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PubMed2005

Plasmid p256 from Lactobacillus plantarum represents a new type of replicon in lactic acid bacteria, and contains a toxin-antitoxin-like plasmid maintenance system.

Lactobacillus plantarum NC7 harbours a single 7.2 kb plasmid called p256. This report describes the complete nucleotide sequence and annotation of p256, as well as the identification of the minimal replicon of the plasmid. Based on sequence features in the unusually small (0.7 kb) minimal replicon, and the absence of a gene for a replication-relevant protein, p256 seems to represent a hitherto unknown type of theta replicon in lactic acid bacteria (LAB), with a relatively low copy-number. In addition, a putative toxin-antitoxin (TA) locus was identified. Experiments with variants of p256 indicated that the TA system was involved in plasmid maintenance. Furthermore, controlled expression of the TA genes stabilized vectors derived from the p256 replicon. To the authors' knowledge, this is the first time a TA locus with a demonstrated plasmid maintenance function has been identified in LAB. Transformation of several LAB with plasmids derived from p256 indicated that it has a narrow host range. Several effective expression vectors based on the p256 replicon have been constructed.

Amino Acid Sequence↗

PubMed2002

Recognition of variant Rifin antigens by human antibodies induced during natural Plasmodium falciparum infections.

Antibodies from individuals living in areas where malaria is endemic are known to react with parasite-derived erythrocyte surface proteins. The major immunogenic and clonally variant surface antigen described to date is Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP-1), which is encoded by members of the multicopy var gene family. We report here that rifin proteins (RIF proteins), belonging to the largest known family of variable infected erythrocyte surface-expressed proteins, are also naturally immunogenic. Recombinant RIF proteins were used to analyze the antibody responses of individuals living in an area of intense malaria transmission. Elevated anti-rifin antibody levels were detected in the majority of the adult population tested, whereas the prevalence of such antibodies was much lower in malaria-exposed children. Despite the high degree of diversity between rif sequences and the high gene copy number, it appears that P. falciparum infections can induce antibodies that cross-react with several variant rifin molecules in many parasite isolates in a given community, and the immune response is most likely to be stable over time in a hyperendemic area. The protein was localized by fluorescence microscopy on the membrane of ring and young trophozoite-infected erythrocytes with antibodies from human immune sera with specificities for recombinant RIF protein.

Adolescent↗

PubMed1994

Structural analysis of two length variants of the rDNA intergenic spacer from Eruca sativa.

Restriction enzyme analysis of the rRNA genes of Eruca sativa indicated the presence of many length variants within a single plant and also between different cultivars which is unusual for most crucifers studied so far. Two length variants of the rDNA intergenic spacer (IGS) from a single individual E. sativa (cv. Itsa) plant were cloned and characterized. The complete nucleotide sequences of both the variants (3 kb and 4 kb) were determined. The intergenic spacer contains three families of tandemly repeated DNA sequences denoted as A, B and C. However, the long (4 kb) variant shows the presence of an additional repeat, denoted as D, which is a duplication of a 224 bp sequence just upstream of the putative transcription initiation site. Repeat units belonging to the three different families (A, B and C) were in the size range of 22 to 30 bp. Such short repeat elements are present in the IGS of most of the crucifers analysed so far. Sequence analysis of the variants (3 kb and 4 kb) revealed that the length heterogeneity of the spacer is located at three different regions and is due to the varying copy numbers of repeat units belonging to families A and B. Length variation of the spacer is also due to the presence of a large duplication (D repeats) in the 4 kb variant which is absent in the 3 kb variant. The putative transcription initiation site was identified by comparisons with the rDNA sequences from other plant species.

Base Sequence↗

PubMed2026

Protocol for Duplex Sequencing of Mitochondrial DNA in Single Human Oocytes.

Oocytes are densely packed with mitochondria, the energy-producing organelles that contain their own genome, mitochondrial DNA (mtDNA). Each cell contains multiple copies of mtDNA, with copy number varying among tissue types. Oocytes possess the highest mtDNA copy number, containing hundreds of thousands of mtDNA molecules per cell. Because mitochondria are inherited exclusively through the maternal lineage, accurate detection of mtDNA variants is essential for studies of inheritance, aging, and disease. The presence of multiple mtDNA copies allows wild-type and mutant molecules to coexist within the same cell, a condition known as heteroplasmy, in which low-frequency and de novo variants may occur at frequencies below 1%. Conventional next-generation sequencing (NGS) lacks sufficient accuracy to reliably distinguish these rare variants from errors introduced during library preparation and sequencing. Here, we present a protocol for enriching mtDNA from single human oocytes using Exonuclease V to remove linear DNA, followed by duplex sequencing library preparation for highly accurate mtDNA analysis. This workflow enables error-corrected sequencing of individual oocytes, facilitating reliable detection of low-frequency mtDNA variants and analysis of heteroplasmy and de novo mutagenesis. The protocol provides a reproducible approach for investigating mitochondrial genome variation in single oocytes using Illumina-compatible sequencing platforms.

Humans↗

PubMedDate not supplied

[Chromosomal anomalies in children with undifferentiated forms of metal retardation based on molecular cytogenetic research data].

The results of cytogenetic diagnosis of 1333 children with mental retardation and congenital malformations are presented. Chromosomal aberrations and chromosomal variants (1qh+, 9qh+, 16qh+) have been detected in 181 (13.9%) cases analyzed and in 158 (11.85%) cases, respectively. Molecular cytogenetic methods have been used for diagnosis or detalization of diagnosis in 42 cases from 181 (23.2%). Independent variation (or polymorphism) of copies number of "classical" and alpha-satellite DNA sequences, forming pericentromeric regions (1qh+, 9qh+, 16qh+) has been detected in patients with chromosomal variants.

Child↗

PubMed1992

Evolution of a B2 tagged sequence from a long-range repeat family in the genus Mus.

A long-range repeat family of more than 50 kb repeat size is clustered in Chromosomes (Chr) 1 of Mus musculus and M. spretus. In M. musculus this long-range repeat family shows considerable variation of copy-number frequency and contains coding regions for at least two genes. In an intron of a gene, which is part of the repeat, a B2 small interspersed repetitive element (SINE) is inserted at identical positions. The B2 element is present in all copies of the long-range repeat family; it was presumably a component of the ancestral single-copy precursor sequence that gave rise by amplification to the repeat family. Copies of the long-range repeat family vary with respect to the number of TAAA tandem repeats in the A-rich 3' end region of the B2 element. As inferred from polymerase chain reaction (PCR) data, presence and frequency of repeat number variants in the (TAAA)n block are strain and species specific. The B2 element and its flanking regions were sequenced from two copies of the long-range repeat family. Sequence divergence between the two copies (only non-CG base substitutions and deletions/insertions) was determined to be 2.6%. Based on the drift rate in human Alu elements and a correction for the higher drift rates in rodents, an estimate for the divergence time of 1.7 million years was calculated. Since the long-range repeat family is present in M. musculus and M. spretus, it must have evolved by amplification before the separation of the two species about 1-4 million years ago.

Animals↗

PubMed2001

Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria.

We have developed a series of powerful and versatile conditional-replication, integration, and modular (CRIM) plasmids. CRIM plasmids can be replicated at medium or high copy numbers in different hosts for making gene (or mutant) libraries. They can be integrated in single copies into the chromosomes of Escherichia coli and related bacteria to study gene function under normal physiological conditions. They can be excised from the chromosome, e.g., to verify that phenotypes are caused by their presence. Furthermore, they can be retrieved singly or en masse for subsequent molecular analyses. CRIM plasmids are integrated into the chromosome by site-specific recombination at one of five different phage attachment sites. Integrants are selected as antibiotic-resistant transformations. Since CRIM plasmids encode different forms of resistance, several can be used together in the same cell for stable expression of complex metabolic or regulatory pathways from diverse sources. Following integration, integrants are stably maintained in the absence of antibiotic selection. Each CRIM plasmid has a polylinker or one of several promoters for ectopic expression of the inserted DNA. Their modular design allows easy construction of new variants with different combinations of features. We also report a series of easily curable, low-copy-number helper plasmids encoding all the requisite Int proteins alone or with the respective Xis protein. These helper plasmids facilitate integration, excision ("curing"), or retrieval of the CRIM plasmids.

Attachment Sites, Microbiological↗

PubMed1983

Mechanism of mutation at the aprt locus in Chinese hamster ovary cells: analysis of heterozygotes and hemizygotes.

A two-step model to explain the high frequency of mutation at the diploid adenine phosphoribosyltransferase (aprt) locus in CHO cells has been proposed previously (Simon et al., Mol. Cell. Biol. 2:1126-1133, 1982). This model indicates that two distinct classes of aprt heterozygotes can be isolated. Class 1 heterozygotes, the most abundant class, were defined as those which arose spontaneously and were capable of undergoing mutation to the APRT- phenotype only at a low frequency (putative point mutation). Class 2 heterozygotes arose from a mutation and gave rise at a high frequency to APRT- cells. This high-frequency event has been identified as a deletion of the wild-type allele (A. E. Simon and M. W. Taylor, Proc. Natl. Acad. Sci. U.S.A. 80:810-814, 1983). In this paper we report further analysis of class 1 heterozygotes with respect to genetic structure, gene products, and karyotype. Our study indicated that class 1 heterozygotes contain two different types of mutants. About half have only one copy of the aprt gene and an unaltered karyotype, indicating that a deletion (similar to the high-frequency second-step event observed for class 2 heterozygotes) rather than a loss of the chromosome was responsible for the generation of the aprt+/- genotype. The remainder of the previously designated class 1 heterozygotes still contained two copies of the aprt gene (within the limits of the quantitation technique used) and arose presumably by a point mutation. One of this group, D423, was characterized with respect to aprt gene products and found to produce an electrophoretic variant in addition to the wild-type protein. APRT- mutants derived from D423 retained the same number of aprt gene copies as D423 and still synthesized a protein that comigrated with wild type, unlike APRT- mutants derived from class 2 heterozygotes. D423 and the other heterozygotes with two aprt genes therefore did not fit into either class 1 or 2 and are now designated class 3. The model we present suggests that only one of the two aprt alleles present in wild-type cells can undergo the deletion.

2-Aminopurine↗

PubMed2006

Chromosome XII context is important for rDNA function in yeast.

The rDNA cluster in Saccharomyces cerevisiae is located 450 kb from the left end and 610 kb from the right end of chromosome XII and consists of approximately 150 tandemly repeated copies of a 9.1 kb rDNA unit. To explore the biological significance of this specific chromosomal context, chromosome XII was split at both sides of the rDNA cluster and strains harboring deleted variants of chromosome XII consisting of 450 kb, 1500 kb (rDNA cluster only) and 610 kb were created. In the strain harboring the 1500 kb variant of chromosome XII consisting solely of rDNA, the size of the rDNA cluster was found to decrease as a result of a decrease in rDNA copy number. The frequency of silencing of URA3 inserted within the rDNA locus was found to be greater than in a wild-type strain. The localization and morphology of the nucleolus was also affected such that a single and occasionally (6-12% frequency) two foci for Nop1p and a rounded nucleolus were observed, whereas a typical crescent-shaped nucleolar structure was seen in the wild-type strain. Notably, strains harboring the 450 kb chromosome XII variant and/or the 1500 kb variant consisting solely of rDNA had shorter life spans than wild type and also accumulated extrachromosomal rDNA circles. These observations suggest that the context of chromosome XII plays an important role in maintaining a constant rDNA copy number and in physiological processes related to rDNA function in S.cerevisiae.

Cell Nucleolus↗

PubMed2026

Metab8D: a metabolic regulome network from multiomics and machine learning.

To explore multiomic regulation of the metabolome, we used machine learning to predict metabolomic variation across ~1000 different cancer cell lines with matched omics data from eight biomolecular classes: genomic copy number variation, mutations, DNA methylation, histone post-translational modifications (PTMs), transcriptomics and RNA splice variants, non-coding transcriptomics (miRNA and lncRNA), proteomics, and phosphoproteomics. Overall, the metabolome is tightly associated with the transcriptome, with coding and non-coding RNAs emerging as top predictors. Peripheral metabolites are predictable via levels of corresponding enzymes, while those in central metabolism require combinatorial predictors in signaling and redox pathways, and may not reflect corresponding pathway expression. We reconstruct multiomic interaction subnetworks for highly predictable metabolites, and YAP1 signaling emerged as a top global predictor across four omic layers. We prioritize predictive multiomic features for single-cell and spatial metabolomics assays. Top predictors were enriched for synthetic-lethal interactions and synergistic combination therapies that target compensatory metabolic modulators.

Machine Learning↗

PubMed2003

Predicting human minisatellite polymorphism.

We seek to define sequence-based predictive criteria to identify polymorphic and hypermutable minisatellites in the human genome. Polymorphism of a representative pool of minisatellites, selected from human chromosomes 21 and 22, was experimentally measured by PCR typing in a population of unrelated individuals. Two predictive approaches were tested. One uses simple repeat characteristics (e.g., unit length, copy number, nucleotide bias) and a more complex measure, termed HistoryR, based on the presence of variant motifs in the tandem array. We find that HistoryR and percentage of GC are strongly correlated with polymorphism and, as predictive criteria, reduce by half the number of repeats to type while enriching the proportion with heterozygosity >/=0.5, from a background level of 43% to 59%. The second approach uses length differences between minisatellites in the two releases of the human genome sequence (from the public consortium and Celera). As a predictor, this similarly enriches the number of polymorphic minisatellites, but fails to identify an unexpectedly large number of these. Finally, typing of the highly polymorphic minisatellites in large families identified one new hypermutable minisatellite, located in a predicted coding sequence. This may represent the first coding human hypermutable minisatellite.

Chromosomes, Human, Pair 21↗

PubMed2002

A variable minisatellite sequence in the chloroplast genome of Sorbus L. (Rosaceae: Maloideae).

The chloroplast genome is now known to be more variable than was once thought. Reports of RFLP (restriction fragment length polymorphism) and sequence variation, as well as variation in chloroplast microsatellites, are common. Here, data are presented on the variability of a minisatellite sequence in the chloroplast genome of Sorbus species. RFLP analysis of a PCR product comprising the region between the trnM and rbcL genes of nine Sorbus species identified seven size variants. Sequencing revealed the observed size polymorphism to be due to differences in the number of copies of an imperfect 9-bp motif. A more intensive survey of the variability of the minisatellite was undertaken in populations of Sorbus aucuparia. The potential uses of such regions in chloroplast DNA are discussed and a possible mechanism for the evolution of the minisatellite is presented.

Base Sequence↗

PubMed2004

Protective activity of monoclonal antibodies to genome-derived neisserial antigen 1870, a Neisseria meningitidis candidate vaccine.

Genome-derived neisserial Ag (GNA) 1870 is a meningococcal vaccine candidate that can be subdivided into three variants based on amino acid sequence variability. Variant group 1 accounts for approximately 60% of disease-producing group B isolates. The Ag went unrecognized until its discovery by genome mining because it is expressed in low copy number by most strains. To investigate the relationship between Ab binding to GNA1870 and complement-mediated protective functions, we prepared a panel of four murine IgG mAbs against rGNA1870 (variant 1) and evaluated their activity against nine genetically diverse encapsulated Neisseria meningitidis strains expressing subvariants of variant 1 GNA1870. Based on flow cytometry with live encapsulated bacteria, surface accessibility of the epitopes recognized by the mAbs appeared to be low in most strains. Yet mAb concentrations <1 to 5 micro g/ml were sufficient to elicit bactericidal activity with human complement and/or activate C3b deposition on the bacterial surface. Certain combinations of mAbs were highly bactericidal against strains that were resistant to bactericidal activity of the respective individual mAbs. The mAbs conferred passive protection against bacteremia in infant rats challenged by strains resistant to bacteriolysis, and the protective activity paralleled the ability of the mAb to activate C3b deposition. Thus, despite low GNA1870 surface exposure, anti-GNA1870 variant 1 Abs are bactericidal and/or elicit C3b deposition and confer protection against bacteremia caused by encapsulated N. meningitidis strains expressing GNA1870 subvariant 1 proteins. The data support GNA1870 as a promising vaccine candidate for prevention of meningococcal group B disease caused by GNA1870 variant 1 strains.

Amino Acid Sequence↗

PubMed2024

Whole exome sequencing analysis of 167 men with primary infertility.

BACKGROUND: Spermatogenic failure is one of the leading causes of male infertility and its genetic etiology has not yet been fully understood. METHODS: The study screened a cohort of patients (n = 167) with primary male infertility in contrast to 210 normally fertile men using whole exome sequencing (WES). The expression analysis of the candidate genes based on public single cell sequencing data was performed using the R language Seurat package. RESULTS: No pathogenic copy number variations (CNVs) related to male infertility were identified using the the GATK-gCNV tool. Accordingly, variants of 17 known causative (five X-linked and twelve autosomal) genes, including ACTRT1, ADAD2, AR, BCORL1, CFAP47, CFAP54, DNAH17, DNAH6, DNAH7, DNAH8, DNAH9, FSIP2, MSH4, SLC9C1, TDRD9, TTC21A, and WNK3, were identified in 23 patients. Variants of 12 candidate (seven X-linked and five autosomal) genes were identified, among which CHTF18, DDB1, DNAH12, FANCB, GALNT3, OPHN1, SCML2, UPF3A, and ZMYM3 had altered fertility and semen characteristics in previously described knockout mouse models, whereas MAGEC1,RBMXL3, and ZNF185 were recurrently detected in patients with male factor infertility. The human testis single cell-sequencing database reveals that CHTF18, DDB1 and MAGEC1 are preferentially expressed in spermatogonial stem cells. DNAH12 and GALNT3 are found primarily in spermatocytes and early spermatids. UPF3A is present at a high level throughout spermatogenesis except in elongating spermatids. The testicular expression profiles of these candidate genes underlie their potential roles in spermatogenesis and the pathogenesis of male infertility. CONCLUSION: WES is an effective tool in the genetic diagnosis of primary male infertility. Our findings provide useful information on precise treatment, genetic counseling, and birth defect prevention for male factor infertility.

Humans↗

PubMed2004

Small cell astrocytoma: an aggressive variant that is clinicopathologically and genetically distinct from anaplastic oligodendroglioma.

BACKGROUND: Small cell glioblastoma (GBM) is a variant with monomorphous, deceptively bland nuclei that often is misdiagnosed as anaplastic oligodendroglioma. METHODS: To elucidate its clinicopathologic and genetic features, the authors studied 71 adult patients (median age, 57 years), including 22 patients who were identified from a set of 229 GBMs (10%) that had been characterized previously by epidermal growth factor receptor (EGFR)/EGFR-vIII variant immunohistochemistry. Tumors also were analyzed by fluorescence in situ hybridization for 1p, 19q, 10q, and EGFR copy numbers. RESULTS: Radiologically, 37% of tumors that were not selected for grade showed minimal to no enhancement. Similarly, 33% of tumors had no endothelial hyperplasia or necrosis histologically, qualifying only as anaplastic astrocytoma (Grade III) using World Health Organization criteria. Nevertheless, such tumors progressed rapidly, with mortality rates that were indistinguishable from their Grade IV counterparts. The median survival for 37 patients who were followed until death was 11 months. Oligodendroglioma-like histology included chicken-wire vasculature (86%), haloes (73%), perineuronal satellitosis (58%), and microcalcifications (45%), although mucin-filled microcystic spaces were lacking. No small cell astrocytomas had 1p/19q codeletions, whereas EGFR amplification and 10q deletions were present in 69% and 97% of small cell astrocytomas, respectively. The tumors expressed EGFR and EGFR-vIII more commonly than nonsmall cell GBMs (83% vs. 35% [P < 0.001]; 50% vs. 21% [P < 0.001] respectively). CONCLUSIONS: Small cell astrocytoma is an aggressive histologic variant that behaved like primary GBM, even in the absence of endothelial hyperplasia and necrosis. Despite considerable morphologic overlap with anaplastic oligodendroglioma, clinicopathologic and genetic features were distinct. Fifty percent of small cell astrocytomas expressed the constitutively activated vIII mutant form of EGFR, and molecular testing for 10q deletion improved the diagnostic sensitivity over EGFR alone.

Adult↗

PubMed2026

Hurdles to horizontal gene transfer: species-specific effects of synonymous variation and plasmid copy number determine antibiotic resistance phenotype.

Could codon composition condition the immediate success and the orientation of horizontal gene transfer? Horizontal gene transfer represents a change in the genome of expression of the transferred gene, and experimental evidence has accumulated indicating that the codon composition of a sequence is an important determinant of its compatibility with the translation machinery of the genome in which it is expressed. This suggests that codon composition influences the phenotype and the fitness conferred by a transferred gene and thus the immediate success of the transfer. To directly test this hypothesis, we characterized the resistance conferred by synonymous variants of a gentamicin resistance gene in three bacterial species: Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. The strongest determinant of the resistance level conferred was the species in which the resistance gene was transferred, very likely because of important differences in the copy number of the plasmid carrying the gene. Significant differences in resistance were also found between synonymous variants within each of the three species, but more importantly, there was a strong interaction between species and variant: variants conferring high resistance in one species confer low resistance in another. However, the similarity in codon usage between the synonymous variants and the host genome only explained part of the phenotypic differences between variants in one species, P. aeruginosa. Further investigation of alternative explanations did not reveal common universal mechanisms across our three bacterial species. We conclude that codon composition can be a determinant of post-horizontal gene transfer success. However, there are multiple paths leading from synonymous sequence to phenotype, and sensitivity to these different paths is species-specific.

Gene Transfer, Horizontal↗

PubMed2003

Extensive normal copy number variation of a beta-defensin antimicrobial-gene cluster.

Using a combination of multiplex amplifiable probe hybridization and semiquantitative fluorescence in situ hybridization (SQ-FISH), we analyzed DNA copy number variation across chromosome band 8p23.1, a region that is frequently involved in chromosomal rearrangements. We show that a cluster of at least three antimicrobial beta-defensin genes (DEFB4, DEFB103, and DEFB104) at 8p23.1 are polymorphic in copy number, with a repeat unit >/=240 kb long. Individuals have 2-12 copies of this repeat per diploid genome. By segregation, microsatellite dosage, and SQ-FISH chromosomal signal intensity ratio analyses, we deduce that individual chromosomes can have one to eight copies of this repeat unit. Chromosomes with seven or eight copies of this repeat unit are identifiable by cytogenetic analysis as a previously described 8p23.1 euchromatic variant. Analysis of RNA from different individuals by semiquantitative reverse-transcriptase polymerase chain reaction shows a significant correlation between genomic copy number of DEFB4 and levels of its messenger RNA (mRNA) transcript. The peptides encoded by these genes are potent antimicrobial agents, especially effective against clinically important pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, and DEFB4 has been shown to act as a cytokine linking the innate and adaptive immune responses. Therefore, a copy number polymorphism involving these genes, which is reflected in mRNA expression levels, is likely to have important consequences for immune system function.

Alleles↗

PubMed2002

Identification and evolution of an IS6110 low-copy-number Mycobacterium tuberculosis cluster.

A cohort of 56 patients infected with related strains of Mycobacterium tuberculosis, the S75 group, was identified in a New Jersey population-based study of all isolates with a low number of copies of the insertion element IS6110. Genotyping was combined with surveillance data to identify the S75 group and to elucidate its recent evolution. The S75 group had similar demographic and geographic characteristics. Seventeen persons (30%) were linked epidemiologically. The S75 group was segregated from other low-copy-number isolates on the basis of several independent molecular methods. This group included 3 IS6110 genotype variants: BE, H6, and C28, containing 1, 2, and 3 IS6110 insertions, respectively. IS6110 insertion site mapping and comparative sequence analysis strongly suggest a stepwise acquisition of IS6110 elements from BE to H6 to C28. S75 represents a locally produced strain cluster that has recently evolved. The combination of multiple molecular tools with traditional epidemiology provides novel insights into dissemination, local transmission, and evolution of M. tuberculosis.

DNA Transposable Elements↗