Purdue University PURDUE AGRICULTURE
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Eukaryotic cell differentiation is generally considered to be a property of metazoa (multicellular organisms), yet dramatic examples of regulated cellular changes also occur in protozoa. Our laboratory is investigating two such examples; one is the formation of the macronucleus during sexual reproduction in ciliated protozoa and the other is the morphological differentiation of Leishmania parasites as they move from insect vector to human hosts. The recent availability of genome sequences for all three of the protozoa used in these studies (Paramecium, Tetrahymena and Leishmania) have opened new lines of investigation and created exciting research opportunities.

Ciliated protozoa contain two types of nuclei, micronuclei and macronuclei, which arise from a single diploid fertilization nucleus during sexual reproduction. During the formation of the macronucleus, the germline chromosomes are amplified, fragmented and specific DNA sequences are eliminated. In Paramecium the coding regions of many micronuclear genes are interrupted by internal eliminated sequences (IES) that must be removed precisely to form the open reading frames in the macronuclear genome. These IESs range in size from 26 bp to over 1 kb and are always flanked by the dinucleotide 5'-TdA-3'. The isolation of mutant cell lines that are unable to remove a specific IES have revealed nucleotides located within the first 6 base pairs of the eliminated sequence which are important for IES excision. Interestingly, this region contains a consensus sequence that has similarity to the termini of the mariner/Tc1 family of transposable elements. The possible relationship between this wide spread family of transposons and Paramecium IESs will require detailed knowledge of the cis and trans acting elements necessary for excision. An in vivo assay for IES excision is being used to dissect the cis acting sequences required for precise and efficient removal of these short DNA elements. Reverse genetic approaches that disrupt the expression of genes encoding developmentally regulated transcripts in Paramecium and Tetrahymena thermophila are being used to identify proteins involved in DNA rearrangement and macronuclear development.

Recently, we began a second project with Dr. Jonathan LeBowitz to investigate the cellular differentiation of Leishmania mexicana, a protozoan parasite that infects humans. Although leishmaniasis is a rare disease in the United States, internationally it is a significant problem that is prevalent in many tropical and desert regions including South America, India and the Persian Gulf.  The flagellated insect form of the parasite differentiates into a non-flagellated form after infection of a human host. A remarkable feature of Leishmania is that it has little or no transcriptional regulation; gene expression is regulated at the post-transcriptional level.  Initial studies by the LeBowitz group showed that the abundance of some mRNAs between the two stages of the life cycle is regulated by a short 9-nucleotide element in the 3’ untranslated region. We performed whole genome microarrays to identify the entire population of regulated mRNAs between the two stages and we demonstrated that a subset of these mRNAs are controlled by the same 9-nucleotide element. Future investigations will capitalize on genomics, proteomics and bioinformatics approaches to identify candidate regulatory genes that will be the focus of additional study.

 

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