Ancestral whole-genome duplication in the marine chelicerate horseshoe crabs.

This corrects the article DOI: 10.1038/hdy.2015.89.

Ancestral whole-genome duplication in the marine chelicerate horseshoe crabs.

Whole-genome duplication (WGD) results in new genomic resources that can be exploited by evolution for rewiring genetic regulatory networks in organisms. In metazoans, WGD occurred before the last common ancestor of vertebrates, and has been postulated as a major evolutionary force that contributed to their speciation and diversification of morphological structures. Here, we have sequenced genomes from three of the four extant species of horseshoe crabs-Carcinoscorpius rotundicauda, Limulus polyphemus and Tachypleus tridentatus. Phylogenetic and sequence analyses of their Hox and other homeobox genes, which encode crucial transcription factors and have been used as indicators of WGD in animals, strongly suggests that WGD happened before the last common ancestor of these marine chelicerates >135 million years ago. Signatures of subfunctionalisation of paralogues of Hox genes are revealed in the appendages of two species of horseshoe crabs. Further, residual homeobox pseudogenes are observed in the three lineages. The existence of WGD in the horseshoe crabs, noted for relative morphological stasis over geological time, suggests that genomic diversity need not always be reflected phenotypically, in contrast to the suggested situation in vertebrates. This study provides evidence of ancient WGD in the ecdysozoan lineage, and reveals new opportunities for studying genomic and regulatory evolution after WGD in the Metazoa.

Fighting outbreaks with bacterial genomics: case review and workflow proposal.

BACKGROUND: Disease outbreak investigation is a key aspect of public health. Whole-genome sequencing of bacterial pathogen based on new generation high-throughput sequencing technologies has facilitated outbreak investigations recently. Whilst the approach has become more affordable and accessible to research and clinical laboratories, a system for adequate and efficient analyses of genome data in the context of bacterial outbreak investigations is missing. METHODS: We performed a literature review of timely genomic investigations performed during the course of bacterial outbreaks that are based on new generation sequencing technologies. Currently available bioinformatics tools for genomic analyses are also reviewed here. RESULTS: Genomic investigations in early stages of bacterial outbreaks have shown to provide timely information on evolutionary origin, transmission route, pathogenic potential, and resistance information of the outbreak strains and allow development of strain-specific typing methods. A systematic genomic analytical workflow is proposed here for the first time to facilitate efficient extraction of epidemiologically useful information from genome data of bacterial pathogens in future bacterial outbreak investigations. CONCLUSION: With the continuous reduction of genome sequencing cost and development of user-friendly analytical tools, it is expected that high-throughput genome sequencing will be applied routinely for timely genomic analysis in bacterial outbreaks in the near future.

Cataloging and profiling genes expressed in Lentinula edodes fruiting body by massive cDNA pyrosequencing and LongSAGE.

This study investigated the molecular mechanism of the fruiting body development and sporulation in the cap of the Shiitake mushroom, Lentinula edodes. Although there has been much research into L. edodes, there remain significant gaps in our knowledge of how the species reproduces. In order to provide molecular resources and to understand the molecular mechanism of the fruiting body development in basidiomycete comprehensively, we searched for the genes which are important for fruiting body development and sporulation in the cap of mature fruiting body of L. edodes by using the whole-genome approach. Massive cDNA pyrosequencing was used to generate >7000 sequence contigs from mature fruiting bodies. We used Gene Ontology to categorize the contigs to form the catalog of genes expressed at the stage of the mature fruiting body. We also assigned the contigs into the KEGG pathways. The catalog of expressed genes indicates that the mature fruiting bodies (1) sense the external environment, (2) transmit signals to express genes through regulatory systems, (3) produce many proteins, (4) degrade unwanted proteins, (5) perform extensive biosynthesis, (6) generate energy, (7) regulate the internal environment, (8) transport molecules, (9) carry out cell division, and (10) differentiate and develop. After establishing the catalog of expressed genes in L. edodes, we used the LongSAGE approach to analyze the expression levels of genes found in mature fruiting bodies before (FB) and after (FBS) spores appeared. Gene-expression patterns according to GO categories were similar in these two stages. We have also successfully identified genes differentially expressed in FB and FBS. Fold-changes in expression levels of selected genes based on LongSAGE tag counts were similar to those obtained by real-time RT-PCR. The consistency between real-time RT-PCR and LongSAGE results indicates reliability of the LongSAGE results. Overall, this study provides valuable information on the fruiting processes of L. edodes through a combination of massive cDNA pyrosequencing and LongSAGE sequencing, and the knowledge thereby obtained may provide insight into the improvement of the yield of commercially grown Shiitake mushrooms.

Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus).

The mushroom Coprinopsis cinerea is a classic experimental model for multicellular development in fungi because it grows on defined media, completes its life cycle in 2 weeks, produces some 10(8) synchronized meiocytes, and can be manipulated at all stages in development by mutation and transformation. The 37-megabase genome of C. cinerea was sequenced and assembled into 13 chromosomes. Meiotic recombination rates vary greatly along the chromosomes, and retrotransposons are absent in large regions of the genome with low levels of meiotic recombination. Single-copy genes with identifiable orthologs in other basidiomycetes are predominant in low-recombination regions of the chromosome. In contrast, paralogous multicopy genes are found in the highly recombining regions, including a large family of protein kinases (FunK1) unique to multicellular fungi. Analyses of P450 and hydrophobin gene families confirmed that local gene duplications drive the expansions of paralogous copies and the expansions occur in independent lineages of Agaricomycotina fungi. Gene-expression patterns from microarrays were used to dissect the transcriptional program of dikaryon formation (mating). Several members of the FunK1 kinase family are differentially regulated during sexual morphogenesis, and coordinate regulation of adjacent duplications is rare. The genomes of C. cinerea and Laccaria bicolor, a symbiotic basidiomycete, share extensive regions of synteny. The largest syntenic blocks occur in regions with low meiotic recombination rates, no transposable elements, and tight gene spacing, where orthologous single-copy genes are overrepresented. The chromosome assembly of C. cinerea is an essential resource in understanding the evolution of multicellularity in the fungi.

Isolation and transcript analysis of two-component histidine kinase gene Le.nik1 in Shiitake mushroom, Lentinula edodes.

Le.nik1, a two-component histidine kinase gene of Lentinula edodes, the Shiitake mushroom, was identified. The relationship between this two-component signal transduction system and mushroom development was studied. We used a modified RNA arbitrarily-primed PCR (RAP-PCR) method to isolate Le.nik1 as a differentially expressed gene during L. edodes development. We determined the 6.29kb full-length cDNA sequence of Le.nik1. It had high sequence homology to Neurospora crassa nik1, which encoded a histidine kinase essential for development and osmotic response. In L. edodes, the expression level of Le.nik1 was highest during primordium formation and fruiting body maturation. The transcripts were localized predominantly in the developing hymenophores, or mushroom gills, which may indicate the role of a two-component signal transduction system in cell differentiation during mushroom development. Mannitol stress influenced transcript expression of Le.nik1, suggesting that it may be involved in osmo-sensing and regulation. To our knowledge, this is the first report on the two-component system in mushrooms and the first analysis on the distribution of Le.nik1 transcript in the course of fruiting body formation and in parts of fruiting bodies.

Overexpression of prostate stem cell antigen is associated with gestational trophoblastic neoplasia.

AIMS: Hydatidiform mole (HM) is the most common type of gestational trophoblastic disease. A proportion of patients with HM develop gestational trophoblastic neoplasia (GTN) requiring chemotherapy. The aim was to identify differentially expressed genes that are associated with development of GTN. METHODS AND RESULTS: Using cDNA microarray, differential expression of prostate stem cell antigen (PSCA) was identified in HMs that developed GTN compared with those that spontaneously regressed. Significant overexpression of PSCA RNA (P = 0.037) and protein (P < 0.05) in aggressive HM was verified by real-time polymerase chain reaction (PCR) and immunohistochemical analysis in 10 first-trimester placentas, 36 HM that subsequently regressed and 11 HM that developed GTN. A high level of PSCA expression was also found in three choriocarcinomas and three placental site trophoblastic tumours. A positive correlation was observed between PSCA expression and proliferation and apoptotic indices as assessed by Ki67 (P = 0.01), mcm7 (P = 0.001) and M30 (P = 0.016), as well as p53 (P < 0.01), p21(WAF1/CIP1) (P < 0.01) and mdm2 (P = 0.002) expression. CONCLUSIONS: Overexpression of PSCA is associated with development of GTN in HM. PSCA probably plays a role in the regulation of cell growth through p53-related signaling pathways.

Le.MAPK and its interacting partner, Le.DRMIP, in fruiting body development in Lentinula edodes.

Development in shiitake mushroom, Lentinula edodes, is a unique process and studies of the molecular basis of this process may lead to improvement in mushroom cultivation. Previous studies have identified a number of signal transduction genes related to mushroom development, but those genes have not been well characterized. The present work characterized a developmentally regulated MAP kinase, Le.MAPK, and its interaction with a novel gene, Le.DRMIP in the signal transduction pathway. The expression profiles of these two genes reveal their importance in fruiting body initiation and development; the Le.DRMIP transcript is localized predominantly in the developing young fruiting body and gills, which further signifies its role in cell differentiation during mushroom development.

Differential gene expression identified in complete hydatidiform mole by combining suppression subtractive hybridization and cDNA microarray.

Complete hydatidiform mole (CHM) is a type of gestational trophoblastic disease with pure paternal chromosome contribution and unpredictable malignant potential. As an attempt to assess the molecular pathogenesis of CHM, suppression subtractive hybridization (SSH) combined with cDNA microarray was used to compare the gene expression pattern of CHM compared with normal first-trimester placenta of similar gestational ages. cDNA microarray analysis using tissue-specific chips constructed with subtracted cDNA libraries identified 13 differentially expressed gene transcripts. Quantitative real-time polymerase chain reaction (PCR) confirmed up-regulation of human chorionic gonadotropin beta subunit (CGB) (P=0.0008) and KIAA1200 (P=0.0005), a G-protein regulator, as well as down-regulation of osteopontin (SPP1) (P<0.0001) in 14 genotyped CHM when compared with 15 normal placentas. These candidate genes may contribute toward understanding the mechanism involved with the development and progression of CHM.

Inhibition of ATPases by Cleistocalyx operculatus. A possible mechanism for the cardiotonic actions of the herb.

The water extract of the buds of Cleistocalyx operculatus, Roxb. (CO), a herb commonly used as an ingredient for tonic drinks in southern China, was shown to increase the contractility and decrease the frequency of contraction in an isolated rat heart perfusion system. CO was found to inhibit Na+/K(+)-ATPase activities in rat heart sarcolemma, as well as in a purified enzyme from porcine cerebral cortex. CO also inhibited Ca(2+)-dependent ATPase in mouse heart homogenate and in mouse heart sarcoplasmic reticulum at a similar dose. These enzyme inhibitory actions provide a possible explanation for the positive inotropic and negative chronotropic actions of CO on the perfused rat heart. This study suggests the presence of ATPase inhibitory compounds in CO with specificities different from that of ouabain.

Inotropic and chronotropic actions of Ilex latifolia inhibition of adenosine-5 '-triphosphatases as a possible mechanism.

Ilex latifolia is widely used as an ingredient to prepare traditional beverage drinks in southern China. In fact, various Ilex species have been used in Chinese folk medicine to treat coronary heart diseases. The mode of action is believed to be mediated by their coronary vasodilative effects. In this study, the water extract of the leaves of Ilex latifolia (IK-TP) was shown to increase the contractility and decrease the frequency of contraction in an isolated rat heart perfusion system. IK-TP was found to inhibit Na+/K+-ATPase activities in rat heart sarcolemma, rat brain microsomes and a purified enzyme from porcine cerebral cortex. IK-TP also inhibited Ca2+-dependent ATPase at a similar dose. Following exposure of the isolated rat heart to IK-TP at a dose that produces pronounced cardiac effects, inhibition of Na+/K+-ATPase activity can be readily detected in the heart. This study suggests the presence of ATPase inhibitory compounds in Ilex latifolia with specificities different from that of ouabain.

Molecular cloning, characterization, and differential expression of a glucoamylase gene from the basidiomycetous fungus Lentinula edodes.

The complete nucleotide sequence of putative glucoamylase gene gla1 from the basidiomycetous fungus Lentinula edodes strain L54 is reported. The coding region of the genomic glucoamylase sequence, which is preceded by eukaryotic promoter elements CAAT and TATA, spans 2,076 bp. The gla1 gene sequence codes for a putative polypeptide of 571 amino acids and is interrupted by seven introns. The open reading frame sequence of the gla1 gene shows strong homology with those of other fungal glucoamylase genes and encodes a protein with an N-terminal catalytic domain and a C-terminal starch-binding domain. The similarity between the Gla1 protein and other fungal glucoamylases is from 45 to 61%, with the region of highest conservation found in catalytic domains and starch-binding domains. We compared the kinetics of glucoamylase activity and levels of gene expression in L. edodes strain L54 grown on different carbon sources (glucose, starch, cellulose, and potato extract) and in various developmental stages (mycelium growth, primordium appearance, and fruiting body formation). Quantitative reverse transcription PCR utilizing pairs of primers specific for gla1 gene expression shows that expression of gla1 was induced by starch and increased during the process of fruiting body formation, which indicates that glucoamylases may play an important role in the morphogenesis of the basidiomycetous fungus.

Cloning and characterization of two hydrophobin genes differentially expressed during fruit body development in Lentinula edodes.

Hydrophobins play important roles in morphogenesis and pathogenesis in fungi and fruit development in mushrooms. Two genes encoding hydrophobins (Le.hyd1 and Le.hyd2) were isolated during sequencing of random clones from a primordial cDNA library of Lentinula edodes. The nucleotide sequences of these two genes were determined. These two genes are 760 and 738 bp in length and the deduced amino acid sequences are homologous to various fungal hydrophobins with characteristic cysteine spacing. These hydrophobin genes are Class I hydrophobins judging by their conserved domains and hydropathy patterns. The transcript level of Le.hyd1 is high in primordium and that of Le.hyd2 is high in dikaryotic mycelial tissues. Poor expression of these two genes in monokaryotic parents indicates that these two genes are under mating-type regulation. We thus suggest that differential expression of these two L. edodes hydrophobins during fruit development may contribute to their distinct roles in fruiting of this mushroom.

Identification by RNA fingerprinting of genes differentially expressed during the development of the basidiomycete Lentinula edodes.

As part of an ongoing project to understand the molecular mechanisms of fruit body development in Lentinula edodes (Shiitake mushroom), RNA fingerprinting by arbitrarily primed PCR (RAP-PCR) was used to identify differentially expressed genes in RNA populations from four stages of L. edodes development vegetative mycelium, primordium, young fruit body and mature fruit body. From 30 RNA fingerprints, we cloned and sequenced 33 RAP fragments after their differential expression patterns had been verified by reverse Northern dot-blot hybridization. Thirteen RAP fragments show high sequence similarity to known gene products which are involved in (1) transport across the plasma membrane (drug efflux pump and sugar transporter); (2) cell cycle control (cyclin B); (3) signal transduction and transcriptional regulation (mitogen-activated protein kinase, Cdc39/Not1, PriA, Jun-D); (4) intracellular molecule trafficking (ubiquitin, plasma membrane proton ATPase, and alpha-adaptin); (5) mitochondrial biogenesis (mitochondrial processing peptidase beta-subunit, mitochondrial glycerol-3-phosphate dehydrogenase); and (6) intermediary metabolism (fructose 1,6 bisphosphatase). The transcript levels for plasma membrane proton ATPase and alpha-adaptin remained constant, whereas the other eleven genes were differentially expressed during L. edodes. development. The expression profiles of the genes suggest that transport across the plasma membrane is important in the mycelial stage. Specific signal transduction and transcriptional controls may play important roles during the initiation of primordia and the formation of young fruiting bodies. When the mushroom matures, expression of genes involved in metabolic pathways becomes prominent. The isolation of these genes indicates their involvement in homobasidiomycete development and suggests new directions for molecular studies on mechanisms of mushroom development.

Characterization, molecular cloning, and differential expression analysis of laccase genes from the edible mushroom Lentinula edodes.

The effect of different substrates and various developmental stages (mycelium growth, primordium appearance, and fruiting-body formation) on laccase production in the edible mushroom Lentinula edodes was studied. The cap of the mature mushroom showed the highest laccase activity, and laccase activity was not stimulated by some well-known laccase inducers or sawdust. For our molecular studies, two genomic DNA sequences, representing allelic variants of the L. edodes lac1 gene, were isolated, and DNA sequence analysis demonstrated that lac1 encodes a putative polypeptide of 526 amino acids which is interrupted by 13 introns. The two allelic genes differ at 95 nucleotides, which results in seven amino acid differences in the encoded protein. The copper-binding domains found in other laccase enzymes are conserved in the L. edodes Lac1 proteins. A fragment of a second laccase gene (lac2) was also isolated, and competitive PCR showed that expression of lac1 and lac2 genes was different under various conditions. Our results suggest that laccases may play a role in the morphogenesis of the mushroom. To our knowledge, this is the first report on the cloning of genes involved in lignocellulose degradation in this economically important edible fungus.

Identification and molecular characterization of Charybdis feriatus tropomyosin, the major crab allergen.

BACKGROUND: Crab sensitivity is one of the most common seafood allergies. However, to date, there has been no report on the molecular characterization of crab allergens and no comparative analysis with other seafood allergens. OBJECTIVE: This study was undertaken to clone, identify, and determine the primary structure of a major IgE-reactive molecule in crab. METHODS: We constructed an expression cDNA library from a common crab, Charybdis feriatus. This library was then screened with the use of sera from subjects with a well-documented history of type I hypersensitivity reactions upon ingestion of crab. An IgE-reactive clone was chosen and subcloned into plasmids for nucleotide sequence determination and expression in Escherichia coli. RESULTS: We identified a 1-kb cDNA designated as Cha f 1. Expression of Cha f 1 produces a 34-kd recombinant protein reactive to the IgE antibodies from patients with crab allergies but not from control subjects. Cha f 1 has an opening reading frame of 264 amino acids and demonstrates marked homology to the shrimp tropomyosin Met e 1. Absorption of allergic sera with Cha f I removes IgE reactivity to crab extract. Moreover, absorption of allergic sera with recombinant shrimp Met e 1 tropomyosin removes IgE reactivity to Cha f 1. CONCLUSIONS: This 34-kd protein, designated as Cha f 1, is the first identified major allergen of crab. Nucleotide and amino acid comparison shows that this protein is the crab tropomyosin. The molecular basis of shrimp and crab allergy is readily demonstrated at the nucleotide and amino acid level.

Identification of genes differentially expressed in nasopharyngeal carcinoma by messenger RNA differential display.

We have applied the mRNA differential display method to compare and analyze mRNAs prepared from five normal nasopharyngeal epithelial cell cultures and five nasopharyngeal carcinoma cell lines. A total of 24 differential display experiments was performed using different combinations of PCR primers. Sixty-nine cDNA fragments differentially expressed in either normal or malignant nasopharyngeal epithelial cells were identified. Subsequent cloning and sequencing of these differentially expressed cDNA fragments resulted in the identification of seventeen distinct sequences. Seven of these sequences were shown to be novel cDNA sequences not previously reported. Ten of the remaining cDNA fragments showed sequence homology to previously reported genes. Differential expression of four of these seventeen cDNA fragments in normal nasopharyngeal epithelial cells was confirmed by reverse Northern hybridization. One of these cloned cDNA fragments is a novel cDNA sequence while the other three matched to previously reported cDNA sequences involved in cell growth and migration. Homologous sequences identified to be differentially expressed in normal nasopharyngeal epithelial cells in this study are: human 26 kDa cell surface protein (TAPA-1) mRNA, NF-E2 like basic leucine zipper transcriptional activator and the human bullous pemphigoid antigen. The mRNA differential display is a useful tool to identify candidate genes involved in the pathogenesis of nasopharyngeal carcinoma.

Cloning and characterization of the gene encoding beta subunit of mitochondrial processing peptidase from the basidiomycete Lentinula edodes.

We have isolated the gene encoding the beta subunit of mitochondrial processing peptidase (beta-MPP) from the shiitake mushroom Lentinula edodes. It is a nuclear gene with two small introns. Comparison with known beta-MPP genes revealed that the L. edodes gene is most closely related with that from Neurospora crassa, with 60.8% identities and 87% similarity in the amino-acid sequences. The deduced L. edodes beta-MPP peptide sequence contains the inverse zinc-binding motif (H-X-E-H) that has been found in a large family of zinc-binding metalloproteinases including bacterial proteinases, insulin degrading enzymes and beta-MPPs. The two histidines are thought to contribute two of the three residues for zinc binding. The expression of L. edodes beta-MPP is higher during the development of the fruiting bodies, suggesting that higher mitochondrial activities may be required to meet the energy demand in the rapid growth of the fruiting bodies.

IgE reactivity against a cross-reactive allergen in crustacea and mollusca: evidence for tropomyosin as the common allergen.

BACKGROUND: Although cross-reactivity between mollusks and other crustaceans in shrimp-sensitive subjects has been reported, the mechanism of this allergenic cross-reactivity has not been studied in detail. OBJECTIVE: To investigate this cross-reactivity in vitro, we have taken advantage of a complementary DNA that expresses tropomyosin, the immunodominant shrimp allergen. METHODS: Serum IgE from nine patients with known anaphylaxis to shrimp and five normal volunteers were analyzed by immunoblotting against 13 distinct crustaceans and mollusks. As additional antigens, muscle preparations were isolated from grasshopper, cockroach, fruit fly, chicken, and mouse. RESULTS: Sera from all patients, but not control subjects, reacted specifically with a 38 kd protein in all crustaceans and mollusks studied. In addition, 8 of 9 sera from patients, but from none of the normal control subjects, recognized proteins of various other molecular weights among the mollusk extracts studied. The 38 kd protein was identified as tropomyosin and was shown to share immunodominant epitopes among all species of crustaceans and mollusks tested by specific absorption studies. Moreover, sera from all nine subjects with shrimp allergy demonstrated IgE reactivities against grasshopper, cockroach, and fruit fly but not chicken or murine muscle. CONCLUSION: The allergic epitopes on tropomyosin are conserved among invertebrates including not only shellfish but also insects. This latter observation suggests that persons sensitive to shrimp should undergo further study for potential cross-reactive inhalant or ingested insect sensitivity.