Mixed anhydrides at the intersection between peptide and RNA autocatalytic sets: evolution of biological coding.

We present a scenario for the origin of biological coding, a semiotic relationship between chemical information stored in one location that links to chemical information stored in a separate location. Coding originated from cooperation between two, originally separate, collectively autocatalytic sets (CASs), one for nucleic acids and one for peptides. Upon interaction, a series of RNA folding-directed processes led to their joint cooperativity. The aminoacyl adenylate was the first covalent association made by these two CASs and solidified their interdependence, and is a palimpsest of this era, a relic of the original semiotic relationship between RNA and proteins. Coding was driven by selection pressure to eliminate waste in CASs. Eventually a 1 : 1 relationship between single amino acids and short RNA pieces was established, i.e. the 'genetic code'. The two classes of aaRS enzymes are remnants of the complementary information in two RNA strands, as postulated by Rodin and Ohno. Every stage in the evolution of coding was driven by the downward selection on the components of a system to satisfy the Kantian whole. Coding was engendered because there were two chemically distinct classes of polymers needed for open-ended evolution; systems with only one polymer cannot exhibit this characteristic. Coding is thus synonymous with life as we know it.

Molecular, clinical and neuropsychological study in 31 patients with Kabuki syndrome and KMT2D mutations.

Kabuki syndrome (KS-OMIM 147920) is a rare developmental disease characterized by the association of multiple congenital anomalies and intellectual disability. This study aimed to investigate intellectual performance in children with KS and link the performance to several clinical features and molecular data. We recruited 31 children with KMT2D mutations who were 6 to 16 years old. They all completed the Weschler Intelligence Scale for Children, fourth edition. We calculated all indexes: the Full Scale Intellectual Quotient (FSIQ), Verbal Comprehension Index (VCI), Perceptive Reasoning Index (PRI), Processing Speed Index (PSI), and Working Memory Index (WMI). In addition, molecular data and several clinical symptoms were studied. FSIQ and VCI scores were 10 points lower for patients with a truncating mutation than other types of mutations. In addition, scores for FSIQ, VCI and PRI were lower for children with visual impairment than normal vision. We also identified a discrepancy in indexes characterized by high WMI and VCI and low PRI and PSI. We emphasize the importance of early identification and intensive care of visual disorders in patients with KS and recommend individual assessment of intellectual profile.

The molecular underpinnings of genetic phenomena.

Epiphenomena are those processes that ostensibly have no precedent at lower levels of scientific organization. In this review, it is argued that many genetic processes, including ploidy, dominance, heritability, pleiotropy, epistasis, mutational load and recombination, all are at least analogous to biochemical events that were requisite features of the RNA world. Most, if not all, of these features of contemporary whole organisms and populations may have their ultimate evolutionary roots in the chemical repertoire of catalytic RNAs. Some of these phenomena will eventually prove to be not only analogous but homologous to ribozyme activities.

The elucidation of novel SH2 binding sites on PLD2.

Our laboratory has recently reported that the enzyme phospholipase D2 (PLD2) exists as a ternary complex with PTP1b and the growth factor receptor bound protein 2 (Grb2). Here, we establish the mechanistic underpinnings of the PLD2/Grb2 association. We have identified residues Y(169) and Y(179) in the PLD2 protein as being essential for the Grb2 interaction. We present evidence indicating that Y(169) and Y(179) are located within two consensus sites in PLD2 that mediate an SH2 interaction with Grb2. This was demonstrated with an SH2-deficient GSTGrb2 R86K mutant that failed to pull-down PLD2 in vitro. In order to elucidate the functions of the two neighboring tyrosines, we created a new class of deletion and point mutants in PLD2. Phenylalanine replacement of Y(169) (PLD2 Y169F) or Y(179) (PLD2 Y179F) reduced Grb2 binding while simultaneous mutation completely abolished it. The role of the two binding sites on PLD2 was found to be functionally nonequivalent: Y(169) serves to modulate the activity of the enzyme, whereas Y(179) regulates total tyrosine phosphorylation of the protein. Interestingly, binding of Grb2 to PLD2 occurs irrespectively of lipase activity, since Grb2 binds to catalytically inactive PLD2 mutants. Finally, PLD2 residues Y(169) and Y(179) are necessary for the recruitment of Sos, but only overexpression of the PLD2 Y179F mutant resulted in increased Ras activity, p44/42(Erk) phosphorylation and enhanced DNA synthesis. Since Y(169) remains able to modulate enzyme activity and is capable of binding to Grb2 in the PLD2 Y179F mutant, we propose that Y(169) is kept under negative regulation by Y(179). When this is released, Y(169) mediates cellular proliferation through the Ras/MAPK pathway.

Conservation of MHC class II DOA sequences among carnivores.

We obtained the nucleotide sequence for most of the major histocompatibility complex (MHC) class II DOA locus for Weddell, leopard, northern elephant, and southern elephant seals and from the coyote and compared them to all known DOA data available to date. We found generally low levels of interspecific polymorphisms, providing further support for stabilizing selection acting on the DOA locus. This suggests that DO gene products play a substantial functional role in the regulation of antigen presentation. A seven-amino-acid motif of VWRLPEF was found to be conserved across all DOA sequences and may be a DO-specific recognition element.

Recessive dystrophic epidermolysis bullosa associated with mesangioproliferative glomerulonephritis and multifocal necrotizing leucoencephalopathy of the pons.

We report a woman with recessive dystrophic epidermolysis bullosa (RDEB) in whom there was prolonged sepsis and death at age 22 years. Autopsy revealed multiple epidermolytic skin lesions with chronic ulceration, mesangioproliferative glomerulonephritis and multifocal necrotizing leucoencephalopathy (MNL) of the pons. The latter two conditions may have been mediated by sepsis-associated cytokines. Although mesangioproliferative glomerulonephritis has previously been described in association with RDEB, to our knowledge this is the first report of MNL in a patient with RDEB.

Genetic consequences of a severe population bottleneck in the Guadalupe fur seal (Arctocephalus townsendi).

Population bottlenecks may lead to diminished genetic variability and correlative effects on fitness. The Guadalupe fur seal was nearly exterminated by commercial sealers during the late 18th and early 19th centuries. To determine the genetic consequences of this population bottleneck, we compared the variation at a 181 bp section of the mitochondrial DNA (mtDNA) control region from the bones of 26 prebottleneck fur seals versus variation in the extant population. We found 25 different mtDNA genotypes in the prebottleneck fur seals and only 7 genotypes among 32 extant fur seals, including only one of the ancient genotypes. These data demonstrate a substantial loss of genetic variability correlating with the recent population bottleneck. We also found from several genetic measures that the prehistoric population of Guadalupe fur seals was robust and that it had been increasing at some time during the late prehistoric period. Continued recovery of this species may, however, owe more to more immediate nongenetic factors, such as poaching and local availability of food resources during the breeding season and consequent effects on pup survival, than on the reduced genetic variability.

Major histocompatibility complex class II DOA sequences from three Antarctic seal species verify stabilizing selection on the DO locus.

To provide additional support for the sequence conservation and hence the regulatory role of the MHC class II DOA locus, we obtained the nucleotide sequences of exon 2 and exon 3, along with the intervening intron, of the Ross seal, and sequences from the exon 2 region from the Weddell and leopard seals. These are the first reports of the sequences of this locus from a carnivore species. The results demonstrate strong conservation among mammals for the exon sequence and produce a gene genealogy that is consistent in topology with a species tree.

Holoprosencephaly associated with an apparent isolated 2q37.1-->2q37.3 deletion.

A female infant survived 5(1/2) hours after delivery at 33 weeks gestation. Autopsy showed a lobar variant of holoprosencephaly (HPE). Cytogenetic analysis revealed a 2q37.1-->2q37.3 deletion. This case represents the fourth reported case of HPE associated with partial monosomy 2q37 and the first with an apparent isolated 2q37 deletion. Chromosome segment 2q37.1-->2q37.3 may harbor yet another locus important in forebrain development, which, when disrupted, can lead to brain malformations within the HPE spectrum.

Molecular evolution: Please release me, genetic code.

The genetic code is no longer universal, even in non-mitochondrial genomes. Recent studies have implicated the eukaryotic release factor eRF1 in mediating coding changes that are not as inconceivable as once thought. Specific residues in eRF1 proteins can be correlated with specific code changes in a wide variety of taxa.

An empirical genetic assessment of the severity of the northern elephant seal population bottleneck.

A bottleneck in population size of a species is often correlated with a sharp reduction in genetic variation. The northern elephant seal (Mirounga angustirostris) has undergone at least one extreme bottleneck, having rebounded from 20-100 individuals a century ago to over 175,000 individuals today. The relative lack of molecular-genetic variation in contemporary populations has been documented, but the extent of variation before the late 19th century remains unknown. We have determined the nucleotide sequence of a 179 base-pair segment of the mitochondrial DNA (mtDNA) control region from seals that lived before, during and after a bottleneck low in 1892. A 'primerless' PCR was used to improve the recovery of information from older samples. Only two mtDNA genotypes were present in all 150+ seals from the 1892 bottleneck on, but we discovered four genotypes in five pre-bottleneck seals. This suggests a much greater amount of mtDNA genotypic variation before this bottleneck, and that the persistence of two genotypes today is a consequence of random lineage sampling. We cannot correlate the loss of mtDNA genotypes with a lowered mean fitness of individuals in the species today. However, we show that the species historically possessed additional genotypes to those present now, and that sampling of ancient DNA could elucidate the genetic consequences of severe reductions in population size.

Multi-locus genetic evidence for rapid ecologically based speciation in Daphnia.

The process of speciation involves the divergence of two or more subpopulations of a parent species into independent evolutionary trajectories. To study this process in natural populations requires a detailed knowledge of the genetic and ecological characteristics of the parent species and an understanding of how its populations can lose evolutionary cohesion. The cosmopolitan and speciose genus Daphnia provides many of these features by existing in multiple freshwater habitat types, particularly permanent lakes and temporary ponds, each of which presents distinct ecological challenges. We assayed the genetic composition of 20 temporary pond populations of members of the Daphnia pulex species complex in north-western Oregon and compared them to published data on related lake and pond populations. We collected molecular genetic data from 13 allozyme loci, from six microsatellite loci, and from the control region of the mitochondrial DNA. By assaying over 400 individual Daphnia for these data, we were able to compile composite genotypes not only of individual Daphnia but of each pond population as a whole. In these ponds, we discovered two distinct genotypic constellations, one which bears resemblance to the lake-dwelling taxon D. pulicaria, and one which bears resemblance to the pond-dwelling taxon, D. pulex. Using published genetic data from these and other species as a frame of reference, we characterized 13 of these ponds as being 'pond-like', three as being 'lake-like', and four as being 'mixed'. Unlike studies performed elsewhere, however, these ponds do not exhibit high probabilities of interspecific hybridization. Over 95% of all individuals have either a lake-like or a pond-like genotype at all three genetic systems, suggesting the two forms do not represent hybridized vs. nonhybridized genotypes. Because both types can be found in the same ponds at the same time in gametic disequilibrium, we also discount the possibility that they are two extremes of a single species that is highly genetically subdivided. With these genetic data, and with supporting life-history and ecological data previously gathered on these pond populations, we conclude that the most likely description of this system is of a taxon caught in the act of speciating, with new pond-adapted populations periodically stemming from lake-adapted sources during river flooding events.

The genotypic landscape during in vitro evolution of a catalytic RNA: implications for phenotypic buffering.

The Tetrahymena group I ribozyme catalyzes the cleavage of a phosphodiester linkage in specific sequences of RNA. This phenotype can be used in an in vitro selection-amplification process to evolve variants that are capable of RNA catalysis in the presence of Ca(2+) as the sole available cation. With sufficient genotypic characterization of the population as it evolves, we have a rare opportunity of observing how the information stored in an evolving population responds to selective pressures, such as the requisite of catalyzing RNA cleavage in the absence of Mg(2+) or Mn(2+). In the present work, we examine the population dynamics of this system using sequence information from previous experimental work. We focus on two issues: How does the information content of the population evolve? and Is the system evolving as an adaptive walk on a rugged landscape? To investigate these questions, information theoretical parameters are examined. The evolution of the population is visualized by mapping the genotypic frequency distribution onto a two-dimensional projection of sequence space. The projection was generated using Hamming distances from the wild-type, starting sequence and a catalytically successful, evolved sequence. The evolution of the information content of the system was measured by calculating the grammar complexity of the observed sequences, which showed a very slight increase over 12 generations. This result is consistent with the system performing a search for a local optimum. The dynamics of the population in this sequence space is consistent with an adaptive walk on an uncorrelated, or "rugged," genotypic landscape, despite the observation that the phenotypic progress of the population appears smooth. The relative insensitivity of the phenotypic landscape to the variegation of the genotypic landscape suggests that the former is buffered against variation in the latter through various epigenetic-like mechanisms.

Genetic differentiation among Oregon lake populations of the Daphnia pulex species complex.

Gene flow among invertebrate populations inhabiting bodies of nonflowing freshwater such as ponds or lakes must at some stage involve transport across habitat unsuitable for adult stages. Consequently the potential for interpopulational differentiation is high in these species, yet empirical studies of lake populations of Cladocerans such as Daphnia have failed to reveal high levels of genetic distinctiveness among populations and have led to much speculation about how these populations exchange genes and remain cohesive evolutionary units. In this study we surveyed 42 Oregon lake populations of Daphnia from the D. pulex species complex for genetic variation within the mitochondrial DNA control region. We have used this data to test the relative abilities of various ecological factors to explain the observed patterns in genetic differentiation among lakes. Despite limited genetic variation detected among our samples--11 very similar RFLP-defined mtDNA genotypes from 388 individuals--analyses of nucleotide variance using analogs to Wright's F statistics indicate that when multilake populations are defined in terms of the river drainage basin to which they belong, strong and significant amounts of among-population genetic variation can be detected at this locus (F(ST) estimates between 0.5 and 0.6). In contrast, we fail to detect consistent significant among-population variation when populations are defined on the basis of regional physical geography, bird migratory flyways, or lake trophic status. The manner in which the data are compiled, that is, whether RFLPs or nucleotide sequences are used, has little effect on the overall conclusions, yet it is clear that nucleotide sequence data would lower the standard errors of F(ST) estimates. We propose that periodic widescale flooding during the late Pleistocene may be an important mechanism to homogenize genetic differences among lake Daphnia continent-wide south of the southern-most extent of Pleistocene glaciation.

Modulation of RTX cytotoxicity by thymidine and dipyridamole in vitro: implications for chemotherapy.

PURPOSE: The cytotoxicity of the thymidylate synthase (TS) inhibitor raltitrexed (RTX) is reversed by supraphysiologic thymidine concentrations. Dipyridamole (DP) blocks thymidine salvage (uptake) and potentiates several chemotherapeutic agents in vitro. The purpose of this study was to determine whether DP is capable of potentiating RTX cytotoxicity in the presence of physiologic thymidine concentrations. METHODS: We examined the effect of DP on RTX cytotoxicity in the presence of various physiologic thymidine concentrations in eight colorectal adenocarcinoma cell lines by colony formation assay, and in p53-defective HL60 S and p53-competent HL60 SN3 promyelocytic leukemia cells by a tetrazolium reduction-based assay. RESULTS: In WiDr colon adenocarcinoma cells the cytotoxicity of 1.0 microM RTX (4 h) varied from 0% to >99% within the reported range of human serum thymidine concentrations from 500 to <50 nM, respectively. DP potentiated RTX cytotoxicity 2- to >93-fold within this range. Free DP concentrations of 10 to 20 nM, achievable with oral dosing, potentiated RTX at thymidine concentrations up to 100 nM. Potentiation at higher physiologic thymidine concentrations of >/=200 nM required DP concentrations of at least 50 nM, or about twice the steady-state DP concentration obtainable from oral and intravenous dosing, but well within that obtainable by intraperitoneal infusion. Maximal potentiation required 72 to 120 h of DP exposure. DP also potentiated RTX in the absence of exogenous thymidine in six of eight colon cell lines and HL60 S and SN3 cells suggesting a second, nonthymidine salvage-dependent mechanism of potentiation. CONCLUSIONS: Clinically achievable free DP concentrations potentiated RTX cytotoxicity within the range of physiologic serum thymidine concentrations. RTX/DP or DP analogue-based combination therapy should, therefore, be considered for clinical trial. Serum or tumor thymidine concentration determinations may aid in identification of patients likely to respond to TS and nucleoside salvage inhibitors versus alternate, non-TS-directed therapies.

Toxic optic neuropathy after concomitant use of melatonin, zoloft, and a high-protein diet.

Melatonin is a neuromodulating hormone found in the pineal gland and retina. It is involved in light-dark circadian rhythms and mediates retinal processes in a manner antagonistic to that of dopamine. Zoloft (sertraline) is an antidepressant drug that blocks the reuptake of serotonin at the neural synapse. Serotonin is the natural precursor of melatonin. A 42-year-old woman sought treatment for visual acuity loss, dyschromatopsia, and altered light adaptation. Neuro-ophthalmologic examination was otherwise normal except for evolving bilateral cecocentral scotomas. She had taken Zoloft for 4 years and began a high-protein diet with melatonin supplementation 2 weeks before onset of visual symptoms. Visual acuity and color vision improved within 2 months after melatonin and the high-protein diet were discontinued. Combined use of melatonin, Zoloft, and a high-protein diet may have resulted in melatonin/dopamine imbalance in the retina, manifesting as a toxic optic neuropathy. Physicians and patients should be alerted to this potential drug interaction.

Non-unity molecular heritability demonstrated by continuous evolution in vitro.

INTRODUCTION: When catalytic RNA is evolved in vitro, the molecule's chemical reactivity is usually the desired selection target. Sometimes the phenotype of a particular RNA molecule cannot be unambiguously determined from its genotype, however. This can occur if a nucleotide sequence can adopt multiple folded states, an example of non-unity heritability (i.e. one genotype gives rise to more than one phenotype). In these cases, more rounds of selection are required to achieve a phenotypic shift. We tested the influence of non-unity heritability at the molecular level by selecting for variants of a ligase ribozyme via continuous evolution. RESULTS: During 20 bursts of continuous evolution of a 152-nucleotide ligase ribozyme in which the Mg2+ concentration was periodically lowered, a nine-error variant of the starting 'wild-type' molecule became dominant in the last eight bursts. This variant appears to be more active than the wild type. Kinetic analyses of the mutant suggest that it may not possess a higher first-order catalytic rate constant, however. Examination of the multiple RNA conformations present under the continuous evolution conditions suggests that the mutant is superior to the wild type because it is less likely to misfold into inactive conformers. CONCLUSIONS: The evolution of genotypes that are more likely to exhibit a particular phenotype is an epiphenomenon usually ascribed only to complex living systems. We show that this can occur at the molecular level, demonstrating that in vitro systems may have more life-like characteristics than previously thought, and providing additional support for an RNA world.

Conservation biology: genes are not enough.

A study of correlated genotypic and phenotypic changes over a 2400-year period in a cave population of pocket gophers bolsters the idea that small, isolated populations can not only persist in a fluctuating environment, but may be able to adapt without genetic input from elsewhere.

1H MRS in acute traumatic brain injury.

The objective of this study was to demonstrate 1H MR spectroscopy (MRS) changes in cerebral metabolites after acute head trauma. Twenty-five patients (12 children, 13 adults) were examined with quantitative 1H MRS after closed head injury. Clinical grade (Glasgow Coma Scale [GCS]) and outcome (Rancho Los Amigos Medical Center Outcome Score [ROS]) were correlated with quantitative neurochemical findings. N-acetylaspartate (NAA), a neuronal and axonal marker, was reduced (P < .03-.001). In children, a reduced NAA/creatine plus phosphocreatine (Cr) level and the presence of detectable lipid/lactate predicted bad outcome (sensitivity, 89%; specificity, 89%). The first MRS examination of all patients correlated with ROS versus NAA (r = .65, P < .0001). Although most patients showed MRS abnormalities, striking heterogeneity of 1H MRS characterized the individual patients. 1H MRS identifies multiple patterns of diffuse brain injury after blunt head trauma. There was a strong correlation between MRS and outcome. Future prospective studies will be needed to determine the clinical usefulness of MRS in predicting outcome from closed head injury.