Long ITRs were found in some NCLDVs with linear genome, such as poxvirus ( Tulman et al., 2006, 2004 Mitsuhashi et al., 2014), ASFV ( Meireles and Costa, 1994), and chlorovirus ( Strasser et al., 1991 Quispe et al., 2017). Inverted terminal repeat (ITR) is a DNA structure found at the end of some linear replicons and essential for the stability and replication of the replicon ( Blackburn, 1991). It was proposed that genome duplication played an important role in shaping the genome of mimivirus since one-third of mimivirus genes had at least one paralog ( Suhre, 2005). Mimivirus had a large and complex genome with a relatively conserved central region and highly diversified terminal areas ( Arslan et al., 2011 Yoosuf et al., 2012). Using different gene or gene set may result in slightly different topology in the phylogenic tree, but the lineage classification remains unchanged ( Arslan et al., 2011 Yoosuf et al., 2012 Assis et al., 2017 Abrahão et al., 2018 Takahashi et al., 2021). New lineage has been proposed due to the isolation of new viruses ( Takahashi et al., 2021). Members of Mimiviridae can be divided into several lineages, including lineage A (represented by APMV, La Scola et al., 2003), B (Acanthamoeba polyphaga moumouvirus, Yoosuf et al., 2012), C (Megavirus chiliensis, Arslan et al., 2011), and Tupanvirus ( Abrahão et al., 2018), based on the phylogenetic analysis of conserved genes, like major capsid protein, family B DNA polymerase, D5-like primase-helicase, etc. The amoeba giant viruses were classified into the Nucleocytoplasmic Large DNA Viruses group (NCLDV) together with Poxviridae, Asfarviridae, Phycodnaviridae, Ascoviridae, and Iridoviridae ( Iyer et al., 2001 Yutin et al., 2009). These viruses have extraordinarily large virions size, as well as large and complex genomes harboring not only hundreds of orphan open reading frames (ORFans), but also many proteins barely seen in other viruses, like translation-related proteins, repeat-containing proteins, etc. Following APMV, new classes of giant viruses have been identified in amoeba and other organisms, such as Pandoravirus celtis ( Legendre et al., 2019), Bodo saline virus (BsV) ( Deeg et al., 2018), Yasminevirus ( Bajrai et al., 2019), Tupanvirus ( Abrahão et al., 2018), Medusavirus ( Yoshikawa et al., 2019), etc. A number of small ORFs below 50 bp were also found in the sequence analysis.The first report of amoeba giant virus, Acanthamoeba polyphaga Mimivirus (APMV), changed our perception and understanding about virus greatly ( La Scola et al., 2003 Raoult et al., 2004 Suhre, 2005). The ORF3 of pCYLM02 corresponds to the plasmid sequence from 2323 to 2976 and codes for a putative protein of 217 amino acids long. The ORF2 of pCYLM02 has 243 amino acids and is represented in the plasmid sequence from 3045 to 3776 bp. The analysis of 4359 bp plasmid pCYLM02 sequence revealed 7 ORFs which are longer than 50 amino acids in length. Among them, ORF1 shows high sequence homology to the bacterial replication initiator family protein as evident from BLASTP analysis. The sequence of pCYLM01 contained 4 potential open reading frames (ORFs) that have amino acids in the range of 59–299.
The following plasmids pCYLM01 and pCYLM02 were subjected to restriction digestion using HindIII restriction enzyme and cloned into pBlueScriptSK(-) vector.
Cylindrospermum stagnale isolated (and deposited in NRMCF 3001) from soil showed presence of four plasmids namely pCYLM01, pCYLM02, pCYLM03, and pCYLM04. Isolation and characterization of extra chromosomal DNA in cyanobacteria might help to understand its survival mechanism. It has an extra chromosomal DNA, whose role is not yet defined well. Cyanobacteria play a vital role in supplying nitrogen into the soil and aquatic ecosystem.