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PubMed1999

Phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay.

Viruses in the genus Coronavirus are currently placed in three groups based on antigenic cross-reactivity and sequence analysis of structural protein genes. Consensus polymerase chain reaction (PCR) primers were used to obtain cDNA, then cloned and sequenced a highly conserved 922 nucleotide region in open reading frame (ORF) 1b of the polymerase (pol) gene from eight coronaviruses. These sequences were compared with published sequences for three additional coronaviruses. In this comparison, it was found that nucleotide substitution frequencies (per 100 nucleotides) varied from 46.40 to 50.13 when viruses were compared among the traditional coronavirus groups and, with one exception (the human coronavirus (HCV) 229E), varied from 2.54 to 15.89 when compared within these groups. (The substitution frequency for 229E, as compared to other members of the same group, varied from 35.37 to 35.72.) Phylogenetic analysis of these pol gene sequences resulted in groupings which correspond closely with the previously described groupings, including recent data which places the two avian coronaviruses--infectious bronchitis virus (IBV) of chickens and turkey coronavirus (TCV)--in the same group [Guy, J.S., Barnes, H.J., Smith L.G., Breslin, J., 1997. Avian Dis. 41:583-590]. A single pair of degenerate primers was identified which amplify a 251 bp region from coronaviruses of all three groups using the same reaction conditions. This consensus PCR assay for the genus Coronavirus may be useful in identifying as yet unknown coronaviruses.

Amino Acid Sequence↗
PubMed2005

Genome-wide analysis of the functions of a conserved surface on the corepressor Tup1.

The general transcriptional repressor Tup1 is responsible for the regulation of a large, diverse set of genes in Saccharomyces cerevisiae, and functional homologues of Tup1 have been identified in many metazoans. The crystal structure for the C-terminal portion of Tup1 has been solved and, when sequences of Tup1 homologues from fungi and metazoans were compared, a highly conserved surface was revealed. In this article, we analyze five point mutations that lie on this conserved surface. A statistical analysis of expression microarrays demonstrates that the mutant alleles are deficient in the repression of different subsets of Tup1-regulated genes. We were able to rank the mutant alleles of TUP1 based on the severity of their repression defects measured both by the number of genes derepressed and by the magnitude of that derepression. For one particular class of genes, the mutations on the conserved surface disrupted recruitment of Tup1 to the repressed promoters. However, for the majority of the genes derepressed by the Tup1 point mutants, recruitment of Tup1 to the regulated promoters is largely unaffected. These mutations affect the mechanism of repression subsequent to recruitment of the complex and likely represent a disruption of a mechanism that is conserved in fungi and metazoans. This work demonstrates that the evolutionarily conserved surface of Tup1 interacts with two separate types of proteins-sequence-specific DNA-binding proteins responsible for recruiting Tup1 to promoters as well as components that are likely to function in a conserved repression mechanism.

Alanine↗
PubMed1998

Adenylosuccinate synthetase genes: molecular cloning and phylogenetic analysis of a highly conserved archaeal gene.

Adenylosuccinate synthetase (PurA) catalyzes the first step in the de novo AMP synthesis and has been extensively studied in both Bacteria and Eukarya. We cloned the purA gene from the hyperthermophilic archaeon, Pyrococcus furiosus. The gene appears to be individually transcribed and encodes a protein of 339 amino acids. The amino acid sequence comparison with other archael PurAs found from recent genome analyses indicated that two deletions, one central and the other C-terminal, are a common feature of archaeal PurAs. None of the 21 PurA homologues analyzed from Eukarya and Bacteria exhibited this feature. Amino acid sequences of PurAs in Archaea showed 64% average identities which were significantly higher than the 50% and 55% calculated for Bacteria and Eukarya, respectively. Several residues conserved in PurAs of both Eukarya and Bacteria and shown to be of catalytic importance are missing in the archaeal PurAs. Phylogenetic analysis using PurA as the marker grouped life into 3 domains, hence it was consistent with results derived from 16-18S ribosomal RNA sequences. The topology within the three domains, in general, portrayed the hitherto accepted evolutionary relationship among the organisms utilized. PurA can, thus, serve as an additional marker to evaluate phylogenetic inferences drawn from sequence data from rRNA and other conserved genes. The presence of two unique deletions in both euryarchaeal and crenarchaeal PurAs, but not in those of Bacteria and Eukarya, is a strong evidence confirming the common lineage of these two subdomains of Archaea.

Adenylosuccinate Synthase↗
PubMed2004

ConSSeq: a web-based application for analysis of amino acid conservation based on HSSP database and within context of structure.

SUMMARY: A web-based application to analyze protein amino acids conservation-Consensus Sequence (ConSSeq) is presented. ConSSeq graphically represents information about amino acid conservation based on sequence alignments reported in homology-derived structures of proteins. Beyond the relative entropy for each position in the alignment, ConSSeq also presents the consensus sequence and information about the amino acids, which are predominant at each position of the alignment. ConSSeq is part of the STING Millennium Suite and is implemented as a Java Applet. AVAILABILITY: http://sms.cbi.cnptia.embrapa.br/SMS/STINGm/consseq/, http://trantor.bioc.columbia.edu/SMS/STINGm/consseq/, http://mirrors.rcsb.org//SMS/STINGm/consseq/, http://www.es.embnet.org/SMS/STINGm/consseq/ and http://www.ar.embnet.org/SMS/STINGm/consseq/

Amino Acids↗
PubMed2003

[Re-irradiation after salvage mastectomy for local recurrence after a conservative treatment: a retrospective analysis of twenty patients (Nancy: 1988-2001)].

PURPOSE: To retrospectively assess the efficacy of post-mastectomy re-irradiation for local relapse of breast cancer. PATIENTS AND METHODS: Twenty patients, initially treated by conservative surgery and radiotherapy (50 Gy in 25 fractions over 5 weeks) were treated from 1998 to 2001 for a local relapse by salvage mastectomy and re-irradiation (either electron or photon beams). Mean age was 53 years (31-71). Reasons for re-irradiation were that the local relapses were inflammatory (4 pts), multifocal (5 pts), cutaneous (5 pts), involved the nipple (3 pts) or because the surgical margins (either muscle or skin) were involved (3 pts). The median dose of re-irradiation was 45 Gy (33-65) in 15 fractions over 33 days. Mean follow-up was 48 months (5-97). RESULTS: Fifteen patients remained free of a second local recurrence and 10 were still alive, without metastasis. Neither the dose of re-irradiation nor the irradiated surfaces were prognostic factors of local control (P = 0.877 and P = 0.424). Five patients developed radiation-induced pneumonitis without functional respiratory impairment. The incidence of pneumonitis seemed to be related to the biological dose of re-irradiation (P = 0.037). Other late complications occurred such as pigmentation changes (12 pts), telangiectasia (8 pts), chondritis (2 pts), parietal fibrosis (7 pts), rib fractures (4 pts), severe pain (11 pts) and lymphedema (2 pts). The increase in biological equivalent dose was highly statistically linked with the occurrence of disabling pain (P = 0.0123). CONCLUSION: Parietal re-irradiation achieves good and lasting local control with an acceptable rate of acute complications but with a risk of disabling late sequelae such as severe pain.

Adult↗
PubMed2001

Regional and developmental expression of Epm2a gene and its evolutionary conservation.

Lafora's disease, an autosomal recessive progressive myoclonus epilepsy, is caused by mutations in the EPM2A gene encoding a dual-specificity phosphatase (DSP) named laforin. Here, we analyzed the developmental and regional expression of murine Epm2a and discussed its evolutionary conservation. A phylogenetic analysis indicated that laforin is evolutionarily distant from other DSPs. Southern zoo blot analysis suggested that conservation of Epm2a gene is limited to mammals. Laforin orthologs (human, mouse, and rat) display more than 94% similarity. All missense mutations known in Lafora disease patients affect conserved residues, suggesting that they may be essential for laforin's function. Epm2a is expressed widely in various organs but not homogeneously in brain. The levels of Epm2a transcripts in mice brains increase postnatally, attaining its highest level in adults. The most intense signal was detected in the cerebellum, hippocampus, cerebral cortex, and the olfactory bulb. Our results suggest that Epm2a is functionally conserved in mammals and is involved in growth and maturation of neural networks.

Aging↗
PubMed2001

Capsid structure of Kaposi's sarcoma-associated herpesvirus, a gammaherpesvirus, compared to those of an alphaherpesvirus, herpes simplex virus type 1, and a betaherpesvirus, cytomegalovirus.

The capsid of Kaposi's sarcoma-associated herpesvirus (KSHV) was visualized at 24-A resolution by cryoelectron microscopy. Despite limited sequence similarity between corresponding capsid proteins, KSHV has the same T=16 triangulation number and much the same capsid architecture as herpes simplex virus (HSV) and cytomegalovirus (CMV). Its capsomers are hexamers and pentamers of the major capsid protein, forming a shell with a flat, close-packed, inner surface (the "floor") and chimney-like external protrusions. Overlying the floor at trigonal positions are (alpha beta(2)) heterotrimers called triplexes. The floor structure is well conserved over all three viruses, and the most variable capsid features reside on the outer surface, i.e., in the shapes of the protrusions and triplexes, in which KSHV resembles CMV and differs from HSV. Major capsid protein sequences from the three subfamilies have some similarity, which is closer between KSHV and CMV than between either virus and HSV. The triplex proteins are less highly conserved, but sequence analysis identifies relatively conserved tracts. In alphaherpesviruses, the alpha-subunit (VP19c in HSV) has a 100-residue N-terminal extension and an insertion near the C terminus. The small basic capsid protein sequences are highly divergent: whereas the HSV and CMV proteins bind only to hexons, difference mapping suggests that the KSHV protein, ORF65, binds around the tips of both hexons and pentons.

Amino Acid Sequence↗
PubMed2001

Evolutionary conservation of the folding nucleus.

Here, we present statistical analysis of conservation profiles in families of homologous sequences for nine proteins whose folding nucleus was determined by protein engineering methods. We show that in all but one protein (AcP) folding nucleus residues are significantly more conserved than the rest of the protein. Two aspects of our study are especially important: (i) grouping of amino acid residues into classes according to their physical-chemical properties and (ii) proper normalization of amino acid probabilities that reflects the fact that evolutionary pressure to conserve some amino acid types may itself affect concentration of various amino acid types in protein families. Neglect of any of those two factors may make physical and biological "signals" from conservation profiles disappear.

Conserved Sequence↗
PubMed2002

Microarray analysis of orthologous genes: conservation of the translational machinery across species at the sequence and expression level.

BACKGROUND: Genome projects have provided a vast amount of sequence information. Sequence comparison between species helps to establish functional catalogues within organisms and to study how they are maintained and modified across phylogenetic groups during evolution. Microarray studies allow us to determine groups of genes with similar temporal regulation and perhaps also common regulatory upstream regions for binding of transcription factors. The integration of sequence and expression data is expected to refine our current annotations and provide some insight into the evolution of gene regulation across organisms. RESULTS: We have investigated how well the protein subcellular localization and functional categories established from clustering of orthologous genes agree with gene-expression data in Saccharomyces cerevisiae. An increase in the resolution of biologically meaningful classes is observed upon the combination of experiments under different conditions. The functional categories deduced by sequence comparison approaches are, in general, preserved at the level of expression and can sometimes interact into larger co-regulated networks, such as the protein translation process. Differences and similarities in the expression between cytoplasmic-mitochondrial and interspecies translation machineries complement evolutionary information from sequence similarity. CONCLUSIONS: Combination of several microarray experiments is a powerful tool for the identification of upstream regulatory motifs of yeast genes involved in protein synthesis. Comparison of these yeast co-regulated genes against the archaeal and bacterial operons indicates that the components of the protein translation process are conserved across organisms at the expression level with minor specific adaptations.

Archaea↗
PubMed2004

Analysis of sequence, map position, and gene expression reveals conserved essential genes for iron uptake in Arabidopsis and tomato.

Arabidopsis (Arabidopsis thaliana) and tomato (Lycopersicon esculentum) show similar physiological responses to iron deficiency, suggesting that homologous genes are involved. Essential gene functions are generally considered to be carried out by orthologs that have remained conserved in sequence and map position in evolutionarily related species. This assumption has not yet been proven for plant genomes that underwent large genome rearrangements. We addressed this question in an attempt to deduce functional gene pairs for iron reduction, iron transport, and iron regulation between Arabidopsis and tomato. Iron uptake processes are essential for plant growth. We investigated iron uptake gene pairs from tomato and Arabidopsis, namely sequence, conserved gene content of the regions containing iron uptake homologs based on conserved orthologous set marker analysis, gene expression patterns, and, in two cases, genetic data. Compared to tomato, the Arabidopsis genome revealed more and larger gene families coding for the iron uptake functions. The number of possible homologous pairs was reduced if functional expression data were taken into account in addition to sequence and map position. We predict novel homologous as well as partially redundant functions of ferric reductase-like and iron-regulated transporter-like genes in Arabidopsis and tomato. Arabidopsis nicotianamine synthase genes encode a partially redundant family. In this study, Arabidopsis gene redundancy generally reflected the presumed genome duplication structure. In some cases, statistical analysis of conserved gene regions between tomato and Arabidopsis suggested a common evolutionary origin. Although involvement of conserved genes in iron uptake was found, these essential genes seem to be of paralogous rather than orthologous origin in tomato and Arabidopsis.

Amino Acid Sequence↗
PubMed2007

Estimation of the stress related to conservative scoliosis therapy: an analysis based on BSSQ questionnaires.

BACKGROUND: Adolescent girls treated with a brace for scoliosis are submitted to prolonged stress related to both the disease and the therapy. Currently proposed quality of life questionnaires are focused on the outcome of therapy. Bad Sobernheim Stress Questionnaire (BSSQ) enables monitoring of patients being under treatment with a brace or exercises. The aim of the study was to assess the stress level in conservatively managed scoliotic girls using BSSQ. MATERIALS AND METHODS: 111 girls, aged 14.2 +/- 2.2 years, mean Cobb angle of the primary curve 42.8 degrees +/- 17.0 degrees and mean Bunnell angle of 11.4 degrees +/- 4.5 degrees were examined with two versions of BSSQ (Deformity and Brace). The analysis considered the type of treatment, curve location, correlation of the total score with age, Cobb angle and Bunnell rotation angle. RESULTS: The BSSQ Deformity revealed the median of 17 points in patients managed with exercises (from 4 to 24 points), 18 in patients managed with a brace (from 8 to 24 points) and 12 in patients before surgery (from 3 to 21 points). Braced patients who completed both questionnaires (n = 50) revealed significantly higher score with BSSQ Deformity (median = 18) comparing to BSSQ Brace (median = 9). There was a correlation between the total score of BSSQ Deformity and the Cobb angle (r = -0.34), Bunnell primary curve rotation (r = -0.34) and Bunnell sum of rotation (r = -0.33) but not with the age of patients. CONCLUSION: Scoliotic adolescents managed with exercises and brace suffered little stress from the deformity. The brace increased the level of stress over the stress induced by the deformity. The stress level correlated with clinical deformity (Bunnell angle), radiological deformity (Cobb angle) and the type of treatment (exercises, bracing, surgery). Bad Sobernheim Stress Questionnaires are simple and helpful in the management of girls treated conservatively for idiopathic scoliosis.

Journal Article↗
PubMed2004

Diversity and locus specificity of chicken MHC B class I sequences.

The major histocompatibility complex B (MHC B) region in a standard haplotype of Leghorn chickens contains two closely linked class I loci, B-FI and B-FIV. Few sequences of B-FI alleles are available, and therefore alleles of the two loci have not been compared with regard to sequence diversity or locus specificity. Here, we report eight new B-F alpha 1/alpha 2-coding sequences from broiler chicken MHC B haplotypes, and a unique recombinant between the two B-F loci. The new sequences were combined with existing B-F sequences from Leghorn and broiler haplotypes for analysis. On the basis of phylogenetic analysis and conserved sequence motifs, B-F sequences separated into two groups (Groups A and B), corresponding to B-FIV and B-FI locus, respectively. Every broiler haplotype had one B-F sequence in Group A and the second B-F sequence, if it existed, clustered in Group B. Group B (presumptive B-FI locus) sequences identified in broiler haplotypes resembled the human MHC class I HLA-C locus in their distinctive pattern of allelic polymorphism. Compared with B-FIV, B-FI alleles were less polymorphic and possessed a conserved locus-specific motif in the alpha1 helix, but nevertheless demonstrated evidence of diversifying selection. One B-FI alpha 1/alpha 2-coding nucleotide sequence was completely conserved in four different broiler haplotypes, but each allele differed in the exon encoding the alpha 3 domain.

Amino Acid Sequence↗
PubMed2026

Genome-wide characterization of the bZIP gene family in Rattus norvegicus and expression profiling analysis during brain development.

BACKGROUND: The brown rat (Rattus norvegicus) serves as a cornerstone model organism in biomedical research, particularly for understanding physiological homeostasis and stress responses. The basic leucine zipper (bZIP) transcription factor family is a pivotal regulatory network involved in growth, organogenesis, and neurodevelopment. Despite its importance, a systematic characterization of the bZIP gene family in rats has remained elusive. RESULTS: In this study, we performed a genome-wide identification of 61 RnbZIP genes, which were categorized into 10 distinct subfamilies based on phylogenetic relationships and chromosomal localization. Structural analysis revealed conserved motif arrangements within subfamilies, while collinearity analysis identified significant gene duplication events-predominantly tandem and segmental duplications-that have driven the evolutionary expansion of the RnbZIP family. Quantitative analysis showed that members within the same subfamily shared 45%-92% sequence similarity (calculated using the BLOSUM62 scoring matrix), and all duplicated gene pairs underwent strong purifying selection (Ka/Ks&#x2009;<&#x2009;1). Comparative genomics across seven rodent species further underscored the evolutionary conservation and divergence of these factors. Expression profiling across diverse organs and brain developmental stages indicated that RnbZIP genes exhibit high tissue specificity. Notably, 10 candidate genes, including RnbZIP01, RnbZIP02, and RnbZIP08, demonstrated dynamic expression patterns during brain maturation, suggesting their essential roles in neurodevelopmental processes. CONCLUSIONS: Our findings provide a comprehensive structural and evolutionary framework for the RnbZIP gene family, highlighting their potential regulatory functions in rat organogenesis and brain development. This study establishes a valuable resource for further functional characterization of specific bZIP members in mammalian neurological systems.

Animals↗
PubMed1996

Structure conservation in lipoxygenases: structural analysis of soybean lipoxygenase-1 and modeling of human lipoxygenases.

Lipoxygenases are a class of non-heme iron dioxygenases which catalyze the hydroperoxidation of fatty acids for the biosynthesis of leukotrienes and lipoxins. The structure of the 839-residue soybean lipoxygenase-1 was used as a template to model human 5-, 12-, and 15-lipoxygenases. A distance-based algorithm for placing side chains in a low homology environment (only the four iron ligands were fixed during side chain placement) was devised. Twenty-six of the 56 conserved lipoxygenase residues were grouped in four distinct regions of the enzyme. These regions were analyzed to discern whether the side chain interactions could be duplicated in the models or whether alternate conformers should be considered. The effects of site directed mutagenesis variants were rationalized using the models of the human lipoxygenases. In particular, variants which shifted positional specificity between 12- and 15-lipoxygenase activity were analyzed. Analysis of active site residues produced a model which accounts for observed lipoxygenase positional specificity and stereospecificity.

Algorithms↗
PubMed1997

Localization and expression analysis of a novel conserved brain expressed transcript, Brx/BRX, lying within the Xic/XIC candidate region.

The X inactivation center candidate region (Xic/XIC in mouse and human) is poorly characterized for the presence of transcription units. Only two conserved genes have been isolated to date, Xist/XIST and Cdx4/CDX4. The other known gene lying within this region, Tsx, has been identified so far only in rodents by analyzing the complete genomic sequence of a 94-kb region distal to Xist. Here, we report the characterization of an additional gene lying within this 94-kb sequenced region. Brx, for Brain X-linked gene, is a rare transcript preferentially expressed in the brain. It is normally X-inactivated in the mouse. Localisation of BRX, its human homolog has shown the gene to be located within the orthologous but inverted human CDX4-XIST segment. These results suggest that the gene order of the region encompassing the Cdx4-Xist interval in the mouse is similar in human. Comparison of the Xist-Brx and Brx-Cdx4 regions in mouse and human indicates that these intervals are three times longer in human than in mouse. BRX is a new potential candidate for one of the X-linked mental retardation syndromes mapped within the pericentromeric region of the human X Chromosome (Chr).

Amino Acid Sequence↗
PubMed1989

Analysis of a hearing conservation program data base: factors other than workplace noise.

The hearing conservation records of a large, multiple-facility printing company were obtained. The records were analyzed for factors associated with Standard Threshold Shift. These factors included hearing levels, employee age and sex, occupational and nonoccupational noise exposure histories, and medical history. The analysis revealed that statistically significant factors associated with Standard Threshold Shift were from medical and nonoccupational noise exposure histories, and not occupational noise exposure.

Adult↗