scholarly journals Polymorphism in the Collagen-Like Region of the Bacillus anthracis BclA Protein Leads to Variation in Exosporium Filament Length

2003 ◽  
Vol 185 (5) ◽  
pp. 1555-1563 ◽  
Author(s):  
Patricia Sylvestre ◽  
Evelyne Couture-Tosi ◽  
Michèle Mock
Keyword(s):  
Bacillus Anthracis ◽  
Gene Sequence ◽  
Polymorphic Marker ◽  
Open Reading Frame ◽  
Spore Surface ◽  
Filament Length ◽  
Reading Frame ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Carboxy Terminal

ABSTRACT We recently identified a Bacillus anthracis glycoprotein which is a structural constituent of the exosporium filaments (P. Sylvestre, E. Couture-Tosi, and M. Mock, Mol. Microbiol. 45:169-178, 2002). This Bacillus collagen-like protein (BclA) contains an internal collagen-like region (CLR) of GXX repeats which includes a large proportion of GPT triplets. Here, we report that the polymorphic marker Ceb-Bams13, for which there are nine alleles (P. Le Flèche et al., BMC Microbiol. 1:2, 2001), maps within the open reading frame encoding BclA. The bclA gene in 11 B. anthracis strains representative of seven Ceb-Bams13 alleles was sequenced and compared to the Ames bclA gene sequence. The amino- and carboxy-terminal sequences surrounding the CLR are conserved. The CLR itself is highly polymorphic: it contains between 17 and 91 GXX repeats and one to eight copies of the 21-amino-acid sequence (GPT)5GDTGTT, named the BclA repeat. The length of the filament on the spore surface differed between the strains. We exchanged the bclA gene between strains with different CLRs and examined the spore surfaces by electron microscopy analysis. The length of the BclA CLR is responsible for the variation in filament length.

scholarly journals vrrB, a Hypervariable Open Reading Frame in Bacillus anthracis

2000 ◽  
Vol 182 (14) ◽  
pp. 3989-3997 ◽  
Author(s):  
James M. Schupp ◽  
Alexandra M. Klevytska ◽  
Guenevier Zinser ◽  
Lance B. Price ◽  
Paul Keim
Keyword(s):  
Amino Acid ◽  
Bacillus Anthracis ◽  
Bacterial Species ◽  
Variable Number Tandem Repeat ◽  
Genomic Variation ◽  
Open Reading Frame ◽  
Protein Sequence Analysis ◽  
Adaptive Genetic Variation ◽  
Variable Regions ◽  
Reading Frame

ABSTRACT Bacillus anthracis appears to be the most molecularly homogeneous bacterial species known. Extensive surveys of worldwide isolates have revealed vanishingly small amounts of genomic variation. The biological importance of the resting-stage spore may lead to very low evolutionary rates and, perhaps, to the lack of potentially adaptive genetic variation. In contrast to the overall homogeneity, some gene coding regions contain hypervariability that is translated into protein variation. During marker analysis of diverse strains, we have discovered a novel ca. 750-nucleotide open reading frame (ORF) that contains in-frame, variable-number tandem-repeat sequences. Four distinct variable regions exist within vrrB, giving rise to 11 distinct alleles in eight different length categories among B. anthracis strains. This ORF putatively codes for a 241- to 265-amino-acid protein, rich in glutamine (13.2%), glycine (23.4%), and histidine (23.0%). The variable-region amino acids of the vrrB ORF are strongly hydrophilic. Coupled with putative transmembrane domains flanking the variable regions, this suggests a membrane-anchored cytosolic or extracellular location for the putative protein. Sequence analysis of the complete ORFs from three Bacillus cereus strains shows maintenance of the ORF across species boundaries, including strong conservation of the amino acid sequence and the capacity to vary among strains. The presence of 11 different alleles of the vrrB locus is in stark contrast to the near homogeneity of B. anthracis. Evolution of hypervariable genes can negate the lack of genetic variability in species such as B. anthracisand provide select rapid evolution in other more variable species.

scholarly journals Mouse Hepatitis Virus 3C-Like Protease Cleaves a 22-Kilodalton Protein from the Open Reading Frame 1a Polyprotein in Virus-Infected Cells and In Vitro

1998 ◽  
Vol 72 (3) ◽  
pp. 2265-2271 ◽  
Author(s):  
Xiao Tao Lu ◽  
Amy C. Sims ◽  
Mark R. Denison
Keyword(s):  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Open Reading Frame ◽  
Cleavage Sites ◽  
Reading Frame ◽  
Amino Terminal ◽  
Infected Cells ◽  
Carboxy Terminal ◽  
A Site

ABSTRACT The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.

scholarly journals Cloning and Expression of the algL Gene, Encoding the Azotobacter chroococcum Alginate Lyase: Purification and Characterization of the Enzyme

1999 ◽  
Vol 181 (5) ◽  
pp. 1409-1414 ◽  
Author(s):  
Ana Peciña ◽  
Alberto Pascual ◽  
Antonio Paneque
Keyword(s):  
Gene Sequence ◽  
Azotobacter Vinelandii ◽  
Alginate Lyase ◽  
Azotobacter Chroococcum ◽  
Open Reading Frame ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Reading Frame ◽  
Encoding Gene

ABSTRACT The alginate lyase-encoding gene (algL) ofAzotobacter chroococcum was localized to a 3.1-kbEcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.

scholarly journals Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes.

1989 ◽  
Vol 9 (7) ◽  
pp. 2989-2999 ◽  
Author(s):  
H M Traglia ◽  
N S Atkinson ◽  
A K Hopper
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Open Reading Frame ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Reading Frame ◽  
Genomic Deletions ◽  
Single Amino Acid Change ◽  
Carboxy Terminal

The yeast gene RNA1 has been defined by the thermosensitive rna1-1 lesion. This lesion interferes with the processing and production of all major classes of RNA. Each class of RNA is affected at a distinct and presumably unrelated step. Furthermore, RNA does not appear to exit the nucleus. To investigate how the RNA1 gene product can pleiotropically affect disparate processes, we undertook a structural analysis of wild-type and mutant RNA1 genes. The wild-type gene was found to contain a 407-amino-acid open reading frame that encodes a hydrophilic protein. No clue regarding the function of the RNA1 protein was obtained by searching banks for similarity to other known gene products. Surprisingly, the rna1-1 lesion was found to code for two amino acid differences from wild type. We found that neither single-amino-acid change alone resulted in temperature sensitivity. The carboxy-terminal region of the RNA1 open reading frame contains a highly acidic domain extending from amino acids 334 to 400. We generated genomic deletions that removed C-terminal regions of this protein. Deletion of amino acids 397 to 407 did not appear to affect cell growth. Removal of amino acids 359 to 397, a region containing 24 acidic residues, caused temperature-sensitive growth. This allele, rna1-delta 359-397, defines a second conditional lesion of the RNA1 locus. We found that strains possessing the rna1-delta 359-397 allele did not show thermosensitive defects in pre-rRNA or pre-tRNA processing. Removal of amino acids 330 to 407 resulted in loss of viability.

scholarly journals The Drosophila Gene for Antizyme Requires Ribosomal Frameshifting for Expression and Contains an Intronic Gene for snRNP Sm D3 on the Opposite Strand

1998 ◽  
Vol 18 (3) ◽  
pp. 1553-1561 ◽  
Author(s):  
Ivaylo P. Ivanov ◽  
Karl Simin ◽  
Anthea Letsou ◽  
John F. Atkins ◽  
Raymond F. Gesteland
Keyword(s):  
Open Reading Frame ◽  
P Element ◽  
Terminal Part ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Muscle Cell Differentiation ◽  
Amino Terminal ◽  
Opposite Strand ◽  
Carboxy Terminal ◽  
Transposon Insertions

ABSTRACT Previously, a Drosophila melanogaster sequence with high homology to the sequence for mammalian antizyme (ornithine decarboxylase antizyme) was reported. The present study shows that homology of this coding sequence to its mammalian antizyme counterpart also extends to a 5′ open reading frame (ORF) which encodes the amino-terminal part of antizyme and overlaps the +1 frame (ORF2) that encodes the carboxy-terminal three-quarters of the protein. Ribosomes shift frame from the 5′ ORF to ORF2 with an efficiency regulated by polyamines. At least in mammals, this is part of an autoregulatory circuit. The shift site and 23 of 25 of the flanking nucleotides which are likely important for efficient frameshifting are identical to their mammalian homologs. In the reverse orientation, within one of the introns of the Drosophila antizyme gene, the gene for snRNP Sm D3 is located. Previously, it was shown that two closely linked P-element transposon insertions caused the gutfeelingphenotype of embryonic lethality and aberrant neuronal and muscle cell differentiation. The present work shows that defects in either snRNP Sm D3 or antizyme, or both, are likely causes of the phenotype.

scholarly journals Isolation of a Minireplicon of the Virulence Plasmid pXO2 of Bacillus anthracis and Characterization of the Plasmid-Encoded RepS Replication Protein

2004 ◽  
Vol 186 (9) ◽  
pp. 2717-2723 ◽  
Author(s):  
Eowyn Tinsley ◽  
Asma Naqvi ◽  
Agathe Bourgogne ◽  
Theresa M. Koehler ◽  
Saleem A. Khan
Keyword(s):  
Bacillus Anthracis ◽  
Replication Initiation ◽  
Open Reading Frame ◽  
Virulence Plasmid ◽  
Origin Of Replication ◽  
Mobility Shift ◽  
Reading Frame ◽  
Amino Terminal ◽  
Central Regions ◽  
Putative Origin

ABSTRACT A minireplicon of plasmid pXO2 of Bacillus anthracis was isolated by molecular cloning in Escherichia coli and shown to replicate in B. anthracis, Bacillus cereus, and Bacillus subtilis. The pXO2 replicon included (i) an open reading frame encoding the putative RepS replication initiation protein and (ii) the putative origin of replication. The RepS protein was expressed as a fusion with the maltose binding protein (MBP) at its amino-terminal end and purified by affinity chromatography. Electrophoretic mobility shift assays showed that the purified MBP-RepS protein bound specifically to a 60-bp region corresponding to the putative origin of replication of pXO2 located immediately downstream of the RepS open reading frame. Competition DNA binding experiments showed that the 5′ and central regions of the putative origin were important for RepS binding. MBP-RepS also bound nonspecifically to single-stranded DNA with a lower affinity.

Sequence Analysis of the Riemerella anatipestifer OmpAMotB Gene(ORF 648bp) by Bioinformatics

2013 ◽  
Vol 647 ◽  
pp. 381-385
Author(s):  
Pan Xu ◽  
An Chun Cheng ◽  
Ming Shu Wang ◽  
De Kang Zhu ◽  
Xiao Jia Wang
Keyword(s):  
Amino Acids ◽  
Protein Sequence ◽  
Gene Sequence ◽  
Gc Content ◽  
Gene Bank ◽  
Open Reading Frame ◽  
High Similarity ◽  
Reading Frame ◽  
Bioinformatics Software ◽  
Very High

The OmpA/MotB gene from RA by our lab was sequenced. And the molecular characteristic of this gene was analyzed with bioinformatics software. The result indicated that an open reading frame(ORF) containing 648bp nucleotides was preliminarily identified by aligning with gene bank database by software of BlastN and ORF Finder. The GC content of RA OmpA/MotB gene was 36.88% and encoded a 215 amino acids in this peptide. And from the analysis results we know that this gene sequence didn’t contain a successive at least two rare codons string. Phylogenetic tree of the amino acids sequences showed this gene has not very high similarity with the other 15 OmpA/MotB protein sequence.

scholarly journals Regions of the Varicella-Zoster Virus Open Reading Frame 63 Latency-Associated Protein Important for Replication In Vitro Are Also Critical for Efficient Establishment of Latency

2005 ◽  
Vol 79 (8) ◽  
pp. 5069-5077 ◽  
Author(s):  
Jeffrey I. Cohen ◽  
Tammy Krogmann ◽  
Sebastien Bontems ◽  
Catherine Sadzot-Delvaux ◽  
Lesley Pesnicak
Keyword(s):  
Amino Acids ◽  
Varicella Zoster Virus ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Localization Signal ◽  
Carboxy Terminal

ABSTRACT Varicella-zoster virus (VZV) open reading frame 63 (ORF63) is one of the most abundant transcripts expressed during VZV latency in humans, and ORF63 protein has been detected in human ganglia by several laboratories. Deletion of over 90% of the ORF63 gene showed that the protein is required for efficient establishment of latency in rodents. We have constructed viruses with a series of mutations in ORF63. While prior experiments showed that transfection of cells with a plasmid expressing ORF63 but lacking the putative nuclear localization signal of the protein resulted in increased expression of the protein in the cytoplasm, we found that ORF63 protein remained in the nucleus in cells infected with a VZV ORF63 nuclear localization signal deletion mutant. This mutant was not impaired for growth in cell culture or for latency in rodents. Replacement of five serine or threonine phosphorylation sites in ORF63 with alanines resulted in a virus that was impaired for replication in vitro and for latency. A series of ORF63 carboxy-terminal mutants showed that the last 70 amino acids do not affect replication in vitro or latency in rodents; however, the last 108 amino acids are important for replication and latency. Thus, regions of ORF63 that are important for replication in vitro are also required for efficient establishment of latency.

scholarly journals CapE, a 47-Amino-Acid Peptide, Is Necessary for Bacillus anthracis Polyglutamate Capsule Synthesis

2005 ◽  
Vol 187 (22) ◽  
pp. 7765-7772 ◽  
Author(s):  
Thomas Candela ◽  
Michèle Mock ◽  
Agnès Fouet
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Bacillus Anthracis ◽  
Staphylococcus Epidermidis ◽  
Open Reading Frame ◽  
Synthesis System ◽  
Reading Frame ◽  
Protein Requirements ◽  
Amino Acid Peptide ◽  
Small Open Reading Frame

ABSTRACT Polyglutamate is found in various bacteria, but displays different functions depending on the species and their environment. Here, we describe a minimal polyglutamate synthesis system in Bacillus anthracis. In addition to the three genes previously described as sufficient for polyglutamate synthesis, this system includes a small open reading frame, capE, belonging to the cap operon. The polyglutamate system's requirement for the five cap genes, for capsulation and anchoring, was assayed in nonpolar mutants. The capA, capB, capC, and capE genes are all necessary and are sufficient for polyglutamate synthesis by B. anthracis. capD is required for polyglutamate anchoring to the peptidoglycan. The 47-amino-acid peptide encoded by capE is localized in the B. anthracis membrane. It is not a regulator and it is required for polyglutamate synthesis, suggesting that it has a structural role in polyglutamate synthesis. CapE appears to interact with CapA. Bacillus subtilis ywtC is similar to capE and we named it pgsE. Genes similar to capE or pgsE were found in B. subtilis natto, Bacillus licheniformis, and Staphylococcus epidermidis, species that produce polyglutamate. All the bacterial polyglutamate synthesis systems analyzed show a similar genetic organization and, we suggest, the same protein requirements.

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