Biomedical subjects

Niles Lehman

Publications and source records attributed to Niles Lehman.

12 recordsLinked to original sources

PubMed2006

Accumulation of deleterious mutations in small abiotic populations of RNA.

The accumulation of slightly deleterious mutations in populations leads to the buildup of a genetic load and can cause the extinction of populations of small size. Mutation-accumulation experiments have been used to study this process in a wide variety of organisms, yet the exact mutational underpinnings of genetic loads and their fitness consequences remain poorly characterized. Here, we use an abiotic system of RNA populations evolving continuously in vitro to examine the molecular events that can instigate a genetic load. By tracking the fitness decline of ligase ribozyme populations with bottleneck sizes between 100 and 3000 molecules, we detected the appearance and subsequent fixation of both slightly deleterious mutations and advantageous mutations. Smaller populations went extinct in significantly fewer generations than did larger ones, supporting the notion of a mutational meltdown. These data suggest that mutation accumulation was an important evolutionary force in the prebiotic RNA world and that mechanisms such as recombination to ameliorate genetic loads may have been in place early in the history of life.

Base Sequence↗

PubMed2006

Pinniped phylogeny and a new hypothesis for their origin and dispersal.

The relationships and the zoogeography of the three extant pinniped families, Otariidae (sea lions and fur seals), Odobenidae (one extant species, the walrus), and Phocidae (true seals), have been contentious. Here, we address these topics in a molecular study that includes all extant species of true seals and sea lions, four fur seals and the walrus. Contrary to prevailing morphological views the analyses conclusively showed monophyletic Pinnipedia with a basal split between Otarioidea (Otariidae+Odobenidae) and Phocidae. The northern fur seal was the sister to all remaining otariids and neither sea lions nor arctocephaline fur seals were recognized as monophyletic entities. The basal Phocidae split between Monachinae (monk seals and southern true seals) and Phocinae (northern true seals) was strongly supported. The phylogeny of the Phocinae suggests that the ancestors of Cystophora (hooded seal) and the Phocini (e.g. harp seal, ringed seal) adapted to Arctic conditions and ice-breeding before 12 MYA (million years ago) as supported by the white natal coat of these lineages. The origin of the endemic Caspian and Baikal seals was dated well before the onset of major Pleistocene glaciations. The current findings, together with recent advances in pinniped paleontology, allow the proposal of a new hypothesis for pinniped origin and early dispersal. The hypothesis posits that pinnipeds originated on the North American continent with early otarioid and otariid divergences taking place in the northeast Pacific and those of the phocids in coastal areas of southeast N America for later dispersal to colder environments in the N Atlantic and the Arctic Basin, and in Antarctic waters.

Animals↗

PubMed2006

Calcium(II)-dependent catalytic activity of the Azoarcus ribozyme: testing the limits of resolution for in vitro selection.

Group I intron ribozymes isolated from natural sources have a strict dependence on the divalent metal cations Mg(II) or Mn(II) for catalytic activity. However, mutant versions of the Tetrahymena ribozyme have been previously isolated in the laboratory that show demonstrable activity in 10 mM CaCl(2) as the only supplied salt. Here, we sought to discover similar variants of another group I intron that is likely more evolutionarily specialized. We used in vitro selection to isolate a Ca(II)-dependent variant of the naturally-occurring form of the Azoarcus ribozyme, which is half the size of the Tetrahymena ribozyme and possesses an extremely high G+C content of 71%. A mutation of G to A at position 118 was selected in multiple independent trials. Activity of the mutant is very poor in Ca(II) and can only be observed after RT-PCR, highlighting the power of in vitro selection to isolate molecules with rare and low-level activities. The mutation likely confers an alternate but rare folded conformation that permits accommodation of Ca(II) ions and catalysis. This work also serves to caution that although a selection may be successful, isolates may not be catalytically proficient enough to provide useful levels of activity.

Amino Acid Substitution↗

PubMed2006

Self-assembly of a group I intron from inactive oligonucleotide fragments.

The Azoarcus group I ribozyme was broken into four fragments, 39-63 nucleotides long, that can self-assemble into covalently contiguous ribozymes via RNA-directed recombination events. The fragments have no activity individually yet can cooperate through base pairing and tertiary interactions to produce stable trans complexes at 48 degrees C. These complexes can then catalyze a sequence of energy-neutral recombination reactions utilizing other oligomers as substrates, assembling covalent versions of the ribozyme. Up to 17% of the original fragments are converted into approximately 200 nucleotide products in 8 hr. Assembly occurs primarily by only one of many possible pathways, and the reaction is driven in the correct and forward direction by the burial of key base-pairing regions in stems after recombination. Autocatalysis, and hence self-replication, is inferred by a reaction rate increase upon doping the reaction with full-length RNA.

Azoarcus↗

PubMed2006

Detection of high levels of recombination generated during PCR amplification of RNA templates.

Recombination during the PCR amplification of DNA templates can be a serious problem for those seeking to genotype heterogeneous populations, yet a boon to those seeking to enhance variation during in vitro evolution. Here, the extent to which PCR generates chimeric full-length products was estimated using a powerful restriction fragment-length polymorphism (RFLP) assay involving the use of fluorescently labeled PCR primers. Three different RNA-encoding DNA templates were assayed: (i) one for a group I ribozyme, (ii) one for a 16S ribosomal RNA (rRNA), and (iii) one for a messenger RNA (mRNA). In all cases, the observed frequency of chimeric PCR products exceeded 20%, and longer templates appear to produce more chimeric products. Although two of these templates have the potential to form secondary structures during the PCR, this tendency does not seem to heighten recombination frequency. These results corroborate previous studies that show that the production of chimeras can be best attenuated to a certain extent by varying the extension times in PCR.

In Situ Hybridization, Fluorescence↗

PubMed2005

RNA-directed construction of structurally complex and active ligase ribozymes through recombination.

RNA-directed recombination can be used to catalyze a disproportionation reaction among small RNA substrates to create new combinations of sequences. But the accommodation of secondary and tertiary structural constraints in the substrates by recombinase ribozymes has not been explored. Here, we show that the Azoarcus group I intron can recombine oligoribonucleotides to construct class I ligase ribozymes, which are catalytically active upon synthesis. The substrate oligonucleotides, ranging in size from 58 to 104 nucleotides (nt), along with the 152-nt ligase ribozymes they reconstitute, can contain significant amounts of secondary structure. However, substrate recognition by the Azoarcus ribozyme depends on the existence of a single accessible CAU triplet for effective recombination. A biphasic temperature reaction profile was designed such that the sequential recombination/ligation events could take place in a thermocycler without human intervention. A temperature-dependent pH shift of the reaction buffer contributes to the success of the net reaction. When the substrate for the ligase ribozyme is introduced into the reaction mixture, as much as 11% can be observed being converted to product by the recombined ligase in the same reaction vessel. Recombination followed by ligation can also occur under isothermal conditions at 37 degrees C. Tertiary structure formation of the ligase upon construction can provide some protection from cleavage by the Azoarcus ribozyme when compared to the constituent substrates. These data suggest that RNA-directed recombination can, in fact, articulate complex ribozymes, and that there are logical rules that can guide the optimal placement of the CAU recognition sequence.

Adenosine Monophosphate↗

PubMed2005

Recombination during in vitro evolution.

Recombination, the swapping of large portions of genetic information between and among parental genotypes, can be applied to in vitro evolution experiments on functional nucleic acids. Both homologous and heterologous recombination can be achieved using standard laboratory techniques. In many cases, recombination can allow for the discovery of a ribozyme or DNAzyme phenotype that would not likely be encountered by reliance on point mutations alone. In addition, recombination can often aid in the discovery of global optima in sequence space and/or lessen the number of generations it would take to reach optima. Recombination is most efficiently used in combination with point mutations and applied after the first couple of rounds of selection but before high-fitness genotypes dominate the selection. The "recombination zone" describes that region of sequence space-defined by the residues that will ultimately participate in the function of the winning nucleic acid(s)-where recombination is expected to be the most beneficial in the search for high-fitness genotypes.

Cell Nucleus↗

PubMed2004

Major histocompatibility complex variation at three class II loci in the northern elephant seal.

Northern elephant seals were hunted to near extinction in the 19th century, yet have recovered remarkably and now number around 175,000. We surveyed 110 seals for single-strand conformation polymorphism (SSCP) and sequence variation at three major histocompatibility (MHC) class II loci (DQA, DQB and DRB) to evaluate the genetic consequences of the population bottleneck at these loci vs. other well-studied genes. We found very few alleles at each MHC locus, significant variation among breeding sites for the DQA locus, and linkage disequilibrium between the DQB and DRB loci. Northern elephant seals are evidently inbred, although there is as yet no evidence of correlative reductions in fitness.

Amino Acid Sequence↗

PubMed2004

Assessing the likelihood of recurrence during RNA evolution in vitro.

Recurrence is the possibility of resulting in the same endpoint multiple times when a living system is allowed to evolve repeatedly starting from a given initial point. This concept is of concern to both evolutionary theoreticians and molecular biologists who use nucleic acid selection techniques to mimic biotic and computorial processes in the test tube. Using the continuous in vitro evolution methodology, many replicate experimental evolutionary lineages with populations of catalytic RNA were performed to gain insight into the parameters that could affect recurrence. The likelihood that the same genotype will result in parallel trials of an evolution experiment in vitro depends on several factors, including the phenotype under selection, the size and composition of the initial diverse pool of nucleic acids used in the experiment, the degree of mutation possible during the experiment, the shape of the fitness landscape through which the population evolves, and the strategies used to invoke selection and to search the landscape, among others. By considering these factors, it can be predicted that recurrence is more likely when a small, wild-type-based starting pool is used with efficient selection and search strategies involving little online mutagenesis within a rugged adaptive landscape with a strong local optimum. The recurrence experiments performed here on the 150-nucleotide ligase ribozyme demonstrate that it repeatedly jumps from one peak in a fitness landscape to another, apparently hurdling a deep fitness valley. These predictions can and should be tested by additional multiple replicates of actual evolution experiments in the laboratory.

Base Sequence↗

PubMed2003

A case for the extreme antiquity of recombination.

Recombination is usually assumed to be a mode of reproduction that evolved long after asexual reproduction in response to specific genetic and environmental circumstances. Here the argument is made that recombination was an evolutionary development as ancient as the origins of life. To support this proposition four lines of evidence are given, in particular, the need for primordial genomes to acquire substantial length and to escape from Muller's Ratchet.

Genome↗

PubMed2003

Generalized RNA-directed recombination of RNA.

RNA strand exchange through phosphor-nucleotidyl transfer reactions is an intrinsic chemistry promoted by group I intron ribozymes. We show here that Tetrahymena and Azoarcus ribozymes can promote RNA oligonucleotide recombination in either two-pot or one-pot schemes. These ribozymes bind one oligonucleotide, cleave following a guide sequence, transfer the 3' portion of the oligo to their own 3' end, bind a second oligo, and catalyze another transfer reaction to generate recombinant oligos. Recombination is most effective with the Azoarcus ribozyme in a single reaction vessel in which over 75% of the second oligo can be rapidly converted to recombinant product. The Azoarcus ribozyme can also create a new functional RNA, a hammerhead ribozyme, which can be constructed via recombination and then immediately promote its own catalysis in a homogeneous milieu, mimicking events in a prebiotic soup.

Animals↗

PubMed2003

Expanded divalent metal-ion tolerance of evolved ligase ribozymes.

Class I ligases are artificial ribozymes that catalyze the joining of two single-stranded RNAs. These ribozymes are between 120 and 160 nucleotides in length, making them intermediate in size for catalytic RNAs. Previous characterization of the b1-207 ribozyme suggests that it behaves similar to larger ribozymes in terms of divalent metal-ion dependence. This molecule displays a strong preference for magnesium for catalysis, and is inactive in any other metal except manganese, which actually inhibits its operation in magnesium. Here, we sought to examine the metal-ion usages of two ligases that were obtained through continuous evolution in vitro from the b1-207 sequence framework. We found an expanded catalytic range for the E(100)(#3) and B(16)(#19) ribozymes, as they are both catalytically active in calcium and strontium, and less inhibited by manganese. Though not selected for activity in these salts, the evolved ribozymes exhibit several adaptations to in vitro catalysis, and their ability to accommodate metals other than magnesium can be viewed as an example of a molecular exaptation.

Base Sequence↗