ndei  (New England Biolabs)

Journal: bioRxiv

Article Title: Characterization of the NSE6 subunit of the Physcomitrium patens PpSMC5/6 complex

doi: 10.1101/2022.07.28.501545

Figure Lengend Snippet: Detail analysis of the NSE5-NSE6 interaction. (A) Schematic representation of the different PpNSE6 constructs. The CANIN domain (aa130 – 370) is shown in red. (B) The indicated Gal4AD-PpNSE6 constructs were tested in Y2H for binding to the Gal4BD-PpNSE5(aa1-526). The intact CANIN domain was essential for the PpNSE5-PpNSE6 interaction. (C) Gal4AD-PpNSE6(1-370) mutants were tested in Y2H for binding to Gal4BD-PpNSE5(aa109-526), Gal4BD-PpSMC5(aa280-790), and Gal4BD-PpSMC6(aa226-955) constructs. The Ppnse6/R293A mutation (bold) specifically disturbed the PpNSE6-PpNSE5 interaction, while the Ppnse6/E294A mutation (italic) affected protein structure. All Y2H protein-protein interactions were scored by the growth of the yeast PJ69 transformants on the plates without Leu, Trp, His (-L,T,H), and with indicated concentration of 3-Amino-1,2,4-triazole (AT). Control plates were lacking only Leu and Trp (-L,T). Empty pGBKT7 and pGADT7 vectors were used as negative controls.

Article Snippet: The PpNSE6 construct with aa75-370 was first cloned into the pGBKT7 Nde I-Bam HI sites with NEBuilder, then cleaved out with Nde I-Bam HI and ligated into the Nde I-BamHI sites of pGADT7.

Techniques: Construct, Binding Assay, Mutagenesis, Concentration Assay

Journal: Virology Journal

Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein

doi: 10.1186/1743-422X-9-279

Figure Lengend Snippet: TMV-CP DNA fragments (1% agarose gel). ( A ) The whole TMV-CP fragments with BamH I and Xho I restriction enzyme cutting sites which have been cloned in PGEX-6P-1. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a positive control with DNA template. Lane 3 is a negative control without DNA template. ( B ) The whole TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a negative control without DNA template. Lane 3 is a positive control with DNA template. ( C ) The truncation of four amino acids from the C-terminus of TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. Lane 1 is a negative control without DNA template, whereas lane 2 is a positive control with DNA template. Lane 3 is the amplified PCR product that ran at approximately 500 bp compared with the DNA marker (lane M).

Article Snippet: Both plasmid pET28a (Novagen) and CP were digested with Nde I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in pET28a (pET28a-WT-His-TMV-CP12 , pET28a-TR-His-TMV-CP19 , pET28a-TR-His-TMV-CP62 , and pET28a-TR-His-TMV-CP68 ).

Techniques: Agarose Gel Electrophoresis, Clone Assay, Amplification, Polymerase Chain Reaction, Marker, Positive Control, Negative Control

Journal: Journal of Bacteriology

Article Title: Gene Conservation and Loss in the mutS-rpoS Genomic Region of Pathogenic Escherichia coli

doi:

Figure Lengend Snippet: Restriction maps of the mutS-rpoS chromosomal region. Approximate locations of restriction sites for five restriction enzymes: Eco RV (E), Nde I (N), Acc I (A), Csp 45 (C), and Nsp I (Ns). The pattern of restriction sites is conserved among strains of each pathogenic group with the exception of the second Nsp I site in mutS [(Ns)*], which is present in EPEC 2 strains but absent in EHEC 2 strains. A distinct Nsp I map was obtained for 921-B4 (not shown). The novel DNA segment found in EPEC and EHEC strains is located at the 3′ end of rpoS and is highlighted with the gray bar.

Article Snippet: To estimate the length of the mutS-rpoS genomic region and to map the length variation, the long PCR amplicons were digested with five restriction enzymes, Eco RV and Nde I (New England Biolabs, Beverly, Mass.), Csp 45 (Promega, Madison, Wis.), and Acc I and Nsp I (GIBCO-BRL).

Techniques:

Journal: Genetics

Article Title: The Length of the Shortest Telomere as the Major Determinant of the Onset of Replicative Senescence

doi: 10.1534/genetics.113.152322

Figure Lengend Snippet: Telomere length distribution by simulation and single-telomere Southern blot. (A) The protein-counting mechanism is based on the measurement of telomere length by Rap1/Rif1/Rif2 binding to telomeric repeats and an inhibition of telomerase elongation depending on the number/concentration of these complexes. (B) Trajectories of 10 independent telomeres over 100 divisions. Depending on the length, a telomere (blue trajectory) can shorten by length a over several consecutive divisions (solid arrowhead) and then be elongated by a random length b (shaded arrowhead). (C) Telomere length distribution at equilibrium. Equation 1 was iterated 500 times starting with 100,000 telomeres drawn from an uniform distribution between 200 and 400 bp, and the resulting telomere length distribution was plotted (bin size, 3 bp). (D) Representative single-telomere Southern blot. Six independent TLC1 wild-type spores derived from TLC1/tlc1 Δ heterozygous diploids were grown for 24 hr on YPD plates and then transferred to liquid culture for another 24 hr at exponential growth (for a total of ∼30 population doublings). The Southern blot was performed on Nde I- and Bst EII-digested genomic DNA using simultaneously oT355 (green) and oT360 (red) fluorescent probes, designed to detect I-L and VI-R telomeres, respectively. (E) Single-telomere Southern blot with oT355 and oT360 fluorescent probes performed on telomerase-positive (wild type, WT) or negative ( tlc1 Δ) cells. TLC1/tlc1 Δ diploid cells were sporulated, and the four spores from a tetrad were grown for 24 hr on a plate and then in liquid YPD medium for the total indicated population doublings (PD). (F) Comparison of experimental and simulated data of telomere length distribution. Plot of 117 experimental measurements of telomere length from single-telomere Southern blot as in D (black line, bin size, 20 bp) and 1000 simulated values L i ¯ ∈ [ | 1 : 1000 | ] (colored lines, bin size, 20 bp) for 20, 30, …, 70 divisions. The latter were obtained as follows: after drawing randomly 100 initial lengths L i 0 from the theoretical distribution in C, we applied the dynamics of Equation 1 to 1000 telomeres with initial length L i 0 to get, after 50 divisions, a mean length L i ¯ . This was done for each plot, corresponding to 20, 30,…, or 70 divisions (blue to orange lines).

Article Snippet: Nde I, Bst EII, and Bst NI enzymes were purchased from New England Biolabs.

Techniques: Southern Blot, Binding Assay, Inhibition, Concentration Assay, Derivative Assay