scholarly journals Characterization of genomic PIG-A gene: a gene for glycosylphosphatidylinositol-anchor biosynthesis and paroxysmal nocturnal hemoglobinuria

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3126-3131 ◽  
Author(s):  
Y Iida ◽  
J Takeda ◽  
T Miyata ◽  
N Inoue ◽  
J Nishimura ◽  
...  
Keyword(s):  
Blood Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Somatic Mutations ◽  
Surface Expression ◽  
Sequence Information ◽  
Chain Reaction ◽  
Nucleotide Sequence Information ◽  
Flanking Region ◽  
Polymerase Chain

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by the presence of abnormal subpopulations of blood cells that are deficient in surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins. Recent studies showed that the gene termed PIG-A, which participates in the first step of GPI-anchor biosynthesis, is mutated in the abnormal blood cells from patients with PNH. In this study the genomic PIG-A gene was cloned and characterized to obtain nucleotide sequence information for analyzing somatic mutations of PIG-A in patients with PNH. The PIG-A gene is at least 17 kb long and has six exons. The exon-intron boundaries and 583 bp of the 5′ flanking region were sequenced. The 5′ flanking region has no TATA-like sequence, but includes four CAAT boxes, two AP-2 sequences, and a CRE sequence, some of which are present in regions necessary for the promoter activity. We report pairs of oligonucleotide primers for polymerase chain reaction that should be useful to amplify and analyze various regions of the PIG-A gene in patients with PNH.

scholarly journals Characterization of genomic PIG-A gene: a gene for glycosylphosphatidylinositol-anchor biosynthesis and paroxysmal nocturnal hemoglobinuria

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3126-3131
Author(s):  
Y Iida ◽  
J Takeda ◽  
T Miyata ◽  
N Inoue ◽  
J Nishimura ◽  
...  
Keyword(s):  
Blood Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Somatic Mutations ◽  
Surface Expression ◽  
Sequence Information ◽  
Chain Reaction ◽  
Nucleotide Sequence Information ◽  
Flanking Region ◽  
Polymerase Chain

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by the presence of abnormal subpopulations of blood cells that are deficient in surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins. Recent studies showed that the gene termed PIG-A, which participates in the first step of GPI-anchor biosynthesis, is mutated in the abnormal blood cells from patients with PNH. In this study the genomic PIG-A gene was cloned and characterized to obtain nucleotide sequence information for analyzing somatic mutations of PIG-A in patients with PNH. The PIG-A gene is at least 17 kb long and has six exons. The exon-intron boundaries and 583 bp of the 5′ flanking region were sequenced. The 5′ flanking region has no TATA-like sequence, but includes four CAAT boxes, two AP-2 sequences, and a CRE sequence, some of which are present in regions necessary for the promoter activity. We report pairs of oligonucleotide primers for polymerase chain reaction that should be useful to amplify and analyze various regions of the PIG-A gene in patients with PNH.

scholarly journals Peripheral blood cells are predominantly chimeric of affected and normal cells in patients with paroxysmal nocturnal hemoglobinuria: simultaneous investigation on clonality and expression of glycophosphatidylinositol-anchored proteins

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 853-859 ◽  
Author(s):  
H Ohashi ◽  
T Hotta ◽  
A Ichikawa ◽  
T Kinoshita ◽  
R Taguchi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Southern Hybridization ◽  
Peripheral Blood Cells ◽  
Chain Reaction ◽  
Normal Cells ◽  
Clonality Analysis ◽  
Polymerase Chain

To investigate clonal compositions of hematologic cells in paroxysmal nocturnal hemoglobinuria (PNH), we analyzed peripheral blood (PB) cells of 12 female patients with PNH, by clonality analysis using X-chromosome inactivation and assessment of expression of glycophosphatidylinositol-anchored proteins (GPI-APs) by flow cytometry. Southern hybridization showed that granulocytes were monoclonal in three and polyclonal in eight patients, respectively, whereas lymphocytes were polyclonal in all nine patients examined. Expressions of CD16 and CD59 on granulocytes varied greatly in seven patients examined. Clonality analysis of granulocytes by the polymerase chain reaction showed that CD59-and CD59low+ cells were monoclonal, whereas CD59+ cells were polyclonal. It was shown that PB cells are predominantly chimeric of clonal (GPI-AP-or GPI-APlow+) and nonclonal (GPI-AP+) cells in PNH, and that degrees of chimerism differ greatly from patient to patient.

scholarly journals Peripheral blood cells are predominantly chimeric of affected and normal cells in patients with paroxysmal nocturnal hemoglobinuria: simultaneous investigation on clonality and expression of glycophosphatidylinositol-anchored proteins

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 853-859 ◽  
Author(s):  
H Ohashi ◽  
T Hotta ◽  
A Ichikawa ◽  
T Kinoshita ◽  
R Taguchi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Southern Hybridization ◽  
Peripheral Blood Cells ◽  
Chain Reaction ◽  
Normal Cells ◽  
Clonality Analysis ◽  
Polymerase Chain

Abstract To investigate clonal compositions of hematologic cells in paroxysmal nocturnal hemoglobinuria (PNH), we analyzed peripheral blood (PB) cells of 12 female patients with PNH, by clonality analysis using X-chromosome inactivation and assessment of expression of glycophosphatidylinositol-anchored proteins (GPI-APs) by flow cytometry. Southern hybridization showed that granulocytes were monoclonal in three and polyclonal in eight patients, respectively, whereas lymphocytes were polyclonal in all nine patients examined. Expressions of CD16 and CD59 on granulocytes varied greatly in seven patients examined. Clonality analysis of granulocytes by the polymerase chain reaction showed that CD59-and CD59low+ cells were monoclonal, whereas CD59+ cells were polyclonal. It was shown that PB cells are predominantly chimeric of clonal (GPI-AP-or GPI-APlow+) and nonclonal (GPI-AP+) cells in PNH, and that degrees of chimerism differ greatly from patient to patient.

scholarly journals Somatic mutations of PIG-A in Thai patients with paroxysmal nocturnal hemoglobinuria

Blood ◽  
1995 ◽  
Vol 86 (5) ◽  
pp. 1736-1739 ◽  
Author(s):  
P Pramoonjago ◽  
W Wanachiwanawin ◽  
S Chinprasertsak ◽  
K Pattanapanayasat ◽  
J Takeda ◽  
...  
Keyword(s):  
Blood Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Somatic Mutations ◽  
Surface Expression ◽  
Chromosomal Gene ◽  
Gpi Anchor ◽  
Hematopoietic Stem ◽  
Single Base ◽  
Base Substitutions ◽  
Cell Disorder

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder characterized by clonal blood cells that are deficient in the surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins. In the affected cells, the X-chromosomal gene PIG-A, which participates in biosynthesis of the GPI anchor, is somatically mutated. Analyses of Japanese, British, and American patients with PNH have shown somatic mutations of PIG-A in all of them, indicating that PIG-A is responsible for PNH in most, if not all, patients in those countries. Twenty-nine of the reported somatic mutations are small, mostly involving 1 or 2 bases, except for one with a 4-kb deletion. Here we describe an analysis of PIG-A in neutrophils from 14 patients from Thailand where PNH is thought to be more common. We found small somatic PIG-A mutations in all patients. These consisted of six single base deletions, one each of 2-, 3-, 5- and 10-base deletions, two single base insertions and two base substitutions. Thus, the small somatic mutation in the PIG-A gene is also responsible for PNH in Thailand. However, base substitutions were rarer (2 of 14) than in Japan (8 of 16), and deletions of multiple bases were more common, suggesting various causes of mutation.

scholarly journals Characterization of Xanthomonas axonopodis pv. phaseoli isolates

2008 ◽  
Vol 34 (3) ◽  
pp. 228-231 ◽  
Author(s):  
Willian Mário de Carvalho Nunes ◽  
Maria Júlia Corazza ◽  
Silvana Aparecida Crestes Dias de Souza ◽  
Siu Mui Tsai ◽  
Eiko Eurya Kuramae
Keyword(s):  
Xanthomonas Campestris ◽  
Random Amplified Polymorphic Dna ◽  
Hypersensitive Reaction ◽  
Chain Reaction ◽  
Xanthomonas Axonopodis ◽  
Paulo State ◽  
Total Dna ◽  
Polymerase Chain ◽  
Bacterial Plant Pathogen

A simple, quick and easy protocol was standardized for extraction of total DNA of the bacteria Xanthomonas axonopodis pv. phaseoli. The DNA obtained by this method had high quality and the quantity was enough for the Random Amplified Polymorphic DNA (RAPD) reactions with random primers, and Polymerase Chain Reaction (PCR) with primers of the hypersensitivity and pathogenicity gene (hrp). The DNA obtained was free of contamination by proteins or carbohydrates. The ratio 260nm/380nm of the DNA extracted ranged from 1.7 to 1.8. The hrp gene cluster is required by bacterial plant pathogen to produce symptoms on susceptible hosts and hypersensitive reaction on resistant hosts. This gene has been found in different bacteria as well as in Xanthomonas campestris pv. vesicatoria (9). The primers RST21 and RST22 (9) were used to amplify the hrp gene of nine different isolates of Xanthomonas axonopodis pv. phaseoli from Botucatu, São Paulo State, Brazil, and one isolate, "Davis". PCR amplified products were obtained in all isolates pathogenic to beans.

Characterization of Giardia duodenalis by polymerase-chain-reaction fingerprinting

1998 ◽  
Vol 84 (9) ◽  
pp. 707-714 ◽  
Author(s):  
Wieger L. Homan ◽  
Margriet Gilsing ◽  
Hafida Bentala ◽  
Louis Limper ◽  
Frans van Knapen
Keyword(s):  
Polymerase Chain Reaction ◽  
Giardia Duodenalis ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Rhodococcus equi Infections in Goats: Characterization of Virulence Plasmids

2017 ◽  
Vol 55 (2) ◽  
pp. 273-276 ◽  
Author(s):  
Lauren W. Stranahan ◽  
Quinci D. Plumlee ◽  
Sara D. Lawhon ◽  
Noah D. Cohen ◽  
Laura K. Bryan
Keyword(s):  
Polymerase Chain Reaction ◽  
Lymph Nodes ◽  
Rhodococcus Equi ◽  
Caseous Lymphadenitis ◽  
Chain Reaction ◽  
Disseminated Infection ◽  
Virulence Plasmids ◽  
Visceral Organs ◽  
Polymerase Chain

Rhodococcus equi is an uncommon cause of systemic pyogranulomatous infections in goats with macroscopic similarities to caseous lymphadenitis caused by Corynebacterium pseudotuberculosis. Caprine cases have previously been reported to be caused by avirulent R. equi strains. Six cases of R. equi infection in goats yielding 8 R. equi isolates were identified from 2000 to 2017. Lesions varied from bronchopneumonia, vertebral and humeral osteomyelitis, and subcutaneous abscesses, to disseminated infection involving the lungs, lymph nodes, and multiple visceral organs. Isolates of R. equi from infected goats were analyzed by polymerase chain reaction for R. equi virulence-associated plasmid ( vap) genes. Seven of 8 isolates carried the VapN plasmid, originally characterized in bovine isolates, while 1 isolate lacked virulence plasmids and was classified as avirulent. The VapN plasmid has not been described in isolates cultured from goats.

Kato–Katz thick smears as a DNA source of soil-transmitted helminths

2018 ◽  
Vol 94 ◽  
Author(s):  
K.J.L. Monteiro ◽  
D.A. Calegar ◽  
F.A. Carvalho-Costa ◽  
L.H. Jaeger ◽  
Keyword(s):  
Evolutionary Genetics ◽  
Large Collection ◽  
Chain Reaction ◽  
Rural Regions ◽  
Dna Recovery ◽  
Polymerase Chain ◽  
N 93 ◽  
Initial Processing ◽  
Specific Species

AbstractDespite the reduction in the prevalence of soil-transmitted helminthiases in many regions of the world, morbidity rates remain high in some rural regions. The Kato–Katz technique is a simple, inexpensive and field-applicable tool commonly used for the diagnosis and worm-burden characterization of these infections. Molecular studies have revolutionized our understanding of the epidemiology and evolutionary genetics of parasites. In this study we recovered helminthic DNA from Kato–Katz slides (n = 93) prepared in 2011 in the Brazilian Amazon. We achieved DNA recovery by polymerase chain reaction (PCR) in 84% of cases for Ascaris sp. and 75% of cases for hookworms. The sequencing confirmed the specific species of the amplicons. The slides stored for a few years could be analysed using this methodology, allowing access to DNA from a large collection of samples. We must consider the Kato–Katz thick smears as a source of helminth DNA. This can significantly reduce logistical difficulties in the field in terms of obtaining, preserving, transporting and initial processing of samples.

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