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PubMedDate not supplied

Mitochondrial DNA variation in the European otter (Lutra lutra) and the use of spatial autocorrelation analysis in conservation.

To add genetic information to the international conservation efforts on European otters Lutra lutra, we investigated the genetic population structure in and around a known "source" population of the otter, the Oberlausitz (OL) in eastern Germany. This was complemented by a first survey of genetic variation levels in the Central European otter population. Sequence analysis of 300bp of the mitochondrial control region in 76 specimens from the eastern German study region and 53 individuals from several other European populations revealed a low level of genetic variation, with only 5 haplotypes present and nucleotide diversities within populations ranging from 0.00% to 0.17%. Apart from eastern Germany, one haplotype was by far the most abundant one, from which other, only locally occurring types, could be derived by a single point mutation. This suggests a single Pleistocene refugium from which the analyzed European regions have been reinvaded after the glaciations. Within eastern Germany, two abundant haplotypes were found. Their occurrence differed significantly among subregions of eastern Germany. The uneven distribution of a locally restricted but abundant haplotype could be explained by isolation-by-distance and might reflect emigration from the OL source population to surrounding regions. This suggests that vital local populations can indeed serve as "sources" for the invasion of surrounding areas. Given a suitable genetic marker, we suggest a spatial autocorrelation analysis to monitor the genetic effect of such an emigration from a source population.

Animals↗
PubMed2005

Restriction analysis of conserved and variable regions of VP2 gene of Indian isolates of bluetongue virus serotype 1.

Bluetongue virus (BTV) is a member of Orbivirus genus in family Reoviridae. The virus genome is composed of 10 double-stranded RNA segments. The RNA segment L2 encodes an outer capsid viral protein VP2, which is the main determinant of neutralization and serotype-specific immune response. BTV serotype 1 (BTV-1) specific novel primer pair was designed using VP2 gene sequences available in GenBank to amplify 1240-1844 bp region because two hypervariable and three conserved regions have been reported within these 604 nucleotides. This primer pair successfully amplified cell culture adapted six Indian isolates of BTV-1 from different geographical regions of the country. The 604 bp PCR product of VP2 gene of all six BTV-1 yielded two fragments of 273 and 331 bp when digested with Taq1 restriction enzyme. This indicated that there is only one TaqI site at 1513 bp (within 1240-1844 bp region) of VP2 gene of BTV-1 Indian isolates. The in silico restriction analysis revealed that in BTV-1 South African isolate (BTV-1SA) there is no TaqI site while in BTV-1 Australian isolates (BTV-1AUS), there are two TaqI sites (at 1513 and 1567 bp) within 1240-1844 bp region of VP2 gene. The earlier reported VP2 gene based primer pair for BTV-1 was used in the present study to amplify 2242-2933 bp region of six BTV-1 Indian isolates as three conserved regions have been reported within these 691 nucleotides. The digestion of 691 bp PCR products with XmnI yielded three fragments of 364, 173 and 154 bp with all the six Indian isolates of BTV-1 suggesting that there are two XmnI sites within 2242-2933 bp region of VP2 gene. A single XmnI site was observed in silico in BTV-1AUS and BTV-1SA isolates at different positions within this region. The in vitro and in silico restriction profile analyses of partial VP2 gene sequences using TaqI and XmnI restriction enzymes indicated a close relationship of Indian isolates of BTV-1 with BTV-1AUS isolates but not with BTV-1SA isolate.

Bluetongue virus↗
PubMed2005

Probing the sterol binding site of soybean sterol methyltransferase by site-directed mutagenesis: functional analysis of conserved aromatic amino acids in Region 1.

Soybean sterol methyltransferase (SMT) in the presence of AdoMet catalyzes the transmethylation of the delta24-bond of the sterol side chain to produce phytosterols with a methyl(lene) or ethyl(idene) group at C-24. The function of six aromatic amino acids associated with the putative active center of the SMT, i.e., Region 1 that extends from Phe82 to Phe93 in soybean SMT, was studied by site-directed mutagenesis and heterologous expression in BL21(DE3) bacterial cells. The enzyme-generated products were characterized kinetically and by GC-MS analysis. Substitution of the aromatic amino acids at positions 82, 83, 85, 87, 91, and 93 with a leucine residue produced mutant SMTs with varying activities. The mutants converted cycloartenol to 24(28)-methylene cycloartanol [C1-activity] from a few percent to as much as 95% of the control activity. In contrast, none of the leucine mutants were found to catalyze 24(28)-methylene lophenol [C2-activity], suggesting a loss of function associated with the second C1-transfer activity. In contrast to the loss of the second C1-transfer activity of the Phe82Leu, replacement of the Phe82 residue to isoleucine had minimal effect on the first or second C1-transfer activities, suggesting that the increased bulk (branching) in the leucine side chain contributes to significant perturbations in the active site that generate inaccurate positioning of the substrate side chain disfavoring the second C1-transfer activity. Replacement of Tyr83 to phenylalanine resulted in an increase of the specificity constant (kcat/Km) for the substrate of the second C1-transfer activity by a factor of 5 compared to control and an increase of delta24(28)Z-ethylidene sterol formation in the 24-ethyl sterol product set, suggesting that loss of steric bulk from the phenolic hydroxyl group on tyrosine generates a less precise fit of the delta24(28) sterol side chain into the active site favoring the second C1-transfer activity and prompting reaction channeling during catalysis. Circular dichroism spectra, equilibrium dialysis studies of AdoMet, and chromatographic information of the wild-type and Tyr83 mutants confirmed retention of the overall conformation of the enzyme during the experiments. Together, these findings suggest that the amino acids of Region 1 provide a tight substrate orientation imposed by hydrophobic interactions between the sterol side chain and the SMT active site contacts and control the production and processing of the transmethylation pathways governed by the first and second C1-transfer activities.

Amino Acid Sequence↗
PubMed2000

Genetic analysis and conservation of 31 surviving individuals of a rare New Zealand tree, Metrosideros bartlettii (Myrtaceae).

Metrosideros bartlettii (Myrtaceae) is a distinctive and extremely rare tree, endemic to New Zealand, first discovered in 1975. Prior to this study, a total of 19 adult individuals of the species had been reported; these are located in three small forest remnants in the far north of the North Island of New Zealand. Here we describe a total of 31 adult M. bartlettii at the three sites, including 12 individuals newly discovered by us. We analyse the genetic diversity of the species, using microsatellites to examine the chloroplast genome and amplified fragment length polymorphisms (AFLPs) to monitor nuclear variation. The results clearly demonstrate that M. bartlettii is a unique species, distinct from its two closest relatives M. robusta and M. excelsa. Analysis of genetic diversity within the 31 remaining individuals of M. bartlettii showed an average heterozygosity (< H >) of 0.18 and a proportion of polymorphic genes (< P >) of 0.44. Population structure, as shown by 286 AFLP loci, varied between the three geographical sites; the site with fewest individuals, containing two trees, showed some separation from the populations at the other two locations. These two latter sites, by contrast, had highly overlapping AFLP population diversity profiles. The implications of these results for conservation of the species are discussed.

Conservation of Natural Resources↗
PubMed1998

Functional analysis of conserved histidines in ADP-glucose pyrophosphorylase from Escherichia coli.

Two absolutely conserved histidines and a third highly conserved histidine are noted in 11 bacterial and plant ADP-glucose pyrophosphorylases. These histidines were individually mutagenized in the E. coli enzyme to glutamine in order to determine their function. Glutamine mutations at residues 143 and 156 produced functional enzymes in cell extracts with slightly lower than wild-type specific catalytic activities and with same heat stability characteristics of the wild-type enzyme. Substitution of residue 83 with glutamine however produced an enzyme having decreased thermal stability. Additional mutageneses at residue 83 with asparagine, arginine, or aspartate gave rise to enzymes having a progressively decreasing trend in thermal stability. These mutants are more susceptible to proteolysis than wild-type enzyme. Kinetic analysis of H83Q and H83N indicates that histidine 83 is not involved in the catalytic mechanism or in substrate binding but possibly in maintenance of the active catalytic structure.

Base Sequence↗
PubMed2002

Mutational analysis of conserved hydrophobic amino acid residues in the N-terminal region of DnaA protein.

DnaA is the initiator of chromosomal DNA replication in E. coli. We previously reported that conserved hydrophobic amino acid residues in the N-terminal region of DnaA (I26 and L40) are essential for DNA replication in vivo and in vitro using mutant DnaA proteins (DnaAI26S and DnaAL40S). In this study, we introduced further random mutations to find intragenic suppressors for dnaAI26S or dnaAL40S. By direct DNA sequence, a mutation which causes substitution of the Ser (Ile, in the wild-type DnaA) with Phe (DnaAI26F or DnaAL40F) was found in all of the suppressors. Site-directed mutational analysis showed that DnaAI26L, and DnaAL40I, but not DnaAI26S or DnaAL40S, were active for oriC DNA replication in cells. Furthermore, purified DnaAI26F but not DnaAI26S was active for oriC DNA replication in a crude extract. These results strongly suggest that hydrophobic amino acid residues in these positions of DnaA (I26 and L40) are important for the function of this protein as an initiator of DNA replication both in vivo and in vitro.

Amino Acid Sequence↗
PubMed1976

[Working time analysis in conservative dentistry (dental filling therapy)].

The purpose of the present paper was to determine the time required for filling therapy. The authors deal briefly with the subdivision of time adopted for the present study. Conclusions as to planning in dental practice may be drawn from the results obtained only if values determined by different dentists are available for comparison.

Dental Restoration, Permanent↗
PubMed1993

Analysis of conserved binding proteins for nuclear localization sequences.

Correct targeting of nuclear proteins is mediated by nuclear localization sequences (NLS) which permit specific binding to the nucleus and subsequent translocation across the nuclear envelope via the nuclear pore complex. It is proposed that nuclear import is facilitated by NLS-receptors which reside in the cytoplasm and at the nuclear pore. These NLS-receptors could facilitate an early step of nuclear protein import, i.e. targeting and binding of nuclear proteins at the nuclear pore. We have generated anti-idiotype antibodies against the SV40 T-antigen nuclear localization sequence that allowed us to study NLS-binding proteins in a variety of different organisms. Proteins of similar size are recognized by these antibodies in yeast, Drosophila, rat and human cells. Cytological analysis indicates that the NLS-binding proteins reside in part at nuclear pores. One of the proteins recognized by anti-idiotype antibodies is identical to a previously identified NLS-binding protein. Using isolated yeast nuclei we demonstrate that the anti-idiotype antibodies compete for binding of nuclear proteins in vitro. We show that the yeast mutant npl3, which is defective in nuclear protein localization, has an altered distribution of antigens recognized by these anti-idiotype antibodies, at the semi-permissive temperature. Our results suggest that a set of proteins common to various eukaryotes recognizes nuclear localization sequences.

Amino Acid Sequence↗
PubMed1992

[Review of results and clinical analysis of conservative partial surgery associated with postoperative radiotherapy in operable breast cancers stages I amd II].

Over the last 25 years, conservative forms of treatment have been used for the locoregional treatment of early breast cancer, stages I and II (< 3 cm in diameter). At present, the same carcinological results are obtained with conservative surgery and radiation therapy as with radical surgery, and the aesthetic and functional results have improved. Several parameters should be taken into account, ie tumour volume, breast volume, multifocal nature and histologic type. A number of questions still remain unanswered namely: what is the most appropriate conservative surgery (quadrantectomy, tumourectomy or local excision)? What is the most appropriate total irradiation dose (50 Gy over 5 weeks: or 45 Gy over 4.5 weeks)? What is the value of radiation boost in patients with negative resection margins? What is the value of radiation boost in patients with a high local risk of recurrence? What is the most appropriate radiation technique for boost in the primary tumour bed (electrons or iridium implants)? Does the external irradiation of regional lymph nodes improve survival rate? Is it possible to extend conservative treatment to a 4-cm diameter tumour? What is the impact of adjuvant systemic chemotherapy and/or hormonotherapy on the risk of isolated breast cancer recurrence?

Adult↗
PubMed1992

Analysis of conserved domains and sequence motifs in cellular regulatory proteins and locus control regions using new software tools for multiple alignment and visualization.

With the tremendous expansion of molecular sequence data in recent years, multiple alignment is arguably one of the two most important analytic techniques (the other being fast database searching). A number of useful approaches to this problem have previously been developed, but often they are limited to only a subset of multiple-alignment applications and cannot easily deal with the complex structural organization seen in an increasing number of sequences. For example, a single sequence may contain several domains of different evolutionary origins, and the multiplicities and relative ordering of these domains may be quite different among related sequences. Here we describe an integrated set of interactive Unix tools that combines several multiple-alignment techniques with traditional "dot-plot" visualization to provide a flexible environment for approaching complex sequence analysis problems. We apply these tools to the identification and characterization of "catalytic" domains in ras and rho/rac GTPase-activating proteins, to "Src homology" (SH2, SH3) domains in cytoplasmic signaling proteins, to repetitive sequence motifs in the alpha and beta subunits of protein prenyltransferases, and to regulatory DNA sequences in the locus control region of the beta-globin gene cluster.

Alkyl and Aryl Transferases↗
PubMed1996

Functional analysis of conserved motifs in EcoP15I DNA methyltransferase.

EcoP15I DNA methyltransferase recognizes the sequence 5'-CAGCAG-3' and transfers a methyl group to N-6 of the second adenine residue in the recognition sequence. All N-6 adenine methyltransferases contain two highly conserved sequences, FxGxG (motif I), postulated to form part of the S-adenosyl-L-methionine binding site and (D/N/S)PP(Y/F) (motif IV) involved in catalysis. We have altered the second glycine residue in motif I to arginine and serine, and substituted tyrosine in motif IV with tryptophan in EcoP15I DNA methyltransferase, using site-directed mutagenesis. The mutant enzymes were overexpressed, purified and characterized by biochemical methods. The mutations in motif I completely abolished AdoMet binding but left target DNA recognition unaltered. Although the mutation in motif IV resulted in loss of enzyme activity, we observed enhanced crosslinking of S-adenosyl-L-methionine and DNA. This implies that DNA and AdoMet binding sites are close to motif IV. Taken together, these results reinforce the importance of motif I in AdoMet binding and motif IV in catalysis. Additionally, limited proteolysis and UV crosslinking experiments with EcoP15I DNA methyltransferase imply that DNA binds in a cleft formed by two domains in the protein. Methylation protection analysis provides evidence for the fact that EcoP15I DNA MTase makes contacts in the major groove of its substrate DNA. Interestingly, hypermethylation of the guanine residue next to the target adenine residue indicates that the protein probably flips out the target adenine residue.

Base Sequence↗
PubMed2001

Novel posttranslational activation of the LYS2-encoded alpha-aminoadipate reductase for biosynthesis of lysine and site-directed mutational analysis of conserved amino acid residues in the activation domain of Candida albicans.

The alpha-aminoadipate pathway for lysine biosynthesis is present only in fungi. The alpha-aminoadipate reductase (AAR) of this pathway catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic-delta-semialdehyde by a complex mechanism involving two gene products, Lys2p and Lys5p. The LYS2 and LYS5 genes encode, respectively, a 155-kDa inactive AAR and a 30-kDa phosphopantetheinyl transferase (PPTase) which transfers a phosphopantetheinyl group from coenzyme A (CoA) to Lys2p for the activation of Lys2p and AAR activity. In the present investigation, we have confirmed the posttranslational activation of the 150-kDa Lys2p of Candida albicans, a pathogenic yeast, in the presence of CoA and C. albicans lys2 mutant (CLD2) extract as a source of PPTase (Lys5p). The recombinant Lys2p or CLD2 mutant extract exhibited no AAR activity with or without CoA. However, the recombinant 150-kDa Lys2p, when incubated with CLD2 extract and CoA, exhibited significant AAR activity compared to that of wild-type C. albicans CAI4 extract. The PPTase in the CLD2 extract was required only for the activation of Lys2p and not for AAR reaction. Site-directed mutational analysis of G882 and S884 of the Lys2p activation domain (LGGHSI) revealed no AAR activity, indicating that these two amino acids are essential for the activation. Replacement of other amino acid residues in the domain resulted in partial or full AAR activity. These results demonstrate the posttranslational activation and the requirement of specific amino acid residues in the activation domain of the AAR of C. albicans.

Aldehyde Oxidoreductases↗
PubMed1992

Mutational analysis of conserved N-linked glycosylation sites of human immunodeficiency virus type 1 gp41.

Amino acid substitutions were introduced into four conserved N-linked glycosylation sites of the human immunodeficiency virus type 1 envelope transmembrane glycoprotein, gp41, to alter the canonical N-linked glycosylation sequences. One altered site produced a severe impairment of viral infectivity, which raises the possibility that N-linked sugars at this site may have an important role in the human immunodeficiency virus type 1 life cycle.

Animals↗
PubMed1993

Mutational analysis of conserved tyrosines in the NS-1 protein of the parvovirus minute virus of mice.

The NS-1 gene of the parvovirus minute virus of mice encodes a multifunctional protein essential for viral DNA replication and gene expression. In addition to possessing DNA helicase and ATPase activities, NS-1 forms a covalent linkage with the 5' ends of viral DNA and is a strong candidate for the site-specific nicking-closing enzyme postulated to be involved in the resolution of concatemers and terminal hairpin structures that arise during parvoviral DNA replication. Since the covalent linkage between NS-1 and the 5' terminus of MVM DNA resists alkali and mild acid treatment, a tyrosine phosphodiester is likely to be involved. To map domains responsible for this activity, mutations converting tyrosine to phenylalanine were introduced into the NS-1 gene using oligonucleotide-directed mutagenesis and their effect on the DNA replication and transcriptional activation functions of NS-1 was examined in transient in vivo transfection assays. Replacement of Tyr-188, Tyr-197, Tyr-210, Tyr-310, Tyr-422, or Tyr-550 with phenylalanine greatly reduced the ability of NS-1 to complement the replication of the target genome ins 20B in COS-7 cells. However, a Ser-545 to Thr-545 substitution in the Phe-550 mutant restored DNA replication activity. Replacement of 5 other tyrosines in NS-1 with phenylalanine either enhanced (Phe-6), had a moderate inhibitory effect (Phe-209) or had no effect (Phe-47, Phe 227 and Phe-543) on its DNA replication activity. Two of the 11 phenylalanine substitution mutations, Phe-188 and Phe-197, also greatly reduced the ability of NS-1 to transactivate the p38 promoter and displayed a dominant negative phenotype with respect to transactivation. Since the remaining tyrosines in MVM NS-1, Tyr-152, Tyr-252, Tyr-374, and Tyr-595, are not conserved among the NS-1 proteins encoded by porcine and feline parvoviruses, they are presumed to be nonessential for the normal functioning of NS-1. The results point to a role for either Tyr-188, Tyr-197, Tyr-210, Tyr-310, or Tyr-422 in forming a covalent linkage with viral DNA and further suggest a regulatory role for several tyrosines in other DNA replication and transcriptional activation functions of NS-1.

Amino Acid Sequence↗
PubMed2001

Mutational analysis of conserved glycines 42 and 256 in Cephalosporium acremonium isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is critical for the catalytic conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N in the penicillin and cephalosporin biosynthetic pathway. Two conserved glycine residues in Cephalosporium acremonium IPNS (cIPNS), namely glycine-42 and glycine-256, were identified by multiple sequence alignment and investigated by site-directed mutagenesis to study the effect of the substitution on catalysis. Our study showed that both the mutations from glycine to alanine or to serine reduced the catalytic activity of cIPNS and affected its soluble expression in a heterologous host at 37 degrees C. Soluble expression was restored at a reduced temperature of 25 degrees C, and thus, it is possible that these glycine residues may have a role in maintaining the local protein structure and are critical for the soluble expression of cIPNS.

Acremonium↗
PubMed2006

Systematic analysis of conservation relations in Escherichia coli genome-scale metabolic network reveals novel growth media.

A biochemical species is called producible in a constraints-based metabolic model if a feasible steady-state flux configuration exists that sustains its nonzero concentration during growth. Extreme semipositive conservation relations (ESCRs) are the simplest semipositive linear combinations of species concentrations that are invariant to all metabolic flux configurations. In this article, we outline a fundamental relationship between the ESCRs of a metabolic network and the producibility of a biochemical species under a nutrient media. We exploit this relationship in an algorithm that systematically enumerates all minimal nutrient sets that render an objective species weakly producible (i.e., producible in the absence of thermodynamic constraints) through a simple traversal of ESCRs. We apply our results to a recent genome scale model of Escherichia coli metabolism, in which we traverse the 51 anhydrous ESCRs of the metabolic network to determine all 928 minimal aqueous nutrient media that render biomass weakly producible. Applying irreversibility constraints, we find 287 of these 928 nutrient sets to be thermodynamically feasible. We also find that an additional 365 of these nutrient sets are thermodynamically feasible in the presence of oxygen. Since biomass producibility is commonly used as a surrogate for growth in genome scale metabolic models, our results represent testable hypotheses of alternate growth media derived from in silico analysis of the E. coli genome scale metabolic network.

Algorithms↗
PubMed1990

Mutational analysis of conserved nucleotides in a self-splicing group I intron.

We have constructed all single base substitutions in almost all of the highly conserved residues of the Tetrahymena self-splicing intron. Mutation of highly conserved residues almost invariably leads to loss of enzymatic activity. In many cases, activity could be regained by making additional mutations that restored predicted base-pairings; these second site suppressors in general confirm the secondary structure derived from phylogenetic data. At several positions, our suppression data can be most readily explained by assuming non-Watson-Crick base-pairings. In addition to the requirements imposed by the secondary structure, the sequence of the intron is constrained by "negative interactions", the exclusion of particular nucleotide sequences that would form undesirable secondary structures. A comparison of genetic and phylogenetic data suggests sites that may be involved in tertiary structural interactions.

Animals↗
PubMed2004

Identification, characterization and phylogenic analysis of conserved genes within the p74 gene region of Choristoneura fumiferana granulovirus genome.

The genes located within the p74 gene region of the Choristoneura fumiferana granulovirus (ChfuGV) were identified by sequencing an 8.9 kb BamHI restriction fragment on the ChfuGV genome. The global guanine-cytosine (GC) content of this region of the genome was 33.02%. This paper presents the ORFs within the p74 gene region along with their transcriptional orientations. This region contains a total of 15 open reading frames (ORFs). Among those, 8 ORFs were found to be homologues to the baculoviral ORFs: Cf-i-p , Cf-vi, Cf-vii, Cf-viii (ubiquitin), Cf-xi (pp31), Cf-xii (lef-11), Cf-xiii (sod) and Cf-xv-p (p74). To date, no specific function has been assigned to the ORFs: Cf-i, Cf-ii, Cf-iii, Cf-iv, Cf-v, Cf-vi, Cf-vii, Cf-ix and Cf-x. The most noticeable ORFs located in this region of the ChfuGV genome were ubiquitin, lef-11, sod, fibrillin and p74. The phylogenetic trees (constructed using conceptual products of major conserved ORFs) and gene arrangement in this region were used to further examine the classification of the members of the granulovirus genus. Comparative studies demonstrated that ChfuGV along with the Cydia pomonella granulovirus (CpGV), Phthorimaea operculella granulovirus (PhopGV), Adoxophyes orana granulovirus (AoGV) and Cryptophlebia leucotreta granulovirus (ClGV) share a high degree of amino acids sequence and gene arrangement preservation within the studied region. These results support a previous report, which classified a granuloviruses into 2 distinct groups: Group I: ChfuGV, CpGV, PhopGV and AoGV and Group II: Xestia c-nigrum granulovirus (XcGV) and Plutella xylostella granulovirus (PxGV). The phylogenetic and gene arrangement studies also placed ClGV as a novel member of the Group I granuloviruses.

Bacterial Proteins↗