Showing papers on "Restriction map published in 2012"

Role of SAMHD1 nuclear localization in restriction of HIV-1 and SIVmac

Abstract: Background: SAMHD1 is a nuclear protein that blocks lentiviral infection before reverse transcription in macrophages and dendritic cells. The viral accessory protein Vpx overcomes the SAMHD1-mediated lentiviral block by inducing its proteasomal degradation. Results: Here, we identified the nuclear localization signal (NLS) of SAMHD1, and studied its contribution to restriction of HIV-1 and SIVmac. By studying the cellular distribution of different SAMHD1 variants, we mapped the nuclear localization of SAMHD1 to residues 11 KRPR 14 . Mutagenesis of these residues changed the cellular distribution of SAMHD1 from the nucleus to the cytoplasm. SAMHD1 mutants that lost nuclear localization restricted HIV-1 and SIV as potently as the wild type protein. Interestingly, SAMHD1 mutants that localized to the cytoplasm were not degraded by nuclear Vpx alleles. Therefore, nuclear Vpx alleles require nuclear localization of SAMHD1 in order to induce its degradation. In agreement, SIVmac viruses encoding Vpx did not overcome the restriction imposed by the cytoplasmic variants of SAMHD1. Conclusions: We mapped the NLS of SAMHD1 to residues 11 KRPR 14 and studied the contribution of SAMHD1 nuclear localization to restriction of HIV-1 and SIV. These experiments demonstrate that cytoplasmic variants of SAMHD1 potently block lentiviral infection and are resistant to Vpx-mediated degradation. The nuclear Vpx alleles studied here are only capable of degrading a nuclearly localized SAMHD1 suggesting that Vpx-mediated degradation of SAMHD1 is initiated in the nucleus.

Generation of Single Domain Antibody Fragments Derived from Camelids and Generation of Manifold Constructs

Abstract: Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.

Crystal structure and mechanism of action of the N6-methyladenine-dependent type IIM restriction endonuclease R.DpnI.

Abstract: DNA methylation-dependent restriction enzymes have many applications in genetic engineering and in the analysis of the epigenetic state of eukaryotic genomes. Nevertheless, high-resolution structures have not yet been reported, and therefore mechanisms of DNA methylation-dependent cleavage are not understood. Here, we present a biochemical analysis and high-resolution DNA co-crystal structure of the N(6)-methyladenine (m6A)-dependent restriction enzyme R.DpnI. Our data show that R.DpnI consists of an N-terminal catalytic PD-(D/E)XK domain and a C-terminal winged helix (wH) domain. Surprisingly, both domains bind DNA in a sequence- and methylation-sensitive manner. The crystal contains R.DpnI with fully methylated target DNA bound to the wH domain, but distant from the catalytic domain. Independent readout of DNA sequence and methylation by the two domains might contribute to R.DpnI specificity or could help the monomeric enzyme to cut the second strand after introducing a nick.

Optical mapping and sequencing of the Escherichia coli KO11 genome reveal extensive chromosomal rearrangements, and multiple tandem copies of the Zymomonas mobilis pdc and adhB genes.

Abstract: Escherichia coli KO11 (ATCC 55124) was engineered in 1990 to produce ethanol by chromosomal insertion of the Zymomonas mobilis pdc and adhB genes into E. coli W (ATCC 9637). KO11FL, our current laboratory version of KO11, and its parent E. coli W were sequenced, and contigs assembled into genomic sequences using optical NcoI restriction maps as templates. E. coli W contained plasmids pRK1 (102.5 kb) and pRK2 (5.4 kb), but KO11FL only contained pRK2. KO11FL optical maps made with AflII and with BamHI showed a tandem repeat region, consisting of at least 20 copies of a 10-kb unit. The repeat region was located at the insertion site for the pdc, adhB, and chloramphenicol-resistance genes. Sequence coverage of these genes was about 25-fold higher than average, consistent with amplification of the foreign genes that were inserted as circularized DNA. Selection for higher levels of chloramphenicol resistance originally produced strains with higher pdc and adhB expression, and hence improved fermentation performance, by increasing the gene copy number. Sequence data for an earlier version of KO11, ATCC 55124, indicated that multiple copies of pdc adhB were present. Comparison of the W and KO11FL genomes showed large inversions and deletions in KO11FL, mostly enabled by IS10, which is absent from W but present at 30 sites in KO11FL. The early KO11 strain ATCC 55124 had no rearrangements, contained only one IS10, and lacked most accumulated single nucleotide polymorphisms (SNPs) present in KO11FL. Despite rearrangements and SNPs in KO11FL, fermentation performance was equal to that of ATCC 55124.

Characterization of a novel Klebsiella pneumoniae sequence type 476 carrying both bla KPC-2 and bla IMP-4.

Abstract: Carbapenemase-producing Klebsiella pneumoniae has recently spread rapidly throughout China. In this study, we characterized a carbapenem-resistant K. pneumoniae isolate that produced both KPC-2 and IMP-4 type carbapenemases. A clinical isolate of K. pneumoniae, resistant to both meropenem and imipenem, was recovered from a urine sample. Antibiotic susceptibility was determined using the broth microdilution method and Etest (bioMerieux, France). Pulsed-field gel electrophoresis and multilocus sequence typing (MLST) were used for gene type analysis. blaKPC and the encoding genes of ESBLs and plasmid-mediated AmpC enzymes were polymerase chain reaction (PCR) amplified and sequenced. Plasmids were analyzed by transformation, enzyme restriction and Southern blot. PCR analysis revealed that the isolate was simultaneously carrying blaKPC-2, blaIMP-4, blaTEM-1, and blaOKP-B genes. MLST assigned the isolate to a novel sequence type, ST476. blaKPC-2-harbouring plasmids of the isolate and comparative strains had similar EcoRI and HindIII restriction maps, while IMP-4-harbouring plasmids had variable HindIII restriction maps. Coexistence of blaKPC-2 and blaIMP-4 was probably due to blaIMP-4-harbouring plasmid transmission into KPC-2-producing K. pneumoniae (ST476). The concomitant presence of these genes is alarming and poses both therapeutic and infection control problems.

Characterization of MspNI (G/GWCC) and MspNII (R/GATCY), novel thermostable Type II restriction endonucleases from Meiothermus sp., isoschizomers of AvaII and BstYI.

Abstract: MspNI and MspNII, isoschizomers of prototype Type II restriction endonucleases AvaII and BstYI, were extracted from an extreme thermophile bacterium belonging to the genus Meiothermus, isolated from the hot sulphur springs in north Himalayan region of India where temperature and pH ranged from 60 to 80°C and 7.5 to 8.5, respectively. The two enzymes were purified to homogeneity using Cibacron-Blue 3GA Agarose, Q-Sepharose and SP-Sepharose chromatography and were homodimers with subunit molecular weights of 27 and 45 kDa, respectively. Restriction mapping and run-off sequencing of MspNI and MspNII cleaved pBR322 DNA showed that they recognized and cleaved 5′-G/GWCC-3′ and 5′-R/GATCY-3′ sites, respectively. MspNI and MspNII worked optimally at 60 and 70°C, 6 and 5 mM MgCl2, respectively and showed no star activity in organic solvents. Both were resistant to sequence methylation and were stable up to 25 PCR cycles.

Using ITS2 PCR‐RFLP to generate molecular markers for authentication of Sophora flavescens Ait.

Abstract: BACKGROUND:DriedrootofSophoraflavescensAit.isamedicinalmaterialoccasionallymisusedoradulteratedbyotherspecies similar in appearance. In this study the internal transcribed spacer (ITS) regions of DNA samples of S. flavescens Ait. collected from different areas of Taiwan were amplified by polymerase chain reaction (PCR) and compared. The effectiveness of using ITS2 PCR restriction fragment length polymorphism (RFLP)-generated markers to differentiate S. flavescens Ait. from possible adulterants was also evaluated. RESULTS: The S.flavescens Ait. samples collected from different areas were extremely low in ITS sequence variability at species level. ITS2 PCR-RFLP coupled with restriction enzymes Sac I, Sac II, Xho Io rPvu I produced specific fragments for all tested variants. ITS2 PCR-RFLP coupled with Sac II was further performed to identify mixtures of DNA extracts of S.flavescens Ait. and Sophoratomentosa L. in various ratios. The developed ITS2 PCR-RFLP markers could detect mixed DNA samples of S.flavescens Ait./S.tomentosa L. up to a ratio of 10:1. CONCLUSION: The present study demonstrates the usefulness of ITS2 PCR-RFLP coupled with pre-selected restriction enzymes forpracticalandaccurateauthenticationofS.flavescensAit.ThetechniqueisalsosuitableforanalysingS.flavescensAit.mixed with other adulterants. c � 2011 Society of Chemical Industry

DNA of a circular minichromosome linearized by restriction enzymes or other reagents is resistant to further cleavage: an influence of chromatin topology on the accessibility of DNA

Abstract: The accessibility of DNA in chromatin is an essential factor in regulating its activities. We studied the accessibility of the DNA in a ∼170 kb circular minichromosome to DNA-cleaving reagents using pulsed-field gel electrophoresis and fibre-fluorescence in situ hybridization on combed DNA molecules. Only one of several potential sites in the minichromosome DNA was accessible to restriction enzymes in permeabilized cells, and in growing cells only a single site at an essentially random position was cut by poisoned topoisomerase II, neocarzinostatin and γ-radiation, which have multiple potential cleavage sites; further sites were then inaccessible in the linearized minichromosomes. Sequential exposure to combinations of these reagents also resulted in cleavage at only a single site. Minichromosome DNA containing single-strand breaks created by a nicking endonuclease to relax any unconstrained superhelicity was also cut at only a single position by a restriction enzyme. Further sites became accessible after ≥95% of histones H2A, H2B and H1, and most non-histone proteins were extracted. These observations suggest that a global rearrangement of the three-dimensional packing and interactions of nucleosomes occurs when a circular minichromosome is linearized and results in its DNA becoming inaccessible to probes.

Identification of the Replication Region of a 111-kb Circular Plasmid from Rhodococcus opacus B-4 by λ Red Recombination-Based Deletion Analysis

Abstract: The replication region of the 111-kb circular plasmid pKNR from Rhodococcus opacus B-4 was identified. A PCR-based deletion analysis using the λ Red recombination technique followed by restriction digestion and PCR-amplification analyses revealed that a 2.5-kb fragment covering one putative open reading frame (ORF) was involved in the replication of pKNR. The product of this ORF showed significant similarity to a functionally unknown protein encoded in the replication region of the 70-kb circular plasmid of Clavibacter michiganensis and to ones in other bacterial large circular plasmids. These observations suggest that the product of the identified ORF and its orthologs can serve as novel replication proteins for large circular bacterial plasmids.

The cloning of non-structural-1 (NS1) gene of H9N2 subtype of avian influenza virus in pGEX-4T-1 and pMAL-c2X plasmids and expression in Escherichia coli DH5α strain

Abstract: Avian influenza is a viral contagious disease that affects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including trade embargoes and interfering with serologic surveillance in differentiation between infected and vaccinated animals (DIVA strategy). Several distinct DIVA strategies have been presented to conquer these limitations. In this study, the open reading frame of NS1 gene of a H9N2 subtype of AI virus was amplified by polymerase chain reaction. After extraction and purification of NS1 gene from agarose gel, it was inserted into two different pGEX-4T-1 and pMAL-c2X plasmids and transferred in DH5α strain of Escherichia coli by using electroporation procedure. The E. coli colonies possessing recombinant NS1 gene were screened using PCR, restriction mapping and sequencing analysis. The expressed rNS1 protein was purified using affinity chromatography based on MBP (pMALc2X) and GST (pGEX-4T-1). The MBP-NS1 and GSTNS1 proteins on SDS-PAGE had bands with molecular weight of 68 and 52 kDa respectively. Western blotting with MBP-NS1 protein showed positive reaction using antisera obtained from chickens challenged with a H9N2 subtype strain. But, the most sera prepared from H9N2 vaccinated chickens were negative in WB. These findings indicated that the MBP-rNS1 protein of 26 kDa expressed by pMAL-c2X plasmid can be used in a DIVA for differentiation of AI infected and vaccinated chickens.

Construction of a synthetic vector for preparation of a 100 base pair DNA ladder

Abstract: DNA size markers are widely used to estimate the size of DNA samples on agarose or polyacrylamide gel electrophoresis (PAGE). DNA markers can be prepared by mixing PCR products with definite sizes. Alternatively, they are prepared by restriction enzyme digestion of the genomic DNA of bacteriophages or natural and synthetic DNA plasmids. The present study describes engineering of a synthetic plasmid which produces a 100 bp DNA ladder, a popular DNA size marker, upon digestion with a single restriction enzyme. Our strategy consisted on sequential cloning of ten PCR products of 100 to 1000 bp in plasmid pTZ57R, using the BamHI and BglII restriction enzymes and releasing the fragments from the recombinant plasmid by enzyme EcoRV. This strategy could be applied to construct various complex synthetic vectors to produce different DNA ladders.

pSDTV vector: a modification of the pBluescript SK+ plasmid in order to perform PCR-fragments TA-cloning using Eam1105I restriction endonuclease.

Abstract: Some of the approaches for cloning PCR products obtained with conventional Taq-polymerases which do not involve modifications of the ends of the vector or the insert are based on the use of restriction enzymes which can generate 3' thymine single nucleotide overhangs, such as Eam1105I (AhdI). Due to the presence of Eam1105I restriction site within the β-lactamase gene, this is not achievable with a number of the most widely used cloning vectors descending from the pUC family, for which the selection is based on the ampicillin resistance. In this report we describe the construction of a vector for TA-cloning, based on the abolishment of the Eam1105I recognition site within the β-lactamase gene by site-directed mutagenesis, and the introduction of a stuffer flanked by Eam1105I target sites within the polylinker of the pBluescript SK+ plasmid.

DNA and Chromatin Fiber-Based Plant Cytogenetics

Abstract: Development of the fluorescence in situ hybridization (FISH) technique revolutionized cytogenetic research. FISH on prepared chromosomes has become the most commonly used technique in plant molecular cytogenetics, especially as a physical mapping tool in plant genome research. Despite its popularity, chromosome-based FISH analysis is limited in its capacity to distinguish DNA probes that separated by less than a few megabases. Development of FISH methods based on extended DNA fibers has dramatically increased the resolving power of this technique to the point where one can identify clones separated by only a few kilobases. In addition to the conventional fiber-FISH analysis, specialized techniques have been developed to prepare DNA or chromatin fibers that are suitable for restriction mapping (optical mapping) or immunofluorescence assays. Fiber-FISH and its derivatives are now used extensively in various mapping and genome research projects.

PCR-RFLP of 16S rRNA Gene for Identification of Speciation of Meat of Porcine Origin in Processed and Admixed Meat

Abstract: The PCR-RFLP using 16S rRNA gene was explored to test its effectiveness in identification of speciation of meat from porcine in raw, cooked as well as admixed meat. Critical perusal of the restriction maps of ∼591-bp 16S rRNA fragment sequences from pig as well as other livestocks identified RsaI specific to pig, yielding 343-bp and 251-bp fragments in pig, while in other livestock species such as cattle, buffalo, goat and sheep, no restriction enzyme site was found. In all the samples of raw pork, the restriction digestion of 594-bp fragment with RsaI yielded the 343-bp and 251-bp fragments, while no restriction digestion in all other livestock species, i.e. cattle, buffalo, goat and sheep. PCR-RFLP with RsaI (In cursive) of 594-bp fragment amplified from three different types of cooked meat DNA samples from pig, i.e. cooking at 72°C for 30 min, steam cooking at 90°C for 30 min and autoclaving at 120°C/15 lb/30 min, yielded a similar restriction enzyme digestion profile as with raw meat DNA, suggesting no effect of cooking on PCR-RFLP. Similarly, PCR-RFLP was able to differentiate the origin of meat in mixed meat samples quite consistently up to the level of 25%; however, at the level below 10%, results were not consistent.

NKG2 Subfamily C (KLRC)

Abstract: NKG2 receptors are type II C-type, lectin-like, integral membrane glycoproteins, which are expressed on the cell surface as heterodimers with CD94, which is an invariant type II C-type, lectin-like polypeptide. CD94 lacks a cytoplasmic tail and therefore, cannot transduce signals. It is however essential for the expression of NKG2 receptors. Four distinct genes, A/B, C, E/H, and F, encode the NKG2 receptors. Of these receptors, CD94/NKG2A is an inhibitory one, as it contains a long cytoplasmic tail with two ITIMs. Others have short cytoplasmic tails, and each associates noncovalently with a homodimer of DAP-12, as in the case of activating KIRs. The NKG2 family of genes (HGMW-approved symbol KLRC) contains at least six members (NKG2-A, -B, -C, -E, -F and -H) which are localized to human chromosome 12p12.3-p13.2, in the same region where CD69 genes have been mapped. In addition, the human CD94 and NKR-P1A genes map to the short arm of chromosome 12. The physical distance spanned by NK gene complex (NKC) in humans ranges between 0.7 and 2.4 megabases (Renedo et al. 1997). The NKG2 and CD94 genes are localized in a small region (< 350 kb) and mapped in the following order: (NKG2-C/NKG2-A)/NKG2-E/NKG2-F/NKG2-D/CD94. Sequence analysis of 62 kb spanning the NKG2-A, -E, -F, and -D loci allowed the identification of two LINE elements that could have been involved in the duplication of the NKG2 genes. Presence of one MIR and one L1ME2 element at homologous positions in the NKG2-A and NKG2-F genes is consistent with the existence of rodent NKG2 gene(s). The 5′-ends of the NKG2-A transcripts were mapped into two separate regions showing the existence of two separate transcriptional control regions upstream of the NKG2-A locus and defining putative promoter elements for these genes (Plougastel and Trowsdale 1998). Restriction mapping and sequencing revealed the NKG2-C, -D, -E, and -F genes to be closely linked to one another, and of the same transcriptional orientation. The NKG2-C, -E, and -F genes, despite being highly similar, are variable at their 3′ ends. It was found that NKG2-C consists of six exons, whereas NKG2-E has seven, and the splice acceptor site for the seventh exon occurs in an Alu repeat. NKG2-F consists of only four exons and part of exon IV is in some cases spliced to the 5′ end of the NKG2-D transcript. NKG2-D has only a low similarity to the other NKG2 genes Glienke et al. (1998). The murine NKG2-D-like sequence also maps to the murine NK complex near CD94 and Ly49 family members.

SSDNA Cutter v0.0: A new in silico RFLP tool in C

Abstract: Summary: SSDNA Cutter v0.0 is a new in silico RFLP tool written in C. It has a significant utility in studying population diversity as it has the capability of restriction digestion of a group of linear DNA se-quences and then segregate the restriction patterns into separate groups as per similarity in the restriction maps. The software has an inbuilt database for 20 restriction enzymes and it is flexible so that the user can add up to 100 more restriction enzymes to the list. The interface is easy, simple and interactive which enables the user to obtain restriction pattern groups which are quite similar to Operational Taxonomic Units obtained from phylogenetic trees. Availability: The software and the source code is currently available from the authors on request without any cost. We intend to release it in a public domain soon. SSDNA Cutter v0.0 is licensed under the GNU General Public License.

Structure prediction of a putative restriction endonuclease (AbaSORF13P) from Acinetobacter baumannii SDF

Abstract: Restriction enzymes represent a class of endonucleases that cleave DNA in a specific manner. They are widely used in recombinant DNA technology and known as molecular scissors. In this communication, a putative restriction endonucleases from (Acinetobacter baumannii SDF) was identified in NCBI database, and characterized for biophysical properties, sequence homology and a structural modeling. This putative restriction enzyme (AbaSORF13P) shows Molecular Mass equal to 41kDa with high content of aromatics amino acids. In homology search, the enzyme was found to be a member of PLD superfamily. It showed very high sequence similarity with known restriction enzymes BmrI and BfiI. The tertiary structure of the enzyme was determined on the basis homology modeling at SWISS-MODEL workspace using BfiI as the template. The enzyme was found to the made up of two subunits joined together in an asymmetric manner. These studies confirm, restriction endonuclease like behavior of the hypothetical protein, and these findings will be very important in characterization of the enzyme in vitro.