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Chitinase enzymes hydrolyse the polysaccharide chitin, an abundant architectural component in invertebrates and fungi. Most mammals encode at least two endochitinases (CHIT1 and CHIA/AMCase), as well as several homologues encoding catalytically inactive chitinase-like proteins or chilectins (all GH18 family proteins). It is becoming increasingly apparent that chitinases and chilectins play an important role in inflammation and their over-expression is correlated with numerous pathological conditions. We have conducted a detailed phylogenomic study of this gene family in order to understand its evolutionary history and the selection forces at work. The family has undergone extensive expansion, initiating with a duplication event at the root of the vertebrate tree generating the ancestors of CHIT1 and CHIA. Our analyses indicate that two further duplications of ancestral CHIA predate the divergence of bony fishes, one leading to a newly identified paralogous group (we have termed CHIO). In fish these sequences fall into two clades bearing the hallmarks of the teleost-specific genome duplication (referred to as 3R). In tetrapods, additional duplications predate and postdate the amphibian/mammalian split and relics of some exist as pseudogenes in the human genome. Expansion and selection of chilectins is pronounced in mammals and CHI3L1 (with a proposed function in immunity) is found in most mammals but not other vertebrates, while CHI3L2 is also evident in reptiles. Notably oviductin (OVGP1) became basic and gained a glycosylated tail with its evolving role in the mammalian reproductive system. In each case, retention of the sugar-binding barrel structure has constrained positive selection to limited sites.

Asymmetric compositional and mutation bias between the two strands occurs in mitochondrial genomes, and an asymmetric mechanism of mtDNA replication is a potential source of this bias. Some evidence indicates that during replication the heavy strand is subject to a gradient of time spent in a single-stranded state (D (ssH)) and a gradient of mutational damage. The nucleotide composition bias among genes varies with D (ssH). Consequently, partial genome duplications (PGD) will alter the skew for genes located downstream of the duplication, relatively to nascent light strand synthesis, and in the same way, gene rearrangements (GRr) will affect genes by changing their skews. We examined cases where there had been PGD or GRr and determined whether this left a trace in the form of unusual patterns of base composition. We compared the skew of genes differently located on the mtDNA genome of previously published whole mtDNA genomes from amphibians, a group that shows considerable levels of both GRr and PGD. After observing a significant correlation between AT and GC skew with D (ssH) at fourfold redundant sites, we ran our analysis and detected 31.3% of the species with GRr and/or PGD. By comparing the nucleotide composition at fourfold redundant sites in normal and "abnormal" species, we found that A/C variation occurs and is associated with GRr/PGD. These results show that by analyzing the nucleotide skews of only three genes, it may be possible to predict some mitochondrial GRr and/or PGD without knowing the complete mtDNA genome sequence.

The natriuretic peptide system is a complex family of peptides and receptors that is primarily linked to the maintenance of osmotic and cardiovascular homeostasis. A natriuretic peptide system is present in each vertebrate class but there are varying degrees of complexity in the system. In agnathans and chondrichthyians, only one natriuretic peptide has been identified, while new data has revealed that multiple types of natriuretic peptides are present in bony fish. However, it seems in tetrapods that there has been a reduction in the number of natriuretic peptide genes, such that only three natriuretic peptides are present in mammals. The peptides act via a family of guanylyl cyclase receptors to generate the second messenger cGMP, which mediates a range of physiological effects at key targets such as the gills, kidney and the cardiovascular system. This review summarises the current knowledge of the natriuretic peptide system in non-mammalian vertebrates and discusses the physiological actions of the peptides., (Copyright 2004 Springer-Verlag)

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 alpha and beta 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 TRalpha in gills, intestine, and muscles of Necturus as well as in the liver of Ambystoma mexicanum, whereas TRbeta expression was only detected in Ambystoma mexicanum but not in Necturus. Such a differential expression pattern of TRalpha and TRbeta 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.

Ascidians exhibit two different modes of development. A tadpole larva is formed during urodele development, whereas the larval phase is modified or absent during anural development. Anural development is restricted to a small number of species in one or possibly two ascidian families and is probably derived from ancestors with urodele development. Anural and urodele ascidians constitute a model system in which to study the evolution of development, but the phylogeny of anural development has not been resolved. Classification based on larval characters suggests that anural species are monophyletic, whereas classification according to adult morphology suggests they are polyphyletic. In the present study, we have inferred the origin of anural development using rDNA sequences. The central region of 18S rDNA and the hypervariable D2 loop of 28S rDNA were amplified from the genomic DNA of anural and urodele ascidian species by the polymerase chain reaction and sequenced. Phylogenetic trees inferred from 18S rDNA sequences of 21 species placed anural developers into two discrete groups corresponding to the Styelidae and Molgulidae, suggesting that anural development evolved independently in these families. Furthermore, the 18S rDNA trees inferred at least four independent origins of anural development in the family Molgulidae. Phylogenetic trees inferred from the D2 loop sequences of 13 molgulid species confirmed the 18S rDNA phylogeny. Anural development appears to have evolved rapidly because some anural species are placed as closely related sister groups to urodele species. The phylogeny inferred from rDNA sequences is consistent with molgulid systematics according to adult morphology and supports the polyphyletic origin of anural development in ascidians.

We have isolated sequences belonging to Ty1-copia group retrotransposons from the genomes of an amphibian (Pyxicephalus adspersa) and three reptiles (Conolophus subscristatus, Amblyrynchus cristatus and Pytas mucosus). Two different sequences were found in the amphibian (Tpa1 and Tpa2). Each is present in several copies per genome and absent from the genomes of two other amphibian species. The C. subcristatus sequence Tcs1 is present in multiple copies in both its host genome (Galapagos land iguana) and the genome of the related Galapagos marine iguana (A. cristatus). There is little or no polymorphism in Tcs1 insertions between different individual animals, suggesting that this sequence is not transposing rapidly in either iguana genome. The P. mucosus sequence Tpm1 shows a discontinuous distribution in snake species, suggesting that it has either been lost from many lineages during vertical germline transmission or has been transferred horizontally in some snake species. Phylogenetic comparisons of all these sequences with each other and with other members of this retrotransposon group from other animals and plants show that sequences within a particular vertebrate species are most closely related to each other, consistent with a vertical transmission model for their evolution.

A discriminant analysis on the basis of the physicochemical properties of amino acid residues is developed to investigate the accumulation pattern of amino acid substitutions in a family of proteins. The application of this analysis to vertebrate hemoglobins reveals the following new results. (1) The major components of teleost fish and amphibian hemoglobins showing the Root effect are sharply discriminated from mammalian hemoglobins in several regions of the alpha and beta chains, whereas shark, minor components of teleost fish and amphibian, reptile, and bird hemoglobins showing no Root effect exhibit a gradual change to mammalian hemoglobin in a straightforward way. This result suggests at least two lines of molecular evolution in vertebrate hemoglobins. (2) The nonadult hemoglobin chains are allocated to the latter line, i.e., tadpole, zeta, and pi chains are similar to shark and trout I chains, and epsilon and gamma chains are similar to some of the reptile chains. (3) In any case, most of the amino acid residues causing the discrimination are located near the sites that carry the amino acid residues conserved well throughout all classes of vertebrates, suggesting that modifications adapting to the respective living conditions or respiratory organs have taken place effectively near the amino acid residues essential for the manifestation of cooperative oxygen binding. (4) The amino acid residues at other sites are changed from one to another species even within the same class, showing a constant substitution rate as a whole. These amino acid substitutions may be nearly neutral, being under a weak functional constraint. The number of sites allowing such neutral substitutions is rather small, less than one-half of all the sites in the adult hemoglobins of bird and mammal, whereas it amounts to two-thirds in teleost fish hemoglobins.

The compositional properties of DNAs from 122 species of fishes and from 18 other cold-blooded vertebrates (amphibians and reptiles) were compared with those from 10 warm-blooded vertebrates (mammals and birds) and found to be substantially different. Indeed, DNAs from cold-blooded vertebrates are characterized by much lower intermolecular compositional heterogeneities and CsCl band asymmetries, by a much wider spectrum of modal buoyant densities in CsCl, by generally lower amounts of satellites, as well as by the fact that in no case do buoyant densities reach the high values found in the GC-richest components of DNAs from warm-blooded vertebrates. In the case of fish genomes, which were more extensively studied, different orders were generally characterized by modal buoyant densities that were different in average values as well as in their ranges. In contrast, different families within any given order were more often characterized by narrow ranges of modal buoyant densities, and no difference in modal buoyant density was found within any single genus (except for the genus Aphyosemion, which should be split into several genera). The compositional differences that were found among species belonging to different orders and to different families within the same order are indicative of compositional transitions, which were shown to be essentially due to directional base substitutions. These transitions were found to be independent of geological time. Moreover, the rates of directional base substitutions were found to be very variable and to reach, in some cases, extremely high values, that were even higher than those of silent substitutions in primates. The taxonomic and evolutionary implications of these findings are discussed.

DNA preparations obtained from 122 species of fishes, 5 species of amphibians, and 13 species of reptiles were investigated in their compositional properties by analytical equilibrium centrifugation in CsCl density gradients. These species represented 21 orders of Osteichthyes, 3 orders of Chondrichthyes, 2 orders of amphibians, and 3 orders of reptiles. Modal buoyant densities of fish DNAs ranged from 1.696 to 1.707 g/cm3, the vast majority of values falling, however, between 1.699 and 1.704 g/cm3, which is the range covered by the DNAs of amphibians and reptiles. In all cases, DNA bands in CsCl were only weakly asymmetrical and only very rarely were accompanied by separate satellite bands (mostly on the GC-rich side). Intermolecular compositional heterogeneities were low in the vast majority of cases, and, like CsCl band asymmetries, at least partially due to cryptic or poorly resolved satellites. The present findings indicate, therefore, that DNAs from cold-blooded vertebrates are characterized by a number of common properties, namely a very wide spectrum of modal buoyant densities, low intermolecular compositional heterogeneities, low CsCl band asymmetries, and, in most cases, small amounts of satellite DNAs. In the case of fish DNAs a negative correlation was found between the GC level and the haploid size (c value) of the genome. If polyploidization is neglected, this phenomenon appears to be mainly due to the fact that increases and decreases in GC are associated with contraction and expansion phenomena, respectively, of intergenic noncoding sequences, which are GC poor relative to coding sequences.

The intrachromosomal distribution of non-telomeric sites of the (TTAGGG)n telomeric repeat was determined for 100 vertebrate species. The most common non-telomeric location of this sequence was in the pericentric regions of chromosomes. A variety of species showed relatively large amounts of this sequence present within regions of constitutive heterochromatin. We discuss possible relationships between the non-telomeric distribution of the (TTAGGG)n sequence and the process of karyotype evolution, during which these sites may provide potential new telomeres.

The chromosomes of 26 species of Anura from variously highly envolved groups were analysed with the fluorescent GC-specific antibiotics mithramycin and chromomycin A3 as well as with the AT-specific quinacrine. The mithramycin- and chromomycin A3-stainings generally resulted in a pattern of the constitutive heterochromatin opposite to the one obtained with quinacrine stain. The weaker a heterochromatic region fluoresces with quinacrine, the stronger is the intensity of the fluorescence achieved with mithramycin and chromomycin A3. Some of the telomeric and interstitial heterochromatic regions, however, exhibit no enhanced fluorescence with any of the fluorochromes. The nucleolar constrictions of the nucleolus organizer regions (NORs) displayed the brightest mithramycin- and chromomycin A3-fluorescence in the karyotypes and interphase nuclei of all species examined. The contrast of the brightly fluorescing GC-rich heterochromatin and of the NORs is considerably enhanced, when the non-fluorescent AT-specific oligopeptide distamycin A is employed as a counterstain. No banding patterns were observed with the fluorochromes in the euchromatic regions of the metaphase chromosomes; this attributed to the strong spiralization of the anuran chromosomes. A cytochemical classification of the various chromatin types in the anuran chromosomes is discussed on the basis of the differential labelings found on the constitutive heterochromatin by means of the fluorochromes.

Speciation in eukaryotes is often accompanied by massive changes in nuclear DNA amount resulting from the accumulation or deletion of DNA base sequences within chromosomes. Our evidence shows that, among related species, the DNA lost or gained during divergence is of a remarkably consistent and restricted composition in respect of the ratio of repetitive to non-repetitive base sequences. It is argued that the restriction may in part at least be imposed by natural selection, in the sense that the only changes tolerable are confined to DNA fractions of particular composition and organisation.

The karyotypes of 14 species of Anura from 9 genera of the suborders Amphicoela, Aglossa, Opisthocoela and Anomocoela were analysed with various banding techniques and conventional cytogenetic methods. The 18S + 28S and 5S ribosomal RNA genes were localized by means of in situ hybridization. No Q-, R- and G-banding patterns in the euchromatic segments of the metaphase chromosomes could be demonstrated in any of the species; this does not seem to be caused by a higher degree of spiralization of the amphibian chromosomes, but by the special DNA organization in these organisms. In most karyotypes, constitutive heterochromatin is present at centromeres, telomeres and nucleolus organizer regions (NORs), but rarely in interstitial positions. The heterochromatic regions are either quinacrine positive and mithramycin negative or vice versa. All species examined possess only one homologous pair of NORs: these display the brightest mithramycin fluorescence in the karyotypes. Many specimens exhibited unequal labelling of the two NORs both after silver and mithramycin staining as well as after in situ hybridization with 3H-18S + 28S rRNA. In four species, between one and six chromosome pairs with homologous 5S rRNA sites could be identified. The 5S rRNA genes and the 18S + 28S rRNA genes are closely linked in two species. In the male meiosis of the Amphicoela and Opisthocoela, there are intersitial, subterminal and terminal chiasmata in the bivalents, whereas only terminal chiasmata are observed in the bivalents of the Aglossa and Anomocoela. No heteromorphic sex-specific chromosomes could be demonstrated in any of the species. The differential staining techniques revealed that the chromosomal structure in these four suborders is largely the same as in the highly evolved anuran suborders Procoela and Diplasiocoela.

Fixed metaphase chromosomes of several species of Amphibia were treated with various restriction endonucleases and subsequently stained with Giemsa. Metaphases of man and chicken were examined in parallel under the same experimental conditions for comparison. The restriction enzymes always induce subsets of the C-banding patterns present in the amphibian karyotypes. The heterochromatic regions can be either resistant or sensitive to the restriction enzyme. The modified C-banding patterns revealed by different restriction endonucleases in the karyotype of the same species can be either extremely dissimilar or almost completely congruent. Correspondingly, the action of the same restriction enzyme on the karyotypes of different species may vary greatly. There is only rarely a correlation between the type of C-banding patterns produced by different restriction endonucleases and their specific base pair recognition sequences. In contrast to mammalian and avian chromosomes, restriction enzymes induce no multiple G-banding patterns in amphibian chromosomes. This is attributed to the difference in organization of the DNA in the genomes of poikilothermic vertebrates. The possible mechanisms of restriction endonuclease banding and the various uses of this technique for amphibian chromosomes are discussed.

The position of specific constitutive heterochromatic chromosome regions within the elongated sperm nuclei of eight species of Anura was examined with Q- and C-banding. These species differ widely with regard to the number, size and position of the brightly fluorescing heterochromatic regions. The empirical frequency distributions determined for the heterochromatic regions relative to the longitudinal axis of the sperm nuclei were compared with random frequency distributions calculated on the basis of two spatial models. None of the specifically stained heterochromatic regions occupy any definite preferential position within the sperm nuclei. In two instances, a specific sequence of the heterochromatic regions within the sperm nuclei could be excluded. The type of chromosomal arrangement within the elongated sperm nuclei of Anura is discussed on the basis of the distribution patterns obtained.