Amphibian Genome - Xenopus

Main features:

1 Protein coding genes > 20,000 protein-coding genes, including orthologs of at least 1700 human disease genes. 

2 More than one-third of the genome consists

of transposable elements, with unusually prevalent DNA transposons. 

3 Like that of other tetrapods, the

genome of X. tropicalis contains gene deserts enriched for conserved noncoding elements. 

4 The genome

exhibits substantial shared synteny with human and chicken over major parts of large chromosomes,

broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.

5 X. laevis has a large paleotetraploid genome with an estimated size of 3.1 billion

bases (Gbp) on 18 chromosomes  and a generation time of 1 to 2 years. In contrast, the much smaller diploid western clawed frog, X. tropicalis, has a small genome, about 1.7 Gbp on 10 chromosomes (3), matures in only 4 months, and

requires less space than its larger cousin.

6) As a group, amphibians are phylogenetically well positioned for comparisons to other vertebrates, having diverged from the amniote lineage (mammals, birds, reptiles) some 360 million years ago. 

7) More than one-third of the frog genome consists of transposable elements (TEs), higher than the 9% TE density in the chicken genome but comparable to the 40 to 50% density in mammalian genomes. 

TRANSPOSONS

8a) Recently active TEs (1 to 5 million years ago) are more common in frogs than in mammals or birds, and their prevalence is comparable to that in fish, insects, nematodes, and plants. 

b) Among these is an unusually high diversity of very young families of L1 non-LTR (long terminal repeat) retrotransposons, Penelope, and DIRS retrotransposons.

In contrast to those of other vertebrates, most recognizable frog TEs (72%) are DNA transposons, rather than the retrotransposons that dominate other genomes. 

c) Among these families (11, 12), we identified Kolobok as a previously uncharacterized superfamily of DNA transposons. The genome also contains LTR retrotransposons of all major superfamilies, with higher diversity than in all other studied eukaryotes

d) Although most are ubiquitous, Copia, BEL, and Gypsy elements are not found in

birds and mammals, suggesting that this subset became immobile after divergence from the amphibian lineage.

GENES AND FAMILIES

9) X. tropicalis genome contains 20,000 to 21,000 protein-coding genes. These

include orthologs of 79% of identified human disease genes.

10) The genome contains 1850 tandem expanded gene families with between 2

and 160 copies, accounting for nearly 24% of protein-coding loci. 

11) The largest expansion comprises tetrapod-specific olfactory receptors (class

II) occupying the first 1.7 Mb on scaffold_24.

12) Other large expansions include protocadherins, bitter-taste receptors, and vomeronasal (pheromone) receptors (table S9).

COLINEARITY

13) The X. tropicalis genome displays long stretches of gene colinearity with human and chicken (Fig. 2). Of the 272 largest scaffolds (totaling half the assembly), 267 show such colinearity

(4). Sixty percent of all gene models on these scaffolds can be directly associated with a human and/or chicken ortholog by conserved synteny. Patches of strict conserved colinearity are interrupted by large-scale inversions within the same linkage groups, and more rarely by chromosome breakage and fusion events, similar to the findings reported for the human and chicken genome (Fig. 2) (5) and in agreement with persistent conservation of linkage groups across chordates (13).

CHROMOSOMAL REARRANGEMENTS

14) To identify lineages pecific fusion- and breakage-events In The tetrapod ancestry of human and chicken chromosome 1  a core ofmore than 150 Mb of sequence spanning the centromere of human chromosome 1 [chicken chromosome 8, frog linkage group (LG) VII] has remained largely intact during ~360million years of evolution since the tetrapod ancestor (Fig. 2A). 

SYNTENY

15) Detailed shared synteny is interrupted by large-scale inversions, but gene order is frequently conserved over stretches of tens of megabases. Human chromosome 1 is seen to have grown by three lineage-specific mammalian fusions. In contrast, there are several mammalian-specific breakpoints (Fig. 2B). The genomic material on the entire q arm of chicken shows linkage conservation to frog LG VI, whereas the human counterparts are scattered over regions of chromosomes 2, 3, 11, 13, 21, and X. The p arm indicates two mammalian breaks, suggesting that regions of chromosomes 7, 12, and 22 were once part of the same chromosome. By extending this analysis to all human and chicken chromosomes, we identified 22 human fusion and 21 fission events, versus only four fusions and one break in chicken. Clearly, the mammalian lineage has undergone considerably more rearrangement than that of the sauropsids, although the total chromosome count appears to have remained fairly constant. The segments analyzed here are distributed on 23 human and 22 chicken chromosomes, consistent with a derivation from24 or 25 ancestral amniote chromosomes. The chicken microchromosomes are unresolved by this analysis, however, preventing determination of the exact ancestral chromosome number.

Both the vertebrate and eumetazoan ancestors have been suggested to have had about a dozen large chromosomes 

16) The current analysis indicates that the amniote ancestor had twice as many, suggesting substantial chromosome breakage on the amniotic stem.

CNS

17) Frog genes adjacent to conserved noncoding sequences (CNS) are enriched or depleted in several gene ontology categories, including sensory perception of smell, response to stimulus, and regulation of transcription, among others.

GENE DESERTS

18) Gene deserts (defined as the top 3% of the longest intergenic regions) cover 17% of the genome and vary between 201 kbp and 1.2 Mbp. The 683 gene deserts contain almost 25%of CNSs. In mammalian genomes, these gene deserts have been found to harbor cis-regulatory elements .

19) several mammalian-Xenopus CNSs at the Six3 locus were assayed for enhancers regulating its eye- and forebrain-specific expression. The analysis suggests that frog-mammal comparisonsmay be more suitable than fish-mammal comparisons for identifying conserved cis-regulatory elements.

DEVLOPMENTAL GENES

20) Developmental pathways controlling early vertebrate axis specification were first implicated by work in Xenopus (2), but some interesting amphibian modifications can be found. For example, a Wnt ligand required for dorsal development, named Wnt11b in X. tropicalis, has been lost from mammals, but is found in the chick and zebrafish (as silberblick) (18). Despite its retention in these vertebrates, there is no evidence to support a maternal role in axis formation similar to that in Xenopus. Similarly, a tbx16 homolog, vegT, is retained in frog, fish, and chick, but is uniquely used in Xenopus for the establishment of the endoderm and mesoderm.

Q. What differences do you find in devlopmental pathways of frog as compared to other vertevrates?

1) Wnt11b is functional only in frog. It is present in othr except mammals but is not functional.

2) vegT helps in formation of endoderm and mesoderm.

21) X. tropicalis also shows multiplications of genes deployed at the blastula and gastrula stages. For example, mammals have a single nodal gene, whereas X. tropicalis has more than six.  Synteny relationships reveal that nodal4 on scaffold 204 is orthologous to the single human nodal, whereas a cluster of more than six nodals on scaffold 34 is orthologous to the chicken nodal. Further analysis suggests that these two nodal loci arose in one of the whole-genome duplications at the base of vertebrate evolution and that the birds and mammals subsequently lost different nodal genes, whereas the lizard Anolis carolinensis has retained both copies.

22) The theme of duplication is reiterated by several transcription factors that act during gastrulation. The transcriptional activator siamois, expressed in the organizer, is triplicated locally in the genome; so far this gene is unique to the frog. The ventx genes are expressed at the same time, but opposite the organizer, and are present in six linked copies.

IMMUNITY GENES

23) Conservation of the vertebrate immune system is highlighted by mammalian and Xenopus genome comparisons. Although orthology is usually obvious, synteny has been an important tool to identify diverged genes. For example, a diverged CD8 beta retains proximity to CD8 alpha, and CD4 neighbors Lag3 and B protein.

Similarly, an interleukin-2/interleukin-21–like sequencewas identified in a syntenic region between the tenr and centrin4 genes. The immunoglobulin repertoire provides further links between vertebrate immune systems. The IgW immunoglobulin was thought to be unique to shark/lungfish, but an orthologous IgD isotype in frog provides a connection between the fish and amniote gene families.

24) Unique antimicrobial peptides play an important role in skin secretions that are absent in birds, reptiles, and mammals. Antimicrobial peptides (caerulein, levitide, magainin, PGLa/PYLa, PGQ, xenopsin), neuromuscular toxins (e.g., xenoxins), and neuropeptides (e.g., thyrotropin-releasing hormone) are secreted by granular glands,

and the first group represents an important defense against pathogens. Antimicrobial peptides are clustered in at least seven transcription units >350 kbp on scaffold 811, with no intervening genes.