Identification and characterization of SHI family genes from Brassica rapa L. ssp. pekinensis

SHI (short internodes) is a negative regulator of gibberellin-induced cell elongation. Extensive searches in the Brassica rapa genome allowed for the prediction of at least six different SHI-related genes on six chromosomes in the genome. Genome structural examination revealed that these genes had one intron each in their corresponding open reading frames. Protein structure comparisons using the CLUSTALW program and based on alignments of all BrSRS (B. r apa SHI-related sequence) proteins revealed broad conservation of the RING finger-like zinc finger and IGGH motifs. According to the phylogenetic relationship based on deduced amino acid sequences, the six BrSRS proteins were most closely related to Arabidopsis SRS (AtSRS) proteins; however, BrSRS proteins were dispersed in the phylogenetic tree. Semi-quantitative RT-PCR analysis indicated that the six BrSRS genes exhibited different expression patterns in various tissues and responded differently to growth phytohormones. The differences among the six BrSRS genes with respect to gene structure and expression pattern suggest that these genes may play diverse physiological roles in the developmental process of B. rapa.     

A proof of the DBRF-MEGN method, an algorithm for deducing minimum equivalent gene networks

Background

We previously developed the DBRF-MEGN (difference-based regulation finding-minimum equivalent gene network) method, which deduces the most parsimonious signed directed graphs (SDGs) consistent with expression profiles of single-gene deletion mutants. However, until the present study, we have not presented the details of the method's algorithm or a proof of the algorithm.

Results

We describe in detail the algorithm of the DBRF-MEGN method and prove that the algorithm deduces all of the exact solutions of the most parsimonious SDGs consistent with expression profiles of gene deletion mutants.

Conclusions

The DBRF-MEGN method provides all of the exact solutions of the most parsimonious SDGs consistent with expression profiles of gene deletion mutants.     

Coccidioides posadasii infection alters the expression of pulmonary surfactant proteins (SP)-A and SP-D

Background

Patients with asthma demonstrate circadian variations in the airway inflammation and lung function. Pinealectomy reduces the total inflammatory cell number in the asthmatic rat lung. We hypothesize that melatonin, a circadian rhythm regulator, may modulate the circadian inflammatory variations in asthma by stimulating the chemotaxins expression in the lung epithelial cell.

Methods

Lung epithelial cells (A549) were stimulated with melatonin in the presence or absence of TNF-α(100 ng/ml). RANTES (Regulated on Activation Normal T-cells Expressed and Secreted) and eotaxin expression were measured using ELISA and real-time RT-PCR, eosinophil chemotactic activity (ECA) released by A549 was measured by eosinophil chemotaxis assay.

Results

TNF-α increased the expression of RANTES (307.84 ± 33.56 versus 207.64 ± 31.27 pg/ml of control, p = 0.025) and eotaxin (108.97 ± 10.87 versus 54.00 ± 5.29 pg/ml of control, p = 0.041). Melatonin(10^-10 to 10^-6M) alone didn't change the expression of RNATES (204.97 ± 32.56 pg/ml) and eotaxin (55.28 ± 6.71 pg/ml). However, In the presence of TNF-α (100 ng/ml), melatonin promoted RANTES (410.88 ± 52.03, 483.60 ± 55.37, 559.92 ± 75.70, 688.42 ± 95.32, 766.39 ± 101.53 pg/ml, treated with 10^-10, 10^-9, 10^-8, 10^-7,10^-6M melatonin, respectively) and eotaxin (151.95 ± 13.88, 238.79 ± 16.81, 361.62 ± 36.91, 393.66 ± 44.89, 494.34 ± 100.95 pg/ml, treated with 10^-10, 10^-9, 10^-8, 10^-7, 10^-6M melatonin, respectively) expression in a dose dependent manner in A549 cells (compared with TNF-α alone, P < 0.05). The increased release of RANTES and eotaxin in A549 cells by above treatment were further confirmed by both real-time RT-PCR and the ECA assay.

Conclusion

Taken together, our results suggested that melatonin might synergize with pro-inflammatory cytokines to modulate the asthma airway inflammation through promoting the expression of chemotaxins in lung epithelial cell.     

Genome-scale identification of Caenorhabditis elegans regulatory elements by tiling-array mapping of DNase I hypersensitive sites

Background

High throughput techniques have generated a huge set of biological data, which are deposited in various databases. Efficient exploitation of these databases is often hampered by a lack of appropriate tools, which allow easy and reliable identification of genes that miss functional characterization but are correlated with specific biological conditions (e.g. organotypic expression).

Results

We have developed a simple algorithm (DGSA = Database-dependent Gene Selection and Analysis) to identify genes with unknown functions involved in organ development concentrating on the heart. Using our approach, we identified a large number of yet uncharacterized genes, which are expressed during heart development. An initial functional characterization of genes by loss-of-function analysis employing morpholino injections into zebrafish embryos disclosed severe developmental defects indicating a decisive function of selected genes for developmental processes.

Conclusion

We conclude that DGSA is a versatile tool for database mining allowing efficient selection of uncharacterized genes for functional analysis.     

Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

Background

Combination of CHD (chromo-helicase-DNA binding protein)-specific polymerase chain reaction (PCR) with electrophoresis (PCR/electrophoresis) is the most common avian molecular sexing technique but it is lab-intensive and gel-required. Gender determination often fails when the difference in length between the PCR products of CHD-Z and CHD-W genes is too short to be resolved.

Results

Here, we are the first to introduce a PCR-melting curve analysis (PCR/MCA) to identify the gender of birds by genomic DNA, which is gel-free, quick, and inexpensive. Spilornis cheela hoya (S. c. hoya) and Pycnonotus sinensis (P. sinensis) were used to illustrate this novel molecular sexing technique. The difference in the length of CHD genes in S. c. hoya and P. sinensis is 13-, and 52-bp, respectively. Using Griffiths' P2/P8 primers, molecular sexing failed both in PCR/electrophoresis of S. c. hoya and in PCR/MCA of S. c. hoya and P. sinensis. In contrast, we redesigned sex-specific primers to yield 185- and 112-bp PCR products for the CHD-Z and CHD-W genes of S. c. hoya, respectively, using PCR/MCA. Using this specific primer set, at least 13 samples of S. c. hoya were examined simultaneously and the Tm peaks of CHD-Z and CHD-W PCR products were distinguished.

Conclusion

In this study, we introduced a high-throughput avian molecular sexing technique and successfully applied it to two species. This new method holds a great potential for use in high throughput sexing of other avian species, as well.     

A special issue on DNA damage response and genome stability

A novel, cancer-fighting function was recently discovered for Smad ubiquitination regulatory factor 2 (Smurf2).     

Dramatic inhibition of osteoclast sealing ring formation and bone resorption in vitro by a WASP-peptide containing pTyr294 amino acid

Wiskott Aldrich Syndromeprotein (WASP) has a unique regulatory role in sealing ring formation and bone resorption in osteoclasts. Here, using the TAT-transduction method, we show the possible role of WASP domain(s) in sealing ring formation and bone resorption. Transduction of TAT-fused full-length WASP peptide induced Arp2/3 complex formation, F-actin content, sealing ring formation and bone resorption. Transduction of WASP peptides containing basic, verpolin-central, pTyr294, and proline-rich regions inhibited the processes listed above at various levels. The ability to resorb bone by WASP peptides containing basic, verpolin-central, and proline-rich regions was reduced and the resorbed area matched the size of the sealing ring. However, osteoclasts transduced with WASP peptide containing pTyr294aa demonstrated the following: a) a considerable decrease in the interaction and phosphorylation of c-Src with endogenous WASP; b) total loss of sealing ring-like structures; c) formation of actin-rich patches at the peripheral edge that contains filopodia-like projections; d) reduced capacity for bone resorption in vitro. These findings suggest that modulation of phosphorylation state of pTyr294aa assists in integrating multiple signaling molecule and pathways that partake in the assembly of sealing ring.     

The COG database: an updated version includes eukaryotes

Background

The availability of multiple, essentially complete genome sequences of prokaryotes and eukaryotes spurred both the demand and the opportunity for the construction of an evolutionary classification of genes from these genomes. Such a classification system based on orthologous relationships between genes appears to be a natural framework for comparative genomics and should facilitate both functional annotation of genomes and large-scale evolutionary studies.

Results

We describe here a major update of the previously developed system for delineation of Clusters of Orthologous Groups of proteins (COGs) from the sequenced genomes of prokaryotes and unicellular eukaryotes and the construction of clusters of predicted orthologs for 7 eukaryotic genomes, which we named KOGs after eukaryotic orthologous groups. The COG collection currently consists of 138,458 proteins, which form 4873 COGs and comprise 75% of the 185,505 (predicted) proteins encoded in 66 genomes of unicellular organisms. The eukaryotic orthologous groups (KOGs) include proteins from 7 eukaryotic genomes: three animals (the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and Homo sapiens), one plant, Arabidopsis thaliana, two fungi (Saccharomyces cerevisiae and Schizosaccharomyces pombe), and the intracellular microsporidian parasite Encephalitozoon cuniculi. The current KOG set consists of 4852 clusters of orthologs, which include 59,838 proteins, or ~54% of the analyzed eukaryotic 110,655 gene products. Compared to the coverage of the prokaryotic genomes with COGs, a considerably smaller fraction of eukaryotic genes could be included into the KOGs; addition of new eukaryotic genomes is expected to result in substantial increase in the coverage of eukaryotic genomes with KOGs. Examination of the phyletic patterns of KOGs reveals a conserved core represented in all analyzed species and consisting of ~20% of the KOG set. This conserved portion of the KOG set is much greater than the ubiquitous portion of the COG set (~1% of the COGs). In part, this difference is probably due to the small number of included eukaryotic genomes, but it could also reflect the relative compactness of eukaryotes as a clade and the greater evolutionary stability of eukaryotic genomes.

Conclusion

The updated collection of orthologous protein sets for prokaryotes and eukaryotes is expected to be a useful platform for functional annotation of newly sequenced genomes, including those of complex eukaryotes, and genome-wide evolutionary studies.     

CLE polypeptide signaling gene expression in Arabidopsis embryos

The CLAVATA3 (CLV3)/ESR-related (CLE) family of small polypeptides mediate intercellular signaling events in plants. The biological roles of several CLE family members have been characterized, but the function of the majority still remains elusive. We recently performed a systematic expression analysis of 23 Arabidopsis CLE genes to gain insight into the developmental processes they may potentially regulate during vegetative and reproductive growth. Our study revealed that each Arabidopsis tissue expresses one or more CLE genes, suggesting that they might play roles in many developmental and/or physiological processes. Here we determined the expression patterns of nine Arabidopsis CLE gene promoters in mature embryos and compared them to the known expression patterns in seedlings. We found that more than half of these CLE genes have similar expression profiles at the embryo and seedling stages, whereas the rest differ dramatically. The implications of these findings in understanding the biological processes controlled by these CLE genes are discussed.Key words: arabidopsis, CLE, embryo, polypeptide, signalingThe CLE genes encode small, secreted polypeptides characterized by a highly conserved 14 amino-acid region at their carboxyl termini called the CLE domain.¹ To date 32 family members have been identified in Arabidopsis, yet only three have been assigned functions: CLV3, CLE40 and CLE41 have been implicated in stem cell homeostasis in shoot, root and vascular meristems, respectively.^2–5 Overexpression studies indicated that CLE genes may regulate additional biological processes as diverse as root and shoot growth, phyllotaxis, apical dominance and leaf shape and size control.^6,7 This hypothesis is consistent with our recent expression analysis of Arabidopsis A-type CLE genes,⁸ in which we found that all examined tissues expressed one or more CLE genes, in overlapping patterns. Each CLE promoter exhibited a highly distinct and specific activity profile, and many showed complex expression dynamics during vegetative and reproductive growth.Consistent with their roles in meristem maintenance, CLV3 and CLE40 are expressed early in embryogenesis when meristem initiation and organization take place.^3,5 Yet there are no other reports of CLE gene expression in Arabidopsis embryos, and therefore it is not known to what extent this family of small peptides regulates intercellular signaling events during embryogenesis. We addressed this question by analyzing the expression patterns of selected CLE promoters in mature embryos and compared them with those in 11-day-old seedlings. We chose nine CLE genes whose promoters are active in different tissues of the seedling.⁸ Transgenic dried seeds carrying a single CLE promoter sequence driving the expression of the uidA reporter gene were imbibed in water for four days, the embryos dissected out of their seed coats, and beta-glucuronidase (GUS) reporter assays performed.⁹ Stained embryos were cleared with chloral hydrate¹⁰ and visualized using a Zeiss Axiophot microscope.Five of the CLE genes analyzed showed similar promoter expression patterns in mature embryos and in seedlings. In embryos, the CLE11, 13, 16 and 17 promoters drove GUS activity in specific patterns in the root. CLE11 and CLE13 promoter activity was detected in the root cap and root apical meristem (Fig. 1A and B), CLE16 promoter activity was observed in the root cap and above the root apical meristem (Fig. 1C), and CLE17 promoter activity was seen weakly in the root apical meristem (Fig. 1D). Each of these CLE genes exhibited a similar expression pattern in seedling roots.⁸ CLE17 was additionally expressed in the embryo shoot apex and at the cotyledon margins (Fig. 1D). Similarly, in seedlings CLE17 was expressed in the vegetative shoot apex, and at the margins of the cotyledons and fully expanded leaves.⁸ In embryos, CLE27 promoter activity was strong in the hypocotyl, as well as in the medial region of the cotyledons along the main vein (Fig. 1E). In seedlings, CLE27 was strongly expressed in the hypocotyl and exhibited patchy expression in both cotyledons and leaves.⁸ Our analysis reveals that the expression of these CLE genes is established early during development and remains constant at later stages, suggesting that they may perform the same function throughout the Arabidopsis life cycle.Open in a separate windowFigure 1GUS reporter activity driven by the promoters of (A) CLE11, (B) CLE13, (C) CLE16, (D) CLE17, (E) CLE27, (F) CLE1, (G) CLE12, (H) CLE18 and (I) CLE25 in mature Arabidopsis embryos. Arrowhead indicates GUS activity in the root cap and the arrow indicates GUS activity in the root apical meristem. Scale bar, 100 µm.Remarkably, the other four CLE promoters drove embryo expression patterns that were strongly divergent from what was observed in seedlings. We found that the CLE1 promoter was active in the embryo throughout the hypocotyl and in the central region of the cotyledons (Fig. 1F), but was observed in seedlings solely in the vasculature of fully differentiated roots and at the root tips.⁸ CLE12 promoter activity in embryos was observed throughout the hypocotyl and the cotyledons (Fig. 1G), whereas in seedlings it was detected weakly in the leaf vasculature and more strongly in the root vasculature.⁸ In contrast, the CLE18 and CLE25 promoters did not drive reporter activity in mature embryos (Fig. 1H and I), despite being broadly and strongly expressed in seedlings.⁸These four CLE gene promoters show dynamic shifts in their activity between different developmental stages. From our data we infer that CLE1 activity in hypocotyls and cotyledons is required solely during embryogenesis, and that the gene then acquires a distinct function in post-embryonic root development. Similarly CLE12 appears to acquire a post-embryonic function in the root vasculature, and its broad activity in the embryonic leaves becomes restricted to the leaf vasculature following germination. Finally, the absence of CLE18 and CLE25 promoter activity in mature embryos suggests that they may be dispensable for embryo formation, and might either specifically regulate post-embryonic signaling events in certain tissues or be involved in mediating responses to environmental stimuli to which embryos are not subjected. Alternatively, they may be expressed earlier during embryogenesis and become repressed during seed dormancy.Our spatio-temporal expression analysis of a small group of CLE genes in mature embryos and seedlings indicates that the majority of these signaling molecules exert their roles beginning early in development, potentially contributing to tissue patterning and organization. Yet whereas some appear to contribute to the same biological processes throughout the plant life cycle, others seem to function in different tissues at different developmental stages. In addition, each CLE promoter studied here is active in vegetative and/or reproductive tissues that are not present in embryos, such as trichomes (CLE16 and CLE17) and style (CLE1).⁸ This observation suggests that CLE genes are widely recruited to new tissue-specific signaling functions during the course of plant development.     

The isolation and identification of some toxic constituents ofAspergillus wentii wehmer

Extraction of a maize culture of a toxinogenic strain ofA. wentii led to the isolation and characterization of three anthraquinones, three bianthrones, a xanthone and a benzophenone. The structures were derived from spectroscopic data and were supported by chemical degradation. Of these, emodin, 1,6-di-0-methylemodin, 5-0-methylsulochrine and 1,3-di-0-methylemodin bianthrone were mildly toxic to ducklings.     

Myofibril-Inducing RNA (MIR) is essential for tropomyosin expression and myofibrillogenesis in axolotl hearts

The Mexican axolotl, Ambystoma mexicanum, carries the naturally-occurring recessive mutant gene 'c' that results in a failure of homozygous (c/c) embryos to form hearts that beat because of an absence of organized myofibrils. Our previous studies have shown that a noncoding RNA, Myofibril-Inducing RNA (MIR), is capable of promoting myofibrillogenesis and heart beating in the mutant (c/c) axolotls. The present study demonstrates that the MIR gene is essential for tropomyosin (TM) expression in axolotl hearts during development. Gene expression studies show that mRNA expression of various tropomyosin isoforms in untreated mutant hearts and in normal hearts knocked down with double-stranded MIR (dsMIR) are similar to untreated normal. However, at the protein level, selected tropomyosin isoforms are significantly reduced in mutant and dsMIR treated normal hearts. These results suggest that MIR is involved in controlling the translation or post-translation of various TM isoforms and subsequently of regulating cardiac contractility.     

Development of GEBRET: a web-based analysis tool for retroelements in primate genomes

Retroelements play important roles in primate evolution. Specifically, human endogenous retroviruses (HERVs) and Alu elements are primate-specific retroelements. In addition, SVA elements belong to the youngest family of hominid non-long terminal repeat (LTR) retrotransposons. Retroelements can affect adjacent gene expression, supplying cis-regulatory elements, splice sites, and poly-A signals. We developed a database, GEnome-wide Browser for RETroelement (GEBRET, http://neobio.cs.pusan.ac.kr/~gebre/), for comparing the distribution of primate-specific retroelements and adjacent genes. GEBRET database components include 47,381 HERVs, 53,924 Alus and 4639 SVAs in five primate genomes of human, chimpanzee, orangutan, rhesus macaque, and marmoset. Host genes located upstream of a retroelement were also visualized and classified as five categories (0.0, 0.5, 1.0, 2.0, and 3.0Kb). Our results suggest that retroelements preferentially integrate into the distal promoter region relative to the core promoter region. GEBRET database is designed to investigate the distribution of retroelements (HERVs, Alus and SVAs) in the primate genomes that have been sequenced. Our software will be useful in the field to study the impact of retroelements on primate genome evolution.