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.

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.

High performance Nanogold™-in situ hybridzation and its use in the detection of hybridized and PCR-amplified microscopical preparations

1996 ◽  
Vol 54 ◽  
pp. 896-897
Author(s):  
G. W. Hacker ◽  
I. Zehbe ◽  
J. Hainfeld ◽  
A.-H. Graf ◽  
C. Hauser-Kronberger ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Messenger Rna ◽  
High Performance ◽  
Detection System ◽  
Direct Methods ◽  
Genetic Alterations ◽  
Chain Reaction ◽  
Protein Encoding ◽  
Polymerase Chain

In situ hybridization (ISH) with biotin-labeled probes is increasingly used in histology, histopathology and molecular biology, to detect genetic nucleic acid sequences of interest, such as viruses, genetic alterations and peptide-/protein-encoding messenger RNA (mRNA). In situ polymerase chain reaction (PCR) (PCR in situ hybridization = PISH) and the new in situ self-sustained sequence replication-based amplification (3SR) method even allow the detection of single copies of DNA or RNA in cytological and histological material. However, there is a number of considerable problems with the in situ PCR methods available today: False positives due to mis-priming of DNA breakdown products contained in several types of cells causing non-specific incorporation of label in direct methods, and re-diffusion artefacts of amplicons into previously negative cells have been observed. To avoid these problems, super-sensitive ISH procedures can be used, and it is well known that the sensitivity and outcome of these methods partially depend on the detection system used.

scholarly journals Neurodegenerative diseases: a hotbed for splicing defects and the potential therapies

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Dunhui Li ◽  
Craig Stewart McIntosh ◽  
Frank Louis Mastaglia ◽  
Steve Donald Wilton ◽  
May Thandar Aung-Htut
Keyword(s):  
Alternative Splicing ◽  
Neurodegenerative Diseases ◽  
Messenger Rna ◽  
Muscular Atrophy ◽  
Single Gene ◽  
Synaptic Function ◽  
Coding Regions ◽  
Splicing Defects ◽  
The Impact ◽  
Splicing Patterns

AbstractPrecursor messenger RNA (pre-mRNA) splicing is a fundamental step in eukaryotic gene expression that systematically removes non-coding regions (introns) and ligates coding regions (exons) into a continuous message (mature mRNA). This process is highly regulated and can be highly flexible through a process known as alternative splicing, which allows for several transcripts to arise from a single gene, thereby greatly increasing genetic plasticity and the diversity of proteome. Alternative splicing is particularly prevalent in neuronal cells, where the splicing patterns are continuously changing to maintain cellular homeostasis and promote neurogenesis, migration and synaptic function. The continuous changes in splicing patterns and a high demand on many cis- and trans-splicing factors contribute to the susceptibility of neuronal tissues to splicing defects. The resultant neurodegenerative diseases are a large group of disorders defined by a gradual loss of neurons and a progressive impairment in neuronal function. Several of the most common neurodegenerative diseases involve some form of splicing defect(s), such as Alzheimer’s disease, Parkinson’s disease and spinal muscular atrophy. Our growing understanding of RNA splicing has led to the explosion of research in the field of splice-switching antisense oligonucleotide therapeutics. Here we review our current understanding of the effects alternative splicing has on neuronal differentiation, neuronal migration, synaptic maturation and regulation, as well as the impact on neurodegenerative diseases. We will also review the current landscape of splice-switching antisense oligonucleotides as a therapeutic strategy for a number of common neurodegenerative disorders.

An Analysis of WNT5A expression in Wilms’ Tumour

2018 ◽  
Author(s):  
Nicholas Laughton
Keyword(s):  
Messenger Rna ◽  
Published Data ◽  
Expression Data ◽  
Wilms Tumour ◽  
Chain Reaction ◽  
Wnt5a Expression ◽  
Extracellular Signal ◽  
Developing Kidney ◽  
Polymerase Chain ◽  
Pax2 Expression

Wilms’ tumour is a pediatric tumour of the kidney that appears to be the result of abherrent embryonal renal development. The paired­box (PAX) gene family has previously been implicated in Wilm’s tumorogenesis. In this study, Nickel­Agarose Chromatin Enrichment (NACE) was used to identify genes whose expression is regulated by the ranscription cofactor Pax2. Of the genes identified by NACE, the extracellular signal metabolite WNT5A was chosen fro further study. The expression of WNT5A was measured in a set of tumour samples using quantitative real time polymerase chain reaction (qRT­PCR) and compared to a human fetal kidney control. Of the 38 samples tested, 76% showed significantly lower levelsof cytosolic messenger RNA (mRNA). This data, in conjunction with published data on Pax2 expression, suggests Pax2 inhibits the expression of WNT5A. When compared with histological reports for the tumours we examined, the expression data implies that WNT5A may have a role in regulation of tubule growth in the developing kidney

Characterization of alpha-adrenoceptor gene expression in arterial and venous smooth muscle

1993 ◽  
Vol 265 (5) ◽  
pp. H1501-H1509 ◽  
Author(s):  
P. Ping ◽  
J. E. Faber
Keyword(s):  
Smooth Muscle ◽  
Messenger Rna ◽  
Vena Cava ◽  
Pcr Analysis ◽  
Relative Distribution ◽  
Alpha Adrenoceptor ◽  
Aortic Media ◽  
Unique Restriction ◽  
Polymerase Chain

Six genes coding for three unique alpha 1- (1A, 1B, 1C) and three unique alpha 2- (2A, 2B, 2C) adrenergic receptor (AR) subtypes have been cloned. Ligand binding and contractile studies have demonstrated that both alpha 1- and alpha 2-ARs can exist on vascular smooth muscle (VSM) cells, although less is known about the relative distribution and specific subtypes in different vascular segments. In the present study polymerase chain reaction (PCR) analysis was used to characterize the species of alpha-AR messenger RNA (mRNA) present in freshly isolated rat thoracic aortic media and vena cava and in cultured VSM cells (passage 2) derived from both sources. To prevent possible contamination of VSM mRNA, aortic media was separated from adventitia, and vessels were denuded of endothelial cells. Oligonucleotide primers specific for each of the six adrenergic genes were synthesized and used to probe for the presence of alpha-AR mRNA species after reverse transcription of total cellular RNA to cDNA. PCR-amplified AR transcripts were distinguished by the size of amplified DNA fragments and unique restriction endonuclease cleavage. Expression of alpha 1C- or alpha 2C-mRNA was not detected in vascular tissues or cultured VSM cells, although the alpha 2C-primers detected the expected alpha 2C expression in cerebral cortex. Only alpha 1A-mRNA was detected in aortic adventitia. VSM from aorta expressed alpha 1A-, alpha 1B-, and alpha 2A-mRNA, and this pattern was preserved in cultured aortic VSM. Vena cava also expressed both alpha 1A and alpha 1B; however only alpha 2B-mRNA was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

MSX1, PAX9, and TGFA Contribute to Tooth Agenesis in Humans

2004 ◽  
Vol 83 (9) ◽  
pp. 723-727 ◽  
Author(s):  
A.R. Vieira ◽  
R. Meira ◽  
A. Modesto ◽  
J.C. Murray
Keyword(s):  
Dna Sequence ◽  
Cleft Lip ◽  
Dna Analysis ◽  
Ethnically Diverse ◽  
Tooth Agenesis ◽  
Single Strand Conformational Polymorphism ◽  
Coding Regions ◽  
Cleft Lip Palate ◽  
Transmission Distortion ◽  
Polymerase Chain

In this study, we sought to determine the association between tooth agenesis and DNA sequence variation in the genes MSX1 and PAX9 in an ethnically diverse human population. Since cleft lip/palate is also associated with both tooth agenesis and the gene TGFA, we included TGFA in the analysis as well. Cheek swab samples were obtained for DNA analysis from 116 case/parent trios. Probands had at least one developmentally missing tooth, excluding third molars. Genotyping was performed by single-strand conformational polymorphism or kinetic polymerase chain-reaction assays. Transmission distortion of the marker alleles and DNA sequence analysis was performed. Results showed that tooth agenesis is associated with markers of the genes MSX1 and TGFA. No mutations were found in MSX1 or PAX9 coding regions. There were statistically significant data suggesting that MSX1 interacts with PAX9. These findings suggest that MSX1, PAX9, and TGFA play a role in isolated dental agenesis.

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