scholarly journals Identification of P1 gene domain containing epitope(s) mediating Mycoplasma pneumoniae cytoadherence.

1988 ◽  
Vol 167 (2) ◽  
pp. 718-723 ◽  
Author(s):  
S F Dallo ◽  
C J Su ◽  
J R Horton ◽  
J B Baseman
Keyword(s):  
Amino Acids ◽  
Mycoplasma Pneumoniae ◽  
Synthetic Peptide ◽  
Fusion Proteins ◽  
Genomic Dna ◽  
Expression Vector ◽  
Genomic Library ◽  
Vaccine Candidate ◽  
Single Copy

A genomic library of Mycoplasma pneumoniae was constructed by cloning sheared genomic DNA into the expression vector lambda gt11. Recombinant clones were screened using anti-M. pneumoniae mAbs reactive with adhesin P1 epitopes that mediate cytadherence. 10 clones with different size inserts were isolated. These clones possessed P1 sequences localized to the COOH terminus of the P1 gene. All clones produced fusion proteins that reacted with acute and convalescent sera of patients infected with M. pneumoniae. Interestingly, one clone, P1-7, contained an epitope that was confined to a region of 13 amino acids present in the M. pneumoniae genome as a single copy. The identification of this cytadherence-related epitope permits the production of a synthetic peptide that can be used as a rational vaccine candidate and serodiagnostic probe.

scholarly journals Sir3p Domains Involved in the Initiation of Telomeric Silencing in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 150 (3) ◽  
pp. 977-986 ◽  
Author(s):  
Yangsuk Park ◽  
John Hanish ◽  
Arthur J Lustig
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Fusion Proteins ◽  
Initiation Site ◽  
Negative Control ◽  
Model System ◽  
Mutant Protein ◽  
Regulatory Domain ◽  
C Terminus ◽  
Necessary And Sufficient

Abstract Previous studies from our laboratory have demonstrated that tethering of Sir3p at the subtelomeric/telomeric junction restores silencing in strains containing Rap1-17p, a mutant protein unable to recruit Sir3p. This tethered silencing assay serves as a model system for the early events that follow recruitment of silencing factors, a process we term initiation. A series of LexA fusion proteins in-frame with various Sir3p fragments were constructed and tested for their ability to support tethered silencing. Interestingly, a region comprising only the C-terminal 144 amino acids, termed the C-terminal domain (CTD), is both necessary and sufficient for restoration of silencing. Curiously, the LexA-Sir3N205 mutant protein overcomes the requirement for the CTD, possibly by unmasking a cryptic initiation site. A second domain spanning amino acids 481-835, termed the nonessential for initiation domain (NID), is dispensable for the Sir3p function in initiation, but is required for the recruitment of the Sir4p C terminus. In addition, in the absence of the N-terminal 481 amino acids, the NID negatively influences CTD activity. This suggests the presence of a third region, consisting of the N-terminal half (1-481) of Sir3p, termed the positive regulatory domain (PRD), which is required to initiate silencing in the presence of the NID. These data suggest that the CTD “active” site is under both positive and negative control mediated by multiple Sir3p domains.

scholarly journals Investigation of Protein Export in Bifidobacterium breve UCC2003

2003 ◽  
Vol 69 (12) ◽  
pp. 6994-7001 ◽  
Author(s):  
Laura E. MacConaill ◽  
Gerald F. Fitzgerald ◽  
Douwe van Sinderen
Keyword(s):  
Fusion Proteins ◽  
Genomic Library ◽  
Shuttle Vector ◽  
Staphylococcal Nuclease ◽  
Cell Fractionation ◽  
Reporter System ◽  
Bifidobacterium Breve ◽  
Positive Effects ◽  
Encoding Gene ◽  
Export Signal

ABSTRACT The molecular interactions between the bifidobacterial cell and its natural environment, namely, the gastrointestinal tract of its host, are particularly important in understanding the presumed positive effects of Bifidobacterium on the health status of the host. In this study an export-specific reporter system, designed for use in gram-positive organisms and based on the use of the staphylococcal nuclease (Nuc) as a reporter, was employed to identify exported proteins in Bifidobacterium breve UCC2003. A B. breve genomic library of translational fusions to the Nuc-encoding gene devoid of its own export signal was established in the shuttle vector pFUN (I. Poquet, S. D. Ehrlich, and A. Gruss, J. Bacteriol. 180:1904-1912, 1998) and screened for bifidobacterial export signals. Sequence analysis of the fusion proteins obtained that displayed a nuclease-producing phenotype in both Lactococcus lactis and B. breve predicted the presence of a classical signal peptide and/or single or multiple transmembrane domains, thus indicating that some of the export signals in B. breve are comparable to those used in L. lactis. Cell fractionation studies, zymograms, nuclease assays, and Western blotting were employed to confirm the function of the predicted signals and to determine the location and activity of the exported fusion proteins in B. breve and/or L. lactis.

Peptide Synthesis through Cell-Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release

ChemBioChem ◽  
2016 ◽  
Vol 17 (10) ◽  
pp. 908-912 ◽  
Author(s):  
Mantas Liutkus ◽  
Samuel A. Fraser ◽  
Karine Caron ◽  
Dannon J. Stigers ◽  
Christopher J. Easton
Keyword(s):  
Amino Acids ◽  
Peptide Synthesis ◽  
Fusion Proteins ◽  
Free Expression ◽  
Cleavage Sites ◽  
Peptide Release

scholarly journals Arabinanase A from Pseudomonas fluorescens subsp. cellulosa exhibits both an endo- and an exo- mode of action

1997 ◽  
Vol 323 (2) ◽  
pp. 547-555 ◽  
Author(s):  
Vincent A. McKIE ◽  
Gary W. BLACK ◽  
Sarah J. MILLWARD-SADLER ◽  
Geoffrey P. HAZLEWOOD ◽  
Judith I. LAURIE ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Mode Of Action ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Genomic Library ◽  
Glycosyl Hydrolase ◽  
Single Copy ◽  
Terminal Sequence ◽  
Reading Frame ◽  
Significant Release

Pseudomonas fluorescens subsp. cellulosa expressed arabinanase activity when grown on media supplemented with arabinan or arabinose. Arabinanase activity was not induced by the inclusion of other plant structural polysaccharides, and was repressed by the addition of glucose. The majority of the Pseudomonas arabinanase activity was extracellular. Screening of a genomic library of P. fluorescens subsp. cellulosa DNA constructed in Lambda ZAPII, for recombinants that hydrolysed Red-dyed arabinan, identified five arabinan-degrading plaques. Each of the phage contained the same Pseudomonas arabinanase gene, designated arbA, which was present as a single copy in the Pseudomonas genome. The nucleotide sequence of arbA revealed an open reading frame of 1041 bp encoding a protein, designated arabinanase A (ArbA), of Mr 39438. The N-terminal sequence of ArbA exhibited features typical of a prokaryotic signal peptide. Analysis of the primary structure of ArbA indicated that, unlike most Pseudomonas plant cell wall hydrolases, it did not contain linker sequences or have a modular structure, but consisted of a single catalytic domain. Sequence comparison between the Pseudomonas arabinanase and proteins in the SWISS-PROT database showed that ArbA exhibits greatest sequence identity with arabinanase A from Aspergillus niger, placing the enzyme in glycosyl hydrolase Family 43. The significance of the differing substrate specificities of enzymes in Family 43 is discussed. ArbA purifed from a recombinant strain of Escherichia coli had an Mr of 34000 and an N-terminal sequence identical to residues 32–51 of the deduced sequence of ArbA, and hydrolysed linear arabinan, carboxymethylarabinan and arabino-oligosaccharides. The enzyme displayed no activity against other plant structural polysaccharides, including branched sugar beet arabinan. ArbA produced almost exclusively arabinotriose from linear arabinan and appeared to hydrolyse arabino-oligosaccharides by successively releasing arabinotriose. ArbA and the Aspergillus arabinanase mediated a decrease in the viscosity of linear arabinan that was associated with a significant release of reducing sugar. We propose that ArbA is an arabinanase that exhibits both an endo- and an exo- mode of action.

scholarly journals A conserved C-terminal peptide of sorghum phosphoenolpyruvate carboxylase promotes its proteolysis, which is prevented by Glc-6P or the phosphorylation state of the enzyme

Planta ◽  
2021 ◽  
Vol 254 (3) ◽  
Author(s):  
Jacinto Gandullo ◽  
Rosario Álvarez ◽  
Ana-Belén Feria ◽  
José-Antonio Monreal ◽  
Isabel Díaz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Phosphoenolpyruvate Carboxylase ◽  
Synthetic Peptide ◽  
Phosphorylation State ◽  
Allosteric Effector ◽  
Cathepsin Proteases ◽  
Photosynthesis Pathway

Abstract Main conclusion A synthetic peptide from the C-terminal end of C4-phosphoenolpyruvate carboxylase is implicated in the proteolysis of the enzyme, and Glc-6P or phosphorylation of the enzyme modulate this effect. Abstract Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that performs a variety of functions in plants. Among them, it is primarily responsible for CO2 fixation in the C4 photosynthesis pathway (C4-PEPC). Here we show that proteolysis of C4-PEPC by cathepsin proteases present in a semi-purified PEPC fraction was enhanced by the presence of a synthetic peptide containing the last 19 amino acids from the C-terminal end of the PEPC subunit (pC19). Threonine (Thr)944 and Thr948 in the peptide are important requirements for the pC19 effect. C4-PEPC proteolysis in the presence of pC19 was prevented by the PEPC allosteric effector glucose 6-phosphate (Glc-6P) and by phosphorylation of the enzyme. The role of these elements in the regulation of PEPC proteolysis is discussed in relation to the physiological context.

scholarly journals Biochemical Analysis of Lpt3, a Protein Responsible forPhosphoethanolamine Addition to Lipooligosaccharide ofPathogenic Neisseria

2006 ◽  
Vol 188 (3) ◽  
pp. 1039-1048 ◽  
Author(s):  
Ellen T. O'Connor ◽  
Andrzej Piekarowicz ◽  
Karen V. Swanson ◽  
J. McLeod Griffiss ◽  
Daniel C. Stein
Keyword(s):  
Genomic Dna ◽  
Biochemical Analysis ◽  
Expression Vector ◽  
Inner Core ◽  
Genomic Sequence ◽  
Mass Spectrometry Analysis ◽  
Dna Encoding ◽  
Spectrometry Analysis ◽  
Transposon Insertion ◽  
Genomic Dna Sequence

ABSTRACT The inner core of neisserial lipooligosaccharide (LOS) contains heptose residues that can be decorated by phosphoethanolamine (PEA). PEA modification of heptose II (HepII) can occur at the 3, 6, or 7 position(s). We used a genomic DNA sequence of lpt3, derived from Neisseria meningitidis MC58, to search the genomic sequence of N. gonorrhoeae FA1090 and identified a homolog of lpt3 in N. gonorrhoeae. A PCR amplicon containing lpt3 was amplified from F62ΔLgtA, cloned, mutagenized, and inserted into the chromosome of N. gonorrhoeae strain F62ΔLgtA, producing strain F62ΔLgtAlpt3::Tn5. LOS isolated from this strain lost the ability to bind monoclonal antibody (MAb) 2-1-L8. Complementation of this mutation by genetic removal of the transposon insertion restored MAb 2-1-L8 binding. Mass spectrometry analysis of LOS isolated from the F62ΔLgtA indicated that this strain contained two PEA modifications on its LOS. F62ΔLgtAlpt3::Tn5 lacked a PEA modification on its LOS, a finding consistent with the hypothesis that lpt3 encodes a protein mediating PEA addition onto gonococcal LOS. The DNA encoding lpt3 was cloned into an expression vector and Lpt3 was purified. Purified Lpt3 was able to mediate the addition of PEA to LOS isolated from F62ΔLgtAlpt3::Tn5.

scholarly journals Triplex affinity capture of a single copy clone from a yeast genomic library

1992 ◽  
Vol 20 (13) ◽  
pp. 3524-3524 ◽  
Author(s):  
Takashi Ito ◽  
Cassandra L. Smith ◽  
Charles R. Cantor
Keyword(s):  
Genomic Library ◽  
Single Copy ◽  
Affinity Capture ◽  
Triplex Affinity Capture

Molecular and cytogenetic analysis of repetitive DNA in pea (Pisum sativum L.)

Genome ◽  
2001 ◽  
Vol 44 (4) ◽  
pp. 716-728 ◽  
Author(s):  
Pavel Neumann ◽  
Marcela Nouzová ◽  
Jirí Macas
Keyword(s):  
Pisum Sativum ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Genomic Dna ◽  
Tandem Repeats ◽  
Genomic Library ◽  
Chromosome Morphology ◽  
Plant Genome ◽  
Genomic Repeats ◽  
Dispersed Repeats

A set of pea DNA sequences representing the most abundant genomic repeats was obtained by combining several approaches. Dispersed repeats were isolated by screening a short-insert genomic library using genomic DNA as a probe. Thirty-two clones ranging from 149 to 2961 bp in size and from 1000 to 39 000/1C in their copy number were sequenced and further characterized. Fourteen clones were identified as retrotransposon-like sequences, based on their homologies to known elements. Fluorescence in situ hybridization using clones of reverse transcriptase and integrase coding sequences as probes revealed that corresponding retroelements were scattered along all pea chromosomes. Two novel families of tandem repeats, named PisTR-A and PisTR-B, were isolated by screening a genomic DNA library with Cot-1 DNA and by employing genomic self-priming PCR, respectively. PisTR-A repeats are 211–212 bp long, their abundance is 2 × 104 copies/1C, and they are partially clustered in a secondary constriction of one chromosome pair with the rest of their copies dispersed on all chromosomes. PisTR-B sequences are of similar abundance (104 copies/1C) but differ from the "A" family in their monomer length (50 bp), high A/T content, and chromosomal localization in a limited number of discrete bands. These bands are located mainly in (sub)telomeric and pericentromeric regions, and their patterns, together with chromosome morphology, allow discrimination of all chromosome types within the pea karyotype. Whereas both tandem repeat families are mostly specific to the genus Pisum, many of the dispersed repeats were detected in other legume species, mainly those in the genus Vicia.Key words: repetitive DNA, plant genome, retroelements, satellite DNA, Pisum sativum.

scholarly journals Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361

2014 ◽  
Vol 80 (12) ◽  
pp. 3576-3584 ◽  
Author(s):  
Gaoyan Wang ◽  
David C. Manns ◽  
John J. Churey ◽  
Randy W. Worobo
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Gene Cluster ◽  
Expression Vector ◽  
Expression System ◽  
Structural Genes ◽  
Cry Protein ◽  
Content Type ◽  
Thurincin H

ABSTRACTThurincin H is an antimicrobial peptide produced byBacillus thuringiensisSF361. With a helical back bone, the 31 amino acids of thurincin H form a hairpin structure maintained by four pairs of very unique sulfur-to-α-carbon thioether bonds. The production of thurincin H depends on a putative gene cluster containing 10 open reading frames. The gene cluster includes three tandem structural genes (thnA1,thnA2, andthnA3) encoding three identical 40-amino-acid thurincin H prepeptides and seven other genes putatively responsible for prepeptide processing, regulation, modification, exportation, and self-immunity. A homologous thurincin H expression system was developed by transforming a thurincin H-deficient host with a novel expression vector, pGW133. The host, designatedB. thuringiensisSF361 ΔthnA1ΔthnA2ΔthnA3, was constructed by deletion of the three tandem structural genes from the chromosome of the natural thurincin H producer. The thurincin H expression vector pGW133 was constructed by cloning the thurincin H native promoter,thnA1, and a Cry protein terminator into theEscherichia coli-B. thuringiensisshuttle vector pHT315. Thirty-three different pGW133 variants, each containing a different point mutation in thethnA1gene, were generated and separately transformed intoB. thuringiensisSF361 ΔthnA1ΔthnA2ΔthnA3. Those site-directed mutants contained either a single radical or conservative amino acid substitution on the thioether linkage-forming positions or a radical substitution on all other nonalanine amino acids. The bacteriocin activities ofB. thuringiensisSF361 ΔthnA1ΔthnA2ΔthnA3carrying different pGW133 variants against three different indicator strains were subsequently compared.

Predicted structure of the bovine calcitonin gene-related peptide and the carboxy-terminal flanking peptide of bovine calcitonin precursor

1991 ◽  
Vol 6 (2) ◽  
pp. 147-152 ◽  
Author(s):  
K. Collyear ◽  
S. I. Girgis ◽  
G. Saunders ◽  
I. MacIntyre ◽  
G. Holt
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Genomic Library ◽  
Amino Acid Peptide ◽  
Related Peptide ◽  
Calcitonin Gene ◽  
Significant Homology ◽  
Human Counterpart ◽  
Carboxy Terminal

ABSTRACT We have isolated from a bovine genomic library a clone which contains the calcitonin (CT) and CT gene-related peptide (CGRP) sequences, using probes representing the human CT and CGRP sequences. Sequence analysis has identified the nucleotide sequence coding for bovine CT, its C-terminal flanking peptide and bovine CGRP. The deduced amino acid sequence of bovine CGRP revealed a significant homology with other CGRPs so far reported. It differs by only one amino acid from rat CGRPα and porcine CGRP, and by three and four amino acids from human CGRPβ and α respectively. Bovine CT has, however, only 14 out of 32 residues in common with human CT. As in the human CT precursor, the C-terminal flanking peptide of bovine CT precursor is a 21 amino acid peptide. It shares only 11 residues in common with its human counterpart. This study thus provides further evidence that CGRP, in contrast to CT and its C-terminal flanking peptide, is a highly conserved molecule.

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