Identification and functional analysis of a novel BTB-domain gene family.

In a screen for novel genes involved in chick hindbrain development, clone cVll was isolated, encoding a gene expressed in the hindbrain. Sequence analysis established that cVll is a novel gene, now designated cBTBD6, which encodes protein containing three functional domains: BTB, BACK and PHR. Human, mouse and zebrafish cBTBD6 orthologs were cloned, as well as highly related BTBD3 genes that form a distinct subgroup of the same family of genes. Within each subgroup there is a single copy of human, chick and mouse BTBD6 and BTBD3 and two copies of zebrafish orthologs. Spatio- temporal analysis of BTBD6 and BTBD3 expression patterns in chick and zebrafish revealed that, while BTBD3 othologs have divergent expressions, cBTBD6 shares some aspects of its expression pattern with its counterparts in zebrafish, zBTBD6a and zBTBD6b. In particular, both cBTBD6 and zBTBD6a are expressed in the developing central nervous system, suggesting a potential involvement in neurogenesis. This potential function was investigated in zebrafish. Comparative analysis of zBTBD6a expression with markers of neurogenesis revealed that zBTBD6a is expressed in cells during neuronal differentiation, starting from early stages labelled by proneural gene neurogl until later phases marked by isll, when zBTBD6a expression starts to be downregulated. Knockdown experiments demonstrated that zBTBD6a promotes neurogenesis and acts downstream of neurogl and upstream of neurod and neurod4 in the neurogenesis cascade. I found that zBTBD6a interacts with Cul-3, a component of the ubiquitin ligase complex, and the PHR motif and to a lesser extent the BTB domain are required for this interaction. Overexpression experiments showed that the presence of both BIB and PHR is required for zBTBD6a function. Since several BTB- containing proteins have been recently reported to be implicated in targeting proteins for ubiqutination, it is proposed that zBTBD6a positively regulates neurogenesis by mediating ubiquitination of one or more proteins that regulate the formation of neurons

The biodiversity of phytate cycling in soils

Phytic acid, myo-inositol hexakisphosphate, InsP6, is the major storage form of phosphate in seeds and grains that constitute a major part of the diets of monogastric animals such as swine and poultry. Monogastrics lack enough enzymes in the right part of the gut to digest dietary phytate. Consequently, phytases, a group of enzymes capable of releasing inorganic phosphate from phytate, are added to commercial poultry and swine diets. These adjunct phytases are a major sector of the global enzyme market with an estimated value of $5 billion in 2015. There is continued commercial interest in the discovery and development of more effective and cost-effective enzymes. The soil environment is microbially diverse and therefore offers significant potential for the isolation of novel phytases. In this thesis, I have developed new methods for the culture-dependent isolation of phytases from different soil environments by first analysing phytase activity of the soil microbiome using HPLC. The isolation of a multiple inositol polyphosphate phosphatase, MINPP, from Acinetobacter sp. represents one of the first phytases of its kind to be isolated from the soil environment. This study provides a robust characterisation of the protein, identifying an outstanding long-term stability at room temperature and activity from 37.6-101.3% over 755 days. The expression of the phytase was examined using β-galactosidase and qPCR assays which showed that while expression was enhanced in the presence of impure phytate by β-galactosidase, it was significantly repressed in the qPCR experiment. Additionally, a long-term phytase isolation experiment was performed using well-characterised Rothamsted soils. Of the sixty-six isolates that were re-streaked from mixed plates, seventeen showed a diverse array of phytase degradation profiles, highlighting the diversity of phytase activity in the soil environment. I have also undertaken metagenomic analysis to examine the diversity of phytases in environmental and enteric environments. This highlighted the dominance of MINPP genes in enteric environments above all other known classes of phytases. In soil and aquatic metagenomes, the relative abundances were significantly less than in enteric environments and here the Multiple Inositol Polyphosphate Phosphatase (MINPP) gene was not the overwhelming majority, instead the Beta-Propeller Phytase (BPPhy), Histidine Acid Phytase (HAPhy) and Protein Tyrosine Phosphatase-like Phytase (PTPhy) were equal to or even higher in abundances. Additionally, in this analysis I also examined the prospect of horizontal gene transfer in the MINPP dataset using the program T-rex. Here I identified multiple HGT events occurring between both enteric and environmental bacteria, with one transfer occurring between environments

Laboratory directed research and development annual report. Fiscal year 1994

The Department of Energy Order DOE 5000.4A establishes DOE`s policy and guidelines regarding Laboratory Directed Research and Development (LDRD) at its multiprogram laboratories. This report represents Pacific Northwest Laboratory`s (PNL`s) LDRD report for FY 1994. During FY 1994, 161 LDRD projects were selected for support through PNL`s LDRD project selection process. Total funding allocated to these projects was $13.7 million. Consistent with the Mission Statement and Strategic Plan provided in PNL`s Institutional Plan, the LDRD investments are focused on developing new and innovative approaches in research related to our {open_quotes}core competencies.{close_quotes} Currently, PNL`s core competencies have been identified as integrated environmental research; process science and engineering; energy systems development. In this report, the individual summaries of LDRD projects (presented in Section 1.0) are organized according to these core competencies. The largest proportion of Laboratory-level LDRD funds is allocated to the core competency of integrated environmental research. Projects within the three core competency areas were approximately 91.4$35K or less. The projects described in this report represent PNL`s investment in its future and are vital to maintaining the ability to develop creative solutions for the scientific and technical challenges faced by DOE and the nation. The report provides an overview of PNL`s LDRD program, the management process used for the program, and project summaries for each LDRD project

Spacelab Science Results Study

Beginning with OSTA-1 in November 1981 and ending with Neurolab in March 1998, a total of 36 Shuttle missions carried various Spacelab components such as the Spacelab module, pallet, instrument pointing system, or mission peculiar experiment support structure. The experiments carried out during these flights included astrophysics, solar physics, plasma physics, atmospheric science, Earth observations, and a wide range of microgravity experiments in life sciences, biotechnology, materials science, and fluid physics which includes combustion and critical point phenomena. In all, some 764 experiments were conducted by investigators from the U.S., Europe, and Japan. The purpose of this Spacelab Science Results Study is to document the contributions made in each of the major research areas by giving a brief synopsis of the more significant experiments and an extensive list of the publications that were produced. We have also endeavored to show how these results impacted the existing body of knowledge, where they have spawned new fields, and if appropriate, where the knowledge they produced has been applied

Patterns of tooth replacement in osteichthyans: variations on a theme

Nonmammalian tooth-bearing vertebrates usually replace their teeth throughout life. Much about how a replacement pattern is generated has been learned from zebrafish. However, to understand general mechanisms of tooth replacement, advantage can be taken from studying other, “nonmodel” species. We have mapped the patterns of tooth replacement in widely divergent aquatic osteichthyans using 2D charts, in which one axis is time, the other linear spacing along the tooth row. New teeth that are generated simultaneously are considered part of the same odontogenic wave. Using this approach, it appears that a similar, general pattern underlies very distinctive dentitions in distantly related species. A simple shift in spacing of odontogenic waves, or in distance between subsequent tooth positions along a row (or both), can produce dramatically different dentitions between life stages within a species, or between closely related species. Examples will be presented from salmonids, cyprinids, and cichlids. Our observations suggest that lines linking subsequent positions may have more biological significance than replacement waves (usually linking alternate positions), often used to explain the generation of patterns. The presence of a general pattern raises questions about common control mechanisms. There is now increasing evidence, at least for the zebrafish, to support a role for stem cells in continuous tooth renewal and control of replacement patterns