Sorting out inherent features of head-to-head gene pairs by evolutionary conservation

BACKGROUND: A ‘head-to-head’ (h2h) gene pair is defined as a genomic locus in which two adjacent genes are divergently transcribed from opposite strands of DNA. In our previous work, this gene organization was found to be ancient and conserved, which subjects functionally related genes to transcriptional co-regulation. However, some of the biological features of h2h pairs still need further clarification. RESULTS: In this work, we assorted human h2h pairs into four sequentially inclusive sets of gradually incremental conservation, and examined whether those previously asserted features were conserved or sharpened in the more conserved h2h pair sets in order to identify the inherent features of the h2h gene organization. The features of TSS distance, expression correlation within h2h pairs and among h2h genes, transcription factor association and functional similarities of h2h genes were examined. Our conservation-based analyses found that the bi-directional promoters of h2h gene pairs are most likely shorter than 100 bp; h2h gene pairs generally have only significant positive expression correlation but not negative correlation, and remarkably high positive expression correlations exist among h2h genes, as well as between h2h pairs observed in our previous study; h2h paired genes tend to share transcription factors. In addition, expression correlation of h2h pairs is positively related with the TF-sharing and functional coordination, while not related with TSS distance. CONCLUSIONS: Our findings remove the uncertainties of h2h genes about TSS distance, expression correlation and functional coordination, which provide insights into the study on the molecular mechanisms and functional consequences of the transcriptional regulation based on this special gene organization

Functional genomic analysis of developmental control gene action in the embryonic nervous system of "Drosophila"

In the past two decades, developmental genetic analysis of the molecular control elements involved in early embryonic brain patterning has uncovered the existence of structurally and functionally homologous genes that have comparable, and indeed interchangeable functions in vertebrates and invertebrates. The cephalic gap gene family orthodenticle(otd)/Otx is expressed in the anterior brain of Drosophila and mouse. These genes play an important role during the formation of the anterior brain since mutation of otd/Otx2 causes the loss of entire rostral brain in both phyla. Reciprocal gene replacement experiments have demonstrated the functional equivalence of otd and Otx genes. The homeotic genes are expressed in a virtually co-linear anteroposterior pattern in the developing posterior brain of Drosophila and mouse, where they are required for the patterning of the region and the specification of segmental neuronal identity. These findings indicate the evolutionary conservation of cephalic gap gene and homeotic gene action in embryonic brain development and propose the conserved genetic network composed of genes controlled by these genes. In order to gain more information about the molecular basis of the genetic network underlying the observation of evolutionary conservation of key developmental control gene action, it is interesting and important to investigate the downstream targets of these control genes. To this end, this thesis takes advantage of the sequenced genome of Drosophila and the availability of high-density oligonucleotide array techniques to identify downstream genes at a genome wide level: As an initial part of this thesis, microarray analysis of diffe rential gene expression after heat shock revealed substantial changes in gene expression level for known heat -shock genes and identified numerous heat shock-inducible genes. These results demonstrated that high-density oligonucleotide arrays are sensitive, efficient, and quantitative instruments for the analysis of large -scale gene expression in Drosophila embryos. Based on this, in two subsequent parts of this thesis, this functional genomic approach was used to probe for candidate target genes of otd and labial(lab). In a first part, microarray experiments focused on the lab gene. High-density oligonucleotide arrays with probe sets representing 1,513 identified and sequenced genes were used to analyze differential gene expression following lab overexpre ssion in Drosophila embryos. A number of novel candidate downstream target genes for lab were identified, suggesting that LAB differentially regulates a limited and distinct set of embryonically expressed Drosophila genes. This provides preliminary information for further mechanism-orientated experiments. In a second part, microarray experiments focused on otd/Otx genes. In order to understand the functional equivalence of the Drosophila otd gene and the vertebrate Otx gene and gain insights into potential downstream genes of otd gene in the fly, a first genome wide quantitative transcript imaging experiment was carried out. This experiment was designed to study differential gene expression in flies in which either the Drosophila otd gene or the human Otx2 gene was overexpressed under the control of heat shock. These experiments indicated that 93 genes, approximately one third of the otd-regulated transcripts, also respond to overexpression of the human Otx2 gene in Drosophila. We postulate that these transcripts are common downstream targets of the fly otd gene and the human Otx2 gene in Drosophila which are likely to represent the molecular basis of the functional equivalence of otd and Otx2 gene action in Drosophila. A final part of the thesis was aimed at reducing false positive results of microarray experiments. For this, methods were developed using the magnetic cell sorting technique to isolate specific cell population from Drosophila embryos for specific expression profiling. These methods were the n applied to identify new candidate downstream genes of the gene glial cells missing (gcm) which is a key regulator during gliogenesis. The GAL4-UAS system was used to direct expression of a transmembrane protein, mCD8-GFP, exclusively to the neuroectoderm of stage 11 embryos, which permitted a high rate of purification of viable cells from the neuroectoderm as assayed by both cellular and molecular methods. Based on the sorted neuroectodermal cells, differential gene expression was analyzed in wildtype embryos versus embryos in which gcm was misexpressed throughout the neuroectoderm. Follow-up validation studies of genes identified as differentially expressed by in situ hybridization revealed a rate of confirmation for the sorted cellbased microarray experiments of more than 80%. This strongly contrasts to the high false positive rate revealed by microarray experiments based on wholemount embryos. Our results strongly suggest that reduction of cell heterogeneity through cell sorting techniques leads to a marked increase in the ability of microarrays to reveal differential gene expression in the developing nervous system

The pleistocene species pump past its prime:Evidence from European butterfly sister species

This is the final version. Available on open access from Wiley via the DOI in this recordData availability statement: Read data are available from the ENA at PRJEB43082. Sequence alignments for the COI barcode locus were obtained from the dataset DS-EUGENMAP (dx.doi.org/10.5883/DS-EUGENMAP) on BOLD at www.boldsystems.org and were originally produced by Dincӑ et al., (2021). The script used for calculating diversity and divergence is available at https://github.com/samebdon/orthodiver/blob/master/orthodiver.py.The Pleistocene glacial cycles had a profound impact on the ranges and genetic make-up of organisms. Whilst it is clear that the contact zones that have been described for many sister taxa are secondary and have formed during the last interglacial, it is unclear when the taxa involved began to diverge. Previous estimates based on small numbers of loci are unreliable given the stochasticity of genetic drift and the contrasting effects of incomplete lineage sorting and gene flow on gene divergence. Here we use genome-wide transcriptome data to estimate divergence for 18 sister species pairs of European butterflies showing either sympatric or contact zone distributions. We find that in most cases species divergence predates the mid-Pleistocene transition or even the entire Pleistocene period. We also show that although post divergence gene flow is restricted to contact zone pairs, they are not systematically younger than sympatric pairs. This suggests that contact zones are not limited to the initial stages of the speciation process, but can involve notably old taxa. Finally, we show that mitochondrial and nuclear divergence are only weakly correlated and mitochondrial divergence is higher for contact-zone pairs.Biotechnology & Biological Sciences Research Council (BBSRC)Natural Environment Research Council (NERC)European Union Horizon 202

Darwin’s wind hypothesis: does it work for plant dispersal in fragmented habitats?

Using the wind-dispersed plant Mycelis muralis, we examined how landscape fragmentation affects variation in seed traits contributing to dispersal. Inverse terminal velocity (Vt−1) of field-collected achenes was used as a proxy for individual seed dispersal ability. We related this measure to different metrics of landscape connectivity, at two spatial scales: in a detailed analysis of eight landscapes in Spain and along a latitudinal gradient using 29 landscapes across three European regions. In the highly patchy Spanish landscapes, seed Vt−1 increased significantly with increasing connectivity. A common garden experiment suggested that differences in Vt−1 may be in part genetically based. The Vt−1 was also found to increase with landscape occupancy, a coarser measure of connectivity, on a much broader (European) scale. Finally, Vt−1 was found to increase along a south–north latitudinal gradient. Our results for M. muralis are consistent with ‘Darwin’s wind dispersal hypothesis’ that high cost of dispersal may select for lower dispersal ability in fragmented landscapes, as well as with the ‘leading edge hypothesis’ that most recently colonized populations harbour more dispersive phenotypes.

Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire.

Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition

Frogs Hiding in Plain Sight: Phylogenetic Systematics of Myanmar’s Occidozyga Species Complex, and the Identification of a Novel Species

Different species can be difficult to distinguish from one another when they are morphologically similar. Such cryptic species are the reason many anuran species go undetected. For this study, the taxonomic identity of the Occidozyga complex across Myanmar was investigated. An integrated approach was used combining molecular, morphological and phylogeographic data to better assess its taxonomy. Results indicate the presence of three new candidate species within Occidozyga, and three evolutionarily significant unit (ESU) lineages. Two mitochondrial gene fragments (16S, COI) and one nuclear gene fragment (Rhodopsin) were examined from DNA isolated from forty-seven preserved specimens from the California Academy of Science (CAS). Spatial data from collection localities for specimens was integrated into phylogeographic analyses. Additionally, morphological data was analyzed for morphometrics and principal component analysis (PCA) from 259 specimens at the CAS and National Museum of Natural History (NMNH). Significant molecular differentiation was observed, uncovering a novel species from central Tanintharyi. Additional evolutionary significant units were identified. From these findings, we advocate for adequate protection of each of these distinct evolutionary lineages

Graph-based modeling and evolutionary analysis of microbial metabolism

Microbial organisms are responsible for most of the metabolic innovations on Earth. Understanding microbial metabolism helps shed the light on questions that are central to biology, biomedicine, energy and the environment. Graph-based modeling is a powerful tool that has been used extensively for elucidating the organising principles of microbial metabolism and the underlying evolutionary forces that act upon it. Nevertheless, various graph-theoretic representations and techniques have been applied to metabolic networks, rendering the modeling aspect ad hoc and highlighting the conflicting conclusions based on the different representations. The contribution of this dissertation is two-fold. In the first half, I revisit the modeling aspect of metabolic networks, and present novel techniques for their representation and analysis. In particular, I explore the limitations of standard graphs representations, and the utility of the more appropriate model---hypergraphs---for capturing metabolic network properties. Further, I address the task of metabolic pathway inference and the necessity to account for chemical symmetries and alternative tracings in this crucial task. In the second part of the dissertation, I focus on two evolutionary questions. First, I investigate the evolutionary underpinnings of the formation of communities in metabolic networks---a phenomenon that has been reported in the literature and implicated in an organism's adaptation to its environment. I find that the metabolome size better explains the observed community structures. Second, I correlate evolution at the genome level with emergent properties at the metabolic network level. In particular, I quantify the various evolutionary events (e.g., gene duplication, loss, transfer, fusion, and fission) in a group of proteobacteria, and analyze their role in shaping the metabolic networks and determining the organismal fitness. As metabolism gains an increasingly prominent role in biomedical, energy, and environmental research, understanding how to model this process and how it came about during evolution become more crucial. My dissertation provides important insights in both directions

Partial differential equations for self-organization in cellular and developmental biology

Understanding the mechanisms governing and regulating the emergence of structure and heterogeneity within cellular systems, such as the developing embryo, represents a multiscale challenge typifying current integrative biology research, namely, explaining the macroscale behaviour of a system from microscale dynamics. This review will focus upon modelling how cell-based dynamics orchestrate the emergence of higher level structure. After surveying representative biological examples and the models used to describe them, we will assess how developments at the scale of molecular biology have impacted on current theoretical frameworks, and the new modelling opportunities that are emerging as a result. We shall restrict our survey of mathematical approaches to partial differential equations and the tools required for their analysis. We will discuss the gap between the modelling abstraction and biological reality, the challenges this presents and highlight some open problems in the field