Crystal Structures of the Plant Phospholipase A1 Proteins Reveal a Unique Dimerization Domain.

Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the ester bonds they cleave off: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). Among the four phospholipase groups, PLA1 has been less studied than the other phospholipases. Here, we report the first molecular structures of plant PLA1s: AtDSEL and CaPLA1 derived from Arabidopsis thaliana and Capsicum annuum, respectively. AtDSEL and CaPLA1 are novel PLA1s in that they form homodimers since PLAs are generally in the form of a monomer. The dimerization domain at the C-terminal of the AtDSEL and CaPLA1 makes hydrophobic interactions between each monomer, respectively. The C-terminal domain is also present in PLA1s of other plants, but not in PLAs of mammals and fungi. An activity assay of AtDSEL toward various lipid substrates demonstrates that AtDSEL is specialized for the cleavage of sn-1 acyl chains. This report reveals a new domain that exists only in plant PLA1s and suggests that the domain is essential for homodimerization.

Deep sequencing of 1320 genes reveals the landscape of protein-truncating variants and their contribution to psoriasis in 19,973 Chinese individuals.

Protein-truncating variants (PTVs) have important impacts on phenotype diversity and disease. However, their population genetics characteristics in more globally diverse populations are not well defined. Here, we describe patterns of PTVs in 1320 genes sequenced in 10,539 healthy controls and 9434 patients with psoriasis, all of Han Chinese ancestry. We identify 8720 PTVs, of which 77% are novel, and estimate 88% of all PTVs are deleterious and subject to purifying selection. Furthermore, we show that individuals with psoriasis have a significantly higher burden of PTVs compared to controls (P = 0.02). Finally, we identified 18 PTVs in 14 genes with unusually high levels of population differentiation, consistent with the action of local adaptation. Our study provides insights into patterns and consequences of PTVs.

NMR spectroscopy uncovers direct interaction between BAF60A and p53.

The BRG1-associated factor 60A (BAF60A), an SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1, has been known to be important for transcriptional activation and inhibition through the alteration of the DNA nucleosome. Although the association between BAF60A and p53 plays a critical role in tumor suppression, the interaction mode is still unclear. Here, we report the detailed interactions between BAF60A and p53 by both NMR spectroscopy and pull-down analysis. Both N-terminal region (BAF60ANR) and the SWIB domain (BAF60ASWIB) of BAF60A directly interact with the tetramerization domain of p53 (p53TET). NMR data show that Ile315, Met366, Ala388, and Tyr390 of BAF60ASWIB are mostly involved in p53TET binding. The calculated dissociation constant (KD) value between BAF60ASWIB and p53TET revealed relatively weak binding affinity, at approximately 0.3 ± 0.065 mM. Our data will enhance detailed interaction mechanism to elucidate the molecular basis of p53-mediated integration via BAF60A interaction.

A flexible method for estimating the fraction of fitness influencing mutations from large sequencing data sets.

A continuing challenge in the analysis of massively large sequencing data sets is quantifying and interpreting non-neutrally evolving mutations. Here, we describe a flexible and robust approach based on the site frequency spectrum to estimate the fraction of deleterious and adaptive variants from large-scale sequencing data sets. We applied our method to approximately 1 million single nucleotide variants (SNVs) identified in high-coverage exome sequences of 6515 individuals. We estimate that the fraction of deleterious nonsynonymous SNVs is higher than previously reported; quantify the effects of genomic context, codon bias, chromatin accessibility, and number of protein-protein interactions on deleterious protein-coding SNVs; and identify pathways and networks that have likely been influenced by positive selection. Furthermore, we show that the fraction of deleterious nonsynonymous SNVs is significantly higher for Mendelian versus complex disease loci and in exons harboring dominant versus recessive Mendelian mutations. In summary, as genome-scale sequencing data accumulate in progressively larger sample sizes, our method will enable increasingly high-resolution inferences into the characteristics and determinants of non-neutral variation.

A Genome-wide Scan for Selective Sweeps in Racing Horses.

Using next-generation sequencing, we conducted a genome-wide scan of selective sweeps associated with selection toward genetic improvement in Thoroughbreds. We investigated potential phenotypic consequence of putative candidate loci by candidate gene association mapping for the finishing time in 240 Thoroughbred horses. We found a significant association with the trait for Ral GApase alpha 2 (RALGAP2) that regulates a variety of cellular processes of signal trafficking. Neighboring genes around RALGAP2 included insulinoma-associated 1 (INSM1), pallid (PLDN), and Ras and Rab interactor 2 (RIN2) genes have similar roles in signal trafficking, suggesting that a co-evolving gene cluster located on the chromosome 22 is under strong artificial selection in racehorses.

Exploring the genetic signature of body size in Yucatan miniature pig.

Since being domesticated about 10,000-12,000 years ago, domestic pigs (Sus scrofa domesticus) have been selected for traits of economic importance, in particular large body size. However, Yucatan miniature pigs have been selected for small body size to withstand high temperature environment and for laboratory use. This renders the Yucatan miniature pig a valuable model for understanding the evolution of body size. We investigate the genetic signature for selection of body size in the Yucatan miniature pig. Phylogenetic distance of Yucatan miniature pig was compared to other large swine breeds (Yorkshire, Landrace, Duroc and wild boar). By estimating the XP-EHH statistic using re-sequencing data derived from 70 pigs, we were able to unravel the signatures of selection of body size. We found that both selections at the level of organism, and at the cellular level have occurred. Selection at the higher levels include feed intake, regulation of body weight and increase in mass while selection at the molecular level includes cell cycle and cell proliferation. Positively selected genes probed by XP-EHH may provide insight into the docile character and innate immunity as well as body size of Yucatan miniature pig.

A genome-wide scan for signatures of directional selection in domesticated pigs.

BACKGROUND: Animal domestication involved drastic phenotypic changes driven by strong artificial selection and also resulted in new populations of breeds, established by humans. This study aims to identify genes that show evidence of recent artificial selection during pig domestication. RESULTS: Whole-genome resequencing of 30 individual pigs from domesticated breeds, Landrace and Yorkshire, and 10 Asian wild boars at ~16-fold coverage was performed resulting in over 4.3 million SNPs for 19,990 genes. We constructed a comprehensive genome map of directional selection by detecting selective sweeps using an F ST-based approach that detects directional selection in lineages leading to the domesticated breeds and using a haplotype-based test that detects ongoing selective sweeps within the breeds. We show that candidate genes under selection are significantly enriched for loci implicated in quantitative traits important to pig reproduction and production. The candidate gene with the strongest signals of directional selection belongs to group III of the metabolomics glutamate receptors, known to affect brain functions associated with eating behavior, suggesting that loci under strong selection include loci involved in behaviorial traits in domesticated pigs including tameness. CONCLUSIONS: We show that a significant proportion of selection signatures coincide with loci that were previously inferred to affect phenotypic variation in pigs. We further identify functional enrichment related to behavior, such as signal transduction and neuronal activities, for those targets of selection during domestication in pigs.

Cetaceans evolution: insights from the genome sequences of common minke whales.

BACKGROUND: Whales have captivated the human imagination for millennia. These incredible cetaceans are the only mammals that have adapted to life in the open oceans and have been a source of human food, fuel and tools around the globe. The transition from land to water has led to various aquatic specializations related to hairless skin and ability to regulate their body temperature in cold water. RESULTS: We present four common minke whale (Balaenoptera acutorostrata) genomes with depth of ×13 ~ ×17 coverage and perform resequencing technology without a reference sequence. Our results indicated the time to the most recent common ancestors of common minke whales to be about 2.3574 (95% HPD, 1.1521 - 3.9212) million years ago. Further, we found that genes associated with epilation and tooth-development showed signatures of positive selection, supporting the morphological uniqueness of whales. CONCLUSIONS: This whole-genome sequencing offers a chance to better understand the evolutionary journey of one of the largest mammals on earth.

Solution structure of CEH-37 homeodomain of the nematode Caenorhabditis elegans.

The nematode Caenorhabditis elegans protein CEH-37 belongs to the paired OTD/OTX family of homeobox-containing homeodomain proteins. CEH-37 shares sequence similarity with homeodomain proteins, although it specifically binds to double-stranded C. elegans telomeric DNA, which is unusual to homeodomain proteins. Here, we report the solution structure of CEH-37 homeodomain and molecular interaction with double-stranded C. elegans telomeric DNA using nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that CEH-37 homeodomain is composed of a flexible N-terminal region and three α-helices with a helix-turn-helix (HTH) DNA binding motif. Data from size-exclusion chromatography and fluorescence spectroscopy reveal that CEH-37 homeodomain interacts strongly with double-stranded C. elegans telomeric DNA. NMR titration experiments identified residues responsible for specific binding to nematode double-stranded telomeric DNA. These results suggest that C. elegans homeodomain protein, CEH-37 could play an important role in telomere function via DNA binding.

1H, 15N, and 13C resonance assignments and secondary structure of the SWIRM domain of human BAF155, a chromatin remodeling complex component.

Mammalian SWI/SNF complexes are evolutionary conserved, ATP-dependent chromatin remodeling units. BAF155 in the SWI/SNF complex contains several highly conserved domains, including SANT, SWIRM, and leucine zipper domains. The biological roles of the SWIRM domain remain unclear; however, both structural and biochemical analyses of this domain have suggested that it could mediate protein-protein or protein-DNA interactions during the chromatin remodeling process. The human BAF155 SWIRM domain was cloned into the Escherichia coli expression vector pMAL-c2X and purified using affinity chromatography for structural analysis. We report the backbone (1)H, (15)N, and (13)C resonance assignments and secondary structure of this domain using nuclear magnetic resonance (NMR) spectroscopy and the TALOS+ program. The secondary structure consists of five α-helices that form a typical histone fold for DNA interactions. Our data suggest that the BAF155 SWIRM domain interacts with nucleosome DNA (Kd = 0.47 µM).

Peeling back the evolutionary layers of molecular mechanisms responsive to exercise-stress in the skeletal muscle of the racing horse.

The modern horse (Equus caballus) is the product of over 50 million yrs of evolution. The athletic abilities of the horse have been enhanced during the past 6000 yrs under domestication. Therefore, the horse serves as a valuable model to understand the physiology and molecular mechanisms of adaptive responses to exercise. The structure and function of skeletal muscle show remarkable plasticity to the physical and metabolic challenges following exercise. Here, we reveal an evolutionary layer of responsiveness to exercise-stress in the skeletal muscle of the racing horse. We analysed differentially expressed genes and their co-expression networks in a large-scale RNA-sequence dataset comparing expression before and after exercise. By estimating genome-wide dN/dS ratios using six mammalian genomes, and FST and iHS using re-sequencing data derived from 20 horses, we were able to peel back the evolutionary layers of adaptations to exercise-stress in the horse. We found that the oldest and thickest layer (dN/dS) consists of system-wide tissue and organ adaptations. We further find that, during the period of horse domestication, the older layer (FST) is mainly responsible for adaptations to inflammation and energy metabolism, and the most recent layer (iHS) for neurological system process, cell adhesion, and proteolysis.

Gene expression profile of human peripheral blood mononuclear cells induced by Staphylococcus aureus lipoteichoic acid.

Lipoteichoic acid (LTA) is a major virulence factor of Gram-positive bacteria including Staphylococcus aureus. Despite its pivotal role in causing sepsis, the systemic immune responses to LTA in human cells are poorly understood. Here, we produced highly-pure and structurally-intact LTA from S. aureus and examined the gene expression profile of LTA-stimulated human peripheral blood mononuclear cells (PBMCs). The LTA preparation did not contain any detectable biologically-active impurities and stimulated Toll-like receptor 2. Protein expression profiling using a cytokine array kit and ELISA revealed expression of MCP-1/CCL2, IL-6, and IL-1ß. We performed transcriptional profiling of PBMCs in response to S. aureus LTA using an Affymetrix genechip microarray. A total of 208 genes were significantly (fold change>1.5 and P<0.05) altered, with 157 up-regulated and 51 down-regulated genes in response to S. aureus LTA treatment. The up-regulated genes were involved in recognition (30 genes), cellular adhesion (6 genes), signal transduction (42 genes), co-stimulation (4 genes), chemokines, cytokines and their receptors (51 genes), apoptosis (9 genes), and negative regulation (15 genes). The down-regulated genes were involved in recognition (12 genes), antigen processing and presentation (9 genes), signal transduction (27 genes), and chemotaxis (3 genes). The microarray results were validated using real-time RT-PCR with 21 up-regulated genes and 9 down-regulated genes. Our results provide a more comprehensive overview of the transcriptional changes in PBMCs in response to S. aureus LTA, and contribute to the understanding of the pathophysiological role of S. aureus LTA during the systemic inflammatory response.

Change in gene expression of mouse embryonic stem cells derived from parthenogenetic activation.

BACKGROUND: We previously established parthenogenetic mouse embryonic stem cells (ESCs) and this study was subsequently conducted for elucidating the influence of oocyte parthenogenesis on gene expression profile of ESCs. METHODS: Gene expression of parthenogenetic ESC (pESC)-1 or pESC-2 was separately compared with that of two normally fertilized ESC (nfESC) lines (B6D2F1 and R1 strains), and quantification of mRNA expression was conducted for validating microarray data. RESULTS: In two sets of comparison, reaction of 11 347 and 15 454 gene probes were altered by parthenogenesis, while strain difference changed the expression of 15 750 and 14 944 probes. Level of correlation coefficient was higher in the comparisons between normal fertilization and parthenogenesis (0.974-0.985) than in the comparisons between strains of nfESCs (0.97-0.971). Overall, the expression of 3276-3329 genes was changed after parthenogenesis, and 88% (96/109) of major functional genes differentially (P < 0.01) expressed in one comparison set showed the same change in the other. When we monitored imprinted genes, expression of nine paternal and eight maternal genes were altered after parthenogenesis and 88% (14/16) of these was confirmed by mRNA quantification. CONCLUSIONS: The change in gene expression after parthenogenesis was similar to, or less than, the change induced by a strain difference under a certain genetic background. These results may suggest the clinical feasibility of parthenogenesis-derived, pluripotent cells.

Organization and evolution of mitochondrial gene clusters in human.

Currently, the spatial patterns of mitochondrial genes and how the genomic localization of (pseudo)genes originated from mitochondrial DNA remain largely unexplained. The aim of this study was to elucidate the organization of mitochondrial (pseudo)genes given their evolutionary origin. We used a keyword finding method and a bootstrapping method to estimate parameter values that represent the distribution pattern of mitochondrial genes in the nuclear genome. Almost half of mitochondrial genes showing physical clusters were located in the pericentromeric and subtelomeric regions of the chromosome. Most interestingly, the size of these clusters ranged from 0.085 to 3.2 Mb (average+/-SD 1.3+/-0.73 Mb), which coincides with the size of the evolutionary pocket, or the average size of evolutionary breakpoint regions. Our findings imply that the localization of mitochondrial genes in the human genome is determined independent of adaptation.

Identification and gene expression profiling of the Pum1 and Pum2 members of the Pumilio family in the chicken.

Members of the Pumilio (Pum) family of RNA-binding proteins act as translational repressors and are required for germ cell development and asymmetric division. We identified the chicken Pum1 and Pum2 genes and analyzed their expression patterns in various tissues. Comparative sequence analysis of the Pum1 and Pum2 proteins from the drosophila, chicken, mouse, and human revealed a high degree of evolutionary conservation in terms of the levels of homology of the peptide sequences and the structure of Pumilio homology domain (PUM-HD), C-terminal RNA-binding domain, with similar spacing between the adjacent Pum eight tandem repeats. In addition, phylogenetic patterns of pumilio family showed that Pum 1 and 2 of chicken are more closely related to those of mouse and human than other species and Pum1 is more conserved than Pum2. Using real-time RT-PCR, the expression levels of the Pum1 and Pum2 genes were found to be highest in hatched female gonads, and high-level expression of Pum2 was detected in 12-day and hatched gonads among the various chicken embryonic tissues tested. In adult tissues, the expression levels of Pum1 and Pum2 were expressed at higher levels in the testis and muscle than in any other tissue. The characteristics of the tissue-specific expression of Pum genes suggest that Pum1 and Pum2 have effects crucially in particular stage during development of chicken gonads depending on sexual maturation.

Gene expression profiling of the uterus with embryos cloned by somatic cell nuclear transfer on day 30 of pregnancy.

Cloning by somatic cell nuclear transfer (SCNT) in pigs has great value for research and biomedical applications. However, cloning pigs is inefficient, and cloning procedures often lead to the birth of abnormal offspring because of the inadequate nuclear remodeling of donor cells as well as inadequate subsequent development. To understand the problems of the cloning process, it is necessary to understand how the uterus interacts with cloned embryo during pregnancy and supports placentation and fetal development. In this study, we compared gene expression profiles of the uterus with SCNT embryos to those of the uterus with normal embryos by natural mating. We obtained the uterine endometrial tissues on day 30 of pregnancy and conducted gene expression profiling using the Platinum Pig 13K oligonucleotide microarrays. Of the 13,610 genes analyzed, expression of 351 genes significantly increased or decreased in the uterine tissues with SCNT embryos compared to those with normal embryos. The differentially regulated genes included enzymes involved in steroidogenesis and extracellular matrix remodeling and uterine secretory proteins. Analyses of real-time reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization of selected genes confirmed the validity of the gene expression patterns observed in the microarray analysis. Results of this study showed that the transcriptional profile of the genes in the uterus with SCNT embryos was regulated differently indicating that the maternal responsiveness to the SCNT embryos was impaired, resulting in the altered gene expression in the uterus and, in turn, abnormal placental and fetal development and increased embryonic loss.

A set of stage-specific gene transcripts identified in EK stage X and HH stage 3 chick embryos.

BACKGROUND: The embryonic developmental process in avian species is quite different from that in mammals. The first cleavage begins 4 h after fertilization, but the first differentiation does not occur until laying of the egg (Eyal-Giladi and Kochav (EK) stage X). After 12 to 13 h of incubation (Hamburger and Hamilton (HH) stage 3), the three germ layers form and germ cell segregation in the early chick embryo are completed. Thus, to identify genes associated with early embryonic development, we compared transcript expression patterns between undifferentiated (stage X) and differentiated (HH stage 3) embryos. RESULTS: Microarray analysis primarily showed 40 genes indicating the significant changes in expression levels between stage X and HH stage 3, and 80% of the genes (32/40) were differentially expressed with more than a twofold change. Among those, 72% (23/32) were relatively up-regulated at stage X compared to HH stage 3, while 28% (9/32) were relatively up-regulated at HH stage 3 compared to stage X. Verification and gene expression profiling of these GeneChip expression data were performed using quantitative RT-PCR for 32 genes at developmental four points; stage X (0 h), HH stage 3 (12 h), HH stage 6 (24 h), and HH stage 9 (30 h). Additionally, we further analyzed four genes with less than twofold expression increase at HH stage 3. As a result, we identified a set of stage-specific genes during the early chick embryo development; 21 genes were relatively up-regulated in the stage X embryo and 12 genes were relatively up-regulated in the HH stage 3 embryo based on both results of microarray and quantitative RT-PCR. CONCLUSION: We identified a set of genes with stage-specific expression from microarray Genechip and quantitative RT-PCR. Discovering stage-specific genes will aid in uncovering the molecular mechanisms involved the formation of the three germ layers and germ cell segregation in the early chick embryos.

MPSS profiling of embryonic gonad and primordial germ cells in chicken.

The massively parallel signature sequencing (MPSS) provides a greater depth of coverage than expressed sequence tag scan or microarray and provides a comprehensive expression profile. We used the MPSS technology to uncover gene expression profiling in the early embryonic gonads and primordial germ cells (PGCs) in the chicken. Total numbers of sequenced signatures were 1,012,533 and 995,676 for the PGCs and gonad, respectively. Using a noise distribution model, we found that 1.67% of all signatures are expressed at a higher level in PCGs and 2.81% of all signatures are expressed at a higher level in the gonad. The MPSS data are presented via an interactive web interface available at http://snugenome.snu.ac.kr/MPSS. The MPSS data have been submitted to the Gene Expression Omnibus of the National Center for Biotechnology Information (accession number GSM137300 and GSM137301 for PGCs and gonad, respectively).

BcSNPdb: bovine coding region single nucleotide polymorphisms located proximal to quantitative trait loci.

Bovine coding region single nucleotide polymorphisms located proximal to quantitative trait loci were identified to facilitate bovine QTL fine mapping research. A total of 692,763 bovine SNPs was extracted from 39,432 UniGene clusters, and 53,446 candidate SNPs were found to be a depth >3. In order to validate the in silico SNPs experimentally, 186 animals representing 14 breeds and 100 mixed breeds were analyzed. Genotyping of 40 randomly selected candidate SNPs revealed that 43% of these SNPs ranged in frequency from 0.009 to 0.498. To identify non-synonymous SNPs and to correct for possible frameshift errors in the ESTs at the predicted SNP positions, we designed a program that determines coding regions by protein-sequence referencing, and identified 17,735 nsSNPs. The SNPs and bovine quantitative traits loci informations were integrated into a bovine SNP data: BcSNPdb (http://snugenome.snu.ac.kr/BtcSNP/). Currently there are 43 different kinds of quantitative traits available. Thus, these SNPs would serve as valuable resources for exploiting genomic variation that influence economically and agriculturally important traits in cows.

ChickGCE: a novel germ cell EST database for studying the early developmental stage in chickens.

We established a database to study germ cells during the early developmental stage in the chicken. The ChickGCE database provides integrated expressed sequence tag (EST) data from chicken testis, ovary, embryonic gonads, and primordial germ cells. We gathered data on 10,294 ESTs from approximately 1000 embryonic gonads, and we experimentally determined 10,851 ESTs from primordial germ cells purified from 7955 embryonic gonads by magnetically activated cell sorting. The EST testis and ovary datasets were retrieved from the public database of The Institute for Genomic Research (TIGR). The EST data were clustered and assembled into unique sequences, contigs, and singletons. The ChickGCE database provides functional annotation, identification, and putative embryonic germ-cell-specific novel transcripts based on the Gene Ontology database, as well as statistical analyses of expression patterns and pair-wise comparisons of two types of tissue- and germ-cell-specific alternative splicing events in the chicken. The new database is accessible online and queries can be answered using several search options, including tissue database searches, keywords, clone IDs, expected values, and BLAST search scores.