1. Linear plasmid pSLA2 G.Thirupugal

2. Introduction-pSLA2 Name:pSLA2 Original host:Streptomyces rochei Host range:Narrow host range Copy number:low copy number(1 or 2) Size:17Kb Geographical location:Japan

3. Genome map of plasmid pSLA2 1.Telomeres are present in both the ends. 2.Two rep genes are located in internal region of DNA. 3.Two cis acting sites are located in the rep genes 4.cis1 located within the region of the rep1 gene. 5.cis2 located within the region of rep2 gene.

4. Rep1 and Rep2 functions 1.Rep1 and Rep2 proteins are essential for plasmid DNA replication and are encoded by the same transcript. 2.Rep1 is a DNA binding protein,where binding is specific to the pSLA2 replication origin. BssHIIBsaAIBamHI BstxI AgeI BbsI ApaI SphI Rep1Rep2 pSLA2

5. 3.Rep2 contains two highly conserved NTP-binding motifs typical of ATPase domains of DNA helicases. 4.Rep2 was enhanced by the presence of single-stranded DNA. 5.Site directed mutagenesis showed that both the Rep1 and Rep2 proteins are essential for pSLA2 DNA replication.

6. Cis- acting loci for pSLA2 Bcl1 BamH Sph1 Asp718 cis1cis2 Bgl II pSLA2 17Kb  Two cis acting loci was identified by the plasmid pSLA2.  It is required for replication.  Cis1 a segment located within the 864bp protein coding region of the rep1 gene.  Cis2 a 430bp DNA segment positioned downstream of the rep2 protein coding region.

7.  Cis1 contains the plasmid pSLA2 replication origin.  The sequence characteristics of cis1is four directed repeat followed by several runs of CT or GA rich sequences.  Cis2 act like partition locus of plasmid pSC101 and the locus of staphylococcus plasmid pT181.

8. 1.Linear plasmid pSLA2 have protein attached to covalently to their 5’ DNA termini. 2.The linear plasmid replicates bi-directional towards the telomeres from a site located near the center of the molecule. 3.The replication of mode pSLA2 the ability of lacking telomeres to replicate as circular molecules. The replication of linear plasmid pSLA2

9. 4.The replication of pSLA2 is initiated at a series of direct repeats located within a gene specifying an essential DNA- binding protein. 5.That this gene (rep1) is part of a bi-cistronic Rep1 operon that can function in trans to promote plasmid replication from the centrally located origins. 6.The second gene(rep2)in this operon encodes a protein that has single stranded DNA dependent ATPase activity resembles prokaryotic DNA helicases.

10. 7.The origin of replication region of this plasmid replicon thus has features in common with the origin of temperate bacteriophages of E.coli, Salmonella typhimurium, and Bacillus subtlest. 8.Suggesting that pSLA2 may occupy an evolutionary intermediate position between plasmids and phages.

11. Replication-pSLA2 A B C 13 bp

12. Significance of plasmid pSLA2 They produces polyketide antibiotics a)Lankacidin b)lankamycin These antibiotics are used for treatment of swine dysentery caused by Treponema hydrodysenteriae. This plasmid also encodes biosynthesis of carotenoid.