HET-E and HET-D belong to a new subfamily of WD40 proteins involved in vegetative incompatibility specificity in the fungus Podospora anserina

Vegetative incompatibility, which is very common in filamentous fungi, prevents a viable heterokaryotic cell from being formed by the fusion of filaments from two different wild-type strains. Such incompatibility is always the consequence of at least one genetic difference in specific genes (het genes). In Podospora anserina, alleles of the het-e and het-d loci control heterokaryon viability through genetic interactions with alleles of the unlinked het-c locus. The het-d2(Y) gene was isolated and shown to have strong similarity with the previously described het-e1(A) gene. Like the HET-E protein, the HET-D putative protein displayed a GTP-binding domain and seemed to require a minimal number of 11 WD40 repeats to be active in incompatibility. Apart from incompatibility specificity, no other function could be identified by disrupting the het-d gene. Sequence comparison of different het-e alleles suggested that het-e specificity is determined by the sequence of the WD40 repeat domain. In particular, the amino acids present on the upper face of the predicted beta-propeller structure defined by this domain may confer the incompatible interaction specificity.     

WD-repeat instability and diversification of the <Emphasis Type="Italic">Podospora anserina hnwd</Emphasis> non-self recognition gene family

Background  

Genes involved in non-self recognition and host defence are typically capable of rapid diversification and exploit specialized genetic mechanism to that end. Fungi display a non-self recognition phenomenon termed heterokaryon incompatibility that operates when cells of unlike genotype fuse and leads to the cell death of the fusion cell. In the fungus Podospora anserina, three genes controlling this allorecognition process het-d, het-e and het-r are paralogs belonging to the same hnwd gene family. HNWD proteins are STAND proteins (signal transduction NTPase with multiple domains) that display a WD-repeat domain controlling recognition specificity. Based on genomic sequence analysis of different P. anserina isolates, it was established that repeat regions of all members of the gene family are extremely polymorphic and undergoing concerted evolution arguing for frequent recombination within and between family members.     

The fungus-specific HET domain mediates programmed cell death in Podospora anserina

Vegetative incompatibility is a programmed cell death reaction that occurs when fungal cells of unlike genotypes fuse. Genes defining vegetative incompatibility (het genes) are highly polymorphic, and most if not all incompatibility systems include a protein partner bearing the fungus-specific domain termed the HET domain. The nonallelic het-C/het-E incompatibility system is the best-characterized incompatibility system in Podospora anserina. Cell death is triggered by interaction of specific alleles of het-C, encoding a glycolipid transfer protein, and het-E, encoding a HET domain and a WD repeat domain involved in recognition. We show here that overexpression of the isolated HET domain from het-E results in cell death. This cell death is characterized by induction of autophagy, increased vacuolization, septation, and production of lipid droplets, which are hallmarks of cell death by incompatibility. In addition, the HET domain lethality is suppressed by the same mutations as vegetative incompatibility, but not by the inactivation of het-C. These results establish the HET domain as the mediator of cell death by incompatibility and lead to a modular conception of incompatibility systems whereby recognition is ensured by the variable regions of incompatibility proteins and cell death is triggered by the HET domain.     

Model systems of invertebrate allorecognition

Nearly all colonial marine invertebrates are capable of allorecognition--the ability to distinguish between self and genetically distinct members of the same species. When two or more colonies grow into contact, they either reject each other and compete for the contested space or fuse and form a single, chimeric colony. The specificity of this response is conferred by genetic systems that restrict fusion to self and close kin. Two selective pressures, intraspecific spatial competition between whole colonies and competition between stem cells for access to the germline in fused chimeras, are thought to drive the evolution of extensive polymorphism at invertebrate allorecognition loci. After decades of study, genes controlling allorecognition have been identified in two model systems, the protochordate Botryllus schlosseri and the cnidarian Hydractinia symbiolongicarpus. In both species, allorecognition specificity is determined by highly polymorphic cell-surface molecules, encoded by the fuhc and fester genes in Botryllus, and by the alr1 and alr2 genes in Hydractinia. Here we review allorecognition phenomena in both systems, summarizing recent molecular advances, comparing and contrasting the life history traits that shape the evolution of these distinct allorecognition systems, and highlighting questions that remain open in the field.     

Eighteen new tetranucleotide microsatellite DNA markers for Coregonus lavaretus cloned from an alpine lake population

We developed 18 polymorphic microsatellite markers for Coregonus lavaretus from genomic libraries enriched for (GACA)(n) and (GATA)(n) repeat sequences. Emphasis was placed on developing highly polymorphic, perfect repeats. These loci were screened in 69 individuals from two alpine populations in Austria. Allelic variation was high with nine to 37 alleles per locus and expected heterozygosities ranging from 0.37 to 0.95. The high level of polymorphism revealed by these loci will be relevant for population studies in context to the evolutionary history of this species.     

A high frequency of length polymorphisms in repeated sequences adjacent to Alu sequences

We describe a new class of DNA length polymorphism that is due to a variation in the number of tandem repeats associated with Alu sequences (Alu sequence-related polymorphisms). The polymerase chain reaction was used to selectively amplify a (TTA)n repeat identified in the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene from genomic DNA of 41 human subjects, and the size of the amplified products was determined by gel electrophoresis. Seven alleles were found that differed in size by integrals of three nucleotides. The allele frequencies ranged from 1.5% to 52%, and the overall heterozygosity index was 62%. The polymorphic TTA repeat was located adjacent to a repetitive sequence of the Alu family. A homology search of human genomic DNA sequences for the trinucleotide TTA (at least five members in length) revealed tandem repeats in six other genes. Three of the six (TTA)n repeats were located adjacent to Alu sequences, and two of the three (in the genes for beta-tubulin and interleukin-1 alpha) were found to be polymorphic in length. Tandemly repetitive sequences found in association with Alu sequences may be frequent sites of length polymorphism that can be used as genetic markers for gene mapping or linkage analysis.     

Variable numbers of tandem repeat loci in genetically homogeneous Haemophilus influenzae strains alter during persistent colonisation of cystic fibrosis patients

Serial sputum isolates of Haemophilus influenzae (n = 69) were obtained from eight patients suffering from cystic fibrosis. For two of these patients all strains were analysed for polymorphism in the major outer membrane protein profile. For all patients the strains were genetically characterised by random amplification of polymorphic DNA analysis. All strains were included in a survey for polymorphism in regions containing moieties of repetitive DNA as well. A single locus containing trinucleotide repeat units, three loci harbouring tetranucleotides, one region comprising pentanucleotide units and two hexanucleotide repeat unit-containing loci were analysed for repeat number variability. Most of the regions were previously shown to be directly adjacent to or even within virulence genes. All regions behaved as genuine variable number of tandem repeat loci in the sense that genetic polymorphism based on the presence of varying numbers of repeat units could be demonstrated among different strains. Interestingly, several of the repeats showed variation in the absence of the variability as assessed by major outer membrane protein or random amplification of polymorphic DNA analysis. These observations indicate that the repeat loci may vary independently from major chromosomal polymorphism. Consequently, H. influenzae appears to modify its virulence gene regions of the chromosome during persistent colonisation of the lung in cystic fibrosis patients.     

Conservation of (GA)n microsatellite loci between Quercus species

Microsatellite (simple sequence repeat, SSR) amplification was performed in eight different members of the Fagaceae family by using sets of primers developed from sessile oak, Quercus petraea . In total, 136 cases of heterologous amplification were carried out, and 66% resulted in interpretable amplification products. From these, 12 PCR amplification products were sequenced and all 12 contained a sequence homologous to the original locus from Q. petraea . Although SSR primers worked even across different genera, with increasing evolutionary distance there was a clear tendency for decreasing ability to successfully amplify loci and a decreasing proportion of polymorphism amongst those markers which could be amplified. Two of the loci, ssrQpZAG46 and ssrQpZAG110, were polymorphic in all Quercus species tested. Only at one locus, ssrQpZAG58, a specific PCR product could be amplified in all species analysed. For four loci found in two species, we observed significant interspecies differences in the size range of the amplified alleles. Sequence analysis of two alleles showed that the size differences are not only due to variations in the number of (GA) repeats but also to an insertion of approximately 80 nucleotides in the flanking region. Our findings prove the usefulness of SSR markers within and amongst closely related genera of plants.     

Repeat polymorphisms within gene regions: phenotypic and evolutionary implications

We have developed an algorithm that predicted 11,265 potentially polymorphic tandem repeats within transcribed sequences. We estimate that 22% (2,207/9,717) of the annotated clusters within UniGene contain at least one potentially polymorphic locus. Our predictions were tested by allelotyping a panel of approximately 30 individuals for 5% of these regions, confirming polymorphism for more than half the loci tested. Our study indicates that tandem-repeat polymorphisms in genes are more common than is generally believed. Approximately 8% of these loci are within coding sequences and, if polymorphic, would result in frameshifts. Our catalogue of putative polymorphic repeats within transcribed sequences comprises a large set of potentially phenotypic or disease-causing loci. In addition, from the anomalous character of the repetitive sequences within unannotated clusters, we also conclude that the UniGene cluster count substantially overestimates the number of genes in the human genome. We hypothesize that polymorphisms in repeated sequences occur with some baseline distribution, on the basis of repeat homogeneity, size, and sequence composition, and that deviations from that distribution are indicative of the nature of selection pressure at that locus. We find evidence of selective maintenance of the ability of some genes to respond very rapidly, perhaps even on intragenerational timescales, to fluctuating selective pressures.     

Locus-specific conservation of the HLA class I introns by intra-locus homogenization

 The protein-coding sequences of the major histocompatibility complex (MHC) genes are characterized by extraordinarily high polymorphism, apparently maintained by balancing selection, which favors diversity in the peptide-binding domains of the MHC glycoproteins. Here we report that the introns flanking the polymorphic exons of the human MHC class I loci HLA-A, -B, and -C genes have been relatively conserved and have become locus-specific apparently as a result of recombination and subsequent genetic drift, leading to homogenization within loci over evolutionary time. Thus, HLA class I genes have been shaped by contrasting evolutionary forces maintaining polymorphism in the exons and leading to conservation in the introns. This study provides the first extensive analysis of the introns of a highly polymorphic gene family. Received: 10 April 1997 / Revised: 15 July 1997     

A comprehensive survey of human Y-chromosomal microsatellites

We have screened the nearly complete DNA sequence of the human Y chromosome for microsatellites (short tandem repeats) that meet the criteria of having a repeat-unit size of > or = 3 and a repeat count of > or = 8 and thus are likely to be easy to genotype accurately and to be polymorphic. Candidate loci were tested in silico for novelty and for probable Y specificity, and then they were tested experimentally to identify Y-specific loci and to assess their polymorphism. This yielded 166 useful new Y-chromosomal microsatellites, 139 of which were polymorphic, in a sample of eight diverse Y chromosomes representing eight Y-SNP haplogroups. This large sample of microsatellites, together with 28 previously known markers analyzed here--all sharing a common evolutionary history--allowed us to investigate the factors influencing their variation. For simple microsatellites, the average repeat count accounted for the highest proportion of repeat variance (approximately 34%). For complex microsatellites, the largest proportion of the variance (again, approximately 34%) was explained by the average repeat count of the longest homogeneous array, which normally is variable. In these complex microsatellites, the additional repeats outside the longest homogeneous array significantly increased the variance, but this was lower than the variance of a simple microsatellite with the same total repeat count. As a result of this work, a large number of new, highly polymorphic Y-chromosomal microsatellites are now available for population-genetic, evolutionary, genealogical, and forensic investigations.     

Isolation, characterization, and mapping of the mouse and human WDR8 genes, members of a novel WD-repeat gene family

The Trp-Asp (WD) motif has been shown to exist in a number of proteins. Genes containing repeats of the WD motif compose a large gene family associated with a variety of cellular functions and can be divided into a number of functional subfamilies. By means of the differential display method using ttw, a mouse model for the early stage of ectopic ossification, we have identified a novel mouse gene, Wdr8 (WD repeat domain 8), which contains two WD repeats, together with its human orthologue. The human and mouse WDR8 genes encode 460 and 462 amino acids, respectively, with 89% identity, and are expressed in almost all tissues, including bone and cartilage, and in bone-forming cells, including osteoblasts and chondrocytes. Wdr8 expression in cartilage was differentially displayed by stimuli for ectopic ossification in ttw and was observed strongly only at a transition period from hypertrophic to mineralizing stages in ATDC5, a chondrogenic cell line that exhibits endochondral ossification, suggesting a potential role for Wdr8 in the process of ossification. The WDR8 protein is highly conserved among a variety of species, but is distinctly different from other WD-repeat proteins, indicating that it represents a novel subfamily of the WD-repeat gene family.     

The GH18 family of chitinases: their domain architectures, functions and evolutions

The glycoside hydrolase 18 (GH18) family of chitinases is a multigene family that plays various roles, such as ecdysis, embryonic development, allergic inflammation and so on. Efforts are still needed to reveal their functional diversification in an evolutionary and systematic manner. We collected 85 GH18 genes from eukaryotic representatives. The domain architectures of GH18 proteins were analyzed and several conserved patterns were identified. It was observed that some (11 proteins) GH18 members in Ecdysozoa or fungi possess repeats of catalytic domains and/or chitin-binding domains (ChtBs). The domain repeats are likely to meet requirements for higher efficiency of chitin degradation in chitin-containing species. On the contrary, all vertebrate GH18 proteins contain no more than one catalytic domain or ChtB. The results from homologous analysis, domain architectures, exon arrangements and synteny loci supported two evolutionary paths for the GH18 family. One path experienced gene expansion and contraction several times during evolution, covering most of GH18 members except CHID1 (stabilin-1 interacting partner) and its homologs. Proteins in this path underwent frequent domain gain and loss, as well as domain recombination, that could achieve versatility in function. The other path is comparatively conserved. The CHID1 gene evolved without gene duplication except in Danio rerio. Domain architectures of CHID1 orthologs are all identical. The diverse phylogeny of the GH18 family in arthropod is also presented.     

Evolution of genes and repeats in the Nimrod superfamily

The recently identified Nimrod superfamily is characterized by the presence of a special type of EGF repeat, the NIM repeat, located right after a typical CCXGY/W amino acid motif. On the basis of structural features, nimrod genes can be divided into three types. The proteins encoded by Draper-type genes have an EMI domain at the N-terminal part and only one copy of the NIM motif, followed by a variable number of EGF-like repeats. The products of Nimrod B-type and Nimrod C-type genes (including the eater gene) have different kinds of N-terminal domains, and lack EGF-like repeats but contain a variable number of NIM repeats. Draper and Nimrod C-type (but not Nimrod B-type) proteins carry a transmembrane domain. Several members of the superfamily were claimed to function as receptors in phagocytosis and/or binding of bacteria, which indicates an important role in the cellular immunity and the elimination of apoptotic cells. In this paper, the evolution of the Nimrod superfamily is studied with various methods on the level of genes and repeats. A hypothesis is presented in which the NIM repeat, along with the EMI domain, emerged by structural reorganizations at the end of an EGF-like repeat chain, suggesting a mechanism for the formation of novel types of repeats. The analyses revealed diverse evolutionary patterns in the sequences containing multiple NIM repeats. Although in the Nimrod B and Nimrod C proteins show characteristics of independent evolution, many internal NIM repeats in Eater sequences seem to have undergone concerted evolution. An analysis of the nimrod genes has been performed using phylogenetic and other methods and an evolutionary scenario of the origin and diversification of the Nimrod superfamily is proposed. Our study presents an intriguing example how the evolution of multigene families may contribute to the complexity of the innate immune response.     

Linkage mapping of the highly informative DNA marker D21S156 to human chromosome 21 using a polymorphic GT dinucleotide repeat

A (GT)n repeat within the anonymous DNA sequence D21S156 was shown to be highly polymorphic in DNA from members of the 40 CEPH families. At least 12 alleles of this locus were recognized by electrophoresis on polyacrylamide gels of DNA amplified by the polymerase chain reaction (PCR) using primers flanking the (GT)n repeat. The polymorphism information content was 0.82. PCR amplification of DNA from somatic cell hybrid lines mapped D21S156 to human chromosome 21 and linkage analysis localized this marker close to the loci ETS2, D21S3, and HMG14 on chromosomal band 21q22.3. This polymorphism is highly informative and can serve as an anchor locus for human chromosome 21.     

Comparative Genetics of Functional Trinucleotide Tandem Repeats in Humans and Apes

Several human neurodegenerative disorders are caused by the expansion of polymorphic trinucleotide repeat regions. Many of these loci are functional short tandem repeats (STRs) located in brain-expressed genes, and their study is thus relevant from both a medical and an evolutionary point of view. The aims of our study are to infer the comparative pattern of variation and evolution of this set of loci in order to show species-specific features in this group of STRs and on their potential for expansion (therefore, an insight into evolutionary medicine) and to unravel whether any human-specific feature may be identified in brain-expressed genes involved in human disease. We analyzed the variability of the normal range of seven expanding STR CAG/CTG loci (SCA1, SCA2, SCA3-MJD, SCA6, SCA8, SCA12, and DRPLA) and two nonexpanding polymorphic CAG loci (KCNN3 and NCOA3) in humans, chimpanzees, gorillas, and orangutans. The study showed a general conservation of the repetitive tract and of the polymorphism in the four species and high heterogeneity among loci distributions. Humans present slightly larger alleles than the rest of species but a more relevant difference appears in variability levels: Humans are the species with the largest variance, although only for the expanding loci, suggesting a relationship between variability levels and expansion potential. The sequence analysis shows high levels of sequence conservation among species, a lack of correspondence between interruption patterns and variability levels, and signs of conservative selective pressure for some of the STR loci. Only two loci (SCA1 and SCA8) show a human specific distribution, with larger alleles than the rest of species. This could account, at the same time, for a human-specific trait and a predisposition to disease through expansion.This article contains online supplementary material.     

Length of uninterrupted repeats determines instability at the unstable mouse expanded simple tandem repeat family MMS10 derived from independent SINE B1 elements

Mouse expanded simple tandem repeats (ESTRs) provide highly informative loci for analyzing spontaneous and induced germline mutation. We have conducted an extensive sequence database search and identified 17 new members of the highly unstable rodent-specific ESTR family called MMS10. This family has arisen by independent expansions of a common GGCAGA repeat unit from within a subset of both ancestral and modern SINE B1 elements during the course of mouse evolution. Analysis of the interspersion patterns of variant repeats along alleles of 20 of these MMS10 loci revealed two distinct classes of tandem arrays: one composed of uninterrupted GGCAGA repeats and the second with generally larger arrays interrupted by variant units. Surveys of allelic diversity at 11 representative members of these two classes of loci in various laboratory strains and BXD recombinant inbred lines revealed that the level of repeat instability was positively correlated with the length of uninterrupted repeats. Turnover processes at MMS10 loci, therefore, appear similar to the type of mechanism observed at human microsatellites. The MMS10 family thus provides a potentially useful murine model for studying dynamic mutation at simple tandem repeats.     

Instability of interstitial telomeric sequences in the human genome

The length variability of four human interstitial telomeric sequences (ITs) is described. Three of the ITs contain short telomeric stretches ranging between 53 and 84 bp and are localized in 21q22, 2q31, and 7q36; the fourth IT derives from the subtelomeric domain of chromosome 6p and contains a tract of a few hundred basepairs of exact and degenerate repeats. Using primers flanking the repeats, we amplified the genomic DNA from unrelated individuals and from family members, and we found that all the loci are polymorphic. At the 21q22 IT locus, two equally frequent alleles were found, while the number of alleles at the 2q31, 7q36, and 6pter IT loci was 8, 6, and 4, respectively. Sequence analysis revealed that in the three loci containing short ITs the alleles differ from one another for multiples of the hexanucleotide; it is likely that the mechanism leading to the polymorphism is DNA polymerase slippage. These loci were also unstable in gastric tumor cells characterized by microsatellite instability. At the 6pter IT locus, the four alleles range in length from about 500 to about 700 bp; this variability is probably due to unequal exchange or gene conversion. Our data indicate that stretches of exact internal telomeric repeats can be highly unstable, like microsatellites with shorter units, and that they can be useful polymorphic markers for linkage analysis, for forensic applications, and for the detection of genetic instability in tumors.