Acknowledgments

I would like to thank Dr. Karel Schubert for his willingness to work with me over the last few years. The opportunity to stay in an educational atmosphere when grades might merit otherwise, has helped me through several family difficulties. I also thank Dr. Scott Russell and Dr. John Skvarla for their willingness to work with me and provide advice. Thanks are also given to the Schubert Lab for providing space to work, moral support and a variety of mentors. Thanks is given to the Botany- Microbiology department of The University of Oklahoma for allowing me to continue my graduate education. Special thanks also are provided for my wife Linda Sanders Hocker who is greatly responsible for providing me with the strength needed to finish this project.

Title Page

Acknowledgments

Table of Contents

Table of Figures

List of Tables

Abstract

Chapter 1: Introduction

Chapter 2: Materials And Methods

Chapter 3: Results

Chapter 4: Discussion

Chapter 5: Conclusions

References

Appendix A: Estimated Cost of CbMM Media:

Table of Figures

Figure 1. Life Cycle of C. elegans.

Figure 2. Population Growth Trend of C. elegans in CbMM Media.

Figure 3. Population Growth Pattern of C. elegans in Stock HS-YE-HLE Media.

Figure 4. Difference in Accuracy for the Different Counting Methods.

Figure 5. Experiment # 2 Average Population for C. elegans in M-9 Buffer with the Addition of URP-Extract.

Figure 6. Change in Nematode Population Growth Rate in CbMM Increasing Concentration of KRS-1297-5&6 Experiment #5.

Figure 7. Anatomical Variations Seen in C. elegans Cultures.

Figure 8. Factors Affecting Nematode Reproduction Pathways.

Figure 9. Example Rate Chart.

List of Tables

Table 1. Change in Nematode Populations in Cultures Containing URP-Extracts Experiment # 1.

Table 2. Change in Nematode Population in Cultures with KRS-1086 Extracts Experiment #1.

Table 3. Change in Nematode Population in Cultures Containing Fractions after Heat Treatment Experiment # 2.

Table 4. Change in Nematode Population in Cultures with KRS-Fractions Experiment # 3.

Table 5. Change in Nematode Population in Cultures with KRS-1297 Fractions Experiment # 4.

Table 6. Change in Nematode Population in Cultures with Individual Fractions From KRS-1297, Experiment # 4.

Table 7. Change in Nematode Population in Cultures Containing Fractions of KRS-1297-5&6, after Ultra-filtration Experiment # 5.

Table 8. Agglutination Activity and Protein Concentrations of KRS-1297-5&6 Fractionated by Ultrafiltration in Centricon-50 Filters Experiment # 5.

Abstract

An assay system to identify bioactive proteins and other active molecules which inhibit nematode reproduction was developed. The system used a defined media to test the effects of known and unknown extracts on the reproduction potential of the free-living, bacteriophagous nematode Caenorhabditis elegans. C. elegans has been used as a model system to test for nematitoxins. This assay was developed as an initial test to select proteins inhibiting nematode reproduction. Future development of these proteins may help reduce damage caused by plant parasitic nematodes. Nematodes were initially produced in an axenic culture of soy protein, yeast extract and heated liver extract. Nematodes were washed and placed in a defined media for the assay. Extracts were added to the chemically defined media and the nematode populations were monitored. The populations were recorded after inoculation and every three to four days for a three week period. The average populations and rates of population growth of replicate treatments were compared to those of controls. Water or a buffer solution was used as a control depending on the extract used. Two extracts tested inhibited reproduction up to 70%. Inhibition of population growth was dependent on protein concentration. The active fractions agglutinated rabbit, pig and human red blood cells which is a characteristic of lectins. Although fractions were highly purified by size-fractionation chromatography, multiple bands were revealed after SDS-PAGE analysis. Preliminary results suggest that the active material may be a protein and possibly a lectin.

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©Copyright by James Randolph Hocker 1997

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