Random sheared fosmid library as a new genomic tool to accelerate complete finishing of rice (Oryza sativa spp. Nipponbare) genome sequence: sequencing of gap-specific fosmid clones uncovers new euchromatic portions of the genome

The International Rice Genome Sequencing Project has recently announced the high-quality finished sequence that covers nearly 95% of the japonica rice genome representing 370 Mbp. Nevertheless, the current physical map of japonica rice contains 62 physical gaps corresponding to approximately 5% of the genome, that have not been identified/represented in the comprehensive array of publicly available BAC, PAC and other genomic library resources. Without finishing these gaps, it is impossible to identify the complete complement of genes encoded by rice genome and will also leave us ignorant of some 5% of the genome and its unknown functions. In this article, we report the construction and characterization of a tenfold redundant, 40 kbp insert fosmid library generated by random mechanical shearing. We demonstrated its utility in refining the physical map of rice by identifying and in silico mapping 22 gap-specific fosmid clones with particular emphasis on chromosomes 1, 2, 6, 7, 8, 9 and 10. Further sequencing of 12 of the gap-specific fosmid clones uncovered unique rice genome sequence that was not previously reported in the finished IRGSP sequence and emphasizes the need to complete finishing of the rice genome. Fosmid library reported here is publicly available from our web site     

Hyaluronic acid production in Bacillus subtilis

The hasA gene from Streptococcus equisimilis, which encodes the enzyme hyaluronan synthase, has been expressed in Bacillus subtilis, resulting in the production of hyaluronic acid (HA) in the 1-MDa range. Artificial operons were assembled and tested, all of which contain the hasA gene along with one or more genes encoding enzymes involved in the synthesis of the UDP-precursor sugars that are required for HA synthesis. It was determined that the production of UDP-glucuronic acid is limiting in B. subtilis and that overexpressing the hasA gene along with the endogenous tuaD gene is sufficient for high-level production of HA. In addition, the B. subtilis-derived material was shown to be secreted and of high quality, comparable to commercially available sources of HA.     

Novel stent for dacryocystorhinostomy (DCR) and other surgical applications

In summary, this project proposes to explore the feasibility of a new temporary implant and surgical technique to improve the DCR procedure. This is a significant improvement over current DCR practice for the following primary reasons: (1) it eliminates the risk of injury to both the eye and upper lacrimal system; (2) it permits tear flow into the nose immediately following surgery, potentially 6 months earlier than current practice and procedure, which frequently does not re-establish normal tear flow until stents are removed. Plans have been made to implant stents of different sizes and proportions into cadavers in order to test implantation and removal of the stent via external and intranasal approaches.     

Differential inhibition of inducible T cell cytokine secretion by potent iron chelators

Effector functions and proliferation of T helper (Th) cells are influenced by cytokines in the environment. Th1 cells respond to a synergistic effect of interleukin-12 (IL-12) and interleukin-18 (IL-18) to secrete interferon-gamma (IFN-gamma). In contrast, Th2 cells respond to interleukin-4 (IL-4) to secrete IL-4, interleukin-13 (IL-13), interleukin-5 (IL-5), and interleukin-10 (IL-10). The authors were interested in identifying nonpeptide inhibitors of the Th1 response selective for the IL-12/IL-18-mediated secretion of IFN-gamma while leaving the IL-4-mediated Th2 cytokine secretion relatively intact. The authors established a screening protocol using human peripheral blood mononuclear cells (PBMCs) and identified the hydrazino anthranilate compound 1 as a potent inhibitor of IL-12/IL-18-mediated IFN-gamma secretion from CD3(+) cells with an IC(50) around 200 nM. The inhibitor was specific because it had virtually no effect on IL-4-mediated IL-13 release from the same population of cells. Further work established that compound 1 was a potent intracellular iron chelator that inhibited both IL-12/IL-18- and IL-4-mediated T cell proliferation. Iron chelation affects multiple cellular pathways in T cells. Thus, the IL-12/IL-18-mediated proliferation and IFN-gamma secretion are very sensitive to intracellular iron concentration. However, the IL-4-mediated IL-13 secretion does not correlate with proliferation and is partially resistant to potent iron chelation.     

2,5-Diketopiperazines as potent and selective oxytocin antagonists 1: Identification, stereochemistry and initial SAR

This paper covers efforts to discover orally active potent and selective oxytocin antagonists. Screening pooled libraries identified a novel series of 2,5-diketopiperazine derivatives with antagonist activity at the human oxytocin receptor. We report the initial structure-activity relationship investigations and the determination of the stereochemistry of the most potent compounds.     

Novel triazole based inhibitors of Ras farnesyl transferase

A novel series of potent inhibitors of Ras farnesyl transferase possessing a 1,2,4-triazole pharmacophore is described. These inhibitors were discovered from a parallel synthesis effort and were subsequently optimized to in vitro IC₅₀ value of less than 1 nM.     

Rodent malaria parasites Plasmodium chabaudi and P. vinckei do not increase their rates of gametocytogenesis in response to mosquito probing

Several vector-borne infectious agents facultatively alter their life history strategies in response to local vector densities. Some evidence suggests that malaria parasites invest more heavily in transmission stage production (gametocytogenesis) when vectors are present. Such a strategy could rapidly increase malaria transmission rates, particularly when adult mosquitoes begin to appear after dry seasons. However, in contrast to a recent experiment with a rodent malaria (Plasmodium chabaudi), we found no change in gametocytogenesis in either P. chabaudi or in another rodent malaria, P. vinckei, when their mouse hosts were exposed to mosquitoes. Positive results in the earlier study may have been because mosquito-feeding caused anaemia in hosts, a known promoter of gametocytogenesis. The substantial evidence that malaria and a variety of other parasites facultatively alter transmission strategies in response to a variety of environmental influences makes our results surprising.     

Construction of a bacterial artificial chromosome library from the spikemoss Selaginella moellendorffii: a new resource for plant comparative genomics

Background

The lycophytes are an ancient lineage of vascular plants that diverged from the seed plant lineage about 400 Myr ago. Although the lycophytes occupy an important phylogenetic position for understanding the evolution of plants and their genomes, no genomic resources exist for this group of plants.

Results

Here we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from the lycophyte Selaginella moellendorffii. Based on cell flow cytometry, this species has the smallest genome size among the different lycophytes tested, including Huperzia lucidula, Diphaiastrum digita, Isoetes engelmanii and S. kraussiana. The arrayed BAC library consists of 9126 clones; the average insert size is estimated to be 122 kb. Inserts of chloroplast origin account for 2.3% of the clones. The BAC library contains an estimated ten genome-equivalents based on DNA hybridizations using five single-copy and two duplicated S. moellendorffii genes as probes.

Conclusion

The S. moellenforffii BAC library, the first to be constructed from a lycophyte, will be useful to the scientific community as a resource for comparative plant genomics and evolution.     

Proteome analysis of early somatic embryogenesis in Picea glauca

Forestry is a valuable natural resource for many countries. Rapid production of large quantities of genetically improved and uniform seedlings for restocking harvested lands is a key component of sustainable forest management programs. Clonal propagation through somatic embryogenesis has the potential to meet this need in conifers and can offer the added benefit of ensuring consistent seedling quality. Although in commercial use, mass production of conifers through somatic embryogenesis is relatively new and there are numerous biological unknowns regarding this complex developmental pathway. To aid in unravelling the embryo developmental process, two-dimensional electrophoresis was employed to quantitatively assess the expression levels of proteins across four stages of somatic embryo maturation in white spruce (0, 7, 21 and 35 days post abscisic acid treatment). Forty-eight differentially expressed proteins have been identified, which display a significant change in abundance as early as day 7 of embryo development. These proteins are involved in a variety of cellular processes, many of which have not previously been associated with embryo development. The identification of these proteins was greatly assisted by the availability of a substantial expressed sequence tag (EST) resource developed for white, sitka and interior spruce. The combined use of these spruce ESTs in conjunction with GenBank accessions for other plants improved the rate of protein identification from 38% to 62% when compared with GenBank alone using automated, high-throughput techniques. This underscores the utility of EST resources in a proteomic study of any species for which a genome sequence is unavailable.