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Lawrence A Klobutcher

Publications and source records attributed to Lawrence A Klobutcher.

10 recordsLinked to original sources
PubMed2006

The Tetrahymena thermophila phagosome proteome.

In vertebrates, phagocytosis occurs mainly in specialized cells of the immune system and serves as a primary defense against invading pathogens, but it also plays a role in clearing apoptotic cells and in tissue remodeling during development. In contrast, unicellular eukaryotes, such as the ciliate Tetrahymena thermophila, employ phagocytosis to ingest and degrade other microorganisms to meet their nutritional needs. To learn more about the protein components of the multistep process of phagocytosis, we carried out an analysis of the Tetrahymena phagosome proteome. Tetrahymena cells were fed polystyrene beads, which allowed for the efficient purification of phagosomes. The protein composition of purified phagosomes was then analyzed by multidimensional separation coupled with tandem mass spectrometry. A total of 453 peptides were identified that resulted in the identification of 73 putative phagosome proteins. Twenty-eight of the proteins have been implicated in phagocytosis in other organisms, indicating that key aspects of phagocytosis were conserved during evolution. Other identified proteins have not previously been associated with phagocytosis, including some of unknown function. Live-cell confocal fluorescence imaging of Tetrahymena strains expressing green fluorescent protein-tagged versions of four of the identified phagosome proteins provided evidence that at least three of the proteins (including two with unknown functions) are associated with phagosomes, indicating that the bulk of the proteins identified in the analyses are indeed phagosome associated.

Animals↗
PubMed2006

Distinct paths to stop codon reassignment by the variant-code organisms Tetrahymena and Euplotes.

The reassignment of stop codons is common among many ciliate species. For example, Tetrahymena species recognize only UGA as a stop codon, while Euplotes species recognize only UAA and UAG as stop codons. Recent studies have shown that domain 1 of the translation termination factor eRF1 mediates stop codon recognition. While it is commonly assumed that changes in domain 1 of ciliate eRF1s are responsible for altered stop codon recognition, this has never been demonstrated in vivo. To carry out such an analysis, we made hybrid proteins that contained eRF1 domain 1 from either Tetrahymena thermophila or Euplotes octocarinatus fused to eRF1 domains 2 and 3 from Saccharomyces cerevisiae. We found that the Tetrahymena hybrid eRF1 efficiently terminated at all three stop codons when expressed in yeast cells, indicating that domain 1 is not the sole determinant of stop codon recognition in Tetrahymena species. In contrast, the Euplotes hybrid facilitated efficient translation termination at UAA and UAG codons but not at the UGA codon. Together, these results indicate that while domain 1 facilitates stop codon recognition, other factors can influence this process. Our findings also indicate that these two ciliate species used distinct approaches to diverge from the universal genetic code.

Animals↗
PubMed2005

The Bacillus subtilis spore coat provides "eat resistance" during phagocytic predation by the protozoan Tetrahymena thermophila.

Bacillus spores are highly resistant to many environmental stresses, owing in part to the presence of multiple "extracellular" layers. Although the role of some of these extracellular layers in resistance to particular stresses is known, the function of one of the outermost layers, the spore coat, is not completely understood. This study sought to determine whether the spore coat plays a role in resistance to predation by the ciliated protozoan Tetrahymena, which uses phagocytosis to ingest and degrade other microorganisms. Wild-type dormant spores of Bacillus subtilis were efficiently ingested by the protozoan Tetrahymena thermophila but were neither digested nor killed. However, spores with various coat defects were killed and digested, leaving only an outer shell termed a rind, and supporting the growth of Tetrahymena. A similar rind was generated when coat-defective spores were treated with lysozyme alone. The sensitivity of spores with different coat defects to predation by T. thermophila paralleled the spores' sensitivities to lysozyme. Spore killing by T. thermophila was by means of lytic enzymes within the protozoal phagosome, not by initial spore germination followed by killing. These findings suggest that a major function of the coat of spores of Bacillus species is to protect spores against predation. We also found that indigestible rinds were generated even from spores in which cross-linking of coat proteins was greatly reduced, implying the existence of a coat structure that is highly resistant to degradative enzymes.

Animals↗
PubMed2005

Sequencing of random Euplotes crassus macronuclear genes supports a high frequency of +1 translational frameshifting.

Programmed translational frameshifts have been identified in genes from a broad range of organisms, but typically only a very few genes in a given organism require a frameshift for expression. In contrast, a recent analysis of gene sequences available in GenBank from ciliates in the genus Euplotes indicated that >5% required one or more +1 translational frameshifts to produce their predicted protein products. However, this sample of genes was nonrandom, biased, and derived from multiple Euplotes species. To test whether there truly is an abundance of frameshift genes in Euplotes, and to more accurately assess their frequency, we sequenced a random sample of 25 cloned genes/macronuclear DNA molecules from Euplotes crassus. Three new candidate +1 frameshift genes were identified in the sample that encode a membrane occupation and recognition nexus (MORN) repeat protein, a C(2)H(2)-type zinc finger protein, and a Ser/Thr protein kinase. Reverse transcription-PCR analyses indicate that all three genes are expressed in vegetatively proliferating cells and that the mRNAs retain the requirement of a frameshift. Although the sample of sequenced genes is relatively small, the results indicate that the frequency of genes requiring frameshifts in E. crassus is between 3.7% and 31.7% (at a 95% confidence interval). The current and past data also indicate that frameshift sites are found predominantly in genes that likely encode nonabundant proteins in the cell.

Animals↗
PubMed2004

Evolution of programmed ribosomal frameshifting in the TERT genes of Euplotes.

A number of recent studies indicate that programmed + 1 ribosomal frameshifting is frequently required for the expression of genes in species of the genus Euplotes. In E. crassus, three genes encoding the telomerase reverse transcriptase (TERT) subunit have been previously found to possess one or two + 1 frameshift sites. To examine the origin of frameshift sites within the Euplotes group, we have isolated segments of the TERT gene from five Euplotes species. Coupled with phylogenetic analysis, the results indicate that one frameshift site in the TERT gene arose late in the evolution of the group. In addition, a novel frameshift site was identified in the TERT gene of E. minuta, a species where frameshifting has not been previously reported. Coupled with other studies, the results indicate that frameshift sites have arisen during the diversification of the euplotids. The results also are discussed in regard to the mutations necessary to generate frameshift sites, and the specialization of TERT protein function that has apparently occurred in E. crassus.

Amino Acid Sequence↗
PubMed2003

Tec3, a new developmentally eliminated DNA element in Euplotes crassus.

More than 100,000 interstitial segments of DNA (internal eliminated sequences [IESs]) are excised from the genome during the formation of a new macronucleus in Euplotes crassus. IESs include unique sequence DNA as well as two related families of transposable elements, Tec1 and Tec2. Here we describe a new class of E. crassus transposons, Tec3, which is present in 20 to 30 copies in the micronuclear genome. Tec3 elements have long inverted terminal repeats and contain a degenerate open reading frame encoding a tyrosine-type recombinase. One characterized copy of Tec3 (Tec3-1) is 4.48 kbp long, has 1.23-kbp inverted terminal repeats, and resides within the micronuclear copy of the ribosomal protein L29 gene (RPL29). The 23 bp at the extreme ends of this element are very similar to those in other E. crassus IESs and, like these other IESs, Tec3-1 is excised during the polytene chromosome stage of macronuclear development to generate a free circular form with an unusual junction structure. In contrast, a second cloned element, Tec3-2, is quite similar to Tec3-1 but lacks the terminal 258 bp of the inverted repeats, so that its ends do not resemble the other E. crassus IES termini. The Tec3-2 element appears to reside in a large segment of the micronuclear genome that is subject to developmental elimination. Models for the origins of these two types of Tec3 elements are presented, along with a discussion of how some members of this new transposon family may have come to be excised by the same machinery that removes other E. crassus IESs.

Animals↗
PubMed2002

Genome remodeling in ciliated protozoa.

The germline genomes of ciliated protozoa are dynamic structures, undergoing massive DNA rearrangement during the formation of a functional macronucleus. Macronuclear development involves chromosome fragmentation coupled with de novo telomere synthesis, numerous DNA splicing events that remove internal segments of DNA, and, in some ciliates, the reordering of scrambled gene segments. Despite the fact that all ciliates share similar forms of DNA rearrangement, there appears to be great diversity in both the nature of the rearranged DNA and the molecular mechanisms involved. Epigenetic effects on rearrangement have also been observed, and recent work suggests that chromatin differentiation plays a role in specifying DNA segments either for rearrangement or for elimination.

Animals↗
PubMed2002

De novo telomere addition to spacer sequences prior to their developmental degradation in Euplotes crassus.

During sexual reproduction, Euplotes crassus precisely fragments its micronuclear chromosomes and synthesizes new telomeres onto the resulting DNA ends to generate functional macronuclear minichromosomes. In the micronuclear chromosomes, the macronuclear-destined sequences are typically separated from each other by spacer DNA segments, which are eliminated following chromosome fragmentation. Recently, in vivo chromosome fragmentation intermediates that had not yet undergone telomere addition have been characterized. The ends of both the macronuclear-destined and eliminated spacers were found to consist of six-base, 3' overhangs. As this terminal structure on the macronuclear-destined sequences serves as the substrate for de novo telomere addition, we sought to determine if the spacer DNAs might also undergo telomere addition prior to their elimination. Using a polymerase chain reaction approach, we found that at least some spacer DNAs undergo de novo telomere addition. In contrast to macronuclear-destined sequences, heterogeneity could be observed in the position of telomeric repeat addition. The observation of spacer DNAs with telomeric repeats makes it unlikely that differential telomere addition is responsible for differentiating between retained and eliminated DNA. The heterogeneity in telomere addition sites for spacer DNA also resembles the situation found for telomeric repeat addition to macronuclear-destined sequences in other ciliate species.

Animals↗
PubMedDate not supplied

Assessing the effectiveness of coding and non-coding regions in antisense ribosome inhibition of gene expression in Tetrahymena.

In Tetrahymena thermophila, an "antisense ribosome" technology has been developed for inhibiting gene expression and generating novel mutants. Short segments of genes are inserted in antisense orientation into an rDNA vector in a region corresponding to an external loop of the folded rRNA. DNA segments derived from the 5'-ends of genes have proven most effective in reducing cognate gene expression. To investigate the efficacy of other genic regions, we generated Tetrahymena cell lines with antisense ribosome constructs containing 100-bp DNA segments derived from the 5'-ends, 3'-ends, and internal coding regions of two non-essential genes, granule lattice protein 1 and macronuclear histone H1. The 5'- and 3'-end constructs inhibited gene expression, but antisense ribosomes derived exclusively from coding regions had little effect.

Animals↗