Evolutionary Analysis And Studies Of Hoxall Gene In Vertebrates

Hox genes, containing a 180 bp homeobox, encode helix-turn-helix transcription factors. Hox proteins can bind DNA, and regulate the axial patterning during embryonic development of animals. The evolution of morphological characters is thought to be mediated through the evolution of genes controlling embryonic development in animal phylogenesis. Hoxa-11gene plays an important role in the development of fish fins and tetrapod limbs. Therefore, studies on Hoxall gene can help us to understand both the genetic control in animal development and the fin-limb transition in vertebrate evolution.Degenerate primers for PCR, based on reported Hoxall genes from human and mouse, were designed to amplify fragments of Hoxall genes from ten vertebrates, Danio rerio, Pseudosciaena crocea, Lateolabrax japonicus, Oreochromis niloticus, Fugu rubripes, Ophiocephalus argus, Latimeria chalumnae, Xenopus tropicalis, Calotes jerdoni and Gallus gallus. Hoxall genes from 16 vertebrates were analyzed, containing 10 cloned Hoxall genes mentioned above and 6 Hoxall genes from GenBank, Homo sapiens, Mus musculus, Bos taurus, Polyodon spathula, Heterodontus francisci, and Oryzias latipes, in representatives of the three major gnathostome lineages: cartilaginous fish, ray-finned fish and lobe-finned fish. Hoxall genes from the three lineages have high similarity in both nucleotide and amino acid sequences.In nucleotide level, both the coding sequence and 5' intron sequence are very conservative. Two phylogenetic footprints, 35 bp and 17 bp in length respectively, were found in 5' intron sequence. 35 bp nucleotide fragment can bind proteins from HeLa cell extract and chicken embryonic cell extract, which means that the 35 bp nucleotide fragment may be a regulatory element. Relative rates of Hoxall coding sequences among vertebrates were analyzed by Tajima test. The results showed that there was no significant different between horn shark (Heterodontus francisci) and Mississippi paddlefish (Polyodon spathula) lineages. However, the evolutionary rate in higher teleost lineages was significantly higher than that in horn shark and Mississippi paddlefish lineages. Among higher teleost sampled, the zebrafish (Danio rerio) lineage had the highest rate. The difference of relative rates among teleost was a gene-, and not lineage-, specific phenomenon. The highrate in higher teleost might be relative to an additional Hox cluster duplication during teolest evolution. In addition to the coding sequence, relative rates of intron sequences also had significant difference among vertebrate lineages. Exons and intron of Hoxall gene belonged to a co-evolutionary unit in teleost and mammalian lineages, and evolved independently in frog (Xenopus tropicalis) and lizard (Calotes jerdoni) lineages. The results of both Z test and Fisher exact test showed that Darwin's positive selection acted on Hoxall coding sequence, and the positive selection caused a large change to Hoxall N-terminal amino acid sequence.Hoxall protein was divided into three regions, N-terminal conservative region, intermediate changeable region and C-terminal conservative region according to Clustal W alignment. The similarity of Hoxall amino acid sequences from different vertebrates was very high in both N- and C-terminal conservative regions. The number of amino acids was nearly equal from horn shark to human in the two conservative regions. The similarity was low, and the number of amino acids was changeable in the intermediate changeable region. Therefore, the evolutionary model of different regions was various in Hoxall. Polyalanine and closely neighboring polyhistone were first found in the Hoxall changeable region in Percomorpha fishes.Hoxall proteins from all sampled vertebrates had an obvious hydrophilic region, and its position remained relatively stable. It contained the majority of the changeable region and the little minority of the C-terminal conservative region. In the hydrophilic region, the number of amino acids was gradually increased from fish to mammals, which reflected a lengthened tendency of the hydrophilic region in tetrapod lineage. In a similar way, alanine content was also gradually increased in the hydrophilic region from fish to mammals. Horn shark had the lowest alanine content, about 3.03%;the next was coelacanth (Latimeria chalumnae), about 4.55%;other teleosts ranged from 5.88% to 8.82%;frog {Xenopus tropicalis), lizard (Calotes jerdoni) and chick (Gallus gallus) were 10.81%, 15.38% and 17.50% respectively;mammals were more than 20%.