Plasmid Map Research Articles

PlasMapper 3.0-a web server for generating, editing, annotating and visualizing publication quality plasmid maps.

PlasMapper 3.0 is a web server that allows users to generate, edit, annotate and interactively visualize publication quality plasmid maps. Plasmid maps are used to plan, design, share and publish critical information about gene cloning experiments. PlasMapper 3.0 is the successor to PlasMapper 2.0 and offers many features found only in commercial plasmid mapping/editing packages. PlasMapper 3.0 allows users to paste or upload plasmid sequences as input or to upload existing plasmid maps from its large database of>2000 pre-annotated plasmids (PlasMapDB). This database can be searched by plasmid names, sequence features, restriction sites, preferred host organisms, and sequence length. PlasMapper 3.0 also supports the annotation of new or never-before-seen plasmids using its own feature database that contains common promoters, terminators, regulatory sequences, replication origins, selectable markers and other features found in most cloning plasmids. PlasMapper 3.0 has several interactive sequence editors/viewers that allow users to select and view plasmid regions, insert genes, modify restriction sites or perform codon optimization. The graphics for PlasMapper 3.0 have also been substantially upgraded. It now offers an interactive, full-color plasmid viewer/editor that allows users to zoom, rotate, re-color, linearize, circularize, edit annotated features and modify plasmid images or labels to improve the esthetic qualities of their plasmid map and textual displays. All the plasmid images and textual displays are downloadable in multiple formats. PlasMapper 3.0 is available online at https://plasmapper.ca.

156. A Plasmid Manager, a Comprehensive Database for Archiving Plasmid DNA Sequence

A freely available plasmid database that is interoperable with popular freeware (such as a Plasmid Editor, or ApE [1]) has been developed by the Center of Information Technology (CIT) for the NIDA Optogenetics and Transgenic Technology Core (OTTC). As a core facility at the National Institute on Drug Abuse, the NIDA OTTC group has accrued a wealth of plasmid DNA data that can no longer be efficiently analyzed without a comprehensive cataloging system. The OTTC Plasmid Manager provides an intuitive plasmid database with analytical and search capabilities, offering a versatile platform for the storage and organization of plasmid sequences and their associated metadata. The database has been designed with informatics and visualization support to enhance the efficiency and analytic capabilities of the user, with a user-friendly interface. Annotations performed in ApE can be imported directly into the database, and unlike most commercially available and free DNA editing software, A Plasmid Manager (APM) also includes a mechanism to standardize the annotations of uploaded sequences, facilitating the direct comparison of multiple plasmids at the feature level. The visualization component generates a graphic representation of the plasmid map together with its features and annotations, allowing the visual comparison of multiple plasmids side-by-side. The contents of the database can be browsed and searched using a variety of criteria, including plasmid name, database accession number and associated features. This innovative plasmid database presents a new platform for molecular biologists to catalog plasmids relevant to their laboratory, saving time and enhancing the analytical capabilities of researchers.

591. The OTTC Plasmid Manager: A Comprehensive Database for Archiving Plasmid DNA Sequence

A freely available plasmid database that is interoperable with popular freeware (such as a Plasmid Editor, or ApE [1]) has been developed for our research group. Our group has accrued a wealth of data that can no longer be efficiently analyzed without a comprehensive cataloging system. The OTTC Plasmid Manager provides an intuitive plasmid database with analytical and search capabilities, offering a versatile platform for the storage and organization of plasmid sequences and their associated metadata. The database has been designed with informatics and visualization support to enhance the efficiency and analytic capabilities of the user, with a user-friendly interface. Annotations performed in ApE can be imported directly into the database, and unlike most commercially available and free DNA editing software, the Plasmid Manager also includes a mechanism to standardize the annotations of uploaded sequences, facilitating the direct comparison of multiple plasmids at the feature level. The visualization component generates a graphic representation of the plasmid map together with its features and annotations, allowing the visual comparison of multiple plasmids side-by-side. The contents of the database can be browsed and searched using a variety of criteria, including plasmid name, database accession number and associated features. This innovative plasmid database presents a new platform for molecular biologists to catalog plasmids relevant to their laboratory, saving time and enhancing the analytical capabilities of researchers.[1] Davis W. A Plasmid Editor. Retrieved September 9, 2014, from http://biologylabs.utah.edu/wayned/ape/

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details). To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3΄TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details). To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3Î"TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

An in silico DNA cloning experiment for the biochemistry laboratory

This laboratory exercise introduces students to concepts in recombinant DNA technology while accommodating a major semester project in protein purification, structure, and function in a biochemistry laboratory for junior- and senior-level undergraduate students. It is also suitable for forensic science courses focused in DNA biology and advanced high school biology classes. Students begin by examining a plasmid map with the goal of identifying which restriction enzymes may be used to clone a piece of foreign DNA containing a gene of interest into the vector. From the National Center for Biotechnology Initiative website, students are instructed to retrieve a protein sequence and use Expasy's Reverse Translate program to reverse translate the protein to cDNA. Students then use Integrated DNA Technologies' OligoAnalyzer to predict the complementary DNA strand and obtain DNA recognition sequences for the desired restriction enzymes from New England Biolabs' website. Students add the appropriate DNA restriction sequences to the double-stranded foreign DNA for cloning into the plasmid and infecting Escherichia coli cells. Students are introduced to computational biology tools, molecular biology terminology and the process of DNA cloning in this valuable single session, in silico experiment. This project develops students' understanding of the cloning process as a whole and contrasts with other laboratory and internship experiences in which the students may be involved in only a piece of the cloning process/techniques. Students interested in pursuing postgraduate study and research or employment in an academic biochemistry or molecular biology laboratory or industry will benefit most from this experience.

Study on the map of plasmid of Y. pestis in the R. opimus natural plague foci in Junggar Basin

Objective To investigate the type of plasmid map of Y. pestis in the R. opimas natural plague loci in Junggar Basin. Methods A total of 39 plasmid DNA of Y. pestis which were isolated from the natural plague loci of Junggar Basin, Tianshan Mountain, Kunlun Mountain, Qinghai-Tibet Plateau and In-ner Mongolia were extracted by the methods of Kado and Liu. The plasmid map was analyzed by the methods of agarose gel eleetrophoretogram. Results Two types of plasmid map were found in 26 Y. pestis which were isolated from Junggar Basin. Of them 23 were 6 × 106, 45 × 106 and 65 × 106 type of plasmid map, and 3 were 6 × 106, 45 × 106 and 72 × 106 type. Conclusion There are two types of plasmid map in the R. opi-mus natural plague loci in Junggar Basin. One type, which is the dominant type in this area, is 6 × 106, 45 × 106 and 65 × 106 type. This type is also similar to the dominant plasmid map type of the nature plague loci of Tianshan Mountain, Kunlun Mountain, Qinghai-Tibet Plateau and Inner Mongolia. The other type is 6 × 106, 45 × 106 and 72 × 106 type, and this type is new plasmid map type of Y. pestis in our country. Key words: Yersinia pestis; Plasmid; Junggar Basin

Construction of Targeting Vector for Expressing Human GDNF in Cattle Mammary Gland

Glial cell line-derived neurotrophic factor (GDNF) is a type of neurotrophic factor with significant potential in treatment of Parkinson's disease. Combining gene targeting of animal somatic cells with nuclear transfer technique has provided a powerful method to produce transgenic animal mammary gland bioreactor. The aim of this study was to construct a gene-targeting vector for the human gdnf gene knockin at the bovine beta-casein gene locus so that human GDNF protein can be produced in the mammary gland of the gene-targeted bovine. The constructed vector contains the 2.2 kb 5' homologous arm and the 5.7 kb 3' homologous arm. The human gdnf cDNA was located at the downstream of the 5' homologous arm. The neo gene placed between the 5' and 3' homologous arms as positive selection marker gene. The HSV-tk gene and DsRed2 gene were located outside the homologous recombinant area as negative selection marker genes, respectively. The recombinant plasmids were identified by restriction fragment analysis and partial DNA sequencing. The results show that the structure of the final constructed vector accords with the designed plasmid map. In order to analyze the bioactivity of the vector, the plasmid DNA was transfected into human mammary tumor cell line Bcap-37 by lipofectamine. Reverse transcription polymerase chain reaction and Western-blotting analysis showed that the transfected cells produced human GDNF mRNA and protein. The results show that the constructed targeting vector pNRTCNbG has bioactivity to efficiently express GDNF in mammary gland cells. At the same time, it is first time to confirm that human mammary tumor cell line Bcap-37 is valid for bioactivity analysis of mammary gland specific expression vector.

Complete nucleotide sequencing and analysis of the 65-kb highly conjugativeEnterococcus faeciumplasmid pMG1: identification of the transfer-related region and the minimum region required for replication

pMG1 (65.1 kb) is a highly conjugative, pheromone-independent Enterococcus faecium plasmid that carries a Tn4001-like transposon encoding gentamicin resistance. The complete nucleotide sequence (65 029 bp) of the pMG1 plasmid was determined and 73 ORFs lying in the same transcription orientation were identified. Sixty-one of the 73 ORFs showed a high degree of similarity (90-100% identity at the amino acid level) to the ORFs of the pHTbeta plasmids. Like the pHTbeta plasmid, 22 of the pMG1 plasmid ORFs showed homology with ORFs present on the pXO2 plasmid (96.2 kb), which is the virulence plasmid essential for capsular formation by Bacillus anthracis. Analysis of tra mutants created by Tn917 insertion and Northern analysis of transcripts indicated that ORFs 15-49, lying in the 31.7 kb region between 13.6 and 45.3 kb on the plasmid map, were related to transfer. This region was designated as the Tra I region of pMG1. A 5.9-kb HindIII fragment that replicates autonomously in Enterococcus faecalis was cloned and analysis of this fragment by deletion and in vitro insertion mutations showed that ORF10 (rep) and the inverted repeat sequence in the noncoding region between ORF8 and ORF9 were necessary for pMG1 replication.

190 THE REGULATION OF CHONDROCYTE DIFFERENTIATION BY RHO GTPASES AND THE ACTIN CYTOSKELETON

193 – Figure 1. Left: IL-1shRNA plasmid map showing coding region for IL-1shRNA loop. Right: IL-1β expression in chondrocytes conditioned by LPS exposure (LPS) and transduced with pSIREN-IL-1shRNA (pIL-1), pIRES-puro3-IGF (pIGF), or pIRES-puro3-IGF-IL-1shRNA (pIL1sh+IGF). LPS induced a robust IL-1 response, which was significantly reduced by IL-1 RNA interference (pIL-1shRNA or pIL-1sh+IGF). Chondrocytes transduced with pIGF continued to express elevated IL-1 mRNA under the influence of LPS. to study chondrocyte mechanobiological response to complex loading patterns.

Detection of Cloned strR, an Antibiotic Regulatory Gene, using RFLP and Nested PCR

The genetics of streptomycin production is well characterized in Streptomyces griseus. More than 25 clustered genes encode proteins involved in biosynthesis, regulation and transport functions. StrR, the pathway specific transcriptional activator or regulator that located in this cluster, then induces transcription of other streptomycin production genes by binding multiple sites in the gene cluster. We aim to put strR gene in to different multicopy and integrated expression vector specifically designed for Streptomyces. To start with, the isolated strR gene was ligated into pBluescript (pBs) vector and transformed into different strains of Escherichia coli. The correct structure of the recombinant plasmid, isolated from transformed E. coli, was confirmed using gel electrophoresis, PCR and double digested with restriction enzymes BamHI and EcoRI. Finally the plasmid map, named pFDstrR. This unique vector has a much expanded Multiple Cloning Site (MCS), which makes it suitable for different purposes of gene cloning and also site directed mutagenesis or gene targeting. This gene will be lifted up and transfer into different varieties of Streptomyces specific vectors in order to make different transgenic or genetically manipulated Streptomyces.

AiO, combining DNA/protein programs and oligo-management.

AiO (All in One) is a program for Windows, that combines typical DNA/protein features such as plasmid map drawing, finding of ORFs, translate, backtranslate and high quality printing with a number of databases. These databases allow the management of oligonucleotides, oligonucleotide-manufacturers, restriction enzymes, structural DNA and program users in a multi-user/multi-group environment. An AiO specific website, with the possibility to download is at: http://134.99.88.55/aio/ Examples with screen shots- http://134.99.88.55/aio/ : Manual (in PDF format)-http://134.99.88.55/aio/manual.pdf

Refined, Circular Restriction Map of the Bacillus thuringiensis subsp. israelensis Plasmid Carrying the Mosquito Larvicidal Genes

All the genetic elements responsible for the mosquito larval toxicity of Bacillus thuringiensis subsp. israelensis are located on one of its largest plasmids, nicknamed pBtoxis. Two linkage groups (with sizes of about 75 and 55 kb) have previously been mapped partially with respect to SacI and BamHI restriction sites (Ben-Dov et al., 1996), but linking them to a single circular plasmid unambiguously was impossible with the available data. To finalize the plasmid map, another rare cutting restriction endonuclease, AlwNI, was used in addition. The two linkage groups and the fragments generated by AlwNI were aligned on the circular plasmid, and known insertion sequences were localized on the refined map. Pulsed-field electrophoresis revealed that the total size of pBtoxis (137 kb) was larger than thought before.

Organization of biogenesis genes for aggregative adherence fimbria II defines a virulence gene cluster in enteroaggregative Escherichia coli.

Several virulence-related genes have been described for prototype enteroaggregative Escherichia coli (EAEC) strain 042, which has been shown to cause diarrhea in human volunteers. Among these factors are the enterotoxins Pet and EAST and the fimbrial antigen aggregative adherence fimbria II (AAF/II), all of which are encoded on the 65-MDa virulence plasmid pAA2. Using nucleotide sequence analysis and insertional mutagenesis, we have found that the genes required for the expression of each of these factors, as well as the transcriptional activator of fimbrial expression AggR, map to a distinct cluster on the pAA2 plasmid map. The cluster is 23 kb in length and includes two regions required for expression of the AAF/II fimbria. These fimbrial biogenesis genes feature a unique organization in which the chaperone, subunit, and transcriptional activator lie in one cluster, whereas the second, unlinked cluster comprises a silent chaperone gene, usher, and invasin reminiscent of Dr family fimbrial clusters. This plasmid-borne virulence locus may represent an important set of virulence determinants in EAEC strains.

In vitro replication of mitochondrial plasmid mp1 from the higher plant Chenopodium album (L.): a remnant of bacterial rolling circle and conjugative plasmids?

According to the endosymbiotic theory, mitochondrial genomes evolved from the chromosome of an α-proteobacterium-like ancestor and developed during evolution an extraordinary variation in size, structure and replication. We studied in vitro DNA replication of the mitochondrial circular plasmid mp1 (1309 bp) from the higher plant Chenopodium album (L.) as a model system that replicates in a manner reminiscent of bacterial rolling circle plasmids. Several mp1 subclones were tested for their ability to support DNA replication using a newly developed in vitro system. Neutral/neutral two-dimensional gel electrophoresis of the in vitro products revealed typical simple Y patterns of intermediates consistent with a rolling circle type of replication. Replication activity was very high for a Bam HI-restricted total plasmid DNA clone, a 464 bp BamHI/ KpnI fragment and a 363 bp Bam HI/ SmaI fragment. Further subcloning of a 148 bp BamHI/ EcoRI fragment resulted in the strongest in vitro DNA replication activity, while a 1161 bp-template outside of this region resulted in a substantial loss of activity. Electron microscopic studies of in vitro DNA replication products from the highly active clones also revealed sigma-shaped molecules. These results support our in vivo data for the presence of a predominant replication origin between positions 628 and 776 on the plasmid map. This sequence shares homology with double-stranded rolling circle origin ( dso) or transfer origin ( oriT) nicking motifs from bacterial plasmids. mp1 is the first described rolling circle plasmid in eukaryotes.

Restriction map of the Salmonella enteritidis virulence plasmid and its homology with the plasmid of Salmonella typhimurium

A restriction map of the 61 kb virulence plasmid of Salmonella enteritidis was constructed and compared with the Salmonella typhimurium virulence plasmid map. Polymerase chain reaction was used to detect a region homologous to rck gene in the S. enteritidis plasmid which was localized in the map by hybridization. Regions homologous to traT and vagD/vagC loci were not detected in this plasmid by this method.

Characterization of transfer regions within the HII incompatibility group plasmid pHH1508a.

A circular map of the 208-kilobase IncHII plasmid pHH1508a was constructed by using the restriction endonucleases XbaI, XhoI, and NotI. Tn5 insertion mutagenesis showed that transfer genes were distributed widely over the plasmid map. Analysis of the deletion mutant pDT1178 indicated that genes required for conjugative pilus production are located in a 96.0-kilobase region of the plasmid.

PLASMID MAP: a microcomputer program for display and storage of plasmid data

We describe a plasmid map program which runs on an IBM PC microcomputer and facilitates the drawing of circular plasmid maps. The user enters information from the keyboard in the form of restriction enzyme sites, genes and their locations, and other plasmid markers such as promoters, origins, or other sites. This information can then be stored in a file for future reference. The plasmid map can be displayed on the screen, printed on a dot-matrix printer, or plotted on a Hewlett Packard HP7475A plotter.

Lysis protein encoded by plasmid ColA-CA31. Gene sequence and export.

A gene, cal, coding for a polypeptide needed for the release of colicin A from Escherichia coli cells has been identified by transposon insertion. The cal gene was located on the ColA plasmid map adjacent to cai, the gene coding for colicin A immunity protein, and therefore 592 bases downstream from caa, the structural gene for colicin A. Transcription of cal is in the same direction as caa, that is in the opposite direction to cai. Its sequence has been determined and the predicted amino acid composition features a basic N-terminal end followed by a serie of hydrophobic residues similar to the signal sequence in precursors of exported proteins. The C-terminal part also contains a core of hydrophobic residues. The overall amino acid sequence of the cal protein is homologous to that of lytic proteins encoded by the related plasmids pColE1, and pCloDF13. The cal protein has been identified on urea-SDS-polyacrylamide gels by selective labelling with various radioactive amino acids and its synthesis is co-induced with that of colicin A. The cal protein undergoes slow processing with loss of the N-terminal "signal" region and the mature form is released into the medium together with colicin A.

Localization of genes responsible for replication and immunity to colicin A on plasmid ColA-CA31.

The region containing the origin and regulatory sites for replication as well as the immunity gene (iaa) have been localized on the plasmid ColA-CA31. The region involved in replication functions of ColA can be hybridized with that of ColE1. It is located between 1 and 1 kb on the plasmid map previously published (Morlon et al. 1982a). A 0.50 Kb HincII fragment of ColA can be weakly hybridized to the ColE1 immunity region. This fragment contains iaa since directed in vitro mutagenesis at an internal restriction site can abolish the immunity to colicin A; however, it does not contain the entire iaa. Knowing the localization of regions involved in autonomous DNA replication and immunity, a mini-ColA plasmid was constructed that contains these two regions. The mini-ColA of 2.8 Kb can be amplified in the presence of chloramphenicol and confers the immunity to transformants. It thus constitutes a useful cloning vector. Expression of ColA and of the various constructed plasmids in the maxicell system suggests that the immunity protein has a molecular weight of about 18-20 Kd.