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  • Cold Spring Harbor Laboratory  (15)
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  • Cold Spring Harbor Laboratory  (15)
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  • 1
    Online Resource
    Online Resource
    Generating Yeast One-Hybrid DNA-Bait Strains
    Cold Spring Harbor Laboratory ; 2018
    In:  Cold Spring Harbor Protocols Vol. 2018, No. 7 ( 2018-07), p. pdb.prot094961-
    Details
    In: Cold Spring Harbor Protocols, Cold Spring Harbor Laboratory, Vol. 2018, No. 7 ( 2018-07), p. pdb.prot094961-
    Abstract: Generating DNA-bait strains for gateway-compatible yeast one-hybrid (Y1H) screens involves three steps. The first is to generate an Entry clone containing the DNA-bait of interest. Gateway cloning is used to clone larger baits, such as promoters, into pDONR-P4-P1R. (An alternative set of steps is also presented in this protocol that describes the creation of Entry clones by annealing primers and performing conventional ligation into pMW#5—a strategy best suited for smaller DNA-baits up to 100 bp.) The second is to transfer this DNA-bait from the Entry clone to the two Y1H reporter Destination vectors, pMW#2 ( HIS3 ) and pMW#3 ( LacZ ). A two-step process is used because Entry clones generate a versatile resource that can be used for transfer of DNA-baits into a variety of vectors, for instance, upstream of the green fluorescent protein-encoding ORF to study spatiotemporal expression patterns. The final step is to integrate the HIS3 and LacZ reporter constructs into the genome of the Y1H yeast strain, YM4271. The entire process takes 24–32 d, plus sequence confirmation if necessary.
    Type of Medium: Online Resource
    ISSN: 1940-3402 , 1559-6095
    URL: Article
    DOI: 10.1101/pdb.prot094961
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2018
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  • 2
    Online Resource
    Online Resource
    Gateway-Compatible Yeast One-Hybrid and Two-Hybrid Assays
    Cold Spring Harbor Laboratory ; 2018
    In:  Cold Spring Harbor Protocols Vol. 2018, No. 7 ( 2018-07), p. pdb.top094953-
    Details
    In: Cold Spring Harbor Protocols, Cold Spring Harbor Laboratory, Vol. 2018, No. 7 ( 2018-07), p. pdb.top094953-
    Abstract: In the first section of this introduction, we provide background information for yeast two-hybrid (Y2H) assays that provide a genetic method for the identification and analysis of binary protein–protein interactions and that are complementary to biochemical methods such as immunoprecipitation. In the second section, we discuss yeast one-hybrid (Y1H) assays that provide a “gene-centered” (DNA-to-protein) genetic method to identify and study protein–DNA interactions between cis -regulatory elements and transcription factors (TFs). This method is complementary to “TF-centered” (protein-to-DNA) biochemical methods such as chromatin immunoprecipitation.
    Type of Medium: Online Resource
    ISSN: 1940-3402 , 1559-6095
    URL: Article
    DOI: 10.1101/pdb.top094953
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2018
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  • 3
    Online Resource
    Online Resource
    Generating an Open Reading Frame (ORF) Entry Clone and Destination Clone
    Cold Spring Harbor Laboratory ; 2018
    In:  Cold Spring Harbor Protocols Vol. 2018, No. 1 ( 2018-01), p. pdb.prot094938-
    Details
    In: Cold Spring Harbor Protocols, Cold Spring Harbor Laboratory, Vol. 2018, No. 1 ( 2018-01), p. pdb.prot094938-
    Abstract: This protocol describes using the Gateway recombinatorial cloning system to create an Entry clone carrying an open reading frame (ORF) and then to transfer the ORF into a Destination vector. In this example, BP recombination is used to clone an ORF from a cDNA source into the Donor vector pDONR 221. The ORF from the resulting Entry clone is then transferred into the Destination vector pDEST-15; the product (the Destination clone) will express the ORF as an amino-terminal GST-fusion. The technique can be used as a guide for cloning any other DNA fragment of interest—a promoter sequence or 3′ untranslated region (UTR), for example—with substitutions of different genetic material such as genomic DNA, att sites, and vectors as required. The series of constructions and transformations requires 9–15 d, not including time that may be required for sequence confirmation, if desired/necessary.
    Type of Medium: Online Resource
    ISSN: 1940-3402 , 1559-6095
    URL: Article
    DOI: 10.1101/pdb.prot094938
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2018
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  • 4
    Online Resource
    Online Resource
    Generating Yeast Two-Hybrid Bait Strains
    Cold Spring Harbor Laboratory ; 2018
    In:  Cold Spring Harbor Protocols Vol. 2018, No. 7 ( 2018-07), p. pdb.prot094979-
    Details
    In: Cold Spring Harbor Protocols, Cold Spring Harbor Laboratory, Vol. 2018, No. 7 ( 2018-07), p. pdb.prot094979-
    Abstract: Generating DNA-binding domain (DB)-bait strains for Gateway-compatible yeast two-hybrid (Y2H) screens involves three steps. The first is to generate an Entry clone containing a DNA fragment encoding the protein of interest (e.g., an open reading frame, ORF). The second is to transfer this DNA fragment from the Entry clone to the Y2H Destination vector, pDEST32. The final step is to transform this construct into the Y2H yeast strain, MaV103. This protocol takes 24–37 d plus sequence confirmation, if necessary, to complete.
    Type of Medium: Online Resource
    ISSN: 1940-3402 , 1559-6095
    URL: Article
    DOI: 10.1101/pdb.prot094979
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2018
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  • 5
    Online Resource
    Online Resource
    Colony Lift Colorimetric Assay for β-Galactosidase Activity
    Cold Spring Harbor Laboratory ; 2016
    In:  Cold Spring Harbor Protocols Vol. 2016, No. 12 ( 2016-12), p. pdb.prot088963-
    Details
    In: Cold Spring Harbor Protocols, Cold Spring Harbor Laboratory, Vol. 2016, No. 12 ( 2016-12), p. pdb.prot088963-
    Abstract: In this protocol, we present a qualitative assay for monitoring the level of expression of β-galactosidase, an enzyme encoded by the LacZ gene, in yeast. This is useful both for determining autoactivity of LacZ expression in yeast DNA “bait” strains and for assessing LacZ reporter gene activation mediated by a transcription factor “prey” interaction with a DNA bait of interest in yeast one-hybrid (Y1H) assays. In this colorimetric assay, yeast are lysed in liquid nitrogen and then assayed for β-galactosidase expression using the colorless compound X-gal, which turns blue in the presence of this enzyme.
    Type of Medium: Online Resource
    ISSN: 1940-3402 , 1559-6095
    URL: Article
    DOI: 10.1101/pdb.prot088963
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2016
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  • 6
    Online Resource
    Online Resource
    Using Multisite LR Cloning to Generate a Destination Clone
    Cold Spring Harbor Laboratory ; 2018
    In:  Cold Spring Harbor Protocols Vol. 2018, No. 1 ( 2018-01), p. pdb.prot094946-
    Details
    In: Cold Spring Harbor Protocols, Cold Spring Harbor Laboratory, Vol. 2018, No. 1 ( 2018-01), p. pdb.prot094946-
    Abstract: This protocol describes using the Gateway recombinatorial cloning system to simultaneously transfer a promoter and an open reading frame (ORF) from two different Entry clones into the same Destination vector using LR enzymes. A multisite cloning reaction transfers the inserts from multiple Entry clones into a single Destination vector. This type of recombination is much less efficient than transferring a single DNA fragment; however, the variety of Destination clones that can be generated in this manner is vast. In this example protocol, we describe using pDEST-MB14 to make a Destination clone that features a promoter fragment fused upstream to an ORF that is cloned in-frame with a carboxy-terminal green fluorescent protein (GFP) moiety encoded by the plasmid backbone. This method can be used as a guide for other multisite cloning reactions.
    Type of Medium: Online Resource
    ISSN: 1940-3402 , 1559-6095
    URL: Article
    DOI: 10.1101/pdb.prot094946
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2018
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  • 7
    Online Resource
    Online Resource
    Identifying Interactors from an Activation Domain Prey Library
    Cold Spring Harbor Laboratory ; 2018
    In:  Cold Spring Harbor Protocols Vol. 2018, No. 7 ( 2018-07), p. pdb.prot094987-
    Details
    In: Cold Spring Harbor Protocols, Cold Spring Harbor Laboratory, Vol. 2018, No. 7 ( 2018-07), p. pdb.prot094987-
    Abstract: In yeast hybrid assays, the process of identifying preys that interact with the bait of interest involves several steps. First, in this protocol, the bait yeast strain is transformed with a library of activation domain (AD)-prey clones and plated on selective media containing 3-aminotriazole (3AT). This selects transformants containing an AD-prey clone that induces HIS3 reporter expression. Second, these “HIS-positive” colonies are analyzed for LacZ induction (and, optionally, URA3 induction in yeast two-hybrid (Y2H) assays). Third, yeast PCR is used on these “double-positive” colonies to amplify the insert from the AD-prey plasmid. Fourth, some of this PCR product is used to perform a gap-repair retest to confirm the interaction in fresh bait-strain yeast, and the remainder is used for DNA sequencing to determine prey identity for those that successfully retest. Finally, interactions are carefully examined to filter out likely false-positive interactions. This protocol takes 20–43 d plus sequence confirmation to complete.
    Type of Medium: Online Resource
    ISSN: 1940-3402 , 1559-6095
    URL: Article
    DOI: 10.1101/pdb.prot094987
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2018
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  • 8
    Online Resource
    Online Resource
    Performing Yeast One-Hybrid Library Screens
    Cold Spring Harbor Laboratory ; 2016
    In:  Cold Spring Harbor Protocols Vol. 2016, No. 12 ( 2016-12), p. pdb.prot088955-
    Details
    In: Cold Spring Harbor Protocols, Cold Spring Harbor Laboratory, Vol. 2016, No. 12 ( 2016-12), p. pdb.prot088955-
    Abstract: Yeast one-hybrid (Y1H) assays are used to identify which transcription factor (TF) “prey” molecules can bind a DNA fragment of interest that is used as “bait”. Y1H assays involve introducing plasmids that encode TFs into a yeast “bait strain” in which the DNA fragment of interest is integrated upstream of one or more reporters, and activation of these reporters indicates that a TF–DNA interaction has occurred. These plasmids express each TF as a hybrid protein (hence the “one-hybrid” name) fused to the activation domain (AD) of the yeast TF Gal4. The AD moiety activates reporter expression even if the TF to which it is fused typically functions as a repressor. Here, we describe how to perform a Y1H screen of a library of cDNA fragments cloned into a pPC86 plasmid expressing the protein encoded by the cDNA as an AD fusion. The method assumes availability of either commercially available libraries or libraries generated in house using mRNA extracted from a tissue of interest. We also assume that users have access to a yeast bait strain that possesses the DNA fragment of interest integrated upstream of two different reporters— HIS3 , an auxotrophic marker, and LacZ , a colorimetric marker that changes colorless X-gal into a blue compound. Briefly, the screen involves transforming the AD-cDNA library into the yeast bait strain, identifying colonies that show activation of both reporters, retesting the interaction in a freshly grown bait strain, and sequencing the cDNA insert to identify the interacting TF.
    Type of Medium: Online Resource
    ISSN: 1940-3402 , 1559-6095
    URL: Article
    DOI: 10.1101/pdb.prot088955
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2016
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  • 9
    Online Resource
    Online Resource
    Genome-scale spatiotemporal analysis of Caenorhabditis elegans microRNA promoter activity
    Cold Spring Harbor Laboratory ; 2008
    In:  Genome Research Vol. 18, No. 12 ( 2008-12), p. 2005-2015
    Details
    In: Genome Research, Cold Spring Harbor Laboratory, Vol. 18, No. 12 ( 2008-12), p. 2005-2015
    Abstract: The Caenorhabditis elegans genome encodes more than 100 microRNAs (miRNAs). Genetic analyses of miRNA deletion mutants have only provided limited insights into miRNA function. To gain insight into the function of miRNAs, it is important to determine their spatiotemporal expression pattern. Here, we use miRNA promoters driving the expression of GFP as a proxy for miRNA expression. We describe a set of 73 transgenic C. elegans strains, each expressing GFP under the control of a miRNA promoter. Together, these promoters control the expression of 89 miRNAs (66% of all predicted miRNAs). We find that miRNA promoters drive GFP expression in a variety of tissues and that, overall, their activity is similar to that of protein-coding gene promoters. However, miRNAs are expressed later in development, which is consistent with functions after initial body-plan specification. We find that miRNA members belonging to families are more likely to be expressed in overlapping tissues than miRNAs that do not belong to the same family, and provide evidence that intronic miRNAs may be controlled by their own, rather than a host gene promoter. Finally, our data suggest that post-transcriptional mechanisms contribute to differential miRNA expression. The data and strains described here will provide a valuable guide and resource for the functional analysis of C. elegans miRNAs.
    Type of Medium: Online Resource
    ISSN: 1088-9051
    URL: Article
    DOI: 10.1101/gr.083055.108
    RVK:
    XA 10000
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2008
    detail.hit.zdb_id: 1483456-X
    SSG: 12
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  • 10
    Online Resource
    Online Resource
    Zymolyase-Treatment and Polymerase Chain Reaction Amplification from Genomic and Plasmid Templates from Yeast
    Cold Spring Harbor Laboratory ; 2016
    In:  Cold Spring Harbor Protocols Vol. 2016, No. 12 ( 2016-12), p. pdb.prot088971-
    Details
    In: Cold Spring Harbor Protocols, Cold Spring Harbor Laboratory, Vol. 2016, No. 12 ( 2016-12), p. pdb.prot088971-
    Abstract: Here, we present a protocol for amplifying DNA fragments from the genome of, or plasmids transformed into, yeast strains that require the use of the lytic enzyme zymolyase to break open the yeast cells by digesting the cell wall. Yeast strains requiring such treatment include YM4271 and Y1HaS2, whereas other yeast strains (e.g., MaV103) might not require treatment with Zymolyase.
    Type of Medium: Online Resource
    ISSN: 1940-3402 , 1559-6095
    URL: Article
    DOI: 10.1101/pdb.prot088971
    Language: English
    Publisher: Cold Spring Harbor Laboratory
    Publication Date: 2016
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