Genetic and phylogenetic consequences of island biogeography.

Island biogeography theory predicts that the number of species on an island should increase with island size and decrease with island distance to the mainland. These predictions are generally well supported in comparative and experimental studies. These ecological, equilibrium predictions arise as a result of colonization and extinction processes. Because colonization and extinction are also important processes in evolution, we develop methods to test evolutionary predictions of island biogeography. We derive a population genetic model of island biogeography that incorporates island colonization, migration of individuals from the mainland, and extinction of island populations. The model provides a means of estimating the rates of migration and extinction from population genetic data. This model predicts that within an island population the distribution of genetic divergences with respect to the mainland source population should be bimodal, with much of the divergence dating to the colonization event. Across islands, this model predicts that populations on large islands should be on average more genetically divergent from mainland source populations than those on small islands. Likewise, populations on distant islands should be more divergent than those on close islands. Published observations of a larger proportion of endemic species on large and distant islands support these predictions.

How to make a biological switch.

Some biological regulatory systems must "remember" a state for long periods of time. A simple type of system that can accomplish this task is one in which two regulatory elements negatively regulate one another. For example, two repressor proteins might control one another's synthesis. Qualitative reasoning suggests that such a system will have two stable states, one in which the first element is "on" and the second "off", and another in which these states are reversed. Quantitative analysis shows that the existence of two stable steady states depends on the details of the system. Among other things, the shapes of functions describing the effect of one regulatory element on the other must meet certain criteria in order for two steady states to exist. Many biologically reasonable functions do not meet these criteria. In particular, repression that is well described by a Michaelis-Menten-type equation cannot lead to a working switch. However, functions describing positive cooperativity of binding, non-additive effects of multiple operator sites, or depletion of free repressor can lead to working switches.

The use of modified primers to eliminate cycle sequencing artifacts.

Cycle sequencing is the workhorse of DNA sequencing projects, allowing the production of large amounts of product from relatively little template. This cycling regime, which is aimed at linear growth of the desired products, can also produce artifacts by exponential amplification of minor side-products. These artifacts can interfere with sequence determination. In an attempt to allow linear but prevent exponential growth of products, and thus eliminate artifacts, we have investigated the use of primers containing modified residues that cannot be replicated by DNA polymerase. Specifically, we have used primers containing 2'- O -methyl RNA residues or abasic residues. Oligomers consisting of six DNA residues and 20 2'- O -methyl RNA residues, with the DNA residues located at the 3'-end, primed as efficiently as DNA primers but would not support exponential amplification. Oligonucleotides containing fewer DNA residues were not used as efficiently as primers. DNA primers containing a single abasic site located six residues from the 3'-end also showed efficient priming ability without yielding exponential amplification products. Together these results demonstrate that certain types of modified primers can be used to eliminate artifacts in DNA sequencing. The technique should be particularly useful in protocols involving large numbers of cycles, such as direct sequencing of BAC and genomic DNA.

Divergence of the hyperthermophilic archaea Pyrococcus furiosus and P. horikoshii inferred from complete genomic sequences.

Divergence of the hyperthermophilic Archaea, Pyrococcus furiosus and Pyrococcus horikoshii, was assessed by analysis of complete genomic sequences of both species. The average nucleotide identity between the genomic sequences is 70-75% within ORFs. The P. furiosus genome (1.908 mbp) is 170 kbp larger than the P. horikoshii genome (1.738 mbp) and the latter displays significant deletions in coding regions, including the trp, his, aro, leu-ile-val, arg, pro, cys, thr, and mal operons. P. horikoshii is auxotrophic for tryptophan and histidine and is unable to utilize maltose, unlike P. furiosus. In addition, the genomes differ considerably in gene order, displaying displacements and inversions. Six allelic intein sites are common to both Pyrococcus genomes, and two intein insertions occur in each species and not the other. The bacteria-like methylated chemotaxis proteins form a functional group in P. horikoshii, but are absent in P. furiosus. Two paralogous families of ferredoxin oxidoreductases provide evidence of gene duplication preceding the divergence of the Pyrococcus species.

Should we expect substitution rate to depend on population size?

The rate of nucleotide substitution is generally believed to be a decreasing function of effective population size, at least for nonsynonymous substitutions. This view was originally based on consideration of slightly deleterious mutations with a fixed distribution of selection coefficients. A realistic model must include the occurrence and fixation of some advantageous mutations that compensate for the loss of fitness due to deleterious substitutions. Some such models, such as so-called "fixed" models, also predict a population size effect on substitution rate. An alternative model, presented here, predicts the near absence of a population size effect on substitution rate. This model is based on concave log-fitness functions and a fixed distribution of mutational effects on the selectively important trait. Simulations of an instance of the model confirm the approximate insensitivity of the substitution rate to population size. Although much experimental evidence has been claimed to support the existence of a population size effect, the body of evidence as a whole is equivocal, and much of the evidence that is supposed to demonstrate such an effect would also suggest that it is very small. Perhaps the proposed model applies well to some genes and not so well to others, and genes therefore vary with regard to the population size effect.

Enzyme-linked fluorescent detection for automated multiplex DNA sequencing.

Initiatives to sequence DNA on a large scale have created a need for increased throughput and decreased costs. One scheme for increasing throughput, multiplex sequencing, involves the processing of a mixture of sequencing templates followed by sequential hybridization to reveal the individual sequence ladders on a membrane. Because multiplex sequencing has not been fully automated, and has not seemed automatable, few sequencing efforts have attempted to exploit it. We describe here a scheme for the automation of multiplex sequencing. Probe hybridized to target DNA is detected via spatially localized enzyme-linked fluorescence. Light output is high enough that imaging is possible with simple instrumentation. Direct imaging within an automated hybridization apparatus is made feasible so that the entire process will be automatic once a multiplex membrane is produced. The technique has the potential to increase severalfold the throughput of automated sequencing instruments required for sequencing the human genome.

Rapid cycle allele-specific amplification: studies with the cystic fibrosis delta F508 locus.

Rapid cycle DNA amplification is a polymerase chain reaction technique with improved product specificity and cycle times of 20-60 s, allowing complete 30-cycle reactions in 10-30 min. The presence or absence of the delta F508 deletion and wild-type allele was determined in 104 cystic fibrosis patients by rapid cycle DNA amplification. In separate allele-specific assays, sequences on both sides of the delta F508 locus were amplified with the 3' end of a discriminating primer at the delta F508 locus, with either a 3-bp or a 1-bp mismatch. With rapid cycling (35-s cycles), single-base discrimination was achieved over a broad range of annealing temperatures (50 degrees C or lower); with conventional cycling and "hot starts" (160-s cycles), only annealing temperatures of 61-62 degrees C sufficiently discriminated between alleles. With rapid cycling, genotype could still be assessed with annealing temperatures as low as 25 degrees C. We conclude that faster temperature cycling can improve the results of allele-specific amplification.