Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation

Eukaryotic viruses can maintain latency in dividing cells as extrachromosomal nuclear plasmids. Segregation and nuclear retention of DNA is, therefore, a key issue in retaining copy number. The E2 enhancer protein of the papillomaviruses is required for viral DNA replication and transcription. Viral mutants that prevent phosphorylation of the bovine papillomavirus type 1 (BPV) E2 protein are transformation-defective, despite normal viral gene expression and replication function. Cell colonies harboring such mutants show sectoring of viral DNA and are unable to maintain the episome. We find that transforming viral DNA attaches to mitotic chromosomes, in contrast to the mutant genome encoding the E2 phosphorylation mutant. Second-site suppressor mutations were uncovered in both E1 and E2 genes that allow for transformation, maintenance, and chromosomal attachment. E2 protein was also found to colocalize to mitotic chromosomes, whereas the mutant did not, suggesting a direct role for E2 in viral attachment to chromosomes. Such viral hitch-hiking onto cellular chromosomes is likely to provide a general mechanism for maintaining nuclear plasmids.

Segregation of Two Spectrin Isoforms: Polarized Membrane-binding Sites Direct Polarized Membrane Skeleton Assembly

Spectrin isoforms are often segregated within specialized plasma membrane subdomains where they are thought to contribute to the development of cell surface polarity. It was previously shown that ankyrin and β spectrin are recruited to sites of cell–cell contact in Drosophila S2 cells expressing the homophilic adhesion molecule neuroglian. Here, we show that neuroglian has no apparent effect on a second spectrin isoform (αβH), which is constitutively associated with the plasma membrane in S2 cells. Another membrane marker, the Na,K-ATPase, codistributes with ankyrin and αβ spectrin at sites of neuroglian-mediated contact. The distributions of these markers in epithelial cells in vivo are consistent with the order of events observed in S2 cells. Neuroglian, ankyrin, αβ spectrin, and the Na,K-ATPase colocalize at the lateral domain of salivary gland cells. In contrast, αβH spectrin is sorted to the apical domain of salivary gland and somatic follicle cells. Thus, the two spectrin isoforms respond independently to positional cues at the cell surface: in one case an apically sorted receptor and in the other case a locally activated cell–cell adhesion molecule. The results support a model in which the membrane skeleton behaves as a transducer of positional information within cells.

An alteration in concatameric structure is associated with efficient segregation of plasmids in transfected Plasmodium falciparum parasites

Transfection of the human malaria parasite Plasmodium falciparum is currently performed with circularised plasmids that are maintained episomally in parasites under drug selection but which are rapidly lost when selection pressure is removed. In this paper, we show that in instances where gene targeting is not favoured, transfected plasmids can change to stably replicating forms (SRFs) that are maintained episomally in the absence of drug selection. SRF DNA is a large concatamer of the parental plasmid comprising at least nine plasmids arranged in a head-to-tail array. We show as well that the original unstable replicating forms (URFs) are also present as head-to-tail concatamers, but only comprise three plasmids. Limited digestion and γ irradiation experiments revealed that while URF concatamers are primarily circular, as expected, SRF concatamers form a more complex structure that includes extensive single-stranded DNA. No evidence of sequence rearrangement or additional sequence was detected in SRF DNA, including in transient replication experiments designed to select for more efficiently replicating plasmids. Surprisingly, these experiments revealed that the bacterial plasmid alone can replicate in parasites. Together, these results imply that transfected plasmids are required to form head-to-tail concatamers to be maintained in parasites and implicate both rolling-circle and recombination-dependent mechanisms in their replication.

Segregation of Heterotrimeric G Proteins in Cell Surface Microdomains: Gq Binds Caveolin to Concentrate in Caveolae, whereas Gi and Gs Target Lipid Rafts by Default

Select lipid-anchored proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins and nonreceptor tyrosine kinases may preferentially partition into sphingomyelin-rich and cholesterol-rich plasmalemmal microdomains, thereby acquiring resistance to detergent extraction. Two such domains, caveolae and lipid rafts, are morphologically and biochemically distinct, contain many signaling molecules, and may function in compartmentalizing cell surface signaling. Subfractionation and confocal immunofluorescence microscopy reveal that, in lung tissue and in cultured endothelial and epithelial cells, heterotrimeric G proteins (Gi, Gq, Gs, and Gβγ) target discrete cell surface microdomains. Gq specifically concentrates in caveolae, whereas Gi and Gs concentrate much more in lipid rafts marked by GPI-anchored proteins (5′ nucleotidase and folate receptor). Gq, apparently without Gβγ subunits, stably associates with plasmalemmal and cytosolic caveolin. Gi and Gs interact with Gβγ subunits but not caveolin. Gi and Gs, unlike Gq, readily move out of caveolae. Thus, caveolin may function as a scaffold to trap, concentrate, and stabilize Gq preferentially within caveolae over lipid rafts. In N2a cells lacking caveolae and caveolin, Gq, Gi, and Gs all concentrate in lipid rafts as a complex with Gβγ. Without effective physiological interaction with caveolin, G proteins tend by default to segregate in lipid rafts. The ramifications of the segregated microdomain distribution and the Gq-caveolin complex without Gβγ for trafficking, signaling, and mechanotransduction are discussed.

Neutral behavior of shared polymorphism

Several cases have been described in the literature where genetic polymorphism appears to be shared between a pair of species. Here we examine the distribution of times to random loss of shared polymorphism in the context of the neutral Wright–Fisher model. Order statistics are used to obtain the distribution of times to loss of a shared polymorphism based on Kimura’s solution to the diffusion approximation of the Wright–Fisher model. In a single species, the expected absorption time for a neutral allele having an initial allele frequency of ½ is 2.77 N generations. If two species initially share a polymorphism, that shared polymorphism is lost as soon as either of two species undergoes fixation. The loss of a shared polymorphism thus occurs sooner than loss of polymorphism in a single species and has an expected time of 1.7 N generations. Molecular sequences of genes with shared polymorphism may be characterized by the count of the number of sites that segregate in both species for the same nucleotides (or amino acids). The distribution of the expected numbers of these shared polymorphic sites also is obtained. Shared polymorphism appears to be more likely at genetic loci that have an unusually large number of segregating alleles, and the neutral coalescent proves to be very useful in determining the probability of shared allelic lineages expected by chance. These results are related to examples of shared polymorphism in the literature.

Postembryonic segregation of the germ line in sea urchins in relation to indirect development.

The four small micromeres of the sea urchin embryo contribute only to the coelomic sacs, which produce major components of the adult body plan during postembryonic development. To test the proposition that the small micromeres are the definitive primordial germ cell lineage of the sea urchin, we deleted their 4th cleavage parents, and raised the deleted embryos through larval life and metamorphosis to sexual maturity. Almost all of the experimental animals produced functional gametes, excluding the possibility that the germ cell lineage arises exclusively and obligatorily from descendants of the small micromeres; rather, the germ cell lineage arises during the postembryonic development of the rudiment. A survey of the literature indicates that there is no known case of an embryonic primordial germ cell lineage in a bilaterian species that displays maximal indirect development.

Segregation of DNA polynucleotide strands into sister chromatids and the use of endoreduplicated cells to track sister chromatid exchanges induced by crosslinks, alkylations, or x-ray damage.

The method of Matsumoto and Ohta [Matsumoto, K. & Ohta, T. (1992) Chromosoma 102, 60-65; Matsumoto, K. & Ohta, T. (1995) Mutat. Res. 326, 93-98] to induce large numbers of endoreduplicated Chinese hamster ovary cells has now been coupled with the fluorescence-plus-Giemsa method of Perry and Wolff [Perry, P. & Wolff, S. (1974) Nature (London) 251, 156-158] to produce harlequin endoreduplicated chromosomes that after the third round of DNA replication are composed of a chromosome with a light chromatid and a dark chromatid in close apposition to its sister chromosome containing two light chromatids. Unless the pattern is disrupted by sister chromatid exchange (SCE), the dark chromatid is always in the center, so that the order of the chromatids is light-dark light-light. The advent of this method, which permits the observation of SCEs in endoreduplicated cells, makes it possible to determine with great ease in which cell cycle an SCE occurred. This now allows us to approach several vexing questions about the induction of SCEs (genetic damage and its repair) after exposure to various types of mutagenic carcinogens. The present experiments have allowed us to observe how many cell cycles various types of lesions that are induced in DNA by a crosslinking agent, an alkylating agent, or ionizing radiation, and that are responsible for the induction of SCEs, persist before being repaired and thus lose their ability to inflict genetic damage. Other experiments with various types of mutagenic carcinogens and various types of cell lines that have defects in different DNA repair processes, such as mismatch repair, excision repair, crosslink repair, and DNA-strand-break repair, can now be carried out to determine the role of these types of repair in removing specific types of lesions.

Bipolar localization of Bacillus subtilis topoisomerase IV, an enzyme required for chromosome segregation

In Bacillus subtilis, parE and parC were shown to be essential genes for the segregation of replicated chromosomes. Disruption of either one of these genes resulted in failure of the nucleoid to segregate. Purified ParE and ParC proteins reconstituted to form topoisomerase IV (topo IV), which was highly proficient for ATP-dependent superhelical DNA relaxation and decatenation of interlocked DNA networks. By immunofluorescence microscopy and by directly visualizing fluorescence by using green fluorescence protein fusions, we determined that ParC is localized at the poles of the bacteria in rapidly growing cultures. The bipolar localization of ParC required functional ParE, suggesting that topo IV activity is required for the localization. ParE was found to be distributed uniformly throughout the cell. On the other hand, fluorescence microscopy showed that the GyrA and GyrB subunits of gyrase were associated with the nucleoid. Our results provide a physiologic distinction between DNA gyrase and topo IV. The subcellular localization of topo IV provides physical evidence that it may be part of the bacterial segregation machinery.

The Role of Topoisomerase II in Meiotic Chromosome Condensation and Segregation in Schizosaccharomyces pombe

Topoisomerase II is able to break and rejoin double-strand DNA. It controls the topological state and forms and resolves knots and catenanes. Not much is known about the relation between the chromosome segregation and condensation defects as found in yeast top2 mutants and the role of topoisomerase II in meiosis. We studied meiosis in a heat-sensitive top2 mutant of Schizosaccharomyces pombe. Topoisomerase II is not required until shortly before meiosis I. The enzyme is necessary for condensation shortly before the first meiotic division but not for early meiotic prophase condensation. DNA replication, prophase morphology, and dynamics of the linear elements are normal in the top2 mutant. The top2 cells are not able to perform meiosis I. Arrested cells have four spindle pole bodies and two spindles but only one nucleus, suggesting that the arrest is nonregulatory. Finally, we show that the arrest is partly solved in a top2 rec7 double mutant, indicating that topoisomerase II functions in the segregation of recombined chromosomes. We suggest that the inability to decatenate the replicated DNA is the primary defect in top2. This leads to a loss of chromatin condensation shortly before meiosis I, failure of sister chromatid separation, and a nonregulatory arrest.

Geographical structuring in the mtDNA of Italians.

Geographical patterns of mtDNA variation were studied in 12 Italian samples (1072 individuals) by two different spatial autocorrelation methods. Separate analyses of the frequencies of 12 restriction morphs show North-South clines, differences between Sardinia and the mainland populations, and the effects of isolation by distance. A recently developed autocorrelation statistic summarizing molecular similarity at all sites (AIDA; autocorrelation index for DNA analysis) confirms the presence of a clinical pattern; differences between random pairs of haplotypes tend to increase with their geographical distance. The partition of gene diversity, however, reveals that most variability occurs within populations, whereas differences between populations are minor (GST = 0.057). When the data from the 12 samples are pooled, two descriptors of genetic variability (number of polymorphic sites and average sequence difference between pairs of individuals) do not behave as expected under neutrality. The presence of clinal patterns, Tajima's tests, and a simulation experiment agree in suggesting that population sizes increased rapidly in Italy and Sicily but not necessarily so in Sardinia. The distribution of pairwise sequence differences in the Italian peninsula (excluding Sardinia) permits a tentative location of the demographic increase between 8000 and 20,500 years ago. These dates are consistent with archaeological estimates of two distinct expansion processes, occurring, respectively, in the Neolithic and after the last glacial maximum in the Paleolithic. Conversely, there is no genetic evidence that such processes have had a major impact on the Sardinian population.

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.