Chromosomes

Eukaryotic DNA is arranged into chromosomes. It has long been assumed that each chromosome contains a single very long, linear DNA molecule though this has been demonstrated in only a few species. For example in yeast pulsed field gel electrophoresis (which is a technique for separating very long pieces of DNA) shows that the number of individual DNA molecules in the nucleus is approximately the same as the number of chromosomes.

These chromosomes have three functions:

1. they replicate once per cell cycle - during the S phase of interphase

2. they segregate to daughter cells at mitosis

3. they package DNA into managable units

Chromosomes possess within their DNA, specialised nucleotide sequences which are necessary for these functions.

Replication Sequences

Origins of replication

These are the sites where replication begins and there are usually a large number of these on each eukaryotic chromosome.

The DNA sequences which comprise origins of replication have been isolated from yeast. These have been called autonomously replicating sequences or ARSs. An ARS is defined as a region of DNA which is necessary and sufficient to ensure replication of a circular piece of DNA once per S phase.

ARS sequences were identified by cloning random fragments of yeast chromosomal DNA into a plasmid vector and then introducing them into yeast cells. If this plasmid contains a drug resistance marker and the yeast cells are plated on a medium containing the drug - only those cells containing a replicating plasmid will be able to form a colony. About a dozen different ARSs have been isolated in this way and it has been calculated that there are approximately 400 ARSs spread over the 17 chromosomes of yeast.

Mutational analysis has identified a region of about 50 bp that is required for proper ARS function. When the nucleotide sequence of different ARSs was compared it was found that there is an 11 bp sequence found in all of them - this suggests that this sequence is important, and indeed mutations in this region (region A) abolish ARS activity. However, the 11 bp sequence on its own cannot act as an ARS. Additional DNA sequences (B1-B3) are necessary for its function - this DNA shows no similarity between different ARSs but it often contains imperfect matches to the consensus sequence.

A complex of 6 proteins, called the origin recognition complex or ORC, binds to the ARS. The function of these proteins has yet to be determined. One important function must be the initial unwinding of the two strands of the DNA helix. It may be significant that the conserved core sequence is AT rich - AT base pairs are weaker than GC base pairs and so are more easily broken.

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