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At least 19 recordsLinked to original sources
PubMed2026

Genomic and evolutionary analysis reveals dynamic variations of MKK3 gene, a key regulator for seed dormancy in barley.

Barley (Hordeum vulgare L.) is an important crop in the world, and its seed dormancy is primarily controlled by a mitogen-activated protein kinase kinase 3 (MKK3) gene. Although kinase activity of MKK3 and its roles in barley post-domestication have been widely studied, the pre-domestication evolution of MKK3 and the spread of nondormant alleles among global barley varieties remain largely unexplored. In this study, we analyzed MKK3 sequences in barley and its wild progenitor (Hordeum spontaneum K. Koch) and identified two polymorphic miniature inverted-repeat transposable elements (MITEs). Comparative analyses indicated that the insertions/excision of the MITEs predated the current estimates of barley domestication. Examination of the barley pangenomes coupled with droplet digital polymerase chain reaction revealed extensive copy number variation of MKK3 and suggested that transposons likely contributed to tandem amplification of the MKK3 gene on chromosome 5H. Additionally, approximately 1-Kb MKK3 sequences were found on chromosomes 1H and 6H. Further analysis indicated that these short MKK3 sequences were captured by a CACTA transposon that also contained fragments from four other expressed genes. The acquisition of MKK3 was estimated to be between 1.9 and 2.5 million years ago. Together, these findings illuminate the dynamic pre-domestication evolution of the MKK3 gene and identify three divergent MKK3 haplotype groups including a unique lineage predominant in Ethiopian germplasm. This study highlights the contribution of transposons to structural diversification and evolutionary differentiation of the MKK3 locus and provides helpful information for understanding the complex history of MKK3 gene in barley and also for improving preharvest sprouting tolerant varieties under distinct natural conditions.

Hordeum↗
PubMed2025

Ectopic recombination: a novel mechanism of EPSPS gene amplification in glyphosate-resistant Chloris truncata.

Amplification of 5-enolpyruvylshikimate-3-P synthase (EPSPS) gene confers resistance to the herbicide glyphosate in the tetraploid Chloris truncata in Australia. To study the mechanism of amplification, the genomic organization of the EPSPS gene was investigated using fluorescence in situ hybridization (FISH) in one susceptible (Ct-S) and two resistant (Ct-R1 and Ct-R2) biotypes of C. truncata. FISH analysis revealed faint signals of the EPSPS gene on the telomeric regions of a single pair of homologous chromosomes in Ct-S plants. However, much brighter hybridization signals of the EPSPS gene were detected on three pairs of homologous chromosomes in Ct-R1 and on four pairs in the Ct-R2 plants. Thus, there was gene amplification on the native EPSPS locus as well as spread of EPSPS loci to additional chromosomes. All loci were detected in terminal regions which are hotspots of recombination. This local as well as ectopic EPSPS amplification to specific regions of chromosomes is a novel mechanism resistance to herbicides. We hypothesize that, during the bouquet stage of meiosis, telomeres come together forming a bouquet and this may provide an opportunity for ectopic recombination, supported by FISH analyses in interphase nuclei. Overall, the gene amplification appears to have occurred in two steps. First, there was tandem EPSPS amplification at the native locus, possibly via unequal recombination. Second, the amplified locus underwent ectopic recombination and spread to two additional chromosomes in Ct-R1 and three additional chromosomes in Ct-R2 plants.

Glyphosate↗
PubMed2026

The Evolutionary Maintenance of Amino Acid Prototrophy in Escherichia coli.

Escherichia coli is a prototroph and can synthesize all twenty proteinogenic amino acids when required to grow in minimal medium. There are approximately sixty protein-coding genes individually essential for amino acid synthesis. This is a large mutational target for the accumulation of detrimental mutations. E. coli can rewire biosynthetic pathways in response to mutational damage, but the limits of this capacity are poorly understood. Here, to address evolutionary robustness, we asked whether and how the phenotypes of irreversible mutations causing auxotrophy could be suppressed or bypassed in the absence of horizontal gene transfer (HGT). Spontaneous suppressors could be selected for only ten of fifty-nine mutants tested (detection limit ∼7 × 10-11). Mechanisms of suppression included regional amplifications, mutations increasing gene or operon expression, mutations relaxing enzyme specificity, and mutations causing biochemical pathway diversions. Overall, the data show that spontaneous suppression of auxotrophy caused by an irreversible mutation is an evolutionary survival mechanism relevant only to a minority of the genes essential for amino acid synthesis. As a consequence, the essential genetic foundations for amino acid prototrophy are expected to be degraded over time by mutations (Muller's ratchet) and metabolic rewiring alone will be insufficient to counteract this effect. This implies that maintaining phenotypes, including prototrophy in E. coli, and potentially other bacterial species, is likely to be reliant on HGT of housekeeping genes to counteract the effects of inevitable mutational inactivation. Accordingly, chromosomal HGT in bacteria may be critical for survival across diverse environmental niches.

Escherichia coli↗
PubMed1977

Identification and characterization of a small sequence located at two sites on the amplifiable tetracycline resistance plasmid pAMalpha1 in Streptococcus faecalis.

Streptococcus faecalis DT-11 harbors the 6.0-megadalton plasmid pAMalpha1, which determines resistance to tetracycline (TC). When this strain is grown in the presence of TC for a number of generations, a reversible gene amplification occurs, generating tandem repeats of a 2.65-megadalton segment of the plasmid. On the basis of heteroduplex studies between various forms of pAMalpha1 and fragments generated by the Escherichia coli restriction endonuclease EcoRI, we have obtained direct evidence for the presence of a small sequence designated RS1 (for recombination sequence) located on both sides of the TC resistance determinant. Corresponding points on the two sequences are separated by 2.65 megadaltons of deoxyribonucleic acid. The two RS1 sequences have the same polarity and are of a size corresponding to about 380 nucleotide base pairs. The data presented serve as strong support for amplification models that are based on recombinational events involving the RS1 sequences.

Base Sequence↗
PubMed1979

Characterisation of Tn1721, a new transposon containing tetracycline resistance genes capable of amplification.

R plasmid pRSD1 contains tetracycline resistance (tet) genes in a 3.55 Mdal-region capable of amplification by forming tandem repeats (Mattes, Burkardt and Schmitt, Molec. gen. Genet., 1979). The repetitious tet element is itself part of a 7.2 Mdal-transposon, named Tn1721, as demonstrated by the following criteria; (i) Tn1721 has been translocated to phage lambda. The resulting hybrid phage lambda tet contains the 7.2 Mdal-insertion to the right of the attachment site, but not continguous with it indicating translocation of the element by non-homologous recombination. In addition, lambda tet has sustained a 3.4 Mdal-deletion adjacent to the insertion. (ii) Further transposition of Tn1721 to the 21.5 Mdal-plasmid R388 resulted in R388::Tn1721 derivatives, two of which were characterised. They contain Tn1721 inserted into different sites but in the same orientation as shown by restriction and heteroduplex analyses. These translocation of Tn1721 were not accompanied by deletions of DNA. (iii) The insertion plasmid pRSD102(R388::Tn1721) has conserved the capacity of the original plasmid pRSD1 to amplify the 3.55 Mdal-tet region. It has been concluded that Tn1721 constitutes a novel transposon encompassing a tet region capable of selective amplification. The model proposed for Tn1721 contains three short repeats. Two direct repeats, flanking the 3.55 Mdal tet region, provide sequence homology for amplification. The third repeat (located distally to tet) is inverted and provides the basis for transposition of the 7.2 Mdal-element.

Coliphages↗
PubMed2026

NOTCH3 Internal Tandem Duplication Defines a Novel Oncogenic Activation Mechanism of NOTCH Signaling.

NOTCH signaling is activated in tumors through multiple mechanisms, including mutations, gene rearrangements, and gene amplification. We report a novel activation mechanism, an internal tandem duplication (ITD) near the NOTCH3 negative regulatory region (NRR), found in a myogenic mesenchymal neoplasm. This 17-amino acid residue duplication disrupts the tightly autoinhibited structure surrounding the S2 cleavage site, resulting in ligand-independent S2 cleavage and constitutive pathway activation, as demonstrated by increased expression of the NOTCH3 target gene HES1. Cells expressing NOTCH3-ITD showed increased nuclear localization of the receptor and exhibited malignant phenotypes, including enhanced proliferation and migration. Together, these findings support the oncogenic role of NOTCH3-ITD.

Receptor, Notch3↗
PubMed2026

Novel and High-Throughput Method of Isolating Single Fetal Cells Using FACS for NIPT.

OBJECTIVE: To evaluate fluorescence activated cell sorting (FACS) as a method of single-cell isolation of rare circulating fetal cells from maternal blood for use in cell-based non-invasive prenatal testing (cbNIPT). METHOD: Blood samples (30 mL) were collected from 75 'low-risk' pregnant women (gestational age 10-15 weeks). Fetal cells were enriched and stained using magnetic activated cell sorting. Following enrichment, single fetal cells were sorted in individual PCR tubes by FACS. After cell lysis, verification of fetal cell origin was performed using short tandem repeat (STR) analysis with the GlobalFiler PCR Amplification kit. RESULTS: An average of 13.7 cells were sorted using FACS. STR analysis identified 8.2 fetal cells on average, representing 60.2% of the sorted cells. The four-step single-cell isolation procedure facilitated an overall enrichment of approximately 16-million-fold. One sample did not render any fetal cell, corresponding to 1.3% of the samples. CONCLUSION: FACS, which is typically used for segregation of large populations of cells, can be used for single-cell isolation of rare fetal cells in an automated setup. This not only helps in making cell isolation faster and high throughput but also provides fetal cells for a more comprehensive genetic analysis of the fetus.

Humans↗
PubMed1979

Repetition of tetracycline resistance determinant genes on R plasmid pRSD1 in Escherichia coli.

The 30 megadalton (Mdal)-conjugative, fi- plasmid pRSD1 determines inducible tetracycline resistance (Tc) in Escherichia coli. As shown by restriction analysis, a 3.5 Mdal-EcoRI fragment of pRSD1 spliced into the small plasmid pRSD2124 comprises the entire Tc determinant (tet) region. A restriction map of pRSD1 is presented which includes the location of the tet region and of an "underwound" loop not related to Tc (Burkardt et al., 1978). Selective amplification of tet genes is demonstrated by three lines of evidence. (i) The resistance level of cell harbouring pRSD1 increases approximately tenfold by induction with 10 microgram/ml of tetracycline. Further growth in the presence of 100 microgram/ml of the drug ("tetracycline stress") selects for cells with even higher resistance levels (about 300 microgram/ml) in rec+ cells. In a recA strain, a smaller proportion of cells attains these high resistance levels suggesting the involvement of host recombination. (ii) Electron micrographs of pRSD1-DNA isolated from tetracycline-stressed cells reveal a heterogeneous population of circular DNA molecules ranging between 1.7 and 21.6 micron. The distribution of contour lengths shows a discrete pattern ascribed to the presence of autonomous single- and multiple-copy Tc determinants and to intact plasmids containing zero to six tet regions in tandem repeats. (iii) This interpretation is supported by heteroduplex and restriction analyses which demonstrate the presence of multiple copies of the 3.5 Mdal-element encompassing the tet region in pRSD1 molecules selected by tetracycline stress. It has been concluded that gene amplification leading to tandem repetition of the tet region ensues in pRSD1. Such plasmids confer increased tetracycline resistance and can, thefore, be selected by high doses of the drug.

Chromosome Mapping↗
PubMed1979

Amplification of chloramphenicol resistance transposons carried by phage P1Cm in Escherichia coli.

We have characterized a number of P1Cm phages which contain the resistance genes to chloramphenicol and fusidic acid as IS1-flanked Cm transposons. Restriction cleavage and electron microscopic analysis showed that these Cm transposons were carried as monomers (M) or tandem dimers (D). Lysogens of P1Cm (D) are more resistant to chloramphenicol than those of its P1Cm (M) presumably as a result of an increased gene dosage. Amplification of the Cm transposons to tandem multimers was frequently observed in P1Cm (D) lysogens grown in the presence of high concentrations of chloramphenicol or fusidic acid and was also detected in P1Cm (M) lysogens. The degree of amplification varied in different clones which suggests that cells containing spontaneously amplified Cm transposons were selected by high doses of the antibiotics. The dimeric as well as the amplified Cm transposons carried in P1Cm lysogens grown in the absence of chloramphenicol displayed considerable stability. Mechanisms for the amplification of the IS1-flanked transposons are discussed.

Chloramphenicol↗
PubMed2026

Non-invasive embryo assessment: Cell-free DNA-based genetic testing and amino acid metabolomics in relation to morphology: A case-control study.

BACKGROUND: Cell-free DNA (cfDNA) in spent culture medium (SCM) offers a non-invasive option for preimplantation genetic testing, but its low concentration and fragmentation reduce clinical reliability. Combining genetic assessment with metabolomic profiling may provide complementary information about embryo competence. OBJECTIVE: This study assessed pre-analytical cfDNA processing workflows and examined whether SCM amino acid metabolic patterns could act as practical markers of embryo quality. MATERIALS AND METHODS: In this case-control study (2021-2023), 90 embryos were evaluated using fluorescence in situ hybridization or array comparative genomic hybridization. SCM samples underwent rapid boiling, silica-based purification, or whole-genome amplification (WGA). Sex determination was performed using quantitative polymerase chain reaction (qPCR). For cfDNA quality control and aneuploidy screening, the multiplex IRFiling kit and quantitative fluorescent polymerase chain reaction (QF-PCR) were used. Amino acid profiles across embryonic developmental stages and quality grades were quantified via liquid chromatography-tandem mass spectrometry. RESULTS: Rapid boiling resulted in complete failure of DNA amplification. Conversely, silica-based purification yielded 70.0% concordance for qPCR-based sexing and 56.7% for QF-PCR. WGA achieved the highest efficacy (73.3% qPCR and 56.7% QF-PCR concordance), although quality control checks flagged occasional misclassifications. LC-MS/MS profiling revealed significantly elevated alanine and arginine levels in tripronuclear embryos. Furthermore, high-quality blastocysts exhibited elevated glutamic acid levels alongside a pronounced overall depletion of extracellular amino acids compared to low-quality counterparts and controls. CONCLUSION: WGA improves cfDNA detectability and qPCR accuracy compared with boiling or purification, but remains inadequate as a standalone screening approach. SCM amino acid profiling provides informative, complementary metabolic signatures of developmental competence, supporting a multimodal strategy for non-invasive embryo assessment.

Amino acid metabolism↗
PubMed2024

Universal Identification of Pathogenic Viruses by Liquid Chromatography Coupled with Tandem Mass Spectrometry Proteotyping.

Accurate and rapid identification of viruses is crucial for an effective medical diagnosis when dealing with infections. Conventional methods, including DNA amplification techniques or lateral-flow assays, are constrained to a specific set of targets to search for. In this study, we introduce a novel tandem mass spectrometry proteotyping-based method that offers a universal approach for the identification of pathogenic viruses and other components, eliminating the need for a priori knowledge of the sample composition. Our protocol relies on a time and cost-efficient peptide sample preparation, followed by an analysis with liquid chromatography coupled to high-resolution tandem mass spectrometry. As a proof of concept, we first assessed our method on publicly available shotgun proteomics datasets obtained from virus preparations and fecal samples of infected individuals. Successful virus identification was achieved with 53 public datasets, spanning 23 distinct viral species. Furthermore, we illustrated the method's capability to discriminate closely related viruses within the same sample, using alphaviruses as an example. The clinical applicability of our method was demonstrated by the accurate detection of the vaccinia virus in spiked saliva, a matrix of paramount clinical significance due to its non-invasive and easily obtainable nature. This innovative approach represents a significant advancement in pathogen detection and paves the way for enhanced diagnostic capabilities.

Tandem Mass Spectrometry↗
PubMed1977

Selective amplification of genes on the R plasmid, NR1, in Proteus mirabilis: an example of the induction of selective gene amplification.

The drug-resistance plasmid, NR1, is a 37-micron circular DNA molecule that contains two components: the resistance transfer factor (29 micron) carrying the transfer genes and the genes for tetracycline resistance, and the r-determinant (8 micron) carrying the genes for resistance to several other antibiotics including chloramphenicol (Cm). In Proteus mirabilis, these two components are capable of independent replication, or they may replicate as a composite molecule. When cells of P. mirabilis containing NR1 are cultured in medium containing Cm at 250 microgram/ml a growth lag of 20-35 hr ensues. During this lag, Cm induces the selective amplification of the r-determinant, including the gene for resistance to Cm. The amplification results from the excision of the r-determinant from the R plasmid, the independent replication of the r-determinant to give polymeric as well as monomeric r-determinants, and the eventual reintegration of multiple tandem copies of the r-determinant with the resistance transfer factor to form a new R plasmid with multiple copies of the r-determinant. This mechanism represents a new level of control of gene expression in bacterial systems--namely, the induction of selective gene amplification.

Cell Division↗
PubMed2026

A Novel Complete F8 Tandem Duplication Causing Elevated Factor VIII Activity and Associated with Venous Thromboembolism.

Background Coagulation factor VIII (FVIII) is a critical component of the intrinsic coagulation pathway. While elevated FVIII levels are an established risk factor for venous thromboembolism (VTE), genetic variants in the F8 gene directly causing such elevations remain scarce. Here, we report a novel complete F8 tandem duplication identified in a female patient with splanchnic venous thrombosis (SVT). Methods We performed genetic testing using a thrombophilia panel targeting 35 genes involved in thrombosis and haemostasis to detect both point variants and copy number variations (CNVs). Family co-segregation analysis and phenotypic assays for FVIII and von Willebrand factor (VWF) were conducted. The structural basis of the identified F8 copy number gain was elucidated using optical genome mapping (OGM). Full-length F8 mRNA amplification, quantitative PCR, plasma FVIII Western blotting, and X-chromosome inactivation analysis were performed to assess the functional consequences of the duplication. Thrombin generation test (TGT) was employed to assess the hypercoagulable state. Results Genetic testing identified three copies of all 26 exons of the F8 gene in the proband, which was also detected in her mother (CNVs = 3) and son (CNVs = 2). One-stage clotting and chromogenic assays confirmed persistently elevated FVIII activity in the proband and her mother, accompanied by increased FVIII antigen levels. The OGM analysis confirmed a 229 kb tandem duplication including the F8 gene on one of the proband's X chromosomes. The junction regions exhibited high sequence homology and were rich in repetitive sequences, which precluded precise breakpoint mapping. Full-length F8 mRNA amplification revealed no aberrant transcripts, whereas quantitative PCR showed increased F8 mRNA expression in all carriers. Plasma FVIII Western blotting indicated FVIII heavy and light chains of expected molecular weights with increased band intensity in carriers. X-chromosome inactivation analysis in female carriers showed no significant skewing. TGT in two available carriers showed increased thrombin generation compared with a normal control at both low (1 pM) and high (5 pM) tissue factor concentrations. Conclusion We identified a novel complete F8 tandem duplication associated with increased FVIII expression and a hypercoagulable phenotype in a female patient with SVT. These findings support F8 gene dosage gain as a rare gain-of-function mechanism contributing to elevated FVIII levels and thrombophilia, while variation in VWF levels and acquired risk factors may modify thrombotic penetrance.

coagulation factor VIII↗
PubMed1975

The organization, expression, and evolution of antibody genes and other multigene families.

The multigene family is a unit of chromosomal organization. Its gene members are closely linked, homologous in sequence, and have overlapping functions. Multigene families can be divided into three catagories: simple-sequence, multiplicational, and informational-by a variety of structural and functional criteria. Multigene families exhibit two novel evolutionary features-coincidental evolution and rapid change in family size-that suggest that they all share one or more evolutionary mechanisms. Natural selection cannot act directly upon individual genes in a family because of their identical or overlapping functions; hence selection must operate upon the family as a whole or upon blocks of genes within the family. The mechanism(s) for coincidental evolution expands out variant genes within a family so they can be acted upon by natural selection and, accordingly, permits multigene families to evolve adaptively. The control mechanisms in multiplicational families appear to promote the rapid expression of many gene copies. In contrast, the regulatory mechanisms of informational families promote the selection, expression, and amplification of appropriate units of information. The close linkage of the genes in a family appears to be a consequence of the fact that their control and evolutionary mechanisms may only operate on tandemly linked genes. New multigene families may evolve from a single gene or from other multigene families. In addition to evolving new functions, the latter mode of evolution generates a new multigene family whose members are preadapted to interact with those of the old family. These family interactions can lead to the evolution of more sophisticated molecular machines or to the regulation of one family by a second. Multigene families may be large or small. The three catagories of multigene families allow potential multigene families to be identified, and they suggest specific experimental approaches for the study of new families. Some of the most interesting genetic systems under the investigation today are known or potential informational multigene families. This is not fortuitous in that many of the most interesting aspects of phenotype are complex ones with correspondingly complex genetic, evolutionary, and regulatory requirements. One of the frontiers in modern genetics is the identification, characterization, and understanding of informational multigene families.

Alleles↗
PubMed2025

Tracking Somatic Mutations for Lineage Reconstruction.

The human genome is composed of distinct genomic regions that are susceptible to various types of somatic mutations. Among these, Short Tandem Repeats (STRs) stand out as the most mutable genetic elements. STRs are short repetitive polymorphic sequences, predominantly situated within noncoding sectors of the genome. The intrinsic repetition characterizing these sequences makes them highly mutable in vivo. Consequently, this characteristic provides the chance to unravel the natural developmental history of human viable cells retrospectively. However, STRs also introduce stutter noise in vitro amplification, which makes their analysis challenging. Here we describe our integrated biochemical-computational platform for single-cell lineage analysis. It consists of a pipeline whose inputs are single cells and whose output is a lineage tree of input cells.

Humans↗
PubMed2026

Pilot study of allele-specific multi-InDel markers for the detection of extremely unbalanced DNA mixtures.

Mixtures are common in forensic casework, and they represent one of the most challenging types of biological evidence. Traditional short tandem repeat analyses are often associated with limitations when dealing with extremely unbalanced mixtures because alleles from minor contributors can easily be masked by those of major contributors. Consequently, researchers have developed new technologies and methods for improving the analysis of mixtures, spanning upstream DNA extraction and downstream software analysis. Among these, strategies combining allele-specific amplification with compound markers have drawn particular interest because of their ability to selectively detect minor contributors in complex mixtures. In this study, we screened multi-InDels across the entire genome, designed allele-specific primers compatible with the capillary electrophoresis platform, and further explored their potential in unbalanced DNA mixtures and cell-free fetal DNA (cffDNA). Ultimately, a set comprising 10 multi-InDels was developed, and this included two groups of primers that separately amplified the long alleles (L primer set) and short alleles (S primer set). The results demonstrated that each primer pair could detect the minor component at a 1:1000 mixture ratio, whereas the L and S primer sets successfully detected the minor contributors at mixture ratios of 1:200 and 1:500, respectively. Furthermore, in the cffDNA analysis, 60 of 78 informative markers were successfully detected, with the complete detection of all informative markers achieved in 18 mother-child reference pairs. Overall, allele-specific amplification-based multi-InDel markers enabled the sensitive detection of minor contributors, providing a potential strategy for the analysis of unbalanced two-person mixtures.

Allelic-specific amplification↗
PubMed2024

[Pathogenic large duplication in TP53 as a hereditary predisposing factor in breast cancer].

AIM: Germline pathogenic variants of TP53 are associated with Li-Fraumeni syndrome and represent a high risk for hereditary breast and ovarian cancer. We identified a germline, multi-exon heterozygous duplication variant in TP53, NM_000546.6:dup(ex2-5), in a young triple-negative breast cancer patient. We aimed to assign its pathogenicity. METHODS: DNA and RNA (cDNA) level specific amplification tests and sequencings were performed to identify the genomic duplication breakpoint and to detect the presence of defective transcripts. RESULTS: cDNA tests revealed aberrant transcript, which causes a shift in the reading frame. Allelic imbalance was also observed, indicating degradation of the defective RNA product. By locating the breakpoints at the DNA level, we determined that 6975 bp was repeated in tandem and in the same orientation. We also revealed the possible mechanism of the structural rearrangement. CONCLUSIONS: We established that the duplication identified at DNA level manifested in the mRNA and coded for a non-functional protein. Based on these data, we were able to classify this duplication variant as pathogenic, which affects the patient's therapeutic options and justifies genetic screening of family members.

Adult↗