Molecular cloning of cDNA and genomic sequences coding for the 35-kilodalton subunit of the galactose-inhibitable lectin of pathogenic Entamoeba histolytica.
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Amino Acid Sequence↗
Biomedical subjects
E Tannich
Publications and source records attributed to E Tannich.
DQB1*WA1--a new DQB1 allele identified in West Africa.
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Africa, Western↗
Sensitive and specific serodiagnosis of invasive amebiasis by using a recombinant surface protein of pathogenic Entamoeba histolytica.
A recombinantly expressed protein, recEh-P1, representing part of an immunodominant surface antigen of pathogenic Entamoeba histolytica, was used for serodiagnosis of invasive amebiasis. Expression was performed under the control of a T7-RNA promoter by using a modified procaryotic expression vector, designated pHisT7. This vector allowed high-yield expression of recEh-P1 fused to a stretch of sequence containing eight histidine residues, which facilitated purification by metal chelate affinity chromatography on Ni2+ columns under highly denatured conditions. Purified recEh-P1 was found to be water soluble after prolonged dialysis and was used as the antigen for the detection of antiamebic serum antibodies by immunoblotting and enzyme-linked immunosorbent assay. In both tests all sera of patients with invasive amebiasis reacted to recEh-P1 whereas none of those collected from healthy controls, including individuals with noninvasive amebiasis, or from patients suffering from bacterial or protozoan infections unrelated to E. histolytica did so.
Amino Acid Sequence↗
Host tissue destruction by Entamoeba histolytica: molecules mediating adhesion, cytolysis, and proteolysis.
Entamoeba histolytica, the protozoan parasite causing human amoebiasis, has recently been found to comprise two genetically distinct forms, potentially pathogenic and constitutively nonpathogenic ones. Host tissue destruction by pathogenic forms is believed to result from cell functions mediated by a lectin-type adherence receptor, a pore-forming peptide involved in host cell lysis, and abundant expression of cysteine proteinase(s). Isolation and molecular cloning of these amoeba products have provided the tools for structural analyses and manipulations of cell functions including comparisons between pathogenic and nonpathogenic forms.
Animals↗
Recent progress in the molecular biology of Entamoeba histolytica.
Entamoeba histolytica, a protozoan parasite causing human amoebiasis, has recently been found to comprise two genetically distinct forms, potentially pathogenic and constitutively nonpathogenic ones. Host tissue destruction by pathogenic forms is believed to result from cell functions mediated by a lectin-type adherence receptor, a pore-forming peptide involved in host cell lysis, and abundant expression of cysteine proteinases. Comparisons of these products from pathogenic and nonpathogenic E. histolytica suggest that they have evolved to serve functions in free-living or commensal behaviour. Isolation of the corresponding genes have provided the tools for detailed structural studies and manipulations of amoeba cell functions.
Animals↗
Recombinant expression, purification and biochemical characterization of a superoxide dismutase from Entamoeba histolytica.
A recombinant iron-containing superoxide dismutase (recFeSOD) of Entamoeba histolytica was produced in a prokaryotic expression system. Purified recFeSOD was found to be enzymatically active as determined by (i) inhibition of ferri-cytochrome c reduction, (ii) dismutation of superoxide anions generated by human neutrophils and (iii) inhibition of nitroblue tetrazolium reduction. The enzymatic properties of recFeSOD were similar to those of the native protein in trophozoite extracts. In an ELISA using recFeSOD as antigen, 96% of sera from patients having invasive amebiasis were reactive whereas none of the healthy controls or of patients suffering from malaria, bacterial or viral infections were reactive. Only sera of Toxoplasma-, Leishmania- or Trypanosoma-infected individuals exhibited partial cross-reactivity to recFeSOD.
Amino Acid Sequence↗
[Recent findings on the pathogenicity of Entamoeba histolytica].
Entamoeba histolytica is the causative agent of human amoebiasis. During recent years, research in amoebiasis has concentrated on two subjects: 1. the dual manifestation of the infection as harmless colonization of the intestinal cavity or pathogenic tissue invasion and 2. the molecular analysis of functions of E. histolytica that are considered essential for pathogenicity. Besides epidemiological studies and isoenzyme analyses, molecular genetics have revealed additional evidence that two genetically distinct forms of E. histolytica do exist, named "pathogenic" and "nonpathogenic" forms, respectively. Both can infect humans but only the "pathogenic" form is able to invade the tissue and cause disease whereas the "nonpathogenic" is not. Questions remain open about the mechanism that triggers "pathogenic" E. histolytica to become invasive and about the molecules that are involved. Current data indicate that at least three functions of the amoebae are considered essential for pathogenic tissue invasion. Pathogenicity is viewed as a result of 1. adherence of the amoeba to host cells, predominantly mediated by a galactose- and N-acetylgalactosamine-inhibitable lectin, 2. killing of host cells by a pore-forming peptide known as amoebapore, and 3. proteolysis of the host's extracellular matrix mediated by cysteine proteinases. Structural detailed molecular analysis including cloning of the corresponding genes have led to a better understanding of the function of these proteins.
Animals↗
Direct sequencing of variable HLA gene segments after in vitro amplification and allele separation by temperature-gradient gel electrophoresis.
Previously unrecognized variants of human leukocyte antigens (HLA) are currently being analyzed by in vitro amplification and sequencing of the variable gene segments. In heterozygous individuals, molecular cloning is required to separate the two concomitantly amplified haplotypic gene segments. A method is presented which facilitates the procedure of separating the two haplotypic gene segments by using a temperature-gradient gel electrophoresis (TGGE). The procedure comprises PCR amplification of the variable HLA gene segments, allele separation by TGGE, re-amplification of each of the separated allelic segments, and direct DNA sequencing using the PCR primers.
Amino Acid Sequence↗
Homologous cysteine proteinases of pathogenic and nonpathogenic Entamoeba histolytica. Differences in structure and expression.
A cDNA clone derived from the gene encoding a cysteine proteinase of pathogenic Entamoeba histolytica was isolated using an antiserum to the purified enzyme. This clone was used to identify the homologous clone in a cDNA library from nonpathogenic E. histolytica. Sequence analysis and comparison of the predicted amino acid sequences revealed a sequence divergence of 16%. Southern blot analyses indicated that (i) pathogenic isolates may contain more genes coding for these or related enzymes than nonpathogenic isolates, (ii) the structure and organization of these genes are conserved within each group of amoebae, and (iii) none of the genes is found in both pathogenic and nonpathogenic E. histolytica, underlining the notion that the two groups are genetically distinct. Northern blot analyses suggested that the cysteine proteinase is expressed by pathogenic isolates in substantially higher amounts than by nonpathogenic isolates. Overexpression of this enzyme may be an important factor in the pathogenicity of E. histolytica.
Amino Acid Sequence↗
Primary structure of the 170-kDa surface lectin of pathogenic Entamoeba histolytica.
The adherence of Entamoeba histolytica to colonic mucins and to host cells appears to be predominantly mediated by a 170-kDa surface lectin of the amoebae. By using an antiserum to the purified lectin, the corresponding cDNA was isolated from an expression library of the pathogenic E. histolytica isolate HM-1:IMSS. Northern blot analysis indicated a transcript of approximately 4 kilobases, and Southern blot analyses suggested that multiple genes may encode the lectin or closely related proteins in HM-1:IMSS trophozoites. The cDNA-deduced amino acid sequence revealed an N-terminal signal peptide and a mature protein of 1270 amino acids corresponding to a molecular mass of 143 kDa, which comprises a short C-terminal cytoplasmic domain with potential phosphorylation sites, a transmembrane region, and a large extracellular portion with nine potential asparagine-linked glycosylation sites. The extracellular portion may be separated into a cysteine-poor domain and a cysteine-rich domain, the latter of which shows in part repetitive structural elements with a low degree of sequence homology to wheat germ agglutinin and to pDd63, a developmentally expressed protein of Dictyostelium discoideum.
Amino Acid Sequence↗
Pathogenic and nonpathogenic Entamoeba histolytica: identification and molecular cloning of an iron-containing superoxide dismutase.
Superoxide dismutase (SOD) activity was determined in the cell lysate of the axenically cultured Entamoeba histolytica isolate HM-1:IMSS. Under anaerobic culture conditions, 18.7 (+/- 4.9) units SOD activity (mg protein)-1 were found. By inhibition studies the activity was attributed to an iron-containing type of SOD (FeSOD). Using degenerate oligonucleotide primers derived from regions highly conserved in prokaryotic FeSOD sequences, a genomic DNA fragment was amplified by the polymerase chain reaction. The fragment was used to isolate FeSOD specific cDNA clones from a pathogenic and a nonpathogenic E. histolytica isolate. A comparison of the 2 sequences revealed 5% nucleotide differences resulting in a single amino acid exchange. The primary structure showed the characteristics of an iron-containing type of SOD with a homology of approximately 55% with other FeSOD sequences. The enzyme was found to be encoded by single copy genes in both the pathogenic and the nonpathogenic E. histolytica, but restriction fragment lengths differed between the 2 groups. In 5 isolates studied, no correlation was found between pathogenic behavior of the amebae and the expression of FeSOD-related mRNA.
Amino Acid Sequence↗
Differentiation of pathogenic from nonpathogenic Entamoeba histolytica by restriction fragment analysis of a single gene amplified in vitro.
We previously reported the identification of homologous cDNA clones derived from a pathogenic isolate and a nonpathogenic isolate of Entamoeba histolytica, which had been designated cEh-P1 and cEh-NP1, respectively. Sequence analysis of both clones had revealed 10% nucleic acid substitutions, which were dispersed over the entire sequence. This genetic difference had been found to be conserved between all four pathogenic and all five nonpathogenic laboratory strains of E. histolytica tested. On the basis of nucleic acid substitutions, we have now developed a sensitive assay to distinguish pathogenic from nonpathogenic forms of E. histolytica by using fresh clinical isolates. Comparing the sequence of cEh-P1 and cEh-NP1, we identified a 482-bp segment that contained identical 5' and 3' ends but differed in internal cleavage sites for restriction endonucleases. By using oligonucleotide primers corresponding to the 5' and 3' ends of this segment, the corresponding gene was amplified by the polymerase chain reaction. Endonuclease digestion of the amplified DNA yielded restriction fragments that are characteristic for pathogenic and nonpathogenic forms. This assay allows the detection and classification of fewer than 10 amoebae within a few hours. The differentiation of 48 isolates into pathogenic and nonpathogenic strains by using this method corresponded to the clinical status of the infected individuals and to the classification obtained by isoenzyme determination. The results further support the concept that pathogenic and nonpathogenic strains of E. histolytica constitute distinct subspecies.
Animals↗
Deletion mutagenesis in M13 by polymerase chain reaction using universal sequencing primers.
A simple procedure is described for the efficient deletion of large DNA sequences. The method involves a combination of oligonucleotide-directed mutagenesis in bacteriophage M13 and amplification of the mutagenized product by polymerase chain reaction. In contrast to other protocols employing polymerase chain reaction, synthesis of only one specific primer is required. The efficiency of heteroduplex formation between mutagenic primers directing large deletions and single-stranded template is discussed.
Bacteriophages↗
[Molecular biology in the differentiation between pathogenic and apathogenic isolates of Entamoeba histolytica].
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Amebiasis↗
A novel human muscle factor related to but distinct from MyoD1 induces myogenic conversion in 10T1/2 fibroblasts.
We have isolated the cDNA encoding a novel human myogenic factor, Myf-5, by weak cross-hydridization to the mouse MyoD1 probe. Nucleotide sequence analysis and the identification of the corresponding gene indicate that human Myf-5 is a member of a small gene family which also contains the human homologue to MyoD1. Although structurally related to the mouse factor, the human Myf-5 constitutes a different protein which nevertheless is capable of inducing the myogenic phenotype in embryonic C3H mouse 10T1/2 'fibroblasts'. The existence of more than one MyoD1-like protein in human skeletal muscle is further suggested by the detection of several similar but distinct cDNA clones. The phenotypic conversion of 10T1/2 cells by the human factor is recognized by the capacity of the cells to form multinucleated syncytia and synthesize sarcomeric myosin heavy chains. Moreover, transient expression of Myf-5 in 10T1/2 cells leads to the activation of a co-transfected muscle-specific CAT reporter gene which by itself is transcriptionally silent in the non-muscle cell background. The deduced amino acid sequence of clone Myf-5 reveals a region which is highly similar to myc proteins and the developmental factors from Drosophila encoded by the achaete scute locus and the twist gene. The myc homology region and a preceding cluster of basic amino acids are located in a larger sequence domain with strong similarity to the mouse myogenic factor MyoD1. Two additional short segments with high serine and threonine content are conserved between the two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Genomic DNA differences between pathogenic and nonpathogenic Entamoeba histolytica.
cDNA libraries were constructed from pathogenic (HM-1:IMSS) and nonpathogenic (SAW 1734) isolates of Entamoeba histolytica. A cDNA clone (cEH-P1) specific for pathogenic amoebae was identified by screening with a pool of sera from patients with invasive amoebiasis that had been absorbed with nonpathogenic amoebae. This clone was used for the identification of a homologous clone (cEH-NP1) in the cDNA from nonpathogenic amoebae. Sequence analysis and comparison of the predicted amino acid sequences for both clones disclosed 12% evolutionary divergence in structure. Hybridization of both cDNA probes to genomic DNA from four pathogenic and five nonpathogenic E. histolytica isolates revealed two distinct Southern blot patterns, one characteristic for pathogenic amoebae and the other for nonpathogenic amoebae. Further, the complex pattern of restriction fragments hybridizing to an actin cDNA probe was also different between pathogenic and nonpathogenic isolates but was conserved within each group of amoebae. The results indicate that pathogenic isolates of E. histolytica are genetically distinct from nonpathogenic isolates.
Amino Acid Sequence↗
Promoter upstream elements of the chicken cardiac myosin light-chain 2-A gene interact with trans-acting regulatory factors for muscle-specific transcription.
A segment of the 5'-flanking region of the chicken cardiac myosin light-chain gene extending from nucleotide -64 to the RNA start site is sufficient to allow muscle-specific transcription. In this paper, we characterize, by mutational analysis, sequence elements which are essential for the promoter activity. Furthermore, we present evidence for a negative-acting element which is possibly involved in conferring the muscle specificity. Nuclear proteins specifically bind to the DNA elements, as demonstrated by gel mobility shift assays and DNase I protection footprinting. The significance of the DNA-protein interactions for the function of the promoter in vivo is demonstrated by competition experiments in which protein-binding oligonucleotides were microinjected into nuclei of myotubes, where they successfully competed for the protein factors which are required to trans activate the MLC2-A promoter. The ability to bind nuclear proteins involves two closely spaced AT-rich sequence elements, one of which constitutes the TATA box. The binding properties correlate well with the capacity to activate transcription in vivo, since mutations in this region of the promoter concomitantly lead to loss of binding and transcriptional activity.
Animals↗