scholarly journals ISCce1 and ISCce2, Two Novel Insertion Sequences in Clostridium cellulolyticum

2003 ◽  
Vol 185 (3) ◽  
pp. 714-725 ◽  
Author(s):  
Hédia Maamar ◽  
Pascale de Philip ◽  
Jean-Pierre Bélaich ◽  
Chantal Tardif
Keyword(s):  
Amino Acid ◽  
Insertion Site ◽  
Open Reading Frame ◽  
Inverted Repeats ◽  
Target Sequence ◽  
Insertion Sequences ◽  
Reading Frame ◽  
Clostridium Cellulolyticum ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT Two new insertion sequences, ISCce1 and ISCce2, were found to be inserted into the cipC gene of spontaneous mutants of Clostridium cellulolyticum. In these insertional mutants, the cipC gene was disrupted either by ISCce1 alone or by both ISCce1 and ISCce2. ISCce1 is 1,292 bp long and has one open reading frame. The open reading frame encodes a putative 348-amino-acid protein with significant levels of identity with putative proteins having unknown functions and with some transposases belonging to the IS481 and IS3 families. Imperfect 23-bp inverted repeats were found near the extremities of ISCce1. ISCce2 is 1,359 bp long, carries one open reading frame, and has imperfect 35-bp inverted repeats at its termini. The open reading frame encodes a putative 398-amino-acid protein. This protein shows significant levels of identity with transposases belonging to the IS256 family. Upon transposition, both ISCce1 and ISCce2 generate 8-bp direct repeats of the target sequence, but no consensus sequences could be identified at either insertion site. ISCce1 is copied at least 20 times in the genome, as assessed by Southern blot analysis. ISCce2 was found to be mostly inserted into ISCce1. In addition, as neither of the elements was detected in seven other Clostridium species, we concluded that they may be specific to the C. cellulolyticum strain used.

scholarly journals The Legionella pneumophila iraAB Locus Is Required for Iron Assimilation, Intracellular Infection, and Virulence

2000 ◽  
Vol 68 (3) ◽  
pp. 1069-1079 ◽  
Author(s):  
V. K. Viswanathan ◽  
Paul H. Edelstein ◽  
C. Dumais Pope ◽  
Nicholas P. Cianciotto
Keyword(s):  
Amino Acid ◽  
Legionella Pneumophila ◽  
Open Reading Frame ◽  
Host Cells ◽  
Reading Frame ◽  
Intracellular Infection ◽  
Acid Protein ◽  
Iron Deficient ◽  
Iron Assimilation ◽  
Amino Acid Protein

ABSTRACT Legionella pneumophila, a facultative intracellular parasite of human alveolar macrophages and protozoa, causes Legionnaires' disease. Using mini-Tn10 mutagenesis, we previously isolated a L. pneumophila mutant that was hypersensitive to iron chelators. This mutant, NU216, and its allelic equivalent, NU216R, were also defective for intracellular infection, particularly in iron-deficient host cells. To determine whether NU216R was attenuated for virulence, we assessed its ability to cause disease in guinea pigs following intratracheal inoculation. NU216R-infected animals yielded 1,000-fold fewer bacteria from their lungs and spleen compared to wild-type-130b-infected animals that had received a 50-fold-lower dose. Moreover, NU216R-infected animals subsequently cleared the bacteria from these sites. While infection with 130b resulted in high fever, weight loss, and ruffled fur, inoculation with NU216R did not elicit any signs of disease. DNA sequence analysis revealed that the transposon insertion in NU216R lies in the first open reading frame of a two-gene operon. This open reading frame (iraA) encodes a 272-amino-acid protein that shows sequence similarity to methyltransferases. The second open reading frame (iraB) encodes a 501-amino-acid protein that is highly similar to di- and tripeptide transporters from both prokaryotes and eukaryotes. Southern hybridization analyses determined that theiraAB locus was largely limited to strains of L. pneumophila, the most pathogenic of the Legionellaspecies. A newly derived mutant containing a targeted disruption ofiraB showed reduced ability to grow under iron-depleted extracellular conditions, but it did not have an infectivity defect in the macrophage-like U937 cells. These data suggest thatiraA is critical for virulence of L. pneumophila while iraB is involved in a novel method of iron acquisition which may utilize iron-loaded peptides.

The SIR1 gene of Saccharomyces cerevisiae and its role as an extragenic suppressor of several mating-defective mutants

1991 ◽  
Vol 11 (4) ◽  
pp. 2253-2262
Author(s):  
E M Stone ◽  
M J Swanson ◽  
A M Romeo ◽  
J B Hicks ◽  
R Sternglanz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Gene Product ◽  
Open Reading Frame ◽  
Histone H4 ◽  
Reading Frame ◽  
Acid Protein ◽  
Extragenic Suppressor ◽  
Mating Type Genes ◽  
Amino Acid Protein

The SIR1 gene product of Saccharomyces cerevisiae is one of several proteins involved in repressing transcription of the silent mating-type genes. Strains with mutations in the genes coding for these proteins are defective in mating due to derepression of the silent loci. We have found that overexpression of the SIR1 gene suppresses the mating defects of several of these mutants, including nat1 and ard1 mutants (the products of these two genes are responsible for N-terminal acetylation of a subset of yeast proteins), certain sir3 mutants, and a histone H4 mutant. The SIR1 gene has been sequenced and found to contain an open reading frame coding for a 678-amino-acid protein.

scholarly journals The SIR1 gene of Saccharomyces cerevisiae and its role as an extragenic suppressor of several mating-defective mutants.

1991 ◽  
Vol 11 (4) ◽  
pp. 2253-2262 ◽  
Author(s):  
E M Stone ◽  
M J Swanson ◽  
A M Romeo ◽  
J B Hicks ◽  
R Sternglanz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Gene Product ◽  
Open Reading Frame ◽  
Histone H4 ◽  
Reading Frame ◽  
Acid Protein ◽  
Extragenic Suppressor ◽  
Mating Type Genes ◽  
Amino Acid Protein

The SIR1 gene product of Saccharomyces cerevisiae is one of several proteins involved in repressing transcription of the silent mating-type genes. Strains with mutations in the genes coding for these proteins are defective in mating due to derepression of the silent loci. We have found that overexpression of the SIR1 gene suppresses the mating defects of several of these mutants, including nat1 and ard1 mutants (the products of these two genes are responsible for N-terminal acetylation of a subset of yeast proteins), certain sir3 mutants, and a histone H4 mutant. The SIR1 gene has been sequenced and found to contain an open reading frame coding for a 678-amino-acid protein.

scholarly journals The bglA Gene of Aspergillus kawachii Encodes Both Extracellular and Cell Wall-Bound β-Glucosidases

1999 ◽  
Vol 65 (12) ◽  
pp. 5546-5553 ◽  
Author(s):  
Kazuhiro Iwashita ◽  
Tatsuya Nagahara ◽  
Hitoshi Kimura ◽  
Makoto Takano ◽  
Hitoshi Shimoi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Enzyme Assays ◽  
Partial Amino Acid Sequence ◽  
Reading Frame ◽  
Acid Protein ◽  
Aspergillus Kawachii ◽  
Amino Acid Protein

ABSTRACT We cloned the genomic DNA and cDNA of bglA, which encodes β-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound β-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound β-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal β-glucosidases classified in subfamily B. We expressed the bglA cDNA inSaccharomyces cerevisiae and detected the recombinant β-glucosidase in the periplasm fraction of the recombinant yeast.A. kawachii can produce two extracellular β-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound β-glucosidase.A. kawachii in which the bglA gene was disrupted produced none of the three β-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that thebglA gene encodes both extracellular and cell wall-bound β-glucosidases in A. kawachii.

scholarly journals Cloning of a Mucin-Desulfating Sulfatase Gene fromPrevotella Strain RS2 and Its Expression Using aBacteroides Recombinant System

2000 ◽  
Vol 182 (11) ◽  
pp. 3002-3007 ◽  
Author(s):  
Damian P. Wright ◽  
Catriona G. Knight ◽  
Shanthi G. Parkar ◽  
David L. Christie ◽  
Anthony M. Roberton
Keyword(s):  
Amino Acid ◽  
Sequence Data ◽  
Signal Sequence ◽  
Active Form ◽  
Open Reading Frame ◽  
Expression Vectors ◽  
Gene Encoding ◽  
Reading Frame ◽  
Gene Coding ◽  
Acid Protein

ABSTRACT A gene encoding the mucin-desulfating sulfatase inPrevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into aBacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.

scholarly journals New lnu(C) Gene Conferring Resistance to Lincomycin by Nucleotidylation in Streptococcus agalactiae UCN36

2005 ◽  
Vol 49 (7) ◽  
pp. 2716-2719 ◽  
Author(s):  
Adeline Achard ◽  
Corinne Villers ◽  
Vianney Pichereau ◽  
Roland Leclercq
Keyword(s):  
Amino Acid ◽  
Streptococcus Agalactiae ◽  
Open Reading Frames ◽  
Inverted Repeats ◽  
E Coli ◽  
Acid Protein ◽  
Total Dna ◽  
Plasmid Puc18 ◽  
Reading Frames ◽  
Amino Acid Protein

ABSTRACT Streptococcus agalactiae UCN36 was resistant to lincomycin (MIC = 16 μg/ml) but susceptible to clindamycin (MIC = 0.12 μg/ml) and erythromycin (MIC = 0.06 μg/ml). A 4-kb HindIII fragment was cloned from S. agalactiae UCN36 total DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to lincomycin. The sequence analysis of the fragment showed the presence of a 1,724-bp element delineated by imperfect inverted repeats (22 of 25 bp) and inserted in the operon for capsular synthesis of S. agalactiae UCN36. This element carried two open reading frames (ORF). The deduced amino acid sequence of the upstream ORF displayed similarity with transposases from anaerobes and IS1. The downstream ORF, lnu(C), encoded a 164-amino-acid protein with 26% to 27% identity with the LnuAN2, LnuA, and LnuA′ lincosamide nucleotidyltransferases reported for Bacteroides and Staphylococcus, respectively. Crude lysates of E. coli AG100A containing the cloned lnu(C) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl2. Mass spectrometry experiments demonstrated that the LnuC enzyme catalyzed adenylylation of lincomycin.

scholarly journals Isolation and Sequencing of a Plant cDNA Encoding a Bifunctional Methylenetetrahydrofolate Dehydrogenase : Methenyltetrahydrofolate Cyclohydrolase Protein

Pteridines ◽  
1999 ◽  
Vol 10 (4) ◽  
pp. 171-177 ◽  
Author(s):  
Liangfu Chen ◽  
Frank E. Nargang ◽  
Edwin A. Cossin
Keyword(s):  
Plant Cells ◽  
Open Reading Frame ◽  
Cdna Expression Library ◽  
Leaf Extracts ◽  
Expression Library ◽  
Cdna Insert ◽  
Reading Frame ◽  
Acid Protein ◽  
Methylenetetrahydrofolate Dehydrogenase ◽  
Amino Acid Protein

Summary In plant cells, the interconversion of formyl- and methylene-tetrahydrofolates is catalyzed by a bifunctional protein possessing methenyltetrallydrofolate cydohydrolase (EC 3.5.4.9) and methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. The present work reports the isolation and sequencing of a cDNA that encodes this protein. Polydonal antibodies, raised against purified pea cytosolic dehydrogenase:cyclohydrolase, were used to screen a λgt 11 cDNA expression library, constructed from leaf extracts of this species. The screen identified a phage containing a cDNA insert with an open reading frame encoding a 294 amino acid protein (Mr 31,344). The deduced primary structure of this protein contained most of the conserved regions found in other dehydrogenase:cyclohydrolase proteins including the corresponding domains of the trifunctional C1-THF synthases of mammalian and yeast origins.

Isolation and characterization of the muscle-specific isoform of creatine kinase from the zebrafish, Danio rerio

2001 ◽  
Vol 79 (6) ◽  
pp. 779-782 ◽  
Author(s):  
Gregory Harder ◽  
Ross McGowan
Keyword(s):  
Creatine Kinase ◽  
Amino Acid ◽  
Danio Rerio ◽  
Cdna Sequence ◽  
Reading Frame ◽  
Isolation And Characterization ◽  
Acid Protein ◽  
Amino Acid Levels ◽  
Amino Acid Protein

We have isolated and characterized a cDNA sequence corresponding to the zebrafish muscle-specific isoform of creatine kinase. The sequence is 1552 bases in length and contains an open reading frame capable of producing a 381 amino acid protein. The sequence is very similar to muscle-specific creatine kinases isolated from other species at both the nucleotide and amino acid levels but contains some differences from a previously reported zebrafish clone.Key words: creatine kinase, muscle isoform, zebrafish, Danio rerio.

scholarly journals Novel β-Lactamase Genes from Two Environmental Isolates of Vibrio harveyi

2000 ◽  
Vol 44 (5) ◽  
pp. 1309-1314 ◽  
Author(s):  
Jeanette W. P. Teo ◽  
Antonius Suwanto ◽  
Chit Laa Poh
Keyword(s):  
Amino Acid ◽  
Vibrio Harveyi ◽  
Amino Acid Sequences ◽  
Gene Probe ◽  
Reading Frame ◽  
Class A ◽  
Acid Protein ◽  
Low Levels ◽  
High Level ◽  
Amino Acid Protein

ABSTRACT Two ampicillin-resistant (Ampr) isolates ofVibrio harveyi, W3B and HB3, were obtained from the coastal waters of the Indonesian island of Java. Strain W3B was isolated from marine water near a shrimp farm in North Java while HB3 was from pristine seawater in South Java. In this study, novel β-lactamase genes from W3B (bla VHW-1) and HB3 (bla VHH-1) were cloned and their nucleotide sequences were determined. An open reading frame (ORF) of 870 bp encoding a deduced protein of 290 amino acids (VHW-1) was revealed for the bla gene of strain W3B while an ORF of 849 bp encoding a 283-amino-acid protein (VHH-1) was deduced forbla VHH-1. At the DNA level, genes for VHW-1 and VHH-1 have a 97% homology, while at the protein level they have a 91% homology of amino acid sequences. Neither gene sequence showed homology to any other β-lactamases in the databases. The deduced proteins were found to be class A β-lactamases bearing low levels of homology (<50%) to other β-lactamases of the same class. The highest level of identity was obtained with β-lactamases from Pseudomonas aeruginosa, i.e., PSE-1, PSE-4, and CARB-3, and Vibrio cholerae CARB-6. Our study showed that both strains W3B and HB3 possess an endogenous plasmid of approximately 60 kb in size. However, Southern hybridization analysis employingbla VHW-1 as a gene probe demonstrated that thebla gene was not located in the plasmid. A total of nine ampicillin-resistant V. harveyi strains, including W3B and HB3, were examined by pulsed-field gel electrophoresis ofNotI-digested genomic DNA. Despite a high level of intrastrain genetic diversity, thebla VHW-1 probe hybridized only to an 80- or 160-kb NotI genomic fragment in different isolates.

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