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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