Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo

Previous gene-transfer experiments have identified a 2500-nucleotide 5' domain of the CyIIIa cytoskeletal actin gene, which contains cis-regulatory sequences that are necessary and sufficient for spatial and temporal control of CyIIIa gene expression during embryogenesis. This gene is activated in late cleavage, exclusively in aboral ectoderm cell lineages. In this study, we focus on interactions demonstrated in vitro between sequences of the regulatory domain and proteins present in crude extracts derived from sea urchin embryo nuclei and from unfertilized eggs. Quantitative gel-shift measurements are utilized to estimate minimum numbers of factor molecules per embryo at 24 hr postfertilization, when the CyIIIa gene is active, at 7 hr, when it is still silent, and in the unfertilized egg. We also estimate the binding affinity preferences (K_r) of the various factors for their respective sites, relative to their affinity for synthetic DNA competitors. At least 14 different specific interactions occur within the regulatory regions, some of which produce multiple DNA-protein complexes. Values of K_r range from approximately 2 x 10^4 to approximately 2 x 10^6 for these factors under the conditions applied. With one exception, the minimum factor prevalences that we measured in the 400-cell 24-hr embryo nuclear extracts fell within the range of 2 x 10^5 to 2 x 10^6 molecules per embryo, i.e., a few hundred to a few thousand molecules per nucleus. Three developmental patterns were observed with respect to factor prevalence: Factors reacting at one site were found in unfertilized egg cytoplasm at about the same level per egg or embryo as in 24-hr embryo nuclei; factors reacting with five other regions of the regulatory domain are not detectable in egg cytoplasm but in 7-hr mid-cleavage-stage embryo, nuclei are already at or close to their concentrations in the 24-hr embryo nuclei; and factors reacting with five additional regions are not detectable in egg cytoplasm and are low in 7-hr embryo nuclei, i.e., ⩽10% per embryo of the level they attain in 24-hr embryo nuclei. The rise in concentration of factors of the latter class could provide the proximal cause for the temporal activation of the CyIIIa gene at the early blastula stage

Intersecting batteries of differentially expressed genes in the early sea urchin embryo

We determined the distribution of cis-regulatory sites, previously identified in the control domain of the CyIIIa gene, in three other genes displaying diverse spatial patterns of expression in the sea urchin embryo. Competitive gel-shift reactions were carried out using probes from the CyIIIa gene, with competitor fragments isolated from the previously defined control domains of the other genes. CyIIIa is expressed only in aboral ectoderm lineages; the other genes studied were Spec1, also expressed in aboral ectoderm; CyI, expressed in many different cell types; and SM50, expressed only in skeletogenic mesenchyme. All four genes are activated at about the same time in late cleavage. Where competitive interactions indicated a functionally comparable binding site (in vitro), a sequence homology was sought, and in most cases could be identified. An interesting pattern of putative regulatory site usage emerges: Of 10 CyIIIa interactions tested, three only were unique to the CyIIIa gene with respect to the set of four genes tested; one believed on previous evidence to be a temporal regulator was shared by all four genes, and the remainder were shared in various subsets of the four genes

Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo

Previous gene-transfer experiments have identified a 2500-nucleotide 5' domain of the CyIIIa cytoskeletal actin gene, which contains cis-regulatory sequences that are necessary and sufficient for spatial and temporal control of CyIIIa gene expression during embryogenesis. This gene is activated in late cleavage, exclusively in aboral ectoderm cell lineages. In this study, we focus on interactions demonstrated in vitro between sequences of the regulatory domain and proteins present in crude extracts derived from sea urchin embryo nuclei and from unfertilized eggs. Quantitative gel-shift measurements are utilized to estimate minimum numbers of factor molecules per embryo at 24 hr postfertilization, when the CyIIIa gene is active, at 7 hr, when it is still silent, and in the unfertilized egg. We also estimate the binding affinity preferences (K_r) of the various factors for their respective sites, relative to their affinity for synthetic DNA competitors. At least 14 different specific interactions occur within the regulatory regions, some of which produce multiple DNA-protein complexes. Values of K_r range from approximately 2 x 10^4 to approximately 2 x 10^6 for these factors under the conditions applied. With one exception, the minimum factor prevalences that we measured in the 400-cell 24-hr embryo nuclear extracts fell within the range of 2 x 10^5 to 2 x 10^6 molecules per embryo, i.e., a few hundred to a few thousand molecules per nucleus. Three developmental patterns were observed with respect to factor prevalence: Factors reacting at one site were found in unfertilized egg cytoplasm at about the same level per egg or embryo as in 24-hr embryo nuclei; factors reacting with five other regions of the regulatory domain are not detectable in egg cytoplasm but in 7-hr mid-cleavage-stage embryo, nuclei are already at or close to their concentrations in the 24-hr embryo nuclei; and factors reacting with five additional regions are not detectable in egg cytoplasm and are low in 7-hr embryo nuclei, i.e., ⩽10% per embryo of the level they attain in 24-hr embryo nuclei. The rise in concentration of factors of the latter class could provide the proximal cause for the temporal activation of the CyIIIa gene at the early blastula stage

Molecular evolution, diversity and adaptation of H7N9 viruses in China during 2013-2017

The substantial increase in prevalence and emergence of antigenically divergent or highly pathogenic influenza A(H7N9) viruses during 2016–17 raises concerns about the epizootic potential of these viruses. We investigated the evolution and adaptation of H7N9 viruses by analyzing available data and newly generated virus sequences isolated in Guangdong Province, China, during 2015–2017. Phylogenetic analyses showed that circulating H7N9 viruses belong to distinct lineages with differing spatial distributions. Hemagglutination inhibition assays performed on serum samples from patients infected with these viruses identified 3 antigenic clusters for 16 strains of different virus lineages. We used ancestral sequence reconstruction to identify parallel amino acid changes on multiple separate lineages. We inferred that mutations in hemagglutinin occur primarily at sites involved in receptor recognition or antigenicity. Our results indicate that highly pathogenic strains likely emerged from viruses circulating in eastern Guangdong Province during March 2016 and are associated with a high rate of adaptive molecular evolution

First Complete Genome Sequences of Zika Virus Isolated from Febrile Patient Sera in Ecuador

Here, we present the complete genome sequences of two Zika virus (ZIKV) strains, EcEs062_16 and EcEs089_16, isolated from the sera of febrile patients in Esmeraldas City, in the northern coastal province of Esmeraldas, Ecuador, in April 2016. These are the first complete ZIKV genomes to be reported from Ecuador

An Endogenous Foamy-like Viral Element in the Coelacanth Genome

Little is known about the origin and long-term evolutionary mode of retroviruses. Retroviruses can integrate into their hosts' genomes, providing a molecular fossil record for studying their deep history. Here we report the discovery of an endogenous foamy virus-like element, which we designate ‘coelacanth endogenous foamy-like virus’ (CoeEFV), within the genome of the coelacanth (Latimeria chalumnae). Phylogenetic analyses place CoeEFV basal to all known foamy viruses, strongly suggesting an ancient ocean origin of this major retroviral lineage, which had previously been known to infect only land mammals. The discovery of CoeEFV reveals the presence of foamy-like viruses in species outside the Mammalia. We show that foamy-like viruses have likely codiverged with their vertebrate hosts for more than 407 million years and underwent an evolutionary transition from water to land with their vertebrate hosts. These findings suggest an ancient marine origin of retroviruses and have important implications in understanding foamy virus biology

Identification of Evolutionary Trajectories Shared across Human Betacoronaviruses

Comparing the evolution of distantly related viruses can provide insights into common adaptive processes related to shared ecological niches. Phylogenetic approaches, coupled with other molecular evolution tools, can help identify mutations informative on adaptation, although the structural contextualization of these to functional sites of proteins may help gain insight into their biological properties. Two zoonotic betacoronaviruses capable of sustained human-to-human transmission have caused pandemics in recent times (SARS-CoV-1 and SARS-CoV-2), although a third virus (MERS-CoV) is responsible for sporadic outbreaks linked to animal infections. Moreover, two other betacoronaviruses have circulated endemically in humans for decades (HKU1 and OC43). To search for evidence of adaptive convergence between established and emerging betacoronaviruses capable of sustained human-to-human transmission (HKU1, OC43, SARS-CoV-1, and SARS-CoV-2), we developed a methodological pipeline to classify shared nonsynonymous mutations as putatively denoting homoplasy (repeated mutations that do not share direct common ancestry) or stepwise evolution (sequential mutations leading towards a novel genotype). In parallel, we look for evidence of positive selection and draw upon protein structure data to identify potential biological implications. We find 30 candidate mutations, from which 4 (codon sites 18121 [nsp14/residue 28], 21623 [spike/21], 21635 [spike/25], and 23948 [spike/796]; SARS-CoV-2 genome numbering) further display evolution under positive selection and proximity to functional protein regions. Our findings shed light on potential mechanisms underlying betacoronavirus adaptation to the human host and pinpoint common mutational pathways that may occur during establishment of human endemicity

Parasites, pathogens and commensals in the “low-impact” non-native amphipod host Gammarus roeselii

Background: Whilst vastly understudied, pathogens of non-native species (NNS) are increasingly recognised as important threats to native wildlife. This study builds upon recent recommendations for improved screening for pathogens in NNS by focusing on populations of Gammarus roeselii in Chojna, north-western Poland. At this location, and in other parts of continental Europe, G. roeselii is considered a well-established and relatively ‘low-impact’ invader, with little understanding about its underlying pathogen profile and even less on potential spill-over of these pathogens to native species. Results: Using a combination of histological, ultrastructural and phylogenetic approaches, we define a pathogen profile for non-native populations of G. roeselii in Poland. This profile comprised acanthocephalans (Polymorphus minutus Goese, 1782 and Pomphorhynchus sp.), digenean trematodes, commensal rotifers, commensal and parasitic ciliated protists, gregarines, microsporidia, a putative rickettsia-like organism, filamentous bacteria and two viral pathogens, the majority of which are previously unknown to science. To demonstrate potential for such pathogenic risks to be characterised from a taxonomic perspective, one of the pathogens, a novel microsporidian, is described based upon its pathology, developmental cycle and SSU rRNA gene phylogeny. The novel microsporidian Cucumispora roeselii n. sp. displayed closest morphological and phylogenetic similarity to two previously described taxa, Cucumispora dikerogammari Ovcharenko, 2010 and Cucumispora ornata Bojko, 2015. Conclusions: In addition to our discovery extending the host range for the genus Cucumispora Ovcharenko, 2010 outside of the amphipod host genus Dikerogammarus Stebbing, we reveal significant potential for the co-transfer of (previously unknown) pathogens alongside this host when invading novel locations. This study highlights the importance of pre-invasion screening of low-impact NNS and, provides a means to document and potentially mitigate the additional risks posed by previously unknown pathogens