Highly conserved linkage homology between birds and turtles: Bird and turtle chromosomes are precise counterparts of each other

The karyotypes of birds, turtles and snakes are characterized by two distinct chromosomal components, macrochromosomes and microchromosomes. This close karyological relationship between birds and reptiles has long been a topic of speculation among cytogeneticists and evolutionary biologists; however, there is scarcely any evidence for orthology at the molecular level. To define the conserved chromosome synteny among humans, chickens and reptiles and the process of genome evolution in the amniotes, we constructed comparative cytogenetic maps of the Chinese soft-shelled turtle (Pelodiscus sinensis) and the Japanese four-striped rat snake (Elaphe quadrivirgata) using cDNA clones of reptile functional genes. Homology between the turtle and chicken chromosomes is highly conserved, with the six largest chromosomes being almost equivalent to each other. On the other hand, homology to chicken chromosomes is lower in the snake than in the turtle. Turtle chromosome 6q and snake chromosome 2p represent conserved synteny with the chicken Z chromosome. These results suggest that the avian and turtle genomes have been well conserved during the evolution of the Arcosauria. The avian and snake sex Z chromosomes were derived from different autosomes in a common ancestor, indicating that the causative genes of sex determination may be different between birds and snakes.Matsuda and Nishida-Umehara contributed equally to this work.     

Molecular structures of centromeric heterochromatin and karyotypic evolution in the Siamese crocodile (Crocodylus siamensis) (Crocodylidae, Crocodylia)

Crocodilians have several unique karyotypic features, such as small diploid chromosome numbers (30–42) and the absence of dot-shaped microchromosomes. Of the extant crocodilian species, the Siamese crocodile (Crocodylus siamensis) has no more than 2n = 30, comprising mostly bi-armed chromosomes with large centromeric heterochromatin blocks. To investigate the molecular structures of C-heterochromatin and genomic compartmentalization in the karyotype, characterized by the disappearance of tiny microchromosomes and reduced chromosome number, we performed molecular cloning of centromeric repetitive sequences and chromosome mapping of the 18S-28S rDNA and telomeric (TTAGGG) _n sequences. The centromeric heterochromatin was composed mainly of two repetitive sequence families whose characteristics were quite different. Two types of GC-rich CSI-HindIII family sequences, the 305 bp CSI-HindIII-S (G+C content, 61.3%) and 424 bp CSI-HindIII-M (63.1%), were localized to the intensely PI-stained centric regions of all chromosomes, except for chromosome 2 with PI-negative heterochromatin. The 94 bp CSI-DraI (G+C content, 48.9%) was tandem-arrayed satellite DNA and localized to chromosome 2 and four pairs of small-sized chromosomes. The chromosomal size-dependent genomic compartmentalization that is supposedly unique to the Archosauromorpha was probably lost in the crocodilian lineage with the disappearance of microchromosomes followed by the homogenization of centromeric repetitive sequences between chromosomes, except for chromosome 2.     

Reciprocal chromosome painting between white hawk (<Emphasis Type="Italic">Leucopternis albicollis</Emphasis>) and chicken reveals extensive fusions and fissions during karyotype evolution of accipitridae (Aves,Falconiformes)

Evolutionary cytogenetics can take confidence from methodological and analytical advances that promise to speed up data acquisition and analysis. Drastic chromosomal reshuffling has been documented in birds of prey by FISH. However, the available probes, derived from chicken, have the limitation of not being capable of determining if breakpoints are similar in different species: possible synapomorphies are based on the number of segments hybridized by each of chicken chromosome probes. Hence, we employed FACS to construct chromosome paint sets of the white hawk (Leucopternis albicollis), a Neotropical species of Accipitridae with 2n = 66. FISH experiments enabled us to assign subchromosomal homologies between chicken and white hawk. In agreement with previous reports, we found the occurrence of fusions involving segments homologous to chicken microchromosomes and macrochromosomes. The use of these probes in other birds of prey can identify important chromosomal synapomorphies and clarify the phylogenetic position of different groups of Accipitridae.     

Molecular cytogenetic map of the central bearded dragon, Pogona vitticeps (Squamata: Agamidae)

Reptiles, as the sister group to birds and mammals, are particularly valuable for comparative genomic studies among amniotes. The Australian central bearded dragon (Pogona vitticeps) is being developed as a reptilian model for such comparisons, with whole-genome sequencing near completion. The karyotype consists of 6 pairs of macrochromosomes and 10 pairs microchromosomes (2n?=?32), including a female heterogametic ZW sex microchromosome pair. Here, we present a molecular cytogenetic map for P. vitticeps comprising 87 anchor bacterial artificial chromosome clones that together span each macro- and microchromosome. It is the first comprehensive cytogenetic map for any non-avian reptile. We identified an active nucleolus organizer region (NOR) on the sub-telomeric region of 2q by mapping 18S rDNA and Ag-NOR staining. We identified interstitial telomeric sequences in two microchromosome pairs and the W chromosome, indicating that microchromosome fusion has been a mechanism of karyotypic evolution in Australian agamids within the last 21 to 19 million years. Orthology searches against the chicken genome revealed an intrachromosomal rearrangement of P. vitticeps 1q, identified regions orthologous to chicken Z on P. vitticeps 2q, snake Z on P. vitticeps 6q and the autosomal microchromosome pair in P. vitticeps orthologous to turtle Pelodiscus sinensis ZW and lizard Anolis carolinensis XY. This cytogenetic map will be a valuable reference tool for future gene mapping studies and will provide the framework for the work currently underway to physically anchor genome sequences to chromosomes for this model Australian squamate.     

Globin gene structure in a reptile supports the transpositional model for amniote α- and β-globin gene evolution

The haemoglobin protein, required for oxygen transportation in the body, is encoded by α- and β-globin genes that are arranged in clusters. The transpositional model for the evolution of distinct α-globin and β-globin clusters in amniotes is much simpler than the previously proposed whole genome duplication model. According to this model, all jawed vertebrates share one ancient region containing α- and β-globin genes and several flanking genes in the order MPG-C16orf35-(α-β)-GBY-LUC7L that has been conserved for more than 410 million years, whereas amniotes evolved a distinct β-globin cluster by insertion of a transposed β-globin gene from this ancient region into a cluster of olfactory receptors flanked by CCKBR and RRM1. It could not be determined whether this organisation is conserved in all amniotes because of the paucity of information from non-avian reptiles. To fill in this gap, we examined globin gene organisation in a squamate reptile, the Australian bearded dragon lizard, Pogona vitticeps (Agamidae). We report here that the α-globin cluster (HBK, HBA) is flanked by C16orf35 and GBY and is located on a pair of microchromosomes, whereas the β-globin cluster is flanked by RRM1 on the 3′ end and is located on the long arm of chromosome 3. However, the CCKBR gene that flanks the β-globin cluster on the 5′ end in other amniotes is located on the short arm of chromosome 5 in P. vitticeps, indicating that a chromosomal break between the β-globin cluster and CCKBR occurred at least in the agamid lineage. Our data from a reptile species provide further evidence to support the transpositional model for the evolution of β-globin gene cluster in amniotes.     

Assessment of compositional heterogeneity within and between eukaryotic genomes

Using large amounts of long genomic sequences, we studied the compositional patterns of eukaryotic genomes. We developed a simple measure, the compositional heterogeneity (or variability) index, to compare the differences in compositional heterogeneity between long genomic sequences. The index measures the average difference in GC content between two adjacent windows normalized by the standard error expected under the assumption of random distribution of nucleotides in a window. We report the following findings: (1) The extent of the compositional heterogeneity in a genomic sequence strongly correlates with its GC content in all multicellular eukaryotes studied regardless of genome size. (2) The human genome appears to be highly compositionally heterogeneous both within and between individual chromosomes; the heterogeneity goes much beyond the predictions of the isochore model. (3) All genomes of multicellular eukaryotes examined in this study are compositionally heterogeneous, although they also contain compositionally uniform segments, or isochores. (4) The true uniqueness of the human (or mammalian) genome is the presence of very high GC regions, which exhibit unusually high compositional heterogeneity and contain few long homogeneous segments (isochores). In general, GC-poor isochores tend to be longer than GC-rich ones. These findings indicate that the genomes of multicellular organisms are much more heterogeneous in nucleotide composition than depicted by the isochore model and so lead to a looser definition of isochores.     

Avian comparative genomics: reciprocal chromosome painting between domestic chicken (Gallus gallus) and the stone curlew (Burhinus oedicnemus, Charadriiformes)—An atypical species with low diploid number

The chicken is the most extensively studied species in birds and thus constitutes an ideal reference for comparative genomics in birds. Comparative cytogenetic studies indicate that the chicken has retained many chromosome characters of the ancestral avian karyotype. The homology between chicken macrochromosomes (1–9 and Z) and their counterparts in more than 40 avian species of 10 different orders has been established by chromosome painting. However, the avian homologues of chicken microchromosomes remain to be defined. Moreover, no reciprocal chromosome painting in birds has been performed due to the lack of chromosome-specific probes from other avian species. Here we have generated a set of chromosome-specific paints using flow cytometry that cover the whole genome of the stone curlew (Burhinus oedicnemus, Charadriiformes), a species with one of the lowest diploid number so far reported in birds, as well as paints from more microchromosomes of the chicken. A genome-wide comparative map between the chicken and the stone curlew has been constructed for the first time based on reciprocal chromosome painting. The results indicate that extensive chromosome fusions underlie the sharp decrease in the diploid number in the stone curlew. To a lesser extent, chromosome fissions and inversions occurred also during the evolution of the stone curlew. It is anticipated that this complete set of chromosome painting probes from the first Neoaves species will become an invaluable tool for avian comparative cytogenetics.     

Characterization of chromosome structures of Falconinae (Falconidae, Falconiformes, Aves) by chromosome painting and delineation of chromosome rearrangements during their differentiation

Karyotypes of most bird species are characterized by around 2n = 80 chromosomes, comprising 7–10 pairs of large- and medium-sized macrochromosomes including sex chromosomes and numerous morphologically indistinguishable microchromosomes. The Falconinae of the Falconiformes has a different karyotype from the typical avian karyotype in low chromosome numbers, little size difference between macrochromosomes and a smaller number of microchromosomes. To characterize chromosome structures of Falconinae and to delineate the chromosome rearrangements that occurred in this subfamily, we conducted comparative chromosome painting with chicken chromosomes 1–9 and Z probes and microchromosome-specific probes, and chromosome mapping of the 18S–28S rRNA genes and telomeric (TTAGGG) _n sequences for common kestrel (Falco tinnunculus) (2n = 52), peregrine falcon (Falco peregrinus) (2n = 50) and merlin (Falco columbarius) (2n = 40). F. tinnunculus had the highest number of chromosomes and was considered to retain the ancestral karyotype of Falconinae; one and six centric fusions might have occurred in macrochromosomes of F. peregrinus and F. columbarius, respectively. Tandem fusions of microchromosomes to macrochromosomes and between microchromosomes were also frequently observed, and chromosomal locations of the rRNA genes ranged from two to seven pairs of chromosomes. These karyotypic features of Falconinae were relatively different from those of Accipitridae, indicating that the drastic chromosome rearrangements occurred independently in the lineages of Accipitridae and Falconinae.     

Molecular and cytogenetic characterization of site-specific repetitive DNA sequences in the Chinese soft-shelled turtle (<Emphasis Type="Italic">Pelodiscus sinensis</Emphasis>, Trionychidae)

A novel family of repetitive DNA sequences that are components of constitutive heterochromatin were cloned from BglI-digested genomic DNA of the Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae), and characterized by filter hybridization and chromosome in-situ hybridization. The BglI-family of repetitive sequences were classified into four types by their genome organization and chromosomal distribution as follows: the repeated sequences located on (1) two pairs of microchromosomes, (2) four pairs of microchromosomes,(3) about half the number of microchromosomes and (4) the interstitial region of the short arm of chromosome 2. The presence of microchromosome-specific repetitive sequences has also been reported in the Struthioniformes and Galliformes, suggesting that turtle chromosomes retain some similarity to the chromosome structure as well as the karyotypes of avian species     

Compositional Mapping of Mouse Chromosomes and Identification of the Gene-Rich Regions

The mouse genome is a mosaic of isochores, consisting of long (>300 kb), compositionally homogeneous DNA segments that can be divided into two GC-poor families, L1 and L2, representing 56% of the genome, and two GC-rich families, H1 and H2, representing 26% and 7% of the genome, respectively, the remaining 11% being formed by satellite and ribosomal DNAs. (GC is the molar fraction of guanine + cytosine in DNA.) The mouse genome differs from the human genome (which is representative of most mammalian genomes) because it shows a narrower compositional spectrum of isochores and it has a karyotype formed exclusively by acrocentric chromosomes. The chromosomal distribution of the four isochore families, as investigated here by in situ hybridization of single-copy sequences from compositional DNA fractions, has shown that G(iemsa) bands are essentially composed of GC-poor isochores, whereas R(everse) bands comprise three subsets of bands: R′ bands, containing GC-poor isochores and GC-rich isochores of the H1 family, and T and T′ bands, containing all H2 isochores (in addition to other isochores), the former containing a higher proportion of H2 isochores than the latter. Mouse T and T′ bands are generally syntenic with, and are compositionally related to, human T and T′ bands and have the highest gene concentrations. These findings indicate that the distribution of isochore families and genes in chromosomal bands is basically similar in mouse and in human genomes, in spite of their remarkable differences and their extremely large phylogenetic distance.     

Chromosomal polymorphism and comparative painting analysis in the zebra finch

The zebra finch (Taeniopygia guttata) is often studied because of its interesting behaviour and neurobiology. Genetic information on this species has been lacking, making analysis of informative mutants difficult. Here we report on an improved cytological method for preparation of metaphase chromosomes suitable for fluorescent in situ hybridization of adult birds. We found that individual chicken chromosome paints usually hybridized to single zebra finch chromosomes, indicating only minor chromosomal rearrangements since the evolutionary divergence of these two species, and suggesting that the genomic location of chicken genes will predict the location of zebra finch orthologues. Chicken chromosome 1 appears to have split into two macrochromosomes in zebra finches, and chicken chromosome 4 paint hybridizes to a zebra finch macrochromosome and a microchromosome. This pattern was confirmed by mapping the androgen receptor (AR), which is located on chicken chromosome 4 but on a zebra finch microchromosome. We detected a telocentric/submetacentric polymorphism of chromosome 6 in our colony of zebra finches, and found that the polymorphism was inherited in a Mendelian pattern     

The Effect of BCG on Thoracic Duct Lymphocytes

We showed that GC-content of nucleotide sequences coding for linear B-cell epitopes of herpes simplex virus type 1 (HSV1) glycoprotein B (gB) is higher than GC-content of sequences coding for epitope-free regions of this glycoprotein (G + C = 73 and 64%, respectively). Linear B-cell epitopes have been predicted in HSV1 gB by BepiPred algorithm (www.cbs.dtu.dk/services/BepiPred). Proline is an acrophilic amino acid residue (it is usually situated on the surface of protein globules, and so included in linear B-cell epitopes). Indeed, the level of proline is much higher in predicted epitopes of gB than in epitope-free regions (17.8% versus 1.8%). This amino acid is coded by GC-rich codons (CCX) that can be produced due to nucleotide substitutions caused by mutational GC-pressure. GC-pressure will also lead to disappearance of acrophobic phenylalanine, isoleucine, methionine and tyrosine coded by GC-poor codons. Results of our “in-silico directed mutagenesis” showed that single nonsynonymous substitutions in AT to GC direction in two long epitope-free regions of gB will cause formation of new linear epitopes or elongation of previously existing epitopes flanking these regions in 25% of 539 possible cases. The calculations of GC-content and amino acid content have been performed by CodonChanges algorithm (www.barkovsky.hotmail.ru).     

Karyotype evolution in monitor lizards: cross-species chromosome mapping of cDNA reveals highly conserved synteny and gene order in the Toxicofera clade

The water monitor lizard (Varanus salvator macromaculatus (VSA), Platynota) has a chromosome number of 2n?=?40: its karyotype consists of 16 macrochromosomes and 24 microchromosomes. To delineate the process of karyotype evolution in V. salvator macromaculatus, we constructed a cytogenetic map with 86 functional genes and compared it with those of the butterfly lizard (Leiolepis reevesii rubritaeniata (LRE); 2n?=?36) and Japanese four-striped rat snake (Elaphe quadrivirgata (EQU); 2n?=?36), members of the Toxicofera clade. The syntenies and gene orders of macrochromosomes were highly conserved between these species except for several chromosomal rearrangements: eight pairs of VSA macrochromosomes and/or chromosome arms exhibited homology with six pairs of LRE macrochromosomes and eight pairs of EQU macrochromosomes. Furthermore, the genes mapped to microchromosomes of three species were all located on chicken microchromosomes or chromosome 4p. No reciprocal translocations were found in the species, and their karyotypic differences were caused by: low frequencies of interchromosomal rearrangements, such as tandem fusions, or centric fissions/fusions between macrochromosomes and between macro- and microchromosomes; and intrachromosomal rearrangements, such as paracentric inversions or centromere repositioning. The chromosomal rearrangements that occurred in macrochromosomes of the Varanus lineage were also identified through comparative cytogenetic mapping of V. salvator macromaculatus and V. exanthematicus. Morphologic differences in chromosomes 6–8 between the two species could have resulted from pericentric inversion or centromere repositioning.     

Avian genomes: different karyotypes but a similar distribution of the GC-richest chromosome regions at interphase

The chicken karyotype, like that of the vast majority of avian species, shows a large number of dot-shaped microchromosomes that are characterized, like most telomeric regions of the macrochromosomes, by the highest GC levels and the highest gene densities. In interphase nuclei, these gene-dense regions are centrally located, and are characterized by an open chromatin structure (a similar situation also exists in mammals). Avian species belonging to the Accipitridae family (diurnal raptors) show a karyotype with no very large chromosomes, and with only a very small number of microchromosomes. To identify the GC-rich (and gene-rich) regions of the chromosomes and nuclei from Accipitridae, we performed heterologous in-situ hybridizations using chicken GC-richest isochores as probes. Our results clearly show that the gene-rich regions are prevalently located in the few microchromosome pairs and in the telomeric regions of the middle-sized chromosomes, as well as in the interior of the interphase nuclei. This result is consistent with a common organization of the genome in the nuclei of warm-blooded vertebrates. Indeed, in spite of the different size and morphology of the chromosomes, the gene-dense regions are always located in the interior of the nuclei.     

Compositional mapping of chicken chromosomes and identification of the gene-richest regions

Compositional chromosomal mapping, namely the assessment of the GC level of chromosomal bands, led to the identification, in the human chromosomes, of the GC-richest H3⁺ bands and of the GC-poorest L1⁺ bands, which were so called on the basis of the isochore family predominantly present in the bands. The isochore organization of the avian genome is very similar to those of most mammals, the only difference being the presence of an additional, GC-richest, H4 isochore family. In contrast, the avian karyotypes are very different from those of mammals, being characterized, in most species, by few macrochromosomes and by a large number of microchromosomes. The compositional mapping of chicken mitotic and meiotic chromosomes by in-situ hybridization of isochore families showed that the chicken GC-richest isochores are localized not only on a large number of microchromosomes but also on almost all telomeric bands of macrochromosomes. On the other hand, the GC-poorest isochores are generally localized on the internal regions of macrochromosomes and are almost absent in microchromosomes. Thus, the distinct localization of the GC-richest and the GC-poorest bands observed on human chromosomes appears to be a general feature of chromosomes from warm-blooded vertebrates.     

Human chromosomal banding by in situ hybridization of isochores

Further to the classical methods that involve different chromosome treatments followed by staining, in situ hybridization of isochores represents a novel approach to chromosomal banding. Isochores are long compositionally homogeneous DNA segments that, in the human genome, belong to five families, two GC-poor families (L1 and L2) representing 30% and 33% of the genome, respectively, and three GC-rich families (H1, H2 and H3) representing 24%, 7.5% and 4-5- of the genome, respectively. Gene concentration increases with increasing GC levels, reaching an up to 20-fold higher level in H3 compared to L1 isochores. In situ hybridization of DNA from different isochore families on metaphase chromosomes allow to distinguish different sets of Giemsa and Reverse bands. In addition, it also provides information on the chromosomal distribution of genes.     

Novel tools for characterising inter and intra chromosomal rearrangements in avian microchromosomes

Avian genome organisation is characterised, in part, by a set of microchromosomes that are unusually small in size and unusually large in number. Although containing about a quarter of the genome, they contain around half the genes and three quarters of the total chromosome number. Nonetheless, they continue to belie analysis by cytogenetic means. Chromosomal rearrangements play a key role in genome evolution, fertility and genetic disease and thus tools for analysis of the microchromosomes are essential to analyse such phenomena in birds. Here, we report the development of chicken microchromosomal paint pools, generation of pairs of specific microchromosome BAC clones in chicken, and computational tools for in silico comparison of the genomes of microchromosomes. We demonstrate the use of these molecular and computational tools across species, suggesting their use to generate a clear picture of microchromosomal rearrangements between avian species. With increasing numbers of avian genome sequences that are emerging, tools such as these will find great utility in assembling genomes de novo and for asking fundamental questions about genome evolution from a chromosomal perspective.     

Comparison of the chicken and turkey genomes reveals a higher rate of nucleotide divergence on microchromosomes than macrochromosomes

A distinctive feature of the avian genome is the large heterogeneity in the size of chromosomes, which are usually classified into a small number of macrochromosomes and numerous microchromosomes. These chromosome classes show characteristic differences in a number of interrelated features that could potentially affect the rate of sequence evolution, such as GC content, gene density, and recombination rate. We studied the effects of these factors by analyzing patterns of nucleotide substitution in two sets of chicken-turkey sequence alignments. First, in a set of 67 orthologous introns, divergence was significantly higher in microchromosomes (chromosomes 11-38; 11.7% divergence) than in both macrochromosomes (chromosomes 1-5; 9.9% divergence; P = 0.016) and intermediate-sized chromosomes (chromosomes 6-10; 9.5% divergence; P = 0.026). At least part of this difference was due to the higher incidence of CpG sites on microchromosomes. Second, using 155 orthologous coding sequences we noted a similar pattern, in which synonymous substitution rates on microchromosomes (13.1%) were significantly higher than were rates on macrochromosomes (10.3%; P = 0.024). Broadly assuming neutrality of introns and synonymous sites, or constraints on such sequences do not differ between chromosomal classes, these observations imply that microchromosomal genes are exposed to more germ line mutations than those on other chromosomes. We also find that dN/dS ratios for genes located on microchromosomes (average, 0.094) are significantly lower than those of macrochromosomes (average, 0.185; P = 0.025), suggesting that the proteins of genes on microchromosomes are under greater evolutionary constraint.     

Comparison of the lignin-degrading white rot fungi Phanerochaete chrysosporium and Sporotrichum pulverulentum at the DNA level

Summary DNA-hybridisation studies showed a close relationship between Phanerochaete chrysosporium ME446, most used in lignin degradation studies, and Sporotrichum pulverulentum Novobranova, the other standard lignin degrading strain. Two other strains of P. chrysosporium were both less related. We show that P. chrysosporium ME446 and S. pulverulentum Novobranova both have a GC-content of 59% for chromosomal DNA with the rRNA genes present as an AT-rich satellite; the mitochondrial DNA has a GC-content of 33%. The genome size estimated for P. chrysosporium ME446 is about 4–5 × 10⁷ bp.     

Genomic characterization of two novel reptilian papillomaviruses, Chelonia mydas papillomavirus 1 and Caretta caretta papillomavirus 1

In this paper we describe the characterization of the genomes of two sea turtle papillomaviruses, Chelonia mydas PV (CmPV-1) and Caretta caretta PV (CcPV-1). The isolation and sequencing of the first non-avian reptilian PVs extend the evolutionary history of PVs to include all amniotes. PVs have now been described in mammals, birds and non-avian reptiles. The chelonian PVs form a distinct clade most closely related to the avian PVs. Unlike the avian PVs, both chelonian PVs have canonical E6 and E7 ORFs, indicating that these genes were present in the common ancestor to mammalian and non-mammalian amniote PVs. Rates of evolution among the non-mammalian PVs were generally slower than those estimated for mammalian PVs, perhaps due to lower metabolic rates among the ectothermic reptiles.