Phylogenetic and familial estimates of mitochondrial substitution rates: study of control region mutations in deep-rooting pedigrees

We studied mutations in the mtDNA control region (CR) using deep-rooting French-Canadian pedigrees. In 508 maternal transmissions, we observed four substitutions (0.0079 per generation per 673 bp, 95% CI 0.0023-0.186). Combined with other familial studies, our results add up to 18 substitutions in 1,729 transmissions (0.0104), confirming earlier findings of much greater mutation rates in families than those based on phylogenetic comparisons. Only 12 of these mutations occurred at independent sites, whereas three positions mutated twice each, suggesting that pedigree studies preferentially reveal a fraction of highly mutable sites. Fitting the data through use of a nonuniform rate model predicts the presence of 40 (95% CI 27-54) such fast sites in the whole CR, characterized by the mutation rate of 274 per site per million generations (95% CI 138-410). The corresponding values for hypervariable regions I (HVI; 1,729 transmissions) and II (HVII; 1,956 transmissions), are 19 and 22 fast sites, with rates of 224 and 274, respectively. Because of the high probability of recurrent mutations, such sites are expected to be of no or little informativity for the evaluation of mutational distances at the phylogenetic time scale. The analysis of substitution density in the alignment of 973 HVI and 650 HVII unrelated European sequences reveals that the bulk of the sites mutate at relatively moderate and slow rates. Assuming a star-like phylogeny and an average time depth of 250 generations, we estimate the rates for HVI and HVII at 23 and 24 for the moderate sites and 1.3 and 1.0 for the slow sites. The fast, moderate, and slow sites, at the ratio of 1:2:13, respectively, describe the mutation-rate heterogeneity in the CR. Our results reconcile the controversial rate estimates in the phylogenetic and familial studies; the fast sites prevail in the latter, whereas the slow and moderate sites dominate the phylogenetic-rate estimations.     

The DNA damage checkpoint pathways exert multiple controls on the efficiency and outcome of the repair of a double-stranded DNA gap

A DNA gap repair assay was used to determine the effect of mutations in the DNA damage checkpoint system on the efficiency and outcome (crossover/non-crossover) of recombinational DNA repair. In Saccharomyces cerevisiae gap repair is largely achieved by homologous recombination. As a result the plasmid either integrates into the chromosome (indicative of a crossover outcome) or remains extrachromosomal (indicative of a non-crossover outcome). Deletion mutants of the MEC1 and RAD53 checkpoint kinase genes exhibited a 5-fold decrease in gap repair efficiency, showing that 80% of the gap repair events depended on functional DNA damage checkpoints. Epistasis analysis suggests that the DNA damage checkpoints affect gap repair by modulating Rad51 protein-mediated homologous recombination. While in wild-type cells only ~25% of the gap repair events were associated with a crossover outcome, Mec1-deficient cells exhibited a >80% crossover association. Also mutations in the effector kinases Rad53, Chk1 and Dun1 were found to affect crossover association of DNA gap repair to various degrees. The data suggest that the DNA damage checkpoints are important for the optimal functioning of recombinational DNA repair with multiple terminal targets to modulate the efficiency and outcome of homologous recombination.     

Recombination: Holliday junction resolution and crossover formation

The heterodimeric nuclease Mus81-Eme1 has been proposed to be a Holliday junction resolvase and has now been found to be responsible for nearly all meiotic crossovers in fission yeast. The intriguing substrate preference of this enzyme for nicked Holliday junctions opens the possibility that crossover formation may not always involve double Holliday junctions.     

Holliday junctions in the eukaryotic nucleus: resolution in sight?

The Holliday junction is a key recombination intermediate whose resolution generates crossovers. Interplay between recombination, repair and replication has moved the Holliday junction to the center stage of nuclear DNA metabolism. Holliday junction resolvases in the eukaryotic nucleus have long eluded identification. The endonucleases Mus81/Mms4-Eme1 and XPF-MEI-9/MUS312 are structurally related to the archaeal resolvase Hjc and were found to be involved in crossover formation in budding yeast and flies, respectively. Although these endonucleases might represent one class of eukaryotic resolvases, their substrate preference opens up the possibility that junctions other than classical Holliday junctions might contribute to crossovers. Holliday junction resolution to non-crossover products can also be achieved topologically, for example, by the action of RecQ-like DNA helicases combined with topoisomerase III.     

Metabolic pathway promiscuity in the archaeon Sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase

The hyperthermophilic Archaeon Sulfolobus solfataricus metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate (KDG) aldolase then catalyzes the cleavage of 2-keto-3-deoxygluconate to glyceraldehyde and pyruvate. The gene encoding glucose dehydrogenase has been cloned and expressed in Escherichia coli to give a fully active enzyme, with properties indistinguishable from the enzyme purified from S. solfataricus cells. Kinetic analysis revealed the enzyme to have a high catalytic efficiency for both glucose and galactose. KDG aldolase from S. solfataricus has previously been cloned and expressed in E. coli. In the current work its stereoselectivity was investigated by aldol condensation reactions between D-glyceraldehyde and pyruvate; this revealed the enzyme to have an unexpected lack of facial selectivity, yielding approximately equal quantities of 2-keto-3-deoxygluconate and 2-keto-3-deoxygalactonate. The KDG aldolase-catalyzed cleavage reaction was also investigated, and a comparable catalytic efficiency was observed with both compounds. Our evidence suggests that the same enzymes are responsible for the catabolism of both glucose and galactose in this Archaeon. The physiological and evolutionary implications of this observation are discussed in terms of catalytic and metabolic promiscuity.     

Ventilatory support: a dynamical systems approach

Misunderstanding of the dynamical behavior of the ventilatory system, especially under assisted ventilation, may explain the problems encountered in ventilatory support monitoring. Proportional assist ventilation (PAV) that theoretically gives a breath by breath assistance presents instability with high levels of assistance. We have constructed a mathematical model of interactions between three objects: the central respiratory pattern generator modelled by a modified Van der Pol oscillator, the mechanical respiratory system which is the passive part of the system and a controlled ventilator that follows its own law. The dynamical study of our model shows the existence of two crucial behaviors, i.e. oscillations and damping, depending on only two parameters, namely the time constant of the mechanical respiratory system and a cumulative interaction index. The same result is observed in simulations of spontaneous breathing as well as of PAV. In this last case, increasing assistance leads first to an increase of the tidal volume (V_T), a further increase in assistance inducing a decrease in V_T, ending in damping of the whole system to an attractive fixed point. We conclude that instabilities observed in PAV may be explained by the different possible dynamical behaviors of the system rather than changes in mechanical characteristics of the respiratory system.     

Y-chromosome analysis in Egypt suggests a genetic regional continuity in Northeastern Africa

The geographic location of Egypt, at the interface between North Africa, the Middle East, and southern Europe, prompted us to investigate the genetic diversity of this population and its relationship with neighboring populations. To assess the extent to which the modern Egyptian population reflects this intermediate geographic position, ten Unique Event Polymorphisms (UEPs), mapping to the nonrecombining portion of the Y chromosome, have been typed in 164 Y chromosomes from three North African populations. The analysis of these binary markers, which define 11 Y-chromosome lineages, were used to determine the haplogroup frequencies in Egyptians, Moroccan Arabs, and Moroccan Berbers and thereby define the Y-chromosome background in these regions. Pairwise comparisons with a set of 15 different populations from neighboring European, North African, and Middle Eastern populations and geographic analysis showed the absence of any significant genetic barrier in the eastern part of the Mediterranean area, suggesting that genetic variation and gene flow in this area follow the "isolation-by-distance" model. These results are in sharp contrast with the observation of a strong north-south genetic barrier in the western Mediterranean basin, defined by the Gibraltar Strait. Thus, the Y-chromosome gene pool in the modern Egyptian population reflects a mixture of European, Middle Eastern, and African characteristics, highlighting the importance of ancient and recent migration waves, followed by gene flow, in the region.     

Isolation and functional analysis of a Brassica juncea gene encoding a com-ponent of auxin efflux carrier

Polar auxin transport plays a divergent role in plant growth and developmental processes including root and embryo development, vascular pattern formation and cell elongation. Recently isolated Arabidopsis pin gene family was believed to encode a component of auxin efflux carrier (G(?)lweiler et al, 1998). Based on the Arabidopsis pin1 sequence we have isolated a Brassica juncea cDNA (designated Bjpin1), which encoded a 70-kDa putative auxin efflux carrier. Deduced BjPIN1 shared 65% identities at protein level with AtPINl and was highly homologous to other putative PIN proteins of Arabidopsis (with highest homology to AtPIN3). Hydrophobic analysis showed similar structures between BjPINl and AtPIN proteins. Presence of 6 exons (varying in size between 65 bp and 1229 bp) and 5 introns (sizes between 89 bp and 463 bp) in the genomic fragment was revealed by comparing the genomic and cDNA sequences. Northern blot analysis indicated that Bjpin1 was expressed in most of the tissues tested, with a relatively h     

No mutagenic or recombinogenic effects of mobile phone fields at 900 MHz detected in the yeast Saccharomyces cerevisiae

Both actively growing and resting cells of the yeast Saccharomyces cerevisiae were exposed to 900-MHz fields that closely matched the Global System for Mobile Communication (GSM) pulsed modulation format signals for mobile phones at specific absorption rates (SAR) of 0.13 and 1.3 W/kg. Two identical anechoic test chambers were constructed to perform concurrent control and test experiments under well-controlled exposure conditions. Using specific test strains, we examined the genotoxic potential of mobile phone fields, alone and in combination, with a known genotoxic compound, the alkylating agent methyl methansulfonate. Mutation rates were monitored by two test systems, a widely used gene-specific forward mutation assay at CAN1 and a wide-range assay measuring the induction of respiration-deficient (petite) clones that have lost their mitochondrial function. In addition, two further assays measured the recombinogenic effect of mobile phone fields to detect possible effects on genomic stability: First, an intrachromosomal, deletion-formation assay previously developed for genotoxic screening; and second, an intragenic recombination assay in the ADE2 gene. Fluctuation tests failed to detect any significant effect of mobile phone fields on forward mutation rates at CAN1, on the frequency of petite formation, on rates of intrachromosomal deletion formation, or on rates of intragenic recombination in the absence or presence of the genotoxic agent methyl methansulfonate.