Sequence analysis of a compound coding-region microsatellite in Candida albicans resolves homoplasies and provides a high-resolution tool for genotyping

Sequence diversity at a coding-region microsatellite locus of two diploid Candida species was surveyed. Twenty-one alleles from fourteen strains of Candida albicans and three alleles from two strains of the closely related Candida dubliniensis were sequenced. Results show independent length variation in two contiguous hexanucleotide repeats, one non-contiguous hexanucleotide repeat, and two non-contiguous trinucleotide repeats within a 120 bp coding region. A neighboring, non-repetitive 120 bp region showed no variation. The information density of sequence polymorphisms in this region provides a powerful tool for genotyping microorganisms in epidemiological studies, yielding detailed resolution of closely related strains, and clearly distinguishing the two species studied here. The individual length-variable repeat regions are very short (2–8 repeats), demonstrating that even very short microsatellites can show high levels of length variability when surrounded by similarly repetitive DNA. Extensive homoplasy was discovered among the C. albicans alleles, with the majority of overall length categories consisting of alleles with more than one sequence. Our results show that microsatellite length alone should not be used to assume either sequence identity or identity by descent. Microsatellite length mutations appear to have generated the high degree of both inter- and intraspecific polymorphism seen at the ERK1 locus, and form an island of variability in an otherwise well-conserved gene.     

Candida glabrata and Candida albicans; dissimilar tissue tropism and infectivity in a gnotobiotic model of mucosal candidiasis

Germ-free transgenic epsilon 26 (Tgepsilon26) mice, deficient in both natural killer (NK)- and T-cells, were inoculated (orally) with each of two Candida glabrata (BG2 or BG1003) or Candida albicans (CAF2-1 or SC5314) strains. Candida glabrata- or C. albicans-colonized mice exhibited similar numbers of viable Candida in the alimentary tract. Neither C. glabrata nor C. albicans caused systemic candidiasis of endogenous (alimentary tract) origin. Candida albicans invaded oroesophageal (tongue, palate, esophagus) and keratinized gastric tissues, evoked hyperkeratosis and a prominent, chronic, granulocyte-dominated, inflammatory response in all infected tissues, stimulated the production of splenic granulocytes and was lethal for the mice within 3-5 weeks after oral colonization. The two C. glabrata strains colonized the alimentary tract and penetrated into the keratinized (cardia-antrum) gastric tissues, but in contrast to C. albicans, were unable to infect oroesophageal tissues. Furthermore, C. glabrata strains were not lethal for the Tgepsilon26 mice, and did not evoke an inflammatory response in colonized gastric tissues or stimulate the production of splenic granulocytes. This 'stealth-like' behavior could explain the ability of C. glabrata to persist in infected tissues and survive as a commensal in the alimentary tract.     

The development of simple sequence repeat markers for Magnaporthe grisea and their integration into an established genetic linkage map

Although microsatellite or simple sequence repeat (SSR) markers have several advantages, few have been developed in fungi. The goal of this study was to identify and characterize SSR-containing loci in the filamentous ascomycete Magnaporthe grisea, the causal agent of rice blast disease, and to add these markers to an integrated genetic map of this species [Theor. Appl. Genet. 95 (1997) 20]. We have constructed and screened a microsatellite-enriched small-insert genomic library as well as exploited both publicly available and one proprietary databases for identification of M. grisea SSR containing sequences. Twenty-four out of 49 primer pairs designed to amplify SSR, produced unambiguous polymorphic products in our test population of six isolates. The number of alleles at each locus ranged from two to six when assayed on 3% agarose gels. Twenty-three of the primer pairs amplified polymorphic products between Guy11 and 2539, the parents of a cross from which a genetic map for M. grisea has been established. Genetic analysis showed that all the markers segregated in the expected 1:1 ratio and map positions were determined for all 23 loci.     

Utility of barley and wheat simple sequence repeat (SSR) markers for genetic analysis of Hordeum chilense and tritordeum

A selection of 36 wheat and 35 barley simple sequence repeat markers (SSRs) were studied for their utility in Hordeum chilense. Nineteen wheat and nineteen barley primer pairs amplified consistent H. chilense products. Nine wheat and two barley SSRs were polymorphic in a H. chilense mapping population, producing codominant markers that mapped to the expected homoeologous linkage groups in all but one case. Thirteen wheat and 10 barley primer pairs were suitable for studying the introgression of H. chilense into wheat because they amplified H. chilense products of distinct size. Analysis of wheat/H. chilense addition lines showed that the H. chilense products derived from the expected homoeologous linkage groups. The results showed that wheat and barley SSRs provide a valuable resource for the genetic characterization of H. chilense, tritordeums and derived introgression lines. Received: 20 November 2000 / Accepted: 12 April 2001     

The bottle gourd genome provides insights into Cucurbitaceae evolution and facilitates mapping of a Papaya ring‐spot virus resistance locus

Bottle gourd (Lagenaria siceraria) is an important vegetable crop as well as a rootstock for other cucurbit crops. In this study, we report a high‐quality 313.4‐Mb genome sequence of a bottle gourd inbred line, USVL1VR‐Ls, with a scaffold N50 of 8.7 Mb and the longest of 19.0 Mb. About 98.3% of the assembled scaffolds are anchored to the 11 pseudomolecules. Our comparative genomic analysis identifies chromosome‐level syntenic relationships between bottle gourd and other cucurbits, as well as lineage‐specific gene family expansions in bottle gourd. We reconstructed the genome of the most recent common ancestor of Cucurbitaceae, which revealed that the ancestral Cucurbitaceae karyotypes consisted of 12 protochromosomes with 18 534 protogenes. The 12 protochromosomes are largely retained in the modern melon genome, while have undergone different degrees of shuffling events in other investigated cucurbit genomes. The 11 bottle gourd chromosomes derive from the ancestral Cucurbitaceae karyotypes followed by 19 chromosomal fissions and 20 fusions. The bottle gourd genome sequence has facilitated the mapping of a dominant monogenic locus, Prs, conferring Papaya ring‐spot virus (PRSV) resistance in bottle gourd, to a 317.8‐kb region on chromosome 1. We have developed a cleaved amplified polymorphic sequence (CAPS) marker tightly linked to the Prs locus and demonstrated its potential application in marker‐assisted selection of PRSV resistance in bottle gourd. This study provides insights into the paleohistory of Cucurbitaceae genome evolution, and the high‐quality genome sequence of bottle gourd provides a useful resource for plant comparative genomics studies and cucurbit improvement.     

The complete chloroplast genome sequence of Mahonia bealei (Berberidaceae) reveals a significant expansion of the inverted repeat and phylogenetic relationship with other angiosperms

Mahonia bealei (Berberidaceae) is a frequently-used traditional Chinese medicinal plant with efficient anti-inflammatory ability. This plant is one of the sources of berberine, a new cholesterol-lowering drug with anti-diabetic activity. We have sequenced the complete nucleotide sequence of the chloroplast (cp) genome of M. bealei. The complete cp genome of M. bealei is 164,792 bp in length, and has a typical structure with large (LSC 73,052 bp) and small (SSC 18,591 bp) single-copy regions separated by a pair of inverted repeats (IRs 36,501 bp) of large size. The Mahonia cp genome contains 111 unique genes and 39 genes are duplicated in the IR regions. The gene order and content of M. bealei are almost unarranged which is consistent with the hypothesis that large IRs stabilize cp genome and reduce gene loss-and-gain probabilities during evolutionary process. A large IR expansion of over 12 kb has occurred in M. bealei, 15 genes (rps19, rpl22, rps3, rpl16, rpl14, rps8, infA, rpl36, rps11, petD, petB, psbH, psbN, psbT and psbB) have expanded to have an additional copy in the IRs. The IR expansion rearrangement occurred via a double-strand DNA break and subsequence repair, which is different from the ordinary gene conversion mechanism. Repeat analysis identified 39 direct/inverted repeats 30 bp or longer with a sequence identity ≥ 90%. Analysis also revealed 75 simple sequence repeat (SSR) loci and almost all are composed of A or T, contributing to a distinct bias in base composition. Comparison of protein-coding sequences with ESTs reveals 9 putative RNA edits and 5 of them resulted in non-synonymous modifications in rpoC1, rps2, rps19 and ycf1. Phylogenetic analysis using maximum parsimony (MP) and maximum likelihood (ML) was performed on a dataset composed of 65 protein-coding genes from 25 taxa, which yields an identical tree topology as previous plastid-based trees, and provides strong support for the sister relationship between Ranunculaceae and Berberidaceae. Molecular dating analyses suggest that Ranunculaceae and Berberidaceae diverged between 90 and 84 mya, which is congruent with the fossil records and with recent estimates of the divergence time of these two taxa.     

Agent-based model of diffusion of N-acyl homoserine lactones in a multicellular environment of Pseudomonas aeruginosa and Candida albicans

Experimental incapacity to track microbe–microbe interactions in structures like biofilms, and the complexity inherent to the mathematical modelling of those interactions, raises the need for feasible, alternative modelling approaches. This work proposes an agent-based representation of the diffusion of N-acyl homoserine lactones (AHL) in a multicellular environment formed by Pseudomonas aeruginosa and Candida albicans. Depending on the spatial location, C. albicans cells were variably exposed to AHLs, an observation that might help explain why phenotypic switching of individual cells in biofilms occurred at different time points. The simulation and algebraic results were similar for simpler scenarios, although some statistical differences could be observed (p?<?0.05). The model was also successfully applied to a more complex scenario representing a small multicellular environment containing C. albicans and P. aeruginosa cells encased in a 3-D matrix. Further development of this model may help create a predictive tool to depict biofilm heterogeneity at the single-cell level.     

X-ray structure of the cyclomaltohexaicosaose triiodide inclusion complex provides a model for amylose-iodine at atomic resolution

Cyclomaltohexaicosaose (CA26) is folded into two 1(2)/(3) turns long V-helices that are oriented antiparallel. Crystals of complexes of CA26 with NH(4)I(3) and Ba(I(3))(2) are brown and X-ray analyses show that I(3)(-) units are located in the approximately 5 A wide central channels of the V-helices. In the complex with NH(4)I(3), two CA26 molecules are stacked to form 2 x 1(2)/(3) turns long channels harbouring 3 I(3)(-) at 3.66-3.85 A inter I(3)(-) distance (shorter than van der Waals distance, 4.3 A), whereas in the Ba(I(3))(2) complex, CA26 are not stacked and only one I(3)(-) each fills the V-helices. Glucose...I contacts are formed with C5-H, C3-H, C6-H and (at the ends of the V-helices) with O6 in (+) gauche orientation. By contrast, O2, O3, O4 and O6 in the preferred (-) gauche orientation do not interact with I because these distances are >/=4.01 A and exceed the van der Waals I...O sum of radii by about 0.5 A except for one O2...I distance of 3.68 A near the end of one V-helix. Raman spectra indicate that the complexes share the presence of I(3)(-) with blue amylose-iodine.     

Dolphin genome provides evidence for adaptive evolution of nervous system genes and a molecular rate slowdown

Cetaceans (dolphins and whales) have undergone a radical transformation from the original mammalian bodyplan. In addition, some cetaceans have evolved large brains and complex cognitive capacities. We compared approximately 10 000 protein-coding genes culled from the bottlenose dolphin genome with nine other genomes to reveal molecular correlates of the remarkable phenotypic features of these aquatic mammals. Evolutionary analyses demonstrated that the overall synonymous substitution rate in dolphins has slowed compared with other studied mammals, and is within the range of primates and elephants. We also discovered 228 genes potentially under positive selection (dN/dS > 1) in the dolphin lineage. Twenty-seven of these genes are associated with the nervous system, including those related to human intellectual disabilities, synaptic plasticity and sleep. In addition, genes expressed in the mitochondrion have a significantly higher mean dN/dS ratio in the dolphin lineage than others examined, indicating evolution in energy metabolism. We encountered selection in other genes potentially related to cetacean adaptations such as glucose and lipid metabolism, dermal and lung development, and the cardiovascular system. This study underlines the parallel molecular trajectory of cetaceans with other mammalian groups possessing large brains.     

The structure of human 4F2hc ectodomain provides a model for homodimerization and electrostatic interaction with plasma membrane

4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in amino acid transport and cell fusion, adhesion, and transformation. The structure of the ectodomain of human 4F2hc has been solved using monoclinic (Protein Data Bank code 2DH2) and orthorhombic (Protein Data Bank code 2DH3) crystal forms at 2.1 and 2.8 A, respectively. It is composed of a (betaalpha)(8) barrel and an antiparallel beta(8) sandwich related to bacterial alpha-glycosidases, although lacking key catalytic residues and consequently catalytic activity. 2DH3 is a dimer with Zn(2+) coordination at the interface. Human 4F2hc expressed in several cell types resulted in cell surface and Cys(109) disulfide bridge-linked homodimers with major architectural features of the crystal dimer, as demonstrated by cross-linking experiments. 4F2hc has no significant hydrophobic patches at the surface. Monomer and homodimer have a polarized charged surface. The N terminus of the solved structure, including the position of Cys(109) residue located four residues apart from the transmembrane domain, is adjacent to the positive face of the ectodomain. This location of the N terminus and the Cys(109)-intervening disulfide bridge imposes space restrictions sufficient to support a model for electrostatic interaction of the 4F2hc ectodomain with membrane phospholipids. These results provide the first crystal structure of heteromeric amino acid transporters and suggest a dynamic interaction of the 4F2hc ectodomain with the plasma membrane.     

Assembly and annotation of a draft genome sequence for Glycine latifolia,a perennial wild relative of soybean

Glycine latifolia (Benth.) Newell & Hymowitz (2n = 40), one of the 27 wild perennial relatives of soybean, possesses genetic diversity and agronomically favorable traits that are lacking in soybean. Here, we report the 939‐Mb draft genome assembly of G. latifolia (PI 559298) using exclusively linked‐reads sequenced from a single Chromium library. We organized scaffolds into 20 chromosome‐scale pseudomolecules utilizing two genetic maps and the Glycine max (L.) Merr. genome sequence. High copy numbers of putative 91‐bp centromere‐specific tandem repeats were observed in consecutive blocks within predicted pericentromeric regions on several pseudomolecules. No 92‐bp putative centromeric repeats, which are abundant in G. max, were detected in G. latifolia or Glycine tomentella. Annotation of the assembled genome and subsequent filtering yielded a high confidence gene set of 54 475 protein‐coding loci. In comparative analysis with five legume species, genes related to defense responses were significantly overrepresented in Glycine‐specific orthologous gene families. A total of 304 putative nucleotide‐binding site (NBS)‐leucine‐rich‐repeat (LRR) genes were identified in this genome assembly. Different from other legume species, we observed a scarcity of TIR‐NBS‐LRR genes in G. latifolia. The G. latifolia genome was also predicted to contain genes encoding 367 LRR‐receptor‐like kinases, a family of proteins involved in basal defense responses and responses to abiotic stress. The genome sequence and annotation of G. latifolia provides a valuable source of alternative alleles and novel genes to facilitate soybean improvement. This study also highlights the efficacy and cost‐effectiveness of the application of Chromium linked‐reads in diploid plant genome de novo assembly.     

Proliferation of intracellular structure corresponding to reduced affinity of fluconazole for cytochrome P-450 in two low-susceptibility strains of Candida albicans isolated from a Japanese AIDS patient

Three Candida albicans isolates, TIMM 3164, 3165 and 3166 with reduced fluconazole susceptibility, were isolated from two Japanese AIDS patients. We earlier reported that a reduced intracellular accumulation of fluconazole in these isolates played an important role in the resistance mechanism of fluconazole, but we did not exclude the involvement of other factors. We here examined characteristics related to cytochrome P-450 (CYP), especially sterol 14alpha-demethylase encoded by the ERG11 gene which is the target molecule for fluconazole. In TIMM 3164 and 3165, the ergosterol synthesis by cell-free extracts was somewhat less susceptible to fluconazole, due to a decrease in fluconazole affinity for CYP. The nucleotide substitutions in the ERG11 gene were identified to result in three amino acid changes of K143R, E266D and V488I in TIMM 3164, and of E266D, V404L and V488I in TIMM 3165. These amino acid substitutions might contribute to the decreased affinity for CYP in both isolates. However, a single amino acid change, E266D, observed in TIMM 3166 was unrelated to the decreased affinity for CYP. The most prominent finding on the ultrastructure of TIMM 3164 and 3165 was the development of mesh membrane structures of the endoplasmic reticula, which is a location related to sterol synthesis. This phenomenon was not observed in the cells of TIMM 3166 or the susceptible control strains of ATCC 90028 and 10231. In addition to the reduced intracellular accumulation, the decreased affinity of fluconazole for CYP in TIMM 3164 and 3165 is assumed to be associated with the fluconazole-resistance phenotype.     

Mercator: a fast and simple web server for genome scale functional annotation of plant sequence data

Next‐generation technologies generate an overwhelming amount of gene sequence data. Efficient annotation tools are required to make these data amenable to functional genomics analyses. The Mercator pipeline automatically assigns functional terms to protein or nucleotide sequences. It uses the MapMan ‘BIN’ ontology, which is tailored for functional annotation of plant ‘omics’ data. The classification procedure performs parallel sequence searches against reference databases, compiles the results and computes the most likely MapMan BINs for each query. In the current version, the pipeline relies on manually curated reference classifications originating from the three reference organisms (Arabidopsis, Chlamydomonas, rice), various other plant species that have a reviewed SwissProt annotation, and more than 2000 protein domain and family profiles at InterPro, CDD and KOG. Functional annotations predicted by Mercator achieve accuracies above 90% when benchmarked against manual annotation. In addition to mapping files for direct use in the visualization software MapMan, Mercator provides graphical overview charts, detailed annotation information in a convenient web browser interface and a MapMan‐to‐GO translation table to export results as GO terms. Mercator is available free of charge via http://mapman.gabipd.org/web/guest/app/Mercator .     

Founder control and coexistence in a simple model of asymmetric competition for light

Size asymmetry in plant light acquisition complicates predictions of competitive outcomes in light-limited communities. We present a mathematically tractable model of asymmetric competition for light and discuss its implications for predicting outcomes of competition during establishment in two-, three-, and many-species communities. In contrast to the resource-reduction model of symmetric competition for a single resource, the model we present predicts that outcomes of asymmetric competition for light will sometimes depend on the timing of establishment and the consequent hierarchy among species in canopy position. Competitive outcomes in the model depend on the minimum light requirements (L(c)) and self-shading of species lower in the canopy compared to the light available (L(out)(*)) beneath species higher in the canopy. Succession progresses towards species with decreasing values for L(c), but arrested successions occur when initial dominants have relatively high values for L(c) but low values for L(out)(*), leading to founder control. A theoretically limitless number of species may coexist in competition for light when dominance is founder controlled. These model predictions have implications for an array of applied ecological questions, including methods to control invasive species in light-limited restored ecosystems.     

Draft genome sequence of Sinorhizobium meliloti RU11/001, a model organism for flagellum structure,motility and chemotaxis

Sinorhizobium meliloti of the order Rhizobiales is a symbiotic nitrogen-fixing bacterium nodulating plants of the genera Medicago, Trigonella and Melilotus and hence is of great agricultural importance. In its free-living state it is motile and capable of modulating its movement patterns in response to chemical attractants. Here, the draft genome consisting of a circular chromosome, the megaplasmids pSymA and pSymB and three accessory plasmids of Sinorhizobium meliloti RU11/001, a model organism for flagellum structure, motility and chemotaxis, is reported.     

Molecular cloning and characterization of a Candida albicans gene (EFB1) coding for the elongation factor EF-1β

Abstract The formation of H₂ by chemolithoautrophically growing Oligotropha carboxidovorans has been identified as the result of the oxidation of CO mediated by the cytoplasmic species of the molybdenum-containing CO dehydrogenase multienzyme complex as follows: CO + H ₂ O → CO ₂+ H ₂. Purified CO dehydrogenase was shown to carry hydrogen uptake and formation activities in addition to its catabolic function which is the oxidation of CO. Among the electron donors supporting H₂ formation were CO, NADH, reduced flavins and reduced viologen dyes. The reduction of protons to H₂ by cytoplasmic CO dehydrogenase is interpreted as a detoxification reaction for electrons to prevent cell damage in O. carboxidovorans .     

The complete mitochondrial genome of the onychophoran Epiperipatus biolleyi reveals a unique transfer RNA set and provides further support for the ecdysozoa hypothesis

Onychophora (velvet worms) play a crucial role in current discussions on position of arthropods. The ongoing Articulata/Ecdysozoa debate is in need of additional ground pattern characters for Panarthropoda (Arthropoda, Tardigrada, and Onychophora). Hence, Onychophora is an important outgroup taxon in resolving the relationships among arthropods, irrespective of whether morphological or molecular data are used. To date, there has been a noticeable lack of mitochondrial genome data from onychophorans. Here, we present the first complete mitochondrial genome sequence of an onychophoran, Epiperipatus biolleyi (Peripatidae), which shows several characteristic features. Specifically, the gene order is considerably different from that in other arthropods and other bilaterians. In addition, there is a lack of 9 tRNA genes usually present in bilaterian mitochondrial genomes. All these missing tRNAs have anticodon sequences corresponding to 4-fold degenerate codons, whereas the persisting 13 tRNAs all have anticodons pairing with 2-fold degenerate codons. Sequence-based phylogenetic analysis of the mitochondrial protein-coding genes provides a robust support for a clade consisting of Onychophora, Priapulida, and Arthropoda, which confirms the Ecdysozoa hypothesis. However, resolution of the internal ecdysozoan relationships suffers from a cluster of long-branching taxa (including Nematoda and Platyhelminthes) and a lack of data from Tardigrada and further nemathelminth taxa in addition to nematodes and priapulids.