Cloning, expression, and sequence analysis of a cytolytic enterotoxin gene from Aeromonas hydrophila

1993 ◽  
Vol 39 (5) ◽  
pp. 513-523 ◽  
Author(s):  
Ashok K. Chopra ◽  
Clifford W. Houston ◽  
Johnny W. Peterson ◽  
Gui-F. Jin
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Dna Sequence ◽  
Aeromonas Hydrophila ◽  
Biological Activities ◽  
Chinese Hamster Ovary Cells ◽  
Sodium Dodecyl ◽  
Leader Peptide ◽  
Species Variation ◽  
Enterotoxin Gene

The structural gene and regulatory element for a cytolytic enterotoxin of a diarrheal isolate, SSU, of Aeromonas hydrophila was cloned and its DNA sequence was determined. A complementary, mixed synthetic oligonucleotide based on the first 10 NH2-terminal amino acid residues of the Aeromonas cytolytic enterotoxin was used as a probe to screen a genomic library constructed in bacteriophage EMBL3. Cell lysates of Escherichia coli (λCH4), containing the cytolytic enterotoxin gene, lysed rabbit red blood cells and destroyed Chinese hamster ovary cells, caused fluid secretion in rat ileal loops, and were lethal to mice when injected intravenously. All biological activities associated with the cytolytic enterotoxin were neutralized by rabbit homologous polyclonal antibodies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent Western blot analysis of the cell lysate of E. coli (λCH4) revealed a protein band of approximately 52 kDa, using antisera to the cytolytic enterotoxin or antibodies generated against a synthetic peptide to the toxin. DNA sequence analysis of a 2.8-kb SalI-BamHI fragment revealed the presence of one large open reading frame (1479 bp) that would encode a protein of 54.5 kDa, a precursor form of the cytolytic enterotoxin, with a 23 amino acid leader peptide. Despite a significant amount of homology at the DNA and amino acid levels between our cytolytic enterotoxin and two aerolysins of Aeromonas species, variation in the restriction maps of these three toxin genes was prominent. Likewise, considerable divergence in DNA sequence was observed upstream of the structural genes for the reported aerolysins and our cytolytic enterotoxin, suggesting that these structurally similar toxin molecules may be regulated differently. Finally, our data showed that the cytolytic enterotoxin from a diarrheal isolate, SSU, of A. hydrophila exhibited characteristics that were unique compared with those of the reported aerolysins.Key words: Aeromonas hydrophila, cytolytic enterotoxin, aerolysin, cholera toxin.

scholarly journals Novel Genetic and Phenotypic Heterogeneity in Bordetella bronchiseptica Pertactin

2001 ◽  
Vol 69 (3) ◽  
pp. 1917-1921 ◽  
Author(s):  
Karen B. Register
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Dna Sequence ◽  
Outer Membrane Protein ◽  
Phenotypic Heterogeneity ◽  
Bordetella Bronchiseptica ◽  
Amino Acid Repeat ◽  
Sequence Heterogeneity

ABSTRACT The Bordetella bronchiseptica outer membrane protein pertactin is believed to function as an adhesin and is an important protective immunogen. Previous sequence analysis of the pertactin gene identified two regions predicted to encode amino acid repeat motifs. Recent studies have documented DNA sequence heterogeneity in both regions. The present study describes additional variants in these regions, which form the basis for six novel pertactin types. Immunoblotting demonstrated phenotypic heterogeneity in pertactin consistent with the predicted combined sizes of the repeat regions. A revised system for classifying B. bronchiseptica pertactin variants is proposed.

scholarly journals Biological activities of bovine cardiac-muscle troponin C C-terminal peptide (residues 84-161)

1982 ◽  
Vol 207 (2) ◽  
pp. 185-192 ◽  
Author(s):  
N V Barskaya ◽  
N B Gusev
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Cardiac Muscle ◽  
Sodium Dodecyl Sulphate ◽  
Binding Sites ◽  
Binding Constant ◽  
Troponin I ◽  
Biological Activities ◽  
Sodium Dodecyl ◽  
Troponin C

1. Bovine cardiac-muscle troponin C was digested at cysteine residues 35 and 84, and the C-terminal peptide (residues 84-161) was isolated. 2. The C-terminal peptide contains two Ca2+-binding sites. These sites bind Ca2+ with a binding constant of 2.0×10(8) M-1. In the presence of 2 mM-Mg2+ the binding constant for Ca2+ is decreased to 3.70×10(7) M-1. The corresponding constants for native troponin C are 5.90×10(7) M-1. and 2.90×10(7) M-1 respectively. 3. Electrophoretic mobility of the C-terminal peptide is increased in the presence of 0.1 mM-CaCl2 as compared with the mobility in the presence of 2mM-EDTA. The same phenomenon was observed when electrophoresis was performed in the presence of 6 M-urea or 0.1% sodium dodecyl sulphate. 4. When saturated with Ca2+, the C-terminal peptide forms complexes with bovine cardiac-muscle troponin I both in the absence and in the presence of 6 M-urea. This complex is dissociated on removal of Ca2+. 5. The data suggest that the C-terminal peptide of troponin C contains two Ca2+/Mg2+-binding sites and interacts with troponin I. Thus, despite the 30% difference in amino acid composition, the properties of bovine cardiac-muscle troponin C C-terminal peptide are similar to those of rabbit skeletal-muscle troponin C C-terminal peptide.

scholarly journals Substitution of Val for Met at residue 239 of platelet glycoprotein Ib alpha in Japanese patients with platelet-type von Willebrand disease

Blood ◽  
1995 ◽  
Vol 85 (3) ◽  
pp. 727-733 ◽  
Author(s):  
H Takahashi ◽  
M Murata ◽  
T Moriki ◽  
H Anbo ◽  
T Furukawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Dna Sequence ◽  
Amino Acid Substitution ◽  
Genomic Dna ◽  
Von Willebrand Disease ◽  
Dna Sequence Analysis ◽  
Platelet Glycoprotein ◽  
Normal Individuals ◽  
Von Willebrand

Genomic DNA was studied from four patients with platelet-type von Willebrand disease (vWD) from two Japanese families previously reported. The entire coding region of platelet glycoprotein (GP) Ib alpha, a component of the platelet receptor for von Willebrand factor (vWF), was examined by polymerase chain reaction (PCR) followed by direct DNA sequence analysis. A single point mutation was found in all patients resulting in substitution of Val (GTG) for Met (ATG) at residue 239 of GPIb alpha. All patients were heterozygous for the mutation, whereas none of the unaffected family members had an amino acid substitution at residue 239. Because the nucleotide substitution destroys an NIa III restriction site on GPIb alpha, PCR products were subjected to digestion with this enzyme; DNA fragments from both normal and mutant alleles were detected in all affected individuals. In allele- specific PCR, DNA was amplified from patients' genomic DNA using either adenine- or guanine-containing primers, whereas only adenine-containing primer successfully amplified DNA from normal individuals. Cloning of amplified DNA into bacteriophage M13mp19 and subsequent DNA sequence analysis confirmed the mutation in these families. The absence of the amino acid substitution at residue 239 of GPIb alpha in the normal individuals tested, together with the linkage of this substitution to the phenotypic expression of disease in these two families and in a family recently described suggest that this amino acid change is a molecular basis for platelet-type vWD, and the substitution may produce a quite similar phenotype to the one reported previously (Gly to Val at residue 233 of GPIb alpha).

scholarly journals Analysis of rodent platelet glycoprotein IIb: evidence for evolutionarily conserved domains and alternative proteolytic processing

Blood ◽  
1990 ◽  
Vol 75 (6) ◽  
pp. 1282-1289 ◽  
Author(s):  
M Poncz ◽  
PJ Newman
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Dna Sequence ◽  
Genomic Dna ◽  
Amino Acid Identity ◽  
Dna Sequence Analysis ◽  
Amino Acid Residues ◽  
Acid Identity ◽  
Heavy Chains ◽  
Genomic Dna Sequence

Abstract Recently, the full-length primary amino acid sequence for human glycoproteins (GP) IIb and IIIa have been derived from their respective cDNAs. Potential functional domains within these proteins have been proposed based primarily on homology with similar domains in other proteins having known biologic function. To further understand the relationship between structure and function of the platelet fibrinogen receptor, we have begun comparative studies of the human GPIIb/IIIa receptor with the corresponding rodent receptor. The rodent rGPIIb/IIIa receptor differs from the human receptor, having low affinity for R.G.D- containing oligopeptides and not binding at all to the C-terminus of the gamma chain of human fibrinogen. We describe the structure of rodent platelet GPIIb derived from a combination of peptide sequencing, and cDNA and partial genomic DNA sequence analysis. The initial transcript is 1037 amino acid residues, having 78% amino acid identity with its 1039 residue human analog. Both heavy chains have the N- terminal sequence L.N.L.D, agreeing with the consensus derived from other integrin family alpha heavy chains. All 18 cysteine residues occur at positions conserved in human GPIIb and the vitronectin receptor alpha subunit VNR alpha. The putative calcium-binding domains of the GPIIbs have a high level of amino acid identity (92%), supporting the supposition that these regions have a critical biologic role. The final 48 C-terminal amino acid residues of the heavy chain of rodent GPIIb share only 56% identity with its human counterpart, and the proposed cleavage site of human GPIIb into its heavy and light chains is not present in the rodent sequence. Although we demonstrate that rodent GPIIb is split into two subunits during its maturation, this process either involves a different recognition sequence in the rodent or occurs at a different site. Finally, partial genomic DNA sequence analysis indicates that there are at least two rodent GPIIb genes: a normal gene, containing introns in positions similar to those in the human gene, and a processed pseudogene. The human haploid genome contains only a single GPIIb gene.

On the Primary Structure of the A Chains

1975 ◽  
Author(s):  
B. Hessel
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Peptide Mapping ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Amino Acid Sequence Analysis ◽  
Sequences Analysis ◽  
A Chain ◽  
Fibrinogen Molecule

During plasma digestion of fibrinogen a degradation product with molecular weight (MW) of about 50,000 appear very early in the digest. As revealed by sodium dodecyl sulphate (SDS) gel electrophoresis this product has its origin in the A chain. Upon further hydrolysis the 50,000 MW fragment is degraded to a 25,000 MW fragment. Both the 50,000 and 25,000 MW fragments have been isolated. After treatment with cyanogenbromide (CNBr) of the 50,000 MW fragment and separation of the peptides obtained, a fragment with MW of 30,000 and two fragments of smaller MW were isolated. As revealed by amino acid sequence analysis and peptide mapping the 30,000 MW fragment was identical to a hydrophilic sulfurcontaining peptide (Hi2-DSK), isolated after CNBr-treatment of the whole fibrinogen molecule. The amino acid sequence analysis of the fragments is in progress. Amino acid sequences analysis of the 25,000 MW fragment gave the following sequence: Ala-Len-Thr- Asp-Met-Pro- Gln-Met- Arg-Met-Glu-Leu-Glu-. From residue number 11 it appears to be identical to the Hi2-DSK fragment. However, the 25,000 MW fragment is shorter at the COOH-terminal end.

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