Naegleria's mitotic spindles are built from unique tubulins and highlight core spindle features.

Naegleria gruberi is a unicellular eukaryote whose evolutionary distance from animals and fungi has made it useful for developing hypotheses about the last common eukaryotic ancestor. Naegleria amoebae lack a cytoplasmic microtubule cytoskeleton and assemble microtubules only during mitosis and thus represent a unique system for studying the evolution and functional specificity of mitotic tubulins and the spindles they assemble. Previous studies show that Naegleria amoebae express a divergent α-tubulin during mitosis, and we now show that Naegleria amoebae express a second mitotic α- and two mitotic ß-tubulins. The mitotic tubulins are evolutionarily divergent relative to typical α- and ß-tubulins and contain residues that suggest distinct microtubule properties. These distinct residues are conserved in mitotic tubulin homologs of the "brain-eating amoeba" Naegleria fowleri, making them potential drug targets. Using quantitative light microscopy, we find that Naegleria's mitotic spindle is a distinctive barrel-like structure built from a ring of microtubule bundles. Similar to those of other species, Naegleria's spindle is twisted, and its length increases during mitosis, suggesting that these aspects of mitosis are ancestral features. Because bundle numbers change during metaphase, we hypothesize that the initial bundles represent kinetochore fibers and secondary bundles function as bridging fibers.

First identification of Naegleria species and Vahlkampfia ciguana in Nile water, Cairo, Egypt: Seasonal morphology and phylogenetic analysis.

BACKGROUND/PURPOSE: In Egypt, there is a scarcity of data concerning Naegleria (N.) family, with a shortage of phylogenetic studies. This study's aim was molecular detection, sequencing and phylogenetic analysis of morphologically identified Nagleria and to determine natural seasonal distribution of Nagleria species in water sources of Greater Cairo, Egypt. METHODS: A total of 120 water samples were collected during each season over a year. Every water sample was filtrated and cultured on non-nutrient agar (NNA). Morphologically positive Nagleria-like isolates were subjected to Nagleria genus and species-specific PCR targeting rDNA gene, PCR products were sequenced and obtained sequences were phylogenetic analyzed. RESULTS: Nile River water was the only source found to contained Naegleria. For the first time in Egypt, Vahlkampfia ciguana and the Naegleria species N.australiensis, N.philippinensis and N.neojejuensis were identified from the Nile water. The pathogenic Naegleria fowleri, previously reported in Egypt, was however not detected in this study. CONCLUSION: Interestingly, there were no seasonal variations in prevalence of Naegleria spp.; yet, there was seasonal diversity in the water samples of the same site. These newly discovered Vahlkampfiidae in Egyptian aquatic environments indicate the need for further phylogenetic investigations using bigger sample sizes in order to determine their potential risk for human health.

Identification and characterisation of a cryptic Golgi complex in Naegleria gruberi.

Although the Golgi complex has a conserved morphology of flattened stacked cisternae in most eukaryotes, it has lost the stacked organisation in several lineages, raising the question of what range of morphologies is possible for the Golgi. In order to understand this diversity, it is necessary to characterise the Golgi in many different lineages. Here, we identify the Golgi complex in Naegleria, one of the first descriptions of an unstacked Golgi organelle in a non-parasitic eukaryote, other than fungi. We provide a comprehensive list of Golgi-associated membrane trafficking genes encoded in two species of Naegleria and show that nearly all are expressed in mouse-passaged N. fowleri cells. We then study distribution of the Golgi marker (Ng)CopB by fluorescence in Naegleria gruberi, identifying membranous structures that are disrupted by Brefeldin A treatment, consistent with Golgi localisation. Confocal and immunoelectron microscopy reveals that NgCOPB localises to tubular membranous structures. Our data identify the Golgi organelle for the first time in this major eukaryotic lineage, and provide the rare example of a tubular morphology, representing an important sampling point for the comparative understanding of Golgi organellar diversity.This article has an associated First Person interview with the first author of the paper.

Morphology and Phylogenetic Analyses of Three Novel Naegleria Isolated from Freshwaters on Jeju Island, Korea, During the Winter Period.

The genus Naegleria is one of the best known heterolobosean groups, and is the causative agent of primary amoebic meningoencephalitis. This group is rarely studied in temperate regions during winter. Here, three novel Naegleria were isolated from freshwaters on Jeju Island, Korea, during winter. Two isolates were amoeboflagellates, and one of the three amoebae did not undergo enflagellation. All amoebae had eruptive pseudopodia, and the layer of refractile granules around a large nucleus. They formed a cyst with ~2 pores in the cyst stage. The amoeboflagellate form had two flagella and no division in the flagellate stage, and no cytostome. These features are very similar to typical Naegleria. Furthermore, our isolates were able to grow at > 30 °C, suggesting that they had different thermophilicity from Naegleria in polar regions. All amoebae were largely encysted at 5 or 10 °C, indicating that they were likely encysted during winter. Based on the 18S rRNA gene and the ITS1-5.8S rRNA gene-ITS2 sequences, the phylogenetic analyses consistently revealed that the isolates are members of the Naegleria group. However, the isolates differ from other species in both phylogenetic trees. Thus, Naegleria in cold habitats appeared to have a high degree of novelty, but their thermophilicity may be dependent on locality.

Amphotericin B induces apoptosis-like programmed cell death in Naegleria fowleri and Naegleria gruberi.

Naegleria fowleri and Naegleria gruberi belong to the free-living amoebae group. It is widely known that the non-pathogenic species N. gruberi is usually employed as a model to describe molecular pathways in this genus, mainly because its genome has been recently described. However, N. fowleri is an aetiological agent of primary amoebic meningoencephalitis, an acute and fatal disease. Currently, the most widely used drug for its treatment is amphotericin B (AmB). It was previously reported that AmB has an amoebicidal effect in both N. fowleri and N. gruberi trophozoites by inducing morphological changes that resemble programmed cell death (PCD). PCD is a mechanism that activates morphological, biochemical and genetic changes. However, PCD has not yet been characterized in the genus Naegleria. The aim of the present work was to evaluate the typical markers to describe PCD in both amoebae. These results showed that treated trophozoites displayed several parameters of apoptosis-like PCD in both species. We observed ultrastructural changes, an increase in reactive oxygen species, phosphatidylserine externalization and a decrease in intracellular potassium, while DNA degradation was evaluated using the TUNEL assay and agarose gels, and all of these parameters are related to PCD. Finally, we analysed the expression of apoptosis-related genes, such as sir2 and atg8, in N. gruberi. Taken together, our results showed that AmB induces the morphological, biochemical and genetic changes of apoptosis-like PCD in the genus Naegleria.

Rapid centriole assembly in Naegleria reveals conserved roles for both de novo and mentored assembly.

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.

Identification of a cell cycle-dependent duplicating complex that assembles basal bodies de novo in Naegleria.

During the differentiation of the amoeba Naegleria pringsheimi into a flagellate, a transient complex containing γ-tubulin, pericentrin-like protein, and myosin II (GPM complex) is formed, and subsequently a pair of basal bodies is assembled from the complex. It is not understood, however, how a single GPM is formed nor how the capability to form this complex is acquired by individual cells. We hypothesized that the GPM is formed from a precursor complex and developed an antibody that recognizes Naegleria (Ng)-transacylase, a component of the precursor complex. Immunostaining of differentiating cells showed that Ng-transacylase is concentrated at a site in the amoeba and that γ-tubulin is transiently co-concentrated at the site, suggesting that the GPM is formed from a precursor, GPMp, which contains Ng-transacylase and is already present in the amoeba. Immunostaining of growing N. pringsheimi with Ng-transacylase antibody revealed the presence of one GPMp in interphase cells, but two GPMps in mitotic cells, suggesting that N. pringsheimi maintains one GPMp per cell by duplicating and segregating the complex according to its cell cycle. Our results demonstrate the existence of a cell cycle-dependent duplicating complex that provides a site for the de novo assembly of the next generation of basal bodies.

Ancestral centriole and flagella proteins identified by analysis of Naegleria differentiation.

Naegleria gruberi is a single-celled eukaryote best known for its remarkable ability to form an entire microtubule cytoskeleton de novo during its metamorphosis from an amoeba into a flagellate, including basal bodies (equivalent to centrioles), flagella and a cytoplasmic microtubule array. Our publicly available full-genome transcriptional analysis, performed at 20-minute intervals throughout Naegleria differentiation, reveals vast transcriptional changes, including the differential expression of genes involved in metabolism, signaling and the stress response. Cluster analysis of the transcriptional profiles of predicted cytoskeletal genes reveals a set of 55 genes enriched in centriole components (induced early) and a set of 82 genes enriched in flagella proteins (induced late). The early set includes genes encoding nearly every known conserved centriole component, as well as eight previously uncharacterized, highly conserved genes. The human orthologs of at least five genes localize to the centrosomes of human cells, one of which (here named Friggin) localizes specifically to mother centrioles.

Naegleria gruberi de novo basal body assembly occurs via stepwise incorporation of conserved proteins.

Centrioles and basal bodies are discrete structures composed of a cylinder of nine microtubule triplets and associated proteins. Metazoan centrioles can be found at mitotic spindle poles and are called basal bodies when used to organize microtubules to form the core structure of flagella. Naegleria gruberi, a unicellular eukaryote, grows as an amoeba that lacks a cytoplasmic microtubule cytoskeleton. When stressed, Naegleria rapidly (and synchronously) differentiates into a flagellate, forming a complete cytoplasmic cytoskeleton de novo, including two basal bodies and flagella. Here, we show that Naegleria has genes encoding conserved centriole proteins. Using novel antibodies, we describe the localization of three centrosomal protein homologs (SAS-6, gamma-tubulin, and centrin-1) during the assembly of the flagellate microtubule cytoskeleton. We also used these antibodies to show that Naegleria expresses the proteins in the same order as their incorporation into basal bodies, with SAS-6 localizing first, followed by centrin and finally gamma-tubulin. The similarities between basal body assembly in Naegleria and centriole assembly in animals indicate that mechanisms of assembly, as well as structure, have been conserved throughout eukaryotic evolution.

Recognition of Naegleriae ameba surface protein epitopes by anti-human CD45 antibodies.

Phagocytosis is a highly conserved mechanism exhibited by both free-living amebas and mammalian blood cells. Similarities demonstrated by either cell type during engulfment of the same bacterial species may imply analogous surface proteins involved in receptor-mediated endocytosis. The increased availability of anti-human leukocyte antibodies or clusters of differentiation (CD) markers used in conjunction with flow cytometric (FCM) and/or immunohistochemical (IHC) analysis provides investigators with a relatively easy method to screen different cell populations for comparable plasma membrane proteins. In this study, we incubated Naegleria and Acanthamoeba amebas with several directly conjugated anti-human leukocyte monoclonal antibodies (mAb) for similarly recognized amebic epitopes. CD marker selection was based upon a recognized role of each mAb in phagocyte activation and/or uptake of bacteria. These included CD14, CD45, and CD206. In FCM, only one CD45 antibody demonstrated strong reactivity with both Naegleria fowleri and Naegleria gruberi that was not expressed in similarly tested Acanthamoeba species. Additional testing of N. gruberi by IHC demonstrated reactivity to a different CD45 antibody. Our results suggest a possible utility of using anti-human leukocyte antibodies to screen amebic cells for similarly expressed protein epitopes. In doing so, several important items must be considered when selecting potential mAbs for testing to increase the probability of a positive result.

Detection of free living amoebae, Acanthamoeba and Naegleria, in swimming pools, Malaysia.

This study reports the detection of Acanthamoeba and Naegleria species in 14 swimming pools around Petaling Jaya and Kuala Lumpur, Malaysia. Sampling was carried out at 4 sites (the platforms (P), wall (W), 1 meter from the wall (1) and middle (2)) of each swimming pool. These free living amoebae (FLA) were detected under light and inverted microscopes after being cultured on the surface of non-nutrient agar lawned with Escherichia coli. Acanthamoeba species were detected in higher number of culture plates from all sampling sites of all the swimming pools. While Naegleria, were detected in fewer culture plates at 3 sampling sites (absent at site P) of 8 swimming pools. This suggested that the thick double-walled cysts of Acanthamoeba were more resistant, thus remaining viable in the dry-hot areas of the platforms and in chlorinated water of the swimming pools whereas Naegleria cysts, that are fragile and susceptible to desiccation, preferred watery or moist areas for growth and proliferation. The prevalence of both FLA was highest at site W (76.2%), followed by site 1 (64.7%), lowest at site 2 (19.4%), and could be detected at all 3 sampling levels (top, middle and bottom) of these 3 sites. The surface of site W might act as a bio-film that accumulated all kinds of microbes providing sufficient requirement for the FLA to develop and undergo many rounds of life cycles as well as moving from top to bottom in order to graze food. Other factors such as human activities, the circulating system which was fixed at all swimming pools, blowing wind which might carry the cysts from surroundings and the swimming flagellate stage of Naegleria could also contribute to the distribution of the FLA at these sampling sites. Both FLA showed highest growth (80.4%) at room temperature (25-28 ºC) and lesser (70.0%) at 37 ºC which might be due to the overgrowth of other microbes (E. coli, fungi, algae, etc). While at 44 ºC, only Acanthamoeba species could survive thus showing that our swimming pools are free from potentially pathogenic Naegleria species. However, further study is needed in order to confirm the virulence levels of these amoebae isolates.

De novo formation of basal bodies during cellular differentiation of Naegleria gruberi: progress and hypotheses.

Under defined laboratory conditions, Naegleria gruberi undergo an amoeba-to-flagellate differentiation. During this differentiation, N. gruberi changes its shape from an amorphous amoeba to a regular shaped flagellate and forms de novo a flagellar apparatus, which is composed of two basal bodies, two flagella, a flagellar rootlet, and cytoplasmic microtubules. The entire process is accomplished within 2h after initiation of differentiation and more than 95% of cells in the population undergo this differentiation. This rapid and synchronous differentiation of N. gruberi provides us with a unique system in which we can study the process of de novo basal body assembly. In this review, I summarize recent findings associated with de novo basal body assembly and propose a hypothesis to explain how N. gruberi assemble two basal bodies per cell, which is what happens in the majority of cells.

Isolation and genotyping of potentially pathogenic Acanthamoeba and Naegleria species from tap-water sources in Osaka, Japan.

Here, we carried out a survey to determine the prevalence of free-living amoebae (FLA) in tap-water sources from rivers and water treatment plants located in Osaka Prefecture, Japan. A total of 374 raw water samples were collected from 113 sampling points. The samples were filtrated and transferred to non-nutrient agar plates seeded with a heat-killed suspension of Escherichia coli and incubated for 2 to 7 days at 30 degrees C or 42 degrees C. The plates were examined by microscopy to morphologically identify FLA families, and polymerase chain reaction and sequence analysis were then performed to define the species of the detected Naegleria and Acanthamoeba isolates. A total of 257 of 374 samples (68.7%) were positive for FLA by microscopy, and among these there were 800 FLA isolates, including Acanthamoeba and Naegleria species. Sequence analysis identified five Acanthamoeba spp. isolates of the known pathogenic T4 genotype and 43 Naegleria australiensis isolates, a reported pathogen to mice and also of concern as a potential pathogen to humans. Our results suggest a wide distribution of FLA, including potential pathogenic species, in tap-water sources of western Japan.

NgUNC-119, Naegleria homologue of UNC-119, localizes to the flagellar rootlet.

The UNC-119 family of proteins is ubiquitous in animals. The expression of UNC-119 is prominent in neural tissues including photoreceptor cells. Homologues of UNC-119 are also found in ciliated (or flagellated) single-celled organisms; however, the cellular distribution of this protein in protists is unknown. We cloned and characterized a homologue of unc-119 from the ameboflagellate Naegleria gruberi (Ngunc-119) and identified the cellular distribution of the protein. The Ngunc-119 open reading frame contained 570 nucleotides encoding a protein of 189 amino acids with a predicted molecular weight of 22.1 kDa, which is similar to that of Paramecium UNC-119 and Trypanosoma UNC-119. These three proteins are 46-48% identical in their amino acid sequences. The smaller NgUNC-119 corresponds to the conserved C-terminal 3/4 of the UNC-119 from multi-cellular organisms. The amino acid sequence of NgUNC-119 is 43-50% identical to that of the conserved C-terminal regions. NgUNC-119 was not found in growing amoebae but accumulated rapidly after the initiation of differentiation into flagellates. Indirect immunofluorescence staining of differentiating N. gruberi showed that NgUNC-119 begins to concentrate at a spot near the nucleus of differentiating cells and then elongates into a filamentous structure. Purification and indirect immunofluorescence staining of the Naegleria flagellar rootlet suggested that NgUNC-119 is a component of the flagellar rootlet.

The role of actin, actomyosin and microtubules in defining cell shape during the differentiation of Naegleria amebae into flagellates.

Differentiation of Naegleria amebae into flagellates was used to examine the interaction between actin, actomyosin and microtubules in defining cell shape. Amebae, which lack microtubules except during mitosis, differentiate into flagellates with a fixed shape and a complex microtubule cytoskeleton in 120 min. Based on earlier models of ameboid motility it has been suggested that actomyosin is quiescent in flagellates. This hypothesis was tested by following changes in the cytoskeleton using three-dimensional reconstructions prepared by confocal microscopy of individual cells stained with antibodies against actin and tubulin as well as with phalloidin and DNase I. F-actin as defined by phalloidin staining was concentrated in expanding pseudopods. Most phalloidin staining was lost as cells rounded up before the onset of flagellum formation. Actin staining with a Naegleria-specific antibody that recognizes both F- and G-actin was confined to the cell cortex of both amebae and flagellates. DNase I demonstrated G-actin throughout all stages. Most of the actin in the cortex was not bound by phalloidin yet was resistant to detergent extraction suggesting that it was polymerized. The microtubule cytoskeleton of flagellates was intimately associated with this actin cortex. Treatment of flagellates with cytochalasin D produced a rapid loss of flagellate shape and the appearance of phalloidin staining while latrunculin A stabilized the flagellate shape. These results suggest that tension produced by an actomyosin network is required to maintain the flagellate shape. The rapid loss of the flagellate shape induced by drugs, which specifically block myosin light chain kinase, supports this hypothesis.

[Infections caused by free-living amebas. Historical commentaries, taxonomy and nomenclature, protozoology and clinicopathologic features]. / Infecciones por amebas de vida libre. Comentarios históricos, taxonomía y nomenclatura, protozoología y cuadros anátomo-clínicos.

Infections caused by free-living amebae constitute one of emergent opportunistic infections with greatest medical interest. Although infrequently, they have been described in almost all world, its diagnosis depends on a high index of suspicion, especially in morpho-pathologic and laboratory studies. Exciting historical features of infections due to free-living amebae, its taxonomy and the present nomenclature are briefly reviewed. An analysis of the protozoology of the most frequent agents is done and, based on the author's own experience and the published one, already established anatomo-clinical entities are described: the primary amebic meningoencephalitis, granulomatous amebic encephalitis, Acanthamoeba keratitis, cutaneous acanthamoebiasis, disseminated infection and other rare isolated locations.

Transient concentration of a gamma-tubulin-related protein with a pericentrin-related protein in the formation of basal bodies and flagella during the differentiation of Naegleria gruberi.

The distribution of two proteins in Naegleria gruberi, N-gammaTRP (Naegleria gamma-tubulin-related protein) and N-PRP (Naegleria pericentrin-related protein), was examined during the de novo formation of basal bodies and flagella that occurs during the differentiation of N. gruberi. After the initiation of differentiation, N-gammaTRP and N-PRP began to concentrate at the same site within cells. The percentage of cells with a concentrated region of N-gammaTRP and N-PRP was maximal (68%) at 40 min when the synthesis of tubulin had just started but no assembled microtubules were visible. When concentrated tubulin became visible (60 min), the region of concentrated N-gammaTRP and N-PRP was co-localized with the tubulin spot and then flagella began to elongate from the region of concentrated tubulin. When cells had elongated flagella, the concentrated N-gammaTRP and N-PRP were translocated to the opposite end of the flagellated cells and disappeared. The transient concentration of N-gammaTRP coincided with the transient formation of an F-actin spot at which N-gammaTRP and alpha-tubulin mRNA were co-localized. The concentration of N-gammaTRP and formation of the F-actin spot occurred without the formation of microtubules but were inhibited by cytochalasin D. These observations suggest that the regional concentration of N-gammaTRP and N-PRP is mediated by actin filaments and might provide a site of microtubule nucleation for the assembly of newly synthesized tubulins into basal bodies and flagella.

Stable intermediates and holdpoints in the rapid differentiation of Naegleria.

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.

Independent mechanisms are utilized for the coordinate and transient accumulation of two differentiation-specific mRNAs during differentiation of Naegleria gruberi amoebae into flagellates.

During the differentiation of Naegleria gruberi amoebae into flagellates, four differentiation-specific (DS) mRNAs are transiently and coordinately accumulated. Three of the four DS mRNAs, Class II, III, and IV, encode alpha-tubulin, beta-tubulin, and flagellar calmodulin, respectively. The protein product of the Class I mRNA has not been identified. We examined the effects of inhibition of protein synthesis on transcription and accumulation of beta-tubulin mRNA and Class I mRNA to understand the mechanism of coordinate regulation. Inhibition of protein synthesis at the beginning of differentiation completely blocked transcription of the beta-tubulin gene. Addition of cycloheximide at 30 or 40 min after initiation of differentiation inactivated transcription of the beta-tubulin gene in less than 10 min as judged by nuclear run-on experiments. However, once differentiation had proceeded for more than 50 min, inhibition of protein synthesis did not inactivate transcription of beta-tubulin mRNA was more active in cycloheximide-treated cells than in control cells. Cycloheximide treatment at the initiation of the differentiation also blocked transcription of the Class I gene. However, addition of the drug after 30 min had no significant effect on the transcription of the Class I gene. Cycloheximide treatment also increased the half-lives of beta-tubulin and Class I mRNA drastically. These data suggest that: (1) the transient accumulation of the two DS mRNAs during differentiation are regulated by changing both the rate of transcription and the stability of the mRNAs; (2) protein synthesis is required for the transcriptional and post-transcriptional regulations; (3) the transcriptional regulation mechanisms of the beta-tubulin gene and that of the Class I gene are distinct; and (4) the transcription of the beta-tubulin gene is regulated by different mechanisms during differentiation.

A flagellar calmodulin gene of Naegleria, coexpressed during differentiation with flagellar tubulin genes, shares DNA, RNA, and encoded protein sequence elements.

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.