| Plasmid pHH205 is a halophilic archaeal plasmid isolated from Halobacterium halobium J7 by our laboratory. In this study, a detailed restriction map was constructed using multiple groups of restriction endonuclease analysis. According to this map, seven overlapped fragments containing the whole plasmid were determined and cloned into vector respectively for sequencing. A primer-walking strategy was then used to sequence these seven fragments. The whole sequence of pHH205 was obtained by connecting these seven pieces of sequences with each other. The complete nucleotide sequence is 16341 bp, and it has been deposited in the GenBank database under accession number AY048850.The G+C content of the plasmid is 61.1%, but it is not average in the whole sequence. A number of repeat sequences were found in pHH20S, while no insertion sequence that frequently occurred in halophilic archaeal plasmids was found. Thirty-eight large open reading frames (ORFs) were identified in both strands, and there was no distinct coding region or non-coding region. Gene overlapping and multiple start codons like bacterial genome structure were found among these ORFs. The codon bias of coding sequences is consistent with that of genome of Halobacterium sp. NRC-1. These results showed some of the common properties of pHH205 and other plasmids and chromosome of halophilic Archaea, and revealed some special features of the plasmid.Databank searches for homology revealed that most of the ORFs of pHH20S have no significant similarities to known proteins. A predicted protein, encoded by ORF31, shows 20-41% similarity to eleven hypothetical proteins of Halobacteriumsp. NRC-1 and other archaeal genome. The protein must be a conservative protein of Archaea, and it is annotated as a 'predicted nucleic acid-binding protein, containing a PIN domain'. It is predicted that the protein is relevant with archaeal control of DNA replication and transcription in extreme environment The actual function of the protein is required for further studies.The replication origin of pHH205 was determined using a GC-skew analysis method. Several fragments containing the replication origin were cloned into replicon probe vector pUC-MEV constructed in this study. After screening a 4.8 kb Pvull-SnaEl fragment were determined to be the minimum replication region of the plasmid. It is different from the known two kinds of replication mechanism of archaeal plasmids on the structure of replication region, which suggested that pHH205 have a special mechanism of replication and control. Recombinant plasmid pHSPl containing the minimum replication region of pHH205 is an . coli-halophilic Archaea shuttle vector, which can be used for genetics and molecular biological researches of halophilic Archaea.Several methods failed to cure of pHH205 from its host, which did not only confirm that the plasmid is highly stable, but also suggest that it carry some essential factors for the host Studies on ORF31 revealed that this archaeal conservative gene is essential for the host, but does not exist hi the chromosome. This result shows that pHH205 carrying essential gene for survival of its host, which is different from traditional conception of plasmid, but similar to some known archaeal plasmids.Thus, by sequencing and function analysis of pHH205, this study revealed it is a novel plasmid with special features. It not only provides a new cloning and shuttle vector with high stability for archaeal researches which is short of vectors at present, but also give some new thoughts of the traditional concept, genesis and evolution of plasmid, and would contribute to ulteriorly reveal the genesis and essence of archaeal plasmid, and its location hi phylogenetic development...
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