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Mouse A9 cells, deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8), were fused with normal chick erythrocytes and selected in hypoxanthine-aminopterin-thymidine medium for cells with hypoxanthine phosphoribosyltransferase activity. Recovered hybrid cells produced the chick hypoxanthine phosphoribosyltransferase exclusively, as demonstrated by electrophoretic mobility and immunoprecipitation tests, even though no chick chromosomes or chick cell-surface antigens could be identified in the hybrids. Surprisingly, the expression of the chick hypoxanthine phosphoribosyl-transferase activity in the mouse/chick hybrids required the presence of aminopterin in the growth medium; in its absence, enzyme synthesis decreased markedly. Because of the rapid and reversible modulation of hypoxanthine phosphoribosyltransferase activity, the hybrid cells could proliferate equally well in media containing hypoxanthine-aminopterin-thymidine or 8-azaguanine. Cellular selection was definitely ruled out as a possible cause. These results confirm previous reports that specific genetic information can be selectively transferred from one cell to another of a distant species. Furthermore, they demonstrate that an avian gene, whose activity is normally expressed constitutively, can become facultative when integrated into a mammalian cell. This seems to be the first instance where heterologous gene activity has been shown to be reversibly modulated in hybrid cells.

Virtually all land plants are coated in a cuticle, a waxy polyester that prevents nonstomatal water loss and is important for heat and drought tolerance. Here, we describe a likely genetic basis for a divergence in cuticular wax chemistry between Sorghum bicolor, a drought tolerant crop widely cultivated in hot climates, and its close relative Zea mays (maize). Combining chemical analyses, heterologous expression, and comparative genomics, we reveal that: 1) sorghum and maize leaf waxes are similar at the juvenile stage but, after the juvenile-to-adult transition, sorghum leaf waxes are rich in triterpenoids that are absent from maize; 2) biosynthesis of the majority of sorghum leaf triterpenoids is mediated by a gene that maize and sorghum both inherited from a common ancestor but that is only functionally maintained in sorghum; and 3) sorghum leaf triterpenoids accumulate in a spatial pattern that was previously shown to strengthen the cuticle and decrease water loss at high temperatures. These findings uncover the possibility for resurrection of a cuticular triterpenoid-synthesizing gene in maize that could create a more heat-tolerant water barrier on the plant's leaf surfaces. They also provide a fundamental understanding of sorghum leaf waxes that will inform efforts to divert surface carbon to intracellular storage for bioenergy and bioproduct innovations. [ABSTRACT FROM AUTHOR]

The article presents the author's insights on the action taken by the publication "PNAS" after it clearly violated the U.S. National Institute of Health (NIH) embargo policy in relation to the Study of Addiction, Genetics and Environment (SAGE). It notes that "PNAS" has published online an article about PKNOX2 gene, but violated the policy after one of the study's authors has signed a Data Use Certification with NIH. It also examines the implications of the case to the scientific community.

DNA is organized into chromatin-like structures that support the maintenance and regulation of genomes. A unique and poorly understood form of DNA organization exists in chloroplasts, which are organelles of endosymbiotic origin responsible for photosynthesis. Chloroplast genomes, together with associated proteins, form membrane-less structures known as nucleoids. The internal arrangement of the nucleoid, molecular mechanisms of DNA organization, and connections between nucleoid structure and gene expression remain mostly unknown. We show that Arabidopsis thaliana chloroplast nucleoids have a unique sequence-specific organization driven by DNA binding to the thylakoid membranes. DNA associated with the membranes has high protein occupancy, has reduced DNA accessibility, and is highly transcribed. In contrast, genes with low levels of transcription are further away from the membranes, have lower protein occupancy, and have higher DNA accessibility. Membrane association of active genes relies on the pattern of transcription and proper chloroplast development. We propose a speculative model that transcription organizes the chloroplast nucleoid into a transcriptionally active membrane-associated core and a less active periphery. [ABSTRACT FROM AUTHOR]

Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed Veloclmmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the Veloclmmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials. [ABSTRACT FROM AUTHOR]

Genetic humanization, which involves replacing mouse genes with their human counterparts, can create powerful animal models for the study of human genes and diseases. One important example of genetic humanization involves mice humanized for their Ig genes, allowing for human antibody responses within a mouse background (HumAb mice) and also providing a valuable platform for the generation of fully human antibodies as therapeutics. However, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which they were genetically humanized. Heretofore, most genetic humanizations have involved disruption of the endogenous mouse gene with simultaneous introduction of a human transgene at a new and random location (so-called KO-plus-transgenic humanization). More recent efforts have attempted to replace mouse genes with their human counterparts at the same genetic location (in situ humanization), but such efforts involved laborious procedures and were limited in size and precision. We describe a general and efficient method for very large, in situ, and precise genetic humanization using large compound bacterial artificial chromosome-based targeting vectors introduced into mouse E5 cells. We applied this method to genetically humanize 3-Mb segments of both the mouse heavy and κ light chain Ig loci, by far the largest genetic humanizations ever described. This paper provides a detailed description of our genetic humanization approach, and the companion paper reports that the humoral immune systems of mice bearing these genetically humanized loci function as efficiently as those of WT mice. [ABSTRACT FROM AUTHOR]

Congenital scoliosis (CS), affecting approximately 0.5 to 1 in 1,000 live births, is commonly caused by congenital vertebral malformations (CVMs) arising from aberrant somitogenesis or somite differentiation. While Wnt/ß- catenin signaling has been implicated in somite development, the function of Wnt/planar cell polarity (Wnt/PCP) signaling in this process remains unclear. Here, we investigated the role of Vangl1 and Vangl2 in vertebral development and found that their deletion causes vertebral anomalies resembling human CVMs. Analysis of exome sequencing data from multiethnic CS patients revealed a number of rare and deleterious variants in VANGL1 and VANGL2, many of which exhibited loss- of- function and dominant- negative effects. Zebrafish models confirmed the pathogenicity of these variants. Furthermore, we found that Vangl1 knock- in (p.R258H) mice exhibited vertebral malformations in a Vangl gene dose- and environment- dependent manner. Our findings highlight critical roles for PCP signaling in vertebral development and predisposition to CVMs in CS patients, providing insights into the molecular mechanisms underlying this disorder. [ABSTRACT FROM AUTHOR]

The epidermis serves as a protective barrier in animals. After epidermal injury, barrier repair requires activation of many wound response genes in epidermal cells surrounding wound sites. Two such genes in Drosophila encode the enzymes dopa decarboxylase (Ddc) and tyrosine hydroxylase (ple). In this paper we explore the involvement of the Toll/NF-κB pathway in the localized activation of wound repair genes around epidermal breaks. Robust activation of wound-induced transcription from ple and Ddc requires Toll pathway components ranging from the extracellular ligand Spätzle to the Dif transcription factor. Epistasis experiments indicate a requirement for Spätzle ligand downstream of hydrogen peroxide and protease function, both of which are known activators of wound-induced transcription. The localized activation of Toll a few cell diameters from wound edges is reminiscent of local activation of Toll in early embryonic ventral hypoderm, consistent with the hypothesis that the dorsal–ventral patterning function of Toll arose from the evolutionary cooption of a morphogen-responsive function in wound repair. Furthermore, the combinatorial activity of Toll and other signaling pathways in activating epidermal barrier repair genes can help explain why developmental activation of the Toll, ERK, or JNK pathways alone fail to activate wound repair loci. [ABSTRACT FROM AUTHOR]

The article focuses on a paper which examined the genetic legacy of a struggling species and the implications for a phoenix-like recovery from its genetic remnants. Topics include that early migration in Chinook does indeed have a simple genetic basis influenced by GREB1L gene; role of GREB1L gene in migration timing by collecting detailed phenotypic and genetic data; and presenting findings as evidence for a single evolutionary origin of the spring-run allele in Chinook salmon.

Proper expression of odor receptor genes is critical for the function of olfactory systems. In this study, we identified exitrons (exonic introns) in four of the 39 Odorant receptor (Or) genes expressed in the Drosophila antenna. Exitrons are sequences that can be spliced out from within a protein-coding exon, thereby altering the encoded protein. We focused on Or88a, which encodes a pheromone receptor, and found that exitron splicing of Or88a is conserved across five Drosophila species over 20 My of evolution. The exitron was spliced out in 15% of Or88a transcripts. Removal of this exitron creates a non-coding RNA rather than an RNA that encodes a stable protein. Our results suggest the hypothesis that in the case of Or88a, exitron splicing could act in neuronal modulation by decreasing the level of functional Or transcripts. Activation of Or88a-expressing olfactory receptor neurons via either optogenetics or pheromone stimulation increased the level of exitron-spliced transcripts, with optogenetic activation leading to a 14-fold increase. A fifth Or can also undergo an alternative splicing event that eliminates most of the canonical open reading frame. Besides these cases of alternative splicing, we found alternative polyadenylation of four Ors, and exposure of Or67c to its ligand ethyl lactate in the antenna downregulated all of its 3' isoforms. Our study reveals mechanisms by which neuronal activity could be modulated via regulation of the levels of Or isoforms. [ABSTRACT FROM AUTHOR]

One important goal of circadian medicine is to apply time-of-day dosing to improve the efficacy of chemotherapy. However, limited knowledge of how the circadian clock regulates DNA repair presents a challenge to mechanism-based clinical application. We studied time-series genome-wide nucleotide excision repair in liver and kidney of wild type and three different clock mutant genotypes (Cry1-/-Cry2-/-, Per1-/-Per2-/-, and Bmal1-/-). Rhythmic repair on the nontranscribed strand was lost in all three clock mutants. Conversely, rhythmic repair of hundreds of genes on the transcribed strand (TSs) persisted in the livers of Cry1-/-Cry2-/- and Per1-/-Per2-/- mice. We identified a tissue-specific, promoter element-driven repair mode on TSs of collagen and angiogenesis genes in the absence of clock activators or repressors. Furthermore, repair on TSs of thousands of genes was altered when the circadian clock is disrupted. These data contribute to a better understanding of the regulatory role of the circadian clock on nucleotide excision repair in mammals and may be invaluable toward the design of time-aware platinum-based interventions in cancer. [ABSTRACT FROM AUTHOR]

Homosporous lycophytes (Lycopodiaceae) are a deeply diverged lineage in the plant tree of life, having split from heterosporous lycophytes (Selaginella and Isoetes) ~400 Mya. Compared to the heterosporous lineage, Lycopodiaceae has markedly larger genome sizes and remains the last major plant clade for which no chromosome-level assembly has been available. Here, we present chromosomal genome assemblies for two homosporous lycophyte species, the allotetraploid Huperzia asiatica and the diploid Diphasiastrum complanatum. Remarkably, despite that the two species diverged ~350 Mya, around 30% of the genes are still in syntenic blocks. Furthermore, both genomes had undergone independent whole genome duplications, and the resulting intragenomic syntenies have likewise been preserved relatively well. Such slow genome evolution over deep time is in stark contrast to heterosporous lycophytes and is correlated with a decelerated rate of nucleotide substitution. Together, the genomes of H. asiatica and D. complanatum not only fill a crucial gap in the plant genomic landscape but also highlight a potentially meaningful genomic contrast between homosporous and heterosporous species. [ABSTRACT FROM AUTHOR]

Posted on March 12, 2021 Carolyn Beans Plant breeders are masters of genome tuning, spending years developing crops with the genes to resist disease, drought, and pests. In this issue https://doi.org/10.1073/iti1221118 | 3 of 3 JOURNAL CLUB Highlighting recent, timely papers selected by Academy member labs March 23, 2021 A new approach might one day use ordinary crop sprayers like this one to quickly deliver genes designed to respond to environmental challenges such as drought. [Extracted from the article]

The article focuses on the effects of epigenetic modifications of the brain for human behavior. Topics discussed include the link between behavioral neuroscience and epigenetic chemistry, the role of genes and ventromedial hypothalamic neurons for brain development, and aspects related to brain modification such as DNA methylation, noncoding RNAs, and genomic imprinting.

Transcriptional divergence of duplicated genes after whole genome duplication (WGD) has been described in many plant lineages and is often associated with subgenome dominance, a genome-wide mechanism. However, it is unknown what underlies the transcriptional divergence of duplicated genes in polyploid species that lack subgenome dominance. Soybean is a paleotetraploid with a WGD that occurred 5 to 13 Mya. Approximately 50% of the duplicated genes retained from this WGD exhibit transcriptional divergence. We developed accessible chromatin region (ACR) datasets from leaf, flower, and seed tissues using MNase-hypersensitivity sequencing. We validated enhancer function of several ACRs associated with known genes using CRISPR/Cas9-mediated genome editing. The ACR datasets were used to examine and correlate the transcriptional patterns of 17,111 pairs of duplicated genes in different tissues. We demonstrate that ACR dynamics are correlated with divergence of both expression level and tissue specificity of individual gene pairs. Gain or loss of flanking ACRs and mutation of cis-regulatory elements (CREs) within the ACRs can change the balance of the expression level and/or tissue specificity of the duplicated genes. Analysis of DNA sequences associated with ACRs revealed that the extensive sequence rearrangement after the WGD reshaped the CRE landscape, which appears to play a key role in the transcriptional divergence of duplicated genes in soybean. This may represent a general mechanism for transcriptional divergence of duplicated genes in polyploids that lack subgenome dominance. [ABSTRACT FROM AUTHOR]

We present a method for creating a network of gene co-occurrences from the literature and partitioning it into communities of related genes. The way in which our method identifies communities makes it likely that the component genes of each community will be related by their function. The method processes a large database of article abstracts, synthesizing information from many sources to shed light on groups of genes that have been shown to interact. It is a tool to be used by researchers in the biomedical sciences to swiftly search for known interactions and to provide insight into unexplored connections. The partitioning procedure is designed to be particularly applicable to large networks in which individual nodes may play a role in more than one community. In this paper, we explain the details of the method, in particular the partitioning process. We also apply the method to produce communities of genes related to colon cancer and show that the results are useful.

Seventy-five years ago, a convincing demonstration that the genes were physically aligned along the chromosome was lacking. Harriet Creighton (1909-2004) and Barbara McClintock (1902-1992) [reighton, H. B & McClintock, 8. (1931) Proc. Natt. Acad. Scl. USA 17, 492-4971 showed by an elegantly simple experiment in 1931 that exchange between genes was accompanied by exchange of cytological, i.e., physical, parts of chromosomes. The work has been acclaimed as one of the great experiments in biology. Creighton's doctoral dissertation under McClintock's mentorship provided the basis for the landmark paper, which was unique in merging cytological with genetic data. A companion paper by McClintock, printed and bound back-to-back with the joint paper, set the essential stage with data on the cytological and genetic features that Creighton applied. Following directly from this work, and leading to today's recognition that the genome is a graspable entity, was the knowledge that the genes could be studied as components of a linear structure, the chromosome. Here, we review the data surrounding the Creighton and McClintock paper and provide a perspective on the significance of their findings. [ABSTRACT FROM AUTHOR]

The newly evolved gene Heterochromatin Protein 6 (HP6), which has been previously classified as essential, challenged the dogma that functions required for viability are only seen in genes with a long evolutionary history. Based on previous RNA-sequencing analysis in Drosophila germ cells, we asked whether HP6 might play a role in germline development. Surprisingly, we found that CRISPR-generated HP6 mutants are viable and fertile. Using previously generated mutants, we identified an independent lethal allele and an RNAi off-target effect that prevented accurate interpretation of HP6 essentiality. By reviewing existing data, we found that the vast majority of young genes that were previously classified as essential were indeed viable when tested with orthologous methods. Together, our data call into question the frequency with which newly evolved genes gain essential functions and suggest that using multiple independent genetic methods is essential when probing the functions of young genes. [ABSTRACT FROM AUTHOR]

The endothelium is a major target of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Exposure of endothelial cells (EC) to proinflammatory stimuli leads to an increase in mitochondrial metabolism; however, the function and regulation of elevated mitochondrial metabolism in EC in response to proinflammatory cytokines remain unclear. Studies using high-resolution metabolomics and 13C-glucose and 13C-glutamine labeling flux techniques showed that pyruvate dehydrogenase activity (PDH) and oxidative tricarboxylic acid cycle (TCA) flux are elevated in human umbilical vein ECs in response to overnight (16 h) treatment with TNFα (10 ng/mL). Mechanistic studies indicated that TNFα mediated these metabolic changes via mitochondrial-specific protein degradation of pyruvate dehydrogenase kinase 4 (PDK4, inhibitor of PDH) by the Lon protease via an NF-κB-dependent mechanism. Using RNA sequencing following siRNA-mediated knockdown of the catalytically active subunit of PDH, PDHE1α (PDHA1 gene), we show that PDH flux controls the transcription of approximately one-third of the genes that are up-regulated by TNFα stimulation. Notably, TNFα-induced PDH flux regulates a unique signature of proinflammatory mediators (cytokines and chemokines) but not inducible adhesion molecules. Metabolomics and ChIP sequencing for acetylated modification on lysine 27 of histone 3 (H3K27ac) showed that TNFα-induced PDH flux promotes histone acetylation of specific gene loci via citrate accumulation and ATP-citrate lyase-mediated generation of acetyl CoA. Together, these results uncover a mechanism by which TNFα signaling increases oxidative TCA flux of glucose to support TNFα-induced gene transcription through extramitochondrial acetyl CoA generation and histone acetylation. [ABSTRACT FROM AUTHOR]

Current knowledge of cancer genomics remains biased against noncoding mutations. To systematically search for regulatory noncoding mutations, we assessed mutations in conserved positions in the genome under the assumption that these are more likely to be functional than mutations in positions with low conservation. To this end, we use whole-genome sequencing data from the International Cancer Genome Consortium and combined it with evolutionary constraint inferred from 240 mammals, to identify genes enriched in noncoding constraint mutations (NCCMs), mutations likely to be regulatory in nature. We compare medulloblastoma (MB), which is malignant, to pilocytic astrocytoma (PA), a primarily benign tumor, and find highly different NCCM frequencies between the two, in agreement with the fact that malignant cancers tend to have more mutations. In PA, a high NCCM frequency only affects the BRAF locus, which is the most commonly mutated gene in PA. In contrast, in MB, >500 genes have high levels of NCCMs. Intriguingly, several loci with NCCMs in MB are associated with different ages of onset, such as the HOXB cluster in young MB patients. In adult patients, NCCMs occurred in, e.g., the WASF-2/AHDC1/FGR locus. One of these NCCMs led to increased expression of the SRC kinase FGR and augmented responsiveness of MB cells to dasatinib, a SRC kinase inhibitor. Our analysis thus points to different molecular pathways in different patient groups. These newly identified putative candidate driver mutations may aid in patient stratification in MB and could be valuable for future selection of personalized treatment options. [ABSTRACT FROM AUTHOR]

Nearly all eukaryotes carry DNA transposons of the Robertson's Mutator (Mu) superfamily, a widespread source of genome instability and genetic variation. Despite their pervasive impact on host genomes, much remains unknown about the evolution of these transposons. Transposase recognition of terminal inverted repeats (TIRs) is thought to drive and constrain coevolution of MuDR transposase genes and TIRs. To address the extent of this relationship and its impact, we compared separate phylogenies of TIRs and MuDR gene sequences from Mu elements in the maize genome. Five major clades were identified. As expected, most Mu elements were bound by highly similar TIRs from the same clade (homomorphic type). However, a subset of elements contained dissimilar TIRs derived from divergent clades. These "heteromorphs" typically occurred in multiple copies indicating active transposition in the genome. In addition, analysis of internal sequences showed that exchanges between elements having divergent TIRs produced new mudra and mudrb gene combinations. In several instances, TIR homomorphs had been regenerated within a heteromorph clade with retention of distinctive internal MuDR sequence combinations. Results reveal that recombination between divergent clades facilitates independent evolution of transposase (mudra), transposase-binding targets (TIRs), and capacity for insertion (mudrb) of active Mu elements. This mechanism would be enhanced by the preference of Mu insertions for recombination-rich regions near the 5' ends of genes. We suggest that cycles of recombination give rise to alternating homo-and heteromorph forms that enhance the diversity on which selection for Mu fitness can operate. [ABSTRACT FROM AUTHOR]

Gene recognition is one of the most important problems in computational molecular biology. Previous attempts to solve this problem were based on statistics, and applications of combinatorial methods for gene recognition were almost unexplored. Recent advances in large-scale cDNA sequencing open a way toward a new approach to gene recognition that uses previously sequenced genes as a clue for recognition of newly sequenced genes. This paper describes a spliced alignment algorithm and software tool that explores all possible exon assemblies in polynomial time and finds the multiexon structure with the best fit to a related protein. Unlike other existing methods, the algorithm successfully recognizes genes even in the case of short exons or exons with unusual codon usage; we also report correct assemblies for genes with more than 10 exons. On a test sample of human genes with known mammalian relatives, the average correlation between the predicted and actual proteins was 99%. The algorithm correctly reconstructed 87% of genes and the rare discrepancies between the predicted and real exon-intron structures were caused either by short (less than 5 amino acids) initial/terminal exons or by alternative splicing. Moreover, the algorithm predicts human genes reasonably well when the homologous protein is nonvertebrate or even prokaryotic. The surprisingly good performance of the method was confirmed by extensive simulations: in particular, with target proteins at 160 accepted point mutations (PAM) (25% similarity), the correlation between the predicted and actual genes was still as high as 95%.

In an earlier paper it was shown that linguistic families of languages spoken by a set of 38 populations associate rather strongly with an evolutionary tree of the same populations derived from genetic data. While the correlation was clearly high, there was no evaluation of statistical significance; no such test was available at the time. This gap has now been filled by adapting to this aim a procedure based on the consistency index, and the level of significance is found to be much stronger than 10(-3). Possible reasons for coevolution of strictly genetic characters and the strictly cultural linguistic system are discussed briefly. Results of this global analysis are compared with those obtained in independent local analysis.

Echolocation, the detection of objects by means of sound waves, has evolved independently in diverse animals. Echolocators include not only mammals such as toothed whales and yangochiropteran and rhinolophoid bats but also Rousettus fruit bats, as well as two bird lineages, oilbirds and swiftlets. In whales and yangochiropteran and rhinolophoid bats, positive selection and molecular convergence has been documented in key hearing-related genes, such as prestin (SLC26A5), but few studies have examined these loci in other echolocators. Here, we examine patterns of selection and convergence in echolocation-related genes in echolocating birds and Rousettus bats. Fewer of these loci were under selection in Rousettus or birds compared with classically recognized echolocators, and elevated convergence (compared to outgroups) was not evident across this gene set. In certain genes, however, we detected convergent substitutions with potential functional relevance, including convergence between Rousettus and classic echolocators in prestin at a site known to affect hair cell electromotility. We also detected convergence between Yangochiroptera, Rhinolophidea, and oilbirds in TMC1, an important mechanosensory transduction channel in vertebrate hair cells, and observed an amino acid change at the same site within the pore domain. Our results suggest that although most proteins implicated in echolocation in specialized mammals may not have been recruited in birds or Rousettus fruit bats, certain hearing-related loci may have undergone convergent functional changes. Investigating adaptations in diverse echolocators will deepen our understanding of this unusual sensory modality. [ABSTRACT FROM AUTHOR]

Arthropod silk is vital to the evolutionary success of hundreds of thousands of species. The primary proteins in silks are often encoded by long, repetitive gene sequences. Until recently, sequencing and assembling these complex gene sequences has proven intractable given their repetitive structure. Here, using high-quality long-read sequencing, we show that there is extensive variation--both in terms of length and repeat motif order--between alleles of silk genes within individual arthropods. Further, this variation exists across two deep, independent origins of silk which diverged more than 500 Mya: the insect clade containing caddisflies and butterflies and spiders. This remarkable convergence in previously overlooked patterns of allelic variation across multiple origins of silk suggests common mechanisms for the generation and maintenance of structural protein-coding genes. Future genomic efforts to connect genotypes to phenotypes should account for such allelic variation. [ABSTRACT FROM AUTHOR]

The evolution of genomes in all life forms involves two distinct, dynamic types of genomic changes: gene duplication (and loss) that shape families of paralogous genes and extension (and contraction) of low-complexity regions (LCR), which occurs through dynamics of short repeats in protein-coding genes. Although the roles of each of these types of events in genome evolution have been studied, their co-evolutionary dynamics is not thoroughly understood. Here, by analyzing a wide range of genomes from diverse bacteria and archaea, we show that LCR and paralogy represent two distinct routes of evolution that are inversely correlated. The emergence of LCR is a prominent evolutionary mechanism in fast evolving, young protein families, whereas paralogy dominates the comparatively slow evolution of old protein families. The analysis of multiple prokaryotic genomes shows that the formation of LCR is likely a widespread, transient evolutionary mechanism that temporally and locally affects also ancestral functions, but apparently, fades away with time, under mutational and selective pressures, yielding to gene paralogy. We propose that compensatory relationships between short-term and longer-term evolutionary mechanisms are universal in the evolution of life. [ABSTRACT FROM AUTHOR]

Coevolution at the gene level, as reflected by correlated events of gene loss or gain, can be revealed by phylogenetic profile analysis. The optimal method and metric for comparing phylogenetic profiles, especially in eukaryotic genomes, are not yet established. Here, we describe a procedure suitable for large-scale analysis, which can reveal coevolution based on the assessment of the statistical significance of correlated presence/absence transitions between gene pairs. This metric can identify coevolution in profiles with low overall similarities and is not affected by similarities lacking coevolutionary information. We applied the procedure to a large collection of 60,912 orthologous gene groups (orthogroups) in 1,264 eukaryotic genomes extracted from OrthoDB. We found significant cotransition scores for 7,825 orthogroups associated in 2,401 coevolving modules linking known and unknown genes in protein complexes and biological pathways. To demonstrate the ability of the method to predict hidden gene associations, we validated through experiments the involvement of vertebrate malate synthase-like genes in the conversion of (S)-ureidoglycolate into glyoxylate and urea, the last step of purine catabolism. This identification explains the presence of glyoxylate cycle genes in metazoa and suggests an anaplerotic role of purine degradation in early eukaryotes. [ABSTRACT FROM AUTHOR]

Global transcriptomic and proteomic analyses of T cells have been rich sources of unbiased data for understanding T-cell activation. Lack of full concordance of these datasets has illustrated that important facets of T-cell activation are controlled at the level of translation. We undertook translatome analysis of CD8 T-cell activation, combining polysome profiling and microarray analysis. We revealed that altering T-cell receptor stimulation influenced recruitment of mRNAs to heavy polysomes and translation of subsets of genes. A major pathway that was compromised, when TCR signaling was suboptimal, was linked to ribosome biogenesis, a rate-limiting factor in both cell growth and proliferation. Defective TCR signaling affected transcription and processing of ribosomal RNA precursors, as well as the translation of specific ribosomal proteins and translation factors. Mechanistically, IL-2 production was compromised in weakly stimulated T cells, affecting the abundance of Myc protein, a known regulator of ribosome biogenesis. Consequently, weakly activated T cells showed impaired production of ribosomes and a failure to maintain proliferative capacity after stimulation. We demonstrate that primary T cells respond to various environmental cues by regulating ribosome biogenesis and mRNA translation at multiple levels to sustain proliferation and differentiation. [ABSTRACT FROM AUTHOR]

The three mammalian TET di oxygenases oxidize the methyl group of 5-methylcytosine in DNA, and the oxidized methylcytosines are essential intermediates in all known pathways of DNA demethylation. To define the in vivo consequences of complete TET deficiency, we inducibly deleted all three Tet genes in the mouse genome. Tet1/2/3-inducible TKO (iTKO) mice succumbed to acute myeloid leukemia (AML) by 4 to 5 wk. Single-cell RNA sequencing of Tet iTKO bone marrow cells revealed the appearance of new myeloid cell populations characterized by a striking increase in expression of all members of the stefin/cystatin gene cluster on mouse chromosome 16. In patients with AML, high stefin/cystatin gene expression correlates with poor clinical outcomes. Increased expression of the clustered stefin/cystatin genes was associated with a heterochromatin-to-euchromatin compartment switch with readthrough transcription downstream of the clustered stefin/cystatin genes as well as other highly expressed genes, but only minor changes in DNA methylation. Our data highlight roles for TET enzymes that are distinct from their established function in DNA demethylation and instead involve increased transcriptional readthrough and changes in three-dimensional genome organization. [ABSTRACT FROM AUTHOR]

Human ETS Related Gene, ERG, a master transcription factor, turns oncogenic upon its out-of-context activation in diverse developmental lineages. However, the mechanism underlying its lineage-specific activation of Notch (N), Wnt, or EZH2—three well-characterized oncogenic targets of ERG—remains elusive. We reasoned that deep homology in genetic tool kits might help uncover such elusive cancer mechanisms in Drosophila. By heterologous gain of human ERG in Drosophila, here we reveal Chip, which codes for a transcriptional coactivator, LIM-domain-binding (LDB) protein, as its novel target. ERG represses Drosophila Chip via its direct binding and, indirectly, via E(z)-mediated silencing of its promoter. Downregulation of Chip disrupts LIM–HD complex formed between Chip and Tailup (Tup)—a LIM–HD transcription factor—in the developing notum. A consequent activation of N-driven Wg signaling leads to notum-to-wing transdetermination. These fallouts of ERG gain are arrested upon a simultaneous gain of Chip, sequestration of Wg ligand, and, alternatively, loss of N signaling or E(z) activity. Finally, we show that the human LDB1, a homolog of Drosophila Chip, is repressed in ERG-positive prostate cancer cells. Besides identifying an elusive target of human ERG, our study unravels an underpinning of its lineage-specific carcinogenesis. [ABSTRACT FROM AUTHOR]

A growing body of work has addressed human adaptations to diverse environments using genomic data, but few studies have connected putatively selected alleles to phenotypes, much less among underrepresented populations such as Amerindians. Studies of natural selection and genotype-phenotype relationships in underrepresented populations hold potential to uncover previously undescribed loci underlying evolutionarily and biomedically relevant traits. Here, we worked with the Tsimane and the Moseten, two Amerindian populations inhabiting the Bolivian lowlands. We focused most intensively on the Tsimane, because long-term anthropological work with this group has shown that they have a high burden of both macro and microparasites, as well as minimal cardiometabolic disease or dementia. We therefore generated genome-wide genotype data for Tsimane individuals to study natural selection, and paired this with blood mRNA-seq as well as cardiometabolic and immune biomarker data generated from a larger sample that included both populations. In the Tsimane, we identified 21 regions that are candidates for selective sweeps, as well as 5 immune traits that show evidence for polygenic selection (e.g., C-reactive protein levels and the response to coronaviruses). Genes overlapping candidate regions were strongly enriched for known involvement in immune-related traits, such as abundance of lymphocytes and eosinophils. Importantly, we were also able to draw on extensive phenotype information for the Tsimane and Moseten and link five regions (containing PSD4, MUC21 and MUC22, TOX2, ANXA6, and ABCA1) with biomarkers of immune and metabolic function. Together, our work highlights the utility of pairing evolutionary analyses with anthropological and biomedical data to gain insight into the genetic basis of health-related traits. [ABSTRACT FROM AUTHOR]

Understanding gene functions in marine invertebrates has been limited, largely due to the lack of suitable assay systems. Such a system requires investigative methods that are reproducible and can be quantitatively evaluated, such as a cell line, and a strong promoter that can drive high expression of a transgene. In this study, we established primary cell culture from a marine bivalve mollusc, Mizuhopecten yessoensis. Using scallop primary cells, we optimized electroporation conditions for transfection and carried out a luciferase-based promoter activity assay to identify strong promoter sequences that can drive expression of a gene of interest. We evaluated potential promoter sequences from genes of endogenous and exogenous origin and discovered a strong viral promoter derived from a bivalve-infectious virus, ostreid herpesvirus-1 (OsHV-1). This promoter, we termed OsHV-1 promoter, showed 24.7-fold and 16.1-fold higher activity than the cytomegalovirus immediate early (CMV IE) promoter and the endogenous EF1α promoter, the two most commonly used promoters in bivalves so far. Our GFP assays showed that the OsHV-1 promoter is active not only in scallop cells but also in HEK293 cells and zebrafish embryos. The OsHV-1 promoter practically enables functional analysis of marine molluscan genes, which can contribute to unveiling generegulatory networks underlying astonishing regeneration, adaptation, reproduction, and aging in marine invertebrates. [ABSTRACT FROM AUTHOR]