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PubMed2002

Mutational analysis of the conserved cysteines of the rat P2X2 purinoceptor.

P2X receptors are ATP-gated cation channels that are widely expressed in the brain. The extracellular domains of all seven P2X receptors contain 10 conserved cysteines, which could form disulfide bonds or binding sites for transition metals that modulate P2X receptors. To test whether these cysteines are critical for receptor function, we studied wild-type rat P2X(2) receptors and 10 mutant P2X(2) receptors, each containing an alanine substituted for a cysteine. Nine mutants were functional but had reduced maximum currents compared with wild-type P2X(2) expressed in either Xenopus oocytes or human embryonic kidney (HEK) 293 cells. The 10th mutant (C224A) did not respond to ATP when expressed in oocytes and gave very small currents in HEK 293 cells. Seven mutants (C113A, C124A, C130A, C147A, C158A, C164A, and C214A) showed rightward shifts (9- to 30-fold) in their ATP concentration-response relationships and very little potentiation by zinc. In contrast, C258A and C267A had EC(50) values similar to those of wild-type P2X(2) and were potentiated by zinc. Acidic pH potentiated wild-type and all mutant receptor currents. Despite the loss of zinc potentiation in seven mutants, these cysteines are unlikely to be exposed in the zinc-binding site, because [2-(trimethylammonium)ethyl] methanethiosulfonate bromide did not prevent zinc potentiation of wild-type receptor currents. On the basis of correlations in the maximum current, EC(50), zinc potentiation, and pH potentiation, we suggest that the following cysteine pairs form disulfide bonds: C113-C164, C214-C224, and C258-C267. We also suggest that C124, C130, C147, and C158 form two disulfide bonds, but we are unable to assign specific cysteine pairs to these two bonds.

Adenosine Triphosphate↗
PubMed1991

Functional analysis of a conserved amino-terminal region of HSP70 by site-directed mutagenesis.

Hsp70 proteins have been highly conserved throughout evolution. As a first step in a structure-function analysis of hsp70, we constructed and analysed the consequences of mutations in a portion of the SSA1 gene, a member of the Saccharomyces cerevisiae HSP70 multigene family, that encodes a nearly invariant region near the amino terminus. Analysis of strains expressing SSA1 proteins with alterations at positions 8, 11 and 15 showed that these conserved residues within this region are critical for normal functioning of the protein. SSA1 protein containing either of two changes at position 15 was able to slightly complement the inviability of an ssa1ssa2ssa4 strain, but was inactive in other complementation assays. The other mutant proteins tested were unable to complement any tested phenotype. Effective interallelic complementation of several phenotypes was observed when a mutant protein substituted at position 8 was expressed in the same cell with either of two proteins carrying substitutions at position 15, suggesting that hsp70 acts as a multimer. Evidence from previous studies suggests that hsp70 proteins engage in ATP-driven cycles of binding and release from peptides. The ability of the mutant proteins to bind ATP and a peptide was tested. The Ssa1p carrying a substitution at position 8, which inhibits growth of cells carrying wild-type SSA proteins, showed a defect in release from a peptide relative to wild type. Two mutations, one each at position 8 and 15, resulted in accumulation of phosphorylated isoforms which may be a normal, transient hsp70 intermediate.

Adenosine Triphosphate↗
PubMed1995

Use of RAPD analysis in devising conservation strategies for the rare and endangered Grevillea scapigera (Proteaceae).

Extensive human impact in south western Australia has resulted in a high incidence of rarity throughout the highly endemic flora of the region. Grevillea scapigera (Proteaceae) is a typical example, with 27 plants (represented by four extant populations) remaining in the wild. In order to devise an appropriate strategy for the conservation of this species, its population genetics were studied using RAPD analysis, which enabled the discrimination of individual plants and the detection of a relatively high amount of variability (V = 0.32) within G. scapigera. This variability was found to be evenly distributed within the plants analysed despite the clear distinction between most populations (87% of the variability being attributable to single plant difference and 13% to population difference). Finally, RAPD analysis was used to select a small group of plants that captured maximum genetic variability to be used in the recovery program of the species. Because of the low genetic difference between populations, the mixing of these selected plants during the recovery process should not create genetic imbalances. The methods used in this study provide a useful model for future projects involving the recovery of rare flora.

Australia↗
PubMed2003

Analysis of the conservation of synteny between Fugu and human chromosome 12.

BACKGROUND: The pufferfish Fugu rubripes (Fugu) with its compact genome is increasingly recognized as an important vertebrate model for comparative genomic studies. In particular, large regions of conserved synteny between human and Fugu genomes indicate its utility to identify disease-causing genes. The human chromosome 12p12 is frequently deleted in various hematological malignancies and solid tumors, but the actual tumor suppressor gene remains unidentified. RESULTS: We investigated approximately 200 kb of the genomic region surrounding the ETV6 locus in Fugu (fETV6) in order to find conserved functional features, such as genes or regulatory regions, that could give insight into the nature of the genes targeted by deletions in human cancer cells. Seven genes were identified near the fETV6 locus. We found that the synteny with human chromosome 12 was conserved, but extensive genomic rearrangements occurred between the Fugu and human ETV6 loci. CONCLUSION: This comparative analysis led to the identification of previously uncharacterized genes in the human genome and some potentially important regulatory sequences as well. This is a good indication that the analysis of the compact Fugu genome will be valuable to identify functional features that have been conserved throughout the evolution of vertebrates.

Animals↗
PubMed2004

Prediction of functional sites in proteins using conserved functional group analysis.

A detailed knowledge of a protein's functional site is an absolute prerequisite for understanding its mode of action at the molecular level. However, the rapid pace at which sequence and structural information is being accumulated for proteins greatly exceeds our ability to determine their biochemical roles experimentally. As a result, computational methods are required which allow for the efficient processing of the evolutionary information contained in this wealth of data, in particular that related to the nature and location of functionally important sites and residues. The method presented here, referred to as conserved functional group (CFG) analysis, relies on a simplified representation of the chemical groups found in amino acid side-chains to identify functional sites from a single protein structure and a number of its sequence homologues. We show that CFG analysis can fully or partially predict the location of functional sites in approximately 96% of the 470 cases tested and that, unlike other methods available, it is able to tolerate wide variations in sequence identity. In addition, we discuss its potential in a structural genomics context, where automation, scalability and efficiency are critical, and an increasing number of protein structures are determined with no prior knowledge of function. This is exemplified by our analysis of the hypothetical protein Ydde_Ecoli, whose structure was recently solved by members of the North East Structural Genomics consortium. Although the proposed active site for this protein needs to be validated experimentally, this example illustrates the scope of CFG analysis as a general tool for the identification of residues likely to play an important role in a protein's biochemical function. Thus, our method offers a convenient solution to rapidly and automatically process the vast amounts of data that are beginning to emerge from structural genomics projects.

Algorithms↗
PubMed2000

New insights into the role of CcmC, CcmD and CcmE in the haem delivery pathway during cytochrome c maturation by a complete mutational analysis of the conserved tryptophan-rich motif of CcmC.

Maturation of c-type cytochromes in Escherichia coli is a complex process requiring eight membrane proteins encoded by the ccmABCDEFGH operon. CcmE is a mediator of haem delivery. It binds haem transiently at a conserved histidine residue and releases it for directed transfer to apocytochrome c. CcmC, an integral membrane protein with six transmembrane helices, is necessary and sufficient to incorporate haem covalently into CcmE. CcmC contains a highly conserved tryptophan-rich motif, WGXXWXWD, in its second periplasmic loop. Here, we present the results of a systematic mutational analysis of this motif. Changes of the non-conserved T121 and W122 to A resulted in wild-type CcmC activity. Changes of the single amino acids W119A, G120A, W123A, W125I and D126A or of the spacing within the motif by deleting V124 (DeltaV124) inhibited the covalent haem incorporation into CcmE. Enhanced expression of ccmD suppressed this mutant phenotype by increasing the amounts of CcmC and CcmE polypeptides in the membrane. The DeltaV124 mutant showed the strongest defect of all single mutants. Mutants in which six residues of the tryptophan-rich motif were changed showed no residual CcmC activity. This phenotype was independent of the level of ccmD expression. Our results demonstrate the functional importance of the tryptophan-rich motif for haem transfer to CcmE. We propose that the three membrane proteins CcmC, CcmD and CcmE interact directly with each other, establishing a cytoplasm to periplasm haem delivery pathway for cytochrome c maturation.

Amino Acid Motifs↗
PubMed2006

Obstetric outcomes after conservative treatment for intraepithelial or early invasive cervical lesions: systematic review and meta-analysis.

BACKGROUND: Conservative methods to treat cervical intraepithelial neoplasia and microinvasive cervical cancer are commonly used in young women because of the advent of effective screening programmes. In a meta-analysis, we investigated the effect of these procedures on subsequent fertility and pregnancy outcomes. METHODS: We searched for studies in MEDLINE and EMBASE and classified them by the conservative method used and the outcome measure studied regarding both fertility and pregnancy. Pooled relative risks and 95% CIs were calculated with a random-effects model and interstudy heterogeneity was assessed with Cochrane's Q test. FINDINGS: We identified 27 studies. Cold knife conisation was significantly associated with preterm delivery (<37 weeks; relative risk 2.59, 95% CI 1.80-3.72, 100/704 [14%] vs 1494/27 674 [5%]), low birthweight (<2500 g; 2.53, 1.19-5.36, 32/261 [12%] vs 905/13 229 [7%]), and caesarean section (3.17, 1.07-9.40, 31/350 [9%] vs 22/670 [3%]). Large loop excision of the transformation zone (LLETZ) was also significantly associated with preterm delivery (1.70, 1.24-2.35, 156/1402 [11%] vs 120/1739 [7%]), low birthweight (1.82, 1.09-3.06, 77/996 [8%] vs 49/1192 [4%]), and premature rupture of the membranes (2.69, 1.62-4.46, 48/905 [5%] vs 22/1038 [2%]). Similar but marginally non-significant adverse effects were recorded for laser conisation (preterm delivery 1.71, 0.93-3.14). We did not detect significantly increased risks for obstetric outcomes after laser ablation. Although severe outcomes such as admission to a neonatal intensive care unit or perinatal mortality showed adverse trends, these changes were not significant. INTERPRETATION: All the excisional procedures to treat cervical intraepithelial neoplasia present similar pregnancy-related morbidity without apparent neonatal morbidity. Caution in the treatment of young women with mild cervical abnormalities should be recommended. Clinicians now have the evidence base to counsel women appropriately.

Adult↗
PubMed2002

Functional analysis of a conserved region of the baculovirus envelope fusion protein, LD130.

The envelope fusion protein from a baculovirus pathogenic for Lymantria dispar was characterized. N-terminal sequence analysis determined that it was cleaved downstream of predicted signal peptide and furin cleavage motifs. Mutation of the furin motif resulted in a protein that was not cleaved and did not mediate fusion. Mutagenesis of three charged amino acids in a conserved sequence with the features of a fusion peptide resulted in significant reduction of the ability of the constructs to mediate fusion. None of the mutations inhibited transport of the proteins to the cell surface. In addition, the mutations of the predicted fusion peptide region yielded no inhibition of cleavage. No difference in cleavage was detected between constructs expressed in Spodoptera frugiperda or L. dispar cells.

Animals↗
PubMed2005

Finding biologically relevant protein domain interactions: conserved binding mode analysis.

Proteins evolved through the shuffling of functional domains, and therefore, the same domain can be found in different proteins and species. Interactions between such conserved domains often involve specific, well-determined binding surfaces reflecting their important biological role in a cell. To find biologically relevant interactions we developed a method of systematically comparing and classifying protein domain interactions from the structural data. As a result, a set of conserved binding modes (CBMs) was created using the atomic detail of structure alignment data and the protein domain classification of the Conserved Domain Database. A conserved binding mode is inferred when different members of interacting domain families dock in the same way, such that their structural complexes superimpose well. Such domain interactions with recurring structural themes have greater significance to be biologically relevant, unlike spurious crystal packing interactions. Consequently, this study gives lower and upper bounds on the number of different types of interacting domain pairs in the structure database on the order of 1000-2000. We use CBMs to create domain interaction networks, which highlight functionally significant connections by avoiding many infrequent links between highly connected nodes. The CBMs also constitute a library of docking templates that may be used in molecular modeling to infer the characteristics of an unknown binding surface, just as conserved domains may be used to infer the structure of an unknown protein. The method's ability to sort through and classify large numbers of putative interacting domain pairs is demonstrated on the oligomeric interactions of globins.

Algorithms↗
PubMed2000

Outcomes analysis for hearing conservation programs.

In health care, an increasing interest in accountability for outcomes, proof of quality care, and cost effectiveness is forcing many practitioners, including audiologists, in the direction of using outcomes analysis to provide proof of performance in their practices. Critical to the conduct of outcomes analysis are the selection of valid outcomes metrics and application of appropriate statistical processes. Modeling of clinical protocols is a useful exercise for developing outcomes data and data management standards. The authors describe the implications for wider use of outcomes modeling once the Health Insurance Portability and Accountability Act (HIPAA) administrative simplification law is implemented. This legislation will set up a wholly new administrative data set that has strong potential to provide outcomes metrics to a variety of Audiology practices. The modeling exercise described here was originally intended to derive assessment outcomes for evaluating hearing conservation program effectiveness in military populations from an administrative database implemented within the Military Health System starting in 1997. The outcomes modelers, however, soon realized the broader applicability of modeling techniques for other special populations and Audiology practices, particularly with the oncoming startup of HIPAA legislative mandates. The modeling exercise demonstrates a process of structuring standard health-care codes to produce outcomes data for epidemiologic and cost analyses, thereby providing better information to guide health-care practices toward improving quality and cost effectiveness. This information in time series should also provide a record of continuous quality improvement. The authors present a general hearing surveillance protocol for evaluating occupational hearing loss. The protocol is used as a specific instance to demonstrate the shaping of an outcomes metrics model. This same process can be applied more generally when used to model outcomes data from other audiology practices. Implementation of the administrative data model described here began at more than 400 Department of Defense (DOD) medical treatment facilities worldwide as of October 1, 1999. The exercise of developing similar outcomes metrics for newborn hearing screening programs, school hearing conservation programs, and other clinical and rehabilitative audiology practices in the civilian sector remains to be done.

Adult↗
PubMed2005

Comparative genomic and expression analysis of the conserved NTPDase gene family in Xenopus.

The purines, ATP and adenosine, are important signaling molecules in the nervous system. ATP is sequentially degraded to adenosine by the ectonucleotidase proteins. The NTPDase (or CD39) family is a subfamily of these enzymes, which consists of nine members in mammals. In Xenopus embryos, we have shown that ATP, and its antagonist adenosine, regulate the rundown of swimming and we therefore proposed that ectonucleotidase proteins are key regulators of locomotor activity. Here, we report the cloning of all nine members of the NTPDase family in Xenopus laevis and Xenopus tropicalis. Our phylogenetic analysis shows that this family is highly conserved between the frog species and also during vertebrate evolution. In the adult frog, NTPDase genes are broadly expressed. During development, all NTPDase genes, except for NTPDase8, are expressed and display a distinct specific expression pattern, suggesting potentially different functions of these proteins during embryogenesis of X. laevis.

Amino Acid Sequence↗
PubMed2004

Mutational analysis of evolutionarily conserved ACTH residues.

alpha-Melanocyte stimulating hormone (MSH) and adrenocorticotropin (ACTH)1-24, the minimal ACTH sequence required for full activity, differ only by the 10 C-terminal amino acids of ACTH1-24. Interestingly, these ten C-terminal residues have been highly conserved throughout vertebrate evolution. To understand the functional constraints of these 10 amino acids we analyzed the effects of mutating these residues on steroidogenic activity in vivo and in vitro. Alanine substitutions of some of the first four amino acid residues (the basic core residues KKRR, 15-18) greatly reduces ACTH activity in vitro and in vivo; replacement of mutant alanines at residues 15 and 17 with glutamine residues partially restores ACTH activity. Thus, for ACTH receptor binding and activation, the amino acid residues 15-18 are important for their side chains. Surprisingly, conversion of the five C-terminal residues (20-24) to alanines increases ACTH activity in vivo over that of native ACTH. With respect to receptor binding and activity, the last five amino acid residues are important only for the peptide length they contribute; however, with respect to serum stability, their side chains are significant.

Adrenal Glands↗
PubMed1995

Amplification of bacterial DNA using highly conserved sequences: automated analysis and potential for molecular triage of sepsis.

OBJECTIVE: The clinical diagnosis of sepsis remains difficult, particularly in the young child, and would be improved by a rapid and reliable method for identification of bacteria in blood and other body fluids. Polymerase chain reaction (PCR) amplification of highly conserved DNA sequences found in all bacteria would permit fast and sensitive determination of the presence of bacteria in clinical specimens. METHODOLOGY: A primer pair for highly conserved regions of bacterial DNA encoding 16S ribosomal RNA (rDNA) was utilized for PCR amplification. PCR products were analyzed by gel electrophoresis, and, after modification of the primers, by an automated 96-well plate reader. RESULTS: rDNA was amplified from 12 different species of bacteria, including Gram-negative and -positive organisms. No signal was observed when total human DNA was used as template. Colorimetric analysis of amplified sequences using a 96-well format was also successful. CONCLUSIONS: We conclude that a single primer pair designed to anneal to a highly conserved region of bacterial DNA can amplify DNA specimens from a variety of different bacteria, while not amplifying human DNA. Such a molecular genetics approach can be fully automated with existing robotic technology. Because of speed, sensitivity, and cost, molecular triage of patients with signs and symptoms of possible bacterial infection will decrease morbidity and mortality among those with unrecognized bacteremia who are managed as outpatients, and will dramatically reduce hospital expenses from individuals who are admitted and are not bacteremic.

Autoanalysis↗
PubMed2000

Comparative genome analysis reveals extensive conservation of genome organisation for Arabidopsis thaliana and Capsella rubella.

Genome colinearity has been studied for two closely related diploid species of the Brassicaceae family, Arabidopsis thaliana and Capsella rubella. Markers mapping to chromosome 4 of A. thaliana were found on two linkage groups in Capsella and colinear segments spanning more than 10 cM were revealed. Detailed analysis of a 60 kbp region in A. thaliana and its counterpart in C. rubella showed virtually complete conservation of gene repertoire, order and orientation. The comparison of orthologous genes revealed very similar exon-intron structures and sequence identities of 90% or more were found for exon sequences. This extensive genome colinearity at the genetic and molecular level allows the efficient transfer of data from the well-studied A. thaliana genome to other species in the Brassicaceae family, substantially facilitating genome analysis studies for species of this family.

Amino Acid Sequence↗
PubMed1997

Structural and mutational analysis of a conserved gene (DGSI) from the minimal DiGeorge syndrome critical region.

The majority of patients with DiGeorge syndrome (DGS), velocardiofacial syndrome (VCFS), conotruncal anomaly face syndrome (CTAFS) and some individuals with familial or sporadic conotruncal cardiac defects have hemizygous deletions of chromosome 22. Most patients with these disorders share a common large deletion, spanning > 1.5 Mb within 22q11.21-q11.23. Recently, the smallest region of deletion overlap has been narrowed to a 250 kb area, the minimal DGS critical region (MDGCR), which includes the locus D22S75 (N25). We have isolated and characterized a novel, highly conserved gene, DGSI, within the MDGCR. DGSI has 10 exons and nine introns encompassing 1702 bp of cDNA sequence and 11 kb of genomic DNA. The encoded protein has 476 amino acids with a predicted mol. wt of 52.6 kDa. The intron-exon boundaries have been analyzed and conform to the consensus GT/AG motif. The corresponding murine Dgsi has been isolated and localized to proximal mouse chromosome 16. The mouse gene contains the same number of exons and introns, and the predicted protein has 479 amino acids with 93.2% identity to that of the human DGSI gene. By database searching, both genes have significant homology to a Caenorhabditis elegans hypothetical protein, F42H10.7. Further, mutation analysis has been performed in 16 patients, who have no detectable 22q11.2 deletion and some of the characteristic clinical features of DGS/VCFS. We have detected eight sequence variants in DGSI. These occurred in the 5'-untranslated region, the coding region and the intronic regions adjacent to the intron-exon boundaries of the gene. Seven of the eight variants were also present in normal controls or unaffected family members, suggesting they may not be of etiologic significance.

Amino Acid Sequence↗
PubMed2004

Genetic architecture of flowering time in maize as inferred from quantitative trait loci meta-analysis and synteny conservation with the rice genome.

Genetic architecture of flowering time in maize was addressed by synthesizing a total of 313 quantitative trait loci (QTL) available for this trait. These were analyzed first with an overview statistic that highlighted regions of key importance and then with a meta-analysis method that yielded a synthetic genetic model with 62 consensus QTL. Six of these displayed a major effect. Meta-analysis led in this case to a twofold increase in the precision in QTL position estimation, when compared to the most precise initial QTL position within the corresponding region. The 62 consensus QTL were compared first to the positions of the few flowering-time candidate genes that have been mapped in maize. We then projected rice candidate genes onto the maize genome using a synteny conservation approach based on comparative mapping between the maize genetic map and japonica rice physical map. This yielded 19 associations between maize QTL and genes involved in flowering time in rice and in Arabidopsis. Results suggest that the combination of meta-analysis within a species of interest and synteny-based projections from a related model plant can be an efficient strategy for identifying new candidate genes for trait variation.

Chromosome Mapping↗
PubMedDate not supplied

Analysis of the conserved cysteine periodicity of Paramecium variable surface antigens.

The major surface antigens expressed by free-living and parasitic protozoa commonly contain repeating cysteine motifs. Despite the common occurrence of these repeats their functional significance remains largely unexplored. In this paper we investigate the conserved cysteine repeats within the variable surface antigens of Paramecium tetraurelia. We show that deletion of 2 entire repeating units or portions of repeats near the N-terminus does not prevent expression of the A51 variable surface antigen. Alteration of a single cysteine to serine residue also has no effect on A51 expression. In contrast, deletions near the C-terminus of the protein have identified a small segment within the repeats that is required for expression on the surface. The required region contains a number of conserved amino acid residues, yet site-directed mutagenesis of two residues (serine and threonine to alanine) did not prevent expression. These studies demonstrate the feasibility of using deletion analysis to identify regions critical for the expression of cysteine-rich surface antigens. The relationship of these results to the structure and expression of cysteine-rich surface proteins in other protozoa is discussed.

Amino Acid Sequence↗