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PubMed1990

Determination of the cis sequence involved in catabolite repression of the Bacillus subtilis gnt operon; implication of a consensus sequence in catabolite repression in the genus Bacillus.

The mechanism underlying catabolite repression in Bacillus species remains unsolved. The gluconate (gnt) operon of Bacillus subtilis is one of the catabolic operons which is under catabolite repression. To identify the cis sequence involved in catabolite repression of the gnt operon, we performed deletion analysis of a DNA fragment carrying the gnt promoter and the gntR gene, which had been cloned into the promoter probe vector, pWP19. Deletion of the region upstream of the gnt promoter did not affect catabolite repression. Further deletion analysis of the gnt promoter and gntR coding region was carried out after restoration of promoter activity through the insertion of internal constitutive promoters of the gnt operon before the gntR gene (P2 and P3). These deletions revealed that the cis sequence involved in catabolite repression of the gnt operon is located between nucleotide positions +137 and +148. This DNA segment contains a sequence, ATTGAAAG, which may be implicated as a consensus sequence involved in catabolite repression in the genus Bacillus.

Bacillus subtilis↗
PubMed1995

Two-dimensional 1H NMR study of a tetradecapeptide with the consensus sequence Arg5-Asp-Val-Arg-Gly9: structural effects of the outside substitution Ser12 by Ala12.

Conformation of a tetradecapeptide with a RXVRG consensus sequence, Arg5-Asp-Val-Arg-Gly9, found in several precursors of antibacterian peptides, was investigated in dimethylsulfoxide solution by proton NMR spectroscopy. Complete resonance assignments and conformational parameters were obtained through correlated (COSY) and nuclear Overhauser (NOESY) techniques. The 3J(alpha H, beta H) coupling constants and the intramolecular NOE, NH...beta H, were used to analyse the conformers around the C alpha-C beta bond and, in four cases, to obtain stereospecific assignments. Use of restraints derived from NOE connectivities and 3J(NH, alpha H) coupling constants allows the determination of a range of phi and psi dihedral angles for all the residues in the sequence. The present NMR results provide favourable evidence for the formation of two bends in the consensus sequence of the tetradecapeptide. The first one has most of the features of a Glu4-Val7 beta-turn (low temperature coefficient of the Val7NH chemical shift, Arg5 alpha H...Val7NH and Asp6NH...Val7NH NOE correlations). The second one exhibits only the Asp6 alpha H...Arg7NH and Val7NH...Arg8NH NOE interactions. These consensus sequence organizations proposed were confirmed by molecular modeling based on low potential energy structure on the [4-9] fragment with high agreement of NOE data. Overall, the substitution of Ser12 by Ala12 shifts the conformation of the hydrophobic moiety [10-14] towards a quite random coil structure in this fragment and strongly destabilizes the folded structures of the consensus domain where only one NH (Val7) is solvent-shielded opposed to three (Asp6 to Arg8) in the [Ser12] tetradecapeptide. These conformational changes could be related to the processing enzyme activities on these model oligopeptides.

Alanine↗
PubMed1990

Transcription in vivo directed by consensus sequences of E.coli promoters: their context heavily affects efficiencies and start sites.

We studied in vivo transcription and gene expression directed by a series of synthetic sequences, bearing the consensus hexamer (CH) pair of E.coli promoters in various contexts. The results demonstrate that, for the contexts tested, the CH pair supports transcription activity and gene expression, whether the spacer linking them is AT or GC rich, or is as short as 14 bp or as large as 26 bp (standard size 17 bp). However, we find that the context influences transcription efficiency by as much as an order of magnitude, and is able to scatter transcription start sites over a region of as much as 30 bp, including start sites within a CH or even between the two sequences of the CH pair. The results demonstrate that, although the CH pair can be sufficient for directing transcription by E.coli RNAP, important determinants for promoter activity are at least in part contained in the context of the consensus sequences; they advocate a synergic interplay of signals borne by the CH pair and its context, extending over all parts of the promoter sequence. A two-step model is proposed, in which properly located consensus sequences provide RNAP with facilities required for stereospecific docking along the promoter sequence; the result would be a sharp change in the local environment of the double helix inducing local isothermal unwinding. The size of the loop (related to the AT constraint in the promoter) and the extend of the environmental change required for unwinding would determine the rate of transcriptionally competent complex formation, positioning and grouping of start sites.

Base Sequence↗
PubMed2003

Twelve new MotA-dependent middle promoters of bacteriophage T4: consensus sequence revised.

Bacteriophage T4 middle-mode transcription requires Escherichia coli RNA polymerase, phage-encoded transcriptional activator MotA and co-activator AsiA that form a complex at a middle promoter DNA. T4 middle promoters have been defined by a consensus sequence deduced from the list of 14 middle promoters identified in earlier studies. To date, 33 middle promoters have been mapped on the T4 genome. Of these, 12 contain differences even at the highly conserved positions of the consensus sequence. In the T4 prereplicative gene cluster between genes e and rpbA, we have identified 12 new middle promoters, most of which contain differences from the consensus sequence deduced previously. Analysis of base conservation in the different sequence positions of new middle promoters, as well as those identified previously, revealed some new features of middle T4 promoters. We propose to define these promoters by a MotA box (a/t)(a/t)(a/t)TGCTTtA centred at the position -30, the sequence TAtaAT centred at -10 relative to the transcriptional start site, and the spacer region of 12(+/-1) base-pairs between them.

Bacteriophage T4↗
PubMed2002

Generation of a consensus sequence from prevalent and incident HIV-1 infections in West Africa to guide AIDS vaccine development.

We considered several key issues regarding the development of a DNA-based human immunodeficiency virus type 1 (HIV-1) vaccine: (1) should the candidate vaccine construct be derived from incident or prevalent HIV-1 strains; and (2) should circulating plasma virus, archived HIV-1 provirus recovered from peripheral blood mononuclear cells, or both be included? To address these questions, we collected circulating HIV-1 strains from infected individuals residing in Abidjan, Côte d'Ivoire. From a panel of 23 strains, 22 were HIV-1 subtype A in gag, 19 of which phylogenetically clustered with the recombinant HIV-1, CRF02-AG strains from West Africa. The mosaic genome of CRF02-AG was confirmed by sequencing the protease gene. A consensus gag p24 protein sequence was generated and 147 of 148 codons were identical to CRF02-AG (IbNG). Regardless of the sequence origin (RNA, provirus, incident, or prevalent), the gag p24 consensus sequences were highly representative of these distinct virologic compartments. These data suggest that the consensus sequence generated from incident and prevalent infections may provide an appropriate sequence for a DNA vaccine and is largely representative of the major circulating viral strain.

Africa, Western↗
PubMed1986

Splice site consensus sequences are preferentially accessible to nucleases in isolated adenovirus RNA.

The conformation of RNA sequences spanning five 3' splice sites and two 5' splice sites in adenovirus mRNA was probed by partial digestion with single-strand specific nucleases. Although cleavage of nucleotides near both 3' and 5' splice sites was observed, most striking was the preferential digestion of sequences near the 3' splice site. At each 3' splice site a region of very strong cleavage is observed at low concentrations of enzyme near the splice site consensus sequence or the upstream branch point consensus sequence. Additional sites of moderately strong cutting near the branch point consensus sequence were observed in those sequences where the splice site was the preferred target. Since recognition of the 3' splice site and branch site appear to be early events in mRNA splicing these observations may indicate that the local conformation of the splice site sequences may play a direct or indirect role in enhancing the accessibility of sequences important for splicing.

Adenovirus Early Proteins↗
PubMed1987

Efficient Tn10 transposition into a DNA insertion hot spot in vivo requires the 5-methyl groups of symmetrically disposed thymines within the hot-spot consensus sequence.

Transposon Tn10 inserts preferentially at particular insertion "hot spots" that share a symmetrical 6-base-pair consensus sequence: 5' GCTNAGC 3'. The protein that recognizes this sequence is not known but is likely to be the Tn10-encoded transposase protein. We present evidence that the 5-methyl groups of the two thymines in this sequence are essential for efficient transposon insertion; in their absence the sequence is still recognized, but at lower efficiency. We have reached this conclusion by examination of a specific hot spot whose sequence is 5' GCCAGGC 3'. The innermost cytosines of this sequence happen to be substrates for methylation at their 5 positions by the bacterial dcm-encoded methylase. We find that Tn10 transposes into this site 15 times more frequently in a Dcm+ host than in a Dcm- host; in the Dcm- host, insertions still occur, but at a low frequency. Thus, at this site, the absence of pyrimidine 5-methyl groups at the third positions of the consensus sequence is sufficient to convert a strong insertion hot spot into a weaker but still recognizable hot spot. This observation supports the general proposition, suggested previously by comparisons among consensus sequences, that the presence or absence of these 5-methyl groups is one major feature that can make the difference between a strong and a weak Tn10 insertion hot spot.

Base Sequence↗
PubMed1988

A consensus sequence for cleavage by vertebrate DNA topoisomerase II.

Topoisomerase II, purified from chicken erythrocytes, was reacted with a large number of different DNA fragments and cleavages were catalogued in the presence and absence of drugs that stabilize the cleavage intermediate. Cleavages were sequenced to derive a consensus for topoisomerase II that predicts catalytic sites. The consensus is: (sequence; see text) where N is any base and cleavage occurs at the indicated mark between -1 and +1. The consensus accurately predicts topoisomerase II sites in vitro. This consensus is not closely related to the Drosophila consensus sequence, but the two enzymes show some similarities in site recognition. Topoisomerase II purified from human placenta cleaves DNA sites that are essentially identical to the chicken enzyme, suggesting that vertebrate type II enzymes share a common catalytic sequence. Both viral and tissue specific enhancers contain sites sharing strong homology to the consensus and endogenous topoisomerase II recognizes some of these sites in vivo.

Animals↗
PubMed1993

A Y-box consensus sequence is required for basal expression of the human multidrug resistance (mdr1) gene.

Basal transcription of the human multidrug resistance (mdr1) promoter was studied by chloramphenicol acetyltransferase (CAT) reporter fusion gene analysis in two parental and doxorubicin-resistant human tumor cell lines. Deletion of mdr1 DNA sequences to -89 relative to the start of transcription (at +1) had little effect on expression. Deletion of nucleotide sequences from -89 to -70, however, resulted in a 5-10-fold reduction in mdrCAT expression. DNase I footprint analysis demonstrated that the region from -85 to -70 was protected from nuclease digestion using nuclear extracts from these cell lines. The sequence between -82 and -73 is perfectly homologous with the 10-base pair Y-box consensus sequence found in the promoters of all major histocompatibility complex class-II (MHC II) genes. The Y-box sequence in MHC II genes is required for accurate and efficient transcription and contains the sequence CCAAT in the reverse orientation (Dorn, A., Durand, B., Marfing, C., Le Meur, M., Benoist, C., and Mathis, D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6249-6253). Mutations in the reverse CCAAT sequence of the Y-box consensus substantially reduced expression of an mdrCAT vector and eliminated nucleoprotein binding in an electrophoretic mobility shift assay. These results suggest that proteins which bind to the putative Y-box consensus sequence are critical for basal transcriptional regulation of the human mdr1 gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Consensus sequences for good and poor removal of uracil from double stranded DNA by uracil-DNA glycosylase.

We have purified uracil DNA-glycosylase (UDG) from calf thymus 32,000-fold and studied its biochemical properties, including sequence specificity. The enzyme is apparently closely related to human UDG, since it was recognised by a polyclonal antibody directed towards human UDG. SDS-PAGE and western analysis indicate an apparent M(r) = 27,500. Bovine UDG has a 1.7-fold preference for single stranded over double stranded DNA as a substrate. Sequence specificity for uracil removal from dsDNA was examined for bovine and Escherichia coli UDG, using DNA containing less than one dUMP residue per 100 nucleotides and synthetic oligonucleotides containing one dUMP residue. Comparative studies involving about 40 uracil sites indicated similar specificities for both UDGs. We found more than a 10-fold difference in rates of uracil removal between different sequences. 5'-G/CUT-3' and 5'-G/CUG/C-3' were consensus sequences for poor repair whereas 5'-A/TUAA/T-3' was a consensus for good repair. Sequence specificity was verified in double stranded oligonucleotides, but not in single stranded ones, suggesting that the structure of the double stranded DNA helix has influence on sequence specificity. Rate of uracil removal appeared to be slightly faster from U:A base pairs as compared to U:G mis-matches. The results indicate that sequence specific repair may be a determinant to be considered in mutagenesis.

Animals↗
PubMed1999

Comparison of ZP3 protein sequences among vertebrate species: to obtain a consensus sequence for immunocontraception.

The deduced ZP3 amino acid (aa) sequences of 13 vertebrate species namely mouse, hamster, rabbit, pig, porcine, cow, dog, cat, human, bonnet, marmoset, carp, and frog were compared using the PILEUP and PRETTY alignment programs (GCG, Wisconsin, USA). The published aa sequences obtained from 13 vertebrate species indicated the overall evolutionarily conservation in the N-terminus, central region, and C-terminus of the ZP3 polypeptide. More variations of ZP3 polypeptide sequences were seen in the alignments of carp and frog from the 11 mammalian species making the leader sequence more prominent. The canonical furin proteolytic processing signal at the C-terminus was found in all the ZP3 polypeptide sequences except of carp and frog. In the central region, the ZP3 deduced aa sequences of all the 13 vertebrate species aligned well, and six relatively conserved sequences were found. There are 11 conserved cysteine residues in the central region across all species including carp and frog, indicating that these residues have longer evolutionary history. The ZP3 aa sequence similarities were examined using the GAP program (GCG). The highest aa similarities are observed between the members of the same order within the class mammalia, and also (95.4%) between pig (ungulata) and rabbit (lagomorpha). The deduced ZP3 aa sequences per se may not be enough to build a phylogenetic tree.

Amino Acid Sequence↗
PubMed1996

Molecular analysis of a novel schizosaccharomyces pombe gene containing two RNP consensus-sequence RNA-binding domains.

Proteins containing RNP consensus-sequence RNA-binding domains (CS-RBDs) play diverse roles in many aspects of RNA metabolism. Using a PCR strategy, we cloned portions of six new Schizosaccharomyces pombe genes encoding RBD proteins, including a putative homolog of the mammalian splicing factor SAP49. The genomic locus corresponding to a second PCR product, designated rnp24a, was cloned and characterized in detail. Sequence analysis revealed that the Rnp24 protein is highly charged and contains a second RBD with an unusually long Loop-3 sequence. Strains containing a disrupted copy of the rnp24 gene display neither loss of viability nor any discernible growth defects under a variety of conditions, suggesting that the function of Rnp24p overlaps with that of another fission yeast protein. Although database searches did not identify proteins that share extensive amino-acid identity with Rnp24p, phylogenetic analysis suggests that its closest relatives are metazoan hnRNP proteins. The lack of an observable phenotype in S. pombe cells lacking Rnp24p is consistent with this classification, since hnRNP proteins in higher cells include several distinct subfamilies with similar sequences and RNA-binding specificities.

Amino Acid Sequence↗
PubMed1993

Structural classification of the P450 superfamily based on consensus sequence comparison.

The novel method for classification of P450s has been proposed basing on consecutive multiple alignments of consensus sequences. A code can be assigned to each P450 amino acid sequence reflecting its relative distance from the corresponding consensus sequences at three hierarchical levels. Thus, only the principle of sequence similarity is chosen for classification without involving such criteria as enzymatic properties, phylogenetic assignment or chronology.

Algorithms↗
PubMed1996

A plant viral coat protein RNA binding consensus sequence contains a crucial arginine.

A defining feature of alfalfa mosaic virus (AMV) and ilarviruses [type virus: tobacco streak virus (TSV)] is that, in addition to genomic RNAs, viral coat protein is required to establish infection in plants. AMV and TSV coat proteins, which share little primary amino acid sequence identity, are functionally interchangeable in RNA binding and initiation of infection. The lysine-rich amino-terminal RNA binding domain of the AMV coat protein lacks previously identified RNA binding motifs. Here, the AMV coat protein RNA binding domain is shown to contain a single arginine whose specific side chain and position are crucial for RNA binding. In addition, the putative RNA binding domain of two ilarvirus coat proteins, TSV and citrus variegation virus, is identified and also shown to contain a crucial arginine. AMV and ilarvirus coat protein sequence alignment centering on the key arginine revealed a new RNA binding consensus sequence. This consensus may explain in part why heterologous viral RNA-coat protein mixtures are infectious.

Alfalfa mosaic virus↗
PubMed2006

A fibrillin-1-fragment containing the elastin-binding-protein GxxPG consensus sequence upregulates matrix metalloproteinase-1: biochemical and computational analysis.

Mutations in the gene for fibrillin-1 cause Marfan syndrome (MFS), a common hereditary disorder of connective tissue. Recent findings suggest that proteolysis, increased matrix metalloproteinase activity, and fragmentation of fibrillin-rich microfibrils in tissues of persons with MFS contribute to the complex pathogenesis of this disorder. In this study we show that a fibrillin-1 fragment containing a EGFEPG sequence that conforms to a putative GxxPG elastin-binding protein (EBP) consensus sequence upregulates the expression and production of matrix metalloproteinase (MMP)-1 by up to ninefold in a cell culture system. A mutation of the GxxPG consensus sequence site abrogated the effects. This is the first demonstration of such an effect for ligands other than elastin fragments. Molecular dynamics analysis of oligopeptides with the wildtype and mutant sequence support our biochemical results by predicting significant alterations of structural characteristics such as the potential for forming a type VIII beta-turn that are thought to be important for binding to the EBP. These results suggest that fibrillin-1 fragments may regulate MMP-1 expression, and that the dysregulation of MMPs related to fragmentation of fibrillin might contribute to the development of MFS. Our Gene Ontology (GO) analysis of the human proteome shows that proteins with multiple GxxPG motifs are highly enriched for GO terms related to the extracellular matrix. Matrix proteins with multiple GxxPG sites include fibrillin-1, -2, and -3, elastin, fibronectin, laminin, and several tenascins and collagens. Some of these proteins have been associated with disorders involving alterations in MMP regulation, and the results of the present study suggest a potential mechanism for these observations.

Amino Acid Sequence↗
PubMed1992

RNA-dependent RNA polymerase consensus sequence of the L-A double-stranded RNA virus: definition of essential domains.

The L-A double-stranded RNA virus of Saccharomyces cerevisiae makes a gag-pol fusion protein by a -1 ribosomal frameshift. The pol amino acid sequence includes consensus patterns typical of the RNA-dependent RNA polymerases (EC 2.7.7.48) of (+) strand and double-stranded RNA viruses of animals and plants. We have carried out "alanine-scanning mutagenesis" of the region of L-A including the two most conserved polymerase motifs, SG...T...NT..N (. = any amino acid) and GDD. By constructing and analyzing 46 different mutations in and around the RNA polymerase consensus regions, we have precisely defined the extent of domains and specific residues essential for viral replication. Assuming that this highly conserved region has a common secondary structure among different viruses, we predict a largely beta-sheet structure.

Amino Acid Sequence↗
PubMed2003

Crystal structure of Escherichia coli thioesterase I/protease I/lysophospholipase L1: consensus sequence blocks constitute the catalytic center of SGNH-hydrolases through a conserved hydrogen bond network.

Escherichia coli thioesterase I (TAP) is a multifunctional enzyme possessing activities of thioesterase, esterase, arylesterase, protease, and lysophospholipase. In particular, TAP has stereoselectivity for amino acid derivative substrates, hence it is useful for the kinetic resolution of racemic mixtures of industrial chemicals. In the present work, the crystal structure of native TAP was determined at 1.9A, revealing a minimal SGNH-hydrolase fold. The structure of TAP in complex with a diethyl phosphono moiety (DEP) identified its catalytic triad, Ser10-Asp154-His157, and oxyanion hole, Ser10-Gly44-Asn73. The oxyanion hole of TAP consists of three residues each separated from the other by more than 3.5A, implying that all of them are highly polarized when substrate bound. The catalytic (His)C(epsilon1)-H...O=C hydrogen bond usually plays a role in the catalytic mechanisms of most serine hydrolases, however, there were none present in SGNH-hydrolases. We propose that the existence of the highly polarized tri-residue-constituted oxyanion hole compensates for the lack of a (His)C(epsilon1)-H...O=C hydrogen bond. This suggests that members of the SGNH-hydrolase family may employ a unique catalytic mechanism. In addition, most SGNH-hydrolases have low sequence identities and presently there is no clear criterion to define consensus sequence blocks. Through comparison of TAP and the three SGNH-hydrolase structures currently known, we have identified a unique hydrogen bond network which stabilizes the catalytic center: a newly discovered structural feature of SGNH-hydrolases. We have defined these consensus sequence blocks providing a basis for the sub-classification of SGNH-hydrolases.

Amino Acid Sequence↗
PubMed1992

The plastome-encoded zfpA gene of a moss contains procaryotic as well as eucaryotic promoter consensus sequences and its RNA abundance is modulated by cytokinin.

Plastid DNA of the moss Physcomitrella patens has been sequenced. An open reading frame (ORF 315) was identified downstream from rbcL, between trnR-CCG and psaI. This ORF shares homology with zfpA, a putative regulatory gene in Pisum sativum. The moss ORF is preceded by a Shine-Dalgarno sequence, two plastid promoter consensus sequences, and three TATA boxes. A specific probe detected three transcripts of low abundance in the wild-type moss and a cytokinin-sensitive chloroplast mutant. Steady state levels of zfpA transcripts were different in the two genotypes. In mutant protonemata treated with cytokinin, steady state levels of the largest transcript decreased significantly.

Amino Acid Sequence↗