Duplication and retention biases of essential and non-essential genes revealed by systematic knockdown analyses.
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ABSTRACT:
SUBMITTER: Woods S
PROVIDER: S-EPMC3649981 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The Paramecium aurelia complex is a group of 15 species that share at least three past whole-genome duplications (WGDs). The macronuclear genome sequences of P. biaurelia and P. sexaurelia are presented and compared to the published sequence of P. tetraurelia. Levels of duplicate-gene retention from the recent WGD differ by > 10% across species, with P. sexaurelia losing significantly more genes than P. biaurelia or P. tetraurelia. In addition, historically high rates of gene conversion have homogenized WGD paralogs, probably extending the paralogs' lifetimes. The probability of duplicate retention is positively correlated with GC content and expression level; ribosomal proteins, transcription factors, and intracellular signaling proteins are overrepresented among maintained duplicates. Finally, multiple sources of evidence indicate that P. sexaurelia diverged from the two other lineages immediately following, or perhaps concurrent with, the recent WGD, with approximately half of gene losses between P. tetraurelia and P. sexaurelia representing divergent gene resolutions (i.e., silencing of alternative paralogs), as expected for random duplicate loss between these species. Additionally, though P. biaurelia and P. tetraurelia diverged from each other much later, there are still more than 100 cases of divergent resolution between these two species. Taken together, these results indicate that divergent resolution of duplicate genes between lineages acts to reinforce reproductive isolation between species in the Paramecium aurelia complex.
| S-EPMC4199370 | biostudies-literature
Project description:Plants are immobile and often face stressful environmental conditions, prompting the evolution of genes regulating environmental responses. Such evolution is achieved largely through gene duplication and subsequent divergence. One of the most important gene families involved in regulating plant environmental responses and development is the AP2/ERF superfamily; however, the evolutionary history of these genes is unclear across angiosperms and in major angiosperm families adapted to various ecological niches. Specifically, the impact on gene copy number of whole-genome duplication events occurring around the time of the origins of several plant families is unknown. Here, we present the first angiosperm-wide comparative study of AP2/ERF genes, identifying 75 Angiosperm OrthoGroups (AOGs), each derived from an ancestral angiosperm gene copy. Among these AOGs, 21 retain duplicates with increased copy number in many angiosperm lineages, while the remaining 54 AOGs tend to maintain low copy number. Further analyses of multiple species in the Brassicaceae family indicated that family-specific duplicates experienced differential selective pressures in coding regions, with some paralogs showing signs of positive selection. Further, cis regulatory elements also exhibit extensive divergence between duplicates in Arabidopsis. Moreover, comparison of expression levels suggested that AP2/ERF genes with frequently retained duplicates are enriched for broad expression patterns, offering increased opportunities for functional diversification via changes in expression patterns, and providing a mechanism for repeated duplicate retention in some AOGs. Our results represent the most comprehensive evolutionary history of the AP2/ERF gene family, and support the hypothesis that AP2/ERF genes with broader expression patterns are more likely to be retained as duplicates than those with narrower expression profiles, which could lead to a higher chance of duplicate gene subfunctionalization. The greater tendency of some AOGs to retain duplicates, allowing expression and functional divergence, may facilitate the evolution of complex signaling networks in response to new environmental conditions.
| S-EPMC6399210 | biostudies-literature
Project description:Gene duplication is one of the major driving forces shaping genome and organism evolution and thought to be itself regulated by some intrinsic properties of the gene. Comparing the essential genes among mouse and human, we observed that the essential genes avoid duplication in mouse while prefer to remain duplicated in humans. In this study, we wanted to explore the reasons behind such differences in gene essentiality by cross-species comparison of human and mouse. Moreover, we examined essential genes that are duplicated in humans are functionally more redundant than that in mouse. The proportion of paralog pseudogenization of essential genes is higher in mouse than that of humans. These duplicates of essential genes are under stringent dosage regulation in human than in mouse. We also observed slower evolutionary rate in the paralogs of human essential genes than the mouse counterpart. Together, these results clearly indicate that human essential genes are retained as duplicates to serve as backed up copies that may shield themselves from harmful mutations.
| S-EPMC4353620 | biostudies-literature
Project description:Studies of essential genes in bacteria are often hampered by the lack of accessible genetic tools. This is also the case for Lactobacillus plantarum, a key species in food and health applications. Here, we develop a clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for knockdown of gene expression in L. plantarum The two-plasmid CRISPRi system, in which a nuclease-inactivated Cas9 (dCas9) and a gene-specific single guide RNA (sgRNA) are expressed on separate plasmids, allows efficient knockdown of expression of any gene of interest. We utilized the CRISPRi system to gain initial insights into the functions of key cell cycle genes in L. plantarum As a proof of concept, we investigated the phenotypes resulting from knockdowns of the cell wall hydrolase-encoding acm2 gene and of the DNA replication initiator gene dnaA and of ezrA, which encodes an early cell division protein. Furthermore, we studied the phenotypes of three cell division genes which have recently been functionally characterized in ovococcal bacteria but whose functions have not yet been investigated in rod-shaped bacteria. We show that the transmembrane CozE proteins do not seem to play any major role in cell division in L. plantarum On the other hand, RNA-binding proteins KhpA and EloR are critical for proper cell elongation in this bacterium.IMPORTANCE L. plantarum is an important bacterium for applications in food and health. Deep insights into the biology and physiology of this species are therefore necessary for further strain optimization and exploitation; however, the functions of essential genes in the bacterium are mainly unknown due to the lack of accessible genetic tools. The CRISPRi system developed here is ideal to quickly screen for phenotypes of both essential and nonessential genes. Our initial insights into the function of some key cell cycle genes represent the first step toward understanding the cell cycle in this bacterium.
| S-EPMC6429040 | biostudies-literature
Project description:CRISPR interference (CRISPRi) has facilitated the study of essential genes in diverse organisms using both high-throughput and targeted approaches. Despite the promise of this technique, no comprehensive arrayed CRISPRi library targeting essential genes exists for the model bacterium Escherichia coli, or for any Gram-negative species. Here, we built and characterized such a library. Each of the ∼500 strains in our E. coli library contains an inducible, chromosomally integrated single guide RNA (sgRNA) targeting an essential (or selected nonessential) gene and can be mated with a pseudo-Hfr donor strain carrying a dcas9 cassette to create a CRISPRi knockdown strain. Using this system, we built an arrayed library of CRISPRi strains and performed population and single-cell growth and morphology measurements as well as targeted follow-up experiments. These studies found that inhibiting translation causes an extended lag phase, identified new modulators of cell morphology, and revealed that the morphogene mreB is subject to transcriptional feedback regulation, which is critical for the maintenance of morphology. Our findings highlight canonical and noncanonical roles for essential genes in numerous aspects of cellular homeostasis. IMPORTANCE Essential genes make up only ∼5 to 10% of the genetic complement in most organisms but occupy much of their protein synthesis and account for almost all antibiotic targets. Despite the importance of essential genes, their intractability has, until recently, hampered efforts to study them. CRISPRi has facilitated the study of essential genes by allowing inducible and titratable depletion. However, all large-scale CRISPRi studies in Gram-negative bacteria thus far have used plasmids to express CRISPRi components and have been constructed in pools, limiting their utility for targeted assays and complicating the determination of antibiotic effects. Here, we use a modular method to construct an arrayed library of chromosomally integrated CRISPRi strains targeting the essential genes of the model bacterium Escherichia coli. This library enables targeted studies of essential gene depletions and high-throughput determination of antibiotic targets and facilitates studies targeting the outer membrane, an essential component that serves as the major barrier to antibiotics.
| S-EPMC8510551 | biostudies-literature
Project description:Background: The maintenance, regulation, and dynamics of heterochromatin in the human malaria parasite, Plasmodium falciparum, has drawn increasing attention due to its regulatory role in mutually exclusive virulence gene expression and the silencing of key developmental regulators. The advent of genome-wide analyses such as chromatin-immunoprecipitation followed by sequencing (ChIP-seq) has been instrumental in understanding chromatin composition; however, even in model organisms, ChIP-seq experiments are susceptible to intrinsic experimental biases arising from underlying chromatin structure. Methods: We performed a control ChIP-seq experiment, re-analyzed previously published ChIP-seq datasets and compared different analysis approaches to characterize biases of genome-wide analyses in P. falciparum. Results: We found that heterochromatic regions in input control samples used for ChIP-seq normalization are systematically underrepresented in regard to sequencing coverage across the P. falciparum genome. This underrepresentation, in combination with a non-specific or inefficient immunoprecipitation, can lead to the identification of false enrichment and peaks across these regions. We observed that such biases can also be seen at background levels in specific and efficient ChIP-seq experiments. We further report on how different read mapping approaches can also skew sequencing coverage within highly similar subtelomeric regions and virulence gene families. To ameliorate these issues, we discuss orthogonal methods that can be used to characterize bona fide chromatin-associated proteins. Conclusions: Our results highlight the impact of chromatin structure on genome-wide analyses in the parasite and the need for caution when characterizing chromatin-associated proteins and features.
| S-EPMC10445928 | biostudies-literature
Project description:BackgroundAlthough the overlap of transcriptional units occurs frequently in eukaryotic genomes, its evolutionary and biological significance remains largely unclear. Here we report a comparative analysis of overlaps between genes coding for well-annotated proteins in five metazoan genomes (human, mouse, zebrafish, fruit fly and worm).ResultsFor all analyzed species the observed number of overlapping genes is always lower than expected assuming functional neutrality, suggesting that gene overlap is negatively selected. The comparison to the random distribution also shows that retained overlaps do not exhibit random features: antiparallel overlaps are significantly enriched, while overlaps lying on the same strand and those involving coding sequences are highly underrepresented. We confirm that overlap is mostly species-specific and provide evidence that it frequently originates through the acquisition of terminal, non-coding exons. Finally, we show that overlapping genes tend to be significantly co-expressed in a breast cancer cDNA library obtained by 454 deep sequencing, and that different overlap types display different patterns of reciprocal expression.ConclusionOur data suggest that overlap between protein-coding genes is selected against in Metazoa. However, when retained it may be used as a species-specific mechanism for the reciprocal regulation of neighboring genes. The tendency of overlaps to involve non-coding regions of the genes leads to the speculation that the advantages achieved by an overlapping arrangement may be optimized by evolving regulatory non-coding transcripts.
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Project description:Nuclear genome duplication is normally restricted to once per cell division, but aberrant events that allow excess DNA replication (EDR) promote genomic instability and aneuploidy, both of which are characteristics of cancer development. Here we provide the first comprehensive identification of genes that are essential to restrict genome duplication to once per cell division. An siRNA library of 21,584 human genes was screened for those that prevent EDR in cancer cells with undetectable chromosomal instability. Candidates were validated by testing multiple siRNAs and chemical inhibitors on both TP53+ and TP53- cells to reveal the relevance of this ubiquitous tumor suppressor to preventing EDR, and in the presence of an apoptosis inhibitor to reveal the full extent of EDR. The results revealed 42 genes that prevented either DNA re-replication or unscheduled endoreplication. All of them participate in one or more of eight cell cycle events. Seventeen of them have not been identified previously in this capacity. Remarkably, 14 of the 42 genes have been shown to prevent aneuploidy in mice. Moreover, suppressing a gene that prevents EDR increased the ability of the chemotherapeutic drug Paclitaxel to induce EDR, suggesting new opportunities for synthetic lethalities in the treatment of human cancers.
| S-EPMC5085202 | biostudies-literature
Project description:Members of the genus Ranavirus (family Iridoviridae) are large double-stranded (ds) DNA viruses that are found world-wide infecting fish, amphibian and reptile ectothermic hosts. Ranavirus genomes range from 105 to 155kbp in length and they are predicted to encode around 90-125 genes. Currently, our knowledge of the function of ?50% of these genes is known or inferred based on homology to orthologous genes characterized in other systems; however, the function of the remaining open reading frames (ORFS) is unknown. Therefore, in order to begin to uncover the function of unknown ORFs in ranaviruses we developed a standardized approach to generate a recombination cassette for any ORF in Ambystoma tigrinum virus (ATV). Our standardized approach quickly and efficiently assembles recombination cassettes and recombinant ATV. We have used this approach to identify two essential, one semi-essential and two non-essential genes in ATV.
| S-EPMC4985175 | biostudies-literature
Project description:Among major vertebrate groups, anurans (frogs and toads) are understudied with regard to their visual systems, and little is known about variation among species that differ in ecology. We sampled North American anurans representing diverse evolutionary and life histories that likely possess visual systems adapted to meet different ecological needs. Using standard molecular techniques, visual opsin genes, which encode the protein component of visual pigments, were obtained from anuran retinas. Additionally, we extracted the visual opsins from publicly available genome and transcriptome assemblies, further increasing the phylogenetic and ecological diversity of our dataset to 33 species in total. We found that anurans consistently express four visual opsin genes (RH1, LWS, SWS1, and SWS2, but not RH2) even though reported photoreceptor complements vary widely among species. The proteins encoded by these genes showed considerable sequence variation among species, including at sites known to shift the spectral sensitivity of visual pigments in other vertebrates and had conserved substitutions that may be related to dim-light adaptation. Using molecular evolutionary analyses of selection (dN/dS) we found significant evidence for positive selection at a subset of sites in the dim-light rod opsin gene RH1 and the long wavelength sensitive cone opsin LWS. The function of sites inferred to be under positive selection are largely unknown, but a few are likely to affect spectral sensitivity and other visual pigment functions based on proximity to previously identified sites in other vertebrates. We also found the first evidence of visual opsin duplication in an amphibian with the duplication of the LWS gene in the African bullfrog, which had distinct LWS copies on the sex chromosomes suggesting the possibility of sex-specific visual adaptation. Taken together, our results indicate that ecological factors, such as habitat and life history, as well as behavior, may be driving changes to anuran visual systems.
| S-EPMC8820127 | biostudies-literature