scholarly journals Genetic Organization of Plasmid ColJs, Encoding Colicin Js Activity, Immunity, and Release Genes

2001 ◽  
Vol 183 (13) ◽  
pp. 3949-3957 ◽  
Author(s):  
David Šmajs ◽  
George M. Weinstock
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Fusion Protein ◽  
Molecular Mass ◽  
Open Reading Frames ◽  
Unique Region ◽  
Immunity Protein ◽  
N Terminus ◽  
Reading Frames ◽  
Histidine Tag

ABSTRACT The 5.2-kb ColJs plasmid of a colicinogenic strain ofShigella sonnei (colicin type 7) was isolated and sequenced. pColJs was partly homologous to pColE1 and to pesticin-encoding plasmid pPCP1, mainly in the rep,mob, and cer regions. A 1.2-kb unique region of pColJs showed significantly different G+C content (34%) compared to the rest of pColJs (53%). Within the unique region, seven open reading frames (ORFs) were identified. ORF94 was shown to code for colicin Js activity (cja), a 94-amino-acid polypeptide (molecular mass, 10.4 kDa); ORF129 (cji) was shown to code for the 129-amino-acid colicin Js immunity protein (molecular mass, 14.3 kDa); and ORF65 was shown to be involved in colicin Js release by producer bacteria (cjl) coding for a 65-amino-acid polypeptide (molecular mass, 7.5 kDa). In contrast to the gene order in other colicin operons, the cjl gene was found upstream from cja. Moreover, the promoter upstream from cjl was similar to promoters described upstream from several colicin activity genes. The cji gene was found to be located downstream from cja with a transcription polarity opposite to that of the cjl andcja genes. The cja, cji, and cjl genes were not similar to other known colicin genes. Colicin Js was purified as an inactive fusion protein with an N-terminal histidine tag. Activity of the purified fusion form of colicin Js was restored after cleavage of the amino acids fused to the colicin Js N terminus.

The N-end rule of selective protein turnover and its implications [abstract only]

1987 ◽  
Vol 317 (1187) ◽  
pp. 471-471
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Fusion Protein ◽  
Protein Turnover ◽  
Secreted Proteins ◽  
Gene Encoding ◽  
Intracellular Proteins ◽  
N Terminus

When a chimeric gene encoding a ubiquitin: β-galactosidase fusion protein is expressed in the yeast Saccharomyces cerevisiae , ubiquitin is efficiently cleaved off the nascent fusion protein, yielding a deubiquitinated β-galactosidase (βgal). With one exception, this cleavage takes place irrespective of the nature of the amino acid residue of βgal at the ubiquitin-βgal junction. This result, in effect, allows one to expose different residues at the N-termini of the otherwise identical βgal proteins produced in vivo . The βgal proteins thus designed exhibit a striking diversity of in vivo half-lives, from more than 10h to less than 3 min, depending on the nature of the amino acid exposed at the N-terminus of βgal. The N-terminal location of an amino acid is essential for its effect on βgal half-life. The set of individual amino acids can thus be ordered with respect to the half-lives that they confer on βgal when present at its N-terminus (the ‘N-end rule’). The known N-terminal residues in long-lived intracellular proteins from both prokaryotes and eukaryotes are exclusively of the stabilizing class as predicted by the N-end rule. In contrast, a majority of the N-terminal residues in compartmentalized (e.g. secreted) proteins are of the destabilizing class. The N-end rule may thus underlie both the diversity of protein half-lives in vivo and the selective destruction of otherwise normal but miscompartmentalized proteins. The N-end may also account for the function of the previously described post-translational addition of single amino acids to protein N-termini. Thus the recognition of an N-terminal residue in a protein may mediate both the metabolic stability of the protein and the potential for regulation of its stability.

Sequence and characterization of a novel chromosomal aminoglycoside phosphotransferase gene, aph (3')-IIb, in Pseudomonas aeruginosa.

1996 ◽  
Vol 40 (5) ◽  
pp. 1254-1256 ◽  
Author(s):  
H Hächler ◽  
P Santanam ◽  
F H Kayser
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Amino Acid Identity ◽  
Open Reading Frames ◽  
Acid Identity ◽  
Aminoglycoside Phosphotransferase ◽  
Psti Fragment ◽  
Reading Frames

A novel, probably chromosomally encoded, aminoglycoside phosphotransferase gene was cloned on a 2,996-bp PstI fragment from Pseudomonas aeruginosa and designated aph (3')-IIb. It coded for a protein of 268 amino acids that showed 51.7% amino acid identity with APH (3')-II [APH(3') is aminoglycoside-3' phosphotransferase] from Tn5. Two other open reading frames on the cloned fragment showed homology to a signal-transducing system in P. aeruginosa.

scholarly journals Extensive translation of small Open Reading Frames revealed by Poly-Ribo-Seq

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Julie L Aspden ◽  
Ying Chen Eyre-Walker ◽  
Rose J Phillips ◽  
Unum Amin ◽  
Muhammad Ali S Mumtaz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Open Reading Frames ◽  
Small Peptides ◽  
Functional Roles ◽  
Genome Wide ◽  
A Genome ◽  
Non Coding Rnas ◽  
Reading Frames ◽  
Small Open Reading Frames

Thousands of small Open Reading Frames (smORFs) with the potential to encode small peptides of fewer than 100 amino acids exist in our genomes. However, the number of smORFs actually translated, and their molecular and functional roles are still unclear. In this study, we present a genome-wide assessment of smORF translation by ribosomal profiling of polysomal fractions in Drosophila. We detect two types of smORFs bound by multiple ribosomes and thus undergoing productive translation. The ‘longer’ smORFs of around 80 amino acids resemble canonical proteins in translational metrics and conservation, and display a propensity to contain transmembrane motifs. The ‘dwarf’ smORFs are in general shorter (around 20 amino-acid long), are mostly found in 5′-UTRs and non-coding RNAs, are less well conserved, and have no bioinformatic indicators of peptide function. Our findings indicate that thousands of smORFs are translated in metazoan genomes, reinforcing the idea that smORFs are an abundant and fundamental genome component.

Acquisition of NFKB1-selective DNA binding by substitution of four amino acid residues from NFKB1 into RelA

1993 ◽  
Vol 13 (7) ◽  
pp. 3850-3859
Author(s):  
T A Coleman ◽  
C Kunsch ◽  
M Maher ◽  
S M Ruben ◽  
C A Rosen
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Fusion Protein ◽  
Binding Specificity ◽  
Single Amino Acid ◽  
Nf Kappa B ◽  
Dna Binding Specificity ◽  
Dna Motif ◽  
Single Amino Acid Change

The subunits of NF-kappa B, NFKB1 (formerly p50) and RelA (formerly p65), belong to a growing family of transcription factors that share extensive similarity to the c-rel proto-oncogene product. The homology extends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and DNA binding. It is now generally accepted that homodimers of either subunit are capable of binding DNA that contains a kappa B site originally identified in the immunoglobulin enhancer. Recent studies have demonstrated that the individual subunits of the NF-kappa B transcription factor complex can be distinguished by their ability to bind distinct DNA sequence motifs. By using NFKB1 and RelA subunit fusion proteins, different regions within the RHD were found to confer DNA-binding and multimerization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RelA displayed preferential binding to an NFKB1-selective DNA motif while dimerizing with the characteristics of RelA. Within the NFKB1 portion of this fusion protein, a single amino acid change of His to Arg altered the DNA-binding specificity to favor interaction with the RelA-selective DNA motif. Furthermore, substitution of four amino acids from NFKB1 into RelA was able to alter the DNA-binding specificity of the RelA protein to favor interaction with the NFKB1-selective site. Taken together, these findings demonstrate the presence of a distinct subdomain within the RHD involved in conferring the DNA-binding specificity of the Rel family of proteins.

scholarly journals Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm.

1995 ◽  
Vol 128 (1) ◽  
pp. 51-60 ◽  
Author(s):  
M Way ◽  
M Sanders ◽  
C Garcia ◽  
J Sakai ◽  
P Matsudaira
Keyword(s):  
Amino Acid ◽  
Actin Filaments ◽  
Limited Proteolysis ◽  
Open Reading Frames ◽  
Actin Binding ◽  
Molar Ratio ◽  
Domain Organization ◽  
Ring Canals ◽  
Drosophila Protein ◽  
Reading Frames

The acrosomal process of Limulus sperm is an 80-microns long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin filaments at 13-A resolution shows that scruin is organized into two equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organization of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Protease-sensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOH-terminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an approximately 50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four beta-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin.

scholarly journals Genome Sequences of Two Dual-Serotype-Specific Echovirus 20 Strains from Nigeria

2019 ◽  
Vol 8 (43) ◽  
Author(s):  
T. O. C. Faleye ◽  
O. M. Adewumi ◽  
D. Klapsa ◽  
M. Majumdar ◽  
J. Martin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Complete Genome ◽  
Neutralization Assay ◽  
Open Reading Frames ◽  
Genome Sequences ◽  
Overlapping Open Reading Frames ◽  
Reading Frames

Here, we describe nearly complete genome sequences (7,361 nucleotides [nt] and 6,893 nt) of two echovirus 20 (E20) isolates from Nigeria that were simultaneously typed as CVB and E20 (dual serotype) by neutralization assay. Both include two overlapping open reading frames (ORFs) of 67 and 2,183 amino acids that encoded a recently described gut infection-facilitating protein and the classic enterovirus proteins, respectively.

scholarly journals Identification and Analysis of Genes Involved in Anaerobic Toluene Metabolism by Strain T1: Putative Role of a Glycine Free Radical

1998 ◽  
Vol 64 (5) ◽  
pp. 1650-1656 ◽  
Author(s):  
Peter W. Coschigano ◽  
Thomas S. Wehrman ◽  
L. Y. Young
Keyword(s):  
Free Radical ◽  
Amino Acid ◽  
Molecular Mass ◽  
Cysteine Residue ◽  
Open Reading Frames ◽  
Site Directed Mutagenesis ◽  
Pyruvate Formate Lyase ◽  
Calculated Molecular Mass ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. ThetutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.

scholarly journals Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens.

2003 ◽  
Vol 50 (1) ◽  
pp. 269-278
Author(s):  
Amr M Shabaan ◽  
Magdy M Mohamed ◽  
Mohga S Abdallah ◽  
Hayat M Ibrahim ◽  
Amr M Karim
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Schistosoma Mansoni ◽  
Molecular Mass ◽  
Vaccine Development ◽  
Complete Sequence ◽  
Cdna Clones ◽  
Western Blots ◽  
Calculated Molecular Mass ◽  
Reading Frame

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.

scholarly journals The Extracellular Transport Signal of the Vibrio cholerae Endochitinase (ChiA) Is a Structural Motif Located between Amino Acids 75 and 555

2002 ◽  
Vol 184 (8) ◽  
pp. 2225-2234 ◽  
Author(s):  
Jason P. Folster ◽  
Terry D. Connell
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Vibrio Cholerae ◽  
Structural Information ◽  
Amino Acid Sequences ◽  
Structural Motif ◽  
Terminal Branch ◽  
C Terminus ◽  
N Terminus ◽  
Extracellular Transport

ABSTRACT ChiA, an 88-kDa endochitinase encoded by the chiA gene of the gram-negative enteropathogen Vibrio cholerae, is secreted via the eps-encoded main terminal branch of the general secretory pathway (GSP), a mechanism which also transports cholera toxin. To localize the extracellular transport signal of ChiA that initiates transport of the protein through the GSP, a chimera comprised of ChiA fused at the N terminus with the maltose-binding protein (MalE) of Escherichia coli and fused at the C terminus with a 13-amino-acid epitope tag (E-tag) was expressed in strain 569B(chiA::Kanr), a chiA-deficient but secretion-competent mutant of V. cholerae. Fractionation studies revealed that blockage of the natural N terminus and C terminus of ChiA did not prevent secretion of the MalE-ChiA-E-tag chimera. To locate the amino acid sequences which encoded the transport signal, a series of truncations of ChiA were engineered. Secretion of the mutant polypeptides was curtailed only when ChiA was deleted from the N terminus beyond amino acid position 75 or from the C terminus beyond amino acid 555. A mutant ChiA comprised of only those amino acids was secreted by wild-type V. cholerae but not by an epsD mutant, establishing that amino acids 75 to 555 independently harbored sufficient structural information to promote secretion by the GSP of V. cholerae. Cys77 and Cys537, two cysteines located just within the termini of ChiA(75-555), were not required for secretion, indicating that those residues were not essential for maintaining the functional activity of the ChiA extracellular transport signal.

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