Organic Codes: A Unifying Concept for Life

Although the knowledge about biological systems has advanced exponentially in recent decades, it is surprising to realize that the very definition of Life keeps presenting theoretical challenges. Even if several lines of reasoning seek to identify the essence of life phenomenon, most of these thoughts contain fundamental problem in their basic conceptual structure. Most concepts fail to identify either necessary or sufficient features to define life. Here, we analyzed the main conceptual frameworks regarding theoretical aspects that have been supporting the most accepted concepts of life, such as (i) the physical, (ii) the cellular and (iii) the molecular approaches. Based on an ontological analysis, we propose that Life should not be positioned under the ontological category of Matter. Yet, life should be better understood under the top-level ontology of “Process”. Exercising an epistemological approach, we propose that the essential characteristic that pervades each and every living being is the presence of organic codes. Therefore, we explore theories in biosemiotics and code biology in order to propose a clear concept of life as a macrocode composed by multiple inter-related coding layers. This way, as life is a sort of metaphysical process of encoding, the living beings became the molecular materialization of that process. From the proposed concept, we show that the evolutionary process is a fundamental characteristic for life’s maintenance but it is not necessary to define life, as many organisms are clearly alive but they do not participate in the evolutionary process (such as infertile hybrids). The current proposition opens a fertile field of debate in astrobiology, epistemology, biosemiotics, code biology and robotics.

     

Inference of homologous recombination in bacteria using whole-genome sequences

Bacteria and archaea reproduce clonally, but sporadically import DNA into their chromosomes from other organisms. In many of these events, the imported DNA replaces an homologous segment in the recipient genome. Here we present a new method to reconstruct the history of recombination events that affected a given sample of bacterial genomes. We introduce a mathematical model that represents both the donor and the recipient of each DNA import as an ancestor of the genomes in the sample. The model represents a simplification of the previously described coalescent with gene conversion. We implement a Monte Carlo Markov chain algorithm to perform inference under this model from sequence data alignments and show that inference is feasible for whole-genome alignments through parallelization. Using simulated data, we demonstrate accurate and reliable identification of individual recombination events and global recombination rate parameters. We applied our approach to an alignment of 13 whole genomes from the Bacillus cereus group. We find, as expected from laboratory experiments, that the recombination rate is higher between closely related organisms and also that the genome contains several broad regions of elevated levels of recombination. Application of the method to the genomic data sets that are becoming available should reveal the evolutionary history and private lives of populations of bacteria and archaea. The methods described in this article have been implemented in a computer software package, ClonalOrigin, which is freely available from http://code.google.com/p/clonalorigin/.     

Mechanisms of recent genome size variation in flowering plants

BACKGROUND AND AIMS: Plant nuclear genomes vary tremendously in DNA content, mostly due to differences in ancestral ploidy and variation in the degree of transposon amplification. These processes can increase genome size, but little is known about mechanisms of genome shrinkage and the degree to which these can attenuate or reverse genome expansion. This research focuses on characterizing DNA removal from the rice and Arabidopsis genomes, and discusses whether loss of DNA has effectively competed with amplification in these species. METHODS: Retrotransposons were analyzed for sequence variation within several element families in rice and Arabidopsis. Nucleotide sequence changes in the two termini of individual retrotransposons were used to date their time of insertion. KEY RESULTS: An accumulation of small deletions was found in both species, caused by unequal homologous recombination and illegitimate recombination. The relative contribution of unequal homologous recombination compared to illegitimate recombination was higher in rice than in Arabidopsis. However, retrotransposons are rapidly removed in both species, as evidenced by the similar apparent ages of intact elements (most less than 3 million years old) in these two plants and all other investigated plant species. CONCLUSIONS: Differences in the activity of mechanisms for retrotransposon regulation or deletion generation between species could explain current genome size variation without any requirement for natural selection to act on this trait, although the results do not preclude selection as a contributing factor. The simplest model suggests that significant genome size variation is generated by lineage-specific differences in the molecular mechanisms of DNA amplification and removal, creating major variation in nuclear DNA content that can then serve as the substrate for fitness-based selection.     

Genomic deletions of the Drosophila melanogaster Hsp70 genes

Homologous recombination can produce directed mutations in the genomes of a number of model organisms, including Drosophila melanogaster. One of the most useful applications has been to delete target genes to generate null alleles. In Drosophila, specific gene deletions have not yet been produced by this method. To test whether such deletions could be produced by homologous recombination in D. melanogaster we set out to delete the Hsp70 genes. Six nearly identical copies of this gene, encoding the major heat-shock protein in Drosophila, are found at two separate but closely linked loci. This arrangement has thwarted standard genetic approaches to generate an Hsp70-null fly, making this an ideal test of gene targeting. In this study, ends-out targeting was used to generate specific deletions of all Hsp70 genes, including one deletion that spanned approximately 47 kb. The Hsp70-null flies are viable and fertile. The results show that genomic deletions of varied sizes can be readily generated by homologous recombination in Drosophila.     

Stimulation of RecA-mediated D-loop formation by oligonucleotide-directed triple-helix formation: guided homologous recombination (GOREC)

Oligonucleotide-directed triple helix formation provides an elegant rational basis for gene-specific DNA targeting and has been widely used to interfere with gene expression ("antigene" strategies) and as a molecular tool for biological studies. Various strategies have been developed to introduce sequence modifications in genomes. However, the low efficiency of the overall process in eucaryotic cells impairs efficient recovery of recombinant genomes. Since one limiting step in homologous recombination is the targeting to the homologous sequence, we have tested the contribution of an oligonucleotide-directed triple helix formation on the RecA-dependent association of an oligonucleotide and its homologous target on duplex DNA (D-loop formation). For this study, the recombinant ssDNA fragment was noncovalently associated to a triple helix-forming oligonucleotide. The physicochemical and biochemical characteristics of the triple helix and D-loop structures formed by the complex molecules in the presence or in the absence of RecA protein were determined. We have demonstrated that the triple helix-forming oligonucleotide increases the efficiency of D-loop formation and the RecA protein speeds up also the triple helix formation. The so-called "GOREC" (for guided homologous recombination) approach can be developed as a novel tool to improve the efficiency of directed mutagenesis and gene alteration in living organisms.     

Impact of homologous and non-homologous recombination in the genomic evolution of Escherichia coli

ABSTRACT: BACKGROUND: Escherichia coli is an important species of bacteria that can live as a harmless inhabitantof the guts of many animals, as a pathogen causing life-threatening conditions or freely inthe non-host environment. This diversity of lifestyles has made it a particular focus ofinterest for studies of genetic variation, mainly with the aim to understand how acommensal can become a deadly pathogen. Many whole genomes of E. coli have beenfully sequenced in the past few years, which offer helpful data to help understand how thisimportant species evolved. RESULTS: We compared 27 whole genomes encompassing four phylogroups of Escherichia coli (A,B1, B2 and E). From the core-genome we established the clonal relationships between theisolates as well as the role played by homologous recombination during their evolutionfrom a common ancestor. We found strong evidence for sexual isolation between three lineages (A+B1, B2, E), which could be explained by the ecological structuring of E. coliand may represent on-going speciation. We identified three hotspots of homologousrecombination, one of which had not been previously described and contains the aroCgene, involved in the essential shikimate metabolic pathway. We also described the roleplayed by non-homologous recombination in the pan-genome, and showed that thisprocess was highly heterogeneous. Our analyses revealed in particular that the genomes ofthree enterohaemorrhagic (EHEC) strains within phylogroup B1 have converged fromoriginally separate backgrounds as a result of both homologous and non-homologousrecombination. CONCLUSIONS: Recombination is an important force shaping the genomic evolution and diversification ofE. coli, both by replacing fragments of genes with an homologous sequence and also byintroducing new genes. In this study, several non-random patterns of these events wereidentified which correlated with important changes in the lifestyle of the bacteria, andtherefore provide additional evidence to explain the relationship between genomicvariation and ecological adaptation.     

Inverted terminal repeat sequences are important for intermolecular recombination and circularization of adeno-associated virus genomes

The relatively small package capacity (less than 5 kb) of adeno-associated virus (AAV) vectors has been effectively doubled with the development of dual-vector heterodimerization approaches. However, the efficiency of such dual-vector systems is limited not only by the extent to which intermolecular recombination occurs between two independent vector genomes, but also by the directional bias required for successful transgene reconstitution following concatemerization. In the present study, we sought to evaluate the mechanisms by which inverted terminal repeat (ITR) sequences mediate intermolecular recombination of AAV genomes, with the goal of engineering more efficient vectors for dual-vector trans-splicing approaches. To this end, we generated a novel AAV hybrid-ITR vector characterized by an AAV-2 and an AAV-5 ITR at opposite ends of the viral genome. This hybrid genome was efficiently packaged into either AAV-2 or AAV-5 capsids to generate infectious virions. Hybrid AV2:5 ITR viruses had a significantly lower capacity to form circular intermediates in infected cells than homologous AV2:2 and AV5:5 ITR vectors despite their similar capacity to express an encoded enhanced green fluorescent protein (EGFP) transgene. To examine whether the divergent ITR sequences contained within hybrid AV2:5 ITR vectors could direct intermolecular recombination in a tail-to-head fashion, we generated two hybrid ITR trans-splicing vectors (AV5:2LacZdonor and AV2:5LacZacceptor). Each delivered one exon of a beta-galactosidase minigene flanked by donor or acceptor splice sequences. These hybrid trans-splicing vectors were compared to homologous AV5:5 and AV2:2 trans-splicing vector sets for their ability to reconstitute beta-galactosidase gene expression. Results from this comparison demonstrated that hybrid ITR dual-vector sets had a significantly enhanced trans-splicing efficiency (6- to 10-fold, depending on the capsid serotype) compared to homologous ITR vectors. Molecular studies of viral genome structures suggest that hybrid ITR vectors provide more efficient directional recombination due to an increased abundance of linear-form genomes. These studies provide direct evidence for the importance of ITR sequences in directing intermolecular and intramolecular homologous recombination of AAV genomes. The use of hybrid ITR AAV vector genomes provides new strategies to manipulate viral genome conversion products and to direct intermolecular recombination events required for efficient dual-AAV vector reconstitution of the transgene.     

Trypanosoma brucei homologous recombination is dependent on substrate length and homology, though displays a differential dependence on mismatch repair as substrate length decreases

Homologous recombination functions universally in the maintenance of genome stability through the repair of DNA breaks and in ensuring the completion of replication. In some organisms, homologous recombination can perform more specific functions. One example of this is in antigenic variation, a widely conserved mechanism for the evasion of host immunity. Trypanosoma brucei, the causative agent of sleeping sickness in Africa, undergoes antigenic variation by periodic changes in its variant surface glycoprotein (VSG) coat. VSG switches involve the activation of VSG genes, from an enormous silent archive, by recombination into specialized expression sites. These reactions involve homologous recombination, though they are characterized by an unusually high rate of switching and by atypical substrate requirements. Here, we have examined the substrate parameters of T. brucei homologous recombination. We show, first, that the reaction is strictly dependent on substrate length and that it is impeded by base mismatches, features shared by homologous recombination in all organisms characterized. Second, we identify a pathway of homologous recombination that acts preferentially on short substrates and is impeded to a lesser extent by base mismatches and the mismatch repair machinery. Finally, we show that mismatches during T. brucei recombination may be repaired by short-patch mismatch repair.     

人精母细胞重组频率和年龄相关性的分析

减数分裂遗传重组对同源染色体的正确分离和单倍体的正确形成起至关重要的作用,但人们对人精母细胞减数分裂遗传重组机制了解的还很少。通过免疫荧光染色技术标记减数分裂I联会复合体上的MLH1(DNA错配修复蛋白)位点可以检测人精母细胞的重组。文章对10例可育男性进行分析,发现每个细胞中重组位点数平均为49.4士4.4,范围为33~63,具有显著的个体差异,只有0.4%(1/220)的常染色体SC上缺少MLH1位点。进一步通过Spearman相关性分析,分析了年龄因素与个体间重组位点差异的相关性,结果提示年龄因素对常染色体及性染色体的重组均无影响。     

PCR-mediated recombination in amplification products derived from polyploid cotton

PCR recombination describes a process of in vitro chimera formation from non-identical templates. The key requirement of this process is the inclusion of two partially homologous templates in one reaction, a condition met when amplifying any locus from polyploid organisms and members of multigene families from diploid organisms. Because polyploids possess two or more divergent genomes (”homoeologues”) in a common nucleus, intergenic chimeras can form during the PCR amplification of any gene. Here we report a high frequency of PCR-induced recombination for four low-copy genes from allotetraploid cotton (Gossypium hirsutum). Amplification products from these genes (Myb3, Myb5, G1262 and CesA1) range in length from 860 to 4,050 bp. Intergenomic recombinants were formed frequently, accounting for 23 of the 74 (31.1%) amplicons evaluated, with the frequency of recombination in individual reactions ranging from 0% to approximately 89%. Inspection of the putative recombination zones failed to reveal sequence-specific attributes that promote recombination. The high levels of observed in vitro recombination indicate that the tacit assumption of exclusive amplification of target templates may often be violated, particularly from polyploid genomes. This conclusion has profound implications for population and evolutionary genetic studies, where unrecognized artifactually recombinant molecules may bias results or alter interpretations. Received: 28 February 2001 / Accepted: 8 June 2001     

Poly(ADP-ribose) Polymerase (PARP-1) in Homologous Recombination and as a Target for Cancer Therapy

Poly(ADP-ribose) polymerase (PARP-1) binds to DNA breaks to facilitate DNA repair. However, the role of PARP-1 in DNA repair appears to not be critical since PARP-1 knockout mice are viable, fertile and do not develop early onset tumours. Cells isolated from these mice show an increased level of homologous recombination. There is an intricate link between homologous recombination and PARP-1 and a possible role for PARP-1 in DNA double-strand break repair. Although PARP-1 appears not to be required for homologous recombination itself, it regulates the process through its involvement in the repair of DNA single-strand breaks (SSBs). SSBs persisting into the S phase of the cell cycle collapse replication forks, triggering homologous recombination for replication restart. We discuss the recent discoveries on the use of PARP-1 inhibitors as a targeted cancer therapy for recombination deficient cancers, such as BRCA2 tumours.     

PERSPECTIVE: THE EVOLUTIONARY BIOLOGY OF AGING,SEXUAL REPRODUCTION,AND DNA REPAIR

Three recent books on the evolutionary biology of aging and sexual reproduction are reviewed, with particular attention focused on the provocative suggestion by Bernstein and Bernstein (1991) that senescence and genetic recombination are related epiphenomena stemming from the universal challenge to life posed by DNA damages and the need for damage repair. Embellishments to these theories on aging and sex are presented that consider two relevant topics neglected or underemphasized in the previous treatments. The first concerns discussion of cytoplasmic genomes (such as mtDNA), which are transmitted asexually and therefore do not abide by the recombinational rules of nuclear genomes; the second considers the varying degrees of cellular and molecular autonomy which distinguish unicellular from multicellular organisms, germ cells from somatic cells, and sexual from asexual genomes. Building on the Bernsteins' suggestions, two routes to immortality for cell lineages appear to be available to life: an asexual strategy (exemplified by some bacteria), whereby cell proliferation outpaces the accumulation of DNA damages, thereby circumventing Muller's ratchet; and a sexual strategy (exemplified by germlines in multicellular organisms), whereby recombinational repair of DNA damages in conjunction with cell proliferation and gametic selection counter the accumulation of nuclear DNA damages. If true, then elements of both the recombinational strategy (nuclear DNA) and replacement strategy (cytoplasmic DNA) may operate simultaneously in the germ-cell lineages of higher organisms, producing at least some gametes that are purged of the DNA damages accumulated during the lifetime of the somatic parent. For multicellular organisms, production of functionally autonomous and genetically screened gametic cells is a necessary and sufficient condition for the continuance of life.     

Efficient mutagenesis of the rhodopsin gene in rod photoreceptor neurons in mice

Dominant mutations in the rhodopsin gene, which is expressed in rod photoreceptor cells, are a major cause of the hereditary-blinding disease, autosomal dominant retinitis pigmentosa. Therapeutic strategies designed to edit such mutations will likely depend on the introduction of double-strand breaks and their subsequent repair by homologous recombination or non-homologous end joining. At present, the break repair capabilities of mature neurons, in general, and rod cells, in particular, are undefined. To detect break repair, we generated mice that carry a modified human rhodopsin-GFP fusion gene at the normal mouse rhodopsin locus. The rhodopsin-GFP gene carries tandem copies of exon 2, with an ISceI recognition site situated between them. An ISceI-induced break can be repaired either by non-homologous end joining or by recombination between the duplicated segments, generating a functional rhodopsin-GFP gene. We introduced breaks using recombinant adeno-associated virus to transduce the gene encoding ISceI nuclease. We found that virtually 100% of transduced rod cells were mutated at the ISceI site, with ～85% of the genomes altered by end joining and ～15% by the single-strand annealing pathway of homologous recombination. These studies establish that the genomes of terminally differentiated rod cells can be efficiently edited in living organisms.     

Recombination between poly[d(GT).d(CA)] sequences in simian virus 40-infected cultured cells.

CVI cells were transfected with oversized simian virus 40 (SV40) genomes that could be reduced to packageable size by alternative homologous recombination pathways involving either two polydeoxyguanylic-thymidylic acid X polydeoxycytidylic-adenylic acid (poly[d(GT).d(CA)]; abbreviated hereafter as poly(GT)] tracts or two tracts of homologous SV40 sequence. Plaque-forming viruses rescued by this procedure were found to contain genomes formed by homologous and nonhomologous recombination events. Half of the viable viral DNA molecules recovered were the result of recombination between two tracts of poly(GT). Approximately 20% of the rescued viral genomes were produced by homologous recombination between tracts of SV40 DNA. Nonhomologous recombination involving SV40 sequences was also a major pathway of deletion, producing ca. 30% of the viral plaques. Tracts of poly(GT) generated by recombination were variable in length, suggesting that recombination between poly(GT) tracts was usually unequal. On a per-nucleotide basis, poly(GT) recombination occurred eight times more frequently than did recombination between homologous SV40 DNA. This eightfold difference is the maximum recombinatory enhancement attributable to poly(GT) sequences. Although DNA sequence analysis showed that tracts of poly(GT) generated by recombination retained the alternating G-T repeat motif throughout their length, the contribution of the nonhomologous pathway to poly(GT) recombination cannot be ruled out, and the relative proclivity of a given length of d(GT).d(CA) sequence to undergo homologous recombination is probably less than eight times greater than that of an SV40 sequence of the same length.     

青花菜Ogu不育胞质分子鉴定和序列分析

雄性不育是农作物利用杂种优势、进行轮回选择和群体改良的重要手段,在农作物生产中具有巨大的利用价值。该研究为了鉴定青花菜细胞质雄性不育材料的不育胞质类型,以期今后为青花菜种质资源的收集、利用及分子标记辅助育种提供新的不育标记。根据Gen Bank中orf138基因保守序列设计特异引物,对20个青花菜种质资源基因组DNA进行PCR扩增。结果表明:特异引物P1/P2在12个青花菜雄性不育基因型中均扩增出392 bp的片段,在8个可育基因型中未扩增出条带,与田间育性鉴定结果相符。获得青花菜Ogu胞质雄性不育的特异基因orf138序列,Gen Bank中的登录号为HQ149728;用Blastn在Gen Bank中进行同源性比对分析,发现12个不育材料的特异片段与已报道的萝卜Ogu CMS所具有的Ogu orf138基因(Genbank登录号:Z18896.1)同源度高达100%。序列同源比对发现orf138基因存在变异位点。研究结果可为青花菜雄性不育细胞质的分子鉴定、进一步阐明胞质雄性不育败育机理,以及指导青花菜新型不育系的创建和杂种优势高效利用提供理论依据。     

Properties of the Circular Plasmid-like DNA Bl from Mitochondria of Cytoplasmic Male-sterile Rice

Mitochondrial DNA from suspension-cultured cells of the cytoplasmicmale-sterile rice line, A-58 CMS, was shown to contain fourminicircular DNAs. We chose for further examination the largestminicircular DNA, designated Bl. A molecular clone containingthe complete sequence of Bl was constructed and used to probemitochondrial and nuclear genomes by Southern hybridization.No evidence was found for the existence of integrated copiesof Bl in the main mitochondrial genomes of either male-sterileor fertile rice. Sequences homologous to Bl, however, were foundin nuclear genomes of both the male-sterile and the fertilerice. The complete nudeotide sequence of Bl (2,135 bp) was determined,and found to contain sequences homologous to those in the 1,913bp plasmid-like DNA of maize. (Received May 15, 1987; Accepted July 20, 1987)     

Evidence of intra-segmental homologous recombination in influenza A virus

The evolution of influenza viruses is remarkably dynamic. Influenza viruses evolve rapidly in sequence and undergo frequent reassortment of different gene segments. Homologous recombination, although commonly seen as an important component of dynamic genome evolution in many other organisms, is believed to be rare in influenza. In this study, 256 gene segments from 32 influenza A genomes were examined for homologous recombination, three recombinant H1N1 strains were detected and they most likely resulted from one recombination event between two closely rated parental sequences. These findings suggest that homologous recombination in influenza viruses tends to take place between strains sharing high sequence similarity. The three recombinant strains were isolated at different time periods and they form a clade, indicating that recombinant strains could circulate. In addition, the simulation results showed that many recombinant sequences might not be detectable by currently existing recombinant detection programs when the parental sequences are of high sequence similarity. Finally, possible ways were discussed to improve the accuracy of the detection for recombinant sequences in influenza.     

Integration of a vector containing a repetitive LINE-1 element in the human genome.

Mammalian cells contain numerous nonallelic repeated sequences, such as multicopy genes, gene families, and repeated elements. One common feature of nonallelic repeated sequences is that they are homeologous (not perfectly identical). Our laboratory has been studying recombination between homeologous sequences by using LINE-1 (L1) elements as substrates. We showed previously that an exogenous L1 element could readily acquire endogenous L1 sequences by nonreciprocal homologous recombination. In the study presented here, we have investigated the propensity of exogenous L1 elements to be involved in a reciprocal process, namely, crossing-overs. This would result in the integration of the exogenous L1 element into an endogenous L1 element. Of over 400 distinct integration events analyzed, only 2% involved homologous recombination between exogenous and endogenous L1 elements. These homologous recombination events were imprecise, with the integrated vector being flanked by one homologous and one illegitimate junction. This type of structure is not consistent with classical crossing-overs that would result in two homologous junctions but rather is consistent with one-sided homologous recombination followed by illegitimate integration. Contrary to what has been found for reciprocal homologous integration, the degree of homology between the exogenous and endogenous L1 elements did not seem to play an important role in the choice of recombination partners. These results suggest that although exogenous and endogenous L1 elements are capable of homologous recombination, this seldom leads to crossing-overs. This observation could have implications for the stability of mammalian genomes.     

Homologous recombination is very rare or absent in human influenza A virus

To determine the extent of homologous recombination in human influenza A virus, we assembled a data set of 13,852 sequences representing all eight segments and both major circulating subtypes, H3N2 and H1N1. Using an exhaustive search and a nonparametric test for mosaic structure, we identified 315 sequences (approximately 2%) in five different RNA segments that, after a multiple-comparison correction, had statistically significant mosaic signals compatible with homologous recombination. Of these, only two contained recombinant regions of sufficient length (>100 nucleotides [nt]) that the occurrence of homologous recombination could be verified using phylogenetic methods, with the rest involving very short sequence regions (15 to 30 nt). Although this secondary analysis revealed patterns of phylogenetic incongruence compatible with the action of recombination, neither candidate recombinant was strongly supported. Given our inability to exclude the occurrence of mixed infection and template switching during amplification, laboratory artifacts provide an alternative and likely explanation for the occurrence of phylogenetic incongruence in these two cases. We therefore conclude that, if it occurs at all, homologous recombination plays only a very minor role in the evolution of human influenza A virus.