Fundamental and applied aspects of comparative genomics of vertebrates

The Human Genome Project stimulated the development of efficient strategies and relevant hardware for complete genome sequencing. The comparative genomic approach extends the possibilities of using the sequencing data to identify new genes or conserved regulatory regions by means of nucleotide sequence alignment of the particular regions of the mouse and human genomes, or to trace the evolutionary events resulting in the genome structure of modern mammals. The review focuses on the use of new molecular cytogenetic methods along with computer-aided analysis of the genomes in vertebrates. Several factors hindering data analysis are considered. The currently available information on gene evolution rate inferred from comparative genomic data is presented. The origin and evolution of the genomes of several species are discussed.     

Hemoglobin function in the vertebrates: An evolutionary model

Comparative data on quaternary structure, cooperativity, Bohr effect and regulation by organic phosphates are reviewed for vertebrate hemoglobins. A phylogeny of hemoglobin function in the vertebrates is deduced. It is proposed that from the monomeric hemoglobin of the common ancestor of vertebrates, a deoxy dimer, as seen in the lamprey, could have originated with a single amino acid substitution. The deoxy dimer has a Bohr effect, cooperativity and a reduced oxygen affinity compared to the monomer. One, or two, additional amino acid substitutions could have resulted in the origin of a tetrameric deoxy hemoglobin which dissociated to dimers on oxygenation. Gene duplication, giving incipient alpha and beta genes, probably preceded the origin of a tetrameric oxyhemoglobin. The origin of an organic phosphate binding site on the tetrameric hemoglobin of an early fish required only one, or two, amino acid substitutions. ATP was the first organic phosphate regulator of hemoglobin function. The binding of ATP by hemoglobin may have caused the original elevation in the concentration of ATP in the red blood cells by relieving end product inhibition of ATP synthesis. The switch from regulation of hemoglobin function by ATP to regulation by DPG may have been a consequence of the curtailment of oxidative phosphorylation in the red blood cell. The basic mechanisms by which ATP and DPG concentrations can respond to strss on the oxygen transport system were present before the origin of an organic phosphate binding site on hemoglobin. A switch from ATP regulation to IP5 regulation occurred in the common ancestor of birds.     

Metallothionein primary structure in amphibians: Insights from comparative evolutionary analysis in vertebrates

Metallothioneins are cysteine-rich, low-molecular weight metal-binding proteins ubiquitously expressed in living organisms. In the last past years, the increasing amount of vertebrate non-mammalian metallothionein sequences available have disclosed for these proteins differences in the primary structure that have not been supposed before. To provide a more up-to-date view of the metallothioneins in non-mammalian tetrapods, we decided to increase the still scarce knowledge concerning the primary structure and the evolution of metallothioneins in amphibians. Our data demonstrate an unexpected diversity of metallothionein sequences among amphibians, accompanied by remarkable features in their phylogeny. Phylogenetic analysis also reveals the complexity of vertebrate metallothionein evolution, made by both ancient and more recent events of gene duplication and loss.     

Isochores and the evolutionary genomics of vertebrates

The nuclear genomes of vertebrates are mosaics of isochores, very long stretches (>300kb) of DNA that are homogeneous in base composition and are compositionally correlated with the coding sequences that they embed. Isochores can be partitioned in a small number of families that cover a range of GC levels (GC is the molar ratio of guanine+cytosine in DNA), which is narrow in cold-blooded vertebrates, but broad in warm-blooded vertebrates. This difference is essentially due to the fact that the GC-richest 10-15% of the genomes of the ancestors of mammals and birds underwent two independent compositional transitions characterized by strong increases in GC levels. The similarity of isochore patterns across mammalian orders, on the one hand, and across avian orders, on the other, indicates that these higher GC levels were then maintained, at least since the appearance of ancestors of warm-blooded vertebrates. After a brief review of our current knowledge on the organization of the vertebrate genome, evidence will be presented here in favor of the idea that the generation and maintenance of the GC-richest isochores in the genomes of warm-blooded vertebrates were due to natural selection.     

Complicated evolutionary patterns of microRNAs in vertebrates

MicroRNAs (miRNAs) are a class of ∼22 nt long endogenous non-coding RNAs that play important regulatory roles in diverse organisms. Up to now, little is known about the evolutionary properties of these crucial regulators. Most miRNAs were thought to be phylogenetically conserved, but recently, a number of poorly-conserved miRNAs have been reported and miRNA innovation is shown to be an ongoing process. In this work, through the characterization of an miRNA super family, we studied the evolutionary patterns of miRNAs in vertebrates. Recently generated miRNAs seem to evolve rapidly during a certain period following their emergence. Multiple lineage-specific expansions were observed. Homolgous premiRNAs may produce mature products from the opposite stem arms following tandem duplications, which may have important contribution to miRNA innovation. Our observations of miRNAs’ complicated evolutionary patterns support the notion that these key regulatory molecules may play very active roles in evolution.     

Cyclostome embryology and early evolutionary history of vertebrates

Modern agnathans include only two groups, the lampreys and thehagfish, that collectively comprise the group Cyclostomata.Although accumulating molecular data support the cyclostomesas a monophyletic group, there remain some unsettled questionsregarding the evolutionary relationships of these animals inthat they differ greatly in anatomical and developmental patternsand in their life histories. In this review, we summarize recentdevelopmental data on the lamprey and discuss some questionsrelated to vertebrate evolutionary development raised by thelimited information available on hagfish embryos. Comparisonof the lamprey and gnathostome developmental patterns suggestssome plesiomorphic traits of vertebrates that would have alreadybeen established in the most recent common ancestor of the vertebrates.Understanding hagfish development will further clarify the,as yet, unrecognized ancestral characters that either the lampreysor hagfishes may have lost. We stress the immediate importanceof hagfish embryology in the determination of the most plausiblescenario for the early history of vertebrate evolution, by addressingquestions about the origins of the neural crest, thyroid, andadenohypophysis as examples.     

comparative study of opiate and enkephalin receptors on lower vertebrates and higher vertebrates

1. The presence of opiate (μ) and enkephalin (δ) receptors have been characterized in the most primitive amphibians (Urodeles).2. The ratio of the two types of receptors (δ:μ) is the same for Pleurodeles and Axolotl, but is lower than the ratio found in rat brain.3. Thyroxine-induced metamorphosis of the Axolotl does not affect the presence and the number of the major population of “opiate” receptors, the μ receptors.     

Hemoglobins and hemocyanins: comparative aspects of structure and function.

Comparative studies of protein structure and function can be quite interesting by themselves, and even more interesting when interpreted with respect to an animal's physiology. In the case of fish hemoglobins, some success in the latter has been achieved but there are still many unsolved problems. It appears that comparative physiology and biochemistry have entered an era where results from comparative studies can shed a great deal of light on biochemical mechanisms in general. The trout hemoglobin system is an example. Distinctive hemoglobins in this system are presently being used as high resolution probes of the ligand-binding mechanism. Characterization of the multiple, structurally distinct subunits of the 60S Limulus hemocyanin molecule may similarly aid in understanding its function. Our studies suggest the possibility of using Limulus hemocyanin and other hemocyanins as structural homologs and analogs of more complex macromolecular arrays. The rapid development of molecular structural data from X-ray crystallographers combined with the vast data of comparative physiology and biochemistry makes this one of the most exciting areas in present day science.     

Temperature and respiratory function in ectothermic vertebrates

Pulmonary ventilation is adjusted to maintain balance between O₂ demands and CO₂ elimination, which is essential for acid–base status in land ectothermic vertebrates. Rising temperatures cause increases in O₂ consumption (Q₁₀ effect) and decreases in the O₂ affinity of hemoglobin (a rightward shift in the oxygen–hemoglobin dissociation curve). These changes in air-breathing ectotherms are not proportional, i.e., the increased ventilation is relatively smaller than the change in metabolic rate. Therefore, the ratio between ventilation and metabolic rate is reduced, and consequently blood pH changes inversely with temperature. The combination of high temperatures and hypoxia exposure results in an amplified increase of ventilation, which may be explained by the balance between increased O₂-demand and decreased O₂-supply as well as increased O₂-chemoreceptors sensitivity. High temperature also increases pulmonary diffusing capacity. Global warming is expected to have significant impacts on the world’s climate, with temperature changes affecting living organisms, in relation to their physiology and distribution. These physiological mechanisms and their capacity to respond appropriately to temperature illustrate the complexity of the relationship between ambient temperature and the respiratory function in ectothermic vertebrates, which are particularly susceptible to change in their environment.     

Gut-islet endocrinology-some evolutionary aspects

Immunological and biological studies have shown that many of the mammalian gastroenteropancreatic (GEP) hormones have counterparts in lower vertebrates. Hormonal localization in cyclostomes and fishes suggests that insulin was phylogenetically the first islet hormone, followed by somatostatin, glucagon and, last, pancreatic polypeptide (PP). Some of the GEP peptides are present in the central and peripheral nervous system of lower vertebrates as well as mammals. GEP hormone-like substances resembling insulin, somatostatin, glucagon, PP, gastrin, secretin, VIP, substance P and enkephalin also occur in protostomian invertebrates (Annelida, Arthropoda, Mollusca), particularly in their nervous system. These findings indicate that the vertebrate hormones may have originated in neural tissue before the development of the vertebrate line of evolution.     

Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation

Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide‐encoding genes in vertebrates, providing an entry point to define their function in vivo.