Evolutionary analyses of orphan genes in mouse lineages in the context of de novo gene birth

Gene birth is the process through which new genes appear. For a long time it was argued that the natural way of generating new genes was from copies of existing genes, and the possibility of de novo gene emergence was neglected. However, recent evidence has forced to reconsider old models and de novo gene birth gained recognition as a widespread phenomenon. De novo gene birth is the process by which a non-genic sequence is able to gain gene-like features through few mutations. The following work is a compilation of analyses that seek to highlight the importance and prevalence of de novo gene birth in genomes, suggesting that this is a process that is present at all times and which becomes very relevant upon ecological shifts. In the first chapter, I showed through phylostratigraphic analyses that new genes are substantially simpler than older, a trend which was consistent for several features and organisms, and suggestive of a frequent emergence of new genes through non-duplicative processes. In addition to this, I detected a strong association between gene birth and high transcriptional activity and chromosomal proximity. As part of this work, I was also able to use phylostratigraphy to evaluate a different model of gene birth, overprinting of alternative reading frames. In the following chapters of this dissertation, I made use of high-throughput sequencing of transcriptomes and genomes to ask questions about the origin and change of genes at closer time divergences than ever before, ranging from nearly 3000 years to 10 million years of divergence. I was able to detect the theoretically predicted effects of short time scale comparisons on the rate of protein evolution. Also, I contribute evidence that genes of different ages show different selective constraints even after only a few thousand years of divergence. Finally, in the last part of this thesis I evaluated the role of transcription in gene birth dynamics. Transcription seems to be a predominant feature of genomes, as most of the genome showed some level of transcription. In terms of de novo gene birth, I was able to identify 663 candidate loci from presence and absence of transcription. Analyses of these candidate loci indicated that gains are rather stable, meaning that subsequent losses were rarely found. In agreement with previous studies, I confirmed the role of testis as a driver of new genes. These results indicate that transcription is not a limiting factor in the emergence of new genes, and that our knowledge about the key regulatory elements of transcription and their turnover is still limited to explain why new genes seem to arise at a higher rate than they decay.Contents ......................................................................................................................................... 3 Summary of the thesis .................................................................................................................... 6 Zusammenfassung der Dissertation............................................................................................... 7 Acknowledgements ....................................................................................................................... 10 General introduction..................................................................................................................... 12 A brief historic perspective on the concepts of gene birth .................................................... 12 Gene duplication is the main source of new genes .............................................................. 12 Orphan genes and the genomics era .................................................................................... 14 Phylostratigraphy and the continuous emergence of new genes ......................................... 16 Not all genes come from other genes ................................................................................... 17 Considering gene birth from molecular and evolutionary perspectives ................................... 19 Overprinting: true innovation from existing genes .................................................................... 20 The life cycle of genes .............................................................................................................. 22 Overview................................................................................................................................... 24 Chapter 1: Phylogenetic patterns of emergence of new genes support a model of frequent de novo evolution ............................................................................................................................... 26 Introduction............................................................................................................................... 26 Results...................................................................................................................................... 27 Phylostratigraphy of mouse genes ........................................................................................ 27 Genomic features across ages.............................................................................................. 29 Chromosomal distribution ...................................................................................................... 33 Association with transcriptionally active sites ....................................................................... 33 Testis expressed genes......................................................................................................... 35 Alternative reading frames..................................................................................................... 36 Discussion ................................................................................................................................ 39 De novo evolution versus duplication-divergence ................................................................ 40 Regulatory evolution .............................................................................................................. 40 Overprinting ........................................................................................................................... 41 Conclusion................................................................................................................................ 42 Methods .................................................................................................................................... 43 Phylostratigraphy ................................................................................................................... 43 Gene structure analyses........................................................................................................ 43 Transcription associated regions........................................................................................... 44 Expression data for testis ...................................................................................................... 44 Secondary reading frames .................................................................................................... 44 Acknowledgements ................................................................................................................... 45 Chapter 2: Sequencing of genomes and transcriptomes of closely related mouse species....... 46 Introduction............................................................................................................................... 46 Using wild mice to understand gene birth at the transcriptome level ................................... 46 Phylogeographic distribution of the samples ........................................................................ 47 Methods .................................................................................................................................... 49 Biological material.................................................................................................................. 49 Transcriptome sequencing .................................................................................................... 49 Genome sequencing.............................................................................................................. 49 Raw data processing ............................................................................................................. 50 Transcriptome read mapping, annotation and quantification................................................ 50 Genome read mapping .......................................................................................................... 51 Available resources ................................................................................................................... 51 Chapter 3: Differential selective constrains across phylogenetic ages and their impact on the turnover of protein-coding genes. ................................................................................................. 53 Introduction............................................................................................................................... 53 Methods .................................................................................................................................... 53 Transcriptome assembly ....................................................................................................... 53 Generation of ortholog pairs and rate analyses .................................................................... 54 Overlapping genes................................................................................................................. 54 Reading frame polymorphism detection and annotation ...................................................... 55 Statistical analyses ................................................................................................................ 55 Results...................................................................................................................................... 55 Rate differences between genes of different ages ............................................................... 55 Overlapping genes are an unlikely source of bias ................................................................ 57 Impact of reading frame polymorphisms across phylogenetic time...................................... 59 Discussion ................................................................................................................................ 64 Acknowledgements ................................................................................................................... 66 Chapter 4: A transcriptomics approach to the gain and loss of de novo genes in mouse lineages...................................................................................................................................................... 67 Introduction............................................................................................................................... 67 How is a gene made? ............................................................................................................ 67 The early phase of new gene emergence............................................................................. 69 Pervasive transcription and junk-DNA as raw material for new genes ................................ 70 Methods .................................................................................................................................... 71 Transcriptome presence/absence matrix and mapping of gains and losses ....................... 71 Results...................................................................................................................................... 73 How much of the mouse genome has evidence of transcription? ........................................ 73 Genome-wide transcription: gain and loss dynamics ........................................................... 74 Phylogenetic patterns in genome-wide transcription ............................................................ 75 How much of the genome is transcribed in a lineage specific way? .................................... 77 Identification of cases of de novo transcripts ........................................................................ 81 Quantification of gain rates for curated genes ...................................................................... 84 What are the dynamics of transcription loss in known genes?............................................. 86 Where are new genes expressed?........................................................................................ 88 Discussion ................................................................................................................................ 89 Pervasive transcription can provide material for new genes ................................................ 89 Asymmetry in gains and losses of transcription.................................................................... 92 From transcribed protogenes to de novo genes ................................................................... 93 Differences in expression levels ............................................................................................ 95 Testis as a niche for new genes ............................................................................................ 95 Conclusion................................................................................................................................ 96 Concluding remarks ...................................................................................................................... 97 Perspectives................................................................................................................................. 98 References ................................................................................................................................... 99 Chapter contributions .................................................................................................................. 114 Appendices ................................................................................................................................ 115 Appendix A. Phylostratigraphic maps ..................................................................................... 115 Appendix B. Curation data from orphan genes ...................................................................... 115 Appendix C. Functional annotation clusters based on known genes with loss of expression ................................................................................................................................................ 117 Appendix D. Transcriptome information and statistics ........................................................... 118 Curriculum Vitae.......................................................................................................................... 119 Affidavit....................................................................................................................................... 12

Phylogenetic patterns of emergence of new genes support a model of frequent de novo evolution

Background: New gene emergence is so far assumed to be mostly driven by duplication and divergence of existing genes. The possibility that entirely new genes could emerge out of the non-coding genomic background was long thought to be almost negligible. With the increasing availability of fully sequenced genomes across broad scales of phylogeny, it has become possible to systematically study the origin of new genes over time and thus revisit this question. Results: We have used phylostratigraphy to assess trends of gene evolution across successive phylogenetic phases, using mostly the well-annotated mouse genome as a reference. We find several significant general trends and confirm them for three other vertebrate genomes (humans, zebrafish and stickleback). Younger genes are shorter, both with respect to gene length, as well as to open reading frame length. They contain also fewer exons and have fewer recognizable domains. Average exon length, on the other hand, does not change much over time. Only the most recently evolved genes have longer exons and they are often associated with active promotor regions, i.e. are part of bidirectional promotors. We have also revisited the possibility that de novo evolution of genes could occur even within existing genes, by making use of an alternative reading frame (overprinting). We find several cases among the annotated Ensembl ORFs, where the new reading frame has emerged at a higher phylostratigraphic level than the original one. We discuss some of these overprinted genes, which include also the Hoxa9 gene where an alternative reading frame covering the homeobox has emerged within the lineage leading to rodents and primates (Euarchontoglires). Conclusions: We suggest that the overall trends of gene emergence are more compatible with a de novo evolution model for orphan genes than a general duplication-divergence model. Hence de novo evolution of genes appears to have occurred continuously throughout evolutionary time and should therefore be considered as a general mechanism for the emergence of new gene functions

All sequential allotment rules are obviously strategy-proof

Altres ajuts: UNSL (032016) ; CONICET (PIP112-200801-00655) ; Agencia I+D+I (PICT2017-2355)For division problems with single-peaked preferences, we show that all sequential allotment rules, a large subfamily of strategy-proof and efficient rules, are also obviously strategy-proof. Although obvious strategy-proofness is in general more restrictive than strategy-proofness, this is not the case in this setting

Human biogeography and faunal exploitation in Diamante River basin, central western Argentina

A biogeographic model used to describe human peopling of southern Mendoza, central western Argentina, proposed an intensification process activated by an increase in population growth rate during the Late Holocene. During this process, high-ranked resources at the surroundings of residential camps were depleted, and hunter–gatherers broadened their diet by incorporating a larger number of low-ranked prey and domesticated plant resources. In this paper, we evaluate an alternative hypothesis, focusing on zooarchaeological data from the Diamante River basin. The results show that faunal resource intensification does not appear to have occurred in the Diamante River basin during the Late Holocene. Faunal consumption in Diamante River basin mainly reflects the local fauna in each ecological zone. The data do not show a lack of higher ranked resources. We suggest it is more likely that the demographic increase was not significant enough to cause an impact on the faunal resources. The archaeological evidence should be improved and analysed in smaller scales to continue with the intensification debate.Fil: Otaola, Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Provincia de Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Universidad Nacional de Cuyo. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales; ArgentinaFil: Giardina, Miguel Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Provincia de Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Universidad Nacional de Cuyo. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales; ArgentinaFil: Franchetti, Fernando Ricardo. University of Pittsburgh at Johnstown; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

An imbalance between apoptosis and proliferation contributes to follicular persistence in polycystic ovaries in rats

<p>Abstract</p> <p>Background</p> <p>Cystic ovarian disease is an important cause of infertility that affects bovine, ovine, caprine and porcine species and even human beings. Alterations in the ovarian micro-environment of females with follicular cysts could alter the normal processes of proliferation and programmed cell death in ovarian cells. Thus, our objective was to evaluate apoptosis and proliferation in ovarian cystic follicles in rats in order to investigate the cause of cystic follicle formation and persistence.</p> <p>Methods</p> <p>We compared the number of in situ apoptotic cells by TUNEL assay, expression of active caspase-3 and members of Bcl-2 family by immunohistochemistry; and cell proliferation by the expression of the proliferation markers: PCNA and Ki-67.</p> <p>Results</p> <p>The proliferation index was low in granulosa of tertiary and cystic follicles of light exposed rats when compared with tertiary follicles of control animals, while in theca interna only cystic follicles presented low proliferation index when compared with tertiary follicles (p < 0.05). The granulosa of cysts exhibited a similar cell DNA fragmentation to early atretic follicles. In the granulosa and theca interna, active caspase-3 shown similar immunostaining levels in tertiary and cystic follicles (p < 0.05). The granulosa cells presented high expression of Bcl-2, Bcl-xL and Bcl-w in the tertiary and cystic follicles with diminishing intensity in the atretic follicles, except with Bcl-w where the intensity was maintained in the atretic follicles (p < 0.05). The expression of Bax was weak in the healthy and cystic follicles. In the theca interna, Bcl-2 expression was the same as the pattern found in the granulosa; no differences were found between tertiary and cystic follicles from both groups for Bcl-xL and Bcl-w. The expression of Bax in this layer was higher in the tertiary follicles of the treated animals (p < 0.05) while the values for cystic follicles were similar to those in the tertiary follicles of controls. The theca externa showed low expression of the pro and anti-apoptotic proteins.</p> <p>Conclusion</p> <p>These results show that the combination of weak proliferation indices and low apoptosis observed in follicular cysts, could explain the cause of the slow growth of cystic follicles and the maintenance of a static condition without degeneration, which leads to their persistence. These alterations may be due to structural and functional modifications that take place in these cells and could be related to hormonal changes in animals with this condition.</p

An imbalance between apoptosis and proliferation contributes to follicular persistence in polycystic ovaries in rats

Background: Cystic ovarian disease is an important cause of infertility that affects bovine, ovine, caprine and porcine species and even human beings. Alterations in the ovarian micro-environment of females with follicular cysts could alter the normal processes of proliferation and programmed cell death in ovarian cells. Thus, our objective was to evaluate apoptosis and proliferation in ovarian cystic follicles in rats in order to investigate the cause of cystic follicle formation and persistence. Methods: We compared the number of in situ apoptotic cellsby TUNEL assay, expression of active caspase-3 and members of Bcl-2 family by immunohistochemistry; and cell proliferation by the expression of the proliferation markers: PCNA and Ki-67. Results: The proliferation index was low in granulosa of tertiary and cystic follicles of light exposed rats when compared with tertiary follicles of control animals, while in theca interna only cystic follicles presented low proliferation index when compared with tertiary follicles (p < 0.05). The granulosa of cysts exhibited a similar cell DNA fragmentation to early atretic follicles. In the granulosa and theca interna, active caspase-3 shown similar immunostaining levels in tertiary and cystic follicles (p < 0.05). The granulosa cells presented high expression of Bcl-2, Bcl-xL and Bcl-w in the tertiary and cystic follicles with diminishing intensity in the atretic follicles, except with Bcl-w where the intensity was maintained in the atretic follicles (p < 0.05). The expression of Bax was weak in the healthy and cystic follicles. In the theca interna, Bcl-2 expression was the same as the pattern found in the granulosa; no differences were found between tertiary and cystic follicles from both groups for Bcl-xL and Bcl-w. The expression of Bax in this layer was higher in the tertiary follicles of the treated animals (p < 0.05) while the values for cystic follicles were similar to those in the tertiary follicles of controls. The theca externa showed low expression of the pro and anti-apoptotic proteins. Conclusion: These results show that the combination of weak proliferation indices and low apoptosis observed in follicular cysts, could explain the cause of the slow growth of cystic follicles and the maintenance of a static condition without degeneration, which leads to their persistence. These alterations may be due to structural and functional modifications that take place in these cells and could be related to hormonal changes in animals with this conditionFacultad de Ciencias Veterinaria

Insights into the genome and proteome of <i>Sphingomonas paucimobilis</i> strain 20006FA involved in the regulation of polycyclic aromatic hydrocarbon degradation

In order to study the mechanisms regulating the phenanthrene degradation pathway and the intermediate-metabolite accumulation in strain S. paucimobilis 20006FA, we sequenced the genome and compared the genome-based predictions to experimental proteomic analyses. Physiological studies indicated that the degradation involved the salicylate and protocatechuate pathways, reaching 56.3% after 15 days. Furthermore, the strain degraded other polycyclic aromatic hydrocarbons (PAH) such as anthracene (13.1%), dibenzothiophene (76.3%), and fluoranthene. The intermediate metabolite 1-hydroxy2-naphthoic acid (HNA) accumulated during phenanthrene catabolism and inhibited both bacterial growth and phenanthrene degradation, but exogenous-HNA addition did not affect further degradation. Genomic analysis predicted 126 putative genes encoding enzymes for all the steps of phenanthrene degradation, which loci could also participate in the metabolism of other PAH. Proteomic analysis identified enzymes involved in 19 of the 23 steps needed for the transformation of phenanthrene to trichloroacetic-acid intermediates that were upregulated in phenanthrene cultures relative to the levels in glucose cultures. Moreover, the protein-induction pattern was temporal, varying between 24 and 96 h during phenanthrene degradation, with most catabolic proteins being overexpressed at 96 h—e. g., the biphenyl dioxygenase and a multispecies (2Fe–2S)-binding protein. These results provided the first clues about regulation of expression of phenanthrene degradative enzymes in strain 20006FA and enabled an elucidation of the metabolic pathway utilized by the bacterium. To our knowledge the present work represents the first investigation of genomic, proteomic, and physiological studies of a PAH-degrading Sphingomonas strain.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Insights into the genome and proteome of <i>Sphingomonas paucimobilis</i> strain 20006FA involved in the regulation of polycyclic aromatic hydrocarbon degradation

In order to study the mechanisms regulating the phenanthrene degradation pathway and the intermediate-metabolite accumulation in strain S. paucimobilis 20006FA, we sequenced the genome and compared the genome-based predictions to experimental proteomic analyses. Physiological studies indicated that the degradation involved the salicylate and protocatechuate pathways, reaching 56.3% after 15 days. Furthermore, the strain degraded other polycyclic aromatic hydrocarbons (PAH) such as anthracene (13.1%), dibenzothiophene (76.3%), and fluoranthene. The intermediate metabolite 1-hydroxy2-naphthoic acid (HNA) accumulated during phenanthrene catabolism and inhibited both bacterial growth and phenanthrene degradation, but exogenous-HNA addition did not affect further degradation. Genomic analysis predicted 126 putative genes encoding enzymes for all the steps of phenanthrene degradation, which loci could also participate in the metabolism of other PAH. Proteomic analysis identified enzymes involved in 19 of the 23 steps needed for the transformation of phenanthrene to trichloroacetic-acid intermediates that were upregulated in phenanthrene cultures relative to the levels in glucose cultures. Moreover, the protein-induction pattern was temporal, varying between 24 and 96 h during phenanthrene degradation, with most catabolic proteins being overexpressed at 96 h—e. g., the biphenyl dioxygenase and a multispecies (2Fe–2S)-binding protein. These results provided the first clues about regulation of expression of phenanthrene degradative enzymes in strain 20006FA and enabled an elucidation of the metabolic pathway utilized by the bacterium. To our knowledge the present work represents the first investigation of genomic, proteomic, and physiological studies of a PAH-degrading Sphingomonas strain.Centro de Investigación y Desarrollo en Fermentaciones Industriale

DLX5/6 GABAergic Expression Affects Social Vocalization: Implications for Human Evolution

DLX5 and DLX6 are two closely related transcription factors involved in brain development and in GABAergic differentiation. The DLX5/6 locus is regulated by FoxP2, a gene involved in language evolution and has been associated with neurodevelopmental disorders and mental retardation. Targeted inactivation of Dlx5/6 in mouse GABAergic neurons (Dlx5/6VgatCre mice) results in behavioral and metabolic phenotypes notably increasing lifespan by 33%. Here, we show that Dlx5/6VgatCre mice present a hyper-vocalization and hyper-socialization phenotype. While only 7% of control mice emitted more than 700 vocalizations/10 min, 30% and 56% of heterozygous or homozygous Dlx5/6VgatCre mice emitted more than 700 and up to 1,400 calls/10 min with a higher proportion of complex and modulated calls. Hyper-vocalizing animals were more sociable: the time spent in dynamic interactions with an unknown visitor was more than doubled compared to low-vocalizing individuals. The characters affected by Dlx5/6 in the mouse (sociability, vocalization, skull, and brain shape..) overlap those affected in the "domestication syndrome". We therefore explored the possibility that DLX5/6 played a role in human evolution and "self-domestication"comparing DLX5/6 genomic regions from Neanderthal and modern humans. We identified an introgressed Neanderthal haplotype (DLX5/6-N-Haplotype) present in 12.6% of European individuals that covers DLX5/6 coding and regulatory sequences. The DLX5/6-N-Haplotype includes the binding site for GTF2I, a gene associated with Williams-Beuren syndrome, a hyper-sociability and hyper-vocalization neurodevelopmental disorder. The DLX5/6-N-Haplotype is significantly underrepresented in semi-supercentenarians (&gt;105 years of age), a well-established human model of healthy aging and longevity, suggesting their involvement in the coevolution of longevity, sociability, and speech