Discovery of Red-Shifting Mutations in Firefly Luciferase Using High-Throughput Biochemistry.

Photinus pyralis luciferase (FLuc) has proven a valuable tool for bioluminescence imaging, but much of the light emitted from the native enzyme is absorbed by endogenous biomolecules. Thus, luciferases displaying red-shifted emission enable higher resolution during deep-tissue imaging. A robust model of how protein structure determines emission color would greatly aid the engineering of red-shifted mutants, but no consensus has been reached to date. In this work, we applied deep mutational scanning to systematically assess 20 functionally important amino acid positions on FLuc for red-shifting mutations, predicting that an unbiased approach would enable novel contributions to this debate. We report dozens of red-shifting mutations as a result, a large majority of which have not been previously identified. Further characterization revealed that mutations N229T and T352M, in particular, bring about unimodal emission with the majority of photons being >600 nm. The red-shifting mutations identified by this high-throughput approach provide strong biochemical evidence for the multiple-emitter mechanism of color determination and point to the importance of a water network in the enzyme binding pocket for altering the emitter ratio. This work provides a broadly applicable mutational data set tying FLuc structure to emission color that contributes to our mechanistic understanding of emission color determination and should facilitate further engineering of improved probes for deep-tissue imaging.

Novel population genetics in ciliates due to life cycle and nuclear dimorphism.

Our understanding of population genetics comes primarily from studies of organisms with canonical life cycles and nuclear organization, either haploid or diploid, sexual, or asexual. Although this template yields satisfactory results for the study of animals and plants, the wide variety of genomic organizations and life cycles of unicellular eukaryotes can make these organisms behave differently in response to mutation, selection, and drift than predicted by traditional population genetic models. In this study, we show how each of these unique features of ciliates affects their evolutionary parameters in mutation-selection, selection-drift, and mutation-selection-drift situations. In general, ciliates are less efficient in eliminating deleterious mutations-these mutations linger longer and at higher frequencies in ciliate populations than in sexual populations--and more efficient in selecting beneficial mutations. Approaching this problem via analytical techniques and simulation allows us to make specific predictions about the nature of ciliate evolution, and we discuss the implications of these results with respect to the high levels of polymorphism and high rate of protein evolution reported for ciliates.

Ambushing the Ambush Hypothesis: predicting and evaluating off-frame codon frequencies in prokaryotic genomes.

BACKGROUND: In this paper, we address the evidence for the Ambush Hypothesis. Proposed by Seligmann and Pollock, this hypothesis posits that there exists a selection for off-frame stop codons (OSCs) to counteract the possible deleterious effects of translational frameshifts, including the waste of resources and potential cytotoxicity. Two main types of study have been used to support the hypothesis. Some studies analyzed codon usage and showed that codons with more potential to create OSCs seem to be favored over codons with lower potential; they used this finding to support the Ambush Hypothesis. Another study used 342 bacterial genomes to evaluate the hypothesis directly, finding significant excesses of OSCs in these genomes. RESULTS: We repeated both analyses with newer datasets and searched for other factors that could explain the observed trends. In the first case, the relative frequency of codons with the potential to create OSCs is directly correlated with the GC content of organisms, as stop codons are GC-poor. When evaluating the frequency of OSCs directly in 1,976 bacterial genomes we also detected a significant excess. However, when comparing the excess of OSCs with similarly obtained results for the frequency of out-of-frame sense codons, some sense codons have a more significant excess than stop codons. CONCLUSIONS: Two avenues of study have been used to support the Ambush Hypothesis. Using the same methods as these previous studies, we demonstrate that the evidence in support of the Ambush Hypothesis does not hold up against more rigorous testing.

An alternative look at code evolution: using non-canonical codes to evaluate adaptive and historic models for the origin of the genetic code.

The canonical code has been shown many times to be highly robust against point mutations; that is, mutations that change a single nucleotide tend to result in similar amino acids more often than expected by chance. There are two major types of models for the origin of the code, which explain how this sophisticated structure evolved. Adaptive models state that the primitive code was specifically selected for error minimization, while historic models hypothesize that the robustness of the code is an artifact or by-product of the mechanism of code evolution. In this paper, we evaluated the levels of robustness in existing non-canonical codes as well as codes that differ in only one codon assignment from the standard code. We found that the level of robustness of many of these codes is comparable or better than that of the standard code. Although these results do not preclude an adaptive origin of the genetic code, they suggest that the code was not selected for minimizing the effects of point mutations.

1+1 = 3: a fusion of 2 enzymes in the methionine salvage pathway of Tetrahymena thermophila creates a trifunctional enzyme that catalyzes 3 steps in the pathway.

The methionine salvage pathway is responsible for regenerating methionine from its derivative, methylthioadenosine. The complete set of enzymes of the methionine pathway has been previously described in bacteria. Despite its importance, the pathway has only been fully described in one eukaryotic organism, yeast. Here we use a computational approach to identify the enzymes of the methionine salvage pathway in another eukaryote, Tetrahymena thermophila. In this organism, the pathway has two fused genes, MTNAK and MTNBD. Each of these fusions involves two different genes whose products catalyze two different single steps of the pathway in other organisms. One of the fusion proteins, mtnBD, is formed by enzymes that catalyze non-consecutive steps in the pathway, mtnB and mtnD. Interestingly the gene that codes for the intervening enzyme in the pathway, mtnC, is missing from the genome of Tetrahymena. We used complementation tests in yeast to show that the fusion of mtnB and mtnD from Tetrahymena is able to do in one step what yeast does in three, since it can rescue yeast knockouts of mtnB, mtnC, or mtnD. Fusion genes have proved to be very useful in aiding phylogenetic reconstructions and in the functional characterization of genes. Our results highlight another characteristic of fusion proteins, namely that these proteins can serve as biochemical shortcuts, allowing organisms to completely bypass steps in biochemical pathways.

Aminoacyl-tRNA synthetases in human health and disease.

The Aminoacyl-tRNA Synthetases (aaRSs) are an evolutionarily ancient family of enzymes that catalyze the esterification reaction linking a transfer RNA (tRNA) with its cognate amino acid matching the anticodon triplet of the tRNA. Proper functioning of the aaRSs to create aminoacylated (or "charged") tRNAs is required for efficient and accurate protein synthesis. Beyond their basic canonical function in protein biosynthesis, aaRSs have a surprisingly diverse array of non-canonical functions that are actively being defined. The human genome contains 37 genes that encode unique aaRS proteins. To date, 56 human genetic diseases caused by damaging variants in aaRS genes have been described: 46 are autosomal recessive biallelic disorders and 10 are autosomal dominant monoallelic disorders. Our appreciation of human diseases caused by damaging genetic variants in the aaRSs has been greatly accelerated by the advent of next-generation sequencing, with 89% of these gene discoveries made since 2010. In addition to these genetic disorders of the aaRSs, anti-synthetase syndrome (ASSD) is a rare autoimmune inflammatory myopathy that involves the production of autoantibodies that disrupt aaRS proteins. This review provides an overview of the basic biology of aaRS proteins and describes the rapidly growing list of human diseases known to be caused by genetic variants or autoimmune targeting that affect both the canonical and non-canonical functions of these essential proteins.

Soil bacterial assemblage responses to wildfire in low elevation southern California habitats.

Understanding how wildfires and modification in plant assemblages interact to influence soil bacteria assemblages is a crucial step in understanding how these disturbances may influence ecosystem structure and function. Here, we resampled soil from three study sites previously surveyed in spring 2016 and 2017 and compared soil bacterial assemblages prior to and six months after (spring 2019) the 2018 Woolsey Fire in the Santa Monica Mountain National Recreation Area using Illumina sequencing of the 16S rRNA gene. All sites harbored both native California sage scrub and a non-native (grassland or forbland) habitat, allowing us to examine how fire influenced bacterial assemblages in common southern California habitats. Most results contrasted with our a-priori hypotheses: (1) richness and diversity increased following the fire, (2) heat/drought resistant and sensitive bacteria did not show consistent and differing patterns by increasing and decreasing, respectively, in relative abundance after the fire, and (3) bacterial assemblage structure was only minimally impacted by fire, with no differences being found between 2017 (pre-fire) and 2019 (post-fire) in three of the six habitats sampled. As sage scrub and non-native grasslands consistently harbored unique bacterial assemblages both before and following the fire, modifications in plant compositions will likely have legacy effects on these soils that persist even after a fire. Combined, our results demonstrate that bacterial assemblages in southern California habitats are minimally affected by fire. Because direct impacts of fire are limited, but indirect impacts, e.g., modifications in plant compositions, are significant, plant restoration efforts following a fire should strive to revegetate sage scrub areas to prevent legacy changes in bacterial composition.

Detection of fused genes in eukaryotic genomes using gene deFuser: analysis of the Tetrahymena thermophila genome.

BACKGROUND: Fused genes are important sources of data for studies of evolution and protein function. To date no service has been made available online to aid in the large-scale identification of fused genes in sequenced genomes. We have developed a program, Gene deFuser, that analyzes uploaded protein sequence files for characteristics of gene fusion events and presents the results in a convenient web interface. RESULTS: To test the ability of this software to detect fusions on a genome-wide scale, we analyzed the 24,725 gene models predicted for the ciliated protozoan Tetrahymena thermophila. Gene deFuser detected members of eight of the nine families of gene fusions known or predicted in this species and identified nineteen new families of fused genes, each containing between one and twelve members. In addition to these genuine fusions, Gene deFuser also detected a particular type of gene misannotation, in which two independent genes were predicted as a single transcript by gene annotation tools. Twenty-nine of the artifacts detected by Gene deFuser in the initial annotation have been corrected in subsequent versions, with a total of 25 annotation artifacts (about 1/3 of the total fusions identified) remaining in the most recent annotation. CONCLUSIONS: The newly identified Tetrahymena fusions belong to classes of genes involved in processes such as phospholipid synthesis, nuclear export, and surface antigen generation. These results highlight the potential of Gene deFuser to reveal a large number of novel fused genes in evolutionarily isolated organisms. Gene deFuser may also prove useful as an ancillary tool for detecting fusion artifacts during gene model annotation.

Consequences of stop codon reassignment on protein evolution in ciliates with alternative genetic codes.

Tetrahymena thermophila and Paramecium tetraurelia are ciliates that reassign TAA and TAG from stop codons to glutamine codons. Because of the lack of full genome sequences, few studies have concentrated on analyzing the effects of codon reassignment in protein evolution. We used the recently sequenced genome of these species to analyze the patterns of amino acid substitution in ciliates that reassign the code. We show that, as expected, the codon reassignment has a large impact on amino acid substitutions in closely related proteins; however, contrary to expectations, these effects also hold for very diverged proteins. Previous studies have used amino acid substitution data to calculate the minimization of the genetic code; our results show that because of the lasting influence of the code in the patterns of substitution, such studies are tautological. These different substitution patterns might affect alignment of ciliate proteins, as alignment programs use scoring matrices based on substitution patterns of organisms that use the standard code. We also show that glutamine is used more frequently in ciliates than in other species, as often as expected based on the presence of the 2 new reassigned codons, indicating that the frequencies of amino acids in proteomes is mostly determined by neutral processes based on their number of codons.

Tandem stop codons in ciliates that reassign stop codons.

Tandem stop codons are extra stop codons hypothesized to be present downstream of genes to act as a backup in case of read-through of the real stop codon. Although seemingly absent from Escherichia coli, recent studies have confirmed the presence of such codons in yeast. In this paper we will analyze the genomes of two ciliate species--Paramecium tetraurelia and Tetrahymena thermophila--that reassign the stop codons TAA and TAG to glutamine, for the presence of tandem stop codons. We show that there are more tandem stop codons downstream of both Paramecium and Tetrahymena genes than expected by chance given the base composition of the downstream regions. This excess of tandem stop codons is larger in Tetrahymena and Paramecium than in yeast. We propose that this might be caused by a higher frequency of stop codon read-through in these species than in yeast, possibly because of a leaky termination machinery resulting from stop codon reassignment.

Selection for tandem stop codons in ciliate species with reassigned stop codons.

The failure of mRNA translation machinery to recognize a stop codon as a termination signal and subsequent translation of the 3' untranslated region (UTR) is referred to as stop codon readthrough, the frequency of which is related to the length, composition, and structure of mRNA sequences downstream of end-of-gene stop codons. Secondary in-frame stop codons within a few positions downstream of the primary stop codons, so-called tandem stop codons (TSCs), serve as backup termination signals, which limit the effects of readthrough: polypeptide product degradation, mislocalization, and aggregation. In this study, ciliate species with UAA and UAG stop codons reassigned to code for glutamine are found to possess statistical excesses of TSCs at the beginning of their 3' UTRs. The overrepresentation of TSCs in these species is greater than that observed in standard code organisms. Though the overall numbers of TSCs are lower in most species with alternative stop codons because they use fewer than three unique stop codons, the relatively great overrepresentation of TSCs in alternative-code ciliate species suggests that there exist stronger selective pressures to maintain TSCs in these organisms compared to standard code organisms.

Patterns of codon usage in two ciliates that reassign the genetic code: Tetrahymena thermophila and Paramecium tetraurelia.

We used the recently sequenced genomes of the ciliates Tetrahymena thermophila and Paramecium tetraurelia to analyze the codon usage patterns in both organisms; we have analyzed codon usage bias, Gln codon usage, GC content and the nucleotide contexts of initiation and termination codons in Tetrahymena and Paramecium. We also studied how these trends change along the length of the genes and in a subset of highly expressed genes. Our results corroborate some of the trends previously described in Tetrahymena, but also negate some specific observations. In both genomes we found a strong bias toward codons with low GC content; however, in highly expressed genes this bias is smaller and codons ending in GC tend to be more frequent. We also found that codon bias increases along gene segments and in highly expressed genes and that the context surrounding initiation and termination codons are always AT rich. Our results also suggest differences in the efficiency of translation of the reassigned stop codons between the two species and between the reassigned codons. Finally, we discuss some of the possible causes for such translational efficiency differences.

MDS_IES_DB: a database of macronuclear and micronuclear genes in spirotrichous ciliates.

Ciliated protozoa have two kinds of nuclei: Macronuclei (MAC) and Micronuclei (MIC). In some ciliate classes, such as spirotrichs, most genes undergo several layers of DNA rearrangement during macronuclear development. Because of such processes, these organisms provide ideal systems for studying mechanisms of recombination and gene rearrangement. Here, we describe a database that contains all spirotrich genes for which both MAC and MIC versions are sequenced, with consistent annotation and easy access to all the features. An interface to query the database is available at http://oxytricha.princeton.edu/dimorphism/database.htm.

Amitotic chromosome loss predicts distinct patterns of senescence and non-senescence in ciliates.

Over time and repeated asexual divisions, many ciliate species display the characteristics of senescence, reduced fecundity and increased mortality. Their only path to recovery is sexual conjugation or autogamy. While more traditional models of cellular aging have been proposed, one of the most accepted explanations relies on the faulty mechanism by which ciliates duplicate their somatic nucleus, a process referred to as amitosis. Amitosis involves the random segregation of chromosomes with no consideration for homology. Over subsequent divisions, chromosome copy numbers will fluctuate until an entire chromosome is lost, resulting in death. Via simulations of this process, we find that senescence and death via chromosome loss is not the only possible result of amitosis. Random chromosome loss is less damaging to populations than previously thought, and strict adherence to the model predicts that Paramecium tetraurelia would not senesce. A combination of the reciprocal nature of amitosis and lethal selection against low-copy number chromosomes is responsible for this startling prediction. Additionally, our results provide an alternate explanation to recent evidence for selection on chromosome copy number in Tetrahymena thermophila and peculiar patterns of senescence in Tetrahymena pyriformis.

Sequence features of Oxytricha trifallax (class Spirotrichea) macronuclear telomeric and subtelomeric sequences.

We sequenced and analyzed the subtelomeric regions of 1356 macronuclear "nanochromosomes" of the spirotrichous ciliate Oxytricha trifallax. We show that the telomeres in this species have a length of 20 nt, with minor deviations; there is no correlation between telomere lengths at the two ends of the molecule. A search for open reading frames revealed that the 3' and 5' untranslated regions are short, with a median length of approximately 130 nt, and that surprisingly there are no detectable differences between sequences upstream and downstream of genes. Our results confirm a previously reported purine bias in the first approximately 80 nucleotides of the subtelomeric regions, but with this larger data set we curiously detected a 10 bp periodicity in the bias; we relate this finding to the possible regulatory and structural functions these regions must serve. Palindromic sequences in opposing subtelomeric regions, although present in most sequences, are not statistically significant.

A model for the evolution of extremely fragmented macronuclei in ciliates.

While all ciliates possess nuclear dimorphism, several ciliates - like those in the classes Phyllopharyngea, Spirotrichea, and Armophorea - have an extreme macronuclear organization. Their extensively fragmented macronuclei contain upwards of 20,000 chromosomes, each with upwards of thousands of copies. These features have evolved independently on multiple occasions throughout ciliate evolutionary history, and currently no models explain these structures in an evolutionary context. In this paper, we propose that competition between two forces - the limitation and avoidance of chromosomal imbalances as a ciliate undergoes successive asexual divisions, and the costs of replicating massive genomes - is sufficient to explain this particular nuclear structure. We present a simulation of ciliate cell evolution under control of these forces, allowing certain features of the population to change over time. Over a wide range of parameters, we observe the repeated emergence of this unusual genomic organization found in nature. Although much remains to be understood about the evolution of macronuclear genome organization, our results show that the proposed model is a plausible explanation for the emergence of these extremely fragmented, highly polyploid genomes.