Truncation of Glycoprotein (GP) IIIa (▵ 616-762) Prevents Complex Formation With GPIIb: Novel Mutation in Exon 11 of GPIIIa Associated With Thrombasthenia

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4712-4720 ◽  
Author(s):  
Milagros Ferrer ◽  
Jianming Tao ◽  
Gema Iruı́n ◽  
Matilde Sánchez-Ayuso ◽  
José González-Rodrı́guez ◽  
...  
Keyword(s):  
Complex Formation ◽  
Novel Mutation ◽  
Molecular Genetic ◽  
Coding Region ◽  
Chain Reaction ◽  
Molecular Genetic Study ◽  
Nonsense Mutations ◽  
Glanzmann Thrombasthenia ◽  
Polymerase Chain ◽  
Exon 11

Abstract This work reports the molecular genetic study of a patient who suffered from Glanzmann thrombasthenia (GT). Structural analysis of the glycoprotein (GP) IIb and GPIIIa genes showed the presence of a homozygous G1846→T transversion in exon 11 of GPIIIa that changes Glu616→Stop. Cytometric and immunochemical analysis indicated that platelet GPIIb-IIIa was absent in the proband but present at normal levels in the heterozygous relatives. The following observations indicate that this mutation is responsible for the thrombasthenic phenotype of the proband. (1) We failed to detect mutations other than [T1846]GPIIIa in the coding region of both GPIIb and GPIIIa genes. (2) The G1846→T mutation was observed in either parent and a brother of the proband, but none of 100 unrelated individuals carried this defect. (3) Pulse-chase and immunoprecipitation analysis of GPIIb-IIIa complexes in cells transiently cotransfected with cDNAs encoding normal GPIIb and [T1846]GPIIIa showed neither maturation of GPIIb nor complex formation and surface exposure of GPIIb-▵GPIIIa. These observations indicate that the sequence from Glu616 to Thr762 in GPIIIa is essential for heterodimerization with GPIIb. Polymerase chain reaction-based analysis demonstrated the presence of normal levels of full-length GPIIIa-mRNA in the proband and in heterozygous relatives. In addition, a shortened transcript, with a 324-nucleotide deletion, resulting from in-frame skipping of exons 10 and 11, was detectable upon reamplification of the DNA. Thus, unlike other nonsense mutations, [T1846]GPIIIa does not lead to abnormal processing or reduction in the number of transcripts with the termination codon.

Truncation of Glycoprotein (GP) IIIa (▵ 616-762) Prevents Complex Formation With GPIIb: Novel Mutation in Exon 11 of GPIIIa Associated With Thrombasthenia

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4712-4720 ◽  
Author(s):  
Milagros Ferrer ◽  
Jianming Tao ◽  
Gema Iruı́n ◽  
Matilde Sánchez-Ayuso ◽  
José González-Rodrı́guez ◽  
...  
Keyword(s):  
Complex Formation ◽  
Novel Mutation ◽  
Molecular Genetic ◽  
Coding Region ◽  
Chain Reaction ◽  
Molecular Genetic Study ◽  
Nonsense Mutations ◽  
Glanzmann Thrombasthenia ◽  
Polymerase Chain ◽  
Exon 11

This work reports the molecular genetic study of a patient who suffered from Glanzmann thrombasthenia (GT). Structural analysis of the glycoprotein (GP) IIb and GPIIIa genes showed the presence of a homozygous G1846→T transversion in exon 11 of GPIIIa that changes Glu616→Stop. Cytometric and immunochemical analysis indicated that platelet GPIIb-IIIa was absent in the proband but present at normal levels in the heterozygous relatives. The following observations indicate that this mutation is responsible for the thrombasthenic phenotype of the proband. (1) We failed to detect mutations other than [T1846]GPIIIa in the coding region of both GPIIb and GPIIIa genes. (2) The G1846→T mutation was observed in either parent and a brother of the proband, but none of 100 unrelated individuals carried this defect. (3) Pulse-chase and immunoprecipitation analysis of GPIIb-IIIa complexes in cells transiently cotransfected with cDNAs encoding normal GPIIb and [T1846]GPIIIa showed neither maturation of GPIIb nor complex formation and surface exposure of GPIIb-▵GPIIIa. These observations indicate that the sequence from Glu616 to Thr762 in GPIIIa is essential for heterodimerization with GPIIb. Polymerase chain reaction-based analysis demonstrated the presence of normal levels of full-length GPIIIa-mRNA in the proband and in heterozygous relatives. In addition, a shortened transcript, with a 324-nucleotide deletion, resulting from in-frame skipping of exons 10 and 11, was detectable upon reamplification of the DNA. Thus, unlike other nonsense mutations, [T1846]GPIIIa does not lead to abnormal processing or reduction in the number of transcripts with the termination codon.

Characterization of the Original Christmas Disease Mutation (Cysteine 206 → Serine): From Clinical Recognition to Molecular Pathogenesis

1992 ◽  
Vol 67 (01) ◽  
pp. 063-065 ◽  
Author(s):  
Sherryl A M Taylor ◽  
Jacalyn Duffin ◽  
Cherie Cameron ◽  
Jerome Teitel ◽  
Bernadette Garvey ◽  
...  
Keyword(s):  
Nucleotide Substitution ◽  
Novel Mutation ◽  
Factor Ix ◽  
Causative Mutation ◽  
Coding Region ◽  
Body Of Knowledge ◽  
Polymerase Chain ◽  
Splice Junctions

SummaryChristmas disease was first reported as a distinct clinical entity in two manuscripts published in 1952 (1, 2). The eponym associated with this disorder, is the surname of the first patient examined in detail and reported by Biggs and colleagues in a paper describing the clinical and laboratory features of seven affected individuals (3). This patient has severe factor IX coagulant deficiency (less than 0.01 units/ml) and no detectable circulating factor IX antigen (less than 0.01 units/ml). Coding sequence and splice junctions of the factor IX gene from this patient have been amplified in vitro through the polymerase chain reaction (PCR). One nucleotide substitution was identified at nucleotide 30,070 where a guanine was replaced by a cytosine. This mutation alters the amino acid encoded at position 206 in the factor IX protein from cysteine to serine. The non conservative nature of this substitution, the absence of this change in more than 200 previously sequenced factor IX genes and the fact that the remainder of the coding region of this gene was normal, all provide strong circumstantial evidence in favour of this change being the causative mutation in this patient. The molecular characterization of this novel mutation in the index case of Christmas disease, contributes to the rapidly expanding body of knowledge pertaining to Christmas disease pathogenesis.

A Study of the Efficiency of Modern Domestic Disinfectants in the System of TB Control Activities

2015 ◽  
Vol 2 (2) ◽  
pp. 26-31 ◽  
Author(s):  
A. Paliy ◽  
A. Zavgorodniy ◽  
B. Stegniy ◽  
A. Gerilovych
Keyword(s):  
Molecular Genetic ◽  
Critical Role ◽  
Bactericidal Effect ◽  
Chain Reaction ◽  
Tb Control ◽  
Source Of Infection ◽  
Polymerase Chain ◽  
And Control ◽  
Chemical Groups ◽  
Test Objects

Due to the absence of elaborated effi cient means for specifi c prevention of bovine tuberculosis, it is ex- tremely important to detect and eliminate the source of infection and to take veterinary and sanitary preven- tive measures. Here the critical role is attributed to disinfection, which breaks the epizootic chain due to the elimination of pathogenic microorganisms in the environment and involves the application of disinfectants of different chemical groups. Aim. To study the tuberculocidal properties of new disinfectants DZPT-2 and FAG against atypical mycobacteria Mycobacterium fortitum and a TB agent Mycobacterium bovis. Methods. The bacteriological and molecular-genetic methods were used. Results. It was determined that DZPT-2 prepara- tion has bactericidal effect on M. fortuitum when used in the concentration of 2.0 % of the active ingredient (AI) when exposed for 5–24 h, while disinfectant FAG has a bactericidal effect in the concentration of 2.0 % when exposed for 24 h. Disinfectant DZPT-2 in the concentration of 2.0 % of the AI, when exposed for 5–24 h, and FAG preparation in the concentration of 2.0 %, when exposed for 24 h, and with the norm of consump- tion rate of 1 cubic decimeter per 1 square meter disinfect the test-objects (batiste, wood, glazed tile, metal, glass), contaminated with the TB agent M. bovis. Conclusions. Disinfecting preparations of DZPT-2 in the concentration of 2.0 % of AI when exposed for 5 h and FAG in the concentration of 2.0 % when exposed for 24 h may be used in the complex of veterinary and sanitary measures to prevent and control TB of farm ani- mals. The possibility of using the polymerase chain reaction as an additional method of estimating tuberculo- cide activity of disinfectants was proven.

scholarly journals Deteksi Transgen (Glu-1Dx5) pada Populasi Padi (Oryza sativa L.) Putative Transgenik Kultivar Fatmawati

Agrikultura ◽  
2010 ◽  
Vol 21 (1) ◽  
Author(s):  
Nono Carsono ◽  
Sri Nurlianti ◽  
Inez Nur Indrayani ◽  
Ade Ismail ◽  
Tri Joko Santoso ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Oryza Sativa ◽  
Dna Polymerase ◽  
Genomic Dna ◽  
Oryza Sativa L ◽  
Dna Purification ◽  
Coding Region ◽  
Chain Reaction ◽  
Taq Dna Polymerase ◽  
Polymerase Chain

Transformasi gen Glu-1Dx5, pengendali utama karakter elastisitas dan daya mengembang adonan dari gandum, telah berhasil ditransfer ke dalam genom tanaman padi kultivar Fatmawati dengan menggunakan penembakan partikel, dengan tujuan untuk memperbaiki kualitas adonan tepung beras. Galur-galur harapan telah diperoleh, tetapi karena telah mengalami penyerbukan sendiri selama 1-2 generasi yang menyebabkan transgen mengalami segregasi, maka diperlukan upaya pendeteksian transgen pada populasi putative transgenik ini. Upaya ini dapat dilakukan, antara lain dengan menggunakan teknik Polymerase Chain Reaction (PCR) yang memungkinkan perbanyakan fragmen DNA yang spesifik (gen) secara cepat dalam jumlah banyak.  Percobaan ini bertujuan untuk mendapatkan tanaman padi transgenik yang memiliki gen Glu-1Dx5 pada dua generasi yang sedang bersegregasi. DNA genom dari 149 tanaman padi (generasi T1 sebanyak 14 tanaman, generasi T2 sebanyak 134 tanaman, dan satu tanaman non-transgenik) telah diekstraksi menggunakan Genomic DNA Purification Kit dari Fermentas. Plasmid pK+Dx5 digunakan sebagai positif kontrol, selain itu digunakan juga enzim Taq DNA polymerase dari Go Green Taq® Master Mix (Promega) dan 2 primer spesifik yang mengamplifikasi coding region dari Glu-1Dx5 (2,5 kb). Hasil percobaan menunjukkan, tanaman padi yang memiliki gen Glu-1Dx5 pada generasi T2-7 sebanyak 26 tanaman, T2-11 : 12 tanaman, T2-12 : 3 tanaman, T2-40 : 3 tanaman dan T2-45 : 5 tanaman. Seluruh tanaman generasi T1 tidak memiliki insert. Hasil ini menunjukkan bahwa gen Glu-1Dx5 sudah terintegrasi ke dalam genom tanaman padi kultivar Fatmawati dan diwariskan dari satu generasi ke generasi berikutnya.

scholarly journals Association of the I/D polymorphism of angiotensinconverting enzyme gene with the development of essential hypertension

2019 ◽  
Vol 34 (3) ◽  
pp. 87-96
Author(s):  
O. S. Pavlova ◽  
S. E. Ogurtsova ◽  
M. M. Liventseva ◽  
T. H. Lakotko ◽  
I. Y. Korobko ◽  
...  
Keyword(s):  
Essential Hypertension ◽  
Molecular Genetic ◽  
Recessive Model ◽  
Control Group ◽  
Molecular Genetic Study ◽  
Ace Gene ◽  
Paternal Line ◽  
Male Patients ◽  
Polymerase Chain ◽  
The Impact

Objective. To determine the impact of the I/D polymorphism of the angiotensin-converting enzyme (ACE) gene on the development of essential hypertension, taking into account gender differences.Material and Methods. Clinical data were assessed and a molecular genetic study was performed in 602 people including 401 patients with essential hypertension and 201 individuals of the control group, representing the Belarusian ethnic group. Genotyping was performed using the method of polymerase chain reaction and restriction fragment length polymorphism.Results. The distribution of genotypes of the I/D polymorphism of the ACE gene did not differ between patients with hypertension and normotensive individuals: II, ID, and DD genotypes were detected in 100 (24.9%), 192 (47.9%), and 109 (27.2%) patients and in 52 (25.9%), 108 (53.7%), and 41 (20.4%) people of the comparison group, respectively. Differences were found between the distribution of DD genotype in men with hypertension and in the control group, where the frequencies were 28.4% and 17.3% (p  =  0.04), respectively, in contrast to no differences in women: 25.8% and 23.3% (p  =  0.64), respectively. Carrying the DD genotype in men compared with the ID and DD genotypes (recessive model) of the I/D polymorphism of the ACE gene increased the probability of developing essential hypertension by 1.9 times (OR   =   1.89; 95% CI  =  1.04-3.44). The analysis of the prevalence of risk factors depending on the I/D polymorphism of the ACE gene showed that male patients with the DD genotype more often had burdened heredity in regard to the development of premature cardiovascular diseases (23 patients (37.7%)) compared with the individuals with II and ID genotypes: 13 (21.7%) and 14 (14.9%) patients, respectively (χ2  =  1.16; p  =   0.005), and mainly through the paternal line.Conclusions. Development of essential hypertension is associated with the carriership of the mutant DD genotype of I/D polymorphism of the ACE gene in men. 

On the Expression of Several Lhc Genes in Garden Cress (Lepidium sativum L.)

1994 ◽  
Vol 49 (11-12) ◽  
pp. 802-810 ◽  
Author(s):  
Harald Brunner ◽  
Wolfhart Rüdiger
Keyword(s):  
Lepidium Sativum ◽  
Published Data ◽  
Coding Region ◽  
Chain Reaction ◽  
Transcript Levels ◽  
Coding Regions ◽  
B1 Gene ◽  
Polymerase Chain ◽  
Lhc Genes ◽  
Lepidium Sativum L

The polymerase chain reaction was used to prepare gene-specific probes for several Lhc genes coding for chlorophyll a/b-binding proteins of cress (Lepidium sativum L.). Due to the presence of about 150 basepairs of the coding region, the isolated clones could be attributed to Lhc a3 (1 clone), Lhc b1 (5 clones), Lhc b2 (1 clone) and Lhc b3 (1 clone) genes. Probes prepared from the 3′ non-coding regions of the clones did not cross-hybridize; they were specific for 3 different Lhc b1 transcripts and one each of Lhc b2, Lhc b3 and Lhc a 3 transcripts. The transcript levels were higher in leaves than in cotyledons of light-grown seedlings; they decreased significantly in cotyledons from week 1 to week 4. The levels of 2 Lhc b1 transcripts (detected with probes cd 1 and cd 2) changed from 1 week old cotyledons (30% c d l, 28% cd 2) to 3 months old leaves (14% c d l), 44% cd 2), stems (11% c d l, 56% cd 2) and fruits (15% cd 1, 62% cd 2, all values percent of total transcripts), whereas transcript levels of another Lhc b1 gene (detected with probe cd 3) and of a Lhc a 3 gene remained nearly constant. The level of Lhc b2 and Lhc b3 transcripts were 1 - 2 orders of magnitude smaller than those of the other Lhc transcripts. The data obtained with cress plants are compared with published data from other plants.

Minimal intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA of lake trout (Salvelinus namaycush)

Genome ◽  
1994 ◽  
Vol 37 (4) ◽  
pp. 664-671 ◽  
Author(s):  
Lang Zhuo ◽  
S. L. Sajdak ◽  
R. B. Phillips
Keyword(s):  
Polymerase Chain Reaction ◽  
Intraspecific Variation ◽  
Ribosomal Dna ◽  
Internal Transcribed Spacer ◽  
Lake Trout ◽  
Intergenic Spacer ◽  
Coding Region ◽  
Chain Reaction ◽  
Internal Transcribed Spacer Region ◽  
Polymerase Chain

Intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA (rDNA) in lake trout was examined by restriction mapping and sequencing of these regions amplified by the polymerase chain reaction. The length of the first internal transcribed spacer region (ITS-1) was 566 bases and the second internal transcribed spacer region (ITS-2) was 368 bases in lake trout. When the 1.4-kb region including the ITS-1, the 5.8S coding region, and the ITS-2 was amplified from 12 individuals from four populations and digested with eight different enzymes only one intraindividual polymorphism was found that occurred in each population. When the amplified ITS-1 region was sequenced from an additional 10 individuals from five populations, no interindividual variation was found in the sequence. A 6-kb portion of the rDNA repeat unit including 1.6 kb of the 18S coding region, the 5′ external spacer region (5′ ETS), and part of the adjacent intergenic spacer was cloned and a restriction map was prepared for these regions in lake trout. No intraspecific variation was found in the region adjacent to the 18S rDNA, which includes the 5′ ETS, although intraspecific and intraindividual length variation was found in the intergenic spacer region 3–6 kb from the 18S. Sequencing of a 609-b segment of the 5′ ETS adjacent to the 18S coding region revealed the presence of two 41-b repeats. The 198-b sequence between the repeats had some similarity to the 18S coding region of other fishes. Primers were designed for amplification of 559 b of the 5′ ETS using the polymerase chain reaction. No intraspecific variation in this region in lake trout was found when the DNA amplified from this region in 12 individuals from four populations was digested with eight restriction enzymes.Key words: ribosomal DNA, internal transcribed spacer regions, 5′ external spacer region, transcribed spacer, lake trout.

Sex determination in ostrich (Struthio camelus) using DNA markers

2010 ◽  
Vol 90 (3) ◽  
pp. 357-360 ◽  
Author(s):  
M. Alipanah ◽  
A. Torkamanzehi ◽  
H. Taghavi
Keyword(s):  
Polymerase Chain Reaction ◽  
Sexual Dimorphism ◽  
Sex Determination ◽  
Sex Chromosome ◽  
Molecular Genetic ◽  
Bird Species ◽  
Rapid Identification ◽  
Chain Reaction ◽  
Struthio Camelus ◽  
Polymerase Chain

Production of bird species such as ostrich (Struthio camelus) has been gaining increasing importance in Iran as well as many other countries. Ostrich, similar to many other species of birds, lacks sexual dimorphism, making it difficult to differentiate between males and females, especially at an early age, which can be problematic in breeding programs. Recently developed molecular genetic methods that utilize polymerase chain reaction (PCR) based techniques can facilitate rapid identification of the bird’s sex in these species using a DNA sample, which can be easily extracted from blood or feather pulps. We successfully applied a PCR-based RFLP technique and sex chromosome primers for sex determination in a sample of 30 Ostrich chicks using DNA extracted from blood and feather pulps. Both DNA samples (blood and feather pulps) provided useful results. However, using feather pulps from 1-day-old chicks can provide an easy and inexpensive method for sex determination in ostrich. Key words: Ostrich (struthio camelus), sex determination, sexual dimorphism, polymerase chain reaction, RFLP

Central neurocytoma: morphological, flow cytometric, polymerase chain reaction, fluorescence in situ hybridization, and karyotypic analyses

1999 ◽  
Vol 90 (2) ◽  
pp. 348-354 ◽  
Author(s):  
Venita Jay ◽  
Vern Edwards ◽  
Eelco Hoving ◽  
James Rutka ◽  
Laurence Becker ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
In Situ Hybridization ◽  
Ganglion Cells ◽  
Molecular Genetic ◽  
Central Neurocytoma ◽  
Molecular Genetic Analysis ◽  
Fish Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

✓ The results of cytogenetic and molecular genetic analysis of a central neurocytoma are presented. Central neurocytomas are intriguing neoplasms that exhibit primarily neuronal, but also glial characteristics, which indicate an origin from a pluripotential neuroglial precursor. The authors describe an intraventricular neurocytoma in an 11-year-old boy that showed anaplastic features with widespread necrosis and mitoses, as well as extensive calcification and foci that exhibited marked neuronal differentiation with clusters of ganglion cells. Immunohistochemical examination showed prominent synaptophysin and neurofilament positivity and focal glial fibrillary acidic protein positivity. Electron microscopy revealed abundant neuritic processes with microtubules and dense core granules as well as mature ganglion cells. Flow cytometry studies revealed increased S (7.8%) and G2M (9.7%) phase components. Molecular and cytogenetic studies were undertaken to assess whether there were similarities to two other tumor types that exhibit neuronal differentiation, the neuroblastoma and medulloblastoma. Polymerase chain reaction and fluorescence in situ hybridization (FISH) analysis revealed no evidence of amplification of the MYCN oncogene or chromosome 1p deletion, which are common in neuroblastomas. Chromosomal analysis by G banding revealed a complex karyotype, with counts in the near-diploidy range (45–48). Two chromosomes 1 appeared normal on G banding and FISH analysis, with p58 signals present on the distal p arm of both chromosomes 1; however, three additional copies of distal 1q were present in rearrangements with 4 and 7. Although the histological findings indicate a kinship to the neuroblastoma and medulloblastoma, the central neurocytoma appears to have a different karyotypic profile, although more cases need to be assessed using molecular genetic analysis.

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