Gene conversion results in the equalization of genome copies in the polyploid haloarchaeon Haloferax volcanii

Haloferax volcanii is highly polyploid and contains about 20 copies of the major chromosome. A heterozygous strain was constructed that contained two different types of genomes: the leuB locus contained either the wild-type leuB gene or a leuB:trpA gene introduced by gene replacement. As the trpA locus is devoid of the wild-type trpA gene, growth in the absence of both amino acids is only possible when both types of genomes are simultaneously present, exemplifying gene redundancy and the potential to form heterozygous cells as one possible evolutionary advantage of polyploidy. The heterozygous strain was grown (i) in the presence of tryptophan, selecting for the presence of leuB, (ii) in the presence of leucine selecting for leuB:trpA and (iii) in the absence of selection. Both types of genomes were quantified with real-time PCR. The first condition led to a complete loss of leuB:trpA-containing genomes, while under the second condition leuB-containing genomes were lost. Also in the absence of selection gene conversion led to a fast equalization of genomes and resulted in homozygous leuB-containing cells. Gene conversion leading to genome equalization can explain the escape from 'Muller's ratchet' as well as the ease of mutant construction using polyploid haloarchaea.     

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.     

Long-Term Quiescent Fibroblast Cells Transit into Senescence

Cellular senescence is described to be a consequence of telomere erosion during the replicative life span of primary human cells. Quiescence should therefore not contribute to cellular aging but rather extend lifespan. Here we tested this hypothesis and demonstrate that cultured long-term quiescent human fibroblasts transit into senescence due to similar cellular mechanisms with similar dynamics and with a similar maximum life span as proliferating controls, even under physiological oxygen conditions. Both, long-term quiescent and senescent fibroblasts almost completely fail to undergo apoptosis. The transition of long-term quiescent fibroblasts into senescence is also independent of HES1 which protects short-term quiescent cells from becoming senescent. Most significantly, DNA damage accumulates during senescence as well as during long-term quiescence at physiological oxygen levels. We suggest that telomere-independent, potentially maintenance driven gradual induction of cellular senescence during quiescence is a counterbalance to tumor development.     

Quantitative model of cell cycle arrest and cellular senescence in primary human fibroblasts

Primary human fibroblasts in tissue culture undergo a limited number of cell divisions before entering a non-replicative "senescent" state. At early population doublings (PD), fibroblasts are proliferation-competent displaying exponential growth. During further cell passaging, an increasing number of cells become cell cycle arrested and finally senescent. This transition from proliferating to senescent cells is driven by a number of endogenous and exogenous stress factors. Here, we have developed a new quantitative model for the stepwise transition from proliferating human fibroblasts (P) via reversibly cell cycle arrested (C) to irreversibly arrested senescent cells (S). In this model, the transition from P to C and to S is driven by a stress function γ and a cellular stress response function F which describes the time-delayed cellular response to experimentally induced irradiation stress. The application of this model based on senescence marker quantification at the single-cell level allowed to discriminate between the cellular states P, C, and S and delivers the transition rates between the P, C and S states for different human fibroblast cell types. Model-derived quantification unexpectedly revealed significant differences in the stress response of different fibroblast cell lines. Evaluating marker specificity, we found that SA-β-Gal is a good quantitative marker for cellular senescence in WI-38 and BJ cells, however much less so in MRC-5 cells. Furthermore we found that WI-38 cells are more sensitive to stress than BJ and MRC-5 cells. Thus, the explicit separation of stress induction from the cellular stress response, and the differentiation between three cellular states P, C and S allows for the first time to quantitatively assess the response of primary human fibroblasts towards endogenous and exogenous stress during cellular ageing.