Date of Award

2000

Degree Type

Thesis

Degree Name

MS Biology

Department

Biology

Advisor

Carolyn Bentivegna

Committee Member

Maria MacWilliams

Committee Member

Eliot Krause

Committee Member

Sulie L. Chang

Keywords

mRNA, Microsatellite mRNA, Chironomous riparius

Abstract

The expression of a unique mRNA containing a microsatellite sequence was studied in C. riparius using differential display. First, the rnicrosatellite fragment was sequenced using Sanger's Sequencing method. The microsatellite cDNA contained 11 "ACA" repeats, 12 "AC" repeats, and 5 "CTC" repeats. Second, the affects of cadmium and heat shock on rnicrosatellite mRNA expression were studied. In cadmium experiments, larvae were exposed to low cadmium concentrations (0, 0.04, 0.4, and 4.0 mM) for 24 and 48 h and high cadmium concentrations (0, 1.0, 2.0, 4.0, 8.0, and 16.0 mM) for 48 h. The effects of cadmium on fed larvae at 48 h was also investigated. Results showed that microsatellite mRNA expression in C. riparius was unaffected by cadmium at either time point without food (p = 0.732, 0.885, 0.702, respectively). Feeding did not significantly alter microsatellite mRNA expression in C. riparius (p = 0.857). In heat shock experiments, the starved larvae were exposed to 20, 28, 32, and 37 °ҴC at 30 min. Results show that the level of microsatellite mRNA expression was significantly reduced by 37 °C compared to 20 °C (p - 0.002). The levels of microsatellite mRNA was significantly increased when the temperature was elevated from 20 to 28 °C (p = 0.03). The 32 and 37 °C groups were significantly lower than the 28 °C group (p = 0.014 and 0.002, respectively). Previous work in C. ripartus has shown that ribosomal L8 mRNA expression is consistent at all the temperatures tested (Govinda et al., 2000). Differential display was run using the same mRNA samples as used for the microsatellite mRNA heat shock experiment. Results showed that ribosomal mRNA was consistently expressed in heat shock mRNA at all temperatures tested (p = 0.173). In actinomycin D experiments, a concentration of96 nM actinomycin D down regulated microsatellite mRNA transcription in C. riparius (p = 0.054). The following was learned from this project: 1) Results from the heat shock experiment showed that differential display is a sensitive method to detect gene expression. 2) Results from cadmium studies suggested that microsatellite mRNA can be used as a negative control in future cadmium toxicity tests in C. riparius. 3) Results from heat shock studies indicated that this type of stress does modify microsatellite mRNA expression in C. riparius ..

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