Barcode fusion genetics-protein-fragment complementation assay (BFG-PCA): tools and resources that expand the potential for binary protein interaction discovery

Barcode fusion genetics (BFG) utilizes deep sequencing to improve the throughput of protein–protein interaction (PPI) screening in pools. BFG has been implemented in Yeast two-hybrid (Y2H) screens (BFG-Y2H). While Y2H requires test protein pairs to localize in the nucleus for reporter reconstruction, dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA) allows proteins to localize in broader subcellular contexts and proves to be largely orthogonal to Y2H. Here, we implemented BFG to DHFR-PCA (BFG-PCA). This plasmid-based system can leverage ORF collections across model organisms to perform comparative analysis, unlike the original DHFR-PCA that requires yeast genomic integration. The scalability and quality of BFG-PCA were demonstrated by screening human and yeast interactions for >11 000 bait-prey pairs. BFG-PCA showed high-sensitivity and high-specificity for capturing known interactions for both species. BFG-Y2H and BFG-PCA capture distinct sets of PPIs, which can partially be explained based on the domain orientation of the reporter tags. BFG-PCA is a high-throughput protein interaction technology to interrogate binary PPIs that exploits clone collections from any species of interest, expanding the scope of PPI assays.     

Mapping DNA damage‐dependent genetic interactions in yeast via party mating and barcode fusion genetics

Condition‐dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State‐of‐the‐art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double‐mutant strains, does not scale readily to multi‐condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG‐GI), by which double‐mutant strains generated via en masse “party” mating can also be monitored en masse for growth to detect genetic interactions. By using site‐specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG‐GI enables multiplexed quantitative tracking of double mutants via next‐generation sequencing. We applied BFG‐GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4‐nitroquinoline 1‐oxide (4NQO), bleomycin, zeocin, and three other DNA‐damaging environments. BFG‐GI recapitulated known genetic interactions and yielded new condition‐dependent genetic interactions. We validated and further explored a subnetwork of condition‐dependent genetic interactions involving MAG1, SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53.     

A surface display yeast two-hybrid screening system for high-throughput protein interactome mapping

Despite the wide acceptance of yeast two-hybrid (Y2H) system for protein-protein interaction analysis and discovery, conventional Y2H assays are not well suited for high-throughput screening of the protein interaction network (“interactome”) on a genomic scale due to several limitations, including labor-intensive agar plating and colony selection methods associated with the use of nutrient selection markers, complicated reporter analysis methods associated with the use of LacZ enzyme reporters, and incompatibility of the liquid handling robots. We recently reported a robust liquid culture Y2H system based on quantitative analysis of yeast-enhanced green fluorescent protein (yEGFP) reporters that greatly increased the analysis throughput and compatibility with liquid handling robots. To further advance its utility in high-throughput complementary DNA (cDNA) library screening, we report the development of a novel surface display Y2H (sdY2H) library screening system with uniquely integrated surface display hemagglutination (sdHA) antigen and yEGFP reporters. By introduction of a surface reporter sdHA into the yEGFP-based Y2H system, positive Y2H targets are quickly isolated from library cells by a simple magnetic separation without a large plating effort. Moreover, the simultaneous scoring of multiple reporters, including sdHA, yEGFP, and conventional nutrient markers, greatly increased the specificity of the Y2H assay. The feasibility of the sdY2H assay on large cDNA library screening was demonstrated by the successful recovery of positive P53/T interaction pairs at a target-to-background ratio of 1:1,000,000. Together with the massive parallel DNA sequencing technology, it may provide a powerful proteomic tool for high-throughput interactome mapping on a genomic scale.     

A method for reverse interactome analysis: High‐resolution mapping of interdomain interaction network in Dam1 complex and its specific disorganization based on the interaction domain expression

An experimental methodology that facilitates functional analysis of numerous protein–protein interactions, which have been found in genome‐wide interactome researches, has long been awaited. We propose herein an antagonistic inhibition‐based approach. The antagonizing polypeptide is generated in the course of interaction domain mapping based on yeast 2‐hybrid (Y2H) screening coupled with in vitro convergence of the Y2H‐selected fragments, which is performed in a formatted procedure. Using the coupled methodology, we first performed a high‐resolution mapping of an interdomain interaction network within budding yeast's Dam1 complex. Dam1 complex is a kinetochore protein complex composed of 10 essential subunits including Spc34p and Spc19p. The high‐resolution mapping revealed the overall network structure within the complex for the first time: Dam1 components form into two separated subnetworks on N‐terminal scaffolding domains of Spc34p and Spc19p, and the coiled‐coil interaction in their C‐terminal domains connects the subnetworks. Secondly, we show that the domain fragments converged in the high‐resolution mapping acted as potent inhibitors for the endogenous interactions when episomally overexpressed. The in vivo Dam1 interaction targeting with the fragments conferred a similar phenotype on the host cells; a critical and irreversible damage, which was accompanied with disturbed budding and chromosome mis‐segregation as a result of disorganized spindle. These phenotypes were strongly related to the cellular function of the Dam1 complex. The results and approach we demonstrated herein not only shed light on the Dam1 molecular architecture but also pave the road to reverse‐interactome analysis and discoveries of novel drugs that target disease‐related protein–protein interactions. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Predicting co-complexed protein pairs using genomic and proteomic data integration

Background  

Identifying all protein-protein interactions in an organism is a major objective of proteomics. A related goal is to know which protein pairs are present in the same protein complex. High-throughput methods such as yeast two-hybrid (Y2H) and affinity purification coupled with mass spectrometry (APMS) have been used to detect interacting proteins on a genomic scale. However, both Y2H and APMS methods have substantial false-positive rates. Aside from high-throughput interaction screens, other gene- or protein-pair characteristics may also be informative of physical interaction. Therefore it is desirable to integrate multiple datasets and utilize their different predictive value for more accurate prediction of co-complexed relationship.     

SPIKEPIPE: A metagenomic pipeline for the accurate quantification of eukaryotic species occurrences and intraspecific abundance change using DNA barcodes or mitogenomes

The accurate quantification of eukaryotic species abundances from bulk samples remains a key challenge for community ecology and environmental biomonitoring. We resolve this challenge by combining shotgun sequencing, mapping to reference DNA barcodes or to mitogenomes, and three correction factors: (a) a percent‐coverage threshold to filter out false positives, (b) an internal‐standard DNA spike‐in to correct for stochasticity during sequencing, and (c) technical replicates to correct for stochasticity across sequencing runs. The SPIKEPIPE pipeline achieves a strikingly high accuracy of intraspecific abundance estimates (in terms of DNA mass) from samples of known composition (mapping to barcodes R² = .93, mitogenomes R² = .95) and a high repeatability across environmental‐sample replicates (barcodes R² = .94, mitogenomes R² = .93). As proof of concept, we sequence arthropod samples from the High Arctic, systematically collected over 17 years, detecting changes in species richness, species‐specific abundances, and phenology. SPIKEPIPE provides cost‐efficient and reliable quantification of eukaryotic communities.     

Improving yeast two-hybrid screening systems.

Yeast two-hybrid (Y2H) screening methods are an effective means for the detection of protein-protein interactions. Optimisation and automation has increased the throughput of the method to an extent that allows the systematic mapping of protein-protein interactions on a proteome-wide scale. Since two-hybrid screens fail to detect a great number of interactions, parallel high-throughput approaches are needed for proteome-wide interaction screens. In this review, we discuss and compare different approaches for adaptation of Y2H screening to high-throughput, the limits of the method and possible alternative approaches to complement the mapping of organism-wide protein-protein interactions.     

A method for rapid and sensitive negative staining of proteins in SDS‐PAGE using 2′,7′‐dichlorofluorescein

We report here a rapid and sensitive technique for negative visualization of protein in 1D and 2D SDS‐PAGE by using 2′, 7′‐dichlorofluorescein (DCF), which appeared as transparent and colorless bands in an opaque gel matrix background. For DCF stain, down to 0.1–0.2 ng protein could be easily visualized within 7 min by only two steps, and the staining is fourfold more sensitive than that of Eosin Y (EY) negative stain and glutaraldehyde (GA) silver stain, and eightfold more sensitive than that of the commonly used imidazole‐zinc (IZ) negative stain. Furthermore, DCF stain provided good reproducibility, linearity, and MS compatibility compared with those of IZ stain. In addition, the potential staining mechanism was investigated by colorimetric experiment and molecular docking, and the results demonstrated that the interaction between DCF and protein occurs mainly via van der waals force, electrostatic interaction, and hydrogen bonding.     

A DNA mini‐barcode for land plants

Small portions of the barcode region – mini‐barcodes – may be used in place of full‐length barcodes to overcome DNA degradation for samples with poor DNA preservation. 591,491,286 rbcL mini‐barcode primer combinations were electronically evaluated for PCR universality, and two novel highly universal sets of priming sites were identified. Novel and published rbcL mini‐barcode primers were evaluated for PCR amplification [determined with a validated electronic simulation (n = 2765) and empirically (n = 188)], Sanger sequence quality [determined empirically (n = 188)], and taxonomic discrimination [determined empirically (n = 30 472)]. PCR amplification for all mini‐barcodes, as estimated by validated electronic simulation, was successful for 90.2–99.8% of species. Overall Sanger sequence quality for mini‐barcodes was very low – the best mini‐barcode tested produced sequences of adequate quality (B₂₀ ≥ 0.5) for 74.5% of samples. The majority of mini‐barcodes provide correct identifications of families in excess of 70.1% of the time. Discriminatory power noticeably decreased at lower taxonomic levels. At the species level, the discriminatory power of the best mini‐barcode was less than 38.2%. For samples believed to contain DNA from only one species, an investigator should attempt to sequence, in decreasing order of utility and probability of success, mini‐barcodes F (rbcL1/rbcLB), D (F52/R193) and K (F517/R604). For samples believed to contain DNA from more than one species, an investigator should amplify and sequence mini‐barcode D (F52/R193).     

The impact of mating systems and dispersal on fine‐scale genetic structure at maternally,paternally and biparentally inherited markers

For decades, studies have focused on how dispersal and mating systems influence genetic structure across populations or social groups. However, we still lack a thorough understanding of how these processes and their interaction shape spatial genetic patterns over a finer scale (tens—hundreds of metres). Using uniparentally inherited markers may help answer these questions, yet their potential has not been fully explored. Here, we use individual‐level simulations to investigate the effects of dispersal and mating system on fine‐scale genetic structure at autosomal, mitochondrial and Y chromosome markers. Using genetic spatial autocorrelation analysis, we found that dispersal was the major driver of fine‐scale genetic structure across maternally, paternally and biparentally inherited markers. However, when dispersal was restricted (mean distance = 100 m), variation in mating behaviour created strong differences in the comparative level of structure detected at maternally and paternally inherited markers. Promiscuity reduced spatial genetic structure at Y chromosome loci (relative to monogamy), whereas structure increased under polygyny. In contrast, mitochondrial and autosomal markers were robust to differences in the specific mating system, although genetic structure increased across all markers when reproductive success was skewed towards fewer individuals. Comparing males and females at Y chromosome vs. mitochondrial markers, respectively, revealed that some mating systems can generate similar patterns to those expected under sex‐biased dispersal. This demonstrates the need for caution when inferring ecological and behavioural processes from genetic results. Comparing patterns between the sexes, across a range of marker types, may help us tease apart the processes shaping fine‐scale genetic structure.     

Molecular aspects of the interaction between Mason—Pfizer monkey virus matrix protein and artificial phospholipid membrane

The Mason–Pfizer monkey virus is a type D retrovirus, which assembles its immature particles in the cytoplasm prior to their transport to the host cell membrane. The association with the membrane is mediated by the N‐terminally myristoylated matrix protein. To reveal the role of particular residues which are involved in the capsid‐membrane interaction, covalent labelling of arginine, lysine and tyrosine residues of the Mason–Pfizer monkey virus matrix protein bound to artificial liposomes containing 95% of phosphatidylcholine and 5% phosphatidylinositol‐(4,5)‐bisphosphate (PI(4,5)P₂) was performed. The experimental results were interpreted by multiscale molecular dynamics simulations. The application of these two complementary approaches helped us to reveal that matrix protein specifically recognizes the PI(4,5)P₂ molecule by the residues K20, K25, K27, K74, and Y28, while the residues K92 and K93 stabilizes the matrix protein orientation on the membrane by the interaction with another PI(4,5)P₂ molecule. Residues K33, K39, K54, Y66, Y67, and K87 appear to be involved in the matrix protein oligomerization. All arginine residues remained accessible during the interaction with liposomes which indicates that they neither contribute to the interaction with membrane nor are involved in protein oligomerization. Proteins 2016; 84:1717–1727. © 2016 Wiley Periodicals, Inc.     

Spaceflight‐induced enhancement of 2‐keto‐L‐gulonic acid production by a mixed culture of Ketogulonigenium vulgare and Bacillus thuringiensis

Two bacterial strains used for industrial production of 2‐keto‐L‐gulonic acid (2‐KLG), Ketogulonigenium vulgare 2 and Bacillus thuringiensis 1514, were loaded onto the spacecraft Shenzhou VII and exposed to space conditions for 68 h in an attempt to increase their fermentation productivities of 2‐KLG. An optimal combination of mutants B. thuringiensis 320 and K. vulgare 2194 (KB2194‐320) was identified by systematically screening the pH and 2‐KLG production of 16 000 colonies. Compared with the coculture of parent strains, the conversion rate of L‐sorbose to 2‐KLG by KB2194‐320 in shake flask fermentation was increased significantly from 82·7% to 95·0%. Furthermore, a conversion rate of 94·5% and 2‐KLG productivity of 1·88 g l^?1 h^?1 were achieved with KB2194‐320 in industrial‐scale fermentation (260 m³ fermentor). An observed increase in cell number of K2194 (increased by 47·8%) during the exponential phase and decrease in 2‐KLG reductase activity (decreased by 46·0%) were assumed to explain the enhanced 2‐KLG production. The results suggested that the mutants KB2194‐320 could be ideal substitutes for the currently employed strains in the 2‐KLG fermentation process and demonstrated the feasibility of using spaceflight to breed high‐yielding 2‐KLG‐producing strains for vitamin C production.

Significance and Impact of the Study

KB2194‐320, a combination of two bacterial strains bred by spaceflight mutation, exhibited significantly improved 2‐KLG productivity and hence could potentially increase the efficiency and reduce the cost of vitamin C production by the two‐step fermentation process. In addition, a new pH indicator method was applied for rational screening of K2, which dramatically improved the efficiency of screening.     

Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs

Chloroplast genomes supply indispensable information that helps improve the phylogenetic resolution and even as organelle‐scale barcodes. Next‐generation sequencing technologies have helped promote sequencing of complete chloroplast genomes, but compared with the number of angiosperms, relatively few chloroplast genomes have been sequenced. There are two major reasons for the paucity of completely sequenced chloroplast genomes: (i) massive amounts of fresh leaves are needed for chloroplast sequencing and (ii) there are considerable gaps in the sequenced chloroplast genomes of many plants because of the difficulty of isolating high‐quality chloroplast DNA, preventing complete chloroplast genomes from being assembled. To overcome these obstacles, all known angiosperm chloroplast genomes available to date were analysed, and then we designed nine universal primer pairs corresponding to the highly conserved regions. Using these primers, angiosperm whole chloroplast genomes can be amplified using long‐range PCR and sequenced using next‐generation sequencing methods. The primers showed high universality, which was tested using 24 species representing major clades of angiosperms. To validate the functionality of the primers, eight species representing major groups of angiosperms, that is, early‐diverging angiosperms, magnoliids, monocots, Saxifragales, fabids, malvids and asterids, were sequenced and assembled their complete chloroplast genomes. In our trials, only 100 mg of fresh leaves was used. The results show that the universal primer set provided an easy, effective and feasible approach for sequencing whole chloroplast genomes in angiosperms. The designed universal primer pairs provide a possibility to accelerate genome‐scale data acquisition and will therefore magnify the phylogenetic resolution and species identification in angiosperms.     

Low rates of X‐Y recombination,not turnovers,account for homomorphic sex chromosomes in several diploid species of Palearctic green toads (Bufo viridis subgroup)

Contrasting with birds and mammals, most ectothermic vertebrates present homomorphic sex chromosomes, which might be due either to a high turnover rate or to occasional X‐Y recombination. We tested these two hypotheses in a group of Palearctic green toads that diverged some 3.3 million years ago. Using sibship analyses of sex‐linked markers, we show that all four species investigated share the same pair of sex chromosomes and a pattern of male heterogamety with drastically reduced X‐Y recombination in males. Phylogenetic analyses of sex‐linked sequences show that X and Y alleles cluster by species, not by gametolog. We conclude that X‐Y homomorphy and fine‐scale sequence similarity in these species do not stem from recent sex‐chromosome turnovers, but from occasional X‐Y recombination.     

OsASR2 regulates the expression of a defence‐related gene,Os2H16, by targeting the GT‐1 cis‐element

The GT‐1 cis‐element widely exists in many plant gene promoters. However, the molecular mechanism that underlies the response of the GT‐1 cis‐element to abiotic and biotic stresses remains elusive in rice. We previously isolated a rice short‐chain peptide‐encoding gene, Os2H16, and demonstrated that it plays important roles in both disease resistance and drought tolerance. Here, we conducted a promoter assay of Os2H16 and identified GT‐1 as an important cis‐element that mediates Os2H16 expression in response to pathogen attack and osmotic stress. Using the repeated GT‐1 as bait, we characterized an abscisic acid, stress and ripening 2 (ASR2) protein from yeast‐one hybridization screening. Sequence alignments showed that the carboxy‐terminal domain of OsASR2 containing residues 80–138 was the DNA‐binding domain. Furthermore, we identified that OsASR2 was specifically bound to GT‐1 and activated the expression of the target gene Os2H16, as well as GFP driven by the chimeric promoter of 2 × GT‐1‐35S mini construct. Additionally, the expression of OsASR2 was elevated by pathogens and osmotic stress challenges. Overexpression of OsASR2 enhanced the resistance against Xanthomonas oryzae pv. oryzae and Rhizoctonia solani, and tolerance to drought in rice. These results suggest that the interaction between OsASR2 and GT‐1 plays an important role in the crosstalk of the response of rice to biotic and abiotic stresses.     

Enhanced production of l‐lactic acid by Lactobacillus thermophilus SRZ50 mutant generated by high‐linear energy transfer heavy ion mutagenesis

The aim of this study was to improve l ‐lactic acid production of Lactobacillus thermophilus SRZ50. For this purpose, high efficient heavy‐ion mutagenesis technique was performed using SRZ50 as the original strain. To enhance the screening efficiency for high yield l ‐lactic acid producers, a scale‐down from shake flask to microtiter plate was developed. The results showed that 24‐well U‐bottom MTPs could well alternate shake flasks for L. thermophilus cultivation as a scale‐down tool due to its a very good comparability to the shake flasks. Based on this microtiter plate screening method, two high l ‐lactic acid productivity mutants, A59 and A69, were successfully screened out, which presented, respectively, 15.8 and 16.2% higher productivities than that of the original strain. Based on fed‐batch fermentation, the A69 mutant can accumulate 114.2 g/L l ‐lactic acid at 96 h. Hence, the proposed traditional microbial breeding method with efficient high‐throughput screening assay was proved to be an appropriate strategy to obtain lactic acid‐overproducing strain.     

Development and application of a DNA microarray-based yeast two-hybrid system

The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein–protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms.     

A ROCK inhibitor promotes keratinocyte survival and paracrine secretion,enhancing establishment of primary human melanocytes and melanocyte–keratinocyte co‐cultures

Primary melanocytes isolated from skin and expanded in culture have been widely used for laboratory research and clinical applications. The conventional method to isolate primary melanocytes from skin usually requires about 3–4 weeks of culture for melanocytes to grow sufficiently to passage. Considering that melanocytes comprise only 3%–7% of epidermal cells in normal human skin, it would be extremely helpful to increase the isolation efficiency and shorten the initial culture time to quickly meet various application needs. Here, we report that adding Y‐27632, a Rho kinase inhibitor, into the initial culture medium for 2 days can dramatically increase the yield of melanocytes. We found that Y‐27632 can promote keratinocyte attachment and survival in the melanocyte culture system, resulting in not only better recovery, but also increased proliferation of melanocytes by a paracrine signaling pathway. More specifically, Y‐27632 significantly induced keratinocyte expression of stem cell factor, which played an important role in enhancing the growth of melanocytes. In summary, Y‐27632 could profoundly enhance the yield of primary melanocytes in the initial culture through paracrine effects on keratinocytes.