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  • 98
    New England Biolabs mfei
    Visualisation of LPS and <t>PCR</t> investigation of potential OAg loci in H. parainfluenzae . (A) Proteinase K treated H. parainfluenzae cell lysates were fractionated by tricine SDS-PAGE and silver-stained. Strain numbers are listed above each lane. The intense low molecular mass band in each lane is LPS that does not contain OAg (LPS core only), whilst the ladders/smears of bands represent LPS elaborated with OAg of increasing chain length. Band spacing depends on the size of the O-unit. 12.5 μl of lysate at OD 260 = 5 (strains 13 and 15) or OD 260 = 10 (strains 20, 30 and T3T1) was loaded. Weaker OAg-like banding patterns were observed for strains 2, 8, 10, 14, 16, 17, 18 and Hy6. (B) Long range PCR products were obtained using primers to glnA and pepB , which flank the OAg locus. The outside lanes contain a DNA ladder with sizes as indicated. Numbers above each lane indicate the H. parainfluenzae strain of the template gDNA. Top panel: 0.4 μl of each PCR reaction separated by agarose gel electrophoresis. Products of a high molecular mass are visible for all 18 true H. parainfluenzae strains; hybrid strains Hy6 and Hy11 did not yield products and are not shown. Lower panel: <t>MfeI</t> restriction digests of the same long range PCR products, with fragments > 1 kb visible.
    Mfei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 519 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mfei/product/New England Biolabs
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    93
    Thermo Fisher muni
    Visualisation of LPS and <t>PCR</t> investigation of potential OAg loci in H. parainfluenzae . (A) Proteinase K treated H. parainfluenzae cell lysates were fractionated by tricine SDS-PAGE and silver-stained. Strain numbers are listed above each lane. The intense low molecular mass band in each lane is LPS that does not contain OAg (LPS core only), whilst the ladders/smears of bands represent LPS elaborated with OAg of increasing chain length. Band spacing depends on the size of the O-unit. 12.5 μl of lysate at OD 260 = 5 (strains 13 and 15) or OD 260 = 10 (strains 20, 30 and T3T1) was loaded. Weaker OAg-like banding patterns were observed for strains 2, 8, 10, 14, 16, 17, 18 and Hy6. (B) Long range PCR products were obtained using primers to glnA and pepB , which flank the OAg locus. The outside lanes contain a DNA ladder with sizes as indicated. Numbers above each lane indicate the H. parainfluenzae strain of the template gDNA. Top panel: 0.4 μl of each PCR reaction separated by agarose gel electrophoresis. Products of a high molecular mass are visible for all 18 true H. parainfluenzae strains; hybrid strains Hy6 and Hy11 did not yield products and are not shown. Lower panel: <t>MfeI</t> restriction digests of the same long range PCR products, with fragments > 1 kb visible.
    Muni, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    New England Biolabs mfei hf
    Oligomerization of the KSHV and MHV-68 LANA CTDs. A : Oligomeric assemblies of kLANA (top) and mLANA (below) CTD dimers as found in the respective crystals. Inter-chain contact areas within and between dimers are indicated. B : Details on the oligomerization sites of kLANA (left) and mLANA CTDs (right), viewed from the center of the ring (kLANA, monoclinic crystal) and the top of the linear chain (mLANA). Color scheme corresponds to Figure 1A . C : Flow profiles (black graph) and molecular weight (red graph) of kLANA(1013-1149) (top) and mLANA(124-260) (bottom) in asymmetric field flow fractionation. D : Oligomerization assay with kLANA mutants. Top left: Western blot detecting FL kLANA wt or mutants bound to GST-fused kLANA wt or mutant CTDs. Bottom left: Ponceau S –stained WB membrane showing GST-LANA(934–1162) used in this assay. (e.v.) empty vector, (-) GST only, (MUT) mutant GST-LANA CTD always corresponding to the FL LANA mutant indicated above. Right: Expression of FL LANA proteins in eukaryotic cells. The aberrant running behavior of some mutants might be due to different posttranslational modification. E : EMSA with LBS1+2 oligonucleotide and GST-LANA(934-1162) oligomerization deficient mutants. (wt+comp.) control with 10× excess of unlabeled probe. Right: Expression of GST-LANA CTD proteins; Coomassie stained SDS PAGE gel. F : Transient replication assay with oligomerization mutants and pGTR4 vector in HeLa cells. Panel I: Southern blot of replicated <t>DNA,</t> remaining after digest with <t>MfeI</t> and DpnI. Panel II: Southern blot of input DNA linearized with MfeI; pEGFP does not replicate and serves as an internal control. Assay was performed in duplicates. Panel III: LANA expression. Panel IV: Actin loading control. (-) empty vector control.
    Mfei Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs mfei restriction endonuclease
    Purity and initial characterization of the <t>β-GT</t> enzyme. (A) Coomassie blue-stained SDS–PAGE gel showing 8 μg (80 units) of recombinant β-GT enzyme. (B) Glucosylation of 5-hmC DNA (T4 phage gt −/– DNA) with recombinant β-GT protects it from cleavage by <t>MfeI.</t> A time course of the glucosylation reaction followed by MfeI digestion is shown. The arrow indicates T4 phage gt −/– DNA.
    Mfei Restriction Endonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher mfei
    Genetic structure of Tn 6215 and site of host chromosome integration. In recipient strain <t>CD062,</t> a putative transcriptional regulator gene was uninterrupted. In donor strain CD80 and transductant strain CD062E1, Tn 6215 was integrated into the putative transcriptional regulator gene and the site of integration, TCC, was duplicated at either end. Predicted functional modules of the 13-kb transposon are color coded as follows: red, resistance; yellow, mobilization and replication; green, stability; gray, recombination; and white, indeterminate. Restriction sites are indicated as follows: B, BsrGI; H, Hyp99I; and M, <t>MfeI.</t> Probes used in this study are shown as hatched bars and are shown under their target regions. Homologues in other mobile elements and plasmids are indicated by colored outlines of putative genes as follows: purple, Tn 5397 ; brown, pMV158; blue, CTn BST ; pink, CTn DOT ; orange, pSM19035; and turquoise, Tn 5398 . Primers P1 to P7 flanking host and transposon junctions are indicated by black arrows.
    Mfei, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 139 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs xhoi mfei
    Genetic structure of Tn 6215 and site of host chromosome integration. In recipient strain <t>CD062,</t> a putative transcriptional regulator gene was uninterrupted. In donor strain CD80 and transductant strain CD062E1, Tn 6215 was integrated into the putative transcriptional regulator gene and the site of integration, TCC, was duplicated at either end. Predicted functional modules of the 13-kb transposon are color coded as follows: red, resistance; yellow, mobilization and replication; green, stability; gray, recombination; and white, indeterminate. Restriction sites are indicated as follows: B, BsrGI; H, Hyp99I; and M, <t>MfeI.</t> Probes used in this study are shown as hatched bars and are shown under their target regions. Homologues in other mobile elements and plasmids are indicated by colored outlines of putative genes as follows: purple, Tn 5397 ; brown, pMV158; blue, CTn BST ; pink, CTn DOT ; orange, pSM19035; and turquoise, Tn 5398 . Primers P1 to P7 flanking host and transposon junctions are indicated by black arrows.
    Xhoi Mfei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher mfei reverse primer
    Genetic structure of Tn 6215 and site of host chromosome integration. In recipient strain <t>CD062,</t> a putative transcriptional regulator gene was uninterrupted. In donor strain CD80 and transductant strain CD062E1, Tn 6215 was integrated into the putative transcriptional regulator gene and the site of integration, TCC, was duplicated at either end. Predicted functional modules of the 13-kb transposon are color coded as follows: red, resistance; yellow, mobilization and replication; green, stability; gray, recombination; and white, indeterminate. Restriction sites are indicated as follows: B, BsrGI; H, Hyp99I; and M, <t>MfeI.</t> Probes used in this study are shown as hatched bars and are shown under their target regions. Homologues in other mobile elements and plasmids are indicated by colored outlines of putative genes as follows: purple, Tn 5397 ; brown, pMV158; blue, CTn BST ; pink, CTn DOT ; orange, pSM19035; and turquoise, Tn 5398 . Primers P1 to P7 flanking host and transposon junctions are indicated by black arrows.
    Mfei Reverse Primer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Addgene inc mfei
    Genetic structure of Tn 6215 and site of host chromosome integration. In recipient strain <t>CD062,</t> a putative transcriptional regulator gene was uninterrupted. In donor strain CD80 and transductant strain CD062E1, Tn 6215 was integrated into the putative transcriptional regulator gene and the site of integration, TCC, was duplicated at either end. Predicted functional modules of the 13-kb transposon are color coded as follows: red, resistance; yellow, mobilization and replication; green, stability; gray, recombination; and white, indeterminate. Restriction sites are indicated as follows: B, BsrGI; H, Hyp99I; and M, <t>MfeI.</t> Probes used in this study are shown as hatched bars and are shown under their target regions. Homologues in other mobile elements and plasmids are indicated by colored outlines of putative genes as follows: purple, Tn 5397 ; brown, pMV158; blue, CTn BST ; pink, CTn DOT ; orange, pSM19035; and turquoise, Tn 5398 . Primers P1 to P7 flanking host and transposon junctions are indicated by black arrows.
    Mfei, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bio Basic Canada mfei
    Genetic structure of Tn 6215 and site of host chromosome integration. In recipient strain <t>CD062,</t> a putative transcriptional regulator gene was uninterrupted. In donor strain CD80 and transductant strain CD062E1, Tn 6215 was integrated into the putative transcriptional regulator gene and the site of integration, TCC, was duplicated at either end. Predicted functional modules of the 13-kb transposon are color coded as follows: red, resistance; yellow, mobilization and replication; green, stability; gray, recombination; and white, indeterminate. Restriction sites are indicated as follows: B, BsrGI; H, Hyp99I; and M, <t>MfeI.</t> Probes used in this study are shown as hatched bars and are shown under their target regions. Homologues in other mobile elements and plasmids are indicated by colored outlines of putative genes as follows: purple, Tn 5397 ; brown, pMV158; blue, CTn BST ; pink, CTn DOT ; orange, pSM19035; and turquoise, Tn 5398 . Primers P1 to P7 flanking host and transposon junctions are indicated by black arrows.
    Mfei, supplied by Bio Basic Canada, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa mfei digested pegfp n1backbone
    Genetic structure of Tn 6215 and site of host chromosome integration. In recipient strain <t>CD062,</t> a putative transcriptional regulator gene was uninterrupted. In donor strain CD80 and transductant strain CD062E1, Tn 6215 was integrated into the putative transcriptional regulator gene and the site of integration, TCC, was duplicated at either end. Predicted functional modules of the 13-kb transposon are color coded as follows: red, resistance; yellow, mobilization and replication; green, stability; gray, recombination; and white, indeterminate. Restriction sites are indicated as follows: B, BsrGI; H, Hyp99I; and M, <t>MfeI.</t> Probes used in this study are shown as hatched bars and are shown under their target regions. Homologues in other mobile elements and plasmids are indicated by colored outlines of putative genes as follows: purple, Tn 5397 ; brown, pMV158; blue, CTn BST ; pink, CTn DOT ; orange, pSM19035; and turquoise, Tn 5398 . Primers P1 to P7 flanking host and transposon junctions are indicated by black arrows.
    Mfei Digested Pegfp N1backbone, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    New England Biolabs ecori mfei neb restriction sites
    Genetic structure of Tn 6215 and site of host chromosome integration. In recipient strain <t>CD062,</t> a putative transcriptional regulator gene was uninterrupted. In donor strain CD80 and transductant strain CD062E1, Tn 6215 was integrated into the putative transcriptional regulator gene and the site of integration, TCC, was duplicated at either end. Predicted functional modules of the 13-kb transposon are color coded as follows: red, resistance; yellow, mobilization and replication; green, stability; gray, recombination; and white, indeterminate. Restriction sites are indicated as follows: B, BsrGI; H, Hyp99I; and M, <t>MfeI.</t> Probes used in this study are shown as hatched bars and are shown under their target regions. Homologues in other mobile elements and plasmids are indicated by colored outlines of putative genes as follows: purple, Tn 5397 ; brown, pMV158; blue, CTn BST ; pink, CTn DOT ; orange, pSM19035; and turquoise, Tn 5398 . Primers P1 to P7 flanking host and transposon junctions are indicated by black arrows.
    Ecori Mfei Neb Restriction Sites, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mfei  (Qiagen)
    99
    Qiagen mfei
    Genetic structure of Tn 6215 and site of host chromosome integration. In recipient strain <t>CD062,</t> a putative transcriptional regulator gene was uninterrupted. In donor strain CD80 and transductant strain CD062E1, Tn 6215 was integrated into the putative transcriptional regulator gene and the site of integration, TCC, was duplicated at either end. Predicted functional modules of the 13-kb transposon are color coded as follows: red, resistance; yellow, mobilization and replication; green, stability; gray, recombination; and white, indeterminate. Restriction sites are indicated as follows: B, BsrGI; H, Hyp99I; and M, <t>MfeI.</t> Probes used in this study are shown as hatched bars and are shown under their target regions. Homologues in other mobile elements and plasmids are indicated by colored outlines of putative genes as follows: purple, Tn 5397 ; brown, pMV158; blue, CTn BST ; pink, CTn DOT ; orange, pSM19035; and turquoise, Tn 5398 . Primers P1 to P7 flanking host and transposon junctions are indicated by black arrows.
    Mfei, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Visualisation of LPS and PCR investigation of potential OAg loci in H. parainfluenzae . (A) Proteinase K treated H. parainfluenzae cell lysates were fractionated by tricine SDS-PAGE and silver-stained. Strain numbers are listed above each lane. The intense low molecular mass band in each lane is LPS that does not contain OAg (LPS core only), whilst the ladders/smears of bands represent LPS elaborated with OAg of increasing chain length. Band spacing depends on the size of the O-unit. 12.5 μl of lysate at OD 260 = 5 (strains 13 and 15) or OD 260 = 10 (strains 20, 30 and T3T1) was loaded. Weaker OAg-like banding patterns were observed for strains 2, 8, 10, 14, 16, 17, 18 and Hy6. (B) Long range PCR products were obtained using primers to glnA and pepB , which flank the OAg locus. The outside lanes contain a DNA ladder with sizes as indicated. Numbers above each lane indicate the H. parainfluenzae strain of the template gDNA. Top panel: 0.4 μl of each PCR reaction separated by agarose gel electrophoresis. Products of a high molecular mass are visible for all 18 true H. parainfluenzae strains; hybrid strains Hy6 and Hy11 did not yield products and are not shown. Lower panel: MfeI restriction digests of the same long range PCR products, with fragments > 1 kb visible.

    Journal: International Journal of Medical Microbiology

    Article Title: Haemophilus parainfluenzae expresses diverse lipopolysaccharide O-antigens using ABC transporter and Wzy polymerase-dependent mechanisms

    doi: 10.1016/j.ijmm.2013.08.006

    Figure Lengend Snippet: Visualisation of LPS and PCR investigation of potential OAg loci in H. parainfluenzae . (A) Proteinase K treated H. parainfluenzae cell lysates were fractionated by tricine SDS-PAGE and silver-stained. Strain numbers are listed above each lane. The intense low molecular mass band in each lane is LPS that does not contain OAg (LPS core only), whilst the ladders/smears of bands represent LPS elaborated with OAg of increasing chain length. Band spacing depends on the size of the O-unit. 12.5 μl of lysate at OD 260 = 5 (strains 13 and 15) or OD 260 = 10 (strains 20, 30 and T3T1) was loaded. Weaker OAg-like banding patterns were observed for strains 2, 8, 10, 14, 16, 17, 18 and Hy6. (B) Long range PCR products were obtained using primers to glnA and pepB , which flank the OAg locus. The outside lanes contain a DNA ladder with sizes as indicated. Numbers above each lane indicate the H. parainfluenzae strain of the template gDNA. Top panel: 0.4 μl of each PCR reaction separated by agarose gel electrophoresis. Products of a high molecular mass are visible for all 18 true H. parainfluenzae strains; hybrid strains Hy6 and Hy11 did not yield products and are not shown. Lower panel: MfeI restriction digests of the same long range PCR products, with fragments > 1 kb visible.

    Article Snippet: Five microlitres of each LR-PCR product was digested with MfeI (NEB).

    Techniques: Polymerase Chain Reaction, SDS Page, Staining, Agarose Gel Electrophoresis

    Synthesis of plasmids containing aph (7″) or aac (3)IV as non-polar hygromycin B and apramycin resistance markers. (A) Schematic of ultramers designed to amplify aph (7″) or aac (3)IV. The 5′ ultramers 5631 and 5633, for aph (7″) or aac (3)IV respectively, include Mfe I, Kpn I and Sma I restriction sites, stop codons in all three reading frames, and a ribosome binding site. The 3′ ultramers 5632 and 5634 include a ribosome binding site, a start codon in-frame with restriction sites for Sma I and Bam HI, and restriction sites for Xba I, Nde I, Pst I and Sph I. (B) The amplified aac (3)IV was introduced by TA cloning into linearized pGEM-T, conserving the restriction sites in the pGEM-T multiple cloning site (MCS). The resulting plasmid is pAC1A. The pGEM sites may also be used for the sub-cloning of the apramycin resistance marker ( Apr R ). MCS sites that cut aac (3)IV are indicated with a superscript ‘A’. (C) All introduced sites in pAC1A were tested by restriction digest. (D) The Mfe I- and Sph I-digested aph (7″) amplification product was cloned into pBAD24 digested with Eco RI ( Mfe I-compatible) and Sph I. The Mfe I site was lost in the resulting plasmid, pAC1H. (E) All restriction sites introduced to pAC1H were tested by digest.

    Journal: PLoS ONE

    Article Title: Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni

    doi: 10.1371/journal.pone.0095084

    Figure Lengend Snippet: Synthesis of plasmids containing aph (7″) or aac (3)IV as non-polar hygromycin B and apramycin resistance markers. (A) Schematic of ultramers designed to amplify aph (7″) or aac (3)IV. The 5′ ultramers 5631 and 5633, for aph (7″) or aac (3)IV respectively, include Mfe I, Kpn I and Sma I restriction sites, stop codons in all three reading frames, and a ribosome binding site. The 3′ ultramers 5632 and 5634 include a ribosome binding site, a start codon in-frame with restriction sites for Sma I and Bam HI, and restriction sites for Xba I, Nde I, Pst I and Sph I. (B) The amplified aac (3)IV was introduced by TA cloning into linearized pGEM-T, conserving the restriction sites in the pGEM-T multiple cloning site (MCS). The resulting plasmid is pAC1A. The pGEM sites may also be used for the sub-cloning of the apramycin resistance marker ( Apr R ). MCS sites that cut aac (3)IV are indicated with a superscript ‘A’. (C) All introduced sites in pAC1A were tested by restriction digest. (D) The Mfe I- and Sph I-digested aph (7″) amplification product was cloned into pBAD24 digested with Eco RI ( Mfe I-compatible) and Sph I. The Mfe I site was lost in the resulting plasmid, pAC1H. (E) All restriction sites introduced to pAC1H were tested by digest.

    Article Snippet: The initial aph (7″) PCR product was instead digested with Mfe I and Sph I (NEB), purified, and ligated to low-copy pBAD24 digested with Eco RI and Sph I.

    Techniques: Binding Assay, Amplification, TA Cloning, Clone Assay, Plasmid Preparation, Subcloning, Marker

    Oligomerization of the KSHV and MHV-68 LANA CTDs. A : Oligomeric assemblies of kLANA (top) and mLANA (below) CTD dimers as found in the respective crystals. Inter-chain contact areas within and between dimers are indicated. B : Details on the oligomerization sites of kLANA (left) and mLANA CTDs (right), viewed from the center of the ring (kLANA, monoclinic crystal) and the top of the linear chain (mLANA). Color scheme corresponds to Figure 1A . C : Flow profiles (black graph) and molecular weight (red graph) of kLANA(1013-1149) (top) and mLANA(124-260) (bottom) in asymmetric field flow fractionation. D : Oligomerization assay with kLANA mutants. Top left: Western blot detecting FL kLANA wt or mutants bound to GST-fused kLANA wt or mutant CTDs. Bottom left: Ponceau S –stained WB membrane showing GST-LANA(934–1162) used in this assay. (e.v.) empty vector, (-) GST only, (MUT) mutant GST-LANA CTD always corresponding to the FL LANA mutant indicated above. Right: Expression of FL LANA proteins in eukaryotic cells. The aberrant running behavior of some mutants might be due to different posttranslational modification. E : EMSA with LBS1+2 oligonucleotide and GST-LANA(934-1162) oligomerization deficient mutants. (wt+comp.) control with 10× excess of unlabeled probe. Right: Expression of GST-LANA CTD proteins; Coomassie stained SDS PAGE gel. F : Transient replication assay with oligomerization mutants and pGTR4 vector in HeLa cells. Panel I: Southern blot of replicated DNA, remaining after digest with MfeI and DpnI. Panel II: Southern blot of input DNA linearized with MfeI; pEGFP does not replicate and serves as an internal control. Assay was performed in duplicates. Panel III: LANA expression. Panel IV: Actin loading control. (-) empty vector control.

    Journal: PLoS Pathogens

    Article Title: A Structural Basis for BRD2/4-Mediated Host Chromatin Interaction and Oligomer Assembly of Kaposi Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus LANA ProteinsMolecular Basis for Oligomeric-DNA Binding and Episome Maintenance by KSHV LANACrystal Structure of the Gamma-2 Herpesvirus LANA DNA Binding Domain Identifies Charged Surface Residues Which Impact Viral Latency

    doi: 10.1371/journal.ppat.1003640

    Figure Lengend Snippet: Oligomerization of the KSHV and MHV-68 LANA CTDs. A : Oligomeric assemblies of kLANA (top) and mLANA (below) CTD dimers as found in the respective crystals. Inter-chain contact areas within and between dimers are indicated. B : Details on the oligomerization sites of kLANA (left) and mLANA CTDs (right), viewed from the center of the ring (kLANA, monoclinic crystal) and the top of the linear chain (mLANA). Color scheme corresponds to Figure 1A . C : Flow profiles (black graph) and molecular weight (red graph) of kLANA(1013-1149) (top) and mLANA(124-260) (bottom) in asymmetric field flow fractionation. D : Oligomerization assay with kLANA mutants. Top left: Western blot detecting FL kLANA wt or mutants bound to GST-fused kLANA wt or mutant CTDs. Bottom left: Ponceau S –stained WB membrane showing GST-LANA(934–1162) used in this assay. (e.v.) empty vector, (-) GST only, (MUT) mutant GST-LANA CTD always corresponding to the FL LANA mutant indicated above. Right: Expression of FL LANA proteins in eukaryotic cells. The aberrant running behavior of some mutants might be due to different posttranslational modification. E : EMSA with LBS1+2 oligonucleotide and GST-LANA(934-1162) oligomerization deficient mutants. (wt+comp.) control with 10× excess of unlabeled probe. Right: Expression of GST-LANA CTD proteins; Coomassie stained SDS PAGE gel. F : Transient replication assay with oligomerization mutants and pGTR4 vector in HeLa cells. Panel I: Southern blot of replicated DNA, remaining after digest with MfeI and DpnI. Panel II: Southern blot of input DNA linearized with MfeI; pEGFP does not replicate and serves as an internal control. Assay was performed in duplicates. Panel III: LANA expression. Panel IV: Actin loading control. (-) empty vector control.

    Article Snippet: 90% of DNA was digested for 72 h with 60 U of MfeI HF (or KpnI) (NEB) and 60 U DpnI (NEB) and the remaining 10% with 40 U MfeI HF (or KpnI) only.

    Techniques: Flow Cytometry, Molecular Weight, Field Flow Fractionation, Western Blot, Mutagenesis, Staining, Plasmid Preparation, Expressing, Modification, SDS Page, Southern Blot, Control Assay

    -tolerant calli. A, Schematic representation of PCR- Mfe tolerance and two silent mutations (T− > A at +1479, C− > T at +1545) are marked by blue and red vertical lines, respectively. The W548L and S627I mutations create novel Mfe -22) used for PCR, and the expected size of PCR-amplified fragments and their Mfe -tolerant calli by PCR- Mfe locus. Heteroduplex of mutated and nonmutated amplicons generated by PCR reaction is partially tolerant to Mfe I-HF and imperfect digested products at W548L appeared as an approximately 700-bp fragment (*).

    Journal: Plant Physiology

    Article Title: Biallelic Gene Targeting in Rice 1Biallelic Gene Targeting in Rice 1 [OPEN]

    doi: 10.1104/pp.15.01663

    Figure Lengend Snippet: -tolerant calli. A, Schematic representation of PCR- Mfe tolerance and two silent mutations (T− > A at +1479, C− > T at +1545) are marked by blue and red vertical lines, respectively. The W548L and S627I mutations create novel Mfe -22) used for PCR, and the expected size of PCR-amplified fragments and their Mfe -tolerant calli by PCR- Mfe locus. Heteroduplex of mutated and nonmutated amplicons generated by PCR reaction is partially tolerant to Mfe I-HF and imperfect digested products at W548L appeared as an approximately 700-bp fragment (*).

    Article Snippet: To confirm the occurrence of in -tolerant rice plants, we performed PCR analysis coupled with Mfe I digestion using Mfe I-HF; a high fidelity version of Mfe I was supplied by New England Biolabs (Ipswich, MA).

    Techniques: Polymerase Chain Reaction, Amplification, Generated

    locus. A, Diagram showing the location of probes and expected band size in Southern-blot analysis using probes A and B. B, Southern-blot analysis of Mfe gene with the W548L mutation. The two bands other than 11.8 kb and 4.8 kb are due to nonspecific hybridization, since these additional bands are also seen in the wild-type lane. C, Southern-blot analysis of Mfe locus. Abbreviations: LB, left border; RB, right border; M , Mfe I.

    Journal: Plant Physiology

    Article Title: Biallelic Gene Targeting in Rice 1Biallelic Gene Targeting in Rice 1 [OPEN]

    doi: 10.1104/pp.15.01663

    Figure Lengend Snippet: locus. A, Diagram showing the location of probes and expected band size in Southern-blot analysis using probes A and B. B, Southern-blot analysis of Mfe gene with the W548L mutation. The two bands other than 11.8 kb and 4.8 kb are due to nonspecific hybridization, since these additional bands are also seen in the wild-type lane. C, Southern-blot analysis of Mfe locus. Abbreviations: LB, left border; RB, right border; M , Mfe I.

    Article Snippet: To confirm the occurrence of in -tolerant rice plants, we performed PCR analysis coupled with Mfe I digestion using Mfe I-HF; a high fidelity version of Mfe I was supplied by New England Biolabs (Ipswich, MA).

    Techniques: Southern Blot, Mutagenesis, Hybridization

    Purity and initial characterization of the β-GT enzyme. (A) Coomassie blue-stained SDS–PAGE gel showing 8 μg (80 units) of recombinant β-GT enzyme. (B) Glucosylation of 5-hmC DNA (T4 phage gt −/– DNA) with recombinant β-GT protects it from cleavage by MfeI. A time course of the glucosylation reaction followed by MfeI digestion is shown. The arrow indicates T4 phage gt −/– DNA.

    Journal: Biochemistry

    Article Title: Biochemical Characterization of Recombinant ?-Glucosyltransferase and Analysis of Global 5-Hydroxymethylcytosine in Unique Genomes

    doi: 10.1021/bi2014739

    Figure Lengend Snippet: Purity and initial characterization of the β-GT enzyme. (A) Coomassie blue-stained SDS–PAGE gel showing 8 μg (80 units) of recombinant β-GT enzyme. (B) Glucosylation of 5-hmC DNA (T4 phage gt −/– DNA) with recombinant β-GT protects it from cleavage by MfeI. A time course of the glucosylation reaction followed by MfeI digestion is shown. The arrow indicates T4 phage gt −/– DNA.

    Article Snippet: After heat inactivation of β-GT, 1 μL (10 units) of MfeI restriction endonuclease (NEB) was added to each sample and each mixture incubated at 37 °C for 1 h to cleave nonglucosylated T4-gt DNA.

    Techniques: Staining, SDS Page, Recombinant

    Genetic structure of Tn 6215 and site of host chromosome integration. In recipient strain CD062, a putative transcriptional regulator gene was uninterrupted. In donor strain CD80 and transductant strain CD062E1, Tn 6215 was integrated into the putative transcriptional regulator gene and the site of integration, TCC, was duplicated at either end. Predicted functional modules of the 13-kb transposon are color coded as follows: red, resistance; yellow, mobilization and replication; green, stability; gray, recombination; and white, indeterminate. Restriction sites are indicated as follows: B, BsrGI; H, Hyp99I; and M, MfeI. Probes used in this study are shown as hatched bars and are shown under their target regions. Homologues in other mobile elements and plasmids are indicated by colored outlines of putative genes as follows: purple, Tn 5397 ; brown, pMV158; blue, CTn BST ; pink, CTn DOT ; orange, pSM19035; and turquoise, Tn 5398 . Primers P1 to P7 flanking host and transposon junctions are indicated by black arrows.

    Journal: mBio

    Article Title: Phage ?C2 Mediates Transduction of Tn6215, Encoding Erythromycin Resistance, between Clostridium difficile Strains

    doi: 10.1128/mBio.00840-13

    Figure Lengend Snippet: Genetic structure of Tn 6215 and site of host chromosome integration. In recipient strain CD062, a putative transcriptional regulator gene was uninterrupted. In donor strain CD80 and transductant strain CD062E1, Tn 6215 was integrated into the putative transcriptional regulator gene and the site of integration, TCC, was duplicated at either end. Predicted functional modules of the 13-kb transposon are color coded as follows: red, resistance; yellow, mobilization and replication; green, stability; gray, recombination; and white, indeterminate. Restriction sites are indicated as follows: B, BsrGI; H, Hyp99I; and M, MfeI. Probes used in this study are shown as hatched bars and are shown under their target regions. Homologues in other mobile elements and plasmids are indicated by colored outlines of putative genes as follows: purple, Tn 5397 ; brown, pMV158; blue, CTn BST ; pink, CTn DOT ; orange, pSM19035; and turquoise, Tn 5398 . Primers P1 to P7 flanking host and transposon junctions are indicated by black arrows.

    Article Snippet: MfeI (Fermentas)-digested gDNA (3 µg) of CD062, CD062R11, CD80, and CD062 transductants and CD062 transconjugants served as the templates for hybridization of digoxigenin (DIG)-labeled probes, at 40°C for the RF amplicon probe and 49°C for the 3′-end probe.

    Techniques: Functional Assay