Rolling Circle Mode Of Replication Research Articles

Arabidopsis thaliana NAC083 protein interacts with Mungbean yellow mosaic India virus (MYMIV) Rep protein

Geminiviral replication initiator protein (Rep) is a key player in geminiviral rolling circle mode of replication. However, the virus exploits various host cellular machineries for its replication. Study of these host factors is important to understand the geminiviral DNA replication in greater details. With this view, we screened for the peptides interacting with the Rep protein of a representative of geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), employing phage display technique. Through this screen, we have identified a host transcription factor, NAC083, as a potential MYMIV-Rep-binding partner. In silico docking studies also suggested possible binding of NAC083 peptide to MYMIV-Rep. We validated the interaction between MYMIV-Rep and Arabidopsis thaliana full-length NAC083 protein using in vitro pull-down assay and yeast two-hybrid analysis. NAC proteins are well-known transcription factors belonging to the largest gene families in plants. This study demonstrates for the first time the interaction of NAC083, a member of NAC transcription factor family, with MYMIV-Rep protein thereby indicating its possible role in MYMIV DNA replication.

Development of plasmid vector and electroporation condition for gene transfer in sporogenic lactic acid bacterium, Bacillus coagulans

Bacillus coagulans is a sporogenic lactic acid bacterium that ferments glucose and xylose, major components of plant biomass, a potential feedstock for cellulosic ethanol. The temperature and pH for optimum rate of growth of B. coagulans (50 to 55 °C, pH 5.0) are very similar to that of commercially developed fungal cellulases (50 °C; pH 4.8). Due to this match, simultaneous saccharification and fermentation (SSF) of cellulose to products by B. coagulans is expected to require less cellulase than needed if the SSF is conducted at a sub-optimal temperature, such as 30 °C, the optimum for yeast, the main biocatalyst used by the ethanol industry. To fully exploit B. coagulans as a platform organism, we have developed an electroporation method to transfer plasmid DNA into this genetically recalcitrant bacterium. We also constructed a B. coagulans/E. coli shuttle vector, plasmid pMSR10 that contains the rep region from a native plasmid (pMSR0) present in B. coagulans strain P4-102B. The native plasmid, pMSR0 (6823 bp), has 9 ORFs, and replicates by rolling-circle mode of replication. Plasmid pNW33N, developed for Geobacillus stearothermophilus, was also transformed into this host and stably maintained while several other Bacillus/ Escherichia coli shuttle vector plasmids were not transformed into B. coagulans. The transformation efficiency of B. coagulans strain P4-102B using the plasmids pNW33N or pMSR10 was about 1.5 × 10 16 per mole of DNA. The availability of shuttle vectors and an electroporation method is expected to aid in genetic and metabolic engineering of B. coagulans.

The oligomeric Rep protein of Mungbean yellow mosaic India virus (MYMIV) is a likely replicative helicase

Geminiviruses replicate by rolling circle mode of replication (RCR) and the viral Rep protein initiates RCR by the site-specific nicking at a conserved nonamer (TAATATT↓ AC) sequence. The mechanism of subsequent steps of the replication process, e.g. helicase activity to drive fork-elongation, etc. has largely remained obscure. Here we show that Rep of a geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), acts as a replicative helicase. The Rep-helicase, requiring ≥6 nt space for its efficient activity, translocates in the 3′→5′ direction, and the presence of forked junction in the substrate does not influence the activity to any great extent. Rep forms a large oligomeric complex and the helicase activity is dependent on the oligomeric conformation (∼24mer). The role of Rep as a replicative helicase has been demonstrated through ex vivo studies in Saccharomyces cerevisiae and in planta analyses in Nicotiana tabacum. We also establish that such helicase activity is not confined to the MYMIV system alone, but is also true with at least two other begomoviruses, viz., Mungbean yellow mosaic virus (MYMV) and Indian cassava mosaic virus (ICMV).

Characterisation of a novel plasmid p9785S from Lactobacillus johnsonii FI9785

Lactobacillus johnsonii FI9785, a strain originally isolated from poultry gastrointestinal tract for its probiotic function as a competitive excluder of pathogens, was found to contain two cryptic plasmids of 3.5 and 25.6 kb. Nucleotide sequence analysis of the entire small plasmid, designated p9785S (3471 bp), indicated a G + C content of 35.8%, and revealed two open reading frames (orfs). The product of orf1 exhibited similarity to the relaxases of mobilizable plasmids, whereas the product of orf2 displayed significant homology to replication proteins of plasmids which use the rolling circle mode of replication. A conserved double-strand origin of replication was also present in p9785S. A definite minus origin was not identified although a region with extensive intrastrand base pairing potential was revealed. A 1.4 kb fragment encoding the chloramphenicol resistance gene was cloned into p9785S and the resulting vector, pFI2431, was stably maintained when introduced into the parent Lactobacillus cells.

Replication of the herpes simplex virus genome: does it really go around in circles?

Herpes simplex virus type 1 (HSV-1) is the prototype and best-studied virus of the α-herpesvirus group. HSV-1 undergoes a rapid productive replication cycle in host epithelial cells in vivo and in susceptible cultured cells in vitro . However, HSV-1 is also neurotropic and establishes life-long latent infections in the sensory neurons of the host, where the genome is in a nonreplicating chromatin-associated state (1), and viral gene expression is largely repressed (2). Although the mechanisms for the establishment of latency are poorly understood, reactivation requires an immediate early protein, termed ICP0, which is also required for efficient initiation of lytic infection (3–7). The 152-kb linear double-stranded HSV-1 genome is divided into long and short regions of unique sequences, termed UL and US, which are bounded by regions of inverted repeats, termed internal repeats. The genome termini are bounded by regions of direct repeats, termed terminal repeats (Fig. 1). Furthermore, the HSV-1 genome undergoes inversions that result from recombination events mediated by the viral DNA replication machinery, which yield four genomic isomers in equimolar amounts (8, 9). In vivo , the HSV-1 genome has been found to exist in at least three different states: linear, circular, and concatemeric. In the virion, genomes are linear, but within hours after infection, end joining has been found to occur, resulting in ``endless genomes'' that have been interpreted to represent circles (10–12). Although the mechanism of genome circularization has not been established, one model predicts that circularization may involve recombination of the terminal repeats, resulting in a θ replication mode for the initial round of replication (13), providing the template for a rolling circle mode of replication (14). Thus, genome circularization has been thought to be a prerequisite for viral DNA replication, although …

Isolation and Characterization of Plasmids from Strains of Streptococcus Thermophilus Bulgarian Yogurt Starters

ABSTRACTTwenty Bulgarian Streptococcus thermophilus strains used in industrial milk fermentation were analyzed for their plasmid content. Six of them were found to contain one or two plasmids. The newly isolated plasmids were divided into three groups based on DNA homology. Six plasmids, sharing homology with pt38 and p2992, were classified within group I, two plasmids were classified into group II and one plasmid, p2991, possessing homology only with itself, was classified in Group III. Hybridization experiments revealed low homology between streptococcal and Lactobacillus plasmids. Using plus-origin- specific (dso) DIG-labeled probes we determined that two of the plasmids (pt38 and p2992) belong to the pC194 plasmid family, that uses the rolling circle mode of replication. One plasmid from group I, pt38 was further characterized. We constructed a restriction map and identified the restriction fragment bearing the dso of replication. pt38 appears to be an excellent candidate for creation of a shuttle vector for gene expression in Escherichia coli and the Streptococcus thermophilus because it is small in size, has five unique restriction sites and known location of the plus-origin of replication.

Molecular Analysis of a Mobilizable Theta-Mode Trimethoprim Resistance Plasmid from Coagulase-Negative Staphylococci

TheStaphylococcus epidermidisplasmid pSK639 is the prototype of a newly described family of small plasmids identified in clinical staphylococcal isolates. pSK639 is 8 kb in length and possesses a composite structure consisting of an IS257-flanked segment mediating trimethoprim resistance (Tpr), and regions responsible for replication and mobilization of the plasmid. Comparative sequence analysis suggests that a pSK639-like plasmid may represent a progenitor of previously identified staphylococcal Tprdeterminants related to the transposon-like structure, Tn4003.In contrast to the small staphylococcal plasmids characterized to date that all utilize a rolling circle mode of replication, the replication region of pSK639 was found to contain features typical of an iteron-controlled theta-mode replicon. pSK639 is the first small plasmid of this type to be identified in the staphylococci.

Herpes simplex virus DNA replication.

The Herpesviridae comprise a large class of animal viruses of considerable public health importance. Of the Herpesviridae, replication of herpes simplex virustype-1 (HSV-1) has been the most extensively studied. The linear 152-kbp HSV-1 genome contains three origins of DNA replication and approximately 75 open-reading frames. Of these frames, seven encode proteins that are required for originspecific DNA replication. These proteins include a processive heterodimeric DNA polymerase, a single-strand DNA-binding protein, a heterotrimeric primosome with 5'-3' DNA helicase and primase activities, and an origin-binding protein with 3'-5' DNA helicase activity. HSV-1 also encodes a set of enzymes involved in nucleotide metabolism that are not required for viral replication in cultured cells. These enzymes include a deoxyuridine triphosphatase, a ribonucleotide reductase, a thymidine kinase, an alkaline endo-exonuclease, and a uracil-DNA glycosylase. Host enzymes, notably DNA polymerase alpha-primase, DNA ligase I, and topoisomerase II, are probably also required. Following circularization of the linear viral genome, DNA replication very likely proceeds in two phases: an initial phase of theta replication, initiated at one or more of the origins, followed by a rolling-circle mode of replication. The latter generates concatemers that are cleaved and packaged into infectious viral particles. The rolling-circle phase of HSV-1 DNA replication has been reconstituted in vitro by a complex containing several of the HSV-1 encoded DNA replication enzymes. Reconstitution of the theta phase has thus far eluded workers in the field and remains a challenge for the future.

Yeast 20 S RNA replicon. Replication intermediates and encoded putative RNA polymerase

The 20 S RNA genome is a circular single-stranded replicon, present in most laboratory yeast strains, whose copy number is induced 10,000-fold by transfer of cells to acetate medium without a carbon source. We have sequenced most of the 20 S RNA genome, and the (+) strand has a long open reading frame with the potential to encode a protein with homology to viral RNA-dependent RNA polymerases. The presence of a typical cAMP-dependent phosphorylation site in the putative RNA polymerase suggests that the acetate amplification of the 20 S RNA genome might be mediated by cAMP, a signal known to transmit the same nutritional status information to the sporulation-control system. Our inability to clone across the gap in the sequence suggests either autocatalytic cleavage of the RNA in the reverse transcriptase reaction, an unusual linkage of 5' and 3' ends of a fundamentally linear molecule, or a structure unusually resistant to reverse transcription. The identity of our sequence with that of the accompanying paper (Rodriguez-Cousino, N., Esteban, L.M., and Esteban, R. (1991) J. Biol. Chem. 266, 12772-12778) for W double-stranded RNA (dsRNA) suggests that W is the replicative form of 20 S RNA. The presence of single-stranded (+) and (-) strands and greater than unit length molecules suggests a rolling circle mode of replication as has been suggested for viroids.

Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1

The nucleotide sequence of the Lactococcus lactis broad-host-range plasmid pWVO1, replicating in both gram-positive and gram-negative bacteria, was determined. This analysis revealed four open reading frames (ORFs). ORF A appeared to encode a trans-acting 26.8-kDa protein (RepA), necessary for replication. The ORF C product was assumed to play a regulatory role in replication. Both RepA and the ORF C product showed substantial sequence similarity with the Rep proteins of the streptococcal plasmid pLS1. In addition, the plus origin of replication was identified on the basis of strong similarity with the plus origin of pLS1. Derivatives of pWVO1 produced single-stranded (ss) DNA in Bacillus subtilis and L. lactis, suggesting that this plasmid uses the rolling-circle mode of replication. In B. subtilis, but not in L. lactis, the addition of rifampicin resulted in increased levels of ssDNA, indicating that in the former organism the host-encoded RNA polymerase is involved in the conversion of the ssDNA to double-stranded plasmid DNA (dsDNA). Apparently, in L. lactis the conversion of ss to ds pWVO1 DNA occurs by a mechanism which does not require the host RNA polymerase.

Genes and regulatory sequences of bacteriophage ∅X174

Fragments of the DNA of bacteriophage ∅X174 were inserted in the plasmids pACYC177 and pBR322, in order to test the in vivo effects of separate phage genes and regulatory sequences. The ∅X174 inserts were identified by recombination and complementation with phage mutants, followed by restriction enzyme analysis. The genes B, C, F and G can be maintained stably in the cell even when there is efficient expression of these viral genes. Recombinant plasmids with the complete genes D and E can only be maintained when the expression of these genes is completely blocked. Expression of complete H and J genes could not yet be demonstrated. The intact gene A was apparently lethal for the host cell, as it was never found in the recombinants. The genes F and G are expressed, even when they are not preceded by one of the well characterized viral or plasmid promoter sequences. Screening of the nucleotide sequence of ∅X174 gives two promoter-like sequences just in front of the two genes. Viral sequences with replication signals (the ∅X174 (+) origin of replication, the initiation site for complementary strand synthesis and the incompatibility sequence) appeared to be functional also when inserted in recombinant plasmids. A plasmid with the ∅X (+) origin can be forced to a rolling circle mode of replication. The A protein produced by infecting phages works in trans on the cloned viral origin. The (−) origin can function as initiation signal for complementary strand synthesis during transduction of single-stranded plasmid DNA. The intracellular presence of the incompatibility sequence on a plasmid prevents propagation of infecting phages.