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Evolutionary and structural analysis of the cytochrome c oxidase subunit I (COI) gene from Haematobia irritans, Stomoxys calcitrans and Musca domestica (Diptera: Muscidae) mitochondrial DNA.

This work describes the molecular characterization of the cytochrome c oxidase subunit I (COI) gene of the mitochondrial DNA from three species of great medical and veterinary importance: the horn fly, Haematobia irritans, the stable fly, Stomoxys calcitrans and the house fly, Musca domestica (Diptera: Muscidae) (Linnaeus). The nucleotide sequence in all species was 1536 bp in size and coded for a 512 amino acid peptide. The nucleotide bias for an A+T-rich sequence is linked to three features: a high A+T content throughout the entire gene, a high A+T content in the third codon position, and a predominance of A+T-rich codons. An anomalous TCG (serine) start codon was identified. Comparative analysis among members of the Muscidae, Scatophagidae, Calliphoridae and Drosophilidae showed high levels of nucleotide sequence conservation. Analysis of the divergent amino acids and COI protein topologies among these three Muscidae species agreed with the evolutionary model suggested for the insect mitochondrial COI protein. The characterization of the structure and evolution of this gene could be informative for further evolutionary analysis of dipteran species.

Amino Acids↗

Comparative analysis of a conserved zinc finger gene cluster on human chromosome 19q and mouse chromosome 7.

Several lines of evidence now suggest that many of the zinc-finger-containing (ZNF) genes in the human genome are arranged in clusters. However, little is known about the structure or function of the clusters or about their conservation throughout evolution. Here, we report the analysis of a conserved ZNF gene cluster located in human chromosome 19q13.2 and mouse chromosome 7. Our results indicate that the human cluster consists of at least 10 related Kruppel-associated box (KRAB)-containing ZNF genes organized in tandem over a distance of 350-450 kb. Two cDNA clones representing genes in the murine cluster have been studied in detail. The KRAB A domains of these genes are nearly identical and are highly similar to human 19q13.2-derived KRAB sequences, but DNA-binding ZNF domains and other portions of the genes differ considerably. The two murine genes display distinct expression patterns, but are coexpressed in some adult tissues. These studies pave the way for a systematic analysis of the evolution of structure and function of genes within the numerous clustered ZNF families located on human chromosome 19 and elsewhere in the human and mouse genomes.

Amino Acid Sequence↗

MAFin: motif detection in multiple alignment files.

MOTIVATION: Whole Genome and Proteome Alignments, represented by the multiple alignment file format, have become a standard approach in comparative genomics and proteomics. These often require identifying conserved motifs, which is crucial for understanding functional and evolutionary relationships. However, current approaches lack a direct method for motif detection within MAF files. We present MAFin, a novel tool that enables efficient motif detection and conservation analysis in MAF files to address this gap, streamlining genomic and proteomic research. RESULTS: We developed MAFin, the first motif detection tool for Multiple Alignment Format files. MAFin enables the multithreaded search of conserved motifs using three approaches: (i) using user-specified k-mers to search the sequences. (ii) with regular expressions, in which case one or more patterns are searched, and (iii) with predefined Position Weight Matrices. Once the motif has been found, MAFin detects the motif instances and calculates the conservation across the aligned sequences. MAFin also calculates a conservation percentage, which provides information about the conservation levels of each motif across the aligned sequences, based on the number of matches relative to the length of the motif. A set of statistics enables the interpretation of each motif's conservation level, and the detected motifs are exported in JSON and CSV files for downstream analyses. AVAILABILITY AND IMPLEMENTATION: MAFin is offered as a Python package under the GPL license as a multi-platform application and is available at: https://github.com/Georgakopoulos-Soares-lab/MAFin.

Software↗

Analysis of sequences and loci of p44 homologs expressed by Anaplasma phagocytophila in acutely infected patients.

Anaplasma phagocytophila is an obligatory intragranulocytic bacterium that causes human granulocytic ehrlichiosis. Immunodominant 44-kDa outer membrane proteins of A. phagocytophila are encoded by a p44 multigene family. In the present study, expression profiles of p44 genes in the blood of acutely infected patients in the year 2000 were characterized. A single p44 gene was predominantly expressed in peripheral blood leukocytes from one patient, while up to 17 different p44 genes were transcribed without a single majority in the other two patients. The cDNA sequences of the central hypervariable region of several p44 genes were identical among the isolates from the three patients and a 1995 A. phagocytophila isolate. A. phagocytophila was isolated by cell culture from all of the three 2000 patients. Genomic Southern blot analysis of the three 2000 and two 1995 A. phagocytophila isolates with probes specific to the most dominant p44 transcript in each patient showed that the p44 loci in the A. phagocytophila genome were conserved. Analysis of the predicted amino acid sequences of 43 different p44 genes including 19 new sequences found in the present study, revealed that five amino acids were absolutely conserved. The hypervariable region was subdivided into five domains, including three extremely hypervariable central domains. These results suggest that variations in the sequences of p44 are not random but are restricted. Furthermore, several p44 genes are not hypermutatable in nature, based on the conservation of gene sequences and loci among isolates obtained 5 years apart.

Acute Disease↗

The recognition and prediction of sigma70 promoters in Escherichia coli K-12.

Based on the conservation analysis of the 683 latest experimentally verified sigma(70)-promoter sequences of Escherichia coli K-12, it is found that the conservative hexamers segments in different sites play a key role of promoter regions, a novel position-correlation scoring matrix (PCSM) algorithm for predicting sigma(70) promoter is presented. The predictive capacity of the algorithm is tested by 10-cross validation test. The results show that the overall prediction accuracies (sensitivity) and specificity are 91% and 81%, respectively. By selecting the 683 experimentally verified sigma(70) promoters as training set and searching for the complete sequence in E. coli K-12 with 4639221bp. Results show that the 100% of the 683 experimentally verified sigma(70) promoters have been identified and some possible promoters are predicted.

Algorithms↗

The economic consequences of available diagnostic and prognostic strategies for the evaluation of stable angina patients: an observational assessment of the value of precatheterization ischemia. Economics of Noninvasive Diagnosis (END) Multicenter Study Group.

OBJECTIVES: The study aim was to determine observational differences in costs of care by the coronary disease diagnostic test modality. BACKGROUND: A number of diagnostic strategies are available with few data to compare the cost implications of the initial test choice. METHODS: We prospectively enrolled 11,372 consecutive stable angina patients who were referred for stress myocardial perfusion tomography or cardiac catheterization. Stress imaging patients were matched by their pretest clinical risk of coronary disease to a series of patients referred to cardiac catheterization. Composite 3-year costs of care were compared for two patients management strategies: 1) direct cardiac catheterization (aggressive) and 2) initial stress myocardial perfusion tomography and selective catheterization of high risk patients (conservative). Analysis of variance techniques were used to compare costs, adjusting for treatment propensity and pretest risk. RESULTS: Observational comparisons of aggressive as compared with conservative testing strategies reveal that costs of care were higher for direct cardiac catheterization in all clinical risk subsets (range: $2,878 to $4,579), as compared with stress myocardial perfusion imaging plus selective catheterization (range: $2,387 to $3,010, p < 0.0001). Coronary revascularization rates were higher for low, intermediate and high risk direct catheterization patients as compared with the initial stress perfusion imaging cohort (13% to 50%, p < 0.0001); cardiac death or myocardial infarction rates were similar (p > 0.20). CONCLUSIONS: Observational assessments reveal that stable chest pain patients who undergo a more aggressive diagnostic strategy have higher diagnostic costs and greater rates of intervention and follow-up costs. Cost differences may reflect a diminished necessity for resource consumption for patients with normal test results.

Angina Pectoris↗

Apolipoprotein A-I domains involved in lecithin-cholesterol acyltransferase activation. Structure:function relationships.

A series of mutant apolipoprotein (apoA-I) constructs were designed and then expressed in cell culture to identify structural domains within the mature native apoA-I protein that participate in the activation of the plasma enzyme, lecithin-cholesterol acyltransferase (LCAT). Evolutionary conservation analysis has shown previously that apoA-I contains eight repeats containing 22 amino acids and two repeats containing 11 amino acids that are highly conserved among species as well as within the apolipoprotein supergene family. These tandem repeats begin at residue 44 and are usually marked by a proline residue, with six of the 22-mer repeats showing high amphipathic alpha-helical character. To determine if specific 11- or 22-amino acid domains are essential for maximal LCAT activation within the entire native protein, each of the 10 repeats was sequentially deleted using a polymerase chain reaction based method of mutagenesis. The wild-type and mutant apoA-I gene constructs were expressed in Chinese hamster ovary (CHO) cells and stable lines established. Wild-type and mutant apoA-I protein were purified from 48-96-h conditioned serum-free medium and characterized by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Wild-type apoA-I showed a single migrating band of 28,000 daltons that corresponded to the mobility of human plasma apoA-I, whereas apoA-I deletion mutants (lacking 22- or 11-mer repeats) showed the corresponding shift to lower molecular size. To measure the relative LCAT activation of all deletion mutant apoA-I proteins relative to wild-type apoA-I, an assay system utilizing small unilamellar vesicles as the lipid substrate was used. The results of these studies suggest that several central amphipathic alpha-helical regions within the mature protein are critical in LCAT activation.

Animals↗

Genomic structure and promoter analysis of phosphoenolpyruvate carboxylase in a C3 plant, Nicotiana sylvestris.

Three genes encoding phosphoenolpyruvate carboxylase were isolated from Nicotiana sylvestris and designated Nsppc1-3. Sequencing of nucleotides showed that the coding sequence and deduced amino acid sequences were highly conserved among the genes, but sequences for noncoding regions including introns and 5'-flanking regions were not conserved. Analysis of the transcript level of the genes by a combination of reverse transcriptase-PCR and restriction fragment polymorphism showed mostNsppc1 in the leaves, stems, roots, and cultured cells of N. sylvestris. beta-Glucuronidase activity was detected histochemically in mesophyll cells in leaves, lateral buds, and vascular bundles in roots of transgenic tobacco harboring a chimeric construct of the Nsppc1 promoter and the gene for beta-glucuronidase. Deletion analysis indicated the presence of a silencer-like element for basal expression in the promoter region of Nsppc1.

Amino Acid Sequence↗

Constructing epigenetic regulatory landscapes of plant lncRNAs-an exploration utilizing the novel specialized platform PERlncDB.

Long non-coding RNAs (lncRNAs), once overlooked as transcriptional byproducts, are now recognized for their crucial roles in plant growth, development, and stress responses, with increasing focus on their epigenetic regulation. However, studies investigating epigenomic signals to explore the functions of lncRNAs in plants remain relatively limited. This study collected a comprehensive dataset of over 160&#x2009;000 high-quality lncRNAs from 19 representative plant species and integrated 6715 ChIP-seq, BS-seq, and RNA-seq datasets to analyze epigenomic patterns at lncRNA loci. Results showed elevated DNA methylation in lncRNA regions. The highest levels occurred in transposable element-associated lncRNAs. Additionally, activating histone modifications at lncRNA loci showed tissue specificity, with epigenetic preferences differed from those at protein-coding gene (PCG) loci. Differential site analysis in epigenetic mutants further highlighted the selective regulation of lncRNA loci by specific epigenetic factors. To facilitate research, we developed PERlncDB, a platform that provides species-specific lncRNA browsing, epigenetic annotation, cross-species conservation analysis, and visualization of epigenomic landscapes. Case studies on MARS and LINC-AP2 emphasized the platform's utility. Conserved epigenetic mechanisms regulating lncRNAs across species, exemplified by a syntenic conserved MET1-regulated lncRNA pair in Arabidopsis and tomato, suggested the stability of regulatory mechanisms underlying lncRNA functions. This work provides critical insights and resources for understanding plant lncRNA epigenetic regulation.

RNA, Long Noncoding↗

TrkB-like immunoreactivity is present on geniculocortical afferents in layer IV of kitten primary visual cortex.

Exogenous administration of the neurotrophins brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5 (NT-4/5), or blockade of their endogenous actions, have been reported to affect the anatomic organization and physiological responses of neurons in developing mammalian primary visual cortex. Experimental alteration of levels of these neurotrophic factors can also influence the morphology of the geniculocortical afferents that project from the lateral geniculate nucleus (LGN) to primary visual cortex. BDNF and NT-4/5 are ligands of the TrkB tyrosine kinase receptor. Although multiple populations of cortical neurons express TrkB, it is not known whether geniculocortical afferents express this receptor on their axon branches in visual cortex. We have anatomically labeled geniculocortical afferents of postnatal day 40 kittens with the anterograde neuronal tracer Phaseolus vulgaris leucoagglutinin (PHA-L) and performed double-label immunofluorescence with a panel of anti-TrkB antibodies. Confocal microscopy and object-based colocalization analysis were used to measure levels of TrkB-like immunoreactivity (IR) on geniculocortical afferents in layer IV of primary visual cortex. By using a conservative analysis involving a comparison of measured colocalization with the amount of colocalization expected based on random overlap of TrkB puncta and PHA-L--labeled afferents, 3 of 5 anti-TrkB antibodies tested showed significant colocalization with the geniculocortical axons. Results for the other two antibodies were indeterminate. The indices obtained for colocalization of TrkB and geniculocortical afferents were also compared with the equivalent index obtained for GAD65, a protein that has a similar overall expression pattern to that of TrkB but is not expressed on geniculocortical axons. This analysis indicated that TrkB was present on geniculocortical axons for all five TrkB antibodies tested. TrkB-like IR was also observed on neuronal somata in the LGN. These results indicate that TrkB receptors on geniculocortical afferents are potential mediators of the actions of BDNF and NT-4/5 in developing visual cortex.

Animals↗

Crystal structure of human vacuolar protein sorting protein 29 reveals a phosphodiesterase/nuclease-like fold and two protein-protein interaction sites.

Vacuolar protein sorting protein 29 (Vps29p), which is involved in retrograde trafficking from prevacuolar endosomes to the trans-Golgi network, performs its biological functions by participating in the formation of a "retromer complex." In human cells, this complex comprises four conserved proteins: hVps35p, hVps29p, hVps26p, and sorting nexin 1 protein (SNX1). Here, we report the crystal structure of hVps29p at 2.1 Angstroms resolution, the first three-dimensional structure of the retromer subunits. This novel structure adopts a four-layered alpha-beta-beta-alpha sandwich fold. hVps29p contains a metal-binding site that is very similar to the active sites of some proteins of the phosphodiesterase/nuclease protein family, indicating that hVps29p may carry out chemically similar functions. Structure and sequence conservation analysis suggests that hVps29p contains two protein-protein interaction sites. One site, which potentially serves as the interface between hVps29p and hVps35p, comprises 5 conserved hydrophobic and 8 hydrophilic residues. The other site is relatively more hydrophilic and may serve as a binding interface with hVps26p, SNX1, or other target proteins.

Amino Acid Sequence↗

Mitochondrial 12S rRNA gene mutations affect RNA secondary structure and lead to variable penetrance in hearing impairment.

Mutations in the mitochondrial DNA are one of the most important causes of sensorineural hearing loss, especially in the 12S ribosomal RNA (rRNA) gene. We have analyzed the mtDNA 12S rRNA gene in a cohort of 443 families with hearing impairment, and have identified the A1555G mutation in 69 unrelated cases. A1555G is not a fully penetrant change, since only 63% of subjects with this change have developed hearing impairment. In addition, only 22% of the 183 A1555G deaf subjects were treated with aminoglycosides. Two novel nucleotide changes (T1291C and T1243C) were identified. T1243C was found in five deafness cases and one control sample. Mutation T1291C was detected in all maternally related individuals of a pedigree and in none of 95 control samples. Conservation analysis and comparison of the 12S rRNA structure with the 16S rRNA of Escherichia coli showed that the T at nucleotide 1243 and A at nucleotide 1555 are conserved positions. Prediction of RNA secondary structure showed changes in all 12S rRNA variants, the most severe being for T1291C. The reported data confirm the high prevalence of mutation A1555G in deafness cases and the major role of the 12S rRNA gene in hearing. The two novel changes reported here might have different contributions as deafness-related variants. T1291C fulfills the criteria of a disease-causing change. As in the case of mutation A1555G, the underlying phenotype of T1291C is not homogeneous for all family members, providing evidence for the implication of environmental and/or additional genetic factors.

Adult↗

Epidemiology and genotypes of HEV in Wuhan.

BACKGROUND: Understanding the genotype and clinical features of the hepatitis E virus (HEV) are important for understanding its characteristics, for evaluating region-specific diagnostic assays, and producing vaccines. OBJECTIVES: To investigate the epidemiology and the genotypes of HEV among outpatients and inpatients in the Department of Infectious Diseases of Tongji Hospital in Wuhan, China. METHODS: Clinical data were elicited from the hospital records of patients who were clinically diagnosed with acute hepatitis between January 2000 and August 2004 (4920 patients). Of these cases, 120 patients with anti-HEV-IgM, IgG-positive were selected to analysis. Conserved genomic sequences of open reading frame 2 (345 bp) in the HEV gene were detected using polymerase chain reaction, 25 of which were cloned and sequenced. Clustal X and Mega software were used for phylogenetic analysis of genotypes strains. RESULTS: The HEV infection rate is gradually increasing in Wuhan. The number of male patients was 3.3-fold greater than the number of female patients found in clinical investigations. People aged 30-59 years are more susceptible to infection, and people are more susceptible in March-June. Twenty-five isolates shared the same genotype, genotype IV, with 82.61-98.55% nucleotide identity. This genotype had 76.52-81.74%, 70.43-73.04%, 76.52-81.16%, and 84.35-88.70% homology with the nucleotide sequence of HEV genotypes I-IV, respectively. Phylogenetic analysis suggested that these 25 isolates represented at least three different subtypes, but there were no significant differences found in the epidemiological features or liver function of patients with the three subtypes. CONCLUSIONS: HEV sequences isolated from patients in Wuhan belong to different subtypes of HEV genotype IV.

Adolescent↗

Amino acid sequence analysis of the annexin super-gene family of proteins.

The annexins are a widespread family of calcium-dependent membrane-binding proteins. No common function has been identified for the family and, until recently, no crystallographic data existed for an annexin. In this paper we draw together 22 available annexin sequences consisting of 88 similar repeat units, and apply the techniques of multiple sequence alignment, pattern matching, secondary structure prediction and conservation analysis to the characterisation of the molecules. The analysis clearly shows that the repeats cluster into four distinct families and that greatest variation occurs within the repeat 3 units. Multiple alignment of the 88 repeats shows amino acids with conserved physicochemical properties at 22 positions, with only Gly at position 23 being absolutely conserved in all repeats. Secondary structure prediction techniques identify five conserved helices in each repeat unit and patterns of conserved hydrophobic amino acids are consistent with one face of a helix packing against the protein core in predicted helices a, c, d, e. Helix b is generally hydrophobic in all repeats, but contains a striking pattern of repeat-specific residue conservation at position 31, with Arg in repeats 4 and Glu in repeats 2, but unconserved amino acids in repeats 1 and 3. This suggests repeats 2 and 4 may interact via a buried saltbridge. The loop between predicted helices a and b of repeat 3 shows features distinct from the equivalent loop in repeats 1, 2 and 4, suggesting an important structural and/or functional role for this region. No compelling evidence emerges from this study for uteroglobin and the annexins sharing similar tertiary structures, or for uteroglobin representing a derivative of a primordial one-repeat structure that underwent duplication to give the present day annexins. The analyses performed in this paper are re-evaluated in the Appendix, in the light of the recently published X-ray structure for human annexin V. The structure confirms most of the predictions and shows the power of techniques for the determination of tertiary structural information from the amino acid sequences of an aligned protein family.

Algorithms↗

Analysis of a dehiscence zone endo-polygalacturonase in oilseed rape (Brassica napus) and Arabidopsis thaliana: evidence for roles in cell separation in dehiscence and abscission zones, and in stylar tissues during pollen tube growth.

The oilseed rape (Brassica napus) endo-polygalacturonase (endo-PG) RDPG1 is involved in middle lamella breakdown during silique opening. We investigated tissue-specific expression of RDPG1 in transgenic Arabidopsis thaliana. Cellular localization of endo-PG protein in Arabidopsis siliques was determined by immuno-electron microscopy. An Arabidopsis orthologue, ADPG1, was isolated and aligned with the sequence of RDPG1. The proximal 5' sequences as well as introns are largely conserved. Analysis of the histological GUS-staining pattern of two RDPG1 promoter-GUS (beta-glucuronidase) constructs in transgenic Arabidopsis revealed that the conserved proximal part of the 5'-flanking region directs expression in dehiscence zones of siliques and anthers, floral abscission zones and stylar tissues during pollen tube growth, branch points between stems and pedicel and expression associated with the apical meristem of seedlings, while the distal part of the RDPG1 5'-flanking region contains elements involved in vascular-associated expression in petals, cotyledons and roots. Subsequent RT-PCR analysis, on RNA from the corresponding rape tissues, confirms the staining pattern revealed in transgenic Arabidopsis, thereby justifying the use of Arabidopsis as a reliable model system for analysis of oilseed rape regulatory sequences.

Arabidopsis↗

Nested-polymerase chain reaction for the detection of Helicobacter pylori infection with novel primers designed by sequence analysis of urease A gene in clinically isolated bacterial strains.

We have established a highly sensitive semi-nested PCR assay for the detection of H. pylori infection using gastric juice samples, which can be aspirated with disposable nasogastric tubes. The primers targeting H. pylori urease A gene were designed based on the sequence conservation analysis of sixteen H. pylori strains isolated from Japanese patients. The efficacy of the PCR assay, designated as the URA-PCR, was confirmed by in vitro and in vivo assessments. Its sensitivity was 97.5% in the gastric juice samples aspirated from forty patients with proven H. pylori infection, and was significantly higher than that obtained with previously described PCR assays.

Base Sequence↗

Comparative gene expression analysis of blood and brain provides concurrent validation of SELENBP1 up-regulation in schizophrenia.

Microarray techniques hold great promise for identifying risk factors for schizophrenia (SZ) but have not yet generated widely reproducible results due to methodological differences between studies and the high risk of type I inferential errors. Here we established a protocol for conservative analysis and interpretation of gene expression data from the dorsolateral prefrontal cortex of SZ patients using statistical and bioinformatic methods that limit false positives. We also compared brain gene expression profiles with those from peripheral blood cells of a separate sample of SZ patients to identify disease-associated genes that generalize across tissues and populations and further substantiate the use of gene expression profiling of blood for detecting valid SZ biomarkers. Implementing this systematic approach, we: (i) discovered 177 putative SZ risk genes in brain, 28 of which map to linked chromosomal loci; (ii) delineated six biological processes and 12 molecular functions that may be particularly disrupted in the illness; (iii) identified 123 putative SZ biomarkers in blood, 6 of which (BTG1, GSK3A, HLA-DRB1, HNRPA3, SELENBP1, and SFRS1) had corresponding differential expression in brain; (iv) verified the differential expression of the strongest candidate SZ biomarker (SELENBP1) in blood; and (v) demonstrated neuronal and glial expression of SELENBP1 protein in brain. The continued application of this approach in other brain regions and populations should facilitate the discovery of highly reliable and reproducible candidate risk genes and biomarkers for SZ. The identification of valid peripheral biomarkers for SZ may ultimately facilitate early identification, intervention, and prevention efforts as well.

Biomarkers↗

Against relative timing invariance in movement kinematics.

The kinematics of stair climbing were examined to test the assertion that relative timing is an invariant feature of human gait. Six male and four female subjects were video-recorded (at 60 Hz) while they climbed a flight of stairs 10 times at each of three speeds. Each gait cycle was divided into three segments by the maximum and minimum angular displacement of the left knee and left foot contact. Gentner's (1987) analysis methods were applied to the individual subject data to determine whether the duration of the segments remained a fixed proportion of gait cycle duration across changes in stair-climbing speed. A similar analysis was performed using knee velocity maxima to partition the gait cycle. Regardless of how the gait cycle was divided, relative timing was not found to remain strictly invariant across changes in speed. This conclusion is contrary to previous studies of relative timing that involved less conservative analysis but is consistent with the wider gait literature. Strict invariant relative timing may not be a fundamental feature of movement kinematics.

Adult↗