clone 5.3  (Vertex Pharmaceuticals)

Journal: Journal of Bacteriology

Article Title: PepD Participates in the Mycobacterial Stress Response Mediated through MprAB and SigE ▿ †

doi: 10.1128/JB.01167-09

Figure Lengend Snippet: Strains, plasmids, and bacteriophages used in this study

Article Snippet: Hygromycin B was purchased from AG Scientific (San Diego, CA). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or bacteriophage Genotype or description Application Reference or source Strains M. tuberculosis H37Rv Laboratory strain ATCC 27294 M. smegmatis mc 2 155 Laboratory strain ATCC 700084 E. coli DH5α [λ − φ80d lacZ ΔM15 Δ( lacZYA-argF ) U169 recA1 endA1 hsdR17 (r K − m K − ) supE44 thi-1 gyrA relA1 ] Cloning Laboratory collection E. coli TOP10 F − mcr A Δ( mrr-hsdRMS - mcrBC ) φ80 lac ZΔM15 Δ lac X74 recA1 araD139 Δ( ara leu ) 7697 galU galK rpsL (StrR) endA1 nupG Cloning Invitrogen E. coli HB101 supE44 hsdS20 (r B − m B − ) recA13 ara-14 proA 2 lacY1 galK2 rpsL20 xyl-5 mtl-1 leuB6 thi-1 E. coli transduction host Laboratory collection E. coli BL21(DE3)/pLysS F − ompT hsdS B(r B − m B − ) gal dcm (DE3)/pLysE (Cam r ) Protein overexpression Novagen Plasmids pCR2.1-TOPO 3.9-kb plasmid for cloning PCR products; Amp r Kan r PCR cloning vector Invitrogen pET-24b 5.3-kb plasmid allowing C-terminal protein fusion to His 6 ; Kan r Protein overexpression vector Novagen pMV306 3.9-kb plasmid for single-copy integration into mycobacterial chromosome; Kan r Complementation vector Laboratory collection pTZ557 pYUB854 containing pepD upstream and downstream regions; Hyg r pepD deletion This study pTZ612 pYUB854 containing sigE upstream and downstream regions; Hyg r sigE deletion This study pTZ758 pET-24b containing pepD ΔTM coding sequence; Kan r Overexpression This study pTZ775 pET-24b containing pepD ΔTMΔPDZ coding sequence; Kan r Overexpression This study pTZ792 pMV306 containing pepD and moaB2 coding sequences; Kan r Complementation This study pTZ797 pET-24b containing pepD ΔTMS317A coding sequence; Kan r Overexpression This study pTZ844 pYUB854 containing mprA upstream and mprB downstream regions; Hyg r mprAB deletion This study pTZ898 pYUB854 containing sacB coding sequence; Hyg r Mycobacterium gene deletion vector This study pTZ903 pMV306 containing pepD S317A and moaB2 coding sequences; Kan r Complementation This study pTZ947 pTZ898 containing mprAB upstream and downstream regions; Hyg r mprAB deletion This study pTZ949 pTZ898 containing pepD upstream and downstream regions; Hyg r pepD deletion This study pTZ1065 pMV306 containing sigE coding sequence; Kan r Complementation This study pYUB854 3.9-kb cosmid for generating gene deletions in Mycobacterium .

Techniques: Plasmid Preparation, Clone Assay, Transduction, Over Expression, PCR Cloning, Sequencing

Journal: PLoS ONE

Article Title: The effects of C5-substituted 2,4-diaminoquinazolines on selected transcript expression in spinal muscular atrophy cells

doi: 10.1371/journal.pone.0180657

Figure Lengend Snippet: Clone 11 NSC-34 cells harboring a reporter gene driven by the 3.4-kb SMN2 promoter were treated with 1 μM D156844, D158872, D157161, D157495 or DMSO (n = 4/drug) for 19 hours prior to fluorescent β-lactamase assay analysis. (A) All 4 compounds significantly increased SMN2 -drived BLA activity. The asterisk (*) denotes a statistically significant (p ≤ 0.05) difference between drug- and vehicle-treated cells. Dose-response curves (1 nM– 10 μM) for D156844 (B) , D158872 (C) , D157161 (D) and D157495 (E) . Each compound tested exhibited a dose-dependent increase in SMN2 -drived BLA activity.

Article Snippet: The clone 11 cell line (Vertex Pharmaceuticals, [ ]) was used for the SMN2 promoter assay and the clone 5.3 (Vertex Pharmaceuticals, [ ]) was used for the SMN2 splicing assay.

Techniques: Lactamase Assay, Activity Assay

Journal: PLoS ONE

Article Title: The effects of C5-substituted 2,4-diaminoquinazolines on selected transcript expression in spinal muscular atrophy cells

doi: 10.1371/journal.pone.0180657

Figure Lengend Snippet: EC 50 s of the C5-substituted 2,4-DAQs on SMN2 -drived BLA activity.

Article Snippet: The clone 11 cell line (Vertex Pharmaceuticals, [ ]) was used for the SMN2 promoter assay and the clone 5.3 (Vertex Pharmaceuticals, [ ]) was used for the SMN2 splicing assay.

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: The effects of C5-substituted 2,4-diaminoquinazolines on selected transcript expression in spinal muscular atrophy cells

doi: 10.1371/journal.pone.0180657

Figure Lengend Snippet: (A) Clone 5.3 NSC-34 cells harbor a reporter gene whose expression is linked to inclusion of SMN2 exon 7. These cells were treated with 1 μM D156844, D158872, D157161, D157495 or DMSO (n = 4/drug) for 19 hours prior to fluorescent β-lactamase assay analysis. These compounds tested did not increase the inclusion of SMN2 exon 7 but D157161 and D157495 actually decreased SMN2 exon 7 inclusion. The asterisk (*) denotes a statistically significant (p ≤ 0.05) difference between drug- and vehicle-treated cells. (B) Qualitative analysis of the effects of D156844, D157161, D158872 and D157495 on SMN2 transcripts in type II SMA fibroblasts. GM03813 cells were treated with 1 μM each compound or DMSO (n = 3/compound) for 5 days and then analyzed for changes in the amounts of FL-SMN and SMNΔ7 transcripts by RT-PCR and agarose gel electrophoresis. The amounts of FL-SMN and SMNΔ7 transcripts were also compared against GM03814 samples. COL3A transcripts were also assayed as a loading control for RT-PCR. These compounds tested did not affect the proportion of FL-SMN relative to SMNΔ7 .

Article Snippet: The clone 11 cell line (Vertex Pharmaceuticals, [ ]) was used for the SMN2 promoter assay and the clone 5.3 (Vertex Pharmaceuticals, [ ]) was used for the SMN2 splicing assay.

Techniques: Expressing, Lactamase Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Control