Spindle assembly checkpoint genes reveal distinct as well as overlapping expression that implicates MDF-2/Mad2 in postembryonic seam cell proliferation in Caenorhabditis elegans

Background

The spindle assembly checkpoint (SAC) delays anaphase onset by inhibiting the activity of the anaphase promoting complex/cyclosome (APC/C) until all of the kinetochores have properly attached to the spindle. The importance of SAC genes for genome stability is well established; however, the roles these genes play, during postembryonic development of a multicellular organism, remain largely unexplored.

Results

We have used GFP fusions of 5' upstream intergenic regulatory sequences to assay spatiotemporal expression patterns of eight conserved genes implicated in the spindle assembly checkpoint function in Caenorhabditis elegans. We have shown that regulatory sequences for all of the SAC genes drive ubiquitous GFP expression during early embryonic development. However, postembryonic spatial analysis revealed distinct, tissue-specific expression of SAC genes with striking co-expression in seam cells, as well as in the gut. Additionally, we show that the absence of MDF-2/Mad2 (one of the checkpoint genes) leads to aberrant number and alignment of seam cell nuclei, defects mainly attributed to abnormal postembryonic cell proliferation. Furthermore, we show that these defects are completely rescued by fzy-1(h1983)/CDC20, suggesting that regulation of the APC/C^CDC20 by the SAC component MDF-2 is important for proper postembryonic cell proliferation.

Conclusion

Our results indicate that SAC genes display different tissue-specific expression patterns during postembryonic development in C. elegans with significant co-expression in hypodermal seam cells and gut cells, suggesting that these genes have distinct as well as overlapping roles in postembryonic development that may or may not be related to their established roles in mitosis. Furthermore, we provide evidence, by monitoring seam cell lineage, that one of the checkpoint genes is required for proper postembryonic cell proliferation. Importantly, our research provides the first evidence that postembryonic cell division is more sensitive to SAC loss, in particular MDF-2 loss, than embryonic cell division.     

Use of C-banding for identifying radiation induced micronuclei

Differentiation of micronuclei (MN) caused by ionizing radiation from those caused by chemicals is a crucial step for managing treatment of individuals exposed to radiation. MN in binucleated lymphocytes in peripheral blood are widely used as biomarkers for estimating dose of radiation, but they are not specific for ionizing radiation. MN induced by ionizing radiation originate predominantly as a result of chromosome breaks (clastogenic action), whereas MN caused by chemical agents are derived from the loss of entire chromosomes (aneugenic action). C-banding highlights centromeres, which might make it possible to distinguish radiation induced MN, i.e., as a byproduct of acentric fragments, from those caused by the loss of entire chromosomes. To test the use of C-banding for identifying radiation induced MN, a blood sample from a healthy donor was irradiated with 3 Gy of Co-60 gamma rays and cultured. Cells were harvested and dropped onto slides, divided into a group stained directly with Giemsa and another processed for C banding, then stained with Giemsa. The frequency of MN in 500 binucleated cells was scored for each method. In preparations stained with Giemsa directly, the MN appeared as uniformly stained structures, whereas after C banding, some MN exhibited darker regions corresponding to centromeres that indicated that they were not derived from acentric fragments. The C-banding technique enables differentiation of MN from acentric chromosomal material. This distinction is useful for improving the specificity of the MN assay as a biomarker for ionizing radiation.     

Multiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia coli O157:H7, and Salmonella serotypes in water samples

Three pathogens, Campylobacter, Salmonella, and Shiga-toxin-producing Escherichia coli, are leading causes of bacterial gastroenteritis in the United States and worldwide. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water and irrigation water. The aim of this research was to develop two types of PCR assays that could detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR (m-PCR) using each specific primer pair simultaneously. Under optimized m-PCR conditions, the assay produced a 90-bp product for Campylobacter jejuni, a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella Typhimurium, and the limitation of detection was approximately 700 copies for all three bacteria. In addition, real-time PCR was performed to quantify the three pathogens using SYBR green fluorescence. The assay was designed so that each target had a different melting temperature [C. jejuni (80.1 °C), E. coli O157:H7 (83.3 °C), and S. Typhimurium (85.9 °C)]. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction.     

Selection and application of Streptococcus bovis as a silage inoculant.

Three strains of Streptococcus bovis, a homolactic bacterium capable of utilizing starch, were evaluated for growth kinetics and ability to decrease the pH of alfalfa silage. A selected strain was evaluated for its competitiveness as an inoculant with Enterococcus faecium, an organism used in inoculants, and for its ability to enhance the effect of a commercial inoculant. Testing was completed over three studies using wilted alfalfa (28 to 34% dry matter) ensiled into laboratory silos. Treatments were control, E. faecium, E. faecium and commercial inoculant, S. bovis, and S. bovis and commercial inoculant. Replicate silos were emptied and analyzed at 0.5, 1, 2, 4, 8, and 40 days for pH, fermentation products, and nitrogen fractions. S. bovis alone lowered the pH quicker and improved silage parameters early in the fermentation compared with E. faecium, the commercial inoculant, and control treatments. When combined with a commercial inoculant, S. bovis lowered pH more quickly than the commercial inoculant alone and E. faecium plus commercial inoculant. At 40 days, S. bovis combination had lower pH and ammonia nitrogen and acetate contents than the E. faecium combination. Starch in the silage was not utilized by S. bovis as had been anticipated. Results indicate that S. bovis was more effective than E. faecium as a silage inoculant and could enhance a commercial inoculant on low-dry-matter alfalfa.     

Cecal Propionic Acid as a Biological Indicator of the Early Establishment of a Microbial Ecosystem Inhibitory toSalmonellain Chicks

Experiments were conducted to evaluate the effects of different competitive exclusion (CE) cultures on the concentration of cecal propionic acid in 3-day-old broiler chicks, and the correlation between cecal propionic acid concentration and protection againstSalmonellacolonization. CE cultures that significantly (P < 0.05) increased cecal propionic acid in 3-day-old chicks decreased (P < 0.05) cecalSalmonellacolonization in 10-day-old chicks compared with the untreated controls. CE cultures that failed to significantly (P > 0.05) increase cecal propionic acid concentrations in 3-day-old chicks failed to protect (P > 0.05) against cecalSalmonellacolonization in 10-day-old chicks compared with untreated controls. A significant (P < .05) correlation (−.88) was found between cecal propionic acid concentration in 3-day-old chicks and cecalSalmonellacolonization in 10-day-old chicks.