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, ()which amounts to 0.01% 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 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 - and -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 - and -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 -tubulin and -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 - and -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 - and -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

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

Construction of pNCaM1

DNA Sequencing

Hybridization Selection of Calmodulin mRNA for Cell-free Translation

()

Hybrid-arrested Cell-free Translation Using Antisense Oligonucleotides

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 and Laemmli sample buffer were added. To the other aliquot, 1 mM EGTA was added instead of CaCl. 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

The DNA Sequence Encodes a Bona Fide Calmodulin

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 -helix, the next four (Ser-Asn-Asn-Glu) to be involved in a -turn, and then the structure returns to an -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

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 -tubulin and -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 - and -Tubulin Genes

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 - and -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 -tubulin (triangles), and for -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 () compared with the abundance of -tubulin () and -tubulin () 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 -tubulin, the insert of pNT1(33) ; and for -tubulin, the insert of pNT1 (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

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

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

()

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 -Tubulin Share a 12-Nucleotide Sequence Element

When element 2 was found, no Naegleria -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 - and -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 -tubulin but not of -tubulin (A, lane3). This indicates that most if not all of the 8-10 expressed -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, -tubulin and CaM-1, are eliminated from the translation product whereas other mRNAs, including -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 -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 -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 - and -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 -Tubulin

()

DISCUSSION

The Single-copy Intronless Gene Encodes a Bona Fide Calmodulin

The Calmodulin Is Flagellar Calmodulin

A Separate, Divergent Gene Encodes CaM-2

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

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 - and -tubulin genes. The 12-nucleotide element that includes the start codon is perfectly matched in -tubulin, and 8/12 and 9/12 in two -tubulin mRNAs. The translated REAIS in the extended amino terminus encoded by CAM1 is similar to the conserved REVIS in -tubulin.

Conclusion

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.

()

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

()

Y. Levy, E. Y. Lai, S. P. Remillard, and C. Fulton, manuscript in preparation.

()

S. P. Remillard, E. Y. Lai, and C. Fulton, manuscript in preparation.

()

E. Y. Lai and C. Fulton, manuscript in preparation.

ACKNOWLEDGEMENTS

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