Comparative compositional mapping of chicken and quail chromosomes

The distribution of various isochore families on mitotic chromosomes of domestic chicken and Japanese quail was studied by the method of fluorescence in situ DNA--DNA hybridization (FISH). DNA of various isochore families was shown to be distributed irregularly and similarly on chromosomes of domestic chicken and Japanese quail. The GC-rich isochore families (H2, H3, and H4) hybridized mainly to microchromosomes and a majority of macrochromosome telomeric regions. In chicken, an intense fluorescence was also in a structural heterochromatin region of the Z chromosome long arm. In some regions of the quail macrochromosome arms, hybridization was also with isochore families H3 and H4. On macrochromosomes of both species, the pattern of hybridization with isochores of the H2 and H3 families resembled R-banding. The light isochores (L1 and L2 families) are mostly detected within macrochromosome internal regions corresponding to G bands, whereas microchromosomes lack light isochores. Although mammalian and avian karyotypes differ significantly in organization, the isochore distribution in genomes of these two lineages of the warm-blooded animals is similar in principle. On macrochromosomes of the two avian species studied, a pattern of isochore distribution resembled that of mammalian chromosomes. The main specific feature of the avian genome, a great number of microchromosomes (about 30% of the genome), determines a compositional specialization of the latter. This suggests the existence of not only structural but also functional compartmentalization of the avian genome.     

Localization of the NotI clones from human chromosome 3 on quail microchromosomes

For the purpose of comparative mapping of quail (Coturnix c. japonica) and human (Homo sapiens) genomes, DNA fragments from human chromosome 3 (HSA3p14-21 and HSA3q13-23) were localized on quail mitotic chromosomes. Using the method of double-color fluorescence DNA-DNA in situ hybridization, these fragments were mapped to two different microchromosomes. Earlier, similar studies were performed using chicken mitotic chromosomes. There it was demonstrated that the clones of interest were distributed among three microchromosomes (GGA12, GGA14, and GGA15). Thus, interspecific difference in the location of human chromosome 3 DNA fragments in the genomes of closely related avian species was discovered. A new confirmation of the hypothesis on the preferable localization of the gene-rich human chromosome regions on avian microchromosomes was obtained. At the same time, a suggestion on the localization of some orthologous genes in the genome of the organism under study was made: ARF4, SCN5A, PHF7, ABHD6, ZDHHC3, MAPKAPK3, ADSYNA (homolog of chicken chromosome 12), DRD2, PP2C-ETA, RAB7, CCKAR, and PKD1 (homolog of chicken chromosome 15). However, localization of the corresponding quail genes needs to be confirmed, as far as the sequences used were only the orthologs of the corresponding chicken genes.     

A comparative cytogenetic study of chromosome homology between chicken and Japanese quail

In order to construct a chicken (Gallus gallus) cytogenetic map, we isolated 134 genomic DNA clones as new cytogenetic markers from a chicken cosmid DNA library, and mapped these clones to chicken chromosomes by fluorescence in situ hybridization. Forty-five and 89 out of 134 clones were localized to macrochromosomes and microchromosomes, respectively. The 45 clones, which localized to chicken macrochromosomes (Chromosomes 1-8 and the Z chromosome) were used for comparative mapping of Japanese quail (Coturnix japonica). The chromosome locations of the DNA clones and their gene orders in Japanese quail were quite similar to those of chicken, while Japanese quail differed from chicken in chromosomes 1, 2, 4 and 8. We specified the breakpoints of pericentric inversions in chromosomes 1 and 2 by adding mapping data of 13 functional genes using chicken cDNA clones. The presence of a pericentric inversion was also confirmed in chromosome 8. We speculate that more than two rearrangements are contained in the centromeric region of chromosome 4. All 30 clones that mapped to chicken microchromosomes also localized to Japanese quail microchromosomes, suggesting that chromosome homology is highly conserved between chicken and Japanese quail and that few chromosome rearrangements occurred in the evolution of the two species.     

The pig genome: compositional analysis and identification of the gene-richest regions in chromosomes and nuclei

The isochore organization of the mammalian genome comprises a general pattern and some special patterns, the former being characterized by a wider compositional distribution of the DNA fragments. The large majority of the mammalian genomes belong to the former, and only some groups, such as the Myomorpha sub-order of Rodentia, belong to the latter. Here we describe the compositional organization of the pig (Sus scrofa) genome that belongs to the general mammalian pattern. We investigated (i) the compositional distribution of the genes by analysis of their GC3 levels (the GC levels at the third codon positions), and (ii) the correlation between the GC3 value of orthologous genes from pig and other vertebrates (human, calf, mouse, chicken, and Xenopus). As expected, the highest gene concentration corresponded to the H3 isochore family, and the highest GC3 correlations were observed in the pig/human and pig/calf comparisons. Then we identified, by in situ hybridization of the GC-richest H3 isochores, the pig chromosomal regions endowed by the highest gene-density that largely corresponded to the telomeric chromosomal bands. Moreover, we observed that these gene-rich bands are syntenic with the previously identified GC-richest/gene richest H3+ bands of the human chromosomes. At the cell nucleus level, we observed that the gene-dense region corresponded to the more internal compartment, as previously found in human and avian cell nuclei.     

Isochore patterns and gene distributions in fish genomes

The compositional approach developed in our laboratory many years ago revealed a large-scale compositional heterogeneity in vertebrate genomes, in which GC-rich and GC-poor regions, the isochores, were found to be characterized by high and low gene densities, respectively. Here we mapped isochores on fish chromosomes and assessed gene densities in isochore families. Because of the availability of sequence data, we have concentrated our investigations on four species, zebrafish (Brachydanio rerio), medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), and pufferfish (Tetraodon nigroviridis), which belong to four distant orders and cover almost the entire GC range of fish genomes. These investigations produced isochore maps that were drastically different not only from those of mammals (in that only two major isochore families were essentially present in each genome vs five in the human genome) but also from each other (in that different isochore families were represented in different genomes). Gene density distributions for these fish genomes were also obtained and shown to follow the expected increase with increasing isochore GC. Finally, we discovered a remarkable conservation of the average size of the isochores (which match replicon clusters in the case of human chromosomes) and of the average GC levels of isochore families in both fish and human genomes. Moreover, in each genome the GC-poorest isochore families comprised a group of "long isochores" (2-20 Mb in size), which were the lowest in GC and varied in size distribution and relative amount from one genome to the other.     

Chromosomal localization of the <Emphasis Type="Italic">NotI</Emphasis> clones from human chromosome 3 on quail microchromosomes

For the purpose of comparative mapping of quail (Coturnix c. japonica) and human (Homo sapiens) genomes, DNA fragments from human chromosome 3 (HSA3p14-21 and HSA3q13-23) were localized on quail mitotic chromosomes. Using the method of double-color fluorescence DNA-DNA in situ hybridization, these fragments were mapped to two different microchromosomes. Earlier, similar studies were performed using chicken mitotic chromosomes. There it was demonstrated that the clones of interest were distributed among three microchromosomes (GGA12, GGA14, and GGA15). Thus, interspecific difference in the location of human chromosome 3 DNA fragments in the genomes of closely related avian species was discovered. A new confirmation of the hypothesis on the preferable localization of the gene-rich human chromosome regions on avian microchromosomes was obtained. At the same time, a suggestion on the localization of some orthologous genes in the genome of the organism under study was made: ARF4, SCN5A, PHF7, ABHD6, ZDHHC3, MAPKAPK3, ADSYNA (homolog of chicken chromosome 12), DRD2, PP2C-ETA, RAB7, CCKAR, and PKD1 (homolog of chicken chromosome 15). However, localization of the corresponding quail genes needs to be confirmed, as far as the sequences used were only the orthologs of the corresponding chicken genes.     

Characterization and chromosomal distribution of a novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica)

A novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica) was isolated from genomic DNA digested with restriction endonuclease, Bg/II. Sequence analysis of three different-size clones revealed the presence of a tandem array of a GC-rich 41 bp repeated element. This sequence was localized by fluorescence in situ hybridization (FISH) primarily to microchromosomes of Japanese quail (2n = 78); approximately 50 of the 66 microchromosomes showed positive signals, although hybridization signals were also detected on chromosomes 4 and W. This satellite DNA did not cross-hybridize with genomic DNA of chicken (Gallus gallus) and Chinese painted quail (Excalfactoria chinensis) under moderately stringent conditions, suggesting that this class of repetitive DNA sequences was species specific and fairly divergent in Galliformes species.     

Cytogenetic assignment of 29 functional genes to chicken microchromosomes by FISH

We assigned 29 functional genes to chicken microchromosomes by fluorescence in situ hybridization (FISH). Two linkage groups in the genetic linkage map of the East Lansing breed were identified in this study by localizing the genes AGRN and H2FA to microchromosomes. The frequency of the genes mapped on 30 pairs of microchromosomes, which account for roughly 30% of the whole chicken genome, was about 40% of the 73 genes randomly mapped in our laboratory. This result confirms the important role of microchromosomes for avian genome function and supports the likelihood of a high gene density on avian microchromosomes.     

Telomeres in Warm-Blooded Vertebrates Are Composed of GC-Rich Isochores

We have hybridized the vertebrate telomeric sequence (TTAGGG)n on DNA compositional fractions from 13 mammalian species and 3 avian species, representing 9 and 3 orders, respectively. Our results indicate that the 50- to 100-kb fragments derived from telomeric regions are composed of GC-rich and GC-richest isochores. Previous works from our laboratory demonstrated that single-copy sequences from the human H3 isochore family (the GC-richest and gene-richest isochore in the human genome) share homology with compositionally correlated compartments of warm-blooded vertebrates. This correlation suggested that the GC-richest isochores are, as in the human genome, the gene-richest regions of warm-blooded vertebrates' genome. Moreover, this evidence suggests that telomeric regions are the most gene-dense region of all warm-blooded vertebrates. The implications of these findings are discussed.     

Investigation of pseudoautosomal and bordering regions in avian Z and W chromosomes with the use of large insert genomic BAC clones

To study pseudoautosomal and bordering regions in the avian Z and W chromosomes, we used seven BAC clones from genomic libraries as DNA probes of fragments of different gametologs of the ATP5A1 gene located close to the proximal border of the pseudoautosomal region (PAR) of sex chromosomes of domestic chicken and Japanese quail. Localization of BAC clones TAM31-b100C09, TAM31-b99N01, TAM31-b27P16, and TAM31-b95L18 in the short arm of Z chromosomes of domestic chicken and Japanese quail (region Zp23-p22) and localization of the BAC clones CHORI-261-CH46G16, CHORI-261-CH33F10, and CHORI-261-CH64F22 on W chromosomes of these species and in the short arm of Z chromosomes (region Zp23-p22) were determined by fluorescence in situ hybridization with the use of W-specific probes. The difference in the localization of the BAC clones on the Z and W chromosomes is probably explained by divergence of the nucleotide sequences of different sex chromosomes located beyond the pseudoautosomal region.     

FISH mapping of 57 BAC clones reveals strong conservation of synteny between Galliformes and Anseriformes

Karyotypes of chicken (Gallus gallus domesticus; 2n = 78) and mallard duck (Anas platyrhynchos; 2n = 80) share the typical organization of avian karyotypes including a few macrochromosome pairs, numerous indistinguishable microchromosomes, and Z and W sex chromosomes. Previous banding studies revealed great similarities between chickens and ducks, but it was not possible to use comparative banding for the microchromosomes. In order to establish precise chromosome correspondences between these two species, particularly for microchromosomes, we hybridized 57 BAC clones previously assigned to the chicken genome to duck metaphase spreads. Although most of the clones showed similar localizations, we found a few intrachromosomal rearrangements of the macrochromosomes and an additional microchromosome pair in ducks. BAC clones specific for chicken microchromosomes were localized to separate duck microchromosomes and clones mapping to the same chicken microchromosome hybridized to the same duck microchromosome, demonstrating a high conservation of synteny. These results demonstrate that the evolution of karyotypes in avian species is the result of fusion and/or fission processes and not translocations.     

Similar integration but different stability of Alus and LINEs in the human genome

Alus and LINEs (LINE1) are widespread classes of repeats that are very unevenly distributed in the human genome. The majority of GC-poor LINEs reside in the GC-poor isochores whereas GC-rich Alus are mostly present in GC-rich isochores. The discovery that LINES and Alus share similar target site duplication and a common AT-rich insertion site specificity raised the question as to why these two families of repeats show such a different distribution in the genome. This problem was investigated here by studying the isochore distributions of subfamilies of LINES and Alus characterized by different degrees of divergence from the consensus sequences, and of Alus, LINEs and pseudogenes located on chromosomes 21 and 22. Young Alus are more frequent in the GC-poor part of the genome than old Alus. This suggests that the gradual accumulation of Alus in GC-rich isochores has occurred because of their higher stability in compositionally matching chromosomal regions. Densities of Alus and LINEs increase and decrease, respectively, with increasing GC levels, except for the telomeric regions of the analyzed chromosomes. In addition to LINEs, processed pseudogenes are also more frequent in GC-poor isochores. Finally, the present results on Alu and LINE stability/exclusion predict significant losses of Alu DNA from the GC-poor isochores during evolution, a phenomenon apparently due to negative selection against sequences that differ from the isochore composition.     

In vivo - in vitro toxicogenomic comparison of TCDD-elicited gene expression in Hepa1c1c7 mouse hepatoma cells and C57BL/6 hepatic tissue

Background

By comparing the quail genome with that of chicken, chromosome rearrangements that have occurred in these two galliform species over 35 million years of evolution can be detected. From a more practical point of view, the definition of conserved syntenies helps to predict the position of genes in quail, based on information taken from the chicken sequence, thus enhancing the utility of this species in biological studies through a better knowledge of its genome structure. A microsatellite and an Amplified Fragment Length Polymorphism (AFLP) genetic map were previously published for quail, as well as comparative cytogenetic data with chicken for macrochromosomes. Quail genomics will benefit from the extension and the integration of these maps.

Results

The integrated linkage map presented here is based on segregation analysis of both anonymous markers and functional gene loci in 1,050 quail from three independent F2 populations. Ninety-two loci are resolved into 14 autosomal linkage groups and a Z chromosome-specific linkage group, aligned with the quail AFLP map. The size of linkage groups ranges from 7.8 cM to 274.8 cM. The total map distance covers 904.3 cM with an average spacing of 9.7 cM between loci. The coverage is not complete, as macrochromosome CJA08, the gonosome CJAW and 23 microchromosomes have no marker assigned yet. Significant sequence identities of quail markers with chicken enabled the alignment of the quail linkage groups on the chicken genome sequence assembly. This, together with interspecific Fluorescence In Situ Hybridization (FISH), revealed very high similarities in marker order between the two species for the eight macrochromosomes and the 14 microchromosomes studied.

Conclusion

Integrating the two microsatellite and the AFLP quail genetic maps greatly enhances the quality of the resulting information and will thus facilitate the identification of Quantitative Trait Loci (QTL). The alignment with the chicken chromosomes confirms the high conservation of gene order that was expected between the two species for macrochromosomes. By extending the comparative study to the microchromosomes, we suggest that a wealth of information can be mined in chicken, to be used for genome analyses in quail.     

Comparative FISH mapping on Z chromosomes of chicken and Japanese quail

Using direct R-banding fluorescence in situ hybridization, we assigned five functional genes-growth hormone receptor (GHR), prolactin receptor (PRLR), spleen tyrosine kinase (SYK), aldolase B (ALDOB), and muscle skeletal receptor tyrosine kinase (MUSK)-to the chicken Z chromosome. SYK and MUSK were newly localized to the chicken Z chromosome in this study. GHR and PRLR were situated close to each other on the short arm of the chicken Z chromosome, as are their counterparts on human chromosome 5. SYK, MUSK, and ALDOB, which have been mapped to human chromosome 9, were localized to the long arm of the chicken Z chromosome. Thus, the present results indicate the presence of conserved synteny between the chicken Z chromosome and human chromosomes 5 and 9. Using the same method, four of the genes (GHR, PRLR, ALDOB, and MUSK) were assigned to the Japanese quail Z chromosome. The locations of these four Z-linked genes were conserved between chicken and Japanese quail. The results support the notion that the avian Z chromosome and the mammalian X chromosome did not evolve from a common ancestral linkage group.     

Chromosome repatterning in three representative parrots (Psittaciformes) inferred from comparative chromosome painting

Parrots (order: Psittaciformes) are the most common captive birds and have attracted human fascination since ancient times because of their remarkable intelligence and ability to imitate human speech. However, their genome organization, evolution and genomic relation with other birds are poorly understood. Chromosome painting with DNA probes derived from the flow-sorted macrochromosomes (1-10) of chicken (Gallus gallus, GGA) has been used to identify and distinguish the homoeologous chromosomal segments in three species of parrots, i.e., Agapornis roseicollis (peach-faced lovebird); Nymphicus hollandicus (cockatiel) and Melopsittacus undulatus (budgerigar). The ten GGA macrochromosome paints unequivocally recognize 14 to 16 hybridizing regions delineating the conserved chromosomal segments for the respective chicken macrochromosomes in these representative parrot species. The cross-species chromosome painting results show that, unlike in many other avian karyotypes with high homology to chicken chromosomes, dramatic rearrangements of the macrochromosomes have occurred in parrot lineages. Among the larger GGA macrochromosomes (1-5), chromosomes 1 and 4 are conserved on two chromosomes in all three species. However, the hybridization pattern for GGA 4 in A. roseicollis and M. undulatus is in sharp contrast to the most common pattern known from hybridization of chicken macrochromosome 4 in other avian karyotypes. With the exception of A. roseicollis, chicken chromosomes 2, 3 and 5 hybridized either completely or partially to a single chromosome. In contrast, the smaller GGA macrochromosomes 6, 7 and 8 displayed a complex hybridization pattern: two or three of these macrochromosomes were found to be contiguously arranged on a single chromosome in all three parrot species. Overall, the study shows that translocations and fusions in conjunction with intragenomic rearrangements have played a major role in the karyotype evolution of parrots. Our inter-species chromosome painting results unequivocally illustrate the dynamic reshuffling of ancestral chromosomes among the karyotypes of Psittaciformes.     

FISH on avian lampbrush chromosomes produces higher resolution gene mapping

Giant lampbrush chromosomes, which are characteristic of the diplotene stage of prophase I during avian oogenesis, represent a very promising system for precise physical gene mapping. We applied 35 chicken BAC and 4 PAC clones to both mitotic metaphase chromosomes and meiotic lampbrush chromosomes of chicken (Gallus gallus domesticus) and Japanese quail (Coturnix coturnix japonica). Fluorescence in situ hybridization (FISH) mapping on lampbrush chromosomes allowed us to distinguish closely located probes and revealed gene order more precisely. Our data extended the data earlier obtained using FISH to chicken and quail metaphase chromosomes 1–6 and Z. Extremely low levels of inter- and intra-chromosomal rearrangements in the chicken and Japanese quail were demonstrated again. Moreover, we did not confirm the presence of a pericentric inversion in Japanese quail chromosome 4 as compared to chicken chromosome 4. Twelve BAC clones specific for chicken chromosome 4p and 4q showed the same order in quail as in chicken when FISH was performed on lampbrush chromosomes. The centromeres of chicken and quail chromosomes 4 seem to have formed independently after centric fusion of ancestral chromosome 4 and a microchromosome.     

Isochore pattern and gene distribution in the chicken genome

We report here investigations on the isochore pattern and the distribution of genes in the chromosomes of chicken. In spite of large differences in genome size and karyotype, the compositional properties and the gene distribution of the chicken genome are very similar to those recently published for the human genome, which is a good representative of most mammalian genomes. In fact, this similarity, which extends to the relative amounts and, also, to a large extent at least, to the average base composition of isochore families, is most interesting in view of the very large distance of mammals and birds for a common ancestor, which goes back to 310–340 million years ago. This raises important questions about genome evolution in vertebrates.