Dynamic methylation adjustment and counting as part of imprinting mechanisms.

Monoallelic expression in diploid mammalian cells appears to be a widespread phenomenon, with the most studied examples being X-chromosome inactivation in eutherian female cells and genomic imprinting in the mouse and human. Silencing and methylation of certain sites on one of the two alleles in somatic cells is specific with respect to parental source for imprinted genes and random for X-linked genes. We report here evidence indicating that: (i) differential methylation patterns of imprinted genes are not simply copied from the gametes, but rather established gradually after fertilization; (ii) very similar methylation patterns are observed for diploid, tetraploid, parthenogenic, and androgenic preimplantation mouse embryos, as well as parthenogenic and androgenic mouse embryonic stem cells; (iii) haploid parthenogenic embryos do not show methylation adjustment as seen in diploid or tetraploid embryos, but rather retain the maternal pattern. These observations suggest that differential methylation in imprinted genes is achieved by a dynamic process that senses gene dosage and adjusts methylation similar to X-chromosome inactivation.

A mammalian 2-5A system functions as an antiviral pathway in transgenic plants.

Resistance to virus infections in higher vertebrates is mediated in part through catalysis of RNA decay by the, interferon-regulated 2-5A system. A functional 2-5A system requires two enzymes, a 2-5A synthetase that produces 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A) in response to double-stranded RNA, and the 2-5A-dependent RNase L. We have coexpressed these human enzymes in transgenic tobacco plants by using a single plasmid containing the cDNAs for both human RNase L and a low molecular weight form of human 2-5A synthetase under control of different, constitutive promoters. Expression of the human cDNAs in the transgenic plants was demonstrated from Northern blots, by specific enzyme assays, and by immunodetection (for RNase L). Infection of leaves, detached or in planta, of the coexpressing transgenic plants by tobacco mosaic virus, alfalfa [correction of alfafa] mosaic virus, or tobacco etch virus resulted in necrotic lesions. In contrast, leaves expressing 2-5A synthetase or RNase L alone and leaves containing the plasmid vector alone produced typical systemic infections. While alfalfa mosaic virus produced lesions only in the inoculated leaves regardless of the concentration of virus in the inoculum, high, but not low, levels of tobacco etch virus inoculum resulted in escape of virus to uninoculated leaves. Nevertheless, there was a substantial reduction of tobacco etch virus yield as measured by ELISA assay in the coexpressing transgenic plants. These results indicate that expression of a mammalian 2-5A system in plants provides resistance to virus infections.

Significant competitive advantage conferred by meiosis and syngamy in the yeast Saccharomyces cerevisiae.

The presumed advantages of genetic recombinations are difficult to demonstrate directly. To investigate the effects of recombination and background heterozygosity on competitive ability, we have performed serial-transfer competition experiments between isogenic sexual and asexual strains of the yeast Saccharomyces cerevisiae. The members of these diploid pairs of strains differed only in being heterozygous (sexual) or homozygous (asexual) at the mating type or MAT locus. Competing pairs had either a completely homozygous or a heterozygous genetic background, the latter being heterozygous at many different loci throughout the genome. A round of meiotic recombination (automixis) conferred a large and statistically significant enhancement of competitive ability on sexual strains with a heterozygous genetic background. By contrast, in homozygous background competitions, meiosis decreased the sexual strains' initial relative competitive ability. In all cases, however, the sexual strains outcompeted their isogenic asexual counterparts, whether meiotic recombination had occurred or not. In some genetic backgrounds, this was due in part to an overdominance effect on competitive advantage of heterozygosity at the MAT locus. The advantage of the sexual strains also increased significantly during the course of the homozygous background competitions, particularly when meiosis had occurred. This latter effect either did not occur or was very weak in heterozygous background competitions. Overall, sexual strains with heterozygous genetic backgrounds had a significantly higher initial relative competitive ability than those with homozygous backgrounds. The advantage of mating type heterozygosity in this organism extends far beyond the ability to recombine meiotically.

Molecular cloning of a protein kinase whose phosphorylation is regulated by genetic adhesion during Chlamydomonas fertilization.

Fertilization in Chlamydomonas is initiated by adhesive interactions between gametes of opposite mating types through flagellar glycoproteins called agglutinins. Interactions between these cell adhesion molecules signal for the activation of adenylyl cyclase through an interplay of protein kinases and ultimately result in formation of a diploid zygote. One of the early events during adhesion-induced signal transduction is the rapid inactivation of a flagellar protein kinase that phosphorylates a 48-kDa protein in the flagella. We report the biochemical and molecular characterization of the 48-kDa protein. Experiments using a bacterially expressed fusion protein show that the 48-kDa protein is capable of autophosphorylation on serine and tyrosine and phosphorylation of bovine beta-casein on serine, confirming that the 48-kDa protein itself has protein kinase activity. This protein kinase exhibits limited homology to members of the eukaryotic protein kinase superfamily and may be an important element in a signaling pathway in fertilization.

Genetic heterogeneity in isogenic homozygous clonal zebrafish.

The C32 isogenic homozygous diploid (IHD) strain of the zebrafish (Danio rerio) was found to be polyallelic at a malate dehydrogenase locus (sMdh-A). A variant allele is thought to have arisen via mutation within the past 10 bisexual generations that have maintained the strain since its last gynogenetic cloning event; this unique allele now predominates at the sMdh-A locus. The estimated mutation rate in this species is sufficiently high that long-term genetic homogeneity of its IHD clones cannot be assumed. Researchers using such bisexually maintained clones should be aware that they are not necessarily using genetically uniform subjects. Genetic uniformity of cloned IHD zebrafish will be maximized if experimental subjects are obtained soon after a cloning event.

Haploidy and androgenesis in Drosophila.

Adrogenesis, development from paternal but not maternal chromosomes, can be induced to occur in some organisms, including vertebrates, but has only been reported to occur naturally in interspecific hybrids of the Sicilian stick insect. Androgenesis has not been described previously in Drosophila. We now report the recovery of androgenetic offspring from Drosophila melanogaster females mutant for a gene that affects an oocyte- and embryo-specific alpha-tubulin. The androgenetic exceptions are X,X diploid females that develop from haploid embryos and express paternal markers on all 4 chromosomes. The exceptional females arise by fusion of haploid cleavage nuclei or failure of newly replicated haploid chromosomes to segregate, rather than fusion of two inseminating sperm. The frequency of androgenetic offspring is greatly enhanced by a partial loss-of-function mutant of the NCD (nonclaret disjunctional) microtubule motor protein, suggesting that wild-type NCD functions is pronuclear fusion. Diploidization of haploid paternal chromosome complements results in complete genetic homozygosity, which could facilitate studies of gene variation and mutational load in populations.

The genome of Arabidopsis thaliana.

Arabidopsis thaliana is a small flowering plant that is a member of the family cruciferae. It has many characteristics--diploid genetics, rapid growth cycle, relatively low repetitive DNA content, and small genome size--that recommend it as the model for a plant genome project. The current status of the genetic and physical maps, as well as efforts to sequence the genome, are presented. Examples are given of genes isolated by using map-based cloning. The importance of the Arabidopsis project for plant biology in general is discussed.

Functional haplodiploidy: a mechanism for the spread of insecticide resistance in an important international insect pest.

The coffee berry borer, Hypothenemus hampei, is the most important insect pest of coffee worldwide and has an unusual life history that ensures a high degree of inbreeding. Individual females lay a predominantly female brood within individual coffee berries and because males are flightless there is almost entirely full sib mating. We investigated the genetics associated with this interesting life history after the important discovery of resistance to the cyclodiene type insecticide endosulfan. Both the inheritance of the resistance phenotype and the resistance-associated point mutation in the gamma-aminobutyric acid receptor gene Rdl were examined. Consistent with haplodiploidy, males failed to express and transmit paternally derived resistance alleles. Furthermore, while cytological examination revealed that males are diploid, one set of chromosomes was condensed, and probably nonfunctional, in the somatic cells of all males examined. Moreover, although two sets of chromosomes were present in primary spermatocytes, the chromosomes failed to pair before the single meiotic division, and only one set was packaged in sperm. Thus, the coffee berry borer is "functionally" haplodiploid. Its genetics and life history may therefore represent an interesting intermediate step in the evolution of true haplodiploidy. The influence of this breeding system on the spread of insecticide resistance is discussed.

Increased activity of p53 in senescing fibroblasts.

The p53 tumor-suppressor protein binds DNA and activates the expression of a 21-kDa protein that inhibits both the activity of cyclin-dependent kinases and the function of proliferating cell nuclear antigen. Since p21 expression has been reported to increase 10- to 20-fold as human diploid fibroblasts lose the ability to replicate, we examined the expression and activity of p53 during replicative aging. Similar levels of total p53 mRNA and protein were expressed in low-passage (young) and high-passage (old) cells but both DNA binding activity in vitro and transcriptional activity of p53 in vivo were increased severalfold in high-passage cells. While the basis of increased p53 activity is presently unclear, it is not correlated with differential phosphorylation or changes in p53-mouse double minute 2 gene product interactions. These results provide evidence for the activation of a protein involved in the control of cell cycle checkpoints during cellular aging, in the absence of increased expression.

Rapid genome change in synthetic polyploids of Brassica and its implications for polyploid evolution.

Although the evolutionary success of polyploidy in higher plants has been widely recognized, there is virtually no information on how polyploid genomes have evolved after their formation. In this report, we used synthetic polyploids of Brassica as a model system to study genome evolution in the early generations after polyploidization. The initial polyploids we developed were completely homozygous, and thus, no nuclear genome changes were expected in self-fertilized progenies. However, extensive genome change was detected by 89 nuclear DNA clones used as probes. Most genome changes involved loss and/or gain of parental restriction fragments and appearance of novel fragments. Genome changes occurred in each generation from F2 to F5, and the frequency of change was associated with divergence of the diploid parental genomes. Genetic divergence among the derivatives of synthetic polyploids was evident from variation in genome composition and phenotypes. Directional genome changes, possibly influenced by cytoplasmic-nuclear interactions, were observed in one pair of reciprocal synthetics. Our results demonstrate that polyploid species can generate extensive genetic diversity in a short period of time. The occurrence and impact of this process in the evolution of natural polyploids is unknown, but it may have contributed to the success and diversification of many polyploid lineages in both plants and animals.

Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution.

The internal transcribed spacers (ITS) of nuclear ribosomal DNA of 33 species of genus Paeonia (Paeoniaceae) were sequenced. In section Paeonia, different patterns of nucleotide additivity were detected in 14 diploid and tetraploid species at sites that are variable in the other 12 species of the section, suggesting that reticulate evolution has occurred. Phylogenetic relationships of species that do not show additivity, and thus ostensibly were not derived through hybridization, were reconstructed by parsimony analysis. The taxa presumably derived through reticulate evolution were then added to the phylogenetic tree according to additivity from putative parents. The study provides an example of successfully using ITS sequences to reconstruct reticulate evolution in plants and further demonstrates that the sequence data could be highly informative and accurate for detecting hybridization. Maintenance of parental sequences in the species of hybrid origin is likely due to slowing of concerted evolution caused by the long generation time of peonies. The partial and uneven homogenization of parental sequences displayed in nine species of putative hybrid origin may have resulted from gradients of gene conversion. The documented hybridizations may have occurred since the Pleistocene glaciations. The species of hybrid origin and their putative parents are now distantly allopatric. Reconstruction of reticulate evolution with sequence data, therefore, provides gene records for distributional histories of some of the parental species.

Genetic supercycles caused by cyclical selection.

Typical behavior of a two-locus genetic system experiencing cyclical selection, includes fixation (in one or both loci) or a stable polymorphic cycle with a period equal to that of environmental changes. By considering the time scale in terms of environmental periods, the last case could be trivially classified as a polymorphic stable point. Here we report on some results showing the complex limiting behavior of diploid population trajectories resulting from selection in a cyclically changing environment. We found that simple cyclical selection could produce genetic supercycles composed of many hundreds of environmental periods.

Organismal duplication, inclusive fitness theory, and altruism: understanding the evolution of endosperm and the angiosperm reproductive syndrome.

For almost a century, events relating to the evolutionary origin of endosperm, a unique embryo-nourishing tissue that is essential to the reproductive process in flowering plants, have remained a mystery. Integration of recent advances in phylogenetic reconstruction, comparative reproductive biology, and genetic theory can be used to elucidate the evolutionary events and forces associated with the establishment of endosperm. Endosperm is shown to be derived from one of two embryos formed during a rudimentary process of "double fertilization" that evolved in the ancestors of angiosperms. Acquisition of embryo-nourishing behavior (with accompanying loss of individual fitness) by this supernumerary fertilization product was dependent upon compensatory gains in the inclusive fitness of related embryos. The result of the loss of individual fitness by one of the two original products of double fertilization was the establishment of endosperm, a highly modified embryo/organism that reproduces cryptically through behavior that enhances the fitness of its associated embryo within a seed. Finally, although triploid endosperm remains a synapomorphy of angiosperms, inclusive fitness analysis demonstrates that the embryo-nourishing properties of endosperm initially evolved in a diploid condition.