Dna sequencing

Comprehensive high-throughput alanine scanning mutagenesis was carried out on an HCV E1E2 expression construct (genotype 1a, strain H77; reference sequence NC_004102) encoding a C-terminal V5 epitope tag. Individual residues of E1 and E2 were mutated to alanine while existing alanine residues were mutated to serine to create a library of clones, each with a single point mutation. Overall, 545 mutants were generated by Integral Molecular, Inc., covering 98.2% of the E1E2 target residues. The sequence of each clone was confirmed by DNA sequencing (Macrogen) and the library clones were arrayed in 384-well format with each well containing one mutation [26 (link)]. Remaining constructs were found to have additional mutations and to complete the library, these 10 alanine mutations (R237A, C272A, Q336A, D346A, T396A, C452A, K562A, Y613A, Y624A, and W712A) were introduced into the H77C E1E2 sequence [67 (link)] using the QuikChange Lightening Site-Directed Mutagenesis kit (Stratagene) and PCR primers for each mutation (Integrated DNA Technologies). The sequence of these clones was confirmed by DNA sequencing (Retrogen).

A luciferase reporter assay system (Promega) was used to measure promoter activity. To construct the 1.5-kb promoter luciferase reporter plasmid for five genes (Dmrt1, Stra8, Tex13, Triml1 and 1700061G19Rik), DNA fragments corresponding to the putative promoters predicted by DBTSS (http://dbtss.hgc.jp./) were prepared by PCR using the pfu DNA polymerase (Enzynomics) with mouse genomic DNA isolated using Dneasy Blood & Tissue kit (Qiagen). The utilized primers are listed in Table S1. Several deleted versions of the Tex13-luciferase reporter plasmids were also prepared. The sequence of each PCR product was confirmed by DNA sequencing (Macrogen), and each fragment of interest was cloned into the multi-cloning site of the promoter-less pGL3-Basic (Promega) plasmid.

The expression vector pDG148 that can replicate in B. cereus and contains the IPTG-inducible Pspac promoter was used to construct plasmids for inducible gene expression. The plasmids derived from pDG148 are listed in Table 1, and primers used are listed in Table 2. The 1107 bp fragment with the complete gerRB gene (BC_0782) was amplified from genomic DNA with primers YW_6 and YW_2 and the amplicon named gerRB. The SGFP2 gene was amplified from plasmid pSGFP2-C1 with primers YW_3 and YW_7. The purified gerRB and SGFP2 fragments were fused with a flexible linker using a two-step fusion PCR. The purified gerRB-SGFP2 fragment was cloned into pDG148 between the Sal I and Sph I sites, producing pDG148-gerRB-SGFP2 (Figure 2B). The correct construction of the recombinant plasmids was confirmed using analysis of double restriction enzyme digestion and DNA sequencing (Macrogen Europe B. V. The Netherlands).

The A. carolinensis tlr5 gene (actlr5) was amplified from genomic DNA (500 ng) by PCR in 50 μl volume containing 1X Phusion polymerase buffer, dNTP’s (0.2 mM each), MgCl₂ (50 mM), Phusion hot start II high fidelity polymerase (1 Unit, Thermo Scientific) and 20 μM of forward (5′-CCGGATCCATGAAAAAGATGCTTCATTATCTCTTC-3′) and reverse (5′-CCGCGGCCGCAAGAGATTGTGACTACTTT-3′) primer (Life Technologies). Underlined sequences in the forward and reverse primer indicate BamHI and NotI restriction sites, respectively. The bold GC in the reverse primer substituted an AG in the tlr5 gene, thereby replacing the terminal stopcodon for a cysteine. PCR conditions were: one cycle for 1 min at 98 °C followed by 35 cycles of 30 s at 98 °C, 30 s at 54 °C, 90 s at 72 °C and one final extension step of 10 min at 72 °C. The PCR product was purified from gel using the GeneJet gel extraction kit (Thermo Scientific) and ligated into a pTracer-CMV2ΔGFP/3×FLAG8 (link) using the BamHI and NotI restriction sites, yielding pTracer 3 × FLAG-actlr5 carrying actlr5 with a C-terminal 3×FLAG tag. The plasmid was propagated in DH5-α. The cloned actlr5 gene sequence was verified by DNA sequencing (Macrogen). The sequence was deposited in Genbank (accession number: KT347095).

All chimeric TM segments were obtained adding the indicated number of extra leucines to the minimized GpA dimerization motif described by 19 (link). DNA coding for the chimeric TM segments were introduced into the ToxRED plasmid (provided by Dr. Berger) using XhoI and HindIII restriction sites (the selected restrictions sites incorporated a Lys residue preceded by a Leu residue at the amino terminus of the constructs) or into modified BiFC plasmids (pBiFC-VN155 and pBiFC-VC173, provided by Dr. Orzáez 57 (link)) fused to the amino- or carboxyl-end of the Venus Fluorescent Protein using NotI site. All plasmid sequences were corroborated by DNA sequencing (Macrogen, www.macrogen.com).

Yeast asn1(A6E)::GFP (asn2Δ) was created for characterization of the protein expression levels and the assembly frequency of Asn1p(A6E)-GFP, in comparison with those of Asn1p(WT)-GFP, which was expressed by the control yeast ASN1(WT)::GFP (asn2Δ), as previously constructed [28 (link)]. For this, yeast asn2Δ, constructed from the very first step, was used as the base strain. The sequence-verified plasmid pFA6a-asn1(A6E)-GFP-kanMX6 was used as DNA template to make the DNA cassette harboring (from 5′ to 3′) 50 bp upstream of ASN1 start codon, asn1(A6E) coding sequence, GFP, kanamycin resistance gene, and 50 bp downstream of ASN1 stop codon. The PCR reaction was performed using the KOD Hot Start DNA Polymerase Kit. Yeast asn2Δ was transformed with the purified DNA cassette of asn1(A6E)::GFP;kanR using lithium acetate–single-stranded DNA–polyethylene glycol and the heat shock method. Yeast transformants were selected on the YPD agar supplemented with G418 (400 μg/mL final concentration) and hygromycin B (200 μg/mL final concentration). The yeast transformants were initially screened under the fluorescence microscope (to check for GFP signals) and their genomic DNA samples were then extracted to prepare PCR products for DNA sequencing (Macrogen).