New efficient extragenic microsatellite markers for hemophilia A carrier state diagnostics

In search of new efficient markers for genetic diagnostics of hemophilia A, two tri-nucleotide microsatellite repeats (STR) at chromosome X loci, which flank coagulation factor VIII gene (F8), namely STR HA472--CTT-repeat, which is localized adjacent to the GAB3 gene 163 bp apart from the 3' end of the F8 gene and STR HA544--repeat (CTT)x(ATT)y located at a distance of 375 bp from the 5' end of the F8 gene were discovered. Detailed analysis using PCR and sequencing has shown that STR HA472 contains two long variable CTT-blocks separated by small spacer CCTCCC. The location of recognition site of restriction endonuclease Mnl1 (CCTC) in the spacer permits to test differentially the polymorphic blocks and thus to increase the analysis informativity. STR HA544 is also represented by two polymorphic blocks (CTT and ATT), for separate amplification of which highly informative PCR amplification assays were elaborated. The study has been done using DNA samples of 212 individuals (125 women) from 48 families with hemophilia A carriers. Our results point to Mendelian inheritance of the markers studied, a high number of allelic variants and high heterozygosity, which was 90% and 100% for HA544 and HA472, respectively. This permitted us to use these data for practical gene diagnostics of the carriers and prenatal diagnostics of hemophilia A. In addition to high informativity STR HA472 and HA544 are highly important for diagnostics as they are located at a shorter distance than other known extragenic polymorphisms of the F8 gene. In contrast to dinucleotide repeats, trinucleotide repeats are readily tested, not requiring high-resolution electrophoretic systems. In addition, they are located on the opposite sites of the F8 gene. This permits to control homologous recombination events in the locus and thus to prevent diagnostic mistakes.     

Analysis of the <Emphasis Type="Italic">Alu</Emphasis>I polymorphism in intron 1 of the human coagulation factor VIII gene: A new marker for the hemophilia a carrier detection

Frequencies of the C/T SNP alleles at position 2403 of the human coagulation factor VIII gene intron 1, containing the AluI restriction endonuclease recognition site, were examined. Genomic DNA samples for the analysis were obtained from the consulted women and their relatives from the families with hemophilia A. A total of 221 unrelated X chromosomes were studied. The two allelic variants were found with similar frequencies of T(Alu+), 0.53 and C(Alu?), 0.47. The heterozygosity index evaluated as equal to 0.50 was correlated with the experimental heterozygote number. The absence of a tight linkage between the AluI SNP and the widely used in the hemophilia A gene diagnostics HindIII polymorphism (C/T SNP at position 103 of intron 19) was demonstrated. Summarized informativity of these two markers for obligate carriers and for those detected in this study constituted 68% (32 out of 47). At the same time using one of the markers, only 40% (HindIII) and 51% (AluI) of the consulted women were informative. The new marker was used in 13 prenatal DNA diagnostics of hemophilia A. A new deletion polymorphism (del TGA, position 2281–2283 of intron 1) was described in close proximity of the AluI SNP with the frequency of about 0.05. among the five other SNP of the factor VIII gene examined (Bme18I, intron 1; HpaII, intron 13; MnlI, exon 14; Bst4CI, exon 25; and MseI, exon 26) no effective diagnostic markers were found. Only the MnlI polymorphism could be recommended for limited usage.     

FLASH-PCR diagnostics of toxigenic fungi of the genus <Emphasis Type="Italic">Fusarium</Emphasis>

A test system for the diagnostics and identification of seven toxigenic fungi causing Fusarium head blight (Fusarium graminearum, F. culmorum, F. poae, F. sporotrichioides, F. langsethiae, F. avenaceum, and F. tricinctum) was developed using PCR. The identification of pathogen is based on the specific amplification of a DNA fragment of the gene of translation elongation factor 1 alpha (tef-1α) and subsequent detection of the results by the fluorescent amplification-based specific hybridization method. The system was tested on 38 isolates of different fungi of the genus Fusarium.     

Heterochromatic DNA repeats in <Emphasis Type="Italic">Drosophila</Emphasis> and unusual gene silencing in yeast cells

The 1.25-kb heterochromatic Stellate repeats of Drosophila melanogaster are capable of stably persisting in transgenic constructs and silencing the white reporter gene (mosaic position effect variegation). This system reveals an unusual form of silencing, which is insensitive to known modifiers of position effect variegation. The unusual form of silencing was studied with yeast Saccharomyces cerevisiae, a simple eukaryotic model. To be transferred into yeast cells, the D. melanogaster Stellate repeats were cloned in the pYAC4 centromeric vector (CEN4, URA3, TRP1, HIS3). The HIS3 and/or URA3 genes could be inactive in plasmids consisting of pYAC4 and the Stellate insert in yeast cells. Deletion of D. melanogaster DNA from the plasmid was found to activate the URA3 and HIS3 genes. It was assumed that the genes were repressed rather than damaged in the presence of the Stellate repeats and that a new form of gene silencing was revealed in.     

Association between the length of the <Emphasis Type="Italic">MUC8-</Emphasis>minisatellite 5 region and susceptibility to chronic obstructive pulmonary disease (COPD)

In asthma and chronic obstructive pulmonary disease (COPD), mucins display disease-related alterations caused by airway mucus obstruction. MUC5AC, MUC5B and MUC8 are known as the major secretory mucins in human airway epithelial cells. Analysis of mucin genes has identified the presence of several features with a variable number of tandem repeats (VNTR; minisatellites) in the central region of each mucin. In our previous study, six minisatellites in the region of the MUC8 gene were identified, and the MUC8-MS5 minisatellite showed the highest heterozygosity among them. In this study, we evaluated the relationship between MUC8-MS5 and susceptibility to asthma and COPD. A case-control study was performed with 229 controls, 123 COPD cases and 77 asthma cases. A significant association (OR 3.96) between short alleles (2/2 repeats) and the occurrence of COPD was observed [95% confidence interval (CI) 1.32–11.88; p?=?0.008]. Hence, the increased frequency of 2/2 homo-short alleles were also found in asthma cases (3.11; CI 0.88–11.05; p?=?0.066), though this association was not statistically significant. These results revealed a genetic association between MUC8 and COPD, and that the specific short minisatellite alleles (2/2) of MUC8-MS5 may be a risk factor for COPD.     

Tubulin evolution in insects: gene duplication and subfunctionalization provide specialized isoforms in a functionally constrained gene family

Background

The completion of 19 insect genome sequencing projects spanning six insect orders provides the opportunity to investigate the evolution of important gene families, here tubulins. Tubulins are a family of eukaryotic structural genes that form microtubules, fundamental components of the cytoskeleton that mediate cell division, shape, motility, and intracellular trafficking. Previous in vivo studies in Drosophila find a stringent relationship between tubulin structure and function; small, biochemically similar changes in the major alpha 1 or testis-specific beta 2 tubulin protein render each unable to generate a motile spermtail axoneme. This has evolutionary implications, not a single non-synonymous substitution is found in beta 2 among 17 species of Drosophila and Hirtodrosophila flies spanning 60 Myr of evolution. This raises an important question, How do tubulins evolve while maintaining their function? To answer, we use molecular evolutionary analyses to characterize the evolution of insect tubulins.

Results

Sixty-six alpha tubulins and eighty-six beta tubulin gene copies were retrieved and subjected to molecular evolutionary analyses. Four ancient clades of alpha and beta tubulins are found in insects, a major isoform clade (alpha 1, beta 1) and three minor, tissue-specific clades (alpha 2-4, beta 2-4). Based on a Homarus americanus (lobster) outgroup, these were generated through gene duplication events on major beta and alpha tubulin ancestors, followed by subfunctionalization in expression domain. Strong purifying selection acts on all tubulins, yet maximum pairwise amino acid distances between tubulin paralogs are large (0.464 substitutions/site beta tubulins, 0.707 alpha tubulins). Conversely orthologs, with the exception of reproductive tissue isoforms, show little sequence variation except in the last 15 carboxy terminus tail (CTT) residues, which serve as sites for post-translational modifications (PTMs) and interactions with microtubule-associated proteins. CTT residues overwhelming comprise the co-evolving residues between Drosophila alpha 2 and beta 3 tubulin proteins, indicating CTT specializations can be mediated at the level of the tubulin dimer. Gene duplications post-dating separation of the insect orders are unevenly distributed, most often appearing in major alpha 1 and minor beta 2 clades. More than 40 introns are found in tubulins. Their distribution among tubulins reveals that insertion and deletion events are common, surprising given their potential for disrupting tubulin coding sequence. Compensatory evolution is found in Drosophila beta 2 tubulin cis-regulation, and reveals selective pressures acting to maintain testis expression without the use of previously identified testis cis-regulatory elements.

Conclusion

Tubulins have stringent structure/function relationships, indicated by strong purifying selection, the loss of many gene duplication products, alpha-beta co-evolution in the tubulin dimer, and compensatory evolution in beta 2 tubulin cis-regulation. They evolve through gene duplication, subfunctionalization in expression domain and divergence of duplication products, largely in CTT residues that mediate interactions with other proteins. This has resulted in the tissue-specific minor insect isoforms, and in particular the highly diverse α3, α4, and β2 reproductive tissue-specific tubulin isoforms, illustrating that even a highly conserved protein family can participate in the adaptive process and respond to sexual selection.

     

Overexpression of <Emphasis Type="Italic">AtGRDP2</Emphasis> gene in common bean hairy roots generates vigorous plants with enhanced salt tolerance

Proteins with glycine-rich repeats have been identified in plants, mammalians, fungi, and bacteria. Plant glycine-rich proteins have been associated to stress response. Previously, we reported that the Arabidopsis thaliana AtGRDP2 gene, which encodes a protein with a glycine-rich domain, plays a role in growth and development of A. thaliana and Lactuca sativa. In this study, we generated composite Phaseolus vulgaris plants that overexpress the AtGRDP2 gene in hairy roots generated by Agrobacterium rhizogenes. We observed that hairy roots harboring the AtGRDP2 gene developed more abundant and faster-growing roots than control hairy roots generated with the wild type A. rhizogenes. In addition, composite common bean plants overexpressing the AtGRDP2 gene in roots were more tolerant to salt stress showing increments in their fresh and dry weight. Our data further support the role of plant GRDP genes in development and stress response.     

Mutational analysis of hemophilia B in Russia: Molecular-genetic study

Hemophilia B is a hereditary X-linked coagulation disorder. This pathology is caused by various defects in the factor IX gene, which is, being about 34 kb long and consisting of eight exons, localized in the Xq27 locus of the X-chromosome long arm. Mutations were revealed in 56 unrelated patients with hemophilia B in this study by using direct sequencing of factor IX gene functionally important fragments. Forty-six mutations were found with prevailing missense mutations (n = 30). The rest of the mutations were nonsense (n = 4) and splicing (n = 4) mutations, large deletions (n = 3), microdeletions (n = 2), microinsertions (n = 2), and promoter mutations (n = 1). Eleven of 46 mutations were previously unknown for human populations.     

Development of an efficient genetic manipulation strategy for sequential gene disruption and expression of different heterologous <Emphasis Type="Italic">GFP</Emphasis> genes in <Emphasis Type="Italic">Candida tropicalis</Emphasis>

The diploid yeast Candida tropicalis, which can utilize n-alkane as a carbon and energy source, is an attractive strain for both physiological studies and practical applications. However, it presents some characteristics, such as rare codon usage, difficulty in sequential gene disruption, and inefficiency in foreign gene expression, that hamper strain improvement through genetic engineering. In this work, we present a simple and effective method for sequential gene disruption in C. tropicalis based on the use of an auxotrophic mutant host defective in orotidine monophosphate decarboxylase (URA3). The disruption cassette, which consists of a functional yeast URA3 gene flanked by a 0.3 kb gene disruption auxiliary sequence (gda) direct repeat derived from downstream or upstream of the URA3 gene and of homologous arms of the target gene, was constructed and introduced into the yeast genome by integrative transformation. Stable integrants were isolated by selection for Ura⁺ and identified by PCR and sequencing. The important feature of this construct, which makes it very attractive, is that recombination between the flanking direct gda repeats occurs at a high frequency (10^?8) during mitosis. After excision of the URA3 marker, only one copy of the gda sequence remains at the recombinant locus. Thus, the resulting ura3 strain can be used again to disrupt a second allelic gene in a similar manner. In addition to this effective sequential gene disruption method, a codon-optimized green fluorescent protein-encoding gene (GFP) was functionally expressed in C. tropicalis. Thus, we propose a simple and reliable method to improve C. tropicalis by genetic manipulation.     

Molecular analyses of <Emphasis Type="Italic">Ficus erecta</Emphasis> and its allies within the subsection <Emphasis Type="Italic">Frutescentiae</Emphasis> (Moraceae)

Ficus (Moraceae) is a keystone group in tropical and subtropical forests with remarkable diversity of species and taxonomical challenges as a consequence of fig–pollinator coevolution. Ficus subsect. Frutescentiae includes about 30 species that are predominantly shrubs or small trees with Terminalia branching. Many of these species are difficult to delimit morphologically, and the group includes a tangle of uncertain taxa and incorrectly applied names. We conducted a phylogenetic analysis with internal and external transcribed spacer data (ITS and ETS) and data from 18 polymorphic microsatellite loci to evaluate the species status of the most perplexing members of this subsection. The results confirm the monophyly of subsect. Frutescentiae, with F. pedunculosa as sister to the rest. The F. erecta complex comprises approximately 17 taxa: F. erecta, F. abelii, F. boninsimae, F. nishimurae, F. iidaiana, F. gasparriniana var. laceratifolia, F. gasparriniana var. viridescens, F. pyriformis, F. stenophylla, F. fusuiensis, F. fengkaiensis, F. sinociliata, F. tannoensis, F. vaccinioides, F. formosana, F. pandurata, and F. periptera. The last five of these were supported as good species, while the others were not well supported by the present evidence. Evidence also supported the status of the non-F. erecta complex species including. F. pedunculosa, F. ischnopoda, F. heteromorpha, and F. variolosa. Ficus filicauda and F. neriifolia are possibly conspecific. The species status of F. potingensis should be restored and it should be treated as a member of section Eriosycea. Identification of the remaining taxa (F. gasparriniana var. esquirolii, F. ruyuanensis, F. daimingshanensis, F. chapaensis, F. changii, F. trivia, and F. tuphapensis) and their relationships to the F. erecta complex were not clarified. As a whole, only ten species in this subsection are confirmed, one is excluded, one is synonymous, and the others are either unresolved or short of samples. There appears to be a consistent genetic background among these unresolved groups, which suggests that repeated hybridization (as a result of pollinator host shifts) has filled up the interspecific gaps during the fig–pollinator coevolution process.     

Distribution and linkage disequilibrium analysis of polymorphisms of <Emphasis Type="Italic">GH1</Emphasis> gene in different populations of pigs associated with body size

Growth hormone (GH) has been considered as a candidate gene for growth and body size in pigs. In this study, polymorphisms of the GH1 gene were evaluated for associations with body size traits in 190 pig individuals. Seventeen single-nucleotide polymorphisms (SNPs) were identified in GH1 gene of the large pig breeds and miniature pig breeds using direct sequencing and genotyped by allele-specific PCR approach. Notably, six (g.237A>G, g.283T>C, g.309A>G, g.318A>G, g.540A>G and g.544A>G) of them were significantly associated with body size, of which three loci (g.283T>C, g.309A>G, g.318A>G) located in the signal-peptide coding region of GH1 gene compose a CGG haplotype for large pigs and TAA haplotype for miniature pigs (P< 0.001), two loci (g.540A>G and g.544A>G) located in the second intron of GH1 gene compose a GG haplotype for large pigs and AA haplotype for miniature pigs (P< 0.001). Our results demonstrate that these SNPs in GH1 gene are associated with the body size of pigs providing genetic basis for pig breeding with the improved economic benefits.     

The biosynthesis of gibberellic acids by the transformants of orchid-associated <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

The genus Fusarium, including multiple strains in the Gibberella fujikuroi species complex (GFC), is well known for its production of diverse secondary metabolites. F. fujikuroi, associated with the “bakanae” disease of rice, is an active producer of gibberellins (GAs), a wide class of plant hormones. In addition to some members of the GFC, the GA biosynthetic gene cluster, or parts of it, occurs also in some isolates of the closely related species of F. oxysporum, which does not belong to the GFC. However, production of GAs has never been observed in any F. oxysporum strain. In this study, we report on the GA biosynthetic activity in an orchid-associated F. oxysporum strain by transforming a cosmid with the entire F. fujikuroi GA gene cluster. Southern and Northern blot analyses confirmed not only the integration of the entire gene cluster into the genome but also the active expression of the seven GA biosynthetic genes under nitrogen-limiting conditions. The transformants produced GAs at levels similar to those of F. fujikuroi. These data show that the regulatory network for expression of GA genes is fully active in the F. oxysporum background.     

Geographic distribution of phylogenetic species of the <Emphasis Type="Italic">Fusarium graminearum</Emphasis> species complex and their 8-ketotrichothecene chemotypes on wheat spikes in Iran

Isolates of the Fusarium graminearum species complex (FGSC, n = 446) were collected from wheat spikes from northern and western regions of Iran with a history of Fusarium head blight (FHB) occurrences. The trichothecene mycotoxin genotypes/chemotypes, the associated phylogenetic species, and geographical distribution of these isolates were analyzed. Two phylogenetic species, Fusarium asiaticum and F. graminearum, were identified and were found to belong to sequence characterized amplified region (SCAR) groups V and I. Isolates from F. asiaticum species lineage 6 were within SCAR group V, whereas F. graminearum species lineage 7 were of SCAR group I. Of the 446 isolates assayed, 274 were F. asiaticum species predominantly of the nivalenol (NIV) genotype, while other isolates were either deoxynivalenol (DON) plus 3-acetyldeoxynivalenol (3-AcDON) or DON plus 15-acetyldeoxynivalenol (15-AcDON) genotype. Based on Tri7 gene sequences, a new subpopulation of 15-AcDON producers was observed among F. asiaticum strains in which 11-bp repeats were absent in the Tri7 sequences. The trichothecene chemotype was confirmed and quantified by high-performance liquid chromatography (HPLC) in 46 FGSC isolates. Isolates produced NIV (33.4–108.2 μg/g) and DON (64.7–473.6 μg/g) plus either 3-AcDON (51.4–142.4 μg/g) or 15-AcDON (24.1–99.3 μg/g). Among FGSC isolates, F. asiaticum produced the highest levels of trichothecenes. Using BIOCLIM based on the climate data of 20-year during 1994–2014, modelling geographical distribution of FGSC showed that F. asiaticum was restricted to warmer and humid areas with a median value of mean annual temperature of about 17.5 °C and annual rainfall of 658 mm, respectively (P < 0.05). In contrast, F. graminearum (only 15-AcDON producers) was restricted to cooler and drier areas, with a median value of the mean annual temperature of 14.4 °C and an annual rainfall of 384 mm, respectively (P < 0.05). Based on climate parameters at anthesis, the recorded distribution of F. graminearum and F. asiaticum was similar to that based on BIOCLIM parameters. Therefore, geographic differences on the wheat-growing areas in Iran have had a significant effect on distribution of FGSC and their trichothecene chemotypes.     

Multi-locus phylogeny and morphology reveal five new species of <Emphasis Type="Italic">Fomitiporia</Emphasis> (Hymenochaetaceae) from China

Five newly identified species of Fomitiporia (F. alpina, F. gaoligongensis, F. hainaniana, F. subrobusta and F. subtropica) and their morphological and molecular characterisation are described in this paper. Fomitiporia alpina sp. nov. is distinguished by its pileate basidiomata, parallel tramal hyphae and large basidiospores (6.5–8 × 6–8 μm), and by its gymnosperm wood-living habitat. Fomitiporia gaoligongensis sp. nov. is distinct from other species due to its semicircular pileus and subglobose to globose basidiospores (6.5–7.6 × 6–7.4 μm). Fomitiporia hainaniana sp. nov. is marked by its resupinate basidiomata, the presence of setae and small globose basidiospores (4–5 × 3.8–4.4 μm). Fomitiporia subrobusta sp. nov. is characterised by its triquetrous basidiomata, small pores (6–9 per mm) with entire and thick dissepiments, and subglobose to obovoid basidiospores (6.2–6.8 × 5.2–6 μm). Fomitiporia subtropica sp. nov. can be differentiated by its resupinate basidiomata, smaller pores (6–10 per mm) and smaller basidiospores (5.2–6 × 4.4–5 μm). Phylogenetic analysis, based on multi-gene comparison of the internal transcribed spacer regions (ITS), nuclear large subunit ribosomal RNA gene regions (nLSU), the translation elongation factor 1-α gene (tef1α) and the second subunit of RNA polymerase II (rpb2), confirmed affinity with the Fomitiporia species and showed association with similar fungi in the genus.