scholarly journals Comparative Molecular Description of a Novel GST Gene in Culex pipiens (Diptera: Culicidae)

2020 ◽  
Vol 57 (5) ◽  
pp. 1440-1446
Author(s):  
Hagar Samy Ali ◽  
Amany Soliman Khaled ◽  
Laila Sayed Hamouda ◽  
Enas Hamdy Ghallab
Keyword(s):  
Culex Pipiens ◽  
Direct Sequencing ◽  
Pairwise Alignment ◽  
Reference Sequence ◽  
Rflp Markers ◽  
Glutathione S Transferase ◽  
Disease Vector ◽  
Diagnostic Time ◽  
Susceptibility Status ◽  
Molecular Description

Abstract Repeated exposure to insecticides, particularly pyrethroids and organophosphates, has resulted in the development of insecticide resistance in the mosquito Culex pipiens, a primary disease vector. Glutathione S-transferase (GST) is involved in the phase II detoxification of numerous xenobiotics, including insecticides. In this study, a GST gene (CPIJ002678) was amplified, sequenced, and used in comprehensive molecular analyses ending up in development of a rapid assay to distinguish more tolerant individuals from susceptible Culex pipiens using the Restriction Fragment Length Polymorphism (RFLP) technique. Field collected Culex pipiens strains from untreated areas, organophosphates-treated areas and a lab strain reared for many generations, all were used in CDC bottle bioassays to evaluate the susceptibility status of the studied individuals to malathion insecticide. Interestingly, both field sites collected groups showed high levels of resistance at the malathion diagnostic time. Gene amplification, and bidirectional direct sequencing results were analyzed. Compared with the reference genome sequence, the pairwise alignment of the amplified sequences showed 96.6% similarity to the reference sequence in the GenBank database. The confirmed gene sequences were assembled and aligned using various bioinformatic softwares. The assembled contigs were used in NEBcutter V2.0 for constructing restriction maps and checked for the availability of differences (if present) between susceptible and more tolerant strains. Specific molecular RFLP markers were successfully recognized to differentiate the more tolerant from the susceptible Culex pipiens phenotypes.

Genetic polymorphisms of EGF 5'-UTR in patients with glioma: A possible predictive marker of outcome.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e13009-e13009
Author(s):  
Emanuela Vattemi ◽  
Giovanna Cipollini ◽  
Cristina Dealis ◽  
Lorena Rossi ◽  
Susanne Baier ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Direct Sequencing ◽  
Predictive Marker ◽  
Reference Sequence ◽  
Published Data ◽  
Sequencing Method ◽  
Number Of Patients ◽  
Predictive Role ◽  
Epidermal Growth

e13009 Background: Epidermal growth factor (EGF) plays an important role in carcinogenesis. An adenine (A) to guanine (G) single nucleotide polymorphism at position 61 in the 5'-untranslated region (5'-UTR) of the EGF gene has been found to be associated with levels of EGF production and contribute to the risk of glioma. However, published data are contradictory. EGF +61G/A polymorphism may contribute to the risk of glioma in different ethnic group. Patients with glioma and GG genotype have been reported to have a risk of poorer otucome than patients with AA genotype. Purpose of this study is to investigate the potential role of this polymorphism in cancer progression and its role as predictive marker of outcome in glioma caucasian patients. Methods: EGF 61A/G polymorphism (rs4444903) was analyzed in glioma patients and was determined by means of Polymerase Chain Reaction and Direct Sequencing method (GenBank reference sequence-accession no. AC005509) from blood samples. Association of this genetic polymorphism with clinical and pathological data of patients was evaluated. Results: We investigated EGF +61G/A polymorphism in 24 glioma patients . EGF +61G allele has been found in 67% of glioma patients (25% G/G genotype and 42% A/G genotype). In astrocytomas, EGF +61G allele represents a 83% frequency; in glioblastomas and in oligodendrogliomas, EGF +61G allele frequency represents respectively 71% and 50%. In WHO IV gliomas, the EGF +61G allele represents a 63% frequency, (27% G/G and 36% A/G ), in WHO III gliomas a 77% frequency (33% G/G and 44% A/G ) and in WHO II gliomas a 50% frequency (100% A/G ). Median PFS of glioblastoma patients was 9 months. 83% of glioblastoma patients with a relapsing disease showed the G/G and A/G genotype. No difference was detected in the others histotypes. Conclusions: Our data conferm previous studies which reported G allele as a risk factor for glioma in Caucasian. G/A and G/G genotypes seem to be more rappresentative in high grade gliomas . Despite limited number of patients, our study supports the predictive role of EGF 61 A/G polymorphism in GBM. Additional large studies are warranted to confirm the role of EGF polymorphism as indipendent prognostic factor in glioma.

scholarly journals Identification of Campylobacter spp. and discrimination from Helicobacter and Arcobacter spp. by direct sequencing of PCR-amplified cpn60 sequences and comparison to cpnDB, a chaperonin reference sequence database

2006 ◽  
Vol 55 (4) ◽  
pp. 393-399 ◽  
Author(s):  
Janet E. Hill ◽  
Ana Paccagnella ◽  
Kee Law ◽  
Pasquale L. Melito ◽  
David L. Woodward ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Reference Sequence ◽  
Universal Primers ◽  
Reference Database ◽  
Robust Method ◽  
Sequence Database ◽  
Bacterial Isolates ◽  
Require Time ◽  
Campylobacter Spp ◽  
Sequence Identities

A robust method for the identification of Campylobacter spp. based on direct sequencing of PCR-amplified partial cpn60 sequences and comparison of these to a reference database of cpn60 sequences is reported. A total of 53 Campylobacter isolates, representing 15 species, were identified and distinguished from phenotypically similar Helicobacter and Arcobacter strains. Pairwise cpn60 sequence identities between Campylobacter spp. ranged from 71 to 92 %, with most between 71 and 79 %, making discrimination of these species obvious. The method described overcomes limitations of existing PCR-based methods, which require time-consuming and complex post-amplification steps such as the cloning of amplification products. The results of this study demonstrate the potential for use of the reference chaperonin sequence database, cpnDB, as a tool for identification of bacterial isolates based on cpn60 sequences amplified with universal primers.

scholarly journals Screening and evaluation of different algal extracts and prospects for control the disease vector mosquito Culex pipiens L

2021 ◽  
Author(s):  
Doaa R. Abdel Haleem ◽  
Neamat H. El Tablawy ◽  
Lamya Ahmed Alkeridis ◽  
Samy Sayed ◽  
Ahmed M. Saad ◽  
...  
Keyword(s):  
Culex Pipiens ◽  
Disease Vector ◽  
Vector Mosquito ◽  
Algal Extracts

scholarly journals Evaluation of Pyrethroid Susceptibility in Culex pipiens of Northern Izmir Province, Turkey

2019 ◽  
Author(s):  
Onur Guntay ◽  
Mehmet Salih Yikilmaz ◽  
Huseyin Ozaydin ◽  
Savas Izzetoglu ◽  
Asli Suner
Keyword(s):  
Culex Pipiens ◽  
Detoxification Enzymes ◽  
Mosquito Larvae ◽  
Single Population ◽  
Important Species ◽  
Phenotypic Resistance ◽  
Control Programs ◽  
Health Control ◽  
Nuisance Species ◽  
Susceptibility Status

Background: Mosquitoes, being a nuisance species, are considered as one of the most important species in public health control programs due to their role as a vector in mosquito-borne diseases observed in humans and animals. We evaluated the susceptibility status of Culex pipiens collected from northern Izmir, Turkey in 2011–16. Methods: Mosquito larvae, collected from three different locations in northern İzmir, were reared in the laboratory. Adult susceptibility bioassays were performed using the WHO insecticide-impregnated papers including deltamethrin 0.05%, permethrin 0.75%, α-cypermethrin 0.05% and cyfluthrin 0.15%. In addition, adult bioassays were performed after the pre-exposure to piperonyl butoxide (PBO) to determine the contribution of P450 detoxification enzymes to the phenotypic resistance. Results: In all of the three populations, high levels of resistance were observed (mortalities<63%) to all of the four pyrethroids. Different pyrethroids but with the same mode of action can exhibit significantly different phenotypic re­sistance in a single population. PBO bioassays also showed that P450 detoxification enzymes can have diverse effects on different pyrethroids. Conclusion: Using just one chemical in a class of insecticide can be misleading for resistance studies

scholarly journals Development of RB1 mutation detection method based on mRNA

2016 ◽  
Vol 14 (2) ◽  
pp. 209-214
Author(s):  
Vũ Phương Nhung ◽  
Lê Thúy Quỳnh ◽  
Nguyễn Đăng Tôn ◽  
Nguyễn Thị Xuân ◽  
Nguyễn Hải Hà
Keyword(s):  
Healthy Individual ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Reference Sequence ◽  
Coding Region ◽  
Blood Leukocytes ◽  
Standard Pattern ◽  
Rb1 Gene ◽  
Large Size ◽  
Pcr Products

Retinoblastoma (Rb) is a malignant tumor of the retina, occurring usually in children before age five. The heritable form acounting for about 40% of Rb making genetic analysis of RB1 gene is a important part of disease management. In previous study, we have successfully employed a method of direct sequencing for RB1 mutation screening from genomic DNA. However, given the large size of this gene and no reported mutation hotspots, the testing can be costly and time consuming method. To overcome this problem, we have developed a method to detect mutation from RB1 mRNA. Total RNA was isolated from blood leukocytes of a healthy individual and a Rb patient, cDNA was subsequently synthesized using reverse-transcriptase PCR. Whole cDNA of RB1 was amplified using six specific primer pairs and sequenced by Sanger method. The PCR products from healthy individual were showed as six specific bands on the agarose gel and could be used as the standard pattern of the normally spilced transcripts. Those of PCR products were sequenced successfuly and data can be aligned with the reference sequence of RB1 cDNA on the Genebanhk to identify nucleotide variants.  We also identified an upnormal splicing of RB1 gene in the Rb patient haboring a c. G1960>C mutation at the end of the exon 19 by amplification of  the RB1 cDNA fragments. Hence using RB1 mRNA test can reveal not only the silent/pathogenic mutations in the coding region of RB1, but also mutations that affect the normal spilcing of RB1 mRNA. This method reduces cost and time, can be used as the first step of RB1 analysis in Rb patients.

scholarly journals Combining protein-based transcriptome assembly, and efficient MinION long read sequencing for targeted transcript sequencing in orphan species. Validation on herbicide targets and low copy number genes in Gymnosperms, Juncaceae and Pteridophyta

2020 ◽  
Author(s):  
Dyfed Lloyd Evans
Keyword(s):  
Protein Sequence ◽  
Copy Number ◽  
Transcriptome Assembly ◽  
Direct Sequencing ◽  
Reference Sequence ◽  
Sequence Information ◽  
Azolla Filiculoides ◽  
A Genome ◽  
Low Copy Number ◽  
Transcript Assembly

AbstractOrphan species that are evolutionarily distant from their closest sequenced/assembled neighbour provide a significant challenge in terms of gene or transcript assembly for functional analysis. This is because 30% sequence divergence from the closest available reference sequence means that, even with a complete genome or transcriptome sequence, mapping-based or reference-based approaches to gene assembly and gene identification break down.A new approach is required for reference-guided gene and transcript assembly in such orphan species, or species that are evolutionarily very divergent from their closest relatives. When annotating genes, the protein sequence is often preferred as it diverges less than the DNA/RNA sequence and it is often simpler to find meaningful homology at the protein level. This greater conservation of protein sequence across evolutionary time also makes proteins a prime candidate for use as the basis for sequence assembly. A protein-based pipeline was developed for transcript assembly between distantly related species. This was tested on three evolutionarily divergent species with little sequence information available for them and for which the closest genome representatives were at least 40 million years divergent as well as one species (Azolla filiculoides) for which a genome assembly is available. All the species have the potential to be weeds and herbicide targets were chosen as functional genes, whilst low copy number genes were chosen for evolutionary studies. Transcriptomic sequences were assembled using a bait and assemble strategy and final assemblies were verified by direct sequencing.

ABO Genotype-Phenotype Discrepancy Due to Tri-Allelic ABO Genotypes and Its Resolution by Cloning-Sequencing and Chimerism Analysis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4337-4337
Author(s):  
Min-Young Lee ◽  
Tae Sung Park ◽  
Jeong Nyeo Lee ◽  
Hye Ran Kim ◽  
Seung Hwan Oh
Keyword(s):  
Conflicts Of Interest ◽  
Direct Sequencing ◽  
Minor Allele ◽  
Reference Sequence ◽  
Confirmatory Test ◽  
Reference Laboratory ◽  
Sequencing Analysis ◽  
Abo Genotype ◽  
Blood Grouping ◽  
Abo Genotypes

Abstract Abstract 4337 We investigated the molecular genetic basis of an ABO blood group discrepancy found incidentally in an individual with no transfusion or transplantation history. During routine preoperative blood grouping, red blood cells (RBCs) had a weak or mixed field reaction (double populations in a gel column) with anti-A, while serum typing showed the anti-B antibody. We sent the sample to a reference laboratory for ABO genotyping using sequencing analysis, and her genotype was reported to have O/O alleles. To resolve the genotype-phenotype discrepancy, we repeated the direct sequencing analysis and finally found a heterozygous insertion/deletion (indel) on exon 6, involving two major alleles and one minor allele. With cloning and sequencing analysis, we isolated the O01, O02, and A102 alleles, respectively. In addition, tri-allelic or tetra-allelic patterns in short tandem repeat (STR) loci were noted. Chimerism is rare, but it can cause genotype-phenotype discrepancies in blood grouping, parentage, or other genetic laboratory tests. The transfusion of chimeric RBCs was even reported to induce acute intravascular hemolysis. It is very difficult to correctly find heterozygous peaks without assuming their existence, especially when caused by a chimera. Consequently, separation by cloning or enrichment of minor alleles such as using the cold polymerase chain reaction is sometimes needed. A familial study of STR markers could also be a confirmatory test. Here, we report an ABO genotype-phenotype discrepancy due to tri-allelic ABO genotypes and its resolution by cloning sequencing and STR analysis. By cloning-sequencing analysis, the two major alleles O01 and O02 and one minor allele A102 were separated. A blank means the same base as the reference sequence. Bases in parentheses indicate minor peaks. Disclosures: No relevant conflicts of interest to declare.

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