Deletion-mutant mtDNA increases in somatic tissues but decreases in female germ cells with age

The proportions of mutant and wild-type mtDNA are crucial in determining the severity of mitochondrial diseases. It has been generally considered that deletion-mutant mtDNA has replication advantages and accumulates with time. Here, we examine the tissue-by-tissue proportions of mutant mtDNA with a 4696-bp deletion (DeltamtDNA) and wild-type mtDNA in mitochondrial disease model mice (mito-mice). Comparison of the proportions of DeltamtDNA in each tissue at various ages showed that the rate of accumulation of DeltamtDNA differed among tissues. The heart, skeletal muscles, kidney, liver, testis, and ovary showed increases in the proportion of DeltamtDNA with age, but the pancreas, spleen, brain, and blood showed only a slight or no increase in proportion. In contrast to the somatic tissues, however, the germ cells of female mito-mice and resultant offspring showed a strong decrease in DeltamtDNA with maternal age. The decrease was so acute that some offspring showed complete disappearance of DeltamtDNA, even though their elder brothers and sisters had high proportions of DeltamtDNA. Female germ cells have a machinery that prevents the inheritance of defective mtDNA to the following generation since germ cells are kept for a long time until they are ovulated.     

Radiation induced inversions in human somatic cells

Peri- and paracentric inversions induced by various types of ionizing radiation (gamma and alpha-rays, neutron and neon beams) are analysed. Their frequencies significantly increase for radiation doses greater than or equal to .5 Gy. Their distribution does not seem to be at random. Pericentric are detected 3 to 4 times more frequently than paracentric inversions. Some identical inversions are recurrently induced. A proportion reproduces inversions detected in human cytogenetics laboratories and a larger proportion, chromosomes of other primate species. It seems that breakages, which numbers are roughly proportional to chromosome lengths, lead to reassociations with a limited number of combinations.     

Analysis of mitochondrial DNA species in interspecific hybrid somatic cells using restriction endonucleases. Identification of recombinant mtDNA molecules

Interspecific hybrid cells were isolated by fusion between thymidine kinase-deficient (TK⁻) mouse B82 cells and hypoxanthine-guanine-phosphoribosyl-transferase-deficient (HGPRT⁻) rat L6TG cells, and cultivating them in selective medium with hypoxanthine-aminopterin-thymidine (HAT). Karyo-type analysis revealed that they contained both mouse and rat chromosomes. Mitochondrial DNA (mtDNA) species of the hybrid cells were identified by digesting them with three kinds of restriction endonucleases, Hae II, EcoR I and Hpa II. Their restriction endonuclease cleavage patterns indicated that a portion of the mtDNAs was of mouse parent cell origin, while the remainings were recombinant molecules, i.e., part of the rat mtDNA sequence could be detected, but not whole rat mtDNA. The molecular weights of hybrid cell mtDNAs were calculated to be almost the same as that of the parent cells (˜10⁷ D).     

Spontaneous chromosome aberrations in human somatic cells

Conclusion In conclusion it is necessary to say that at present, we cannot consider whether there may be a geographical difference in frequency of spontaneous chromosome aberrations in somatic cells. For that purpose, a very abundant experimental material is required, as well as an improvement in methodology: what causes the difference in the results of various investigators; what methodical principles should be used for collection of data.Important factors in the differences of frequencies of spontaneous chromosome aberrations are likely to be the conditions of cultivation and making the preparations, as well as the methods of scoring the chromosome aberrations. The international standardisation of the cultivation conditions and of the estimates of chromosome aberrations is needed for the further study of the rate and reasons of the spontaneous mutation process in somatic cells.     

Improvement of combined FISH and immunofluorescence to trace the fate of somatic stem cells after transplantation.

Fluorescence in situ hybridization (FISH) combined with immunohistochemistry of tissue-specific markers provides a reliable method for characterizing the fate of somatic stem cells in transplantation experiments. Furthermore, the association between FISH and fluorescent gene reporter detection can unravel cell fusion phenomena, which could account for apparent transdifferentiation events. However, despite the widespread use of these techniques, they still require labor-extensive protocol adjustments to achieve correct and satisfactory simultaneous signal detection. In the present paper, we describe an improvement of simultaneous FISH and immunofluorescence detection. We applied this protocol to the identification of transplanted human and mouse hematopoietic stem cells in murine brain and muscle. This technique provides unique opportunities for following the path taken by transplanted cells and their differentiation into mature cell types.     

Genetic knockouts and knockins in human somatic cells

Gene targeting by homologous recombination with exogenous DNA constructs is the most powerful technique available for analysis of mammalian gene function. Over the past several years, the methods used to generate knockout and knockin mice have been modified for use in cultured human cells. The most significant innovation has been the adaptation of recombinant adeno-associated viruses (rAAVs) for such targeting. The stages of rAAV-mediated gene targeting include (i) the design and construction of a DNA targeting vector, (ii) the production of an infectious rAAV stock, (iii) the generation of cell clones that harbor rAAV transgenes, (iv) screening for homologous recombinants and (v) the iterative targeting of multiple alleles. The protocol described herein allows the generation of a cell line with a single altered allele in 3 months. A second allele of the same gene can be targeted in an additional 3 months.     

Epstein-Barr virus in somatic cell hybrids between mouse cells and human nasopharyngeal carcinoma cells.

Somatic cell hybrids between mouse cells and cells derived directly from NPC biopsies were produced in order to study the association of the Epstein-Barr virus (EBV) genome and the expression of Epstein-Barr nuclear antigen (EBNA) with the human chromosome(s). All attempts to correlate the presence of EBV-DNA and the expression of EBNA with the presence of a particular human chromosome(s) showed that the segregation of EBV-DNA or of EBNA and human chromosomes was dysconcordant. The data, therefore, suggest that in the hybrids studied the presence of EBA-DNA is not determined by the presence of a specific human chromosome.     

The relationship between transformation and somatic mutation in human and Chinese hamster cells

The frequencies of transformations of primary human and Chinese hamster fibroblasts have been compared with the spontaneous and induced frequencies of mutation for resistance to thioguanine and ouabain, and for ability to use fructose, using the carcinogens benzo (alpha) pyrene and urethane. Whereas the rates and frequencies of mutation were similar in the two cell systems, transformations to morphologically altered cells was observed only in hamster cells. The frequency of this latter transformation event in hamster cells was abour 10(3) greater than the frequencies of mutation in these cells. The morphologically altered cells formed in the above transformation process cannot grow in agar (aga-) and do not produce tumors when injected into animals. The frequency of transition of these latter cells to aga+ cells which produce tumors in animals is similar to the mutation-like events.     

Compilation of human mtDNA control region sequences.

This paper describes the organisation of a database for human mitochondrial control-region sequences. The data are divided into three ASCII files that contain aligned sequences from the hypervariable region I (HVRI), from the hypervariable region II (HVRII), and the available information about the individuals, from whom the sequences stem. The current collection comprises 4079 HVRI and 969 HVRII sequences. From 728 individuals sequences of both HVRI and HVRII are available. For easy access, the collection is made available to the scientific community via World Wide Web at URL http://www.zi.biologie.uni-muenchen.de/[symbol: see text]meyers/mtdna.html     

The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells

The Piwi-piRNA pathway is active in animal germ cells where its functions are required for germ cell maintenance and gamete differentiation. Piwi proteins and piRNAs have been detected outside germline tissue in multiple phyla, but activity of the pathway in mammalian somatic cells has been little explored. In particular, Piwi expression has been observed in cancer cells, but nothing is known about the piRNA partners or the function of the system in these cells. We have surveyed the expression of the three human Piwi genes, Hiwi, Hili and Hiwi2, in multiple normal tissues and cancer cell lines. We find that Hiwi2 is ubiquitously expressed; in cancer cells the protein is largely restricted to the cytoplasm and is associated with translating ribosomes. Immunoprecipitation of Hiwi2 from MDAMB231 cancer cells enriches for piRNAs that are predominantly derived from processed tRNAs and expressed genes, species which can also be found in adult human testis. Our studies indicate that a Piwi-piRNA pathway is present in human somatic cells, with an uncharacterised function linked to translation. Taking this evidence together with evidence from primitive organisms, we propose that this somatic function of the pathway predates the germline functions of the pathway in modern animals.     

Mechanics regulates fate decisions of human embryonic stem cells

Research on human embryonic stem cells (hESCs) has attracted much attention given their great potential for tissue regenerative therapy and fundamental developmental biology studies. Yet, there is still limited understanding of how mechanical signals in the local cellular microenvironment of hESCs regulate their fate decisions. Here, we applied a microfabricated micromechanical platform to investigate the mechanoresponsive behaviors of hESCs. We demonstrated that hESCs are mechanosensitive, and they could increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates supported maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility might be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. Our results highlighted the important functional link between matrix rigidity, cellular mechanics, and pluripotency of hESCs and provided a novel approach to characterize and understand mechanotransduction and its involvement in hESC function.     

Nuclear complementation restores mtDNA levels in cultured cells from a patient with mtDNA depletion.

We have studied cultured skin fibroblasts from a patient with a fatal mitochondrial disease manifesting soon after birth. These fibroblasts were found to grow only in the presence of pyruvate and uridine, a characteristic of cells lacking mtDNA (rho0 cells). Southern blot and PCR analyses confirmed that the patient's fibroblasts contained less than 2% of control levels of mtDNA. Biochemical analyses indicated that the activities of all the respiratory-chain enzymes were severely decreased in mitochondria isolated from these fibroblasts. In order to elucidate the underlying molecular defect, cell fusions were performed between enucleated fibroblasts from this patient and a human-derived rho0 cell line (rho0 A549.B2). The resulting cybrids were plated in medium lacking pyruvate and uridine, to select for the restoration of respiratory-chain function. Complementation was observed between the nuclear genome of the rho0 A549.B2 cells and the mtDNA of the patient's cells, restoring mtDNA levels and respiratory-chain function in the cybrid cells. These results indicate that mtDNA depletion in our patient is under the control of the nuclear genome.     

Stability of X chromosomal inactivation in human somatic cells transformed by SV-40.

Clones of fibroblasts from a G6PD A heterozygote transformed with SV-40 did not express the G6PD silent allele in the transformed heteroploid cultures. In addition, transformed fibroblasts from a woman heterozygous for both G6PD A and HGPRT deficiency, subjected to selective pressure, did not reveal a single cell expressing either silent allele. Since the incidence of sex chromatin was significantly lower in these cells after transformation, it is likely that the loss of sex chromatin reflects the loss of the inactive X-chromosome at an early stage following transformation.     

The mutation rate in the human mtDNA control region

The mutation rate of the mitochondrial control region has been widely used to calibrate human population history. However, estimates of the mutation rate in this region have spanned two orders of magnitude. To readdress this rate, we sequenced the mtDNA control region in 272 individuals, who were related by a total of 705 mtDNA transmission events, from 26 large Icelandic pedigrees. Three base substitutions were observed, and the mutation rate across the two hypervariable regions was estimated to be 3/705 =.0043 per generation (95% confidence interval [CI].00088-.013), or.32/site/1 million years (95% CI.065-.97). This study is substantially larger than others published, which have directly assessed mtDNA mutation rates on the basis of pedigrees, and the estimated mutation rate is intermediate among those derived from pedigree-based studies. Our estimated rate remains higher than those based on phylogenetic comparisons. We discuss possible reasons for-and consequences of-this discrepancy. The present study also provides information on rates of insertion/deletion mutations, rates of heteroplasmy, and the reliability of maternal links in the Icelandic genealogy database.     

The fate of desmosomal proteins in apoptotic cells

Activation of caspases results in the disruption of structural and signaling networks in apoptotic cells. Recent biochemical and cell biological studies have shown that components of the cadherin-catenin adhesion complex in epithelial adherens junctions are targeted by caspases during apoptosis. In epithelial cells, desmosomes represent a second type of anchoring junctions mediating strong cell-cell contacts. Using antibodies directed against a set of desmosomal proteins, we show that desmosomes are proteolytically targeted during apoptosis. Desmogleins and desmocollins, representing desmosome-specific members of the cadherin superfamily of cell adhesion molecules, are specifically cleaved after onset of apoptosis. Similar to E-cadherin, the desmoglein-3 cytoplasmic tail is cleaved by caspases. In addition the extracellular domains of desmoglein-3 and desmocollin-3 are released from the cell surface by a metalloproteinase activity. In the presence of caspase and/or metalloproteinase inhibitors, both cleavage reactions are almost completely inhibited. As reported previously, the desmosomal plaque protein plakoglobin is cleaved by caspase-3 during apoptosis. Our studies now show that plakophilin-1 and two other major plaque proteins, desmoplakin-1 and -2, are also cleaved by caspases. Immunofluorescence analysis confirmed that this cleavage results in the disruption of the desmosome structure and thus contributes to cell rounding and disintegration of the intermediate filament system.