DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Promoters from kin1 and cor6.6, two Arabidopsis thaliana low-temperature-and ABA-inducible genes,direct strong β-glucuronidase expression in guard cells,pollen and young developing seeds

The ability of most higher plants to withstand freezing can be enhanced by cold acclimation, although the freezing tolerance of plant tissues is also affected by their developmental stage. In addition, low temperature has pleiotropic effects on many plant developmental processes such as vernalization. The interaction between plant development and low temperature implies that some genes are regulated by both environmental factors and developmental cues. Although a number of cold-inducible genes from plants have been identified, information concerning their regulation during plant development is limited. In order to understand their developmental regulation and obtain possible clues as to function, the promoters of kin1 and cor6.6, two cold- and abscisic acid (ABA)-regulated genes from Arabidopsis thaliana, were fused to the -glucuronidase (GUS)-coding sequence and the resulting constructs were used to transform tobacco and A. thaliana. Transgenic plants with either the kin1 or cor6.6 promoter showed strong GUS expression in pollen, developing seeds, trichomes and, most interestingly, in guard cells. During pollen development, maximum GUS activity was found in mature pollen. In contrast, the maximum GUS activity during seed development was during early embryogenesis. These patterns of expression distinguish kin1 and cor6.6 from related lea genes which are strongly expressed during late embryogenesis. There was no major qualitative difference in patterns of GUS expression between kin1 and cor6.6 promoters and the results were similar for transgenic tobacco and Arabidopsis. Considering the results described, as well as those in an accompanying paper Wang et al., 1995, Plant Mol Biol 28: 605–617 (this issue), we suggest that osmotic potential might be a major factor in regulating the expression of kin1 and cor6.6 during several developmental processes. The implication of the results for possible function of the gene products is discussed.     

Microarray-based genomic selection for high-throughput resequencing

We developed a general method, microarray-based genomic selection (MGS), capable of selecting and enriching targeted sequences from complex eukaryotic genomes without the repeat blocking steps necessary for bacterial artificial chromosome (BAC)-based genomic selection. We demonstrate that large human genomic regions, on the order of hundreds of kilobases, can be enriched and resequenced with resequencing arrays. MGS, when combined with a next-generation resequencing technology, can enable large-scale resequencing in single-investigator laboratories.     

Mapping of Quantitative Trait Loci Underlying Cold Tolerance in Rice Seedlings via High-Throughput Sequencing of Pooled Extremes

Low temperature is a major limiting factor in rice growth and development. Mapping of quantitative trait loci (QTLs) controlling cold tolerance is important for rice breeding. Recent studies have suggested that bulked segregant analysis (BSA) combined with next-generation sequencing (NGS) can be an efficient and cost-effective way for QTL mapping. In this study, we employed NGS-assisted BSA to map QTLs conferring cold tolerance at the seedling stage in rice. By deep sequencing of a pair of large DNA pools acquired from a very large F₃ population (10,800 individuals), we obtained ∼450,000 single nucleotide polymorphisms (SNPs) after strict screening. We employed two statistical methods for QTL analysis based on these SNPs, which yielded consistent results. Six QTLs were mapped on chromosomes 1, 2, 5, 8 and 10. The three most significant QTLs on chromosomes 1, 2 and 8 were validated by comparison with previous studies. Two QTLs on chromosomes 2 and 5 were also identified previously, but at the booting stage rather than the seedling stage, suggesting that some QTLs may function at different developmental stages, which would be useful for cold tolerance breeding in rice. Compared with previously reported QTL mapping studies for cold tolerance in rice based on the traditional approaches, the results of this study demonstrated the advantages of NGS-assisted BSA in both efficiency and statistical power.     

Umbilical cord-derived mesenchymal stromal cells modulate monocyte function to suppress T cell proliferation

Mesenchymal stromal cells (MSCs) may be derived from a variety of tissues, with human umbilical cord (UC) providing an abundant and noninvasive source. Human UC-MSCs share similar in vitro immunosuppressive properties as MSCs obtained from bone marrow and cord blood. However, the mechanisms and cellular interactions used by MSCs to control immune responses remain to be fully elucidated. In this paper, we report that suppression of mitogen-induced T cell proliferation by human UC-, bone marrow-, and cord blood-MSCs required monocytes. Removal of monocytes but not B cells from human adult PBMCs (PBMNCs) reduced the immunosuppressive effects of MSCs on T cell proliferation. There was rapid modulation of a number of cell surface molecules on monocytes when PBMCs or alloantigen-activated PBMNCs were cultured with UC-MSCs. Indomethacin treatment significantly inhibited the ability of UC-MSCs to suppress T cell proliferation, indicating an important role for PGE(2). Monocytes purified from UC-MSC coculture had significantly reduced accessory cell and allostimulatory function when tested in subsequent T cell proliferation assays, an effect mediated in part by UC-MSC PGE(2) production and enhanced by PBMNC alloactivation. Therefore, we identify monocytes as an essential intermediary through which UC-MSCs mediate their suppressive effects on T cell proliferation.     

Experimental autoallergic sialadenitis in the LEW rat. I. Parameters of disease induction

Experimental autoallergic sialadenitis (EAS) is an autoimmune mononuclear cell infiltration of the submandibular salivary gland that results in tissue destruction and glandular dysfunction. A previous report has described an animal model of induced EAS in LEW rats following sensitization with allogeneic WF submandibular gland (SMG). The present study extends this observation to an EAS disease model induced following sensitization of LEW rats with syngeneic LEW SMG. Furthermore, we describe the characterization of the mononuclear cells in the glandular infiltrates, evaluate the production of autoantibodies, and establish the parameters important for reproducible induction of EAS. Our results demonstrate that EAS can be induced in a completely syngeneic system and the histopathology of disease induction in the syngeneic and allogeneic model systems is similar. Helper/inducer (CD4+) and suppressor/cytotoxic (CD8+) T-cell subsets are the dominant cell types in the salivary mononuclear cell infiltrate. An anti-duct autoantibody was found in the serum of virtually all LEW rats with EAS. Although closely associated with disease development, the presence of this antibody was not a prerequisite for development of histopathologic disease. Induction of disease in both the syngeneic and allogeneic models of EAS is dependent upon administration of Bordetella pertussis at the time of sensitization. Finally, the histopathology of the cellular infiltrates in both the allogeneic and syngeneic models of EAS resemble those observed in the salivary tissues of Sj?gren's patients. While there are several differences between EAS in the LEW rat and the full expression of Sj?gren's syndrome, EAS may serve as a model to study the salivary gland component of this complex human disease.