Puneet
Origin of Replication in Eukaryotes
The origin of replication is a specific DNA sequence where replication begins.[1] In prokaryotes, DNA replication begins at a singular AT rich origin of replication. Eukaryotes, however, can have multiple that can initiate replication at different times in order to hasten the process of replicating typically larger genomes [2].
Analysis of the budding yeast, Saccharomyces cerevisiae, genome has shed a great deal of light into the mechanisms underlying Eukaryotic DNA replication. Chromosome replication is initiated when autonomously replicating sequences (ARS) are placed in a plasmids.[3] These ARSs are the site of replication initiation. The number of ARSs in the S. cervisae genome has been estimated to be around 400. Each of these ARSs contains a consensus sequence of about 11bp necessary for the recruitment of various replication proteins. [4] Furthermore, single base pair mutations in the ARS consensus sequence putatively block initiation of replication. [5],[6] However, identifying the origin of replication in other Eukaryotes has proven to be far more challenging. The Schizosaccharomyces pombe genome has much larger origins of replication which are aided by other elements that partially contribute to their activity. [7],[8] In metazoans, replication origins are either localized within a few kilobases, or multiple dispersed origins appear throughout initiation zones of 10 to 50 kb.[9]
Higher Order Eukaryotes
For many other eukaryotic systems, almost any DNA sequence appears to be capable of initiating DNA replication. In both Caenorhabditis elegans [10] and Paramecium ,[11], studies demonstrate that almost any cloned plasmid DNA can replicate autonomously, indicating that replication can occur within almost any size DNA sequence in these creatures. Furthermore, systematic searches in cultured animal cells for ARSs similar to those done in budding yeast did not identify specific sequences that enhance replication when reintroduced into cells i.e. a clear origin of replication was not found in other animal cells. [12]Krysan et al. (1993) demonstrated that in human cells, DNA fragments larger than 15 kb were capable of autonomous replication, indicating that autonomous replication in human cells is stimulated by simple sequences which occur fairly often in human DNA.[13] Similar results were obtained in cultured Drosophila and Xenopus cells.[14]
The Pre-RC
Eukaryotic origins of replication direct the formation of a number of protein complexes leading to the assembly of two bidirectional DNA replication forks. Regardless of the species, the pre-replication (pre-RC) complex forms at the origin of replication during the initiation phase of DNA replication. The pre-RC in eukaryotes is composed of the origin recognition protein complex (ORC) which recruits licensing factors such as the regulatory proteins Cdc6 and Cdt1 and various minichromosme maintenance proteins.[15] The ORC and these regulatory proteins are highly conserved in numerous eukaryotes from budding yeast to humans. Regardless of the origin of initiation site in both Xenopus and Drosophila extracts, initiation of replication requires the ATP-dependent DNA binding activity of the origin of replication protein complex. [16]
RIP Mapping
Recently, researchers have been able to detect start sites for DNA synthesis at the nucleotide level through the use of replication initiation point (RIP) mapping. RIP mapping in yeast and human cells indicate that the following may be true: there exists a single, defined start point at which DNA replication is initiated and the ORC binding site tends to lie immediately adjacent to the replication start point.[17]
Chromosome Number of Vaccinum corymbosum
The high bush blue berry (Vaccinum corymbosum) is a tetraploid with 2n=48 chromosomes. [18],[19]The method behind how the chromosome number was determined is not outlined in any paper that I could find.
