|last page||PLNT3140 Introductory Cytogenetics
Lecture 12, part 1 of 2
1. Circularity of prokaryotic chromosomes simplifies the process of DNA replication.
2. Without special provisions for replicating the ends, linear chromosomes would lose some sequence from the ends every cell cycle.
|Eukaryotic chromosomes contain numerous
replication origins, allowing for DNA synthesis to proceed
in parallel at many sites simultaneously.
The electron micrograph at right shows several replicons, each originating from a different origin (arrows).
Each replicon has two replication forks, moving in opposite directions. Ultimately, replication forks meet, until replication of each template strand is complete.
Displayed by hypertext link to http://bio3400.nicerweb.net/Locked/media/ch11/11_14-replication_bubbles.jpg
Functionally, replication origins
are defined by their ability to confer stable inheritance of
a selectable marker from one cell generation to the next.
A series of experiments from Jack
Szostak's lab helped to define the critical components of
chromosomes. These experiments all had the same steps:
1. Transform yeast, deficient in Leucine production (Leu-) with an artificial construct
2. Isolate a colony on complete media (permissive conditions)
3. Grow in complete media for several generations
4. Transfer to minimal media without Leucine
5. Identify clones that grow without Leucine
CHROMOSOMAL ELEMENTS: EXPERIMENT A - Origins of
If you transfect into yeast
a plasmid containing a selectable marker, in this case a
LEU gene (Leucine biosynthesis) into Leu-
yeast, the cells will not grow, even though you have
given them the correct gene. But if you randomly
clone yeast sequences into this same plasmid and
select on minimal media, you can recover a few clones
that are able to grow (ie. synthesize leucine).
The inserts contained in these surviving plasmids are
origins of replication, referred to in yeast as autonomously
replicating sequences (ARS).
Basically the same strategy has been
used to clone centromeric sequences. A plasmid containing a
selectable marker and an ARS sequence will replicate in culture,
but will be lost from some lines of cells in the absence
of selection. Yeast genomic DNA is randomly cloned into
the plasmid containing LEU and ARS, clones can be identified
that stably maintain the plasmid, even in the absence of
selection. These plasmids contain centromere sequences
(CEN). Segregation of CEN plasmids occurs in a Mendellian
fashion. That is, 100% of the progeny are Leu+ if they contain
The attachment of a chromosome to the mitotic spindle fibers occurs at the centromeric sequence.
It is probably not a coincidence that the length of the yeast CEN sequence necessary for binding centromere attachment is 220bp, which spans about 20nm, which is the width of a spindle fiber. DNAse sensitivity studies have indicated that this region is free of nucleosomes, presumably to enable the spindle attachment to occur. CEN sequences are complexed with the proteins of the kinetochore complex, which facilitate attachment to the spindle fibers.
Centromeres tend to be surrounded by highly-repetitive DNAs, referred to as satellite DNAs. Satellite DNAs may confer different coiling properties to the centromeric regions of chromosomes.
Detail of a positive staining centromere region of a C-banded human chromosome. (C banding preferrentially stains constitutive heterochromatin.) The fibrous organization of this region is still apparent but is either covered by, or embedded in, an amorphous matrix. (Magnification x 64,000.)
Chromosomes and Chromatin, Vol. II (1988) Ed. K.W.Adolph. CRC Press. Fig. 14 pg. 67
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|last page||PLNT3140 Introductory
Lecture 12, part 1 of 2