sbfi  (New England Biolabs)


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    Name:
    SbfI
    Description:
    SbfI 2 500 units
    Catalog Number:
    r0642l
    Price:
    302
    Category:
    Restriction Enzymes
    Size:
    2 500 units
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    Structured Review

    New England Biolabs sbfi
    SbfI
    SbfI 2 500 units
    https://www.bioz.com/result/sbfi/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sbfi - by Bioz Stars, 2021-03
    94/100 stars

    Images

    1) Product Images from "Conserved CxnC Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34"

    Article Title: Conserved CxnC Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34

    Journal: Journal of Virology

    doi: 10.1128/JVI.01299-19

    ORF66 is essential in KSHV and required for late gene transcription. (A) (Left) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. (Right) The mutations were confirmed by Sanger sequencing. (B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that the introduction of mutations did not introduce large-scale changes. (C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. ****, P
    Figure Legend Snippet: ORF66 is essential in KSHV and required for late gene transcription. (A) (Left) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. (Right) The mutations were confirmed by Sanger sequencing. (B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that the introduction of mutations did not introduce large-scale changes. (C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. ****, P

    Techniques Used: Sequencing, Recombinant, Introduce, Flow Cytometry

    2) Product Images from "Conserved CxnC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34"

    Article Title: Conserved CxnC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34

    Journal: bioRxiv

    doi: 10.1101/728139

    ORF66 is essential in KSHV and required for late gene transcription A) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. Mutations were confirmed by Sanger sequencing (right). B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that introduction of mutations did not introduce large-scale changes. C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p
    Figure Legend Snippet: ORF66 is essential in KSHV and required for late gene transcription A) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. Mutations were confirmed by Sanger sequencing (right). B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that introduction of mutations did not introduce large-scale changes. C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p

    Techniques Used: Sequencing, Recombinant, Introduce, Flow Cytometry

    ORF24 does not bind to late gene promoters in the absence of ORF30 or ORF66 iSLK cell lines were created using the recombinant BAC16 system. HA tags were added to the endogenous copies of the N-terminus of ORF24 (HA24) or the C-terminus of ORF66 (66HA). In select BACs, ORF24, ORF30, or ORF66 were deleted by the introduction of a stop codon early in the ORF (24S, 30S, and 66S respectively). A) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that recombination did not introduce large-scale changes. B) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p
    Figure Legend Snippet: ORF24 does not bind to late gene promoters in the absence of ORF30 or ORF66 iSLK cell lines were created using the recombinant BAC16 system. HA tags were added to the endogenous copies of the N-terminus of ORF24 (HA24) or the C-terminus of ORF66 (66HA). In select BACs, ORF24, ORF30, or ORF66 were deleted by the introduction of a stop codon early in the ORF (24S, 30S, and 66S respectively). A) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that recombination did not introduce large-scale changes. B) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p

    Techniques Used: Recombinant, Introduce, Flow Cytometry

    3) Product Images from "Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)"

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003302

    Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .
    Figure Legend Snippet: Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .

    Techniques Used: Activity Assay, Clone Assay, Isolation, Agarose Gel Electrophoresis, Marker, Plasmid Preparation

    4) Product Images from "A pentameric protein ring with novel architecture is required for herpesviral packaging"

    Article Title: A pentameric protein ring with novel architecture is required for herpesviral packaging

    Journal: bioRxiv

    doi: 10.1101/2020.07.16.206755

    Construction and validation of mutant viruses. a , Schematic of the genomic locus of ORF68, with the location of introduced mutations depicted in detail below. Sanger sequencing traces for the mutants and corresponding mutant rescues are shown to the right. b , Digestion of recombinant BACs with RsrII and SbfI was used to assess whether large-scale recombination had occurred during mutagenesis. c , Western blot of whole cell lysate (25 μg) from ORF68.stop iSLK cell lines. GAPDH was used as a loading control. ORF6 is an early gene and K8.1 is a late gene. d , Viral DNA replication was measured by qPCR before and after reactivation. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. **, p
    Figure Legend Snippet: Construction and validation of mutant viruses. a , Schematic of the genomic locus of ORF68, with the location of introduced mutations depicted in detail below. Sanger sequencing traces for the mutants and corresponding mutant rescues are shown to the right. b , Digestion of recombinant BACs with RsrII and SbfI was used to assess whether large-scale recombination had occurred during mutagenesis. c , Western blot of whole cell lysate (25 μg) from ORF68.stop iSLK cell lines. GAPDH was used as a loading control. ORF6 is an early gene and K8.1 is a late gene. d , Viral DNA replication was measured by qPCR before and after reactivation. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. **, p

    Techniques Used: Mutagenesis, Sequencing, Recombinant, Western Blot, Real-time Polymerase Chain Reaction

    5) Product Images from "Amplification Biases and Consistent Recovery of Loci in a Double-Digest RAD-seq Protocol"

    Article Title: Amplification Biases and Consistent Recovery of Loci in a Double-Digest RAD-seq Protocol

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106713

    Sequencing depth for single copy ddRAD loci in relation to the corresponding sequence in the zebra finch reference genome. Categories from top to bottom include: loci mapping as expected to predicted SbfI-EcoRI restriction fragments≤328 bp in length; all loci beginning at a genomic location similar but not identical to the canonical SbfI recognition sequence (1–4 mismatches); subset of loci with one mismatch in position 1 or 8 of the SbfI recognition sequence; subset of loci with one mismatch in positions 2 through 7 of the SbfI recognition sequence; loci mapping to a genomic SbfI site without an EcoRI site within 328 bp; and loci mapping to a predicted SbfI-SbfI restriction fragment less than 328 bp in length.
    Figure Legend Snippet: Sequencing depth for single copy ddRAD loci in relation to the corresponding sequence in the zebra finch reference genome. Categories from top to bottom include: loci mapping as expected to predicted SbfI-EcoRI restriction fragments≤328 bp in length; all loci beginning at a genomic location similar but not identical to the canonical SbfI recognition sequence (1–4 mismatches); subset of loci with one mismatch in position 1 or 8 of the SbfI recognition sequence; subset of loci with one mismatch in positions 2 through 7 of the SbfI recognition sequence; loci mapping to a genomic SbfI site without an EcoRI site within 328 bp; and loci mapping to a predicted SbfI-SbfI restriction fragment less than 328 bp in length.

    Techniques Used: Sequencing

    Recovery and sequencing depth for predicted, single-copy ddRAD loci in the empirical zebra finch data. (A) Proportion of predicted loci recovered at three different minimum depth thresholds as a function of predicted fragment length. Each data point represents the proportion of ∼140–220 predicted loci recovered in a given 10 bp size range. Dashed vertical lines represent the upper and lower bounds of the size range isolated from the agarose gel. (B) Sequencing depth for recovered (depth ≥1), single-copy loci in the 32–500 bp size range (includes 5,232 of 5,783 predicted loci in the 38–328 bp size range). (C) The relationship between GC content and sequencing depth for zebra finch ddRAD loci. Data are shown for predicted, single-copy loci recovered at a depth ≥1 in three selected subsets of the overall size range ( n = 502, 466, and 445 loci in the 100–125, 200–225, and 300–325 bp size ranges, respectively). The predicted length and GC content of each locus are based on the full-length fragment in the reference genome, inclusive of the SbfI and EcoRI restriction sites on either end. Note that the y-axis is on a logarithmic scale in (B) and (C).
    Figure Legend Snippet: Recovery and sequencing depth for predicted, single-copy ddRAD loci in the empirical zebra finch data. (A) Proportion of predicted loci recovered at three different minimum depth thresholds as a function of predicted fragment length. Each data point represents the proportion of ∼140–220 predicted loci recovered in a given 10 bp size range. Dashed vertical lines represent the upper and lower bounds of the size range isolated from the agarose gel. (B) Sequencing depth for recovered (depth ≥1), single-copy loci in the 32–500 bp size range (includes 5,232 of 5,783 predicted loci in the 38–328 bp size range). (C) The relationship between GC content and sequencing depth for zebra finch ddRAD loci. Data are shown for predicted, single-copy loci recovered at a depth ≥1 in three selected subsets of the overall size range ( n = 502, 466, and 445 loci in the 100–125, 200–225, and 300–325 bp size ranges, respectively). The predicted length and GC content of each locus are based on the full-length fragment in the reference genome, inclusive of the SbfI and EcoRI restriction sites on either end. Note that the y-axis is on a logarithmic scale in (B) and (C).

    Techniques Used: Sequencing, Isolation, Agarose Gel Electrophoresis

    6) Product Images from "Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)"

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003302

    Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .
    Figure Legend Snippet: Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .

    Techniques Used: Activity Assay, Clone Assay, Isolation, Agarose Gel Electrophoresis, Marker, Plasmid Preparation

    7) Product Images from "Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)"

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003302

    Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .
    Figure Legend Snippet: Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .

    Techniques Used: Activity Assay, Clone Assay, Isolation, Agarose Gel Electrophoresis, Marker, Plasmid Preparation

    Related Articles

    BAC Assay:

    Article Title: A pentameric protein ring with novel architecture is required for herpesviral packaging
    Article Snippet: Modified BACs were purified using a Nucleobond BAC 100 kit (Clontech). .. BAC quality was assessed by digestion with RsrII and SbfI (New England Biolabs). .. Latently infected iSLK cell lines with modified virus were generated by transfection of HEK293T-ORF68 cells with 5 μg BAC DNA using PolyJet reagent (SignaGen).

    Article Title: Conserved CxnC Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34
    Article Snippet: The modified BACs were purified using a Nucleobond BAC 100 kit (Clontech). .. BAC quality was assessed by digestion with RsrII and SbfI (New England Biolabs). .. Latently infected iSLK cell lines with modified virus were generated by transfection of HEK293T cells (either WT cells or cells stably expressing the relevant essential viral ORF) with 5 μg BAC DNA using the PolyJet reagent (SignaGen).

    Article Title: Conserved CxnC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34
    Article Snippet: The modified BACs were purified using a Nucleobond BAC 100 kit (Clontech). .. BAC quality was assessed by digestion with RsrII and SbfI (New England Biolabs). .. Latently infected iSLK cell lines with modified virus were generated by transfection of HEK293T cells (either WT or stably expressing the relevant essential viral ORF) with 5 μg BAC DNA using PolyJet (SignaGen).

    Plasmid Preparation:

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: Fosmids were individually isolated from 12 randomly selected library clones using the FosmidMAXTM DNA purification kit (Lucigen Corporation). .. Each fosmid was digested with SbfI (New England Biolabs), a unique restriction site in the pSMART FOS vector. .. Digestion products were separated by electrophoresis on a 1% agarose gel to estimate the average insert size of cloned DNA.

    Isolation:

    Article Title: Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers
    Article Snippet: Therefore, even as sequencing technology continues to improve, RAD marker sequencing will remain a useful and cost-effective tool for most genetic mapping studies. .. Isolation of RAD markers for Illumina sequencing Genomic DNA (0.1–1 µg; from either individual or pooled samples) was digested for 15 min at 37°C in a 50 µL reaction with 20 units (U) of EcoR I or Sbf I (New England Biolabs [NEB]). ..

    Sequencing:

    Article Title: Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers
    Article Snippet: Therefore, even as sequencing technology continues to improve, RAD marker sequencing will remain a useful and cost-effective tool for most genetic mapping studies. .. Isolation of RAD markers for Illumina sequencing Genomic DNA (0.1–1 µg; from either individual or pooled samples) was digested for 15 min at 37°C in a 50 µL reaction with 20 units (U) of EcoR I or Sbf I (New England Biolabs [NEB]). ..

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    • sbfi  (New England Biolabs)
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    New England Biolabs sbfi
    ORF66 is essential in KSHV and required for late gene transcription. (A) (Left) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. (Right) The mutations were confirmed by Sanger sequencing. (B) Digestion of the recombinant BACs with <t>SbfI</t> or <t>RsrII</t> demonstrates that the introduction of mutations did not introduce large-scale changes. (C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. ****, P
    Sbfi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sbfi/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sbfi - by Bioz Stars, 2021-03
    94/100 stars
    		  Buy from Supplier

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    ORF66 is essential in KSHV and required for late gene transcription. (A) (Left) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. (Right) The mutations were confirmed by Sanger sequencing. (B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that the introduction of mutations did not introduce large-scale changes. (C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. ****, P

    Journal: Journal of Virology

    Article Title: Conserved CxnC Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34

    doi: 10.1128/JVI.01299-19

    Figure Lengend Snippet: ORF66 is essential in KSHV and required for late gene transcription. (A) (Left) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. (Right) The mutations were confirmed by Sanger sequencing. (B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that the introduction of mutations did not introduce large-scale changes. (C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. ****, P

    Article Snippet: BAC quality was assessed by digestion with RsrII and SbfI (New England Biolabs).

    Techniques: Sequencing, Recombinant, Introduce, Flow Cytometry

    ORF66 is essential in KSHV and required for late gene transcription A) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. Mutations were confirmed by Sanger sequencing (right). B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that introduction of mutations did not introduce large-scale changes. C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p

    Journal: bioRxiv

    Article Title: Conserved CxnC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34

    doi: 10.1101/728139

    Figure Lengend Snippet: ORF66 is essential in KSHV and required for late gene transcription A) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. Mutations were confirmed by Sanger sequencing (right). B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that introduction of mutations did not introduce large-scale changes. C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p

    Article Snippet: BAC quality was assessed by digestion with RsrII and SbfI (New England Biolabs).

    Techniques: Sequencing, Recombinant, Introduce, Flow Cytometry

    ORF24 does not bind to late gene promoters in the absence of ORF30 or ORF66 iSLK cell lines were created using the recombinant BAC16 system. HA tags were added to the endogenous copies of the N-terminus of ORF24 (HA24) or the C-terminus of ORF66 (66HA). In select BACs, ORF24, ORF30, or ORF66 were deleted by the introduction of a stop codon early in the ORF (24S, 30S, and 66S respectively). A) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that recombination did not introduce large-scale changes. B) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p

    Journal: bioRxiv

    Article Title: Conserved CxnC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34

    doi: 10.1101/728139

    Figure Lengend Snippet: ORF24 does not bind to late gene promoters in the absence of ORF30 or ORF66 iSLK cell lines were created using the recombinant BAC16 system. HA tags were added to the endogenous copies of the N-terminus of ORF24 (HA24) or the C-terminus of ORF66 (66HA). In select BACs, ORF24, ORF30, or ORF66 were deleted by the introduction of a stop codon early in the ORF (24S, 30S, and 66S respectively). A) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that recombination did not introduce large-scale changes. B) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p

    Article Snippet: BAC quality was assessed by digestion with RsrII and SbfI (New England Biolabs).

    Techniques: Recombinant, Introduce, Flow Cytometry

    Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .

    Journal: The Journal of Biological Chemistry

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

    doi: 10.1074/jbc.RA118.003302

    Figure Lengend Snippet: Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .

    Article Snippet: Each fosmid was digested with SbfI (New England Biolabs), a unique restriction site in the pSMART FOS vector.

    Techniques: Activity Assay, Clone Assay, Isolation, Agarose Gel Electrophoresis, Marker, Plasmid Preparation

    Sequenced RAD marker mapping. (A) A native saltwater stickleback population, Rabbit Slough (RS), have complete lateral plate armor (brackets) while these structures are absent in the derived, freshwater Bear Paw (BP) population. The freshwater fish also have a reduction in pelvic structure (arrow) compared to the oceanic population. These two phenotypes segregate independently in an F 2 mapping cross. Using Sbf I (B) or EcoR I (C), we mapped polymorphic RAD markers from RS (red) and BP (green) parental fish along the 21 stickleback linkage groups. The apparent size differences of the linkage groups between (B) and (C) reflect the fact that the EcoR I recognition sequence occurs more frequently than Sbf I. Red and green bars above the linkage groups are measures of lateral plate linkage in the F 2 progeny, indicating the number of tightly linked markers in the local region. (D) Sequence reads per barcoded F 2 individual used to create (C). Variable numbers of reads were obtained from each of the 96 individuals used in our analysis, reflecting different concentrations of starting DNA template. 68% of individuals had between 50 K and 150 K RAD tags sequenced (∼0.4–1.0× coverage of the ∼150 K tags present in the genome). Only 2 individuals had less than 10,000 reads (red). (E) A close-up of the boxed region from (C) showing recombination breakpoints in six informative low plate F 2 fish on LGIV. Black tick marks are 1 Mb apart in physical distance. (F) F 2 individuals were repooled in silico based on the pelvic structure phenotype (A, arrow). Linkage was determined as in (B, C), mapping the locus for a reduction in pelvic structure to the end of LGVII.

    Journal: PLoS ONE

    Article Title: Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers

    doi: 10.1371/journal.pone.0003376

    Figure Lengend Snippet: Sequenced RAD marker mapping. (A) A native saltwater stickleback population, Rabbit Slough (RS), have complete lateral plate armor (brackets) while these structures are absent in the derived, freshwater Bear Paw (BP) population. The freshwater fish also have a reduction in pelvic structure (arrow) compared to the oceanic population. These two phenotypes segregate independently in an F 2 mapping cross. Using Sbf I (B) or EcoR I (C), we mapped polymorphic RAD markers from RS (red) and BP (green) parental fish along the 21 stickleback linkage groups. The apparent size differences of the linkage groups between (B) and (C) reflect the fact that the EcoR I recognition sequence occurs more frequently than Sbf I. Red and green bars above the linkage groups are measures of lateral plate linkage in the F 2 progeny, indicating the number of tightly linked markers in the local region. (D) Sequence reads per barcoded F 2 individual used to create (C). Variable numbers of reads were obtained from each of the 96 individuals used in our analysis, reflecting different concentrations of starting DNA template. 68% of individuals had between 50 K and 150 K RAD tags sequenced (∼0.4–1.0× coverage of the ∼150 K tags present in the genome). Only 2 individuals had less than 10,000 reads (red). (E) A close-up of the boxed region from (C) showing recombination breakpoints in six informative low plate F 2 fish on LGIV. Black tick marks are 1 Mb apart in physical distance. (F) F 2 individuals were repooled in silico based on the pelvic structure phenotype (A, arrow). Linkage was determined as in (B, C), mapping the locus for a reduction in pelvic structure to the end of LGVII.

    Article Snippet: Isolation of RAD markers for Illumina sequencing Genomic DNA (0.1–1 µg; from either individual or pooled samples) was digested for 15 min at 37°C in a 50 µL reaction with 20 units (U) of EcoR I or Sbf I (New England Biolabs [NEB]).

    Techniques: Marker, Derivative Assay, Fluorescence In Situ Hybridization, Sequencing, In Silico