A novel locus <Emphasis Type="Italic">DFNA59</Emphasis> for autosomal dominant nonsyndromic hearing loss maps at chromosome 11p14.2–q12.3

Autosomal dominant nonsyndromic hearing loss (ADNSHL) accounts for about one-fifth of hereditary hearing loss in humans. In the present study, we have analyzed a three-generation family with 14 of its members manifesting ADNSHL, using a genome-wide linkage mapping approach. We found a novel locus DFNA59 between the D11S929 and D11S480 markers in the chromosome location 11p14.2–q12.3. The highest two-point lod score of 5.72 at recombination fraction = 0 was obtained for D11S4152, D11S4154, D11S1301, D11S905 and D11S1344. The critical genomic region comprising about 37 megabases of DNA is proposed to carry a gene for ADNSHL in the family. About 50 cochlear-expressed genes mapping to the region are strong candidates which we propose to examine to identify the gene responsible for the hearing impairment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Linkage of 35S and 5S rRNA genes in <Emphasis Type="Italic">Artemisia</Emphasis> (family Asteraceae): first evidence from angiosperms

Typically in plants, the 5S and 35S ribosomal DNA (rDNA) encoding two major ribosomal RNA species occur at separate loci. However, in some algae, bryophytes and ferns, they are at the same locus (linked arranged). Southern blot hybridisation, polymerase chain reactions (PCR), fluorescent in situ hybridisation, cloning and sequencing were used to reveal 5S and 35S rDNA genomic organisation in Artemisia. We observed thousands of rDNA units at two–three loci containing 5S rDNA in an inverted orientation within the inter-genic spacer (IGS) of 35S rDNA. The sequenced clones of 26–18S IGS from Artemisia absinthium appeared to contain a conserved 5S gene insertion proximal to the 26S gene terminus (5S rDNA-1) and a second less conserved 5S insertion (5S rDNA-2) further downstream. Whilst the 5S rDNA-1 showed all the structural features of a functional gene, the 5S-rDNA-2 had a deletion in the internal promoter and probably represents a pseudogene. The linked arrangement probably evolved before the divergence of Artemisia from the rest of Asteraceae (>10 Myrs). This arrangement may have involved retrotransposons and once formed spread via mechanisms of concerted evolution. Heterogeneity in unit structure may reflect ongoing homogenisation of variant unit types without fixation for any particular variant. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ecotype-specific and chromosome-specific expansion of variant centromeric satellites in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Despite the conserved roles and conserved protein machineries of centromeres, their nucleotide sequences can be highly diverse even among related species. The diversity reflects rapid evolution, but the underlying mechanism is largely unknown. One approach to monitor rapid evolution is examination of intra-specific variation. Here we report variant centromeric satellites of Arabidopsis thaliana found through survey of 103 natural accessions (ecotypes). Among them, a cluster of variant centromeric satellites was detected in one ecotype, Cape Verde Islands (Cvi). Recombinant inbred mapping revealed that the variant satellites are distributed in centromeric region of the chromosome 5 (CEN5) of this ecotype. This apparently recent variant accumulation is associated with large deletion of a pericentromeric region and the expansion of satellite region. The variant satellite was bound to HTR12 (centromeric variant histone H3), although expansion of the satellite was not associated with comparable increase in the HTR12 binding. The results suggest that variant satellites with centromere function can rapidly accumulate in one centromere, supporting the model that the satellite repeats in the array are homogenized by occasional unequal crossing-over, which has a potential to generate an expansion of local sequence variants within a centromere cluster. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Length Variation in 18S rRNA Expansion Segment 43/e4 of <Emphasis Type="Italic">Daphnia obtusa</Emphasis>: Ancient or Recurring Polymorphism?

Expansion segments in ribosomal DNA (rDNA) can show length variation at the level of the individual, yet our understanding of the evolutionary forces shaping this variation is incomplete. Previous studies of expansion segment 43/e4 of the 18S rRNA gene in Daphnia obtusa have examined this variation in six individuals; however, it is not known if the variation documented at this locus is representative of variation across the species’ geographic range. Furthermore, it is unclear whether length variants found in multiple individuals share common ancestry, or were generated de novo through recombination. We quantified expansion segment length variant frequencies in 134 individual D. obtusa from 33 populations at 15 sites across the species range in the US, and used a phylogeographic approach to determine whether recombination continues to add to the standing crop of variation at this locus. We identified seven length variants across the sampling range, which spans almost 3000 km. Based on the phylogeographic distribution of length variants in the expansion segment, we conclude that they are shared ancient polymorphisms that have persisted despite the operation of molecular mechanisms that cause the concerted evolution of multigene families such as rDNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A novel locus for distal motor neuron degeneration maps to chromosome 7q34-q36

The motor neuron diseases (MND) are a group of related neurodegenerative diseases that cause the relative selective progressive death of motor neurons. These diseases range from slowly progressive forms including hereditary motor neuropathy (HMN), to the rapidly progressive disorder amyotrophic lateral sclerosis (ALS). There is clinical and genetic overlap among these MNDs, implicating shared pathogenic mechanisms. We recruited a large family with a MND that was previously described as juvenile ALS and distal HMN. We identified a novel MND/HMN locus on chromosome 7q34-q36 following a genome-wide scan for linkage in this family. The disease causing mutation maps to a 26.2 cM (12.3 Mb) interval flanked by D7S2513 and D7S637 on chromosome 7q34-q36. Recombinant haplotype analysis including unaffected individuals suggests that the refined candidate interval spans 14.3 cM (6.3 Mb) flanked by D7S2511 and D7S798. One gene in the candidate interval, CDK5, was selected for immediate mutation analysis based upon its known association with an ALS-like phenotype in mice however, no mutations were identified. Identification of genes causing familial MND will lead to a greater understanding of the biological basis of both familial and sporadic motor neuron degeneration including ALS. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phosphorylation site mapping of soluble proteins: bioinformatical filtering reveals potential plastidic phosphoproteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Protein phosphorylation is a major mode of regulation of metabolism, gene expression, and cell architecture. A combination of phosphopeptide enrichment strategies based on TiO₂ and IMAC in addition to our MudPIT strategy revealed the detection of 181 phosphorylation sites which are located on 125 potentially plastidic proteins predicted by GoMiner, TargetP/Predotar in Arabidopsis thaliana. In our study phosphorylation on serine is favored over threonine and this in turn over phosphorylation on tyrosine residues, showing a percentage of 67.4% to 24.3% to 8.3% for pS:pT:pY. Four phosphorylated residues (S208, Y239, T246 and T330), identified by our approach have been fitted to the structure of the activated form of spinach RuBisCO, which are located in close proximity to the substrate binding site for ribulosebisphosphate. Potentially, these phosphorylation sites exert a direct influence on the catalytic activity of the enzyme. Such examples show nicely the value of the presented mass spectrometric dataset for further biochemical applications, since alternative mutation analysis often turns out to be unsuccessful, caused by mutations in essential proteins which result in lethal phenotypes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tricistronic overexpression of cytochrome P450<Subscript><Emphasis Type="Italic">cam</Emphasis></Subscript>, putidaredoxin,and putidaredoxin reductase provides a useful cell-based catalytic system

The catalytic turnover of cytochrome P450 _cam from Pseudomonas putida requires two auxiliary reduction partners, putidaredoxin (Pd) and putidaredoxin reductase (PdR). We report the functional expression in Escherichia coli of tricistronic constructs consisting of P450 _cam encoded by the first cistron and the auxiliary proteins, Pd and PdR by the second and the third. Transformed bacterial whole cells efficiently oxidized (1R)-(+)-camphor to 5-exo-hydroxycamphor and, interestingly, limonene to (−)-perillyl alcohol. These bioengineered E. coli cells possess a heterologous self-sufficient P450 catalytic system that may have advantages in terms of low cost and high yield for the production of fine chemicals. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Screening of terrestrial <Emphasis Type="Italic">Streptomyces</Emphasis> leading to identification of new stereoisomeric anthracyclines

Thirty different Streptomyces strains were isolated from soil samples collected from different places in Egypt. The strains were isolated using Oatmeal agar and ISP medium 2 agar at pH 7.5. The isolate Streptomyces sp. Eg23 was selected for further working up to yield three new streoisomers of anthracycline metabolites. The structures were elucidated as (7R, 9R, 10S)-e-Rhodomycinone (1), (7R, 9R, 10S) Aklavinone (2), and (9R, 10S) 7-Desoxy-z-Rhodomycinone (3) by interpretation of their 1D and 2D NMR spectra. The absolute stereochemistry of 1 was confirmed by modified Mosher’s method. The (7R, 9R, 10S)-e-Rhodomycinone (1) showed activity against human lung cancer cell H460, murine Lewis lung cancer cell LL/2 and human breast cancer cell MCF-7. Compounds 1–3 were known synthetically, but until now have not been isolated as natural products from a microorganism. The results showed that the Streptomyces sp. Eg23 seems to be an interesting target for studying the regio-and stereochemistry of the cyclization step catalysed by polyketide cyclases to produce new anthracyclines through combinatorial biosynthesis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Proteome analysis of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> mutants affected in the proteasome system reveals changes in stress-responsive proteins

Prokaryotic 20S proteasomes are confined to archaebacteria and actinomycetes. Bacterial targets of this compartmentalized multi-subunit protease have not yet been identified and its physiological function in prokaryotes remains unknown. In this study, intracellular and extracellular proteomes of Streptomyces coelicolor A3(2) mutants affected in the structural genes of the 20S proteasome, in the gene encoding the presumed proteasome-accessory AAA ATPase ARC, or in two putative proteasome-associated actinomycete-specific genes (sco1646, sco1647) were analysed, revealing modified patterns of stress-responsive proteins. In addition, the extracellular protease profile of the sco1647 mutant was significantly altered. The most prominent change, common to the four mutants, was a strongly increased level of the non-heme chloroperoxidase SCO0465, coinciding with an increased resistance to cumene hydroperoxide. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The chloroplast genome from a lycophyte (microphyllophyte), <Emphasis Type="Italic">Selaginella uncinata</Emphasis>, has a unique inversion,transpositions and many gene losses

We determined the complete nucleotide sequence of the chloroplast genome of Selaginella uncinata, a lycophyte belonging to the basal lineage of the vascular plants. The circular double-stranded DNA is 144,170 bp, with an inverted repeat of 25,578 bp separated by a large single copy region (LSC) of 77,706 bp and a small single copy region (SSC) of 40,886 bp. We assigned 81 protein-coding genes including four pseudogenes, four rRNA genes and only 12 tRNA genes. Four genes, rps15, rps16, rpl32 and ycf10, found in most chloroplast genomes in land plants were not present in S. uncinata. While gene order and arrangement of the chloroplast genome of another lycophyte, Hupertzia lucidula, are almost the same as those of bryophytes, those of S. uncinata differ considerably from the typical structure of bryophytes with respect to the presence of a unique 20 kb inversion within the LSC, transposition of two segments from the LSC to the SSC and many gene losses. Thus, the organization of the S. uncinata chloroplast genome provides a new insight into the evolution of lycophytes, which were separated from euphyllophytes approximately 400 million years ago. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning of milk-derived bioactive peptides in <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis>

The enzymatic breakdown of milk proteins releases bioactive peptides. Two such peptides are the 11-residue antimicrobial peptide from bovine lactoferrin (BL-11) and the 12-residue hypotensive peptide from α_s1-casein (C-12). These two peptides have now been cloned in Streptococcus thermophilus to develop strains that enhance the functionality and nutritional value of dairy food products. Nucleic acid sequences encoding the peptides were generated by overlapping PCR and were subsequently cloned into a new expression vector under control of the ST₂₂₀₁ promoter. S. thermophilus transformants were successfully identified using GFP as a selectable marker. The presence of the synthetic gene constructs in S. thermophilus was confirmed by PCR. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Directed evolution of a beta-galactosidase from <Emphasis Type="Italic">Pyrococcus woesei</Emphasis> resulting in increased thermostable beta-glucuronidase activity

We performed directed evolution on a chemically synthesized 1,533-bp recombinant beta-galactosidase gene from Pyrococcus woesei. More than 200,000 variant colonies in each round of directed evolution were screened using the pYPX251 vector and host strain Rosetta-Blue (DE3). One shifted beta-galactosidase to beta-glucuronidase mutant, named YG6762, was obtained after four rounds of directed evolution and screening. This mutant had eight mutated amino acid residues. T29A, V213I, L217M, N277H, I387V, R491C, and N496D were key mutations for high beta-glucuronidase activity, while E414D was not essential because the mutation did not lead to a change in beta-glucuronidase activity. The amino acid site 277 was the most essential because mutating H back to N resulted in a 50% decrease in beta-glucuronidase activity at 37°C. We also demonstrated that amino acid 277 was the most essential site, as the mutation from N to H resulted in a 1.5-fold increase in beta-glucuronidase activity at 37°C. Although most single amino acid changes lead to less than a 20% increase in beta-glucuronidase activity, the YG6762 variant, which was mutated at all eight amino acid sites, had a beta-glucuronidase activity that was about five and seven times greater than the wild-type enzyme at 37 and 25°C, respectively. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In vivo functional characterization of the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 60S biogenesis GTPase Nog1

The Saccharomyces cerevisiae Nog1 GTPase is critical for assembly of the large ribosomal subunit. Mutations in conserved residues in the GTP-binding pocket cause defects in cell growth and 60S ribosome assembly but mutant proteins retain their ability to associate with the pre-60S. Association of Nog1 with the pre-60S is independent of guanine nucleotide added to cell extracts. Thus, it appears that nucleotide occupancy does not substantially affect Nog1 association with pre-60S particles. Somewhat surprisingly, neither of the conserved threonines in the G2 motif of the GTPase domain is essential for Nog1 function. Neither the steady-state rRNA levels nor the protein composition (as determined by isobaric labeling and identification by mass spectrometry of peptides) of the pre-60S particles in the nog1P176V mutant are grossly perturbed, although levels of four proteins (Nog1, Nop2, Nop15, and Tif6) are modestly reduced in pre-60S particles isolated from the mutant. Deletion analysis revealed that the C-terminal 168 amino acids are not required for function; however, the N-terminal 126 amino acids are required. Optimal association with pre-60S particles requires sequences between amino acids 347–456. Several conserved charge-to-alanine substitutions outside the GTPase domain display modest growth phenotypes indicating that these residues are not critical for function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inherited maternal effects on the drought tolerance of a natural hybrid aquatic plant, <Emphasis Type="Italic">Potamogeton anguillanus</Emphasis>

We tested whether maternal effects have led to the adaptive divergence of strains of the natural hybrid Potamogeton anguillanus, whose putative parents show contrastingly divergent ecologies. To examine the correlation between phenotypic characters and maternal types, we conducted drought experiments and DNA typing using nuclear and chloroplast genes. In the field, we investigated the distribution of the maternal type along the depth and the inshore-offshore gradient. Hybrids of P. malaianus mothers (M-hybrids) and those of P. perfoliatus mothers (P-hybrids) could not be distinguished morphologically under submerged conditions, but differed in drought tolerance. M-hybrids and P. malaianus formed more terrestrial shoots and exhibited higher survival than P-hybrids and P. perfoliatus in drought experiments. The distribution survey clarified that M-hybrids were dominant in shallow and inshore areas, whereas they were almost absent in deeper and offshore areas. These results indicate that the natural hybrid P. anguillanus differs in adaptive values depending on the maternal type. Bidirectional hybridization and heritable maternal effects may have played important roles in its phenotypic adaptation to local environmental conditions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biotransformation of diosgenin to nuatigenin-type steroid by a newly isolated strain, <Emphasis Type="Italic">Streptomyces virginiae</Emphasis> IBL-14

An actinomycete strain IBL-14 isolated from soil by utilizing diosgenin as the sole carbon and energy source was identified as Streptomyces virginiae. S. virginiae IBL-14 can transform diosgenin to isonuatigenone. To our knowledge, this is the first reported case of producing rare nuatigenin-type spirosteroid (isonuatigenone) from pyrano-spirosteroid (diosgenin) by microbial transformation. From diosgenin to isonuatigenone, the pathway has been confirmed in this study that diosgenin was first converted to diosgenone, and then diosgenone was transformed to isonuatigenone by the C25 tertiary hydroxylation reaction. It appeared to be favorable to accumulate isonuatigenone when diosgenin was added to the onset of the stationary phase of cell growth, and the yield of isonuatigenone was about 28.4% during 48 h from 1.5 mM diosgenin. The C25 tertiary hydroxylation of diosgenone by S. virginiae IBL-14 is a novel and interesting reaction and will be a practical tool in producing natural nuatigenin-type steroids. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Altered ivermectin pharmacology and defective visual system in <Emphasis Type="Italic">Drosophila</Emphasis> mutants for histamine receptor HCLB

The Drosophila gene hclB encodes a histamine-gated chloride channel, which can be activated by the neurotoxin ivermectin when expressed in vitro. We have identified two novel hclB mutants, carrying either a missense mutation (P293S, allele hclB ^T1 ) or a putative null mutation (W111*, allele hclB ^T2 ), as well as a novel splice form of the gene. In survival studies, hclB ^T1 mutants were more sensitive to ivermectin than wild-type, whereas hclB ^T2 were more resistant. Electroretinogram recordings from the two mutants exhibited enlarged peak amplitudes of the transient components, indicating altered synaptic transmission between retinal photoneurons and their target cells. Ivermectin treatment severely affected or completely suppressed these transient components in an allele-specific manner. This suppression of synaptic signals by ivermectin was dose-dependent. These results identify HCLB as an important in vivo target for ivermectin in Drosophila melanogaster, and demonstrate the involvement of this protein in the visual pathway. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

The in vitro activity of a Rad55 homologue from <Emphasis Type="Italic">Sulfolobus tokodaii</Emphasis>, a candidate mediator in RadA-catalyzed homologous recombination

Archaea have recombination proteins similar to those of eukaryote, but many have not been characterized. Here, the characterization of a Rad55 homologue from Sulfolobus tokodaii (stRad55A) was reported. StRad55A protein preferred binding to ssDNA and had ssDNA-dependent ATPase activity. In addition, UV light could induce the expression of this protein, which was different from RadB, a RadA paralog found in euryarchaeota. Most importantly, stRad55A could release the suppression of excessive stSSB (single strand DNA binding protein from S. tokodaii) on the strand exchange catalyzed by stRadA (RadA homologue from S. tokodaii), by interacting directly with both stRadA and stSSB. StRad55A may function as a mediator to accelerate the displacement of stSSB by stRadA. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conserved and novel miRNAs in the legume <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> in response to stress

MicroRNAs (miRNAs) are small RNA molecules recognized as important regulators of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data. We are interested in the identification of miRNAs in Phaseolus vulgaris (common bean) to uncover different plant strategies to cope with adverse conditions and because of its relevance as a crop in developing countries. Here we present the identification of conserved and candidate novel miRNAs in P. vulgaris present in different organs and growth conditions, including drought, abscisic acid treatment, and Rhizobium infection. We also identified cDNA sequences in public databases that represent the corresponding miRNA precursors. In addition, we predicted and validated target mRNAs amongst reported EST and cDNAs for P. vulgaris. We propose that the novel miRNAs present in common bean and other legumes, are involved in regulation of legume-specific processes including adaptation to diverse external cues. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.