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Here, we present a new approach for increasing the rate and lowering the cost of identifying, cataloging, and monitoring global biodiversity. These advances, which we call Closed-Tube Barcoding, are one application of a suite of proven PCR-based technologies invented in our laboratory. Closed-Tube Barcoding builds on and aims to enhance the profoundly important efforts of the International Barcode of Life initiative. Closed-Tube Barcoding promises to be particularly useful when large numbers of small or rare specimens need to be screened and characterized at an affordable price. This approach is also well suited for automation and for use in portable devices.

Centrioles are eukaryotic organelles whose number and position are critical for cilia formation and mitosis. Many cell types assemble new centrioles next to existing ones ("templated" or mentored assembly). Under certain conditions, centrioles also form without pre-existing centrioles (de novo). The synchronous differentiation of Naegleria amoebae to flagellates represents a unique opportunity to study centriole assembly, as nearly 100% of the population transitions from having no centrioles to having two within minutes. Here, we find that Naegleria forms its first centriole de novo, immediately followed by mentored assembly of the second. We also find both de novo and mentored assembly distributed among all major eukaryote lineages. We therefore propose that both modes are ancestral and have been conserved because they serve complementary roles, with de novo assembly as the default when no pre-existing centriole is available, and mentored assembly allowing precise regulation of number, timing, and location of centriole assembly.

Thiaminases, enzymes that cleave vitamin B1, are sporadically distributed among prokaryotes and eukaryotes. Thiaminase I enzymes catalyze the elimination of the thiazole ring moiety from thiamin through substitution of the methylene group with a nitrogenous base or sulfhydryl compound. In eukaryotic organisms, these enzymes are reported to have much higher molecular weights than their bacterial counterparts. A thiaminase I of the single-celled amoeboflagellate Naegleria gruberi is the only eukaryotic thiaminase I to have been cloned, sequenced, and expressed. Here, we present the crystal structure of N. gruberi thiaminase I to a resolution of 2.8 Å, solved by isomorphous replacement and pseudo-two-wavelength multiwavelength anomalous diffraction and refined to an R factor of 0.231 (Rfree, 0.265). This structure was used to solve the structure of the enzyme in complex with 3-deazathiamin, a noncleavable thiamin analog and enzyme inhibitor (2.7 Å; R, 0.233; Rfree, 0.267). These structures define the mode of thiamin binding to this class of thiaminases and indicate the involvement of Asp272 as the catalytic base. This enzyme is able to use thiamin as a substrate and is active with amines such as aniline and veratrylamine as well as sulfhydryl compounds such as l-cysteine and β-mercaptoethanol as cosubstrates. Despite significant differences in polypeptide sequence and length, we have shown that the N. gruberi thiaminase I is homologous in structure and activity to a previously characterized bacterial thiaminase I.

Protists account for the bulk of eukaryotic diversity. Through studies of gene and especially genome sequences the molecular basis for this diversity can be determined. Evident from genome sequencing are examples of versatile metabolism that go far beyond the canonical pathways described for eukaryotes in textbooks. In the last 2-3 years, genome sequencing and transcript profiling has unveiled several examples of heterotrophic and phototrophic protists that are unexpectedly well-equipped for ATP production using a facultative anaerobic metabolism, including some protists that can (Chlamydomonas reinhardtii) or are predicted (Naegleria gruberi, Acanthamoeba castellanii, Amoebidium parasiticum) to produce H(2) in their metabolism. It is possible that some enzymes of anaerobic metabolism were acquired and distributed among eukaryotes by lateral transfer, but it is also likely that the common ancestor of eukaryotes already had far more metabolic versatility than was widely thought a few years ago. The discussion of core energy metabolism in unicellular eukaryotes is the subject of this review. Since genomic sequencing has so far only touched the surface of protist diversity, it is anticipated that sequences of additional protists may reveal an even wider range of metabolic capabilities, while simultaneously enriching our understanding of the early evolution of eukaryotes.

SummaryGenome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naegleria's 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism. The Naegleria genome facilitates substantially broader phylogenomic comparisons of free-living eukaryotes than previously possible, allowing us to identify thousands of genes likely present in the pan-eukaryotic ancestor, with 40% likely eukaryotic inventions. Moreover, we construct a comprehensive catalog of amoeboid-motility genes. The Naegleria genome, analyzed in the context of other protists, reveals a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal, sexual, signaling, and metabolic modules.PaperClip

Centrioles are eukaryotic organelles whose number and position are critical for cilia formation and mitosis. Many cell types assemble new centrioles next to existing ones ("templated" or mentored assembly). Under certain conditions, centrioles also form without pre-existing centrioles (de novo). The synchronous differentiation of Naegleria amoebae to flagellates represents a unique opportunity to study centriole assembly, as nearly 100% of the population transitions from having no centrioles to having two within minutes. Here, we find that Naegleria forms its first centriole de novo, immediately followed by mentored assembly of the second. We also find both de novo and mentored assembly distributed among all major eukaryote lineages. We therefore propose that both modes are ancestral and have been conserved because they serve complementary roles, with de novo assembly as the default when no pre-existing centriole is available, and mentored assembly allowing precise regulation of number, timing, and location of centriole assembly.

The rapidity of the optional 90-min differentiation of Naegleria gruberi from amoebae to flagellates suggests the possibility of a free-running cascade of events from initiating stimulus through gene expression to organelle assembly and cell morphogenesis. Instead our experiments reveal two points early in the differentiation at which the strength of the inducing stimulus is reevaluated by the cells. Two new physical start signals for differentiation, temperature downshift (DeltaT) and mechanical agitation, are shown to regulate differentiation synergistically with each other and with previously defined signals. A DeltaT of -10 degrees C induces complete differentiation directly in the growth environment, whereas smaller DeltaTs initiate differentiation and allow it to progress for a short time, after which the cells "hold" for up to 4 h, awaiting a stimulus to continue differentiation. Our work defines two "holdpoints," optional points in development where progress can stop, awaiting a suitable signal, while cells retain whatever intermediates represent progress. We propose that such holdpoints, which can be detected in this system because of the temporal reproducibility of the differentiation, are likely to be found in other differentiating cells.

During differentiation of Naegleria from vegetative amoebae to temporary flagellates, the microtubular cytoskeleton, including two basal bodies and flagella, is assembled de novo. Centrin is an integral component of these basal bodies [Levy et al., 1996, Cell Motil. Cytoskeleton 33: 298-323]. In many organisms, centrin appears to be a constitutive protein, but in Naegleria centrin gene expression occurs only during differentiation. Centrin mRNA, which has not been detected in amoebae, appears and disappears earlier in differentiation than a coordinately regulated set of differentiation-specific mRNAs encoding flagellar tubulin and calmodulin. Centrin antigen accumulates during differentiation, and then decreases in abundance as the flagellates mature and revert to amoebae. No localization of centrin has been detected in amoebae. During differentiation, centrin becomes localized to the basal bodies as soon as these structures are detected with anti-tubulin antibodies, first as a single dot and finally as two basal bodies. During reversion of flagellates to amoebae, centrin remains localized to the basal bodies for as long as they are present. When assembly of tubulin-containing structures during differentiation is prevented using oryzalin, centrin localization is prevented as well, yet inhibition of assembly does not affect accumulation of centrin antigen. Apparently in Naegleria, the role of centrin is primarily for a differentiation- or flagellate-specific function. The temporary presence of centrin is concurrent with the presence of centriolar basal bodies, which supports the conjecture that in Naegleria centrin may be needed only when these organelles are present.

Centrin, a approximately or equal to 20 kDa calcium-binding protein also known as caltractin, is a component of centrosome-associated algal flagellar roots capable of calcium-mediated contraction, and is also found in the centrosomes of vertebrate cells. Our analysis of a centrin gene from a protist, the amoeboflagellate Naegleria gruberi, reveals conserved features that distinguish centrins from calmodulin. Antibodies to bacterially expressed Naegleria centrin, which also recognize yeast Cdc31p, were employed to localize centrin immunoreactivity in selected organisms possessing specialized microtubule-organizing centers (MTOCs) or accessory structures. There is a striking morphological diversity of such structures. In the simplest associations, as found in Naegleria flagellates and vertebrates tracheal epithelium, centrin is intimately associated with the cylinder of the basal bodies. In cells with unfocused mitotic spindles, Naegleria amoebae and onion root tips, no localization of centrin was detected. In Dictyostelium discoideum and Saccharomyces cerevisiae, which lack centrioles, centrin immunoreactivity was observed as punctate cytoplasmic bodies but not associated with spindle pole MTOCs. In Paramecium multimicronucleatum, centrin immunoreactivity is localized to the infraciliary lattice, previously shown to exhibit calcium-mediated contraction. In Vorticella microstoma, known for the calcium-induced rapid contraction of its stalk, centrin immunoreactivity is localized to the contractile spasmoneme and myonemes. Similar antigens from Paramecium and Vorticella are detected by anti-centrin and anti-spasmin. The pattern of localization of centrin immunoreactivity supports the conjecture that a contractile system involving centrin, initially associated with centriolar structures, was recruited during evolution to build specialized organelles in different organisms and cell types.

Two calmodulins are synthesized during differentiation of Naegleria gruberi from amoebae to flagellates; one remains in the cell body and the other becomes localized in the flagella. The single, intronless, expressed gene for flagellar calmodulin has been cloned and sequenced. The encoded protein is a typical calmodulin with four putative calcium-binding domains, but it has an amino-terminal extension of 10 divergent amino acids preceding conserved calmodulin residue 4. The transcripts encoding flagellar calmodulin and flagellate cell body calmodulin are clearly divergent. Expression of the flagellar calmodulin gene is differentiation-specific; its mRNA appears and then disappears concurrently with those encoding flagellar alpha- and beta-tubulin. Three provocative sequence elements are shared among these unrelated coexpressed genes: (i) a palindromic DNA sequence element is found in duplicate or triplicate upstream to each transcribed region; (ii) a perfect 12-nucleotide match is found near the AUG start codon of flagellar calmodulin and alpha-tubulin; and (iii) the novel amino-terminal extension of flagellar calmodulin contains a 5-amino-acid element similar to the amino terminus of flagellar alpha-tubulin. These shared sequence elements are proposed to have roles in differentiation, possibly in regulation of transcription, mRNA stability, and localization of these proteins to flagella.

One of two similar genes in the unicellular eukaryote Naegleria gruberi is shown to encode calcineurin B (CnB), the regulatory subunit of calcium-calmodulin-regulated protein phosphatase 2B. Over a span of 156 amino acids, excluding divergent N-termini, the encoded sequence shows 62% identity with vertebrate CnB, and also shows sequence elements specific, among calcium-binding proteins, to CnB. In contrast, the sequence shows only 23% identity with N. gruberi flagellar calmodulin. CNB mRNA is readily detected in amoebae; its abundance increases fourfold during differentiation to flagellates, reaches a peak at 50-70 min, when flagella are forming, and then declines. A genomic clone matches an expressed cDNA, except that it is interrupted by two phase I introns. The position of one intron, which separates the divergent N-terminal domain from the four calcium-binding domains (EF hands), is shared with a yeast CNB gene; the other is located in the central helix between the two pairs of calcium-binding loops; features that support an ancient origin. These introns, the first found in protein-coding genes of Naegleria, are flanked by characteristic splice junction sequences. N. gruberi CnB also shares similarities with recoverins. The finding in a protist of a CNB gene that contains two introns separating functional domains, shares similarities to recoverins and shows increased expression during differentiation is provocative. If the phylogeny of major groups derived from ribosomal RNA is accepted, Naegleria is among the earliest branching eukaryotes known to contain canonical pre-mRNA introns.

A gene that directs the programmed synthesis of flagellar β -tubulin during the rapid differentiation of Naegleria gruberi from amoebae to flagellates has been cloned and sequenced. The intronless gene is one of 8 to 10 similar but non-identical genes that are dispersed in the genome. β -Tubulin mRNA homologous to this gene family is expressed transiently during differentiation, and has not been detected in amoebae. The encoded β -tubulin is strongly conserved, with features that closely resemble the β -tubulins of diverse organisms, especially organisms that, like Naegleria , use tubulin to assemble flagellar axonemes. In most sequenced α -tubulins, the encoded carboxy-terminal amino acid is tyrosine, which undergoes post-translational removal and readdition, conserved processes of unknown function. In N. gruberi , unusually, the terminus of α -tubulin is encoded as glutamine while that of β -tubulin is tyrosine. The presence of these divergent termini on subunits of a conserved tubulin provoked us to re-examine aromatic amino acids at the termini of α - and β -tubulins. Although evolution has tinkered extensively with the carboxyterminal domains of tubulin subunits, we find an unexpected conservation. In every organism or cell type for which both tubulin subunits have been sequenced, except the ciliate Stylonychia lemnae , at least one tubulin subunit of some or all tubullin heterodimers terminates in an aromatic amino acid, either tyrosine or phenylalanine. This remarkable conservation of carboxy-terminal aromatic amino acids suggests that these residues serve some crucial function.

Organisms in the genus Naegleria offer special opportunities for research in contemporary biology. the dramatic cell differentiation from amebae to flagellates is unique among eukaryotes in the rapidity, synchrony, reproducibility, homogeneity, and accessibility of a major phenotypic change. Environmental signals initiate a progressive signal transduction pathway in which genes are turned on, including those for several calcium-binding proteins, and newly synthesized proteins become localized in newly assembled organelles, including the centriole-like basal bodies, with the overall consequence that the cell changes its shape, motility, and behavior. This essay reviews research opportunities for which Naegleria excels, as well as interesting aspects of its biology that provide challenges for future investigations. Because these organisms alternate between two major eukaryotic motility forms, their phylogenetic position is also provocative. Although there are hints that Naegleria is capable of sexual reproduction in nature, mating has not yet been observed in the laboratory. In order to fully exploit the opportunities offered by this wonderful experimental system we are working to develop means to do genetic manipulation, in particular via DNA-mediated transformation.

Naegleria gruberi, a free-living protist, has long been treasured as a model for basal body and flagellar assembly due to its ability to differentiate from crawling amoebae into swimming flagellates. The full genome sequence of Naegleria gruberi has recently been used to estimate gene families ancestral to all eukaryotes and to identify novel aspects of Naegleria biology, including likely facultative anaerobic metabolism, extensive signaling cascades, and evidence for sexuality. Distinctive features of the Naegleria genome and nuclear biology provide unique perspectives for comparative cell biology, including cell division, RNA processing and nucleolar assembly. We highlight here exciting new and novel aspects of Naegleria biology identified through genomic analysis.

The successful commercialization of fuel cells will depend on the achievement of competitive system costs and efficiencies. System cost directly impacts the capital equipment component of cost of electricity (COE) and is a major contributor to the O and M component. The replacement costs for equipment (also heavily influenced by stack life) is generally a major contributor to O and M costs. In this project, they worked with the SECA industrial teams to estimate the impact of general manufacturing issues of interest on stack cost using an activities-based cost model for anode-supported planar SOFC stacks with metallic interconnects. An earlier model developed for NETL for anode supported planar SOFCs was enhanced by a linkage to a performance/thermal/mechanical model, by addition of Quality Control steps to the process flow with specific characterization methods, and by assessment of economies of scale. The 3-dimensional adiabatic performance model was used to calculate the average power density for the assumed geometry and operating conditions (i.e., inlet and exhaust temperatures, utilization, and fuel composition) based on publicly available polarizations curves. The SECA team provided guidance on what manufacturing and design issues should be assessed in this Phase I demonstration of cost modeling capabilities. They considered the impact of the following parameters on yield and cost: layer thickness (i.e., anode, electrolyte, and cathode) on cost and stress levels, statistical nature of ceramic material failure on yield, and Quality Control steps and strategies. In this demonstration of the capabilities of the linked model, only the active stack (i.e., anode, electrolyte, and cathode) and interconnect materials were included in the analysis. Factory costs are presented on an area and kilowatt basis to allow developers to extrapolate to their level of performance, stack design, materials, seal and system configurations, and internal corporate overheads and margin goals.

We have constructed a molecular karyotype for two strains of Naegleria gruberi using pulsed field gel electrophoresis. Each strain has about 23 chromosomes, considerably more than any previous estimate. These chromosomes range in size from 400 kilobasepairs to over 2,000 kilobasepairs. In Naegleria, construction of the DNA karyotype depends on assessment of the anomalous electrophoretic mobility of the circular ribosomal RNA genes. We have determined the chromosomal locations of an identified unique gene (flagellar calmodulin) and four identified multigene families (alpha- and beta-tubulin, actin, ubiquitin), as well as three differentially expressed genes of unknown functions. The ca. 12 actin genes are dispersed over at least seven chromosomes, whereas the majority of the more than eight alpha-tubulin genes are confined to a single chromosome. The ubiquitin genes are found on five chromosomes in one strain and seven in the other and the beta-tubulin genes are on three or four. Our observations provide a foundation for molecular genetic studies in this organism.

Actin, the major protein of amebae of Naegleria gruberi, proved to be strongly immunogenic in rabbits. The resulting precipitating antibodies are specific to actin of Naegleria. In a competitive solid-phase radioimmunoassay, these antibodies bound similarly to Naegleria G- and F-actin. Actins from amebae of Acanthamoeba and Dictyostelium, plasmodia of Physarum, sea urchin eggs, and vertebrate muscles gave no competition in the radioimmunoassay. Estimates of the amount of actin in Naegleria amebae ranged from a minimum of 5% of the total cell protein by radioimmunoassay to a maximum of 16% by electrophoresis. The unusual species specificity of these antibodies indicates that Naegleria actin, although conserved in many properties, is different enough to have unique antigenic determinants.

Amebae of Naegleria gruberi differentiate into flagellates when transferred from growth medium to non-nutrient buffer. This differentiation, which requires 48 min at 28 degrees C, is dependent on transcription and translation. Tubulin of the flagellar outer doublets comprises about 0.15% of the protein of flagellate, and only about 1-2% of the total tubulin. An antiserum to flagellar (outer-doublet) tubulin contains antibodies that react selectively with flagellar tubulin. Measurements using this antiserum have shown that 97-98% of the flagellar tubulin antigen appears during differentiation. The appearance of tubulin antigen is sensitive to actinomycin D and cycloheximide. Isotope dilution experiments using [35S]methione demonstrated that at least 70% of the flagellar tubulin is synthesized from amino acids during differentiation. Experiments using both the specific antiserum and isotopes have shown that flagellar tubulin synthesis begins about one-third of the way through differentiation, before any morphological change has occurred. These experiments demonstrate that most, if not all, of the flagellar tubulin is synthesized de novo during differentiation, and that cells selectively use a specific subpopulation of tubulin in assembling the outer doub)lets. The results bring into focus major unsolved questions about the synthesis and assembly of flagellar tubulin.

Outer doublet tubulin has been purified from flagella of differentiated cells of the amebo-flagellate Naegleria gruberi. The flagellar tubulin is similar to other tubulins in molecular weight (55,000), amino acid composition, electrophoretic mobility, and nucleotide composition. However, the protein contains 2 moles of guanine nucleotide per 55,000 g of protein, twice that usually found in tubulin. A rabbit antiserum against flagellar tubulin was shown to contain antibodies specific for tubulin. Purified tubulin iodinated with 125I was used in a radioimmune assay to measure levels of flagellar tubulin antigen in cells. The measurements indicate that the antigen increases 35- to 55-fold during differentiation of amebae to flagellates.

The rapid, synchronous differentiation of N. gruberi from amoebae to flagellates is a useful paradigm to study aspects of cell differentiation, including regulation of the synthesis of proteins that are related to the changes in cell shape and motility, which occur during differentiation. The differentiation requires synthesis of new RNA and protein molecules to accomplish defined morphogenetic events. Specific new proteins, including the tubulins that form the flagellar microtubules, are synthesized at various times during differentiation, and particular mRNA species appear and disappear. The time course of the synthesis of the alpha and beta subunits of flagellar tubulin is paralleled by the programmed appearance and disappearance of flagellar tubulin mRNAs. The evidence supports the hypothesis that the synthesis of flagellar tubulin is regulated by the transcription, and subsequent disappearance, of flagellar tubulin mRNA. Translatable mRNAs for two calmodulin-like calcium-binding proteins appear and disappear contemporaneously with those for flagellar tubulin. During differentiation the synthesis of actin, the major protein of amoebae, is selectively shut down, and translatable actin mRNA rapidly disappears. This description of the orderly appearance, utilization, and disappearance of the mRNAs for actin, calcium-binding proteins, and flagellar tubulin during differentiation provides means and motivation to investigate the mechanisms that regulate these events.

Genes that direct the programmed synthesis of flagellar alpha-tubulin during the differentiation of Naegleria gruberi from amebae to flagellates have been cloned, and found to be novel with respect to gene organization, sequence, and conservation. The flagellar alpha-tubulin gene family is represented in the genome by about eight homologous DNA segments that are exceptionally similar and yet are neither identical nor arrayed in a short tandem repeat. The coding regions of three of these genes have been sequenced, two from cDNA clones and one from an intronless genomic gene. These three genes encode an identical alpha-tubulin that is conserved relative to the alpha-tubulins of other organisms except at the carboxyl terminus, where the protein is elongated by two residues and ends in a terminal glutamine instead of the canonical tyrosine. In spite of the protein conservation, the Naegleria DNA sequence has diverged markedly from the alpha-tubulin genes of other organisms, a counterexample to the idea that tubulin genes are conserved. alpha-Tubulin mRNA homologous to this gene family has not been detected in amebae. This mRNA increases markedly in abundance during the first hour of differentiation, and then decreases even more rapidly with a half-life of approximately 8 min. The abundance of physical alpha-tubulin mRNA rises and subsequently falls in parallel with the abundance of translatable flagellar tubulin mRNA and with the in vivo rate of flagellar tubulin synthesis, which indicates that flagellar tubulin synthesis is directly regulated by the relative rates of transcription and mRNA degradation.

A simplified axenic medium for Naegleria gruberi strain NEG-M contains l -methionine, dextrose, yeast extract, a macromolecular fraction of fetal calf serum, and phosphate buffer. Amoebae cultured in suspension in this medium grow with doubling times of 8–10 h (at 32 °C) to yield 2–4 × 10 6 cells/ml. Amoebae from growing or early stationary phase cultures, transferred to nonnutrient buffer, differentiate synchronously into flagellates. Differentiation occurs reproducibly 80 min after initiation (time for 50% flagellates at 25 °C) if amoebae are taken from a culture maintained at pH ⩾ 6.6.

Amebae of Naegleria gruberi may be grown on nutrient agar in association with Aerobacter aerogenes. When transferred from agar to a defined aqueous environment, the amebae transform into flagellates. Conditions are described which favor the synchronous and reproducible conversion of entire populations of amebae into flagellates. Transformation is measured by fixing aliquots of the transforming population and counting the proportion which have completed a specific change. The time course of appearance of flagellates has been analyzed. At 25°C half of the amebae round up about 55 minutes after suspension, develop visible flagella 6–10 minutes later, and assume a flagellate shape 14–18 minutes after that. The times are reproducible, and more than 90% of the population completes a given change within 25 minutes. The relative rate of transformation is dependent on temperature, but independent of cell population density. The early events of transformation are arrested at 10°C, but at about the time the amebae round up, some critical event occurs after which the appearance and elongation of flagella can continue at the low temperature. The capacity of Naegleria amebae to transform is stably inherited, and the phenotypic changes occur without change in genotype. In addition, although the flagellates are unstable and revert to amebae, in an aqueous environment these amebae retransform and revert repeatedly until they begin to encyst.

For amoebae of Naegleria gruberi NEG-M growing in an axenic medium, one component of the medium, yeast extract, controls whether phenotypic transformation into flagellates occurs. Fractionation of the yeast extract yielded two components that control transformation: electrolytes and an unidentified Factor. Electrolytes prevent transformation if present above a certain concentration. For NaCl and KCl, for example, the critical concentration is 0.08 M . The effect is not specific to particular ions, nor is it due simply to ionic strength. Osmotic pressure produced by nonpenetrating nonelectrolytes like sucrose can also inhibit transformation, but the effect of electrolytes is not due solely to osmotic pressure. The unidentified Factor, a small molecule, probably aromatic (adsorbs reversibly to charcoal), can prevent transformation at a concentration that does not increase the osmotic pressure of the solution, and that is estimated to be not more than 0.001 M . The action of Factor in controlling transformation is much potentiated by electrolytes, such that a combination of one-eighth the electrolyte concentration needed to prevent transformation with one-fourth the Factor concentration completely prevents transformation. The synergistic action with Factor is not specific to a particular ion, but there are differences. For example, Na + is much more effective than Cs + . Factor and osmotic pressure do not act synergistically. Amoebae grown in the presence of extra Factor adapt to it, and will transform in concentrations of Factor and of electrolyte that prevent transformation of unadapted cells. These adapted cells also can transform in regular growth medium. It is concluded that Factor and electrolytes are involved in the normal control of transformation of these axenic Naegleria . The results provide a starting point for studying early events in this cell differentiation.

Amebae of Naegleria gruberi transform into flagellates whose basal bodies have the typical centriole-like structure. The amebae appear to lack any homologous structure, even during mitosis. Basal bodies are constructed during transformation and, in cells transforming synchronously at 25°C, they are first seen about 10 min before flagella are seen. No structural precursor for these basal bodies has been found. These observations are discussed in the light of hypotheses about the continuity of centrioles.

Flagellates of Naegleria gruberi have an interconnected flagellar apparatus consisting of nucleus, rhizoplast and accessory filaments, basal bodies, and flagella. The structures of these components have been found to be similar to those in other flagellates. The development of methods for obtaining the relatively synchronous transformation of populations of Naegleria amebae into flagellates has permitted a study of the development of the flagellar apparatus. No indications of rhizoplast, basal body, or flagellum structures could be detected in amebae. A basal body appears and assumes a position at the cell surface with its filaments perpendicular to the cell membrane. Axoneme filaments extend from the basal body filaments into a progressive evagination of the cell membrane which becomes the flagellum sheath. Continued elongation of the axoneme filaments leads to differentiation of a fully formed flagellum with a typical "9 + 2" organization, within 10 min after the appearance of basal bodies.

The colonial hydroid Cordylophora is a carnivore whose feeding is induced by substances released from captured prey. An active molecule, probably the only one, has been isolated from a fraction of the laboratory food of Cordylophora, brine shrimp larvae, and identified on paper chromatograms as the imino acid proline. Reagent proline induces the feeding reaction at 10-5 M. The reaction is specific in that only two α-imino acids very closely related to proline were found to possess significant activity: azetidine-2-carboxylic acid and pipecolic acid. The response to proline is inhibited by magnesium ions and enhanced by phosphate. Since previous studies have shown that the feeding reactions of Hydra, Physalia, and Campanularia are controlled by reduced glutathione, the phylogenetic implications of the proline control of feeding in Cordylophora are discussed. The feeding reactions of both Cordylophora and Hydra are also induced by proteases, suggesting similar mechanisms of induction in the two hydroids.

The development of pattern has been analyzed in asexual Cordylophora colonies growing under a single set of standard conditions. For the analysis, a colony is considered to be composed of a series of tubes of uniform diameter. On the basis of positional and developmental criteria, these tubes are of three types: stolon tubes, upright tubes, and branch tubes. All types of tubes grow at constant rates once they reach a certain length. However the relative rates for the three types of tubes are different; stolons grow at about twice the rate of uprights, and uprights grow more rapidly than branches. A model was developed integrating the relative growth rates and patterning of the tubes to describe the development of a single stolon (Fig. 10); by following the development of individual young colonies this model was shown to be correct. The linear growth of tubes posed a paradox, since the number of hydranths in an entire colony, as well as the mass and total tube length, had been found to increase exponentially. The model of colony development does not predict exponential growth. Colonies were shown to achieve exponential growth by the addition of secondary stolons at appropriate time intervals. The analysis of colony development poses specific questions for the study of pattern formation.

An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins.

1. Rose and Rose (1941) found that adult Tubularia hydranths agitated in sea water produced a solution, inhibitor water, which prevented regeneration. They and subsequent workers have ascribed to this inhibitor a role in normal physiological dominance. In the present investigation it has been found that considerable bacterial growth occurs in the solution during the preparation of inhibitor water by the usual methods, and that when antibiotics have been added to maintain bacteriostasis no inhibitor can be collected. Experiments have excluded the possibilities that the antibiotics used are preventing the production of the inhibitor or destroying it as it is produced. It has been shown that metabolites produced by bacterial growth in the absence of hydranths inhibit regeneration.2. These data lead to the conclusion that inhibitor water represents the by-products of bacterial growth for which the hydranths serve as source of inoculum and as nutritive medium.

The programmed de novo synthesis of flagellar tubulin during the hour-long differentiation of Naegleria gruberi from amoebae to flagellates is our paradigm for the study of gene expression during cell differentiation. This paper reports the efficient translation of flagellar tubulin mRNA in the wheat germ cell-free system directed by total or polyadenylated RNA extracted from differentiating cells. The tubulin in the in vitro product has a subunit molecular weight of 55,000, separates into α and β subunits under suitable conditions of polyacrylamide gel electrophoresis and co-polymerizes with calf brain tubulin. At least half of the tubulin synthesized in vitro is precipitated by antibodies specific to flagellar tubulin, and the immunoprecipitated tubulin subunits yield peptide maps similar to those of outer doublet tubulin. Flagellar tubulin is the predominant protein synthesized in the cell-free system, and amounts to about 5% of the polypeptides whose synthesis is directed by total RNA from differentiating cells. In contrast, little or no flagellar tubulin is synthesized when the cell-free system is directed by RNA extracted from amoebae prior to differentiation. Translation assays show that at least 92% of the flagellar tubulin mRNA appears during differentiation. The time course of appearance of this mRNA was measured by quantitative immunoprecipitation of the cell-free products. Under conditions where cells form flagella 60 min after initiation of differentiation, translatable flagellar tubulin mRNA was first detected at 20 min, reached a maximum at about 60 min and then declined. An excellent correlation was observed between the amount of translatable flagellar tubulin mRNA and the previously measured rates of flagellar tubulin synthesis in vivo. These results indicate that synthesis of flagellar tubulin is a direct reflection of the abundance of its mRNA, and provide the molecular techniques for dissection of the factors that regulate the rapid appearance of this structural protein during differentiation.

The amebo-flagellate Naegleria gruberi contains three major RNA polymerase activities separable from whole cell extracts by DEAE-Sephadex column chromatography. One is resistant to the durg α-amanitin and the other two are sensitive to it. These separated activities were characterized and then examined during the differentiation of ameba to flagellate, which is dependent on RNA synthesis and which exhibits gross changes in RNA metabolism under the conditions employed. It was found that no detectable changes occurred qualitatively or quantitatively in these polymerases during differentiation. Care was taken to account for total enzyme activity in every fraction of the extraction procedure. It is concluded from the above observation that gross changes in RNA synthesis as well as differential gene activity can occur with absolutely no major fluctuations in the DNA-dependent RNA polymerases.

Amoebae of Naegleria gruberi differentiate to temporary flagellates that have a regular, asymmetric, streamlined body contour. During the hour-long differentiation, amoeboid movement gradually ceases and as a consequence the cells round up. Subsequent elongation to flagellate shape includes the formation of a microtubular cytoskeleton. Both the loss of amoeboid motility and the formation of the flagellate shape require prior transcription and translation, suggesting the possibility that specific syntheses of RNA and protein may be required for each shape change. Flagellates can “revert” to motile amoebae within 20 sec after a suitable stimulus, indicating that the amoeboid motility system remains latent in flagellates. A cell-produced chemical factor extracted from Naegleria, Ψ, triggers a reproducible sequence of rapid shape changes in flagellates when added to their environment. Cells respond to the presence of external Ψ only “transiently,” and the reaction of flagellates to added Ψ requires extracellular Ca+2. Ionophore A23187 produces shape changes in flagellates similar to those produced by Ψ, supporting the conclusion that Ψ is involved in the movement of Ca+2. Normally Ψ is intracellular, and the intracellular distribution of Ψ changes during differentiation. These results lead to and support a working hypothesis to explain the rapid changes in shape and motility in Naegleria. Four elements are postulated: Ca+2; an actin-based amoeboid motility system that depends on free Ca+2 for functioning; a tubulin-based cytoskeleton that assembles and remains assembled only when free Ca+2 is low; and Ψ. The factor Ψ is postulated to regulate the intracellular release of Ca+2. According to the hypothesis, intracellular free Ca+2 is constantly swept up into Ca-reservoirs. Motility of amoebae depends on local release of Ca+2 from these reservoirs, which in turn is caused by the intracellular release of Ψ. During differentiation, Ψ is “compartmentalized” as part of the developmental program, and as a consequence intracellular Ca+2 is swept up into Ca-reservoirs but not released. As free Ca+2 becomes limiting, amoeboid movement stops, and the cells round up. Subsequently, in a process that depends on low free Ca+2, the microtubular cytoskeleton is assembled, and the flagellate shape is formed. During reversion of flagellates to amoebae, release of Ψ from its “compartments” permits local release of Ca+2, which then causes both disassembly of the flagellate cytoskeleton and immediate resumption of amoeboid movement. This testable hypothesis has implications for the study of cell shape, motility, and differentiation.

Cultivation of amebae of the axenic strain of Naegleria gruberi , NEG-M, was achieved in media consisting entirely of chemically defined components. A complete medium that contains 31 components allows growth with yields up to 5 × 10 6 amebae per ml. A minimal medium gives lower yields but defines 22 components that are essential for continuous cultivation: 11 amino acids, 6 vitamins, hematin, guanosine, D-glucose, Mg 2+ , and inorganic phosphate. These media allow precise studies of the metabolism and differentiation of this unusual eukaryote.

Amebae of Naegleria gruberi undergo phenotypic transformation into flagellates when the aqueous environment is changed from axenic growth medium to non-nutrient buffer. This differentiation, which requires 80 min at 25 °C, is dependent on transcription during the first 40 min. The nature of the RNA synthesized during differentiation is the subject of this report. Net synthesis of RNA continues for the first 30 min after differentiation is initiated, but thereafter degradation exceeds synthesis leading to an overall decrease of 6 % in total RNA per cell by 90 min. [14C]-Uracil is incorporated into RNA during differentiation. Incorporation is linear until roughly 10 min before the appearance of flagella, at which time the rate of incorporation declines. Sucrose gradient analysis has been used to compare the RNA synthesized by cells differentiating in buffer versus amebae in growth medium. There is proportionally more synthesis of messenger-like RNA and less of rRNA in differentiating cells. The messenger-like RNA synthesized during differentiation is heterogeneous in size, but very large molecules (> 45 S) have not been found. This rapidlylabeled RNA is first present in the nucleoplasm, and moves rapidly to the cytoplasm, reaching equilibrium (nucleuscytoplasm) within 10 min. In the cytoplasm it is found associated with polysomes, where it has the EDTA and ribonuclease sensitivity expected of mRNA. These observations support the interpretation that the rapidlylabeled RNA is mRNA. It is not yet known whether this mRNA includes the RNA essential for differentiation.

A simple method has been developed for the cultivation of colonies of Cordylophora lacustris. The colonies, attached to microscope slides slanted in beakers, are grown in a culture solution containing five required ions. Artemia larvae are supplied as food. Increase in hydranth number is exponential with a doubling time of about 3 days.

Amebae of Naegleria gruberi can be cultured in suspension in buffer containing a dense suspension of Aerobacter aerogenes . At 30 °C the amebae grow exponentially with a doubling time of 2 h. Flagellates are rare in these cultures, but the amebae from liquid cultures transform into flagellates when washed relatively free of Aerobacter . If a growing culture of amebae is shifted to 38–39 °C, division ceases and the amebae increase in volume. After 100 min at the high temperature, when the volume of the amebae has nearly doubled, if the amebae are returned to 30 °C a single burst of mitosis occurs. Usually 80 to 90% of the amebae divide within one quarter of the normal doubling time, and 60 to 70% of the amebae may be found in mitosis simultaneously. After the single division, the amebae usually return to exponential growth, though occasionally a second synchronous division has been obtained. Amebae sampled at any time during a synchronized division are able to transform into flagellates. Some amebae, sampled just prior to division, complete nuclear but not cytoplasmic division, and become binucleate.

The amoeboflagellate Naegleria was one of the first organisms in which de novo basal body/centriole assembly was documented. When in its flagellate form, this single-celled protist has two flagella that are templated by two basal bodies. Each of these basal bodies is structurally well conserved, with triplet microtubules and well-defined proximal cartwheel structures, similar to most other eukaryotic centrioles. The basal bodies are anchored to the nucleus by a single, long striated rootlet. The Naegleria genome encodes many conserved basal body genes whose expression is induced prior to basal body assembly. Because of the rapid and synchronous differentiation from centriole-less amoebae to temporary flagellates with basal bodies, Naegleria offers one of the most promising systems to study de novo basal body assembly, as well as the mechanisms regulating the number of centrioles assembled per cell.

Methods have beeni developed for controlling the transformation of Tetramitus rostratus, a protozoan able to reproduce either as an ameba or as a flagellate. An essential conidition for transformation is a low concentration of oxygen in the environment.