Episodic Evolution of Protein Hormones: Molecular Evolution of Pituitary Prolactin

Previous studies have shown that pituitary growth hormone displays an episodic pattern of evolution, with a slow underlying evolutionary rate and occasional sustained bursts of rapid change. The present study establishes that pituitary prolactin shows a similar pattern. During much of tetrapod evolution the sequence of prolactin has been strongly conserved, showing a slow basal rate of change (approx 0.27 × 10⁹ substitutions/amino acid site/year). This rate has increased substantially (∼12- to 38-fold) on at least four occasions during eutherian evolution, during the evolution of primates, artiodactyls, rodents, and elephants. That these increases are real and not a consequence of inadvertant comparison of paralogous genes is shown (for at least the first three groups) by the fact that they are confined to mature protein coding sequence and not apparent in sequences coding for signal peptides or when synonymous substitutions are examined. Sequences of teleost prolactins differ markedly from those of tetrapods and lungfish, but during the course of teleost evolution the rate of change of prolactin has been less variable than that of growth hormone. It is concluded that the evolutionary pattern seen for prolactin shows long periods of near-stasis interrupted by occasional bursts of rapid change, resembling the pattern seen for growth hormone in general but not in detail. The most likely basis for these bursts appears to be adaptive evolution though the biological changes involved are relatively small. Received: 31 August 1999 / Accepted: 9 February 2000     

Function Switching as a Basis for Bursts of Rapid Change During the Evolution of Pituitary Growth Hormone

Pituitary growth hormone shows a pattern of molecular evolution in which occasional bursts of rapid change are imposed on a slow basal rate. It is suggested that these bursts of rapid evolution are a consequence of acquisition by this protein hormone of a secondary function, the importance of which varies. As the function of the hormone switches to accommodate the changes in role, its structure will also alter, adapting it to acquisition or loss of the secondary function. Several rounds of such ``function switching' could give a substantial change in structure (the sum of several small changes) with little overall change in function. A similar process could underlie rapid bursts of evolution in other proteins. Received: 15 July 1996 / Accepted: 19 September 1996     

Intron Dynamics and the Evolution of Integrin β-Subunit Genes: Maintenance of an Ancestral Gene Structure in the Coral, Acropora millepora

We have determined the genomic structure of an integrin β-subunit gene from the coral, Acropora millepora. The coding region of the gene contains 26 introns, spaced relatively uniformly, and this is significantly more than have been found in any integrin β-subunit genes from higher animals. Twenty-five of the 26 coral introns are also found in a β-subunit gene from at least one other phylum, indicating that the coral introns are ancestral. While there are some suggestions of intron gain or sliding, the predominant theme seen in the homologues from higher animals is extensive intron loss. The coral baseline allows one to infer that a number of introns found in only one phylum of higher animals result from frequent intron loss, as opposed to the seemingly more parsimonious alternative of isolated intron gain. The patterns of intron loss confirm results from protein sequences that most of the vertebrate genes, with the exception of β4, belong to one of two β subunit families. The similarity of the patterns within each of the β1,2,7 and β3,5,6,8 groups indicates that these gene structures have been very stable since early vertebrate evolution. Intron loss has been more extensive in the invertebrate genes, and obvious patterns have yet to emerge in this more limited data set. Received: 5 March 2001 / Accepted: 17 May 2001     

Molecular Evolution of Prolactin in Primates

Pituitary prolactin, like growth hormone (GH) and several other protein hormones, shows an episodic pattern of molecular evolution in which sustained bursts of rapid change contrast with long periods of slow evolution. A period of rapid change occurred in the evolution of prolactin in primates, leading to marked sequence differences between human prolactin and that of nonprimate mammals. We have defined this burst more precisely by sequencing the coding regions of prolactin genes for a prosimian, the slow loris (Nycticebus pygmaeus), and a New World monkey, the marmoset (Callithrix jacchus). Slow loris prolactin is very similar in sequence to pig prolactin, so the episode of rapid change occurred during primate evolution, after the separation of lines leading to prosimians and higher primates. Marmoset prolactin is similar in sequence to human prolactin, so the accelerated evolution occurred before divergence of New World monkeys and Old World monkeys/apes. The burst of change was confined largely to coding sequence (nonsynonymous sites) for mature prolactin and is not marked in other components of the gene sequence. This and the observations that (1) there was no apparent loss of function during the episode of rapid evolution, (2) the rate of evolution slowed toward the basal rate after this burst, and (3) the distribution of substitutions in the prolactin molecule is very uneven support the idea that this episode of rapid change was due to positive adaptive selection. In the slow loris and marmoset there is no evidence for duplication of the prolactin gene, and evidence from another New World monkey (Cebus albifrons) and from the chimpanzee and human genome sequences, suggests that this is the general position in primates, contrasting with the situation for GH genes. The chimpanzee prolactin sequence differs from that of human at two residues and comparison of human and chimpanzee prolactin gene sequences suggests that noncoding regions associated with regulating expression may be evolving differently from other noncoding regions.     

Expression of the Avian Na,K-ATPase Subunits in Dictyostelium discoideum

This study explored whether Dictyostelium discoideum can be used to express the avian Na,K-ATPase, a heterodimeric membrane protein. Dictyostelium was able to express mRNAs encoding the avian Na,K-ATPase subunits. However, Dictyostelium expressed avian Na,K-ATPase protein when only when a Dictyostelium consensus ribosomal binding sequence, AAAATAAA, was inserted in front of the open reading frames of the α₁- and β₁-subunit cDNAs and the first eight codons following the start-translation codons were changed to Dictyostelium preferred codons. These modified mRNAs appeared to be much less stable than the forms that were not readily translated. Dictyostelium could express the avian β-subunit alone but only expressed the α₁-subunit when the β₁-subunit was co-expressed. Subunit assembly occurred in cells expressing both α₁- and β₁-subunits. The bulk of the exogenously expressed sodium pump subunits remained in an intracellular compartment, presumed to be the endoplasmic reticulum. Dictyostelium exported little or no Na,K-ATPase or free β-subunit to the plasma membrane. Received: 7 July 1998/Revised: 8 October 1998     

Thyroid Hormone Receptor Genes of Neotenic Amphibians

Since thyroid hormones play a pivotal role in amphibian metamorphosis we used PCR to amplify DNA fragments corresponding to a portion of the ligand-binding domain of the thyroid hormone receptor (TR) genes in several neotenic amphibians: the obligatory neotenic members of the family Proteidea the mudpuppy Necturus maculosus and Proteus anguinus as well as two members of the facultative neotenic Ambystoma genus: the axolotl Ambystoma mexicanum and the tiger salamander Ambystoma tigrinum. In addition, we looked for TR genes in the genome of an apode Typhlonectes compressicaudus. TR genes were found in all these species including the obligatory neotenic ones. The PCR fragments obtained encompass both the C and E domains and correspond to α and β genes. Their sequences appear to be normal, suggesting that there is no acceleration of evolutionary rates in the TR genes of neotenic amphibians. This result is not surprising for Ambystomatidae, which are known to respond to T3 (3,3′,5-triiodothyronine) but is not in agreement with biochemical and biological data showing that Proteidea cannot respond to thyroid hormones. Interestingly, by RT-PCR analysis we observed a high expression levels of TRα in gills, intestine, and muscles of Necturus as well as in the liver of Ambystoma mexicanum, whereas TRβ expression was only detected in Ambystoma mexicanum but not in Necturus. Such a differential expression pattern of TRα and TRβ may explain the neoteny in Proteidea. The cloning of thyroid-hormone-receptor gene fragments from these species will allow the molecular study of their failure to undergo metamorphosis. Received: 23 April 1996 / Accepted: 20 January 1997     

Insulin/FGF-binding Ciliary Membrane Glycoprotein from Tetrahymena

Triton X-100 extracted ciliary membrane protein from isolated cilia, prepared from the protozoon Tetrahymena thermophila, were fractionated by affinity chromatography on columns with covalently bound fibroblast growth factor (FGF), insulin, or concanavalin A (ConA), respectively. The eluted proteins were further analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide gels, isoelectric focusing, and by immunoblotting techniques using antibodies against the FGF receptor, platetelet derived growth factor (PDGF) receptor α-subunit, and insulin receptor β-subunit. The particular antibodies were chosen because the peptides PDGF, FGF, insulin, and ConA are chemoattractants in this organism and corresponding binding (receptor) proteins could be expected to be identified. A 66 kDa protein fraction was eluted from the FGF-MiniLeak agarose, insulin-MiniLeak agarose and ConA sepharose. This fraction responded in Western immunoblots to an antibody against the β-subunit of the human insulin receptor, to an antibody against the PDGF receptor (PDGFR) and also to an antibody against the bovine FGF receptor (FGFR) that is known, in other systems, to inhibit FGF binding to its receptor. When analyzed by SDS-PAGE and stained with Coomassie blue the 66 kDa fraction appeared as a single component. However, in some experiments it appeared more heterogeneous when stained with silver indicating the presence of minor components that may be a procedural artifact or isoforms of the same glycoprotein. The 66 kDa protein(s) migrated in isoelectric focusing with a pI of 7.4. The results are discussed in terms of the possible role of the 66 kDa glycoprotein as a protein involved in peptide-mediated cell signalling. Received: 9 June 2000/Revised: 11 January 2001     

Phylogenetic Analyses Reveal Ancient Duplication of Estrogen Receptor Isoforms

To determine the origin and evolutionary significance of a recently discovered isoform of the estrogen receptor (ERβ), we examined the phylogenetic relationship of ERβ to the well-known α isoform (ERα) and other steroid receptors. Our phylogenetic analyses traced the origin of ERβ to a single duplication event at least 450 million years ago. Since this duplication, the evolution of both ER isoforms has apparently been constrained such that 80% of the amino acid positions in the DNA binding domain (DBD) and 53% of the ligand binding domain (LBD) have remained unchanged. Using the phylogenetic tree, we determined the amount of evolutionary change that had occurred in two ER isoforms. The DBD and the LBD had lower rates of evolutionary change compared to the NH₂ terminal domain. However, even with strong selective constraints on the DBD and LBD, our phylogenetic analyses demonstrate two clearly separate phylogenetic histories for ERα and ERβ dating back several hundred million years. The ancient duplication of ER and the parallel evolution of the two ER isoforms suggest that, although ERα and ERβ share a substantial degree of sequence identity, they play unique roles in vertebrate physiology and reproduction. Received: 19 January 1999 / Accepted: 26 May 1999     

Comparisons of the Nucleotide Substitution Process Among Repetitive Segments of the α- and β-Spectrin Genes

The actin–cross-linking protein spectrin is a prominent component of the membrane cytoskeleton. Spectrin is a tetramer of two antiparallel αβ-dimers which share a unique and ancient gene structure. The α-spectrin and β-spectrin genes are composed primarily of tandemly repeated 106-amino-acid segments, each of which forms a triple α-helical coiled coil. Both the genes and the repeats themselves are homologous. The two genes are thought to be the result of a gene duplication event, and each gene is the product of duplications of the 106-amino-acid repeats. In this work we compare the process of molecular evolution across the repeated segments of the α- and β-spectrin genes. We find that the α-spectrin segments have, for the most part, evolved in a homogeneous fashion, while considerable heterogeneity is found among β-spectrin segments. Several segments with unique known functions are found to have evolved differently than the others. On the basis of heterogeneity of the evolutionary process, we suggest that at least one repeat has a unique function that has yet to be documented. We also present new statistical methods for comparing the evolutionary process between different regions of DNA sequences. Received: 27 March 1996 / Accepted: 21 October 1996     

Circular Permutations in the Molecular Evolution of DNA Methyltransferases

Circular permutations of genes during molecular evolution often are regarded as elusive, although a simple model can explain these rearrangements. The model assumes that first a gene duplication of the precursor gene occurs in such a way that both genes become fused in frame, leading to a tandem protein. After generation of a new start codon within the 5′ part of the tandem gene and a stop at an equivalent position in the 3′ part of the gene, a protein is encoded that represents a perfect circular permutation of the precursor gene product. The model is illustrated here by the molecular evolution of adenine-N⁶ DNA methyltransferases. β- and γ-type enzymes of this family can be interconverted by a single circular permutation event. Interestingly, tandem proteins, proposed as evolutionary intermediates during circular permutation, can be directly observed in the case of adenine methyltransferases, because some enzymes belonging to type IIS, like the FokI methyltransferase, are built up by two fused enzymes, both of which are active independently of each other. The mechanism for circular permutation illustrated here is very easy and applicable to every protein. Thus, circular permutation can be regarded as a normal process in molecular evolution and a changed order of conserved amino acid motifs should not be interpreted to argue against divergent evolution. Received: 17 November 1998 / Accepted: 19 February 1999     

Domain evolution in the GH13 pullulanase subfamily with focus on the carbohydrate-binding module family 48

Glycoside hydrolase (GH) family 13 comprises about 30 different specificities. Four of them have been proposed to form the GH13 pullulanase subfamily: pullulanase, isoamylase, maltooligosyl trehalohydrolase and branching enzyme forming the seven CAZy GH13 subfamilies: GH13 8-GH13 14. Recently, a new family of carbohydrate-binding modules (CBMs), the family CBM48 has been established containing the putative starch-binding domains from the pullulanase subfamily, the β-subunit of AMP-activated protein kinase and some other GH13 enzymes with pullulanase and/or α-amylase-pullulanase specificity. Since all of these enzymes are multidomain proteins and the structure for at least one representative of each enzyme specificity has already been determined, the main goal of the present study was to elucidate domain evolution within this GH13 pullulanase subfamily (84 real enzymes) focusing on the CBM48 module. With regard to CBM48 positioning in the amino acid sequence, the N-terminal end of a protein appears to be a predominant position. This is especially true for isoamylases and maltooligosyl trehalohydrolases. Secondary structure-based alignment of CBM modules from CBM48, CBM20 and CBM21 revealed that several residues known as consensus for CBM20 and CBM21 could also be identified in CBM48, but only branching enzymes possess the aromatic residues that correspond with the two tryptophans forming the evolutionary conserved starch-binding site 1 in CBM20. The evolutionary trees constructed for the individual domains, complete alignment, and the conserved sequence regions of the α-amylase family were found to be comparable to each other (except for the C-domain tree) with two basic parts: (i) branching enzymes and maltooligosyl trehalohydrolases; and (ii) pullulanases and isoamylases. Taxonomy was respected only within clusters with pure specificity, i.e. the evolution of CBM48 reflects the evolution of specificities rather than evolution of species. This is a feature different from the one observed for the starch-binding domain of the family CBM20 where the starch-binding domain evolution reflects the evolution of species.     

Close Evolutionary Relatedness of α-Amylases from Archaea and Plants

The amino acid sequences of 22 α-amylases from family 13 of glycosyl hydrolases were analyzed with the aim of revealing the evolutionary relationships between the archaeal α-amylases and their eubacterial and eukaryotic counterparts. Two evolutionary distance trees were constructed: (i) the first one based on the alignment of extracted best-conserved sequence regions (58 residues) comprising β2, β3, β4, β5, β7, and β8 strand segments of the catalytic (α/β)₈-barrel and a short conserved stretch in domain B protruding out of the barrel in the β3 →α3 loop, and (ii) the second one based on the alignment of the substantial continuous part of the (α/β)₈-barrel involving the entire domain B (consensus length: 386 residues). With regard to archaeal α-amylases, both trees compared brought, in fact, the same results; i.e., all family 13 α-amylases from domain Archaea were clustered with barley pI isozymes, which represent all plant α-amylases. The enzymes from Bacillus licheniformis and Escherichia coli, representing liquefying and cytoplasmic α-amylases, respectively, seem to be the further closest relatives to archaeal α-amylases. This evolutionary relatedness clearly reflects the discussed similarities in the amino acid sequences of these α-amylases, especially in the best-conserved sequence regions. Since the results for α-amylases belonging to all three domains (Eucarya, Eubacteria, Archaea) offered by both evolutionary trees are very similar, it is proposed that the investigated conserved sequence regions may indeed constitute the ``sequence fingerprints' of a given α-amylase. Received: 3 June 1998 / Accepted: 20 August 1998     

Extensive Gene Duplication in the Early Evolution of Animals Before the Parazoan–Eumetazoan Split Demonstrated by G Proteins and Protein Tyrosine Kinases from Sponge and Hydra

To know whether genes involved in cell–cell communication typical of multicellular animals dramatically increased in concert with the Cambrian explosion, the rapid evolutionary burst in the major groups of animals, and whether these genes exist in the sponge lacking cell cohesiveness and coordination typical of eumetazoans, we have carried out cloning of the G-protein α subunit (Gα) and the protein tyrosine kinase (PTK) cDNAs from Ephydatia fluviatilis (freshwater sponge) and Hydra magnipapillata strain 105 (hydra). We obtained 13 Gα and 20 PTK cDNAs. Generally animal gene families diverged first by gene duplication (subtype duplication) that gave rise to diverse subtypes with different primary functions, followed by further gene duplication in the same subtype (isoform duplication) that gave rise to isoform genes with virtually identical function. Phylogenetic trees of Gα and PTK families including cDNAs from sponge and hydra revealed that most of the present-day subtypes had been established in the very early evolution of animals before the parazoan–eumetazoan split, the earliest branching among the extant animal phyla, by extensive subtype duplication: for PTK and Gα families, 23 and 9 subtype duplications were observed in the early stage before the parazoan–eumetazoan split, respectively, and after that split, only 2 and 1 subtype duplications were found, respectively. After the separation from arthropods, vertebrates underwent frequent isoform duplications before the fish–tetrapod split. Furthermore, rapid amino acid changes appear to have occurred in concert with the extensive subtype duplication and isoform duplication. Thus the pattern of gene diversification during animal evolution might be characterized by bursts of gene duplication interrupted by considerably long periods of silence, instead of proceeding gradually, and there might be no direct link between the Cambrian explosion and the extensive gene duplication that generated diverse functions (subtypes) of these families. Received: 4 November 1998 / Accepted: 17 November 1998     

Molecular Diversity and Evolution of bla TEM Genes Encoding β-Lactamases Resistant to Clavulanic Acid in Clinical E. coli

The molecular diversity of inhibitor-resistant TEM (IRT) enzymes was explored using a strategy which involved DNA amplification by polymerase chain reaction (PCR), analysis of restriction fragment length polymorphism (RFLP), and direct nucleotide sequencing. The study of plasmid-borne genes from 27 strains, resistant to amoxicillin and β-lactamase-inhibitor combinations, identified mutations resulting in amino acid change at positions 69, 244, 275, and 276 known to be associated with the IRT phenotype and a mutation at nucleotide position 162 in the promoter region. These mutations were found to lie on two different gene sequences, described here as ``TEM-1B like' and ``TEM-2 like' restriction linkage groups. Further analysis, of nucleotide sequences of promoter and coding regions of the β-lactamases, confirmed that a given mutation causing IRT phenotype could be associated with two different gene sequence frameworks and two different causal mutations could lie on identical gene sequence framework. These data argue in favor of convergent phenotypic evolution of IRT enzymes under the selective pressure imposed by the intensive clinical use of β-lactam–β-lactamase inhibitor combinations. Received: 18 March 1996 / Accepted: 15 July 1996     

Effects of 8-cpt-cAMP on the epithelial sodium channel expressed in Xenopus oocytes

Vasopressin stimulates the activity of the epithelial Na channel (ENaC) through the cAMP/PKA pathway in the cortical collecting tubule, or in similar amphibian epithelia, but the mechanism of this regulation is not yet understood. This stimulation by cAMP could not be reproduced with the rat or Xenopus ENaC expressed in Xenopus oocyte. Recently, it was shown that the α-subunit cloned from the guinea-pig colon (αgp) could confer the ability to be activated by the membrane-permeant cAMP analogue 8-chlorophenyl-thio-cAMP (cpt-cAMP) to channels produced by expression of αgp, βrat and γrat ENaC subunits. In this study we investigate the mechanism of this activation. Forskolin treatment, endogenous production of cAMP by activation of coexpressed β adrenergic receptors, or intracellular perfusion with cAMP did not increase the amiloride-sensitive Na current, even though these maneuvers stimulated CFTR (cystic fibrosis transmembrane conductance regulator)-mediated Cl currents. In contrast, extracellular 8-cpt-cAMP increased αgp, βrat and γrat ENaC activity but had no effect on CFTR. Swapping intracellular domains between the cpt-cAMP-sensitive αgp and the cpt-cAMP-resistant αrat-subunit showed that neither the N-terminal nor the C-terminal of α ENaC was responsible for the effect of cpt-cAMP. The mechanisms of activation of ENaC by cpt-cAMP and of CFTR by the cAMP/PKA pathway are clearly different. cpt-cAMP seems to increase the activity of ENaC formed by αgp and βγrat by interacting with the extracellular part of the protein. Received: 19 January 2001/Revised: 27 April 2001     

Evidence That Calcium Release-activated Current Mediates the Biphasic Electrical Activity of Mouse Pancreatic β-Cells

The electrical response of pancreatic β-cells to step increases in glucose concentration is biphasic, consisting of a prolonged depolarization with action potentials (Phase 1) followed by membrane potential oscillations known as bursts. We have proposed that the Phase 1 response results from the combined depolarizing influences of potassium channel closure and an inward, nonselective cation current (I _CRAN) that activates as intracellular calcium stores empty during exposure to basal glucose (Bertram et al., 1995). The stores refill during Phase 1, deactivating I _CRAN and allowing steady-state bursting to commence. We support this hypothesis with additional simulations and experimental results indicating that Phase 1 duration is sensitive to the filling state of intracellular calcium stores. First, the duration of the Phase 1 transient increases with duration of prior exposure to basal (2.8 mm) glucose, reflecting the increased time required to fill calcium stores that have been emptying for longer periods. Second, Phase 1 duration is reduced when islets are exposed to elevated K⁺ to refill calcium stores in the presence of basal glucose. Third, when extracellular calcium is removed during the basal glucose exposure to reduce calcium influx into the stores, Phase 1 duration increases. Finally, no Phase 1 is observed following hyperpolarization of the β-cell membrane with diazoxide in the continued presence of 11 mm glucose, a condition in which intracellular calcium stores remain full. Application of carbachol to empty calcium stores during basal glucose exposure did not increase Phase 1 duration as the model predicts. Despite this discrepancy, the good agreement between most of the experimental results and the model predictions provides evidence that a calcium release-activated current mediates the Phase 1 electrical response of the pancreatic β-cell. Received: 5 June 1996/Revised: 15 August 1996     

Phylogenetic Analysis of Reptilian Hemoglobins: Trees, Rates, and Divergences

Phylogenetic relationships among reptiles were examined using previously published and newly determined hemoglobin sequences. Trees reconstructed from these sequences using maximum-parsimony, neighbor-joining, and maximum-likelihood algorithms were compared with a phylogenetic tree of Amniota, which was assembled on the basis of published morphological data. All analyses differentiated α chains into α^A and α^D types, which are present in all reptiles except crocodiles, where only α^A chains are expressed. The occurrence of the α^D chain in squamates (lizards and snakes only in this study) appears to be a general characteristic of these species. Lizards and snakes also express two types of β chains (βI and βII), while only one type of β chain is present in birds and crocodiles. Reconstructed hemoglobin trees for both α and β sequences did not yield the monophyletic Archosauria (i.e., crocodilians + birds) and Lepidosauria (i.e., Sphenodon+ squamates) groups defined by the morphology tree. This discrepancy, as well as some other poorly resolved nodes, might be due to substantial heterogeneity in evolutionary rates among single hemoglobin lineages. Estimation of branch lengths based on uncorrected amino acid substitutions and on distances corrected for multiple substitutions (PAM distances) revealed that relative rates for squamate α^A and α^D chains and crocodilian β chains are at least twice as high as those of the rest of the chains considered. In contrast to these rate inequalities between reptilian orders, little variation was found within squamates, which allowed determination of absolute evolutionary rates for this subset of hemoglobins. Rate estimates for hemoglobins of lizards and snakes yielded 1.7 (α^A) and 3.3 (β) million years/PAM when calibrated with published divergence time vs. PAM distance correlates for several speciation events within snakes and for the squamate ↔ sphenodontid split. This suggests that hemoglobin chains of squamate reptiles evolved ∼3.5 (α^A) or ∼1.7 times (β) faster than their mammalian equivalents. These data also were used to obtain a first estimate of some intrasquamate divergence times. Received: 15 September 1997 / Accepted: 4 February 1998     

Organization, Structure, and Evolution of the Nonadult Rat β-Globin Gene Cluster

The β-globin gene cluster of Wistar rat was extensively cloned and the embryonic genes were mapped and sequenced. Four overlapping λ Dash recombinant clones cover about 31 kb and contain four nonadult β-globin genes, 5′–ε1–γ1–γ2–ψγ3–3′. The ε1 and γ2 are active genes, since their protein products were detected in the fetal stage of the rat (Iwahara et al., J Biochem 119:360–366, 1996). The γ1 locus might be a pseudogene, since the ATA box in the promoter region is mutated to GTA; however, no other defect is observed. The ψγ3 locus is a truncated pseudogene because a 19-base deletion, which causes a shift of the reading frame, is observed between the second nucleotide of the putative codon 68 and codon 76. A sequence comparison suggests that the ψγ3 might be produced by a gene conversion event of the proto-γ-globin gene set. Possible histories of the evolution of rat nonadult β-globin genes are discussed. Received: 6 August 1998 / Accepted: 12 February 1999     

Sperm Nuclear Basic Proteins (SNBPs) of Agnathans and Chondrichthyans: Variability and Evolution of Sperm Proteins in Fish

We have characterized for the first time SNBPs from the hagfish Eptatratus stouti (Myxini) and the lamprey Lampetra tridentatus (Cephalaspidomorphi) and have found that histones are the major protein components of the sperm of these agnathans. We have also conducted a systematic analysis of SNBPs from different groups of chondrichthyan fishes, including the skate Raja rhina and seven species of sharks. Together with our previous data showing the sporadic nature of SNBP evolution in bony fish (Saperas, N., Ausio, J., Lloris, D. and Chiva, M. [1994] J. Mol. Evol. 39: 282–295), the present study provides a unique insight into the overall evolutionary complexity and variability of the nuclear sperm proteins of fishes. It would appear that despite the discontinuous evolution of these proteins, the macroevolutionary pattern of histone (H type) → protamine-like (PL type) → protamine (P type) has been conserved in fish evolution, as it has in the evolution of other Deuterostomes. Received: 11 June 1996 / Accepted: 6 August 1996