scholarly journals Molecular defect of a phosphoglycerate kinase variant (PGK-Matsue) associated with hemolytic anemia: Leu----Pro substitution caused by T/A- ---C/G transition in exon 3

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1348-1352
Author(s):  
M Maeda ◽  
A Yoshida
Keyword(s):  
Hemolytic Anemia ◽  
Blot Hybridization ◽  
Phosphoglycerate Kinase ◽  
Southern Blot Hybridization ◽  
Enzyme Deficiency ◽  
Nucleotide Sequencing ◽  
Molecular Defect ◽  
Coding Regions ◽  
Variant Gene

We have identified the mutation in a phosphoglycerate kinase variant (PGK-Matsue) associated with severe enzyme deficiency, congenital nonspherocytic hemolytic anemia, and mental disorders. The mRNA coding for PGK was reverse transcribed and amplified by the polymerase chain reaction. Nucleotide sequencing of the variant cDNA showed a point mutation, a T/A----C/G transition in exon 3 of the variant gene. No other mutation was found in all coding regions of PGK-Matsue. The nucleotide change created an additional NciI cleavage site in the variant gene; thus, the NciI fragment types detected by Southern blot hybridization differ in the variant DNA and normal DNA. The mutation should cause Leu----Pro substitution at the 88th position from the NH2- terminal Ser of PGK. Because the Leu----Pro substitution is expected to induce serious perturbation and instability in the protein structure, the severe enzyme deficiency is mainly caused by more rapid in vivo denaturation and degradation of the variant enzyme.

scholarly journals Molecular defect of a phosphoglycerate kinase variant (PGK-Matsue) associated with hemolytic anemia: Leu----Pro substitution caused by T/A- ---C/G transition in exon 3

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1348-1352 ◽  
Author(s):  
M Maeda ◽  
A Yoshida
Keyword(s):  
Hemolytic Anemia ◽  
Blot Hybridization ◽  
Phosphoglycerate Kinase ◽  
Southern Blot Hybridization ◽  
Enzyme Deficiency ◽  
Nucleotide Sequencing ◽  
Molecular Defect ◽  
Coding Regions ◽  
Variant Gene

Abstract We have identified the mutation in a phosphoglycerate kinase variant (PGK-Matsue) associated with severe enzyme deficiency, congenital nonspherocytic hemolytic anemia, and mental disorders. The mRNA coding for PGK was reverse transcribed and amplified by the polymerase chain reaction. Nucleotide sequencing of the variant cDNA showed a point mutation, a T/A----C/G transition in exon 3 of the variant gene. No other mutation was found in all coding regions of PGK-Matsue. The nucleotide change created an additional NciI cleavage site in the variant gene; thus, the NciI fragment types detected by Southern blot hybridization differ in the variant DNA and normal DNA. The mutation should cause Leu----Pro substitution at the 88th position from the NH2- terminal Ser of PGK. Because the Leu----Pro substitution is expected to induce serious perturbation and instability in the protein structure, the severe enzyme deficiency is mainly caused by more rapid in vivo denaturation and degradation of the variant enzyme.

scholarly journals Pyruvate Kinase (PK) Deficiency Hereditary Nonspherocytic Hemolytic Anemia

Blood ◽  
1962 ◽  
Vol 19 (3) ◽  
pp. 267-295 ◽  
Author(s):  
KOUICHI R. TANAKA ◽  
WILLIAM N. VALENTINE ◽  
SHIRO MIWA
Keyword(s):  
Pyruvate Kinase ◽  
Hemolytic Anemia ◽  
Glycolytic Enzyme ◽  
Enzyme Deficiency ◽  
Exact Nature ◽  
Symptomatic Disease ◽  
Genetically Determined ◽  
Recessive Transmission ◽  
Enzymatic Defect

Abstract 1. The erythrocytes of seven patients conforming to the criteria of Type II congenital nonspherocytic hemolytic anemia have been demonstrated to have a specific deficiency in the glycolytic enzyme pyruvate kinase. Other glycolytic enzymes, glucose-6-phosphate and 6-phosphogluconic dehydrogenases, and certain non-glycolytic erythrocyte enzymes are normally active. The leukocytes of these patients possess normal pyruvate kinase activity. 2. Although no inhibitors were detected, the exact nature of the enzymatic defect remains to be elucidated. 3. Family studies provide strong evidence for a genetically determined disorder and are consistent with an autosomal recessive transmission of the defect. A partial enzyme deficiency, not reflected in clinical disease, is present in heterozygotes. The symptomatic disease, though variable in severity, appears to be due to homozygosity for the defect. 4. It is suggested that the enzyme deficiency is pathogenetically related to the premature demise of the red cells in vivo. 5. The name "pyruvate kinase (PK) deficiency hereditary nonspherocytic hemolytic anemia" is proposed for these patients.

A study of the messenger RNA encoding pyruvate kinase of Neurospora crassa

1986 ◽  
Vol 6 (2) ◽  
pp. 201-208
Author(s):  
M. Devchand ◽  
M. Kapoor
Keyword(s):  
Neurospora Crassa ◽  
Pyruvate Kinase ◽  
Messenger Rna ◽  
Blot Hybridization ◽  
Dot Blot ◽  
Kinase Gene ◽  
S1 Nuclease ◽  
Coding Regions

In Neurospora crassa, there is a single pyruvate kinase (PK) consisting of four identical subunits of ∼60k daltons. Northern and dot blot hybridization studies, using most of the yeast pyruvate kinase gene as a probe, suggest the presence of two distinct mRNA species for pyruvate kinase, separable on the basis of the length of their polyadenylated tails, by oligo(dT)cellulose chromatography. These messages are present in polysomes, immuno-precipitated by anti-PK antibodies, indicating probable translation in vivo. Fractions containing both messages were translated in vitro in the heterologous systems as well as in a homologous N. crassa lysate, the newly-synthesized PK being detected by immunoadsorption. Protection studies using S1-nuclease suggest no major structural differences in the 5′-untranslated and most of the coding regions of the two messages.

scholarly journals Genetic Analysis of Type E Botulinum Toxin-Producing Clostridium butyricum Strains

2000 ◽  
Vol 66 (11) ◽  
pp. 4992-4997 ◽  
Author(s):  
Xingmin Wang ◽  
Tsuneo Maegawa ◽  
Tadahiro Karasawa ◽  
Shunji Kozaki ◽  
Kentaro Tsukamoto ◽  
...  
Keyword(s):  
Botulinum Toxin ◽  
Southern Blot ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Nucleotide Sequences ◽  
Clostridium Butyricum ◽  
Nucleotide Sequencing ◽  
Lake Area ◽  
Chromosomal Dna ◽  
E Gene

ABSTRACT Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of thebont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of thebont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for thebont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.

scholarly journals Attacin A Gene from Tricloplusia ni Reduces Susceptibility to Xanthomonas axonopodis pv. citri in Transgenic Citrus sinensis `Hamlin'

2006 ◽  
Vol 131 (4) ◽  
pp. 530-536 ◽  
Author(s):  
Raquel L. Boscariol ◽  
Mariza Monteiro ◽  
Elizabete K. Takahashi ◽  
Sabrina M. Chabregas ◽  
Maria Lucia C. Vieira ◽  
...  
Keyword(s):  
Citrus Sinensis ◽  
Fluorescent Protein ◽  
Blot Hybridization ◽  
Plasmid Vector ◽  
Southern Blot Hybridization ◽  
Citrus Canker ◽  
Green Fluorescent Protein Gene ◽  
Xanthomonas Axonopodis ◽  
Transgenic Lines

Citrus canker, caused by Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al., is one of the main problems affecting citrus production. In order to obtain resistance to phytopathogenic bacteria, insect genes, coding for antimicrobial proteins, have been used in plant genetic transformation. In this study, transgenic Citrus sinensis (L.) Osb. `Hamlin' plants expressing the antimicrobial insect-derived attacin A gene (attA) were obtained by Agrobacterium tumefaciens (Smith and Towns.) Conn-mediated transformation. Initially, the cDNA clone was used to construct a binary plasmid vector (pCattA 2300). The construction included the native signal peptide (SP) responsible for directing the insect protein to the extracellular space where bacteria is supposed to accumulate in vivo. In order to investigate the native SP effectiveness in a plant model system, onion (Allium cepa L.) epidermal cells were transformed, via biobalistics, using plasmids containing the attA gene with or without SP, fused with the green fluorescent protein gene (pattA 1303 and pSPattA 1303). Fluorescence accumulation surrounding the cells was observed only in tissues transformed with the plasmid containing the gene with SP, indicating the protein secretion to the apoplast. Citrus transformation was confirmed by PCR and Southern blot hybridization analysis in 12 regenerated plants. Transcription of attA gene was detected by Northern blot analysis in all transgenic plants. Eight selected transgenic lines were propagated and inoculated with a 106 cfu/mL suspension of the pathogen X. axonopodis pv. citri. Compared to control (non-transformed plant), seven transgenic lines showed a significant reduction in susceptibility to citrus canker. The results obtained here indicate the potential use of antibacterial proteins to protect citrus from bacterial diseases.

Molecular characterization of the First Italian Variant of Phosphoglycerate Kinase Deficiency

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5270-5270
Author(s):  
Paola Bianchi ◽  
Elisa Fermo ◽  
Luana Mandarà ◽  
Cristina Vercellati ◽  
Anna Paola Maria Luisa Marcello ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Conflicts Of Interest ◽  
Phosphoglycerate Kinase ◽  
Glycolytic Enzyme ◽  
Molecular Defect ◽  
Sequencing Analysis ◽  
Coding Region ◽  
Atp Binding Site ◽  
Terminal Domain ◽  
Worldwide Distribution

Abstract Abstract 5270 Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate (1,3-BPG) to ADP to form 3-phosphoglycerate (3-PG) and ATP. It is a small monomeric molecule characterized by two hinge-bent domains, with a highly conserved structure. The N-terminal domain binds 1,3-BPG or 3-PG, whereas the C-terminal domain binds Mg-ADP or Mg-ATP. Two isozymes, PGK1 and PGK2, are present in humans, encoded by two distinct genes. Whereas PGK2 is a testis-specific enzyme, PGK1 is expressed in all the somatic cells. The PGK1 gene is located on the Xq-13.1 chromosome, and encodes a protein of 416 amino acids. Mutations of the PGK1 gene result in enzyme deficiency characterized at clinical level by mild to severe hemolytic anemia, neurological dysfunctions and myopathy. Patients rarely exhibit all three clinical features. Since the first description by Kraus et al, nearly 40 patients with PGK deficiency have been reported, 27 of them characterized at the DNA or protein level. To date, 20 different mutations with worldwide distribution have been described. The aim of the study was to characterize the molecular defect in an Italian patient affected by phosphoglycerate kinase deficiency. The patient, born from unrelated parents with negative family history of neurological defects, showed at birth neonatal jaundice. At the age of four years, in concomitance of an infective episode, he displayed hemolytic anemia (Hb 8.6 g/dL, reticulocytes 19%, unconjugated bilirubin 0.91 mg/dL, LDH 445 u/l, aptoglobin absent) and increased CPK values (2483U/L). The patient showed respiratory distress. The study of red cell glycolytic enzymes displayed a drastic reduction of PGK activity (41.8 UI/gHb ref. values 287–392). We examined again the patient at the age of 25 yrs in occasion of his sister's first pregnancy. The patient displayed compensated hemolytic anemia (Hb 14.1 g/dL, reticulocytes 6.6%) and severe myopathy. Sequencing analysis of the entire coding region and flanking intronic sequences of PGK1 gene showed the presence of a novel missense mutation c.1112 (ATA>AAA) responsible for amino acid substitution Ile371Lys. Although the mutation falls in the third last nucleotide of exon 9, it doesn't alter the splicing as confirmed by patient cDNA analysis that showed a normal transcript. The new mutation is located in a highly conserved region among species close to the ATP binding site and it was not found among the 100 normal alleles examined thus excluding the possibility of a polymorphism. Family study performed in the parents, the two healthy sisters and maternal uncle confirmed the X-linked transmission of the disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification of a Locus Involved in Systemic Dissemination of Yersinia enterocolitica

2001 ◽  
Vol 69 (10) ◽  
pp. 6201-6208 ◽  
Author(s):  
Kristin M. Nelson ◽  
Glenn M. Young ◽  
Virginia L. Miller
Keyword(s):  
Mouse Model ◽  
Yersinia Enterocolitica ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Transcriptional Regulators ◽  
Host Cells ◽  
Systemic Dissemination ◽  
Southern Blot Hybridization Analysis ◽  
Lysr Family

ABSTRACT A putative LysR-type transcriptional activator, Hre20, was identified previously in an in vivo expression technology screen designed to identify factors which are expressed early during infection by Yersinia enterocolitica (G. M. Young and V. L. Miller, Mol. Microbiol. 25:319–328, 1997). An insertion in hre20, now designated rscR, resulted in increased splenic dissemination of bacteria during infection in a BALB/c mouse model. A nonpolar mutation was generated inrscR, and examination of this strain in the BALB/c mouse model demonstrated that the mutation in rscR was responsible for the increased dissemination to the spleen that was seen in the original experiments. RscR is homologous to the LysR family of transcriptional regulators; thus, a screen was undertaken to identify genes regulated by RscR. A strain containing an insertion in the chromosomal rscR gene and carrying rscR on a plasmid under the control of the inducible araBAD promoter was mutagenized with an mTn5Km-2 transposon containing a promoterless lacZY. Eighteen insertions were identified which appeared to respond to levels of RscR, and these were classified into four allelic groups based on Southern blot hybridization analysis. Representative members were sequenced from three allelic groups. Sequencing revealed insertions in an ORF with no known homologues, a homologue of OmpF of Serratia marcescens, and a locus (designated rscBAC) with similarity to thehmwABC locus of Haemophilus influenzae. ThehmwABC locus promotes adherence of H. influenzae to host cells (S. J. Barenkamp and J. W. St. Geme III, Infect. Immun. 62:3320–3328, 1994; J. W. St. Geme III, S. Falkow, and S. J. Barenkamp, Proc. Natl. Acad. Sci. USA 90:2875–2879, 1993). A strain containing a deletion mutant of rscA, the hmwA homologue, exhibits increased splenic dissemination of bacteria during infection in a BALB/c mouse model, similar to the rscR mutant. This suggests that the phenotype of an rscR mutant is due to the loss of RscA.

scholarly journals Molecular abnormalities of a phosphoglycerate kinase variant generated by spontaneous mutation

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2759-2762 ◽  
Author(s):  
M Maeda ◽  
EV Bawle ◽  
R Kulkarni ◽  
E Beutler ◽  
A Yoshida
Keyword(s):  
Messenger Rna ◽  
Spontaneous Mutation ◽  
Phosphoglycerate Kinase ◽  
Cultured Fibroblasts ◽  
Molecular Abnormalities ◽  
Coding Regions ◽  
Variant Gene ◽  
Exon 9 ◽  
Polymerase Chain ◽  
Variant Enzyme

A new case of X chromosome-linked phosphoglycerate kinase (PGK) abnormality is described. The male proband was mentally retarded, had behavior disorders, and displayed episodes of hemolytic anemia. The enzyme activity of red blood cells from the patient was about 10% of normal, and that of the cultured fibroblasts was about 50% of normal cells. The variant PGK was characterized by a lower affinity for the substrates, reduced thermostability, and increased anodal electrophoretic mobility. The pH activity profile of the variant enzyme was different from that of normal. The amount of messenger RNA (mRNA) in the variant fibroblasts was comparable to that of normal fibroblasts. The mRNA coding for PGK was subjected to coupled reverse transcription followed by amplification by the polymerase chain reaction. Nucleotide sequence of the variant cDNA showed a point mutation, T/A----C/G transition, in exon 9 of the variant gene. No other mutation was found in all coding regions of the variant. The mutation should cause Cys----Arg substitution at the 315th position from the NH2-terminal Ser of PGK, and it created an additional Ava II (or isoschimatic) cleavage site in the variant gene. Because the variant gene was not detected in the proband's mother and siblings, it must have been generated by spontaneous mutation during oogenesis.

scholarly journals Molecular abnormalities of a phosphoglycerate kinase variant generated by spontaneous mutation

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2759-2762
Author(s):  
M Maeda ◽  
EV Bawle ◽  
R Kulkarni ◽  
E Beutler ◽  
A Yoshida
Keyword(s):  
Messenger Rna ◽  
Spontaneous Mutation ◽  
Phosphoglycerate Kinase ◽  
Cultured Fibroblasts ◽  
Molecular Abnormalities ◽  
Coding Regions ◽  
Variant Gene ◽  
Exon 9 ◽  
Polymerase Chain ◽  
Variant Enzyme

Abstract A new case of X chromosome-linked phosphoglycerate kinase (PGK) abnormality is described. The male proband was mentally retarded, had behavior disorders, and displayed episodes of hemolytic anemia. The enzyme activity of red blood cells from the patient was about 10% of normal, and that of the cultured fibroblasts was about 50% of normal cells. The variant PGK was characterized by a lower affinity for the substrates, reduced thermostability, and increased anodal electrophoretic mobility. The pH activity profile of the variant enzyme was different from that of normal. The amount of messenger RNA (mRNA) in the variant fibroblasts was comparable to that of normal fibroblasts. The mRNA coding for PGK was subjected to coupled reverse transcription followed by amplification by the polymerase chain reaction. Nucleotide sequence of the variant cDNA showed a point mutation, T/A----C/G transition, in exon 9 of the variant gene. No other mutation was found in all coding regions of the variant. The mutation should cause Cys----Arg substitution at the 315th position from the NH2-terminal Ser of PGK, and it created an additional Ava II (or isoschimatic) cleavage site in the variant gene. Because the variant gene was not detected in the proband's mother and siblings, it must have been generated by spontaneous mutation during oogenesis.

Selection and characterization ofSpiroplasma citrimutants by random transposome mutagenesis

2011 ◽  
Vol 57 (6) ◽  
pp. 525-532 ◽  
Author(s):  
Kikin Mutaqin ◽  
Jana L. Comer ◽  
Astri C. Wayadande ◽  
Ulrich Melcher ◽  
Jacqueline Fletcher
Keyword(s):  
Genetic Manipulation ◽  
Blot Hybridization ◽  
Random Mutagenesis ◽  
Southern Blot Hybridization ◽  
Nucleotide Sequencing ◽  
Spiroplasma Citri ◽  
Additional Tool ◽  
Reductase Gene ◽  
Host Life ◽  
Stable Growth

Phytopathogenic spiroplasmas can multiply in vascular plants and insects. A deeper understanding of this dual-host life could be furthered through the identification by random mutagenesis of spiroplasma genes required. The ability of the EZ::TN™ <DHFR-1> Tnp transposome™ system to create random insertional mutations in the genome of Spiroplasma citri was evaluated. The efficiency of electroporation-mediated transformation of S. citri BR3-3X averaged 28.8 CFUs/ng transposome for 109spiroplasma cells. Many transformants appearing on the selection plates were growth impaired when transferred to broth. Altering broth composition in various ways did not improve their growth. However, placing colonies into a small broth volume resulted in robust growth and successful subsequent passages of a subset of transformants. PCR using primers for the dihydrofolate reductase gene confirmed the transposon’s presence in the genomes of selected transformants. Southern blot hybridization and nucleotide sequencing suggested that insertion was random within the chromosome and usually at single sites. The insertions were stable. Growth rates of all transformants were lower than that of the wild-type S. citri, but none lost the ability to adhere to a Circulifer tenellus (CT-1) cell line. The EZ::TN™ <DHFR-1> Tnp transposome™ system represents an additional tool for genetic manipulation of the fastidious spiroplasmas.

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