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TY - JOUR. T1 - A new usage of functionalized oligodeoxynucleotide probe for site-specific modification of a guanine base within RNA. AU - Onizuka, Kazumitsu. AU - Taniguchi, Yosuke. AU - Sasaki, Shigeki. PY - 2010/1/29. Y1 - 2010/1/29. N2 - Site-specific modification of RNA is of great significance to investigate RNA structure, function and dynamics. Recently, we reported a new method for sequence-and cytosine-selective chemical modification of RNA based on the functional group transfer reaction of the 1-phenyl-2-methylydene-1,3-diketone unit of the 6-thioguanosine base incorporated in the oligodeoxynucleotide probe. In this study, we describe that the functionality transfer rate is greatly enhanced and the selectivity is shifted to the guanine base when the reaction is performed under alkaline conditions. Detailed investigation indicated that the 2-amino group of the enolate form of rG is the reactant of the functionality transfer reaction. As a potential application of this efficient ...
In 2000 H. van den Berg published eleven articles. In 2001 H. van den Berg published five articles, published one book - monograph and published one chapter. In 2002 H. van den Berg published seven articles, published three chapters and published one book - monograph . In 2004 H. van den Berg published four articles. In 2005 H. van den Berg published three articles and published one chapter. In 2006 H. van den Berg published six articles. In 2007 H. van den Berg published seven articles. In 2009 H. van den Berg published one article. In 2010 H. van den Berg published three articles. In 2011 H. van den Berg published six articles. In 2012 H. van den Berg published two articles. In 2013 H. van den Berg published four articles. ...
We report the sequence of a 4.5-kb cDNA clone isolated from a human melanoma library which bears high amino acid sequence identity to the yeast mitochondrial (mt) DNA polymerase (Mip1p). This cDNA contains a 3720-bp open reading frame encoding a predicted 140-kDa polypeptide that is 43% identical to Mip1p. The N-terminal part of the sequence contains a 13 glutamine stretch encoded by a CAG trinucleotide repeat which is not found in the other DNA polymerases gamma (Pol gamma). Multiple amino acid sequence alignments with Pol gamma from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Drosophila melanogaster, Xenopus laevis and Mus musculus show that these DNA polymerases form a family strongly conserved from yeast to man and are only loosely related to the Family A DNA polymerases. ...
A short sequence of predominantly basic amino acids Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val from SV40 Large T is responsible for the normal nuclear location of the protein. Alteration of Lys-128 to each of six different residues other than Arg renders Large T cytoplasmic, whereas single amino acid changes in the surrounding region impair but do not prevent nuclear accumulation. When transposed to the amino terminus of cytoplasmic Large T species, or Escherichia coli β-galactosidase or of chicken muscle pyruvate kinase, the sequence around Lys-128 of Large T is able to direct the recipient protein to the nucleus. This demonstrates that these amino acids can be sufficient for nuclear location and can act as a nuclear location signal. A computer search of over 2500 proteins reveals that some other nuclear proteins (for example, BK virus Large T, SV40 VP2 and adenovirus 72kDa DNA binding protein) contain very similar basic tracts, but so too do some presumed non-nuclear proteins (for example, poliovirus ...
A New Approach for DNA Sequence Similarity Analysis based on Triplets of Nucleic Acid Bases: 10.4018/978-1-60960-064-8.ch006: Similarity analysis of DNA sequences is a fundamental research area in Bioinformatics. The characteristic distribution of L-tuple, which is the tuple of
Alazawi W, Heath H, et al. Stat2 loss leads to cytokine-independent, cell-mediated lethality in LPS-induced sepsis. Proc Natl Acad Sci U S A 110(21):8656-8661, 2013 [95].. Arnold, ES, SC Ling, et al. ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP- 43. Proc Natl Acad Sci U S A 110(8):E736- E745, 2013 [96].. Burdick RC, Hu WS, Pathak VK. Nuclear import of APOBEC3F-labeled HIV-1 preintegration complexes. Proc Natl Acad Sci U S A 110(49):E4780-E4789, 2013 [97].. Chen J, Feigenbaum L, et al. Insulin-dependent diabetes induced by pancreatic beta cell expression of IL-15 and IL-15R alpha. Proc Natl Acad Sci U S A 110(33):13534- 13539, 2013 [98].. Feng MQ, Gao W, et al. Therapeutically targeting glypican-3 via a conformation-specific single-domain antibody in hepatocellular carcinoma. Proc Natl Acad Sci U S A 110(12):E1083-E1091, 2013 [99].. Kim TS, Park JE, Shukla A, Choi S, Murugan RN, Lee JH, Ahn M, Rhee K, ...
Using rigorous statistical methods, we have identified and evaluated unusual properties of the distribution of charged residues within the sequences of eukaryotic cellular transcription factors. Virtually all transcription factors, including GAL4, c-Jun, C/EBP, CREB, Oct-1, Oct-2, Sp1, Egr-1, CTF-1, steroid and thyroid hormone receptors, and others, carry one or more highly significant charge clusters. For the most part these clusters (conserved within families of homologous proteins) are of positive net charge but contain also substantial numbers of acidic residues. Predominantly basic charge clusters are often, but not exclusively, associated with DNA-binding domains, and vice versa. Negative charge clusters of note occur only in the yeast protein PHO4 and in the proteins encoded at the Drosophila loci zeste (zeta) and knrl. This dearth of statistically significant negative charge clusters raises questions with respect to the generality of acidic activation domains. A number of sequences ...
RFC 7035 Relative Location October 2013 reference and relative locations while providing a baseline that is as accurate as possible. Both the baseline and the reference location are defined as either a geodetic location [OGC.GeoShape] or a civic address [RFC4776]. If the baseline location was expressed as a geodetic location, the reference MUST be geodetic. If the baseline location was expressed as a civic address, the reference MUST be civic. Baseline and reference locations MAY also include dynamic location information [RFC5962]. The relative location can be expressed using a point (2- or 3-dimensional) or a shape that includes uncertainty: circle, sphere, ellipse, ellipsoid, polygon, prism, or arc-band. Descriptions of these shapes can be found in [RFC5491]. Optionally, a reference to a map document can be provided. The reference is a URI [RFC3986]. The document could be an image or dataset that represents a map, floor plan, or other form. The type of document the URI points to is described ...
The M2 protein of the influenza A virus is a homotetrameric transmembrane proton channel implicated in several stages of the viral replication process. Each of its 97-residue monomers is known to include a transmembrane α-helix. but the structures of the N- and C-terminal domains have not yet been solved. A significant barrier to an atomic level understanding of the M2 protein is the difficulty associated with expression and purification of the full-length protein, which has primarily been studied in the form of truncated constructs covering the amphipathic helix and a short C-terminal segment. This C-terminal segment, which includes residues 46-62, has been shown for a truncated version of the protein to consist of an amphipathic helix lying on the membrane surface. Here, we present SDSL-EPR structural studies using full-length M2 constructs to examine sites 50-54 in the proposed amphipathic helix region of M2. Using power saturation data for the protein reconstituted into vesicles and CW ...
Аннотация доклада: A new algorithm Zebra and a corresponding web-server have been developed to systematically study diverse protein superfamilies and identify the subfamily-specific positions (SSPs) - conserved only within functional subfamilies but different between them - that seem to be responsible for different substrate specificity, catalytic activity, stability, etc. [1]. It is known from experimental enzymology that mutations in the active site can change enantioselectivity, substrate specificity and catalytic promiscuity more effectively than distant ones. However, both close and distant mutations can be important for activity and stability thus highlighting complexity of evolutionary adaptation. Therefore, to identify functionally important SSPs a novel scoring function is suggested that incorporates structural information as well as physicochemical and residue conservation in protein subfamilies. The algorithm does not require pre-defined subfamilies and can propose ...
Human cellular nucleic acid binding protein (CNBP) is a zinc finger DNA binding protein of unknown function. The human CNBP cDNA was used as a probe to isolate four structurally distinct but highly homologous mouse liver cDNA clones. Each of the mouse clones exhibited extraordinary sequence conservation with human CNBP cDNA, and the predicted mouse amino acid sequence identities with human CNBP protein ranged from 99 to 100%. Genetic mapping of CNBP genes in interspecific and intersubspecific mouse backcrosses revealed two loci that hybridize to CNBP cDNA at high stringency, located on chromosomes 5 and 6. The subcellular distribution of the CNBP protein was characterized with a specific polyclonal antibody generated against a synthetic peptide from the carboxyl terminus. CNBP was found in the cytosol and the endoplasmic reticulum in subcellular fractions from mouse liver, but was undetectable in nuclear fractions. These data suggest that CNBP is a member of a highly conserved family of
The majority of mammalian genes produce multiple transcripts resulting from alternative splicing (AS) and/or alternative transcription initiation (ATI) and alternative transcription termination (ATT). Comparative analysis of the number of alternative nucleotides, isoforms, and introns per locus in genes with different types of alternative events suggests that ATI and ATT contribute to the diversity of human and mouse transcriptome even more than AS. There is a strong negative correlation between AS and ATI in 5′ untranslated regions (UTRs) and AS in coding sequences (CDSs) but an even stronger positive correlation between AS in CDSs and ATT in 3′ UTRs. These observations could reflect preferential regulation of distinct, large groups of genes by different mechanisms: 1) regulation at the level of transcription initiation and initiation of translation resulting from ATI and AS in 5′ UTRs and 2) posttranslational regulation by different protein isoforms. The tight linkage between AS in CDSs ...
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Affects function: The variants effect on the proteins function, in the format Reported/Curator concluded; + indicating the variant affects function, +? probably affects function, +* affects function, not associated with individuals disease phenotype, # affects function, not associated with any known disease phenotype, - does not affect function, -? probably does not affect function, ? effect unknown, . effect not classified ...
Affects function: The variants effect on the proteins function, in the format Reported/Curator concluded; + indicating the variant affects function, +? probably affects function, +* affects function, not associated with individuals disease phenotype, # affects function, not associated with any known disease phenotype, - does not affect function, -? probably does not affect function, ? effect unknown, . effect not classified ...
Genetic susceptibility to autoimmunity is frequently associated with specific MHC alleles. Diabetogenic MHC class II molecules, such as human HLA-DQ8 and mouse I-Ag7, typically have a small, uncharged amino acid residue at position 57 of their β chain (β57); this results in the absence of a salt bridge between β57 and Argα76, which is adjacent to the P9 pocket of the peptide-binding groove. However, the influence of Argα76 on the selection of the TCR repertoire remains unknown, particularly when the MHC molecule binds a peptide with a neutral amino acid residue at position P9. Here, we have shown that diabetogenic MHC class II molecules bound to a peptide with a neutral P9 residue primarily selected and expanded cells expressing TCRs bearing a negatively charged residue in the first segment of their complementarity determining region 3β. The crystal structure of one such TCR in complex with I-Ag7 bound to a peptide containing a neutral P9 residue revealed that a network of favorable ...
Many receptors involved in clathrin-mediated protein transport through the endocytic and secretory pathways of yeast and animal cells share common features. They are all type I integral membrane proteins containing cysteine-rich lumenal domains and cytoplasmic tails with tyrosine-containing sorting signals. The cysteine-rich domains are thought to be involved in ligand binding, whereas the cytoplasmic tyrosine motifs interact with clathrin-associated adaptor proteins during protein sorting along these pathways. In addition, tyrosine-containing signals are required for the retention and recycling of some of these membrane proteins to the trans-Golgi network. Here we report the characterization of an approximately 80-kD epidermal growth factor receptor-like type I integral membrane protein containing all of these functional motifs from Arabidopsis thaliana (called AtELP for A. thaliana Epidermal growth factor receptor-Like Protein). Biochemical analysis indicates that AtELP is a membrane protein ...
TY - JOUR. T1 - Potential bias of fungal 18S rDNA and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil. AU - Anderson, Ian C. AU - Campbell, C. D.. AU - Prosser, James Ivor. PY - 2003. Y1 - 2003. N2 - Four fungal 18S rDNA and internal transcribed spacer (ITS) polymerase chain reaction (PCR) primer pairs were tested for their specificity towards target fungal DNA in soil DNA extracts, and their ability to assess the diversity of fungal communities in a natural grassland soil was compared. Amplified PCR products were cloned, and approximate to 50 clones from each library were sequenced. Phylogenetic analysis and database searches indicated that each of the sequenced cloned DNA fragments was of fungal origin for each primer pair, with the exception of the sequences generated using the 18S rDNA primers nu-SSU-0817 and nu-SSU-1196, where 35 of the 50 sequenced clones represented soil invertebrates. Although some of the primers have previously ...
By directed mutagenesis, we constructed a set of seven TEM-1 derivatives containing single replacements in each one of the amino acids substituted in naturally occurring extended-spectrum TEM beta-lactamases. The exact contribution of each mutation to the resistance phenotype was determined. In addition, mutant enzyme production and stabilities were studied. Five of seven mutations determined to some extent variations in cephalosporin and/or monobactam activity. Dramatic changes in the hydrolysis of ceftazidime and aztreonam occurred when a serine was at position 164. Changes at positions 104, 238, and 240 showed more leaky variation in activity towards cephalosporins and aztreonam. Replacements at positions 237 and 265 caused no variation in susceptibility to cephalosporins. Interestingly, the change from Gln to Lys at position 39 found in TEM-2, classically considered a neutral change, slightly but consistently increased the MIC of ceftazidime and aztreonam. The in vitro construction of ...
Three members of Brassica napus TRANSPARENT TESTA 2 (BnTT2) gene family encoding potential R2R3-MYB regulatory proteins of proanthocyanidin biosynthesis were isolated. BnTT2-1, BnTT2-2, and BnTT2-3 are 1102 bp with two introns, and have a 938-bp full-length cDNA with a 260 amino acid open reading frame. They share 98.2-99.3% nucleotide and 96.5-98.5% amino acid identities to each other, and are orthologous to Arabidopsis thaliana TT2 (AtTT2) with 74.1-74.8% nucleotide and 71.1-71.8% amino acid identities. An mRNA type of BnTT2-2 was found to contain unspliced intron 2 and encode a premature protein. They all have an alternative polyadenylation site. BnTT2-1 and BnTT2-3 also have an alternative transcription initiation site. Aligned with AtTT2, their 5 untranslated regions (UTRs) are astonishingly conserved, and two conserved regions were also found in their 3 UTRs. Oligonucleotide deletion leads to double-start codons of them. Resembling AtTT2, BnTT2 proteins are nuclear-located R2R3-MYB ...
The 54-kDa subunit of the mammalian signal recognition particle (SRP54) binds to the signal sequences of nascent secretory and transmembrane proteins and facilitates their cotranslational targeting to the membrane translocation apparatus in the endoplasmic reticulum (ER). A 48-kDa Escherichia coli protein that shares extensive sequence similarity with SRP54 was identified in homology searches. Recent genetic experiments by Phillips and Silhavy [Phillips, G. J. & Silhavy, T. J. (1992) Nature (London) 359, 744-746] have shown that depletion of this protein, designated Ffh (fifty-four homolog), leads to a significant secretory defect in vivo. We demonstrate here that Ffh is structurally and functionally related to SRP54 by virtue of its ability to mimic closely its mammalian counterpart in several established biochemical assays, thereby suggesting that it plays a direct role in protein export. Ffh assembled efficiently with mammalian SRP components into a chimeric ribonucleoprotein [SRP(Ffh)] and ...
TY - JOUR. T1 - Characterization of a family of related cellular transcription factors which can modulate human immunodeficiency virus type 1 transcription in vitro. AU - Yoon, Jong-Bok. AU - Li, Gen. AU - Roeder, Robert G.. PY - 1994/1/1. Y1 - 1994/1/1. N2 - LBP-1 is a cellular protein which binds strongly to sequences around the human immunodeficiency virus type 1 (HIV-1) initiation site and weakly over the TATA box. We have previously shown that LBP-1 represses HIV-1 transcription by inhibiting the binding of TFIID to the TATA box. Four similar but distinct cDNAs encoding LBP-1 (LBP-1a, -b, -c, and -d) have been isolated. These are products of two related genes, and each gene encodes two alternatively spliced products. Comparison of the amino acid sequence of LBP- 1 with entries in the available protein data bases revealed the identity of LBP-1c to α-CP2, an α-globin transcription factor. These proteins are also homologous to Drosophila melanogaster Elf-1/NTF-1, an essential transcriptional ...
Inverse polymerase chain reaction (Inverse PCR) is a variant of the polymerase chain reaction that is used to amplify DNA with only one known sequence. One limitation of conventional PCR is that it requires primers complementary to both termini of the target DNA, but this method allows PCR to be carried out even if only one sequence is available from which primers may be designed. Inverse PCR is especially useful for the determination of insert locations. For example, various retroviruses and transposons randomly integrate into genomic DNA. To identify the sites where they have entered, the known, internal viral or transposon sequences can be used to design primers that will amplify a small portion of the flanking, external genomic DNA. The amplified product can then be sequenced and compared with DNA databases to locate the sequence which has been disrupted. The inverse PCR method involves a series of restriction digests and ligation, resulting in a looped fragment that can be primed for ...
Chang, E., et al. N-Terminal Amino Acid Sequence Determination of Proteins by N-Terminal Dimethyl Labeling: Pitfalls and Advantages When Compared with Edman Degradation Sequence Analysis. Journal of Biomolecular Technology. 27(2). 07/03/2016.. ...
Proteins (/ˈproʊˌtiːnz/ or /ˈproʊti.ɪnz/) are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific three-dimensional structure that determines its activity.. A linear chain of amino acid residues is called a polypeptide. A protein contains at least one long polypeptide. Short polypeptides, containing less than 20-30 residues, are rarely considered to be proteins and are commonly called peptides, or sometimes oligopeptides. The individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino ...
The invention provides a method for determining an amino acid sequence motif for a phosphorylation site of a protein kinase. In the method of the invention, a protein kinase is contacted with an oriented degenerate peptide library, peptides within the library which are substrates for the kinase are converted to phosphopeptides and the phosphopeptides are separated from non-phosphorylated peptides. The isolated phosphopeptides are sequenced and an amino acid sequence motif for the phosphorylation site is determined based upon the relative abundance of different amino acids residues at each degenerate position. The invention also provides peptide substrates for protein kinase A, cell cycle control kinases, src family kinases, the EGF receptor and p92.sup.c-fps/fes based upon amino acid sequence motifs for the phosphorylation sites of these kinases.
Purified glucocorticoid receptorhormone complex from rat liver cytosol binds specifically to cloned mouse mammary tumor virus long terminal repeats in vitro. Proc Natl Acad Sci USA 1982; 79:5157-61. Moore DD, Marks AR, Buckley DI, Kapler G, Payvar F, Goodman HM. The first intron of the human growth hormone gene contains a binding site for glucocorticoid receptor. Proc Natl Acad Sci USA 1985; 82:699-702. Bechet D. Control of gene expression by steroid hormones. Reprod Nutr Develop 1986; 26:1025-55. Endocrinology 1981; 108:1533-7. King WJ, DeSombre ER, Jensen EV, Greene GL. Comparison of immunocytochemical and steroid-bind:ing assays for estrogen receptor in human breast tumors. Cancer Res 1985; 45:293-304. Pertschuk LP, Eisenberg KB, Carter AC, Fcldman JG. Immunohistologic localization of estrogen receptors in breast cancer with monoclonal antibodies. Cancer 1985; 55:1513-8. Perrot-Applanat M, Groyer-Picard MT, Lorenzo F, et al. Immunocytochemical study with monoclonal antibodies to progesterone ...
A cDNA library prepared from the mouse osteoblastic cell line MC3T3-E1 was screened for the presence of specifically expressed genes by employing a combined subtraction hybridization/differential screening approach. A cDNA was identified and sequenced which encodes a protein designated osteoblast-specific factor 2 (OSF-2) comprising 811 amino acids. OSF-2 has a typical signal sequence, followed by a cysteine-rich domain, a fourfold repeated domain and a C-terminal domain. The protein lacks a typical transmembrane region. The fourfold repeated domain of OSF-2 shows homology with the insect protein fasciclin I. RNA analyses revealed that OSF-2 is expressed in bone and to a lesser extent in lung, but not in other tissues. Mouse OSF-2 cDNA was subsequently used as a probe to clone the human counterpart. Mouse and human OSF-2 show a high amino acid sequence conservation except for the signal sequence and two regions in the C-terminal domain in which in-frame insertions or deletions are observed, ...
Preparation of LPA Receptor cDNA Plasmids and Expression. The entire coding regions of LPA1 (1,095 bp, GenBank accession number Y09479), LPA2 (1,149 bp, GenBank accession number AF011466), and LPA3 (1,148 bp, GenBank accession number AF127138) were amplified from human cDNA library by RT-PCR. The respective amplified fragment was subcloned into the EcoRI site of pEFneo eukaryotic expression vector (Kon et al., 1999; Sato et al., 2000), and each DNA sequence was confirmed. The primers used for the RT-PCR were as follows. The 5′primers contained an EcoRI site, a Kozak sequence (CCACC), and the N-terminal region of the respective receptor. The 3′-primers contained an EcoRI site and a stop codon in addition to the C-terminal region of the respective receptor. CHO cells or RH7777 cells were transfected with pEFneo empty vector alone or the pEFneo vector containing human LPA1, human LPA2, or human LPA3 by electroporation, and the neomycin-resistant cells (G418 sulfate at 1 mg/ml for CHO cells and ...
The complete nucleotide sequence of an isolate of puma lentivirus (PLV-14) was obtained by an inverse polymerase chain reaction (I-PCR) technique and confirmed by conventional PCR. Both methods were used to amplify overlapping regions of proviral DNA, for cloning and sequencing, from genomic DNA isolated from PLV-14 infected Florida puma (Felis concolor coryi) peripheral blood mononuclear cells (PBMC). The provirus has a total length of 9100 nucleotides and the genomic organization of presumed protein coding regions are similar to those seen in other members of the lentivirus family, i.e., three large open reading frames gag, pol, and env as well as smaller intergenic regions that apparently encode regulatory proteins vif and 3′ rev by positional and sequence similarity to those seen in other lentiviruses. Two additional open reading frames were identified in the env region and their function (if any) is unknown. The length of the PLY-14 long terminal repeat (LTR) was found to be shorter than the LTRs
37 CFR 1.822(c)(5) provides that nucleotide sequences shall only be represented by a single strand, in the 5′ to 3′ direction, from left to right. That is, double stranded nucleotides shall not be represented in the sequence listing. A double stranded nucleotide may be represented as two single stranded nucleotides, and any relationship between the two may be shown in the drawings. The procedures for presenting and numbering amino acid sequences are set forth in 37 CFR 1.822(d). Two alternatives are presented for numbering amino acid sequences. Amino acid sequences may be numbered with respect to the identification of the first amino acid of the first mature protein or with respect to the first amino acid appearing at the amino terminal. The numbering procedure for nucleotides is set forth in 37 CFR 1.822(c)(6). Sequences that are circular in configuration are intended to be encompassed by these rules, and the numbering procedures described above remain applicable with the exception that the ...
CombAlign is a new Python code that generates a gapped, multiple structure-based sequence alignment (MSSA) given a set of pairwise structure-based sequence alignments. CombAlign has utility in assisting the user in distinguishing structurally conserved versus divergent regions on a reference protein structure relative to other closely related structures. The method for combining multiple pairwise alignments is straightforward, involving the recording of pre-computed residue-residue correspondences between positions on the reference protein and each compared structure, and insertion of non-redundant gaps, as needed, to reflect amino-acid deletions or structural divergence in the reference relative to one or more compared structures.. CombAlign is not intended for use in applications for which greater benefit would be provided using a multiple structure alignment as generated by the vast majority of open-source programs [20], nor does it propose to address matters of protein evolution or function ...
en] AmiD is the fifth identified N-acetylmuramoyl-l-alanine zinc amidase of Escherichia coli. This periplasmic lipoprotein is anchored in the outer membrane and has a broad specificity. AmiD is capable of cleaving the intact peptidoglycan (PG) as well as soluble fragments containing N-acetylmuramic acid regardless of the presence of an anhydro form or not, unlike the four other amidases, AmiA, AmiB, AmiC, and AmpD, which have some specificity. AmiD function is, however, not clearly established but it could be part of the enzymatic machinery involved in the PG turnover in E. coli. We solved three structures of the E. coli zinc amidase AmiD devoid of its lipidic anchorage: the holoenzyme, the apoenzyme in complex with the substrate anhydro-N-acetylmuramic-acid-l-Ala-gamma-d-Glu-l-Lys, and the holoenzyme in complex with the l-Ala-gamma-d-Glu-l-Lys peptide, the product of the hydrolysis of this substrate by AmiD. The AmiD structure shows a relatively flexible N-terminal extension that allows an easy ...
The serine-proteinase domain is responsible for the proteolytic events that occur during complement activation. The sequences of nine serine proteinases of known crystal structure were compared with the serine-proteinase sequences in the six complement proteins C1r, C1s, C2, factor B, factor I and factor D to assess the degree of structural homology of the latter with the crystal structures. All sequence insertions and deletions were readily located at the protein surface. The internal location of disulphide bridges and the surface location of putative glycosylation sites are compatible with this structure. Secondary-structure predictions for the sequences were fully consistent with the crystal structures. It is concluded that the double subdomain beta-sheet motif is retained in the complement sequences, but that localized differences are observed for factor I, C2 and factor B. ...
Specific cis-acting sequences within the carlavirus potato virus S (PVS) genomic RNA molecule appear to control gene expression at the translational level. Two sequences have been investigated, the untranslated sequence upstream from the initiation codon of the viral coat protein gene, designated VTE and the 5 untranslated leader sequence from the genomic RNA molecule (PVS 5). In vitro and in vivo, either of these sequences enhance the translation of a downstream open reading frame when provided as the untranslated leader in a transcript molecule. Translational enhancement was also detected at the transgenic plant level. Both PVS sequences were deleted in an attempt to identify the core regulatory element responsible for this translational enhancement phenomenon. Results indicate that in vitro and in vivo, the functional motif is contained within the 5 promixal portion of both sequences. When the sequences of these important regions were compared, a homologous block of nucleotides was ...
The role of the cytoplasmic domain of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins in virus replication was investigated. Deletion of residues 840 to 856 at the carboxyl terminus of gp41 reduced the efficiency of virus entry during an early step in the virus life cycle between CD4 binding and formation of the DNA provirus without affecting envelope glycoprotein synthesis, processing, or syncytium-forming ability. Deletion of residues amino terminal to residue 846 was associated with decreased stability of envelope glycoproteins made in COS-1 cells, but this phenotype was cell type dependent. The cytoplasmic domain of gp41 was not required for the incorporation of the HIV-1 envelope glycoproteins into virions. These results suggest that the carboxyl terminus of the gp41 cytoplasmic domain plays a role in HIV-1 entry other than receptor binding or membrane fusion. The cytoplasmic domain of gp41 also affects the stability of the envelope glycoprotein in some cell types. ...
The fine specificity of T cell recognition of peptide analogues of the influenza nucleoprotein epitope, NP 383-391 SRYWAIRTR, was studied using HLA B27-restricted influenza-specific cytotoxic T cell (CTL) clones, of defined T cell receptor (TcR) usage, derived from unrelated individuals following natural infection. Even conservative amino acid substitutions of the peptide residues P4, P7 and P8 influenced CTL recognition. These side chains are probably directly contacted by the TcR. CTL clones which used the TcR V alpha 14 gene segment (but not those using TcR V alpha 12) were also sensitive to P1 substitutions, suggesting that the TcR alpha chain of these clones lies over the N terminus of bound peptide, and that the footprint of certain TcR can span all exposed residues of a peptide bound to a major histocompatibility complex class I molecule. These results, taken together with previous structural and functional data, suggest that, for nonamer peptides bound to HLA B27, P1, P4 and P8 are flag
We have taken a comprehensive approach to the generation of novel DNA binding zinc finger domains of defined specificity. Herein we describe the generation and characterization of a family of zinc finger domains developed for the recognition of each of the 16 possible 3-bp DNA binding sites having the sequence 5-GNN-3. Phage display libraries of zinc finger proteins were created and selected under conditions that favor enrichment of sequence-specific proteins. Zinc finger domains recognizing a number of sequences required refinement by site-directed mutagenesis that was guided by both phage selection data and structural information. In many cases, residues not expected to make base-specific contacts had effects on specificity. A number of these domains demonstrate exquisite specificity and discriminate between sequences that differ by a single base with >100-fold loss in affinity. We conclude that the three helical positions -1, 3, and 6 of a zinc finger domain are insufficient to allow for ...
cDNA clones coding for the β subunit of rat brain type II Ca2+/calmodulin-dependent protein kinase were isolated and sequenced. The clones, including one containing the entire coding region, hybridize at high stringency to a single band of poly(A)+ RNA of length 4.8 kilobases. The subunit coded for by the clones was identified by in vitro transcription of the cDNA followed by translation of the resulting RNA. The DNA sequence of the clones contains a single long open reading frame (1626 nucleotides) coding for a protein of 542 amino acids with a molecular weight of 60,333, the amino-terminal half of which is homologous to several other protein kinases. Potential ATP- and calmodulin-binding domains were identified. Two independent clones contain an identical 45-nucleotide deletion, relative to the clones described above, resulting in a shorter open reading frame coding for a protein of molecular weight 58,000. This suggests that the minor, 58-kDa β subunit of the type II ...
Key publications. Semenza, G.L, Nejfelt, M.K., Chi, S.M. & Antonarakis, S.E. (1991). Hypoxia-inducible nuclear factors bind to an enhancer element located 3 to the human erythropoietin gene. Proc Natl Acad Sci USA, 88, 5680-5684. Wang, G.L., Jiang, B.-H., Rue, E.A. & Semenza, G.L. (1995). Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA, 92, 5510-5514. Maxwell, P.H., Wiesener, M.S., Chang, G.-W., Clifford, S.C., Vaux, E.C., Cockman, M.E., Wykoff, C.C., Pugh, C.W., Maher, E.R. & Ratcliffe, P.J. (1999). The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature, 399, 271-275. Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J.M., Lane, W.S. & Kaelin Jr., W.G. (2001) HIFa targeted for VHL-mediated destruction by proline hydroxylation: Implications for O2 sensing. Science, 292, 464-468. Jaakkola, P., Mole, D.R., Tian, Y.-M., Wilson, M.I., ...
CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which ...
CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which ...
positive regulation of transcription from RNA polymerase II promoter involved in norepinephrine biosynthetic process - Ontology Report - Rat Genome Database
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Glutathione transferase genes (GST) are candidate genes for Parkinsons disease because they are involved with the metabolism of pesticides, dopamine, and glutathione. Recent reports have suggested an association between Parkinsons disease and polymorphisms of GSTP1 1 orGSTM1 andGSTT1.2 Recently we discovered a new polymorphic site in the zeta class G→T (GSTZ1) gene.3 This consists of a C6T transition at nucleotide 245 in exon 5 that results in an amino acid change at position 82 from methionine to threonine. The T substitution occurs in 14% of white people. We have previously reported two other polymorphic sites at nucleotides 94 and 124 in exon 3.4 There are now thought to be four alleles of GSTZ1:Z1*A(A94A124C245),Z1*B(A94G124C245),Z1*C(G94G124C245,) andZ1*D(G94G124T245). Here we investigated the association of Parkinsons disease, pesticide exposure, and theseGSTZ1 polymorphisms.. DNA was extracted from blood samples collected from patients with Parkinsons disease and matched controls as ...
Human immunodeficiency virus type 1 (HIV-1) accessory genes including nef, vif, and vpr are important factors that determine the replication and pathogenesis of HIV-1. The state of activation is also important for the replication of HIV-1. We evaluated the properties of nef-, vif-, and vpr-minus macrophage-tropic HIV-1(JR) CSF in primary CD4+ Th1- or Th2-like cell cultures which had been activated through CD3 molecules in the presence of interleukin-2 (IL-2) and IL-12 (Th1-like culture) or IL-4 (Th2-like culture), respectively. In activated Th1- or Th2-like cultures, replication of nef-minus HIV-1(JR-CSF) was markedly lower than that of wild-type HIV-1. Subsequent analysis by site-directed mutagenesis showed that (i) the presence of an acidic amino acid-rich domain (amino acid residues 72 to 75) in the Nef protein was critical for the enhancement of viral DNA synthesis, resulting in increased virus growth rate, and (ii) prolines that form part of Src homology 3 binding domain were not essential ...
Search PubMed for more publications by Chris Todd Hittinger. RESEARCH PUBLICATIONS (& equal contributions, @corresponding author). Libkind D &, Hittinger CT &, Valério E, Gonçalves C, Dover J, Johnston M, Gonçalves P, Sampaio JP. Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast. Proc Natl Acad Sci USA 108: 14539-44.. Scannell DR&, Zill [email protected], Rokas A, Payen C, Dunham MJ, Eisen MB, Rine J, Johnston M, Hittinger [email protected] 2011. The awesome power of yeast evolutionary genetics: New genome sequences and strain resources for the Saccharomyces sensu stricto genus. G3 Genes Genomes Genet 1: 11-25.. Hittinger CT, Gonçalves P, Sampaio JP, Dover J, Johnston M, Rokas A. 2010. Remarkably ancient balanced polymorphisms in a multi-locus gene network. Nature 464: 54-8.. Hittinger CT, Johnston M, Tossberg JT, Rokas A. 2010. Leveraging skewed transcript abundance by RNA-Seq to increase the genomic depth of the tree of life. Proc Natl Acad Sci USA 107: 1476-81.. Gibbons ...
RNA degradation is important for the regulation of gene expression. Despite the identification of proteins and sequences related to deadenylation-dependent RNA degradation in plants, endonucleolytic cleavage-dependent RNA degradation has not been studied in detail. Here, we developed truncated RNA end sequencing in Arabidopsis thaliana to identify cleavage sites and evaluate the efficiency of cleavage at each site. Although several features are related to RNA cleavage efficiency, the effect of each feature on cleavage efficiency has not been evaluated by considering multiple putative determinants in A. thaliana. Cleavage site information was acquired from a previous study, and cleavage efficiency at the site level (CSsite value), which indicates the number of reads at each cleavage site normalized to RNA abundance, was calculated. To identify features related to cleavage efficiency at the site level, multiple putative determinants (features) were used to perform feature selection using the Least
Analysis of the heteromeric DNA binding protein GABP has revealed the interaction of two distinct peptide sequence motifs normally associated with proteins located in different cellular compartments. The alpha subunit of GABP contains an 85-amino acid segment related to the Ets family of DNA binding proteins. The ETS domain of GABP alpha facilitates weak binding to DNA and, together with an adjacent segment of 37 amino acids, mediates stable interaction with GABP beta. The beta subunit of GABP contains four imperfect repeats of a sequence present in several transmembrane proteins including the product of the Notch gene of Drosophila melanogaster. These amino-terminal repeats of GABP beta mediate stable interaction with GABP alpha and, when complexed with GABP alpha, directly contact DNA. These observations provide evidence for a distinct biochemical role for the 33-amino acid repeats, and suggest that they may serve as a module for the generation of specific dimerization interfaces. ...
TY - JOUR. T1 - The study of biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli. I. Introduction of mutations into alkaline phosphatase gene. AU - Karamyshev, A. L.. AU - Shlyapnikov, M. G.. AU - Khmelnitsky, M. I.. AU - Nesmeyanova, M. A.. AU - Ksenzenko, V. N.. PY - 1994. Y1 - 1994. N2 - Various mutations in E. coli alkaline phosphatase gene were obtained by oligonucleotide-directed mutagenesis. They result in amino acid substitutions in the signal peptide cleavage site [Val for Ala(-1)] and in the N terminus of mature polypeptide chain: Ala for Arg(+1) and Gln for Glu(+4); Gln for Glu(+4). Enzyme activity was observed in all E. coli strains transformed by plasmids with cloned mutant genes. In addition, an amber mutation was introduced into the Arg(+1) position, and the synthesis of mutant alkaline phosphatase was shown in E. coli strains containing suppressor tRNAs specific for Ser, Gln, Tyr, Leu, Ala, Glu, Phe, Gly, His, Pro, and Cys.. AB - Various ...
Secondary structure prediction is a set of techniques in bioinformatics that aim to predict the local secondary structures of proteins based only on knowledge of their amino acid sequence. The secondary structure of proteins is determined by the pattern of hydrogen bonding. A large number of servers and tools are used to predict the secondary structure analysis.. Protein secondary structure refers to the local conformation proteins polypeptide backbone. There are two regular secondary structure states, α-helix (H) and β-strand (E), and one irregular secondary structure type, the coil region (C). Sander developed a secondary structure assignment method Dictionary of Secondary Structure of Proteins (DSSP)3, which automatically assigns secondary structure into eight states (H, E, B, T, S, L, G, and I) according to hydrogen-bonding patterns. These eight states are often further simplified into three states of helix, sheet and coil. The most widely used convention is that helix is designated as G, ...
p>The checksum is a form of redundancy check that is calculated from the sequence. It is useful for tracking sequence updates.,/p> ,p>It should be noted that while, in theory, two different sequences could have the same checksum value, the likelihood that this would happen is extremely low.,/p> ,p>However UniProtKB may contain entries with identical sequences in case of multiple genes (paralogs).,/p> ,p>The checksum is computed as the sequence 64-bit Cyclic Redundancy Check value (CRC64) using the generator polynomial: x,sup>64,/sup> + x,sup>4,/sup> + x,sup>3,/sup> + x + 1. The algorithm is described in the ISO 3309 standard. ,/p> ,p class=publication>Press W.H., Flannery B.P., Teukolsky S.A. and Vetterling W.T.,br /> ,strong>Cyclic redundancy and other checksums,/strong>,br /> ,a href=http://www.nrbook.com/b/bookcpdf.php>Numerical recipes in C 2nd ed., pp896-902, Cambridge University Press (1993),/a>),/p> Checksum:i ...
TY - JOUR. T1 - Genes within genes. T2 - independent expression of phage T4 intron open reading frames and the genes in which they reside.. AU - Gott, J. M.. AU - Zeeh, A.. AU - Bell-Pedersen, D.. AU - Ehrenman, Karen. AU - Belfort, M.. AU - Shub, D. A.. PY - 1988/1/1. Y1 - 1988/1/1. N2 - The td, nrdB, and sunY introns of bacteriophage T4 each contain a long open reading frame (ORF). These ORFs are preceded by functional T4 late promoters and, in the case of the nrdB intron ORF, a functional middle promoter. Expression of phage-encoded intron ORF-lacZ fusions indicates that these T4 genes are highly regulated. The lack of translation of these ORFs from early pre-mRNAs can be accounted for by the presence of secondary structures that are absent from the late RNAs. Because translation of the intron ORFs could disrupt core structural elements required for pre-mRNA splicing, such regulation may be necessary to allow expression of the genes in which they reside.. AB - The td, nrdB, and sunY introns ...
Accurate sequence alignments of distantly related proteins are crucial for the better understanding of proteins at their family/superfamily level. However, such alignments of distantly related proteins are often hard to obtain by automatic multiple sequence alignment programs. Hence, we suggest a protocol that permits the reliable sequence alignment of distantly related proteins whose structural information is available. This protocol employs two stages of structure-based sequence alignments in order to obtain reliable alignments. The method proposed is clearly suited to work for protein structural members with distant relationships. We further propose a novel assessment of the derived alignments using the measurements of the structural variations and the percentage secondary structural equivalences. This structure-based sequence alignment protocol can be employed for a single superfamily or for a large number of structural domain superfamilies in a near-automatic and rapid manner.. Development ...
BACKGROUND: There is an increasing need to develop bioinformatic tools to organise and analyse the rapidly growing amount of nucleotide and amino acid sequence data in organisms ranging from viruses to eukaryotes. FINDING: A simple sequence editor (SSE) was developed to create an integrated environment where sequences can be aligned, annotated, classified and directly analysed by a number of built-in bioinformatic programs. SSE incorporates a sequence editor for the creation of sequence alignments, a process assisted by integrated CLUSTAL/MUSCLE alignment programs and automated removal of indels. Sequences can be fully annotated and classified into groups and annotated of sequences and sequence groups and access to analytical programs that analyse diversity, recombination and RNA secondary structure. Methods for analysing sequence diversity include measures of divergence and evolutionary distances, identity plots to detect regions of nucleotide or amino acid homology, reconstruction of sequence changes,
1 Motivating the need for optimal sequence alignments Note that this actually combines two objectives of optimal sequence alignments: (i) use the score of the alignment o infer homology; (ii) use
Antiprotease targeting : altered specificity of α1-antitrypsin by amino acid replacement at the reactive centre Academic Article ...
The supernumerary subunit g is found in all mitochondrial ATP synthases. Most of the conserved amino acid residues are present in the membrane C-terminal part of the protein that contains a dimerization motif GXXXG. In yeast, alteration of this motif leads to the loss of subunit g and of supramolecular structures of the ATP synthase with concomitant appearance of anomalous mitochondrial morphologies. Disulfide bond formation involving an engineered cysteine in position 109 of subunit g and the endogenous cysteine 28 of subunit e promoted g + g, e + g, and e + e adducts, thus revealing the proximity in the mitochondrial membrane of several subunits e and g. Disulfide bond formation between two subunits g in mitochondria increased the stability of an oligomeric structure of the ATP synthase in digitonin extracts. These data suggest the participation of the dimerization motif of subunit g in the formation of supramolecular structures and is in favor of the existence of ATP synthase associations, in ...
The CLV3/ESR-RELATED (CLE) gene family encodes small secreted peptides (SSPs) and plays vital roles in plant growth and development by promoting cell-to-cell communication. The prediction and classification of CLE genes is challenging because of their low sequence similarity. We developed a machine learning-aided method for predicting CLE genes by using a CLE motif-specific residual score matrix and a novel clustering method based on the Euclidean distance of 12 amino acid residues from the CLE motif in a site-weight dependent manner. In total, 2156 CLE candidates-including 627 novel candidates-were predicted from 69 plant species. The results from our CLE motif-based clustering are consistent with previous reports using the entire pre-propeptide. Characterization of CLE candidates provided systematic statistics on protein lengths, signal peptides, relative motif positions, amino acid compositions of different parts of the CLE precursor proteins, and decisive factors of CLE prediction. The approach
Protein kinase CK2 (CK2) is a ubiquitous serine/threonine kinase with multiple cellular functions in vertebrates including apoptosis, differentiation, proliferation, survival, tumorigenesis, signal transduction, immune regulation and inflammation. In the current study, the catalytic and regulatory subunit homologs of Litopenaeus vannamei protein kinase CK2 (LvCK2α and LvCK2ß) were cloned and characterized. LvCK2α has a full-length cDNA sequence of 1764 bp with a 1053 bp open reading frame (ORF) encoding a putative protein of 351 amino acids, which contains a typical serine/threonine kinase domain. On the other hand, LvCK2ß has a 1394 bp full-length cDNA with an ORF of 663 bp encoding a protein with 221 amino acids, which contains a Casein kinase II regulatory subunit domain. Sequence and phylogenetic analysis revealed that LvCK2 was evolutionary related with the CK2 of invertebrates. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that LvCK2α and LvCK2ß transcripts were ...
TY - JOUR. T1 - Recurrent sequence exchange between homeologous grass chromosomes. AU - Wicker, Thomas. AU - Wing, Rod A.. AU - Schubert, Ingo. N1 - Publisher Copyright: © 2015 John Wiley & Sons Ltd. Copyright: Copyright 2016 Elsevier B.V., All rights reserved.. PY - 2015/11/1. Y1 - 2015/11/1. N2 - All grass species evolved from an ancestor that underwent a whole-genome duplication (WGD) approximately 70 million years ago. Interestingly, the short arms of rice chromosomes 11 and 12 (and independently their homologs in sorghum) were found to be much more similar to each other than other homeologous regions within the duplicated genome. Based on detailed analysis of rice chromosomes 11 and 12 and their homologs in seven grass species, we propose a mechanism that explains the apparently younger age of the duplication in this region of the genome, assuming a small number of reciprocal translocations at the chromosome termini. In each case the translocations were followed by unbalanced ...