SinEx DB 2.0 update 2020: database for eukaryotic single-exon coding sequences.

Single-exon coding sequences (CDSs), also known as 'single-exon genes' (SEGs), are defined as nuclear, protein-coding genes that lack introns in their CDSs. They have been studied not only to determine their origin and evolution but also because their expression has been linked to several types of human cancers and neurological/developmental disorders, and many exhibit tissue-specific transcription. We developed SinEx DB that houses DNA and protein sequence information of SEGs from 10 mammalian genomes including human. SinEx DB includes their functional predictions (KOG (euKaryotic Orthologous Groups)) and the relative distribution of these functions within species. Here, we report SinEx 2.0, a major update of SinEx DB that includes information of the occurrence, distribution and functional prediction of SEGs from 60 completely sequenced eukaryotic genomes, representing animals, fungi, protists and plants. The information is stored in a relational database built with MySQL Server 5.7, and the complete dataset of SEG sequences and their GO (Gene Ontology) functional assignations are available for downloading. SinEx DB 2.0 was built with a novel pipeline that helps disambiguate single-exon isoforms from SEGs. SinEx DB 2.0 is the largest available database for SEGs and provides a rich source of information for advancing our understanding of the evolution, function of SEGs and their associations with disorders including cancers and neurological and developmental diseases. Database URL: http://v2.sinex.cl/.

α-fur, an antisense RNA gene to fur in the extreme acidophile Acidithiobacillus ferrooxidans.

A large non-coding RNA, termed α-Fur, of ~1000 nt has been detected in the extreme acidophile Acidithiobacillus ferrooxidans encoded on the antisense strand to the iron-responsive master regulator fur (ferric uptake regulator) gene. A promoter for α-fur was predicted bioinformatically and validated using gene fusion experiments. The promoter is situated within the coding region and in the same sense as proB, potentially encoding a glutamate 5-kinase. The 3' termination site of the α-fur transcript was determined by 3' rapid amplification of cDNA ends to lie 7 nt downstream of the start of transcription of fur. Thus, α-fur is antisense to the complete coding region of fur, including its predicted ribosome-binding site. The genetic context of α-fur is conserved in several members of the genus Acidithiobacillus but not in all acidophiles, indicating that it is monophyletic but not niche specific. It is hypothesized that α-Fur regulates the cellular level of Fur. This is the fourth example of an antisense RNA to fur, although it is the first in an extreme acidophile, and underscores the growing importance of cis-encoded non-coding RNAs as potential regulators involved in the microbial iron-responsive stimulon.

The ferric iron uptake regulator (Fur) from the extreme acidophile Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is a Gram-negative bacterium that lives at pH 2 in high concentrations of soluble ferrous and ferric iron, making it an interesting model for understanding the biological mechanisms of bacterial iron uptake and homeostasis in extremely acid conditions. A candidate fur(AF) (Ferric Uptake Regulator) gene was identified in the A. ferrooxidans ATCC 23270 genome. Fur(AF) has significant sequence similarity, including conservation of functional motifs, to known Fur orthologues and exhibits cross-reactivity to Escherichia coli Fur antiserum. The fur(AF) gene is able to complement fur deficiency in E. coli in an iron-responsive manner. Fur(AF) is also able to bind specifically to E. coli Fur regulatory regions (Fur boxes) and to a candidate Fur box from A. ferrooxidans, as judged by electrophoretic mobility shift assays. Fur(AF) represses gene expression from E. coli Fur-responsive promoters fiu and fhuF when expressed at high protein levels. However, it increases gene expression from these promoters at low concentrations and possibly from other Fur-regulated promoters involved in iron-responsive oxidative stress responses.

ISAfe1, an ISL3 family insertion sequence from Acidithiobacillus ferrooxidans ATCC 19859.

A 1.3-kb insertion sequence, termed ISAfe1 (U66426), from Acidithiobacillus ferrooxidans ATCC 19859 is described. ISAfe1 exhibits the features of a typical bacterial insertion sequence. It has 26-bp, imperfectly matched, terminal inverted repeats and an open reading frame (ORF) that potentially encodes a transposase (TPase) of 404 amino acids (AAB07489) with significant similarity to members of the ISL3 family of insertion sequences. A potential ribosome-binding site and potential -10 and -35 promoter sites for the TPase ORF were identified, and a +1 transcriptional start site was detected experimentally. A potential outwardly directed -35 site was identified in the right inverted repeat of ISAfe1. A second ORF (ORF B), of unknown function, was found on the complementary strand with significant similarity to ORF 2 of ISAe1 from Ralstonia eutropha. Southern blot analyses demonstrated that ISAfe1-like elements can be found in multiple copies in a variety of A. ferrooxidans strains and that they exhibit transposition. A codon adaptation index (CAI) analysis of the TPase of ISAfe1 indicates that is has a CAI of 0.726 and can be considered well adapted to its host, suggesting that ISAfe1 might be an ancient resident of A. ferrooxidans. Analysis of six of its target sites of insertion in the genome of A. ferrooxidans ATCC 19859 indicates a preference for 8-bp pseudopalindromic sequences, one of which resembles the termini of its inverted repeats. Evidence is presented here that is consistent with the possibility that ISAfe1 can promote both plasmid cointegrate formation and resolution in E. coli.

Amiodarone inhibits cardiac ATP-sensitive potassium channels.

INTRODUCTION: ATP-sensitive K+ channels (K(ATP)) are expressed abundantly in cardiovascular tissues. Blocking this channel in experimental models of ischemia can reduce arrhythmias. We investigated the acute effects of amiodarone on the activity of cardiac sarcolemmal K(ATP) channels and their sensitivity to ATP. METHODS AND RESULTS: Single K(ATP) channel activity was recorded using inside-out patches from rat ventricular myocytes (symmetric 140 mM K+ solutions and a pipette potential of +40 mV). Amiodarone inhibited K(ATP) channel activity in a concentration-dependent manner. After 60 seconds of exposure to amiodarone, the fraction of mean patch current relative to baseline current was 1.0 +/- 0.05 (n = 4), 0.8 +/- 0.07 (n = 4), 0.6 +/- 0.07 (n = 5), and 0.2 +/- 0.05 (n = 7) with 0, 0.1, 1.0, or 10 microM amiodarone, respectively (IC50 = 2.3 microM). ATP sensitivity was greater in the presence of amiodarone (EC50 = 13 +/- 0.2 microM in the presence of 10 microM amiodarone vs 43 +/- 0.1 microM in controls, n = 5; P < 0.05). Kinetic analysis showed that open and short closed intervals (bursting activity) were unchanged by 1 microM amiodarone, whereas interburst closed intervals were prolonged. Amiodarone also inhibited whole cell K(ATP) channel current (activated by 100 microM bimakalim). After a 10-minute application of amiodarone (10 microM), relative current was 0.71 +/- 0.03 vs 0.92 +/- 0.09 in control (P < 0.03). CONCLUSION: Amiodarone rapidly inhibited K(ATP) channel activity by both promoting channel closure and increasing ATP sensitivity. These actions may contribute to the antiarrhythmic properties of amiodarone.

IST1 insertional inactivation of the resB gene: implications for phenotypic switching in Thiobacillus ferrooxidans.

Thiobacillus ferroxidans ATCC 19859 undergoes rapid phenotypic switching between a wild-type state characterized by the ability to oxidize ferrous iron (FeII) and reduced sulfur compounds and a mutant state where it has lost the capacity to oxidize FeII but retains the ability to oxidize sulfur. The mutant has also gained the capacity to swarm. It is proposed that loss of FeII oxidation is due to the reversible transposition of the insertion sequence IST1 into resB encoding a putative cytochrome c-type biogenesis protein. Downstream from resB and co-transcribed with it is resC, encoding another putative cytochrome biogenesis protein. IST1 insertional inactivation of resB could result in the loss of activity of its target c-type cytochrome(s). This putative target cytochrome(s) is proposed to be essential for FeII oxidation but not for sulfur oxidation. Curiously, resB and resC pertain to the proposed system II cytochrome biogenesis pathway whereas gamma Proteobacteria, of which T. ferrooxidans is a member, normally use system I. This could represent an example of lateral gene transfer.

Sequence and expression of the rusticyanin structural gene from Thiobacillus ferrooxidans ATCC33020 strain.

The periplasmic blue copper protein rusticyanin is thought to play an important role in iron oxidation by Thiobacillus ferrooxidans. We present the sequence of the gene, rus, encoding rusticyanin together with about 1.4 kb of upstream and 0.3 kb of downstream DNA. The rus gene is unique to T. ferrooxidans. Evidence is presented that it is the last gene of an operon and that it can be transcribed from its own promoter. In ATCC33020 strain, rusticyanin is synthesized in ferrous iron but also in sulfur growth conditions suggesting that it could play a role in both energetic metabolisms. The rus gene transcribed from a vector promoter in Escherichia coli leads to the production of a processed aporusticyanin in the periplasmic space, indicating that its signal sequence is correctly recognized by the secretion machinery and the signal peptidase of E. coli.

Mimics of the sialyl Lewis X tetrasaccharide. Replacement of the N-acetylglucosamine sugar with simple C2-symmetric 1,2-diols.

Analogues of sialyl Lewis X have been synthesized that feature replacement of the N-acetylglucosamine residue with C2-symmetric diols. The diols used contain different levels of torsional constraint and various functional groups. The cyclohexyl derived compound 27 was equipotent to sLex in vitro (IC50 0.5 mM).

Biological cyanide destruction mediated by microorganisms.

Many microorganisms have an inherent capacity to degrade the toxic organic compounds that enter the environment as a result of pollution and natural activities. Significant degradation of these compounds may take many years and it is frequently necessary to consider methods that can accelerate this process. There have been several demonstrations of enhanced biological degradation of toxic wastes, both in the laboratory and under field conditions. The prospects for enhanced biological cyanide degradation are reviewed. Compared with bench-scale processes, there are very few reports of field-scale processes for cyanide bioremediation. The implementation of such field-scale degradation requires inputs from biology, hydrology, geology, chemistry and civil engineering. A conceptual framework is emerging that can be adapted to develop new processes for bioremediation of toxic organic wastes. In terms of cyanide biodegradation, this framework incorporates identification of microbes, determination of the optimal conditions for degradation, establishment of the metabolic pathways involved in cyanide degradation, identification and localization of the genes involved, identification of suitable microbial strains for practical application and development of practical engineering processes. The present review addresses the progress that has been made in each of these aspects of cyanide biodegradation. It also examines the existing field applications of biological cyanide degradation and makes recommendations for future research.

Synthesis and structure-activity relationships of a series of penicillin-derived HIV proteinase inhibitors: heterocyclic ring systems containing P1' and P2' substituents.

As an extension of our earlier work based upon a single penicillin-derived thiazolidine moiety we have found that the decahydroisoquinoline grouping, also present in Ro 31-8959, is an effective replacement for one of the thiazolidine units in C2 symmetric penicillin-derived dimers. Reaction of racemic epoxide 6 with [3S-[3 alpha, 4a alpha, 8a alpha]]-decahydro-N-(1,1-dimethylethyl)-3- isoquinolinecarboxamide gave diasteroisomers 34a and 34b. The stereochemistry of the hydroxyl grouping of 34a was determined to be (S). Reaction of the amines derived from 34a and 34b with thiazolidine 8a gave 50 and 51, respectively. Compound 50 was a potent inhibitor of HIV proteinase (IC50 = 23 nM) with antiviral activity against HIV-1 in vitro (EC50 C8166 cells = 50 nM). However, a poor pharmacokinetic profile in the dog for compound 50 and its analogues, in keeping with earlier studies on penicillin-derived dimers in three species, precluded their development as potential antivirals.

Transposition of IST2 in Thiobacillus ferrooxidans.

The genome of Thiobacillus ferrooxidans contains at least two different repetitive DNA elements. One of these elements, termed IST2 has been sequenced and shown to exhibit the characteristics of a typical prokaryotic insertion sequence. Furthermore, preliminary evidence has implicated IST2 in genomic rearrangements, although the mechanism of rearrangement, whether by transposition or recombination, has not been established. In this report we provide evidence from detailed restriction enzyme analyses and DNA sequencing data that support a model of transposition, consistent with the notion that IST2 is a mobile insertion sequence.

Discovery and analysis of a series of C2-symmetric HIV-1 proteinase inhibitors derived from penicillin.

In order to identify a suitable peptide substrate for human immunodeficiency virus-1 (HIV-1) proteinase, a range of peptides from various cleavage sites within the gag-pol polyprotein were assayed by HPLC for specific cleavage. The peptide with the optimal combination of favorable kinetics and good solubility was based on the N-terminus cleavage site of HIV-1 proteinase (KQGTVSFNF*PQIT). The HPLC assay, using the above peptide, was developed into a rapid isocratic method in order to analyze inhibition kinetics. An assay suitable for high-throughput screening was developed using a radioactively labeled peptide with the same sequence, coupled to a solid phase. Using this assay, a C2-symmetric HIV-1 proteinase inhibitor derived from penicillin was discovered during random screening of a compound library. A chemical synthesis program developed this structure into a series of potent inhibitors. The lead structures were highly selective for HIV-1 proteinase with good antiviral activity in vitro against HIV and no cytotoxicity. The HPLC assay was used to demonstrate that these compounds are competitive tight-binding inhibitors of HIV-1 proteinase.

Development of biosensors for the detection of mercury and copper ions.

The development of genetically engineered biosensors for copper and mercury ions is described. The biosensors have been constructed by fusing thelux or light emitting genes fromVibrio fischeri with genetic regulating elements that respond to copper ions or mercury ions, derived respectively fromEscherichia coli andSerratia marcescens. The fusions were placed intoE. coli cells which then emitted light in response to copper or mercury ions. Data is presented describing the sensitivity, specificity, and dynamic range of the biosensors to their respective target metal ions. A preliminary description of experiments is provided indicating how these biosensors might be used to investigate the bioavailability of mercury and copper ions in environmental samples.

Synthesis and structure-activity relationships of a series of penicillin-derived HIV proteinase inhibitors containing a stereochemically unique peptide isostere.

A series of HIV-1 proteinase inhibitors was synthesized based upon a single penicillin derived thiazolidine moiety. Reaction of the C-4 carboxyl group with (R)-phenylalaninol gave amide 10 which was a moderately potent inhibitor of HIV-1 proteinase (IC50 = 0.15 microM). Further modifications based on molecular modeling studies led to compound 48 which contained a stereochemically unique statine-based isostere. This was a potent competitive inhibitor (Ki = 0.25 nM) with antiviral activity against HIV-1 in vitro (5 microM). Neither modification to the benzyl group in an attempt to improve interaction with the S2' pocket, nor introduction of a hydrogen bond donating group to interact with residue Gly48' resulted in improved inhibitory or antiviral activity.

Genetic linkage analysis of manic depression in Iceland.

Genetic linkage analysis has been used to study five Icelandic pedigrees multiply affected with manic depression. Genetic markers were chosen from regions which had been implicated by other studies or to which candidate genes had been localized. The transmission model used was of a dominant gene with incomplete penetrance and allowing for a large number of phenocopies, especially for unipolar rather than bipolar cases. Multipoint analysis with linked markers enabled information to be gained from regions spanning large distances. Using this approach we have excluded regions of chromosome 11p, 11q, 8q, 5q, 9q and Xq. Candidate genes excluded include those for tyrosine hydroxylase, the dopamine type 2 receptor, proenkephalin, the 5HT1A receptor and dopamine beta hydroxylase. Nevertheless, we remain optimistic that this approach will eventually identify at least some of the genes predisposing to manic depression.

Segregation and linkage analysis in five manic depression pedigrees excludes the 5HT1a receptor gene (HTR1A).

Five kindreds selected through probands attending an Icelandic hospital were recruited for linkage studies of manic depression. The rates of affection were equal for males and females and the age of onset appeared to be predominantly in early adult life, since prevalence did not rise appreciably with age. A complex segregation analysis was performed using the computer program POINTER to obtain maximum likelihood estimates of the contributions to liability from multifactorial transmission and a single major locus. Likelihood ratios between models supported a role for a single major locus which was dominant and had moderately high penetrance with, in the case of unipolar illness, additional multifactorial transmission. The best-fitting parameters were used to devise a transmission model for linkage analysis. Three markers on chromosome 5 were studied, at D5S76, D5S6 and D5S39. Strongly negative lod scores were obtained which were less than -2 over a distance of 40 cM, which included the region to which the gene for the 5HT1a receptor has been mapped.

Construction and evaluation of a self-luminescent biosensor.

The genes encoding bioluminescence (lux genes), derived from the marine bacterium V. fischeri, have been fused next to the genes encoding mercury detoxification (mer genes), derived from a clinical isolate of S. marcescens. The fusion has been made so that the expression of the light genes comes under the control of the mer regulatory gene and promoter. These genetic elements activate the expression of the light genes in the presence of mercury. The light can readily be collected and quantitated, resulting in a biosensor for the detection of mercury.

Effects of an acute bout of aerobic exercise on cardiovascular and subjective responses during subsequent cognitive work.

The experiment was conducted to test the effects of an acute bout of aerobic exercise on cardiovascular arousal and mood during a subsequent period of cognitive work. Each subject participated in two testing sessions, one that followed exercise and one that did not. In each session the subjects studied for 40 minutes, and arousal during the study period was assessed. The results indicated that prior exercise resulted in higher pulse rate (10%; p = 0.02), slightly lower systolic blood pressure (2%; p = 0.09), and higher feelings of vigor (59%; p = 0.01) than did no-exercise. The results are somewhat inconsistent with anecdotal reports concerning the effects of acute exercise, and possible reasons for the inconsistencies are discussed.

Selection of a specifically blocked mutant of Streptomyces cinnamonensis: isolation and synthesis of 26-deoxymonensin A.

Streptomyces cinnamonensis produces the polyether ionophore antibiotic monensin A. Following a single round of mutagenesis by UV light, a derivative of this strain has been isolated, which secretes a new metabolite identified as 26-deoxymonensin A (3). The structural elucidation of the new metabolite followed from a spectroscopic analysis, and its identity was proven conclusively following a comparison to 26-deoxymonensin A (3) obtained synthetically from monensin A. The preparation of labelled forms of 3 is described, together with incorporation experiments using the parent strain of S. cinnamonensis. Only very low levels of incorporation of 3 into monensin A were observed.

IST2: an insertion sequence from Thiobacillus ferrooxidans.

The genome of Thiobacillus ferrooxidans (strain ATCC 19859) contains at least two families of repeated sequences, termed family 1 and 2. The nucleotide sequence of a family 2 member was determined. It is 1408 base pairs long and has structural features similar to those of insertion sequences (IS elements). Terminal inverted repeats 25 base pairs in length are present. These inverted repeats are imperfect and adjacent to target-site duplications 9 base pairs in length. Several open reading frames were detected (the longest was 888 base pairs). We have named this IS element-like sequence IST2. The ends of a second example of IST2 were analyzed and compared to those of the first. The DNA sequences are identical and similarly sized target-site duplications are present.