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Faculty
Lawrence A. Klobutcher
Associate Dean of the Graduate School
Professor of Molecular, Microbial & Structural Biology
klobutcher@nso2.uchc.edu
Areas of Interest:
The laboratory employs ciliated protozoa as model organisms to study
basic cellular processes. Currently, the following two areas of research
are being pursued:
Phagocytosis in Tetrahymena thermophila. Phagocytosis refers
to the process by which cells are able to ingest large particles (>1
um). In vertebrates, phagocytosis mainly occurs in specialized cells of
the immune system (macrophages, monocyctes, and neutrophils). Such
“professional phagocytes” serve as a primary line of defense by
ingesting invading pathogens, and also activate specific immune
responses. In addition, phagocytosis is important in clearing cell
debris and for tissue remodeling during development. Finally, a number
of microbial pathogens (e.g., Salmonella, Legionella,
Mycobacterium, anthrax, and specific types of yeast) utilize and/or
subvert phagocytosis as a means of entering cells. Learning how
phagocytosis is carried out is thus important for understanding tissue
maintenance and immune defense in humans, as well as the infection
strategies of some disease-causing microorganisms.
Research in a number of experimental systems has made it clear that
phagocytosis is a multistep process that involves hundreds of genes and
proteins. Nonetheless, the identities and functions of phagocytic genes,
and the molecular mechanisms of phagocytosis, are still poorly
understood. We are seeking to further our understanding of phagocytosis
by studying Tetrahymena thermophila, an organisms particularly
amenable to genetic and molecular biological analysis. In nature,
Tetrahymena uses phagocytosis to feed on bacteria and other
microorganisms, but in the laboratory it can be grown on defined culture
medium where phagocytosis is not an essential process. This feature has
allowed us to develop a screening procedure for isolating cells that are
deficient in phagocytosis, which will lead to the identification of new
genes/proteins that are involved in various steps of the process. In
addition, a system for the efficient purification of phagosomes from
Tetrahymena has been developed, and we are pursuing mass
spectrometry approaches to define the complete Tetrahymena
phagosome proteome. This analysis is expected to identify numerous new
genes previously unsuspected of playing a role in phagocytosis. The
genetic tools available in Tetrahymena, coupled with its
favorable cytological features, will allow us to investigate the
localization and function of these novel proteins.
Frequent Frameshifting in Euplotes crassus. Ciliated
protozoa, including members of the genus Euplotes, are unusual in that
they employ alternative genetic codes to specify how mRNAs are
translated into proteins. Previously. the genetic code had been
considered universal and unalterable. The observation that many ciliates
have altered genetic codes in which canonical stop codons are decoded as
sense (stop codon reassignment) shows that even the code is subject to
evolutionary pressure to change. In addition, recent data suggest that
Euplotes genes also have an unusually high frequency of
programmed +1 translational frameshifting. In our own pilot sequencing
survey of 25 randomly selected genes, we observed 3 genes that require a
+1 frameshift, indicating that more than 10% of the genes in the genome
may require such a frameshift for expression. we have also carried out a
phylogenetic analysis on the origin of frameshift sites within the
telomerase reverse transcriptase genes of Euplotes species, and
have found that two frameshift sites have arisen during the evolution of
this group. In other organisms, frameshifting is involved in regulation
of gene expression. The apparent high frequency of frameshift genes in
Euplotes is unprecedented, and suggests that the organism has
particular features that have potentiated the origin of frameshift sites
within genes and that allow for efficient frameshifting.
The mechanism of frameshifting in Euplotes is unknown. The
initial open reading frame of all the Euplotes frameshift genes
terminates with an AAA lysine codon, followed by a stop codon (usually
UAA), and an additional A residue. This AAA-UAA-A motif suggests that
the Euplotes genes may employ a "shifty stop" mode of
frameshifting. There are two features of a typical "shifty stop" site.
First, there is a slippery codon (AAA in Euplotes) that, during
translation, would allow the cognate tRNA to slip forward 1 base and
still maintain two correct base pairs with the mRNA. Second, there is a
poorly recognized termination tetranucleotide (the stop codon plus the
next base) that is thought to slow translation, providing an opportunity
for the usually rare slippage in reading frame. Surprisingly, and
perhaps contrary to this model, UAA-A frequently occurs at natural sites
of translation termination in Euplotes. To explain this apparent
enigma, we have developed a model in which stop codon reassignment is
linked to the mechanism of frameshifting (Klobutcher and Farabaugh,
2002, Cell 111:763). Stop codon reassignment requires changes in
the translation termination factor eRF1 such that it can no longer
recognize the reassigned stop codon (UGA in Euplotes). We
postulate that these changes have also impaired the recognition of the
remaining stop codons, so that translation termination is a slow step.
Thus, the slowing of translation when a stop codon is encountered would
provide an opportunity for a +1 frameshift in the context of a slippery
codon. We plan to test this model by both defining the minimal nucleic
acid sequence element(s) that promotes frameshifting, and by determining
if Euplotes eRF1 has reduced affinity for stop codons. As a
further test of the hypothesis, we plan to determine if a high frequency
of frameshifting is observed in other ciliates that have independently
undergone stop codon reassignment.
Selected Publications:
Jacobs ME, DeSouza LV, Samaranayake H, Pearlman RE, Siu KW,
Klobutcher LA. 2006. The Tetrahymena thermophila phagosome proteome.
Eukaryot Cell. Dec;5(12):1990-2000.
Klobutcher LA, Ragkousi K, Setlow P. 2006. The Bacillus subtilis
spore coat provides "eat resistance" during phagocytic predation by the
protozoan Tetrahymena thermophila. Proc Natl Acad Sci U S A. Jan
3;103(1):165-70.
Klobutcher LA. 2005. Sequencing of random Euplotes crassus
macronuclear genes supports a high frequency of +1 translational
frameshifting. Eukaryot Cell. Dec;4(12):2098-105.
Jacobs ME, Cortezzo DE, Klobutcher LA. 2004 Assessing the
effectiveness of coding and non-coding regions in antisense ribosome
inhibition of gene expression in Tetrahymena. J Eukaryot Microbiol.
Sep-Oct;51(5):536-41.
Mollenbeck M, Gavin MC, Klobutcher LA. 2004. Evolution of programmed
ribosomal frameshifting in the TERT genes of Euplotes. J Mol Evol.
Jun;58(6):701-11.
Jacobs ME, Sanchez-Blanco A, Katz LA, Klobutcher LA. 2003. Tec3, a
new developmentally eliminated DNA element in Euplotes crassus.
Eukaryot Cell. Feb;2(1):103-14.
Klobutcher, L.A., and Farabaugh, P.J. 2002. Shifty ciliates: frequent
programmed translational frameshifting in euplotids. Cell 111,
763-766.
Jahn, C.L., and Klobutcher, L.A. 2002. Genome remodeling in ciliated
protozoa. Ann. Rev. Micro., 56, 489-520.
Mollenbeck, M., and Klobutcher, L.A. 2002. De novo telomere
addition to spacer sequences prior to their developmental degradation in
Euplotes crassus. Nucleic Acids Res. 30, 523-531.
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