Journal:

Article Title: Apyrase, the Product of the Virulence Plasmid-Encoded phoN2 ( apy ) Gene of Shigella flexneri , Is Necessary for Proper Unipolar IcsA Localization and for Efficient Intercellular Spread †

doi: 10.1128/JB.188.4.1620-1627.2006

Figure Lengend Snippet: Bacterial strains and plasmids a

Article Snippet: The locations of 3× FLAG-tagged OspB recombinant protein as well as of IpaB, IpaC, and apyrase proteins are indicated. na, not added. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant genotype or characteristic(s) Source or reference Strains M90T Wild-type S. flexneri serotype 5a 32 HND115 M90T Δ phoN2 ; susceptible This work HND201 M90T Δ ospB ; susceptible This work HND215 M90T Δ ospBphoN2 ; susceptible This work HND43 M90T Δ mxiA ; susceptible This work HND53 M90T Δ virB :: aphA-3 ; Km r This work HND549 M90T ospB -3×FLAG; susceptible This work HND5311 M90T Δ mxiA ospB -3×FLAG; susceptible This work SC560 M90T Δ icsA ::Ω; Sm r 10 M15[pREP4] E. coli K-12 strain carrying plasmid pREP4 Qiagen Plasmids pGEM-T Cloning vector, Ap r Promega pBluescript SK Cloning vector, Ap r Stratagene Inc., La Jolla, CA pBAD28 Arabinose-inducible P BAD expression vector, Ap r Cm r 13 pSUB11 Template plasmid carrying a 3×FLAG epitope and Km r cassette 42 pKD46 λ Red helper plasmid; oriR101 repA101 (Ts) P-araB-gam-bet-exo Ap r 7 pKD4 Template plasmid carrying a Km r gene with FLP recognition target sequence 7 pCP20 FLP helper plasmid; pSC101 replicon (Ts) bla cat Flp (λR p ) cI 857 Ap r Cm r 7 pREP4 lacI , Km r Qiagen pQE30 Cloning vector; Ap r Qiagen pHN301 pQE30 carrying His6- phoN2 ; Ap r This work pHN28 pBAD28 carrying phoN2 ; Ap r Cm r This work pHN111 pBAD28 carrying mxiA ; Ap r Cm r This work pFB1-4 pBluescript SK carrying a mutated phoN2 gene of EIEC strain HN280 presenting the R192P amino acid substitution 36 Open in a separate window a Ap r , ampicillin resistance; Cm r , cloramphenicol resistance; Km r , kanamycin resistance.

Techniques: Plasmid Preparation, Clone Assay, Expressing, Sequencing

Journal:

Article Title: Apyrase, the Product of the Virulence Plasmid-Encoded phoN2 ( apy ) Gene of Shigella flexneri , Is Necessary for Proper Unipolar IcsA Localization and for Efficient Intercellular Spread †

doi: 10.1128/JB.188.4.1620-1627.2006

Figure Lengend Snippet: Formation of plaques on confluent monolayers of HeLa and Caco-2 cells a

Article Snippet: The locations of 3× FLAG-tagged OspB recombinant protein as well as of IpaB, IpaC, and apyrase proteins are indicated. na, not added. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant genotype or characteristic(s) Source or reference Strains M90T Wild-type S. flexneri serotype 5a 32 HND115 M90T Δ phoN2 ; susceptible This work HND201 M90T Δ ospB ; susceptible This work HND215 M90T Δ ospBphoN2 ; susceptible This work HND43 M90T Δ mxiA ; susceptible This work HND53 M90T Δ virB :: aphA-3 ; Km r This work HND549 M90T ospB -3×FLAG; susceptible This work HND5311 M90T Δ mxiA ospB -3×FLAG; susceptible This work SC560 M90T Δ icsA ::Ω; Sm r 10 M15[pREP4] E. coli K-12 strain carrying plasmid pREP4 Qiagen Plasmids pGEM-T Cloning vector, Ap r Promega pBluescript SK Cloning vector, Ap r Stratagene Inc., La Jolla, CA pBAD28 Arabinose-inducible P BAD expression vector, Ap r Cm r 13 pSUB11 Template plasmid carrying a 3×FLAG epitope and Km r cassette 42 pKD46 λ Red helper plasmid; oriR101 repA101 (Ts) P-araB-gam-bet-exo Ap r 7 pKD4 Template plasmid carrying a Km r gene with FLP recognition target sequence 7 pCP20 FLP helper plasmid; pSC101 replicon (Ts) bla cat Flp (λR p ) cI 857 Ap r Cm r 7 pREP4 lacI , Km r Qiagen pQE30 Cloning vector; Ap r Qiagen pHN301 pQE30 carrying His6- phoN2 ; Ap r This work pHN28 pBAD28 carrying phoN2 ; Ap r Cm r This work pHN111 pBAD28 carrying mxiA ; Ap r Cm r This work pFB1-4 pBluescript SK carrying a mutated phoN2 gene of EIEC strain HN280 presenting the R192P amino acid substitution 36 Open in a separate window a Ap r , ampicillin resistance; Cm r , cloramphenicol resistance; Km r , kanamycin resistance.

Techniques:

Journal:

Article Title: Apyrase, the Product of the Virulence Plasmid-Encoded phoN2 ( apy ) Gene of Shigella flexneri , Is Necessary for Proper Unipolar IcsA Localization and for Efficient Intercellular Spread †

doi: 10.1128/JB.188.4.1620-1627.2006

Figure Lengend Snippet: phoN2 mutant strain HND115 forms plaques of approximately wild-type size on Caco-2 cell monolayers when complemented with plasmid pFB1-4. pFB1-4 (Table ​(Table1)1) contains a randomly generated mutant of the apy gene, the orthologue of phoN2 in the EIEC strain HN280, encoding a recombinant apyrase presenting the R192P substitution which inactivates its dNTP-hydrolyzing activity. (A) Western blot analysis with anti-apyrase antibodies of whole-cell extracts of M90T, HND115 (phoN2), and HND115/pFB1-4 (pBluescript SKapyR192P). (B) Plaques formed by M90T, HND115 (phoN2), and HND115/pFB1-4 (pBluescript SKapyR192P). Dishes were photographed after 48 h of infection.

Article Snippet: The locations of 3× FLAG-tagged OspB recombinant protein as well as of IpaB, IpaC, and apyrase proteins are indicated. na, not added. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant genotype or characteristic(s) Source or reference Strains M90T Wild-type S. flexneri serotype 5a 32 HND115 M90T Δ phoN2 ; susceptible This work HND201 M90T Δ ospB ; susceptible This work HND215 M90T Δ ospBphoN2 ; susceptible This work HND43 M90T Δ mxiA ; susceptible This work HND53 M90T Δ virB :: aphA-3 ; Km r This work HND549 M90T ospB -3×FLAG; susceptible This work HND5311 M90T Δ mxiA ospB -3×FLAG; susceptible This work SC560 M90T Δ icsA ::Ω; Sm r 10 M15[pREP4] E. coli K-12 strain carrying plasmid pREP4 Qiagen Plasmids pGEM-T Cloning vector, Ap r Promega pBluescript SK Cloning vector, Ap r Stratagene Inc., La Jolla, CA pBAD28 Arabinose-inducible P BAD expression vector, Ap r Cm r 13 pSUB11 Template plasmid carrying a 3×FLAG epitope and Km r cassette 42 pKD46 λ Red helper plasmid; oriR101 repA101 (Ts) P-araB-gam-bet-exo Ap r 7 pKD4 Template plasmid carrying a Km r gene with FLP recognition target sequence 7 pCP20 FLP helper plasmid; pSC101 replicon (Ts) bla cat Flp (λR p ) cI 857 Ap r Cm r 7 pREP4 lacI , Km r Qiagen pQE30 Cloning vector; Ap r Qiagen pHN301 pQE30 carrying His6- phoN2 ; Ap r This work pHN28 pBAD28 carrying phoN2 ; Ap r Cm r This work pHN111 pBAD28 carrying mxiA ; Ap r Cm r This work pFB1-4 pBluescript SK carrying a mutated phoN2 gene of EIEC strain HN280 presenting the R192P amino acid substitution 36 Open in a separate window a Ap r , ampicillin resistance; Cm r , cloramphenicol resistance; Km r , kanamycin resistance.

Techniques: Mutagenesis, Plasmid Preparation, Generated, Recombinant, Activity Assay, Western Blot, Infection

Journal: bioRxiv

Article Title: Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening

doi: 10.1101/2021.12.23.474055

Figure Lengend Snippet: (A) A schematic diagram of the strategy for cloning the BAC vector encoding the SARS-CoV-2 reporter replicon. PCR fragments F1 to F10 were amplified from subcloning or cassette vectors by PCR using the primers listed in as described in Materials and Methods and then subjected to CPER. The resulting vector was designated pBAC-SCoV2-Rep-Reo. The numbers inside the parentheses represent the nucleotide positions in the SARS-CoV-2/Hu/DP/Kng/19-020 genome. TRSs in the leader sequence of the 5’ UTR (TRS-L) and in the genome body (TRS-B) are indicated as a white arrowhead and a black arrowhead, respectively. The blue line indicates the pSMART-BAC v2.0 vector sequence. Reo, a fusion gene consisting of Renilla luciferase and neomycin phosphotransferase; CMVp, cytomegalovirus promoter; 5’, 5’ UTR; 3’, 3’ UTR; pA, synthetic 25-poly(A); Rz, hepatitis delta virus ribozyme; BGH, bovine growth hormone termination and polyadenylation sequence. Refer to for detailed information on fragment F9 and the intermediate sequence between the ORF1ab and N genes in pBAC-SCoV2-Rep-Reo. (B) Immunostaining of N protein in VeroE6 cells transfected with pBAC-SCoV2-Rep-Reo or mock. These cells were fixed at 24 h post-transfection. Nuclei were stained with DAPI. (C) Western blotting of viral proteins. VeroE6, Huh7 and 293T cells were transfected with the replicon-BAC vector or mock, treated with DMSO (0.1%), IFN-β (100 U/ml) or remdesivir (0.5 μM for 293T; 5 μM for Huh7 and VeroE6) at 6 h post-transfection, and harvested at 54 h post-transfection. The cell lysates were subjected to Western blotting using specific antibodies against the N protein, nsp8 or actin. (D) Northern blotting of sgRNAs. The replicon-BAC vector was transfected into 293T cells as described in (C). The resulting cells were treated with each reagent and subjected to Northern blotting. Ribosomal RNAs were stained with EtBr. (E) Luciferase activities in the cells transfected with the replicon vector or mock. The replicon-BAC vector was transfected into 293T cells as described in (C). The resulting cells were treated with each reagent and subjected to a luciferase assay. Statistical significance was calculated compared to the mock group (*, p < 0.05; **, p < 0.01; ns, no significance).

Article Snippet: In this study, we prepared a BAC vector encoding SARS-CoV-2 reporter replicon RNA with a fusion gene encoding Renilla luciferase and neomycin phosphotransferase II (the replicon-BAC vector), examined the antiviral effects of remdesivir, molnupiravir, type I interferon (IFN), camostat and favipiravir on viral replication in the transient system using the replicon-BAC vector, and established a cell line that stably supports the replication of the replicon.

Techniques: Clone Assay, Plasmid Preparation, Amplification, Subcloning, Sequencing, Luciferase, Immunostaining, Transfection, Staining, Western Blot, Northern Blot

Journal: Toxins

Article Title: Pore-Forming Cardiotoxin VVA2 (Volvatoxin A2) Variant I82E/L86K Is an Atypical Duplex-Specific Nuclease

doi: 10.3390/toxins14060392

Figure Lengend Snippet: Re-VVA2 I82E/L86K cleaves dsDNA non-specifically with cleavage preference in dG-dC-rich regions. ( A ) The schematic diagram of the experimental design for mapping the cleavage sites of nuclease Re-VVA2 I82E/L86K. The cleaved products (pUC19 and pET28a, mainly in RF II) were digested by S1 nuclease and cloned into pre-linearized pBluescript II SK (+) vector by EcoRV. Colonies were selected and sequenced using universal primer M13F. The detailed protocol is stated in the Materials and Methods. ( B ) Cleavage sites of Re-VVA2 I82E/L86K on pUC19 and pET28a were marked on the related plasmid map. Specific cleavage sites are shown in . ( C ) Re-VVA2 I82E/L86K has cleavage preference in dG-dC-rich regions. The sequences around cleavage sites were aligned with WebLogo 3. The proposed cleavage sites were marked on the figure.

Article Snippet: Plasmid: pET28a, pUC19, pBluescript II SK (+) vector (Addgene, Watertown, MA, USA).

Techniques: Clone Assay, Plasmid Preparation

Journal:

Article Title: Modification of Type IV Pilus-Associated Epithelial Cell Adherence and Multicellular Behavior by the PilU Protein of Neisseria gonorrhoeae

doi: 10.1128/IAI.70.7.3891-3903.2002

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: All E. coli strains and their recombinants were maintained in Luria-Bertani medium supplemented with the appropriate antibiotics. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant characteristic(s) Reference or source N. gonorrhoeae strains N400 Derived from VD300, a contains the IPTG-inducible recA6 allele b 11 GF2 (PilF − ) pilF ::m-Tn 3erm 11 GT3 pilT ::m-Tn 3erm 40 GT7 pilT ::m-Tn 3erm 40 GT17 pilT ::m-Tn 3cm at position 73 c This study GT26 pilT ::m-Tn 3erm 40 GT50 pilT ::m-Tn 3erm at position 1105 c This study GT101 pilT dud1 40 GT102 pilT Δ QSL 40 GT103 pilT ::m-Tn 3erm 40 GT104 pilT ind 40 GT105 pilT fs164 40 GTU2 pilT ::m-Tn 3cm pilU ::mTn 3erm This study GTXE1 pilT :: xylE This study GUXE2 pilU :: xylE This study GU2 pilU ::m-Tn 3erm at position 1591 c This study GU4 pilU ::m-Tn 3erm at position 1714 c This study GU5 pilU ::m-Tn 3erm at position 2102 c This study GU21 pilU ::m-Tn 3erm at position 1314 c This study GU33 pilU ::m-Tn 3erm at position 2541 c This study GU127 pilU ::m-Tn 3erm at position 1192 c This study E. coli strains BL21(DE3) F − ompT r B − m B − with prophage λ carring the T7 RNA polymerase gene 32 HB101 F − mcrB mrr hsdS20 (r B − m B − ) recA13 supE44 ara14 galK2 lacY1 proA2 rpsL20 (Sm r ) xyl5 λ − leu mtl1 GIBCO BRL Plasmids pBluescript II SK Cloning vector, Amp r 32 p6/16/11 1.1-kb Sal I fragment in pBluescript II SK This study p11/2/13 3.3-kb EagI/Xba I fragment in pBluescript II SK This study pHSS6 Vector for shuttle mutagenesis, Kan r 27 p1-49 3.3-kb Eag I/ Xba I fragment in pHSS6 This study pT7-5 T7 RNA polymerase promoter λ10, Amp r 32 pT7U pilU in pT7-5 This work pT7U (Δ Sal I) Sal I deletion subclone of pT7U This work Open in a separate window a VD300 is an MS11 derivative ( 15 ). b All remaining Gc strains are derived from N400. c Nucleotide positions correspond to those in GenBank no. {"type":"entrez-nucleotide","attrs":{"text":"S72391","term_id":"632717","term_text":"S72391"}} S72391 .

Techniques: Plasmid Preparation, Derivative Assay, Clone Assay, Mutagenesis

Journal: Biochimica et biophysica acta. Gene regulatory mechanisms

Article Title: Interaction of Positive Coactivator 4 with Histone 3.3 Protein is Essential for Transcriptional Activation of the Luteinizing Hormone Receptor Gene

doi: 10.1016/j.bbagrm.2018.09.002

Figure Lengend Snippet: (A, above) Western blot showing PC4, H3 and acetylated H3 levels in MCF7 cells transfected with scrambled or PC4 siRNA followed by treatment with or without TSA (500ng/ml) for 16 h revealed by PC4, H3 and specific H3 acetylated antibodies to H3 Lys residues (K9, K14, K18, K23, K27 and K36). (A, below) Western blot showing PC4 and H3.3 levels in MCF7 cells treated with scrambled or PC4 siRNA followed by treatment with or without TSA. β-actin is shown as a loading control. Results are representative of three independent experiments. (B) Effect of endogenous PC4 knock-down by siRNA on LHR promoter activity. Cells transfected with scrambled (control) or PC4 siRNA followed by transfection of LHR promoter construct or PGL2 basic vector and later treated with or without TSA prior to measurement of promoter activity. Different superscript letters indicate significant differences (Dunn’s multiple comparison test, P < 0.001). (C) ChIP analysis illustrates relative enrichment of H3K9-Ac onto the LHR promoter of cells transfected with scrambled or PC4 siRNA cultured in the presence or absence of TSA. Different superscript letters indicate significant differences (Dunn’s multiple comparison test, P < 0.05). (D) H3.3 enrichment at the LHR promoter assessed in cells treated with and without TSA following PC4 knockdown. Different superscript letters indicate significant differences (Dunn’s multiple comparison test, P < 0.05). Representative western blot showing knock-down of PC4 protein in MCF7 cells following PC4 siRNA treatment. Data represent mean ± SE of two independent experiments performed in triplicates.

Article Snippet: Expression vectors and cell culture The reporter gene construct containing the LHR promoter was generated by cloning the human LHR gene promoter region (−176 to +1) into the SacI/BglII sites of the pGL2 basic vector [ 12 ]. pCMV6-PC4 was purchased from Origene (Rockville, MD) and used as PCR template for generation of constructs expressing PC4-Flag protein in MCF7 cells. p3XFLAG-PC4 vector was created by inserting PCR-amplified PC4 cDNA into the EcoRI and KpnI sites of the p3XFLAG-CMV-7.1 vector (Sigma) [ 11 ].

Techniques: Western Blot, Transfection, Activity Assay, Construct, Plasmid Preparation, Cell Culture