Nuclear and cytoplasmic contributions to intraspecific divergence in an annual legume

The genetic architecture of trait differentiation was evaluated between two ecologically distinct populations of Chamaecrista fasciculata. Individuals from Maryland and Illinois populations were crossed to create 10 types of seed: Maryland and Illinois parents, reciprocal F1 and F2 hybrids, and backcrosses to Maryland and to Illinois on reciprocal F1 hybrids. Reciprocal crosses created hybrid generation seeds with both Maryland and Illinois cytoplasmic backgrounds. Experimental individuals were grown in a common garden near the site of the Maryland population. In the garden, plants from the Illinois population flowered, set fruit, and died earlier than those from Maryland, likely reflecting adaptations to differences in growing season length between the two populations. Although reproductive components at the flower and whole plant level differed between the two populations, reproductive output as measured by fruit and seed production was similar. Cytoplasmic genes had a subtle but pervasive effect on population differentiation; hybrids with Maryland cytoplasm were significantly differentiated from those with Illinois cytoplasm when all characters were evaluated jointly. The nuclear genetic architecture of population differentiation was evaluated with joint scaling tests. Depending on the trait, both additive and nonadditive genetic effects contributed to population differentiation. Intraspecific genetic differentiation in this wild plant species appears to reflect a complex genetic architecture that includes the contribution of additive, dominance, and epistatic components in addition to subtle cytoplasmic effects.     

Reproductive isolation in Caenorhabditis: terminal phenotypes of hybrid embryos

SUMMARY Several interspecific combinations of the "elegans" group of Caenorhabditis species are cross-fertile. Most F1 hybrids from these crosses arrest during embryogenesis. Developmental defects observed in hybrid embryos include defects in gastrulation initiation, defects in embryonic compaction, and defects in embryonic elongation. These reproductive barriers have arisen multiple times in the evolution of Caenorhabditis.     

Nuclear DNA-like RNA of regions of developing frog embryos

Summary A comparison of equal amounts of labeled nuclear RNA of dorsal ectodermmesoderm and endoderm cells of frog neurulae by DNA-RNA hybridizations revealed more kinds of DNA-like RNA in the ectoderm-mesoderm nuclei.This research was supported by a grant from the National Institutes of Health.     

Ontogeny of lactate dehydrogenase isozymes in chicken-quail hybrid embryos

The ontogeny of lactate dehydrogenase (LDH) isozymes was examined in avian hybrids and compared with the isozyme patterns of the parental species. Hybrids were obtained by crossing female Japanese quali (Coturnix coturnix japonica) with male domestic chickens (Gallus gallus domesticus). By use of starch gel electrophoresis and an enzyme-specific stain, traces of embryonic paternally derived LDH were detected in unincubated hybrid eggs. It was concluded that the embryonic genes coding for the B subunits of LDH are activated during the hours between fertilization and oviposition. In early blastoderms, a great excess of maternally stored LDH is present. In the hybrid, the predominantly maternal pattern of isozymes shifts during embryogenesis to a predominantly paternal pattern. This was considered evidence for differential allelic regulation of LDH inactivation. A progressive trend toward the establishment of the adult distribution of isozymes in various tissues was also observed in the hybrid and quail, and found to be similar to chicken LDH isozyme ontogeny.     

Human-animal cytoplasmic hybrid embryos, mitochondria, and an energetic debate

Scientists are seeking permission to generate human embryonic stem cells to study disease by introducing human genetic material into an animal oocyte. This has raised ethical questions that centre on whether the entities being generated are actually human. The answer to these questions will determine how this area of research will be regulated and whether such work will be legal. The function of the extra-nuclear mitochondrial genome lies at the heart of these issues and forms the focus of this commentary.     

Gene activation of alcohol dehydrogenase in Japanese quail and chicken-quail hybrid embryos

No preferential activation of the maternally derived alcohol dehydrogenase (ADH) allele was found in any of the chicken male x Japanese quail female hybrids examined. ADH activity in the liver was, in fact, found to exist in two different cathodal zonal regions on starch gel electropherograms; the zone II bands appeared at day 5 of incubation in the quail embryo (day 6 in the hybrid embryo) and the zone I bands appeared in 9-day quail embryos (10-day hybrid embryos). By day 13 of incubation, only the faster-migrating zone I bands could be detected in both quail and hybrid embryos.     

The relative contributions of newly synthesized and stored messages to Hl histone synthesis in interspecies hybrid echinoid embryos.

We have measured the ratio of incorporation of 3H-lysine into the maternal and paternal forms of Hl histones synthesized by the interordinal hybrid embryo which results from the fertilization of sand dollar eggs with sea urchin sperm. This ratio has been used to calculate the relative contributions of newly transcribed and stored Hl histones mRNA to the synthesis of Hl histone at five different stages of development. These calculations are based on the assumption that histone mRNA of both parental types is transcribed with equal efficiency from the genome and that these RNAs are translated with equal efficiency in the cytoplasm of the hybrid embryos. On this basis, we have estimated that the contribution of new mRNA represents 80% of total Hl histone synthesis at the 16--32 cell stage, 54% at the hatching blastula stage, 40% at the mesenchyme blastula stage, and 100% after gastrulation. These data are discussed in the light of presently known parameters of histone and histone mRNA synthesis in echinoderm embryos.     

Nuclear and cytoplasmic mitotic cycles continue in Drosophila embryos in which DNA synthesis is inhibited with aphidicolin

We have microinjected aphidicolin, a specific inhibitor of DNA polymerase alpha, into syncytial Drosophila embryos. This treatment inhibits DNA synthesis and, as a consequence, nuclear replication. We demonstrate that under these conditions several cycles of both centrosome replication and cortical budding continue, although the cycles have a longer periodicity than is normally found. As in uninjected embryos, when the cortical buds are present, the embryos have nuclei containing decondensed chromatin surrounded by nuclear membranes as judged by bright annular staining with an anti-lamin antibody. As the buds recede, the unreplicated chromatin condenses and lamin staining becomes weak and diffuse. Thus, both cytoplasmic and nuclear aspects of the mitotic cycle continue following the inhibition of DNA replication in the Drosophila embryo.     

Nuclear and cytoplasmic DNA synthesis in cotton embryos: a correlated light and electron microscope autoradiographic study

Summary To investigate the possibility, implied by an earlier report, that large amounts of degradable DNA are probably present in the cytoplasm of young cotton embryos, an investigation was undertaken to establish the distribution, amount and metabolic stability of DNA in cotton embryos. Several sensitive cytochemical tests failed to detect any but small amounts of extranuclear DNA. Quantitative determination of the nucleic acid content of embryos during embryogenesis showed that the amounts of DNA and RNA remained fairly constant during embryogenesis, with a ratio of RNA to DNA of about 3.5 to 1. Quantitative autoradiography at both the light and electron microscope levels of sections from embryos pulse-labeled with ³H-thymidine showed that the grain density over the nucleus and cytoplasm did not change during a seven-hour period after labeling, nor did the distribution of label in the cytoplasm. Virtually all incorporation was eliminated by the inclusion of iododeoxy-uridine in the medium. Almost all of the nuclear label and at least 90% of the cytoplasmic label after ³H-thymidine incorporation was eliminated by deoxyribonuclease. It was concluded that there are no unusual features related to DNA distribution or metabolism in cotton embryo; i.e., that only small amounts of DNA are present in the cytoplasm and that all of the DNA is metabolically stable.Approximately 40% of the cytoplasmic grains after ³H-thymidine labeling were not associated with either plastids or mitochondria (i.e., were more than 0.1 micron distant). No fully satisfactory explanation for such an apparently high figure could be given.This work was supported by a Public Health Service fellowship 5-F2-GM-22,031-02 from the National Institute of General Medical Sciences, by NSF grant GB 3460, by NIH grant 5-R01-Ca0356-10 and by Miller Institute for Basic Science.     

Nuclear membrane contributions to the Golgi complex

Summary Electron microscopic examination of a variety of rapidly growing or differentiating mammalian and avian cells suggests that many of the Golgi vesicles and saccules arise directly from the outer nuclear membrane. Evidence for this interpretation includes: (1) the presence of a continuum of vesicles which appears to originate from the outer nuclear membrane and to enlarge gradually into saccules in the region of the Golgi membrane complex; (2) the absence of ribosomes on the nuclear blebs and the vesicles formed in these regions along the nuclear envelope; (3) the presence of active nuclear vesiculation near the Golgi region in cells essentially devoid of rough and/or smooth endoplasmic reticulum, and (4) the demonstration of peroxidase activity in the cisternae of the nuclear envelope and in vesicles extending in rows from the nuclear envelope to the Golgi complex. Supported by Research Grants HE 04061-09 (HEM), C-5315 and 1S01 FR-05109-01, Project 10 from the National Institutes of Health, Bethesda, Maryland.     

Activation of human embryonic gene expression in cytoplasmic hybrid embryos constructed between bovine oocytes and human fibroblasts

Cross-species somatic all number transfer (SCNT) provides a potential solution to overcome the problem of oocyte shortage for therapeutic cloning. To further characterize the system, we constructed cytoplasm hybrid embryos between bovine oocytes and human fibroblasts and examined dynamics of human gene activation during preimplantation stages. Data from this study showed that human embryonic genes, OCT4, SOX2, NANOG, E-CADHERIN, as well as beta-ACTIN, were activated by enucleated bovine oocytes. Activation of human genes was correlated with developmental potential of the embryos. The extent of human gene activation varied drastically and was incomplete in a large proportion of the embryos. Activation of human genes in the human-bovine cytoplasm hybrid embryos occurs in a temporal pattern resembling that of the bovine species. Results from this study suggest that human gene products are required for hybrid embryos to develop to later preimplantation stages. Facilitating human genome activation may improve successful rates in cross-species SCNT.     

Nuclear transplantation in mouse embryos

The ability of foreign nuclei to support development in nuclear transplantation manipulations has proven an effective means to assess the consequences of nuclear differentiation. In addition, nuclear transplantation might serve to define the persistence and role of maternally inherited cytoplasmic constituents during embryogenesis. We have extended the use of a technique that enables the efficient transfer of one-cell-stage pronuclei into the cytoplasm of enucleated mouse embryos, and have successfully transferred two-, four-, eight-cell-stage and inner cell mass (ICM) cell nuclei. We have also used this technique as a means to determining that the stage-specific embryonic antigen, SSEA-3, is a cytoplasmic contribution of the unfertilized ovum. The potential value of this technique in determining the developmental capacity of nuclei from various embryonic states, and in determining nuclear/cytoplasmic origins or early embryonic gene products, is discussed.     

Substrate and cofactor binding to fluorescently labeled cytoplasmic malate dehydrogenase

Cytoplasmic malate dehydrogenase (cMDH) is a key enzyme in several metabolic pathways. Though its activity has been examined extensively, there are lingering mechanistic uncertainties involving substrate and cofactor binding. To more completely understand this enzyme's interactions with cofactor and substrate ligands, a fluorescent reporter group was introduced into the enzyme's structure. This was accomplished by selective modification of Cys 110. The reaction placed an aminonaphthaline sulfonic acid group near the enzyme's active site. Substrate, inhibitor, and NAD binding activities were characterized using changes in this label's fluorescence. Results demonstrated that both substrate and cofactor molecules bound to the enzyme in the absence of their companion ligands. This is in contrast to strictly ordered cofactor then substrate binding as has been suggested by kinetic analyses of closely related enzymes. Binding results also indicated that the cofactor, NAD, bound to cMDH in a negatively cooperative manner, but substrates and the inhibitor, hydroxymalonate, bound non-cooperatively. Multiple substrate binding modes were identified and interactions between substrate and cofactor binding were found.     

A genetic component of resistance to fungal infection in frog embryos

The embryo has traditionally been considered to completely rely upon parental strategies to prevent threats to survival posed by predators and pathogens, such as fungi. However, recent evidence suggests that embryos may have hitherto neglected abilities to counter pathogens. Using artificial fertilization, we show that among-family variation in the number of Saprolegnia-infected eggs and embryos in the moor frog, Rana arvalis, cannot be explained by maternal effects. However, analysed as a within-females effect, sire identity had an effect on the degree of infection. Furthermore, relatively more eggs and embryos were infected when eggs were fertilized by sperm from the same, compared with a different, population. These effects were independent of variation in fertilization success. Thus, there is likely to be a significant genetic component in embryonic resistance to fungal infection in frog embryos. Early developmental stages may show more diverse defences against pathogens than has previously been acknowledged.