©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Flagellar Calmodulin Gene of Naegleria, Coexpressed during Differentiation with Flagellar Tubulin Genes, Shares DNA, RNA, and Encoded Protein Sequence Elements (*)

(Received for publication, March 22, 1994; and in revised form, January 3, 1995)

Chandler Fulton (§) Elaine Y. Lai Stephen P. Remillard

From the Department of Biology, Brandeis University, Waltham, Massachusetts 02254

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Most eukaryotes manage diverse calcium-regulated functions through the intermediary of a single calmodulin. Single genes encode calmodulin in fungi(1, 2, 3) , in diverse protists including the water mold Achyla(4) , the cellular slime mold Dictyostelium(5, 6) , the ciliate Paramecium(7) , and the malaria parasite Plasmodium(8) , in the alga Chlamydomonas(9) , in the mollusc Aplysia(10) , and in Drosophila, a metazoan genome where multiple calmodulin genes were methodically sought(11, 12) . Vertebrates from fish to mammals contain multiple calmodulin genes, but these all encode an identical amino acid sequence (13, 14, 15, 16) . Trypanosomes also encode one calmodulin, in this case employing multiple, tandemly repeated genes(17, 18) . Although organisms also contain a multiplicity of related, specialized calcium-binding proteins in the calmodulin superfamily, there are only a few known exceptions to a single authentic calmodulin per organism. Chickens and humans each have a provocative, intronless ``retropseudogene,'' which shows limited, tissue-specific expression of a calmodulin-related protein(19, 20, 21, 22, 23) . Two distinct bona fide calmodulins were found in eggs of the sea urchin Arbacia punctulata but not in sperm of the same species or in eggs of another species, Strongylocentrotus purpuratus(24) . The two Arbacia egg calmodulins, the products of separate genes, differ in four of 148 amino acids(25) . In the plant Arabidopsis thaliana six genes produce four distinct isoforms of calmodulin that differ in, at most, six amino acids(26, 27, 28) .

Among unicellular organisms, an exception to one calmodulin per organism was found in the amoeboflagellate Naegleria gruberi, where two calmodulins are synthesized during rapid differentiation from amoebae to flagellates(29) . The ``major'' differentiation-specific calmodulin, CaM-1, (^1)which amounts to leq0.01% of the total flagellate cell protein, is specifically localized in the flagella. The second calmodulin, CaM-2, present in about one-third the amount of CaM-1 and apparently smaller, is localized in the flagellate cell body. The intracellular segregation of the two calmodulins appears precise; although only small amounts of each calmodulin are present, no CaM-1 was detected in the cell body and no CaM-2 in the flagella(29) . Each of these polypeptides is a bona fide calmodulin by several criteria, most decisively by its ability to activate calmodulin-dependent vertebrate cyclic nucleotide phosphodiesterase in a calcium- and calmodulin-dependent manner and by its ability to be recognized by antibodies to vertebrate calmodulin that specifically react with calmodulins(29) . In addition to the difference in intracellular location, the only other known difference in the two calmodulins is apparent molecular weight; these calmodulins are easily distinguished by mobility on SDS-polyacrylamide gel electrophoresis (M(r) approx 16,000 and 15,300). It is unlikely that one calmodulin is derived post-translationally from the other since both calmodulins are synthesized in the wheat germ cell-free system directed by mRNA from differentiating Naegleria. Several possible origins for the two calmodulins were considered in the initial study(29) , but it was not possible to decide whether they are encoded by one gene or two.

Expression of these two Naegleria calmodulins is differentiation specific. Translatable mRNAs were not detected in amoebae, were first seen after 10-20 min of differentiation, reached maximum abundance at 60 min at the time flagella appear, and then rapidly decreased in abundance(29) . This timetable matches the programmed appearance and disappearance of the flagellar alpha- and beta-tubulin mRNAs(30, 31) . For tubulin, the increase in mRNA abundance involves switching on of transcription(32) , and the decrease is due to the disappearance of the mRNA sequences with a half-life of 8 min(33) . The contemporaneous rise and fall in abundance of flagellar alpha- and beta-tubulin and flagellar calmodulin mRNAs indicates concurrent regulation of these genes, presumably by coordinate regulation both of transcription and of mRNA stability. Concurrent regulation is not a universal feature of this differentiation program(34) . The coordinate regulation of the evolutionarily unrelated calmodulin and tubulin genes suggests that one might find clues to their regulation by comparing the sequences of one gene or gene product to the other. Representative alpha-tubulin and beta-tubulin genes expressed during differentiation have been cloned(33, 35) , and the cloning of a calmodulin gene makes this comparison feasible.

In addition to their concurrent syntheses, the translated products of the flagellar calmodulin gene as well as the flagellar alpha- and beta-tubulin genes become localized in the peninsular flagella. Little is known about how products become localized in eukaryotic flagella and cilia, but proteins might be expected to contain signals to direct this localization.

The finding of two differentiation-specific and precisely localized calmodulins in a unicellular organism raises several questions. One obvious question, the function of these calmodulins, remains a challenge for future investigations. Other questions can be addressed now. What is the difference between the two calmodulins, and are they the products of one gene or two? Do they possess the sequences of authentic calmodulins? Can elements in the sequences be identified that could account for the coordinate regulation of the tubulin and calmodulin genes? What special features of these calmodulins might account for their intracellular localizations and especially for the location of flagellar calmodulin and flagellar tubulins within the flagellum? As a next step toward answering these questions, we here report the cloning and sequencing of a calmodulin gene expressed during differentiation, together with evidence that it encodes flagellar calmodulin (CaM-1). The single intronless gene encodes a typical conserved calmodulin except for a unique amino-terminal extension. This single gene is quite divergent from the distinct (but not yet cloned) gene that encodes the cell body calmodulin (CaM-2) of flagellates. The coordinate expression of these genes is confirmed and quantitated more precisely than was possible using translatable mRNAs. By comparison with the sequences of coexpressed and colocalized flagellar alpha- and beta-tubulin, provocative sequence segments are identified in the gene sequences, in the encoded mRNAs, and in the expressed proteins that are candidates to regulate the coordinate expression of flagellar calmodulin and tubulin genes, the stability of their mRNAs, and the localization of the products in flagella.


MATERIALS AND METHODS

Standard Cell and Molecular Biological Reagents and Techniques

N. gruberi NEG (36) was used in all experiments, and synchronous differentiation of amoebae to flagellates at 25 °C was accomplished as described previously(30) . Isolation of DNA and of RNA from Naegleria, the cDNA and genomic libraries used in this study, and procedures for qualitative and quantitative genomic and RNA blots have been described(33) . In the experiment shown in Fig. 3B, the autoradiogram was scanned and analyzed using an LKB Ultrascan XL.


Figure 3: A single genomic sequence is homologous to calmodulin gene CAM1.A, restriction map of the 2.2-kb BglII insert of genomic clone pNCaM1, with the sequenced region (Fig. 1) boxed and coding region shaded. (The sequence of cDNA clone 22E9 matches between the arrows but also has a useful upstream PvuII site, shown in brackets, that is part of the junction with the vector and thus is not in the genomic clone.) B, genomic Southern blot. Total Naegleria genomic DNA was digested to completion with the indicated restriction endonucleases, and aliquots of 2.0 µg were placed in the indicated lanes of a 0.8% agarose gel. In lane4, 1 copy eq of the CAM1 gene from pNCaM1 was added to the 2.0 µg of genomic DNA as an internal control. In addition, pNCaM1 linearized with BglII in amounts equivalent to the indicated multiples of the CAM1 gene were loaded in lanes5-9 (0.5-4 copy eq of the CAM1 sequence, calculated as (33) ). The sizes of fragments were determined using DNA digested with HindIII in another lane. After electrophoresis, the DNA was transferred onto nitrocellulose and hybridized to the P-labeled 0.34-kb RsaI-EcoRI fragment of the cDNA insert. Inset, copy eq in lanes 5-9 (triangles) used to determine number of copies found in the 2.2-kb fragments in lanes3 (circle) and 4 (square).




Figure 1: The nucleotide and deduced amino acid sequence of the CAM1 gene of N. gruberi strain NEG. Both strands of the genomic DNA clone were sequenced. The cDNA clone, of which at least one strand was sequenced, matched the genomic sequence from the uparrow to the downarrow, after which the cDNA sequence continued with 35 A residues. In the last upstream line before the coding sequence a candidate TATA element is double-underscored, and downstream of the stop codon a candidate polyadenylation signal is underlined. The other underlined elements are provocative matches to sequences in the flagellar tubulin genes and proteins, as discussed in the text.



Minimal Hybridization Stringency Used to Isolate CaM cDNA Clones

In order to find the Naegleria calmodulin cDNA clones using a heterologous probe, we used newly devised permissive hybridization conditions that gave an improved signal to noise ratio. These conditions were based primarily on the studies of Howley et al. ((37) see also (38) ) and of Singh and Jones (39) . Hybridization was in 5 times SSPE(40) , 20% formamide, 0.2% SDS, 100 µg/ml heparin, with Escherichia coli and vector DNA added to minimize background, at 35 °C for 18 h followed by extensive washing of the filters in 5 times SSC at 50 °C.

Construction of pNCaM1

In order to subclone the 2.2-kb BglII fragment containing genomic calmodulin gene CAM1 from the genomic clone with a approx15-kb insert, pBR322 was modified such that the BalI site at nucleotide 1444 was changed to a BglII site. Plasmid pBR322 was digested with BalI, and the flush ends were ligated to phosphorylated BglII linker (New England BioLabs Inc., Beverly, MA; linker d(pCAGATCTG)). The modified vector was linearized with BglII and ligated with the 2.2-kb CAM1 fragment. The resulting clone, pNCaM1, was shown to contain a single copy of the 2.2-kb insert by using partial digestion with BglII. Ladders of multiples of 2.2-kb fragments were not seen, which eliminates the possibility of tandem repeats of the insert in the clone.

DNA Sequencing

The cDNA clone 22E9 was sequenced in one direction by the dideoxynucleotide chain termination method (41) after directionally subcloning fragments into bacteriophage M13mp8, -9, -18, or -19 using E. coli JM103 or 109 as hosts(42) . The insert from pNCaM1 containing the genomic gene was sequenced after subcloning the 2.2-kb BglII fragment into the BamHI site of M13mp18. Two nested sets of exonuclease III deletion clones were generated from the replicative form plasmids using the unique SphI and SalI sites as described by Henikoff(43) . The sequence in Fig. 1ends at the hybrid BglII-BamHI junction.

Hybridization Selection of Calmodulin mRNA for Cell-free Translation

The procedure was based on Ricciardi et al.(44) . Five µg of linearized CaM-1 cDNA clone was spotted onto each of three 1-cm^2 squares of nitrocellulose, denatured by successive treatments with 0.5 N NaOH, 1 M Tris-HCl, pH 8.0, and 6 times SSC(40) , air dried, and then baked in vacuo for 2 h at 80 °C. The three filters were rewetted with sterile distilled water, cut into approx2-mm squares using a razor blade, placed in a 1.5-ml microcentrifuge tube, washed with distilled water, and air dried. Hybridization was carried out by adding 15 µg of poly(A)-containing RNA isolated at 60 min of differentiation (30) in 50% formamide (final concentration) and hybridization buffer (10 mM PIPES, pH 6.4, 0.4 M NaCl, 0.15% SDS). The mixture was incubated at 42 °C overnight. The filters were then washed extensively at 55 °C to remove unhybridized RNA, first with 1 times SSC, 0.5% SDS and then with 2 mM EDTA. The hybridized RNA was finally released by boiling the filters in distilled water followed by quick freezing in a dry ice-ethanol bath. This RNA was used to direct cell-free synthesis in the wheat germ system, using [S]methionine (DuPont NEN, NEG-009T) to label the translation products, as described(30) . Samples of the translation products also were immunoprecipitated as described(30) , using either a monoclonal antibody to Dictyostelium calmodulin, 6D4(45) , kindly supplied by Dr. Margaret Clarke, or an affinity-purified polyclonal antibody to Naegleria centrin as expressed in E. coli. (^2)The translation products were analyzed by SDS-polyacrylamide gel electrophoresis designed to separate the calmodulins(29) , followed by autoradiography(30) .

Hybrid-arrested Cell-free Translation Using Antisense Oligonucleotides

Antisense oligonucleotide-arrested translation was performed as described(46) . The sense and antisense 12-mers used in this study were synthesized and purified twice by high pressure liquid chromatography by Dr. Rolf Heumann. 20 µM oligonucleotide or distilled water was heated in the presence of 0.58 µg of poly(A)-enriched mRNA isolated at 60 min of differentiation (30) in 7 mM Tris-HCl, pH 7.5, at 80 °C for 2 min. The oligomers were then allowed to anneal with the mRNA for 2 h at 4 °C. Cell-free translation was initiated by adding [S]methionine to a wheat germ cell-free translation system(30) . Translation was allowed to proceed for 2 h at 23 °C and subsequently terminated by the addition of NaOH. Trichloroacetic acid-precipitable counts were determined.

To display the heat-stable translation products, an equal volume of distilled water was added to each product, and it was heated at 90 °C for 2 min and then quickly chilled in ice water (0 °C) for 5 min. After centrifugation at 12,000 rpm for 20 min in a Beckman JA-20 rotor at 4 °C, the supernatant was removed and divided equally into two aliquots. To one aliquot, 1 mM CaCl(2) and Laemmli sample buffer were added. To the other aliquot, 1 mM EGTA was added instead of CaCl(2). The samples were immediately mixed on a vortex mixer, placed in boiling water for 2 min, cooled to room temperature, and then loaded onto a 15% Laemmli SDS-polyacrylamide gel as described(29) . Autoradiograms were exposed overnight.


RESULTS

Naegleria Calmodulin DNA Clones

Our first success in isolating a calmodulin gene expressed during Naegleria differentiation was obtained using a heterologous probe, a cDNA clone to Xenopus calmodulin (clone 71 of Chien and Dawid(13) ), under permissive hybridization conditions. We screened our ordered library of cDNA clones prepared to RNA at 60 min of differentiation (33) , when the translatable calmodulin mRNAs are most abundant(29) . Two positive clones were found among 5444; complete sequencing of one (22E9) and partial sequencing of the other revealed that they both encoded the same calmodulin. Clone 22E9 contains a calmodulin cDNA from its 5`-untranslated region to a 36-nucleotide poly(A) tail, but the cDNA is inserted in reverse orientation to that predicted based on the method used to construct the cDNA library(47) ; the clone also contains a second insert of an unidentified DNA immediately after the poly(A) tail of the calmodulin cDNA, a double-insert combination that presumably arose due to a recombinational event. Since we did not wish to depend on the sequence of an aberrant clone, clone 22E9 was used to isolate a genomic DNA clone from a library of Naegleria DNA in EMBL3(33) . The selected genomic clone contained a approx15 kb insert from which we subcloned a 2.2-kb segment that spans the calmodulin coding region (clone pNCaM1).

The DNA Sequence Encodes a Bona Fide Calmodulin

The DNA sequence of the Naegleria genomic calmodulin gene CAM1 from pNCaM1, together with its encoded 17,601-dalton polypeptide, is shown in Fig. 1. The sequenced segment of cDNA clone 22E9 matches the genomic clone perfectly for 534 nucleotides that span the coding region (from arrow to arrow in Fig. 1). In the 5` region a putative TATA box is double-underlined. The deduced start codon has an A at -3 as expected for a eukaryotic translation start(48) . Codon usage is strongly biased, as in the Naegleria tubulin genes(33, 35) . For example, the 22 glutamates in CAM1 are all encoded by GAA, none by GAG. Downstream of the coding region the DNA sequence includes a polyadenylation signal (underlined) preceding the point where the cDNA clone sequence ends in a poly(A) tail immediately following the downarrow in Fig. 1.

After the first 10 amino acid residues, the protein encoded by CAM1 is colinear with calmodulins of other organisms, with no deletions or insertions and only scattered substitutions. The Naegleria sequence is compared with the sequence of vertebrate calmodulin in Fig. 2, using the conventional numbering of calmodulin residues. Over the span of residues 4-148, the encoded Naegleria protein shows 16 differences from vertebrate calmodulin; the vertebrate residues are shown in blackrectangles in Fig. 2. For most residues where the Naegleria sequence differs from vertebrate calmodulin similar substitutions have been found in one or more sequenced non-vertebrate calmodulins. At several positions (Phe-99, Ile-136, Lys-143, and Met-146) the Naegleria sequence rather than the vertebrate sequence has the residue most frequently found in the calmodulins of diverse eukaryotes. Two residues outside the putative Ca-binding loops are unusual; His-49 and Cys-110 substitute for the Gln and Thr found in most other calmodulins.


Figure 2: Amino acid sequence comparison of calmodulins encoded by Naegleria CAM1 (ovals) and by vertebrate calmodulin genes (differences in black rectangles). The residues are numbered based on vertebrate calmodulin, which differs from the numbering in Fig. 1because the Naegleria calmodulin has a distinctive amino-terminal extension (shown in boldface). The four potential Ca-binding loops (I-IV) are shown, including spokes to indicate probable Ca ligands.



The encoded Naegleria protein contains four putative Ca-binding domains typical of calmodulins (Fig. 2). Only one of the differences from vertebrate calmodulins would be expected to affect the Ca binding capacity of the ``EF-hands'' (criteria reviewed in (49) and (50) ). The inwardly directed hydrophobic residues in the E- and F-helices surrounding the loops are all identical to those in vertebrate calmodulin. The calcium-binding residues in the loops are also conserved or show substitutions found in other calmodulins. The most likely candidate to affect calcium binding is the Gly-134 Asn in the fourth domain. This substitution can be expected to perturb the backbone conformation of the loop at this position, where the glycine residue normally makes a sharp turn. A Gly at this position is conserved in almost all calmodulins and most calcium-binding loops of the calmodulin superfamily. Although this is the first time this substitution has been found in any calmodulin sequenced to date, it has been found in the EF-hands of two other proteins. The same substitution is found in the homologous position in domain IV of the basal body-associated calcium-binding protein caltractin (also known as centrin)(51) . This loop has been inferred to bind Ca(49, 51) although its ability to do so is unknown. However, the same substitution is also found in domain III of annelid (Nereis and Perinereis) sarcoplasmic calcium-binding protein(52, 53) , and this loop is known to bind Ca(54) . Only experimental measurements can determine whether each of the four loops in the encoded Naegleria protein actually binds calcium, but the loops are conserved in a fashion that allow us to predict this function with confidence.

The protein encoded by CAM1 seems as conserved as the calmodulins of most protists. For example, from residues 4-148, where Naegleria calmodulin shows 16 substitutions from vertebrate calmodulin (Fig. 2), the calmodulins of Chlamydomonas and Trypanosoma also each show 16 substitutions from vertebrate calmodulin, and that of Dictyostelium shows 12. Overall, among the calmodulins in the GenBank/EMBL or SwissProt data bases, the Naegleria calmodulin shows 85-92% identity to the calmodulins of diverse eukaryotes, including metazoa, metaphytes, the mushroom Pleurotus, Euglena, Dictyostelium, Trypanosoma, the oomycetes (water molds) Achlya and Phytophthora, and several ciliates, 83% to Chlamydomonas, 81% to Aspergillus, and 59% to Saccharomyces.

The most exceptional feature of the protein encoded by CAM1 is its extended amino terminus. In general the amino termini of proteins often are charged, flexible, exposed at the surface, and variable(55) . The first 4 residues of vertebrate calmodulin apparently are mobile, at least to the extent that they are poorly defined in the crystal structure of calmodulin(56) , yet these residues are absolutely conserved in vertebrate calmodulins over >500 million years of ``fish-to-mammal'' evolution, so they probably interact in important ways with other residues of calmodulin itself or those of other proteins. This terminus (also conserved in the calmodulins of invertebrates, plants, water molds, trypanosomes, the cornucopia mushroom Pleurotus, and the ciliates Tetrahymena and Stylonychia) is completely replaced in Naegleria (Fig. 2). The potential structure of the extended terminus of the Naegleria calmodulin was evaluated using the Chou-Fasman algorithm(57) , which predicts a strong potential for the first 6 residues to form an alpha-helix, the next four (Ser-Asn-Asn-Glu) to be involved in a beta-turn, and then the structure returns to an alpha-helix beginning at the Leu-4. Whatever actual structure this amino terminus forms, the presence of charged residues and serines makes it likely that this domain will be found on the outside of the protein, where it might interact with other parts of the calmodulin or with other proteins. Although many calmodulins share the sequence of the amino terminus of vertebrate calmodulin, others have extensions; the calmodulin of Dictyostelium has five amino acids before the conserved Leu-4 (6) and that of Chlamydomonas has six(9) . The calmodulin-related proteins caltractin/centrin of Chlamydomonas(51) and that encoded by cal-1 of Caenorhabditis(58) each has a long amino terminus. However, none of these amino termini are related in sequence to the extension seen in the Naegleria calmodulin.

The genomic DNA of CAM1, its cDNA, and the encoded calmodulin are congruent, so the protein is encoded by a single exon. Introns are rare in protein-coding genes of Naegleria; the only examples so far are two introns in a calcineurin B gene, which have typical splice junction sequences(59) . One possible explanation of the origin of the two calmodulins in Naegleria flagellates was considered to be alternate splicing(29) . Precedence for this is found in the myosin essential light chains of striated muscle, where two isoforms with different amino termini are produced by alternate splicing(60) . We searched the sequences of CAM1, both upstream (nucleotides 1-800; Fig. 1) and downstream (nucleotides 1060-1680), for any possible alternative start or stop codons, as well as for any donor-acceptor junctions that might be utilized to encode a second calmodulin. We found no evidence within this gene for any exon that would provide an alternative amino or carboxyl terminus for this calmodulin. The sequencing results show that CAM1 is an intronless gene that encodes a single conserved calmodulin with a novel amino terminus.

This Calmodulin Is Encoded by a Single Gene

In order to further define the relation of the two calmodulins, we determined the architecture and copy number of homologous calmodulin gene(s) in the genome. Naegleria DNA was digested to completion with each of several restriction endonucleases (BamHI, HpaI, PvuII, PstI) that have 6-base recognition sequences and that lack sites within the 2.2-kb pNCaM1 gene. The digested genomic DNA samples were blotted and then probed with a calmodulin DNA sequence (either the 0.34-kb RsaI-EcoRI fragment of the coding region (Fig. 3A) or the full 2.2-kb insert from pNCaM1). On each blot a single large genomic fragment was detected, with sizes of 14-18 kb. In no case were additional bands detected, even when the blots were hybridized and washed under minimal stringency conditions (the conditions described under ``Materials and Methods''). An example of such a large single band is shown in the leftlane of Fig. 3B, where the genomic DNA was cut using PvuII and a single band of approx15 kb was observed. HindIII cuts in the center of the coding region and again upstream, and thus is expected to yield one fragment of 0.6 kb and a second larger fragment (Fig. 3A); the expected two fragments were observed, of 6.7 and 0.6 kb. BglII cuts the genomic clone to give the 2.2-kb piece that was cloned in pNCaM1 (Fig. 3A); it also cuts the Naegleria genome to give a single band of the same size (Fig. 3B). These results indicate one of the following possibilities: (i) a single flagellar calmodulin gene; (ii) multiple genes conserved over approx15 kb of DNA; or (iii) short tandem repeats of a gene within a approx15-kb segment. If the gene were arrayed in short tandem repeats, as are calmodulin genes in trypanosomes(17) , partial digestion with BglII would produce a ladder of multiples of geq2.2 kb. Progressive digestion with BglII gave an increase in a 2.2-kb fragment and a decrease in heterogenous high molecular weight homologous DNA but no sign of a ladder (data not shown). Thus if there is a tandem repeat the units would have to be conserved over geq15 kb.

Quantitative genomic blots were performed to directly determine the number of homologous calmodulin genes in Naegleria. Two pilot experiments and the final experiment shown in Fig. 3supported the conclusion that CAM1 is a single-copy gene. It was possible to use the same-sized fragment, the 2.2-kb insert of pNCaM1, both to quantitate the standard and to titer the copy number in DNA digested to completion with BglII. As shown in Fig. 3B, the pNCaM1 insert was loaded on an agarose gel adjacent to 2.0-µg aliquots of BglII-digested total Naegleria DNA, with the plasmid DNA in amounts equivalent to from 0.5 to 4 copies of plasmid DNA per 2 µg of Naegleria DNA (based on the copy number calculation of (33) ). As an internal control for the quantitation, a sample of 2 µg of BglII-digested Naegleria DNA and 1 copy eq of plasmid DNA was loaded on a separate lane. The hybridization standard showed a linear relationship between band intensity and DNA loaded (Fig. 3B, inset). The number of homologous calmodulin genes measured 0.98 when the BglII-digested genomic DNA channel was compared with the standard hybridization curve, while the channel containing the mixture of 1 copy eq each of plasmid and genomic DNA measured 1.7 copies. The single bands, the lack of any indication of a tandem repeat, and titrations indicating the presence of a single copy establish that CAM1 is a unique gene in the Naegleria genome. This is also the first single-copy gene defined for Naegleria; for example the alpha-tubulin and beta-tubulin genes are both multicopy(33, 35) . In separate experiments, we determined that the CAM1 gene is located on one of the two largest chromosomes in N. gruberi(61) . Since CAM1 is a single-copy gene, both CaM-1 and CaM-2 must be encoded by this gene or, more likely, CaM-2 must be encoded by a gene sufficiently divergent that it was not detected under the hybridization conditions used.

Calmodulin Gene CAM1 Is Coordinately Expressed with alpha- and beta-Tubulin Genes

Previous experiments in which mRNAs were measured by translation (29) or by hybridization to partially characterized cDNA clones (31) suggested that the abundances of mRNAs for calmodulin(s) and for tubulins increase and then decrease concurrently during Naegleria differentiation. For studies of regulation of gene expression during differentiation, it is important to evaluate the precision with which the unrelated calmodulin and tubulin genes are contemporaneously expressed, that is to determine whether they really are on the same timetable. DNA probes to measure flagellar alpha- and beta-tubulin mRNAs have been described and their suitability for mRNA measurement evaluated(33, 35) .

Calmodulin clone pNCaM-1 recognizes a single-sized mRNA of 0.65 kb on Northern blots of RNA from differentiating cells, as shown in a sample of total 60-min RNA (Fig. 4A). No complementary RNA has been detected in RNA from amoebae (0 min); a similar absence of detectable RNA has been found for both alpha- and beta-tubulin(33, 35) . The abundance of each mRNA has been measured using quantitative RNA dot blots; a set of triplicate dots probed with CAM1 DNA is shown in Fig. 4C. As is seen in the measurements of mRNA abundance shown in Fig. 4B, the mRNA for CAM1 (circles), for alpha-tubulin (triangles), and for beta-tubulin (invertedtriangles), each is first detected within 10 min after the initiation of differentiation, increases to maximum abundance at 60 min, and then declines with an apparent half-life of 8 min. Within the limits of these measurements the rise and fall of these mRNAs expressed by the unrelated calmodulin and tubulin genes appear fully concurrent. The absence of detectable mRNAs at time zero, the rapidity of the rise in abundance, and the subsequent rapidity of the decline make this an unusually quick and striking example of gene expression in a eukaryotic differentiation.


Figure 4: Calmodulin CAM1 mRNA abundance during Naegleria differentiation. A, Northern blot showing the single-sized homologous RNA in total RNA extracted at 60 min of differentiation and the absence of a similar RNA at 0 min. The blot was probed with the 0.34-kb RsaI-EcoRI fragment of the CAM1 gene (Fig. 3A). RNA size was estimated using E. coli rRNA as a standard. B, calmodulin mRNA abundance (circle) compared with the abundance of alpha-tubulin (up triangle) and beta-tubulin (down triangle) mRNAs. Total mRNA was extracted from cells at 10-min intervals during differentiation, as described (30) , and the abundance of each mRNA was measured by quantitative dot hybridization (as (33) ). Probes were P-labeled inserts: for CAM1, the 0.34-kb fragment; for alpha-tubulin, the insert of pNalphaT1(33) ; and for beta-tubulin, the insert of pNbetaT1 (35) . The line was interpolated to show a linear rise in abundance of mRNAs from 10 to 40 min, and the decay curve fit to an exponential decrease with a half-life of 8 min. C, triplicate dots used in determination of the CAM1 mRNA abundance curve in B.



The CAM1 Gene Encodes the Previously Characterized CaM-1

The linearized calmodulin cDNA clone was used to select complementary mRNA from 60-min RNA by hybridization. The hybridization-selected RNA was translated in the wheat germ cell-free system, and the product was compared with those translated from total 60-min RNA (Fig. 5). The selected RNA directs the synthesis of a polypeptide that comigrates with the previously characterized flagellar calmodulin, CaM-1, as translated in total 60-min RNA (29) and also, unexpectedly, a larger calcium-binding polypeptide (Fig. 5, lanes 3 and 4). The CaM-1-sized polypeptide is precipitated by a monoclonal antibody to Dictyostelium calmodulin (lane5), while the larger polypeptide is precipitated by antibodies specific to Naegleria centrin (lane6). The products encoded by the selected mRNAs do not include a product that comigrates with the smaller flagellate cell body calmodulin, CaM-2. The simplest interpretation of these results is that CAM1 encodes CaM-1 and that its nucleotide sequence is sufficiently different from that of the gene encoding CaM-2 that its RNA was not selected under the conditions used, even though these conditions selected centrin/caltractin mRNA.


Figure 5: Selection of mRNAs homologous to CAM1 by hybrid selection followed by translation in the wheat germ cell-free system and immunoprecipitation. The translation products were processed to obtain the heat-stable components as described(29) . Lanes1 and 3 contain 1 mM Ca, and lanes2 and 4 contain 1 mM EGTA. Lanes1 and 2 are the translation products directed by total 60-min RNA; those in lanes3 and 4 are the products of hybrid-selected RNA. Lanes5 and 6 show the hybrid-selected translation products immunoprecipitated using antibodies to Dictyostelium calmodulin (lane5) and to Naegleria centrin (lane6). The positions of CaM-1, CaM-2, and centrin are marked.



Sequence Elements Shared among Coexpressed Genes and Colocalized Gene Products

Given the sequences of the coexpressed flagellar calmodulin and tubulin genes, whose products become localized in the flagella, we compared these sequences and found three elements of particular interest.

The Genes for Flagellar Calmodulin and Flagellar Tubulins Share Upstream Palindromic Elements

Upstream genomic sequences are available for three coordinately expressed genes: CAM1 and, in each case, one of the 8-10 alpha- and beta-tubulin genes that are expressed during differentiation(33, 35) . Comparison of these sequences revealed that upstream in each of these genes there are two to three representatives of a variant of a palindromic 12-nucleotide sequence, 5`-TTTGGCGCCAAA-3`, with the first seven bases perfectly matched in all copies (Table 1). The positions of these sequences in the CAM1 gene are underlined in Fig. 1; in the tubulin genes they are found upstream in comparable positions but sometimes in reverse orientation. In each gene the element nearest the TATA box has the best fit to the perfect palindrome, with the calmodulin gene containing a perfect copy (Table 1). This element is absent in the upstream region of a constitutively expressed Naegleria gene, actin. (^3)



This sequence element is similar to the E2F recognition consensus sequence (Table 1), which has a dyad symmetry and is found in the adenovirus E2 promoter, the E1A enhancer, the c-myc promoter, and in a promoter of a hamster dihydrofolate reductase gene (reviewed in (62) ). It is also similar to the HIP binding site, a similar sequence also found in the dihydrofolate reductase gene that binds different proteins(63) .

The mRNAs for Flagellar Calmodulin and alpha-Tubulin Share a 12-Nucleotide Sequence Element

In the transcribed portion of the gene, there is a 12-nucleotide match between the CAM1 gene and the three sequenced alpha-tubulin genes, in each case surrounding the start codon, 5`-AUACAAAAUGAG-3` (Table 2). There are matches to this sequence, termed element 2, in the data bases, but most are not similarly positioned, and only one seems potentially related to this series, a similarly located sequence in the Drosophila beta1-tubulin gene(66) .



When element 2 was found, no Naegleria beta-tubulin sequence was available, so we attempted to use antisense hybrid arrest of translation, i.e. arrest of cell-free translation by hybridization of mRNA to complementary oligonucleotides(67) , to determine which mRNAs contain this element. The antisense oligonucleotide, as well as the sense control, are shown in Table 2. Tubulin and calmodulin translation products were displayed on separate gel systems. For tubulin (Fig. 6A), a control of background wheat germ translation, without added RNA, is shown in A, lane1, and translation products directed by 60-min total RNA in A, lane2, with considerable synthesis of both alpha- and beta-tubulin as previously reported(30) . Addition of the sense oligonucleotide with the total RNA does not affect the translation products (A, lane4), but the antisense oligonucleotide eliminates the translation of alpha-tubulin but not of beta-tubulin (A, lane3). This indicates that most if not all of the approx8-10 expressed alpha-tubulin genes share this element. In the case of calmodulin, the translation products of 60-min RNA show the two calmodulins described previously(29) , including the Ca-induced mobility shift (Fig. 6B, lanes1 and 2). These translation products are unaffected by the addition of the sense oligonucleotide (B, lanes 5 and 6), but the antisense oligonucleotide eliminated the CaM-1 band from the translation product (B, lanes3 and 4). We conclude from these experiments that two mRNAs known to have element 2, alpha-tubulin and CaM-1, are eliminated from the translation product whereas other mRNAs, including beta-tubulin and CaM-2, are not eliminated and thus presumably do not have a perfect match to element 2. We subsequently determined the sequences of two beta-tubulin genes (35) and found that these encode 8/12 and 9/12 matches to element 2 (Table 2).


Figure 6: Antisense oligonucleotide blocks translation of alpha-tubulin and of CaM-1. Total 60-min RNA was used to direct translation in the wheat germ system, and the products were evaluated either by A, an 8-12% urea/SDS-polyacrylamide gel(30) , which shows alpha- and beta-tubulin (as marked) and other larger translation products, or by B, a 15% Laemmli gel, a procedure that displays small heat-stable translation products, including CaM-1 and CaM-2 as marked. In B, odd-numbered lanes contain Ca and even-numbered lanes contain EGTA. In A, lane1 shows the translation without added RNA, lane 2 shows translation with 60-min RNA, lane 3 with the same RNA plus the antisense oligonucleotide (Table 2), and lane 4 with the same RNA and the sense oligonucleotide. In B, lanes1 and 2 show translation with 60-min RNA, lanes3 and 4 with antisense oligonucleotide, and lanes5 and 6 with sense oligonucleotide.



The Encoded Amino Terminus of Flagellar Calmodulin Is Similar to the Amino Terminus of alpha-Tubulin

Perhaps the most remarkable sequence motif is found near the amino termini of the encoded proteins (Table 3). The extended amino terminus encoded by CAM1 contains a segment of five amino acids, REAIS, similar to a conserved sequence found at the amino terminus of the alpha-tubulins, REVIS(33) . These segments differ by a single, conservative substitution. Naegleria beta-tubulin (35) contains the MREI segment involved in autoregulation of tubulin genes in vertebrates (68) but probably not in Naegleria(^4)and overall shares two amino acids of the REAIS element plus two conservative substitutions.




DISCUSSION

The Single-copy Intronless Gene Encodes a Bona Fide Calmodulin

Calmodulin is so conserved among organisms that any change has to be considered potentially significant. This is certainly true of exceptional changes, such as the change encoded in CAM1 of the almost universally conserved Thr-110 to Cys (Fig. 2) and also of the extended amino terminus. In comparison to the calmodulins of other protists, the calmodulin encoded by Naegleria's CAM1 seems comparably conserved, including the four EF-hand domains, and thus appears to be a bona fide calmodulin with an amino-terminal extension. This gene is single copy, and its expression is differentiation-specific, features that offer opportunities for studying the structure and function of this calmodulin by mutation.

The Calmodulin Is Flagellar Calmodulin

The evidence presented here indicates that CAM1 encodes flagellar calmodulin, CaM-1, as defined previously(29) . The gene product is developmentally regulated (Fig. 4) on the same timetable as was found for translatable mRNA encoding CaM-1. The cloned DNA hybrid-selects mRNA that directs the translation of a calmodulin previously shown to be CaM-1 (Fig. 5). An antisense oligonucleotide complementary to a region near the translation start codon of CAM1 eliminates the translation of CaM-1 (Fig. 6B). We believe this evidence is convincing, but definitive proof that CAM1 encodes flagellar calmodulin must await direct sequencing of a portion of the calmodulin isolated from flagellates.

A Separate, Divergent Gene Encodes CaM-2

CaM-2 is almost certainly encoded by a separate gene from the one that encodes CaM-1. When translation in the cell-free wheat germ system is directed by mRNA from differentiating Naegleria, both CaM-1 and CaM-2 are synthesized in the same proportion that the two calmodulins are found in flagellates(29) , so whatever is different between them either is already encoded by the mRNAs or is added post-translationally in the heterologous cell-free system. The results presented herein show clearly that the difference is encoded. When the cloned DNA was used to hybrid-select mRNA for translation, it selected mRNA that translated to a calmodulin with the electrophoretic mobility and calcium-induced mobility shift of CaM-1 but no calmodulin with the mobility of CaM-2 even under conditions where the DNA also selected centrin/caltractin mRNA. Thus the two calmodulin mRNAs do not share a sufficient region of similar sequence to be co-selected by hybridization. In addition, this result shows the cell-free system can translate one calmodulin without producing the other (e.g. by post-translational modification). The dissimilarity of sequence also argues against the two mRNAs being the result of alternate splicing, which would produce exons with substantial segments of identity. Alternate splicing is also an unlikely possibility given the intronless gene and lack of any candidate sequences for separate exons. It appears CAM1 can only encode CaM-1. Finally, when the antisense oligonucleotide was used to block translation, it blocked translation of CaM-1 (and of alpha-tubulin) but not of CaM-2 (or beta-tubulin). Thus CaM-2 lacks this 12-nucleotide element, and in addition it can be translated in the wheat germ system when translation of CaM-1 is arrested by an antisense oligonucleotide. Overall these results argue strongly that CaM-2 is not encoded in any fashion in the CaM-1 gene, but proof awaits the isolation and characterization of a separate CaM-2 gene. Until then, of the three possible explanations for the two differentiation-specific calmodulins in Naegleria ((i) two different genes, two transcripts, two calmodulins; (ii) one gene, alternate splicing, so two calmodulins are translated; and (iii) one gene product is translated, then post-translational modification produces two calmodulins) only the first explanation is consistent with the results presented in this paper.

Overall, the results indicate two separate genes that are quite divergent, even though they both appear to encode authentic calmodulins (29) . There is precedence for such divergence. Three human genes, all of which encode the same calmodulin polypeptide, have diverged to the point of 81% identity(14) . Is it reasonable to anticipate that the DNA sequences encoding CaM-1 and CaM-2 might have diverged sufficiently that they would not be detected in genomic DNA by hybridization to a DNA probe (e.g. using gene CAM1) at the minimal stringency that would give a clear signal above noise? In order to assess this possibility, on paper we changed each codon in CAM1 (Fig. 1) to the synonymous codon with the largest possible number of nucleotide changes (from 0 to 3), without considering codon preferences. It proved possible to encode the same calmodulin after changing 168 of the 465 coding nucleotides, i.e. with retention of only 63.9% identity. A calmodulin identical to vertebrate calmodulin can be encoded by a DNA that shares only 57.7% identity with CAM1. Such 58-64% identities are probably below the boundary at which DNA-DNA hybrids could be detected under the conditions we used (cf. (37) ). Thus it appears feasible for Naegleria to encode another calmodulin without the gene being detected by DNA-DNA hybridization to CAM1.

Such extreme divergence as proposed for the CaM-2 gene would require strong selective pressure, away from preferred codon usage, to minimize homology. One possible reason for such divergence at the gene level while retaining a conserved protein sequence might be to avoid homogenization of differences between the two calmodulin genes by intergene recombination, since divergence of sequences can markedly reduce the frequency of recombination between homologous DNA segments (69) . Even the three mammalian calmodulin genes, which all encode a common calmodulin, are diverged to a considerable extent(14) , suggesting there may be a reason for divergence in mammals too. The evidence for a ``hidden'' CaM-2 gene seems compelling; the challenge now is to find it. Obviously, our results also raise the possibility of undetected multiple calmodulins in other organisms.

Provocative Sequence Elements in CAM1 and Its Products

Events involving the flagellar tubulins and flagellar calmodulin are orchestrated in concert during Naegleria differentiation: (i) a marked increase in abundance of mRNA during the first hour, apparently due to new transcription; (ii) a rapid decrease in mRNA abundance thereafter, with a half-life of 8 min, which suggests the possibility of targeted decay; and (iii) localization of product in the peninsular flagella. We examined the available sequences to see if putative signals for these processes might be found. We found three distinctive motifs, as summarized in Fig. 7, each likely to fulfill some function. The upstream elements with dyad symmetry shared by CAM1 and representative co-expressed alpha- and beta-tubulin genes (Table 1) are candidates to have a function in regulating transcription. Element 2, which surrounds the start codon, is provocative, although the true comparison is smaller than 12 nucleotides because four of the nucleotides are included in the transcription start sequence ANNAUG. One possible function for element 2 might be to regulate mRNA stability, as described for an element positioned in a comparable region of a yeast mRNA(70) . The amino-terminal REAIS, shared with the REVIS in alpha-tubulin, is unique among calmodulins. Both these proteins are transported to flagella, and an appealing speculation is that these sequences serve as a zip code for a transport system. If this speculation were true, this element need not be shared with beta-tubulin, since beta-tubulin presumably travels with alpha-tubulin in the tubulin heterodimer. Currently we do not know if this unique amino terminus remains on the CaM-1; it is conceivable that it is removed by post-translational processing. If the shared amino acid sequence near the amino terminus is used for a regulatory purpose, such as a zip code to direct the proteins to the flagella, the coincidence of this element on CaM-1 and the tubulins should prove useful in dissecting how it works, even if this particular adaptation proves unique to Naegleria. Obviously we desire to test how these elements might orchestrate events during this differentiation.


Figure 7: Provocative sequence elements shared among Naegleria flagellar calmodulin and tubulin genes and their products, shown as they appear in the CAM1 gene, its mRNA, and its encoded CaM-1 protein. These sequence elements are also underlined in Fig. 1. The upstream palindrome, found twice in CAM1, once as a perfect match and once as a 10/12 match, is also found in the alpha- and beta-tubulin genes. The 12-nucleotide element that includes the start codon is perfectly matched in alpha-tubulin, and 8/12 and 9/12 in two beta-tubulin mRNAs. The translated REAIS in the extended amino terminus encoded by CAM1 is similar to the conserved REVIS in alpha-tubulin.



Conclusion

We sought calmodulin in differentiating cells because of evidence for a role of intracellular Ca in differentiation(71) . To our surprise we found two differentiation-specific calmodulins(29) . We here report a description of one of these calmodulins, deduced to be flagellar calmodulin, as well as indirect evidence that the other differentiation-specific calmodulin is encoded by a separate, divergent gene. We report sequence elements that are candidates to be involved in regulation of transcription, mRNA stability, and localization of proteins in flagella. Provocative though these sequences are, any such putative signals remain tantalizing candidates until they can be dissected using the power of genetics. For this the ability to obtain DNA-mediated transformation of Naegleria is essential; work toward this crucial goal is in progress.


FOOTNOTES

*
This work was supported by National Science Foundation Grants MCB-9005589 and MCB-9307759. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U04381[GenBank].

§
To whom correspondence should be addressed: Dept. of Biology, Brandeis University, 415 South St., Waltham, MA 02254-9110. Tel.: 617-736-3150; Fax: 617-736-3107; fulton{at}binah.cc.brandeis.edu.

(^1)
The abbreviations used are: CaM-1, flagellar calmodulin; CaM-2, flagellate cell body calmodulin; kb, kilobase(s); PIPES, 1,4-piperazinediethanesulfonic acid.

(^2)
Y. Levy, E. Y. Lai, S. P. Remillard, and C. Fulton, manuscript in preparation.

(^3)
S. P. Remillard, E. Y. Lai, and C. Fulton, manuscript in preparation.

(^4)
E. Y. Lai and C. Fulton, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Igor Dawid and Yueh-hsiu Chien for the Xenopus calmodulin cDNA clone, Rolf Heumann for the 12-mer oligonucleotides, Daniel Sussman for scanning the autoradiogram of Fig. 3B, Margaret Clarke for the anti-calmodulin antibody, G. D. Fasman for predicting the structure of the amino terminus, Hayden Coon for initial computer searches, and Yaron Levy for sharing his expertise and anti-centrin antiserum with us as well as for helpful suggestions about the manuscript and for printing the photographs.


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