Production and characterization of novel ssrna bacteriophage. Therefore, this approach should be used for different diagnostic nats, as demonstrated in several rtpcr assays. Complete genome sequence of bacteriophage t5 sciencedirect. The first step in the rna sequencing has been taken with the studies performed on bacteriophage ms2 by walter et al. Preparation and evaluation of ms2 bacteriophagelike. You can put as many letters on a sequence line as you want, and a fasta file can have as many lines as necessary to include your phage genome sequence. Type if crisprcas provides protection from dna, but not. The rna genome maturation protein a complex is released from the capsid upon host receptor binding pubmed. X174, the first triumph of the new, rapid sequencing methods developed in the laboratories of walter gilbert and fred sanger 553,743. Ms2 is a member of a family of closely related bacterial viruses that includes bacteriophage f2, bacteriophage q. Use of bacteriophage ms2 as an internal control in viral. Specifically, the contrast variation technique was employed to determine the molecular weight of the individual components of the ms2 virion protein shell and genomic rna and the spatial relationship of the genomic rna to its. The first genome, that of rna bacteriophage ms2, was sequenced in 1976, in a truly heroic feat of direct determination of an rna sequence 225.
Another icosahedral virus that has been used extensively as a bioconjugation target is bacteriophage ms2, or simply ms2. Ms2 is a singlestranded rna bacteriophage that infects its host via adsorption to bacterial pili. Ms2 tagging is a technique based upon the natural interaction of the ms2 bacteriophage coat protein with a stemloop structure from the phage genome, which is used for biochemical purification of rnaprotein complexes and partnered to gfp for detection of rna in living cells. The ms2 genome sequence was absent from the human specimens, cell cultures, and veterinary samples. The term was derived from bacteria and the greek phagein, meaning to devour. The cdna of encoding ap and cp sequences of ms2 were reversely transcribed from ms2 genome mrnas purchased from sigma by primers ms2f and ms2r using a onestep rtpcr kit takara, shiga, japan and cloned into tvector pmd 19 simple. Bacteriophage ms2 has been the first known gene in 1972 and the first sequenced genome in 1976 bergallo et al. A secondary structure model has also been proposed. The potential of the rna phage ms2 to accommodate extra amino acids in its major coat protein has been examined.
Daniel larsons lab contains the insert ms2 coat protein fused to egfp and is published in elife. Recircularized genomes were then transfected into suitable host cells. The complete genome sequence of phage p8625 is 32,894 bp long with 51. Its capsid consists of a 27 nm hollow sphere, which, like many other t 3 viruses, is formed from 180 sequence identical monomers. Consensus sequence and structure of the minimal hammerhead ribozyme. Walter recruited very talented phd students, and his team set out to determine the genome sequence of the escherichia coli bacteriophage ms2. Interactions of escherichia coli rna with bacteriophage. Asymmetric cryoem reconstruction of phage ms2 reveals. The present article describes the process of preparation and use of such pcv ms2 phagelike particles ms2 plp in rtqpcr detection and quantification of enteric rna viruses. While numerous bacillus phages have been isolated up till now only very few bacillus cereus phages have been characterized in detail. Frontiers preparation of ms2 phagelike particles and. Some nucleotide sequences from the rnapolymerase gene journal. Figure 3 shows the sequence coverage obtained for three viruses.
In situ structures of the genome and genomedelivery apparatus in ssrna bacteriophage ms2. Find msds or sds, a coa, data sheets and more information. Bacteriophage ms2 is a 275 a rna virus that infects male escherichia coli. Their products are necessary for phage maturation, encapsidation, lysis of the host, and phage rna replication, respectively.
Ms2 is also used as a pathogen simulant in the research, development, and testing including open air tests of methods, systems, and devices for the detection of. In rna bacteriophages, gene expression is controlled at the level of translation. An introduction to phage whole genome sequencing and. Escherichia virus ms2 is an icosahedral, positivesense singlestranded rna virus that infects the bacterium escherichia coli and other members of the enterobacteriaceae. This was accomplished by walter fiers and his team, building upon their earlier milestone in 1972 of the first gene to be completely sequenced, the ms2 coat protein. In 1961, ms2 was isolated by alvin john clark and recognized as an rnacontaining phage very similar to bacteriophage f2 in 1976, the ms2 genome was the first genome to be completely sequenced.
In the ms2 crystal structure, this part of the coat protein forms a loop that extends from the outer surface of the icosahedral virion. The invention discloses a plasmid expressing ms2 phage capsid protein and maturase. Viral genome sequencing by random priming methods bmc. Here the authors visualize the ms2 virion with asymmetric cryoem reconstruction, revealing that. Realtime fluorogenic reverse transcriptionpcr assays for.
Addendum bacteriophage ms2 genomic rna encodes an assembly instruction manual for its capsid peter g. Interactions of escherichia coli rna with bacteriophage ms2. Pdf bacteriophage ms2 rna is 3569 nucleotides long. Pdf complete nucleotide sequence of bacteriophage ms2. Abe76i, is a highly specific rabbit polyclonal antibody that targets escherichia phage ms2 bacteriophage ms2 capsid protein and has been tested in immunoprecipitation and western blotting.
The first sequenced genome was that of the 3569nucleotide singlestranded rna ssrna bacteriophage ms2. Marian watermans lab contains the insert ms2 coat protein and is published in mol cell proteomics. These are some of the smallest known genomes with only four and. Information from sequence entries show organism modifiers. May, 2019 protein shells assembled from viral coat proteins are an attractive platform for development of new vaccines and other tools such as targeted bioimaging and drug delivery agents. We have also found that the method can result in complete genome sequence of viruses ranging in size from 3,00015,000 kb in a single experimental procedure. The singlestranded rna genome of bacteriophage ms2 is 3,569 nt long and contains 4 genes fig. It is proved through experiments that a novel ms2 phage capsid protein gene sequence and a novel maturase gene. This is the first report of the complete genome sequence of a lytic phage that infects the verrucomicrobia, for which the name verrucophage is proposed.
Accession numbers of all the entries listed below may be downloaded as a text file for use in downloading using the sequence version archive. Cn105969786a plasmid expressing ms2 phage capsid protein. In 1972, walter fiers university of ghent, belgium was the first to establish the complete nucleotide sequence of a gene and in 1976, of the viral genome of bacteriophage ms2. Bacteriophage ms2 genomic rna encodes an assembly instruction. Whole phage genome sequencing and annotation phages can usually be mixed into a single index or pool if they are not similar to each other different hosts different morphotypes phage 1 phage 2 phage 3 phage 1 contig a. Finally, when our design and assembly process resulted in viable phage, we picked plaques and sequenced genomic dna from clonal lineages. At the rna level, the insert forms a large loop at the top of an. Complete nucleotide sequence of bacteriophage ms2 rna. The viral genomes resource is a collection of viral genomic sequences that is a part of the entrez genomes, which provides curated sequence data and annotations of complete genomes to the scientific community. Antienterobacterio phage ms2 coat protein anti enterobacteriophage ms2 coat protein, cat.
Genome sequence and analysis of a broadhost range lytic. Comparison of uvinduced inactivation and rna damage in. Frontiers preparation of ms2 phagelike particles and their. Involved in viral genome encapsidation through the interaction between a capsid protein dimer and the multiple packaging signals present in the rna genome pubmed. Bacteriophage ms2 an overview sciencedirect topics.
Biological properties, such as ribosome binding and codon interactions can now be disc. Ms2 bacteriophage capsid studied using allatom molecular. A moredetailed, tabdelimited list is also available. The rest of the line is a description of the sequence free format. To simulate the pooled libraries, genomes were randomly sampled with replacement from specific sets of phage genomes, either using all phage genomes or only those confined to a particular host e. Despite the recent accumulation of vast amounts of dna and rna sequence data, only 12 representative ssrna phage genome sequences are available from the ncbi genome database june 2019. T4 biology and its genomic sequence provide the bestunderstood model for modern functional genomics and proteomics. An introduction to phage whole genome sequencing and annotation. According to the basic theory of armored rna technology, almost any exogenous rna in a suitable size can be into downstream of the truncated ms2 phage genome and nonspecifically assembled into the expressed ms2 phage capsid with resulting formation of target rna armored by viruslike particles vlps pasloske et al. Bacteriophages are composed of proteins that encapsulate a dna or rna genome, and may have structures that are either simple. However, it was not known whether ms2 cp binds any li rna and what role such interactions might play. Assessing illumina technology for the highthroughput.
Capsid protein selfassembles to form an icosahedral capsid with a t3 symmetry, about 26 nm in diameter, and consisting of 89 capsid proteins dimers 178 capsid proteins pubmed. Type if crisprcas provides protection from dna, but not rna. The 121,752bp genome sequence of bacteriophage t5 was determined. Smallangle neutron scattering sans has been used to extend the structural characterization of the ms2 phage by examining its physical characteristics in solution.
The result of this calculation revealed that the concentration of ms2 plp in a suspension of od 260 was 1 is 2. Amino acid residues thr45, which is universally conserved among the small rna phages, and thr59 are part of the specific rna binding pocket and interact directly with the rna. Pdf complete nucleotide sequence of bacteriophage ms2 rna. Biological properties, such as ribosome binding and codon interactions can now be discussed on a molecular basis. Source enterobacteria phage ms2 organism enterobacteria phage ms2 viruses. We have several phage species isolated from sewage and we need to sequence their dna. The xbaisite was used for insertion of the ribozyme sequence.
Bacteriophage t4 genome microbiology and molecular biology. Jun 20, 2014 construction and evolution of a ribozyme in phage ms2. A maturation protein a escherichia phage ms2 a gene. The structural characteristics of the capsid and the dynamics of the surrounding solution were examined using molecular dynamics simulation. Bacteriophage ms2 is used in place of pathogenic viruses in a wide variety of studies that range from testing of compounds for disinfecting surfaces to studying environmental transport and fate of pathogenic viruses in groundwater. In conclusion, the use of ms2 phage to control clinical diagnostic nat was demonstrated.
Bacteriophage ms2 genomic rna encodes an assembly instruction manual for its capsid peter g. The difficulty in detecting rna phages in metagenomic datasets raises questions as to their abundance. Citeseerx document details isaac councill, lee giles, pradeep teregowda. The epitope sequence 1160 came from the obycha2010 strain genbank. In the ms2 crystal structure, this part of the coat protein forms a loop that extends from the outer surface of the. Summary phage t4 has provided countless contributions to the paradigms of genetics and biochemistry. Accordingly, a pentapeptide was encoded in the genome as an nterminal extension. It is one of the bestcharacterized phage in the t5like viruses genus of the siphoviridae family. Binds to sequences located toward each end of the genome, hence circularizing it pubmed. Apr 19, 2019 the allatom model of an ms2 bacteriophage particle without its genome the capsid was built using highresolution cryoelectron microscopy em measurements for initial conformation. Rna folding kinetics regulates translation of phage ms2. Ranson a, roman tuma, and reidun twarockb aastbury center for structural molecular biology. The method available here is the illumina hiseq or illumina miseq.
More recently, the technique has been used to monitor the appearance of rna in living cells, at the site of. Nov 01, 2000 these binding data have been supported both by the crystal structure of ms2 cp complexed with the rna 17,18 and by the randomized sequence selex with closely related phage r17 coat protein. Variations on gene expression, including overlapping genes, internal translation initiation, spliced. Viruslike particles vlps derived from the singlestranded rna ssrna bacteriophage coat proteins cps have been important and successful contenders in the area due to their simplicity and robustness. The maturation protein is required for the typical attachment of the phage to the side of the bacterial pili pubmed. Its complete genome sequence of 168,903 bp encodes about 300 gene products. In ms2 assembly of phage particles results from an interaction between a coat protein dimer and a stemloop of the rna genome the operator hairpin. The rna genomematuration protein a complex is released from the capsid upon host receptor binding pubmed. The nucleotide sequence has been established for the third and last gene, which codes for the replicase protein. The nucleotide sequence has been established for the third and last gene, which codes for the. Its capsid consists of a 27 nm hollow sphere, which, like many other t 3 viruses, is formed from 180 sequenceidentical monomers.
The ssrna phage ms2 is a nonenveloped virus with a positivesense monopartite genome of 3569 nt and was the first biological entity to have its entire genome sequenced 14. Here we present data on the large, virulent, broadhostrange b. This phage was chosen because it provided a means to isolate high amounts of intact rna, as it is sheltered by the phage coat, and because the ms2 genome serves as a mrna for this virus. Complete genome sequence of the broad host range single. The linear and doublestranded dna genome is nicked at specific. Bacteriophage t4 genome microbiology and molecular. The complete genome sequences of 11 leviviridae phages have been deposited in. Pa14 provides sequencespecific elimination of invading dna 1,2.
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