Extrachromosomal Replicons

Plasmids are extrachromosomal DNA molecules that can be passed horizontally and vertically via bacterial lineages. Plasmids have been shown to code for a variety of bacterial phenotypes, the most concerning of which is antibiotic resistance.

Plasmid-mediated drug resistance gene transfer has resulted in the fast spread of resistant strains, posing a public health threat.

While the rapid development of bacteria caused by horizontal plasmid transfer has dominated this topic in the past, evolutionary interactions that impact the long-term population dynamics of bacteria and plasmids are receiving more attention.

Replicon:

Replicons are virus-like particles that enter a target cell and undergo limited transcription and translation in order to generate encoded proteins, but do not produce infectious progeny. Replicons are made up of a virus genome that has been modified to add a new protein and delete part of the parent virus’s genes. These genomic constructs frequently lack the genes for their envelope spike and are transfected into packaging cell lines that provide a viral envelope in a transient manner.

This enables the construction of a virus-like particle with the envelope’s cellular specificity. Replicons are a safer platform than recombinant viruses since they can’t move beyond the cells they “infect.” Many wild-type viruses have properties that would make them unsuitable for use as an infectious recombinant virus.

Importance in Medicine:

The medical significance of plasmids that encode for antibiotic resistance and certain virulence features has been widely documented, demonstrating the critical function that these bacterial genetic components play in nature.

Plasmids rely on host-encoded factors for replication, despite the fact that they encode specific molecules essential for replication initiation. Plasmid replication begins at ori, a preset cis-site, and can proceed in one of two ways: by rolling circle or by theta replication.

Plasmid properties like copy quantity and incompatibility are determined by plasmid-encoded elements essential for replication, such as antisense RNA molecules and DNA repeating sequences situated adjacent to ori.

The Replicon Model:

The replicon model predicts that chromosome replication happens in discrete units called replicons, which was first proposed by F. Jacob in 1963 based on observations on bacterial (Escherichia coli) cells.

 Each replicon comprises a unique DNA sequence known as the ‘origin,’ as well as a DNA-binding protein known as the ‘initiator,’ whose primary function is to recognise the origin and recruit the extra factors needed to open the double-stranded chromosomal DNA and begin synthesis.

 In essence, this paradigm still applies to all creatures, although there are a few key differences. Prokaryotic cells and unicellular eukaryotic species, like the budding yeast Saccharomyces cerevisiae, have both a fixed origin sequence and unique initiator proteins in their replicons.

Types of replication origins include:

There are many different types of replication origins, therefore for the purpose of simplicity, we’ve overlooked those found in eukaryotic cells and viruses and only looked at those found in bacteria. ColE1, pMB1 (which comes in a few somewhat different but well-known versions), pSC101, R6K, and 15A are some of the more prevalent ones.

Not all replication origins are created equal. Depending on how they are regulated, some will make a large number of plasmid copies while others will only produce a few. Depending on whether or not the ori is positively regulated by RNA or proteins, replication regulation is termed as “relaxed” or “stringent.”

The copy number of a plasmid has to do with the balance of positive and negative regulation, and it can be modified via replicon mutations. The pMB1 ori, for example, maintains roughly 20 copies per cell, whereas the pUC ori, which differs only by two mutations, can create up to 700 copies per cell.

Factors that influence plasmids copies:

Although the ori sequence and regulation have a significant impact on plasmid copy quantity, other external factors also play a role.

These points are very important to remember if you intend to purify your plasmid DNA:

Insertion:

If plasmids have big inserts or genes that produce a harmful output, bacteria tend to keep fewer copies of them.

The E. coli strain is as follows:

Most E. coli strains can be used to replicate plasmids, however endA- E. coli strains provide the highest yields.

Conditions for growth:

Copy number is affected by aeration, temperature, culture volume, antibiotic, and medium.you have to be sure the growth conditions aren’t working against you. Some ori’s are temperature sensitive; others can be “tricked” into amplifying more copies by adding chloramphenicol – make sure your growth conditions aren’t working against you.

The inoculum for culturing:

Freshly streaked bacteria have larger copy numbers; for best results, subculture from a single colony rather than glycerol stocks, agar stabs, or liquid cultures.

Conclusion:

Plasmids are replicons that exist in bacteria as separate extrachromosomal genetic components. They are normally much smaller than the bacterial chromosome, ranging in size from less than 5 to several hundred kbp, while some bacteria have plasmids as large as 2 Mbp.