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PubMed2026

Mitochondrial Haplotype Shapes the Trajectory of Ovarian Aging in Genetically Heterogeneous Rats.

Ovarian aging leads to permanent reproductive senescence and systemic hormonal changes that predispose women to age-associated comorbidities. Despite these observations, the intrinsic mechanisms driving age-related ovarian decline are poorly defined. Mitochondrial DNA (mtDNA) mutations and instability are strongly associated with aging; however, it remains unknown if naturally occurring mitochondrial genetic variation influences the trajectory of ovarian aging. To address this, we compared two genetically heterogeneous rat cohorts (OKC-HETB and OKC-HETW) that differ in mitochondrial haplotype on a randomized but equivalently distributed nuclear background. The OKC-HETW haplotype was associated with accelerated loss of primordial follicles and pathological remodeling marked by fibrosis, macrophage infiltration, and multinucleated giant cells. These tissue-level pathologies were paralleled by mitochondrial dysfunction, characterized by decreased respiratory complex activity, ATP production, and mtDNA copy number. Mechanistically, we identified a haplotype-specific defect in mitochondrial genome maintenance. Although TFAM expression was normal, and total TFAM protein was elevated, OKC-HETW ovaries showed reduced mitochondrial TFAM abundance, TFAM-mtDNA binding, and TOMM20, suggesting that impaired TOMM20-mediated import is associated with compromised mitochondrial genomic stability. Longitudinal transcriptomic and proteomic analyses further indicate that mitochondrial haplotype influences the rate of ovarian aging, with OKC-HETW ovaries showing accelerated activation of inflammatory and fibrotic pathways alongside suppressed proteostasis and mitochondrial function. These defects corresponded to impairments in ovulation and a trend toward worsening oocyte quality. Collectively, our findings identify mitochondrial haplotype as a heritable modifier of ovarian aging rate that acts in concert with the nuclear genome, and a putative target for preserving ovarian function and female healthspan.

Animals↗

PubMed1999

Role of genomic rearrangements in producing new ribotypes of Salmonella typhi.

Salmonella typhi is the only species of Salmonella which grows exclusively in humans, in whom it causes enteric typhoid fever. Strains of S. typhi show very little variation in electrophoretic types, restriction fragment length polymorphisms, cell envelope proteins, and intervening sequences, but the same strains are very heterogeneous for ribotypes which are detected with the restriction endonuclease PstI. In addition, the genome of S. typhi has been proven to undergo genomic rearrangement due to homologous recombination between the seven copies of rrn genes. The relationship between ribotype heterogeneity and genomic rearrangement was investigated. Strains of S. typhi which belong to 23 different genome types were analyzed by ribotyping. A limited number of ribotypes were found within the same genome type group; e. g., most strains of genome type 3 belonged to only two different ribotypes, which result from recombination between rrnH and rrnG operons. Different genome type groups normally have different ribotypes. The size and identity of the PstI fragment containing each of the seven different rrn operons from S. typhi Ty2 were determined, and from these data, one can infer how genomic rearrangement forms new ribotypes. It is postulated that genomic rearrangement, rather than mutation, is largely responsible for producing the ribotype heterogeneity in S. typhi.

Bacterial Typing Techniques↗

PubMed1983

An African trypanosome variant surface glycoprotein gene whose expression is not activated by duplication.

A variant surface glycoprotein (VSG) of Trypanosoma brucei is encoded by a gene whose expression is not governed by duplication-transposition. There are two copies of this gene. The 5' flanking regions of the two genes are indistinguishable by restriction mapping, although each possesses approximately 5-10 Kbp of DNA which is devoid of restriction sites. All restriction enzymes tested appeared to cut genomic DNA at a uniform distance 3' of the gene. This, coupled with the observed sensitivity of both genes to BAL 31, indicates that they lie near chromosomal termini. Length variation occurs 3' of these genes in bloodstream clones and their procyclic derivatives, although the number of length variants is conserved. This suggests that length variation alone does not control VSG switching or gene expression and that constraints exist on the extent to which 3' flanking regions can vary in length.

Animals↗

PubMed2005

A genetic-algorithm approach to simulating human immunodeficiency virus evolution reveals the strong impact of multiply infected cells and recombination.

It has been previously shown that the majority of human immunodeficiency virus type 1 (HIV-1)-infected splenocytes can harbour multiple, divergent proviruses with a copy number ranging from one to eight. This implies that, besides point mutations, recombination should be considered as an important mechanism in the evolution of HIV within an infected host. To explore in detail the possible contributions of multi-infection and recombination to HIV evolution, the effects of major microscopic parameters of HIV replication (i.e. the point-mutation rate, the crossover number, the recombination rate and the provirus copy number) on macroscopic characteristics (such as the Hamming distance and the abundance of n-point mutants) have been simulated in silico. Simulations predict that multiple provirus copies per infected cell and recombination act in synergy to speed up the development of sequence diversity. Point mutations can be fixed for some time without fitness selection. The time needed for the selection of multiple mutations with increased fitness is highly variable, supporting the view that stochastic processes may contribute substantially to the kinetics of HIV variation in vivo.

Algorithms↗

PubMed1989

Mechanisms of genetic variability in Halobacterium halobium: the purple membrane and gas vesicle mutations.

Several phenotypic variants of Halobacterium halobium arise spontaneously at extremely high frequencies (up to 1%) and are readily identified by inspection of bacterial colonies. Two mutant types, those lacking the buoyant gas vesicles or the photosynthetic purple membrane, have been studied in detail by phenotypic and molecular genetic analysis. In the wild-type NRC-1 strain, the bop gene, encoding the purple membrane protein bacterio-opsin, is found on the bacterial chromosome, while the gas vesicle protein genes, gvpA and gvpC, are present on pNRC100, a multicopy plasmid of approximately 150 kilobase pairs. The gvpA and gvpC genes are on a single transcription unit, while the major bop mRNA is monocistronic. Essentially all of the purple membrane deficient mutants contain insertion sequence (IS) elements into or upstream of the bop gene. Two elements, ISH 1 and ISH 2, account for most (80-90%) of the purple membrane mutations, but at least three other elements, ISH 23, ISH 26, and ISH S1, have also been implicated. The gas vesicle mutants are more heterogeneous, with many displaying partial phenotypes. Three major classes of gas vesicle mutants are distinguishable: class I and class III mutants are the result of large deletions in pNCR100; however, while class I mutants are partially gas vesicle deficient and contain a correspondingly reduced number of gvpAC operon copies, class III mutants contain no detectable copies of the gas vesicle genes and are essentially completely gas vesicle deficient. Class II mutants, like the purple membrane mutants, contain IS elements into or upstream of the gas vesicle genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Archaeal Proteins↗

PubMed2005

Quantitative TaqMan PCR for detection of Pneumocystis jiroveci.

We developed a quantitative real-time PCR assay for detection and quantification of Pneumocystis jiroveci in bronchoalveolar lavage (BAL) specimens based on primers and probe targeting the gene encoding beta-tubulin. The assay was able to detect 50 DNA copies per ml of a standard plasmid containing the target sequence. The intra- and interassay coefficients of variation were 0.46%-4.27% and 0.05-2.00% over 5 log(10) values. Fifty-seven controls of human, viruses, bacteria and fungi DNA samples were amplified and found negative. Fifty-three BAL samples sent to the laboratory for diagnosis of pneumocystosis were prospectively investigated by real-time PCR and direct microscopic examinations (DME) using Giemsa stain and direct immunofluorescence. All PCR negative samples were negative by microscopy. Among the 24 (45%) BAL found PCR positive, 8 were positive by microscopy (35%). The copy numbers of the target gene were between 4.4 x 10(3) and 2.8 x 10(6) per ml for the microscopically positive samples and between 8 and 9.2 x 10(3) per ml for the microscopically negative samples. In conclusion, we developed a rapid, sensitive and specific real time PCR for the diagnosis and quantification of Pneumocystis jiroveci in BAL samples.

Base Sequence↗

PubMed2000

Ty1-copia retrotransposon-like elements in chickpea genome: their identification, distribution and use for diversity analysis.

Ty1-copia retrotransposon-like elements were amplified from Cicer species using primers derived from the conserved region of the reverse transcriptase gene. Two fragments, of size approximately 280bp and approximately 650 bp, were obtained, which on sequencing showed homology for the Ty1-copia reverse transcriptase region. Interestingly, the approximately 650 bp fragment showed two reverse transcriptase regions, one from Ty1-copia and the other from Tto1 element fused together. The copy number was high in the cultivated Cicer arietinum genome compared with the wild Cicer reticulatum. Genetic diversity among the Cicer species was investigated using the conserved primers which grouped the wild species and the cultivated C. arietinum separately.

Amino Acid Sequence↗

PubMed2002

Hologram recording in polyvinyl alcohol/acrylamide photopolymers by means of pulsed laser exposure.

The variation in transmittance produced when a photopolymer is irradiated with a pulsed laser is analyzed and experimental results obtained when diffraction gratings are stored using pulsed exposure are presented. In either case, the influence of the energy of the irradiation pulse, the number of pulses, and the pulse repetition rate were studied. The photopolymer used was an acrylamide/polyvinyl alcohol dry film with a yellow eosin-thiethanol-amine mixture as a photoinitiator system. The recording of the gratings was performed by use of a holographic copying process. The samples were exposed and holograms recorded with a collimated beam from a frequency-doubled Nd:YAG (532 nm) Q-switched laser. Our initial results show that it is possible to obtain diffraction gratings with a diffraction efficiency of 60% and a refractive index modulation up to 2.8 x 10(-3). The energetic sensitivities achieved are close to those obtained with the same material and continuous irradiation without a preprocessing of the gratings.

Journal Article↗

PubMed2005

Quantitative change in mitochondrial DNA content in various mouse tissues during aging.

In order to systematically characterize age-related changes in the mtDNA content of various tissues during aging, we analyzed the mtDNA content of eight tissues from mice at five different ages from young to senescent by quantitative real-time PCR analysis. Obvious variations of mtDNA content among the tissues were detected: There was a 20-fold range in 2-week-old mice and a 50-fold range in 15-month-old mice. The mtDNA contents of the heart, lung, kidney, spleen and skeletal muscle increased gradually with age, whereas those of bone marrow and brain showed no age-related pattern. The expression patterns of mitochondrial transcription factor A (mtTFA) and mitochondrial single-strand DNA binding protein (mtSSB), possible regulatory factors of the mtDNA copy number, were not necessarily linked with the age-related pattern of the mtDNA content, suggesting the existence of other factors that affect the mtDNA content. The Western blot analysis of mtDNA-encoded cytochrome c oxidase subunit III (MTCO3) demonstrated that the expression levels of this protein in the heart and skeletal muscle increase with age in parallel with the mtDNA content. These findings confirm that the mtDNA content of tissues changes during aging.

Aging↗

PubMed2005

Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE.

The analysis of microbial communities in environmental samples requires accurate and reproducible methods for extraction of DNA from sample matrices that have different physical and chemical characteristics. Even with the same sample type, variations in laboratory methods can result in different DNA yields. To circumvent this problem, we have developed an easy and inexpensive way to normalize the quantities of DNA that involves the addition of an internal standard prepared from plasmid DNA. The method was evaluated by comparing DNA yields using different DNA extraction procedures, after which the DNA was used for microbial community analysis by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S ribosomal RNA (rRNA) and for quantification of 16S rRNA gene copy numbers in environmental samples by real-time PCR. Our results show that use of the internal standard allows normalization of the resulting data and more accurate quantification of gene copy numbers in soil samples. These methods should also have broad application for various other types of environmental samples.

DNA Primers↗

PubMed2005

Establishment of real-time polymerase chain reaction-based assay for quantitation of Epstein-Barr virus DNA in healthy donors and in patients with EBV associated lymphoma.

Epstein-Barr virus (EBV) is associated with several malignancies including nasopharyngeal carcinoma and lymphoma in immunocompromised patients. Quantitative monitoring of EBV DNA in these patients has recently become essential for management of the disease. In the present study the authors developed a rapid and reliable real-time PCR to quantify the EBV DNA in peripheral blood mononuclear cell (PBMC) using hybridization probe technique. The real-time primers and probes in this real-time PCR system were designed based on EBNA-1 sequence. The newly-established real-time PCR demonstrated its high sensitivity (as few as 10 copies of EBV could be detected) and specificity. The intra- and inter-assay variations of the assay were shown to be within a 0.5-log10-difference range. A total of 2 EBV-seronegative, 14 EBV-seropositive healthy donors and 4 patients with PCNSL were enrolled into the study. Our results revealed the median of EBV-DNA in lymphoma patients (7886 copies/10(6) PBMC or 15,150 copies /microg DNA) was higher than that of healthy donors (<10 copies/l0(6) PBMC or <10 copies/microg DNA) with statistic significance (P < 0.01). Assessment of this assay in larger number of donors and patients will provide clinical cut-off values which are essential for monitoring and diagnosis of EBV-associated diseases.

Adult↗

PubMed2004

Intratypic variability of a tandem repeat locus within the DNA polymerase gene of human herpes simplex virus type 2.

In this study, the irntratypic variability of a tandem repeat locus within the DNA polymerase (pol) gene of human herpes simplex virus type 2 (HSV2) was uncovered. The locus contained variable numbers of tandem dodecanucleotide (5'-GAC GAG GAC GGG-3') repetitive units. Our result showed that approximately 95% of analyzed HSV2 clinical isolates and the current GenBank HSV2 strains contained two copies of the repetitive units. From genital herpes specimens, three new HSV2 strains, which respectively contained 1, 3, and 4 copies of the repetitive units, were identified. This variable number of tandem repeat (VNTR) locus is absent in HSV1, and thus it also contributes to the intertypic variability of HSV1 and HSV2. The intratypic variability of the locus may be useful for HSV2 strain genotyping and this application is discussed.

DNA-Directed DNA Polymerase↗

PubMed1974

Biochemical and genetic basis of tetracycline resistance in Staphylococcus aureus.

Both the genetic and biochemical basis of tetracycline resistance in a number of staphylococcal strains was investigated. The strains examined could be classified into three groups: (i) those possessing a high basal level of resistance and in which resistance could be induced to higher levels (macro-inducible); (ii) those which had a high uninduced level of resistance, but which were virtually uninducible (macro-constitutive); (iii) one derivative which had a low basal level of resistance and was also uninducible (micro-constitutive). Resistance in macro-constitutive strains was plasmid mediated and typical of organisms possessing wild-type plasmids. The macro-constitutive pattern of resistance appeared to be correlated with a chromosomal location for the resistance genes, whereas the micro-constitutive pattern was correlated with loss of a region from the wild-type plasmid. Analysis of membrane proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested that a number of membrane polypeptides became unstable in staphylococci possessing high-level tetracycline resistance. In particular, the absence of a polypeptide of 22,000 daltons was always associated with high-level resistance. There was no evidence that multiple gene copies are required for expression of tetracycline resistance in Staphylococcus aureus.

Gene Expression Regulation, Bacterial↗

PubMed2006

Small gene effect and exercise training-induced cardiac hypertrophy in mice: an Ace gene dosage study.

Small gene effects influence complex phenotypes in a context dependent manner. Here we evaluated whether increasing dosage of the angiotensin I converting enzyme (Ace) gene influence exercise-induced cardiac hypertrophy. Mice harboring one, two, three, and four copies of the Ace gene were assigned to sedentary (S1-4) and swimming exercise-trained (T1-4) groups (1.5 h twice daily, 5 days/wk, 4 wk). Exercising resulted in comparable bradycardia and elevated skeletal muscle citrate synthase activity, while blood pressure remained unchanged. Left ventricle mass index and cardiomyocyte diameter were similar among sedentary mice and the magnitude of their increase associated to exercising was not influenced by the Ace genotype (T1: 12.6 and 17.9%, T2: 15.2 and 13.8%, T3: 16.9 and 20%, T4: 17 and 19%, respectively). Plasma renin activity (PRA) levels were higher in one vs. three or four copies mice (4.89 +/- 0.5 vs. 2.43 +/- 0.6 and 2.12 +/- 01.1 ng/ml Ang I, P < 0.05), while cardiac ACE activity was higher in three vs. two or one copy mice (5,946 +/- 590.8 vs. 2,951.5 +/- 328.3 and 3,504.1 +/- 258.9 microF x min(-1) x ml(-1), P < 0.05). With exercise, PRA remained unchanged in each group, while cardiac immunostaining for Ang II reached comparable levels. In summary: 1) exercise training led to similar aerobic adaptation regardless of the Ace genotype, and 2) higher number of Ace gene copies per se, which alters cardiac ACE activity, did not influence basal cardiac mass or, most importantly, the magnitude of swimming-induced cardiac hypertrophy. Collectively, these data indicate that small isolated genetic disturbances in ACE cardiac levels can be well compensated under physiological perturbations.

Angiotensin II↗

PubMed2004

Functional interactions between receptors in bacterial chemotaxis.

Bacterial chemotaxis is a model system for signal transduction, noted for its relative simplicity, high sensitivity, wide dynamic range and robustness. Changes in ligand concentrations are sensed by a protein assembly consisting of transmembrane receptors, a coupling protein (CheW) and a histidine kinase (CheA). In Escherichia coli, these components are organized at the cell poles in tight clusters that contain several thousand copies of each protein. Here we studied the effects of variation in the composition of clusters on the activity of the kinase and its sensitivity to attractant stimuli, monitoring responses in vivo using fluorescence resonance energy transfer. Our results indicate that assemblies of bacterial chemoreceptors work in a highly cooperative manner, mimicking the behaviour of allosteric proteins. Conditions that favour steep responses to attractants in mutants with homogeneous receptor populations also enhance the sensitivity of the response in wild-type cells. This is consistent with a number of models that assume long-range cooperative interactions between receptors as a general mechanism for signal integration and amplification.

Bacterial Proteins↗

PubMed1981

Linkage and evolutionary diversification of developmentally regulated multigene families: tandem arrays of the 401/18 chorion gene pair in silkmoths.

The coordinately expressed silkmoth chorion genes, 401 and 18, are closely linked as a pair, in divergent orientation. Analysis of overlapping clones (chromosomal "walk") demonstrated that each of the multiple copies of this gene pair is embedded within a larger deoxyribonucleic acid unit, which is tandemly repeated in a few arrays or possibly a single array. Southern analysis and examination of clones from a single individual moth demonstrated that the repeat units are extensively polymorphic in restriction sites, length, and possibly number, no differential amplification was evident during choriogenesis. Intron and 5'-flanking sequences were shown to be specific for the 401/18 gene pair and not to be present elsewhere in the genome. The spatial distribution of variations in the genes and their flanking sequences were examined.

Animals↗

PubMed2003

Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays.

BACKGROUND: Long oligonucleotide microarrays are potentially more cost- and management-efficient than cDNA microarrays, but there is little information on the relative performance of these two probe types. The feasibility of using unmodified oligonucleotides to accurately measure changes in gene expression is also unclear. RESULTS: Unmodified sense and antisense 70-mer oligonucleotides representing 75 known rat genes and 10 Arabidopsis control genes were synthesized, printed and UV cross-linked onto glass slides. Printed alongside were PCR-amplified cDNA clones corresponding to the same genes, enabling us to compare the two probe types simultaneously. Our study was designed to evaluate the mRNA profiles of heart and brain, along with Arabidopsis cRNA spiked into the labeling reaction at different relative copy number. Hybridization signal intensity did not correlate with probe type but depended on the extent of UV irradiation. To determine the effect of oligonucleotide concentration on hybridization signal, 70-mers were serially diluted. No significant change in gene-expression ratio or loss in hybridization signal was detected, even at the lowest concentration tested (6.25 microm). In many instances, signal intensity actually increased with decreasing concentration. The correlation coefficient between oligonucleotide and cDNA probes for identifying differentially expressed genes was 0.80, with an average coefficient of variation of 13.4%. Approximately 8% of the genes showed discordant results with the two probe types, and in each case the cDNA results were more accurate, as determined by real-time PCR. CONCLUSIONS: Microarrays of UV cross-linked unmodified oligonucleotides provided sensitive and specific measurements for most of the genes studied.

Arabidopsis↗

PubMed1997

Repetitive sequence families in Alces alces americana.

High-resolution derivative melting was used to obtain detailed distributions of local (G + C) contents in a number of ruminant DNAs. Profiles over low (G + C) regions [20-36% (G + C)] are congruent for all ruminants. This region represents 45-50% of the nuclear DNA content and primarily contains intergenic and intron sequences. The high (G + C) region, where most coding sequences are found [38-68% (G + C)], is marked by satellite bands denoting the presence of transcriptionally inert, tandemly repetitive sequence families. These bands can be analyzed for the abundance, base composition, and sequence divergence of satellite families with relatively high precision. Band patterns are unique to each species; even closely related species can be readily distinguished by their base distribution profiles. Variations in nuclear DNA contents in ruminants, determined by flow cytometry, are primarily due to variations in abundances of these repetitive sequence families. Thus, A. alces (moose) is found to have 8.85 +/- 0.2 pg DNA/cell, 25% more than the average in ruminants, while the base distribution curve indicates the presence of an unusually abundant satellite of 52.6% (G + C). The size (1 kb) and sequence of this satellite corresponds to satellite-I of other cervids, and in consequence it is designated Alces-I. The sequence of a cloned repeat of Alces-I has a length of 968 bp, a (G + C) content of 52.6%, and contributes 35%, or almost 3 million copies to the nuclear DNA, exceeding by approximately 300% the average array size of this repeat family in related cervids. In situ hybridization indicates the repeat is distributed throughout centromeric regions of all 62 acrocentric autosomes. Alces-I has much greater-than-expected numbers of GG, GA, and AG and far fewer numbers of TA and CG duplets, characteristics of all tandem repeats. The sequence is judged to be orthologous with satellite-I sequences from Rangifer tarandus (caribou), Capreolus capreolus (roe deer), Muntiacus muntjac (Chinese muntjac) and Muntiacus reevesi (Indian muntjac), as well as Antilocapra americana (pronghorn), and the bovids Bos taurus and Ovis aries. A tentative tree for the five cervids is in excellent agreement with one proposed on the basis of morphological characteristics. Differences from a consensus sequence indicate transversions exceed transitions by almost twofold, suggesting that substitutions occur randomly, or nearly so.

Animals↗