We report here on the genome sequence of Pasteurella multocida Razi 0002 of avian origin, isolated in Iran. The genome has a size of 2,289,036 bp, a GC content of 40.3%, and is predicted to contain 2,079 coding sequences.

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The reverse transcriptase encoded by the non-long terminal repeat retrotransposon R2 has been shown to be able to jump from the 5-end of one RNA template (the donor) to the 3-end of a second RNA template (the acceptor) in the absence of preexisting sequence identity between the two templates. These jumps between RNA templates have similarity to the end-to-end template jumps described for the RNA-directed RNA polymerases encoded by certain RNA viruses. Here we describe for the first time the mechanism by which such end-to-end template jumps can occur. Most template jumps by the R2 reverse transcriptase are brought about by the enzyme's ability to add nontemplated (overhanging) nucleotides to the cDNA when it reaches the end of the donor RNA. The enzyme then anneals these overhanging nucleotides to sequences at the 3-end of the acceptor RNA. The annealing is most efficient if it involves the terminal nucleotide(s) of the acceptor RNA but can occur to sites at least 5 nucleotides from the 3-end. These end-to-end jumps are similar to steps proposed to be part of the integration reaction of non-long terminal repeat retrotransposons and can explain chimeric integration products derived from multiple RNA templates.One of the most abundant classes of mobile elements found in eukaryotic genomes is non-LTR 1 retrotransposons. These elements, also referred to as LINE-like elements or retroposons, use a relatively simple mechanism of reverse transcribing RNA templates into DNA for insertion into the genome. In the first step, the target chromosomal DNA is cleaved by an element-encoded endonuclease. The RNA-directed DNA polymerase encoded by the element then uses the free end of the cleaved target DNA as the primer to initiate reverse transcription starting at the 3Ј-end of the element's RNA transcript. This process has been termed target (DNA)-primed reverse transcription (TPRT) (1). Whereas the cleavage that initiates the TPRT reaction could potentially be single-stranded (i.e. a DNA nick) or double-stranded (i.e. a DNA break), in the two systems studied in vitro the cleavage has been shown to be a DNA nick (1, 2). It has been suggested that a short region of the RNA template can anneal to the DNA immediately downstream of the nicked site, thus aiding the initiation of reverse transcription (3). However, such annealing is not required in the most extensively characterized element, the R2 element from Bombyx mori (4 -6).We are conducting a systematic study of the catalytic properties of the reverse transcriptase (RT) encoded by the R2 element (7,8). Perhaps the most dramatic difference between R2 RT and the RT encoded by LTR retrotransposons and retroviruses is the ability of R2 RT to jump from the 5Ј-end of one RNA template to the 3Ј-end of a second RNA template. This continuous polymerization of cDNA on noncontinuous RNA templates differs dramatically from the template switching reaction associated with retroviral cDNA synthesis. Retroviral template switches occur through catalytic removal of the donor RNA from the cDNA s...

R2 is a site-specific non-long terminal repeat (non-LTR) retrotransposon encoding a single polypeptide with reverse transcriptase, DNA endonuclease and nucleic acid-binding domains. The current model of R2 retrotransposition involves an ordered series of cleavage and polymerization steps carried out by at least two R2 protein subunits, one bound upstream and one bound downstream of the integration site. The role in the retrotransposition reaction of two conserved DNA-binding motifs, a C2H2 zinc finger (ZF) and a Myb motif, located within the N-terminal domain of the protein are explored in this report. These motifs do not appear to play a role in RT or the ability of the protein to bind the R2 RNA transcript. Methylation and missing nucleoside interference-based DNA footprints using polypeptides to the N-terminal domain suggest the ZF and Myb motifs bind to regions −3 to −1 and +10 to +15 with reference to the insertion site. Mutations in these DNA sites or of the N-terminal protein domain blocked binding and the activity of the downstream subunit. Mutations of the protein domain also affected binding of the upstream subunit but not its function, suggesting the primary path to DNA target recognition by R2 involves both upstream and downstream subunits.

R2 elements are non-long terminal repeat (non-LTR) retrotransposons with a single open reading-frame encoding reverse transcriptase, DNA endonuclease and nucleic acid-binding domains. The elements are specialized for insertion into the 28 S rRNA genes of many animal phyla. The R2-encoded activities initiate retrotransposition by sequence-specific cleavage of the 28 S gene target site and the utilization of the released DNA 3' end to prime reverse transcription (target primed reverse transcription). The activity of the R2 polymerase on RNA templates has been shown to differ from retroviral reverse transcriptases (RTs) in a number of properties. We demonstrate that the R2-RT is capable of efficiently utilizing single-stranded DNA (ssDNA) as a template. The processivity of the enzyme on ssDNA templates is higher than its processivity on RNA templates. This finding suggests that R2-RT is also capable of synthesizing the second DNA strand during retrotransposition. However, R2-RT lacks the RNAse H activity that is typically used by retroviral and LTR-retrotransposon RTs to remove the RNA strand before the first DNA strand is used as template. Remarkably, R2-RT can displace RNA strands that are annealed to ssDNA templates with essentially no loss of processivity. Such strand displacement activity is highly unusual for a DNA polymerase. Thus the single R2 protein contains all the activities needed to make a double-stranded DNA product from an RNA transcript. Finally, during these studies we found an unexpected property of the highly sequence-specific R2 endonuclease domain. The endonuclease can non-specifically cleave ssDNA at a junction with double-stranded DNA. This activity suggests that second-strand cleavage of the target site may not be sequence specific, but rather is specified by a single-stranded region generated when the first DNA strand is used to prime reverse transcription.

R2 is a retrotransposable element that specifically inserts into the 28 S rRNA genes of arthropods. The element encodes a single protein with endonuclease activity that cleaves the 28 S gene target site and reverse transcriptase (RT) activity that uses the cleaved DNA to prime reverse transcription. Here we compare various properties of the R2 RT activity with those of the well characterized retroviral RT, avian myeloblastosis virus (AMV). In processivity assays using heterogeneous RNA templates, R2 RT can synthesize cDNA over twice the length of that synthesized by AMV RT and can synthesize cDNA over 4 times longer than AMV RT in assays with poly(rA) templates. The higher processivity of R2 RT compared with retroviral RTs is a result of the slower rate of dissociation of the enzyme from RNA templates. The elongation rates of the two enzymes are similar. Finally, a highly distinct property of the R2 RT, compared with retroviral enzymes, is its ability to displace RNA strands annealed to RNA templates during cDNA synthesis. We suggest that both the higher processivity and displacement properties of R2 RT compared with retroviral RT result from the greater affinity of the R2 protein for the RNA template upstream of its active site.The processivity of an enzyme can be defined as the number of rounds of catalysis that can be performed before the enzyme dissociates from the substrate (1). The most processive nucleic acid polymerases are the large multisubunit polymerases involved in genomic DNA replication that can polymerize for tens of thousands of bp before dissociation. The high processivity of these complexes can be explained because they form a toroid around the DNA substrate (sliding clamp model) that prevents the complex from dissociation (2). Most other nucleic acid polymerases form a structure resembling a right hand, in which the palm contains the catalytic residues of the active site while the fingers and thumb grasp the template loosely to allow lateral movement along the length of the template (3).Retroviral reverse transcriptases (RTs) 1 have a hand structure (4) and exhibit low levels of processivity for a replicative DNA polymerase (5, 6). In typical in vitro assays with templates of random base composition, retroviral RTs can extend a cDNA strand for only a few hundred nucleotides before dissociating. This low processivity has been suggested to be of advantage to the virus because it promotes recombination between RNA templates, thus allowing faster rates of evolution for avoidance of the vertebrate host's immune system (7). Despite this low processivity, production of full-length reverse transcripts occurs because polymerization occurs within a viral particle, allowing efficient reassociation of the reverse transcriptase with the RNA template (8).Two other classes of mobile elements in eukaryotes also use RTs to make DNA copies of their RNA transcript. One class, the LTR retrotransposons, assume structures and utilize mechanisms of reverse transcription and integration that are highly similar to th...

Several elements of oligoribonucleotide structure are important for efficient hydrolysis. We have found that the following factors influence oligoribonucleotide hydrolysis: (i) single-stranded structure of RNA flanking the scissile phosphodiester bond, (ii) the substituent on atom C-5 of the uridine adjacent to the cleaved internucleotide bond, (iii) the position of the scissile UA phosphodiester bond within a hairpin loop, (iv) the concentration of formamide, urea, ethanol and sodium chloride.

Single-stranded oligoribonucleotides containing UA and CA phosphodiester bonds can be hydrolyzed specifically under non-enzymatic conditions in the presence of spermidine, a biogenic amine found in a wide variety of organisms. In the present study, the rate of oligonucleotide and tRNA(i)(Met)hydrolysis was measured in the presence of spermidine and other biogenic amines. It was found that spermine [H(3)N(+)(CH(2))(3)(+)NH(2)(CH(2))(4)(+)NH(2)(CH(2))(3)(+)NH(3)] and putrescine [H(3)N(+)(CH(2))(4)(+)NH(3)] can replace spermidine [H(3)N(+)-(CH(2))(4)(+)NH(2)(CH(2))(3)(+)NH(3)] to induce the hydrolysis. For all three polyamines, a bell-shaped cleavage rate versus concentration relationship was observed. The maximum rate of hydrolysis was achieved at 0.1, 1.0 and 10 mM spermine, spermidine and putrescine, respectively. Moreover, we found that the hydrolysis requires at least two linked amino groups since two aminoalcohols, 2-aminoethanol and 3-aminopropanol, were not able to induce the cleavage of the phospho-diester bond. The optimal cleavage rate of the oligo-ribonucleotides was observed when amino groups were separated by tri- or tetramethylene linkers. The methylation of the amino groups reduced the ability of diamines to induce oligoribonucleotide hydrolysis. Non-enzymatic cleavage of tRNA(i)(Met)from Lupinus luteus and tRNA(i)(Met)from Escherichia coli demonstrate that both RNAs hydrolyze as expected from principles derived from oligoribonucleotide models.