Genetic And Evolutionary Study On Alpha Satellite DNA In Arctic Fox, Silver Fox, Domestic Dog And Raccoon Dog

In higher eukaryotes, genomic DNAs comprise a lot of repetitive DNA sequence- es. According to their structural organization, highly repetitive sequences have been divided into two classes,one class comprises the interspersed sequence families throughout the genome, the interspersed sequence families can be subdivided into the long interspersed elements (LINEs) and the short interspersed elements (SINEs);the other class comprises the tandem repeat elements, can be subdivided into the satellites, mimisatellites and microsatellites. Tandem repeat elements or satellite DNAs that consist of large numbers of monomeric units of 100-1000bp, organized in long tandem arrays, usually a single centromeric satellite DNA family is predominant, accounting for 10% or more of the genome. The satellite DNAs constitute much of the heterochromatin of chromosomes, predominately that of centromeres and telomeres.The Canidae is a family of dog-like carnivores that includes 16 genera and 36 species. Considerable interest in the evolutionary relationships of canids had resulted in analyses based on morphological data, karyotype, allozymes and DNA molecular. Great progress had been made in evolutionary relationships of canids in the past ten years, however, several phylogenetic issues remain unresolved, especially, the relatio- nship among Bat-eared fox, Raccoon dog, and Gray fox and the other canids is not clear. Analyses of nuclear, mitochondrial, and morphologic data had resulted in different placements of these taxa within the canid phylogeny. Canidae species fall into two categories with respect to their chromosome composition: those with high numbered largely acrocentric karyotypes and others with a low numbered principally metacentric karyotypes. Extensive chromosomes homology is apparent among acroce- ntric chromosome arms within Canidae species. This indicates that an ancestral episode of extensive centric fission leading to an ancestral canid genome organization that was subsequently reorganized by multiple chromosome fusion events in some Canidae species.The study was designed to clone and analyze the alpha satellite DNAs of Silver fox , Arctic fox, domestic dog and Raccoon dog. This would provide theoretical basis in future studies on the evolutionary relationships of canids and molecular mechanisms of Robertsonian translocation.Genomic DNAs from Arctic fox were cleaved with restriction enzymes BamH I,Xho I,EcoR V,Kpn I and Sac I respectively. Three prominent ethidium-bromide stanined bands of approximately 750 bp, 1500 bp and 2100 bp were readily distingui- shed through the background smear of DNA-digested with Kpn I and Sac I, after separation of DNA fragments by agarose gel electrophoresis. DNA fragments were recovered and cloned into pUC18 plasmid vector, then propagated in E.coli DH5αbacteria. The recombinant plasmids were designed as AKpn, AKpnm and ASac. Nucl- eotide sequences were analyzed and compared with DNAstar version 6.13, DNAMAN version 5.0 and MEGA version 3.1. Forty-four sequences were obtained, including sixteen AKpn, four AKpnm and twenty-four ASac. Sequence analyses revealed that these sequences had 90% homology between AKpn and ASac. Each AKpnm sequence consisted of two intact AKpn monomers. The satellite monomer units in length was 737 bp, with an average G+C content of 51.9%. To determine the genomic organizat- io n of satellite DNA, Dig-11-dUTP-labeled ASac1 was used as probe to Southern blots containing different endonuclease-digested genomic DNAs of Arctic fox and genomic DNA of different species with Sac I. Southern blot hybridizations revealed a 700 bp ladder patterns that were characteristic of satellite DNA, and the satellite DNAs were specific for Canidae. The satellite DNAs were 75% homology to the dog satellite DNA sequence or centromere-associated nucleotide sequences cloned by Fanning in 1987. In order to distinguish from another satellite DNAs of canidae genome, the satellite was designed as alpha satellite DNA. The monomer consisted of three subrepeats of approximately 245 bp in length with 49-55% homology.Alpha satellite DNAs of Silver fox and domestic dog were cloned with restriction enzyme Sac I.The procedures were performed as described before. Nine alpha satellite monomer units were obtained for Silver fox. Sequence analysis revealed that the satellite monomer units in length was 737 bp, with an average G+C content of 52.7%. Each sequence had 90% homology with anothers. Twenty-one alpha satellite monom- er units were obtained for Domestic dog, the satellite monomer units in length was 738 bp, with an average G+C content of 50.8%. The monomer consists of three subrepeats of approximately 245 bp in length.While genomic DNAs of Raccoon dog were cleaved with restriction enzymes BamH I,Xho I,EcoR V,Kpn I and Sac I respectively, however, no prominent ethidium-bromide stanined bands were in the smear background after separation of DNA fragments by agarose gel electrophoresis. A pair of primers, that is RSP1(5′-GA- GCTCAACTCTCXGXAGTA-3′) and RSP2(5′-ACCCTXAGTCCCTCAXAXTT-3′), were designed according to the alpha satellite monomers of Arctic fox, Silver fox and Domestic dog. PCR products were separated, purified and cloned with the pUCm-T vector. Fourteen RSP sequences were obtained, the cloned sequences were variable in length, ranging from 495 to 1815 bp. But approximately the 500 bp of the 3′side of all the RSP sequences was similar, with 90% identity except the four RSP4 sequences. The 500 bp fragment of all the RSP sequence had 75% homology to the satellite monomers of Arctic fox, Silver fox and Domestic dog. Sequence analyses indicated that the alpha satellite monomers of Raccoon dog cannot be deduced only by PCR amplification.A random sheared cosmid library of Raccoon dog was successfully constructed. Genomic DNA was sheared by aspirating and expelling the DNA with a 200μL pipette tip, the ends of the sheared DNA were polished using by T4 DNA Polymerase, and phosphorylated by T4 Polynucleotide Kinase. The sheared and end-repaired DNA was run on a 1% agarose gel, DNA fragments in the size range of 25-45 kb were recovered, purified and ligated to CopyControl pCC1FOS vector, then packaged into phage particles. The packaged cosmid clones were titered by serial dilutions and infected to EPI300. The titer of the original library was 4.5×105 cfu/mL, analyses of randomly selected fosmid clones indicated that 100% of the clones contained inserts with an average size of approximately 25 kb.Dig-11-dUTP-labeled RSP5.1 was used as probe to screen alpha satellite DNA from the library by colony in situ hybridization, 30 candidate cosmid clones contain- ing alpha satellite DNA had been obtained. Cosmid DNAs of twelve candidate clones were cleaved with restriction enzyme Sac I, two prominent ethidium-bromide stanined bands of approximately 500 bp and 700 bp were obtained from the twelve clones, me- anwhile there were some clones containing bands of approximately 800 bp and 1100 bp. Southern blot hybridizations revealed that all bands except 500 bp in size contain- ed hybridizational signals. Cosmid DNAs of RCL25 was digested to incompletion with restriction enzyme Sac I, three bands of approximately 700 bp,1100 bp and 1500 bp were cloned, the fragments of 800 bp and 1100 bp from RCL33 ,and 1100 bp from RCL2.2 were cloned, too.Sequence analyses of eighteen clones indicated that alpha satellite monomer of Raccoon dog in size was 1626 bp, Three fragment including 667 bp, 475 bp and 502 bp would be obtained if satellite monomer were digested to completion with Sac I.The fragments had lowly similarity to each others, only about 50%, but the sequences of the same size from different clones had high similarity, about 90%. The monomer consisted of seven subrepeats, six subrepeats approximately 245 bp in length, one was only 140 bp.The monomers of alpha satellite family varied in size and in G+C content between canidae species, 737 bp and 52.1% for Arctic fox, 737 bp and 52.7% for Silver fox, 738 bp and 50.8% for Domestic dog, 1626 bp and 57.4% for Raccoon dog, 880 bp and 54.0% for Gray fox, respectively.There was polymorphism between the monomers of alpha satellite within the species. The substitution spectra were different in the four species, 5.92% for Domest- ic dog, 4.12% for Arctic fox, 4.12% for Silver fox, 4.3% for Raccoon dog, respectively. Substitutions involving G or C residues are much more common in the Arctic fox, and Raccoon dog than Domestic dog and Silver fox.Genomic DNAs of Arctic fox, Silver fox and Silver-Arctic fox, were cleaved with Sac I, respectively. The satellite bands of Arctic fox were much more intense than Silver fox and Silver-Arctic fox, however, the bands of Silver-Arctic fox were much more intense than Silver fox. Sequence analyses indicated that the satellite monomers were highly identical between the two species. But the arctic fox karyotype was highly different with the silver fox karyotype. The difference resulted from an ancestral episode of extensive centric fission or centric fussion, so, it was speculated that alpha satellite of the canidae could be involved in centric fission or centric fussion.Based on the alpha satellite DNA data from the five canidae species, the phyloge- netic relations were speculated.Relationship between Arctic fox and Silver fox was more closer than others, meanwhile, Raccoon dog, Domestic dog and Gray fox should be basal to all other canidae.