-
Scientific Reports Aug 2022Tobacco is an important commercial crop and a rich source of alkaloids for pharmaceutical and agricultural applications. However, its yield can be reduced by up to 70%...
Tobacco is an important commercial crop and a rich source of alkaloids for pharmaceutical and agricultural applications. However, its yield can be reduced by up to 70% due to virus infections, especially by a potyvirus Potato virus Y (PVY). The replication of PVY relies on host factors, and eukaryotic translation initiation factor 4Es (eIF4Es) have already been identified as recessive resistance genes against potyviruses in many plant species. To investigate the molecular basis of PVY resistance in the widely cultivated allotetraploid tobacco variety K326, we developed a dual guide RNA CRISPR/Cas9 system for combinatorial gene editing of two clades, eIF4E1 (eIF4E1-S and eIF4E1-T) and eIF4E2 (eIF4E2-S and eIF4E2-T) in the eIF4E gene family comprising six members in tobacco. We screened for CRISPR/Cas9-induced mutations by heteroduplex analysis and Sanger sequencing, and monitored PVY accumulation in virus challenged regenerated plants by DAS-ELISA both in T0 and T1 generations. We found that all T0 lines carrying targeted mutations in the eIF4E1-S gene displayed enhanced resistance to PVY confirming previous reports. More importantly, our combinatorial approach revealed that eIF4E1-S is necessary but not sufficient for complete PVY resistance. Only the quadruple mutants harboring loss-of-function mutations in eIF4E1-S, eIF4E1-T, eIF4E2-S and eIF4E2-T showed heritable high-level resistance to PVY in tobacco. Our work highlights the importance of understanding host factor redundancy in virus replication and provides a roadmap to generate virus resistance by combinatorial CRISPR/Cas9-mediated editing in non-model crop plants with complex genomes.
Topics: CRISPR-Cas Systems; Mutation; Plant Diseases; Potyvirus; Solanum tuberosum; Nicotiana
PubMed: 36028578
DOI: 10.1038/s41598-022-18923-0 -
Journal of Clinical Microbiology Oct 2009Porphyromonas gingivalis is implicated in the etiology of chronic periodontitis. Genotyping studies suggest that genetic variability exists among P. gingivalis strains;...
Porphyromonas gingivalis is implicated in the etiology of chronic periodontitis. Genotyping studies suggest that genetic variability exists among P. gingivalis strains; however, the extent of variability remains unclear and regions of variability remain largely unidentified. To assess P. gingivalis strain diversity, we previously used heteroduplex analysis of the ribosomal operon intergenic spacer region (ISR) to type strains in clinical samples and identified 22 heteroduplex types. Additionally, we used ISR sequence analysis to determine the relatedness of P. gingivalis strains to one another and demonstrated a link between ISR sequence phylogeny and the disease-associated phenotype of the strains. In the current study, heteroduplex analysis of the ISR was used to determine the worldwide genetic variability and distribution of P. gingivalis, and microarray-based comparative genomic hybridization (CGH) analysis was used to more comprehensively examine the variability of major heteroduplex type strains by using the entire genome. Heteroduplex analysis of clinical samples from geographically diverse populations identified 6 predominant geographically widespread heteroduplex types (prevalence, > or = 5%) and 14 rare heteroduplex types (prevalence, <2%) which are found in one or a few locations. CGH analysis of the genomes of seven clinically prevalent heteroduplex type strains identified 133 genes from strain W83 that were divergent in at least one of the other strains. The relatedness of the strains to one another determined on the basis of genome content (microarray) analysis was highly similar to their relatedness determined on the basis of ISR sequence analysis, and a striking correlation between the genome contents and disease-associated phenotypes of the strains was observed.
Topics: Cluster Analysis; Comparative Genomic Hybridization; DNA, Bacterial; DNA, Ribosomal Spacer; Genetic Variation; Genotype; Geography; Heteroduplex Analysis; Humans; Periodontitis; Phylogeny; Porphyromonas gingivalis
PubMed: 19675220
DOI: 10.1128/JCM.00569-09 -
BioTechniques Jul 2019SNPs and single base pair (SBP) insertion/deletions (indels) are not only the most abundant genetic markers for genetic mapping and breeding selection, but also always...
SNPs and single base pair (SBP) insertion/deletions (indels) are not only the most abundant genetic markers for genetic mapping and breeding selection, but also always occur in the mutants generated from chemical mutagenesis or CRISPR/Cas9-mediated genome editing. Most of the current SNP and SBP indel genotyping methods are time-consuming and/or require special equipment or reagents. Here, we describe an improved heteroduplex analysis method, named iHDA, that can readily discriminate SNP and SBP indel alleles with specially designed DNA probes that harbor a couple of nucleotides adjacent to the SNP site. By hybridizing with the same probe, SNP and SBP indel alleles form different heteroduplexes, differing in bulge size, which show different mobility on a polyacrylamide gel. Therefore, iHDA is an easy, fast and inexpensive method for SNP and SBP indel genotyping.
Topics: Base Pairing; CRISPR-Cas Systems; DNA, Plant; Genotyping Techniques; Heteroduplex Analysis; INDEL Mutation; Oryza; Polymorphism, Single Nucleotide; Time Factors
PubMed: 31124706
DOI: 10.2144/btn-2019-0012 -
Frontiers in Oncology 2020γδT cell lymphoma (γδ TCL) is a class of hematopoietic malignancy that expresses the γδ T cell receptor (TCR) with a low incidence. Determining the clonal...
γδT cell lymphoma (γδ TCL) is a class of hematopoietic malignancy that expresses the γδ T cell receptor (TCR) with a low incidence. Determining the clonal proliferation of γδT cells is important for the diagnosis of such malignancies. Few studies have used flow cytometry to detect VδTCR and its subtypes (Vδ1 and Vδ2) at the protein level, although it is a practical method for determining the neoplastic γδT cells. A TCRVδ-based 10-color protocol was designed for the detection of malignant proliferation of γδT subtype cells by multiparameter flow cytometry, and the diagnostic results were compared with the gene rearrangement results. All 19 cases of γδ TCL were positive for cluster of differentiation 3 (CD3) and TCR γδ and presented with abnormal distribution patterns of Vδ1 and Vδ2, of which 16 of the 19 cases showed a restricted Vδ1 staining pattern and the remaining three cases lacked the expression of either Vδ1 or Vδ2. Among the 10 normal controls and 11 patients with reactively higher CD4 and CD8 double-negative ratio, the percentage of Vδ2 positive events (range: 16.4-99.0%) was significantly higher than that of Vδ1 (range: 0-50.5%; < 0.0001), and all cases had a normal Vδ distribution pattern. To detect clonality, there was no difference in the detection rate between the TCRVδ analysis and the gene scanning techniques ( = 1.000) with a high degree of coincidence (Kappa = 0.850, < 0.001). The heteroduplex analysis was less sensitive than the other methods but was more specific (100%) than the gene scanning techniques, and the TCRVδ subtype analysis had the highest sensitivity, specificity, positive predictive value, and negative predictive value. Compared with molecular methods, immunophenotyping is able to distinguish the T cell lineage. The γδT panel, based on the TCRVδ antibody by flow cytometry, could be advantageous for the rapid identification of suspected γδTCL.
PubMed: 32612945
DOI: 10.3389/fonc.2020.00844 -
Genetics Aug 2019Recombination between divergent DNA sequences is actively prevented by heteroduplex rejection mechanisms. In baker's yeast, such antirecombination mechanisms can be...
Recombination between divergent DNA sequences is actively prevented by heteroduplex rejection mechanisms. In baker's yeast, such antirecombination mechanisms can be initiated by the recognition of DNA mismatches in heteroduplex DNA by MSH proteins, followed by recruitment of the Sgs1-Top3-Rmi1 helicase-topoisomerase complex to unwind the recombination intermediate. We previously showed that the repair/rejection decision during single-strand annealing recombination is temporally regulated by MSH (utomolog) protein levels and by factors that excise nonhomologous single-stranded tails. These observations, coupled with recent studies indicating that mismatch repair (MMR) factors interact with components of the histone chaperone machinery, encouraged us to explore roles for epigenetic factors and chromatin conformation in regulating the decision to reject repair recombination between divergent DNA substrates. This work involved the use of an inverted repeat recombination assay thought to measure sister chromatid repair during DNA replication. Our observations are consistent with the histone chaperones CAF-1 and Rtt106, and the histone deacetylase Sir2, acting to suppress heteroduplex rejection and the Rpd3, Hst3, and Hst4 deacetylases acting to promote heteroduplex rejection. These observations, and double-mutant analysis, have led to a model in which nucleosomes located at DNA lesions stabilize recombination intermediates and compete with MMR factors that mediate heteroduplex rejection.
Topics: Histone Chaperones; Histone Code; Histone Deacetylases; Homologous Recombination; Molecular Chaperones; Nucleosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Silent Information Regulator Proteins, Saccharomyces cerevisiae; Sirtuin 2
PubMed: 31221666
DOI: 10.1534/genetics.119.302395 -
BioTechniques Jul 2002In transgenic animal models, the conservation of DNA sequences between the transgene and the host wild-type gene can complicate the evaluation of the expression of each...
In transgenic animal models, the conservation of DNA sequences between the transgene and the host wild-type gene can complicate the evaluation of the expression of each gene. The potential for gene silencing may complicate matters further. Here we report the use of RT-PCR heteroduplex analysis to differentiate the expression of a transgene and its homologous wild-type, even when these genes are very similar in their respective DNA sequences. We designed RT-PCR primers to amplify identically sized 243-bp fragments within the DNA binding domain of the p53 gene from both human and mouse mRNA samples. Ten samples from human p53 (273H) transgenic mice and 10 samples from wild-type controls were tested. Heteroduplex bands were formed in all transgenic samples but were absent from all wild-type samples. In addition, RT-PCR heteroduplex analysis was able in one sample to differentiate a silenced transgene from its wild-type allele, without the assistance of sequencing or labeling. In summary, the RT-PCR heteroduplex analysis is easy to use and has the ability to screen a large number of samples in a short time. The RT-PCR heteroduplex analysis is especially useful for the detection of expression when a transgene and the host homologous endogenous allele are too conserved in sequence to design species-specific RT-PCR primers.
Topics: Animals; Animals, Genetically Modified; Base Sequence; Gene Expression; Gene Expression Regulation; Genes, p53; Heteroduplex Analysis; Humans; Mice; Models, Animal; Molecular Sequence Data; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Transgenes
PubMed: 12139258
DOI: 10.2144/02331st02 -
Philosophical Transactions of the Royal... Mar 2000The functional units of immune response are lymphocyte clones. Analysis of lymphocyte life span in vivo shows that the overall turnover of CD4 and CD8 lymphocytes does... (Review)
Review
The functional units of immune response are lymphocyte clones. Analysis of lymphocyte life span in vivo shows that the overall turnover of CD4 and CD8 lymphocytes does not differ greatly. Recently, molecular methods have been developed which allow a global analysis of T-cell clones responding to an antigen in vivo. We have used a sensitive, modified heteroduplex analysis to follow T-cell clones responding to Epstein-Barr virus in acute infectious mononucleosis (AIM). Strikingly, all the many large clones detected in freshly isolated AIM blood were found within the CD8 fraction. CD4 clonal populations responding to the soluble recall antigen tetanus toxoid could only be detected after in vitro re-stimulation. These data imply that CD4 responses may be more polyclonal than those of CD8 cells and that the size of CD4 clones is more tightly regulated. Several molecular mechanisms may contribute to this. Up-regulation of telomerase allows very large expansions of CD8 cells to occur without exhaustion of proliferative capacity.
Topics: Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Division; Cell Survival; Clone Cells; Humans; T-Lymphocyte Subsets
PubMed: 10794061
DOI: 10.1098/rstb.2000.0580 -
Methods in Molecular Biology (Clifton,... 2022Immunoglobulin (IG) clonality assessment is a widely used supplementary test for the diagnosis of suspected lymphoid malignancies. The specific rearrangements of the...
Immunoglobulin (IG) clonality assessment is a widely used supplementary test for the diagnosis of suspected lymphoid malignancies. The specific rearrangements of the immunoglobulin (IG) heavy and light chain genes act as a unique hallmark of a B-cell lymphoma, a feature that is used in clonality assessment. The widely used BIOMED-2/EuroClonality IG clonality assay, visualized by GeneScanning or heteroduplex analysis, has an unprecedented high detection rate because of the complementarity of this approach. However, the BIOMED-2/EuroClonality clonality assays have been developed for the assessment of specimens with optimal DNA quality. Further improvements for the assessment of samples with suboptimal DNA quality, such as from formalin-fixed paraffin-embedded (FFPE) specimens or specimens with a limited tumor burden, are required. The EuroClonality-NGS Working Group recently developed a next-generation sequencing (NGS)-based clonality assay for the detection of the IG heavy and kappa light chain rearrangements, using the same complementary approach as in the conventional assay. By employing next-generation sequencing, both the sensitivity and specificity of the clonality assay have increased, which not only is very useful for diagnostic clonality testing but also allows robust comparison of clonality patterns in a patient with multiple lymphoma's that have suboptimal DNA quality. Here, we describe the protocols for IG-NGS clonality assessment that are compatible for Ion Torrent and Illumina sequencing platforms including pre-analytical DNA isolation, the analytical phase, and the post-analytical data analysis.
Topics: Clone Cells; DNA; Gene Rearrangement; Genes, Immunoglobulin; High-Throughput Nucleotide Sequencing; Humans; Immunoglobulins; Lymphoma, B-Cell; Sequence Analysis, DNA
PubMed: 35622318
DOI: 10.1007/978-1-0716-2115-8_2 -
Blood Jun 1994The possibility to detect markers of T-cell clonality at the T-cell receptor (TCR) beta and gamma loci in skin biopsy samples has proven to be helpful for the often...
The possibility to detect markers of T-cell clonality at the T-cell receptor (TCR) beta and gamma loci in skin biopsy samples has proven to be helpful for the often difficult clinical and immunohistochemical diagnosis of cutaneous T-cell lymphoma (CTCL). However, particularly at the early stage of the neoplastic infiltration, an emerging clonal pattern at Southern may be obscured by the germline TCR configuration of the predominant dermal and epidermal cell component. Additionally, multiple TCR gamma rearranged bands of variable intensity are often observed, either in the presence or in the absence of a major clone. To overcome these difficulties, we have investigated the T-lymphocyte clonality in selected patients with variable signs of CTCL by means of heteroduplex analysis of the amplified TCR gamma VJ junctions, separated in nondenaturing polyacrylamide gel. This technique has several advantages over standard Southern blot because it is simple, rapid, not radioactive, and likely more sensitive than other polymerase chain reaction-based procedures. In particular, the cases with uncertain or contradictory TCR beta and gamma patterns were solved by the heteroduplex analysis, showing homoduplex or heteroduplex bands of clonal nature. The direct sequence of the VJ junctions, easily obtained from the homoduplex or heteroduplex bands, allowed us to confirm the same clonal marker in two apparently different skin lesions and in different biopsy samples obtained from the same patients, either at the same or different time points, thus emphasizing the utility of this method in monitoring CTCL clinical progression.
Topics: Adult; Aged; Aged, 80 and over; Base Sequence; Female; Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor; Humans; Immunophenotyping; Lymphoma, T-Cell, Cutaneous; Male; Middle Aged; Molecular Sequence Data; Nucleic Acid Heteroduplexes
PubMed: 8193362
DOI: No ID Found -
Human Heredity Jan 2022The CHEK2 gene is known to be an important signal transducer involved in DNA repair, apoptosis, or cell cycle arrest in response to DNA damage. The mutations in this...
INTRODUCTION
The CHEK2 gene is known to be an important signal transducer involved in DNA repair, apoptosis, or cell cycle arrest in response to DNA damage. The mutations in this gene have been associated with a wide range of cancers, both sporadic and hereditary. Germline CHEK2 mutations are linked to an increased risk of breast cancer. Therefore, the aim of this study was to identify the prevalence of CHEK2 variants in BRCA1/2 and PALB2 negative early-onset patients with breast cancer and/or ovarian cancer in a Turkish population for the first time.
METHODS
The study included 95 patients with BRCA1/2 and PALB2 negative early-onset breast cancer and/or ovarian cancer and also 60 unaffected women. All the intron/exon boundaries and coding exons of CHEK2 were subjected to mutational analysis by heteroduplex analysis and DNA sequencing.
RESULTS
A total of 16 CHEK2 variants were found in breast cancer patients within the Turkish population. CHEK2 c.1100delC mutation studied in the CHEK2 gene most frequently was not detected in our study. The prevalence of variants of uncertain significance in CHEK2 was found to be 7.3% (n= 7) in BRCA1/2 and PALB2 mutation negative Turkish patients with early-onset breast and/or ovarian cancer.
DISCUSSION/CONCLUSION
The present study may shed light on alternative variations that could be significant for understanding the prevalence and clinical suitability of the CHEK2 gene.
PubMed: 34991090
DOI: 10.1159/000521369