Antigen polymorphism in Borrelia hermsii, a clonal pathogenic bacterium.

The relapsing fever spirochete, Borrelia hermsii, escapes immune selection by alternating expression of surface lipoprotein alleles. The switch results from a duplicative transposition of one of several surface lipoprotein-encoding nucleotide sequences into the singular expression site. These nucleotide sequences constitute a large gene family whose diversity originated, in some cases, before the major divergences of Borrelia species. We have examined the B. hermsii vsp subfamily of alleles, which are carried on linear plasmids within each cell and maintained in several diverse copies as an antigenic archive. Each encodes a distinct serotype-specific protein. We sequenced more than 90% of the alleles within a single strain-B. hermsii strain HS1. A preponderance of allelic mosaicism suggests that intragenic recombination, coupled with selection imposed by host immune response, has driven diversification of the archived ensemble of vsp alleles. The recombinational diversification of vsp alleles generates change in the associated serotypes of the magnitude (30-40% amino acid differentiation) necessary for overcoming cross-reactivity of neutralizing antibodies. We conclude that evolution of vsp has occurred by punctuated occurrence of allelic differentiation, rather than by gradual selection of incremental point mutations that do not meet the threshold for antigenic diversity.

Post-genomics and the neutral theory: variation and conservation in the tumor necrosis factor-alpha promoter.

In the post-genomics era, molecular evolutionary geneticists have come to possess the molecular, statistical, and computational tools for estimating the relative importance of selection and random genetic drift in virtually any gene in almost any organism. We have examined single-nucleotide polymorphisms (SNPs) and nucleotide divergence across a region of approximately 1 kb in the promoter of the human tumor necrosis factor alpha (TNF-alpha) gene. TNF-alpha, which plays an important role in lymphocyte biology and in the pathogenesis of infectious and autoimmune diseases, is tightly regulated at the level of transcription through sequence-specific binding of transcription factors to cognate binding sites in a relatively small region of the 5' non-coding region of the gene. Analysis of the promoter region in 207 human chromosomes revealed nine SNPs, none of which were located in regions known to be important in transcriptional activation. Comparison with one promoter sequence in each of seven species of primates revealed 162 nucleotide sites occupied by a monomorphic nucleotide in the human sample but occupied by a different nucleotide in at least one of the primate sequences (a 'fixed human difference'). The fixed human differences were found outside the regions known to be important in transcriptional activation, and their large number suggests that they might be effectively neutral (Ns<<1). With regard to the human SNPs, although the hypothesis Ns approximately 0 cannot be rejected, the sample configurations suggest that the substitutions might be mildly deleterious. We emphasize the analytical insight to be gained from interspecific comparisons: through the interspecific comparisons, 3.1% of the total sequence information yielded 94.7% of the variable nucleotides. This combined approach, using interspecific comparisons and human polymorphism together with data from functional analyses, provides valuable insights into the evolutionary history and regulation of a key gene in the human immune response.

Molecular population genetic analysis of the streptokinase gene of Streptococcus pyogenes: mosaic alleles generated by recombination.

To understand the mechanisms governing molecular evolution of the streptokinase gene (skn), a 384 bp DNA fragment encoding two variable regions of the molecule was characterized in 47 isolates of Streptococcus pyogenes. The results reveal that alleles of the streptokinase gene have a mosaic structure, and provide strong evidence for intragenic recombination. Moreover, organisms that are well differentiated in overall chromosomal character have identical skn alleles, which suggests that horizontal gene transfer and recombination have participated in the evolution of this locus. No simple relationship between skn allele and serum opacity factor production or specific disease was identified. The predicted amino acid sequences of highly divergent skn alleles are strikingly similar in hydrophilicity and hydrophobicity profiles, distribution of amphipathic and flexible regions, surface probability plots, and antigenic indices, indicating that despite extensive nucleotide polymorphism in the two skn variable regions, selective pressure has constrained overall structural divergence. These results add to an important emerging theme that intragenic recombination plays a critical role in diversifying genes coding for streptococcal virulence factors.

Selection intensity for codon bias.

The patterns of nonrandom usage of synonymous codons (codon bias) in enteric bacteria were analyzed. Poisson random field (PRF) theory was used to derive the expected distribution of frequencies of nucleotides differing from the ancestral state at aligned sites in a set of DNA sequences. This distribution was applied to synonymous nucleotide polymorphisms and amino acid polymorphisms in the gnd and putP genes of Escherichia coli. For the gnd gene, the average intensity of selection against disfavored synonymous codons was estimated as approximately 7.3 x 10(-9); this value is significantly smaller than the estimated selection intensity against selectively disfavored amino acids in observed polymorphisms (2.0 x 10(-8)), but it is approximately of the same order of magnitude. The selection coefficients for optimal synonymous codons estimated from PRF theory were consistent with independent estimates based on codon usage for threonine and glycine. Across 118 genes in E. coli and Salmonella typhimurium, the distribution of estimated selection coefficients, expressed as multiples of the effective population size, has a mean and standard deviation of 0.5 +/- 0.4. No significant differences were found in the degree of codon bias between conserved positions and replacement positions, suggesting that translational misincorporation is not an important selective constraint among synonymous polymorphic codons in enteric bacteria. However, across the first 100 codons of the genes, conserved amino acids with identical codons have significantly greater codon bias than that of either synonymous or nonidentical codons, suggesting that there are unique selective constraints, perhaps including mRNA secondary structures, in this part of the coding region.

Dynamics and species richness of tropical rain forests.

We present a worldwide analysis of humid tropical forest dynamics and tree species richness. New tree mortality, recruitment, and species richness data include the most dynamic and diverse mature tropical forests known. Twenty-five sites show a strong tendency for the most species-rich forests to be dynamic and aseasonal. Mean annual tree mortality and recruitment-turnover-is the most predictive factor of species richness, implying that small-scale disturbance helps regulate tropical forest diversity. Turnover rates are also closely related to the amount of basal area turnover in mature tropical forests. Therefore the contribution of small-scale disturbance to maintaining tropical forest diversity may ultimately be driven by ecosystem productivity.

Population genetics of polymorphism and divergence.

Frequencies of mutant sites are modeled as a Poisson random field in two species that share a sufficiently recent common ancestor. The selective effect of the new alleles can be favorable, neutral, or detrimental. The model is applied to the sample configurations of nucleotides in the alcohol dehydrogenase gene (Adh) in Drosophila simulans and Drosophila yakuba. Assuming a synonymous mutation rate of 1.5 x 10(-8) per site per year and 10 generations per year, we obtain estimates for the effective population size (N(e) = 6.5 x 10(6)), the species divergence time (tdiv = 3.74 million years), and an average selection coefficient (sigma = 1.53 x 10(-6) per generation for advantageous or mildly detrimental replacements), although it is conceivable that only two of the amino acid replacements were selected and the rest neutral. The analysis, which includes a sampling theory for the independent infinite sites model with selection, also suggests the estimate that the number of amino acids in the enzyme that are susceptible to favorable mutation is in the range 2-23 at any one time. The approach provides a theoretical basis for the use of a 2 x 2 contingency table to compare fixed differences and polymorphic sites with silent sites and amino acid replacements.

Genetic differences at four DNA typing loci in Finnish, Italian, and mixed Caucasian populations.

Highly polymorphic segments of the human genome containing variable numbers of tandem repeats (VNTRs) have been widely used to establish DNA profiles of individuals for use in forensics. Methods of estimating the probability of occurrence of matching DNA profiles between two randomly selected individuals have been subject to extensive debate regarding the possibility of significant substructure occurring within the major races. We have sampled two Caucasian subpopulations, Finns and Italians, at four commonly used VNTR loci to determine the extent to which the subgroups differ from each other and from a mixed Caucasian database. The data were also analyzed for the occurrence of linkage disequilibrium among the loci. The allele frequency distributions of some loci were found to differ significantly among the subpopulations in a manner consistent with population substructure. Major differences were also found in the probability of occurrence of matching DNA profiles between two individuals chosen at random from the same subpopulation. With respect to the Finnish and Italian subpopulations, the conventional product rule for estimating the probability of a multilocus VNTR match using a mixed Caucasian database consistently yields estimates that are artificially small. Systematic errors of this type were not found using the interim ceiling principle recently advocated in the National Research Council's report [National Research Council (1992) DNA Technology in Forensic Science (Natl. Acad. Sci., Washington)]. The interim ceiling principle is based on currently available racial or ethnic databases and sets an arbitrary lower limit on each VNTR allele frequency. In the future the ceiling frequencies are expected to be established from more adequate data acquired for relevant VNTR loci from multiple subpopulations.

Optimized strategies for sequence-tagged-site selection in genome mapping.

The physical mapping of complex genomes is based on the construction of a genomic library and the determination of the overlaps between the inserts of the mapping clones in order to generate an ordered, cloned representation of nearly all the sequences present in the target genome. Evaluation of the relative efficiency of experimental procedures used to accomplish this goal must minimally include a comparison of the fraction of the genome covered by the ordered arrays (or "contigs"), the average size of the contigs, and the cost, in terms of time and resources, required to generate the map. Sequence-tagged-site (STS) content mapping is one strategy that has been proposed and is being utilized for this type of experiment. This paper describes three STS selection schemes and presents computer simulations of contig-building experiments based on these procedures. The results of these simulations suggest that a nonrandom STS strategy that uses paired probes requires one-third to one-fourth as many STS assays as are required in random and nonpaired approaches, and also results in a map that has both greater genome coverage and a larger average contig size. This strategy promises to reduce the time and cost required to build a high-quality physical map.

IS103, a new insertion element in Escherichia coli: characterization and distribution in natural populations.

IS103 is a previously unknown insertion sequence found in Escherichia coli K12. We have sequenced IS103 and find that it is a 1441-bp element that consists of a 1395-bp core flanked by imperfect 23-bp inverted repeats. IS103 causes a 6-bp duplication of the target sequence into which it inserts. There is a single copy of IS103 present in wild-type E. coli K12 strain HfrC. In strain X342 and its descendents there are two additional copies, one of which is located within the bglF gene. IS103 is capable of excising from within bglF and restoring function of that gene. IS103 exhibits 44% sequence identity with IS3, suggesting that the two insertion sequences are probably derived from a common ancestor. We have examined the distribution of IS103 in the chromosomes and plasmids of the ECOR collection of natural isolates of E. coli. IS103 is found in 36 of the 71 strains examined, and it strongly tends to inhabit plasmids rather than chromosomes. Comparison of the observed distribution of IS103 with distributions predicted by nine different models for the regulation of transposition according to copy number and of the effects of copy number on fitness suggest that transposition of IS103 is strongly regulated and that it has only minor effects on fitness. The strong clustering of IS103 within one phylogenetic subgroup of the E. coli population despite its presence on plasmids suggests that plasmids tend to remain within closely related strains and that transfer to distantly related strains is inhibited.

Why do unrelated insertion sequences occur together in the genome of Escherichia coli?

Natural isolates of Escherichia coli are polymorphic for the presence or absence of insertion sequences. Among the ECOR reference collection of 71 natural isolates studied for the number of copies of the insertion sequences IS1, IS2, IS3, IS4, IS5 and IS30, the number of strains containing no copies of the insertion sequences were 11, 28, 23, 43, 46 and 36, respectively. Significant correlations occur in the ECOR strains in the presence or absence of unrelated insertion sequences in the chromosome and plasmid complements. Strains containing any insertion sequence are more likely to contain additional, unrelated insertion sequences than would be expected by chance. We suggest that the positive correlations result from horizontal transfer mediated by plasmids. A branching-process model for the plasmid-mediated transmission of insertion sequences among hosts yields such a correlation, even in the absence of interactions affecting transposition or fitness. The predictions of the model are quantitatively in agreement with the observed correlations among insertion sequences.

Confidence interval for the number of selectively neutral amino acid polymorphisms.

A statistical approach to the analysis of DNA sequences has been developed, which provides a confidence interval estimate for the proportion of naturally occurring amino acid polymorphisms that are selectively neutral. When applied to the gnd gene coding for 6-phosphogluconate dehydrogenase in a sample of seven natural isolates of Escherichia coli, the method indicates that the proportion of observed amino acid polymorphisms that are selectively neutral is unlikely to be greater than 49% (upper 95% confidence limit). On the other hand, the observations are also consistent with a model in which all of the observed amino acid substitutions are mildly deleterious with an average selection coefficient approximating 1.6 X 10(-7). Various models for the distribution of configurations at silent sites are also investigated.

Distribution and abundance of insertion sequences among natural isolates of Escherichia coli.

A reference collection of 71 natural isolates of Escherichia coli (the ECOR collection) has been studied with respect to the distribution and abundance of transposable insertion sequences using DNA hybridization. The data include 1173 occurrences of six unrelated insertion sequences (IS1, IS2, IS3, IS4, IS5 and IS30). The number of insertion elements per strain, and the sizes of DNA restriction fragments containing them, is highly variable and can be used to discriminate even among closely related strains. The occurrence and abundance of pairs of unrelated insertion sequences are apparently statistically independent, but significant correlations result from stratifications in the reference collection. However, there is a highly significant positive association among the insertion sequences considered in the aggregate. Nine branching process models, which differ in assumptions regarding the regulation of transposition and the effect of copy number on fitness, have been evaluated with regard to their fit of the observed distributions. No single model fits all copy number distributions. The best models incorporate no regulation of transposition and a moderate to strong decrease in fitness with increasing copy number for IS1 and IS5, strong regulation of transposition and a negligible to weak decrease in fitness with increasing copy number for IS3, and less than strong regulation of transposition for IS2, IS4 and IS30.

Joint distribution of insertion elements IS4 and IS5 in natural isolates of Escherichia coli.

A reference collection of natural isolates of Escherichia coli has been studied in order to determine the distribution, abundance and joint occurrence of DNA insertion elements IS4 and IS5. Among these isolates, 36% were found to contain IS4 and 30% were found to contain IS5. Among strains containing IS4 the mean number of copies per strain was 4.4 +/- 0.8; the comparable figure for IS5 was 3.7 +/- 1.0. Although the presence of the elements among the isolates was independent, among those isolates containing both IS4 and IS5, there was a significant negative correlation in the number of copies of the elements. The reference collection was also studied for the presence of the DNA sequences flanking the single copy of IS4 in the chromosome of E. coli K12. Homologous sequences were found in only 26% of the isolates. The sequences flanking the IS4 invariably occur together, and their presence is significantly correlated with the presence of IS4. In eight of the strains that carry these flanking sequences, an IS4 is located between them, and the sequences are present at the homologous position as in the K12 strain. We suggest that IS4 and its flanking sequences share a common mechanism of dissemination, such as plasmids, and we present evidence that they are included in a much larger transposable element.