scholarly journals Differences in gene copy number carried by different MHC ancestral haplotypes. Quantitation after physical separation of haplotypes by pulsed field gel electrophoresis.

1990 ◽  
Vol 171 (6) ◽  
pp. 2101-2114 ◽  
Author(s):  
W J Zhang ◽  
M A Degli-Esposti ◽  
T J Cobain ◽  
P U Cameron ◽  
F T Christiansen ◽  
...  
Keyword(s):  
Autoimmune Disease ◽  
Gel Electrophoresis ◽  
Copy Number ◽  
Gene Copy Number ◽  
Pulsed Field Gel Electrophoresis ◽  
Gene Copy ◽  
Functional Genes ◽  
Specific Content ◽  
Physical Separation ◽  
Ancestral Haplotypes

We have examined the hypothesis that MHC ancestral haplotypes have a specific content of genes regulating the extent of autoimmune reactions. Gene copy number was quantitated by objective densitometry after PFGE was used to separate heterozygous AHs of different lengths. Initially we analyzed examples of known gene copy number at the C4 and 21 hydroxylase loci and showed that the approach provides predictable results. We then studied heterozygotes containing one characterized and one uncharacterized AH with particular attention to the gene copy number at the C4, Cyp21, and DRB loci. Each AH studied has a characteristic gene copy number at each locus studied. The same may be true of TNF, but other possibilities must be considered. AHs are markers for extensive chromosomal segments including particular numbers of several functional genes. Since AHs mark susceptibility to autoimmune disease, differences in gene copy number may be implicated.

Enhanced Detection of DNA Sequences Using End-Point PCR Amplification and Online Gel Electrophoresis (GE)-ICP-MS: Determination of Gene Copy Number Variations

2014 ◽  
Vol 86 (22) ◽  
pp. 11028-11032 ◽  
Author(s):  
T. Iglesias González ◽  
M. Espina ◽  
L. M. Sierra ◽  
J. Bettmer ◽  
E. Blanco-González ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Gel Electrophoresis ◽  
Copy Number ◽  
Gene Copy Number ◽  
Pcr Amplification ◽  
Copy Number Variations ◽  
Gene Copy ◽  
End Point ◽  
Icp Ms

scholarly journals Reduced Susceptibility of Haemophilus influenzae to the Peptide Deformylase Inhibitor LBM415 Can Result from Target Protein Overexpression Due to Amplified Chromosomal def Gene Copy Number

2007 ◽  
Vol 51 (3) ◽  
pp. 1004-1010 ◽  
Author(s):  
Charles R. Dean ◽  
Shubha Narayan ◽  
Joel Richards ◽  
Denis M. Daigle ◽  
Stacy Esterow ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Gel Electrophoresis ◽  
Copy Number ◽  
Efflux Pump ◽  
Gene Copy Number ◽  
Target Protein ◽  
Peptide Deformylase ◽  
Gene Copy ◽  
Two Dimensional Gel Electrophoresis ◽  
Altered Expression

ABSTRACT Previous genetic analysis of Haemophilus influenzae revealed two mechanisms associated with decreased susceptibility to the novel peptide deformylase inhibitor LBM415: AcrAB-TolC-mediated efflux and Fmt bypass, resulting from mutations in the pump repressor gene acrR and in the fmt gene, respectively. We have isolated an additional mutant, CDS23 (LBM415 MIC, 64 μg/ml versus 4 μg/ml against the parent strain NB65044) that lacks mutations in the acrR or fmt structural genes or in the gene encoding Def, the intracellular target of LBM415. Western immunoblot analysis, two-dimensional gel electrophoresis, and tryptic digestion combined with mass spectrometric identification showed that the Def protein was highly overexpressed in the mutant strain. Consistent with this, real-time reverse transcription-PCR revealed a significant increase in def transcript titer. No mutations were found in the region upstream of def that might account for altered expression; however, pulsed-field gel electrophoresis suggested that a genetic rearrangement of the region containing def had occurred. Using a combination of PCR, sequencing, and Southern blot analyses, it was determined that the def gene had undergone copy number amplification, explaining the high level of target protein expression. Inactivation of the AcrAB-TolC efflux pump in this mutant increased susceptibility 16-fold, highlighting the role of efflux in exacerbating the overall reduced susceptibility resulting from target overexpression.

scholarly journals Estimating Copy-Number Proportions: The Comeback of Sanger Sequencing

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 283
Author(s):  
Eyal Seroussi
Keyword(s):  
Copy Number ◽  
Sanger Sequencing ◽  
Cytosine Methylation ◽  
Direct Sequencing ◽  
Information Source ◽  
Gene Copy Number ◽  
Cost Effective ◽  
Gene Copy ◽  
Base Editing ◽  
Recent Developments

Determination of the relative copy numbers of mixed molecular species in nucleic acid samples is often the objective of biological experiments, including Single-Nucleotide Polymorphism (SNP), indel and gene copy-number characterization, and quantification of CRISPR-Cas9 base editing, cytosine methylation, and RNA editing. Standard dye-terminator chromatograms are a widely accessible, cost-effective information source from which copy-number proportions can be inferred. However, the rate of incorporation of dye terminators is dependent on the dye type, the adjacent sequence string, and the secondary structure of the sequenced strand. These variable rates complicate inferences and have driven scientists to resort to complex and costly quantification methods. Because these complex methods introduce their own biases, researchers are rethinking whether rectifying distortions in sequencing trace files and using direct sequencing for quantification will enable comparable accurate assessment. Indeed, recent developments in software tools (e.g., TIDE, ICE, EditR, BEEP and BEAT) indicate that quantification based on direct Sanger sequencing is gaining in scientific acceptance. This commentary reviews the common obstacles in quantification and the latest insights and developments relevant to estimating copy-number proportions based on direct Sanger sequencing, concluding that bidirectional sequencing and sophisticated base calling are the keys to identifying and avoiding sequence distortions.

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.

Relationship between paralytic shellfish toxin content and sxtA gene copy number in different growth phases of Gymnodinium catenatum (Dinophyceae)

Toxicon ◽  
2021 ◽  
Author(s):  
Armando Mendoza-Flores ◽  
Ignacio Leyva-Valencia ◽  
Francisco E. Hernández-Sandoval ◽  
Clara E. Galindo-Sánchez ◽  
Christine J. Band-Schmidt ◽  
...  
Keyword(s):  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Growth Phases ◽  
Toxin Content ◽  
Paralytic Shellfish Toxin ◽  
Paralytic Shellfish ◽  
Shellfish Toxin ◽  
Gymnodinium Catenatum
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