Amount Of Mitochondrial DNA Research Articles

An Increase in Mitochondrial DNA Promotes Nuclear DNA Replication in Yeast

Coordination between cellular metabolism and DNA replication determines when cells initiate division. It has been assumed that metabolism only plays a permissive role in cell division. While blocking metabolism arrests cell division, it is not known whether an up-regulation of metabolic reactions accelerates cell cycle transitions. Here, we show that increasing the amount of mitochondrial DNA accelerates overall cell proliferation and promotes nuclear DNA replication, in a nutrient-dependent manner. The Sir2p NAD+-dependent de-acetylase antagonizes this mitochondrial role. We found that cells with increased mitochondrial DNA have reduced Sir2p levels bound at origins of DNA replication in the nucleus, accompanied with increased levels of K9, K14-acetylated histone H3 at those origins. Our results demonstrate an active role of mitochondrial processes in the control of cell division. They also suggest that cellular metabolism may impact on chromatin modifications to regulate the activity of origins of DNA replication.

Mitochondrial DNA integrity and copy number in sperm from infertile men

To determine the integrity and copy number of mitochondrial DNA (mtDNA) in spermatozoa from infertile men. We also compared mtDNA integrity to nuclear DNA integrity. Observational. Tertiary care center. Semen samples were collected from 57 men, whose wives were undergoing in vitro fertilization, including 24 men with normal semen parameters. Quantitative long polymerase chain reaction (PCR) and real-time PCR were used to measure mtDNA integrity and copy number in spermatozoa. We also measured nuclear DNA integrity of spermatozoa with flow cytometry after acridine orange staining. A significant increase in mtDNA copy number and a decrease in mtDNA integrity were observed in the spermatozoa samples from patients with abnormal semen parameters compared to the patients with normal semen parameters. The integrity and copy number of mtDNA were significantly correlated with sperm count. Mitochondrial DNA quantity and quality may serve as useful diagnostic markers of sperm quality in infertile men.

Influence of polymorphism of glutathione S-transferase T1 on Chinese infertile patients with varicocele

To investigate the influence of glutathione S-transferase T1 (GSTT1) gene polymorphism on Chinese infertile patients with varicocele, 63 infertile patients with varicocele and 54 healthy fertile controls were recruited in this case-control study. Our results show that oxidative damage may be the cause of infertility in patients with varicocele, and GSTT1 null genotype predisposes to over oxidative damage to spermatocytes of infertile patients with varicocele.

Convergence of multiple signaling pathways is required to coordinately up-regulate mtDNA and mitochondrial biogenesis during T cell activation

The quantity and activity of mitochondria vary dramatically in tissues and are modulated in response to changing cellular energy demands and environmental factors. The amount of mitochondrial DNA (mtDNA), which encodes essential subunits of the oxidative phosphorylation complexes required for cellular ATP production, is also tightly regulated, but by largely unknown mechanisms. Using murine T cells as a model system, we have addressed how specific signaling pathways influence mitochondrial biogenesis and mtDNA copy number. T cell receptor (TCR) activation results in a large increase in mitochondrial mass and membrane potential and a corresponding amplification of mtDNA, consistent with a vital role for mitochondrial function for growth and proliferation of these cells. Independent activation of protein kinase C (via PMA) or calcium-related pathways (via ionomycin) had differential and sub-maximal effects on these mitochondrial parameters, as did activation of naïve T cells with proliferative cytokines. Thus, the robust mitochondrial biogenesis response observed upon TCR activation requires synergy of multiple downstream signaling pathways. One such pathway involves AMP-activated protein kinase (AMPK), which we show has an unprecedented role in negatively regulating mitochondrial biogenesis that is mammalian target of rapamycin (mTOR)-dependent. That is, inhibition of AMPK after TCR signaling commences results in excessive, but uncoordinated mitochondrial proliferation. Thus mitochondrial biogenesis is not under control of a single master regulatory circuit, but rather requires the convergence of multiple signaling pathways with distinct downstream consequences on the organelle’s structure, composition, and function.

Simultaneously Monitoring the Superoxide in the Mitochondrial Matrix and Extramitochondrial Space by Micellar Electrokinetic Chromatography with Laser-Induced Fluorescence

Respiring mitochondria produce superoxide and release it into both sides of the mitochondrial inner membrane: the mitochondrial matrix and the extramitochondrial space. These two pools of superoxide are expected to have very distinctive effects on cellular function. Currently, separate measurements are required to measure superoxide in both pools, which complicates the comparison of superoxide's effects and roles in physiology and pathology. In this study, we describe a highly sensitive strategy to monitor simultaneously these two pools of superoxide in respiring isolated rat skeletal muscle mitochondria using hydroethidine. The oxidation of hydroethidine by superoxide forms the membrane-impermeable 2-hydroxyethidium in these two superoxide pools that can be separated by differential centrifugation. Two technical limitations in using 2-hydroxyethidium as a superoxide reporter are (i) the uncontrolled fluorescence enhancement due to intercalation of 2-hydroxyethidium with variable amounts of mitochondrial DNA and (ii) the spectral interference of ethidium fluorescence. These complications were eliminated by digestion of mitochondrial DNA with DNase and by separation of ethidium and 2-hydroxyethidium using cationic micellar electrokinetic capillary chromatography with laser-induced fluorescence, respectively. Using this method, which has subattomole limits of detection, we compared the levels of 2-hydroxyethidium in normally respiring and antimycin A-treated mitochondria and demonstrated that the strategy can be extended to observe how menadione induces superoxide generation in mitochondria.

Standard and Light-Cycler PCR methods for animal DNA species detection in animal feedstuffs

Abstract In this work four species-specific primers and probes were designed and evaluated for the detection and quantification of bovine, ovine, swine and chicken mitochondrial DNA in feeds. PCR primers were optimized using conventional and Real Time PCR, to detect short species-specific sequences amplifiable from heat treated material. Both methods confirmed the high specificity of the primers designed. Real time quantitative PCR assay allowed the detection of as few as 0.01 ng and 0.05 ng of ovine and bovine genomic DNA, respectively. The detection limit for swine and chicken genomic DNA was 0.5 ng. Sensitivity levels observed in DNA extracted from meat samples processed according to EU legislation were different compared to those in genomic DNAs previously described. They resulted in swine 5 fg of MBM DNA, in chicken 25 ng, in ovine and bovine 50 ng. We confirmed the efficiency and specificity of primers in RT-PCR to detect 0.5% of bovine, ovine, swine and chicken MBM in contaminated feedstuffs. Industrial relevance The variant Creutzfeldt Jakob disease is a rare and fatal human neurodegenerative condition clearly linked with the bovine spongiform encephalopathies (BSE) of cattle. The ban of using animal derived protein in animal feeds has efficiently controlled the development of the BSE epidemic. The work presented by Frezza and collaborators is an application of the real time polymerase chain reaction (a standard procedure used in molecular biology also known as RT‐PCR) to identify specific DNA of four animal species (bovine, ovine, swine and chicken). This method is applied to the analysis of feeds to detect and eventually estimate the amount of animal derived proteins. The difficult aim to detect DNA derived from heat‐treated material was successfully reached using as target short mitochondrial DNA sequences. The method presented could have important application not only in the control of feed production but also in many fields of the food industry as quality and process control.

Adipose Mitochondrial Biogenesis Is Suppressed in db/db and High-Fat Diet–Fed Mice and Improved by Rosiglitazone

The objective of this study was to further establish and confirm the relationship of adipose mitochondrial biogenesis in diabetes/obesity and the effects of rosiglitazone (RSG), a peroxisome proliferator-activated receptor (PPAR) gamma agonist, by systematically analyzing mitochondrial gene expression and function in two mouse models of obesity and type 2 diabetes. Using microarray technology, adipose mitochondrial gene transcription was studied in db/db, high-fat diet-fed C57BL/6 (HFD) and respective control mice with or without RSG treatment. The findings were extended using mitochondrial staining, DNA quantification, and measurements of citrate synthase activity. In db/db and HFD mice, gene transcripts associated with mitochondrial ATP production, energy uncoupling, mitochondrial ribosomal proteins, outer and inner membrane translocases, and mitochondrial heat-shock proteins were decreased in abundance, compared with db/+ and standard-fat diet-fed control mice, respectively. RSG dose-dependently increased these transcripts in both db/db and HFD mice and induced transcription of mitochondrial structural proteins and cellular antioxidant enzymes responsible for removal of reactive oxygen species generated by increased mitochondrial activity. Transcription factors, including PPAR coactivator (PGC)-1beta, PGC-1alpha, estrogen-related receptor alpha, and PPARalpha, were suppressed in both models and induced by RSG. The effects of RSG on adipose mitochondrial genes were confirmed by quantitative RT-PCR and further supported by mitochondrial staining, mitochondrial DNA quantification, and citrate synthase activity. Adipose mitochondrial biogenesis was overwhelmingly suppressed in both mouse models of diabetes/obesity and globally induced by RSG. These findings suggest an important role of adipose mitochondria in diabetes/obesity and the potential for new treatment approaches targeting adipose mitochondria.

Exceptionally High Density of NUMTs in the Honeybee Genome

The available genome sequences of 4 insects (the fruit fly, the African malaria mosquito, the flour beetle, and the honeybee) are used to compare the amount of mitochondrial DNA transferred to the nuclear genome (NUMTs). The data from the beetle and the bee show frequent transfer of NUMTs, whereas NUMTs in the 2 other insects are rare. The density of NUMTs in the honeybee (>1.0 bp transferred DNA per 1 kb of the nuclear sequence) is the highest in any animal studied, about ten times higher than in humans and comparable to the densities in plant genomes. The density of NUMTs in the beetle (0.056 bp/kb) is of the same order of magnitude as that in humans. The analysis of the honeybee genome indicates that NUMTs originate from all parts of the mitochondrial genome, that about two-thirds of the nuclear copies result from secondary transpositions within the nuclear genome, that the copies are significantly associated to "mariner" type transposons, and that the NUMTs consist mainly of short and fragmented copies.

A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA

We developed a modular real-time (rt) PCR system for absolute quantification of human nuclear (n) and mitochondrial (mt) DNA. For determination of the number of amplifiable template molecules with a minimum length required for downstream genotyping and assessment of the PCR-relevant degradation grade of the template DNA, primers yielding differently sized PCR products (nDNA: 79, 156, and 246 bp; mtDNA: 102, 143, 283, and 404 bp) and TaqMan hybridization probes were used for amplification and on-line product detection. DNase-degraded DNA served as model to demonstrate the effects of DNA fragmentation on rtPCR quantification and subsequent genotyping. Introduction of cloned internal amplification positive controls (IPCs) – generated by in vitro mutagenesis of primer-binding sites of the wild-type nDNA and mtDNA targets – enabled functionality-testing of the reaction mixture and detection of PCR inhibitors in DNA extracts, without a need for additional TaqMan probes. A hematin model was used to test the ability of the quantitative real-time (rtq) PCR system to predict the effects of inhibitors in downstream PCR-based genotyping.

Quantification of total mitochondrial DNA and mitochondrial common deletion in the frontal cortex of patients with schizophrenia and bipolar disorder

Data published during the last decade are suggestive of a role for mitochondrial dysfunction in the pathogenesis of schizophrenia, bipolar disorder and other psychiatric diseases. In order to determine if the mitochondrial deficits reported in the literature are caused by abnormalities in the mitochondrial DNA of psychiatric patients, we quantified mitochondrial DNA (mtDNA) levels and the 5 kb common mitochondrial deletion (CD) in postmortem frontal cortex tissue. The mitochondrial CD and mtDNA levels were measured in tissue obtained from the frontal cortex (Brodmann Area 46) of 144 individuals (45 patients with schizophrenia, 40 patients with bipolar disorder, 44 controls, and 15 patients with major depression). These variables were measured using newly developed SYBR green and TaqMan real time PCR assays. Both the TaqMan and the SYBR green assays gave similar results. There was no statistically significant difference for the quantity of the common mitochondrial deletion between controls and patients. We also did not detect a difference in the mtDNA levels amongst the diagnosis groups. There were statistically significant differences for the evaluated parameters for smokers, schizophrenic patients on antipsychotic drugs at time of death, and bipolar patients with antidepressant use and alcohol abuse. Based on this study and other reports, we conclude that neither the common mitochondrial deletion nor changes in mitochondrial DNA levels are likely to account for the mitochondrial changes associated with bipolar disorder or schizophrenia. The effect of premortem agonal factors and medication on mitochondrial dysfunction still needs further elucidation.

Secondary mitochondrial dysfunction in propionic aciduria: a pathogenic role for endogenous mitochondrial toxins

Mitochondrial dysfunction during acute metabolic crises is considered an important pathomechanism in inherited disorders of propionate metabolism, i.e. propionic and methylmalonic acidurias. Biochemically, these disorders are characterized by accumulation of propionyl-CoA and metabolites of alternative propionate oxidation. In the present study, we demonstrate uncompetitive inhibition of PDHc (pyruvate dehydrogenase complex) by propionyl-CoA in purified porcine enzyme and in submitochondrial particles from bovine heart being in the same range as the inhibition induced by acetyl-CoA, the physiological product and known inhibitor of PDHc. Evaluation of similar monocarboxylic CoA esters showed a chain-length specificity for PDHc inhibition. In contrast with CoA esters, non-esterified fatty acids did not inhibit PDHc activity. In addition to PDHc inhibition, analysis of respiratory chain and tricarboxylic acid cycle enzymes also revealed an inhibition by propionyl-CoA on respiratory chain complex III and alpha-ketoglutarate dehydrogenase complex. To test whether impairment of mitochondrial energy metabolism is involved in the pathogenesis of propionic aciduria, we performed a thorough bioenergetic analysis in muscle biopsy specimens of two patients. In line with the in vitro results, oxidative phosphorylation was severely compromised in both patients. Furthermore, expression of respiratory chain complexes I-IV and the amount of mitochondrial DNA were strongly decreased, and ultrastructural mitochondrial abnormalities were found, highlighting severe mitochondrial dysfunction. In conclusion, our results favour the hypothesis that toxic metabolites, in particular propionyl-CoA, are involved in the pathogenesis of inherited disorders of propionate metabolism, sharing mechanistic similarities with propionate toxicity in micro-organisms.

A low dose of ethidium bromide leads to an increase of total mitochondrial DNA while higher concentrations induce the mtDNA 4997 deletion in a human neuronal cell line

Ethidium bromide (EtBr) is widely used to deplete mitochondrial DNA (mtDNA) and produce mitochondrial DNA-less cell lines. However, it frequently fails to deplete mtDNA in mouse cells. In this study we show by using a highly sensitive real-time PCR, that low doses of EtBr (10 μM) did lead to a three-fold increase of the total amount of mitochondrial DNA in a human neuronal cell line (Ntera 2). A higher dose of EtBr (25 μM) led to the expected decrease of mtDNA until day 22 when the cells almost died. Cell growth and mtDNA content could be restored after additional 22 days of non-EtBr treatment. The highest concentration of 50 μM also led to a significant increase of mtDNA. The cells died when they had only about 10% of mtDNA left, indicating a mtDNA threshold for cell survival. Additionally, the so-called common 4977 bp deletion could be induced by prolonged exposure to ethidium bromide. Whereas the higher doses led to significant higher amounts of deleted mtDNA.

Nuclear and mitochondrial DNA quantification of various forensic materials

Due to the different types and quality of forensic evidence materials, their DNA content can vary substantially, and particularly low quantities can impact the results in an identification analysis. In this study, the quantity of mitochondrial and nuclear DNA was determined in a variety of materials using a previously described real-time PCR method. DNA quantification in the roots and distal sections of plucked and shed head hairs revealed large variations in DNA content particularly between the root and the shaft of plucked hairs. Also large intra- and inter-individual variations were found among hairs. In addition, DNA content was estimated in samples collected from fingerprints and accessories. The quantification of DNA on various items also displayed large variations, with some materials containing large amounts of nuclear DNA while no detectable nuclear DNA and only limited amounts of mitochondrial DNA were seen in others. Using this sensitive real-time PCR quantification assay, a better understanding was obtained regarding DNA content and variation in commonly analysed forensic evidence materials and this may guide the forensic scientist as to the best molecular biology approach for analysing various forensic evidence materials.

RETRACTED: Quantification of mitochondrial DNA using real-time polymerase chain reaction in patients with premature ovarian failure

To quantify mitochondrial DNA using real-time PCR in women with premature ovarian failure (POF) and a control group. Prospective study. Genome Research Center for Reproductive Medicine and Infertility, Korea Ministry of Health & Welfare. Thrity patients with POF and 30 control individuals. The mitochondrial DNA content was quantified using real-time PCR. The effectiveness of the assay was determined by relative quantification using the comparative threshold cycle (CT) method. Relative quantification of mitochondrial DNA content. The mitochondrial DNA content was significantly lower in the POF group than in the control group (0.58 +/- 0.38 vs. 1.15 +/- 0.67; P < .01). In both groups, there was a significant positive correlation between the mitochondrial DNA/28S rRNA ratio and mitochondrial DNA CT (control group: r = 0.774; P < .001; POF group: r = 0.556; P = .001) and a significant negative correlation between the mitochondrial DNA/28S rRNA ratio and 28S rRNA CT (control group: r = -0.677; P < .001; POF group: r = -0.627; P = .001). This study has established the clinical feasibility of quantifying amounts of mitochondrial DNA, relative to an internal standard, using real-time PCR. Further studies are warranted to elucidate the roles of apoptosis and mitochondrial function in the pathogenesis of POF.

A Duplex Real-Time qPCR Assay for the Quantification of Human Nuclear and Mitochondrial DNA in Forensic Samples: Implications for Quantifying DNA in Degraded Samples

A duplex real-time qPCR assay was developed for quantifying human nuclear and mitochondrial DNA in forensic samples. The nuclear portion of the assay utilized amplification of a approximately 170-190 bp target sequence that spans the repeat region of the TH01 STR locus, and the mitochondrial portion of the assay utilized amplification of a 69 bp target sequence in the ND1 region. Validation studies, performed on an ABI 7000 SDS instrument using TaqMan detection, demonstrated that both portions of the duplex assay provide suitable quantification sensitivity and precision down to 10-15 copies of each genome of interest and that neither portion shows cross-reactivity to commonly encountered non-human genomes. As part of the validation studies, a series of DNase-degraded samples were quantified using three different methods: the duplex nuclear-mitochondrial qPCR assay, the ABI Quantifiler Human DNA Quantification Kit qPCR assay, which amplifies and detects a 62 bp nuclear target sequence, and slot blot hybridization. For non-degraded and moderately degraded samples in the series, all three methods were suitably accurate for quantifying nuclear DNA to achieve successful STR amplifications to yield complete profiles using the ABI AmpFlSTR Identifiler kit. However, for highly degraded samples, the duplex qPCR assay provided better estimates of nuclear template for STR amplification than did either the commercial qPCR assay, which overestimated the quantity of STR-sized DNA fragments, leading to an increased proportion of undetected alleles at the larger STR loci, or slot blot hybridization, which underestimated the quantity of nuclear DNA, leading to an increased proportion of STR amplification artifacts due to amplification of excess template.

Lack of age-related increase of mitochondrial DNA amount in brain, skeletal muscle and human heart

During the ageing process, an increase of mitochondrial DNA (mtDNA) deletions and other mutations have been reported. These structural alterations of mtDNA are assumed to cause a reduction in the respiratory chain activity and may contribute to the ageing process. Therefore, the question arises if the accumulation of deleted mtDNA is compensated in vivo by an increase of mtDNA synthesis via a feedback mechanism. We designed two human mtDNA-specific oligonucleotide probes for quantitative mtDNA analysis of 5 different tissues from 50 individuals aged from 8 weeks to 93 years. The amount of mtDNA was approximately 1.1 ± 0.5% (4617 ± 2099 copies) in the caudate nucleus, 1.0 ± 0.5% (4198 ± 2099 copies) in the frontal lobe cortex, 0.3 ± 0.2% (1259 ± 840 copies) in the cerebellar cortex, 1.0 ± 0.4% (4198 ± 1679 copies) in skeletal muscle and 2.2 ± 1.3% (9235 ± 5457 copies) in heart muscle. We did not observe any significant change in the absolute copy number during ageing in five different tissues, and therefore, found no evidence for the postulated feedback mechanism. Our study indicates that mtDNA copy number is tissue-specific and depends on the energy demand of the tissue.

Mitochondrial DNA copy number threshold in mtDNA depletion myopathy

The authors measured the absolute amount of mitochondrial DNA (mtDNA) within single muscle fibers from two patients with thymidine kinase 2 (TK2) deficiency and two healthy controls. TK2 deficient fibers containing more than 0.01 mtDNA/microm3 had residual cytochrome c oxidase (COX) activity. This defines the minimum amount of wild-type mtDNA molecules required to maintain COX activity in skeletal muscle and provides an explanation for the mosaic histochemical pattern seen in patients with mtDNA depletion syndrome.

Metabolic and Mitochondrial Effects of Switching Antiretroviral-Experienced Patients to Enfuvirtide, Tenofovir and Saquinavir/Ritonavir

Objective Investigate the metabolic and mitochondrial effects of switching a highly active antiretroviral therapy (HAART) regimen with a high mitochondrial toxicity profile to a HAART with a theoretically low mitochondrial toxicity. Patients and Methods Six consecutive HAART-experienced patients receiving at least one dideoxy-nucleoside reverse transcriptase inhibitor (NRTI) switched to enfuvirtide plus tenofovir plus saquinavir/ritonavir (T20+TDF+SQV/r). Blood samples were collected at baseline, 12 and 24 weeks after the switch, and viral load (VL) and lymphocyte CD4+ T-cell count were determined. Metabolic parameters consisted of fasting serum triglycerides, cholesterol (total and fractions), glucose, insulin, C-peptide and lactate. Mitochondrial assessment consisted on mitochondrial DNA (mtDNA) quantification, COX-II mitochondrial protein expression rate, mitochondrial respiratory chain complex III and IV activities, and oxygen consumption in peripheral blood mononuclear cells. For baseline mitochondrial comparisons, we included six HIV-infected patients naive for ART. Results Switched patients exhibited a mean increase of 26 CD4+ T-cells/mm3 and a mean decrease of 1.1 log in VL ( P=NS for both). Lactate, lipids and glycaemia remained stable during the study; only insulin levels increased significantly ( P&lt;0.05). Switched patients exhibited, at baseline, low mitochondrial measurements, being significant only for complex III and IV activities with respect to naive patients ( P&lt;0.05 for both). MtDNA content did not rise significantly during the study. However, we observed increases in COX-II mitochondrial protein synthesis (124%, P&lt;0.05), complex III activity (127%, P&lt;0.05), complex IV activity (86%, P=0.37) and oxygen consumption (194%, P&lt;0.05). Conclusion Switching a HAART-containing dideoxy-NRTI to T20+TDF+SQV/r minimally alters metabolic parameters and exerts beneficial effects on mitochondrial function at 24 weeks. Mitochondrial improvement should be considered as an additional advantage of this rescue therapy.

Mitochondrial DNA Quantity Increases with Histopathologic Grade in Premalignant and Malignant Head and Neck Lesions

Mitochondria are highly susceptible to oxidative damage. Although mitochondrial function decreases with oxidative damage, overall mitochondrial DNA (mtDNA) content increases to compensate for general mitochondrial dysfunction. We performed quantitative polymerase chain reaction for genes specific to mitochondrial and nuclear genomes to investigate relative mitochondrial abundance in a spectrum of dysplastic head and neck lesions. DNA from mild, moderate, and severe dysplasias, as well as invasive tumors and normal mucosal cells, was extracted. Using quantitative polymerase chain reaction, mitochondrial to nuclear DNA ratios were determined by quantification of cytochrome c oxidase subunit 1 (CoxI) and beta-actin genes. Mean CoxI/beta-actin DNA ratios for mild, moderate, and severe premalignant lesions were 0.0529, 0.0607, and 0.1021, respectively. The mean ratio for the normal mucosal cells contained in saliva was 0.0537, whereas the mean ratio for tumors was 0.1667. As a whole, our experimental model demonstrated significance (P = 0.0358). Comparisons between individual categories showed borderline significance when compared with the normal group, with P values of 0.0673, 0.0747, and 0.0824 for moderate and severe dysplasia and invasive tumor, respectively. Head and neck squamous cell carcinomas arise through premalignant intermediates and may be merely morphologic manifestations of accumulated genetic alterations. In keeping with this molecular tumor progression model, our study shows that mtDNA increases according to histopathologic grade, a phenomenon that may be a feedback mechanism that compensates for a generalized decline in respiratory chain function. Therefore, high mtDNA content may be another marker of genetic alteration, a measure of relative DNA injury, and a surrogate measure of histopathologic grade.

A Tribute to the Xenopus laevis Oocyte and Egg

When I was asked to reflect as part of the celebration of the 100-year anniversary of the Journal of Biological Chemistry I recalled a talk by Seymour Cohen at the Federation meetings in Atlantic City about 1960. He began by thanking bacteriophage for providing him with so many wonderful research problems. I decided in the same spirit to pay homage to the Xenopus laevis egg and oocyte, two states of a cell that has played and continues to play a central role in most of the disciplines of modern biology including biochemistry and molecular biology. Many of us owe a debt of gratitude to this cell. The egg is the most important and interesting cell in the repertoire of any organism. In X. laevis this single cell has a diameter of about 1.3 mm. The oocyte is permeable to small molecules, but after it undergoes meiosis and becomes an unfertilized egg it is impermeable to these same molecules. The oocyte is an active site of RNA and protein synthesis but not DNA replication, whereas the unfertilized egg is poised to replicate every 10 min after it is fertilized. During the cleavage period the embryo is transcriptionally silent. An oocyte can be cultured for days, but once ovulated the egg degenerates rapidly if it is not fertilized. The oocyte is the largest single cell in the body yet it has many of the same structures as somatic cells. These compartments are so exaggerated in size that they are accessible to cell biologists for manipulation and visualization. The huge oocyte nucleus is called a germinal vesicle (GV). The premeiotic tetraploid chromosomes are expanded into a “lampbrush” configuration so that they can be studied with a light microscope. The X. laevis egg and its possibilities for experimental manipulation were introduced to modern biology in the late 1950s by John Gurdon (Fig. 1), then a graduate student in the laboratory of Michail Fischberg in the Department of Zoology at Oxford University. Gurdon’s thesis was to reproduce with the South African “clawed toad” X. laevis the famous nuclear transplantation experiments that Briggs and King had perfected with the American leopard frog, Rana pipiens (1). Embryonic nuclei were injected into eggs whose own nuclei had been removed or destroyed. These early cloning experiments addressed the question of whether a differentiated and specialized somatic cell nucleus was still capable of expressing its full genetic repertoire. Briggs and King found that nuclei from R. pipiens embryos older than gastrula never promoted normal development, suggesting that the genetic material might change in cells as they specialize during embryonic development. This result was challenged by John Gurdon’s demonstration that nuclei from X. laevis embryos are more permissive. Even a small fraction of nuclei derived from differentiated intestinal epithelial cells supported normal development (2). This crucial scientific question of whether irreversible changes occur in the genome of highly specialized cells was addressed more precisely by experimental manipulation than by biochemistry or genetics. The concepts behind these experiments have influenced the stem cell field. The topic of whether the genome is altered in specialized somatic cells was revisited just this year by the same strategy of nuclear transplantation. Postmitotic nuclei from olfactory neurons support the development of normal fertile mice when transplanted into enucleated mouse eggs (3). THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 44, Issue of October 29, pp. 45291–45299, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.