Showing papers on "Restriction map published in 2008"

Scaffolding and validation of bacterial genome assemblies using optical restriction maps

Abstract: Motivation: New, high-throughput sequencing technologies have made it feasible to cheaply generate vast amounts of sequence information from a genome of interest. The computational reconstruction of the complete sequence of a genome is complicated by specific features of these new sequencing technologies, such as the short length of the sequencing reads and absence of mate-pair information. In this article we propose methods to overcome such limitations by incorporating information from optical restriction maps. Results: We demonstrate the robustness of our methods to sequencing and assembly errors using extensive experiments on simulated datasets. We then present the results obtained by applying our algorithms to data generated from two bacterial genomes Yersinia aldovae and Yersinia kristensenii. The resulting assemblies contain a single scaffold covering a large fraction of the respective genomes, suggesting that the careful use of optical maps can provide a cost-effective framework for the assembly of genomes. Availability: The tools described here are available as an open-source package at ftp://ftp.cbcb.umd.edu/pub/software/soma Contact: mpop@umiacs.umd.edu

Molecular approaches for identification and characterization of lactic acid bacteria

Abstract: The last few years have produced a revolution in the development of very sensitive, rapid, automated, molecular detection methods for a variety of various species of lactic acid bacteria (LAB) associated with food and dairy products Nowadays many such strains of LAB are considered probiotics The genome-based methods are useful in identifying bacteria as a complementary or alternative tool to phenotypical methods Over the years, identification methodologies using primers that target different sequences, such as the 16S ribosomal RNA (rRNA)-encoding gene, the 16S-23S rRNA intergenic spacer region, the 23S rRNA-encoding, recA and ldhD genes; randomly amplified polymorphic DNA, restriction fragment length polymorphism, denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, amplification rDNA restriction analysis, restriction enzyme analysis, rRNA, pulse field gel electrophoresis and amplification fragment length polymorphism have played a significant role in probiotic bacteriology Hence, the aim of this review is to provide an overview of some rapid and reliable polymerase chain reaction-based molecular methods used for identifying and differentiating closely related species and strains of LAB associated with food and industry

The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases

Abstract: Restriction endonucleases are the basic tools of molecular biology. Many restriction endonucleases show relaxed sequence recognition, called star activity, as an inherent property under various digestion conditions including the optimal ones. To quantify this property we propose the concept of the Fidelity Index (FI), which is defined as the ratio of the maximum enzyme amount showing no star activity to the minimum amount needed for complete digestion at the cognate recognition site for any particular restriction endonuclease. Fidelity indices for a large number of restriction endonucleases are reported here. The effects of reaction vessel, reaction volume, incubation mode, substrate differences, reaction time, reaction temperature and additional glycerol, DMSO, ethanol and Mn(2+) on the FI are also investigated. The FI provides a practical guideline for the use of restriction endonucleases and defines a fundamental property by which restriction endonucleases can be characterized.

Restriction enzyme mining for SNPs in genomes.

Abstract: Many different single nucleotide polymorphisms (SNPs) genotyping methods have been developed recently. However, most of them are expensive. Using restriction enzymes for SNP genotyping is a cost-effective method. However, restriction enzyme mining for SNPs in a genome sequence is still challenging for researchers who do not have a background in genomics and bioinformatics. In this review, the basic bioinformatics tools used for restriction enzyme mining for SNP genotyping are summarized and described. The objectives of this paper include: i) the introduction of SNPs, genotyping and PCR-restriction fragment length polymorphism (RFLP); ii) a review of components for genotyping software, including tools for primer design only or restriction enzyme mining only; iii) a review of software providing the flanking sequence for primer design; iv) recent advances in PCR-RFLP tools and natural and mutagenic PCR-RFLP; v) highlighting the strategy for restriction enzyme mining for SNP genotyping; vi) a discussion of potential problems for multiple PCR-RFLP. The different implications for restriction enzymes on sense and antisense strands are also discussed. Our PCR-RFLP freeware, SNP-RFLPing, is included in this review to illustrate many characteristics of PCR-RFLP software design. Future developments will include further sophistication of PCR-RFLP software in order to provide better visualization and a more interactive environment for SNP genotyping and to integrate the software with other tools used in association studies.

Increasing cloning possibilities using artificial zinc finger nucleases.

Abstract: The ability to accurately digest and ligate DNA molecules of different origins is fundamental to modern recombinant DNA research. Only a handful of enzymes are capable of recognizing and cleaving novel and long DNA sequences, however. The slow evolution and engineering of new restriction enzymes calls for alternative strategies to design novel and unique restriction enzymes capable of binding and digesting specific long DNA sequences. Here we report on the use of zinc finger nucleases (ZFNs)-hybrid synthetic restriction enzymes that can be specifically designed to bind and cleave long DNA sequences-for the purpose of DNA recombination. We show that novel ZFNs can be designed for the digestion of specific sequences and can be expressed and used for cloning purposes. We also demonstrate the power of ZFNs in DNA cloning by custom-cloning a target DNA sequence and assembling dual-expression cassettes on a single target plasmid, a task that rarely can be achieved using type-II restriction enzymes. We demonstrate the flexibility of ZFN design and the ability to shuffle monomers of different ZFNs for the digestion of compatible recognition sites through ligation of compatible ends and their cleavage by heterodimer ZFNs. Of no less importance, we show that ZFNs can be designed to recognize and cleave existing DNA sequences for the custom-cloning of native target DNA molecules.

RFLP analysis of Hordeum species relationships1

Abstract: Detailed ribosomal DNA restriction maps were prepared for eight representative plants from Hordeum L. Section Hordeum (two H. bulbosum, H. glaucum, a diploid H. leporinum, a tetraploid H. leporinum, H. murinum, H. spontaneum, H. vulgare) in order to explore species relationships. The four restriction enzymes (EcoRI,SacI, BamHI and HindIII) used in our earlier study were supplemented with six additional ones (BglI, ClaI, KpnI, Pvu II, Sst II and XhoI). Southern blots prepared, using the restriction enzymes singly and in combinations, were probed with the wheat ribosomal probe pTA7l, and hybridization patterns analyzed. A comparison of the derived maps with known maps for wheat, pea and pumpkin showed strong conservation of restriction sites, especially in the ribosomal coding region. The maps suggest that duplication of coding region DNA and its insertion into the spacer region as well as amplification of subrepeats are responsible for variation in rDNA repeat length. Phylogenetic analysis indicated the early divergence of ancestral wheat and Hordeum species, followed by the divergence of the progenitors of H. spontaneum/H. vulgare and the remaining Hordeum species and, most recently, between those of H. glaucum and H. murinum.

Improved gene correction efficiency with a tailed duplex DNA fragment.

Abstract: A 606-base single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, corrects a hygromycin resistance and enhanced green fluorescent protein (Hyg-EGFP) fusion gene more efficiently than a PCR fragment, which is the conventional type of DNA fragment used in gene correction. Here, a tailed duplex, obtained by annealing an oligonucleotide to the ss DNA fragment, was used in the correction. The tailed duplex may be a good substrate for the RAD51 protein, an important enzyme in homologous recombination, which could be the gene correction pathway. The annealing of the oligonucleotides enhanced the correction efficiency of the Hyg-EGFP gene, especially when annealed in the 3'-region of the ss DNA fragment. Both the length and backbone structure of the oligonucleotides affected the gene correction efficiency. This type of gene correction device was also effective for another target gene, the rpsL gene. The results obtained in this study indicate that tailed duplex DNA fragments are effective nucleic acids for gene correction.

Construction of infectious clones for DNA viruses: mastreviruses.

Abstract: To characterize a virus at the molecular and biological levels, it is necessary to produce an infectious clone. For most of the Geminiviridae, cloning of the genome is relatively easy because of their small genomes and the presence of the virus double-stranded (replicative) DNA form in infected plants. Indeed, the presence of conserved sequences between species in the genera Begomovirus, Curtovirus, and Topocuvirus allows the PCR amplification of most genomes using degenerate "universal" primers. Unlike the other genera, no universal primers are reported that are suitable for all mastreviruses and alternative, more time-consuming methods must be used. This chapter describes a method that has proven successful for the preparation and testing of infectious clones for a wide range of mastreviruses. It has been designed to ensure its applicability for laboratories throughout the world. Methods are presented for the isolation of total plant DNA and the purification of the replicative (cccDNA) form of the virus using a commercially available plasmid purification kit. Restriction enzyme digestion of the purified DNA using a restriction enzyme with a unique site in the viral genome allows the cloning of a full-length copy of the genome into a high copy number vector, thereby providing a template for sequence analysis and further cloning. The only efficient method for confirming infectivity of mastrevirus clones is using agroinoculation (also termed agroinfection). This requires the production of a multimeric copy of the genome in a T-DNA binary vector, transformation of specific Agrobacterium strains with the binary vector clone, and inoculation of specific regions of seedlings, or seeds, of the appropriate host species. These specific requirements are described and discussed.

Presence of a Family of Plasmids (29 to 65 Kilobases) with a 26-Kilobase Common Region in Different Strains of the Sulfur-Oxidizing Bacterium Acidithiobacillus caldus

Abstract: Three large cryptic plasmids from different isolates of Acidithiobacillus caldus were rescued by using an in vitro transposition system that delivers a kanamycin-selectable marker and an Escherichia coli plasmid origin of replication. The largest of the plasmids, the 65-kb plasmid pTcM1, was isolated from a South African A. caldus strain, MNG. This plasmid was sequenced and compared to that of pTcF1 (39 kb, from strain "f," South Africa) and pC-SH12 (29 kb, from strain C-SH12, Australia). With the exception of a 2.7-kb insertion sequence, pC-SH12 appears to represent the DNA common to all three plasmids and includes a number of accessory genes plus the plasmid "backbone" containing the replication region. The two larger plasmids carry, in addition, a number of insertion sequences of the ISL3 family and a composite transposon related to the Tn21 subfamily containing a highly mosaic region within the borders of the inverted repeats. Genes coding for arsenic resistance, plasmid mobilization, plasmid stability, and a putative restriction-modification system occur within these mosaic regions.

Restriction fragment length polymorphism analysis of the C1-inhibitor gene in hereditary C1-inhibitor deficiency.

Abstract: Four out of 12 kindreds with Type I hereditary angio-oedema (HAE) were shown to have unique disease-related restriction fragment length polymorphism (RFLPs) in one allele of the C1-inhibitor gene. These RFLPs were used to localise the gene mutations responsible for them in each family. The four mutations affected exon 4, exon 6, exon 7 and exon 8, respectively. Mutations in exon 6 and exon 8 have not been described previously in Type I HAE. The other two mutations which comprised an exon 4 deletion and an exon 7 deletion have already been documented by other investigators. In each family the mutation was seen to cosegregate with the disease. Detection of a disease-related RFLP in 30% of the Type I HAE kindred tested is higher than other published studies, and reflects the larger number of restriction enzymes employed. These results suggest that Type I HAE is likely to be associated with a multiplicity of gene mutations as is seen in other genetic diseases. A new C1-inhibitor gene-related RFLP in the normal population was also characterised. This may be useful as an indirect marker of the mutant C1-inhibitor allele in certain families with Type I HAE.

A physical map of human Alu repeats cleavage by restriction endonucleases

Abstract: Alu repetitive elements are the abundant sequences in human genome. Diversity of DNA sequences of these elements makes difficult the construction of theoretical patterns of Alu repeats cleavage by restriction endonucleases. We have proposed a method of restriction analysis of Alu repeats sequences in silico. Simple software to analyze Alu repeats database has been suggested and Alu repeats digestion patterns for several restriction enzymes' recognition sites have been constructed. Restriction maps of Alu repeats cleavage for corresponding restriction enzymes have been calculated and plotted. Theoretical data have been compared with experimental results on DNA hydrolysis with restriction enzymes, which we obtained earlier. Alu repeats digestions provide the main contribution to the patterns of human chromosomal DNA cleavage. This corresponds to the experimental data on total human DNA hydrolysis with restriction enzymes.

Application of physical and genetic map of Rhizobium leguminosarum bv. trifolii TA1 to comparison of three closely related rhizobial genomes.

Abstract: A combined physical and genetic map of Rhizobiumleguminosarum biovar trifolii TA1 (RtTA1) genome was constructed and used in comparison of chromosomal organization with the closely related R. leguminosarum bv. viciae 3841 (Rlv) and Rhizobium etli CNF42 (Rhe). This approach allowed evaluation of chromosome and genome plasticity and provided important insights into R. leguminosarum lineage diversity. MssI, SmiI, PacI, and I-CeuI restriction endonucleases were chosen for the analysis, generating fragments with suitable size distributions for RtTA1 genome mapping. The fragments were assembled into a physical map using a combination of complementary methods, including multiple and partial digests of genomic DNA, hybridization with homologous gene probes, and cross-Southern hybridization. About 100 genetic markers were located on the RtTA1 restriction map. Comparison of genetic maps of RtTA1, Rlv, and Rhe revealed extensive chromosomal colinearity despite differences in the physical maps. The comparison provides bases for comprehensive analysis of the evolution of R. leguminosarum genome, indicating that, at least on the chromosomal level, no major rearrangements had occurred after the evolutionary divergence of R. leguminosarum biovars.

Methods, computer-accessible medium, and systems for generating a genome wide haplotype sequence

Abstract: Methods, computer-accessible medium, and systems for generating a genome wide probe map and/or a genome wide haplotype sequence are provided. In particular, a genome wide prob map can be generated by obtaining a plurality of detectable oligonucleotide probes hybridized to at least one double stranded nucleic acid molecule cleaved with at least one restriction enzyme, and detecting the location of the detectable oligonucleotide probes. For example, genome wide haplotype sequence can be generated by analyzing at least one genome wide restriction map in conjunction with at least one genome wide probe map to determine distances between restriction sites of the at least one genome wide restriction map and locations of detectable oligonucleotide probes of the at least one genome wide probe map and defining a consensus map indicating restriction sites based on each of the at least one genome wide restriction map and locations of detectable oligonucleotide probes based on each of the at least one genome wide probe map.

Points of Recombination in Epstein-Barr Virus (EBV) Strain P3HR-1-Derived Heterogeneous DNA as Indexes to EBV DNA Recombinogenic Events In Vivo

Abstract: Deletions and rearrangements in the genome of Epstein-Barr virus (EBV) strain P3HR-1 generate subgenomic infectious particles that, unlike defective interfering particles in other viral systems, enhance rather than restrict EBV replication in vitro. Reports of comparable heterogeneous (het) DNA in EBV-linked human diseases, based on detection of an abnormal juxtaposition of EBV DNA fragments BamHI W and BamHI Z that disrupts viral latency, prompted us to determine at the nucleotide level all remaining recombination joints formed by the four constituent segments of P3HR-1-derived het DNA. Guided by endonuclease restriction maps, we chose PCR primer pairs that approximated and framed junctions creating the unique BamHI M/B1 and E/S fusion fragments. Sequencing of PCR products revealed points of recombination that lacked regions of extensive homology between constituent fragments. Identical recombination junctions were detected by PCR in EBV-positive salivary samples from human immunodeficiency virus-infected donors, although the W/Z rearrangement that induces EBV reactivation was frequently found in the absence of the other two. In vitro infection of lymphoid cells similarly indicated that not all three het DNA rearrangements need to reside on a composite molecule. These results connote a precision in the recombination process that dictates both composition and regulation of gene segments altered by genomic rearrangement. Moreover, the apparent frequency of het DNA at sites of EBV replication in vivo is consistent with a likely contribution to the pathogenesis of EBV reactivation.

Methods of identifying an organism from a heterogeneous sample

Abstract: This disclosure features methods of identifying at least one organism from a heterogeneous sample. The methods include: (a) obtaining a nucleic acid from at least one organism in a heterogeneous sample; (b) imaging said nucleic acid; (c) obtaining a restriction map of said nucleic acid; and (d) correlating the restriction map of said nucleic acid with a restriction map database, thereby identifying at least one organism in the heterogeneous sample.

Quick identification of Type I restriction enzyme isoschizomers using newly developed pTypeI and reference plasmids

Abstract: Although DNA-recognition sequences are among the most important characteristics of restriction enzymes and their corresponding methylases, determination of the recognition sequence of a Type-I restriction enzyme is a complicated procedure. To facilitate this process we have previously developed plasmid R-M tests and the computer program RM search. To specifically identify Type-I isoschizomers, we engineered a pUC19 derivative plasmid, pTypeI, which contains all of the 27 Type-I recognition sequences in a 248-bp DNA fragment. Furthermore, a series of 27 plasmids (designated 'reference plasmids'), each containing a unique Type-I recognition sequence, were also constructed using pMECA, a derivative of pUC vectors. In this study, we tried those vectors on 108 clinical E. coli strains and found that 48 strains produced isoschizomers of Type I enzymes. A detailed study of 26 strains using these 'reference plasmids' revealed that they produce seven different isoschizomers of the prototypes: EcoAI, EcoBI, EcoKI, Eco377I, Eco646I, Eco777I and Eco826I. One strain EC1344 produces two Type I enzymes (EcoKI and Eco377I).

Creation of a genetic engineering design with the choline oxidase gene for improved expression in plant

Abstract: Class-IIS restriction endonucleases were used for precise joining of DNA fragments encoding part of the sequence of the choline oxidase gene, signal sequence, and translation enhancer for creating constructs.

The Efficient Strategy of Plasmid Rescue from Tn5 Mutants Derived from Bradyrhizobium japonicum Is-1, Based on Whole Genome Sequence Information of Strain USDA110

Abstract: We previously reported that Tn5 mutants derived from Bradyrhizobium japonicum Is–1 were isolated. In this report, Tn5–encoded kanamycin resistance cassette and its flanking sequence were concurrently cloned into cosmid vector from Tn5 mutants. In general, the genomic southern blot analysis is often performed to select appropriate restriction enzyme before performing the plasmid rescue from cosmid clone. Therefore plasmid rescue experiment consumes the time, especially when more than one Tn5 mutant was obtained. To improve this situation, tentative restriction map of strain Is–1 was constructed based on whole genome sequence information of strain USDA110. Plasmid rescue was performed with restriction enzymes selected according to this map and these enzymes were appropriate for plasmid rescue. This strategy is thought to be effective for saving the time of plasmid rescue in B. japonicum strains other than USDA110.

Restriction endonuclease Asi256I recognizes and cuts the nucleotide sequence 5′-GATC-3′

Abstract: A strain producing a restriction endonuclease was isolated from soil samples and identified as the Arthrobacter sp. strain Ck256. The enzyme produced by this strain was termed Asi256I. The isolation procedure for this enzyme was described, and the optimal conditions for its function were determined. It was shown that the restriction endonuclease Asi256I is a true isoschizomer of MboI, it has a temperature optimum of 6°C, and can be used in molecular-biological and genetic-engineering studies performed at low temperatures.

Design, construction and cloning of pCAMBIA-MiAMP1 vector for enhancing disease resistance in plants using Agrobacterium- mediated transformation

Abstract: Objective: To design, construct and clone a pCAMBIA-MiAMP1 vector that can be used to enhance disease resistance in plants through Agrobacterium-mediated transformation. Methodology and results: The cDNA sequence encoding MiAMP1 antimicrobial peptide as a defense resistance gene, which had been cloned into the cloning site of pGEM-T T-vector, was obtained from Australia. The pGEM-T-MiAMP1 was transformed into E.coli and the identification of transformed clones among antibiotic resistant bacteria carried out by lacZ expression using blue/white screening system. The presence of MiAMP1 gene was confirmed using PCR analysis with primers M13, restriction enzymes NcoI and BamHI and DNA sequencing. Based on the sequence, the orientation and restriction map of target gene in plasmid was defined. After designing the specific PCR primers flanking the coding region of the MiAMP1 gene and amplifying the complete cDNA with additional restriction sites, either PCR product, or the binary vector pCAMBIA1305.1 were digested with restriction enzymes NcoI and BglII. Subsequently, the insert and the vector were ligated with T4 DNA ligase to produce binary vector pCAMBIA-MiAMP1 for genetic transformation of plants. This construction contains the full coding region of the MiAMP1 antimicrobial peptide and is flanked at its 5' end by the strong constitutive promoter of CaMV35S and at its 3' end by the polyadenylation sequence of NOS polyA. The designed expression vector also contains other elements that are useful for plant transformation such as a kanamycin resistance gene (npt II) and a hygromycin resistance gene (hpt II) that can be used as markers for selecting transformed plants, while the GUSPlus™ reporter gene contains the intron. The presence of MiAMP1 gene in the pCAMBIA-MiAMP1 construction was confirmed among antibiotic resistant colonies by PCR method, restriction enzymes analysis and DNA sequencing. The designed construction was transferred into Agrobacterium tumefaciens strain AGL0 by tri-parental mating method with the helper plasmid pRK2013. Conclusion and application of findings : pCAMBIA-MiAMP1 construction can be used to genetically transform plants to enhance resistance to fungi e.g. Leptosphaeria maculans and Sclerotinia sclerotiorum. Currently, it is being used in Agrobacterium-mediated transformation of canola and sunflower.

Arsenic resistance genes in nocardioform bacteria

Abstract: Arsenic contamination is becoming a global dilemma making it imperative to study natural biomechanisms involved in arsenic detoxification in humans and other living organisms to gain insight in ways how arsenic poisoning can be treated Arsenic resistance genes are being detected in ever increasing microorganisms with diverse gene organizations Here, in this report the presence of ars genes in Rhodococcus rhodochrous, Arthrobacter oxydans, and Gordonia rubropertincta was investigated Genomic libraries of these AsR actinomycete species were constructed and then screened for arsenic resistance and unique carbon source utilization genes More than one hundred positive arsenate tolerant clones were obtained from a partially PstI digested library of Gordonia rubropertincta of which ten random clones (pKL1-10) clearly showed the arsenic resistance phenotype in an AsS Gram-positive recipient strain Nine of these ten clones demonstrated a conserved ∼35 kbp DNA fragment with only clone 1 containing a 5100 bp nucleotide segment Restriction mapping and subcloning was performed on the designated pKL335 and pKL1 recombinant plasmid constructs after which the minimum DNA required for conferring arsenic resistance were sent for sequencing Only the pKL316 and pKL120 subclones were successfully sequenced as putative secondary structures prevented the sequencing of the rest of pKL335 and pKL1 Four and three putative cistrons were predicted from the edited contiguous sequences of pKL335(ORF1-4) and pKL120(ORF5-7), respectively Blastn,x,p and CD alignment searches revealed that ORF1 was a putative Acr3-type ArsB homolog, ORF2,3,4 and 7 showed significant homology with Trx-type ArsCs, and ORF5 and 6 are flavin-binding monooxygenase and thioredoxin reductase analogs, respectively From the alignment data it can be deduced that pKL335 carries an arsRBCCC operon structure, such as C efficiens YS-314, and that pKL1 probably comprises a unique arsRBCCCTO gene organization This is only the third report of a thioredoxin reductase and a flavoprotein detected within an arsenic resistant gene cluster Multiple ArsCs, an ArsT/TrxB and an ArsO/FMO-like homolog seems to be a unique feature amongst high GC-rich Gram(+) bacteria