Somatic embryogenesis of tissue cultures of Papaver somniferum and Papaver orientale and its relationship to alkaloid and lipid metabolism

Transfer from complete to 2,4-D free Gamborg's B5-medium efficiently induced somatic embryogenesis in Papaver tissue cultures (P. somniferum and P. orientale). Embryogenesis was preceded by a strong temporary accumulation of triacylglycerols. In both tissue cultures large amounts of sanguinarine type alkaloids were present, which disappeared during regeneration in the P. orientale cultures but persisted in the P. somniferum cultures. In the P. somniferum cultures protopine and morphine type alkaloids (morphine, codeine, thebaine) appeared about 45 days after exchanging the medium. Thebaine was the main alkaloid in the P. somniferum embryoids accumulating up to 0.2 % of dry weight.     

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.     

Inactivation of aphidicolin by plant cells

Plant cells are endowed with an aphidicolin inactivating activity. Data on cultured cells show that the rate of inactivation depends on the cell type, Daucus carota cells being the most effective among the other tested materials (Oryza sativa and Nicotiana plumbaginifolia). Also germinating seedling of Haplopappus gracilis and of Citrullus vulgaris inactivate aphidicolin. Inactivation, which may lead to unexpected results when a prolonged incubation with the drug is required, as in the case of the induction of synchrony of the cell cycle by aphidicolin, can be controlled by appropriately choosing the experimental conditions.     

Agarose plating and a bead type culture technique enable and stimulate development of protoplast-derived colonies in a number of plant species

Two novel techniques improve division and colony formation from protoplasts:

1. Plating in agarose stimulates colony formation of protoplasts from a wide range of species. Protoplasts from Nicotiana tabacum developed to colonies from lower initial population densities in agarose than in agar or liquid. Protoplasts from Hyoscyamus muticus which do not divide in agar divided and formed colonies in agarose at higher efficiencies than in liquid medium.
2. Culture of gel embedded protoplasts in large volumes of liquid medium on a gyrotatory shaker (‘bead culture’) further improved plating efficiencies in some species (e.g. Lycopersicon esculentum and Crepis capillaris) and enabled sustained proliferation of protoplasts which had not previously developed beyond the few cell colony stage (Brassica rapa and a mutator gene variety of Petunia hybrida).

The combination of ‘agarose plating’ and ‘bead culture’ dramatically improved plating efficiencies of protoplasts in all species tested.     

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.     

Backbone resonance assignments of the α sub-domain of Brevibacillus thermoruber Lon protease

Lon is an ATPases associated with diverse cellular activities protease and belongs to a unique group that binds DNA. The α sub-domain of Lon protease is responsible for DNA-binding, but the structural information for its DNA-recognition mode is still limited. Here, we report ¹H, ¹⁵N and ¹³C backbone assignment for the α sub-domain from Brevibacillus thermoruber Lon protease as the basis for the elucidation of its structure and interactions with DNA, necessary for understanding the allosteric regulatory mechanism of the enzymatic function.     

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.     

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).     

Roles of glycosaminoglycans and glycanmimetics in tumor progression and metastasis

Various tumor cells exhibit structural alterations in the sulfated modifications to glycosaminoglycans (GAGs). The altered expression of chondroitin sulfate (CS) and heparan sulfate (HS) on the surfaces of tumor cells is known to play a key role in malignant transformation and tumor metastasis. The receptor molecule for the CS chains containing E-disaccharide units (CS-E) expressed on Lewis lung carcinoma (LLC) cells was recently revealed to be Receptor for Advanced Glycation End-products (RAGE). RAGE is also involved in the development of various pathological conditions including aging, infection, pulmonary fibrosis, diabetes, and Alzheimer’s disease, by binding to a wide range of ligands. RAGE binds strongly not only to CS-E, but also to HS-expressing LLC cells. Recombinant RAGE bound CS-E and HS with high affinity. Furthermore, in a mouse model, the colonization of the lungs by LLC cells was inhibited by intravenously injected CS-E, an anti-CS-E antibody, or an anti-RAGE antibody. These findings demonstrated that RAGE is at least one of the critical receptors for CS and HS chains expressed on the tumor cell surface and is involved in experimental lung metastasis, and also that CS/HS and RAGE are potential molecular targets for the treatment of pulmonary metastasis. We, hence, reviewed these findings and also several chemically synthesized small GAGmimetics that exhibit potent anti-metastatic and/or anti-tumor activities.     

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.     

Somatic embryogenesis in cell cultures of Glycine species

This report describes the development of procedures for the production of somatic embryos in cell cultures of Glycine species including soybean. The conditions for callus induction and initiation of rapidly growing cell suspension cultures were defined. Methods for inducing embryogenesis were tested on 16 lines of several Glycine species and cultivars of soybean. The SB-26 Culture of a G. soja gave the best results and was used in the experiments. Embryogenesis required the presence of picloram or 2,4-D. AMO 1618, CCC, PP-333 and Ancymidol enhanced the embryogenesis frequency. Plants of the G. soja (SB-26) were grown to maturity from seed-derived shoot tips. Characteristics of the plants are discussed.     

siMBa—a simple graphical user interface for the Bayesian phylogenetic inference program MrBayes

MrBayes is a program that uses a Bayesian framework for inferring phylogenetic relationships. As MrBayes is a command-line-driven program, users acquainted to programs with graphical user interfaces will not find it easy to operate, especially as it requires a complex input format for the data to be analysed. We thus developed siMBa (simple MrBayes), a simple graphical user interface for MrBayes. This tool gives the user interactive control over most of the parameters and also facilitates the input of a multiple sequence alignment, as any widely used format can be used. siMBa is coded in Perl using the Tk module. Executables are provided for Windows, Linux, and Macintosh. The Perl codes, along with executables for different operating system, are freely available to download from [http://www.thines-lab.senckenberg.de/simba].     

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.     

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.