecules that seek sequences they are complementary and Anti-
a way to see something specific without having to see everything else
o ie) 2 (maternal and paternal) copies of a _______ chromosome
“Detectable markers” for specific chromosomes
There are 2 ways to see the whole chromosome
1) use one probe molecule as long as your chromosome (SOOO MANY BASE PAIRS
IN LEGNTH) to see the whole chromosome (not cost efficient because you pay by
2) break the probe molecule into pieces that overlap
o if you need to identify the whole chromosome
use a series of overlapping probes which collectively cover the whole
sequence of the chromosome
Chromosome Painting: an extension of FISH
Using fluorescence to identify the entire length of a mitotic chromosome using
o In a diploididentifies the maternal and paternal copy of the same
Use chromosome painting on different chromosomes using different colors on each
o You can identify all of the different chromosomes in the spread
Organization of Interphase Chromosomes
Yes, interphase chromosomes do occupy their own territories (organized)
o You can use chromosome painting on interphase chromosomes as well as
Nuclei are divided into discrete functional domains that play an important role in
regulating gene expression in replication 2) Is the Nucleus just a bag of discrete domains??? (hell no)
Chromosomal domains (or territories) are localized regions of function
o Interchromosomal domains
Open space with no DNA
Where the machinery is
o Chromosomal territories
Where the DNA is
2) a) Is DNA Replication Organized into discrete regions?
Domains provide localized regions of function, so DNA replication occurs in specific
o To find this, you must look at where the DNA actually starts to replicate and
make note of the pattern in which the ori is.
Incorporate fluorescently-labeled dTTP (unique to replication) during
replication to study where it is occurring
Initiate FISH on a DTTP and wherever you find accumulations of the