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PubMedDate not supplied

DNMT1 gene copy number does not influence susceptibility to development of malignant lymphoproliferative disease.

BACKGROUND: For many years, the methylating enzyme DNMT1 has been thought to critically influence neoplastic transformation. To determine if DNMT1 is involved in the early events of malignant transformation of lymphoid cells, we investigated whether allele-specific variation in the gene copy number of DNMT1 influences susceptibility to the development of malignant lymphoproliferative disease (LPD) associated with DNA hypermethylation. MATERIALS AND METHODS: DNMT1 gene copy number was assessed by subjecting DNA from DNMT1A/DNMT1B heterozygous patients suffering from benign LPD or malignant LPD featuring Myf-3 hypermethylation to Southern blotting and densitometric analysis. RESULTS: The distribution of DNMT1B to DNMT1A intensity ratios did not significantly differ between patients suffering from benign and patients suffering from malignant LPD and there was no association between increasing Myf-3 hypermethylation and increased DNMT1 gene copy number. CONCLUSION: In contrast to findings in mice, the results of this study exclude a role for DNMT1 gene copy number in susceptibility to neoplastic transformation in humans.

Cell Transformation, Neoplastic↗
PubMed2003

Fingerprints as evidence for a genetic profile: morphological study on fingerprints and analysis of exogenous and individual factors affecting DNA typing.

Material recovered from 374 fingerprints left by eleven laboratory workers on three different substrates (glass, wood, metal) at a standard pressure time of 30 s, with and without preliminary handwashing, was submitted to morphological, quantitative, and type analysis. Morphological and agarose-gel electrophoresis analysis showed that a non-negligible amount of epidermal corneal cells presented apoptotic alterations. The quantity of DNA recovered from fingerprints ranged between 0.04 to 0.2 ng, and in a significant number of experiments no DNA was detected. Handwashing reduced the amount of DNA recovered from fingerprints. The "shedder status" of the donor was a very important factor, causing inter-individual variations in the amount of DNA left by fingerprints. Spurious alleles from laboratory-based and secondary transfer contamination, stutters, and other artifacts described when analyzing low-copy-number DNA and capable of affecting correct profiles were observed.

Alleles↗
PubMed1990

Recurrent mutation of immunoglobulin and c-myc genes and differential expression of cell surface antigens occur in variant cell lines derived from a Burkitt lymphoma.

The phenotypic and molecular genetic characteristics of 4 variant sublines of the Burkitt lymphoma cell line Namalwa have been examined. The sublines are DNA-fingerprint-identical and derived from a monoclonal tumour, as shown by a rearrangement of the T-cell-receptor beta-chain gene common to the 4 sublines. There is non-co-ordinate expression of MHC class-I MHC class-II, surface immunoglobulin and a number of antigens recognized by CD MAbs on the different sublines. These different phenotypes of the cells are reminiscent of B cells arrested in varying states of cellular maturity. On Southern blots there are different patterns of restriction fragments hybridizing with Ig heavy- and light-chain gene probes among the sublines, indicating that multiple rearrangements or mutations of Ig genes have occurred in the cells. Different patterns of hybridizing fragments among the sublines were also found by using c-myc probes, implying the existence of different mutations of the c-myc locus. The c-myc mutation found in one of the sublines mapped to the 5' flanking sequence and in another 3' to the c-myc locus. Using the J17BS8 probe, which detects a restriction fragment length polymorphism in the 3' flanking region of the c-myc gene, a 4-fold variation in the gene copy number among the subline was found and one of the sublines was shown to be hemizygous for c-myc. Examination of DNA from early cultures of Namalwa cells showed that the alternations in Ig and c-myc structure had occurred on prolonged culture of the cells.

Antibodies, Monoclonal↗
PubMed2026

Prognostic Role of Global DNA Methylation in Renal Cancer Reveals Decitabine Treatment Benefit.

BACKGROUND: Renal cancer presents a significant global health challenge due to its rising incidence and mortality rates. Often undetected in early stages, it complicates diagnosis and treatment. Current therapies face resistance and limited effectiveness, especially in advanced stages. The diverse subtypes of renal cancer highlight the need for new biomarkers and risk assessment tools for targeted treatments. OBJECTIVE: This study aims to assess the prognostic significance of global DNA methylation (GM) levels in renal cancer, identify new biomarkers, and evaluate the therapeutic potential of the DNA methyltransferase inhibitor decitabine. METHODS: Data on RNA sequencing, gene mutations, DNA methylation, and clinical outcomes were collected from TCGA and GEO databases. We calculated global DNA methylation scores (GMS) and categorized patients into high, intermediate, and low GMS groups. Survival analysis and genomic analyses were conducted to explore the relationships between GMS, clinical outcomes, and tumor characteristics. RESULTS: Higher GMS was identified as an independent prognostic factor associated with worse outcomes in renal cancer. Patients with elevated GMS showed increased mutations, copy number variations, and a more aggressive tumor phenotype. Treatment with decitabine was observed to reduce tumor hypermethylation and downregulate cell cycle pathway activity, indicating potential therapeutic benefits. CONCLUSION: Global DNA methylation plays a significant role in renal cancer prognosis. GMS may serve as valuable biomarkers for prognosis and personalized treatment strategies. Decitabine shows potential efficacy for high GMS patients, particularly through its impact on cell cycle regulation, underscoring the importance of personalized approaches in cancer treatment.

Humans↗
PubMed1991

Sequence and expression of potato U2 snRNA genes.

Plant UsnRNA multigene families show a high degree of sequence variation among individual gene members. The potato U2snRNA gene family consists of between twenty-five and forty genes. Four potato U2snRNA gene variants have been isolated. Despite the sequence variation in coding and flanking regions, all maintain the conserved U2snRNA secondary structure and all contain the plant UsnRNA promoter elements: the upstream sequence element (USE) and TATA-like box in the -70 and -30 regions respectively. In RNase A/T1 protection analyses, one of the genes, PotU2-22, protected high levels of full length U2snRNA transcripts in potato leaf, stem, root and tuber RNA. Thus, PotU2-22 or genes with identical coding regions, are highly expressed in these potato organs and therefore represent a major subset of functional U2snRNA genes. Similar expression levels of the PotU2-22 sequence variant were also found in four genetically different potato cultivars and also in tobacco, a species closely related to potato, suggesting conservation of the coding regions of expressed U2snRNA genes. A second gene, PotU2-4, protected very low levels of full length transcripts while a third gene, PotU2-11, was not expressed in the potato organs analysed. The relative expression levels of the gene variants may reflect individual gene differences in, for example, the USE and TATA regulatory elements, or variations in gene copy number.

Animals↗
PubMed2002

Transposable elements and the evolution of genome size in eukaryotes.

It is generally accepted that the wide variation in genome size observed among eukaryotic species is more closely correlated with the amount of repetitive DNA than with the number of coding genes. Major types of repetitive DNA include transposable elements, satellite DNAs, simple sequences and tandem repeats, but reliable estimates of the relative contributions of these various types to total genome size have been hard to obtain. With the advent of genome sequencing, such information is starting to become available, but no firm conclusions can yet be made from the limited data currently available. Here, the ways in which transposable elements contribute both directly and indirectly to genome size variation are explored. Limited evidence is provided to support the existence of an approximately linear relationship between total transposable element DNA and genome size. Copy numbers per family are low and globally constrained in small genomes, but vary widely in large genomes. Thus, the partial release of transposable element copy number constraints appears to be a major characteristic of large genomes.

DNA Transposable Elements↗
PubMed1992

Chromosome-size variation in Giardia lamblia: the role of rDNA repeats.

Giardia lamblia trophozoites contain at least five sets of chromosomes that have been categorized by chromosome-specific probes. Pulsed field separations of G. lamblia chromosomes also demonstrated minor bands in some isolates which stained less intensely with ethidium than the major chromosomal bands. Two of the minor bands of the E11 clone of the ISR isolate, MBa and MBb, were similar to each other and to chromosomal band I by hybridization to total chromosomal DNA and by hybridization of specific probes. In order to determine the extent of this similarity, I have developed a panel of probes for many of the Pacl restriction fragments and have shown that most of the Pacl and Notl fragments found in MBa are also present in MBb. The differences are found in both telomeric regions. At one end, MBb contains a 300 kb region not found in MBa. At the other end of MBb is a 160 kb region containing the rDNA repeats which is bounded on one end by the telomeric repeat and on the other by sites for multiple enzymes that do not digest the rDNA repeats. The corresponding region of MBa is 23 kb in size. The size difference is consistent with the eightfold greater number of rDNA repeats in MBb than MBa and suggests that 30% of the size difference is accounted for by different numbers of copies of the rDNA repeat. MBa of another ISR clone (ISR G5) is 150 kb larger in size than MBa of ISR E11. The data suggest that MBa and MBb are homologous chromosomes of different sizes and that a portion of the size difference is accounted for by different copy numbers of the rDNA repeat.

Animals↗
PubMed1994

Binding of nuclear factors to a satellite DNA of retroviral origin with marked differences in copy number among species of the rodent Ctenomys.

The major satellite DNA of the subterranean rodent Ctenomys, named RPCS, contains several consensus sequences characteristic of the U3 region of retroviral long terminal repeats (LTRs), such as a polypurine tract, CCAAT boxes, binding sites for the CCAAT/enhancer-binding protein (C/EBP), a TATA box and putative polyadenylation signals. RPCS presents an enormous variation in abundance between species of the same genus: while C. australis or C. talarum have approximately 3 x 10(6) copies per genome, C. opimus has none. A sequence (RPCS-I) with identity to the SV40-enhancer core element, present in all the repeating units of the satellite is specifically protected in DNase I footprintings. Competitions of band-shift assays with different transcription factor binding sites indicate that binding to RPCS-I is specific and involves CCAAT proteins related to NF-1, but not to C/EBP. By the use of quantitative protein/DNA binding assays we determined that, despite of their conspicuous difference in RPCS copy number, C. talarum and C. opimus have equivalent amounts and identical quality of RPCS-binding proteins. These results are consistent with the observation, by in situ hybridization, that RPCS is clustered in heterochromatic regions, where it might have restricted accessibility to transcription factors in vivo. This is the first report of the binding of transcription factors to a satellite DNA of retroviral origin.

Animals↗
PubMed1983

Nucleotide sequence of the genetically labile repeated elements 5' to the origin of mouse rRNA transcription.

We have determined the complete nucleotide sequence of a cloned Balb/c mouse rDNA NTS fragment containing 13 tandem copies of a 135 bp subrepeating segment. This repetitious region (VrDNA) lies close to the origin of ribosomal RNA transcription. Analyses of these VrDNA subrepeats from Balb/c and a related species, Mus pahari, reveal regions of inverted repeat DNA as well as large poly T tracts, either of which may be significant to the generation of the high levels of VrDNA copy number variation found in wild and inbred mice and/or the modulation of rRNA synthesis. Unlike the highly homogeneous subrepeats in the Xenopus laevis NTS repetitious regions, the VrDNA subrepeats differ from one another on the average by about 13%. Sequence analysis and Southern hybridization studies have also shown that, unlike the Xenopus and Drosophila NTS, extensive duplications of sequences found surrounding the mouse rRNA initiation site are found neither in the VrDNA region nor 6 kb further upstream in the NTS.

Animals↗
PubMed2025

Epstein-Barr Virus-Associated Gastric Cancer: A Histopathologic Study With Comprehensive Molecular Profiling.

A subset of gastric cancers (GCs) is linked to Epstein-Barr virus (EBV) infection. This study aims to characterize the histopathological and molecular features of EBV-associated GCs (EBVaGCs), focusing on predictive biomarkers and genomic and transcriptomic analysis. A total of 35 primary EBVaGCs were considered. The presence of EBV was confirmed with in situ hybridization. Immunohistochemical analyses for HER2, PD-L1, claudin 18.2, and mismatch repair proteins were performed. Genomic and transcriptomic profiles were assessed using AmoyDx Master Panel, which can identify single-nucleotide variants, InDels, and copy number variations on 571 hot genes, as well as microsatellite status, tumor molecular burden, and homologous recombination deficiency at the DNA level; however, at the RNA level, it identifies rearrangements/fusions in 45 genes and also quantifies the expression of 2396 cancer-related transcripts. The following histotypes were identified: carcinoma with lymphoid stroma (CLS; 69%), tubular (20%), and mixed (11%). Most cases were associated with atrophic gastritis (71%), and only 11% with dysplasia. The vast majority (94%) of EBVaGCs expressed EBV-encoded RNA in all tumor cells. Mismatch repair deficiency and HER2 overexpression were each observed in 6% of cases, whereas all tumors had a PD-L1-combined positive score ≥10. Sixty-six percent of cases showed moderate/strong claudin 18.2 expression in ≥75% of cancer cells. The most frequently altered genes were PIK3CA (41%) and ARID1A (17%). Transcriptomic analysis revealed substantial differential gene expression between EBVaGCs and EBV-negative controls, with upregulation of genes involved in antigen presentation, natural killer cell-mediated cytotoxicity, and cytokine-cytokine receptor interaction in EBVaGCs. Within EBVaGC, CLS showed higher expression of immune-related transcripts and higher PD-L1 expression than other histotypes. This study establishes EBVaGC as a distinct molecular class, with a distinctive profile of genomic alterations and expression of predictive biomarkers, and also with a unique immune microenvironment with enhanced cytotoxic activity. The findings highlight EBV's role in early tumor development and EBVaG-CLS as a distinct subgroup within EBVaGC, characterized by unique morphologic features and a pronounced immune activation profile.

Humans↗
PubMed1996

Detection of cytomegalovirus in blood donors by PCR using the digene SHARP signal system assay: effects of sample preparation and detection methodology.

Cytomegalovirus (CMV) is an important cause of transfusion-associated morbidity and mortality; however, only 0.4 to 12% of the blood products obtained from seropositive blood donors transmit infection. The effects of three commercially available whole-blood sample preparation kits on the detection of CMV PCR products by a semiquantitative adaptation of the Digene SHARP Signal System Assay (DSSSA) in samples from volunteer blood donors was assessed. Of 101 samples from seropositive blood donors, CMV was detected in 0 (0%) of the samples extracted with a QIAamp blood kit (QIAGEN), 1 (1%) of the samples extracted with an Amplicor whole-blood specimen preparation kit (Roche), and 8 (8%) of the samples extracted with an Isoquick nucleic acid extraction kit (modified by the addition of carrier tRNA) (Microprobe). CMV DNA was not detected in samples from seronegative blood donors (n = 13). Nested PCR of selected samples confirmed the detection of CMV in the sane eight samples extracted with the modified Isoquick nucleic acid extraction kit and detected an additional nine CMV-positive samples (n = 50). Samples from volunteer blood donors contain low copy numbers of CMV DNA. PCR amplification of such specimens can result in analytical sampling errors, giving results similar to the variations in titers recognized during determinations of the 50% tissue culture infective dose. The detection of CMV in blood samples from volunteer blood donors by PCR is a function of sample preparation, amplification conditions, and detection methodology. Accurate assessments of the clinical utility of CMV DNA detection by nucleic acid amplification for blood product screening and patients will require highly standardized and quantitative methodology.

Blood Donors↗
PubMed1991

Overamplification of macronuclear linear DNA molecules during prolonged vegetative growth of Oxytricha nova.

During prolonged vegetative growth of a clonal line of Oxytricha nova, several macronuclear linear DNA molecules increased greatly in copy number over the rest of the approx. 24,000 kinds of molecules comprising the macronuclear genome. One of the amplified sequences was the linear DNA molecule encoding rRNA (rDNA). We have cloned and sequenced the other, smaller, amplified molecules and found that they comprise a gene family, with different allelic versions of one of the family members being amplified. Thus, increased replication is a general property of the molecules comprising this gene family. To date, no function has been assigned to these genes; thus, whether the amplification of these sequences has functional significance is unknown. The rDNA molecule and the two small amplified sequences increased 11-, 24- and 107-fold, respectively, during clonal growth of this line, eventually comprising up to 15% of the macronuclear DNA molecules. Seven other macronuclear DNA molecules did not vary substantially in copy number at different times during the clonal growth of this strain. Analysis of cell-to-cell differences in copy numbers in this clonally aged strain indicated more extensive variation than is evident when large populations from different times are compared.

Amino Acid Sequence↗
PubMed2000

[Mitochondrial medicine for internists].

Human mitochondrial DNA (mtDNA) is a small, circular molecule, encoding for the translational machinery of the mitochondrion, as well as for 13 structural proteins that are all subunits of the respiratory chain. Point mutations, deletions, and copy-number variations are now functionally and genetically linked to human disease. Despite the fact that mtDNA is solely transmitted from the mother to the offspring, e.g. is maternally inherited, some mutations may occur spontaneously or may be acquired due to defects in nuclear DNA, e.g. are inherited in a mendelian fashion. The internist encounters predominantly myopathies, cardiomyopathies, lactic acidosis or diabetes mellitus but mtDNA-changes are also present with neurologic, hematologic and renal symptoms. Acquired mtDNA alterations are responsible for important drug side effects, such as ifosfamide, carboplatin, doxorubicin or nucleoside-analog reverse-transcriptase inhibitors. A specific mtDNA point-mutation predisposes to aminoglycoside-induced sensorineural deafness.

DNA Mutational Analysis↗
PubMed2006

Biological characteristics in bladder cancer depend on the type of genetic instability.

PURPOSE: Malignant tumors show an inherent genetic instability that can be classified as microsatellite instability (MSI) or chromosomal instability (CIN). To elucidate the differences in biological characteristics of bladder cancer between the two types of genetic instability, the expression of the mismatch repair (MMR) proteins, Aurora-A and p53 proteins, the number of centrosomes, numerical aberrations of chromosomes and 20q13, and DNA ploidy were examined in 100 human urothelial carcinomas of the bladder. EXPERIMENTAL DESIGN: Expressions of the MLH1, MSH2, Aurora-A, and p53 proteins and the numbers of centrosomes were immunohistochemically assessed. Numerical aberrations of chromosomes 7, 9, 17, and 20q13 spots were evaluated by fluorescence in situ hybridization, and DNA ploidy was assessed by laser scanning cytometry. RESULTS: The expression levels of the MMR related-proteins decreased in 9 of 100 tumors. Tumors with low MLH1 or MSH2 expression (designated as MSI cancers) were not linked with centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, 20q13 gain, DNA aneuploidy, and disease progression. MSI cancers showed a favorable prognosis. CIN cancers (49 cases), defined as tumors with a large intercellular variation in centromere copy numbers, were associated more frequently with centrosome amplification, Aurora-A overexpression, increased p53 immunoreactivity, and 20q13 gain than the others (51 cases). Tumors with disease progression were included in the CIN cancer group. CONCLUSIONS: The present observations suggest that there are differences in the biological characteristics of the two types of genetic instability.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Low copy number and limited variability of proviral DNA in alveolar macrophages from HIV-1-infected patients: evidence for genetic differences in HIV-1 between lung and blood macrophage populations.

BACKGROUND: We investigated the human immunodeficiency virus (HIV) proviral DNA sequence and copy number in alveolar macrophages (AM) and peripheral blood monocytes (PBM) from 10 HIV-positive patients without any active concurrent pulmonary disease to understand the nature of HIV-1 infection in vivo in the lung microenvironment. MATERIALS AND METHODS: The 10 seropositive patients without active pulmonary disease were selected based on chest roentegenography and pathological/cytological test of bronchoalveolar (BAL) fluid. In order to determine accurate proviral copy numbers, AM and PBM were isolated to 99 and 94% purity, respectively, and quantitative polymerase chain reaction (PCR), with a sensitivity to detect three copies of HIV proviral DNA per 10(5) cells, was applied. For analysis of genetic variation in HIV-1, PCR-amplified HIV-1 DNA from AM and PBM of five patients were subcloned and 2-12 clones from each sample underwent DNA sequence analysis of HIV-1 gp120 V3-V5. Heteroduplex mobility assays were performed to confirm the results of the sequence analysis. RESULTS: The proviral copy number in AM or PBM were less than 20 copies/10(5) cells in all patients, and five patients had less than the detection limit. There was no significant difference in HIV copy number between AM and PBM. No correlation was found between PBM/AM HIV copy number and CD4+ lymphocyte count in the peripheral blood. Sequence analysis revealed that the mean intrapatient genetic similarity in AM was 97.5 +/- 0.18% (n = 107), which was significantly higher than that in PBM (96.2 +/- 0.26% (n = 94), p < 0.001), suggesting that variability of HIV-1 DNA in AM was relatively limited. Divergence occurred when AM derived HIV-1 sequence was compared with PBM derived sequence from the same patient (95.8 +/- 0.17% (n = 223) p < 0.001). Phylogenetic analysis of DNA sequence demonstrated complete separation of HIV lineages from lung and blood in four of five patients. CONCLUSIONS: The results suggest the HIV-1 infection in AM is restricted in vivo with low viral burden and homogenous genotype. We propose that the pulmonary microenvironment may limit the extent of HIV-1 infection.

Adult↗
PubMed2005

Organization and evolution of highly repeated satellite DNA sequences in plant chromosomes.

A major component of the plant nuclear genome is constituted by different classes of repetitive DNA sequences. The structural, functional and evolutionary aspects of the satellite repetitive DNA families, and their organization in the chromosomes is reviewed. The tandem satellite DNA sequences exhibit characteristic chromosomal locations, usually at subtelomeric and centromeric regions. The repetitive DNA family(ies) may be widely distributed in a taxonomic family or a genus, or may be specific for a species, genome or even a chromosome. They may acquire large-scale variations in their sequence and copy number over an evolutionary time-scale. These features have formed the basis of extensive utilization of repetitive sequences for taxonomic and phylogenetic studies. Hybrid polyploids have especially proven to be excellent models for studying the evolution of repetitive DNA sequences. Recent studies explicitly show that some repetitive DNA families localized at the telomeres and centromeres have acquired important structural and functional significance. The repetitive elements are under different evolutionary constraints as compared to the genes. Satellite DNA families are thought to arise de novo as a consequence of molecular mechanisms such as unequal crossing over, rolling circle amplification, replication slippage and mutation that constitute "molecular drive".

Centromere↗
PubMed2005

Variation of the Mycobacterium tuberculosis PE_PGRS 33 gene among clinical isolates.

PE_PGRS 33, one of about 60 PE_PGRS genes in the Mycobacterium tuberculosis genome, encodes a surface-expressed protein that may be involved in the antigenic variation of M. tuberculosis strains and evasion of the host immune system. While genetic differences between the PE_PGRS 33 genes of H37Rv and CDC 1551 have been noted, genetic variation in this gene among clinical isolates has not been evaluated. In order to gain a better understanding of the genetic basis for the role of PE_PGRS in antigenic variation and evasion of the host immune system, we investigated the genetic diversity of the PE_PGRS 33 gene among 123 clinical M. tuberculosis isolates from a population-based study, using PCR and DNA sequencing. The 123 isolates belonged to principal genetic groups 1, 2, and 3 and had IS 6110 copy numbers ranging from 1 to 22. Eighty-four (68.3%) of the 123 isolates were found to have at least one sequence variation in the PE_PGRS 33 gene, relative to that of H37Rv. Twenty-five different sequence variations were observed and included three insertions (ranging from 9 to 87 bp), nine deletions (ranging from 1 to 273 bp), one insertion-and-deletion event, and 12 single-nucleotide polymorphisms (six synonymous and six nonsynonymous). Analysis of the relationships among the different PE_PGRS 33 gene sequence variations suggests that polymorphisms in the gene are shifting along evolutionary lineages. The observed genetic diversity of the PE_PGRS 33 gene supports its role in antigenic variation and can serve as a basis for future investigations of the function of the PE_PGRS 33 gene among clinical isolates.

Antigens, Bacterial↗
PubMed2000

The dynamics of repeated elements: applications to the epidemiology of tuberculosis.

We propose a stepwise mutation model to describe the dynamics of DNA fingerprint variation in Mycobacterium tuberculosis. The genome of M. tuberculosis carries insertion sequences (IS6110) that are relatively stable over time periods of months but have an observable transposition rate over longer time scales. Variability in copy number and genomic location of (IS6110) can be harnessed to generate a DNA fingerprint for each strain, by digesting the genome with a restriction enzyme and using a portion of the element as a probe for Southern blots. The number of bands found for a given genome approximates the number of copies of IS6110 it carries. A large data set of such fingerprints from tuberculosis (TB) cases in San Francisco provides an observed distribution of IS6110 copy number. Implementation of the model through deterministic and stochastic simulation indicates some general features of IS/TB dynamics. By comparing observations with outcomes of the model, we conclude that the IS/TB system is very heterogeneous and far from equilibrium. We find that the transposition parameters have a much stronger effect than the epidemic parameters on copy number distribution.

DNA Fingerprinting↗