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PubMed2003

Architecture of the ParF*ParG protein complex involved in prokaryotic DNA segregation.

The mechanism by which low copy number plasmids are segregated at cell division involves the concerted action of two plasmid-encoded proteins that assemble on a centromere-like site. This study explores the topology of the DNA segregation machinery specified by the parFG locus of TP228, a partition system which is phylogenetically distinct from more well-characterized archetypes. A variety of genetic, biochemical and biophysical strategies revealed that the ParG protein is dimeric. ParF, which is more closely related to the cell division regulator MinD than to the prototypical ParA partition protein of plasmid P1, is instead multimeric and its polymeric state appears to be modulated by ATP which correlates with the proposed ATP-binding activity of ParF. ParG interacts in a sequence-specific manner with the DNA region upstream of the parFG locus and this binding is modulated by ParF. Intriguingly, the ParF and ParG proteins form at least two types of discrete complex in the absence of this region suggesting that the assembly dynamics of these proteins onto DNA is intricate.

Adenosine Triphosphate↗

PubMed1996

Mitochondrial dysfunction in spontaneous inbred turkey cardiomyopathy.

Mitochondrial enzyme activities were examined in cardiac tissues of turkeys with spontaneous inbred cardiomyopathy. Marked declines in specific enzyme activities were noted for respiratory complexes III and V ranging from 65-90% of the control values. No significant differences in complexes I, IV and citrate synthase nor in mitochondrial DNA copy number were detected. These results suggest that specific mitochondrial enzyme defects occur in cardiac tissues during spontaneous inbred turkey cardiomyopathy.

Animals↗

PubMed2026

Prenatal diagnosis and genetic counseling of a de novo 10q11.22q11.23 duplication associated with a normal development at 12 months of age.

BACKGROUND: Copy number variants are an important source of genomic variations, ranging from pathogenic to benign. The 10q11.22q11.23 region contains complex low-copy repeats that predispose to recurrent deletions and duplications via nonallelic homologous recombination. While some reports associate duplications of this region with developmental delay, intellectual disability, and autism spectrum disorders, emerging evidence suggests that such duplications may also be observed in phenotypically normal individuals, indicating incomplete penetrance and variable expressivity. CASE PRESENTATION: A 35-year-old pregnant woman with an unremarkable obstetric history underwent amniocentesis at 20 weeks of gestation. Conventional karyotyping and copy number variation sequencing (CNV-seq) were performed. CNV-seq revealed a de novo 4.56 Mb duplication at 10q11.22q11.23. The duplication was classified as a variant of uncertain significance. After extensive genetic counseling, the parents elected to continue the pregnancy. At 40 weeks of gestation, a female infant was delivered by cesarean section with normal birth parameters. A comprehensive physical examination at birth revealed no abnormalities. At the 12-month follow-up, the infant demonstrated normal growth parameters and age-appropriate neurodevelopmental milestones, with no evidence of dysmorphic features, developmental delay, or other clinical concerns. CONCLUSION: This report describes a prenatal case of a de novo 10q11.22q11.23 duplication with a normal development at 12 months of age. Our findings contribute to the growing body of literature suggesting that duplications in this pericentromeric region may exhibit incomplete penetrance and variable expressivity, and in some cases, may represent benign familial or de novo variants without apparent clinical consequences.

10q11.22q11.23 duplication↗

PubMed2006

Rai1 duplication causes physical and behavioral phenotypes in a mouse model of dup(17)(p11.2p11.2).

Genomic disorders are conditions that result from DNA rearrangements, such as deletions or duplications. The identification of the dosage-sensitive gene(s) within the rearranged genomic interval is important for the elucidation of genes responsible for complex neurobehavioral phenotypes. Smith-Magenis syndrome is associated with a 3.7-Mb deletion in 17p11.2, and its clinical presentation is caused by retinoic acid inducible 1 (RAI1) haploinsufficiency. The reciprocal microduplication syndrome, dup(17)(p11.2p11.2), manifests several neurobehavioral abnormalities, but the responsible dosage-sensitive gene(s) remain undefined. We previously generated a mouse model for dup(17)(p11.2p11.2), Dp(11)17/+, that recapitulated most of the phenotypes observed in human patients. We have now analyzed compound heterozygous mice carrying a duplication [Dp(11)17] in one chromosome 11 along with a null allele of Rai1 in the other chromosome 11 homologue [Dp(11)17/Rai1(-) mice] in order to study the relationship between Rai1 gene copy number and the Dp(11)17/+ phenotypes. Normal disomic Rai1 gene dosage was sufficient to rescue the complex physical and behavioral phenotypes observed in Dp(11)17/+ mice, despite altered trisomic copy number of the other 18 genes present in the rearranged genomic interval. These data provide a model for variation in copy number of single genes that could influence common traits such as obesity and behavior.

Animals↗

PubMed2005

Suppressors of Bir1p (Survivin) identify roles for the chromosomal passenger protein Pic1p (INCENP) and the replication initiation factor Psf2p in chromosome segregation.

Fission yeast Bir1p/Cut17p/Pbh1p, the homolog of human Survivin, is a conserved chromosomal passenger protein that is required for cell division and cytokinesis. To study how Bir1p promotes accurate segregation of chromosomes, we generated and analyzed a temperature-sensitive allele, bir1-46, and carried out genetic screens to find genes that interact with bir1(+). We identified Psf2p, a component of the GINS complex required for DNA replication initiation, as a high-copy-number suppressor of the bir1-46 growth defect. Loss of Psf2p function by depletion or deletion or by use of a temperature-sensitive allele, psf2-209, resulted in chromosome missegregation that was associated with mislocalization of Bir1p. We also found that the human homolog of Psf2p, PSF2, was required for proper chromosome segregation. In addition, we observed that high-copy-number expression of Pic1p, the fission yeast homolog of INCENP (inner centromere protein), suppressed bir1-46. Pic1p exhibited a localization pattern typical of chromosomal passenger proteins. Deletion of pic1(+) caused chromosome missegregation phenotypes similar to those of bir1-46. Our data suggest that Bir1p and Pic1p act as part of a conserved chromosomal passenger complex and that Psf2p/GINS indirectly affects the localization and function of this complex in chromosome segregation, perhaps through an S-phase role in centromere replication.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Detection and quantification of human immunodeficiency virus RNA in patient serum by use of the polymerase chain reaction.

Human immunodeficiency virus (HIV) RNA was detected and quantified in the serum of HIV-seropositive individuals using the polymerase chain reaction (PCR) and a nonisotopic enzyme-linked affinity assay. Of 55 HIV-infected patients who were not receiving therapy, serum HIV RNA was detected in 9 of 19 who were asymptomatic, 11 of 16 with AIDS-related complex (ARC), and 18 of 20 with AIDS, with copy numbers ranging from 10(2) to greater than or equal to 5 x 10(4) 200 microliters of serum based on a relationship between absorbance and known copy number of gag gene RNA. Linear regression analysis demonstrated a correlation between infectious titer in 42 patient sera cocultured with donor peripheral blood mononuclear cells (PBMC) and PCR product absorbance (r = .70, P less than .01). Serum HIV RNA detected by PCR also correlated with serum p24 antigen positivity, CD4 counts less than 400/mm3, and the presence of HIV-related symptoms or disease. Quantification of infectious HIV RNA in cell-free serum by PCR may be useful as a marker for for disease progression or in monitoring antiviral therapy.

AIDS-Related Complex↗

PubMed2026

Chromosome-scale genome remodeling in tumor evolution: Copy number alterations and structural variants as two sides of the same coin.

Chromosome-scale genomic rearrangements are a dominant force in tumor evolution. Copy-number alterations (CNAs) and structural variants (SVs) constitute two complementary axes of this process. Although detection technologies now deliver near-comprehensive catalogs, technical resolution has outpaced conceptual integration. In this review, we frame CNAs and SVs as inextricable facets of chromosomal aberrations. They reshape cancer genomes through altered gene dosage and three-dimensional regulatory rewiring. CNAs quantify the gene-dosage imbalance, yet arise through mechanistically distinct routes. Segmental CNAs typically require chromosomal breakage, and therefore often coincide with SV junctions. By contrast, whole-chromosome aneuploidy and whole-genome doubling (WGD) primarily reflect mitotic or cytokinetic failure and can occur without local breakpoints, while nevertheless reshaping the karyotypic landscape and seeding subsequent structural complexity. SVs, in turn, range from unbalanced events that alter copy number to ostensibly balanced exchanges that predominantly rewire regulatory architecture. Despite their diverse and sometimes catastrophic architectures, SVs are ultimately rooted in double-strand break formation and error-prone resolution. By integrating CNAs and SVs within a unified mechanistic and functional framework, we aim to convert catalogs into concepts and distill the organizing principles that govern tumor genome evolution.

Humans↗

PubMed1987

Plasmid stability in immobilized and free recombinant Escherichia coli JM105(pKK223-200): importance of oxygen diffusion, growth rate, and plasmid copy number.

Stability of the plasmid pKK223-200 in Escherichia coli JM105 was studied for both free and immobilized cells during continuous culture. The relationship between plasmid copy number, xylanase activity, which was coded for by the plasmid, and growth rate and culture conditions involved complex interactions which determined the plasmid stability. Generally, the plasmid stability was enhanced in cultured immobilized cells compared with free-cell cultures. This stability was associated with modified plasmid copy number, depending on the media used. Hypotheses are presented concerning the different plasmid instability kinetics observed in free-cell cultures which involve the antagonistic effects of plasmid copy number and plasmid presence on the plasmid-bearing/plasmid-free cell growth rate ratio. Both diffusional limitation in carrageenan gel beads, which is described in Theoretical Analysis of Immobilized-Cell Growth, and compartmentalized growth of immobilized cells are proposed to explain plasmid stability in immobilized cells.

Culture Media↗

PubMed1995

The level of the pUB110 replication initiator protein is autoregulated, which provides an additional control for plasmid copy number.

Plasmids control their copy number by limiting the amount of the initiator for DNA replication. The plasmid pUB110 initiator protein is termed RepU. Expression of the pUB110 repU gene is controlled by two antisense RNAs that interfere with repU mRNA translation. Genetic evidence suggests that Rep protein levels may be regulated by additional uncharacterized mechanisms. The repU gene product was radiolabeled and purified by monitoring the radioactive label. RepU overproduction was performed in cells containing the plasmid leading strand replication origin (dso), to allow for a putative inactivation of RepU. Polypeptides with apparent molecular masses of 42 (RepU*) and 39 (RepU) kDa were purified, both having the N-terminal sequence expected for the repU gene. The RepU/RepU* protein mixture bound specifically to dso. At low protein concentrations, about six RepU/RepU* protomers bound to the dso region. At higher concentrations, an extended nucleoprotein complex was formed. The promoter for the repU gene was localized downstream of the dso region. The results suggest that the extended RepU/RepU*-dso DNA complex interferes with repU promoter utilization. This provides an additional copy number control by limiting RepU concentration. Our results suggest that during replication the RepU protein might be converted into an inactive RepU-RepU* hetero-oligomer, further limiting the amount of RepU protein available for replication initiation.

Amino Acid Sequence↗

PubMed1998

The tomato Cf-5 disease resistance gene and six homologs show pronounced allelic variation in leucine-rich repeat copy number.

The tomato Cf-2 and Cf-5 genes confer resistance to Cladosporium fulvum and map to a complex locus on chromosome 6. The Cf-5 gene has been isolated and is predicted to encode a largely extracytoplasmic protein containing 32 leucine-rich repeats (LRRs), resembling the previously isolated Cf-2 gene, which has 38 LRRs. Three haplotypes of this locus from Lycopersicon esculentum, L. pimpinellifolium, and L. esculentum var cerasiforme were compared, and five additional homologs of Cf-5 were sequenced. All share extensive sequence identity, particularly within the C-terminal portions of the predicted proteins. In striking contrast to the Cf-9 gene family, six of seven homologs in the Cf-2/Cf-5 gene family vary in LRR copy number, ranging from 25 to 38 LRRs. Cf-5 and one adjacent homolog differ by only two LRRs. Recombination events that vary the LRR copy number in this region could provide a mechanism for the generation of new specificities for recognition of different ligands. A recombination breakpoint between the Cf-2 and Cf-5 loci was fully characterized and shown to be intragenic.

Alleles↗

PubMed2002

Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets.

The lack of a convenient high-resolution strain-typing method has hampered the application of molecular epidemiology to the surveillance of bacteria of the Mycobacterium tuberculosis complex, particularly the monitoring of strains of Mycobacterium bovis. With the recent availability of genome sequences for strains of the M. tuberculosis complex, novel PCR-based M. tuberculosis-typing methods have been developed, which target the variable-number tandem repeats (VNTRs) of minisatellite-like mycobacterial interspersed repetitive units (MIRUs), or exact tandem repeats (ETRs). This paper describes the identification of seven VNTR loci in M. tuberculosis H37Rv, the copy number of which varies in other strains of the M. tuberculosis complex. Six of these VNTRs were applied to a panel of 100 different M. bovis isolates, and their discrimination and correlation with spoligotyping and an established set of ETRs were assessed. The number of alleles varied from three to seven at the novel VNTR loci, which differed markedly in their discrimination index. There was positive correlation between spoligotyping, ETR- and VNTR-typing. VNTR-PCR discriminates well between M. bovis strains. Thirty-three allele profiles were identified by the novel VNTRs, 22 for the ETRs and 29 for spoligotyping. When VNTR- and ETR-typing results were combined, a total of 51 different profiles were identified. Digital nomenclature and databasing were intuitive. VNTRs were located both in intergenic regions and annotated ORFs, including PPE (novel glycine-asparigine-rich) proteins, a proposed source of antigenic variation, where VNTRs potentially code repeating amino acid motifs. VNTR-PCR is a valuable tool for strain typing and for the study of the global molecular epidemiology of the M. tuberculosis complex. The novel VNTR targets identified in this study should additionally increase the power of this approach.

Alleles↗

PubMed1997

Relational mapping of MYCN and DDXI in band 2p24 and analysis of amplicon arrays in double minute chromosomes and homogeneously staining regions by use of free chromatin FISH.

MYCN amplification has been observed in diverse neuronal tumors including neuroblastoma, retinoblastoma, and small cell carcinoma of the lung, and has been correlated with a poor prognosis in advanced-stage neuroblastomas. Recent studies have shown a co-amplification of DDXI, a DEAD box gene, and MYCN in retinoblastoma and neuroblastoma. DDXI has been mapped to within a megabase of the MYCN gene in band 2p24. In the present study, the relational map of DDXI and MYCN by fluorescence in situ hybridization (FISH) mapping to metaphase cells and extended free chromatin fibers indicated that DDXI is telomeric to MYCN. Dual-color FISH analysis of amplicons within arrays of extended chromatin fibers was performed to examine the physical relationship of MYCN and DDXI within double minute chromosomes (dmins) and homogeneously staining regions (hsrs). No regular reiterated amplicon repeat unit was present in the hsrs, but detailed analysis of the configurations of DDXI and MYCN within each array indicated that multiple rearrangements generated a complex hsr amplicon structure. Similarly, analysis of a cell line bearing dmins showed that a composite amplicon structure involving deletions and/or duplications of MYCN and DDXI is a feature of dmin formation. These data are consistent with a molecular mechanism involving many rearrangements during the evolution of gene amplification, resulting in complex amplicon structures with distinct changes in relative gene copy number and considerable variation in intragenic distances between coamplified genes.

Blotting, Southern↗

PubMed1989

Loss of bovine papillomavirus DNA replication control in growth-arrested transformed cells.

The bovine papillomavirus type 1 (BPV-1) genome replicates as a plasmid within the nuclei of BPV-1-transformed murine C127 cells at a constant multiple copy number, and spontaneous amplification of the viral DNA is rarely observed. We report here that a mutant BPV-1 plasmid within a contact-inhibited C127 cell line replicated as a stable multicopy plasmid in exponentially growing cells but amplified to a high level in confluent cell culture. In situ hybridization analysis revealed that most of the mutant viral DNA amplification occurred in a minor subpopulation of cells within the culture. These consisted of giant nondividing cells with greatly enlarged nuclei, a cell form which was specifically induced in stationary-phase cultures. These observations indicated that expression of a viral DNA replication factor was cell growth stage specific. Consistent with this hypothesis, considerable amplification of wild-type BPV-1 DNA associated with characteristic giant cell formation was observed in typical wild-type virus-transformed C127 cultures following a period of growth arrest achieved by serum deprivation. Further observations indicated that induction of the giant-cell phenotype was dependent on BPV-1 gene expression and implicated a viral E1 replication factor in this process. Moreover, heterogeneity in virus genome copy numbers within the giant-cell population suggested a complex regulation of induction of DNA synthesis in these cells. It appears that this process represents a mechanism employed by the virus to ensure maximal viral DNA synthesis within a growth-arrested cell. Fundamental questions concerning the integration of the virus-cell control circuitry in proliferating and resting cells are discussed.

Animals↗

PubMed2006

Complex T cell memory repertoires participate in recall responses at extremes of antigenic load.

The CD8 T cell memory response to the HLA-A2-restricted influenza epitope M1(58-66) can be an instructive model of immune memory to a nonevolving epitope of a frequently encountered pathogen that undergoes clearance. This memory repertoire can be complex, composed of a large number of clonotypes represented at low copy numbers, while maintaining a focus on the use of VB17 T cell receptors with identified Ag recognition motifs. Such a repertoire structure might provide a panoply of clonotypes whose differential avidity for the epitope would allow responses under varying antigenic loads. This possibility was tested experimentally by characterizing the responding repertoire in vitro while varying influenza Ag concentration over five orders of magnitude. At higher and lower Ag concentrations there was increased cell death, yet a focused but diverse response could still be observed. Thus, one of the characteristics of complex memory repertoires is to provide effector function at extremes of Ag load, a characteristic that is not generally considered in vaccination development but may be important in measuring its efficacy.

Antigens, Viral↗

PubMed2003

Quantitation of antibody-bound and unbound Sin Nombre virus in the plasma of patient with hantavirus pulmonary syndrome.

Hantavirus pulmonary syndrome (HPS) is a rare but acute fulminant disease caused by Sin Nombre virus and other related hantaviruses. We reported earlier that HPS patients had high levels of viremia and the levels of viremia correlated with the severity of the disease. To determine whether the viruses are free in plasma or bound to antibodies to form immune complexes, we established a method to quantitate viral genomic RNA in antibody-bound viruses and in unbound viruses separately. To isolate immune complexes from plasma we used protein L, which binds to all classes of immunoglobulins and all subclasses of IgG, and we measured viral RNA copy number in antibody-bound viruses and unbound viruses by quantitative RT-PCR. We analyzed plasma samples collected on admission from 15 patients with HPS in the acute phase. The percentage of viral RNA in immune complexes versus total viral RNA varied from 9 to 91% among patients, and correlated with the total viral RNA copy numbers (P<0.05) and the neutralizing antibody titers of plasma (P<0.05). This quantitative method may be useful to examine diseases where immune complex formation or antibody-dependent enhancement may be involved in pathogenesis.

Antibodies, Viral↗

PubMed2026

"Tissue-specific mitochondrial dysfunction in keratoconus: An integrated structural, genomic, and functional analysis".

PURPOSE: Keratoconus (KC) is a progressive corneal ectasia characterized by stromal thinning, conical protrusion, and irregular astigmatism, leading to visual impairment. Although oxidative stress is implicated in KC, the role of mitochondrial dysfunction remains unclear. We evaluated mitochondrial structural, genomic, and functional abnormalities in corneal tissues and blood from KC patients. METHODS: This prospective study enrolled 110 KC patients and 55 controls. Transmission electron microscopy (TEM) and immunohistochemistry (IHC) were performed on epithelial and stromal tissues from 10 KC to 5 control corneas assessing mitochondrial morphology, oxidative phosphorylation (OXPHOS) complexes and pro-apoptotic protein NOXA. Whole mitochondrial DNA (mtDNA) sequencing and relative mtDNA copy number analysis were performed on paired blood and corneal tissues from 50 KC patients and 35 controls including both epithelial and stromal samples. Gene expression of mitochondrial biogenesis and oxidative stress-related genes was analysed by qRT-PCR in corneal epithelium from independent 50 KC patients and 15 controls. RESULTS: TEM revealed cristolysis, membrane disruption, and reduced mitochondrial density in KC corneas. IHC showed reduced expression of OXPHOS complexes and increased NOXA expression (p < 0.05). Sequencing identified 1107 mtDNA variants, with more variants in corneal tissues than matched blood (929 vs. 576; p = 0.0002). Recurrent likely pathogenic variants were enriched in complex I-encoding genes (ND4, ND5). KC corneas showed reduced mtDNA copy number, downregulated POLRMT, upregulated NOX4, and significant downregulation of multiple antioxidant genes (p < 0.0001). CONCLUSION: KC patients exhibit tissue-specific mitochondrial abnormalities and impaired oxidative stress regulation, supporting a role for mitochondrial dysfunction in disease pathogenesis and highlighting potential therapeutic targets.

Corneal pathology↗

PubMed2003

Genome amplification of chromosome 20 in breast cancer.

Recurrent gain and amplification of the long arm of chromosome 20 (20q) has been observed in a wide variety of cancers. This suggests that a gene or genes encoded on 20q play important roles in contributing to the cancer phenotype when overexpressed. In the quest to discover cancer genes, this region of the genome has been exhaustively studied, and the results demonstrate remarkable complexity. Multiple regions of low and high-level 20q copy number gain correlate with poor clinical prognosis and appear to contribute to the cancer phenotype, especially aspects of immortalization, genome instability, apoptosis, and increased proliferation. Gene discovery efforts have revealed a number of interesting candidate genes on chromosome 20 that may contribute to oncogenic progression. The study of 20q serves as a model for positional cloning enthusiasts, demonstrating the path typically taken when moving from initial discovery of an important genomic abnormality to identification of genes likely to be significant players in disease progression. This review will summarize approximately a decade of study on 20q and is structured as moving from an introduction to the techniques used in 20q analyses, to the details of 20q genomic complexity and its involvement with cancer, and finally to a detailed gene-specific look at this region.

Breast Neoplasms↗

PubMed1977

Number and distribution of polyadenylated RNA sequences in yeast.

The poly(A)-containing RNA, isolated from the budding yeast Saccharomyces cerevisiae, has been characterized with regard to the number and distribution of sequences by a kinetic analysis of RNA-cDNA hybridization. In agreement with results previously obtained on metazoan eucaryotes (Bishop et al., 1974), discrete complexity classes were observed. There exist low, medium, and high complexity classes which contain approximately 20, 400, and 2400 sequences, respectively. This measurements of the number of sequences has been verified by hybridization with single copy DNA. 20% of the single copy fraction of the yeast genome is rendered double-stranded by poly(A)-containing RNA. Assuming asymmetric transcription, this is equivalent to approximately 4000 poly(A)-containing sequences, verifying the results obtained with RNA-cDNA hybridization. In addition, the first-order kinetics of the hybridization with single copy DNA verified the notion that most of the sequence complexity is present at the same intracellular concentration. The same number and distribution of sequences were found in poly(A)-containing polysomal RNA and in total RNA, suggesting that most or all of the sequence complexity is on polysomes and is adenylated. The results indicate that RNA-cDNA hybridization is an accurate method for determining sequence complexity values and that yeast, grown under vegetative conditions, has 3000-4000 different mRNA sequences.

Base Sequence↗