Unlinked Clusters Research Articles

Genomic epidemiology of Salmonella Typhi in Central Division, Fiji, 2012 to 2016

SummaryBackgroundTyphoid fever is endemic in some Pacific Island Countries including Fiji and Samoa yet genomic surveillance is not routine in such settings. Previous studies suggested imports of the global H58 clade of Salmonella enterica var Typhi (Salmonella Typhi) contribute to disease in these countries which, given the MDR potential of H58, does not auger well for treatment. The objective of the study was to define the genomic epidemiology of Salmonella Typhi in Fiji.MethodsGenomic sequencing approaches were implemented to study the distribution of 255 Salmonella Typhi isolates from the Central Division of Fiji. We augmented epidemiological surveillance and Bayesian phylogenomic approaches with a multi-year typhoid case-control study to define geospatial patterns among typhoid cases.FindingsGenomic analyses showed Salmonella Typhi from Fiji resolved into 2 non-H58 genotypes with isolates from the two dominant ethnic groups, the Indigenous (iTaukei) and non-iTaukei genetically indistinguishable. Low rates of international importation of clones was observed and overall, there were very low levels an antibiotic resistance within the endemic Fijian typhoid genotypes. Genomic epidemiological investigations were able to identify previously unlinked case clusters. Bayesian phylodynamic analyses suggested that genomic variation within the larger endemic Salmonella Typhi genotype expanded at discreet times, then contracted.InterpretationCyclones and flooding drove ‘waves’ of typhoid outbreaks in Fiji which, through population aggregation, poor sanitation and water safety, and then mobility of the population, spread clones more widely. Minimal international importations of new typhoid clones suggest that targeted local intervention strategies may be useful in controlling endemic typhoid infection. These findings add to our understanding of typhoid transmission networks in an endemic island country with broad implications, particularly across Pacific Island Countries.FundingThis work was supported by the Coalition Against Typhoid through the Bill and Melinda Gates Foundation [grant number OPP1017518], the Victorian Government, the National Health and Medical Research Council Australia, the Australian Research Council, and the Fiji Ministry of Health and Medical Services.

Vaccination Status and Attitude among Measles Cluster Cases in Austria, 2019.

On 21 January 2019, public health authorities of two neighboring Austrian provinces reported an increase in measles cases. We investigated this occurrence to identify clusters of epidemiologically linked cases and the associated vaccination status in order to generate hypotheses on those factors explaining the size of the measles clusters. Probable cases were residents of the provinces of Styria or Salzburg with clinical presentation of measles after 1 January 2019 who were linked to a confirmed case using RNA virus detection. We collected data on age, rash onset, certificate-based vaccination status and reasons for being unvaccinated. Contact history was used to identify chains of transmission. By 11 March, we identified 47 cases, with 40 (85.1%) in unvaccinated patients. A cluster of 35 cases with a median age of seven years (IQR: 1–11) occurred between 9 January and 20 February in the province of Styria due to one transmission chain with four case generations. Of 31 vaccine-eligible cases, 25 (80.6%) were unvaccinated, of which 13 refused vaccination. Between 10 January and 1 March, we identified 12 cases as part of five unlinked clusters in the province of Salzburg. Each of these five clusters consisted of two generations: the primary case and the successive cases (median age: 22 years, IQR: 11–35). Eleven of 12 cases occurred in unvaccinated patients, with none of the 11 having a vaccination-refusing attitude. An extended measles cluster in a vaccination-refusing community, compared to five short-lived clusters concurrently occurring in the neighboring province, illustrates how vaccine refusal may hamper control of transmission.

Linking household surveys and facility assessments: a comparison of geospatial methods using nationally representative data from Malawi

BackgroundLinking facility and household surveys through geographic methods is a popular technique to draw conclusions about the relationship between health services and population health outcomes at local levels. These methods are useful tools for measuring effective coverage and tracking progress towards Universal Health Coverage, but are understudied. This paper compares the appropriateness of several geospatial methods used for linking individuals (within displaced survey cluster locations) to their source of family planning (at undisplaced health facilities) at a national level.MethodsIn Malawi, geographic methods linked a population health survey, rural clusters from the Woman’s Questionnaire of the 2015 Malawi Demographic and Health Survey (MDHS 2015), to Malawi’s national health facility census to understand the service environment where women receive family planning services. Individuals from MDHS 2015 clusters were linked to health facilities through four geographic methods: (i) closest facility, (ii) buffer (5 km), (iii) administrative boundary, and (iv) a newly described theoretical catchment area method. Results were compared across metrics to assess the number of unlinked clusters (data lost), the number of linkages per cluster (precision of linkage), and the number of women linked to their last source of modern contraceptive (appropriateness of linkage).ResultsThe closest facility and administrative boundary methods linked every cluster to at least one facility, while the 5-km buffer method left 288 clusters (35.3%) unlinked. The theoretical catchment area method linked all but one cluster to at least one facility (99.9% linked). Closest facility, 5-km buffer, administrative boundary, and catchment methods linked clusters to 1.0, 1.4, 21.1, and 3.3 facilities on average, respectively. Overall, the closest facility, 5-km buffer, administrative boundary, and catchment methods appropriately linked 64.8%, 51.9%, 97.5%, and 88.9% of women to their last source of modern contraceptive, respectively.ConclusionsOf the methods studied, the theoretical catchment area linking method loses a marginal amount of population data, links clusters to a relatively low number of facilities, and maintains a high level of appropriate linkages. This linking method is demonstrated at scale and can be used to link individuals to qualities of their service environments and better understand the pathways through which interventions impact health.

Linking Molybdenum-Sulfur Clusters for Electrocatalytic Hydrogen Evolution.

Controlling the spatial arrangement of molecular catalysts on electrodes is critical to developing an optimal electrocatalyst. Mo-S clusters have shown great promise in catalyzing hydrogen evolution for the generation of carbon-free fuel from water. Here we report a synthetic approach to organize these molecular clusters into ordered dimers, cages, and chains through the use of organic linkers, as solved by single-crystal X-ray diffraction. We find that the linkage through the coordination bond between thiolate and Mo3S7 leads to (1) a 40-fold enhancement in turnover frequency compared with the unlinked cluster and (2) the periodic arrangement of clusters on the electrode with control over their distance, orientation, and density, thus enabling hydrogen evolution at high catalyst loading. The materials developed here require an overpotential of only 89 mV to achieve a current density of 10 mA cm-2,outperforming other Mo-S catalysts (both molecular and solid-state).

Genomic organization and spatio-temporal expression of the hemoglobin genes in European sea bass (Dicentrarchus labrax)

Hemoglobins (Hb) play a critical role in satisfying the oxygen demand of vertebrate aerobic metabolism. The present study reports the characterization of the European sea bass (Dicentrarchus labrax) Hb genes, including genomic organization, phylogeny, and spatio-temporal gene expression. These Hb genes are divided into two unlinked clusters, the “MN” cluster containing eleven genes (five Hbα genes named MN-Hbα1-5 and six Hbβ genes named MN-Hbβ1–6) and the “LA” cluster consisting of three genes (two Hbα genes named LA-Hbα1-2 and one Hbβ gene named LA-Hbβ1). Comparative analysis of Hb amino acid sequences indicates that most of the important amino acid residues involved in hemoglobin-oxygen binding, particularly in the Bohr and Root effects, are generally well conserved, except in MN-Hbβ3. Six genes were mainly expressed during early life (MN-Hbα3-5, MN-Hbβ4–6), while the others were predominantly expressed at juvenile–adult stages. Adult fish expressed Hb genes at high levels in the head kidney and spleen; the main organs involved in blood formation. The Hb genes expressed in non-hematopoietic organs (intestine, gills, heart, brain, and liver) may facilitate oxygen homeostasis or be involved in antimicrobial defense. Stage- and tissue-specific gene expression patterns, together with the sequence features of the different Hb proteins, suggest a broad range of roles in European sea bass.

Evolution of Mating Systems in Basidiomycetes and the Genetic Architecture Underlying Mating-Type Determination in the Yeast Leucosporidium scottii.

In most fungi, sexual reproduction is bipolar; that is, two alternate sets of genes at a single mating-type (MAT) locus determine two mating types. However, in the Basidiomycota, a unique (tetrapolar) reproductive system emerged in which sexual identity is governed by two unlinked MAT loci, each of which controls independent mechanisms of self/nonself recognition. Tetrapolar-to-bipolar transitions have occurred on multiple occasions in the Basidiomycota, resulting, for example, from linkage of the two MAT loci into a single inheritable unit. Nevertheless, owing to the scarcity of molecular data regarding tetrapolar systems in the earliest-branching lineage of the Basidiomycota (subphylum Pucciniomycotina), it is presently unclear if the last common ancestor was tetrapolar or bipolar. Here, we address this question, by investigating the mating system of the Pucciniomycotina yeast Leucosporidium scottii. Using whole-genome sequencing and chromoblot analysis, we discovered that sexual reproduction is governed by two physically unlinked gene clusters: a multiallelic homeodomain (HD) locus and a pheromone/receptor (P/R) locus that is biallelic, thereby dismissing the existence of a third P/R allele as proposed earlier. Allele distribution of both MAT genes in natural populations showed that the two loci were in strong linkage disequilibrium, but independent assortment of MAT alleles was observed in the meiotic progeny of a test cross. The sexual cycle produces fertile progeny with similar proportions of the four mating types, but approximately 2/3 of the progeny was found to be nonhaploid. Our study adds to others in reinforcing tetrapolarity as the ancestral state of all basidiomycetes.

The Novel Lipopeptide Poaeamide of the Endophyte Pseudomonas poae RE*1-1-14 Is Involved in Pathogen Suppression and Root Colonization.

Endophytic Pseudomonas poae strain RE*1-1-14 was originally isolated from internal root tissue of sugar beet plants and shown to suppress growth of the fungal pathogen Rhizoctonia solani both in vitro and in the field. To identify genes involved in its biocontrol activity, RE*1-1-14 random mutagenesis and sequencing led to the identification of a nonribosomal peptide synthetase (NRPS) gene cluster predicted to encode a lipopeptide (LP) with a 10-amino-acid peptide moiety. The two unlinked gene clusters consisted of three NRPS genes, designated poaA (cluster 1) and poaB and poaC (cluster 2), spanning approximately 33.7 kb. In silico analysis followed by chemical analyses revealed that the encoded LP, designated poaeamide, is a structurally new member of the orfamide family. Poaeamide inhibited mycelial growth of R. solani and different oomycetes, including Phytophthora capsici, P. infestans, and Pythium ultimum. The novel LP was shown to be essential for swarming motility of strain RE*1-1-14 and had an impact on root colonization of sugar beet seedlings The poaeamide-deficient mutant colonized the rhizosphere and upper plant cortex at higher densities and with more scattered colonization patterns than the wild type. Collectively, these results indicate that Pseudomonas poae RE*1-1-14 produces a structurally new LP that is relevant for its antagonistic activity against soilborne plant pathogens and for colonization of sugar beet roots.

Integration of chemotaxis, transport and catabolism in Pseudomonas putida and identification of the aromatic acid chemoreceptor PcaY.

Aromatic and hydroaromatic compounds that are metabolized through the β-ketoadipate catabolic pathway serve as chemoattractants for Pseudomonas putida F1. A screen of P. putida F1 mutants, each lacking one of the genes encoding the 18 putative methyl-accepting chemotaxis proteins (MCPs), revealed that pcaY encodes the MCP required for metabolism-independent chemotaxis to vanillate, vanillin, 4-hydroxybenzoate, benzoate, protocatechuate, quinate, shikimate, as well as 10 substituted benzoates that do not serve as growth substrates for P. putida F1. Chemotaxis was induced during growth on aromatic compounds, and an analysis of a pcaY-lacZ fusion revealed that pcaY is expressed in the presence of β-ketoadipate, a common intermediate in the pathway. pcaY expression also required the transcriptional activator PcaR, indicating that pcaY is a member of the pca regulon, which includes three unlinked gene clusters that encode five enzymes required for the conversion of 4-hydroxybenzoate to tricarboxylic acid cycle intermediates as well as the major facilitator superfamily transport protein PcaK. The 4-hydroxybenzoate permease PcaK was shown to modulate the chemotactic response by facilitating the uptake of 4-hydroxybenzoate, which leads to the accumulation of β-ketoadipate, thereby increasing pcaY expression. The results show that chemotaxis, transport and metabolism of aromatic compounds are intimately linked in P. putida.

Abstract 3053: Epigenetic control of cell phenotypes in the stem cell compartments of human prostate and prostate cancer: Implications for enhancement of prostate cancer therapies

Abstract There is increasing evidence that the phenotype of heterogeneous prostate tumors can be determined to some extent by reversible (non-mutagenic/epigenetic) genetic mechanisms. In the complex microenvironment of a developing cancer, this is an excellent survival strategy. The target cell (and molecular targets) for primary therapeutic intervention in prostate cancer is frequently determined by hormone responsiveness. We have shown that, in cells fractionated from human cancers (relative to cell lines or multiply passaged xenografts), the human prostate cancer-inducing cell (TIC) is a primitive, more basal-like cell with stem cell characteristics and limited hormone sensitivity. The TICs, and indeed normal tissue stem cells, have proven remarkably resistant to both radiotherapy and chemotherapy, as a result of their unique stem cell-like chromatin configuration, which can be ‘unlocked’ using common epigenetic control disruptors (e.g. HDAC inhibition), resulting in differentiation and the enhancement of therapeutic efficacy. This result prompted us to explore the nature of epigenetic control in multiple cellular fractions of both basal and luminal cells purified from established prostate cell lines, primary tumor cultures, primary xenografts of prostate cancer and small directed biopsies of freshly biopsied prostate tumor tissues. The data indicate a remarkable difference between multiple ‘standard’ cell line models of prostate cancer and cell types from near-patient models. Chromatin status in terms of histone modification, genomic methylation patterns, microRNA expression are all cell-type specific, in contrast to the variable data reported in extracts of unfractionated human tumor biopsies, reflecting tissue heterogeneity. The TIC fraction has retained (or acquired) the bivalent chromatin of embryonic stem cells in a fully undifferentiated state. Dominant epigenetic signals are associated with cellular differentiation and maintenance of the proliferating luminal phenotype, which can mask underlying changes responsible for tumor induction, stem cell maintenance and metastatic capability. Therapeutic targeting of the former simply shrinks tumors without affecting metastatic induction and patient survival. Unlinked clusters of coregulated genes define the differentiation process, and contain novel molecular targets such as stem cell maintenance genes, which could render all cells in a cancer susceptible to current therapies as part of a combination treatment strategy. The lower concentrations of stem cell modifying agents (e.g. retinoic acid and HDAC inhibitors), when used as phenotype modifiers rather than toxins, in combination with more established (cytotoxic) therapies, are more likely to be well tolerated in patients, compared to the toxic clinical doses of current ‘differentiating’ agents. Citation Format: Norman J. Maitland, Davide Pellacani, Jayant K. Rane, Fiona M. Frame, Anne T. Collins. Epigenetic control of cell phenotypes in the stem cell compartments of human prostate and prostate cancer: Implications for enhancement of prostate cancer therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3053. doi:10.1158/1538-7445.AM2014-3053

Channel catfish hemoglobin genes: Identification, phylogenetic and syntenic analysis, and specific induction in response to heat stress

Hemoglobins transport oxygen from gill to inner organs in fish, and this process is affected by temperature, one of the major environmental factors for fish. The hemoglobin gene clusters have been well studied in humans and several model fish species, but remain largely unknown in catfish. Here, eight α- and six β-hemoglobin genes were identified and characterized in channel catfish. Genomic synteny analysis showed that these hemoglobin genes were separated into two unlinked clusters, the MN cluster containing six α- and six β-hemoglobin genes, and the LA cluster consisting of two α-hemoglobin genes. Channel catfish hemoglobin genes were ubiquitously expressed in all the 10 tested tissues from healthy fish, but exhibited higher expression level in spleen, head kidney, and trunk kidney. In response to heat stress, hemoglobin genes, especially MN Hbα4, MN Hbα5, MN Hbα6, MN Hbβ4, MN Hbβ5, MN Hbβ6, LA Hbα1, and LA Hbα2, presumably the embryonic hemoglobin genes, were drastically up-regulated in the gill and head kidney of heat-tolerant fishes, but not in these tissues of the heat-intolerant fish, suggesting the importance of the embryonic hemoglobin genes in coping with the low oxygen conditions under heat stress.

Germ-cell hyaluronidases: their roles in sperm function

Hyaluronidases (hyases) are a family of enzymes that catalyse the breakdown of hyaluronic acid (HA), which is abundant in the extracellular matrix. Two unlinked gene clusters encode these six proteins: three each in the somatic (or ubiquitous) acid-active subgroup and the neutral-active germ-cell subgroup. This review analyses the data on the expression and role of hyases in gamete biology and fertilization, using electronic databases until October 2010. Evidence indicates that hyases are membrane proteins with multifunctional essential, enzymatic and non-enzymatic, roles (cumulus penetration, zona binding and HA receptor) in fertilization. While sperm adhesion molecule-1 (SPAM1), which has neutral and acidic (bimodal) activity, is the widely conserved mammalian sperm hyase, it co-exists with an acidic hyase in murine and human spermatozoa. Thus, sperm function depends on the concerted activity of both germ cell and 'somatic' hyases. Some hyases are in low abundance in the ovary, somatic testicular cells, the male accessory organs and the male and female genital tracts where they are secreted and acquired by spermatozoa. The latter opens up the possibility of treating hyase-deficient spermatozoa via assisted reproductive technology. The findings challenge the existing classification of hyases, and support the notion that hyase activities are polygenic traits controlled by as many as five hyase genes in mice. Multiple sperm hyases may function cooperatively in a quantitative system and/or serve redundant roles. Unsolved problems include functional redundancy, which can be addressed by double gene-knockouts, and identifying the murine hyase(s) involved in zona binding or whether this role shows species specificity.

Improved differentiation of Mycobacterium tuberculosis isolates in the north of England using additional variable number tandem repeat loci

Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) genotyping of over 3300 Mycobacterium tuberculosis isolates from the north of England has identified large clusters of strains which share common profiles. However, many apparent clusters identified when typed using the existing 15 loci lack clear epidemiological links. This study seeks to discover whether or not six additional VNTR loci can increase the discriminatory power of the existing MIRU-VNTR 15-loci technique. Two hundred and six M. tuberculosis isolates were genotyped, including 57 isolates from 20 epidemiologically linked clusters and 149 from unlinked patients belonging to six large MIRU-VNTR-defined clusters. The discriminatory power of the six additional loci was high (Hunter Gaston Discriminatory Index [HGDI]: 0.952). Five of the six loci were highly discriminative (h>0.6); however, locus 2401 was less discriminative (h=0.5). The additional VNTR loci were able to subtype all six unlinked common MIRU-VNTR clusters into 56 subclusters, significantly differentiating unrelated strains in a set previously incorrectly clustered using 15 MIRU-VNTR loci. The largest cluster size was 14 (9.3%) when typed using the six additional VNTR loci, compared to 30 (20%) when typed using the original 15 MIRU-VNTR loci. The same loci were also found to be stable as a result of their inability to subdivide any of the epidemiologically linked clusters. This study has demonstrated that expanding the MIRU-VNTR panel beyond the 15 previously used loci significantly increases the discriminatory power of the technique and thus provides a valuable tool in the epidemiological monitoring of this disease.

Genomic organization and gene expression of the multiple globins in Atlantic cod: conservation of globin-flanking genes in chordates infers the origin of the vertebrate globin clusters

BackgroundThe vertebrate globin genes encoding the α- and β-subunits of the tetrameric hemoglobins are clustered at two unlinked loci. The highly conserved linear order of the genes flanking the hemoglobins provides a strong anchor for inferring common ancestry of the globin clusters. In fish, the number of α-β-linked globin genes varies considerably between different sublineages and seems to be related to prevailing physico-chemical conditions. Draft sequences of the Atlantic cod genome enabled us to determine the genomic organization of the globin repertoire in this marine species that copes with fluctuating environments of the temperate and Arctic regions.ResultsThe Atlantic cod genome was shown to contain 14 globin genes, including nine hemoglobin genes organized in two unlinked clusters designated β5-α1-β1-α4 and β3-β4-α2-α3-β2. The diverged cod hemoglobin genes displayed different expression levels in adult fish, and tetrameric hemoglobins with or without a Root effect were predicted. The novel finding of maternally inherited hemoglobin mRNAs is consistent with a potential role played by fish hemoglobins in the non-specific immune response. In silico analysis of the six teleost genomes available showed that the two α-β globin clusters are flanked by paralogs of five duplicated genes, in agreement with the proposed teleost-specific duplication of the ancestral vertebrate globin cluster. Screening the genome of extant urochordate and cephalochordate species for conserved globin-flanking genes revealed linkage of RHBDF1, MPG and ARHGAP17 to globin genes in the tunicate Ciona intestinalis, while these genes together with LCMT are closely positioned in amphioxus (Branchiostoma floridae), but seem to be unlinked to the multiple globin genes identified in this species.ConclusionThe plasticity of Atlantic cod to variable environmental conditions probably involves the expression of multiple globins with potentially different properties. The interspecific difference in number of fish hemoglobin genes contrasts with the highly conserved synteny of the flanking genes. The proximity of globin-flanking genes in the tunicate and amphioxus genomes resembles the RHBDF1-MPG-α-globin-ARHGAP17-LCMT linked genes in man and chicken. We hypothesize that the fusion of the three chordate linkage groups 3, 15 and 17 more than 800 MYA led to the ancestral vertebrate globin cluster during a geological period of increased atmospheric oxygen content.

Sequence of a Turkey BAC Clone Identifies MHC Class III Orthologs and Supports Ancient Origins of Immunological Gene Clusters

Two genetically unlinked gene clusters currently define the turkey major histocompatibility complex (MHC). Previous studies identified turkey bacterial artificial chromosome (BAC) clones hypothesized as orthologs of the MHC-B and MHC-Y regions of the chicken. Physical mapping assigned these clones to the same microchromosome (MGA18) and sequencing of the MHC-B BAC found near synteny with a portion of the chicken B-locus. This study examines the sequence of the second MHC BAC clone that was hypothesized, based on subclone sequences, to be orthologous to the MHC-Y. Sequencing of this clone identified a class I locus and orthologs of additional genes found in the mammalian class III region. Approximately 50% of the BAC insert is comprised of sequence corresponding to the centromeric repeat, MGASat2. This turkey MHC BAC sequence is unique from sequences assigned to the MHC-Y in the chicken. Based on sequence comparisons, the class I gene appears to be a nonfunctional pseudogene. The class III genes (BAT1, BAT3, STK19, and a G4-like locus) represent the second class III gene cluster identified in the galliform genome. This cluster appears to be of ancient origin and provides insight into the evolution of the avian MHC.

The mating type locus (MAT) and sexual reproduction of Cryptococcus heveanensis: insights into the evolution of sex and sex-determining chromosomal regions in fungi.

Mating in basidiomycetous fungi is often controlled by two unlinked, multiallelic loci encoding homeodomain transcription factors or pheromones/pheromone receptors. In contrast to this tetrapolar organization, Cryptococcus neoformans/Cryptococcus gattii have a bipolar mating system, and a single biallelic locus governs sexual reproduction. The C. neoformans MAT locus is unusually large (>100 kb), contains >20 genes, and enhances virulence. Previous comparative genomic studies provided insights into how this unusual MAT locus might have evolved involving gene acquisitions into two unlinked loci and fusion into one contiguous locus, converting an ancestral tetrapolar system to a bipolar one. Here we tested this model by studying Cryptococcus heveanensis, a sister species to the pathogenic Cryptococcus species complex. An extant sexual cycle was discovered; co-incubating fertile isolates results in the teleomorph (Kwoniella heveanensis) with dikaryotic hyphae, clamp connections, septate basidia, and basidiospores. To characterize the C. heveanensis MAT locus, a fosmid library was screened with C. neoformans/C. gattii MAT genes. Positive fosmids were sequenced and assembled to generate two large probably unlinked MAT gene clusters: one corresponding to the homeodomain locus and the other to the pheromone/receptor locus. Strikingly, two divergent homeodomain genes (SXI1, SXI2) are present, similar to the bE/bW Ustilago maydis paradigm, suggesting one or the other homeodomain gene was recently lost in C. neoformans/C. gattii. Sequencing MAT genes from other C. heveanensis isolates revealed a multiallelic homeodomain locus and at least a biallelic pheromone/receptor locus, similar to known tetrapolar species. Taken together, these studies reveal an extant C. heveanensis sexual cycle, define the structure of its MAT locus consistent with tetrapolar mating, and support the proposed evolutionary model for the bipolar Cryptococcus MAT locus revealing transitions in sexuality concomitant with emergence of a pathogenic clade. These studies provide insight into convergent processes that independently punctuated evolution of sex-determining loci and sex chromosomes in fungi, plants, and animals.

Cloning and Comparative Analyses of the Zebrafish Ugt Repertoire Reveal Its Evolutionary Diversity

UDP-glucuronosyltransferases (Ugts) are a supergene family of phase II drug-metabolizing enzymes that catalyze the conjugation of numerous hydrophobic small molecules with the UDP-glucuronic acid, converting them into hydrophilic molecules. Here, we report the identification and cloning of the complete zebrafish Ugt gene repertoire. We found that the zebrafish genome contains 45 Ugt genes that can be divided into three families: Ugt1, Ugt2, and Ugt5. Both Ugt1 and Ugt2 have two unlinked clusters: a and b. The Ugt1a, Ugt1b, Ugt2a, and Ugt2b clusters each contain variable and constant regions, similar to that of the protocadherin (Pcdh), immunoglobulin (Ig), and T-cell receptor (Tcr) clusters. Cloning the full-length coding sequences confirmed that each of the variable exons is separately spliced to the set of constant exons within each zebrafish Ugt cluster. Comparative analyses showed that both a and b clusters of the zebrafish Ugt1 and Ugt2 genes have orthologs in other teleosts, suggesting that they may be resulted from the “fish-specific” whole-genome duplication event. The Ugt5 genes are a novel family of Ugt genes that exist in teleosts and amphibians. Their entire open reading frames are encoded by single large exons. The zebrafish Ugt1, Ugt2, and Ugt5 genes can generate additional transcript diversity through alternative splicing. Based on phylogenetic analyses, we propose that the ancestral tetrapod and teleost Ugt1 clusters contained multiple Ugt1 paralogs. After speciation, these ancestral Ugt1 clusters underwent lineage-specific gene loss and duplication. The ancestral vertebrate Ugt2 cluster also underwent lineage-specific duplication. The intronless Ugt5 open reading frames may be derived from retrotransposition followed by gene duplication. They have been expanded dramatically in teleosts and have become the most abundant Ugt family in these lineages. These findings have interesting implications regarding the molecular evolution of genes with diversified variable exons in vertebrates.

Electron correlation and properties of many-electron systems

Different computational methods of quantum chemistry, which are currently employed in accurate calculations of atomic and molecular properties, are discussed and their advantages and disadvantages are investigated in calculations of electric properties of Be, FH, and H2O. A complete fourth-order many-body perturbation theory (MBPT) approach based on the coupled Hartree–Fock (CHF) solution for the perturbed problem reveals that in several cases the fourth-order correlation correction to the CHF result is dominated by those terms which involve triply substituted intermediate states. Moreover, in some cases a complete fourth-order MBPT result appears to be inferior to those obtained in approximate MBPT treatments which involve only certain classes of intermediate states. Hence, a complete fourth-order MBPT approach may not be sufficient for accurate predictions of atomic and molecular properties. The applicability of the complete active space (CAS) SCF method is studied in calculations of electric properties of FH. Although this method offers some advantages over the MBPT approach, its usefulness may be limited by the requirement of rather large active orbital spaces which have to be used in order to achieve sufficiently high accuracy. The CAS SCF results can be improved by using multireference CI methods with reference functions built of the CAS SCF natural orbitals. A good performance of the MBPT method limited to singly and doubly substituted intermediate states suggests that reasonable results can also be obtained in similar configuration interaction (CI) calculations, provided the latter are corrected for the erratic treatment of unlinked clusters. It is shown that the unlinked cluster corrections due to Davidson and Siegbahn result in reasonable estimates of the total correlation contribution to different properties. Because of its fairly routine character the CI method limited to single and double substitutions may in this way provide useful data with relatively small computational effort.

Ionization energies of water from PNO-CI calculations

A new configuration interaction method using a non-orthogonal set of pseudonatural orbitals (PNO-CI) is applied to the ground state of water and five states of the ionized molecule. Including only double substitutions, the computed upper bound for the ground state is 0.0631 a.u. above the experimental non-relativistic value. An approximate treatment of unlinked clusters lowers the energy by 0.0145 a.u. Thus above 90% of the total correlation energy is accounted for. The computed vertical ionization energies (IE's) agree with experiment to within 2%. An analysis of the IE's shows the causes of anomalous behavior of correlation energies and supports a suggestion for useful limited CI calculations of vertical IE's. The equilibrium geometries of the three lowest states of the ion are determined by SCF calculations and estimates for the adiabatic IE's are given.

Global diversity and evidence for coevolution of KIR and HLA

The killer immunoglobulin-like receptor (KIR) gene cluster shows extensive genetic diversity, as do the HLA class I loci, which encode ligands for KIR molecules. We genotyped 1,642 individuals from 30 geographically distinct populations to examine population-level evidence for coevolution of these two functionally related but unlinked gene clusters. We observed strong negative correlations between the presence of activating KIR genes and their corresponding HLA ligand groups across populations, especially KIR3DS1 and its putative HLA-B Bw4-80I ligands (r = -0.66, P = 0.038). In contrast, we observed weak positive relationships between the various inhibitory KIR genes and their ligands. We observed a negative correlation between distance from East Africa and frequency of activating KIR genes and their corresponding ligands, suggesting a balance between selection on HLA and KIR loci. Most KIR-HLA genetic association studies indicate a primary influence of activating KIR-HLA genotypes in disease risk; concomitantly, activating receptor-ligand pairs in this study show the strongest signature of coevolution of these two complex genetic systems as compared with inhibitory receptor-ligand pairs.

Upregulated hoxC4 induces CD14 expression during the differentiation of acute promyelocytic leukemia cells

Homeobox (hox) genes encode transcription factors which are critically involved in embryonic development. The 39 different hox genes are organized into four unlinked chromosomal gene clusters (hoxA-D) in mammals and a number of studies have suggested that hox genes play important roles in human leukemogenesis. Acute promyelocytic leukemia (APL) cells carrying the PML-RARa fusion protein, respond well by differentiating in their response to an all-trans-retinoic acid (ATRA) treatment. We examined the expression of the individual hox genes during this differentiation. Besides the constitutive expression of hoxA9 and no expression of hoxB7, the expression of hoxC4 increased significantly during differentiation of NB4 cells (PML-RAR(alpha)+). We also examined the expression of hoxC4 in bone marrow cells from APL patients and found that the hoxC4 expression was reproducibly induced during an ATRA treatment. To further examine this finding, HoxC4 was stably expressed in NB4 cells by retroviral transduction. The NB4 cells expressing HoxC4 showed differentiated phenotypes including a CD14 expression, which strongly suggests that upregulated hoxC4 is functionally involved in NB4 cell differentiation in response to ATRA. Upregulation of CD14 is at the transcription level and mediated by the homeodomain of the HoxC4.