DNA sequencing of lysA gene of a rumen protozoan species

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In connection with the fact that the head investigator had discovered an ability of the rumen protozoa to synthesize lysine from diaminopirnelate (DAP), this project was conducted to screen a gene for coding DAPdecarboxylase to produce lysine (lysA) from a cDNA library of Entodinium caudsturn, one of the popular ruinen protozoa, and to determine its DNAsequence, in order to clarify whether or not the enzyme could be produced by the protozoa themselves. At first, mono-faunated goats which contain only E.caudatum as protozoa in the rinnen were prepared. Then the cDNA library was made from the protozoa collected from the goats and was used for screening lysA using lysine-requiring mutant of Escherichia coli. However, the result was negative, In the next step, the library was again used for screening lysA with digoxigenin using a probe made from the nuceotide sequences with high homology common to the lysAs of six sorts of bacteria. As a result a positive plaque was found and its DNA seque … More nce was determined. The result of the analysis of the DNA sequence, however, revealed high homologies not with lysA but with sperm-tail specific proteins and surface antigen of Paramecium. Then the nuceotide sequences with high homology common to the lysAs of six sorts of bacteria was again used as primers to examine PCR products using the cDNA library as a template, but no products were obtained. Since every trial resulted in failure as mentioned above, lysA seemed not to exist in the cDNA library. Finally we marked endosymbionts in the cells of E.caudatwn and examined the distribution of DAP decarboxylase activity in the cells. it was shown that the precipitates after centrifugation at 8,000 and 37,000 x g had high activity of the enzyme. Observations of the precipitates by a transmission electron microscope revealed that a lot of oval bacterium-like particles were included in both precipitates and the particles also existed in vesicles in the intact cells. DNA (20,000 bp) has been extracted from the particles. We already started screening lysA again from the DNA and expect to attain the initial objective. Less