Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Media composition influences yeast one- and two-hybrid results.

ABSTRACT: Although yeast two-hybrid experiments are commonly used to identify protein interactions, the frequent occurrence of false negatives and false positives hampers data interpretation. Using both yeast one-hybrid and two-hybrid experiments, we have identified potential sources of these problems: the media preparation protocol and the source of the yeast nitrogen base may not only impact signal range but also effect whether a result appears positive or negative. While altering media preparation may optimize signal differences for individual experiments, media preparation must be reported in detail to replicate studies and accurately compare results from different experiments.

SUBMITTER: Liu Y

PROVIDER: S-EPMC3177868 | biostudies-other | 2011 Aug

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Media composition influences yeast one- and two-hybrid results.

Liu Ying Y Merchant Zabeena Z Hsiao Hao-Ching HC Gonzalez Kim L KL Matthews Kathleen S KS Bondos Sarah E SE

Biological procedures online 20110815

Although yeast two-hybrid experiments are commonly used to identify protein interactions, the frequent occurrence of false negatives and false positives hampers data interpretation. Using both yeast one-hybrid and two-hybrid experiments, we have identified potential sources of these problems: the media preparation protocol and the source of the yeast nitrogen base may not only impact signal range but also effect whether a result appears positive or negative. While altering media preparation may ...[more]

PMID: 21843345

Similar Datasets

Yeast Two-Hybrid: State of the Art.

Project description:Genome projects are approaching completion and are saturating sequence databases. This paper discusses the role of the two-hybrid system as a generator of hypotheses. Apart from this rather exhaustive, financially and labour intensive procedure, more refined functional studies can be undertaken. Indeed, by making hybrids of two-hybrid systems, customised approaches can be developed in order to attack specific function-related problems. For example, one could set-up a "differential" screen by combining a forward and a reverse approach in a three-hybrid set-up. Another very interesting project is the use of peptide libraries in two-hybrid approaches. This could enable the identification of peptides with very high specificity comparable to "real" antibodies. With the technology available, the only limitation is imagination.

| S-EPMC140126 | biostudies-literature

Culture media composition influences patient-derived organoids ability to predict therapeutic response in gastrointestinal cancers

Project description:In this study, we have generated patient-derived organoids (PDOs) from primary and metastatic lesions of gastrointestinal cancers, including pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and cholangiocarcinoma and evaluated their ability to predict donor tumor drug response. Specifically, we examined individual PDO responses to therapeutic agents with the observed clinical responses of donor patients to the same treatments and demonstrated an approximately 80% concordance rate. Importantly, we found a profound influence of culture media in PDOs phenotypes, we observed a significant difference in response to common PDAC chemotherapies, distinct morphologies, and transcriptomes between media in the same PDOs. These studies demonstrate the important role of culture media when using PDOs to inform patient care and predict response across a spectrum of GI cancers. We compared transcriptome data from pancreatic ductal adenocarcinoma patient-derived organoids in two media (WNT and PaTOM).

2022-10-12 | GSE212014 | GEO

Yeast two-hybrid screens: improvement of array-based screening results by N- and C-terminally tagged fusion proteins.

Project description:Matrix-based yeast two-hybrid screens are an alternative to library-based screens. Recent improvements of matrix screens (also called array screens), use various pooling strategies as well as novel vectors that increase its efficiency while decreasing the false-negative rate, thus increasing reliability. In this chapter, we describe a screening strategy that systematically combines N- and C-terminal fusion proteins using a recently developed vector system.

| S-EPMC7120008 | biostudies-literature

A comparison and optimization of yeast two-hybrid systems.

Project description:Two-hybrid (Y2H) assays are available in a variety of different versions, including bacterial, yeast, and mammalian systems. However, even when done exclusively in yeast, multiple different host strains, vectors, reporter genes, or protocols can be used. Here we systematically compare protein-protein interactions (PPIs) from several previously published Y2H datasets. PPIs of a human gold-standard dataset were generated by Y2H assays as well as other methods such as LUMIER or protein fragment complementation assays (PCAs). Different Y2H methods detect substantially different subsets of these PPIs, even when protocols are standardized. In order to maximize the number of interactions found and to minimize the number of false positive interactions we recommend to combine multiple vectors and protocols. While the combined results of all 18 methods detected about 92% of a gold-standard interaction set, a combination of just three Y2H assays detected up to 78% of these protein pairs, or up to 83% when a fourth assay was included. These findings indicate that three or four separate assays may be sufficient to detect the majority of protein-protein interactions in many systems.

| S-EPMC5737776 | biostudies-literature

Yeast two-hybrid junk sequences contain selected linear motifs.

Project description:Yeast two-hybrid (Y2H) screenings result in identification of many out-of-frame (OOF) clones that code for short (2-100 amino acids) peptides with no sequence homology to known proteins. We hypothesize that these peptides can reveal common short linear motifs (SLiMs) responsible for their selection. We present a new protocol to address this issue, using an existing SLIM detector (TEIRESIAS) as a base method, and applying filters derived from a mathematical model of SLiM selection in OOF clones. The model allows for initial analysis of likely presence of SLiM(s) in a collection of OOF sequences, assisting investigators with the decision of whether to invest resources in further analysis. If SLiM presence is detected, it estimates the length and number of amino acid residues involved in binding specificity and the amount of noise in the Y2H screen. We demonstrate that our model can double the prediction sensitivity of TEIRESIAS and improve its specificity from 0 to 1.0 on simulated data and apply the model to seven sets of experimentally derived OOF clones. Finally, we experimentally validate one SLiM found by our method, demonstrating its utility.

| S-EPMC3201885 | biostudies-literature

Studying protein complexes by the yeast two-hybrid system.

Project description:Protein complexes are typically analyzed by affinity purification and subsequent mass spectrometric analysis. However, in most cases the structure and topology of the complexes remains elusive from such studies. Here we investigate how the yeast two-hybrid system can be used to analyze direct interactions among proteins in a complex. First we tested all pairwise interactions among the seven proteins of Escherichia coli DNA polymerase III as well as an uncharacterized complex that includes MntR and PerR. Four and seven interactions were identified in these two complexes, respectively. In addition, we review Y2H data for three other complexes of known structure which serve as "gold-standards", namely Varicella Zoster Virus (VZV) ribonucleotide reductase (RNR), the yeast proteasome, and bacteriophage lambda. Finally, we review an Y2H analysis of the human spliceosome which may serve as an example for a dynamic mega-complex.

| S-EPMC3517932 | biostudies-literature

Construction and characterization of a high-quality cDNA library of Cymbidium faberi suitable for yeast one- and two-hybrid assays.

Project description:BACKGROUND:Cymbidium faberi is one of the oldest cultivars of oriental orchids, with an elegant flower fragrance. In order to investigate the molecular mechanism and the functions of related proteins in the methyl jasmonate (MeJA) signaling pathway, one of the main components of flower fragrance in C. faberi, yeast one- and two-hybrid three-frame cDNA libraries were constructed. RESULTS:In this study, a modified cDNA library used for yeast one- and two-hybrid screening was successfully constructed, with a recombinant efficiency of 95%. The lengths of inserted fragments ranged from 750~3000?bp, and the library capacity reached 6?×?109?CFU/ ?g of cDNA insert, which was suitable for the requirements of subsequent screening. Finally, a homologous protein related with pathogenesis was screened out by the bait vector of CfbHLH36, which may participate in the MeJA signaling pathway. CONCLUSION:The yeast one- and two-hybrid library of C. faberi provides large amounts of useful information for the functional genomics research in C. faberi, and this method could also be applied to other plants to screen DNA-protein and protein-protein interactions.

| S-EPMC6966867 | biostudies-literature

Yeast one-hybrid assays for gene-centered human gene regulatory network mapping.

Project description:Gateway-compatible yeast one-hybrid (Y1H) assays provide a convenient gene-centered (DNA to protein) approach to identify transcription factors that can bind a DNA sequence of interest. We present Y1H resources, including clones for 988 of 1,434 (69%) predicted human transcription factors, that can be used to detect both known and new interactions between human DNA regions and transcription factors.

| S-EPMC3263363 | biostudies-literature

Isolation of plant transcription factors using a modified yeast one-hybrid system.

Project description:BACKGROUND:The preparation of expressional cDNA libraries for use in the yeast two-hybrid system is quick and efficient when using the dedicated Clontechtrade mark product, the MATCHMAKER Library Construction and Screening Kit 3. This kit employs SMART technology for the amplification of full-length cDNAs, in combination with cloning using homologous recombination.Unfortunately, such cDNA libraries prepared directly in yeast can not be used for the efficient recovery of purified plasmids and thus are incompatible with existing yeast one-hybrid systems, which use yeast transformation for the library screen. RESULTS:Here we propose an adaptation of the yeast one-hybrid system for identification and cloning of transcription factors using a MATCHMAKER cDNA library. The procedure is demonstrated using a cDNA library prepared from the liquid part of the multinucleate coenocyte of wheat endosperm. The method is a modification of a standard one-hybrid screening protocol, utilising a mating step to introduce the library construct and reporter construct into the same cell. Several novel full length transcription factors from the homeodomain, AP2 domain and E2F families of transcription factors were identified and isolated. CONCLUSION:In this paper we propose a method to extend the compatibility of MATCHMAKER cDNA libraries from yeast two-hybrid screens to one-hybrid screens. The utility of the new yeast one-hybrid technology is demonstrated by the successful cloning from wheat of full-length cDNAs encoding several transcription factors from three different families.

| S-EPMC1402289 | biostudies-literature

Novel cell surface luciferase reporter for high-throughput yeast one-hybrid screens.

Project description:Gene-centered yeast one-hybrid (Y1H) screens provide a powerful and effective strategy to identify transcription factor (TF)-promoter interactions. While genome-wide TF ORFeome clone collections are increasingly available, screening protocols have limitations inherent to the properties of the enzymatic reaction used to identify interactions and to the procedure required to perform the assay in a high-throughput format. Here, we present the development and validation of a streamlined strategy for quantitative and fully automated gene-centered Y1H screens using a novel cell surface Gaussia luciferase reporter.

| S-EPMC5737895 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data