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Stratagene deoxynucleoside triphosphates
Deoxynucleoside Triphosphates, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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deoxynucleoside triphosphates - by Bioz Stars, 2020-07
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Clone Assay:

Article Title: The Escherichia coli Citrate Carrier CitT: a Member of a Novel Eubacterial Transporter Family Related to the 2-Oxoglutarate/Malate Translocator from Spinach Chloroplasts
Article Snippet: .. The PCR mixture contained 500 ng of genomic DNA of E. coli JM83, 0.5 μM each primer, 0.2 mM deoxynucleoside triphosphates, 1× buffer for cloned Pfu DNA polymerase, and 2.5 U of Pfu DNA polymerase (Stratagene). ..

Amplification:

Article Title: Novel Nickel Resistance Genes from the Rhizosphere Metagenome of Plants Adapted to Acid Mine Drainage ▿Novel Nickel Resistance Genes from the Rhizosphere Metagenome of Plants Adapted to Acid Mine Drainage ▿ †
Article Snippet: .. Subcloning was accomplished by PCR amplification using the following reaction mixture: 25 ng of plasmid DNA, 500 μM of each of the four deoxynucleoside triphosphates, 2.5 U of Pfu Turbo DNA polymerase (Stratagene), and 100 nM of each forward and reverse primers (see Table S1 in the supplemental material) up to a total volume of 50 μl. ..

Isolation:

Article Title: Integration Site Preference of Xenotropic Murine Leukemia Virus-Related Virus, a New Human Retrovirus Associated with Prostate Cancer ▿
Article Snippet: .. Ten micrograms of genomic DNA isolated from prostate peripheral zones (containing tumor cells) as described previously ( ) was mixed with 50 pM bXMRV7550, 0.2 mM deoxynucleoside triphosphates, and PicoMaxx polymerase (Stratagene) under the following conditions: 3 min of preincubation at 94°C, followed by 80 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 4 min. Two units of fresh PicoMaxx enzyme was added to the reaction mixture after 40 cycles. .. Biotinylated DNA was isolated from the PCR product by binding to 200 mg of streptavidin-agarose Dynabeads, and the remaining procedure was identical to that described earlier for XMRV-infected DU145 cells.

Subcloning:

Article Title: Novel Nickel Resistance Genes from the Rhizosphere Metagenome of Plants Adapted to Acid Mine Drainage ▿Novel Nickel Resistance Genes from the Rhizosphere Metagenome of Plants Adapted to Acid Mine Drainage ▿ †
Article Snippet: .. Subcloning was accomplished by PCR amplification using the following reaction mixture: 25 ng of plasmid DNA, 500 μM of each of the four deoxynucleoside triphosphates, 2.5 U of Pfu Turbo DNA polymerase (Stratagene), and 100 nM of each forward and reverse primers (see Table S1 in the supplemental material) up to a total volume of 50 μl. ..

Purification:

Article Title: The Serine Protease Motif of EspC from Enteropathogenic Escherichia coli Produces Epithelial Damage by a Mechanism Different from That of Pet Toxin from Enteroaggregative E. coli
Article Snippet: .. Amplifications were performed by using 0.2 mM deoxynucleoside triphosphates, 0.2 μM each primer, 500 ng of the purified plasmid pJLM174 ( ) as a template, 2.5 mM MgCl2 , 1× buffer, and 1 U of Pfu Turbo DNA polymerase (Stratagene, Inc.). .. The reaction mixture was subjected to initial denaturation for 2 min at 94°C, followed by 35 amplification cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, 30 s; the final step was carried out at 72°C for 5 min. PCR products were purified, and a 3′ adenine was added to each fragment by one-step incubation at 72°C for 20 min using 0.2 mM deoxynucleoside triphosphates, 2.5 mM MgCl2 , 1× buffer, and 0.2 U of Taq DNA polymerase (Promega).

Polymerase Chain Reaction:

Article Title: Novel Nickel Resistance Genes from the Rhizosphere Metagenome of Plants Adapted to Acid Mine Drainage ▿Novel Nickel Resistance Genes from the Rhizosphere Metagenome of Plants Adapted to Acid Mine Drainage ▿ †
Article Snippet: .. Subcloning was accomplished by PCR amplification using the following reaction mixture: 25 ng of plasmid DNA, 500 μM of each of the four deoxynucleoside triphosphates, 2.5 U of Pfu Turbo DNA polymerase (Stratagene), and 100 nM of each forward and reverse primers (see Table S1 in the supplemental material) up to a total volume of 50 μl. ..

Article Title: The Escherichia coli Citrate Carrier CitT: a Member of a Novel Eubacterial Transporter Family Related to the 2-Oxoglutarate/Malate Translocator from Spinach Chloroplasts
Article Snippet: .. The PCR mixture contained 500 ng of genomic DNA of E. coli JM83, 0.5 μM each primer, 0.2 mM deoxynucleoside triphosphates, 1× buffer for cloned Pfu DNA polymerase, and 2.5 U of Pfu DNA polymerase (Stratagene). ..

Article Title: Development and Characterization of a Green Fluorescent Protein-Based Bacterial Biosensor for Bioavailable Toluene and Related Compounds †
Article Snippet: .. PCR conditions were as follows: 95°C for 5 min followed by 50 cycles of 95°C for 30 s, 68°C for 30 s, and 72°C for 2 min, followed by 72°C for 7 min. Pfu DNA polymerase and deoxynucleoside triphosphates were from Stratagene (La Jolla, Calif.). .. Purified PCR product was treated with T4 polynucleotide kinase and ligated into the Sma I site of pGEM-7 (Promega) in a nondirectional manner to create pG-7.1, and a Bam HI fragment containing the promoter was subcloned into pGEM-7 to create pG-7.2 (Table ).

Plasmid Preparation:

Article Title: Novel Nickel Resistance Genes from the Rhizosphere Metagenome of Plants Adapted to Acid Mine Drainage ▿Novel Nickel Resistance Genes from the Rhizosphere Metagenome of Plants Adapted to Acid Mine Drainage ▿ †
Article Snippet: .. Subcloning was accomplished by PCR amplification using the following reaction mixture: 25 ng of plasmid DNA, 500 μM of each of the four deoxynucleoside triphosphates, 2.5 U of Pfu Turbo DNA polymerase (Stratagene), and 100 nM of each forward and reverse primers (see Table S1 in the supplemental material) up to a total volume of 50 μl. ..

Article Title: The Serine Protease Motif of EspC from Enteropathogenic Escherichia coli Produces Epithelial Damage by a Mechanism Different from That of Pet Toxin from Enteroaggregative E. coli
Article Snippet: .. Amplifications were performed by using 0.2 mM deoxynucleoside triphosphates, 0.2 μM each primer, 500 ng of the purified plasmid pJLM174 ( ) as a template, 2.5 mM MgCl2 , 1× buffer, and 1 U of Pfu Turbo DNA polymerase (Stratagene, Inc.). .. The reaction mixture was subjected to initial denaturation for 2 min at 94°C, followed by 35 amplification cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, 30 s; the final step was carried out at 72°C for 5 min. PCR products were purified, and a 3′ adenine was added to each fragment by one-step incubation at 72°C for 20 min using 0.2 mM deoxynucleoside triphosphates, 2.5 mM MgCl2 , 1× buffer, and 0.2 U of Taq DNA polymerase (Promega).

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    Stratagene c neoformans ade2 gene
    Spontaneous frr1 mutations confer rapamycin and FK506 resistance in C. <t>neoformans</t> . (A) FRR1 wild-type (H99, JEC20, and JEC21) and isogenic frr1 :: <t>ADE2</t> , frr1-1 , frr1-2 , frr1-3 , and FKR1-1 mutant strains were grown on YPD medium containing rapamycin (1 μg/ml), FK506 (1 μg/ml), or CsA (100 μg/ml). R and S, drug resistant and sensitive, respectively. (B) Southern analysis of genomic DNA from isogenic wild-type FRR1 and frr1 rapamycin-FK506-resistant mutant strains. Genomic DNA was cleaved with Eco RI (R), Hin dIII (H), or Pst I (P), electrophoresed in a 0.8% agarose gel, transferred to nitrocellulose, and hybridized to a 700-bp FRR1 gene probe. Positions of DNA size markers are shown on the left. (C) PCR amplification of the frr1 mutant locus in strains C20F1, C20F2, and C21F3. Genomic DNA was PCR amplified with a pair of primers directed against the N-terminal region of the FKBP12 gene (primers 1 and 2) or against the C-terminal region of the FKBP12 gene (primers 3 and 4). The longer PCR products in lanes 5 and 6 resulting from strains C20F1 and C20F2 were cloned and sequenced, revealing novel DNA sequences of ∼2,200 and ∼780 bp that have inserted into the FKBP12 locus in strains C20F1 and C20F2 and are flanked by FKBP12 gene sequence in both cases. Lane M, markers. (D) Rapamycin-resistant mutants fail to express FKBP12. Protein extracts from wild-type and isogenic rapamycin-resistant mutants were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and probed with rabbit polyclonal antiserum against yeast FKBP12. Strains were FRR1 wild-type serotype A strain H99 and the isogenic frr1 :: ADE2 mutant strain, wild-type FRR1 serotype D strain JEC20 and the isogenic frr1-1 (C20F1) and frr1-2 (C20F2) mutant strains, and wild-type FRR1 serotype D strain JEC21 and the isogenic FKR1-1 (C21F2) and frr1-3 (C21F3) mutant strains. One hundred micrograms of protein from the same extracts was analyzed by Western blotting with antiserum against the C. neoformans cyclophilin A protein (CypA) as a loading control.
    C Neoformans Ade2 Gene, supplied by Stratagene, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    c neoformans ade2 gene - by Bioz Stars, 2020-07
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    93
    Stratagene deoxynucleoside triphosphates dntps
    Spontaneous frr1 mutations confer rapamycin and FK506 resistance in C. <t>neoformans</t> . (A) FRR1 wild-type (H99, JEC20, and JEC21) and isogenic frr1 :: <t>ADE2</t> , frr1-1 , frr1-2 , frr1-3 , and FKR1-1 mutant strains were grown on YPD medium containing rapamycin (1 μg/ml), FK506 (1 μg/ml), or CsA (100 μg/ml). R and S, drug resistant and sensitive, respectively. (B) Southern analysis of genomic DNA from isogenic wild-type FRR1 and frr1 rapamycin-FK506-resistant mutant strains. Genomic DNA was cleaved with Eco RI (R), Hin dIII (H), or Pst I (P), electrophoresed in a 0.8% agarose gel, transferred to nitrocellulose, and hybridized to a 700-bp FRR1 gene probe. Positions of DNA size markers are shown on the left. (C) PCR amplification of the frr1 mutant locus in strains C20F1, C20F2, and C21F3. Genomic DNA was PCR amplified with a pair of primers directed against the N-terminal region of the FKBP12 gene (primers 1 and 2) or against the C-terminal region of the FKBP12 gene (primers 3 and 4). The longer PCR products in lanes 5 and 6 resulting from strains C20F1 and C20F2 were cloned and sequenced, revealing novel DNA sequences of ∼2,200 and ∼780 bp that have inserted into the FKBP12 locus in strains C20F1 and C20F2 and are flanked by FKBP12 gene sequence in both cases. Lane M, markers. (D) Rapamycin-resistant mutants fail to express FKBP12. Protein extracts from wild-type and isogenic rapamycin-resistant mutants were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and probed with rabbit polyclonal antiserum against yeast FKBP12. Strains were FRR1 wild-type serotype A strain H99 and the isogenic frr1 :: ADE2 mutant strain, wild-type FRR1 serotype D strain JEC20 and the isogenic frr1-1 (C20F1) and frr1-2 (C20F2) mutant strains, and wild-type FRR1 serotype D strain JEC21 and the isogenic FKR1-1 (C21F2) and frr1-3 (C21F3) mutant strains. One hundred micrograms of protein from the same extracts was analyzed by Western blotting with antiserum against the C. neoformans cyclophilin A protein (CypA) as a loading control.
    Deoxynucleoside Triphosphates Dntps, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/deoxynucleoside triphosphates dntps/product/Stratagene
    Average 93 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    deoxynucleoside triphosphates dntps - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    92
    Stratagene deoxynucleoside triphosphates
    Spontaneous frr1 mutations confer rapamycin and FK506 resistance in C. <t>neoformans</t> . (A) FRR1 wild-type (H99, JEC20, and JEC21) and isogenic frr1 :: <t>ADE2</t> , frr1-1 , frr1-2 , frr1-3 , and FKR1-1 mutant strains were grown on YPD medium containing rapamycin (1 μg/ml), FK506 (1 μg/ml), or CsA (100 μg/ml). R and S, drug resistant and sensitive, respectively. (B) Southern analysis of genomic DNA from isogenic wild-type FRR1 and frr1 rapamycin-FK506-resistant mutant strains. Genomic DNA was cleaved with Eco RI (R), Hin dIII (H), or Pst I (P), electrophoresed in a 0.8% agarose gel, transferred to nitrocellulose, and hybridized to a 700-bp FRR1 gene probe. Positions of DNA size markers are shown on the left. (C) PCR amplification of the frr1 mutant locus in strains C20F1, C20F2, and C21F3. Genomic DNA was PCR amplified with a pair of primers directed against the N-terminal region of the FKBP12 gene (primers 1 and 2) or against the C-terminal region of the FKBP12 gene (primers 3 and 4). The longer PCR products in lanes 5 and 6 resulting from strains C20F1 and C20F2 were cloned and sequenced, revealing novel DNA sequences of ∼2,200 and ∼780 bp that have inserted into the FKBP12 locus in strains C20F1 and C20F2 and are flanked by FKBP12 gene sequence in both cases. Lane M, markers. (D) Rapamycin-resistant mutants fail to express FKBP12. Protein extracts from wild-type and isogenic rapamycin-resistant mutants were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and probed with rabbit polyclonal antiserum against yeast FKBP12. Strains were FRR1 wild-type serotype A strain H99 and the isogenic frr1 :: ADE2 mutant strain, wild-type FRR1 serotype D strain JEC20 and the isogenic frr1-1 (C20F1) and frr1-2 (C20F2) mutant strains, and wild-type FRR1 serotype D strain JEC21 and the isogenic FKR1-1 (C21F2) and frr1-3 (C21F3) mutant strains. One hundred micrograms of protein from the same extracts was analyzed by Western blotting with antiserum against the C. neoformans cyclophilin A protein (CypA) as a loading control.
    Deoxynucleoside Triphosphates, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/deoxynucleoside triphosphates/product/Stratagene
    Average 92 stars, based on 60 article reviews
    Price from $9.99 to $1999.99
    deoxynucleoside triphosphates - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

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    Spontaneous frr1 mutations confer rapamycin and FK506 resistance in C. neoformans . (A) FRR1 wild-type (H99, JEC20, and JEC21) and isogenic frr1 :: ADE2 , frr1-1 , frr1-2 , frr1-3 , and FKR1-1 mutant strains were grown on YPD medium containing rapamycin (1 μg/ml), FK506 (1 μg/ml), or CsA (100 μg/ml). R and S, drug resistant and sensitive, respectively. (B) Southern analysis of genomic DNA from isogenic wild-type FRR1 and frr1 rapamycin-FK506-resistant mutant strains. Genomic DNA was cleaved with Eco RI (R), Hin dIII (H), or Pst I (P), electrophoresed in a 0.8% agarose gel, transferred to nitrocellulose, and hybridized to a 700-bp FRR1 gene probe. Positions of DNA size markers are shown on the left. (C) PCR amplification of the frr1 mutant locus in strains C20F1, C20F2, and C21F3. Genomic DNA was PCR amplified with a pair of primers directed against the N-terminal region of the FKBP12 gene (primers 1 and 2) or against the C-terminal region of the FKBP12 gene (primers 3 and 4). The longer PCR products in lanes 5 and 6 resulting from strains C20F1 and C20F2 were cloned and sequenced, revealing novel DNA sequences of ∼2,200 and ∼780 bp that have inserted into the FKBP12 locus in strains C20F1 and C20F2 and are flanked by FKBP12 gene sequence in both cases. Lane M, markers. (D) Rapamycin-resistant mutants fail to express FKBP12. Protein extracts from wild-type and isogenic rapamycin-resistant mutants were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and probed with rabbit polyclonal antiserum against yeast FKBP12. Strains were FRR1 wild-type serotype A strain H99 and the isogenic frr1 :: ADE2 mutant strain, wild-type FRR1 serotype D strain JEC20 and the isogenic frr1-1 (C20F1) and frr1-2 (C20F2) mutant strains, and wild-type FRR1 serotype D strain JEC21 and the isogenic FKR1-1 (C21F2) and frr1-3 (C21F3) mutant strains. One hundred micrograms of protein from the same extracts was analyzed by Western blotting with antiserum against the C. neoformans cyclophilin A protein (CypA) as a loading control.

    Journal: Molecular and Cellular Biology

    Article Title: Rapamycin Antifungal Action Is Mediated via Conserved Complexes with FKBP12 and TOR Kinase Homologs in Cryptococcus neoformans

    doi:

    Figure Lengend Snippet: Spontaneous frr1 mutations confer rapamycin and FK506 resistance in C. neoformans . (A) FRR1 wild-type (H99, JEC20, and JEC21) and isogenic frr1 :: ADE2 , frr1-1 , frr1-2 , frr1-3 , and FKR1-1 mutant strains were grown on YPD medium containing rapamycin (1 μg/ml), FK506 (1 μg/ml), or CsA (100 μg/ml). R and S, drug resistant and sensitive, respectively. (B) Southern analysis of genomic DNA from isogenic wild-type FRR1 and frr1 rapamycin-FK506-resistant mutant strains. Genomic DNA was cleaved with Eco RI (R), Hin dIII (H), or Pst I (P), electrophoresed in a 0.8% agarose gel, transferred to nitrocellulose, and hybridized to a 700-bp FRR1 gene probe. Positions of DNA size markers are shown on the left. (C) PCR amplification of the frr1 mutant locus in strains C20F1, C20F2, and C21F3. Genomic DNA was PCR amplified with a pair of primers directed against the N-terminal region of the FKBP12 gene (primers 1 and 2) or against the C-terminal region of the FKBP12 gene (primers 3 and 4). The longer PCR products in lanes 5 and 6 resulting from strains C20F1 and C20F2 were cloned and sequenced, revealing novel DNA sequences of ∼2,200 and ∼780 bp that have inserted into the FKBP12 locus in strains C20F1 and C20F2 and are flanked by FKBP12 gene sequence in both cases. Lane M, markers. (D) Rapamycin-resistant mutants fail to express FKBP12. Protein extracts from wild-type and isogenic rapamycin-resistant mutants were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and probed with rabbit polyclonal antiserum against yeast FKBP12. Strains were FRR1 wild-type serotype A strain H99 and the isogenic frr1 :: ADE2 mutant strain, wild-type FRR1 serotype D strain JEC20 and the isogenic frr1-1 (C20F1) and frr1-2 (C20F2) mutant strains, and wild-type FRR1 serotype D strain JEC21 and the isogenic FKR1-1 (C21F2) and frr1-3 (C21F3) mutant strains. One hundred micrograms of protein from the same extracts was analyzed by Western blotting with antiserum against the C. neoformans cyclophilin A protein (CypA) as a loading control.

    Article Snippet: The FRR1 gene was disrupted by inserting a 3,000-bp Kpn I/ Sma I fragment spanning the C. neoformans ADE2 gene (blunted with T4 DNA polymerase and deoxynucleoside triphosphates) into an Rsr II site within the FRR1 gene in a 5-kb Eco RI genomic fragment cloned in pBluescript (Stratagene), yielding plasmid pMCC1 bearing the frr1 :: ADE2 disruption allele.

    Techniques: Mutagenesis, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Clone Assay, Sequencing, SDS Page, Western Blot

    FKBP12 is not required for virulence of C. neoformans . (A) Rabbits were immunosuppressed with steroids and inoculated intrathecally with wild-type FRR1 C. neoformans M049 expressing FKBP12 (○) (wild-type M049 ADE2 reconstituted) and the isogenic frr1 :: ADE2 mutant strain lacking FKBP12 (●). CSF was removed on days 4, 7, 10, and 14 following inoculation, and the number of surviving C. neoformans cells (expressed as the mean log 10 CFU per milliliter of CSF from eight rabbits at each time point) was determined by serial dilution and plating on YPD medium with growth for 3 days at 30°C. Error bars indicate the standard error of the mean. (B) Mice (10 each) were injected in the lateral tail vein with 10 7 cells of the FRR1 wild-type strain H99 or the isogenic frr1 :: ADE2 mutant strain lacking FKBP12. Survival was monitored and plotted with respect to time.

    Journal: Molecular and Cellular Biology

    Article Title: Rapamycin Antifungal Action Is Mediated via Conserved Complexes with FKBP12 and TOR Kinase Homologs in Cryptococcus neoformans

    doi:

    Figure Lengend Snippet: FKBP12 is not required for virulence of C. neoformans . (A) Rabbits were immunosuppressed with steroids and inoculated intrathecally with wild-type FRR1 C. neoformans M049 expressing FKBP12 (○) (wild-type M049 ADE2 reconstituted) and the isogenic frr1 :: ADE2 mutant strain lacking FKBP12 (●). CSF was removed on days 4, 7, 10, and 14 following inoculation, and the number of surviving C. neoformans cells (expressed as the mean log 10 CFU per milliliter of CSF from eight rabbits at each time point) was determined by serial dilution and plating on YPD medium with growth for 3 days at 30°C. Error bars indicate the standard error of the mean. (B) Mice (10 each) were injected in the lateral tail vein with 10 7 cells of the FRR1 wild-type strain H99 or the isogenic frr1 :: ADE2 mutant strain lacking FKBP12. Survival was monitored and plotted with respect to time.

    Article Snippet: The FRR1 gene was disrupted by inserting a 3,000-bp Kpn I/ Sma I fragment spanning the C. neoformans ADE2 gene (blunted with T4 DNA polymerase and deoxynucleoside triphosphates) into an Rsr II site within the FRR1 gene in a 5-kb Eco RI genomic fragment cloned in pBluescript (Stratagene), yielding plasmid pMCC1 bearing the frr1 :: ADE2 disruption allele.

    Techniques: Expressing, Mutagenesis, Serial Dilution, Mouse Assay, Injection

    Disruption of the C. neoformans FRR1 gene encoding FKBP12. (A) Diagram of the frr1 :: ADE2 gene disruption. The restriction map of the genomic FRR1 gene is shown. The FRR1 gene was disrupted by inserting a blunt-ended ADE2 gene into an Rsr II restriction site. R, Eco RI; H, Hin dIII. (B) Confirmation of the frr1 :: ADE2 disruption by Southern analysis. Genomic DNA from the isogenic FRR1 wild-type strain M049 (H99 Δ ade2 ) and four frr1 :: ADE2 disruption mutant strains was cleaved with Eco RI (R) and Hin dIII (H), electrophoresed in a 0.8% agarose gel, and transferred to nitrocellulose. The membrane was hybridized to a random-primed 32 P-labelled 700-bp gel-purified fragment spanning the FRR1 gene. Positions of DNA size markers are shown on the left. Note that there is a Hin dIII site in the ADE2 marker; hence, two fragments arise from the frr1 :: ADE2 allele. In addition, integration of tandem copies of the frr1 :: ADE2 disruption allele results in more intense hybridization with the frr1 :: ADE2 allele than with the wild-type (WT) locus. (C) An frr1 :: ADE2 mutant strain is rapamycin and FK506 resistant. Wild-type FRR1 strain M049 and an isogenic frr1 disruption mutant lacking FKBP12 were grown for 72 h on YPD medium containing 1 μg of rapamycin or FK506 per ml at 37°C.

    Journal: Molecular and Cellular Biology

    Article Title: Rapamycin Antifungal Action Is Mediated via Conserved Complexes with FKBP12 and TOR Kinase Homologs in Cryptococcus neoformans

    doi:

    Figure Lengend Snippet: Disruption of the C. neoformans FRR1 gene encoding FKBP12. (A) Diagram of the frr1 :: ADE2 gene disruption. The restriction map of the genomic FRR1 gene is shown. The FRR1 gene was disrupted by inserting a blunt-ended ADE2 gene into an Rsr II restriction site. R, Eco RI; H, Hin dIII. (B) Confirmation of the frr1 :: ADE2 disruption by Southern analysis. Genomic DNA from the isogenic FRR1 wild-type strain M049 (H99 Δ ade2 ) and four frr1 :: ADE2 disruption mutant strains was cleaved with Eco RI (R) and Hin dIII (H), electrophoresed in a 0.8% agarose gel, and transferred to nitrocellulose. The membrane was hybridized to a random-primed 32 P-labelled 700-bp gel-purified fragment spanning the FRR1 gene. Positions of DNA size markers are shown on the left. Note that there is a Hin dIII site in the ADE2 marker; hence, two fragments arise from the frr1 :: ADE2 allele. In addition, integration of tandem copies of the frr1 :: ADE2 disruption allele results in more intense hybridization with the frr1 :: ADE2 allele than with the wild-type (WT) locus. (C) An frr1 :: ADE2 mutant strain is rapamycin and FK506 resistant. Wild-type FRR1 strain M049 and an isogenic frr1 disruption mutant lacking FKBP12 were grown for 72 h on YPD medium containing 1 μg of rapamycin or FK506 per ml at 37°C.

    Article Snippet: The FRR1 gene was disrupted by inserting a 3,000-bp Kpn I/ Sma I fragment spanning the C. neoformans ADE2 gene (blunted with T4 DNA polymerase and deoxynucleoside triphosphates) into an Rsr II site within the FRR1 gene in a 5-kb Eco RI genomic fragment cloned in pBluescript (Stratagene), yielding plasmid pMCC1 bearing the frr1 :: ADE2 disruption allele.

    Techniques: Mutagenesis, Agarose Gel Electrophoresis, Random Primed, Purification, Marker, Hybridization