Long Periods Of Evolutionary Time Research Articles

Functional Conservation of Cis-Regulatory Elements of Heat-Shock Genes over Long Evolutionary Distances

Transcriptional control of gene regulation is an intricate process that requires precise orchestration of a number of molecular components. Studying its evolution can serve as a useful model for understanding how complex molecular machines evolve. One way to investigate evolution of transcriptional regulation is to test the functions of cis-elements from one species in a distant relative. Previous results suggested that few, if any, tissue-specific promoters from Drosophila are faithfully expressed in C. elegans. Here we show that, in contrast, promoters of fly and human heat-shock genes are upregulated in C. elegans upon exposure to heat. Inducibility under conditions of heat shock may represent a relatively simple “on-off” response, whereas complex expression patterns require integration of multiple signals. Our results suggest that simpler aspects of regulatory logic may be retained over longer periods of evolutionary time, while more complex ones may be diverging more rapidly.

Molecular Evidence for Ancient Asexuality in Timema Stick Insects

Asexuality is rare in animals in spite of its apparent advantage relative to sexual reproduction, indicating that it must be associated with profound costs [1-9]. One expectation is that reproductive advantages gained by new asexual lineages will be quickly eroded over time [3, 5-7]. Ancient asexual taxa that have evolved and adapted without sex would be "scandalous" exceptions to this rule, but it is often difficult to exclude the possibility that putative asexuals deploy some form of "cryptic" sex, or have abandoned sex more recently than estimated from divergence times to sexual relatives [10]. Here we provide evidence, from high intraspecific divergence of mitochondrial sequence and nuclear allele divergence patterns, that several independently derived Timema stick-insect lineages have persisted without recombination for more than a million generations. Nuclear alleles in the asexual lineages displayed significantly higher intraindividual divergences than in related sexual species. In addition, within two asexuals, nuclear allele phylogenies suggested the presence of two clades, with sequences from the same individual appearing in both clades. These data strongly support ancient asexuality in Timema and validate the genus as an exceptional opportunity to attack the question of how asexual reproduction can be maintained over long periods of evolutionary time.

Protein?Protein and Protein?DNA Dosage Balance and Differential Paralog Transcription Factor Retention in Polyploids

Most eukaryotes have an evolutionary history of repeated polyploidization fol-lowed by fractionation (or diploidization; Makino and McLysaght, 2010; Jiao et al., 2011). The progression to the near dip-loid level is not random with regard to the classes of genes that are retained (Freeling et al., 2008; Freeling, 2009; Makino and McLysaght, 2010). Typically, the genes that are preferentially retained are involved with macromolecular machines or heavily con-nected in the interactome. This differential progression of genic retention is unlikely to be only due to changes in protein function of the different members of a duplicate pair in processes referred to as subfunctionali-zation (subdivision of function) and neo-functionalization (gain of a novel function; Freeling, 2009). There are two arguments why this should be the case. First, the same classes of genes that are preferentially retained following whole genome duplica-tion are preferentially underrepresented in segmental duplications (Freeling, et al., 2008; Makino and McLysaght, 2010). Both processes will produce duplicate genes that are available for divergence but the recipro-cal distribution suggests that other factors are operative. Secondly, the duplicates that are retained for longer periods of evolution-ary time very often eventually decay to the diploid state indicating that there has been no bona fide subdivision of function that would maintain both copies. It should be noted, however, that subdivision or gain of function has certainly been documented for duplicate genes in evolution and the reten-tion of regulatory genes for longer peri-ods of evolutionary time provides greater opportunity for these changes in function to accumulate.The types of genes that are preferen-tially retained following whole genome duplications and depleted in segmental copy number changes are quite similar to those shown to exhibit dosage effects in aneuploids (Birchler, 1979; Birchler and Newton, 1981; Guo and Birchler, 1994; Birchler et al., 2001). An analogy can be made to the generalized lack of effects on gene expression by whole genome changes but a regular and consistent set of modu-lations that occur in aneuploids (Birchler and Newton, 1981; Guo and Birchler, 1994; Guo et al., 1996). This set of observations led to the concept that the stoichiometry of members of regulatory macromolecular complexes involved in the control of tran-scription was important in affecting the expression of the target genes ( Birchler and Newton, 1981; Birchler et al., 2001 ). These types of dosage effects can often be reduced to the action of single genes ( Birchler et al., 2001) and indeed heterozygous mutations of transcription factors were recognized to produce human clinical conditions ( Veitia, 2002, 2003, 2004). The stoichiometry of members of macromolecular complexes was postulated to explain this (semi-) dominance (Veitia, 2002). An issue perti-nent to this discussion is the relationship of gene copy number to protein expression level. For instance, in a study in diploid yeast, knockouts of every gene were exam-ined for protein concentration (Springer et al., 2010). Only 5% showed no correla-tion and 80% of genes showed a strong correlation, i.e., 50% expression of normal. The connection between gene dosage and the phenotype can be traced back to clas-sical genetics in which it was known that changes in whole ploidy would produce some level of morphological change but alterations in the copy number of portions of the genome could be quite detrimental or indeed lethal ( Birchler and Veitia, 2007 ). Thus, the change in stoichiometry of dos-age balanced gene products would have negative fitness consequences manifested in the phenotype and be selected against (Papp et al., 2003; Birchler et al., 2005; Veitia et al., 2008).Biophysical evidence suggests that the more interaction partners a particular pro-tein has, the less likely it is to be involved with a duplication event, indicating further that macromolecular complexes require a balance of subunits to maintain good fitness (Liang et al., 2008 ). Examinations of protein databases also indicate that proteins with many interactions display lower expres-sional noise and are underrepresented in copy number variants (Schuster-Bockler et al., 2010). Thus, from the biochemical level to the phenotype, there is evidence for a balance of gene products involved in such complexes, which provides implications in biophysics, evolution, gene expression, and quantitative trait analysis. This synthesis is referred to as the Gene Balance Hypothesis (Birchler and Veitia, 2007, 2010). To reit-erate, the underlying theme of the above synthesis is that the amounts of different subunits and mode of assembly of multi-subunit complexes will affect the final yield and that this fact will impact the phenotype. One of the tenets of this concept is that dur -ing the assembly of multi-subunited com-plexes, a relative excess of one subunit might lead to the production of potentially inac-tive subcomplexes. Such a circumstance will produce a different quantity of the whole complex under consideration and affect the functional output.Schnable et al. (2011) highlight another aspect for the study of retained genes fol-lowing ancient tetraploidy. These authors

Invertebrate immune diversity

The arms race between hosts and pathogens (and other non-self) drives the molecular diversification of immune response genes in the host. Over long periods of evolutionary time, many different defense strategies have been employed by a wide variety of invertebrates. We review here penaeidins and crustins in crustaceans, the allorecognition system encoded by fuhc, fester and Uncle fester in a colonial tunicate, Dscam and PGRPs in arthropods, FREPs in snails, VCBPs in protochordates, and the Sp185/333 system in the purple sea urchin. Comparisons among immune systems, including those reviewed here have not identified an immune specific regulatory “genetic toolkit”, however, repeatedly identified sequences (or “building materials” on which the tools act) are present in a broad range of immune systems. These include a Toll/TLR system, a primitive complement system, an LPS binding protein, and a RAG core/Transib element. Repeatedly identified domains and motifs that function in immune proteins include NACHT, LRR, Ig, death, TIR, lectin domains, and a thioester motif. In addition, there are repeatedly identified mechanisms (or “construction methods”) that generate sequence diversity in genes with immune function. These include genomic instability, duplications and/or deletions of sequences and the generation of clusters of similar genes or exons that appear as families, gene recombination, gene conversion, retrotransposition, alternative splicing, multiple alleles for single copy genes, and RNA editing. These commonly employed “materials and methods” for building and maintaining an effective immune system that might have been part of that ancestral system appear now as a fragmented and likely incomplete set, likely due to the rapid evolutionary change (or loss) of host genes that are under pressure to keep pace with pathogen diversity.

Focus on polyploidy

Focus on polyploidy

The human protein coevolution network

Coevolution maintains interactions between phenotypic traits through the process of reciprocal natural selection. Detecting molecular coevolution can expose functional interactions between molecules in the cell, generating insights into biological processes, pathways, and the networks of interactions important for cellular function. Prediction of interaction partners from different protein families exploits the property that interacting proteins can follow similar patterns and relative rates of evolution. Current methods for detecting coevolution based on the similarity of phylogenetic trees or evolutionary distance matrices have, however, been limited by requiring coevolution over the entire evolutionary history considered and are inaccurate in the presence of paralogous copies. We present a novel method for determining coevolving protein partners by finding the largest common submatrix in a given pair of distance matrices, with the size of the largest common submatrix measuring the strength of coevolution. This approach permits us to consider matrices of different size and scale, to find lineage-specific coevolution, and to predict multiple interaction partners. We used MatrixMatchMaker to predict protein-protein interactions in the human genome. We show that proteins that are known to interact physically are more strongly coevolving than proteins that simply belong to the same biochemical pathway. The human coevolution network is highly connected, suggesting many more protein-protein interactions than are currently known from high-throughput and other experimental evidence. These most strongly coevolving proteins suggest interactions that have been maintained over long periods of evolutionary time, and that are thus likely to be of fundamental importance to cellular function.

The Strength of Selection Against the Yeast Prion [PSI+

The [PSI(+)] prion causes widespread readthrough translation and is rare in natural populations of Saccharomyces, despite the fact that sex is expected to cause it to spread. Using the recently estimated rate of Saccharomyces outcrossing, we calculate the strength of selection necessary to maintain [PSI(+)] at levels low enough to be compatible with data. Using the best available parameter estimates, we find selection against [PSI(+)] to be significant. Inference regarding selection on modifiers of [PSI(+)] appearance depends on obtaining more precise and accurate estimates of the product of yeast effective population size N(e) and the spontaneous rate of [PSI(+)] appearance m. The ability to form [PSI(+)] has persisted in yeast over a long period of evolutionary time, despite a diversity of modifiers that could abolish it. If mN(e) < 1, this may be explained by insufficiently strong selection. If mN(e) > 1, then selection should favor the spread of [PSI(+)] resistance modifiers. In this case, rare conditions where [PSI(+)] is adaptive may permit its persistence in the face of negative selection.

Consequences of inbreeding depression due to sex-linked loci for the maintenance of males and outcrossing in branchiopod crustaceans

Androdioecy, where males co-occur with hermaphrodites, is a rare sexual system in plants and animals. It has a scattered phylogenetic distribution, but it is common and has persisted for long periods of evolutionary time in branchiopod crustaceans. An earlier model of the maintenance of males with hermaphrodites in this group, by Otto et al. (1993), considered the importance of male-hermaphrodite encounter rates, sperm limitation, male versus hermaphrodite viability and inbreeding depression suffered by selfed progeny. Here I advance this model in two ways: (1) by exploring the conditions that would allow the invasion of hermaphrodites into a dioecious population and that of females into an androdioecious population; and (2) by incorporating a term that accounts for the potential effects of genetic load linked to a dominant hermaphrodite-determining allele in androdioecious populations. The new model makes plausible sense of observations made in populations of the species Eulimnadia texana, one of a number of related species whose common ancestor evolved hermaphroditism (and androdioecy) from dioecy. In particular, it offers an explanation for the long evolutionary persistence of androdioecy in branchiopods and suggests reasons for why dioecy has not re-evolved in the clade. Finally, it provides a rather unusual illustration of the implications of the degeneration of loci linked to a sex-determining locus.

Peptide vocabulary analysis reveals ultra-conservation and homonymity in protein sequences.

A new algorithm is presented for vocabulary analysis (word detection) in texts of human origin. It performs at 60%-70% overall accuracy and greater than 80% accuracy for longer words, and approximately 85% sensitivity on Alice in Wonderland, a considerable improvement on previous methods. When applied to protein sequences, it detects short sequences analogous to words in human texts, i.e. intolerant to changes in spelling (mutation), and relatively context-independent in their meaning (function). Some of these are homonyms of up to 7 amino acids, which can assume different structures in different proteins. Others are ultra-conserved stretches of up to 18 amino acids within proteins of less than 40% overall identity, reflecting extreme constraint or convergent evolution. Different species are found to have qualitatively different major peptide vocabularies, e.g. some are dominated by large gene families, while others are rich in simple repeats or dominated by internally repetitive proteins. This suggests the possibility of a peptide vocabulary signature, analogous to genome signatures in DNA. Homonyms may be useful in detecting convergent evolution and positive selection in protein evolution. Ultra-conserved words may be useful in identifying structures intolerant to substitution over long periods of evolutionary time.

Rapid lineage accumulation in a non-adaptive radiation: phylogenetic analysis of diversification rates in eastern North American woodland salamanders (Plethodontidae: Plethodon).

Adaptive radiations have served as model systems for quantifying the build-up of species richness. Few studies have quantified the tempo of diversification in species-rich clades that contain negligible adaptive disparity, making the macroevolutionary consequences of different modes of evolutionary radiation difficult to assess. We use mitochondrial-DNA sequence data and recently developed phylogenetic methodologies to explore the tempo of diversification of eastern North American Plethodon, a species-rich clade of woodland salamanders exhibiting only limited phenotypic disparity. Lineage-through-time analysis reveals a high rate of lineage accumulation, 0.8 species per million years, occurring 11-8 million years ago in the P. glutinosus species group, followed by decreasing rates. This high rate of lineage accumulation is exceptional, comparable to the most rapid of adaptive radiations. In contrast to classic models of adaptive radiation where ecological niche divergence is linked to the origin of species, we propose that phylogenetic niche conservatism contributes to the rapid accumulation of P. glutinosus-group lineages by promoting vicariant isolation and multiplication of species across a spatially and temporally fluctuating environment. These closely related and ecologically similar lineages persist through long-periods of evolutionary time and form strong barriers to the geographic spread of their neighbours, producing a subsequent decline in lineage accumulation. Rapid diversification among lineages exhibiting long-term maintenance of their bioclimatic niche requirements is an under-appreciated phenomenon driving the build-up of species richness.

Functional tests of enhancer conservation between distantly related species.

Expression patterns of orthologous genes are often conserved, even between distantly related organisms, suggesting that once established, developmental programs can be stably maintained over long periods of evolutionary time. Because many orthologous transcription factors are also functionally conserved, one possible model to account for homologous gene expression patterns, is conservation of specific binding sites within cis-regulatory elements of orthologous genes. If this model is correct, a cis-regulatory element from one organism would be expected to function in a distantly related organism. To test this hypothesis, we fused the green fluorescent protein gene to neuronal and muscular enhancer elements from a variety of Drosophila melanogaster genes, and tested whether these would activate expression in the homologous cell types in Caenorhabditis elegans. Regulatory elements from several genes directed appropriate expression in homologous tissue types, suggesting conservation of regulatory sites. However, enhancers of most Drosophila genes tested were not properly recognized in C. elegans, implying that over this evolutionary distance enough changes occurred in cis-regulatory sequences and/or transcription factors to prevent proper recognition of heterospecific enhancers. Comparisons of enhancer elements of orthologous genes between C. elegans and C. briggsae revealed extensive conservation, as well as specific instances of functional divergence. Our results indicate that functional changes in cis-regulatory sequences accumulate on timescales much shorter than the divergence of arthropods and nematodes, and that mechanisms other than conservation of individual binding sites within enhancer elements are responsible for the conservation of expression patterns of homologous genes between distantly related species.

Robustness, Flexibility, and the Role of Lateral Inhibition in the Neurogenic Network

Background: Many gene networks used by developing organisms have been conserved over long periods of evolutionary time. Why is that? We showed previously that a model of the segment polarity network in Drosophila is robust to parameter variation and is likely to act as a semiautonomous patterning module. Is this true of other networks as well?Results: We present a model of the core neurogenic network in Drosophila. Our model exhibits at least three related pattern-resolving behaviors that the real neurogenic network accomplishes during embryogenesis in Drosophila. Furthermore, we find that it exhibits these behaviors across a wide range of parameter values, with most of its parameters able to vary more than an order of magnitude while it still successfully forms our test patterns. With a single set of parameters, different initial conditions (prepatterns) can select between different behaviors in the network's repertoire. We introduce two new measures for quantifying network robustness that mimic recombination and allelic divergence and use these to reveal the shape of the domain in the parameter space in which the model functions. We show that lateral inhibition yields robustness to changes in prepatterns and suggest a reconciliation of two divergent sets of experimental results. Finally, we show that, for this model, robustness confers functional flexibility.Conclusions: The neurogenic network is robust to changes in parameter values, which gives it the flexibility to make new patterns. Our model also offers a possible resolution of a debate on the role of lateral inhibition in cell fate specification.

Differential expression of the actin-binding proteins, alpha-actinin-2 and -3, in different species: implications for the evolution of functional redundancy.

The alpha-actinins are a multigene family of four actin-binding proteins related to dystrophin. The two skeletal muscle isoforms of alpha-actinin (ACTN2 and ACTN3) are major structural components of the Z-line involved in anchoring the actin-containing thin filaments. In humans, ACTN2 is expressed in all muscle fibres, while ACTN3 expression is restricted to a subset of type 2 fibres. We have recently demonstrated that alpha-actinin-3 is absent in approximately 18% of individuals in a range of human populations, and that homozygosity for a premature stop codon (577X) accounts for most cases of true alpha-actinin-3 deficiency. Absence of alpha-actinin-3 is not associated with an obvious disease phenotype, raising the possibility that ACTN3 is functionally redundant in humans, and that alpha-actinin-2 is able to compensate for alpha-actinin-3 deficiency. We now present data concerning the expression of ACTN3 in other species. Genotyping of non-human primates indicates that the 577X null mutation has likely arisen in humans. The mouse genome contains four orthologues which all map to evolutionarily conserved syntenic regions for the four human genes. Murine Actn2 and Actn3 are differentially expressed, spatially and temporally, during embryonic development and, in contrast to humans, alpha-actinin-2 expression does not completely overlap alpha-actinin-3 in postnatal skeletal muscle, suggesting independent function. Furthermore, sequence comparison of human, mouse and chicken alpha-actinin genes demonstrates that ACTN3 has been conserved over a long period of evolutionary time, implying a constraint on evolutionary rate imposed by continued function of the gene. These observations provide a real framework in which to test theoretical models of genetic redundancy as they apply to human populations. In addition we highlight the need for caution in making conclusions about gene function from the phenotypic consequences of loss-of-function mutations in animal knockout models.

The Wilhelmine E. Key 1994 invitational Lecture. Plant genetic diversity and the struggle to measure selection.

The fundamental research program of population genetics has been to seek a quantitative assessment of the role of the various forces of evolution in shaping patterns of genetic variation. This goal has been pursued on both empirical and theoretical fronts. The introduction of biochemical and molecular techniques into population genetics more than 25 years ago revealed vast stores of genetic variation within populations. This level of genetic diversity is difficult to reconcile with balancing selection, and as a consequence, recent thinking has emphasized the role of mutation and genetic random drift as the primary determinants of genetic diversity. The resulting neutral theory of molecular evolution has dominated population genetic thought for more than 20 years. Nonadaptive theories have also emphasized the role of deleterious mutations in driving evolutionary change. New insights into the relative importance of selection and genetic random drift can now be obtained from samples of DNA sequences of genes drawn from within species. The elaboration of coalescence theory, together with the accumulation of data on gene genealogies, permits an integration over relatively long periods of evolutionary time. The ability to integrate over long periods of evolutionary time permits the detection of small selection intensities and it reveals some information about the mode of selection. When the genealogy is consistent with a neutral process, the effective population size can be estimated, as can the age of the coalescent, thus providing new empirical approaches to the estimation of these important parameters. Applications of these approaches in plant population genetics are still in their infancy, but they have already provided new insights into effective population sizes and they are beginning to illustrate how selection for domestication has affected plant genomes.

Mutation rate varies among alleles at a microsatellite locus: phylogenetic evidence.

The understanding of the mutational mechanism that generates high levels of variation at microsatellite loci lags far behind the application of these genetic markers. A phylogenetic approach was developed to study the pattern and rate of mutations at a dinucleotide microsatellite locus tightly linked to HLA-DQB1 (DQCAR). A random Japanese population (n = 129) and a collection of multiethnic samples (n = 941) were typed at the DQB1 and DQCAR loci. The phylogeny of DQB1 alleles was then reconstructed and DQCAR alleles were superimposed onto the phylogeny. This approach allowed us to group DQCAR alleles that share a common ancestor. The results indicated that the DQCAR mutation rate varies drastically among alleles within this single microsatellite locus. Some DQCAR alleles never mutated during a long period of evolutionary time. Sequencing of representative DQCAR alleles showed that these alleles lost their ability to mutate because of nucleotide substitutions that shorten the length of uninterrupted CA repeat arrays; in contrast, all mutating alleles had relatively longer perfect CA repeat sequences.

The Wilhelmine E. Key 1991 Invitational Lecture. The evolutionary history of the P family of transposable elements.

Similar to other transposable genetic elements, P elements occasionally exhibit non-Mendelian inheritance because of their ability to move, from one genomic site in their host species to another, during certain phases of their life cycle. The biological range of this capacity for transposition is almost always restricted to new sites within the same nuclear genome, but exceptionally it appears that interspecific horizontal transfer of P elements can occur. Although the P family appears to have had an ancient origin, its present natural distribution appears to be patchy and phylogenetically restricted to a limited number of Dipteran species. The most likely explanation of the observed restriction is the requirement of transposition for a host-encoded factor whose range is itself similarly restricted. Occasional horizontal transfer of P elements into a new host species, together with the normal mode of vertical transfer, may be the mechanism that ensures the survival of this, and other transposable element families, over long periods of evolutionary time.

琉球列島北部におけるアユの分布ならびにその遺伝的・形態的特徴

The ayu population in the middle of the Ryukyu Archipelago was recently found to differ considerably from that in the mainland of Japan, and was described as a distinct subspecies (Plecoglossus altivelis ryukyuensis). Little is, however, known about the border between the distributional ranges of nominotypical and the Ryukyuan subspecies. Observation carried out in the northern Ryukyus showed that the ayu also occurs in a few streams with relatively long middle reaches on Yaku Island, but that none inhabit Nakanoshima Island. Variations in meristic characters of specimens from Yaku Island were within those of the nominotypical subspecies. Allozyme analyses by starch gel electrophoresis showed that the ayu from Yaku Island was genetically much closer to the nominotypical subspecies from Kyushu and Honshu than to the Ryukyuan subspecies from Amami-oshima Island. We thus conclude that the ayu on Yaku Island represents the southernmost population of the nominotypical subspecies in the area of the Japan-Ryukyu Archipelago, and that the ayu on Amami-oshima Island forms the northernmost population of the Ryukyuan subspecies. Complete allele substitution detected in five enzyme loci, as well as the absence of any particular genetic similarity, between the populations of Yaku and Amami-oshima Islands, reinforces the previous hypothesis that the two subspecies have been isolated from each other genetically for a long period of evolutionary time.

MHC and Captive Breeding: A Rebuttal

In a recent Comment (Conservation Biology 5(2):249251), Austin Hughes advocated a radical reorientation of all captive breeding programs. His suggestion is that the major goal of captive breeding programs should be to maintain an even distribution of MHC in a captive population. This will, he points out, result in a skewed distribution away from an equal representation of founders, with concomitant increased inbreeding. That is, Hughes's strategy will entail greater loss of allelic diversity at other loci, but he dismisses these loci as neutral and unimportant. We agree that allelic diversity at the MHC locus is important and that Hughes's optimization ideas should not be summarily dismissed. However, we point out several errors in Hughes's analysis. First, he has grossly miscalculated extinction probabilities for loss of alleles under genetic drift. For example, he says that for N, = 200, the chance of fixing in 20 generations an allele initially at 90% frequency is 0.85. We calculate this probability as 0.03. Possibly, Hughes has used an Ne of 20. In any case, he is exaggerating the problem of allele loss by at least an order of magnitude. In a related calculation, Hughes looks at the loss of heterozygosity in a triallelic locus and shows, correctly, that a 10% loss of heterozygosity can occur in different ways, one involving allele loss and others not. We point out that these cases are not, however, equally probable. We have calculated the extinction probability of an allele initially at frequency of 10% as 0.15 at the point in the process at which the population heterozygosity has dropped by 10%; thus, 85% of the time the allele would still be present in such a population, though of course not in the frequency at which it started. Second, Hughes dismisses as neutral the entire nonMHC fraction of the genome (see Comment by Vrijenhoek and Leberg 1991). He cavalierly banishes a great many genes in the process. The number of gene loci in vertebrates has been variously estimated, but a generally agreed-on number is approximately 100,000. The number of environments the gene products of these loci face may be considerably greater than that; Walter Gilbert (personal communication) has estimated that because of alternative splicing in different tissues the number of different gene products produced by the genome may be more like 106. There is no way of knowing at the moment whether a polymorphism that has no effect when it is expressed in one tissue might produce a selective differential when it is expressed in another, but it does not seem unreasonable to expect this to happen occasionally. While it is also dangerous to extend to the entire genome estimates of the number of loci that carry nonsynonymous substitutions at appreciable frequency, judging by the data from soluble enzymes and blood groups it appears that about a third of them do. Let us suppose that in only 1% of these 3 x 105 potentially selective situations the polymorphic loci are selectively maintained. This is still 3000 loci, only a tiny fraction of which are known. MHC is the one we are currently aware of that is most obviously subject to balancing selection, but it verges on hubris to suggest that there may not be others equally or more important. Consider the A and B alleles at the ABO locus, which differ by four amino acids and specify very different glycosyltransferase activities (Yamamoto et al. 1990); these alleles, which have persisted for long periods of evolutionary time, appear unlikely to be neutral. Consider also the Rh

Two divergent routes of evolution gave rise to the DRw13 haplotypes.

The HLA class II genes and haplotypes have evolved over a long period of evolutionary time by mechanisms such as gene conversion, reciprocal recombination and point mutation. The extent of the diversity generated is most clearly evident in an analysis of the HLA class II alleles present within DRw13 haplotypes. This study uses cDNA sequencing to examine the first domains of DRB1, DRB3, DQA1, and DQB1 alleles from several American black individuals expressing seven different DRw13 haplotypes, five with undefined HLA-D specificities (i.e., not Dw18 or Dw19). Two new DRw13 alleles described in this study are the first examples of convergent evolution of DR alleles in which gene conversion has apparently combined segments of DRB1 alleles encoding DRw11 and DRw8 to generate two new DRB1 alleles, DRB1*1303 and DRB1*1304, that encode molecules bearing serologic determinants of a third allele, DRw13. These new DRw13 alleles are found embedded in haplotypes of DRw11 origin distinct from haplotypes encoding previously identified DRw13 alleles, DRB1*1301 and DRB1*1302. These data suggest that two evolutionary pathways may have given rise to two subgroups of alleles encoding molecules that share DRw13 serologic determinants yet which possess different structural and, likely, functional motifs. Reciprocal gene recombination events resulting in different DR, DRw52 and DQ allele combinations also appear to have played a crucial role in augmenting the level of diversity found in DRw13 haplotypes. Recombination has resulted in the association of one of the new DRw13 alleles with a DQw2 allele normally found associated with DR7 and the association of the DRw52c-associated DRw13 allele (DRB1*1302) with three different DQw1 alleles. The seven DRw13 haplotypes that have resulted from the effect of recombination on haplotypes formed by the two pathways of DRw13 allelic diversification have resulted in different repertoires of class II molecules and, most likely, different immune response profiles in individuals with these haplotypes.

Duplication of large genomic regions during the evolution of vertebrate homeobox genes.

The phylogenetic relationships of 21 murine Antp-class (Drosophila mutation Antennapedia-type class) homeobox genes have been analyzed, and several groups of related genes have been identified. The murine Antp-class homeobox genes are localized within four gene clusters. The similar structural organization of the four gene clusters strongly suggests that genes within a group of related Antp-class homeobox genes are derived from duplications of large genomic regions. After the duplication, the gross structures of the homeobox gene clusters have been maintained over a long period of evolutionary time, indicating that the specific organization of genes within a cluster may be of functional importance.