Immunocytochemical Detection of Dentin Matrix Proteins in Primary Teeth from Patients with Dentinogenesis Imperfecta Associated with Osteogenesis Imperfecta

Dentinogenesis imperfecta determines structural alterations of the collagen structure still not completely elucidated. Immunohisto-chemical analysis was used to assay type I and VI collagen, various non-collagenous proteins distribution in human primary teeth from healthy patients or from patients affected by type I dentinogenesis imperfecta (DGI-I) associated with osteogenesis imperfecta (OI). In sound primary teeth, an organized well-known ordered pattern of the type I collagen fibrils was found, whereas atypical and disorganized fibrillar structures were observed in dentin of DGI-I affected patients. Expression of type I collagen was observed in both normal and affected primary teeth, although normal dentin stained more uniformly than DGI-I affected dentin. Reactivity of type VI collagen was significantly lower in normal teeth than in dentin from DGI-I affected patients (P<0.05). Expressions of dentin matrix protein-1 (DMP1) and osteopontin (OPN) were observed in both normal dentin and dentin from DGI-I affected patients, without significant differences, being DMP1 generally more abundantly expressed. Immuno labeling for chondroitin sulfate (CS) and biglycan (BGN) was weaker in dentin from DGI-I-affected patients compared to normal dentin, this decrease being significant only for CS. This study shows ultra-structural alterations in dentin obtained from patients affected by DGI-I, supported by immunocytochemical assays of different collagenous and non-collagenous proteins.Key words: Osteogenesis imperfecta, dentinogenesis imperfecta, immuno-electron microscopy, collagen, non-collagenous proteins     

A screen for recessive speciation genes expressed in the gametes of F1 hybrid yeast

Diploid hybrids of Saccharomyces cerevisiae and its closest relative, Saccharomyces paradoxus, are viable, but the sexual gametes they produce are not. One of several possible causes of this gamete inviability is incompatibility between genes from different species—such incompatible genes are usually called “speciation genes.” In diploid F1 hybrids, which contain a complete haploid genome from each species, the presence of compatible alleles can mask the effects of (recessive) incompatible speciation genes. But in the haploid gametes produced by F1 hybrids, recessive speciation genes may be exposed, killing the gametes and thus preventing F1 hybrids from reproducing sexually. Here I present the results of an experiment to detect incompatibilities that kill hybrid gametes. I transferred nine of the 16 S. paradoxus chromosomes individually into S. cerevisiae gametes and tested the ability of each to replace its S. cerevisiae homeolog. All nine chromosomes were compatible, producing nine viable haploid strains, each with 15 S. cerevisiae chromosomes and one S. paradoxus chromosome. Thus, none of these chromosomes contain speciation genes that were capable of killing the hybrid gametes that received them. This is a surprising result that suggests that such speciation genes do not play a major role in yeast speciation.     

Genetic Mechanisms of Rare Matings of the Yeast SACCHAROMYCES CEREVISIAE Heterozygous for Mating Type

Yeast heterozygous for mating type lacks the ability to conjugate as judged by the mass-mating technique and accordingly is designated "non-mater". However, the non-mater shows rare mating ability with a frequency of less than 10^-6. In the present study, the RD auxotroph mating method was mainly employed with the intention of examining the rare mating ability of various non-maters, using lactate ethanol minimal medium as a selective medium for hybridization. Crosses of aα x a, aα x a, aaα x a, aαα x a, etc. resulted in the production of respective hybrids with a relatively high frequency of about 10^-6 to 10^-7, whereas crosses of aaα x a, aαα x α, aaαα x a, aaαα x α, etc. resulted in hybrids with an extremely low frequency of about less than 10^-8. Genetic analyses revealed that the rare matings were mostly caused by the presence of cells derived from the non-maters in which mating type had converted to a homozygous genotype. Mitotic recombination was shown to be a likely explanation for most of the conversion, judging from associated exchange of an outside marker, thr4. By successive employment of the RD auxotroph mating method, it was possible to produce a series of polyploid yeasts, triploids to octoploids. The DNA content and the cell volume were observed to increase parallel to the elevated ploidy states.     

The parasexual cycle in Candida albicans provides an alternative pathway to meiosis for the formation of recombinant strains

Candida albicans has an elaborate, yet efficient, mating system that promotes conjugation between diploid a and α strains. The product of mating is a tetraploid a/α cell that must undergo a reductional division to return to the diploid state. Despite the presence of several “meiosis-specific” genes in the C. albicans genome, a meiotic program has not been observed. Instead, tetraploid products of mating can be induced to undergo efficient, random chromosome loss, often producing strains that are diploid, or close to diploid, in ploidy. Using SNP and comparative genome hybridization arrays we have now analyzed the genotypes of products from the C. albicans parasexual cycle. We show that the parasexual cycle generates progeny strains with shuffled combinations of the eight C. albicans chromosomes. In addition, several isolates had undergone extensive genetic recombination between homologous chromosomes, including multiple gene conversion events. Progeny strains exhibited altered colony morphologies on laboratory media, demonstrating that the parasexual cycle generates phenotypic variants of C. albicans. In several fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, the conserved Spo11 protein is integral to meiotic recombination, where it is required for the formation of DNA double-strand breaks. We show that deletion of SPO11 prevented genetic recombination between homologous chromosomes during the C. albicans parasexual cycle. These findings suggest that at least one meiosis-specific gene has been re-programmed to mediate genetic recombination during the alternative parasexual life cycle of C. albicans. We discuss, in light of the long association of C. albicans with warm-blooded animals, the potential advantages of a parasexual cycle over a conventional sexual cycle.     

Adaptive gene expression divergence inferred from population genomics

Detailed studies of individual genes have shown that gene expression divergence often results from adaptive evolution of regulatory sequence. Genome-wide analyses, however, have yet to unite patterns of gene expression with polymorphism and divergence to infer population genetic mechanisms underlying expression evolution. Here, we combined genomic expression data—analyzed in a phylogenetic context—with whole genome light-shotgun sequence data from six Drosophila simulans lines and reference sequences from D. melanogaster and D. yakuba. These data allowed us to use molecular population genetics to test for neutral versus adaptive gene expression divergence on a genomic scale. We identified recent and recurrent adaptive evolution along the D. simulans lineage by contrasting sequence polymorphism within D. simulans to divergence from D. melanogaster and D. yakuba. Genes that evolved higher levels of expression in D. simulans have experienced adaptive evolution of the associated 3′ flanking and amino acid sequence. Concomitantly, these genes are also decelerating in their rates of protein evolution, which is in agreement with the finding that highly expressed genes evolve slowly. Interestingly, adaptive evolution in 5′ cis-regulatory regions did not correspond strongly with expression evolution. Our results provide a genomic view of the intimate link between selection acting on a phenotype and associated genic evolution.     

Odorant-binding proteins OBP57d and OBP57e affect taste perception and host-plant preference in Drosophila sechellia

Despite its morphological similarity to the other species in the Drosophila melanogaster species complex, D. sechellia has evolved distinct physiological and behavioral adaptations to its host plant Morinda citrifolia, commonly known as Tahitian Noni. The odor of the ripe fruit of M. citrifolia originates from hexanoic and octanoic acid. D. sechellia is attracted to these two fatty acids, whereas the other species in the complex are repelled. Here, using interspecies hybrids between D. melanogaster deficiency mutants and D. sechellia, we showed that the Odorant-binding protein 57e (Obp57e) gene is involved in the behavioral difference between the species. D. melanogaster knock-out flies for Obp57e and Obp57d showed altered behavioral responses to hexanoic acid and octanoic acid. Furthermore, the introduction of Obp57d and Obp57e from D. simulans and D. sechellia shifted the oviposition site preference of D. melanogaster Obp57d/e^KO flies to that of the original species, confirming the contribution of these genes to D. sechellia's specialization to M. citrifolia. Our finding of the genes involved in host-plant determination may lead to further understanding of mechanisms underlying taste perception, evolution of plant–herbivore interactions, and speciation.     

The genome sequence of Caenorhabditis briggsae: a platform for comparative genomics

The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.     

Asymmetrical reinforcement and Wolbachia infection in Drosophila

Reinforcement refers to the evolution of increased mating discrimination against heterospecific individuals in zones of geographic overlap and can be considered a final stage in the speciation process. One the factors that may affect reinforcement is the degree to which hybrid matings result in the permanent loss of genes from a species' gene pool. Matings between females of Drosophila subquinaria and males of D. recens result in high levels of offspring mortality, due to interspecific cytoplasmic incompatibility caused by Wolbachia infection of D. recens. Such hybrid inviability is not manifested in matings between D. recens females and D. subquinaria males. Here we ask whether the asymmetrical hybrid inviability is associated with a corresponding asymmetry in the level of reinforcement. The geographic ranges of D. recens and D. subquinaria were found to overlap across a broad belt of boreal forest in central Canada. Females of D. subquinaria from the zone of sympatry exhibit much stronger levels of discrimination against males of D. recens than do females from allopatric populations. In contrast, such reproductive character displacement is not evident in D. recens, consistent with the expected effects of unidirectional cytoplasmic incompatibility. Furthermore, there is substantial behavioral isolation within D. subquinaria, because females from populations sympatric with D. recens discriminate against allopatric conspecific males, whereas females from populations allopatric with D. recens show no discrimination against any conspecific males. Patterns of general genetic differentiation among populations are not consistent with patterns of behavioral discrimination, which suggests that the behavioral isolation within D. subquinaria results from selection against mating with Wolbachia-infected D. recens. Interspecific cytoplasmic incompatibility may contribute not only to post-mating isolation, an effect already widely recognized, but also to reinforcement, particularly in the uninfected species. The resulting reproductive character displacement not only increases behavioral isolation from the Wolbachia-infected species, but may also lead to behavioral isolation between populations of the uninfected species. Given the widespread occurrence of Wolbachia among insects, it thus appears that there are multiple ways by which these endosymbionts may directly and indirectly contribute to reproductive isolation and speciation.     

Complete Mitochondrial Genome and Phylogeny of Pleistocene MammothMammuthus primigenius

Phylogenetic relationships between the extinct woolly mammoth(Mammuthus primigenius), and the Asian(Elephas maximus) and African savanna(Loxodonta africana) elephants remain unresolved. Here, we report the sequence of the complete mitochondrial genome (16,842 base pairs) of a woolly mammoth extracted from permafrost-preserved remains from the Pleistocene epoch—the oldest mitochondrial genome sequence determined to date. We demonstrate that well-preserved mitochondrial genome fragments, as long as ~1,600–1700 base pairs, can be retrieved from pre-Holocene remains of an extinct species. Phylogenetic reconstruction of the Elephantinae clade suggests thatM. primigenius andE. maximus are sister species that diverged soon after their common ancestor split from theL. africana lineage. Low nucleotide diversity found between independently determined mitochondrial genomic sequences of woolly mammoths separated geographically and in time suggests that north-eastern Siberia was occupied by a relatively homogeneous population ofM. primigenius throughout the late Pleistocene.     

Als3 is a Candida albicans invasin that binds to cadherins and induces endocytosis by host cells

Candida albicans is the most common cause of hematogenously disseminated and oropharyngeal candidiasis. Both of these diseases are characterized by fungal invasion of host cells. Previously, we have found that C. albicans hyphae invade endothelial cells and oral epithelial cells in vitro by inducing their own endocytosis. Therefore, we set out to identify the fungal surface protein and host cell receptors that mediate this process. We found that the C. albicans Als3 is required for the organism to be endocytosed by human umbilical vein endothelial cells and two different human oral epithelial lines. Affinity purification experiments with wild-type and an als3Δ/als3Δ mutant strain of C. albicans demonstrated that Als3 was required for C. albicans to bind to multiple host cell surface proteins, including N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. Furthermore, latex beads coated with the recombinant N-terminal portion of Als3 were endocytosed by Chinese hamster ovary cells expressing human N-cadherin or E-cadherin, whereas control beads coated with bovine serum albumin were not. Molecular modeling of the interactions of the N-terminal region of Als3 with the ectodomains of N-cadherin and E-cadherin indicated that the binding parameters of Als3 to either cadherin are similar to those of cadherin–cadherin binding. Therefore, Als3 is a fungal invasin that mimics host cell cadherins and induces endocytosis by binding to N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. These results uncover the first known fungal invasin and provide evidence that C. albicans Als3 is a molecular mimic of human cadherins.     

Homozygosity for a Missense Mutation in SERPINH1, which Encodes the Collagen Chaperone Protein HSP47, Results in Severe Recessive Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is characterized by bone fragility and fractures that may be accompanied by bone deformity, dentinogenesis imperfecta, short stature, and shortened life span. About 90% of individuals with OI have dominant mutations in the type I collagen genes COL1A1 and COL1A2. Recessive forms of OI resulting from mutations in collagen-modifying enzymes and chaperones CRTAP, LEPRE1, PPIB, and FKBP10 have recently been identified. We have identified an autosomal-recessive missense mutation (c.233T>C, p.Leu78Pro) in SERPINH1, which encodes the collagen chaperone-like protein HSP47, that leads to a severe OI phenotype. The mutation results in degradation of the endoplasmic reticulum resident HSP47 via the proteasome. Type I procollagen accumulates in the Golgi of fibroblasts from the affected individual and a population of the secreted type I procollagen is protease sensitive. These findings suggest that HSP47 monitors the integrity of the triple helix of type I procollagen at the ER/cis-Golgi boundary and, when absent, the rate of transit from the ER to the Golgi is increased and helical structure is compromised. The normal 3-hydroxylation of the prolyl residue at position 986 of the triple helical domain of proα1(I) chains places the role of HSP47 downstream from the CRTAP/P3H1/CyPB complex that is involved in prolyl 3-hydroxylation. Identification of this mutation in SERPINH1 gives further insight into critical steps of the collagen biosynthetic pathway and the molecular pathogenesis of OI.     

Helicobacter pylori evolution: lineage- specific adaptations in homologs of eukaryotic Sel1-like genes

Geographic partitioning is postulated to foster divergence of Helicobacter pylori populations as an adaptive response to local differences in predominant host physiology. H. pylori's ability to establish persistent infection despite host inflammatory responses likely involves active management of host defenses using bacterial proteins that may themselves be targets for adaptive evolution. Sequenced H. pylori genomes encode a family of eight or nine secreted proteins containing repeat motifs that are characteristic of the eukaryotic Sel1 regulatory protein, whereas the related Campylobacter and Wolinella genomes each contain only one or two such “Sel1-like repeat” (SLR) genes (“slr genes”). Signatures of positive selection (ratio of nonsynonymous to synonymous mutations, d_N/d_S = ω > 1) were evident in the evolutionary history of H. pylori slr gene family expansion. Sequence analysis of six of these slr genes (hp0160, hp0211, hp0235, hp0519, hp0628, and hp1117) from representative East Asian, European, and African H. pylori strains revealed that all but hp0628 had undergone positive selection, with different amino acids often selected in different regions. Most striking was a divergence of Japanese and Korean alleles of hp0519, with Japanese alleles having undergone particularly strong positive selection (ω_J > 25), whereas alleles of other genes from these populations were intermingled. Homology-based structural modeling localized most residues under positive selection to SLR protein surfaces. Rapid evolution of certain slr genes in specific H. pylori lineages suggests a model of adaptive change driven by selection for fine-tuning of host responses, and facilitated by geographic isolation. Characterization of such local adaptations should help elucidate how H. pylori manages persistent infection, and potentially lead to interventions tailored to diverse human populations.     

Distribution and Frequency of Tandem Duplications of the rII Region of Bacteriophage T4d

Genetic analyses of 49 duplications of the rII region of bacteriophage T4D suggests that there is a non-random relationship between the end points of duplicated segments, that relaxed packaging restrictions have little if any effect on the distribution of duplications, that segregation is 3–4 times more frequent than normal recombination for the same interval, and that non-tandem duplications are rare. Extrapolation of the r1231 x rJ101 cross data suggests that the minimum frequency of duplications/genome is 1.7 x 10^-6, but possibly 3.4 x 10^-4.     

The genetic system controlling recombination in the silkworm

The nature of recombination modifiers was investigated in Bombyx mori lines selected for high (H) and low (L) recombination rates between the p^S and Y loci in chromosome 2. Since the mean recombination rates for the H x L and L x H F₁ crosses were approximately intermediate between those of high and low lines, the cytoplasmic maternal effect and difference in the activity of recombination modifiers between marked and unmarked second chromosomes were not detected. The H x (L x H), H x (H x L), L x (L x H) and L x (H x L) backcrosses indicated the presence of additive and dominance effects of marked and unmarked second chromosomes and the remaining chromosomes.——Recombination rates between the p^S and Y loci in chromosome 2 and half-nonrecombination rates between the pe and re loci in chromosome 5 of high and low lines indicated that these recombination modifiers caused changes in the recombination frequency between p^S and Y in chromosome 2, but not between pe and re in chromosome 5.——There were no differences in viability between individuals having the second chromosomes of the recombinant types [p^S +, p^Y (H); p^S +, + Y (L)] and those of the nonrecombinant types [p^S Y, p + (H); p^S Y, + + (L)] in both high and low lines. Mean recombination rates measured in cis [p^S Y/p + (H); p^S Y/+ + (L)] and trans [p^S +/p Y (H); p^S +/+ Y (L)] males were the same in the high but not in the low line. No segregation of a single recombination modifier was indicated by the distribution of recombination rates measured in trans males [p^S +/p Y (H); p^S +/+ Y (L)] of high and low lines. Accordingly, the recombination modifiers distributed on chromosome 2 in the heterozygous condition were not gross chromosomal aberrations, but polygenic factors in the low line.     

The evolution of spinnable cotton fiber entailed prolonged development and a novel metabolism

A central question in evolutionary biology concerns the developmental processes by which new phenotypes arise. An exceptional example of evolutionary innovation is the single-celled seed trichome in Gossypium (“cotton fiber”). We have used fiber development in Gossypium as a system to understand how morphology can rapidly evolve. Fiber has undergone considerable morphological changes between the short, tightly adherent fibers of G. longicalyx and the derived long, spinnable fibers of its closest relative, G. herbaceum, which facilitated cotton domestication. We conducted comparative gene expression profiling across a developmental time-course of fibers from G. longicalyx and G. herbaceum using microarrays with ~22,000 genes. Expression changes between stages were temporally protracted in G. herbaceum relative to G. longicalyx, reflecting a prolongation of the ancestral developmental program. Gene expression and GO analyses showed that many genes involved with stress responses were upregulated early in G. longicalyx fiber development. Several candidate genes upregulated in G. herbaceum have been implicated in regulating redox levels and cell elongation processes. Three genes previously shown to modulate hydrogen peroxide levels were consistently expressed in domesticated and wild cotton species with long fibers, but expression was not detected by quantitative real time-PCR in wild species with short fibers. Hydrogen peroxide is important for cell elongation, but at high concentrations it becomes toxic, activating stress processes that may lead to early onset of secondary cell wall synthesis and the end of cell elongation. These observations suggest that the evolution of long spinnable fibers in cotton was accompanied by novel expression of genes assisting in the regulation of reactive oxygen species levels. Our data suggest a model for the evolutionary origin of a novel morphology through differential gene regulation causing prolongation of an ancestral developmental program.     

Frequency of gamma-Ray Induced Specific Locus and Recessive Lethal Mutations in Mature Germ Cells of the Zebrafish, BRACHYDANIO RERIO

Specific locus and recessive lethal mutations are induced by γ-rays with approximately first order kinetics in the zebrafish (Brachydanio rerio) with frequencies of 4 x 10^-5 r^-1 and 4 x 10^-3 r^-1, respectively. The surprisingly low ratio (100:1) of recessive lethals to specific locus mutations may be due to the induction of large deficiencies by γ-rays.     

A Comparison of Mutation Rates for Specific Loci and Chromosome Regions in Dysgenic Hybrid Males of DROSOPHILA MELANOGASTER

The mutation rates of specific loci and chromosome regions were estimated for two types of dysgenic hybrid males. These came from crosses between P or Q males and M females in the P-M system of hybrid dysgenesis. The M x P hybrids were the more mutable for each of the loci and chromosome regions tested. The Beadex locus was highly mutable in these hybrids but did not mutate at all in the sample of gametes from the M x Q hybrids. The singed locus had 75% of the mutability of Beadex in the M x P hybrids; it was also mutable in the M x Q hybrids. The white locus was only slightly mutable in the M x P hybrids and not at all mutable in the M x Q hybrids. The mutations in singed and white probably arose from the insertion of P elements into these loci; the mutations at Beadex probably involved the action of a P element located near this locus on the X chromosome of the P strain that was used in the experiments. Mutations in two chromosome regions, one including the zeste-white loci and the other near the miniature locus, were much more frequent in the M x P hybrids than in the M x Q hybrids. These mutations also probably arose from P element insertions. The implication is that insertion mutations occur infrequently in the M x Q hybrids, possibly because most of the P elements they carry are defective. In M x P hybrids, there is variation among loci with respect to P elements mutagenesis, indicating that P elements possess a degree of insertional specificity.     

Type III effector diversification via both pathoadaptation and horizontal transfer in response to a coevolutionary arms race

The concept of the coevolutionary arms race holds a central position in our understanding of pathogen–host interactions. Here we identify the molecular mechanisms and follow the stepwise progression of an arms race in a natural system. We show how the evolution and function of the HopZ family of type III secreted effector proteins carried by the plant pathogen Pseudomonas syringae are influenced by a coevolutionary arms race between pathogen and host. We surveyed 96 isolates of P. syringae and identified three homologs (HopZ1, HopZ2, and HopZ3) distributed among ~45% of the strains. All alleles were sequenced and their expression was confirmed. Evolutionary analyses determined that the diverse HopZ1 homologs are ancestral to P. syringae, and have diverged via pathoadaptive mutational changes into three functional and two degenerate forms, while HopZ2 and HopZ3 have been brought into P. syringae via horizontal transfer from other ecologically similar bacteria. A PAML selection analysis revealed that the C terminus of HopZ1 is under strong positive selection. Despite the extensive genetic variation observed in this family, all three homologs have cysteine–protease activity, although their substrate specificity may vary. The introduction of the ancestral hopZ1 allele into strains harboring alternate alleles results in a resistance protein-mediated defense response in their respective hosts, which is not observed with the endogenous allele. These data indicate that the P. syringae HopZ family has undergone allelic diversification via both pathoadaptive mutational changes and horizontal transfer in response to selection imposed by the host defense system. This genetic diversity permits the pathogen to avoid host defenses while still maintaining a virulence-associated protease, thereby allowing it to thrive on its current host, while simultaneously impacting its host range.     

Functional divergence caused by ancient positive selection of a Drosophila hybrid incompatibility locus

Interspecific hybrid lethality and sterility are a consequence of divergent evolution between species and serve to maintain the discrete identities of species. The evolution of hybrid incompatibilities has been described in widely accepted models by Dobzhansky and Muller where lineage-specific functional divergence is the essential characteristic of hybrid incompatibility genes. Experimentally tractable models are required to identify and test candidate hybrid incompatibility genes. Several Drosophila melanogaster genes involved in hybrid incompatibility have been identified but none has yet been shown to have functionally diverged in accordance with the Dobzhansky-Muller model. By introducing transgenic copies of the X-linked Hybrid male rescue (Hmr) gene into D. melanogaster from its sibling species D. simulans and D. mauritiana, we demonstrate that Hmr has functionally diverged to cause F1 hybrid incompatibility between these species. Consistent with the Dobzhansky-Muller model, we find that Hmr has diverged extensively in the D. melanogaster lineage, but we also find extensive divergence in the sibling-species lineage. Together, these findings implicate over 13% of the amino acids encoded by Hmr as candidates for causing hybrid incompatibility. The exceptional level of divergence at Hmr cannot be explained by neutral processes because we use phylogenetic methods and population genetic analyses to show that the elevated amino-acid divergence in both lineages is due to positive selection in the distant past—at least one million generations ago. Our findings suggest that multiple substitutions driven by natural selection may be a general phenomenon required to generate hybrid incompatibility alleles.     

Incipient Genome Differentiation in Gossypium. II. Comparison of 12 Chromosomes in G. HIRSUTUM, G. MUSTELINUM and G. TOMENTOSUM Using Heterozygous Translocations

Eight homozygous translocation lines (TT) of G. hirsutum marking 3 chromosomes of the A genome and 9 chromosomes of the D genome were crossed with G. hirsutum, G. mustelinum and G. tomentosum, all homozygous for the standard end arrangements (tt). Chiasma frequencies in the G. hirsutum Tt controls were compared with those in the G. hirsutum x G. mustelinum and the G. hirsutum x G. tomentosum Tt hybrids. Both nucleus-wide and region-specific chiasma frequencies were compared.—Some genome differentiation appears to have arisen between G. hirsutum and G. mustelinum. The G. hirsutum x G. mustelinum hybrids had a 1.8 to 1.9% reduction in the nucleus-wide chiasma frequency. Four of the eight TT lines showed a 3.4 to 10.5% reduction in chiasmata in the hybrid translocation quadrivalents, suggesting that chromosomes 1, 21, 23 and 24 may have undergone localized genome differentiation. The two species may differ naturally in the end arrangement of two chromosomes, since a quadrivalent not due to experimentally introduced translocations was observed in 13% of the PMC's of two G. hirsutum x G. mustelinum hybrids.—Very little genome differentiation has occurred between G. hirsutum and G. tomentosum. In the G. hirsutum x G. tomentosum hybrids, the nucleus-wide estimates showed only a very small (0.1 to 0.2%), though statistically significant, lowering of the chiasma frequency, and there was no reduction in chiasma frequency in the more sensitive readings for specific translocation quadrivalents.