The high mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates in vivo

Abf2p is a high mobility group (HMG) protein found in yeast mitochondria that is required for the maintenance of wild-type (ρ+) mtDNA in cells grown on fermentable carbon sources, and for efficient recombination of mtDNA markers in crosses. Here, we show by two-dimensional gel electrophoresis that Abf2p promotes or stabilizes Holliday recombination junction intermediates in ρ+ mtDNA in vivo but does not influence the high levels of recombination intermediates readily detected in the mtDNA of petite mutants (ρ−). mtDNA recombination junctions are not observed in ρ+ mtDNA of wild-type cells but are elevated to detectable levels in cells with a null allele of the MGT1 gene (Δmgt1), which codes for a mitochondrial cruciform-cutting endonuclease. The level of recombination intermediates in ρ+ mtDNA of Δmgt1 cells is decreased about 10-fold if those cells contain a null allele of the ABF2 gene. Overproduction of Abf2p by ≥ 10-fold in wild-type ρ+ cells, which leads to mtDNA instability, results in a dramatic increase in mtDNA recombination intermediates. Specific mutations in the two Abf2p HMG boxes required for DNA binding diminishes these responses. We conclude that Abf2p functions in the recombination of ρ+ mtDNA.

Crossover isomer bias is the primary sequence-dependent property of immobilized Holliday junctions

Recombination of genes is essential to the evolution of genetic diversity, the segregation of chromosomes during cell division, and certain DNA repair processes. The Holliday junction, a four-arm, four-strand branched DNA crossover structure, is formed as a transient intermediate during genetic recombination and repair processes in the cell. The recognition and subsequent resolution of Holliday junctions into parental or recombined products appear to be critically dependent on their three-dimensional structure. Complementary NMR and time-resolved fluorescence resonance energy transfer experiments on immobilized four-arm DNA junctions reported here indicate that the Holliday junction cannot be viewed as a static structure but rather as an equilibrium mixture of two conformational isomers. Furthermore, the distribution between the two possible crossover isomers was found to depend on the sequence in a manner that was not anticipated on the basis of previous low-resolution experiments.

Expanding the functional human mitochondrial DNA database by the establishment of primate xenomitochondrial cybrids

The nuclear and mitochondrial genomes coevolve to optimize approximately 100 different interactions necessary for an efficient ATP-generating system. This coevolution led to a species-specific compatibility between these genomes. We introduced mitochondrial DNA (mtDNA) from different primates into mtDNA-less human cells and selected for growth of cells with a functional oxidative phosphorylation system. mtDNA from common chimpanzee, pigmy chimpanzee, and gorilla were able to restore oxidative phosphorylation in the context of a human nuclear background, whereas mtDNA from orangutan, and species representative of Old-World monkeys, New-World monkeys, and lemurs were not. Oxygen consumption, a sensitive index of respiratory function, showed that mtDNA from chimpanzee, pigmy chimpanzee, and gorilla replaced the human mtDNA and restored respiration to essentially normal levels. Mitochondrial protein synthesis was also unaltered in successful “xenomitochondrial cybrids.” The abrupt failure of mtDNA from primate species that diverged from humans as recently as 8–18 million years ago to functionally replace human mtDNA suggests the presence of one or a few mutations affecting critical nuclear–mitochondrial genome interactions between these species. These cellular systems provide a demonstration of intergenus mtDNA transfer, expand more than 20-fold the number of mtDNA polymorphisms that can be analyzed in a human nuclear background, and provide a novel model for the study of nuclear–mitochondrial interactions.

Genetic instability of cancer cells is proportional to their degree of aneuploidy

Genetic and phenotypic instability are hallmarks of cancer cells, but their cause is not clear. The leading hypothesis suggests that a poorly defined gene mutation generates genetic instability and that some of many subsequent mutations then cause cancer. Here we investigate the hypothesis that genetic instability of cancer cells is caused by aneuploidy, an abnormal balance of chromosomes. Because symmetrical segregation of chromosomes depends on exactly two copies of mitosis genes, aneuploidy involving chromosomes with mitosis genes will destabilize the karyotype. The hypothesis predicts that the degree of genetic instability should be proportional to the degree of aneuploidy. Thus it should be difficult, if not impossible, to maintain the particular karyotype of a highly aneuploid cancer cell on clonal propagation. This prediction was confirmed with clonal cultures of chemically transformed, aneuploid Chinese hamster embryo cells. It was found that the higher the ploidy factor of a clone, the more unstable was its karyotype. The ploidy factor is the quotient of the modal chromosome number divided by the normal number of the species. Transformed Chinese hamster embryo cells with a ploidy factor of 1.7 were estimated to change their karyotype at a rate of about 3% per generation, compared with 1.8% for cells with a ploidy factor of 0.95. Because the background noise of karyotyping is relatively high, the cells with low ploidy factor may be more stable than our method suggests. The karyotype instability of human colon cancer cell lines, recently analyzed by Lengnauer et al. [Lengnauer, C., Kinzler, K. W. & Vogelstein, B. (1997) Nature (London) 386, 623–627], also corresponds exactly to their degree of aneuploidy. We conclude that aneuploidy is sufficient to explain genetic instability and the resulting karyotypic and phenotypic heterogeneity of cancer cells, independent of gene mutation. Because aneuploidy has also been proposed to cause cancer, our hypothesis offers a common, unique mechanism of altering and simultaneously destabilizing normal cellular phenotypes.

Plasmon analyses of Triticum (wheat) and Aegilops: PCR–single-strand conformational polymorphism (PCR-SSCP) analyses of organellar DNAs

To investigate phylogenetic relationships among plasmons in Triticum and Aegilops, PCR–single-strand conformational polymorphism (PCR-SSCP) analyses were made of 14.0-kb chloroplast (ct) and 13.7-kb mitochondrial (mt)DNA regions that were isolated from 46 alloplasmic wheat lines and one euplasmic line. These plasmons represent 31 species of the two genera. The ct and mtDNA regions included 10 and 9 structural genes, respectively. A total of 177 bands were detected, of which 40.6% were variable. The proportion of variable bands in ctDNA (51.1%) was higher than that of mtDNA (28.9%). The phylogenetic trees of plasmons, derived by two different models, indicate a common picture of plasmon divergence in the two genera and suggest three major groups of plasmons (Einkorn, Triticum, and Aegilops). Because of uniparental plasmon transmission, the maternal parents of all but one polyploid species were identified. Only one Aegilops species, Ae. speltoides, was included in the Triticum group, suggesting that this species is the plasmon and B and G genome donor of all polyploid wheats. ctDNA variations were more intimately correlated with vegetative characters, whereas mtDNA variations were more closely correlated with reproductive characters. Plasmon divergence among the diploids of the two genera largely paralleled genome divergence. The relative times of origin of the polyploid species were inferred from genetic distances from their putative maternal parents.

Mechanisms of Human Mitochondrial DNA Maintenance: The Determining Role of Primary Sequence and Length over Function

Although the regulation of mitochondrial DNA (mtDNA) copy number is performed by nuclear-coded factors, very little is known about the mechanisms controlling this process. We attempted to introduce nonhuman ape mtDNA into human cells harboring either no mtDNA or mutated mtDNAs (partial deletion and tRNA gene point mutation). Unexpectedly, only cells containing no mtDNA could be repopulated with nonhuman ape mtDNA. Cells containing a defective human mtDNA did not incorporate or maintain ape mtDNA and therefore died under selection for oxidative phosphorylation function. On the other hand, foreign human mtDNA was readily incorporated and maintained in these cells. The suicidal preference for self-mtDNA showed that functional parameters associated with oxidative phosphorylation are less relevant to mtDNA maintenance and copy number control than recognition of mtDNA self-determinants. Non–self-mtDNA could not be maintained into cells with mtDNA even if no selection for oxidative phosphorylation was applied. The repopulation kinetics of several mtDNA forms after severe depletion by ethidium bromide treatment showed that replication and maintenance of mtDNA in human cells are highly dependent on molecular features, because partially deleted mtDNA molecules repopulated cells significantly faster than full-length mtDNA. Taken together, our results suggest that mtDNA copy number may be controlled by competition for limiting levels of trans-acting factors that recognize primarily mtDNA molecular features. In agreement with this hypothesis, marked variations in mtDNA levels did not affect the transcription of nuclear-coded factors involved in mtDNA replication.

LC2, the Chlamydomonas Homologue of the t Complex-encoded Protein Tctex2, Is Essential for Outer Dynein Arm Assembly

Tctex2 is thought to be one of the distorter genes of the mouse t haplotype. This complex greatly biases the segregation of the chromosome that carries it such that in heterozygous +/t males, the t haplotype is transmitted to >95% of the offspring, a phenomenon known as transmission ratio distortion. The LC2 outer dynein arm light chain of Chlamydomonas reinhardtii is a homologue of the mouse protein Tctex2. We have identified Chlamydomonas insertional mutants with deletions in the gene encoding LC2 and demonstrate that the LC2 gene is the same as the ODA12 gene, the product of which had not been identified previously. Complete deletion of the LC2/ODA12 gene causes loss of all outer arms and a slow jerky swimming phenotype. Transformation of the deletion mutant with the cloned LC2/ODA12 gene restores the outer arms and rescues the motility phenotype. Therefore, LC2 is required for outer arm assembly. The fact that LC2 is an essential subunit of flagellar outer dynein arms allows us to propose a detailed mechanism whereby transmission ratio distortion is explained by the differential binding of mutant (t haplotype encoded) and wild-type dyneins to the axonemal microtubules of t-bearing or wild-type sperm, with resulting differences in their motility.

Polymer Models of Meiotic and Mitotic Chromosomes

Polymers tied together by constraints exhibit an internal pressure; this idea is used to analyze physical properties of the bottle-brush–like chromosomes of meiotic prophase that consist of polymer-like flexible chromatin loops, attached to a central axis. Using a minimal number of experimental parameters, semiquantitative predictions are made for the bending rigidity, radius, and axial tension of such brushes, and the repulsion acting between brushes whose bristles are forced to overlap. The retraction of lampbrush loops when the nascent transcripts are stripped away, the oval shape of diplotene bivalents between chiasmata, and the rigidity of pachytene chromosomes are all manifestations of chromatin pressure. This two-phase (chromatin plus buffer) picture that suffices for meiotic chromosomes has to be supplemented by a third constituent, a chromatin glue to understand mitotic chromosomes, and explain how condensation can drive the resolution of entanglements. This process resembles a thermal annealing in that a parameter (the affinity of the glue for chromatin and/or the affinity of the chromatin for buffer) has to be tuned to achieve optimal results. Mechanical measurements to characterize this protein–chromatin matrix are proposed. Finally, the propensity for even slightly chemically dissimilar polymers to phase separate (cluster like with like) can explain the apparent segregation of the chromatin into A+T- and G+C-rich regions revealed by chromosome banding.

Multiple independent transpositions of mitochondrial DNA control region sequences to the nucleus

Transpositions of mtDNA sequences to the nuclear genome have been documented in a wide variety of individual taxa, but little is known about their taxonomic frequency or patterns of variation. We provide evidence of nuclear sequences homologous to the mtDNA control region in seven species of diving ducks (tribe Aythyini). Phylogenetic analysis places each nuclear sequence as a close relative of the mtDNA haplotypes of the specie(s) in which it occurs, indicating that they derive from six independent transposition events, all occurring within the last ≈1.5 million years. Relative-rate tests and comparison of intraspecific variation in nuclear and mtDNA sequences confirm the expectation of a greatly reduced rate of evolution in the nuclear copies. By representing mtDNA haplotypes from ancestral populations, nuclear insertions may be valuable in some phylogenetic analyses, but they also confound the accurate determination of mtDNA sequences. In particular, our data suggest that the presumably nonfunctional but more slowly evolving nuclear sequences often will not be identifiable by changes incompatible with function and may be preferentially amplified by PCR primers based on mtDNA sequences from related taxa.

Functional integrity of mitochondrial genomes in human platelets and autopsied brain tissues from elderly patients with Alzheimer’s disease

To determine whether pathogenic mutations in mtDNA are involved in phenotypic expression of Alzheimer’s disease (AD), the transfer of mtDNA from elderly patients with AD into mtDNA-less (ρ0) HeLa cells was carried out by fusion of platelets or synaptosomal fractions of autopsied brain tissues with ρ0 HeLa cells. The results showed that mtDNA in postmortem brain tissue survives for a long time without degradation and could be rescued in ρ0 HeLa cells. Next, the cybrid clones repopulated with exogenously imported mtDNA from patients with AD were used for examination of respiratory enzyme activity and transfer of mtDNA with the pathogenic mutations that induce mitochondrial dysfunction. The presence of the mutated mtDNA was restricted to brain tissues and their cybrid clones that formed with synaptosomes as mtDNA donors, whereas no cybrid clones that isolated with platelets as mtDNA donors had detectable mutated mtDNA. However, biochemical analyses showed that all cybrid clones with mtDNA imported from platelets or brain tissues of patients with AD restored mitochondrial respiration activity to almost the same levels as those of cybrid clones with mtDNA from age-matched normal controls, suggesting functional integrity of mtDNA in both platelets and brain tissues of elderly patients with AD. These observations warrant the reassessment of the conventional concept that the accumulation of pathogenic mutations in mtDNA throughout the aging process is responsible for the decrease of mitochondrial respiration capacity with age and with the development of age-associated neurodegenerative diseases.

Apical sorting of a voltage- and Ca2+-activated K+ channel α-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation

The voltage- and Ca2+-activated K+ (KV,Ca) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel KV,Ca α-subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., KV,Ca β-subunits) were not required. hSlo seems to display properties for lipid-raft targeting, as judged by its buoyant distribution in sucrose gradients after extraction with either detergent or sodium carbonate. The evidence indicates that the hSlo protein possesses intrinsic information for transport to the apical cell surface through a mechanism that may involve association with lipid rafts and that is independent of glycosylation of the channel itself or an associated protein. Thus, this particular polytopic model protein shows that glycosylation-independent apical pathways exist for endogenous membrane proteins in Madin-Darby canine kidney cells.

Synthesis of mitochondrial DNA in permeabilised human cultured cells

The mechanisms that underlie the maintenance of and increase in mutant mitochondrial DNA (mtDNA) are central to our understanding of mitochondrial disease. We have therefore developed a technique based on saponin permeabilisation that allows the study of mtDNA synthesis in intact cells. Permeabilisation of cells has been extensively used in an established method both for studying transcription and DNA replication in the nucleus and for measuring respiratory chain activities in mitochondria. We have quantitatively studied incorporation of radiolabelled DNA precursors into mtDNA in human cell lines derived from controls and from patients with mitochondrial DNA disease. Total cell DNA is extracted, restriction digested and Southern blotted, newly synthesised mtDNA being proportional to the label incorporated in each restriction band. A rate of synthesis can then be derived by estimating the relative steady-state mtDNA after probing with full-length mtDNA. Where co-existing mutant and wild-type mtDNA (heteroplasmy) can be distinguished using restriction digestion, their rates of synthesis can be compared within a single cell line. This will be particularly useful in elucidating the pathophysiology of mtDNA diseases in which the distribution of mutant and wild-type mtDNA in cell lines in patient tissues may evolve with time.

Detection of mitochondrial single nucleotide polymorphisms using a primer elongation reaction on oligonucleotide microarrays

We have developed a novel allele-specific primer elongation protocol using a DNA polymerase on oligonucleotide chips. Oligonucleotide primers carrying polymorphic sites at their free 3́end were covalently bound to glass slides. The generation of single-stranded targets of genomic DNA containing single nuclotide polymorphisms (SNPs) to be typed was achieved by an asymmetric PCR reaction or exonuclease treatment of phosphothioate (PTO)-modified PCR products. In the presence of DNA polymerase and all four dNTPs, with Cy3-dUTP replacing dTTP, allele-specific extension of the immobilized primers took place along a stretch of target DNA sequence. The yield of elongated products was increased by repeated reaction cycles. We performed multiplexed assays with many small DNA targets, or used single targets of up to 4.4 kb mitochondrial DNA (mtDNA) sequence to detect multiple SNPs in one reaction. The latter approach greatly simplifies preamplification of SNP-containing regions, thereby providing a framework for typing hundreds of mtDNA polymorphisms.

Global air-sea flux of CO2: An estimate based on measurements of sea–air pCO2 difference

Approximately 250,000 measurements made for the pCO2 difference between surface water and the marine atmosphere, ΔpCO2, have been assembled for the global oceans. Observations made in the equatorial Pacific during El Nino events have been excluded from the data set. These observations are mapped on the global 4° × 5° grid for a single virtual calendar year (chosen arbitrarily to be 1990) representing a non-El Nino year. Monthly global distributions of ΔpCO2 have been constructed using an interpolation method based on a lateral advection–diffusion transport equation. The net flux of CO2 across the sea surface has been computed using ΔpCO2 distributions and CO2 gas transfer coefficients across sea surface. The annual net uptake flux of CO2 by the global oceans thus estimated ranges from 0.60 to 1.34 Gt-C⋅yr−1 depending on different formulations used for wind speed dependence on the gas transfer coefficient. These estimates are subject to an error of up to 75% resulting from the numerical interpolation method used to estimate the distribution of ΔpCO2 over the global oceans. Temperate and polar oceans of the both hemispheres are the major sinks for atmospheric CO2, whereas the equatorial oceans are the major sources for CO2. The Atlantic Ocean is the most important CO2 sink, providing about 60% of the global ocean uptake, while the Pacific Ocean is neutral because of its equatorial source flux being balanced by the sink flux of the temperate oceans. The Indian and Southern Oceans take up about 20% each.

The representation of the ipsilateral visual field in human cerebral cortex

Previous studies of cortical retinotopy focused on influences from the contralateral visual field, because ascending inputs to cortex are known to be crossed. Here, functional magnetic resonance imaging was used to demonstrate and analyze an ipsilateral representation in human visual cortex. Moving stimuli, in a range of ipsilateral visual field locations, revealed activity: (i) along the vertical meridian in retinotopic (presumably lower-tier) areas; and (ii) in two large branches anterior to that, in presumptive higher-tier areas. One branch shares the anterior vertical meridian representation in human V3A, extending superiorly toward parietal cortex. The second branch runs antero-posteriorly along lateral visual cortex, overlying motion-selective area MT. Ipsilateral stimuli sparing the region around the vertical meridian representation also produced signal reductions (perhaps reflecting neural inhibition) in areas showing contralaterally driven retinotopy. Systematic sampling across a range of ipsilateral visual field extents revealed significant increases in ipsilateral activation in V3A and V4v, compared with immediately posterior areas V3 and VP. Finally, comparisons between ipsilateral stimuli of different types but equal retinotopic extent showed clear stimulus specificity, consistent with earlier suggestions of a functional segregation of motion vs. form processing in parietal vs. temporal cortex, respectively.