The Lithbea Domain.

The tree of life is the evolutionary metaphor for the past and present connections of all cellular organisms. Today, to speak of biodiversity is not only to speak of archaea, bacteria, and eukaryotes, but they should also consider the "new biodiversity" that includes viruses and synthetic organisms, which represent the new forms of life created in laboratories. There is even a third group of artificial entities that, although not living systems, pretend to imitate the living. To embrace and organize all this new biodiversity, I propose the creation of a new domain, with the name Lithbea (from life-on-the-border entites) The criteria for inclusion as members of Lithbea are: i) the acellular nature of the living system, ii) its origin in laboratory manipulation, iii) showing new biological traits, iv) the presence of exogenous genetic elements, v) artificial or inorganic nature. Within Lithbea there are two subdomains: Virworld (from virus world) which includes all viruses, regarded as lifeless living systems, and classified according to the International Committee on Taxonomy of Viruses (ICTV), and ii) Humade (from human-made) which includes all synthetic organisms and artificial entities. The relationships of Lithbea members to the three classical woesian domains and their implications are briefly discussed.

Reflections upon a new definition of life.

What is life? Multiple definitions have been proposed to answer this question, but unfortunately, none of them has reached the consensus of the scientific community. Here, the strategy used to define what life is was based on first establishing which characteristics are common to all living systems (organic nature, entropy-producing system, self-organizing, reworkable pre-program, capacity to interact and adapt, reproduction and evolution) and from them constructing the definition taking into account that reproduction and evolution are not essential for life. On this basis, life is defined as an interactive process occurring in entropy-producing, adaptive, and informative (organic) systems. An unforeseen consequence of the inseparable duality between the system (living being) and the process (life) is the interchangeability of the elements of the definition to obtain other equally valid alternatives. In addition, in the light of this definition, cases of temporarily lifeless living systems (viruses, dormant seeds, and ultracold cells) are analyzed, as well as the status of artificial life entities and the hypothetical nature of extraterrestrial life. All living systems are perishable because the passage of time leads to increasing entropy. Life must create order by continuously producing disorder and exporting it to the environment and so we move and stay in the phase transition between order and chaos, far from equilibrium, thanks to the input of energy from the outside. However, the passage of time eventually leads us to an end in which life disappears and entropy increases.

What is life?

BACKGROUND: Many traditional biological concepts continue to be debated by biologists, scientists and philosophers of science. The specific objective of this brief reflection is to offer an alternative vision to the definition of life taking as a starting point the traits common to all living beings. RESULTS AND CONCLUSIONS: Thus, I define life as a process that takes place in highly organized organic structures and is characterized by being preprogrammed, interactive, adaptative and evolutionary. If life is the process, living beings are the system in which this process takes place. I also wonder whether viruses can be considered living things or not. Taking as a starting point my definition of life and, of course, on what others have thought about it, I am in favor of considering viruses as living beings. I base this conclusion on the fact that viruses satisfy all the vital characteristics common to all living things and on the role they have played in the evolution of species. Finally, I argue that if there were life elsewhere in the universe, it would be very similar to what we know on this planet because the laws of physics and the composition of matter are universal and because of the principle of the inexorability of life.

What are the principles that govern life?

We know that living matter must behave in accordance with the universal laws of physics and chemistry. However, these laws are insufficient to explain the specific characteristics of the vital phenomenon and, therefore, we need new principles, intrinsic to biology, which are the basis for developing a theoretical framework for understanding life. Here I propose what I call the seven commandments of life (the Vital Order, the Principle of Inexorability, the reformulated Central Dogma, the Tyranny of Time, the Evolutionary Imperative, the Conservative Rule, the Cooperating Thrust) as a set of principles that help us explain the vital phenomenon from an evolutionary perspective. In a metaphorical way, we can consider life like an endless race in which living beings are the runners, who are changing as the race goes on (the evolutionary process), and the commandments the rules.

Preliminary methylation analysis of prothymosin α genomic sequences.

Prothymosin α is a mammalian nuclear protein involved in cell proliferation and differentiation. Here, we carried out the first study of the methylation status of ProTα genomic sequences in cell lines during differentiation as well as in tumoral tissues. We found that there is hypermethylation in all cell lines analyzed with a pattern that is characteristic of each cell type revealing specific genomic reorganizations. The decrease of ProTα mRNA during differentiation was not accompanied by changes in the methylation status. Remarkably, we found that there is hypomethylation in gastrointestinal tumors when compared with the peritumoral tissue. The biological implications of these findings are discussed.

Effect of monovalent cations and G-quadruplex structures on the outcome of intramolecular homologous recombination.

Homologous recombination is a very important cellular process, as it provides a major pathway for the repair of DNA double-strand breaks. This complex process is affected by many factors within cells. Here, we have studied the effect of monovalent cations (K+, Na+, and NH4+) on the outcome of recombination events, as their presence affects the biochemical activities of the proteins involved in recombination as well as the structure of DNA. For this purpose, we used an in vitro recombination system that includes a protein nuclear extract, as a source of recombination machinery, and two plasmids as substrates for intramolecular homologous recombination, each with two copies of different alleles of the human minisatellite MsH43. We found that the presence of monovalent cations induced a decrease in the recombination frequency, accompanied by an increase in the fidelity of the recombination. Moreover, there is an emerging consensus that secondary structures of DNA have the potential to induce genomic instability. Therefore, we analyzed the effect of the sequences capable of forming G-quadruplex on the production of recombinant molecules, taking advantage of the capacity of some MsH43 alleles to generate these kinds of structure in the presence of K+. We observed that the MsH43 recombinants containing duplications, generated in the presence of K+, did not include the repeats located towards the 5'-side of the G-quadruplex motif, suggesting that this structure may be involved in the recombination events leading to duplications. Our results provide new insights into the molecular mechanisms underlying the recombination of repetitive sequences.

Evolution of a complex minisatellite DNA sequence.

Minisatellites are tandem repeats of short DNA units widely distributed in genomes. However, the information on their dynamics in a phylogenetic context is very limited. Here we have studied the organization of the MsH43 locus in several species of primates and from these data we have reconstructed the evolutionary history of this complex minisatellite. Overall, with the exception of gibbon, MsH43 has an organization that is asymmetric, since the distribution of repeats is distinct between the 5' and 3' halves, and heterogeneous since there are many different repeats, some of them characteristic of each species. Inspection of the MsH43 arrays showed the existence of many duplications and deletions, suggesting the implication of slippage processes in the generation of polymorphism. Concerning the evolutionary history of this minisatellite, we propose that the birth of MsH43 may be situated before the divergence of Old World Monkeys since we found the existence of some MsH43 repeat motifs in prosimians and New World Monkeys. The analysis of MsH43 in apes revealed the existence of an evolutionary breakpoint in the pathway that originated African great apes and humans. Remarkably, human MsH43 is more homologous to orang-utan than to the corresponding sequence in gorilla and chimpanzee. This finding does not comply with the evolutionary paradigm that continuous alterations occur during the course of genome evolution. To adjust our results to the standard phylogeny of primates, we propose the existence of a wandering allele that was maintained almost unaltered during the period that extends between orang-utan and humans.

Function of prothymosin alpha in chromatin decondensation and expression of thymosin beta-4 linked to angiogenesis and synaptic plasticity.

Prothymosin alpha (ProTalpha) is an abundant highly acidic protein found in the nuclei of virtually all mammalian cells. The expression of this protein is increased in proliferating mammalian cells. However, the function of this molecule is still controversial. Here I present a model explaining the role of this protein in chromatin decondensation through its interaction with histone H1. beta-thymosins are a family of small actin-binding peptides widely distributed in eukaryotic cells. Here I will focus on thymosin beta-4, the most abundant member of this family. In particular, I will discuss its expression in the mammalian development of cardiovascular and nervous systems as well as its implications in neuronal plasticity.

DNA end-joining driven by microhomologies catalyzed by nuclear extracts.

In a previous work we used an in vitro system for the generation and analysis of double-strand breaks (DSBs) using nuclear extracts from rat testes as a source of DSB activity. Since the recombination process can be triggered by the formation of DSB, in the present study we developed a strategy to isolate and characterize recombinant molecules using the same in vitro system. Our results indicate that the mechanism for the formation of recombinants was non-homologous end-joining driven by microhomologies. The procedure described here represents an alternative to investigate the mechanisms of DNA end-joining and other forms of DNA repair.

Heteroduplex analysis of minisatellite variability.

Minisatellites are tandem repeat arrays of middle size (5-100 bp) repeat units widely distributed in eukaryotic genomes. They have been related to several important features of human genome biology, including gene regulation, chromosomal fragile sites, and imprinting. In this report, we have critically assessed and employed heteroduplex analysis (HA) for the identification of different human minisatellite MsH43 alleles. This minisatellite is organized as a repeat array of 5-6 bp units spanning 0.5 kbp. Our results demonstrate that this procedure is an easy, rapid, and reliable method to document allelic diversity for this locus. This work suggests that HA will also be a useful tool for studying the polymorphism of other minisatellites with small repeat units.

Generation of DNA double-strand breaks by two independent enzymatic activities in nuclear extracts.

We have reported the existence in rat nuclear extracts of a specific cleavage activity on a DNA fragment containing the human minisatellite MsH42 region (minisatellite plus its flanking sequences). Here, we have developed a system to analyse the nature of the cleavage products from the MsH42 region generated by the nuclear extracts. Our results demonstrated the formation of DNA double-strand breaks (DSB) in the MsH42 region by two different enzymatic activities, and that their distribution along this fragment changes depending on the presence of Mg2+. In the assays with Mg2+, the DSB were located in the minisatellite and its 3'-flanking region, showing preference for G-rich stretches. Oligonucleotide mutagenesis analysis confirmed that this enzymatic activity has a strong preference for G-tracts and that the recognition site is polarized towards the 3' end. Moreover, this activity cuts GC palindromes efficiently. In contrast, in the experiments without Mg2+, most DSB were mapped within the minisatellite flanking sequences. The analysis with oligonucleotides showed that G-tracts are recognized by this endonuclease activity, but with differences in the cleavage behaviour with respect to the reactions observed with Mg2+. The existence of two separate activities (Mg2+-dependent and Mg2+-independent) for the production of DSB was confirmed by analysing the effect of EGTA, N-ethyl maleimide, ionic strength, and by preincubations of the nuclear extracts at different temperatures. The tissue distribution of both DSB-producing activities was also different. The in vitro system used in the present work may be a useful tool for studying the formation of DSB and for investigation of the mechanisms of DNA repair.

Inhibition of DNA synthesis by K+-stabilised G-quadruplex promotes allelic preferential amplification.

PCR preferential amplification consists of the inefficient amplification of one allele in a heterozygous sample. Here, we report the isolation of a GC-rich human minisatellite, MsH43, that undergoes allelic preferential amplification during PCR. This effect requires the existence of a (TGGGGC)(4) motif that is able to form a G-quadruplex in the presence of K(+). This structure interferes with the DNA synthesis of the alleles harbouring this motif during PCR The present results are the first demonstration that the formation of G-quadruplex can be one of the mechanisms involved in some kinds of preferential amplification.

A paradox in the in vitro end-joining assays.

Much work has been focused on the pathways that restore the integrity of the genome after different kinds of lesions, especially double-strand breaks. A classical method to investigate double-strand break repair is the incubation of a DNA substrate with cell-free extracts. In these end-joining assays, the DNA is efficiently ligated by the proteins present in the extract, generating circular molecules and/or multimers. In contrast, using a similar in vitro system, we detected DNA cleavage rather than end ligation. When comparing our results with previous works, a paradox emerges: lower amounts of DNA become multimerized instead of degraded and higher amounts of DNA are degraded rather than multimerized. Here, we have demonstrated that when the DNA/protein ratio is low enough, the DNA-binding proteins of the nuclear extract protect the DNA substrate, avoiding DNA degradation and vice versa. Therefore, the variation of the DNA/protein ratio is enough to switch the outcome of the experiment from a DNA cleavage assay to a typical end-joining assay.

Birth and evolutionary history of a human minisatellite.

One of the most exciting challenges in human biology is the understanding of how our genome was constructed during evolution. Here we explore the evolutionary history of the low polymorphic human minisatellite MsH42 and its flanking sequences. We show that the evolutionary birth of MsH42 took place within an intron, early in primate lineage evolution, more than 40 MYA. Then, single base-pair changes and duplications/deletions of repeat blocks by mispairing were probably the main forces governing the generation of this minisatellite and its polymorphism throughout primate evolution. Moreover, we detected several phylogenetic footprints at both sides of MsH42. We believe that our findings will contribute to the understanding of low-variability minisatellite evolution.

Recombination analysis of the human minisatellite MsH42 suggests the existence of two distinct pathways for initiation and resolution of recombination at MsH42 in rat testes nuclear extracts.

We have previously described a GC-rich human minisatellite, termed MsH42, which exists in two allelic forms, long and short. Here, we have identified a third allele of medium length and localized the MsH42 locus in the chromosome 15q25.1 inside an intron belonging to a gene of unknown function. The recombinogenic potential of the three alleles was assayed in vitro incubating pBR322-based constructs containing two copies of the minisatellite MsH42 with its flanking sequences, in the presence of rat testes nuclear extracts. This assay system was configured to monitor only reciprocal exchange type events and not gene conversion. All MsH42 allelic sequences enhanced intramolecular homologous recombination promoting high rates (approximately 76%) of equal crossover, the long allele showing the highest recombinogenic activity. Removal of the MsH42 long allele flanking sequences, which are identical in the three alleles, provoked a decrease in the enhancement of recombination and in the frequency of equal crossovers, suggesting that these sequences are important for the recombinogenic activity and for the correct pairing between homologous sequences. The occurrence of some complex recombination events within the minisatellite MsH42 suggests the existence of processes related to polymerase slippage and unwinding with reinvasion during the repair synthesis. Our findings point toward the existence of two distinct biochemical pathways for initiation and resolution of recombination at the minisatellite MsH42. Finally, the in vitro recombination system employed in this study could provide an approach to dissect processes of repetitive DNA instability and recombination.

The beta-thymosins, small actin-binding peptides widely expressed in the developing and adult cerebellum.

The beta-thymosins are a highly conserved family of small polar peptides known to bind monomeric actin and inhibit its polymerization. The beta-thymosins show a high degree of sequence conservation among all vertebrate classes and they have been also identified in some invertebrate phyla. The most abundant beta-thymosins in mammals are thymosin beta4 (Tbeta4) and thymosin beta10 (Tbeta10), two ubiquitous small (43 amino acids) peptides sharing a high degree of sequence homology. Both beta-thymosins are present in virtually all mammalian tissues and cells studied, showing distinct patterns of expression in several tissues. The beta-thymosins are expressed in the developing and mature nervous system, indicating their participation with other actin-binding peptides in the control of actin polymerization. In the rat cerebellum the temporal and cellular patterns of expression of Tbeta4 and Tbeta10 are different, suggesting that each beta-thymosin could play a specific physiological function during cerebellum development. The possible roles of beta-thymosins in the developing mammalian cerebellum are discussed.