scholarly journals Identification of a novel murine glutathione S-transferase class mu gene

1998 ◽  
Vol 330 (2) ◽  
pp. 623-626 ◽  
Author(s):  
Wieke C. C. de BRUIN ◽  
René H. M. te MORSCHE ◽  
J. M. Muriel WAGENMANS ◽  
C. Jeroen ALFERINK ◽  
J. Alan TOWNSEND ◽  
...  
Keyword(s):  
Cdna Probe ◽  
Glutathione S Transferase ◽  
Chain Reaction ◽  
Specific Primers ◽  
Dna Library ◽  
Isolation And Characterization ◽  
Signature Sequences ◽  
Polymerase Chain ◽  
Conceptual Translation

Screening of a genomic mouse DNA library with a glutathione S-transferase class mu cDNA probe resulted in the identification of mGSTM5, a novel member of the murine glutathione S-transferase class mu gene family. Here we present the sequence of the promoter region, the exon-intron organization of the gene and the isolation and characterization of its complete cDNA. Conceptual translation of the cDNA sequence revealed that several amino acid positions have been changed in ‘invariant’ mu class signature sequences in mGSTM5. Reverse transcriptase polymerase chain reaction using gene specific primers revealed that mGSTM5 is uniquely expressed in mouse liver, stomach and small intestine.

a Novel Method for the Detection of Minimal Residual Disease in B-Cell Malignancies: Ion Semiconductor Sequencing for the Evaluation of Igh Gene Rearrangements

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2983-2983
Author(s):  
Silvia Gimondi ◽  
Alessandra Cavanè ◽  
Antonio Vendramin ◽  
Giulia Biancon ◽  
Paolo Corradini ◽  
...  
Keyword(s):  
B Cell ◽  
Multiplex Pcr ◽  
Residual Disease ◽  
Chain Reaction ◽  
B Cell Malignancies ◽  
Specific Primers ◽  
Namalwa Cell ◽  
Polymerase Chain ◽  
Minimal Residual

Abstract Background: Minimal residual disease (MRD) detection is of high clinical relevance in patients with B-cell malignancies and is generally a surrogate parameter to evaluate treatment response and long-term prognosis. IgH gene rearrangements can be used as molecular marker in approximately 80% of lymphoma and myeloma patients since they represent lineage-specific markers and the complementarity determining region 3 (CDR3) is unique to each clone. To date, allele specific oligonucleotide polymerase chain reaction (ASO-PCR) and real-time quantitative polymerase chain reaction (RQ-PCR) are considered the most sensitive and widely applicable methods for MRD detection. A major disadvantage of ASO-PCR and RQ-PCR assays, is the use of specific primers and probes for every individual patient. Clone-specific primers and probes are not only expensive but also time-consuming to design and to test, which limits their wide applicability in the clinical setting. The recent major improvements in next generation sequencing (NGS) technologies, provide the opportunity to identify and quantify clonotypes with consensus primers combining the benefits of high sensitivity and universal applicability. The present work was designed to overcome ASO-PCR and RQ-PCR limitations by developing a feasible method for rearranged IgH genes amplification, NGS and analysis using Ion Torrent Personal Genome Machine (IT-PGM). Methods: To define a multiplex PCR protocol, DNA from 7 CLL patients, previously shown to bare a family specific clonal VDJ rearrangement, was amplified with a pool of the seven different family-specific IgH-V primers, and a consensus JH primer (Voena et al., Leukemia 1997). After Sanger sequencing, results were compared to the ones obtained with singleplex PCR protocol. Once validated, the multiplex PCR protocol was used to amplify DNA from patients and serially diluted (up to 10-8 ) DNA from Namalwa cell line (bearing a known IgH rearrangement) and subsequently sequenced on the IT-PGM using the 316 Ion-chip. NGS data were analyzed by using the IMGT-High V-Quest web server tool and the statistical software R. RQ-PCR was used to quantify the specific VDJ rearrangement in the serially diluted Namalwa DNA solutions and in DNA from patients as previously described (Farina et al, Haematologica 2009). RQ-PCR data were analyzed through a relative quantification procedure. Results: The multiplex PCR reactions we have tested, demonstrated the same specificity as the standard singleplex PCR protocol and therefore was used to construct the DNA library required for IT-PGM-based sequencing. The IT-PGM sequencing output is represented by at least 400000 reads per sample with a minimum average coverage of the VDJ repertoire of 500x. The IMGT-High V-quest tool allows a user-friendly web based analysis and a deep molecular characterization of the IgH recombinatorial repertoire. Namalwa clonal CRD3 sequences were detected up to a dilution of 10-5 without the need for specific CDR3 primers. Comparability of NGS and ASO RQ-PCR results was assessed. The use of CDR3 specific primers, along with the specific IgH-V family fluorescent probe, enabled the identification of clonal VDJ rearrangements with a sensitivity up to 10-5 (2/3 replicates) and 10-6 (just 1/3 replicates) in Namalwa Cell Line. Similar results were obtained when we characterized the IgH recombination repertoire of two CLL patients over time. Conclusions: IgH sequencing with the IT-PGM platform showed at least the same level of sensitivity as ASO RQ-PCR, without the need for patient-specific reagents. It also allows specific and detailed molecular characterization of the clonal rearrangements and could be easily incorporated into clinical laboratories for routine testing of MRD in B-cell malignancies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Karakterisasi Molekuler Papaya ringspot virus tipe P pada Tanaman Mentimun di Jawa

2018 ◽  
Vol 14 (3) ◽  
pp. 75
Author(s):  
Listihani Listihani ◽  
Tri Asmira Damayanti ◽  
Sri Hendrastuti Hidayat ◽  
Suryo Wiyono
Keyword(s):  
Ringspot Virus ◽  
Mosaic Symptom ◽  
Papaya Ringspot Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Specific Primers ◽  
Central Java ◽  
Dna Fragment ◽  
Polymerase Chain

Moleculer Characterization of Papaya ringspot virus type P on Cucumber in JavaInfection of Papaya ringspot virus (PRSV) on cucumber plants showing mosaic symptom was detected using specific antibody.  Further investigation was conducted to determine molecular characters and status of PRSV infecting cucumber in Java.  Infection of PRSV was detected from leaf samples collected from the field using dot immunobinding assay (DIBA).  Disease frequency caused by PRSV infection reached 81.11%, 95.86%, 91.66%, and 92.3% in East Java, Central Java, Yogyakarta, and West Java, respectively.  Characterization of PRSV isolates was conducted by reverse transcription polymerase chain reaction (RT-PCR) using specific primers for PRSV-P and PRSV-W, followed by cloning, and DNA sequencing.  DNA fragment of 470 bp was successfully amplified using specific primers for PRSV-P from several samples from Nganjuk, Brebes, Kulon Progo, and Subang; but no amplification was achieved using specific primers for PRSV-W.  Nucleotide and amino acid analysis showed high homology among PRSV-P isolates from Nganjuk, Brebes, Kulon Progo, and Subang, i.e. 98.6%-99.7% and 99.3%-100%, respectively.  This is an indication of a low genetic variation among PRSV-P from Java. Further phylogenetic analysis indicated that PRSV-P isolate cucumber is in the same cluster with PRSV-P isolate papaya from Bali, Indonesia.  This is the first report of PRSV-P infecting cucumber in Indonesia.

Molecular cloning and characterization of a glutathione S-transferase in the tropical liver fluke, Fasciola gigantica

2009 ◽  
Vol 84 (1) ◽  
pp. 55-60 ◽  
Author(s):  
A. Jedeppa ◽  
O.K. Raina ◽  
S. Samanta ◽  
G. Nagar ◽  
N. Kumar ◽  
...  
Keyword(s):  
Prokaryotic Expression ◽  
Liver Fluke ◽  
Nitrilotriacetic Acid ◽  
Fasciola Gigantica ◽  
Glutathione S Transferase ◽  
Chain Reaction ◽  
Prokaryotic Expression Vector ◽  
Polymerase Chain ◽  
Dot Elisa

AbstractGlutathione S-transferase from an Indian isolate of Fasciola gigantica of buffalo origin was isolated and characterized. Total RNA was transcribed to cDNA by reverse transcription and an amplicon of 657 bp glutathione S-transferase gene was obtained by polymerase chain reaction (PCR). The present isolate showed 99.1% sequence homology with the published sequence of the F. giganticaGST gene of cattle origin, with six nucleotide changes causing an overall change of four amino acids. Glutathione S-transferase protein was expressed in Escherichia coli using a prokaryotic expression vector pPROEXHTb. The recombinant protein was purified under non-denaturing and denaturing conditions by nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography. Recombinant GST protein detected F. gigantica infection in naturally infected buffaloes by dot-ELISA.

scholarly journals Isolation and characterization of genes differentially expressed during conidiation of Neurospora crassa.

1985 ◽  
Vol 5 (4) ◽  
pp. 849-855 ◽  
Author(s):  
V Berlin ◽  
C Yanofsky
Keyword(s):  
Neurospora Crassa ◽  
Cdna Probe ◽  
Genomic Sequences ◽  
Dna Library ◽  
Isolation And Characterization ◽  
Polyadenylated Rna ◽  
Length Polymorphisms ◽  
Genomic Dna Library ◽  
Asexual Cycle

A Neurospora crassa genomic DNA library was screened with a cDNA probe enriched in sequences expressed in conidiating cultures. Clones were isolated that preferentially hybridized to this probe versus a second cDNA probe complementary to polyadenylated RNA isolated from mycelia. Twelve clones contained unique sequences that hybridized to 22 transcripts, 19 of which accumulated preferentially in conidiating cultures. Eight transcripts were present in higher levels in conidiating cultures than in mycelia. Eleven transcripts were detected only in conidiating cultures and first appeared at different times during the asexual cycle. We mapped genomic sequences homologous to the 11 clones by conventional crosses using restriction fragment-length polymorphisms as genetic markers. The sequences homologous to genes expressed preferentially in conidiating cultures are distributed on six of the seven chromosomes. Clones that map to the same chromosome are linked. No recombination occurred between genomic sequences homologous to three clones, suggesting that the genes contained in these clones may constitute a gene cluster.

scholarly journals Isolation and Characterization of Micro-organisms with Industrial Importance From Sisal Bole Rots

2013 ◽  
Vol 34 (1) ◽  
pp. 26-35
Author(s):  
Consolatha J.N. Mhaiki ◽  
Enock Masanja ◽  
Jamidu H.Y. Katima ◽  
Gunaraths Rajarao ◽  
Gunnel Dalhammar
Keyword(s):  
Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Isolation And Characterization ◽  
Brevundimonas Diminuta ◽  
Industrial Use ◽  
Brevundimonas Vesicularis ◽  
Polymerase Chain ◽  
The Individual ◽  
Micro Organisms

Investigation of microorganisms naturally acclimatized to Agave hybrid H 11648 (sisal bole rot) was conducted, with the aim of isolating and characterizing Aspergillus niger strains for industrial use. Microorganism were identified morphologically and then confirmation made by polymerase chain reaction (PCR). Results showed the existence of four major groups, listed in order of abundances as follows; Aspergilli (36.0±0.8) %, Penicillin (28.0±0.1) %, Yeast (15.0±1.6) %and Fusarium (10.0±0.12) %. The main groups of Aspergilli strains were A. nidulans, A. tamari and A. niger in ratios (3:2:2), respectively. Several endo-spore forming non-enteric gram (-) rods and coccid bacteria identified by API20 NE identification systemincluded,Brevundimonas diminuta sp, Shewanella putrefaciens sp, Brevundimonas vesicularis sp and Pasteurella sp. Results showed that sisal bole rot stems hosts a high bio-diversity of microorganism species other than A. niger. Exploitation of the individual strains is recommended. This could eventually produce strains forprecursors of industrially and therapeutically metabolites.

Isolation and characterization of genes differentially expressed during conidiation of Neurospora crassa

1985 ◽  
Vol 5 (4) ◽  
pp. 849-855
Author(s):  
V Berlin ◽  
C Yanofsky
Keyword(s):  
Neurospora Crassa ◽  
Cdna Probe ◽  
Genomic Sequences ◽  
Dna Library ◽  
Isolation And Characterization ◽  
Polyadenylated Rna ◽  
Length Polymorphisms ◽  
Genomic Dna Library ◽  
Asexual Cycle

A Neurospora crassa genomic DNA library was screened with a cDNA probe enriched in sequences expressed in conidiating cultures. Clones were isolated that preferentially hybridized to this probe versus a second cDNA probe complementary to polyadenylated RNA isolated from mycelia. Twelve clones contained unique sequences that hybridized to 22 transcripts, 19 of which accumulated preferentially in conidiating cultures. Eight transcripts were present in higher levels in conidiating cultures than in mycelia. Eleven transcripts were detected only in conidiating cultures and first appeared at different times during the asexual cycle. We mapped genomic sequences homologous to the 11 clones by conventional crosses using restriction fragment-length polymorphisms as genetic markers. The sequences homologous to genes expressed preferentially in conidiating cultures are distributed on six of the seven chromosomes. Clones that map to the same chromosome are linked. No recombination occurred between genomic sequences homologous to three clones, suggesting that the genes contained in these clones may constitute a gene cluster.

Isolation and characterization of microsatellite loci for Euphorbia palustris (Euphorbiaceae)

Genome ◽  
2009 ◽  
Vol 52 (12) ◽  
pp. 1037-1039 ◽  
Author(s):  
Walter Durka
Keyword(s):  
Microsatellite Loci ◽  
Habitat Destruction ◽  
Perennial Species ◽  
Chain Reaction ◽  
Natural Habitats ◽  
Isolation And Characterization ◽  
Polymerase Chain ◽  
Polymorphic Microsatellite ◽  
Two Populations

Swamp spurge ( Euphorbia palustris , Euphorbiaceae) is a large perennial species of wet grassland and swamps. Its natural habitats are fragmented and isolated both naturally and owing to habitat destruction by human activity. Thus the species is endangered and legally protected in Germany. This report describes seven novel polymorphic microsatellite loci that will be helpful to characterize genetic variation and to analyze the population genetic structure and levels of gene flow within and among populations. All loci were amplified within one multiplex polymerase chain reaction for two populations, yielding between 3 and 13 alleles per locus and high levels of heterozygosity. Trans-species amplification is reported for four Euphorbia species.

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