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

BACKGROUND: The preparation of expressional cDNA libraries for use in the yeast two-hybrid system is quick and efficient when using the dedicated Clontech™ 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

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Identification of functional DRE/CRT element in the <i>TdCor410b</i> promoter using transient expression assay.

<p>(A), Identification of functional drought-responsive DRE/CRT elements by 5′ deletion analysis of the <i>TdCor410b</i> promoter, using <i>trans</i>-activation of the <i>GUS</i> reporter gene in a transient expression assay. The full-length <i>TdCor410b</i> promoter and six promoter deletions were linked to the <i>GUS</i> reporter gene and co-transformed <i>via</i> particle bombardment into cell suspension cultures with either pUbi-GFP (negative control) or pUbi-TaDREB3 (transcription activator). A schematic representation of the 5′ terminal deletions of the promoter fused to the <i>GUS</i> gene is shown in the left part of the figure: asterisk (*) denotes the predicted DRE/CRT site. A negative control (basal levels of full-length promoter activity) is shown at the top of the right panel as an empty box. Error bars represent standard deviation (P<0.05) for 3 – 4 independent measurements. (B) Influence of point mutations in the identified functional CRT element on <i>TdCor410b</i> promoter activation, as demonstrated by a transient expression assay. D7 denotes a −263 promoter deletion containing the non-mutated CRT element (positive control), D8 and M5 denote promoter deletions without the CRT element (negative controls), and M1–M4 denote the D7 deletion with different single base pair substitutions to T.</p

Expression of <i>TaCor410b</i> and five <i>ERF</i> genes in a variety of wheat tissues in the absence of stress.

<p>Levels of expression were detected by Q-PCR and are shown as normalised transcription levels in arbitrary units (n = 4± SD (P<0.05)).</p

Key residues of AP2 domains that underlie selectivity of <i>cis</i>-elements binding, and regulation of the <i>TdCor410b</i> promoter activity.

<p>(A) Multiple sequence alignment of selected AP2 domains using PROMALS3D (44). Representative sequences are coloured according to predicted secondary structures (red: α-helix, blue: β-strand). The black box indicates the boundaries of the AP2 domains. The positions of highly conserved Pro residues in the ERF sequences and of variable non-proline residues in the DREB sequences are highlighted in yellow and green, respectively. The positions of two Pro residues conserved in selected cereal ERF sequences are highlighted in cyan, while the positions of the corresponding Arg residues are highlighted in grey. Consensus of secondary structure elements indicates the position of β-sheets (black arrows) and of an α-helix (purple). The degree of conservation of residues is shown above the sequences by black and brown numbers with a conservation index of 5 and higher. (B) Influence of conserved proline residue substitutions in the AP2 domain of TaERF4a on recognition of the GCC-box. TaDREB3 was used as a negative control and TaERF5a as a positive control of interaction with the GCC-box. Mutation of Pro26 to Arg26 (underlined) has no influence on interaction of the TaERF4a variant with the <i>cis</i>-element. Mutation of Pro42 to Arg42 (underlined and boxed in blue) lead to restoration of interaction and consequent growth of yeast on the selective (-Leu, -His, + 5 mM 3-AT) medium. (C) Regulation of the activity of the <i>TdCor410b</i> promoter and of the artificial promoter with substitution of the CRT element for a tandem of three GCC-boxes by representatives of each isolated ERF subfamily, and variants of TaERF4a with mutations in the AP2 domain. TFs were tested in a transient expression assay in a wheat cell culture. Either pTdCor410b-GUS or 3×GCCbox-GUS constructs were co-bombarded with pUbi-GFP (GFP; negative control), pUbi-TaERF4a (TaERF4a), pUbi-TaERF4a mutated at Pro26 (TaERF4a m1), pUbi-TaERF4a mutated at Pro42 (TaERF4a m2), pUbi-TaERF4a mutated at Pro26 and Pro42 (TaERF4a m1+2), pUbi-TaERF6 (TaERF6), or pUbi-TaERF5a (TaERF5a).</p

A schematic representation of the regulation of abiotic and biotic stress-responsive genes by ERFs and DREBs/CBFs, through three types of stress-responsive <i>cis</i>-acting elements.

<p>A schematic representation of the regulation of abiotic and biotic stress-responsive genes by ERFs and DREBs/CBFs, through three types of stress-responsive <i>cis</i>-acting elements.</p