Journal: BMC Genomics

Article Title: Different nucleosomal architectures at early and late replicating origins in Saccharomyces cerevisiae

doi: 10.1186/1471-2164-15-791

Figure Lengend Snippet: The NDR is modified along S phase. (A) , ARS603 is represented by a red rectangle and flanking genes, YFL034W and YFL033C, by blue ones. Vertical bars indicate sites for digestion with restriction enzymes PvuII and HindIII. Green rectangle represents the end-terminal probe. The nucleosomal profile from the G1 sample is presented below. (B) , Naked DNA and chromatin from wild-type cells collected at 20, 40 60 and 80 min after α-factor arrest and release into YPD were digested with increasing amounts of MNase (indicated with triangles) and subsequently digested with PvuII and HindIII. Samples were electrophoresed, transferred onto a membrane and hybridized. A ladder of polynucleosomes is observed with a prominent region of hypersensitivity to MNase corresponding to the NDR (indicated in brackets in A , B ). The black arrow and asterisk point to the changes in chromatin organization observed in the NDR at later time points.

Article Snippet: For each sample, 2.5 μg of DNA were digested with HindIII and PvuII enzymes (Fermentas), separated by electrophoresis in a 1.5% agarose gel, blotted and hybridized to an specific end-terminal probe.

Techniques: Modification

Journal: eLife

Article Title: A universal vector concept for a direct genotyping of transgenic organisms and a systematic creation of homozygous lines

doi: 10.7554/eLife.31677

Figure Lengend Snippet: The pAVOIAF{#1–#2–#3–#4} vector. ( A ) Vector map of pAVOIAF{#1–#2–#3–#4}. The vector is based on the pUC57-Kan vector, from which only the kanamycin resistance cassette and the origin of replication remain. The four-slot cloning site together with the 3’ and 5’ piggyBac terminal repeats is located between the AatII and PciI sites. The light gray band on the inside indicates the transgene. ( B ) Scheme of the four-slot cloning site. Each slot consists of an 18 bp spacer that translates into the amino acids Phe-Arg-Glu-Asp-Asp-Tyr and thus was termed FREDDY spacer. The slots can be accessed individually by unique restriction enzyme site pairs (XmaI/SpeI for #1, HindIII/XbaI for #2, XhoI/NheI for #3 and AflII/AvrII for #4). They are embedded into five PmeI restriction enzyme sites that allow a simple one-enzyme control digestion to determine the size of the sequences that were inserted into the slots. For convenience, the downstream restriction enzyme sites for each slot (SpeI for #1, XbaI for #2, NheI for #3 and AvrII #4) result in identical sticky ends, facilitating cloning procedures that cannot utilize the suggested restriction enzyme site pairs. Extends of the genetic elements are not to scale. ORF, open-reading frame.

Article Snippet: Molecular biology: the pGS[#P’#O(LA)-mEmerald] and pAGOC{#P’#O(LA)-mEmerald} vectors A hybrid sequence, consisting of (i) a HindIII site, (ii) the modular fluorescent protein expression cassette as described above and (iii) a XbaI site, was de novo synthetized and inserted into the unique SfiI site of pMK-RQ (Thermo Fisher Scientific).

Techniques: Plasmid Preparation, Clone Assay

Journal: Journal of Virology

Article Title: Roles of the Phosphorylation of Herpes Simplex Virus 1 UL51 at a Specific Site in Viral Replication and Pathogenicity

doi: 10.1128/JVI.01035-18

Figure Lengend Snippet: Strategy to construct a self-excisable HSV-1(F) BAC clone. (A) Lines 1 and 2, schematic diagrams of E. coli plasmid pYEbac102 and pYEbac102Cre, respectively, contained in GS1783; line 3, schematic diagrams of recombinant virus YK312 (ΔBAC). (B) Schematic diagram of the genome structures of wild-type HSV-1(F) and relevant domains of the recombinant viruses. Line 1, sequence arrangement of the HSV-1(F) genome. Line 2, location of the EcoRV-HindIII fragment used as a labeled probe in panel C. Line 3, an enlarged portion of the EcoRI J and D fragments of HSV-1(F). Lines 4 and 6, double-headed arrow represents the fragment region. Lines 5 and 7, summary of DNA fragments from recombinant viruses with or without the BAC sequence and the poly(A) signals. (C) Southern blot analysis of EcoRI-digested DNA isolated from YK312 (ΔBAC)- or YK304 (BAC)-infected cells using the EcoRV-HindIII fragment of pRB442 as a probe. The letters on the right refer to the designations of the DNA fragments generated by restriction endonuclease EcoRI cleavage.

Article Snippet: This was then cloned into the EcoRI site of pcDNA3.1(+) (Thermo Fisher Scientific). pcDNA-FEM-KanS was constructed by PCR amplification of the domain containing the FEM tag, I-SceI site, and kanamycin resistance gene from pBS-FEM-KanS and cloning it into the EcoRI and HindIII sites of pcDNA3.1/myc-His(−)-A (Thermo Fisher Scientific). pcDNA-FEM was constructed by digesting pcDNA-FEM-KanS with NheI and self-ligating to remove the I-SceI site and kanamycin resistance gene. pcDNA-FEM-UL51 was constructed by amplifying the entire UL51 open reading frame (ORF) from pYEbac102 by PCR and cloning it into pcDNA-FEM in frame with FEM. pBS1007, used to generate the recombinant virus YK639 (ΔUL51-repair), was described previously ( ). pRB442, used to generate the probe for Southern blotting, contains a 6.3-kbp KpnI-HindIII fragment of HSV-1(F), which encompasses the left end of the UL region, including the region between 0.0415 and 0.084 map units, as described previously ( ).

Techniques: Construct, BAC Assay, Plasmid Preparation, Recombinant, Sequencing, Labeling, Southern Blot, Isolation, Infection, Generated

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Oral Administration of Lactococcus lactis Expressing Synthetic Genes of Myelin Antigens in Decreasing Experimental Autoimmune Encephalomyelitis in Rats

doi: 10.12659/MSM.892764

Figure Lengend Snippet: Control digestions of recombinant plasmid DNA using HindIII (Fermentas) enzyme. (A) pIL253:PptcB:MOG35-55, (B) pIL253:PptcB:MBP85-97, (C) pIL253:PptcB:PLP139-151. Expected DNA fragments after restriction analysis for correct constructs: 3897 bp, 845 bp (marked by red arrows), 1kb DNA ladder (M) 1kb DNA Ladder (Fermentas) used as DNA size reference marker.

Article Snippet: From confirmed proper recombinant cells, plasmid DNA was isolated using the Plasmid Mini Kit (A & A Biotechnology) and subjected to restriction analysis with HindIII enzyme (Fermentas).

Techniques: Recombinant, Plasmid Preparation, Construct, Marker

Journal: PLoS ONE

Article Title: A Selectable and Excisable Marker System for the Rapid Creation of Recombinant Poxviruses

doi: 10.1371/journal.pone.0024643

Figure Lengend Snippet: Confirmation of genomic composition of 3 independent recombinant VV-ΔTk viruses. ( A ) An ethidium bromide stained DNA gel of genomic HindIII restriction digests of viral DNA isolated from parental VV (Wyeth Strain), 3 clones of VV-ΔTk- yfp-gpt and 3 clones of VV-ΔTk'. Arrows indicate the Tk insertion site (VV-Wyeth), and the unique bands that result from insertion of the yfp-gpt cassette (VV-ΔTk- yfp-gpt ). ( B ) Southern hybridization of the DNA gel in A identifying the yfp insert present in the genome of the VV-ΔTk- yfp-gpt clones, but not in parental VV-Wyeth or the VV-ΔTk' clones. ( C ) DNAStar sequence alignment at the yfp-gpt insertion site of DNA isolated from the 3 VV-ΔTk' clones post Cre passage.

Article Snippet: Briefly, 7 µg of DNA was digested overnight with HindIII restriction enzyme (Invitrogen) at 37°C.

Techniques: Recombinant, Staining, Isolation, Clone Assay, Hybridization, Sequencing