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PubMed1990

Chick integrin alpha V subunit molecular analysis reveals high conservation of structural domains and association with multiple beta subunits in embryo fibroblasts.

We have cloned and characterized a chick homologue of the human vitronectin receptor alpha subunit (alpha v) whose primary sequence is 83% identical with its human counterpart but less than 40% identical with any other known integrin alpha subunit. Comparison of the chick and human sequences reveals several highly conserved regions, including the cytoplasmic domain. The putative ligand binding domain contains alpha v-specific residues that may contribute to ligand binding specificity. These are concentrated in three regions that are located before and between the first three Ca2+ binding domains. Polyclonal antibodies raised against two peptides deduced from the putative cytoplasmic and extracellular domains of the chick alpha v sequence recognize specifically integrin heterodimers in chick embryo fibroblasts. At least three putative beta subunits coimmunoprecipitate with the chick alpha v subunit. In addition to a protein with the same molecular weight as beta 3 (94K), protein bands of Mr 84K and 110K are also coprecipitated. By successive immunodepletions, we demonstrate that this latter Mr 110K subunit is beta 1, which appears to be one of the alpha v-associated subunits in chick embryo fibroblasts.

Amino Acid Sequence↗
PubMedDate not supplied

Analysis of the conserved N-terminal domains in major ampullate spider silk proteins.

Major ampullate silk, also known as dragline silk, is one of the strongest biomaterials known. This silk is composed of two proteins, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2). Only partial cDNA sequences have been obtained for these proteins, and these sequences are toward the C-terminus. Thus, the N-terminal domains have never been characterized for either protein. Here we report the sequence of the N-terminal region of major ampullate silk proteins from three spider species: Argiope trifasciata, Latrodectus geometricus, and Nephila inaurata madagascariensis. The amino acid sequences are inferred from genomic DNA clones. Northern blotting experiments suggest that the predicted 5' end of the transcripts are present in fibroin mRNA. The presence of more than one Met codon in the N-terminal region indicates the possibility of translation of both a long and a short isoform. The size of the short isoform is consistent with the published, cDNA based, N-terminal sequence found in flagelliform silk. Analyses comparing the level of identity of all known spider silk N-termini show that the N-terminus is the most conserved part of silk proteins. Two DNA sequence motifs identified upstream of the putative transcription start site are potential silk fibroin promoter elements.

Amino Acid Sequence↗
PubMedDate not supplied

An analysis of the conserved residues between halobacterial retinal proteins and G-protein coupled receptors: implications for GPCR modeling.

An alignment of the transmembrane domains of halobacterial retinal proteins (including bacteriorhodopsin) and G-protein coupled receptors (GPCRs) is presented based on the commonality of conserved residues between families. Due to the limited sequence homology displayed by these proteins, an alternative strategy is proposed for sequence alignment that correlates residues within secondary structure elements. The nonsequential alignment developed identifies three proline and two aspartates residues that share common positions and, in the former case, similar functions in the transmembrane domain. The alignment is further applied to model the packing of transmembrane helices 5 and 6 of the beta-adrenergic receptor based on the backbone coordinates of bacteriorhodopsin helices 3 and 2, respectively. Unlike models derived from standard sequential alignments, the approach developed here allows the key structural features conferred by the proline residues to be captured during model building. The structure described is also compared with available site directed mutagenesis results as well as existing GPCR models. In addition to the implications to model building, the commonality observed suggests a potential relationship among the GPCRs and retinal proteins.

Amino Acid Sequence↗
PubMed2004

Analysis of the conserved P9-G10.1 metal-binding motif in hammerhead ribozymes with an extra nucleotide inserted between A9 and G10.1 residues.

Hammerhead ribozymes (Rz) have catalytically important tandem G:A pairs in the core region, and we recently demonstrated that the P9-G10.1 motif (a sheared-type G:A pair with a guanine residue on the 3' side of the adenine residue) with several flanking base pairs is sufficient for capture of divalent cations, such as Mg(2+) and Cd(2+) ions that are important to maintain full activities (Tanaka et al. J. Am. Chem. Soc. 2002, 124, 4595-4601; Tanaka et al. J. Am. Chem. Soc. 2004, 126, 744-752). We also found that mutant hammerhead ribozymes that have an additional G residue inserted between A9 and G10.1 residues (the metal-binding P9-G10.1 motif) have significant catalytic activities. In this study, we demonstrate that the hammerhead ribozymes are capable of maintaining the catalytically competent structure even when the tandem, sheared-type G:A pairs were perturbed by an insertion of an additional nucleotide, whereas the chirality of the phosphorothioate at the P9 position significantly influenced the enzymatic activity for both the natural and G-inserted ribozymes.

Adenine↗
PubMed2006

In vivo enhancer analysis of human conserved non-coding sequences.

Identifying the sequences that direct the spatial and temporal expression of genes and defining their function in vivo remains a significant challenge in the annotation of vertebrate genomes. One major obstacle is the lack of experimentally validated training sets. In this study, we made use of extreme evolutionary sequence conservation as a filter to identify putative gene regulatory elements, and characterized the in vivo enhancer activity of a large group of non-coding elements in the human genome that are conserved in human-pufferfish, Takifugu (Fugu) rubripes, or ultraconserved in human-mouse-rat. We tested 167 of these extremely conserved sequences in a transgenic mouse enhancer assay. Here we report that 45% of these sequences functioned reproducibly as tissue-specific enhancers of gene expression at embryonic day 11.5. While directing expression in a broad range of anatomical structures in the embryo, the majority of the 75 enhancers directed expression to various regions of the developing nervous system. We identified sequence signatures enriched in a subset of these elements that targeted forebrain expression, and used these features to rank all approximately 3,100 non-coding elements in the human genome that are conserved between human and Fugu. The testing of the top predictions in transgenic mice resulted in a threefold enrichment for sequences with forebrain enhancer activity. These data dramatically expand the catalogue of human gene enhancers that have been characterized in vivo, and illustrate the utility of such training sets for a variety of biological applications, including decoding the regulatory vocabulary of the human genome.

Animals↗
PubMedDate not supplied

[Premature rupture of fetal membranes at term: sequelae of conservative management. An analysis of a personal patient sample].

Few procedures are less standardised than the procedure in case of pre-labour rupture of the membranes at term (PROM). We propose that management should be reviewed regularly on the basis of one's own data and be modified accordingly if necessary. For the duration of three months we analysed 400 pregnancies retrospectively. Patients with PROM were observed expectantly for 24 hours. If there were no spontaneous uterine contractions, labour was induced, depending on the degree of cervical dilatation. 10 percent of the cases studied had PROM. Of these a high proportion of 73 percent were primigravida, likewise 73 percent had an unripe cervix. The average time between PROM and delivery was 27 h. 50 percent of the babies were born 24 h after PROM. If delivery occurred more than 24 h after PROM, the rate of caesarean section (15 vs. 30 percent), the rate of forceps deliveries (11 vs. 20 percent), the rate of amnionitis (16 vs. 35 percent) and the number of admissions to the newborn-ICU (16 vs. 25 percent) almost doubled. The patients were examined vaginally relatively often prior to delivery (up to 18 times, with a mean of 8 times). We therefore recommend active management 6-8 h after PROM, should there be no onset of spontaneous uterine contractions. This is particularly beneficial to primigravida with an unripe cervix.

Adolescent↗
PubMed1992

Anchoring pockets in human histocompatibility complex leukocyte antigen (HLA) class I molecules: analysis of the conserved B ("45") pocket of HLA-B27.

Dissection of the peptide binding grooves of seven subtypes of human histocompatibility leukocyte antigen (HLA)-B27 into the six specificity pockets defined by the 2.6-A structure of HLA-A*0201 revealed just one pocket, the B ("45") pocket, that is conserved among all the HLA-B27 subtypes. Functional studies of mutant HLA-B*2705 molecules with point substitutions in residues of the B pocket show that this structure, and the glutamine residue at position 45 in particular, plays a critical role in cell surface expression, peptide binding, and in the presentation of both exogenous and endogenous peptides by HLA-B*2705. We predict that the B pocket of HLA-B*2705 interacts with an amino acid side chain that anchors peptides in the binding groove, and that this peptide motif is present in most endogenously processed peptides that bind to all seven subtypes of HLA-B27.

Amino Acid Sequence↗
PubMed2005

Genetic diversity within and among four South European native horse breeds based on microsatellite DNA analysis: implications for conservation.

In the present study, genetic analyses of diversity and differentiation were performed on four Basque-Navarrese semiferal native horse breeds. In total, 417 animals were genotyped for 12 microsatellite markers. Mean heterozygosity was higher than in other horse breeds, surely as a consequence of management. Although the population size of some of these breeds has declined appreciably in the past century, no genetic bottleneck was detected in any of the breeds, possibly because it was not narrow enough to be detectable. In the phylogenetic tree, the Jaca Navarra breed was very similar to the Pottoka, but appeared to stand in an intermediate position between this and the meat breeds. Assuming that Pottoka is the breed less affected by admixture, the others gradually distanced themselves from it through varying influences from outside breeds, among other factors. In a comparative study with other breeds, the French breeds Ardanais, Comtois, and Breton were the closest to the four native breeds. Three different approaches for evaluating the distribution of genetic diversity were applied. The high intrabreed variability of Euskal Herriko Mendiko Zaldia (EHMZ) was pointed out in these analyses. In our opinion, cultural, economic, and scientific factors should also be considered in the management of these horse breeds.

Animals↗
PubMed1995

Mutational analysis of a conserved motif of Agrobacterium tumefaciens VirD2.

The VirD2 polypeptide from Agrobacterium tumefaciens, in the presence of VirD1, introduces a site- and strand-specific nick at the T-DNA borders. A similar reaction at the origin of transfer (oriT) of plasmids is essential for plasmid transfer by bacterial conjugation. A comparison of protein sequences of VirD2 and its functional homologs in bacterial conjugation and in rolling circle replication revealed that they share a conserved 14 residue segment, HxDxxx(P/u)HuHuuux [residues 126-139 of VirD2; Ilyina, T.V. and Koonin, E.V. (1992) Nucleic Acids Res. 20, 3279-3285]. A mutational approach was used to test the role of these residues in the endonuclease activity of VirD2. The results demonstrated that the two invariant histidine residues (H133 and H135) are essential for activity. Mutations at three sites, histidine 126, aspartic acid 128 and aspartic acid 130, that are conserved in a subfamily of the plasmid mobilization proteins, led to the loss of VirD2 activity. Aspartic acid at position 130, could be substituted with glutamic acid and to a much lesser extent, with tyrosine. In contrast, another conserved residue, asparagine 139, tolerated many different amino acid substitutions. The non-conserved residues, arginine 129, proline 132 and leucine 134, were also found to be important for function. Isolation of null mutations that map throughout this conserved domain confirm the hypothesis that this region is essential for function.

Agrobacterium tumefaciens↗
PubMed2001

Analysis of a conserved hydrophobic pocket important for the thermostability of Bacillus pumilus chloramphenicol acetyltransferase (CAT-86).

Site-directed mutagenesis was carried out on Bacillus pumilus chloramphenicol acetyltransferase (CAT-86) to determine the effects of substitution at a conserved hydrophobic pocket identified earlier as important for thermostability. Mutations were introduced that would substitute residues at consensus positions 33, 191 and 203 in the enzyme, both individually and in combination. Two mutants, SDM1 (CAT-86 Y33F, A203V) and SDM5 (CAT-86 A203I), were more thermostable than wild-type and two mutants, SDM4 (CAT-86 I191V) and SDM7 (CAT-86 A203G), were less stable. Reconstruction of the residues of this hydrophobic pocket to that of a more thermostable CAT-R387 enzyme pocket (as a Y33F, I191V, A203V triple mutant) increased the thermostability of the enzyme above the wild-type, but its stability was less than that of SDM1 and SDM5. The K(m) values of the mutant enzymes for chloramphenicol and acetyl-CoA were essentially unaltered (in the ranges 15-30 and 26-35 microM respectively) and the specific activity of purified enzyme was in the range 270-710 units/mg protein. The possible effects of the amino acid substitutions on the CAT-86 structure were determined by homology modelling. A reduction in conformational strain and optimized hydrophobic interactions are predicted to be responsible for the increased thermostability of the SDM1 and SDM5 mutants.

Amino Acid Sequence↗
PubMed1994

Analysis of the conserved Asp(114) residue of the dopamine D2 receptor in schizophrenic patients.

The factors that influence response to antipsychotics treatment in chlorpromazine remain difficult to delineate but are thought to include genetic factors. Site-directed mutagenesis studies have demonstrated that substitution of the conserved residues Asp(113) to an Asn or Glu greatly reduces the binding affinity of propranolol in the beta-adrenergic receptor and the substitution of an Asp(114) has similar effects in the dopamine D2 receptor. In this study we have found the Asp(114) in the dopamine D2 receptor to be unaltered in 72 unrelated schizophrenic individuals including 12 patients classified according to their response to chlorpromazine.

Aspartic Acid↗
PubMed2000

Crystallization and preliminary X-ray analysis of the conserved domain IV of Escherichia coli 4.5S RNA.

4.5S RNA forms with Ffh protein the prokaryotic signal recognition particle (SRP), a highly conserved ribonucleoprotein complex essential for protein secretion. It also independently binds to elongation factor G (EF-G) in the ribosome and has a function in a subset of translocation events that is transient but required for viability. Crystals of three different constructs encompassing the conserved domain IV of 4.5S RNA, containing the recognition elements for both Ffh and EF-G, were obtained. Native X-ray diffraction data were collected for two crystal forms under cryogenic cooling conditions. The best crystals are of a 45 nt construct, diffract anisotropically to 2.6 A resolution using synchrotron radiation and belong to space group P3(2)21, with unit-cell parameters a = b = 69.1, c = 84.6 A and a single RNA molecule per asymmetric unit.

Bacterial Proteins↗
PubMed2003

Making the most of two crystals: structural analysis of a conserved hypothetical protein using native gel screening and SAD phasing.

The protein PAE2307 is a member of a protein family of unknown function which is conserved among a number of bacterial and archaeal species. The protein was overexpressed in Escherichia coli, purified and crystallized in two crystal forms. The prevalent form was twinned, but the other diffracted to 1.45 A resolution. The non-twinned crystals proved difficult to reproduce, so screening of potential heavy-atom derivatives by native polyacrylamide gel electrophoresis was used to establish suitable derivatization conditions. This process enabled the production of a K(2)Pt(NO2)4 derivative that was used to collect a single-wavelength anomalous diffraction (SAD) data set from the only available crystal. Phase information of high quality was obtained, enabling the calculation of an interpretable electron-density map.

Amino Acid Sequence↗
PubMed2004

Crystallization and preliminary X-ray analysis of a conserved hypothetical protein PAE2754 from Pyrobaculum aerophilum and of a double Leu-->Met mutant engineered for MAD phasing.

Structural genomics offers a potential route to the discovery of protein function. As part of a structural genomics project focused on the hyperthermophilic crenarchaeon Pyrobaculum aerophilum, a conserved hypothetical protein, PAE2754, has been expressed in Escherichia coli, purified and crystallized. Because of the difficulties of preparing interpretable heavy-atom derivatives with limited resolution and 8-12 molecules in the asymmetric unit, two leucine residues were selected for mutation to methionine. The double mutant L65M/L80M was created, expressed incorporating SeMet and crystallized. The crystals are monoclinic, space group P2(1), with unit-cell parameters a = 56.4, b = 193.3, c = 60.5 A, beta = 94.6 degrees and eight molecules (two tetramers) in the asymmetric unit. The crystals diffract to 2.75 A resolution and are suitable for MAD phasing.

Bacterial Proteins↗
PubMed2005

Crystallization and preliminary X-ray crystallographic analysis of a conserved domain in plants and prokaryotes from Pyrococcus horikoshii OT3.

A plant- and prokaryote-conserved domain (PPC) has previously been found in AT-hook motif nuclear localized protein 1 (AHL1) localized in the nuclear matrix of Arabidopsis thaliana (AtAHL1). AtAHL1 has a DNA-binding function. Mutation analyses of AtAHL1 has previously revealed that the hydrophobic region of the PPC domain is essential for its nuclear localization. In this study, the PPC of the hyperthermophilic archaebacterium Pyrococcus horikoshii (PhPPC) was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 53.69, c = 159.2 A. Data were obtained at 100 K, with diffraction being observed to a resolution of 1.7 A. A complete data set from crystals of the SeMet-substituted protein was also obtained.

Amino Acid Motifs↗
PubMed2006

Cloning, purification and preliminary crystallographic analysis of a conserved hypothetical protein, SA0961 (YlaN), from Staphylococcus aureus.

SA0961 is an unknown hypothetical protein from Staphylococcus aureus that can be identified in the Firmicutes division of Gram-positive bacteria. The gene for the homologue of SA0961 in Bacillus subtilis, ylaN, has been shown to be essential for cell survival, thus identifying the protein encoded by this gene as a potential target for the development of novel antibiotics. SA0961 was cloned and the protein was overexpressed in Escherichia coli, purified and subsequently crystallized. Crystals of selenomethionine-labelled SA0961 diffract to beyond 2.4 angstroms resolution and belong to the monoclinic space group P2(1), with unit-cell parameters a = 31.5, b = 42.7, c = 62.7 angstroms, beta = 92.4 degrees and two molecules in the asymmetric unit. A full structure determination is under way to provide insights into the function of this protein.

Amino Acid Sequence↗
PubMed2005

Analysis of evolutionary conservation in CD1d molecules among primates.

The hereditary conservation in the genetically encoded CD1D sequences of various primates was analyzed. Genomic CD1D sequences of 17 rhesus macaques with distinct origins, eight Indian and nine Chinese, were examined and differences of only one or two nucleotides were detected and the consensus sequence of rhesus CD1D was determined. CD1D consensus sequences of three African green monkeys (AGMs) and the rhesus monkeys were then compared to study the evolutionary differences among interspecies. The CD1D consensus sequence determined from AGMs apparently differed by seven nucleotides from the rhesus consensus sequence, and nucleotide difference induced only three amino acid changes within Exon3, corresponding to the alpha2 domain of CD1d having a hydrophobic ligand-binding pocket. Such changes in the alpha2 domain may alter the characteristics of the SIV-derived glycolipid/lipid antigens presented by each CD1d molecule to innate natural killer T cells. In addition, the CD1D genomic sequences of three chimpanzees (chimps) were determined. To our surprise, although Exon2 and Exon3 reflecting antigen-binding alpha1 and alpha2 domains in chimps' CD1D were identical to that in humans except one amino acid, three amino acids within Exon4, reflecting alpha3 domain, were distinct from humans, and one of them was identical to those in rhesus and AGM CD1D. On the basis of the findings, the evolutionary relationship of the CD1d molecules among the various primates and their HIV-1/SIV susceptibility will be discussed.

Amino Acid Sequence↗