Gene Conversion and Evolution of Daphniid Hemoglobins (Crustacea, Cladocera)

The extracellular hemoglobins of cladocerans derive from the aggregation of 12 two-domain globin subunits that are apparently encoded by four genes. This study establishes that at least some of these genes occur as a tandem array in both Daphnia magna and Daphnia exilis. The genes share a uniform structure; a bridge intron separates two globin domains which each include three exons and two introns. Introns are small, averaging just 77 bp, but a longer sequence (2.2–3.2 kb) separates adjacent globin genes. A survey of structural diversity in globin genes from other daphniids revealed three independent cases of intron loss, but exon lengths were identical, excepting a 3-bp insertion in exon 5 of Simocephalus. Heterogeneity in the extent of nucleotide divergence was marked among exons, largely as a result of the pronounced diversification of the terminal exon. This variation reflected, in part, varying exposure to concerted evolution. Conversion events were frequent in exons 1–4 but were absent from exons 5 and 6. Because of this difference, the results of phylogenetic analyses were strongly affected by the sequences employed in this construction. Phylogenies based on total nucleotide divergence in exons 1–4 revealed affinities among all genes isolated from a single species, reflecting the impact of gene conversion events. In contrast, phylogenies based on total nucleotide divergence in exons 5 and 6 revealed affinities among orthologous genes from different taxa. Received: 8 March 1999 / Accepted: 14 July 1999     

Ferritin gene organization: Differences between plants and animals suggest possible kingdom-specific selective constraints

Ferritin, a protein widespread in nature, concentrates iron ∼10¹¹–10¹²-fold above the solubility within a spherical shell of 24 subunits; it derives in plants and animals from a common ancestor (based on sequence) but displays a cytoplasmic location in animals compared to the plastid in contemporary plants. Ferritin gene regulation in plants and animals is altered by development, hormones, and excess iron; iron signals target DNA in plants but mRNA in animals. Evolution has thus conserved the two end points of ferritin gene expression, the physiological signals and the protein structure, while allowing some divergence of the genetic mechanisms. Comparison of ferritin gene organization in plants and animals, made possible by the cloning of a dicot (soybean) ferritin gene presented here and the recent cloning of two monocot (maize) ferritin genes, shows evolutionary divergence in ferritin gene organization between plants and animals but conservation among plants or among animals; divergence in the genetic mechanism for iron regulation is reflected by the absence in all three plant genes of the IRE, a highly conserved, noncoding sequence in vertebrate animal ferritin mRNA. In plant ferritin genes, the number of introns (n= 7) is higher than in animals (n= 3). Second, no intron positions are conserved when ferritin genes of plants and animals are compared, although all ferritin gene introns are in the coding region; within kingdoms, the intron positions in ferritin genes are conserved. Finally, secondary protein structure has no apparent relationship to intron/exon boundaries in plant ferritin genes, whereas in animal ferritin genes the correspondence is high. The structural differences in introns/exons among phylogenetically related ferritin coding sequences and the high conservation of the gene structure within plant or animal kingdoms suggest that kingdom-specific functional constraints may exist to maintain a particular intron/exon pattern within ferritin genes. In the case of plants, where ferritin gene intron placement is unrelated to triplet codons or protein structure, and where ferritin is targeted to the plastid, the selection pressure on gene organization may relate to RNA function and plastid/nuclear signaling. Received: 25 July 1995 / Accepted: 3 October 1995     

Distribution and evolution of introns in drosophila amylase genes

While the two amylase genes of Drosophila melanogaster are intronless, the three genes of D. pseudoobscura harbor a short intron. This raises the question of the common structure of the Amy gene in Drosophila species. We have investigated the presence or absence of an intron in the amylase genes of 150 species of Drosophilids. Using polymerase chain reaction (PCR), we have amplified a region that surrounds the intron site reported in D. pseudoobscura and a few other species. The results revealed that most species contain an intron, with a variable size ranging from 50 to 750 bp, although the very majoritary size was around 60–80 bp. Several species belonging to different lineages were found to lack an intron. This loss of intervening sequence was likely due to evolutionarily independent and rather frequent events. Some other species had both types of genes: In the obscura group, and to a lesser extent in the ananassae subgroup, intronless copies had much diverged from intron-containing genes. Base composition of short introns was found to be variable and correlated with that of the surrounding exons, whereas long introns were all A-T rich. We have extended our study to non-Drosophilid insects. In species from other orders of Holometaboles, Lepidoptera and Hymenoptera, an intron was found at an identical position in the Amy gene, suggesting that the intron was ancestral. Received: 23 October 1995 / Accepted: 5 March 1996     

Molecular evolution of nitrate reductase genes

To understand the evolutionary mechanisms and relationships of nitrate reductases (NRs), the nucleotide sequences encoding 19 nitrate reductase (NR) genes from 16 species of fungi, algae, and higher plants were analyzed. The NR genes examined show substantial sequence similarity, particularly within functional domains, and large variations in GC content at the third codon position and intron number. The intron positions were different between the fungi and plants, but conserved within these groups. The overall and nonsynonymous substitution rates among fungi, algae, and higher plants were estimated to be 4.33 × 10⁻¹⁰ and 3.29 × 10⁻¹⁰ substitutions per site per year. The three functional domains of NR genes evolved at about one-third of the rate of the N-terminal and the two hinge regions connecting the functional domains. Relative rate tests suggested that the nonsynonymous substitution rates were constant among different lineages, while the overall nucleotide substitution rates varied between some lineages. The phylogenetic trees based on NR genes correspond well with the phylogeny of the organisms determined from systematics and other molecular studies. Based on the nonsynonymous substitution rate, the divergence time of monocots and dicots was estimated to be about 340 Myr when the fungi–plant or algae–higher plant divergence times were used as reference points and 191 Myr when the rice–barley divergence time was used as a reference point. These two estimates are consistent with other estimates of divergence times based on these reference points. The lack of consistency between these two values appears to be due to the uncertainty of the reference times. Received: 10 April 1995 / Accepted: 10 September 1995     

Molecular evolution of the genes encoding receptor tyrosine kinase with immunoglobulinlike domains

Receptor tyrosine kinases (RTK) with five, three, or seven immunoglobulinlike domains in their extracellular regions are classified as subclasses III, IV, and V, respectively. Conservation of the exon/intron structure of the downstream part of the human KIT, FMS, and FLT3 genes that encode RTK of subclass III together with the particular chromosomal localization of these genes suggests that RTKIII genes have evolved from a common ancestor by cis and trans duplications. To strengthen this model of evolution and to determine if it can be extended to RTKIV and V genes, we constructed a phylogenetic tree of RTKIII, IV, and V on the basis of a multiple alignment of their catalytic tyrosine kinase domain sequences and determined the exon/intron structure of PDGFRA (subclass III), FGFR4 (subclass IV), and FLT4 (subclass V) genes in their downstream part. Phylogenetic analyses with amino acid or nucleotide sequences both resulted in one most parsimonious tree. The phylogenetic trees obtained indicate that all three subclasses are well individuated and that RTKIII and RTKV are closer to each other than RTKIV. Furthermore, RTKIII and FLT4 (subclass V) genes possess the same exon/intron structure in their downstream part while the structure of the RTKIV genes is very similar to that of RTKIII and FLT4. Both approaches are in complete agreement and indicate that RTKIII, IV, and V genes most probably evolved from a common ancestor already in pieces by successive duplications involving entire genes.Correspondence to: F. Agnès     

Mutation Pattern Variation Among Regions of the Primate Genome

We sequenced three argininosuccinate-synthetase-processed pseudogenes (ΨAS-A1, ΨAS-A3, ΨAS-3) and their noncoding flanking sequences in human, orangutan, baboon, and colobus. Our data showed that these pseudogenes were incorporated into the genome of the Old World monkeys after the divergence of the Old World and New World monkey lineages. These pseudogene flanking regions show variable mutation rates and patterns. The variation in the G/C to A/T mutation rate (u) can account for the unequal GC contents at equilibrium: 34.9, 36.9, and 41.7% in the pseudogene ΨAS-A1, ΨAS-A3, and ΨAS-3 flanking regions, respectively. The A/T to G/C mutation rate (v) seems stable and the u/v ratios equal 1.9, 1.7, and 1.4 in the flanking regions of ΨAS-A1, ΨAS-A3, and ΨAS-3, respectively. These ``regional' variations of the mutation rate affect the evolution of the pseudogenes, too. The ratio u/v being greater than 1.0 in each case, the overall mutation rate in the GC-rich pseudogenes is, as expected, higher than in their GC-poor flanking regions. Moreover, a ``sequence effect' has been found. In the three cases examined u and v are higher (at least 20%) in the pseudogene than in its flanking region—i.e., the pseudogene appears as mutation ``hot' spots embedded in ``cold' regions. This observation could be partly linked to the fact that the pseudogene flanking regions are long-standing unconstrained DNA sequences, whereas the pseudogenes were relieved of selection on their coding functions only around 30–40 million years ago. We suspect that relatively more mutable sites maintained unchanged during the evolution of the argininosuccinate gene are able to change in the pseudogenes, such sites being eliminated or rare in the flanking regions which have been void of strong selective constraints over a much longer period. Our results shed light on (1) the multiplicity of factors that tune the spontaneous mutation rate and (2) the impact of the genomic position of a sequence on its evolution. Received: 10 February 1997 / Accepted: 21 April 1997     

Evolution of Mhc–DRB Introns: Implications for the Origin of Primates

Introns are generally believed to evolve too rapidly and too erratically to be of much use in phylogenetic reconstructions. Few phylogenetically informative intron sequences are available, however, to ascertain the validity of this supposition. In the present study the supposition was tested on the example of the mammalian class II major histocompatibility complex (Mhc) genes of the DRB family. Since the Mhc genes evolve under balancing selection and are believed to recombine or rearrange frequently, the evolution of their introns could be expected to be particularly rapid and subject to scrambling. Sequences of intron 4 and 5 DRB genes were obtained from polymerase chain reaction-amplified fragments of genomic DNA from representatives of six eutherian orders—Primates, Scandentia, Chiroptera, Dermoptera, Lagomorpha, and Insectivora. Although short stretches of the introns have indeed proved to be unalignable, the bulk of the intron sequences from all six orders, spanning >85 million years (my) of evolution, could be aligned and used in a study of the tempo and mode of intron evolution. The analysis has revealed the Mhc introns to evolve at a rate similar to that of other genes and of synonymous sites of non-Mhc genes. No evidence of homogenization or large-scale scrambling of the intron sequences could be found. The Mhc introns apparently evolve largely by point mutations and insertions/deletions. The phylogenetic signals contained in the intron sequences could be used to identify Scandentia as the sister group of Primates, to support the existence of the Archonta superorder, and to confirm the monophyly of the Chiroptera. Received: 26 October 1998 / Accepted: 21 December 1998     

Dynamic Insertion–Deletion of Introns in Deuterostome EF-1α Genes

To test the validity of intron–exon structure as a phylogenetic marker, the intron–exon structure of EF-1α genes was investigated for starfish, acornworms, ascidians, larvaceans, and amphioxus and compared with that of vertebrates. Of the 11 distinct intron insertion sites found within the coding regions of the deuterostome EF-1α genes, 7 are shared by several taxa, while the remainder are unique to certain taxa. Examination of the shared introns of the deuterostome EF-1α gene revealed that independent intron loss or intron insertion must have occurred in separate lineages of the deuterostome taxa. Maximum parsimony analysis of the intron–exon data matrix recovered five parsimonious trees (consistency index = 0.867). From this result, we concluded that the intron–exon structure of deuterostome EF-1α has evolved more dynamically than previously thought, rendering it unsuitable as a phylogenetic marker. We also reconstructed an evolutionary history of intron insertion–deletion events on the deuterostome phylogeny, based on several molecular phylogenetic studies. These analyses revealed that the deuterostome EF-1α gene has lost individual introns more frequently than all introns simultaneously.     

Gene arrangement of the killer cell inhibitory receptor family on human chromosome 19q13.4 detected by fiber-FISH

 Human chromosome 19q13.4 has recently been revealed to be a remarkable region harboring multiple receptor genes of the immunoglobulin (Ig) superfamily differentially expressed on hematopoietic cell lineages. Over the past few years, more than 50 cDNAs have been cloned for the natural killer cell inhibitory receptor (KIR) gene family, which possess two or three Ig-like domains in the extracellular region. In this study, using two genomic DNA probes containing intron sequences of genes corresponding to the two- and three-domain types, we applied two-color-fluorescence in situ hybridization on stretched DNA fiber preparations (fiber-FISH). As a result, 11 positions homologous to KIR genes were found as a cluster within a range of approximately 120 kilobases on a chromatin fiber from human chromosome 19. Received: 7 January 1998 / Revised: 25 February 1998     

Expression of the Dentin Matrix Protein 1 Gene in Birds

The emergence of jawed vertebrates was predicated on the appearance of several innovations, including tooth formation. The development of teeth requires the participation of several specialized genes, in particular, those necessary for the formation of hard tissues—dentin, enamel, and cementum. Some vertebrates, most conspicuously birds, secondarily lost the tooth-forming ability. To determine the fate of some of the tooth-forming genes in the birds, we tested a domestic fowl cDNA library for the expression of the dentin matrix protein 1 (DMP1) gene. The library was prepared from the poly(A⁺) RNA isolated from the jaws of 11- to 13-day-old embryos and the testing was carried out by the polymerase chain reaction with degenerate primers designed on the basis of the available mammalian and reptile sequences. A chicken homologue of the DMP1 gene identified by this approach was shown to be expressed in the jaws and long bones, the same two tissues as in mammals. The chicken DMP1 gene has an exon/intron organization similar to that of its mammalian and reptile counterparts. The chicken gene contains three short highly conserved segments, the rest of the gene being poorly alignable or not alignable with its mammalian or reptilian homologues. The distribution of similarities and dissimilarities along the gene is indicative of a mode of evolution in which only short segments are kept constant, while the rest of the gene is relatively free to vary as long as the proportion of certain amino acid residues is retained in the encoded polypeptide. The DMP1 gene may have been retained in birds because of its involvement in bone formation. Received: 5 April 1999 / Accepted: 9 August 1999     

Sequence Peculiarity of Gnetalean Legumin-Like Seed Storage Proteins

The development of seeds as a specialized organ for the nutrition, protection, and dispersal of the next generation was an important step in the evolution of land plants. Seed maturation is accompanied by massive synthesis of storage compounds such as proteins, starch, and lipids. To study the processes of seed storage protein evolution we have partially sequenced storage proteins from maturing seeds of representatives from the gymnosperm genera Gnetum, Ephedra, and Welwitschia—morphologically diverse and unusual taxa that are grouped in most formal systems into the common order Gnetales. Based on partial N-terminal amino acid sequences, oligonucleotide primers were derived and used for PCR amplification and cloning of the corresponding cDNAs. We also describe the structure of the nuclear gene for legumin of Welwitschia mirabilis. This first gnetalean nuclear gene structure contains introns in only two of the four conserved positions previously characterized in other spermatophyte legumin genes. The distinct phylogenetic status of the gnetalean taxa is also reflected in a sequence peculiarity of their legumin genes. A comparative analysis of exon/intron sequences leads to the hypothesis that legumin genes from Gnetales belong to a monophyletic evolutionary branch clearly distinct from that of legumin genes of extant Ginkgoales and Coniferales as well as from all angiosperms. Received: 5 June 1997 / Accepted: 31 March 1998     

Multiple Ig-like featuring genes divergent within and among individuals of the marine sponge Geodia cydonium

The receptor tyrosine kinase of the marine sponge Geodia cydonium features two extracellular Ig-like domains in which we recently documented RT-PCR polymorphism among individuals. Genomic-PCR analysis presented here revealed 14 unique sequences from four sponges, differing predominantly in the sequence of an intron which splits the Ig-like domains. Nevertheless, analysis of putative coding regions in 19 distinct clones (156–159 aa) from seven sponges revealed 69 positions of nucleotide substitutions, 67.6% of them non-synonymous, translating into 43 positions of divergent residues. Excluding aa deletions, these 19 sequences share pairwise aa identities of 89–99%. In three sponges, four or five unique Ig-like coding regions were scored. PCR amplification across this intron revealed multiple bands, polymorphic among five of six sponges. Further substantiated by Southern and Northern blots, it is evident that the genome of G. cydonium harbors multicopies of moderately divergent Ig-like domains. Presently, this only appears paralleled by the human KIR multigene family of NK cells MHC class I-specific receptors, which consist of two or three moderately divergent extracellular Ig-like domains.     

Intron Conservation in a UV-Specific DNA Repair Gene Encoded by Chlorella Viruses

Large dsDNA-containing chlorella viruses encode a pyrimidine dimer-specific glycosylase (PDG) that initiates repair of UV-induced pyrimidine dimers. The PDG enzyme is a homologue of the bacteriophage T4-encoded endonuclease V. The pdg gene was cloned and sequenced from 42 chlorella viruses isolated over a 12-year period from diverse geographic regions. Surprisingly, the pdg gene from 15 of these 42 viruses contain a 98-nucleotide intron that is 100% conserved among the viruses and another 4 viruses contain an 81-nucleotide intron, in the same position, that is nearly 100% identical (one virus differed by one base). In contrast, the nucleotides in the pdg coding regions (exons) from the intron-containing viruses are 84 to 100% identical. The introns in the pdg gene have 5′-AG/GTATGT and 3′-TTGCAG/AA splice site sequences which are characteristic of nuclear-located, spliceosomal processed pre-mRNA introns. The 100% identity of the 98-nucleotide intron sequence in the 15 viruses and the near-perfect identity of an 81-nucleotide intron sequence in another 4 viruses imply strong selective pressure to maintain the DNA sequence of the intron when it is in the pdg gene. However, the ability of intron-plus and intron-minus viruses to repair UV-damaged DNA in the dark was nearly identical. These findings contradict the widely accepted dogma that intron sequences are more variable than exon sequences. Received: 13 May 1999 / Accepted: 20 August 1999     

Evolution of Orthologous Intronless and Intron-Bearing Globin Genes in Two Insect Species

While globin genes ctt-2β and ctt-9.1 in Chironomus thummi thummi each have a single intron, all of the other insect globin genes reported so far are intronless. We analyzed four globin genes linked to the two intron-bearing genes in C. th. thummi. Three have a single intron at the same position as ctt-2β and ctt-9.1; the fourth is intronless and lies between intron bearing genes. Finally, in addition to its intron, one gene (ctt-13RT) was recently interrupted by retrotransposition. Phylogenetic analyses show that the six genes in C. th. thummi share common ancestry with five globin genes in the distantly related species C. tentans, and that a 5-gene ancestral cluster predates the divergence of the two species. One gene in the ancestral cluster gave rise to ctn-ORFB in C. tentans, and duplicated in C. th. thummi to create ctt-11 and ctt-12. From parsimonious calculations of evolutionary distances since speciation, ctt-11, ctt-12, and ctn-ORFB evolved rapidly, while ctn-ORFE in C. tentans evolved slowly compared to other globin genes in the clusters. While these four globins are under selective pressure, we suggest that most chironomid globin genes were not selected for their unique function. Instead, we propose that high gene copy number itself was selected because conditions favored organisms that could synthesize more hemoglobin. High gene copy number selection to produce more of a useful product may be the basis of forming multigene families, all of whose members initially accumulate neutral substitutions while retaining essential function. Maintenance of a large family of globin genes not only ensured high levels of hemoglobin production, but may have facilitated the extensive divergence of chironomids into as many as 5000 species. Received: 31 December 1996 / Accepted: 16 May 1997     

Mhc Allelic Diversity and Modern Human Origins

Thirty complete coding sequences of human major histocompatibility complex (Mhc) class II DRB alleles, spanning 237 codons, were analyzed for phylogenetic information using distance, parsimony, and likelihood approaches. Allelic genealogies derived from different parts of the coding sequence (exon 2, the 5′ and 3′ ends of exon 2, respectively, and exons 3–6) were compared. Contrary to prior assertions, a rigorous analysis of allelic genealogies in this gene family cannot be used to justify the claim that the lineage leading to modern humans contained on average at least 100,000 individuals. Phylogenetic inferences based upon the exon 2 region of the DRB loci are complicated by selection and recombination, so this part of the gene does not provide a complete and accurate view of allelic relationships. Attempts to reconstruct human history from genetic data must use realistic models which consider the complicating factors of nonequilibrium populations, recombination, and different patterns of selection. Received: 19 February 1997 / Accepted: 12 June 1997     

The Dentin Matrix Protein 1 Gene of Prototherian and Metatherian Mammals

Mineralization of tooth dentin (the deposition of hydroxyapatite crystals in and around collagen type I fibers of the extracellular matrix) requires the involvement of several genes, among them the gene coding for the dentin matrix protein 1, DMP1. We determined the exon–intron organization of the cattle DMP1 gene and used this information to amplify by the polymerase chain reaction homologous gene fragments from the genomic DNA of two species of metatherian (marsupial) mammals and one prototherian (monotreme) species. The translated proto- and metatherian protein sequences are highly divergent from the eutherian sequences but retain the general characteristics of the DMP1 (high acidity, serine-richness, multiple glycosylation sites, and the presence of the RGD cell attachment tripeptide). They therefore appear to be functional even though, evolutionarily, teeth are in a regression phase in prototherians. It is possible, therefore, that DMP1 is also involved in other functions besides dentinogenesis. The DMP1 gene appears to evolve rapidly and apparently tolerates non-frame-shifting insertions/deletions throughout the coding sequence. Received: 22 February 1998 / Accepted: 11 July 1998     

The Heat-Shock Gene hsp83 of Drosophila auraria: Genomic Organization, Nucleotide Sequence, and Long Antiparallel Coupled ORFs (LAC ORFs)

The genomic organization of the hsp83 gene of Drosophila auraria, a far-eastern endemic species belonging to the montium subgroup of the melanogaster species group, is presented here. Based on in situ hybridization on polytene chromosomes, cDNA and genomic clone mapping, nucleotide sequencing, and genomic Southern analysis, hsp83 is shown to be present as a single-copy gene at locus 64B on the 3L chromosome arm in D. auraria. This gene is organized into two exons separated by a 929-bp intron. The first exon represents the mRNA leader sequence and is not translated, while the coding region, having a length of 2,151 bp, is solely included in the second exon. Nucleotide sequence comparisons of D. auraria hsp83 with homologous sequences from other organisms show high conservation of the coding region (88–92% identity) in the genus Drosophila, in addition to the conserved genomic organization of two-exons–one-intron, of comparable size and arrangement. A phylogenetic tree based on the protein sequences of homologous genes from representative organisms is in accord with the accredited phylogenetic position of D. auraria. In the hsp83 gene region, a second case of long antiparallel coupled open reading frames (LAC ORFs) for this species was found. The antiparallel to the hsp83 gene ORF is 1,554 bases long, while the two ORFs overlap has a size of 1,548 bp. The anti-hsp83 ORF does not show significant homology to any known gene sequences. In addition, no similar LAC ORF structures were found in homologous gene regions of other organisms. Received: 18 April 1997 / Accepted: 1 August 1997     

Expression of exogenous genes under the control of endogenous HSP70 and CAB promoters in the Closterium peracerosum-strigosum-littorale complex

A unicellular charophyte alga, Closterium peracerosum–strigosum–littoralecomplex (C. psl. complex), has been studied in order to obtainbasic information regarding sexual reproduction in plants. Systemsfor gene introduction and transient expression were developedfor endogenous genes using phleomycin resistance (ble) and Chlamydomonasgreen fluorescent protein (cgfp) genes as selection markers.These genes have codon usage similar to that of genes in theC. psl. complex. To drive these genes strongly into C. psl.complex cells, two native promoters of the C. psl. complex genome—CpHSP70and CpCAB1—were linked to a ble::cgfp fusion gene andintroduced into the cells by particle bombardment. Following2 d of incubation, we found 500 cells expressing GFP under thecontrol of the CpHSP70 promoter, which were identified followingheat shock treatment at 42°C, and 100 cells expressing GFPunder the control of the CpCAB1 promoter, which were observedin lit conditions. In contrast, the GFP signal was only detectedin two cells when ble::cgfp under control of the cauliflowermosaic virus 35S promoter was introduced. The ble::cgfp fusionprotein was detected in the nucleus, whereas the single cgfpprotein was detected in the cytoplasm. Our results indicatethat the newly isolated native promoters of CpHSP70 and CpCAB1are useful tools for inducing exogenous gene expression underheat shock and lit conditions, respectively. In addition, thisstrategy can be used for transient assays, such as the intracellularlocalization of unknown gene products in the C. psl. complex.     

Molecular Evolution of Calmodulin and Calmodulin-Like Genes in the Cephalochordate Branchiostoma

Calmodulin is a calcium-binding EF-hand protein that is an activator of many enzymes as well as ion pumps and channels. Due to its multiple targets and its central role in the cell, understanding the evolutionary history of calmodulin genes should provide insights into the origin of genetic complexity in eukaryotes. We have previously isolated and characterized a calmodulin gene from the early-diverging chordate Branchiostoma lanceolatum (CaM1). In this paper, we report the existence of a second calmodulin gene (CaM2) as well as two CaM-like genomic fragments (CaML-2, CaML-3) in B. lanceolatum and a CaM2 and three CaM-like genes (CaML-1, CaML-2, CaML-3) in B. floridae. The CaM-like genes were isolated using low-stringency PCR. Surprisingly, the nucleotide sequences of the B. lanceolatum CaM1 and CaM2 cDNAs differ by 19.3%. Moreover, the CaM2 protein differs at two positions from the amino acid sequence of CaM1; the latter is identical to calmodulins in Drosophila melanogaster, the mollusc Aplysia californica, and the tunicate Halocynthia roretzi. The two B. lanceolatum CaM-like genes are more closely related to the CaM2 than to the CaM1 gene. This relationship is supported by the phylogenetic analyses and the identical exon/intron organization of these three genes, a relationship unique among animal CaM sequences. These data demonstrate the existence of a CaM multigene family in the cephalochordate Branchiostoma, which may have evolved independently from the multigene family in vertebrates. Received: 2 November 1999 / Accepted: 25 April 2000