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An orally administered, fixed‐dose coformulation of sodium phenylbutyrate‐taurursodiol (PB‐TURSO) significantly slowed functional decline in a randomized, placebo‐controlled, phase 2 trial in ALS (CENTAUR). Herein we report results of a long‐term survival analysis of participants in CENTAUR. In CENTAUR, adults with ALS were randomized 2:1 to PB‐TURSO or placebo. Participants completing the 6‐month (24‐week) randomized phase were eligible to receive PB‐TURSO in the open‐label extension. An all‐cause mortality analysis (35‐month maximum follow‐up post‐randomization) incorporated all randomized participants. Participants and site investigators were blinded to treatment assignments through the duration of follow‐up of this analysis. Vital status was obtained for 135 of 137 participants originally randomized in CENTAUR. Median overall survival was 25.0 months among participants originally randomized to PB‐TURSO and 18.5 months among those originally randomized to placebo (hazard ratio, 0.56; 95% confidence interval, 0.34‐0.92; P = .023). Initiation of PB‐TURSO treatment at baseline resulted in a 6.5‐month longer median survival as compared with placebo. Combined with results from CENTAUR, these results suggest that PB‐TURSO has both functional and survival benefits in ALS.

BackgroundCoformulated sodium phenylbutyrate/taurursodiol (PB/TURSO) was shown to prolong survival and slow functional decline in amyotrophic lateral sclerosis (ALS).ObjectiveDetermine whether PB/TURSO prolonged tracheostomy/ventilation-free survival and/or reduced first hospitalisation in participants with ALS in the CENTAUR trial.MethodsAdults with El Escorial Definite ALS ≤18 months from symptom onset were randomised to PB/ TURSO or placebo for 6 months. Those completing randomised treatment could enrol in an open-label extension (OLE) phase and receive PB/TURSO for ≤30 months. Times to the following individual or combined key events were compared in the originally randomised treatment groups over a period spanning trial start through July 2020 (longest postrandomisation follow-up, 35 months): death, tracheostomy, permanent assisted ventilation (PAV) and first hospitalisation.ResultsRisk of any key event was 47% lower in those originally randomised to PB/TURSO (n=87) versus placebo (n=48, 71% of whom received delayed-start PB/TURSO in the OLE phase) (HR=0.53; 95% CI 0.35 to 0.81; p=0.003). Risks of death or tracheostomy/PAV (HR=0.51; 95% CI 0.32 to 0.84; p=0.007) and first hospitalisation (HR=0.56; 95% CI 0.34 to 0.95; p=0.03) were also decreased in those originally randomised to PB/TURSO.ConclusionsEarly PB/TURSO prolonged tracheostomy/PAV-free survival and delayed first hospitalisation in ALS.Trial registration numberNCT03127514;NCT03488524.

BACKGROUND: Sodium phenylbutyrate and taurursodiol have been found to reduce neuronal death in experimental models. The efficacy and safety of a combination of the two compounds in persons with amyotrophic lateral sclerosis (ALS) are not known. METHODS: In this multicenter, randomized, double-blind trial, we enrolled participants with definite ALS who had had an onset of symptoms within the previous 18 months. Participants were randomly assigned in a 2:1 ratio to receive sodium phenylbutyrate–taurursodiol (3 g of sodium phenylbutyrate and 1 g of taurursodiol, administered once a day for 3 weeks and then twice a day) or placebo. The primary outcome was the rate of decline in the total score on the Amyotrophic Lateral Sclerosis Functional Rating Scale–Revised (ALSFRS-R; range, 0 to 48, with higher scores indicating better function) through 24 weeks. Secondary outcomes were the rates of decline in isometric muscle strength, plasma phosphorylated axonal neurofilament H subunit levels, and the slow vital capacity; the time to death, tracheostomy, or permanent ventilation; and the time to death, tracheostomy, permanent ventilation, or hospitalization. RESULTS: A total of 177 persons with ALS were screened for eligibility, and 137 were randomly assigned to receive sodium phenylbutyrate–taurursodiol (89 participants) or placebo (48 participants). In a modified intention-to-treat analysis, the mean rate of change in the ALSFRS-R score was −1.24 points per month with the active drug and −1.66 points per month with placebo (difference, 0.42 points per month; 95% confidence interval, 0.03 to 0.81; P = 0.03). Secondary outcomes did not differ significantly between the two groups. Adverse events with the active drug were mainly gastrointestinal. CONCLUSIONS: Sodium phenylbutyrate–taurursodiol resulted in slower functional decline than placebo as measured by the ALSFRS-R score over a period of 24 weeks. Secondary outcomes were not significantly different between the two groups. Longer and larger trials are necessary to evaluate the efficacy and safety of sodium phenylbutyrate–taurursodiol in persons with ALS. (Funded by Amylyx Pharmaceuticals and others; CENTAUR Clinicaltrials.gov number, NCT03127514.)

This review commemorates the 40th anniversary of DNA sequencing, a period in which we have already witnessed multiple technological revolutions and a growth in scale from a few kilobases to the first human genome, and now to millions of human and a myriad of other genomes. DNA sequencing has been extensively and creatively repurposed, including as a 'counter' for a vast range of molecular phenomena. We predict that in the long view of history, the impact of DNA sequencing will be on a par with that of the microscope.

In this Review, the year of publication of reference 54 should be 2005, not 2015. In Box 2, "1982: GenBank ( https://www.ncbi.nlm.nih.gov/genbank/statistics/ )" should read "1982: Genbank/ENA/DDBJ" and "2007: NCBI Short Read Archive" should read "2007: NCBI and ENA Short Read Archives"; this is because the launches of these American, European and Japanese databases were coordinated. These errors have not been corrected.

We study the intron–exon structures of 684 groups of orthologs from seven diverse eukaryotic genomes and provide maximum likelihood estimates for rates and numbers of intron losses and gains in these same genes for a variety of lineages. Rates of intron loss vary from ≈2 × 10 –9 to 2 × 10 –10 per year. Rates of gain vary from 6 × 10 –13 to 4 × 10 –12 per possible intron insertion site per year. There is an inverse correspondence between rates of intron loss and gain, leading to a 20-fold variation among lineages in the ratio of the rates of the two processes. The observed rates of intron gain are insufficient to explain the large number of introns estimated to have been present in the plant–animal ancestor, suggesting that introns present in early eukaryotes may have been created by a fundamentally different process than more recently gained introns.

The relationship between three biologically important groups, arthropods, nematodes, and deuterostomes, remains unresolved. It is unknown whether arthropods are more closely related to nematodes (consistent with the “ecdysozoa” hypothesis) or to deuterostomes (consistent with “coelomata”). We present a method in which we use the pattern of spliceosomal intron conservation to develop a series of inequalities that characterize each possible relationship. We find that only the ecdysozoa grouping satisfies these predictions, with P < 10 –6 . Simulations show that our method, unlike some previous methods, is largely insensitive to rate variation between branches.

We use the pattern of intron conservation in 684 groups of orthologs from seven fully sequenced eukaryotic genomes to provide maximum likelihood estimates of the number of introns present in the same orthologs in various eukaryotic ancestors. We find: ( i ) intron density in the plant–animal ancestor was high, perhaps two-thirds that of humans and three times that of Drosophila ; and ( ii ) intron density in the ancestral bilateran was also high, equaling that of humans and four times that of Drosophila . We further find that modern introns are generally very old, with two-thirds of modern bilateran introns dating to the ancestral bilateran and two-fifths of modern plant, animal, and fungus introns dating to the plant–animal ancestor. Intron losses outnumber gains over a large range of eukaryotic lineages. These results show that early eukaryotic gene structures were very complex, and that simplification, not embellishment, has dominated subsequent evolution.

We studied intron loss in 684 groups of orthologous genes from seven fully sequenced eukaryotic genomes. We found that introns closer to the 3′ ends of genes are preferentially lost, as predicted if introns are lost through gene conversion with a reverse transcriptase product of a spliced mRNA. Adjacent introns tend to be lost in concert, as expected if such events span multiple intron positions. Directly contrary to the expectations of some, introns that do not interrupt codons (phase zero) are more, not less, likely to be lost, an intriguing and previously unappreciated result. Adjacent introns with matching phases are not more likely to be retained, as would be expected if they enjoyed a relative selective advantage. The findings of 3′ and phase zero intron loss biases are in direct contradiction to an extremely recent study of fungi intron evolution. All patterns are less pronounced in the lineage leading to Caenorhabditis elegans , suggesting that the process of intron loss may be qualitatively different in nematodes. Our results support a reverse transcriptase-mediated model of intron loss.

We compared intron–exon structures in 1,560 human–mouse orthologs and 360 mouse–rat orthologs. The origin of differences in intron positions between species was inferred by comparison with an outgroup, Fugu for human–mouse and human for mouse–rat. Among 10,020 intron positions in the human–mouse comparison, we found unequivocal evidence for five independent intron losses in the mouse lineage but no evidence for intron loss in humans or for intron gain in either lineage. Among 1,459 positions in rat–mouse comparisons, we found evidence for one loss in rat but neither loss in mouse nor gain in either lineage. In each case, the intron losses were exact, without change in the surrounding coding sequence, and involved introns that are extremely short, with an average of 200 bp, an order of magnitude shorter than the mammalian average. These results favor a model whereby introns are lost through gene conversion with intronless copies of the gene. In addition, the finding of widespread conservation of intron–exon structure, even over large evolutionary distances, suggests that comparative methods employing information about gene structures should be very successful in correctly predicting exon boundaries in genomic sequences.

For nearly 15 years, it has been widely believed that many introns were recently acquired by the genes of multicellular organisms. However, the mechanism of acquisition has yet to be described for a single animal intron. Here, we report a large-scale computational analysis of the human, Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis thaliana genomes. We divided 147,796 human intron sequences into batches of similar lengths and aligned them with each other. Different types of homologies between introns were found, but none showed evidence of simple intron transposition. Also, 106,902 plant, 39,624 Drosophila, and 6021 C. elegans introns were examined. No single case of homologous introns in nonhomologous genes was detected. Thus, we found no example of transposition of introns in the last 50 million years in humans, in 3 million years in Drosophila and C. elegans, or in 5 million years in Arabidopsis. Either new introns do not arise via transposition of other introns or intron transposition must have occurred so early in evolution that all traces of homology have been lost.

We study the distribution of exons in eukaryotic genes to determine whether one can detect the reuse of exon sequences and to use the frequency of such reuse to estimate how many ancestral exon sequences there might have been. We use two databases of exons. One contained 56,276 internal exons from putatively unrelated genes (less than 20% sequence identity) and the second contained 8917 internal exons from regions of these genes that are homologous and colinear with prokaryotic genes; these are ancient conserved regions (ACRs). At the 95% significance level we find 3500 exon-sequence matches in the large database and 500 matches in the ACR database. These matches correspond to groups of similar sequences. The size-rank relationship for these groups follows a power law, the size falling off as the inverse square root of the rank. This form of the power law distribution leads us to make an estimate for the size of a possible universe of ancestral exons. Using the data corresponding to the ACR regions, that universe is estimated to be about 15,000-30,000 in size.

We purge large databases of animal, plant, and fungal intron-containing genes to a 20% similarity level and then identify the most similar animal-plant, animal-fungal, and plant-fungal protein pairs. We identify the introns in each BLAST 2.0 alignment and score matched intron positions and slid (near-matched, within six nucleotides) intron positions automatically. Overall we find that 10% of the animal introns match plant positions, and a further 7% are "slides." Fifteen percent of fungal introns match animal positions, and 13% match plant positions. Furthermore, the number of alignments with high numbers of matches deviates greatly from the Poisson expectation. The 30 animal-plant alignments with the highest matches (for which 44% of animal introns match plant positions) when aligned with fungal genes are also highly enriched for triple matches: 39% of the fungal introns match both animal and plant positions. This is strong evidence for ancestral introns predating the animal-plant-fungal divergence, and in complete opposition to any expectations based on random insertion. In examining the slid introns, we show that at least half are caused by imperfections in the alignments, and are most likely to be actual matches at common positions. Thus, our final estimates are that approximately equal 14% of animal introns match plant positions, and that approximately equal 17-18% of fungal introns match animal or plant positions, all of these being likely to be ancestral in the eukaryotes.

In ancient genes whose products have known 3-dimensional structures, an excess of phase zero introns (those that lie between the codons) appear in the boundaries of modules, compact regions of the polypeptide chain. These excesses are highly significant and could support the hypothesis that ancient genes were assembled by exon shuffling involving compact modules. (Phase one and two introns, and many phase zero introns, appear to arise later.) However, as more genes, with larger numbers of homologs and intron positions, were examined, the effects became smaller, dropping from a 40% excess to an 8% excess as the number of intron positions increased from 570 to 3,328, even though the statistical significance remained strong. An interpretation of this behavior is that novel inserted positions appearing in homologs washed out the signal from a finite number of ancient positions. Here we show that this is likely to be the case. Analyses of intron positions restricted to those in genes for which relatively few intron positions from homologs are known, or to those in genes with a small number of known homologous gene structures, show a significant correlation of phase zero intron positions with the module structure, which weakens as the density of attributed intron positions or the number of homologs increases. These effects do not appear for phase one and phase two introns. This finding matches the expectation of the mixed model of intron origin, in which a fraction of phase zero introns are left from the assembly of the first genes, while other introns have been added in the course of evolution.

Background Charcot-Marie-Tooth type 1A disease (CMT1A) is a rare orphan inherited neuropathy caused by an autosomal dominant duplication of a gene encoding for the structural myelin protein PMP22, which induces abnormal Schwann cell differentiation and dysmyelination, eventually leading to axonal suffering then loss and muscle wasting. We favour the idea that diseases can be more efficiently treated when targeting multiple disease-relevant pathways. In CMT1A patients, we therefore tested the potential of PXT3003, a low-dose combination of three already approved compounds (baclofen, naltrexone and sorbitol). Our study conceptually builds on preclinical experiments highlighting a pleiotropic mechanism of action that includes downregulation of PMP22. The primary objective was to assess safety and tolerability of PXT3003. The secondary objective aimed at an exploratory analysis of efficacy of PXT3003 in CMT1A, to be used for designing next clinical development stages (Phase 2b/3). Methods 80 adult patients with mild-to-moderate CMT1A received in double-blind for 1 year Placebo or one of the three increasing doses of PXT3003 tested, in four equal groups. Safety and tolerability were assessed with the incidence of related adverse events. Efficacy was assessed using the Charcot-Marie-Tooth Neuropathy Score (CMTNS) and the Overall Neuropathy Limitations Scale (ONLS) as main endpoints, as well as various clinical and electrophysiological outcomes. Results This trial confirmed the safety and tolerability of PXT3003. The highest dose (HD) showed consistent evidence of improvement beyond stabilization. CMTNS and ONLS, with a significant improvement of respectively of 8% (0.4% - 16.2%) and 12.1% (2% - 23.2%) in the HD group versus the pool of all other groups, appear to be the most sensitive clinical endpoints to treatment despite their quasi-stability over one year under Placebo. Patients who did not deteriorate over one year were significantly more frequent in the HD group. Conclusions These results confirm that PXT3003 deserves further investigation in adults and could greatly benefit CMT1A-diagnosed children, usually less affected than adults. Trial registration EudraCT Number: 2010-023097-40. ClinicalTrials.gov Identifier: NCT01401257. The Committee for Orphan Medicinal Products issued in February 2014 a positive opinion on the application for orphan designation for PXT3003 (EMA/OD/193/13). Electronic supplementary material The online version of this article (doi:10.1186/s13023-014-0199-0) contains supplementary material, which is available to authorized users.

Do introns delineate elements of protein tertiary structure? This issue is crucial to the debate about the role and origin of introns. We present an analysis of the full set of proteins with known three-dimensional structures that have homologs with intron positions recorded in GenBank. A computer program was generated that maps on a reference sequence the positions of all introns in homologous genes. We have applied this program to a set of 665 nonredundant protein sequences with defined three-dimensional structures in the Protein Data Bank (PDB), which yielded 8,217 introns in 407 proteins. For the subset of proteins corresponding to ancient conserved regions (ACR), we find that there is a correlation of phase-zero introns with the boundary regions of modules and no correlation for the phase-one and phase-two positions. However, for a subset of proteins without prokaryotic counterparts (131 non-ACR proteins), a set of presumably modern proteins (or proteins that have diverged extremely far from any ancestral form), we do not find any correlation of phase-zero intron positions with three-dimensional structure. Furthermore, we find an anticorrelation of phase-one intron positions with module boundaries: they actually have a preference for the interior of modules. This finding is explicable as a preference for phase-one introns to lie in glycines, between G|G sequences, the preference for glycines being anticorrelated with the three-dimensional modules. We interpret this anticorrelation as a sign that a number of phase-one introns, and hence many modern introns, have been inserted into G|G “protosplice” sequences.

To aid studies of molecular evolution and to assist in gene prediction research, we have constructed an Exon-Intron Database (EID) in FASTA format. Currently, the database is derived from GenBank release 112, and it contains 51 289 protein-coding genes (287 209 exons) that harbor introns, along with extensive descriptions of each gene and its DNA and protein sequences, as well as splice motif information. There is 17% redundancy inherited from GenBank-a purge at the 99% identity level reduced the database to 42 460 genes (243 589 exons). We have created subdatabases of genes whose intron positions have been experimentally determined. One such database, constructed by comparing genomic and mRNA sequences, contains 11 242 genes (62 474 exons). A larger database of 22 196 genes (105 595 exons) was constructed by selecting on keywords to eliminate computer-predicted genes. By examining the two nucleotides adjacent to the intron boundary, we infer that there is a 2% rate of errors or other deviations from the standard GTellipsisAG motif in nuclear genes. This criterion can be used to eliminate 4921 genes from the overall database. Various tools are provided to enable generation of user-specific subsets of the EID. The EID distribution can be obtained from http://mcb.harvard.edu/gilbert/EID

We test the hypothesis that introns were used to construct the first genes from small exons, whose protein products represent compact elements of structure. For any three-dimensional structure, a computer program analyzes the structure into a set of modules, segments of the polypeptide chain bounded in space by a maximum diameter, separated by a set of ‘boundary regions’. The ‘boundary regions’ are such that if the gene were divided by an intron in each ‘boundary region’, the protein would be divided into modules less than the specified diameter. Using a set of 32 ancient proteins, which have no introns in prokaryotes, we examine the intron positions in their eukaryotic homologs and show that the introns are correlated with modules of diameter 21, 28 and 33 A, with P values below 0.001.

We have identified and characterized a novel protein from adult zebrafish retina, which we named ES1. Database search revealed that the ES1 gene has significant similarity to two genes with unknown functions: theEscherichia colisigma cross-reacting protein 27a (scrp27a) and the human KNP-I/GT335.In situhybridization and immunohistochemistry experiments showed that both ES1 mRNA and protein are expressed specifically in adult photoreceptor cells. ES1 seems to be a cytoplasmic protein. An ES1-like antigen was also detected in photoreceptor cells of goldfish with anti-ES1 antibodies. The retina specific expression and the evolutionary conservation suggest that ES1 protein may be important for maintaining normal retina structure and function.

Establishment of the vertebrate body plan requires a variety of signaling molecules. In a search for tyrosine kinases expressed in early zebrafish embryos, a model system for the study of vertebrate development, we discovered Jak1 kinase to be maternally encoded and the mRNA evenly distributed among the cells of blastula-stage embryos. Injection of RNA-encoding dominant-negative Jak1 kinases reduces a specific cell migration, epiboly, and results in the reduction of goosecoid expression and of anterior structures. This work establishes that, in addition to its role in signal transduction of cytokines in adult tissues, Jak1 kinase has a role in early vertebrate development.

We have identified a new family of Tc1-like transposons in the zebrafish, Danio rerio. The sequence of a candidate active transposon, deduced from sample Tzf elements, shows limited resemblance to the previously described Tdr1 elements of zebrafish. Both the Tzf and the Tdr elements are extremely abundant in zebrafish. We describe here a general strategy for detecting transposition events in a complex genome and demonstrate its utility by selectively monitoring hundreds of potentially active Tzf copies in the zebrafish genome against a background of other related elements. We have followed members of a zebrafish pedigree, using this two-dimensional transposon display strategy, to identify the first examples of active transposition of such elements in vertebrates.

Since most of the examples of "exon shuffling" are between vertebrate genes, the view is often expressed that exon shuffling is limited to the evolutionarily recent lineage of vertebrates. Although exon shuffling in plants has been inferred from the analysis of intron phases of plant genes [Long, M., Rosenberg, C. & Gilbert, W. (1995) Proc. Natl. Acad. Sci. USA 92, 12495-12499] and from the comparison of two functionally unknown sunflower genes [Domon, C. & Steinmetz, A. (1994) Mol. Gen. Genet. 244, 312-317], clear cases of exon shuffling in plant genes remain to be uncovered. Here, we report an example of exon shuffling in two important nucleus-encoded plant genes: cytosolic glyceraldehyde-3-phosphate dehydrogenase (cytosolic GAPDH or GapC) and cytochrome c1 precursor. The intron-exon structures of the shuffled region indicate that the shuffling event took place at the DNA sequence level. In this case, we can establish a donor-recipient relationship for the exon shuffling. Three amino terminal exons of GapC have been donated to cytochrome c1, where, in a new protein environment, they serve as a source of the mitochondrial targeting function. This finding throws light upon an old important but unsolved question in gene evolution: the origin of presequences or transit peptides that generally exist in nucleus-encoded organelle genes.

Two issues in the evolution of the intron/exon structure of genes are the role of exon shuffling and the origin of introns. Using a large data base of eukaryotic intron-containing genes, we have found that there are correlations between intron phases leading to an excess of symmetric exons and symmetric exon sets. We interpret these excesses as manifestations of exon shuffling and make a conservative estimate that at least 19% of the exons in the data base were involved in exon shuffling, suggesting an important role for exon shuffling in evolution. Furthermore, these excesses of symmetric exons appear also in those regions of eukaryotic genes that are homologous to prokaryotic genes: the ancient conserved regions. This last fact cannot be explained in terms of the insertional theory of introns but rather supports the concept that some of the introns were ancient, the exon theory of genes.

RNA editing by extensive uridine addition and deletion creates over 90% of the amino acid codons in the cytochrome-c oxidase subunit III (COIII) transcript in Trypanosoma brucei and Herpetomonas, whereas editing of the COIII transcripts in Leishmania tarentolae and Crithidia fasciculata generates only 6% of the amino acid codons and is limited to the 5' ends. Is extensive RNA editing a primitive or derived character? We constructed a phylogenetic tree based on nuclear small-subunit and mitochondrial large- and small-subunit ribosomal RNA sequences for nine species of kinetoplastid protozoa. Our results suggest that extensive editing is a primitive genetic phenomenon that has disappeared in recent evolutionary time and also that there have been multiple losses of the digenetic lifestyle by loss of the vertebrate host in parasite evolution.

We discuss some of the arguments for introns arising early or late in evolution. We outline the exon theory of genes and discuss the series of discoveries of introns in the gene (TPI) encoding triosephosphate isomerase (TPI) that have filled out a series of better fits to the Go plot, culminating in the 1986 prediction of an intron position that was finally discovered in 1992. We present a statistical argument that the 11-intron structure of TPI (based on attributing all of the introns to an ancestral gene and interpreting three cases of very close intron positions as examples of sliding) has a clear relationship to the protein structure. The exons of this 11-intron TPI are a better approximation to Mitiko Go's modules (Go, 1981) than are 99.9% of all alternative exon patterns corresponding to 11 introns placed randomly in the gene, and better than 96% of all alternative patterns in which the lengths of the exons are preserved while the introns are moved. We combine four tests relating exons to protein structure: (i) whether the exons are compact modules, (ii) whether the exons contain most of the close contacts in the protein, (iii) whether the exon configuration maximized buried surface area along the backbone, and (iv) whether the exons maximize their content of hydrogen bonds. On a joint measure for these tests, the native exon structure with 11 introns fits these tests better than 99.4% of all alternative structures obtained by permuting the exon lengths and intron positions.(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied partially edited molecules for the cytochrome-c oxidase subunit III (COIII) transcript from two species of the insect trypanosome Herpetomonas. We found unexpected patterns of editing, in which editing does not proceed strictly 3' to 5', in 24 of 61 partially edited clones. A comparison of the partially edited molecules between the two kinetoplastid species revealed an 8- to 10-nt shift in precisely defined editing boundaries, sites at which editing pauses before binding of the next guide RNA after formation of a stable duplex between a guide RNA and mRNA. This suggests that the region of base pairing between individual guide RNAs and the COIII transcript is not strictly conserved in kinetoplastids, implying gradual evolution of the editing process.

If genes have been assembled from exon subunits, the frequency with which exons are reused leads to an estimate of the size of the underlying exon universe. An exon database was constructed from available protein sequences, and homologous exons were identified on the basis of amino acid identity; statistically significant matches were determined by Monte Carlo methods. It is estimated that only 1000 to 7000 exons were needed to construct all proteins.

Single-stranded complex guanine-rich DNA sequences from chromosomal telomeres and elsewhere can associate to form stable parallel four-stranded structures termed G4-DNA by a process that is anomalously dependent on the particular alkali metal cation that is present. The anomaly, which is not found in the formation of G4-DNA by oligonucleotides containing short, single runs of three or more guanines, is caused by potassium cations excessively stabilizing fold-back intermediate structures, or pathway by-products.

We study the distribution of exons in eukaryotic genes to determine whether one can detect the reuse of exon sequences and to use the frequency of such reuse to estimate how many ancestral exon sequences there might have been. We use two databases of exons. One contained 56,276 internal exons from putatively unrelated genes (less than 20% sequence identity) and the second contained 8917 internal exons from regions of these genes that are homologous and colinear with prokaryotic genes; these are ancient conserved regions (ACRs). At the 95% significance level we find 3500 exon-sequence matches in the large database and 500 matches in the ACR database. These matches correspond to groups of similar sequences. The size-rank relationship for these groups follows a power law, the size falling off as the inverse square root of the rank. This form of the power law distribution leads us to make an estimate for the size of a possible universe of ancestral exons. Using the data corresponding to the ACR regions, that universe is estimated to be about 15,000-30,000 in size.

The hypothesis that some (but not all) introns were used to construct ancient genes by exon shuffling of modules at the earliest stages of evolution is supported by the finding of an excess of phase-zero intron positions in the boundary regions of such modules in 276 ancient proteins (defined as common to eukaryotes and prokaryotes). Here we show further that as phase-zero intron positions are shared by distant taxa, and thus are truly phylogenetically ancient, their excess in the boundaries becomes greater, rising to an 80% excess if shared by four out of the five taxa: vertebrates, invertebrates, fungi, plants, and protists.