Rescue of a failed polymerase chain reaction catalyzed by vent or deep vent DNA polymerase with 10% dimethyl sulfoxide.
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Base Sequence↗
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Spliceosomal introns in a deep-branching eukaryote: the splice of life.
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Animals↗
Population structure among African and derived populations of Drosophila simulans: evidence for ancient subdivision and recent admixture.
Previous studies based on allozyme variation have found little evidence for genetic differentiation in Drosophila simulans. On the basis of DNA sequence variation at two nuclear loci in four African populations of D. simulans, we show that there is significant structure to D. simulans populations within Africa. Variation at one of the loci, vermilion, appears to be neutral and supports an eastern African origin for European and American populations. Samples from the West Indies, Europe, and North America had a nucleotide diversity lower than that of African populations at vermilion and show nonequilibrium haplotype distributions at both vermilion and G6pd, consistent with a hypothesis of recent bottleneck and possibly also admixture in the history of these populations. Directional selection, previously documented at G6pd, appears to have occurred within the coalescence time of the species, obscuring deep population history.
Africa↗
Population genetic structuring of the king weakfish, Macrodon ancylodon (Sciaenidae), in Atlantic coastal waters of South America: deep genetic divergence without morphological change.
Phylogeographic patterns in Macrodon ancylodon sampled from 12 locations across all its range were investigated using mitochondrial DNA cytochrome b sequences, and analysed together with patterns of morphometric differentiation. Populations of the North Brazil and the Brazil currents, with warmer waters, form a clade (tropical clade) separated by 23 fixed mutations from the populations that inhabit regions of colder waters influenced by the Brazil and Malvinas currents (subtropical clade). No gene flow exists between the tropical and subtropical clades, and most likely also between the two groups of the tropical clade. Distribution of these clades and groups is correlated with flow of currents and their temperatures, and is facilitated by larval retention and low adult migration. Despite differentiation at the molecular level, fishes analysed from all these current-influenced regions are morphometrically homogeneous. Throughout its range M. ancylodon inhabits the same, or very similar niche; thus, stabilizing selection probably promotes the retention of highly conserved morphology despite deep genetic divergence at the mitochondrial DNA cytochrome b.
Animals↗
Peptide-triggered conformational switch in HIV-1 RRE RNA complexes.
We have used NMR spectroscopy to determine the solution structure of a complex between an oligonucleotide derived from stem IIB of the Rev responsive element (RRE-IIB) of HIV-1 mRNA and an in vivo selected, high affinity binding Arg-rich peptide. The peptide binds in a partially alpha-helical conformation into a pocket within the RNA deep groove. Comparison with the structure of a complex between an alpha-helical Rev peptide and RRE-IIB reveals that the sequence of the bound peptide determines the local conformation of the RRE peptide binding site. A conformational switch of an unpaired uridine base was revealed; this points out into the solvent in the Rev peptide complex, but it is stabilized inside the RNA deep groove by stacking with an Arg side chain in the selected peptide complex. The conformational switch has been visualized by NMR chemical shift mapping of the uridine H5/H6 atoms during a competition experiment in which Rev peptide was displaced from RRE-IIB by the higher affinity binding selected peptide.
Amino Acid Sequence↗
Functional correlates of pallidal stimulation for Parkinson's disease.
We measured regional cerebral blood flow with H2 15O and positron emission tomography (PET) scanning at rest and during a motor task to study the mechanism of motor improvement induced by deep brain stimulation of the internal globus pallidus in Parkinson's disease. Six right-handed patients with Parkinson's disease were scanned while performing a predictable paced sequence of reaching movements and while observing the same screen displays and tones. PET studies were performed ON and OFF stimulation in a medication-free state. Internal globus pallidus deep brain stimulation improved off-state United Parkinson's Disease Rating Scale motor ratings (37%, p < 0.002) and reduced timing errors (movement onset time, 55%, p < 0.01) as well as spatial errors (10%, p < 0.02). Concurrent regional cerebral blood flow recordings revealed a significant enhancement of motor activation responses in the left sensorimotor cortex (Brodmann area [BA] 4), bilaterally in the supplementary motor area (BA 6), and in the right anterior cingulate cortex (BA 24/32). Significant correlations were evident between the improvement in motor performance and the regional cerebral blood flow changes mediated by stimulation. With internal globus pallidus deep brain stimulation, improved movement initiation correlated with regional cerebral blood flow increases in the left sensorimotor cortex and ventrolateral thalamus and in the contralateral cerebellum. By contrast, improved spatial accuracy correlated with regional cerebral blood flow increases in both cerebellar hemispheres and in the left sensorimotor cortex. These results suggest that internal globus pallidus deep brain stimulation may selectively improve different aspects of motor performance. Multiple, overlapping neural pathways may be modulated by this intervention.
Adult↗
Evidence of balancing selection at the HLA-G promoter region.
HLA-G is a class Ib HLA gene with unique tissue expression pattern and immunomodulatory properties. Polymorphisms in the HLA-G promoter region have been associated with miscarriage and asthma, whereas expression levels have been associated with a wide range of pathologic conditions as well as survival of embryos after in vitro fertilization and of organs after transplantation. Here, we characterize the sequence variation and haplotype structure of the HLA-G promoter and flanking sequences in 44 African Americans, 47 European Americans and 43 Han Chinese by haplotype-specific PCR and sequencing. In all three populations, we observed high levels of nucleotide variation, an excess of intermediate-frequency alleles, and a genealogy with two common haplotypes separated by deep branches, features that are suggestive of balancing selection acting in this region. Comparisons to HLA-A and a pseudogene, HLA-J, suggested that the observed pattern of sequence variation in the HLA-G promoter region is not likely due to other selected HLA genes. We suggest that the mechanism for this selection is related to the highly regulated expression pattern of HLA-G and that high- and low-expressing promoters may be favored under temporally and/or spatially varying selective pressures.
Black or African American↗
Biases in phylogenetic estimation can be caused by random sequence segments.
We consider the effects of fully or partially random sequences on the estimation of four-taxon phylogenies. Fully or partially random sequences occur when whole subsets of sequences or some sites for subsets of sequences are independent of sequence data for the other taxa. Random sequences can be a consequence of misalignment or because sites evolve at very fast rates in some portions of a tree, a situation that occurs especially in analyses involving deep divergence times. One might reasonably speculate that random sites will only add noise to the estimation of a phylogeny. We show that in the case that a random sequence is added to a three-taxa alignment, it is more likely to be a neighbor of the sequence corresponding to the longest branch in the three-taxon tree. Surprisingly, when only about half of the sites show randomness, a long-branch-repels form of small sample bias occurs, and when a minority of sites show randomness this becomes a long-branch-attraction bias again. The most serious bias, one that does not vanish with increasing sequence length, occurs when more than one sequence is partially random. If there is a large amount of overlap in the random sites for two sequences, those two sequences will be attracted to each other; otherwise, they will repel each other. Random sequences or sites can, therefore, cause complicated biases in phylogenetic inference. We suggest performing analyses with and without potentially saturated sequences and/or misaligned sites, to check that these biases are not affecting the inferred branching pattern.
Animals↗
DNA sequence variation in a non-coding region of low recombination on the human X chromosome.
DNA sequence variation has become a major source of insight regarding the origin and history of our species as well as an important tool for the identification of allelic variants associated with disease. Comparative sequencing of DNA has to date focused mainly on mitochondrial (mt) DNA, which due to its apparent lack of recombination and high evolutionary rate lends itself well to the study of human evolution. These advantages also entail limitations. For example, the high mutation rate of mtDNA results in multiple substitutions that make phylogenetic analysis difficult and, because mtDNA is maternally inherited, it reflects only the history of females. For the history of males, the non-recombining part of the paternally inherited Y chromosome can be studied. The extent of variation on the Y chromosome is so low that variation at particular sites known to be polymorphic rather than entire sequences are typically determined. It is currently unclear how some forms of analysis (such as the coalescent) should be applied to such data. Furthermore, the lack of recombination means that selection at any locus affects all 59 Mb of DNA. To gauge the extent and pattern of point substitutional variation in non-coding parts of the human genome, we have sequenced 10 kb of non-coding DNA in a region of low recombination at Xq13.3. Analysis of this sequence in 69 individuals representing all major linguistic groups reveals the highest overall diversity in Africa, whereas deep divergences also exist in Asia. The time elapsed since the most recent common ancestor (MRCA) is 535,000+/-119,000 years. We expect this type of nuclear locus to provide more answers about the genetic origin and history of humans.
Africa↗
Extremely barophilic bacteria isolated from the Mariana Trench, Challenger Deep, at a depth of 11,000 meters.
Two strains of obligately barophilic bacteria were isolated from a sample of the world's deepest sediment, which was obtained by the unmanned deep-sea submersible Kaiko in the Mariana Trench, Challenger Deep, at a depth of 10,898 m. From the results of phylogenetic analysis based on 16S rRNA gene sequences, DNA-DNA relatedness study, and analysis of fatty acid composition, the first strain (DB21MT-2) appears to be most highly similar to Shewanella benthica and close relatives, and the second strain (DB21MT-5) appears to be closely related to the genus Moritella. The optimal pressure conditions for growth of these isolates were 70 MPa for strain DB21MT-2 and 80 MPa for strain DB21MT-5, and no growth was detected at pressures of less than 50 MPa with either strain. This is the first evidence of the existence of an extreme-barophile bacterium of the genus Moritella isolated from the deep-sea environment.
Bacteria↗
Clot-blood contrast in fast gradient-echo magnetic resonance imaging.
RATIONALE AND OBJECTIVES: Contrast between clot and blood in magnetic resonance imaging (MRI) at 1.5T using fast gradient-echo pulse sequences (fast GRE), with 8 ms < TR < 20 mseconds was studied both in vitro and in clinical human deep venous thrombosis (DVT) to assess whether good contrast could be obtained at such short repetition times and at clinically relevant flow rates. METHODS: In vitro studies used an apparatus that contained flowing MnCl2[aq] (water adjusted with manganese chloride to have T1, T2 similar to blood) and an immobilized clot (T1, T2 similar to those in DVT) for flow velocities between 0 and 16.5 cm/sec. Seven patients with DVT were imaged with the fast GRE sequences to observe the clot-blood contrast in vivo. RESULTS: Peak contrast-to-noise ratio (CNR) was achieved using flip angles between 20 degrees and 40 degrees (increasing with flow velocity) with or without radiofrequency "spoiling," consistent with a natural spoiling effect of flow. The CNR between MnCI2[aq] and clot decreased less than 10% as TR was reduced 56% from 18 mseconds to 8 mseconds (30 degrees flip angle). In four patients with nonocclusive DVT, fast GRE imaging provided good contrast while in occlusive cases (three patients) the contrast was not as good as conventional GRE sequences with longer TR values (TR = 33 mseconds). CONCLUSION: A fast GRE sequence with TR = 8 mseconds, TE = 3 mseconds, and a flip angle = 40 degrees is a promising approach to speeding up the detection of nonocclusive clinical DVT.
Blood↗
The antigenic and catalytically active formamidase of Paracoccidioides brasiliensis: protein characterization, cDNA and gene cloning, heterologous expression and functional analysis of the recombinant protein.
Paracoccidioides brasiliensis is a well-characterized pathogen of humans. To identify proteins involved in the fungus-host interaction, P. brasiliensis yeast proteins were separated by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Immunoreactive bands were detected with pooled sera of patients with P. brasiliensis infection. A protein species with a molecular mass of 45 kDa was subsequently purified to homogeneity by preparative gel electrophoresis. The amino acid sequence of four endoproteinase Lys-C-digested peptides indicated that the protein was a formamidase (FMD) (E.C. 3.5.1.49) of P. brasiliensis. The complete cDNA and a genomic clone (Pbfmd) encoding the isolated FMD were isolated. An open reading frame predicted a 415-amino acid protein. The sequence contained each of the peptide sequences obtained from amino acid sequencing. The Pbfmd gene contained five exons interrupted by four introns. Northern and Southern blot analysis suggested that there is one copy of the gene in P. brasiliensis and that it is preferentially expressed in mycelium. The complete coding cDNA was expressed in Escherichia coli to produce a recombinant fusion protein with glutathione S-transferase (GST). The purified recombinant protein was recognized by sera of patients with proven paracoccidioidomycosis and not by sera of healthy individuals. The recombinant 45-kDa protein was shown to be catalytically active; FMD activity was detected in P. brasiliensis yeast and mycelium.
Amidohydrolases↗
Molecular cloning and complete nucleotide sequence of the repeated unit and flanking gene of the scallop Pecten maximus mitochondrial DNA: putative replication origin features.
In the bivalve mollusc Pecten maximus, the size of the mitochondrial DNA molecules ranges from 20 to 25.8 kbp. This variability is mainly correlated with the occurrence of a variable domain composed with two to five 1.6-kbp repeated units tandemly arrayed in the genome. DNA fragments spanning the 1,586-base-pair-long repeated element and the nearest flanking gene have been cloned and sequenced. This sequence was analyzed regarding its base composition and potential secondary structures. The repeated unit domain was positioned and oriented with regard to the known flanking gene. It ends 2 base pairs upstream relative to the beginning of the tRNAgly gene. The peculiar properties of the repeated unit were compared with those of the 1,442-bp repeated element found in the mitochondrial genome of the deep sea scallop Placopecten magellanicus. This comparison provided evidence for the absence of nucleotide conservation, except for a small sequence engaged in a secondary structure, but argued for a strong pressure maintaining domains with specific nucleotide content. A possible role for the conserved sequence is discussed.
Animals↗
Evidence for chemoautotrophic symbiosis in a Mediterranean cold seep clam (Bivalvia: Lucinidae): comparative sequence analysis of bacterial 16S rRNA, APS reductase and RubisCO genes.
Symbioses between lucinid clams (Bivalvia: Lucinidae) and autotrophic sulphide-oxidizing bacteria have mainly been studied in shallow coastal species, and information regarding deep-sea species is scarce. Here we study the symbiosis of a clam, resembling Lucinoma kazani, which was recently collected in sediment cores from new cold-seep sites in the vicinity of the Nile deep-sea fan, eastern Mediterranean, at depths ranging from 507 to 1691 m. A dominant bacterial phylotype, related to the sulphide-oxidizing symbiont of Lucinoma aequizonata, was identified in gill tissue by comparative 16S rRNA gene sequence analysis. A second phylotype, related to spirochete sequences, was identified twice in a library of 94 clones. Comparative analyses of gene sequences encoding the APS reductase alpha subunit and ribulose-1,5-bisphosphate carboxylase oxygenase support the hypothesis that the dominant symbiont can perform sulphide oxidation and autotrophy. Transmission electron micrographs of gills confirmed the dominance of sulphide-oxidizing bacteria, which display typical vacuoles, and delta(13)C values measured in gill and foot tissue further support the hypothesis for a chemoautotrophic-sourced host carbon nutrition.
Animals↗
Diversity and origin of Desulfovibrio species: phylogenetic definition of a family.
The different nutritional properties of several Desulfovibrio desulfuricans strains suggest that either the strains are misclassified or there is a high degree of phenotypic diversity within the genus Desulfovibrio. The results of partial 16S rRNA and 23S rRNA sequence determinations demonstrated that Desulfovibrio desulfuricans ATCC 27774 and "Desulfovibrio multispirans" are closely related to the type strain (strain Essex 6) and that strains ATCC 7757, Norway 4, and El Agheila Z are not. Therefore, these latter three strains of Desulfovibrio desulfuricans are apparently misclassified. A comparative analysis of nearly complete 16S rRNA sequences in which we used a least-squares analysis method for evolutionary distances, an unweighted pair group method, a signature analysis method, and maximum parsimony was undertaken to further investigate the phylogeny of Desulfovibrio species. The species analyzed were resolved into two branches with origins deep within the delta subdivision of the purple photosynthetic bacteria. One branch contained five deep lineages, which were represented by (i) Desulfovibrio salexigens and Desulfovibrio desulfuricans El Agheila Z; (ii) Desulfovibrio africanus; (iii) Desulfovibrio desulfuricans ATCC 27774, Desulfomonas pigra, and Desulfovibrio vulgaris; (iv) Desulfovibrio gigas; and (v) Desulfomicrobium baculatus (Desulfovibrio baculatus) and Desulfovibrio desulfuricans Norway 4. A correlation between 16S rRNA sequence similarity and percentage of DNA relatedness showed that these five deep lineages are related at levels below the minimum genus level suggested by Johnson (in Bergey's Manual of Systematic Bacteriology, vol. 1, 1984). We propose that this branch should be grouped into a single family, the Desulfovibrionaceae. The other branch includes other genera of sulfate-reducing bacteria (e.g., Desulfobacter and Desulfococcus) and contains Desulfovibrio sapovorans and Desulfovibrio baarsii as separate, distantly related lineages.
Base Sequence↗
Active learning of enhancers and silencers in the developing neural retina.
Deep learning is a promising strategy for modeling cis-regulatory elements. However, models trained on genomic sequences often fail to explain why the same transcription factor can activate or repress transcription in different contexts. To address this limitation, we developed an active learning approach to train models that distinguish between enhancers and silencers composed of binding sites for the photoreceptor transcription factor cone-rod homeobox (CRX). After training the model on nearly all bound CRX sites from the genome, we coupled synthetic biology with uncertainty sampling to generate additional rounds of informative training data. This allowed us to iteratively train models on data from multiple rounds of massively parallel reporter assays. The ability of the resulting models to discriminate between CRX sites with identical sequence but opposite functions establishes active learning as an effective strategy to train models of regulatory DNA. A record of this paper's transparent peer review process is included in the supplemental information.
Retina↗
Mitochondrial gene rearrangement in the sea cucumber genus Cucumaria.
A novel mitochondrial tRNA gene arrangement is described for two species of sea cucumber. The mitochondrial tRNA gene cluster common to sea stars, sea urchins, and the sea cucumber Parastichopus californicus has been significantly modified in the genus Cucumaria as a result of dispersal of the tRNA genes into two separate areas of the genome. The tRNA genes in the novel clusters are interspersed with short unassigned sequences (UASs). Alignment of the two separated novel clusters indicates that the rearrangement was most likely the result of a tandem duplication of approximately 7 kb, encompassing the putative control region, the tRNA cluster, NADH dehydrogenase subunits 1 and 2, the large ribosomal RNA (lrRNA), cytochrome oxidase subunit I, and tRNAArg. Subsequently, deletion of the duplicated lrRNA and protein-coding genes occurred. In addition, the degeneration of one of each of the duplicated tRNA gene pairs has resulted in the interspersed UAS segments observed in each cluster. In contrast, the second copy of the putative control region has been maintained with a very high degree of sequence conservation, suggesting either some functional constraint or concerted evolution for the duplicated element. Analysis of gene organization in other sea cucumber species may provide (1) important insights into the mechanism of mitochondrial gene rearrangements and (2) an informative character set for deep-level phylogenetic analysis of this echinoderm class.
Amino Acid Sequence↗
Culture-independent identification of periodontitis-associated Porphyromonas and Tannerella populations by targeted molecular analysis.
Periodontitis is the commonest bacterial disease of humans and is the major cause of adult tooth loss. About half of the oral microflora is unculturable; and 16S rRNA PCR, cloning, and sequencing techniques have demonstrated the high level of species richness of the oral microflora. In the present study, a PCR primer set specific for the genera Porphyromonas and Tannerella was designed and used to analyze the bacterial populations in subgingival plaque samples from inflamed shallow and deep sites in subjects with periodontitis and shallow sites in age- and sex-matched controls. A total of 308 clones were sequenced and found to belong to one of six Porphyromonas or Tannerella species or phylotypes, one of which, Porphyromonas P3, was novel. Tannerella forsythensis was found in significantly higher proportions in patients than in controls. Porphyromonas catoniae and Tannerella phylotype BU063 appeared to be associated with shallow sites. Targeted culture-independent molecular ecology studies have a valuable role to play in the identification of bacterial targets for further investigations of the pathogenesis of bacterial infections.
Adult↗