The origin of the eukaryotic cell.

Molecular sequence data are beginning to provide important insights into the evolutionary origin of eukaryotic cells. Global phylogenies of numerous protein sequences indicate that the eukaryotic cell nucleus is a chimera, which has received major contributions from both a Gram-negative eubacterium and an archaebacterium. Recent studies also indicate that the formation of the nuclear envelope and the endoplasmic reticulum was accompanied by duplication of genes for the molecular chaperone proteins (e.g. hsp70, hsp90), which facilitate protein transport across membranes. Based on these observations, it is suggested that the ancestral eukaryotic cell arose by a unique endosymbiotic event involving engulfment of an eocyte archaebacterium by a Gram-negative eubacterial host.

Evolution of allosteric control in glycogen phosphorylase.

In relation to the primary sequence and three-dimensional structure of rabbit muscle glycogen phosphorylase, we have carried out a comparative sequence analysis of phosphorylases from human, rat, Dictyostelium, yeast, potato and Escherichia coli. Based on sequence similarity, a large region of the protein is shared by these enzymes extending from alpha-helix-1 to the last alpha-helix-33. Conserved residues are equally distributed between the N and C-terminal domains and occur primarily in buried residues. Phylogenetic analysis indicates that the two isozymes within either E. coli, potato or Dictyostelium are more closely related to each other than they are to other phosphorylases. Yeast phosphorylase is most closely related to the Dictyostelium isozymes. Mammalian muscle and brain isozymes are more closely related to each other than to the liver isozyme and the muscle isozyme is evolving at the slowest rate. All phosphorylases exhibit high conservation of active site and pyridoxal phosphate binding residues. Most phosphorylases also exhibit high conservation of sugar binding residues in the glycogen storage site. Phosphorylation and AMP binding site residues are poorly conserved in non-mammalian phosphorylases. In contrast, glucose-6-P binding residues are highly conserved in four of the seven non-mammalian enzymes. Analysis of interacting pairs of dimer contact residues indicates that they can be grouped into three relatively independent networks. One network contains phosphorylation and AMP binding residues and is poorly conserved in non-mammalian enzymes. A second network contains glucose-6-P binding residues and is highly conserved in enzymes containing a conserved glucose-6-P binding site. A third, conserved network contains residues within the tower helix and gate loop. A model for the evolution of allostery in phosphorylase is proposed, suggesting that glucose-6-P inhibition was an early control mechanism. The later creation of primarily distinct ligand binding sites for AMP/phosphorylation control may have allowed the establishment of a separate dimer contact network for propagating conformational changes leading to activation rather than inhibition of enzyme activity.

The sampling distribution of linkage disequilibrium.

The probabilities of obtaining particular samples of gametes with two completely linked loci are derived. It is assumed that the population consists of N diploid, randomly mating individuals, that each of the two loci mutate according to the infinite allele model at a rate mu and that the population is at equilibrium. When 4N mu is small, the most probable samples of gametes are those that segregate only two alleles at either locus. The probabilities of various samples of gametes are discussed. The results show that most samples with completely linked loci have either a very small or a very large association between the alleles of each locus. This causes the distribution of linkage disequilibrium to be skewed and the distribution of the correlation coefficient to be bimodal. The correlation coefficient is commonly used as a test statistic with a chi square distribution and yet has a bimodal distribution when the loci are completely linked. Thus, such a test is not likely to be accurate unless the rate of recombination between the loci and/or the effective population size are sufficiently large enough so that the loci can be treated as unlinked.

A sampling theory of selectively neutral alleles in a subdivided population.

Ewens' sampling distribution is investigated for a structured population. Samples are assumed to be taken from a single subpopulation that exchanges migrants with other subpopulations. A complete description of the probability distribution for such samples is not a practical possibility but an equilibrium approximation can be found. This approximation extracts the information necessary for constructing a continuous approximation to the complete distribution using known values of the distribution and its derivatives in randomly mating populations. It is shown that this approximation is as complete a description of a single biologically realistic subpopulation as is possible given standard uncertainties about the actual size of the migration rates, relative sizes of each of the subpopulations and other factors that might affect the genetic structure of a subpopulation. Any further information must be gained at the expense of generality. This approximation is used to investigate the effect of population subdivision on Watterson's test of neutrality. It is known that the infinite allele, sample distribution is independent of mutation rate when made conditional on the number of alleles in the sample. It is shown that the conditional, infinite allele, sample distribution from this approximation is also independent of population structure and hence Watterson's test is still approximately valid for subdivided populations.

Linkage disequilibrium in a finite population that is partially selfing.

The linkage disequilibrium expected in a finite, partially selfing population is analyzed, assuming the infinite allele model. Formulas for the expected sum of squares of the linkage disequilibria and the squared standard linkage disequilibrium are derived from the equilibrium values of sixteen inbreeding coefficients required to describe the behavior of the system. These formulas are identical to those obtained with random mating if the effective population size N(e) = (1-(1/2)S)N and the effective recombination value r(e) = (1-S)r/(1-(1/2)S), where S is the proportion of selfing, are substituted for the population size and the recombination value. Therefore, the effect of partial selfing at equilibrium is to reduce the population size by a factor 1-(1/2)S and the recombination value by a factor (1-S)/(1-(1/2)S).

Variance and covariance of homozygosity in a structured population.

The variance of homozygosity for a K-allele model with n partially isolated subpopulations is derived numerically using identity coefficients. The variance of homozygosity within a subpopulation is shown to depend strongly upon the migration rates between subpopulations but is not strongly influenced by the number of alleles possible at a locus. The variance of homozygosity within a subpopulation, given the value of expected homozygosity, is approximately equal to the value of the variance of homozygosity given by Stewart's formula for a single population. If the population is presumed to be panmictic, but is actually subdivided, and the gametes are sampled at random from the total population, the apparent variance of homozygosity depends on the number of alleles possible. With small migration rates and K large, the apparent variance of homozygosity is much smaller than in a single population with the same expected homozygosity. However, when K is small, the variance of homozygosity is approximately given by Stewart's formula. The transient behavior of the variance of homozygosity shows that a large number of generations may be required to approach equilibrium values.

The effect of combining alleles into electrophoretic classes on detecting linkage disequilibrium.

When alleles are combined into few detectable classes, linkage correlations are underestimated most of the time. The probability that the linkage correlation will be underestimated is a function of the actual degree of correlation and the evenness of the allelic distribution, but is mainly determined by the distribution of alleles into distinguishable classes. With only two alleles per class this probability will usually be higher than 0.7. Also, the consistency in the sign of the linkage disequilibrium over many populations may escape detection. An increase of sample size by one order of magnitude or more may be required to compensate for the loss in detection power. It follows that the available electrophoretic studies of linkage correlations, although negative in their majority, do not suggest that epistatic interactions and linkage disequilibria are rare in natural populations.

Patterns of somatic mutations in immunoglobulin variable genes.

The mechanism responsible for somatic mutation in the variable genes of antibodies is unknown and may differ from previously described mechanisms that produce mutation in DNA. We have analyzed 421 somatic mutations from the rearranged immunoglobulin variable genes of mice to determine if the nucleotide substitutions differ from those generated during meiosis and if the presence of nearby direct and inverted repeated sequences could template mutations around the variable gene. The results reveal a difference in the pattern of substitutions obtained from somatic mutations vs. meiotic mutations. An increased frequency of T:A to C:G transitions and a decreased frequency of mutations involving a G in the somatic mutants compared to the meiotic mutants is indicated. This suggests that the mutational processes responsible for somatic mutations in antibody genes differs from that responsible for mutation during meiosis. An analysis of the local DNA sequences revealed many direct repeats and palindromic sequences that were capable of templating some of the known mutations. Although additional factors may be involved in targeting mutations to the variable gene, mistemplating by nearby repeats may provide a mechanism for the enhancement of somatic mutation.

Simple sequence is abundant in eukaryotic proteins.

All proteins of Saccharomyces cerevisiae have been compared to determine how frequently segments from one protein are present in other proteins. Proteins that are recently evolutionarily related were excluded. The most frequently present protein segments are long, tandem repetitions of a single amino acid. For some of these segments, up to 14% of all proteins in the genome were found to have similar peptides within them. These peptide segments may not be functional protein domains. Although they are the most common shared feature of yeast proteins, their ubiquity and simplicity argue that their probable function may be to simply serve as spacers between other protein motifs.

Expressed sequence tags from the Yukon ecotype of Thellungiella reveal that gene expression in response to cold, drought and salinity shows little overlap.

Thellungiella salsuginea (also known as T. halophila) is a close relative of Arabidopsis that is very tolerant of drought, freezing, and salinity and may be an appropriate model to identify the molecular mechanisms underlying abiotic stress tolerance in plants. We produced 6578 ESTs, which represented 3628 unique genes (unigenes), from cDNA libraries of cold-, drought-, and salinity-stressed plants from the Yukon ecotype of Thellungiella. Among the unigenes, 94.1% encoded products that were most similar in amino acid sequence to Arabidopsis and 1.5% had no match with a member of the family Brassicaceae. Unigenes from the cold library were more similar to Arabidopsis sequences than either drought- or salinity-induced sequences, indicating that latter responses may be more divergent between Thellungiella and Arabidopsis. Analysis of gene ontology using the best matched Arabidopsis locus showed that the Thellungiella unigenes represented all biological processes and all cellular components, with the highest number of sequences attributed to the chloroplast and mitochondria. Only 140 of the unigenes were found in all three abiotic stress cDNA libraries. Of these common unigenes, 70% have no known function, which demonstrates that Thellungiella can be a rich resource of genetic information about environmental responses. Some of the ESTs in this collection have low sequence similarity with those in Genbank suggesting that they may encode functions that may contribute to Thellungiella's high degree of stress tolerance when compared with Arabidopsis. Moreover, Thellungiella is a closer relative of agriculturally important Brassica spp. than Arabidopsis, which may prove valuable in transferring information to crop improvement programs.

Somatic immunoglobulin sequence divergence and its implications for studies of evolutionary divergence.

The divergence of immunoglobulin genes due to somatic mutation provides a natural example of DNA sequence divergence. This divergence was examined to gain insight into the processes of evolution and the determinants of the variance-to-mean ratio of sequence divergence. Normally, this ratio is found to be larger than expected (1.0 under Poisson assumptions) for the evolutionary divergence or most genes. Although not significantly less than one, all seven groups of immunoglobulin amino acid sequences have ratios smaller than expected, contrary to the evolutionary pattern generally observed. The substitutions in the immunoglobulin genes appear to be highly nonrandom and an excess of parallel changes (the major nonrandom feature of these mutations) is shown to cause smaller ratios. Because convergent or parallel mutations are often observed in the evolutionary divergence of genes, this suggests that forces causing the large observed ratios may actually have to be more powerful than previously expected. Further, since selection is one of the likely causes of parallel mutations, it should be noted that selection could significantly decrease the variance-to-mean ratio. The high frequency of parallel mutations and their resulting effects, as observed in the immunoglobulin genes, suggest that only poor inferences of sequence divergence can be made without actual knowledge of the ancestral sequence.

Multiple substitutions create biased estimates of divergence times and small increases in the variance to mean ratio.

Analysis of mutational processes has demonstrated that mutations usually occur as non-random events with many factors that influence the fidelity of DNA replication. One such unusual pattern of mutation shows that some mutational events will create more than one sequence alteration. This possibility is not generally considered in estimates of sequence divergence and yet affects both the mean and variance of these estimates. Theoretical results and simulation results are presented to examine how extensive the effects of multiple alterations resulting from single mutational events may be on sequence divergence. It is shown that estimates of the divergence times are biased but that this bias is not large unless the number of sequence alterations per event are unrealistically large. The number of alterations per event required to achieve a given bias is determined. The variance is increased by multiple alterations above the variance expected for the same mean number of single alterations, but not up to the levels that are observed in nature. The resulting increase in the variance to mean ratio changes with the amount of divergence, from an initially high ratio followed by a slow decline to one.

Estimates of DNA and protein sequence divergence: an examination of some assumptions.

Some of the assumptions underlying estimates of DNA and protein sequence divergence are examined. A solution for the variance of these estimates that allows for different mutation rates and different population sizes in each species and for an arbitrary structure in the initial population is obtained. It is shown that these conditions do not strongly affect estimates of divergence. In general, they cause the variance of divergence to be smaller than a binomial variance. Thus, the binomial variance that is usually assumed for these estimates is safely conservative. It is shown that variability in the mutation rate among sites can have an effect as large as or larger than variability in the mutation rate among bases. Variability in the mutation rate among bases and among sites causes the number of substitutions between two sequences to be underestimated. Protein and DNA sequences from several species are collected to estimate the variability in mutation rates among sites. When many homologous sequences are known, standard methods to estimate this variability can be used. The estimates of this variability show that this factor is important when considering the spectrum of spontaneous mutations and is strongly reflected in the divergence of sequences. Smaller variability is found for the third position of codons than for the first and second codon positions. This may be because of less selective constraints on this position or because the third position has been saturated with mutations for the sequences examined.

Evolution of simple sequence in proteins.

The proteins of Saccharomyces cerevisiae contain a high proportion of low-complexity, simple sequences. These are protein segments composed almost exclusively or largely of a single repetitive amino acid polymer and are the most commonly shared feature between proteins. We have examined a survey of other species to determine how widespread this phenomenon might be. This was done by comparing how frequently segments from one protein are present in other proteins. Any recently evolutionarily related proteins were excluded. It was found that the most commonly shared features of eukaryotic proteins were repetitive but that prokaryotes did not contain such shared, extensively redundant repeats. The proportion of eukaryotic proteins that contain a significantly repetitive fraction changes dramatically from species to species. In addition the individual amino acids present in these repeats change between species. This suggests that the primary sequence of the repeats may not be important for their function. Further tests of the yeast repeats confirmed that these repeats evolve more quickly than the remainder of the protein sequence within which they are embedded. These results show that these rapid evolving, simple sequence repeats are in fact the most commonly shared pattern between all of the genomic proteins of eukaryotes.

Protein-based phylogenies support a chimeric origin for the eukaryotic genome.

The phylogenetic position of the archaebacteria and the place of eukaryotes in the history of life remain a question of debate. Recent studies based on some protein-sequence data have obtained unusual phylogenies for these organisms. We therefore collected the protein sequences that were available with representatives from each of the major forms of life: the gram-negative bacteria, gram-positive bacteria, archaebacteria, and eukaryotes. Monophyletic, unrooted phylogenies based on these sequence data show that seven of 24 proteins yield a significant gram-positive-archaebacteria clade/gram-negative-eukaryotic clade. The phylogenies for these seven proteins cannot be explained by the traditional three-way split of the eukaryotes, archaebacteria, and eubacteria. Nine of the 24 proteins yield the traditional gram-positive-gram-negative clade/archaebacteria-eukaryotic clade. The remaining eight proteins give phylogenies that cannot be statistically distinguished. These results support the hypothesis of a chimeric origin for the eukaryotic cell nucleus formed from the fusion of an archaebacteria and a gram-negative bacteria.

Evolution of HSP70 gene and its implications regarding relationships between archaebacteria, eubacteria, and eukaryotes.

The 70-kDa heat-shock protein (HSP70) constitutes the most conserved protein present in all organisms that is known to date. Based on global alignment of HSP70 sequences from organisms representing all three domains, numerous sequence signatures that are specific for prokaryotic and eukaryotic homologs have been identified. HSP70s from the two archaebacterial species examined (viz., Halobacterium marismortui and Methanosarcina mazei) have been found to contain all eubacterial but no eukaryotic signature sequences. Based on several novel features of the HSP70 family of proteins (viz., presence of tandem repeats of a 9-amino-acid [a.a.] polypeptide sequence and structural similarity between the first and second quadrants of HSP70, homology of the N-terminal half of HSP70 to the bacterial MreB protein, presence of a conserved insert of 23-27 a.a. in all HSP70s except those from archaebacteria and gram-positive eubacteria) a model for the evolution of HSP70 gene from an early stage is proposed. The HSP70 homologs from archaebacteria and gram-positive bacteria lacking the insert in the N-terminal quadrants are indicated to be the ancestral form of the protein. Detailed phylogenetic analyses of HSP70 sequence data (viz., by bootstrap analyses, maximum parsimony, and maximum likelihood methods) provide evidence that archaebacteria are not monophyletic and show a close evolutionary linkage with the gram-positive eubacteria. These results do not support the traditional archaebacterial tree, where a close relationship between archaebacterial and eukaryotic homologs is observed. To explain the phylogenies based on HSP70 and other gene sequences, a model for the origin of eukaryotic cells involving fusion between archaebacteria and gram-negative eubacteria is proposed.

The rat adenine phosphoribosyltransferase sequence shows evolutionary rate variation among exons in rodents.

The complete genomic sequence of the rat APRT gene is described and compared with published mammalian sequences. The rat APRT gene organization is typical of other rodent APRTs with five exons, one large intron of 993 bp, and three smaller introns averaging 145 bp. Because complete sequences for mouse and Chinese hamster APRT are also known, it is possible to compare the evolutionary rates of change in the exons with those of the introns. The latter provide one possible estimate of underlying rates of change. It is shown that the APRT exons have differential rates of evolution in rodents and have had a recent and rapid burst of substitutions within the mouse lineage. Rates of change in the exons do not appear to be strongly correlated with the rates of change in the introns.

Patterns of bacterial gene movement.

Lateral gene transfer has emerged as an important force in bacterial evolution. A substantial number of genes can be inserted into or deleted from genomes through the process of lateral transfer. In this study, we looked for atypical occurrence of genes among related organisms to detect laterally transferred genes. We have analyzed 50 bacterial complete genomes from nine groups. For each group we use a 16s rRNA phylogeny and a comparison of protein similarity to map gene insertions/deletions onto their species phylogeny. The results reveal that there is poor correlation of genes inserted, deleted, and duplicated with evolutionary branch length. In addition, the numbers of genes inserted, deleted, or duplicated within the same branch are not always correlated with each other. Nor is there any similarity within groups. For example, in the Rhizobiales group, the ratio of insertions to deletions in the evolutionary branch leading to Agrobacterium tumefaciens str. C58 (Cereon) is 0.52, but it is 39.52 for Mesorhizobium loti. Most strikingly, the number of insertions of foreign genes is much larger in the external branches of the trees. These insertions also greatly outnumber the occurrence of deletions, and yet the genome sizes of these bacteria remain roughly constant. This indicates that many of the insertions are specific to each organism and are lost before related species can evolve. Simulations of the process of insertion and deletion, tailored to each phylogeny, support this conclusion.

Evidence for local DNA influences on patterns of substitutions in the human alpha-interferon gene family.

The evolutionary history of genes can be used to examine patterns of spontaneous mutation if the sequences are sufficiently extensive to provide reliable data. Many human alpha-interferon genes have been sequenced and they form a large multigene family including several pseudogenes. A phylogenetic history for 15 human interferon sequences was reconstructed and their ancestral sequences inferred using a maximum parsimony method. This evolutionary history provided a record of more than 738 spontaneous mutations that have occurred in man's recent evolution. Of these mutations, more than 267 base substitution and deletion-insertion events were analyzed to determine the possible effects of nearby DNA sequences. Many substitutions occur at the end of long runs of identical bases and some dinucleotide pairs may mutate more often than others. Because templating by local DNA sequences has been implicated in prokaryotic mutation, the sequences were also examined for nearby repeats that include the substituted nucleotide and hence are potentially capable of templating the substitution. The majority of sequence alterations examined have either a similar direct repeat or palindrome nearby. Often such templates can account for simultaneous multiple mutations. These results suggest that sequence-directed events may occur occasionally in eukaryotes and that neighbouring DNA sequences can influence both the occurrence and types of mutations in several different ways.

Evolutionary rate variation within Mus APRT.

Rodents are thought to have relatively high rates of evolution, twice as fast as the rates for mammals in other orders. However, the uniformly high rates of evolution inferred for the order Rodentia from Mus musculus and Rattus norvegicus are not consistently found for other rodent species. Using a maximum likelihood phylogenetic algorithm (DNAML), we show here that Mus spicilegus has a fivefold different rate of evolution in 1100 bp around the adenine phosphoribosyltransferase gene (APRT) since its divergence from a common ancestor with Mus musculus. A greater than threefold difference in rates is also found in a comparison of the number of evolutionary events directly detected from the APRT sequences of these two closely related Mus species. The evolutionary events can be directly detected, since M. spicilegus, M. musculus, and the four rodent outgroup species used to determine the ancestral sequence are so closely related. One of the major differences between M. spicilegus and M. musculus that might affect evolutionary rate is the degree of commensalism with man. The Mus species therefore provide a useful model for testing various hypotheses for the causes of rate variations between genes, and possibly, between lineages.