GAGA-associated factor fosters loop formation in the Drosophila genome.

The impact of genome organization on the control of gene expression persists as a major challenge in regulatory biology. Most efforts have focused on the role of CTCF-enriched boundary elements and TADs, which enable long-range DNA-DNA associations via loop extrusion processes. However, there is increasing evidence for long-range chromatin loops between promoters and distal enhancers formed through specific DNA sequences, including tethering elements, which bind the GAGA-associated factor (GAF). Previous studies showed that GAF possesses amyloid properties in vitro, bridging separate DNA molecules. In this study, we investigated whether GAF functions as a looping factor in Drosophila development. We employed Micro-C assays to examine the impact of defined GAF mutants on genome topology. These studies suggest that the N-terminal POZ/BTB oligomerization domain is important for long-range associations of distant GAGA-rich tethering elements, particularly those responsible for promoter-promoter interactions that coordinate the activities of distant paralogous genes.

Rubisco Accumulation Factor1-like (RAFL) interacts with RAF1 to mediate Rubisco assembly in Arabidopsis.

Rubisco catalysis a rate-limiting step in photosynthesis. It is a complex of eight large (RbcL) and eight small (RbcS) subunits. The biogenesis of Rubisco requires assembly chaperones. One of the key Rubisco assembly chaperones, Rubisco accumulation factor1 (RAF1), assembled as a dimer, acts downstream of chaperonin-assisted RbcL folding by stabilizing RbcL antiparallel dimers for assembly into RbcL8 complexes. In maize, lacking RAF1 causes Rubisco deficient and seedling lethal. A RAF1 homologue, RAF1-like (RAFL), has been detected in Arabidopsis. We found RAFL shares 61.98 % sequence similarity with RAF1. They have similar conserved domains, predicted 3D structures and gene expression pattern. Phylogenetic tree analysis showed that RAFL and RAF1 only present in analyzed dicots, while only one copy of RAF presented in monocots, mosses and green algae. Combined analysis by three different protein-protein interaction methods showed that RAFL interacts with RAF1 both in vivo and in vitro. Taken together, we conclude that RAFL and RAF1 are close paralogous genes, and they can form heterodimer and/or homodimers to mediate Rubisco assembly in Arabidopsis.

Gene duplication as a major force driving the genome expansion in some giant viruses.

IMPORTANCE: Giant viruses are noteworthy not only due to their enormous particles but also because of their gigantic genomes. In this context, a fundamental question has persisted: how did these genomes evolve? Here we present the discovery of cedratvirus pambiensis, featuring the largest genome ever described for a cedratvirus. Our data suggest that the larger size of the genome can be attributed to an unprecedented number of duplicated genes. Further investigation of this phenomenon in other viruses has illuminated gene duplication as a key evolutionary mechanism driving genome expansion in diverse giant viruses. Although gene duplication has been described as a recurrent event in cellular organisms, our data highlights its potential as a pivotal event in the evolution of gigantic viral genomes.

Whole-Genome Duplication and Yeast's Fruitful Way of Life.

Studies on the fate of Saccharomyces cerevisiae paralogous gene pairs that arose through a whole-genome duplication event have shown diversification of retained duplicated genes. Paralogous functional specialization often results in improved function and/or novel function that could contribute to the adaptation of the organism to a new lifestyle. Here, we analyze and discuss particular case studies of paralogous functional diversification that could have played a role in the acquisition of yeast fermentative metabolism.

Retrofitting massively parallel sequencing (MPS) for HLA-DQA1 and polymarker (PM) in forensic casework.

Genotype profiling has played a major role in forensics for decades. The technology for detection and discrimination has advanced substantially, from serology to DNA sequence analysis. Currently, there may be situations where there is a need for re-analysis of forensic DNA data that was produced using methodology that is no longer available. An example of this is the allele-specific oligonucleotide hybridization assays used in the 1990s. In the study presented herein, we have developed a multiplex system combining PCR and massively parallel sequencing (MPS) technologies to identify DNA polymorphisms. Our results are consistent with those found in the widely utilized AmpliType PM + DQA1 Amplification and Typing Kit originally marketed by Perkin Elmer. During the course of our studies, it became apparent that paralogous genes for two of the loci, GYPA and HBG2 (formerly HBGG), could have confounded the interpretation of the original assays, and we describe the technical solutions we developed to overcome ambiguity in genotype assignment. This study results in a novel resource enabling the re-analysis of DNA profiling results produced decades past using current day technology.

Genome-wide analysis of AP2/ERF transcription factors family in Brassica napus.

The AP2/ERF transcription factor family plays an important role in different biological processes such as growth, development and response to abiotic and biotic stresses in plants. The genome-wide analysis identified 531 AP2/ERF genes in Brassica napus (oilseed rape or canola) that ranged from 333 to 6440 bp in genomic and 273-2493 bp in coding DNA sequence length. We classified BnAP2/ERF proteins into five subfamilies including AP2 (58 genes), ERF (250 genes), DREB/CBF (194 genes), RAV (26 genes), and Soloist (3 genes). Furthermore, AP2/ERF proteins were subdivided into 15 groups according to the AP2/ERF classification in Arabidopsis. The number of exons in BnAP2/ERF genes was from one to eleven and most of these genes in the same subfamily had the same exon-intron pattern. The results also indicated that the composition of conserved motifs in most proteins in each group was similar. The intron-exon patterns and the composition of conserved motifs validated the BnAP2/ERF transcription factors phylogenetic classification. Based on the results of genome distribution, BnAP2/ERF genes were located unevenly on the 19 B. napus chromosomes. The results indicated that gene duplication may play an important role in genome expansion of B. napus. Furthermore, genome evolution of B. napus using orthologous and paralogous identification was studied. We found 278, 380 and 366 orthologous gene pairs between B. napus with A. thaliana, B. rapa and B. oleracea, respectively. The results of this study will be useful in investigation of functional role and molecular mechanisms of BnAP2/ERF transcription factors genes in response to different stresses.

The 'TranSeq' 3'-end sequencing method for high-throughput transcriptomics and gene space refinement in plant genomes.

High-throughput RNA sequencing has proven invaluable not only to explore gene expression but also for both gene prediction and genome annotation. However, RNA sequencing, carried out on tens or even hundreds of samples, requires easy and cost-effective sample preparation methods using minute RNA amounts. Here, we present TranSeq, a high-throughput 3'-end sequencing procedure that requires 10- to 20-fold fewer sequence reads than the current transcriptomics procedures. TranSeq significantly reduces costs and allows a greater increase in size of sample sets analyzed in a single experiment. Moreover, in comparison with other 3'-end sequencing methods reported to date, we demonstrate here the reliability and immediate applicability of TranSeq and show that it not only provides accurate transcriptome profiles but also produces precise expression measurements of specific gene family members possessing high sequence similarity. This is difficult to achieve in standard RNA-seq methods, in which sequence reads cover the entire transcript. Furthermore, mapping TranSeq reads to the reference tomato genome facilitated the annotation of new transcripts improving >45% of the existing gene models. Hence, we anticipate that using TranSeq will boost large-scale transcriptome assays and increase the spatial and temporal resolution of gene expression data, in both model and non-model plant species. Moreover, as already performed for tomato (ITAG3.0; www.solgenomics.net), we strongly advocate its integration into current and future genome annotations.

Genome-wide diversity in temporal and regional populations of the betabaculovirus Erinnyis ello granulovirus (ErelGV).

BACKGROUND: Erinnyis ello granulovirus (ErelGV) is a betabaculovirus infecting caterpillars of the sphingid moth E. ello ello (cassava hornworm), an important pest of cassava crops (Manihot esculenta). In this study, the genome of seven field isolates of the virus ErelGV were deep sequenced and their inter- and intrapopulational sequence diversity were analyzed. RESULTS: No events of gene gain/loss or translocations were observed, and indels were mainly found within highly repetitive regions (direct repeats, drs). A naturally occurring isolate from Northern Brazil (Acre State, an Amazonian region) has shown to be the most diverse population, with a unique pattern of polymorphisms. Overall, non-synonymous substitutions were found all over the seven genomes, with no specific gathering of mutations on hotspot regions. Independently of their sizes, some ORFs have shown higher levels of non-synonymous changes than others. Non-core genes of known functions and structural genes were among the most diverse ones; and as expected, core genes were the least variable genes. We observed remarkable differences on diversity of paralogous genes, as in multiple copies of p10, fgf, and pep. Another important contrast on sequence diversity was found on genes encoding complex subunits and/or involved in the same biological processes, as late expression factors (lefs) and per os infectivity factors (pifs). Interestingly, several polymorphisms in coding regions lie on sequences encoding specific protein domains. CONCLUSIONS: By comparing and integrating information about inter- and intrapopulational diversity of viral isolates, we provide a detailed description on how evolution operates on field isolates of a betabaculovirus. Our results revealed that 35-41% of the SNPs of ErelGV lead to amino acid changes (non-synonymous substitutions). Some genes, especially non-core genes of unknown functions, tend to accumulate more mutations, while core genes evolve slowly and are more conserved. Additional studies would be necessary to understand the actual effects of such gene variations on viral infection and fitness.

Purifying selection acts on coding and non-coding sequences of paralogous genes in Arabidopsis thaliana.

BACKGROUND: Whole-genome duplications in the ancestors of many diverse species provided the genetic material for evolutionary novelty. Several models explain the retention of paralogous genes. However, how these models are reflected in the evolution of coding and non-coding sequences of paralogous genes is unknown. RESULTS: Here, we analyzed the coding and non-coding sequences of paralogous genes in Arabidopsis thaliana and compared these sequences with those of orthologous genes in Arabidopsis lyrata. Paralogs with lower expression than their duplicate had more nonsynonymous substitutions, were more likely to fractionate, and exhibited less similar expression patterns with their orthologs in the other species. Also, lower-expressed genes had greater tissue specificity. Orthologous conserved non-coding sequences in the promoters, introns, and 3' untranslated regions were less abundant at lower-expressed genes compared to their higher-expressed paralogs. A gene ontology (GO) term enrichment analysis showed that paralogs with similar expression levels were enriched in GO terms related to ribosomes, whereas paralogs with different expression levels were enriched in terms associated with stress responses. CONCLUSIONS: Loss of conserved non-coding sequences in one gene of a paralogous gene pair correlates with reduced expression levels that are more tissue specific. Together with increased mutation rates in the coding sequences, this suggests that similar forces of purifying selection act on coding and non-coding sequences. We propose that coding and non-coding sequences evolve concurrently following gene duplication.

Generation, Transfer, and Loss of Alternative Oxidase Paralogues in the Aspergillaceae Family.

Alternative oxidase (Aox) is a terminal oxidase operating in branched electron transport. The activity correlates positively with overflow metabolisms in certain Aspergilli, converting intracellular glucose by the shortest possible path into organic acids, like citrate or itaconate. Aox is nearly ubiquitous in fungi, but aox gene multiplicity is rare. Nevertheless, within the family of the Aspergillaceae and among its various species of industrial relevance-Aspergillus niger, A. oryzae, A. terreus, Penicillium rubens-paralogous aox genes coexist. Paralogous genes generally arise from duplication and are inherited vertically. Here, we provide evidence of four independent duplication events along the lineage that resulted in aox paralogues (aoxB) in contemporary Aspergillus and Penicillium taxa. In some species, three aox genes are co-expressed. The origin of the A. niger paralogue is different than that of the A. terreus paralogue, but all paralogous clades ultimately arise from ubiquitous aoxA parent genes. We found different patterns of uncorrelated gene losses reflected in the Aspergillus pedigree, albeit the original aoxA orthologues persist everywhere and are never replaced. The loss of acquired paralogues co-determines the contemporary aox gene content of individual species. In Aspergillus calidoustus, the two more ancient paralogues have, in effect, been replaced by two aoxB genes of distinct origins.

ß-tubulin paralogue tubC is frequently misidentified as the benA gene in Aspergillus section Nigri taxonomy: primer specificity testing and taxonomic consequences.

ß-tubulin (benA, tub-2) and calmodulin (caM) are crucial genes in the taxonomy of Aspergillus section Nigri. Widely used ß-tubulin primers are not specific for the benA gene for some taxa and preferentially amplify the tubC paralogue. Sequences of the tubC paralogue are widely combined with benA sequences in recent taxonomical works as well as other works, resulting in incongruent trees. In this study we newly provide benA sequences for several ex-type strains, which were characterised using the tubC gene only. We designed a highly specific forward primer to benA designated Ben2f for use in Aspergillus section Nigri, and tested specificity of numerous primer combinations to ß-tubulin paralogs. The primer pairs with the highest specificity to the benA gene and functional across species in section Nigri includes Ben2f/Bt2b, Ben2f/T22 and T10/T22. We also provide tools based on codon usage bias analysis that reliably distinguish both paralogues. Exon/intron arrangement is the next distinctive characteristic, although this tool is not valid outside section Nigri. The species identity of taxa from the A. aculeatus clade used in previous molecular studies was revised using combined molecular data (ITS, benA, caM). These data together with two different PCR-fingerprinting methods indicated that A. japonicus should be treated as a synonym of A. violaceofuscus. Similarly, A. fijiensis is reduced to synonymy with A. brunneoviolaceus.

Paralogous Genes Involved in Embryonic Development: Lessons from the Eye and Other Tissues.

During evolution, gene duplications lead to a naturally increased gene dosage. Duplicated genes can be further retained or eliminated over time by purifying selection pressure. The retention probability is increased by functional diversification and by the acquisition of novel functions. Interestingly, functionally diverged paralogous genes can maintain a certain level of functional redundancy and at least a partial ability to replace each other. In such cases, diversification probably occurred at the level of transcriptional regulation. Nevertheless, some duplicated genes can maintain functional redundancy after duplication and the ability to functionally compensate for the loss of each other. Many of them are involved in proper embryonic development. The development of particular tissues/organs and developmental processes can be more or less sensitive to the overall gene dosage. Alterations in the gene dosage or a decrease below a threshold level may have dramatic phenotypic consequences or even lead to embryonic lethality. The number of functional alleles of particular paralogous genes and their mutual cooperation and interactions influence the gene dosage, and therefore, these factors play a crucial role in development. This review will discuss individual interactions between paralogous genes and gene dosage sensitivity during development. The eye was used as a model system, but other tissues are also included.

The activity of the stress modulated Arabidopsis ubiquitin ligases PUB46 and PUB48 is partially redundant.

The Arabidopsis ubiquitin ligases PUB46, PUB47 and PUB48 are encoded by paralogus genes. Single gene pub46 and pub48 mutants display increased drought sensitivity compared to wild type (WT) suggesting that each has specific biological activity. The high sequence homology between PUB46 and PUB48 activity suggested that they may also share some aspects of their activity. Unfortunately, the close proximity of the PUB46 and PUB48 gene loci precludes obtaining a double mutant required to study if they are partially redundant by crossing the available single mutants. We thus applied microRNA technology to reduce the activity of all three gene products of the PUB46-48 subfamily by constructing an artificial microRNA (aMIR) targeted to this subfamily. Expressing aMIR46-48 in WT plants resulted in increased drought-sensitivity, a phenotype resembling that of each of the single pub46 and pub48 mutants, and enhanced sensitivity to methyl viologen, similar to that observed for the pub46 mutant. The WT plants expressing aMIR46-48 plants also revealed reduced inhibition by ABA at seed germination, a phenotype not evident in the single mutants. Expressing aMIR46-48 in pub46 and pub48 mutants further enhanced the drought sensitivity of each parental single mutant and of WT expressing aMIR46-48. These results suggest that the biological activities of PUB46 and PUB48 in abiotic stress response are partially redundant.

An overview of online resources for intra-species detection of gene duplications.

Gene duplication plays an important role in evolutionary mechanism, which can act as a new source of genetic material in genome evolution. However, detecting duplicate genes from genomic data can be challenging. Various bioinformatics resources have been developed to identify duplicate genes from single and/or multiple species. Here, we summarize the metrics used to measure sequence identity among gene duplicates within species, compare several computational approaches that have been used to predict gene duplicates, and review recent advancements of a Basic Local Alignment Search Tool (BLAST)-based web tool and database, allowing future researchers to easily identify intra-species gene duplications. This article is a quick reference guide for research tools used for detecting gene duplicates.

Divergent expression of paralogous genes by modification of shared enhancer activity through a promoter-proximal silencer.

The duplication of genes and their associated cis-regulatory elements, or enhancers, is a key contributor to genome evolution and biological complexity. Moreover, many paralogs, particularly tandem duplicates, are fixed for long periods of time under the control of shared enhancers. However, in most cases, the mechanism by which gene expression and function diverge following duplication is not known. Here, we dissect the regulation and function of the paralogous nubbin/pdm2 genes during wing development in Drosophila melanogaster. We show that these paralogs play a redundant role in the wing and that their expression relies on a single shared wing enhancer. However, the two genes differ in their ability to respond to this enhancer, with nub responding in all wing progenitor cells and pdm2 only in a small subset. This divergence is a result of a pdm2-specific silencer element at the pdm2 promoter that receives repressive input from the transcription factor Rotund. Repression through this silencer also depends on nub, allowing pdm2 to fully respond to the wing enhancer when nub expression is perturbed and functional compensation to occur. Thus, expression divergence downstream of a shared enhancer arises as a consequence of silencing the promoter of one paralog.

HSDFinder: A BLAST-Based Strategy for Identifying Highly Similar Duplicated Genes in Eukaryotic Genomes.

Gene duplication is an important evolutionary mechanism capable of providing new genetic material for adaptive and nonadaptive evolution. However, bioinformatics tools for identifying duplicate genes are often limited to the detection of paralogs in multiple species or to specific types of gene duplicates, such as retrocopies. Here, we present a user-friendly, BLAST-based web tool, called HSDFinder, which can identify, annotate, categorize, and visualize highly similar duplicate genes (HSDs) in eukaryotic nuclear genomes. HSDFinder includes an online heatmap plotting option, allowing users to compare HSDs among different species and visualize the results in different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional categories. The external software requirements are BLAST, InterProScan, and KEGG. The utility of HSDFinder was tested on various model eukaryotic species, including Chlamydomonas reinhardtii, Arabidopsis thaliana, Oryza sativa, and Zea mays as well as the psychrophilic green alga Chlamydomonas sp. UWO241, and was proven to be a practical and accurate tool for gene duplication analyses. The web tool is free to use at http://hsdfinder.com. Documentation and tutorials can be found via the GitHub: https://github.com/zx0223winner/HSDFinder.

The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-functionalized Paralogs Involved in Chloroplast Development.

The Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2, the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3, HvCMF7, or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL, and their barley homologs and to the function of the putative cTPs of CIA2 and CIL.

Two paralogous znf143 genes in zebrafish encode transcriptional activator proteins with similar functions but expressed at different levels during early development.

BACKGROUND: ZNF143 is an important transcriptional regulator protein conserved in metazoans and estimated to bind over 2000 promoter regions of both messenger RNA and small nuclear RNA genes. The use of zebrafish is a useful model system to study vertebrate gene expression and development. Here we characterize znf143a, a novel paralog of znf143b, previously known simply as znf143 in zebrafish. This study reveals a comparison of quantitative and spatial expression patterns, transcriptional activity, and a knockdown analysis of both ZNF143 proteins. RESULTS: ZNF143a and ZNF143b have a fairly strong conservation with 65% amino acid sequence identity, and both are potent activators in transient transfection experiments. In situ hybridization analyses of both znf143 mRNAs show that these genes are expressed strongly in regions of the brain at 24 h post fertilization in zebrafish development. A transient knockdown analysis of znf143 expression from either gene using CRISPR interference revealed similar morphological defects in brain development, and caused brain abnormalities in up to 50% of injected embryos. Although present in the same tissues, znf143a is expressed at a higher level in early development which might confer an evolutionary benefit for the maintenance of two paralogs in zebrafish. CONCLUSIONS: znf143a encodes a strong activator protein with high expression in neural tissues during early embryogenesis in zebrafish. Similar to its paralogous gene, znf143b, both znf143 genes are required for normal development in zebrafish.

An Overview of Duplicated Gene Detection Methods: Why the Duplication Mechanism Has to Be Accounted for in Their Choice.

Gene duplication is an important evolutionary mechanism allowing to provide new genetic material and thus opportunities to acquire new gene functions for an organism, with major implications such as speciation events. Various processes are known to allow a gene to be duplicated and different models explain how duplicated genes can be maintained in genomes. Due to their particular importance, the identification of duplicated genes is essential when studying genome evolution but it can still be a challenge due to the various fates duplicated genes can encounter. In this review, we first describe the evolutionary processes allowing the formation of duplicated genes but also describe the various bioinformatic approaches that can be used to identify them in genome sequences. Indeed, these bioinformatic approaches differ according to the underlying duplication mechanism. Hence, understanding the specificity of the duplicated genes of interest is a great asset for tool selection and should be taken into account when exploring a biological question.

Genome-Wide Identification and Comparative Analysis of ARF Family Genes in Three Apiaceae Species.

The family Apiaceae includes many important vegetables and medicinal plants. Auxin response factors (ARFs) play critical roles in regulating plant growth and development. Here, we performed a comprehensive analysis of the ARF gene family in three Apiaceae species, celery, coriander, and carrot, and compared the results with the ARF gene family of lettuce, Arabidopsis, and grape. We identified 156 ARF genes in all six species and 89 genes in the three Apiaceae species, including 28, 34, and 27 in celery, coriander, and carrot, respectively. The paralogous gene number in coriander was far greater than that in carrot and celery. Our analysis revealed that ARF genes of the three Apiaceae species in 34 branches of the phylogenetic tree underwent significant positive selection. Additionally, our findings indicated that whole-genome duplication played an important role in ARF gene family expansion. Coriander contained a greater number of ARF genes than celery and carrot because of more gene duplications and less gene losses. We also analyzed the expression of ARF genes in three tissues by RNA-seq and verified the results by quantitative real-time PCR. Furthermore, we found that several paralogous genes exhibited divergent expression patterns. Overall, this study provides a valuable resource for exploring how ARF family genes regulate plant growth and development in other plants. Since this is the first report of the ARF gene family in Apiaceae, our results will serve as a guide for comparative and functional analyses of ARF and other gene families in Apiaceae.