Showing papers on "Restriction map published in 2014"

The other face of restriction: modification-dependent enzymes

Abstract: The 1952 observation of host-induced non-hereditary variation in bacteriophages by Salvador Luria and Mary Human led to the discovery in the 1960s of modifying enzymes that glucosylate hydroxymethylcytosine in T-even phages and of genes encoding corresponding host activities that restrict non-glucosylated phage DNA: rglA and rglB (restricts glucoseless phage). In the 1980’s, appreciation of the biological scope of these activities was dramatically expanded with the demonstration that plant and animal DNA was also sensitive to restriction in cloning experiments. The rgl genes were renamed mcrA and mcrBC (modified cytosine restriction). The new class of modification-dependent restriction enzymes was named Type IV, as distinct from the familiar modification-blocked Types I–III. A third Escherichia coli enzyme, mrr (modified DNA rejection and restriction) recognizes both methylcytosine and methyladenine. In recent years, the universe of modification-dependent enzymes has expanded greatly. Technical advances allow use of Type IV enzymes to study epigenetic mechanisms in mammals and plants. Type IV enzymes recognize modified DNA with low sequence selectivity and have emerged many times independently during evolution. Here, we review biochemical and structural data on these proteins, the resurgent interest in Type IV enzymes as tools for epigenetic research and the evolutionary pressures on these systems.

Editorial: NAR Surveys the Past, Present and Future of Restriction Endonucleases

Abstract: In this issue, Nucleic Acids Research presents five Survey and Summary articles that describe the historical development of studies on restriction endonucleases and summarize much of our current understanding of this diverse and complex group of enzymes. The first of these articles, entitled ‘Highlights of the DNA cutters: a short history of the restriction enzymes’ (1), describes seminal studies on bacteriophage host restriction, details subsequent work on type I and type III enzymes that established the restriction-modification (RM) paradigm and summarizes other landmark events that led to restriction enzymes becoming a main driving force in the development of modern biotechnology and molecular medicine. Other Survey and Summary articles in this issue describe three of the major types of RM systems as they are understood today (2–4). The different types of RM systems—of which there are currently four—vary in their cofactor dependence, in the spatial relationship of DNA binding and cleavage sites and in the way in which endonuclease and modification activities are physically and mechanistically coupled to one another. The last of the Survey and Summary articles in this issue discusses RM systems in the broader context of toxin–antitoxin genetic systems, which exist in great variety throughout the microbial world (5). With this issue, NAR enters its 42nd year of publication. The journal was founded during the period when restriction endonucleases began to be widely adopted for molecular biology research. An article in one of our first issues presented an early restriction map of the SV40 virus, which was an essential precursor to the sequencing of the viral genome (6). Another article from journal’s early years described a widely adopted method for restriction enzyme purification (7). In subsequent years, we published semiannual updates describing the popular REBASE database. Indeed, >10% of the articles published since the journal’s inception are indexed as dealing with RM systems in a significant fashion. The current collection of Survey and Summary articles thus builds on our long tradition of coverage of this field. It is important to recognize that the structural protein folds, catalytic motifs and catalytic mechanisms that are found in restriction endonucleases and methyltransferases are also observed in a wide variety of other enzymes, including those responsible for the transfer of mobile DNA elements, the repair of DNA lesions and the maintenance of genome integrity. Several decades of detailed studies of restriction endonucleases (and of their corresponding modification enzymes) have motivated and informed investigations in these other areas of biology. At the same time, RM systems have provided the reagents required for the development of rapid and inexpensive genome sequencing technologies, powerful strategies for genome engineering, new methods for therapeutic gene modification and correction and novel protein- and cell-based medicines. We hope that, as well as being interesting and informative, the current articles will remind our readers of the importance of basic research for the development of future therapies and new applications in biotechnology. In retrospect, the contribution of restriction endonucleases to modern molecular biology and biomedicine seems obvious and inevitable. However, it should be remembered that the original studies of phage restriction were driven purely by intellectual curiosity, and were supported by research infrastructures that valued discovery for discovery’s sake. The critical importance of such studies for creating new technologies for medicine and technology, as illustrated by >60 years of restriction endonuclease research, can be used as an example for all who believe in the importance of research and development for future generations.

Flexible and scalable genotyping-by-sequencing strategies for population studies

Abstract: Many areas critical to agricultural production and research, such as the breeding and trait mapping in plants and livestock, require robust and scalable genotyping platforms. Genotyping-by-sequencing (GBS) is a one such method highly suited to non-human organisms. In the GBS protocol, genomic DNA is fractionated via restriction digest, then reduced representation is achieved through size selection. Since many restriction sites are conserved across a species, the sequenced portion of the genome is highly consistent within a population. This makes the GBS protocol highly suited for experiments that require surveying large numbers of markers within a population, such as those involving genetic mapping, breeding, and population genomics. We have modified the GBS technology in a number of ways. Custom, enzyme specific adaptors have been replaced with standard Illumina adaptors compatible with blunt-end restriction enzymes. Multiplexing is achieved through a dual barcoding system, and bead-based library preparation protocols allows for in-solution size selection and eliminates the need for columns and gels. A panel of eight restriction enzymes was selected for testing on B73 maize and Nipponbare rice genomic DNA. Quality of the data was demonstrated by identifying that the vast majority of reads from each enzyme aligned to restriction sites predicted in silico. The link between enzyme parameters and experimental outcome was demonstrated by showing that the sequenced portion of the genome was adaptable by selecting enzymes based on motif length, complexity, and methylation sensitivity. The utility of the new GBS protocol was demonstrated by correctly mapping several in a maize F2 population resulting from a B73 × Country Gentleman test cross. This technology is readily adaptable to different genomes, highly amenable to multiplexing and compatible with over forty commercially available restriction enzymes. These advancements represent a major improvement in genotyping technology by providing a highly flexible and scalable GBS that is readily implemented for studies on genome-wide variation.

Restriction Analysis of β-Tubulin Gene for Differentiation of the Common Pathogenic Dermatophytes

Abstract: Background: Identification of dermatophytes at the species level, relying on macro- and microscopic properties of the colonies is time-consuming, questioned in many circumstances, and requires considerable expertise. In this study, we examined the potency of a new genetic marker, β-tubulin (BT2) gene, for differentiation of dermatophytes in an in silico and experimental restriction fragment length polymorphism (RFLP) profile. Methods: The BT2 sequences of dermatophyte species were retrieved from GenBank and analyzed using bioinformatics softwares to choose suitable restriction enzyme(s). Forty reference culture collections and 100 clinical isolates were PCR-amplified using the primers T1 and Bt2b and consequently subjected to virtual RFLP analysis. The dermatophytes were identified according to specific lengths of bands in agarose gel electrophoresis. Results: After digestion of partially amplified β-tubulin gene with the restriction enzyme FatI, three dermatophyte species, that is, Microsporum gypseum, M. canis, and Trichophyton verrucosum yielded unique restriction maps while the remaining species including T. interdigitale, T. rubrum, T. tonsurans, T. schoenleinii, and T. violaceum, were identified by further restriction digestion by Alw21I, MwoI, and HpyCH4V endonucleases. The length of RFLP products was same as of those expected by computer analysis. Conclusion: The two-step BT2 restriction mapping used in this study is an effective tool for reliable differentiation of the clinically relevant species of dermatophytes. © 2014 Wiley Periodicals, Inc.

Physical Map of the Bacillus subtilis 168 Chromosome

Abstract: This chapter summarizes information on the physical and genetic maps of the chromosome, the integration of the physical and genetic maps, and the unique features of the B. subtilis genome. Two basic physical maps of bacterial genomes have been developed: (i) a long-range restriction map generated by sequence-specific restriction endonucleases that cleave the bacterial chromosome infrequently and (ii) a detailed restriction map derived from the assembly of overlapping cloned DNA segment. The physical map of the B. subtilis 168 chromosome has been constructed by the first method. The strategy and ideas for its construction are unique and specific to B. subtilis 168, and described briefly. In this chapter, the author uses the map constructed by Itaya and Tanaka because it is complete and sufficiently accurate. Diversity of the B. subtilis genome based on the physical map can be used to evaluate the frequency of DNA rearrangements.

DYZ1 arrays show sequence variation between the monozygotic males.

Abstract: Monozygotic twins (MZT) are an important resource for genetical studies in the context of normal and diseased genomes. In the present study we used DYZ1, a satellite fraction present in the form of tandem arrays on the long arm of the human Y chromosome, as a tool to uncover sequence variations between the monozygotic males. We detected copy number variation, frequent insertions and deletions within the sequences of DYZ1 arrays amongst all the three sets of twins used in the present study. MZT1b showed loss of 35 bp compared to that in 1a, whereas 2a showed loss of 31 bp compared to that in 2b. Similarly, 3b showed 10 bp insertion compared to that in 3a. MZT1a germline DNA showed loss of 5 bp and 1b blood DNA showed loss of 26 bp compared to that of 1a blood and 1b germline DNA, respectively. Of the 69 restriction sites detected in DYZ1 arrays, MboII, BsrI, TspEI and TaqI enzymes showed frequent loss and or gain amongst all the 3 pairs studied. MZT1 pair showed loss/gain of VspI, BsrDI, AgsI, PleI, TspDTI, TspEI, TfiI and TaqI restriction sites in both blood and germline DNA. All the three sets of MZT showed differences in the number of DYZ1 copies. FISH signals reflected somatic mosaicism of the DYZ1 copies across the cells. DYZ1 showed both sequence and copy number variation between the MZT males. Sequence variation was also noticed between germline and blood DNA samples of the same individual as we observed at least in one set of sample. The result suggests that DYZ1 faithfully records all the genetical changes occurring after the twining which may be ascribed to the environmental factors.

T7 lytic phage-displayed peptide libraries: construction and diversity characterization.

Abstract: In this chapter, we describe the construction of T7 bacteriophage (phage)-displayed peptide libraries and the diversity analyses of random amino acid sequences obtained from the libraries. We used commercially available reagents, Novagen's T7Select system, to construct the libraries. Using a combination of biotinylated extension primer and streptavidin-coupled magnetic beads, we were able to prepare library DNA without applying gel purification, resulting in extremely high ligation efficiencies. Further, we describe the use of bioinformatics tools to characterize library diversity. Amino acid frequency and positional amino acid diversity and hydropathy are estimated using the REceptor LIgand Contacts website http://relic.bio.anl.gov. Peptide net charge analysis and peptide hydropathy analysis are conducted using the Genetics Computer Group Wisconsin Package computational tools. A comprehensive collection of the estimated number of recombinants and titers of T7 phage-displayed peptide libraries constructed in our lab is included.

Simple and Cost-Effective Restriction Endonuclease Analysis of Human Adenoviruses

Abstract: Restriction endonuclease analyses (REAs) constitute the only inexpensive molecular approach capable of typing and characterizing human adenovirus (HAdV) strains based on the entire genome. However, the application of this method is limited by the need for time-consuming and labor-intensive procedures. We herein developed a simple and cost-effective REA for assessing HAdV. The method consists of (1) simple and cost-effective DNA extraction, (2) fast restriction endonuclease (RE) digestion, and (3) speedy mini agarose gel electrophoresis. In this study, DNA was isolated according to the kit-based method and 21.0 to 28.0 μg of viral DNA was extracted from prototypes (HAdV-1, HAdV-3, HAdV-4, and HAdV-37) in each flask. The amount of DNA ranged from 11.4 to 57.0 μg among the HAdV-3 () isolates. The obtained viral DNA was found to be applicable to more than 10 types of REAs. Fast-cut restriction endonucleases (REs) were able to digest the DNA within 15 minutes, and restriction fragments were easily separated via horizontal mini agarose gel electrophoresis. The whole procedure for 10 samples can be completed within approximately six hours (the conventional method requires at least two days). These results show that our REA is potentially applicable in many laboratories in which HAdVs are isolated.