(1/475) A novel H2A/H4 nucleosomal histone acetyltransferase in Tetrahymena thermophila.
Recently, we reported the identification of a 55-kDa polypeptide (p55) from Tetrahymena macronuclei as a catalytic subunit of a transcription-associated histone acetyltransferase (HAT A). Extensive homology between p55 and Gcn5p, a component of the SAGA and ADA transcriptional coactivator complexes in budding yeast, suggests an immediate link between the regulation of chromatin structure and transcriptional output. Here we report the characterization of a second transcription-associated HAT activity from Tetrahymena macronuclei. This novel activity is distinct from complexes containing p55 and putative ciliate SAGA and ADA components and shares several characteristics with NuA4 (for nucleosomal H2A/H4), a 1.8-MDa, Gcn5p-independent HAT complex recently described in yeast. A key feature of both the NuA4 and Tetrahymena activities is their acetylation site specificity for lysines 5, 8, 12, and 16 of H4 and lysines 5 and 9 of H2A in nucleosomal substrates, patterns that are distinct from those of known Gcn5p family members. Moreover, like NuA4, the Tetrahymena activity is capable of activating transcription from nucleosomal templates in vitro in an acetyl coenzyme A-dependent fashion. Unlike NuA4, however, sucrose gradient analyses of the ciliate enzyme, following sequential denaturation and renaturation, estimate the molecular size of the catalytically active subunit to be approximately 80 kDa, consistent with the notion that a single polypeptide or a stable subcomplex is sufficient for this H2A/H4 nucleosomal HAT activity. Together, these data document the importance of this novel HAT activity for transcriptional activation from chromatin templates and suggest that a second catalytic HAT subunit, in addition to p55/Gcn5p, is conserved between yeast and Tetrahymena. (+info)
(2/475) Gene knockouts reveal separate functions for two cytoplasmic dyneins in Tetrahymena thermophila.
In many organisms, there are multiple isoforms of cytoplasmic dynein heavy chains, and division of labor among the isoforms would provide a mechanism to regulate dynein function. The targeted disruption of somatic genes in Tetrahymena thermophila presents the opportunity to determine the contributions of individual dynein isoforms in a single cell that expresses multiple dynein heavy chain genes. Substantial portions of two Tetrahymena cytoplasmic dynein heavy chain genes were cloned, and their motor domains were sequenced. Tetrahymena DYH1 encodes the ubiquitous cytoplasmic dynein Dyh1, and DYH2 encodes a second cytoplasmic dynein isoform, Dyh2. The disruption of DYH1, but not DYH2, resulted in cells with two detectable defects: 1) phagocytic activity was inhibited, and 2) the cells failed to distribute their chromosomes correctly during micronuclear mitosis. In contrast, the disruption of DYH2 resulted in a loss of regulation of cell size and cell shape and in the apparent inability of the cells to repair their cortical cytoskeletons. We conclude that the two dyneins perform separate tasks in Tetrahymena. (+info)
(3/475) Molecular cloning and expression of a stress-responsive mitogen-activated protein kinase-related kinase from Tetrahymena cells.
To identify genes responsive to cold stress, we employed the differential display mRNA analysis technique to isolate a novel gene from Tetrahymena thermophila which encodes a protein kinase of 430 amino acids. A homolog of this kinase with 90% amino acid sequence identity was also found in T. pyriformis. Both kinases contain 11 subdomains typical of protein kinases. Sequence analysis revealed that the predicted amino acid sequences resemble those of mitogen-activated protein kinase (MAPK), especially p38 and stress-activated protein kinase which are known to be involved in various stress responses. However, it should be noted that the tyrosine residue in the normally conserved MAPK phosphorylation site (Thr-X-Tyr) is replaced by histidine (Thr226-Gly-His228) in this MAPK-related kinase (MRK). The recombinant MRK expressed in Escherichia coli phosphorylated myelin basic protein (MBP) and became autophosphorylated. However, the mutated recombinant protein in which Thr226 was replaced by Ala lost the ability to phosphorylate MBP, suggesting that Thr226 residue is essential for kinase activity. The MRK mRNA transcript in T. thermophila increased markedly upon temperature downshift from 35 to 15 degrees C (0.8 degrees C/min). Interestingly, osmotic shock either by sorbitol (100-200 mM) or NaCl (25-100 mM) also induced mRNA expression of the MRK in T. pyriformis. In addition, the activity of the kinase as determined by an immune complex kinase assay using MBP as a substrate was also induced by osmotic stress. This is the first demonstration of a MAPK-related kinase in the unicellular eukaryotic protozoan Tetrahymena that is induced by physical stresses such as cold temperature and osmolarity. The present results suggest that this MRK may function in the stress-signaling pathway in Tetrahymena cells. (+info)
(4/475) Phosphorylation of histone H3 is required for proper chromosome condensation and segregation.
Phosphorylation of histone H3 at serine 10 occurs during mitosis in diverse eukaryotes and correlates closely with mitotic and meiotic chromosome condensation. To better understand the function of H3 phosphorylation in vivo, we created strains of Tetrahymena in which a mutant H3 gene (S10A) was the only gene encoding the major H3 protein. Although both micronuclei and macronuclei contain H3 in typical nucleosomal structures, defects in nuclear divisions were restricted to mitotically dividing micronuclei; macronuclei, which are amitotic, showed no defects. Strains lacking phosphorylated H3 showed abnormal chromosome segregation, resulting in extensive chromosome loss during mitosis. During meiosis, micronuclei underwent abnormal chromosome condensation and failed to faithfully transmit chromosomes. These results demonstrate that H3 serine 10 phosphorylation is causally linked to chromosome condensation and segregation in vivo and is required for proper chromosome dynamics. (+info)
(5/475) A nonessential HP1-like protein affects starvation-induced assembly of condensed chromatin and gene expression in macronuclei of Tetrahymena thermophila.
Heterochromatin represents a specialized chromatin environment vital to both the repression and expression of certain eukaryotic genes. One of the best-studied heterochromatin-associated proteins is Drosophila HP1. In this report, we have disrupted all somatic copies of the Tetrahymena HHP1 gene, which encodes an HP1-like protein, Hhp1p, in macronuclei (H. Huang, E. A. Wiley, R. C. Lending, and C. D. Allis, Proc. Natl. Acad. Sci. USA 95:13624-13629, 1998). Unlike the Drosophila HP1 gene, HHP1 is not essential in Tetrahymena spp., and during vegetative growth no clear phenotype is observed in cells lacking Hhp1p (DeltaHHP1). However, during a shift to nongrowth conditions, the survival rate of DeltaHHP1 cells is reduced compared to that of wild-type cells. Upon starvation, Hhp1p becomes hyperphosphorylated concomitant with a reduction in macronuclear volume and an increase in the size of electron-dense chromatin bodies; neither of these morphological changes occurs in the absence of Hhp1p. Activation of two starvation-induced genes (ngoA and CyP) is significantly reduced in DeltaHHP1 cells while, in contrast, the expression of several growth-related or constitutively expressed genes is comparable to that in wild-type cells. These results suggest that Hhp1p functions in the establishment and/or maintenance of a specialized condensed chromatin environment that facilitates the expression of certain genes linked to a starvation-induced response. (+info)
(6/475) Telomerase RNA function in recombinant Tetrahymena telomerase.
Telomerase is a ribonucleoprotein reverse transcriptase specialized for use of a sequence within its integral RNA component as the template for DNA synthesis. Telomerase adds telomeric simple sequence repeats to single-stranded primers in vitro or chromosome ends in vivo. We have investigated the sequences and structures of recombinant Tetrahymena thermophila telomerase RNA necessary for physical association and activity with the catalytic protein subunit expressed in rabbit reticulocyte lysate. In contrast with previous results using another reconstitution method, we find that phylogenetically conserved primary sequences and a phylogenetically nonconserved secondary structure are essential for telomerase RNA function. Telomerase RNA binding to the catalytic protein subunit requires sequences 5' of the template and is highly sequence specific. Other telomerase RNA sequences are required for enzyme activity and proper template use but not for protein interaction affinity. In addition, we demonstrate that the production of active recombinant telomerase requires a factor in rabbit reticulocyte lysate that promotes ribonucleoprotein assembly. These studies demonstrate multiple functions for the telomerase RNA and indicate that recombinant telomerase activity requires more than the catalytic protein and RNA components of the enzyme that have been identified to date. (+info)
(7/475) Identification and mutation of phosphorylation sites in a linker histone. Phosphorylation of macronuclear H1 is not essential for viability in tetrahymena.
Linker histone phosphorylation has been suggested to play roles in both chromosome condensation and transcriptional regulation. In the ciliated protozoan Tetrahymena, in contrast to many eukaryotes, histone H1 of macronuclei is highly phosphorylated during interphase. Macronuclei divide amitotically without overt chromosome condensation in this organism, suggesting that requirements for phosphorylation of macronuclear H1 may be limited to transcriptional regulation. Here we report the major sites of phosphorylation of macronuclear H1 in Tetrahymena thermophila. Five phosphorylation sites, present in a single cluster, were identified by sequencing 32P-labeled peptides isolated from tryptic peptide maps. Phosphothreonine was detected within two TPVK motifs and one TPTK motif that resemble established p34(cdc2) kinase consensus sequences. Phosphoserine was detected at two non-proline-directed sites that do not resemble known kinase consensus sequences. Phosphorylation at the two noncanonical sites appears to be hierarchical because it was observed only when a nearby p34(cdc2) site was also phosphorylated. Cells expressing macronuclear H1 containing alanine substitutions at all five of these phosphorylation sites were viable even though macronuclear H1 phosphorylation was abolished. These data suggest that the five sites identified comprise the entire collection of sites utilized by Tetrahymena and demonstrate that phosphorylation of macronuclear H1, like the protein itself, is not essential for viability in Tetrahymena. (+info)
(8/475) Flanking regulatory sequences of the Tetrahymena R deletion element determine the boundaries of DNA rearrangement.
In the ciliate Tetrahymena thermophila, thousands of DNA segments of variable size are eliminated from the developing somatic macronucleus by specific DNA rearrangements. It is unclear whether rearrangement of the many different DNA elements occurs via a single mechanism or via multiple rearrangement systems. In this study, we characterized in vivo cis-acting sequences required for the rearrangement of the 1.1-kbp R deletion element. We found that rearrangement requires specific sequences flanking each side of the deletion element. The required sequences on the left side appear to span roughly a 70-bp region that is located at least 30 bp from the rearrangement boundary. When we moved the location of the left cis-acting sequences closer to the eliminated region, we observed a rightward shift of the rearrangement boundary such that the newly formed deletion junction retained its original distance from this flanking region. Likewise, when we moved the flanking region as much as 500 bp away from the deletion element, the rearrangement boundary shifted to remain in relative juxtaposition. Clusters of base substitutions made throughout this critical flanking region did not affect rearrangement efficiency or accuracy, which suggests a complex nature for this regulatory sequence. We also found that the right flanking region effectively replaced the essential sequences identified on the left side, and thus, the two flanking regions contain sequences of analogous function despite the lack of obvious sequence identity. These data taken together indicate that the R-element flanking regions contain sequences that position the rearrangement boundaries from a short distance away. Previously, a 10-bp polypurine tract flanking the M-deletion element was demonstrated to act from a distance to determine its rearrangement boundaries. No apparent sequence similarity exists between the M and R elements. The functional similarity between these different cis-acting sequences of the two elements is firm support for a common mechanism controlling Tetrahymena rearrangement. (+info)