Hypomethylated sequences: Characterization of the duplicate soybean genome

Soybean is believed to be a diploidized tetraploid generated from an allotetraploid ancestor. In this study, we used hypomethylated genomic DNA as a source of probes to investigate the genomic structure and methylation patterns of duplicated sequences. Forty-five genomic clones from Phaseolus vulgaris and 664 genomic clones from Glycine max were used to examine the duplicated regions in the soybean genome. Southern analysis of genomic DNA using probes from both sources revealed that greater than 15% of the hypomethylated genomic regions were only present once in the soybean genome. The remaining ca. 85% of the hypomethylated regions comprise duplicated or middle repetitive DNA sequences. If only the ratio of single to duplicate probe patterns is considered, it appears that 25% of the single-copy sequences have been lost. By using a subset of probes that only detected duplicated sequences, we examined the methylation status of the homeologous genomes with the restriction enzymes MspI and HpaII. We found that in all cases both copies of these regions were hypomethylated, although there were examples of low-level methylation. It appears that duplicate sequences are being eliminated in the diploidization process. Our data reveal no evidence that duplicated sequences are being silenced by inactivation correlated with methylation patterns.     

Experimental evaluation of realized niche models for predicting responses of plant species to a change in environmental conditions

Abstract. We estimated, using logistic regression techniques, the realized niches of the four dominant species in an experimental marsh complex located in the Delta Marsh, Manitoba, Canada. These models were then used to predict the probability of occurrence of these species in selected elevation ranges when water levels were raised in 1985 either 0, 30 or 60 cm above the long-term normal water level. These realized-niche models were calculated using elevation and species data collected in 1980. After having been eliminated by two years of deep flooding, the emergent vegetation in this complex had been re-established during a drawdown beginning in either 1983 or 1984. Our hypothesis was that from 1985 to 1989 the frequencies of occurrence of species in selected elevation ranges would converge to their probabilities predicted from the 1980 logistic models. This was not borne out by our results. Actual frequencies and predicted probabilities of occurrence of a species were similar at best less than 40% and then mostly in the control (0 cm) treatment. The realized-niche models were not adequate to predict the distribution of emergents after an increase in water level in the short term because the emergent species did not migrate upslope. Emergent species in the medium and high treatments either (1) died out - Scolochloa festucacea and Scirpus lacustris - after 3 yr because they could not survive permanent flooding, (2) stayed where they were - Phragmites australis - because they were unable to move upslope through clonal growth, or (3) became more widespread - Typha glauca only because of the expansion of small local populations already established in 1985 in areas dominated formerly by other species.     

Cell Cycle Arrest of Proliferating Neuronal Cells by Serum Deprivation Can Result in Either Apoptosis or Differentiation

Abstract: Apoptotic cell death plays a critical role in the development of the nervous system. The death of mature nondividing neurons that fail to receive appropriate input from the target field has been extensively studied. However, the mechanisms mediating the extensive cell death occurring in areas of the developing brain where proliferating neuroblasts differentiate into mature nondividing neurons have not been analyzed. We show here that the cell cycle arrest of a proliferating cell of neuronal origin by removal of serum results in either apoptotic cell death or differentiation to a mature nondividing neuronal cell. The proportion of cells undergoing death or differentiation is influenced in opposite directions by treatment of the cells with cyclic AMP and retinoic acid. This suggests that following the withdrawal of signals stimulating neuroblast cell division, neuronal cells either can cease to suppress a constitutive suicide pathway and hence die by apoptosis or, alternatively, can differentiate into a mature neuronal cell. Regulation of the balance between apoptosis and neuronal differentiation could therefore play a critical role in controlling the numbers of mature neurons that form.     

DNA Fragmentation Induced by Cytotoxic T Lymphocytes Can Result in Target Cell Death

Cytotoxic T lymphocyte (CTL)-mediated lysis is accompanied by fragmentation of target cell DNA into an oligonucleosome ladder, a hallmark of apoptosis. Is this a fortuitous coincidence, or could CTL be inducing lysis by activation of the suicide signal? In this report we demonstrate that CTL-mediated target cell death can be blocked with the drug aurintricarboxylic acid (ATA). The abrogation of death correlates with the inhibition of DNA fragmentation. While ATA prevented DNA fragmentation, it failed to significantly alter protein, RNA, or DNA synthesis in the cell lines over the dose range used. In addition, there was no inhibition of cell-cell interaction or granule exocytosis during CTL-mediated killing. ATA also significantly inhibited the cytolysis and DNA fragmentation mediated by isolated cytolytic granules, as well as the granular protein fragmentin. We developed an assay in which target cells could be separated from CTL after binding and programming for lysis. Once they had received the "kiss of death," target cells could be rescued from lysis (as indicated by inhibition of DNA fragmentation and increased target cell viability) by treatment with ATA. These results suggest that ATA blocks target cell death by inhibition of DNA fragmentation, and further, that chromatin degradation is a cause rather than a result of cell death in CTL-mediated lysis.     

Stress and disturbance in the phytoplankton community of a shallow,hypertrophic lake

The validity of Connell's intermediate disturbance hypothesis in phytoplankton communities was tested on data from a hypertrophic, shallow lake, Hjarbæk Fjord, Denmark.The present data from Hjarbæk Fjord demonstrate the difficulties in distinguishing stress from disturbance in a phytoplankton community, and show that great changes in the phytoplankton community can take place within few days.A collapse of blue-green algae in late June 1986 caused remineralization of nutrients and resulted in a rapid increase of fast-growing small chlorococcal green algae and phytoplankton species diversity, without any external disturbances acting on the lake. External disturbances in the form of wind action and brackish water intrusion occurred several days after the onset of these events. Carbon depletion and pH 11.0 were severe stress factors on the phytoplankton community. They were induced by calm, warm weather, but eventually acted as a kind of disturbance to the normally well circulated lake.     

Deprotection of methylphosphonate oligonucleotides using a novel one-pot procedure.

Deprotection of methylphosphonate oligonucleotides with ethylenediamine was evaluated in a model system. Methylphosphonate sequences of the form 5'-TTTNNTTT, where N was either N4-bz-dC, N4-ibu-dC, N2-ibu-O6-DPC-dG, N2-ibu-dG, N6-bz-dA, or T, were used to determine the extent of modifications that occur during deprotection. Up to 15% of N4-bz-dC was found to transaminate at the C4 position when treated with ethylenediamine. A similar displacement reaction with ethylenediamine was observed at the O6 position of N2-ibu-O6-DPC-dG, and to a much lesser extent of N2-ibu-dG. Side reactions were not observed when oligonucleotides containing N4-ibu-dC, N6-bz-dA, or T were treated with ethylenediamine. A novel method of deprotecting methylphosphonate oligonucleotides was developed from these studies. The method incorporates a brief treatment with dilute ammonia for 30 minutes followed by addition of ethylenediamine for 6 hours at room temperature to complete deprotection in a one-pot format. The solution is then diluted and neutralized to stop the reaction and prepare the crude product for chromatographic purification. This method was used to successfully deprotect a series of oligonucleotides at the 1, 100, and 150 mumole scales. These deprotection results were compared to a commonly used two-step method and found to be superior in yield of product by as much as 250%.