MCB 150 Exam III Outline.docx

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Molecular and Cell Biology
MCB 150
Brad Mehrtens

Exam III Outline Lecture 21 Main Questions  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? 1) 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- Parallel DNA  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 the base)  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 multiple probes o In a diploididentifies the maternal and paternal copy of the same chromosome  Use chromosome painting on different chromosomes using different colors on each macro chromosome 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 mitotic chromosomes  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 places 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
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