Perturbation of Parentally Biased Gene Expression during Interspecific Hybridization

Interspecific hybridization often induces epigenetic remodeling that leads to transposon activation, gene expression changes, and loss of imprinting. These genomic changes can be deleterious and contribute to postzygotic hybrid incompatibility. In Arabidopsis, loss of genomic imprinting of PHERES1 and presumed failure of Polycomb Repressive Complex contributes to seed inviability observed in A. thaliana X A. arenosa interspecific hybrids. We used this species pair to further analyze the relationship between parentally biased gene expression and postzygotic hybrid incompatibility using two A. thaliana accessions, Col-0 and C24, with differential seed survival. We found that parentally biased expression was perturbed to a similar degree in both A. thaliana hybrids for PHERES1, HDG3, and six other normally paternally expressed genes. We propose that early genome remodeling and loss of imprinting of seed development genes induces lethality in both compatible and incompatible hybrids.     

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 °C to 5 °C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.     

Initiation of lytic DNA replication in Epstein-Barr virus: search for a common family mechanism

Herpesviruses are a complex family of dsDNA viruses that are a major cause of human disease. All family members share highly related viral replication proteins, such as DNA polymerase, ssDNA-binding proteins and processivity factors. Consequently, it is generally thought that lytic replication occurs through a common and conserved mechanism. However, considerable evidence indicates that proteins controlling initiation of DNA replication vary greatly among the herepesvirus subfamilies. In this article, we focus on some of the known mechanisms that regulate Epstein-Barr virus lytic-cycle replication, and compare this to other herpesvirus family members. Our reading of the literature leads us to conclude that diverse viral mechanisms generate a common nucleoprotein prereplication structure that can be recognized by a highly conserved family of viral replication enzymes.     

Inhibitors of RNA synthesis and passage of chick embryo fibroblasts through the G1 period

Events that are essential for progression through the G1 period begin immediately or shortly after resting chick embryo cells are given fresh medium with serum. The following observations support the contention that the critical events include the production of non-ribosomal RNAs: (1) Addition to the “shift-up” medium of either of two inhibitors of RNA formation, comptothecin or 5, 6-dichloro?1-β-D-ribofuranosylbenzimidazole, delays the onset of DNA replication by about the length of time the cells are exposed to the drugs. (2) Although entry into the S phase is delayed by the inhibitors, the slopes of the DNA response curves are identical to that of control cultures. (3) Neither drug reduces significantly the rate of overall protein synthesis. Observations (2) and (3) are taken to mean that expansion of the G1 period is not due to cell damage. (4) A third inhibitor of RNA synthesis, cordycepin, also delays passage of stimulated cells throgh the G1 phase, but, in this case, the length of the delay period is greater than that of the exposure period. (5) A low dose of actinomycin D does not impede movement towards the S phase, even though the synthesis of preribosomal RNA is considerably reduced. The possibility is considered that the essential G1 molecules are mRNAs.     

Computerized image analysis of Ki-67 in ductal breast carcinoma in situ.

OBJECTIVE: To develop and determine the staining protocols and computerized image analysis methods that are the most effective combination for performing quantitative analysis of Ki-67. STUDY DESIGN: We compared conventional bright-field light microscopy and refractive optical enhancement methods in combination with various immunodetection and filter enhancement methods, including immunogold in combination with epipolarization refractive optics and enzymatic conversion of chromogenic substrates in combination with optical filter enhancement. Initial Ki-67 tests were performed on lymph node tissues and cultured human breast cells and then applied to 200 ductal carcinoma in situ (DCIS) samples. DCIS acini were digitally acquired, and a region of interest was manually outlined in each one with a digital stylus to include only the cellular component; then the Ki-67 staining index was quantified by segmentation analysis. RESULTS: Although combining epipolarization analysis with immunohistogold staining was the most sensitive detection method, nonspecific binding was too high. The streptavidin-horseradish-peroxidase enzymatic conversion of 3,3'-diaminobenzidine (DAB) in combination with optical enhancement filters was the most effective method tested. Ki-67 stain was associated with dense fibrillar structures of the nucleoli in the less intensely staining nuclei and was most intense in paired nuclei. CONCLUSION: The method of measuring Ki-67 expression by DAB staining combined with optical enhancement filters and quantification via computer-assisted image analysis techniques produced objective and reproducible results. As such, this method can offer (1) less intraobserver and interobserver variability, (2) a digital archival record, and (3) a baseline for digital exchange of information between studies.     

Nearest-neighbor tree species combinations in tropical forest: the role of chance, and some consequences of high diversity

In three permanent inventory plots comprising 12.4 ha of undisturbed forest at La Selva, Costa Rica, all stems ≥10 cm dbh were mapped and identified to species. There were 1628, 1478 and 1954 trees in the plots, representing 168, 166 and 171 species respectively. We determined the species of each nearest-neighbor pair of trees, and asked whether the occurrence of species pairs conforms to a simple random mixing model. If trees are randomly mixed in terms of species, the expected frequency of any nearest neighbor species combination is a function of the relative abundance of the two species. Departures from random mixing could arise from species interactions, differential responses to habitat, or both. The number of possible ij species combinations increases approximately as the square of the number of species. For the 168 species in plot 1, for example, there are 14 196 possible combinations. We compared the expected frequency of each species combination in the three plots (42 736 combinations in all) with observed frequencies. Over 98% of the combinations had observed frequencies of zero and expected frequencies close to zero. A consequence of high diversity is low density of most individual species, and exceedingly low frequencies of the vast majority of species combinations. For each of the 805 combinations with observed frequencies >0, we used simulation to generate a distribution of expected frequencies. We used a t-test to compare the observed frequency with the mean of the simulated distribution for each combination. Only 40 combinations (0.09% of the possible species combinations in the plots) departed from expected frequencies; 39 combinations were more common, and one less common than expected. The overwhelming majority of nearest neighbor species combinations occur at frequencies predictable from their individual abundances.