Comparative Genomics Research Articles

Quorum sensing regulates virulence factors in the coral pathogen Vibrio coralliilyticus.

The bacterial pathogen Vibrio coralliilyticus causes disease in coral species worldwide. The mechanisms of V. coralliilyticus coral colonization, coral microbiome interactions, and virulence factor production are understudied. In other model Vibrio species, virulence factors like biofilm formation, toxin secretion, and protease production are controlled through a density-dependent communication system called quorum sensing (QS). Comparative genomics indicated that V. coralliilyticus genomes share high sequence identity for most of the QS signaling and regulatory components identified in other Vibrio species. Here, we identify an active QS signaling pathway in two V. coralliilyticus strains with distinct infection etiologies: type strain BAA-450 and coral isolate OCN008. In V. coralliilyticus, the inter-species AI-2 autoinducer signaling pathway in both strains controls expression of the master QS transcription factor and LuxR/HapR homolog VcpR to regulate >300 genes, including protease production, biofilm formation, and two conserved type VI secretion systems (T6SSs). Activation of T6SS1 by QS results in the secretion of effectors and enables interbacterial competition and killing of prey bacteria. We conclude that the QS system in V. coralliilyticus is functional and controls the expression of genes involved in relevant bacterial behaviors typically associated with host infection.IMPORTANCEVibrio coralliilyticus infects many marine organisms, including multiple species of corals, and is a primary causative agent of tissue loss diseases and bacterial-induced bleaching. Here, we investigated a common cell-cell communication mechanism called quorum sensing, which is known to be intimately connected to virulence in other Vibrio species. Our genetic and chemical studies of V. coralliilyticus quorum sensing uncovered an active pathway that directly regulates the following key virulence factors: proteases, biofilms, and secretion systems. These findings connect bacterial signaling in communities to the infection of corals, which may lead to novel treatments and earlier diagnoses of coral diseases in reefs.

Discovery of Evolutionary Loss of the Ubiquitin-Like Autophagy-Related ATG12 System in a Lineage of Apicomplexa

The autophagy-related ubiquitin-like conjugation systems, the ATG8 and ATG12 systems, are universally conserved in eukaryotes. However, the covalent bond in the ATG12 system has recently been shown to be evolutionarily lost in Apicomplexa. Here, we show that all genes associated with the ATG12 system are absent in piroplasmida, a lineage within Apicomplexa. Comparative genomics of ATGs further shows that piroplasm ATG3 has lost the region necessary for ATG12 binding. However, our in vitro functional analysis using recombinant proteins demonstrated that ATG3 retained the ability to interact with ATG8 in Babesia bovis, a model species in piroplasmida. These findings provide evidence that the ATG8 system is functional, while the ATG12 system is completely lost in the common ancestor of piroplasmida and highlight the evolutionary flexibility of the ATG12 system in Apicomplexa.

Comparative analysis under the family Tetranychidae with two new mitochondrial genomic resources of Stigmaeopsis nanjingensis and Stigmaeopsis continentalis (Acari: Tetranychidae)

Stigmaeopsis (Acari: Tetranychidae) species with social behaviors are difficult to be identified by morphological characteristics. The lack of nucleic acid resources as well as difficulties in sample acquisition have hindered phylogenetic studies and comparative mitochondrial genome analyses. In this study, we sequenced and annotated the mitochondrial genomes of Stigmaeopsis nanjingensis (Ma & Yuan, 1980) and S. continentalis Saito & Lin, 2018 using Illumina platform and homologous structures. The new resources integrated with public representative species under the family Tetranychidae to support the comparative and phylogenetic analysis. We obtained 37 mitochondrial genes from S. nanjingensis and S. continentalis, respectively. In phylogenetic results, all species of family Tetranychidae are well-clustered and divided into three clades. Genes and relative synonymous codon usage showed strong AT bias within Tetranychidae. Atp8 was the most variable gene and cox1 was the most conserved gene among 14 spider mite species verified by nucleotide content and bias, genes pairwise identities and the nucleotide diversities.

Localization and Molecular Cloning of the ASMT Gene for Melatonin Synthesis in Pigs

Melatonin is synthesized in multiple tissues and organs of pigs, and existing studies have shown the presence of the melatonin-synthesizing enzyme ASMT protein. However, the genomic information for the ASMT gene has been lacking. The aim of this study was to locate the genomic information of the ASMT gene in pigs using comparative genomics analysis and then obtain the coding region information through molecular cloning. First, using the NCBI Genome Data Viewer, we found that in most animals, the AKAP17A gene is often located next to the ASMT gene, with both genes arranged in the same direction. Similarly, the P2RY8 gene is commonly adjacent to the ASMTL gene, also in the same orientation. We also discovered that the ASMTL gene is frequently adjacent to the ASMT gene and arranged in the opposite direction. Using the “three-point localization” principle, we inferred the position of the ASMT gene based on the coordinates of AKAP17A and ASMTL in pigs. Our results revealed that on the pig X chromosome, a gene called LOC110258194 is located next to the AKAP17A and ASMTL genes, and its arrangement aligns with the ASMT gene in other species. Additionally, Ensembl contains a gene, ENSSSCG00000032659, at the same position, with completely overlapping exons, though it is not annotated as ASMT. Further analysis using the TreeFam tool from EMBL-EBI and the CDD tool from NCBI revealed that LOC110258194 and ENSSSCG00000032659 do not contain the typical Maf domain of ASMTL and, thus, should not be annotated as ASMTL, but rather as the ASMT gene. Using a slow-down PCR method for high-GC content genes, we successfully cloned the full CDS region of the pig ASMT gene and identified a new transcript missing Exon 6 and Exon 7. This transcript was submitted to NCBI and assigned the GenBank accession number MW847601. Our results represent the first successful localization of the ASMT gene in pigs, the first cloning of the ASMT gene’s coding region, and the first discovery of a new transcript of the pig ASMT gene.

Novel genomic features in entomopathogenic fungus Beauveria bassiana ILB308: accessory genomic regions and putative virulence genes involved in the infection process of soybean pest Piezodorus guildinii.

Biological control methods involving entomopathogenic fungi like Beauveria bassiana have been shown to be a valuable approach in integrated pest management as an environmentally friendly alternative to control pests and pathogens. Identifying genetic determinants of pathogenicity in B. bassiana is instrumental for enhancing its virulence against insects like the resistant soybean pest Piezodorus guildinii. This study focused on comparative genomics of different B. bassiana strains and gene expression analyses to identify virulence genes in the hypervirulent strain ILB308, especially in response to infection of P. guildinii and growth on hydrocarbon HC15, a known virulence enhancer. Strain ILB308 showed the highest number of virulence-related features, such as candidate virulence proteins, effectors, small secreted proteins and biosynthetic gene clusters. ILB308 also had a high percentage of unique DNA sequences, including six accessory scaffolds. Gene expression analysis at 4 days post inoculation revealed upregulation of known virulence factors, including Tudor domain proteins, LysM motif-containing proteins, subtilisin-like proteases and novel genes encoding secreted effectors and heat-labile enterotoxins. Growth on HC15 led to the upregulation of genes associated with oxidoreductase activity related to cuticular alkane degradation and fermentation metabolism/antioxidant responses in the hemolymph. The low number of known B. bassiana virulence genes points to novel or unknown mechanisms acting on the interaction between P. guildinii and strain ILB308. The presence of accessory genomic regions and unique virulence genes in ILB308 may contribute to its higher virulence. These genes could be considered as potential targets for enhancing fungal virulence through genetic manipulation. © 2025 Society of Chemical Industry.

Translational and clinical comparison of whole genome and transcriptome to panel sequencing in precision oncology

Precision oncology offers new cancer treatment options, yet sequencing methods vary in type and scope. In this study, we compared whole-exome/whole-genome (WES/WGS) and transcriptome sequencing (TS) with broad panel sequencing by resequencing the same tumor DNA and RNA as well as normal tissue DNA for germline assessment, from 20 patients with rare or advanced tumors, who were originally sequenced by WES/WGS ± TS within the DKFZ/NCT/DKTK MASTER program from 2015 to 2020. Molecular analyses resulted in a median number of 2.5 (gene panel) to 3.5 (WES/WGS ± TS) treatment recommendations per patient. Our results showed that approximately half of the therapy recommendations (TRs) of both sequencing programs were identical, while approximately one-third of the TRs in WES/WGS ± TS relied on biomarkers not covered by the panel. Eight of 10 molecularly informed therapy implementations were supported by the panel, the remaining two were based on biomarkers absent from the panel, highlighting the potential additional clinical benefit of WGS and TS.

Distinct Virulence Mechanisms of Burkholderia gladioli in Onion Foliar and Bulb Scale Tissues.

Slippery skin of onion caused by Burkholderia gladioli pv. alliicola (Bga) is a common bacterial disease reported from onion growing regions around the world. Despite the increasing attention in recent years, our understanding of the virulence mechanisms of this pathogen remains limited. In this study, we characterized the predicted genetic determinants of virulence in Bga strain 20GA0385 using reverse genetics approach. Using the closely related rice pathogen, B. glumae as a reference, comparative genomics analysis was performed to identify Bga candidate virulence factors and regulators. Marked and unmarked deletion mutants were generated using allelic exchange and the mutants were functionally validated using in vitro and in vivo assays. The role of mutants in pathogenic phenotypes was analyzed using onion foliar/seedling necrosis assays, the Red Scale Necrosis (RSN) assay and in planta bacterial population counts. The phytotoxin toxoflavin was a major contributor to foliar necrosis and bacterial populations whereas the type II and type III secretion system (T2SS/T3SS) were dispensable for foliar symptoms. In onion scale tissue, the T2SS single mutant gspC and its double and triple mutant derivatives all contributed to scale lesion area. Neither the lipocyclopeptide icosalide, toxoflavin, nor T3SS were required for scale symptoms. Our results suggest the quorum sensing tofIMR system in Bga regulates, toxoflavin, T2SS, and T3SS, contributing to onion symptom production. We show different virulence factors contribute to onion tissue-specific virulence patterns in Bga and that decreases in scale symptoms often do not result in decreased Bga populations in onion tissue.

Timescale and genetic linkage explain the variable impact of defense systems on horizontal gene transfer.

Prokaryotes have evolved a wide repertoire of defense systems to prevent invasion by mobile genetic elements (MGE). However, because MGE are vehicles for the exchange of beneficial accessory genes, defense systems could consequently impede rapid adaptation in microbial populations. Here, we study how defense systems impact horizontal gene transfer (HGT) in the short and long terms. By combining comparative genomics and phylogeny-aware statistical methods, we quantified the association between the presence of 7 widespread defense systems and the abundance of MGE in the genomes of 196 bacterial and 1 archaeal species. We also calculated the differences in the rates of gene gain and loss between lineages that possess and lack each defense system. Our results show that the impact of defense systems on HGT is highly taxon- and system-dependent, and in most cases not statistically significant. Timescale analysis reveals that defense systems must persist in a lineage for a relatively long time to exert an appreciable negative impact on HGT. In contrast, for shorter evolutionary timescales, frequent co-acquisition of MGE and defense systems results in a net positive association of the latter with HGT. Given the high turnover rates experienced by defense systems, we propose that the inhibitory effect of most defense systems on HGT is masked by their strong linkage with MGE. These findings help explain the contradictory conclusions of previous research by pointing at mobility and within-host retention times as key factors that determine the impact of defense systems on genome plasticity.

Deciphering recent transposition patterns in plants through comparison of 811 genome assemblies.

Transposable elements (TEs) are significant drivers of genome evolution, yet their recent dynamics and impacts within and among species, as well as the roles of host genes and non-coding RNAs in the transposition process, remain elusive. With advancements in large-scale pan-genome sequencing and the development of open data sharing, large-scale comparative genomics studies have become feasible. Here, we performed complete de novo TE annotations and identified active TEs in 310 plant genome assemblies across 119 species and seven crop populations. Using 811 high-quality genomes, we detected 13 844 553 TE-induced structural variants (TE-SVs), providing unprecedented resolution in delineating recent TE activities. Our integrative analysis revealed a mutual evolutionary relationship between TEs and host genomes. On one hand, host genes and ncRNAs are involved in the transposition process, as evidenced by their colocalization and coactivation with TEs, and may play a role in chromatin regulation. On the other hand, TEs drive genetic innovation by promoting the duplication of host genes and inserting into regulatory regions. Moreover, genes influenced by active TEs are linked to plant growth, nutrient absorption, storage metabolism and environmental adaptation, aiding in crop domestication and adaptation. This TE dynamics atlas not only reveals evolutionary and functional features linked to transposition activity but also highlights the role of TEs in crop domestication and adaptation, paving the way for future exploration of TE-mediated genome evolution and crop improvement strategies.

Comparative Genomics Reveals Evidence of the Genome Reduction and Metabolic Potentials of Aliineobacillus hadale Isolated from Challenger Deep Sediment of the Mariana Trench

Comparative Genomics Reveals Evidence of the Genome Reduction and Metabolic Potentials of Aliineobacillus hadale Isolated from Challenger Deep Sediment of the Mariana Trench

Comparative genomic analysis of Fusarium oxysporum f. sp. lycopersici reveals telomeric duplications of a lineage-specific region carrying SIX8 and PSL1 and genome-wide expansion of Foxy transposable elements.

Fusarium oxysporum f. sp. lycopersici (Fol), the causal agent of tomato wilt disease, is a soil-borne, vascular-colonizing fungal pathogen that severely impacts tomato production in most growing regions worldwide. Despite the availability of over thirty Fol genome sequences in public databases, only one chromosome-scale assembly exists, comprising the low sequence-coverage Fol4287 reference genome generated using Sanger sequencing. Thus, genome structural variation and comparative genomics analyses of Fol remain largely unexplored. Here, using third generation Nanopore long-read sequencing in combination with high-throughput chromosome conformation capture (Hi-C) data, we have independently constructed a high-quality assembly and annotation of the Fol007 race 2 genome that consists of 15 pseudochromosomes with 25 telomeres, including 10 telomere-to-telomere chromosomes. The Fol007 genome is predicted to contain 29,148 protein-encoding genes, including all known SIX genes except for SIX4, which is absent in Fol race 2. Compared to the genome of Fusarium verticillioides, the Fol007 genome contains four complete lineage-specific (LS) chromosomes, including chromosome 5 (Chr5), chromosome 13 (Chr13), and two small chromosomes, Chr14 and Chr15. Comparative genomic analysis between the newly assembled Fol007 genome and Fol4287_2010 deposited in the GenBank database reveals that the completely assembled Chr13 of Fol007 carrying all known SIX genes (except SIX4 and SIX8) and three novel candidate effector genes is relatively stable and corresponds to pathogenicity chromosome Chr14 in Fol4287_2010. It also reveals a telomeric duplication of an LS region carrying SIX8 and PSL1 on core chromosomes Chr6, Chr7 and Chr11, and LS chromosome Chr15, as well as the absence of segmental duplications on LS chromosomes of the Fol007 genome. Furthermore, compared to the genomes of Fol race 1 and non-pathogenic Fo strains, an active and specific Foxy transposable element, responsible for the inactivation of a second copy of SIX13, was identified and found to have expanded in the genomes of Fol race 2 and 3 strains. This element may contribute to Fusarium oxysporum genome evolution and has potential as a genetic marker for studying phylogenetic relationships among formae speciales of Fusarium oxysporum. These findings provide a basis for further genetic and genomic understanding of Fol evolution and virulence mechanisms employed by Fol and contribute another reference genome for Fusarium study more broadly.

Evaluation of the cross-immunity between Mycobacterium tuberculosis and Mycobacterium abscessus in vitro

Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium abscessus (M. abscessus) are important pathogens that can cause lung diseases. Given the abundance of shared antigens between these two pathogens, evaluating the cross-immunization between Mycobacterium tuberculosis and Mycobacterium abscessus has implications for the assessment of tuberculosis vaccines based on nontuberculous mycobacteria (NTM). The whole-cell proteins of Mycobacterium abscessus were lysed via ultrasonication and then were subcutaneously injected into BALB/c mice either alone or mixed with adjuvant for three times at a 10-day interval. After the final immunization, cross-immune antigens were analysed via genomic comparison and Mycobacterium tuberculosis proteome microarrays. BALB/c mice splenic lymphocytes were stimulated with TB-PPD to assess the cross-immunity of the cellular immune response. The effect of cross-immunity on the growth of Mycobacterium tuberculosis was evaluated using a Mycobacterium tuberculosis growth inhibition assay. Despite the presence of 1,953 homologous gene clusters between Mycobacterium tuberculosis and Mycobacterium abscessus, only 302 Mycobacterium tuberculosis antigens exhibited cross-immunoreactivity after three immunizations. Compared with the PBS group, TB-PPD stimulation significantly increased the secretion of TNF-α, IL-4, and IL-6 by sensitized mouse splenic lymphocytes, and significantly affected the proliferation of IL-2+CD4 T and TNF-α+CD4 T cells in the immunized group (P < 0.05), but had no impact on IFN-γ and IFN-γ+ CD4 T cells. Furthermore, there was no significant difference in the proliferation of Mycobacterium tuberculosis between the immunized group and the PBS group in spleen cells. These data indicate that proteins from Mycobacterium abscessus are highly immunogenic in mice. However, the cross-immune response between Mycobacterium abscessus and Mycobacterium tuberculosis was inadequate to effectively inhibit the proliferation of Mycobacterium tuberculosis.

A phylogenetic approach to comparative genomics.

Comparative genomics, whereby the genomes of different species are compared, has the potential to address broad and fundamental questions at the intersection of genetics and evolution. However, species, genomes and genes cannot be considered as independent data points within statistical tests. Closely related species tend to be similar because they share genes by common descent, which must be accounted for in analyses. This problem of non-independence may be exacerbated when examining genomes or genes but can be addressed by applying phylogeny-based methods to comparative genomic analyses. Here, we review how controlling for phylogeny can change the conclusions of comparative genomics studies. We address common questions on how to apply these methods and illustrate how they can be used to test causal hypotheses. The combination of rapidly expanding genomic datasets and phylogenetic comparative methods is set to revolutionize the biological insights possible from comparative genomic studies.

BetaAlign: a deep learning approach for multiple sequence alignment.

Multiple sequence alignments are extensively used in biology, from phylogenetic reconstruction to structure and function prediction. Here, we suggest an out-of-the-box approach for the inference of multiple sequence alignments, which relies on algorithms developed for processing natural languages. We show that our AI-based methodology can be trained to align sequences by processing alignments that are generated via simulations, and thus different aligners can be easily generated for datasets with specific evolutionary dynamics attributes. We expect that natural-language processing solutions will replace or augment classic solutions for computing alignments, and more generally, challenging inference tasks in phylogenomics. The multiple sequence alignment (MSA) problem is a fundamental pillar in bioinformatics, comparative genomics, and phylogenetics. Here we characterize and improve BetaAlign, the first deep learning aligner, which substantially deviates from conventional algorithms of alignment computation. BetaAlign draws on natural language processing (NLP) techniques and trains transformers to map a set of unaligned biological sequences to an MSA. We show that our approach is highly accurate, comparable and sometimes better than state-of-the-art alignment tools. We characterize the performance of BetaAlign and the effect of various aspects on accuracy; for example, the size of the training data, the effect of different transformer architectures, and the effect of learning on a subspace of indel-model parameters (subspace learning). We also introduce a new technique that leads to improved performance compared to our previous approach. Our findings further uncover the potential of NLP-based methods for sequence alignment, highlighting that AI-based algorithms can substantially challenge classic approaches in phylogenomics and bioinformatics. Datasets used in this work are available on HuggingFace (Wolf et al., 2020) at: https://huggingface.co/dotan1111. Source code is available at: https://github.com/idotan286/SimulateAlignments. Supplementary data are available at Bioinformatics online.

Comparative genomics of Leptospira santarosai reveals genomic adaptations in bovine genital strains

Bovine genital leptospirosis (BGL) is a silent and chronic reproductive syndrome associated with reproductive failures that result in animal suffering and substantial financial losses for farmers. Important aspects of the interactions between the host and the pathogen during chronic leptospirosis have been well described in the kidney, but little is known about the genital infection mechanisms. The present study sheds light on the pathophysiology of BGL based on comparative genomic analysis of renal versus genital isolates of Leptospira santarosai genomes, an endemic species on Latin America. A significant number of genes were exclusive of the genital strains, with emphasis on genes associated with cell wall/membrane/envelope biogenesis, mobilome: prophages and transposons, and signal transduction mechanisms. Overall, these gene clusters play crucial roles in bacterial colonization and evasion of the immune response, which can reflect leptospiral tissue tropism to the genital niche. We provide new insights into the pathophysiology of an important and neglected syndrome in bovine, helping to elucidate the evolution of adaptation of leptospires in the genital tract of cows.

Four novel nontuberculous mycobacteria species: Mycobacterium wendilense sp. nov., Mycobacterium burgundiense sp. nov., Mycobacterium kokjensenii sp. nov. and Mycobacterium holstebronense sp. nov. revived from a historical Danish strain collection.

Four novel nontuberculous mycobacteria were discovered from a historical strain collection at the International Reference Laboratory of Mycobacteriology at Statens Serum Institut in Copenhagen, Denmark. Phylogenetic analysis combining the 16S rrs, internal transcribed spacer and 23S rrl elements, as well as a single-copy core-gene (hsp65, rpoB+C, secA and tuf) analysis of these freeze-dried mycobacteria, clinically isolated from gastric lavage samples between 1948 and 1955, showed to be associated with type strains grouping within the Terra and Fortuitum-Vaccae clade. Phenotypic characteristics, biochemical properties and fatty acid and mycolic acid profiles supported the classification as novel strains. A genomic comparison to the closest related type strain was done by calculating average nucleotide identity and in silico DNA:DNA hybridization values, which showed 87.9% and 33.0% for Mu0050, 85.2% and 27.4% for Mu0053, 85.3% and 27.6% for Mu0083 and 93.3% and 50.1% for Mu0102, respectively. The names proposed for the new species are Mycobacterium wendilense sp. nov. (Mu0050T=ITM 501390T=CCUG 77525T), Mycobacterium burgundiense sp. nov. (Mu0053T=ITM 501391T=CCUG 77526T), Mycobacterium kokjensenii sp. nov. (Mu0083T=ITM 501392T=CCUG 77527T) and Mycobacterium holstebronense sp. nov. (Mu0102T=ITM 501393T=CCUG 77528T).

Telomere-to-telomere genome and resequencing of 254 individuals reveal evolution, genomic footprints in Asian icefish, Protosalanx chinensis.

The Asian icefish, Protosalanx chinensis, has undergone extensive colonization in various waters across China for decades due to its ecological and physiological significance as well as its economic importance in the fishery resource. Here, we decoded a telomere-to-telomere (T2T) genome for P. chinensis combining PacBio HiFi long reads and ultra-long ONT (nanopore) reads and Hi-C data. The telomere was identified in both ends of the contig/chromosome. The expanded gene associated with circadian entrainment suggests that P. chinensis may exhibit a high sensitivity to photoperiod. The contracted genes' immune-related families and DNA repair associated with positive selection in P. chinensis suggested the selection pressure during adaptive evolution. The population genetic analysis reported the genetic diversity and genomic footprints in 254 individuals from 8 different locations. The natural seawater samples can be the highest diversity and different from other freshwater and introduced populations. The divergent regions' associated genes were found to be related to the osmotic pressure system, suggesting adaptations to alkalinity and salinity. Thus, the T2T genome and genetic variation can be valuable resources for genomic footprints in P. chinensis, shedding light on its evolution, comparative genomics, and the genetic differences between natural and introduced populations.

Genome-resolved analysis of Serratia marcescens SMTT infers niche specialization as a hydrocarbon-degrader.

Bacteria that are chronically exposed to high levels of pollutants demonstrate genomic and corresponding metabolic diversity that complement their strategies for adaptation to hydrocarbon-rich environments. Whole genome sequencing was carried out to infer functional traits of Serratia marcescens SMTT recovered from soil contaminated with crude oil. The genome size (Mb) was 5,013,981 with a total gene count of 4,842. Comparative analyses with carefully selected S. marcescens strains, two of which are associated with contaminated soil, show conservation of central metabolic pathways in addition to intra-specific genetic diversity and metabolic flexibility. Genome comparisons also indicated an enrichment of genes associated with multidrug resistance and efflux pumps for SMTT. The SMTT genome contained genes that enable the catabolism of aromatic compounds via the protocatechuate para-degradation pathway, in addition to meta-cleavage of catechol (meta-cleavage pathway II); gene enrichment for aromatic compound degradation was markedly higher for SMTT compared to the other S. marcescens strains analyzed. Our data presents a valuable genetic inventory for future studies on strains of S. marcescens and provides insights into those genomic features of SMTT with industrial potential.

Promoter Analysis and Dissection Using Reporter Genes, Comparative Genomics, and Gel Shift Assays in Phytophthora.

Transcriptional regulation allows cells to execute developmental programs, maintain homeostasis, and respond to intra- and extracellular signals. Central to these processes are promoters, which in eukaryotes are sequences upstream of genes that bind transcription factors (TFs) and which recruit RNA polymerase to initiate mRNA synthesis. Valuable tools for studying promoters include reporter genes, which can be used to indicate when and where genes are activated. Moreover, functional regions within promoters (typically TF binding sites) can be identified by integrating reporter assays with promoter mutagenesis. These sites may also be revealed through comparative genomics, or by the DNA-protein binding procedure known as a gel shift or electrophoretic mobility shift assay (EMSA). The latter can also be used to test if a specific TF binds a DNA target or assess the binding kinetics or affinity of the complex. In this chapter, we describe procedures for expressing reporter genes in Phytophthora, assaying reporter activity, identifying functional sites within promoters, and testing purified TFs or proteins within nuclear extracts for DNA binding.

Complete Genome Characterization of Canine Adenovirus From Türkiye With Next-Generation Sequencing.

Determining the complete genome sequence data of adenoviruses has recently become greatly important due to their use by scientists as vectors in cancer studies and other fields, including vaccine development. However, the GenBank database currently has few complete genome sequences of adenoviruses, which are known for their large genomes. To address this gap, we analysed next-generation sequencing data obtained from our previous study to provide the complete genome sequence of the canine adenovirus-2 strain. For the obtained canine adenovirus-2 strain (OQ596341), comparative genomics, recombination and phylogenetic analysis were conducted. This sequence was compared and phylogenetically analysed with the 20 complete genome sequences of canine adenovirus previously reported in GenBank worldwide, as well as partial E3 ORFA sequences obtained from Türkiye. The nucleotide similarity rates of the sequence obtained from this study with other CAdV-2 whole genomes are over 99.04%. The gene alignment results reveal that the OQ596341 was found to be closely related to the AC000020 reference genome and LC557011. There are two recombination events related to the genome in this study. Comparisons with other complete genome sequences revealed several previously unseen mutations. These mutations include H34Y in the E1A gene; P55A in the E1B 55K gene; D13N and D202N in the IVa2 gene; K679R, V934I and K989N in the Pol gene; E205K in the pTP gene; T455A in the pIIIa gene; A310V in the V gene; G151R in the protease gene; E268K in the 100K gene; G66S and G141S in the 33K gene; T14A, E250K, D287N and I293T in the E3 ORFA gene; and L193F in the E434K gene. Moreover, a comparison with partial sequences obtained from Türkiye revealed the E250K mutation in the E3 ORFA gene, which we report for the first time in Türkiye. The complete CAdV-2 genome sequence obtained in the present study is the first sequence from Europe. Comparative analysis with other genomes revealed some unique mutations. This study is the first to report the E250K amino acid change in the E3 ORFA gene in Türkiye. We anticipate that this data can be used in future CAdV-2 vaccine development studies. Further studies are recommended to evaluate the impact of these mutations on viral tropism and other host interactions.