scholarly journals Quantitative analysis of gene amplification in insecticide-resistant Culex mosquitoes

2000 ◽  
Vol 346 (1) ◽  
pp. 17-24 ◽  
Author(s):  
Michael G. PATON ◽  
S. H. P. Parakrama KARUNARATNE ◽  
Elsa GIAKOUMAKI ◽  
Neil ROBERTS ◽  
Janet HEMINGWAY
Keyword(s):  
Translational Control ◽  
Copy Number ◽  
Gene Copy Number ◽  
Absolute Quantification ◽  
Gene Copy ◽  
Common Mechanism ◽  
Control Mechanisms ◽  
Dot Blot ◽  
Culex Mosquitoes ◽  
Pcr Product

The amplification of carboxylesterase structural genes followed by their overexpression is the most common mechanism of resistance to organophosphorus insecticides in Culex mosquitoes. Most resistant Culex quinquefasciatus mosquitoes have co-amplified estα21 and estβ21 genes. Recently, Southern, DNA dot-blot analysis and phosphorimaging technology were used to quantify the est gene copy number in aphids and mosquitoes. Although more accurate than autoradiography, this method relies on probe hybridization, which can be variable. We have directly measured gene and mRNA copy number by using real-time quantitative PCRs in mosquitoes. The acquisition of fluorescence from incorporation of the double-strand-specific dye SYBR GreenI into a PCR product once per cycle is used to provide an absolute quantification of the initial template copy number. Thus it has been possible to show that estα21 and estβ21 are co-amplified approx. 80-fold in the genome of the resistant PelRR strain of C. quinquefasciatus. The two genes, although co-amplified in a 1:1 ratio, are differentially transcribed: the estβ21 gene from this amplicon has greater transcription than estα21 in all individual mosquito larvae tested, with an average ratio of 10:1. Purified esterases from mosquito homogenates were found in a ratio of 3:1, which, combined with the quantitative mRNA data, suggests the operation of both transcriptional and translational control mechanisms to regulate the expression of the amplified genes in C. quinquefasciatus insecticide-resistant mosquitoes.

scholarly journals Nongenotoxic ABCB1 activator tetraphenylphosphonium can contribute to doxorubicin resistance in MX-1 breast cancer cell line

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Raimonda Kubiliute ◽  
Indre Januskeviciene ◽  
Ruta Urbanaviciute ◽  
Kristina Daniunaite ◽  
Monika Drobniene ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Protein Level ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Common Mechanism ◽  
Dna Hypomethylation ◽  
Mesenchymal Transition ◽  
Molecular Features

AbstractHyperactivation of ABC transporter ABCB1 and induction of epithelial–mesenchymal transition (EMT) are the most common mechanism of acquired cancer chemoresistance. This study describes possible mechanisms, that might contribute to upregulation of ABCB1 and synergistically boost the acquisition of doxorubicin (DOX) resistance in breast cancer MX-1 cell line. DOX resistance in MX-1 cell line was induced by a stepwise increase of drug concentration or by pretreatment of cells with an ABCB1 transporter activator tetraphenylphosphonium (TPP+) followed by DOX exposure. Transcriptome analysis of derived cells was performed by human gene expression microarrays and by quantitative PCR. Genetic and epigenetic mechanisms of ABCB1 regulation were evaluated by pyrosequencing and gene copy number variation analysis. Gradual activation of canonical EMT transcription factors with later activation of ABCB1 at the transcript level was observed in DOX-only treated cells, while TPP+ exposure induced considerable activation of ABCB1 at both, mRNA and protein level. The changes in ABCB1 mRNA and protein level were related to the promoter DNA hypomethylation and the increase in gene copy number. ABCB1-active cells were highly resistant to DOX and showed morphological and molecular features of EMT. The study suggests that nongenotoxic ABCB1 inducer can possibly accelerate development of DOX resistance.

Resistance to methotrexate due to gene amplification in a patient with acute leukemia.

1984 ◽  
Vol 2 (1) ◽  
pp. 16-20 ◽  
Author(s):  
M D Carman ◽  
J H Schornagel ◽  
R S Rivest ◽  
S Srimatkandada ◽  
C S Portlock ◽  
...  
Keyword(s):  
Gene Amplification ◽  
Copy Number ◽  
Cdna Probe ◽  
Leukemia Cell ◽  
Gene Copy Number ◽  
Leukemia Cell Line ◽  
Gene Copy ◽  
Acute Myelocytic Leukemia ◽  
Human Leukemia ◽  
Dot Blot

A patient is described with acute myelocytic leukemia refractory to conventional therapy, who also became highly resistant to methotrexate (MTX) after repeated courses of this drug. Leukemia cells from this patient were found to contain an elevated level of dihydrofolate reductase (DHFR) activity, with no change in the affinity of the enzyme for MTX. A sensitive "dot blot" assay revealed a fourfold increase in the gene copy number of DHFR. Southern blot analysis with a human DHFR cDNA probe confirmed this increase in the gene copy number, and demonstrated a similar restriction pattern with Eco R1, Hind III, and Pst 1 as seen with a highly amplified human leukemia cell line, K562. Additional DHFR fragments were detected, not seen in the K562 blot, suggesting the presence of pseudogenes, or a result of gene rearrangements occurring as part of the amplification process. Resistance to MTX in this patient was therefore ascribed to gene amplification and overproduction of DHFR.

scholarly journals Genome instability drives epistatic adaptation in the human pathogen Leishmania

2021 ◽  
Vol 118 (51) ◽  
pp. e2113744118
Author(s):  
Giovanni Bussotti ◽  
Laura Piel ◽  
Pascale Pescher ◽  
Malgorzata A. Domagalska ◽  
K. Shanmugha Rajan ◽  
...  
Keyword(s):  
Translational Control ◽  
Copy Number ◽  
Biomarker Discovery ◽  
Genome Instability ◽  
Gene Dosage ◽  
Gene Copy Number ◽  
Copy Number Variations ◽  
Gene Copy ◽  
Snorna Gene ◽  
Ideal System

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches, we provide evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover posttranscriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in small nucleolar RNA (snoRNA) gene dosage with changes in ribosomal RNA 2′-O-methylation and pseudouridylation, suggesting translational control as an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression and genome-independent compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery and may be relevant to other fast-evolving eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer.

scholarly journals Screening and Absolute Quantification of a β-lactamase Resistance Gene NDM-1 in Lake Sediment

2021 ◽  
Author(s):  
Rajeev Ranjan ◽  
Shashidhar Thatikonda
Keyword(s):  
Lake Sediment ◽  
Copy Number ◽  
Quantitative Polymerase Chain Reaction ◽  
Horizontal Transmission ◽  
Gene Copy Number ◽  
Absolute Quantification ◽  
Gene Copy ◽  
Qpcr Analysis ◽  
Wide Range ◽  
Template Dna

Abstract The extensive usage of antibiotics in humans and veterinary medicine and their discharge into the aquatic environment hasten the growth, selection, and horizontal transmission of ARGs in a given bacterial community. New Delhi Metallo-β-lactamase-1(NDM-1) is an enzyme that hydrolyzes a wide range of β-lactams antibiotics, including carbapenems. The rapid distribution of NDM-1 harboring bacteria accounts for a significant public health menace worldwide. The presence of the NDM-1 inhibited the potential of β–lactam antibiotics for treating infections caused by bacterial strains carrying such resistances, leaving minimal treatment options available. NDM-1 harboring bacteria have been detected in clinical specimens and environmental compartments where bacterial infections are ubiquitous. In this study, identification and absolute quantification of NDM-1 in sixteen lake sediment samples collected in and around Hyderabad, India, was carried out using a real-time quantitative polymerase chain reaction (qPCR) and results were expressed in gene copy number/ng (nanogram) of template DNA. 13 samples (out of 16) shown a positive signal for NDM-1 during qPCR analysis. Durgamcheru lake, Kandi lake, and Singur dam showed a negative signal for the NDM-1 during qPCR analysis among the tested samples. The remaining sampling locations tested positive with the highest gene copy number/ng of template DNA observed in the Amberpet STP (71.8). Hierarchical clustering analysis was performed to categorize the sampling location into different clusters based on pollution sources and observed results expressed in the form of a dendrogram.

scholarly journals Levels of ornithine decarboxylase genomic sequences, heterogeneous nuclear RNA and mRNA in human myeloma cells resistant to α-difluoromethylornithine

1991 ◽  
Vol 278 (3) ◽  
pp. 871-874 ◽  
Author(s):  
J M Hyttinen ◽  
M Halmekytö ◽  
L Alhonen ◽  
J Jänne
Keyword(s):  
Ornithine Decarboxylase ◽  
Reverse Transcription ◽  
Copy Number ◽  
Gene Dosage ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Dot Blot ◽  
Semiquantitative Assessment ◽  
Myeloma Cells ◽  
Human Myeloma Cells

We previously isolated and characterized a human myeloma cell line overproducing ornithine decarboxylase (ODC) due to gene amplification [Leinonen, Alhonen-Hongisto, Laine, Jänne & Jänne (1987) Biochem. J. 242, 199-203]. We have now employed the PCR combined with reverse transcription to determine semiquantitatively ODC gene dosage and the amounts of heterogeneous nuclear (hn) RNA and of mature mRNA of the enzyme in parental and alpha-difluoromethylornithine-resistant human myeloma cells. Experiments with dilution series revealed that the ODC gene copy number and the amount of both hnRNA and mRNA were increased to the same extent (about 100-fold) in the resistant cells. Similar dot-blot analyses of ODC-specific genomic DNA and total RNA indicated that the ODC gene copy number was increased by a factor of 380 and the amount of ODC mRNA by a factor of 700. Our results indicate that the PCR combined with reverse transcription is at least as useful as blot analyses to give semiquantitative assessment of the amounts of specific DNA or RNA sequences. In addition, the use of the PCR enables the analysis of minute sample amounts in extremely short time.

scholarly journals Determination of gene dosage at the PMP22 and androgen receptor loci by quantitative PCR

1998 ◽  
Vol 44 (4) ◽  
pp. 724-730 ◽  
Author(s):  
Renee A Poropat ◽  
Garth A Nicholson
Keyword(s):  
Copy Number ◽  
Gene Dosage ◽  
Point Mutations ◽  
Gene Copy Number ◽  
Fluorescent Labeling ◽  
Gene Copy ◽  
Target Sequence ◽  
Hereditary Neuropathy ◽  
Deletion Event ◽  
Pcr Product

Abstract Although many genetic diseases are caused by the presence of point mutations in respective genes, an increasing number of diseases are known to be caused by gene copy number changes. We report the development of a rapid and reliable PCR-based method for quantitation of gene copy number with sufficient sensitivity to detect single copy changes without the use of radioactive or fluorescent labeling. The sensitivity of this technique has been demonstrated by the detection of the DNA duplication or deletion occurring in two inherited peripheral neuropathies, Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), that are caused by a reciprocal duplication or deletion event on chromosome 17p11.2–12. This method relies on the comparison of the amount of PCR product generated from a potentially duplicated or deleted target sequence with the amount of product generated from a disomic reference gene. The value of this ratio (target PCR product:reference PCR product) indicates whether the target sequence is duplicated, deleted, or unchanged. Using primers from within a duplicated or deleted region (PMP22 gene and EW401) and from within a reference region (NF1 gene), we tested 50 CMT1A, 30 HNPP, and 50 unaffected individuals for the presence of a DNA duplication or deletion. Target:reference ratios of 1.58, 1.02, and 0.56 were detected for the CMT1A, unaffected, and HNPP groups, respectively. Thus, differentiation of the three groups of individuals was on the basis of gene copy number. This technique was successfully used to detect the difference in the X chromosome copy number between males and females (target:reference ratios of 1.1 and 2.3, respectively). This approach to the detection of DNA duplications and deletions is sensitive, accurate, and has potential applications in the quantitation of changes in gene copy number associated with diseases characterized by such chromosomal alterations.

scholarly journals Genome instability drives epistatic adaptation in the human pathogen Leishmania

2021 ◽  
Author(s):  
Giovanni Bussotti ◽  
Laura Piel ◽  
Pascale Pescher ◽  
Malgorzata Anna Domagalska ◽  
K. Shanmugha Rajan ◽  
...  
Keyword(s):  
Translational Control ◽  
Copy Number ◽  
Biomarker Discovery ◽  
Genome Instability ◽  
Gene Dosage ◽  
Gene Copy Number ◽  
Copy Number Variations ◽  
Gene Copy ◽  
Snorna Gene ◽  
Ideal System

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania that exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches we provide first evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations that can inform on pathways driving fitness gain in a given environment. We further uncover post-transcriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in snoRNA gene dosage to changes in rRNA 2′-O-methylation and pseudouridylation, proposing translational control as an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression, and genome-independent, compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selection. Epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery, and may be relevant to other fast evolving, eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer.

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