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PubMed2000

Strain identification of Trichophyton rubrum by specific amplification of subrepeat elements in the ribosomal DNA nontranscribed spacer.

Trichophyton rubrum is the commonest cause of dermatophytosis of skin and nail tissue. Molecular characterization of the T. rubrum ribosomal DNA nontranscribed-spacer region revealed two novel tandemly repetitive subelements (TRSs): TRS-1, containing a 27-bp palindromic sequence, and TRS-2. Specific amplification of TRS-1 produced strain-characteristic banding patterns (PCR types), with 21 TRS-1 PCR types recognized from 101 clinical isolates. Four simple patterns representing 1 to 4 copies of TRS-1 accounted for 75 (75%) of all 101 strains, whereas more complex patterns were observed for 21 (20%) of the 101 isolates. The copy number of TRS-2 was 0 to 3 repeats per cistron, with a majority of isolates having two copies of this element. Eleven isolates were polymorphic for TRS-2, and in combination, 23 separate PCR types were recognized by amplification of both TRS-1 and TRS-2. The PCR patterns from both elements were stable and reproducible. Elements with homology to TRS-1 were present in three phylogenetically related species, Trichophyton violaceum, Trichophyton gourvilii, and Trichophyton soudanense, but these elements were not identified in other dermatophyte taxa. There was no clear correlation of PCR type with specimen (skin or nail tissue), but certain PCR types appeared to show a bias in geographic distribution. This new method of typing T. rubrum will enable important questions about pathogenesis and epidemiology of this fungus to be addressed.

Base Sequence↗
PubMed2006

Genetic mechanisms of TP53 loss of heterozygosity in Barrett's esophagus: implications for biomarker validation.

BACKGROUND AND AIMS: 17p (TP53) loss of heterozygosity (LOH) has been reported to be predictive of progression from Barrett's esophagus to esophageal adenocarcinoma, but the mechanism by which TP53 LOH develops is unknown. It could be (a) DNA deletion, (b) LOH without copy number change, or (c) tetraploidy followed by genetic loss. If an alternative biomarker assay, such as fluorescence in situ hybridization (FISH), provided equivalent results, then translation to the clinic might be accelerated, because LOH genotyping is presently limited to research centers. METHODS: We evaluated mechanisms of TP53 LOH to determine if FISH and TP53 LOH provided equivalent results on the same flow-sorted samples (n = 43) representing established stages of clonal progression (diploid, diploid with TP53 LOH, aneuploid) in 19 esophagectomy specimens. RESULTS: LOH developed by all three mechanisms: 32% had DNA deletions, 32% had no copy number change, and 37% had FISH patterns consistent with a tetraploid intermediate followed by genetic loss. Thus, FISH and LOH are not equivalent (P < 0.000001). CONCLUSIONS: LOH develops by multiple chromosome mechanisms in Barrett's esophagus, all of which can be detected by genotyping. FISH cannot detect LOH without copy number change, and dual-probe FISH is required to detect the complex genetic changes associated with a tetraploid intermediate. Alternative biomarker assay development should be guided by appreciation and evaluation of the biological mechanisms generating the biomarker abnormality to detect potential sources of discordance. FISH will require validation in adequately powered longitudinal studies before implementation as a clinical diagnostic for esophageal adenocarcinoma risk prediction.

Adenocarcinoma↗
PubMed1979

Analysis of chromosomes, nucleic acids, and polypeptides in hamster cells transformed by herpes simplex virus type 2.

Syrian hamster embryo fibroblasts were oncogenically transformed by UV-inactivated Herpes simplex type 2. Eighteen clones were isolated shortly after transformation occurred. Two clones and their tumor derivatives were studied using several techniques. The karyotype analysis revealed different chromosome patterns in the two clones and a tendency toward hypodiploidy in the tumor derivatives. All of these cell lines were shown by molecular hybridization to contain 40% of the HSV-genome in several copies. The viral DNA sequence complexity was retained in the tumor derivatives, but a decrease in the copy number was observed. Viral RNA's were detected by in situ hybridization in all the lines that were tested. Viral antigens could be observed in these transformed cells by immunofluorescence. Finally, polypeptide analysis showed three differences between normal and transformed cells.

Animals↗
PubMed2025

Evolutionary Diversification and Functions of the Candidate Male Killing Gene wmk.

Symbiont-mediated male killing (MK) is a mechanism that selectively eliminates male offspring, often by disrupting sex-specific developmental processes. In Drosophila melanogaster, the WO-mediated killing gene wmk from Wolbachia prophage WO transgenically reproduces the MK phenotype, yet how the gene evolves and functions across diverse Wolbachia has not been systematically investigated. We analyzed 32 Wolbachia genomes available in the NCBI database to study wmk homologs across different arthropod hosts, reproductive parasitism functions, and Wolbachia supergroups. First, we report at least five distinct wmk phylogenetic clusters (Types I to V), often organized in multigenic dyads or triads. Second, among MK Wolbachia, there is a significantly higher number of wmk genes and diversity in Lepidoptera strains than in Drosophila strains, which exclusively harbor wmk Types I and III. Third, there are three patterns of wmk sequence and genomic organizational changes in Drosophila MK strains that associate with different evolutionary trajectories underpinning the MK phenotype. Fourth, single and combinatory transgenic expression of Types I and III in D. melanogaster uncovers male-biased lethality associated with Type I; however, dual expression of the Types together elicits a major reduction in offspring number. Fifth, wmk genes have low expression level across D. melanogaster developmental stages relative to the cifA and cifB genes, which could explain why cytoplasmic incompatibility is expressed in this system. These findings establish a complex and phylogenetically informed genetic basis of wmk-induced lethality, highlighting the role of gene copy number and expression, wmk Types, and host background in shaping the phenotype.

Animals↗
PubMed1990

DNA metabolism gene CDC7 from yeast encodes a serine (threonine) protein kinase.

The yeast Cdc7 protein is indispensable to initiation of nuclear DNA replication, based on the phenotype of the conditional, temperature-sensitive (ts) cdc7 mutants at the restrictive temperature. This protein has likewise been implicated in commitment to meiotic DNA recombination and induced mutagenesis, which may result from error-prone DNA repair. Our previous work revealed sequence similarity between the Cdc7 protein and known protein kinases. To determine whether it possesses kinase activity, we have immunoprecipitated the protein from Cdc7-overproducing yeast cells by using polyclonal antibodies raised against a nondenatured beta-galactosidase-Cdc7 fusion protein. In this report, we demonstrate that Cdc7 immune complexes are capable of phosphorylating mammalian histone H1 on serine and/or threonine residues. Immune complexes derived from cells harboring the cdc7-2 ts mutant gene on a high copy number plasmid possess a thermolabile kinase activity. Thus, we postulate that Cdc7 may regulate the various DNA metabolic pathways by phosphorylating one or more target substrates. Because Cdc7 kinase acts downstream of Cdc28/cdc2 kinase function at "start," the transition from G1 to S phase in the cell cycle may be the result of a cascade of protein phosphorylation.

Antibodies↗
PubMed1996

Transcriptional regulation of the proton-translocating ATPase (atpIBEFHAGDC) operon of Escherichia coli: control by cell growth rate.

The F0F1 proton-translocating ATPase complex of Escherichia coli, encoded by the atpIBEFHAGDC operon, catalyzes the synthesis of ATP from ADP and Pi during aerobic and anaerobic growth when respiratory substrates are present. It can also catalyze the reverse reaction to hydrolyze ATP during nonrespiratory conditions (i.e., during fermentation of simple sugars) in order to maintain a electrochemical proton gradient across the cytoplasmic membrane. To examine how the atp genes are expressed under different conditions of cell culture, atpI-lacZ operon fusions were constructed and analyzed in single copy on the bacterial chromosome or on low-copy-number plasmids. Expression varied over a relatively narrow range (about threefold) regardless of the complexity of the cell growth medium, the availability of different electron acceptors or carbon compounds, or the pH of the culture medium. In contrast to prior proposals, atp operon expression was shown to occur from a single promoter located immediately before atpI rather than from within it. The results of continuous-culture experiments suggest that the cell growth rate rather than the type of carbon compound used for growth is the major variable in controlling atp gene expression. Together, these studies establish that synthesis of the F0F1 ATPase is not greatly varied by modulating atp operon transcription.

Bacterial Outer Membrane Proteins↗
PubMed1988

The methanoreductosome: a high-molecular-weight enzyme complex in the methanogenic bacterium strain Gö1 that contains components of the methylreductase system.

The methanogenic bacterium strain Gö1 harbors a high-molecular-weight enzyme complex containing methyl coenzyme M methylreductase as revealed by immunoelectron microscopy. This complex consists of a spherelike, hollow head piece, in the wall of which a number of copies of the methyl coenzyme M methylreductase are located. It is named Rc (c indicates collector). Intimately bound to it is a group of additional subunits of unknown composition referred to as Rm (m indicates mediator). Electron microscopy of negatively stained samples indicated that Rm contains a functional pore or channel which connects the internal volume of Rc with the outside. The RcRm complex is named Rs (s indicates spherelike). This complex was often found detached from the inside of the cytoplasmic membrane when membrane vesicles were investigated. However, Rs was also seen attached to a third component of the complex located in the membrane, the attachment being mediated by Rm. This membrane part of the complex is designated Rt (t indicates translocator). It consists of subunits with unknown composition. When Rs is attached to the membrane, the pore in Rm appears to be plugged by Rt. This indicates that the internal volume in Rc is in contact, via the pore in Rm, with Rt. The RcRmRt complex is referred to as methanoreductosome. Functional implications of the structural organization of the methylreductase system are discussed in view of methane formation and the creation of a transmembrane proton gradient used by the cell for ATP synthesis.

Cell Membrane↗
PubMed2002

Role of short tandem repeat DNA in forensic casework in the UK--past, present, and future perspectives.

The analysis of short tandem repeat (STR) DNA sequences is of fundamental importance to forensic science because they have become the recognized standard in constructing national public databases. Consequently, considerable effort has been expended in developing multiplexed (one tube) reactions that analyze several loci in combination. The implementation of STRs in casework cannot take place without a full understanding of the systems used. The purpose of validation is to characterize multiplexes when one is challenged with forensic samples. For example, mixtures are often encountered that may be particularly difficult to interpret against a background of allelic artifacts. By increasing the number of PCR amplification cycles, it is possible to dramatically boost the sensitivity of the system so that just a handful of cells may be successfully analyzed. However, interpretation is much more complex because the origin of DNA profiles may be less certain and complicated by issues such as contamination, the potential for innocent transfer and a predominance of mixtures. This review provides a brief historical background of the development of STRs in forensic casework that culminated in the creation of national DNA databases. The development of guidelines to interpret complex DNA profiles, such as mixtures, is outlined. Finally, the recent innovation of low copy number DNA profiling is explained along with the special considerations needed to report in court.

DNA↗
PubMed1994

Effect of different plasmids on colonization of mouse tissues by the aromatic amino acid dependent Salmonella typhimurium SL 3261.

The stability of plasmids pBR322, pUC19 and pBluescript and their effect on bacterial colonization of mice was determined. S. typhimurium SL 3261 carrying high copy number plasmids colonized spleen and liver tissues poorly compared to low copy number plasmids. After inoculation into mice, the stability of the plasmids appeared to be inversely related to the plasmid's size and complexity. Mouse-passaging a pBluescript-based recombinant plasmid expressing the Protective Antigen of Bacillus anthracis selected for a mutant S. typhimurium strain (designated G3) that colonized at high levels and more stably maintained plasmids than S. typhimurium SL 3261. S. typhimurium G3 down-regulated the copy number of ColE1 plasmids. The significance of these data for vaccine design is discussed.

Animals↗
PubMed2026

Optical genome mapping enhanced by refined variant interpretation in pediatric acute lymphoblastic leukemia.

Reliable detection of structural variants (SVs) and copy number variations (CNVs) is crucial in the contemporary diagnostics of pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, limitations of commonly used conventional and molecular cytogenetic methods may hinder the accurate genetic characterization of patients. Optical genome mapping (OGM) offers a reliable alternative by enabling high-resolution, genome-wide detection of CNVs and SVs. Chromosomal aberrations were screened using OGM in 51 children with B-ALL. The results were compared with those of karyotyping, fluorescence in situ hybridization (FISH), digital multiplex ligation-dependent probe amplification (digitalMLPA), and targeted RNA sequencing (RNA-seq). OGM data showed high congruency with karyotyping and FISH findings, detecting clinically relevant variants beyond G-banding results and unraveling a complex KMT2A fusion undetected by FISH. Gene fusions involved in complex ETV6::RUNX1 translocations, but not detected by RNA-seq, were confirmed using FISH. Normalization of OGM copy number values with DNA-index-improved concordance with FISH-derived copy numbers in near-tri/tetraploid cases. In the peripheral regions of OGM variants (fringe-zones), a novel evaluation strategy called 'FriZone' was applied, which significantly improved the concordance between OGM and digitalMLPA. In addition, a co-segregation analysis revealed strong associations between ETV6::RUNX1 fusion and deletions of ETV6, RAG2, and NR3C2. OGM uncovered complex rearrangements undetected by widely used methods in 15% of cases, improving genetic classification and risk stratification in 10% of the patients. The FriZone analysis and normalization by DNA-index provide a refined, more accurate approach to OGM variant interpretation, facilitating the efficient application of OGM in clinical diagnostics. &#xa9; 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Humans↗
PubMed2003

Crystal structure of the plasmid maintenance system epsilon/zeta: functional mechanism of toxin zeta and inactivation by epsilon 2 zeta 2 complex formation.

Programmed cell death in prokaryotes is frequently found as postsegregational killing. It relies on antitoxin/toxin systems that secure stable inheritance of low and medium copy number plasmids during cell division and kill cells that have lost the plasmid. The broad-host-range, low-copy-number plasmid pSM19035 from Streptococcus pyogenes carries the genes encoding the antitoxin/toxin system epsilon/zeta and antibiotic resistance proteins, among others. The crystal structure of the biologically nontoxic epsilon(2)zeta(2) protein complex at a 1.95-A resolution and site-directed mutagenesis showed that free zeta acts as phosphotransferase by using ATPGTP. In epsilon(2)zeta(2), the toxin zeta is inactivated because the N-terminal helix of the antitoxin epsilon blocks the ATPGTP-binding site. To our knowledge, this is the first prokaryotic postsegregational killing system that has been entirely structurally characterized.

Amino Acid Sequence↗
PubMed2003

Hexamerization of RepA from the Escherichia coli plasmid pKL1.

The Escherichia coli plasmid pKL1 is one of the smallest bacterial plasmids. It encodes a single, autoregulating structural gene, repA, responsible for replication and copy number control. The oligomerization of RepA was previously proposed as the basis of a strategy for pKL1 copy number control. To elucidate the oligomerization properties of RepA in solution, RepA was expressed in E. coli; purified by ion exchange and hydrophobic chromatography; and examined in solution by spectrapolarimetry, light scattering, sedimentation velocity, and equilibrium ultracentrifugation. RepA behaved as a concentration-dependent equilibrium of dimers and hexamers. Conformational parameters of the RepA hexameric complex were determined. These results support the proposed autogenous regulatory model whereby RepA hexamers negatively regulate repA expression thereby affecting the copy number control of pKL1. RepA of pKL1 is the first plasmid replication initiation protein documented to be in dimeric-hexameric forms.

Base Sequence↗
PubMed1999

The reaction center-LH1 antenna complex of Rhodobacter sphaeroides contains one PufX molecule which is involved in dimerization of this complex.

The PufX membrane protein is essential for photosynthetic growth of Rhodobacter sphaeroides wild-type cells. PufX is associated with the reaction center-light harvesting 1 (RC-LH1) core complex and plays a key role in lateral ubiquinone/ubiquinol transfer. We have determined the PufX/RC stoichiometry by quantitative Western blot analysis and RC photobleaching. Independent of copy number effects and growth conditions, one PufX molecule per RC was observed in native membranes as well as in detergent-solubilized RC-LH1 complexes which had been purified over sucrose gradients. Surprisingly, two gradient bands with significantly different sedimentation coefficients were found to have a similar subunit composition, as judged by absorption spectroscopy and protein gel electrophoresis. Gel filtration chromatography and electron microscopy revealed that these membrane complexes represent a monomeric and a dimeric form of the RC-LH1 complex. Since PufX is strictly required for the isolation of dimeric core complexes, we suggest that PufX has a central structural role in forming dimeric RC-LH1 complexes, thus allowing efficient ubiquinone/ubiquinol exchange through the LH1 ring surrounding the RC.

Bacterial Proteins↗
PubMed2006

Array-CGH and multipoint FISH to decode complex chromosomal rearrangements.

BACKGROUND: Recently, several high-resolution methods of chromosome analysis have been developed. It is important to compare these methods and to select reliable combinations of techniques to analyze complex chromosomal rearrangements in tumours. In this study we have compared array-CGH (comparative genomic hybridization) and multipoint FISH (mpFISH) for their ability to characterize complex rearrangements on human chromosome 3 (chr3) in tumour cell lines. We have used 179 BAC/PAC clones covering chr3 with an approximately 1 Mb resolution to analyze nine carcinoma lines. Chr3 was chosen for analysis, because of its frequent rearrangements in human solid tumours. RESULTS: The ploidy of the tumour cell lines ranged from near-diploid to near-pentaploid. Chr3 locus copy number was assessed by interphase and metaphase mpFISH. Totally 53 chr3 fragments were identified having copy numbers from 0 to 14. MpFISH results from the BAC/PAC clones and array-CGH gave mainly corresponding results. Each copy number change on the array profile could be related to a specific chromosome aberration detected by metaphase mpFISH. The analysis of the correlation between real copy number from mpFISH and the average normalized inter-locus fluorescence ratio (ANILFR) value detected by array-CGH demonstrated that copy number is a linear function of parameters that include the variable, ANILFR, and two constants, ploidy and background normalized fluorescence ratio. CONCLUSION: In most cases, the changes in copy number seen on array-CGH profiles reflected cumulative chromosome rearrangements. Most of them stemmed from unbalanced translocations. Although our chr3 BAC/PAC array could identify single copy number changes even in pentaploid cells, mpFISH provided a more accurate analysis in the dissection of complex karyotypes at high ploidy levels.

Cell Line, Tumor↗
PubMedDate not supplied

Genomic gain of the epidermal growth factor receptor harboring band 7p12 is part of a complex pattern of genomic imbalances in oral squamous cell carcinomas.

BACKGROUND: To determine the association between changes of genomic gene dose and clinical parameters in squamous cell carcinomas of the oral cavity, comparative genomic hybridization seemed suited not only to detect genomic imbalances in these tumors, but also particularly to examine the role of gain of 7p12, the band harboring the epidermal growth factor receptor (EGFR) in this context. METHODS: Total genomic DNA obtained from 35 oral squamous cell carcinomas was subjected to comparative genomic hybridization (CGH) and detected patterns of genomic imbalances were associated with various clinical parameters. RESULTS: The examined tumors exhibited five and up to 47 DNA copy number alterations (CNAs). Nineteen of these showed a gain of chromosome band 7p12. A highly complex but strikingly consistent pattern of genomic imbalances (average, 32 CNAs per tumor) was associated with this alteration, among which gains clearly dominated over losses of genomic material. Comparable patterns, however, could also be found in a few tumors with a high number of CNAs (average, 26) but without the 7p gain. Low numbers of imbalances always were accompanied by low consistency of CNA patterns and none of these cases showed enh(7p12). No significant differences with respect to pT class or grade of tumors were found between enh(7p)-positive and -negative tumors. Stage IV and lymph node affection were slightly more frequent among enh(7p12)-positive than in -negative cases. Relapse occurred in 63% in 7p12-positive vs. 25% in the negative group. Average disease-free survival of tumors without 7p gain clearly exceeded that of tumors with gain of 7p (36.8 vs. 21.3). However, some of these associations could also be found if comparison was based on number of CNAs. By means of hierarchical cluster analysis, we were able to show that different patterns of CNAs can be separated from each other in tumors with or without 7p alterations, and that these patterns predict short- or long-term survival of patients. CONCLUSIONS: Previously described associations of gains of 7p12, the chromosomal band harboring the EGFR gene with clinical parameters can reasonably be estimated only within the context of the pattern and complexity of the genomic imbalances accompanying this chromosomal loss in examined tumors.

Adult↗
PubMed1987

Differential regulation of T cell receptor gamma genes in immature thymocyte populations.

Immature thymocytes that lack both Lyt-2 (CD8) and L3T4 (CD4) expression can respond rapidly to stimulation with phorbol ester and calcium ionophore by expressing some gene products characteristic of mature, activated T cells. Here we studied the effect of such short-term stimulation on the number of copies per cell of RNA for components of the T cell receptor complex. Although, upon stimulation, mRNAs for T cell receptor beta chain accumulated to higher levels, the cells did not rapidly increase their expression of alpha-chain transcripts from rearranged or germ-line genes. Transcripts from the C gamma 1 (C gamma 13.4) and C gamma 2 (C gamma 10.5) genes were differentially regulated. The rarer C gamma 1 transcripts were strongly induced, while the initially abundant C gamma 2 transcripts showed a modest decrease in transcripts per cell within 24 h. Thus, the ratio of these two transcripts could be shifted dramatically prior to any significant change in the cellular composition of the population. These results suggest regulatory processes that may contribute to the observed expression of gamma products in vitro or in normal development.

Animals↗
PubMedDate not supplied

Hypertrophic cardiomyopathy with mitochondrial DNA depletion and respiratory enzyme defects.

We report the case of a child with severe hypertrophic cardiomyopathy, with decreased activity levels of cardiac mitochondrial respiratory complex I and III, and with a pronounced reduction in cardiac mitochondrial DNA copy number level. Mitochondrial DNA depletion has not been previously reported in hypertrophic cardiomyopathy and it may play a role in its pathogenesis.

Cardiomyopathy, Hypertrophic↗
PubMed2005

Strategies for revealing lower abundance proteins in two-dimensional protein maps.

One of the most challenging contemporary research endeavors is the mapping of proteins and establishing their linkages to normal and pathological conditions. The availability of current proteomics technologies has greatly facilitated the separation and identification of proteins in a complex protein mixture by standard two-dimensional gel electrophoresis and subsequent MALDI-TOF mass spectrometry. Due to the huge differences in the distribution of proteins in complex proteomes of humans, the detection and identification of proteins expressed in low copy number is a major challenge. The low abundance of important physiologically relevant proteins has rendered their analyses almost impossible without some means of prior purification and enrichment from tissue lysates or biological fluids. It is the current limits of detection of the methods that are used that prevents the detection of these proteins not the proteins themselves. More importantly, considering the frequency at which post-translational modifications of proteins occur, the separation of protein isoforms is essential to understand biological changes, and two-dimensional gel electrophoresis remains the only technique that can offer sufficient resolution to address this issue at a functional level. Cellular fractionation techniques followed by specific affinity probes for tracking target proteins have been developed to deplete the proteome of high abundance proteins in order to increase the sample loading for achieving greater sensitivity for proteins present in low abundance. Those applications can entail the removal of one protein or a class of proteins that interferes with the resolution of proteins in a 2-DE map. Moreover, the use of better solubilizing detergents in combination with an overlapping narrow immobilized pH gradients, results in higher resolution by stretching the protein pattern in the first dimension. In this review we will discuss strategies to remove high abundance proteins that can result in the visualization and detection of low abundance proteins in biological samples. The potential use of these strategies, as a means of developing diagnostic tools for early screening of diseases and identification of drug targets for therapeutic intervention, will also be discussed.

Animals↗